Datasets:
ManuelAlv
/
Cancer

Dataset card Data Studio Files Community
1
input
stringlengths
600
32.7k
label
stringclasses
3 values
"colorectal cancer crc remains the third most prevalent cancer type and leading cause of cancerrelated deaths with million cases and deaths worldwide during the occurrence and progression of crc result from a wide array of cellular transformation processes which include genetic and epigenetic mutations that drive uncontrolled cell proliferation and escape from apoptosis2“ chemotherapy and surgery remain the major therapeutic treatment for crc patients5 fluoropyrimidinebased chemotherapy eg 5fluorouracil has been used as the firstline systemic chemotherapy of treating advanced crc for over a half century6 however most patients receiving chemotherapy finally develop drug resistance which is considered to be the major reason for crc therapy failure7 furthermore even though chemotherapy has significant antitumor activity the side effects can affect the quality of a patient's life which makes the new therapeutic approaches urgentdrug design development and therapy “ sun this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomtermsphp and incorporate the creative commons attribution “ non commercial unported v30 license httpcreativecommonslicensesbync30 by accessing the work you hereby accept the terms noncommercial uses of the work are permitted without any further permission from dove medical press limited provided the work is properly attributed for permission for commercial use of this work please see paragraphs and of our terms wwwdovepresscomtermsphp 0csun dovepresstraditional chinese medicines such as dendrobium have been shown to exert anticancer activity in many kinds of cancers89 erianin 2methoxy5[2345trimethoxy phenylethyl]phenol figure 1a a natural compound derived from dendrobium candidum shows various pharmacological activities and therapeutic potential to inhibit multiple cancers in vivo and in vitro10“ li demonstrated that erianin inhibited the proliferation of acute promyelocytic leukemia hl60 cells by regulating the expression of bcl2 and bax10 in addition erianin caused moderate growth delay in xenografted human hepatoma bel7402 and melanoma a37511 furthermore erianin induced cell cycle g2mphase arrest and apoptosis via the jnk signalling pathway in osteosarcoma and bladder cancer1213 erianin can also inhibit cell invasion metastasis and angiogenesis in lung cancer and breast cancer by the figure erianin inhibited crc cells growth a chemical structure of erianin b and c sw480 and hct116 cells were treated with indicated concentration b and time c of erianin cell viability was assessed by cck8 assay p ˂ p ˂ d and e ncm460 cells were treated with indicated concentration d and time e of erianin cell viability was assessed by cck8 assay f sw480 and hct116 cells were performed colony formation assay after being treated with indicated concentration of erianinsubmit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun regulation of ido mpp and timp expressions1415 interestingly besides the function on cell growth apoptosis and migration erianin was found to strongly affect the serum levels of cytokines and immune response in liver cancer16 more importantly in addition to the anticancer effects previous a study also suggested that erianin had no major anrelated toxicities12however to the best of our knowledge neither the mechanism nor the effect of erianin on colorectal cancer has been reported hence in this study we evaluate the antitumor potential and molecular mechanisms of erianin in human colorectal cancer sw480 and hct116 cells and provide a theoretical basis of erianin application for colorectal cancer therapymaterials and methodsmaterialsantibodies against cleaved parp cat bak cat bax cat bcl2 cat bclxl cat catenin cat cyclin d1 cat cmyc cat hdac2 cat and gapdh cat were purchased from cell signaling technologies danvers ma usa antibody against αtubulin cat t6199 was purchased from sigma aldrich co st louis mo usaerianin was purchased from shanghai yuanye bio technology co ltd china and dissolved in dmso wntcatenin signaling inhibitor wnt974 was purchased from medchemexpress monmouth junction nj usa and dissolved in dmsocell culturethe human colorectal cancer cell lines sw480 and hct116 were purchased from american type culture collection atcc manassas va usa cells were maintained in rpmi1640 medium supplemented with fbs thermo fisher scientific waltham ma usa uml penicillin and µgml streptomycin thermo fisher scientific and cells were cultured at °c with co2cell viability and colony formation assaycell viability was assessed with the cell counting kit cck8 dojindo japan according to the manufactorer™s instructionsfor the colony formation assay crc cells cells well were seeded in a sixwell plate and maintained in medium for “ days subsequently the colonies were fixed with paraformaldehyde and stained with crystal violet and the number of clones was counted using an inverted microscopekit quantitative realtime pcr qrtpcrtotal rna from crc cells was isolated using rna isolation kit omega norcross ga usa according to the manufacturer™s protocol total rna µg was used as the template for cdna synthesis by using iscripttm reverse transcription super mix biorad laboratories inc hercules ca usa before the samples were analyzed using sybr green master mix on a realtime pcr system biorad laboratories inc the primer sequences used were as follows cmyc forward 5ʹ aaacacaaacttgaaca gctac3ʹ reverse 5ʹ atttgaggcagtttacatt atgg3ʹ cyclin d1 forward 5ʹaggcggatgagaac aagcaga3ʹ reverse 5ʹcaggcttgactccagaag gg3ʹ cd47 forward 5ʹggcaatgacgaaggaggt ta3ʹ reverse 5ʹatccggtggtatggatgaga3ʹ and gapdh forward 5ʹcacccactcctccacctttg3ʹ and reverse 5ʹccaccaccctgttgctgtag3ʹ the 2δδcq method was used to calculate the relative expression levelswestern blottingfor western blotting μg cellular protein extracts were separated in sdspage gel and were then transferred to nitrocellulose membranes emd millipore burlington ma usa the membrane was blocked with nonfat milk and incubated with primary antibodies overnight at ° then the membranes were incubated with secondary antibody and the proteins were visualized using super signal west pico chemiluminescent substrate thermo fisher scientifictransit transfectionplasmid pegfpn1betacatenin was purchased from addgene watertown ma usa lipofectamine thermo fisher scientific carlsbad ca usa was used for transit transfection according to the instructionscatenin sirna was purchased from sigmaaldrich co lipofectamine rnaimax thermo fisher scientific was used for transfection according to the instructiondrug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepresscell cycle analysisafter treated with vehicle or indicated drugs crc cells were harvested by trypsinization fixed with ethanol and retained at ˆ’°c overnight after cells were centrifuged and washed with pbs they were resuspended in propidium iodide pi solution containing rnase μgml in the dark at room temperature for min and then studied in a flow cytometercaspase37 activity assayapoone„¢ homogeneous caspase37 assay promega corporation madison wi usa was used to measure caspase37 activity briefly apoone® homogeneous caspase37 reagent μlwell was added to a 96well plate and the plate was then placed on a shaker for five minutes “ rpm before incubating for h at room temperature the reading of each well was measured by spectrofluorometerapoptosis assay by annexin vannexin vfitc staining was used to detect the extent of apoptosis induced by erianin briefly crc cells were treated with erianin for h and were then collected and resuspended in μl annexin vbinding buffer and μl pi for minat room temperature in the dark then the cells were finally analyzed by the flow cytometry bd facs calibur with an emission filter of nm for pi red and “ nm for fitc greenapoptosis assay by dapithe effect of erianin on apoptosis induction was evaluated by dapi staining assay crc cells × were seeded in a 96well plate after treatment the cells were washed three times with pbs and paraformaldehyde was added to each well for fixation after permeabilization with triton x100 solution dapi solution was added the cells with condensed and fragmented chromatin were analyzed by echo fluorescence microscopycellular thermal shift assayfor cellular thermal shift assay crc cells were pretreated with μm mg132 for one hour and then incubated with erianin for four hours after washing with icecold pbs cells were aliquot into pcr tubes μl each and incubated at different temperatures for four minutes after being frozen and thawed twice using liquid nitrogen cells were centrifuged and proteins were analyzed by western blottingtopfop luciferase reporter assaythe transcriptional activity of catenin was assessed using the topfop dualluciferase reporter system dual glo„¢ luciferase assay system promega the renilla luciferase plasmid prltk promega which controls for transfection efficiency was cotransfected with catenin responsive firefly luciferase reporter plasmid topflash emd millipore or the negative control fopflash emd millipore using the lipofectamine thermo fisher scientific cells were harvested after h in culture and the luciferase activity was determined by the luciferase assay system promega using a microplate luminometer berthold bad wildbad germanyflow cytometry analysiserianin treated crc cells were washed and resuspended in μl facs buffer and stained with fitcconjugated anticd47 bd biosciences san jose ca usa antibodies all samples were incubated for minutes at °c and then washed twice with facs buffer flow cytometry analyses were performed on bd facs canto iiin vitro phagocytosis assayfor phagocytosis assay thp1 derived macrophages were seeded in a sixwell tissue culture plate erianintreated crc cells were washed and labeled with μm of carboxyfluorescein succinimidyl ester cfse thermo fisher scientific after incubating macrophages in serum free medium for two hours cfselabeled crc cells were added to the macrophages for another two hours at °c macrophages were then washed and imaged with an inverted microscope the phagocytosis efficiency was calculated as the number of macrophages containing cfse labeled crc cells per macrophageschromatin immunoprecipitation chip assaychip assays were performed using the simplechip® enzymatic chromatin ip kit cell signaling technologies according to manufacturer's instructions using the antibodies against h3k27ac immunoprecipitated dna was analyzed by qrtpcr using the following primers cd47 promoter fragment f ²aggatgaatgatgtggcctgt3² and r ² caaacaggcattagcagcgt3² fragment f submit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun ²ggggatgtgttggatacgct3² and r ² ctctg cgttcggctcgtcta3² fragment f ²agggaag agcagagcgagta3² and r ² ttgctttcactcc caccctc3² fragment f ²agagagaggacag tggggc3² and r ² ccagtcgcaggctccaga3² fragment f ² gccgcgtcaacagca3² and r ² aaaggcatcattcttggaaattgt3²with ¨° sw480 cells per mouse suspended in vivo xenograftnodscid shanghai slac laboratory animal co ltd china mice were injected subcutaneously in right flank in µl pbs and mixed with an equal volume of matrigel animals with tumors volume mm3 were divided into two groups n6 and treated with either placebo or mgkg erianin for continuously three weeks by intraperitoneal injection tumor size were measured at the indicated times all the animalrelated procedures were approved by the animal care and use committee of the changchun university of chinese medicine all animal experiments were conducted according to the nih guide for the care and use of laboratory animalsstatistical analysisdata were presented as mean ±sd from three independent experiments p value was determined using paired student™s ttest and a p value ˂ was deemed to indicate statistical significanceresultserianin inhibited crc cell growthfigure 1a illustrates the chemical structure of erianin to investigate the inhibitory effect of erianin on crc cell viability we treated two crc cell lines sw480 and hct116 with different concentrations of erianin and nm for and h as shown in figure 1b and c erianin treatment significantly inhibited the viability of crc cells in a dose and timedependent manner importantly erianin did not show cytotoxic effects on normal human colon mucosal epithelial cell line ncm460 figure 1d and e in addition consistent with the shortterm growth assay our colony forming unit assay also showed that erianin inhibited the colony formation ability of sw480 and hct116 cells figure 1ferianin elevated cell cycle arrest and apoptosisto verify the causal relation of cell viability inhibition the cell cycle distribution was analyzed erianin increased cell number at g2m phase but decreased cell number at s and g0g1 phases after 24h incubation with indicated concentration in sw480 and hct116 cells figure 2a and b to explore the effect of erianin on apoptosis we examined the activity of caspase the protein level of cleaved parp bax bak bcl and bclxl as shown in figure 2c“e the activity of caspase protein level of cleaved parp bak and bax pro apoptosis increased as the concentration of erianin increased in contrast the protein level of bcl2 and bclxl anti apoptotic decreased after erianin treatment figure 2e annexin v flow cytometry and dapi staining further confirmed that erianin could induce cell apoptosis figure 2f and gerianin inhibited catenin translocationincreasing evidence revealed that the wntcatenin pathway plays critical role in colorectal cancer tumorigenesis we hypothesized that erianin might have effect in modulating the wntcatenin pathway first we investigated the effect of erianin on catenin phosphorylation as shown in figure 3a no obvious change was observed on catenin phosphorylation level we then evaluated the effect of erianin on catenin translocation as shown in figure 3b“e catenin expression in cytoplasm was increased whereas expression in the nucleus was decreased with the treatment of erianin in a dose and timedependent manner to further explore the effect of erianin on catenin transcription activity we performed topfop dual luciferase assay we found that topfop relative luciferase activity was significantly decreased after erianin treatment both in sw480 and hct116 cells figure 3f and gerianin bound catenin directlysince erianin inhibited catenin translocation to the nuclear without changing its phosphorylation level we hypothesized that erianin might bind catenin directly to determine whether erianin physically binds catenin we performed a cellular thermal shift assay the results from this experiment indicated that erianin treatment increased the thermal stability of catenin when cells were pretreated with the proteasome inhibitor mg132 for one hour figure 4a and b in contrast erianin treatment had no effect on the thermal stability of gapdh a loading control figure 4a and b these results strongly suggested a specific physical interaction between erianin and catenindrug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressfigure erianin elevated cell cycle arrest and apoptosis a and b sw480 and hct116 cells were treated with erianin for h and then analyzed by pi staining to determine cell cycle phase distribution c sw480 and hct116 cells were treated with erianin for h the relative caspase37 activity was measured using apoone„¢ homogenous caspase37 assay p ˂ p ˂ d and e the protein level of cleaved parp1 bak bax bcl2 and bclxl were analyzed by western blotting after treated with indicated concentration of erianin f and g sw480 and hct116 cells were treated with erianin for h apoptosis was assessed using annexinv flow cytometry analysis f or dapi staining gsubmit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun figure erianin inhibited catenin translocation a the protein level of indicated proteins was analyzed by western blotting after being treated with indicated concentration of erianin for h b“e the protein level of catenin in cytosol and nucleus was analyzed by western blotting after treated with erianin for indicated concentration b and c and time d and e f and g sw480 and hct116 cells were treated with erianin for indicated concentration f and time g the transcriptional activity of catenin was assessed by topfop luciferase reporter assay p ˂ p ˂erianin inhibited the expression of cmyc and cyclin d1as cmyc and cyclin d1 are the direct targets of the wnt catenin pathway we then evaluated the mrna and protein level of cmyc and cyclin d1 unsurprisingly both mrna and protein level of these two proteins were significantly decreased after erianin figure 5a“c interestingly no synergetic effect was observed when combining erianin with wntcatenin signaling inhibitor wnt974 which indicated that erianin regulates cmyc treatment drug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressfigure erianin interacted with catenin a and b sw480 a and hct116 b cells were treated with μm mg132 for one hour followed by four hours incubation with nm erianin before performing thermal shift assay the lower panel shows the charts of percentages of nondenatured protein fractionand cyclin d1 via wntcatenin signaling figure 5d furthermore the inhibitory effect of erianin on cmyc and cyclin d1 expression and cell viability could be reversed by catenin overexpression figure 5e and f which indicated that erianin regulates crc cell growth via catenincd47 mediated phagocytosis we used an in vitro assay by coculturing thp1 derived macrophage with crc cell lines sw480 or hct116 as shown in figure 6g and h treatment of erianin markedly promote colorectal cancer cell phagocytosis by macrophages these results suggest that erianin treatment can attenuate cd47 expression and ultimately promote phagocytosis of crc cellserianin decreased cd47 expression and increased phagocytosisthe immune checkpoint protein cd47 is included in the list of wntcatenin target molecules with a role in immunity escape17 since catenin depletion by sirna inhibited the expression of cd47 figure 6a we then sought to know whether erianin regulates the expression of cd47 first we explored the effects of erianin on cd47 mrna protein and cell surface level in both sw480 and hct116 cells erianin treatment significantly decreased the mrna protein and cell surface level of cd47 figure 6b“d promoter analysis by ucsc genome browser demonstrates that h3k27 acetyl marks are enriched in cd47 promoter regions figure 6e next our chip assay demonstrated that h3k27ac enrichment specifically near promoter region f3f5 was significantly decreased with erianin treatment figure 6f to investigate the effect of erianin on erianin inhibited tumor growth in vivoto investigate the possibility of erianin as a potential therapy in crc we tested the function of erianin on tumor growth in a mouse model the mouse model was established by s c injection of sw480 cells into nodscid mice after three weeks treatment we analyzed the tumor size and weight as shown in figure 7a“c the tumor size and weight from the erianin treatment group were significantly lower than that from the control group in addition after days of bearing tumor the weight of the mice had no significant change figure 7dto examine the impact of therapy on catenin and its downstream signaling localization of catenin protein level of cd47 cmyc bcl2 and bax three representative tumors from each group were analyzed using western blotting as shown in figure 7e and f catenin expression in cytoplasm was increased whereas expression in nucleus was decreased with the treatment of erianin the submit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun figure erianin inhibited the expression of cmyc and cyclin d1 a“c after treated with indicated concentration and time of erianin mrna and protein level of cmyc and cyclin d1 were analyzed by qrtpcr and western blotting p ˂ d sw480 cells were treated with erianin orand wnt974 for h protein levels of cmyc and cyclin d1 were analyzed by western blotting e and f sw480 cells were treated with erianin for h followed by overexpression with catenin plasmid for h protein levels of cmyc and cyclin d1 were analyzed by western blotting e and cell viability was assessed by cck8 assay f p ˂protein level of cd47 cmyc and bcl2 decreased while bax increased after erianin treatment these data indicated that erianin inhibited tumor growth via catenin in vivodiscussioncrc is one of the most malignant and commonly diagnosed solid tumors all around the world18“ although crc incidence rates have declined somewhat chemotherapies are inefficient in most crc patients due to resistance2122 thus the development of acquired therapeutic drugs researching novel and safe treatment strategies is essential for improving the prognosis of crc patients in recent years natural medicinal plants are receiving more and more attention and considered to be important sources of treatment23 novel dendrobium is considered as one of the most important herbs in the orchidaceae family and shows diverse pharmacological functions including anticancer neuroprotective antidiabetic and immunemodulating activities24 erianin derived from dendrobium is one of the most for cancer drug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressfigure erianin decreased cd47 expression and increased phagocytosis a sw480 cells were transfected with nontarget nt or catenin sirna for h protein levels of indicated protein weres measured by western blotting b“d sw480 and hct116 cells were treated with erianin for indicated dose the mrna level b protein level c and cell surface cd47 d were detected by qrtpcr and flow e the ucsc genome browser revealed the enrichment of h3k27ac on cd47 promoter f the enrichment of h3k27ac on cd47 promoter f1f6 was detected by chip assay g and h sw480 and hct116 were treated with indicated concentration of erianin for h representative images showed the effect of erianin on phagocytosis g and bar graphs showed quantitative analysis of phagocytosis h p ˂ p ˂submit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun figure erianin inhibited tumor growth in vivo a typical photos of tumors from the control and erianin treated groups b and c erianin decreased tumor volume and weight p ˂ d mice body weight of control and erianin treated groups was measure at indicated time e the protein level of catenin in cytosol and nucleus in three representative tumors from mouse to mouse of each group were analyzed by western blotting f the protein level of indicated protein in three representative tumors from mouse to mouse of each group were analyzed by western blottingdrug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressnoteworthy constituents that have been used as an antipyretic and an analgesic in traditional chinese medicine25 recently several studies have proved that erianin shows significant antitumour activity in a variety of human cancer cells10ˆ’ consistent with literature in this study we found that erianin had a significant antiproliferative effect against crc cells the inhibitory effect caused by erianin may result from induction of apoptosis and arrest of cell cycle at g2m since the effect of erianin on crc cells has never been studied before we further confirm its antitumor activity in a mouse model which indicated that erianin significantly inhibited tumor growth in vivoseveral signaling pathways including egfrmapk pi3kakt or wntcatenin have been linked to crc genesis and progression26 as the aberrant activation is present in almost all crc cases wntcatenin signaling is prominent among these pathways27 inactivated mutations in the apc gene leads to stabilization and ensuing nuclear translocation of catenin to facilitate tcflef dependent transcription of wntcatenin signaling target genes such as cmyc and cyclin d1 to drive cell proliferation survival and metastasis28“ to understand the mechanisms of action of erianin we assessed the effect of erianin on wntcatenin pathway interestingly we found that erianin treatment had no effect on catenin phosphorylation but inhibited the translocation of catenin in the nucleus which suggested to us that erianin physically interacts with catenin our cellular thermal shift assay confirmed this hypothesis the thermal stability of catenin increased after erianin treatment as catenin downstream targets the expression level of cmyc and cyclin d1 significantly decreased after erianin treatmentcd47 a transmembrane glycoprotein expresses ubiquitously and mediates a œselfdonoteatme signal on normal cells however cd47 is often upregulated in tumor cells to evade innate immunity31“ anticd47 antibodies which block cd47 sirpα interactions and promote macrophage mediated phagocytosis of tumor cells has shown promise in several solid tumors31 in colorectal cancer cd47 promotes colon cancer cell migration and metastasis34 in addition upregulated immuneescape pathways such as cd47 sirpα are responsible for immune escape and survival in circulating tumor cells of colorectal cancer35 myc an oncogene identified as a wntcatenin target gene was reported to control cd47 transcription therefore mutations in components of the wntcatenin signaling pathway which induced
Colon_Cancer
" postmortem studies can provide important information for understanding new diseases and smallautopsy case series have already reported different findings in covid19 patientsmethods we evaluated whether some specific postmortem features are observed in these patients and if thesechanges are related to the presence of the virus in different ans complete macroscopic and microscopicautopsies were performed on different ans in covid19 nonsurvivors presence of sarscov2 was evaluatedwith immunohistochemistry ihc in lung samples and with realtime reversetranscription polymerase chainreaction rtpcr test in the lung and other ansresults pulmonary findings revealed earlystage diffuse alveolar damage dad in out of patients andmicrothrombi in small lung arteries in patients latestage dad atypical pneumocytes andor acute pneumoniawere also observed four lung infarcts two acute myocardial infarctions and one ischemic enteritis were observedthere was no evidence of myocarditis hepatitis or encephalitis kidney evaluation revealed the presence ofhemosiderin in tubules or pigmented casts in most patients spongiosis and vascular congestion were the mostfrequently encountered brain lesions no specific sarscov2 lesions were observed in any an ihc revealedpositive cells with a heterogeneous distribution in the lungs of of the patients rtpcr yielded a widedistribution of sarscov2 in different tissues with patients showing viral presence in all tested ans ie lungheart spleen liver colon kidney and brains in autopsies revealed a great heterogeneity of covid19associated an injury and theremarkable absence of any specific viral lesions even when rtpcr identified the presence of the virus in many anskeywords covid19 sarscov2 autopsy rtpcr immunohistochemistry correspondence isabellesalmonerasmeulbacbe1department of pathology erasme hospital universit libre de bruxellesulb route de lennik brussels belgium2centre universitaire inter rgional d™expertise en anatomie pathologiquehospitali¨re curepath chirec chu tivoli ulb rue de borfilet 12a jumet belgiumfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cremmelink critical care page of severeacuteincluding coronavirusesrespiratorysyndrome coronavirus sarscov and middle eastrespiratory syndrome coronavirus merscov causesevere acute respiratory failure which is associated withhigh mortality rates the novel sarscov2 strainexhibits phylogenetic similaritiesto sarscov andcauses coronavirus disease covid19 which hasresulted in more than deaths worldwide so faras the pandemic has progressed the pathophysiology ofthis viral infection has become clearer in particular ithas been shown that sarscov2 can directly alter cellfunction by a link to the angiotensin converting enzyme ace2 receptor which is almost ubiquitous in thehuman body nevertheless the mechanisms behind the high mortalityand severe an dysfunction associated with covid19remain poorly understood controversies exist regardingthe occurrence of fatal complications such as pulmonaryembolism or diffuse endothelial injury [ ] as well as onthe roles of direct viral cellular injury or concomitantcomorbidities in the fatality of this disease in this setting autopsy is of great importance to helpphysicians understand the biological characteristics andthe pathogenesis of covid19 most of the previously reported postmortem findings focused on lung morphologyand few data are available on complete postmortemanalyses of other ans [ ] the aim of this study wastherefore to investigate the presence of specific features ofviral injury as well as the distribution of the virus in different ans of patients who died from covid19methodsstudy designin this postmortem study we included the first adultpatients years who died in our hospital either in acovid19 unit or an intensive care unit from march with confirmed sarscov2 infection ie positivertpcr assay on nasopharyngeal swab andor bronchoalveolar lavage specimen exclusion criteria were lack offamily consent and a delay of more than days after deathbefore postmortem examination the study protocol wasapproved by the local ethics committee p2020218data collectionrelevantwe collected demographics comorbiditiesclinical dataincluding duration between symptomonset or hospitalization and death the results of chestcomputed tomography scan andif available microbiological tests and medical treatments eg hydroxychloroquine antivirals or antibiotics and use of ansupport acute respiratory distress syndrome ardsand acute kidney injury aki were defined accordingto standard definitions [ ]postmortem procedurethe belgian public health institute sciensano guidelines were integrated into our postmortem procedure the cadavers were kept in the refrigerator at °cand autopsies were performed to h after death toensure the safety of the autopsy team personal protective equipment consisted of two superposed disposablelatex gloves plastic sleeves ffp3 mask scrub hat clearface visor surgical gown plus plastic apron and rubberboots in the postmortem room dirty and clean circulations were used in the airlocks to allow decontaminationall analyses were performed at normal pressurespleen bone marrow kidney bladderusing standard surgical pathology processing completesets of tissue samples were collected for diagnosis andbiobanking the material was biobanked by biobanqueh´pital erasmeulb be_bera1 cub h´pital erasmebbmrieric the banked material consists of samplesper an including the trachea thyroid lymph nodesheartliverstomach colon and brain for the lungs we collected sixsamples per lobe ie a total of samples except fortwo patients who had undergone lobectomy for cancerand from whom only samples were taken for safetyreasons complete brain removal was not allowed butwith the help of a neurosurgeon in cases we used anew safe procedure with drills and protective devicesto avoid air dispersion to obtain between and samples from different brain regions as detailed inthe additional file additional material formalinfixed paraffinembedded ffpetissues underwentstandard processing to provide hematoxylin and eosinhestained sections special stains and immunohistochemistry ihc were used for lung masson™s trichromeperiodic acidschiff [pas] gomorigrocott anticmvihc antihsv ihc antipneumocystis j ihc and kidneypas masson™s trichromejones methenamine silversamplesmorphological analysismorphological analysis was performed on he stainedglass slides using the secundos digital platform tribvnhealth care chatillon francefor digital diagnosisafter the acquisition of whole slide digital scans — magnification using a nanozoomer ht slide scanner hamamatsu hamamatsu city japansarscov2 detection by immunohistochemistrysince no antibody against sarscov2 has been validatedfor ihc on ffpe tissues we selected an antisarsnucleocapsid protein antibody standard ihc was appliedas previously described to 4μmthick postmortem lungsections one sample for each lung lobe per patient to display sarsnucleocapsid protein invitrogen pa141098dilution on dako omnis agilent technologies 0cremmelink critical care page of santa clara ca usa using the envision flexdetection system according to the manufacturer™sprotocol the sections were counterstained withhematoxylin negative tissue controls were obtainedfrom patients who had an autopsy before the covid pandemic semiquantitative ihc evaluation wasperformed by two senior pathologists nd mr as follows negative ˆ’ between one and five positive cellsper whole slide scattered cells more than five cellsper whole slide but no foci isolated cells andwith foci more than cells in one — field sarscov2 detection by rrtpcrtotal nucleic acid was extracted from ffpe tissues usingthe maxwell rsc dna ffpe kit reference as1450promega corporation madison wi usa and thepromega maxwell extractor following the protocol described by the manufacturer onestep rtpcr assaysspecific for the amplification of sarscov2 e envelopeprotein gene were adapted from a published protocol briefly μl of rna ng was amplified in μl reaction mixture containing μl of taqman fastvirus 1step master mix life technologies μm ofeach forward acaggtacgttaatagttaatagcgt and reverse atattgcagcagtacgcacacaprimers and μm of probe famacactagccatccttactgcgcttcgbbq the amplification condition was °c for min for reverse transcriptionfollowed by °c for s and then cycles of °c for s and °c for s a clinical sample highly positivefor sarscov2 was diluted and used as a positive control in each analysis a clinical sample obtainedfrom a patient who was autopsied before the covid19pandemic was used as a negative control the quality ofthe rna from the samples showing negative results wasassessed by amplification of the human met rna according to a validated iso15189 accredited methodused as a routine diagnostic method in our laboratorystatistical analysisdata are reported as counts percentage or medians[interquartile ranges iqrs] all data were analyzedusing graphpad prism version graphpad software san diego ca usaresultsstudy cohortthe main characteristics of the study cohort malesout of median age [“] years are given intable the time period between the onset of symptoms and death ranged from to days median days and between admission and death from to days median days all except two patients had atleast one comorbidity including hypertension n diabetes n cerebrovascular disease n coronaryartery disease n and solid cancer n none ofthe patients had tested positive on admission for therespiratory syncytial virus or influenza a and b viruseseleven of the patients were treated with mechanicalventilation eleven patients died in the icu and on themedical ward the main causes of death were respiratoryfailure n and multiple an failure n laboratory data are reported in additional file table s1macroscopic findingsone patient had had a left pneumonectomy and onepatient a right bilobectomy the lungs were typicallyheavy and the lung parenchyma had a diffuse firmconsistency with redtan and patchy darkred areas ofhemorrhage thrombi were found in the large pulmonary arteries in patients and lung infarction in patientspleural adhesions associated with pleural effusions were observed in cases we observed cardiomegaly in and hepatomegaly in patients the kidneys were often enlargedwith a pale cortex and petechial aspect but no hemorrhageor infarct the gut had advanced postmortem autolysiswith no evidence of specific lesions except for one patientwho had ischemic enteritis in the patients for whombrain samples were available one had had a recently drainedsubdural hematoma and another a cerebral hemorrhagemicroscopic findingsfile as shown in figs and and additionaltable s2the main pulmonary findings includedearlystage diffuse alveolar damage dad which consisted ofinterstitial and intraalveolar edema withvariable amounts of hemorrhage and fibrin depositioninterstitial mononuclearhyaline membranes minimalinflammatoryii pneumocytehyperplasia microthrombi were noted in the smallpulmonary arteries in patients ten of the patientsalso had advanced dad lesions ie fibroblastic proliferation within the interstitium and in the alveolar spaces patients had evidence of acute pneumonia or bronchopneumonia had atypical pneumocytes and three hadsyncytial multinucleated giant cells we observed no viralinclusions or squamous metaplasiaand typeinfiltrateall the patients who survived more than weeksn had late dad lesions there was no relationship between the delay from onset of symptoms todeath orfrom hospitalization to death and thepresence of other histological lesions including bronchopneumonia pneumonia microthrombiischemiclesions pulmonary emboli or pulmonary infarct in of the patients who had not received mechanicalventilationthe delay between hospitalization anddeath was less than days in this group only casehad microthrombi the other patients had longer 0cremmelink critical care page of table characteristics of the study populationidct scancomorbiditiesagesexrrtpcrpostime todeathantemorteman failureardsakiposnegnegmfmfmcadcvddiabeteshypertensioncadcrfliver cirrhosiscopdcancerhypertensioncancercvdcopdcancerggoposmaggopospostreatmentscause of deathmechanicalventilationantibioticshydroxychloroquineantibioticscorticosteroidsmechanical ventilationhydroxychloroquinelopinavirritonavirantibioticsmechanical ventilationhydroxychloroquineantibioticsmechanical ventilationecmorrthydroxychloroquinelopinavirritonavirantibioticsmechanical ventilationecmohydroxychloroquineremdesivircorticosteroidsantiobioticshydroxychloroquineantibioticscardiogenicshockmofrespiratory failurerespiratory failurerespiratory failuremesentericischemiamofrespiratory failurerespiratory failureantibioticsseptic shockmofmechanical ventilationrrthydroxychloroquinelopinavirritonavirantibioticsmechanical ventilationecmorrthydroxychloroquineoseltamivirantibioticshydroxychloroquineantibioticsrespiratory failurerespiratory failuresudden deathmechanical ventilationhydroxychloroquineantibioticsmofhydroxychloroquinerespiratory failurehydroxychloroquineantibioticsrespiratory failureardsakihypoxichepatitisardsakiardsardsakihypoxichepatitisardsakiardsakihypoxichepatitisakiardsakiardsakiardsardsakihypoxichepatitisardsardsakihypoxichepatitismhypertensioncrfbcposmnonebcposmfhypertensioncadcvdcrfdiabeteshypertensiondiabetesemphysemaposggoposmhypertensiondiabetesggobcposmmmfdiabetesliver cirrhosiscancerdiabeteshypertensioncaddiabetesdiabeteshypertensiondiabetesbcposggoposggobcggobcpospos 0cremmelink critical care page of table characteristics of the study population continuedidcomorbiditiesct scanagesexmggolpposrrtpcrtime todeathfmhypertensiondiabetesliver transplanthypertensioncvdggobcposggobclpposantemorteman failureardsakipulmonaryembolismardsakipulmonaryembolismardsakipulmonaryembolismtreatmentscause of deathmechanical ventilationrrthydroxychloroquineremdesivirantibioticsmechanical ventilationrrthydroxychloroquineantibioticsmechanical ventilationecmorrthydroxychloroquineantibioticsseptic shockmofseptic shockmofseptic shockmoftime to death time from admission to death days cause of death was reported by the attending physician m male f female rrtpcr reverse transcription realtime polymerase chain reaction used as diagnostic laboratory test neg negative pos positive cad coronary artery disease cvd cerebrovascular disease lp lobarpneumonia ggo groundglass opacity ma minor abnormalities bc bilateral consolidation copd chronic obstructive pulmonary disease crf chronic renal failureards acute respiratory distress syndrome aki acute kidney injury ecmo extracorporeal membrane oxygenation rrt renal replacement therapy mof multiplean failuredelays between hospitalization and death daysthey had no microthrombififteen patients had signs of chronic ischemic cardiomyopathy of different severities and patients had signsof acute myocardial infarction there was no evidence ofcontraction bands or myocarditis histological evaluationof the kidneys was limited because of moderate to severepostmortem autolysis occasional hemosiderin granuleswere observed in the tubular epithelium in patientsand pigmented casts in in the medulla edematousexpansion of the interstitial space without significant inflammation was observed in patients chronic renal lesions ie nodular mesangial expansion and arteriolarhyalinosis glomerulosclerosis or chronic pyelonephritisfig main histological findings green finding present gray finding absent black unavailable 0cremmelink critical care page of fig pulmonary histological findings a earlystage diffuse alveolar damage dad hyaline membrane he — magnification with a zoom ona giant cell — magnification b fibrin thrombi in a pulmonary artery he — magnification c latestage dad fibroblastic proliferationhe — magnification d latestage dad fibroblastic proliferation trichrome staining — magnification e acute pneumonia he — magnification f antisarscov immunohistochemistry ihcpositive cells — magnificationwere also observed no microthrombi were identifiedbut one patient had a thrombus in an interlobar arteryliver examination revealed congestive hepatopathyand steatosis but no patchy necrosis hepatitis or lobular lymphocytic infiltrate the histological changes in theabdominal ans including the esophagus stomachand colon are reported in additional file table s2most of the findings were related to chronic underlyingdiseases except for one case of ischemic enteritisbrain samplesshowed cerebral hemorrhage orhemorrhagic suffusion n ischemic necrosisn edema andor vascular congestion n anddiffuse or focal spongiosis n we found no evidence of viral encephalitis or vasculitis isolated neuronalnecrosis or perivascular lymphocytic infiltrationfocalsarscov2 detection in the lungs by ihcsarscov2 was identified by ihc in the lungs of ofthe patients fig howeverthere was largevariability in the distribution of sarscov2positivecells in the lung parenchymasarscov2 detection by rtpcrsarscov2 rna was detected in at least one anfrom every patient fig in the lung rtpcr waspositive in patients with threshold cycle ct valuesvarying from to viral rna was alsodetected in the heart n the liver n thebowel n the spleen n and the kidney n as well as in of the cerebral samples ct valuesfor nonpulmonary ans ranged from to eight patients had positive rtpcr in all tested ansabnormalitiesdiscussionthis postmortem study showed several histopathologicalin covid19 nonsurvivorshowever none of the findings was specific for direct viralinjury even though sarscov2 was detected in all examined ans using rtpcr we decided to performcomplete autopsies rather than other techniques such aspostmortem core biopsies so as to obtain a better overview of all ans especially the lungs we collected samples from each lobe this approach enabled us to 0cremmelink critical care page of fig detection of sarscov2 by immunohistochemistry ihc in ffpe post mortem lung samples of patients semiquantitative evaluationœˆ’ negative result œ scattered positive cells between and positive cellswhole slide œ positive isolated cells cellswhole slide butno foci œ foci of positive cells more than positive cells in one — field na not availabledocument the considerable heterogeneity of histologicallesions and of sarscov2 spread through the bodythe diagnosis of sarscov2related an injury ischallenging postmortem histologicalfindings wereheterogeneous and often associated with chronic underlying diseases in a previous autopsy study in covid19patients the authors reported that dad associatedwith viral pneumonia was almost impossible to distinguishfrom that caused by bacterial pneumonia no obviousintranuclear or intracytoplasmic viralinclusions wereidentified in another report desquamation of pneumocytes and hyaline membrane formation are frequentlydescribed in ards of many different causes especially inearlyphase ards the presence of multinucleatedcells with nuclear atypia is used to diagnose herpes virusinfection in daily practice as in previous reports [ ]we also observed the presence of multinucleated cellswithin lung alveoli in three patients however the significance of multinucleated cells is unclear and may not bespecific of sarscov2 infection finally some ofthe microscopic features of these patients are compatiblewith an changes secondary to shock or systemicinflammation and no histological finding could be specifically ascribed to sarscov2in the absence of typical postmortem viral featuresour results show that rtpcr is feasible on ffpe blocksand could be used in postmortem analyses to identifythe presence of sarscov2 in multiple ans and tounderstand the spread of the virus within the humanbody the discordant rtpcr and ihc results fordetection of sarscov2 in the lungs may be explainedby the different sensitivity of these assays which washigher for the rtpcr whereas lowlevel viral replication might not be detected by ihc moreover ihc wasbased on the only available antibodies which aretargeted against sarscov new antibodies againstsarscov2 need to be developed to improve theaccuracy of ihc in the analysis of tissue samples fromsuspected or confirmed covid19 patientsmost of the previous postmortem studies in covid19patients were conducted using needle biopsies and weretherefore rather limited in terms of sampling our completeautopsy analysis identified considerable heterogeneity ofsarscov2 spread through the human body and providesa more accurate description of macroscopic and microscopic an alterations as for previous coronavirusdiseases [ ] the lungs are the most affected ans incovid19 however dad findings werehighly 0cremmelink critical care page of fig molecular detection of sarscov2 rna in postmortem samples detection of sarscov2 by reverse transcription realtime polymerasechain reaction rtpcr in ffpe postmortem tissues of patients œ positive result œˆ’ negative result œna tissue not available nc noninformative test result due to lowquality rnaheterogeneous including both earlyonset and additionallate lesions this finding could be explained by the heterogeneity of the pulmonary injury including compliant lungsin the early phase and a more dense and nonrecruitablelung in the late phase as some patients died outsidethe icu without receiving mechanical ventilation we couldnot estimate lung compliance before death the heterogeneity could also reflect different treatments eg fluid administration or corticosteroids or different complications asan example half of the patients had concomitant acutepneumonia and it is difficult to conclude whether the dadreflected the natural timecourse of the viral disease or wassecondary to superimposed complications such as nosocomial infections in a recent report needle postmortem biopsies suggested that covid19 is not associated withdad but rather with an acute fibrinous and anizingpneumonia afop consequently requiring corticoid treatment a diagnosis of afop is based on the absence ofhyaline membranes and the presence of alveolar fibrin ballshowever hyaline membranes are heterogeneously distributed in the lung parenchyma with dad and complete lunganalysis not just biopsies are necessary to exclude theirpresence moreover afop may be a fibrinous variant ofdad the limitation of lung biopsy was also shown inanother study in which only of lung samples werepositive for sarscov2 using rtpcr when compared to almost in our series in addition we did notfind specific œendothelitis as previously reported in a smallcase series considering the heterogeneity of postmortem covid19 associated lesions molecular and ihcassessments are mandatory in the histological analysis ofcovid19 tissue samplespatients with covid19 often have altered coagulation and a prothrombotic status with the possible development of acute pulmonary embolism pe in ourstudy three patients had pe already diagnosed beforedeath four patients had pulmonary infarction in a previous study acute pe was considered as the main causeof death in four patients however the inclusion ofpatients who died before hospital admission and the lackof specific thromboprophylaxis during the hospital staymay account for the differences in the severity of pewhen compared to our study although we frequentlyobserved the presence of microthrombi in the lung parenchyma this feature is also reported in other forms ofards regardless of etiology [ ] as such whetherdiffuse pulmonary thrombosis is a main contributor ofthe fatal course of severe hypoxemia in covid19 0cremmelink critical care page of patients remains to be further studied in a systematicreview of pathologicalfindings in covid19 polak identified a timeline in the histopathologicalfindings in the lung with epithelial dad denudationand reactive pneumocytes atypia and vascular microvascular damage thrombi intraalveolar fibrin depositschanges present at all stages of the disease but fibroticchanges interstitialfibrous changes only appearingabout weeks after the onset of symptoms few patientshad fibrosis at early stages and in these cases it waslikely because of preexisting lung disease our resultsare consistent with those of polak except forthe lack of late fibrotic changes which may be related tothe use of antiinflammatory drugs at high doses fornearly all our patients we did not observe specific viral an injury such asmyocarditis hepatitis or encephalitis the cases ofœacute cardiac injury reported in covid19 clinicalstudies do not necessarily translate into myocarditisor acute myocardialischemia only two had acutemyocardial ischemia similar to data reported in septicpatients ie elevated troponin without overt cardiacischemia however using rtpcr we found thevirus in almost all the examined ans this suggeststhat the virus can bind to most cells probably via theace2 receptor which is ubiquitous but may notdirectly cause an injury as extrapulmonary directviral injury eg encephalitis hepatitis or myocarditishas only been reported in very few cases we suggest thatsarscov2 infection may be just the trigger for anoverwhelming host response which could secondarilyresult in covid19associated an dysfunction asrtpcr mightitremains unclear whether this represents active viral replication into the tissues or previous cellular infectionwithout clinically relevant significance just detect residual viral genomethis study has several limitations i we only includedpatients who had had a positive rtpcr on nasopharyngeal swab andor bronchoalveolar lavage to ensurethat only true positive cases were enrolled we decidednot to include three patients who had had thoracic ctscan findings suggestive of covid19 but had negativertpcr results this limitation in our study reflects thedifficulty of diagnosing covid19 on a clinical basis iithe sample size was relatively small and autopsies wereonly carried out from to h after death this delaydid not allow us to properly analyze the gastrointestinaltract and kidneys which showed signs of autolysis inparticular acute tubular injury in the proximal tubuleswas indistinguishable from autolysis iii we could notdetermine the timecourse andor sequence of anspread of the virus and no specific hypothesis regardinghow sarscov2 spreads eg hematogenously couldbe identified and iv the time to death differed frompatient to patient as did the course of the disease andtreatments received which limits a precise clinicalpathological correlation of histological findings related tocovid19 finally we did not evaluate specific mechanisms involved in the pathogenesis of an injurythese results underline the heterogeneity of an injuriesduring covid19 disease and the absence of specificsarscov2 lesions using rtpcr sarscov2 couldbe detected in all ans even those without evidentmicroscopic lesionssupplementary informationsupplementary information accompanies this paper at httpsdoi101186s13054020032185additional file critical careautopsycovid additional materialprocedure to obtain brain samplesadditional file critical careautopsycovid additional table s1laboratory findings on the day of admissionadditional file critical careautopsycovid additional table s2detailed histological findings in all patientsabbreviationsace2 angiotensin converting enzyme afop acute fibrinous andanizing pneumonia aki acute kidney injury ards acute respiratorydistress syndrome covid19 coronavirus disease ct threshold cycledad diffuse alveolar damage ffpe formalinfixed paraffinembeddedhe hematoxylin and eosin ihc immunohistochemistry iqrs interquartileranges merscov middle east respiratory syndrome coronaviruspas periodic acidschiff pe pulmonary embolism rtpcr realtime reversetranscription polymerase chain reaction sarscov severe acute respiratorysyndrome coronavirusacknowledgmentsthe authors thank nathalie lijsen christophe valleys gees lacroixbarbara alexiou dominique penninck nicole haye and audrey verrellen fortechnical and logistic supports prof frdric schuind and dr djameleddineyahiacherif for neurosurgical procedure egor zindy diapath ulb forproofreading the paper and dr mariepaule van craynest for trainees™supervisionauthors™ contributionsis had the idea for and designed the study and had full access to all thedata in the study and takes responsibility for the integrity of the data andthe accuracy of the data analysis is ft jlv and cd drafted the paper mrcv ll pl mlr cm alt jcg lp rdm sd sr nd lp and od collected thedata mr nd and rdm did the analysis and all authors critically revised themanuscript for important intellectual content and gave final approval for theversion to be published all authors agree to be accountable for all aspectsof the work in ensuring that questions related to the accuracy or integrity ofany part of the work are appropriately investigated and resolvedfundingthis study received financial support from fonds y bo«l brussels belgiumfonds erasme pour la recherche mdicale brussels belgium and œappel  projet spcial covid19 ulb brussels belgium the cmmi is supported bythe european regional development fund and the walloon region ofbelgium walloniabiomed grant no project œcmmiulbsupport the center for microscopy and molecular imaging and its diapathdepartment cd is a senior research associate with the fnrs belgiannational fund for scientific research 0cremmelink critical care page of availability of data and materialsthe data that support the findings of this study are available from thecorresponding author on reasonable request participant data without namesand identifiers will be made available after approval from the correspondingauthor and local ethics committee the research team will provide an emailaddress for communication once the data are approved to be shared withothers the proposal with a detailed description of study objectives andstatistical analysis plan will be needed for the evaluation of the reasonabilityto request for our data additional materials may also be required during theprocess d'haene n melndez b blanchard o de n¨ve n lebrun l vancampenhout c design and validation of a genetargeted nextgeneration sequencing panel for routine diagnosis in gliomas cancersbasel corman vm landt o kaiser m molenkamp r meijer a chu dk detection of novel coronavirus 2019ncov by realtime rtpcr eurosurveill de hemptinne q remmelink m brimioulle s salmon i vincent jl ards aclinicopathological confrontation chest “ menter t haslbauer jd nienhold r savic s hopfer h deigendesch n ethics approval and consent to participatethe study protocol was approved by the local ethics committee erasmehospital p2020218 the ethical committee has waived the need for writteninformed consentpostmortem examination of covid19 patients reveals diffuse alveolardamage with severe capillary congestion and variegated findings of lungsand other ans suggesting vascular dysfunction histopathology epub ahead of print httpsdoi101111his14134franks tj chong py chui p galvin jr lourens rm reid ah lungpathology of severe acute respiratory syndrome sars a study of autopsy cases from singapore hum pathol “ nicholls jm poon ll lee kc ng wf lai st leung cy lungpathology of fatal severe acute respiratory syndrome lancet “ hwang d chamberlain d poutanen s low de asa sl butany j pulmonarypathology of severe acute re
Colon_Cancer
alcoholic liver disease ald is a chronic alcoholinduced disorder of the liver for which there are few effectivetherapies for severe forms of ald and for those who do not achieve alcohol abstinence in this study we used asystematic drugrepositioning bioinformatics approach querying a large compendium of geneexpression profiles toidentify candidate us food and drug administration fda“approved drugs to treat ald one of the top compoundspredicted to be therapeutic for ald by our approach was dimethyl fumarate dmf an nuclear factor erythroid related factor nrf2 inducer we experimentally validated dmf in liver cells and in vivo our work demonstrates thatdmf is able to significantly upregulate the nrf2 protein level increase nrf2 phosphorylation and promote nrf2nuclear localization in liver cells dmf also reduced the reactive oxygen species ros level lipid peroxidation andferroptosis furthermore dmf treatment could prevent ethanolinduced liver injury in ald mice our results provideevidence that dmf might serve as a therapeutic option for ald in humans and support the use of computationalrepositioning to discover therapeutic options for aldintroductionoxidative stress is implicated in the development ofdiverse liver disorders such as alcoholic liver diseaseald12 ald encompasses a variety of chronic liverdiseasesincluding liver steatosis fatty liver hepatitiscombined with ‚ammation fibrosis cirrhosis andultimately hepatocellular carcinoma hcc3 althoughalcohol abstinence is effective for patients with mild aldsteatosis there are few effective therapies for severeforms of ald and for those who do not achieve alcoholabstinence corticosteroid is the only treatment option toimprove the shortterm survival of severe alcoholiccorrespondence yongheng chen yonghenc163com orting liu liuting818126com1department of oncology nhc key laboratory of cancer proteomics statelocal joint engineering laboratory for anticancer drugs national center feriatrics clinical research xiangya hospital central south university changsha hunan china2department of gastroenterology xiangya hospital central south university changsha hunan chinathese authors contributed equally ye zhang shuang zhaoedited by m agostinihepatitis ah patients4 however many of these patientsdo not respond to this treatment and experience severeadverse effects such as infection5 therefore there is anurgent need to develop novel targeted therapeutics totreat severe forms of ald or patients who fail to achievealcohol abstinence the computational repositioning offood and drug administration fdaapproved drugs is apromising and efficient avenue for discovering new uses6given the high costs possible side effects high failurerate and long testing periods for developing new medicines an fdaapproved compound was known to begenerally safe in humans and available for clinical use7 itis possible to identify safe drugs with potentialforrepurposing in other conditions by using computationalstrategies which can eliminate the need for a phase isafety trial and expedite phase ii efficacy trials analysis ofinteractions between genes and fdaapproved drugsallow the pursuit of new indications for treating diseaseswith no fdaapproved pharmacotherapiesrecent advancements in computing and the dramaticexpansion of available highthroughput datasets have the authors open access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate ifchanges were made the images or other third party material in this are included in the ™s creative commons license unless indicated otherwise in a credit line to the material ifmaterial is not included in the ™s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this license visit httpcreativecommonslicensesby40official of the cell death differentiation association 0cenabled the development of drug repurposing to identifynovel treatment options for ald thus in this study weaimed to identify a new therapeutic option with potential forrepositioning in ald we used a systematic computationalapproach based on both public geneexpression patterns inald and the interactions between genes and fdaapproved drugs interestingly we identified nuclear factorerythroid 2related factor nrf2 as a novel therapeutictarget in ald8 nrf2 is a basic leucine zipper bziptranscription factor that regulates the expression of certainproteins which protect cells against oxidative stress underunstressed conditions nrf2 is kept in the cytoplasm bykelch likeechassociated protein keap1 and cullin3upon oxidative stress nrf2 is phosphorylated at ser40 andreleases from keap1 then translocates into the nucleus inthe nucleus nrf2 forms a heterodimer with one of thesmall maf proteins maff mafg and mafk binds tothe antioxidant response element are in the promoterregions of many antioxidative enzymes and regulates thetranscription of these enzymes such as glutamate“cysteineligase catalytic gclc and heme oxygenase1 ho1more surprisingly we found that the fdaapproved nrf2inducer9 dimethyl fumarate dmf which has not previously been described to have a therapeutic associationwith ald was determined to have a strong therapeuticpotential for repositioning in ald we evaluated the efficacy of dmf for ald in liver cells and in vivo using anethanolinduced mouse model concordant with our computational prediction the experimental results demonstratethat dmf is able to significantly ameliorate ethanolinducedliver injury compared to untreated groupsresultscomputational repositioning of fdaapproved drugs foraldto identify efficient therapeutic strategies for patientswith liver diseases we downloaded drug datasets thatcontain both clinical application and animal test fromgene expression omnibus wwwncbinlmnihgovgeogse accession number gse28619 and then weused a bioinformatics approach to testthe drugrepositioning potential of fdaapproved drugs for aldfrom this approach we computed the activity score ofcandidate drugs and compared geneexpression profiles inresponse to these drugs in ald then we annotated theknown gene targets of the topscoring candidates andqueried fdaapproved drugbank using gene targets as aninput which displayed an output of a list of chemicalcompounds notably ald cells are known to abnormallyexpress molecules in the antioxidant response pathwaythus we aimed to study one of the five topscored candidate genes nrf2 among nrf2compound interactionsthe main use of dmf is previously tested with some success in multiple sclerosis patients with relapsing formsofficial of the cell death differentiation associationfocused on thesuggesting that dmf used in the clinic may affect the aldgeneexpression signature this analysis led us to focus ondrugs targeting molecules fig 1a the majority of knownphysiologic or pharmacological nrf2 inducers are electrophilic molecules that covalently modify by oxidation oralkylation cysteine residues presentin the thiolrichkeap1 protein10 dmf is one of the known nrf2 inducers which has been tested for the treatment of multiplesclerosis and approved in for its drug bioavailabilityand efficacy11 currently mmf has been used to develop asecond generation of nrf2 inducers as prodrugs12therefore wefumarateregulationmechanism of nrf2 in liver disorders the generation oftoxic metabolites by ethanol such as lipidperoxidationproducts contributes to the pathogenesis of alcoholic liverinjury fumarates prevent ros accumulation via the nrf2pathway in liver cells therefore we used an ald mousemodel six mice a group and hepatic fibrosis rat modelnine rats a group to examine the role of fumarates in vivohepatic lipid accumulation was distinctively increased inethanolfed rats in order to address the role of dmf inhepatic lipid accumulation we administered ald micewith dmf at mgkgday or mgkgday for daysin order to address the role of dmf in hepatic fibrosis weadministered hepatic fibrosis rats with dmf at mgkgday or mgkgday for weeks dmf ameliorated thehepatic steatosis induced by ethanol as observed in liversections stained with hematoxylin and eosin he fig 1band supplementary fig s1a at the same time the highlycrosslinked collagen fraction increased significantly during ethanolinduced fibrosis progression while collagendeposition was partly reduced under dmf treatment fig1c and supplementary fig s1b to substantiate thefinding that dmf increases the activity of nrf2 pathwayto inhibit ald we collected liver sections from normaland ald mice and checked nrf2 and gclc proteinlevels in the mouse model we performed immunohistochemistry ihc and western blotting for nrf2 andgclc results revealed that dmf treatment significantlyincreased nrf2 and gclc protein levels in ald mouseliver when compared to the matched control groups fig1d“f and supplementary fig s1c ddmf and mmf activate the nrf2 pathway in liver cellsregulator ofnrf2 is an essentialthe antioxidantresponse pathway which promotes the expression of various genes in response to oxidative stress1314 fumaratesprotect neurons and astrocytes against ros damage15 todetermine whether dmf or mmf regulates the nrf2protein level in liver cells we cultured hepg2 and lo2cells under the treatment of μm dmf and mmf fordifferent lengths of time and found that both dmf andmmf increased the protein level of nrf2 in a timedependent manner fig 2a and supplementary fig s2a 0czhang et al cell death and disease page of fig see legend on next pageofficial of the cell death differentiation association 0czhang et al cell death and disease page of see figure on previous pagefig computational repositioning of food and drug administration fdaapproved drugs for alcoholic liver disease ald a schematicrepresentation of the bioinformatics workflow for the repositioning approach used to identify potential candidate drugs and genes for the treatmentof ald b dimethyl fumarate dmf prevents ethanolinduced hepatic steatosis mice were fed with the control diet or ethanol diet containing vv ethanol respectively followed by treatment with mgkg dmf or mgkg dmf by oral gavage for days tissue sections from the mouseliver were prepared for hematoxylin and eosin he staining scale bars are μm c dmf decreases ethanolinduced hepatic fibrosis mice were fedas in b tissue sections from the mouse liver were prepared for collagen staining scale bars are μm d e dmf increases endogenous nrf2 andgclc to activate the nrf2 signaling pathway in the mouse liver immunohistochemical staining of nrf2 and gclc proteins in mouse liver tissuesliver tissue sections from different groups were stained immunohistochemically with antinrf2 antibody d or antigclc antibody e as indicateddata shown are from one mouse from each group scale bars are μm f nrf2 and gclc in mouse liver sections were compared against actb bywestern blotting the statistical analysis of all samples is shownfurther results revealed that the nrf2 protein level wasupregulated with increased dmf and mmf concentrationsfig 2b and supplementary fig s2b phosphorylationserine40 is required for nrf2 activation1617 to confirmthe activation of nrf2 we treated hepg2 or lo2 cellswith dmf and mmf respectively as indicated thendetermined the level of phosphorylated nrf2 protein bywestern blotting results showed that dmf and mmftreatment significantly increased the phosphorylation levelof nrf2 when we adjusted the sample loading to keep thenrf2 level constant fig 2c and supplementary fig s2cindicating that nrf2 was activatedin addition wechecked the protein levels of nrf2regulated genes15 ourdata showed that dmf and mmf treatment promoted theexpression of gclc and ho1 protein levels fig 2a bmoreover nrf2 knockdown dramatically decreasedgclc and ho1 protein upon either normal condition orfumarates treatment fig 2d and supplementary fig s2dcollectively our results demonstrate that fumarates activate the nrf2 pathway in liver cellsonce phosphorylated nrf2 can translocate into thenucleus and activate transcription of various detoxification and antioxidant enzymes upon exposure to stresses18to examine whether fumarates regulated nrf2 nuclearlocalization in liver cells we treated hepg2 or lo2 cellswith dmf and mmf at different concentrations for hfig 2e then cells were lysed and subjected to cytosolicand nuclear fraction extraction we found that dmf fig2e left pannel and mmf fig 2e right pannel promotednrf2 nuclear accumulation in a dosedependent mannermoreover we performed immunofluorescence in livercells confocal microscopy data showed that nrf2expression and nuclear localization were enhanced inhepg2 cells upon dmf and mmf treatment fig 2ftaken together our data provide evidence that fumaratesactivate nrf2 and promote its translocation from cytoplasm to the nucleusdmf and mmf reduce the ros level by activating nrf2 inliver cellsthe relative levels of gsh and gssg are associatedwith various disease aging and cell signaling events19“official of the cell death differentiation associationto illustrate the potency offumarates as antioxidantagents we performed the reaction to convert total glutathione and the oxidized form gssg to the reducedform gsh then we measured both total glutathioneand gssg in the luminescent reaction scheme with thegsh probe the results showed that dmf and mmfinduced a dosedependent increase of intracellular gshfig 3a b doxorubicin dox an effective anticanceragent can induce the generation of ros which then leadsto oxidative damage of cellular and mitochondrial membranes2223 27dichlorofluorescin diacetate dcfhdais a specific indicator of ros formation24 and has beenused widely as a fluorescence probe in cells2526 confocalmicroscopy data revealed that ros were accumulated inhepg2 cells with the presence of dox while dmf andmmf blocked the doxinduced accumulation of rosfig 3c and supplementary fig s3a then we performedsirna transfection in hepg2 cells to knock down nrf2and observed a significant increase of ros upon doxtreatment even in the presence of dmf and mmf fig3d and supplementary fig s3b moreover we usedmitotracker® red cmxros kit an agent which can bepassively transported through the cell membrane anddirectly gathered on the active mitochondria to test theeffect of fumarates on the mitochondrial ros level wefound a significant reduction of h2o2 or ethanolinducedmitochondrial ros under fumarates treatment fig 3eand supplementary fig s3cthese results suggest aresistant effect of fumarates in response to ros by activating the nrf2 pathwaydmf and mmf reduce rosinduced lipid peroxidation andferroptosis in liver cellsrecent studies showed accumulation of ros can lead tolipid peroxidation and ferroptosis27 therefore we speculated that fumarates regulate rosinduced ferroptosis toexamine ferroptosis in dox or ethanoltreated cells weexamined the levels of hepatic malondialdehyde mdaand nadpnadph content2829 consistent with rosinduced ferroptosis we found that dox or ethanoltreatment significantly increased lipid peroxidation fig4a b and decreased nadph content fig 4c we 0czhang et al cell death and disease page of fig see legend on next pageofficial of the cell death differentiation association 0czhang et al cell death and disease page of see figure on previous pagefig dimethyl fumarate dmf and mmf activate the nrf2 pathway in liver cells a dmf or mmf treatment increases endogenous nrf2gclc and ho1 protein level in a timedependent manner hepg2 or lo2 cells were either untreated or treated with μm dmf or mmf for differentlengths of time followed by being lysed and subjected to western blotting with the indicated antibodies b dmf or mmf treatment increasesendogenous nrf2 gclc and ho1 protein level in a dosedependent manner hepg2 or lo2 cells were either untreated or treated with dmf ormmf at the indicated concentrations for h actb is shown as a loading control c dmf or mmf increases the nrf2 s40 phosphorylation levelhepg2 or lo2 cells were treated as in b analyzed by western blotting with nrf2 phospho s40 antibody and normalized against nrf2 proteinthe sample loading was adjusted to keep the nrf2 level constant d nrf2 knockdown decreases gclc and ho1 protein levels under normal orfumarates condition hepg2 or lo2 cells were transfected with sinrf2 or negative control nrf2 gclc and ho1 protein levels were determined bywestern blotting e dmf or mmf promotes nrf2 nuclear accumulation after treated with μm dmf left panel or mmf right pannel for hhepg2 or lo2 cells were subjected to cytosolic and nuclear fractionation and nrf2 protein levels were determined by western blotting histone3h3 and αtubulin were used as nuclear and cytoplasmic markers respectively while actb was used as a wholecell lysate maker f hepg2 cells weretreated with dmso μm dmf or μm mmf for h as indicated then paraformaldehyde fixed blocked and processed for immunofluorescencewith dapi blue or antibody against nrf2 green nrf2 staining is shown on the left and the merged nrf2 and dapi on the right bar μm relativenrf2 fluorescence intensity was calculated using imagej software the ratio was quantified mean values were calculated from the individualdistributions in ten cells per conditionobserved a decrease of mda levels and a restoration ofnadph when we added fumarates into liver cells pretreated with dox or ethanol fig 4a“c more evidencewas obtained when we detected the protein level of gpx4an important ferroptosis regulator which can inhibit cellmembrane phospholipid peroxidation results showedthat compared with dmso treatment gxp4 was substantially decreased under ethanolstimulated conditionindicating a promoting role of ethanol in liver lipid peroxidation and ferroptosis however we observed arestoration of the gxp4 protein level when we addedferrostatin1 an inhibitor of ferroptosis into hepg2 andlo2 cells pretreated with ethanol fig 4d and supplementary fig s4a a similar result was detected in mouseliver primary cells ethanol treatment lead to a significantdecrease offerrostatin1restored gpx4 protein pretreated with ethanol fig 4eand supplementary fig s4b in addition we treated livercells with erastin an inducer of ferroptosis which playsthe opposite role to ferrostatin1 in ferroptosis and foundfumarates led to an accumulation of gxp4 and nrf2protein even in the presence of ethanol or erastin fig 4ef we also detected lipid peroxidation with c11bodipy undecanoic acid by measuring the fluorescenceintensity in red color consistent with our previousresults an increase of ros production was observedunderthe treatment of ethanol and erastin whileferrostatin1 or fumarates can inhibit lipid peroxidationinduced by ethanol supplementary fig s4c suggestinga preventive effect of fumarates in rosinduced lipidperoxidation and ferroptosisendogenous gpx4 whiledmf inhibits ethanolinduced lipid peroxidation andferroptosis in vivothese results strongly suggest that dmf prevents rosinduced liver injury and ferroptosis via activating thenrf2 pathway we therefore studied the role of dmf inrosinduced ferroptosis in mice hepatocytes treated withofficial of the cell death differentiation associationethanol or not compared to the untreated group andferrostatin1 treated group groups treated by ferroptosisinducer erastin and ethanol had smaller ruptured mitochondria fig 5a these cellular morphological featuresare characteristic of ferroptosis however dmf ameliorated the ferroptosis induced by ethanol as observed bytransmission electron microscopymore evidence was obtained when we performed western blotting and ihc compared with the normal micethe protein levels of 4hne which indicated an increasedlipidperoxidationinduced ferroptosis were higher in aldmouse livers while the gpx4 protein level was lower incontrast dmf treatment could block lipidperoxidationinduced ferroptosis by decreasing the protein levels of4hne and increasing the protein levels of gpx4 in vivofig 5b“f these data further validate fumarates as inhibitors of the lipidperoxidationinduced ferroptosisdiscussionusing a computational repositioning of existing drugsbased on the publicly available geneexpression data todiscover therapies for ald we inferred that the nrf2inducer dmf could serve as a therapeutic option for aldand performed experimental validations which demonstrated the efficacy of dmf in ameliorating ald in livercells and in the mouse model the precise mechanism ofaction for dmf is unknown but it is known to activatethe nrf2 antioxidant pathway although dmf has notpreviously been suggested as a therapy for ald previousstudy has shown that nrf2 prevents alcoholinducedfulminant liver injury30 in this study we found thatfumarates activate the nrf2 signaling pathway promoting nrf2 phosphorylation and nuclear localization inliver cells nrf2 further activates the transcription ofgenes encoding various detoxification and antioxidantenzymes in response to rosoxidative stress is implicated in the development ofdiverse liver disorders such as ald nonalcoholic fatty 0czhang et al cell death and disease page of fig see legend on next pageofficial of the cell death differentiation association 0czhang et al cell death and disease page of see figure on previous pagefig dimethyl fumarate dmf and mmf reduce the ros level by activating nrf2 in liver cells a b dmf or mmf enhances cellular redoxpotential by increasing gsh level hepg2 cells were treated with or without different concentrations of dmf a or mmf b for h and thenassessed for cellular gsh and gssg levels denotes p ns denotes no significance error bars represent mean ± sd for triplicate experiments cfumarates block dox or ethanolinduced ros accumulation hepg2 cells were pretreated with dox or ethanol for h followed by treatment with μm dmf or mmf for another h as indicated cells were loaded with dcfhda μm and incubated for min at °c in the darkfluorescence images were acquired by a confocal microscope bar μm d nrf2 knockdown accumulates ros damage in liver cells either with orwithout fumarates hepg2 cells were transfected with sinrf2 and treated as in c fluorescence images were obtained e fumarates block h2o2 orethanolinduced mitochondrial ros accumulation lo2 cells were pretreated with h2o2 or ethanol for h followed by the treatment with μmdmf or mmf for another h as indicated cells were incubated with mitotracker® red cmxros red at °c in the dark images were acquired byfluorescence microscope bar μmliver disease nafld and hcc2 elevated cellularstresses which are induced by alcohol hepatic viruses ordrugs play a vital role in the initiation and progression ofmultiple liver pathologies31“ certain stressed conditions can cause the accumulation of cellular rosuncontrolled production of ros results in oxidativestress on tissues and cells and causes lipid peroxidation34the nrf2 antioxidant pathway is a highly conservedsignal transduction pathway that allows cells tissues andans to survive under oxidative stress conditions35 ourstudy showed that fumarates activate the nrf2 signalingpathway reduce the cellular ros level and protect livercells from ethanolinduced oxidative injuryferroptosis is an iron and rosdependent form of celldeath which is characterized by the accumulation of lipidlevels3637 ros accumulationhydroperoxides to lethalcould directly react with unsaturated fatty acids whichmay lead to a destruction of the mitochondrial membrane a massive release of substances promoting apoptosisand increased ferroptosis dysregulation offerroptosis has been implicated in various pathologicalprocesses including cancer neurodegenerative diseasesacute renal failure druginduced hepatotoxicity ischemiareperfusion injury and tcell immunity3839 our studyshowed that fumarates upregulate the protein level ofgpx4 a gshdependent enzyme that reduces lipidhydroperoxides while decrease lipid peroxidation andferroptosis and thus ameliorate ethanolinduced liverinjury in the ald mouse model fig in addition thesefindings support that fumarates could also be effective inother ferroptosisassociated diseasesin recent years drug repurposing has gained more andmore attention for accelerating drug development40given the high costs possible side effects high failure rateand long testing periods for developing new medicines7drug repurposing provides an attractive approach to meetthe need for improved diseases treatment for exampledisulfiram an old alcoholaversion drug has emerged as acandidate for treating highrisk breast cancer7 hippeastrine hydrobromide hh which has been used to preventavian ‚uenza h5n1 has become a promising drug forinhibiting zika virus zikvinfection41 topiramate aofficial of the cell death differentiation associationsafe and effective drug for treating neurological diseasesis capable of ameliorating ‚ammatory bowel disease42in this study we demonstrate that computational repositioning of fdaapproved drugs by analyzing publicgeneexpression data can be used to infer drug therapiesfor ald and offer experimental evidence that the nrf2inducer dmf is capable of ameliorating disease pathophysiology in the ald mouse model dmf was alreadyestablished as a safe and effective drug for treating multiple sclerosis43 additional clinical investigation will beneeded to test whether dmf could benefit patients suffering from aldmaterials and methodscell culture and treatmentcell culture was performed as previously described44hepg2 or lo2 cells were cultured in dmemhigh glucose medium hyclone sh3002201 or rpmi mediummodified hyclone sh3080901 supplemented with fetal bovine serum gibco penicillin andstreptomycin gibco at °c in a humidifiedatmosphere containing co2 for fumarate treatmentcells were first cultured in the medium which containedfetal bovine serum then dmf sigmaaldrich and mmf sigmaaldrich of different concentrations were added into the medium the treatmentsto increase cell oxidative stress and ferroptosis wereperformed by adding ethanolsigmaaldrich e7023 mm doxorubicindox solarbio d8740 μm anderasitin selleck s7242 μm to the culture medium for hthen we treated liver cells with fumarates orferrostatin1 sigmaaldric sml0583 μm for another h all the concentrations are final concentrations in theculture mediumwestern blottingwestern blotting was performed as previously mentioned4546 hepg2 or lo2 cells were lysed in ripa lysisbufferbeyotime p0013b containing protease andphosphatase inhibitors cell debris was removed by centrifugation while celllysates were boiled for minand centrifuged at °c before loading on or 0czhang et al cell death and disease page of fig see legend on next pageofficial of the cell death differentiation association 0czhang et al cell death and disease page of see figure on previous pagefig dimethyl fumarate dmf and mmf reduce rosinduced lipid peroxidation and ferroptosis in liver cells a b fumarates obviouslyreverse dox or ethanolinduced lipid peroxidation hepg2 a and lo2 b cells were pretreated with μm dox mm ethanol or μm erastinfor h followed by μm ferrostatin1 or μm fumarates for h thereafter cells were lysed and subjected to lipid peroxidation malondialdehydemda assay c fumarates reverse dox or ethanolinduced ferroptosis lo2 cells were pretreated with μm dox mm ethanol or μm erastinfor h followed by μm ferrostatin1 or μm fumarates for h thereafter cells were lysed and subjected to nadpnadph assay denotesp denotes p and ns denotes no significance error bars represent mean ± sd for triplicate experiments d“f fumarates block lipidperoxidation and ferroptosis in liver cells hepg2 cells lo2 cells d f or mouse liver primary cells e were pretreated with mm ethanol or μmerastin for h as indicated followed by μm ferrostatin1 or μm fumarates for another h thereafter cells were lysed and subjected to westernblotting for nrf2 and gxp4 with actb as loading control the statistical analysis of all samples is shown fsdspage gels then proteins were transferred ontopvdf membranes merck millipore ltd ipvh00010 forwestern blotting analysis the primary antibodies tophosphors40 nrf2 abcam ab76026 workingdilution nrf2 proteintech 163961ap working dilution gclc proteintech 126011ap working dilution ho1 proteintech 107011ap working dilution αtubulin proteintech 660311lg working dilution gxp4 abcam ab125066 working dilution histone3 proteintech 1ap working dilution actbβactin proteintech 205361ap working dilution werecommercially obtainedrna interferenceknocking down of nrf2 was performed by rnainterference following the manufacturer™s instructions forlipofectamine rnaimax reagent invitrogen the knockdown efficiency was determined by westernblotting synthetic sirna oligo nucleotides were obtainedcommercially from genepharma co ltd list of effectivesequences is as follows sinrf21 ²gguugagacuaccaugguutt3²sinrf22 ²ccagaacacucaguggaautt3²sinrf23 ²gccuguaaguccuggucautt3²negative control ²uucuccgaacgugucacgutt3²cytoplasmic and nuclear extractsfor nrf2 nuclear translocation experiments cells werecultured in the medium which contained fetal bovineserum then dmf and mmf of different concentrationswere added into the medium for h in all 10cmdiameter plates of hepg2 and lo2 cells were lysed andcytosolic and nuclear fractions were separated followingthe protocol provided by the nuclear and cytoplasmicextraction kit manufacturer active motif inc the nuclear pellets were washed three times with phosphate buffered saline containing freshly added proteaseand phosphatase inhibitors the cytosolic supernatantwas centrifuged to remove any nuclear contamination andtransferred to a new tube both the cytosolic and nuclearfractions were boiled separately in sds sample buffer andanalyzed by western blotofficial of the cell death differentiation associationgsh analysishepg2 cells were plated into white and flatbottom well plates and cultured for h at °c then we treatedcells with dmso or fumarates and incubated for another h for fluorescent gsh assay we first washed cells withhanks™ balanced salt solution solarbio h1045500 thendetermined the levels of reduced and oxidized gsh bygshgssg assay kit promega v6611 according to themanufacturer™s protocol totalrelative luminescenceunits rlu are graphed as means ± sd denotes p ns denotes no significance graphed data represents oneof three experimental repeatsmeasurement of cell lipid peroxidation and nadpnadphassayin thefumarates erasitin μm orliver cells were plated into 60mm dishes and culturedfor h at °c the treatments to increase cell lipidperoxidation were performed by adding ethanol mmdoxorubicindox μm and erasitin μm to theculture medium for h then we treated liver cells with orwithoutferrostatin1 μm for another h all the concentrations are finalconcentrationslipidperoxidation assay and nadpnadph assay we firstwashed cells with °c precooled phosphate bufferedsaline then determined the levels of cell lipid peroxidation by mda assay kit beyotime s0131 and nadpnadph quantitation colorimetric kit biovision k347according to the manufacturer™s protocol the totalhepatic mda content and nadpnadph levels aregraphed as means ± sd graphed data represent one ofthree experimental repeatsculture medium forimmunofluorescence staininghepg2 cells were plated into glass bottom cell culturedishes nest and pretreated with or withoutdox for h followed by addition of dmf and mmf intothe medium thereafter cells were first fixed with paraformaldehyde biosharp then permeabilized in triton x100 amresco blocked by bovine serum albumin amresco in pbs buffersigmaaldrich p5368 and lastly incubated with theindicated primary nrf2 antibody working dilution 0czhang et al cell death and disease page of fig see legend on next pageofficial of the cell death differentiation association 0czhang et al cell death and disease page of see figure on previous pagefig dimethyl fumarate dmf inhibits ethanolinduced lipid peroxidation and ferroptosis in vivo a dmf prevents ethanolinducedferroptosis mice were fed as indicated on the final day morning the mice were given alcohol liquid gkg or maltodextrin control by gavageand sacrificed after h in addition ferrostatin1 mgkg and erastin mgkg were provided min before
Colon_Cancer
the molecular heterogeneity of renal cell carcinoma rcc complicates the therapeutic interventions for advancedmetastatic disease and thus its management remains a significant challenge this study investigates the role of thelncrna cdkn2bas1 and mir1413p interactions in the progression and metastasis of kidney cancer human renalcancer cell lines achn and caki1 normal rptec cells tissue cohorts and a series of in vitro assays and in vivo mousemodel were used for this study an overexpression of cdkn2bas1 was observed in rcc compared to normal samplesin tcga and our inhouse sfvamc tissue cohorts reciprocally we observed reduced expression of mir141 in rcccompared to normal in the same cohorts cdkn2bas1 shares regulatory mir141 binding sites with ccnd1 andccnd2 genes direct interactions of cdkn2bas1mir141cyclin d1“d2 were confirmed by rna immunoprecipitationand luciferase reporter assays indicating that cdkn2bas1mir141cyclin d1“d2 acts as a cerna network in rccfunctionally attenuation of cdkn2bas1 andor overexpression of mir141 inhibited proliferation clonogenicitymigrationinvasion induced apoptosis in vitro and suppressed tumor growth in xenograft mouse model furtheroverexpression of cdkn2bas1 is positively correlated with poor overall survival of rcc patients expression of mir141also robustly discriminated malignant from nonmalignant tissues and its inhibition in normal rptec cells induced procancerous characteristics cdkn2bas1 attenuation or mir141 overexpression decreased ccnd1ccnd2 expressionresulting in reduced rac1ppxn that are involved in migration invasion and epithelial“mesenchymal transition thisstudy for the first time deciphered the role of cdkn2bas1mir141cyclin d axis in rcc and highlights this networkas a promising therapeutic target for the regulation of emt driven metastasis in rccintroductionrenal cell carcinoma rcc is one of the most commoncancers in the usa accounting for nearly deathsand new cases in surgery is the first line oftreatment resulting in successful resection and longtermdiseasefree status with an overall survival rate of morecorrespondence rajvir dahiya rdahiyaucsfedu1department of urology veterans affairs medical center san francisco anduniversity of california san francisco san francisco ca usa2department of surgery university of miami miller school of medicine miamifl usafull list of author information is available at the end of the these authors contributed equally pritha dasgupta priyanka kulkarniedited by e candithan however in approximately of localizedrcc cases recurrence occurs with distant metastasis2the obstinate nature of rcc to current treatment regimens is a primary cause of poor prognosis in patients withmetastatic recurrence lack of sensitivity to both chemotherapytherapeuticoptions difficult3“ it is therefore of utmost importanceto improve our understanding of rcc pathogenesis byidentifying new biomarkers that lead to better predictionand therapeutic intervention of aggressive rcc6and immunotherapy makesemerging lines of evidence suggest that cancer aggressiveness is associated with epithelial“mesenchymal transition emt7 it is a wellorchestrated process involved in the authors open access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate ifchanges were made the images or other third party material in this are included in the ™s creative commons license unless indicated otherwise in a credit line to the material ifmaterial is not included in the ™s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this license visit httpcreativecommonslicensesby40official of the cell death differentiation association 0cdasgupta cell death and disease page of tumor invasion and metastasis comprising characteristicphenotypic changes through transition from polarizedimmotile epithelial cells to motile mesenchymal cells8emt changes in cellular morphology and migratoryproperties are governed by numerous factors9 increase inmesenchymal properties accompanied by augmentedexpression of mesenchymal markers like ncadherbronectinvimentin and matrix metalloproteinasemmps and decreased expression of epithelial markerslike ecadherin αecatenin claudin etc10“ are common emt phenomena often the progression of cancerthrough emt is significantly induced by the interaction ofcyclind with its binding partner cdk4 which act astranscriptional regulators controlling cell proliferationand migration14“ it is well known that cyclind regulates the ratelimiting step in cell cycle progression fromg1 to s phase accumulating evidence also suggest thatabnormal cyclindcdk4overexpression promotestumor growth and metastasis17 but how this correlateswith tumor metastasis or controls cell adherence andinvasion is poorly understoodreports show that noncoding rnas are involved in thefactors involved in emt18 micrornasregulation ofmirnas a naturally occurring class of small noncodingrna molecules of nucleotides long19 are known toregulate gene expression via both translational inhibitionand mrna degradation20 whereas long noncoding rnaslncrnas with more than nucleotides can also actas regulators for tumorsuppressive mirnas in differentcancers21“ recently a class oflncrnas have beencategorized as competing endogenous rna cernawhich involves crosstalk among lncrnas mrnas andtheir shared mirnas thus a novel regulatory mechanismis hypothesized suggesting that lncrnas and mrnascommunicate with each other by competing for commonmirna response elements24“in this context we describe the novel role of lncrnacdkn2bas1 and mir1413p mir141 in the regulation of cyclind to govern the metastatic progression ofrcc to our knowledge this is the first report to directlydemonstrate that cdkn2bas1mir141 interaction is acrucial component in rcc progression and metastasisthrough the cyclindrac paxillin pathwaymaterials and methodscell lines and cell culturethe normal rptec atcc number crl4031 andrenalcancer achn atcc number crl1611and caki1 atcc number htb46 cell lines were purchased from the atcc manassas va these humanderived celllines were authenticated by dna shorttandem repeat analysis by atcc cell line experimentswere performed within “ months of their procurementresuscitation achn cells were cultured in mem mediaofficial of the cell death differentiation associationcaki1 cells in and mccoy 5a medium and rptec cellsin dmemf12 medium atcc® „¢ all mediawere supplemented with fbs and 1x antibioticspenicillin and streptomycin cell lines were maintainedat °c and humidified atmosphere of co2mirnasirna transfectionsto induce overexpression or knockdown cells were transiently transfected with either mirvana mirna mimic nmoll or antimir mirna inhibitor nmollthermo fisher scientific and nmoll of sirna sigmaaldrich using lipofectamine rnai max thermo fisherscientific according to the manufacturers™ protocol toverify transfection efficiency mirvana mirna mimicnegative control mirna inhibitor control and sirnacontrol were used respectively in each transfection experiment at the same concentration all transfection experimentswere carried out for hclinical specimensformaldehydefixedparaffinembedded ffpe tissue specimens from patients undergoing radical nephrectomy wereobtained from the san francisco veterans affairs medicalcenter sfvamc written informed consent was obtainedfrom all patients and the study was in accordance withinstitutional guidelines irb approval no allpatient samples were pathologically confirmed for clear cellrcc ccrcc and slides were reviewed by a boardcertifiedpathologist for the identification of tumor foci and adjacentnormal tissue apart from sfvamc cohort tcgakirctcgakich tcgakirp icgc and geo cohorts forrcc from online databases were also used to check theexpression levelsrnamirna extraction and quantitative realtime pcrqrtpcrtotal rna was extracted from microdissected ffpetissues and cell lines using mirneasy ffpe and mirneasymini kits qiagen respectively in accordance to manufacturer™s instructions mature mirna and mrnas wereassayed by qrtpcr using quantstudio flexreal timepcr system applied biosystem using fast sybr®green master mixtaqman universal pcr master mixprobes and primers applied biosystems inc foster cityca usa following manufacturer™s protocol humangapdh and rnu48 were used as endogenous controlsand relative expression of rnamirna were calculatedusing comparative ct threshold cycle primer sequencesare provided in supplementary table t1dna methylation analysis insilico in cell lines and 5azacdr treatmentdna hypermethylation of the mir141 promoter innormal and rcc samples was first confirmed in the 0cdasgupta cell death and disease page of tcga database using wanderer software27 in order toconfirm the methylation status of the mir141 promoterin rcc cell lines we extracted dna from achn andcaki1 using dneasy tissue kit qiagen sodium bisulphite modification was done using ez dna methylationgold kit zymo research orange ca usa followingthe manufacturer™s protocol bisulfitetreated dna wasanalyzed by methylationspecific quantitative polymerasechain reaction msqpcr with primer pairs specific formethylated and unmethylated regions of the mir141promoter msqpcr was performed as described earlier28 for each sample the percent of methylation wascalculated by the difference of ct in methylated samplectm and ct in unmethylated sample ctu the primers sequences are mentioned in supplementary table achn and caki1 cells were treated daily with μmoll5azadeoxycytidine 5azacdrfor h29 and total rna was isolated using a mirneasy minikit qiagen to check mir141 expressionsigmaaldrichcell viability clonability migratory invasion andapoptosis assayscell viability was measured at and h using acelltiter aqueous solution cell proliferation assay kitpromega madison wi following the manufacturer™sinstructions for colony formation assay cells were seeded at a low density cellsplate after h oftransfection and were allowed to grow until visible colonies were formed plates were then stained with giemsafollowed by crystal violet and colonies were countedculture inserts of 8µm pore size transwell costar wereused for migration and invasion assay inserts were coatedwith matrigel bd biosciences µgwell for invasionbriefly h posttransfection cells were counted andplaced on inserts at × cellsml for migration and × for invasion in serumfree medium and wereallowed to migrateinvade for “ h at °c cellsmigrated or invaded through the pores were fixed stainedwith crystal violet crystal violet was solubilizedwith methanol and quantified at nm by a kineticmicroplate reader spectra max molecular devicesfacs analysis for apoptosis was done h posttransfection using annexin vfitc and 7aad kit beckmanin accordance with the manufacturer™scoulter incinstructions cold pbs washed cells were resuspended in1x binding buffer and stained with annexin vfitc7aad viability dye after min of incubation at roomtemperature in the dark stained cells were analyzed usingbd facsverse bd pharmingendualluciferase reporter assaythe wild type wt and offtarget ot luciferasereporter constructs were made by ligating annealed custom oligonucleotides containing putative target bindingofficial of the cell death differentiation associationsites and corresponding nontarget mutant sites into thepmirglo reporter vector luciferase constructs µgwere cotransfected into achn and caki1 cells along with nmoll mir141 mimic or controlmir using transfection reagent jetprime polyplustransfection illkirchfrance luciferase activities were measured using thedualluciferase assay promega madison wi h posttransfection relative luciferase activity was calculated bynormalizing firefly luciferase to renilla luminescencerna immunoprecipitation rip assaysimultaneous binding of mir141 to lncrna andmrna was confirmed by rip assay an imprint rip kitwas used following the manufacturer™s protocol sigmaaldrich st louis mo usa igg control and ago2antibodies were used forimmunoprecipitation theimmunoprecipitated rna fraction was reverse transcribed to cdna using high capacity cdna reversetranscription kit thermo fisher fold enrichment oflncrna and mrna to ago2 with respect to igg wascalculated using quantitative rtpcrwestern blot and immunofluorescence analysistotal protein extraction was performed as describedpreviously18 proteins were then separated by nupage“ bistris protein gels invitrogen and subsequentlytransferred onto nitrocellulose membraneresulting blots were blocked using odyssey blockingbuffer licor and subsequently probed with primaryand secondary antibodies blots were scanned using anodyssey infrared imaging system scan and quantificationwas carried out with the licor odyssey® scanner andsoftware licor biosciences the primary antibodiesused are listed in supplementary table for immunofluorescencetransfected achn andcaki1 cells were fixed in paraformaldehyde for minfollowed by blocking 1x pbs5 normal goat serum triton x100 for h at room temperature cellswere then incubated overnight in fold diluted primary antibody at °c cells were then reprobed with fold diluted secondary antibody for h and counterstained with µgml of ²6diamidino2phenylindoledapi for min cells were then mounted on a slideusing prolong gold antifade reagent images were captured using zeiss microscope model axio imagerd2transientlyrenal cancer xenograftswe studied the antitumorigenic effects of mir141 inestablished tumors using a renal cancer xenograft nudemouse model as previously described630 male nude mice“ weekold n charles river lab were subcutaneously injected with × caki1 cells oncepalpable tumors were formed mice were randomized intwo groups for the treatment and control groups five in 0cdasgupta cell death and disease page of each synthetic mirna mir141 mimicmircon of μg was complexed with μl siportamine transfection reagent ambion in μl pbs and deliveredintratumorally in 3day intervals tumor volume wascalculated according to the formula x2 y2 where x yx width y length experiments were terminated days after the last treatment day tumor measurements and statistical analysis were performed byresearchers who were blinded for the control and treatment groups all animal care was in accordance withinstitutional guidelines iacuc approval no statistical analysisall quantified data represents an average of at leastthree independent experiments or as indicated statisticalanalyses were performed using graphpad prism andmedcalc error bars represent ± standard error meansem the mann“whitney u test was used to assessthe difference between mirna expressions in tumornormal adjacent tissue significant differences betweenthe groups were determined using the student™s ttest alltests were performed either one tailed or two tailed andresults were considered statistically significant at p ‰¤ receiver operating curves roc were performed toevaluate the potential of mir141 to differentiate betweenmalignant and nonmalignant samples using medcalcsoftware showing area under curve auc and confidence interval kaplan“meier analyses for overall survival with respectto mir141 methylation levels intcgakirc cohort were generated using softwareœezrhttpsdoi101038bmt2012244 tumormeasurements and statistical analyses for all experimentswere performed blindly for the control and treatment groupsresultslncrna cdkn2bas1 is oncogenic and is a direct target ofmir141initially we found cdkn2bas1 is an oncogeniclncrna in rcc based on tcga fig 1a icgc andgeo databases fig s1 tcga cohort also revealed thathigh cdkn2bas1 expression increases from lower gradeand stage to higher grade and stage fig 1b moreoverhigher expression is significantly p correlated tooverall survival fig 1c in agreement with these datacohorts significantly higher cdkn2bas1 expression wasalso seen in rcc cell lines achn caki1 as compared tonormal rptec fig 1d and sfvamc cohort fig 1epatient and tumor characteristics are summarized insupplementary table t3 roc analysis shows an auc of p ci “ fig 1f suggesting the diagnostic potential of cdkn2bas1 to discriminate between normal and tumor tissues we usedcomputational algorithms and identified putative mir141binding sites in the cdkn2bas1 sequence fig 1g toofficial of the cell death differentiation associationexamine potential mir141cdkn2bas1 interactionexperimentally we performed luciferase reporter assayboth achn and caki1 cells cotransfected with mir141and cdkn2bas1 wild type binding site revealed a consistent reduction ofluciferase activity suggesting thatmir141 directly interacts and regulates cdkn2bas1fig 1h thus all these data suggest that clinicallyimportant cdkn2bas1 is an oncogenic lncrna in rccand is a novel target of mir141cdkn2bas1 inhibition by sirna suppressestumorigenicity in rcctransient transfection of achn and caki1 cells withcdkn2bas1 sirnas for h showed significant reduction in cdkn2bas1 expression fig 2a cdkn2bas1knockdown in both cell lines significantly inhibited cellproliferation figs 2b s2a and clonogenic survivalfigs 2c s2b with a significant increase in apoptosis fig2d e decreased cell migrationinvasion figs 2f s2c dwith simultaneous changes in emt markers such as anincrease in epithelial markers αecatenin and claudinand decrease in mesenchymal markers vimentin andfibronectin were also observed figs 2g s3expression of mir141 and its clinical importance in renalcarcinomaand samples lowersince our results confirmed cdkn2bas1 as a directtarget of mir141 we examined mir141 status andclinical importance in rcc expression of mir141 wassignificantly downregulated in rcc cell lines fig 3aand in tumor samples fig 3b“e compared to normal celllinesignificantlydecreased with increasing grade stages and in metastaticcompared to nonmetastatic tumors fig 3c“e patientand tumor characteristics are summarized in supplementary table t3 roc analysis showed an auc of p ci “ fig 3f suggesting thatmir141 can be used as a potential diagnostic parameterto discriminate between normal and tumor tissuesexpressionsepigenetic regulation of the mir141 locuswe identified a genomic site rich in cpg island locatedupstream of the mir141 in chromosome 12p13 in thetcga cohort we observed hypermethylation of mir141promoter in tumor tissues as compared to normalfigs 3g s4a which is significantly associated with poorpatient survival fig 3h similarly rcc cell lines achnand caki1 also showed hypermethylation compared tonormal rptec cells fig 3i further we treated achnand caki1 cell lines with demethylating agent 5azacdrand observed decrease in methylation fig s4b withconcomitant increase in mir141 expression fig 3jindicating possible epigenetic regulation a significantdecrease in the expression of methylation regulatory 0cdasgupta cell death and disease page of fig lncrna cdkn2bas1 is oncogenic in renal cancer and is a direct target of mir141 a expression levels of cdkn2bas1 among kircnormal tumor kich normal tumor and kirp normal tumor patient samples in tcga cohort using wanderersoftware pvalue calculated by mann“whitney twotailed test b expression of cdkn2bas1 in tcgakirc cohort among different grades normal grade “ grade “ and stages normal stage i“ii and stage iii“iv c overall survival in tcgakirc cohort as performedby kaplan“meier analysis using ualcan software d relative expression levels of lncrna cdkn2bas1 in rcc cell lines achn and caki1 e expressioncdkn2bas1 in matched pairs of rcc tissue samples from sfvamc cohort pvalue calculated by mann“whitney twotailed test f receiver operatingcurve roc analysis on sfvamc cohort showing ability of lncrna cdkn2bas1 to differentiate between malignant and nonmalignant samples sfvamccohort g predicted binding sites of mir141 in cdkn2bas1 sequence h luciferase assays showing decreased reporter activity after cotransfection witheither wildtype wt offtarget ot cdkn2bas1 or luciferase control constructs ev with mirconmir141 in achn and caki1 cellsgenes such as dnmtl dnmt3a and dnmt3b werealso noted after 5azacdr treatment compared to control dmso in both achn and caki1 cell lines31mir141 overexpression phenocopies functional effectsobtained with cdkn2bas1 inhibition in vitro andsuppresses tumorigenicity and in vivowe sought to determine if cdkn2bas1 causes itsantitumorigenic effects through mir141 we checkedthe effect of mir141 overexpression in rcc cellstransient transfection of mir141 mimic in achn andcaki1 cells for h led to over expression of mir141compared to control mircon fig 4a also overexpression of mir141 significantly reduced cdkn2bas1 expression fig 4b indicating a reciprocal correlation between mir141 and cdkn2bas1 a significant decrease in cell proliferation over time fig 4cand marked decreasefig 4din clonogenicityofficial of the cell death differentiation association 0cdasgupta cell death and disease page of fig knockdown of lncrna cdkn2bas1 reduces tumorigenicity in renal cancer a relative expression of cdkn2bas1 in achn and caki1 cellstransfected with cdkn2bas1 sirnas b cell proliferation assessed by mts assay after knockdown of cdkn2bas1 in both cell lines with sirna2 cgraphical representation showing knockdown of cdkn2bas1 with sirna2 significantly decreased colony formation in achn and caki1 cellsd e achn and caki1 cell lines showing significant induction of apoptosis early late compared to control after knockdown of cdkn2bas1f reduced migration and invasion in cdkn2bas1 sirna transfected cells compared to control treatment g changes in emt related proteins in bothachn and caki1after knockdown of cdkn2bas1compared to controls were also observed further westudied the therapeutic potential of mir141 in amouse xenograft model a significant decrease intumor growth was observed by intratumoral delivery ofmir141 mimic compared to control over the course ofexperiment average tumor volume in the controlgroup was mm3 compared to mm3 in micethat received mir141 mimic fig 4e in additionmir141 overexpression significantly induced apoptosis with a concomitant decrease in the viable population in both rcc celllines compared to controlfig 5a this proapoptotic role was supported by theinduction of cleaved caspase3 cleaved polyadpribose polymerase parp an increase in bax and adecrease in bcl2 at protein levels fig 5b a significantdecrease in migration fig 5c and invasion fig 5dwas also observed in both rcc cell lines with mir141overexpression we also examined emt markers aschange in migration and invasion are directly associated with emt our results showed an increase inepithelial markers αecatenin and claudin with concomitant decrease in mesenchymal markers fibronectinand vimentin at both protein fig 5e and mrnaofficial of the cell death differentiation associationfig s5 levels taken together overexpression of mir phenocopies the functional effects of cdkn2bas1 inhibition in vitro and tumor growth suppressioneffects in vivolike cdkn2bas1aremir141cdkn2bas1 interaction negatively regulatescyclind and its downstream effectors in rcccyclind1cyclind2alsooncogenic in rcc fig s6 and are direct targets of mir as discussed earlier lncrnas can act as cernas tocarry out their regulatory functions32“ we observedthat cdkn2bas1 shared regulatory mir141 bindingsites with cyclind1cyclind2 fig 6a and therebysponges mir141 allowing cyclind1d2 to be expressedin tumors to determine potential mir141cdkn2bas1cyclind interaction experimentally we performedrip assay both achn and caki1 cells over expressingmir141 revealed significant enrichment of cyclind1cyclind2 and cdkn2bas1 with ago2 as compared toigg control fig 6b moreover decreased luciferaseactivity also confirmed direct binding of mir141 tocyclind in mir141overexpressing achn andcaki1 cells compared to controls fig 6c we also found 0cdasgupta cell death and disease page of fig expression clinical significance and epigenetic regulation of mir141 in renal cancer a mir141 expression levels in achn caki1 andrptec cells b expression levels of mir141 in kirctcga cohort normal and tumor c expression levels of mir141 in normal n nonmetastatic n metastatic n kirc patient samples in tcga cohort d expression levels of mir141 in kirctcga cohort amongdifferent grades normal grade “ grade “ and stages normal stage i“ii and stage iii“iv e relative mir expression in rcc tissue vs matched adjacent normal regions n among different grades normal grade grade “ andin different stages normal stage i“ii stage iii“iv as assessed by qrtpcr sfvamc cohort f receiver operating curve roc analysisshowing ability of mir141 to differentiate between malignant and nonmalignant samples g methylation for kirc patient samples in tcgakirccohort for probe cg02624246 h overall survival with tcgakirc methylation as performed by kaplan“meier analysis i methylation status of mir141promoter in rcc cell lines compared to nonmalignant rptec as assessed by msqpcr j expression of mir141 in 5azacdr treated and untreatedachn and caki1 cell lines results are representative of three independent experiments p value calculated by student t test bar mean ± standarderror mean semthat overexpression of mir141 or inhibition of cdkn2bas1 significantly decreased cyclind1d2 expression atboth the mrna fig 6d e and protein levels figs 6f“hs8a b this effect was significantly attenuated by mir inhibitor fig s7 indicating that cyclind expressionis dependent on the interaction between mir141 andcdkn2bas1 we further observed a decrease in rac1 asmall gtpase and a reduction in the phosphorylation ofpaxillin a focal adhesion protein at mrna levelsfigs s3 s5 and protein levels figs 6g i“j s8c“f inboth mir141 overexpressed and cdkn2bas1 inhibitedrcc cell lines which in turn are involved in regulatingcellular migrationinvasion cumulatively these resultsindicate that suppression of cdkn2bas1 by mir141inhibits renal cell proliferation invasion and migration byinhibiting cyclind rac1 and phosphorylation of paxillinofficial of the cell death differentiation association 0cdasgupta cell death and disease page of fig mir141 overexpression mimics the knockdown effect of lncrna cdkn2bas1 in vitro and reduces tumorigenicity in vivo a relativeexpression of mir141 in achn and caki1 cells transfected with mir141 mimic and control b significant decrease in cdkn2bas1 expressioncompared to control in both cell lines overexpressed with mir141 c rcc cell proliferation after transfecting with mir141 mimic and control asassessed by mts assay d colony formation and its graphical representation in mir141 overexpressing achn and caki1 cells compared to controlse pictures of excised tumors are taken at the termination of experiment day graph represents tumor volume after intratumoral injection ofcontrol or mir141 mimic into established tumors injection was started at day and was followed for days each mouse in both groups mirconand mir1413pmimic received a total of eleven injections intermittently data represent the mean of each group and error bars are sem results arerepresentative of three independent experiments p value calculated by student t test bar mean ± standard error mean semattenuation of mir141 exerts tumorigenic attributes innormal rptec cellswe next determined whether attenuation of mir141induces tumorigenic characteristics in normal rpteccells by targeting cdkn2bas1 and cyclind transienttransfection of mir141 inhibitor indeed showed a significant decrease in mir141 expression fig 7a and anincrease in cdkn2bas1 expression fig 7b along withother procancerous phenotypes such as increased cellproliferation fig 7c colony formation fig 7d migration and invasion fig 7e as compared to controlsadditionally a significant increase in cyclind1 cyclind2 rac1 and paxillin pxn expressions were observed inmir141 inhibited rptec cells fig 7f a noticeableincrease in prometastatic fibronectin and vimentin with aconcomitant decrease in antimetastatic claudin andαecatenin genes were also observed in mir141 inhibited rptec cells compared to controls fig 7gdiscussionprior studies have shown the regulatory role of noncoding rnas in tumorigenesis especially in the emtofficial of the cell death differentiation associationpathway leading to cancer aggressiveness cdkn2bas1also known as anril is located at chromosome 9p21cdkn2bas1 is reported to be upregulated in tumortissues and function as an oncogenic lncrna in pancreatic ovarian and laryngeal squamous cell carcinoma36“ human mir141 is located at chromosome12p1331 and is transcribed from a mir200 familyclusterinterestingly expression of mir141 is controversial since it exhibits either oncogenic39“ or tumorsuppressive roles42“ in specific types of cancer theprime goal of the present study was to understand therole of cdkn2bas1mir141 interactions in regulatingrcc progression and metastasisin this study we identify cdkn2bas1 to be a crucialoncogenic lncrna that plays an important role in renalcarcinogenesis cdkn2bas1significantly overexpressed in rcc and the expression increases fromlower to higher grades and stages lncrna cdkn2bas1directly interacted with mir141 as it was found to be anovel target of mir141 in contrast to cdkn2bas1 weobserved significant attenuation of mir141 expression inrcc cell lines and tumor samples compared to normalis 0cdasgupta cell death and disease page of fig ectopic mir141 expression induces apoptosis and inhibits migrationinvasion in renal cancer cells a apoptosis assessed by flowcytometric analysis of annexinvfitc 7aad“stained achn and caki1 cells transfected with mirconmir141mimic graph represents totalapoptosis early late b immunoblots showing apoptotic proteins in mirconmir141mimic treated achn and caki1 cells with gapdh asendogenous control c migration assay and d invasion assay as seen in pictures and graphical representation of both achn and caki1 cells after mir overexpression compared to control e immunoblot assay showing emt related proteins in mirconmir141mimic treated achn andcaki1 cells with βactin as endogenous control graphs are average of three independent experiments p value calculated by student t testbar mean ± standard error mean semcell line or matched normal samples as it is known thatextensive dna hypermethylation of cpg islands is highlycorrelated to activation of cancerspecific genes45 wechecked the methylation status of mir141 in normal andrcc tissues interestingly insilico analysis showed thepresence of cpg island in the promoter region of mir and we also found hypermethylation of mir141 intcga samples as compared to normal this hypermethylation is also found to be significantly associatedwith poor survival of patients similar results were alsoobserved in rcc cell lines compared to a normal rpteccell line functionally inhibition of cdkn2bas1 andoroverexpression of mir141 significantly inhibits thetumorigenic characteristics such as cell proliferationclonogenicity migration and invasion whereas inducesanticancer apoptotic phenotype in rcc in vitro in vivodata show suppression of tumor growth by mir141overexpression conversely attenuation of mir141 innormal rptec cells induced precancerous characteristicsindicated by increased proliferation migration andinvasionfrom a clinical point of view noncoding rnas signatures are powerful tools for early cancer diagnosisofficial of the cell death differentiation associationmaking them attractive candidates as diagnostic andprognostic biomarkers184647 our results revealed thathigher expression of cdkn2bas1 is positively correlatedwith poor overall survival probability of rcc patientsindicating its prognostic capability cdkn2bas1 canalso discriminate normal from tumor samples showing itsdiagnostic potential similarly mir141 expression canalso robustly distinguish between cancerous from noncancerous samples and hence has potential to be a diagnostic biomarker for rcc collectivelythese resultshighlight the biomarker potential of cdkn2bas1mir expression in rcc although it needs to be confirmedin a larger independent sample cohortinterestingly we found tha
Colon_Cancer
" triple negative breast cancer tnbc remains recalcitrant to most targeted therapy approaches however recent clinical studies suggest that inducing tumor damage can render tnbc responsive to immunotherapy we therefore tested a strategy for immune sensitization of murine tnbc 4t1 tumors through combination of focused ultrasound fus thermal ablation and a chemotherapy gemcitabine gem known to attenuate myeloid derived suppressor cells mdscsmethods we applied a sparse scan thermally ablative fus regimen at the tumor site in combination with systemically administered gem we used flow cytometry analysis to investigate the roles of monotherapy and combinatorial therapy in mediating local and systemic immunity we also tested this combination in rag1ˆ’ˆ’ mice or t cell depleted wild type mice to determine the essentiality of adaptive immunity further we layered programmed cell death protein pd1 blockade onto this combination to evaluate its impact on tumor outgrowth and survivalresults the immune modulatory effect of fus monotherapy was insufficient to promote a robust t cell response against 4t1 consistent with the dominant mdsc driven immunosuppression evident in this model the combination of fusgem significantly constrained primary tnbc tumor outgrowth and extended overall survival of mice tumor control correlated with increased circulating antigen experienced t cells and was entirely dependent on t cell mediated immunity the ability of fusgem to control primary tumor outgrowth was moderately enhanced by either neoadjuvant or adjuvant treatment with anti pd1 thermally ablative fus in combination with gem restricts primary tumor outgrowth improves survival and enhances immunogenicity in a murine metastatic tnbc model this treatment strategy promises a novel option for potentiating the role of fus in immunotherapy of metastatic tnbc and is worthy of future clinical evaluationtrial registration numbers nct03237572 and nct04116320 metastatic breast cancer brca particularly the triple negative breast cancer tnbc phenotype is resistant to most chemical and molecularly targeted therapeutic approaches interestingly tnbc is often infiltrated with immune cells and the presence of these cells has been shown to have a favorable prognosis in patients treated with neoadjuvant chemotherapy1 early studies in the use of immunotherapies targeting the pd1programmed death ligand pd l1 checkpoint inhibitory axis showed some efficacy2“ in tnbc compared with other brca subtypes which are generally recalcitrant to checkpoint blockade activity in the tnbc subtype may be related to the relatively high immune infiltration and correlated with the higher mutational burden observed in tnbc greater immunotherapy efficacy in tnbc has been recently observed with the use of antibodies targeting the pd1pd l1 checkpoint inhibitory axis in combination with nab paclitaxel5 this outcome suggests that inducing tumor damage augments antitumor immunity either by promoting antigen availability or disrupting the immunosuppressive tumor microenvironment tme found in tnbcamong the potential networks in tnbc that could constrain the activity of antitumor immunity is the presence of immunosuppressive myeloid cell subsets these have the capacity to impair adaptive immunity and promote tumor growth and metastasis among these cell types myeloid derived suppressor cells mdscs prevail as a heterogeneous population of immature myeloid cells which serve the eponymous role of suppressing the antitumor immune response limiting both t cell activation and effector functions6 increased levels of this cell type have been demonstrated in tumor tissues of patients with primary brca while those with metastatic disease bear the highest abundance of circulating mdscs8 studies have sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access shown that approaches that either stimulate myeloid cells with inflammatory mediators or eliminate mdsc can improve antitumor immunity9“to this end the central premise put forth in this study is that focused ultrasound fus”a safe noninvasive and nonionizing strategy for localized acoustic energy deposition into tissues”can synergize with immunotherapy in a murine model of metastatic tnbc fus is capable of rapidly heating tumors to thermally ablative temperatures its extracorporeal application obviates the need for catheterization injection or implantation fus can be targeted with millimeter precision under mri or ultrasound guidance thereby allowing for thermal damage and destruction of tumor tissue without compromising healthy intervening or peripheral tissues the bioeffects of fus hold distinct implications for tumor antigenicity immune cell activation and trafficking13 thermally active fus regimes have elicited antitumor immune responses in implantable models of melanoma15 pancreatic16 prostate17“ colon20 kidney21 and brca23 pertaining to the challenge of myeloid cell immunosuppression in tnbc thermally ablative fus has been shown to induce the expression of heat shock proteins24 and proinflammatory cytokines including interleukin il12 interferonÎ ifnÎ and tumor necrosis factorα tnfα from a variety of cancer cell lines and after in vivo treatment of tumors26 whether the ability of fus to induce these inflammatory mediators is sufficient to overcome myeloid suppression in the context of brca is currently under debate with some studies showing activation of antigen presenting cells and t cell recruitment in patients with brca treated with thermally ablative fus28 while others show that additional innate stimuli are needed to support antitumor immunity23 notably some studies have suggested that a sparse scan thermal ablation regimen more effectively recruits and activates dendritic cells dcs and antitumor immunity than total thermal ablation perhaps by limiting thermal denaturation of tumor antigens and innate stimuli31based on the improved myeloid cell maturation that occurs with sparse scan regimens we herein tested the ability of a sparse scan partial thermal ablation fus regimen as a monotherapy to promote antitumor immunity in an aggressive syngeneic model of metastatic murine tnbc with extensive granulocytic mdsc involvement that is recalcitrant to anti pd1 while some activity is evident with the partial ablation approach significantly greater control was achieved by targeting mdsc inhibition in combination with thermally ablative fus this control was completely dependent on the adaptive immune responsemoreover we demonstrate that layering anti pd1 immune checkpoint blockade onto this combinatorial regimen moderately improves tumor growth restriction these data suggest that in disease settings where myeloid allied approaches to attenuate myeloid immunosuppression may be employed to reveal the full immunotherapeutic immunosuppression predominates potential of thermally ablative fus once immunosuppressive myeloid cells are accounted for fus treatment can promote adaptive immunity that in turn potentiates immune checkpoint blockademethodscell line maintenance4t1 and e0771 cell lines were maintained in rpmi l glut or dulbecco™s modified eagle™s medium dmem gl d glucose l glutamine respectively supplemented with fetal bovine serum fbs at °c and co2 thawed cells were cultured for up to three passages and maintained in logarithmic growth phase for all experiments cells tested negative for mycoplasmaeight week old to week old female balbc or c57bl6 mice were obtained from nci charles river nci crl or the jackson laboratory female balbc rag1ˆ’ˆ’ mice were obtained from the jackson laboratory 4t1 or e0771 cells — were subcutaneously implanted into the right flank of mice mice were housed on a hour12 hour lightdark cycle and supplied food ad libitum tumor outgrowth was monitored via digital caliper measurements tumor volume was calculated as follows volume length—width22 approximately days 4t1 or days e0771 following tumor implantation mice were randomized into groups in a manner that ensured matching mean starting tumor volume across experimental groupsin vivo ultrasoundguided fus partial thermal ablationmice were treated with fus either days 4t1 cohorts or days e0771 postimplantation on treatment day mice were anesthetized with intraperitoneal injection of ketamine mgkg zoetis and dexdomitor mgkg pfizer in sterilized saline mouse flanks were shaved and depilated following which ultrasound guided fus thermal ablation was performed using one of the two systems system and treatment details are provided in online supplementary materials and methods mice that did not receive fus treatment consistently underwent anesthesia and depilation of the flank additionally these mice underwent a ˜sham™ treatment consisting of exposure to the °c degassed water bath exposure for min following ˜sham™ or fus treatment all mice were moved to a heating pad and given antisedan for anesthesia reversal and recoverygemcitabine therapygemcitabine gem mgmouse in µl volume mylan diluted in saline and filter sterilized through a µm syringe filter was administered intraperitoneally once a week on the day of fus treatment following which administration was repeated for an additional weeks administration of gem doses was based on existing literature demonstrating the use of gem for inhibition of mdscs in 4t112 the initial dose of gem was administered immediately prior to ˜sham™ or fus treatment sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0cmice that did not receive gem received an intraperitoneal injection of ˜vehicle™ treatment µl of sterile saline at the time points specifiedpd1 blockade therapyfor checkpoint inhibitor therapy the rat anti mouse pd1 antibody αpd1 rmp114 diluted in sterilized saline was administered intraperitoneally every days for a total of five doses µg per mouse treatment was initiated on day ˜early αpd1™ or day ˜delayed αpd1™t cell depletionst cell depletion antibodies”anti cd8 clone bio x cell and anti cd4 gk15 clone bio x cell”were diluted in sterilized saline and administered intraperitoneally every to days starting at day days post fus for a total of seven doses µg of each antibody for a total µg per mouseimmunohistochemistryon day sham or fus exposed tumors were excised and fixed in neutral buffered formalin sigma fixed tumors were paraffin embedded sectioned and stained for hematoxylin and eosin digital images of stained slides were acquired using the vectra automated quantitative pathology imaging system akoya biosciences whole slide screening and image capture were subsequently performed using phenochart akoya biosciencesflow cytometrymice were bled at days and via tail vein and samples were rbc lysed hybri max sigma and stained for flow cytometry analysis at days post tumor implantation tissues were obtained from euthanized tumor bearing animals for immune response assessment in order to gain resolution into tissue resident versus vascular immune cell populations mice were injected intravenously with rat anti mouse cd45 fitc clone f11 bd biosciences min prior to euthanasia 4t1 tumors spleens cardiac blood axillary and brachial tumor draining lymph nodes tumor dlns pooled and nondraining inguinal lymph nodes were harvested processed and stained for flow cytometry analysis additional details are provided in online supplementary materials and methodssamples were acquired on an attune nxt flow cytometer thermofisher scientific and data were analyzed with flowjo treestar or fcs express de novo software a representative gating strategy for granulocytic myeloid derived suppressor cell g mdsc and cd44 t cells is provided in online supplementary figure statistical analysisall statistical analyses were performed in graphpad prism graphpad software a detailed description of statistical methods for each experiment is provided in the corresponding figure legendopen accessanimal study approvalall animal work was performed under a protocol approved by the animal care and use committee at the university of virginia and conformed to the national institutes of health guidelines for the use of animals in researchresultspartial thermal ablation of established tnbc tumors promotes peripheral dc activation but has limited impact on the presence of t cells and other myeloid cell subsetsto achieve partial thermal ablation of 4t1 tumors we used an ultrasound guided fus system equipped with a single element therapeutic transducer driven at mhz figure 1a online supplementary figure a grid of sonications was overlaid on the ultrasound visible tumor and ablated in a raster pattern under b mode ultrasound guidance figure 1b“c the exceptionally small focus of this system rendered a low ablation fraction “ of total tumor volume immediately following ablation tumors displayed evidence of coagulative necrosis in the ablated zone with surrounding periablative margins figure 1d one week following fus partial thermal ablation tumors and secondary lymphoid organs were excised for immunological characterization by flow cytometry figure 1b fus partial thermal ablation of 4t1 tumors conferred a significant increase fold in the absolute number of cd11c hi dcs within the axillary tumor draining lymph node adln of mice figure 1e while this was accompanied by a nearly threefold elevation in the absolute number of cd86 dcs within the adln figure 1f the percentage of dcs expressing cd86 did not change figure 1g increased numbers of dcs”and cd86 dcs in particular”suggest fus is promoting the maturation or trafficking of these cells in the dlns where they could encounter and activate t cells however this did not translate to tumor growth restriction data not shown we also did not observe significant differences in the absolute number of activated t cells in 4t1 tumors figure 1h or dlns data not shown following fus exposure suggesting limitations in the ability of fus activated dc to further drive an antitumor t cell responseimmune profiling by flow cytometry revealed that irrespective of fus exposure of the intratumoral cd45 immune cell population is comprised of cd11b myeloid cells figure 1i similarly approximately of the circulating immune cell population in 4t1 tumor bearing mice is comprised of myeloid cells a striking fold elevation in circulating myeloid burden compared with naive mice online supplementary figure notably ly6g granulocytic myeloid derived suppressor cells g mdscs significantly dominated the immune cell repertoire within 4t1 tumors relative to other myeloid including f480 macrophages ly6c cell subsets monocytic myeloid derived suppressor cells m mdscs and cd11c hi dcs figure 1j fus partial thermal ablation did not significantly alter the absolute number per sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access figure partial thermal ablation of established tnbc tumors promotes peripheral dc activation but has limited impact on the presence of t cells and other myeloid cell subsets a design overview of a custom ultrasound guided fus system consisting of a mhz single element transducer orthogonally co registered to an mhz linear ultrasound imaging array the tumor bearing flank of each anesthetized mouse was acoustically coupled to ultrasound transducers via degassed water bath maintained at °c ˜sham™ mice were similarly positioned but did not undergo sonications b schematic illustration of fus partial thermal ablation scheme and study layout for evaluation of immune sequelae in 4t1 tumor bearing mice a grid of sonications was applied in a raster pattern onto the b mode ultrasound visible tumor in total two planes of sonication spaced mm apart were applied to each tumor grid points were spaced mm apart within a single plane one week following thermal ablation tumors and secondary lymphoid organs were excised for sham n6 or fus treated n5 mice and processed for flow cytometry c representative b mode ultrasound images of ectopic 4t1 tumors either before top or during bottom fus exposure sonication grid depicting targets red points is superimposed on b mode image during treatment subsequent to thermal ablation hyperechoic signatures yellow arrow are occasionally observed d representative he staining of either sham 4t1 tumors or those resected immediately following fus partial thermal ablation zoomed insets depict the transition from necrotic to intact tumor tissue within the periablative zone scale bars400 µm and µm on left and right inset respectively e absolute number of cd11c hi dcs in the axillary tumor draining lymph node adln of 4t1 tumor bearing mice p00136 vs sham f absolute number of cd86 cd11c hi dcs in the adln p00063 vs sham g percentage of cd86 subset out of total cd11c hi dcs within adln h absolute number of intratumoral cd44 cd8 and cd44 cd4 t cells and regulatory t cells tregs per gram tumor i percentage of cd11b myeloid cells out of total cd45 immune cells across tumor spleen adln inguinal dln idln and nontumor draining axillary and inguinal lns ndlns p005 vs all other groups irrespective of fus exposure specifically tumor vs spleen p00226 tumor spleen vs all other organs p00001 j absolute number of intratumoral myeloid cells cd11c hi dcs f480 macrophages ly6c monocytic myeloid derived suppressor cells m mdscs ly6g granulocytic myeloid derived suppressor cells g mdscs per gram 4t1 tumor p00001 vs all other cell types irrespective of fus exposure all data represented as mean±sem significance assessed by unpaired t test f“h or two way analysis of variance followed by tukey multiple comparison correction i“k ˜ns™not significant dcs dendritic cells fus focused ultrasound hifu high intensityfocused ultrasoundsheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0cgram tumor of these myeloid cell subsets these observations led us to formulate the hypothesis that widespread immunosuppressive mechanisms associated with the 4t1 tme must be addressed in order to facilitate the t cell response to fusfus partial thermal ablation in combination with gem constrains primary tnbc tumor outgrowth and extends overall survivalour observation of the overwhelming mdsc burden following 4t1 tumor implantation warranted implementation of an allied therapeutic strategy in order to counter this immunosuppressive barrier to this end we tested a combinatorial paradigm incorporating gem a myelosuppressive chemotherapy demonstrated to inhibit mdscs transiently in the 4t1 model without consequence to t cell phenotype or function12to evaluate the efficacy of fus and gem in combination we used a preclinical ultrasound guided fus system to achieve partial thermal ablation of established 4t1 tumors 14d after tumor implantation average tumor volume of mm3 in combination with the single session of fus thermal ablation we initiated gem therapy mgmouse which was then readministered weekly for a total of three gem doses figure 2a combinatorial therapy synergized to produce significant constraint of 4t1 tumor outgrowth compared with sham and monotherapy groups figure 2b“cby termination of treatments at day 4t1 tumors exposed to fusgem combination saw nearly — and — reductions in average volume compared with sham or gem exposed tumors respectively figure 2b two dimensional tumor projections at day postimplantation saw a nearly fold reduction in area from sham to combinatorial therapy setting figure 2d“e in a fraction of mice treated with fusgem we observed complete regression of 4t1 tumors although transient figure 2c tumor outgrowth eventually rebounded after termination of treatments 4t1 tumor bearing mice receiving fusgem treatment additionally saw the greatest extension in overall survival with and increases in median survival time compared with sham and gem groups respectively hrs and for fusgem relative to sham and gem groups respectively figure 2f we additionally observed that fusgem significantly constrained outgrowth in a separate c57bl6 metastatic mammary carcinoma model e0771 online supplementary figure to further the clinical relevancy of these findings we applied this combinatorial strategy with the research grade analog of a clinical ultrasound guided fus system theraclion echopulse that is already ce marked for applications in breast fibroadenoma thyroidparathyroid gland and varicose vein ablation and currently in use for multiple clinical trials leveraging fus thermal ablation in combination with cancer immunotherapy we observed that partial thermal ablation using the theraclion visualization and treatment unit mhz in combination open accesswith gem controlled 4t1 tumor outgrowth to a degree comparable with that observed with the custom in house system online supplementary figure these findings lend credence to the notion that the impact of combining gem with fus may be conserved across partial thermal ablation regimens moreover they demonstrate that the efficacy of fus partial thermal ablation in combination with gem can be recapitulated on a system with a larger focus and in line image guidance that is currently in use clinicallycombination of fus partial thermal ablation with gem increases the levels of circulating t cellslymphocytes”in particular cd8 and cd4 t cells”play an important role in responding to tumor antigen and generating a durable antitumor response based on the extended protective effect observed in mice treated with fusgem flow cytometry analysis was performed to evaluate the contribution of t cells in generating systemic and local tumor control we sampled the circulating immune cell repertoire in 4t1 tumor bearing mice via serial tail bleeds days and prior to readministration of gem and a terminal cardiac bleed at the time of spleen harvest day figure 3a combinatorial therapy significantly elevated absolute number of cd8 and cd4 t cells in the circulation at days and figure 3b“c and e“f moreover a trend threefold to fivefold increase in circulating t cells was noted in the fus group relative to sham figure 3b“c and e“f from days to systemic cd44 expressing antigen experienced t cell populations both cd8 and cd4 saw a steady significant increase after combinatorial therapy figure 3d and g a similar modest trend was noted for the fus monotherapy group relative to sham and gem figure 3d and g these changes were concordant with a decrease in circulating myeloid cd11b cells in gem recipient groups demonstrating the ability of gem to partially alleviate circulating myeloid burden figure 3hsplenomegaly is a common signature that arises in parallel with the leukemoid reaction to 4t1 tumors that is the expansion of immunosuppressive myeloid cells during tumor progression we observed that combinatorial therapy most significantly reverses splenomegaly online supplementary figure 6a“b consistent with this observation immunological characterization of spleens revealed a significant decrease in cd11b myeloid cells”a “ reduction in fusgem spleens relative to sham or monotherapy figure 3i while there appeared to be a trend toward more cd11b cells in the monotherapy groups compared with the sham this difference was not significant and there was no difference between these groups in terms of absolute cd11b cell numbers within the spleen data not shown the decrease in myeloid cells in the combination treatment group was accompanied by a significant corresponding elevation in lymphocytes in the spleen following fusgem treatment relative to these sham and gem groups combination therapy elevated splenic cd8 t lymphocytes by fold and sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access figure combination of focused ultrasound fus partial thermal ablation with gemcitabine gem constrains primary triple negative breast cancer outgrowth and extends overall survival a overview of experimental design for evaluation combination of fus with serial gem treatment in murine mammary carcinoma b average 4t1 tumor outgrowth in sham n7 fus monotherapy n5 gem monotherapy n10 and combinatorial fusgem therapy groups n10 data are represented up to select time points corresponding with mouse dropout due to humane endpoints all data represented as mean±sem significance assessed on outgrowth up to day by repeated measures mixed effects model implementing restricted maximum likelihood method followed by tukey multiple comparison correction p005 vs all other groups specifically sham vs fusgem p00001 fus vs fusgem p00001 shamgem vs fusgem p00026 c 4t1 tumor outgrowth from individual mice in sham fus shamgem or fusgem groups data represent outgrowth from initiation of treatments at day up to removal of mouse from study for meeting a humane endpoint d representative images of 4t1 tumors excised at day scale bar1 cm e quantification of 2d tumor areas from images in previous panel f kaplan meier curve depicting overall survival of sham treatment n9 fus monotherapy n6 gem monotherapy n10 and combinatorial fusgem therapy n10 recipient mice significance assessed by log rank mantel cox test p005 vs all other groups specifically sham vs fus p02154 sham vs fusgem p00001 sham vs shamgem p00050 fus vs fusgem p00021 fus vs shamgem p00312 fusgem vs shamgem p00041sheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen accessfigure combination of focused ultrasound fus partial thermal ablation with gemcitabine gem increases the levels of circulating t cells a overview of experimental design to understand the impact of fus andor gem treatment on circulating immune cells b“c absolute number of circulating cd8 t cells at day b and day c d percentage of circulating cd8 t cells expressing cd44 from days to e“f absolute number of circulating cd4 t cells at day e and day f g percentage of circulating cd4 t cells expressing cd44 from days to h percentage of cd11b myeloid cells out of total cd45 immune cell in circulation from days to i“k percentage of myeloid cells i cd8 t cells j and cd4 t cells k out of total cd452 immune cells all data represented as mean±sem all data representative of sham n6“ fus monotherapy n4“ gem monotherapy n9 and combinatorial fusgem therapy n6“ groups significance assessed by analysis of variance followed by tukey multiple comparison correction for b c e f or fisher™s least significant difference lsd without multiple comparisons correction for i“k significance for d g and h assessed by repeated measures mixed effects model implementing restricted maximum likelihood method followed by fisher™s lsd without multiple comparisons correction p005 vs all other groups unless otherwise indicated p001 p0001 vs groups indicatedsheybani a0nd et a0al j immunother cancer 20208e001008 101136jitc2020001008 0copen access fold figure 3j and cd4 t lymphocytes by fold and fold figure 3k these elevations were accompanied by a modest increase in percentage of foxp3 regulatory t cells tregs online supplementary figure 6e additionally increases in percentage of nk and b cells were noted twofold to fivefold online supplementary figure 6c“d these findings indicate that combinatorial therapy with fusgem promotes a systemic lymphocyte response that is discrete from the effects of either intervention alone which may account for reduced mortality associated with pulmonary metastasescombinatorial fusgem therapy does not promote robust local antitumor t cell responsesgiven the robust systemic immune signatures within the blood and spleen following fusgem we assayed 4t1 tumors at a time point within the window of tumor growth restriction and subsequent to termination of treatments ie day to interrogate whether tumor control correlates with an increase in the effector functions of the intratumoral t cell response figure 4a approximately hours prior to euthanasia mice received intravenous brefeldin a injection to inhibit cytokine secretion for subsequent intracellular cytokine staining by flow cytometry immune characterization of tumors at days postimplantation”that is days subsequent to final gem administration”revealed no significant changes in absolute number of antigen experienced cd44 cd8 or cd4 t lymphocytes figure 4b“c moreover the polyfunctionality of these t cells as denoted by ifnÎ and granzyme b expression was not significantly altered figure 4d“e however intratumoral functional changes were noted in the myeloid compartment gem monotherapy modestly increased il 12p40 production by dcs fold but this was not conserved in the combinatorial therapy group figure 4f moreover while fus monotherapy generated a trend in elevated tnfα production by intratumoral g mdscs gem recipient groups saw a significant increase threefold relative to sham figure 4g these findings indicate that changes in the myeloid compartment in response to monotherapy and combination therapy may contribute to tumor control but are unlikely to drive the protective response entirely interestingly intratumoral t cell representation correlates poorly with circulating lymphocytes suggesting a transitory immune response that either cannot be fully characterized at this time point or is hampered by additional modes of immunosuppressionprotection conferred by combination of fus and gem is dependent on adaptive immunitysince our findings revealed no obvious advantage or function of adaptive immunity in the local tme we next investigated the overarching role of the adaptive immune system in protection offered by combinatorial therapy with fusgem to this end we utilized an rag1ˆ’ˆ’ model that is deficient in t and b cells to address the hypothesis that mature t andor b cells play a role in the observed response wild type wt or rag1ˆ’ˆ’ mice bearing 4t1 tumors were randomized into groups in a manner that preserved similarity in average initial tumor volumes mice were subsequently treated with either gem monotherapy or the combination of fusgem the tumor growth inhibition offered by fusgem was entirely lost in rag1ˆ’ˆ’ mice relative to their wt counterparts with average 4t1 tumor volume in rag1ˆ’ˆ’ mice being over fivefold higher than that of wt mice on termination of treatments figure 5a of note despite a trend toward loss of protection in rag1ˆ’ˆ’ mice tumor outgrowth in response to gem monotherapy did not significantly stratify between wt and rag1ˆ’ˆ’ settings figure 5a we also observed a complete loss of fusgem mediated survival benefit over gem monotherapy in the rag1ˆ’ˆ’ setting figure 5b while these results demonstrate that an intact adaptive immune response is required for both the overall survival benefit and restriction of primary tumor offered by fusgem therapy they do not delineate the relative roles of t andor b cellsthus to address the hypothesis that the protective effect of fusgem is specifically dependent on cd4 and cd8 t cells we depleted these populations via serial coinjections of cd8 depleting and cd4 depleting antibodies in 4t1 tumor bearing wt mice on a fusgem figure 5c depletions were maintained between day and day and flow cytometry analysis of circulating immune cells at day confirmed that the target t cell populations were effectively depleted in all mice online supplementary figure consistent with the tumor escape observ
Colon_Cancer
" atopic dermatitis ad is a worldwide chronic skin disease which burden public health sea buckthornsbt hippophae rhamnoides l elaeagnaceae oil as a traditional herbal medicine has been used for diseasetreatment for many years the effects of sbt oil on ad mouse model induced by repeated administration of dinitrochlorobenzene dncb in balbc mice was evaluated in this studymethods mice were divided into four groups including the normal control group ad model group ad modelgroup treated with sbt oil mlkg and ad model group treated with sbt oil mlkg same volume at differentconcentrations of sbt oil was applied daily on the latter two groups by gavage for days following ad modelinduction the function of skin barrier and the production of il4 ifnÎ tnfα and tslp were examined afteranimal sacrifice the migration and mature of langerhans cell lcs in lymph node was further assessed by flowcytometryresults sbt oil alleviated dermatitis scores decreased ear thickness prevented infiltration of mast cell reducedlymph node weight and depressed activity of th2 cells sbt oil also reduced the expression of il4 ifnÎ tnfα andtslp in ear tissue ige level in serum and mrna relative expression of il4 ifnÎ tnfα in lymph node moreoversbt oil inhibited the migration of lcs cells from local lesions to lymph node and it™s mature in lymph nodes these results suggest sbt oil had a beneficial effect either systemic or regional on dncbinduced admice via maintain the balance of th1th2 and may be a potential complementary candidate for ad treatmentkeywords atopic dermatitis sea buckthorn oil 24dinitrochlorobenzene cytokine ad is a chronic inflammatory skin disease characterizedwith eczematous pruritic rash which has high morbidityin children and could be recurrent in adulthood [ ]as a general public health disease the prevalence of adhas increased in recent years [ ] ad affects nearly correspondence hongquanguansinacom houdiandong163com xinxin wang and sijia li contributed equally to this work5college of integrated traditional chinese and western medicine liaoninguniversity of traditional chinese medicine chongshan road no79shenyang liaoning pr chinafull list of author information is available at the end of the of children and of adults worldwide and the incidents become higher and higher although thepathogenesis of ad is not explicit utterly genetic riskenvironmental factors skin barrier dysfunction and immune dysregulation are thought to play important rolesduring the pathogenesis of ad [“] as for immunedysregulation th2 skewing seems to be the key point ofad pathogenesis [ ] immunological disorder ofth1th2 balance due to strong type immune responses characterized by over infiltration of mast cellincreased production of th2 cytokines and ige level in the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwang bmc complementary medicine and therapies page of serum plays crucial role in the onset and process of adthese th2 cytokines subsequently induce the release ofother proinflammatory cytokines such as ifnÎ throughactivating of th1 cells in clinical practicedue to the heavy burden ad placed on society and patients [ ] treatment approaches are needed imperativelyregional emollient andsystemic corticosteroids were generally used to cure ad[ ] however experts ofinternational eczemacouncil reached on a that although the useof corticosteroids for ad is widespread it is also discouraged due to the side effects and the risk of reboundin consideration of potential fearful side effect of topicalsteroid and immunosuppressive application [ ]there is a strong enthusiasm in seeking alternative andcomplementary medication to treat ad recently seeking new potential candidate from natural materials forad management attracted greatly attention [ “]sbt is a wild deciduous shrub or dwarf tree belongingto the elaeagnaceae family which has been used in tibetan mongolian and chinese traditional medicine extensively for disease management [“] according tomany researchers sbt has various medicinal effects suchas antitumor antistress antiinflammatory antiulcerantioxidant healing regulation of cardiovascular andimmune system [“] sbt oil which contains richfatty acids tocopherols ω3 and ω6 etc is a main bioactive part of sbt and it has been proved to have beneficial effect on skin inflammation conditions and have theability to improve the composition of fatty acid in skin[ ] therefore this study carried out to explore thebeneficial effects of sbt oil on dncbinduced admouse model and its possible mechanismmethodssbt oilsbt oil was provided by liaoning dongning pharmceutical co ltd the oil was extracted from thedried press residue including berry flesh seeds andpeel of sbt juice processing by aseptic supercriticalcarbon dioxide process analysis of samples was performed using a hp5ms capillary column m — mm μm agilentinc santaclara ca usa in a gcms 5975c agilent technologies inc sample was injected into the columnand ran using split mode split ratio the helium carrier gas was programmed to maintain atechnologiesconstantflow rate of mlmin oven was initially °c for min then finally raised to °c at °cmin fatty acids were identified by a reference standard mixture fame supelco bellefonte pa usa analyzed under the same operating conditions as thoseemployed for fame of the samples the componentsin sbt oil are exhibited in table animals and animal treatmentfemale healthy specific pathogenfree balbc miceaged weeks weighted ± g were provided byliaoning changsheng biotechnology co ltd benxichina all mice were housed in groups of mice percage waiting to be grouped in a specific pathogenfreeenvironment in h lightdark cycle and allowed free towater and food mice were acclimatized for week before ad model induction mice were randomly dividedinto groups the normal control group ad modelgroup ad model group treated with sbt oil mlkgand ad model group treated with sbt oil mlkgeach group with mice the dorsal skin of each mousewas shaved following dncb μl1 sensitizationthree times in total from day to day and the skin ofleft ear was challenged by dncb μl05 seventimes in total from day to day ad model grouptreated with sbt oil mlkg was given ml sbt oilplus ml olive oil per mice ad model group treatedwith sbt oil mlkg was given ml sbt oil permice oil was applied by intragastric administration oncea day from day15 to day olive oil ml was givenfor the normal control group and ad model group atthe same time at the same time the thickness of left earwas measured every days all animals were sacrificedat day and samples including blood left ear tissuesand submaxillary lymph nodes were collected the micewere anesthetized with avertin solution g tribromoethanol powder dissolved into ml 2methyl2butanol and ml pbs at °c which was filtered usinga nalgene μm filter thermo fisher scientific incwaltham ma usa and sacrificed via exsanguination the fullscale procedures of ad model inductionand treatment are shown in fig all experimental procedures performed were approved by the ethical committee of experimental animal careat liaoninguniversity of traditional chinese medicine shenyangpr chinatable major fatty acids and contents of sitosterol and βcarotene in sbt oilfatty acids myristic acidc140palmitic acidc160palmitoleicacidc161stearic acidc180oleic acidc181linoleic acidc182linolenic acidc183sitosterolmggβcarotenemgg 0cwang bmc complementary medicine and therapies page of fig general schematic diagram for ad model induction and sbt oil treatmentevaluation of ad severitydermatitis severity was assessed by ear thickness anddermatitis scores through the method of blind ear thickness was measured every days since day with a digitalthickness gauge mitutoyo co kanagawa japan dermatitis scores was calculated according to main characteristics drynesscrusting hemorrhageerythema erosionexcoriation and edema each one was marked on ascale from none mild moderate to severethe overall dermatitis score was consist of the sum of individual scores which range from to μm thick werehistopathological analysisthe left ear samples of mice were collected on day then fixed in formalin and embeded in paraffin thesectionseither withhematoxylin and eosin he for visualizing dermal andepidermal thickness or with toluidine blue tb for visualizing mast cell numbers the mast cells were countedin sections of power fields at — magnificationstainedimmunohistochemistryin short after deparaffinization and rehydration the eartissue slides were treated with hydrogen peroxidemethanol for inhibiting endogenous peroxidase and withhigh pressure for antigen retrieval then the slides wereincubated with sheep serum for min at °c withprimary antibodies abcam for overnight at °c withsecondary antibodies provided by zhongshanjinqiaobeijing china for h at °c at last the slides werestained with diaminobenzidine dab provided byzhongshanjinqiao beijing china for coloration resultwas analyzed by imagejrealtime polymerase chain reaction rtpcrmouse submaxillary lymph nodes were collected andweighted while sacrifice and total rna was extractedfrom lymph node tissues using trizol reagent invitrogen thermo fisher scientificfollowing theincmanufacturer™s protocols and reverse transcribed withprime scripttmrt reagent kit takara biotechnologyco ltd dalian china realtime polymerase chain reaction analyses were performed under the protocols ofsybr®premix ex taqtm ii takara biotech co ltddalian china and the primers used in this study weredesigned as shown in table relative quantities of alltargets in test samples were normalized to their corresponding gapdh levels the 2δδct method was usedto calculate relative expression quantifyflow cytometrythe antibodies used for flow cytometry were providedby bd usa and the scheme was performed follow induction about — “ — cells of submaxillarylymph node was collected in ep tube centrifuged at rpm and °c for min one hundred μl pbs wasmixed with cells after discarding the supernatant thenanother μl pbs containing fluorescent antibodieswas added for staining at °c for min and the processwas kept out of the sun five hundred μl pbs was addedand blended repeatedly for washing then centrifuged at rpm and °c for min the supernatant was discarded carefully and another μl pbs was added andmixed finally the mixture was transferred to flow tubefor flow cytometrytable primers used for rtpcrgeneil4ifnÎtnfαgapdhprimer sequenceforward ²acaggagaagggacgccat3²reverse ²gaagccctacagacgagctca3²forward ² tgagctcattgaatgcttgg ˆ’²reverse ² ggccatcagcaacaacataa ˆ’²forward ²ggaaaggacggactggtgta3²reverse ² tgccactggtctgtaatcca ˆ’²forward ²tggtgaaggtcggtgtgaac3²reverse ²actgtgccgttgaatttgcc3² 0cwang bmc complementary medicine and therapies page of statistical analysisthe data is presented as mean ± sd the significance ofdifferences of different groups was evaluated by oneway analysis of variance anova followed by the dunnett t test p005 was considered statistically significantresultssbt oil has a beneficial effect on skin against thedevelopment of dncbinduced ad models in balbcmiceto investigate the effect of sbt oil on adlike skinlesions in our model sbt oil mlkg10 mlkg wasapplied by gastric administration once a day followingthe ad model induction by dncb showed in fig topical application of dncb including sensitizationand challenge induced adlike skin lesions presenting as erythemaitching and hemorrhage companiedby abnormal scratching marks and dryness dermatitisseverity was assessed by ear thickness and dermatitisscores ear thickness was measured every days sinceday and the dermatitis scores was evaluated according to main characteristics as described previously after sbt oil administration for days theear thickness and dermatitis scores were significantlydecreased in a dosedependent manner compared tothe ad model group fig fig effects of sbt oil on ad model skin induced by dncb a examples of characteristic of adlike skin lesions b the ear thickness of the micec the dermatitis scores were summarized by the sum of scores according to various ad symtoms p p 005vs the control group p p vs the ad model group 0cwang bmc complementary medicine and therapies page of sbt oil contributed to the skin barrier repair anddecreased infiltration of mast cell in ad model miceinduced by dncbto evaluate the effect of sbt oil on adlike skin lesions histologically he and tb staining were performed on tissue slides repetitive application ofdncb induced dermal thickening epidermal hyperplasia and increased mast cellinfiltration in admodel group while according to he staining slidesthe dermal and epidermalthickness were both decreased and the epidermal hyperplasia was suppressedafter sbt oil administration for days which relatedto dosage fig 3a b according to tb staining slidesthe infiltration numbers of mast cell were also decreased in mice treated with sbt oil fig 3b csbt oil decreased the lymph node weights in ad modelmice induced by dncbthe submaxillary lymph nodes were collected andweighted after mice sacrifice to estimate whether sbtoil play a part in the process of ad induction the results indicated an increase in submaxillary lymph nodeweights in ad model group which was decreased bysbt oil administration fig sbt oil inhibited the expression of il4 ifnÎ tnfα andtslp in ear tissue in ad model mice induced by dncbin order to evaluate the effects of sbt oil on regulationof th1th2 cytokines the expression of il4 ifnÎtnfα and tslp in ad model mice was examined byimmunohistochemistry staining on ear tissue slides results demonstrated an upregulation expression of il4fig effects of sbt oil on histological ear skin tissue a he staining — thickness in hestained tissue mast cell number in tb stained tissue c tb staining — epidermis mast cell dermis b dermal and epidermal 0cwang bmc complementary medicine and therapies page of fig weights of submaxillary lymph node a submaxillary lymph node b lymph node weight a normal control group b ad model groupc treated with sbt oil mlkg d treated with sbt oil mlkg p p vs the control group p p vs the admodel groupifnÎ tnfα and tslp in ad model group which wasinhibited dosedependent by application of sbt oilfig sbt oil downregulated ige level in serum and mrnarelative expression of il4 ifnÎ and tnfα in lymphnodewe next measured ige levelin serum by elisa andmrna relative expression of il4 ifnÎ and tnfα inlymph node by rtpcr we found that ige levelinserum was increased in ad model mice induced bydncb the increase was suppressed significantly ingroups treated with sbt oil in a dosedependent manner the same as above mrna relative expression ofil4 ifnÎ and tnfα in lymph node was increased inad model group and decreased in groups treated withsbt oil fig sbt oil decreased numbers of lcs in draining lymph nodeand the expressions of cd86 ox40l and mhcii on lcsin order to assess the effect of sbt oil on the maturityand migration of lcs cell in submaxillary lymph nodewe detected cell numbers expressing cd207cd326cd86 cd80 ox40l and mhc ii by flow cytometryresults as shown in fig indicated that the expressionsof cd207cd326 cd86 ox40l and mhciion lcs cellin submaxillary lymph node were all increased in admodel groups induced by dncb and decreased ingroups treated with sbt oildiscussionad is a skin inflammatory disease induced by haptenand mediated by t cells clinically the main characteristics of ad are erythema edema papule blisterbleb bullous reaction and even necrosis the pathological changes of ad were infiltration of inflammatorystudy weand tissuecellsedemain thisemployed dncbinduced ad model using balbcmice which has been proposed as an appropriate representative of human ad because of similar symptoms including skin erosion hemorrhage epidermalhyperplasia mast cellinfiltration and increased igelevel in serum etc sbt oil as a traditional herbal extracts have been proved diversified pharmacologicalactions such as antiinflammatory relieve the pressure protecting vascular endothelial cell and immunomodulatory effects [ ] the main constituents ofsbt oilinclude fatty acids such as myristic acidpalmitic acid palmitoleic acid oleic acid etc sitosterol and βcarotene fatty acids are crucial components of cell membranes and play important role inbiologicalfunction of cells some of the fattyacids are required for innate immune activation andpathogen defense sitosterol is the main constituent of many plants and vegetables and has the abilityto modulate the functions of macrophages and antiinflammation [ ] βcarotenealso has beenshown to suppress the cellular and tissue response toinflammation [ ] in view of the immunoregulation and antiinflammatory actions of sbt oil weassessed the antiad effects of sbt oil in vivotopical application of dncb followed schedule including sensitization and challenge induced adlikeskin lesions presenting as erythemaitching andhemorrhage companied by abnormal scratching marksand dryness the ear thickness and dermatitis scoreswere all significantly increased in ad model groupcompared to control group after sbt oil administration for daysthickness and dermatitisscores in groups treated with sbt oil were significantly decreased in a dosedependent manner compared to the ad model group which indicate thatsbt oil administration suppressed the development ofadlike skin lesionsthe ear 0cwang bmc complementary medicine and therapies page of fig effects of sbt oil on expression of il4 ifnÎ tnfα and tslp in ear tissue a immunohistochemical staining of il4 ifnÎ tnfα and tslpin ear tissue b aod analysis of il4 ifnÎ tnfα and tslp p vs the control group p vs the ad model groupad is recognized as a th2midiated allergic disease withover expression of th2 cytokines and increased serum igelevel being the antibody synthesized by plasma cellsige plays an essential role in some hypersensitivity suchas ad allergic asthma and allergic rhinitis ige has thecapability of elevating the production of th2 cytokinesth2 cytokines il4 induced immunoglobulin switching inplasma cells and resulting in upregulation of serum igelevel mast cell as one of granular leukocytes can releasemany cytokines to mediate inflammatory reaction and immune regulation these cytokines also participate inpathological manifestations of many allergic disorders including ad the infiltration of mast cell which wasactivated by ige is one of the key features of adlike skin 0cwang bmc complementary medicine and therapies page of fig effect of sbt oil on ige level in serum and mrna relative expression of il4 ifnÎ and tnfαin lymph node a ige level in serum b mrnarelative expression of il4 ifnÎ and tnfα in lymph node p p vs the control group p p vs the ad model grouplesions [ ] cytokines released from activated mastcells attract eosinophils into the skin which give rise toskin tissue damage histologically according to tb staining slides the numbers of mast cell infiltration in ear skintissue of ad model mice were increased by application ofdncb and were inhibited remarkably by sbt oil the results indicated that sbt oil has beneficial effects on suppression of skin tissue mast cell accumulation in dncbinduced ad mice our tb staining results indicated thatmast cells in skin tissue were scarce in control group whileabundance in ad model group which highly conform tothe pathological changes of ad the mast cell numberwas reduced remarkably after sbt oil administration insbt oil treated group compared with ad model groupthese results suggest that sbt oil has inhibiting effect ofmast cell infiltrationaccording to studies published the over expression ofth2 cytokines go hand in hand with tslp tslp whichcan strongly promote the differentiation of th0 cells toth2 phenotype through activation of dendritic celldcs was determined as a crucial factor in the induction of th2 skewing in ad the expression of tslphas been shown to be enhanced markedly in keratinocytes of ad lesions both in ad patients and in mousemodels il4 can in turn induce the synthesis oftslp by keratinocytes importantly the migration ofdcs to draining lymph node was triggered by tslpmore interesting th1 cytokines ifnÎ which can activate keratinocytes was found also elevated in ad ifnÎand tnfα can synergistically stimulate the release ofcytokines and chemokines in chronic stage of ad inour study sbt oiltreatment reduced the increasedserum ige level which was induced by dncb application moreover sbt oil treatment also reduced dncbinduced increases in expression of il4 ifnÎ tnfαand tslp in ear tissue and mrna relative expression ofil4 ifnÎ and tnfα in lymph node these resultssuggest that sbt oil ameliorated ad symptoms partlythrough the activity suppression of th1th2 cells according to the downregulated effects sbt oil did to thetslp expression in ear tissue we speculated that sbtoil may have the ability to suppress both the activationand migration of dcs cell in order to clarify our speculationflow cytometry was used to do further studyabout lcsdraining lymph node plays an important role in thepathogenesis of ad the weight and volume of lymphnode will increase company with strengthened functionwhen it is active we investigated the local lymph nodesthrough different means first of all the submaxillarylymph node weights of dncbinduced ad model micewere increased significantly which were markedly reducedafter intragastric application of sbt oil for days in adosedependent manner we further assessed the expressions of cd207cd326 mhc class ii cd80 cd86 onlcs and ox40l on cd4 t cells in lymph node by flowcytometry because the complex immune reaction of adwas mainly taken place in lymph node langerin cd207a type ii transmembrane protein is a ctype lection oflcs lcs are virtual mediators of immune surveillance and tolerance which resided at epidermis as dc subpopulation antigens both external and internal werecaptured by lcs and presented to th0 cells within theskin draining lymph node cd207 is the only surface antigen just restricted to lcs epithelial cell adhesionmolecule epcamcd326 a cell surface protein is highlyexpressed on lcs and appears to stimulate lcs migration since cd207cd326 as the main symbol of lcs 0cwang bmc complementary medicine and therapies page of fig effect of sbt oil on the maturity and migration of lc cell a the scatter diagram which indicate the proportion of cells in lymph nodeexpressing cd207cd326 mhc ii cd86 cd80 and ox40l b the histogram of abovementioned results p p 005vs the controlgroup p p vs the ad model groupmigrated into lymph nodethe proportion changesshowed that sbt oil suppressed the migration of lcs cellfrom skin lesion to draining lymph node after degradingproteins derive from extracellular environment were takenup by endocytosis or phagocytosis and captured by mhcclass ii molecules then result in peptideloaded mhc iiand migrate to the surface of antigen presenting cell waiting for recognition by cd4 t cells finally activate adaptive immune response the increased cell proportionof mhc class ii indicated the uptrend tendency of mature 0cwang bmc complementary medicine and therapies page of lcs in lymph node in dncb induced ad model micecompared with normal control mice while this uptrendtendency was inhibited by sbt oil application in micetreated with sbt oil constant epidermal lcs are immature normally and barely express costimulatory moleculessuch as cd80 and cd86 while upon lcsmaturation the expression of these costimulatory molecules was enhanced in this study sbt oil down regulatedthe expression of cd86 on lcs in lymph node which wasenhanced by dncb in ad model mice it suggested theeffect of sbt oil on inhibiting lcs maturationox40cd134 is a transmembrane protein of tumor necrosis factor receptor superfamily member which mainlyexpressed on activated cd4 t cells and upregulatedwithin inflammatory lesions on the antigenactivated tcells [ ] tslp can stimulate the expression of ox40ligand ox40l on lcs lcs expressing ox40l migratefrom skin lesion to local lymph node and induce the transformation of th0 cells to th2 cells on one hand the increased proportion of ox40l cells in lymph node ofdncb induced ad mice was suppressed by sbt oil administration which confirmed the effect of sbt oil on activation of cd4 t cells on the other hand sbt oilconduced to the normal function of lcs through renovating the keratinocyte and suppressing tslp release as aresultthe abnormal th2 skewing inflammation wasinhibited by sbt oil administrationall in all our results suggest that sbt oil inhibitedboth the migration of lcs to lymph node and its maturation in lymph node thereby inhibited the transformation of th0 cells to th2 cells and finally limited theoccurrence of th2 type inflammatory responsein summary our study results attested that sbt oil application suppressed dncbinduced adlike symptomsby downregulating serum ige level and the productionof cytokines and chemokines and regulated th1th2balance in addition our results also indicated that sbtoil treatment inhibited the migration of lcs to draininglymph node and its maturation taken together sbt oilhas excellent therapeutic effect on inflammatory skindiseases and might be a potential complementary candidate for ad treatment in further studiesit will beworthwhile to explore the mechanism of sbt oil and itsactive constituent in the treatment of adabbreviationsad atopic dermatitis dab diaminobenzidine dcs dendritic cell dncb dinitrochlorobenzene he hematoxylin and eosin ifnÎ interferonÎige immunoglobulin e il4 interleukin4 lcs langerhans cell mhcii majorhistocompatibility complex ii rtpcr realtime polymerase chain reactionsbt sea buckthorn tb toluidine blue th1 thelper th2 thelper tnfα tumor necrosis factorα tslp thymic stromal lymphopoietinacknowledgementsnot applicableauthors™ contributionsxxw carried out the experiment and drafting of the manuscript sjl and jplassisted to accomplish the experiment dnk and xwh carried out statisticalanalysis pl and mx did the interpretation work hqg and ddh revised theresearch and manuscript ddh designed the experiment and submitted themanuscript all the authors read and approved the final manuscriptfundingthis project was supported by the national natural science foundation ofchina grant no which was granted to diandong hou the resultsindicated in this manuscript were main achievements of the projectavailability of data and materialsall data and materials are contained and described within the manuscriptethics approval and consent to participateall experimental procedures were conducted according to the guidelinesprovided by the ethical committee of experimental animal care at liaoninguniversity of traditional chinese medicine shenyang pr chinaconsent for publicationnot applicablecompeting intereststhe authors state no potential conflict of interestauthor details1liaoning university of traditional chinese medicine shenyang liaoning prchina 2basic medical and forensic medicine baotou medical collegebaotou inner mongolia pr china 3neurosurgery department northernhospital of inner mongolia baotou inner mongolia pr china 4liaoningdongning pharmceutical co ltd fuxin liaoning pr china 5college ofintegrated traditional chinese and western medicine liaoning university oftraditional chinese medicine chongshan road no79 shenyang liaoning pr chinareceived december accepted june referencesklonowska j new cytokines in the pathogenesis of atopic dermatitisnew therapeutic targets int j mol sci choopani r treatment of atopic dermatitis from the perspective oftraditional persian medicine presentation of a novel therapeutic approach jevid based complementary altern med “brunner pm guttmanyassky e leung dy the immunology of atopicdermatitis and its reversibility with broadspectrum and targeted therapiesj allergy clin immunol 20171394ss65“leung dym new insights into atopic dermatitis j clin investig “kim je molecular mechanisms of cutaneous inflammatory disorderatopic dermatitis int j mol sci kim j molecular mechanism of atopic dermatitis induction followingsensitization and challenge with 24dinitrochlorobenzene in mouse skintissue toxicol res “hou dd sea buckthorn hippophae rhamnoides l oil improvesatopic dermatitislike skin lesions via inhibition of nfkappab and stat1activation skin pharmacol physiol “campana r molecular aspects of allergens in atopic dermatitis curropin allergy clin immunol “brandt eb sivaprasad u th2 cytokines and atopic dermatitis j clin cellimmunol de vuyst e atopic dermatitis studies through in vitro models frontmed lausanne park jg tabetri tabebuia avellanedae ethanol extract amelioratesatopic dermatitis symptoms in mice mediat inflamm 0cwang bmc complementary medicine and therapies page of liu r βsitosterol modulates macrophage polarization and attenuatesrheumatoid inflammation in mice pharm biol “ vollmer d west v lephart e enhancing skin health by oral administrationof natural compounds and minerals with implications to the dermalmicrobiome int j mol sci cicero afg colletti a effects of carotenoids on health are all the sameresults from clinical trials curr pharm des “ito t il33 promotes mhc class ii expression in murine mast cellsimmun inflamm dis “ dubois a regulation of th2 responses and allergic inflammationthrough bystander activation of cd8 t lymphocytes in early life jimmunol “ girtsman t natural foxp3 regulatory t cells inhibit th2 polarizationbut are biased toward suppression of th17driven lung inflammation jleukoc biol “ wallmeyer l tslp is a direct trigger for t cell migration in filaggrindeficient skin equivalents sci rep leyvacastillo jm tslp produced by keratinocytes promotes allergensensitization through skin and thereby triggers atopic march in mice jinvest dermatol “feinberg h trimeric structure of langerin j biol chem “ zhang x tim4 is differentially expressed in the distinct subsets ofdendritic cells in skin and skindraining lymph nodes and controls skinlangerhans cell homeostasis oncotarget “kumkate s cd207 langerhans cells constitute a minor population ofskinderived antigenpresenting cells in the draining lymph node followingexposure to schistosoma mansoni int j parasitol “ gaiser mr cancerassociated epithelial cell adhesion moleculeepcam cd326 enables epidermal langerhans cell motility and migrationin vivo proc natl acad sci u s a 201210915e889“ natarajan k the role of molecular flexibility in antigen presentationand t cell receptormediated signaling front immunol weinberg ad science gone translational the ox40 agonist storyimmunol rev “kinnear g a diametric role for ox40 in the response of effectormemory cd4 t cells and regulatory t cells to alloantigen j immunol“publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsshrestha s burden of atopic dermatitis in the united states analysis ofhealthcare claims data in the commercial medicare and medicaldatabases adv ther “ arima k burden of atopic dermatitis in japanese adults analysis ofdata from the national health and wellness survey j dermatol “ megna m systemic treatment of adult atopic dermatitis a reviewdermatol ther heidelb “ glatz m emollient use alters skin barrier and microbes in infants at riskfor developing atopic dermatitis plos one 2018132e0192443 drucker a
Colon_Cancer
"although the clinical development of immune checkpoint inhibitors icis therapy has ushered in a new era of antitumor therapy with sustained responses and significant survival advantages observed in multiple tumors mostpatients do not benefit therefore more and more attention has been paid to the identification and developmentof predictive biomarkers for the response of icis and more indepth and comprehensive understanding has beencontinuously explored in recent years predictive markers of icis efficacy have been gradually explored from theexpression of intermolecular interactions within tumor cells to the expression of various molecules and cells intumor microenvironment and been extended to the exploration of circulating and host systemic markers with thedevelopment of highthroughput sequencing and microarray technology a variety of biomarker strategies havebeen deeply explored and gradually achieved the process from the identification of single marker to thedevelopment of multifactorial synergistic predictive markers comprehensive predictivemodels developed byintegrating different types of data based on different components of tumorhost interactions is the direction offuture research and will have a profound impact in the field of precision immunooncology in this review wedeeply analyze the exploration course and research progress of predictive biomarkers as an adjunctive tool totumor immunotherapy in effectively identifying the efficacy of icis and discuss their future directions in achievingprecision immunooncologykeywords neoplasm immune checkpoint inhibitor predictive biomarker tumor mutation burden programmeddeath ligand1 immune checkpoint inhibitors icis therapy has usheredin a new era of antitumor therapy with sustained responses and significant survival advantages observed inmultiple tumors antiprogrammed cell death1programmed cell deathligand pd1pdl1 antibody hasbeen approved for secondline or firstline treatment in avariety of malignant neoplasms including melanoma lungcancer renal cell carcinoma rcc head and neck squamous cell carcinoma hnscc and gastroesophageal correspondence cuijwjlueducncancer center the first hospital of jilin university xinmin streetchangchun jilin chinacancer [ ] however despite the breakthrough in clinical treatment with icis most patients do not benefitpembrolizumab or nivolumab has an objective responserate orr of “ in firstline melanoma and insecondline nonsmall cell lung cancer nsclc [“]therefore in recent years more and more attentions havebeen paid to the identification and development of predictive biomarkers for the efficacy of icis and more indepth and comprehensive understanding has also beenobtained in recent yearsincluding new data on biomarkers of tumor genome and neoantigen tumor immune microenvironmentbiopsybiomarkers hostrelated factors and all of which havephenotypeliquid the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cbai biomarker research page of technologyimmunohistochemicalmade many new advances in the corresponding fieldswith the development and continuous improvement ofmultiplexhighthroughput sequencing and microarray technology a variety of biomarker strategies have emerged and graduallyrealized the process from the identification of singlemarker to the development of multifactorial synergisticpredictive markers the development of predictive biomarkers contributes to revealing the therapeutic mechanisms of icis and the interaction mechanisms betweentumor and host immunity achieving decisionmaking ofindividualized antitumorimmunotherapy monitoringefficacy and disease development guiding clinical trial design as well as for further understanding of drug resistance mechanisms and tumor prognosis in this review wedeeply analyze the exploration course and research progress of predictive biomarkers as an adjunctive tool totumor immunotherapy in effectively identifying the efficacy of icis it should be pointed out here that when reading and collating we try to read and include all therelevant s in the process of selecting s we include the authoritative s published in highlevel s or the latest research results and objectively describe and analyze their roles in this field as well as discuss the reasons that different research results may beinvolvedadvances of multiple predictive biomarkers toicis efficacyi tumor genome and neoantigen biomarkerstumor mutation burdensignificant correlations between high tumor mutationburden tmb and response to icis have been reported inseveral cancer types including urothelial carcinoma small cell lung cancer sclc nsclc [“]melanoma and human papilloma virus hpvnegative hnscc a metaanalysis of cancer typesshowed that the mean response rate was positively correlated with log tmb the national comprehensivecancer network nccn guidelines have adopted tmbas the recommended test for patients with nsclc receiving immunotherapy although the results in some clinicalstudies of rcc hpvpositive hnscc and melanoma receiving antipd1 after recurrence showedthat tmb alone also did not clearly distinguish respondersand predict os it is still exciting that multiple studies inthe american society of clinical oncology ascomeeting have confirmed the predictive value of tmb inimmunization or combination therapy keynote061study [ ] condor study eagle study epoc1704 study etc consolidating its status oftmb as an independent predictor and in april theus food and drug administration fda prioritized theapproval of tmb as a companion diagnostic biomarkerfor pembrolizumabnonetheless the cutoff values of tmb were defineddifferently across studies and assay platforms such asatezolizumab mtmb in urothelial cancer pembrolizumab mtmb in nsclc and atezolizumab‰¥ ‰¥ or ‰¥ mtmb in nsclc [“] andnivolumab plus ipilimumab ‰¥ mtmb in nsclc which needs further study to confirm the optimalcutoff value in different tumors moreover the ngspanels have approved by the fda that can be used to estimate tmb include the mskimpact and foundationone cdx panel the detection results of which arehighly consistent with whole exome sequencing wes[ ] and other solutions are under development astudy detecting tmb cutoff value at mtmb in nsclc patients with the foundationone platformcontaining a gene panel found that compared withtmbl patients overall survival os and dcr was significantly improved in tmbh patients treated withantipd1l1 drug both wes and targeted ngs a422cancergene panel performed in patients withnsclc treated with antipd1l1 demonstrated thattmbh population has a significantly better durableclinical benefitdcb and progressionfree survivalpfs these findings demonstrate the feasibility ofcomprehensive genomic profiling cgp but the designof optimal next generation sequencing ngs panel thatis more accurate comprehensive and costeffective isstill not clear in addition given that btmb was identified as a predictor of pfs but failed to differentiate patients with os benefits researchers consider the need toexplore other more precise factors eg allele frequencyaf a study that developed a new btmb algorithm intwo independent cohorts poplar and oak showedthat modified btmb low af btmb lafbtmb mutation counts with an af was significantly associated with favorable hr 95ci “ p pfs hr 95ci “ p andorr p after immunotherapy but required tobe prospectively validated finally static biomarkersare insufficient to accurately predict response due to thecomplexity of tumorimmune interactions a recent analysis of tumor genomewide dynamic detection in pretreatment and ontreatment melanomasfound thatpretreatment tmb was only associated with os in untreated patients while early 4week ontreatment changein tmb δtmb was strongly associated with antipd1response and os in the entire cohort the detectionof δtmb is helpful for early evaluating the response totherapy of patient but its clinical usability limited by thedifficulty in obtaining tissue samples and high price whileliquid biopsy discussed below might better 0cbai biomarker research page of in addition epigenetic changes are associated withtmb the latest study investigated the association between tmb and dna methylation dnam to explorepotential complimentary biomarkers for nsclc immunotherapies the results showed that high tmbnsclcs had more dnam aberrance and copy numbervariations cnvs showing certain value in predictingefficacy such as hox gene methylation status and tmb thus the correlated exploration of epigenetics hasattracted more attention in recent years and liquidbiopsybased epigenetic studies may become a future research direction exploration in chinese nsclc patientsshowed that nsclcs with high tmb had dnam aberrance and cnvs some insertion and deletion indelmutations can lead to frameshifts and more immunogenic neoantigens in the pancancer analysis of cancer types evaluated in the cancer genome atlastcga rcc had the highest indel mutation load andframeshift indel mutations were found to produce threetimes more candidate neoantigens per mutation thannonsynonymousnssnvs somatic copy number alterations scnas are another feature of the genomic landscape of tumors andpancancer tcga analysis revealed an inverse correlation between scnas atthe singlearm or wholechromosomelevel and immune infiltration in tumortypes tested and this result was subsequently replicated in a larger study of tcga single nucleotide variantsdna damage response pathwaysgenetic variation involved in dna mismatch repairmmr pathway can lead to microsatellite instabilitymsi a specific type of high tmb tumors and increased numbers of cd8 tumor infiltrating lymphocytestils pd1tils and indoleamine 23dioxygenaseido tumor cells have been shown in mmr deficiencydmmr colorectal cancer recently five clinical trials keynote016 including multipletumor types have shown that patients with dmmrmsih can achieve durable responses to pembrolizmabbased on this pembrolizumab is approved by the usfda for the treatment of any advanced solid tumor withdmmrmsih and nivolumab in combination with ipilimumab has also shown promising response in dmmrmsih colorectal cancer in addition dmmr canalso cause mutations in the dna polymerase gene epsilondelta polepold1increasing the mutationload and neoantigen load analysis of polepold1mutations in patients with different cancer typesshowed that patients with these mutations had significantly higher tmb and os therefore it may be an infordependentinidentifying patients who benefitaddition pathways of base excision repairberand prognostic markerfrom icis risk factorhomologous recombination repair hrr mmr in thedna damage response ddr signaling network contribute more significantly to tmb or neoantigens whichhave the highest levels when comutated it hadbeen identified that comutations in the ddr pathwaysof hrr and mmr or hrr and ber defined as comutare associated with increased levels of tmb neoantigenload and immune gene expression signatures comutpatients showed a higher orr and longer pfs or os indicating that comut can be used as predictors of response to icis and provide a potentially convenientmethod for future clinical practice specific mutated gene pathways in tumor cellsit is worth noting that alterations of signaling pathwaysin tumor cells affect the responsiveness to immunotherapy patients with mutations in the interferon ifnγpathway genes ifngr12 jak12 and irf1 are poorlyresponsive to icis treatment and confer resistance a study found that in patients receiving immunotherapytumor cells can downregulate or alter ifnγ signalingpathways such as lossoffunction alleles of genes encoding for jak12 and changes in stat1 to escape the influence of ifnγ resulting in poor efficacy andresistance recent studies suggest that inactivating mutations in a mammalian analog of the chromatin remodeling swisnf complex and unique genes of the pbafcomplex pbrm1 arid2 and brd7 lead to sensitivitiesto icis [ ] loss of function of the pbaf complexincreased chromatin accessibility to transcription regulator elements of ifnγ“inducible genes within tumorcells and subsequently increased production of cxcl9cxcl10 chemokines leading to more efficient recruitment of effector t cells into tumors in human cancers expression of arid2 and pbrm1 are related toexpression of t cell cytotoxicity genes which confirmedin pbrm1deficient murine melanomas with strongly infiltrated by cytotoxic t cells and responsive to immunotherapy [ ]in addition doublestranded rnadsrna editing enzyme adenosine deaminase acting onrna adar1 protein can block the ifnγ signalingpathway and lead to poor icis efficacy and resistanceloss of function of adar1 in tumor cells can reduce atoi editing of interferoninducible rna species and leadto dsrna ligand sensing by pkr and melanomadifferentiationassociated protein mda5 this resultsin growth inhibition and tumor inflammation respectively and profoundly sensitizes tumors to immunotherapy finally demethylation positively regulates thetranscriptional activity of some immunerelated genesincluding pdl1 and ifn signaling pathway genes sensitizingto anticytotoxic tlymphocyteassociatedprotein4 ctla4 therapy it 0cbai biomarker research page of in addition to the ifnγrelated signaling pathway alterations in other tumor genome such as tumor oncogenes and suppressor genes pathways and pathwaysrelated to tumor cell proliferation and infiltration canalso affect immunotherapy efficacy epidermal growthfactor receptor egfr and anaplastic lymphoma kinasealk mutations have been shown to be associated withreduced response rates to icis and low tmb and therefore the fda does not recommend firstline icistreatment in patients with egrf or alk positive tumors[ ] certain types of mutations in mdm2mdm4and arid1a can predict nonresponse to icis in hightmb tumors nsclc with kras and stk11 comutated was associated with reduced response andshorter survival in three independent cohorts of patientstreated with antipd1 therapy and stk11 deficiency was an independent indicator of poor antipd1response in nsclc with kras mutant however at the american association for cancer research aacrmeeting of patients in the keynote042 studynct02220894 update data were tested for stk11 andkeap1 and the results showed that patients could benefit from pembrolizumab regardless of stk11 and keap1status but patients with stk11 mutations did not respond well to chemotherapy but given that only ofall patients had mutation detection the results may beaffected in initial data from studies using targeted ngspanels suggested that duration of icistreatment was associated with certain braf and m terations butnot tmb status notch signaling pathway is associated with the occurrence development and prognosisof tumors especially with the biological function of cancer stem cells recent breakthrough findings have distinguished deleterious notch mutation showing that itcan be used as a potential predictor of favorable ici response in nsclc potentially via greater transcription ofgenes related to dna damage response and immune activation another tumorspecific inheritance thatmay influence icis efficacy is the aberrant expression ofendogenous retroviruses ervs pancancer analysisidentified a positive correlation of transcript expressionof ervs with tcell activity in various tumors andpatient prognosis furthermore with the improvement of precision detection technology the accurateanalysis of negative mutation sites helps to identify thepossibly effective ones for example the analysis of studydata of secondline pd1l1 inhibitor therapy found thatthe mpfs of patients with kras g12c or g12v was significantly better than that of patients with kras mutations at other sites in addition several pancancer biomarkers are recentlyapproved by the fda for example given the effectiveorr of and a disease control rate dcr of in secondline cholangiocarcinoma patients treated withanalysispemigatinib a new targeted therapy the recent fda approval of pemigatinib for the treatment of previouslytreated patients with locally advanced or metastatic cholangiocarcinoma with fibroblast growth factor receptor fgfr2 fusion or rearrangement and the comprehensive genomicassay foundationone cdxdeveloped by foundation medicine as a companiondiagnostic also exciting is the recent fda approval ofthe targeted anticancer drug capmatinib for the treatment of metastatic nsclc with met exon skippingmetex14 mutations including firstline patients andpreviously treated patients also using foundationonecdx as a companion diagnostic to help detect specificmutations present in tumor tissueimmunogenicity ofneoantigen loadneoantigen load the number of mutations actually targeted by t cells may be directly related to the responseto icis [“] a retrospective study showed thatclonal neoantigen burden was associated with the longeros in primary lung adenocarcinomas p traditionallycomputational neoantigen predictionshave focused on major histocompatibility complexmhc binding of peptides based on anchor residueidentities however neoantigen loads identified by thismethod are generally not superior to overall tmb inpredicting icis efficacy or survival in recent practice this neoantigen can be assessed by the difference inpredicted mhci binding affinity between the wildtypepeptide and the corresponding mutant peptide knownas the differential agretopicity index dai reflectingclinically relevanttumor peptide a high dai value indicates that the mutant peptidesignificantly increases binding affinity to mhc compared to the wildtype sequence and can generate moreimmune responses studies on previously published cohorts treated with three icis have shown that dai outperforms tmb and the traditionally defined neoantigenload in predicting survival [ ] in additionlowneoantigen intratumour heterogeneity might also be important for icis response analysis of the lung adenocarcinoma tcga database found that combining highmutational load and low intratumoral neoantigen heterogeneity was significantly associated with osand longer lasting clinical benefit than either variablealone anotherreported method for assessingneoantigen foreignness is based on sequence homologyof experimentally validated immunogenic microbial epitopes in the immune epitope database iedb butit does not account for all possible human leukocyteantigen hla contexts in addition the detection forneoantigen can be reflected from different levels such aspeptides or genomes a study developed the neopepseealgorithm using a machine learning approach incorporating 0cbai biomarker research page of integration of nine immunogenicity features and gene mutation expression levels and its application to melanoma and leukemia patients could improve the sensitivityand specificity of neoantigen prediction recently it has alsobeen shown that promoter hypermethylation of neoantigengenes may be an important mechanism for immune editingand tumor immune evasion indicating that combineddetection of tumor genome and epigenetics may providemore information for immunotherapy efficacyii tumor immune microenvironment phenotypebiomarkerscells is also considered separately as one of the biomarkersto distinguish the benefit population called immune positive score ips herbst showed that response toatezolizumab treatment was significantly associated withhigh levels of pdl1 expression on the surface of tils before treatment but not with pdl1 expression on tumorcells p finally other inhibitory immune pathways may affect the response to icis therapy including tcelllymphocyte activationgene3 lag3 and vdomain ig suppressor of tcell activation vista which can be used as potential biomarkers for icis responseimmunoglobulin3 tim3pdl1 expressiongiven that multiple studies in a variety of tumors havedemonstrated a positive correlation between pdl1 expression and response to icis or os even in firstlinecombination therapy [“] pembrolizumab is currently approved by the fda for use in patients with pdl1 pdl1 ‰¥ of tumor cells in firstline treatmentand ‰¥ in secondline treatment nsclc and pdl1immunohistochemistry ihc as a companion diagnosticfor antipd1 therapy in nsclc patients [ ] however some studies have not detected a significant correlation between pdl1 expression and response to icis[ ] and pdl1 negative patients can still benefitclinically with treatment with ici or combination treatment with icis with orrs ranging from to therefore pdl1 cannot yet be a comprehensive and independent biomarker in clinical practice in assessing efficacy with following challenges still existing firstlypdl1 assay and antibody are not standardized secondly pdl1 expression is temporally and spatiallyheterogeneous a study of metastatic nsclctreated with icis showed that pdl1 varies substantiallyacross different anatomic sites and during clinicalcourse being highest in adrenal liver and lymph nodemetastases and lower in bone and brain metastases andthe predictive value of pdl1 at different biopsy sites forthe benefit of icis in nsclc may vary higher pdl1 inlung or distant metastasis specimens was significantly associated with higher response rate pfs and os whilepdl1 in lymph node metastasis biopsy was not associated with either response or survival thirdly positive score and cutoff value of pdl1 expression is notstandardized at present pdl1 positive scoremainly focuses on the pdl1 expression level of tumorcells that is tumor proportion score tps but pdl1is also expressed on immune cells such as lymphocytesand macrophages and stromal cells thus the investigators introduce the concept of combined positive scorecps which is the proportion score of the sum of pdl1 expressed by tumor cells and tumorassociated immune cells in addition pdl1 expression on immuneresponseto icisimmunetreatmentbiomarkers of tumorinfiltrating immune cellsoverall immune status of tumor microenvironmentthe pattern of tumor immune infiltration can be broadlyclassified into immuneinflamed immuneexcluded andimmunedesert immuneinflamed is characterizedby the presence of cd8 and cd4 t cells in the tumorparenchyma accompanied by the expression of immunecheckpoint molecules indicating a potential antitumor immuneexcluded is characterized by the presence ofdifferent immune cell types in the aggressive margin orstroma of tumor but cannot infiltration into tumor parenchyma [ ] analysis of pretreatment samples forantipd1pdl1 revealed a relatively high abundance ofcd8t cells at the invasive margin in responders andserial sampling during treatment showed an increasedinfiltration of cd8t cells into tumor parenchyma while immunedesert phenotype is characterized by theabsence of abundant t cells in the parenchyma orstroma of tumors and poor response to icitreatment recentlyimmunoscore has been proposed as avalid marker for characterizing the immune status oftumor microenvironment tme classifying tumors aswell as predicting treatment response and prognosis which involves the density of two lymphocyte populations cd8 and memory [cd45ro] t cells in thecenter and invading margin of tumor mlecnik evaluated immunoscore in specimens of stagei“iv colorectal tumor and confirmed that it was significantly associated with pfs dfs and os and multivariate analysis also showed the superiority of immunoscorein predicting disease recurrence and survival the valueof immunoscore to predicting icis efficacy is being validated internationally in clinical trials of melanoma andnsclc a wider assessment of active immune responses withintme by immune gene expression profiling might effectively predict clinical benefit to icis strategies analysisof total rna and genes that were substantially differentbetween the patient groups in pretreatment tumor biopsies revealed atleast a 25fold increase in the 0cbai biomarker research page of expression of immunerelated genes in clinically active patientsincluding cytotoxic t cell markers egcd8a perforin granzyme b th1 cytokines or chemokines mhcii and other immunerelated genes egnkg7 ido1 ascierto screened morethan immunerelated genes in patients with recurrent breast cancer “ years after treatment and thosewithout recurrence more than years later and foundthat five genes igk gbp1 stat1 igll5 and oclnwere highly overexpressed in patients with recurrencefree survival in addition ifnγinduced immune genesignatures may be effective biomarkers for predicting theclinical benefit of treatment with icis the study developed ifnγ scores combining multiple immune variablesbased on gene signatures which were then extendedto gene signatures in a validation set of melanomapatients including genes encoding ifnγ granzymes ab perforin ido1 and other immunerelated genesboth gene scores showed significant associations withbest overall response rate and pfs optimized cutoffvalues for ifnγ scores based on receiver operatingcharacteristic curve roc curve can achieve a positivepredictive value of for responders and a negativepredictive value of for nonresponders immune cells with specific phenotypes in tmethe phenotype of tils also influences the efficacy oficis the study used singlecell mrna sequencingscrnaseq data analysis to identify two major cd8tcell phenotypes within melanoma memorylike andexhausted the proportion of which is strongly correlated with response to icis the research furtherfound that the transcription factor tcf7 is selectivelyexpressed in memorylike t cells so the ratio ofcd8tcf7 to cd8tcf7tils is strongly correlatedwith improved response and survival in melanoma patients treated with antipd1 balatoni found that of immune cells in tme were positivelyassociated with os after treatment including cd4 andcd8 t cells foxp3 t cells cd20 b cells cd134and cd137 cells and nkp46 cells and different immune cells at different sites were differently associatedwith clinical outcomes researchers found that only asmall proportion of cd8 tilsin tumors couldrecognize tumor mutationassociated antigens while another population bystander cells was insensitive anddifferential cd39 expression was the key molecule thatdistinguished the two populations analysis of peripheral blood from a patient with colorectal cancer whoresponded rapidly to pembrolizumab treatment showedhigh expression of cd39 on cd8 tils indicating thatcd39cd8til may be a promising predictive biomarker the fact of very low level of cd39 expression on cd8tils in of egfrmutant nsclc isconsistent with their low response rate to antipd1immunotherapyin addition a study showed that fc domain glycan ofthe drug and fcγ receptor fcγr expressed by the hostbone marrow cells could determine the ability of pd1tumorassociated macrophages tams to capture antipd1 drugs from the surface of t cells which leads topd1 inhibitor resistance and the association oftams and poor antipd1 response was reported inmelanoma cohorts antipd1 response was associated with an increase in cd8t cells and natural killercells nk cells and a decrease in macrophages andhigh intratumoral myeloid markers were associated witha nearly 6fold decrease in mpfs after antipdl1 therapy in rcc emphasizing the inhibitory role of myeloidcells in response to icis in conclusion immunecells in tme show a great promise in the developmentof predictive biomarkers for icisimmunerepertoirediversity of immune repertoires in tmeeffective t cell responses involve the activation and expansion of specific antigenreactive t cell clones so diversity ofin intratumoral orperipheral may correlate with icis responses and can bequantified as richness and clonality however theresults seem to be complex with some studies finding apositive correlation between til clonality and the response to icis before or after treatment whileothers showing that only an increase in til clonalityduring treatment is associated with the response to antipd1 [ ] others show that intratumoral t cellclonality is not associated with survival while peripheralt cell clonality is inversely associated with pfs and os tumeh further investigated whetherbaseline tils have a narrow t cell receptor tcr repertoire focusing on tumorspecific immune responsesand whether this narrow tcr repertoire correlates withpembrolizumab responses they found that respondingpatient had more restricted usage of the tcr beta chainie a more clonal less diverse population than patientswith progressive disease and showed a 10times increasein these clones after treatmentimplying a tumorspecific response to treatment in these patients notablybaseline tcr clonality was not highly correlated withtil density suggesting that some patients with restricted tcr clonality specific for tumor antigens maystill benefit from antipd1 therapy even though tildensity is low recently researchers have proposed theimmune repertoire irindex the average frequency ofshared tcr clones in t clones in tils and peripheralpd1cd8 t cells they found that neoantigenstimulated tcr agreed with irindex and patients withhigh irindex had better immune activation and highergene expression profiles geps score subsequently they 0cbai biomarker research page of confirmed the predictive value of irindex to icis efficacy dcrpfs but considering that it is difficult tosort out pd1cd8 t cells in tumor tissue based ontwo separate patient cohorts a research confirmed thattcr repertoire diversity and clonality of peripheral pd1cd8t cells may serve as noninvasive predictors ofclinical outcomes after icis in patients with nsclc the viewpoints of t cell diversity and tcr clonality as markers of icis efficacy need to be further validated in a large patient populationiiiliquid biopsy biomarkersperipheral blood cell biomarkersperipheral blood is a noninvasive source to explore potential biomarkers for icis and although associationswith clinical benefit and survival have been observed itseffectiveness has not been validated in prospective studies analysis of melanoma treated with ipilimumabshowed that improved os and pfs were associated withbaseline values of peripheral blood components including low absolute neutrophil countlow neutrophiltolymphocyte ratio nlr low absolute monocyte countlow frequency of myelogenous suppressor cells high frequency of foxp3 treg cells high lymphocyte frequencyhigh eosinophil count and clinical benefit also associated with the dynamic changes of blood markers duringincluding decreased foxp3treg concentratreatmenttions and increased lymphocyte and eosinophil counts reports in patients with melanoma treated withpembrolizumab and in patients with nsclc treatedwith nivolumab have shown that nlr is associated withworse tumor response [ ] multivariate analysis inmelanoma patients treated with antipd1 antibodiesshowed that nlr was the only factor associated withworse orr and shorter pfs indicating that nlr is astrong predictor of worse outcome in patients treatedwith ici low baseline lactate dehydrogenase ldhlevels high relativeabsolute eosinophil counts and relative lymphocyte counts were associated with prolongedos in antipd1 and ctla4 treated melanoma given that previous studies have proposed the importance of baseline derived nlr dnlr and ldhlevels as prognostic markers a recent study proposed acomposite prognostic index that comprehensively takesthe two factors into account lung immune prognosticindex lipi which characterized risk groups goodintermediate and poor the analysis of patients with advanced nsclc in randomized trialss
Colon_Cancer
the hypoxic tumour is a chaotic landscape of struggle and adaption against the adversity of oxygen starvationhypoxic cancer cells initiate a reprogramming of transcriptional activities allowing for survival metastasis andtreatment failure this makes hypoxia a crucial feature of aggressive tumours its importance to cancer and otherdiseases was recognised by the award of the nobel prize in physiology or medicine for research contributing toour understanding of the cellular response to oxygen deprivation for cancers with limited treatment options forexample those that rely heavily on radiotherapy the results of hypoxic adaption are particularly restrictive to treatmentsuccess a fundamental aspect of this hypoxic reprogramming with direct relevance to radioresistance is the alterationto the dna damage response a complex set of intermingling processes that guide the cell for good or for badtowards dna repair or cell death these alterations compounded by the fact that oxygen is required to inducedamage to dna during radiotherapy means that hypoxia represents a persistent obstacle in the treatment of manysolid tumours considerable research has been done to reverse correct or diminish hypoxia™s power over successfultreatment though many clinical trials have been performed or are ongoing particularly in the context of imagingstudies and biomarker discovery this research has yet to inform clinical practice indeed the only hypoxia interventionincorporated into standard of care is the use of the hypoxiaactivated prodrug nimorazole for head and neck cancerpatients in denmark decades of research have allowed us to build a picture of the shift in the dna repair capabilitiesof hypoxic cancer cells a literature consensus tells us that key signal transducers of this response are upregulatedwhere repair proteins are downregulated however a complete understanding of how these alterations lead toradioresistance is yet to comefacts— hypoxia is present in almost every solid tumour— hypoxia is a major barrier to effective radiotherapyand is associated with radioresistance— the hypoxic tumour is highly heterogenous withregions of chronic and acute hypoxia altered ph andimmune ltration— differences in gene expression and protein functioncan occur between acute or chronic and mild orsevere hypoxiacorrespondence mahvash tavassoli mahvashtavassolikclacuk1head and neck oncology group centre for host microbiome interactionking™s college london hodgkin building london se1 1ul ukedited by i amelio— all dna damageresponsehomologousddr pathwaysincludingnonhomologous end joining missmatch repair andthe fanconi anaemia pathways have been shown tosuffer alterations in hypoxiarecombination— activation of ddr transducer protein atm is seenin severe hypoxia in the absence of classical atmactivatingstranddna breaksfeaturesdoublesuchas— atr is also activated most likely in response tohypoxiainduced replication stress— however downregulation of dna repair effectorproteins such as rad51 and brca12 is seen— results of ddr reprogramming include geneticinstability aberrant cell cycle and apoptotic control the authors open access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate ifchanges were made the images or other third party material in this are included in the ™s creative commons license unless indicated otherwise in a credit line to the material ifmaterial is not included in the ™s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this license visit httpcreativecommonslicensesby40official of the cell death differentiation association 0cbegg and tavassoli cell death discovery page of open questions— precisely how do alterations to the ddr in hypoxialead to radioresistance for example when genomicinstability and generation of radioresistant clonestakes several cell divisions to set in how does adecrease in dna repair ability lead to increasedradioresistance— what aspects of the hypoxic response could betargeted to radiosensitise or more effectively treattumours particularly in the context of ddr forexampleupregulated ddrtransducers such as atm atr and dnapkcs orinduce synthetic lethality following downregulationof dna repair effectorscan wetarget— do different types of cancers have different patternsof ddr alteration within hypoxic tumours thisparticularly needs further research as tissues havebeen shown to have different oxygen pressureslevels of hypoxia and hypoxic heterogeneity— can we use the data on this subject to develop abiomarkerhypoxiainducedradioresistance as we have done using hypoxia as asingle parametersignatureof— how can we monitor hypoxic tumours during thecourse of a patient™s disease to help guide treatmentand— how can wereportingreliableensureinterpretation of in vitro and in vivo dataintroductionofprogramstranscriptionalhypoxia is present in almost every solid tumour aninevitability of cancer™s characteristic disanised andfunctionally inefficient vasculature rapid growth anddemanding metabolism1 the result is a comprehensiverewritingupdownregulating certain genes and proteins allowing cells toevade apoptosis and migrate to areas with better oxygenperfusion crucially this microenvironmentally inducedintracellular shift also results in genomic instability gialterations to dna repair and resistance to cell killing bycancer therapies after decades of research it has becomeclear that the relevance of hypoxia for both oncogenesisand treatment resistance is inescapablein the context of radiotherapy rt a link betweenresistance and low intratumoral oxygen pressure has beenknown since the publication of a study modelling oxygenflow in lung tumours years ago2 for some cancerssuch as head and neck squamous cell carcinomashnsccs hypoxia is a major contributing factor to localrt failure3“ advancesin developing technologiesallowing for more precise delivery of rtimaging oftumours and sensitisation to treatment while protectingnormal tissues have led to improved locoregional controland quality ofsincelife for patients78 howeverofficial of the cell death differentiation associationtreatment for many cancers like hnsccs which incidentally are also some of the most hypoxic depends onrt the hypoxic problem remains particularly pertinent9in recent years efforts have been made to correct orreverse hypoxia including administering hyperbaric oxygen therapy to patients1011 reducing cellular oxygenconsumption12 and increasing blood vesselfunctionality1314 to more precisely tackle resistance induced byhypoxia researchers have also soughtthethreshold of treatability of hypoxic tumours by use ofsensitizers15“ as a third arm in our battle plan researchhas also gone into developing methods to detect hypoxiaincluding the use of specialised imaging techniques petct scanning combined with hypoxiadetecting radionuclides often studied in tandem with genetic signaturesseeking to genotypically define these tumours1819to lowerhowever very few of these advancements have allowedus to overcome hypoxiainduced radioresistance rrthough research activity in this area remains strong amore complete understanding of how the hypoxic environment contributes to rr particularly by modulation ofpotentially targetable dna damage response ddrpathways is warranted this review will outline our current knowledge of the molecular processes that underpinhypoxic rr particularly in the reprogramming of the ddrradiotherapy mechanism of action”therequirement of oxygenseminal work by gray and colleagues during the 1950sproved that the efficacy of rt was dependent on theavailability of oxygen within the tissue22021 radiationinduces damage through the direct and indirect generation of double stranded breaks dsbs in dna in thepresence of oxygen damage induced is “ times morelikely to end in cell death22 this effect is best explainedby the oxygen fixation hypothesis where radicals produced directly or indirectly by ionizing radiation ir areoxidised to dna in the presence of oxygen23 making thedamage irreversible24 thistothe hypothesis with the notion that these lesions cannotbe restored to an undamaged state as the damage isœï¬xed to dna by oxidisation25 thus without oxygendamage induced is transient and hypoxic cells experiencefar reduced radiationassociated damagelast pointis crucialthough crucial the requirement of oxygen to inducedamage is not where the story ends for rr as it does notfully explain the level of rr we observe this is evidencedby the fact that restoration of oxygen to tumours forexample through applying hyperbaric oxygen does notrestore radiosensitivity26 importantlyit also does notaccount for changes that occur with respect to dnarepair which have been shown to be crucial in impactingthe radiation response27 as these changes are retainedpast the point of radiotherapy administration 0cbegg and tavassoli cell death discovery page of the landscape of the hypoxic tumourin vivo the hypoxic region exists on a gradient ofoxygen pressures with oxygen levels throughout the tissue ranging between severe hypoxia “ mildhypoxia and anoxia with around consideredphysoxia see table for definitions tissue oxygenpressures are usually measured in mmhg however sincethe majority of research on hypoxia and the ddr hasbeen performed in vitro where oxygen levels are measured in percent for the purpose of this review o2 willbe referred to predominantly the difference betweenin vivo and in vitro measurements of oxygen is importantthough with tissue normoxia physoxia classified ataround o2 or mmhg and in vitro normoxia beingaround o2 fig the hypoxic tumour is a space of restricted proliferationparticularly when oxygen levels are o2 cell cyclearrest and decreased protein synthesis juxtaposed againstaccelerated aggressivity microenvironmental interactionsand altered ph28“ at the most oxygendepleted borderexists the barren land of necrosis with the highly proliferating and comparatively treatmentsensitive aerobiccells closest to the blood vessel fig tumour hypoxia does not develop in a linear fashionand is highly heterogenous and changeable the hypoxictumour is dynamic with fluctuating vessel functionalityand cycling oxygen levels creating regions of acute andchronic hypoxia31 where part of the tissue may sufferacute hypoxia after temporary occlusion of a blood vesselsocalled perfusion limited in which oxygendeprivationcycles last sometimes minutes sometimes hours beforesubsequent reoxygenation chronic hypoxia is diffusionlimited where oxygen levels become a factor of distancefrom the blood vessel31 compounded with this is thediffering rates of oxygen consumption and responsivenessto oxygen availability in cells within the tissue to be ableto fully understand and ultimately treat the hypoxictumour it must be remembered that the changeability ofoxygen concentrations in the hypoxic tumour also predictably ‚uences its behaviour and response to treatment in the context of radiotherapy cells with o2 levels is where we see most resistance so called radiobiological hypoxia24 whether this is acute and thereforefollowed by reoxygenation or chronic oxygen deprivedfor more than h can have marked differences on theensuing genomic and proteomic changes that ultimatelyallow for hypoxic survival32 thus within one tumourdifferent regions are likely to have a completely differentresponse to the same dose of radiotherapyitaside from the oxygen status the involvement of otherenvironmentalfeatures affected by hypoxia must beconsidered an additional outcome of hypoxic adaption isthe concomitant phenotypic shift of the microenvironmentis now accepted that hypoxia can induce‚ammation33 demonstrated even by patients whodevelop mountain sickness after prolonged periods athigh altitude34 hypoxic tumours are known to havehigher ltration of protumour immune cells such asm2 macrophages35 a feature known to be involved inrr3637 the same could also be said with respect to thehypoxicinduction of highly rt resistant cancer stemcells38 though this subject needs further research thelikelihood of an interplay between intracellular geneticreprogramming as a result of hypoxic adaption and themicroenvironment in mediating radioresponse is stronggenetic reprogramming”the hifswithin this chaotic showground of heterogeneity andat the core of cellular adaption to hypoxia are the alteredgenetic pathways that push for survival against adversitycommonly dysregulated genes include glut1 involvedin altered glucose metabolism vegf involved intable glossary of terms multiple classifications of the terms used to describe hypoxia exist throughout the literaturethis represents a general consensus and what is used in this reviewtermhypoxianormoxiaphysoxiaanoxiasevere hypoxiamild hypoxiaacute hypoxiachronic hypoxiadefinitionreduced oxygen levels usually ‰¤ o2 mmhg in in vitro studiesnormal atmospheric oxygen used in in vitro studies o2 mghgphysiological levels of oxygen in tissues between mmhg tissue specific see fig complete absence of oxygen o2 o2“ o2incubation in hypoxic conditions “ hincubation in hypoxic conditions hradiobiological hypoxiaoxygen levels where the efficacy of radiotherapy is half maximal mmhg04 o2official of the cell death differentiation association 0cbegg and tavassoli cell death discovery page of fig approximate oxygen levels reported in different tissues in mmhg used in in vivo experiments and o2 used in in vitroexperiments note that normal tissue normoxia or physoxia is considerably less than the o2 used in vitro as normoxia adapted frommckeown139 liu140 and graham26fig the heterogeneity of the hypoxic tumour tumours suffer from reduced oxygen availability due to the disanised nature of thevasculature where occlusion of a blood vessel bv occurs tumours are said to be under perfusion limited hypoxia pl hypoxia where lack ofoxygen is a function of distance from the vessel cells experience diffusion limited dl hypoxia when these states are temporary h it is said tobe acute or chronic when h within hypoxia tumour cells undergo considerable genetic reprogramming contributing to therapy resistance andmetastatic behaviourofficial of the cell death differentiation association 0cbegg and tavassoli cell death discovery page of neoangiogenesis and lox involved in remodelling ofthe extracellular matrix7in the nobel prize in physiology or medicine wasawarded to three scientists gregg semenza williamkaelin and sir peter ratcliffe for their contributions toour understanding of cellular oxygensensing mechanisms39 this included the discovery of a group of transcription factors regulated by hypoxia that allow forcellular adaption40 these hypoxiainducible factor hifproteins hif13 are transcription factors composed oftwo subunits the α subunits reside in the cytoplasm andto rapid degradation “ min41 underare subjectnormal circumstances this degradation is mediated bythe actions of prolylhydroxylasesphd14 whichhydroxylate hifα at the oxygendependentdegradationdomains oddd of note phd2 and phd3 are themselves transcriptional targets of hif alluding to possiblenegative feedback systems in place though conflictingresults suggest this system doesn™t always function effectively to constrain cancer growth42“ subsequentlyhydroxylation by phds recruits the vonhippellandauvhl protein45 this alongside other proteins forms ane3 ubiquitin ligase complex ubiquitinating hifα forproteasomal degradationin hypoxia due to the lack of molecular oxygen neededfor hydroxylation4246 this degradation cascade does nottake place and hifα subunits translocate to the nucleusto associate with hifβ subunits47 the hif complex ininteraction with its coactivators p300 and crebbindingprotein cbp then binds to hypoxia response elementshresin dna to initiate transcription of hiftarget genesintheincludingcontrolledadditional layers of hif regulation also exist to keepthis pathway in check such as factor inhibiting hif fihwhich hydroxylates hif subunits at asparagine residuesblocking their association with p300cbp48 some evidence has shown that hifs also undergo other posttranslational modifications including phosphorylation andacetylation as a further method of regulation4247 however as with many such processes in cancer it can beaberrantlyhypoxiaindependent stabilisation of hifα by oncogenes such asegfr and mtor4950 and depletion of hifregulatoryfactors4251 the hif proteins themselves can interactwith a number of factors relevant in cancer such as p53mutants present in human papillomavirus hpvnegativehnsccs and nonsmall celllung cancers nsclcsresulting in transcriptional control of protumorigenicgenes52 since both hifs and p53 compete for binding ofp300cbp to enact transcriptional control the hifs havea unique relationship with this highly cancerrelevantprotein53 inactivation of p53™s transcriptional abilities hasbeen observed54 though again conflicting results exist forthis55official of the cell death differentiation associationmost of the work investigating hifdirected transcriptional changes in hypoxia has focussed on the actions ofthe bestknown hif hif1 however both hif2 andhif3 also play a role in hypoxic transcriptional control42interestingly relative expression of the hifs has beenshown to differ between hypoxic tissues demonstratingthat each may have specific functions56 in some casesthey may indeed work in concert as hif2 has beenshown to be induced when hif1 is depleted50hifs are master regulators of the hypoxic response andconcurrent with the notion that hypoxic tumours areradioresistant depletion of hif1α in tumour modelsradiosensitises cells41 one study showed that intermittenthypoxia showed less radiationinduced cell death bothin vitro and in mice via stabilisation of hif1α57 thisinvestigation also found that intermittent hypoxia had amore significant effect than chronic hypoxia hif1α hasalso been shown to function via the hif1α“myc pathway in which hif1α competes with the transcriptionactivator myc for sp1 binding in the target gene promoter to downregulate mismatch repair mmr genesmsh2 and msh6 in o2however how exactly hifs contribute to rr of thehypoxic tumour be itthrough their transcriptionalfunctions or interactions with other proteins is so farunresolved notably some radio and chemotherapiesthemselves upregulate or stabilise hifs41though genetic reprogramming in hypoxia can lead to anumber of alterations for the purpose of this review wewill focus on those associated with rr and ddr formore general reviews see schito60 and tsai61reprogramming of the ddrthe ddr is a complex process consisting of overlapping and interconnected pathways initiated by differentforms of dna damage arguably one ofthe mostimportant homeostatic processes it allows us to withstand constant and numerous dna damageinducinginsults the result of this protection ensures that onlyreliable genomes are passed on to the next cellular generation for cancer considering both the power ofmutagenesis in driving oncogenic potential and the factthat many cancer therapies function by inducing dnadamage the ddr has considerable relevance for therapyresistance and tumour progressionrepair of dna is a tale of three acts firstly the damagepropagates a signal that recruits sensors to the site ofdamage secondly the signal is amplified by transducersand thirdly response pathways are initiated by effectorsfor each part of the process welldefined though notexclusive sets of proteins act as sensors transducers andeffectors respectively28 shrouding these repair processesare signals to stall the cell cycle initiated by chk1 orchk2activated cdc25 and p21to allow time for 0cbegg and tavassoli cell death discovery page of clearance of this damage and initiation of apoptoticpathways for example as initiated by atm™s interactionwith p53 if the repair is unsuccessful6263for the repair of radiationinduced dsbs two primarypathways are put to use homologous recombination hrand nonhomologous end joining nhej the formerconsidered less errorprone uses sister chromatids torepair dna and as such can only take place during g2sphase of the cell cycle nhej predominates in g1 but canoccur at any stage of the cell cycle and often results in thegeneration of insertiondeletion mutations which havethe potential to lead to more oncogenic alterations hr ismediated primarily by the recruitment to sites of damageof master transducer of the dsb response atm ataxiatelangiectasia mutated a phosphoinositide3kinaserelated protein kinase pikk following detection by themrn complex composed of mre11rad50nbs1which initiates activity of effectors including rad51 andbrca1 nhej occurs following sensing of damage by theku proteins ku70 and ku80 and signal transduction ofku in complex with dnapkcs dnadependent proteinkinase catalytic subunit”together forming dnapk andsubsequent activity of effectors dna ligase iv ligivand xrcc464alteration of ddr pathways has been seen across manycancers compared to normal tissue perhaps the mostwellknown are the mutations in brca12 in aggressivehereditary breast and ovarian cancers65 understandablywhere hypoxia represents an exaggerated form ofaggressive tumours the ddr pathways in hypoxia operatedifferently to those in normoxia indeed this is true forevery aspect of the ddr process dna damage in theform of dsbs is reduced in conditions of hypoxia o2and hypoxia alone does not induce dsbs2466 researchhas shown that different members of the ddr pathwayscan be either activated or downregulated in conditions oflow oxygen see tables and for reported alterationsto hr nhej and mismatch repair mmr pathwayscrucially whether the cells are in acute or chronichypoxia or h and at what level of oxygen depletion may define the ensuing response despite this delineation there lacks within the literature proper reportingof experiments carried out in either acute or chronic mildor severe hypoxia with interchangeability in use of termssee table for a consensus of parameters used with thesedefinitionssensorsthe mre11rad50nbs1 mrn complex is responsible for sensing dna damage and initiating both the hrand nhej pathways by recruitment of transducers such asatm via nbs167 while the mrn complex is consideredthe main sensor responsible for recruiting and activatingatm followingdamage atrip atrinteractingofficial of the cell death differentiation associationprotein and ku7080 are sensorsresponsible forrecruitment of atr and dnapkcs respectively67 fig though there are many overlapping interactions atminatminteracting protein with roles in replication stressrs genome stability and the base excision repair berpathway has also been shown to recruit atm independent of dna damage68repression of the mrn machinery has been seen inchronic hypoxia days in a medulloblastoma modelwith transcriptomic downregulation of both mre11a andnbs1 resulting in downregulation of etoposideinducedatm and p5369 nbs1 has also been shown to stabilisehif1α particularly in response to ir70 while hif1α hasbeen shown to downregulate nbs1 the authors of onestudy where reduction of nbs1 was seen after h in o2 noted that this repression resulted in the induction ofγh2ax and 53bp1 foci in hypoxia suggesting the presence of dna breaks59 interestingly all components ofthe mrn were found to be downregulated both at themrna and protein level in nsclcs harbouring egfrmutations incubated in severe hypoxia o2 thisdownregulation in egfrmutated cells correlated withtheir increased sensitivity to egfrinhibiting drugs71sensing of damaged dna is a crucial step in theinitiation of repair and begins with changes to the chromatin72 γh2ax a phosphorylated variant of histoneh2ax is induced by mrn activation and accumulates atsites of damage in the chromatin preceding recruitmentand necessary for retention of key ddr signalling proteinsincluding mrn and atm73 studies also show thatγh2ax is crucial for retaining mediators such as 53bp1p53 binding protein mdc1 mediator of dnadamage checkpoint and brca1 at sites of damage66h2ax is primarily phosphorylated by atm but can alsobe phosphorylated by atr and dnapkcs63 indeed aswell as by radiation and chemotherapies γh2ax has alsobeen shown to be induced by hypoxia following replication fork stalling this phosphorylation has been shown inchronic severe hypoxia to occur in a hif atr or atmdependent manner74“ crucially some evidence hasshown the phosphorylation of h2ax present only inproliferating cells7778 the downstream effects of thisactivation have been linked to other consequences ofhypoxic regulation including angiongenesis79 via induction of vegf80 experimentally resolution of γh2ax fociafter irradiation is often used as a marker of dsb repair astheory dictates that the phosphorylation should disappearafter damage is repaired however in hypoxia this heavilyrelied upon protocol may necessitate further finetuningku70 and ku80 together forming a heterodimericcomplex tether damaged dna at breaks and are keysensors of dsbs responsible for recruitment of dnapkcs as part of the nhej pathway63 the ku complex hasbeen shown to be both upregulated and downregulated by 0cbegg and tavassoli cell death discovery page of table a nonexhaustive list showing alterations to sensors transducers and effectors of the homologoursrecombination hr pathways in hypoxiaprotein role in ddrmechanism of alterationalteration conditions and consequencesreferencenbs1sensor of dsbs in hr activated atmas part of the mrn complexcid129 pasb domain of hif1αmre11sensor of dsbs in hr activated atmas part of the mrn complexcid129 atmtransducer of hr in dsb repaircid129 autophosphorylation atser1981atrtransducer of dna repair induced byreplication stresscid129 rad51effector of dsb repair in hrrad52effector of dsb repair in hrrad54 motor protein effector of dsbrepair in hrbrca1effector of dsb repair in hrcid129 e2f4p130cid129 lsd1cid129 ezh2cid129 mir210cid129 mir373cid129 mir210cid129 cid129 e2f4p130cid129 h3k4 demethylation via lsd1cid129 downregulated in chronic mild hypoxia days o2cid129 downregulation in acute mild h o2cid129 resulted in induction of γh2ax and 53bp1 focicid129 downregulated in chronic mild hypoxia days o2cid129 activated in acute hypoxia o2cid129 increased expression and activity o2“ hcid129 mediated by src and ampk signallingcid129 activated in acute o2cid129 resulted in phosphorylated p53 andaccumulation and growth arrestcid129 downregulation in chronic severe hypoxia o2 “ h and or “ hcid129 decreased radioresistancecid129 increased genomic instabilitycid129 downregulation in o2 hcid129 decreased mrna expression o2“ hcid129 downregulated o2 hcid129 decreased mrna expression o2“ hcid129 downregulation in chronic severe hypoxia o2 hcid129 decreased mrna expression o2“ hcid129 downregulation in o2 hcid129 decreased radioresistancecid129 decreased expression o2 “ hcowman69to59cowman69hashimoto88bencokova28hammond75meng119bindra oliveira82meng119crosby118meng119meng119lu117bindra120oliveira82meng119brca2effector of dsb repair in hrcid129 hypoxia in different studies81 one study found downregulation of ku80 after h in mild hypoxia o282another using severe hypoxia o2 found upregulation of ku70ku80 in a431 cells alongside many othermembers of the nhej pathway and proteins generallyinvolved in metastatic progression83 a study in humanand mouse hepatoma cells found upregulation of the kuheterodimer upon incubation in hypoxia o2 or withhypoxia mimics and downregulation associated with hif1βdeficient cells84 alternative subpathways of nhejalso exist possibly as insurance for when classical nhejmediators are inoperative howeverthe impact ofhypoxia on these pathways has not been extensivelystudiedofficial of the cell death differentiation associationtransducersatm and atr are two of the most important proteinsinvolved in transduction of the ddr as pikk familymembers they phosphorylate a number of proteinsinvolved in propagating the signal and repairing dna aspart of both the hr and nhej pathways as well asundergoingtheresponse until dna is repairedautophosphorylationto maintainbroadly speaking atm has been shown to be activatedparticularly in acute hypoxia as shown in the study bybencokova et al the pattern of activation in this contextdoes not match rtinduced atm activation which follows mrn recruitment to dsbs85 as atm phosphorylation does not correlate with the presence of dsbs 0cbegg and tavassoli cell death discovery page of table a nonexhaustive list showing alterations to sensors transducers and effectors of the nonhomologous endjoining nhej pathways in hypoxiaproteinrole in ddrmechanism of alterationalteration conditions and consequencesreferenceku70ku80sensor in nhej pathwaysrecruits dnapkcsin complex with ku80sensor in nhej pathwaysrecruits dnapkcsin complex with ku70cid129 cid129 dnapkcstransducer of nhej pathwaycid129 autophosphorylation atser2056dna ligiv effector of nhej repairxrcc4effector of nhej repaircid129 cid129 cid129 decreased mrna expression o2 “ hcid129 upregulation o2 hcid129 downregulation in cervical tumour sectionscid129 upregulation o2 “ hcid129 upregulation o2 hcid129 downregulation o2 hcid129 downregulation in cervical tumour sectionscid129 upregulation o2 “ hcid129 decreased mrna expression o2 “ hcid129 increased expression and activity o2 “ hcid129 activated in mild hypoxia “ o2 led to positiveregulation of hif1 and upregulation of glut1cid129 decreased mrna expression o2 “ hcid129 decreased mrna expression o2 “ hmeng119ren83lara81um84oliveira82ren83lara81um84meng119hashimoto88bouquet103meng119meng119table a nonexhaustive list showing alterations to sensors transducers and effectors of the mismatch repair pathwayin hypoxiaprotein role in ddrmechanism of alterationmlh1dimerises to pms2 to form themutlα complex in mmrpms2dimerises to mlh1 to form themutlα complex in mmrmsh2dimerises with msh6 forms themutsα complex in mmrmsh6dimerises with msh6 forms themutsα complex in mmrcid129 mad1maxcid129 mntmaxcid129 dec12cid129 mir155cid129 lsd1cid129 hdaccid129 hypoacetylationhypermethylation on h3cid129 cid129 mycmaxcid129 hif1α via sp1cid129 mir155cid129 hcid129 p53cid129 hif1α via sp1cid129 mir155cid129 hdaccid129 p53cid129 hypoacetylationhypermethylation on h3official of the cell death differentiation associationalteration conditions andconsequencescid129 downregulation “ h o2cid129 downregulation in h o2cid129 increased expression “ h o2resulting in genomic instability instem cellscid129 downregulation at protein level“ h o2cid129 resulting in genomic instability instem cellscid129 downregulation “ h o2referencebindra121127mihaylova128nakamura129rodriguezjimenez145lu115mihaylova128rodriguezjimenez145bindra121127koshiji58cid129 downregulation “ h o2cid129 increased expression “ h o2resulting in genomic instability instem cellskoshiji58rodriguezjimenez145 0cbegg and tavassoli cell death discovery page of often reduced or absent in severe hypxoia28 the authorsof this study emphasized that atm activation was specificto the level of hypoxia only phosphorylated at o2and hifindependent since phosphorylation was maintained even in hifknockout cells activation of atmwas attributed to autophosphorylation the result of whichwas an activation of targets much like dna damageinduced atm activation but dependent on the activityof cell cycle regulator mdc128 this study did not analyserr but the results suggest that atm activation byhypoxia is likely enacted as a means of halting the cellcycle in order to allow for dna repairthe pattern of atm activation in hypoxia however isnot clearcut and may depend on cancer type atm canbe regulated by a number of factors as part of the hypoxicresponse including mrn or atmin but also by posttranslational and epigenetic factors86 one study foundthat atm was downregulated along with hif1α by amicrorna mir18 resulting in radiosensitivity87the study by hashimoto et al88 showed atm activation alongside activation of a number of other key ddr orcancerrelated proteins including dnapkcs akt andegfr and decreased expres
Colon_Cancer
recently the current pandemic of coronavirus disease covid characterized by a pulmonary infection in humans is caused by a novel virus strain from family coronaviridae known as severe acute respiratory syndrome coronavirus sarscov2 the previous outbreak of severe acute respiratory syndrome sars in “ and middle east respiratory syndrome mers in has demonstrated the lethality of coronaviruses when they cross the species barrier and infect humans so far six coronaviruses infecting humans have been identified and the novel coronavirus is the seventh one described to date as being responsible for a respiratory infection sarscov and merscov and the new sarscov2 belong to the betacoronavirus family [“] the coronaviruses have the largest genome around k among the rna viruses sarscov2 was closely related from “ identity to two batderived severe acute respiratory syndrome sarslike coronaviruses batslcovzc45 and batslcovzxc21 but it was more distant from sarscov from “ and merscov about furthermore the performed bioinformatic analysis showed that the nucleotide sequence of sarscov2 is similar to those of other betacoronaviruses with nucleotide identities of ‰¥ there are currently no effective licensed therapies for human coronaviruses hcov infections and existing treatment strategies are generally limited to symptomatic treatment and supportive care email addresses kuzunovahqtchaikapharmacom k uzunova efilipovahqtchaikapharmacom e filipova vpavlovahqtchaikapharmacom corresponding author v pavlova tvekovmuplevenabvbg t vekov 101016jbiopha2020110668 received may received in revised form august accepted august biomedicinepharmacotherapy1312020110668availableonline24august2020075333222020theauthorspublishedbyelseviermassonsasthisisanopenaccessundertheccbyncndlicensehttpcreativecommonslicensesbyncnd40 0csuch as solidarity who recovery k uzunova in the absence of a specific treatment for this novel virus the effort of researchers is focused on understanding and controlling the disease and on preventing and controlling the replication and spread of the virus to devise therapeutic strategies to counteract the sarscov2 infection numerous potential treatment options are being evaluated in ongoing clinical trials many antiviral and immunological treatments being investigated against coronaviruses are summarized by who in landscape analysis of therapeutics as of march the realtime dashboard of completed ongoing and planned clinical trials for covid includes drugs and promising therapies such as remdesevir lopinavirritonavir hydroxychloroquine il6 inhibitors tocilizumab and sarilumab convalescent plasma therapy stemcell transfusion vaccine candidates traditional chinese medicines which are of top interventions of the presented network among them remdesivir an analogue of adenosine seems to have a more promising future due to proven in vitro and in vivo antiviral efficacy till the beginning of june promising therapies involving lopinavirritonavir and chloroquine or hydroxychloroquine were part of treatment guidelines in many countries but currently they are excluded from covid19 treatment protocols because of uncertainty regarding their risks and benefits and it is recommended that they should be used only in the context of clinical trials [“] in spite of its known in vitroin vivo efficacy and safety profiles some trials evaluating these drugs for covid19 infection treatment uk ntc04381936 and discovery inserm ntc04315948 discontinued hydroxychloroquine and lopinavirritonavir arms the interim trial results showed that hydroxychloroquine and lopinavirritonavir produced little or no reduction in the mortality of hospitalized covid19 patients when compared to standard of care nevertheless some countries worldwide continue to recommend chloroquinehydroxychloroquine as a treatment option [“] the existing drugs that target viral proteins associated with enzymatic activities or blocking viral replication machinery or host proteins involved in viral life cycle regulating the function of the immune system or other cellular processes in host cells have great potential and are available on the market our review aims to highlight the potential molecular mechanisms of the therapeutic options available for the cure of other health conditions and their repurposing for the treatment of this novel coronavirus sars cov2 selected treatments of sarscov2 remdesivir gs5734 “ polymerase inhibitor deltacoronavirus genus pdcov which have the most divergent rdrp of known cov as compared to sars and merscov an in silico test of the covid19 rdrp built model suggested the effectiveness of remdesivir as a potent drug sarscov and sarscov2 both belong to the betacoronaviruses of the b lineage and the rdrp amino acid sequences of the two viruses are identical whereas merscov belongs to the betacoronaviruses of the c lineage and is only identical with sarscov2 another in vitro and in vivo proof came from sheahan who examined if gs5734 could inhibit replication of sarscov and mers cov in primary human airway epithelial hae cell cultures they found out a dosedependent reduction in replication with average ic50 values of μm sarscov and μm merscov moreover the compound inhibits a broad range of diverse cov including circulating human zoonotic bat cov and prepandemic zoonotic cov with both prophylactic and therapeutic 1dpi dosing of gs5734 a reduction in replication below a diseasecausing threshold in mouse model of sars cov pathogenesis was demonstrated therapeutic gs5734 substantially reduced the sarscov induced weight loss in infected animals and significantly suppressed virus lung titers p thus demonstrating that therapeutic administration of gs5734 can reduce disease and suppress replication during an ongoing infection furthermore remdesivir has the potential to block sarscov2 infection in vitro at lowmicromolar concentration and in treatment of merscov and sarscov infections in vivo it demonstrated a significant improvement of pulmonary pathology in mice the rnadependent rna polymerase rdrpmediated mechanism of cov inhibition by gs5734 is proven even in the setting of intact exoribonuclease exonmediated proofreading using the model coronavirus murine hepatitis virus mhv it was demonstrated that gs5734 dramatically inhibited viral replication and viral rna synthesis in wildtype wt virus while an nsp14 exon mutant lacking proofreading demonstrated increased susceptibility to gs5734 45fold more active this suggests that gs5734 is recognized at least partially by a functional exon but that the exon activity is not sufficient to prevent potent inhibition of cov replication the results provide strong evidence that rdrp is the target for remdesivir and support the hypothesis that gs5734 directly inhibits viral rna synthesis the mechanism of inhibition of rdrp of merscov by remdesivir was studied by gordon et al they coexpressed the merscov nonstructural proteins nsp5 nsp7 nsp8 and nsp12 rdrp in insect cells as a part of a polyprotein coexpression of the mers nsp5 protease with nsp7 nsp8 and nsp12 in insect cells yielded a stable complex composed of nsp8 and nsp12 the triphosphate form of the inhibitor rdvtp is utilized as a substrate and competes with its natural counterpart atp and they observed that incorporation of the nucleotide analogue was significantly more efficient once added into the growing rna chain the inhibitor does not cause immediate chain termination the presence of the ²hydroxyl group allows the addition of three more nucleotides until rna synthesis is arrested at position i3 therefore the main possible mechanism of action is delayed rna chain termination recently the same authors obtained almost identical results with sarscov merscov and sarscov2 rdrps they provided evidence that all three coronavirus rdrp complexes terminated rna synthesis at position i3 almost all viruses encode polymerases in the central steps of replication and transcription therefore polymerases are becoming the most attractive and suitable targets for antiviral development there are two major types of polymerase inhibitors i nucleoside and nucleotide substrate analogs and ii allosteric inhibitors nucleoside analogs are first triphosphated by the host cell to produce the active inhibitor and then act as an inhibitor by competing with the natural nucleoside triphosphates and terminating the growing viral nucleic acids to date most of the approved antiviral drugs for antihiv therapy utilize this mechanism remdesivir is a nucleotide analogue with a proved mechanism of action as an inhibitor of rnadependent rna remdesivir rdv is an investigational compound with a broad spectrum of antiviral activities against rna viruses including sarscov and merscov gs5734 was originally developed for the treatment of the ebola virus disease gs5734 the single sp isomer of the 2ethylbutyl lalaninate phosphoramidate prodrug effectively bypasses the rate limiting first phosphorylation step of the nuc nucleoside ribose analogue the mechanism of action of nuc requires intracellular anabolism to the active triphosphate metabolite ntp which is expected to interfere with the activity of viral rnadependent rna polymerases rdrp gs5734 selectively inhibits ebola virus replication by targeting its rdrp and inhibiting viral rna synthesis following efficient intracellular conversion to ntp in nonhuman primates this compound shows a broad spectrum of antiviral activities against several rna viruses including respiratory syncytial virus rsv junín virus lassa fever virus and middle east respiratory syndrome virus but was inactive against alphaviruses or retroviruses furthermore remdesivir dosedependently inhibits endemic human cov229e and covoc43 replications which typically cause upper respiratory infection in children but can cause more severe lower respiratory infection in adults with underlying respiratory conditions ie asthma copd and the elderly as well as a member of the biomedicinepharmacotherapy13120201106682 0c lopinavirritonavir “ protease inhibitor the proteases encoded by most viruses play a crucial role in the viral life cycle the protease inhibitors pis bind competitively to the substrate site of the viral protease this enzyme is responsible for the post translational proteolysis of a polyprotein precursor and the release of functional viral proteins allowing them to function correctly and individually in replicationtranscription and maturation inhibition results in the production of immature virus ps coronavirus proteases of which there are two in sarscov a papainlike cysteine proteinase plpro nsp3 and a 3clike proteinase 3clpro or mpro nsp5 and three in several other coronaviruses cleave the orf1 polypeptide as it is translated enabling the formation of the viral replication complex the substratebinding pockets are highly conserved among cov 3clpro suggesting the possibility for a widespectrum inhibitor design targeting this region in the 3clpro of all covs it is postulated that the 3clproinhibiting activity of lopinavirritonavir contributes at least partially to its anticov effects in silico binding studies of the drugs using the identified crystal structure of mpro and employing the hex program to conduct the docking of the ligands to the sarscov main proteinase revealed that lopinavir and ritonavir could basically bind to the active site of sars main proteinase but the efficacy of lopinavirritonavir was predicted to be poor according to the latest report of the structure of 3clpro from sarscov2 pdb code 6lu7 and the available structure of 3clpro from sarscov pdb code 1uk4 the two main proteases differ by only amino acids comparing ligand binding free energies for the main proteases has confirmed that good binders for sarscov are in general and sarscov k uzunova polymerases this mechanism is probably involved in an antiviral activity against sarscov2 biochemical data provided by gordon suggested a unifying mechanism of inhibition of sarscov merscov and sarscov2 fig and future emerging covs may be similarly susceptible to the inhibition by remdesivir comparable replication with also good binders for sarscov2 3clpro protease inhibitors a class of drugs best known for success against hiv block the final step of virion assembly in the treatment of human immunodeficiency virus infection with proven efficacy the combination of lopinavir with ritonavir is widely used as a boosted protease inhibitor in the treatment of hiv infection because of low oral bioavailability of lopinavir and its extensive metabolism by the cyp3a4 isoenzyme lopinavir needs to be coadministered with ritonavir to achieve drug concentrations high enough to inhibit viral replication [ “] so far the reported results from studies in different cell lines animal models and patients for lopinavirritonavir are not so convincing in their inhibition action in human coronaviruses screening the library of fdaapproved drugs for antimerscov activity in cell culture has identified four compounds chloroquine chlorpromazine loperamide and lopinavir which inhibit merscov replication effective concentration ec50 3cid0 μmoll in vitro lopinavir inhibited mers cov efficacy ec50 μm and a maximal protective effect were observed at a dose of μm it was previously shown that lopinavir but not ritonavir inhibit sarscov chymotrypsinlike 3cl protease at the concentration of μm moreover it was suggested that lopinavir blocks a postentry step in the merscov replicative cycle in vitro the detectable antiviral activities of ribavirin rimantadine lopinavir and baicalin were shown by using the frhk4 cell line and in vero e6 cells infected with sarscov2 lopinavir inhibit replication with ec50 at μm during the sars outbreak treatment with lopinavir in combination with ritonavir was explored with some success in nonrandomized clinical trials patients with sarscov treated with lopinavirritonavir showed a progressive decrease of viral load and reduction of the composite adverse outcome at day recently the antiviral activity of remdesivir and ifn was found to be superior to that of lopinavirritonavirifn against merscov in vitro and in vivo the efficacy of lopinavirritonavir with or without ribavirin is evaluated in sarscov2 patients under randomized control trials currently it was demonstrated that this combination has no benefits in adult patients with severe covid19 although protease inhibitors are a common class of medication used in the treatment of hiv1 infection their efficacy in human coronavirus infections is not convincing moreover several antihiv pis are also known to influence other intracellular pathways it was demonstrated that hiv protease inhibitors indinavir saquinavir and lopinavir independently from any viral infection can hinder lymphocyte apoptosis by influencing mitochondrial homeostasis in view of the weak antiviral activity of protease inhibitors further studies should be done to ascertain whether the clinical benefit could be attributed to their antiapoptotic rather than their antiviral activity hence even if the molecular target of lopinavirritonavir is the main protease 3clpro in sarscov2 infected cells fig there are no biochemical and molecular studies confirming the interaction and associating this with clinical efficacy of the protease inhibitor chloroquinehydroxychloroquine chloroquine chq was introduced into clinical practice in as a prophylactic treatment for malaria hydroxychloroquine hcq differs from chloroquine by the presence of a hydroxyl group at the end of the side chain the nethyl substituent is hydroxylated currently chq and its hydroxyl form hcq are used as antiinflammatory agents for the treatment of rheumatoid arthritis lupus erythematosus and amoebic hepatitis in addition chq has been studied as a potent antiviral agent against hiv1aids [“] hcov229e sarscov [ ] influenza a h5n1virus influenza a and b and many other rna and dna viruses many recent reports and published studies suggested that chq and hcq were associated with reduced fig inhibition of viral infection by lopinavirritonavir and remdesivir biomedicinepharmacotherapy13120201106683 0ck uzunova progression of the covid19 and decreased duration of the symptoms [“] there are in fact overall more than trials currently underway around the world on its impact either as a prophylactic or treatment for covid19 it is noteworthy that the usefulness of hydroxychloroquine and chloroquine is intensively investigated chloroquine was found to exert an antiviral effect during pre and postinfection conditions suggesting to have both prophylactic and therapeutic advantages timeofaddition assay demonstrated that chq functioned at both entry and postentry stages of the sarscov2 infection in vero e6 cells however it did not reduce viral replication in sarscov infected mice hydroxychloroquine is significantly more potent than chq in vitro ec50 values and μm respectively and has a lower potential for drugdrug interactions than chloroquine pharmacokinetic models demonstrate that hydroxychloroquine sulfate is significantly superior days in advance to chloroquine phosphate in inhibiting sarscov2 in vitro and was demonstrated to be much less toxic than chq in animals on the other hand data presented by liu demonstrated that the antiviral effect of hcq against sarscov2 infection was comparable with chq in vitro cc50 μm and μm for chq and hcq respectively moreover they suggested that both chq and hcq blocked the transport of sarscov2 from early endosomes ees to endolysosomes els and caused noticeable sizemorphological changes in ees and els they surmised that endosome maturation might be blocked at intermediate stages of endocytosis resulting in failure of further transport of virions to the ultimate releasing site hydroxychloroquine shares the same mechanism of action as chloroquine apart from the probable role of chq and hcq as antiviral agents their mechanisms of action are not fully understood and it was demonstrated that they have multiple effects on mammalian cells ace2 is known to be a cell receptor for sarscov the high similarities of the amino acid sequences and predicted protein structures of the receptorbinding domain of sarscov2 and sarscov suggest that sarscov2 may efficiently use human ace2 as a receptor for cellular entry and employ the cellular serine protease tmprss2 for s protein priming zhou confirmed that sarscov2 used the ace2 receptor to enter cells and did not use other coronavirus receptors such as aminopeptidase n apn and dipeptidyl peptidase dpp4 so the primary mechanism by which cell infection is prevented by these drugs may be at the stage of binding with the surface receptor and endosomemediated viral entry two independent in vitro studies confirmed that chq inhibits the replication of the sarscov chloroquine inhibits the early stage of sarscov replication in vero e6 cells with a effective concentration of ± μml the antiviral activity of chq was indicative at the time point at virus attachment or penetration vincent established that the drug might interfere with terminal glycosylation of the cellular receptor ace2 when chq was added prior to infection the impairment of terminal glycosylation of ace2 may result in reduced binding affinities between ace2 and sarscov spike protein and negatively influence the initiation of sarscov infection when chq or nh4cl were added after infection these agents could rapidly raise the ph and interrupt ongoing fusion events between the virus and endosomes thus inhibiting the infection on the basis of in vitro experiments they suggested that the primary mechanism by which infection was prevented was the poor affinity of sarscov spike protein to underglycosylated ace2 in vitro studies with hiv infected cells also identified that inhibition of glycosylation might be a possible mechanism of action of chq chq inhibits hiv replication at a postintegration stage resulting in the production of immature virions it was demonstrated that the sole mechanism explaining the antihiv activity of chq was a decrease in the infectivity of the newly produced virus associated with defective production of the heavily glycosylated 2g12 epitope of gp120 according to in vitro results the antiretroviral effects of chq are attributable to the inhibition of viral p glycosylation these effects appeared to be specific since the chq concentrations effective in vitro neither affected any other step in hiv1 replication nor were cytotoxic thus there is direct evidence that chq is an inhibitor of glycosylation of gp120 and these alterations may be responsible for the decreased infectivity of hiv grown in the presence of chloroquine when added after the initiation of infection these drugs might affect the endosomemediated fusion and subsequent virus replication sarscov pseudoviruses may enter cells via receptordependent clathrin and caveolaeindependent phsensitive endocytosis likely through a process involving lipid rafts a later study however suggests that the entry of coronaviruses into the host cells occurs through clathrinmediated endocytosis murine hepatitis virus mhv a prototypic member of the cov family requires trafficking to lysosomes for proteolytic cleavage at the fp proximal position of its spike s protein membrane fusion to occur many authors indicated that s protein cleavage is an important step for fusion activity and subsequent internalization of the sarscov virus genome into cells [“] adding chq prior to infection results to inhibition of endosome maturation and strongly decreased mhv infection and fusion which was not observed when the drug was added at hpi indicating that the compound mainly affects mhv entry chloroquine is a weak base that is known to increase the ph of lysosomal and transgolgi network tgn vesicles leading to the dysfunction of enzymes necessary for proteolytic processing and posttranslational modifications of newly synthesized viral proteins chloroquine is able to prevent the processing of prm protein to m protein in flavivirusinfected mammalian and mosquito cells by raising the ph of the postgolgi vesicles in which this cleavage occurs as a result virions from infected cells which had been treated with acidotropic amines late in the virus replication cycle contained prm protein rather than m protein and this reduced the infectivity of the virus the chloroquinemediated rise in endosomal ph modulates iron metabolism in a variety of cell types decreasing in intracellular concentration of iron affects the function of several cellular enzymes involved in pathways leading to the replication of cellular dna and to the expression of different genes [“] autophagy is a lysosomedependent degradative pathway chq and its analogue hcq are known clinically relevant autophagy inhibitors chq is a weak base that inhibits lysosomal acidification which prevents the fusion of autophagosomes with lysosomes and subsequent autophagic degradation inhibition of autophagy with chq stimulates superoxide generation ubiquitinconjugated protein accumulation and apoptosis in a colon cancer xenograft model chq treatment clearly inhibited autophagy in mouse lung and efficiently ameliorated acute lung injury and dramatically improved the survival rate in mice infected with live avian influenza a h5n1 virus h5n1 virusinduced autophagic cell death in alveolar epithelial cells through a pathway involving the kinase akt the tumor suppressor protein tsc2 and the mammalian target of rapamycin and autophagyblocking agents might be useful as prophylactics and therapeutics against infection of humans by the h5n1 virus furthermore prentice suggested that authophagy was induced by the coronavirus mouse hepatitis virus mhv and was required for formation of double membranebound mhv replication complexes which significantly enhanced the efficiency of replication replication of the virus was impaired in atg5 knockout embryonic stem cells the same authors also examined the sarscovs and found out similar colocalization of the key viral replication proteins with endogenous lc3 a protein marker for autophagosome it could be assumed that autophagy inhibitors like chq could inhibit virus replication at present the exact role of autophagy in cov infection remains debatable and there is much evidence suggesting that the endocytic pathway plays a key role in mediating viral entry for many covs including sarscovs merscovs and possibly sarscov2 the antiinflammatory properties of chqhcq should also be considered several studies have suggested that multiple an failure biomedicinepharmacotherapy13120201106684 0chas not yet been identified in sarscov2 infected patients and probably multiple pathways could be involved fig conclusion the sarscov2 is the cause of the coronavirus disease covid19 that has been declared a global pandemic by the world health anization who in despite some clinical characteristics that differentiate covid19 from sarscov merscov and seasonal influenza the pathogen sarscov2 has the same phylogenetic similarity to sarscov and mers cov most of the encoded proteins exhibited high sequence identity between sarscov2 and the related batderived coronaviruses batslcovzc45 and batslcovzxc21 a notable difference was a longer spike protein encoded by sarscov2 compared with the bat sarslike coronaviruses sarscov and merscov in addition sarscov2 was distinct from sarscov in a phylogeny of the complete rnadependent rna polymerase rdrp gene moreover the receptorbinding domain of sarscov2 which directly engages the ace2 receptor for cell entry was more closely related to those of sarscovs “ amino acid identity since the outbreak researchers have released many agents that could have potential efficacy against covid19 there is currently no clinically proven specific antiviral agent available for sarscov2 infection like sarscov and merscov certain agents like chloroquine hydroxychloroquine lopinavirritonavir and remdesivir are being used in ongoing clinical trials all over the world with hopes to further delineate their role in the treatment and prophylaxis of covid19 furthermore due to their availability and using for decades and proven safety records it is reasonable to suggest that they may be appropriate for treatment of covid19 remdesivir an adenosine analogue with wellstudied mechanism of action in cov infections can target the rnadependent rna polymerase and block viral rna synthesis and has been a promising antiviral drug antiviral studies in cell culture and animal models the available human safety data as well as the clear mechanism of action characterize rdv as a directacting antiviral since some authors found that lopinavir“ritonavir treatment did not significantly accelerate clinical improvement hence antiviral effects as an inhibitor of the sarscov main 3cl protease should be further investigated although chq and hcq are wellknown drugs for the treatment of k uzunova observed in fatal cases are most likely associated with not only the direct viral infection and destruction of susceptible cells eg endothelial cells but also the effects of proinflammatory cytokines chemokines and other mediators released from infected and activated cells such as monocytes and macrophages the clinical worsening of individuals with sars in week is apparently unrelated to uncontrolled sars coronavirus replication but may be related to immunopathological damage another study reveals that the presence of viral elements within endothelial cells and the accumulation of inflammatory cells led to endotheliitis in several ans as a direct consequence of viral involvement and to host inflammatory response moreover chq has immunomodulatory effects suppressing the productionrelease of tumour necrosis factorα and interleukin6 which mediate the inflammatory complications of several viral diseases chloroquinehcq was reported to inhibit the production of soluble mature tnf in macrophage cell line inhibit tnfα receptor in human histocytic u937 cells inhibit tnfα ifnγ and il6 in peripheral blood mononuclear cells pbmc reduce tnfα production and lipopolysaccharide lpsinduced il1 release in human monocytic cells it is suggested that chq exerts antiinflammatory and immunomodulatory effects predominantly by pretranslational and nonlysosomotropic mechanisms chloroquineinduced inhibition of tnf and il6 production is not mediated through a lysosomotropic mechanism and chloroquine probably acts on tnf secretion by disrupting iron homeostasis besides its antiviral activity and due to its suppressive effects on the productionrelease of tnfα and interleukin chqhcq may be effectively used in the treatment of viral infections characterized by symptoms associated with inflammatory processes andor immunehyperactivation antiinflammatory effects of chq remain poorly understood regulation of proinflammatory cytokines chq can also act on the immune system through cell signaling chq inhibits the activation of p38 mapk in hcov229einfected cells and evokes the activation of erk independently of infection these results suggested that chq may inhibit cov replication by suppressing the p38 activation additionally chq strongly inhibited phosphorylation of mitogenactivated protein kinase mapk p38 and to a lesser extent cjun nterminal kinase and extracellular signalregulated kinase ½ chloroquine could also inhibit innate immune responses trough downregulation of tlr9 signaling pathways requiring endocytosis and acidification of endosomes within plasmacytoid dendritic cells pdcs and act as novel antagonists to chemokine receptor cxcr4 that suppress pancreatic cancer cell proliferation on the other hand another hypothesized mechanism of chq is via the inhibition of antigen degradation and improving the crosspresentation efficiency of dcs in vitro in vivo evidence suggested that a short course of treatment with chq followed by a booster dose of a soluble antigen immunization can efficiently enhance human cd8 t cell responses and single vaccination with inactivated influenza virus combined with chloroquine treatment elicits a higher t cell immunity in mice regulation of nlrp3 inflammasome activation may offer a promising therapeutic approach by inhibiting or slowing down the process of acute respiratory distress syndrome hcq is a known nlrp3 inhibitor and its potential clinical effectiveness is certainly based on the downregulation of il1 expression the major proinflammatory cytokine interleukin1beta il1 is elevated in plasma from hospitalized covid19 patients and its associated signaling pathway seems to drive sarscov2 pathogenicity il1 secretion is primarily initiated by inflamm
Colon_Cancer
surfactin from bacillus subtilis displays antiproliferative effect via apoptosis induction cell cycle arrest and survival cell membrane protein cell “ meeting of pharmaceutical society of japan p signaling suppression febs lett “ domonad arch microbiol “ dorn e hellwig m reineke w knackmuss h j isolation and characterization of a 3chlorobenzoate degrading pseu chang yj phylogenetic analysis of aerobic freshwater and marine enrichment cultures efficient in hydrocarbon degradation effect of profiling method j microbiol methods “ thomas a t in a0vitro anticancer activity of microbial isolates from diverse habitats braz j pharm sci “ miao l the antiinflammatory potential of portulaca oleracea l purslane extract by partial suppression on nfκb and mapk activation food chem “ elseedi h r the traditional medical uses and cytotoxic activities of sixtyone egyptian plants discovery of an active cardiac glycoside from urginea maritima j ethnopharmacol “ thakur a n antiangiogenic antimicrobial and cytotoxic potential of spongeassociated bacteria mar biotechnol “ liu k liu p c li r wu x dual aoeb staining to detect apoptosis in osteosarcoma cells compared with flow cytometry dassonneville l cytotoxicity and cell cycle effects of the plant alkaloids cryptolepine and neocryptolepine relation to drug dengler r immunocytochemical and flow cytometric detection of proteinase myeloblastin in normal and leukaemic med sci monit basic res “ induced apoptosis eur j pharmacol “ myeloid cells br j haematol “ hsu sm raine l fanger h x use of avidinbiotinperoxidase complex abc in immunoperoxidase techniques a comparison between abc and unlabeled antibody pap procedures j histochem cytochem “ elgarawani i m ameliorative effect of cymbopogon citratus extract on cisplatininduced genotoxicity in human leukocytes j singh n p mccoy m t tice r r schneider e l a simple technique for quantitation of low levels of dna damage in biosci appl res “ individual cells exp cell res “ acknowledgementswe are very grateful to the swedish research links grant vr stockholm sweden grant number for generous financial support dr sam khalifa thanks the department of molecular biosciences wennergrens institute stockholm university swedenauthor contributionshs ss and ielg planned and designed the experiments ielg and na performed analyzed and wrote the biological and molecular experiments hs and dm performed analyzed and wrote the phytochemical experiments ha performed the microbiological experiments ha and ielg wrote the manuscript ielg sk and oe revised the manuscript all authors read and approved the final manuscriptcompeting interests the authors declare no competing interestsadditional informationsupplementary information is available for this paper at 101038s4159 correspondence and requests for materials should be addressed to imeg a0or a0hresreprints and permissions information is available at wwwnaturecomreprintspublisher™s note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsopen access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate if changes were made the images or other third party material in this are included in the ™s creative commons license unless indicated otherwise in a credit line to the material if material is not included in the ™s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this license visit httpcreat iveco mmons licen sesby40 the authors scientific reports 101038s41598020709458vol0123456789wwwnaturecomscientificreports 0c'
Colon_Cancer
tenascinc tnc is an extracellular matrix ecm glycoprotein that plays an important rolein cell proliferation migration and tumour invasion in various cancers tnc is one of themain protein overexpressed in breast cancer indicating a role for this ecm molecule in cancer pathology in this study we have evaluated the tnc lossofffunction in breast cancercells in our approach we used dsrna sharing sequence homology with tnc mrna calledatnrna we present the data showing the effects of atnrna in mdamb231 cells bothin monolayer and threedimensional culture cells treated with atnrna were analyzed forphenotypic alterations in proliferation migration adhesion cell cycle multicaspase activation and the involvement in epithelial to mesenchymal transition emt processes as complementary analysis the oncogenomic portals were used to assess the clinical implication oftnc expression on breast cancer patient™s survival showing the tnc overexpression associated with a poor survival outcome our approach applied first in brain tumors and then inbreast cancer cell lines reveals that atnrna significantly diminishes the cell proliferationmigration and additionally reverses the mesenchymal cells phenotype to the epithelial onethus tnc could be considered as the universal target in different types of tumors wheretnc overexpression is associated with poor prognosisa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation wawrzyniak d grabowska m głodowiczp kuczyński k kuczyńska b fedorukwyszomirska a downregulation oftenascinc inhibits breast cancer cells developmentby cell growth migration and adhesionimpairment one e0237889 101371 pone0237889editor lucia r languino thomas jeffersonuniversity united statesreceived may accepted august published august copyright wawrzyniak this is anopen access distributed under the terms ofthe creative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportingintroductioninformation filesfunding this work was supported by the ministryof science and higher education of the republic ofpoland by know program kk was supported byncbr program powr03020000i03216competing interests the authors have declaredthat no competing interests existthe tumor microenvironment is composed of the surrounding stromal cells such as endothelialcells in blood vessels immune cells fibroblasts and the extracellular matrix ecm [ ] during carcinogenesis is often perturbed and deregulated while during embryonic development isstrictly controlled to maintain homeostasis in tumors the composition of the ecm differsfrom that of normal tissue and enables new interactions that affect the function of cancer cellsand are critical in modulating invasion associated with cell migration and growth the tumorassociated ecm presents several tumorassociated antigens that are generally more abundant one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentand possibly more stable than those of the cell surface [“] consequently these proteins represent possible valuable targets for tumor imaging and therapy [ ] ecm proteins such as fibronectin fn and tenascin have isoforms that are expressed in a tissue specific manner generatedby alternative splicing of their primary transcripts one of the most consistent isoform changesin the ecm of many tumors is the upregulation of the glycoprotein tenascinc tnc tncalongside tenascinx tnx tenascinr tnr and tenascinw tnn are members of wellconserved among vertebrates tenascin family tn [“] numerous isoforms of tnc can beproduced through alternative splicing of nine fibronectin type iii regions between repeats and at the premrna level there is a considerable amount of literature on the contribution of different splicingdependent tnc domains in specific biological functions changes in thetnc isoforms expression pattern have been then described in a number of malignancies andtheir nature appears to be tumortype specific recent studies have demonstrated that somesplice variants are specific to diseased tissues [“] in breast tissues expression of two tncvariants one containing domain d and the other both b and d was found to be associated withinvasive phenotype tnc promotes cell migration angiogenesis inhibit focal contact formation and also act as a cell survival factor [“] its importance was found in the development and progression of different types of neoplasm including colon and breast cancerfibrosarcoma lung cancer melanoma squamous cell carcinoma bladder cancer and prostaticadenocarcinoma [ ] tnc is also highly expressed in highgrade gliomas which correlatesas well with the invasiveness of glioma cells [“] in the brain it is important for the development of neural stem cells [ ] and moreover is suspected to be a potential marker for glioblastoma multiforme gbm stem cells gsc previously we have shown that tnc is overexpressed in gbm and can be a good target inrnai approach with 164nt long dsrna complementary to the mrna of tnc which wecalled atnrna we conducted the experimental therapy for gbm patients the discoverythat tnc presents a dominant epitope in glioblastoma prompted us to investigate the potentialof atnrna to block the tnc expression and its effect on the growth of human breast cancers where tnc overexpression was also established and linked with the highest malignancyinvasion capability and metastasis ability this view is supported by mock who showedthat gbm patients with antibodies against the egflike repeats of tnc antibody targetvcedgftgpdcae have a significantly better prognosis than other patients thus weassumed that in the light of the satisfactory results of brain tumors experimental therapy breastcancer could be the next possible object of interest to establish the atnrna approachhere we demonstrate that atnrna approach can be successfully used in breast cancercells impairing the basic hallmarks of tumor cells with the performed analysis of proliferation migration rate multicaspases induction pathway cell cycle analysis spheroids viabilityand the involvement of tnc in emt induction we have then interrogated the impact of tncon breast cancer growth showing its potency to be also the promising therapeutic target inbreast cancer treatmentresultsoncogenomic in silico analysis reveals the tnc correlation with poorsurvival of breastcancer patientsto look deeper into the tnc function we performed the analysis of genomewide breast cancerdata with available oncogenomic portals such as gepia the human protein atlas cbioportaland ppisurv based on the status of three important receptors conventionally used for breastcancer subtyping ie estrogen receptor er progesterone receptor pr and human epithelialreceptor her2 breast cancer is classified as luminal a luminal b her2 positive and triple one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentnegative œbasallike triplenegative and her2overexpressing breast cancer yields a poorpatient prognosis because of a high incidence of metastases disease progression and resistanceto current chemotherapy regimens we first compared the expression level of tenascincin subtypes of breast cancer using gepia program fig a in s1 file mrna level of tncwere higher in triplenegative and her2 subtypes compared to the luminal a and luminal bsubtypes which have a better prognosis for patient survival therefore we chose mdamb231cells as a model for in vitro experiments because it is the most invasive cell line from breast cancer models mdamb231 cell genome clusters with the basal subtype of breast cancer sincethe cells also lack the growth factor receptor her2 they represent a good model of triplenegative breast cancer what is important adams showed that only invasive cell linessuch as mdamb231 or mdamb468 express tenascinc whereas the tumor cell lines witha low invasive capacity mcf7 and t47d do notas a next step we compared the expression levels of tenascin genes tnc tnn tnrtnxb in invasive breast cancer using gepia program fig 1a mrna level of tnc washighly expressed in breast cancer tissue brca interestingly expression levels of tnn andtnxb were significantly lower in breast cancer tissues fig 1a there was no significant difference in tnr gene expression between breast cancer and nontumor tissueswe also examined the expression of tenascin proteins in normal and malignant tissues byquerying data from the human protein atlas tnc in most cases and partly tnn wereexpressed at medium levels whereas tnr was not detected fig 1b and 1c tnc and tnrlevels were undetectable in samples from normal breast adipocytes glandular and myoepithelial cells taken together our results demonstrate that mrna and protein levels of tnc is relatively higher in invasive breast cancer tissues than those in normal tissuesthe cbioportal analysis enabled to look for the mutations in the tnc gene it appeared thattnc gene mutations measured for breast cancer patients are present as somatic mutation only in cases since these mutations seem to be irrelevant for breast cancer wedid not perform any further analysiswith ppisurv portal we looked through the transcriptomic data to correlate the tncexpression with the different clinical parameters such as survival or prognosis of the canceras the initial step of analysis we performed the alignment of tnc and other proteins from thetenascin family such as tenascinx tnx looking for the homology between these two proteinsat the top of that we made the analysis of the homology between the tnc and tnx with the relation to atnrna sequence the alignment of these two ecm proteins shows that they shareonly limited number of nucleotides query cover and for short and long transcriptionalvariants in the protein nterminus region respectively the sequence alignment analysis clearlyshow the atnrna matching exclusively to the tnc sequence identity fig 2appisurv analysis based on the kaplanmeier statistics showed very clearly the strong correlation with tnc expression and patients survival the high expression level has a greatimpact on the shorter survival for the patients thus suggesting also that tnc can be also considered as the prognostic factor for breast cancers p fig 2b at the same time weanalyzed also the available data for the tnx the results showed an inverse correlation oftnxb mrna expression and survival p fig 2c analysis was carried out on thegroup of patient n for tnc and n for tnxbatnrna mediates the downregulation of tnc mrna and proteinexpression in breast cancer cellsto achieve downregulation of tnc expression in breast cancer cell line transfection withvarious concentration of atnrna and nm was performed h after one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig tenascin is highly expressed in breast invasive carcinoma tcgabrca a messenger rna levels of tnc tenascinctnn tenascinw tnr tenascinr tnxb tenascinx genes in specimens from patients with invasive carcinoma of the breast one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentvs nontumor samples rna sequencing data were retrieved from the database of tcga and analysed using the gepia geneexpression profiling interactive analysis online web server httpgepiacancerpkucn the red boxes represent cancer specimensgrey boxes represent healthy breast specimens significance value � p b summary of tenascin expression patterns in breastcancer tissues and healthy breast determined by immunohistochemical staining data were retrieved from the human protein atlasdatabase case numbers of invasive breast cancer are shown na not available nd not detectable in œhealthy breast column œmeans that tnc and tnr are not detected in nontumor samples results in normal breast are based on immunohistochemical stainingof a single sample c representative images of immunohistochemical staining for tnc tnn and tnr in breast healthy tissue andinvasive breast carcinoma specimens the images shown here are of the tissue sections from tissue microarray arrays tmas stainedwith appropriate antibodies tnc“cab004592 tnn“cab010907 tnr“cab022343 all the images were taken at × magnification101371 pone0237889g001transfection the expression level of tnc was examined by qrtpcr analyses significantdownregulation of tnc mrna expression was observed compared to control treated withscrambled rna the level of tnc was decreased from at a concentration of nmatnrna up to for cells treated with nm atnrna in comparison to the controlp fig 3afig the oncogenomic analysis of the survival association with tenascinc and tenascinx in breast cancer asequence alignment of tenascinc versus tenascinx with their relation to atnrna the sequence alignment analysisclearly show the atnrna matching exclusively to the tnc sequence identity relation of tnc b and tnxbc gene expression to survival of breast cancer patients survival analysis performed with the use of a dataset breastcancer geo gse7390 and gse3494 deposited in and tools available from the ppisurv web portal tnxbl longtranscription variant tnxbs short transcription variant101371 pone0237889g002 one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig expression level of tnc and immune response genes after atnrna treatment in mdamb231 cell line a relative expression level of theexpression of tnc oas1 oas3 rig1 ifi16 and tlr3 established by qrtpcr relative expression was calculated using the“δδcp method statisticalevaluation of atnrna versus scrambled sirnas ccontrol cells was performed using oneway anova followed by tukey™s posthoc test effect of poly ic μgml on immune response genes oas1 oas3 rig1 ifi16 tlr3 in the figure presented as purple bars the results for hprtnormalized expressionof mrna are expressed as fold change of target gene expression relative to the control without poly ic treatment which is defined as b the proteinexpression levels of tnc and hprt c western blot analysis reveals efficient tnc silencing in mdamb231 cells with atnrna compared to cells treatedwith sirnas ccontrol the data represents the means ± sd from independent experiments significance value � p �� p ��� p 101371 pone0237889g003the qrtpcr analysis was also supported by direct analysis of the protein expression levelwe have observed already a decrease in tnc protein expression upon 50nm atnrnatreatment the highest concentration 100nm used led to the dramatic drop of the protein one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentexpression measured as the of the decrease fig 3b and 3c these observations were fullyconsistent with relative tnc expression level measured by qrtpcrinterferon response to atnrnato establish interferon induction in breast cancer mdamb231 cultured cells we looked forinterferon stimulated genes isg including oas1 oas3 rig1 tlr3 and ifi16 genes theanalysis was carried out with the qrtpcr fig 3a changes after atnrna measured byqrtpcr were not significant as shown basically for all of the genes in the concentrationrange of “ nm in parallel a synthetic form of dsrna polyipolyc poly ic wastransfected as a positive control poly ic has been used extensively as a tlr3 ligand to induceantiviral response [“] we showed that transfection with poly ic μgml efficientlyinduced the expression of immune response genes oas1 oas3 rig1 ifi16 tlr3 inmdamb231 cells this enhancement in mrna expression was “7fold higher in poly ictreated cells than in untreated cells fig 3a purple barstnc knockdown inhibits cells proliferation and leads to the changes inmigration rate and adhesion potential of breast cancer cellsin order to investigate the involvement of tnc on breast cancer cells proliferation mdamb cell line was treated with atnrna and the realtime cell proliferation assay was performed the cells ability to proliferate was measured for h we have noticed time and concentrationdependent decrease in proliferation rate the most effective concentration ofatnrna was nm with decrease from “ after and h respectively fig 4anoteworthy nm and nm of atnrna was already sufficient concentration for the efficient inhibition of breast cancer cells proliferation the dosedependent effect of atnrna inmdamb231 proliferation potential resulted in standard sigmoidal doseresponses with ic50of ± nm after h ± nm after h and ± nm after h of treatmentfig 4bto get more insight into the downregulation of tnc expression by atnrna on themobility of breast cancer cells realtime measurements of migration was carried out wefound that downregulation of tnc expression by atnrna significantly impaired the cellmigration in breast cancer cell lines fig 4c the results were quantitatively assessed during h of experiment and showed that mdamb231 cells transfected with atnrna had thelowest motility beginning from h post transfection it was established that atnrnadelayed the migration of mdamb231 cells by ± h ± h ± h and ± h with and 100nm concentration respectively notably the most effective concentration which affected the migration potential of the cells was 10nm when compared to the control the observed delay was ± hsince tnc is implicated also in cellmatrix attachment we further looked at the adhesionability of atnrna treated cells the cells were conducted to realtime adhesion assay withxcelligence system compared with the controls tnc knockdown resulted in increased cellsadhesion on average fig 4dtnc promotes apoptosis and is involved in cell cycle regulation in breastcancer cellsto determine additionally the effect of atnrna on the cell cycle progression themdamb231 cells were treated with different concentrations of atnrna for “ h andthe cell cycle analysis was assessed by muse1 cell analyzer cells transfected with atnrna one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig activity of atnrna in proliferation migration and adhesion proliferation of breast cancer in culture was monitored in realtime using xcelligencesystem a impedance was recorded every min but to improve the clarity of the graphs only every fourth readout was plotted data show the mean ± sd ofthree independent measurements b dosedependent effects of atnrna on proliferation was evaluated using nonlinear regression by fitting experimentalvalues to sigmoidal bellshaped equation c migration of mdamb231 cancer cells was studied using xcelligence system serumdepleted cells weretransfected with increasing concentrations of atnrna from to nm or scrambled sirnas ccontrol impedance ci values of each experimentalcondition was recorded over time plotted against time fitted to fourparameter logistic nonlinear regression model and et50 was calculated for each atnrnaconcentration to generate doseresponse curves et50 value was normalized to the data obtained for untreated cells and plotted as normalized half maximaleffective time et50 of cell migration against atnrna concentrations d adhesion of mdamb231 cell line was observed in realtime using xcelligencesystem graph shows the final impendence values minus the initial values for the respective samples differences between ci values for atnrna treated andcontrol cells were statistically evaluated using oneway anova followed by tukey™s posthoc test symbols above the bars significance value ��� p compared to cells treated with scrambled sirnas ccontrol101371 pone0237889g004showed the cell cycle distribution with the concentrationdependent decrease in cell numberin g0g1 phase increased in s phase and unchanged in g2m phase compared to the cellstransfected with unspecific control rna fig 5a atnrna impacted the cell cycle by almostdoubling the cells sphase fraction from to for the highest atnrna concentrationthus resulted with the cells arrest in s phase the cell cycle analysis proved the nontoxic effectof atnrna since we did not observe the increase in g1 population s phase arrest persistsfollowing up to h of atnrna treatment fig 5bthus to examine whether atnrnainduced apoptosis would be associated with the caspases activation the expression levels and activity of caspases such as caspase1 and in the atnrnatreated mdamb231 cells were assessed using muse1 cell one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig effect of atnrna on breast cancer cell cycle phase analysis mdamb231 cells were transiently transfected with increasing concentrations ofatnrna from to nm or scrambled sirna ccontrol for a and h b the cells were then fixed and added with propidium iodide pirnase a staining solution and analyzed in muse1 cell analyzer using modfit lttm software a percentage of cell distribution in each cell cycle phase wassummarized and shown the cell cycle distribution profile image is shown as a representative result of three independent experiments101371 pone0237889g005analyzer as shown in fig 6a breast cancer cells treated with atnrna exhibited enhancedmulticaspase activity in a concentrationdependent manner the multicaspase activity was ± ± ± and ± respectively at and nm atnrna concentration compared with the control fig 6b thus we observed almost5fold increase of the population of the apoptotic cells with the lowest atnrna concentration whereas almost 12fold with the highest oneatnrna has an impact on spheroids integrityto visualize the involvement of tnc in tumor formation the 3d culture model was appliedsince 3d cell culture models mimic better the in vivo behavior of cells in tumour tissues andare excellent surrogates to predict tumorigenic potential in vivo the ability to spheroid maintenance of breast cancer cells was assessed after atnrna treatment we have observed that one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig effect of atnrna on multiple caspase activation caspase1 and in mdamb231 cell linebreast cancer cells were transiently transfected with increasing concentrations of atnrna from to nm orscrambled sirnas ccontrol for h the transfected cells were then incubated with muse1 multicaspase reagent followedby analysis of the percentage of cell population in live caspase caspasedead and dead in muse1 cell analyzer a thepercentage of live caspase caspasedead and dead cells profile image is shown as a representative result from one of threeindependent experiments b the graphical representation of percentage of live and exhibiting caspase activity cellpopulation transfected with atnrna and scrambled sirnas statistical evaluation of atnrna versus scrambled cells one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmenttreated with scrambled sirnas was performed using oneway anova followed by tukey™s posthoc test significance value��� p compared to scrambled control ccontrol error bars represent sd101371 pone0237889g006downregulation of tnc led to the disintegration of the spontaneously forming spheroids ofmdamb231 the clearly visible effect on the spheroid viability was observed even with thelowest atnrna concentration the increased concentrations of dsrna had a great impacton spheroids integrity resulting in structure disintegration at the highest concentration of nm fig 7a the atnrna application influenced the spheroid volume and shape displaying the total shrinking of the compact structure into the small fragments with the highestatnrna concentration fig 7ato have a better insight into the mdamb231 spheroids structure and the atnrnaimpact on their viability the confocal microscopy imaging was assessed the analysis of fluorescent labelling with greenfluorescent calceinam of living and dead cells within the spheroid with the livedead viabilitycytotoxicity kit was carried out as revealed by the image ofthe untreated mdamb231 cells compact multicellular spheroids were obtained fluorescence images revealed the overall morphology of the mdamb231 spheroids fig 7b thecell density in the core of the untreated spheroid was high and no dead cells were identifiedthe similar pictures were obtained for the control furthermore all conditions with differentatnrna concentrations resulted with losing the spheroid density increased dimensionsand appearing a higher population of dead cells it is worthy of note that the spheroids treatedwith increasing atnrna concentrations did not display a smooth contour following h oftreatment and subsequently their round shape was markedly altered by the treatments afterthe treatment with 50nm concentrations the spheroids showed the strong overrepresentationof dead cells fig 7b thus the 100nm concentration of atnrna was most likely too highfor the cells viability and we were not able to keep the spheroids in shape that would allow forthe imagingtnc is involved in emt processesas the consequence of these finding we analyzed the expression level of proteins involved inemt processes we took into account two main emt markers ecadherin and vimentinwestern blot analysis shows the significant increase of ecadherin level followed by the dropof the expression of vimentin protein fig 8a these observations were concentrationdependent showing the highest efficacy for atnrna at the concentration of 100nm for ecadherin expression similarly for vimentin we have observed the highest decrease of expressionupon atnrna transfection at 100nm concentration fig 8bdiscussiontnc is the main ecm protein of various tumors and its overexpression is repeatedlyobserved in breast cancer cells both in vitro and in vivo indicating a role for this extracellularmatrix glycoprotein in neoplastic pathology moreover its high expression correlates withworsened patient survival prognosis in several cancer types in breast cancers severalstudies demonstrate that high expression of tnc is not only an indicator of poor prognosisbut also correlates with metastasis to distinct ans such as lymph nodes liver and lung [“] tnc plays a substantial role in emt that is believed to be a key mechanism in cancer progression whereby cancer cells acquire more aggressive behavior [ ] in human breast cancer specimens tnc is coexpressed with the mesenchymal marker vimentin themechanistic role of tnc in the process of emt remains poorly defined however studies one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentfig effects of atnrna on viability and spheroid structure in mdamb231 cells a monolayer cultures weretransfected with indicated amounts of atnrna oligonucleotides and after hrs cellular spheroids of mdamb cells were generated from cells in perfecta3d1 96well hanging drop plate and cultured for up to hoursscale bars μm scrambled sirnas cscr b the viability of the atnrna transfected cells within spheroidsusing livedead cell imaging kit left and middle panels present live cells green and dead cells red respectively one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmentthe right panels show merge of two fluorescent images scrambled sirnas cscr concentrations ofatnrna used for transfection fluorescence images were taken using leica tcs sp5 confocal laser scanningmicroscope and plan apo × na oilimmersion objective scale bars μm101371 pone0237889g007suggest that tnc can induce an emt like phenotype in mcf7 breast cancer cells via theαvβ6 and αvβ1 integrins [ ] many studies on various cancer tissues have demonstrateddownregulation of epithelial markers including ecadherin plakoglobin and cytokeratin aswell as the upregulation of mesenchymal markers such as ncadherin and vimentin andexpression of emt transcription factors snai and twist since these changes towardsmesenchymal phenotype could correlate with invasiveness metastatic potential and poorpatient™s outcome we have investigated the effect of tnc knockdown on the expression levelsof emt markers our results show that down regulation of tnc reverses the malignant phenotype of the cancer cells as the experimental result we observed the downregulation of mesenchymal marker”vimentin followed by the upregulation of epithelial marker”ecadherinthis indicates atnrna as a potential therapeutic agent which could switch the mesenchymal phenotype of breast cancer cells to the epithelial one inhibiting the ability to metastasisand invasion additionally it has been also shown that tnc as the ecm component plays alsofig the effect of tnc downregulation on emt process of mdamb231 cells a the protein expression levelsof ecadherin vimentin and gapdh b western blot analysis of atnrna effects on the emt process revealed asignificant increase in ecadherin level followed by the drop of the expression of vimentin protein the data representsthe means ± sd from independent experiments101371 pone0237889g008 one 101371 pone0237889 august one 0cdownregulation of tenascinc inhibits breast cancer cells developmenta role in cell to cell or cellmatrix attachment most probably inhibiting the cells™ migration inapproach with atnrna it seems that tnc in breast cancer cell line plays an antiadhesiverole which would affect the cell migration and invasion ability in addition to emt processesthus tnc downregulation seems to enhances the adhesiveness of cancer cells showing thedirect involvement of tnc in cell adhesive properties targeting the tnc in potential therapymight be also highly beneficial since it has been already established that tnc maintain a stemcell niche in the brain tumors thus could promote the tumor cell invasion therefore its overexpression largely contributes to radiochemotherapy resistance and tumor recurrence infact it has been shown that targeting gbm invasion increases tumor sensitivity to temozolomide tnc also promotes stromal events such as the angiogenic switch and the formationof more but leaky blood vessels involving wnt signaling and inhibition of dickkopf1 dkk1in a neuroendocrine tumor model we observed significant atnrna“mediated downregulation of tnc was in concordance with the observed changes in proliferation and migration rates the results show thatthe atnrna transfected cells lose their ability to migrate thus showing the involvement oftnc in breast cancer invasiveness our data indicate also that the downregulation of tncexpression in mdamb231 cancer cell lines inhibits proliferation along with induction of celldeath we were able to determine the tnc impact on the apoptosis by measuring level of bothcaspases initiating intracellular events caspase2 and effector caspases3 and orend demonstrated that tnc causes cdk2 inactivation and blocks cell cycle progression from g1 phase to s phase of anchoragedependent fibroblasts by interfering withfibronectin“syndecan4 interactions for the proliferation of anchoragedependent cellsattachment to the ecm is required detachment of fibroblasts by a pure tenascinc substratum results in g1phase arrest by inactivation of the cdk2 complex in a ckidependent manner in contrast to fibroblasts proliferation of most tumor cells is stimulated by tnc this shows that the effect of tnc on proliferation is cell typespecific and suggests that in cancer cells the cdk2 complex is not repressed in the presence of tnc in leukemia breast carcinoma and glioma were found subpopulations of stemlike cells that support tumor growth in such cells adhesion on the tnc substratum may override the g0g1 and gls cellcycle checkpoints which may explain the increased proliferation rate the g1s transitionis enforced by cyclin ecdk2 activation via syndecan4 related signalling it has been shownthat tnc in tumor cells binds to the syndecan4 binding site in fibronectin thereby blockingsyndecan4 ligation releasing the tumor cells from the suppressive effect of fibronectin ontheir proliferation this would a
Colon_Cancer
"annual meeting of the european associationfor the study of diabetes“ september sindex of oral presentationsop diabetes complications new insights from cutting edge epidemiologyop news on the insulin secretion frontop insulin sensitivity and biomarkersop central actions in diabetesop glucoselowering therapies and the liverop uncomplicating the pathogenesis of diabetes complications inhumansop smoke on the water is bat still hotop charting human beta cell failure in type diabetesop novel agents in type diabetesop developing better insulinsop from diagnostics to the endstage of diabetic kidney diseaseop nafld is it all about the liverop diabetic retinopathy see what's newop taking the long view of diabetesop pregnancy in diabetes prediction and outcomesop signals and networks in beta cell failureop broken heart in diabetesop unlocking the potential of digital healthop decoding the heritable basis of type diabetesop feeding the pipeline from drugs to surgeryop sglt2 inhibitors at the heart of the matterop new treatments for nafld hope or hypeop addressing potential new treatments of diabetic kidney diseaseop glucagon and hormones beyondop incretin based therapiesop unusual forms of diabetesop macrovascular complications and beyondop linking inflammation to metabolismop what's new in automated insulin deliveryop understanding the mechanisms of diabetic kidney diseaseop novel aspects of diabetic neuropathyop reducing the burden of hypoglycaemiaop what exercise doesop back to the future risk markers in diabetesop diet not only quantity mattersop on the road to human islet failure in type diabetesop a deep dive into the mechanisms of diabetesop triggers and drivers of beta cell failure in type diabetesop gastroentero pancreatic factors anoids mice and menop new aspects of novel therapiesop fatty mattersop diabetes care is expensiveop developing beta cellsop modelling metabolism lessons from animalsop diabetic foot new developments in wound healingop challenges in delivering diabetes care new solutionsop thinking about diabetes complications in the brainop insulin secretion in various subgroupsindex of poster sessionsps diabetes and early deathps living with chronic diabetes complicationsps micro and macrovascular complications of diabetesps global view on diabetes complicationsps type diabetes treatment irlps unusual forms of diabetesps molecular insights into glucose abnormalitiesps pathophysiology of glucose homeostasisps the inner workings of the pancreasps islets and antibodies in type diabetesps markers and phenotypes of glucose traitsps global aspects on the epidemiology of type diabetesps risk factors for type diabetesps prevalence of type diabetes around the worldps risk factors in type diabetesps islet transplants revisitedps islets in type diabetes new playersps beta cells under stressps to live and let die a beta cell perspectiveps job description insulin secretionps further down the road to human islet failure in type diabetesps sitting and exercising does it allps the ins and outs of carbohydrate metabolismps pregnancy in vitro and in vivo studiesps pregnancy epidemiologyps pregnancy who is at riskps incremental studies on gut hormonesps the fundamentals of insulin resistanceps studies on insulin resistanceps treatment of hyperglycaemia in pregnancyps pancreatic hormonesps insulin secretion in mice and menps something more about obesityps more about metabolismps inflammation in type diabetesps models of prediabetes and diabetesps models of obesity and insulin resistanceps lipid metabolismps adipokine signallingps drugs and environment in obesityps weight loss interventionsps brain mattersps sglt2 inhibitors clinical aspectsps different aspects of sglt2 inhibitorsps basic aspects of incretinbased therapiesps clinical outcome of incretinbased therapies 0cdiabetologiaps glycaemic control and incretinbased therapiesps various clinical aspects of incretinbased therapiesps various aspects of nutrition and dietps oral therapies metformin sensitizers and other nonsecretagoguesps novel agents to treat diabetes and its consequencesps novel glucoselowering agents in type diabetesps key issues in improving outcomes in people with diabeteseducation and costsps how to improve diabetes careps the impact of new basal insulinsps insulin therapy real world studiesps insulin therapy fast acting insulin analoguesps the challenges of insulin therapy in type diabetesps different aspects of insulin therapyps the continued advance of continuous glucose monitoringps insulin pump therapyps automated insulin deliveryps the varied use of technologies in type diabetesps novel applications of technology in diabetesps novel therapies to reduce hypoglycaemiaps mechanisms and clinical consequences of hypoglycaemiain diabetesps emerging topics in hypoglycaemiaps investigating diabetes distress and depressionps aspects of quality of life and well beingps digital health in type diabetesps is telehealth the answer to improving care in diabetesps predicting prognosis of diabetic kidney diseaseps clinical aspects of diabetic kidney diseaseps the rock and role of experimental kidney diseaseps new tools to view diabetic retinopathyps diabetic retinopathy screening and interventionps focus on diabetic foot ulcersps hypertension and vascular diseaseps cure the pain of diabetic neuropathyps understanding clinical neuropathyps from artificial intelligence to treatment of diabetic footps from biomarkers to genetics of diabetic kidney diseaseps treatment of nafld and diabetes from food to pharmacologyps mechanisms and prevalence of nafldps lipids everywhere lipid metabolism in the liver and the heartps all about coronary arteries and diabetesps lipids and glucose not so good for the heartps cardiac complications of mice rats and cellsps atherosclerotic complications stemming from cells to the kidneyps stiff arteries and how to avoid themps cardiac function and dysfunctionps cardiovascular complications in humans through and throughps diabetes and neoplasiaps contemplating cognitive dysfunction in diabetesps endothelial cell circulation and the heartps tradition no nontraditional complications of diabetes 0cdiabetologiaop diabetes complications new insightsfrom cutting edge epidemiologycirculating metabolites significantly improve the prediction of renaldysfunction in type diabetesm scarale1 s de cosmo1 c prehn2 f schena3 j adamski2 vtrischitta4 c menzaghi11fondazione irccs œcasa sollievo della sofferenza san giovannirotondo italy 2helmholtz zentrum m¼nchen germany 3universityof bari bari italy 4sapienza university roma italy and aims chronic kidney disease ckd mainly indicated by a reduced glomerular filtration rate gfr remains one of theleading causes of reduced lifespan in patients with type diabetest2d discovering novel biomarkers able to predict low gfr will helpidentify highrisk patients to be targeted to more aggressive and burdensome preventive and treatment strategiesmaterials and methods we measured serum metabolites byabsoluteidqtm p180 kit biocrates life sciences ag innsbruckaustria and investigated their association with egfr calculated with theckdepi formula in a discovery sample of patients with t2d cases and controls with egfr60 and ‰¥70mlmin173m2 respectively a threshold p value of 28x104 ie followingbonferroni's rule was used as statistical significance in a model comprising age sex smoking bmi hba1c diabetes duration albumintocreatinine ratio acr and ongoing treatments metabolites associatedin the discovery sample were validated threshold p value of 005numberof surviving validation metabolites in a second cohort comprising diabetic patients cases and controls for egfr60 or ‰¥70mlmin173m2 respectively standardized values of each validated metabolitesweighted for the effect size ie observed in the discovery samplewere then summed up in a metabolic score metscore to be used as agfr prediction tool to this purpose metscore was used on top of anestablished clinical model comprising sex age bmi hba1c and acrand then discrimination [δ area under the receiver operating characteristic roc curve auc and the relative integrated discriminationimprovement ridi] and reclassification [the categoryfree net reclassification index cnri] measures were evaluatedresults thirteen metabolites six acylcarnitines six biogenic amines andone amino acid were independently associated to low egfr [ors range for 1sd increase p range 13x107 20x104] in the discoverysample all of them but one a biogenic amine were validated in thereplication sample [ors range for 1sd increase p range32x1018 43x106 below the threshold of 0051242x103] theauc of the abovementioned clinical model was and in the discovery the replication and the pooled sample respectively the addition of metscore on top of the clinical model improvedboth discrimination and reclassification measures in the discovery δauc4 p14x103 ridi29 p20x1011 ½cnri54p15x1014 the replication δ auc39 p16x103 ridi28p38x108 ½cnri30 p22x1010 and the pooled samples δauc39 p40x106 ridi29 p22x1017 ½cnri35p19x108conclusion we have discovered and validated metabolites that arestrongly associated with low egfr in patients with t2d a metscorecomprising these metabolites improves an established clinical prediction model of low egfr in terms of both discrimination and reclassification encouraged by these findings we are now investigating the ability ofmetscore to improve prediction of gfr decline in prospective cohorts oft2d with the aim of improving risk stratification and therefore refiningprevention efforts of kidney dysfunction in diabetic patientssupported by italian ministry of health rf201302356459disclosure m scarale noneassociation between insulinlike growth factor binding protein2 andinsulin sensitivity metformin and mortality in patients with newlydiagnosed type diabetesmr kristiansen12 js nielsen12 i brandslund3 da olsen3 jvstidsen2 sk nicolaisen4 r hjortebjerg25 j frystyk561danish centre for strategic research in type diabetes dd2odense 2steno diabetes center odense odense 3irs lillebaelthospital biochemistry and immunology vejle 4department ofclinical epidemiology aarhus 5department of clinical researchuniversity of southern denmark odense 6department ofendocrinology odense university hospital odense denmark and aims insulinlike growth factor binding protein2igfbp2 is engaged in metabolism circulating concentrations ofigfbp2 are positively correlated to insulin sensitivity overexpressionof igfbp2 protects against obesity and diabetes in mice and metforminincreases igfbp2 gene expression indicating that igfbp2 is a target ofmetformin action interestingly igfbp2 appears to predict mortalityindependently of insulin sensitivity this study aimed to investigate theassociation between indices of insulin sensitivity metformin treatmentand mortality in patients with newly diagnosed type diabetes t2dmaterials and methods in this crosssectional study we included newlydiagnosed patients with t2d enrolled in the danish centre for strategicresearch in type diabetes dd2 cohort patients were continuouslyenrolled from to throughout denmark and followed usingdanish healthcare registries unbound fractions of igfbp2 were determinedin serum from fasting drug na¯ve n864 and metformin treated ‰¥ twoprescriptions months prior enrollment patients n558 using an inhouseassay developed on the simoa platform values are given as medians iqrassociation was analyzed using a pearson™s regressioncox regression amultivariable model was used to adjust for age bmi and homasresults a total of patients with median age of medianbmi of and median diabetes duration of yearswere included igfbp2 level was positively correlated with homasr2026 and p0005 and inversely correlated with cpeptide r2018and p0005 both associations persisted following adjustments for ageand bmi the igfbp2 level in metformin treated patients was slightlylower ngml than in drug na¯ve patients ngml p0026 a total of patients suffered from one or morecomorbidities from charlson comorbidity index their igfbp2 levelswere higher than patients with no comorbidity vs ngml p0001 during a median of years offollowup a total of patients died igfbp2 level was significantly higher at baseline in patients that died vs not died vs ngml p0001 igfbp2 was associated withmortality with a hazard ratiohr ci per doubling in proteinconcentration of p0001 this association was notobserved when analyzing patients without comorbidities but was significant in patients with other comorbidities hr p0001conclusion this is the first larger study to confirm that igfbp2 isassociated with indices of insulin sensitivity but is not largely affectedby metformin treatment interestingly increased igfbp2 level is associated with high mortality rates but the association was mainly driven bythe presence of comorbidities at baselinesupported by university of southern denmark and region of southerndenmarkdisclosure mr kristiansen nonebuilding clinical risk score systems for predicting allcause andcardiovascularspecific mortality among type diabetes patientscs liu1 tc li2 cc lin1 ci li11china medical university hospital taichung 2china medicaluniversity taichung taiwan 0c and aims no prior prediction model for mortality considered the effect of glycemic variability and blood pressure variabilitywhich have been broadly reported as the important clinical predictors ofmortality especially in diabetes patients the aim of this study was todevelop and validate risk score systems with considering the effects ofglycemic and blood pressure variability on allcause and cardiovascularspecific mortality in persons with type dmmaterials and methods this is a retrospective cohort study consistingof type diabetic patients aged years during allparticipants were randomly allocated into two groups derivation andvalidation sets in ratio and were followed up until death or august cox proportional hazards regression were used to develop allcauseand cardiovascularspecific mortality prediction model prediction modelperformance was assessed by the area under the receiver operating characteristics curve aurocresults overall deaths were identified after a mean of years offollowup the prediction accuracy measured by auroc of and 15year allcause mortality based on a model containing the identifiedtraditional risk factor biomarkers and variability in fasting plasmaglucose and hba1c and diastolic blood pressure variability were and respectively in derivation set and the corresponding values forcardiovascularspecific mortality were and respectively the predictionaccuracy in the validation set for allcause mortality were and respectively and for cardiovascularspecific mortality were and respectivelyconclusion our prediction model considering glycemic and blood pressure variability had good accuracy of prediction of cardiovascularspecific and allcause mortality in patients with type diabetessupported by ministry of science and technology of taiwandisclosure c liu noneincident cardiovascular disease by clustering of favourable riskfactors in type diabetes the eurodiab prospectivecomplications studys soulimane1 yd vogtschmidt12 m toeller3 b balkau4 nchaturvedi5 jh fuller6 ss soedamahmuthu121department of medical and clinical psychology center of research onpsychological and somatic disorders corps tilburg universitynetherlands 2institute for food nutrition and health university ofreading reading uk 3heinrichheineuniversity d¼sseldorfd¼sseldorf germany 4clinical epidemiology universit© parissaclayuvsq inserm cesp villejuif france 5institute of cardiovascularscience university college of london london uk 6department ofepidemiology and public health eurodiab london uk and aims the incidence of cardiovascular diseases cvdis up to eight times higher in people with type diabetes t1d greaterclustering of adverse risk factors is thought to contribute to excess cvdrisks in type diabetes though not explored in t1d the aim of this studywas to examine a cvd risk reduction for those in the most favourablethird of individual risk factors compared to the least favourable two thirdsand b cvd risk reduction by clustering of favourable cvd risk factorsmaterials and methods we analysed data of participants from theeurodiab prospective complications study a european t1d cohortrecruited in countries between were men with a meanage of ± years we studied seven cvd risk factors namely hba1csmoking bmi combined systolic and diastolic bp ldl cholesterolphysical activity pa and diet table cox proportional hazards analyses were used to calculate hazard ratios hr [95ci] of incident cvdfor each cvd risk factor adjusted for age sex retinopathy comparingthose in the most favourable tertiles with the least favourable two tertilesdiabetologiawe then scored each individual by the number of risk factors for whichthey occupied the most favourable tertilesresults there were incident cvd cases after a mean followup of± years multivariable cox models showed that participants withthe most favourable hba1c57 [39mmolmol] had a significantlylower cvd risk hr [95ci] [] than the least favourabletwo tertiles nonsignificant inverse associations were found withfavourable bmi [] pa [] diet score[] and bp [] no associations were foundwith smoking or ldlcholesterol greater clustering of favourablecvd risk factors was associated with a lower risk of cvd in univariatemodels with a significant linear trend in multivariate models the resultswere partly attenuated with the lowest hr of [ ] in peoplewith clustering of favourable cvd risk factors tableconclusion greater clustering of favourable cvd risk factors was associated with a lower risk of incident cvd in people with t1d with a doseresponse relationship hba1c remained the most protective factor againstcvd in t1d targeting combined risk factors could be more effective inpreventing cvd risk than targeting single risk factorssupported by welcome trust the european community and diabetesukdisclosure s soulimane nonebidirectional association between type diabetes and obstructivesleep apnoea a metaepidemiological studyt karagiannis1 e athanasiadou1 a tsapas12 e bekiari11clinical research and evidencebased medicine unit aristotleuniversity of thessaloniki thessaloniki greece 2harris manchestercollege university of oxford oxford uk and aims individual epidemiological studies suggest acomplex relationship between type diabetes and obstructive sleepapnea we aimed to assess whether there is a bidirectional associationbetween the two conditions by conducting a metaanalysis of longitudinalcohort studiesmaterials and methods we included cohort studies that evaluated theassociation between type diabetes and obstructive sleep apnea in eitherdirection published until january we pooled cohortspecific estimates by means of random and fixed effects metaanalyses and calculatedodds ratios ors with confidence intervals cis to measure theassociation of prevalent obstructive sleep apnea with incident type diabetes and of prevalent type diabetes with incident obstructive sleepapnearesults out of records identified through the search cohortstudies were included in the metaepidemiological analysis ten studiesevaluated the association between prevalent obstructive sleep apnea andincident type diabetes one study assessed the association betweenprevalent type diabetes and incident obstructive sleep apnea while fourstudies evaluated a bidirectional association duration of study followupranged between and years median years the random effectsmetaanalysis for prevalent obstructive sleep apnea and incident type diabetes patients yielded an or of ci to 0cdiabetologiaresults were consistent in the fixed effects metaanalysis figureprevalent type diabetes increased the odds of incident obstructive sleepapnea patients with an or of ci to and ci to for the randomeffects and fixedeffects metaanalysis respectively metaanalyses of effect estimates adjusted forconfounding factors were similar to those of the main analysisconclusion pooled evidence from large cohort studies suggests thatpresence of obstructive sleep apnea at baseline is associated withincreased risk for developing type diabetes while presence of type diabetes is associated with increased risk for developing obstructive sleepapnea thus effective management of either condition could preventdevelopment of the otherfigure odds ratio for developing type diabetes in patients with obstructive sleep apnea versus those without obstructive sleep apneaalzheimer hr [ ic ] vascular dementia hr [ ic ] and nonvascular dementia hr [ ic ] when a 3years landmark analysis was conducted the associations remained similar for vascular and nonvascular dementia but disappeared for alzheimer™s diseasesconclusion the association of t2d with neurodegenerative diseasesdiffer by type of dementia the strongest detrimental association wasobserved for vascular dementia moreover t2d patients with polycaemic control have an increased risk of developing vascular andnonvascular dementiadisclosure c celismorales nonesupported by greece and the european social fund esfdisclosure t karagiannis noneglycated haemoglobin type diabetes and the links to dementia andits major sub types findings from the swedish national diabetesregisterc celismorales1 s franz©n2 am svensson3 n sattar1 sgudbjornsdottir21institute of cardiovascular and medical sciences university ofglasgow glasgow uk 2department of molecular and clinicalmedicine university of gothenburg gothenburg sweden 3swedishnational diabetes register gothenburg sweden and aims type diabetes t2d has been associated withhigh dementia risk however the links to different dementia subtypes isunclear we examined to what extent t2d associated with alzheimervascular and non vascular dementia incidence and whether such associations differed by glycaemic controlmaterials and methods in this swedish national diabetes registerstudy we included patients with t2d and matchedcontrols the outcomes were incidence of alzheimer vascular and nonvascular dementia the association of t2d with dementia was stratifiedby baseline glycated haemoglobin hba1c concentrations cox regression was used to study the excess risk of outcomesresults the followup median years t2d patientsand controls developed dementia the strongest association was observed for vascular dementia here patients with t2d had ahr of [ ci ] compared to controls the association oft2d with nonvascular dementia was more modest hr [ ci ] however risk of alzheimer was lower in t2d patientscompared to controls hr [ ci ] when the analyseswere stratified by circulating concentrations of hba1c a doseresponseassociation was observed compare to patients with t2d with hba1c mmolmol those with hba1c mmolmol had a higher risk of 0cop news on the insulin secretion frontwhat makes beta cells 1st responders and are they temporallyconsistentv kravets we schleicher jm dwulet am davis rkpbenningerbioengineering university of colorado aurora usa and aims calcium ca2 uptake drives glucosestimulated insulin secretion from the pancreatic cells functionalsubpopulations of cells disproportionally control the oscillatory phaseof ca2 uptake which is disrupted with ageing and in diabetes less isknown about cells which impact the 1st phase of ca2 uptake disruptedin early diabetes here we determine whether œ1stresponder cells thatlead the 1st phase of ca2 uptake are the same as œhub cells that coordinate oscillatory ca2 2nd phase we study what makes cell a1st responder and whether 1st responders are a transient state or a distincttemporally stable subpopulationmaterials and methods we used mipcreer gcamp6s mouse modelwhich expresses ca2sensitive gfp specifically in cells weperformed simultaneous recording of ca2 dynamics and gap junctionpermeability in individual islets we stimulated islets with glucosekatp channel blocker glibenclamide and kcl based on ca2 dynamicswe defined the of cells responding to the glucose stimulation soonerthan the rest of the islet as œ1st responders and the of cellsresponding slower as œlast responders we tested their temporal consistency over and hours we used laser ablation to remove specificcells from the islet we performed computational modelling of the isletelectrophysiologyresults we found that ca2 wave coordination of the 1st responders wasnot greater than the isletaverage and hence they are not overlapping withhighlycoordinated œhub cells in fact according to our gap junctionpermeability data 1st responders had lower than average electricalcoupling p00157 furthermore our computational model showedlower electrical coupling conductance in both 1st and last respondersp00447 p00279 this may be explained by our finding that1st responders are located at the islet™s periphery at ± of the islet™sradius we found 1st responders to be consistent under glibenclamidestimulation cells which respond first to the glucose remained in the15th percentile of the time response distribution when stimulated withglibenclamide sem this is consistent with our computationalresults 1st responders had lower katp conductance hence highermembrane depolarization probability p00086 glucose elevationswith 1h period showed that 1st responders remained consistent withreaction time within the of the reaction time distribution for all cellswith an elevation period of hours their reaction time shifted to thesecond quartile of the distribution and with hours to the medianunlike 1st responders last responder cells were not consistent at any timeinterval ablation of the 1st responders discoordinated but did notdisrupt the ca2 response of the islet a different cell took over the roleof the 1st responder postablation this new 1st responder was a cell whichoriginally preablation was within a leading 7th percentile of the timeresponse distribution sem conclusion in conclusion 1st responders are distinct from œhub cellsubpopulation have higher membrane depolarization probability and areless strongly coupled to other cells after the laser ablation of a1st responder new 1st responder taking on it™s role comes from a poolof original leading cells while initially consistent over a short 1h periodof time 1st responders may be losing temporal consistency over longertime periodssupported by nr01 dk102950 dk106412 jdrf 3pdf2019741andisclosure v kravets nonediabetologiabetaarrestin is absolutely required for the potentiation of insulinsecretion by gipma ravier1 j obeid1 m leduc1 s costes1 p gilon2 s dalle1 gbertrand11igf univ montpellier cnrs inserm montpellierfrance 2universit© catholique de louvain brussels belgium and aims the scaffold protein betaarrestin2 arrb2 isknown to uncouple g protein coupled receptors gpcrs from the gprotein and to recruit new signaling pathways such as the erk12pi3k fak‹¯ in non beta cells arrb2 interacts with a wide rangeof gpcrs but its interaction with the gip receptor gipr is still unclearour aim is to determine if arrb2 is involved in the signaling of thegipr in pancreatic beta cellsmaterials and methods the experiments were carried out in beta cellsfrom fivemonthold arrb2 and arrb2 male mice camp productioncampsepac endogenous pka akar3 and erk12 ekaractivations [ca2] in the cytosol [ca2]c fura2lr and in the endoplasmic reticulum [ca2]er d4er were assessed by live cell imagingin mouse pancreatic beta cells epac2 epac2gfp recruitmentbeneath the plasma membrane was monitored by total internal reflectionfluorescence microscopy factin depolymerisation was evaluated byphalloidin staining alexa fluor 488conjugated phalloidin and thep h o s p h o r y l a t i o n o f f o c a l a d h e s i o n k i n a s e f a k b yimmunofluorescenceresults insulin secretion from arrb2 islets was reduced by compared to arrb2 islets in response to gip 100pm10nm p001when arrb2 arrb2gfp was reexpressed in arrb2 beta cells insulin secretion in response to gip was restored to a similar level thanin arrb2 islets surprisingly upon gip stimulation 10pm10nm thecamp production pka activation and epac2 recruitment were similarin arrb2and arrb2 beta cells both [ca2]c and [ca2]er remainedcomparable finally the activation of erk12 was also similarin arrb2 and arrb2 beta cells by contrast the factin depolymerisationinduced by 10nm gip was significantly reduced p001 in arrb2 beta cells pi3kγ and fak have been reported to be involved in factindepolymerisation in response to gip and glucose respectively and to berequired for optimal insulin secretion as expected the pi3kγ inhibitoras604850 1μmoll reduced factin depolymerisation p001by gip stimulation in arrb2 beta cells but no additional effect wasobserved in arrb2 beta cells moreover gipinduced fak activationwas also reduced by in arrb2 beta cellsconclusion our study revealed that arrb2 is required for the potentiation of insulin secretion by gip through factin depolymerisation probably via fak activation and pi3kγ recruitment but independently fromthe canonical camp signalling pka and epac2 and the erk12 pathway therefore any variation in the expression of arrb2 as observed indiabetic states should functionally affect the incretin effect produced bygipsupported by soci©t© francophone du diabete sfddisclosure ma ravier nonepancreatic beta cellselective deletion of the mitofusins and mfn1and mfn2 impairs glucosestimulated insulin secretion in vitro andin vivoga rutter1 e geiadou1 t rodriguez2 c muralidharan3 mmartinez3 p chabosseau1 a tomas1 g carrat1 a di gregorio2 ileclerc1 ak linnemann31cell biology functional genomics faculty of medicine imperialcollege london london uk 2national heart and lung instituteimperial college london london uk 3center for diabetes andmetabolic diseases indiana university school of medicineindianapolis usa 0cdiabetologia and aims mitochondrial metabolism of glucose is essential for the initiation of insulin release from pancreatic beta cellsalthough altered in subjects with type diabetes whether mitochondrialultrastructure and the proteins controlling the fission and fusion of theseanelles are important for glucose recognition is unclear here wegenerated mice with beta cellselective adultrestricted deletionof mfn1 and mfn2 essential for mitochondrial fusion and studied theimpact on insulin secretion and glucose homeostasis in vivo and in vitromaterials and methods c57bl6 mice bearing mfn1 and mfn2 alleleswith floxp sites were crossed to transgenic animals carrying aninducible cre recombinase under pdx1 promoter control pdxcreertrecombination was achieved by daily tamoxifen injections for one weekislets were isolated and used for live beta cell fluorescence imaging ofcytosolic cal520 or mitochondrial rgeco free ca2 concentrationand membrane potential tetramethyl rhodamine methyl ester tmrmusing spinning disc confocal microscopy nikon ti2 mitochondrialnetwork characteristics were quantified using super resolution fluorescence zeiss lsm and transmission electron microscopy intravitalimaging was performed in mice injected with an adenoassociated virusto express the cytosolic ca2 sensor gcamp6s selectively in beta cellsunder the control of the rat insulin promoter using multiphoton microscopy leica tcs sp8 dive blood flow through the islet was visualisedsimultaneously after injection of fluorescent albumin647results mitochondrial length was sharply to ± of controlsp00001 reduced in the mfn12 ko mice and these animals displayedhigher fasting glycaemia than control littermates at weeks vs mmoll p005 in vivo an increase in circulating glucose levelswas also observed p005 at min and p001 at min and wasassociated with a substantial fivefold decrease in plasma insulin min p00001 postintraperitoneal glucose injection mitochondrialca2 accumulation and membrane potential were significantly reducedp001 in response to high glucose in the ko animals examined byintravital imaging of the exteriorised pancreas antiparallel changes incytosolic ca2 and mitochondrial membrane potential observed incontrol animals were largely suppressed after mfn12 deletionconclusion mitochondrial fusion and fission cycles are essential in thebeta cell to maintain normal mitochondrial bioenergetics and glucosesensing both in vitro and in the living mouse such cycles may bedisrupted in some forms of diabetes to impair mitochondrial functionand consequently insulin secretio
Colon_Cancer
" pathogenic axin2 variants cause absence of permanent teeth hypodontia sparse hair and eyebrows ectodermal dysplasia and gastrointestinal polyps and cancer inheritance is autosomal dominant withvariable penetrance only twenty five patients have been reported from five families a mayo clinic pilot programtested newly diagnosed cancer patients for pathogenic germline variants in hereditary cancer genesincluding axin2 we found only one patient with a pathogenic axin2 variantcase presentation the proband was a yearold female who came to otolaryngology clinic complaining ofrightsided nasal obstruction biopsy of identified nasal polyp revealed olfactory neuroblastomaesthesioneuroblastoma surgical resection with gross total tumor resection was followed by radiation therapy thepatient enrolled in a clinical pilot of genetic testing and a pathogenic variant in axin2 c1822del pleu608phefs81nm_0046553 was found she was seen in medical genetics clinic and found to have a personal history ofhypodontia her eyebrows hair and nails were all normal she underwent upper endoscopy and colonoscopy afour mm gastric adenoma was found and removeds this is the first case reported on a patient with a pathogenic germline axin2 variant and an olfactoryneuroblastoma or a gastric adenoma we propose that these could be features of the axin2 phenotype the knownassociation between gastric adenomas and familial adenomatous polyposis the other wntbetacatenin disordersupports the hypothesis that pathogenic axin2 variants increase risk as well as the odds of a chance cooccurrenceof a pathogenic axin2 variant and an olfactory neuroblastoma are so rare it is worth exploring potential causationwe are building a clinical registry to expand understanding of the axin2 phenotype and request any clinicianscaring for patients with pathogenic axin2 variants to contact uskeywords axin2 hereditary cancer syndrome hereditary polyposis hereditary colorectal cancer hypodontiaolfactory neuroblastoma gastric adenomas correspondence macklinsarahmayoedu1department of clinical genomics mayo clinic san pablo road southjacksonville fl usafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cmacklin mantia bmc medical genetics page of axin2 axin1 apc and gsk3 beta comprise thebetacatenin destruction complex of the canonical wntsignaling pathway mim604025 somatic pathogenicvariants in apc and the other members of the betacatenin destruction complex occur in most colorectalcancers and in many other cancers germline pathogenicvariants of the beta catenin destruction complex geneshave only been reported in axin2 and apcaxin2 pathogenic variants are associated with theabsence of permanent teeth hypodontia sparse hairand eye brows ectodermal dysplasia and gastrointestinal gi polyps and cancer inheritance is autosomaldominant with variable penetrance four of five pathogenic axin2 variants reported result in a frameshift withpremature termination trp663 arg656 asn666ser658 one is a missense substitution arg463cys[“]axin2 pathogenic variants are rare only patientshave been reported in five families [“] the first familywas published years ago in there were individuals in a generation finnish pedigree and anadditional de novo case included all cases had absenceof teeth with of missing or more teeth nine ofthe patients had had colonoscopy and the findingsranged from normal to hyperplastic polyps and adenomas without dysplasia adenomas with severe dysplasia and adenocarcinoma in the second family there were confirmed axin2pathogenic variant carriers three sisters and the daughter of one sister all four had oligodontia all threeof the sisters had colorectal neoplasia one had polyposis adenomas with two surgeries resulting in a subtotal colectomy another had metachronous colon cancers at and years of age and breast cancer at age the last sister had a history of colon polyps withoutfurther details available the daughter had completedcolonoscopy and an upper endoscopy at years of ageshe had cystic fundic gland stomach polyps this is theonly axin2 family with ectodermal dysplasia two family members had absent eyebrows and sparse hair andanother had sparse eyebrows the mother of the threesisters had oligodontia absent eyebrows and sparse hairwith no known history of malignancy she died at years of age and never had genetic testingthe third family was spanish family of four a fathertwo daughters and a son three of the four had beendiagnosed with colon cancer at ages and yearsof age one of the daughters completed colonoscopy atage which was negative none of the family membershad oligodontia or otherectodermaldysplasiafeatures ofthe most recent family was an australian mother andher three children the mother was the proband shecame to a medical genetics clinic for her history of over adenomatous polyps aftertesting positive forpathogenic axin2 variant she was discovered to havehistory of oligodontia her daughter and two sons bothall had oligodontia and colonic polyposis the daughterwas diagnosed with colon cancer at years of age anddied at we report the patient with a novel axin2 pathogenic germline variant given the rarity of the disordereach additional case report is useful in defining the natural history and informing patient care this is the firstaxin2 germline pathogenic variant case reported withan olfactory neuroblastoma and also the first case with agastric adenomaitoutside of a medical genetics clinicis veryunlikely the axin2 phenotype would be recognizedby a clinician this family was identified through amayo clinic pilot program testing patients newly diagnosed with all types of cancer for germline variantsincluding axin2 intein cancer related genesrcept study in and patients weretested only one pathogenic axin2 germline variantwas foundcase presentationthe proband was a yearold female she was referred to the mayo clinic for evaluation and treatmentof a rightsided nasal mass at initial consultation inthe otolaryngology clinic the patient complained of a month history of rightsided nasal symptoms including progressive congestion decreased sense of smelland epistaxis she also reported mild right posteriororbital pain with slight right eye swelling occipitalpain upper gum pain and progressive decrease insense of tastethe patient had a normal physical exam except forlymphadenopathy on nasal endosunilateral cervicalcopy a grade polyp was noted in the middle meatusan intranasal biopsy was performed identifying cellsconsistent with a neuroendocrine malignancy mostlikely olfactory neuroblastoma esthesioneuroblastomasubsequent magnetic resonance imaging mri showeda tumor in her right nasal cavity with thickening of theright frontal sinus ethmoids and sphenoid sinus therewas contact with the dura as well as medial orbital walland maxillary sinus [fig 1a] given the tumor™s proximity to the skull base a neurosurgery consultation wasobtainedthe patient was brought to the operating room about months after she first sought medical attention and months after her symptoms began the patient had apurelytumor whichincluded bilateral maxillary antrostomies total ethmoidectomies frontal sinus draf iii procedure and duralresection ofendoscopicthe 0cmacklin mantia bmc medical genetics page of fig mri imaging of the olfactory neuroblastoma pre a and post b operative sagittal brain mris from our proband the arrow on the leftimage indicates the olfactory neuroblastoma neuroesthioblastomabiopsy with skull base reconstruction using a left nasoseptal flap the total operative time was min andthe patient was discharged home on postoperative day with no complicationsthe operative pathology report confirmed the diagnosis of olfactory neuroblastoma hyams grade and kadish stage c [fig ] the pathologist confirmed tumor inthe vidian canal skull base right sphenoid and rightfrontal sinus no definitive tumor was identified in theright maxillary margin but the impression was that themargin was close mri the day following surgery showedgross total resection of the tumor [fig 1b]thirty days after surgery the patient began radiationshe was treated to a dose of gy in fractions atthe time this paper was submitted the patient was over days postsurgery the patient™s most recent imagingwas days after surgery and showed no recurrencegermline genetic testing was ordered through a commercial genetic testing company and included analysis of genes a pathogenic variant was detected in axin2c1822del pleu608phefs81 nm_0046553 two variants of uncertain significance were also found nf1c6982c t parg2328cys and rad50 c1336a gplys446glu other genes tested were alk apc atmbap1 bard1 blm bmpr1a brca1 brca2 brip1casr cdc73 cdh1 cdk4 cdkn1b cdkn1ccdkn2a p14arf cdkn2a p16ink4a cebpachek2 ctnna1 dicer1 dis3l2 egfr epcamfh flcn gata2 gpc3 grem1 hoxb13 hraskit max men1 met mitf mlh1 msh2 msh3msh6 mutyh nbn nf2 nthl1 palb2 pdgfraphox2b pms2 pold1 pole pot1 prkar1aptch1 pten rad51c rad51d rb1 recql4 retrunx1sdhdsdhaf2sdhcsdhasdhbfig pathology from olfactory neuroblastoma histopathological images from the olfactory neuroblastoma esthesioneuroblastoma removedfrom our proband at surgery images shown are from the tumor tissue resected from the right frontal nasal sinus panel a shows a nest of tumorcells with the characteristic œsalt and pepper appearance hematoxylin and eosin staining 40x panel b shows intense immunohistochemicalstaining for synaptophysin supporting the diagnosis of neuroblastoma 20x several other immunostains were done but not shown tumor cellswere also positive for chromogranin and negative for cam ema and cd45 gfap is essentially negative in the matrix 0cmacklin mantia bmc medical genetics page of fig panoramic dental radiograph panoramic dental radiographfrom our proband with a pathogenic axin2 variant there are teeth a complete set of adult is teeth the patient is lacking herupper lateral incisors bottom second premolars and third molarswisdom teethsmad4 smarca4 smarcb1 smarce1 stk11sufu terc tert tmem127 tp53 tsc1 tsc2vhl wrn and wt1the patient completed genetic testing before startingradiation treatment she was not seen in the medicalgenetics clinic prior to testing but did watch an educational video on hereditary cancer genetic testing shewas informed of the results by telephone and scheduledinto the medical genetics clinicin genetics clinic she shared history of hypodontialacking her upper lateral incisors bottom second premolars and wisdom teeth [fig ] her upper cuspids hadbeen capped to appear more similar to lateral incisorson physical examination eyebrows hair pattern andnails were all normal the patient had no dysmorphicfeatures the patient had no gastrointestinal symptomsand had never had an upper endoscopy or colonoscopyboth upper endoscopy and colonoscopy were requesteddue to association of germline axin2 variants with colorectal neoplasia in the body of the stomach there was asmall mm polyp [fig ] it was removed and reportedby the pathologist as a gastric adenoma foveolar typeboth the stomach and duodenum were normal a mmhyperplastic polyp was removed from the ascendingcolonthere was no maternal history of hypodontia or gipolyps [fig ] paternal history was incomplete and nopaternal relatives were available for axin2 testing thepatient did recall her father lacked several lower teethand had a removable lower bridge later in life he wasdiagnosed with a malignant tumor in his cerebellumfurther pathology details unavailable a paternal aunthad been diagnosed with breast cancer no further paternal history was available the proband has three children two have typical teeth the third had a typicalnumber of teeth but her upper lateral incisors were described as œpegged and had been cappedfig gastric adenoma endoscopic photograph taken duringupper endoscopy from our proband with a pathogenic axin2variant shown is a mm polyp found in the body of the stomach itwas removed with cold forceps and sent to pathology and reportedas a gastric adenoma photograph taken with narrow band imagingand near focus using an olympus endoscopediscussion and we propose that gastric adenomas and olfactory neuroblastoma could be features of the axin2 phenotype asthe total number of patients reported to have pathogenicvariants in axin2 is small this hypothesis is difficult toprove from the current case report population howeverthere are other pieces of evidence that support a relationship between germline pathogenic variants in axin2and gastric adenomas and olfactory neuroblastomasgastric adenomas are an established feature of the otherfig proband™s pedigree pedigree of our proband carrying apathogenic axin2 variant the proband™s father was not availablefor testing 0cmacklin mantia bmc medical genetics page of wntbetacatenin disorder familial adenomatous polyposis fap gastric adenomas are seen in about ofthose with fap and are very uncommon in the generalpopulation the subtype of gastric adenoma found in the patient foveolar would be even moreuncommon in the general population neuroblastomas both olfactory and nonolfactory arenot associated with the fap phenotype as gastric adenomas are but other lines support a causative associationwith germline pathogenic axin2 variants the probandhad negative germline testing for other genes associatedwith neuroblastoma bard1 and chek2 axin2pathogenic variants and olfactory neuroblastoma areboth so uncommon that further evaluation of a potentialrelationship is warranted only cases of olfactoryneuroblastoma werereported in the surveillanceepidemiology and end results seer tumor registrybetween and dysregulation of the wntbetacatenin pathway hasbeen reported in neuroblastoma [ ] the zebra fishapc mutant animal model has been reportedinabstract form to have high rate of esthesioneuroblastoma olfactory neuroblastoma have not beenreported in the wellstudied apc min1 mousemodeldata on germline axin2 variants in patients witholfactory neuroblastomas is very limited we were unable to find a reported series of patients with olfactoryneuroblastoma and clinical axin2 germline genetic testing four molecular profiling studies of olfactory neuroblastomas have been published three used gene panelsnot including axin2 [“] one used whole exomesequencing and other technologies on samples there was no comment on axin2 mutations howeversomatic deletions of either the dmd or lama2 lociwere present of the samples [gallia] in a reviewpaper on genetic patterns in olfactory neuroblastoma nopartial chromosomal deletions were reported involving17q241 given the very limited data on axin2 and olfactory neuroblastomas data from related tumors that are betterstudied was reviewed neuroblastomas originate from sympathetic nerve cells and olfactory neuroblastomas fromolfactory sensory cells both the sympathetic nerve cells andolfactory sensory cells are derived from the neural crestgermline axin2 variants in patients with neuroblastoma have been previously reported [table ] threeof patients with neuroblastoma studied with wholeexome sequencing had axin2 variants there were novariants in controls one of these changes was anon frameshift deletion and the three others were nonsynonymous single nucleotide variants other than thediagnosis of neuroblastoma only age of diagnosis andlocation of neuroblastoma was reported so it is unknown if any of these four cases had features or familyhistory of axin2 related disease such as hypodontiabased on our experience and review of the literaturewe offer this tentative clinical guidance for the management of pathogenic axin2 carriers 0f upper endoscopy and colonoscopy beginning at years of age if there are concerning symptoms suchas rectal bleeding colonoscopy should be doneearlier subsequent upper endoscopy andcolonoscopy should be done at least every years ifpolyps are found then procedures may need to bedone more frequently than every years 0f yearly history and physical assessment if patients haveany nasal or facial symptoms we recommend referralto an ear nose and throat specialist and imagingthe small number of known cases with germlineaxin2 variants makes it challenging to accurately assessrisks for these individuals we would ask that any clinicians treating patients with axin2 pathogenic variantscontact us our group working with genetic testinglaboratories and other clinical groupsis building anaxin2 patient registrytable reported germline axin2 variants in patients with neuroblastomapublicationn diagnosisvariationlocationagelasorsaab monthsadrenalmissensecdnachangec684g cproteinchangepl228f monthsadrenalnonframeshiftdeletionc1144_1149delp382_383del months notmissensec1151a g pe384gspecifiedmissensec1878 t g ps626r yearsthis publicationmacklincalasorsa va exome and deep sequencing of clinically aggressive neuroblastoma reveal somatic mutations that affect key pathways involved in cancerprogression oncotarget apr “ bnm_004655 cnm_0046553frameshift deletion c1822delolfactorypl608“exon exacclinvarsiftpolyphen2absent absentabsent absentdeleterious damaging““absent variant ofuncertainsignificancetoleratedbenignabsent826eabsent pathogenic “deleterious benign 0cmacklin mantia bmc medical genetics page of abbreviationsgi gastrointestinal mri magnetic resonance imaging fap familialadenomatous polyposis seer surveillance epidemiology and end resultscarrying an exon nonsense variant in the axin2 gene familial cancer httpsdoi101007s10689019001200ngamruengphong s boardman la heigh ri krishna m roberts meriegertjohnson dl gastric adenomas in familial adenomatous polyposisare common but subtle and have a benign course hered cancer clinpract httpsdoi10118618974287124 wood ld salaria sn cruise mw giardiello fm montgomery ea upper gitract lesions in familial adenomatous polyposis fap enrichment of pyloricgland adenomas and other gastric and duodenal neoplasms am j surgpathol httpsdoi101097pas0000000000000146jethanamest d morris lg sikora ag kutler di esthesioneuroblastoma apopulationbased analysis of survival and prognostic factors archotolaryngol head neck surg httpsdoi101001archotol1333topcagic j feldman r ghazalpour a swensen j gatalica z vranic scomprehensive molecular profiling of advancedmetastatic olfactoryneuroblastomas doi101371journalpone0191244 ecollection li y ohira m yong z xiong t wen l yang c genomic analysis“integrated wholeexome sequencing of neuroblastomas identifies geneticmutations in axon guidance pathway doihttpsdoi1018632oncotarget18079 greenwood ve museles n weinberg q glasgow e abstract highrates of esthesioneuroblastoma in adenomatous polyposis coli apc mutantzebrafish suggest a new modifier mutation altering tissue specificity of wntβcatenindependent hyperproliferation doihttpsdoi10115815387445am20134296 gay lm kim s fedorchak k kundranda m odia y nangia c comprehensive genomic profiling of esthesioneuroblastoma revealsadditional treatment options oncologist httpsdoi101634theoncologist20160287 mintzer dm zheng s nagamine m newman j benito mesthesioneuroblastoma olfactory neuroblastoma with ectopic acthsyndrome a multidisciplinary case presentation from the joan karnellcancer center of pennsylvania hospital oncologist httpsdoi101634theoncologist20090016lazo de la vega l mchugh jb cani ak kunder k walocko fm liu cj comprehensive molecular profiling of olfactory neuroblastoma identifiespotentially targetable fgfr3 amplifications mol cancer res httpwwwpubmedncbinlmnihgovtermlazodelavegalcauthor_id gallia gl zhang m ning y haffner mc batista d binder za genomicanalysis identifies frequent deletions of dystrophin in olfactoryneuroblastoma nat commun httpsdoi101038s4146701807578z czapiewski p kunc m haybaeck j genetic and molecular alterations inolfactory neuroblastoma implications for pathogenesis prognosis andtreatment oncotarget httpsdoi1018632oncotarget9683lasorsa va formicola d pignataro p cimmino f calabrese fm mora j exome and deep sequencing of clinically aggressive neuroblastomareveal somatic mutations that affect key pathways involved in cancerprogression oncotarget httpsdoi1018632oncotarget8187publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsacknowledgementsnot applicableauthors™ contributionssm acquired relevant data and drafted the work kc acquired and analyzedrelevant data ad acquired and analyzed relevant data sk acquired andanalyzed relevant data qz acquired and analyzed relevant data shsubstantively revised the work njs substantively revised the work drjacquired and analyzed relevant data and substantively reviewed the workall authors read and approved the final manuscriptfundingthe mayo clinic cancer genomics service line biorepository is funded bythe mayo clinic center for individualized medicine the cost of genetictesting was covered by the mayo clinic center for individualized medicineno other funding required for this reportavailability of data and materialsthe raw datasets generated andor analyzed during the current study arenot publicly available in order to protect participant confidentialitynm_0046553 can be accessed through the national center forbiotechnology information at httpswwwncbinlmnihgovnuccorenm_ accessed ethics approval and consent to participateour proband provided written consent to participate in a clinical researchpilot the mayo clinic cancer genomics service line biorepository whichwas approved by the mayo clinic institutional review board id “consent for publicationproband provided written consent for publicationcompeting intereststhe authors declare that they have no competing interestsauthor details1department of clinical genomics mayo clinic san pablo road southjacksonville fl usa 2department of medicine division of diagnostic consultative medicine mayo clinic san pablo road southjacksonville fl usa 3department of neurologic surgery mayo clinic san pablo road south jacksonville fl usa 4department ofotolaryngology mayo clinic san pablo road south jacksonville fl usa 5department of radiation oncology mayo clinic sanpablo road south jacksonville fl usa 6department of laboratorymedicine and pathology mayo clinic san pablo road southjacksonville fl usa 7department of gastroenterology mayo clinic e mayo boulevard phoenix az usa 8department ofgastroenterology mayo clinic san pablo road south jacksonville fl usareceived october accepted august referenceslammi l arte s somer m jarvinen h lahermo p thesleff i mutationsin axin2 cause familial tooth agenesis and predispose to colorectal canceram j hum genet httpsdoi101086386293 marvin ml mazzoni sm herron cm edwards s gruber sb petty em axin2associated autosomal dominant ectodermal dysplasia and neoplasticsyndrome am j med genet a httpsdoi101002ajmga33927rivera b perea j sánchez e villapún m sáncheztomé e mercadillo f a novel axin2 germline variant associated with attenuated fap withoutsigns of oligondontia or ectodermal dysplasia eur j hum genet httpsdoi101038ejhg2013146beard c purvis r winship im macrae fa buchanan dd phenotypicconfirmation of oligodontia colorectal polyposis and cancer in a family 0c"
Colon_Cancer
"autophagy is an evolutionary cellular program that serves for thebreakdown of cytoplasmic components within lysosomes [“] inidentified autophagy in's electron microscopic studies firstmammalian cells but the molecular pathways were not understooduntil the discovery of autophagy genes atgs in yeast by performinggenetic screening it is a cytoprotective rather than a selfdestructive process it is extensively accepted as a main regulator of innateand adaptive immune mechanisms the change in which completelyimpact the pathogenesis of disease and the processes that are influencedby autophagy includes the regulation of inflammation antigen presentation and bacterial clearance moreover autophagy aids in themaintenance of fundamental anelle populations such as mitochondria which is necessary for cellular bioenergetics and homeostasis homeostasis of the cell is been accomplished by maintaining the biosynthesis and turnoverthere are two broader protein degrading systems in eukaryoticcells first is the ubiquitinproteasome system responsible for the selective breakdown of most shortlived proteins and second is the lysosomalsystem the primary anelle called lysosome ineukaryotes is known for degradation through its acid hydrolases inunfavourable nutrient deprivation condition autophagy arbitrates aregulated phagocytosis via lysosomes autophagy is mediated byautophagosome that is thought to be an on selective degradation systemas it engulfs some of the cytoplasmic contents the ubiquitin“proteasome system concedes only ubiquitinated proteins for degradation andit marks a remarkable contrast to autophagosome process autophagosomes a doublemembered vesicle that engulfs durableproteins impaired anelles intracellular pathogenic anisms andtransports it to the lysosomes that are fused to form autolysosome andthe inner vesicle along with its cargo is been degraded at the time ofstarvation the remaining macromolecules are again recycled to thecytosol for reuse the precise mechanism in cargo recognition isuncertain but this process involves ubiquitination autophagicprocess is separated into distinct steps which includes induction recognition selection of cargo formation of vesicle then occurs the fusion of autophagosomevacuole followed by the degradation of thecargo and release into the cytosol various atg proteins are indulged inthis process and it consists of the central autophagic machinery autophagy encompasses different process by which cells delivercytoplasmicaredegradation theylysosomalsubstratesforŽ corresponding author at department of biotechnology psg college of arts and science civil aerodrome post coimbatore indiaemail address rashmiffrajangmailcom rr rasmi all authors deserve contribution equally101016jlfs2020118308received june received in revised form august accepted august available online august elsevier inc all rights reserved 0cs vishnupriya life sciences macroautophagy chaperonemediated autophagy cma and microautophagy all the three processes of autophagy are morphologically distinct in microautophagy the lysosomal membrane invaginations or protrusions are needed to seize cargo once thecargo is captured the uptake directly happens at the limiting membrane of the lysosome that includes intact anelles cma uses chaperones to sequester cargo proteins that have a pentapeptide motifthese proteins are unfolded and translocated directly across the lysosomal membrane via lamp2a receptor macroautophagy involves requisition of cargo vesicle formation and its subsequenttransport to the lysosome in recent years deletion of the autophagy related genes atg invarious model anisms has proved that autophagy plays decisive rolein adaptive responses to stress cellular differentiation and development an oncogenic event may establish by the partial minimization inthe autophagic capacity atg6beclin one of the phylogeneticallypremeditated autophagy genes is often subdued at one locus in humancancers studies in mice have shown that beclin is a haplo insufficienttumor suppressor autophagic programmed cell death was primarily depicted in actively developing tissues several conjugationsystems comprising of the atg genes are available that take part inautophagasomal elongation one such system is the lc3 microtubule associated protein light chain and atg8 conjugation system lc3 is the mammalian conjugative protein ortholog of yeastprotein atg8 the lipid derivative phosphatidylethanolamine bindsto lc3 to form lc3ii an important molecular marker of autophagylc3ii remains on the mature autophagosome until it fuses with thelysosomes fig the beclin complex gives rise to an incipientautophagosome membrane and it assemble around cargo in a vesiclethat combines with a lysosome forming autolysosome that is degradedby acid hydrolases present in the lysosomes lung injury clinical implicationsthe primary an of gas exchange is the pair of lungs theymediate inspiration of oxygen and elimination of mono and dioxides ofcarbon lung also serves as an attractive target for the entry of thepathogens regulation of the pulmonary functions is mediated by intricate cells of endothelial and epithelial lining dendritic cells alveolarmacrophages and fibroblasts all these pulmonary cells are highlyheterogeneous in nature they together in association respond to thelung injury by provoking inflammatory and immune responses airway epithelial cells express pattern recognition receptors prrsalong with toll like receptors ctype lectins rigi and inflammasome components which are involved in the innate response against microbes microbial cell wall constituents likelipopolysaccharide are sensed by prrs and induce an inflammatoryresponse alveolar epithelium comprises type i and type ii alveolarcells apart from these the mucus layer and the physical barrier madeby the epithelium contribute to the first line of defense lung injury is described as any damage to the associated tissues and compartments of the pulmonary system the concept of lung injury influenced by the microenvironment can be demonstrated via series ofchanges in cell deformability and manifestation of intercellular adhesive molecules the major causes of lung injury are atelectasisalveolar instability volutrauma barotrauma infections and oxygentoxicity the pathogenicity of acute and chronic lung injury iscorrelated with the release of proteases free radicals and growth regulatory proteins by the alveolar macrophages neutrophil takescare of the extreme ranges of lungs searching for pathogens therebyfig mechanism of autophagy ulk1pi3kmtor signalling pathway binds to endoplasmic reticulum and activates dcedp1 that initiates beclin and atg512conjugation system which forms isolation membrane this is followed by the sequestration process that leads to autophagosomal formation the autophagosomefuses with lysosomes to form autolyososome that leads to degradation 0cs vishnupriya eliminating via phagocytosis they undergo transendothelial andtransepithelial migration primary host defense mechanism oflungs include the immunity conferred by surfactant proteins spnamely spa and spd they link both innate and adaptive immunity byregulating the responses of innate immune cells antigen presentingcells apcs and tcells they can act as potential biomarkers of lunginjury the implication of autophagy over lung injury is tortuousas its function varies highly with the cell types specific to the pulmonary disorders it provides both defensive and injurious outcomes of lung injury lung injury “ types and associated pathologies acute lung injury ali and acute respiratory distress syndromeardsacute lung injury and acute respiratory distress syndrome arecommon grievous diseases among critically illpatients and are evidential sources of mortality and morbidity they are expressed alongwith hypercapnia and hypoxemia ards is outlined as the most seriousform of ali ali is characterized by inflammation of lung surfacesresulting in the disruption of alveolarcapillary membrane followed bytransmigration of neutrophils significant event in the procession of aliand ards and outbreak of cytotoxic mediators immune responsemediated by several components like tcells macrophages naturalkiller nk cells chemokines and proinflammatory cytokines also has apredominant role in the progression of the acute injury and results inimmune reconstitution inflammatory syndrome iris immune cellsinvolved in the immune response like macrophages and monocytes arethought to be involved in the pathophysiology of iris elevatedlevels of cd14 cd16ˆ’ monocyte population and an resulting increase in the proinflammatory cytokines il6 tnfα and c reactiveprotein crp are also observed in tuberculosis tbiris patients lung endothelial biomarkers like vwf and epithelial biomarkers likespd are the diagnostic targets of ali sepsis pancreatitis pneumoniatrauma transfusions aspiration and inhalation of toxic gases are someof the clinical factors of ali and ards histological patterns ofali and ards have the chances of demonstrating diffuse alveolar damage alveolar haemorrhage eosinophilic pneumonia and acute fibrinous pneumonia associated with ards may also result frompathogens like fungal viral bacterial and parasitic most commonpathogen strains include streptococcus pneumoniae respiratory virusesstaphylococcus aureus fungal pathogens like pneumocystis jirovecii andaspergillus fumigatus legionella pneumophila and enteric gramnegativeanisms among bacteria the virulence capability of pseudomonas aeruginosa is one of the prime determinant of the severity of thelung injury disease progress takes place in three degrees namelyacute phase exudative subacute phase proliferative and chronicphase fibrotic radiological features during the acute phasesignify twosided patchy groundglass densities relating to interstitialedema and hyaline membranes the important constituent of innateimmune system the pattern recognition receptors prrs are affiliatedwith the advancement of ali and ards the ligands of prrs includepathogenassociated molecular patterns pamps which induce inflammatory signalling events and the damageassociated molecularpatterns damps which induce neutrophilmediated tissue damageincreased incidences of mitochondrial damps result in high mortalityrates a common type of ali is the transfusionrelated acute lunginjury trali trali is defined as adult respiratory distress syndromeoccurring with transfusionsis manifested by pulmonary insufficiency severe hypoxia and dyspnea it is often reported as theneutrophil pmnmediated syndrome a particular study has reported that the potential risk factors for ali and ards may be associated with larger tidal volume vt and higher airway pressure pawduring one lung ventilation olv in postpneumonectomy aliardspatients and also in patients who developed aliards longtermeffects after acute lung injury are related to neurological impairmentsitlife sciences namely neuromuscular dysfunction neurocognitive dysfunction andneuropsychological dysfunction some of the minor impacts includeheterotopic ossification stiae and frozen joints chronic lung injury cli and bronchopulmonary dysplasia bpdchronic lung injury is characterized by a condition called pleuroparenchymal fibroelastosis ppfe in which a rapid multiplication ofsubpleural intestinal elastic fibres majorly in the upper lobes predominates along with other clinicopathological conditions following chronic lung injury cells undergoing apoptosis is cleared by aprocess called efferocytosis it is a type of phagocytosis confined only tothe cells that undergo apoptosis to maintain the cellturnover in pulmonary airways carried out mainly by the macrophages while immature dendritic cells mesenchymal cells and epithelial cells can alsoperform efferocytosis recently sarscov2 manifested as severerespiratory infection resulted in number of deaths worldwide commonrisk factors associated with death in sarscov2 patients identified arehypertension diabetes cardiovascular disease or chronic lung disease the most prevailing chronic lung injury ofinfants is thebronchopulmonary dysplasia bpd occurs when preterm infants suffering from various respiratory syndromes like meconium aspirationsyndrome and neonatal pneumonia are subjected to treatment withsupplemental oxygen and extensive mechanical ventilators it is proventhat the common risk factors of bpd increases with a fall in birthweight prematurity and gestational period apart from theabove other perinatal risk factors with which bpd is associated with areintrauterine growth restrictions race or ethnicity chorioamnionitis and genetic risk factors it is characterized bysaccular formation and elastic fibre aggregation of distal air spacesinflation and edema barotrauma and pulmonary oxygen toxicity arepathologies of bpd bpd is multifactorial in nature studiesdeclare that surviving patients of bpd have established pulmonarydysfunction incidence adults with bpd history are strictly prohibitedto cigarette smoking another form of bpd called the new bpdwhen surfactants are used as treatment new bpd is depicted by pulmonary hypertension and abnormalities in vasculopulmonary development glucocorticoids and tgfbeta are the efficient modulators thatinitiate bpd injury bpd infants have significantly lower levels ofvascular endothelial growth factor and platelet endothelial cell adhesion molecules chronic obstructive pulmonary disease copd and asthmacopd is a general illness worldwide it is defined as the completeirreversible state of airflow limitation characterized by weak inflammatory response of the lungs emphysema chronic obstructivebronchiolitis fibrosis blocking and narrowing of airways deprivationof lung parenchyma and elasticity immune cells like tlymphocyteswith cd8 dominance blymphocytes macrophages and neutrophilsare the regulators of copd acute provocation of copd is definedas the continuous deterioration from steady state facilitating an alteration in typical medication for rudimentary copd systemicinflammation responses are a result of leukotriene b4 tnfalpha interleukin il8 and proteases a decrease in the ratio of cd4 to cd8is a typical feature of pulmonary inflammatory responses additionalimpacts of copd are cardiovascular diseases nervous effects and osteoskeletal effects smoking is an important cause of systemicoxidative stresses immoderate inflammatory responses and emphysema as manifested as copd implications copd being an hetergoenousdisease patients with exacerbations are found to be associated withbacterial infections like moraxella catarrhalis streptococcus pneumoniaand haemophilus influenza apart from bacteria other microbes likevirus and fungi also comtribute to the lung microbiota and pathogenesisof lung microbiota through initiation of chronic inflammation it hasbeen found that bacteria and viruses fungi can promote local andsystemic inflammation that may contribute to the pathogenesis ofcopd specific biomarkers of copd observed are creactive 0cs vishnupriya protein crp il6 il10 and ccl18parc skeletal muscle losstakes place in copd wasting of the cell mass is evidenced to be theresult of tnfα participation in the pathogenesis of copd muscleglutamate reduction is linked with lactic acidosis in copd patientsending in muscle wastage similar to copd asthma is characterized by pulmonary obstruction and similar immune responses onlyvariation from copd is that the airway obstruction in asthma is reversible and it does not affect lung parenchyma dendritic cells are themodulators of th2 cells playing an important immune response ofasthma severe asthma is equal to the effects of copd illustrated by theincrease in neutrophils tumor necrosis factor tnf cxcl8 and decreased reception to corticosteroids while reversible copd is potentially to have subsequent asthma and copd ventilatorinduced lung injury vili and ventilatorassociated lunginjury valithe prevailing lung injury cases with ards when given ventilatorassistance mechanically in a clinical setup may develop additional lunginjuries ending up in ventilatorassociated lung injury vali similarlyin experimental models lung injury can be provoked by external application of injurious ventilation procedures contributing to ventilatorinduced lung injury vili thus vali can adversely aggravate thehealth of the ards patients at low lung volumes of ventilation atelectrauma occurs at high lung volumes of ventilation barotrauma andpulmonary edema occur biomarkers studied via experimental modelsof vali include several proinflammatory molecules like tnfα il1βil8 and antiinflammatory molecules like il10 il6 and stnfr1 respectively [“] positive endrespiratory pressure peep and tidalvolume applied have direct influence on vali and are to be studiedspecifically during ali and edema it is reported that peep when provokes overinflation the extent of edema also increases mechanism ofsurfactant inactivation is seen in alveolar microvessels with an increasein fluid filtration further greater the lung volume higher is thetransmural pressure in them a retrospective cohort study revealedthat height and gender of the patients should be taken into considerations along with establishing limitations to large tidal volumes beforesetting up a ventilator experiments explain that decrease in thepeak pulmonary arterial pressure or respiratory frequency can lessenthe grimness effects of vali it is proven that hypercapnic acidosisaffords protection to vili addressable implications of vali arebarotrauma volutrauma atelectrauma biotrauma and oxytoxic effects cellular pathology includes physical disruption of cells and tissuesand activation of cytotoxic responses leucocytes are raised to likelyinteract with the endothelium as the increasing intraalveolar pressurefastens the transit time of them inferences for current medicalpractices involve lungprotective ventilation survival of patients can beencouraged by prone positioning future clinical practices involveprecisioned ventilationindividualized tidal volumes using drivingpressure individualized peep and extracorporeal strategies pulmonary fibrosis pf and cystic fibrosis cfidiopathicpulmonary fibrosis ipf is a degenerative interstitial fibrosing disease prevalent worldwide it is also termed as cryptogenicfibrosing alveolitis a typical honeycomblike structure of asymmetricairspaces covered by dense fibrosis is observed ipf is followed by interstitial pneumonia which is featured by deficit inflammation and withabsence of homogeneous participation of lung tissues studies havebeen made since ages which revealed several inherited forms of pulmonary fibrosis mutations in various genes were associated to pfnamely sftpc sftpa2 tert terc and muc5b incidence withrheumatoid arthritis and scleroderma are more likely to form pf thereis a chance of clearance of alveolar basement membrane and occurrenceof hyperplastic epithelial cells ipf ensures migration of fibroblastsinto the fibrinrich exudates thus a chemoattractant activity is createdin the airspaces after lung injury this process is proven to be regulatedthe proby lysophosphatidic acid experiment proves thatlife sciences inflammatory cytokines il1β directly regulates the initiation of acuteand chronic inflammation making it a valuable target of ipf cystic fibrosis is characterized as a most common autosomal recessivegenetic disorder caused by the mutation in cftr gene transmembraneprotein genecystic fibrosis transmembrane conductance receptor pathology of the disease involves bronchiolitis obstruction of pathways endobronchiolar infection impaired ciliary actions atelectasisfinally leading to secondary alveolar injury bacterial infections of saureus p aeruginosa and h influenzae causes cf it is the pulmonarymacrophages and polymorphonuclear neutrophils that forms the defense against infections while lymphocytemediated mucosal injury isobserved in cf patients cf patients have increased flow of tnfαil8 and il1β submucosal glands of the lungs are the crucialhosts of cftr genes as the number expressed are high hence cfcontributes to epithelial airway lining abnormalities with respect to thechanges taking place in the submucosal glands respectively the macromolecular secretions of the submucosal glands have changes in theircomposition viscosity and greatly impacts on the mucociliary clearance radiationinduced lung injury riliseveral complications of the lung malignancies require treatmentsinvolving radiation therapy rt extensive reports claim that rt maylead to a state of rili the threedimensional dosimetric predictors canbe emphasized for the risk of symptomatic rili the alveolarcapillary subunit forms the most radiosensitive complex of the lungs thusthe rili is also called as the diffuse alveolar damage radiation exposure provokes the production of reactive oxygen species creatinghigh toxicity levels in the lung parenchyma this may ultimately lead tolung fibrosis which develops one to six months after rt accompaniedby dyspnea in addition to fibrosis rili also develops radiationinduced pneumonitis gradually after months to years almost all thepatients undergoing rt have the chance of developing fibrosis radiationinduced pneumonitis is characterized by cough occasional fevernonspecific symptoms of dyspnea and chest pain with or without deformities in pulmonary functional tests while radiationinduced pulmonary fibrosis is characterized by cough differential levels of dyspnea chest pain or symptomless and stable scarring of the lung tissueswhen detected radiographically it has been stated that the incidence and occurrence of rili is directly influenced by dose and volume determinants of rt several studies provide information onrili that result in provoking inflammatory responses a biphasicmanifestation of cytokines is observed in the lung tissues once exposedto rt one such study carried out to assess the cytokine productionin c3hhen irradiated mice revealed a spatial change in the expression of proinflammatory cytokines among various cellular compartments of the lungs further it was noted that the bronchoalveolar lavage cells responded immediately while the interstitial cells contributedonly in the later stages during pneumonitis profiling of cytokinesnamely the interleukins interferons monocyte chemotactic protein1tumor necrosis factors macrophage inflammatory proteins and granulocyte colonystimulating factors can be performed to analyse the developmental risks of symptomatic radiationinduced lung injury in vitro studies experiments state that the application of melatonin andcarnosine compounds reduced the reactive oxygen species and inflammatory cytokines produced following rili regulators of autophagy in lung injurynumerous regulators of autophagy play a vital part in the development of lung injury with regard to autophagy the following are theimportant regulators fig the mechanism by which these autophagy regulators act is shown in the table lc3biithe microtubuleassociated protein light chain lc3 is the 0cs vishnupriya life sciences fig figure depicts the autophagy regulators in regard to lung injury various conditions like hypoxia starvation environmental stress and cigarette smoke leadsto autophagy that in turn causes lung injury activation of class ipi3kmtor pathway takes place under hypoxic and starvation conditions while environmentalstress results in provoking beclin 1vps34 pathway that induces the sequestration of the phagophore formation cigarette smoke induces ros accumulation which inturn causes egr1 e2f signalling to activate lc3ii along with sqtm1p62 and atg512 conjugation system the ros generated may also leads to apoptosis theautophagosome fuses with lysosomes and forms autophagolysosome that causes pulmonary arterial hypertension and lung injuryprinciple autophagic protein expressed on the doublemembraned autophagosome nearly eight homologues of lc3 proteins are studied inmammals the amino acid composition of these homologues classifiesthem into two subfamilies first one comprising of lc3a splicingvariants lc3b and lc3c taking part in autophagosomal membraneelongation while the second one comprising of gabarap gabarapl1 gabarapl2 and gabarapl3 taking part in the maturation ofautophagosome generally lc3b occurs in cytosol as lc3bi by theproteolytic cleavage of cterminal of lc3b which then unites withphosphatidylethanolamine to form lc3bii to assemble on the autophagosomal membrane conjugation of phosphatidylethanolamine withlc3bii can be revoked by the activity of atg4 thus it is clear thatoccurrence of lc3bii is crucial in the process of autophagy lc3bii levels are analysed through immunoblotting while limitationsare degradation of lc3bii by autophagy itself and the nonindication ofautophagic flux at distinct time points this can be overcome by comparison studies with lc3bi and using lysosomal protease inhibitors antilc3b antibodies can be applied to detect autophagy invarious cell types application of antilc3b to glioblastoma tissuesdemonstrated a positive detection of lc3b levels both in vitro and invivo suggesting a latent monitoring system of lc3b severalstudies state that hyperoxic conditions can result in ali and ards thisfurther triggers the morphological biomarker of autophagy lc3bii toaccumulate thereby determining the fate of cell clearance experimentsperformed in hyperoxiainduced human bronchial epithelial cells andcultured epithelial cells beas2b clearly stated that the expression oflc3bii was high mediated by the apoptotic regulators similarexperiment carried out in the hyperoxiainduced lung injury in c57bl mice inferred the involvement of apoptotic pathways in the activationof lc3bii interaction of lc3bii with fas proteins was observedmarking the importance of lc3bii in the management of ali pulmonary hypertension is a main cause of copd manifestation inlungs that affects vascular architecture when chronic hyperoxia wasinduced in the lung tissue extracts of patients with pulmonarytable regulators of autophagy in lung injury and their mechanismregulatorslc3biibeclin p62hif1bnip3mtormechanismelongation of autophagosome and its maturation requires atg8map1lc3 protein lc3i combines with phosphatidylethanolamine forming lc3ii essential for autophagosome formationthe initiation of the isolation membrane that forms autophagosome after the sequestration process is regulated by beclin and pi3kp62 along with sqstm1 is the receptor for polyubiquitinated substrates it helps in the transportation of cargo into the autophagosome by bindingwith lc3ii for degradationduring hypoxic conditions hif1 induces bnip3 that in turn brings about cell survival through autophagy and provoke cell death by apoptosisthe mammalian homologue atg13 is phosphorylated by mtor that binds ulk1 proteins fip200 phosphorylates ulk and initiates the isolationmembrane formation in autophagyreference no 0cs vishnupriya hypertension the upregulation of lc3b prevailed indicating the regulatory role of lc3b in vascular cell proliferation and mediatingadaptive cellular responses smoking results in various implications of lung injury in due course a multimeric protein complexcomprising of lc3bcaveolin1fas occurs under basal state extensivestudies reveal that smoking triggers lc3b to initiate the dissociation ofcaveolin1 from fas protein thus facilitating apoptotic pathways accordingly emphysema a destructive expression of copd isworsened by cigarette smoking in vitro studies in lung tissues of miceon exposure to cigarette smoking ensured the driving role of lc3b inregulating apoptotic mechanisms and finally developing emphysemarespectively all these experiments prove the comprehensive bridgebetween autophagy and apoptosis highly regulated by the expressionlc3b lc3bi and lc3bii bind to microtubule associated protein1b map1b wherein overexpression of map1b decreases the levels oflc3bii protein kinase c is known to cease autophagy by interferingwith autophagosomal formation both in vitro and in vivo studiesconfirmed that the lc3b phosphorylation by protein kinase c takesplace consistently in lungs the emergence of autophagy either asa protective role or maladaptive response due to sepsis was studied in acecal ligation and puncture clp induced septic mice it was ensuredautophagy to be a protective response yet an overexpression of lc3bii in the later stages of sepsis leads to ali describing a maladaptive role lung injury can be provoked by ischemiareperfusion in whichautophagy is stated as the safeguarding mechanism by moderatelymaintaining the level of lc3bii also the ischemiareperfusioninduced lung injury is positively governed by the erk12 signallingpathway that regulates the cellular expression of lc3bii respectively nanoparticles of zinc oxide on exposure to lungs may induceali further zinc oxide nanoparticles resulted in the raise of autophagosomal structures followed by the accumulation of lc3bii proteinsthus zno nanoparticlesinduced ali is autophagy dependent 3methyladenine a classical autophagy inhibitor reduced the manifestationof lc3bii and lowered the release of zinc particles thereby stoppingzno nanoparticlesrelated toxicity of lungs similarly the artificially synthesized polyamidoamine dendrimers pamam used as aneffective drugdelivery system may sometimes result in pamam nanoparticlesinduced ali the levels of lc3bii biomarkers were highindicating the autophagic responses beclin beclin was first identified by beth levine as becn1atg6 inchromosome 17q21 in the year and it is the major autophagyregulating gene it is a coiledcoil protein of molecular weight kda comprising of amino acids and acts together with bcl2an antiapoptotic protein beclin is an indispensable autophagypromoting gene that is homologue to the mammalian yeastatg6 gene which regulates cell survival of different types and is involved in the constitution of autophagosomes the initiation ofthe anization of autophagosomes is regulated by class iii phsophoinositide 3kinase pi3k and autophagy related gene beclin beclin has got a novel bcl2 homology region3 bh3 domainthe bh3 domain in beclin1 can bind to bcl family proteins that initiateapoptotic signalling and prevents the beclin 1mediated autophagy byremoving beclin from hvps34 either phosphorylation or ubiquitination of beclin or bcl2 can disunite bcl2 from beclin andincrease the activity of vps34 kinase which brings about increase infunction during autophagy autophagyassociated protein beclin1 binds to lc3i that adapts to its membranebound form lc3iiand it cooperates with the ubiquitinbinding protein p62sequestosome sqstm1 the first an that fails during sepsis is lungs thefamiliar complications of sepsis are ali and ards ali activated various autophagy related proteins like lc3ii beclin and lysosomerelated protein lamp2 and rab7 expressions in sepsisinduced ali to evaluate the function of autophagy in severe sepsis an experiment is carried out using endotoxemia that frequently uses septiclife sciences shock and clp which is a clinical polymicrobial sepsis model herebecn1ˆ’ mice was susceptible to clpinduced sepsis in cysticfibrosis transmembrane conductance regulator cftr autophagy bybeclin overexpression cystamine or antioxidants and the restorationof beclin recovers the localization of beclin to endoplasmic reticulum and regresses the cf airway phenotype both in vitro and in vivoin scnn1btransgenic and cftr f508del homozygous mice and also inhuman cf nasal biopsies in lpsinduced ali there are threedistinct complexes of beclin1vps34 have been identified the firstcomplex contains beclin1 vps34 vps15 and atg14l second complexcontains beclin1 vps34 vps15 and ultraviolet irradiation resistanceassociated gene uvarag and the final complex contains beclin1vps34 vps15 uvrag and rubicon among these complex the onethat contains atg14l is concerned in the formation of autophagosomewhile others are in the autophagosome and endosome maturationbeclin forms the bridge in the recruitment of inducers and suppressorsof autophagy and simultaneously behaves as a key modulator in autophagosome formation mesenchymal stem cells mscs increases the translation level of beclin but not its transcription ratemscs might alleviate lpsali via downregulation of mir142a5p thatpermits pulmonary epithelial cells pecs to proceed with beclin mediated cell autophagy in lung disease the bacterial stu
Colon_Cancer
"autophagy is an evolutionary cellular program that serves for thebreakdown of cytoplasmic components within lysosomes [“] inidentified autophagy in's electron microscopic studies firstmammalian cells but the molecular pathways were not understooduntil the discovery of autophagy genes atgs in yeast by performinggenetic screening it is a cytoprotective rather than a selfdestructive process it is extensively accepted as a main regulator of innateand adaptive immune mechanisms the change in which completelyimpact the pathogenesis of disease and the processes that are influencedby autophagy includes the regulation of inflammation antigen presentation and bacterial clearance moreover autophagy aids in themaintenance of fundamental anelle populations such as mitochondria which is necessary for cellular bioenergetics and homeostasis homeostasis of the cell is been accomplished by maintaining the biosynthesis and turnoverthere are two broader protein degrading systems in eukaryoticcells first is the ubiquitinproteasome system responsible for the selective breakdown of most shortlived proteins and second is the lysosomalsystem the primary anelle called lysosome ineukaryotes is known for degradation through its acid hydrolases inunfavourable nutrient deprivation condition autophagy arbitrates aregulated phagocytosis via lysosomes autophagy is mediated byautophagosome that is thought to be an on selective degradation systemas it engulfs some of the cytoplasmic contents the ubiquitin“proteasome system concedes only ubiquitinated proteins for degradation andit marks a remarkable contrast to autophagosome process autophagosomes a doublemembered vesicle that engulfs durableproteins impaired anelles intracellular pathogenic anisms andtransports it to the lysosomes that are fused to form autolysosome andthe inner vesicle along with its cargo is been degraded at the time ofstarvation the remaining macromolecules are again recycled to thecytosol for reuse the precise mechanism in cargo recognition isuncertain but this process involves ubiquitination autophagicprocess is separated into distinct steps which includes induction recognition selection of cargo formation of vesicle then occurs the fusion of autophagosomevacuole followed by the degradation of thecargo and release into the cytosol various atg proteins are indulged inthis process and it consists of the central autophagic machinery autophagy encompasses different process by which cells delivercytoplasmicaredegradation theylysosomalsubstratesforŽ corresponding author at department of biotechnology psg college of arts and science civil aerodrome post coimbatore indiaemail address rashmiffrajangmailcom rr rasmi all authors deserve contribution equally101016jlfs2020118308received june received in revised form august accepted august available online august elsevier inc all rights reserved 0cs vishnupriya life sciences macroautophagy chaperonemediated autophagy cma and microautophagy all the three processes of autophagy are morphologically distinct in microautophagy the lysosomal membrane invaginations or protrusions are needed to seize cargo once thecargo is captured the uptake directly happens at the limiting membrane of the lysosome that includes intact anelles cma uses chaperones to sequester cargo proteins that have a pentapeptide motifthese proteins are unfolded and translocated directly across the lysosomal membrane via lamp2a receptor macroautophagy involves requisition of cargo vesicle formation and its subsequenttransport to the lysosome in recent years deletion of the autophagy related genes atg invarious model anisms has proved that autophagy plays decisive rolein adaptive responses to stress cellular differentiation and development an oncogenic event may establish by the partial minimization inthe autophagic capacity atg6beclin one of the phylogeneticallypremeditated autophagy genes is often subdued at one locus in humancancers studies in mice have shown that beclin is a haplo insufficienttumor suppressor autophagic programmed cell death was primarily depicted in actively developing tissues several conjugationsystems comprising of the atg genes are available that take part inautophagasomal elongation one such system is the lc3 microtubule associated protein light chain and atg8 conjugation system lc3 is the mammalian conjugative protein ortholog of yeastprotein atg8 the lipid derivative phosphatidylethanolamine bindsto lc3 to form lc3ii an important molecular marker of autophagylc3ii remains on the mature autophagosome until it fuses with thelysosomes fig the beclin complex gives rise to an incipientautophagosome membrane and it assemble around cargo in a vesiclethat combines with a lysosome forming autolysosome that is degradedby acid hydrolases present in the lysosomes lung injury clinical implicationsthe primary an of gas exchange is the pair of lungs theymediate inspiration of oxygen and elimination of mono and dioxides ofcarbon lung also serves as an attractive target for the entry of thepathogens regulation of the pulmonary functions is mediated by intricate cells of endothelial and epithelial lining dendritic cells alveolarmacrophages and fibroblasts all these pulmonary cells are highlyheterogeneous in nature they together in association respond to thelung injury by provoking inflammatory and immune responses airway epithelial cells express pattern recognition receptors prrsalong with toll like receptors ctype lectins rigi and inflammasome components which are involved in the innate response against microbes microbial cell wall constituents likelipopolysaccharide are sensed by prrs and induce an inflammatoryresponse alveolar epithelium comprises type i and type ii alveolarcells apart from these the mucus layer and the physical barrier madeby the epithelium contribute to the first line of defense lung injury is described as any damage to the associated tissues and compartments of the pulmonary system the concept of lung injury influenced by the microenvironment can be demonstrated via series ofchanges in cell deformability and manifestation of intercellular adhesive molecules the major causes of lung injury are atelectasisalveolar instability volutrauma barotrauma infections and oxygentoxicity the pathogenicity of acute and chronic lung injury iscorrelated with the release of proteases free radicals and growth regulatory proteins by the alveolar macrophages neutrophil takescare of the extreme ranges of lungs searching for pathogens therebyfig mechanism of autophagy ulk1pi3kmtor signalling pathway binds to endoplasmic reticulum and activates dcedp1 that initiates beclin and atg512conjugation system which forms isolation membrane this is followed by the sequestration process that leads to autophagosomal formation the autophagosomefuses with lysosomes to form autolyososome that leads to degradation 0cs vishnupriya eliminating via phagocytosis they undergo transendothelial andtransepithelial migration primary host defense mechanism oflungs include the immunity conferred by surfactant proteins spnamely spa and spd they link both innate and adaptive immunity byregulating the responses of innate immune cells antigen presentingcells apcs and tcells they can act as potential biomarkers of lunginjury the implication of autophagy over lung injury is tortuousas its function varies highly with the cell types specific to the pulmonary disorders it provides both defensive and injurious outcomes of lung injury lung injury “ types and associated pathologies acute lung injury ali and acute respiratory distress syndromeardsacute lung injury and acute respiratory distress syndrome arecommon grievous diseases among critically illpatients and are evidential sources of mortality and morbidity they are expressed alongwith hypercapnia and hypoxemia ards is outlined as the most seriousform of ali ali is characterized by inflammation of lung surfacesresulting in the disruption of alveolarcapillary membrane followed bytransmigration of neutrophils significant event in the procession of aliand ards and outbreak of cytotoxic mediators immune responsemediated by several components like tcells macrophages naturalkiller nk cells chemokines and proinflammatory cytokines also has apredominant role in the progression of the acute injury and results inimmune reconstitution inflammatory syndrome iris immune cellsinvolved in the immune response like macrophages and monocytes arethought to be involved in the pathophysiology of iris elevatedlevels of cd14 cd16ˆ’ monocyte population and an resulting increase in the proinflammatory cytokines il6 tnfα and c reactiveprotein crp are also observed in tuberculosis tbiris patients lung endothelial biomarkers like vwf and epithelial biomarkers likespd are the diagnostic targets of ali sepsis pancreatitis pneumoniatrauma transfusions aspiration and inhalation of toxic gases are someof the clinical factors of ali and ards histological patterns ofali and ards have the chances of demonstrating diffuse alveolar damage alveolar haemorrhage eosinophilic pneumonia and acute fibrinous pneumonia associated with ards may also result frompathogens like fungal viral bacterial and parasitic most commonpathogen strains include streptococcus pneumoniae respiratory virusesstaphylococcus aureus fungal pathogens like pneumocystis jirovecii andaspergillus fumigatus legionella pneumophila and enteric gramnegativeanisms among bacteria the virulence capability of pseudomonas aeruginosa is one of the prime determinant of the severity of thelung injury disease progress takes place in three degrees namelyacute phase exudative subacute phase proliferative and chronicphase fibrotic radiological features during the acute phasesignify twosided patchy groundglass densities relating to interstitialedema and hyaline membranes the important constituent of innateimmune system the pattern recognition receptors prrs are affiliatedwith the advancement of ali and ards the ligands of prrs includepathogenassociated molecular patterns pamps which induce inflammatory signalling events and the damageassociated molecularpatterns damps which induce neutrophilmediated tissue damageincreased incidences of mitochondrial damps result in high mortalityrates a common type of ali is the transfusionrelated acute lunginjury trali trali is defined as adult respiratory distress syndromeoccurring with transfusionsis manifested by pulmonary insufficiency severe hypoxia and dyspnea it is often reported as theneutrophil pmnmediated syndrome a particular study has reported that the potential risk factors for ali and ards may be associated with larger tidal volume vt and higher airway pressure pawduring one lung ventilation olv in postpneumonectomy aliardspatients and also in patients who developed aliards longtermeffects after acute lung injury are related to neurological impairmentsitlife sciences namely neuromuscular dysfunction neurocognitive dysfunction andneuropsychological dysfunction some of the minor impacts includeheterotopic ossification stiae and frozen joints chronic lung injury cli and bronchopulmonary dysplasia bpdchronic lung injury is characterized by a condition called pleuroparenchymal fibroelastosis ppfe in which a rapid multiplication ofsubpleural intestinal elastic fibres majorly in the upper lobes predominates along with other clinicopathological conditions following chronic lung injury cells undergoing apoptosis is cleared by aprocess called efferocytosis it is a type of phagocytosis confined only tothe cells that undergo apoptosis to maintain the cellturnover in pulmonary airways carried out mainly by the macrophages while immature dendritic cells mesenchymal cells and epithelial cells can alsoperform efferocytosis recently sarscov2 manifested as severerespiratory infection resulted in number of deaths worldwide commonrisk factors associated with death in sarscov2 patients identified arehypertension diabetes cardiovascular disease or chronic lung disease the most prevailing chronic lung injury ofinfants is thebronchopulmonary dysplasia bpd occurs when preterm infants suffering from various respiratory syndromes like meconium aspirationsyndrome and neonatal pneumonia are subjected to treatment withsupplemental oxygen and extensive mechanical ventilators it is proventhat the common risk factors of bpd increases with a fall in birthweight prematurity and gestational period apart from theabove other perinatal risk factors with which bpd is associated with areintrauterine growth restrictions race or ethnicity chorioamnionitis and genetic risk factors it is characterized bysaccular formation and elastic fibre aggregation of distal air spacesinflation and edema barotrauma and pulmonary oxygen toxicity arepathologies of bpd bpd is multifactorial in nature studiesdeclare that surviving patients of bpd have established pulmonarydysfunction incidence adults with bpd history are strictly prohibitedto cigarette smoking another form of bpd called the new bpdwhen surfactants are used as treatment new bpd is depicted by pulmonary hypertension and abnormalities in vasculopulmonary development glucocorticoids and tgfbeta are the efficient modulators thatinitiate bpd injury bpd infants have significantly lower levels ofvascular endothelial growth factor and platelet endothelial cell adhesion molecules chronic obstructive pulmonary disease copd and asthmacopd is a general illness worldwide it is defined as the completeirreversible state of airflow limitation characterized by weak inflammatory response of the lungs emphysema chronic obstructivebronchiolitis fibrosis blocking and narrowing of airways deprivationof lung parenchyma and elasticity immune cells like tlymphocyteswith cd8 dominance blymphocytes macrophages and neutrophilsare the regulators of copd acute provocation of copd is definedas the continuous deterioration from steady state facilitating an alteration in typical medication for rudimentary copd systemicinflammation responses are a result of leukotriene b4 tnfalpha interleukin il8 and proteases a decrease in the ratio of cd4 to cd8is a typical feature of pulmonary inflammatory responses additionalimpacts of copd are cardiovascular diseases nervous effects and osteoskeletal effects smoking is an important cause of systemicoxidative stresses immoderate inflammatory responses and emphysema as manifested as copd implications copd being an hetergoenousdisease patients with exacerbations are found to be associated withbacterial infections like moraxella catarrhalis streptococcus pneumoniaand haemophilus influenza apart from bacteria other microbes likevirus and fungi also comtribute to the lung microbiota and pathogenesisof lung microbiota through initiation of chronic inflammation it hasbeen found that bacteria and viruses fungi can promote local andsystemic inflammation that may contribute to the pathogenesis ofcopd specific biomarkers of copd observed are creactive 0cs vishnupriya protein crp il6 il10 and ccl18parc skeletal muscle losstakes place in copd wasting of the cell mass is evidenced to be theresult of tnfα participation in the pathogenesis of copd muscleglutamate reduction is linked with lactic acidosis in copd patientsending in muscle wastage similar to copd asthma is characterized by pulmonary obstruction and similar immune responses onlyvariation from copd is that the airway obstruction in asthma is reversible and it does not affect lung parenchyma dendritic cells are themodulators of th2 cells playing an important immune response ofasthma severe asthma is equal to the effects of copd illustrated by theincrease in neutrophils tumor necrosis factor tnf cxcl8 and decreased reception to corticosteroids while reversible copd is potentially to have subsequent asthma and copd ventilatorinduced lung injury vili and ventilatorassociated lunginjury valithe prevailing lung injury cases with ards when given ventilatorassistance mechanically in a clinical setup may develop additional lunginjuries ending up in ventilatorassociated lung injury vali similarlyin experimental models lung injury can be provoked by external application of injurious ventilation procedures contributing to ventilatorinduced lung injury vili thus vali can adversely aggravate thehealth of the ards patients at low lung volumes of ventilation atelectrauma occurs at high lung volumes of ventilation barotrauma andpulmonary edema occur biomarkers studied via experimental modelsof vali include several proinflammatory molecules like tnfα il1βil8 and antiinflammatory molecules like il10 il6 and stnfr1 respectively [“] positive endrespiratory pressure peep and tidalvolume applied have direct influence on vali and are to be studiedspecifically during ali and edema it is reported that peep when provokes overinflation the extent of edema also increases mechanism ofsurfactant inactivation is seen in alveolar microvessels with an increasein fluid filtration further greater the lung volume higher is thetransmural pressure in them a retrospective cohort study revealedthat height and gender of the patients should be taken into considerations along with establishing limitations to large tidal volumes beforesetting up a ventilator experiments explain that decrease in thepeak pulmonary arterial pressure or respiratory frequency can lessenthe grimness effects of vali it is proven that hypercapnic acidosisaffords protection to vili addressable implications of vali arebarotrauma volutrauma atelectrauma biotrauma and oxytoxic effects cellular pathology includes physical disruption of cells and tissuesand activation of cytotoxic responses leucocytes are raised to likelyinteract with the endothelium as the increasing intraalveolar pressurefastens the transit time of them inferences for current medicalpractices involve lungprotective ventilation survival of patients can beencouraged by prone positioning future clinical practices involveprecisioned ventilationindividualized tidal volumes using drivingpressure individualized peep and extracorporeal strategies pulmonary fibrosis pf and cystic fibrosis cfidiopathicpulmonary fibrosis ipf is a degenerative interstitial fibrosing disease prevalent worldwide it is also termed as cryptogenicfibrosing alveolitis a typical honeycomblike structure of asymmetricairspaces covered by dense fibrosis is observed ipf is followed by interstitial pneumonia which is featured by deficit inflammation and withabsence of homogeneous participation of lung tissues studies havebeen made since ages which revealed several inherited forms of pulmonary fibrosis mutations in various genes were associated to pfnamely sftpc sftpa2 tert terc and muc5b incidence withrheumatoid arthritis and scleroderma are more likely to form pf thereis a chance of clearance of alveolar basement membrane and occurrenceof hyperplastic epithelial cells ipf ensures migration of fibroblastsinto the fibrinrich exudates thus a chemoattractant activity is createdin the airspaces after lung injury this process is proven to be regulatedthe proby lysophosphatidic acid experiment proves thatlife sciences inflammatory cytokines il1β directly regulates the initiation of acuteand chronic inflammation making it a valuable target of ipf cystic fibrosis is characterized as a most common autosomal recessivegenetic disorder caused by the mutation in cftr gene transmembraneprotein genecystic fibrosis transmembrane conductance receptor pathology of the disease involves bronchiolitis obstruction of pathways endobronchiolar infection impaired ciliary actions atelectasisfinally leading to secondary alveolar injury bacterial infections of saureus p aeruginosa and h influenzae causes cf it is the pulmonarymacrophages and polymorphonuclear neutrophils that forms the defense against infections while lymphocytemediated mucosal injury isobserved in cf patients cf patients have increased flow of tnfαil8 and il1β submucosal glands of the lungs are the crucialhosts of cftr genes as the number expressed are high hence cfcontributes to epithelial airway lining abnormalities with respect to thechanges taking place in the submucosal glands respectively the macromolecular secretions of the submucosal glands have changes in theircomposition viscosity and greatly impacts on the mucociliary clearance radiationinduced lung injury riliseveral complications of the lung malignancies require treatmentsinvolving radiation therapy rt extensive reports claim that rt maylead to a state of rili the threedimensional dosimetric predictors canbe emphasized for the risk of symptomatic rili the alveolarcapillary subunit forms the most radiosensitive complex of the lungs thusthe rili is also called as the diffuse alveolar damage radiation exposure provokes the production of reactive oxygen species creatinghigh toxicity levels in the lung parenchyma this may ultimately lead tolung fibrosis which develops one to six months after rt accompaniedby dyspnea in addition to fibrosis rili also develops radiationinduced pneumonitis gradually after months to years almost all thepatients undergoing rt have the chance of developing fibrosis radiationinduced pneumonitis is characterized by cough occasional fevernonspecific symptoms of dyspnea and chest pain with or without deformities in pulmonary functional tests while radiationinduced pulmonary fibrosis is characterized by cough differential levels of dyspnea chest pain or symptomless and stable scarring of the lung tissueswhen detected radiographically it has been stated that the incidence and occurrence of rili is directly influenced by dose and volume determinants of rt several studies provide information onrili that result in provoking inflammatory responses a biphasicmanifestation of cytokines is observed in the lung tissues once exposedto rt one such study carried out to assess the cytokine productionin c3hhen irradiated mice revealed a spatial change in the expression of proinflammatory cytokines among various cellular compartments of the lungs further it was noted that the bronchoalveolar lavage cells responded immediately while the interstitial cells contributedonly in the later stages during pneumonitis profiling of cytokinesnamely the interleukins interferons monocyte chemotactic protein1tumor necrosis factors macrophage inflammatory proteins and granulocyte colonystimulating factors can be performed to analyse the developmental risks of symptomatic radiationinduced lung injury in vitro studies experiments state that the application of melatonin andcarnosine compounds reduced the reactive oxygen species and inflammatory cytokines produced following rili regulators of autophagy in lung injurynumerous regulators of autophagy play a vital part in the development of lung injury with regard to autophagy the following are theimportant regulators fig the mechanism by which these autophagy regulators act is shown in the table lc3biithe microtubuleassociated protein light chain lc3 is the 0cs vishnupriya life sciences fig figure depicts the autophagy regulators in regard to lung injury various conditions like hypoxia starvation environmental stress and cigarette smoke leadsto autophagy that in turn causes lung injury activation of class ipi3kmtor pathway takes place under hypoxic and starvation conditions while environmentalstress results in provoking beclin 1vps34 pathway that induces the sequestration of the phagophore formation cigarette smoke induces ros accumulation which inturn causes egr1 e2f signalling to activate lc3ii along with sqtm1p62 and atg512 conjugation system the ros generated may also leads to apoptosis theautophagosome fuses with lysosomes and forms autophagolysosome that causes pulmonary arterial hypertension and lung injuryprinciple autophagic protein expressed on the doublemembraned autophagosome nearly eight homologues of lc3 proteins are studied inmammals the amino acid composition of these homologues classifiesthem into two subfamilies first one comprising of lc3a splicingvariants lc3b and lc3c taking part in autophagosomal membraneelongation while the second one comprising of gabarap gabarapl1 gabarapl2 and gabarapl3 taking part in the maturation ofautophagosome generally lc3b occurs in cytosol as lc3bi by theproteolytic cleavage of cterminal of lc3b which then unites withphosphatidylethanolamine to form lc3bii to assemble on the autophagosomal membrane conjugation of phosphatidylethanolamine withlc3bii can be revoked by the activity of atg4 thus it is clear thatoccurrence of lc3bii is crucial in the process of autophagy lc3bii levels are analysed through immunoblotting while limitationsare degradation of lc3bii by autophagy itself and the nonindication ofautophagic flux at distinct time points this can be overcome by comparison studies with lc3bi and using lysosomal protease inhibitors antilc3b antibodies can be applied to detect autophagy invarious cell types application of antilc3b to glioblastoma tissuesdemonstrated a positive detection of lc3b levels both in vitro and invivo suggesting a latent monitoring system of lc3b severalstudies state that hyperoxic conditions can result in ali and ards thisfurther triggers the morphological biomarker of autophagy lc3bii toaccumulate thereby determining the fate of cell clearance experimentsperformed in hyperoxiainduced human bronchial epithelial cells andcultured epithelial cells beas2b clearly stated that the expression oflc3bii was high mediated by the apoptotic regulators similarexperiment carried out in the hyperoxiainduced lung injury in c57bl mice inferred the involvement of apoptotic pathways in the activationof lc3bii interaction of lc3bii with fas proteins was observedmarking the importance of lc3bii in the management of ali pulmonary hypertension is a main cause of copd manifestation inlungs that affects vascular architecture when chronic hyperoxia wasinduced in the lung tissue extracts of patients with pulmonarytable regulators of autophagy in lung injury and their mechanismregulatorslc3biibeclin p62hif1bnip3mtormechanismelongation of autophagosome and its maturation requires atg8map1lc3 protein lc3i combines with phosphatidylethanolamine forming lc3ii essential for autophagosome formationthe initiation of the isolation membrane that forms autophagosome after the sequestration process is regulated by beclin and pi3kp62 along with sqstm1 is the receptor for polyubiquitinated substrates it helps in the transportation of cargo into the autophagosome by bindingwith lc3ii for degradationduring hypoxic conditions hif1 induces bnip3 that in turn brings about cell survival through autophagy and provoke cell death by apoptosisthe mammalian homologue atg13 is phosphorylated by mtor that binds ulk1 proteins fip200 phosphorylates ulk and initiates the isolationmembrane formation in autophagyreference no 0cs vishnupriya hypertension the upregulation of lc3b prevailed indicating the regulatory role of lc3b in vascular cell proliferation and mediatingadaptive cellular responses smoking results in various implications of lung injury in due course a multimeric protein complexcomprising of lc3bcaveolin1fas occurs under basal state extensivestudies reveal that smoking triggers lc3b to initiate the dissociation ofcaveolin1 from fas protein thus facilitating apoptotic pathways accordingly emphysema a destructive expression of copd isworsened by cigarette smoking in vitro studies in lung tissues of miceon exposure to cigarette smoking ensured the driving role of lc3b inregulating apoptotic mechanisms and finally developing emphysemarespectively all these experiments prove the comprehensive bridgebetween autophagy and apoptosis highly regulated by the expressionlc3b lc3bi and lc3bii bind to microtubule associated protein1b map1b wherein overexpression of map1b decreases the levels oflc3bii protein kinase c is known to cease autophagy by interferingwith autophagosomal formation both in vitro and in vivo studiesconfirmed that the lc3b phosphorylation by protein kinase c takesplace consistently in lungs the emergence of autophagy either asa protective role or maladaptive response due to sepsis was studied in acecal ligation and puncture clp induced septic mice it was ensuredautophagy to be a protective response yet an overexpression of lc3bii in the later stages of sepsis leads to ali describing a maladaptive role lung injury can be provoked by ischemiareperfusion in whichautophagy is stated as the safeguarding mechanism by moderatelymaintaining the level of lc3bii also the ischemiareperfusioninduced lung injury is positively governed by the erk12 signallingpathway that regulates the cellular expression of lc3bii respectively nanoparticles of zinc oxide on exposure to lungs may induceali further zinc oxide nanoparticles resulted in the raise of autophagosomal structures followed by the accumulation of lc3bii proteinsthus zno nanoparticlesinduced ali is autophagy dependent 3methyladenine a classical autophagy inhibitor reduced the manifestationof lc3bii and lowered the release of zinc particles thereby stoppingzno nanoparticlesrelated toxicity of lungs similarly the artificially synthesized polyamidoamine dendrimers pamam used as aneffective drugdelivery system may sometimes result in pamam nanoparticlesinduced ali the levels of lc3bii biomarkers were highindicating the autophagic responses beclin beclin was first identified by beth levine as becn1atg6 inchromosome 17q21 in the year and it is the major autophagyregulating gene it is a coiledcoil protein of molecular weight kda comprising of amino acids and acts together with bcl2an antiapoptotic protein beclin is an indispensable autophagypromoting gene that is homologue to the mammalian yeastatg6 gene which regulates cell survival of different types and is involved in the constitution of autophagosomes the initiation ofthe anization of autophagosomes is regulated by class iii phsophoinositide 3kinase pi3k and autophagy related gene beclin beclin has got a novel bcl2 homology region3 bh3 domainthe bh3 domain in beclin1 can bind to bcl family proteins that initiateapoptotic signalling and prevents the beclin 1mediated autophagy byremoving beclin from hvps34 either phosphorylation or ubiquitination of beclin or bcl2 can disunite bcl2 from beclin andincrease the activity of vps34 kinase which brings about increase infunction during autophagy autophagyassociated protein beclin1 binds to lc3i that adapts to its membranebound form lc3iiand it cooperates with the ubiquitinbinding protein p62sequestosome sqstm1 the first an that fails during sepsis is lungs thefamiliar complications of sepsis are ali and ards ali activated various autophagy related proteins like lc3ii beclin and lysosomerelated protein lamp2 and rab7 expressions in sepsisinduced ali to evaluate the function of autophagy in severe sepsis an experiment is carried out using endotoxemia that frequently uses septiclife sciences shock and clp which is a clinical polymicrobial sepsis model herebecn1ˆ’ mice was susceptible to clpinduced sepsis in cysticfibrosis transmembrane conductance regulator cftr autophagy bybeclin overexpression cystamine or antioxidants and the restorationof beclin recovers the localization of beclin to endoplasmic reticulum and regresses the cf airway phenotype both in vitro and in vivoin scnn1btransgenic and cftr f508del homozygous mice and also inhuman cf nasal biopsies in lpsinduced ali there are threedistinct complexes of beclin1vps34 have been identified the firstcomplex contains beclin1 vps34 vps15 and atg14l second complexcontains beclin1 vps34 vps15 and ultraviolet irradiation resistanceassociated gene uvarag and the final complex contains beclin1vps34 vps15 uvrag and rubicon among these complex the onethat contains atg14l is concerned in the formation of autophagosomewhile others are in the autophagosome and endosome maturationbeclin forms the bridge in the recruitment of inducers and suppressorsof autophagy and simultaneously behaves as a key modulator in autophagosome formation mesenchymal stem cells mscs increases the translation level of beclin but not its transcription ratemscs might alleviate lpsali via downregulation of mir142a5p thatpermits pulmonary epithelial cells pecs to proceed with beclin mediated cell autophagy in lung disease the bacterial stu
Colon_Cancer
" national economies are increasingly facing the challenge of having to finance the prevention andtreatment of human diseases and of having to compensate for the resulting loss of economic production physicalinactivity is demonstrably closely related to the risk of developing certain disease group physical inactivity results indirect and indirect burdens that the present study intends to quantify in hungary for the period between and methods based on the data of the hungarian public finances this study determines the direct and indirect costsincurred by hungary due to illnesses and through the par method it quantifies the financial burden of physicalinactivity incurred by the hungarian treasuryresults the total financial burden of illnesses in hungary showed a decreasing tendency from to eventhough the year saw an increase in costs compared to similarly while total public expenditure on illnessesassociated with physical inactivity increased by when compared to the total amount attributable to medicalconditions stemming from physical inactivity still showed a decrease of billion huf in the overall period the biggesteconomic burden is posed by cardiovascular diseases hypertension and type diabetess the increase in the economic burden associated with physical inactivity can be attributed to thecombined effect of two factors changes in total expenditure on specific disease groups which showed an increase inthe period under review and changes in the physical activity levels of the hungarian population which showed animprovement over the period under review initiatives in hungary aimed at encouraging an active lifestyle fromchildhood onwards should be continued since “ beyond the initial impact that has already been felt to some extent inrecent years these initiatives will come to their full fruition in the coming decadeskeywords physical inactivity economic burden parmethod direct costs indirect burden population attributable risk the fundamental change towards a more sedentary lifestyle has a serious impact on people™s health physical inactivity is one of the most important global issues of thetwentyfirst centuryleading to an increased risk ofchronic diseases such as type diabetes cardiovascular correspondence davidpaaretkptehu1university of pecs faculty of health sciences pecs hungaryfull list of author information is available at the end of the disease certain types of cancer rectal colon breastobesity and osteoporosis these diseases may even become the cause of death the world health anizationwho has also identified these medical conditions as themost burdensome noncommunicable diseases of today™sdeveloped world regular moderate physical activity reduces the risk of the most common of these diseases andcontributes to an increased sense of wellbeing [ ] acinactivity ranks as thecording to the who physical the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0c¡cs bmc public health 20suppl page of fourth most significant mortality factor in the world with million deaths a year worldwide [ ]another study suggests that there is a lower likelihoodof health problems among people engaging in regularphysical activity than among those leading a sedentarylifestyle furthermore there is convincing evidence thatregular physical activity increases life expectancy and reduces the likelihood of developing coronary and cardiovascular problems of suffering a stroke or developingcolon cancer inactive and sedentary lifestyles directly affect metabolism bone mineral composition and magnify the healtheffects of cardiovascular disease furthermore thereis epidemiological evidence to suggest that a sedentarylifestyle increases the risk of cancer obesity metabolicand psychosocial problems according to oecd data the average life expectancyof hungarians at birth in was years which is years below the oecd average actually one of the lowest on the list for men this value is years forwomen years both showing an increasing trend in recent years the hungarian government has made anumber of efforts to bring about significant changes in theinactive lifestyle of the hungarian population these include measures to increase the number of physical education lessons and to improve the conditions in pe lessonsat school also the development and construction of sportsfacilitiesincreased funding for sports associations andeven the use of corporate tax incentives for sporting purposes while improving the conditions alone does notresult in a change in the attitudes of the population towards sport it is certainly a prerequisite procedures that quantify the burden on the hungarianeconomy resulting from physicalinactivity are one ofthe ways of measuring the effectiveness of state intervention [ ] this study aims to contribute to this bodyof research and proposes to analyse a longer timespectrummethodsto analyze the economic burden of physical inactivity weneed to start with the burden of diseases on the nationaleconomy as physical inactivity plays a vital role in the onset of several diseases and leads to various causes of deathat a national level diseases have direct and indirect costsdirect costs of diseases include treatments medications sickpay allowances and associated ancilliary coststhat are directly related to the illness the direct costs inhungary are mainly financed by the national health insurance fund nhif since it is called the national health insurance fund administration nhifa but we must not disregard the cost of sick leave and private costs outside of nhifnhifa financing the latterof which are directly borne by members of societyamong the indirect burdens we include items that constitute a loss to the economy or to society as a result ofthe loss of work caused by a disease there was a significant change in this area during the research periodwhile in and there was a longterm loss ofproduction only in jobs on the skillsshortage list or invery special cases by the number of job vacanciesin hungary reached thousand while by july thisnumber rose to thousand people which is of theworkforce our calculations were based on the following assumptions in and in a labor marketcharacterised by an oversupply of labor a frictional unemployment of months groupbased performance expectations and the market of goods and services beingoversupplied people on average worked days a yearand loss was calculated based on gdp per capita thestudy that inspired our calculations had a similarcalculation but we replaced its assumptions with theabovementioned assumptions and we broadened andtightened formulas and corrected data that had becomefact since then however when calculating the results we had to change the assumptions about the labormarket from the previouslyoutlined conditions as by hungarian labor market had become characterizedwith overdemand therefore we had to increase frictiontime as well monthsanother economic burden is presenteesim which isthe term used for the phenomenon when a sick individual goes to work which results in poorer performanceand thus loss of productionour main goalin this research was to quantify theeconomic burden of diseases for the years and and more specifically the costs to thenational economy directly attributable to physicalinactivity in the market years and during our research we treated as relevant secondarydata eurobarometer and and nhifnhifa data from and [“]in the course of our resreach we examined the typesof medical conditions related to physical inactivity andtheir possible complications the factual data for whichwas obtained from nhif and nhifawith the help of par method population attributable risk the most commonly used method in international research we were able to obtain quantitativemeasurements that were used uniformly in the analysisof data for all three yearspar ¼ pexp 02 rrˆ’¾ ¾ pexp 02 rrˆ’°°¾ 02 wherepexp prevalence refers to the section of the populationwhere a given risk is present 0c¡cs bmc public health 20suppl page of rr relative risk describes the risk associated with asedentary lifestylewhen using the index it is necessary to break downthe population into physically active and inactive sections and then by determining the relative risk rate wecan estimate the number and cost of illnesses stemmingfrom a physically inactive lifestyle the physical activity indicators ofthe hungarianpopulation showed fluctuations during the period underreview the situation was the worst in when wesaw of the population as physically inactive in ouropinion the health protection effect does not manifestitself in the case of those who never excercise or only doso “ times a month by this figure dropped to and at the same time the ratio of those engaging inexercise at least times per week increased threefold inthe following years a more negative trend was observed as the rate of inactive people rose to although this is still significantly better than the basefigure for fig with the help of metaanalysis we calculated the relative risk ratio rr an indicator which is prevalent ininternational literature in order to estimate the futureexpenditure stemming from physical inactivity for all affected disease groups such as cardiovascular diseasestroke hypertension colon cancertype diabetesosteoporosis depression gastrointestinal complicationsobesity high triglyceride diseases and deliberate selfharm [“]the rr is the proportion of the applicaple diseasesamong people with inactive lifestyles divided by the proportion of the applicable diseases among people with active lifestyles on the basis of the rr values it is possibleto quantify the par indicator by disease group for eachyear table in order to allow the data to be compared over timethe data on the burden of illnesses stemming from physicalinactivity for and was recalculated to prices while the total amount of burden imposedby illnesses was recalculated to prices using thefig the ratio of physical activity and inactivity in hungary in “ sourcespecial eurobarometer special eurobarometer specialeurobarometer 0c¡cs bmc public health 20suppl page of table the cumulative relative risk rate and par values for theexamined disease types in “disease typesheart and coronary diseasespar par rrpar strokehypertensioncolon cancertype diabetesosteoporosisdepressiongastrointestinal complicationsobesityhigh triglyceridesdeliberate selfharmsource katzmarzyk aldoori ewing andersen schuch domestic producer and consumer price index of thehungarian central statistical office hcso the economic burden ofresultsat pricesillnessesamounted to more than billion forints huf in of which the direct burden was billion forintsdirect costs accounted for of the burden of illnessesand the billion huf sickness benefit represented justover of total direct costs indirect burden representeda significantly lower percentage amounting to over billion huf the economic burden imposed by sicknessin was of hungary™s gdpby the economic burden of diseases fell to billion huf at prices direct costs accounted for of the total burden of illnesses that year less than of which amounting to billion huf was forsickness allowance expenditues indirect burdens decreased to billion huf the burden of sicknessamounted to of the gdp in by the economic burden of diseases fell to billion huf at prices direct costs accounted for of the total burden that year and of it weresick allowances amounting to billion forints indirectburdens fell to billion huf the burden of sicknessdecreased to of the gdp in by the economic burden of illnesses increasedcompared to but it was still below the initial figure huf billion and it decreased in comparison with the gdp the share of direct costs dropped significantly to within which the sickness benefitrepresented to the value of billion forints atthe same time indirect burdens increased significantlyto billion huf all in all the burden of sickness decreased to of the gdp in between and the economic burden of diseases fell by billion huf which is a total decrease of corresponding to an average annual decrease of and in the meantime the country™s gdp increasedsignificantly altogether at current prices obviously the decrease is due to a number of reasons butthe effect of the increase in physical activity is an important factor among them table in the years examined in the case of disease groupslinked to physical inactivity the burden of illnesses onthe state budget excluding sickness allowance amounted to billion huf and billion hufrespectively of which the lowest value was in however only a part of these can be directly linked tophysical inactivity as many other risk factors play a rolein the development of these diseases as regards therelative weight of each disease group cardiovascular disease is the biggest burden followed by hypertension atthe same time type diabetes was only ranked the fifthfor costs in the first year but by it became thethird largest item only slightly behind high blood pressure expenditure on stroke obesity and deliberate selfharm were almost negligible compared to other diseasegroups table based on the results it can be stated that in theexpenditures in the state budgetfor the diseasegroups examined drastically decreased by approximately billion huf compared to the initial starting positionof billion huf but by the expenditures hadsurpassed the base total from by more than billion huf compared to only type two diabetesand osteoporosis showed an increase and respectively compared to although the latter is dueto the relatively low total expenditure for all other disease groups the level of expenditure declined in absoluteterms resulting in a significant decrease of billionhuf in total expenditurehowever in the case of the picture is more varied if we examine the relative position of certain diseasegroups compared to type diabetes showed themost significant increase to the tune of more than billion huf the other diseases lag behind in terms ofexpenditure cardiovascular diseases and colon cancerare next with an increase of “ billion forints inaddition there is an increase in the costs associated withosteoporosis stagnation or decrease was observed forthe other disease groups but this could not compensatefor the increase in the costs of the aforementioned diseases the most significant drop in expenditure is observed in hypertension billion huf and hightriglyceride diseases billion huf table focusing on the direct burden of physical inactivitywe can conclude that “ of the total expenditure ofthe disease groups is directly attributable to physical 0c¡cs bmc public health 20suppl page of table economic burdens of diseases in hungary “ in huf million in real terms in direct costs statefinancedeconomic burdens of diseasesin hungary “medicationmedical aidsgeneral practitioner servicesdental servicesoutpatient carect mrimedical centers exluding vd clinicsdialysishome careinpatient carehighcost medical procedurespatient transportspa treatmentsgovernmental health careexpendituresick leavedisability rehabilitation treatmentcharged tonhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifnhifanhifnhifain totalprivate costsprivate health care expenditureindirect costsexpenditure associated withsick leavehealth insurance managementand other costsfriction due to sickness leading toloss of productionof which reduced pay due to sickpay and sick leaveof which tax loss for the statefriction due to disability leadingto loss of productionindividualemployernhifaemployer individualstateindividualstatesocietypresenteeism costsin totalemployerinactivity the major part is the cost of cardiovasculardiseases and hypertension and these were closelyfollowed by type diabetes by due to the factthat the total expenditure for stroke obesity and deliberate selfharm was also insignificant compared to otherdisease groups their expenditure related to physical inactivity is insignificant in the case of deliberate selfharm the costs cannot be measured even in the order ofone hundred thousand forints table compared to the decrease for the year ofthe direct costs stemming from physical inactivity is larger in proportion than the decrease of total expenditurethis is true of each disease group and for those twogroups type diabetes and osteoporosis where therewas an actual increase in costs the increase was less forrespectivelyphysical inactivityrelated expenses than for overall expenses the largest drop in monetary terms can be observed in the case of hypertension and cardiovasculardiseases over billion huf but there was a decreaseof and billion hufin hightriglyceriderelated diseases and colon cancer howeverpercentagewise nhif achieved the highest cost reduction for high triglycerides and colon cancer closely followed by a decrease in stroke expenditure and deliberate selfharm although inthe last two categories the low sum total spent alsomakes this decrease appear larger percentagewise thanwould be the case with larger totalscompared to the expenditure related to physicalinactivity in shows a decrease of billion huf 0ctotal amountinactivitytotal amountinactivitytotal amountinactivitycardiovascular diseasesstrokehypertensioncolon cancertype diabetesosteoporosisdepressiondigestive disordersobesityhigh triglyceridesdeliberate selfharmtotal¡cs bmc public health 20suppl page of table total cost incured by nhifa nhif of those disease groups that are associated with physical inactivity and costs directlyattributtable to physical inactivity itself in terms of prices million hufdisease typesat the level of the individual disease groups the amountsvary the most significant decline in absolute terms is inthe high blood pressure and high triglyceriderelated illness groups however the burden of type diabetes increased significantly and there was an increase in coloncancer and osteoporosis disease groups the direction andextent of the changes are mostly comparable to the totalexpenditure amounts at the overall level of the diseasegroups although the changes in the physical inactivity ratenaturally lead to differences in the specific values this isso much so that the total expenditure amounts increasedat the level of all disease groups by but overall theexpenditure related to physical inactivity shows a decreaseof table discussionwe can clearly conclude similarly to other international researches [ “] that physical activityand forms of recreational exercise have a protectiveeffect eg a preventive effect against certain types ofchronic diseases cardiovascularlocomotor disordersdiabetes and certain types of tumors the decreasein physical inactivity has a positive effect on productivity as fewer people avail themselves of sick leave atable changes in total expenditure as reported by nhifa nhif and changes in expenditure directly stemming from physicalinactivity compared to the base level expenditure in in real terms adjusted to prices million hufdisease typescardiovascular diseasesstrokehypertensioncolon cancertype diabetesosteoporosisdepressiondigestive disordersobesityhigh triglyceridesdeliberate selfharmtotaltotal amountˆ’ ˆ’ ˆ’ˆ’ ˆ’ ˆ’ ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ ˆ’ˆ’ˆ’ˆ’ ˆ’ˆ’ˆ’ ˆ’inactivityˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ˆ’ ˆ’ ˆ’ ˆ’ˆ’ ˆ’ˆ’ ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ total amountˆ’ ˆ’ˆ’ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ˆ’ ˆ’ˆ’ˆ’ˆ’ˆ’inactivityˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ˆ’ ˆ’ ˆ’ ˆ’ˆ’ ˆ’ ˆ’ ˆ’ˆ’ˆ’ ˆ’ ˆ’ 0c¡cs bmc public health 20suppl page of study of economic development over the past centuryhas concluded that the advancement of the population™s health status is responsible for about “ ofeconomic growth [“]in our comparative study we used four samplingpoints between and to demonstrate the burden of diseases at the level of the national economy forthe various loadcarriers in the period under review theeconomic burden decreased significantly overall from of the gdp to the weight of indirect burden increased however as in the currently demanddominated labormarket it is more difficult to replacelost workforce in the period of analysis the number ofemployees in hungary increased with which increased the amount of sick leave and number of sicknessdays but their gdp contribution was significantly higheralthough associated costs and burdens increased innominal terms they decreased in relation to the gdpa large part of diseases™ burdens are borne by the stateand society followed by households andemployers the proportions are similar to ding in european countries included hungary although we estimate that the burdens on employers arehigher and the burdens on households are lower inhungarysince the physical activity rate of the hungarianpopulation has been fluctuating but overall there is animproving tendency which is also apparent in the savings potential of the examined expenditures categoriescompared to the gdp the amount of spending dependsheavily apart from the physical inactivity rate on thenumber of employees as well as those people who arenot employed can not have sick leavefor exampleoverall government spending depends on the budget ofthe country which is connected to the overall economicsituationcardiovascular diseases accounts for most of the costof physical inactivity in hungary which coincides withmattli in switzerland however of directinactivityto depression inswitzerland while nhifa™s depression costs account foronly “ in hungaryattributablecostsarein hungary a number of measures have recently beentaken in order to integrate physical activity and sportinto people™s daily lives such measures include theintroduction of everyday physical education in schoolsor the extensive development of sports infrastructure however the effects of these measures will have amore pronounced effect in the long run several studieshave shown that high physical activity in childhood isnot yet measurable in terms of economic returns lessfrequent use of health care and a lower cost associatedwith using them as some effects “ such as the high costof sports injuries high rates of childhood illness “ haveresearch data confirm the facta negative bearing on the rate of return on investmenthowever a longterm change of attitude and opennessto physical activity at later stages in life are where thesemeasures bear a profit so any effort to support childinterhood sports is rewarding [ ] in additionnationalthatthoseparents who are themselves engaged in sport or currently do so are more likely to prefer sporting activitiesamong their children it is important to draw attentionto the fact even minimal physical activity has a healthimproving effect at any stage of life that is whysport and health policies at all times should promoterecreational activities for all ages not just young peoplewe would like to expand the scope of our current research if we could also examine how the patient numbers varied each year by disease group unfortunatelythe data was not available this would be of particularinterest for the year as the expenditure on illnessesshowed a significant increase in real terms compared to which may be due to the fact that more patientsreceived care and treatment but may also be due an increase of the normative provision per person by the government possibly to provide better quality careif we posit based on the eurobarometer data that thephysical activity rate improved compared to wecould also assume that fewer people were treated for theanalysed medical conditions in which would basically have a downward effect on total expenditure at thesame time however the picture is somewhat shaded bythe fact that even if the attitude of the population towards regular physical activity has changed in the lastfew years it is not certain that the number of illnesseswould decrease significantly in such a short period asthe negative effects of a sedentary lifestyle led for decades would not be easily offset by a few years ofexcerciseladen lifestyle this is especially true forolder age groups that is to say a reduction in the number of patients is not realised yet in patient care at thesame time the use of rapidlydeveloping medical technologies is also increasing the financial burden on thebudget as the higher costs of new technologies makemedical care per patient more expensive on the otherhandit should not be fotten that healing can bemade more effective and can lead to higher returns onhuman capitalthe study examined the development and compositionof direct and indirect burdens of disease in hungary andthe costs of physical inactivity to the state budget theseburdens fell in the examined periode which was associated with an increase of gdphowever there was an increase in the economic burinactivity which can beden associated with physical 0c¡cs bmc public health 20suppl page of attributed to the combined effect of two factors changesin total expenditure on specific disease groups whichshowed an increase in the period under review andchanges in the physical activity levels of the hungarianpopulation which showed an improvement over theperiod under review initiatives in hungary aimed at encouraging an active lifestyle from childhood onwardsshould be continued since “ beyond the initial impactthat has already been felt to some extent in recent years these initiatives will come to their full fruition in thecoming decadesabbreviationsct computed tomography gdp gross domestic product hcso hungariancentral statistical office huf hungarian forint mri magnetic resonanceimaging nhif national health insurance fund nhifa national healthinsurance fund administration oecd anisation for economic cooperation and development par population attributable risk rr relativerisk vd veneral disease who world health anizationacknowledgementsthe authors acknowledge to the nhifa™s colleagues for their help incollecting the dataset especially to mr zsolt kiss director general to drmihaly palosi head of department to petra fadgyasfreyler head ofdepartment and to valentina beitl analistthe authors would like to express their special thanks to prof attila fabianformer vice state secretary for his cooperative help during the datacollectionabout this supplementthis has been published as part of bmc public health volume supplement level and determinants of physical activity in the v4countries part the full contents of the supplement are available online athttpsbmcpublichealthbiomedcentralcomssupplementsvolume20supplement1authors™ contributionspa was the leader of the complete research coordinated the different coauthors™ work systematized the dataset summarised the literature related tothe relative risk ratios of illnesses calculated the par indices and contributedto the s dp has made calculations of par indices the direct costs ofphysical inactivity in the nhifa budget and contributed to the sfrom its results ms has made calculations of the total burdens direct andindirect of illnesses in hungary and contributed to the s from itsresults mh and psz summarised the related literature to the section ak and tsz have revised the results and contributed to the sall authors read and approved the final manuscriptfundingthe research was carried out and the publication costs funded by thesupport of hrdop36216201700003 cooperative research network ineconomy of sport recreation and health the authors declare that thefunding body does not have any role in the design of the study andcollection analysis and interpretation of data and in writing the manuscriptavailability of data and materialsthe data of the state financed direct costs that support the findings of thisstudy are available from national health insurance fund administration butrestrictions apply to the availability of these data which were used underlicense for the current study and so are not publicly available data arehowever available from the authors upon reasonable request and withpermission of national health insurance fundthe datasets of the private ind indirect costs used and analysed during thecurrent study are available from the corresponding author on reasonablerequestethics approval and consent to participatethe ethical approval was granted for the study by ethics committee ofuniversity of p©cs nr participants were informed about theresearch aim and methods before signing the informed consent form theinvestigation conforms to the principles outlined in the declaration ofhelsinkiconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1university of pecs faculty of health sciences pecs hungary 2university ofphysical education budapest hungary 3corvinus university of budapestcorvinus business school budapest hungaryreceived march accepted march published august referencessebestyen a boncz i molnar a korosi l kovi r kriszbacher i olah apentek m sandor j relationship between surgical intervention type and days mortality of elderly femoral neck fracture in the prsence of differentcomorbidities value health 2009123a66kruk j health and economic costs of physical inactivity asian pac j cancerprev “reiner m niermann c jekauc d woll a longterm health benefits ofphysical activitya systematic review of longitudinal studies bmc publichealth pratt m norris j lobelo f roux l wang g the cost of physical inactivitymoving into the 21st century br j sports med “ who global recommendations on physical activity for health switzerlandgeneva who blair sn cheng y holder js is physical activity or physical fitness moreimportant in defining health benefits med sci sports exerc s379“tremblay ms colley rc saunders tj healy gn owen n physiological andhealth implications of a sedentary lifestyle appl physiol nutr metab “rishiraj n inactivity a bad œhabit costing our productive lifestyle int j physmed rehabil oecd health status in edited by development ofecoa ¡cs p h©cz r pa¡r d stocker m a fitts©g m©rt©ke a fizikai inaktivit¡snemzetgazdas¡gi terhei magyarorsz¡gon k¶zgazdas¡gi szemle “ gabnai z m¼ller a b¡cs z b¡ba ©b the economic burden of physicalinactivity at national level [a fizikai inaktivit¡s nemzetgazdas¡gi terhei]eg©szs©gfejleszt©s health dev “ acs p stocker m fuge k paar d olah a kovacs a economic and publichealth benefits the result of increased regular physical activity eur j integrmed “ hcso in hcs o editor edn stadat time series of annual data labour market distribution of job vacancies koll¡nyi z imecs o az eg©szs©g“befektet©s budapest demosmagyarorsz¡g special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan powell ke population attributable risk of physical inactivity phys actcardiovasc health “katzmarzyk pt gledhill n shephard rj the economic burden of physicalinactivity in canada cmaj “ 0c¡cs bmc public health 20suppl page of aldoori wh giovannucci el rimm eb wing al willett wc use ofacetaminophen and nonsteroidal antiinflammatory drugs a prospectivestudy and the risk of symptomatic diverticular disease in men arch fammed “ andersen lb schnohr p schroll m hein ho allcause mortality associatedwith physical activity during leisure time work sports and c
Colon_Cancer
" it is estimated that around “ of patients with early stage colon cancer benefit from adjuvantchemotherapy we are currently not capable of upfront selection of patients who benefit from chemotherapywhich indicates the need for additional predictive markers for response to chemotherapyit has been shown that the consensus molecular subtypes cmss defined by rnaprofiling have prognostic andorpredictive value due to postoperative timing of chemotherapy in current guidelines tumor response tochemotherapy per cms is not known which makes the differentiation between the prognostic and predictive valueimpossible therefore we propose to assess the tumor response per cms in the neoadjuvant chemotherapy settingthis will provide us with clear data on the predictive value for chemotherapy response of the cmsscontinued on next page correspondence jpmedemaamsterdamumcnljhjm van krieken jnm ijzermans jp medema and m koopman havejoint last authorship3laboratory for experimental oncology and radiobiology center forexperimental and molecular medicine cancer center amsterdamamsterdam umc university of amsterdam meibergdreef azamsterdam the netherlands5oncode institute amsterdam umc university of amsterdam amsterdamthe netherlandsfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cberg bmc cancer page of continued from previous pagemethods in this prospective single arm multicenter intervention study patients with resectable microsatellite stablect3“4nxm0 colon cancer will be treated with two courses of neoadjuvant and two courses of adjuvant capecitabine andoxaliplatin the primary endpoint is the pathological tumor response to neoadjuvant chemotherapy per cms secondaryendpoints are radiological tumor response the prognostic value of these responses for recurrence free survival andoverall survival and the differences in cms classification of the same tumor before and after neoadjuvant chemotherapythe study is scheduled to be performed in “ dutch hospitals the first patient was included in february discussion patient selection for adjuvant chemotherapy in early stage colon cancer is far from optimal the cmsclassification is a promising new biomarker but a solid chemotherapy response assessment per subtype is lacking in thisstudy we will investigate whether cms classification can be of added value in clinical decision making by analyzing thepredictive value for chemotherapy response this study can provide the results necessary to proceed to future studies inwhich neo adjuvant chemotherapy may be withhold in patients with a specific cms subtype who show no benefitfrom chemotherapy and for whom possible new treatments can be investigatedtrial registration this study has been registered in the netherlands trial register nl8177 at “ wwwtrialregisternltrial8177 the study has been approved by the medical ethics committee utrecht mec18712keywords colon cancer consensus molecular subtypes neoadjuvant chemotherapy surgery colon cancer is one of the most common types of cancer in the netherlands with an incidence of around patients in approximately of patients present with local disease stage iiii curativesurgery followed by adjuvant systemic chemotherapy isstandard of care in patients with microsatellite stablemss highrisk stage ii and stage iii colon cancer despite this intensive treatment “ of the patients develop metastatic disease these patients do not benefitenough from the current adjuvant systemic therapymoreover it is estimated that will not develop metastases after surgery alone and are therefore overtreated with adjuvant chemotherapy identifying the patients at risk of developing metastases as well as thoseresponding to therapy is a clear unmet need in coloncancer care the development of new prognostic andpredictive markers for chemotherapy response is therefore of utmost importancemany efforts have been undertaken to stratify crc patients into biologically and clinically distinct subtypesone of these led to the development of the consensusmolecular subtypes cmss which is based on rna expression profiling of tumor tissue and which is currentlyconsidered to be the most robust molecular stratificationin crc cms1 is characterized by hypermutationmicrosatellite instability msi and strong immune infiltration cms2 the canonical subtype has marked wntand myc signaling activation cms3 is enriched forkrasmutations and shows evident metabolic deregulation cms4 the mesenchymal subtype is characterizedby prominent tgf activation stromalinvasion andangiogenesis activation subtyping in a large heterogeneous patient cohort n with stage i to iv colorectal cancer showed significant differences in prognosiswith cms4 as the poorprognosis subtype confirmingthe clinical relevance of the intrinsic processes implicated in each cms these results support the idea that the cmss mighthave predictive value for response to chemotherapy dueto the postoperative timing of chemotherapy in currentguidelines the tumor response to chemotherapy is notassessable which makes a distinction between the prognostic value and predictive value of the subtypes impossible only a randomized controlled trialin whichpatients would be randomized in either surgery plus adjuvant chemotherapy or surgery alone would make thisdistinction possible howeverthis causes ethical dilemmas because chemotherapy would be withhold in patients who might actually benefit yet a solid responseassessment per subtype is necessary for implementationin clinical decisionmaking we therefore propose totreat patients with two neoadjuvant and two adjuvantcourses of capox and determine the response in tumorresected specimensapplying neoadjuvant chemotherapy may have severaladvantages the possibility of response monitoring earlyeradication of micrometastases and more complete resections neoadjuvant treatment is already standard ofcare for different gi malignancies including esophagealgastric and rectal cancers [“] the foxtrot collaborative group was the first to set up a neoadjuvant trialin patients with locally advanced resectablecolon cancer and concluded that preoperative chemotherapy is feasible with acceptable toxicity and perioperative morbidity after this pilot studytheyconducted a randomized phase trial investigating theeffect of neoadjuvant chemotherapy in patients with act3“ n02 m0 colon cancer patients were randomized between weeks of neoadjuvant combined with 0cberg bmc cancer page of weeks of adjuvant folfoxcapox or weeks of adjuvant folfoxcapox neoadjuvant chemotherapywas safe with less major surgical complications significant downstaging and a reduced risk of incomplete resection although the primary endpoint of the studyfreedom from recurrent or persistent disease after years was not met the risk of a recurrence after yearswas reduced to with perioperative chemotherapycompared to with adjuvant chemotherapy onlyhr “ p in the proposed study we will investigate the predictivevalue of the cms classification on chemotherapy response in a neoadjuvant setting including pathologicalresponse and radiological response and their correlationwith rfs and os this allows us to determine therapyefficacy in individual patients and per subtypeobjectivethe primary aim of this study is the evaluation of thepathological tumor response to neoadjuvant systemicchemotherapy per cms in patients with mss high riskstage ii and stage iii colon cancermethodsstudy designconnection ii is a prospective multicenter interventional cohort study that will be performed as a substudyofthe prospective dutch colorectal cancer cohortplcrc plcrc is a nationwide cohort study of thedutch colorectal cancer group dccg facilitating scientific research to improve the outcome and quality oflife of patients with colorectal cancer we aim to include patients in “ dutch hospitals that participatein plcrcin connection ii patients with a mss ct3“4nxm0 colon tumor will be treated with two courses ofneoadjuvant and two courses of adjuvant capecitabineand oxaliplatin capox fig and table the cmsclassification will be determined on both the pretreatment biopsies and the resection specimen at least multiregion biopsies will be taken pretreatment toensure a sample with vital tumor and sufficient rnaquality tumor response will be assessed on the resection specimen using the tumor response grading trgsystem as proposed by dworak radiologicalresponse evaluation will be centrally performed by dedicated radiologists on sequential ct scans made at baseline and after two courses of neoadjuvant chemotherapybut before resection pathologists and radiologists will beblinded for the cms classificationoptionally blood samples are taken for circulatingtumor dna ctdna analysis and plasma storage atfour time points at baseline after neoadjuvant treatment after surgery and after completion of the adjuvantchemotherapy followup will be performed until years postsurgery data on local recurrences metastasesand survival will be documentedstudy populationpatients diagnosed with resectable ct3“4nxm0 coloncancer are eligible for the connection ii trial baselinectscans of all patients will be reviewed by dedicated radiologists in the treating hospitals with special focus ontumor staging msi status will be determined on biopsymaterial to exclude patients with an msi tumor patients are eligible when they meet the followingconsent for the connection ii studycriteria 0f able and willing to provide written informed 0f informed consent signed for plcrc components 0f mss based on pretreatment biopsy by immunohis 0f fit to undergo neoadjuvant chemotherapy with˜clinical data™ and ˜future studies™tochemistry ihccapecitabine oxaliplatin and subsequent surgeryjudged by the primary treating physician 0f adequate bone marrow liver and renal functionpatients will be excluded if any of the following criteriaare met 0f any other malignant disease within the preceding years apart from nonmelanotic skin cancerfig flow diagram of clinical study patients with an msi tumor will be excluded from this study at time points blood samples will becollected for ctdna analyses and future biomarker studies 0cweekcheck in and exclusionsign informed consentblood withdrawal for ctdna plasmactscansurgerycapoxrecord medical history xxx axxxcxbx fxgc1d1c2d1xxc3d1exxdc4dxxberg bmc cancer page of table study flowchart of clinical studystudy proceduresinclusion neoadjuvantsurgeryadjuvant chemotherapyfollow upchemotherapy xxxdocument concomitant medicationtherapiesa blood withdrawal may be done at screening or immediately before cycle day b blood withdrawal to be performed after cycle week and before surgeryc blood withdrawal to be performed before cycle day d blood withdrawal to be performed approximately weeks after surgerye cycle day should ideally start within “ weeks after surgery at the latest weeks after surgeryf ct should be performed after completion of cycle and before surgeryg surgery should ideally be performed “ weeks after cycle day but has to be performed weeks after cycle day xcarcinoma in situ and early stage disease with a recurrence risk of less than 0f colonic obstruction that cannot be defunctioned by 0f pregnant or lactating womena stomamain study parameterendpointthe primary endpoint is the pathological tumor responseto neoadjuvant chemotherapy per cms the pathologicalresponse will be centrally scored on hestained slidesfrom the resection specimen using the tumor responsegrading system according to dworak [ ] based on results from the foxtrot study a good response will bedefined as trg2 trg3 or trg4 poor response as trg1or trg0 the cms classification will be determined onthe pretreatment biopsies and on the resection specimens rna will be isolated from ffpe material and analyzed on the ncounter sprint profiler a reliable androbust platform for samples with degraded rna such asffpe samples [ ]secondary study parametersendpoints 0f additional pathological markers to assess the tumorresponse the modified ryan scheme trs andexpression of ki67 and caspase3 and morphological cytostaticcytotoxic effects on hestained tissue slides 0f pathological response per trg and trs categoryseparately for the different cms subtypes 0f radiological tumor response to neoadjuvantchemotherapy 0f recurrence free survival rfs at two and threeyears rfs is defined as the time elapsed betweenthe diagnosis of the primary tumour and either thedate of any recurrence of disease time of death orthe date of the last followup visit at which a patientwas considered to have no recurrence 0f overall survival os at five and ten years 0f therapyinduced cms differences 0f prognostic and predictive value of cytotoxiclymphocytes cytolym and cancerassociated fibroblasts caf infiltration scores 0f diagnostic accuracy of ctdna measurements formonitoring treatment response to neoadjuvanttreatment and detection of residual disease 0f exploration of proteome profiles for monitoringtreatment response to neoadjuvant treatment anddetection of residual disease 0f percentages of pathological complete r0pathological microscopic incomplete r1 andpathologically macroscopic incomplete r2resections 0f surgical complication rate ie wound infections andanastomotic leakstatistical analysisprimary study endpointthe primary study endpoint is the pathological tumorresponse per cms using the trg system according todworak pathologic tumor regression rates withcorresponding confidence intervals will be analyzedper cms subgroup using the wilson method 0cberg bmc cancer page of secondary study endpointscategorical data pathological tumor response accordingto the modified ryan scheme are compared using chisquare analysis or fisher™s exact test and are shown asnumbers relative and absolute rates continuous datacytolym and caf infiltration scoresradiologicaltumor response pathological response by percentage ofki67 and caspase3 positive neoplastic cells are compared using nonparametric ttest or mannwhitney utest where appropriate and are shown as mean andstandard deviation or median and interquartile range“ pvalues are twotailed and results areconsidered significantthe os at and years and rfs at and years willbe calculated and depicted by means of the kaplanmeier technique and will be compared using the stratified logrank test hazard ratios and confidence intervals will be calculated with a stratified coxproportional hazard analysis the rfs will be analyzedper cms subgroup per trg and radiological responseall estimates will be accompanied by confidenceintervalssample size calculationwe based our sample size calculation on the desiredprecision with which we will be able to estimate thepathological response rates to neoadjuvant chemotherapy within each cms subtype this precision is quantified by the margin of error the radius of the confidence interval which we set at a maximum of this margin of error is achieved with patients inthe least prevalent cms subgroup namely cms3 andan anticipated pathologic responses yielding a response rate of with a 95ci of “ based onthe currently observed ratios of subtypes derived fromthe large consensus dataset after exclusion of the msitumors which holds cms1 tumors for most part wewill need a total of mss patients cms2 cms3 cms4 with this sample size we anticipatemaximum margins of error of and forcms2 cms3 and cms4 respectively and overallthe above depends on the assumption that the response rates will not be higher than within eachcms subgroup if response rates will actually be closerto the maximum margin of error will increasethe sample size hence indicates that for the analysis patients will be needed for whom followup andsubtype is known we expect a loss in patients dueto loss of followupinsufficient quality of the biopsymaterial or failure to faithfully assign patients to a subgroup based on the rna expression profiles resulting ina total of patients needed to have sufficient data forboth the primary and secondary outcomesdata collection and data managementdata collection and data management will be performedby the netherlands comprehensive cancer anizationiknl they have broad experience with continuousdata collection based on high quality electronic case reportforms ecrfs which guarantees complete andtimely recording handling and storage of data and documents all local and central data managers are registeredand the electronic database trias is iso certifieddata will be documented in line with ˜good clinicalpractice gcp™ and dutch legal requirements major violations of the protocol will be recordedmonitoringno data and safety monitoring board dsmb will beassigned since patients are subjected to an interventionwith a low postoperative morbidity that is already beingperformed in routine clinical practice no interim analyses will be performedauditingindependent monitoring of the study is performed by aqualified monitor of iknl the monitor plan is basedon the judgement of the irb that study participation isof low to moderate risk monitoring will be performedby investigating the electronic trial database and performing site visits each participating site will be visitedat least once with repeat visits to sites where performance is a concern the quality assessment will focus onthe safety wellbeing and rights of the patients the quality of the documented data in the ecrf and their traceability to source documents and the completeness of theregulatory binder after each monitor visit the trialmonitor reports feedback to the project leader study coordinator and local investigatoradverse eventsthe treatment with capox in this study is standard ofcare therefore ae and sae are not expected to be different as both the treatment with capox and the surgery are part of the standard of care only two specificsaes are defined which are possibly related to the adjusted study schedule information will be collected onpatients who prematurely stop chemotherapy treatmentand of patients who are not able to undergo plannedsurgery due to progressive diseaseobstructionthe following two saes will be reported 0f if the surgery has to be postponed for more than 0f if patients can not complete all the neoadjuvantweeks after the start of cycle of capoxchemotherapy courses 0cberg bmc cancer page of the study coordinator will report these saes to thethat apaccredited institutional review board irbproved the study protocoldiscussioncolon cancer is one of the most common types of cancer in the netherlands the standard of care for patientswith mss high risk stage ii and stage iii colon cancercurrently consists ofsurgery followed by systemicchemotherapy patient selection for adjuvant chemotherapy is still far from optimal approximately wouldnever develop metastases after surgery alone and istherefore overtreated with adjuvant chemotherapymoreover “ still develop metastatic disease despite this intensive treatment leaving merely “ thatin fact benefit from adjuvant chemotherapy this illustrates the evident need for additional predictive markersfor chemotherapy benefitone potential marker is the cms classification whichis based on the integration of six different molecularclassification systems based on rna expression profiling the cms classification divides crc patients intofour subtypes with distinctive biological features guinney showed a clear relapse free survival and overallsurvival advantage for cms1“ compared to cms4 in aheterogeneous patient cohort with stage iiv crc withdivergent treatment schemes besides the prognostic value literature provides somesupport for a predictive value of cms for response tosystemic treatment in a retrospective analysis of thensabp c07 trial on patients n with stage iiicolon cancer only cms2 was associated with benefitfrom oxaliplatin treatment patients with cms4 tumorsdid not benefit from addition of oxaliplatin treatment the mesenchymal subtype showed no benefit from5fu monotherapy compared to no systemic therapy ina nonrandomized retrospective analysis of stage iiicrc patients although being a promising molecular marker a solidchemotherapy response assessment per subtype has notbeen performed and it remains unknown whether thedifference in longterm outcome between cms1“ andcms4 originates from differences in prognosis or response to therapythis makes it impossible to know whether patientswith the poorprognosis subtype cms4 have an impaired survival due to the aggressive nature of the tumoror due to a limited response to chemotherapy therefore it is unknown whether these patients should receivechemotherapy or not this also holds true for the othersubtypes although cms1“ show superior outcomes tocms4 it is unknown whether this is due to a favorabletumor biology or due to a substantial response tochemotherapy wesolidtherefore believethatachemotherapy response assessment per subtype is animportant and essential step to distinguish betweenprognosis and prediction and to incorporate the cmssin clinical decisionmakingadministering neoadjuvant chemotherapy in the suggested study population was proven safe and feasible inthe foxtrot study [ ] importantly the pathological tumor response was evidently associated with recurrence free survival patients with a complete responsetrg4 developed no recurrences after years of followup compared to of patients that showed no regression at all trg0 this illustrates that the responseto chemotherapy of the primary tumor may indeed be areliable measurement for chemotherapy efficiencythe primary endpoint of the proposed study is thepathological tumor response which will be centrallyscored using the trg by dworak a highly reproduciblescoring system which is often used and clinically meaningful evidently tumor response monitoring usinghistology requires invasive procedures as a secondaryendpoint radiological response will be scored by a central board of radiologists and compared to the pathologicaltumor response to evaluate this noninvasivetechnique as a response modality both the histologicaland radiological response will be correlated to rfs andos to assess their prognostic valueinvolvementthe proposed neoadjuvant approach requires reliableclinical tnm staging to minimize the risk of overtreating patients with stage i or low risk stage ii colon cancer a metaanalysis analyzing the accuracy of t and nstaging on ct imaging showed that t1“ can be reliablydistinguished from t3“ sensitivity and specificity while nodalis unreliable with apooled sensitivity and specificity of and respectively therefore only t stage will be used to selectpatients second only patients with an mss status willbe included which will be determined on the biopsiesfollowing the latest recommendations of the update ofthe esmo guideline to refrain from adjuvant chemotherapy in highrisk stage ii msi colon cancer patientsas the possible clinical benefit is too low this wasalso seen in the foxtrot trial where msi status wasassociated with a significantly higher rate of poorno response vs p using the proposed selection of patients with a mss ct3“4nxm0colon tumor up to of patients is estimated to beovertreated [ ]results from this study in which we analyze both thepathological and radiological tumor response per cmswill lead to improved patient stratification and clearerinsight into which patients benefit from chemotherapythis will allow us to identify the group of patients thatreceives chemotherapy appropriately and the group ofpatients that may not benefit from the current treatment 0cberg bmc cancer page of regimen future studiesshould focus on whetherchemotherapy can be withheld in this patient group oron the development of new therapies to improve patientoutcomeabbreviationscaf cancer associated fibroblast capox capecitabin and oxaliplatincms consensus molecular subtype ctdna circulating tumor dnacytolym cytotoxic lymphocytes dccg dutch colorectal cancer groupesmo european society of medical oncology mec medical ethicscommittee msi microsatellite instable mss microsatellite stable os overallsurvival plcrc prospective dutch colorectal cancer cohort rfs recurrencefree survival trg tumor response gradingacknowledgementsnot applicableauthors™ contributionsib sw jr gv rb1 cj se dv mk1 eh wg mo rb2 jk ji jm mk2 authors make substantial contributions toconception and design andor acquisition of data andor analysis andinterpretation of data authors participate in drafting the orrevising it critically for important intellectual content authors give finalapproval of the version to be published served as scientific advisorfundingthe connection ii trial is funded by the dutch cancer society alped™huzes the dutch cancer society is a nonprofit society that funds cancerresearch and has had no direct influence in the structuring of the trial andwill also not benefit financially from the outcomeavailability of data and materialsthe datasets used andor analyzed during the current study are availablefrom the principal investigator on reasonable request results will becommunicated via plcrc presentations at international conferences and viapublications in peer reviewed journalsethics approval and consent to participatethe study has been approved by the medical ethics committee utrechtmec18712 the medical ethics committee utrecht belongs to the umcutrecht and the prinses máxima center reference number slrc19009541the study will be conducted according to the principles of the declarationof helsinki 10th version fortaleza and in concordance with the dutchmedical research improving human subjects act wmo and otherapplicable guidelines regulations and actsauthorships will be defined following the international committee ofmedical journal editors guidelines the patients treating physicians local investigator or research nurse of theparticipating hospitals will follow ichgcp and other applicable regulationsin informing the patient and obtaining consent this includes explaining theconnectionii study to the patient providing himher with informationsuch as the expected efficacy and possible side effects and that refusal toparticipate will not influence further options for therapy before informedconsent may be obtained the investigator should provide the patient ampletime and opportunity to inquire about details of the trial and to decidewhether or not to participate in the trial all questions about the trial shouldbe answered to the satisfaction of the patient only after written informedconsent the patient will be included in this study the inclusion has to takeplace shortly after diagnosis to prevent delay in treatmentpatients are well informed that participation in voluntary and that they maywithdraw at any point during the studyconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interests nielsen t wallden b schaper c ferree s liu s gao d analyticalvalidation of the pam50based prosigna breast cancer prognostic geneauthor details1department of surgery erasmus mc university medical center rotterdamrotterdam the netherlands 2department of medical oncology universitymedical center utrecht utrecht university utrecht the netherlands3laboratory for experimental oncology and radiobiology center forexperimental and molecular medicine cancer center amsterdamamsterdam umc university of amsterdam meibergdreef azamsterdam the netherlands 4department of pathology radboud universitymedical centre nijmegen the netherlands 5oncode institute amsterdamumc university of amsterdam amsterdam the netherlands 6netherlandscomprehensive cancer anisation department of research utrecht thenetherlands 7department of medical oncology amsterdam umc locationvumc amsterdam the netherlands 8julius center for health sciences andprimary care university medical center utrecht utrecht university utrechtthe netherlands 9department of radiology the netherlands cancer instituteamsterdam the netherlands 10department of surgery university medicalcenter utrecht utrecht university utrecht the netherlandsreceived may accepted july referencesnederlandse kankerregistratie nkr iknl retrieved from wwwiknlnlnkrcijfers in may guinney j dienstmann r wang x de reynies a schlicker a soneson c the consensus molecular subtypes of colorectal cancer nat med“van hagen p hulshof mc van lanschot jj steyerberg ew van bergehenegouwen mi wijnhoven bp preoperative chemoradiotherapy foresophageal or junctional cancer n engl j med “bosset jf mercier m triboulet jp conroy t seitz jf surgical resection withand without chemotherapy in oesophageal cancer lancet “author reply cunningham d allum wh stenning sp thompson jn van de velde cjnicolson m perioperative chemotherapy versus surgery alone forresectable gastroesophageal cancer n engl j med “sebagmontefiore d stephens rj steele r monson j grieve r khanna s preoperative radiotherapy versus selective postoperativechemoradiotherapy in patients with rectal cancer mrc cr07 and ncicctgc016 a multicentre randomised trial lancet “roh ms yothers ga connell mjo beart rw pitot hc shields af theimpact of capecitabine and oxaliplatin in the preoperative multimodalitytreatment in patients with carcinoma of the rectum nsabp r04 j clinoncol foxtrot collaborative g feasibility of preoperative chemotherapy for locallyadvanced operable colon cancer the pilot phase of a randomisedcontrolled trial lancet oncol “ matthew t seymour dm and on behalf of the international foxtrot trialinvestigators foxtrot an international randomised controlled trial in patients pts evaluating neoadjuvant chemotherapy nac for colon cancerj clin oncol 20193715_suppl3504 coebergh van den braak rrj van rijssen lb van kleef jj vink gr berbeem van berge henegouwen mi nationwide comprehensive gastrointestinal cancer cohorts the 3p initiative acta oncol “ dworak o keilholz l hoffmann a pathological features of rectal cancerafter preoperative radiochemotherapy int j colorectal dis “ veldmanjones mh brant r rooney c geh c emery h harbron cg evaluating robustness and sensitivity of the nanostring technologiesncounter platform to enable multiplexed gene expression analysis ofclinical samples cancer res “ weissenberg e tnm staging of colorectal carcinoma ajcc 8th ed song n poguegeile kl gavin pg yothers g kim sr johnson nl clinical outcome from oxaliplatin treatment in stage iiiii colon canceraccording to intrinsic subtypes secondary analysis of nsabp c07nrgoncology randomized clinical trial jama oncol “lee j sohn i do ig kim km park sh park jo nanostringbasedmultigene assay to predict recurrence for gastric cancer patients aftersurgery plos one 20149e90133 0cberg bmc cancer page of signature assay and ncounter analysis system using formalinfixed paraffinembedded breast tumor specimens bmc cancer roepman p schlicker a tabernero j majewski i tian s moreno v colorectal cancer intrinsic subtypes predict chemotherapy benefit deficientmismatch repair and epithelialtomesenchymal transition int j cancer“ nerad e lahaye mj maas m nelemans p bakers fc beets gl diagnostic accuracy of ct for local staging of colon cancer a systematicreview and metaanalysis ajr am j roentgenol “ committee eg eupdate early colon cancer treatment recommendations murakami k west n westwood a hemmings g bottomley d davis j the relationship between dna mismatch repair and response to folfoxbased preoperative chemotherapy in the international phase iii foxtrottrial journal of patholog
Colon_Cancer
" exercise has been extensively utilised as an effective therapy for overweight and obesityassociatedchanges that are linked to health complications several preclinical rodent studies have shown that treadmillexercise alongside an unhealthy diet improves metabolic health and microbiome composition furthermorechronic exercise has been shown to alter hypothalamic and adipose tissue gene expression in dietinduced obesityhowever limited work has investigated whether treadmill exercise commenced following exposure to anobesogenic diet is sufficient to alter microbiome composition and metabolic healthmethods to address this gap in the literature we fed rats a highfathighsugar westernstyle cafeteria diet andassessed the effects of weeks oftreadmill exercise on adiposity dietinduced gut dysbiosis as well ashypothalamic and retroperitoneal white adipose tissue gene expression fortyeight male spraguedawley rats wereallocated to either regular chow or cafeteria diet and after weeks half the rats on each diet were exposed tomoderate treadmill exercise for weeks while the remainder were exposed to a stationary treadmillresults microbial species diversity was uniquely reduced in exercising chowfed rats while microbiome compositionwas only changed by cafeteria diet despite limited effects of exercise on overall microbiome composition exerciseincreased inferred microbialreduced fat mass and altered adipose andhypothalamic gene expression after controlling for diet and exercise adipose il6 expression and liver triglycerideconcentrations were significantly associated with global microbiome compositions moderate treadmill exercise induced subtle microbiome composition changes in chowfed rats but didnot overcome the microbiome changes induced by prolonged exposure to cafeteria diet predicted metabolic functionof the gut microbiome was increased by exercise the effects of exercise on the microbiome may be modulated byobesity severity future work should investigate whether exercise in combination with microbiomemodifyinginterventions can synergistically reduce diet and obesityassociated comorbiditieskeywords obesity microbiome exercise hypothalamus white adipose tissue cafeteria dietfunctions involved in metabolism correspondence mmorrisunsweduau1department of pharmacology school of medical sciences unsw sydneynsw australiafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cleigh nutrition metabolism page of introductionoverweight and obesity leads to reductions in physicaland mental health and quality of life resulting in increased direct and indirect costs to the global economy along with gross metabolic changes obesity is associated with altered fecal microbial species diversity and composition separate studies involving transferof obese human fecal microbiome samples induced fatgain in na¯ve mice and supplementation withakkermansia muciniphila improved insulin sensitivityand reduced body weightin overweight and obesepeople providing some evidence for a potential roleof diet and obesityassociated gut microbiota changes inadiposity and metabolic dysfunctionweight loss through lifestyle intervention is an effective strategy for reducing obesityrelated comorbidities one such intervention is moderate exercise a practical and sustainable approach for people with overweight and obesity while exercising at this intensityis unlikely to cause weight loss independent of caloricrestriction it confers cardiovascular and metabolic benefits and assists with weight maintenance [ ] furthermore regular exercise is known to improve glucoseregulation and insulin sensitivity as well as reducingcardiovascular disease and cancer risk andthere is increasing interest in the effects of exercise onthe gut microbiotathe first study to indicate an effect of exercise on fecalmicrobiome showed that elite professional athletes exhibited a distinct microbiome composition with increased microbial species diversity however sinceathletes consume a distinct diet from healthy people inthe community further work has been undertaken in rodents to identify the specific effects of exercise on fecalmicrobiome a recent systematic review of primarily rodent studies concluded that while there was no consistent effect of exercise on microbial species richnessexercise increases the relative abundance of firmicutes the different types of exercise used forced versusvoluntary have been shown to exert different effects onfecal microbiome composition in mice [ ] whichmay in part explain the inconsistent findingsfurthermore there were considerable differences inexercise duration used “ weeks which may contribute to the range of responses observed microbiomecompositional changes were observed in mice maintained on a healthy diet following weeks of moderatetreadmill exercise and after weeks in mice fed ahighfat diet in contrast eight weeks of lowtomoderateconfer microbiomecompositional changes in mice fed a highfat diet furthermore most studies examining the effects oftreadmill exercise on fecal microbiome in dietinducedobesity used a design wherecoexercise wasexercisedidnotadministered with highfat diet making it difficult totranslate the findings to people in terms of implementing exercise after a history of unhealthy eating andobesityhere we sought to examine whether moderate treadmill exercise in rats could exert benefits to gut microbiome composition following exposure to either ahealthy or a highfat highsugar westernstyle cafeteriadiet we aimed to investigate whether any changes ingut microbiome were associated with altered gene expression in white adipose tissue wat and the hypothalamus which are known to be affected by bothobesogenic diets and exercisematerials and methodssubjects and diet manipulationthis protocol was approved by the animal care andethics committee of unsw sydney in accordance withthe australian guidelines for the use and care of animalsfor scientific purposes national health and medicalresearch councilfortyeight male spraguedawley rats“ weeks g animal resource centre australia werehoused 3box °c h lightdark and handleddaily for one week while maintained on standard chow kjg premium rat and mouse maintenance dietgordon™s specialty stock feeds australia and water adlibitumfollowingacclimatization weightmatched groupswere randomly allocated to chow plus water or cafeteria diet n rats n cages per group ad libitumwhich comprised sucrosesolution alongsidecommerciallyproduced cakes cookies and savoury foods in addition to chow and water body weight and h food intake were measured twice weekly and food intake was calculated assuming equalintake per rat ineach cage body composition was measured during week by echomri900 echomri llc usacafsedcex cafeteria sedentarytreadmill exercisefollowing weeks half the rats in each dietary conditionwere allocated to treadmill exercise generating weightmatched groups chow sedentary csed chowexerciseandcafeteria exercise cafex for weeks until the day before sacrifice moderate exercise consisted of mmin for min five days a week at zero incline the firstweek comprised two training sessions “ mminafter which time spent at mmin was gradually increased sedentary rats were placed in stationary treadmills during the exercise protocol exercising rats wereclosely monitored three cafex rats showed signs of fatigue distress or vocalisation and were removed fromthe treadmill and thereafter exercised at a lower 0cleigh nutrition metabolism page of intensity mmin for min these rats were analysed together with moderately exercised cafex ratssample collectionat weeks of diet rats were deeply anaesthetized ketaminexylazine mgkg intraperitoneally bodyweight nasoanal length girth and blood glucose weremeasured following induction of anesthesia rats weredecapitated for trunk bloodthe hypothalamus within coronal block defined byrostrocaudal limits of circle of willis was rapidly dissected and collected retroperitoneal and gonadal watand liver were dissected and weighed one fecal pelletwas removed from the distal colon tissue and feceswere snap frozen in liquid nitrogen and stored at °cprotein and triglyceride measurementsplasma leptin and insulin concentrations were measuredusing commercial kits cat90040 and cat90060crystalchem inc usa plasma and liver triglyceridecontent were measured spectrophotometrically usingtriglyceride reagentroche diagnostics australia ptyltd australia at °c alongside a standard curvegenerated from glycerol standard g77935ml sigmaaldrich pty ltd australia livers were extracted byhomogenization in chloroformmethanol and incubated overnight nacl was added and samples werevortexed and centrifuged g for min the lowerphase was then evaporated at °c under nitrogen gasdried extract was redissolved in absolute ethanol andmeasured spectrophotometrically retroperitoneal watil6 content was determined using a duoset il6 ratelisa dy506 rd systems inc following manufacturers™ recommendationsstatistical analysesdata were analyzed using twoway betweensubjectsanova while measures over time were analysedusing 3way mixed anova posthoc pairwise comparisons were performed using a tukey adjustmentwhere appropriate thsd and presented in the associated figures and tables only when p pearson™scorrelations were used to identify associations allanalyses were completed using ibm spss statistics australiafecal dna extraction microbiome community sequencingand statistical analysesdna extraction was performed using the powerfecaldna isolation kit mobio laboratories usa microbial community composition was assessed by illuminaamplicon sequencing — bp miseq chemistry v4region 515f806r primer pair using a standard protocol sequence data were analyzed using mothur using modified commands from miseq sop including alignment with the silva database singletonremoval chimera checking with uchime and classification against the latest rdp training set sequence depthwas normalized by subsampling to total clean readsper samplecompletedusing calypso withotuoperationaltaxonomic unitcorrelationswerethebenjaminihochberg false discovery rate fdr procedure used to control for multiple tests fdrcorrected deseq2 was performed using the phyloseq r package for the negative binomial walk test indeseq2 otu abundances were analyzed usingspss with kruskalwallisfollowed by nonparametric bonferronidunn posthoc testing whereappropriate otus ofinterest were identified usingsina aligner testsrna extraction and gene expression assaysrna was extracted from hypothalamus and retroperitoneal wat using tri reagent sigmaaldrich pty ltdaustralia following dnase i treatment catalogue merck australia or μg of rna wat andhypothalamus respectively were reverse transcribed toproduce cdna high capacity reverse transcriptasekit thermofisher scientific usa gene expression wasassessed using taqman inventoried gene expression assays life technologies australia pty ltd australia seesupplementary table genes of interest were normalized against the geometric mean of the two most stablehousekeeping genes gapdh and hprt1 for wat hprt1and b2m for hypothalamus identified by the normfinder package analysis of relative gene expressionwas performed using the δδct method normalized toan independent calibrator made from all samplesdblmalpha diversity metrics distancedbased linear modellingpermutational multivariate anovapermanova nonmetric multidimensional scalingnmds and canonical analysis of principal coordinateswere completed using primer v6 primere ltdplymouth united kingdom all primer analysesutilized a braycurtis similarity matrix constructed atthe otu levelphylogenetic investigation of communities by reconstruction of unobserved states picrust was performed using galaxy web to predict putative functionsthrough metagenomic prediction from the 16s otudata using greengenes for taxonomic classification pathway counts were compared across groupsusing fdrcorrected kruskalwallis tests followed bynonparametric bonferronidunn posthoc testing whereappropriate 0cleigh nutrition metabolism page of resultsenergy intake body weight and composition and watgene expressionover the 7week study cafeteriafed rats ate more thantwice the energy consumed by chowfed groups fig 1acsed kjrat cex kjrat cafsed kjrat cafex kjrat when energy intake wasstratified into pre and duringexercise intervention asignificantinteraction between time and diet wasobserved fig 1b f p which appeared to be due to increased cafeteria diet intake whilerats were exercising although this comparison did notreach statistical significance significant interactions between time and diet were also identified for fat andfig shortterm treadmill exercise reduces body weight gain and fat mass and alters wat expression without affecting energy intake a energyintake over the study and b average weekly energy intake before and during treadmill exercise intervention c body weight of the study and dbody weight gain during treadmill exercise intervention e fat mass as a percentage of body weight and f absolute lean mass data expressedas mean ± sem n for cage data n “ for individual data data were analyzed by twoway anova followed by tukeyadjusted posthoctesting ap relative to csed bp relative to cex cp relative to cafsed 0cleigh nutrition metabolism page of carbohydrate intakes supplementary figure 1a f p and supplementary figure 1c f p respectively where both macronutrient intakes increased in the cafeteriafed rats during exercise protein intake while unaffected by time orexercise was elevated by cafeteria diet supplementaryfigure 1b f p sucrose intake increased over time supplementary figure 1d f p but was not affected by exerciseall rats gained body weight over time fig 1c andcafeteriafed rats gained significantly more than chowfed controls prior to f p and during fig 1d f p exercise exercisesignificantly reduced weight gain overall f p thsd posthoc comparisons showed thatcompared to csed and cex cafsed p and p and cafex p and p gained significantly more weight over the exercise intervention ofnote cafsed rats gained significantly more weight thancafex rats over the exercise intervention p body composition data following weeks of treadmillexercise showed that relative fat mass was significantlyincreased by cafeteria diet fig 1e f p absolute fat mass presented in table and reduced by exercise f p thsdposthoc comparisons revealed that relative fat mass inboth cafeteriafed groups were significantly greater thanboth chowfed groups lean mass however showed onlyan overall diet effect fig 1f f p with significantly more lean mass in cafeteriafed ratsthan chowfed ratscafeteria di so increased nasoanallength girthplasma insulin and triglycerides with no effect of exercise table plasma leptin levels were significantly increased by diet f p and reducedby exercise f p unfasted bloodglucose did not differ between groups table in line with plasma leptin retroperitoneal and epidydimal fat pad weights were significantly greater in cafeteria than chowfed rats f p andf p for retroperitoneal and epidydimal fat pads respectively and were significantly reduced by exercise overall f p and f p for retroperitoneal and epidydimalfat pads respectively see table microbial species diversity and microbiome compositionmicrobial species diversity was assessed using shannon™sdiversity index microbial species richness and microbialspecies evenness shannon™s diversity and evenness weresignificantly reduced by cafeteria diet overall f p fig 2a and f p fig 2c respectively and thsd posthoc comparisonsshowed that cafex rats exhibited reduced evenness relative to cex p and reduced shannon™s diversityrelative to csed p no significant differenceswere observed for bacterial species richness fig 2bmicrobiome composition at the otu level was signifipseudof p cantly affected by diet and cage pseudof p butnot by exercise when assessed using 4way permanova permutations and confirmed with nontable anthropometric measures at tissue collection and plasma measuresmeasurecafsedcsedcexterminal body weight gnasoanal length cmgirth cmliver scoreheart weight g ± ± ± 262ab ± ± ± ± ± ± ± ± cafex ± 124ab ± 01b ± 04ab ± 022ab ± 003ab ± 681ab ± 047ab ± 021ab ± ± 043ab ± 121ab ± 025ab ± 241abmain effects pvalueexercisediet interaction ± ± 06ab ± 017ab ± 007ab ± 1627ab ± 043ab ± 054ab ± ± 032ab ± 143ab ± 032ab ± 252ababsolute fat mass g echomri ± ± rp fat pad weight bw ± ± epidydimal fat pad weight bw ± ± blood glucose mmoll ± ± plasma insulin ngmlplasma leptin ngml ± ± ± ± plasma triglycerides mmoll ± ± liver triglycerides mgg tissueblood and plasma measures performed unfasted data expressed as mean ± sem n “ data were analyzed using twoway anova followed by posthocmultiple comparisons with a tukey hsd correction liver score was analysed using a kruskalwallis test followed by nonparametric bonferronidunnposthoc testingap relative to csed bp relative to cex ± ± 0cleigh nutrition metabolism page of fig see legend on next page 0cleigh nutrition metabolism page of see figure on previous pagefig impact of cafeteria diet and treadmill exercise intervention on fecal microbiota and inferred microbiome function at weeks a shannon™sdiversity b microbial species richness and c evenness data expressed as mean ± sem n “ data were analyzed by twoway anovafollowed by tukeyadjusted posthoc testing d nonmetric multidimensional scaling braycurtis permutations showing similarity betweenfecal microbiota samples at weeks e muribaculum_otu72 identified by deseq2 adjusted p as differentially abundant with exercise inchowfed rats f relative abundance of otu72 at weeks data expressed as boxandwhisker plots min iqr max n “ data were analyzedusing kruskalwallis test followed by nonparametric dunnbonferroni posthoc testing g amino acid metabolism h overall energy metabolism idglutamine and dglutamate metabolism and j one carbon pool by folate predicted using picrust from fecal microbiome data at weeks dataare expressed as boxandwhisker plots min iqr max n “ were analyzed by kruskalwallis tests fdradjusted overall pvalue to accountfor multiple relevant pathways included in analysis followed by nonparametric bonferronidunn posthoc comparisons posthoc symbols ap relative to csed bp relative to cex cp relative to cafsedmetric multidimensional scaling fig 2d supplementary figure shows groups differences in microbiomecomposition at the phylum leveldeseq2 analyses were used to identify otus differentially enriched with exercise exposure amongst the top otus while exercise was not associated with differentially expressed otus in cafeteriafed rats muribaculum_otu72 was significantly depleted in cex ratsrelative to csed fig 2e relative abundance in fig 2fthis otu was originally classified as akkermansia whenaligned with the rdp reference library muribaculum_otu72 was putatively identified as an unknown bacterium from the genus muribaculum using sina aligner alignment identitypredicted microbiome functionto determine whether the subtle microbiome composition changes observed with exercise affected microbiome function we inferred microbiome function usingpicrust following an fdr correction amino acidmetabolism fig 2g h p overallenergy metabolism fig 2h h p dglutamine and dglutamate metabolism fig 2i h p and one carbon pool by folate fig 2jh p exhibited overall group differences amino acid metabolism was significantly elevatedin cafex relative to csed p and cafsed p rats while the other processes were significantlyelevated in both exercised groups relative to csedwat and hypothalamic gene expressionexamination of wat inflammatory signaling andbrowning genes fig 3a revealed a significant interaction effect for ucp1 f p whilethsd posthoc comparisons showed that cafex ratsexhibited elevated ucp1 expression relative to cafsedp lep expression was significantly elevatedby cafeteria diet consumption f p while lepr expression was increased with exercise overall f p no significantdifferences wereproinflammatorymarkers or adipoq gene expressionobservedinhypothalamic gene expression was analyzed to determine if weeks of moderate exercise was sufficient toreverse dietinduced changes in expression of genes involved in feeding fig 3b blood brain barrier integrityand proinflammatory signaling fig 3c a significantinteraction effect was observed for npy gene expressionin the hypothalamus f p andthsd showed that cafsed rats exhibited downregulatednpy relative to csed p cex p andcafex rats p no significant differences wereobserved for pomc agrp npy1r lepr or insr crh geneexpression was significantly increased in cafeteriafedrats overall f p but was unaffected by exercise f p posthocthsd analysis revealed that cafex rats exhibited significantly upregulated crh relative to csed p andcex p both cln5 and glut1 were significantly increased incafeteriafed rats overall f p andf p respectively while no groupdifferences were apparent for cln5 using thsd comparisons glut1 expression was significantly elevated incafsed rats relative to csed controls p no significant differences were observed in ocln tjp1 or anyof the proinflammatory genes assessedassociations between variables of interest andmicrobiomewhen variables were assessed for their unique contribution to the variance in overall microbiome compositionseveral adiposity measures as well as retroperitonealwat il6 gene expression and hypothalamic crh andnpy expression were identified as significant predictorsof global microbiomecomposition supplementarytable when the contribution of variables of interestto overall microbiome composition was assessed whilecontrolling for diet and treadmill exercise both livertriglyceride concentration r2 p andretroperitoneal wat il6 gene expression r2 p were significant predictors of microbiomecomposition attable completemodel predicts of the variance in microbiomecompositionthe otu level 0cleigh nutrition metabolism page of fig gene expression in retroperitoneal wat hypothalamus and associations with gut microbiome changes a adipokine metabolic andinflammatory gene expression in retroperitoneal wat b feeding and stressrelated gene expression in the hypothalamus c bloodbrain barrierand proinflammatory gene expression in the hypothalamus adipoq adiponectin agrp agoutirelated protein cln5 claudin5 crh corticotrophinreleasing hormone glut1 glucose transporter il6 interleukin il10 interleukin10 il1b interleukin beta insr insulin receptor lep leptin leprleptin receptor npy neuropeptide y npy1r neuropeptide y receptor ocln occludin pomc proopiomelanocortin tjp1 tight junction protein tnf tumour necrosis factor ucp1 uncoupling protein data expressed as mean ± sem n “ data were analyzed by twoway anovafollowed by tukeyadjusted posthoc testing ap relative to csed bp relative to cex cp relative to cafsedporphyromonadaceae unclassified_otu106 wasincreased with cafeteria diet fig 4a h p and significantly associated with wat il6 geneexpression fig 4b interestingly this otu was alsocorrelated with hypothalamic npy genenegativelyexpression fig 4c il6 protein in wat exhibited asignificant interaction effect f p fig 4d and was positively associated with porphyromonadaceae unclassified_otu106 fig 4efollowing asimilar although nonsignificant trend to that observedwith il6 gene expression like il6 gene expression watil6 content was a significant independent predictor ofoverall microbiome composition supplementary table but was not a significant predictor in the final model 0cleigh nutrition metabolism page of table the shared contributions of diet exercise and variables of biological relevance on the variance observed in microbiomecomposition using distancebased linear modellingvariabledietpseudofpvaluessr2cumulative r2exerciseliver triglyceride contentwat il6 expressionsequential multiple regression captured by the braycurtis similarity matrix at the otu level max permutations involves interrogating the conditionalcontribution of each variable in order of entry into the model to determine whether variables contribute significantly to the variance explained in the presence ofother variables here diet and exercise conditions were added before any metabolic predictors were considered and the final model containing only statisticallysignificant covariates is shown metabolic predictors included in the sequential regression were selected based on their predictive value while trying to eliminatevariables with high covariance n “after controlling for diet and exercise sina alignerputatively identified porphyromonadaceae unclassified_otu106 as a strain of bacteroides eggerthii a gramnegative bacterium known to hydrolyze carbohydratesincluding simple sugars discussionwe found that weeks of moderate treadmill exercisereduced fat mass and plasma leptin concentrations andaltered wat expression ofsome adipokine andmetabolismassociated genes overall microbiome composition and microbial species diversity was changed bycafeteria diet but not by exercise however predictedmicrobial functions associated with metabolism were increased by exercise cafeteria dietinduced changes inhypothalamic npy and glut1 gene expression werereturned to control levels by exerciseexercise induced modest changes in gut microbiomecomposition that were statistically significant in chowfed rats only and the relative abundance of otu72 wassignificantly reduced in chowfed rats that exercisedcex rats relative to sedentary controls csed whilecafex rats exhibited reduced microbial species diversitythis reduction appeared dependent on cafeteria dietexposure rather than exercise to date work examiningthe effects of exercise on microbial species diversity hasproduced inconsistent results some rodent studieshave shown that exercise is associated with reductions infecal microbial species richness while others havereported no such effect after weeks of treadmill exercise we did not observechanges in overall microbiome composition our dataare in line with findings in humans showing thatchanges in microbiome composition are dependent onobesity status such that more severe obesity is associated with smaller effects of exercise since thecafeteria diet used here tends to produce a more severe metabolic phenotype than purified highfat diets[“] which to our knowledge have been used in allstudies investigating the interrelationship between exercise and diet on microbiome compositionthe dietinduced effects on microbiome composition here may bemore resistant to the effects of exercise than previouslyreported additionally a number of rodent studies report no differences in overall microbiome compositionwith exercise [ ] and there is evidence that this effect may be moderated by age which may have contributed to the inconsistent findings in the literaturepredicted microbial functions associated with metabolism specifically overall energy metabolism amino acidmetabolism one carbon pool by folate and dglutamineand dglutamate metabolism were increased in exercised rats this is in line with metagenomic resultswhere fecal microbiome from male elite athletes exhibited increased amino acid biosynthesis and overall energy metabolism relative to sedentary normalweightcontrols while picrust analysis produces predicted functional data unlike metagenomic analysis thisis an interesting finding that warrants followup to determine if and how exercise shifts the metabolic profileof the gut microbiome and whether any such shift is affected by exercise intensity and duration furthermoreconfirming these results across a range of diets would beuseful to determine whether the shift in microbial function with exercise is modulated by the macro andmicronutrients availablehere a moderate exercise intervention reduced fatmass and plasma leptin concentrations and increasedwat lepr gene expression in both exercised groupsand ucp1 gene expression in exercised cafeteriafedcafex rats uniquely increased ucp1 in wat depots isa marker of adipocyte beiging known to be promoted by exercise and is most likely related toexerciseinduced fat loss while there were no significanteffects of exercise on wat proinflammatory gene expression after controlling for diet and exercise wat il6expression wassignificantly associated with globalmicrobiome compositionwat is one of the major sources of il6 in obesehumans and mice [ ] which is a key component of the lowgrade systemic inflammation observedin overweight and obesity and is associated with 0cleigh nutrition metabolism page of fig relationship between otu106 and hypothalamic npy and wat il6 expression a relative abundance of otu106 data expressed as boxandwhisker plots min iqr max n “ data were analyzed using kruskalwallis test followed by nonparametric dunnbonferroni posthoctesting scatterplots for otu106 abundance and b il6 gene expression in wat and c npy gene expression in the hypothalamus showingoverall lines of best fit n d wat il6 protein content data expressed as mean ± sem n “ data were analyzed by twoway anovafollowed by tukeyadjusted posthoc testing e scatterplot for otu106 abundance and il6 protein content in wat showing overall lines of bestfit n ap relative to csed bp relative to cex cp relative to cafsedinsulin resistance wat il6 expression was stronglyassociated with the relative abundance of a strain ofbacteroides eggerthii otu106 a gramnegative sugarscavenging bacterium which is enriched in obesechildren relative to normalweight controls theassociations between wat il6 expression global microbiome composition and otu106 abundance are therefore likely to be due to the effects of cafeteria diet onboth adipose inflammatory processes and the gut microbiome however probiotic treatment with a strain of 0cleigh nutrition metabolism page of bifidobacterium in mice fed a highfat diet reducedwat macrophage infiltration and plasma il6 concentration indicating that changes to the gut microbiome may contribute to wat inflammatory signalingand il6 production furtherstudies determiningwhether specific bacterial species can modulate watil6 production are warranted as interventions thatcould reduce wat il6 expression in obesity may provide an avenue for preventing insulin resistance and type diabetesin contrast to the overall effect of exercise on watgene expression hypothalamic genes disrupted by cafeteria diet were typically normalized with exercise withno differences observed in chowfed rats npy wasdownregulated in cafsed rats as shown previously but normalized to control levels with exercise which isin line with other rodent work showing increased hypothalamic npy mrna and protein in response to bothacute and chronic exercise [“] hypothalamic glut1gene expression was increased in cafsed rats and reduced to control levels with exercise this is in contrastto studies showing that acute exercise increased glut1protein expression across the rat brain and prolonged exercise increases whole brain resting glucoseuptake in people further work investigating acuteand chronic exerciseinduced changes in glut1 expression in the hypothalamus and other brain regions isrequiredactivatescafeteria dietinduced crh upregulation in the hypothalam
Colon_Cancer
" prognostic markers play an essential role in the proper management of communityacquiredpneumonia this research work aimed to evaluate the association of rdw and or mpv with mortality andmorbidity in patients with cap to improve the yield of already used prognostic scoresresults the current study enrolled patients with communityacquired pneumonia cap out of them patients improved while died it was noticed that each of delta mpv and rdw p hadpositive significant correlation with psi and curb65 delta mpv and rdw was significantly higher in patients whodied ± vs ± p for delta mpv and ± vs ± p for rdw initial rdw and rising mpv during hospitalization for cap is associated with more severe clinicalcharacteristics and high mortality moreover the use of rdw and delta mpv in patients admitted with cap canimprove the performance of prognostic scaleskeywords communityacquired pneumonia red cell distribution width mean platelet volume communityacquired pneumonia cap is the fourthleading cause of death all over the world and plays animportant role of morbidity and mortality [“] scoringsystems have an essential role in the management of patients with cap and currently there are several severityscores in use such as pneumonia severity index psicurb65 however these severity scores have some limitations and variations for example the curb65 andcrb65 are crude scores for rapid assessment of thehighrisk patients while psi is believed to be useful foridentifying lowrisk patients therefore there is effort toimprove the prognostic value of these severity scores correspondence randaezzeldin98gmailcom32561department of chest diseases faculty of medicine assiut university assiutegyptfull list of author information is available at the end of the several biomarkers have been checked and verified foruse in cap which could improve the prognostic performance of severity scores [ ] however some ofthese biomarkers are nonspecific and not sensitiveothers are expensive and are not always available immediately mean platelet volume mpv is done as a routine laboratory test that is measured in complete bloodcount and it is considered a marker of platelet functionand activation [ ] a single elevated mpv measurement has been found to be associated with increasedmorbidity and mortality in various patient populations[ ] patients hospitalized with communityacquiredpneumonia cap are found to be at an increased risk ofdeath in the hospital and following discharge [“]the prognostic significance of mpv has been studied inonly two small studies on cap patients which werebased on single mpv determinations [ ] the clinical characteristics and prognosis oftimedependent the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 0cfarghly the egyptian of bronchology page of mpv changes have not been investigated in the cappopulation we hypothesized that the mpv may reflectplatelet activity and may be associated with an impairedhost response according to this hypothesis an increasing mpv may be associated with poor outcomes andmay predict inhospital mortality in icu patients withsevere pneumonia to test our hypothesis we examinedmpv alterations in patients with severe pneumonia whohad been admitted or transferred to the icured cell distribution width rdw is defined as a coefficient of variation of circulating red cells it is affectedby changes of red cell volume and is measured in theroutine complete blood count cbc few years earlyrdw used in the clinical practice to diagnose differenttypes of anemia moreover elevated rdw had a prognostic role in the outcome of some diseases like cardiovascular disease rheumatoid arthritis colon cancer andmetabolic syndrome [ ] furthermorefew researches have reported rdw as a prognostic predictorof mortality in different populations the exactmechanism of variation in rdw is still unknown but itis mostly associated with the process of oxidative stressand inflammation which reflects the prognostic role ofrdw to our knowledge rdw use does not implyany additional cost because it is routinely provided aspart of the whole blood count by hemocytometryseveral studies support the hypothesis that rdw maybe a useful parameter for gathering either diagnostic orprognostic information on a variety of cardiovascularand thrombotic disorders [ ] although the link between rdw and cardiovascular disease is unclear in recent years rdw has been associated with capoutcomes especially with 30day and 90day mortalityand complicated hospitalization [ ] this researchwork aimed to validate the role of rdw and mpv aspromising markers of mortality and morbidity in patients with cap to improve the yield of already used severity scoresmethodsthis prospective study included adult years ofage patients admitted to chest department and ricuof assiut university hospital with a diagnosis of capbetween october and october patients wereprospectively recruited within h of their arrival capwas defined as an acute disease with a radiological infiltrate that was not previously present and not due to another known cause and was associated with symptomsof lower respiratory tract infection exclusion criteriawere severe immunodepression hiv infection or severe hematological diseasesimmunosuppressivetherapy prednisone or equivalent dose of mg dailyfor weeks or any immunosuppressive regime therapy azathioprine cyclosporine cyclophosphamide andor other immunosuppressant drugs leukopenia leukocyte per mm3 or neutropenia neutrophils per mm3 andor chemotherapy in the previousyear pulmonary abscess radiological cavitation aspiration pneumonia and obstructive pneumoniapossible nosocomial origin days from hospital discharge and known active neoplasia all patientswere followed up during their hospital stay and thosewith a definitive diagnosis other than cap were excluded all of the patients were followed up until beingdischarged our study primary outcome variable was inhospital mortality of patients with cap the secondaryfig outcome of patients with cap 0cfarghly the egyptian of bronchology page of table baseline characteristics of both groupsimproved n died n ± ± p valuetable correlation between curb65 and psi in correlation todelta mpv and rdwdelta mpvrppsicurb65 rdwrp date expressed as r strength of correlation p significance of correlation pvalue was significant if mpv mean platelet volume rdw red celldistribution width psi pneumonia severity indexage yearsexmalefemalesmokingcurrent smokingnonestopped smokerexsmokerscomorbid diseasescurb65psiclassiiiiiivvlaboratory datardw delta mpvwbcs — 109lplatelets — 109lpao2 fio2hospital stay dayneed to mvtransfer to icuradiological findings ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ph pao2 urea nainhaled or oral corticoids and antibiotics were recordedon admission the following data were also recordeddays of duration of disease cap signs and symptomsfever cough sputum dyspnea pleural pain vital constants at the ed temperature respiratory and heartrates arterial pressure and oxygen saturation analyticdataglucose hematocrithemoglobin red cell distribution width rdw andmean platelet volume mpv number of lobes involvedand type of radiological condensation alveolar interstitial or mixed and complications respiratory cardiologic renal neurologic digestive and others noninvasive mechanical ventilation nimv need of icuand invasive mechanical ventilation imv psi andcurb65 scores were calculated for all patients all patients were admitted to the hospital for ‰¥ hsample collection and processingblood was collected from anticubital fossa by experienced phlebotomists using a standardized atraumaticprotocol using clean venipuncture and minimum tourniquet pressure needles used were “ gauge specimens were maintained at room temperature “ °cnot placed on ice refrigerator or water bath tubes keptcapped upright at room temperature not exposed to vibration excessive mixing or agitation specimens containing any evidence of clotting were discarded twosamples of ml of venous blood in standard tubes contain ethylenediamine tetraacetic acid edta anticoagulant for complete blood count cbc the first one atthe time of admission and the second one after daysof admission the cbc sample was examined within has recommended by bcsh guideline to avoidbias due to excessive platelet swelling and to minimizevariation due to sample aging mpv and other bloodcount parameters were measured by an automatedanalyzer advia 2120i jermany with lh controlsystem in our laboratory the range of normal mpvvalues is “ fl for analysis of timedependentmpv changes patients were categorized according toδmpv mpv on discharge minus mpv on admissioninto patients with no rising mpv δmpv fl andpatients with rising mpv δmpv ‰¥ fl rdw wasunilobar pneumonia multilobar pneumonia effusion positive blood culture positive sputum culture data expressed as mean sd frequency percentage p value was significantif mv mechanical ventilation icu intensive care unitoutcome variables were length of hospital stay intensivecare unit icu admission and mechanical ventilator requirement this study protocol was approved by thelocal ethics committee and informed consent was obtained from all patients or next of kindata collectionage sex tobacco and alcohol consumption comorbidconditions diabetes liver chronic kidney hearth andcerebrovascular diseases nonactive neoplasia bronchiectasis and previous cap and previous therapies 0cfarghly the egyptian of bronchology page of fig correlation between delta mpv and psireported as a part of the cbc results rdw is the standard deviation of mcv and was measured in as percentage a single rdw had been recorded from cbc ofpatients on admission in our laboratory the range ofnormal rdw values is statistical analysisdata was collected and analyzed those using spss statistical package for the social science version ibmand armonk ny continuous data was expressed inform of mean ± sd or median range while nominaldata was expressed in form of frequency percentageindependent risk factors of mortality had been determined by multivariate regression analysisreceiver operator curve was used to determine cutoffof rdw and delta mpv for prediction of inhospitalmortality in patient with communityacquired pneumonia pearson correlation was used to determine correlation between psi and curb65 with rdw and deltampv level of confidence was kept at hence pvalue was significant if resultsthe currentstudy included with communityacquired pneumonia cap out of them patients improved while died as shown in fig the characteristic data of the enrolled patients are summarized in table mean age of improved patients was ± years majority of them were malesand of them were smokers mean age of patients who died was ± years and ofthem were females it was noticed that patientswho died were smokers it was noticed that patients who improved and patients who diedhad coexisting comorbidities with significant differencesbetween both groups it was noticed that class of psi wasii iii iv and v in and respectively of improved patients and in and of patients died respectively mean psi was significantly higher in patientswho died ± vs12339 ± p moreover curb65 was significantly higher in patientswho died in comparison to improved patients ± vs ± p fig correlation between delta mpv and curb65 0cfarghly the egyptian of bronchology page of fig correlation between rdw and psidelta mpv and rdw was significantly higher in patients who died ± vs ± p fordelta mpv and ± vs ± p for rdw our research also detected that comorbiddiseases transfer to icu and need for mechanical ventilation were highly frequent in patients who died moreover patients who died had longer duration of hospitalstay on radiological findings pleural effusion and unilobar pneumonia were presented in and ofpatients who died vs and of improved patients respectively while multilobar pneumonia wasmore frequent in patients who died blood culture waspositive only in of patients who improved vs of patients who diedthe current study also discovered that each of δ mpvand rdw had positive significant correlation with psiand curb65 p as shown in table and figs and based on the current study table the following variables were predictors of inhospital mortality in patients withcap with adjusted r2 was curb65 or ci “ p psi or ci “ p rdw or ci “ p delta mpv or ci “ p it was worthwhile to notice that rdw at cutoff point had sensitivity and specificity for predictionof mortality in patient with cap with area under curve while delta mpv at cutoff point had sensitivityand specificity for prediction of death in patient withcap with area under curve as shown in table andfig discussionin the recent few years cap had been considered one ofthe leading causes of death worldwide thereforefig correlation between rdw and curb65 0cfarghly the egyptian of bronchology page of table predictors of inhospital mortality in patients with capp valueodd™s ratioage confidence interval“““““““sexcomorbiditiescurb65psirdwdelta mpvp value was significant if mpv mean platelet volume rdw red celldistribution width psi pneumonia severity index capcommunityacquired pneumoniaaugmentation of the conventional severity scoreslikethe psi and curb65 became a must to identify patients with high risk for a complex course as the predictive performance of these scores alone may be limitedseveral researches have detected that discovering newbiomarkers could augment the validity of these severityscores [ ]in this prospective study we planned to assess the validity of the mean platelet volume change and rdw asbiomarkers for assessing the severity of cap it wasworthwhile to know that no previous study has beendone at assiut university hospital to assess those twobiomarkers in patients with cap the main potentialmechanism for rising mpv in patient population is severe inflammation caused by pneumonia in severe infection increased release of thrombopoietin and variousinflammatory cytokines such as interleukin1 and and tumor necrosis factorα result in increased thrombopoiesis and enhanced expression of younger largeplatelets into the blood circulation [ “] on theother hand rising mpv may be attributed to increasedthrombocyte consumption in the peripheral tissue andspleeninduced by severe inflammatory status [ ] communityacquired pneumonia is an infectiousdisease that results in inflammatory and oxidative stresstable performance of rdw and delta mpv in prediction ofmortality in capsensitivityspecificitypositive predictive valuenegative predictive valuecutoff pointrdw delta mpv area under curvep valuep value was significant if rdw red cell distribution width mpv meanplatelet volume cap communityacquired pneumonia to the host if these stresses are severe mortality will beincreased the underlying pathophysiological mechanisms for a relationship of rising mpv with poor prognosis and mortality are not fully understood the mainpotential explanation can be increased platelet activation[ ] larger thrombocytes are known to be functionally metabolically and enzymatically more active thansmaller ones the greater activation of enlarged plateletsresults in increased release of procoagulant substancessuch as thromboxane a2 βthromboglobulin and surface proteins [ ] consequent hyperaggregability ofplatelets extended vasoconstriction and endothelial dysfunction may contribute to an increased shortterm riskof cardiovascular thrombosis and death in patients withrising mpv [ ]our research detected that a high rdw and risingmpv were significantly related with increased risk ofdeath in patients with cap as delta mpv and rdw wassignificantly higher in patients who died ± vs ± p for delta mpv and ± vs ± p for rdw our results are inagreement with braun who detected thatrdw was associated with high risk of death and disability in young patients admitted with cap in this retrospective study brawn in a cohort of patients of years or older hospitalized due to cap demonstratedthat elevated rdw was independently associated with complicated hospitalization length of stay days and admission to icu and 90day mortality irrespective of hemoglobin levels in line with the results of our study lee also identified a high rdwis a prognostic factor for 30day cap mortality ageis significant prognostic factor in various diseasesincluding cap in our study the mean age of improvedpatients with cap was ± years while meanage of patients who died was ± years thusour findings support rdw as a significant prognosticfactor in patients with cap across all ages these resultsare in agreement with braun their finding is similar to our results however they only includedpatients who were younger than years which theycited as a limitation of their study this study revealedthat each of psi and curb65 had positive significantcorrelation with delta mpv and rdw p ourresults are also in line with gorelik whostated that rising mpv during hospitalization for cap isassociated with a more severe clinical profile and mortality than no rise in mpv they found that mpv valuesabove the cutoff at discharge were associated with anincreased risk of mechanical ventilation and death during hospitalization and shortened survival following discharge based on the current study the following variableswere predictors of inhospital mortality in patients with 0cfarghly the egyptian of bronchology page of fig diagnostic performance of delta mpv and rdw in prediction of inhospital mortalitycap with adjusted r2 was curb65 or ci “ p psi or ci “ p rdw or ci “ p delta mpv or ci “ p indeed in our patient population a risein mpv was associated with higher pneumonia severityscoresin our study the mortality prediction of both the psiand curb65 was improved by the addition of rdwand delta mpv as severity criteria the exact mechanisms of an association of rdw with the mortality ofcap need to be determined it has been suggested thatinflammation and oxidative stress affect red cell homeostasis a previous study revealed that rdw displayed astrong graded association with inflammatory biomarkersin general outpatient populations and anotherstudy indicated that serum antioxidant levels includingselenium and carotenoids were associated with rdw inolder women in our data patients with a higherrdw had a tendency toward higher severity indexscores and the overall mortality was also higher in patients with a higher rdwthere are some limitations of our research work firstthe study included one group of cap patients who wereadmitted in our assiut chest department and ricuthus it cannot be generalized to all patients with capsecond larger studies of large numbers of patients needto be done to investigate the value of mean plateletvolume change and rdw as prognostic markers incommunityacquired pneumoniasrdw and rising mpv during hospitalization for cap isassociated with more severe clinical characteristics andhigh mortality we suggestthat repeated mpv andrdw determination throughout hospitalization may improve risk stratification for cap patientsabbreviationscap communityacquired pneumonia cbc complete blood count crb confusion respiratory rate blood pressure age ‰¥ curb65 confusionurea respiratory rate blood pressure age ‰¥ δmpv mean platelet volumechange ed emergency department icu intensive care unit rdw blood celldistribution widthacknowledgementsto all work team who do their best to do this research in a perfect way andto all patients involved in this study and their parentsauthors™ contributionssf and ra carried out historytaking and physical examination of all participants in addition to obtaining blood samples participated in the sequencealignment and drafted the manuscript they also analyzed and interpretedthe patients™ data re and da carried out laboratory investigations and participated in the revision of the manuscript all authors read and approved thefinal manuscriptfundingno fund was taken from any institute or companyavailability of data and materialsdata and materials are available 0cfarghly the egyptian of bronchology page of ethics approval and consent to participatethe study obtained approval from the ethical committee at the faculty ofmedicine assiut university and a written consent was taken from theparticipants no reference number is usually given for approved studies inour universityconsent for publicationconsent for publication was taken from all authorscompeting intereststhe authors declare that they have no competing interestsauthor details1department of chest diseases faculty of medicine assiut university assiutegypt 2department of clinical pathology faculty of medicine assiutuniversity assiut egyptreceived april accepted august references mandell la wunderink rg anzueto a infectious diseasessociety of americaamerican thoracic society consensus guidelines on themanagement of communityacquired pneumonia in adults clin infect dis44suppl 2s27“s72almirall j bolibar i vidal j epidemiology of community acquiredpneumonia in adults a populationbased study eur respir j “armstrong gl conn la pinner rw trends in infectious diseasemortality in the united states during the 20th century jama “ menendez r martinez r reyes s biomarkers improve mortalityprediction by prognostic scales in communityacquired pneumonia thorax“lee jh kim j kim k albumin and creactive protein haveprognostic significance in patients with communityacquired pneumonia jcrit care “chu sg becker rc berger pb mean platelet volume as apredictor of cardiovascular risk a systematic review and metaanalysis jthromb haemost “noris p melazzini f balduini cl new roles for mean platelet volumemeasurement in the clinical practice platelets “ar©valolorido jc carreterog³mez j ¡lvarezoliva a guti©rrezmonta±o cfern¡ndezrecio jm najarrodiez f mean platelet volume in acutephase of ischemic stroke as predictor of mortality and functional outcomeafter 1year j stroke cerebrovasc dis “ wasilewski j desperak p hawranek m prognostic implicationsof mean platelet volume on short and longterm outcomes among patientswith nonstsegment elevation myocardial infarction treated withpercutaneous coronary intervention a singlecenter large observationalstudy platelets “ dabbah s hammerman h markiewicz w relation between redcell distribution width and clinical outcomes after acute myocardialinfarction am j cardiol 105312e331sangoi mb da silva sh da silva je relation between red bloodcell distribution width and mortality alter acute myocardial infarction int jcardiol 146278e280 montagnana m cervellin g meschi t the role of red blood celldistribution width in cardiovascular and thrombotic disorders clin chemlabmed 504635e641lee jh chung hj kim k red cell distribution width as aprognostic marker in patients with communityacquired pneumonia am jemerg med 31172e79 braun e domany e kenig y elevated red cell distribution widthpredicts poor outcome in young patients with community acquiredpneumonia crit care 154r194 harrison p mackie i mumford a briggs c liesner r winter m machin s guidelines for the laboratory investigation of heritable disorders ofplatelet function br j haematol “ bello s fandos s lasierra ab minchole e panadero c simon al de pabloog menendez r torres a red blood cell distribution width [rdw]and longterm mortality after communityacquired pneumonia acomparison with proadrenomedullin respir med 1091193e1206 ware j corken a khetpal r platelet function beyond hemostasis andthrombosis curr opin hematol “ becchi c al malyan m fabbri lp marsili m boddi v boncinelli s mean platelet volume trend in sepsis is it a useful parameter minervaanestesiol “kitazawa t yoshino y tatsuno k ota y yotsuyanagi h changes inthe mean platelet volume levels after bloodstream infection haveprognostic value intern med “kamath s blann ad lip gy platelet activation assessment andquantification eur heart j “ gorelik o tzur i barchel d almozninosarafian d swarka m feldman iblcohen n izhakian s a rise in mean platelet volume duringhospitalization for communityacquired pneumonia predicts poorprognosis a retrospective observational cohort study bmc pulmonarymedicine lippi g targher g montagnana m relation between red bloodcell distribution width and inflammatory biomarkers in a large cohort ofunselected outpatients arch pathol lab med “semba rd patel kv ferrucci l serum antioxidants andinflammation predict red cell distribution width in older women thewomen's health and aging study i clin nutr “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations musher dm thorner ar communityacquired pneumonia n engl jmed “jain s self wh wunderink rg communityacquired pneumoniarequiring hospitalization among us adults n engl j med “eurich dt marrie tj minhassandhu jk tenyear mortality aftercommunityacquired pneumonia a prospective cohort am j respir critcare med “karadagoncel e ozsurekci y kara a karahan s cengiz ab ceyhan m the value of mean platelet volume in the determination ofcommunity acquired pneumonia in children ital j pediatr golcuk y golcuk b bilge a irik m dikmen o combination of meanplatelet volume and the curb65 score better predicts 28day mortality inpatients with communityacquired pneumonia am j emerg med “felker gm allen la pocock sj red cell distribution width as anovel prognostic marker in heart failure data from the charm programand the duke databank j am coll cardiol “ ani c ovbiagele b elevated red blood cell distribution width predictsmortality in persons with known stroke j neurol sci ““ patel kv semba rd ferrucci l red cell distribution width andmortality in older adults a metaanalysis j gerontol a biol sci med sci“ 0c"
Colon_Cancer
the hypoxic tumour is a chaotic landscape of struggle and adaption against the adversity of oxygen starvationhypoxic cancer cells initiate a reprogramming of transcriptional activities allowing for survival metastasis andtreatment failure this makes hypoxia a crucial feature of aggressive tumours its importance to cancer and otherdiseases was recognised by the award of the nobel prize in physiology or medicine for research contributing toour understanding of the cellular response to oxygen deprivation for cancers with limited treatment options forexample those that rely heavily on radiotherapy the results of hypoxic adaption are particularly restrictive to treatmentsuccess a fundamental aspect of this hypoxic reprogramming with direct relevance to radioresistance is the alterationto the dna damage response a complex set of intermingling processes that guide the cell for good or for badtowards dna repair or cell death these alterations compounded by the fact that oxygen is required to inducedamage to dna during radiotherapy means that hypoxia represents a persistent obstacle in the treatment of manysolid tumours considerable research has been done to reverse correct or diminish hypoxia™s power over successfultreatment though many clinical trials have been performed or are ongoing particularly in the context of imagingstudies and biomarker discovery this research has yet to inform clinical practice indeed the only hypoxia interventionincorporated into standard of care is the use of the hypoxiaactivated prodrug nimorazole for head and neck cancerpatients in denmark decades of research have allowed us to build a picture of the shift in the dna repair capabilitiesof hypoxic cancer cells a literature consensus tells us that key signal transducers of this response are upregulatedwhere repair proteins are downregulated however a complete understanding of how these alterations lead toradioresistance is yet to comefacts— hypoxia is present in almost every solid tumour— hypoxia is a major barrier to effective radiotherapyand is associated with radioresistance— the hypoxic tumour is highly heterogenous withregions of chronic and acute hypoxia altered ph andimmune ltration— differences in gene expression and protein functioncan occur between acute or chronic and mild orsevere hypoxiacorrespondence mahvash tavassoli mahvashtavassolikclacuk1head and neck oncology group centre for host microbiome interactionking™s college london hodgkin building london se1 1ul ukedited by i amelio— all dna damageresponsehomologousddr pathwaysincludingnonhomologous end joining missmatch repair andthe fanconi anaemia pathways have been shown tosuffer alterations in hypoxiarecombination— activation of ddr transducer protein atm is seenin severe hypoxia in the absence of classical atmactivatingstranddna breaksfeaturesdoublesuchas— atr is also activated most likely in response tohypoxiainduced replication stress— however downregulation of dna repair effectorproteins such as rad51 and brca12 is seen— results of ddr reprogramming include geneticinstability aberrant cell cycle and apoptotic control the authors open access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate ifchanges were made the images or other third party material in this are included in the ™s creative commons license unless indicated otherwise in a credit line to the material ifmaterial is not included in the ™s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this license visit httpcreativecommonslicensesby40official of the cell death differentiation association 0cbegg and tavassoli cell death discovery page of open questions— precisely how do alterations to the ddr in hypoxialead to radioresistance for example when genomicinstability and generation of radioresistant clonestakes several cell divisions to set in how does adecrease in dna repair ability lead to increasedradioresistance— what aspects of the hypoxic response could betargeted to radiosensitise or more effectively treattumours particularly in the context of ddr forexampleupregulated ddrtransducers such as atm atr and dnapkcs orinduce synthetic lethality following downregulationof dna repair effectorscan wetarget— do different types of cancers have different patternsof ddr alteration within hypoxic tumours thisparticularly needs further research as tissues havebeen shown to have different oxygen pressureslevels of hypoxia and hypoxic heterogeneity— can we use the data on this subject to develop abiomarkerhypoxiainducedradioresistance as we have done using hypoxia as asingle parametersignatureof— how can we monitor hypoxic tumours during thecourse of a patient™s disease to help guide treatmentand— how can wereportingreliableensureinterpretation of in vitro and in vivo dataintroductionofprogramstranscriptionalhypoxia is present in almost every solid tumour aninevitability of cancer™s characteristic disanised andfunctionally inefficient vasculature rapid growth anddemanding metabolism1 the result is a comprehensiverewritingupdownregulating certain genes and proteins allowing cells toevade apoptosis and migrate to areas with better oxygenperfusion crucially this microenvironmentally inducedintracellular shift also results in genomic instability gialterations to dna repair and resistance to cell killing bycancer therapies after decades of research it has becomeclear that the relevance of hypoxia for both oncogenesisand treatment resistance is inescapablein the context of radiotherapy rt a link betweenresistance and low intratumoral oxygen pressure has beenknown since the publication of a study modelling oxygenflow in lung tumours years ago2 for some cancerssuch as head and neck squamous cell carcinomashnsccs hypoxia is a major contributing factor to localrt failure3“ advancesin developing technologiesallowing for more precise delivery of rtimaging oftumours and sensitisation to treatment while protectingnormal tissues have led to improved locoregional controland quality ofsincelife for patients78 howeverofficial of the cell death differentiation associationtreatment for many cancers like hnsccs which incidentally are also some of the most hypoxic depends onrt the hypoxic problem remains particularly pertinent9in recent years efforts have been made to correct orreverse hypoxia including administering hyperbaric oxygen therapy to patients1011 reducing cellular oxygenconsumption12 and increasing blood vesselfunctionality1314 to more precisely tackle resistance induced byhypoxia researchers have also soughtthethreshold of treatability of hypoxic tumours by use ofsensitizers15“ as a third arm in our battle plan researchhas also gone into developing methods to detect hypoxiaincluding the use of specialised imaging techniques petct scanning combined with hypoxiadetecting radionuclides often studied in tandem with genetic signaturesseeking to genotypically define these tumours1819to lowerhowever very few of these advancements have allowedus to overcome hypoxiainduced radioresistance rrthough research activity in this area remains strong amore complete understanding of how the hypoxic environment contributes to rr particularly by modulation ofpotentially targetable dna damage response ddrpathways is warranted this review will outline our current knowledge of the molecular processes that underpinhypoxic rr particularly in the reprogramming of the ddrradiotherapy mechanism of action”therequirement of oxygenseminal work by gray and colleagues during the 1950sproved that the efficacy of rt was dependent on theavailability of oxygen within the tissue22021 radiationinduces damage through the direct and indirect generation of double stranded breaks dsbs in dna in thepresence of oxygen damage induced is “ times morelikely to end in cell death22 this effect is best explainedby the oxygen fixation hypothesis where radicals produced directly or indirectly by ionizing radiation ir areoxidised to dna in the presence of oxygen23 making thedamage irreversible24 thistothe hypothesis with the notion that these lesions cannotbe restored to an undamaged state as the damage isœï¬xed to dna by oxidisation25 thus without oxygendamage induced is transient and hypoxic cells experiencefar reduced radiationassociated damagelast pointis crucialthough crucial the requirement of oxygen to inducedamage is not where the story ends for rr as it does notfully explain the level of rr we observe this is evidencedby the fact that restoration of oxygen to tumours forexample through applying hyperbaric oxygen does notrestore radiosensitivity26 importantlyit also does notaccount for changes that occur with respect to dnarepair which have been shown to be crucial in impactingthe radiation response27 as these changes are retainedpast the point of radiotherapy administration 0cbegg and tavassoli cell death discovery page of the landscape of the hypoxic tumourin vivo the hypoxic region exists on a gradient ofoxygen pressures with oxygen levels throughout the tissue ranging between severe hypoxia “ mildhypoxia and anoxia with around consideredphysoxia see table for definitions tissue oxygenpressures are usually measured in mmhg however sincethe majority of research on hypoxia and the ddr hasbeen performed in vitro where oxygen levels are measured in percent for the purpose of this review o2 willbe referred to predominantly the difference betweenin vivo and in vitro measurements of oxygen is importantthough with tissue normoxia physoxia classified ataround o2 or mmhg and in vitro normoxia beingaround o2 fig the hypoxic tumour is a space of restricted proliferationparticularly when oxygen levels are o2 cell cyclearrest and decreased protein synthesis juxtaposed againstaccelerated aggressivity microenvironmental interactionsand altered ph28“ at the most oxygendepleted borderexists the barren land of necrosis with the highly proliferating and comparatively treatmentsensitive aerobiccells closest to the blood vessel fig tumour hypoxia does not develop in a linear fashionand is highly heterogenous and changeable the hypoxictumour is dynamic with fluctuating vessel functionalityand cycling oxygen levels creating regions of acute andchronic hypoxia31 where part of the tissue may sufferacute hypoxia after temporary occlusion of a blood vesselsocalled perfusion limited in which oxygendeprivationcycles last sometimes minutes sometimes hours beforesubsequent reoxygenation chronic hypoxia is diffusionlimited where oxygen levels become a factor of distancefrom the blood vessel31 compounded with this is thediffering rates of oxygen consumption and responsivenessto oxygen availability in cells within the tissue to be ableto fully understand and ultimately treat the hypoxictumour it must be remembered that the changeability ofoxygen concentrations in the hypoxic tumour also predictably ‚uences its behaviour and response to treatment in the context of radiotherapy cells with o2 levels is where we see most resistance so called radiobiological hypoxia24 whether this is acute and thereforefollowed by reoxygenation or chronic oxygen deprivedfor more than h can have marked differences on theensuing genomic and proteomic changes that ultimatelyallow for hypoxic survival32 thus within one tumourdifferent regions are likely to have a completely differentresponse to the same dose of radiotherapyitaside from the oxygen status the involvement of otherenvironmentalfeatures affected by hypoxia must beconsidered an additional outcome of hypoxic adaption isthe concomitant phenotypic shift of the microenvironmentis now accepted that hypoxia can induce‚ammation33 demonstrated even by patients whodevelop mountain sickness after prolonged periods athigh altitude34 hypoxic tumours are known to havehigher ltration of protumour immune cells such asm2 macrophages35 a feature known to be involved inrr3637 the same could also be said with respect to thehypoxicinduction of highly rt resistant cancer stemcells38 though this subject needs further research thelikelihood of an interplay between intracellular geneticreprogramming as a result of hypoxic adaption and themicroenvironment in mediating radioresponse is stronggenetic reprogramming”the hifswithin this chaotic showground of heterogeneity andat the core of cellular adaption to hypoxia are the alteredgenetic pathways that push for survival against adversitycommonly dysregulated genes include glut1 involvedin altered glucose metabolism vegf involved intable glossary of terms multiple classifications of the terms used to describe hypoxia exist throughout the literaturethis represents a general consensus and what is used in this reviewtermhypoxianormoxiaphysoxiaanoxiasevere hypoxiamild hypoxiaacute hypoxiachronic hypoxiadefinitionreduced oxygen levels usually ‰¤ o2 mmhg in in vitro studiesnormal atmospheric oxygen used in in vitro studies o2 mghgphysiological levels of oxygen in tissues between mmhg tissue specific see fig complete absence of oxygen o2 o2“ o2incubation in hypoxic conditions “ hincubation in hypoxic conditions hradiobiological hypoxiaoxygen levels where the efficacy of radiotherapy is half maximal mmhg04 o2official of the cell death differentiation association 0cbegg and tavassoli cell death discovery page of fig approximate oxygen levels reported in different tissues in mmhg used in in vivo experiments and o2 used in in vitroexperiments note that normal tissue normoxia or physoxia is considerably less than the o2 used in vitro as normoxia adapted frommckeown139 liu140 and graham26fig the heterogeneity of the hypoxic tumour tumours suffer from reduced oxygen availability due to the disanised nature of thevasculature where occlusion of a blood vessel bv occurs tumours are said to be under perfusion limited hypoxia pl hypoxia where lack ofoxygen is a function of distance from the vessel cells experience diffusion limited dl hypoxia when these states are temporary h it is said tobe acute or chronic when h within hypoxia tumour cells undergo considerable genetic reprogramming contributing to therapy resistance andmetastatic behaviourofficial of the cell death differentiation association 0cbegg and tavassoli cell death discovery page of neoangiogenesis and lox involved in remodelling ofthe extracellular matrix7in the nobel prize in physiology or medicine wasawarded to three scientists gregg semenza williamkaelin and sir peter ratcliffe for their contributions toour understanding of cellular oxygensensing mechanisms39 this included the discovery of a group of transcription factors regulated by hypoxia that allow forcellular adaption40 these hypoxiainducible factor hifproteins hif13 are transcription factors composed oftwo subunits the α subunits reside in the cytoplasm andto rapid degradation “ min41 underare subjectnormal circumstances this degradation is mediated bythe actions of prolylhydroxylasesphd14 whichhydroxylate hifα at the oxygendependentdegradationdomains oddd of note phd2 and phd3 are themselves transcriptional targets of hif alluding to possiblenegative feedback systems in place though conflictingresults suggest this system doesn™t always function effectively to constrain cancer growth42“ subsequentlyhydroxylation by phds recruits the vonhippellandauvhl protein45 this alongside other proteins forms ane3 ubiquitin ligase complex ubiquitinating hifα forproteasomal degradationin hypoxia due to the lack of molecular oxygen neededfor hydroxylation4246 this degradation cascade does nottake place and hifα subunits translocate to the nucleusto associate with hifβ subunits47 the hif complex ininteraction with its coactivators p300 and crebbindingprotein cbp then binds to hypoxia response elementshresin dna to initiate transcription of hiftarget genesintheincludingcontrolledadditional layers of hif regulation also exist to keepthis pathway in check such as factor inhibiting hif fihwhich hydroxylates hif subunits at asparagine residuesblocking their association with p300cbp48 some evidence has shown that hifs also undergo other posttranslational modifications including phosphorylation andacetylation as a further method of regulation4247 however as with many such processes in cancer it can beaberrantlyhypoxiaindependent stabilisation of hifα by oncogenes such asegfr and mtor4950 and depletion of hifregulatoryfactors4251 the hif proteins themselves can interactwith a number of factors relevant in cancer such as p53mutants present in human papillomavirus hpvnegativehnsccs and nonsmall celllung cancers nsclcsresulting in transcriptional control of protumorigenicgenes52 since both hifs and p53 compete for binding ofp300cbp to enact transcriptional control the hifs havea unique relationship with this highly cancerrelevantprotein53 inactivation of p53™s transcriptional abilities hasbeen observed54 though again conflicting results exist forthis55official of the cell death differentiation associationmost of the work investigating hifdirected transcriptional changes in hypoxia has focussed on the actions ofthe bestknown hif hif1 however both hif2 andhif3 also play a role in hypoxic transcriptional control42interestingly relative expression of the hifs has beenshown to differ between hypoxic tissues demonstratingthat each may have specific functions56 in some casesthey may indeed work in concert as hif2 has beenshown to be induced when hif1 is depleted50hifs are master regulators of the hypoxic response andconcurrent with the notion that hypoxic tumours areradioresistant depletion of hif1α in tumour modelsradiosensitises cells41 one study showed that intermittenthypoxia showed less radiationinduced cell death bothin vitro and in mice via stabilisation of hif1α57 thisinvestigation also found that intermittent hypoxia had amore significant effect than chronic hypoxia hif1α hasalso been shown to function via the hif1α“myc pathway in which hif1α competes with the transcriptionactivator myc for sp1 binding in the target gene promoter to downregulate mismatch repair mmr genesmsh2 and msh6 in o2however how exactly hifs contribute to rr of thehypoxic tumour be itthrough their transcriptionalfunctions or interactions with other proteins is so farunresolved notably some radio and chemotherapiesthemselves upregulate or stabilise hifs41though genetic reprogramming in hypoxia can lead to anumber of alterations for the purpose of this review wewill focus on those associated with rr and ddr formore general reviews see schito60 and tsai61reprogramming of the ddrthe ddr is a complex process consisting of overlapping and interconnected pathways initiated by differentforms of dna damage arguably one ofthe mostimportant homeostatic processes it allows us to withstand constant and numerous dna damageinducinginsults the result of this protection ensures that onlyreliable genomes are passed on to the next cellular generation for cancer considering both the power ofmutagenesis in driving oncogenic potential and the factthat many cancer therapies function by inducing dnadamage the ddr has considerable relevance for therapyresistance and tumour progressionrepair of dna is a tale of three acts firstly the damagepropagates a signal that recruits sensors to the site ofdamage secondly the signal is amplified by transducersand thirdly response pathways are initiated by effectorsfor each part of the process welldefined though notexclusive sets of proteins act as sensors transducers andeffectors respectively28 shrouding these repair processesare signals to stall the cell cycle initiated by chk1 orchk2activated cdc25 and p21to allow time for 0cbegg and tavassoli cell death discovery page of clearance of this damage and initiation of apoptoticpathways for example as initiated by atm™s interactionwith p53 if the repair is unsuccessful6263for the repair of radiationinduced dsbs two primarypathways are put to use homologous recombination hrand nonhomologous end joining nhej the formerconsidered less errorprone uses sister chromatids torepair dna and as such can only take place during g2sphase of the cell cycle nhej predominates in g1 but canoccur at any stage of the cell cycle and often results in thegeneration of insertiondeletion mutations which havethe potential to lead to more oncogenic alterations hr ismediated primarily by the recruitment to sites of damageof master transducer of the dsb response atm ataxiatelangiectasia mutated a phosphoinositide3kinaserelated protein kinase pikk following detection by themrn complex composed of mre11rad50nbs1which initiates activity of effectors including rad51 andbrca1 nhej occurs following sensing of damage by theku proteins ku70 and ku80 and signal transduction ofku in complex with dnapkcs dnadependent proteinkinase catalytic subunit”together forming dnapk andsubsequent activity of effectors dna ligase iv ligivand xrcc464alteration of ddr pathways has been seen across manycancers compared to normal tissue perhaps the mostwellknown are the mutations in brca12 in aggressivehereditary breast and ovarian cancers65 understandablywhere hypoxia represents an exaggerated form ofaggressive tumours the ddr pathways in hypoxia operatedifferently to those in normoxia indeed this is true forevery aspect of the ddr process dna damage in theform of dsbs is reduced in conditions of hypoxia o2and hypoxia alone does not induce dsbs2466 researchhas shown that different members of the ddr pathwayscan be either activated or downregulated in conditions oflow oxygen see tables and for reported alterationsto hr nhej and mismatch repair mmr pathwayscrucially whether the cells are in acute or chronichypoxia or h and at what level of oxygen depletion may define the ensuing response despite this delineation there lacks within the literature proper reportingof experiments carried out in either acute or chronic mildor severe hypoxia with interchangeability in use of termssee table for a consensus of parameters used with thesedefinitionssensorsthe mre11rad50nbs1 mrn complex is responsible for sensing dna damage and initiating both the hrand nhej pathways by recruitment of transducers such asatm via nbs167 while the mrn complex is consideredthe main sensor responsible for recruiting and activatingatm followingdamage atrip atrinteractingofficial of the cell death differentiation associationprotein and ku7080 are sensorsresponsible forrecruitment of atr and dnapkcs respectively67 fig though there are many overlapping interactions atminatminteracting protein with roles in replication stressrs genome stability and the base excision repair berpathway has also been shown to recruit atm independent of dna damage68repression of the mrn machinery has been seen inchronic hypoxia days in a medulloblastoma modelwith transcriptomic downregulation of both mre11a andnbs1 resulting in downregulation of etoposideinducedatm and p5369 nbs1 has also been shown to stabilisehif1α particularly in response to ir70 while hif1α hasbeen shown to downregulate nbs1 the authors of onestudy where reduction of nbs1 was seen after h in o2 noted that this repression resulted in the induction ofγh2ax and 53bp1 foci in hypoxia suggesting the presence of dna breaks59 interestingly all components ofthe mrn were found to be downregulated both at themrna and protein level in nsclcs harbouring egfrmutations incubated in severe hypoxia o2 thisdownregulation in egfrmutated cells correlated withtheir increased sensitivity to egfrinhibiting drugs71sensing of damaged dna is a crucial step in theinitiation of repair and begins with changes to the chromatin72 γh2ax a phosphorylated variant of histoneh2ax is induced by mrn activation and accumulates atsites of damage in the chromatin preceding recruitmentand necessary for retention of key ddr signalling proteinsincluding mrn and atm73 studies also show thatγh2ax is crucial for retaining mediators such as 53bp1p53 binding protein mdc1 mediator of dnadamage checkpoint and brca1 at sites of damage66h2ax is primarily phosphorylated by atm but can alsobe phosphorylated by atr and dnapkcs63 indeed aswell as by radiation and chemotherapies γh2ax has alsobeen shown to be induced by hypoxia following replication fork stalling this phosphorylation has been shown inchronic severe hypoxia to occur in a hif atr or atmdependent manner74“ crucially some evidence hasshown the phosphorylation of h2ax present only inproliferating cells7778 the downstream effects of thisactivation have been linked to other consequences ofhypoxic regulation including angiongenesis79 via induction of vegf80 experimentally resolution of γh2ax fociafter irradiation is often used as a marker of dsb repair astheory dictates that the phosphorylation should disappearafter damage is repaired however in hypoxia this heavilyrelied upon protocol may necessitate further finetuningku70 and ku80 together forming a heterodimericcomplex tether damaged dna at breaks and are keysensors of dsbs responsible for recruitment of dnapkcs as part of the nhej pathway63 the ku complex hasbeen shown to be both upregulated and downregulated by 0cbegg and tavassoli cell death discovery page of table a nonexhaustive list showing alterations to sensors transducers and effectors of the homologoursrecombination hr pathways in hypoxiaprotein role in ddrmechanism of alterationalteration conditions and consequencesreferencenbs1sensor of dsbs in hr activated atmas part of the mrn complexcid129 pasb domain of hif1αmre11sensor of dsbs in hr activated atmas part of the mrn complexcid129 atmtransducer of hr in dsb repaircid129 autophosphorylation atser1981atrtransducer of dna repair induced byreplication stresscid129 rad51effector of dsb repair in hrrad52effector of dsb repair in hrrad54 motor protein effector of dsbrepair in hrbrca1effector of dsb repair in hrcid129 e2f4p130cid129 lsd1cid129 ezh2cid129 mir210cid129 mir373cid129 mir210cid129 cid129 e2f4p130cid129 h3k4 demethylation via lsd1cid129 downregulated in chronic mild hypoxia days o2cid129 downregulation in acute mild h o2cid129 resulted in induction of γh2ax and 53bp1 focicid129 downregulated in chronic mild hypoxia days o2cid129 activated in acute hypoxia o2cid129 increased expression and activity o2“ hcid129 mediated by src and ampk signallingcid129 activated in acute o2cid129 resulted in phosphorylated p53 andaccumulation and growth arrestcid129 downregulation in chronic severe hypoxia o2 “ h and or “ hcid129 decreased radioresistancecid129 increased genomic instabilitycid129 downregulation in o2 hcid129 decreased mrna expression o2“ hcid129 downregulated o2 hcid129 decreased mrna expression o2“ hcid129 downregulation in chronic severe hypoxia o2 hcid129 decreased mrna expression o2“ hcid129 downregulation in o2 hcid129 decreased radioresistancecid129 decreased expression o2 “ hcowman69to59cowman69hashimoto88bencokova28hammond75meng119bindra oliveira82meng119crosby118meng119meng119lu117bindra120oliveira82meng119brca2effector of dsb repair in hrcid129 hypoxia in different studies81 one study found downregulation of ku80 after h in mild hypoxia o282another using severe hypoxia o2 found upregulation of ku70ku80 in a431 cells alongside many othermembers of the nhej pathway and proteins generallyinvolved in metastatic progression83 a study in humanand mouse hepatoma cells found upregulation of the kuheterodimer upon incubation in hypoxia o2 or withhypoxia mimics and downregulation associated with hif1βdeficient cells84 alternative subpathways of nhejalso exist possibly as insurance for when classical nhejmediators are inoperative howeverthe impact ofhypoxia on these pathways has not been extensivelystudiedofficial of the cell death differentiation associationtransducersatm and atr are two of the most important proteinsinvolved in transduction of the ddr as pikk familymembers they phosphorylate a number of proteinsinvolved in propagating the signal and repairing dna aspart of both the hr and nhej pathways as well asundergoingtheresponse until dna is repairedautophosphorylationto maintainbroadly speaking atm has been shown to be activatedparticularly in acute hypoxia as shown in the study bybencokova et al the pattern of activation in this contextdoes not match rtinduced atm activation which follows mrn recruitment to dsbs85 as atm phosphorylation does not correlate with the presence of dsbs 0cbegg and tavassoli cell death discovery page of table a nonexhaustive list showing alterations to sensors transducers and effectors of the nonhomologous endjoining nhej pathways in hypoxiaproteinrole in ddrmechanism of alterationalteration conditions and consequencesreferenceku70ku80sensor in nhej pathwaysrecruits dnapkcsin complex with ku80sensor in nhej pathwaysrecruits dnapkcsin complex with ku70cid129 cid129 dnapkcstransducer of nhej pathwaycid129 autophosphorylation atser2056dna ligiv effector of nhej repairxrcc4effector of nhej repaircid129 cid129 cid129 decreased mrna expression o2 “ hcid129 upregulation o2 hcid129 downregulation in cervical tumour sectionscid129 upregulation o2 “ hcid129 upregulation o2 hcid129 downregulation o2 hcid129 downregulation in cervical tumour sectionscid129 upregulation o2 “ hcid129 decreased mrna expression o2 “ hcid129 increased expression and activity o2 “ hcid129 activated in mild hypoxia “ o2 led to positiveregulation of hif1 and upregulation of glut1cid129 decreased mrna expression o2 “ hcid129 decreased mrna expression o2 “ hmeng119ren83lara81um84oliveira82ren83lara81um84meng119hashimoto88bouquet103meng119meng119table a nonexhaustive list showing alterations to sensors transducers and effectors of the mismatch repair pathwayin hypoxiaprotein role in ddrmechanism of alterationmlh1dimerises to pms2 to form themutlα complex in mmrpms2dimerises to mlh1 to form themutlα complex in mmrmsh2dimerises with msh6 forms themutsα complex in mmrmsh6dimerises with msh6 forms themutsα complex in mmrcid129 mad1maxcid129 mntmaxcid129 dec12cid129 mir155cid129 lsd1cid129 hdaccid129 hypoacetylationhypermethylation on h3cid129 cid129 mycmaxcid129 hif1α via sp1cid129 mir155cid129 hcid129 p53cid129 hif1α via sp1cid129 mir155cid129 hdaccid129 p53cid129 hypoacetylationhypermethylation on h3official of the cell death differentiation associationalteration conditions andconsequencescid129 downregulation “ h o2cid129 downregulation in h o2cid129 increased expression “ h o2resulting in genomic instability instem cellscid129 downregulation at protein level“ h o2cid129 resulting in genomic instability instem cellscid129 downregulation “ h o2referencebindra121127mihaylova128nakamura129rodriguezjimenez145lu115mihaylova128rodriguezjimenez145bindra121127koshiji58cid129 downregulation “ h o2cid129 increased expression “ h o2resulting in genomic instability instem cellskoshiji58rodriguezjimenez145 0cbegg and tavassoli cell death discovery page of often reduced or absent in severe hypxoia28 the authorsof this study emphasized that atm activation was specificto the level of hypoxia only phosphorylated at o2and hifindependent since phosphorylation was maintained even in hifknockout cells activation of atmwas attributed to autophosphorylation the result of whichwas an activation of targets much like dna damageinduced atm activation but dependent on the activityof cell cycle regulator mdc128 this study did not analyserr but the results suggest that atm activation byhypoxia is likely enacted as a means of halting the cellcycle in order to allow for dna repairthe pattern of atm activation in hypoxia however isnot clearcut and may depend on cancer type atm canbe regulated by a number of factors as part of the hypoxicresponse including mrn or atmin but also by posttranslational and epigenetic factors86 one study foundthat atm was downregulated along with hif1α by amicrorna mir18 resulting in radiosensitivity87the study by hashimoto et al88 showed atm activation alongside activation of a number of other key ddr orcancerrelated proteins including dnapkcs akt andegfr and decreased expres
Colon_Cancer
coutard b valle c de lamballerie x canard b seidah ng and decroly e the spike glycoprotein of the new coronavirus 2019ncovcontains a furinlike cleavage site absent in cov of the same clade antiviral res 101016jantiviral2020104742 cao z wu y faucon e and sabatier jm sarscov2 covid19 keyroles of the ˜reninangiotensin™ systemvitamin d impacting drugand vaccine developments infect disord drug targets “ 1021741871526520999200505174704 zhang z chen l zhong j gao p and oudit gy ace2ang17 signaling and vascular remodeling sci china life sci “s1142701446933 paz ocaranza m riquelme ja garcia l jalil je chiong m santos ras counterregulatory reninangiotensin system incardiovascular disease nat rev cardiol “ 101038s4156901902448 ziegler cgk allon sj nyquist sk mbano im miao vn tzouanas cn sarscov2 receptor ace2 is an interferonstimulatedgene in human airway epithelial cells and is detected in specific cell subsets across tissues cell “101016jcell202004035 marrero mb schieffer b paxton wg heerdtt l berkt bc delafontaine p direct stimulation of jakstat pathway by theangiotensin ii at1 receptor j med chem “ perrotta f matera mg cazzola m and bianco a severe respiratory sarscov2 infection does ace2 receptor matter respir med 101016jrmed2020105996 cheng h wang y and wang g anprotective effect of angiotensinconverting enzyme and its effect on the prognosis of covid19 jmed virol “ 101002jmv25785 de lang a osterhaus adme and haagmans bl interferon Î and interleukin4 downregulate expression of the sars coronavirusreceptor ace2 in vero e6 cells virology “ the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20200904 mcgonagle d sharif k o™regan a and bridgewood c the role of cytokines including interleukin6 in covid19 induced pneumonia andmacrophage activation syndromelike disease autoimmun rev linkinghubelseviercomretrievepiis1568997220300926101016jautrev2020102537 ye q wang b and mao j the pathogenesis and treatment of the ˜cytokine storm™ in covid19 j infect “linkinghubelseviercomretrievepiis0163445320301651 101016jjinf202003037 behrens em and koretzky ga review cytokine storm syndrome looking toward the precision medicine era arthritis rheumatol “ 101002art40071 guo xj and thomas pg new fronts emerge in the ‚uenza cytokine storm semin immunopathol “s002810170636y ronco c and reis t kidney involvement in covid19 and rationale for extracorporeal therapies nat rev nephrol “101038s4158102002847 wu c chen x cai y xia j zhou x xu s risk factors associated with acute respiratory distress syndrome and death in patientswith coronavirus disease pneumonia in wuhan china jama intern med “ 101001jamainternmed20200994 jose rj and manuel a covid19 cytokine storm the interplay between ‚ammation and coagulation lancet respir med “ murakami m kamimura d and hirano t pleiotropy and specificity insights from the interleukin family of cytokines immunity “ 101016jimmuni201903027 o™shea jj schwartz dm villarino av gadina m mcinnes ib and laurence a the jakstat pathway impact on human disease andtherapeutic intervention annu rev med “ 101146annurevmed051113024537 lawrence t the nuclear factor nf k b pathway in ‚ammation cold spring harb perspect biol “ catanzaro m fagiani f racchi m corsini e govoni s and lanni c immune response in covid19 addressing a pharmacologicalchallenge by targeting pathways triggered by sarscov2 signal transduct target ther 101038s4139202001911 li q and verma im nfκb regulation in the immune system nat rev immunol “ 101038nri910 tergaonkar v correa rg ikawa m and verma im distinct roles of iκb proteins in regulating constitutive nfκb activity nat cell biol “ 101038ncb1296 tergaonkar v nfκb pathway a good signaling paradigm and therapeutic target int j biochem cell biol “linkinghubelseviercomretrievepiis1357272506001245 101016jbiocel200603023 correa rg matsui t tergaonkar v rodriguezesteban c izpisuabelmonte jc and verma im zebrafish ikappab kinase negativelyregulates nfkappab activity curr biol “ 101016jcub200506023 wong et and tergaonkar v roles of nfκb in health and disease mechanisms and therapeutic potential clin sci “portlandpresscomclinsci116645168639rolesofnfκbinhealthanddiseasemechanismsand101042cs20080502 morrison dk map kinase pathways cold spring harb perspect biol a011254 101101cshperspecta011254 mizutani t fukushi s saijo m kurane i and morikawa s phosphorylation of p38 mapk and its downstream targets in sarscoronavirusinfected cells biochem biophys res commun “ 101016jbbrc200405107 grimes jm and grimes kv p38 mapk inhibition a promising therapeutic approach for covid19 j mol cell cardiol “101016jyjmcc202005007 cuadrado a and nebreda ar mechanisms and functions of p38 mapk signalling biochem j “101042bj20100323 han j lee jd tobias ps and ulevitch rj endotoxin induces rapid protein tyrosine phosphorylation in 70z3 cells expressing cd14 jbiol chem “ jiang y chen c li z guo w gegner ja lin s characterization of the structure and function of a new mitogenactivated proteinkinase p38 j biol chem “ 101074jbc2713017920 li z jiang y ulevitch rj and han j the primary structure of p38Î a new member of p38 group of map kinases biochem biophys rescommun “ linkinghubelseviercomretrievepiis0006291x96916629 101006bbrc19961662 jiang y gram h zhao m new l gu j feng l characterization of the structure and function of the fourth member of p38 groupmitogenactivated protein kinases p38δ j biol chem “ 101074jbc2724830122 zarubin t and han j activation and signaling of the p38 map kinase pathway cell res “ 101038sjcr7290257 cuevas bd abell an and johnson gl role of mitogenactivated protein kinase kinase kinases in signal integration oncogene “ httpwwwnaturecoms1210409 101038sjonc1210409 alonso g ambrosino c jones m and nebreda ar differential activation of p38 mitogenactivated protein kinase isoforms depending onsignal strength j biol chem “ 101074jbcm007835200 brancho d mechanism of p38 map kinase activation in vivo genes dev “ 101101gad1107303 parker cg hunt j diener k mcginley m soriano b keesler ga identification of stathmin as a novel substrate for p38 deltabiochem biophys res commun “ linkinghubelseviercomretrievepiis0006291x98992506101006bbrc19989250 rouse j cohen p trigon s morange m alonsollamazares a zamanillo d a novel kinase cascade triggered by stress and heatshock that stimulates mapkap kinase2 and phosphorylation of the small heat shock proteins cell “linkinghubelseviercomretrievepii0092867494902771 1010160092867494902771 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20200904 tan y rouse j zhang a cariati s cohen p and comb mj fgf and stress regulate creb and atf1 via a pathway involving p38 mapkinase and mapkap kinase2 embo j “ 101002j146020751996tb00840x xu w wang x tocker am huang p reith mea liuchen ly functional characterization of a novel series of biased signalingdopamine d3 receptor agonists acs chem neurosci “ 101021acschemneuro6b00221 kotlyarov a neininger a schubert c eckert r birchmeier c volk hd mapkap kinase is essential for lpsinduced tnfαbiosynthesis nat cell biol “ httpwwwnaturecomsncb0699 10103810061 kontoyiannis d pasparakis m pizarro tt cominelli f and kollias g impaired onoff regulation of tnf biosynthesis in mice lacking tnfaurich elements immunity “ linkinghubelseviercomretrievepiis1074761300800382101016s1074761300800382 saccani s pantano s and natoli g p38dependent marking of ‚ammatory genes for increased nfκb recruitment nat immunol “ 101038ni748 dienz o and rincon m the effects of il6 on cd4 t cell responses clin immunol “ 101016jclim200808018 velazquezsalinas l verdugorodriguez a and rodriguez ll the role of interleukin during viral infections front microbiol “ zhang c wu z li jw zhao h and wang gq the cytokine release syndrome crs of severe covid19 and interleukin6 receptoril6r antagonist tocilizumab may be the key to reduce the mortality int j antimicrob agents 101016jijantimicag2020105954 matthay ma ware lb and zimmerman ga the acute respiratory distress syndrome j clin invest “101172jci60331 zhang h rostami mr leopold pl mezey jg o™beirne sl strulovicibarel y expression of the sarscov2 ace2 receptor inthe human airway epithelium am j respir crit care med “ 101164rccm2020030541oc tian s xiong y liu h niu l guo j liao m pathological study of the novel coronavirus disease covid19 throughpostmortem core biopsies mod pathol “ 101038s413790200536x sungnak w huang n b ´ecavin c berg m queen r litvinukova m sarscov2 entry factors are highly expressed in nasalepithelial cells together with innate immune genes nat med “ 101038s4159102008686 lukassen s chua rl trefzer t kahn nc schneider ma muley t sars cov2 receptor ace and tmprss are primarilyexpressed in bronchial transient secretory cells embo j “ 1015252embj20105114 leung jm yang cx tam a shaipanich t hackett tl singhera gk ace2 expression in the small airway epithelia of smokersand copd patients implications for covid19 eur respir j 10118313993003006882020 dhawale vs amara vr karpe pa malek v patel d and tikoo k activation of angiotensinconverting enzyme ace2 attenuatesallergic airway ‚ammation in rat asthma model toxicol appl pharmacol “ 101016jtaap201606026 bradding p richardson m hinks tsc howarth ph choy df arron jr ace2 tmprss2 and furin gene expression in the airwaysof people with asthma implications for covid19 j allergy clin immunol “ 101016jjaci202005013 vareille m kieninger e edwards mr and regamey n the airway epithelium soldier in the fight against respiratory viruses clinmicrobiol rev “ 101128cmr0001410 sabroe i parker lc dower sk and whyte mkb the role of tlr activation in ‚ammation j pathol “101002path2264 frieman m and baric r mechanisms of severe acute respiratory syndrome pathogenesis and innate immunomodulation microbiol mol biolrev “ 101128mmbr0001508 lafferty ei qureshi st and schnare m the role of tolllike receptors in acute and chronic lung ‚ammation j ‚amm httpwwwjournal‚ammationcomcontent7157 10118614769255757 kawai t and akira s signaling to nfκb by tolllike receptors trends mol med “101016jmolmed200709002 darby wj mcnutt kw and todhunter en niacin nutr rev “ 101111j175348871975tb05075x yoshikawa t hill te yoshikawa n popov vl galindo cl garner hr dynamic innate immune responses of human bronchialepithelial cells to severe acute respiratory syndromeassociated coronavirus infection plos one e8729101371journalpone0008729 dediego ml nietotorres jl reglanava ja jimenezguardeno jm fernandezdelgado r fett c inhibition ofnfkappabmediated ‚ammation in severe acute respiratory syndrome coronavirusinfected mice increases survival j virol “101128jvi0257613 imai y kuba k neely gg yaghubianmalhami r perkmann t van loo g identification of oxidative stress and tolllike receptor signaling as a key pathway of acute lung injury cell “ 101016jcell200802043 ding y feng q chen j and song j tlr4nfκb signaling pathway gene single nucleotide polymorphisms alter gene expression levelsand affect ards occurrence and prognosis outcomes medicine baltimore e16029 101097md0000000000016029 totura al whitmore a agnihothram s sch ¨afer a katze mg heise mt tolllike receptor signaling via trif contributes to aprotective innate immune response to severe acute respiratory syndrome coronavirus infection mbio “101128mbio0063815 olejnik j hume aj and m ¨uhlberger e tolllike receptor in acute viral infection too much of a good thing plos pathog “101371journalppat1007390 okumura a rasmussen al halfmann p feldmann f yoshimura a feldmann h suppressor of cytokine signaling is aninducible host factor that regulates virus egress during ebola virus infection j virol “ 101128jvi0173615 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20200904 o™brien tr thomas dl jackson ss prokuninaolsson l donnelly rp and hartmann r weak induction of interferon expression bysarscov2 supports clinical clin infect dis 101093cidciaa453 chang yj liu cyy chiang bl chao yc and chen cc induction of il8 release in lung cells via activator protein1 byrecombinant baculovirus displaying severe acute respiratory syndromecoronavirus spike proteins identification of two functional regions j immunol “ 104049jimmunol173127602 wang w ye l ye l li b gao b zeng y upregulation of il6 and tnfα induced by sarscoronavirus spike protein in murinemacrophages via nfκb pathway virus res “ 101016jvirusres200702007 zhang x wu k wang d yue x song d zhu y nucleocapsid protein of sarscov activates interleukin6 expression throughcellular transcription factor nfκb virology “ 101016jvirol200704009 toews gb cytokines and the lung eur respir j suppl “ 101183090319360100266001 russell b moss c rigg a and van hemelrijck m covid19 and treatment with nsaids and corticosteroids should we be limiting theiruse in the clinical setting ecancermedicalscience “ 103332ecancer20201023 severgnini m takahashi s rozo lm homer rj kuhn c jhung jw activation of the stat pathway in acute lung injury am jphysiol lung cell mol physiol “ 101152ajplung003492003 zhao j yu h liu y gibson sa yan z xu x protective effect of suppressing stat3 activity in lpsinduced acute lung injury amj physiol lung cell mol physiol l868“l880 101152ajplung002812016 pedroza m schneider dj karmoutyquintana h coote j shaw s corrigan r interleukin6 contributes to ‚ammation andremodeling in a model of adenosine mediated lung injury plos one e22667 101371journalpone0022667 goldman jl sammani s kempf c saadat l letsiou e wang t pleiotropic effects of interleukin6 in a œtwohit murine modelof acute respiratory distress syndrome pulm circ “ 101086675991 rincon m and irvin cg role of il6 in asthma and other ‚ammatory pulmonary diseases int j biol sci “107150ijbs4874 swanson kv deng m and ting jpy the nlrp3 ‚ammasome molecular activation and regulation to therapeutics nat rev immunol “ 101038s4157701901650 zhao c and zhao w nlrp3 ‚ammasome ” a key player in antiviral responses front immunol “ orzalli mh smith a jurado ka iwasaki a garlick ja kagan jc an antiviral branch of the il1 signaling pathway restrictsimmuneevasive virus replication an antiviral branch of the il1 signaling pathway restricts immuneevasive virus replication mol cell 825e5“840e5 101016jmolcel201807009 shi c and nabar nr sarscoronavirus open reading frame8b triggers intracellular stress pathways and activates nlrp3 ‚ammasomescell death discov 101038s4142001901817 siu k yuen k castanorodriguez c ye z yeung m fung s severe acute respiratory syndrome coronavirus orf3a proteinactivates the nlrp3 ‚ammasome by promoting traf3dependent ubiquitination of asc faseb j “101096fj201802418r kusov y tan j alvarez e enjuanes l and hilgenfeld r a gquadruplexbinding macrodomain within the œsarsunique domain isessential for the activity of the sarscoronavirus replicationtranscription complex virology “101016jvirol20150601 chang ys ko bh ju jc chang hh huang sh and lin cw sars unique domain sud of severe acute respiratory syndromecoronavirus induces nlrp3 ‚ammasomedependent cxcl10mediated pulmonary ‚ammation int j mol sci “103390ijms21093179 sun dz song cq xu ym and dong xs role of the mapk pathway in human lung epitheliallike a549 cells apoptosis induced byparaquat genet mol biol e20190137 10159016784685gmb20190137 arcaroli j yum hk kupfner j park js yang ky and abraham e role of p38 map kinase in the development of acute lung injury clinimmunol “ 101006clim20015108 li sw wang cy jou yj yang tc huang sh wan l sars coronavirus papainlike protease induces egr1dependentupregulation of tgf1 via rosp38 mapkstat3 pathway sci rep “ yoshida k kuwano k hagimoto n watanabe k matsuba t fujita m map kinase activation and apoptosis in lung tissues frompatients with idiopathic pulmonary fibrosis j pathol “ 101002path1208 qian f deng j wang g ye rd and christman jw pivotal role of mitogenactivated protein kinaseactivated protein kinase in‚ammatory pulmonary diseases curr protein pept sci “ 1021741389203716666150629121324 renda t baraldo s pelaia g bazzan e turato g papi a increased activation of p38 mapk in copd eur respir j “1011830903193600036707 mercer ba and d™armiento jm emerging role of map kinase pathways as therapeutic targets in copd int j chron obstruct pulmon dis “ feng y fang z liu b and zheng x p38mapk plays a pivotal role in the development of acute respiratory distress syndrome clinics “ 106061clinics2019e509 mizutani t signaling pathways of sarscov in vitro and in vivo mol biol “ 9783642036835˙ mizutani t signal transduction in sarscovinfected cells ann ny acad sci “ 101196annals1408006 cheng vcc hung ifn tang bsf chu cm wong mml chan kh viral replication in the nasopharynx is associated withdiarrhea in patients with severe acute respiratory syndrome clin infect dis “ 101086382681 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20200904 arabi ym alomari a mandourah y alhameed f sindi aa alraddadi b critically ill patients with the middle east respiratorysyndrome a multicenter retrospective cohort study crit care med “ 101097ccm0000000000002621 wong sh lui rns and sung jjy covid19 and the digestive system j gastroenterol hepatol “101111jgh15047 luo s zhang x and xu h don™t overlook digestive symptoms in patients with novel coronavirus disease covid19 clingastroenterol hepatol “ 101016jcgh202003043 xiao f tang m zheng x liu y li x and shan h evidence for gastrointestinal infection of sarscov2 gastroenterology 1831e3“1833e3 101053jgastro202002055 young be ong swx kalimuddin s low jg tan sy loh j epidemiologic features and clinical course of patients infected withsarscov2 in singapore jama jamanetworkcomjournalsjamafull2762688 101001jama20203204 xu y li x zhu b liang h fang c gong y characteristics of pediatric sarscov2 infection and potential evidence for persistentfecal viral shedding nat med “ 101038s4159102008174 tang a tong z wang h dai y li k liu j detection of novel coronavirus by rtpcr in stool specimen from asymptomatic childchina emerg infect dis “ httpwwwnccdcgoveid266200301 htm 103201eid2606200301 zhang w du rh li b zheng xs yang xl hu b molecular and serological investigation of 2019ncov infected patientsimplication of multiple shedding routes emerg microbes infect “ 1010802222175120201729071 yeo c kaushal s and yeo d enteric involvement of coronaviruses is faecaloral transmission of sarscov2 possible lancetgastroenterol hepatol “ 101016s2468125320300480 van doremalen n bushmaker t morris dh holbrook mg gamble a williamson bn aerosol and surface stability ofsarscov2 as compared with sarscov1 n engl j med “ 101056nejmc2004973 setti l passarini f de gennaro g barbieri p perrone mg borelli m airborne transmission route of covid19 why meters6feet of interpersonal distance could not be enough int j environ res public health 103390ijerph17082932 perlman s and netland j coronaviruses postsars update on replication and pathogenesis nat rev microbiol “101038nrmicro2147 m ¨onkem ¨uller k fry l and rickes s covid19 coronavirus sarscov2 and the small bowel rev esp enferm dig “ wang d hu b hu c zhu f liu x zhang j clinical characteristics of hospitalized patients with novelcoronavirusinfected pneumonia in wuhan china jama jamanetworkcomjournalsjamafull2761044101001jama20201585 pan l mu m yang p sun y wang r yan j clinical characteristics of covid19 patients with digestive symptoms in hubei chinaam j gastroenterol “ 1014309ajg0000000000000620 budden kf gellatly sl wood dla cooper ma morrison m hugenholtz p emerging pathogenic links between microbiota andthe gutlung axis nat rev microbiol “ 101038nrmicro2016142 camargo smr singer d makrides v huggel k pos km wagner ca tissuespecific amino acid transporter partners ace2 andcollectrin differentially interact with hartnup mutations gastroenterology 872e3“882e3linkinghubelseviercomretrievepiis0016508508018933 101053jgastro200810055 hashimoto t perlot t rehman a trichereau j ishiguro h paolino m ace2 links amino acid malnutrition to microbial ecology andintestinal ‚ammation nature “ 101038nature11228 br ¨oer a juelich t vanslambrouck jm tietze n solomon ps holst j impaired nutrient signaling and body weight control in naneutral amino acid cotransporter slc6a19deficient mouse j biol chem “ 101074jbcm111241323 ma xm and blenis j molecular mechanisms of mtormediated translational control nat rev mol cell biol “101038nrm2672 bostancklo ˘glu m temporal correlation between neurological and gastrointestinal symptoms of sarscov2 ‚amm bowel dis “101093ibdizaa131 louveau a smirnov i keyes tj eccles jd rouhani sj peske jd structural and functional features of central nervous systemlymphatic vessels nature “ 101038nature14432 jessen na munk asf lundgaard i and nedergaard m the glymphatic system a beginner™s guide neurochem res “s1106401515816 wu y xu x chen z duan j hashimoto k yang l nervous system involvement after infection with covid19 and othercoronaviruses brain behav immun “ 101016jbbi202003031 liu k pan m xiao z and xu x neurological manifestations of the coronavirus sarscov2 pandemic j neurol neurosurgpsychiatry “ 101136jnnp2020323177 xydakis ms dehganimobaraki p holbrook eh geisthoff uw bauer c hautefort c smell and taste dysfunction in patients withcovid19 lancet infect dis s14733099 “ 101016s1473309920302930 moriguchi t harii n goto j harada d sugawara h takamino j a first case of meningitisencephalitis associated withsarscoronavirus2 int j infect dis “ 101016jijid202003062 baig am khaleeq a ali u and syeda h evidence of the covid19 virus targeting the cns tissue distribution hostvirus interaction andproposed neurotropic mechanisms acs chem neurosci “ 101021acschemneuro0c00122 helms j kremer s merdji h clerejehl r schenck m kummerlen c neurologic features in severe sarscov2 infection nengl j med “ 101056nejmc2008597 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20200904 panizmondolfi a bryce c grimes z gordon re reidy j lednicky j central nervous system involvement by severe acuterespiratory syndrome coronavirus2 sarscov2 j med virol “ 101002jmv25915 zhou l zhang m wang j and gao j sarscov2 underestimated damage to nervous system travel med infect dis linkinghubelseviercomretrievepiis1477893920301101 101016jtmaid2020101642 panciani pp saraceno g zanin l renisi g signorini l battaglia l sarscov2œthreesteps infection model and csfdiagnostic implication brain behav immun “ 101016jbbi202005002 desfes m le coupanec a dubeau p bourgouin a lajoie l dub ´e m human coronaviruses and other respiratory virusesunderestimated opportunistic pathogens of the central nervous system viruses 103390v12010014 fazakerley jk and walker r virus demyelination j neurovirol “ 10108013550280390194046 murray rs cai gy hoel k zhang jy soike kf and cabirac gf coronavirus infects and causes demyelination in primate centralnervous system virology “ 101016004268229290757g lane te and buchmeier mj murine coronavirus infection a paradigm for virusinduced demyelinating disease trends microbiol “101016s0966842x97817684 stohlman sa and hinton dr viral induced demyelination brain pathol “ 101111j175036392001tb00384x li z liu t yang n han d mi x li y neurological manifestations of patients with covid19 potential routes of sarscov2neuroinvasion from the periphery to the brain front med “ s1168402007865 netland j meyerholz dk moore s cassell m and perlman s severe acute respiratory syndrome coronavirus infection causes neuronaldeath in the absence of encephalitis in mice transgenic for human ace2 j virol “ 101128jvi0073708 bohmwald k galvez n r´Ä±os m and kalergis am neurologic alterations due to respiratory virus infections front cell neurosci 103389fncel201800386 montalvan v lee j bueso t de toledo j and rivas k neurological manifestations of covid19 and other coronavirus infections asystematic review clin neurol neurosurg 101016jclineuro2020105921 zhou f yu t du r fan g liu y liu z clinical course and risk factors for mortality of adult inpatients with covid19 in wuhanchina a retrospective cohort study lancet “ linkinghubelseviercomretrievepiis0140673620305663101016s0140673620305663 varga z flammer aj steiger p haberecker m andermatt r zinkernagel as endothelial cell infection and endotheliitis incovid19 lancet “ 101016s0140673620309375 bostanciklio ˘glu m sarscov2 entry and spread in the lymphatic drainage system of the brain brain behav immun “101016jbbi202004080 li yc bai wz and hashikawa t the neuroinvasive potential of sarscov2 may play a role in the respiratory failure of covid19 patientsj med virol “ 101002jmv25728 li y bai w hirano n hayashida t taniguchi t sugita y neurotropic virus tracing suggests a membranouscoatingmediatedmechanism for transsynaptic communication j comp neurol “ 101002cne23171 reichard rr kashani kb boire na constantopoulos e guo y and lucchinetti cf neuropathology of covid19 a spectrum ofvascular and acute disseminated encephalomyelitis ademlike pathology acta neuropathol “s00401020021662 kanberg n ashton nj andersson lm yilmaz a lindh m nilsson s neurochemical evidence of astrocytic and neuronal injurycommonly found in covid19 neurology nneurologycontentneurologyearly20200616wnl0000000000010111fullpdf101212wnl0000000000010111 xiao l haack kkv and zucker ih angiotensin ii regulates ace and ace2 in neurons through p38 mitogenactivated protein kinase andextracellular signalregulated kinase signaling am j physiol cell physiol c1073“c1079 101152ajpcell003642012 tang n li d wang x and sun z abnormal coagulation parameters are associated with poor prognosis in patients with novel coronaviruspneumonia j thromb haemost “ 101111jth14768 mehta p mcauley df brown m sanchez e tattersall rs manson jj covid19 consider cytokine storm syndromes andimmunosuppression lancet 101016s0140673620306280 koralnik ij and tyler kl covid19 a global threat to the nervous system ann neurol “ 101002ana25807 toscano g palmerini f ravaglia s ruiz l invernizzi p cuzzoni mg guillainbarr ´e syndrome associated with sarscov2 nengl j med nejmc2009191 101056nejmc2009191 madjid m safavinaeini p solomon sd and vardeny o potential effects of coronaviruses on the cardiovascular system jama cardiol “ jamanetworkcomjournalsjamacardiologyfull2763846 epelman s shrestha k troughton rw francis gs sen s klein al soluble angiotensinconverting enzyme in human heartfailure relation with myocardial function and clinical outcomes j cardiac fail “ 101016jcardfail200901014 wild ps dimmeler s and eschenhagen t an epidemiological study exploring a possible impact of treatment with ace inhibitors orangiotensin receptor blockers on ace2 plasma concentrations j mol cell cardiol “ 101016jyjmcc202003018 wu z and mcgoogan jm characteristics of and important lessons from the coronavirus disease covid19 outbreak in china jama jamanetworkcomjournalsjamafull2762130 101001jama20202648 zheng yy ma yt zhang jy and xie x covid19 and the cardiovascular system nat rev cardiol “101038s4156902003605 ruan q yang k wang w jiang l and song j clinical predictors of mortality due to covid19 based on an analysis of data of patients from wuhan china intensive care med “ s0013402005991x the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20200904 cheng r and leedy d covid19 and acute myocardial injury the heart of the matter or an innocent bystander heart “101136heartjnl2020317025 zhu l carretero oa xu j harding p ramadurai n gu x activation of angiotensin ii type receptor suppresses tnfαinducedicam1 via nfκb possible role of ace2 am j physiol heart circ physiol h827“h834 101152ajpheart008142014 dzimiri n receptor crosstalk implications for cardiovascular function disease and therapy eur j biochem “101046j14321033200203181x wheelerjones cpd cell signalling in the cardiovascular system an overview heart “101136hrt2005072280 houliston ra pearson jd and wheelerjones cpd agonistspecific cross talk between erks and p38 mapk regulates pgi synthesis inendothelium am j physiol physiol c1266“c1276 101152ajpcell20012814c1266 adam lp franklin mt raff gj and hathaway dr activation of mitogenactivated protein kinase in porcine carotid arteries circ res “ 10116101res762183 nebreda ar and porras a p38 map kinases beyond the stress response trends biochem sci “linkinghubelseviercomretrievepiis0968000400015954 101016s0968000400015954 wang y huang s sah vp ross j brown jh han j cardiac muscle cell hypertrophy and apoptosis induced by distinct membersof the p38 mitogenactivated protein kinase family j biol chem “ 101074jbc27342161 saurin at martin jl heads rj foley c mockridge jw wright mj the role of differential activation of p38mitogenactivatedprotein kinase in preconditioned ventricular myocytes faseb j “ 101096fj990671com podewski ek hilfikerkleiner d hilfiker a morawietz h lichtenberg a wollert kc alterations in janus kinase jaksignaltransducers and activators of transcription stat signaling in patients with endstage dilated cardiomyopathy circulation “10116101cir000005754582749ff patel ab and verma a covid19 and angiotensinconverting enzyme inhibitors and angiotensin receptor blockers what is the evidencejama “ jamanet
Colon_Cancer
continuously increasing with development of the economy and the environment [“] the prognosis for hcc patients remains extremely poor although significant progress has been achieved strategies for early diagnosis are urgently needed because the majority of patients with hcc are diagnosed in very late stages however the molecular mechanism of hcc has not been clearly defined circular rnas circrnas are a new class of rna molecules that have functions as regulators of parental gene transcription in alternative splicing and as mirna sponges through use of rna deep sequencing gtechnology numerous circrnas have been identified as the predominant regulatory elements in diseases moreover accumulating evidence shows that circrnas play pivotal roles in many diseases in particular abnormally expressed circrnas are involved in tumor progression including cell proliferation migration and invasion [“] in addition some research indicates that circrnas level are closely correlated wit specific phenotypes and tumorigenesis in hcc [“] nevertheless the research concerning circrnas is frankly in its infancy which greatly hinders the application of circrnas as biomarkers for diagnosis of hcc in clinicsrelated research shows that circrnas possess great potential to be used for diagnosis of hcc recent studies have found that hsa_circ_0067934 plays oncogenic roles by accelerating cell proliferation and metastasis in glioblastoma gbm circsmarca5 was significantly elevated and thereby suppressed cell apoptosis and arrested cell cycle in prostate cancer in addition previous studies have shown that downregulation of hsa_circ_0005986 facilitated cell proliferation by promoting the g0g1 to s phase transition in hcc similarly alteration in expression of circrnas correlated with development and metastasis of malignant tumors these data suggest that circrnas may be of greater benefit in clinical diagnosis of hcc however reliable circrna biomarkers for hcc are still lacking therefore this review synthetically integrates available data on the role of circrna in hcc progression and attempts to provide crucial clues for investigating the molecular mechanism regarding hccoverview of circrnacircrnas are a category of singlestranded closedcircle molecules which take part in multifaceted biological regulation recently research has verified that the majority of circrnas are synthesized by backspliced exons and that others are formed from intron intergenic and untranslated regions utr therefore biogenesis of circrnas can be divided into eicirnas exonintron circrnas ecircrnas circular exonic rnas and cirnas circular intronic rnas meanwhile over circrnas have been identified and this type of transcript has been considered a new form of gene expression generally the structure of the transcription is inverted and the order of genomic exons is altered and these exons are spliced over time the biological functions of circrnas gradually have been recognized including roles in embryonic development maintainenance of homeostasis and promotion of tumor progression figure properties of circrnascircrnas recently have attracted great attention related to their pathological role in disease development compared with linear rnas circrnas have special properties including biological roles and clinical use circrnas are mainly enriched in certain body fluids comprising blood saliva and urine they are covalently closed loop structures degradation of most rna is highly dependent on rna exonuclease or rnase hence circrnas remain highly stable based on their high resistance to enzyme degradation moreover studies have shown that expression of circrnas is tissuespecific and correlated with different phases of development and they exhibit different expression patterns at different developmental stages roles of circrnasaccumulating evidence shows that circrnas play a crucial role in the pathogenesis of diseases as a result of their complex biological functions generally the molecular functions of circrnas mainly include being sponges of mirna acting as rnabinding proteins performing alternative splicing of premrnas regulating transcription and translation and potentially encoding proteins these properties are described in detail belowsponges of mirnathe different types of circrnas have different mirna binding sites some circrnas negatively regulate mirnas by absorbing and specifically binding to mirnas then decreasing mirna activity and elevating expression of mirnarelated target genes researchers have shown that cirs7 inhibits mir7 function and positively mediates mir7 target genes acting as a molecular sponge in addition functional analyses have indicated that circrnas constitute an entire molecular regulatory network which specifically regulates degradation of mirnas as mirna sponges this work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd e9238322indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832premrna™e1e2abcbasepairinge3dguriche4™ecrichpretrnarna bindingproteinsrbp™™™™™™™™gnicilpskcablariat splicingaecircrnaelcirnacirnatrnatricrnafigure1 biogenesis of circular rnas a lariatdriven circularization the ™ hydroxyl of the upstream exon reacts with the ™ phosphate of the downstream exon to form a covalent linkage then producing a lariat including exons and introns the ™ hydroxyl of the ™ intron interacts with the ™ phosphate of the ™intron to form an ecircrna following an interaction between the ™ hydroxyl of the ™ exon and the ™ phosphate of the ™ exon b rnabinding protein rbpdriven circularization rbps accelerate interaction of the downstream intron and upstream intron thereby promoting formation of ecircrna c basepairingdriven circularization the downstream introns and upstream introns are paired depends on inverserepeatingcomplementary sequences formation of ecircrnaeicirna was derived from the introns are removedretained d biosynthesis of cirna formation of cirnas mainly based on a 7nt gurich element and an 11nt crich element to escape debranching and exonucleolytic degradation e formation of tricrna trna splicing enzymes divide pretrna into two parts tricrnas are generated by a ™“™ phosphodiester bond and the other part generates trnascircrnasbinding proteinsrna binding proteins rbps are a broad class of proteins involved in gene transcription translation and interaction studies suggest that distribution of rbps is widespread in many tissue types furthermore rbps participate in development of disorders by regulating posttranscriptional regulation of rnas rbps assemble ribonucleoprotein complexes to bind rna sequences thereby affecting the function of the target rnas previous research has shown that circrnas serve as protein decoys to harbor binding sites of specific proteins and block protein activity circfoxo3 induces cell cycle arrest resulting in defective cdk2 gene function by binding to p21 and cdk2 moreover circrna ciacgas binds to cgas protein and suppresses enzymatic activity of cgas thereby preventing cgas from recognizing selfdna e9238323indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0czou rc research progress on circrnas in hcc med sci monit e923832circrnas regulate alternative splicing transcription and translationcellular localization of most circrnas is cytoplasmic which is the basis for the biological function of mirna and protein decoys several studies have suggested that circrnas participate in rna splicing assembly and biosynthesis recently research has shown that circrnas may play pivotal roles in regulating alternative splicing transcription and translation in addition the exon of the splicing factor may form a circrna by affecting formation of linear rna eicirnas interact with the u1 small nuclear ribonucleoproteinsnrnp thereby regulating parental gene transcription by binding to rna polymerase ii interestingly translation of circrnas is mediated by ires and n6methyladenosine m6a and translation efficiency of circrna is regulated by the level of m6a modification moreover circfbxw7 effectively inhibits glioma proliferation and cell cycle progression by antagonizing usp28induced cmyc stabilization potential to encode proteinscircrnas are implicated in numerous physiological processes and pathogenesis of diseases strong evidence indicates that circrnas can encode proteins by mimicking dna rolling circle amplification related studies indicate that circrna circppp1r12a plays a key molecular role by encoding a functional protein circppp1r12a73aa which promotes proliferation migration and invasion of colon cancer circanril interacts with pescadillo zebrafish homolog pes1 to mediate ribosome biogenesis and prerrna processing in vascular macrophages and smooth muscle cells these studies have significantly increased the knowledge base about the biological functions of circrnascircrnas in diseasescircrnas are involved in processes that lead to development of various disorders such as neuronal and cardiovascular diseases and cancers circrnas participate in regulating gene transcription and protein expression and are indirectly and directly associated with time and regionspecific variations as mentioned previously abnormal expression of circrnas is implicated in neurological disorders atherosclerosis and ribosomal rna maturation reportedly are regulated by circanril simultaneously some studies have suggested that circrnas upregulation significantly affects sprouting and proliferation of vascular endothelial cells and elicits vascular dysfunction recently several experiments have implicated circrnas in pathogenesis of cancer via activation of a series of cascade reactions however the underlying mechanism for the effect of circrnas in initiation and progression of tumors has not been fully clarified to date related studies have revealed that certain circrnas are highly expressed in tumor tissues and overexpression of circrnas promotes tumor proliferation and deterioration an investigation revealed that hsa_circ_002059 was downregulated in gastric cancer while hsa_circ_0004018 was upregulated in hcc meanwhile tumorspecific circrnas candidates were screened in lung adenocarcinoma tissue by microarrays and circrnas were identified downregulated and upregulated of the circrnas hsa_circ_0013958 clearly was positive correlated with lymph node metastasis and tnm stage these findings indicate that circrnas have important roles in tumor progression and may have potential for broad applicatoins in medicine scienceoverview of hcchcc is one of the most prevalent tumors worldwide with diagnoses and approximately deaths annually epidemiological survey data indicate that morbidity and mortality from hcc are gradually increasing risk factors for hcc include diabetes mellitus obesity smoking alcohol consumption older age male sex chronic hbv liver cirrhosis and chronic hepatitis c virus hcv the primary risk factors include liver cirrhosis viral hepatitis alcohol intake and obesity worldwide approximately hcc patients are infected with hepatitis b virus hbv or hcv in addition alcohol abuse is a crucial factor for onset of hcc [“] obesity hypertension and diabetes are closely linked with development of hcc but specific correlations remain unknown moreover regular screening has been widely applied for early detection and to ensure effective treatment of hcc most commonly good results have been achieved with regular screening with ultrasonography blood alphafetoprotein content testing mri and ct generally surgical resection and chemotherapy are mainstays of therapy in patients with hcc yet some tumors cannot be fully removed which results in tumor growth invasion and metastasis local and systemic metastases are the main reasons for the unsatisfactory prognosis in patients with hcc therefore more effective therapeutic approaches need to be developedroles of circrnas in hccnumerous studies have documented the important role that circrnas play in tumorigenesis metastasis and invasion research has shown that circrnas are localized in the nucleus and interfere with transcription and promote alternative this work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd e9238324indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832hsa_circ_0001649circzkscan1circitchwntbetacatenincircmto1mir9p21hsa_circ_00059836mir1295phmgb1 ragenfκbmir7hsa_circ_101368hsa_circ_001569cdr1ashsa_circ_0000673figure the function of circrnas in hcc carcinogenesis this graph demonstrates the role of circrnas in hcc carcinogenesis including positive and negative effects respectivelytable brief summary of circrnas as biomarkers for hccnamediseaseconclusiondoicirs7hsa_circ_0003570hsa_circ_0005075hepatocellular carcinomahepatocellular carcinomahepatocellular carcinomacirs7 was one of the independent factors and may be a promising biomarker for hepatic mvi and a novel therapy target for restraining mvi101007s0043201622567hsa_circ_0003570 expression levels were associated with hcc clinicopathological characteristics101002jcla22239hsa_circ_0005075 promotes proliferation migration and invasiveness of hcc via mir431 regulation101016jbiopha201801150splicing circpvt1 is overexpressed in gastric cancer tissues compared with nontumor tissues and circpvt1 acts as an oncogene to mediate expression of mir4975p however studies concerning the role of circrnas in development and progression of hcc remain in their infancytumor inhibitioncurrently circrnas are considered promising diagnostic biomarkers and ideal therapeutic targets for hcc studies have revealed that circitch inhibits tumor proliferation by suppressing the wntbetacatenin pathway expression of circitch has been positively correlated with good survival outcome in patients with hcc analysis of the circrnas expression profile in human hcc tissues showed that circmto1 was markedly decreased in hcc tissues and that expression of circmto1 was positively correlated with survival rate circmto1 reportedly inhibits hcc progress by sponging mir9 and thereby increasing p21 expression meanwhile overexpression of hsa_circ_0001649 negatively affects invasion and proliferation and promotes apoptosis of hcc cells downregulation of zkscan1 and circzkscan1 enhances cell proliferation and promotes progression of hcc tumor promotionin patients with hcc cdr1was more abundant in tumor specimens than in adjacent normal tissues cdr1as effectively suppresses the invasion and proliferation of hcc cells by targeting mir7 some reports have shown that hsa_circ_0000673 is significantly upregulated in hcc tissues and hsa_circ_0000673 downregulation markedly inhibits proliferation and invasion of hcc cells in vitro meanwhile a positive correlation was found between circ_001569 expression level and tumor size advanced tnm stages and unfavorable prognosis in patients with hcc circrna101368 was abundantly expressed in hcc tissue which correlated with poorer prognosis in addition circrna101368 inhibited cell migration by reducing protein levels in nfkb rage and hmgb1 figure e9238325indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0czou rc research progress on circrnas in hcc med sci monit e923832biomarkerconclusionsprevious studies have shown that circrnas are closely related to development of tumors clinicopathological features in patients with hcc are correlated to with levels of expression of cirs7 and its targeted mrnas global circrna expression profile analysis showed that hsa_circ_0005075 exhibited significant differences in tumor tissue versus adjacent tissues in patients with hcc expression of hsa_circ_0005075 also was related to tumor proliferation and metastasis therefore an increasing number of circrnas have been identified as diagnostic markers as summarized in table given the high incidence and mortality fo hcc worldwide it is one of the most serious diseases threatening human health increasing attention is being paid due to this serious situation evidence is increasing to support the close association between circrnas progression of hcc circrnas may play an important role in the occurrence and development of tumors however the molecular mechanism underlying the relationship between circrnas and hcc has not been fully elucidated therefore indepth research is needed on the potential regulatory relationships and to uncover regulatory patterns between circrnas and hcc so that new diagnostic markers for hcc can be developedreferences bray f ferlay j soerjomataram i global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries cancer j clin “ feng rm zong yn cao sm xu rh current cancer situation in china good or bad news from the global cancer statistics cancer commun lond jemal a bray f center mm global cancer statistics cancer j clin “ li r jiang j shi h circrna a rising star in gastric cancer cell mol life sci “ salzman j gawad c wang pl circular rnas are the predominant transcript isoform from hundreds of human genes in diverse cell types plos one e30733 lukiw wj circular rna circrna in alzheimer™s disease ad front genet liu y yang y wang z insights into the regulatory role of circrna in angiogenesis and clinical implications atherosclerosis “ zhao y alexandrov pn jaber v lukiw wj deficiency in the ubiquitin conjugating enzyme ube2a in alzheimer™s disease ad is linked to deficits in a natural circular mirna7 sponge circrna cirs7 genes basel shen f liu p xu z circrna_001569 promotes cell proliferation through absorbing mir“ in gastric cancer j biochem “ song t xu a zhang z circrna hsa_circrna_101996 increases cervical cancer proliferation and invasion through activating tpx2 expression by restraining mir8075 j cell physiol “ min l wang h zeng y circrna_104916 regulates migration apoptosis and epithelial“mesenchymal transition in colon cancer cells front biosci landmark ed “ verduci l strano s yarden y blandino g the circrnamicrorna code emerging implications for cancer diagnosis and treatment mol oncol “ wei j wei w xu h circular rna hsa_circrna_102958 may serve as a diagnostic marker for gastric cancer cancer biomark “ li p chen s chen h using circular rna as a novel type of biomarker in the screening of gastric cancer clin chim acta “ xin j zhang xy sun dk upregulated circular rna hsa_circ_0067934 contributes to glioblastoma progression through activating pi3kakt pathway eur rev med pharmacol sci “ kong z wan x zhang y androgenresponsive circular rna circsmarca5 is upregulated and promotes cell proliferation in prostate cancer biochem biophys res commun “ fu l chen q yao t hsa_circ_0005986 inhibits carcinogenesis by acting as a mir1295p sponge and is used as a novel biomarker for hepatocellular carcinoma oncotarget “ zhu x wang x wei s hsa_circ_0013958 a circular rna and potential novel biomarker for lung adenocarcinoma febs j “ zhang q wang w zhou q roles of circrnas in the tumour microenvironment mol cancer qu z jiang c wu j ding y exosomes as potent regulators of hcc malignancy and potential biotools in clinical application int j clin exp med “ memczak s jens m elefsinioti a circular rnas are a large class of animal rnas with regulatory potency nature “ cocquerelle c mascrez b hetuin d bailleul b missplicing yields circular rna molecules faseb j “ zhao x cai y xu j circular rnas biogenesis mechanism and function in human cancers int j mol sci qu s yang x li x circular rna a new star of noncoding rnas cancer lett “ bahn jh zhang q li f the landscape of microrna piwiinteracting rna and circular rna in human saliva clin chem “ hsu mt cocaprados m electron microscopic evidence for the circular form of rna in the cytoplasm of eukaryotic cells nature “ yu x odenthal m fries jw exosomes as mirna carriers formationfunctionfuture int j mol sci hanan m soreq h kadener s circrnas in the brain rna biol “ constantin l circular rnas and neuronal development adv exp med biol “ van rossum d verheijen bm pasterkamp rj circular rnas novel regulators of neuronal development front mol neurosci ebert ms sharp pa microrna sponges progress and possibilities rna “ ebert ms neilson jr sharp pa microrna sponges competitive inhibitors of small rnas in mammalian cells nat methods “ hansen tb jensen ti clausen bh natural rna circles function as efficient microrna sponges nature “ hsiao ky lin yc gupta sk noncoding effects of circular rna ccdc66 promote colon cancer growth and metastasis cancer res “ janga sc mittal n construction structure and dynamics of posttranscriptional regulatory network directed by rnabinding proteins adv exp med biol “ du ww yang w liu e foxo3 circular rna retards cell cycle progression via forming ternary complexes with p21 and cdk2 nucleic acids res “ xia p wang s ye b a circular rna protects dormant hematopoietic stem cells from dna sensor cgasmediated exhaustion immunity “701e7 li z huang c bao c exonintron circular rnas regulate transcription in the nucleus nat struct mol biol “this work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd e9238326indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832 yang y fan x mao m extensive translation of circular rnas driven by n6methyladenosine cell res “ yang y gao x zhang m novel role of fbxw7 circular rna in repressing glioma tumorigenesis j natl cancer inst “ abe n hiroshima m maruyama h rolling circle amplification in a prokaryotic translation system using small circular rna angew chem int ed engl “ zheng x chen l zhou y a novel protein encoded by a circular rna circppp1r12a promotes tumor pathogenesis and metastasis of colon cancer via hippo“yap signaling mol cancer holdt lm stahringer a sass k circular noncoding rna anril modulates ribosomal rna maturation and atherosclerosis in humans nat commun gokul s rajanikant gk circular rnas in brain physiology and disease adv exp med biol “ idda ml munk r abdelmohsen k gorospe m noncoding rnas in alzheimer™s disease wiley interdiscip rev rna luo q chen y long noncoding rnas and alzheimer™s disease clin interv aging “ li cy ma l yu b circular rna hsa_circ_0003575 regulates oxldl induced vascular endothelial cells proliferation and angiogenesis biomed pharmacother “ chen j cui l yuan j circular rna wdr77 target fgf2 to regulate vascular smooth muscle cells proliferation and migration by sponging mir biochem biophys res commun “ “ kristensen ls hansen tb veno mt kjems j circular rnas in cancer opportunities and challenges in the field oncogene “ fu l yao t chen q screening differential circular rna expression profiles reveals hsa_circ_0004018 is associated with hepatocellular carcinoma oncotarget “ massarweh nn elserag hb epidemiology of hepatocellular carcinoma and intrahepatic cholangiocarcinoma cancer control salem r gilbertsen m butt z increased quality of life among hepatocellular carcinoma patients treated with radioembolization compared with chemoembolization clin gastroenterol hepatol “65e1 ozer ed suna n boyacioglu as management of hepatocellular carcinoma prevention surveillance diagnosis and staging exp clin transplant 15suppl “ lou w liu j ding b identification of potential mirnamrna regulatory network contributing to pathogenesis of hbvrelated hcc j transl med yang t hu ly li zl liver resection for hepatocellular carcinoma in nonalcoholic fatty liver disease a multicenter propensity matching analysis with hbvhcc j gastrointest surg “ nishibatake km minami t tateishi r impact of directacting antivirals on early recurrence of hcvrelated hcc comparison with interferonbased therapy j hepatol “ toyoda h kumada t tada t the impact of hcv eradication by directacting antivirals on the transition of precancerous hepatic nodules to hcc a prospective observational study liver int “ zhao j o™neil m vittal a prmt1dependent macrophage il6 production is required for alcoholinduced hcc progression gene expr “ vandenbulcke h moreno c colle i alcohol intake increases the risk of hcc in hepatitis c virusrelated compensated cirrhosis a prospective study j hepatol “ fabris c toniutto p falleti e mthfr c677t polymorphism and risk of hcc in patients with liver cirrhosis role of male gender and alcohol consumption alcohol clin exp res “ vernon g baranova a younossi zm systematic review the epidemiology and natural history of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis in adults aliment pharmacol ther “ bruix j reig m sherman m evidencebased diagnosis staging and treatment of patients with hepatocellular carcinoma gastroenterology “ zhang bh yang bh tang zy randomized controlled trial of screening for hepatocellular carcinoma j cancer res clin oncol “ verduci l ferraiuolo m sacconi a the oncogenic role of circpvt1 in head and neck squamous cell carcinoma is mediated through the mutant p53yaptead transcriptioncompetent complex genome biol yu j xu qg wang zg circular rna csmarca5 inhibits growth and metastasis in hepatocellular carcinoma j hepatol “ wang m yu f li p circular rnas characteristics function and clinical significance in hepatocellular carcinoma cancers basel guo w zhang j zhang d et al polymorphisms and expression pattern of circular rna circitch contributes to the carcinogenesis of hepatocellular carcinoma oncotarget “ han d li j wang h circular rna circmto1 acts as the sponge of microrna9 to suppress hepatocellular carcinoma progression hepatology “ qin m liu g huo x hsa_circ_0001649 a circular rna and potential novel biomarker for hepatocellular carcinoma cancer biomark “ yao z luo j hu k zkscan1 gene and its related circular rna circzkscan1 both inhibit hepatocellular carcinoma cell growth migration and invasion but through different signaling pathways mol oncol “ xu l zhang m zheng x the circular rna cirs7 cdr1as acts as a risk factor of hepatic microvascular invasion in hepatocellular carcinoma j cancer res clin oncol “ yu l gong x sun l the circular rna cdr1as act as an oncogene in hepatocellular carcinoma through targeting mir7 expression plos one e0158347 jiang w wen d gong l circular rna hsa_circ_0000673 promotes hepatocellular carcinoma malignance by decreasing mir7673p targeting set biochem biophys res commun “ liu h xue l song c overexpression of circular rna circ_001569 indicates poor prognosis in hepatocellular carcinoma and promotes cell growth and metastasis by sponging mir4115p and mir4325p biochem biophys res commun “ li s gu h huang y circular rna 101368mir200a axis modulates the migration of hepatocellular carcinoma through hmgb1rage signaling cell cycle “ “ shang x li g liu h comprehensive circular rna profiling reveals that hsa_circ_0005075 a new circular rna biomarker is involved in hepatocellular carcinoma development medicine baltimore e3811 yao t chen q shao z circular rna as a new biomarker for hepatocellular carcinoma metastasis j clin lab anal e22572e9238327indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0c'
Colon_Cancer
overexpression of epithelial cell adhesion molecule epcam has been associated with chemotherapeutic resistanceleads to aggressive tumor behavior and results in an adverse clinical outcome the molecular mechanism by whichepcam enrichment is linked to therapeutic resistance via nrf2 a key regulator of antioxidant genes is unknown wehave investigated the link between epcam and the nrf2 pathway in light of therapeutic resistance using head andneck squamous cell carcinoma hnscc patient tumor samples and cell lines we report that epcam was highlyexpressed in nrf2positive and hpvnegative hnscc cells in addition cisplatinresistant tumor cells consisted of ahigher proportion of epcamhigh cells compared to the cisplatin sensitive counterpart epcamhigh populationsexhibited resistance to cisplatin a higher efficiency in colony formation sphere growth and invasion capacity anddemonstrated reduced reactive oxygen species ros activity furthermore nrf2 expression was significantly higher inepcamhigh populations mechanistically expression of nrf2 and its target genes were most prominently observed inepcamhigh populations silencing of epcam expression resulted in the attenuation of expressions of nrf2 and sod1concomitant with a reduction of sox2 expression on the other hand silencing of nrf2 expression rendered epcamhighpopulations sensitive to cisplatin treatment accompanied by the inhibition of colony formation sphere formation andinvasion efficiency and increased ros activity the molecular mechanistic link between epcam expression andactivation of nrf2 was found to be a concerted interaction of interleukin6 il6 and p62 silencing of p62 expressionin epcamhigh populations resulted in the attenuation of nrf2 pathway activation suggesting that nrf2 pathwayactivation promoted resistance to cisplatin in epcamhigh populations we propose that therapeutic targeting the nrf2epcam axis might be an excellent approach to modulate stress resistance and thereby survival of hnscc patientsenriched in epcamhigh populationscorrespondence syed s islam drsyedsislamgmailcom1department of biochemistry and molecular biology university of chittagongchittagong bangladesh2department of pathology mcgill university montreal qc canadafull list of author information is available at the end of the edited by b zhivotovskyintroductionhead and neck squamous cell carcinoma hnsccaffects more than patients per year12 resistanceto chemotherapeutic drugs limits the overall treatment the authors open access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate ifchanges were made the images or other third party material in this are included in the ™s creative commons license unless indicated otherwise in a credit line to the material ifmaterial is not included in the ™s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this license visit httpcreativecommonslicensesby40official of the cell death differentiation association 0coutcome in hnscc patients3 response to chemotherapeutic drugs is partly mediated by the keap1nrf2 signaling system4 nrf2nfe2l2 nuclear factor erythroid2like is a key transcription factor which in the normalbasal state functions as cytoprotective response to oxidative and electrophilic stress under oxidative stressstate nrf2 dissociates from cytoplasmic inhibitor keap1kelch like echassociated protein translocate into thenucleus and activates nrf2 transcriptional genes andprotects cells against oxidative stress mediates detoxification and participates in atpdependent drug efflux5abnormalities of keap1nrf2 pathwaylead to amechanism of oncogenesis and chemo and radioresistance in a variety of cancers including hnscc4inhibition of nrf2 expression by sirna augmentedcarboplatininduced tumor growth inhibition in a xenograft mouse model6 recent studies have indicated thatkeap1nrf2 pathway is engaged in sustaining csc cancerstem celllike properties in cancers and causes resistanceto therapeutic agentscscs exhibit enhanced selfrenewal properties lead todisease recurrence and most importantly exhibit thestrongest therapeutic resistance within the tumor cellspopulation7“ elevated expression of nrf2 target genescontribute to therapeutic resistance and facilitate survivalof cscs10 several cell surface markers such as cd44cd133 cd24 cd49f and aldh have been proposed forthe detection and isolation of cscs from tumors1112many studies also emphasized the potential use of epithelial cell adhesion molecule epcam as a marker ofcscs due to its ubiquitous overexpression in tumors13epcam was originally identified as a novel tumorspecificcell surface antigen and overexpressed in a large numberof cancers14“ and involved in cell migration proliferation and differentiation18 due to its wide expressionepcam may be a potential target for molecular intervention for therapeutically resistant tumors and requiresfurther investigationa recentstudy reported that nrf2 knockdowninhibits the selfrenewal capacity of glioma stemcells19 furthermore nrf2 signaling is activated inspheroids in breast and colon cancer cells where highnrf2 activity in spheroids has correlated with therapeutic resistance20 however it is unknown how thenrf2 pathway and epcam interact and play roles inthe development of chemotherapeutic resistance inview of the importance of epcam and nrf2 signalingin the development of chemoresistance and the limited understanding of the link between epcam andthe nrf2 pathway we investigated the potential role ofnrf2 signaling in cscs with special emphasis onepcamenriched cells that leads to chemotherapeuticresistanceofficial of the cell death differentiation associationresultscancer stem cell markers are upregulated in hpvnegativeand nrf2 overexpressing hnscc tumorsgiven the role of nrf2 signaling in chemotherapeuticresistance and csc survival2122 we first explored theexpression of several prominent csc markers in hnsccusing cases from tcga dataset we used normalizedmrna zscores and compared several csc markers withinnrf2high and nrf2low tumors statistically significantdifferences were obtained for all csc markers comparingthe nrf2high and nrf2low groups with the most significant relationship found in epcam p fig 1asince hpv human papillomavirus has emerged as anovel risk factor for hnsccs23 we therefore comparednrf2 expression in hpvpositive and hpvnegative patientgroups from our own archived tumor samples no significant differences were noted between the hpv groupsand nrf2 expression p fig 1b a significantexpression difference was noted in epcam cd49f andstemness factor sox2 p p p fig 1cin hpvnegative versus hpvpositive groups in additioncd44 p cd49fp epcam p and sox2 p showed significantly higherexpressions in the nrf2high group fig 1d csc markersincluding sox2 were significantly increased in the tumortissue compared with matched normal tissues fig 1eepcam is expressed in cisplatin resistant cells and epcaminhibition sensitizes cells to cisplatin and inhibits hnscccell proliferationtumors cisplatin resistantnext we evaluated epcam transcript levels from agroup of cisplatin resistant n and sensitive n hnscc patienttumors showed relatively high expression of epcamcompared with cisplatin sensitive tumors fig2a this finding led us to test the cisplatin resistancein vitro for which we established a line of cisplatinresistant fadu cells termed as fadures we established acisplatinresistant fadures cells by maintaining parentalfadu cells in a series of cisplatin concentrations for weeks before these cells were stably grown in μmcisplatin as shown in supplementary fig s1a fadurescells exhibited higher resistance to cisplatin treatmentcompared to parental fadu cells we then treated faducells μm of cisplatin for days and analyzed the epcamexpression by western blot fadu cells maintained in μm of cisplatin showed higher epcam expression incontrast to untreated parental cells supplementary figs1b to assess the role of epcam in cisplatin resistanceuntreated patient tumor cells scc15 and fadu cellswere transfected with siepcam and siscramble for hwashed and followed by cisplatin treatment for an additional h and assessed for cell viability supplementary 0cnoman et al cell death and disease page of fig cancer stem cell markers cscs are upregulated in hpvnegativenrf2 overexpressing hnscc tumors a cd44 cd133 epcam andcd49f were compared between nrf2high and low groups using the tcga dataset ratios were calculated by dividing the mrna expression of thenrf2high group by that of the nrf2low group b nrf2 expression was compared between hpv and hpvˆ’ group using our own dataset n ratio was calculated by dividing the expression intensity of the hpv group by that of the hpvˆ’ group c cscs expression comparedbetween hpv and hpvˆ’ group ratio was calculated by dividing the expression intensity of the hpv group by that of the hpvˆ’ group dexpression of cscs was compared between nrf2high and nrf2low group ratios were calculated by dividing the expression intensity of the nrf2high group by that of the nrf2low group e cancer stem markers were compared between hnscc and matched normal tissues ratio was calculatedby dividing the mrna expression of the tumor sample by that of the matched normal samples in all cases whiskers indicate the maximum and theminimum values pvalues were calculated using student™s t testfig s2 whereas parental cells were found to be somewhat resistantto cisplatin treatment knockdown ofepcam with siepcam enhanced the sensitivity to cisplatin treatment fig 2b to examine the resistancefurther cells were treated with different concentrations ofcisplatin and determined the ec50 fig 2c furthermoresilencing epcam significantly reduced epcam transcriptlevel and inhibited cell proliferation fig 2d echemotherapeutic resistance is associated with increasednrf2 transcriptional activity and epcam overexpressionit was reported that inhibition of nrf2 reverses theresistance to cisplatin of hnscc cells22 to further assessthe role of nrf2 in chemotherapeutic resistance wecompared nrf2 target genes in cisplatin sensitive n and resistant n hnscc patients™ tumor cells by realtime quantitative polymerase chain reaction qrtpcrthe unsupervised heat map analysis showed that nrf2target genes sod2 slc3a1 akrc1 gclc ho1nqo1 and sod1 were highly upregulated in thecisplatinresistant tumor cells compared to the cisplatinsensitive tumor counterparts fig 3a suggesting thatcisplatin treatment potentially plays a significant role innrf2 pathway activation to establish the link betweennrf2 and epcam in resistance freshly isolated cisplatinresistant n and sensitive n patient tumor cellswere subjected to flow cytometry analysis and quantifiedthe epcam expression approximately epcampositive cell population was found in cisplatin resistanttumors while onlycella epcampositiveofficial of the cell death differentiation association 0cnoman et al cell death and disease page of fig epcam is expressed in cisplatin resistant cells and epcam inhibition sensitizes hnscc cells to cisplatin and inhibits hnscc cellproliferation a expression of epcam mrna in hnscc patients™ cisplatin resistant and sensitive tumor cells b doseresponse and cell viability ofhnscc patient tumor cells top panel scc15 middle panel and fadu bottom panel cells cell viability of siepcam and scrambled sirnatransfected cells were monitored following exposures of cells to different concentrations of cisplatin c ec50 of cisplatin in parental siscrambled andsiepcam transfected patient tumor cells top panel scc15 middle panel and fadu bottom panel cells the ec50 differences between siscrambleand siepcam cells were compared d relative epcam mrna expression in hnscc patient tumor cells top panel scc15 middle panel and fadubottom panel cells following transfection of cells by siscrambled and siepcam pvalues were calculated using student™s t test e cell proliferationwas determined following transfection of cells by siscrambled and siepcam siscrambled and siepcam cell growth was compared on day nsdenote not significant p p p population was detectable in cisplatin sensitive tumorsfig 3b based on these epcam cell fractions in resistantand sensitive groups we hereafter termed these twopopulations epcamhigh and epcamlow immunostainingfor epcam in cisplatin resistance n tissues showedenhanced expression of epcam fig 3c fluorescenceactivated cell sorting facs sorted epcamhigh cellofficial of the cell death differentiation associationfraction was highly resistant to cisplatin compared to theepcamlow cell fraction fig 3d etumorsresistantfunctionallyto cisplatin showedenhanced expression of epcam coupled with theincreased level of sox2 and abcg5 fig 3f indicatingenrichment of epcam coincident with stemlike anddrug resistant features in cells we then analyzed nrf2 0cnoman et al cell death and disease page of fig chemotherapeutic resistance is associated with increased nrf2 transcriptional activity and epcam overexpression a heat map ofhierarchically clustering based on the expression of nrf2 pathway target genes reveals distinct expressions in cisplatin resistant and sensitive patienttumor cells significantly upregulated gene expression intensity marked as red and downregulated genes are marked as blue b hnscc patient tumorcells were isolated from treatment resistant and sensitive patients and epcam positive cells identified by flow cytometry c immunofluorescenceimages of epcam expression were captured from the cultured cisplatin resistant and sensitive hnscc patient tumor cells scale bar µm d cellviability determination in parental epcamhigh and epcamlow cells after treating the cells with different cisplatin concentrations e apoptotic celldetermination in epcamhigh and epcamlow cells after cisplatin µm treatment f epcam sox2 and abcg5 protein levels were determined fromcisplatin resistant and sensitive patient tumor cells by western blotting g transcript levels of epcam and nrf2 in hnscc patient tumor cells assessedby qrtpcr values represents ±sd for three independent experiments h“i transcript levels of epcam and nrf2 in scc15 and fadu cells assessed byqrtpcr values represents ±sd for three independent experiments j nrf2 and sod1 protein expression in cisplatintreated scc15 and fadu cellsand epcam transcript levels by qrtpcr and found thatboth transcripts were increased in cisplatinresistanttumor cells fig 3g suggesting that resistance to cisplatin is due in part to an increased level of nrf2 transcriptional activity and epcam overexpression toconfirm this finding in cell lines scc15 and fadu cellswere treated with cisplatin μm for days and wereassessed for the level of nrf2 and epcam transcriptscisplatin treatment significantly increased the nrf2 andepcam expression levels fig 3h“j immunoblot analysis confirmed that both nrf2 and sod1 expression werehigher in cisplatin treated cells fig 3jnrf2 pathway is predominantly activated in epcamhighcells and epcam knockdown inactivates the nrf2arepathwaytumorto explore if the nrf2 pathway is exclusively activatedin epcamhigh cells freshly isolated cisplatinresistant andsensitive patientcells were facs sortedepcamhigh cells were predominantly detected in thecisplatinresistant cell fraction compared to sensitive cellsfig 4a cells were treated either with cisplatin or vehiclefor days and analyzed by flow cytometry the resultscorroborated the results obtained in patient tumor cellsfig 4a next we cultured cisplatin treated fadu cells inofficial of the cell death differentiation association 0cnoman et al cell death and disease page of fig nrf2 pathway is predominantly activated in epcamhigh cells and epcam knockdown inactivates the nrf2are pathway a epcamhighand epcamlow cells were determined in hnscc patient tumor cells scc15 and fadu cells using flow cytometry b total cellular protein levels ofepcam nrf2 and sod1 were determined in epcamhigh and epcamlow cells by western blot analysis c nrf2 transcript levels in epcamhigh andepcamlow cells p compared to epcamlow group d sod1 nqo1 and akrc1 transcript levels in epcamhigh and epcamlowcells p compared to epcamlow group e fadu cells were transfected with siepcam and scrambled sirna and epcam nrf2 sod1 and sox2 transcript levelswere determined by qrtpcr analysis in epcamhigh cells f g protein levels of epcam nrf2 sod1 and sox2 were determined by western blotanalysis in fadu and scc15 hnscc cells after silencing epcam in epcamhigh cells h a luciferase assay was used to detect reporter gene activity fromares i immunostaining of hnscc cells stained with epcam green nrf2 red and dapi blue after epcam knockdown scale bar μmgrowth supplemented csc medium for days facssorted for epcamhigh and epcamlow cells were recultured in csc medium for an additional daysepcamhigh cells overexpressed epcam nrf2 and sod1proteins and nrf2 transcripts fig 4b c in additionepcamhigh cells overexpressed sod1 nqo1 andakrc1 transcripts fig 4d these results indicated thatthe nrf2 signaling pathway is exclusively activated in theepcamhigh cells to determine whether the elevated nrf2in epcamhigh cells is epcam dependent welevelsilenced epcam expression by siepcam and observedthat epcam nrf2 sod1 and sox2 proteins and transcripts were attenuated in epcamhigh cells fig 4e“gthese observations prompted us to hypothesize thatepcam might regulate the expression of antioxidantfactors via the nrf2are antioxidant response elementsofficial of the cell death differentiation association 0cnoman et al cell death and disease page of fig nrf2 inhibition in epcamhigh cells sensitizes cells to cisplatin treatment coupled with the abrogation of production of reactiveoxygen species ros a epcamhigh and epcamlow cells were determined in hnscc patient tumor cisplatinresistant and untreated tumor cellsusing flow cytometry legend 1untreated and 2cisplatin resistant patient tumor cells b protein levels of nrf2 and sod1 were measured insinrf2 silenced and siscrambled epcamhigh cells by western blot c epcam protein was measured in sinrf2 silenced and siscramble cells by westernblot d sox2 and abcg5 protein levels were determined in sinrf2 silenced and siscrambled epcamhigh cells by western blot e cell viability wasanalyzed after incubation of cisplatin for h in sinrf2 and siscrambled epcamhigh cells e f ros activity was measured from e patient tumor cellsand f fadu cells facs sorted epcamhigh and epcamlow cells after cisplatin or sinrf2rna treatment values represent ±sd from triplicate sampledwells p compared with untreated groupspathway this hypothesis was tested by transfection offadu and scc15 cells with an are luciferase reporterhnscc cells with or without epcam knockdown weretransiently transfected with an are luciferase reporterplasmid at h post transfection the cells were assayedfor luciferase activity epcam knockdown decreased theluciferase reporter activity with a comparable decreasedstaining intensity in epcam and nrf2 fig 4h i theseresults suggest that the inhibitory effects of epcamknockdown on cell growth and cisplatin resistance correlates with the degree of nrf2 activation in csclikeepcamhigh cellsnrf2 inhibition in epcamhigh cells sensitizes cells tocisplatin treatment coupled with the abrogation ofproduction of reactive oxygen speciesan increasing number of reports suggest that cisplatinmediated csc enrichment and resulting resistance substantially limits the positive outcome of the disease24furthermore in a group of hnscc patient tumors highexpression of epcam has been reported to correlate withtherapeutic resistance2526 to explore the possible functionallink between chemotherapeutic resistance andepcam we first sorted epcamhigh cells by flow cytometry from cisplatin and vehicletreated fadu cells andfound that higher percentage of epcamhigh cells vs fig 5a in cisplatin treated cells knockdown ofnrf2 in epcamhigh cells attenuated the expression ofnrf2 and nrf2 target gene sod1 proteins fig 5b concomitant with the attenuation in expression of epcamsox2 and abcg5 fig 5c d additionally nrf2 silencingin epcamhigh cells showed a significant increased sensitivity to cisplatin treatment fig 5evarious antioxidant enzymes are induced by nrf2pathway activation that reduce the intracellular ros levelresulting in cells becoming drug resistant27 hence wespeculated that chemotherapeutic resistance might likelybe due to the reduction of ros in epcamhigh cells toaddress this possibility we used two approaches to analyze mitochondrial ros generation first we sortedepcamhigh and epcamlow cells from treatment naivepatient tumor cells and fadu cells treated cells withofficial of the cell death differentiation association 0cnoman et al cell death and disease page of cisplatin and measured the mitochondrial ros using afluorescent indicator ros activity was decreased at and μm cisplatin concentrations in epcamhigh cellswhile increased in epcamlowcells fig 5f suggesting thattherapeutic resistance was partly caused by reducing rossecondly we knocked down nrf2 by sinrf2rna incisplatintreated fadu cells and measured the ros levelros levels steadily increased in both epcamhigh andepcamlow cells fig 5gnrf2 inhibition eliminates colonyforming capacity spheregrowth and invasion capacity in epcamhigh cellswe hypothesize that cells overexpressing epcam mayacquire higher colony forming capacity increased spheregrowth and invasion capacity to test this fadu andscc15 cells were grown in growth factor supplementedcsc medium for days to allow epcam enrichmentfacs sorted and quantified for the percent of epcamhighand epcamlow cells sorted cells were evaluated for thedegree of colonyforming capacity sphere formation andinvasiveness epcamhigh populations are highly efficientin forming colonies sphere growth and invasive capacitycompared to epcamlow cells fig 6a“c knockdown ofnrf2 in epcamhigh cells demonstrated reduced colonyformation sphere growth and invasive capacity as compared to scramble sirna treated cells fig 6d“finterleukin6 and p62 are involved in the activation of thenrf2 pathway and resistance to cell death in epcamhighcellsaccumulating evidence indicates that both interleukin il6 and the nrf2mediated antioxidant pathwaycontribute to chemotherapeutic resistance in oral squamous cell carcinoma2829 to confirm the role of il6 inthe activation of nrf2 in epcamhigh cells we assessed il mrna transcripts from a group of hnscc patienttumors treated either with chemotherapy cisplatin n doxorubicin n or chemoradiotherapy crt n or tumors obtained after debulking surgery n without treatment il6 mrna was increased in thechemotherapy and chemoradiotherapy tumors comparedwith matched adjacent normal and surgery alone tumorfig 7a to determine the effects ofil6 on theexpression of nrf2 fadu cells were treated with eithercisplatin μm or il6 pgml alone or in a combination of cisplatin and il6 and assessed for nrf2expression by immunofluorescence labeling a detectableincrease in nrf2 expression in the cytoplasm and nucleuswas observed in the cisplatintreated cells fig 7baddition of il6 significantly increased the cytoplasmicand nuclear nrf2 expression fig 7b western blot analysis showed that il6 treatment activated expression ofnrf2 in cisplatin treated cells fig 7c no changes inkeap1 mrna and protein expression levels wereofficial of the cell death differentiation associationobserved fig 7d next we determined whether il6plays role in preventing or reducing ros activity undercisplatin and il6 treatment conditions we found thattreatment with il6 alone reduces ros generation whilecells treated with cisplatin and il6 in combination further reduces the level of ros fig 7e tocilizumab is ahumanized antihuman il6 receptor monoclonal antibody which has been shown to controls resistance toradiation by suppressing oxidative stress via nrf2 pathway28 cisplatintreated cells undergoing il6 and tocilizumab ngml treatment were analyzed by westernblot for the expressions of sod1 and nrf2 il6 alonetreatment enhanced sod1 expression via the nrf2 pathway while tocilizumab inhibited the expression fig 7fin addition il6 treatment significantly reduced the rosproduction while tocilizumab inhibited fig 7g suggesting that il6 is likely involved in the activation of nrf2and plays a role in therapeutic resistance by reducing rosactivityto analyze the possible involvement of p62 in nrf2activation in the chemotherapeutic resistant epcamhighcells p62 protein was analyzed by western blotting p62protein in the epcamhigh cells was increased concomitant with an increase in microtubuleassociatedprotein 1a1b light chainii lc3b fig 7h it appearslikely that the increase in p62 is directly related toepcam expression fig 7h knockdown of epcamdiminished p62 expression suggesting a correlationbetween epcam and p62 fig 7i accordingly epcamsilencing in fadu cells depleted the growth of spheresfig 7i silencing of p62 by p62sirna revealed theinhibition of nrf2 p62 and sod1 fig 7j furthermorep62 knockdown also diminished the efficiency of spheregrowth fig 7j interestingly keap1 expression levelincreased following p62mediated silencing fig 7j theexpression of epcam remained unchanged after p62mediated silencing suggesting epcammediated p62upregulation in these cells fig 7j k the expression levelof lc3b was also reduced during p62mediated silencingfig 7j k silencing of p62 further caused the reductionin expression of nrf2 target genes sod2 ho1 andakrc1 fig 7l all together these results suggested thatnrf2 activation in epcamhigh csclike cells were associated with the increased levels of il6 and p62 inhnscc cellsdiscussionin this study we have shown the role of the nrf2pathway activation because the cellular response to electrophilic agents is partially mediated by this pathway andlikely plays a significant role in therapeutic resistancethrough activation of nrf2 enrichment of cscs andlowering of ros activity we report that increased nrf2activity is associated with the enrichment of cscs and 0cnoman et al cell death and disease page of fig nrf2 inhibition eliminates colony forming capacity sphere growth and invasion capacity in epcamhigh cells a colonyforming assaywas carried out and quantified from sorted epcamhigh and epcamlow cell populations b sphereforming efficiency was determined and quantifiedusing sphere formation assay from epcamhigh and epcamlow cell populations c invasive potential of epcamhigh and epcamlow cells wasdetermined and quantified by transwell invasion assay pvalues were calculated using student™s t test p p p d“fnumbers of soft agar colonies formed d sphere formation e and invasion capacity f were quantified in sinrf2rna and siscramble epcamhighcells scale bar μm values represent mean ± sd from three independent experiments p p p pvalues werecalculated using student™s t testdemonstrated a previously unknown link betweenepcam and the nrf2 pathway a leading cause of chemotherapeutic resistancerecent studies have highlighted the association betweenthe nrf2 pathway and cscs for examplein neuralstemprogenitor cells nrf2 overexpression modulatedofficial of the cell death differentiation association 0cnoman et al cell death and disease page of fig il6 interleukin6 and p62 are involved in the activation of the nrf2 pathway and cell death resistance in epcamhigh cells a realtime pcr analysis of il6 expression in hnscc tumor tissues from matched adjacent normal n untreated surgery only n cisplatintreated n doxorubicin n and chemoradiotherapy n treated tumor tissues transcripts levels were normalized to betaactin b theimmunofluorescence images of cytoplasmic and nuclear nrf2 in fadu cells after 5day cisplatin treatment with or without pgml il6 legend cytoplasmic and 2nuclear nrf2 scale bar μm c nrf2 protein levels after 5day posttreatment with cisplatin or combination of cisplatin and pgml of il6 analyzed by western blotting d keap1 mrna and protein expression in fadu cells 5day posttreatment with cisplatin alone orwith pgml of il6 e ros level was determined in fadu and scc15 cells after treating cells with cisplatin il6 and combination of il6 andcisplatin f after h cisplatin treatment with vehicle or pgml il6 or combination of il6 and ngml tocilizumab cell lysates were subjectedto western blotting and the levels of sod1 and nrf2 proteins were determined g after h cisplatin treatment with vehicle or pgml il6 or il6with ngml tocilizumab the ros production was analyzed h p62 and lc3b were measured in epcamlow and epcamhigh fadu cells by westernblotting i p62 protein was determined in epcamlow and epcamhigh fadu cells following scrambled sirna and epcamsirna transfectionquantification and representative images of spheres formed by siscrambled and epcamsirna transfected epcamhigh cells are presented scale bar μm values represent three separate experiments p compared with siscramble group j epcamhigh cells were transfected withscrambled or p62sirna and protein levels of nrf2 p62 keap1 sod1 epcam and lc3b were assessed quantification analysis and representativeimages of spheres formed by siscrambled and p62sirna transfected epcamhigh cells are presented scale bar μm values represent threeseparate experiments p compared with sicontrol group k l transcript levels of epcam lc3b sod1 ho1 and akrc1 were determined insiscrambled and p62sirna transfected cells by qrtpcr values represent mean ± sd from three separate experiments p p valuescompared with sicontrol groupneurosphere formation efficiency as well as neural differentiation30 in addition nrf2 knockdown in primaryhuman glioblastoma cells decreased the selfrenewalcapacity of glioma stem cells31 as additional evidencecscschemotherapies and are considered alternative causes ofconventionalare highlyresistanttotumor relapse and aggressiveness cd44 cd133 cd24and aldh activity are frequently used for the detectionand isolation of cscs from tumor tissues and manycancer cell lines epcam has evolved as a potential cscmarker due to its involvement in cell signaling migrationmetastasis and therapeutic resistance the link betweenofficial of the cell death differentiation association 0cnoman et al cell death and disease page of in the clinicalepcam and acquisition of csclike properties is supported by epcam inhibition activation of wntbetacatenin signaling enriched the epcam cell populationwhereas rna interferencebased blockage of epcamattenuated csc activities in cancer cells32 these reportshighlight a critical role for epcam in the development ofcsclike featuressetting epcamexpression is associated with an unfavorable prognosis inbreast cancer33 furthermore low ros levels are correlated with the maintenance of a subpopulation of drugresistant cscs within tumors34 since the mechanisticinsights into the functions of epcam have only beenrecently explored the relationship between epcam andan association with regulation of the nrf2 pathway hasnever been described moreover thus far no studies haveexplored the association between the nrf2 pathway andepcam expression in the context of csclike featuresand drug resistance as a molecular mechanism of differential antioxidant capacity and stress resistance ofcscs we identified a direct association between epcamand nrf2 signaling with respect to drug resistance andenrichment of csclike features in hnscc cellsseveral noteworthy findings have emerged from ourstudy first epcam was highly expressed in hpvnegative tumors nrf2positive tumors were highly enriched in a epcam cell population and epcam was highlyexpressed in hnscc tumors compared to normalcounterparts these observations suggest a direct association between epcam and the nrf2 pathway in concordance we found that nrf2 and its target genes weresignificantly upregulated in the cisplatinresistant hnscctumors compared to cisplatin sensitive tumors thefunctional implication is that nrf2 activation led to theinduction of stemness and drug resistance features byoverexpressing sox2 and abcg5 proteins in epcamhighcell population while knockdown of epcam by sirnaattenuated the expression of nrf2 sod1 and sox2secon
Colon_Cancer
the bacteroides fragilis b fragilis produce biofilm for colonisation in the intestinal tract can cause a series of inflammatory reactions due to b fragilis toxin bft which can lead to chronic intestinal inflammation and tissue injury and play a crucial role leading to colorectal cancer crc the enterotoxigenic b fragilis etbf forms biofilm and produce toxin and play a role in crc whereas the nontoxigenic b fragilis ntbf does not produce toxin the etbf triggers the expression of cyclooxygenase cox2 that releases pge2 for inducing inflammation and control cell proliferation from chronic intestinal inflammation to cancer development it involves signal transducers and activators of transcription stat3 activation stat3 activates by the interaction between epithelial cells and bft thus regulatory tcell tregs will activates and reduce interleukin il2 amount as the level of il2 drops thelper th17 cells are generated leading to increase in il17 levels il17 is implicated in early intestinal inflammation and promotes cancer cell survival and proliferation and consequently triggers il6 production that activate stat3 pathway additionally bft degrades ecadherin hence alteration of signalling pathways can upregulate spermine oxidase leading to cell morphology and promote carcinogenesis and irreversible dna damage patient with familial adenomatous polyposis fap disease displays a high level of tumour load in the colon this disease is caused by germline mutation of the adenomatous polyposis coli apc gene that increases bacterial adherence to the mucosa layer mutatedapc gene genotype with etbf increases the chances of crc development therefore the colonisation of the etbf in the intestinal tract depicts tumour aetiology can result in risk of hostility and effect on human health keywords bacteroides fragilis colon cancer stat3 pathway bacteroides fragilis toxin inflammationintroductionbacteroides species are nonspore forming anaerobe and gramnegative bacteria there are more than different species of bacteroides these bacteria act as normal flora in the intestine to maintain healthy intestinal microflora in humans bacteroides fragilis b fragilis has two classes nontoxigenic b fragilis ntbf and enterotoxigenic b fragilis etbf the differences between ntbf and etbf are the presence of b fragilis toxin bft gene and its ability to produce biofilm bft product is a kda zincdependent metalloprotease toxin also known as fragilysin or bft “ bft plays an important role in intestinal inflammation and tissue injury by damaging the tight junction and increasing intestinal permeability furthermore it has been proven that tissue inflammation and injury promote cancer formation simultaneously the biofilm produced by b fragilis induces carcinogenesis fortunately only etbf encompasses bft and can produce biofilms hence ntbf does not harm the intestinal tract malays j med sci jul“aug “wwwmjmsusmmy penerbit universiti sains malaysia this work is licensed under the terms of the creative commons attribution cc by httpcreativecommonslicensesby40 0cmalays j med sci jul“aug “in the united states colorectal cancer crc is the third most common cancer in both genders it is also the second most common cancerrelated death especially for older patients who are ‰¥ years old in the american cancer society stated that there were new cases of crcs that led to the death of people moreover crc is the fourth leading cancer resulting in deaths worldwide inflammatory bowel disease ibd and genetic mutations are factors predisposing an individual towards colon cancer this indicates that crc has a high mortality rate “microbes are capable in promoting cancer development through several routes such as activation of chronic inflammation alteration of tumour microenvironment and production of toxins that damage dna when there is chronic etbf colonisation in the intestine it stimulates chronic intestinal inflammation triggering signal transducers and activators of transcription stat3 activation which contributes to interleukin il17 production il17 is involved in colon inflammation bft produced by etbf causes the alteration of signalling pathways and production of reactive oxygen species ros that leads to dna damage and cleavage of ecadherin in the below review we have provided a general information regarding bft produced by etbf triggering crc developmentliterature reviewcolon cancer associated with microbespromote nutrition in the human gastrointestinal tract there are nearly trillion microbes out of which make up normal flora in the intestine meanwhile the normal flora is characterised into beneficial and harmful microbes beneficial microbes including production of vitamins in the intestine and prevent disease formation however harmful microbes produce carcinogenic toxin and substances in the intestine these harmful substances may cause cancer there are many types of bacteria that stimulate a variety of cancer formation through their respective site of inflammation eg bacteria such as enterococcus faecalis e faecalis colibactinproducing escherichia coli e coli and etbf are involved in crc development however the mechanisms between each bacterium in contributing to crc formation are different for instance e faecalis damages the dna through ros colibactinproducing e coli produces colibactin that damages the dna and etbf produces bft that contributes to inflammation and immunecell infiltration intestinal dysbiosis inflammation and colon cancergenetic normal flora is advantageous to a person as it maintains intestinal health and gut homeostasis however as the bacteria such as etbf in the gut undergoes dysbiosis it brings harmful effects to the person according to deng et al a correlation was observed between microbiota imbalance and cancer progression while liu et al claimed is associated with that crc development intestinal microecology disorder imbalance among microbiota leads to bacterial infection that can progress to chronic inflammation one of the main environmental risk factors contributing to crc development is chronic intestinal inflammation chronic inflammation alters cellular microenvironment enhances gene mutation inhibits apoptosis and induces neovascularisation and cell proliferation that causes precancerous conditions eventually leading cancer simultaneously chronic inflammation alterations that directly affect the stat3 pathway and promoting there are three stages involved in tumour development namely initiation promotion and progression during initiation and progression cancer cells and microbes interact both producing genetic inflammatory“immunological factors that are responsible for their survival and replication in tumour progression tumour cells interact with the inflammatory cells in the tumour microenvironment these tumour cells inflammatory“immunological factors to attract the inflammatory cells and the stromal cells simultaneously activate both stromal cells start to produce various soluble factors including growth factors and protease these soluble factors play an important role in facilitating the growth differentiation and survival of tumour cells hence it promotes tumour progression and promotion additionally cytokines or microbes promote cancer by changing genetic sequence during gene mutation epithelial cells inflammatory and activated carcinogenesis cytokines chemokines causes and secrete wwwmjmsusmmy 0creplicate rapidly and develop into a hyperplastic epithelium which progresses into adenomas and then towards adenocarcinomas both adenomas and adenocarcinomas affect the growth rate of colonic epithelial cells and improve the cells™ toleration towards apoptosis and abnormal cells escape from the immune cells furthermore these invade submucosa turning into cancer when the growth of malignant cells continues the tumour continues to spread in the colon thus carcinogenesis becomes more efficientadenocarcinomas begin to ibd is an example of chronic intestinal inflammation that is associated with etbf pathogenic bacteria are capable of stimulating infection inflammation and carcinogenesis whereas the relationship between ibd and crc is well established surprisingly patients with ibd show a high level of immunoglobulin ig g antibodies il6 vascular endothelial growth factor vegf and tumour necrosis factor tnf igg antibodies are responsible for killing bacteria moving into the intestinal lumen simultaneously il6 and vegf are responsible for stat3 activation ibd is also known as ulcerative colitis uc and crohn™s disease cd this chronic intestinal inflammation increases the risk of colitisassociated crc the probability of which depends on multiple casual factors including severity duration of inflammation in the intestine and gut microbiota imbalance “ patients with uc or cd have “ folds higher incidence of crc when compared to healthy individuals it is also stated that patients with uc and cd have and respectively higher risks of crc compared to a normal healthy person this indicates that patients with uc tend to be more susceptible to crc than those with cd furthermore it is evident that the large intestine tends to have a higher risk of crc compared to the small intestine which can be attributed to the higher amount of bacteria simultaneously people with ibd and crc have a higher quantity of etbf in the intestine or stool examination compared to healthy persons additionally etbf are biofilm producers they can reduce or redistribute ecadherin in the colonic epithelial cells trigger the production of il6 by epithelial cells activate stat3 pathway and enhance cells proliferation at the site of crypt epithelial in normal colon mucosa this shows that biofilms are associated with the risk of colon cancer development review article formation of colon cancercox enzymes involved in inflammation carcinogenesis and biomarkerchronic inflammation is a principal factor that contributes to carcinogenesis prostaglandin is a paracrine hormone that plays an important role in inflammation cyclooxygenase cox is the ratelimiting enzyme responsible for producing prostaglandins cox1 and cox2 are the isoforms of cox enzymes that break down arachidonic acid into prostaglandins cox2 plays an important role in maintaining environment for the development of cancer inflammation cox2 is normally expressed in epithelial and stromal cells and the expression level is increased in both inflammation and cancer due to the presence of proinflammatory cytokines additionally bft triggers colonic epithelial cells to express cox2 but not cox1 cox2 releases prostaglandin e2 pge2 that triggers pain and inflammation at the site of tissue injury simultaneously pge2 controls cell proliferation by binding at the cell receptor and activating oncogenic signalling pathways thus it is proven that cox2 plays an important role in carcinogenesis and cancer progression by promoting cell proliferation angiogenesis and cancer stem cell formation inhibiting cell apoptosis and heightening metastatic potential through producing pge2 “the in certain studies it is stated that aspirin and nonsteroidal antiinflammatory drugs have the ability to inhibit the activity of cox enzyme which reduces inflammatory response thus it delays crc occurrence fortunately cox1 and cox2 act as biomarkers for screening purposes the biomarker is defined as any substance structure or process that is measurable in the body to determine the incidence of a disease it is commonly detected in circulation and body fluids cox1 is present in most cells thus it is not a specific biomarker however cox2 is only detected when is stimulated by trauma release of cytokines and stimulation of arachidonate metabolism by a toxin such as bft thus cox2 acts as a useful biomarker to detect “ cox2 is also a useful biomarker for colorectal carcinogenesis screening the level of cox2 biomarker in the blood is dependent upon epithelial cell proliferation apoptosis inhibition and neoangiogenesis patients with crc have high levels of cox2 compared to normal individuals “ indicating more aggressive growth rate and higher mortality rate this inflammatory responses inflammation the wwwmjmsusmmy 0cmalays j med sci jul“aug “suggests that cox2 expression is correlated to the aggressiveness of growth rate and mortality rate etbf activates stat3immune is activated to eliminate regulation of etbf is associated with ibd due to the abnormal response to bacteria the systemic adaptive immune response foreign antigens in the body this action eventually reduces intestinal mucosal tolerance although immune cells kill foreign antigens neutrophils and th17 cells contribute to inflammation and tumourigenesis transcription factors are known as stat protein family comprising seven members each stat protein responds to its specific cytokines they play an important role in regulating immune responses by controlling th cell types generation for instance the activation and generation of th17 cells require transcription factor stat3 protein the roles of stat3 protein include promotion of cell proliferation cell survival inflammation cellular transformation metastasis of cancer blood vessel formation and tumourpromoting moreover stat3 intrinsic pathway for cancer inflammation it induces genes in tumour cells that are responsible for inflammation within a tumour cell it exhibits an overly expressed stat3 pathway inflammation is a major factors etbf has the ability to activate stat3 rapidly in both colonic epithelial cells and colonic mucosal immune cells through phosphorylation and nuclear translocation however stat3 activation first occurs in colonic mucosal immune cells followed by colonic epithelial cells to activate stat3 in immune cells epithelial cells should respond in the production of cytokines such as il6 il10 and il23 besides cytokines growth including vegf and fibroblast growth factor fgf2 are also involved in activating stat3 when etbf and bft first interact with colonic epithelial cells they stimulate early stat3 activation in colonic mucosal immune cells this stat3 activation continuously rises slowly until it reaches the peak level the peak indicates that etbf activates the immune system due to barrier dysfunction during etbfinduced colitis it activates both stat3 and th17 immune response in the colonic mucosa stat3 activation induces prooncogenic inflammatory pathways and increases the permeability of mucosa although stat3 activation is longterm and lasts for months it highly increases the chance of getting a tumour as a result of chronic inflammation additionally stat3 activation promotes the accumulation of tumour regulatory tcell tregs and blocks the generation of antitumour immune responses which give an adverse effect to the body this abnormal persistent stat3 activation increases the cancer cell tolerance prevents rejection of cancer by the immune system reduces the effectiveness of immunotherapy and enhances the effectiveness of oncogenesis activated stat3 predominantly detected in human cancers is constitutively activated and depicts its association with neoplasms patients with ibd tend to show stat3 activation and a high level of th17 cells and il17 the level of activated stat3 in patients with ibd and dysplasia is different from patients with ibd and without dysplasia patients with ibd and dysplasia show a higher level of activated stat3 compared to those without dysplasia simultaneously the level of activated stat3 increases together with the continuum of dysplasia to colitisassociated cancer it is clear that b fragilis can either be toxigenic or nontoxigenic the latter does not activate stat3 because it does not produce bft therefore ntbf does not contribute to colon cancer development but etbf does are tregs th17 and il17 good or badreduces intestinal il17 this process in a normal healthy condition tregs play an important role in inflammatory responses and immune homeostasis they express high levels of il2 receptor and produce endogenous il2 which inhibits the production of intestinal inflammation and prevents carcinogenesis however when etbf colonises a particular site of the colon it produces a large amount of bft damaging the intestinal mucosa to initiate etbftriggered colitis with the activation of the stat3 pathway this leads to direct contact between tregs and etbf and promotes tregs activation activated tregs lack the ability to produce endogenous il2 “ instead of producing endogenous il2 tregs consume exogenous il2 for their survival the consumption of exogenous il2 by tregs reduces the levels of exogenous il2 and produces an environment that favours the growth of th17 cells as the levels of il2 drop th17 cells are no longer inhibited and undergo expansion to produce a large quantity of naïve tcells this naïve subset of tcells then differentiates into th17 cells in excess wwwmjmsusmmy 0ccarcinogenesis this shows that colonisation of etbf promotes the accumulation of both tregs and th17 cells “ th17 cells start to produce large amounts of cytokines including tnf and il17 these cytokines promote cell survival and proliferation during injuries although th17 cells heal an injured site they turn into pathogenic th17 cells when deregulated these pathogenic th17 cells initiate chronic inflammatory condition il17 produced by pathogenic th17 cells are involved in an early inflammatory stage of the injuries it promotes tumour cell survival proliferation tumour neovascularisation and metastasis which allow “ additionally tumour cells and fibroblasts are stimulated by il17 to produce high amounts of angiogenic factors for angiogenesis il17 can activate stat3 pathway indirectly through il6 when il17 binds to il17 receptorbearing tumour cells it stimulates il6 production that is highly important for stat3 pathway activation as mentioned above this stat3 pathway activation contributes to several characteristics such as cancer proliferation antiapoptosis and angiogenesis that favour carcinogenesis in the colon this shows that there is a relationship between stat3 pathway and tregs in contributing to crc formation when etbf is accumulating in the intestinal tract as shown in figure to some extent stat5 and stat6 have been reported to be involved in inhibiting antitumour immunity when all stat3 and are activated together it highly enhances the tumourigenesis effect cleavage of ecadherin stimulate cell proliferationapart from inflammation bft alters the structure and function of colon epithelial cells by degrading ecadherin ecadherin is a 120kda glycoprotein that is the major structural protein in zonula adherens and is also known to be a tumour suppressor and zonula adherence protein this protein is responsible for the epithelial polarity in normal conditions the expression of ecadherin is linked to cellular functions including apoptosis and homotypic cell“cell “ unfortunately when ecadherin interacts with bft in the intestinal epithelial cells it degrades ecadherin rapidly in an atpindependent manner this cleavage promotes colonic injury inflammation and loss of membraneassociation resulting in morphological enhances cellular metastatic potential it is proven that changes and adhesion review article formation of colon canceretbfbftintestinal lumenepithelial cells†“ il2intestinal immune system†‘ th17tnfcell proliferation†‘ il17tregsinflammationcarcinogenesisil17 receptorbearing tumour cellsstat3 activationfigure the mechanism through abnormal systemof intestinal carcinogenesis immune factordependent the cleavage of ecadherin correlates with the changes of cell morphologies simultaneously degradation of ecadherin also promotes the binding of nuclear localisation of βcatenin and tcell transcriptional activator this binding promotes gene regulation and transcription additionally βcatenin plays an important role in wingless and int wnt signalling pathway promoting cell proliferation and epithelialmesenchymal transition and enhancing the expression of protooncogene in primary colorectal tumours cells in the centre of the tumour exhibit the presence of βcatenin and ecadherin however when the cells move away from the centre of the tumour they exhibit high amounts of nuclear βcatenin and the junction of ecadherin is lost important role ecadherin plays an in maintaining the morphology of cells there is a relationship with the ecadherin and the apical factin ring of the intestinal epithelial cells™ secretion when the loss of ecadherin increases the integrity of the apical factin is lost resulting in the increase in cell volume and chloride secretion and cell and epithelial barriers become wwwmjmsusmmy 0cmalays j med sci jul“aug “more permeable this contributes to intestinal inflammation diarrhoea and colon carcinogenesisalteration of the signalling pathway of colorectal cancerbft is involved in many colonic epithelial cell signal transductions when bft disturbs or activates the signalling pathway it brings adverse effects to the body and can lead to colorectal tumourigenesis figure the colonic epithelial cell signal transduction transpires through the nuclear factor kappalightchainenhancer of activated bcells nfκb wnt and mitogenactivated protein kinase mapk signalling pathways bft can stimulate nfκb pathway in the intestinal epithelial cells with the expression of heme oxygenase1 ho1 and cytokines to induce mucosal inflammation this pathway has the ability to enhance the survival of neoplastic cells by preventing them from undergoing apoptosis leading to tumour formation furthermore in figure it shows that when nfκb of intestinal epithelial cells is activated for a long time it induces the activity of nitric oxide synthase that breaks down larginine to produce nitric oxide which can damage cellular dna wnt signalling pathway is important to maintain the structures of the intestinal epithelium however wnt signalling pathway contributes negatively and affects cells which are extremely important for colorectal carcinogenesis and progression as wnt signalling pathway is activated it weakens tight junctions and reduces cellular adhesion this allows the cancer cells to undergo migration and metastasis hence cancerous cells can migrate to another ans spermine oxidase is a catabolic enzyme that increases ros which can be upregulated by bft in normal conditions figure ros acts as an important mediator in multiple cell signalling pathways and immune response that is produced naturally within biological systems it consists of superoxide hydroxyl radical and hydrogen peroxide however as the amount of ros becomes excessive it imparts negative effects in the disruption of redox homeostasis figure this excessive ros induces oxidative stress it oxidises cellular components including dna lipids and proteins within the cells etbfbftbftepithelial cellactivation of signaling pathwaynfκbwntnitric oxide synthase — reduce cell adhesion — weaken tight junction — cancer cell migratenitric oxidedamage cellular dnafigure the role of the signalling pathways when epithelial cells contact bftspermine oxidase smorelative oxygen species ros “ mediatorimmune responsemultiple cell signalling pathwaysfigure normal condition of the smo and ros that helps in immune response and cell signalling pathwayswwwmjmsusmmy 0conce the cellular components are oxidised it generates irreversible damage to host cells additionally ros plays an important role in the survival of cancer cells enhancing the effectiveness of carcinogenesis and aggravating cancer formed in the body “spermine oxidase smo “ upregulated†‘ relative oxygen species ros “ mediatordnalipidproteinirreversible damagecarcinogenesisfigure the adverse effect of smo contacted bftfamilial adenomatous polyposisfactors contributes the combination of both genetic and environmental to crc formation it is estimated that of crc development is due to genetic predisposition wherein nearly of all crcs are attributed to familial adenomatous polyposis fap fap is an autosomal dominant inherited disorder that describes the development of numerous colorectal adenomatous polyps these polyps are able to develop in the teenager™s colon meanwhile the number of polyps formed in the colon depends on the age of a person which means the number of polyps is directly proportional to the age of a person if these polyps are not removed from the colon they may transform from benign to malignant developing crc the source of fap disease is mainly due in the adenomatous to germline mutation polyposis coli apc gene “ this apc mutation occurs due to frameshifts insertions or deletions that may introduce a premature stop codon during the halfway through the transcription process these earlyintroduced premature stop codons in the gene sequence lead to incompletetruncated apc protein formation thus the normal function of apc protein is lost eventually facilitating carcinogenesis review article formation of colon canceradditionally germline mutations along with somatic mutations of the normal allele or loss of the normal allele lead to inactivation of apc once apc is inactivated it precisely commences carcinogenesis in normal conditions apc pathway acts as a gatekeeper controlling a part of wnt signalling pathway unfortunately when apc is mutated the function of apc pathway is lost or inactivated this inactivation of the apc pathway results in the activation of wnt signalling pathway this characteristic is mainly found in crc moreover apc mutation has the ability to alter bacteria“host epithelial interaction where it allows the bacteria to attach onto the mucosa if a person has the apcmutated gene and is exposed to etbf the chances of developing crc are high concurrently high amount of tumour load is displayed in the person™s colon conclusionthe human gastrointestinal tract contains its own bacterial flora that benefit humans daily b fragilis is one of them and consists of two classes namely ntbf and etbf the differences between both the classes is the presence of bft etbf is able to produce bft that can disrupt the intestinal environment and promotes inflammation simultaneously bft degrade ecadherin and causes inflammation ibd is a chronic intestinal inflammation associated with etbf and can induce crc however patients with cd have lower risk of developing crc as compared to those with uc patients with ibd exhibit stat3 activation due to the stimulation of immune response that favours th17 cell generation as the levels of th17 cell increase it brings a huge disadvantage to the intestinal tract due to the production of il17 furthermore il17 stimulates the production of il6 that is required to activate stat3 this indicates that the stat3 pathway activates for a long time longterm stat3 activation blocks antitumour immune response which supports the growth of cancer cells thus stat3 th17 and il17 are highly important in carcinogenesis concurrently the production of proinflammatory cytokines at the site of inflammation triggers the production of cox2 enzymes that release pge2 cox2 is also known for its carcinogenic abilities due to the production of pge2 that controls cell proliferation additionally bft affects signal transductions such as wnt wwwmjmsusmmy 0cmalays j med sci jul“aug “nfκb and mapk signalling pathways and induces tumourigenesis considering that bft induces inflammation activates stat3 and alters signalling pathways it can be concluded that bft produced by etbf plays an important role in colon carcinogenesis boleij a hechenbleikner em goodwin ac badani r stein em lazarev mg et al the bacteroides fragilis toxin gene is prevalent the colon mucosa of colorectal cancer in patients clin infect dis “ httpsdoi101093cidciu787acknowledgementsnoneconflict of interestnonefundsnoneauthors™ contributionsconception and design hkkdrafting of the article with supervision cwtcritical revision of the article for important intellectual content fdfinal approval of the article hkk fdcorrespondencedr fabian davamaniphd microbiology university of madrasfaculty of biomedical scienceschool of health sciences international medical university kuala lumpur malaysia tel fax email fabian_davamaniimuedumyreferences snezhkina av krasnov gs lipatova av sadritdinova af kardymon ol fedorova ms et al the dysregulation of polyamine metabolism in colorectal cancer is associated with overexpression of cmyc and cebpβ rather than enterotoxigenic bacteroides fragilis infection oxid med cell longev “ httpsdoi10115520162353560 sears cl geis al housseau f bacteroides fragilis from carcinogenesis j clin symbiont invest “ httpsdoi101172jci72334subverts mucosal to biology colon lv y ye t wang h zhao j chen w wang x et al suppression of colorectal tumorigenesis by recombinant bacteroides fragilis enterotoxin2 in vivo world j gastroenterol “ httpsdoi103748wjgv23i4603 pierce jv bernstein hd genomic diversity of enterotoxigenic strains of bacteroides fragilis plos one 2016116e0158171 httpsdoi 101371journalpone0158171 sun j kato i gut microbiota inflammation and colorectal cancer genes dis “ httpsdoi101016jgendis201603004 erdrich j zhang x giovannucci e willett w proportion of colon cancer attributable to lifestyle in a cohort of us women cancer causes control “ httpsdoi101007s105520150619z mughinigras l schaapveld m kramers j mooij s neefjesborst ea van pelt w et al increased colon cancer risk after severe salmonella infection plos one 2018131e0189721 httpsdoi101371journalpone0189721 francescone r hou v grivennikov si microbiome inflammation and cancer cancer j “ httpsdoi101097ppo0000000000000048 wick ec rabizadeh s albesiano e wu x wu s chan j et al stat3 activation in murine enterotoxigenic bacteroides inflamm bowel dis “ httpsdoi101097mib0000000000000019induced by fragili
Colon_Cancer
" recent impressive advances in cancer immunotherapy have been largely derived from cellular immunity the role of humoral immunity in carcinogenesis has been less understood based on our previous observations we hypothesize that an immunoglobulin subtype igg4 plays an essential role in cancer immune evasionmethods the distribution abundance actions properties and possible mechanisms of igg4 were investigated with human cancer samples and animal tumor models with an extensive array of techniques both in vitro and in vivoresults in a cohort of patients with esophageal cancer we found that igg4 containing b lymphocytes and igg4 concentration were significantly increased in cancer tissue and igg4 concentrations increased in serum of patients with cancer both were positively related to increased cancer malignancy and poor prognoses that is more igg4 appeared to associate with more aggressive cancer growth we further found that igg4 regardless of its antigen specificity inhibited the classic immune reactions of antibody dependent cell mediated cytotoxicity antibody dependent cellular phagocytosis and complement dependent cytotoxicity against cancer cells in vitro and these effects were obtained through its fc fragment reacting to the fc fragments of cancer specific igg1 that has been bound to cancer antigens we also found that igg4 competed with igg1 in reacting to fc receptors of immune effector cells therefore locally increased igg4 in cancer microenvironment should inhibit antibody mediated anticancer responses and help cancer to evade local immune attack and indirectly promote cancer growth this hypothesis was verified in three different immune potent mouse models we found that local application of igg4 significantly accelerated growth of inoculated breast and colorectal cancers and carcinogen induced skin papilloma we also tested the antibody drug for cancer immunotherapy nivolumab which was igg4 in nature with a stabilizing s228p mutation and found that it significantly promoted cancer growth in mice this may provide an explanation to the newly appeared hyperprogressive disease sometimes associated with cancer immunotherapy there appears to be a previously unrecognized immune evasion mechanism with igg4 playing an essential role in cancer microenvironment with implications in cancer diagnosis and immunotherapyintroductionwhile new immune therapy for cancer focuses mostly on manipulating cellular immunity humoral immunity also holds great promise for cancer therapy1 igg4 is a unique antibody that has the lowest concentration among igg subtypes in healthy individuals and its function has not been well understood3“ igg4 was regarded as a ˜blocking antibody™ because of its reduced ability to trigger effector immune reactions6 therefore whatever molecules igg4 reacts to the subsequent immune reaction was subdued8 igg4 has a unique structure of fab arm exchange fae in which the two heavy and light chains of two different antibodies with different specificities are exchanged resulting in an asymmetric bispecific antibody with reduced ability to bind to antigen and to form immune complexes9 another unique feature of igg4 is that it can react to other iggs via its fc fragment and the significance of this property has not been well understood evidence suggests that fae and fc fc reactivity may involve the same molecular structure on igg4 molecule10 davies et al11 resolved the crystal structure of igg4 fc fragment revealing a unique molecular conformation supporting its fc binding property recent interests in igg4 related diseases unveiled a wide range of pathologies with a common phenomenon of often increased igg4 concentration in the serum and igg4 postive plasma cells in the affected ans accompanied by local inflammation and fibrosis but its pathogenic mechanism is still poorly understood13“to cite wang a0h xu a0q zhao a0c et a0al an immune evasion mechanism with igg4 playing an essential role in cancer and implication for immunotherapy for immunotherapy of cancer 20208e000661 101136jitc2020000661 –º additional material is published online only to view please visit the online http dx jitc hw qx cz and zz are joint first authorsaccepted july authors or their employers re use permitted under cc by nc no commercial re use see rights and permissions published by bmjfor numbered affiliations see end of correspondence toprofessor jiang gu qq comwang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen access the possible functions of igg4 in cancer and also in the immune system have not been well elucidated increases of igg4 positive plasma cells were reported in gastrocarcinoma16 extrahepatic cholangiocarcinoma17 and melanoma18 the above studies were performed on limited number of cases without convincing explanation of mechanism or significance wu 19 reported that serum igg4igg ratio could predict recurrence of hepatocellular carcinoma after surgery the most extensive study on igg4 and cancer was performed by karagiannis 20 who investigated the possible effect of cancer specific igg4 in inhibiting cancer immunity in melanomas and suggested competition between cancer specific igg4 and igg1 in binding to cancer antigens as the cause for the inhibition a recent report raised the concept of cancer educated b cells and toxic igg produced by these cells in facilitating lymph node metastasis for breast cancer in a mouse model21 we performed a multidimensional investigation of igg4 in a wide array of patients with cancer and tissues with both in vitro and in vivo experiments extensive new evidence led us to hypothesize that there is a potent humoral immune editing mechanism in cancer microenvironment with igg4 playing an essential role we propose that fc fc reaction could be the basic mechanism of this immune inhibition we validated this in three immune potent animal models this property was also found applicable to cancer immunotherapy drug nivolumab which was igg4 in nature our study points to a so far little appreciated mechanism of immune evasion in cancermaterials and methodskey resourcesdetailed information about key resources including antibodies biological samples chemicals assay kits cell lines and software are shown in online supplementary table experimental model and subject detailspatients and healthy volunteerswe collected tissue and blood samples from over patients with cancer in shantou university affiliated tumor hospital and the east guangdong provincial pathological consultation center details of the human samples are shown in online supplementary table immunohistochemistrydetails of the protocols and the antibodies are shown in online supplementary datasds techniquethe expressions of igg1 igg2 igg3 and igg4 in esophageal cancer were detected at the histological level the stain decolorize stain sds technology22 was performed on the same slide sequentially with four different antibodies to demonstrate the four antigens the distribution pattern abundance and relationship of the four antigens were processed with an image software photoshop to achieve an integrated figure the proximity of different cell types on the tissue sections was measured with an image analyzing software image pro plus v60 details of the protocol are presented in the online supplementary dataimmunofluorescence double stainingtwo antibodies from different species were incubated on the same tissue section primary antibodies were detected with goat antimouse 555antirabbit igg or goat antimouse 488antirabbit igg alexa fluor life sciences and immunoreactivity was visualized with a fluorescence microscope antigens were detected and demonstrated with two fluorescence signals in red and green and the blue signal of ' diamidino2 phenylindole dapi as for cell nuclei images were acquired with the evos fl fluorescence microscope life technologies usaigg4 immunohistochemistryto verify that igg4 could react to igg1 that had been immobilized to tissue sections we used human pancreas and brain and antibodies to insulin glucagon and neurofilament as models detailed protocol of this experiment is shown in the online supplementary dataimmunoglobulin preparationsfab and fc fragments of igg igg1 and igg4 were prepared with papain digestion in the presence of cysteine iggs were cleaved at a position above the hinge region by papain at °c for hours after purification with protein a affinity chromatography pure fab and fc fragments were isolated with elution buffer igg fc fragment was washed down from protein a column after centrifugation and concentration measurement igg preparations were collected and stored at °c before usewestern blotigg subclasses were resolved on sodium dodecyl sulfate polyacrylamide gels under reducing conditions and then transferred onto nitrocellulose membranes ge healthcare life sciences the membranes were blocked for hour with bovine serum albumin bsa in tris buffered saline with tween20 ph and then incubated with primary antibody biotin labeling kit anaspec overnight at °c it was then incubated with secondary antibody for hour at room temperature finally reaction density was measured with odyssey western at nm and nm detection channelserum igg4 and igg assessmentsera samples collected from patients with esophageal cancer and healthy volunteers were sent to golden field medical test company guangzhou china and roche immune turbidimetry method was used to wang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0cmeasure serum igg4 and total igg concentrations all serum samples were stored at ˆ’°c freezer immediately before analysis all quantitative data were treated statisticallymeasurement of igg4 concentrations in tumor and tumoradjacent normal tissues with elisaconcentrations of igg4 in pairs of esophageal cancer and adjacent normal tissues cm from the edge of the cancer mass were measured with elisa detailed protocol of this experiment is shown in the online supplementary datacell culture for fc receptor binding assaysu937 cell line was bought from procell life science technology china cl0239 and cells were cultured in rpmi gibco c22400500bt supplemented with fetal bovine serum gibco “ and uml penicillin streptomycin gibco “ at ×106ml in ml cell culture bottleprotein preparationsdetails of the protocols for protein preparation separation and papain digestion are shown in online supplementary datafluorescence activating cell sorter facs for fc receptor assaysdetails of the relevant protocols are shown in online supplementary dataadcc adcp and cdc teststhe classic antibody dependent cell mediated cytotoxicity adcc antibody dependent cellular phagocytosis adcp and complement dependent cytotoxicity cdc tests were performed based on previously reported protocols23“ non specific igg4 instead of cancer specific igg4 was used to inhibit these reactions igg1 was used as control details of the protocols are shown in online supplementary dataanimal modelsbreast cancer and colon cancer modelsbalbc mice were obtained from vital river technical beijing china mice aged between and weeks and weighed ± g were used in all experiments all mice were inoculated with 4t1 mouse breast cancer cells or ct26 mouse colon cancer cells subcutaneously under the left forearm × 4t1 cells per mouse to build cancer models the mice were divided into different groups and were treated with igg1 or igg4 derived from intravenous igg ivig ivig without igg4 nivolumab and fc of nivolumab respectively details of the protocols are shown in online supplementary datacarcinogeninduced skin tumor modelwe employed a well established carcinogen induced skin tumor model to study the effect of igg4 and ivig without igg4 in comparison with controls phosphate buffered open accesssaline pbs detailed protocol is shown in online supplementary dataquantification and statistical analysisdata were analyzed in graphpad prism all reported p values were derived from two sided comparisons with values of less than considered to be statistically significantresultsigg4 was significantly increased in the serum of patients with cancer and this increase was related to cancer stage and patient prognosiswe first measured the concentrations of igg1 igg2 igg3 and igg4 in sera of patients with esophageal cancer n82 igg4 was significantly increased in patients with cancer in comparison with normal healthy subjects n70 the concentration of igg4 was increased from about to the ratio of igg4iggtotal was also significantly increased the statistical significance of both reached p00001 the increase of igg4 was also positively correlated to the stages of cancer with late stage cancers having more obvious increases higher igg4 serum concentrations were associated with worse prognosis figure 1a“digg4containing lymphocytes and igg4 concentration were significantly increased in cancer microenvironment and this increase was associated with cancer cell infiltrationigg4 positive lymphocytes were significantly increased in cancer microenvironment in comparison with tissue more distant to the cancer mass igg4 positive lymphocytes were barely detectable in tumor adjacent normal tissue figure 1h on the same tissue sections employing the sds technique22 to simultaneously visualize the four subtypes of igg with four distinct colors we found that one plasma cell only contained one igg subtype that is igg1 igg2 igg3 and igg4 were all contained in their own plasma cell populations separately figure 1i the marked increase of igg4 containing plasma cells in comparison with other subtypes was clearly visualized on cancer tissue sections igg4 positive plasma cells appeared to accumulate more in tissues with extensive cancer cell infiltration than in other areas figure 1eh in addition igg4 positive cells are often in close proximity to cells containing igg1 and igg2 but not to igg3 this phenomenon was not seen among other igg subtypes apart from igg4 quantitative data of the proximity of different cell types are presented in online supplementary figure s2 with the multiple immunostaining method we also demonstrated that igg1 containing and igg4 containing lymphocytes were distinct from cd3 positive t lymphocytes in the same region of the cancer tissue figure 1j in addition we measured the concentrations of igg4 in cancer tissue and cancer adjacent normal tissue n46 pairs the average concentration of igg4 in cancer tissue was about four times higher than that in the adjacent wang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen access figure significant increase of igg4 and igg4iggtotal in serum and igg4 positive b lymphocytes in esophageal cancer a igg4 in serum of esophageal squamous cell cancer escc n82 was significantly higher when compared with healthy controls n70 p00001 b igg4iggtotal ratio in escc n82 was significantly higher than that in matching healthy adults n70 p00001 c igg4 in stage …³ n18 was significantly higher than those in stages …° and …± n16 p001 d igg4iggtotal in serum of stage iv escc n18 was significantly higher than those in stages …° and …± n16 p005 e scatter diagram of igg4 positive cell numbers in cancer cancer adjacent normal tissue adjacent and normal tissues igg4 positive lymphocytes in and around the esophageal cancer mass n110 are significantly more abundant than those in the adjacent normal tissue n60 and normal lymphoid tissues n63 p0001 for both increases of igg4 positive lymphocytes were most abundant in areas of cancer cell proliferation f the increase of igg4 positive cell numbers was related to the prognoses of the patients more igg4 positive cells were associated with worse outcome p005 g igg4 concentration in cancer tissue n46 was significantly higher than that in adjacent normal tissue n46 p001 h immunohistochemistry of igg4 in esophageal cancer tissues from left to right are igg4 in cancer tissues cancer adjacent tissue and normal lymphoid tissue tonsil it clearly demonstrates that igg4 positive lymphocytes red were markedly increased left in comparison with normal lymphoid tissue right and with tumor adjacent normal tissue middle scale bar µm i demonstration of four subpopulations of igg containing plasma cells with multiple immunostaining sds method in cancer each subclass has its own distribution pattern and one plasma cell only produces one subclass of igg igg1 yellow igg2 green igg3 purple igg4 red j on the same tissue section a triple immunostaining was performed with the sds method to demonstrate the distribution and relationship among cd3 positive t cells yellow igg1 positive b cells greens and igg4 positive b cells red each cell type has its own distribution and no overlap between different cell types is observed sds stain decolorize stainwang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0cnormal tissue and the difference between these two groups was statistically significant p001 figure 1gigg1 extracted from patients with cancer reacted to cancer cells of the same patients but igg4 extracted from the same patients did notwe extracted igg1 and igg4 from the serum of patients with esophageal cancer with breast cancer and with colon cancer with respective specific antibody columns we then labeled the antibodies with biotin and tested the reactivity of these extracted antibodies to cancer tissue sections of the same patients in all cases igg1 reacted to cancer cells from the same patients but igg4 did not figure 2a it appeared that the increased igg4 in cancer microenvironment and in the patient™s serum was not reactive to cancer antigens while igg1 extracted from the same patients reacted to the cancer antigensigg4 reacted to cancerspecific igg1 that was bound to cancer cellsalthough igg4 extracted from patients with cancer did not react to cancer antigen it did react to cancer specific igg1 that was bound to cancer antigen on tissue sections as shown in figure 2b when cancer specific igg1 that was not labeled with biotin was applied to cancer tissue sections followed by biotin labeled igg4 the cancer cells became positive while when the same biotin labeled igg4 was applied to the same cancer tissue section without prior application of igg1 it did not react this reaction of igg4 to cancer specific igg1 was not via the antigen specific variable region of igg4 as such igg4 was neither specifically against igg1 nor was it specifically against cancer antigen with its antigen recognizing fab variable region as shown in figure 2a the only explanation was that igg4 reacted to igg1 through its fc region this was validated by subsequent experiments with western blot analysis as shown in figure 3a“ein western blot igg4 was found to react to other iggs igg1 igg2 igg3 and igg4 via an fcfc mechanism and this reaction was across species but igg4 did not react to other ig subtypes igm ige iga or igdto test if and how igg4 could react to igg1 we performed western blot with igg4 from different sources extracted from patients with cancer from normal adults and commercially purchased igg4 was found to react to igg1 igg2 igg3 and igg4 at the molecular weight mw of about kd and this reaction was not seen when igg1 igg2 or igg3 was used as the antibody and igg4 as the target molecule running on the gel the above phenomenon was observed in western blot of both reducing and non reducing conditions figure 3a online supplementary figure s3 however human igg4 did not react to human igm iga igd or ige online supplementary figure s4 nevertheless we found that this reaction was across species that is human igg4 reacted to iggs of human mouse rabbit and goat online supplementary figure s5 we open accessigg4 extracted from a patient with cancer reacted figure to cancer bound igg1 and blocked antibody mediated cancer immunity a upper panel these photos serve as an example of the reactivity of igg1 and igg4 extracted from patients with cancer igg1 from the serum of a patient with breast cancer was labeled with biotin and stained a frozen cancer tissue section of the same patient cancer cells were positively stained by igg1 left the cancer cells were confirmed by their characteristic histopathology of he staining middle igg4 from serum of the same patient labeled with biotin and applied on the same cancer on a consecutive section was completely negative right lower panel another breast cancer positively stained by igg1 from the patient™s serum left the cancer cells were identified by positive immunostaining of cytokeratin ck on a consecutive section middle igg4 from the same patient was not reactive to the same cancer on a consecutive section right b the upper panel illustrates the principle of the experimental reactions and the middle and lower panels show staining results from two patients with breast cancer left igg1 from a patient with cancer positively reacted to frozen cancer tissue of the same patient brown cells middle igg4 from the same patient with cancer applied to consecutive sections but did not react to the same cancer right however when unlabeled igg1 was applied to the same cancer tissue section followed by biotin labeled igg4 the cancer cells were positively stained brown cellsfurther digested human igg4 and igg1 into fab and fc fragments with papain it was found that it was the fc fragment of igg4 reacting to fc of igg1 figure 3b“e this reaction was easy to occur as only min incubation wang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen access igg4 reacted to igg1 in western blot and tissue figure sections in an fc fc fashion a in western blot non cancer specific igg4 from a patient with breast cancer reacted to igg1 and igg4 from the same patient with cancer right panel arrows however when igg1 and igg4 were run on the gel and biotin labeled igg1 was used as the primary antibody no band was seen left panel these are the same antibodies used in figure a02ab providing support to explain the reaction between igg4 and igg1 seen on cancer tissue b western blot demonstrated that igg4 reacted with igg1 igg2 igg3 and igg4 c igg4 reacted with igg fc fragment but not with fab arm d igg4 reacted with igg1 fc fragment but not with fab arm e biotin labeled igg4 fc fragment reacted to igg1 and igg4 fc fragments but not with igg1 or igg4 fab biotin labeled igg4 fab did not react to igg1 or igg4 fc or fab these results demonstrate that it is the fc region of igg4 that reacted to fc of igg1resulted in a clear band therefore the positive reaction obtained by sequential applications with cancer specific igg1 followed by non cancer specific igg4 on cancer tissue figure 2b had to take place between the fc of igg4 and the fc of igg1 as shown in figure the fcfc reaction between igg4 and igg1 bound to tissue sections was further tested and validated with a number of antibodies and tissue types apart from cancerfollowing the same logic we tested the reactivity between the fc fragments of igg4 and igg1 already demonstrated in western blot on tissue sections we used igg1 primary antibodies to insulin and glucagon in normal human pancreas and antibody to neurofilament in human brain for this test the same principle was established with these normal tissues detailed results and figures are shown in online supplementary figure s6igg4 competed with igg1 to bind to fc receptors of pbmc and macrophageswe performed immunoglobulin and fc receptor binding assays with peripheral blood monocytes pbmc and with a human monocyte cell line u937 procell life science technology china cl0239 igg1 and igg4 were extracted from the serum of patients with cancer and pbmcs were isolated from the same human subjects the extracted and purchased igg1 and igg4 were labeled with biotin or fitc in the igg1 and igg4 binding assay we found that igg1 and igg4 competed with one another in binding to pbmc and this reaction could be completely blocked by fc receptor blocker this competition was concentration dependent that is as the concentration of igg4 increased more igg1 bound to pbmc was replaced the reverse was also true that is igg1 could also replace igg4 in this competition assay this phenomenon was demonstrated on cytospin slides of pbmc preparation online supplementary figure s7in addition flow cytometry was performed to examine the binding properties of igg1 and igg4 to fc receptors of monocytes the ability of igg1 and igg4 to bind to all three subtypes of fc gamma receptor fcγr”fcγr…° cd64 fcγr…± cd32 and fcγr…² cd16”was examined with corresponding antibodies we found that igg4 could compete with igg1 in binding to the fc receptor of monocytes u937 we further found that the binding force of igg1 was about twice as strong as that of igg4 for individual receptor subtypes igg1 could bind to all three receptor subtypes that is fcγr…° cd64 fcγr…± cd32 fcγr…² cd16 in contrast igg4 could only bind to fcγr…° cd64 although their binding sites were different igg4 could completely block igg1 we also found that it was necessary for a relatively high concentration of igg4 to be present in the solution in order to compete with igg1 in binding to fc receptors online supplementary figure s8igg4 inhibited the classic immune reactions of adcc adcp and cdc mediated via cancerspecific igg1 and effector immune cellscomplementswe first verified that non cancer specific igg4 indeed reacted to igg1 cetuximab used in adcc adcp and cdc figure 4a we then found that igg4 inhibited adcc elicited cytotoxicity with cancer specific igg1 antibody and pbmc figure 4b the igg4 used in our test was not directed against cancer antigen or to lymphocytes non cancer specific igg1 could also inhibit adcc but to a much lesser extent also reached statistical significance we obtained evidence to show that inhibition of adcc appeared to take place at the site of the cancer specific antibody that is igg4 reacted to the igg1 antibody bound to cancer cells figure 2b and the site of immune effector fc receptors the latter effect could wang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen accessfigure non cancer specific igg4 inhibited classic adcc adcp and cdc reactions against cancer but had no direct effect on cancer cell growth a on western blot the chimeric antibody cetuximab igg1 against egfr was run on the gel and igg4 and igg1 at concentrations of and µgml were used as the primary antibodies igg4 reacted to cetuximab at a concentration dependent manner but igg1 did not react b left in a classic adcc experiment cetuximab igg1 was incubated with an egfr expressing lung cancer cell line a549 and then with pbmc from normal healthy adult cancer cell activity was significantly reduced n12 non cancer specific igg1 and hsa were used as controls showing that they had no direct effect on the cancer cells n12 middle when non cancer specific igg4 was added to the mixture the effect of cetuximab was significantly reversed demonstrating an inhibitory effect of igg4 in adcc n12 non cancer specific igg1 had a much smaller but also significant effect in inhibiting adcc action n12 right when fc receptor blocker was incubated with pbmc the effect of cytotoxicity was blocked c“e adcp was performed with a lung cancer cell line a549 expressing egfr as the targets human peripheral monocyte derived macrophages as the effector cells and the antibody cetuximab igg1 against egfr as the mediating antibody the tumor cells were stained with cfda se fluorescence probes green macrophages derived from pbmc were stained with dii fluorescent probes orange blue fluorescence is the nuclei stained with dapi d higher magnification of c the orange colored macrophages were in close contact with green tumor cells tumor debris ingested by macrophages appeared yellow in the cytoplasm of macrophages e bar chart showing the effect of adcp and its inhibition by igg4 f left in µgml rituximab mediated adcp model giemsa staining results of phagocytosis of raji cells by macrophages after the addition of µgml igg1 and igg4 respectively right igg4 significantly inhibited rituximab mediated adcp in phagocytosis by macrophage but igg1 could not inhibit the adcp effect scale bar30 µm p005 p001 p0001 g in a classic cdc experiment cetuximab anti egfr antibody was incubated with an egfr expressing lung cancer cell line a549 and then with complement co from serum of a normal healthy adult the cancer cell activity was significantly reduced h when non cancer specific igg4 was added to the mixture in the above cdc experiment the effect of cetuximab was significantly reversed i igg4 and igg1 were incubated with kyse150 for hours and no effect on cell growth was found adcc antibody dependent cell mediated cytotoxicity adcp antibody dependent cellular phagocytosis cdc complement dependent cytotoxicity cfse da carboxyfluorescein diacetate succinimidyl ester dapi ' diamidino2 phenylindole egfr epidermal growth factor receptor hsa human serum albumin pbmc peripheral blood mononuclear cellwang a0h et a0al j immunother cancer 20208e000661 101136jitc2020000661 0copen access be abolished with the addition of fc receptor blocker human trustain fcx biolegend china to the pbmcwe also performed an adcp experiment employing human monocyte derived macrophages and esophageal cancer cells cetuximab igg1 was used as the mediating antibody this was performed employing a coculture and cell counting method and fitc labeled antibody flow cytometry two models were employed and both methods showed that non cancer specific igg4 was able to reduce the effect of adcp mediated by cancer specific igg1 figure 4c“fin a classic cdc assay we used cancer cell line a549 atcc usa c4215 as the target cancer cells cetuximab as the cancer specific igg1 mediating antibody and human plasma as the source of complements and demonstrated the destructive effect on cancer cells we then used non cancer specific igg4 or igg1 to inhibit the effect we found that the cdc effect was partially inhibited by non cancer specific igg4 but not by igg1 figure 4ghfor comparison we added igg4 or igg1 at various concentrations in cancer cell culture for different periods of time and found no direct effect of these proteins on cancer cell growth figure 4iigg4 including nivolumab significantly accelerated breast cancer cell and colon cancer cell growth in two immune potent mouse models in vivothe above results point to a mechanism that igg4 plays an important role in local immune evasion by blocking immune responses mediated by cancer specific igg antibodies to further examine this mechanism mediated by such antibodies we performed in vivo studies to verify this hypothesis with immune competent mouse models in one model we injected non cancer specific igg4 into a location where breast cancer cells were inoculated subcutaneously in this group of mice cancer cell growth was significantly increased resulting in a much larger cancer mass by days in comparison with other groups injections of pbs or igg without igg4 figure 5ab as there is no direct effect of igg4 on cancer cell growth figure 4i these results unequivocally confirmed that igg4 can inhibit local immune reaction and thereby promote cancer growth in vivo through immune evasionin a separate but similar experiment of a colon cancer mouse model we injected antibody to programmed cell death1 pd1 nivolumab which is a widely used drug in cancer immune therapy and is also an igg4 isotype with s228p mutation which replaces a serine residu
Colon_Cancer
"the kinetics and localization of the reactions of metabolism are coordinated by the enzymes that catalyze themthese enzymes are controlled via a myriad of mechanisms including inhibitionactivation by metabolitescompartmentalization thermodynamics and nutrient sensingbased transcriptional or posttranslational regulationall of which are influenced as a network by the activities of metabolic enzymes and have downstream potential toexert direct or indirect control over protein abundances considering many of these enzymes are active only whenone or more vitamin cofactors are present the availability of vitamin cofactors likely yields a systemsinfluence overtissue proteomes furthermore vitamins may influence protein abundances as nuclear receptor agonistsantioxidants substrates for posttranslational modifications molecular signal transducers and regulators ofelectrolyte homeostasis herein studies of vitamin intake are explored for their contribution to unraveling vitamininfluence over protein expression as a body of work these studies establish vitamin intake as a regulator of proteinabundance with the most powerful demonstrations reporting regulation of proteins directly related to the vitaminof interest however as a whole the field has not kept pace with advances in proteomic platforms and analyticalmethodologies and has not moved to validate mechanisms of regulation or potential for clinical applicationkeywords proteomics big data vitamin metabolism precision nutrition molecular nutritionintroductionregulatory mechanismscellular metabolism is a system of chemical reactions inwhich cells harness the energy stored in the chemicalbonds of substrate molecules to perform their biologicalfunctions maintain homeostasis or to synthesize buildingblocks for structural maintenance or cellular division thekinetics of these reactions are dependent on the activity ofthe proteins which catalyze them thus proteins are keymodulators of metabolismmetabolic activity also exerts network control over itselfby a diverse array of mechanisms which finely tune proteinexpression responses via nutrient sensing machineries products or intermediates of a metabolic pathway caninhibit or activate metabolic enzymes eg malate inhibitsthe succinate dehydrogenase complex and fructose correspondence nv83cornelledudivision of nutritional sciences cornell university ithaca ny usa26bisphosphate activates phosphofructokinase theoxidative status of a cell can drive the directionality ofredox reactions and impact abundances of redox reactioncatalyzing proteins eg the keap1nrf2 network responds to oxidative stress by upregulating expression ofantioxidantfunctioning proteins splicevariant or isozyme expression can impact relative pathway utilization atmetabolic network nodes eg splice variants and isozymesof pyruvate and lactate dehydrogenase respectively impactthe bridge between glycolysis and the tricarboxylic acidtca cycle [ ] additionally local metabolite concentrations and thermodynamics can dictate the directionalityof reactions catalyzed by compartmentspecific isozymeseg reductive activity of isocitrate dehydrogenase can beconfined to the cytosolspecific isozyme the impactsof the abovementioned regulations are closely monitoredby nutrient sensing proteins which initiate molecularevents altering protein activation and expression eg the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cjeong and vacanti nutrition metabolism page of serinethreonine kinase ampactivated protein kinasemammalian target of rapamycin and sterol regulatoryelementbinding protein are part of overlapping proteinnetworks that orchestrate proteinexpression and posttranslational modification responses to nutrient availability[ ] considering that many metabolic enzymes do notfunction in isolation and as detailed in the sections thatfollow require vitamin cofactors to stabilize intermediatesdonateaccept electrons shuttle substrates and hold reactants in close proximity vitamin status is a critical consideration when examining proteinmediated regulation ofmetabolism and the impacts of metabolism on proteinexpressionin addition to their potential regulatory roles ascofactors vitamins orchestrate other direct or indirectmechanisms influencing protein abundance retinoicacid vitamin a interacts with nuclear receptors impacting gene transcription ascorbic acid vitamin cimpacts oxidative status and associated protein networks and is reported to exhibit epigenetic regulation overprotein expression vitamin d regulates calcium signaling machinery activates nuclear receptors and exertshormonal regulation over protein expression [ ]and niacin vitamin b3 and biotin vitamin b7 can beincorporated as posttranslational modifications impacting protein function [ ]herein studies on systemic intake dietary injectionoral gavage of vitamins and their impacts on tissueproteomes are examined and their contributions tounraveling vitaminbased regulation of protein expression and tissue function are explored the currentwork is intended to provide informationto understand each vitamin™s figs and molecularfunctions and highlight its role as a cofactor or substrate in the reactions of central metabolism fig tables s1 s2 s3 s4 s5 s6 s7 s8 s9 s10 s11 s12and s13 finally this work is intended as a resourcefor identifying regulation of proteins related to vitaminmetabolism in published works the public domain ofproteomic data sets is ever expanding but is rarelysearched for effects related to vitamin metabolism tothat end all proteins are specified by their hugogene nomenclature committee hgnc gene symbolor the hgnc gene symbol of the human orthologwhen identified in another species and proteins requiring a vitamin as a cofactor or substrate are tabulated tables s1 s2 s3 s4 s5 s6 s7 s8 s9 s10s11 s12 and s13proteomics platformsproteomics platforms ofthe discussed studies areprovided to place them on a technological timelineplatforms are described with the terms orbitrap qtofquadrupole timeofflight tripletof triple “ time offlight qqq triplequadrupole 2dgems twodimensional gel electrophoresis “ mass spectrometry and2dge in brief orbitrap platforms are the workhorses ofmodern proteomics because their high achievable massresolutions combined with high sensitivity are bestsuited for maximizing the number of proteins identifiedin a complex sample [ ] though qtof and tripletof instruments capable of maintaining mass resolutionfig fat soluble vitamin structures 0cjeong and vacanti nutrition metabolism page of fig water soluble vitamin structuresat higher scan speeds hold a substantial influence inthis arena within the categories of orbitrap qtof andtripletof there are major technological advances notdiscussed here qqq platforms are best suited for quantifying a predetermined list of proteins lower scan speedsand mass resolution render them less capable thanorbitrap qtof or tripletof systems for nontargetedapplications advances in nanoflow liquid chromatography coupled directly to mass spectrometry haveimproved proteomic depth by orders of magnitude overthat achievable by 2dgems where the upstream selection of protein spots predates the modern definition ofnontargeted proteomics similarlyidentifying differentially intense protein spots using 2dge alone is considered an important milestone in the development ofproteomics but is rarely discussed outside the topic of thefield™s historyvitamin regulation of tissue proteomesvitamin avitamin a exists in alcohol aldehyde acid and esterforms known as retinol retinal retinoic acid and retinylesters respectively fig several carotenoids areprecursors to vitamin a including α and βcarotene βcarotene is converted to two molecules of retinalby beta carotene oxygenases bco1 or bco2 retinal is an important component of rhodopsin rho aprotein in rod cells responsible for detecting low levelsof light thus night blindness is telltale characteristic of vitamin a deficiency retinoic acid serves as asignaling molecule acting through nuclear retinoic acidrara rarb rarg and retinoid x rxra rxrbrxrg receptors which regulate growth and differentiation [ ] cellular and anismal trafficking ofvitamin a is dependent on retinolretinoic acid bindingproteins rbp family crabp1 crabp2 and retinolesterification via lecithin retinol acyltransferase lrat retinal is oxidized to retinol via aldehyde dehydrogenases aldh family and retinol is oxidized to retinoicacid by retinol dehydrogenases rdh and dhrs families in addition to inducing night blindness vitamin adeficiency adversely impacts cellular growth bone development and antibodybased immune responses in an orbitrapbased study of mouse embryo headstoxic levels of prenatal retinoic acid exposure intendedto model an established risk factor for craniofacial birthdefects are reported to induce abundance alterations inproteins associated with craniofacial development and 0cjeong and vacanti nutrition metabolism page of fig schematic of vitamin involvement in reactions of central carbon metabolism the depicted lipid bilayer represents the inner mitochondiralmembrane abbreviations defined in the abbreviations section vitamins specified by alphanumeric designations 0cjeong and vacanti nutrition metabolism page of neural crest processes in a parallel tripletofbased study of gerbil plasma and 2dgemsbased studyof gerbil liver and white adipose tissue a few dozen protein abundances linked to a handful of biological processesare reported to respond to dietary retinol βcarotene lutein or lycopene though process or pathway enrichmentanalyses are not reported as the authors discuss plasmawas not depleted of common highly abundant proteinsupstream of analysis by mass spectrometry which areknown to adversely impact data quality in anorbitrapbased study of plasma from nepalese childrendozens of proteins are associated with circulating carotenoid abundances potentiating development oflowcostantibodybased tests for carotenoid deficiencies apair of 2dgemsbased studies link tissue function toprotein abundance responses to vitamin a status in micebrains and bovine muscle vitamin b1thiamine vitamin b1 is composed of linked pyrimidineand thiazole rings decorated with methyl amine andalkylhydroxyl functional groups fig thiamineistransported through the plasma membrane viathiamine transporters slc19a2 and slc19a3 andthen twice phosphorylated on the alkylhydroxyl functional group by thiamine pyrophosphokinase tpk1rendering it active as thiamine diphosphate tdp tdp is a cofactor for enzymes catalyzing the oxidativedecarboxylation of ketoacids including the pyruvatedehydrogenase complex pdha pdhb pdhx dlatdld the oxoglutarate dehydrogenase complex ogdhdlst dld and the branched chain keto acid dehydrogenase complex bckdha dbt dld it is also acofactor for transketolase tkt in the nonoxidativebranch of the pentose phosphate pathway independent from its role as a cofactor thiamine is believed toregulate ion transport activity in the nervous system vitamin b1 deficiency is marked by a broad range ofneurological respiratory and cardiovascular pathophysiologies and is termed beriberi symptoms of beriberi aredifficult to directly link to the molecular functions ofvitamin b1 in a 2dgemsbased study of type diabetic andhealthy control subjects authors report treatment withthiamine reduces albumin alb abundance in urine indicating the vitamin serves a protective role of kidneyfunction in a qtofbased study of rat thalamiunder thiamine deficiency glyceraldehyde3phosphatedehydrogenase gapdh is the most upregulated protein fold while regulated proteins are most enrichedin the synaptic vesicle cycle pathway according to thekegg database proteomic changes are accompaniedby diminished performances on cognitive tests vitamin b2riboflavin vitamin b2 is composed of an isoalloxazinering and a bound ribitol fig it is activated byriboflavin kinase rfk forming flavin mononucleotidefmn and by flavin adenine dinucleotide synthase flad1 forming flavin adenine dinucleotide fad bound fmn or fad serves as an electron carrierfor redoxreactioncatalyzing proteins flavoproteinsincludingcomplexsdha sdhb sdhc sdhd the pyruvate dehydrogenase complex pdha pdhb pdhx dlat dldacylcoa dehydrogenases acads and methylenetetrahydrofolate reductase mthfr dehydrogenasethesuccinateriboflavin deficiency in humans predominantly occursin combination with that of other nutrients howeveranimal studies link it to impaired fetal and intestinaldevelopment [ ]iron absorption and lipidmetabolism [ ]in a qtofbased study of duck livers riboflavin deficiency is accompanied by a reduced abundance of smallchainspecific acylcoenzyme a dehydrogenases acadsfor which riboflavin serves as a cofactor and concordantelevation of hepatic small chain fattyacid lipid contentdramatic decreases in protein abundance are reported forinpp1 involved in inositol signaling thrsp purportedregulator of lipid metabolism bdh2 a regulator of lipidmetabolism fxn involved in mitochondrial ironsulfurcomplex assembly and ndufs1 a subunit of electrontransport chain complex i in a qtofbased study ofmaternal riboflavin deficiency reductions in fetal duck hepatic tca cycle betaoxidation and electron transport chainproteins are reported with idh3a being the lone memberof these pathways whose abundance increases vitamin b3niacin vitamin b3 is inclusive of nicotinic acid and nicotinamide fig which are converted to their mononucleotide forms by nicotinate phosphoribosyltransferase naprt and nicotinamide phosphoribosyltransferase namptrespectively both forms of the mononucleotide aresubsequently converted to their adenosine dinucleotideforms by nicotinamidenicotinic acid mononucleotidenmnat1 nmnat2 nmnat3adenylyltransferasesnicotinamide adenine dinucleotide nad is a cofactorform of the vitamin whereas nicotinic acid dinucleotide issubsequently converted to nad by nad synthase nadsyn1 nad is reduced to nadh by oxidative reactions of glycolysis the tca cycle and βoxidation andsubsequently serves as a redox equivalent carrier to theelectron transport chain and to regenerate reducedascorbic acid vitamin c glutathione and thioredoxin nad can also be phosphorylated by nadkinases nadk nadk2 to form a distinct redox shuttlingcofactor nadp nadp is reduced by reactions in the 0cjeong and vacanti nutrition metabolism page of oxidative pentose phosphate pathway g6pd pgd andother enzymes eg me1 me3 idh1 idh2 to nadphnadph provides reducing equivalents for biosynthetic reactions in fatty acid cholesterol and deoxyribonucleotidesynthesis outside its role as a reducing equivalentshuttle nad provides adenine dinucleotide phosphateadp ribose for synthesis of the second messenger cyclicadenosine monophosphate camp via the activity of adenylate cyclases adcy family nad also providesadpribose and polyadpribose for post translationalmodifications of proteins via activity of adpribosyl transferases art family and adp ribose polymerases parpfamily [ ] camp and protein polyadpribosylationare important mediators of cell signaling and proteinexpression niacin is synthesized from tryptophan butin small quantities relative to a healthydietary intake deficiency known as pellagraismarked by dermatitis and severe gastrointestinalneurological pathophysiologies which are fatalif untreated no proteomic studies on systemic intakeof vitamin b3 were found at the time of writing thisreviewvitamin b5pantothenic acid vitamin b5 is composed of a moleculeof pantoic acid bound to βalanine fig itsprimary metabolic function is as an acylcarrier pantothenic acid is a substrate in the first reaction of coenzyme a coa biosynthesis catalyzed by pantothenatekinases pank1 pank2 pank3 pank4 coa isa substrate for enzymes catalyzing the oxidative decarboxylation of ketoacids including the pyruvate dehydrogenase complex pdha pdhb pdhx dlat dldthe oxoglutarate dehydrogenase complex ogdh dlstdld and the branched chain keto acid dehydrogenasecomplex bckdha dbt dld [“] acyl speciesare activated by conjugation with coa and are substratesin or products of glycolysis the tca cycle fattyacidsynthesisβoxidation cholesterol synthesis ketogenesisbranchedchain amino acid catabolism and proteinacetylationoglcnacylation finally ™phosphopanthetheine product of pank proteins™ activities is acofactor of the acyl carrier protein domain of fatty acidsynthase fasn vitamin b5 deficiency is rare andusually accompanied by that of other nutrients burning of the feet and numbness in the toes is a characteristic manifestation along with variety of other symptoms no proteomic studies on systemic intake ofvitamin b5 were found at the time of writing this reviewvitamin b6vitamin b6 has aldehyde alcohol and amine forms fig of which the phosphorylated aldehyde form pyridoxalphosphate acts as a cofactor to over enzymes allthree forms of vitamin b6 are phosphorylated by pyridoxalkinase pdxk both the phosphorylated alcohol andamine forms pyridoxine phosphate and pyridoxaminephosphate are converted to pyridoxal phosphate bypyridoxine phosphate oxidase pnpo pyridoxalphosphate is a cofactor for enzymes catalyzing decarboxylase reactions in gammaaminobutyric acid gad1 gad2 and serotonindopamine biosynthesis ddc aswell as for enzymes catalyzing transamination reactionseg got1 got2 gpt gpt2 cysteine synthesiscth heme synthesis alas1 alas2 carnitinesynthesis3hydroxy6ntrimethyllysine aldolase geneunidentified andsphingolipid synthesis sptlc1 sptlc2 pyridoxalphosphate is also an important cofactor for enzymes ofonecarbon metabolism shmt1 and shmt2 andglycogen catabolism pygl and pygm vitamin b6deficiency is rare because of its availability in many foodsand pathophysiologies can be diverse niacin synthesis kynuin a tripletofbased study of streptozotocininduceddiabetic rat hippocampi pyridoxamine treatment prevented longterm recognition memory impairment andregulated protein abundances in a number of diversepathways notably upregulating half of the proteins involved in ubiquinol biosynthesis in a 2dgemsbased study of mice hippocampi the abundances ofphosphoglycerate mutase pgam1 and cannabinoidreceptorinteracting protein cnrip1 are reported tobe elevatedreduced respectively upon administration ofpyridoxine proteomic changes are accompanied by improved novel object recognition the plasma membrane byvitamin b7biotin vitamin b7 is composed of a fusedring structurebound to a valeric acid side chain fig it istransported acrossthesodiumdependent solute carriers slc5a6 and slc19a3[ ] as a cofactorposttranslational modificationbiotin covalently binds lysine residues it is a cofactor for pyruvate carboxylase pc acetylcoa carboxylase acaca propionylcoa carboxylase pcca andthe methylcrotonylcoa carboxylase complex mccc1mccc2 histones are also biotinylated regulatinggene expression the posttranslational modification occurs via the activity of holocarboxylase synthetasehlcs biotin deficiency is rare and has wide ranging pathophysiologies eating raw egg whites can preventitsabsorption leading to deficiency because of its affinityfor avidin a chemical in egg whites that is denaturedupon cooking this observation led to the vitamin™seventual discovery no proteomic studies onsystemic intake of vitamin b7 were found at the time ofwriting this review 0cjeong and vacanti nutrition metabolism page of vitamin b9the term folate vitamin b9 is inclusive of a group ofcompounds composed of a pteridine ring linked to paraaminobenzoic acid with a mono or polyglutamate tailfig in its reduced form tetrahydrofolate aonecarbon unit crosslinks as ch or ch2 aminegroups on the ring structure and aminobenzoic acid orbinds the secondary amine as a formyl group on theaminobenzoic acid group [ ] this onecarbonunit is utilized in the synthesis of purines and thymidineconversion of homocysteine to methionine interconversion of serine and glycine and catabolism of histidinereactions collectively termed onecarbon metabolism[ ] at the cellular level onecarbon metabolismis tightly regulated by compartmentalization [ ] while wholebody folate homeostasis is predominantly maintained by the liver through the enterohepaticcycle folate deficiency induces megaloblastic macrocyticanemia and fetal neural tube defects purportedly via itsadverse impact on nucleotide synthesis [ ] lowintake of folate is also linked to cardiovascular disease[ ] neurodegenerative disease [ ] alzheimer™s disease [ ] and cancer [“] in an orbitrapbased study of follicle fluid of womenundergoing in vitro fertilization the folate supplementedgroup is reported to have elevated abundances of apolipoproteins from high density lipoproteins and reducedreactive protein c crp the study is performed onwomen who did not become pregnantin aqtofbased study of a folatedeficiencyinduced intestinal neoplasia mouse model the combinatorial impactsof folate deficiency and methylene tetrahydrofolate reductase heterozygous deletion mthfr are reported toimpact protein abundances spanning diverse cellularfunctions however of samples are discarded as outliers and the simultaneous examination of mthfr anddietary folate deficiency does not allow proteomic adaptations to be attributed to either in isolation in a2dgemsbased study of adult rats aortic calmodulincalm1 calcium signaling protein abundances arepositively correlated with folate dose while abundancesof triose phosphate isomerase tpi1 glycolysis transgelin tagln cytoskeleton and glutathione stransferasealpha gsta3 reductive detoxification respond inversely in an 2dgemsbased study of rat liversprdx6 and gpx1 are reported to be elevated whilecofilin cfl1 is reported to be depleted under folatedeficiency other studies report protein abundancedifferences due to folate intake in rat urinary exosomesqqqbased human plasma 2dgems fetal brain tissue from pregnant mice fed ethanol2dgems pregnant rat livers 2dgems fetal rat livers 2dgems adult rat livers andbrains 2dgems and livers of piglets born tofolate deficient mothers 2dgems vitamin b12cobalamin vitamin b12 encompasses a group of molecules with four linked pyrrole ring derivatives forming a corrin ring and a cobalt atom bound at thecenter of the corrin ring the cobalt atom also binds a56dimethylbenzimidazole nucleotide and a functionalgroup fig the identity of the functionalgroup distinguishes the vitamin b12 compounds ascyanocobalamin hydroxycobalamin hydrocobalaminnitrocobalamin ™deoxyadenosylcobalamin also called adenosylcobalamin and methyl cobalamin [ ] methylcobalamin serves as a coenzyme in the conversion ofhomocysteine to methionine by methionine synthase mtrin the cytosol and adenosylcobalamin is required forconversion of lmethylmalonylcoa to succinylcoa bymethylmalonylcoa mutase mut in mitochondria vitamin b12 deficiency is closely related to folatedeficiency and can lead to megaloblastic anemia by impairment in the activity of methionine synthase mtr 5methyl tetrahydrofolate cannot be converted toonecarbon donors required for purine and thymidinesynthesis without vitamin b12 as a cofactor thus interfering with dna synthesis and erythrocyte production vitamin b12 deficiency is also linked to neurological disorders independent of anemia ruoppolo and colleagues performed a 2dgemsbased study of lymphocytes isolated from methylmalonicacidemia with homocystinuria cobalamin deficiency typec mmachc patients an inborn error in metabolismmarked by inactivity of the mmachc gene productreceiving a standard treatment of hydroxycobalaminbetaine folate and carnitine protein products of me2glud1 and gpd2 genes involved in anaplerosis andredox equivalent shuttling are upregulated while variant of protein pyruvate kinase muscle isozyme pkmand lactate dehydrogenase b ldhb are downregulatedrelative to lymphocytes isolated from healthy control donors in a 2dgebased study of adult rat cerebralspinal fluid protein abundance shifts are reported topeak after several months on a cobalamin deficient dietmodest shifts or after a total gastrectomy more severeshifts and return to near control values at later timepoints in a 2dgemsbased study glutathione stransferase p gstp1 abundances are diminished andglutathione peroxidase gpx1 abundances are elevated in rat pup kidneys under maternal vitamin b12deficient and maternal folate deficient conditions suggesting maternal dietary intake of these vitamins impacts offspring kidney redox homeostasis mechanisms 0cjeong and vacanti nutrition metabolism page of in a similar 2dgemsbased study of maternal vitaminb12 deficiencythe same group reports that severaldozen rat kidney pup proteins revert to control levelsupon administration of vitamin b12 at birth additionally diminished abundance of betaoxidation proteinsin kidneys of pups born to vitamin b12 deficientmothers is accompanied by elevated ppara apositive regulator offatty acid oxidation suggestingattempted compensation at the cellular levelvitamin cvitamin c ascorbic acid is absorbed at the brushborder and distributed to cells throughout the body bythe sodiumdependent plasma membrane solute carriersslc23a1 and slc23a2 the oxidized form of vitamin c dehydroascorbate is also transported via plasmamembrane glucose transporters slc2a1 slc2a3 andslc2a4 also known as glut1 glut3 and glut4 and reduced intracellularly to ascorbic acid byglutathione and the activity of thioredoxin reductases txnrd1 txnrd2 or txnrd3 vitamin c is a cofactor in the function of prolyl andlysyl hydroxylases which consume oxygen and alphaketoglutarate to form the hydroxylated amino acid residueand succinate the fe2 of these enzymes is restoredfrom fe3 by oxidation of vitamin c in the presenceof oxygen prolyl hydroxylases egln1 egln2 egln3also known as phd2 phd1 phd3 respectively hydroxylate the hif1a protein providing a necessary signal for itsdegradation and preventing a hypoxic response at the cellular level prolyl and lysyl hydroxylase activities arealso necessary for posttranslational modifications to formfunctional collagen lysyl hydroxylases includeplod1 plod2 and plod3 vitamin c serves anearly identical function in reducing fe3 as a cofactor fortrimethyllysine dioxygenase tmlh which catalyzes thefirst reaction in carnitine biosynthesis carnitine isessential for fatty acid catabolism in the mitochondria asonly fatty acyl carnitines formed via the activity of carnitine palmitoyl transferases cpt1a cpt1b and cpt1ccross the inner mitochondrial membrane through thesolute carrier slc25a20 vitamin c similarly servesas a cofactor for tyrosine hydroxylase th which catalyzes the first reaction in catecholamine eg dopamineepinephrine and norepinephrine synthesis additionally vitamin c serves and as a general antioxidant vitamin c deficiency leads to the condition knownas scurvy with symptoms largely attributed to malformedconnective tissue due to improperly folded collagen in a orbitrapbased study on a pig model of hemorrhagicshock vitamin c administration is reported to impactplasma protein abundances in the complement pathwayand those in polytrauma related processes including thestabilization of adamts13 abundance an importantregulator of clot formation an orbitrapbased studyof endoplasmic reticulum enriched fractions of livers inwerner syndrome mouse models identifies around adozen proteins whose abundances are impacted by administration of vitamin c a qtofbased study ofzebrafish reports upregulation of glutamate dehydrogenase glud1 and downregulation of pyruvate kinasemuscle isozyme pkm upon administration of vitamin cin a vitamin e deficient in a qqqbased study of human plasma ascorbic acid concentrationis reported to be inversely related to vitamin d bindingprotein gc abundance 2dgemsbased studiesidentify protein abundance regulations in mouse modelsof sarcoma metastases in the liver and tumor nodules of adenocarcinoma due to administration of vitaminc another 2dgemsbased study reports polypeptide abundance shifts in hemodialysis patient plasma uponvitamin c supplementation vitamin dvitamins d2 and d3 are respectively distinguished by theirergosterol and cholesterol backbones though onlyvitamin d3 is synthesized in animals both can be converted to active forms exposure of 7dehydrocholesterolan intermediate in cholesterol synthesis to ultravioletradiation in the skin and subsequent isomerization produces cholecalciferol vitamin d3 fig whether dehydrocholesterol is derived from cholesterol via activityof 7dehydrocholesterol reductase dhcr7 or synthesizedde novo in the skin is disputed 7dehydrocholesterolis successively hydroxylated by activity of cytochrome p450enzymes eg cyp2r1 and cyp27b1 in the liver and kidney to its active 125oh2 cholecalciferol [125oh2d3]form transport of vitamin d and its metabolitesoccurs bound to vitamin d binding protein gc ergocalciferol is the vitamin d2 equivalent of cholecalciferol and is activated analogously 125oh2d3 influences cellular function via nuclearreceptordependent and nuclear receptorindependentmechanisms the former involves 125oh2d3boundvitamin d receptor vdr forming a heterodimer complex with a retinoid x receptor rxra rxrb rxrgand subsequently binding vitamin d response elementsregulating transcription of genes largely involved modulating calcium and phosphorous transport andmaintaining homeostasis by regulating their absorptionin the kidneysintestines and bones [ ] therapidonsetreceptorindependent of 125oh2d3 are mediated by a membraneassociated rapid response steroid binding protein identifiedextracellularimpactsnuclear 0cjeong and vacanti nutrition metabolism page of as pdia3 and diversely impact cell growth survivaland immune response deficiency in vitamin d impairs bone mineralizationcausing rickets in infantschildren and osteomalacia inadults vitamin d deficiency is also linked tocardiovascular diseases [ ] cancer [ ]neurologicalimpairments [ ] and autoimmunediseases [ ] though underlying mechanisms arenot completely understoodin an orbitrapbased study of mouse fetal and postnatal lung tissue maternal vitamin d deficiency is reflectedin total proteome adaptations which are unexpectedlystrongest at postnatal day opposed to fetal time pointsimpacted proteins include several associated with lungdevelopment an orbitrapbased study of a mousebrain tissue model of remyelination in multiple sclerosisreports calcium binding protein abundances to be upregulated upon treatment with 125oh2d3 consistentwith the vitamin's regulatory role over calcium absorption in an orbitrapbased study of serum fromoverweight adults vitamin d deficiency is reported todifferentially affect abundances of proteins related toblood coagulation in males and females howeverabundances of these proteins are likely impacted by theproduction of serum from whole blood the authors report quantifying proteins table an impressiveanalytical depth for serum in a 2dgebased studyvitamin d deficient children are reported to have diminished serum abundances of adiponectin adipoq in a separate 2dgebased study the same groupreports fetuinb fetub to be elevated in the plasma ofobese vitamin d deficient children compared with theirvitamin d sufficient counterparts however theauthors do not directly identify fetub and rely on comparison of their findings to those of another study two 2dgemsbased studies of rat left ventricular andaortic tissueidentify proteins whose abundances respo
Colon_Cancer
colorectal cancer crc is a malignant tumor in the gastrointestinal tract and it arises from theinner wall of the large intestine the colon crc is the third most common cancer worldwideaccounting for roughly million new cases per year and ˆ¼ deaths per year whichmakes it the fourth most common cause of cancerrelated death globally and remains a hugechallenge “ in order to identify eï¬ective molecular targets for crc diagnosis and potentialedited byzhonghua taofudan university shanghai cancercenter chinareviewed byshengli liuniversity of texas health sciencecenter at houston united statesrossano lattanziouniversity of studies g d™annunziochieti and pescara italycorrespondencejianhua wangwangjianhuaman163comyansong pudoctor_puyahoocom these authors have contributedequally to this workspecialty sectionthis was submitted tocancer geneticsa section of the frontiers in oncologyreceived september accepted june published august citationliu y cao j zhu yn ma ymurtaza g li y wang jh and pu ys c1222c deletion in exon ofabl1 is involved in carcinogenesisand cell cycle control of colorectalcancer through irs1pi3kaktpathway front oncol 103389fonc202001385frontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerinterventions for crc therapy indepth studies on the regulatorymechanism of crc progression should be conductedthe pathogenesis of crc accompanies with genetic orepigenetic changes numerous genes and pathways such aswnt tgf egfr“ras erk“mapk pi3k and p53 havebeen demonstrated to be associated with crc “ abl1a protooncogene of cabl encodes a nonreceptor tyrosinekinase plays an important role in carcinogenesis regulatingcell adhesion proliferation diï¬erentiation and apoptosis studies have characterized abl1 as an oncogene thatpromotes breast cancer cell proliferation and induces anchorageindependent growth under p53 deficiency in breast cancercells “ craig reported that inhibition of abl1by imatinib reduced the proliferation of lymphoma cells andprevented tumor formation in mice however the roleand mechanism of abl1 in crc development and progressionremain largely unclearthe aim of this study was to elucidate the role of abl1using highthroughput dna sequencing technology to obtaininformation on colon cancer gene mutation we analyzedthe variation in the expression of abl1 among patients withcrc and in crc celllines we additionally determinedthe eï¬ect of downregulating abl1 on the proliferation cellcycle progression and apoptosis of crc cells further theeï¬ects of knockout of abl1 in tumor and the molecularmechanisms of activated and suppressed downstream signalingpathways were assayed to elicit the mechanisms involved incrc carcinogenesismaterials and methodsadmitted atclinicalpathological data ofpatients and samplesthefortyeight patients with crc wereshaanxi people™s hospitalshaanxi china colorectalcancer was confirmed by histopathology or biopsy basedon which thesubjectswere evaluated formalxed paraffinembedded ffpetissues were used as the study material tumor contentsin the ffpe tissues werethedepartment of histopathology shaanxi people™s hospitaland only ffpe tissue blocks with tumor contentwere qualified the study was approved by the ethicscommittee of shaanxi people™s hospital written informedconsent was obtained from allsubjects participating inthis studythethoroughlychecked atcell culture and rna interferencecrc cell lines ncm460 lovo sw620 sw480 and hct116were purchased from the cell bank of the chinese academyof sciences shanghai china the cells were cultured inrp1640 medium supplemented with heatactivated fetalbovine serum gibco gaithersburg md and penicillinstreptomycin gibco at —¦c with co2abl1 protein phosphatase catalyticsubunit alphappp3ca and tgf1 knockdown kd lentiviruses weregenerated using pfugwgfprnai vector by insertingshabl1 shppp3ca and shtgf1 sequence empty pfugwgfp vector was used as vector control shctrl in crc cells or ncmice the rnai sequence of abl1 ppp3ca and tgf1 were²cgttctatatcatcactga3² ²atatacgcgttctgaatactt3² ²gattatcga catggagctg3² respectivelysw480 and hct116 cells were plated in a 24wellplate andincubated at —¦c with co2 for h a multiplicity ofinfection of was added to infect sw480 and hct116 cellsovernight the infection medium was then replaced with normalcomplete growth medium cells without infection were used ascorresponding controlsproliferation and colony formation assayproliferation rate was determined using bromodeoxyuridinebrdu cell proliferation elisa kit abcam boston ma theoptical density of each sample was measured at nm using asynergy h1 microplate reader biotek winooski vtfor the clonogenic assay sw480 and hct116 cells wereplated onto 6wellplate and incubated in culture medium for days the cells were then fixed with pfa and stained with crystal violet sigmaaldrich st louis mo for h at roomtemperature the total number of colonies was counted wheneach clone contained more than cells size “ mmflow cytometric analysisfor cell cycle analysis cells were fixed in pfa for minat —¦c and treated with propidium iodide pi µgmlsigmaaldrich at room temperature for min in dark atotal of cells were analyzed by flow cytometry using abd facscalibur system becton“dickinson el paso tx thedistribution of cell cycle phases was estimated using modfitlt in mac v30 software apoptosis was further determinedby annexin v fitcconjugated thermo fischer scientificmiami ok and pi staining cells were immediately counted byflow cytometrydna extraction and sequencingfortyeight specimens of colorectal cancer tissue with clinicalliver metastases were collected dna was isolated using allprep dna ffpe kit qiagen germantown md genomic dnawas extracted by fully automated purification using promegamaxwell promega madison wi the dna concentrationwas measured fluorimetrically using the qubit dna highsensitivity kit thermo fisher scientific waltham ma anion torrent semiconductor chip sequencer was used to sequencecommon gene mutations in the tumorsin vivo studybalbc nude mice female aged weeks were purchased fromshanghai ling chang biological technology co ltd shanghaichina the mice were housed in spflevel laboratories with freeaccess to food and water and accommodated for week prior toany experiments the animal study was performed in accordancewith iacuc guidelines shabl1hct116 kd or shctrlhct nc cells — µl were subcutaneously injected tothe left flank of the mice at day posttransplantation micewere sacrificed and tumors were excised and weighed the tumorfrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancervolume was calculated using digital calipers with the followingformula tumor volume volume length width22ingenuity pathway analysisto elucidate the role and action mechanism of abl1 incrc after abl1 kd high throughput realtime pcr arraywas performed by shanghai genechem co ltd shanghaichina and the data were analyzed using ingenuity pathwayanalysis ipa software to elucidate the aï¬ected molecules andsignal pathwaysimmunohistochemistryfor crc 180point tissue microarray hcolade180sur07which contained crc tissuesfrom patients and thecorresponding adjacent tissues table s4 was purchased fromshanghai outdo biotech co ltd shanghai china briefly thetissue microarray block was constructed by embedding a singletissue core mm in diameter was taken from each region informalxed paraffinembedded crc or adjacent tissue blockusing a tissue microarrayer beecher instruments silver springmd usa and was set to a blank recipient block predrilled with mm holesthe tissue microarray blocks and paraffinembedded tumorsections were cut into 7µm sections for immunohistochemicalihc analysis slides were deparaffinized and rehydrated aspreviously described followed by antigen retrievalincitrate buï¬er mm citric acid tween ph for min in —¦c water bath after washing with pbsslides were incubated with pbst with bovine serumalbumin sigmaaldrich for h slides were then incubatedovernight at —¦c with antiabl1 antibody ab15130abcam cambridge ma and developed using mouse andrabbit specific hrpdab detection ihc kit ab64264 abcamfollowing the manufacturer™s instructionsthe selection of cutoï¬ value to dichotomize the expressionlevels of abl1 was based on previously reported method []briefly the high expression level of abl1 was defined from twocriteria dab staining showed equal or darker color comparedto positive control the population of abl1positive cells washigher than all cases were independently evaluated anddiagnosed by two senior pathologists y m and l y who wereblinded to the pathologic diagnosis cases with any disagreementwere reviewed simultaneously by the original two pathologistsand a senior pathologist j w until they reach a consensuswestern blotthe western blotting assay was performed by wellestablishedprotocols as previously described primary antibodiesused in this study were antiabl1 antibody ab85947abcam cambridge ma antibcl2 antibody bcl10c4biolegend san diego ca antibclxl antibody sc santa cruz biotechnology dallas tx antibaxantibody 2d2 biolegend antiactin antibody 2f11 biolegend antigapdh antibody ff26af9biolegend antip27 antibody sc56338 santa cruzbiotechnology anticyclind1 antibody sc8396 santacruz biotechnology antiirs1 antibody ab52167abcam antiakt2 antibody ab175354 abcam antippp3ca antibody ab52761 abcam antitgf1antibody ab92486 abcam antimap2k2 antibody sc81473 santa cruz biotechnology antipi3kp11aantibody ab151549 abcam secondary antibodiesused were antimouse igg hrpconjugated secondary antibody sc516102 santa cruz biotechnology and antirabbitigg hrpconjugated secondary antibody sc2357 santacruz biotechnologyreverse transcriptionpolymerase chainreactionthe mrna level was measured using realtime polymerasechain reaction briefly total rna was extracted from culturedcells using trizol reagent thermo fisher scientific andcdna synthesis was performed using the quantitect reversetranscription kit qiagen the primers used were as followsabl1andantisense ²acaccctcccttcgtatctcag3² gadphsense ²tgacttcaacagcgacaccca3² antisense ²caccctgttgctgtagccaaa3² the realtime pcr wascarried out by using rt2 sybr rcid13 green qpcr mastermixesqiagen according to the manufacturer™s instructions all pcrswere performed in triplicate 01 01ct method was used to calculatethe relative expression levels²catcacgccagtcaacagtct3²sensestatistical analysisstatistical analyses were performed using spss spss incchicago il χ 2test was used to investigate the possiblerelationships between abl1 expression and clinic pathologicalcharacteristics mann“whitney utest was used to compare thediï¬erence in abl1 protein expression between paired coloncancer and adjacent normal colon tissues survival analysiswas performed using the kaplan“meier curve and the logranktest the values are expressed as mean ± sd comparisonsbetween two groups were conducted using student™s ttest allexperiments were carried out in triplicate results with p were considered statistically significantresultshighly expressed abl1 in crc tissue isassociated with poor clinical outcometo verify the existence of abl1 in crc tissues we comparedcrc tissues and their adjacent noncancerous tissues by ihcstaining our results showed that the immunostaining of abl1was significantly higher in crc tissues compared with adjacentnormal colon tissues p figures 1a“d table ourwestern blot and realtime pcr results confirmed the muchhigher expression level of abl1 in crc tissues compared tonormal tissues figures 1ef similarly the expression of abl1was significantly increased in diï¬erent crc cell lines comparedwith that in the normal colon cell line ncm460 figures 1ghremarkably the expression of abl1 was significantly p increased in the advanced stages stage iiiiiiv of crccompared with the early stages stage i and noncancerousfrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerfigure abl1 is highly expressed colorectal cancer tissue and cell lines a“d representative ihc staining of abl1 in normal ab and crc cd tissuesac x bd x e western blot analysis of abl1 expression in crc tumor t and adjacent normal n tissues f realtime pcr analysis of abl1expression in crc tumor crc and adjacent normal n tissues p g western blot analysis of abl1 expression in crc cell lines h realtime pcranalysis of abl1 expression in crc cell lines relative expression was normalized to ncm460 cells p i realtime pcr analysis of abl1 expression incrc tumors at different clinical stages relative expression was normalized to adjacent normal n tissues p table expression of abl1 in colon cancer and adjacent tissuestable c1222c deletion in exon of abl1variablesnoexpression levelspvariablesexpression levelsor95 cipneglowhighno no mutation mutationnormalcolon cancergenderfemaleadjacent normal colon tissues neg negativetissues figure 1i with a median followup of monthsranging from to months our survival analysis showed thatthe patients with high abl1 expression death had a lowersurvival rate compared to patients with low abl1 expression death p figure s5 these results suggested thatabl1 is a potential oncogene abl1 and that its expression waspositively associated with the clinical stage in patients with crcc1222c deletion in exon of abl1 inrelation to the tnm stageprevious studies have shown the mutations of the abl1 geneare of major clinical relevance to study the possiblemutations in patients with crc we performed dna sequencingour results indicated that a mutation of abl1 was presentin males and females of patients with crc females and males this mutation occurred in exon ofthe abl1 gene and all mutations were found to be deletion““ maletnm stage““ofthe c1222c nucleotide sequence within this exon theincidence rate of mutation was in females and malestable figure additionally the analysis of patients withmutations and the corresponding stages revealed that patientswere in stages “ and in stages “ the tnm stage was asignificant risk factor for c1222c deletion the results suggestedthat c1222c deletion is involved in crc carcinogenesisinterference of abl1 decreased theproliferation and enhanced the apoptosisof sw480 and hct116 cellsto further investigate the role of abl1 in crc carcinogenesiswe used lentivirus vector to downregulate abl1 expression insw480 and hct116 cells after infection both cell lines showedfrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerfigure structural domain and c1222c deletion in exon of the abl1 genefigure depletion of abl1 decreases the proliferation of crc cells a representative fluorescent images showing gfppositive cells after infection images weretaken under x b western blot detects the abl1 expression in cells transfected infected with shctrl or shabl1 c representative images of colony formationassay using crc cells infected with shctrl or shabl1 cells without infection was used as control con d quantification of colony numbers data are shown asmean ± sd p compared with control e brdu assay detects the proliferation of sw480 and hct116 cell lines infected with shctrl or shabl1 cellswithout infection was used as control con p compared with control“ of average gfppositive rate figure 3a our westernblot results showed a significant decrease of abl1 protein levelfigure 3b indicating a successful downregulation after rnainterference to evaluate the proliferation of crc cells afterabl1 depletion we performed a clonogenic assay figure 3ccompared with the control group the number of clones inthe shabl1 group was obviously decreased figure 3d ourbrdu proliferation assay confirmed that the proliferation offrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerfigure depletion of abl1 causes cell cycle arrest and apoptosis in sw480 and hct116 cells ab representative cell cycle analysis of sw480 a andhct116 b infected with shctrl or shabl1 cells without infection was used as control con c quantification of cell cycle distribution in g1 s g2m phases p p compared to control group d western blot analysis of p27 and cyclin d1 expressions in crc cells infected with shctrl or shabl1 cellswithout infection was used as control configure depletion of abl1 increases the apoptosis of crc cells flow cytometry detected apoptosis of sw480 a and hct116 b cells infected with shctrl orshabl1 cells without infection was used as control con c quantification of apoptotic cells data are shown as mean ± sd p compared with controld western blot analysis of bax bclxl and bcl2 expression in crc cells infected with shctrl or shabl1 cells without infection was used as control conshabl1 cells was obviously reduced compared with that ofthe control cells figure 3e additionally our flow cytometryresults showed more cells were arrested in s phase afterabl1 depletion while fewer cells in g2m phases were foundfigures 4a“c indicating downregulation of abl1 inhibitedcell cycle progression of crc cells to validate these data wedetected p27 a negative regulator of cell cycle progression andfound its expression was significantly increased in cells infectedwith shabl1 vector figure 4d on the contrary cyclin d1 wasdecreased in abl1depleted crc cells figure 4dnext we performed flow cytometric analysis to examinethe apoptosis of abl1depleted crc cells figures 5ab wefrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerfigure depletion of abl1 inhibits crc tumor growth in vivo a representative image of mice injected with hct116 cells infected with shctrl nc or shabl1kd b representative tumor images showing depletion of abl1 decreased the size of crc tumors body weight c tumor volume d and tumor weight e weremeasured at day after inoculation p compared to nc groupfound downregulation of abl1 significantly increased apoptosisin crc cells as compared with the control group p figure 5c the expression of the apoptosisrelated proteinbcl2associated x bax was obviously increased while bcelllymphomaextralarge bclxl and bcell lymphoma bcl2were remarkably decreased in the shabl1 group figure 5dtaken together these results suggest depletion of abl1 increasesthe apoptosis of crc cellsdepletion of abl1 inhibited crc tumrowth in vivoin order to examine the involvement of abl1 in regulatingcrc tumor growth we inoculated hct116 cells infected withshctrl nc group or shabl1 kd group into balbc nudemice figure 6a as shown in figure 6b the tumor growth wasremarkably inhibited in the kd group compared with the ncgroup interestingly we also observed a significant bodyweightincrease in the kd group figure 6c as compared to controlabl1 depletion caused a significant reduction in tumor volumefigure 6d the average tumor weight in kd xenografts wasobviously lower than that in the nc group ± mgvs ± mg p figure 6e taken together ouranimal experiments demonstrated that abl1 knockdown couldinhibit crc tumor growth in vivoabl1 interference inhibited tgf1 via thepi3kaktirs1 pathway and ppp3cato elucidate the molecular pathways regulated by abl1 in crcwe performed high throughput pcr array from xenografts inabl1 kd or nc mice our ipa results identified upregulatedgenes and downregulated genes in xenografts from kdmice compared with those from nc mice further analysisrevealed that these diï¬erentially expressed genes were involvedin multiple biological functions and pathogenesis of multiplediseases figure s1as shown in figures s2 s3 and tables s1“s3 the tgfand pi3kakt pathways were inhibited by depletion of abl1the associated molecules of the two pathways including tlr4akt2 il4r camk2d ppp3cb map2k2 pdia3 irs1itpr3 abl1 atf4 ppp3ca ca2 cpt1a csrp1 ctsv fn1lamp2 ptgs2 runx2 s100a4 and spp1 were mapped withabl1 gene to show a predicted interaction network figure 7ato verify this interaction we examined the levels of proteins intgf and pi3kakt pathways in xenografts of nc and kd micefigure 7b our western blot results showed that knockdown offrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerfigure abl1 interacts with pi3kakt and tgf1 pathways a molecule network generated by ipa showing interactions among abl1 pi3kakt and tgf1pathways up and downregulated genes are shown in red and green respectively b western blot analysis of predicted interactive proteins in xenografts fromabl1 knockdown kd or control nc mice the gene of œ is detected by multiple probes and it is statistically significantabl1 significantly decreased irs1 akt2 ppp3ca and tgf1 expression while did not change the expression of map2k2compared with those in the nc groupto further verify the involvement of tgf1 in the regulationof pi3kakt pathway we generated a tgf1depletion hct cell line by lentivirus infection figure 8a our western blotresult showed that the expression of tgf1 was significantlydownregulated after infection figure 8b as expected thekey proteins in pi3kakt pathways were deactivated upontgf1depletion figure 8c including irs1 phosphopi3kand akt according to the findings in the previous study thedownregulated gene ppp3ca found in abl1 kd mouse isinvolved in the regulation of the pi3kakt pathway wenext investigated the interaction between ppp3ca and abl1by establishing a ppp3ca knockdown cell line figures 8dewe found the depletion of ppp3ca significantly decreasedthe expression of abl1 figure 8f to validate the regulatoryrole of ppp3ca we also examined the abl1 expression inppp3ca overexpressed cells and found the expression of abl1was elevated by upregulated ppp3ca figure s4 indicatingppp3ca is a positive regulator of abl1discussionas a ubiquitously expressed nonreceptor tyrosine kinase abl1has been reported to be associated with glioblastoma and breastcancer in this study we examined the role of abl1 incrc progression the results indicated that abl1 might play animportant role in crc which is associated with the mutation andexpression of the abl1 genewe found the expression of abl1 was remarkably elevatedin crc tissues and cell lines figure which is correspondedto the survival rate among patients with crc figure s5indicating abl1 is a potential oncogene in crc “moreover the mutation of abl1 was also elevated in crcpatients based on the results from previous studies the mutationrate of the abl1 gene is relatively higher in men than in womenpatients with crc worldwide in a previous study theabl1 gene was found to be mutated in of patients withcrc at sir ganga ram hospital delhi india in the presentstudy the mutation rate was much higher in crcpatients accepted in our hospital the diï¬erent races of patientsfrom diï¬erent regions of the world reported in the two studiesfrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerfigure tgf1 knockdown deactivates pi3kakt pathway in hct116 cells a representative images of hct116 cells infected with gfpcontaining lentivirusexpressing shrna against tgf1 gfp positive lentivirus with scramble shrna was used as control images were captured under 200x magnification bknockdown of tgf1 was confirm by western blot proteins were extracted from hct116 cells at h postinfection actin was used as loading control cwestern blot examined irs1pi3kakt pathway in hct116 cells after knockdown of tgf1 d western blot examined irs1pi3kakt pathway in hct116 cellsinfected with gfpcontaining lentivirus expressing shrna against ppp3ca gfp positive lentivirus with scramble shrna was used as control e representativefluorescent images of hct116 cells at h postinfection images were captured under 200x magnification f expression of abl1 after ppp3ca knockdown wasdetermined by western blot proteins were extracted from hct116 cells at h postinfection actin was used as loading controlcould be a possible reason causes the diï¬erence of abl1 mutationrates “ gene mutations are often involved in tumorigenesisthe clustered deletions were found in abl1 notch1 retstk11 gna11 and jak3 genes in crc melanoma and nonsmall cell lung cancers additionally the abl1 mutationdata in tcga showed that an average mutation rate of abl1is in coad patients the high mutation rate isconsistent with the findings in this study that abl1 mutationcorrelates with the oncogenesis of crc to the best of ourknowledge the present study presents a novel mutation inexon in which c1222c deletion occurred this deletion wasrelatively higher in female patients than in male patients thehigher distribution of this deletion at the higher tnm stage inpatients with crc suggests that this deletion might be relatedto tumorigenesis of crc however further investigation withlarger sample size is needed to elucidate the relationship andmechanism between c1222c deletion and crc progressionto determine the role of abl1 in crc progression wedownregualted abl1 expression in crc cell lines and foundthat the cell cycle was arrested at s phase figure theseobservations are consistent with previous reports thatthenumber of cells in s phase was increased when abl1 wasinhibited by imatinib or sti571 in u2os hela and a549 cells it is wellreported that p27 inhibits g1s transition ofthe cell cycle while cyclin d1 is a key regulator of cell entryinto the s phase allowing cells to enter the s phase smoothlyfrom the g1 phase our study provides direct evidencethat abl1 interference increased p27 expression and decreased incyclin d1 expression figure which is similar to the increasedp27 expression and decreased cyclin d1 expression found incells treated with nilotinib an abl1specific inhibitor however previous studies showed that when abl1 expressionwas inhibited by nilotinib the number of cells in the g0g1 phasewas increased while the number of cells in s and g2m phases wasdecreased which is contradictory to the finding in this studythat downregulation of abl1 arrested crc cells at s phase thismight be due to the varied function of abl1 in diï¬erent tissuesand cell types abl1 controls cell apoptosis via downstream moleculessuch as puma bax and p73 as well as by changingfrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancermembrane potential depletion of abl1 inducedapoptosis of crc cells observed in this study is consistentwith the findings ofthese studies studies have reportedthat abl inhibitor danusertib treatment significantly decreasedthe expression of bclxl and bcl2 while increasing theexpression of bax similarly we found increasedbax expression and decreased levels of bcl2 and bclxl after downregulation of abl1 in crc cells figure indicating abl1 is involved in the regulation of apoptosis incrc cellsinvolved in cell proliferation migrationfurthermore information obtained using the ipa indicatedthe expressions of numerousthat after abl1 knockdowngenesinvasiondiï¬erentiation death and survival were aï¬ected figures s1“s3 tables s1“s3 the results demonstrated that abl1 mightplay a pivotal role in crc progression especiallythe tgf and pi3kakt pathways were inhibited afterabl1 interferenceit has been reported that abl1 regulates tgf signaling which is associated with tumor progression by modulatingangiogenesis in crc resulting in poor prognostic outcome“ studies have demonstrated thattreatment withan abl1 inhibitor significantly reduced the tgf level similarly we found the expression of tgf1 wassignificantly inhibited after abl1depletion figure thisindicated that abl1 is a positive regulator of tgf signalpathways as one of the tgfaï¬ected downstream signals thepi3kakt pathway plays a crucial role in tumorigenesisitassociated withproliferation apoptosisinvasion and metastasis of cancercells insulin receptor substrates irs including irs1and irs1 are a downstream messenger of the pi3k pathway our study provides novel evidence that abl1 mightinteract with tgf1 via pi3kaktirs1 that is involved incrc progressionexpression of proteinsregulatestheca2aandcalcineurincalmodulindependentserinethreonine protein phosphatase has been reported topromote intestinal tumor development and crc tumorigenesis the expression of calcineurin a specifically increases inhuman crc cell lines in the present study we foundthat ppp3ca which is also known as an alpha isoform ofthe calcineurin catalytic subunit was inhibited afterknockdown of abl1 figure 7b this finding provides novelevidence that abl1 might interact with the ppp3ca oncogenein crc carcinogenesisconclusionin conclusion we found a high level of abl1 expression incrc tissue and cells which was associated with the tnmstages a novel mutation of c1222c deletion in exon of theabl1 gene was found and was associated with the crc stagedepletion of abl1 decreased the growth of crc cell lines bothin vitro and in vivo by inhibiting tgf pathway these resultsdemonstrated novel understandings of the function of abl1during the progression of crc thus provides a clinically viablestrategy for crc therapydata availability statementthe raw data supporting the conclusions of this will bemade available by the authors without undue reservationethics statementthe studies involving human participants were reviewed andapproved by ethics committee of shaanxi people™s hospital thepatientsparticipants provided their written informed consent toparticipate in this study the animal study was reviewed andapproved by ethics committee of shaanxi people™s hospitalauthor contributionsall authors have a significant scientific contribution to all aspectsof this studyfundingstudy wasthissupported by national natural sciencefoundation of china”youth projects grant no shaanxi natural science foundation grant nos 2015jq8321and 2019jm547 shaanxi innovative talents cultivate programgrant no 2017kct28 and operating expenses of basicscientific research project of xi™an jiaotong university grantno xzy012019112supplementary materialthe supplementary materialonline202001385fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389foncatreferences ouerhani s bougatef k soltani i elgaaied ab abbes s menif s theprevalence and prognostic significance of kras mutation in bladder cancerchronic myeloid leukemia and colorectal cancer mol biol rep “ 101007s1103301325128 brenner h kloor m pox cp colorectal cancer lancet “ 101016s0140673613616499 navarro m nicolas a ferrandez a lanas a colorectal cancer populationscreening programs worldwide in an update world j gastroenterol “ 103748wjgv23i203632 favoriti p carbone g greco m pirozzi f pirozzi re corcione fworldwide burden of colorectal cancer a review updates surg “ 101007s133040160359yjauhri m bhatnagar a gupta s shokeen y minhas s aggarwal stargeted molecular profiling of rare genetic alterations in colorectalcancer 101007s1203201608202sequencing med oncolnextgenerationusing peng x luo z kang q deng d wang q peng h foxq1mediates the crosstalk between tgfbeta and wnt signaling pathways inthe progression of colorectal cancer cancer biol ther “ frontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancer rossner f gieseler c morkel m royer hd rivera m blaker h uncoupling of egfrras signaling and nuclear localization of ybx1in colorectal cancer oncogenesis 5e187 101038oncsis alpay k farshchian m tuomelainhibition ofsiljamaki ecancer9e105526 101371 pone0105526cells highly sensitivealetj sandholm j aittokallio krenderscabl kinaseactivityto mitoxantrone plos one tian xq guo ff sun df wang yc yang l chen sl downregulationof znf278 arrests the cell cycle and decreases the proliferation of colorectalcancer cells via inhibition of the erkmapk pathway oncol rep “ 103892or20176031 udden sm moritafujimura y satake m ikawa s cabl tyrosinefate “kinase modulates p53dependent p21 induction and ensuing celldecision in response to dna damage cell signal 101016jcellsig201310005 cai s cheng x liu y lin z zeng w yang c eya1 promotes tumorangiogenesis by activating the pi3k pathway in colorectal cancer exp cell res “ d
Colon_Cancer
four to nine percent of the sequences™ transcription are long noncoding rnas lncrnas inmammalian genomes canzio ji lncrna was regarded as the noise ofgenome transcription and did not have biological functions at first however an increasing numberof studies have reported that lncrna is widely robinson involved in chromosomeedited bylei dengcentral south university chinareviewed byhao linuniversity of electronic science andtechnology of china chinainner mongolia university chinajuan wangcorrespondencenan dudunan05aliyuncomganfeng xiexiegfaliyuncom these authors share first authorshipspecialty sectionthis was submitted tomolecular medicinea section of the frontiers in cell and developmentalbiologyreceived june accepted june published august citationliu z zhang y han x li c yang xgao j xie g and du n identifying cancerrelated lncrnasbased on a convolutional neuralnetwork front cell dev biol 103389fcell202000637frontiers in cell and developmental biology wwwfrontiersinaugust volume 0cliu a method to identify cancerrelated lncrnasgenomicimprintingchromatin modificationsilencingtranscriptional activationinterference andnuclear transport cheng 2018a recently it has beenproven to be associated with many kinds of cancerstranscriptionalthe secondary structure spliced form and subcellularlocalization of most lncrnas are conserved karner which is very important for lncrna to execute functionshowever compared to the functions of micrornas mirnasand proteins the function oflncrna is more difficult todetermine according to the position of lncrna in the genomerelative to proteincoding genes it can be divided into five typessense antisense bidirectional intronic and intergenicmany researchers have found lncrnas play an important rolein cancers avgeris cheng 2018b zhao and neurodegenerative diseases peng and zhao as other biological molecules zhang t bai cheng 2019a liang although manyresearchers have verified many associations between lncrnasand cancers by biological experiments compared with ourknowledge about diseaserelated genes we still do not knowenough about diseaserelated lncrnas considering the timeand money cost of finding diseaserelated lncrnas more andmore researchers tend to use computational methods to identifydiseaserelated lncrnas these methods could be divided intothree categories machine learning methods network methodsand other methodsmachine learning methods build models based on thesimilarities of diseases orlncrnas and their biologicalcharacteristics cheng cheng 2019b zeng zou lan developed thelncrna“disease association prediction ldap which is amethod based on bagging support vector machine svm toidentify lncrna“disease associations they used similarities oflncrnas and diseases as the features yu developedcollaborative filtering naive bayesian classifier cfnbc based onnaive bayesian they integrated mirna“lncrna associationsmirna“disease associations and lncrna“disease associationsto infer more lncrna“disease associations considering thediscriminative contributions of the similarity association andinteraction relationships among lncrnas disease and mirnasxuan 2019a developed a dual convolutional neuralnetwork cnn with attention mechanisms to predict diseaserelated lncrnasnetwork methods are the most common way to identifyassociations between diseases and lncrnas nowadays gu yu zhang j kuang wang l liu thiskind of method would build one or multiple networks toinfer new information wang l built a lncrna“mirna“disease interactive network and used their novel methodœldlmd to predict associations between lncrnas and diseasessumathipala used a multilevel network topologywhich includes lncrna“protein protein“protein interactionprotein“disease relationship to use network diï¬usion algorithmto predict diseaserelated lncrnas the graph convolutionalnetwork gcn and cnn were used on a lncrna“mirna“disease network by xuan 2019b deng builtlncrna similarity network disease similarity network mirnasimilarity network and their associations then they calculatedthe metapath and feature vector for each lncrna“disease pair inthe heterogeneous information networkother methods may borrow the feature extraction methodor similarity conjecture of network methods but the core ofthis method is matrix decomposition or matrix completionlu developed the geometric matrix completionlncrna“disease association gmclda which is a methodbased on geometric matrix completion they calculated diseasesimilarity based on disease ontology do and calculatedthe gaussian interaction profile kernel similarity for lncrnasthen they inferred diseaserelated lncrnas based on theassociation patterns among functionally similar lncrnas andsimilar diseases wang y proposed a weightedmatrix factorization to capture the interintraassociationsbetween diï¬erent types of nodes then they approximated thelncrna“disease association matrix using the optimized matricesand weights to predict diseaserelated lncrnas localityconstrained linear coding label propagation latent dirichletallocation llclplda was developed by xie firstly localconstraint features of lncrnas and diseases wereextracted by localityconstrained linear coding llc thenthey predicted diseaserelated lncrnas by label propagationlp strategyhowever previous methods did not consider the regulatingtarget gene expression of lncrna which is an important functionof lncrna and plays an important role in associations betweenlncrnas and diseases in addition deep learning methods arean important tool and have shown their power in bioinformaticschen lv wei wu zhao 2019abc therefore in this paper we used thisinformation as features of lncrna in addition the expressionof lncrna in diï¬erent tissues were also used as the featuresof lncrna then the deep belief network dbn was used toencode and the cnn was used to classifymethodsfeature extractiontissue expression specificity of long noncodingrnacompared with proteincoding geneslncrna shows strongtissue specificity the specificity of lncrnas in diï¬erent kindsof tissues and cell types has been proven by many biologicalexperiments the diï¬erent expression also plays an importantrole in essential cellular processes sasaki testedthe expression of lncrnas in diï¬erent tissues and found lncrnas exhibited tissuespecific expression and oflncrnas were only expressed in one discrete tissue thereforethe expression of lncrnas in diï¬erent tissues were used asthe featureswe obtained the expression of lncrnas in diï¬erenttissues which included adipose adrenal breast colon heartkidney liver lung lymph node ovary placenta prostate testisand thyroidtherefore the dimension of each lncrna™s expression featureis ˆ— frontiers in cell and developmental biology wwwfrontiersinaugust volume 0cliu a method to identify cancerrelated lncrnastherefore the dimension of each lncrna™s target gene featureis ˆ— deep belief networkthe dbn can eï¬ectively learn complex dependencies betweenvariables zhao 2019d the dbn contains many layers ofhidden variables which can eï¬ectively learn the internal featurerepresentation of the data and can also be used as an eï¬ectivenonlinear dimensionality reduction methodwhen the observable variables are known the joint posteriorprobabilities of the hidden variables are no longer independentof each other so it is difficult to accurately estimate the posteriorprobabilities of all hidden variables the posterior probability ofearly dbn is generally approximated by monte carlo methodbut its efficiency is relatively low which makes its parameterlearning difficult in order to eï¬ectively train the dbn weconvert the sigmoid belief network of each layer to a restrictedboltzmann machine rbm the advantage of this is that theposterior probabilities of the hidden variables are independentof each other which makes it easy to sample in this way thedbn can be regarded as being stacked from top to bottom bymultiple rbms and the hidden layer of the lth rbm is used asthe observable layer of the l 1th rbm further the dbn canbe trained quickly by layerbylayer training that is starting fromthe bottom layer and training only one layer at a time until thelast layer the specific layerbylayer training process is to trainthe rbm of each layer in turn from bottom to top assuming wehave trained the rbm in the first l1 layer we can calculate theconditional probability of the bottomup hidden variablesphihiˆ’ σ bi wihiˆ’where bi is the bias of ith layer of rbm wi is the connectionweight hi is the ith layer of rbmthe process of training dbn is as followsfigure the number of target genes for each long noncoding rnalncrnafigure the distribution of the number of target genes lncrna longnoncoding rnareversetarget gene of long noncoding rnaquantitativechainreaction qrtpcr and western blot were used to testthe diï¬erentexpression genes after knocking down oroverexpressing lncrnastranscriptasepolymerasewe obtained target genes of lncrna from lncrna2targetinput train dataset ˆvn learning rate λjiang as we can see in figure there are kinds of lncrnasone lncrna has more than target genes then we drawthe distribution of the number of target genes correspondingto lncrnaasshown in figure most ofthe target genes arecorresponding to less than five lncrnas therefore if we usedthem to be the features of lncrnas the features would be sparsetherefore we only select the most common target genes to bethe features the genes which are corresponding to more thanfive lncrnas were selected as the features of lncrnas there are kinds of genes then we need to encode these genesf [g1 g2 · · · g45]where g1 denotes the first gene of these genes and f denotesthe feature of lncrna for each lncrna if g1 is the target geneof it then g1 otherwise g1 output weight matrix wl bias al and blfor l 1linitialization wi al bi sample from train dataset ˆh0for i lˆ’sample hi based on phi ˆhiˆ’endset hi1as the train sample to train lth layer ofrbmendsince the dimension of expression feature and target genefeature are diï¬erent we should reduce the dimension of targetgene feature and make it the same as the expression feature™stherefore in this paper two layers of rbm were used to builda dbn modelthe number of nodes oftheand respectively sigmoid function was used astwo layers was thefrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cliu activation functionσ x eˆ’xtherefore the dimension of final features is ˆ— f cid20 g1 g2 · · · g13e1 e2 · · · e13 cid21a method to identify cancerrelated lncrnasconvolutional neural networkthe power of cnn in dealing with bioinformatic problems hasbeen proven by many researchers we selected cnn as theclassifier based on two reasons the dimension of features is ˆ— which can be regarded as an image the outstandingperformance of cnn in image classificationthere are five layers in our cnn model the structure of cnnis shown as table where g1 g2 · · · g13 denotes target gene feature after dbnand e1 e2 · · · e13 denotes the expression of lncrnas in diï¬erent tissuestable the structure of convolutional neural network cnnlayersparameterconvolutional layerpooling layerconvolutional layerpooling layerfully connected layeroutputfilter kernel size activation function tanhpool size activation function tanhfilter kernel size activation function tanhpool size activation function tanhunits activation function tanhunits activation function sigmoidwork framefigure shows the work frame of our method œdbn“cnnthere are three steps of our methods firstly we should extractfeatures of lncrnas there are two parts of features expressionfeature and target gene feature then dbn was used to encodethe target gene feature after encoding the two kinds of featureswere combined together finally cnn was used to classifyresultsdata descriptionthe known associations between lncrna and diseases wereobtained from lncrnadisease database bao wetotally obtained kinds of cancerrelated lncrnas the numberof their corresponding lncrnas is shown as figure as shown in figure people™s understanding of cancerrelated lncrnas varies widely we have known more than lncrnas for some cancers but few lncrnas are known for somecancers to better build our model we only selected cancerswhich have more than related lncrnas therefore kindsof cancers were selectedfigure work frame of deep belief network dbn“convolutional neural network cnn lncrna long noncoding rnafrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cliu a method to identify cancerrelated lncrnasfigure the number of long noncoding rnas lncrnas for each cancertable the performance of deep belief network dbn“convolutional neuralnetwork cnn in cancerscancerarea undercurve aucarea under precisioncurve auprcervical cancerbreast cancercolorectal cancerstomach cancerurinary bladder cancerlung cancerovarian cancerthyroid cancerprostate cancerliver cancerpancreatic cancerovarian epithelial cancergallbladder cancerendometrial cancercolon canceresophageal cancerthetargetgenes oflncrnas were obtained fromlncrna2target database we have discussed about this insection target gene of long noncoding rnafigure the receiver operating characteristic roc curves of the threemethods dbn deep belief network cnn convolutional neural network pcaprincipal component analysisfigure the area under the precision“recall curve aupr of the threemethods dbn deep belief network cnn convolutional neural network pcaprincipal component analysisthe expression oftissues wasobtained from noncodev5 zhao we only usedhuman datalncrnas in diï¬erentthe performance of deep beliefnetwork“convolutional neural networkwe did 10cross validation on each cancer area under the curveauc cheng dao zhang and areaunder the precision“recall curve aupr were used to evaluatethe performance of dbn“cnn the results are shown in table as we can see in table the performance of dbn“cnn isquite diï¬erent in diï¬erent cancers this may be caused by thediï¬erent sample sizes the average auc is and aupr is comparison experimentsto verify the superior of dbn“cnn we compared it with similarmethods since the main function of dbn is to reduce dimensionprincipal component analysis pca has the same functiontherefore instead of using dbn to encode we used pca thistime and cnn was used to classify the features after pca we callthis method pca“cnn in addition we also used the deep neuralnetwork dnn to replace cnn so this comparison method wascalled dbn“dnnwe used these three methods to test on cancers andsummarized the results to get a final auc and aupr for eachmethod the receiver operating characteristic roc curves areshown in figure as shown in figure the blue curve denotes the results ofdbn“cnn the red and black curves denote pca“cnn anddbn“dnn respectively as we can see dbn“cnn performedbest among these three methods the auc of dbn“cnn is which is better than and for pca“cnn anddbn“dnn respectivelyfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cliu a method to identify cancerrelated lncrnasas shown in figure the aupr of dbn“cnn is the highestwith the least standard errorcase studyliu found down syndrome cell adhesion molecule antisense rna dscamas1 is associated with breast cancerby constructing two suppression subtracted cdna librariesmartensuzunova reported the associationbetween h19 and bladder cancer they also pointed out that h19could be the biomarker of bladder cancershi measured the expression level of lncrnasloc554202 in breast cancer tissues and found that loc554202was significantly increased compared with normal control andassociated with advanced pathologic stage and tumor sizeconclusionsincreasing evidence has shown the relationship between lncrnasand cancers lncrnas could be the biomarkers to help diagnosecancer and also help researchers understand the mechanismof cancers compared with people™s knowledge of diseaserelated protein coding genes we knew few about diseaserelated lncrnas however the biological experiments for findingdiseaserelated lncrnas are timeconsuming and expensivetherefore in this paper we proposed a novel method foridentifying cancerrelated lncrnas we called this methodœdbn“cnn which is a fusion of dbn and cnn two kindsof features were used based on the biological background sincelncrnas have tissuespecific expression and the expression ofcancer tissues is diï¬erent from normal tissues the expressionoftissues could provide importantin diï¬erentlncrnasreferencesavgeris m tsilimantou a levis p k tokas t sideris d c stravodimosk loss of gas5 tumour suppressor lncrna an independentmolecular cancer biomarker for shortterm relapse and progression in bladdercancer patients br j cancer “ 101038s4141601803206bai y dai x ye t zhang p yan x gong x plncrnadba repository of plant lncrnas and lncrnarbp protein interactions currbioinform “ bao z yang z huang z zhou y cui q and dong d lncrnadisease an updated database of long noncoding rnaassociateddiseases nucleic acids res d1034“d1037 101093nargky905canzio d nwakeze c l horta a rajkumar s m coï¬ey e l duï¬y ee antisense lncrna transcription mediates dna demethylationto drive stochastic protocadherin α promoter choice cell “653e15 101016jcell201903008chen x shi w and deng l prediction of disease comorbidity usinghetesim scores based on multiple heterogeneous networks curr gene ther “ cheng l computational and biological methods for gene therapy currgene ther cheng l hu y sun j zhou m and jiang q 2018a dincrna afor exploring disease“comprehensive webbased bioinformaticsassociations 101093bioinformaticsbty002ncrna functionbioinformaticstoolkitandcheng l jiang y ju h sun j peng j zhou m 2018busingcrossontologyinfacrontsimilaritiescalculatingtermtheirexecutelncrnasinformation for us to identify cancerrelated lncrnas inadditionregulation function byinteracting with their target genes therefore the target genesof lncrnas can also be the features of lncrnas to encode thefeatures dbn was used to reduce the dimension finally cnnwas used to identify real cancerrelated lncrnas based on thefinal featureto verify the eï¬ectiveness of our method we compareddbn“cnn with pca“cnn and dbn“dnn since pca canalso reduce the dimension of features and dnn can also doclassification the results showed that dbn“cnn performedbest finally case studies have been done to verify the accuracy ofour results we found potential lncrnas for kinds of cancerswhich can be a kind of guidance for researchers finding novelcancerrelated lncrnasdata availability statementthe datasets presented in this study can be found in onlinerepositoryrepositoriesrepositories theandnumbersbethesupplementary materialaccessionnamesfoundcantheofinauthor contributionsnd and gx designed the research zl performed the researchand wrote the manuscript yz and xh acquired the dataand reviewed and edited the manuscript cl xy and jganalyzed the data all authors reviewed the manuscript andprovided commentsinformation flow by a random walk bmc genomics 19suppl 101186s1286401743386cheng l yang h zhao h pei x shi h sun j 2019a metsigdisa manually curated resource for the metabolic signatures of diseases briefbioinform “ 101093bibbbx103cheng l zhao h wang p zhou w luo m li t 2019bcomputational methods for identifying similar diseases molecular therapynucleic acids “ 101016jomtn201909019dao f y lv h zulfiqar h yang h su w gao h acomputational platform to identify origins of replication sites in eukaryotesbrief bioinform 101093bibbbaa017 [epub ahead of print]deng l li w and zhang j ldah2v exploring metapaths acrossmultiple networks for lncrnadisease association prediction ieeeacmtransac comput biol bioinform 101109tcbb20192946257 [epubahead of print]gu c liao b li x cai l li z li k global network randomwalk for predicting potential human lncrnadisease associations sci rep 101038s4159801712763zjij tangj xia kjandjiang rtumorigenesis microenvironment currbioinformlncrna in“jiang q wang j wu x ma r zhang t jin s lncrna2targeta database for diï¬erentially expressed genes after lncrna knockdown oroverexpression nucleic acids res d193“d196 101093nargku1173karner h webb ch carmona s liu y lin b erhard m functional conservation of lncrna jpx despite sequence and structuraldivergence j mol biol “ 101016jjmb201909002frontiers in cell and developmental biology wwwfrontiersinaugust volume 0cliu a method to identify cancerrelated lncrnaskuang l zhao h wang l xuan z and pei t a novel approachbased on point cut set to predict associations of diseases and lncrnas currbioinform “ lan w li m zhao k liu j wu fx pan y ldap a webserver for lncrnadisease association prediction bioinformatics “ 101093bioinformaticsbtw639liang c changlu q he z tongze f and xue z gutmdisorder acomprehensive database for dysbiosis of the gut microbiota in disorders andinterventions nucleic acids res liu d rudland p sibson d and barraclough r identification ofmrnas diï¬erentiallyexpressed between benign and malignant breast tumourcells br j cancer “ 101038sjbjc6600456liu x hong z liu j lin y alfonso rp zou q computational methods for identifying the critical nodes in biologicalnetworks brief bioinform “ 101093bibbbz011lu c yang m li m li y wu f and wang j predicting humanlncrnadisease associations based on geometric matrix completion ieee jbiomed health inform 101109jbhi20192958389 [epub ahead of print] protein function predictionto deep learning proteomics 19e1900119lv z b ao c y and zou qfrom traditionalclassifier 101002pmic201900119martensuzunova e s böttcher r croce c m jenster g visakorpi t andcalin g a long noncoding rna in prostate bladder and kidneycancer eur urol “ 101016jeururo201312003peng j and zhao t reduction in tom1 expression exacerbatesalzheimer™s disease proc natl acad sci usa “ 101073pnas1917589117robinson e k covarrubias s and carpenter s the how and why oflncrna function an innate immune perspective biochim biophys acta generegul mech 101016jbbagrm2019194419sasaki y t sano mideue t kin t asai k and hirose t identification and characterization of human noncoding rnas withtissuespecific expression biochem biophys res commun “ 101016jbbrc200704034sumathipala m maiorino e weiss s t and sharma ashi y lu j zhou j tan x he y ding j long noncodingrna loc554202 regulates proliferation and migration in breast cancer cellsbiochem biophys res commun “ 101016jbbrc201402144network diï¬usion approach to predictlncrna disease associationsusing multitype biological networks lion front physiol 103389fphys201900888wang l xuan z zhou s kuang l and pei t a novel modelassociations based on the lncrna“for predicting lncrnadiseasemirnadisease interactive network curr bioinformwang y yu g wang j fu g guo m and domeniconi c weightedmatrix factorization on multirelational data for lncrnadisease associationprediction methods “ 101016jymeth201906015wei l su r wang b li x zou q and gao x integrationof deep feature representations and handcrafted featuresto improvethe prediction of n 6methyladenosine sites neurocomputing “ 101016jneucom201804082wu b zhang h lin l wang h gao y zhao l a similarity searching system for biological phenotype images using deepconvolutional encoderdecoder architecture curr bioinform “ xie g huang s luo y ma l lin z and sun y llclplda a novelmodel for predicting lncrna“disease associations mol genet genomics “ 101007s00438019015908xuan p cao y zhang t kong r and zhang z2019a dualconvolutional neural networks with attention mechanisms based methodfor predicting diseaserelated lncrna genes front genet 103389fgene201900416xuan p pan s zhang t liu y and sun h 2019b graph convolutionalnetwork and convolutional neural network based method for predictinglncrnadisease associations cells 103390cells8091012yu g fu g lu c ren y and wang j brwlda birandomwalks for predicting lncrnadisease associations oncotarget “ 1018632oncotarget19588yu j xuan z feng x zou q and wang l a novel collaborativefiltering model for lncrnadisease association prediction based on the naïvebayesian classifier bmc bioinform 101186s1285901929850zeng x x wang w deng g s bing j x and zou q prediction ofpotential diseaseassociated micrornas by using neural networks mol thernucleic acids “ 101016jomtn201904010zhangand deng lj zhang z chen zintegratinglncrnadisease associationieeeacm transac comput biol bioinform “multiple heterogeneous networks for novelinference 101109tcbb20172701379zhang t tan p wang l jin n li y zhang l rnalocate aresource for rna subcellular localizations nucleic acids res d135“d138 101093nargkw728zhang z m tan j x wang f dao f y zhang z y and linh early diagnosis of hepatocellular carcinoma using machinelearning method front bioeng biotechnol 103389fbioe2020zhao t cheng l zang t and hu y 2019a peptidemajor histocompatibilitycomplex class i binding prediction based on deep learning with novel featurefront genet 103389fgene201901191and cheng lidentifyingalzheimer™s diseaserelated proteins by lrrgd bmc bioinform 101186s1285901931247zhao t hu y zang t2019bzhao t hu y zang t and cheng l mrtfb regulates the expressionof nomo1 in colon proc natl acad sci usa 101073pnas2000499117zhao t hu y zang t and wang y 2019c integrate gwas eqtland mqtl data to identify alzheimer™s diseaserelated genes front genet 103389fgene201901021zhao t wang d hu y zhang n zang t and wang y 2019d identifyingalzheimer™s diseaserelated mirna based on semiclustering curr gene ther “ zhao y li h fang s kang y wu w hao y noncode an informative and valuable data source of long noncoding rnas nucleicacids res d203“d208 101093nargkv1252zou q xing p wei l and liu b gene2vec gene subsequenceembedding for prediction of mammalian n6methyladenosine sites frommrna rna “ 101261rna069112118conflict of interest the authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestcopyright liu zhang han li yang gao xie and du this is an openaccess distributed under the terms of the creative commons attribution license ccby the use distribution or reproduction in other forums is permitted providedthe original authors and the copyright owners are credited and that the originalpublication in this is cited in accordance with accepted academic practiceno use distribution or reproduction is permitted which does not comply with thesetermsfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0c'
Colon_Cancer
in the uk the death toll from severe covid19 is among the highest worldwide1 severe covid19 is characterised by respiratory failure with socalled cytokine storm occurring in some patient subsets2 pathological correlates are required to understand the pathophysiology of covid19 autopsybased histopathological analysis is crucial in this respect in anticipation of the covid19 pandemic our group produced national guidelines for autopsy performance in suspected covid19 cases3covid19 is caused by infection with severe acute respiratory syndrome coronavirus sarscov245 although sarscov2 and its predecessor sarscov causing severe acute respiratory syndrome [sars] are toll similar on a molecular and clinical level covid19 has a lower death rate for covid19 vs for sars and a substantially higher death deaths worldwide from covid19 as of aug vs from sars than sars due to a higher basic reproduction number1 the postmortem findings in patients with sarscov infection included diffuse alveolar damage dad splenic and nodal lymphocyte depletion haemophagocytosis renal acute tubular injury cerebral oedema microthrombosis and adrenalitis with necrosis with intracellular sarscov detected in the lungs kidney brain and haematological ans6 various autopsy series on covid19 have begun to emerge in the literature7“ here we document the major pathological lancet microbe published online august 101016 s2666524720301154department of cellular pathology northwest london pathology b hanley mbbch prof k n naresh md c roufosse phd j weir frcpath prof r goldin md p viola md m osborn frcpath and department of hepatology p manousou phd imperial college london nhs trust london uk centre for haematology b hanley prof k n naresh and centre for inflammatory diseases c roufosse department of immunology and inflammation department of infectious disease prof g s cooke frcp a abdolrasouli phd o c swann mres l baillon bsc r penn msc prof w s barclay phd and department of metabolism prof m thursz md faculty of medicine imperial college london london uk department of histopathology royal brompton and harefield nhs foundation trust and national heart and lung institute imperial college london london uk prof a g nicholson dm renal and transplant centre hammersmith hospital imperial college healthcare nhs london uk r corbett phd department of neuropathology kings college hospital london uk prof s alsarraj frcpath death investigation committee royal college of pathologists london uk m osborn and nightingale nhs hospital london uk m osborn correspondence to dr brian hanley department of cellular pathology northwest london pathology charing cross hospital campus london w6 8na uk bhanleyimperialacukwwwthelancetcommicrobe published online august 101016s2666524720301154 s 0cresearch in contextevidence before this studycovid19 is a new disease and comprehensive descriptions of the histopathological findings at autopsy are scarce we reviewed the literature available on covid19 autopsy findings up to and including may for this we searched pubmed and google scholar databases with no language restrictions using the search terms œcovid19 œsarscov2 œhistology œautopsy and œpostmortemadded value of this studyour series focused on providing a comprehensive description of the histopathological findings in patients with severe fatal covid19 and correlating these findings with data on viral tropism the most prominent findings included diffuse alveolar damage thrombosis haemophagocytosis and immune cell depletion several novel autopsy findings in patients with covid19 were also described including pancreatitis pericarditis adrenal microinfarction secondary disseminated mucormycosis and brain microglial activationimplications of all the available evidenceour study supports the existing clinical and autopsy literature that identified diffuse alveolar damage thrombosis immune cell depletion and macrophage activation as the most prominent pathological features in covid19 other factors including acute kidney injury pancreatitis pericarditis secondary fungal infections and preexisting liver disease require further investigation the presence of ongoing viral replication in late stage covid19 supports the continued use of antiviral therapy even at a point in illness when immunopathology is dominantsee online for appendixfindings of ten postmortem examinations done on patients with clinically confirmed covid19methodspatient selectionfor this study eligible patients were older than years with premortem sarscov2 infection and covid19 listed clinically as the direct cause of death under part on the medical certificate of cause of death [mccd] consent was obtained for all included patients according to the human tissue authority codes of practice by a member of the trust core postmortem consent team consent rate was · ten of patients exclusion criteria included extended postmortem interval before autopsy days and patients with covid19 contributing but not directly leading to death under part of the mccd patients were from imperial college national health service nhs trust nine patients london uk and royal brompton harefield foundation nhs trust one patient london uk premortem sarscov2 infection was identified using the coronavirus typing multiplextandem pcr highplex system aus diagnostics chesham uk ethical approval for this project was provided by the imperial college healthcare tissue bank r20012autopsy proceduresfull autopsies were done on nine patients pm1“ and one patient underwent percutaneous biopsy sampling heart lungs pancreas kidneys and liver using percutaneous biopsy under ultrasound guidance pm10 full postmortem examinations included standard sampling and were done according to royal college of pathologists guidelines3 eight different regions of the brain were sampled for each full neuropathological examination all tissue samples were fixed in formalin for a minimum of h before embedding histochemical stains and immunohistochemistry were applied according to local protocols appendix p ans were reviewed by subspecialist pathologists in lung agn and pv haem ato pathology and immune pathology knn liver rg gastrointestinal mo neuropathology sas and renal pathology cr integrated interpretation was done by a subspecialty autopsy pathologist bh and mo all cases were reviewed independently by at least two pathologistspcr proceduresfresh tissue for quantitative rtpcr qrtpcr analysis was processed within the biosafety level facilities at st mary™s hospital london uk approved by the uk health and safety executive and in accordance with local rules at imperial college london total rna was obtained from fresh tissue samples by use of trizolchloroform extraction followed by precipitation and purification using the rneasy kit qiagen hilden germany qrtpcr against e gene rdrp and subgenomic rna was done as described elsewhere1617 in patient pm5 total fungal genomic dna was extracted from four to five ribbon slices of a formalinfixed paraffinembedded lung tissue block purified dna was amplified with pcr for panfungal and mucoralesspecific targetsstatistical analysisall data was analysed using spss software version and expressed using median iqr and percentagerole of the funding sourcethe funder of the study had no role in study design data collection data analysis data interpretation or writing of the the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publicationresultsbetween march and april ten patients were included in the study the median age at death was years wwwthelancetcommicrobe published online august 101016s2666524720301154s 0ciqr “ seven of ten patients were men three were women and most patients were white or asian nine hypertension four patients and chronic obstructive pulmonary disease three were the most common contributing factors to death according to mccd all ten patients developed fever and had at least two respiratory symptoms or signs cough shortness of breath reduced oxygen saturations or pleuritic chest pain during their early presentation of eight patients assessed for inflammatory markers all had elevated inflammatory markers these features were either apparent upon presentation to hospital eight of ten patients or developed in an inpatient two patients pm8 and pm9 most patients died within weeks of symptom onset seven patients and were not intubated or ventilated six patients four patients were intubated during their presentation pm2 for days pm5 for days pm6 for less than day and pm7 for days the median bodymass index bmi was in the obese range · iqr ·“· and more patients were obese according to bmi at post mortem five of nine than indicated on the mccd one of ten the median interval between death and postmortem examination was days iqr ·“· although the limited post mortem had a shorter interval less than h after death detailed clinical case vignettes are available in the appendix p and clinical data are summarised also in the appendix p all patients had dad six showed purely exudative phase dad and four showed a mixture of exudative and anising dad appendix p figure three of four patients with anisingphase dad had spent a substantial period on a ventilator days days and days florid acute bronchopneumonia and ventilatorassociated pneumonia were not noted in this series although mild interstitial neutrophilic inflammation three of ten patients and patchy acute bronchopneumonia three patients were observed interstitial macrophages were prominent macrophages were accompanied by scattered plasma cells mild or moderate lymphocyte inflammation was present in all ten patients although focal lymphocyte cuffing of small vessels was noted in six patients we noted that lymphocytes in the lung were predominantly cd4positive t cells cd56positive natural killer cells were rarely found occasionally a patient had small aggregates of small b cells chronic bronchiolitis was seen in most patients nine of ten no granulomas or viral inclusion were seen invasive mucormycosis was noted in one patient pm5 figure and confirmed with mucoralesspecific pcr the mucormycosis was vasculocentric and disseminated involving the hilar lymph nodes heart brain and kidney in the same patientmacroscopic two of nine patients and microscopic eight of nine pulmonary thromboemboli were frequent observations appendix p figure both fibrinrich and plateletrich thrombi were identified in smallsized and mediumsized vessels and within the capillaries in alveolar septa figure external examination findings of deep venous thrombosis were not noted very focal lymphocytic vasculitis was identified in one patientthrombotic features were universal in this cohort and all nine patients who underwent a full autopsy had at least one microthrombosis or macrothrombosis in a major an one of nine patients had a macroscopic acute coronary thrombosis in the right coronary artery whereas five patients had thrombi in the microcirculation of the heart on histological analysis coronary artery disease was negligible or mild in most patients seven of nine acute myocardial ischaemic damage h old was noted in the patient with an acute coronary artery thrombus figure 2a pm1 a mottled myocardium and subendocardial contraction band necrosis was noted in a acebdf 03m µm µmfigure pulmonary pathological findings in patients with covid19a macroscopic subpleural petechial haemorrhage in a 24yearold man pm6 b hyaline membranes indicative of exudative phase diffuse alveolar damage in a 79yearold woman pm9 at 20x magnification c cd61 immunohistochemical staining indicating plateletrich microthrombosis in alveolar capillaries pm6 d squamous metaplasia in a 61yearold man pm1 with exudative phase diffuse alveolar damage at × magnification e interstitial multinucleated giant cells in a 79yearold man pm7 with anising phase diffuse alveolar damage at × magnification the top right insert is of multinucleated giant cells showing positive cd68 staining indicative of macrophage lineage the bottom left insert shows absence of staining for cytokeratins f periodic acid schiff staining indicating wide irregular aseptate and ribbonlike hyphae with openangle branching and a vasculocentric pattern indicative of mucormycosis in a 22yearold man pm5 the insert is a grocott silver stain highlighting mucormycosis at 20x magnificationwwwthelancetcommicrobe published online august 101016s2666524720301154 s 0csecond patient pm2 whether the contraction band necrosis was related to ischaemia or inotropic medication received in the intensive care unit is uncertain appendix p pm1 and pm2 were the two patients with the highest active viral load detected in the heart a single patient had a right atrial thrombus pericarditis was acegbdfh µm µm 03m µmfigure thrombotic features identified at autopsy in patients with covid19a macroscopic right coronary artery thrombosis arrow in a 61yearold man pm1 with exudative phase diffuse alveolar damage b macroscopic pulmonary thromboembolism arrow in a 97yearold man pm8 c thrombus in the lung of a 79yearold woman pm9 on haematoxylin and eosin staining at 20x magnification the insert shows cd61 immunohistochemistry indicating moderate staining for platelets d plateletrich thrombus in the mediumsized vessels surrounding the heart in a 61yearold man pm1 the insert shows strong cd61 staining for platelets periodic acid schiff staining showing a glomerular microaneurysm arrow e and microthrombi within glomerular capillary loops arrow f at 40x magnification indicative of thrombotic microangiopathy in a 97yearold man pm8 macroscopic splenic g and hepatic h infarction in a 22year old man pm5identified in two patients one patient showed florid fibrinous pericarditis containing fungal hyphae pm5 while the other showed only microscopic acute pericarditis appendix p figure the median heart weight was high g and four of nine patients had left ventricular hypertrophy nonbacterial thrombotic marantic endocarditis was noted in one patient pm5 with no known history or autopsy findings consistent with malignancy or chronic disorder associated with nonbacterial thrombotic marantic endocarditis appendix p figure pm5 had disseminated mucormycosis and numerous other thrombotic features appendix p cardiac amyloidosis and right atrial thrombosis were identified in one of ten patients pm8 appendix p lymphocyte depletion involving specific compartments and increased phagocytosis were prominent findings appendix p figure increased phagocytosis of other cells was identified in the sinusoidal macrophages of the red pulp of the spleen in four of seven patients sinus histiocytes of hilar lymph nodes in three of six and bone marrow four of eight phagocytosis was identified in at least one of these ans in six of nine patients bone marrow haemophagocytosis was prominent in two patients pm4 and pm8 and focal in two patients pm7 and pm9 depletion of periarteriolar tcell sheaths within the white pulp was observed figure red pulp was generally congested showing reduced numbers of cd8positive t cells plasma cells were variably prominent and sinusoidal histiocytes showed phagocytosis of red blood cells and other cells to varying extents both igmpositive and iggpositive plasma cells were identified and they were polytypic for lightchain expression figure lymph nodes showed preservation of follicles and relative depletion of paracortical areas medullary areas showed prominence of plasma cells and histiocytes were prominent in the sinuses bone marrow samples showed reactive changes with trilineage hyperplasia and prominence of plasma cells and histiocytes were a common finding a necrotising granuloma was noted in a single hilar lymph node in one patient and acidfast bacilli were noted on ziehl neelson staining appendix p all spleen and lymphoid material examined with immuno histochemistry were negative for epsteinbarr virus and cytomegaloviruspancreatitis was noted in two of eight patients pm5 was a 22yearold man with frank necrotichaemorrhagic pancreatitis and secondary mucor mycosis figure no fungal hyphae were noted in the pancreas pm8 was a 97yearold man who showed no substantial macroscopic pancreatitis although micro scopic acute inflammation within the pancreas and periadrenal fat necrosis was noted figure a third of patients three of nine showed patchy areas of infarcttype adrenocortical necrosis with one patient showing anising microthrombi in adrenal vessels figure no wwwthelancetcommicrobe published online august 101016s2666524720301154s 0cadrenalitis was noted two of nine patients showed chronic inflammation in the thyroid with follicular epithelial cell disruption however the significance of this finding is uncertainmedian combined kidney weight was within normal range at g iqr “ salient renal pathology findings were acute tubular injury in all nine patients underlying moderate cortical scarring of uncertain cause in one patient glomerular microaneurysm and thrombi in one patient figure and rare thrombi in interlobular arteries in four patients pm6 24yearold man of arterial intimal thickening than expected for that age appendix p we observed no evidence of focal and segmental glomerulosclerosis diabetic glomerulopathy or glomerulonephritisdegree had a higher large droplet fatty change was seen in most patients seven of eight cirrhosis or bridging hepatic fibrosis were noted in three patients no liver thrombosis was identified histologically but one patient showed macroscopic liver infarction figure the median liver weight was g iqr “ and three of nine patients showed hepatomegaly liver weighing g two patients pm4 and pm7 showed marked autolysis and were not included in analysismoderate to intense microglial activation was the most prominent pathological feature in the cns five of five patients mild tcell infiltration was noted around blood vessels and capillaries in all five patients but b cells were absent we found ischaemic changes of variable extent in the neurons of the cortex and in the white matter detected by bapp β amyloid precursor protein stain however no necrosis of brain tissue or extensive infiltration of inflammatory cells in brain parenchyma or meninges was observed on histological examination although one of nine patients showed macroscopic haemorrhagic transformation in a large recent cerebral infarction in the distribution of the middle cerebral arterytissues from five patients were analysed for presence of viral genomes against e gene and indications of viral replication against subgenomic rna transcripts by qrtpcr viral rna was present in respiratory tract samples including lung of all five cases analysed in addition two of three patients had detectable viral rna in the nasal epithelium and four of five patients in the trachea evidence of viral genomes outside the respiratory tract was found for all five patients but the distribution and viral loads varied case by case figure 5a viral genomes were also detected using a different qrtpcr targeted at rdrp gene and patterns were consistent between the two sets of primers data not shown a third primer set that detected subgenomic rna indicated virus replication in all tissues examined with variation between patients in levels and distribution figure 5bace µmbdf µm µmfigure other notable autopsy findings in patients with covid19a contained aortic dissection green arrow and fibrinous pericarditis red arrow in a 22yearold man pm5 insert is a haematoxylin and eosin stain image of the pericardium showing fibrinous pericarditis 10x magnification b adrenocortical microinfarcts in a 79yearold woman pm9 with reendothelialising thrombus in small adrenal vessels highlighted by cd34 insert bottom left and haematoxylin and eosin insert top right marantic endocarditis c highlight with haematoxylin and eosin staining bottom left at 10x magnification and necrotising haemorrhagic pancreatitis d in a 22yearold man pm5 with covid19 and a secondary fungal lung infection e periodic acid schiff staining showing a granular cast arrow indicative of acute tubular injury in a 24yearold man pm6 20x magnification f microscopic acute pancreatitis on haematoxylin and eosin staining in a 97yearold man pm8 20x magnificationdiscussionin this series we have described the major pathological findings identified at autopsy in ten patients who died of severe covid19 the most consistent findings were dad thrombosis haemophagocytosis and immune cell depletion although unexpected pathologies that are probably related to sarscov2 infection were also identifieddad was the most consistent and prominent feature in our series and others78 the specific phase of dad probably represents the degree and chronicity of the offending insult sarscov2 infection in relation to the time of death this is similar to previous coronavirus epidemics6 the conclusion by copin and colleagues7 that covid19related lung injury œis not diffuse alveolar damage might relate to their sampling strategy and wwwthelancetcommicrobe published online august 101016s2666524720301154 s 0cadbc µm µm µm µm µmef µm µm µmfigure pathological findings in haematological ans in patients with covid19tcell depletion in the spleen of a 79yearold woman pm9 with covid19 haematoxylin and eosin staining of the spleen at 10x magnification a cd20 staining of spleen indicating presence of b cells b 10x magnification with the insert showing the same region at higher power 20x magnification and cd3 staining of spleen indicating depletion of t cells c 10x magnification with the insert showing the same region at higher power 20x magnification bone marrow phagocytosis in a 97yearold man pm8 with covid19 haematoxylin and eosin staining of a well preserved bone marrow with an arrow indicating presence of phagocytosis d 40x magnification and cd68pgm1 staining of bone marrow indicating presence of phagocytosis 20x [e] and 40x [f] magnificationchronicity five patients had spent approximately weeks on a ventilator barton and colleagues8 described prominent acute bronchopneumonia as the major finding in one of two patients although the authors acknowledge that this was probably affected by aspiration in their patient with muscular dystrophy reports of lung histology in early covid19 also suggest a degree of lymphocytic pneumonia although dad is probably superimposed on this over time in the majority of fatal cases7 pulmonary macrophage infiltration and multinucleated giant cell reactions are prominent similar to other series8“ definite evidence of in covid19 will require quantitative analysis comparing tissues from covid19 patients with dad associated with other conditions and unaffected tissues several cases of invasive pulmonary aspergillosis have been reported in patients with severe covid19 pneumonia18 to our knowledge this is the first description of histologically proven mucormycosis in patients with covid19 and suggests that other human fungal pathogens including members of mucoromycotina can complicate covid19associated infectionslymphocyte depletion tissuerelated numerous clinical features including raised serum ddimer concentrations raised procalcitonin concentrations and imaging findings suggest thrombosis is prominent in patients with covid192 thrombotic features were universal among patients who underwent full autopsies all nine patients had thrombi in at least one major an and have been noted to be prominent in other covid19 autopsy series15 in a retrospective study of autopsies in patients with acute respiratory distress syndrome and dad of various causes only showed thrombi within the small vessels of the lung despite sampling of every lobe of the lung19 another study used postmortem angiography and identified thrombi in nearly all cases of acute respiratory distress syndrome from various causes20 whether thrombosis in covid19 is more common than in other causes of dad remains uncertain however our data support thrombosis as being a striking feature in these patients a study suggested endotheliitis as a prominent feature in patients with severe covid1910 but this was not a prominent feature in our patients importantly limited post mortem or postmortem crosssectional imaging are likely to underrepresent the true extent of thrombosis particularly microthrombosis and its impact on patient death the extent of cardiomegaly fibrointimal thickening of renal blood vessels and obesity in our series supports a contribution of hypertension beyond that noted clinically only four patients had hypertension documented on the mccda raised cytokine profile has been documented in a subset of patients with severe covid192 consistent with this haematological ans in our series showed prominent phagocytosis in several patients which has not been documented in previous series21 of the four patients with bone marrow haemophagocytosis one patient pm7 showed mild transaminitis hyperbilirubinaemia elevated serum ferritin concentrations and fever of ·°c however most clinical data were insufficient to assess the presence of haemophagocytic lympho histiocytosis wwwthelancetcommicrobe published online august 101016s2666524720301154s 0ca substantial feature in covid19 is lymphocyte depletion and this is supported in our series by the spleen and lymph node findings when compared with those with mild disease patients with severe covid19 tend to have a higher neutrophil to lymphocyte ratio and higher cd4positive to cd8positive tcell ratio22 additionally a negative correlation exists between peripheral blood lymphocyte count and viral copy number22 we have corroborated this evidence by documenting a low number of t cells especially cd8positive t cells and foxp3positive regulatory t cells in the spleen and lymph nodes in severe fatal covid19 notably normal plasma cell both igm and igg positive response was present in haematological ans in most patientsthe extent to which anspecific pathologies relate to direct viral replication or consequent immunological and cardiovascular complications is of clinical relevance we report here evidence of viral genomic rna outside the respiratory tract this finding is in agreement with several previous studies that have identified viral genomes by qrtpcr in postmortem tissues including the colon14 spleen14 liver1423 skin24 heart23 and brain25 we also report detection of subgenomic rna a product that is only produced in actively infected cells a report identified low viral load in the brain of three of patients with covid19 but could not detect the virus in subsequent immunohistochemistry and concluded that the viral genomes might have been present in the blood25 although we cannot exclude that the rnas detected in our series were similarly carried to the site of sampling in blood the distribution of rna in different tissues varied widely between postmortem casespm3 and pm4 appear to have died earlier in the disease course days after symptom onset and had higher viral loads in the respiratory tract than other patients whereas pm3 and pm4 died after long stays in intensive care units and had either lower overall viral rna pm5 or higher viral rna outside the respiratory tract pm2 pm1 and pm2 were the only patients in whom we detected viral rna and subgenomic rna in the heart and are the only two patients with evidence of acute myocardial injury moreover unlike the previously mentioned study where virus detected in the brain was times less than that detected in the lung the number of viral genomes detected in external tissues in our series was frequently of similar or even higher levels than that found in the respiratory tree one study has detected sarscov2 infection of the endothelium in the vasculature of the skin and lung by immunofluorescent staining for viral antigens24 it is not obvious what determines spread and tropism of sarscov2 outside the respiratory tract several studies have reported ace2 expression levels that were higher in some ans than in others26 the sites of highest expression did not correlate with areas of most severe pathological involvement in our series we should note that cellular receptor status is one of many things that determines the degree of anr latot g 03 rep seipocenege larivpm1pm2pm3pm4pm5abtcbone marrowbrainheartileumkidneyliverspleentonguetrachealungnasal epitheliumfigure tissue tropism of sarscov2 in postmortem samplesfresh tissues were collected from a subset of postmortem examinations and viral load quantified by use of qrtpcr targeting the viral e gene a detection of viral rna was verified by use of qrtpcr against the viral polymerase gene data not shown tissues were additionally tested for subgenomic viral rna transcripts b dotted lines indicate the limit of detection as ascertained by negative control data are included for a 61yearold man pm1 a 64yearold man pm2 a 69yearold woman pm3 a 78yearold man pm4 and a 22yearold man pm5 qrtpcrquantitative rtpcr sarscov2severe acute respiratory syndrome coronavirus pathological involvement other aspects are likely to play a role such as route of transmission acid lability in the stomach coreceptors expression of proteases required to activate entry of virus into the cell eg tmprss2 possibly other yet unidentified host factors and invivo routes of dissemination the evidence of ongoing replication late in disease supports the use of antiviral therapy even at a point in illness when immunopathology is dominantpancreatic pathology was a major unexpected pathology in this series which had not been previously reported in covid19 autopsies or large clinical covid19 series2 a 22yearold man had haemorrhagic necrotic pancreatitis pm5 although this patient also had disseminated mucormycosis he had evidence of viral persistence in the lung and no fungal hyphae were identified in the pancreas on histological sections the other patient with wwwthelancetcommicrobe published online august 101016s2666524720301154 s 0cpancreatic pathology had microscopic evidence of pancreatitis that could be missed at autopsy without adequate sampling the pancreas is a classic site of unexpected pathology identified at autopsy27 serum amylase is not part of the routine care bundle for covid19 in our trust whether the pancreatitis was related to sarscov2 iatrogenic comorbidities or secondary infection is not clear in our study most cases had evidence of hepatic steatosis which is consistent with clinical findings that obesity is a risk factor for poor outcome in covid19 and liver cirrhosis or bridging fibrosis was prominent in this cohort28 from these data nothing suggests direct viral inflammation of the liverinfection or other causes the interval between time of death and autopsy impaired histological interpretation in many ans and affected antigen preservation postmortem endothelial stripping did affect the endothelial interpretation in our study however a subs
Colon_Cancer
the development of fungal fruiting bodies from a hyphal thallus is inducible under low temperature cold stress the molecular mechanism has been subject to surprisingly few studies analysis of gene expression level has become an important means to study genefunction and its regulation mechanism but identification of reference genes rgs stabilityunder cold stress have not been reported in famous medicinal mushroomforming fungi cordyceps militaris herein candidate rgs had been systematically validated under coldstress in c militaris three different algorithms genorm normfinder and bestkeeper wereapplied to evaluate the expression stability of the rgs our results showed that ubc andubq were the most stable rgs for cold treatments in short and long periods respectively rgs ubc and pp2a and rgs ubq tub and cyp were the suitable rgs for coldtreatments in short and long periods respectively moreover target genes twocomponentsystem histidine kinase genes were selected to validate the most and least stable rgsunder cold treatment which indicated that use of unstable expressed genes as rgs leadsto biased results our results provide a good starting point for accurate reverse transcriptasequantitative polymerase chain reaction normalization by using ubc and ubq in c militarisunder cold stress and better support for understanding the mechanism of response to coldstress and fruiting body formation in c militaris and other mushroomforming fungi in futureresearcha1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation liu yn liu by ma yc yang hl liugq analysis of reference genes stabilityand histidine kinase expression under cold stressin cordyceps militaris one e0236898101371 pone0236898editor shwet kamal icardirectorate ofmushroom research indiareceived april accepted july published august copyright liu this is an open access distributed under the terms of the creativecommons attribution license which permitsunrestricted use distribution and reproduction inany medium provided the original author andsource are crediteddata availability statement all relevant data arewithin the paper and its supporting informationfilesfunding this work was supported by the nationalnatural science foundation of china no and the natural sciencefoundation of hunan province no 2020jj5972the scientific research fund of hunan provincialeducation department china no 17k106 and18b167 the national key r d programs ofintergovernmental international science andtechnology cooperation no 2017yfe0108100introductioncordyceps militaris a famous traditional chinese medicine has been used as a healthy food fora long time in china the c militaris fruiting body has antiinflammatory antitumor antiinfluenza virus and radioprotection functions methods for commercial production of fruiting bodies of this fungus have been established in artificial media [ ] or withinsects such as silkworm bombyx mori pupae but the molecular mechanism of fruitingbody formation is not well understood the essential role of light in fruiting body development one 101371 pone0236898 august one 0cthe project of international cooperation base ofscience and technology innovation on forestresource biotechnology of hunan province no2018wk4008 and aid program for science andtechnology innovative research team in highereducational instituions of hunan province thefunders had no role in the study design datacollection and analysis decision to publish orpreparation of the manuscriptcompeting interests the authors have declaredthat no competing interests existstable reference genes in cordyceps militaris for cold stressin c militaris was demonstrated in previous study inactivation of a bluelight receptene white collar1 wc1 resulted in thicker aerial hyphae disordered development in c militaris further study showed that the fruiting body primordia could form in the drosophilaarabidopsis synechocystis and human dash type cryptochromes crys gene mutantstrains but the fruiting bodies were unable to elongate normally and crydash and cmwc1exhibited interdependent expression under light in c militaris compared to light temperature is another pivotal factor influencing fruiting body formation from a hyphal thallus which represents a transition from simple to complex multicellularity the fruiting body formation of various mushrooms depends on low temperaturecold stress induction for example the fruiting body development was induced by shiftingthe cultivation condition ˚c for mycelia to lower temperature ˚c in flammulina velutipes in pleurotus ostreatus the mycelia were cultured at ˚c for days in a sawdustmedium and then the temperature was lowered to ˚c to induce fruiting body development in c militaris after the mycelia were cultured under static conditions at ˚c on potatodextrose broth medium for days the mycelia were injected into pupae and then the inoculated pupae were kept at ˚c to induce the fruiting bodies however the molecular mechanism of mycelial development to fruiting body under cold induction has been subject tosurprisingly few studies which will seriously restrict the further development of commercialproduction of mushrooms fruiting bodiestwocomponent signal transduction is commonly used by eubacteria archea and eukaryotes as a stimulus“response coupling mechanism to sense and respond to changes in many different environmental conditions especially temperature sensing they consist of ahistidine kinase hk and a response regulator the histidine kinase desk from bacillus subtilis is mechanistically the best understood after a temperature downshift desk gets autophosphorylated at the conserved histidine residue and donates the phosphate group to theconserved aspartate residue of desr the phosphorylated desr activates the transcription ofthe des gene however little is known the role of hk genes in cold stress response andfruiting body formation in c militaris and other mushroomforming fungidue to its high sensitivity specificity and reliable quantification realtime quantitativepolymerase chain reaction qrtpcr is an important tool to understand the complex signaling networks when an anism is submitted to different stimuli [ ] however the resultsof qrtpcr are often inaccurate because of the occurrence of errors in the primer specificitycomplementary dna cdna synthesis and pcr amplification therefore qrtpcrmust be normalized using reference genes that show a stable expression however validationof suitable rgs for expression analysis under cold stress have not been conducted in c militaris and many other mushroomforming fungi in this study we systematically identified candidate rgs in c militaris that were measured using qrtpcr three different algorithmsgenorm normfinder and bestkeeper were applied to evaluate the expression stability of the candidate rgs under cold treatment in c militaris moreover the relative expression levelsof twocomponentsystem hk genes were analyzed under cold stress conditions with threedifferent normalization strategies the rgs screened out in this paper could provide robustness to study the regulatory mechanism of cold induced fruiting body formation from myceliain c militaris and other mushroomforming fungimaterials and methodsstrains and culture conditionsa laboratory and commercial strain of c militaris cgmcc from china generalmicrobiological culture collection center was used the c militaris was cultured at ˚c in one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stressdark with an artificial medium containing g rice g powder of silkworm pupae and25ml nutrient solution glucose g kh2po4 g mgso4 g ammonium citrate g peptone g vitamin b1 mg and 1000ml distilled watercold stressafter cultured for days at ˚c in dark the c militaris mycelia were transferred to ˚c indark for different treatments under cold stress for different time periods including short periods and 8hours and long periods and 8d after treatmentmycelia samples were immediately frozen in liquid nitrogen and stored at ˆ’Ëšc in a deepfreezer until rna isolation untreated mycelia samples were used as the control each treatment was repeated for timesrna isolation and cdna synthesisrna isolation and cdna synthesis were performed as previously described method briefly g mycelia were collected and subsequently were disrupted under liquid nitrogenconditions the rna isolation kit takara china was used to extract total rna treatedwith dnase i to avoid genomic dna contamination μg total rna of each sample wasadded to μl reverse transcription reaction system to synthesize cdna the cdna was subjected to 10fold serial dilution for determining the amplification efficiency of qrtpcr uponcold treatmentsrealtime quantitative polymerase chain reaction conditionsqrtpcr experiments were performed using a previously described method briefly allgenes were amplified by initial heating at ˚c for min followed by cycles of ˚c for s ˚c for s and ˚c for s at the final amplification cycle the specificity of pcr reactions was checked through the use of melting curve analysis “Ëšc in increments of ˚cevery s negative controls were included to ensure the suitability of the assay conditionseach experiment described above was repeated independently at least for timesselection of candidate reference genesin this study genes namely act tub ubc ef1α gapdh pp2a ubq pgk rpsfbox cyp and gtpb were selected as candidate rgs our gene panel contained traditional rgs such as tubulin tub actin act polyubiquitin ubq glyceraldehyde3phosphate dehydrogenase gapdh and translation elongation factor1α ef1α geneschosen on the basis of stable expression in several rna sequencing and quantitative pcrexperiments such as cyclophilin cyp and ubiquitinconjugating enzyme ubc and novel stable rgs in cold stress such as phosphoglycerate kinase pgk and serinethreonine protein phosphatase 2a pp2a details of these genes are provided in table these genes were annotated with a c militaris genome database national center for biotechnology information accession gse28001 and aevu00000000 and used to design primers the specific primers for twocomponentsystem histidine kinase hk genes were listed ins1 tabledata analysis to select the internal reference genethe expression stability of the candidate rgs was analyzed using genorm normfinder and bestkeeper as previously described one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stresstable details on primers used for quantitative reverse transcriptase polymerase chain reaction analysisgeneacttububcef1αannotationaccession noactin cytoskeleton proteinxm_0066695521tubulinxm_0066692031ubiquitinconjugating enzymexm_0066722771elongation factor 1alphaxm_0066659681gapdhglyceraldehyde 3phosphate dehydrogenasexm_0066696971pp2aubqpgkrpsfboxcypgtpbserinethreonine protein phosphatase 2axm_0066730331polyubiquitinxm_0066724691phosphoglycerate kinasexm_0066734081ribosomal protein s25xm_0066653991fbox proteinxm_0066728101cyclophilinxm_0066747781gtpbinding proteinxm_0066746481primer sequences ™™fcaacaacttcctgacgggcrtccttgggcttctgctgacfatgtcgttcgtcgtgaggragagtggcgttgtagggtfaccattgacacgagccagttrgcccatgtaagcctcctcaftatcggaactgtgcctgtrcgttaccacgacggatttfcatccactcctacactgctacrctcaagacgaacagtcaggtfcctcctacagtcgtcatcagcragaaatgtcaaagcgagaftcaaagaagataatggtaacgrgtatgggttctcggaaaggtfgctcaagcccgtcgtttcrccctcctcctcaatgtggfaagtggtctaagggcaaggrttctcctccaggtcggtaafccgatgacaacgacagcgacrgtagttgaccgtggagatgtfttttccgccttattccaccrtccagagcatcaaatccctftaagaagcccaagaagaaaargtcccacaggttcagcgtf forward r reverse101371 pone0236898t001amplification size bpefficiency abbreviationsact actin cytoskeleton protein cdna complementary dna cq quantification cycle cvcoefficient of variation cyp cyclophilin ef1α elongation factor 1alpha fbox fbox protein gapdh glyceraldehyde 3phosphate dehydrogenase gtpb gtpbinding protein hkhistidine kinase m value measurement value pgk phosphoglycerate kinase pp2a serinethreonine protein phosphatase 2a rgs reference genes rps ribosomal protein s25qrtpcr realtime quantitative polymerase chain reaction sd standard deviation tubtubulin ubc ubiquitinconjugating enzyme ubq polyubiquitin vnvn1 pairwisevariationresultsexpression profiling of candidate reference genes in c militaris genes namely act tub ubc ef1α gapdh pp2a ubq pgk rps fbox cyp andgtpb were selected as candidate rgs to determine the most stable rgs under cold treatments we first calculated the pcr efficiency of twelve pairs of primers according to the previously reported method as shown in table qrtpcr efficiency of the genes rangedfrom cyp to ef1α which fell within the acceptable range the quantification cycle cq values of the genes exhibited a high variation ranging and shown in fig the cq values ranged from to and to of short periods fig 1a and long one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stressfig the range of quantification cycle cq values of the candidate reference genes in c militaris under cold stress for short a and long periods b101371 pone0236898g001periods fig 1b respectively rps and pp2a was the most and least transcribed respectivelyacross all the tested samples fig 1a and 1b three different algorithms genorm normfinder and bestkeeper were used to analyze the expression stability of the genes in the nextsectiongenorm analysisthe genorm algorithm calculates the expression stability factor m the m value is defined asthe average pairwise variation of a particular gene with all other reference genes within a givengroup of cdna samples where a low value represents stable and a high value represents unstable expression as determined by genorm fig expression stability m values of the genes both in short and long periods were within the acceptable range m the mvalue ranged from pgk to ubc and pp2a in short periods fig 2a and pgk to tub and ef1α in long periods fig 2b the genes were ranked from thehighest m value least stable to the lowest m value most stable pgk act gapdh tubef1α fbox rps ubq cyp gtpb ubc and pp2a in short periods fig 2a pgk ubcact fbox rps gtpb pp2a cyp gapdh ubq tub and ef1α in long periods fig 2bfig genorm analysis of the average expression stability values m of the candidate reference genes under cold stress for short a and long periods b a higherm value indicates more unstable expression101371 pone0236898g002 one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stresstable gene expression stability under short time cold stress ranked by genorm normfinder and bestkeepergenesubcpp2agtpbcypubqrpsfboxef1αtubgapdhactpgkgenormnormfindermean rankabestkeeperm valuerank orderstability valuerank ordercvsd101371 pone0236898t002normfinder analysisnext the expression stability of the candidate rgs was analyzed using normfinder to confirm the genorm results in short periods ubc and ubq were the most stable their stabilityvalues were and respectively table in long periods cyp and ubq were themost highly ranked their stability values were and respectively table combining the results of genorm and normfinder analysis ubc followed by pp2a gtpb and ubqwere the most stable rgs in short periods table mean rank ubq followed by tub cypand gapdh were the most stable rgs in long periods table mean rankoptimal number of reference gene for normalization across theexperimental setsnext we determine the minimal number of genes for qrtpcr normalization by estimatedpairwise variation vnvn1 in genorm below which was a proposed cutoff vnvn1value adding an additional rg is not required according to this principle the vnvn1value was calculated in short periods the v23 value was therefore ubctable gene expression stability under long time cold stress ranked by genorm normfinder and bestkeepergenestubef1αubqgapdhcyppp2agtpbrpsfboxactubcpgkgenormnormfindermean rankabestkeeperm valuerank orderstability valuerank ordercvsd101371 pone0236898t003 one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stressfig genorm analysis of the pairwise variation v of the candidate reference genes under cold stress for short a and long periods b the pairwise variation vnvn1 measured the effect of adding additional reference genes on the normalization factor for cold stress treatment in c militaris101371 pone0236898g003together with pp2a would be sufficient for purpose fig 3a and table in long periodscompared with v23 v34 value was suggesting that three rgs ubqtub and cyp were identified as available for normalization fig 3b and table bestkeeper analysiswe further used bestkeeper to analyze the stability of candidate rgs by calculating thestandard deviation sd and the coefficient of variation cv of their cq values it has beenreported that any studied gene showing a sd in expression lower than can be considered stable and the most stably expressed gene exhibiting the lowest cv as shown in table inshort periods the top two stable genes ubc and pp2a with lower cv values and respectively and the sd values of ubc and pp2a were and respectively in long periods the top three stable genes ubq tub and cyp with lower cv values and respectively and the sd values of ubq tub and cyp were and respectively table these bestkeeper results also suggested that ubcpp2a and ubqtubcyp were stable in short and long periods respectivelytaken together our results suggesting that two rgs ubcpp2a and three rgs ubqtubcyp were identified as available for qrtpcr normalization under cold incubation duringshort and long cold periods respectively in c militarisquantitative effects of best and least ranked reference genes on target geneexpressionto validate the utility of stable rgs on gene expression analysis three different strategies theleast stable rg pgk the best ranked rg and multiple stable rgs were selected to normalizethe expression of target gene in short and long periods treatments respectively using pgkubc and ubcpp2a for short periods rg and pgk ubq and ubqtubcyp for long periods rg hk genes in cold stress response were used as target genes based on the annotationwith c militaris genome database national center for biotechnology information accessiongse28001 and aevu00000000 genes cmhk19 s1 table were identified as hk homologous genes normalization of the hk genes using the three different strategies showed that asignificant increase in transcription of hk2hk3hk6 and hk1hk3 hk5 were observed one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stressfig relative expression levels of hk genes during cold stress conditions using the least stable gene best rankedgene or multiple stable reference genes for normalization the least stable reference gene pgk best rankedreference gene ubc and multiple reference genes ubcpp2a were used for normalization to analysis of hk2 a hk3b and hk6 c expression levels in short periods the least stable reference gene pgk best ranked reference geneubq and multiple reference genes ubqtubcyp were used for normalization to analysis of hk1 d hk3 e andhk5 f expression levels in long periods the average ct value of multiple reference genes is used to analysis of hkgene expression levels in the strategy of multiple reference genes were used for normalization101371 pone0236898g004under cold incubation during short and long cold periods respectively fig the geneexpression levels of other hk genes were not shown when using the best ranked ubc or themultiple stable ubcpp2a as the rgs in short periods the upregulated trends for hk2 hk3and hk6 genes were consistent and the peak points were observed at h fig 4a“4c whenusing the best ranked ubq or the multiple stable ubqtubcyp as the rgs in long periodsthe upregulated trends for hk1 hk3 and hk5 genes were consistent and the peak points wereobserved at d fig 4d“4f respectively however using the least ranked pgk as the rg inshort periods the peak points were observed at h of hk2 hk3 hk6 respectively fig4a“4c in addition when pgk was applied as the rg in our experiment the transcriptionpatterns of hk genes were higher than those using the best ranked rg and multiple stable rgsfig 4a“4f which indicated that normalization using the least stable pgk as rg resulted inan overestimated relative expression level of the target genes thus our experimental resultssuggest that it is feasible to use any one or more of ubcpp2a and ubqtubcyp genes asthe normalization gene under cold incubation during short and long cold periods respectivelywith c militarisdiscussioncompared with microarray and rna sequencing qrtpcr technique has the advantages ofhigh accuracy high sensitivity good repeatability and low cost for quantifying gene expression one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stresstable the screening results of candidate reference genes of different species under cold stressgenepp2acyppgkef1αactgapdhubcspeciesananas comosuselymus sibiricusananas comosusbeauveria bassianahordeum vulgareatropa belladonnahemarthria compressa and h altissimamorchella sppbeauveria bassianaelymus sibiricusmorchella spparabidopsis pumilaarabidopsis pumila101371 pone0236898t004expression stability stableunstablerefexpression stability in this studystablestablestablestableunstablestablestableunstablestablestableunstablestablestablechen zhang chen zhou cai li lin stable short periodsstable long periodsunstable short and long periodsunstable short periodszhang unstable short and long periodszhou zhang zhang unstable short and long periodsjin jin stable short periods unstable long periods[ ] to ensure the accuracy of qrtpcr results it is necessary to analyze the stability ofinternal rg under a specific experimental condition a previous study in c militaris showedthat polymerase ii large subunit rpb1 gene was the best rg during all developmental stagesexamined while the most common reference genes actin and tub were not suitable internalcontrols in addition the actin also was not suitable rgs under developmental stagestrain and nutrient source in lentinula edodes however suitable rgs for expressionanalysis under cold stress have not been reported in c militaris in this study the expressionstability of candidate rgs in c militaris was systematically analyzed by genorm normfinder and bestkeeper under the cold treatment our results showed that ubc and ubq wereidentified as the most suitable for qrtpcr normalization under cold incubation during shortand long cold periods respectively while the act a traditionally rg was not suitable coldstress in c militaris therefore we suggest to use two or more rgs rpb1 ubc and ubq tostudy the expression levels of the fruiting body developmental genes in c militaris in the followup studya large number of studies have been conducted to identify the suitable rgs under coldtreatment conditions among different species but the most stable rgs are diverse table inpineapple ananas comosus l pp2a and cyp were the stable rgs under cold stress andin hulless barley hordeum vulgare l var nudum hook f pgk was not stable rg undercold stress which were consistent with our results however in hemarthria compressaand h altissima leaf tissue ef1α was the most stable gene under cold stress and inatropa belladonna pgk was a reliable gene for normalizing gene expression under cold stressconditions which were not consistent with our results in entomopathogenic fungusbeauveria bassiana both act and cyp were the most stably expressed gene sets under a variety of stress conditions including cold however in our study act was not stable inour experimental conditions in another mushroomforming ascomycota true morelsmorchella spp two candidate internal control genes act and gapdh were not reliablegene for normalizing gene expression under cold stress conditions which was similar toour findings an interesting result is that in arabidopsis pumila both ubc and gapdhunder cold stress were the most stable rgs table but in our result ubc and gapdhwere stable and not stable rgs in short periods respectively the similar results were alsofound in siberian wild rye elymus sibiricus that pp2a and act presented the highest degreeof expression stability for cold stress but in our result only pp2a was stable rg while one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stressact was found to be unstable the more interesting result is that in our study ubc was thestable rg in short periods but not the stable rg in long periods all of these results indicatethat the expression stability of the same gene differs in different species moreover the expression stability of the same gene is also not the same under the same treatment at different timesin arabidopsis thaliana arabidopsis histidine kinase ahk2 ahk3 and coldinducibletype a arabidopsis response regulators arrs play roles in cold signaling in additionstresssensing in fungi also depends on a signaling cascade comprised of a twocomponentphosphorylation relay plus a subsequent map kinase cascade to trigger gene expression moreover twocomponent pathways are important determinants of pathogenicity in animalpathogens such as candida albicans cryptococcus neoformans and plant pathogensincluding fusarium oxysporum tomato monilinia fructicola brown rot of stone fruit and botrytis cinerea bean tomato and apple in this study the relative expressionlevels of twocomponentsystem hk genes were analyzed under cold stress conditions withthree different normalization strategies the results showed that hk genes were significantlyupregulated after cold stress using stable rgs in normalization however an overestimatedrelative expression level in the hk genes transcription was obtained from qrtpcr usingpgk in normalization fig the relative expression level of target gene is overestimated dueto use unstable gene normalization is also found in other studies [ ] these results indicated that using unstable rgs for qrtpcr normalization will lead to inconsistent resultsmoreover our results suggested that the upregulated hk genes participated in response tocold stress of c militaris however whether or how these hk genes involved in coldinducedfruiting body formation of c militaris needs further studyconclusionswe systematic validated candidate rgs under cold incubation during short and long coldperiods respectively in c militaris our results showed that two rgs ubcpp2a and threergs ubqtubcyp were identified as available for qrtpcr normalization under cold incubation during short and long cold periods respectively in addition our results indicate thatfailure to statistically validate rgs leads to inconsistent results moreover the role of upregulated hk genes in coldinduced fruiting body formation in c militaris needs further studyall in all our results provide a good starting point for accurate qrtpcr normalization in cmilitaris under cold stress and better support for understanding the mechanism of response tocold stress and fruiting body formation in c militaris and other mushroomforming fungi infuture researchsupporting informations1 table details on primers of twocomponentsystem histidine kinas used for qrtpcranalysisdocacknowledgmentsthe authors thank jing zhang yanghong zhang and feifei xue for their help during carryingout the experiments the authors thank xiaoxiao lu and jiashun zhang for their help in dataanalysis we would like to thank muling shi for english language editingauthor contributionsconceptualization yongnan liu biyang liu one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stressfunding acquisition gaoqiang liuvalidation youchu ma gaoqiang liuwriting “ review editing hailong yangreferences won sy park eh antiinflammatory and related pharmacological activities of cultured mycelia andfruiting bodies of cordyceps militaris j ethnopharmacol “ 101016jjep200410009 pmid jin c kim g choi y induction of apoptosis by aqueous extract of cordyceps militaris through activation of caspases and inactivation of akt in human breast cancer mdamb231 cells “lee hh park h sung gh lee k lee t lee i antiinfluenza effect of cordyceps militaris throughimmunomodulation in a dba2 mouse model of microbiology “ 101007s1227501443000 wos000339600200010 pmid jeong mh park ys jeong dh lee cg kim js oh sj in vitro evaluation of cordyceps militarisas a potential radioprotective agent international of molecular medicine “103892ijmm20141901 wos000344423800020 pmid xie cy gu zx fan gj gu fr han yb chen zg production of cordycepin and mycelia by submerged fermentation of cordyceps militaris in mixture natural culture appl biochem biotechnol “ 101007s1201000985672 pmid zheng zl huang ch cao l xie ch han rh agrobacterium tumefaciensmediated transformation asa tool for insertional mutagenesis in medicinal fungus cordyceps militaris fungal bioluk “ 101016jfunbio201012011 wos000289023800008 pmid hong ip kang pd kim ky nam sh lee my choi ys fruit body formation on silkworm by cordyceps militaris mycobiology “ 104489myco2010382128pmid pubmed central pmcid pmc3741563tiantian l caihong d tao y junde s effects of blue light on the growth and bioactive compoundproduction of cordyceps militaris mycosystema yang t guo mm yang hj guo sp dong ch the bluelight receptor cmwc1 mediates fruit bodydevelopment and secondary metabolism in cordyceps militaris appl microbiol biot “ 101007s0025301570476 wos000368103200019 pmid wang f song x dong x zhang j dong c dashtype cryptochromes regulate fruiting body development and secondary metabolism differently than cmwc1 in the fungus cordyceps militaris applmicrobiol biot “ 101007s0025301782767wos000402008300025 pmid krizsan k almasi e merenyi z sahu n viragh m koszo t transcriptomic atlas of mushroomdevelopment reveals conserved genes behind complex multicellularity in fungi p natl acad sci usa “ 101073pnas1817822116 wos000463936900040 pmid wu t hu c xie b zhang l yan s wang w a single transcription factor pdd1 determines development and yield of winter mushroom flammulina velutipes appl environ microbiol 101128aem0173519 pmid sunagawa m magae y isolation of genes differentially expressed during the fruit body development ofpleurotus ostreatus by differential display of rapd fems microbiol lett “ 101016jfemsle200504018 pmid bourret rb silversmith re twocomponent signal transduction annual review of biochemistry “ aguilar ps hernandezarriaga am cybulski le erazo ac mendoza d de molecular basis of thermosensing a twocomponent signal transduction thermometer in bacillus subtilis embo “ vandesompele j de preter k pattyn f poppe b van roy n de paepe a accurate normalization of realtime quantitative rtpcr data by geometric averaging of multiple internal control genesgenome biol 37research0034 101186gb200237research0034 pmid pubmed central pmcid pmc126239 one 101371 pone0236898 august one 0cstable reference genes in cordyceps militaris for cold stress bustin sa benes v nolan t pfaffl mw quantitative realtime rtpcr”a perspective j mol endocrinol “ 101677jme101755 pmid huggett j dheda k bustin s zumla a realtime rtpcr normalisation strategies and considerations genes immunity “lu x liu y zhao l liu y zhao m selection of reliable reference genes for qrtpcr during methyljasmonate salicylic acid and hydrogen peroxide treatments in ganoderma lucidum world j microbiolbiotechnol 101007s112740182476x pmid han bin yang zheng samma kaleem m systematic validation of candidate reference genes forqrtpcr normalization under iron deficiency in arabidopsis biometals “ 101007s1053401396235 pmid gutierrez n gimenez mj palomino c avila cm assessment of candi
Colon_Cancer
" while the impact of family caregiving has been welldocumented many of such studies center oninvestigating external factors such as socioeconomic status accessibility to resources and availability of socialsupport as the primary causation of caregiver wellbeing outcomes this paper explores the motivations that drivefamily caregivers in supporting their family members at the endoflife and critically examines how internalappraisal processes of such motivations can both positively and negatively impact their wellbeingmethods this study adopted an interpretative phenomenological analysis ipa to investigate the motivations andinternal appraisal processes of asian family caregivers in singapore who were tending to a dying family memberqualitative dyadic interview data n was drawn from a larger randomized controlled trial for a novel familydignity intervention fdi for palliative care patients and their families the sampling population consisted ofparticipants aged and above who were identified to be the primary caregivers of older palliative care patientswith a prognosis of less than months data collection was conducted in the homes of patients and familycaregiverscontinued on next page correspondence andyhyhontuedusg1psychology programme school of social sciences nanyang technologicaluniversity singapore singapore2centre for population health sciences lee kong chian school of medicinenanyang technological university singapore singaporefull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ctanho bmc palliative care page of continued from previous pageresults findings revealed six themes that could either nurture or diminish caregiver wellbeing honoringfidelity caregivers were motivated to commit to their caregiving roles in order to avoid regret alleviatingsuffering caregivers were motivated to relieve their family member™s pain enduring attachment caregiverswere motivated to spend time together with their family member preserving gratitude caregivers weremotivated to express their appreciation to their family member by caregiving navigating change caregiverswere motivated to adapt accordingly to changes in the illness trajectory and reconciling with mortalitycaregivers were motivated to respond accordingly to their family member™s prognosis the final theme of thewellbeing determinant is posited as an indication of selfdetermination and is conjectured to influence howcaregiving motivations are appraised by the caregiver fulfilling and enhancing one™s sense of selfdetermination appears central to infusing one™s caregivingmotivations with positive meaning and consequently nurturing one™s wellbeing in the endoflife caregivingjourney these findings are discussed with recommendations for healthcare professionals working with familycaregivers of palliative care patientskeywords palliative care caregiver motivations wellbeing meaning burnout resilience selfdetermination endoflife qualitative research the experience of an endoflife eol family caregivercan be likened to a paradox “ what could evoke a senseof pleasure appreciation and gratitude could also bringabout feelings of anxiety distress and pain while somehave related the caregiving journey to the metaphor ofascending a mountain the expedition of an eol family caregiver usually spans beyond merely a couple ofdays weeks or months they must navigate the peaks ofdiagnosis to prognosis and eventually death and bereavement in what often unfolds into a lifelong climbthe role of the eol family caregiver is often multifaceted and interminable daily duties involve managingmedical regimes traversing the healthcare system andtaking charge of other dependents alongside providingphysical mental and emotional support throughout theillness trajectory [ ] many family caregivers are rarelyequipped with formal or adequate training and nor dothey possess sufficient resources and skills before theyfind themselves embroiled in eol caregiving responsibilities family caregivers must also process and manage amultitude of thoughts and emotions as they come toterms with the changes and sometimes losses in theirpersonal lives this complex experience is not limited to a minorityof people despite strong global advancements in medical technology and healthcare systems older populations remain highly susceptible to chronic and terminalmorbidities that are incapacitating in the unitedstates alone over million caregivers tend to their ailing family members annually while an estimated of patients in europe requiring longterm care areattended to by informal caregivers with the anticipated number of older adults in the world soaring to billion by the year there will certainly be a surging demand for family caregivers to relieve the ensuingresource strains on healthcare settingsthe impact of family caregiving stressors has beenwelldocumented [“] with various studies exploringcaregiver burnout and its effects on the community andsociety complementing these studies are literature thatreveal characteristics of resilience and transformationalgrowth displayed by family caregivers in adversity [“] the common thread that impacts both caregiverburnout and resilience appears to be a lack of or adequate coping a process that requires the individual toconstantly change efforts in both thoughts and behaviorsin order to manage internal or external demands thatare considered stressful despite this indication thatone™s psychological resources are key to maintainingone™s wellbeing many studies often consider externalfactors such as socioeconomic status accessibility toresources and availability of social support as the primary causation for the degree of caregiver wellbeingthus these studies often recommend pragmatic interventions that focus on improving external circumstancesaccordinglyperception emotion and motivation of the familycaregiverwhile it is undoubtedly beneficial to mitigate tangiblestressors one must not lose sight of the magnitude of aperson™sthedemands of caregiving this perception and appraisalembody one™s subjective caregiver burden “ observed inreviews of over caregiver studies to pose deepseatedimplications on caregivers™ quality oflevels ofdepression and anxiety and stress coping [ ]internal perception and appraisal oflife 0ctanho bmc palliative care page of campbell later confirmed this observation in astudy that evaluated multiple variations in the caregiverexperience subjective caregiver burden was repeatedlyidentified as the primary indicator for caregiver stressit was around the same time that folkman andmoskowitz discovered that caregivers experiencepositive emotions alongside negative emotions duringstressful events they put forth the tenet of meaningfocused coping in which caregivers derive mental andemotional sustenance thus effecting positive emotionsin challenging circumstances by deferring to their beliefsvalues and existential goals folkman and moskowitzfound that meaningfocused coping is an intrapsychicprocess of discovering benefits in caregiving reminding oneself of such benefits setting goals thatinspire a sense of mastery and competence realigningpriorities in view of changes and infusing ordinaryevents with positive meaning folkman furtherattested that meaningfocused coping exists alongsidenegative appraisals in a caregiver™s stress process inorderand psychologicalresources during stressful eventsto reinstate physiologicalgiven the importance of psychological appraisal it isreasonable to postulate that gaining insight into a caregiver™s beliefs values and goals that stimulate them incaregiving defined as motivations in this paper wouldyield valuable information that could be used to enhancemeaningfocused coping such interventions would target the essence of a person™s selfconcept “ that is theindividual™s perception of who they are and what theybelieve in “ and transcend current cursory social mentaland emotionalfor caregiverstresssymptom management[ ] a studybridging the research gap in asian caregivingwhile the multidimensional nature of caregiving burdenand coping is not confined to any specific culture thereare distinctive elements that bear influence on the asiancaregiving experience family takes precedence in asiansocieties with strongly inculcated values and expectations of filial piety and filial responsibility placed uponfamily membersconducted insingapore a multiethnic society that is predominantlychinese found that family caregivers who internalizedand prioritized societal expectations as motivations overtheir personal wellbeing most often faced internalconflict and were highly likely to experience difficultiesin maintaining their mental health familial ties and caregiving duties this is corroborated by the evolvingattitudes towards such confucian rules “ younger asiangenerations no longer perceive absolute submission orcomplete obedience to the family as instrumental valuesin a modern and globalized society and it would bevaluable to further understand how these complexitiesmight manifest in a family caregiver™s motivationsthis paper aims to contribute to and grow the currentbody of knowledge for asian eol family caregivers byanswering the following research questions what arethe internalized motivations defined here as unconone™ssciously assimilated beliefs and valuesattitudes or behaviors of asian family caregivers how might these motivations affect the way they respond to caregiving and impact their wellbeing howcan an understanding of these motivations be integratedinto psychosocial interventions to enhance and sustaincaregiver wellbeingintomethodsresearch design and proceduresthe current study draws qualitative dyadic interviewdata n from a larger randomized controlled trialfor a novel family dignity intervention fdi for asianpalliative care patients and their families n thesampling methods inclusion criteria interview procedure and study protocol for the fdi are comprehensivelydescribed by ho briefly the fdi is developedbased on an integrative body of empirical investigationthat focuses on dignified endoflife care in both western and asian contexts [ ] it integrates elements oflogotherapy and narrative life review to provide psychosociospiritual support to patients and families facingmortality and has been piloted for acceptability andfeasibility before being fully adapted into the intervention study in practice fdi comprises a recorded dyadicsemistructured interview with a patient and a familycaregiver conducted in their homes the fdi therapistuses a guided question framework to facilitate joint conversation on shared memories and living wisdoms thatlead to meaningmaking and the expression of appreciation and reconciliation this is done with the ultimategoal of creating a legacy document that tells the life storyof the patient and is bestowed to the rest of the familythrough an open reading each interview lasted between and min and was conducted in english malaymandarin or a chinese dialect hokkien teochew orcantonese these recorded interviews were transcribedverbatim translated into english by a native languagespeaker where applicable and edited into legacy documents transcripts and legacy documents were reviewedand finalized by patients and caregivers to ensure accuracy and authenticitysamplingthe sample drawn for this study consisted of primaryfamily caregivers of older palliative care patients aged and above with mainly a cancer prognosis of lessthan months eleven were spousal caregivers seven 0ctanho bmc palliative care page of were adultchildren caregivers and two were siblingcaregivers the majority were female aged between to with a mean age of years see table for caregiver demographics they were recruited through theinpatient daycare and homecare hospice service unitsof hca hospice care dover park hospice tan tockseng hospital singapore cancer society and methodistwelfare services the inclusion criteria required familycaregivers to be above years old and identified by thepatient to be their primary carer patients and familycaregivers came from varioussocioeconomic s and were predominantly of chinese ethnicityas the fdi focused on patient narratives transcriptsbearing a moderate to sizeable amount of input from thefamily caregiver were selected for data analysisdata analysisstudy adopted an interpretative phenomenothislogical analysis ipato investigate the internalizedmotivations of family caregivers in tending to a dyingfamily member the ipa is an approach in qualitativeresearch that aims to provide insights into how an individual makes meaning out of a phenomenon itis important to note that as the current study drawsqualitative data from the larger fdi study no explicitinternalized caregiver motivationsquestionsaboutwere asked reflections on motivations towards caregiving occurred anically throughout the interviewtranscripts and were identified by the researcher usingipa through a process of data reduction and datareconstructionfirst authors and screened all transcripts andselected those that had adequate inputfrom familycaregivers to be used for analysis authors and thenconducted linebyline coding to develop descriptivethemes and analytical categories conceptualizing newinterpretation of the data this was followed by regularmeetings among all authors for the further refinement ofthemes and categories to encapsulate the meaning andcontent within the cluster of similar codes with theemergent themes and subthemes created via a summarychart all authors reviewed and defined the emergentthemes once consensus was reached operational definitions were created finally relationships between categoriesthemes and subthemes were proposed andmapped with supporting quotes from transcripts to address issues of trustworthiness and credibility emergentthemes were constantly compared and contrasted withinand across groups during regular meetings the finaltheme categorization and definitions were agreed uponby the entire research team and data saturation and investigator triangulation were achieved table demographics of family caregiversidentifierdph14caregiver relationshipchildcaregiver ethnicitychinesepatient™s diagnosislung cadph19dph34dph42dph53dph59dph68hca12hca68hca75hca81hca87hca109hca114hca116hca117mws004scs18ttsh61ttsh65spousespousesiblingspousechildspousespousechildchildchildspousespousespousespousespousesiblingspousechildchildchinesechinesechinesechinesechinesemalayeurasianchinesemalaychinesemalaychinesechinesechinesechinesechinesechinesemalaychineseprostate calung casigmoid calung cagynaecological malignancylung caprostate cacolon cabreast caendometrial carenal caendometrial cabrain canasopharyngeal capancreas cacopdliver calung cagynaecological capatient™s prognosis months“““““““““ 0ctanho bmc palliative care page of didn™t i do this why didn™t i do that™  dph19spousein some instances family caregivers displayed poignant emotion and dedication to their family membersconveying the great extent to which they would goto give theirthe best care andcomfortfamily membersœi wish to care for him till the very end¦ i want thebest for him and i will do what™s best for him i amwilling to sacrifice my soul to make that happen ortake his place if i could dph68 childalleviating suffering n family caregivers displayed awareness and empathytowards their family member™s physical and emotionalsuffering tied in with a desire to relieve their painœthis morning she was upset with me for forcing herto drink the bitter medicine i told her ˜i love you iwouldn't do this if i had a choice i want you todrink this for your own benefit not mine i'm just encouraging you from thedph53spousesidelines™underlining this motivation to alleviate suffering was thefamily caregivers™innate compassion for their familymember an empathic bond so strong that witnessingtheir family member™s suffering caused them deep emotional distressœ¦ it hurts a lot to drain the fluids i™m heartbrokenwhen i see how much pain she is in especially wheni see the tubing being inserted it must hurt somuch hca109 spousefindingsfigure presents the six caregiving motivations and onewellbeing determinant generated from data to form theblessings or burdens of endoflife caregiving bobecmodelthe six caregiving motivations honoring fidelityalleviating suffering enduring attachment preservinggratitude navigating change and reconciling withmortality represent the beliefs values and goals that areassimilated into the eol family caregiver™s daily life eachmotivation is posited to affect the way a caregiver makesmeaning of their role hence leading to a nurturance ofcaregiver wellbeing termed in this paper as blessings ora diminishment in caregiver wellbeing termed in thispaper as burdens the wellbeing determinant which ischaracterized by the caregiver™s sense of control selfempowerment and kinship derived from their experiences serves as an indication of selfdetermination andis theorized to have a positive influence on how the sixcaregiving motivations are appraised by the caregiverthese themes are described in greater detail and demfrom study participantsonstrated by direct quotesbelowhonoring fidelity number of transcripts theme hasoccurred in n family caregivers expressed their faithfulness and commitment to attend to the needs and wishes of their family members for the remainder of their lives fearingregret to see things through till the end this motivatedthem in fulfilling their sense of duty to the utmost oftheir abilitiesœi shouldn™t regret anything whatever i can do forhim i will do my best and [instead of waiting till]he™s in the coffin you know [and then say] ˜oh whyfig the blessings or burdens of endoflife caregiving bobec model 0ctanho bmc palliative care page of enduring attachment n family caregivers experienced a prevailing attachment totheir family member that motivated them in spendingcherished time together and doing all they could toensure that their family member was well taken care ofœi think i try to make him as comfortable as he canbe every medical checkup every appointment wewill keep to it and i will always be there for him[there will never be] any appointment that i am notgoing with him hca117 spousesome family caregivers found that the motivation tosustain this attachment was also driven by feelings ofanxiety about their family members™ wellbeing thesecaregivers felt the need to be within their family members™ physical proximity as much as possibleœi get worried when she™s lying there and sleepingbecause i™m not sure if anything has happened toher i™m much happier when she™s sitting here withme when she™s just lying there i would think ˜ohno what if something has happened to her™ and i™dbe worried dph59 childpreserving gratitude n family caregivers described past circumstances and beliefs that motivated present feelings of gratitude to theirfamily members this consequently influenced their efforts and responses in caregivingœshe was constipated for as long as a week and shedidn™t tell me when she eventually relieved herselfshe made a mess on the bathroom floor as i wascleaning the mess i thought about how she hadcleaned me up when i was little so i didn™t mindhca81 childwhile many family caregivers reported feelings of gratitude stemming from how their family member hadtreated them in the past some indicated that religiousand cultural beliefs had indoctrinated a sense of indebtedness to their family membersœmy mother says i was indebted to my brother in mypast life this is why i have to settle my debt in thislifetime [by caregiving] because he is here to get hiscompensation mws004 siblingnavigating change n family caregivers reflected on their perceptions of thechanges that had taken place in their lives and that oftheir family members throughout the illness trajectorysome caregivers found motivation in helping their familymembers adaptenergy to liftsupportto changes by dedicating time andtheirspirits and provide emotionalœi would bring my father food when i visit while myhusband would share words of encouragement andtalk to him to cheer him up we just want him to behappy so that he wouldn't spend the whole day innegativity ttsh65 childothers saw the changes as a temporary setback andfound motivation in steering their family member backto their previous condition if possibleœsometimes i will move his legs a little to give himthat exercise i hope that he can walk again but itdepends on how strong his willhca116spouseisreconciling with mortality n the knowledge of their family member™s prognosis motivated some family caregivers to make the most of thetime left with their family members “ creating treasuredmemories and remembering their legaciesœall of us just want to cherish the time that we haveleft with her and we want her to help us spend moregood times together we [want to] learn about mygrandmother learn about my mother so that wecan pass on to the next generation share with themthe traits and the role models to look up tottsh61 childother family caregivers perceived their family member™sprognosis to be unacceptable choosing to push for further treatment in order to stall death™s journey to theirdoorsœmy grandmother lived past years old so ithought my mother would live till atleast without any problems i felt really shocked becausei always thought œshe still has more than years istill have time ¦ so we felt that if it was possibleshe should extend her life dph14 childthe wellbeing determinant n family caregivers reflected on the discovery of positivetheir family member™schanges amidstillness one such change was found within strengthenedkinshipthe trials ofœas we grow up it™s a bit harder [to have familygatherings] because we are all working so when thedisease came even though it™s not a good thing not 0ctanho bmc palliative care page of something you will ask for it united us again maybewithout it [we] would have been a bit more separated ttsh61 childœi feel like we are more united now maybe in thepast we didn't really chat with each other¦ theamount of communication we had increased i feelthat our unity has become stronger dph14 childthey expressed pride in overcoming initial fears by taking charge of and learning to perform unfamiliar taskswith a newfound confidence in their abilities to faceboth practical and emotional difficulties family caregivers also demonstrated a sense of selfempowermentand strengthbased reflection in their sharingœi know nothing about going to visit the government¦ or to do this do that but somehow i findmy way there [i am a] much stronger person so ifanything happens to me i think i know i can faceup to it dph19 spouseœthis is how you grow i learnt to grow because of[my husband] you have to face the insurmountablechallenges that come your way i learnt how toshoulder my responsibilities on my own scs18spousediscussionthis is the first known study that investigates and bringsattention to the internalized motivations of eol familycaregivers while the family dignity intervention studyquestions did not specifically query family caregivers ontheir motives these motivationcentred responses occurred spontaneously and abundantly throughout the interviews “ an indication that internalized motivationsare profoundly espoused within eol caregiving attitudesand behaviors the bobec model fig illustrates theduality ofthese caregiving motivations in regard tomeaningfocused coping and intrapsychic strains as well as identifies an important influence on caregiver wellbeingmotivations with cultural influencessome themes revealed cultural undertones that reflectedthe internalization of asian values into family caregivermotivations in their motivation for alleviating sufferingfamily caregivers displayed the desire to do so by practical means such as administering medication to theirfamily member and experiencing distress when suchmethods were not feasible this is in line with the asianculture of preferring to show concern for their familymembers through pragmatic ways in their motivation for preserving gratitude family caregivers demonstrated the significance of filial piety as well as culturalbeliefs about karma and pastlife within theirattitudes towards caregiving finally in their motivationfor reconciling with mortality family caregivers indicated the importance placed on close intergenerationalconnections as well as longevity for their elders motivations as blessings meaningfocused copinghonoring fidelityall eol caregiving motivationsalleviating suffering enduring attachment preservinggratitude navigating change and reconciling with mortality were found to embody the tenets of meaningfocused coping fuelled by these motivationsfamilycaregivers displayed the propensity for benefitfindingand benefitreminding even in witnessing their familymember™s suffering and imminent mortality adaptivegoal processes in adjusting their expectations and aspirations in accordance with their family member™s physicalcondition and prognosis reordering priorities in hopesof making the most of the time left with their familymember and infusing ordinary events both past andpresent with positive meaning that allowed them to feelaffirmed encouraged and grateful in their daily caregiving as such the capacity for imbuing stressful caregiving events with positive meaning and responseswould make these motivations advocates of perceivedœblessings in the eol caregiving journeymotivations as burdens intrapsychic strainsparadoxically the authors found that these eol caregiving motivations also ran parallel to the intrapsychicstrains as postulated by pearlin and colleagues intheir seminal stress process model intrapsychic strainsoccur when the caregiver™s selfconcept is diminisheddue to the chronicity of providing care intrapsychicstrains unique to caregivers were defined as role captivity in which the caregiver feels entrapped within hisor her role whether or not by personal choice the lossof self in which the caregiver experiences a loss of identity and sense of personhood as enmeshment with thepatient ensues perceived low competencein whichthe caregiver does not see the value and skill of the workthey do leading to a sense of helplessness and perceived lack of gain in which the caregiver does not findpersonal growth orcaregivingprocessenrichmentin theshould their eol caregiving motivations personifythese strains it is only a matter of time before familycaregivers experience outcomes of mental and emotionaldistress such as depression anxiety and irritability aswell as a decline in physical health and a disengagementfrom their caregiving roles in short these motivations would most certainly bludgeon the family caregiverwith great ˜burdens™ within the eol caregiving journey 0ctanho bmc palliative care page of the crucial factor selfdeterminationselfdetermination theory suggests that people needto feel a sense of competence gaining mastery of tasksand having selfefficacy relatedness feeling like theybelong and mutually relating to others and autonomycontrol over their own choices behaviors and goals inorder to fuel high quality motivation that helps one tothrive the theme of the wellbeing determinant alignswith this concept in a caregivingcentric phenomenonfamily caregivers contributing to this theme displayedconfidence in carrying out previously unfamiliar caregiving tasks competence affirmed a stronger sense of kinship relatedness with the patient and their families andtook ownership of their caregiving responsibilities andchallenges autonomy encouragingly numerous studieshave proposed that high quality motivations stemmingfrom selfdetermination can elicit outcomes of greaterfortitude higher commitment and more positive emotions and selfconcepts [“]building on said studies the authors propose that family caregivers who feel a sense of competence relatedness and autonomy within their caregiving motivationswould be further inclined to meaningfocused copingsuch as deriving perceived benefits from their caregiving even in difficult events reminding themselves ofthese benefits when faced with similar circumstances setting their own goals in caregiving having the competence and confidence to be flexible and adaptive and finding positivity in normal everyday situations possessing a sense of selfdetermination in the caregivingrole would in essence safeguard one™s caregiving motivations from the intrapsychic strains of perceived entrapment a sense of disempowermentineptitude andfruitlessnessas such the bobec modelidentifies the wellbeingdeterminant as an indicative element of caregiver selfdetermination and a crucial factor as to whether the eolfamily caregiver perceives their journey as one lined withblessings or laden with burdens competencetargeted mediators apart fromgeneral psychoeducation on symptom managementmedical care and selfcare interventions could incorporate mediums to develop and improve selfefficacy these can take the form of personalstrengths journaling facilitating peer support between new and experienced caregivers rolemodelling and goalsetting such interventions canbe created in the form of structured support groupsonline platforms or mobile applications autonomytargeted mediators in addition toproviding adequate and appropriate eol caregivereducation autonomytargeted interventions suchas those involving mindfulness practice and thearts can serve to give caregivers a sense of control as well as help them make meaning out oftheir thoughts emotions and circumstances todate the mindfulcompassion art therapymcat for eol care professionals showsgreat potential to be converted into a programfor eol family caregivers relatednesstargeted mediators dyadic or familyprojects that recall shared memories expressappreciation seek fiveness impart wisdom andcreate generativity such as the fdi are valuable incrafting the bond of relatedness among familycaregivers and their family members such projectsshould be implemented as a foundation ofpsychosocial interventions at the endoflifeinterventions should be offered to family caregiversin a culturallyrelatable manner this can be donethrough emphasizing how these mediators will helpfamily caregivers enhance their practical caregivingwith increased competencesense of control andmeaningmaking as well as enrich their intergenerational familial bonds with conversations that focus onlegacy creationimplications and recommendationsfindings from a number of studies show that fulfillingone™s sense of selfdetermination appears central to sustaining one™s motivation and innate satisfaction in caregiving [“] the findings from this paper indicatethat caregivers are driven by motivations that couldequally contribute to wellbeing nurturance or diminishment at the same time our findings also indicate thatcaregivers possess a caregivercentric sense of selfdetermination the wellbeing determinant that is theorized to have positive affect on their motivations thusthis paper recommends that caregiver support interventions should comprise all of the following mediators inorder to fulfil the need for selfdeterminationlimitations and future directionswhile the anically occurring responses emphasize thesignificance of motivations within eol caregiving theseresponses were not examined further during the fdi interviews due to other pri
Colon_Cancer
lenvatinib inhibits tyrosine kinases including vascular endothelial growth factor vegf receptor fibroblast growth factor receptor platelet derived growth factor receptor alpha ret proto oncogene and kit proto oncogene receptor tyrosine kinase we assessed the efficacy and safety of lenvatinib in patients with metastatic colorectal cancer after failure of standard chemotherapiespatients and methods this was an open label single centre single arm phase study eligible patients had unresectable metastatic colorectal adenocarcinoma refractory or intolerant to fluoropyrimidine irinotecan oxaliplatin trifluridinetipiracil anti vegf therapy and anti epidermal growth factor receptor therapy for tumours with wild type ras patients were treated with oral lenvatinib at mg one time a day in day cycles until disease progression or unacceptable toxicity the primary endpoint was centrally assessed disease control rate secondary endpoints included safety response rate progression free survival and overall survival the planned sample size was patients to expect a disease control rate of with a threshold disease control rate of one sided alpha of and power of results between october and january patients were enrolled and had received or ‰¥ lines of prior chemotherapy for metastatic disease respectively the median number of lenvatinib cycles was range “ the centrally assessed disease control rate was ci to one sided p00001 patients had a partial response and had a stable disease median progression free survival was months ci to median overall survival was months ci to the most common grade ‰¥ adverse events were hypertension thrombocytopenia increased alanine aminotransferase and anorexia eachs lenvatinib showed promising clinical activity and was tolerated in patients with metastatic colorectal cancer after failure of standard chemotherapiestrial registration number umin ctr umin000023446 and jamcct ctr jma iia00261introductionthe combination of cytotoxic chemotherapy with a molecular targeted agent has significantly key questionswhat is already known about this subject –º no studies have previously reported the efficacy and safety of lenvatinib monotherapy in patients with metastatic colorectal cancer refractory to standard chemotherapieswhat does this study add –º lenvatinib showed promising antitumour activity with acceptable toxicity for heavily pretreated patients with metastatic colorectal cancer refractory to standard chemotherapies –º no unexpected safety signals were observed and toxicities were manageable with dose modification interruptions and supportive medicationshow might this impact on clinical practice –º further prospective randomised studies are warranted to evaluate the efficacy of lenvatinib in patients with metastatic colorectal cancer refractory to standard chemotherapiesimproved the survival of patients with unresectable metastatic colorectal cancer1“ from results of recent clinical trials trifluridinetipiracil and regorafenib are recognised as new treatment options for patients with metastatic colorectal cancer refractory or intolerant to standard therapies6 nevertheless the prognosis of patients which are refractory or intolerant to standard chemotherapies is poor and there are still an unmet medical needs for these patients especially for those who are in a good performance status and eligible for further therapieslenvatinib is an oral multitargeted tyrosine kinase inhibitor of the vascular endothelial growth factor receptor vegfr “ fibroblast growth factor receptors “ platelet derived growth factor receptor alpha ret and kit8 preclinical studies have shown that iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0copen accesslenvatinib not only interferes the interaction between cancer cells and endothelial cells but also inhibits tumour growth10 several phase trials of patients with solid tumours in the usa11 europe12 and japan13 showed that the optimum dosage of lenvatinib was mg one time a day in a day cyclea total of patients were enrolled in four phase studies of lenvatinib monotherapy of whom had colorectal cancer disease control rate dcr was achieved in out of patients including one with a partial response which continued for weeks mg two times a day for weeks of a week cycle grade palmar plantar erythrodysesthesia was reportedly much lower in of patients treated with lenvatinib for thyroid cancer in a japanese population of the select trial than that of reported in a japanese population of correct trial using regorafenib for metastatic colorectal cancer15 these results suggested that lenvatinib may have a potential for improving the outcomes of patients with unresectable metastatic colorectal cancer who have already received conventional chemotherapy with a fluoropyrimidine irinotecan and oxaliplatinwe conducted a single centre phase study to evaluate efficacy and safety in patients with metastatic colorectal cancer failing to standard therapiespatients and methodsstudy design and patientsthis study was a single arm phase study conducted at national cancer center hospital tokyo japan the inclusion criteria were histological diagnosis of colorectal adenocarcinoma excluding carcinoma of the appendix and the anal canal unresectable metastatic disease an eastern cooperative oncology group performance status of or an age of “ years no previous treatment with regorafenib or lenvatinib sufficient oral intake adequate an and bone marrow function at least one measurable lesion in accordance with the response evaluation criteria in solid tumors recist version refractory or intolerant to fluoropyrimidine irinotecan oxaliplatin therapy and antiepidermal growth factor receptor therapy for tumours with wild type ras and no systemic therapy for at least weeks weeks if any investigational drug had been administered before study enrolment the exclusion criteria were provided in the online supplementary materialtrifluridinetipiracil anti vegf all patients provided written informed consentprocedurespatients received lenvatinib at mg one time a day in day cycles orally until disease progression or unacceptable toxicity the dose was reduced to mg mg mg mg and mg if a patient had an intolerable grade or grade adverse event treatment was discontinued if a dose interruption was required for more than consecutive daystumour response was assessed by the independent radiological review committee based on the ct or mri performed at baseline every weeks for weeks and every weeks thereafter until confirmed objective disease progression safety assessments including laboratory tests were done at screening days and of cycle and days and of the subsequent cycles urinalysis thyroid function prothrombin time international normalized ratio pt inr and tumour markers both carcinoembryonic antigen and carbohydrate antigen “ were measured at screening and on day of each treatment cycle adverse events were recorded from the first day of the protocol treatment to days after the last dose of study medication and graded using the national cancer institute common terminology criteria for adverse events version blood sampling for biomarker analyses was done at baseline on days and and at the end of treatment plasma levels of angiopoietin2 were measured by the human angiopoietin2 quantikine elisa kit rd systems minneapolis usaoutcomesthe primary endpoint was centrally assessed dcr which was defined as the proportion of patients with a complete response partial response or stable disease persisting for more than weeks from the initiation of study treatment according to recist version a complete response and partial response were needed to be confirmedthe secondary endpoints were the objective response rate orr proportion of patients who had a complete response or partial response progression free survival pfs time from the enrolment until investigator assessed disease progression or death overall survival os time from the enrolment until death due to any cause and adverse events the incidence of adverse events was calculated based on the information of the worst grade of each adverse event experienced in each patient relative dose intensity which is unprespecified outcome was calculated as the proportion of the actual cumulative dose divided by planned cumulative dose mg times treatment daysstatistical analysisfor this single arm study the required sample size of patients provided power to reject the null hypothesis of dcr ‰¤ with expectation that of patients would have a disease control one sided α of considering the possibility of a few ineligible patients we planned to recruit patientsthe final analysis was planned approximately months after enrolment of the last patient we included all eligible patients in the efficacy analysis and all patients receiving a least one dose of lenvatinib in the safety analyses for the primary analysis binomial test was performed and the centrally assessed dcr was estimated with ci using the clopper and pearson method which corresponds to one sided α of we also estimated the investigator assessed dcr a supplementary analysis of the primary iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0ctable baseline patient characteristicscharacteristicstable continuedoverall n characteristicsoverall n open access median range   continued intolerant wild type mutant ras mutational status braf mutational status wild type mutant unknownmsi status mss unkown there is an overlapping this number includes patients with the ras wild type and patient with mutant rasecog eastern cooperative oncology group egfr epidermal growth factor receptor msi microsatellite instability mss microsatellite stableendpoint and orr with cis using the same method we estimated the median time and month and year probability of os and pfs with the kaplan meier method the cis for the median time were calculated using brookmeyer and crowley method the cis of month and year survival probabilities were calculated based on the greenwood™s formula hrs and cis were estimated by cox regression we did subgroup analyses divided by prespecified baseline patient and disease characteristic variables including ras status for dcr pfs and os we also did a prespecified exploratory analysis of potential predictive biomarkers in blood samples we did all analyses with sas v94resultspatient characteristicsbetween october and january patients with unresectable metastatic colorectal cancer were enrolled all patients were eligible and received the study medication table summarises the baseline characteristics of all enrolled patients the median number of previous lines of palliative chemotherapy was range “ and patients had received or ‰¥ prior lines of chemotherapy for metastatic disease respectively the data cut off date was january with median follow up of months iqr “efficacythe centrally assessed dcr was ci to one sided p00001 two patients had a partial response and had a stable disease including unconfirmed pr table figure a total of patients had a reduction in target lesion size from baseline figure time on treatment for all patients is ‰¥ ‰¥ male female months ‰¥ months right sided colon left sided colorectum lung liver lymph node peritoneumage years sex ecog performance status primary site number of metastatic site metastatic an time from start of first line chemotherapy number of previous palliative chemotherapy previous chemotherapy and reason for discontinuation fluoropyrimidine refractory intolerantoxaliplatin irinotecan tas102 trifluridinetipiracil angiogenesis inhibitor anti egfr inhibitor refractory intolerant refractory intolerant refractory refractory intolerant refractory intolerantiwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0copen accesstable best response to treatmentcomplete responsepartial responsestable diseaseprogressive diseasenot evaluabledisease control rate ciresponse rate cicentral assessmentn30 to to investigator assessmentn30 to to shown in online supplementary figures and events for pfs were recorded in all patients and median pfs was months ci to figure all deaths were recorded median os was months ci to with a month and year os of ci to and ci to figure safetypatients received the study treatment for four cycles at median range “ the median relative dose intensity was iqr “ dose interruptions and reductions were required in and patients respectively the major treatment related adverse events ‰¥ for dose reduction were proteinuria patients palmar plantar erythrodysesthesia patients diarrhoea patients hypertension patients fatigue patients and thrombocytopenia patients the reasons for treatment discontinuation of all patients were disease progression in patients and adverse events in patients gastrointestinal perforation and grade proteinuria in of each after treatment with lenvatinib patients received a subsequent treatment online supplementary table most patients only had mild grades “ adverse events table the most common grade ‰¥ adverse events were hypertension patients thrombocytopenia patients increased alanine aminotransferase and anorexia patients each no clear relationship was found between the incidence of lenvatinib associated adverse event of any grade and baseline body surface area online supplementary table serious adverse events occurred in four patients including figure waterfall plot analysis of maximum percentage change from baseline in measurable target lesions response evaluation criteria in solid tumors version central reviewiwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0copen accessfigure kaplan meier curves of a progression free survival pfs by investigator assessment and b overall survival os in all patients n30five treatment associated events anorexia in two and gastrointestinal perforation central venous catheter related bloodstream infection caused by staphylococcus aureus and nausea in each one in each of four patients all patients recovered from these adverse eventssubgroup analysisin patients with wild type ras the median pfs was months ci to and that was months ci to in patients with mutant ras online supplementary figure in patients with wild type ras the median os was months ci ci to and months ci to in patients with mutant ras online supplementary figure plasma angiopoietin2 levels were decreased by lenvatinib treatment in almost all patients and increased at the iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776time of treatment discontinuation online supplementary table with a first quartile cut off point17 the eight patients with a first quartile or lower level of angiopoietin2 had a median os of months ci to months compared with months ci to in the patients with higher than a first quartile level of angiopoietin2 hr ci to online supplementary figure patients with a first quartile or less level of angiopoietin2 had a median pfs of months ci to compared with months ci to in the patients with more than a first quartile level of angiopoietin2 hr ci to online supplementary figure 0copen accesstable treatment related adverse events occurring in ‰¥ patients n30any gradegrade ‰¥treatment related adverse eventhypertensionproteinuriathrombocytopeniafatiguehypothyroidismweight losshoarsenesspalmar plantar erythrodysesthesia syndromeanorexiadiarrhoeamucositis oralserum ast increasedserum creatinine increasedast aspartate transaminase discussionpatients with metastatic colorectal cancer with disease progression after three or more lines of therapy have limited treatment options in this open label single arm phase study of patients with previously treated metastatic colorectal cancer lenvatinib demonstrated manageable toxic effects and promising antitumour activity a total of out of patients had disease control including with partial responses moreover patients experienced reduction in measurable tumour size the overall toxicity profiles were similar to that reported for lenvatinib across a spectrum of advanced malignant neoplasmstwo recent international phase studies reported that regorafenib or trifluridinetipiracil provided significant improvements in dcr pfs and os compared with placebo in patients with metastatic colorectal cancer after failure of standard chemotherapies dcr median pfs months median os months in the correct study and dcr median pfs months median os months in the recourse study6 interestingly the present single arm phase study of lenvatinib revealed favourable dcr and median pfs values in patients with metastatic colorectal cancer compared with those in the regorafenib or trifluridinetipiracil study moreover about half of the patients received post study treatment which led to a favourable osthe lenvatinib safety profile in this study was similar to the published safety profiles of lenvatinib for thyroid cancer and hepatocellular carcinoma in the japanese population18 moreover we found no unexpected or off target safety signals the most common adverse events were hypertension proteinuria thrombocytopenia and fatigue while the most case of grade or hypertension and proteinuria required treatment interruption and dose reduction while the target population for thyroid cancer or hepatocellular carcinoma that showed efficacy for lenvatinib was first line setting20 this study targeted patients receiving salvage line therapy most patients with metastatic colorectal cancer in the salvage line setting had grade or proteinuria and hypertension at baseline because of the long term prior treatment with anti vegfvegfr treatment whereas the occurrence of grade hypertension was significantly higher compared with that of regorafenib in a similar study population in the correct concur and consign trials7 it was manageable by dose reduction or interruption but it may be necessary to consider the starting dose in the future although palmar plantar erythrodysesthesia is a not life threatening toxicity these adverse events have a significant impact on treatment schedules and quality of life in treated patients grade ‰¥ palmar plantar erythrodysesthesia has been observed in and of patients treated with lenvatinib in this study and the select japanese population15 respectively while in patients treated with regorafenib in the correct japanese population16 to date the clear mechanism of palmar plantar erythrodysesthesia by vegf receptor tyrosine kinase inhibitors is not known but it has been reproduced that palmar plantar erythrodysesthesia by lenvatinib is well tolerated overall it is suggested that lenvatinib might be a favourable treatment option in terms of toxicitiesseveral preclinical studies demonstrated that vegf targeted treatment affects immune suppression by promoting the expansion of suppressive immune cell populations such as regulatory t cells and myeloid derived suppressor cells24 several clinical studies suggested that modulation of vegf mediated immune suppression via angiogenesis inhibition could potentially augment the immunotherapeutic activity of anti programmed cell death pd1 antibody26 regorafenib and nivolumab showed antitumour activity in patients with metastatic colorectal cancer including those with microsatellite stable tumours in a phase study28angiopoietin2 a relatively novel regulator of angiogenesis that acts through the tek tyrosine kinase endothelial tie2 receptor has been identified as a potential prognostic biomarker for some types of cancer although the baseline ang2 level was a predictive biomarker in patients with thyroid cancer in the select trial17 it did not become a reliable biomarker of lenvatinib response in this study prior treatment with anti vegfvegfr antibodies probably had an effect on baseline angiopoietin2 levels because the study population was refractory to standard treatment in this study the decrease in angiopoietin2 levels was observed after treatment therefore it may be an indicator of treatment responsethe limitations of our study include its small size which could limit the interpretation of the subgroup analyses iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0cand the absence of a comparison group however the level of clinical benefit in the form of confirmed responses observed in this study was remarkable in the historical context of other clinical trials done in heavily pretreated patients with metastatic colorectal cancer moreover most of the patients in our study had left sided tumours which were known to have a better prognosis compared with right sided tumoursin lenvatinib provided promising activity with prolonged survival relative to the anticipated median pfs in heavily pretreated patients with metastatic colorectal cancer the safety profile of lenvatinib was similar to that in other tumour types with no new safety signals recorded based on these findings further investigation of lenvatinib with anti pd1 antibody or other novel combinations with the potential to build on the benefit of lenvatinib is currently taking place nct03797326 and nct04008797acknowledgements the authors thank the patients and their families the members of the clinical research support office for their support with data collection and running the study and nai incorporated for editing a draft of this manuscriptcontributors all authors conceived and designed the study and drafted and revised the manuscript for publication si no hs yh at kk th nb and yy collected data ak go mk and kn analysed the data and managed data and study progress all authors interpreted the data and approved the final version of the manuscriptfunding the study was supported by the project promoting clinical trials for development of new drugs and medical devices japan medical association from the japan agency for medical research and development grant number jp18lk0201037 and by eisai cocompeting interests si has received research grants from eisai and merck biopharma th has received research grants from eisai and honoraria from merck serono yy has received honoraria from eisaipatient consent for publication not requiredethics approval the study was conducted in accordance with the declaration of helsinki and good clinical practice guidelines the study protocol was approved by the national cancer center institutional review board t4329provenance and peer review not commissioned externally peer revieweddata availability statement data are available upon reasonable request proposals should be directed to siwasa ncc go jp the data will be available for achieving aims in the approved proposalopen access this is an open access article distributed in accordance with the creative commons attribution non commercial cc by nc license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited any changes made are indicated and the use is non commercial see a0http creativecommons licenses by nc orcid idssatoru a0iwasa http orcid yasuhide a0yamada http orcid references saltz lb clarke s díaz rubio e et a0al bevacizumab in combination with oxaliplatin based chemotherapy as first line therapy in metastatic colorectal cancer a randomized phase iii study j clin oncol “ tabernero j yoshino t cohn al et a0al ramucirumab versus placebo in combination with second line folfiri in patients with metastatic colorectal carcinoma that progressed during or after first line therapy with bevacizumab oxaliplatin and a fluoropyrimidine raise a randomised double blind multicentre phase study lancet oncol “open access van cutsem e tabernero j lakomy r et a0al addition of aflibercept to fluorouracil leucovorin and irinotecan improves survival in a phase iii randomized trial in patients with metastatic colorectal cancer previously treated with an oxaliplatin based regimen j clin oncol “ yamazaki k nagase m tamagawa h et a0al randomized phase iii study of bevacizumab plus folfiri and bevacizumab plus mfolfox6 as first line treatment for patients with metastatic colorectal cancer wjog4407g ann oncol “ price tj peeters m kim tw et a0al panitumumab versus cetuximab in patients with chemotherapy refractory wild type kras exon metastatic colorectal cancer aspecct a randomised multicentre open label non inferiority phase study lancet oncol “ mayer rj van cutsem e falcone a et a0al randomized trial of tas102 for refractory metastatic colorectal cancer n engl j med “ grothey a van cutsem e sobrero a et a0al regorafenib monotherapy for previously treated metastatic colorectal cancer correct an international multicentre randomised placebo controlled phase trial lancet “ matsui j yamamoto y funahashi y et a0al e7080 a novel inhibitor that targets multiple kinases has potent antitumor activities against stem cell factor producing human small cell lung cancer h146 based on angiogenesis inhibition int j cancer “ matsui j funahashi y uenaka t et a0al multi kinase inhibitor e7080 suppresses lymph node and lung metastases of human mammary breast tumor mda mb231 via inhibition of vascular endothelial growth factor receptor vegf r and vegf r3 kinase clin cancer res “ wiegering a korb d thalheimer a et a0al e7080 lenvatinib a multi targeted tyrosine kinase inhibitor demonstrates antitumor activities against colorectal cancer xenografts neoplasia “ hong ds kurzrock r wheler jj et a0al phase i dose escalation study of the multikinase inhibitor lenvatinib in patients with advanced solid tumors and in an expanded cohort of patients with melanoma clin cancer res “ boss ds glen h beijnen jh et a0al a phase i study of e7080 a multitargeted tyrosine kinase inhibitor in patients with advanced solid tumours br j cancer “ yamada k yamamoto n yamada y et a0al phase i dose escalation study and biomarker analysis of e7080 in patients with advanced solid tumors clin cancer res “ nakamichi s nokihara h yamamoto n et a0al a phase study of lenvatinib multiple receptor tyrosine kinase inhibitor in japanese patients with advanced solid tumors cancer chemother pharmacol “ kiyota n schlumberger m muro k et a0al subgroup analysis of japanese patients in a phase study of lenvatinib in radioiodine refractory differentiated thyroid cancer cancer sci “ yoshino t komatsu y yamada y et a0al randomized phase iii trial of regorafenib in metastatic colorectal cancer analysis of the correct japanese and non japanese subpopulations invest new drugs “ tahara m schlumberger m elisei r et a0al exploratory analysis of biomarkers associated with clinical outcomes from the study of lenvatinib in differentiated cancer of the thyroid eur j cancer “ takahashi s kiyota n yamazaki t et a0al a phase ii study of the safety and efficacy of a0lenvatinib in patients with advanced thyroid a0cancer future oncol “ yamashita t kudo m ikeda k et a0al reflect a phase trial comparing efficacy and safety of lenvatinib to sorafenib for the treatment of unresectable hepatocellular carcinoma an analysis of japanese subset j gastroenterol “ kudo m finn rs qin s et a0al lenvatinib versus sorafenib in first line treatment of patients with unresectable hepatocellular carcinoma a randomised phase non inferiority trial lancet “ schlumberger m tahara m wirth lj et a0al lenvatinib versus placebo in radioiodine refractory thyroid cancer n engl j med “ li j qin s xu r et a0al regorafenib plus best supportive care versus placebo plus best supportive care in asian patients with previously treated metastatic colorectal cancer concur a randomised double blind placebo controlled phase trial lancet oncol “ van cutsem e martinelli e cascinu s et a0al regorafenib for patients with metastatic colorectal cancer who progressed after standard therapy results of the large single arm open label phase iiib consign study oncologist “iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0copen access ott pa hodi fs buchbinder ei inhibition of immune checkpoints and vascular endothelial growth factor as combination therapy for metastatic melanoma an overview of rationale preclinical evidence and initial clinical data front oncol kato y tabata k kimura t et a0al lenvatinib plus anti pd1 antibody combination treatment activates cd8 t cells through reduction of tumor associated macrophage and activation of the interferon pathway plos one 201914e0212513 taylor mh lee c h makker v et a0al phase ibii trial of lenvatinib plus pembrolizumab in patients with advanced renal cell carcinoma endometrial cancer and other selected advanced solid tumors j clin oncol “ makker v rasco d vogelzang nj et a0al lenvatinib plus pembrolizumab in patients with advanced endometrial cancer an interim analysis of a multicentre open label single arm phase trial lancet oncol “ fukuoka s hara h takahashi n et a0al regorafenib plus nivolumab in patients with advanced gastric gc or colorectal cancer crc an open label dose finding and dose expansion phase 1b trial regonivo epoc1603 jco iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0c'
Colon_Cancer
"gut microbiota composition influences the balance between human health and disease increasing evidencesuggests the involvement of microbial factors in regulating cancer development progression and therapeuticresponse distinct microbial species have been implicated in modulating gut environment and architecture thataffects cancer therapy outcomes while some microbial species offer enhanced cancer therapy response othersdiminish cancer treatment efficacy in addition use of antibiotics often to minimize infection risks in cancer causesintestinal dysbiosis and proves detrimental in this review we discuss the role of gut microbiota in cancerdevelopment and therapy we also provide insights into future strategies to manipulate the microbiome and gutepithelial barrier to augment therapeutic responses while minimizing toxicity or infection riskskeywords intestinal dysbiosis cancer development cancer therapy microbial therapy human intestinal microbiota is essentialfor microbialhomeostasis regulation of metabolism and immune tolerance intestinal dysbiosis occurs when there are alteredratios of healthy microbial flora along with changes in theirdiversity and density such changes may lower mucus layerthickness reduce antimicrobial defense and disrupt theepithelial tightjunction barriers to allow increased translocation of intestinal bacteria and bacterial products intothe systemic circulation and trigger inflammation andimmune responses circulating bacterial products such asendotoxin genotoxin and trimethylamine oxide have beenimplicated in many human disorders including metabolicsyndrome cardiovascular complications atherosclerosisand thrombosis and various neoplastic conditions intestinal dysbiosis may also affect adaptive immunity by correspondence seahhlimyahoocom1division of hematology and oncology suny downstate health sciencesuniversity clarkson avenue room b5495 brooklyn new york usa2division of hematology and oncology department of medicine new yorkmedical college valhalla new york usamodulating the functions of t lymphocytes and promotingtumor immune escapewhile increased translocation of intestinal luminal content is associated with carcinogenesis and poor therapeuticresponse the causeeffect relationship is often bidirectionalin this review we will discuss the role of gut microbes inmodulating tumor immunity intestinal permeability andcancer development next we will highlight the effects ofintestinal dysbiosis and increased permeability in cancertherapy finally we will explore the options to improve guthealth to enhance the efficacy of cancer therapyintestinal immunity and permeabilitythe intestinal architecture and microbiota regulateinnate and adaptive immunity disruption of the architecture andor microbiota affects these functions therelationships between the different players in the intestinal microenvironment is summarized in fig the composition of microbes in the gut dictatesmucus layer thickness and production of antimicrobialsignals in germfree mice mucus layer and effector t the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cdutta and lim biomarker research page of fig interplay between different factors involved in gut immunity and permeability a the intestinal epithelial cells containing paneth cellsgoblet cells enterocytes and enteroendocrine cells coordinate with intraepithelial lymphocytes to generate a functional immune responsepaneth cells secrete antimicrobial peptides and goblet cells produce mucus to cover the epithelial layer this mucus layer prevents adhesion ofmicrobes to the epithelial cells lamina propria situated under the mucus layer contains peyer™s patches and immune cells including antigenpresenting cells apcs like dendritic cells dcs t cells and b cells pattern recognition receptors prrs such as tolllike receptors tlrs onepithelial cells interact with microbederived pathogenassociatedmolecular patterns pamps such as lipopolysaccharide lps to activatemyd88dependent signaling dcs travel to mesenteric lymph nodes mln and promote the differentiation of na¯ve t cells to regulatory t tregcells that migrate to other sites treg cells secrete il10 to elicit an antiinflammatory response b dysbiosis decreases mucus layer thickness andshortchain fatty acid scfas production this affects the secretion of antimicrobial peptides and allows microbes to come in close proximity tothe epithelial cells reduction in scfas influences gut barrier dysfunction as a result the gut luminal content also translocated and spreadedthrough the systemic circulation to trigger local and systemic immune responses in addition to pamps damps released from damaged intestinalepithelium interact with prrs to facilitate expression of macrophages and maturation of dcs mature dcs promote the differentiation of na¯ve tcells to effector t cells such as t helper cells th1 th2 th17 th1 release tnfα and ifnÎ and th17 secrete il17 to recruit polymorphonuclearneutrophils pmns these cytokines create a proinflammatory condition 0cdutta and lim biomarker research page of cells are absent [ ] microbes secrete shortchain fattyacids scfas such as propionate and butyrate thatprevent microbial binding to the epithelial cells and helpmaintain barrier function and immune homeostasisbutyrate promotes tightjunction formation [ ] andactivates peroxisome proliferatoractivated receptor gammapparÎto enhance epithelial oxygen consumptionresulting in reduced emanation of oxygen from the mucosalsurface it helps in maintaining an anaerobic condition inthe gut lumen needed for colonization of obligate anaerobes this intestinal microenvironment determines thecomposition of resident bacterial species for example onlyclostridium lactobacillus and enterococcus are enrichedon the epithelial surface and in the mucus layer whereasbacteroides bifidobacterium streptococcus enterobacteriaceae enterococcus clostridium and lactobacillus are allpredominant in the intestinal lumen dysbiosis increases inflammatory signals that shiftthe metabolism of enterocytes epithelial hypoxia iseliminated and increased oxygenation results in therelease of more oxygen from the mucosal surfacesince only facultative anaerobes can respire oxygendysbiosisinduced shift in epithelial oxygenation altersgut microbial community from obligate to facultativeanaerobes intestinal pathogens such as proteobacteria produce genotoxins like colibactin and cytolethaldistending toxin cdt to induce inflammation andhost deoxyribonucleic acid dna damage that initiates tumor formation dysbiosis also decreasesmucus layer thickness reduces scfa production anddamages mucosal barrier allowing pathogenassociatedmolecular patterns pamps to interact with pattern recognition receptors prrs and activate tolllike receptortlr 24myeloid differentiation primary response protein myd88 signaling pathways in addition changes inmicrobial composition and density triggers epithelial releaseof damageassociated molecular patterns damps such asextracellular adenosine triphosphate atp cytoplasmiccalreticulin high mobility group box hmgb1 proteinsendogenous nucleic acids and intracellular proteins tointeract with prrs prr engagementtriggers a proinflammatory condition that causes tissue damage and localinflammation microbiotadriven tlr immune signalinghas been implicated in cancer formation and modificationof treatment efficacy [“] for example cpg oligodeoxynucleotides that mimic bacterial dna acts as a pamp totrigger a tlr9dependent tlr4 activation and tumor necrosis factor tnfα production by tumorinfiltratingmyeloidderived cells mice bearing el4 lymphomamc38 colon carcinoma and b16 melanoma when treatedwith cpg oligodeoxynucleotides show reduced tumrowth and enhanced survival rate the beneficial effects ofcpg oligodeoxynucleotides were positively associated withthe abundance of alistipes shaii in the gut effects of intestinal microbiota on cancerdevelopmentintestinal microbes can influence local and distantcarcinogenesis through infection and microbial productsor by modulating tumor immunosurveillance this isaccomplished via altering the balance between the rateof cell proliferation and apoptosis triggering chronicinflammation andor immunosuppression or changingthe metabolism of the products produced by host andmicrobes in this section we will discuss how intestinaldysbiosisrelated permeability may contribute to tumorigenesis in different anscolorectal cancerfusobacterium nucleatum a gramnegative mucosaadherent anaerobic bacteria has been implicated in theinitiation and progression of colorectal cancer crc[ ] fada an adhesion molecule on f nucleatumbinds to host ecadherin to enter epithelial cells this activates the wntβcatenin pathway leading toan increased secretion of inflammatory cytokines including il6 il8 and tnfα and upregulation of nuclearfactor kappa light chain enhancer of activated b cellsnfκb that facilitates crc development in addition itattracts myeloidderived suppressor cells and the autotransporter protein fap2 interacts with the human inhibitoryreceptor t cellimmunoreceptor with ig and itimdomains tigit to create a tumor immunosuppressivemicroenvironment f nucleatum may also induce chemoresistance by modulating the tlr4myd88 signalingpathway following 5fluoruracil treatment in crc patients an increased abundance of f nucleatum along with clostridium difficile and species ofstreptococcus campylobacter and leptotrichia has beendemonstrated in tumor tissue and fecal materials [“]f nucleatummediated colorectal carcinogenicity occursdownstream of apc introduction of f nucleatum resultedin rapid onset of colonic tumors in mice deficient in onecopy of adenoma polyposis coli apc apcmin gene both intestinal dysbiosis and loss of apc disruptepithelial tightjunctions and mucus layer [ ] andallow increased infiltration of f nucleatum and other nonresidential microbes to drive crc development the roleof defective gut barrier in crc has been confirmed inmucin 2knockout muc2ˆ’ˆ’ mice in which the lack ofgastrointestinal mucin resulted in spontaneous crc development therefore dysbiosisinduced gut permeabilitymay play an important role in tissue enrichment of fnucleatum and increased risks for crchepatobiliary cancerthe liver is chronically exposed to intestinal microbiotaand its products via the portal vein intestinal dysbiosisand increased permeability enhance translocation of gut 0cdutta and lim biomarker research page of microbiota to trigger inflammation and chronic liver disease that predisposes patients to the development of hepatocellular cancer alteration in bile acid metabolism due tochanges in clostridium spp suppress anticancer immunity in mice eradication of grampositive bacteria by oralvancomycin inhibits secondary bile acid conversion resulting in the upregulation of chemokine cxc motif ligandcxcl16 in liver sinusoidal endothelial cells cxcr16recruits natural killer t nkt cells in the tumor microenvironment and kill tumor cells in a cd1ddependentmanner in addition gut microbiotaderived lipopolysaccharides lps promote tumor progression in liver cancerby activating the tlr4 signaling in a study involving cholangiocarcinoma patients bile ducttissues haddistinct dominance of dietziaceae pseudomonadaceae andoxalobacteraceae members pancreatic cancergut microbiota influences the development of pancreaticcancer through activating tlr4 signaling the stromain pancreatic tumor harbors an abundance of microbiotaespecially bifidobacterium pseudolongum compared tonormal pancreas this helps in creating an immunosuppressive environment by differentially activating distincttlrs in monocytes pancreatic adenocarcinoma has anenrichment of proteobacteria synergistetes and euryarchaeota longer survival is observed in patients with amore diverse intratumor microbial composition primarilyof sachharopolyspora pseudoxanthomonas streptomycesand bacillus clausii tumoral colonization with mycoplasma hyorhinis and gammaproteobacteria is associatedwith gemcitabine resistance antibiotics diminishmyeloidderived suppressor cells and increase antitumorm1 macrophages to promote th1 differentiation of cd4t cells and cd8 t cell activation in the tumor cotreatment of gemcitabine with ciprofloxacin abrogatedgammaproteobacteriainduced chemotherapy resistance the efficacy of immune checkpoint inhibitors icistherapy is also enhanced by antibiotics lung cancerwhile local microbiota is important there are reportsthat gut microbiome may also contribute to lung cancerdevelopment lung cancer patients demonstrated an abundance in intestinal enterococcus and depletion in bifidobacterium and actinobacteria they are also enrichedwith veillonella bacteroides and fusobacterium depletedof dialister enterobacter escherichiashigella fecalibacterium and kluyvera in nonsmall celllung cancernsclc patients butyrate producers such as faecalibacterium prausnitzii clostridium leptum clostridial cluster iruminococcus spp clostridial cluster xiva and roseburiaspp were significantly reduced since butyrate isessential for preserving mucosal homeostasis reduction ofintestinal butyrate producers may imply a compromised intestinal barrier in these patientshematologic malignanciesdysbiosisinduced intestinal permeability affects mucosaassociated lymphoid tissue malt and plays a significantrole in hematologic malignancies composition of intestinalmicrobiota is responsible for maintaining the pool of bonemarrow myeloid cells preleukemic myeloproliferationis driven by microbial signals in teneleven translocation2tet2deficient mice [ ] these mice show increasedinfiltration of inflammatory cells disrupted mucosal barrierand increased translocation of bacteria [ ] it wassuggested that dysfunction of small intestinal barrier andleakage of microbes can occur due to tet2 mutation in occurrence of tet2hematopoietic compartmentmutation intestinal dysbiosis and leaky gut is common inleukemia and lymphomaacute myeloid leukemia aml and acute lymphoblasticleukemia all patients have a compromised intestinalbarrier [“] fecal microbiota in all patients showedlower microbial diversity they were enriched in enterococcaceae porphyromonadaceae and bacteroidetes mainlyb fragilis and depleted in blautia erysipelotrichiales lachnospiraceae and clostridiales members [ ] abundanceof staphylococcaceae and streptococcaceae have also beenreported in pediatric all and adult aml [ ]helicobacter pylori is associated to malt lymphoma and chamydophila psittacito ocular maltlymphoma while borrelia burgdorferi was linked tocutaneous bcell nonhodgkin lymphoma two studiesdid not find significant risk of borrelia burgdorferi in thedevelopment of nonhodgkin lymphoma [ ] abundance of proteobacteria is a predictor for neutropenicfever and enrichments of enterococcaceae and streptococcaceae are strong predictors of infectious complications inall similarly higher gut microbiota diversity inmultiple myeloma is associated with reduced risk fordisease relapse all patients with infectious complications have an abundance of brevundimonas diminuta andagrobacterium tumefaciens whereas faecalibacteriumprausnitzii producer of scfas is completely absent similar findings have been reported in nonhodgkinlymphoma with infectious complications effects of intestinal microbiota on cancer therapythe efficacy of cancer treatment is in parts dependenton normal immune function since gut microbiota playsa crucial role in modulating immune response it is notsurprising that dysbiosis affects treatment outcomesprophylactic antibiotics are commonly used for cancerpatients undergoingallogeneichematopoietic stem cell transplantation allohsct toreduce the risk of neutropeniaassociated infectionchemotherapyand 0cdutta and lim biomarker research page of however antibiotic use causes intestinal dysbiosis thatresults in negative outcomes including poor treatmentresponse and toxicity and the development of clostridium difficile infection cdi in addition to antibioticsopioid analgesics for cancer pain management may alsotrigger dysbiosis opioid analgesics impair intestinal motility and promote bacterial overgrowth resulting in dysbiosisand gut permeability intestinal dysbiosis induces mucosal injury and triggers the release of damps damps have a dual andbidirectional effect on cancer although damps exertimmunosurveillance and immunemediated cell death toeliminate tumor cells and protect against cancer development chronic inflammation induced by damps maypromote tumor initiation damps released by apoptoticcells from cancer therapy may also induce chemoresistance and promote metastasis for example tlr78expressed on tumor cells may bind damps loxoribinefor tlr7 and poly u for tlr8 and promote chemoresistance through the activation of nfκb and the upregulation of bcl2 damps may also activate tlr9on human breast prostate and lung cancer cells to trigger tumor invasion and metastasis [ ] given theclinical significance of dysbiosismediated mucosal injuryand permeability in cancer we will in this section discusshow the treatment outcome by various cancer therapymay be affected by intestinal microflora and permeabilitychemotherapy and radiation therapyintestinal microbial composition and mucosal barrier function influence chemotherapeutic outcome and the effect isbidirectional while dysbiosis can exacerbate chemotherapy drug toxicity and reduce its efficacy chemotherapy canitself cause dysbiosis although prevalence of certain intestinal microbes in the gastrointestinal tract offer beneficialeffects others contribute to chemoresistance and drug toxicity this multiplepathway effect is best covered by timer mechanisms “ translocation of microbes immunomodulation metabolism and enzymatic effects on drugsand reduced microbial diversity these mechanistic effectsalter chemotherapy efficacy and toxicity and risks forinfections for example translocation of microbes due tochemotherapy induceddysbiosis and disruption of mucosal barrier can increase the risk of infection howevercertain chemotherapy drugs such as cyclophosphamideand doxorubicin damage intestinal barrier for the translocation of commensal bacteria into secondary lymphnodes to elicit antitumor immune response vancomycin prophylaxis inhibits antitumor effects of cyclophosphamide in fibrosarcoma inoculated mice irinotecanused for crc treatment is transformed into its active formsn38 by tissue carboxylesterase it is detoxified in theliver by host udpglucuronosyltransferases into inactiveglucuronide sn38g and excreted into the gut via bileducts in the gut bacterial βglucuronidases reconvertssn38g into active sn38 which causes severe intestinaltoxicity and diarrhea streptomycin inhibits irinotecanabsorption and reduces epithelial carboxylesterase activityand diarrhea ciprofloxacin inhibit βglucuronidases and low dose amoxapine βglucuronidases inhibitorsuppress irinotecanassociated diarrhea in rats table provides a selection of chemotherapeutic agents affectingand affected by intestinal microbial composition andpermeabilitylocal pelvic irradiation damages intestinal epitheliumand barrier integrity and produce reactive oxygen speciesirradiation increase alistipes and decrease prevotella inmice in gynecologic cancer patients receiving pelvicradiotherapy firmicutes and fusobacterium were significantly decreased in addition to reduced diversitysignificant enrichment of clostridium iv roseburia andphascolarctobacterium was associated with radiationenteropathy in pelvic cancer patients the effects oftotal body irradiation which is a preparative regimen forallohsct that causes dysbiosis and gastrointestinaltoxicity is discussed in more details in the allohsctsection belowimmunotherapycancer cells often create an immunosuppressive microenvironment to mediate tumor immune escape this immune escape mechanism may be reversed by icis directedat cytotoxic t lymphocyteassociated antigen ctla4programmed death receptor pd1 or pd1 ligandspdl1 since intestinal microbes influence local and systemic antitumor immune reaction by modulating prrspamps and dampsintestinal dysbiosis may impacttreatment outcome figure illustrates how the potentialmechanisms ofthe antitumor immune responses aredownregulated by intestinal dysbiosis the effects of intestinal microbiome on responses to icis have been discussed previously [ ] broadspectrum antibioticsbefore during or after icis therapy alter intestinal microbiome and resulted in lower tumor response rate inferiorprogressionfree survival and reduced overall survival responses to inhibition of ctla4 by ipilimumab inmouse models of mca205 sarcoma ret melanomaand mc38 colon carcinoma were inferior in germfreeor in broadspectrum antibiotic treated mice poorresponses were associated with decrease in intestinalbacteroides thetaiotaomicron bacteroides uniformis andburkholderia cepacia and increase in clostridiales suchdysbiosis was also associated with mucosal damage andcolitis oral feeding with either bacteroides thetaiotaomicron or bacteroides fragilis individually or with acombination of bacteroides fragilis and burkholderiacepacia restored the antitumor effects of ctla4 blockade through augmentation of th1 responses in tumor 0cdutta and lim biomarker research page of table selection of chemotherapeutic agents and the bidirectional effects between the chemotherapy and intestinal microbiotachemotherapy drugcisplatintoxicity infectioncdi ototoxicity effects on gut changes in microbiotadamages mucosal barrier by impairing dnareplication of rapidly proliferating epithelialcells facilitates translocation of gut bacteriacommensal gut bacteria influencesgenotoxicity by inducing reactive oxygenspecies ros production and recruitment ofpathogenic th17 cells in the tumormicroenvironment independently ofimmunity elicited by immunogenic celldeath microbial interventionantibiotics against grampositive bacteriaabrogate antitumor chemotoxicityincrease tumor size and decrease survivalratecisplatin alone show better responsecompared to a combined treatment ofcisplatin and antibiotics in mice with lungcancer the combination treatmentincreased tumor size and decreasedsurvival ratelactobacillus acidophilus restores antitumorefficacy following antibiotic treatment[ ]restoration of gut microbiota andepithelial integrity by fmt andtreatment with dmethionine [ ]prevent infections and ototoxicity withoutaffecting tumor chemotoxicityfmt increases a muciniphilaabundance and reduces cipn oral butyrate supplementation improvesgut barrier by reducing inflammation andmucositis antibiotics reduce mucositis and cytokineproduction but also diminish antitumorefficacy and promote chemotherapyresistance antibiotics against grampositive bacteriareduce th17 responses and subsequentdevelopment of cyclophosphamideresistancereestablishment of e hirae alone restoresantitumor activity e hirae decreases tumorinfiltrating tregsbarnesiella intestinihominis accumulates inthe colon and increases the number ofintratumoral ifnÎproducing Îδt cellse hirae and b intestinihominissynergistically stimulate local and systemicimmunity to improve anticancer effects nod1ˆ’ˆ’nod2ˆ’ˆ’ mice having abundant bintestinihominis demonstrate increased Îδtcells in tumor beds and enhancedcyclophosphamide efficacy paclitaxel5fluoruracilincreases gut permeability as indicated by5fold elevation in circulating lpsbindingprotein and systemic inflammation reduces abundance of roseburiaporphyromonadaceae and akkermanisamuciniphila [ ]reduces clostridium spp and increasesmembers of proteobacteria mainlyenterobacteriaceae damages mucosal barrierchemotherapyinducedperipheral neuropathicpain cipn cdi [ ]mucositis along the entiregastrointestinal tract cdi [ ]cyclophosphamidecdi triggers disruption of gut barrier by alteringbacterial compositiongrampositive bacteria such asenterococcus hirae lactobacillus johnsoniiand l murinus translocate from gut intomesenteric lymph nodes and spleen this enhances immune responses by theproduction of interferon gamma ifnÎ andactivation of th17 cellsdraining lymph nodes and promotion of maturation oftheintratumoral dendritic cells dcscombination treatment of bacteroidesandburkholderia cepacia prevented intestinal damage andrefractory colitisin additionfragilisfecal microbiota analysis of melanoma patients beforeand after ipilimumab treatment showed a change in therelative proportions ofenterotypeclusters cluster a was dominated by prevotella spwhereas clusters b and c by different bacteroides sppfecal microbiota transplantation fmt from patientsinto tumorbearing germfree mice showed that onlyfecal material from cluster c resulted in colonizationthree dominantwith bacteroides thetaiotaomicron or bacteroides fragilisand enhanced ipilimumab response in another study ofipilimumab in mice vancomycin treatment resulted in amore severe manifestation of colitis whereas oraladministration of bifidobacterium ameliorated the sideeffects similarly melanoma patients with increasedabundance of bacteroidaceae rikenellaceae and barnesiellaceae members responded better to ctla4 antibodies however a different study in ipilimumabtreated melanoma patients found that bacteroides spp were associatedwith decreased response whereas faecalibacterium andother firmicutes members improved clinical outcome 0cdutta and lim biomarker research page of fig potential antitumor immune mechanisms induced by intestinal dysbiosis a in the presence of intact mucosal barrier and signals fromcommensal microbiota effector t cell activation is modulated by t cell receptor tcr ligation with major histocompatibility complex mhc classi and costimulation of cd80cd86 and cd28 binding of cytotoxic t lymphocyteassociated antigen ctla4 receptor to antictla4 antibodyon treg impairs its effector tcell inhibitory function it also downregulates ctla4 expression on apc ligation of repressive receptorprogrammed death receptor pd1 and its ligand pdl1 to antipd1 and antipdl1 antibodies respectively activate effector tcell proliferationand function activated effector t cells interact with tumor cells and release cytokines to induce tumor cell death b signals from unfavorablemicrobes due to dysbiosis upregulates ctla4 pd1 and pdl1 expression to inhibit tcell activation ctla4 on treg binds to cd80cd86 onantigen presenting cell apc cd80cd86 on apc also disengages from cd28 and binds to ctla4 on effector t cells pdl1 the ligand of pd1is expressed on antigen presenting cell apc and tumor cells pd1 on effector t cells ligates to pdl1 on apc and tumor cells these activitiesinhibit effector tcell activation reduces immune checkpoint inhibitor ici efficacy and causes tumor escape 0cdutta and lim biomarker research page of patients with higher abundance of faecalibacteriumand improved response to ctla4 antibodies showedhigher incidence of enterocolitis and lower level of treg inperipheral blood thus the beneficial effects of specificand isolated gut microbes may depend on the commensalassociation with other microbial species and may differ between humans and micepd1 blockade may also be modulated by intestinalmicrobiota melanoma patients who responded to pd1blockade had increased abundance of enterococcus faeciumcollinsella aerofaciens bifidobacterium adolescentis klebsiella pneumoniae veillonella parvula parabacteroidesmerdae lactobacillus sp and bifidobacterium longumwhereas in nonresponders the intestinal microbiome wasenriched in ruminococcus obeum and roseburia intestinalis another study found higher abundance of faecalibacterium species in responders and enrichment with bacteroides thetaiotaomicron escherichia coli and anaerotruncuscolihominis in nonresponders clinically nonsmallcell lung cancer nsclc and renal cell carcinoma rccpatients experienced increased resistance to pd1 blockadeafter antibiotic treatment these patients had shorterprogressionfree survival as well as overall survival in thisstudy response to pd1 blockade correlated with higherfecal abundance of akkermansia muciniphila fmt fromresponders to germfree or antibiotictreated mice improved the outcome of pd1 blockade administration ofa muciniphila after fmt from nonresponders restoredresponsesimilarly intestinal microbiota may influence the outcome of chimeric antigen receptor t cell car t therapypatients with complete response to cd19 car ttherapyexhibited enrichment of oscillospiraceae ruminococcacaeae and lachnospiraceae in their intestinal microbiomewhereas patients who did not attain a complete responseshowed increased abundance of peptostreptococcaceae effectiveallogenic hematopoietic stem cell transplantationalthough allohsct issomein treatinghematological malignancies the immunosuppressive agentsbroadspectrum antibiotics and chemoradiation used withthe transplant often induce intestinal dysbiosis gut permeability and impaired systemic immune response highermicrobiota diversity is associated with longterm survivaland lower diversity in gut microflora is associated with reduced overall survival and higher transplantrelated mortalityfollowing allohsct [ ] severe infections that occurdue to intestinal dysbiosis such as cdi and vancomycinresistant enterococci vre infections are also associatedwith higher treatmentrelated mortality [“] allohsctdisrupts the equilibrium of bacterial composition in feceswith a dominance of enterococcus streptococcus and proteobacteria members [ ] and reduces beneficial bacteriasuch as faecalibacterium and ruminococcus higherabundance of blautia was found to be associated with improved overall survival moreover allohsct patientswith reduced risk of relapse had an enrichment of eubacterium limosum ofgraftversushost diseaseone of the major complications of allohsct is thedevelopmentgvhdoccurrence of cdi during allohsct increases the riskof gvhd besides the loss of overall microbial diversityreduction in beneficial faecalibacterium blautia lactobacillus and ruminococcus and increased abundance ofenterococcus and clostridiales was observed in gvhd[ “] patients without gvhd had increasedabundance of parabacteroides and bacteroides in theirpretransplant feces in a preclinical study reducedgvhd and improved overall survival was observed afterthe administration of the probiotics lactobacillus rhamnosus gg alone or in combination with ciprofloxacindue to the preservation of gut mucosal integrity in therecipient mice restoration of normalintestinalmicrobiome by fmt has been found to benefit patientswith steroidrefractory gvhd [ ] multipleclinical trials are currently ongoing to investigate howmanipulation of gut microbiota using dietary intervention and fmt might reduce the risk of gvhdmanipulation of intestinal microbiome and barrierto improve outcome of cancer therapeuticsifintestinal dysbiosis and its associated increased gutpermeability are associated with cancer development andtherapyrelated complications and treatment outcomes itfollows that intervention of the intestinal microbiomeandor gut barrier may alter cancer outcome in thissection we will explore three broad approaches fig that might be investigated nonselective modificationof intestinal microbiome using fmt semiselectivemodification of intestinal microbiome using antibioticsand biologic modification of intestinal barrier we willdiscuss the challenges and obstacles each of the approaches may encounternonselective modification of intestinal microbiome usingfmtmodification of the intestinal microbiome is theoreticallybest accomplished by fmt unmanipulated fmt will notonly replete the dysbiotic intesti
Colon_Cancer
"although the clinical development of immune checkpoint inhibitors icis therapy has ushered in a new era of antitumor therapy with sustained responses and significant survival advantages observed in multiple tumors mostpatients do not benefit therefore more and more attention has been paid to the identification and developmentof predictive biomarkers for the response of icis and more indepth and comprehensive understanding has beencontinuously explored in recent years predictive markers of icis efficacy have been gradually explored from theexpression of intermolecular interactions within tumor cells to the expression of various molecules and cells intumor microenvironment and been extended to the exploration of circulating and host systemic markers with thedevelopment of highthroughput sequencing and microarray technology a variety of biomarker strategies havebeen deeply explored and gradually achieved the process from the identification of single marker to thedevelopment of multifactorial synergistic predictive markers comprehensive predictivemodels developed byintegrating different types of data based on different components of tumorhost interactions is the direction offuture research and will have a profound impact in the field of precision immunooncology in this review wedeeply analyze the exploration course and research progress of predictive biomarkers as an adjunctive tool totumor immunotherapy in effectively identifying the efficacy of icis and discuss their future directions in achievingprecision immunooncologykeywords neoplasm immune checkpoint inhibitor predictive biomarker tumor mutation burden programmeddeath ligand1 immune checkpoint inhibitors icis therapy has usheredin a new era of antitumor therapy with sustained responses and significant survival advantages observed inmultiple tumors antiprogrammed cell death1programmed cell deathligand pd1pdl1 antibody hasbeen approved for secondline or firstline treatment in avariety of malignant neoplasms including melanoma lungcancer renal cell carcinoma rcc head and neck squamous cell carcinoma hnscc and gastroesophageal correspondence cuijwjlueducncancer center the first hospital of jilin university xinmin streetchangchun jilin chinacancer [ ] however despite the breakthrough in clinical treatment with icis most patients do not benefitpembrolizumab or nivolumab has an objective responserate orr of “ in firstline melanoma and insecondline nonsmall cell lung cancer nsclc [“]therefore in recent years more and more attentions havebeen paid to the identification and development of predictive biomarkers for the efficacy of icis and more indepth and comprehensive understanding has also beenobtained in recent yearsincluding new data on biomarkers of tumor genome and neoantigen tumor immune microenvironmentbiopsybiomarkers hostrelated factors and all of which havephenotypeliquid the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cbai biomarker research page of technologyimmunohistochemicalmade many new advances in the corresponding fieldswith the development and continuous improvement ofmultiplexhighthroughput sequencing and microarray technology a variety of biomarker strategies have emerged and graduallyrealized the process from the identification of singlemarker to the development of multifactorial synergisticpredictive markers the development of predictive biomarkers contributes to revealing the therapeutic mechanisms of icis and the interaction mechanisms betweentumor and host immunity achieving decisionmaking ofindividualized antitumorimmunotherapy monitoringefficacy and disease development guiding clinical trial design as well as for further understanding of drug resistance mechanisms and tumor prognosis in this review wedeeply analyze the exploration course and research progress of predictive biomarkers as an adjunctive tool totumor immunotherapy in effectively identifying the efficacy of icis it should be pointed out here that when reading and collating we try to read and include all therelevant s in the process of selecting s we include the authoritative s published in highlevel s or the latest research results and objectively describe and analyze their roles in this field as well as discuss the reasons that different research results may beinvolvedadvances of multiple predictive biomarkers toicis efficacyi tumor genome and neoantigen biomarkerstumor mutation burdensignificant correlations between high tumor mutationburden tmb and response to icis have been reported inseveral cancer types including urothelial carcinoma small cell lung cancer sclc nsclc [“]melanoma and human papilloma virus hpvnegative hnscc a metaanalysis of cancer typesshowed that the mean response rate was positively correlated with log tmb the national comprehensivecancer network nccn guidelines have adopted tmbas the recommended test for patients with nsclc receiving immunotherapy although the results in some clinicalstudies of rcc hpvpositive hnscc and melanoma receiving antipd1 after recurrence showedthat tmb alone also did not clearly distinguish respondersand predict os it is still exciting that multiple studies inthe american society of clinical oncology ascomeeting have confirmed the predictive value of tmb inimmunization or combination therapy keynote061study [ ] condor study eagle study epoc1704 study etc consolidating its status oftmb as an independent predictor and in april theus food and drug administration fda prioritized theapproval of tmb as a companion diagnostic biomarkerfor pembrolizumabnonetheless the cutoff values of tmb were defineddifferently across studies and assay platforms such asatezolizumab mtmb in urothelial cancer pembrolizumab mtmb in nsclc and atezolizumab‰¥ ‰¥ or ‰¥ mtmb in nsclc [“] andnivolumab plus ipilimumab ‰¥ mtmb in nsclc which needs further study to confirm the optimalcutoff value in different tumors moreover the ngspanels have approved by the fda that can be used to estimate tmb include the mskimpact and foundationone cdx panel the detection results of which arehighly consistent with whole exome sequencing wes[ ] and other solutions are under development astudy detecting tmb cutoff value at mtmb in nsclc patients with the foundationone platformcontaining a gene panel found that compared withtmbl patients overall survival os and dcr was significantly improved in tmbh patients treated withantipd1l1 drug both wes and targeted ngs a422cancergene panel performed in patients withnsclc treated with antipd1l1 demonstrated thattmbh population has a significantly better durableclinical benefitdcb and progressionfree survivalpfs these findings demonstrate the feasibility ofcomprehensive genomic profiling cgp but the designof optimal next generation sequencing ngs panel thatis more accurate comprehensive and costeffective isstill not clear in addition given that btmb was identified as a predictor of pfs but failed to differentiate patients with os benefits researchers consider the need toexplore other more precise factors eg allele frequencyaf a study that developed a new btmb algorithm intwo independent cohorts poplar and oak showedthat modified btmb low af btmb lafbtmb mutation counts with an af was significantly associated with favorable hr 95ci “ p pfs hr 95ci “ p andorr p after immunotherapy but required tobe prospectively validated finally static biomarkersare insufficient to accurately predict response due to thecomplexity of tumorimmune interactions a recent analysis of tumor genomewide dynamic detection in pretreatment and ontreatment melanomasfound thatpretreatment tmb was only associated with os in untreated patients while early 4week ontreatment changein tmb δtmb was strongly associated with antipd1response and os in the entire cohort the detectionof δtmb is helpful for early evaluating the response totherapy of patient but its clinical usability limited by thedifficulty in obtaining tissue samples and high price whileliquid biopsy discussed below might better 0cbai biomarker research page of in addition epigenetic changes are associated withtmb the latest study investigated the association between tmb and dna methylation dnam to explorepotential complimentary biomarkers for nsclc immunotherapies the results showed that high tmbnsclcs had more dnam aberrance and copy numbervariations cnvs showing certain value in predictingefficacy such as hox gene methylation status and tmb thus the correlated exploration of epigenetics hasattracted more attention in recent years and liquidbiopsybased epigenetic studies may become a future research direction exploration in chinese nsclc patientsshowed that nsclcs with high tmb had dnam aberrance and cnvs some insertion and deletion indelmutations can lead to frameshifts and more immunogenic neoantigens in the pancancer analysis of cancer types evaluated in the cancer genome atlastcga rcc had the highest indel mutation load andframeshift indel mutations were found to produce threetimes more candidate neoantigens per mutation thannonsynonymousnssnvs somatic copy number alterations scnas are another feature of the genomic landscape of tumors andpancancer tcga analysis revealed an inverse correlation between scnas atthe singlearm or wholechromosomelevel and immune infiltration in tumortypes tested and this result was subsequently replicated in a larger study of tcga single nucleotide variantsdna damage response pathwaysgenetic variation involved in dna mismatch repairmmr pathway can lead to microsatellite instabilitymsi a specific type of high tmb tumors and increased numbers of cd8 tumor infiltrating lymphocytestils pd1tils and indoleamine 23dioxygenaseido tumor cells have been shown in mmr deficiencydmmr colorectal cancer recently five clinical trials keynote016 including multipletumor types have shown that patients with dmmrmsih can achieve durable responses to pembrolizmabbased on this pembrolizumab is approved by the usfda for the treatment of any advanced solid tumor withdmmrmsih and nivolumab in combination with ipilimumab has also shown promising response in dmmrmsih colorectal cancer in addition dmmr canalso cause mutations in the dna polymerase gene epsilondelta polepold1increasing the mutationload and neoantigen load analysis of polepold1mutations in patients with different cancer typesshowed that patients with these mutations had significantly higher tmb and os therefore it may be an infordependentinidentifying patients who benefitaddition pathways of base excision repairberand prognostic markerfrom icis risk factorhomologous recombination repair hrr mmr in thedna damage response ddr signaling network contribute more significantly to tmb or neoantigens whichhave the highest levels when comutated it hadbeen identified that comutations in the ddr pathwaysof hrr and mmr or hrr and ber defined as comutare associated with increased levels of tmb neoantigenload and immune gene expression signatures comutpatients showed a higher orr and longer pfs or os indicating that comut can be used as predictors of response to icis and provide a potentially convenientmethod for future clinical practice specific mutated gene pathways in tumor cellsit is worth noting that alterations of signaling pathwaysin tumor cells affect the responsiveness to immunotherapy patients with mutations in the interferon ifnγpathway genes ifngr12 jak12 and irf1 are poorlyresponsive to icis treatment and confer resistance a study found that in patients receiving immunotherapytumor cells can downregulate or alter ifnγ signalingpathways such as lossoffunction alleles of genes encoding for jak12 and changes in stat1 to escape the influence of ifnγ resulting in poor efficacy andresistance recent studies suggest that inactivating mutations in a mammalian analog of the chromatin remodeling swisnf complex and unique genes of the pbafcomplex pbrm1 arid2 and brd7 lead to sensitivitiesto icis [ ] loss of function of the pbaf complexincreased chromatin accessibility to transcription regulator elements of ifnγ“inducible genes within tumorcells and subsequently increased production of cxcl9cxcl10 chemokines leading to more efficient recruitment of effector t cells into tumors in human cancers expression of arid2 and pbrm1 are related toexpression of t cell cytotoxicity genes which confirmedin pbrm1deficient murine melanomas with strongly infiltrated by cytotoxic t cells and responsive to immunotherapy [ ]in addition doublestranded rnadsrna editing enzyme adenosine deaminase acting onrna adar1 protein can block the ifnγ signalingpathway and lead to poor icis efficacy and resistanceloss of function of adar1 in tumor cells can reduce atoi editing of interferoninducible rna species and leadto dsrna ligand sensing by pkr and melanomadifferentiationassociated protein mda5 this resultsin growth inhibition and tumor inflammation respectively and profoundly sensitizes tumors to immunotherapy finally demethylation positively regulates thetranscriptional activity of some immunerelated genesincluding pdl1 and ifn signaling pathway genes sensitizingto anticytotoxic tlymphocyteassociatedprotein4 ctla4 therapy it 0cbai biomarker research page of in addition to the ifnγrelated signaling pathway alterations in other tumor genome such as tumor oncogenes and suppressor genes pathways and pathwaysrelated to tumor cell proliferation and infiltration canalso affect immunotherapy efficacy epidermal growthfactor receptor egfr and anaplastic lymphoma kinasealk mutations have been shown to be associated withreduced response rates to icis and low tmb and therefore the fda does not recommend firstline icistreatment in patients with egrf or alk positive tumors[ ] certain types of mutations in mdm2mdm4and arid1a can predict nonresponse to icis in hightmb tumors nsclc with kras and stk11 comutated was associated with reduced response andshorter survival in three independent cohorts of patientstreated with antipd1 therapy and stk11 deficiency was an independent indicator of poor antipd1response in nsclc with kras mutant however at the american association for cancer research aacrmeeting of patients in the keynote042 studynct02220894 update data were tested for stk11 andkeap1 and the results showed that patients could benefit from pembrolizumab regardless of stk11 and keap1status but patients with stk11 mutations did not respond well to chemotherapy but given that only ofall patients had mutation detection the results may beaffected in initial data from studies using targeted ngspanels suggested that duration of icistreatment was associated with certain braf and m terations butnot tmb status notch signaling pathway is associated with the occurrence development and prognosisof tumors especially with the biological function of cancer stem cells recent breakthrough findings have distinguished deleterious notch mutation showing that itcan be used as a potential predictor of favorable ici response in nsclc potentially via greater transcription ofgenes related to dna damage response and immune activation another tumorspecific inheritance thatmay influence icis efficacy is the aberrant expression ofendogenous retroviruses ervs pancancer analysisidentified a positive correlation of transcript expressionof ervs with tcell activity in various tumors andpatient prognosis furthermore with the improvement of precision detection technology the accurateanalysis of negative mutation sites helps to identify thepossibly effective ones for example the analysis of studydata of secondline pd1l1 inhibitor therapy found thatthe mpfs of patients with kras g12c or g12v was significantly better than that of patients with kras mutations at other sites in addition several pancancer biomarkers are recentlyapproved by the fda for example given the effectiveorr of and a disease control rate dcr of in secondline cholangiocarcinoma patients treated withanalysispemigatinib a new targeted therapy the recent fda approval of pemigatinib for the treatment of previouslytreated patients with locally advanced or metastatic cholangiocarcinoma with fibroblast growth factor receptor fgfr2 fusion or rearrangement and the comprehensive genomicassay foundationone cdxdeveloped by foundation medicine as a companiondiagnostic also exciting is the recent fda approval ofthe targeted anticancer drug capmatinib for the treatment of metastatic nsclc with met exon skippingmetex14 mutations including firstline patients andpreviously treated patients also using foundationonecdx as a companion diagnostic to help detect specificmutations present in tumor tissueimmunogenicity ofneoantigen loadneoantigen load the number of mutations actually targeted by t cells may be directly related to the responseto icis [“] a retrospective study showed thatclonal neoantigen burden was associated with the longeros in primary lung adenocarcinomas p traditionallycomputational neoantigen predictionshave focused on major histocompatibility complexmhc binding of peptides based on anchor residueidentities however neoantigen loads identified by thismethod are generally not superior to overall tmb inpredicting icis efficacy or survival in recent practice this neoantigen can be assessed by the difference inpredicted mhci binding affinity between the wildtypepeptide and the corresponding mutant peptide knownas the differential agretopicity index dai reflectingclinically relevanttumor peptide a high dai value indicates that the mutant peptidesignificantly increases binding affinity to mhc compared to the wildtype sequence and can generate moreimmune responses studies on previously published cohorts treated with three icis have shown that dai outperforms tmb and the traditionally defined neoantigenload in predicting survival [ ] in additionlowneoantigen intratumour heterogeneity might also be important for icis response analysis of the lung adenocarcinoma tcga database found that combining highmutational load and low intratumoral neoantigen heterogeneity was significantly associated with osand longer lasting clinical benefit than either variablealone anotherreported method for assessingneoantigen foreignness is based on sequence homologyof experimentally validated immunogenic microbial epitopes in the immune epitope database iedb butit does not account for all possible human leukocyteantigen hla contexts in addition the detection forneoantigen can be reflected from different levels such aspeptides or genomes a study developed the neopepseealgorithm using a machine learning approach incorporating 0cbai biomarker research page of integration of nine immunogenicity features and gene mutation expression levels and its application to melanoma and leukemia patients could improve the sensitivityand specificity of neoantigen prediction recently it has alsobeen shown that promoter hypermethylation of neoantigengenes may be an important mechanism for immune editingand tumor immune evasion indicating that combineddetection of tumor genome and epigenetics may providemore information for immunotherapy efficacyii tumor immune microenvironment phenotypebiomarkerscells is also considered separately as one of the biomarkersto distinguish the benefit population called immune positive score ips herbst showed that response toatezolizumab treatment was significantly associated withhigh levels of pdl1 expression on the surface of tils before treatment but not with pdl1 expression on tumorcells p finally other inhibitory immune pathways may affect the response to icis therapy including tcelllymphocyte activationgene3 lag3 and vdomain ig suppressor of tcell activation vista which can be used as potential biomarkers for icis responseimmunoglobulin3 tim3pdl1 expressiongiven that multiple studies in a variety of tumors havedemonstrated a positive correlation between pdl1 expression and response to icis or os even in firstlinecombination therapy [“] pembrolizumab is currently approved by the fda for use in patients with pdl1 pdl1 ‰¥ of tumor cells in firstline treatmentand ‰¥ in secondline treatment nsclc and pdl1immunohistochemistry ihc as a companion diagnosticfor antipd1 therapy in nsclc patients [ ] however some studies have not detected a significant correlation between pdl1 expression and response to icis[ ] and pdl1 negative patients can still benefitclinically with treatment with ici or combination treatment with icis with orrs ranging from to therefore pdl1 cannot yet be a comprehensive and independent biomarker in clinical practice in assessing efficacy with following challenges still existing firstlypdl1 assay and antibody are not standardized secondly pdl1 expression is temporally and spatiallyheterogeneous a study of metastatic nsclctreated with icis showed that pdl1 varies substantiallyacross different anatomic sites and during clinicalcourse being highest in adrenal liver and lymph nodemetastases and lower in bone and brain metastases andthe predictive value of pdl1 at different biopsy sites forthe benefit of icis in nsclc may vary higher pdl1 inlung or distant metastasis specimens was significantly associated with higher response rate pfs and os whilepdl1 in lymph node metastasis biopsy was not associated with either response or survival thirdly positive score and cutoff value of pdl1 expression is notstandardized at present pdl1 positive scoremainly focuses on the pdl1 expression level of tumorcells that is tumor proportion score tps but pdl1is also expressed on immune cells such as lymphocytesand macrophages and stromal cells thus the investigators introduce the concept of combined positive scorecps which is the proportion score of the sum of pdl1 expressed by tumor cells and tumorassociated immune cells in addition pdl1 expression on immuneresponseto icisimmunetreatmentbiomarkers of tumorinfiltrating immune cellsoverall immune status of tumor microenvironmentthe pattern of tumor immune infiltration can be broadlyclassified into immuneinflamed immuneexcluded andimmunedesert immuneinflamed is characterizedby the presence of cd8 and cd4 t cells in the tumorparenchyma accompanied by the expression of immunecheckpoint molecules indicating a potential antitumor immuneexcluded is characterized by the presence ofdifferent immune cell types in the aggressive margin orstroma of tumor but cannot infiltration into tumor parenchyma [ ] analysis of pretreatment samples forantipd1pdl1 revealed a relatively high abundance ofcd8t cells at the invasive margin in responders andserial sampling during treatment showed an increasedinfiltration of cd8t cells into tumor parenchyma while immunedesert phenotype is characterized by theabsence of abundant t cells in the parenchyma orstroma of tumors and poor response to icitreatment recentlyimmunoscore has been proposed as avalid marker for characterizing the immune status oftumor microenvironment tme classifying tumors aswell as predicting treatment response and prognosis which involves the density of two lymphocyte populations cd8 and memory [cd45ro] t cells in thecenter and invading margin of tumor mlecnik evaluated immunoscore in specimens of stagei“iv colorectal tumor and confirmed that it was significantly associated with pfs dfs and os and multivariate analysis also showed the superiority of immunoscorein predicting disease recurrence and survival the valueof immunoscore to predicting icis efficacy is being validated internationally in clinical trials of melanoma andnsclc a wider assessment of active immune responses withintme by immune gene expression profiling might effectively predict clinical benefit to icis strategies analysisof total rna and genes that were substantially differentbetween the patient groups in pretreatment tumor biopsies revealed atleast a 25fold increase in the 0cbai biomarker research page of expression of immunerelated genes in clinically active patientsincluding cytotoxic t cell markers egcd8a perforin granzyme b th1 cytokines or chemokines mhcii and other immunerelated genes egnkg7 ido1 ascierto screened morethan immunerelated genes in patients with recurrent breast cancer “ years after treatment and thosewithout recurrence more than years later and foundthat five genes igk gbp1 stat1 igll5 and oclnwere highly overexpressed in patients with recurrencefree survival in addition ifnγinduced immune genesignatures may be effective biomarkers for predicting theclinical benefit of treatment with icis the study developed ifnγ scores combining multiple immune variablesbased on gene signatures which were then extendedto gene signatures in a validation set of melanomapatients including genes encoding ifnγ granzymes ab perforin ido1 and other immunerelated genesboth gene scores showed significant associations withbest overall response rate and pfs optimized cutoffvalues for ifnγ scores based on receiver operatingcharacteristic curve roc curve can achieve a positivepredictive value of for responders and a negativepredictive value of for nonresponders immune cells with specific phenotypes in tmethe phenotype of tils also influences the efficacy oficis the study used singlecell mrna sequencingscrnaseq data analysis to identify two major cd8tcell phenotypes within melanoma memorylike andexhausted the proportion of which is strongly correlated with response to icis the research furtherfound that the transcription factor tcf7 is selectivelyexpressed in memorylike t cells so the ratio ofcd8tcf7 to cd8tcf7tils is strongly correlatedwith improved response and survival in melanoma patients treated with antipd1 balatoni found that of immune cells in tme were positivelyassociated with os after treatment including cd4 andcd8 t cells foxp3 t cells cd20 b cells cd134and cd137 cells and nkp46 cells and different immune cells at different sites were differently associatedwith clinical outcomes researchers found that only asmall proportion of cd8 tilsin tumors couldrecognize tumor mutationassociated antigens while another population bystander cells was insensitive anddifferential cd39 expression was the key molecule thatdistinguished the two populations analysis of peripheral blood from a patient with colorectal cancer whoresponded rapidly to pembrolizumab treatment showedhigh expression of cd39 on cd8 tils indicating thatcd39cd8til may be a promising predictive biomarker the fact of very low level of cd39 expression on cd8tils in of egfrmutant nsclc isconsistent with their low response rate to antipd1immunotherapyin addition a study showed that fc domain glycan ofthe drug and fcγ receptor fcγr expressed by the hostbone marrow cells could determine the ability of pd1tumorassociated macrophages tams to capture antipd1 drugs from the surface of t cells which leads topd1 inhibitor resistance and the association oftams and poor antipd1 response was reported inmelanoma cohorts antipd1 response was associated with an increase in cd8t cells and natural killercells nk cells and a decrease in macrophages andhigh intratumoral myeloid markers were associated witha nearly 6fold decrease in mpfs after antipdl1 therapy in rcc emphasizing the inhibitory role of myeloidcells in response to icis in conclusion immunecells in tme show a great promise in the developmentof predictive biomarkers for icisimmunerepertoirediversity of immune repertoires in tmeeffective t cell responses involve the activation and expansion of specific antigenreactive t cell clones so diversity ofin intratumoral orperipheral may correlate with icis responses and can bequantified as richness and clonality however theresults seem to be complex with some studies finding apositive correlation between til clonality and the response to icis before or after treatment whileothers showing that only an increase in til clonalityduring treatment is associated with the response to antipd1 [ ] others show that intratumoral t cellclonality is not associated with survival while peripheralt cell clonality is inversely associated with pfs and os tumeh further investigated whetherbaseline tils have a narrow t cell receptor tcr repertoire focusing on tumorspecific immune responsesand whether this narrow tcr repertoire correlates withpembrolizumab responses they found that respondingpatient had more restricted usage of the tcr beta chainie a more clonal less diverse population than patientswith progressive disease and showed a 10times increasein these clones after treatmentimplying a tumorspecific response to treatment in these patients notablybaseline tcr clonality was not highly correlated withtil density suggesting that some patients with restricted tcr clonality specific for tumor antigens maystill benefit from antipd1 therapy even though tildensity is low recently researchers have proposed theimmune repertoire irindex the average frequency ofshared tcr clones in t clones in tils and peripheralpd1cd8 t cells they found that neoantigenstimulated tcr agreed with irindex and patients withhigh irindex had better immune activation and highergene expression profiles geps score subsequently they 0cbai biomarker research page of confirmed the predictive value of irindex to icis efficacy dcrpfs but considering that it is difficult tosort out pd1cd8 t cells in tumor tissue based ontwo separate patient cohorts a research confirmed thattcr repertoire diversity and clonality of peripheral pd1cd8t cells may serve as noninvasive predictors ofclinical outcomes after icis in patients with nsclc the viewpoints of t cell diversity and tcr clonality as markers of icis efficacy need to be further validated in a large patient populationiiiliquid biopsy biomarkersperipheral blood cell biomarkersperipheral blood is a noninvasive source to explore potential biomarkers for icis and although associationswith clinical benefit and survival have been observed itseffectiveness has not been validated in prospective studies analysis of melanoma treated with ipilimumabshowed that improved os and pfs were associated withbaseline values of peripheral blood components including low absolute neutrophil countlow neutrophiltolymphocyte ratio nlr low absolute monocyte countlow frequency of myelogenous suppressor cells high frequency of foxp3 treg cells high lymphocyte frequencyhigh eosinophil count and clinical benefit also associated with the dynamic changes of blood markers duringincluding decreased foxp3treg concentratreatmenttions and increased lymphocyte and eosinophil counts reports in patients with melanoma treated withpembrolizumab and in patients with nsclc treatedwith nivolumab have shown that nlr is associated withworse tumor response [ ] multivariate analysis inmelanoma patients treated with antipd1 antibodiesshowed that nlr was the only factor associated withworse orr and shorter pfs indicating that nlr is astrong predictor of worse outcome in patients treatedwith ici low baseline lactate dehydrogenase ldhlevels high relativeabsolute eosinophil counts and relative lymphocyte counts were associated with prolongedos in antipd1 and ctla4 treated melanoma given that previous studies have proposed the importance of baseline derived nlr dnlr and ldhlevels as prognostic markers a recent study proposed acomposite prognostic index that comprehensively takesthe two factors into account lung immune prognosticindex lipi which characterized risk groups goodintermediate and poor the analysis of patients with advanced nsclc in randomized trialss
Colon_Cancer
methods results and discussion sections yz drafted the methods and results sections of the initial manuscript mvk assisted in manuscript revisions and proof reading all authors provided feedback and insights into the manuscript srt jkv msa yz mvk and sat edited and revised the manuscript and all authors read edited and approved the final version of the manuscriptcompeting interests the authors declare no competing interestsadditional informationsupplementary information is available for this paper at 101038s4159 xcorrespondence and requests for materials should be addressed to jkvreprints and permissions information is available at wwwnaturecomreprintspublisher™s note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsopen access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the ™s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the ™s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreat iveco mmons licen sesby40 the authors scientific reports 101038s4159802071102xvol0123456789wwwnaturecomscientificreports 0c'
Colon_Cancer
" while the impact of family caregiving has been welldocumented many of such studies center oninvestigating external factors such as socioeconomic status accessibility to resources and availability of socialsupport as the primary causation of caregiver wellbeing outcomes this paper explores the motivations that drivefamily caregivers in supporting their family members at the endoflife and critically examines how internalappraisal processes of such motivations can both positively and negatively impact their wellbeingmethods this study adopted an interpretative phenomenological analysis ipa to investigate the motivations andinternal appraisal processes of asian family caregivers in singapore who were tending to a dying family memberqualitative dyadic interview data n was drawn from a larger randomized controlled trial for a novel familydignity intervention fdi for palliative care patients and their families the sampling population consisted ofparticipants aged and above who were identified to be the primary caregivers of older palliative care patientswith a prognosis of less than months data collection was conducted in the homes of patients and familycaregiverscontinued on next page correspondence andyhyhontuedusg1psychology programme school of social sciences nanyang technologicaluniversity singapore singapore2centre for population health sciences lee kong chian school of medicinenanyang technological university singapore singaporefull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ctanho bmc palliative care page of continued from previous pageresults findings revealed six themes that could either nurture or diminish caregiver wellbeing honoringfidelity caregivers were motivated to commit to their caregiving roles in order to avoid regret alleviatingsuffering caregivers were motivated to relieve their family member™s pain enduring attachment caregiverswere motivated to spend time together with their family member preserving gratitude caregivers weremotivated to express their appreciation to their family member by caregiving navigating change caregiverswere motivated to adapt accordingly to changes in the illness trajectory and reconciling with mortalitycaregivers were motivated to respond accordingly to their family member™s prognosis the final theme of thewellbeing determinant is posited as an indication of selfdetermination and is conjectured to influence howcaregiving motivations are appraised by the caregiver fulfilling and enhancing one™s sense of selfdetermination appears central to infusing one™s caregivingmotivations with positive meaning and consequently nurturing one™s wellbeing in the endoflife caregivingjourney these findings are discussed with recommendations for healthcare professionals working with familycaregivers of palliative care patientskeywords palliative care caregiver motivations wellbeing meaning burnout resilience selfdetermination endoflife qualitative research the experience of an endoflife eol family caregivercan be likened to a paradox “ what could evoke a senseof pleasure appreciation and gratitude could also bringabout feelings of anxiety distress and pain while somehave related the caregiving journey to the metaphor ofascending a mountain the expedition of an eol family caregiver usually spans beyond merely a couple ofdays weeks or months they must navigate the peaks ofdiagnosis to prognosis and eventually death and bereavement in what often unfolds into a lifelong climbthe role of the eol family caregiver is often multifaceted and interminable daily duties involve managingmedical regimes traversing the healthcare system andtaking charge of other dependents alongside providingphysical mental and emotional support throughout theillness trajectory [ ] many family caregivers are rarelyequipped with formal or adequate training and nor dothey possess sufficient resources and skills before theyfind themselves embroiled in eol caregiving responsibilities family caregivers must also process and manage amultitude of thoughts and emotions as they come toterms with the changes and sometimes losses in theirpersonal lives this complex experience is not limited to a minorityof people despite strong global advancements in medical technology and healthcare systems older populations remain highly susceptible to chronic and terminalmorbidities that are incapacitating in the unitedstates alone over million caregivers tend to their ailing family members annually while an estimated of patients in europe requiring longterm care areattended to by informal caregivers with the anticipated number of older adults in the world soaring to billion by the year there will certainly be a surging demand for family caregivers to relieve the ensuingresource strains on healthcare settingsthe impact of family caregiving stressors has beenwelldocumented [“] with various studies exploringcaregiver burnout and its effects on the community andsociety complementing these studies are literature thatreveal characteristics of resilience and transformationalgrowth displayed by family caregivers in adversity [“] the common thread that impacts both caregiverburnout and resilience appears to be a lack of or adequate coping a process that requires the individual toconstantly change efforts in both thoughts and behaviorsin order to manage internal or external demands thatare considered stressful despite this indication thatone™s psychological resources are key to maintainingone™s wellbeing many studies often consider externalfactors such as socioeconomic status accessibility toresources and availability of social support as the primary causation for the degree of caregiver wellbeingthus these studies often recommend pragmatic interventions that focus on improving external circumstancesaccordinglyperception emotion and motivation of the familycaregiverwhile it is undoubtedly beneficial to mitigate tangiblestressors one must not lose sight of the magnitude of aperson™sthedemands of caregiving this perception and appraisalembody one™s subjective caregiver burden “ observed inreviews of over caregiver studies to pose deepseatedimplications on caregivers™ quality oflevels ofdepression and anxiety and stress coping [ ]internal perception and appraisal oflife 0ctanho bmc palliative care page of campbell later confirmed this observation in astudy that evaluated multiple variations in the caregiverexperience subjective caregiver burden was repeatedlyidentified as the primary indicator for caregiver stressit was around the same time that folkman andmoskowitz discovered that caregivers experiencepositive emotions alongside negative emotions duringstressful events they put forth the tenet of meaningfocused coping in which caregivers derive mental andemotional sustenance thus effecting positive emotionsin challenging circumstances by deferring to their beliefsvalues and existential goals folkman and moskowitzfound that meaningfocused coping is an intrapsychicprocess of discovering benefits in caregiving reminding oneself of such benefits setting goals thatinspire a sense of mastery and competence realigningpriorities in view of changes and infusing ordinaryevents with positive meaning folkman furtherattested that meaningfocused coping exists alongsidenegative appraisals in a caregiver™s stress process inorderand psychologicalresources during stressful eventsto reinstate physiologicalgiven the importance of psychological appraisal it isreasonable to postulate that gaining insight into a caregiver™s beliefs values and goals that stimulate them incaregiving defined as motivations in this paper wouldyield valuable information that could be used to enhancemeaningfocused coping such interventions would target the essence of a person™s selfconcept “ that is theindividual™s perception of who they are and what theybelieve in “ and transcend current cursory social mentaland emotionalfor caregiverstresssymptom management[ ] a studybridging the research gap in asian caregivingwhile the multidimensional nature of caregiving burdenand coping is not confined to any specific culture thereare distinctive elements that bear influence on the asiancaregiving experience family takes precedence in asiansocieties with strongly inculcated values and expectations of filial piety and filial responsibility placed uponfamily membersconducted insingapore a multiethnic society that is predominantlychinese found that family caregivers who internalizedand prioritized societal expectations as motivations overtheir personal wellbeing most often faced internalconflict and were highly likely to experience difficultiesin maintaining their mental health familial ties and caregiving duties this is corroborated by the evolvingattitudes towards such confucian rules “ younger asiangenerations no longer perceive absolute submission orcomplete obedience to the family as instrumental valuesin a modern and globalized society and it would bevaluable to further understand how these complexitiesmight manifest in a family caregiver™s motivationsthis paper aims to contribute to and grow the currentbody of knowledge for asian eol family caregivers byanswering the following research questions what arethe internalized motivations defined here as unconone™ssciously assimilated beliefs and valuesattitudes or behaviors of asian family caregivers how might these motivations affect the way they respond to caregiving and impact their wellbeing howcan an understanding of these motivations be integratedinto psychosocial interventions to enhance and sustaincaregiver wellbeingintomethodsresearch design and proceduresthe current study draws qualitative dyadic interviewdata n from a larger randomized controlled trialfor a novel family dignity intervention fdi for asianpalliative care patients and their families n thesampling methods inclusion criteria interview procedure and study protocol for the fdi are comprehensivelydescribed by ho briefly the fdi is developedbased on an integrative body of empirical investigationthat focuses on dignified endoflife care in both western and asian contexts [ ] it integrates elements oflogotherapy and narrative life review to provide psychosociospiritual support to patients and families facingmortality and has been piloted for acceptability andfeasibility before being fully adapted into the intervention study in practice fdi comprises a recorded dyadicsemistructured interview with a patient and a familycaregiver conducted in their homes the fdi therapistuses a guided question framework to facilitate joint conversation on shared memories and living wisdoms thatlead to meaningmaking and the expression of appreciation and reconciliation this is done with the ultimategoal of creating a legacy document that tells the life storyof the patient and is bestowed to the rest of the familythrough an open reading each interview lasted between and min and was conducted in english malaymandarin or a chinese dialect hokkien teochew orcantonese these recorded interviews were transcribedverbatim translated into english by a native languagespeaker where applicable and edited into legacy documents transcripts and legacy documents were reviewedand finalized by patients and caregivers to ensure accuracy and authenticitysamplingthe sample drawn for this study consisted of primaryfamily caregivers of older palliative care patients aged and above with mainly a cancer prognosis of lessthan months eleven were spousal caregivers seven 0ctanho bmc palliative care page of were adultchildren caregivers and two were siblingcaregivers the majority were female aged between to with a mean age of years see table for caregiver demographics they were recruited through theinpatient daycare and homecare hospice service unitsof hca hospice care dover park hospice tan tockseng hospital singapore cancer society and methodistwelfare services the inclusion criteria required familycaregivers to be above years old and identified by thepatient to be their primary carer patients and familycaregivers came from varioussocioeconomic s and were predominantly of chinese ethnicityas the fdi focused on patient narratives transcriptsbearing a moderate to sizeable amount of input from thefamily caregiver were selected for data analysisdata analysisstudy adopted an interpretative phenomenothislogical analysis ipato investigate the internalizedmotivations of family caregivers in tending to a dyingfamily member the ipa is an approach in qualitativeresearch that aims to provide insights into how an individual makes meaning out of a phenomenon itis important to note that as the current study drawsqualitative data from the larger fdi study no explicitinternalized caregiver motivationsquestionsaboutwere asked reflections on motivations towards caregiving occurred anically throughout the interviewtranscripts and were identified by the researcher usingipa through a process of data reduction and datareconstructionfirst authors and screened all transcripts andselected those that had adequate inputfrom familycaregivers to be used for analysis authors and thenconducted linebyline coding to develop descriptivethemes and analytical categories conceptualizing newinterpretation of the data this was followed by regularmeetings among all authors for the further refinement ofthemes and categories to encapsulate the meaning andcontent within the cluster of similar codes with theemergent themes and subthemes created via a summarychart all authors reviewed and defined the emergentthemes once consensus was reached operational definitions were created finally relationships between categoriesthemes and subthemes were proposed andmapped with supporting quotes from transcripts to address issues of trustworthiness and credibility emergentthemes were constantly compared and contrasted withinand across groups during regular meetings the finaltheme categorization and definitions were agreed uponby the entire research team and data saturation and investigator triangulation were achieved table demographics of family caregiversidentifierdph14caregiver relationshipchildcaregiver ethnicitychinesepatient™s diagnosislung cadph19dph34dph42dph53dph59dph68hca12hca68hca75hca81hca87hca109hca114hca116hca117mws004scs18ttsh61ttsh65spousespousesiblingspousechildspousespousechildchildchildspousespousespousespousespousesiblingspousechildchildchinesechinesechinesechinesechinesemalayeurasianchinesemalaychinesemalaychinesechinesechinesechinesechinesechinesemalaychineseprostate calung casigmoid calung cagynaecological malignancylung caprostate cacolon cabreast caendometrial carenal caendometrial cabrain canasopharyngeal capancreas cacopdliver calung cagynaecological capatient™s prognosis months“““““““““ 0ctanho bmc palliative care page of didn™t i do this why didn™t i do that™  dph19spousein some instances family caregivers displayed poignant emotion and dedication to their family membersconveying the great extent to which they would goto give theirthe best care andcomfortfamily membersœi wish to care for him till the very end¦ i want thebest for him and i will do what™s best for him i amwilling to sacrifice my soul to make that happen ortake his place if i could dph68 childalleviating suffering n family caregivers displayed awareness and empathytowards their family member™s physical and emotionalsuffering tied in with a desire to relieve their painœthis morning she was upset with me for forcing herto drink the bitter medicine i told her ˜i love you iwouldn't do this if i had a choice i want you todrink this for your own benefit not mine i'm just encouraging you from thedph53spousesidelines™underlining this motivation to alleviate suffering was thefamily caregivers™innate compassion for their familymember an empathic bond so strong that witnessingtheir family member™s suffering caused them deep emotional distressœ¦ it hurts a lot to drain the fluids i™m heartbrokenwhen i see how much pain she is in especially wheni see the tubing being inserted it must hurt somuch hca109 spousefindingsfigure presents the six caregiving motivations and onewellbeing determinant generated from data to form theblessings or burdens of endoflife caregiving bobecmodelthe six caregiving motivations honoring fidelityalleviating suffering enduring attachment preservinggratitude navigating change and reconciling withmortality represent the beliefs values and goals that areassimilated into the eol family caregiver™s daily life eachmotivation is posited to affect the way a caregiver makesmeaning of their role hence leading to a nurturance ofcaregiver wellbeing termed in this paper as blessings ora diminishment in caregiver wellbeing termed in thispaper as burdens the wellbeing determinant which ischaracterized by the caregiver™s sense of control selfempowerment and kinship derived from their experiences serves as an indication of selfdetermination andis theorized to have a positive influence on how the sixcaregiving motivations are appraised by the caregiverthese themes are described in greater detail and demfrom study participantsonstrated by direct quotesbelowhonoring fidelity number of transcripts theme hasoccurred in n family caregivers expressed their faithfulness and commitment to attend to the needs and wishes of their family members for the remainder of their lives fearingregret to see things through till the end this motivatedthem in fulfilling their sense of duty to the utmost oftheir abilitiesœi shouldn™t regret anything whatever i can do forhim i will do my best and [instead of waiting till]he™s in the coffin you know [and then say] ˜oh whyfig the blessings or burdens of endoflife caregiving bobec model 0ctanho bmc palliative care page of enduring attachment n family caregivers experienced a prevailing attachment totheir family member that motivated them in spendingcherished time together and doing all they could toensure that their family member was well taken care ofœi think i try to make him as comfortable as he canbe every medical checkup every appointment wewill keep to it and i will always be there for him[there will never be] any appointment that i am notgoing with him hca117 spousesome family caregivers found that the motivation tosustain this attachment was also driven by feelings ofanxiety about their family members™ wellbeing thesecaregivers felt the need to be within their family members™ physical proximity as much as possibleœi get worried when she™s lying there and sleepingbecause i™m not sure if anything has happened toher i™m much happier when she™s sitting here withme when she™s just lying there i would think ˜ohno what if something has happened to her™ and i™dbe worried dph59 childpreserving gratitude n family caregivers described past circumstances and beliefs that motivated present feelings of gratitude to theirfamily members this consequently influenced their efforts and responses in caregivingœshe was constipated for as long as a week and shedidn™t tell me when she eventually relieved herselfshe made a mess on the bathroom floor as i wascleaning the mess i thought about how she hadcleaned me up when i was little so i didn™t mindhca81 childwhile many family caregivers reported feelings of gratitude stemming from how their family member hadtreated them in the past some indicated that religiousand cultural beliefs had indoctrinated a sense of indebtedness to their family membersœmy mother says i was indebted to my brother in mypast life this is why i have to settle my debt in thislifetime [by caregiving] because he is here to get hiscompensation mws004 siblingnavigating change n family caregivers reflected on their perceptions of thechanges that had taken place in their lives and that oftheir family members throughout the illness trajectorysome caregivers found motivation in helping their familymembers adaptenergy to liftsupportto changes by dedicating time andtheirspirits and provide emotionalœi would bring my father food when i visit while myhusband would share words of encouragement andtalk to him to cheer him up we just want him to behappy so that he wouldn't spend the whole day innegativity ttsh65 childothers saw the changes as a temporary setback andfound motivation in steering their family member backto their previous condition if possibleœsometimes i will move his legs a little to give himthat exercise i hope that he can walk again but itdepends on how strong his willhca116spouseisreconciling with mortality n the knowledge of their family member™s prognosis motivated some family caregivers to make the most of thetime left with their family members “ creating treasuredmemories and remembering their legaciesœall of us just want to cherish the time that we haveleft with her and we want her to help us spend moregood times together we [want to] learn about mygrandmother learn about my mother so that wecan pass on to the next generation share with themthe traits and the role models to look up tottsh61 childother family caregivers perceived their family member™sprognosis to be unacceptable choosing to push for further treatment in order to stall death™s journey to theirdoorsœmy grandmother lived past years old so ithought my mother would live till atleast without any problems i felt really shocked becausei always thought œshe still has more than years istill have time ¦ so we felt that if it was possibleshe should extend her life dph14 childthe wellbeing determinant n family caregivers reflected on the discovery of positivetheir family member™schanges amidstillness one such change was found within strengthenedkinshipthe trials ofœas we grow up it™s a bit harder [to have familygatherings] because we are all working so when thedisease came even though it™s not a good thing not 0ctanho bmc palliative care page of something you will ask for it united us again maybewithout it [we] would have been a bit more separated ttsh61 childœi feel like we are more united now maybe in thepast we didn't really chat with each other¦ theamount of communication we had increased i feelthat our unity has become stronger dph14 childthey expressed pride in overcoming initial fears by taking charge of and learning to perform unfamiliar taskswith a newfound confidence in their abilities to faceboth practical and emotional difficulties family caregivers also demonstrated a sense of selfempowermentand strengthbased reflection in their sharingœi know nothing about going to visit the government¦ or to do this do that but somehow i findmy way there [i am a] much stronger person so ifanything happens to me i think i know i can faceup to it dph19 spouseœthis is how you grow i learnt to grow because of[my husband] you have to face the insurmountablechallenges that come your way i learnt how toshoulder my responsibilities on my own scs18spousediscussionthis is the first known study that investigates and bringsattention to the internalized motivations of eol familycaregivers while the family dignity intervention studyquestions did not specifically query family caregivers ontheir motives these motivationcentred responses occurred spontaneously and abundantly throughout the interviews “ an indication that internalized motivationsare profoundly espoused within eol caregiving attitudesand behaviors the bobec model fig illustrates theduality ofthese caregiving motivations in regard tomeaningfocused coping and intrapsychic strains as well as identifies an important influence on caregiver wellbeingmotivations with cultural influencessome themes revealed cultural undertones that reflectedthe internalization of asian values into family caregivermotivations in their motivation for alleviating sufferingfamily caregivers displayed the desire to do so by practical means such as administering medication to theirfamily member and experiencing distress when suchmethods were not feasible this is in line with the asianculture of preferring to show concern for their familymembers through pragmatic ways in their motivation for preserving gratitude family caregivers demonstrated the significance of filial piety as well as culturalbeliefs about karma and pastlife within theirattitudes towards caregiving finally in their motivationfor reconciling with mortality family caregivers indicated the importance placed on close intergenerationalconnections as well as longevity for their elders motivations as blessings meaningfocused copinghonoring fidelityall eol caregiving motivationsalleviating suffering enduring attachment preservinggratitude navigating change and reconciling with mortality were found to embody the tenets of meaningfocused coping fuelled by these motivationsfamilycaregivers displayed the propensity for benefitfindingand benefitreminding even in witnessing their familymember™s suffering and imminent mortality adaptivegoal processes in adjusting their expectations and aspirations in accordance with their family member™s physicalcondition and prognosis reordering priorities in hopesof making the most of the time left with their familymember and infusing ordinary events both past andpresent with positive meaning that allowed them to feelaffirmed encouraged and grateful in their daily caregiving as such the capacity for imbuing stressful caregiving events with positive meaning and responseswould make these motivations advocates of perceivedœblessings in the eol caregiving journeymotivations as burdens intrapsychic strainsparadoxically the authors found that these eol caregiving motivations also ran parallel to the intrapsychicstrains as postulated by pearlin and colleagues intheir seminal stress process model intrapsychic strainsoccur when the caregiver™s selfconcept is diminisheddue to the chronicity of providing care intrapsychicstrains unique to caregivers were defined as role captivity in which the caregiver feels entrapped within hisor her role whether or not by personal choice the lossof self in which the caregiver experiences a loss of identity and sense of personhood as enmeshment with thepatient ensues perceived low competencein whichthe caregiver does not see the value and skill of the workthey do leading to a sense of helplessness and perceived lack of gain in which the caregiver does not findpersonal growth orcaregivingprocessenrichmentin theshould their eol caregiving motivations personifythese strains it is only a matter of time before familycaregivers experience outcomes of mental and emotionaldistress such as depression anxiety and irritability aswell as a decline in physical health and a disengagementfrom their caregiving roles in short these motivations would most certainly bludgeon the family caregiverwith great ˜burdens™ within the eol caregiving journey 0ctanho bmc palliative care page of the crucial factor selfdeterminationselfdetermination theory suggests that people needto feel a sense of competence gaining mastery of tasksand having selfefficacy relatedness feeling like theybelong and mutually relating to others and autonomycontrol over their own choices behaviors and goals inorder to fuel high quality motivation that helps one tothrive the theme of the wellbeing determinant alignswith this concept in a caregivingcentric phenomenonfamily caregivers contributing to this theme displayedconfidence in carrying out previously unfamiliar caregiving tasks competence affirmed a stronger sense of kinship relatedness with the patient and their families andtook ownership of their caregiving responsibilities andchallenges autonomy encouragingly numerous studieshave proposed that high quality motivations stemmingfrom selfdetermination can elicit outcomes of greaterfortitude higher commitment and more positive emotions and selfconcepts [“]building on said studies the authors propose that family caregivers who feel a sense of competence relatedness and autonomy within their caregiving motivationswould be further inclined to meaningfocused copingsuch as deriving perceived benefits from their caregiving even in difficult events reminding themselves ofthese benefits when faced with similar circumstances setting their own goals in caregiving having the competence and confidence to be flexible and adaptive and finding positivity in normal everyday situations possessing a sense of selfdetermination in the caregivingrole would in essence safeguard one™s caregiving motivations from the intrapsychic strains of perceived entrapment a sense of disempowermentineptitude andfruitlessnessas such the bobec modelidentifies the wellbeingdeterminant as an indicative element of caregiver selfdetermination and a crucial factor as to whether the eolfamily caregiver perceives their journey as one lined withblessings or laden with burdens competencetargeted mediators apart fromgeneral psychoeducation on symptom managementmedical care and selfcare interventions could incorporate mediums to develop and improve selfefficacy these can take the form of personalstrengths journaling facilitating peer support between new and experienced caregivers rolemodelling and goalsetting such interventions canbe created in the form of structured support groupsonline platforms or mobile applications autonomytargeted mediators in addition toproviding adequate and appropriate eol caregivereducation autonomytargeted interventions suchas those involving mindfulness practice and thearts can serve to give caregivers a sense of control as well as help them make meaning out oftheir thoughts emotions and circumstances todate the mindfulcompassion art therapymcat for eol care professionals showsgreat potential to be converted into a programfor eol family caregivers relatednesstargeted mediators dyadic or familyprojects that recall shared memories expressappreciation seek fiveness impart wisdom andcreate generativity such as the fdi are valuable incrafting the bond of relatedness among familycaregivers and their family members such projectsshould be implemented as a foundation ofpsychosocial interventions at the endoflifeinterventions should be offered to family caregiversin a culturallyrelatable manner this can be donethrough emphasizing how these mediators will helpfamily caregivers enhance their practical caregivingwith increased competencesense of control andmeaningmaking as well as enrich their intergenerational familial bonds with conversations that focus onlegacy creationimplications and recommendationsfindings from a number of studies show that fulfillingone™s sense of selfdetermination appears central to sustaining one™s motivation and innate satisfaction in caregiving [“] the findings from this paper indicatethat caregivers are driven by motivations that couldequally contribute to wellbeing nurturance or diminishment at the same time our findings also indicate thatcaregivers possess a caregivercentric sense of selfdetermination the wellbeing determinant that is theorized to have positive affect on their motivations thusthis paper recommends that caregiver support interventions should comprise all of the following mediators inorder to fulfil the need for selfdeterminationlimitations and future directionswhile the anically occurring responses emphasize thesignificance of motivations within eol caregiving theseresponses were not examined further during the fdi interviews due to other pri
Colon_Cancer
recently the current pandemic of coronavirus disease covid characterized by a pulmonary infection in humans is caused by a novel virus strain from family coronaviridae known as severe acute respiratory syndrome coronavirus sarscov2 the previous outbreak of severe acute respiratory syndrome sars in “ and middle east respiratory syndrome mers in has demonstrated the lethality of coronaviruses when they cross the species barrier and infect humans so far six coronaviruses infecting humans have been identified and the novel coronavirus is the seventh one described to date as being responsible for a respiratory infection sarscov and merscov and the new sarscov2 belong to the betacoronavirus family [“] the coronaviruses have the largest genome around k among the rna viruses sarscov2 was closely related from “ identity to two batderived severe acute respiratory syndrome sarslike coronaviruses batslcovzc45 and batslcovzxc21 but it was more distant from sarscov from “ and merscov about furthermore the performed bioinformatic analysis showed that the nucleotide sequence of sarscov2 is similar to those of other betacoronaviruses with nucleotide identities of ‰¥ there are currently no effective licensed therapies for human coronaviruses hcov infections and existing treatment strategies are generally limited to symptomatic treatment and supportive care email addresses kuzunovahqtchaikapharmacom k uzunova efilipovahqtchaikapharmacom e filipova vpavlovahqtchaikapharmacom corresponding author v pavlova tvekovmuplevenabvbg t vekov 101016jbiopha2020110668 received may received in revised form august accepted august biomedicinepharmacotherapy1312020110668availableonline24august2020075333222020theauthorspublishedbyelseviermassonsasthisisanopenaccessundertheccbyncndlicensehttpcreativecommonslicensesbyncnd40 0csuch as solidarity who recovery k uzunova in the absence of a specific treatment for this novel virus the effort of researchers is focused on understanding and controlling the disease and on preventing and controlling the replication and spread of the virus to devise therapeutic strategies to counteract the sarscov2 infection numerous potential treatment options are being evaluated in ongoing clinical trials many antiviral and immunological treatments being investigated against coronaviruses are summarized by who in landscape analysis of therapeutics as of march the realtime dashboard of completed ongoing and planned clinical trials for covid includes drugs and promising therapies such as remdesevir lopinavirritonavir hydroxychloroquine il6 inhibitors tocilizumab and sarilumab convalescent plasma therapy stemcell transfusion vaccine candidates traditional chinese medicines which are of top interventions of the presented network among them remdesivir an analogue of adenosine seems to have a more promising future due to proven in vitro and in vivo antiviral efficacy till the beginning of june promising therapies involving lopinavirritonavir and chloroquine or hydroxychloroquine were part of treatment guidelines in many countries but currently they are excluded from covid19 treatment protocols because of uncertainty regarding their risks and benefits and it is recommended that they should be used only in the context of clinical trials [“] in spite of its known in vitroin vivo efficacy and safety profiles some trials evaluating these drugs for covid19 infection treatment uk ntc04381936 and discovery inserm ntc04315948 discontinued hydroxychloroquine and lopinavirritonavir arms the interim trial results showed that hydroxychloroquine and lopinavirritonavir produced little or no reduction in the mortality of hospitalized covid19 patients when compared to standard of care nevertheless some countries worldwide continue to recommend chloroquinehydroxychloroquine as a treatment option [“] the existing drugs that target viral proteins associated with enzymatic activities or blocking viral replication machinery or host proteins involved in viral life cycle regulating the function of the immune system or other cellular processes in host cells have great potential and are available on the market our review aims to highlight the potential molecular mechanisms of the therapeutic options available for the cure of other health conditions and their repurposing for the treatment of this novel coronavirus sars cov2 selected treatments of sarscov2 remdesivir gs5734 “ polymerase inhibitor deltacoronavirus genus pdcov which have the most divergent rdrp of known cov as compared to sars and merscov an in silico test of the covid19 rdrp built model suggested the effectiveness of remdesivir as a potent drug sarscov and sarscov2 both belong to the betacoronaviruses of the b lineage and the rdrp amino acid sequences of the two viruses are identical whereas merscov belongs to the betacoronaviruses of the c lineage and is only identical with sarscov2 another in vitro and in vivo proof came from sheahan who examined if gs5734 could inhibit replication of sarscov and mers cov in primary human airway epithelial hae cell cultures they found out a dosedependent reduction in replication with average ic50 values of μm sarscov and μm merscov moreover the compound inhibits a broad range of diverse cov including circulating human zoonotic bat cov and prepandemic zoonotic cov with both prophylactic and therapeutic 1dpi dosing of gs5734 a reduction in replication below a diseasecausing threshold in mouse model of sars cov pathogenesis was demonstrated therapeutic gs5734 substantially reduced the sarscov induced weight loss in infected animals and significantly suppressed virus lung titers p thus demonstrating that therapeutic administration of gs5734 can reduce disease and suppress replication during an ongoing infection furthermore remdesivir has the potential to block sarscov2 infection in vitro at lowmicromolar concentration and in treatment of merscov and sarscov infections in vivo it demonstrated a significant improvement of pulmonary pathology in mice the rnadependent rna polymerase rdrpmediated mechanism of cov inhibition by gs5734 is proven even in the setting of intact exoribonuclease exonmediated proofreading using the model coronavirus murine hepatitis virus mhv it was demonstrated that gs5734 dramatically inhibited viral replication and viral rna synthesis in wildtype wt virus while an nsp14 exon mutant lacking proofreading demonstrated increased susceptibility to gs5734 45fold more active this suggests that gs5734 is recognized at least partially by a functional exon but that the exon activity is not sufficient to prevent potent inhibition of cov replication the results provide strong evidence that rdrp is the target for remdesivir and support the hypothesis that gs5734 directly inhibits viral rna synthesis the mechanism of inhibition of rdrp of merscov by remdesivir was studied by gordon et al they coexpressed the merscov nonstructural proteins nsp5 nsp7 nsp8 and nsp12 rdrp in insect cells as a part of a polyprotein coexpression of the mers nsp5 protease with nsp7 nsp8 and nsp12 in insect cells yielded a stable complex composed of nsp8 and nsp12 the triphosphate form of the inhibitor rdvtp is utilized as a substrate and competes with its natural counterpart atp and they observed that incorporation of the nucleotide analogue was significantly more efficient once added into the growing rna chain the inhibitor does not cause immediate chain termination the presence of the ²hydroxyl group allows the addition of three more nucleotides until rna synthesis is arrested at position i3 therefore the main possible mechanism of action is delayed rna chain termination recently the same authors obtained almost identical results with sarscov merscov and sarscov2 rdrps they provided evidence that all three coronavirus rdrp complexes terminated rna synthesis at position i3 almost all viruses encode polymerases in the central steps of replication and transcription therefore polymerases are becoming the most attractive and suitable targets for antiviral development there are two major types of polymerase inhibitors i nucleoside and nucleotide substrate analogs and ii allosteric inhibitors nucleoside analogs are first triphosphated by the host cell to produce the active inhibitor and then act as an inhibitor by competing with the natural nucleoside triphosphates and terminating the growing viral nucleic acids to date most of the approved antiviral drugs for antihiv therapy utilize this mechanism remdesivir is a nucleotide analogue with a proved mechanism of action as an inhibitor of rnadependent rna remdesivir rdv is an investigational compound with a broad spectrum of antiviral activities against rna viruses including sarscov and merscov gs5734 was originally developed for the treatment of the ebola virus disease gs5734 the single sp isomer of the 2ethylbutyl lalaninate phosphoramidate prodrug effectively bypasses the rate limiting first phosphorylation step of the nuc nucleoside ribose analogue the mechanism of action of nuc requires intracellular anabolism to the active triphosphate metabolite ntp which is expected to interfere with the activity of viral rnadependent rna polymerases rdrp gs5734 selectively inhibits ebola virus replication by targeting its rdrp and inhibiting viral rna synthesis following efficient intracellular conversion to ntp in nonhuman primates this compound shows a broad spectrum of antiviral activities against several rna viruses including respiratory syncytial virus rsv junín virus lassa fever virus and middle east respiratory syndrome virus but was inactive against alphaviruses or retroviruses furthermore remdesivir dosedependently inhibits endemic human cov229e and covoc43 replications which typically cause upper respiratory infection in children but can cause more severe lower respiratory infection in adults with underlying respiratory conditions ie asthma copd and the elderly as well as a member of the biomedicinepharmacotherapy13120201106682 0c lopinavirritonavir “ protease inhibitor the proteases encoded by most viruses play a crucial role in the viral life cycle the protease inhibitors pis bind competitively to the substrate site of the viral protease this enzyme is responsible for the post translational proteolysis of a polyprotein precursor and the release of functional viral proteins allowing them to function correctly and individually in replicationtranscription and maturation inhibition results in the production of immature virus ps coronavirus proteases of which there are two in sarscov a papainlike cysteine proteinase plpro nsp3 and a 3clike proteinase 3clpro or mpro nsp5 and three in several other coronaviruses cleave the orf1 polypeptide as it is translated enabling the formation of the viral replication complex the substratebinding pockets are highly conserved among cov 3clpro suggesting the possibility for a widespectrum inhibitor design targeting this region in the 3clpro of all covs it is postulated that the 3clproinhibiting activity of lopinavirritonavir contributes at least partially to its anticov effects in silico binding studies of the drugs using the identified crystal structure of mpro and employing the hex program to conduct the docking of the ligands to the sarscov main proteinase revealed that lopinavir and ritonavir could basically bind to the active site of sars main proteinase but the efficacy of lopinavirritonavir was predicted to be poor according to the latest report of the structure of 3clpro from sarscov2 pdb code 6lu7 and the available structure of 3clpro from sarscov pdb code 1uk4 the two main proteases differ by only amino acids comparing ligand binding free energies for the main proteases has confirmed that good binders for sarscov are in general and sarscov k uzunova polymerases this mechanism is probably involved in an antiviral activity against sarscov2 biochemical data provided by gordon suggested a unifying mechanism of inhibition of sarscov merscov and sarscov2 fig and future emerging covs may be similarly susceptible to the inhibition by remdesivir comparable replication with also good binders for sarscov2 3clpro protease inhibitors a class of drugs best known for success against hiv block the final step of virion assembly in the treatment of human immunodeficiency virus infection with proven efficacy the combination of lopinavir with ritonavir is widely used as a boosted protease inhibitor in the treatment of hiv infection because of low oral bioavailability of lopinavir and its extensive metabolism by the cyp3a4 isoenzyme lopinavir needs to be coadministered with ritonavir to achieve drug concentrations high enough to inhibit viral replication [ “] so far the reported results from studies in different cell lines animal models and patients for lopinavirritonavir are not so convincing in their inhibition action in human coronaviruses screening the library of fdaapproved drugs for antimerscov activity in cell culture has identified four compounds chloroquine chlorpromazine loperamide and lopinavir which inhibit merscov replication effective concentration ec50 3cid0 μmoll in vitro lopinavir inhibited mers cov efficacy ec50 μm and a maximal protective effect were observed at a dose of μm it was previously shown that lopinavir but not ritonavir inhibit sarscov chymotrypsinlike 3cl protease at the concentration of μm moreover it was suggested that lopinavir blocks a postentry step in the merscov replicative cycle in vitro the detectable antiviral activities of ribavirin rimantadine lopinavir and baicalin were shown by using the frhk4 cell line and in vero e6 cells infected with sarscov2 lopinavir inhibit replication with ec50 at μm during the sars outbreak treatment with lopinavir in combination with ritonavir was explored with some success in nonrandomized clinical trials patients with sarscov treated with lopinavirritonavir showed a progressive decrease of viral load and reduction of the composite adverse outcome at day recently the antiviral activity of remdesivir and ifn was found to be superior to that of lopinavirritonavirifn against merscov in vitro and in vivo the efficacy of lopinavirritonavir with or without ribavirin is evaluated in sarscov2 patients under randomized control trials currently it was demonstrated that this combination has no benefits in adult patients with severe covid19 although protease inhibitors are a common class of medication used in the treatment of hiv1 infection their efficacy in human coronavirus infections is not convincing moreover several antihiv pis are also known to influence other intracellular pathways it was demonstrated that hiv protease inhibitors indinavir saquinavir and lopinavir independently from any viral infection can hinder lymphocyte apoptosis by influencing mitochondrial homeostasis in view of the weak antiviral activity of protease inhibitors further studies should be done to ascertain whether the clinical benefit could be attributed to their antiapoptotic rather than their antiviral activity hence even if the molecular target of lopinavirritonavir is the main protease 3clpro in sarscov2 infected cells fig there are no biochemical and molecular studies confirming the interaction and associating this with clinical efficacy of the protease inhibitor chloroquinehydroxychloroquine chloroquine chq was introduced into clinical practice in as a prophylactic treatment for malaria hydroxychloroquine hcq differs from chloroquine by the presence of a hydroxyl group at the end of the side chain the nethyl substituent is hydroxylated currently chq and its hydroxyl form hcq are used as antiinflammatory agents for the treatment of rheumatoid arthritis lupus erythematosus and amoebic hepatitis in addition chq has been studied as a potent antiviral agent against hiv1aids [“] hcov229e sarscov [ ] influenza a h5n1virus influenza a and b and many other rna and dna viruses many recent reports and published studies suggested that chq and hcq were associated with reduced fig inhibition of viral infection by lopinavirritonavir and remdesivir biomedicinepharmacotherapy13120201106683 0ck uzunova progression of the covid19 and decreased duration of the symptoms [“] there are in fact overall more than trials currently underway around the world on its impact either as a prophylactic or treatment for covid19 it is noteworthy that the usefulness of hydroxychloroquine and chloroquine is intensively investigated chloroquine was found to exert an antiviral effect during pre and postinfection conditions suggesting to have both prophylactic and therapeutic advantages timeofaddition assay demonstrated that chq functioned at both entry and postentry stages of the sarscov2 infection in vero e6 cells however it did not reduce viral replication in sarscov infected mice hydroxychloroquine is significantly more potent than chq in vitro ec50 values and μm respectively and has a lower potential for drugdrug interactions than chloroquine pharmacokinetic models demonstrate that hydroxychloroquine sulfate is significantly superior days in advance to chloroquine phosphate in inhibiting sarscov2 in vitro and was demonstrated to be much less toxic than chq in animals on the other hand data presented by liu demonstrated that the antiviral effect of hcq against sarscov2 infection was comparable with chq in vitro cc50 μm and μm for chq and hcq respectively moreover they suggested that both chq and hcq blocked the transport of sarscov2 from early endosomes ees to endolysosomes els and caused noticeable sizemorphological changes in ees and els they surmised that endosome maturation might be blocked at intermediate stages of endocytosis resulting in failure of further transport of virions to the ultimate releasing site hydroxychloroquine shares the same mechanism of action as chloroquine apart from the probable role of chq and hcq as antiviral agents their mechanisms of action are not fully understood and it was demonstrated that they have multiple effects on mammalian cells ace2 is known to be a cell receptor for sarscov the high similarities of the amino acid sequences and predicted protein structures of the receptorbinding domain of sarscov2 and sarscov suggest that sarscov2 may efficiently use human ace2 as a receptor for cellular entry and employ the cellular serine protease tmprss2 for s protein priming zhou confirmed that sarscov2 used the ace2 receptor to enter cells and did not use other coronavirus receptors such as aminopeptidase n apn and dipeptidyl peptidase dpp4 so the primary mechanism by which cell infection is prevented by these drugs may be at the stage of binding with the surface receptor and endosomemediated viral entry two independent in vitro studies confirmed that chq inhibits the replication of the sarscov chloroquine inhibits the early stage of sarscov replication in vero e6 cells with a effective concentration of ± μml the antiviral activity of chq was indicative at the time point at virus attachment or penetration vincent established that the drug might interfere with terminal glycosylation of the cellular receptor ace2 when chq was added prior to infection the impairment of terminal glycosylation of ace2 may result in reduced binding affinities between ace2 and sarscov spike protein and negatively influence the initiation of sarscov infection when chq or nh4cl were added after infection these agents could rapidly raise the ph and interrupt ongoing fusion events between the virus and endosomes thus inhibiting the infection on the basis of in vitro experiments they suggested that the primary mechanism by which infection was prevented was the poor affinity of sarscov spike protein to underglycosylated ace2 in vitro studies with hiv infected cells also identified that inhibition of glycosylation might be a possible mechanism of action of chq chq inhibits hiv replication at a postintegration stage resulting in the production of immature virions it was demonstrated that the sole mechanism explaining the antihiv activity of chq was a decrease in the infectivity of the newly produced virus associated with defective production of the heavily glycosylated 2g12 epitope of gp120 according to in vitro results the antiretroviral effects of chq are attributable to the inhibition of viral p glycosylation these effects appeared to be specific since the chq concentrations effective in vitro neither affected any other step in hiv1 replication nor were cytotoxic thus there is direct evidence that chq is an inhibitor of glycosylation of gp120 and these alterations may be responsible for the decreased infectivity of hiv grown in the presence of chloroquine when added after the initiation of infection these drugs might affect the endosomemediated fusion and subsequent virus replication sarscov pseudoviruses may enter cells via receptordependent clathrin and caveolaeindependent phsensitive endocytosis likely through a process involving lipid rafts a later study however suggests that the entry of coronaviruses into the host cells occurs through clathrinmediated endocytosis murine hepatitis virus mhv a prototypic member of the cov family requires trafficking to lysosomes for proteolytic cleavage at the fp proximal position of its spike s protein membrane fusion to occur many authors indicated that s protein cleavage is an important step for fusion activity and subsequent internalization of the sarscov virus genome into cells [“] adding chq prior to infection results to inhibition of endosome maturation and strongly decreased mhv infection and fusion which was not observed when the drug was added at hpi indicating that the compound mainly affects mhv entry chloroquine is a weak base that is known to increase the ph of lysosomal and transgolgi network tgn vesicles leading to the dysfunction of enzymes necessary for proteolytic processing and posttranslational modifications of newly synthesized viral proteins chloroquine is able to prevent the processing of prm protein to m protein in flavivirusinfected mammalian and mosquito cells by raising the ph of the postgolgi vesicles in which this cleavage occurs as a result virions from infected cells which had been treated with acidotropic amines late in the virus replication cycle contained prm protein rather than m protein and this reduced the infectivity of the virus the chloroquinemediated rise in endosomal ph modulates iron metabolism in a variety of cell types decreasing in intracellular concentration of iron affects the function of several cellular enzymes involved in pathways leading to the replication of cellular dna and to the expression of different genes [“] autophagy is a lysosomedependent degradative pathway chq and its analogue hcq are known clinically relevant autophagy inhibitors chq is a weak base that inhibits lysosomal acidification which prevents the fusion of autophagosomes with lysosomes and subsequent autophagic degradation inhibition of autophagy with chq stimulates superoxide generation ubiquitinconjugated protein accumulation and apoptosis in a colon cancer xenograft model chq treatment clearly inhibited autophagy in mouse lung and efficiently ameliorated acute lung injury and dramatically improved the survival rate in mice infected with live avian influenza a h5n1 virus h5n1 virusinduced autophagic cell death in alveolar epithelial cells through a pathway involving the kinase akt the tumor suppressor protein tsc2 and the mammalian target of rapamycin and autophagyblocking agents might be useful as prophylactics and therapeutics against infection of humans by the h5n1 virus furthermore prentice suggested that authophagy was induced by the coronavirus mouse hepatitis virus mhv and was required for formation of double membranebound mhv replication complexes which significantly enhanced the efficiency of replication replication of the virus was impaired in atg5 knockout embryonic stem cells the same authors also examined the sarscovs and found out similar colocalization of the key viral replication proteins with endogenous lc3 a protein marker for autophagosome it could be assumed that autophagy inhibitors like chq could inhibit virus replication at present the exact role of autophagy in cov infection remains debatable and there is much evidence suggesting that the endocytic pathway plays a key role in mediating viral entry for many covs including sarscovs merscovs and possibly sarscov2 the antiinflammatory properties of chqhcq should also be considered several studies have suggested that multiple an failure biomedicinepharmacotherapy13120201106684 0chas not yet been identified in sarscov2 infected patients and probably multiple pathways could be involved fig conclusion the sarscov2 is the cause of the coronavirus disease covid19 that has been declared a global pandemic by the world health anization who in despite some clinical characteristics that differentiate covid19 from sarscov merscov and seasonal influenza the pathogen sarscov2 has the same phylogenetic similarity to sarscov and mers cov most of the encoded proteins exhibited high sequence identity between sarscov2 and the related batderived coronaviruses batslcovzc45 and batslcovzxc21 a notable difference was a longer spike protein encoded by sarscov2 compared with the bat sarslike coronaviruses sarscov and merscov in addition sarscov2 was distinct from sarscov in a phylogeny of the complete rnadependent rna polymerase rdrp gene moreover the receptorbinding domain of sarscov2 which directly engages the ace2 receptor for cell entry was more closely related to those of sarscovs “ amino acid identity since the outbreak researchers have released many agents that could have potential efficacy against covid19 there is currently no clinically proven specific antiviral agent available for sarscov2 infection like sarscov and merscov certain agents like chloroquine hydroxychloroquine lopinavirritonavir and remdesivir are being used in ongoing clinical trials all over the world with hopes to further delineate their role in the treatment and prophylaxis of covid19 furthermore due to their availability and using for decades and proven safety records it is reasonable to suggest that they may be appropriate for treatment of covid19 remdesivir an adenosine analogue with wellstudied mechanism of action in cov infections can target the rnadependent rna polymerase and block viral rna synthesis and has been a promising antiviral drug antiviral studies in cell culture and animal models the available human safety data as well as the clear mechanism of action characterize rdv as a directacting antiviral since some authors found that lopinavir“ritonavir treatment did not significantly accelerate clinical improvement hence antiviral effects as an inhibitor of the sarscov main 3cl protease should be further investigated although chq and hcq are wellknown drugs for the treatment of k uzunova observed in fatal cases are most likely associated with not only the direct viral infection and destruction of susceptible cells eg endothelial cells but also the effects of proinflammatory cytokines chemokines and other mediators released from infected and activated cells such as monocytes and macrophages the clinical worsening of individuals with sars in week is apparently unrelated to uncontrolled sars coronavirus replication but may be related to immunopathological damage another study reveals that the presence of viral elements within endothelial cells and the accumulation of inflammatory cells led to endotheliitis in several ans as a direct consequence of viral involvement and to host inflammatory response moreover chq has immunomodulatory effects suppressing the productionrelease of tumour necrosis factorα and interleukin6 which mediate the inflammatory complications of several viral diseases chloroquinehcq was reported to inhibit the production of soluble mature tnf in macrophage cell line inhibit tnfα receptor in human histocytic u937 cells inhibit tnfα ifnγ and il6 in peripheral blood mononuclear cells pbmc reduce tnfα production and lipopolysaccharide lpsinduced il1 release in human monocytic cells it is suggested that chq exerts antiinflammatory and immunomodulatory effects predominantly by pretranslational and nonlysosomotropic mechanisms chloroquineinduced inhibition of tnf and il6 production is not mediated through a lysosomotropic mechanism and chloroquine probably acts on tnf secretion by disrupting iron homeostasis besides its antiviral activity and due to its suppressive effects on the productionrelease of tnfα and interleukin chqhcq may be effectively used in the treatment of viral infections characterized by symptoms associated with inflammatory processes andor immunehyperactivation antiinflammatory effects of chq remain poorly understood regulation of proinflammatory cytokines chq can also act on the immune system through cell signaling chq inhibits the activation of p38 mapk in hcov229einfected cells and evokes the activation of erk independently of infection these results suggested that chq may inhibit cov replication by suppressing the p38 activation additionally chq strongly inhibited phosphorylation of mitogenactivated protein kinase mapk p38 and to a lesser extent cjun nterminal kinase and extracellular signalregulated kinase ½ chloroquine could also inhibit innate immune responses trough downregulation of tlr9 signaling pathways requiring endocytosis and acidification of endosomes within plasmacytoid dendritic cells pdcs and act as novel antagonists to chemokine receptor cxcr4 that suppress pancreatic cancer cell proliferation on the other hand another hypothesized mechanism of chq is via the inhibition of antigen degradation and improving the crosspresentation efficiency of dcs in vitro in vivo evidence suggested that a short course of treatment with chq followed by a booster dose of a soluble antigen immunization can efficiently enhance human cd8 t cell responses and single vaccination with inactivated influenza virus combined with chloroquine treatment elicits a higher t cell immunity in mice regulation of nlrp3 inflammasome activation may offer a promising therapeutic approach by inhibiting or slowing down the process of acute respiratory distress syndrome hcq is a known nlrp3 inhibitor and its potential clinical effectiveness is certainly based on the downregulation of il1 expression the major proinflammatory cytokine interleukin1beta il1 is elevated in plasma from hospitalized covid19 patients and its associated signaling pathway seems to drive sarscov2 pathogenicity il1 secretion is primarily initiated by inflamm
Colon_Cancer
section all disclosure information for gie editors can befound online at httpwwwgie contentconflictofinterest cme editors and their disclosures are as followsprasad g iyer md associate editor for cmeconsultingadvisoryspeaking olympus research supporttakeda pharmaamit rastogi md associate editor for cmeconsultingadvisoryspeaking olympuskarthik ravi md cme editordisclosed no relevant financial relationshipswilliam ross md cme editorconsultingadvisoryspeaking boston scientific olympusara sahakian md cme editordisclosed no relevant financial relationshipsbrian weston md cme editordisclosed no relevant financial relationshipsall cme activities including their associated sare copyrighted by the asge gastrointestinal endoscopy volume no wwwgie 0ccme activitycontinuing medical education questions september question objectivedemonstrate triaging advanced gi endoscopy procedures during the covid19 pandemictriaging advanced gi endoscopy procedures during the covid19 pandemicquestion due to the resurgence of covid19 in your area healthofficials issue a mandate to cease elective proceduresbased on the findings of the current study a consensuswas reached that all of the following advanced gi procedures can be safely deferred for weeks exceptpossible answers aea ercp for asymptomatic choledocholithiasisb radiofrequency ablation for barrett™s esophagus withhighgrade dysplasiac endoscopic mucosal resection for barrett™s esophaguswith nodular highgrade dysplasiad endoscopic mucosal resection for a cm adenomatous colon polyp with highgrade dysplasiae eus for incident pancreatic and common bile duct dilation on ct or mri normal liver function testslookup sawhney ms bilal m pohl h triaging advanced gi endoscopy procedures during the covid19 pandemic consensus recommendationsusing the delphi method gastrointest endosc question objectivedescribe the learning curve for ablative therapy for dysplastic barrett™s esophagus and the effect of center volumes onoutcomeslearning curves and the ‚uence of procedural volume for the treatment of dysplastic barrett™sesophagusquestion a 53yearold man returns for surveillance endoscopy ofc3m5 nondysplastic barrett™s esophagus initially diagnosed years prior the patient has been compliant with twicedailyproton pump inhibitor therapy and denies any heartburnacid regurgitation or dysphagia he has no other significantmedical history you perform an egd which again reveals ac3m5 barrett™s segment without any nodularity biopsyspecimens are obtained and subsequently reveal multifocallowgrade dysplasia which is subsequently confirmed by anexpert gi pathologist after a subsequent discussionregarding ablative therapy with radiofrequency ablationrfa versus continued surveillance the patient wishes topursue ablation you discuss referral to a new colleague whojoined your practice several months earlier the colleague isthe first endoscopist to perform rfa at your institution andhas treated a total of patients the patient inquireswhether he would be more likely to have a successfuloutcome if he is referred to a moreexperienced endoscopistat a highvolume center which of the following is truepossible answers ada the patient is more likely to achieve complete remission of intestinal metaplasia crim if he undergoesablation at a center that has treated more than patientsb the patient is more likely to achieve complete remission of dysplasia crd if he undergoes ablation at acenter that has treated more than patientsc the patient is more likely to achieve crim if he undergoes ablation by an endoscopist who has treatedmore than patientsd the patient is more likely to achieve crd if he undergoes ablation by an endoscopist who has treatedmore than patientslookup lipman g markar s gupta a learning curves and the ‚uence of procedural volume for the treatment of dysplastic barrett™s esophagusgastrointest endosc wwwgie volume no gastrointestinal endoscopy 754e1 0ccme examquestion objectiveexplain the utility of a new gi bleeding prediction score for patients with upper gi bleedingthe horibe gi bleeding prediction score a simple score for triage decisionmaking in patients withsuspected upper gi bleedingquestion you are called to the emergency department to see apatient who has just arrived with the complaint of blacktarry stools the emergency department physician has notyet seen the patient but the nurse relays vital signs labresults and history sufficient to calculate a harbingerscore according to the study by horibe in thismonth™s issue this score is used to assess risk of which ofthe followingpossible answers ada deathb need for blood transfusionc need for endoscopic interventiond presence of highrisk stigmatalookup horibe m iwasaki e bazerbachi f horibe gi bleeding prediction score a simple score for triage decisionmaking in patients with suspectedupper gi bleeding gastrointest endosc question objectivereport the longterm efficacy and safety of eusguided hepaticogastrostomy for treatment of malignant biliary obstructionlongterm outcomes of a long partially covered metal stent for eusguided hepaticogastrostomy inpatients with malignant biliary obstructionpossible answers ada success rate of eushgs is less than b adverse events occur in less than of casesc recurrent biliary obstruction occurs in about onethirdof patientsd prior biliary drainage has no impact on the likelihood ofrecurrent biliary obstructionquestion a 58yearold man is transferred to your hospitalformanagement of a pancreatic head mass causing obstructive jaundice and gastric outlet obstruction an endoscopic ultrasound with fineneedle aspiration of the massconfirmed pancreatic adenocarcinoma and a contrastenhanced computed tomography scan demonstratednumerous liver lesions compatible with metastatic disease a duodenal stent was placed which precluded anercp procedure for biliary drainage biliary drainage isnecessary before chemotherapy can be initiated variousoptions are discussed with the patientincluding eusguided hepaticogastrostomy eushgs according tothe current study by nakai which of the following isaccuratelookup nakai y sato t hakuta r longterm outcomes of a long partially covered metal stent for eusguided hepaticogastrostomy in patients withmalignant biliary obstruction with video gastrointest endosc 754e2 gastrointestinal endoscopy volume no wwwgie 0ccme activitycontinuing medical education answers september question correct response erationale for correct responsethe covid19 pandemic has necessitated many elective procedures to be rescheduled in order to mitigate the spreadof infection and conserve vital resources choosing which advanced endoscopic procedures should be performed or safelydeferred during the covid19 pandemic may be a complex decision14 the aim of the current study was to provideguidance for triaging advanced endoscopic procedures using a modified delphi method the delphi method is a validatedand structured technique to obtain expert consensus which is useful in the present situation in which outcome data arelimited156 the following important patient outcomes were used avoidance of deathprolongation of life avoidance of cancercancer progression avoidance of major surgery andor hospitalization and improvement orpalliation of symptoms the following procedural timing categories were used timesensitive emergent schedulewithin week timesensitive urgent schedule within to weeks or nontime sensitive defer for weeks andthen reassess the timinga prespecified consensus threshold of was achieved in of advanced endoscopy indications reviewed by expert gastroenterologists one hundred percent consensus was achieved in of indications in of indications to consensus was achieved the only indication for which consensus could not be achieved was œincidentallyfound pancreatic duct dilation mm and common bile duct dilation mm on ct scan or mri with normal liverfunction teststhis study provides a decisionmaking framework for endoscopists to determine the timing for endoscopic proceduresduring and after the pandemic the decision to perform endoscopy during the covid19 pandemic needs to balance therisks associated with delaying the procedure in the individual patient with the risk of viral exposure to patients and healthcare providerstakehome message triaging advanced gi procedures during the covid19 pandemic may be facilitated by thecurrent consensus recommendations using the delphi method individual practice and patient factors must also beconsideredreferences sawhney ms bilal m pohl h triaging advanced gi endoscopy procedures during the covid19 pandemic consensus recommendations usingthe delphi method gastrointest endosc sultan s lim jk altayer o aga institute rapid recommendations for gastrointestinal procedures during the covid19 pandemic gastroenterology epub mar joint gi society message on covid19 available at httpsgi20200315jointgisocietymessageoncovid19 accessed march gastroenterology professional society guidance on endoscopic procedures during the covid19 pandemic available at httpswebfilesgilinksmediajoint_gi_society_guidance_on_endoscopic_procedure_during_covid19_final_impending_3312020pdf accessed april hsu cc sandford ba the delphi technique making sense of consensus pract assess res eval “ donohoe h stellefson m tennant b advantages and limitations of the edelphi technique implications for health education researchers am j healtheduc question correct response crationale for correct responseendoscopic eradication therapy for dysplastic barrett™s typically uses endoscopic mucosal resection of visible lesionswith subsequent ablation of the flat barrett™s segment with rfa this approach has demonstrated high clinical successwith estimates that crd is achieved in over and crim in over of treated patients1 however studies assessingthe learning curve associated with attaining proficiency with endoscopic therapy of dysplastic barrett™s and the effect ofcase volumes at centers on outcomes have yielded unclear results23 consequently current guidelines suggesting minimum numbers of cases to achieve competence for individual endoscopists or reflecting quality for centers are based onlimited evidence4wwwgie volume no gastrointestinal endoscopy 754e3 0ccme answersin this month™s issue of gie lipman and colleagues5 report results from a retrospective study examining patientstreated in the uk rfa registry to assess crd crim and dysplasia recurrence based on endoscopist experience andcenter case volumes5 a total of consecutive patients were identified from centers which were further divided into patients treated at lowvolume centers patients treated patients treated at mediumvolume centers patients treated and patients treated at highvolume centers patients treated rates of crd and crim at months did not differ based on center volumes however dysplasia recurrence washigher in patients treated at lowvolume centers further analysis using a riskadjustment cumulative risk sum curve wasdone to assess the effect of the learning curve on individual endoscopists a significant reduction of crd was seen at cases whereas a similar reduction of crim was seen at cases takehome message in conclusion this study suggests that the learning curve for rfa may be relatively short withfewer than cases required to achieve competency further center volume may have a very limited effect on outcomesfuture prospective studies are still required to assess the optimal number of training cases required to achieve endoscopicablative competencyreferences haidry rj dunn jm butt ma radiofrequency ablation and endoscopic mucosal resection for dysplastic barrett™s esophagus and early esophagealadenocarcinoma outcomes of the uk national rfa registry gastroenterology pasricha s cotton c hathorn ke effects of the learning curve on efficacy of radiofrequency ablation for barrett™s esophagus gastroenterology fudman di lightdale cj poneros jm positive correlation between endoscopist radiofrequency ablation volume and response rates in barrett™sesophagus gastrointest endosc fitzgerald rc di pietro m ragunath k british society of gastroenterology guidelines on the diagnosis and management of barrett™s oesophagusgut lipman g markar s gupta a learning curves and the ‚uence of procedural volume for the treatement of dysplastic barrett™s esophagus gastrointest endosc question correct response drationale for correct responsefor decades gastroenterologists have been searching for a straightforward method to determine whether a patientpresenting with an upper gastrointestinal bleed is at high risk of needing hospital admission therapeutic interventions ordeath multiple scoring systems have been proposed but systematic reviews have found them all flawed to various degrees12 however one of the betterknown scores proposed by blatchford has been recommended in recentguidelines to assess risk of requiring an intervention34 yet the quest for a more predictive model continues with somegroups turning to machine learning methods for an answer5in this month™s issue of gie horibe 6 report on a simple 3factor assessment to predict the likelihood of findinghighrisk stigmata hrs on endoscopy which they call harbinger because their median time to endoscopy was only hours the prevalence of hrs was quite high up to even in patients deemed to be low risk for stigmata predictablythe intervention rate was more than double that in a large international trial vs comparing harbinger toother scoring systems including blatchford™s the authors found their model to be superior this outperformance couldhave been predicted because the other models were designed to detect risk of other endpoints such as mortality or needfor intervention not hrsin addition the performance of the model was not consistently strong across the study institutions because auc valuesfor harbinger ranged from to between institutions with no overlap in confidence intervals between the topand low scores6 concerns about how harbinger would perform in other settings are reasonable as factors like access toppis varies across marketsin a resourceconstrained world the ability to predict need for interventions would seem more helpful than predictingendoscopic appearance although hrs are linked to the need for interventions the rapid time to endoscopy skews thelinkage by ballooning the prevalence of hrs many of these patients are likely to have far less ominous endoscopic appearances if the endoscopy was done hours after presentationreferences ramaekers r mukarram m smith cam the predictive value of preendoscopic risk scores to predict outcomes in emergency department patientswith upper gastrointestinal bleeding a systematic review acad emerg med de groot nl bosman jh siersema pd prediction scores in gastrointestinal bleeding a systematic review and quantitative analysis endoscopy blatchford o murray wr blatchford m a risk score to predict need for treatment for uppergastrointestinal hemorrhage lancet barkun an almadi m kuipers ej management of nonvariceal upper gastrointestinal bleeding guideline recommendations from the internationalconsensus group ann int med 754e4 gastrointestinal endoscopy volume no wwwgie 0ccme answers shung dl au b taylor ra validation of a machine learning model that outperforms clinical risk scoring systems for upper gastrointestinalbleeding gastroenterology horibe m iwasaki e bazerbachi f horibe gi bleeding prediction score a simple score for triage decisionmaking in patients with suspected uppergi bleeding gastrointest endosc stanley aj laine l dalton hr comparison or risk scoring systems for patients presenting with upper gastrointestinal bleeding international multicenter prospective study bmj 2017356i6432question correct response crationale for correct responsebiliary drainage options may be limited by gastric outlet obstruction or surgically altered anatomy eusguided hepaticogastrostomy eushgs provides an effective alternative to ercp and percutaneous drainage in these complex cases1additionally eushgs has been compared to ercp for primary biliary drainage and outcomes were found to be comparable2 however potential adverse events of eushgs such as stent migration and peritonitis can be severe and longterm data thus far have been lacking3in the current issue of gie nakai and colleagues4 present a retrospective study of patients who underwent eushgs for drainage of malignant biliary obstruction partially covered metallic stents ranging in length from to cm wereused to create the hepaticogastrostomy technical and functional success rates were and respectively adverseevents occurred in of patients with the most common being fever and abdominal pain peritonitis occurred in andcholangitis in of patients no cases of stent dislodgement were reported recurrent biliary obstruction rbo occurredin of patients in a median time of months short length of the intragastric portion of the stent and prior biliarydrainage were both associated with recurrent biliary obstruction of the patients who had rbo underwent successfulreintervention with eushgstakehome message eusguided hepaticogastrostomy is an effective alternative for longterm biliary drainage recurrences can be successfully managed with repeat hepaticogastrostomyreferences nakai y isayama h yamamoto n safety and effectiveness of a long partially covered metal stent for endoscopic ultrasoundguided hepaticogastrostomy in patients with malignant biliary obstruction endoscopy paik wh lee th park dh eusguided biliary drainage versus ercp for the primary palliation of malignant biliary obstruction a multicenter randomized clinical trial am j gastroenterol giovannini m eusguided hepaticogastrostomy endosc ultrasound 20198s35s9 nakai y sato t hakuta r longterm outcomes of a long partially covered metal stent for eusguided hepaticogastrostomy in patients with malignant biliary obstruction with video gastrointest endosc wwwgie volume no gastrointestinal endoscopy 754e5 0c'
Colon_Cancer
covid19 has had an impact on the provision of colorectal cancer care the aim of the crccovid study is to describe the changes in colorectal cancer services in the uk and usa in response to thepandemic and to understand the longterm impactmethods and analysis this study comprises phases phase is a survey of colorectal units that aims toevaluate adherences and deviations from the best practice guidelines during the covid19 pandemicphase is a monthly prospective data collection of service provision that aims to determine the impactof the service modifications on the longterm cancer specific outcomes compared to the national standards phase aims to predict costs attributable to the modifications of the crc services and additionalresources required to treat patients whose treatment has been affected by the pandemic phase aims tocompare the impact of covid19 on the nhs and usa model of healthcare in terms of service provisionand cost and to propose a standardised model of delivering colorectal cancer services for future outbreaksethics and dissemination this study is a service evaluation and does not require hra approval or ethicalapproval in the uk local service evaluation registration is required for each participating centre in theusa ethical approval was granted by the research and development committee the results of thisstudy will be disseminated to stakeholders submitted for peer review publications conference presentations and circulated via social mediaregistration details nil the authors published by elsevier ltd on behalf of surgical associates ltd this is an open access under the cc byncnd license httpcreativecommonslicensesbyncnd40 introductionthe covid19 pandemic has had a significant impact on theprovision of healthcare worldwide as of the 29th june covid19 has resulted in confirmed cases and ‡‘ corresponding author at department of surgery and cancer imperial collegelondon chelsea and westminster and the royal marsden campus unitedkingdomemail address ckontovounisiosimperialacuk c kontovounisiosdeaths in the uk at a local level hospitals have been forcedto make a number of workforce modifications and changes toserviceprovision to combat the crisis and maintain standards ofcare for our patients facetoface consultations have beendissolved or minimized in favour of telephone or virtual clinicsprovision of investigations including ct scans and endoscopieshave been significantly reduced and all benign surgical procedurespostponed furthermore the treatment algorithm for confirmed colorectal cancer cases has proved challenging101016jisjp20200700524683574 the authors published by elsevier ltd on behalf of surgical associates ltdthis is an open access under the cc byncnd license httpcreativecommonslicensesbyncnd40 0ca courtney international of surgery protocols “in the uk over forty thousand patients are diagnosed with colorectal cancer each year deviation from nice colorectal cancercrc guidelines may lead to significantly poorer outcomes however the current model of cancer services delivery cannot be maintained because of both resource limitation and the potential risksto patients and staff during the pandemic there is a lack of highdependency beds which are being utilized for covid19 patientsthere is the risk of exposing colorectal cancer patients the majority of whom are elderly and have significant comorbidities to thevirus during their treatment within the hospital patients requiringneoadjuvant or adjuvant therapy are at particular risk finallystaff safety must also be considered particularly around aerosolgenerating procedures such as endoscopy and laparoscopic surgery intercollegiate general surgery guidance on covid19 outlinedgeneral principles on the provision of a safe surgical service duringthe pandemic however there has been no specific guidance todate on how to best modify colorectal cancer crc service provision during the pandemic in the absence of a national consensusthe onus is on individual hospital trusts and multidisciplinaryteams to make very challenging decisions about individual patientcare lack of a unified approach may have important consequencesat patient and healthcare institution levelsdelay in cancer diagnosis or treatment due to service modification is likely to create an increased demand in resources once thecrisis has passed predicting the economic impact and planningfor this is essentialhow hospitals approach the new constraints on crc care andallocate resources may vary between the uk and usa it is hopedthat gaining insights from both perspectives will improve the problem solving methods and analysis aims and objectivesthe aim of the crc covid study is to describe the changes incolorectal cancer services in the uk and usa in response to thecovid19 pandemic and to understand the longterm impactour primary and secondary objectives relevant to each phase ofthe study are listed in table study designthis is a multicentre service evaluation conducted through aresearch collaborative with the support of the crc covid steeringcommittee all colorectal units continuing to provide cancer services in the uk ireland and the usa have been invited to participate all study and recruitment information is available on thetable primary and secondary study objectiveswebsite crccovid this service evaluation has been endorsedby the royal college of surgeons of england rcsthis service evaluation will be carried out in phases fig phase uses a questionnaire to assess the modificationsadopted by each colorectal unit in order to continue provision ofthe colorectal cancer services during the covid19 pandemic ithas been developed using an iterative process after research ofall relevant guidelines to construct the standard against which services would be evaluatedthe following guidelines relevant to the management of colorectal cancer have been used as standards for this serviceevaluation nice guidelines colorectal cancer [ng151] nice guideline suspected cancer recognition and referral[ng12] association of coloproctology of great britain irelandacpgbi guidelines for the management of cancer of thecolon rectum and anus [“] british society of gastroenterologyassociation of coloproctology of great britain and irelandpublic health england postpolypectomy and postcolorectal cancer resection surveillanceguidelines informal consultations with consultants nurse specialists andpatients have been used to develop the tool and then it has beenmodified after clinician review for face validity flow and relevance the final instrument comprises questionsphase investigates the provision of colorectal cancer servicesduring the covid19 pandemic by evaluating the performance ofeach unit against the national bowel cancer audit outcomes all centres participating in phase will be required to registerthis service evaluation as per local protocol prior to commencement of data collection on redcap this will be the responsibilityof the local leadphase of the study will develop a prediction model of the economic burden of the modifications in cancer service delivery thismodel will be designed jointly by two international businessschools based on previous publications and national statisticsphase will evaluate and compare the impact of the covid19pandemic on the nhs and the usa healthcare using data collectedduring phase and the predictive mode utilized in phase specificdifferences in modifications of crc services will be examined recruitmentphase survey has been distributed to all colorectal consultantsin the uk and ireland through personalised emails social mediaand the rcs the recruitment of colorectal cancer units in thephasephase phase phase phase primary objectiveevaluate adherences and deviations from best practiceguidelines on colorectal cancer during covid19pandemicative crc service deliverysecondary objectives 0f describe modifications to screening process for crc 0f describe modifications to preoperative intraoperative and postoper 0f demonstrate global effect of covid19 pandemic on crc service provi 0f outline consensus recommendations for sustainable modifications to 0f predict the impact of modifications on the incidence and prevalence of 0f plan adjustments to crc service provision after the end of pandemicsion irrespective of the type of healthcare systemdetermine the impact of crc service provision followingmodifications on longterm cancer specific outcomescompared to national standards 0f predict the costs attributable to modifications of crc services during covid19 pandemic 0f predict additional resources required to treat patients whose treatment has been affected by covid19 0f compare the impact of covid19 on the nhs and usa model of healthcare in terms of service provision and cost 0f propose a standardised model of delivering colorectal cancer services for future outbreaksdifferent crc stages in monthscrc services 0ca courtney international of surgery protocols “fig phases of crc covidusa will use similar approach all units recruited into phase arerecruited into phase participation in phase is not mandatory inorder to participate in phase data collectionall surveys and data collection follow the gdpr requirementsand comply with caldicott principles individual patient identifiable data is not collected in this study study data is collectedand managed using redcap electronic data capture tool hostedat the kennedy institute of rheumatology at the university ofoxford redcap research electronic data capture is asecure webbased software platform designed to support datacapture for research studies providing an intuitive interfacefor validated data capture audit trails for tracking data manipulation and export procedures automated export procedures forseamless data downloads to common statistical packages and procedures for data integration and interoperability with externalsources after the close of the phase data collection period all data setswill be checked for missing data where possible centres will begiven an opportunity to rectify missing data centres where of data is missing will be excluded from data analysis and localleads will be notified a nominated data validator will need toensure data accuracy prior to submission if during this processmajor discrepancy is identified within the data set the centre™sdata will be excluded completely from the analysisfor details of the data collected in phase and phase pleaserefer to supplement data analysisphase responses to the survey pertaining to deviations fromdiagnostic and treatment protocols during the covid19 pandemicsee supplement will be converted to a numerical scale where will denote no deviation and will denote complete cessation ofservice provision the scores will be summarized using appropriatesummary statistics and analyzed using unsupervised learningkmeans and hierarchical clustering to identify clusters of homogeneous response to the pandemicphase every month participating centres will report theirdiagnostic and treatment activity see supplement to determine the impact of covid19 on colorectal cancer activity we willuse timeseries methods and data on historical activity and patientoutcomes to estimate a baseline of expected monthly activity that would have taken place in the absence of the pandemicthe baseline and a confidence interval will be estimated atthe national regional and individual nhs trust level the baselineestimate will then be compared to the actual activity as reportedby publicly available data and data collected by this studythe difference between expected and actual activity will providean estimate of the reduction in activityto quantify the impact on patient outcomes associated with theestimated reduction in activity and deviation from standardizedcare protocols we will use estimates of disease progression available in peerreviewed literature [“] a similar methodologywill be used to predict the impact of the pandemic on the incidenceand prevalence of different colorectal cancer stages in the following months under different scenarios predictions will be madeat the regional and national level and depending on data granularity at the trust levelphase will estimate the financial costs of modifications to thecrc service provision due to the covid19 pandemic this willallow prediction of the expenditure and the additional resourcesrequired to resume routine services we will base this on the literature regarding the price of treatment at different disease stages and the information about the cost of resource utilizationconsultations diagnostic tests operating theatre time and hospital stay phase will compare the results from phases “ in theuk with those in the us discussioncancer care and maintaining high standards of diagnosis andtreatment has long been a priority of the nhs and internationalhealth care systems the pandemic has shifted this focus awayfrom the cancer services colorectal cancer patients are particularly 0ca courtney international of surgery protocols “vulnerable to the disruption of their care as diagnosis throughendoscopy was stopped due to concerns about virus aerosolysation this study is important because it is the first study to ask howindividual units had to modify their services and adapt to the newconstraintsin addition to describing the changes and understandingwhether different units had different approaches we wish to gofurther by understanding the effects of diagnostic and treatmentdelay by prospective data collection of cancer cases referrals diagnosis staging and treatment and comparing them to nationally collected audit data these data will allow us to model the economic impact of thedelay and what resources are required to restore cancer servicesto precovid19 standardsthe strengths of this study are in the multimodal approach tothe issues international collaboration and support from the royalcollege of surgeons our diverse team of management and business academics colorectal surgeons nurse specialists and patientadvisors enable us to have a range of approaches to collect andanalyse the datathe main limitation to the study is nonresponder or samplingbias as we require voluntary participation from colorectal teamswe will ensure that we adjust statistical analysis for any underrepresentation we expect that even with minimal participationuseful models can be generated to understand future resourcerequirements at an individual hospital level the methodologyemployed by other units will demonstrate the utility of the modelin summary this is a novel and important multiphase studythat is vital to understand how to best care for cancer patientsand ensure that the effects of the pandemic are mitigated ethics and disseminationthis study is a service evaluation and does not require hraapproval or ethical approval in the uk departmental approvalhas been granted by the university each participating centre mustseek local permission from their local audit department prior tocommencement of data collection in the usa ethical approvalwas granted by the research and development committeedata for phase will be submitted for publication as soon as theresults become available interim data analysis will be presented tothe royal college of surgeons covid19 research collaborativedata for other phases will be submitted for publication once thedata collection has been completed which is anticipated to be afterthe routine service provision resumes all data will be presented atnational and international conferences circumstances permitting guarantornone research registration numbernoneethical approvalnoneauthor contributionsac designed the study wrote the initial proposal drafted themanuscript based on the study proposal and is part of the auditadvisory groupamh ns nd ow sm sr gm nt mg td bs jee ad ptadvised on the study design and the protocol and is part of thesteering committeeck is a project piall authors read commented on and approved the study designthe protocol and the final manuscriptfundingthis research received no specific grant from any fundingagency in the public commercial or notforprofit sectorsdeclaration of competing interestthe authors declare that they have no known competing financial interests or personal relationships that could have appearedto influence the work reported in this paperreferences world health anization the united kingdom who coronovirus diseasecovid19 dashboard covid19whointregioneurocountrygbaccessed june covidsurg collaborative global guidance for surgical care during the covid pandemic br j surg a spinelli g pellino covid19 pandemic perspectives on an unfolding crisisbr j surg “ british society of gastroenterology endoscopy activity and covid19 bsgand jag guidance apr wwwbsgukcovid19adviceendoscopyactivityandcovid19bsgandjagguidance accessed may nhs england nhs improvement letter to chief executives of all nhs trustsand foundation trusts ccg accountable officers gp practices and primary carenetworks and providers of community health services mar wwwenglandnhsukcoronaviruswpcontentuploadssites52202003urgentnextstepsonnhsresponsetocovid19lettersimonstevenspdfaccessed may research uk cancerincidencecancerbowelstatisticswwwcancerresearchukhealthprofessionalcancerstatisticsstatisticsbycancertypebowelcancerincidenceheadingzeroaccessed may royal college of surgeons of england updated intercollegiate general surgeryguidance on covid19 apr wwwrcsengacukcoronavirusjointguidanceforsurgeonsv2 accessed may national institute for health and care excellence nice colorectal cancernice guideline ng151 wwwniceukguidanceng151accessed accessed march national institute for health and care excellence nice suspected cancerrecognition and referral nice guideline ng12 wwwniceukguidanceng12 accessed accessed march c cunningham k leong s clark a plumb s taylor i geh s karandikar bmoran association of coloproctology of great britain ireland acpgbiguidelines for the management of cancer of the colon rectum and anus diagnosis investigations and screening colorectal dis suppl “ s gollins b moran r adams c cunningham s bach as myint a renehans karandikar v goh d prezzi g langman s ahmedzai i geh association ofcoloproctology of great britain ireland acpgbi guidelines for themanagement ofmultidisciplinary management colorectal dis suppl “rectum and anusthe coloncancer of glangman m loughrey n shepherd p quirke association ofcoloproctology of great britain ireland acpgbi guidelines for themanagement of cancer of the colon rectum and anus pathologystandards and datasets colorectal dis suppl “ k leong j hartley s karandikar association of coloproctology of greatbritain ireland acpgbi guidelines for the management of cancer of thecolon rectum and anus follow uplifestyle and survivorshipcolorectal dis suppl “ b moran c cunningham t singh p sagar j bradbury i geh s karandikarassociation of coloproctology of great britain ireland acpgbi guidelinesfor the management of cancer of the colon rectum and anus surgicalmanagement colorectal dis suppl “ md rutter j east cj rees n cripps j docherty s dolwani pv kaye kjmonahan mr novelli a plumb bp saunders s thomasgibson djmtolan s whyte s bonnington a scope r wong b hibbert j marsh bmoores a cross l sharp british society of gastroenterologyassociation ofcoloproctology of great britain and irelandpublic health england postpolypectomy and postcolorectal cancer resection surveillance guidelines gut “ 0ca courtney international of surgery protocols “ healthcare quality improvement partnership hqip national bowel canceraudit annual report an audit of the care received by people with bowelcancer in england and wales v20 wwwnbocaukcontentuploads202001nboca2019v20pdf accessed june pa harris r taylor r thielke j payne n gonzalez jg conde researchelectronic data capture redcap“a metadatadriven methodology andworkflow process for providing translational research informatics support jbiomed inform “ pa harris r taylor bl minor v elliott m fernandez l o™neal l mcleodg delacqua f delacqua j kirby sn duda re consortium the redcapconsortium building an international community of software platformpartners j biomed inform nhs england and nhs improvement cancer waiting times wwwenglandnhsukstatisticsstatisticalworkareascancerwaitingtimesaccessed june national bowel cancer audit nboca datagov weblink accessed junewwwnbocaukresourcesnbocadatagovweblink cancer research uk incisive health saving lives averting costs an analysis ofthe financial implications of achieving earlier diagnosis of colorectal lung andovarian cancer wwwcancerresearchuksitesdefaultfilessaving_lives_averting_costspdf accessed may s sun f klebaner t tian a new model of time scheme for progression ofcolorectal cancer bmc syst biol suppl s2 j emery p vedsted new nice guidance on diagnosing cancer in generalpractice br j gen pract “ hb keshava je rosen mr deluzio aw kim fc detterbeck dj boffawhat if i do nothing the natural history of operable cancer of the alimentarytract eur j surg oncol “ yh lee pt kung yh wang wy kuo sl kao wc tsai effect of length oftime from diagnosis to treatment on colorectal cancer survival a populationbased study plos one e0210465 d roder cs karapetis i olver d keefe r padbury j moore r joshi dwattchow dl worthley cl miller c holden e buckley k powell dburanyitrevarton k fusco t price time from diagnosis to treatment ofcolorectal cancer in a south australian clinical registry cohort how it variesand relates to survival bmj open e031421 cancer research uk bowel cancersurvivalstatistics wwwcancerresearchukhealthprofessionalcancerstatisticsstatisticsbycancertypebowelcancersurvival accessed june kk turaga s girotra are we harming cancer patients by delaying theircancer surgery during the covid19 pandemic ann surg 0c'
Colon_Cancer
dysregulation of ribosome production can lead to a number of developmental disorderscalled ribosomopathies despite the ubiquitous requirement for these cellular machinesused in protein synthesis ribosomopathies manifest in a tissuespecific manner with manyaffecting the development of the face here we reveal yet another connection between craniofacial development and making ribosomes through the protein paired box pax9pax9 functions as an rna polymerase ii transcription factor to regulate the expression ofproteins required for craniofacial and tooth development in humans we now expand thisfunction of pax9 by demonstrating that pax9 acts outside of the cell nucleolus to regulatethe levels of proteins critical for building the small subunit of the ribosome this function ofpax9 is conserved to the anism xenopus tropicalis an established model for humanribosomopathies depletion of pax9 leads to craniofacial defects due to abnormalities inneural crest development a result consistent with that found for depletion of other ribosomebiogenesis factors this work highlights an unexpected layer of how the making of ribosomes is regulated in human cells and during embryonic developmentauthor summarywe are only beginning to understand the complex process of making human ribosomesthe cellular machines critical for all protein synthesis in humans making a ribosomerequires hundreds of regulatory factors to ensure proper cellular growth and development dysregulation of this process can lead to a number of tissue specific disorderstermed ribosomopathies here we have discovered a new role for the protein pairedbox pax9 in making human ribosomes while pax9 has traditionally been known toa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation farleybarnes ki deniz e overton mmkhokha mk baserga sj paired box pax9 the rna polymerase ii transcription factorregulates human ribosome biogenesis andcraniofacial development genet e1008967 101371 pgen1008967editor paul a trainor stowers institute formedical research united statesreceived february accepted june published august copyright farleybarnes this is anopen access distributed under the terms ofthe creative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all rnaseq andrnapii chipseq files are available from the geneexpression omnibus geo database and areaccessible through the geo series accessionnumber gse154764 wwwncbinlmnihgovgeoqueryacccgiaccgse154764 allnumerical data that underlies graphs or summarystatistics is available in the supporting informations4 table genetics 101371 pgen1008967 august genetics 0cfunding this work was supported by the nationalinstitutes of health r01gm115710r01gm122926 and r35gm131687 to sjbr01hd081379 to mkk t32gm007223 to sjb andkif f31de026946 to kif and a pilot grant from theyale cancer center to sjb the funders had no rolein study design data collection and analysisdecision to publish or preparation of themanuscriptcompeting interests the authors have declaredthat no competing interests existpaired box pax9 regulates human ribosome biogenesisplay a role in regulating the levels of proteins required for craniofacial and tooth development in humans we expand this function of pax9 by showing that pax9 acts outside ofthe cell nucleolus to regulate the levels of proteins critical for building the small subunit ofthe ribosome in addition we show that this function is conserved to the model anismxenopus tropicalis this link between pax9™s role in craniofacial development and inribosome biogenesis may lead to new insights into the pathogenesis of these pax9 mutations in humansintroductionwhen ribosome biogenesis is genetically disrupted in humans a number of surprisingly tissuespecific disorders called ribosomopathies arise for example genetic disruption of thetcof1 polr1c or polr1d genes in the ribosomopathy treacher collins syndrome tcsomim results in reduced preribosomal rna prerrna transcription [“] interestingly while these mutations each affect the global process of prerrna transcriptionpatients have specific defects in craniofacial development tcs patients present with hypoplasia of the facial bones micrognathia with or without cleft palate narrowing of the ear canaland bilateral conductive hearing loss [“] modeling the disease in mice has shown that thistissue specificity arises from differential tissue susceptibility to p53 levels p53 levels are stabilized upon disruptions in ribosome biogenesis when free ribosomal proteins bind to mdm2the e3 ligase for p53 [ ] this stabilization of p53 leads to apoptosis of the developing neural crest cells ultimately resulting in the mandibulofacial dysostosis of tcstcs is not the only ribosomopathy to affect craniofacial development for example diamond blackfan anemia dba omim is characterized by anemia low reticulocytecount and elevated erythrocyte adenosine deaminase activity [ ] however dba patientsoften also have craniofacial anomalies and cleft palate reviewed in additionally theribosomopathy acrofacial dysostosis cincinnati type omim is caused by mutationsin polr1a that inhibit prerrna transcription resulting in craniofacial defects amore precise understanding of the role of the factors involved in human ribosome biogenesiscan therefore shed light on the molecular mechanisms underlying aberrant craniofacialdevelopmentthe process of making the cellular machines required for protein synthesis called ribosomebiogenesis includes a large number of factors that work together to control cell growth anddevelopment in humans these proteins are still being defined previous studies have shownthat ribosome biogenesis begins with the transcription of the tandemly repeated ribosomaldna rdna into the 47s polycistronic prerrna fig 1a the prerrna is further modified and processed to create the mature 18s 58s and 28s rrnas these rrnas are incorporated along with the 5s rrna and ribosomal proteins into the small ssu and large lsusubunits of the ribosome the 47s prerrna is transcribed by rna polymerase i rnapiwhile the 5s rrna is transcribed by rna polymerase iii rnapiii various assembly factorsand the ribosomal proteins are transcribed by rna polymerase ii rnapii in addition torequiring all rna polymerases synthesis and assembly of functional ribosomes integrates anumber of cellular signaling pathways to ensure proper regulation of this essential process inresponse to stimulithe complex development of the face is controlled by a number of proteins including several rnapii transcription factors such as paired box pax9 pax9 belongs to a family oftranscription factors that play key roles in anogenesis and neural crest cell development by genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesis genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesisfig pax9 is required for human ribosome biogenesis a ribosome biogenesis at a glance the tandemly repeated ribosomal dna rdna istranscribed into the 47s polycistronic preribosomal rna prerrna by rna polymerase i rnapi this 47s prerrna is processed throughmultiple steps to form the mature 18s 58s and 28s rrnas which are incorporated into the small and large subunits of the ribosome along with the5s rrna and ribosomal proteins ribosomes perform cytoplasmic cellular protein synthesis through the translation of mrnas b pax9 depletionreduces nucleolar number from “ to only in mcf10a cells left panel nuclei stained in hoechst are shown in blue nucleoli are shown in pinkand stained with antifibrillarin antibody as in sigfp top was used as a negative control “ nucleolinucleus and siutp4 middle was usedas a positive control nucleolusnucleus sipax9 is shown at the bottom right panel quantitation of the number of nucleoli per nucleus for sigfptop siutp4 middle or sipax9 bottom c pax9 is not required for rnapi transcription in mcf10a cells a dualluciferase reporter assay wasused to quantify luminescence after sirna depletion of pax9 the plasmids are phrdiresluc firefly to report rnapi transcription and arenilla transfection control as in the ratio of firefly to renilla luciferase was normalized to the sint control n sinol11 was used as apositive control data were analyzed by student™s t test using graphpad prism ���� p � d pax9 is required for pre18s rrnaprocessing in mcf10a cells left schematic of prerrna processing steps in human cells intermediates detected by probe p3 are indicated with ablack box below center northern blot with probe p3 a probe for the 7sl rna was used as a loading control intermediates detected by probe p3 areshown to the right of the northern blot negative controls were mock no sirna and sint nontargeting siutp4 was used as a positive control right quantitation by ramp of probe p3 upper and 7sl lower northern blots graph is mean ± sem n data were analyzed by2way anova using graphpad prism ���� p � ��� p � �� p � and � p � ptp indicates the 47s 45s and 43s processingintermediates e pax9 sirna depletion in mcf10a cells results in an increased ratio of 28s18s by agilent bioanalyzer significance was calculatedby student™s t test in graphpad prism where �� p � f pax9 sirna depletion in mcf10a cells results in decreased global protein synthesis asassessed by the puromycin incorporation assay a representative western blot using an antipuromycin antibody with a β actin loading control isshown to the left protein was harvested after knockdown for hours using the indicated sirnas mock indicates no sirna and mock μmindicates no sirna and half the concentration of puromycin sint nontargeting was used a positive control quantitation of replicates using cellsof different passage numbers is shown to the right significance was calculated by oneway anova in graphpad prism where ���� p � and��� p � g pax9 depletion in mcf10a cells results in decreased 40s 60s and 80s ribosome subunit levels representative polysome profile ofmcf10a cells depleted using sirnas targeting either pax9 red or a nontargeting sint blue control equal amounts of protein were loaded oneach gradient this experiment was performed times using cells of different passage numbers101371 pgen1008967g001controlling gene expression [ ] in humans mutations in pax9 cause tooth agenesis aswell as hair loss [“] and reviewed in indeed pax9 mutations are the most prevalent mutation in patients with nonsyndromic tooth agenesis including oligodontia additionally mice homozygous for a partial deletion of pax9 likely null have craniofacialmalformations including cleft palate skeletal abnormalities and arrested tooth developmentand die a few hours after birth pax9 mice also exhibit a range of cardiac malformations another commonly impacted tissue in ribosomopathies while some research hasbeen done to identify the signaling pathways regulated by pax9 attempts to correct the developmental defects have been only partially successful [“] therefore further studies areneeded to identify all factors regulated by pax9 in order to understand pax9™s role in craniofacial developmentour work ties together pax9 and ribosome biogenesis filling in some gaps in our knowledge of the many cell growth and signaling pathways influenced by pax9 depletion we originally identified pax9 as a potential regulator of ribosome biogenesis in an sirna screen forproteins required to maintain nucleolar number probing pax9™s specific role in makingribosomes in human tissue culture cells we discovered that pax9 is required both for the prerrna processing that produces the small subunit 18s rrna and for global protein synthesiswe employed the genomewide transcriptomics analysis rnaseq to define pax9 dependentmrnas necessary for making ribosomes several of the differentially expressed mrnasincluding several ribosomal proteins were further examined to pinpoint roles for these proteins in prerrna processing and global protein synthesis finally given an established rolefor pax9 in human craniofacial development we sought to model this disease in xenopus tropicalis x tropicalis embryos an established model of human ribosomopathies depletion ofpax9 in x tropicalis did indeed alter craniofacial patterning as well as neural crest development a migratory cell population that plays a major role in establishing craniofacial structurex tropicalis embryos depleted of pax9 also show defective prerrna processing these resultsshed light on the plethora of factors whose expression is regulated by pax9 and open the doorto likely connections between pax9™s role in craniofacial development and human ribosomebiogenesis genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesisresultspax9 depletion disrupts small subunit ribosome biogenesispreviously we performed an sirna screen for new regulators of nucleolar number in humanmcf10a cells we identified pax9 by this screening approach as a protein that whendepleted reduced the number of nucleoli per nucleus from “ to only fig 1b as in cells were fixed after hours of knockdown using pools of sirnas targeting sigfp as a negative control siutp4 as a positive control or sipax9 this transient knockdown approach successfully depleted pax9 s1a and s1b fig the mcf10a cells were then stained with anantibody to fibrillarin fbl a nucleolar protein to detect nucleoli and with hoechst todetect nuclei a cellprofiler pipeline was used to quantify the number of nucleoli per cellnucleus which shifted from “ in sigfp control cells to only in sipax9 and siutp4treatedcells we had previously used oligonucleotide deconvolution to additionally confirm thatpax9 depletion leads to reductions in nucleolar number using this approach individualdepletion of pax9 using of the sirnas from the original pax9 pool reduced the numberof nucleoli from “ to only per cell nucleus [ ] as the sirna screen served as a phenotypic readout of nucleolar function we hypothesized that pax9 plays a role in humanribosome biogenesis through its function as an rnapii transcription factorwe sought to investigate the extent to which pax9 depletion affects human ribosome biogenesis using a panel of assays the first assay probes pax9™s role in rnapi transcriptionusing a dualluciferase reporter system previously published by our laboratory and others [ ] as pax9 is a known rnapii transcription factor it was relevant to test it for a possibleadditional role in rnapi transcription in this reporter assay the ratio of firefly luciferasewhich is under the control of the rdna promoter and measures rnapi transcription wasquantified relative to a renilla luciferase transfection control relative to a nontargeting control sirna sint sirnas targeting pax9 had no significant effect on rnapi transcriptionlevels after hours of knockdown in mcf10a cells fig 1c mock no sirna and sinol11were used as negative and positive controls respectively pax9 is therefore not required forrnapi transcription in mcf10a cellsnorthern blotting was used to define pax9™s role in prerrna processing in humans prerrna processing occurs via a number of different pathways fig 1d left and requires a number of transacting factors we therefore employed different probes to pinpoint any prerrna processing defects occurring after pax9 depletion in mcf10a cells s2a fig after hours of sirna knockdown pax9 depletion resulted in a significant increase in the 30s prerrna intermediate as well as a decrease in the levels of its 21s processing product relative tothe nontargeting sirna sint fig 1d middle and right and s2 fig additionally 41s levelswere decreased relative to the primary processing transcript 47s plus the 45s and 43s processing intermediates herein termed the primary transcript plus or ptp fig 1d middle andright and s2 fig quantitation of the ratios of each intermediate relative to its precursor in theprocessing pathway by ratio analysis of multiple precursors ramp confirmed the statistical significance of these results fig 1d right and s2 fig these effects were also significant relative to a 7sl loading control fig 1d right and s2 fig because the 30s and 21sintermediates are both precursors to the 18s rrna pax9 is required for ssu biogenesis thesame prerrna processing defect was also detected in human embryonic kidney hek293ftand colon carcinoma rko cells depleted of pax9 indicating that pax9™s role in ribosomebiogenesis is conserved among diverse human cell lines s1 figbecause the prerrna processing defects indicate aberrant ssu biogenesis we sought todetermine the extent to which pax9 depletion affects the production of the mature 18s rrnaagilent bioanalyzer quantitation shows an increase in the ratio of 28s to 18s fig 1e genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesiscombined with the northern blot results indicating defects in the processing of the precursorsto the 18s rrna this result is consistent with a predicted reduction in 18s rrna levels takentogether these results argue that pax9 is required likely indirectly for the biogenesis of thesmall subunit of the ribosome which contains the 18s rrnawe also utilized a puromycin incorporation assay to test the extent to which pax9 depletion alters the final product of ribosome biogenesis global cellular translation fig 1f cellswith or without pax9 depletion are treated with a low dose μm of puromycin which isincorporated into all nascent peptides produced during a hour pulse western blottingfor incorporated puromycin shows decreased protein synthesis after hours of pax9 depletion relative to a nontargeting sirna control sint fig 1f mock μm puromycin withno sirna and mock at a halfdose of puromycin μm were used as negative controls fig1f these results confirm that pax9 depletion leads to reduced protein synthesis consistentwith a role for pax9 in ssu biogenesisbecause the above assays indicated a role for pax9 in small subunit prerrna processingand global protein synthesis we also tested the extent to which pax9 depletion is required forribosomal subunit biogenesis and assembly using polysome profiling we determined thatpax9 depletion does result in significantly decreased ssu 40s levels in mcf10a cells fig1g consistent with the reduction in 18s rrna levels seen in fig 1e additionally 60s and80s polysome fractions also showed significant decreases after pax9 knockdown fig 1ginterestingly both here and in previous experiments mcf10a cells do not demonstraterobust polysome fractions using this technique regardless we conclude that pax9 depletion results in decreased ssu biogenesis that impacts the assembly and function of theribosomeas disruptions in ribosome biogenesis result in interruptions in the cell cycle [“] wealso examined the extent to which pax9 depletion changed the distribution of mcf10a cellswithin the cell cycle using flow cytometry s3 fig relative to a sint control depletion ofpax9 for hours resulted in a minor increase in the proportion of cells in g1 phase of thecell cycle but this was not statistically significant s3 fig sirnas targeting the ribosome biogenesis factor nol11 were used as a positive control and depletion of this protein resulted inan increase in the proportion of cells in g2 s3 fig consistent with previous findings inall these assays allowed us to conclude that pax9 regulates human ribosome biogenesisrnaseq analysis upon pax9 depletion reveals decreased levels ofnucleolar mrnas responsible for small subunit maturationas pax9 is a known rnapii transcription factor we hypothesized that pax9 works indirectlyto modulate ssu biogenesis fig 2a this is consistent with a nuclear but not nucleolar localization of pax9 in existing databases [“] pax9 may act directly as a transcription factorfor nucleolar proteins or indirectly for proteins that affect the expression or function of nucleolar proteins to test the hypothesis that pax9 affects nucleolar protein expression through itsfunction as a rnapii transcription factor we used rnaseq in mcf10a cells to define the setof mrnas that were differentially expressed after pax9 depletion relative to a nontargetingcontrol sirna sint pax9 depletion resulted in the differential expression of over mrnas fold change � or � q � fig 2b and s1 table approximately half of these were reduced in their levels consistent with the hypothesis that pax9 acts as a transcription factor to drive their expressionwhen considered as a whole the rnaseq dataset is enriched for several pathways knownto be regulated by pax9 s4a fig ingenuity pathway analysis of the differentiallyexpressed mrnas reveals enrichment of both wntca2 signaling differential expression of genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesis genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesisfig rnaseq transcriptomics analysis in human tissue culture cells reveals changes in the expression levels of over nucleolar mrnas afterpax9 knockdown a schematic of how pax9 would act as a rnapii transcription factor to drive the levels of mrnas required for making the smallsubunit ssu of the ribosome in the cell nucleus pax9 binds to dna to affect the transcription of mrnas that either encode nucleolar proteinsdirect solid arrow or to transcribe mrnas that affect the levels of mrnas encoding nucleolar proteins indirect dotted arrows the resulting mrnasare translated in the cytoplasm into proteins that function in ssu prerrna processing in the nucleolus b rnaseq analysis after pax9 sirnadepletion in mcf10a cells reveals decreased levels of mrnas encoding nucleolar proteins relative to a nontargeting sirna control sint pax9depletion resulted in differential expression of mrnas fold change � or and fdr � of these mrnas had a decreased foldchange � and of those mrnas code for proteins designated as nucleolar in at least one of three databases [“] of the mrnas whoselevels were decreased and that also code for nucleolar proteins were chosen as candidates for followup studies c qrtpcr confirms reduced mrnalevels of the rnaseq candidates after pax9 sirna knockdown in mcf10a cells after depletion using sirnas targeting either pax9 or a nontargeting control sirna sint the levels of the indicated mrnas were quantified by qrtpcr using primers to each target gene relative to a 7slcontrol and sint data are shown as mean ± sem three replicates using cells of different passage numbers with technical replicates each wereperformed significance was calculated by oneway anova using graphpad prism where ���� p � d depletion of of the candidatemrnas rps6es6 rps9us4 rps28es28 and fbl individually results in the same prerrna processing defect as pax9 sirna depletion in mcf10acells representative northern blot after knockdown of the indicated sirnas using probe p3 a probe for the 7sl rna was used as a loading control prerrna processing intermediates detected by probe p3 are shown to the right of the northern blot ptp indicates the 47s 45s and 43s prerrnaprocessing intermediates e quantitation of northern blots using probe p3 as shown in fig 2d using ramp graph is mean ± sem n datawere analyzed using 2way anova in graphpad prism where ���� p � ��� p � and �� p � quantitation relative to the 7sl loadingcontrol is shown in s5 fig f sirna depletion of rnaseq candidates in mcf10a cells results in decreased global protein synthesis after hoursof knockdown with the indicated sirnas mcf10a cells were pulsed with puromycin for hour and protein was harvested western blotting with anantipuromycin antibody as well as a β actin loading control was carried out representative western blots shown to the left mock mock at half theconcentration of puromycin μm and sint nontargeting sirnas were used as negative controls g quantitation of three replicates usingmcf10a cells of different passage numbers of the puromycin incorporation assays following depletion with the indicated sirnas relative to the sint andβ actin loading controls is shown as mean ± sem n significance was calculated by student™s ttest using graphpad prism where ���� p � ���p � and � p � 101371 pgen1008967g002 pathway members p x ˆ’ s4b fig and wntβcatenin signaling differentialexpression of pathway members p x ˆ’ s4c fig in cells depleted of pax9expression levels are increased for many of the mrnas in the wnt signaling pathway this isconsistent with previous results suggesting a role for pax9 in the negative regulation of wntsignaling [ ]as an additional validation of the rnaseq dataset we determined that the genomesequences kb upstream of the start sites of the differentially expressed mrnas containmultiple potential pax9 binding sites making these mrnas candidates for direct transcriptional regulation by pax9 scanning for known pax9 binding sequences [™sgtcacgcwtgantgma3™ ™cgcgtgaccg3™ cd192ains ™gcgtgacca3™ and e5 ™gcggaacgg3™] in the kb upstream of the mrnas using centrimo analysis reveals and potential pax9 binding sites respectively in the kbupstream of the mrnas this number of potential binding sites upstream of the mrnas sites per gene is similar to that observed in pax9 chip experiments in the vertebral column of e125 mice sites per gene additionally centrimo enrichmentanalysis of the sequence kb upstream of each of the differentially expressedmrnas reveals significant enrichment of different dna binding sequences including thepax3 pax5 pax6 and pax7 dna binding domains as multiple pax proteins can bindthe same dna sequence this provides further evidence for pax9 regulation of these mrnas these analyses therefore support the hypothesis that pax9 regulates the levels of themrnas identified in our rnaseq datasetin the rnaseq dataset many of the differentially expressed mrnas have knownroles in nucleolar function for example have appeared in other genomewidesirna screens for nucleolar function s1 table [ ] additionally of the differentially expressed mrnas code for proteins designated as nucleolar in at least of nucleolar databases s1 table [“] this is a significant enrichment in the expected number of nucleolar proteins assuming that nucleolar proteins make up only of the proteins inhuman cells surprisingly expression of most of the mrnas encoding nucleolar proteins genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesis was downregulated upon pax9 depletion indicating that pax9 is requiredto maintain normal levels of many mrnas whose protein products are destined for functionin the nucleolus s1 tableto determine the mechanism of pax9™s function in ssu biogenesis we chose candidatesfrom the rnaseq dataset to follow up on in greater detail the mrna levels for the candidates were all downregulated after pax9 depletion and all code for nucleolar proteins fig 2band s1 table [“] four of the candidates rps6es6 rps9us4 rps28es28 and fblwere chosen on the basis of literature suggesting a role for these proteins in ssu prerrnaprocessing in hela cells [ ] additionally it was pertinent to analyze rpl5ul18 as it hasa known role in the p53dependent nucleolar stress response qrtpcr confirmed thernaseq results with reduced levels of each mrna when pax9 is depleted in mcf10a cellsfig 2c interestingly depletion of either rps9us4 or rps28es28 resulted in a decrease innucleolar number from “ to only in our original sirna screen therefore it is possible that the mechanism through which pax9 depletion results in decreased nucleolar numberrelies upon reduced expression of rps9us4 andor rps28es28 fig 2awe were able to confirm that depletion of of the tested candidates rps6es6 rps9us4 rps28es28 and fbl in mcf10a cells resulted in prerrna processing defects similarto that of pax9 depletion fig 2d and 2e s5 fig only rpl5ul18 did not give the 30sincrease characteristic of pax9 depletion although this was expected given its known role inlsu prerrna processing additionally depletion of several of the candidates individually resulted in significantly decreased global protein synthesis by the puromycin incorporation assay similar to the effect seen after pax9 depletion fig 2f and 2g puromycinincorporation was also reduced after rps6es6 depletion although it was not statistically significant fig 2f and 2g therefore pax9 may function as a transcription factor to directly orindirectly increase the expression of rps6es6 rps9us4 rps28es28 andor fbl fig 2aas the proteins encoded by these mrnas are required for ssu ribosome biogenesis fig their depletion after pax9 knockdown is a plausible mechanism through which pax9 regulates prerrna processing and global protein synthesisrnapii chipseq analysis reveals decreased transcription of mrnasencoding nucleolar proteins after pax9 depletionas the rnaseq analysis confirmed that levels of nucleolar mrnas were decreased afterpax9 depletion fig we sought to map how rnapii distributes on genes using rnapiichipseq as a readout of transcription through this approach we aimed to untangle the effectsof pax9 depletion on rnapii transcription from its effects on mrna stability since rnapiichipseq is able to detect genomewide changes in rnapii occupancy as pax proteins areable to both activate and repress target protein expression we have included genes withboth increased and decreased rnapii occupancy in this analysis approximately mrnaswere differentially occupied by rnapii upon pax9 knockdown compared to the nontargeting control sirna sint in mcf10a cells fold change cutoff � or � and maxtags � s2 table of these had decreased rnapii occupancy consistent with pax9 acting as a transcriptional driver of these mrnasto assess the validity of the rnapii chipseq dataset we again used centrimo to identifypotential pax9 dnabinding sites in the kb upstream of the genes with differentialrnapii occupancy searching for the ™ cgcgtgaccg ™ pax9 binding motif definedin revealed possible sites in the bp upstream of the differentially occupiedgenes also the known pax9 dna binding motifs cd192ains ™gcgtgacca3™ ande5 ™gcggaacgg3™ had and binding sites in these sequences respectively genetics 101371 pgen1008967 august genetics 0cpaired box pax9 regulates human ribosome biogenesisadditionally analysis of motif enrichment ame identified the pax5 and pax6 dnabinding motifs as being significantly enriched in the kb of sequence upstream of the genes p � since multiple pax proteins can bind the same motif this suggests thatthis dataset does contain mrnas that are regulated by pax9 of the differentially occupied genes have also been shown to be differentially regulated by pax9 directly in pax9chipseq experiments on e125 wt vertebral column murine tissue fig 3a and s2 table these analyses confirm the ability of rnapii chipseq to detect changes in rnapiimediated transcription after pax9 knockdownbased on the hypothesis that pax9 acts as an rnapii transcription factor for regulators ofnucleolar function fig we expected to detect changes in the rnapiimediated transcription of a number of mrnas encoding nucleolar proteins after pax9 depletion indeedmrnas coding for nucleolar proteins were enriched with of the genes with differentialrnapii occupancy coding for nucleolar proteins in at least one of three databases s2table [“] this is again higher than would be expected assuming that nucleolar proteins account for approximately of all cellular proteins additionally depletion of onegene with differential rnapii occupancy anln resulted in decreased nucleolar number inour sirna screen similar to the phenotype seen after pax9 depletion fig 1b notably of the genes with differential rnapii occupancy appeared in other genomewide screens for ribosome biogenesis factors s2 table [ ] another targeted screeninvestigated the effects of of the genes top2a and cdca8 on prerrna processingwhen depleted by sirna in hela cells however depletion of neither gave the 30s prerrna increase on northern blots characteristic of pax9 depletion in al
Colon_Cancer
antibioticresistant pathogen strainlevel investigations are beginning to reveal themolecular mechanisms used by vrefm to colonize regions of the human bowelhowever the role of commensal bacteria during vrefm colonizationin particularfollowing antibiotic treatment remains largely unknown we employed amplicon16s rrna gene sequencing and metabolomics in a murine model system to try andinvestigate functional roles of the gut microbiome during vrefm colonization firstorder taxonomic shifts between bacteroidetes and tenericutes within the gut microbial community composition were detected both in response to pretreatment usingceftriaxone and to subsequent vrefm challenge using neural networking approaches to find cooccurrence profiles of bacteria and metabolites we detected keymetabolome features associated with butyric acid during and after vrefm colonization these metabolite features were associated with bacteroides indicative of a transition toward a preantibiotic naive microbiome this study shows the impacts of antibiotics on the gut ecosystem and the progression of the microbiome in responseto colonization with vrefm our results offer insights toward identifying potentialnonantibiotic alternatives to eliminate vrefm through metabolic reengineering topreferentially select for bacteroidesimportance this study demonstrates the importance and power of linking bacterial composition profiling with metabolomics to find the interactions betweencommensal gut bacteria and a specific pathogen knowledge from this researchwillinform gut microbiome engineering strategies with the aim of translatingobservations from animal models to humanrelevant therapeutic applicationscitation mu a carter gp li l isles ns vrbanacaf morton jt jarmusch ak de souza dpnarayana vk kanojia k nijagal b mcconvillemj knight r howden bp stinear tp microbemetabolite associations linked to therebounding murine gut microbiomepostcolonization with vancomycinresistantenterococcus faecium msystems 5e0045220101128msystems0045220editor manuel liebeke max planck institutefor marine microbiologycopyright mu this is an openaccess distributed under the terms ofthe creative commons attribution international licenseaddress correspondence to andre muandremuunimelbeduaureceived may accepted july published august julyaugust volume issue e0045220msystemsasm 0cmu keywords microbiome multiomics metagenomics metabolomics gutmicrobiome vancomycinresistant enterococci colonization antimicrobial resistanceceftriaxonevancomycinresistant enterococcus faecium vrefm is a significant health careassociated pathogen vrefm infections can be difficult to treat due to their intrinsicand acquired resistance to nearly all classes of antibiotics the world healthanization categorizes vrefm as a œhigh priority bacterial pathogen advocatingresearch to stop the global increase in antibiotic resistance recent studies highlightthe importance of the gut microbiota in modulating the growth and virulence of vrefmin the gastrointestinal ecosystem for instance the depletion of normal gut flora usingantibiotics exacerbates the severity of vrefm infection whereas transplant ofcommensal species including a consortium of clostridium bolteae blautia productablautia sartorii and parabacteroides distasonis can drive established vrefm colonization to below levels of culture detection specifically b producta”a colonizer ofthe colon”reduces vrefm growth in vivo by secreting a lantibiotic these observations raise the intriguing possibility that metabolic traits act in concert betweenpathogen and select gut commensals to confer mutual benefits during pathogenpersistence these findings also highlight the greater risk posed to immunocompromised patients when colonized with vrefm for instance allogeneic hematopoietic celltransplantation patients have gastrointestinal tracts that are dominated by vrefm as aresult of losing a large portion of the intestinal commensal microbiota upon receivingbroadspectrum antibiotics as pretreatment hildebrand discovered longtermecological impacts to the gut microbiome with strong bacterial species turnover afterceftriaxone treatment in humans further mice receiving broadspectrum antibioticscombination of metronidazole neomycin and vancomycin showed markedly increased vrefm colonization of the cecum and colon the compromised intestinal innateimmune defenses in these animals allowed proliferation of vrefm caused by theantibiotic exposure and subsequently reduced the expression of antimicrobial molecules produced by bacteria in the intestinal mucosa the problem with vrefm is further complicated by the fact that enterococci aremembers of the gastrointestinal tract microbiota a key reservoir of antimicrobialresistance amr genes and potentially facilitating gene transfer within the gut microbiome for example the vanb resistance gene was detected in human fecalspecimens that did not contain culturable vre and instead demonstrated that isolatescarrying the resistance transposon are anaerobic commensal bacteria eggerthella lentaand clostridium innocuum colonization of and persistence in the gastrointestinaltract therefore presents as a key mechanism for de novo vre and may lead to severeinvasive diseasethe current study aimed to understand the impact of antibiotics on the murine gutmicrobiota and the subsequent colonization pattern of vrefm to this extent wedesigned a murine model timeseries study that consisted of two main perturbativephases i antibiotic pretreatment with ceftriaxone and ii vrefm challenge our 16srrna gene profiling analyses highlighted a firstorder shift in bacterial biodiversitycomposition across time a secondorder clustering of samples associated with theexperimental phases and the transition of the postvrefm colonization gut microbiotaand its metabolome toward resembling an asymptomatic carriagelike microbiomephenotype this research provides support for engineering the metabolic potential ofthe gut microbiome using for example prebiotics as a nonantibiotic alternative fortreating multidrugresistant bacterial infectionsresultsexperimental design the following experimental design was developed to address the hypothesis that there are specific murine gut microbiome factors thatfacilitate vrefm colonization three groups of three c57bl6 mice cocaged wildtypejulyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vretable summary of samples analyzed in this studyday of exptphase of exptannnnnnabxtxabxtxabxwnvreevreevreevreevrelvrelamplicon 16s rrnagene databœ“œ“œ“«º«ºœ“œ“œ“œ“œ“«º«ºœ“œ“œ“metabolomicsbœ“«º«º«º«ºœ“œ“œ“œ“œ“«º«º«º«ºœ“avg no ofobserved sotusc«º«º«º«ºathe key phases of the experiment where n represents naive abxtx represents antibiotic treatment abxwn represents antibiotic weaning vree represents earlyphase postvrefm colonization and vrelrepresents latephase postvrefm colonizationbsymbols œ“ sample processed «º data unavailablecthe average number of sotus observed across all mice for each day of the experimentmales were monitored and fecal samples were collected over a 14day period with twointervention time points including i ceftriaxone treatment administered at 05gliter indrinking water across a 2day period and ii colonization via oral gavage with «» vrefm st796 per mouse postantibiotic treatment at a single time point mice werehoused in groups of five and samples were collected from the same three mice torepresent technical replicates per cage herein each group of cohoused mice will bereferred to as group a group b and group c the remaining two mice per group werereserved for microbiological assays table highlights samples and data sets collectedamplicon 16s rrna gene sequencing revealed firstorder shifts in bacterialcommunity composition amplicon 16s rrna gene sequencing was performed tocapture the bacterial community composition in an effort to track changes in responseto antibiotic pretreatment and vrefm colonization bacterial community profiles wereassessed in fecal samples from nine mice before during and after the two interventionstable a total of of reads reads passed quality control with reads on average per sample and a total of exact variant sequence typesie features with an average of features per sample and an upper bound of features when rarefied to reads alpha rarefaction analysis demonstratedsufficient sequencing depth to capture microbial diversity to saturation see fig s1 inthe supplemental materialthe biodiversity profiles of each sample were compared and showed that keysuboperational taxonomic units sotus were differentially abundant throughout thecourse of the experiment fig there was a shift in the dominance of bacteroidiabacteroidetes light green colored bars during the naive phase of the experiment tomollicutes tenericutes fuschia colored bars in response to ceftriaxone treatment witha return to the predominance of bacteroidia during the late phase of the experimentafter vrefm colonization ie days to of note is the predominance of lactobacillales in mouse to from group a fig the murine gut microbiota responds to antibiotics and microbial communityrichness begins to rebound days after vre colonization principalcoordinateanalysis pcoa of the unweighted unifrac distances was used to assess clusteringof fecal samples based on bacterial composition this assessment showed that the fecalmicrobiota from samples collected from each phase clustered together but were clearlyseparated between phases after exposure to ceftriaxone and challenge with vrefmpostantibiotic treatment fig 2a permutationbased statistical testing demonstratesthe groups are significantly different from one another fig s2 temporal tracking ofjulyaugust volume issue e0045220msystemsasm 0cmu fig biodiversity plot of sotus as relative frequencies at the taxonomic level of class firstorder shifts in microbial communitycomposition as revealed by 16s rrna gene community profiling from a predominance of bacteroidetes to tenericutes and return tobacteroidetes was observed each column displays the relative bacterial community composition in a mouse fecal sample collecteddaily and sorted by the chronology of the experiment ie day of experiment table the columns are further sorted by group iegroup a group b and group c and individual mice within each group mouse mouse and mouse stacked bars are presentedas relative frequencies at the taxonomical level of class however annotations of key taxa are at the phylum level bacteroidetes [green]firmicutes [gray] and tenericutes [fuschia] or order level lactobacillales [yellow]the changing microbiomes against each mouse on the pcoa sample space demonstrated a clear unidirectional trajectory that followed the chronology of the experiment106084m9figshare12775859 procrustes analyses of weighted andunweighted unifrac distances showed that the same general patterns on the samplespace were preserved meaning that there is congruency in global spatial patternsbetween qualitative and quantitative measures of community dissimilarity fig s3analysis of community diversity faith™s phylogenetic diversity index revealed astable and rich microbial community during the naive phase preceding a sharpdecrease following antibiotic treatment and a further decrease immediately followingvrefm colonization fig 2b of note is the responsiveness of the microbiota within h to the removal of antibiotics at the end of day community richness began torebound at approximately days after vrefm colonization ie day with group ademonstrating a higher rate of rebound compared to groups b and c calculating thedistances of dissimilarity unweighted unifrac distances of each mouse microbiotatime point relative to day a proxy for the naive bacterial community phenotyperevealed a small dissimilarity distance for samples collected during the naive phase andan increasing dissimilarity distance following antibiotic treatment day and vrefmcolonization day fig 2c there was a downward trajectory in distance scores daysafter vrefm colonization ie day group a followed a sharper return to a microbiotaresembling day these observations suggest that mice were transitioning toward apersistent carrierlike state and that the rebounding community richness toward levelsrepresentative of the naive phase was by a microbial community structure that resembled the naive phase additional studies where the time frame of postvrefm challengeextends beyond week of monitoring are needed to understand whether the perturbed microbiome will return to resemble an absolute naive state or arrive at a newaltered statemultinomial regression identifies sotus most positively associated with vrefmcolonization multinomial regression using songbird was employed to identify sotusjulyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vrefig diversity analyses a principalcoordinate analysis plot of unweighted unifrac distances data points areprojected onto the sample space and colored by prevrefm colonization red and postvrefm colonization bluenote that circles and ellipses function to highlight the separation of experimental phases and do not indicatestatistical confidence intervals principal coordinate axis explains of the variation observed between thenaive microbiota and those from the postvrefm colonization phase b community richness of the murine gutmicrobiome as measured by faith™s phylogenetic diversity in response to ceftriaxone treatment and challengewith vrefm c community dissimilarity distances as calculated by unweighted unifrac of each time point relativeto day naive phasethat were most positively and negatively associated with the postvrefm colonizationphase fig the five most positively associated sotus were enterococcus bacteroideserysipelotrichaceae catabacter and lachnospiraceae while the five most negativelyassociated were clostridiales adlercreutzia mollicutes peptostreptococcaceae and clostridiales temporal tracking of exact sequence variants esvs demonstrated that theesv feature classified as enterococcus”and identified as the most positively associatedwith the postvrefm colonization phase”was most abundant on days of challengeconfirming that this esv likely was the st796 vrefm colonization challenge anismsfig there were a further eight esv features classified as enterococcus however theywere absent during the days representing vrefm colonization and lacked positiveassociations with the postvrefm colonization phase suggesting that these featuresrepresent murine gut commensal enterococcijulyaugust volume issue e0045220msystemsasm 0cmu fig multinomial regression multinomial regression identified an enterococcus exact sequence variant as the most positivelyassociated with the colonization phase log fold change score of read counts for the enterococcus esv tracked daily acrossthe experiment showing high abundance during the days of vrefm challengemolecular networking identifies differential metabolome profiles duplicatefecal samples from key time points throughout the experiment ie days and were analyzed by datadependent tandem mass spectrometry msms performed on a liquid chromatography quadrupole time of flight lcqtof system tomonitor changes in the murine gut metabolome table polar metabolite analysiswas given preference in an effort to broadly capture primary metabolites that play a keyrole in œmetabolic handoffs that define interspecies interactions analysis of the globalmetabolome profile of each sample was measured based on their overlapping molecules and a pcoa plot using a binary jaccard distance metric through the global naturalproducts social molecular networking gnps platform a separation of metaboliteprofiles along pcoa1 was observed fig 4a metabolomes from the naive andlate vrefm colonization phase tended to cluster together while samples from thepostantibiotic phases including the early vrefm colonization phase clustered togethersupporting pairwise permutational multivariate analysis of variance permanovatesting fig s4 highlights that naive and early vre samples are significantly differentwhile late vre has a lower distance to naive samples compared to antibiotictreatedantibiotic wean and early vre samplesrandom forest analysis of spectrum profiles from lcmsms was used to predictexperimental phase and rank the importance of metabolite association with eachexperimental phase the top metabolite features for each experimental phase arehighlighted in fig 4b unique profiles of metabolite features were observed for eachphase of the experiment importantly the late vrefm colonization phase capturesan unknown metabolite feature with a masstocharge ratio mz of and retention time rt of this metabolite is exclusively present during whatrepresents the transition toward resembling the naive microbiome manual curationoffeature in positiveion mode predicts a molecular formula c5h8n4o3with ±–10 ppm in mass error the major peaks in the msms spectrum for feature are precursor ion [m«¹h]«¹ assumed precursor ion [h2o] product ion [likely c2h2o] [c2h2o«¹h] plus product ionfurther supporting neutral loss of c2h2o given the summation of results the chemicalstructure of feature is likely to contain a nacetylated hydroxyl group peakquantification values indicate its presence during the late phase of vre colonizationfig 4c further manual curation of msms data identified ceftriaxone as feature julyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vrefig metabolomic analyses a emperor plot displaying principalcoordinate analysis of binary jaccard distances of metabolomic profiles samples are colorcoded and the colors represent the naive orange antibiotic treatment red antibiotic weaning blue early vre colonization green and late vrefmcolonization purple phases b random forest classifier identifying metabolite features spectra for each phase of the experiment the heatmap is color codedfrom low ranking score white ie lowest importance to high ranking score dark blue highest importance metabolite features are labeled by theirmasscharge ratios and retention times for the reason that current databases do not capture their chemical structure andor identifications abx tx antibiotictreatment c peak quantification values for feature mz «½ and rt «½ present in abundance during vre colonization late phase dpeak quantification values for ceftriaxone mz «½ and rt «½ tracked across the experiment ceftriaxone values are highest during antibiotictreatment phase and begins to wane during antibiotic weaning phase julyaugust volume issue e0045220msystemsasm 0cmu with an mz of and rt of and mostly abundant during days of antibioticexposure fig 4dbacteroidalesassociated metabolites implicated in latephase postvrefm colonization a distinct profile shift in microbe and metabolite abundances as calculatedby multinomial regression was observed particularly during latephase vrefm colonization fig s6 shallow neural networking analysis with mmvec was used to predictmicrobemetabolite interactions through their cooccurrence probabilities fig sequential biplots captured the shift in experimental phases and highlighted the cooccurrences of microbiota and metabolomic data sets fig 5a to c there was a strongenterococcus effect as indicated by the magnitude of the corresponding arrow and therebounding species during the latephase vrefm colonization are predominantly bacteroidales sotus fig 5c with cooccurring metabolite features mz rt and mz rt metabolite feature mz rt was ranked asbeing highly associated with the postvre colonization phase these results integratemicrobial and metabolite data sets to reveal which microbes may be responsible fordetected metabolites in this instance the metabolite present during the phase representing a transition toward a microbiome approximating the naive state feature mz and rt fig 4b is linked with bacteroidales fig 5adiscussionin this study of the murine gut ecosystem we employed a mouse model ofgastrointestinal tract colonization that replicates the shift in bacterial compositionwhen patients enter the health care system develop an imbalance in their microbiomeas a result of pretreatment eg antibiotic treatment and are subsequently colonizedwith a hospital superbug the resolution of current studies describes a consortiumof commensal microbes that can for example reduce the magnitude of vrefm colonization however understanding the key metabolic shifts relative to the gutmicrobiota remains challenging here we employed amplicon 16s rrna genesequencing and highresolution mass spectrometry metabolomics in an effort towarddetermining microbiotametabolome interactions during vrefm colonization we demonstrated clear changes in the gut microbiome in response to ceftriaxone and vrefmchallengeconceptual and statistical advances in analysis of amplicon 16s rrna gene data whereby otus are clustered at a nucleotide similarity threshold allows for theidentification of exact sequence variants esvs query against an errorcorrecteddatabase can detect multiple esvs that may be classified to the same taxonomicrank for example our analyses identified multiple esvs classified as enterococcihowever when the relative abundances were tracked across the chronology of theexperiment only one enterococcus esv was dominant in relative abundances and mostpositively associated with the days of postvrefm challenge fig this highlights theresolving power to differentiate between commensal and pathogenic strains of enterococci when the composition of the microbial community is considered the factthat this was achievable at the level of amplicon 16s rrna gene sequencing alludes tothe possibility of implementing microbiota screenings as routine diagnostics for patients entering health care systems further firstorder level shifts in microbial community composition was observed in response to ceftriaxone and subsequent vrefmchallenge fig three days after vrefm colonization ie day the microbiomerichness begins to rebound suggesting that mice are transitioning toward a persistentcarrierlike state interestingly the group a cohort exhibited a higher rate of reboundthat may be facilitated by their initially higher microbial community richness andpredominance of lactobacillales on the day of vrefm challenge fig 2b this observation supports the need to prescreen œbaseline microbiota profiles of patients uponadmission into hospital for the reason that it is not necessarily which microbialpopulations are removed postperturbation eg antibiotic pretreatment but insteadwhich populations persist that drives the responding phenotype we can begin toassess patients from across different wards eg intensive care unit oncology neuroljulyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vrefig microbemetabolite vector biplots sequential biplots highlighting the changing metabolitedifferentials across each key phase of the experiment abx tx is the antibiotic treatment phase and abxwean is the period when antibiotics were removed for a 24h period prior to colonization with vrefmeach point on the sample space represents metabolites and arrows represent microbes microbe andcontinued on next pagejulyaugust volume issue e0045220msystemsasm 0cmu ogy and healthy cohorts and build a database of microbiome profiles that can be usedas biomarkers to predict i the susceptibility of patients to develop persistent bacterialcolonization and ii propensity to clear the pathogen once colonized the clinicalimplication is that new patients are screened and identified via betadiversity metaanalyses by these biomarkers and placed in bedding cohorts accordingly therebyimproving infectious disease management and isolation precautions within healthcareassociated ecosystemsthe shortlist of microbes ranked as most negatively associated with the colonizationphase clostridiales adlercreutzia mollicutes peptostreptococcaceae and clostridialesfig are hypothesized to play a role in maintaining the health of the animals indeedamong the microbes identified are known shortchain fatty acid eg butyric acidproducers which supports and expands upon those previously identified bycaballero further the use of deblur to identify esvs facilitates the temporaltracking of their relative abundances to inform selection of primary fecal samples thatwill provide the best probability ie highest relative abundance of culturing targettaxa for downstream screening of probiotic potential however translating animalderived observations from experimental animal models to human clinical situationsremains challenging particularly where the key microbes are rodentspecific microbesone solution may be to integrate metabolomics to reveal shared metabolic capacityamong taxonomically divergent microbes our supervised classifying approaches suggests an altered metabolome composition during the late phase of vrefm challengethat may facilitate the apparent œsuppression of vrefm to levels below the limit ofdetection by culture despite the caveat of poor resolution in current databases to linkmetabolite features to associated chemical structures microbemetabolite vector analysis linked metabolite feature mz «½ and rt «½ to bacteroidesfig our efforts toward manually identifying feature suggests a chemicalformula of c5h8n4o3 and a structure likely to contain a nacetylated hydroxyl group aputative annotation through pubchem search is 3hydroxy4nitrosocyanamido butyramide butyramide is the amide group of butyric acid a shortchain fatty acid thathas been shown to play a key role in colonization resistance against intestinal pathogens “ further research to comprehensively characterize interactions betweenmicrobe and metabolites will be critical to address the gaps in our understanding of thebiochemical parameters that define interspecies microbiome interactions during antibiotic pretreatment and persistent infectionsthe resolution of our results provides the basis in which to begin to identifynonantibiotic alternatives to engineer the gut microbiome through prebiotic interventions eg butyric acid and translating animal studies to humanrelevant therapeuticapplications by delineating taxonomically diverse microbes with shared metaboliccapacity here achieving integrative omics to link microbemetabolite associations ourfindings add support to the incorporation of microbiome profiling approaches intoroutine clinical microbiology particularly in the context of monitoring the impacts ofantibiotic usematerials and methodsmouse gastrointestinal colonization model sixweekold wildtype c57bl6 male mice were usedto establish an animal model of gastrointestinal colonization with vrefm mice were cohoused and hadfree access to food ordinary chow and water and had environmental enrichment eg fun tunnels chewblocks and tissue paper the lightdark cycle was 12h light12h dark and cages were changed weeklyfig legend continuedmetabolite features are fixed upon the sample space with gradient coloring of metabolites indicating thetransition across key phases of the experiment the distance between each point is indicative ofmetabolite cooccurrence frequency and the angle between arrows indicates microbial cooccurrencethe directionality of the arrows describes the variation in the metabolites explained by the microbesrepresented by the arrows for example metabolite feature mz and rt isdemonstrated to cooccur with bacteroides information about the abundances of these cooccurringfeatures are provided as heatmaps in fig s6 in the supplemental materialjulyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vremice were pretreated with gliter ceftriaxone in drinking water for days followed by an antibioticwean period of h mice were then challenged with «» cfu vrefm st796genomic dna extraction and sequencing wholecommunity genomic dna gdna was extractedfrom mouse fecal samples using the qiagen powersoil dna extraction kit formerly mobio following themanufacturer™s protocol a preprocessing step of mechanicallysis was incorporated using a bertintechnologies precellysis machine for one round of a 40s cycle at rpm the v4 region of thebacterial 16s rrna gene was amplified using small subunit forward golaybarcoded and ssu806reverse primers following the earth microbiome project protocol and sequenced using the illuminamiseq platform v2 cycles illumina inc san diego ca usa further primary derived data egbiom tables used to produce results can be found within qiita study id amplicon 16s rrna gene profiling analyses sequence data were processed within the qiitav010 framework for quality control split libraries v q2191 demultiplexing trimming sequencereads to a length of nucleotides nt and picking suboperational taxonomic units sotus usingdeblur v110 to resolve singlenucleotide community sequencing patterns ie feature identification ofsotus [] the output biom files were further processed using qiime2 v20197 for downstreamstatistical analyses alpha rarefaction curves were generated to determine whether each sample hadbeen sequenced to saturation the feature table was subsequently rarefied to reads per sampletaxonomy was assigned using the sklearn classifier and greengenes otus from 515f806rregion of sequences classifier available from docsqiime220184dataresources furthermorerelative abundances of each taxa were visualized as bar plots using the qiime2 taxa plugin a phylogenetic tree was constructed using fragment insertion qiime fragmentinsertion sepp [] to guidephylogeneticaware statistical analyses generated using the qiime2 plugin q2diversity coremetricsphylogenetic key metrics computed by default include both alphadiversity eg shannon™s diversityindex faith™s phylogenetic diversity and evenness and betadiversity eg braycurtis distance andunweighted unifrac distance metrics the unweighted unifrac distance matrix was used tocompute first distances and calculate distances relative to day as the baseline between sequentialstates qiime longitudinal firstdistances ggplot2 r v360 ggplot2tidyverse was used tovisualize the distance scores as line plots emperor was used to visualize principalcoordinate analysisplots of unweighted unifrac distances permutationbased statistical testing permanova on unweighted unifrac distances was used to determine whether samples grouped by phase of experimentwere significantly different from one another q2betagroupsignificance songbird githubcommortonjtsongbird was employed to determine the importance ie fold change of each sotu inrelation to a given metadata variable eg vrefm colonization microbial features from all samples weresplit into training and test sets for supervised learning classifier analyses of input samples wereallocated to train the random forest classifier within qiime2 the estimator method used for sampleprediction the different experimental phases were the response variables while the 16s rrna gene datawere the featuresmetabolite extraction and liquid chromatographytandem mass spectrometry analysis duplicate fecal samples as outlined in table were processed for polar metabolite extraction and analysisdays and feces were metabolically arrested by immediate collection into dry ice andstored at “ °c until further processing metabolite extraction from the fecal samples was undertaken bythe addition of \u242el per sample of methanolwater solution [volvol] containing \u242em [13c]sorbitoland \u242em [13c15n]valine and \u242em [13c]leucine as internal standards fecal samples were homogenizedat rpm for min in a thermomixer maintained at °c mechanically disrupted and incubated fora further min in the thermomixer samples were randomized for metabolite extractionmetabolite analysis of the extracted samples pooled biological quality control pbqc samples and mixtures of authentic standard mixes was performed by liquid chromatographymass spectrometrylcms using hydrophilic interaction column zicphilic and highresolution agilent seriesquadrupole time of flight mass spectrometry qtof ms as described previously pcoa of binaryjaccard distances of test standard mixes and pbqc samples are presented in fig s5 in the supplementalmaterial ions were analyzed in positive mode with full scan range of to mz and in datadependent tandem ms mode to facilitate downstream metabolite identificationmetabolomic analyses
Colon_Cancer
" tumor associated macrophages tam constitute the most abundant immune cells in the tumor stroma initiating pro inflammatory m1 or immunosuppressive m2 responses depending on their polarization status advances in tumor immunotherapy call for a detailed understanding of potential immunogenic mechanisms of irradiation routinely applied in rectal cancer patientsmethods to test the effects of radiotherapy on tam we ex vivo irradiated tissue samples of human rectal cancer and assessed the phenotype by flow cytometry we furthermore evaluated the distribution of leucocyte subsets in tissue sections of patients after short course radiotherapy and compared findings to non pretreated rectal cancer using an immunostaining approach anotypic assays ota consisting of macrophages cancer associated fibroblast and cancer cell lines were used to dissect the immunological consequences of irradiation in macrophagesresults we demonstrate that short course neoadjuvant radiotherapy in rectal cancer patients is associated with a shift in the polarization of tam towards an m1 like pro inflammatory phenotype in addition ex vivo irradiation caused an increase in the phagocytic activity and enhanced expression of markers associated with stimulatory signals necessary for t cell activation in ota we observed that this alteration in macrophage polarization could be mediated by extracellular vesicles ev derived from irradiated tumor cells we identified high mobility group box in ev from irradiated tumor cells as a potential effector signal in that crosstalks our findings highlight macrophages as potential effector cells upon irradiation in rectal cancer by diminishing their immunosuppressive phenotype and activate pro inflammation our data indicate that clinically applied short term radiotherapy for rectal cancer may be exploited to stimulate immunogenic macrophages and suggest to target the polarization status of macrophages to enhance future immunotherapeutic strategiesintroductionsince the introduction of immune checkpoint blockade immunotherapy has become an attractive therapeutic option in cancer1“ irradiation used as standard therapy in a number of solid malignancies induces immunogenic cell death icd by the release of damage associated pattern damp4 studies based on murine models indicated that irradiation induced dna damage of tumor cells elicited an antitumor immune response5 it was shown that alterations of the infiltrating immune cells by irradiation might be augmented when combined with immune modulating drugs6“ a detailed understanding of the impact of radiotherapy on the immune system in humans should allow application of radiotherapy as a part of novel immunotherapeutic concepts however little is known about the detailed regulation of the immunogenic effect of irradiation in clinical settingsmacrophages are one of the most abundant immune cell subsets in tumor tissue9 and play a key role in the cancer microenvironment11 tumor associated macrophages tam are functionally diverse13 and display high plasticity upon immunological stimuli14 the concept of m1 and m2 macrophages was introduced to describe the heterogeneity of this cell subset16 m1 like macrophages possess the capacity to clear infections in support of a t helper type th1 immune response17 whereas m2 like macrophages respond in general to th2 cytokines and are strongly enriched in the tumor microenvironment18 the concept of m1m2 has been challenged and is seen as an oversimplified approach to the phenotype of macrophages but can be viewed as a linear scale on which m1 and m2 present two extremes19 tam recognize damp and respond by producing a variety of cytokines and growth factors to promote innate and stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access adaptive immunity9 in response to these signals macrophages are able to undergo reprogramming with enhanced antitumor features making them an attractive target for anticancer therapies22 there are conflicting results with respect to the effect of irradiation on the macrophage phenotype klug et al demonstrated in a murine model of breast cancer and human pancreatic cancer that low dose irradiation gy induced repolarization of m2 like macrophages to m1 like macrophages via induction of nitric oxide synthase inos24 moreover expression of inos correlated with vessel normalization and an influx of cd8 t cells suggesting a tumor ablative as well as pro inflammatory effect of repolarized macrophages upregulation of inos was also observed in murine prostate cancer which has been irradiated with up to gy25 similarly agonists of the toll like receptor further stimulated the m1 phenotype of macrophages and enhanced the antitumor effects of irradiation in a murine model of breast cancer26 other studies suggested that irradiation of tumors was associated with a more immunosuppressive phenotype of tam27“ jones found that the depletion of macrophages by an anti csf antibody greatly increased tumor ablation upon irradiation gy in murine tumors generated from a colorectal and a pancreatic cell line obviously the effect of irradiation appears to depend on the model irradiation dose tissue as well as on the investigated time point after irradiation however despite the controversial reports an important role for tam in response to radiotherapy seems evidentmore recently extracellular vesicles ev have attracted attention in mediating signals to immune cells ev are rich in molecular cargos and are emerging as critical messengers in the cell to cell crosstalk they contain a variety of small signaling molecules which can be transferred to other cell types to modulate cell functions30“ thus we hypothesized that ev might be involved in macrophages regulation in the tumor microenvironment in irradiated cells using a clinically relevant approach we show that short course irradiation increased the proportion of m1 like tam in human rectal cancer tissue primary short term cultures and anotypic tumor assays ota consisting of tumor cell lines macrophages and cancer associated fibroblasts allowed to further dissect irradiation induced changes in colorectal tam using irradiated tumor cell lines we demonstrate that ev are able to mediate irradiation induced repolarization of macrophagesmethodspatients and tissue materialpatients with clinical t3 rectal cancer patients characteristics online supplementary table received neoadjuvant hyperfractionated radiotherapy over the course of days within the frame of a controlled clinical study34 as a control group we used historical surgical tumor specimen of non pretreated rectal cancer lesions of patients from the same institution with no history of irradiation therapy or cytoablative treatment for immunofluorescence and immunohistochemical stainings surgical paraffin embedded specimen of the cancer lesions were cut in µm sections and mounted on slides for ex vivo cultures and subsequent flow cytometric stainings rectal cancer tissue was obtained from patients with histologically verified rectal cancer with no history of irradiation therapy or cytoablative treatment studies involving patient material were performed according to the declaration of helsinki and approved by the local ethics committee of the medical university of vienna for ota hct116 ccl147 and dld1 ccl221 were purchased from atcc peripheral blood mononuclear cells for macrophage differentiation were isolated from healthy blood donorsprimary ex vivo cultures of leucocytes and flow cytometrytissue samples of non irradiated rectal cancer were minced resuspended in rpmi medium with fetal bovine serum and plated in a mm petri dish irradiation protocol was applied as indicated below after hour of incubation °c co2 a single cell suspension from cancer tissue was prepared briefly tissue was digested with collagenase iv unitsml and dnase i mgml for hour min in rpmi supplemented with fetal bovine serum and hepes buffer solution at °c afterwards cell suspension was rinsed through a µm mesh and leucocytes were isolated by density gradient centrifugation using ficoll gradients for flow cytometry analysis cells were stained with fluorescence antibodies listed in the online supplementary table livedead discrimination was performed with fixable viability dye biolegend samples were acquired on a facsaria iii bd and analyzed with flowjo software v1061irradiation protocola theratron mds nordion radiotherapy unit with a cobalt60 source was used for γirradiation of ota ex vivo cultures of rectal cancer tissue monocyte derived macrophages and cancer cell lines tissue samples of rectal cancer were minced and cultivated petri dishes with cancer tissue were irradiated with × gy after hours of incubation a single cell suspension was prepared ota and cancer cell lines hct116 and dld1 were irradiated with × gy or × gy and subsequently cultivated over the course of hours monocyte derived macrophages were irradiated with gy or gy a negative control gy was always transported to the radiotherapy unit but was not exposed to γirradiationex vivo phagocytosis assaymononuclear cells isolated of rectal cancer lesions were spun down at g for min at °c for ex vivo escherichia coli e coli phagocytosis assay irradiated and non irradiated leucocytes were resuspended in µl phrodo red e coli bioparticles thermo fisher scientific as per the manufacturer™s instruction a same set of cells were resuspended in phosphate buffered saline pbs as controls after hours incubation at °c stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen accessleucocytes were washed and stained for flow cytometry tam cd11b cd14 hla dr viable cd45 cells that were pe high were considered to be phagocytosing for ex vivo tumor cell phagocytosis assay tam were isolated via fluorescence activated cell sorting facs cd11b cd14 hla dr viable cd45 cells from healthy colon mucosa or colorectal cancer lesions and incubated overnight with ev isolated from irradiated × gy and non irradiated gy dld1 cells evtam after washing dld1 were labeled with carboxyfluorescein diacetate succinimidyl ester cfse thermo fisher µm and cocultured with tam at an effector to target cell ratio of the proportion of cfse tam was assessed by flow cytometry after hours of incubation at °cat the air liquid interface in dmem supplemented with egm for seeding of tumor cells nylon discs with collagen cell cylinders were transferred into well plates five hundred microlitres of a tumor cell suspension with × cellsml hct116 or dld1 were added to each well after hours the tumor cells attached to the surface of the collagen gels and the nylon meshes with the tumor cell colonized collagen cell cylinders were put back onto the metal grids and incubated for days with media changes every days prior to further experiments for further assessment ota were embedded in optimal cutting temperature media snap frozen in liquid nitrogen and stored at “°c until processing or fixed and embedded in paraffinimmunofluorescence and immunohistochemical stainingdirectly and indirectly labeled monoclonal antibodies as listed in online supplementary table and dapi as a nuclear marker were used an isotype was used as negative control in brief after incubation with the primary antibodies overnight at °c an appropriate secondary fluorescence labeled antibody was applied for min at room temperature followed by staining of dapi nuclear marker for immunohistochemical staining antibodies were mixed with bovine serum albumin in pbs and applied overnight to sections in a humid chamber at °c for visualization of the cells aec was used as chromogen sections were counter stained with hematoxylin for evaluation of staining results images of whole tissue sections one section per patient were acquired using a z1 axio observer microscope equipped with a ld plan neofluar × objective zeiss for leucocyte evaluation the tumor normal interface mm on tumor and normal zone was defined as regions of interests roi for γh2ax staining only tumor tissue was selected as roi roi were automatically quantified using tissuefaxstissuequest image analysis software tissuegnostics gmbhpreparation of otaota were set up as previously described35 briefly cancer associated fibroblasts caf were isolated and cultured from fresh samples of colon adenocarcinomas for the preparation of macrophages monocytes were isolated using the easysep direct human monocyte isolation kit stemcell technologies according to the manufacturer™s instructions × caf and monocytes per ota were mixed and pelleted by centrifugation at g for min for the collagen gel preparation all steps were performed on ice collagen solutions were prepared by mixing mgml of collagen i rat tail mgml becton dickinson and the fibroblastmonocyte suspension in dulbecco's modified eagle medium dmem supplemented with endothelial cell growth medium mv2 egm promocell a total of µl each of the collagen cell suspension were transferred into silicone gel casting devices after polymerization of the collagen solution the gels were lifted up on nylon mesh discs and transferred onto metal grids in well plates and cultured macrophage differentiationpbmc were obtained by ficoll plaque density gradient centrifugation pbmc were seeded at a concentration of ×106well well plates in rpmi medium monocytes were isolated using the ability of monocytes to adhere to non tissue culture treated plastic culture dishes attached cells were cultivated in rpmi medium with glutamax thermo fisher scientific supplemented with ngml macrophage colony stimulating factor m csf thermo fisher scientific fetal bovine serum uml penicillin uml streptomycin and µgml fungizone in a humidified atmosphere at °c cells were cultivated for days with two medium changes to obtain m1 lpsifnγ polarized macrophages cells were stimulated with ngml lipopolysaccharide lps sigma aldrich and ngml interferonγ ifnγ thermo fisher scientific for hours m2 il4il13 polarized macrophages were generated using ngml interleukin il4 strathmann and ngml il13 biolegendev preparation and characterizationev were isolated by differential centrifugation hct116 and dld1 cells were cultured in mccoy™s 5a medium supplemented with exosome depleted fetal calf serum briefly cancer cell line culture medium was centrifuged at g for min to pellet cells supernatant was transferred to new falcon tubes and then centrifuged at g for min to pellet dead cells and apoptotic bodies rotanta 460rc hettich supernatant was transferred to the high speed centrifugation tubes and large ev were removed by ultracentrifugation at g for min sorvall evolution rc thermo fisher scientific after filtration of the supernatant through a µm syringe filter small ev were pelleted by ultracentrifugation at g for min rotor t1250 suspended in ice cold pbs and collected after another ultracentrifugation at g for min rotor type sorvall wx ultra thermo fisher scientific the ev pellet was resuspended in cold pbs for further use ev count was determined using a nanoparticle tracking analyzer zetaview particlemetrix results were analyzed with zetaview software ev were added to macrophages at a stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access concentration of ×× cells we have submitted all relevant data of our experiments to the ev track knowledgebase ev track id ev20006537western blotcells were scraped from cell culture plates washed in ice cold pbs and lysed in × ripa buffer mm tris“hcl ph mm nacl np40 sodium deoxycholate sds containing × halt protease inhibitor cocktail thermo fisher scientific ev suspensions were mixed with × ripa buffer containing × halt protease inhibitor cocktail to obtain × concentration protein extracts were incubated at °c for min and centrifuged at g for min clear protein extract supernatants were quantified with the bca protein assay kit thermo fisher scientific one to five micrograms of protein were denatured with laemmli buffer loaded into each well of a acrylamide bisacrylamide gel containing sds and electrophoresed with magel for vhours proteins were transferred to a polyvinylidene fluoride membrane using the transblot turbo and the rta ready to assemble transfer kit bio rad after several hours of blocking pvdf membranes were incubated with primary antibodies online supplementary table at °c overnight after washing with tris buffered saline tween detergent membranes were incubated with the secondary antibody anti rabbit immunoglobulin g igg hrp linked antibody cell signaling technology or anti mouse igg hrp linked antibody cell signaling technology at room temperature for hour detection was done with clarity or clarity max western ecl substrate bio rad protein band intensities were quantified with imagequant tl ge healthcareelectron microscopyafter mounting cu mesh r22 holy carbon grids quantifoil with a tweezer into a leica gp leica microsystems grid plunger µl of ev sample solution were applied to grid™s front side and blotted for “ s grids were plunge frozen into liquid ethane for instant vitrification and transferred to a glacios cryo transmission microscope thermo fisher scientific equipped with a x feg images were recorded in low dose mode using the serialem software mastronarde with a falcon3 direct electron detector at magnifications of and with pixel sizes of and respectivelystatistical analysisstatistical analysis was performed using graphpad prism graphpad software statistical significance was determined by student's t test when comparing two groups the two way analysis of variance followed by tukey's multiple comparison test was used when comparing three or more groups significance was set at a p value of less than resultstam in rectal cancer polarize towards m1like phenotype upon irradiationwe first investigated the effect of irradiation on human tam in a primary ex vivo culture of rectal cancer specimen for this purpose minced tumor tissue was irradiated with gy and subsequently cultivated for hours before leucocyte isolation figure 1a the gating strategy for macrophages isolated from viable cd45 mononuclear cells of human rectal cancer is shown in figure 1b tam as defined by cd14cd11bcd68hla dr accounted for over of all viable cd45 leucocytes in rectal cancer figure 1c with over cells per gram of tissue figure 1d there was no significant difference in percentage or absolute numbers of tam in non irradiated versus corresponding ex vivo irradiated tissue samples figure 1cd we then assessed the phenotype of treatment naïve and ex vivo irradiated tam using flow cytometry in naïve rectal cancer lesions tam were characterized by high expression of cd206 cd163 and cd64 and low levels of the chemokine receptor ccr7 indicating that the m2 like macrophage phenotype was present in untreated rectal cancer figure 1e this was also reflected in their cytokine pattern as more tam in untreated rectal cancer samples produced il10 il13 and il4 while few interferonγ ifnγ and tumor necrosis factor alpha tnfα producing tam were found in untreated samples in contrast ex vivo application of gy γirradiation to primary rectal cancer leucocytes resulted in a reduced presence of cd163 tam this phenotype correlated with enhanced levels of tnfα and inos tam as well as diminished detection of il10 and il13 tam hence low dose irradiation of rectal cancer tissue can polarize tam towards a pro inflammatory m1 like phenotype and may therefore directly contribute to antitumor activity by tnfα and inos productionlowdose irradiation reduces pd1 expression and enhances phagocytosis in tam to e colito further assess the distinct functional phenotype of tam on irradiation we investigated the ability of tam to phagocyte as an aspect of direct effector function and activation to model the phagocytic behavior of irradiated tam we incubated leucocytes derived from irradiated ex vivo tumor culture with ph sensitive pe labeled e coli particles in the phagosome the fluorescence of e coli particles increases as demonstrated in a representative example figure 2a phagocytosis by tam was significantly elevated upon irradiation with gy figure 2b demonstrating that the acquired shift towards m1 polarization due to irradiation was also functionally relevant as pd1 was previously shown to inhibit phagocytosis38 we next determined pd1 expression on irradiated ex vivo tumor culture derived tam we found variable pd1 expression on tam in non irradiated rectal cancer and observed a significant decrease of pd1 on tam derived from irradiated cultures figure 2c these stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0cchanges induced by irradiation can be instrumental for phagocytic activity of tamirradiation promotes antigen presentation and cytokines associated with th1 responseone of the hallmarks of m1 polarization of macrophages is the acquisition of antigen presenting features leading to efficient th1 response17 as a first step to determine whether irradiation may result in improved capability of tam to initiate immune responses we analyzed the expression of cd86 on tam derived from irradiated ex vivo tumor cultures irradiation correlated with a significant increase of cd86 on the cell surface of tam as compared with tam derived from non irradiated naïve cultures figure 2d we next investigated hla dr on non irradiated tam versus tam irradiated with gy we observed hla dr low expressing tam and high expressing tam clearly dividing them into two groups figure 2e in non irradiated samples more than of tam expressed high levels of hla dr while irradiation significantly increased the fraction of hla drhigh tam to over figure 2f we next investigated whether irradiation can induce the expression of il12 p70 and il23 p19 as markers for the initiation of adaptive immune responses whereas il23 p19 showed a tendency to be produced by tam on irradiation il12 p70 was significantly induced in irradiated tumor tissue compared with open accesstreatment naïve samples figure 2g together these results suggest that low dose irradiation influences tam polarization in rectal cancers by equipping the cells with an hla drhiil12hi il23hi cytokine profile this observation emphasizes that irradiated tam have a higher probability to participate in antitumor immune responses directly by phagocytosis and cytokine secretion as well as indirectly by exhibiting a broad armamentarium that potentially activates t cells as main players of antitumor adaptive immunityshortcourse irradiation of patients with rectal cancer increases the m1m2 ratio of tamto corroborate our ex vivo data with the in vivo situation on short course irradiation we examined tam polarization and function in rectal cancer patients treated by a routinely applied radiotherapy protocol we made use of a cohort of patients in clinical stage t3 rectal cancer who received neoadjuvant hyperfractionated short course radiotherapy two times gy per day over the course of days figure 3a surgery was performed “ days after radiotherapy in these patients as a control we used surgical specimen from treatment naïve clinical t3 rectal cancer patients importantly the indication for radiotherapy was not correlated with a more severe tumor progression compared with the treatment naïve cohort figure ex vivo γirradiation induces polarization of tumor associated macrophages a tissue samples of naïve rectal cancer were minced and irradiated after incubation a single cell suspension of cancer tissue was prepared b representative example of the gating strategy for macrophages isolated from viable cd45 mononuclear cells of human rectal cancer c percentage of macrophages compared to total of viable cd45 cells in non irradiated and ex vivo irradiated rectal cancer lesions assessed by flow cytometry and presented as mean±sd n10 d mean numbers of macrophages in rectal cancer per gram in non irradiated and ex vivo irradiated rectal cancer presented as mean±sd n10 e expression of intracellular and extracellular markers in macrophages assessed by flow cytometry bars presented as mean percentage of indicated markers±sd of non irradiated and ex vivo irradiated macrophages n10 p005 p0001 p00001 by two tailed paired student™s t test gy gray tam tumor associated macrophages ifnγ interferonγ tnfα tumor necrosis factorα il interleukin inos inducible nitric oxide synthasestary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access irradiation increases phagocytosis and marker associated with antigen presentation and th1 response a figure representative histogram and flow cytometry plots indicating difference in pe fluorescence of non irradiated blue versus irradiated red tam in ex vivo phagocytosis assay total phagocytosis was analyzed by first gating on tam and then gating on pe cells b analysis of tam phagocytosis of phrodo e coli particles data presented as percentage of phagocytosis of non irradiated tam and corresponding irradiated tam gy n5 c percentage of tam positive for pd1 non irradiated versus ex vivo irradiated and representative histogram non irradiatedblue vs irradiatedred data presented as mean±sd n10 d percentage of expression of cd86 in non irradiated versus ex vivo irradiated tam and representative histogram non irradiatedblue vs irradiatedred data presented as mean±sd n10 e representative plots histogram and f expression of hla dr on tam of non irradiated and ex vivo irradiated rectal cancer data given as mean±sd n10 g expression of il12 p70 and il23 p19 data presented as mean percentage±sd of total tam n10 representative example of il12 p70 and il23 p19 expression on tam p005 p0001 by two tailed paired student™s t test th1 t helper type gy gray tam tumor associated macrophages il interleukinstary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen accessfigure short course irradiation in patients modulates the immune infiltrate with induction of macrophage polarization a irradiation protocol of patients with neoadjuvant hyperfractionated × gy per day over the course of five days radiotherapy for clinical t3 rectal cancer surgery was performed the following week b quantitative in situ assessment of infiltrating macrophages cd68 t cells cd3cd56ˆ’ nk cells cd56cd3ˆ’ and nkt cells cd56cd3 in non irradiated n25 and irradiated rectal cancer n45 assessed by multicolor immunofluorescence staining data are given as absolute numbers of positive cells per mm2±sd c analysis of immunohistochemical staining for γh2ax as irradiation response in non irradiated n25 and irradiated tissue sections data are given as mean±sd d representative examples of immunohistochemical staining for γh2ax images below are magnified × e representative immunohistochemical staining of cd68 cells in non irradiated n25 and irradiated tissue sections n45 images below are magnified × f representative example of immunofluorescence multicolor staining of cd68 macrophages pe m1inos fitc m2 cd163 apc g quantitative in situ analysis of immunofluorescence staining of ratio of inos m1 to cd163 m2 tams cd68 macrophages presented as m1m2 in non irradiated n25 compared to irradiated sections n45 data are given as mean±sd h percentage of il10 cells to total cd68 cells data are presented as mean±sd i representative image of multicolor immunofluorescence staining of il10 in macrophages cd68 peil10 fitc of irradiated rectal cancer p0001 p00001 by two tailed unpaired student™s t test gy gray inos inducible nitric oxide synthase tam tumor associated macrophages il interleukinstary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access since the stage in the tnm classification of malignant tumors and cancer differentiation of the two cohorts were comparable online supplementary table to assess the composition of the leucocytic infiltrate and the impact of radiotherapy we evaluated the distribution of macrophages t cells nk cells and nkt cells via immunofluorescence staining and automated image analysis irradiated tumor sections showed significantly less infiltration of cd68 cells which correlated with a tendency for a decrease of cd3 t cells while the amount of nk cells cd56 cd3ˆ’ or nkt cd56 cd3 cells did not show significant differences figure 3b immunostaining of phosphorylated γh2ax was used to confirm radiotherapy induced dna damage in analyzed tumor specimen irradiated tumors revealed a significantly higher amount of γh2ax positive cells than non irradiated tissue figure 3c γh2ax foci were primarily located in the nuclei of tumor cells rather than in the cells of the tumor infiltrating microenvironment figure 3d in both patient cohorts cd68 cells accumulated in regions surrounding tumor cell clusters figure 3eto evaluate the in vivo effect of irradiation on macrophage polarization and to correlate results with ex vivo irradiated tumor specimens we determined the ratio of m1 like tam to m2 like tam by staining for inos and cd163 in cd68 cells irradiation was associated with higher numbers of inos expressing tam while cd163 tam were drastically reduced resulting in a significant increase in the m1m2 ratio figure 3fg similar to the ex vivo cultures we observed a sixfold decrease of il10 tam in tumors from irradiated patients as compared with treatment naive tumors figure 3hi these data are corroborating hallmarks of our ex vivo irradiation protocols with macrophages becoming active players within the tumor microenvironment upon γirradiationirradiation of anotypic cultures promote macrophage activation towards an m1like phenotypeto be able to further dissect the irradiation induced findings in patients tissue sections and ex vivo cultures we used anotypic cultures which were established with colorectal cancer cell lines hct116 or dld1 primary caf and peripheral blood derived macrophages online supplementary figure a in collagen i gels in contrast to primary cultures ota allowed culture periods of up to days to mimic the clinical protocol of short course irradiation ota containing either hct116 or dld1 were exposed to irradiation with two times gy or two times gy over the course of hours compared with non irradiated controls figure 4ab frozen tissue sections of ota were assessed for macrophage marker using immunostaining and automated image analysis the number of macrophages in ota was comparable and not significantly different among the distinct conditions online supplementary figure b non irradiated gy ota harbored macrophages resembling an m2 like phenotype of cd11bcd68 macrophages as indicated by high expression of cd163 figure 4cd low expression of ccr7 figure 4e and modest expression of il10 figure 4fg upon irradiation of ota cd163 and il10 were reduced whereas the expression of the pro inflammatory marker ccr7 was elevated thus the marker profile of ota correlated with those of primary ex vivo tumor cultures and surgical specimen following neoadjuvant irradiation these observa
Colon_Cancer
"the kinetics and localization of the reactions of metabolism are coordinated by the enzymes that catalyze themthese enzymes are controlled via a myriad of mechanisms including inhibitionactivation by metabolitescompartmentalization thermodynamics and nutrient sensingbased transcriptional or posttranslational regulationall of which are influenced as a network by the activities of metabolic enzymes and have downstream potential toexert direct or indirect control over protein abundances considering many of these enzymes are active only whenone or more vitamin cofactors are present the availability of vitamin cofactors likely yields a systemsinfluence overtissue proteomes furthermore vitamins may influence protein abundances as nuclear receptor agonistsantioxidants substrates for posttranslational modifications molecular signal transducers and regulators ofelectrolyte homeostasis herein studies of vitamin intake are explored for their contribution to unraveling vitamininfluence over protein expression as a body of work these studies establish vitamin intake as a regulator of proteinabundance with the most powerful demonstrations reporting regulation of proteins directly related to the vitaminof interest however as a whole the field has not kept pace with advances in proteomic platforms and analyticalmethodologies and has not moved to validate mechanisms of regulation or potential for clinical applicationkeywords proteomics big data vitamin metabolism precision nutrition molecular nutritionintroductionregulatory mechanismscellular metabolism is a system of chemical reactions inwhich cells harness the energy stored in the chemicalbonds of substrate molecules to perform their biologicalfunctions maintain homeostasis or to synthesize buildingblocks for structural maintenance or cellular division thekinetics of these reactions are dependent on the activity ofthe proteins which catalyze them thus proteins are keymodulators of metabolismmetabolic activity also exerts network control over itselfby a diverse array of mechanisms which finely tune proteinexpression responses via nutrient sensing machineries products or intermediates of a metabolic pathway caninhibit or activate metabolic enzymes eg malate inhibitsthe succinate dehydrogenase complex and fructose correspondence nv83cornelledudivision of nutritional sciences cornell university ithaca ny usa26bisphosphate activates phosphofructokinase theoxidative status of a cell can drive the directionality ofredox reactions and impact abundances of redox reactioncatalyzing proteins eg the keap1nrf2 network responds to oxidative stress by upregulating expression ofantioxidantfunctioning proteins splicevariant or isozyme expression can impact relative pathway utilization atmetabolic network nodes eg splice variants and isozymesof pyruvate and lactate dehydrogenase respectively impactthe bridge between glycolysis and the tricarboxylic acidtca cycle [ ] additionally local metabolite concentrations and thermodynamics can dictate the directionalityof reactions catalyzed by compartmentspecific isozymeseg reductive activity of isocitrate dehydrogenase can beconfined to the cytosolspecific isozyme the impactsof the abovementioned regulations are closely monitoredby nutrient sensing proteins which initiate molecularevents altering protein activation and expression eg the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cjeong and vacanti nutrition metabolism page of serinethreonine kinase ampactivated protein kinasemammalian target of rapamycin and sterol regulatoryelementbinding protein are part of overlapping proteinnetworks that orchestrate proteinexpression and posttranslational modification responses to nutrient availability[ ] considering that many metabolic enzymes do notfunction in isolation and as detailed in the sections thatfollow require vitamin cofactors to stabilize intermediatesdonateaccept electrons shuttle substrates and hold reactants in close proximity vitamin status is a critical consideration when examining proteinmediated regulation ofmetabolism and the impacts of metabolism on proteinexpressionin addition to their potential regulatory roles ascofactors vitamins orchestrate other direct or indirectmechanisms influencing protein abundance retinoicacid vitamin a interacts with nuclear receptors impacting gene transcription ascorbic acid vitamin cimpacts oxidative status and associated protein networks and is reported to exhibit epigenetic regulation overprotein expression vitamin d regulates calcium signaling machinery activates nuclear receptors and exertshormonal regulation over protein expression [ ]and niacin vitamin b3 and biotin vitamin b7 can beincorporated as posttranslational modifications impacting protein function [ ]herein studies on systemic intake dietary injectionoral gavage of vitamins and their impacts on tissueproteomes are examined and their contributions tounraveling vitaminbased regulation of protein expression and tissue function are explored the currentwork is intended to provide informationto understand each vitamin™s figs and molecularfunctions and highlight its role as a cofactor or substrate in the reactions of central metabolism fig tables s1 s2 s3 s4 s5 s6 s7 s8 s9 s10 s11 s12and s13 finally this work is intended as a resourcefor identifying regulation of proteins related to vitaminmetabolism in published works the public domain ofproteomic data sets is ever expanding but is rarelysearched for effects related to vitamin metabolism tothat end all proteins are specified by their hugogene nomenclature committee hgnc gene symbolor the hgnc gene symbol of the human orthologwhen identified in another species and proteins requiring a vitamin as a cofactor or substrate are tabulated tables s1 s2 s3 s4 s5 s6 s7 s8 s9 s10s11 s12 and s13proteomics platformsproteomics platforms ofthe discussed studies areprovided to place them on a technological timelineplatforms are described with the terms orbitrap qtofquadrupole timeofflight tripletof triple “ time offlight qqq triplequadrupole 2dgems twodimensional gel electrophoresis “ mass spectrometry and2dge in brief orbitrap platforms are the workhorses ofmodern proteomics because their high achievable massresolutions combined with high sensitivity are bestsuited for maximizing the number of proteins identifiedin a complex sample [ ] though qtof and tripletof instruments capable of maintaining mass resolutionfig fat soluble vitamin structures 0cjeong and vacanti nutrition metabolism page of fig water soluble vitamin structuresat higher scan speeds hold a substantial influence inthis arena within the categories of orbitrap qtof andtripletof there are major technological advances notdiscussed here qqq platforms are best suited for quantifying a predetermined list of proteins lower scan speedsand mass resolution render them less capable thanorbitrap qtof or tripletof systems for nontargetedapplications advances in nanoflow liquid chromatography coupled directly to mass spectrometry haveimproved proteomic depth by orders of magnitude overthat achievable by 2dgems where the upstream selection of protein spots predates the modern definition ofnontargeted proteomics similarlyidentifying differentially intense protein spots using 2dge alone is considered an important milestone in the development ofproteomics but is rarely discussed outside the topic of thefield™s historyvitamin regulation of tissue proteomesvitamin avitamin a exists in alcohol aldehyde acid and esterforms known as retinol retinal retinoic acid and retinylesters respectively fig several carotenoids areprecursors to vitamin a including α and βcarotene βcarotene is converted to two molecules of retinalby beta carotene oxygenases bco1 or bco2 retinal is an important component of rhodopsin rho aprotein in rod cells responsible for detecting low levelsof light thus night blindness is telltale characteristic of vitamin a deficiency retinoic acid serves as asignaling molecule acting through nuclear retinoic acidrara rarb rarg and retinoid x rxra rxrbrxrg receptors which regulate growth and differentiation [ ] cellular and anismal trafficking ofvitamin a is dependent on retinolretinoic acid bindingproteins rbp family crabp1 crabp2 and retinolesterification via lecithin retinol acyltransferase lrat retinal is oxidized to retinol via aldehyde dehydrogenases aldh family and retinol is oxidized to retinoicacid by retinol dehydrogenases rdh and dhrs families in addition to inducing night blindness vitamin adeficiency adversely impacts cellular growth bone development and antibodybased immune responses in an orbitrapbased study of mouse embryo headstoxic levels of prenatal retinoic acid exposure intendedto model an established risk factor for craniofacial birthdefects are reported to induce abundance alterations inproteins associated with craniofacial development and 0cjeong and vacanti nutrition metabolism page of fig schematic of vitamin involvement in reactions of central carbon metabolism the depicted lipid bilayer represents the inner mitochondiralmembrane abbreviations defined in the abbreviations section vitamins specified by alphanumeric designations 0cjeong and vacanti nutrition metabolism page of neural crest processes in a parallel tripletofbased study of gerbil plasma and 2dgemsbased studyof gerbil liver and white adipose tissue a few dozen protein abundances linked to a handful of biological processesare reported to respond to dietary retinol βcarotene lutein or lycopene though process or pathway enrichmentanalyses are not reported as the authors discuss plasmawas not depleted of common highly abundant proteinsupstream of analysis by mass spectrometry which areknown to adversely impact data quality in anorbitrapbased study of plasma from nepalese childrendozens of proteins are associated with circulating carotenoid abundances potentiating development oflowcostantibodybased tests for carotenoid deficiencies apair of 2dgemsbased studies link tissue function toprotein abundance responses to vitamin a status in micebrains and bovine muscle vitamin b1thiamine vitamin b1 is composed of linked pyrimidineand thiazole rings decorated with methyl amine andalkylhydroxyl functional groups fig thiamineistransported through the plasma membrane viathiamine transporters slc19a2 and slc19a3 andthen twice phosphorylated on the alkylhydroxyl functional group by thiamine pyrophosphokinase tpk1rendering it active as thiamine diphosphate tdp tdp is a cofactor for enzymes catalyzing the oxidativedecarboxylation of ketoacids including the pyruvatedehydrogenase complex pdha pdhb pdhx dlatdld the oxoglutarate dehydrogenase complex ogdhdlst dld and the branched chain keto acid dehydrogenase complex bckdha dbt dld it is also acofactor for transketolase tkt in the nonoxidativebranch of the pentose phosphate pathway independent from its role as a cofactor thiamine is believed toregulate ion transport activity in the nervous system vitamin b1 deficiency is marked by a broad range ofneurological respiratory and cardiovascular pathophysiologies and is termed beriberi symptoms of beriberi aredifficult to directly link to the molecular functions ofvitamin b1 in a 2dgemsbased study of type diabetic andhealthy control subjects authors report treatment withthiamine reduces albumin alb abundance in urine indicating the vitamin serves a protective role of kidneyfunction in a qtofbased study of rat thalamiunder thiamine deficiency glyceraldehyde3phosphatedehydrogenase gapdh is the most upregulated protein fold while regulated proteins are most enrichedin the synaptic vesicle cycle pathway according to thekegg database proteomic changes are accompaniedby diminished performances on cognitive tests vitamin b2riboflavin vitamin b2 is composed of an isoalloxazinering and a bound ribitol fig it is activated byriboflavin kinase rfk forming flavin mononucleotidefmn and by flavin adenine dinucleotide synthase flad1 forming flavin adenine dinucleotide fad bound fmn or fad serves as an electron carrierfor redoxreactioncatalyzing proteins flavoproteinsincludingcomplexsdha sdhb sdhc sdhd the pyruvate dehydrogenase complex pdha pdhb pdhx dlat dldacylcoa dehydrogenases acads and methylenetetrahydrofolate reductase mthfr dehydrogenasethesuccinateriboflavin deficiency in humans predominantly occursin combination with that of other nutrients howeveranimal studies link it to impaired fetal and intestinaldevelopment [ ]iron absorption and lipidmetabolism [ ]in a qtofbased study of duck livers riboflavin deficiency is accompanied by a reduced abundance of smallchainspecific acylcoenzyme a dehydrogenases acadsfor which riboflavin serves as a cofactor and concordantelevation of hepatic small chain fattyacid lipid contentdramatic decreases in protein abundance are reported forinpp1 involved in inositol signaling thrsp purportedregulator of lipid metabolism bdh2 a regulator of lipidmetabolism fxn involved in mitochondrial ironsulfurcomplex assembly and ndufs1 a subunit of electrontransport chain complex i in a qtofbased study ofmaternal riboflavin deficiency reductions in fetal duck hepatic tca cycle betaoxidation and electron transport chainproteins are reported with idh3a being the lone memberof these pathways whose abundance increases vitamin b3niacin vitamin b3 is inclusive of nicotinic acid and nicotinamide fig which are converted to their mononucleotide forms by nicotinate phosphoribosyltransferase naprt and nicotinamide phosphoribosyltransferase namptrespectively both forms of the mononucleotide aresubsequently converted to their adenosine dinucleotideforms by nicotinamidenicotinic acid mononucleotidenmnat1 nmnat2 nmnat3adenylyltransferasesnicotinamide adenine dinucleotide nad is a cofactorform of the vitamin whereas nicotinic acid dinucleotide issubsequently converted to nad by nad synthase nadsyn1 nad is reduced to nadh by oxidative reactions of glycolysis the tca cycle and βoxidation andsubsequently serves as a redox equivalent carrier to theelectron transport chain and to regenerate reducedascorbic acid vitamin c glutathione and thioredoxin nad can also be phosphorylated by nadkinases nadk nadk2 to form a distinct redox shuttlingcofactor nadp nadp is reduced by reactions in the 0cjeong and vacanti nutrition metabolism page of oxidative pentose phosphate pathway g6pd pgd andother enzymes eg me1 me3 idh1 idh2 to nadphnadph provides reducing equivalents for biosynthetic reactions in fatty acid cholesterol and deoxyribonucleotidesynthesis outside its role as a reducing equivalentshuttle nad provides adenine dinucleotide phosphateadp ribose for synthesis of the second messenger cyclicadenosine monophosphate camp via the activity of adenylate cyclases adcy family nad also providesadpribose and polyadpribose for post translationalmodifications of proteins via activity of adpribosyl transferases art family and adp ribose polymerases parpfamily [ ] camp and protein polyadpribosylationare important mediators of cell signaling and proteinexpression niacin is synthesized from tryptophan butin small quantities relative to a healthydietary intake deficiency known as pellagraismarked by dermatitis and severe gastrointestinalneurological pathophysiologies which are fatalif untreated no proteomic studies on systemic intakeof vitamin b3 were found at the time of writing thisreviewvitamin b5pantothenic acid vitamin b5 is composed of a moleculeof pantoic acid bound to βalanine fig itsprimary metabolic function is as an acylcarrier pantothenic acid is a substrate in the first reaction of coenzyme a coa biosynthesis catalyzed by pantothenatekinases pank1 pank2 pank3 pank4 coa isa substrate for enzymes catalyzing the oxidative decarboxylation of ketoacids including the pyruvate dehydrogenase complex pdha pdhb pdhx dlat dldthe oxoglutarate dehydrogenase complex ogdh dlstdld and the branched chain keto acid dehydrogenasecomplex bckdha dbt dld [“] acyl speciesare activated by conjugation with coa and are substratesin or products of glycolysis the tca cycle fattyacidsynthesisβoxidation cholesterol synthesis ketogenesisbranchedchain amino acid catabolism and proteinacetylationoglcnacylation finally ™phosphopanthetheine product of pank proteins™ activities is acofactor of the acyl carrier protein domain of fatty acidsynthase fasn vitamin b5 deficiency is rare andusually accompanied by that of other nutrients burning of the feet and numbness in the toes is a characteristic manifestation along with variety of other symptoms no proteomic studies on systemic intake ofvitamin b5 were found at the time of writing this reviewvitamin b6vitamin b6 has aldehyde alcohol and amine forms fig of which the phosphorylated aldehyde form pyridoxalphosphate acts as a cofactor to over enzymes allthree forms of vitamin b6 are phosphorylated by pyridoxalkinase pdxk both the phosphorylated alcohol andamine forms pyridoxine phosphate and pyridoxaminephosphate are converted to pyridoxal phosphate bypyridoxine phosphate oxidase pnpo pyridoxalphosphate is a cofactor for enzymes catalyzing decarboxylase reactions in gammaaminobutyric acid gad1 gad2 and serotonindopamine biosynthesis ddc aswell as for enzymes catalyzing transamination reactionseg got1 got2 gpt gpt2 cysteine synthesiscth heme synthesis alas1 alas2 carnitinesynthesis3hydroxy6ntrimethyllysine aldolase geneunidentified andsphingolipid synthesis sptlc1 sptlc2 pyridoxalphosphate is also an important cofactor for enzymes ofonecarbon metabolism shmt1 and shmt2 andglycogen catabolism pygl and pygm vitamin b6deficiency is rare because of its availability in many foodsand pathophysiologies can be diverse niacin synthesis kynuin a tripletofbased study of streptozotocininduceddiabetic rat hippocampi pyridoxamine treatment prevented longterm recognition memory impairment andregulated protein abundances in a number of diversepathways notably upregulating half of the proteins involved in ubiquinol biosynthesis in a 2dgemsbased study of mice hippocampi the abundances ofphosphoglycerate mutase pgam1 and cannabinoidreceptorinteracting protein cnrip1 are reported tobe elevatedreduced respectively upon administration ofpyridoxine proteomic changes are accompanied by improved novel object recognition the plasma membrane byvitamin b7biotin vitamin b7 is composed of a fusedring structurebound to a valeric acid side chain fig it istransported acrossthesodiumdependent solute carriers slc5a6 and slc19a3[ ] as a cofactorposttranslational modificationbiotin covalently binds lysine residues it is a cofactor for pyruvate carboxylase pc acetylcoa carboxylase acaca propionylcoa carboxylase pcca andthe methylcrotonylcoa carboxylase complex mccc1mccc2 histones are also biotinylated regulatinggene expression the posttranslational modification occurs via the activity of holocarboxylase synthetasehlcs biotin deficiency is rare and has wide ranging pathophysiologies eating raw egg whites can preventitsabsorption leading to deficiency because of its affinityfor avidin a chemical in egg whites that is denaturedupon cooking this observation led to the vitamin™seventual discovery no proteomic studies onsystemic intake of vitamin b7 were found at the time ofwriting this review 0cjeong and vacanti nutrition metabolism page of vitamin b9the term folate vitamin b9 is inclusive of a group ofcompounds composed of a pteridine ring linked to paraaminobenzoic acid with a mono or polyglutamate tailfig in its reduced form tetrahydrofolate aonecarbon unit crosslinks as ch or ch2 aminegroups on the ring structure and aminobenzoic acid orbinds the secondary amine as a formyl group on theaminobenzoic acid group [ ] this onecarbonunit is utilized in the synthesis of purines and thymidineconversion of homocysteine to methionine interconversion of serine and glycine and catabolism of histidinereactions collectively termed onecarbon metabolism[ ] at the cellular level onecarbon metabolismis tightly regulated by compartmentalization [ ] while wholebody folate homeostasis is predominantly maintained by the liver through the enterohepaticcycle folate deficiency induces megaloblastic macrocyticanemia and fetal neural tube defects purportedly via itsadverse impact on nucleotide synthesis [ ] lowintake of folate is also linked to cardiovascular disease[ ] neurodegenerative disease [ ] alzheimer™s disease [ ] and cancer [“] in an orbitrapbased study of follicle fluid of womenundergoing in vitro fertilization the folate supplementedgroup is reported to have elevated abundances of apolipoproteins from high density lipoproteins and reducedreactive protein c crp the study is performed onwomen who did not become pregnantin aqtofbased study of a folatedeficiencyinduced intestinal neoplasia mouse model the combinatorial impactsof folate deficiency and methylene tetrahydrofolate reductase heterozygous deletion mthfr are reported toimpact protein abundances spanning diverse cellularfunctions however of samples are discarded as outliers and the simultaneous examination of mthfr anddietary folate deficiency does not allow proteomic adaptations to be attributed to either in isolation in a2dgemsbased study of adult rats aortic calmodulincalm1 calcium signaling protein abundances arepositively correlated with folate dose while abundancesof triose phosphate isomerase tpi1 glycolysis transgelin tagln cytoskeleton and glutathione stransferasealpha gsta3 reductive detoxification respond inversely in an 2dgemsbased study of rat liversprdx6 and gpx1 are reported to be elevated whilecofilin cfl1 is reported to be depleted under folatedeficiency other studies report protein abundancedifferences due to folate intake in rat urinary exosomesqqqbased human plasma 2dgems fetal brain tissue from pregnant mice fed ethanol2dgems pregnant rat livers 2dgems fetal rat livers 2dgems adult rat livers andbrains 2dgems and livers of piglets born tofolate deficient mothers 2dgems vitamin b12cobalamin vitamin b12 encompasses a group of molecules with four linked pyrrole ring derivatives forming a corrin ring and a cobalt atom bound at thecenter of the corrin ring the cobalt atom also binds a56dimethylbenzimidazole nucleotide and a functionalgroup fig the identity of the functionalgroup distinguishes the vitamin b12 compounds ascyanocobalamin hydroxycobalamin hydrocobalaminnitrocobalamin ™deoxyadenosylcobalamin also called adenosylcobalamin and methyl cobalamin [ ] methylcobalamin serves as a coenzyme in the conversion ofhomocysteine to methionine by methionine synthase mtrin the cytosol and adenosylcobalamin is required forconversion of lmethylmalonylcoa to succinylcoa bymethylmalonylcoa mutase mut in mitochondria vitamin b12 deficiency is closely related to folatedeficiency and can lead to megaloblastic anemia by impairment in the activity of methionine synthase mtr 5methyl tetrahydrofolate cannot be converted toonecarbon donors required for purine and thymidinesynthesis without vitamin b12 as a cofactor thus interfering with dna synthesis and erythrocyte production vitamin b12 deficiency is also linked to neurological disorders independent of anemia ruoppolo and colleagues performed a 2dgemsbased study of lymphocytes isolated from methylmalonicacidemia with homocystinuria cobalamin deficiency typec mmachc patients an inborn error in metabolismmarked by inactivity of the mmachc gene productreceiving a standard treatment of hydroxycobalaminbetaine folate and carnitine protein products of me2glud1 and gpd2 genes involved in anaplerosis andredox equivalent shuttling are upregulated while variant of protein pyruvate kinase muscle isozyme pkmand lactate dehydrogenase b ldhb are downregulatedrelative to lymphocytes isolated from healthy control donors in a 2dgebased study of adult rat cerebralspinal fluid protein abundance shifts are reported topeak after several months on a cobalamin deficient dietmodest shifts or after a total gastrectomy more severeshifts and return to near control values at later timepoints in a 2dgemsbased study glutathione stransferase p gstp1 abundances are diminished andglutathione peroxidase gpx1 abundances are elevated in rat pup kidneys under maternal vitamin b12deficient and maternal folate deficient conditions suggesting maternal dietary intake of these vitamins impacts offspring kidney redox homeostasis mechanisms 0cjeong and vacanti nutrition metabolism page of in a similar 2dgemsbased study of maternal vitaminb12 deficiencythe same group reports that severaldozen rat kidney pup proteins revert to control levelsupon administration of vitamin b12 at birth additionally diminished abundance of betaoxidation proteinsin kidneys of pups born to vitamin b12 deficientmothers is accompanied by elevated ppara apositive regulator offatty acid oxidation suggestingattempted compensation at the cellular levelvitamin cvitamin c ascorbic acid is absorbed at the brushborder and distributed to cells throughout the body bythe sodiumdependent plasma membrane solute carriersslc23a1 and slc23a2 the oxidized form of vitamin c dehydroascorbate is also transported via plasmamembrane glucose transporters slc2a1 slc2a3 andslc2a4 also known as glut1 glut3 and glut4 and reduced intracellularly to ascorbic acid byglutathione and the activity of thioredoxin reductases txnrd1 txnrd2 or txnrd3 vitamin c is a cofactor in the function of prolyl andlysyl hydroxylases which consume oxygen and alphaketoglutarate to form the hydroxylated amino acid residueand succinate the fe2 of these enzymes is restoredfrom fe3 by oxidation of vitamin c in the presenceof oxygen prolyl hydroxylases egln1 egln2 egln3also known as phd2 phd1 phd3 respectively hydroxylate the hif1a protein providing a necessary signal for itsdegradation and preventing a hypoxic response at the cellular level prolyl and lysyl hydroxylase activities arealso necessary for posttranslational modifications to formfunctional collagen lysyl hydroxylases includeplod1 plod2 and plod3 vitamin c serves anearly identical function in reducing fe3 as a cofactor fortrimethyllysine dioxygenase tmlh which catalyzes thefirst reaction in carnitine biosynthesis carnitine isessential for fatty acid catabolism in the mitochondria asonly fatty acyl carnitines formed via the activity of carnitine palmitoyl transferases cpt1a cpt1b and cpt1ccross the inner mitochondrial membrane through thesolute carrier slc25a20 vitamin c similarly servesas a cofactor for tyrosine hydroxylase th which catalyzes the first reaction in catecholamine eg dopamineepinephrine and norepinephrine synthesis additionally vitamin c serves and as a general antioxidant vitamin c deficiency leads to the condition knownas scurvy with symptoms largely attributed to malformedconnective tissue due to improperly folded collagen in a orbitrapbased study on a pig model of hemorrhagicshock vitamin c administration is reported to impactplasma protein abundances in the complement pathwayand those in polytrauma related processes including thestabilization of adamts13 abundance an importantregulator of clot formation an orbitrapbased studyof endoplasmic reticulum enriched fractions of livers inwerner syndrome mouse models identifies around adozen proteins whose abundances are impacted by administration of vitamin c a qtofbased study ofzebrafish reports upregulation of glutamate dehydrogenase glud1 and downregulation of pyruvate kinasemuscle isozyme pkm upon administration of vitamin cin a vitamin e deficient in a qqqbased study of human plasma ascorbic acid concentrationis reported to be inversely related to vitamin d bindingprotein gc abundance 2dgemsbased studiesidentify protein abundance regulations in mouse modelsof sarcoma metastases in the liver and tumor nodules of adenocarcinoma due to administration of vitaminc another 2dgemsbased study reports polypeptide abundance shifts in hemodialysis patient plasma uponvitamin c supplementation vitamin dvitamins d2 and d3 are respectively distinguished by theirergosterol and cholesterol backbones though onlyvitamin d3 is synthesized in animals both can be converted to active forms exposure of 7dehydrocholesterolan intermediate in cholesterol synthesis to ultravioletradiation in the skin and subsequent isomerization produces cholecalciferol vitamin d3 fig whether dehydrocholesterol is derived from cholesterol via activityof 7dehydrocholesterol reductase dhcr7 or synthesizedde novo in the skin is disputed 7dehydrocholesterolis successively hydroxylated by activity of cytochrome p450enzymes eg cyp2r1 and cyp27b1 in the liver and kidney to its active 125oh2 cholecalciferol [125oh2d3]form transport of vitamin d and its metabolitesoccurs bound to vitamin d binding protein gc ergocalciferol is the vitamin d2 equivalent of cholecalciferol and is activated analogously 125oh2d3 influences cellular function via nuclearreceptordependent and nuclear receptorindependentmechanisms the former involves 125oh2d3boundvitamin d receptor vdr forming a heterodimer complex with a retinoid x receptor rxra rxrb rxrgand subsequently binding vitamin d response elementsregulating transcription of genes largely involved modulating calcium and phosphorous transport andmaintaining homeostasis by regulating their absorptionin the kidneysintestines and bones [ ] therapidonsetreceptorindependent of 125oh2d3 are mediated by a membraneassociated rapid response steroid binding protein identifiedextracellularimpactsnuclear 0cjeong and vacanti nutrition metabolism page of as pdia3 and diversely impact cell growth survivaland immune response deficiency in vitamin d impairs bone mineralizationcausing rickets in infantschildren and osteomalacia inadults vitamin d deficiency is also linked tocardiovascular diseases [ ] cancer [ ]neurologicalimpairments [ ] and autoimmunediseases [ ] though underlying mechanisms arenot completely understoodin an orbitrapbased study of mouse fetal and postnatal lung tissue maternal vitamin d deficiency is reflectedin total proteome adaptations which are unexpectedlystrongest at postnatal day opposed to fetal time pointsimpacted proteins include several associated with lungdevelopment an orbitrapbased study of a mousebrain tissue model of remyelination in multiple sclerosisreports calcium binding protein abundances to be upregulated upon treatment with 125oh2d3 consistentwith the vitamin's regulatory role over calcium absorption in an orbitrapbased study of serum fromoverweight adults vitamin d deficiency is reported todifferentially affect abundances of proteins related toblood coagulation in males and females howeverabundances of these proteins are likely impacted by theproduction of serum from whole blood the authors report quantifying proteins table an impressiveanalytical depth for serum in a 2dgebased studyvitamin d deficient children are reported to have diminished serum abundances of adiponectin adipoq in a separate 2dgebased study the same groupreports fetuinb fetub to be elevated in the plasma ofobese vitamin d deficient children compared with theirvitamin d sufficient counterparts however theauthors do not directly identify fetub and rely on comparison of their findings to those of another study two 2dgemsbased studies of rat left ventricular andaortic tissueidentify proteins whose abundances respo
Colon_Cancer
"immunotherapy is revolutionising cancer treatment and has already emerged as standard treatment for patients with recurrent or metastatic gastric cancer gc recent research has been focused on identifying robust predictive biomarkers for gc treated with immune checkpoint inhibitors icis the expression of programmed cell death protein ligand1 pd l1 is considered a manifestation of immune response evasion and several studies have already reported the potential of pd l1 expression as a predictive parameter for various human malignancies meanwhile based on comprehensive molecular characterisation of gc testing for epstein barr virus and microsatellite instability is a potential predictive biomarker culminating evidence suggests that novel biomarkers such as the tumour mutational burden and gene expression signature could indicate the success of treatment with icis however the exact roles of these biomarkers in gc treated with icis remain unclear therefore this study reviews recent scientific data on current and emerging biomarkers for icis in gc which have potential to improve treatment outcomesintroductionthe prognosis for metastatic or recurrent gastric cancer gc remains very poor making it the third leading cause of cancer related death worldwide1 however the recent development of immune checkpoint inhibitors icis targeting the programmed cell death protein1 pd1 and programmed cell death protein ligand1 pd l1 pathways has produced improved outcomes for gc and already been successfully incorporated into clinical practice2 in particular checkpoint inhibition with anti pd1 monoclonal antibodies such as pembrolizumab and nivolumab has led to durable and significant responses in a minority of gcs3“ as a result interest has increased in selecting the right patient population to benefit such icis along with further exploration of immunotherapy notably various tumour related and host related factors with a critical impact on systemic immune functions may influence the response to icis6 moreover a significant proportion of patients do not respond to these therapies and there can also be a threat of unpredictable immune related adverse events aes and even severe toxicity7 therefore identifying more robust predictive biomarkers is critical to optimise treatment with icis while avoiding unnecessary treatment of patients who could develop life threatening or life altering aesgc is a heterogeneous and complex disease9 thus various approaches such as molecular classification have already been proposed for the subhistological exploration of gc as a potential tool for more effective therapeutic strategies the cancer genome atlas research network tcga suggested a comprehensive molecular characterisation of gcs using various platforms and proposed four distinct subtypes epstein barr virus ebv positive microsatellite unstable microsatellite instability msi genomically stable and tumours with chromosomal instability10 more recently the asian cancer research group acrg described four subtypes msi microsatellite stable mssepithelial to mesenchymal transition mssand msstp53 negative11 tp53 positive while the above subtypings show some overlapping molecular aberrations msi was identified as a subtype by both tcga and acrg2 msi is a molecular marker of a defective function of the dna mismatch repair mmr system that recognises and repairs the erroneous insertion deletion and mis incorporation of bases that can arise during dna replication and recombination as well as repairing some forms of dna damage12 it is also well known that msi high msi h tumours exhibit a high tumour mutational burden tmb neoantigen load and immune infiltration making them respond well to icis13 meanwhile the distinct characteristics of ebv positive gc is the overexpression of pd l1 and pd l214 the driving features of pd l1 positivity in ebv positive gc can also be effectively targeted with immunotherapy similar to the msi h subtype interestingly in a recent phase ii trial by kim kang a0bw chau a0i esmo open 20205e000791 101136esmoopen2020000791 0copen accesspembrolizumab showed a promising efficacy in patients with msi h and ebv positive tumours15 accordingly based on a better molecular characterisation of gc this review focuses on the current and emerging biomarkers for icis that would facilitate precision medicineclinical evidences of icis in gcseveral phase ii and iii trials have recently investigated the pd1 and pd l1 blockade in gc as summarised in table “ pembrolizumab is an igg4 human antibody targeting pd1 thereby interfering with the interaction between pd1 and pd l120 in the phase ib keynote trial patients with pd l1 positive gc or gastro oesophageal junction gej cancer were enrolled in both asian and non asian countries21 the objective response rate orr was and durable responses were seen with a median duration of responses of weeks the subsequent phase ii multicohort keynote059 trial cohort enrolled patients with recurrent or metastatic gc and gej cancer who received two pretreated lines of chemotherapy4 here the orr was and median overall survival os was months following these two trials the randomised phase iii keynote061 trial compared pembrolizumb monotherapy with paclitaxel in patients with pd l1 positive gc that had progressed on first line flouropyrimidine and platinum doublet chemotherapy17 while patients with a pd l1 status were initially unselected pd l1 positive patients with a combined positive score cps of or higher were included in the latter part of the study the cps is the number of pd l1 staining cells including tumour cells lymphocytes and macrophages divided by the total number of viable tumour cells while a tumour proportion score tps is the percentage of viable tumour cells showing partial or complete membrane staining relative to all viable tumour cells present in the sample22 approximately of patients were found to have pd l1 positive tumours using the cps pembrolizumab did not meet its primary end point of a longer os and progression free survival pfs in patients with pd l1 positive tumours notwithstanding it is worth noting that patients who expressed pd l1 cps of or higher exhibited a better benefit from treatment with pembrolizumab in post hoc analyses although these subgroup analyses should be interpreted with cautionsubsequently the keynote062 was a large randomised first line clinical trial of patients with advanced gc or gej cancer who were randomly assigned to one of three arms pembrolizumab at mg every weeks for up to years pembrolizumab plus chemotherapy cisplatin and fluorouracil or capecitabine or placebo plus chemotherapy23 pembrolizumab was non inferior to chemotherapy for os in patients with cps or higher no survival benefit was observed with the addition of pembrolizumab to chemotherapy compared with chemotherapy alone in this studynivolumab also an igg4 antibody is very similar in structure to pembrolizumab except that nivolumab binds to the n terminal loop on the pd1 molecule while pembrolizumab binds to the c™d loop24 the phase iii attraction2 ono453812 trial compared nivolumab with a placebo in asian patients with unresectable or recurrent gc that was refractory to or intolerant of at least two previous standard chemotherapy regimens5 the results showed a significantly prolonged os for the nivolumab group with a year os rate of vs more recently the long term survival was reported showing year survival rates of for nivolumab and for placebo25 nivolumab also showed a significant advantage compared with the placebo in terms of pfs and the radiological objective response therefore these results provide randomised evidence that nivolumab is a valid approach to improving the clinical outcomes for patients with gc in a third line and subsequent line setting however the overall clinical value of attraction02 was partially limited by several important issues26 the patient population was only asian and pd l1 positivity reported at a low frequency of as this was assessed as tps rather than cps was not associated with the survival outcomes plus there was no comparative data on quality of life a phase iii checkmate032 study also reported that nivolumab and nivolumab plus ipilimumab provide a durable antitumour activity in heavily pretreated western patients with chemotherapy refractory gc gej cancer and oesophageal cancer19 in particular the clinical benefit of nivolumab monotherapy was consistent with that reported for asian patients in the attraction02 study yet similar to the attraction02 study there was no association of pd l1 positivity according to tps with survival outcomes therefore ongoing randomised controlled trials of icis including pembrolizumab and nivolumab in earlier line treatment need to unify assessment of pd l1 expression and create more accurate profiles of aes in gcavelumab is an igg1 antibody which binds to the front beta sheet of pd l1 and possesses pd1pd l1 blockade activity with antibody dependent cell mediated cytotoxicity adcc28 there are some differences between pd1 inhibition and pd l1 inhibition as pd1 targeting therapeutic antibodies including pembrolizumab and nivolumab block the pd1pd l1 or pd1pd l2 interaction to restore tumor specific t cell reactivity without mediating adcc24 the javelin gastric trial the first study to compare avelumab with standard chemotherapy in third line treatment for gc did not achieve its primary end point of improving os or the secondary end points of pfs and orr18 this negative finding may be attributed to the usage of the active comparator in the control arm in addition there are possible reasons including the different drug biding sites heterogeneity of tumour biology and methodology of pd l1 testing notwithstanding fewer patients experienced aes with avelumab than with chemotherapy although researchers found no evidence of clinical benefit compared with commonly used chemotherapy in any of the examined subgroups including the tumour pd l1 expression status kang a0bw chau a0i esmo open 20205e000791 101136esmoopen2020000791 0csecnerefersodehcaer tonsfp rristnopdne yramirp ichpargoegrrinogerlaboglso sfplaboglsorrrrinasainasanretsewsolabogl tneitaprebmunrecnac cirtsagn i srotibhniii tnopkcehc enummi lgnitauave seduts iii idna ii esahpdetcees l l ebatsmra tnemtaertsutats ldpgnittesesahpeman ydutstegratstnegabamuzilorbmepdetceesnul eni ldrihtretal robmuzilorbmepevitisop dnoceslexatilcapenilbamuovnilobecapldetceesnul eni ldrihtretal roiiiiiiii trohoc etonyekdpbamuzilorbmepetonyekdpbamuzilorbmepi notcarttaonodpbamuovnlixopac roxosbamuovnil bamumilipibamuovnilbamuovnil bamumilipibamuovnilretal rodetceesnuleni ltsrifiiiiii notcarttadp trapdetceesnul eni ldrihtiiietamkcehcdpbamuovnlibamuovnlii¡ecohc s™nacsyhpiibamuevaldetceesnuleni ldrihtiii cirtsagnlevaj i ldpbamuevalevitisop ldp erew stneitapgnoma open access dna s xos etar esnopser rri lavvrus eer fnossergorpi sfp dnagii lnetorphtaed llec demmargorp ldp inetorphtaed llec demmargorp dpi lavvrus llarevo so nitaplil axodna enbacepac iixopacnitaplilaxoskeew yreve iipovni gkg mbamum ilipl i supgkg mbamuovn il skeew yreve povniii gkg m bamumilipl i supgkg mbamuovn  illecyc tnemtaert kee w a fohcae dna syadno mg m nacetoniri ro dna syad no mg m lexatilcap¡kang a0bw chau a0i esmo open 20205e000791 101136esmoopen2020000791 0copen accessthese results might support the potential of avelumab for combination or maintenance therapy notwithstanding the recent javelin gastric trial with maintenance avelumab therapy following initial chemotherapy in gc produced disappointing results29 although there was more efficacy seen with avelumab in an exploratory analysis of pd l1 positive patients with a cps ‰¥ vs months no significant difference with respect to os was observed between the avelumab maintenance and continued chemotherapy groups vs months p0177as discussed above several clinical trials are currently ongoing with various strategies30 the phase iii attraction04 nct02746796 trial is evaluating nivolumab plus standard chemotherapy oxaliplatin plus either s1 or capecitabine versus chemotherapy alone in asian patients16 the phase ii component part of this study showed chemotherapy plus nivolumab led to an orr of and median pfs of months while the subsequent phase iii trial part is evaluating the survival outcomes another phase iii trial checkmate649 nct02872116 has completed recruitment with over patients randomising patients to oxaliplatin based chemotherapy alone or in combination with nivolumab or nivolumab plus ipilimumab32 the third arm of this study terminated recruitment early due to early safety signalantiangiogenic treatment such as ramucirumab and bevacizumab is also generating recent interest as several studies have suggested that simultaneously blocking angiogenesis and pd1 pathways induces synergistic antitumour effects especially involving the control of the tumour microenvironment tme33 researchers including the current authors noted that ramucirumab in combination with pembrolizumab jvdf showed a manageable safety profile with favourable antitumour activity in patients with previously treated gc35 consequently the phase iii nivoram nct02999295 trial has evaluated the safety and tolerability of the addition of ramucirumab to nivolumab in patients with gc as second line therapy7 as an alternative antiangiogenic and multitargeted kinase inhibitor a phase i trial of regorafenib plus nivolumab is also exploring in gc nct0340687136 this trial demonstrated an orr of in gc and icis pretreated patients with gc achieved a partial response pr in patients interestingly of the seven patients who had received prior anti pd1pd l1 treatment three achieved an objective responseanother interesting phase ii trial investigating the role of pembrolizumab plus trastuzumab combined with chemotherapy in patients with previously untreated human epidermal growth factor receptor positive tumours is currently ongoing nct0295453637 preliminary results from this study showed of patients experienced reduction in target lesions after one dose of pembrolizumabtrastuzumab before oxaliplatincapecitabine was introduced in second cycle the high orr of was coupled with median pfs of months this has led to the current phase iii keynote811 study randomising patients to chemotherapy plus trastuzumab with or without pembrolizumb nct0361532638 thus these therapeutic strategies including combination treatment represent a true opportunity in the contemporary treatment of gc and may produce further success when considering integrative genomic databiomarkers for icis in gcthe identification and validation of reliable biomarkers are important to facilitate precise patient selection and increase the clinical benefit from icis an overview of the predictive roles of biomarkers obtained from tissue or blood and their characterisation in the management of gc is briefly summarised in table pdl1 expressiontesting for pd l1 expression by ihc is the current standard in most solid tumours and several studies have already assessed the clinical outcomes according to the pd l1 expression status in gc nevertheless different antibodies are being used for ihc to assess with different performance and different scoring criteria for pd l1 expression a recent multicentre study blueprint pd l1 ihc comparison project attempted to compare the performance of each ihc pd l1 assay in lung cancer39 three assays including 22c3 for pembrolizumab for nivolumab and sp263 for durvalumab were found to be comparable to each other in the staining of tumour tissue whereas sp142 for atezolizumab was found to be less sensitive however further study is required to carefully validate these assays in gcas noted above in the keynote059 trial pd l1 expression was assessed using the pd l1 ihc 22c3 pharmdx assay and measured using a cps4 this trial demonstrated a higher orr vs in patients with high pd l1 expression defined as cps ‰¥ both pfs and os were also more prolonged in this group40 although pembrolizumab in a second line trial keynote061 did not significantly prolong os greater benefits were seen in tumours with higher pd l1 expression cps ‰¥ orr25 cps ‰¥ orr16 cps orr217 however the attraction02 and javelin gastric trials showed no clinical improvement for pd l1 positive tumours as they used tps rather than cps18 these differences may also have been due to the use of different cut off points and scoring systems a lack of standardisation of the assays and testing platforms the heterogeneous nature of pd l1 expression in tumours intratumouralintertumoural heterogeneity and intraobserverinterobserver variability41“ for instance the attraction02 study retrospectively evaluated pd l1 expression using a pd l1 ihc pharmdx assay defined as the tps while pd l1 expression was prospectively assessed in tumour cells tumour associated lymphocytes and macrophages using a 22c3 pharmdx assay in keynote06117 as such pd l1 positivity was only reported in about patients using tps with nivolumab whereas it is generally kang a0bw chau a0i esmo open 20205e000791 101136esmoopen2020000791 0ctable overview of candidate biomarkers associating with response to immune checkpoint inhibitors in gastric cancerbiomarkerssample source methodspd l1tumourihctreatmentrecent results in gastric cancerreferencespembrolizumab expression of pd l1 ‰¥ associated with open accessbetter clinical efficacy orr mrdpembrolizumab expression of pd l1 cps of or higher associated with better clinical efficacy os orrpembrolizumab expression of pd l1 associated with higher response rateebv positivitymsi htumourin situ hybridisationpembrolizumab ebv positivity associated with higher tumour msi testing or ihcnivolumbresponse ratemsi h associated with clinical efficacy orr dcr ospembrolizumab msi h associated with better clinical efficacy orrpembrolizumab msi h associated with better clinical efficacy orr ospembrolizumab msi h associated with better clinical tmbtilstumour or bloodtumourwes or targeted sequencingimage analysing software or manually countedtoripalimabicisefficacy orrtmb associated with better clinical efficacy orr ospresence of tils associated with better clinical efficacy in various solid tumours but very limited data in gcgeptumour multigene profilingpembrolizumab ifn gamma gene signature associated with better clinical efficacy orr pfspembrolizumab t cell inflamed gene signature gut microbiotastoolnlrbloodculture or molecular technique sequencingmetagenomicscomplete blood counticisicisnivolumabassociated with better clinical efficacy orr pfsvarious species associated with enhancement and iraes of icis in various solid tumoursincreased nlr correlated with dcr and osdecreased change of nlr associated with better survivalsixty seven patients had tumours from the stomachcps combined positive score dcr disease control rate ebv epstein barr virus gc gastric cancer gep gene expression profiling icis immune checkpoint inhibitors ifn interferon ihc immunohistochemistry iraes immune related adverse events mrd median response duration msi h microsatellite instability nlr neutrophil to lymphocyte ratio orr overall response rate os overall survival pd l1 programmed cell death protein ligand pfs progression free survival tils tumour infiltrating lymphocytes tmb tumour mutational burdenbetween and using cps ‰¥ as the cut off more recently using the checkmate032 study data tumours were re scored using cps and there was better correlation between nivolumab treatment and survival even at the cps ‰¥ level44 thus one of the coprimary patient population in the first line checkmate649 study that has recently completed recruitment is including a subpopulation of patients with pd l1 cps ‰¥ebv positivityebv status is also emerging as a potential biomarker for personalised treatment strategies in gc14 in situ hybridisation detection of ebv encoded small rna in tumour cells is generally recommended to identify ebv associated gc ebvagc30 the incidence of ebvagc varies from to in different regions with an average of worldwide45 nevertheless this subgroup is associated with better prognosis thus less frequently found in advanced or metastatic setting in particular ebv positive tumours frequently display pd l12 overexpression and occasional immune cell signalling activation10 several research groups found that the level of pd l1 expression ranging from approximately to of ebvagc with kang a0bw chau a0i esmo open 20205e000791 101136esmoopen2020000791 0copen accessvariable results between studies20 pd l1 positivity has also been significantly associated with a poorer prognosis than pd l1 negativity in ebvagc furthermore ebv triggers a significantly higher infiltration of cd8 t cells in tme46 in previous studies of patients with ebv positive cancer the current authors showed that high levels of tumour infiltrating lymphocytes tils were associated with a favourable prognosis while intratumoural pd l1 positivity with a worse prognosis47in the phase i javelin solid tumour trial where avelumab was shown to be beneficial for a patient with metastatic gc it is worth noting that ebv positive tumours with a low mutation burden and msi tumours with a high mutation burden had statistically significantly higher tumour lymphocytic infiltration when compared with mss tumours48 the strength of immune mediated signalling signatures in ebv positive tumours also represents a t cell inflamed tme10 these findings support the concept that icis can be used in patients with gc with ebv by suppressing the pd1 pathway in tumour cells and allowing immune activation a recent phase ii trial by kim demonstrated improved efficacy associated with pembrolizumab in patients with ebv positive tumours15 this study enrolled patients with pretreated gc in a subgroup analysis pembrolizumab monotherapy as salvage treatment showed that all six ebv positive patients with gc attained pr orr100 with a median duration of months however in another study out of patients considered ebv positive were treated with toripalimab50 only one pr was observed with two stable and one progressive diseases patients with pr was also pd l1 positive these contrasting results with pembrolizumab could be due to toripalimab rather than ebvagc as a predictive biomarker for icimicrosatellite instabilityhighmmr deficiency is generally characterised by a failure to repair dna replication associated errors leading to the accumulation of mutations in microsatellite regions of the genome51 these phenomena are known as msi52 currently two different methods have been validated for detecting msi h53 the mmr status is assessed by ihc staining to measure the expression levels of the proteins involved in dna mmr and a polymerase chain reaction based exam also tests the length of repetitive dna that are known as microsatellite in the normal and tumour tissues while there are discrepancies between the ihc of mmr protein expression and msi test results the overall concordance in the two tests is high52 the incidence of msi in gc varies between countries being relatively high in approximately “ of western patients51 msi h gc is commonly associated with intestinal type female sex older age lack of lymph node metastases and onset in the distal stomach54 to date multiple retrospective studies and limited prospective studies have reported on a positive association between msi h and a better prognosis in resectable gc55 for example the magic study reported that patients with msi h tumours have superior survival compared with patients with mssmsi low msi l tumours when treated with surgery alone and conversely have inferior survival to patients with mssmsi l tumours when treated with perioperative chemotherapy plus surgery56 however similar to ebv status patients with msi h had better prognosis thus only “ of patients with metastatic gc would be mmr deficient the prognostic and predictive values of the msi status on the survival of patients with metastatic gc remain a subject of debate52theoretically in the presence of mmr deficiency undetected dna replication errors leading to a tumour with a high mutational burden reproduce various neoantigens that stimulate t cell activation and tumour infiltration by immune cells keynote012 trial reported that msi h tumours showed a partial response in two out of four patients regardless of pd l1 expression21 a subgroup analysis of keynote059 revealed an orr of for patients with msi h gc4 in keynote061 and more recently reported keynote062 there was a substantially enhanced survival benefit in patients treated with pembrolizumab compared with chemotherapy17 similar to the results for ebv positive tumours the clinical study by kim also showed that msi h tumours responded particularly well to pembrolizumab monotherapy orr85715 in the checkmate032 trial that assessed the efficacy of another pd1 monoclonal antibody nivolumab the orr was for the msi h group vs for the msi l group or mss group19 therefore this evidence highlights the potential of msi h as a predictor of the response to icis in gcof note whereas msi hmmr deficiency is the most consistent predictor of efficacy to icis in gc a substantial portion of msi h gc still has unsatisfactory outcomes even with icis the degree of msi and resultant mutation load in part might explain the variable response to pd1 blockade in mmr deficient tumours57 tumours sensitive to pd1 antibodies showed a loss or a reduction in tumour allele frequency of missense non synonymous single nucleotide variant and indel mutations after pd1 treatment suggestive of immune editing of tumour cellstmb and neoantigentmb may be a potential biomarker of outcomes with icis in multiple solid tumour types41 generally cells have a number of repair pathways to maintain their genome stability13 the mutational load acquired by defective dna repair pathways frequently alters protein function and expression resulting in the formation of neoantigens that serve a source of antitumour immune response therefore it is reasonable to hypothesise that tumours with a high mutational load are more likely to produce neoantigens and increase immunogenicity58 in turn this course of reaction induces a more intensified immune response resulting in tumours becoming more sensitive to treatment with icis41 although tumour specific neoantigens with high clonality are more predictable and beneficial for the response to icis accurate measurement kang a0bw chau a0i esmo open 20205e000791 101136esmoopen2020000791 0cof these neoantigens is known to be expensive and time consuming59 in this situation tmb could be a good approach for indirectly evaluating the neoantigen load tmb is defined by the total number of somatic non synonymous mutations per coding area of the tumour dna58 several studies have already demonstrated the predictive impact of tmb in lung cancer and melanoma one early study by yarchoan observed a significant correlation between tmb and orr for anti pd1 or anti pd l1 therapy60 rizvi also reported that patients with tmb 50th percentile exhibited an improved durable clinical benefit rate and pfs versus those with lower tmb61 this benefit was also seen in the checkmate227 study that included patients with advanced non small cell lung cancer nsclc who received a combination of nivolumab and ipilimumab as the first line metastatic setting62 a significantly prolonged pfs was reported for the patients with higher tmb treated with the combination treatment irrespective of the expression of pd l1 likewise a large scale study across multiple cancer types found a significant association between tmb and the clinical outcome63“ these findings can also provide a novel strategy for subgroups with high tmb considering that the measurement of the mutational load is a critical factor for therapeutic successhowever for patients with gc there is still insufficient evaluation and conflicting results on the utility of tmb as a biomarker of the response to icis65 interestingly wang performed a tmb analysis of patients with chemorefractory gc who were treated with toripalimab as a monotherapy in a prospective phase ibii clinical trial50 in this study tmb high tmb h patients responded significantly better than the tmb low patients orr vs p0017 a survival benefit has also been demonstrated for patients with high tmb os vs months p0038 similar correlation was also found between tmb h according to circulating tumour dna and better survival when treated with pembrolizumab in gc15 in light of recent approaches this close relationship between tmb and clinical outcomes also points to the possibility of tmb as a predictive biomarker in patients with gc however in the study with regorafenib plus nivolumab due to small sample size tmb did not correlate with response or pfs to this combination36despite its identified significant predictive role there are still many challenges in precisely estimating tmb first it is difficult to apply the protocol including whole exome sequencing or targeted sequencing panels using next generation sequencing to clinical practice due to various problems such as the sample amount cost sensitivity coverage and analysis time8 second a standardised cut off value for tmb has not yet been clearly established since many studies have reported a wide range of cutoffs for different tumour types58 thus given the variety of tmb cutoffs assays related with tmb in clinical studies should be interpreted cautiously moreover the availability of tumour sampling to detect tmb is commonly limited and tmb may present temporal open accessvariability a novel blood based tmb approach could be considered as an alternative method as the advantages of repeating sampling during treatment could provide information of a dynamic immune reaction8 plus this approach is less invasive and enables investigators to document the evolution of tmb interestingly in a study by gandara et al blood based tmb was correlated with tissue based tmb and showed a longer pfs in patients with metastatic nsclc who received treatment with atezolizumab68 in summary tmb could be a novel and independent biomarker that reflects the therapeutic effects of icis in gc however its accuracy in predicting the efficacy of icis varies among studies and still needs to be explored for gctumour infiltrating lymphocytesthe immune microenvironment of tumours is now recognised as an important determinant for understanding the relationship between a patient™s immune system and their cancer informing prognosis and guiding immunotherapy like icis69 tumour cells are typically surrounded by infiltrating inflammatory cells such as cytotoxic t cells helper t cell subsets regulatory t cells tumor associated macrophages dendritic cells and myeloid lineage leucocytes70 among these differentiated lymphocytes referred to as tils are considered a manifestation of the host immune response against tumour cells and seem to play an important role in various human malignancies71 several studies have already reported the potential of ti
Colon_Cancer
sarcomatoid carcinomas scs are extremely rare aggressivemalignant tumors characterized by distinct cellular morphology the features of this tumor were first described in by snover scs can occur in a wide variety of sitesincluding the respiratory tract digestive tract genitourinary tractbreast and thyroid glands however these tumors are rarein the digestive tract especially in the stomach as of april there are only six cases of gastric sarcomatoid carcinomagsc reported in the english medical literature these previousreports focused on the pathological and clinical manifestationsthem have not systematically described the radiologic appearanceof the tumor due to the more invasive nature and poorerprognosis of gsc than pure gastric adenocarcinoma gacand gastric lymphoma gl it is clinically beneficial to narrowdown the diï¬erential diagnoses by understanding the computedtomography ct characteristics of gsc the present studyanalyzed our experience in diagnosing five patients with gscin terms of the imaging findings and clinical features to thebest of our knowledge our study represents the largest seriesof gscs to datein addition due to the rarity of gsc the diï¬erential diagnosisbetween gsc and other types of malignant gastric tumors hasnot received much attention so we also initially explored thediï¬erential diagnosis of gsc from gac and glmaterials and methodsthe protocol was approved by the medical ethics committeeof zhengzhou universityinformed consent was obtainedfrom all patientspatient selectionfrom august to january we searched the pathologyrecords and the picture archiving and communication systemspacs of our hospital the search terms included stomachand sarcomatoid carcinomas a total of five patients wereidentified as having sc and were enrolled in the present study weretrospectively reviewed all clinical data demographic featureslaboratory findings clinical interventions and the histologicfindings of the five biopsy or operation specimensct evaluationfive gsc patients underwent ct examinations the ctscans were acquired with a 64row multidetector devicediscoveryct750hd ge healthcare waukesha wiunited states conventional axial scanning was performedbefore and after an intravenous iv injection of nonioniciohexol iopromide mgml ge medical systems mlkgand mls through a dualhead pump injector medradwarrendale pa united states the imaging parameters wereas follows tube voltage kv tube current ma fieldof view fov mm matrix — mm and sectionthickness mm finally a 20ml saline flush was performedat a rate of mlsgastric sarcomatoid carcinomacontrastenhanced ct scans were acquired with scanningdelays of s arterial phase ap and s portal venous phasepp after the iv injection of the contrast agent started the ctdose index volume for the three phases was msvimage analysistwo experienced radiologists and years of abdominal ctexperience performed a retrospective analysis of the ct imagesall analyses were performed with an aw47 workstation gehealthcare and the radiologists were blinded to the clinicalinformation of the patients the evaluated parameters includedthe tumor location gastric cardia gastric fundus gastric bodygastric angle and gastric antrum longaxis diameter shapegrowth pattern serosa condition attenuation and enhancementcharacteristics such as the enhancement pattern and degreeof enhancement the enhancement pattern of the tumor wasclassified as homogeneous or heterogeneous based on the apimage the degree of enhancement of the tumor was based ondynamic ct imaging using hu attenuation where œobviousenhancement was defined as hu œmoderate enhancementas hu and œmildly enhancement as huthe gscs were staged with the union for internationalcancer control uicc tnm staging standard all imagingfindings were compared with the postoperative pathologicalfindings the accuracy rate the number of cts coincidentwith the pathological diagnosisthe number of actual pathologicaldiagnoses — pathological evaluationthree patients underwent gastrectomy and two underwentendoscopic biopsy the three gastrectomy specimens measured cm — cm — cm cm — cm — cmcm — cm — cm respectively in two of theseand tumors the mucosal surface of the excised specimen showedulcerative masses of approximately cm — cm — cmand cm — cm the remaining specimen was a soft massmeasuring cm — cm — cm for biopsy multiple sampleswere acquired and the diameter of each sample was cmaccording to the relevant literature the diagnostic criteria fsc were generally as follows the tumor originated fromthe stomach and the tumor consisted of both carcinomatousand sarcomatoid components and the sarcomatoid componentaccounted for more than of the tissue in addition if biopsywas performed the sarcomatoid component can be seen in everysample furthermore sarcomatoid regions express epithelialmarkers such as ck or emathe specimens were fully stretched fixed and soaked in formaldehyde solution for h all biopsy specimenswere analyzed the specimens underwent routine dehydrationparaffin embedding sectioning into µm thick sectionsand hematoxylin eosin he staining immunohistochemicalstaining was performed using a roche benchmark xt automaticimmunohistochemical detector the antibodies used in thisstudy included ae1ae3 ckl ck818 epithelial membraneantigen ema vimentin p40 p63 and antigen ki67 ki67all antibodies listed above were purchased from dako dakoglostrup denmarkfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatable comparison between gsc and gac glage median age rangemain symptomsepigastric discomfortpainintermittent vomitingacute hematemesisbloody stooldysphagialocationcardia and fundusbodyantrumthe longaxis diameter median size rangeshapefocal thickeningdiffuse thickeningmassserosal surfacebare areaclearunclearulcersyesnodensity characteristicsheterogeneoushomogeneousenhancement patterheterogeneoushomogeneouslymph node involvementyesnoliver involvementyesnotherapyresectionchemotherapyresection and chemotherapyneoadjuvant chemotherapyradiation therapygscgacgl “ years “ years “ years “ cm “ cm “ cm comprehensive comparative analysiseach patient with gsc was matched by age ± years year ofdiagnosis and sex to four patients with gac gl patients witheach disease were retrieved from pacs patients with gsc werecompared with those with gac gl in terms of demographicclinical and ct characteristics table resultspatient characteristicsthe patients included four men and one woman ranging inage from to years with a median age of years theclinical and ct features of these patients are summarized intables all patients had nonspecific symptoms includingabdominal discomfort epigastric discomfort nausea or vomitingthe other presenting symptoms included hematemesis or weightloss three patients underwent radical resectionin whichonly one patient was treated with adjuvant chemotherapyafter surgery and two patients chose to deny treatment inaddition we also reviewed the upper gastrointestinal radiographyresults figure the laboratory findings revealed that patient was positivefor tumor abnormal protein tap and patient was positivefor carbohydrate antigen ca125 before treatmenthemoglobin and erythrocyte count decreased in three patientsfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatable clinical and pathological factors of the five gsc patientscasesexage yearscomplaintlocationmaximumdiametertumor markercmanemiatherapymetastasismmfmmsuddenhematemesisepigastricdiscomfortintermittentvomitingepigastric painepigastric painepigastric painlesser curvatureremnant stomachcardia andfunduscardia and fundusfunduscardia and fundusnormaltap ca125 normalna““rrnnonenonerc“““œ yespresentpositive œ“ noabsentnegative f female m male age in years r radical gastrectomy rn remnant gastrectomy c chemotherapyna not availabletable computed tomography features of the five gsc patientscasegross features of the tumorulcersgrowth modedensity characteristicsenhancement patterfocal thickeningfocal thickeningmassfocal thickeningfocal thickening“intracavityintracavityintracavityintracavityintracavityheterogeneousheterogeneoushomogeneousheterogeneousheterogeneousheterogeneousheterogeneoushomogeneousheterogeneousheterogeneousmarginunclearunclearunclearclearunclearœ yespresentpositive œ“ noabsentnegativefigure characteristics of xray examinations of a 65yearold male patient with gsc ab reveals that there is a huge niche with irregular shapes at the smallcurvature of the stomach the niche is located inside the outline of the stomach the niche is surrounded by transparent bands with different widths that is ringembankments with irregular outlines the surrounding mucosa is thickened interrupted and the local gastric cavity is narrowedpatients and and platelet count was elevated in fourpatients patients and pathological featuresmicropathologically the gastric tumor cells showed infiltrativegrowth the cytological characteristics ofthe tumor cellsshowed obvious malignant characteristics microscopicallythe spindle cell structure and the nucleus were obviouslyatypical pleomorphic and enlarged mitotic figures were visiblefigures 2ab on immunohistochemical examination thetumor cells showed positive staining for ae1ae3 cklck818 ema p40 vimentin the ki67 index was higher than figures 2c“i all five tumors were diagnosed as gscin addition the sarcomatoid component showed spindle cellsarcomatoid morphologyct findingsof the cases of gsc were in the gastric fundus and cardiafigure was in the gastric body and was in the gastricfundus of these tumors one was a recurrence in the remnantstomach the ct manifestations of this tumor included localthickening n mass formation n the longaxisfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomafigure histological and immunohistochemical features of gsc ab hematoxylineosin he staining showing tumor cells demonstrated spindleshapedstructures significant atypical nuclei pleomorphic nuclei and giant nuclei mitotic figures visible tumor cells showed infiltrative growth cells were stained withhematoxylin and eosin stain magnification a — b — by immunohistochemistry the tumor cells were positive for ae1ae3 c ckl d ck818 e emaf p40 g and vimentin h moreover of them were positive for ki67 i the final diagnosis was sc [magnification c“i —]diameter of the lesions ranged from to cm mean size cm in addition ulcers with an irregular base and slightlyraised borders were observed in of cases among the threepatients who underwent surgery two lesions invaded the gastricserosa and the remaining lesion invaded the gastric bare areaamong the two patients with biopsyproven gsc one patientexhibited tumor invasion of the gastric bare area the majorchanges in the ct imaging characteristics were an irregularouter layer of the gastric wall and obscuration of the perigastricfat initially the ct findings were interpreted as gac in fourcases and gl in the tumor showed predominantly inhomogeneous densityand the radiodensity values were “ hu in the noncontrastphase heterogeneous enhancement was seen in four casesdue to necrotic or cystic areas and the other tumor revealedhomogeneous enhancement the radiodensity values on the apimages ranged from to hu and to hu in thevenous phase after contrast medium injection two tumorsshowed obvious enhancement and moderate enhancementwas seen in the other three tumors the peak tumor valuewas observed in the portal phase one of the three patientswho underwentlymphsurgery demonstrated evidence ofin one patientthe boundary betweennode involvementthe lesion and the left lobe of the liver was unclear andthe area with low attenuation was confirmed by pathologythe liver withas a metastatic lesion in the rightcircular enhancement the remaining patientshowed noevidence of metastasis among the two patients with biopsyspecimens one patient was identified as having lymph nodemetastasis on ctlobe ofct staging versus pathological stagingof gscnone of the gscs were staged as t1t2 by ct or pathologythe accuracy of ct staging t3 and t4 gsc was and respectively the overall diagnostic accuracy of ctfor determining the t stage of gsc was none of the gscs were staged as n2n3 by ct or pathologythe accuracy of ct in staging n3 and n4 gsc was and respectively the overall diagnostic accuracy of ct fordetermining the n stage of gsc was the comparison of tn staging based on ct and pathology isshown in table frontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomafigure sarcomatoid carcinoma of the stomach in 62yearold women a unenhanced ct image of stomach reveals an intraluminal mass of homogeneousattenuation with an irregular surface at the gastric fundus and cardiac region b“d contrastenhanced ct image shows obvious homogeneous enhancement ofmass with the peak value of the tumor on the portal phase in perigastric lymph nodes an enlarged and significantly enhancement lymph node can be seenb arterial phase of contrast enhancement image c portal phase of contrast enhancement image d portal phase of contrast enhancement coronal imagediscussiontable ct and pathological tn staging for comparisonsarcomatoid carcinoma is an extremely rare and complicatedmalignant tumor composed of malignant epithelial componentsand atypical spindle cells however the spindle cells of scsappear to show evidence of epithelial diï¬erentiationforexample showing epithelial markers or epithelial ultrastructuralinstead of a specific line of mesenchymalcharacteristicsdiï¬erentiation moreoverliteratureemphasizes that the sarcomatous components occupy ofthe elements involved in the present study our patients™tumor cells displayed atypical spindle shapes that expressed theepithelial phenotypethe currentsome ofsarcomatoid carcinomas can occur in almost any an wherecarcinoma can occur in the digestive system the incidencesof scs in the esophagus and liver are relatively high but scsare exceedingly rare in the stomach we could find only sixprevious reports in the english literature table between and patients with sc confirmed by pathologywere retrospectively analyzed with only five tumors occurringin the stomach the average age of the reported patientswas years range “ and that in our series was years range “ a previous study reported that the sexcaseno no no no no ctt4an0t3n0t3n1t3n0t4an1na not available t tumor n nodepathological staget4an1t3n0nanat4an0distribution of male to female gsc patients was and thecorresponding proportion in our patients was “ ithas been noticed that scs are more common in male patientsand sex is a probable risk factor gsc patients may present withepigastric pain or discomfort dysphagia nausea and vomitinghematemesis and emaciation due to thickening of the gastricwall pain or discomfort in the upper abdomen is common thesymptoms can last from a few days to several years withoutobvious specificityin the present study of the cases of gsc were recognizedin the proximal stomach and the remaining tumor was founddistal to the stomach four cases of gsc in the present study hadfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatable clinical and imaging features of six previously reported cases of gcscasegenderageyearslocationsize cmshapeulcersenhanceappearancerecurrencemetastasistherapyprognosis mmffmmremnant stomachgreater curvaturelesser curvaturegastroesophageal junctionremnant stomachdistal stomachpolypoidpolypoidpolypoidulceratedpolypoidmass“““nenenenenehyperne“““““nonesurgerysurgerysurgeryendoscopysurgeryna mo d mo d mo d mo d mo dthe patient succumbed to heart failure before the surgical treatment an autopsy was performed œ yespresentpositive œ“ noabsentnegative hyper hyperdensene no evaluate mo month d dieareregarded asa longaxis diameter less than cm and the remaining tumorhad the largest longaxis diameter among our patients cmthe location distribution and longaxis diameters of the gscs inour patients were similar to those in previous reports “in the actual diagnosis processthe diagnosis of sc has always been difficult for cliniciansand pathologists especially the diï¬erential diagnosis fromcarcinosarcoma carcinosarcomastrulybiphasic neoplasms composed of distinct malignant epithelialcarcinomatous and mesenchymal sarcomatous componentsthe sarcoma components show typical specialized diï¬erentiation howeverthe termsœsarcomatoid carcinoma and œcarcinosarcoma have been usedinterchangeably in some cases therefore the understanding ofthese tumors has been hampered nevertheless we can try tofocus on whether there is a diï¬erence between these tumors froma new perspective the ct finding sc in the stomach have notbeen previously scientific reported or even detailed descriptionthere are only four simple descriptions chunchao reported that a patient with a giant sc presented a mass witha cm diameter in the antrum and body of stomach whichinfiltrated the gastric serosa the hepatic flexure of the colon andgallbladder were also involved on ct contrastenhanced ctimages showed obvious enhancement of the two lesions sato reported a patient with sc of the remnant stomachand the radiographic examination showed an elevated lesionwith a large ulcer at the gastric cardiac lesser curvature thatmeasured cm in diameter the other two reports only describeda soft tissue mass or a large tumor in the dilated stomach on the other hand within in the upper gastrointestinal tractalthough there are fewer reports of carcinosarcoma localized inthe stomach this type of tumor is still more common than sc gastric carcinosarcoma showed an elevated lesion or thickenedgastric walls in “ of all reviewed cases “ tomoaki reported a 79yearold man with gastric carcinosarcomaand his veins showed severe invasion enhanced abdominalct showed irregular thickening and slight enhancement of thegastric wall on the side of the lesser curvature with suspiciousbulky lymph nodes yoshiyuki reported a 70yearoldjapanese woman who presented with a soft tissue mass adjacentto the lesser curvature of the stomach that was lobulated andct revealed an ulcer on the lesion the contrastenhanced ctimages showed heterogeneous enhancement of the mass thefinal pathological diagnosis was gastric carcinosarcoma inthe present study we found that gsc showed local thickeningof the gastric wall and mass formation often accompaniedby ulcers the site of the disease was mostly in the proximalpart of the stomach but these tumors can also occur in theremnant stomach the signal of the tumor was homogeneousor heterogeneous on plain ct scans after contrast mediuminjection of tumors demonstrated heterogeneousenhancement on ap images due to cystic areas or necrosis inthe lesions in this study the enhancement degree of all tumorsreached a peak in the pp after contrast enhancement for thesetumors the enhancement degree in the delayed phase wasnot significantly reduced the overall enhancement mode wasdelayed enhancement in addition ct showed that four patientshad invasion into the gastric serosal region or gastric bare areatwo patients had the characteristics of enlarged perigastric orretroperitoneal lymph nodes and uneven enhancement and onepatient had invasion into the adjacent liver tissue these findingsreflect the metastatic and highly invasive characteristics of gscoverall ct and contrastenhanced ct can clearly show theprimary lesion infiltration range lymph node metastasis anddistant metastasis of gsctomographic diagnosis of gsc has not been attemptedbecause of the rarity of this entity according to the findingsof our study gsc needs to be diï¬erentiated from gac andgl on ct adenocarcinoma is the most common pathologicaltype of gastric tumor and is mainly distributed in the antrumseldomly in the body and fundus of the stomach the incidenceof gac is high in men and the median patient age is years the most common ct signs of gac are localor extensive thickening of the gastric wall mass formationincluding fungoidestype polypoidtype masses rough orsmooth serous surfaces and continuous interruption of themucosal layer tumors involving the mucosal surface can appearenhanced “ s after injecting a contrast agent the peakvalue for tumors invading the muscular layer usually appearsafter “ s and after the mucosal surface is strengthenedthe duration is longer primary gl accounts for “ ofmalignant gastric tumors and is predominantly situated in thegastric antrum gastric body and gastric fundus the incidenceof gl is high among males with a median patient age of yearsthe clinical symptoms included epigastric pain bleeding earlysatiety and fatigue the most common ct manifestations ofgl are diï¬use thickening of the gastric wall or a homogeneousfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatissue mass with slight attenuation or an appearancesoftsimilar to that of the normal gastric wall for gl becauseof hemorrhage necrosis submucosal edema or infarction thegastric wall may be heterogenous on ct gl originates froma submucosal process and gastric mucosa is commonly intactin the early stage but shows interruptions or ulceration in thelater stage after contrast medium injection most gl showedhomogeneous and slight enhancement in the delayed phase lymphoma is considered when distant structures the mesenteryretroperitoneum or other parts of the abdomen have lymphnode metastasis the ct findings may only reflect features of gsc but cannotaccurately diagnose gsc l one explore the origin of thesarcomatous portion immunohistochemistry ihc also failedto conclusively establish the origin of gsc rodrigues usedfluorescence in situ hybridization fish to confirm that sc andadenocarcinoma have a common origin that is the epithelium while primary gl originated from gastric submucosallymphoid tissuethe main treatment for localized lymphomas is eradicationof helicobacter pylori and surgical treatment whereas advanceddisease often requires radiation or chemotherapy alone surgery is the only treatment option for patients with gacadjuvant chemotherapy and chemoradiotherapy are also oftenused targeted therapy isin the exploration stage however there are currently no specific national comprehensivecancer network guidelines for the treatment of only gscbecause the tumor is relatively rare although complete surgicalresection is the most important treatment method for examplewhile chemotherapy is considered in clinical practice whetherchemotherapy can be applied for gsc and the efficacy ofchemotherapy remain controversial domblides firstevaluated the efficacy of immune checkpoint inhibitors icis forsc and found that lung sc patients exhibited high response ratesand prolonged overall survival os with icis this studyprovides a new idea for the treatment of gscbecause gl tends to be confined to the gastric wall forprolonged periods before tumor spread its prognosis is betterthan that of gac previous literature has found that scin the parotid gland lung hypopharynx liver and pancreashave poor prognoses due to metastasis or recurrence with asurvival period of a few months “ similarly gscpatients also died or developed metastasis or recurrence withina few months or it was already in the advanced stage at thefirst diagnosis all these clinical manifestations suggest that gschas a poorer prognosis than gac and gl in additiongsc can metastasize through the blood and lymph nodesand the most common sites of metastasis are the local lymphnodes and liver this conclusion is consistent with ourresearch resultsconclusionthe incidence rate of gsc is extremely low so clinicians andradiologists are not familiar with the features of this tumorbased on systematic research of this rare tumor and comparisonswith common gastric cancers we found that gsc is morecommon in men who are approximately years old and isoften accompanied by ulcers the disease is mostly located in theproximal part of the stomach and can also occur in the remnantstomach with delayed enhancement on contrastenhanced ctimages these characteristics can provide a reference for furtherresearch on gscs in the future however an accurate diagnosisof gsc depends on the combination of clinical imaging andhistopathological features due to the aggressive nature and poorprognosis of the tumor rapid clinical intervention and detailedfollowup with ct are essentialdata availability statementthe original contributions presented in the study are includedin the supplementary material further inquiries can bedirected to the corresponding authorethics statementthe studies involving human participants were reviewed andapproved by the medical ethical committee of the zhengzhouuniversity the patientsparticipants provided their writteninformed consent to participate in this study written informedconsent was obtained from the individuals for the publication ofany potentially identifiable images or data included in this author contributionsyl manuscript preparationliterature research and dataanalysis pl literature research and data analysis kf manuscriptreview and data collection kc guidance of pathologicalknowledge sy guidance of imaging knowledge jj imaging datacollection and analysis wl and xz manuscript editing jgstudy conception and design manuscript review and guarantor ofintegrity of the entire study all authors have read and approvedthe final manuscriptfundingthis work was supported by the national natural and sciencefund of china no references zhu cc li mr lin tl zhao g sarcomatoid carcinoma of the stomach acase report and literature review oncol lett “ doi ol20153460 snover dc levine gd rosaij thymic carcinoma five distinctivehistological variants am j surg pathol “ zhou dk gao bq zhang w qian xh ying lx wang wl sarcomatoidcarcinoma of the pancreas a case report world j clin cases “doi 1012998wjccv7i2236 xie y xiang y zhang d yao x sheng j yang y sarcomatoidthe “ doi 103892mmr2018and review ofthe pancreasreportcaseofcarcinomaliterature mol med repafrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinoma sato a oki e kohso h endo y uchida h hiroshige s sarcomatoidcarcinoma of the remnant stomach report of a case surg today “ doi 101007s0059501204027 nakayama y murayama h iwasaki h iwanaga s kikuchi m ikeda s gastric carcinosarcoma sarcomatoid carcinoma with rhabdomyoblastic andosteoblastic diï¬erentiation pathol int “ doi 101111j144018271997tb04540x robeycaï¬erty ss grignon dj ro jy cleary kr ayala ag ordonezng sarcomatoid carcinoma of the stomach a report of three caseswith immunohistochemical and ultrastructural observations cancer “ doi 101002109701421990040165730co2n ruess da kayser c neubauer j fichtnerfeigl s hopt ut wittel uacarcinosarcoma of the pancreas case report with comprehensive literaturereview pancreas “ doi 101097mpa0000000000000904 fujiie m yamamoto m taguchi k iwanaga a ohgaki k egashira a gastric carcinosarcoma with rhabdomyosarcomatous diï¬erentiation a casereport and review surg case rep doi 101186s407920160176z tanimura h furuta m carcinosarcoma of the stomach am j surg “ doi 101016000296106790325x kitamura s study on carcinosarcoma of stomach gan “ kumagai k kawai k kusano h matsuo k irie j tsuchiyama h a caseof socalled carcinosarcoma of the stomach gan no rinsho “ bekki t fujikuni n tanabe k yonehara s amano h noriyuki t thegastric carcinosarcoma with severe venous invasion a case report surg caserep doi 101186s4079201804218 ikeda y kosugi s nishikura k ohashi m kanda t kobayashi t gastriccarcinosarcoma presenting as a huge epigastric mass gastric cancer “ doi 101007s1012000604054 cid³n eu cuenca ij gastric adenocarcinoma is computed tomography ctuseful in preoperative staging clin med oncol “ doi cmos2641 hallinan jt venkatesh sk gastric carcinoma imaging diagnosis stagingand assessment of treatment response cancer imaging “doi gossios k katsimbri p tsianos e ct features of gastric lymphoma eurradiol “ doi 101007s003300050069 rodrigues dn hazell s miranda s crespo m fisher c de bono js sarcomatoid carcinoma of the prostate erg fluorescence insituhybridization confirms epithelial origin histopathology “doi 101111his12493 levine ms rubesin se pantongragbrown l buck jl herlinger h nonhodgkin™s lymphoma of the gastrointestinal tract radiographic findings ajram j roentgenol “ doi 102214ajr16818976941 russo ae strong ve gastric cancer etiology and management in asia and thewest annu rev med “ doi 101146annurevmed081117 domblides c leroy k monnet i mazi¨res j barlesi f gounant v efficacy of immune checkpoint inhibitors in lung sarcomatoid carcinoma jthor oncol “ doi 101016jjtho202001014 niu x sarcomatoid carcinoma in the parotid gland a review of years ofexperience laryngoscope “ doi 101002lary27474 li s jiang l he q wei w wang y zhang x the prognostic significanceof jmjd3 in primary sarcomatoid carcinoma of the lung a rare subtypeof lung cancer onco targets ther “ doi 102147otts22 dai l fang q li p liu f zhang x oncologic outcomes of patients withsarcomatoid carcinoma of the hypopharynx front oncol doi103389fonc201900950 seo n kim mj rhee h hepatic sarcomatoid carcinoma magnetic resonanceimaging evaluation by using the liver imaging reporting and data system eurradiol “ doi 101007s00330019060528 shi y chen j chen h hong x sarcomatoid carcinoma of the gallbladdera case report j int med res doi conflict of interest the authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestcopyright liu liang feng chen yue ji li zhao and gao this is anopenaccess distributed under the terms of the creative commons attributionlicense cc by the use distribution or reproduction in other forums is permittedprovided the original authors and the copyright owners are credited and that theoriginal publication in this journal is cited in accordance with accepted academicpractice no use distribution or reproduction is permitted which does not complywith these termsfrontiers in oncology wwwfrontiersinaugust volume 0c'
Colon_Cancer
" leukotriene receptor antagonists ltras are broadly used for the management of allergic asthmaand have recently been indicated to inhibit carcinogenesis and cancer cell growth in colorectal cancer crcchemoprevention studies the occurrence of adenoma or crc itself is generally set as the trial endpoint althoughthe occurrence rate of crc is the most confident endpoint it is inappropriate for chemoprevention studies becausecrc incidence rate is low in the general population and needed for longterm monitoring aberrant crypt fociacf defined as lesions containing crypts that are larger in diameter and darker in methylene blue staining thannormal crypts are regarded to be a fine surrogate biomarker of crc therefore this prospective study was designedto explore the chemopreventive effect of ltra on colonic acf formation and the safety of the medicine in patientsscheduled for a poly resection as a pilot trial leading the crc chemoprevention trialmethods this study is a nonrandomized openlabel controlled trial in patients with colorectal acf and polypsscheduled for a polypectomy participants meet the inclusion criteria will be recruited and the number of acf inthe rectum will be counted at the baseline colonoscopic examination next the participants will be assigned to theltra or no treatment group participants in the ltra group will continue mg of oral montelukast for weeksand those in the no treatment group will be observed without the administration of any additional drugs at theend of the 8week ltra intervention period a polypectomy will be conducted to evaluate the changes in thenumber of acf and cell proliferation in the normal colorectal epithelium will be analyzeddiscussion this will be the first study to investigate the effect of ltras on colorectal acf formation in humanstrial registration this trial has been registered in the university hospital medical information network uminclinical trials registry as umin000029926 registered november keywords colorectal cancer chemoprevention leukotriene receptor antagonist aberrant crypt foci correspondence takuma_hyokohamacuacjpdepartment of gastroenterology and hepatology yokohama city universityschool of medicine fukuura kanazawa yokohama kanagawa japan the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0chigurashi bmc cancer page of cancer is a one of the major health issues and a leadingcause of death globally the incidence prevalence andmortality rates of colorectal cancer crc continue torise all over the world [ ] the majority of crc casesare derived from adenomatous polyps and their resection has been shown to reduce the risk of the futuredevelopment of advanced adenomas and crc [ ]thereby preventing crcrelated deaths however patients with polyps adenomatous polyps andor hyperplastic polyps represent a highrisk group for theoccurrence of metachronous colorectal polyps andorcrc then a paradigm is shifting from surveillancefor the early detection of advance adenomas or crcsand resection to novel tactics for prevention is requiredto reduce the mortality rate of this disease a number oflargescale epidemiologic andor clinicaltrials haveassessed the prophylactic agentsincluding vitamin dcalcium fiber and nonsteroidal antiinflammatory drugsnsaids such as aspirin and selective cyclooxygenase cox2 inhibitors in preventing against crc development we previously reported that the nsaidsulindac suppressed the development of sporadic colorectal adenomas to this point nsaids particularlycox2 inhibitors have been proven to offer the greatestpossibility for reducing crc risk either alone or in combination with other drugs while it has been reportedan elevated risk of serious cardiovascular events relatedwith the administration of cox2 inhibitors [ ] inview of these adverse cardiovascular effects and the lackof efficacy of other drugs that initially looked promisingthe development of novel agents that meet both safetyand efficacy in preventing crc is essentialleukotriene receptor antagonists ltras such asmontelukast and zafirlukast are commonly used for thetreatment of allergic asthma [ ] and tsai mj reported that ltras reduced cancer risk in a dosedependent manner in asthma patients it was alsoreported that the cancer incidence rate was significantlylower in ltra users than in nonltra users vs per patientsyear this means that apart fromits role in asthma ltra has also been associated withcarcinogenesis and tumormediated immunosuppression for example the overexpression of cysteinyl leukotriene receptor cyslt1r has been observed in crcand montelukast leads the apoptosis of crc cancer cells[ ] previous in vivo studies have shown the chemopreventive effect of ltras [ ] but the chemopreventive effects of ltra have not been studied in clinicalpractice therefore we designed this study to ivestigatethe chemopreventive effect of ltras in clinical practicein crc chemoprevention trials the occurrence of adenomas or crc itself is generally set as the trial endpointthough the occurrence of crc is the most confidentendpoint it is not recommended for chemopreventionstudies because crc incidence rate is low in the generalpopulation and needed for longterm monitoring furthermore there are ethical concerns about conductinglongterm trials to determine whether a test agent is effective or notaberrant crypt foci acf defined as lesions containing crypts that are larger in diameter and darker inmethylene blue staining than normal crypts [“] areregarded to be a fine surrogate biomarker of crc our group has previously reported that acf is usefulbiomarker for crc [ ] and study endpoint for achemoprevention study [ ] the advantages of chemoprevention studies with the number of colorectalacf as the trial endpoint are that longterm observationis not needed to investigate the agent efficacy and thenumber of acf can be quantitatively estimated therefore we set the acf count as a good endpoint for thisstudy to the knowledge of us this is the first clinicalstudy investigating the use of ltras as chemopreventive agents against colorectal acf in humansmethodsstudy design and settingthis study was designed as a nonrandomized openlabel controlled trial to be conducted in patients withcolorectal acf it will be performed at the departmentof gastroenterology and hepatology yokohama cityuniversity ycu hospital japan the coordinating office will be at the ycu hospital and patient registrationwill be conducted at the ycu center for novel and exploratory clinical trials ynext and data collectionwill be done using electronic data caputureethical considerations and trial registrationthe trial protocol complies with the declaration ofhelsinki and the ethical guidelines for clinical research of the ministry of health labour and welfarejapan ethical approval of this trial was obtainedfrom the ethics committee of ycu hospital on may the study protocol and informed consent documents were approved by the ycu hospital ethics committee this trial has been approved in the clinical trialact in japan and registered in the japan registry of clinical trials jrct as jrcts031180094 and the universityhospital medical information network umin clinicaltrials registry as umin000029926 all study participants will submit a written study participation informedconsent formparticipation criteriawe will recruit the patients with colorectal acf and resectable polyps for this trial the inclusion criteria are asfollows patients with resectable polyps [adenoma 0chigurashi bmc cancer page of hyperplastic polyp and sessile serrated adenomapolypssap] patients with more than five rectal acfand submit written study participation informed consent formthe exclusion criteria are as follows patients withlesions suitable for early removal a history of ltrause within months before study participation a history of malignant disease within years before studyparticipation a history of heart renal liver failure orliver cirrhosis a history offamilial adenomatouspolyposis hereditary nonpolyposis crc or inflammatory bowel disease pregnancy or the possibility ofpregnancy prohibitions of montelukast allergiesto montelukast regular use of nsaids metforminand pioglitazone and participants considered as unsuitable for the study by the researchersinterventionall eligible participants will be assigned to the ltra orno treatment group because this is an openlabel trialpatients will be assigned to the no treatment group afterthe inclusion of patients in the ltra group participantsin the ltra group will receive mg of oral montelukast for weeks and those in the no treatment groupwill be observed without the administration of any additional drugs at the end of the 8week ltra treatmentperiod a polypectomy will be performed to evaluate thechanges in the number of acf and cell proliferation inthe normal colorectal epithelium will be analyzedoutcome measurementsthe primary endpoint will be the change in the numberof colorectal acf after weeks of treatment a magnifying colonoscope pcfq290azi hz290 olympus cotokyo japan will be used in all cases procedure preparation for the colonoscopy will begin day before theprocedure each participant will be informed to take alowresidue diet and mg oral sodium picosulfate onthe evening before the procedure on the day of the procedure each participant will be given ml polyethylene glycol peg if the stools are not clear enough anadditional ml peg will be given to ensure adequatebowel cleansing in most cases conscious sedation withmidazolam “ mg and pentazocine “ mg willbe use at the start of the colonoscopy subcutaneousscopolamine or glucagon will be administered for colonic movements reduction at the time of the first colonoscopy the endoscopists will insert into the cecumand the observe entire colorectum as the endoscope ispulled back one colonic mucosal sample will be collected the number of rectal acf will be counted usinga magnifying endoscope at the end of the 8week ltratreatment period the same endoscopists will performthe polypectomies and countthe number of acfendoscopists will record all procedures on a hard diskdrive and take photograph all acf the number of acfin each participant will first be counted by the endoscopists during the procedure to provide additional validation the number of acf will be recounted by threeblinded expert endoscopists aj ht and ak by observing the recorded hard disk drive cases that these evaluators deem colonoscopy to be inappropriate will beexcluded from the final analysisthe secondary outcomes will be as follows drugsafety adverse events aes will be graded according tothe national cancer institute common toxicity criteriafor adverse events ncictcae version all trialparticipants will be provided with a trial record for thedaily dose of the trial agent and aes participants whodevelop serious ae of grade or higher will be discontinued at that time in the study the effects ofltras on cell proliferation in the rectal mucosa onecolonic mucosal sample will be collected from the samestudy patient by performing a biopsy at the time of thebaseline colonoscopy and polypectomy a biopsy will beobtained from all participants cell proliferation will beevaluated by ki67 staining briefly we will randomly select six crypts and count the number of ki67positivecells per crypt in total cells will be counted at amagnification of × using a brightfield microscopethe results will be presented as the percentage of ki67positive cells all participants will receive laboratory testsand a physical examination at the point of the baselinecolonoscopy and polypectomydrug supplymontelukast capsules will be purchased from kyorincorporation ltd tokyo japan participants will be informed to take one tablet of the study drug every nightbefore bed medication adherence will be monitored bycounting the empty medication sheets returned by theparticipants at the time of their polypectomy the participants will also be interviewed and monitored to confirm thatthey have not used any prohibited drugsaspirin metformin andor other nsaids aes will bemonitored by the investigator and graded according tothe ncictcae version if serious aes or less than drug adherence are confirmed in a participant thisparticipant will be withdrawn from the trialsample size estimation and allocationwe previously reported the administration of mgmetformin per a day for weeks reduced the number ofacf in that trial the mean number of acf per patientdecreased significantly from ± at baseline to ± at weeks p although this study is exploratory research and the accurate chemopreventive efficacy of montelukastis unknown we assume that 0chigurashi bmc cancer page of montelukast may have an effect that is equivalent to of that observed for metformin on the reduction of acfnubmer therefore we estimate that the acf numberwill change by about ˆ’ to ˆ’ on average we determined that a sample size of “ individuals in theltra group was needed to detect a significant reduction in the number of acfs in the ltra group using apaired ttest with a twosided significance level of and power assuming some dropouts we propose to recruit participants in the ltra group to confirmthat the number of acf does not change during thestudy period we propose to recruit patients in the notreatment group after consecutively accumulating patients in the ltra group therefore we propose to recruit patientsstatistical analysisthe change in the number of acf which is the primaryendpoint will be compared before and after the 8weekstudy period between the ltra and no treatment groupsby the paired ttest drug safety will be assessed by thechisquare test and the remaining results will be compared by the ttest or mann“whitney u test between thetwo groups p will be defined as statistically significant all statistical analyses will be conducted using jmp® software sas institute inc cary nc usatrial steering committee and data monitoring committeethe trial steering committee and data monitoringthe ynext thecommittee will be located atmanagement team will perform central monitoring ofthe trial status and data collection every monthstudy flowa study flow is shown in fig discussionto the knowledge of us this is the first clinical trial proposed to investigate the efficacy of ltras on colorectalacf formation ltras are broadly used for the treatment of allergic asthma and rhinitis [ ] and ltrasare reported to decrease the risk of cancer in asthma patients in a dosedependent manner previous basicresearch has reported that cyslt1r is overexpressed incrc and that montelukast induces the apoptosis ofcrc cells [ ] cyslts have recently been focusedon as significant regulators of gut homeostasis with endogenous cyslt production mediating the proliferationand survival of gut mucosal cells recent evidence focuses on the effect of leukotrienec4 ltc4 in accelerating oxidative dna damage if notadequately repaired can contribute to increase mutationrates and genomic instability dna damage andgenomic instability are major drivers of carcinogenesis cyslts also acts as leukocyte chemoattractants inaddition cyslt1 mediates th17 cell migration the storage of which associates with the progression ofinflammationassociated cancers chronic inflammation is a risk factor for cancer initiation and progression as observed in patients with inflammatory bowelfig study flow chart 0chigurashi bmc cancer page of disease furthermore leukotriene d4 ltd4 antagonists suppress chronic inflammation in a rodent modelof acute enteritis and this may be effective in preventinginflammationassociated crc ltras are leukotriene pathway inhibitors and thus they may have potentialas chemotherapy andor chemoprevention agents to reduce the effect of leukotrienes previous in vivo studieshave elucidated the chemopreventive effect of leukotriene pathway inhibitors [ ] and showed the potentialuse of ltras for chemoprevention therefore we willconduct this trial to explore the chemopreventive effectof ltras in clinical settingthis trial may have some limitations as follow firstacf are believed to be a fine surrogate biomarker ofcrc though its biological significance in humans is stillcontroversial in crc chemoprevention studies typically set the occurrence of adenomas or the crc itselfas endpoint of the study though the occurrence of crcis the most appropriate endpoint it is inappropriate forchemoprevention studies because crc incidence rate islow in the general population and needed for longtermmonitoring our group has previously reported thatacf is useful biomarker for crc and conducted a chemoprevention study for colorectal acf [ ] therefore we designed this study using the number of acf asthe primary endpoint to investigate the chemopreventiveeffect of ltras second an intervention duration of weeks may be too short to reliably detect differences between two groups since our group reported in a previous trial that oral administration of metformin for weeks reduced the number of colorectal acf in humans an intervention period of weeks should beenough to assess the changes in the number of acf ifltras have a chemopreventive effectour group previously conducted a shortterm chemoprevention study of metformin for colorectal acfand reported the preventive effect of the agent on theformation of acf then we conducted a oneyearmetformin chemoprevention studycolorectalpolyps we propose to repeat the same steps as inour metformin study for the chemoprevention studyusing montelukastforif ltras were proved to have efficacy for crc prevention the impact would be significant therefore webelieve it will be very interesting to assess whetherltras inhibit the formation of human colorectal acfabbreviationscrc colorectal cancer nsaids nonsteroidal antiinflammatory drugs cox cyclooxygenase2 ltras leukotriene receptor antagonistscyslt1r cysteinyl leukotriene receptor acf aberrant crypt fociycu yokohama city university umin university hospital medicalinformation network ssap sessile serrated adenomapolyp ncictcae national cancer institute common toxicity criteria for adverseevents peg polyethylene glycol aes adverse events ltc4 leukotriene c4ltd4 leukotriene d4acknowledgmentsthe authors would like to thank the staff for their support in recruitingeligible patients and the patients who participated in this study and theirfamily we thank cathel kerr bsc phd and melissa crawford phd fromedanz group httpsenauthorservicesedanzgroupcom for editing a draftof this manuscriptauthors™ contributionsth ja and an conceived the study th and ja equally contributed to thismanuscript ja th ka nm ty tm af and ho will perform the baselinecolonoscopies and polypectomies ja th and ka will conduct the secondcount of acf using a hard disk drive recording to ensure validity tt nm tyaf and ho will recruit participants and followup with them at an outpatientclinic the analysis and interpretation of data will be conducted by ja thand ka all authors have read the final manuscript and approved its submission for publicationfundinga grant for this research from the kanagawa institute of industrial scienceand technology kistec was awarded to th we declare that the fundingbody has no role in the design data collection analysis interpretation andwriting of the studyavailability of data and materialsthe datasets used andor analyzed during the current study will be availablefrom the corresponding author on reasonable requestethics approval and consent to participateethical approval of this trial was obtained from the ethics committee of ycuhospital on may the study protocol and informed consentdocuments were approved by the ycu hospital ethics committee this trialhas been registered in the university hospital medical information networkumin clinical trials registry as umin000029926 all study participants willsubmit a written study participation informed consent formconsent for publicationnot applicablecompeting intereststhe authors declare that they have no conflicts of interestsreceived may accepted august referencestorre la bray f siegel rl global cancer statistics ca cancer jclin “anderson wf umar a brawley ow colorectal carcinoma in black andwhite race cancer metastasis rev “vogelstein b fearon er hamilton sr genetic alterations duringcolorectaltumor development n engl j med “ winawer sj zauber ag ho mn o™brien mj gottlieb ls sternberg ss wayejd schapiro m bond jh panish jf prevention of colorectal cancer bycolonoscopic polypectomy the national polyp study workgroup n engl jmed “citarda f tomaselli g capocaccia r barcherini s crespi m italianmulticentre study group efficacy in standard clinical practice ofcolonoscopic polypectomy in reducing colorectal cancer incidence gut“zauber ag winawer sj o™brien mj lansdorpvogelaar i van ballegooijenm hankey bf shi w bond jh schapiro m panish jf stewart et waye jdcolonoscopic polypectomy and longterm prevention of colorectalcancerdeaths n engl j med “ maisonneuve p botteri e lowenfels ab fiveyear risk of colorectalneoplasia after negative colonoscopy n engl j med “author reply das d arber n jankowski ja chemoprevention of colorectal cancerdigestion “ epub oct review matsuhashi n nakajima a fukushima y yazaki y oka t effects of sulindacon sporadic colorectal adenomatous polyps gut “ 0chigurashi bmc cancer page of drazen jm cox2 inhibitorsa lesson in unexpected problems n engl jmed “ epub feb meyskens fl jr mclaren ce pelot d fujikawabrooks s carpenter pmhawk e kelloff g lawson mj kidao j mccracken j albers cg ahnen djturgeon dk goldschmid s lance p hagedorn ch gillen dl gerner ewdifluoromethylornithine plus sulindac for the prevention of sporadiccolorectal adenoma a randomized placebocontrolled doubleblind trialcancer prev res phila “scott jp petersgolden m antileukotriene agents for the treatment of lungdisease am j respir crit care med “shen z genomic instability and cancer an introduction j mol cell biol“kim hs lee g the cysteinyl leukotriene receptor cysltr1 mediates th17cell migration j immunol bernstein cn blanchard jf kliewer e wajda a cancer risk in patients withinflammatory bowel disease a populationbased study cancer “ nishikawa m hikasa y hori k tanida n shimoyama t effect of leukotrienec4d4 antagonist on colonic damage induced by intracolonic administrationof trinitrobenzene sulfonic acid in rats j gastroenterol “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations dahlen se dahlen b drazen jm asthma treatment guidelines meet the realworld n engl j med “tsai mj wu ph sheu cc hsu yl chang wa hung jy corrigendumcysteinyl leukotriene receptor antagonists decrease cancer risk in asthmapatients sci rep wang d dubois rn eicosanoids and cancer nat rev cancer “ nielsen ck the leukotriene receptor cyslt1 and 5lipoxygenase areupregulated in colon cancer adv exp med biol “ burke l butler ct murphy a moran b gallagher wm o™sullivan j kennedybn evaluation of cysteinyl leukotriene signaling as a therapeutic target forcolorectal cancer front cell dev biol savari s liu m zhang y sime w sjolander a cyslt1r antagonists inhibittumor growth in a xenograft model of colon cancer plos one e73466 bellamkonda k satapathy sr douglas d chandrashekar n selvanesan bcliu m savari s jonsson g sjolander a montelukast a cyslt1 receptorantagonist reduces colon cancer stemness and tumor burden in a mousexenograft model of human colon cancer cancer lett “ roncucci l stamp d medline a cullen jb bruce wr identification andquantification of aberrant crypt foci and microadenomas in the humancolon hum pathol “ roncucci l medline a bruce wr classification of aberrant crypt foci andmicroadenomas in human colon cancer epidemiol biomark prev “ pretlow tp barrow bj ashton ws aberrant crypts putativepreneoplastic foci in human colonic mucosa cancer res “ pretlow tp o™riordan ma pretlow tg stellato ta aberrant crypts in humancolonic mucosa putative preneoplastic lesions j cell biochem suppl “takayama t katsuki s takahashi y ohi m nojiri s sakamaki s kato jkogawa k miyake h niitsu y aberrant crtpt foci of the colon as precursorsof adenoma and cancer n engl j med “sakai e takahashi h kato s uchiyama t hosono k endo h maeda syoneda m taguri m nakajima a investigation of the prevalence andnumber of aberrant crypt foci associated with human colorectal neoplasmcancer epidemiol biomarkers prev “ epub jul ohkubo h takahashi h yamada e sakai e higurashi t uchiyama t hosonok endo h taguri m nakajima a natural history of human aberrant cryptfoci and correlation with risk factors for colorectal cancer oncol rep “takahashi h yoneda m tomimoto a endo h fujisawa t iida h mawatarih nozaki y ikeda t akiyama t yoneda m inamori m abe y saito snakajima a nakagama h life stylerelated diseases of the digestive systemcolorectal cancer as a life stylerelated disease from carcinogenesis tomedical treatment j pharmacol sci “ hosono k endo h takahashi h sugiyama m sakai e uchiyama t suzuki kiida h sakamoto y yoneda k koide t tokoro c abe y inamori mnakagama h nakajima a metformin suppresses colorectal aberrant cryptfoci in a shourtterm clinical trial cancer prev res phila “the world medical association wma declaration of helsinki “ ethicalprinciples for medical research involving human subjects the ministry of health labour and welfare ethics guidelines for clinicalresearch paruchuri s mezhybovska m juhas m sjolander a endogenous productionof leukotriene d4 mediates autocrine survival and proliferation via cyslt1receptor signaling in intestinal epithelial cells oncogene “ dvash e hartal m barak s meir o rubinstein m leukotriene c4 is themajor trigger of stressinduced oxidative dna damage nat commun 0c"
Colon_Cancer
" camp responsive element binding protein creb5 is a transcriptional activator in eukaryotic cells that canregulate gene expression previously we found that creb5 was involved in the occurrence and development of colorectalcancer crc using bioinformatics analysis however the biological roles and underlying regulatory mechanism of creb5 incrc remain unclearmethods realtime pcr western blotting and immunohistochemistry were used to examine creb5 expression in vitroexperiments including migration assay woundhealing assay chicken chorioallantoic membrane assay and human umbilicalvein endothelial cells tube formation assay were used to investigate the effects of creb5 on crc cell migration and tumorangiogenesis ability additionally an orthotopic implantation assay was performed in nude mice to confirm the effects ofcreb5 in vivo furthermore gene set enrichment analysis was performed to explore the potential mechanism of creb5 incrcresults we found that creb5 expression was highly upregulated in crc creb5 overexpression was positively correlatedwith advanced who stages and tnm stages and shorter survival in crc patients moreover creb5 overexpressionpromoted while creb5 silencing reduced the invasiveness and metastatic capacity of crc cells both in vitro and in vivofurthermore creb5 directly interacted with the met promoter and activated the hepatocyte growth factormet signallingpathway importantly inhibition of met reduced the invasion and metastasis of creb5overexpressing crc cells suggestingthat creb5 promotes metastasis mainly through activation of met signalling our study demonstrates a crucial role for creb5 in crc metastasis by directly upregulating met expressioncreb5 may be both a potential prognostic marker and a therapeutic target to effectively overcome metastasis in crckeywords colorectal cancer creb5 invasion metastasis met correspondence llifimmucom liaowt2002gmailcom shuyang wang junfeng qiu and lei liu contributed equally to this work1department of pathology nanfang hospital southern medical universityguangzhou guangdong chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwang of experimental clinical cancer research page of colorectal cancer crc is one of the most commoncancers ranking third in morbidity and tumorrelatedmortality among both men and women worldwidemoreover approximately of crc patients have metastases at the time of diagnosis metastasis is theleading cause of death among crc patients although systemic treatment of metastatic crc hasimproved the 5year survival rate is only “ andbecause ofthis poor prognosis understanding theunderlying mechanism of the metastatic process in crcis criticalfor both early detection of metastases andmore effective treatment the gene camp responsive element binding protein creb5 which is located on chromosome 7p151encodes a transcription activator in eukaryotic cells creb5 belongs to the atfcreb family the membersof which are characterized by a high affinity for campresponse elements cres the targets of the atfcreb family include transcriptional regulators including chromatinmodifying enzymes coactivators and corepressors genes involved in mitochondrial homeostasisand protein import and genes associated with proliferation and cell cycle entry metabolism proteases transporters and chaperones as an atfcreb familymember the creb5 protein contains several importantfunctional domainsincluding nterminal zinc fingerand cterminal bzip domains the latter of which includes a dna binding region and leucine zipper creb5 is a transcription factor that specifically binds tocre as a homodimer or a heterodimer with cjun orcrebp1 and functions as a credependenttransactivator creb5 is physiologically required for embryonic development in mice recent studies revealedthe roles of creb5 in the development and progressionof cancers examination of tcga pan cancer datasetsrevealed frequent creb5 amplification and overexpression in kidney cancers sarcomas lymphomas and lungadenocarcinomas as well as glioblastomas and gliomas experimentalinvestigations showed that creb5was upregulated in ovarian cancers hepatocellularcarcinoma and prostate cancer high creb5expression correlated with a poor prognosis in epithelialovarian cancer and hepatocellular carcinoma creb5 overexpression increased the proliferation of hepatocellular carcinoma moreover overexpression oramplification of creb5 promoted proliferation and mediated resistance to ar inhibition in metastatic castrationresistant prostate cancers in silico analysis showedthat the creb5 regulatory network was involved in crcmetastasis in addition qrtpcr assay revealed thatcreb5 mrna was upregulated in crc tissues and cells in vitro assays revealed that overexpression of creb5resulted in enhanced proliferation and migration andapoptosis inhibition in crc cells these findings suggest that creb5 may play an essential role in the progression of crc howeverthe specific function andmolecular mechanism of creb5 in crc metastasis remain largely unclearactivation of the hgfmet signalling pathway hasbeen reported to lead to the occurrence and metastasisof a variety of tumorsincluding crc breast cancerovarian cancer lung cancer and liver cancer as atyrosine kinase receptor met can be activated bydimerization multimerization and phosphorylation afterbinding to its ligand hepatocyte growth factor hgf activation of hgfmet can initiate downstreamsignalling pathways that drive malignant progression inmany types of tumors met is considered an essentialfactor for early invasion and metastasis of crc and canbe regarded as an important prognostic indicator in the present study we found that creb5 promotes crc invasion and metastasis by increasing metexpression to activate hgfmet signalling these results uncover a new molecular mechanism for cancermetastasis and suggest that creb5 may be a promisingtarget for crc treatmentmaterials and methodspatients and specimensa total of pathological specimens were obtainedfrom colon cancer patients between and atthe department of pathology nanfang hospital southern medical university the medical records of these patients provided information on sex age and thefollowing essential factors tumor pathological characteristics pathologic stage t stage dukes stage lymph nodemetastasis and distant metastasis ten pairs of fresh biopsies collected from crc patients and matched noncancerous mucosaltissue were obtained from theoperating room of nanfang hospital the fresh biopsieswere stored in liquid nitrogen before usein addition a tissue microarray no co802 containing colon cancer tissue specimens and adjacentnoncancerous tissue specimens was purchased fromailina biotechnology company approval for the use ofclinical materials for research purposes was obtainedfrom the southern medical university institutionalboard guangzhou china all samples were collectedand analysed with the prior written informed consent ofthe patientscell culturesthe human crc celllines sw480 ht29 hct15hct116 sw620 ls174t sw837 lovo dld1 andrko were purchased from the american type culturecollection sw620 ht29 and lovo cells were culturedin dmem medium gibco supplemented with 0cwang of experimental clinical cancer research page of foetal bovine serum fbs gibco sw480 hct116hct15 ls174t sw837 and dld1 cells were culturedin rpmi medium gibco with fbs gibcoplasmidscreb5 constructs were generated by cloning pcramplifiedfulllength human creb5 cdna into psinef2puro thefollowing primers were used for cloning including enzymesforward primer ² cgcgaattcatgatttatgaggaatccaa3² ecor i reverse primer ²ccggctagcttaaagaatcggattcaggt3² nhe i for deletion ofcreb5 short hairpin rna shrna sequences creb5²aacaagtcatccagcataa3² creb5shrna1shrna2 ²ggaatatctcgatgcataa3² were separately cloned into a gv248 vector psinef2puro and gv248were purchased from addgene incthe intensity of staining was graded according to thefollowing criteria no staining weak staining lightyellow moderate staining yellow brown and strong staining brown the staining index was calculated as the staining intensity score × the proportion ofpositive tumor cells using this method of assessmentwe evaluated the expression of creb5 in benign breastepithelium and malignant lesions by determining thestaining index with scores of and cutoff values for creb5 were selected on the basis of ameasure of heterogeneity with the logrank statisticaltest with respectto overall survival optimal cutoffvalues were identified a staining index ‰¥ was used todefine tumors with high creb5 expression and anindex ‰¤ was used to define tumors with low creb5expressionrna isolation reverse transcription rt and realtimepcrtotal rna samples from cultured cells and primarytumor tissues were extracted using trizol reagent invitrogen usa according to the manufacturer™s instruction realtime rtpcr was performed at least threetimes in triplicate using sybr green mix toyobojapan and the abi prism sequence detectionsystem applied biosystems usa the data were normalized to the geometric mean of the housekeeping genegapdh and calculated using the 2δδct method primerexpress was used to design the realtime pcr primersand primer sequences for amplification are shown insupplementary table s2luciferase reporter assaygenomic dna extracted from sw480 cells was used as atemplate to amplify met promoter fragments met promoter fragments were obtained by pcr and constructedinto pgl3basic plasmid using a fast ligation kit followingmanufacturer™s instructions sangon b620511“ thesequences of the pcr primers are listed in supplementarytable s4 cells at confluence in a 24well plate weretransfected using lipofectamine fortyeight hoursafter transfection luciferase activity was measured usingthe dualluciferase reporter assay system promega corpmadison wi usa and normalized to renilla luciferasegene expression all the experiments were performed intriplicatewestern blotting analysiswe carried out western blotting as previously described using anticreb5 abcam ab168928 antimetcell signaling technology antipmetcellsignaling technology antiakt cell signalingtechnology antipaktcell signaling technology antierkcell signaling technology antiperk cell signaling technology and antisnail cell signaling technology antiantiαtubulin antibodybodies mouse monoclonalsigma was used as the internal controlimmunohistochemistryimmunohistochemistry ihc staining was performed aspreviously described using creb5 antibody abcamusa ab168928 the degree of ihc staining wasreviewed and scored independently by two observersbased on both the proportion of positively stained tumorcells and the intensity of staining [ ] the proportion of tumor cells was scored as follows no positivetumor cells positive tumor cells “positive tumor cells and positive tumor cellschromatin immunoprecipitation chip assaychip assays were carried out as previously described precleared lysates were incubated with creb5 antibody abcam ab168928 or normal mouse immunoglobulin g cst as a negative control overnightat °c with rotation the human met promoter wasamplified by pcr all chip assays were performed threetimes and the sequences of the pcr primers are listedin supplementary table s3migration assaya boyden chamber with an 8μmpore filter membranewas used for the in vitro migration and invasion assaybriefly cells × in culture medium containing fbs were seeded in the upper chamber and culturemedium with fbs was added in the lower chamberas a chemoattractant the upper side of the filter wasfirst coated with matrigel bd biosciences sanjose ca usa after incubation for h cells on theupper side of the filter were removed with cotton swabscells that migrated to the lower surface of the filter werefixed in paraformaldehyde and stained with giemsa 0cwang of experimental clinical cancer research page of the migratory cells were counted random ×fields per well three independent experiments wereperformed and the data are presented as the mean ±semwoundhealing assaycells were seeded in 6well plates and incubated underpermissive conditions until confluence after serumstarvation for h wounds were created in the confluent cells using a pipette tip wound healing within thescrape line was then observed and photographed at indicated time points each experiment was repeated at leastthree timeschicken chorioallantoic membrane assaya chicken chorioallantoic membrane cam assay wasperformed on the sixth day of development of fertilizedchicken eggs as previously described human umbilical vein endothelial cell tube formationassayfirst μl of matrigel was pipetted into each well of24well plates and polymerized for min at °c human umbilical vein endothelial cells huvecs × in μl of conditional medium were added to eachwell and incubated at °c in co2 for h imageswere obtained under a brightfield microscope and thecapillary tubes were quantified by the counting lengthorthotopic mouse metastatic model to 6weekold balbc athymic nude mice nunuwere obtained from the animal center of southernmedical university guangzhou china all mice werehoused in a sterile environment cells × permouse were orthotopically inoculated into the caecumof anaesthetized nude mice the mice were sacrificedwithin weeks after surgery individual ans were excised and metastases were observed by histological analysis tissues were then fixed with formaldehyde andparaffinembedded and then 5mm sections were cutand stained with haematoxylin and eosin he thenumbers of gross metastatic foci were determined usinga dissection microscope all the mice used in this studywere maintained under specific pathogenfree conditions and all animal experiments were conducted in accordance with standard procedures and approved by theinstitutional use committee for animal carestatistical analysisall statistical analyses were carried out using spss version pearson correlation analysis was used for expression correlation analysis the survival curves of crcpatients in low and highcreb5 expression groups wereanalysed by the kaplanmeier method and the logranktest was used to compare differences p was considered significantresultscreb5 is upregulated in crc and associated with a poorprognosisthe expression of creb5 was analysed in differenttypes of malignant tumors in the public database oncomine wwwconominecom revealing that creb5was upregulated in crc tissues in of crc datasetssupplementary fig s1a additionally a gene set enrichment analysis gsea plot showed significant enrichment of tumorrelated genes and crcrelated genesets in the highcreb5 expression group supplementary fig s1b realtime pcr and western blotting analyses showed that creb5 was differentially expressed incrc cell lines supplementary fig s1cd in additioncreb5 was significantly upregulated in ten crc tissuescompared with adjacent normal intestinal epithelial tissues fig 1a and b ihc showed that creb5 proteinwas weakly expressed in normal tissue but markedly increased in adenocarcinoma cells and was mainly localized in the nucleifig 1c kaplanmeier survivalanalysis showed that crc patients with higher creb5protein expression levels had a poorer prognosis fig1d in addition creb5 expression levels were significantly correlated with the t classification lymph nodemetastasis and distant metastasis p supplementary table s1 creb5 expression was substantiallyhigher in tumors from patients with distant metastasismoreover high creb5 expression was also positivelycorrelated with who stages p supplementarytable s1 these data suggested that creb5 expressionis significantly correlated with advanced stages of crccreb5 activates met signallinggseas of creb5regulated gene signatures revealed thathigher creb5 expression was positively correlated withenrichment of an met signalling pathway signaturegse17538 fig 2a to validate this result we establishedstable creb5overexpressing and creb5knockdowncrc cell lines fig 2b upregulation of creb5 significantly increased while knockdown of creb5 decreasedthe expression of total met at both translational andtranscriptional levels fig 2c and d in addition creb5overexpression markedly increased but creb5 downregulation significantly attenuated the expression of phosphorylated met perk pakt and snail fig 2c moreovermet expression was increased in a dosedependent manner at both translational and transcriptional levels by transiently transfecting sw480 cells with a creb5 expressionvector fig 2e 0cwang of experimental clinical cancer research page of fig creb5 is upregulated in crc and associated with a poor prognosis a and b realtime pcr and western blotting analysis of creb5 expression inpaired human colon cancer tissues and adjacent noncancerous tissues p quantity one software was used to quantify the protein expressionlevels c ihc representative images of creb5 expression in normal intestinal epithelium and crc tissues scale bar μm d the paraffin samples of crc patients were divided into a lowcreb5 expression group n and a highcreb5 expression group n based on ihc results thekaplanmeier method was used to analyse survival curves and the logrank test was used to compare differences p fig creb5 activates the met signalling pathway a gsea of gse17538 in met signalling pathways es p b stable overexpressionand interference cell lines were detected by western blotting and realtime pcr c the expression of met and downstream signalling moleculesin creb5knockdown or creb5overexpressing cells was observed by western blotting d creb5 had an effect on met by realtime pcr in theindicated cells e after transient transfection of different amounts of the creb5overexpression plasmid in sw480 cells the protein and mrnalevels of met were detected by western blotting and realtime pcr respectively p 0cwang of experimental clinical cancer research page of creb5 associates with the met promotergiven that creb5 is a transcriptional factor and upregulatesmet at the transcriptional level we performed a luciferasereporter assay to investigate whether creb5 can increasemet promoter activity a 27kb fragment of the fulllengthmet promoter region was subcloned into a luciferase vector met promoter activity wasincreased by cotransfection with a creb5 expression vector in sw480 cellsbut decreased in hct116 cells expressing creb5 shrna ina dosedependent manner compared with empty vectorsfig 3a to determine the effective regions of the met promoter that creb5 may affect we transfected met promotertruncations fig 3b into hct116 cells expressing eithercreb5 shrna or a scramble control sequence as shown infig 3c luciferase activity was increased in cells carrying thefulllength met promoter and truncations ˆ’ and ˆ’ bp upstream of the transcription start site but not incells carrying truncations ˆ’ to ˆ’ bp or ˆ’ to ˆ’ bp knockdown of creb5 expression bycotransfection of creb5 shrna significantly decreased metpromoter activity fig 3c furthermore we performed chromatin immunoprecipitation chip assays and identified thatthe ˆ’ to ˆ’ 223bp region of the met promoter whichcontains an ap1 motif was a creb5 protein binding sitefig 3d these data identify met as a direct transcriptionaltarget of creb5downregulation of creb5 represses invasiveness andreduces the metastatic potential of crc cellsnext we investigated the role of creb5 in invasivenessand metastasis in crc cells silencing of creb5 significantly compromised the migratory and invasive abilitiesof crc cells fig 4a and b supplementary fig s2a andb the tubule formation and chicken cam assays revealed that knockdown of creb5 strongly inhibited theformation of tubules by huvecs and inhibited angiogenesis in cams fig 4c and d supplementary figs2c orthotopic inoculation assay showed that knockdown of creb5 inhibited liver metastases fig 4eknockdown of creb5 also obviously extended the overall survivaltime of nude mice inoculated with thecrc cell lines fig 4f these results indicate that silencing creb5 inhibits the invasiveness and metastasis of crc cellsinhibition of met attenuates the invasion and metastasisof crc cells by creb5 in vivo and in vitroto determine the functional relationship between creb5and met in the invasion and metastasis of crc weknocked down met using two met shrnas or suppressed met activation using the met inhibitor crizotinib emd silencing or inhibition of met insignificantlysw480creb5or ht29creb5cellsfig creb5 regulates met and binds directly to the met promoter a the met promoter sequence was cloned into pgl3basic vector containing theluciferase reporter gene and then transfected into crc cells with the indicated treatments b schematic diagram of the full and truncated met promoterc the fulllength met promoter or its truncations were cloned into pgl3basic vector containing the luciferase reporter gene and then transfected intohct116 cells with creb5 shrna or empty vector d chip analysis of creb5 binding to the met promoter in sw480 cells p 0cwang of experimental clinical cancer research page of fig downregulation of creb5 inhibits the invasion and metastasis of crc cells in vivo and in vitro a and b woundhealing assay and transwellmigration assay were performed to evaluate the invasive and migratory abilities of crc cells with different treatments in vitro c huvec tubeformation after stimulation with the indicated conditioned medium d representative images of the cam assay histograms show the formationof secondary and tertiary blood vessels after stimulation with the indicated conditional medium scale bar mm e orthotopic transplantationwith the indicated hct116 cells in nude mice n in each group was performed and representative gross images of the livers and intestinesare shown the arrows indicated the tumors liver sections were stained with haematoxylin and eosin he scale bar μm f the kaplanmeiermethod was used to analyse survival curves in the specified treatment groups and the logrank test was used to compare differences p decreased the expression of phosphorylated met perkand pakt as well as snail supplementary fig s3a inaddition the invasive and migratory abilities of sw480creb5 or ht29creb5 cells were partially diminished byinhibition of met fig 5a and b supplementary fig s3band c moreover upregulation of creb5 expression enhanced the capacity of crc cells to induce tube formationand angiogenesis in cams in contrast angiogenesis ability was partially diminished by met inhibition fig 5cand d supplementary fig s3d orthotopic inoculationassay showed that creb5 significantly promoted liver metastases and decreased the overall survival of mice fig 5eand f conversely inhibition of met significantly attenuated the formation of metastatic foci by sw480creb5cells and extended the survival time of mice inoculatedwith sw480creb5 cells fig 5e and fcreb5 expression positively correlates with metexpression in crcto assess a potential link between creb5 and met expression in human crc we analysed tcga crc dataand identified a strong positive correlation between highexpression levels of creb5 and met p r fig 6a in addition analyses of fresh crc tissuesshowed that creb5 expression was positively correlatedwith met expression at both mrna p r and protein levels p r fig 6b and c 0cwang of experimental clinical cancer research page of fig overexpression of creb5 promotes the invasion and metastasis of crc cells but inhibition of met weakens these effects a and b theinvasive and migratory abilities of crc cells in vitro with different treatments were evaluated by woundhealing assay and transwell migrationassay c huvec tube formation after stimulation with the indicated conditional medium d representative images of the cam assay histogramsshow the formation of secondary and tertiary blood vessels after stimulation with the indicated conditional medium scale bar mm eorthotopic transplantation with the indicated sw480 cells in nude mice n in each group was conducted and representative gross images ofthe livers and intestines are shown the arrows indicate the tumors liver sections were stained by he scale bar μm f the kaplanmeiermethod was used to analyse the survival curves of different treatment groups and the logrank test was used to compare differences p p p furthermore ihc revealed that creb5 expression waspositively correlated with met fig 6d p discussionmetastasis of crc is a multistep process requiring the accumulation of genetic andor epigenetic alterations andabnormal expression of genes involved in signal transduction pathwaysincluding oncogenic mutation of krasand activation of the erkmapk pathway wntβcatenin signalling and tgfβ signalling ap1 dnabinding sequences act as crucial response elements fortranscriptional activation by the raserk pathway creb5 is a credependent transactivator downstream ofthe raserk signalling pathway since it interacts with cjun which is one of the ap1 subunits to form a homodimer or a heterodimer along with foxd1 and atf3creb5 formed a transcription factor regulatory networkthat negatively regulates mapk signalling which is suppressed by fzd3 in melanoma previous studies haverevealed that creb5 mrna was upregulated in crc asdemonstrated by bioinformatics analysis or qrtpcrexamination in human cancer tissues [ ] in thecurrent study we demonstrated that creb5 was highlyupregulated in crc at both the mrna and protein levelsoverexpression of creb5 was significantly associatedwith aggressive cellular characteristics of crc eg an 0cwang of experimental clinical cancer research page of fig creb5 positively correlated with met expression in crc a correlation analysis of creb5 and met in tcga crc data r p band c correlation analysis of creb5 and met at the mrna r p and protein levels r p in fresh crc tissues dthe expression of creb5 and met protein in specimens including colon cancer tissue specimens and adjacent normal tissue specimens wasdetected by ihc representative ihc images left and correlation analysis right of creb5 and met expression scale bar μm high expressionof creb5 n high expression of met n low expression of creb5 n low expression of met n p advanced who stage and an advanced tnm stage andpoorer patient outcomes suggesting that creb5 might bean oncogene and a prognostic marker of crc progressionconsistent with our results upregulation of creb5 hasbeen reported to be responsible for poorer outcomes inpatients with epithelial ovarian cancer and hepatocellular carcinoma in prostate cancers creb5 overexpression occursthrough both copy number gain and increased gene expression however examination of tcga colorectalcancer datasets via cbioportal revealed rare amplificationof creb5 suggesting that overexpression of creb5 iscontrolled attranscriptional and posttranscriptionallevels creb5 has been shown to be a downstream target of lncrna snhg5mir1323p and circularrna circvapamir125a in addition fzd3 inhibits transcriptional networks controlled by creb5 however the alternative mechanisms involved inupregulation ofthe creb5 gene and activation ofcreb5mediated signalling require further investigationthe effects of creb5 overexpression on promotingcell proliferation and migration have been demonstratedusing in vitro assays in human hepatocellular carcinomacells and crc cell lines [ ] in the current studywe showed that creb5 overexpression promoted whilecreb5 silencing reduced the invasiveness and metastaticcapacity of crc cells both in vitro and in vivo mechanistically creb5 directly interacted with the met promoter and activated the hgfmet signalling pathwayimportantlyinhibition of met reduced the invasion 0cwang of experimental clinical cancer research page of and metastasis of creb5overexpressing crc cells suggesting that creb5 promotes metastasis mainly throughactivation of met signalling our data provide solid evidence that upregulation of creb5 plays an essential rolein crc metastasis recently overexpression or amplification of creb5 was reported to promote proliferationand mediate resistance to ar inhibition in metastaticcastrationresistant prostate cancers these datasuggest that creb5 may function as a multitaskingregulator in cancer progression and clinical outcomessignallingtransportimmunegrowth factorscreb family members can be phosphorylated via various intracellular signal transduction pathways such asprotein kinase a pka calmodulindependent proteinkinasecamk mitogenactivated protein kinasesmapks and other kinases upon phosphorylationcreb recruits crebbinding protein cbp and binds tothe cres of the promoters of its target genes target genes containing consensus sites for creb bindinginclude those related to metabolism transcription neuropeptidesneurotransmitters cell cyclecellsurvivalregulationdna repairreproductiondevelopmentandstructure specifically knockdown of creb1creb5increased tumor necrosis factor alpha tnfα levelsenhanced the expression of phosphonfκb p65 andnfκb p65 and induced immunosuppression in monocytes in prostate cancer creb5 could improve resistance to enzalutamide with the help of foxa1 andselectively enhance the interaction of ar with targetgenes critical for survival however little is knownabout the downstream targets of creb5 involved in theprogression of crc our results showed that creb5 directly interacted with the met promoter and activatedthe hgfmet signalling pathway which in turn increased the expression of downstream erk and pi3ksignalling cascades meanwhile the expression of snailan essential emt transcription factor was also upregulated via the creb5hgfmet axistranscriptional factor that interacts with the met promoter at the ap1 motif and activates met expressionin our data suggest that creb5 has an essential role in crc metastasis by regulating the protooncogene met interfering with creb5 may representan alternative therapeutic target to prevent or reducemetastasis in crcsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s13046020016730additional file table s1 the relationship between creb5 expressionand clinicopathological parameters table s2 primer sequences used forrealtime pcr ² to ² table s3 sequence of primers for chip assaytable s4 sequence of primers for luciferase reporter assayadditional file figure s1 bioinformatics analysis of creb5expression the expression of creb5 in crc and other malignant tumorswas analyzed by oncomine database the inclusion criteria were that thedifference of creb5 expression between tumor tissue and normal tissuewas more than times and the arrangement of gene position was lessthan with p the outliers in the red and blue boxes representthe number of data sets with high and low expression of creb5respectively the right table of a represents the copa score of creb5 in crc data sets b the two crc chips gse17538 n andgse35896 n from the public database of geo was analyzed bygsea the plot showed significant enrichment of tumorrelated gene setkegg_pathway_in_cancer and colorectal cancerrelated gene setkegg_colorectal_cancer in the creb5 high expression group cand d realtime pcr and western blotting analysis of creb5 endogenous expression in indicated crc cellsadditional file figure s2 representative images of woundhealingassay a transwell migration assay b and huvec tube formation assayc with i
Colon_Cancer
"objectives to describe the benefits and limitations of using individual and combinations of linked english electronic health data to identify incident cancersdesign and setting our descriptive study uses linked english clinical practice research datalink primary care cancer registration hospitalisation and death registration dataparticipants and measures we implemented case definitions to identify first site specific cancers at the most common sites based on the first ever cancer diagnosis recorded in each individual or commonly used combination of data sources between and we calculated positive predictive values and sensitivities of each definition compared with a gold standard algorithm that used information from all linked data sets to identify first cancers we described completeness of grade and stage information in the cancer registration data setresults gold standard cancers were identified positive predictive values of all case definitions were ‰¥ and ‰¥ for the four most common cancers breast lung colorectal and prostate sensitivity for case definitions that used cancer registration alone or in combination was ‰¥ for the four most common cancers and ‰¥ across all cancer sites except bladder cancer using cancer registration alone for case definitions using linked primary care hospitalisation and death registration data sensitivity was ‰¥ for the four most common cancers and ‰¥ for all cancer sites except kidney oral cavity and ovarian cancer when primary care or hospitalisation data were used alone sensitivities were generally lower and diagnosis dates were delayed completeness of staging data in cancer registration data was high from minimum in and in for the four most common cancerss ascertainment of incident cancers was good when using cancer registration data alone or in combination with other data sets and for the majority strengths and limitations of this study –º this is the first study to present comprehensive information on the implications of using different individual and combinations of linked electronic health data sources in england to identify cases of the most common incident cancers –º using a gold standard algorithm that combined all available data from multiple sources as a comparator we were able to estimate both positive predictive values and sensitivity values for a range of pragmatic case definitions –º we described similarities and differences in values between age groups sexes and calendar years the impact of choice of sources on diagnosis dates and mortality rates and completeness of stage and grade in cancer registration data –º a key limitation was that our gold standard algorithm is not validated and may be affected by differences in clinical diagnosis and coding of invasive cancers between data sourcesof cancers when using a combination of primary care hospitalisation and death registration dataintroductionthe clinical practice research datalink cprd provides de identified primary care data linked to additional secondary health data sources under a well governed framework1 use of linked data helps researchers to answer more epidemiological questions and increase study quality through improved exposure outcome and covariate classification2 in the field of cancer epidemiology cprd primary care data linked to hospital episode statistics admitted patient care strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access data hes apc office of national statistics ons mortality and national cancer registration and analysis service ncras cancer registration data are used to analyse factors contributing to the risk of cancer and the consequences of cancer and its treatment use of linked data reduces the sample to the common source population and data coverage period for each included data set and has cost and logistical implications which are greatest for ncras data research teams therefore commonly choose not to use all available linked data3 cancer epidemiology studies can also be conducted using ncras and hes apc data provided by national health service nhs digital and public health england phe without linkage to cprd primary care data4 this provides national coverage at the expense of the detailed health data that are available in primary care recordsvalidation studies assessing concordance between cprd gold hes apc and ncras data have estimated high positive predictive values ppvs for cprd gold data and varying proportions of registered cancers that are not captured in cprd gold and hes apc5“ the most up to date analysis by arhi et al included the five most common cancers and all papers focussed on concordance between cprd gold only and ncras existing evidence therefore does not provide a complete assessment of the benefits and limitations of using different combinations of data sources within the context of practical study designs national data are available describing completeness of data fields within the cancer registry data in each collection year9 and over time for all cancers combined4 missingness for individual years has been associated with age comorbidities and clinical commissioning groups10 we aim to describe and compare the benefits and limitations of using different combinations of linked cprd primary care data hes apc ons mortality and ncras cancer registration data for conducting cancer epidemiology studies our analyses focus on incident cancer ascertainment as it is a common and important outcome in cancer epidemiology and it is more difficult to distinguish between secondary recurrent and primary cancers at a second site in these data sets we have compared definitions of the most common cancers based on the first ever cancer recorded in individual or combinations of data sets with a gold standard definition comparing information from all four data sets we also describe the availability of stage grade and treatment variables over time in the cancer registration data for the cprd linked cohort this reflects real life study design and will help researchers to decide which combination of data sources to use for future studiesmethodsstudy design and settingwe completed a concordance study using linked2 english cprd gold hes apc ons mortality and ncras data cprd gold data were extracted from the january monthly release and the 13th update to cprd™s linked data the study period was from january to december with december matching the end of the ncras coverage periodthe cprd gold database includes de identified records from participating general practices in the uk who use vision software1 general practice staff can record cancer diagnoses using read codes or in free text comments boxes though the latter are not collected by cprd diagnoses will typically be entered duringfollowing a consultation or from written information that is returned to the practice from secondary care cprd gold data are linked to hes apc ons mortality and ncras through a trusted third party for english practices that have agreed to participate in the linkage programme2 hes apc data are collected by nhs digital to co ordinate clinical care in england and calculate hospital payments12 admissions for and related to cancer diagnoses are recorded using international classification of diseases version icd10 codes national cancer registration data are collected by ncras which is part of phe in accordance with the cancer outcomes and services data set13 which has been the national standard for reporting of cancer in england since january data include icd10 codes to identify the cancer site and more detailed information such as stage and grade ons mortality data includes dates and causes of deaths registered in england recorded using icd10 codesparticipants exposures and outcomesour underlying study population included male and female patients registered in cprd gold practices who were eligible for linkage to hes apc ncras and ons mortality data and had at least days of follow up between january and december start of follow up was defined as the latest of the current registration date within the practice and the cprd estimated start of continuous data collection for the practice up to standard date end of follow up was determined as the date the patient left the practice ons mortality date of death or practice last collection dateidentification and classification of cancer codeswe used code lists to classify cancer records in each of cprd gold hes apc and ons mortality data as one of the most common sites other specified cancers history of cancer secondary cancers benign tumours administrative cancer codes unspecified and incompletely specified cancer codes https org data incompletely specified cancer codes could be mapped to cancer site eg icd10 code c689 œmalignant neoplasms of urinary organ unspecified was considered consistent with both bladder and kidney cancer for ncras we accessed coded records for the most common cancers we included cancers recorded in the clinical or referral file for cprd gold cancers recorded in any diagnosis field for hes apc and the underlying or strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure gold standard algorithm to identify incident site specific cancers using all data sources hes hospital episode statistics ncras national cancer registration and analysis service ons office of national statisticsmost immediate cancer cause of death in ons mortality datacancer case definitions based on individual sources and combinations of sourceswe developed alternative cancer case definitions mirroring those commonly used in epidemiology studies based on identifying the first malignant cancer excluding administrative codes and benign tumours recorded in various combinations of data sources ncras alone ncras and hes apc all sources cprd gold hes apc and ons mortality cprd gold alone hes apc alone multiple malignant cancers recorded on the index date in cprd gold or hes apc were reclassified as multiple site cancer and were not considered as individual site cancer records for positive predictive value and sensitivity calculations multiple codes recorded in different sources on the same date were reclassified as the site identified in the ncras data if available and as multiple site cancer if not for each case definition we only examined the first malignant cancer per individual where this occurred within the study period and at least year after the start of follow upgold standard cancer case definitionwe developed a gold standard algorithm that classifies incident records of the most common cancers by comparing the first malignant cancer identified in each individual source figure cancers recorded in ncras alone with no contradictions ie records for first cancers at different sites were considered true cases whereas cancers recorded in hes apc alone or gold alone required internal confirmation within that source in the form of another code for cancer consistent with the same site or with site unspecified within months and no contradictory codes eg for cancers at other sites in this period where cancer records were present in data source we considered a site specific cancer to be a true case a if it was recorded as the first cancer in ncras and the total number of data sources with records for cancer at that site was equal to or greater than the number of data sources with contradictory records ie records for first cancers at different sites or b where the cancer was not present in ncras if there were more data sources in total with records for cancer at that site than data sources with contradictory recordswe used ncras data to identify stage grade and treatment where available in the cancer registry only cohort binary surgery chemotherapy and radiotherapy variables were derived using individual records of treatment from the first year after diagnosisstatistical analysisfor each cancer site and each individual or combined data source we combined our applied study definitions strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access with our gold standard definition to classify each applied study definition as a true positive false positive or false negative recordwe used these categories to calculate sensitivity and positive predictive value overall and stratified by age categories to and calendar year and sex we calculated differences in diagnosis dates for true positives by subtracting the gold standard index date from the index date for each source and combination of sourceswe used kaplan meier methods to describe mortality over time for cancers identified using each definition the ons mortality death date was used for these analyseswe used the ncras only definition to calculate proportions of patients with complete stage and grade and recorded cancer treatment modalities over timepatient public involvementpatients and the public were not involved in conceiving designing or conducting this study and will not be consulted regarding the dissemination of study resultsresultsof research quality patients in the cprd gold january build were eligible for linkage to hes ons mortality and ncras data in set were excluded due to unknown sex of the remainder and had at least year of follow up between january and december and were included in the study population using the gold standard algorithm incident cases of cancer were identified the number of patients identified with each cancer is presented in online supplementary appendix table half n82 of these patients were male aged to aged to and aged or olderfigure presents ppvs for each case definition comparing the first recorded cancer in each combination of data sources with the gold standard algorithm when using ncras data alone to of cancers were confirmed by the algorithm for out of cancer sites the ncras only case definition gave the highest ppv case definitions using data sources not including ncras generally had lower ppvs ranging from to for individual cancer sites for the four most common cancers breast lung colorectal and prostate ppvs were at least for all case definitions minimal differences in ppvs were observed between age groups years and sexes online supplementary appendix figures “figure presents sensitivity values for each case definition sensitivity was generally higher for the case definitions that included ncras data ranging from to for individual cancer sites except bladder cancer identified using ncras data alone and ‰¥ for the four most common cancers breast lung colorectal and prostate sensitivity was also generally high for definitions using a combination of cprd gold hes apc and ons mortality data ranging from to ‰¥ for the four most common cancers sensitivity was lower for case definitions that used cprd gold alone range to for individual cancer sites or hes apc alone range to sensitivity values for cprd gold alone and hes apc alone increased slightly in younger patients and more recent years no differences were observed between men and women online supplementary appendix figures “ post hoc analysis suggested that the low sensitivity of cprd gold only definitions for kidney cancer sensitivity n false negatives was driven by missing n1136 or incompletely specified urinary organ cancer codes n1108 in cprd gold rather than contradictory information about the first cancer record n625 these incompletely specified codes are less likely to be used for bladder cancers n85 than kidney cancers n1108 bladder cancers that were not recorded in ncras data n3445 were commonly recorded in both hes apc and cprd gold n2228 or in hes apc only with a subsequent unspecified or bladder cancer record in hes apc within months n995 table describes the number of days median iqr and 5th95th percentile lag between the date of incident cancers from the gold standard definition and the date of cancer arising from each case definition ie the first record within the specific combinations of data sources used case definitions using ncras alone and combinations of ‰¥ data sources captured cancers close to the gold standard date median lag ‰¤ days for all cancer sites whereas median lags were generally longer for the case definitions using cprd gold alone and hes apc alonefigure describes mortality over time following incident cancer diagnoses ascertained from each case definition minimal differences in mortality were observed between cancers identified from different case definitions where variability was observed cancers identified using cprd gold only had the lowest mortality rates eg kidney cancer and cancers identified using hes apc only or ncras only had higher mortality rates eg prostate cancer and bladder cancer respectivelyfigure describes completeness of grade and stage for cancers identified using ncras only recording of grade was highly variable between cancers with gradual increases in completeness over time completeness of staging information was low in earlier calendar years but improved substantially from around especially for the four most common cancers minimum in and in post hoc logistic regression models adjusted for year and cancer site indicated that completeness of stage and grade were associated with each other and these variables were least complete in patients aged stage data was more complete for higher grade tumours whereas grade data was more complete for lower stage tumours online supplementary appendix figure online supplementary appendix figure describes recording of treatment modalities identified using ncras strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure positive predictive value of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm percentage of incident cancers defined using the first ever record in each combination of sources confirmed by a gold standard algorithm that considers confirmatory and contradictory data from each source cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system nhl non hodgkin's lymphoma cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service ons office of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access figure sensitivity of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm percentage of incident cancers identified using the main gold standard algorithm that considers confirmatory and contradictory data from each source that are identified using the first ever record in each combination of sources cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system nhl non hodgkin's lymphoma cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service onsoffice of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0celitnecrep ht“htrqi inademelitnecreprqi inadem ht“htelitnecrep ht“ht inademrqielitnecrep ht“htcpaseh dlogdrpc ytil atromsnodna cpaseh dlogdrpc cpasehdnasarcn secruos fonoitanbmoc hcae nii iidrocer reve tsrfi ot etad ssongaddradnats dog nammorf syad n ili emti l ebatopen access recnac lanoitan sarcn atad erac tneitapdetti mda scitsitats edospei latipsoh cpaseh knil atadhcraeser ecitcarp lacniilc drpc metsys suovren lartnec sncl tluafed ybnoitinfieddradnats dog eht sa emas eht si etad ssongad sa nwohs tonnoitinfied secruos ll iia setis recnac nommoc tsom ruofdcii nosrev sesaesdi fonoitacfissacliscitsitats lanoitan rof ecfifo snoi atadnoitartsgerrecnac ecvres ssyanadna liinoitartsgeri lanoitanretni eht imorf sedoc gndnopserroc ot gndrocca deredro era setis recnaci snoitinfiedde ilii ppa dna etad ssongaddradnats dog nam neewteb syadil fo rebmun ot ot ““ ot ot ot cl amoeym epitluml ot ci ameakuel““““““““““““ ot ot ot ot ot ot ot ot ot ot ot ot “ot “““““““ ot ot ot ot ot ot ot ““““““““““““““““““““ ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ““““““““““““““ˆ’““““““ ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ““““““““““““““““““ inademrqi ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot elitnecrep ht“ht““““““““““““““sarcn inademrqi ot ot ot ot ot ot ot ot ot ot ot ot ot ot “ ot ot ot ot “““““c ytivac laroc laegahposeoc hcamots cc latcerooclrecnac amonaeml tnangilamc saercnapc gnulc suretuc etatsorpc seiravoc yendkic tsaerbci xvreccc sncnarbic reddablc amohpmyl s'inkgdo hnonc idoryhtc revlistrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access figure mortality following first ever record of cancer in each combination of sources cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service nhl non hodgkin's lymphoma ons office of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure completeness of grade and stage for cancers identified using ncras data only cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites grading information is not applicable to braincns sarcoma or haematological cancers and not required by in the national data standard cosd for prostate cancer core staging is not applicable to haematological and gynaecological cancers other types of staging are recommended by cosd cns central nervous system cosd cancer outcomes and services data set ncras national cancer registration and analysis service nhl non hodgkin's lymphomastrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access only missing records may indicate that the patient did not receive that treatment modality or that the treatment modality was not recordeddiscussionstatement of principal findingswe investigated the use of different sources of electronic health record data to identify incident cancers for all case definitions using individual or combined data sources a minimum of of incident site specific cancers were confirmed using the gold standard algorithm this rose to of the four most common cancers use of cancer registration data alone or in any combination of data sources captured at least of site specific cancers identified by the gold standard algorithm excepting bladder cancer and of cases for the four most common cancers combining cprd gold hes apc and ons mortality data captured at least of site specific cancers excepting kidney oral cavity and ovarian cancers and captured of cases for the four most common cancers sensitivity was much more variable when using primary care or hospital data alone and dropped to when identifying bladder cancers using cancer registration data alone use of primary care or hospital data alone resulted in a small lag in identifying cancers of interest compared with the gold standard dates but other case definitions captured cancers close to the gold standard date finally while we observed minimal changes in ppvs and sensitivities between and completeness of ncras cancer registration stage and grade data increased markedly from onwards for specific cancer types demonstrating that initiatives to improve data can have a profound impact on the quality of the data4 completeness of cancer treatment recording was difficult to assess due to the absence of a missing categorystrengths and weaknesses of the studythe main strength of this study is that we have developed a gold standard algorithm using the entirety of the evidence available from cprd to demonstrate the impact of choice of data sets in identifying incident cancers for real life studies we have also assessed the value of using ncras cancer registration data to measure stage grade and cancer treatment modalitiesa limitation of the study is that our gold standard algorithm is not validated we feel that we were justified in pre weighting ncras data as more reliable that other data sources as ncras is a highly validated data set that matches merges and quality checks data from multiple sources4 we did not consider ncras to be the outright gold standard as it is plausible that ncras does not identify all tumours diagnosed and treated in primary and secondary care for most cancer sites our gold standard algorithm identified a small proportion of cancers that are recorded in hes apc cprd gold or ons mortality data but not in ncras these tumours may have been diagnosed and coded as invasive in primary or secondary care but not by ncras been incorrectly coded in hes apc cprd gold or ons mortality data not have been notified to ncras eg tumours treated in private hospitals or be the result of linkage errors between the data sets the proportion of cancers identified in hes apc but not in ncras is particularly high for bladder cancer this is likely to be the result of difficulties inconsistencies and changes in the pathological definition and coding of cancers over time in ncras which are greatest for bladder cancer4 this explanation is supported by the higher mortality rates that we observed in bladder cancer cases identified in ncras compared with other data sources to identify incident cancers we required months of research quality follow up in cprd gold prior to inclusion in the study previous research has demonstrated that historic data is generally incorporated within the patient record with this time frame15 the identification of first ever cancers will also have been affected by different lengths of follow up data available in linked data sources as ncras data collection started in hes apc in and ons mortality data in and by the inclusion of all diagnostic codes in hes apc assuming that the first ever primary or secondary record identified incident cancer reassuringly ppvs for liver and brain cancer were high for all individual and combinations of data sets suggesting that these were not unduly misclassified as primary incident cancers despite being common sites for metastases requiring internal confirmation within months for cancers recorded in cprd gold alone in our gold standard definition is more likely to discount cancers with poorer prognoses and those recorded in the last months of follow up our data cut only included ncras data for the top cancers earlier cancers at other sites will have been missed in this studyit is also important to note that as the gold standard algorithm uses data recorded after the first record of the cancer site in any source index date it cannot be used to identify outcomes in applied studies and follow up of cohort studies with cancer as an exposure would need to start at least months after diagnosis our first ever cancer record in any source definition would be more appropriate for most studiesstrengths and weaknesses in relation to other studies discussing important differences in resultsthe most up to date study describing concordance between linked cprd gold hes apc and ncras data sets demonstrated that to of the five most common cancers recorded in cprd gold are not confirmed in either hes apc or cancer registration data and to of registered cancers are not recorded in cprd gold8 for cancers recorded in both sources the diagnosis date was a median of to days later in cprd gold than in the cancer registration data using cprd gold alone to identify these strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0ccancers marginally over represented younger healthier patients and identified to fewer deaths in the first years after diagnosis use of hes apc only identified a higher proportion of patients with the correct diagnosis date than cprd gold but over represented older patients and those diagnosed through the emergency route the majority of registered cancers were picked up using both cprd gold and hes apc ranging from for lung cancer to for breast cancer previous research demonstrated similar results with substantial differences between cancer types5 additionally a study using data from to found that using hes data in addition to ncras data identified an additional and of surgically treated colorectal lung and breast cancer cases respectively16our study is consistent with these results and provides more complete and practical evidence of the strengths and limitations of using individual and combinations of linked data sets to identify and characterise the most common incident cancerswe have also demonstrated the added value of using cancer registration data to measure stage and grade of incident cancers from about onwards levels of data completeness of staging information in the cprd extract in were similar to those reported by the united kingdom and ireland association of cancer registries ukaicr9meaning of the study possible explanations and implications for clinicians and policymakersuse of ncras cancer registration data maximised the proportion of cases confirmed as true positive based on all available linked information and captured the highest proportion of true positive cases highly complete staging and grading information is available from this source from approximately case definitions based on a combination of cprd gold hes apc and ons mortality data also had acceptable validity for the majority of cancer sites including the four most common cancersthese findings should be considered when deciding which data sources to include in research studies and which sources to use to define cancer exposures outcomes and covariatesunanswered questions and future researchfurther research is required to investigate the validity of cancer recorded in cprd gold and hes apc that are not recorded in the ncras data and to understand differences in cancer data recording with cprd gold and cprd aurum cprd™s recently launched primary care database based on records from practices that use emis software17 further investigation would be required to confidently identify subtypes of cancer either using codes available in each data set eg colon and rectal cancer or additional information available in hes apc or ncras data use of ncras™s recently open accesslaunched systemic anti cancer therapy sact18 and national radiotherapy data sets will also improve ascertainment of therapies for futu
Colon_Cancer
"cellular recognition of microbial dna is an evolutionarily conserved mechanism by which the innate immunesystem detects pathogens cyclic gmpamp synthase cgas and its downstream effector stimulator of interferongenes sting are involved in mediating fundamental innate antimicrobial immunity by promoting the release oftype i interferons ifns and other inflammatory cytokines accumulating evidence suggests that the activation ofthe cgassting axis is critical for antitumor immunity the downstream cytokines regulated by cgasstingespecially type i ifns serve as bridges connecting innate immunity with adaptive immunity accordingly a growingnumber of studies have focused on the synthesis and screening of sting pathway agonists however chronicsting activation may lead to a protumor phenotype in certain malignancies hence the cgassting signalingpathway must be orchestrated properly when sting agonists are used alone or in combination in this review wediscuss the dichotomous roles of the cgassting pathway in tumor development and the latest advances in theuse of sting agonistskeywords cgassting innate immunity type i interferon sting agonists antitumor response cancerdevelopmentintroductionthe discovery of phagocytosis in advanced the understanding of innate immunity the first line of host defenses protection againston patternrecognition receptors prrs which recognize microbialproducts coordinate antimicrobial defenses and activateinfection dependsagainstinfection byvarious pathogens correspondence zqliucsueducn juyan zheng and junluan mo contributed equally to this work1department of clinical pharmacology hunan key laboratory ofpharmacogenetics and national clinical research center for geriatricdisorders xiangya hospital central south university changsha people™s republic of china2institute of clinical pharmacology engineering research center for appliedtechnology of pharmacogenomics of ministry of education central southuniversity changsha people™s republic of chinafull list of author information is available at the end of the adaptive immunity abnormal rna or dna rnadna hybridization and cyclic dinucleotides derived frommicrobes are usually considered pathogenassociated molecular patterns pamps [ ] cells associated with innate immunity recognize different microbial pampsthrough specific prrs thereby playing key roles in hostresistance to microbial infection the pathways governing rna recognition such as retinoid acid induciblegene i rigilike receptors have been reviewed elsewhere and will not be covered herein in the case of dnarecognition one of the best known prrs is tolllike receptor tlr9 which senses extracellular cpg hypomethylated dna that has entered the cytosol through thephagosomelysosome system in addition the aim2like receptor aim2 inflammasome can be triggered afterthe entry of doublestranded dna dsdna into the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czheng molecular cancer page of cytosolic compartment which induces the proteolyticmaturation of proinflammatory cytokines such as il1and il18 and the activation of gasdermin d leading topyroptosis [“] nevertheless the most notable prr iscgas a direct cytosolic dsdna sensor which was identified by dr chen™s group in once cgas bindsto dsdna the cgassting pathway is activated to further induce the expression of type i ifns and other inflammatory cytokinesthus triggering innate immuneresponses mounting evidence suggests that cgassting signaling not only plays pivotal roles in the hostdefense against microbialinfection but also modulatestumorigenesis hence in this review we summarize themechanism of cgassting activation and elaboratefindings regarding its dual effects on tumor developmentcurrent advances in the use of sting agonists as a novelstrategy for antitumor therapy are also reviewedinsights into the cgassting signal transductioncascadecgas is an innate immune sensor that identifies variouscytosolic dsdnaincluding dna with viral bacterialmitochondrial micronuclei and retroelement originswhich can be mainly divided into pathogenderiveddna and selfdna table in the cytoplasm cgas isactivated by interacting with dsdna in a sequence[“]independent butstructural and biochemical analyses have revealed thatthe cterminal lobe of cgas contains a conserved zinclengthdependent mannerionbinding module that mediates dna binding andcgas dimerization [ ] dna ligands promotecgas activation primarily by inducing conformationalchanges around the catalytic site and in the dnabinding structures of cgasthe gscontaining loopundergoes conformational change to maintain stabilitywhich is a major mechanism of cgas activation bydna in addition to the primary dnabinding sitementioned above the secondary site located beside theprimary site is a helix formed between strands 78and several surfaceexposed loops the proximity ofthe two dnabinding sites in cgas leads to a cgasdna complex assembly in which two cgas moleculesembrace two molecules of dsdna [ ] the cgasdimers are anized in œheadtohead alignment nextto the dna and thus form stable œladderlike networks between one long curved dsdna helix or two independent dsdna strands [ ] in this way eachindividual cgasdsdna complex can be cooperativelystabilized and can lead to stronger enzymatic activitywhich may provide a possible explanation for longerdsdna as more likely to activate cgas in additionlong dna is more efficient than short dna in drivingthe liquidliquid phase separation of cgas and the formation ofcriticallydependent on the concentration of cgas and dna inthe cytoplasm cgas and dsdna are spatially concentratedcgasdimerization and activation [“] once cgas andcgas liquidlike dropletsin liquiddropletsistofacilitatetable classification of the cytosolic dsdna that activates the cgassting signaling axisclassificationselfdnasource of dsdnamicronucleipossible mechanismsrupture of the micronuclei membrane leads to exposureof chromatin dna that is recognized by cgas whichactivates the cgassting pathwayreferences mitochondrionnuclear rnapathogenderived dnadna virushsv1 hsv2 kshv adenovirus vacciniavirus cytomegalovirus papillomavirusmurine gammaherpesvirus retrovirushiv siv murine leukemia virusrna viruswest nile virus dengue virus vsvsarscov2bacterialisteria monocytogenes mycobacteriumtuberculosis listeria shigella francisellachlamydia and neisseriamitochondrial stress induces mtdna leakage into thecytosol thus activating the sting pathway and inducingproduction of cytokinesfacilitated by endogenous retroelements nuclear rnacan be reversely transcribed into dna that activatescgassting signaling dna viruses invade host cells and release pathogenderiveddna to induce sting activation[“]dna intermediates generated from reverse transcription maybe recognized by cgas to stimulate downstream stingsignaling infection with rna viruses might cause cellular damage andcell death which results in the release of cellular dna andfurther activation of the cgassting axis sarscov2 bindingto ace2 can lead to excessive angiotensin ii signaling thatactivates the sting pathway in mice[“]bacteria produce cdns such as cyclic digmp and cyclicdiamp which can directly bind to and activate sting[ “]hsv1 herpes simplex virus hsv2 herpes simplex virus kshv kaposi sarcoma“associated herpesvirus hiv human immunodeficiency virus siv simianimmunodeficiency virus vsv vesicular stomatitis virus cdns cyclic dinucleotides and sarscov2 severe acute respiratory syndrome coronavirus 0czheng molecular cancer page of dsdna interacts structural switches rearrange the catalytic pocket to enable cgas to catalyze the synthesis of²²cyclic gmpamp ²²cgamp with atp andgtp as substrates the first step in this process is theformation of a linear dinucleotide ²pppg ²²pawith atp serving as the donor and ²oh on gtp serving as the acceptor then the intermediate product flipsover in the catalytic pocket placing gtp at the donorposition and amp at the acceptor position to form asecond ²² phosphodiester bond [ ] notablyalthough dsrna or singlestrand dna ssdna is ableto bind to cgas neither can rearrange the catalyticpocket which may explain the exclusive activation ofcgas by dsdna ultimately cgamp acts as a secondmessenger to bind to and activate sting a small endoplasmic reticulum erlocated protein kd withfour putative transmembrane domains [ ] normally in a resting state sting is retained in the er byinteracting with the ca2 sensor stromalinteractionmolecule stim1 the cytosolic ligandbindingdomain lbd of sting exists as the most functionalunit capable of integrating with ²² cgamp or cdnscyclic dinucleotides such as cdiamp cdigmp or ²²cgamp from bacteria upon interaction the obviousclosure of the ligand binding pocket in the lbd is observed which is related to the activation of sting next sting transforms into a tetramer through a highorder oligomerization reaction and is translocated fromthe er to the perinuclear area facilitated by cytoplasmiccoat protein complex ii copii and adpribosylationfactor arf gtpases [ ] in the golgi sting ispalmitoylated atcys88 andcys91 a posttranslational modification necessary forsting activation modified sting recruits thekinase tankbinding kinase tbk1 in turn the cterminal domains of sting are phosphorylated bytbk1 and then phosphorylated sting recruits interferon regulatory factor irf3 which is also phosphorylated by tbk1 and dimerizes ultimately dimerizedirf3 enters the nucleus and exerts its function in thetranscription of type i ifns and interferonstimulatedgenes isgs in parallel sting can also bind toand stimulate iκb kinase ikk to mediate the production of nuclear factorκb nfκbdriven inflammatorygenes upon signal transduction termination sting istransferred to endolysosomes for degradation considering that cgamp can be transferred through gapjunctions or delivered in viralexosome packages cgassting signaling may be activated in the cytoplasmwithout dsdna [ ] moreover newly produced typei ifns activate heterodimer interferon receptors ifnar1 and ifnar2 through paracrine signaling and thusinduce the transcription of isgs [ ] in summaryonce virusderived dna and selfdna are located intwo cysteine residuesthe cytoplasm they can be sensed by cgas and a cgasdsdna complex is formed to catalyze the synthesis of ²²cgamp with atp and gtp then ²²cgamp and bacteriaderived cdns induce sting activation and mediate the release of downstream type iifns tnfα and il6 which are prerequisites for antimicrobial defense and antitumor effects the wholeprocess shows that the dsdnacgassting axis canlead to the activation of both innate and adaptive immunity fig the antitumor functions of the cgasstingsignaling pathwayrecent evidence has revealed the close association of thecgassting pathway with cancer development thissignaling pathway is generally regarded as a potent regulator of cancer immunity a stingmediated immunesupportive microenvironment can hamper malignancyoccurrence stressbytumor cell cytosolic dsdna induces sting activationunder normal circumstances dna is strictly unaffiliatedwith the cytoplasm in eukaryotic cells to avoid autoimmunity however dna leaks aberrantly in tumorcells [ ] cancer cells share common features including genome instability tumor suppressor gene mutation or deletion oxidativeand vigorousmetabolism under these intense states nuclear andmitochondrial dna is fragile and easily damaged whichleads to eventual dna leakage in the forms of micronuclei chromatin fragments andor free telomeric dna[ ] chromosomal instability cin is the primary source of cytoplasmic dna in malignant cells andis generally associated with tumor progression distantmetastasis and therapeutic tolerance excessive proliferation of cancer cells results in unstable genomes usuallychromosomal missegregation during mitosis due to defects in segregation lagging chromosomes generate micronuclei in a cellcycledependent manner the vulnerable membraneof micronuclei easily exposes the inner dna to the cytoplasm and activates the cgassting signaling axis exogenous stimuli such as chemotherapy and irradiation can also cause dna damage in addition to leakednuclear dna oxidative stressinduced mitochondrialdna leakage is another crucial initiator of sting pathway activation several anticancer treatments that precisely attack mitochondrial membranes result in effluxand cell death therefore the permeabilization of mitochondria membranes provides a reasonable explanationfor mitochondrial dna escape [ ] other sourcessuch as apoptotic cellderived dna exosomal dnaexodna and transposable elements have also beencharacterized 0czheng molecular cancer page of fig the cgassting dna sensing signaling pathway various dna derived from virus dying tumor cells or nucleus and mitochondria binds toand activates the cytosolic dna sensor cgas cgas catalyzes the synthesis of ²²cgamp in the presence of atp and gtp then ²²cgamp bindsto the er adaptor sting which also can be activated by cdns derived from bacteria upon activation sting translocates from er to golgicompartments where it activates tbk1 and ikk which phosphorylate irf3 and iκbα respectively then irf3 and iκbα dimerize and enter nucleusinitiating the transcription of type i ifn tnf and il6 the primary roles of these cytokines are reflected in host defense inflammation andantitumor immunitydemonstrated to evoke cgas“sting activation intumor cells [ ]type i ifns mediators of sting and adaptive antitumoreffectscgassting signaling exerts antitumor functions incancer cells both in an autonomous and nonautonomousmanner on the one hand dna damage can provokeacute sting signal transduction and induce cellularsenescence an irreversible cell cycle arrest state whichthwarts the aberrant proliferation of tumor cells throughacquisition ofsecretoryphenotype sasp which is associated with the releaseof abundantinflammatory mediators proteases andgrowth factors [ ] in contrast to undergoingsenescence tumor cells also directly propel apoptosisprocesses by upregulating proapoptosis protein bcl2associated x bax and downregulating the bcl2 apoptosis on the other hand stingsenescenceassociatedtheregulatoractivation in tumor cells not only facilitates the transcription of downstream type i ifns to induce dendriticcell maturation but also recruits supportive immunecells for direct nonspontaneous tumor elimination sting activation in nonmalignant cells causes tumorsuppressive effects as well sting signaling protectsagainst colitisassociated carcinomas cacs induced byazoxymethane aom and dextran sulfate sodiumdss which induce dna damage in intestinal epithelialcells and further trigger sting activation downstreamcytokines of sting signaling such as il1 and il18prevent neoplastic transformation by facilitating woundrepair more importantly sting signaling can also provoke cytotoxic t cell responses to control tumorigenesis necrotic cancer cells are commonly engulfed byantigenpresenting cells especially the basic leucine zippertranscription factor atflike batf3drivenlineage of dendritic cells dcs batf3 dcs take intumorassociated antigens and migrate towardsthe 0czheng molecular cancer page of tumordraining lymph node via the lymphatic systemwhere they crossprime tumorspecific cd8 t cellsthen cd8 t cells undergo activation and clonal expansion in the lymph nodes and are trafficked throughblood vessels to kill tumor cells in turn damaged cancercells release more antigens that are further captured bydcs the whole process forms a positive feedback loopcalled the cancerimmunity cycle tumor eradication can be achieved by multiple processes in thecancerimmunity cycle including tumor antigen captureand presentation and t cell priming and activation withtumor antigenspecific t cell priming and activationrelying on dcs and type i ifn release the involvement of type i ifns in innate immune sensing and adaptive immunity provides a reasonable hypothesis forexploring candidate prr pathways as potential immunomodulators mice lacking tlr9 myeloid differentiationprimary response gene myd88 cytosolic rna sensor mavs or the purinergic receptor p2x7r maintainintact antitumor immunity responses whereas mice deficient in sting or irf3 present with impaired cd8 tcell priming and activation [ ] in fact dying tumorcells can release multiple damageassociated molecularpatterns damps to trigger innate immune responsesin dcs among these released stimuli tumor cellderiveddna is a pivotal inducer in general the phagocytosis ofapoptotic cells causesimmune silence because ofdnasebased degradation nevertheless tumor cellreleased dna can be preserved in the dc endolysosomal compartment through an unknown mechanism cgas recognizes dna invading the cytoplasm andinduces the activation of sting cascades excretion oftype i ifns and expression of isgs additionally undersome physiological conditions such as hypoxia andacidic environments nuclear or mitochondrial dnamight be packaged in exosomes exosomal dnaexodna animates sting signaling once it is absorbedby tumorinfiltrating dcs finallytumor cellderived cgamp can also be transferred to host dcs bythe folate transporter slc19a1 and then directly bindsto sting activating it in dcs a recent study moredirectly demonstrated that cellautonomous sting promoted the maintenance of stem celllike cd8 t cellsand augmented antitumor t cell responses and mechanistically cgasstingmediated type i interferon signaling reinforced the stem cell“like cd8 t celldifferentiation program mainly by restraining akt activity immune cellderived type i ifns have crucial functions in antitumor immunity control on the one handtype i ifns boost cross presentation by various mechanisms first they stimulate the maturation of dcs secondthey slow the endosomelysosome acidificationprocess to prevent engulfed tumor antigen clearance andelevate the expression of mhc i molecules on the cellsurface [ ] finally they accelerate dc migrationtowardslymph nodes where they can crossprimetumorspecific cd8 t cells on the other handtype i ifns drive the expression of multiple chemokinessuch as cxcl9 and cxcl10 both of which are necessary for cytotoxic t lymphocyte ctl transfer and infiltration similarly type i ifns restrain the defaultimmune suppressive action of regulatory t treg cellsby downregulating phosphodiesterase pde4 and upregulating cyclic amp camp consequently typei ifns serve as bridges linking the cgassting pathway with cd8 t cellmediated antitumor immunitythe antitumor mechanisms of the cgassting signaling axis are illustrated in fig indeed previous studies revealed that sting activation can stimulate antitumor immune responses inleukemia melanoma glioma and hepatocellular carcinoma [“] additionally sting expression is downregulated in a wide variety of tumor tissues and celllines according to a pancancer analysis with a smallproportion of tumors approximately bearing silent sting expression lower sting expressionwas found in hepatic carcinoma and gastric cancer compared with its level in corresponding normal tissues andthis lower expression level was correlated with highertumor stage and poorer prognosis [ ] consistentlycompared with that in the mcfg10a mammary epithelial cell line lower sting expression was detected inmalignant breast cancer cellincluding mcf7hbl100 and t47d cells as well as human melanomacell lines and colorectal adenocarcinoma lines [ ] collectivelythat cgassting signaling might act as a tumor suppressor in certain types of cancersthese findings suggestlinessting pathway agonists as cancer therapeuticsthe immunostimulatory potential of the cgasstingpathway makes it an attractive pharmacological targetsince its activation in the tumor microenvironmenttme can induce efficient crosspriming oftumorspecific antigens and facilitate the infiltration of effectort cells recent drug research has focused on the development of sting agonists because of their potential inanticancer therapy [ ] to date various kinds ofsting agonists have been discovered and they aremainly divided into the following categories cyclic dinucleotides and their derivates dmxaa and its analogsand small molecular agonists in addition some conventional antitumor therapeutics can also indirectly activatesting such as chemotherapy radiotherapy rt andtargeted therapy in addition sting agonists areable to enhance the efficacy of other anticancer therapeutic agents when used in combination sting 0czheng molecular cancer page of fig the antitumor immunity effect of the cgassting pathway dna damage leads to the formation of dsdna in tumor cells upon itsstimulation sting signaling is activated and promotes the release of type i ifn which is crucial for dc maturation sting signaling activation indcs is the core step of the whole cancerimmunity cycle which can be initiated through engulfment of dyingdamaged tumor cells exosometransfer and cgamp gap junctions then dcs migrate towards the tumordraining lymph node and crossprime tumor specific cd8 t cells withthe help of type i ifns finally t cells undergo clonal expansion and traffic through the blood vessel to conduct tumor killingagonists and their synergistic use with other remedies isfurther explored in detail belowcyclic dinucleotides cdnscdns constitute a main type of sting agonist whichmainly originate from bacteria the known naturalcdns consist of exogenous cyclic digmp cdigmpcdiamp ²²cgamp and endogenous ²²cgampamong these cdigmp cdiamp and ²²cgampare synthesized by bacteria and identified as secondarymessengers that mediate sting signal transduction inprokaryotic cells while ²²cgamp functions as theinitiator of sting in mammalian cells the antitumor potential of these natural dinucleotides was firstproven by the finding that cdigmp could inhibit theproliferation of human colon cancer cells in vitro andbasal cell proliferation of human cecal adenocarcinomah508 cells was inhibited with μm cdigmp intraperitoneal ip injection of highdose cdigmpdirectly activated caspase3 and triggered t1 tumoripcell apoptosis in vitro nmol of cdigmp reduced thegrowth of t1 tumor cells in vitro by and nmreduced it by while lowdose cdigmp nmol accelerated the adaptive t cell response by converting a subgroup of myeloidderived suppressor cellsmdscs into immune stimulatory cells producing il12injection of ²²cgamp consistentlymgkg expedited dramatic leukemic elimination in eltcl1 transgenic mice bearing chronic lymphocyticleukemia cll and promoted tumor shrinkage of multiple myeloma in vivo from the perspective of endogenous cdns ²²cgamp mgkg was alsoshown to restrain tumorigenesis and improve the survival rate of mice bearing ct26 colon adenocarcinomain a dosagedependent manner relying on dc activationand t cell crosspriming more recently ohkurit further demonstrated that intratumoral it injection of ²²cgamp μg25 μldose on and days after the injection of tumor cells significantly mitigated tumor growth and prolonged the survival of breast 0czheng molecular cancer page of cancer t1luc squamous cell carcinoma mscc1colon cancer ct26 and melanoma b16f10 mousemodels notably the it injection of ²²cgampinhibited not only tumor growth but also lung metastases in mice bearing b16f10 cellderived tumors suggesting that cgampinduced cd8 tcell priming can drivesystemic antitumor immunity to control local and distant tumor growth termedvaccinestingvaxconsidering the superior properties of sting signaling in activating adaptive immunityit is rational toutilize sting agonists such as cdns as cancer vaccineadjuvants to increase tumor immunogenicity fu investigated the in vivo therapeutic efficacy of acancercomprisinggranulocytemacrophage colonystimulating factor gmcsf and bacteriaderived or synthetic cdns theyobserved that after it injection of stingvax with μg of cdns per vaccine dose the volume of b16melanoma tumors was dramatically reduced in a dosedependent manner compared to mice receiving gmcsf cancer vaccine alone stingvaxtreated mice hadmore infiltrating cd8 ifnγ t cells in the tumormicroenvironment the in vivo antitumor effect of stingvax was also verified in models of colon carcinomact26 pancreatic carcinoma panc02 and upper aerodigestive squamous cell carcinoma sccfvii although natural cdns are able to produce robust antitumor immunity their chemical features might hindertheir future application in the clinical setting first native cdns are easily degraded by enzymes inside the cellor in the bloodstream second their negatively chargedproperty hydrophilicity and phosphate moieties severelyimpede cdns from penetrating cell membranes to activate cytosolic sting leading to low bioavailability andpoor retention of the cdns in specific cells and tissuesthird unintentional toxicities and narrow therapeuticwindows are also unavoidable thus new strategies toimprove therapeutic efficacy and reduce adverse effectsare urgently needed including drug delivery carrier engineering original structural modification and nonnucleotide agonist screening regarding agonistdelivery smith reported that biopolymer implantscodelivering cdigmp μg and chimeric antigen receptor t cart cells resulted in significant tumor regression in mice bearing pancreatic tumors moreoveriv administration of cdigmpysk05lip equivalent to μg of cdigmp aysk05liposome delivery system encapsulating cdigmp led to a tremendous decrease in metastatic lesionsin a b16f10 mouse melanoma model with nearly ofthe injected mice showing resistance against tumor relapsethe adaptive immune responsememory was successfully induced chen alsofound thatliposomalindicating thatinjection ofintravenousintravenousivnanopdelivered cgamp cgampnp could activate the sting axis more effectively than solublecgamp and converted the immunosuppressive tme toa tumoricidal state in a transplanted b16f10 cell melanoma model and in a genetically engineered triplenegative breast cancer model moreover a recentstudy creatively suggested that modified bacteria mightbe exploited as a selective carrier of sting agonistsintroduction of a dinucleotide cyclasecoding gene intothe escherichia coli nissle strain was an attempt at realizing this effect however advancements to the systemare needed tobysnakeapartdigestionresistancecompoundatoms the modifiedfrom improving delivery methods cdnswith superior properties are currently being synthesized and tested for instance to prevent enzymatichydrolysis of cgamp the nonbridging oxygen atomsin cgamp phosphodiester linkages were replaced by²²sulfurcgsasmp showed resistance against degradation byenpp1 a major ²²cgamp hydrolasetherebyleading to a longer halflife and sustained high affinity for human sting hsting syntheticdithio mixedlinkage cdns with both rp rp r rand rp sp r s dithio diastereomers possessed notonlyvenomphosphodiesterase but also enhanced affinityforsting a novel superior modified product ml rrs2 cda also termed adus100 had the potencyto activate all hsting variants and mouse stingmsting adus100 had higher efficiency in activating sting signaling than endogenous or exogenous cdns mainly because of its enhanced stabilityand lipophilicity its powerful tumor elimination effect was extensively demonstrated in multiple murinemodelsincluding b16 melanoma t1 breast cancer and ct26 colon cancer with all treated animalsshowing significant and durable tumorregressionafter itinjection of adus100 three mg doseswhen tumor volumes reached mm3 theremarkableforhsting laid the foundation for its clinical use related clinicaltrials of adus100 are outlined intable in addition to adus100 some other novelsting agonists have been well designed iacs8779and iacs8803 are two highly potent ²²thiophosphate cdn analogs that induced striking systemicantitumorin a b16 melanoma murineinjection μg on and daysmodel after itposttumor implantation compared with adus100or cgamp the characteristics and preclinicalapplications of all these mentioned cnds are summarized in table because of the structural modification and optimization of delivery strategiestheapplication range and efficacy of cdns have beenand high affinityresponsescurativeeffect 0czheng molecular cancer page of table characteristics and preclinical applications of different sting agonistsclassificationcharacteristicsapplicationmodelsnatural cdnagonistscdigmppoor membrane permeabilitysuitable for various codeliverytechnologiescolon cancer h508cells t1 metastaticbreast cancertreatmentinformation μm nmol ip nmol ip nmol ip²²cgamp²²cgamphigher binding affinity formsting than for hstinghigher affinity for hsting thanits lineage isomers binds tovarious sting nucleotidepolymorphisms observed inhumans easily degraded byphosphodiesteraseimpermeable to the cellmembranechronic lymphocyticleukemia mgkg ipmultiple myeloma mgkg ipct26 colonadenocarcinoma mgkgbreast cancer t1lucsquamous cellcarcinomasmscc1 μg25 μldose it μg25 μldose itcolon cancer ct26 μg25 μldose itmelanoma b16f10 μg25 μldose ittherapeutic effects references[ ] [ ]inhibitsproliferation tumorregression tumorregressionaccelerates tcellresponseleukemiaeliminationsuppressesgrowthrestrainstumorigenesisimproves survivalratedelays tumrowthdelays tumrowthdelays tumrowthdelays tumrowthstingvaxsyntheticcdnagonistspotent in vivo antitumor efficacyin multiple therapeutic modelsof established cancercgampnpsbiopolymer scaffolds cdigmp and car t cellscdigmpysk05lip²²cgsasmpadus100iacs8779iacs8803noncdnagonistsfaaliposomal nanops npsdeliver cgamp intracellularlymore effectively than realizedwith soluble cgamperadicates tumors moreeffectively than systemicdeliveryysk05 is a lipid that can efficientlydeliver cdigmp to the cytosolpossesses high fusogenic activitywhich enhances endosomalescapemore resistant to degradation byenpp1 tenfold more potent atinducing ifn secretion potentialuse as a cancer vaccine adjuvantimproves stability and lipophilicityhigher affinity for hsting thannatural cdn agonists capable toactivate all hsting variants andmstingstimulates a superior systemicantitumor response thanadus100 and cgampcauses hemorrhagic necrosisfailed in a phase i clinical trialdue to species specificity μg cdns itreduces tumorvolume b16 melanomacolon carcinomact26pancreaticcarcinoma panc02b16f10 melanomaivtnbccreates atumoricidal state pancreatic cancer μg cdigmptumor regression b16f10 mousemelanoma μg cdigmp ivdecreasesmetastasisthp1 monocytesb16 melanomathree mg doses it t1 breast cancerthree mg doses itmc26 colon cancerthree mg doses itdurable tumorregressiondurable tumorregressiondurable tumorregression b16 melanoma μg on day and posttumor implantationantitumorresponse murine colontumorsextensive tumorrejection[ ]dmxaafirst discovered as a vascularrat mammary mgkg iphigh anticancer[ 0czheng molecular cancer page of table characteristics and preclinical applications of different sting agonists continuedclassificationcharacteristicsapplicationmodelstreatmentinformationinduces proinflammatory cytokinesin a stingdependent mannerhuman fibroblastsantiviral activity selectively induces stingdependentsynthesis and secretion of bioactiveifns no evidence of binding directlyto stingactivates sting in œopenconformation submicromolarlevels induce sting activationand ifn productionhuman fibroblastsantiviral activity colon tumors mgkg iv of a treatedgroup remainedtumor free faa flavone acetic acid dmxaa 56dimethylxanthenone4acetic acid cma 10carboxymethyl9acrid
Colon_Cancer
" inflammatory bowel disease ibd is the collective term for chronic immunemediated diseases ofunknown multifactorial etiology arising from the interplay between genetic and environmental factors andincluding two main disease manifestations ulcerative colitis uc and crohn™s disease in the last few decadesnaturally occurring alkaloids have gained interest because of their substantial antiinflammatory effects in severalanimal models of disease studies on mouse models of ibd have demonstrated the antiinflammatory action of themain tobacco alkaloid nicotine in addition anatabine a minor tobacco alkaloid also present in peppers tomatoand eggplant presents antiinflammatory properties in vivo and in vitro in this study we aimed to evaluate theantiinflammatory properties of nicotine and anatabine in a dextran sulfate sodium dss mouse model of ucresults oral administration of anatabine but not nicotine reduced the clinical symptoms of dssinduced colitisthe result of gene expression analysis suggested that anatabine had a restorative effect on global dssinducedgene expression profiles while nicotine only had limited effects accordingly map findings revealed that anatabinereduced the colonic abundance of dssassociated cytokines and increased il10 abundances our results support the amelioration of inflammatory effects by anatabine in the dss mouse modelof uc and suggest that anatabine constitutes a promising therapeutic agent for ibd treatmentkeywords plantderived alkaloid mouse model nicotine colitis correspondence pedroantonioruizcastropmicomblainephillipspmicom juliahoengpmicom pedro a ruiz castro ulrike kogel giuseppe lo sasso blaine w phillips andalain sewer contributed equally to this work1philip morris international rd philip morris products sa quai jeanrenaud neuch¢tel switzerland2philip morris international research laboratories pte ltd science parkroad the kendall science park ii singapore singapore the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cruiz castro of inflammation page of crohn™s disease cd and ulcerative colitis uc the mainclinical phenotypes of inflammatory bowel diseases ibdare chronic relapsing inflammatory disorders that affectthe gastrointestinal tract ibd are thought to result froman inappropriate and continuing inflammatory responseto commensal microbes in a genetically susceptible hostenvironmental triggers such as increased hygiene druguse stress smoking and diet influence the onset of thedisease over the past decades naturally occurring alkaloids from plant or medicinal herb sources have sparkedconsiderable interest because of their significant antiinflammatory and antioxidant properties [“]alkaloids”a class of natural bioactive compoundsderived from amino acids that contain one or moreheterocyclic nitrogen atoms”are produced by a widerange of living anisms such as bacteria fungi plantsand animals in plants alkaloids are produced assecondary metabolites in response to environmentalbiotic or abiotic interactions and they confer protectionthrough a range of insecticidal antimicrobial and pharmacological attributes the antiinflammatory activities ofplantderived alkaloids have been documented in severalanimal models of disease including respiratory distress spinal cord injury hepatic fibrosis cancer and ibd [ ] the protective effects of alkaloids havebeen attributed to amelioration of inflammatory responsesand colonic oxidative stress [ ] promotion of epithelial barrier function and positive regulation of gutmicrobiota pyridine alkaloids present in tobacco nicotiana tabacum have been the subject of intensive research nicotinethe major alkaloid in tobacco accounts for approximately of the total alkaloid content of tobacco while thestructurally related nornicotine and anatabine are themost abundant minor pyridine alkaloids accounting for to of total alkaloids other pyridine alkaloids intobacco such as anabasine anabaseine and cotinine arepresent in smaller amounts nicotine and all minortobacco alkaloids have been shown to be pharmacologically active upon binding to several nicotinic acetylcholinereceptors nachrs tobacco nachr agonists suchas nicotine anatabine anabasine anabaseine and cotininedisplay protective effects in animal models of several inflammatory conditions including sepsis parkinson™sdisease alzheimer™s disease and ibd several in vitro and in vivo studies have shown a clearnicotinedependent positive effect on inflammatory processes [ ] in a previous study oral nicotine administration reduced the severity of dssinduced colitis andreduced colonic tnfα synthesis while subcutaneous injection or minipump infusion had no effect highlightingthe crucial role of administration route for the protectiveeffects of nicotine in dss colitis nicotine has alsobeen shown to attenuate the severity of dss colitis andexpression of il6 in cd4t cells a recent studysuggested that nicotine ameliorates dssinduced inflammation through inhibition of signaltransducer andactivator oftranscription stat3 in gutinfiltratedlymphocytes and intestinal epithelial cells recentlynicotine was shown to cause a decrease in leukocyte recruitment disease activity index dai and histologicalscore in dss colitis and block tnfmediated expressionof mucosal vascular addresin cell adhesion molecule1 inendothelial cells these authors concluded that nicotinesuppressesinflammation through downregulation ofadhesion molecules in the gut nonetheless clinicalstudies on the efficacy and tolerability of nicotine haveshown thattherapeutic application of nicotine fortreatment of uc is limited because of frequent adverseeffects and nicotine inconsistent efficacy in maintainingremission in uc patients [ ]anatabine is found in plants of the solanacea familywhich includes tobacco peppers tomato and eggplant little is known about the biological properties of anatabinealthough several studies have suggested that this alkaloid is apotential candidate compound for antiinflammatory drugdevelopment [ ] anatabine was marketed in the us asa dietary supplement under the name anatabloc in aninternetbased survey approximately of all users ratedthe effect of anatabine supplementation as good or excellentfor joint pain stiffness and functionality anatabine hasbeen shown to inhibit lipopolysaccharide lpsinducedproinflammatory gene expression as well as nfκb andstat3 phosphorylation in human neuroblastoma shsy5y hek293 human microglia and human bloodmononuclear cells as well as in the brain and spleen ofmouse models of autoimmune encephalomyelitis andalzheimer™s disease in shsy5y cells anatabine alsoreduced the expression of betasecretase ”the rate limitingenzyme for amyloid peptide production which is a majorhallmark of alzheimer™s disease”through inhibition of nfκb activation in this study we aimed to assess the antiinflammatoryeffects of the tobacco alkaloids nicotine and anatabine inthe established dss mouse model of uc our resultsshow that oral administration of anatabine but notnicotine ameliorates the clinical manifestations of dsstreatment in mice the results of gene expression analysisindicated that anatabine had a partial restorative effect onglobal dssinduced gene expression profiles while nicotineonly had minimal effects moreover multianalyte profilingmap showed that anatabine but not nicotine suppressesthe production of il6 il1α tnfα granulocytecolonystimulating factor gcsf and keratinocyte chemoattractant kc while increasing il10 expression in the colon ofdsstreated mice for an overview of the study conceptand analytical procedures see œonline resource  0cruiz castro of inflammation page of resultsanatabine has a protective effect in the dss mousemodel of colitisto study the antiinflammatory properties of nicotineand anatabine c57bl6 mice were provided with nicotine or anatabine in drinking water for a total of dayscolitis was induced by oral administration of dssin drinking water ad libitum during days “ fig 1adsstreated mice developed colitis as evident from thebody weight loss fig 1b increased colon weightlengthratio fig 1c increased dai fig 1d increased stooloccult blood score fig 1e increased intestinal bleedingscore fig 1fincreased diarrhea score fig 1g andincreased colon inflammation score fig 1h in micefig clinical parameters of dsstreated mice administered nicotine or anatabine a mice were orally administered nicotine or anatabine or mgkg for a total of days colitis was induced by oral administration of dss in drinking water ad libitum during days “ b bodyweight changes in mice during the colitis induction and recovery phases body weight changes were calculated as percentage relative to thestarting day of dss treatment day c colon weightlength ratio are represented as mgcm of colon d dai was calculated according to weightloss colon weightlength ratio and intestinal bleeding e stool occult blood score f intestinal bleeding score g diarrhea score h coloninflammatory status data are shown as mean ± sem p p nic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkg 0cruiz castro of inflammation page of not subjected to dss treatment nicotine and anatabine administration had no significant effect on these parametershowever mice treated concomitantly with anatabine anddss showed a decrease in colitis severity in particular inmice that received concomitant anatabine and dss treatment anatabine improved body weight recovery at mgkg p fig 1b caused a decrease in global dai relative to that in dsstreated mice at daily dose of p and mgkg p fig 1c and reduced the stooloccult blood score at mgkg p fig 1e interestingly nicotine but not anatabine improved the intestinalbleeding score relative to that in dssadministered mice at mgkg p fig 1f the diarrhea score fig 1gand the colon inflammation score fig 1h were notaffected by nicotine or anatabine no variation in waterconsumption was registered across the different experimental groups online resource anatabine reduces dssinduced gene expression changesin the distal colon on a global levelto complement these pathological findings we analyzed the colon transcriptome dsselicited lesions inmost mouse inbred strains including c57bl6 micehave been shown to be more pronounced in the distalcolon [ ] therefore we focused our study on thatportion of the large intestine principal componentanalysis clearly captured the effect of dss treatmenton the first principal component explainedvariance fig 2a while nicotine treatment did notproduce a pronounced effect on the first principalcomponent in the dsstreatment context anatabinetreatmentin combination with dss produced aresponse more similar to that observed in the watercontrols no dssindicating that anatabine had anameliorating effect on dssinduced gene expressionchanges the results of our differential gene expressionanalysis suggested substantial perturbation of the colontranscriptomefdr fig 2b“c application of dss in drinking watercaused significant changes in nearly genes incolon tissues addition of nicotine to dsscontainingdrinking water slightly increased the number of differentially expressed genes in contrast addition of anatabine decreased the number of differentially expressedgenes upon dss treatment by approximately fig 2b and c this alleviating effect of anatabinetreatment on dssinduced gene expression profileswas also apparent in the global gene expression heatmap which showed a global reduction of expressionfold changes upon anatabine treatment fig 2d and eof note in the absence of dss anatabine treatmentdid not result in differential expression of genes andnicotine treatment alone had minor effects fdr pvalue online resource upon dsstreatmentanatabine reduces dssinduced inflammatory geneexpression in the distal colonto gain a more mechanistic understanding of the effectsof nicotine and anatabine treatment we further investigated gene expression changes at the level of functionalgene sets and a ucrelevant causal network model gsaofthe reactome database showed changes acrossmultiple functional categories fig 3b the effects onfunctional categories in dsstreated mice administerednicotine appeared rather similar to those in mice treatedwith dss alone whereas administration of anatabineresulted in a general repression of dssmediated effectswe also evaluated the interaction terms betweennicotineanatabine and dss treatment to more directlyfocus on the modulating effect of anatabine and nicotinetreatment on dss effects fig 3a online resource the following six biological categories showed significant interaction terms upon anatabine and dss treatmentsupporting asignificant suppression of dssinduced effects in thepresence of anatabine œimmune system œextracellularmatrix anization œprotein localization œmetabolism œhemostasis and œsignal transduction fig 3bof these we further explored œimmune system œextracellular matrix anization and œsignal transductionas the most relevant categories in ibdana 20dss fdr allfiginteractiontermsp the hierarchical anization of the reactome database allowed us to investigate the underlying functionalchanges in more detail the œimmune system categoryincludes œadaptive immune system œcytokine signaling in immune system and œinnate immune systemin particular within these immune categories œcytokinesignaling egincluding il6 family signaling andœtolllike receptor cascades were modulated withsignificant3conline resource online resource within thereactome œsignal transduction category œsignaling byreceptor tyrosine kinasesby rhogtpases œsignaling by transforming growth factortgfbeta family members œmapk family signalingcascades œintegrin signaling œsignaling by erythropoietin and œsignaling by gpcr were found to be significantly impacted online resource online resource within the œextracellular matrix anization category almost all subcategories were perturbed includingœdegradation of the extracellular matrix œelastic fiberformation and œextracellular matrix proteoglycansonline resource online resource œsignalingœdegradation ofthe extracellular matrix includesbiological processes such as activation of matrix metalloproteinases mmps and collagen degradationto further follow up on the effects of nicotine andanatabine on inflammatory signaling we evaluatedthe perturbation of the ucrelevant tlril1rtnfr 0cruiz castro of inflammation page of fig transcriptomics results of colon biopsy samples a principal component pc analysis the plot displays all samples in the reduced pc1“pc2plane covering of the total data variance it allows us to examine the relationships between the various groups and treatmentsremarkably pc1 captured the pure dss effect black arrow while pc2 captured the pure exposure effects of anatabine and nicotine blue andred arrows respectively b volcano plots for individual gene differential expressions the horizontal axis represents the log2 differential expressionœfold changes and the vertical axis represents its statistical significance as log10 fdr c number of differentially expressed genes for theselected pairwise comparisons the bar plot displays the number of genes with positive or negative fold changes with corresponding statisticallysignificant fdr ‰¤ note that the lower fdr values observed for the œana dss comparisons do not necessarily prefigure a reduction ofbiological effects because the statistics underlying the fdrp values also depend on the gene expression variance within the experimentalgroups d highlevel heatmap of the statistically significant differentially expressed genes for the selected pairwise comparisons in order toprovide a comprehensible display of the large — foldchange matrix we first normalized its rows to their maximum absolute values andthen reordered them by hierarchical clustering complete linkage method on the basis of their euclidean distances e scatter plot from thecomparisons between the pure and exposuremodified dss effects by using the differential gene expression results the horizontal axis of thescatter plots represents the fold changes of the pure dss effect œwater dss vs water pairwise comparison whereas the vertical axis containsthe corresponding values in case of anatabine or nicotine exposure œanatabinenicotine dss vs water pairwise comparisons in an idealcase included as a reference [first plot] the bestfitted straight line coincides with the diagonal indicated in green and its slope is equal to the transcriptomics effects of anatabine or nicotine exposure were quantified by the slope of the bestfitted straight lines indicated on each plotnic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkgnetwork model by using an established networkenrichment approach we inferred the activationstates of the network nodes on the basis of the observedgene expression changes and calculated the overall network perturbation amplitude for each group comparisonfig 3c dss treatment had a strong activating effect onthis signaling network including activation of several coresignaling nodes such as il1rassociated kinase irak1irak4 and myeloid differentiation primary response myd88 online resource œheatmap leadingnodes of note the network perturbation induced bydss treatment was reduced only in the presence ofanatabine with this the results of network analysisfurther supported the ameliorating effect of anatabineon dssmediated inflammation whereas no similareffect was apparent upon nicotine treatment 0cruiz castro of inflammation page of fig biological interpretation of the transcriptomics results a schematic representation of the effects of anatabinenicotine exposure as amodification of the pure dss effect the measured combined œanatabinenicotine dss effect captured by the pairwise comparisons œanatabinenicotine dss vs water can be viewed as the sum of the pure effects of the two factors œdss treatment and œanatabinenicotine exposuretogether with the adjusting twofactor interaction term œanatabinenicotinedss anatabinenicotine exposure is synergistic with dss treatmentif the interaction term has the same sign as the pure dss effect eg both are positive as in the schema in contrast the relationship isantagonistic if the interaction term has an opposite sign to the pure dss effect b gsa results for the top reactome pathway categories theheatmap displays the gsa scores normalized rowwise to their maximum absolute values as well as their competitive q1 statistical significancethe fdrs were calculated only among the top reactome pathway categories which are sufficiently distinct in their gene content to assumeindependence of their enrichment results c hierarchical representation of the gsa results for all pathways contained in the top reactomeœimmune system category and for the twofactor œana 20dss interaction the color map corresponds to the normalized gsa scores containedin the interval [ˆ’ ] whereas their statistical significance competitive q1 p values ‰¤ is indicated by black circles around the nodes theactual labels of the nodes ie the reactome pathway names can be found in online resource the treelike structure of the reactomepathway collection enables topdown investigation within the relevant pathway categories by identifying increasingly specific biologicalprocesses ie involving fewer and fewer genes along the longest paths connecting the statistically significant pathways d npa results for thetlr“il1r“tnfr network model the bar plot displays the npa values for the selected contrasts and their statistical significance is indicated bythe three colored asterisks note that by definition the positive npa values depend on the square of the input genelevel fold changes andtherefore might amplify the differences between the contrasts without preserving the additive relationships among them nic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkgexpressionvalues”from theto validate the observations obtained by microarraytranscriptomics we quantified the expression of six œleadingedge genes”genes of the gene set with the highestœimmunedifferentialsystem œextracellular matrix anization and œsignaltransduction reactome categories using realtime quantitative pcr qpcr selected genes included il33 œsignaltransduction and œimmune system categoriesil6œsignal transduction and œimmune system categoriesmmp13 œextracellular matrix anization categoryserpine1 œsignal transduction and œextracellular matrixanization categories thbs1 thrombospondin œsignal transduction and œextracellular matrix anizationcategories and timp1 tissue inhibitor of metalloproteinase œextracellular matrix anization and œimmunesystem categories qpcr results showed a clear decreasein dssinduced expression of il33 il6 mmp13 serpine1thbs1 and timp1 expression in the presence of anatabineat mgkg thereby confirming the findings obtainedfrom the microarray data fig 0cruiz castro of inflammation page of fig validation of microarray transcriptomic results using qpcr a boxandwhisker plots for the distribution of the qpcr expression levels δcqof the selected genes the boxes represent the quartiles while the whiskers extend to the most extreme data point which is no more than times the interquartile range from the box the horizontal brackets indicate the statistical significance of the corresponding comparisons mean pvalue smaller than respectively b scatter plots comparing the mouse colon differential expression values obtained bymicroarray horizontal axis and qpcr vertical axis the following similarity metrics were indicated œbeta is the slope of the best interceptfreelinear fit between microarray and qpcr values œr2 is the coefficient of determination measuring the œgoodnessoffit and œpval is the pvalueassociated to the null hypothesis beta nic nicotine mgkg ana anatabine mgkganatabine decreases dssinduced proinflammatorycytokine production and promotes il10 expressionnext we sought to evaluate the impact of nicotine andanatabine on the expression of colonic inflammatorycytokines by mapin line with the previous dataanatabine but not nicotine significantly reduced theabundance of dssassociated inflammatory cytokines including il6 kc tnfα il1α and gcsf whereas itincreased the levels of the antiinflammatory cytokineil10 at a daily dose of mgkg fig interestinglyanatabine also increased the abundance of il21 andshowed a clear tendency towards increasing colonic il 0cruiz castro of inflammation page of fig map results for colon biopsies statistical assessment of the differences observed in the abundance of selected cytokines between thestudy experimental groups and water control fold changes in the treatment groups nicotine and anatabine at and mgkg dss relative towater control are illustrated with colors ranging from blue decrease to red increase statistically significant differences on the basis of raw pvalues no adjustment has been made for multiple testing grey cells highlight missing estimates on the observed differences due to lackof cytokine quantifications nic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkg1 levels fig taken together the map data confirmthe antiinflammatory effects of anatabine in dssinduced colitisdiscussionour study shows that oral administration of anatabinebut not nicotine reduces the clinical manifestations ofdssinduced colitis in a mouse model in line with thesefindings anatabine demonstrated a global downregulatory effect on dssinduced gene expression changes inthe colon whereas the effects of nicotine were morelimited in particular the results of gene expression profiling further supported the reduction of inflammatorysignaling processes upon anatabine treatment includingsuppression of il6 signaling as shown by gsa findingsand tlr signaling as shown by the results of networkperturbation analysis and gsa map also showed asignificant decrease in the abundance of il6 kc tnfαil1α and gcsf and an increase in the expression of il and the antiinflammatory cytokine il10several studies have reported on the antiinflammatoryeffects of nicotine on the dss mouse model of uc subcutaneous administration of nicotine was shown toameliorate tissue injury in dss colitis and il6 expression in cd4t cells via α7nachrs nicotine wasalso shown to decrease the activation of stat3 throughinduction of mir124 in gutinfiltrated lymphocytes andintestinal epithelial cells strikingly alsharari 0cruiz castro of inflammation page of observed that oral but not subcutaneous injection ofnicotine ameliorated intestinalinflammation and colonic tnfα expression in dsstreated mice in spiteof the reported beneficial effects our results do not support a protective effect of orally administered nicotineon dss colitis of note we found that nicotine significantly reduced dssassociated intestinal bleeding whichwas the only clinical parameter affected by this tobaccoalkaloid the vasoconstrictor effects of nicotine are wellstablished in the gut mucosa nicotine decreasesblood flow and cigarette smoking decreases rectalblood flow to normal levels in patients with uc however how changes in blood flow affect the pathophysiology of uc is still unclear the possible therapeuticuse of nicotine to induce remission in uc patients hasbeen evaluated in five clinical studies [“] and twometaanalyses [ ] these studies have demonstrated avariable efficacy of nicotine therapy in induction of remission with several studies showing no effect [ ]moreover a high frequency of adverse events increasedthe withdrawal rate in the nicotine group in some studiesthus limiting the therapeutic benefit of nicotine nucleotidebindingto the best of our knowledge the present study isthe first to assess the impact of anatabine on experimental colitis gene expression analysis of distal colonbiopsy specimens highlighted the antiinflammatoryproperties of anatabine in multiple functional categories including œimmune responses œsignal transduction and œextracellular matrix anization which inturn encompassed several tlr and cytokine signalingpathways genes contributing to the downregulation oftlr cascades included tlr2 tlr4 and tlr6 and anumber of downstream signaling factors shared byseveral tlrs including myd88 ripk2 irak3 irak4and nod1oligomerizationdomaincontaining protein as well as the nuclearfactors elk1 fos cyclic adenosine monophosphatecamp response elementbinding protein creb1activating transcription factor atf1 and atf2 seeonline resource of the respective gene lists for thecytokine signaling pathways the contributing genes included il6 and il6 receptor α ifnγ il4 cxcl10il22 receptor α2 il2 receptor α tnf receptor superfamily member 1a il17α and il17 receptor α jak1jak2 stat3 and stat4 members of the nfκbsignaling pathway also contributed to the overall reducincluding nfκb p65tion in inflammatory cascadesp105 and p100 subunits iκbα and the nfκb activating protein tab3 in agreement with the results oftranscriptional analysis map findings showed a significant decrease in dssassociated il6 kc tnfα il1αand gcsf protein expression and an increase in il10expression in the presence of anatabine strikinglywhile tlr downstream factors modulated by anatabineare shared by most tlrs the majority of cytokineassociated signaling molecules are specific for eachpathway the seemingly pleiotropic regulatory effects ofanatabine suggest that this alkaloid reduces inflammation by inhibiting the expression of several factors involved in different proinflammatory signaling cascadesprevious studies using in vitro and in vivo diseasemodels have demonstrated the antiinflammatory effectsof anatabine [ ] paris showed that this pyridine alkaloid reduced the plasma levels of il1 il6and il17a as well as the expression of il1 infγ andtnfα in the spleen of experimental autoimmune encephalomyelitis mice these authors also showedthat anatabine suppressed stat3 and nfκb phosphorylation in the spleen and brain of these mice anatabine also prevented lps and tnfαinduced nfκb andstat3 phosphorylation in shsy5y and hek cellshuman microglia and human blood mononuclear cells additionally anatabine prevented lpsinduced il1 expression in human whole blood as well as il1il6 and tnfα production in the plasma kidney andspleen in the lps mouse model phosphorylatedstat3 tnfα and il6 were also downregulated in thepresence of anatabine in a transgenic mouse model ofalzheimer™s disease our results on the effects of anatabine in the dssmouse model of uc are also in line with the findings ofa substantial number of studies demonstrating the protective effects of natural alkaloids in several animalmodels of colitis [ ] intraperitoneal administrationof the minor tobacco alkaloid and nicotinic receptoragonist anabaseine was shown to reduce tissue damagemyeloperoxidase activity and colonic tnfα expressionin a tnbs mouse model of colitis these mice alsoshowed reduced nfκb activation in lamina propriamononuclear cells while mice administered a nicotinicreceptor antagonist presented worse colitis symptomsthan those treated with tnbs alone the algaealkaloid caulerpin reduces dss colitis by suppressingnfκb activation and subsequently inhibiting the colonicproduction of tnfα ifnγ il6 ifnγ and il17 oral administration of berberine also ameliorates dssinduced colitis and downregulates the expression oftnfα ifnγ kc and il17 in colonic macrophages the plantderived alkaloid nmethylcytisine andthe tea alkaloid theophylline mitigate colitis by downregulating tnfα il1 and il6 expression in dss andacetic acid models of colitis respectively [ ] induction ofthe antiinflammatory cytokine il10 in thepresence of natural alkaloids has also been reportedthus indirubin ameliorates dssinduced colitis by suppressing the expression of colonic tnfα ifnγ and il2and upregulating il10 additionally indole alkaloids caulerpin and isatin have been shown to increase 0cruiz castro of inflammation page of the expression of il10 in dss and tnbs models of ibdrespectively [ ] of note our results show anincrease in the abundance of il21 and a tendencytowards increase in colonic il1 levels in the presenceof anatabine although il21 expression is increased inmany chronic inflammatory disorders genetic deficiencyof il21 is associated with ibd and inhibition of il21in the early phases of some inflammatory disorders exacerbates disease development suggesting the dual role ofil21 in the control of immune responses il21also promotes il22 expression in mucosal tcellsthrough a mechanism involving stat3 retinoidrelatedorphan receptor γt and aryl hydrocarbon receptorthereby helping protect immunodeficient mice from dsscolitis interestingly il21 has been recently shownto induce il1 production in dendritic cells through astat3dependent but nfκbindependent mechanismthereby suggesting a link between il21 and il1 mounting evidence suggests that alkaloids”in particular isoquinoline alkaloids present in traditional medicineherbs”exertthroughregulation of nfκb and stat3 signaling pathways forexample sanguinarine and cavidine suppress the expression of nfκb p65 subunit thereby reducing colonictnfα and i
Colon_Cancer
inanic arsenic ias is the chemical form of as commonlyfound in drinking water atsdr and in some foodscubadda chronic exposure to ias has been associatedwith risk of skin bladder lung and liver cancers iarc aswell as with other diseases naujokas including diabetes maull cardiovascular abhyankar moon saquib states respiratorysanchez and neurological diseases caito andaschner parvez humans and most other mammalian species have developed a mechanism for detoxifying iasthat involves the sequential conversion of ias to monomethylasmas and mas to dimethylas dmas thomas both methylation steps are catalyzed by orthologs of a singleenzyme arsenic oxidation state methyltransferase as3mtlin the methylation of ias by as3mt not onlyaddress correspondence to miroslav st½blo department of nutritionuniversity of north carolina at chapel hill chapel hill nc usa telephone email styblomeduncedu or beverly hkoller department of genetics university of north carolina at chapel hillchapel hill nc usa telephone emailbkolleremailuncedusupplemental material is available online 101289ehp6943this document was reviewed by the center for computational toxicology andexposure office of research and development us environmental protectionagency and approved for publication approval does not signify that thecontents reflect the views of the agency nor does mention of trade names orcommercial products constitute endorsement or recommendation for usethe authors declare they have no actual or potential competing financialinterestspublished august received february revised july accepted july note to readers with disabilities ehp strives to ensure that all content is accessible to all readers however some figures and supplementalmaterial published in ehp s may not conform to standards due tothe complexity of the information being presented if you need assistanceaccessing content please contact ehponlineniehsnihgov our staï¬will work with you to assess and meet your accessibility needs within working dayspromotes wholebody clearance of as drobn¡ hughes but also produces methylated intermediates that contain highly reactive and toxic trivalent as asiii watanabe andhirano masiii and dmasiii unlike their pentavalent counterparts masvand dmasv exceed ias in potency as cytotoxinsgenotoxins and enzyme inhibitors thomas hencemasiii and dmasiii may be critical determinants of toxic and carcinogenic eï¬ects associated with chronic exposure to ias alteredcapacity to methylate ias has been linked to an increased risk ofdiseases associated with ias exposure ahsan pierce vahter in most mammals the gene encoding as3mt is present asa single copy ˆ¼ kb from the gene encoding bloc1 relatedcomplex subunit borcs7 to date the methylation of ias isthe only known function of as3mt however recent studies haveidentified the as3mtborcs7 locus as conferring risk for schizophrenia duarte compared with healthy individualsexpression of borcs7 and of the humanspecific splicing variantof as3mt as3mtd2d3 has been found to be consistently higher inthe brains of patients with schizophrenia li notablyexpression levels of as3mt and borcs7 were found to be weaklycorrelated li suggesting that the two genes may sharetranscriptional regulatory elements although published datashowed that ias exposure can aï¬ect as3mt expression in micest½blo the role of ias exposure in the expression ofborcs7 or as3mtd2d3 which is thought to lack ias methylationactivity has never been studiedlaboratory studies of the mechanistic basis of iasassociateddiseases have been hindered by substantial diï¬erences between laboratory animals and humans in their capacity to metabolize anddetoxify ias rats unlike humans sequester significant portions ofingested ias in erythrocytes in the form of dmasiii lu mice the species most commonly used in mechanistic studies diï¬erfrom humans displaying very high rates of as methylation thisspecies diï¬erence is associated with faster rates of urinary clearanceof methylated metabolites and the predominance of dmas as themain urinary metabolite of ias in mice vahter interspeciesdiï¬erences in as metabolism may contribute to difficultiesenvironmental health perspectives august 0cencountered replicating some of the eï¬ects of ias exposure reportedin humans including the carcinogenic eï¬ects in laboratory micethus producing an ias methylation phenotype in mice that resembles the phenotype found in humans could create a better animalmodel to study the role of ias metabolism in adverse health eï¬ectsof chronic ias exposure to generate such a model we have humanized the entire borcs7as3mt locus in 129s6 mice by syntenicreplacement in this process the segment of mouse dna carryingthese two genes was removed and replaced with the syntenic regionof human dna in mouse embryonic stem es cells using homologous recombination this ensured that the junctions between thehuman and mouse dna are known to the base pair level mice weregenerated from the es cells with the expectation that they wouldexpress the human as3mt and borcs7 proteinsthe present study describes in detail the creation of thehumanized mouse strain expression of human as3mt andborcs7 in tissues of the humanized mice and metabolism ofias in these mice after a single dose and during a subchronic exposure to ias in drinking watermethodsrationale for humanizing the borcs7as3mt locuswe chose to excise the 61kb segment of mouse dna carrying theas3mt and borc7 genes and replaced it with the correspondingpromoter of as3mt abuts59kb segment of human dna the untranslated region of borc7 and the weak correlation inthe the expression pattern of the genes suggests possible shared transcriptional regulatory elements li in addition the current human transcript databases for example the university ofcalifornia santa cruz genome browser ucsc lists a putative readthrough borcs7as3mt transcript lacking the finalexon of borcs7 and the initial exon of as3mt this suggests thepossibility that at least some as3mt activity may be linked to transcription driven by the borcs7 promoter thus the inclusion ofborcs7 in the humanized region increases the likelihood that theelements directing the tissue species diï¬erences in the expressionof as3mt are included in the humanized regionassembly of borcs7as3mt displacerthe borcs7as3mt displacer construct was assembled using astandard recombineering approach figure the mouse arms ofhomology were derived from the 129s7ab22 bmq bac librarysource bioscience plc nottingham uk the short homologyarm was derived from bac bmq455l14 and the long arm ofhomology from bac bmq345l20 the segment of humangenomic dna containing the borcs7 and as3mt loci wasderived from the human tile path bac rp11753c18 bacpacresources center children™s hospital oakland research instituteoakland ca the single nucleotide polymorphisms snps previously associated with interindividual diï¬erences in ias metabolism apata or schizophrenia risk li which were included in the this borcs7as3mt segment aredescribed in table s1 a dna segment bp extendingfrom chr1946683032 to were deleted from the mousegenome and replaced with a 59261bp segment of human dnaextending from chr10102845563 to the haplotypeof the as3mt gene carried in the displacer construct is 1a wood the resistance marker gene used for selection of escells in which the displacer construct underwent genomic integration consisted of a phosphoglycerate kinase pgkneo cassetteflanked by mutant loxp sites the marker gene can be excised leaving only a nonfunctional lox site in its placeall polymerase chain reactions pcrs carried out during thedisplacer assembly used taq polymerase bio basic with an initial denaturation temperature of °c for s followed by cycles of denaturation for s at °c annealing for s at a°c and extension at °c with an extension time of min per bp of product length all redet recombination reactionswere carried out using a commercially available kit gene bridgescat no k001 all predesigned primers used during the construction of the displacer vector were from sigmaaldrich and arelisted in table s2 in descriptions of specific assays each of theseprimers is listed by the corresponding number as stated above the short arm of the displacer construct wasderived from the bmq455l15 bac library this was accomplished by first replacing the borcs7as3mt region of the bac withan ampicillin resistance marker by redet recombination theampicillin resistance marker was amplified from the pbluescriptii sk cloning vector stratagene primers and the homology arms referred to as the red arm primers and and the ds arm primers and respectively were generated by pcr amplification ofsegments of the bmq455l15 bac the homology arms werefused to the ampicillin resistance gene by overlap pcrabhuman locuscyp17a1borcs7as3mtmouse locuscyp17a1borcs7as3mt kb deletedcnnm2cnnm2cborcs7 as3mt displacerddisplaced mouse locuscyp17a1floxedneofloxedneoborcs7as3mtcnnm2homologousrecombinationborcs7as3mtcnnm2 kb insertedfigure scheme for humanization of the mouse borcs7as3mt locus a human borcs7as3mt locus showing the relative positions of the borcs7 andas3mt genes as well as the closest flanking genes b mouse borcs7as3mt locus with flanking genes c schematic of the borcs7as3mt displacer construct d humanized locus prior to cremediated marker excision the names of human genes are capitalizedenvironmental health perspectives august 0cwas used to insert the human borcs7as3mt segment and neomycin resistance cassette by redet recombination into thebmq354l20 bac carrying the short arm and ampicillin genesimultaneously displacing the ampicillin gene this reactionyielded the final borcs7as3mt displacer vector which wasdigested with noti to release the bac backbone before transfection into es cellsgeneration of mouse es cellsthe parental es cell line phnx43 used in these studies was generated as follows male and female 129s6svevtac mice taconicbioscience were mated females were checked every morning forcopulatory plugs indicative of successful mating three days laterfemales were euthanized by lethal carbon dioxide co2 exposurefollowed by physical euthanasia and the 35d embryos blastocysts were collected by flushing the uterus with co2independentmedium gibco cat no supplemented with bovine serum albumin sigmaaldrich cat no a3675 individualembryos were collected from the lavage fluid with a pipette andplaced in 2cm2 wells that had been seeded h earlier withprimary mouse embryo fibroblasts mefs gibco cat nogsc6005m blastocysts and mefs were cultured in gibcoknockout„¢ ko medium cat no supplementedwith esqualified fetal bovine serum fbs gibco cat no and 2mm lglutamine gibco cat no after “ d in culture when the inner cell mass of the embryosreached ˆ¼ mm in diameter the embryos were removed and disrupted with a pipette and placed in a new well seeded with mefsthis process of serial expansion of a single inner cell mass wascontinued until a single inner cell mass was expanded to at least colonies of cells cells were then further expanded by disruptionand exposure to trypsin for min at °c after expansionand cryopreservation in dimethyl sulfoxide sigmaaldrichcat no d2660 a sample of the cell line was subjected to karyotype analysis in karyologic inc rtp nc the cell line used inthis study had a xy normal male mouse karyotypeexpression of human borcs7as3mt in mouse es cellssinglecell suspensions of the parental es cell were prepared bytreatment with trypsin as described above in a volume of ml — cells were electroporated in the presence of lgof dna the apparatus used was a btx electro cell manipulator with cuvette btx cheshire analytical the settingsused were v lf r8 ohms after electroporation the cells were distributed onto seven 100mm tissue cultureplates corning cat no in gibco ko medium supplemented with esqualified fbs gibco cat no and mm lglutamine gibco cat no after hselection was initiated with the addition of geneticin„¢ final concentration mgml gibco cat no after d individual neomycin resistant colonies became visible cells fromˆ¼ colonies were pooled and used for rna isolation to confirmthat the human genes were expressed see details below once thiswas established individual colonies from the remaining plateswere transferred individually by pipette to a 96cell plate each colony was trypsinized and a portion used for pcr analysis to identify those in which the dna had integrated by homologousrecombination as described below the remaining cells wereallowed to grow and cultures carrying a targeted allele as determined by the assay detailed below were transferred sequentially to and 60cm2 tissue culture plates at this point some cellswere cryopreserved while a portion of each was used for karyotypeanalysis and to confirm the expression of human as3mt by probebased droplet digital pcr ddpcr using commercially availablethe displacer short arm together with the inserted ampicillingene was subcloned from the modified bmq455l15 bac into aplasmid vector by redet recombination the plasmid vectorwas prepared by ligation of four pcr products the vector backbone carrying a cole1 origin of replication and a spectinomycingene was derived by amplification of the pfloxxerx plasmid previously assembled in koller™s laboratory see table s3 primers and the bmq455l15 bac was used as atemplate for the other three pcrs us arm primers arm primers and andand short ds arm primers and all pcr productswere gel purified on a agarose gel using a commerciallyavailable kit qiagen cat no digested with bbsi nebcat no r3539s and mlui neb cat no r0198s and columnpurified qiagen cat no fifty nanograms of each pcrproduct was combined and ligated neb cat no m0202s in a20ll reaction for h at °c the ligation reaction was heatinactivated at °c for min and then ll of the reaction wastransformed into chemically competent e coli dh5afcellszymo research cat no t3002 according to the manufacturer™s directions and plated on luria broth agar plates supplemented with spectinomycin at lgml the resulting plasmidvector was digested with mlui and used for redet recombination with the modified bmq455l15 bac the resulting plasmidwas in turn digested with bbsi to release the displacer short armand ampicillin resistance gene flanked by the us and dshomology arms this bbsi restriction fragment was introducedinto the bmq345l20 bac by redrecombination to create abac vector in which the ampicillin resistance marker wasflanked by the displacer arms of homologythe segment of the human borcs7as3mt locus included inthe displacer vector was prepared for excision from the rp11753c18 bac in two steps in the first step a neomycin resistancegene was inserted into the bac upstream of the borcs7 gene toaccomplish this the neo gene flanked by mutant loxp sites wasligated to homology arms referred to as blackred and purplerespectively the neomycin resistance gene was excised from thevector psfiijt15neojtz17sfii previously assembled in koller™slaboratory see table s4 by digestion with sfii neb cat nor0123s the blackred homology arm was amplified primers and using the bmq455l15 bac as a templateand the purple homology arm was amplified primers and using the rp11753c18 bac as a template all fragments were gel purified as described above and the blackred andpurple homology arm pcrs were digested with bgli neb catno r0143s and then column purified as described above theloxpflanked neomycin resistance gene was ligated to the homology arms as described above in a 20ll reaction containing a totalof lg of dna with the three fragments at an equimolar ratiotwo microliters of the ligation reaction was used for a redetreaction with the rp11753c18 bac in the second step an ampicillin gene was inserted downstream of the as3mt gene in therp11753c18 bac carrying the neomycin resistance marker toaccomplish this homology arms referred to as bluegreen andyellow respectively were added to the ampicillin resistance genethe bmq455l14 bac was used as the template for the bluegreen pcr primers and pbluescript ii sk was used as the template for the ampicillinyellow pcr primers and the two pcr products were gel purifiedand then combined in an overlap pcr primers and the resulting pcr product was gel purified and ngwas used for a redet reaction with the rp11735c18 bac carrying the neomycin resistance cassetteapproximately ng of the modified rp11753c18 bacwas digested with mlui in a 20ll digest and ll of the digestenvironmental health perspectives august 0cprimers applied biosystems hs00960526 here a 20ll reaction mixture was pipetted into a dg8„¢ cartridge along with llof droplet generation oil for probes bio rad and loaded into theqx200 droplet generator bio rad according to the manufacturer™s instructions after droplet generation the droplets weretransferred to an eppendorf twintec® 96well plate and placed in ac100 touch thermal cycler bio rad using the manufacturer™srecommended cycling conditions after cycling the plate was readin the qx200 droplet reader bio rad quantasoft„¢ softwareversion a portion of each of the es colonies ˆ¼ cells was disruptedby incubation for min at °c in lysis buï¬er consisting of trisedta [ mm trisma base thermo fisher scientific cat nobp152 mm ethylenediaminetetraacetic acid edta thermofisher scientific cat no volvol triton„¢ x100millipore sigma cat no and mgml proteinase kroche cat no ] lysates from all colonies werescreened for homologous recombination by pcr using primersscreen f and see table s4 with gxl polymerase takarabio according to the manufacturer™s suggested protocol the pcrprogram used was °c initial denaturation for s followed by cycles with s denaturation at °c s annealing at °c minat °c and a final 10min extension at °c pcr products wereanalyzed by agarose gel electrophoresis agarose bio basiccat no d0012 in — trisacetateedta running buï¬er the sizeof the pcr product was determined by comparison with 2logladder neb cat no n3200ltwentyseven clones with homologous recombination wereidentified and were subjected to further analysis because of thishigh recombination frequency it was not necessary to use clustered regularly interspaced short palindromic repeats and crisprassociated protein 9crisprcas9 to stimulate recombinationevents rna was prepared from pools of the cells and examinedfor the expression of the human genes rna was also preparedfrom a number of the individual clones after expansion brieflycells were lysed directly in the culture dish and homogenized byrepeated pipetting rna was isolated using rna bee or stat60teltest according to the manufacturer™s instructions rna yieldand quality were assessed using a nanodrop thermofisher one to two micrograms total rna was reverse transcribedwith the high capacity reverse transcription kit appliedbiosystems expression of the human as3mt was examined byqualitative pcr qpcr on a c100 touch thermal cycler biorad using commercially available primers applied biosystemshs00960526generation of mice expressing human borcs7as3mtfor injection into blastocysts the es cells carrying the humanborcs7as3mt locus were again trypsinized to generate singlecell suspensions collection of recipient blastocysts blastocystinjection and transfer to pseudopregnant dams was carried out bythe unc chapel hill animal models core following proceduresdescribed by longenecker and kulkarni and by koller to maintain the mutation on the 129s6svevtac background the chimeric mice were bred to 129s6svevtac micetaconic bioscience the resulting f1 mice were identifiedby taq as wildtype wt homozygotes for the mouse as3mtborcs7 locus wtwt or heterozygous wths or homozygoushshs for the human as3mtborcs7 locus the primers usedwere common and endo for the endogenous locus and common anddisplaced for the displaced locus see table s2 the pcr assayswere carried out using taq polymerase bio basic with an initialdenaturation temperature of °c for s followed by cycles ofdenaturation for s at °c annealing for s at a °c andextension at °c for s with a final extension of min the f2generation”consisting of hshs homozygotes hswt heterozygotes and wtwt homozygotes”was produced by mating of f1hswt heterozygotes the hshs hswt and wtwt oï¬springfrom the f2 generation were housed together one litter per cagecunder controlled conditions with 12h lightdark cycle at ± and ± relative humidity the parental mice and mice in thef1 and f2 generations were fed purina labdiet 5v5r and drankdeionized water ad libitumall proceduresinvolving mice were approved by theuniversity of north carolina institutional animal care and usecommittee 0ecollection of tissues for mrna expression analysistissues for mrna expression analysis were collected from f2hswt mice both males and females measurement of rnaexpression in hswt mice which were heterozygous for thehuman and mouse gene allowed direct comparison of expressionin the same tissuerna sample minimizing the eï¬ects of physiological variation between animals or quality of sample whichcould result in subtle diï¬erences in rna quality and cdnaformationmice were euthanized by exposure to co2 followed by physical euthanasia the following tissues were collected whole brainparts of brain cortices pons medulla hippocampus cerebellumpituitary spinal cord adrenals liver spleen kidney duodenumjejunum colon stomach uterus ovaries testes heart and skinfrom back of neck neuronal and glial cells were also dissectedbrain parts and spinal cord were collected from male mice at dof age neuronal cells were isolated from embryonic day e17embryos and glial cells from postnatal day p1 pups all othertissues were collected from to 12wkold mice three mice wereused for each tissue blood was collected from lethally anesthetized mice via cardiac puncture with an edtacoated syringe justprior to thoracotomy to isolate mononuclear cells from blood ml of whole blood were layered onto ml of histopaque® sigma and centrifuged at room temperature for min with nobrakes at — g the upper layer was discarded and the interfacecontaining the mononuclear cells was transferred to a 15ml conical tube the cells were then washed twice with phosphatebuï¬ered saline and then lysed with rna beeneuronal cell isolation and culture brains were harvestedfrom eight e17 embryos and placed in a petri dish containinghanks™s balanced salt solution hbss gibco meninges wereremoved cortices dissected and placed in a new petri dish containing hbss cortices were transferred to a 15ml conical tube containing ml hbss and centrifuged at — g for min at °cthe tissue was digested with ml tryple express gibco catno and dnase i roche cat no for min at °c the digested cortices were centrifuged at — gfor min at °c the tryple dnase i was aspirated and thecortices were washed for min with ml fbs to inactivate thetrypsin the tissue was centrifuged again and resuspended in complete dulbecco™s modified eagle medium dmem gibco with fbs vwr glutamax gibco with penicillinstreptomycingibco and 2mercaptoethanol sigma the cortices were titurated times with a 10ml pipette and then times with aflamedtip pasteur pipette the cells were then passed through a100lm cell strainer into a clean conical tube cells were then pelleted by centrifugation at — g for min the cell pellet wasresuspended in ml complete dmem and plated in mm culture dishes coated with lgml poly dlysine sigma — cells per dish after h 1lm cytosine bdarabinofuranosidearac sigma cat no c6645 was added using a mediachange without exposing the cells to air the cells were harvestedfor rna isolation on day environmental health perspectives august 0cmixed glial cell culture brains were harvested from six p1pups and placed in a dish containing hbss meninges wereremoved cortices dissected and placed in a conical tube containing cold dmem cortices were centrifuged at — g for min at°c and media replaced with ll cold dmem the corticeswere titurated times with a flamedtip pasteur pipette coated inserum the cells were then passed through a 100lm cell strainerinto a fresh conical tube cells were pelleted by centrifugation at — g for min the cell pellet was resuspended in ml complete dmem and ml of the suspension was plated on three100mm culture plates the medium was changed h later andthen daily the cells were harvested for rna on day isolation of adrenal cortex and medulla to obtain samplesenriched for each of these two distinct functional regions of thegland the adrenal cortex was removed from medulla of male andfemale adrenal gland by microdissection enrichment was quantitated by qpcr analysis of genes known to be expressed in onlyone of these two regions chgb in the medulla and cyp11b1 inthe cortex the human protein atlas using appliedbiosystem taqman probes mm00483287 and mm01204952respectively rna was isolated from cells and tissues using themethod described for es cells however the tissues were first homogenized in stat60 in a desktop homogenizer fastprep mp bio using ceramic beadsanalysis of as3mt as3mtd2d3 borcs7 as3mt andborcs7 expression in collected tissues and cellsboth ddpcr and qpcr were used to detect and quantify as3mtas3mtd2d3 borcs7 as3mt and borcs7 mrna the specificmethod is indicated in each figure legend ddpcr is minimallyinfluenced by diï¬erences in the efficiency of the mouse and humanprimer sets quan and therefore provides a means ofdirect comparison of expression between the endogenous mouselocus and the humanized locus the distribution of the as3mttranscript in the tissues was compared with as3mt protein distribution described by the human protein atlas for the human tissues sybr green qpcr was used to compare rna expression inwhole brain cortex hippocampus pituitary cerebellum pons medulla spinal cord glial cells neurons adrenals ovaries testesheart kidney liver colon jejunum spleen and blood ddpcr wasused to assess rna expression in es cells whole brain liver adrenals spinal cord ovaries testes spleen and heart for all assays“ lg total rna was reverse transcribed with the high capacityreverse transcription kit applied biosystemsfor sybr green qpcrthe relative expression of fulllength as3mtthe d2d3 variant and the borcs7as3mtfusion transcript were assessed on a quantstudio6 flex„¢ realtime pcr system applied biosystems sybr green reactions were run using itaq universal sybr green supermixbio rad nm of each primer and ngll of cdnaprimers used for quantification of as3mt isoforms were d2d3fand d2d3r for the d2d3 isoform and fullf and fullr for thefulllength isoform see table s2 for analysis of borcs7as3mt readthrough transcripts expression in mouse tissues lgrna was reverse transcribed and sybr green quantitative pcrwas run using primers gaacagtcatcggatctacagga3and and itaq sybrgreen universal supermix bioradgaacagtcatcggatctacagga3for ddpcr absolute concentration of as3mt as3mt borcs7and borcs7 was acquired using the ddpcr supermix forprobes no dutp bio rad and taqman gene expressionassays as3mt mm00491075_m1 as3mt hs00960526_g1borcs7 mm01205060_m1 and borcs7 hs00376014_m1all from applied biosystems the ddpcr analysis was carriedout as described aboveevaluation of the metabolism of a single dose of iasthe metabolism of ias was examined in to 22wkold hshsmale n and female n f2 oï¬spring and their wtwtmale n and female n littermates the mice weregiven a single dose of ias sodium arsenite pure sigmaaldrich in deionized water diw by gavage lg askg ofbody weight immediately after dosing each mouse was placedin a metabolic cage mousecage and urine and feces were collected in 24h intervals for d during these d all mice drankdiw ad libitum the mice were fasted during the first h buthad free access to purified laboratory diet envigo teklad catno ain93g during the second and the third days our published data showed that ias content in this type of diet rangesfrom to ˆ¼ lg askg douillet huang murko the 24h urine and feces samples werefrozen in dry ice and stored at ˆ’c 0eevaluation of the metabolism of ias during subchronicexposureafter collection of urine and feces following the singledoseadministration the hshs and wtwt mice were again cagedtogether one litter per cage and maintained on the purified dietand diw for wk to allow for clearance of as from the bodywhich was confirmed by measuring total as tas levels inurine see the œresults section for details both wt andhshs mice now to 27wkold were then exposed to iassodium arsenite pure sigmaaldrich in drinking water lg asl for wk spot urine samples ˆ¼ to llwere collected weekly body weights were recorded before andafter the exposure after wk all mice were sacrificed by cervical dislocation without anesthesia and tissues were collectedincluding the liver kidneys pancreas spleen heart lung adrenals kidneys bladder visceral fat calf muscle testes or ovaries and cecum and colon all tissues were flashfrozen in dryice and stored at ˆ’c 0eanalysis of as species in urine feces and tissuesspeciation analysis of as was carried out in 24h urines and fecescollected after the single dose of ias and in spot urine samplescollected during subchronic ias exposure the speciation analysis was also performed in livers and kidneys collected after subchronic exposure at sacrifice ten percent homogenates of liverand kidney were prepared in icecold diw wtvol usingwheaton potterelvehjem“style tissue grinders with a ptfepestle and wheaton overhead stirrer apparatus dwk lifesciences feces were snap frozen in liquid nitrogen and pulverized to powder using a steel mortar and pestle placed in dry icethe powder was mixed with 2n ultrapure phosphoric acid thermo fisher and digested in a mars5 microwave cemcorp for h at °c this method eliminates the biologicalmatrix but does not alter as speciation currier the digestates were neutralized by sodium hydroxide sigmaaldrich to ph the urines tissue homogenates and neutralized digestates were treated with lcysteine sigmaaldrich at room temperature for h to reduce pentavalent asspecies to their trivalent counterparts prior to the analysiscurrier the cysteinetreated samples were analyzed by hydridegeneration atomic absorption spectrometrycoupled with a cryotrap hgctaas as previously describedcurrier hern¡ndezzavala this analysis determined the concentrations of ias mas and dmas tasconcentration in urine feces and tissues was calculated as thesum of ias mas and dmas for assessment of the efficiencyof ias metabolism after a single oral dose the amount of tasenvironmental health perspectives august 0cwas expressed as the percentage of the dose by comparing theamount of elemental as administered as ias to each mousewith the amount of tas in 24h urine and feces samples collected from that mousethe instrumental limits of detection lod for ias mas anddmas using this method are and pg as respectivelyhern¡ndezzavala an imputed value of was usedfor measurements below the lod hgctaas is also capableof detecting and quantifying trimethylarsine oxide tmasohern¡ndezzavala a product of ias metabolism byrat as3mt waters however because tmaso israrely detected in human urine and because tmaso was not aproduct of ias methylation by recombinant human as3mt inour published study ding we did not includetmaso analysis in this studystatistical analysisoneway analysis of variance with studentnewmankeuls ortukey™s multiple comparison posttest was used to assess diï¬erences in gene expression and in the concentrations and proportions of as species among wtwt and hshs mice student™s ttest was applied for comparison of mouse and human geneexpression in a single tissue of hswt mice and for comparisonof as species concentrations or proportions between male andfemale mice and between hshs and wtwt mice of the samesex the statistical analyses were performed using prism software graphpad software diï¬erences with p were considered statistically significantresultsexpression of borcs7 and as3mt in hswt miceexpression of as3mt and borcs7 were directly compared withthose of endogenous mouse genes in tissues collected from f2male hswt mice that carried one copy of the mouse locus andone copy of the human locus figure 2a using both conventionalqpcr and when appropriate ddpcr expression of humanas3mt and borcs7 was easily detected in all tissues expressingthe corresponding mouse genes although the level of expressionof the human and mouse genes diï¬ered figure 2bc notable inthis analysis was
Colon_Cancer
" camp responsive element binding protein creb5 is a transcriptional activator in eukaryotic cells that canregulate gene expression previously we found that creb5 was involved in the occurrence and development of colorectalcancer crc using bioinformatics analysis however the biological roles and underlying regulatory mechanism of creb5 incrc remain unclearmethods realtime pcr western blotting and immunohistochemistry were used to examine creb5 expression in vitroexperiments including migration assay woundhealing assay chicken chorioallantoic membrane assay and human umbilicalvein endothelial cells tube formation assay were used to investigate the effects of creb5 on crc cell migration and tumorangiogenesis ability additionally an orthotopic implantation assay was performed in nude mice to confirm the effects ofcreb5 in vivo furthermore gene set enrichment analysis was performed to explore the potential mechanism of creb5 incrcresults we found that creb5 expression was highly upregulated in crc creb5 overexpression was positively correlatedwith advanced who stages and tnm stages and shorter survival in crc patients moreover creb5 overexpressionpromoted while creb5 silencing reduced the invasiveness and metastatic capacity of crc cells both in vitro and in vivofurthermore creb5 directly interacted with the met promoter and activated the hepatocyte growth factormet signallingpathway importantly inhibition of met reduced the invasion and metastasis of creb5overexpressing crc cells suggestingthat creb5 promotes metastasis mainly through activation of met signalling our study demonstrates a crucial role for creb5 in crc metastasis by directly upregulating met expressioncreb5 may be both a potential prognostic marker and a therapeutic target to effectively overcome metastasis in crckeywords colorectal cancer creb5 invasion metastasis met correspondence llifimmucom liaowt2002gmailcom shuyang wang junfeng qiu and lei liu contributed equally to this work1department of pathology nanfang hospital southern medical universityguangzhou guangdong chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwang of experimental clinical cancer research page of colorectal cancer crc is one of the most commoncancers ranking third in morbidity and tumorrelatedmortality among both men and women worldwidemoreover approximately of crc patients have metastases at the time of diagnosis metastasis is theleading cause of death among crc patients although systemic treatment of metastatic crc hasimproved the 5year survival rate is only “ andbecause ofthis poor prognosis understanding theunderlying mechanism of the metastatic process in crcis criticalfor both early detection of metastases andmore effective treatment the gene camp responsive element binding protein creb5 which is located on chromosome 7p151encodes a transcription activator in eukaryotic cells creb5 belongs to the atfcreb family the membersof which are characterized by a high affinity for campresponse elements cres the targets of the atfcreb family include transcriptional regulators including chromatinmodifying enzymes coactivators and corepressors genes involved in mitochondrial homeostasisand protein import and genes associated with proliferation and cell cycle entry metabolism proteases transporters and chaperones as an atfcreb familymember the creb5 protein contains several importantfunctional domainsincluding nterminal zinc fingerand cterminal bzip domains the latter of which includes a dna binding region and leucine zipper creb5 is a transcription factor that specifically binds tocre as a homodimer or a heterodimer with cjun orcrebp1 and functions as a credependenttransactivator creb5 is physiologically required for embryonic development in mice recent studies revealedthe roles of creb5 in the development and progressionof cancers examination of tcga pan cancer datasetsrevealed frequent creb5 amplification and overexpression in kidney cancers sarcomas lymphomas and lungadenocarcinomas as well as glioblastomas and gliomas experimentalinvestigations showed that creb5was upregulated in ovarian cancers hepatocellularcarcinoma and prostate cancer high creb5expression correlated with a poor prognosis in epithelialovarian cancer and hepatocellular carcinoma creb5 overexpression increased the proliferation of hepatocellular carcinoma moreover overexpression oramplification of creb5 promoted proliferation and mediated resistance to ar inhibition in metastatic castrationresistant prostate cancers in silico analysis showedthat the creb5 regulatory network was involved in crcmetastasis in addition qrtpcr assay revealed thatcreb5 mrna was upregulated in crc tissues and cells in vitro assays revealed that overexpression of creb5resulted in enhanced proliferation and migration andapoptosis inhibition in crc cells these findings suggest that creb5 may play an essential role in the progression of crc howeverthe specific function andmolecular mechanism of creb5 in crc metastasis remain largely unclearactivation of the hgfmet signalling pathway hasbeen reported to lead to the occurrence and metastasisof a variety of tumorsincluding crc breast cancerovarian cancer lung cancer and liver cancer as atyrosine kinase receptor met can be activated bydimerization multimerization and phosphorylation afterbinding to its ligand hepatocyte growth factor hgf activation of hgfmet can initiate downstreamsignalling pathways that drive malignant progression inmany types of tumors met is considered an essentialfactor for early invasion and metastasis of crc and canbe regarded as an important prognostic indicator in the present study we found that creb5 promotes crc invasion and metastasis by increasing metexpression to activate hgfmet signalling these results uncover a new molecular mechanism for cancermetastasis and suggest that creb5 may be a promisingtarget for crc treatmentmaterials and methodspatients and specimensa total of pathological specimens were obtainedfrom colon cancer patients between and atthe department of pathology nanfang hospital southern medical university the medical records of these patients provided information on sex age and thefollowing essential factors tumor pathological characteristics pathologic stage t stage dukes stage lymph nodemetastasis and distant metastasis ten pairs of fresh biopsies collected from crc patients and matched noncancerous mucosaltissue were obtained from theoperating room of nanfang hospital the fresh biopsieswere stored in liquid nitrogen before usein addition a tissue microarray no co802 containing colon cancer tissue specimens and adjacentnoncancerous tissue specimens was purchased fromailina biotechnology company approval for the use ofclinical materials for research purposes was obtainedfrom the southern medical university institutionalboard guangzhou china all samples were collectedand analysed with the prior written informed consent ofthe patientscell culturesthe human crc celllines sw480 ht29 hct15hct116 sw620 ls174t sw837 lovo dld1 andrko were purchased from the american type culturecollection sw620 ht29 and lovo cells were culturedin dmem medium gibco supplemented with 0cwang of experimental clinical cancer research page of foetal bovine serum fbs gibco sw480 hct116hct15 ls174t sw837 and dld1 cells were culturedin rpmi medium gibco with fbs gibcoplasmidscreb5 constructs were generated by cloning pcramplifiedfulllength human creb5 cdna into psinef2puro thefollowing primers were used for cloning including enzymesforward primer ² cgcgaattcatgatttatgaggaatccaa3² ecor i reverse primer ²ccggctagcttaaagaatcggattcaggt3² nhe i for deletion ofcreb5 short hairpin rna shrna sequences creb5²aacaagtcatccagcataa3² creb5shrna1shrna2 ²ggaatatctcgatgcataa3² were separately cloned into a gv248 vector psinef2puro and gv248were purchased from addgene incthe intensity of staining was graded according to thefollowing criteria no staining weak staining lightyellow moderate staining yellow brown and strong staining brown the staining index was calculated as the staining intensity score × the proportion ofpositive tumor cells using this method of assessmentwe evaluated the expression of creb5 in benign breastepithelium and malignant lesions by determining thestaining index with scores of and cutoff values for creb5 were selected on the basis of ameasure of heterogeneity with the logrank statisticaltest with respectto overall survival optimal cutoffvalues were identified a staining index ‰¥ was used todefine tumors with high creb5 expression and anindex ‰¤ was used to define tumors with low creb5expressionrna isolation reverse transcription rt and realtimepcrtotal rna samples from cultured cells and primarytumor tissues were extracted using trizol reagent invitrogen usa according to the manufacturer™s instruction realtime rtpcr was performed at least threetimes in triplicate using sybr green mix toyobojapan and the abi prism sequence detectionsystem applied biosystems usa the data were normalized to the geometric mean of the housekeeping genegapdh and calculated using the 2δδct method primerexpress was used to design the realtime pcr primersand primer sequences for amplification are shown insupplementary table s2luciferase reporter assaygenomic dna extracted from sw480 cells was used as atemplate to amplify met promoter fragments met promoter fragments were obtained by pcr and constructedinto pgl3basic plasmid using a fast ligation kit followingmanufacturer™s instructions sangon b620511“ thesequences of the pcr primers are listed in supplementarytable s4 cells at confluence in a 24well plate weretransfected using lipofectamine fortyeight hoursafter transfection luciferase activity was measured usingthe dualluciferase reporter assay system promega corpmadison wi usa and normalized to renilla luciferasegene expression all the experiments were performed intriplicatewestern blotting analysiswe carried out western blotting as previously described using anticreb5 abcam ab168928 antimetcell signaling technology antipmetcellsignaling technology antiakt cell signalingtechnology antipaktcell signaling technology antierkcell signaling technology antiperk cell signaling technology and antisnail cell signaling technology antiantiαtubulin antibodybodies mouse monoclonalsigma was used as the internal controlimmunohistochemistryimmunohistochemistry ihc staining was performed aspreviously described using creb5 antibody abcamusa ab168928 the degree of ihc staining wasreviewed and scored independently by two observersbased on both the proportion of positively stained tumorcells and the intensity of staining [ ] the proportion of tumor cells was scored as follows no positivetumor cells positive tumor cells “positive tumor cells and positive tumor cellschromatin immunoprecipitation chip assaychip assays were carried out as previously described precleared lysates were incubated with creb5 antibody abcam ab168928 or normal mouse immunoglobulin g cst as a negative control overnightat °c with rotation the human met promoter wasamplified by pcr all chip assays were performed threetimes and the sequences of the pcr primers are listedin supplementary table s3migration assaya boyden chamber with an 8μmpore filter membranewas used for the in vitro migration and invasion assaybriefly cells × in culture medium containing fbs were seeded in the upper chamber and culturemedium with fbs was added in the lower chamberas a chemoattractant the upper side of the filter wasfirst coated with matrigel bd biosciences sanjose ca usa after incubation for h cells on theupper side of the filter were removed with cotton swabscells that migrated to the lower surface of the filter werefixed in paraformaldehyde and stained with giemsa 0cwang of experimental clinical cancer research page of the migratory cells were counted random ×fields per well three independent experiments wereperformed and the data are presented as the mean ±semwoundhealing assaycells were seeded in 6well plates and incubated underpermissive conditions until confluence after serumstarvation for h wounds were created in the confluent cells using a pipette tip wound healing within thescrape line was then observed and photographed at indicated time points each experiment was repeated at leastthree timeschicken chorioallantoic membrane assaya chicken chorioallantoic membrane cam assay wasperformed on the sixth day of development of fertilizedchicken eggs as previously described human umbilical vein endothelial cell tube formationassayfirst μl of matrigel was pipetted into each well of24well plates and polymerized for min at °c human umbilical vein endothelial cells huvecs × in μl of conditional medium were added to eachwell and incubated at °c in co2 for h imageswere obtained under a brightfield microscope and thecapillary tubes were quantified by the counting lengthorthotopic mouse metastatic model to 6weekold balbc athymic nude mice nunuwere obtained from the animal center of southernmedical university guangzhou china all mice werehoused in a sterile environment cells × permouse were orthotopically inoculated into the caecumof anaesthetized nude mice the mice were sacrificedwithin weeks after surgery individual ans were excised and metastases were observed by histological analysis tissues were then fixed with formaldehyde andparaffinembedded and then 5mm sections were cutand stained with haematoxylin and eosin he thenumbers of gross metastatic foci were determined usinga dissection microscope all the mice used in this studywere maintained under specific pathogenfree conditions and all animal experiments were conducted in accordance with standard procedures and approved by theinstitutional use committee for animal carestatistical analysisall statistical analyses were carried out using spss version pearson correlation analysis was used for expression correlation analysis the survival curves of crcpatients in low and highcreb5 expression groups wereanalysed by the kaplanmeier method and the logranktest was used to compare differences p was considered significantresultscreb5 is upregulated in crc and associated with a poorprognosisthe expression of creb5 was analysed in differenttypes of malignant tumors in the public database oncomine wwwconominecom revealing that creb5was upregulated in crc tissues in of crc datasetssupplementary fig s1a additionally a gene set enrichment analysis gsea plot showed significant enrichment of tumorrelated genes and crcrelated genesets in the highcreb5 expression group supplementary fig s1b realtime pcr and western blotting analyses showed that creb5 was differentially expressed incrc cell lines supplementary fig s1cd in additioncreb5 was significantly upregulated in ten crc tissuescompared with adjacent normal intestinal epithelial tissues fig 1a and b ihc showed that creb5 proteinwas weakly expressed in normal tissue but markedly increased in adenocarcinoma cells and was mainly localized in the nucleifig 1c kaplanmeier survivalanalysis showed that crc patients with higher creb5protein expression levels had a poorer prognosis fig1d in addition creb5 expression levels were significantly correlated with the t classification lymph nodemetastasis and distant metastasis p supplementary table s1 creb5 expression was substantiallyhigher in tumors from patients with distant metastasismoreover high creb5 expression was also positivelycorrelated with who stages p supplementarytable s1 these data suggested that creb5 expressionis significantly correlated with advanced stages of crccreb5 activates met signallinggseas of creb5regulated gene signatures revealed thathigher creb5 expression was positively correlated withenrichment of an met signalling pathway signaturegse17538 fig 2a to validate this result we establishedstable creb5overexpressing and creb5knockdowncrc cell lines fig 2b upregulation of creb5 significantly increased while knockdown of creb5 decreasedthe expression of total met at both translational andtranscriptional levels fig 2c and d in addition creb5overexpression markedly increased but creb5 downregulation significantly attenuated the expression of phosphorylated met perk pakt and snail fig 2c moreovermet expression was increased in a dosedependent manner at both translational and transcriptional levels by transiently transfecting sw480 cells with a creb5 expressionvector fig 2e 0cwang of experimental clinical cancer research page of fig creb5 is upregulated in crc and associated with a poor prognosis a and b realtime pcr and western blotting analysis of creb5 expression inpaired human colon cancer tissues and adjacent noncancerous tissues p quantity one software was used to quantify the protein expressionlevels c ihc representative images of creb5 expression in normal intestinal epithelium and crc tissues scale bar μm d the paraffin samples of crc patients were divided into a lowcreb5 expression group n and a highcreb5 expression group n based on ihc results thekaplanmeier method was used to analyse survival curves and the logrank test was used to compare differences p fig creb5 activates the met signalling pathway a gsea of gse17538 in met signalling pathways es p b stable overexpressionand interference cell lines were detected by western blotting and realtime pcr c the expression of met and downstream signalling moleculesin creb5knockdown or creb5overexpressing cells was observed by western blotting d creb5 had an effect on met by realtime pcr in theindicated cells e after transient transfection of different amounts of the creb5overexpression plasmid in sw480 cells the protein and mrnalevels of met were detected by western blotting and realtime pcr respectively p 0cwang of experimental clinical cancer research page of creb5 associates with the met promotergiven that creb5 is a transcriptional factor and upregulatesmet at the transcriptional level we performed a luciferasereporter assay to investigate whether creb5 can increasemet promoter activity a 27kb fragment of the fulllengthmet promoter region was subcloned into a luciferase vector met promoter activity wasincreased by cotransfection with a creb5 expression vector in sw480 cellsbut decreased in hct116 cells expressing creb5 shrna ina dosedependent manner compared with empty vectorsfig 3a to determine the effective regions of the met promoter that creb5 may affect we transfected met promotertruncations fig 3b into hct116 cells expressing eithercreb5 shrna or a scramble control sequence as shown infig 3c luciferase activity was increased in cells carrying thefulllength met promoter and truncations ˆ’ and ˆ’ bp upstream of the transcription start site but not incells carrying truncations ˆ’ to ˆ’ bp or ˆ’ to ˆ’ bp knockdown of creb5 expression bycotransfection of creb5 shrna significantly decreased metpromoter activity fig 3c furthermore we performed chromatin immunoprecipitation chip assays and identified thatthe ˆ’ to ˆ’ 223bp region of the met promoter whichcontains an ap1 motif was a creb5 protein binding sitefig 3d these data identify met as a direct transcriptionaltarget of creb5downregulation of creb5 represses invasiveness andreduces the metastatic potential of crc cellsnext we investigated the role of creb5 in invasivenessand metastasis in crc cells silencing of creb5 significantly compromised the migratory and invasive abilitiesof crc cells fig 4a and b supplementary fig s2a andb the tubule formation and chicken cam assays revealed that knockdown of creb5 strongly inhibited theformation of tubules by huvecs and inhibited angiogenesis in cams fig 4c and d supplementary figs2c orthotopic inoculation assay showed that knockdown of creb5 inhibited liver metastases fig 4eknockdown of creb5 also obviously extended the overall survivaltime of nude mice inoculated with thecrc cell lines fig 4f these results indicate that silencing creb5 inhibits the invasiveness and metastasis of crc cellsinhibition of met attenuates the invasion and metastasisof crc cells by creb5 in vivo and in vitroto determine the functional relationship between creb5and met in the invasion and metastasis of crc weknocked down met using two met shrnas or suppressed met activation using the met inhibitor crizotinib emd silencing or inhibition of met insignificantlysw480creb5or ht29creb5cellsfig creb5 regulates met and binds directly to the met promoter a the met promoter sequence was cloned into pgl3basic vector containing theluciferase reporter gene and then transfected into crc cells with the indicated treatments b schematic diagram of the full and truncated met promoterc the fulllength met promoter or its truncations were cloned into pgl3basic vector containing the luciferase reporter gene and then transfected intohct116 cells with creb5 shrna or empty vector d chip analysis of creb5 binding to the met promoter in sw480 cells p 0cwang of experimental clinical cancer research page of fig downregulation of creb5 inhibits the invasion and metastasis of crc cells in vivo and in vitro a and b woundhealing assay and transwellmigration assay were performed to evaluate the invasive and migratory abilities of crc cells with different treatments in vitro c huvec tubeformation after stimulation with the indicated conditioned medium d representative images of the cam assay histograms show the formationof secondary and tertiary blood vessels after stimulation with the indicated conditional medium scale bar mm e orthotopic transplantationwith the indicated hct116 cells in nude mice n in each group was performed and representative gross images of the livers and intestinesare shown the arrows indicated the tumors liver sections were stained with haematoxylin and eosin he scale bar μm f the kaplanmeiermethod was used to analyse survival curves in the specified treatment groups and the logrank test was used to compare differences p decreased the expression of phosphorylated met perkand pakt as well as snail supplementary fig s3a inaddition the invasive and migratory abilities of sw480creb5 or ht29creb5 cells were partially diminished byinhibition of met fig 5a and b supplementary fig s3band c moreover upregulation of creb5 expression enhanced the capacity of crc cells to induce tube formationand angiogenesis in cams in contrast angiogenesis ability was partially diminished by met inhibition fig 5cand d supplementary fig s3d orthotopic inoculationassay showed that creb5 significantly promoted liver metastases and decreased the overall survival of mice fig 5eand f conversely inhibition of met significantly attenuated the formation of metastatic foci by sw480creb5cells and extended the survival time of mice inoculatedwith sw480creb5 cells fig 5e and fcreb5 expression positively correlates with metexpression in crcto assess a potential link between creb5 and met expression in human crc we analysed tcga crc dataand identified a strong positive correlation between highexpression levels of creb5 and met p r fig 6a in addition analyses of fresh crc tissuesshowed that creb5 expression was positively correlatedwith met expression at both mrna p r and protein levels p r fig 6b and c 0cwang of experimental clinical cancer research page of fig overexpression of creb5 promotes the invasion and metastasis of crc cells but inhibition of met weakens these effects a and b theinvasive and migratory abilities of crc cells in vitro with different treatments were evaluated by woundhealing assay and transwell migrationassay c huvec tube formation after stimulation with the indicated conditional medium d representative images of the cam assay histogramsshow the formation of secondary and tertiary blood vessels after stimulation with the indicated conditional medium scale bar mm eorthotopic transplantation with the indicated sw480 cells in nude mice n in each group was conducted and representative gross images ofthe livers and intestines are shown the arrows indicate the tumors liver sections were stained by he scale bar μm f the kaplanmeiermethod was used to analyse the survival curves of different treatment groups and the logrank test was used to compare differences p p p furthermore ihc revealed that creb5 expression waspositively correlated with met fig 6d p discussionmetastasis of crc is a multistep process requiring the accumulation of genetic andor epigenetic alterations andabnormal expression of genes involved in signal transduction pathwaysincluding oncogenic mutation of krasand activation of the erkmapk pathway wntβcatenin signalling and tgfβ signalling ap1 dnabinding sequences act as crucial response elements fortranscriptional activation by the raserk pathway creb5 is a credependent transactivator downstream ofthe raserk signalling pathway since it interacts with cjun which is one of the ap1 subunits to form a homodimer or a heterodimer along with foxd1 and atf3creb5 formed a transcription factor regulatory networkthat negatively regulates mapk signalling which is suppressed by fzd3 in melanoma previous studies haverevealed that creb5 mrna was upregulated in crc asdemonstrated by bioinformatics analysis or qrtpcrexamination in human cancer tissues [ ] in thecurrent study we demonstrated that creb5 was highlyupregulated in crc at both the mrna and protein levelsoverexpression of creb5 was significantly associatedwith aggressive cellular characteristics of crc eg an 0cwang of experimental clinical cancer research page of fig creb5 positively correlated with met expression in crc a correlation analysis of creb5 and met in tcga crc data r p band c correlation analysis of creb5 and met at the mrna r p and protein levels r p in fresh crc tissues dthe expression of creb5 and met protein in specimens including colon cancer tissue specimens and adjacent normal tissue specimens wasdetected by ihc representative ihc images left and correlation analysis right of creb5 and met expression scale bar μm high expressionof creb5 n high expression of met n low expression of creb5 n low expression of met n p advanced who stage and an advanced tnm stage andpoorer patient outcomes suggesting that creb5 might bean oncogene and a prognostic marker of crc progressionconsistent with our results upregulation of creb5 hasbeen reported to be responsible for poorer outcomes inpatients with epithelial ovarian cancer and hepatocellular carcinoma in prostate cancers creb5 overexpression occursthrough both copy number gain and increased gene expression however examination of tcga colorectalcancer datasets via cbioportal revealed rare amplificationof creb5 suggesting that overexpression of creb5 iscontrolled attranscriptional and posttranscriptionallevels creb5 has been shown to be a downstream target of lncrna snhg5mir1323p and circularrna circvapamir125a in addition fzd3 inhibits transcriptional networks controlled by creb5 however the alternative mechanisms involved inupregulation ofthe creb5 gene and activation ofcreb5mediated signalling require further investigationthe effects of creb5 overexpression on promotingcell proliferation and migration have been demonstratedusing in vitro assays in human hepatocellular carcinomacells and crc cell lines [ ] in the current studywe showed that creb5 overexpression promoted whilecreb5 silencing reduced the invasiveness and metastaticcapacity of crc cells both in vitro and in vivo mechanistically creb5 directly interacted with the met promoter and activated the hgfmet signalling pathwayimportantlyinhibition of met reduced the invasion 0cwang of experimental clinical cancer research page of and metastasis of creb5overexpressing crc cells suggesting that creb5 promotes metastasis mainly throughactivation of met signalling our data provide solid evidence that upregulation of creb5 plays an essential rolein crc metastasis recently overexpression or amplification of creb5 was reported to promote proliferationand mediate resistance to ar inhibition in metastaticcastrationresistant prostate cancers these datasuggest that creb5 may function as a multitaskingregulator in cancer progression and clinical outcomessignallingtransportimmunegrowth factorscreb family members can be phosphorylated via various intracellular signal transduction pathways such asprotein kinase a pka calmodulindependent proteinkinasecamk mitogenactivated protein kinasesmapks and other kinases upon phosphorylationcreb recruits crebbinding protein cbp and binds tothe cres of the promoters of its target genes target genes containing consensus sites for creb bindinginclude those related to metabolism transcription neuropeptidesneurotransmitters cell cyclecellsurvivalregulationdna repairreproductiondevelopmentandstructure specifically knockdown of creb1creb5increased tumor necrosis factor alpha tnfα levelsenhanced the expression of phosphonfκb p65 andnfκb p65 and induced immunosuppression in monocytes in prostate cancer creb5 could improve resistance to enzalutamide with the help of foxa1 andselectively enhance the interaction of ar with targetgenes critical for survival however little is knownabout the downstream targets of creb5 involved in theprogression of crc our results showed that creb5 directly interacted with the met promoter and activatedthe hgfmet signalling pathway which in turn increased the expression of downstream erk and pi3ksignalling cascades meanwhile the expression of snailan essential emt transcription factor was also upregulated via the creb5hgfmet axistranscriptional factor that interacts with the met promoter at the ap1 motif and activates met expressionin our data suggest that creb5 has an essential role in crc metastasis by regulating the protooncogene met interfering with creb5 may representan alternative therapeutic target to prevent or reducemetastasis in crcsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s13046020016730additional file table s1 the relationship between creb5 expressionand clinicopathological parameters table s2 primer sequences used forrealtime pcr ² to ² table s3 sequence of primers for chip assaytable s4 sequence of primers for luciferase reporter assayadditional file figure s1 bioinformatics analysis of creb5expression the expression of creb5 in crc and other malignant tumorswas analyzed by oncomine database the inclusion criteria were that thedifference of creb5 expression between tumor tissue and normal tissuewas more than times and the arrangement of gene position was lessthan with p the outliers in the red and blue boxes representthe number of data sets with high and low expression of creb5respectively the right table of a represents the copa score of creb5 in crc data sets b the two crc chips gse17538 n andgse35896 n from the public database of geo was analyzed bygsea the plot showed significant enrichment of tumorrelated gene setkegg_pathway_in_cancer and colorectal cancerrelated gene setkegg_colorectal_cancer in the creb5 high expression group cand d realtime pcr and western blotting analysis of creb5 endogenous expression in indicated crc cellsadditional file figure s2 representative images of woundhealingassay a transwell migration assay b and huvec tube formation assayc with i
Colon_Cancer
malignant mesothelioma is an aggressive cancer associated with asbestos exposure with median survival time of to months following diagnosis given that mesothelial cells also line the peritoneum and pericardium malignant mesothelioma can present in unusual sites and in patients with nonrespiratory complaints a 73yearold male presented to the emergency department for worsening intermittent diffuse abdominal pain for the past months with associated unintentional 40pound weight loss early satiety and diarrhea he denied exposure to asbestos computed tomography imaging revealed multiple masses concerning for malignancy including the primary retroperitoneal mass a mass involving the terminal ileum and a mass in the right upper lung esophagogastroduodenoscopy demonstrated significant mass effect within the stomach without signs of endoluminal infiltration computed tomography“guided biopsy of the retroperitoneal abdominal and intramuscular paraspinal masses was performed stage iv epithelioid mesothelioma was confirmed when hematoxylin and eosin staining revealed pleomorphic malignancy nuclei containing a vesicular chromatin pattern and prominent nucleoli and immunohistochemical staining was positive for ck oscar cytokeratin gata3 calretinin ema and ck56 he was started on cisplatin pemetrexed and bevacizumab but developed severe abdominal pain with pneumoperitoneum and bowel perforation month later and expired shortly thereafter to our knowledge this represents a highly atypical presentation of malignant mesothelioma considering the involvement of the retroperitoneum with diffuse lesions in the abdominopelvic cavity and thorax sparing the lung pleurae this case also calls attention to the occurrence of malignant mesothelioma in patients without known asbestos exposure and the crucial role of pathology in diagnosing atypical presentationskeywordsepithelioid mesothelioma neoplasm asbestos esophagogastroduodenoscopy retroperitonealintroductionmalignant mesothelioma mm is a rare cancer originating from mesothelial cells forming the linings of the pleura to of mm cases12 peritoneum to of cases23 and pericardium of cases456 typically the pathophysiology of mm is believed to occur in patients with asbestos exposure leading to chronic inflammation of the pleura67 subsequently mm presents in a patient with shortness of breath with findings on imaging of nodular thickening of the pleura pleural effusion or a mass7 in cases involving the peritoneum a more common initial presentation would be ascites with computed tomography ct findings including irregular thickening of the peritoneum lymph node enlargement and possible metastasis to the chest table regardless of location published guidelines detail the importance of first looking at the histology of a biopsy prior to immunohistochemical ihc and molecular testing to confirm suspicion of mm from the histology mm is classified into epithelioid sarcomatoid or biphasic mixed with epithelioid mm remaining the most common with its polygonal cells resembling reactive mesothelial cells other histologic features include scalloped cell borders with increased cytoplasm prominent and enlarged nucleoli and nuclear atypia10 the presence of a solid mass separate from the pleura and peritoneum with histologic features of mm eliminates the requirement for stromal invasion for the diagnosis910immunohistochemical testing should use markers of mesothelial origin such as cytokeratin markers and alternative tumor markers to narrow the differential diagnosis based on histology10 this will vary by histology and location 1mercyone des moines medical center des moines ia usa2des moines university des moines ia usareceived may revised june accepted july corresponding authordustin j uhlenhopp mercyone des moines medical center 6th avenue des moines ia usa email duhlenhoppmercydesmoinescreative commons non commercial cc bync this is distributed under the terms of the creative commons attributionnoncommercial license creativecommonslicensesbync40 which permits noncommercial use reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages ussagepubcomenusnamopenaccessatsage 0c of investigative medicine high impact case reportstable typical characteristics of malignant abdominal mesothelioma1819characteristicspercentage of casespathological subtypes epithelial sarcomatous mixedasbestos exposure male femalepresenting symptoms ascitesa abdominal pain asthenia weight loss anorexia fevera diarrhea vomitingact scan findings ascites abdominal mass peritoneal thickening mesenterial thickeningadditional laboratory findings thrombocytosis 000mm3 anemia male gdl female gdlmaletofemale ratiomedian overall survivalaverage age of onset months yearsabbreviation ct computed tomographyamost significant symptoms on presentation associated with deathof the mm epithelioid markers are needed to rule out carcinomas and peritoneal markers are needed to rule out adenocarcinomas and cancers causing peritoneal carcinomatosis810 key markers for mesothelioma are positive ihc staining for calretinin d240 podoplanin and cytokeratin in this case report we present an unusual case of mm with metastatic disease in a patient with no known asbestos exposure and a history of abdominal pain and weight loss found to have multiple lesions and diffuse lymphadenopathy on imagingcase descriptiona 73yearold male presented to the emergency department with worsening intermittent diffuse abdominal pain and 40pound weight loss over the past months associated with increased flatulence early satiety and frequent exive nonbloody diarrhea the patient described the pain as cramping that typically worsened in severity at night he denied feverchills dyspnea cough epistaxis hematemesis hemoptysis hematochezia and melena the patient was a longtime science instructor at a local community college prior to transitioning to the swedish importexport business with no significant exposure history including asbestos he had a prior history of right middle cerebral artery stroke and was a former smoker with a 60packyear smoking historyinitial testing revealed normal electrolytes and liver function panel elevated lactic acid mmoll leukocytosis white blood cell 12100mm3 and anemia hematocrit abdominal ct with oral and intravenous iv contrast revealed a homogenous retroperitoneal mass measuring — — cm with displacement of the gastroesophageal junction and lesser curvature of stomach as well as a soft tissue mass involving the terminal ileum measuring — — cm both were concerning for neoplasm additional findings included diffuse lymphadenopathy in the retroperitoneum likely signifying metastatic disease no evidence of obstruction was noted see figure esophagogastroduodenoscopy confirmed concerns for extrinsic compression of gastroesophageal junction and fundus of stomach suggesting an underlying mass no endoluminal gastric masslesions were appreciated see figure colonoscopy was attempted but scope was not able to advance past the sigmoid colon safely due to colonic stricturecomputed tomography chest with iv contrast identified moderate diffuse emphysema a right upper lung lobe spiculated mass measuring cm and indeterminate small left upper and lower lobe nodules see figure diffuse retrocrural and gastroesophageal lymphadenopathy was noted in addition to the retroperitoneal lymphadenopathygiven the concern for lymphoma versus alternative neoplastic disease ctguided biopsy was performed on the retroperitoneal abdominal mass and an intramuscular paraspinal mass hematoxylin and eosin staining revealed pleomorphic malignancy nuclei containing a vesicular chromatin pattern and prominent nucleoli see figure cells were predominantly polygonal with occasional spindling noted ihc staining was positive for ck oscar cytokeratin gata3 calretinin ema and ck56 staining was negative for pax8 tte1 p40 p63 d240 wt1 moc31 cd34 mari cd45 dog desmin cd117 sox10 actin ca9 and myogenthese findings were suggestive of epithelioid mm with metastasis to the terminal ileum and lung a diagnosis that was confirmed by mayo clinic laboratories noting this is a particularly atypical presentation of mesotheliomathe patient opted for treatment of stage iv mesothelioma with plans to pursue a 21day cycle of cisplatin pemetrexed and bevacizumab for up to cycles however he presented to the emergency department days after starting the second cycle with severe abdominal pain of hours duration he was found to have a moderate pneumoperitoneum with bowel perforation worsening intraabdominal mesothelioma and several small bilateral acute renal parenchymal infarcts the patient and his family opted for comfort care and he passed the following day 0cuhlenhopp et al figure abdominal computed tomography with oral and intravenous contrast demonstrating a homogenous retroperitoneal neoplastic mass measuring — — cm green arrows on left image with displacement of the gastroesophageal junction and lesser curvature of stomach a soft tissue mass involving the terminal ileum measuring — — cm and a 17cm midmesentery mass are also noted green arrows on right imagefigure esophagogastroduodenoscopy revealed extrinsic compression of the fundus suggesting an underlying mass or lesion no endoluminal gastric masseslesions were appreciated computed tomography“guided biopsy later revealed this to be epithelioid mesothelioma originating from the retroperitoneal spacediscussionthe diagnosis of mm carries a poor prognosis and requires careful review by pathology our case details the workup necessary to diagnosis mm in a patient with an atypical presentation of intermittent abdominal pain found to have figure computed tomography chest with intravenous contrast identified moderate diffuse emphysema a right upper lung lobe spiculated mass measuring cm green arrows and indeterminate small left upper and lower lobe nodules as this did not involve the pleura as is traditionally seen this was felt to be a metastasis of the primary retroperitoneal mass with metastasis to lungs and ileumsignificant metastatic disease prior to identification of a primary malignancyreview of the case reveals the importance of tissue analysis and the continued public health concern of mesothelioma 0c of investigative medicine high impact case reportsongoing research is investigating whether these genetic changes have a role in disease development in cases without asbestos exposure610 in the case of malignant peritoneal mesothelioma there is a higher fraction of mm without an identifiable environmental exposure which raises the question whether the disease process and role of genetics vary by the location of mm14 in the future these will hopefully lead to the development of immunotherapies that are able to target these pathways and improved disease prognosis6known asbestos exposure was not a factor in our case and should not be used to rule out mesothelioma10 over of mm cases are attributable to asbestos exposure with of malignant peritoneal mesothelioma attributed to asbestos1516 recent studies detail that despite the restriction of asbestos products in the united states for over years the role of asbestos in chronic inflammation and dna damage will continue to cause malignancy615 in the united states alone there were approximately new mm cases in with current estimates anticipating a peak incidence of mm worldwide in given the continued use of asbestos worldwide and time to disease development6715 this case highlights the importance of considering mesothelioma in the differential diagnosis of abdominal pain regardless of asbestos exposure historydeclaration of conflicting intereststhe authors declared no potential conflicts of interest with respect to the research authorship andor publication of this fundingthe authors received no financial support for the research authorship andor publication of this ethics approvalour institution does not require ethical approval for reporting individual cases or case seriesinformed consentverbal informed consent was obtained from the patient for their anonymized information to be published in this orcid idsdustin j uhlenhopp orcid0000000315092203ann saliares orcid0000000281484435tagore sunkara orcid0000000195369027references robinson bm malignant pleural mesothelioma an epidemiological perspective ann cardiothorac surg doi103978jissn2225319x20121104 hui m harshavardhana kr uppin sg pericardial mesothelioma presenting as chronic constrictive pericarditis a series of three cases from a single institution indian j pathol microbiol doi104103ijpmijpm_711_17figure hematoxylin and eosin stain of the retroperitoneal mass — malignant mesothelioma that is somewhat pleomorphic but mostly epithelioid polygonal cells with some spindling are seen cells have pleomorphic nuclei occasionally multinucleated with moderate amount of eosinophilic cytoplasm nuclei have a vesicular chromatin pattern with distinct nucleoli there is no obvious glandular or squamous differentiationmm carries a median survival time of to months following diagnosis7 this poor prognosis is partially due to the fact that over of cases are not discovered until the distant metastasis stage11 while most cases of epithelioid mm have an improved prognosis compared with sarcomatoid mm it can possess pleomorphic features that indicate highly aggressive cancer and shortened expected survival time71012 treatment consists of chemotherapy radiation and surgery although no therapy is curative7in our case ihc staining had an important role in developing the diagnosis of epithelioid mm with pleomorphic features it distinguished the disease from reactive mesothelial hyperplasia and other malignancies with possibly better prognoses10 the right lung lobe mass is not the typical presentation for mm involving the thorax and complicates the clinical picture the presentation of the retroperitoneal mass coupled with diffuse metastatic lesions and atypical involvement of the thorax make this patient™s presentation particularly unique calretinin gata3 and ck56 were positive in the tissue obtained from the retroperitoneal and paraspinal muscle masses which implicated a mesothelial origin for the malignancy calretinin is a strong indicator of epithelioid mm and is useful in distinguishing mm from adenocarcinoma10 recent reports estimate a positive staining for gata3 in mesothelioma in of cases and note a presence in epithelioid mm in one third of cases1013 another important ihc markers for mm is d240 which was negative in our patientwhile not completed in our patient other markers present in mm include the deletion of p16 occurring in of epithelioid mm and the presence of a bap1 mutation101214 0cuhlenhopp et al lev½ m boubl­kov¡ l b¼chler t Å¡imÅ¡a j treatment of malignant peritoneal mesothelioma klin onkol doi1014735amko2019333 sardar mr kuntz c patel t et al primary pericardial mesothelioma unique case and literature review tex heart inst j mutsaers se the mesothelial cell int j biochem cell biol napolitano a carbone m malignant mesothelioma time to translate trends cancer bibby ac tsim s kanellakis n et al malignant pleural mesothelioma an update on investigation diagnosis and treatment eur respir rev doi10118316000617 liang yf zheng gq chen yf song h yin wj zhang l ct differentiation of diffuse malignant peritoneal mesothelioma and peritoneal carcinomatosis j gastroenterol hepatol gill rr imaging of mesothelioma in a tannapfel ed malignant mesothelioma recent results in cancer research vol springer husain an colby tv ordonez ng et al guidelines for pathologic diagnosis of malignant mesothelioma update of the consensus statement from the international mesothelioma interest group arch pathol lab med howlader n noone am krapcho m et al seer cancer statistics review accessed february seercancergovcsr1975_2016 arif q husain an malignant mesothelioma diagnosis arch pathol lab med miettinen m mccue pa sarlomorikala m et al gata3 a multispecific but potentially useful marker in surgical pathology a systematic analysis of epithelial and nonepithelial tumors am j surg pathol attanoos rl churg a galateausalle f gibbs ar roggli vl malignant mesothelioma and its nonasbestos causes arch pathol lab med craighead je epidemiology of mesothelioma and historical background in a tannapfel ed malignant mesothelioma recent results in cancer research vol springer spirtas r heineman ef bernstein l et al malignant mesothelioma attributable risk of asbestos exposure occup environ med bray f ferlay j soerjomataram i siegel rl torre la jemal a global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries ca cancer j clin doi103322caac21492 manzini vde p recchia l cafferata m et al malignant peritoneal mesothelioma a multicenter study on cases ann oncol doi101093annoncmdp shih ca ho sp tsay fw lai kh hsu pi diffuse malignant peritoneal mesothelioma kaohsiung j med sci doi101016jkjms201305003 0c'
Colon_Cancer
" excessive perioperative fluid administration may result in iatrogenic endothelial dysfunction andtissue edema transducing inflammatory markers into the bloodstream colloids remain longer in the circulationrequiring less volume to reach similar hemodynamic endpoints compared to crystalloids thus we tested thehypothesis that a goaldirected colloid regimen attenuates the inflammatory response compared to a goaldirectedcrystalloid regimemethods patients undergoing moderate to highrisk open abdominal surgery were randomly assigned to goaldirected lactated ringer™s solution n or a hydroxyethyl starch n fluid regimen our primaryoutcome was perioperative levels of pro and antiinflammatory cytokines secondary outcome was perioperativelevels of white blood cell count wbc creactive protein crp procalcitonin pct and lipopolysaccharidebindingprotein lbp measurements were performed preoperatively immediate postoperatively on postoperative day onetwo and fourresults the areas under the curve of interleukin il p il p il p and tumor necrosisfactor α p levels did not differ significantly between the groups wbc crp and pct values were alsocomparable lbp although significantly higher in the crystalloid group remained in the normal range patientsassigned to crystalloids received a median iqr amount of ml “ of crystalloid patients assigned tocolloids received ml “ of crystalloid and ml “ of colloid cytokine and inflammatory marker levels did not differ between goaldirected crystalloid and colloidadministration after moderate to highrisk abdominal surgerytrial registration clinicaltrialsgov nct00517127 registered 16th august correspondence barbarakabonmeduniwienacat1department of anaesthesia general intensive care medicine and painmedicine medical university of vienna spitalgasse vienna austriafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cobradovic bmc anesthesiology page of is crucialintroductionvolume replacementin the perioperativeperiod and has great impact on postoperative outcome fluid restriction may cause hypotension and hypoperfusion leading to an dysfunction on the otherhand excessive fluid administration leads to destructionof the endothelial surface layer and consequently to tissue edema with harmful side effects [ ]fluidgdtbasedtherapygoaldirectedonoptimization of flowrelated hemodynamic parametersimproves clinical outcome in low to highrisk surgicalpatients compared to fixed fluid protocols [ ] specifically gdt enhances cardiac performance and gutmicrocirculation while avoiding iatrogenic hyperhydration [ ] in addition to hypervolemia the inflammatoryaggravatesdegradation of the endothelial barrier the socalled glycocalyx inflammation leads to cytokine release andmay thus worsen outcome for example high postoperative interleukin il levels are independently associatedwith postoperative complications response dueto surgicaltraumaso far in most previously performed gdt studieshemodynamic algorithms were based on colloid bolusadministration to improve hemodynamic variables colloids better maintain the intravascular oncotic pressure and provide a higher volume effect when used incase of hypovolemia goaldirected colloid administration reduces intraoperative fluid requirement and improves cardiac performance compared to crystalloids[ ] whether this translates into better outcomespecifically in a decreased postoperative inflammatoryresponse is still a matter of research the comparisonbetween colloid versus crystalloid based fluid regimenswas still lacking therefore we tested the primary hypothesis that perioperative levels of pro and antiinflammatory cytokines il il il and tumor necrosis factor alpha tnf α are reduced by goaldirectedcolloid versus crystalloid administration during the firstfour postoperative days in patients undergoing moderateto highrisk open abdominal surgery in addition wemeasured white blood cell wbc count creactive protein crp procalcitonin pct and lipopolysaccharidebinding protein lbp levelsmaterials and methodsthis prospective randomized controlled trial was conducted at the department of anesthesia intensive caremedicine and pain medicine medical university ofvienna vienna austria the institutional review boardof the medical university of vienna approved it as partof a large multicenter outcome trial evaluating the effectof goaldirected crystalloid and colloid on postoperativecombined morbidity and complications the ethicalcommittee of medical university of vienna viennaaustria provided ethical approval for this trial the trialwas conducted in accordance with the declaration ofhelsinki and good clinical practice and registered atclinicaltrialsgov nct00517127 and eudract “ a written informed consent was obtainedfrom all patients the authors have followed the applicable consort guidelinesfor this single center substudy consecutive eligiblepatients were included patients aged to yearsundergoing elective moderate to highrisk open abdominal surgery with american society of anesthesiologistsasa physical status iiii were included we excludedpatients with severe obesity body mass index bmi kgmˆ’ cardiac insufficiency ejection fraction ef coronary artery disease with angina severe chronicobstructive pulmonary disease autoimmune diseases coagulopathies renal insufficiency creatinine clearance mlminˆ’ or renal replacement therapy symptoms of infection or sepsis and preoperative crp higher than mgdlˆ’ allpatientsreceivedprotocolpreoperativelyantimicrobialprophylaxis using a single dose of a 2nd generationcephalosporine according to our clinicalstandardsanesthetic management wasstandardized standardmonitoring included electrocardiography ecg invasiveblood pressure surveillance pulse oximetry and esophageal core temperature monitoring a central venouscatheter was inserted when deemed clinically necessarywe used balanced anesthesia with sevoflurane none ofour patients received locoregional anesthesia accordingto patients™ requirements additional fentanyl and nondepolarizing neuromuscular blocking were administeredventilatory rate was adjusted to maintain endtidal carbon dioxide partial pressure etco2 of “ mmhgnormothermia was maintained with forced air warmingpatients were randomized to crystalloid lactatedringer™s solution or colloid hydroxyethyl starch voluven fresenius kabi germany grouprandomization was based on computergenerated codesto conceal allocation sealed opaque envelopes wereopened only shortly before induction of anesthesiaall patients were given “ mlkgˆ’ oflactatedringer™s solution during induction of anesthesia followedby “ mlkgˆ’ per hour for maintenance normalized toideal body weight ibw throughout surgery we calculated ibw according to the robinson formula thereafter the randomized fluid crystalloid or colloidwas esophageal dopplerguided cardiac q deltex medical group plc chichester uk according to a standardalgorithm this method is based on corrected aorticflow time ftc as well as stroke volume sv and allowsdistinguishing whether a patient is a fluid responder or 0cobradovic bmc anesthesiology page of not if mean arterial pressure map was below mmhg and no signs of hypovolemia were detected vasopressors were administratedpatients were transferred to postanesthetic care unitpacu or intensive care unit icu at the discretion ofthe attending anesthesiologist fluid management wasstandardized for the first postoperative hours in whichpatients received mlkgˆ’ ibw crystalloid per hourmeasurementsdemographic and morphometric data were recorded aswell as asa score medical history type of surgery andpreoperative laboratory values duration of anesthesiaand surgery were recorded we also recorded intraoperative fluid requirements estimated blood loss transfusion requirements and urinary output for evaluation ofanesthetic management the total amount of fentanylendtidal sevoflurane concentration core temperatureandnotedhemodynamic parameters such as map heart ratehr ftc sv and cardiac output co were recorded at10min intervals the application of phenylephrine usewas notedicu admission werepostoperativethe primary outcomes were the areas under the curveaucs of postoperative levels of pro and antiinflammatory cytokines il il il and tnf α andtheir differences between the crystalloid and the colloidgroup secondary outcomes were aucs of wbc crppct and lpb and their differences between the groupsall blood samples for parameteranalyses were obtainedbefore surgery as baseline values t0 immediately postoperatively t1 as well as on postoperative days onetwo and four t2 t3 and t4 respectively for analysisof il il il and tnf α blood samples were centrifuged within h at g for min and plasma wasimmediately stored at ˆ’ °c for later enzymelinkedimmunosorbent assay elisa analyses the serum concentrations of il il il and tnf α were determined according to the manufacturer™sinstructionshuman sil6 instant elisa human il8nap1 instant elisa and human sil10 instant elisaebioscience vienna austria wwwebiosciencecom human tnf α duoset rd systems minneapolis minnesota wwwrndsystemscom for that purpose opticaldensity was measured with a victor microplate readerat a wavelength of nm multiple testing of sampleson different plates revealed an intraassay variability of for il for il for il for tnf α andan interassay variability of for il for il for il and for tnf αfor investigation of wbc crp pct and lpb separateblood samples were obtained their analysis took placeimmediately after blood sampling as routine laboratoryanalysessample size calculation and statistical analysissample size calculations for our trial were based on thestudy of steppan and colleagues they observed amean standard deviation sd of pgmlˆ’ in il h after surgery in abdominal surgery patients assuming a similar coefficient of variation sdmean for each of the four cytokines primarily plannedfor evaluation in our study we calculated a total of patients in order to obtain a power to detect a reduction in any of the cytokines at an overall significance level with powergroups were primarily compared for balance in patients™ demographic data intraoperative characteristicsand postoperative variables absolute standardized differences asd were calculated for patients™ baseline covariates subsequent measurements ofintraoperativeparameters were first averaged within each patient andthen averaged among the patients in each treatmentgroup for descriptive analysis normal distribution wasassessed with qq plots and kolmogorowsmirnow testsnormally distributed variables were with unpaired twotailed ttests otherwise the in case of normally distributed values wilcoxon ranksum test was used for notnormally distributed continuous data paired comparisons between baseline data and postoperative data wereperformed with paired sample ttest or wilcoxonsignedrank test as applicable nominal data were analyzed with chisquare or fisher™s exact test for low expected cell counts data were presented as means ± sdmedians iqr or as numbers percentage as applicableadjustment for multiple testing was performed with thebonferroni method a p value was considered statistically significantanalysis was conducted with spss software version armonk ny ibm corp r for macintosh version321 r core team r a language and environmentfor statistical computing r foundation forstatistical computing vienna austria was used to calculate asdresultsa total of patients were included between november and october in the colloid group and in the crystalloid group fig at t0 all values weremeasured overall in the crystalloid group and inthe colloid group of the preplanned blood sampleswere collected and analyzedpatient™s baseline characteristics did not differ exceptfor height bmi with a slightly higher bmi in the colloidgroup type of surgery and crp also higher in the colloid group table duration of anesthesia and surgerywere comparable between both groups patients assignedto crystalloid administration received a median of ml “ crystalloids whereas patients assigned 0cobradovic bmc anesthesiology page of fig consort patient flow chartto the colloid group received ml “ ofcrystalloid solution and ml “ of colloidsblood loss transfusion requirements and urinary outputdid not differ between the groups anesthetic management map and hr did not differ between the groupsftc sv and co were significantly higher in the colloidgroup compared to the crystalloid group ftc ms“ versus ms “ p sv ml“ versus ml to p and co ± lminˆ’ versus ± lminˆ’ p thenumber of patients requiring vasopressor support wascomparable between groups the incidence of postoperative icu admissions did not differ in the crystalloid versus in the colloid group p table baseline values of il il il and tnf α in thecrystalloid group were comparable to values in the colloid group il pgmlˆ’ “ versus pgmlˆ’ “ p il pgmlˆ’ “ versus pgmlˆ’ “ p il pgmlˆ’ “ versus pgmlˆ’ “ p tnf α pgmlˆ’ “ versus pgmlˆ’ “ p immediatepostoperative values of il and were significantlyhigher compared to baseline values in both groupsp for all measurements while tnf α did notshow any significant increase in the crystalloid p and the colloid group p fig aucs of il il il and tnf α did not differ significantly between the groups table wbc values at baseline were glˆ’ “ in thecrystalloid versus glˆ’ “in the colloidgroup p crp pct and lbp baseline valueswere also comparable in both groupscrp mgdlˆ’ “ versus mgdlˆ’ “p pct ngmlˆ’ “ versus ngmlˆ’ “ p lbp mcglˆ’ “ versus mcglˆ’ “ p immediate postoperative values of wbc and pctwere significantly higher compared to the baseline valuesin both groups p for all measurements fig 0cobradovic bmc anesthesiology page of table baseline characteristicsage yrsweight kgheight cmbmi kgmˆ’ gender no menwomenasa score no iiiiiimedical history no pulmonary diseasecardiovascular diseasediabetes type idiabetes type iitype of surgery no colorectalliverpancreaticcrystalloidsn ± ± ± ± colloidsn ± ± ± ± asd preoperative laboratory valuescrp mgdlˆ’ ± ± patient characteristics data are presented as means ± sd or as counts for thecategorical outcomesabbreviations asd absolute standardized differences absolute difference inmeans or proportions divided by the pooled sd asd values of and represent small median and large differencesbmi body mass index m male f female asa american society ofanesthesiologists crp creactive protein sd standard deviationthe aucs for wbc crp pct and lbp for the timeperiods from t1 to t4 did not differ significantly between the groups fig table however lbpshowed significantly higher levels in the crystalloidgroup in the immediate postoperative period comparedto the colloid group mcglˆ’ “ versus mcglˆ’ “ p at all other postoperativetime points there were no significant differences betweenthe groups fig discussionthis trial is a substudy of a large multicenter randomized trial evaluating the effect of goaldirected crystalloidversus goaldirected colloid fluid administration on acomposite of serious complications after moderate tohighrisk open abdominal surgery the overall trial concluded that colloids did not decrease the composite ofmajorincomplicationsare ourresultsconcordance as they did not show any differences inperioperative pro and antiinflammatory markers between a crystalloid and a colloid fluid regimendespite multimodal care and enhanced recovery programs it still remains challenging to blunt the inflammatory response to surgery systemic inflammationafter abdominal surgery impairs outcome and thereforemany attempts have been made to alter the inflammatory response several factors influence the perioperative inflammatoryresponse such as the underlying disease type and invasiveness of surgery as well as type of anesthesia [“] themost important factor is the magnitude of surgical traumaand tissue damage which induce proliferation and activation of immune competent cells in turn triggering cytokine and inflammatory marker release so far veryfew trials have specifically investigated the influence offluid therapy and differences in terms of the type of fluidon the extent of inflammatory marker releaseto investigate the potential influence of goaldirected hydroxyethyl starch versus a lactated ringer™s solution fluid regimen on inflammatory response pro il il and tnf α and antiinflammatory cytokine il serum levels were measured during the perioperativeperiod additionally we measured wbc crp pct andlbpgenerally the most commonly measured biomarkersare crp and wbc if levels of crp are above mgdlˆ’ after postoperative day four a postoperative infection can be suspected crp levels in our studygroups increased on the first postoperative day droppingon the fourth postoperative day to nearly mgdlˆ’ inboth study groupswbcs are an imprecise marker to detect postoperativecomplications after major abdominal surgery amore sensitive parameter in predicting postoperativecomplications after major abdominal surgery is il surgical trauma and hypoperfusion of the colon aremain sources of il release in colorectal surgery noblett demonstrated that gdt during elective colorectal surgery significantly reduced il levels in comparison to a control group yates showed no differencesof il and il levels between goaldirected colloidand crystalloid fluid therapy during the first h in asubgroup of patients undergoing colorectal surgery although patients in the crystalloid group received significant more volume amount as compared to the colloid groupthere was no significant difference inhemodynamic variables our patients showed similar courses of il and il levels in the immediatepostoperative period in contrast to the trial of yateswho measured cytokine levels up to the first h aftersurgery we extended our measurement period to fourpostoperative days we showed comparable circulating 0cobradovic bmc anesthesiology page of table intraoperative dataduration of anesthesia minduration of surgery minfluid managementtotal fluid intake mlacrystalloid mlcolloid mlestimated blood loss mltransfusion yesno urinary output mlanesthesia managementfentanyl mcgtwa et sevoflurane core temperature °cicu admission yesno hemodynamictwa map mmhgtwa hr beatsminˆ’twa ftc mstwa sv mltwa co lminˆ’ phenylephrine yesno crystalloidsn ± ± “ “ “ “ “ [ ] “ ± ± ± “ “ ± colloidsn ± ± “ “ “ “ [ ] “ “ ± ± ± “ “ ± p value ° ° °° °°intraoperative data are presented as means ± sd medians iqr or as counts for the categorical outcomes means were compared with an unpaired twosided ttests ormannwhitneyu tests as appropriate medians with wilcoxon ranksum tests and counts with chisquare or fisher™s exact tests ° represents statisticalsignificance p abbreviations et end tidal icu intensive care unit twa time weighted average map mean arterial pressure hr heart rate ftc corrected flow time sv strokevolume co cardiac output sd standard deviationa total fluid intake includes baseline fluid boluses antibiotics analgesics and additional fluid administered at the discretion of attending anesthesiologistfig ad pro and antiinflammatory cytokines il il il and tnf α over time a il b il c il and d tnf α data arepresented as medians iqr abbreviations il “ interleukin tnf α “ tumor necrosis factor alpha pod “ postoperative day 0cobradovic bmc anesthesiology page of table areas under the curve of inflammatory markerspcrystalloidsn “ “ “colloidsn “ “ “ “ “ “ “ “auc il pgmlˆ’ 1dauc il pgmlˆ’ 1dauc il pgmlˆ’ 1dauc tnf α pgmlˆ’ 1d “auc wbc glˆ’1dauc crp mgdlˆ’ 1dauc pct ngmlˆ’ 1dauc lbp mcglˆ’ 1dtable areas under the curve of il il il and tnf α as well as wbccrp pct and lbp are presented as medians iqr medians were comparedwith wilcoxon ranksum testsabbreviations il interleukin tnf α tumor necrosis factor alpha wbc whiteblood cells pct procalcitonin lbp lipipopolysaccharidebinding protein “ “ “ “il and il levels between a gdt crystalloid and colloid administration these surrogates of inflammatoryresponse imply that gut perfusion during surgery waswell preserved with both types of fluid and suggest thatthe type of fluid might be of minor importance as longas the fluid is administered in a goaldirected fashionthe fact that tnf α levels in both groups remainedstable over the entire measured period further supportsour theory the course of tnf α levels during the perioperative period was in accordance with the study ofin which fluid therapy was guided withszakmanypicco versusin majorvenous pressurecentralabdominal surgery in patients at risk for postoperativecomplications as tnf α per se triggers glycocalyxdegradation we anticipate that tnf α did not influence glycocalyx shedding and thus possible fluid shifts inour study populationpct is an early predictive marker for systemic inflammation after abdominal surgery values above ngmlˆ’ are associated with postoperative complicationssuch as pneumonia or anastomotic leakage in ourstudy median pct levels did not exceed ngmlˆ’ atany measured time point these results are in concordance with our main study where infectious complications rate were held low and did not differ between thegroups moreover as pct production can also beinduced by tissue hypoperfusion we might assume thatgoaldirected fluid administration contributed to lowpct values by optimizing cardiac performance furthermore we measured lbp a prognostic markerfor bacterial infections patients in the crystalloidgroup showed significantly higher levels immediatelyafter surgery however the measured values remainedwithin the normal range therefore this difference ismost likely not to of clinical importancethe vascular endothelium is one of the earliest sitesinvolved in the inflammatory response syndrome an adequate perioperative fluid management has a major impact on the integrity of the glycocalyx with goaldirected fluid management individualized and time appropriate fluid resuscitation can be achieved enablingfig ad inflammatory markers wbc crp pct and lbp over time a wbc b crp c pct and d lbp data are presented as medians iqrabbreviations wbc “ white blood cells crp “ creactive protein pct “ procalcitonin lbp “ lipopolysaccharidebinding protein pod “postoperative day ˜… represents significant difference in lbp in the immediate postoperative period between the crystalloid and the colloidgroup p 0cobradovic bmc anesthesiology page of preservation of endothelial surface layer and sufficientan perfusion thus improving postoperative outcomesafter major surgery patients in the colloid group received significantly lessfluid ml confirming the previously publishedfluid sparing effect of colloids however clinical significance of this difference may be questionable during aperioperative period of nearly five hours further ourhemodynamic data showed significantly higher values ofsv and co with colloid administration though the absolute difference of ml in sv most likely has onlylimited clinical relevance it might very well be that thesignificant differences in sv and co are the result ofour number of patients included in this substudyassurrogatesfirst limitation of our study is that we measured inflammatory markers that reflect the inflammatory responseand not direct markers ofglycocalyx degradation like syndecan1 therefore wecannot draw any s about the preservation ofthe endothelial surface layer in our patients secondlywe did not control postoperative fluid management during the postoperative followup period a further limitation isthe time between patient enrolment andsubmission of our current results due to the fact thatthe main trial has been published recently a delay of oursubmission occurred nevertheless our results canstill be extrapolated to current clinical practicein summary goaldirected hydroxyethyl starch administration did not attenuate the inflammatory responseexpressed by cytokine levels of il il il and tnfα in patients undergoing moderate to highrisk open abdominal surgery wbc crp and ptc values did not differ between the different fluid regimes as wellabbreviationsasa american society of anesthesiologists asd absolute standardizeddifference auc area under the curve bmi body mass index co cardiacoutput crp creactive protein ecg electrocardiography ef ejectionfraction elisa enzymelinked immunosorbent assay etco2 endtidalcarbon dioxide ftc corrected flow time gdt goaldirected therapyhr heart rate ibw ideal body weight icu intensive care unitil interleukin iqr interquartile range lbp lipopolysaccharidebindingprotein map mean arterial pressure pacu postanesthetic care unitpct procalcitonin picco pulse contour cardiac output spss statisticalpackage for the social sciences sv stroke volume tnf α tumor necrosisfactor alpha twa time weighted average wbc white blood cell countacknowledgementsassistance with this we thank bianca tudor md department ofanesthesia intensive care medicine and pain medicine medical university ofvienna spitalgasse vienna austria for her support in laboratoryskills performing elisasauthors™ contributionsall authors have read and approved the manuscript mo patientrecruitment data acquistion and prepratation of the manuscript ak studyprotocol writing and preparation of the manuscript bk study protocolwriting and preparation of the manuscript statistical analyisis gr dataanalysis revision of the manuscript ok data analysis preparation of themanuscript oz patient recruitment data acquisition data managementab patient recruitment data acquisition revsion the manuscript cr dataacquistion revision of the manuscript as patient recruitment revision ofthe manuscript ef study protocol writing and preparation of the manuscriptfundingmedical university of viennapartially funded by fresenius kabi deltex medical provided oesophagealdoppler monitors and disposablesthe sponsors were not involved in protocol development data acquisitionor data analysisavailability of data and materialsthe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestbarbarakabonmeduniwienacatethics approval and consent to participatethe main trial was approved by the local ethics committee of the medicaluniversity of vienna in ek and was registered atclinicaltrialsgov nct00517127 and eudract “ a writteninformed consent was obtained from all patients prior to participationconsent for publicationnot applicablecompeting intereststhe authors declare no competing interestsauthor details1department of anaesthesia general intensive care medicine and painmedicine medical university of vienna spitalgasse vienna austria2department of outcomes research and general anesthesiologyanesthesiology institute euclid avenue cleveland clinic cleveland ohusa 3department of anesthesiology and general intensive care franziskushospital nikolsdorfergasse vienna austria 4department ofgynecology klinik ottakring montleartstrasse vienna austria5department of surgery medical university of vienna spitalgasse vienna austriareceived july accepted august referencesbrandstrup b tonnesen h beierholgersen r effects of intravenousfluid restriction on postoperative complications comparison of twoperioperative fluid regimens a randomized assessorblinded multicentertrial ann surg “ myles ps bellomo r corcoran t restrictive versus liberal fluidtherapy for major abdominal surgery new engl j med “chappell d jacob m hofmannkiefer k conzen p rehm m a rationalapproach to perioperative fluid management anesthesiology “lowell ja schifferdecker c driscoll df benotti pn bistrian brpostoperative fluid overload not a benign problem crit care med “sun y chai f pan c romeiser jl gan tj effect of perioperative goaldirected hemodynamic therapy on postoperative recovery following majorabdominal surgerya systematic review and metaanalysis of randomizedcontrolled trials critical care calvovecino jm ripollesmelchor j mythen mg effect of goaldirected haemodynamic therapy on postoperative complications in lowmoderate risk surgical patients a multicentre randomised controlled trialfedora trial br j anaesth “noblett se snowden cp shenton bk han af randomized clinical trialassessing the effect of doppleroptimized fluid management on outcomeafter elective colorectal resection br j surg “ 0cobradovic bmc anesthesiology page of alphonsus cs rodseth rn the endothelial glycocalyx a review of thevascular barrier anaesthesia “ pearse rm harrison da macdonald n effect of a perioperativecardiac outputguided hemodynamic therapy algorithm on outcomesfollowing major gastrointestinal surgery a randomized clinical trial andsystematic review jama “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationskimberger o arnberger m brandt s goaldirected colloidadministration improves the microcirculation of healthy andperianastomotic colon anesthesiology “kolarova h ambruzova b svihalkova sindlerova l klinke a kubala lmodulation of endothelial glycocalyx structure under inflammatoryconditions mediat inflamm rettig tc verwijmeren l dijkstra im boerma d van de garde emnoordzij pg postoperative interleukin6 level and early detection ofcomplications after elective major abdominal surgery ann surg “ gan tj soppitt a maroof m goaldirected intraoperative fluidadministration reduces length of hospital stay after major surgeryanesthesiology “jacob m chappell d rehm m clinical update perioperative fluidmanagement lancet “feldheiser a pavlova v bonomo t balanced crystalloid comparedwith balanced colloid solution using a goaldirected haemodynamicalgorithm br j anaesth “ orbegozo cortes d gamarano barros t njimi h vincent jl crystalloidsversus colloids exploring differences in fluid requirements by systematicreview and metaregression anesth analg “kabon b sessler di kurz a effect of intraoperative goaldirectedbalanced crystalloid versus colloid administration on major postoperativemorbidity a randomized trial anesthesiology “ robinson jd lupkiewicz sm palenik l lopez lm ariet m determination ofideal body weight for drug dosage calculations am j hosp pharm “steppan j hofer s funke b sepsis and major abdominal surgery leadto flaking of the endothelial glycocalix j surg res “ wilmore dw from cuthbertson to fasttrack surgery years of progress inreducing stress in surgical patients ann surg “ desborough jp the stress response to trauma and surgery br j anaesth“ veenhof aa sietses c von blomberg bm the surgical stress responseand postoperative immune function after laparoscopic or conventional totalmesorectal excision in rectal cancer a randomized trial int j color dis “ gilliland he armstrong ma carabine u mcmurray tj the choice ofanesthetic maintenance technique influences the antiinflammatory cytokineresponse to abdominal surgery anesth analg “ wichmann mw huttl tp winter h immunological effects oflaparoscopic vs open colorectal surgery a prospective clinical study archsurg “ watt dg han pg mcmillan dc routine clinical markers of themagnitude of the systemic inflammatory respon
Colon_Cancer
chronic respiratory diseases are highly prevalent worldwide and will continue to rise in theforeseeable future despite intensive efforts over recent decades the development of novel and effectivetherapeutic approaches has been slow however there is new and increasing evidence that communities ofmicroanisms in our body the human microbiome are crucially involved in the development andprogression of chronic respiratory diseases understanding the detailed mechanisms underlying this crosstalk between host and microbiota is critical for development of microbiome or hosttargeted therapeuticsand prevention strategies here we review and discuss the most recent knowledge on the continuousreciprocal interaction between the host and microbes in health and respiratory disease furthermore wehighlight promising developments in microbiomebased therapies and discuss the need to employ moreholistic approaches of restoring both the pulmonary niche and the microbial communityreceived nov accepted after revision april copyright ers this version is distributed under the terms of the creative commons attribution noncommercial licence 10118313993003023202019eur respir j 0crespiratory basic science r gosens introductionin the past decade we have learned that the lungs previously considered sterile in fact harbour a dynamicecosystem of diverse bacteria fungi and viruses this lung microbiota is detectable in health [ ]altered in disease predictive of disease outcomes [ ] and correlates with variations in host immunity[ ] recent insights based on studies in both humans and mice are that the baseline immunetone of the lungs even in health is closely linked to the local microbial milieu this hypothesis thatthe œimmune tone in the airways and alveoli is at least in part regulated by the microbiota is a radicaldeparture from our conventional dichotomous understanding of lung immunity dormant in healthactivated in infection next to known changes in the inflammatory milieu of the lungs most lung diseasessuch as asthma copd cystic fibrosis idiopathic pulmonary fibrosis ipf andrecently lung cancer have been associated with a microbial dysbiosis in the lung however it isunknown if changes in the bacterial composition drive disease pathogenesis or if they are rather areflection of alterations of the ecological niche [ ] thus it is of utmost importance to understand theunderlying molecular mechanisms at the host“microbiome interface to develop novel targeted therapies orpreventative approacheshere we discuss the impact of host“microbiome crosstalk on respiratory health and disease whilefocusing on how the local microbiota and the host interact at the epithelial surface furthermore wereview current therapeutic approaches and suggest a more holistic approach for treating lung disease inthe future this review is a followup of presentations and discussions at and after the ers researchseminar œcrosstalk in the lung microenvironment implications for understanding chronic lung diseaseberlin february the mucosal niche in the lungall external interfaces of the human body such as gut skin reproductive and respiratory tracts arecolonised by a distinct microbial flora the microbial communities differ at the various body sites dueto local factors eg oxygen carbon dioxide ph nutrients host defence factors inhaled pollutants thatshape the niche as examples the lumen of the lower gastrointestinal tract represents a lowoxygennutrientrich environment and is thus commonly populated by highabundance communities of anaerobicanisms by contrast the skin is a lownutrient environment directly exposed to environmental oxygenand is thus more commonly populated by relatively sparse communities of oxygentolerant bacteria thusthe local environment is a crucial determinant of the formation of microbial communities early indevelopment but also at later stages antimicrobial peptidesand otherclearance production ofconsequently the lung microenvironment creates a special ecological niche for microbes that differs fromother body sites and will presumably influence nichespecific colonisation accordingly airway epithelialcells are a principle contributor in shaping this niche as they are strategically located to be the first contacttissues various mechanisms arebetween inhaled substances including microanisms and hostemployed by the airway epithelium in host defences against infectionincluding its barrier functionmucociliaryandantiinflammatory mediators and ability to transport eg polymeric iga and igm from the basal to theapical side of the epithelium through the polymeric immunoglobulin receptor pigr [“] it is likelythat similar mechanisms contribute to the formation of nichespecific communities along the respiratorytract and such local conditions are crucial for allowing beneficial microbiota to persist at epithelialsurfaces the involvement of such host“microbiota interactions in disease is wellillustrated in cysticfibrosis in cystic fibrosis mutational dysfunction of the cystic fibrosis transmembrane regulator cftrprotein results in a reduction of anion mostly bicarbonate and chloride exchange across the epithelialsurface resulting in a dehydrated surface and sticky mucus this mucus cannot be readily cleared from theairways which helps to explain why cftr mutations are associated with alterations of the residentmicrobiota including frequent colonisation with staphylococcus aureus and pseudomonas aeruginosa asrecently reviewed in however we are still lacking knowledge on the underlying mechanisms of theniche alterations in more complex genetic lung diseases such as asthma and copdsubstances proadditionallyallowing it to mount appropriate responses or develop tolerance [ ]the epithelium transmits environmental and microbial signals to the immune systemvarious mechanisms of sensing microbial presence include pattern recognition receptors such as thetolllike receptors and other processes not mediated via classical receptormediated signalling egthe integrated stress response upon sensing microbial environmental or endogenous challengesepithelial cells mount active responses by increasing defence molecules such as antimicrobial peptidescytokines and chemokines these enhance the defence against respiratory pathogens while simultaneouslychanging the ecological niche of complex microbial communities indicating that the properties of theecological niche encountered by microanisms changes as a result of environmental exposures10118313993003023202019 0crespiratory basic science r gosens anatomical differences along the respiratory tract such as differences in epithelial cell composition shapethis ecological niche while the epithelium contributes to local conditions that shape the microbiotaepithelial exposure to microbes and their products has marked effects on its function consequentlyhuman epithelia and the microbiota have developed interactions that are of mutual benefit responding topathogens and tolerating innocuous substances this concept is important to understand how inhaledenvironmental triggers regulate immunity since microbial components contribute to the response toenvironmental exposures such as farm and geogenic earthderived dust however how theepithelium integrates these microbial and nonmicrobial signals into a finetuned response is incompletelyunderstoodin order to transmit signals from the environment epithelial cells use sophisticated communicationsystems with other lung cell types the airway epithelium and that of gut and skin plays key roles intransmitting signals from microanisms and environmental stimulito instruct antigenpresentingdendritic cells to direct immunity towards inflammation or tolerance epithelial cells interact not onlywith other immune cells such as macrophages neutrophils innate lymphoid cells and tcells but alsowith structural cells such as fibroblasts airway smooth muscle cells and endothelial cells via a plethora ofdifferent mechanisms figure [ ] this array of interactions with environmental triggersmicroanisms and lung cells allows epithelial cells to orchestrate host defence and immunity and tomaintain epithelial integrity and mediate pathologic airway remodelling figure establishment and maintenance of the lung microbiotathe establishment of the lung microbiota likely occurs in the first days and weeks of life although earlyhighprofile reports suggested the presence of a œplacental microbiome that could influence prepartumlung development subsequent wellcontrolled studies have failed to detect bacterial signals distinctfrom contaminating dna present in negative controls the lung microbiota of newborn mice isbelow the limit of detection via quantitative pcr and increase in total burden in the following days andweeks [ ] in human infants the composition of the respiratory microbiome seems to mature in apredictable wellcharacterised pattern during the first year oflife besides the special nichecharacteristics of the lung earlylife respiratory microbiota are influenced by mode of delivery vaginalversus caesarean method of feeding breast versus bottle and exposures siblings daycare attendance provocatively earlylife respiratory microbiota may predict subsequent susceptibility to respiratorytract infections [ ] suggesting roles of the local microbes in immune homeostasis and resistance topathogens in contrast being exposed to an increased microbial diversity during childhood promotes thedevelopment of balanced immunity and is protective against inflammatory responses to allergens andasthma development [ ] this protection is partly associated with distinct farm dust which in vitroincreases epithelial barrier function and antiviral defences once established the composition of the lung microbiome is determined by three ecological factorsimmigration elimination and relative growth rates of community members figure in health lungcommunities are sparse and dynamic largely determined by the equilibrium between immigration viamicroaspiration and elimination via cough mucociliary clearance and immune defences littleevidence supports the presence of resident lung bacteria in health that reproduce and survive selectivepressures [ ] nevertheless the transient and dynamic communities detected in health are largelyviable and exert a detectable effect on the immune constitution of the lower respiratory tract [ ]the establishment of a diverse respiratory microbial flora is influenced by many different factors there isa finetuned balance between tolerance of commensal or œbeneficial bacteria at the epithelial surface andthe development of active immune responses to pathogens notably this balance is regulated by both hostand microbederived signals sudden shifts in this tightly controlled equilibrium such as outgrowth ofspecific pathogens or for example virally induced damage to the airway epithelium destroying the barrierand inducing local immune responses can have detrimental effects on both the host and the microbiomeand might therefore contribute to the pathogenesis of respiratory diseasesrole of the environment in shaping the lung microbiomeenvironmental influences play pivotal roles in the development of lung diseases this might be due to directeffects on the host epithelial barrier and immune responses but most known risk factors such as cigarettesmoke air pollution [ ] viral infections and di so directly impact the microbiota [ “]thus a combination of both might be critical for disease developmentfor example one of the most studied inhaled toxins cigarette smoke supposedly has a detrimental effecton both the host and microbial communities prolonged cigarette smoke exposure contributes to increasedbaseline inflammation in the airways and epithelial remodelling towards goblet cell hyperplasia and areduction in cilia and ciliary activity and can reduce the antimicrobial defence provided by the airway10118313993003023202019 0crespiratory basic science r gosens mucociliaryclearingmicroaspirationairway regionbacterial metabolitesouter membrane vesiclesquorumsensing moleculesalveolar regionantimicrobial peptidesciliary beatingmucuscytokineschemokinesgrowth factorsextracellular vesiclescritically altered in chronic lung diseasebacteriarespiratory virusclub cellciliated cell goblet cell alveolar type cell alveolar type cellfibroblastmucus layer basal celldendritic cell macrophagesmooth muscle cellcapillaryfigure host“microbiome crosstalk in the lung microenvironment the lung microenvironment consists of different cell types depending onthe location in the proximaltodistal airway tree the epithelial layer in larger airways is constantly exposed to a variety of different microbes ofthe local microbiota while the composition of the latter is influenced by host factors such as elimination via mucociliary clearance it alsodepends on the competition among the microbial inhabitants it is now evident that there is a complex crosstalk between host and microbes inthis environment accordingly bacterial metabolites or outer membrane vesicles can influence the host status while antimicrobial peptides orcytokines can shape the composition of the microbiota as virtually all of these single factors are altered in chronic respiratory disease it is ofutmost importance to appreciate and assess the complexity of this system in future studiesepithelium in patients with copd it has been shown to be associated with reduced lung bacilli andincreased haemophilus influenzae and streptococcus pneumoniae but the largest study to date inœhealthy smokers has not found a significant effect of cigarette smoke on the lung microbiome thus it is intriguing to speculate that cigarette smoke primarily affects the host system which over timeand upon disease copd development in susceptible individuals may also affect the microbiomeanother example of environmental influence on respiratory disease is diet which has profound effects onboth microbiota and health even in the short term the intake of dietary fibre induces similarbeneficial microbiota changes in the lung and gut while lowfibre diets change the gut microbialcommunity over multiple generations in mice highfibre diets have beneficial effects in pregnant miceand suppress allergic airway disease aad in mothers and their offspring this also highlights theimportance of crosstalk between the gut and lung highfibre diets induce the production of shortchainfatty acids by gut bacteria which are transported systemically to the lung where they exertantiinflammatory actions and ameliorate aad in mice in contrast a lipidrich di ters themicrobiota and promotes metabolic inflammation and has been associated with premetastatic nichedevelopment in lung cancer however the detailed mechanisms of action remain unclearalong with bacteria common major respiratory viruses including respiratory syncytial virus rhinoviruscoronaviruses influenza and adenoviruses are part of the respiratory microbiome and contribute tothe pathogenesis of chronic respiratory diseases this may result from complex interactions of viruses withthe host™s immune system and the microbiota including other pathogens these interactions can influencethe prevalence of bacterial pathogens by increasing the expression of adhesion molecules on therespiratory epithelium damaging respiratory epithelial cells compromising barrier functionimpairingmucociliary clearance and altering host immunity and the lung microenvironment [“] interestinglyit has been suggested that viruses and bacteriophages induce consistent and reproducible changes inrespiratory microbiomes in chronic disease but not the healthy state and have been shown to increasemicrobial diversity in the nasopharynx [ ] furthermore fungi such as aspergillus spp contributeto the pathogenesis of chronic respiratory diseases as pathogens on their own but also by activation of theimmuneinteractions with microbiota particularly withnontuberculous mycobacteria [ ]through theirsystemand probablyimportantly the absence of certain members from the microbiome can have detrimental influences on lunghealth this is illustrated by the relative disappearance of parasitic worms which have been a constant partof our gut microbiome and have even been found in fossils from the period of jawed fish in fact our10118313993003023202019 0crespiratory basic science r gosens œmodern immune system developed in the continuous presence of helminths which may explaintheir important regulatory role in immunity typically worms induce type2 immune responses which areconsidered instrumentalin host defence against these parasitic infections however worm infectionsinduce regulatory responses that are aimed to suppress immune responses directed at worm antigenswhich allows worms to live in their host for years additionally this regulatory response has a bystandereffect by promoting allergen tolerance this is illustrated by studies in mouse models in whichvarious helminth infections have been shown to provide protection against the development of allergicasthma the range of helminthinduced protective mechanisms includes the inhibition of interleukinil33 in the airways and the induction of regulatory tcells [ ] bcells [ ] andmacrophages intriguingly the presence of helminths also affects the composition of the bacterialcommunity which might be important for the protection against allergic airway disease in mice niche and microbiome alterations in respiratory diseasein disease the ecology of the lower respiratory tract changes dramatically the airways and alveolinormally inhospitable to bacterial reproduction are radically altered by the influx of nutrientrichoedema and mucus establishment of stark oxygen gradients surge of bacterial growthpromotinginflammatory responses [ ] and impairment of local host defences [ ] thus it is unsurprisingthat crosssectional studies have identified altered lung microbiomes in established lung diseases aschanges in ecological niches supposedly result in different microbial communities due to selective pressurethe composition of the lung microbiome then becomes increasingly determined by the relative growthrates of its constituentswhile there are some studies on the role of the microbiome in ipf and lung cancer mostknowledge is based on studies of chronic inflammatory respiratory diseases such as asthma copd andcystic fibrosis generally those patients have increased susceptibility to infections and exacerbations thatagain affect the microbiome [ ] h influenzae moraxella catarrhalis and s pneumoniae areassociated with the development of severe asthma and copd they modify the lung microbiome anddrive inflammation oxidative stress symptoms and exacerbations [ “] which may form a viciouscircle perpetuating the disease m catarrhalis and hinfluenzae in particular induce neutrophilicinflammation more severe disease and steroid resistance [“] furthermorein asthma alteredrespiratory microbiome profiles are associated with asthma phenotype and severity responses toallergens and treatment in cystic fibrosis and bronchiectasis haemophilus and pseudomonas spp increasingly dominate the lungmicrobiota [ ] genetic association studies in phe508del cystic fibrosisaffected homozygousindividuals have shown associations of single nucleotide polymorphisms with important disease featuresrelated to bacterial colonisation for example gene variants of human leukocyte antigen class ii genesimplicating a role for tcell and bcell immunity associate with age of onset of persistent p aeruginosainfection indicating the role of the host™s immune system in selecting a distinct microbiota like the bacterial composition ofthe lung microbiome antiviral responses are altered in chronicrespiratory diseases in asthma where airway epithelial repair in response to viral infection is aberrant dueto changes in genes regulating epithelial barrier function and repair the airway epithelium hasreduced innate immunity to common viruses such as rhinovirus [ ] this is due to reduced type iand iii interferon and increased mirna levels [ ] transforming growth factor tgfβ a cytokinecommonly upregulated in asthma suppresses airway epithelialinnate immune responses throughsuppressor of cytokine signalling socs1 and socs3 contributing to impaired antiviral immunity this impaired antiviral immunity may also alter the bacterial microbiome and contribute to coinfectionsby bacteria rhinovirus infection can also impair the phagocytosis of bacteria by alveolarmacrophages which can lead to bacterial outgrowth in copd murine studies showed thatinfluenzainfected epithelial progenitors of the distal lung exhibited severely impaired renewal capacity dueto virusinduced blockade of βcatenindependent fgfr2b signalling this could contribute toimpaired repair of the distal lung in copd although its involvement in human disease is still unclearthus viral and bacterial infections and the inability to resolve them may aggravate impaired airway andalveolar repair combined with a heightened airway andor alveolar immune tone aberrant repairmechanisms following pathogen exposures in vulnerable individuals might represent a mechanism for howhost“microbiome interactions contribute to disease progression it is currently unclear if alterations in thelung microbiome can also precede lung structural changes and inflammation and thereby contribute toonset of diseaseas discussed earlier the relationship between lung dysbiosis and lung inflammation is bidirectionaldisordered lung communities trigger epithelial and luminal inflammation further altering growth conditionsof the lung microenvironment chronic inflammation and perpetuating dysbiosis [ ] a common10118313993003023202019 0crespiratory basic science r gosens isthe outgrowth oftheinflammatory lung conditionsfinding acrossinflammationassociatedproteobacteria phylum and in mice the lung microbiome has been shown to be associated withpulmonary levels of the innate cytokine il1α additionally alterations in the microbiome of the gutmight influence the immune tone of the lung which is shown by the development of more severeexperimental asthma in germfree mice [ ] or mice with disturbed microbiomes due to earlylifeantibiotic treatment [ ] in contrast the absence of microbiota in germfree mice or due toantibiotic treatment has recently been shown to protect mice with kras mutations and p53 loss from lungcancer development according to the authors this effect was due to the induction of il1β andil23 through the microbiota that led to il17 production by γδt cells and tumorigenesisin humans copd patients are two to three times more likely to have crohn™s colitis and of peoplewith inflammatory bowel disease also have pulmonary inflammation [“] the gastrointestinal tracthas by far the highest microbial content and thus an interaction of gut microbiota and their metaboliteswith the gastric mucosa affects systemic immunity which may in turn impactthe lung consequently alterations in gut microbiota and physiology might contribute to respiratory disease andvice versa possibly via the gut“lung axis however it is important to note that germfree breeding and tosome extent antibiotic treatment will affect both the gut and the lung microbiome so it is still notcompletely clear whether these effects can fully be explained by gut“lung crosstalk or if they areinfluenced by aberrant local microbiotaimmune crosstalk along this line in mice the lung immune tonehas been suggested to be more correlated to lung bacteria than gut bacteria in contrast in mice colitisinduced pulmonary pathology was associated with increased inflammation andgramnegative bacterial endotoxins in the lung that probably emanate from the gut furthermorecigarette smokeinduced experimental copd models [“] indicate that reduced gas exchange andhypoxia in the lung is associated with hypoxia in the gut causing colon remodelling cell deathinflammation and impaired barrier function additionally activation of nodlike receptors bybacteria in the gut increase production of reactive oxygen species by alveolar macrophages suggesting thatthe gastrointestinal microbiome contributes to oxidative stress in the lung conversely oralapplication of beneficial probiotic bacteria bifidobacterium and lactobacillusbased reduced airwayinflammation and emphysema in cigarette smokeexposed mice a large canadian study reported that four bacterial genera lachnospira veillonella faecalibacterium androthia were reduced in the faeces of human infants that subsequently developed asthma and inoculating ahuman faecal microbiome supplemented with these four taxa into germfree mice partially protected theirf1 progeny from experimentally induced aad thus there are encouraging studies in mousemodels highlighting cause and effect of the gut“lung axis in respiratory disease however findings firstneed to be validated in humans before proposing this crosstalk as a treatable trait for respiratory diseasein summary at the host“microbiome interface disordered communities are probably both cause andeffects of host inflammation however given the close reciprocal interactions between the microbiome andhost at all times it might be impossible to determine the real cause of the very first changes in diseasedevelopment as host and microbiome factors are coinciding and closely intertwinedimplications for the development of novel therapiesdue to the strong and dynamic interdependency between host and microbiome in local niches it isunsurprising that most drugs used in clinical practice that were designed to target the host also affect themicrobiome accordingly inhaled corticosteroids and proton pump inhibitors affect the lung microbiota[“] as well as subsequent pneumonia risk [ ] macrolide antibiotics broadly effective acrosschronic lung conditions such as copd affect both lung microbiota and host immunity perhapsmost provocatively baseline differences in lung microbiota appear to predict patient responsiveness totherapies such as inhaled corticosteroids suggesting that variation in lung microbiota may representan untapped phenotype of œprecision medicine in the lung this opens new possibilities to exploit thisimportant crosstalk in therapeutic interventions but in order to do so we first need to improve ourunderstanding of the molecular mechanismsincreasing evidence of the importance of the microbiome raises the concept of restoring œdiseasedmicrobiomes to prevent or treat diseases using microbiotadirected therapies or hosttargeted therapiessuch as probiotics metabolites lung microbiota transplantation or vitamin d therapy which we discusshere in more detailmany clinical studies have investigated the efficacy of probiotic bacteria which are supposedly beneficialfor the host to prevent chronic diseases such as asthma or allergic rhinitis however these studies havelargely produced contradictory outcomes which might be due to the fact that the used probioticswere not selected based on potential mechanistic effects and may not be ideal the microbiome is a10118313993003023202019 0crespiratory basic science r gosens complex ecosystem comprised of a variety of different inhabitants and influenced by many externalhostderived factors thus the addition of single strains may not make a profound difference thus thetransfer of whole microbiomes via faecal microbiota transplantation fmt could be a more promisingapproach the introduction of healthy microbiota into diseased hosts has restored immunity andphysiology demonstrating that intestinal microbiota and their products can modulate host immunitylocally and systemically and that fmt can replace diseaserelated microbiomes with healthy ones fmt is remarkably successfulin treating antibioticresistant clostridium difficileinducedcolitis and is now being used as treatment in selected patients [ ] the new microbiomeengrafts quickly and lasts for at least a month indicating a potential difficulty in inducing longtermbeneficial changes in the microbiome via only targeting the microbial side while there are encouragingdata questions remain whether fmt may also affectlung health and whether lung microbiotatransplantation is feasible furthermore recently severe complications of fmt have been reported due totransfer of drugresistant bacteria thus it is essential to determine whether such approaches that sofar only transiently change the microbiome can be used for the required longterm treatments of chroniclung diseases that coincide with a variety of structural changes aberrant mucociliary clearance and manymore such as[“]specific microbial moleculeslipopolysaccharide lps andalong with living bacteriapeptidoglycan can induce or modulate inflammatory responsesin addition culturesupernatants of probiotic bacteria display antiinflammatory effects which have been ascribed to thepresence of secreted immunemodulatory metabolites for example culture supernatants of certainprobiotic bifidobacterium species decreased the secretion of type cytokines from immune cell lines andthe expression of costimulatory molecules on primary dendritic cells a likely mechanism is quorumsensing quorum sensing is a means of communication among bacteria of the same species to coordinateeffector functions such as biofilm formation sporulation or toxin secretion the bestdescribed quorumsensing molecules are acyl homoserine lactones ahls several ahls are targeted by the host tointerfere with growth of pathogens and are in turn exploited by bacteria to regulate host geneexpression for their benefit some ahls can bind to distinct bitter taste receptors expressed on the airwayepithelium and innate and adaptive immune cells [ ] thereby modulating barrier andimmune functions the moststudied ahl 3oc12hsl can activate phagocytesto increasephagocytosis expression of adhesion receptors and chemotaxis [ ] but is itself cleaved andinactivated by airway epithelial cells another example of bacterialderived modulators of the host™s immune responses are outer membranevesicles omvs omvs are spherical bilayered membrane vesicles released from the surface of bothgramnegative and grampositive bacteria and contain much of the biological material from the parentbacterium but in a nonreplicative form [ ] evidence suggests that the release of omvs providesbacteria with competitive advantages when exposed to acute and chronic hostassociated stressors innate and adaptive immune responses [“] antimicrobialthey may protect bacteria againstpeptides and antibiotics [ ] moreover omvs contain factors eg siderophores aiding in theacquisition of nutrients in an environment devoid of crucial elements such as iron besidessupporting the survival of the parent bacteria omvs may also play role in the progression of pulmonarydiseases bacteria frequently associated with copd exacerbations are known to release omvs furthermore macrophages stimulated with omvs derived from prominent airway pathogens such asp aeruginosa h influenzae or m catarrhalis release higher amounts of tumour necrosis factorα and il6 legionelladerived omvs significantly enhanced bacterial replication in macrophages andbacteriafree p aeruginosa omvs have been shown to potently induce pulmonary inflammation in mice strengthening the idea that omvs exert diseasepromoting activities in addition omvs have beenshown to induce tolerance and hyporesponsivenessthereby facilitating bacterial adherence to andinternalisation by macrophages which may contribute to clearance of the infection [ ] thusdespite our increasing knowledge on omvs and their potential role in interkingdom communicationthere is a need for further research to better understand their pathogenic properties and possibletherapeutic or prophylactic implications eg novel vaccinesan interesting alternative to fmt or probiotic bacteria might be to use immunemodulatory microbialmetabolites or œbeneficial omvs such chemically defined bacterial substances could be produced at largescale under controlled conditions applied in defined effective doses both systemically or locally and mayhave fewer adverse sideeffects ie in immunocompromised patients compared to live bacteria several studies have already used defined bacterial metabolites to treat aad in mice lps from escherichiacoli o111 bacterial polysaccharide a oligodeoxynucleotides with bacterial cpg motifs flagellin b shortchain fatty acids dtryptophan and the neutro
Colon_Cancer
"dysregulation of bcl2 is a pathophysiology observed in haematological malignancies forimplementation of available treatmentoptions it is preferred to know the relative quantificationof bcl2 mrna with appropriate reference genes for the choice of reference genes”i reference genes were selected by assessing variation of genes from rnaseq datasets of haematological malignancies followed by filtering based on their go biological processannotations and proximity of their chromosomal locations to known disease translocationsselected genes were experimentally validated across various haematological malignancy samples followed by stability comparison using genorm normfinder bestkeeper and reffinderii commonly used reference genes were obtained from literature through extensive systematic review levels of bcl2 mrna was assessed by qpcr normalized either by novel reference genes from this study or gapdh the most cited reference gene in literature andcompared the analysis showed ptcd2 ppp1r3b and fbxw9 to be the most unregulatedgenes across lymphnodes bone marrow and pbmc samples unlike the reference genesused in literature bcl2 mrna level shows a consistent higher expression in haematologicalmalignancy patients when normalized by these novel reference genes as opposed togapdh the most cited reference gene these reference genes should also be applicable inqpcr platforms using taqman probes and other model systems including cell lines and rodentmodels absence of sample from healthynormal individual in diagnostic cases call for carefulselection of reference genes for relative quantification of a biomarker by qpcrbcl2 can beused as molecular diagnostics only if normalized with a set of reference genes with stable yetlow levels of expression across different types of haematological malignanciesintroductionoverexpression of bcl2 bcell lymphoma a mitochondrial membrane protein has beenobserved in several haematological malignancies due to genetic and epigenetic mechanismsa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation dwivedi n mondal s p k s t ssachdeva k bathula c relativequantification of bcl2 mrna for diagnostic usageneeds stable uncontrolled genes as reference one e0236338 101371 pone0236338editor pedro v baptista universidade nova delisboa portugalreceived may accepted july published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0236338copyright dwivedi this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportinginformation files one 101371 pone0236338 august one 0cfunding the study is funded by glue grantscheme number btpr23078med2912532017by department of biotechnology httpdbtindiagovin govt of india awarded to sd and md thefunders had no role in study design data collectionand analysis decision to publish or preparation ofthe manuscriptcompeting interests the authors have declaredthat no competing interests existbcl2 molecular diagnostics with novel reference genesresulting in evasion of apoptosis giving the malignant cells a longer life span and survival benefits at times of nutrient deficiency hypoxia and growth factor deprivation [“] estimationof level of bcl2 along with other antiapoptotic genes are essential to avail efficient treatmentoptions by rchop regimen of cyclophosphamide doxorubicin vincristine and prednisone and rituximab or venetoclax in different haematological malignancies [ ] byvisualization of chromosomal aberrations using karyotyping or fish fluorescence insituhybridization bcl2 levels can be inferred indirectly detection of expression of bcl2protein by immunohistochemistry a standard pathological testing procedure for dlbcl hasnot been adopted in the clinics for bone marrow tissues of liquid cancers due to sample inconsistency and challenging procedure of capturing low concentrations of biomarkers western blotting for the very nature of the method cannot be adopted for high throughputpathological testing elisa for detection of bcl2 in human plasma remains limited sinceonly one splice isoform of the mitochondrial membrane protein is available in soluble formthus bringing down the effectiveness of the assay bcl2 at the mrna level can be determined without ambiguity by next generation sequencing nanostring and microarray though increasing time and expense of pathological testing in clinical trials relative quantification by qpcr quantitative polymerase chain reactioncan be successfully used due tothe availability of appropriate controls in untreated or normal groups [ ] although beingtime and costeffective it suffers misinterpretation in pathological setting since the relativequantification depends only on the rg reference gene used due to the absence of normalsamplesnormalization with a rg which shows varying expression across samples can often lead towrong s as seen with the use of glyceraldehyde3phosphate dehydrogenasegapdh as rg in gene expression studies of pulmonary tuberculosis and cd8 tcellsunder inactivated or activated condition similarly abl protooncogene abl1 therecommended rg for gene expression studies with leukemic patients was found to haveextremely low expression in neutrophils making it unsuitable as rg for the specific casesuch discrepancies have prompted researchers to analyze gene expression across multiple tissues or pancancer database like tcga to propose normalization factors using multiple rg candidatesthis study through a systematic review of literature in haematological malignancies concluded that mostly conventionally used œhousekeeping genes are still being deployed s1table and s1 fig despite their varied expression based on cell type developmental stage andexperimental conditions with rare exceptions [ ] none of the genes thus identified could be used to relatively quantify bcl2 as molecular diagnostics since compared to thefpkm fragments per kilobase of transcript per million mapped reads value of the antiapoptotic genes across databases s2 fig most of the rgs from the literature are not onlyhigher but also varied significantly s3 and s4 figs with few exceptions inspired by genomewide search for rgs from publicly available rnaseq or microarray data in human and otheranisms [“] we report here a set of novel candidate rgs obtained from an unbiasedsearch of genes in haematological malignancies to be used to normalize bcl2 andother antiapoptotic genes in qpcr as molecular diagnosticsmaterials and methodsethics statementthe study was performed in compliance with ethical practices and was approved by narayanahealth academics ethics committee narayana health hospitals ethics approval numbernhhaeccl2017152a one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genessystematic review of commonly used rgsliterature search was carried out in pubmed databasepubmed as detailed in s5 figaccording to prisma preferred reporting items for systematic reviews and metaanalysesguidelines selection of stable genes proteincoding genes identified from publicly available datasets table using ensembldb annotation package within r statistical software were categorised into four quartiles based on their median expression values across all samples geneswith median expression in middle two quartiles q2 and q3 in all datasets were consideredas q1 and q4 representing extreme ends of the expression spectrum are not preferred as rgcandidates for normalization of molecular diagnostic markersto determine the stability of a gene following statistical measures were employed“i cv �xsx where �x and σx are mean and standard deviation of a variable x respectively and ii normality pvalue as measured by shapirowilks test where a pvalue less than signifies thatthe distribution is away from normal cv although used most frequently isn™t a robustmeasure as it is affected by outliers to solve this a third parameter was used mad medianabsolute deviation medianjx 00 xj where x is the median of x after normalization withmedian mad is a better measure for understanding the spread of the distribution as itdepends on medians a parameter less prone to deviations by outlierslow or comparable statistical variation across samples represented by low values of cvand mad and a normal distribution high value of normality pvalue or low values of “pvalue are characteristics of an ideal rg therefore genes with median expression values inmiddle quartiles q2 and q3 were shortlisted and clustered based on their cv mad and “pvalue normalized to their respective zscores using pam partitioning around medsalgorithm required optimal number of clusters was calculated using silhouette graphicalmethod for each tissue sample the gene cluster with the lowest med value of parameters was selected and the genes at the intersection of the four clusters were shortlisted the list was further filtered by analysing and eliminating genes based on stop words in theirgo gene ontology annotation such as transcription factors nuclear receptor or other nuclearlocalization dna binding activity response to external stimuli translational and transcriptionalactivation since genes with such characteristics regulated by environmental conditions areunsuitable as rg candidates next genes were ranked in ascending order of their mean euclidqffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffifficv þ mad2 þ ð1 00 pþ2ean distance d ¼all parameters replaced by their zscores in thisthreeparameter hyperspace for each dataset average of d across four datasets was taken to calculate the mean euclidean distance �d genes with �d median were selected for furthertable list of rnaseq databasesdatasetdiseasetcgalamlamltargetaml paediatric amlgdcdlbcdlbclmmrfmmmultiplemyeloma� both primary and recurrent tumor only 1st visit recordstissuebloodbonemarrowlymphnodesbonemarrowsamples n sourcedownload location� tcga research networkwwwcancergovtcgaschmitz multiple myeloma researchfoundationgdccancergovaboutdatapublicationsdlbclresearchthemmrf fpkm data for gdcdlbc dataset was available as log2 transformed normalized value which was converted to fpkm101371 pone0236338t001 one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesanalysis locus of genes associated with pathogenic translocations were identified [ ] andcandidate rgs in close proximity of such loci within bands in the same arm of chromosomewere eliminated by an automated method further only genes with nonzero fpkm value in allsamples from four datasets were retained then each gene was given a composite quartile ranking cqr the sum of quartile indices from each dataset and genes with cqr value median expression in 2nd quartile in at least two datasets were shortlisted s6 figdesign of primersbcl2 primers bcl2 has two known splice isoforms membranebound bcl2α and aless studied soluble bcl2β lacking the transmembrane domain at the ™ cterminal most reported primers amplified only bcl2α or larger amplicon s2 table hence new primers were designed table rg primers primers for shortlisted genes were designed table s3 table using primerbank and idt sample detailsrna was isolated from peripheral blood or bone marrow samples from patient or normalindividuals s7 fig with their informed consent ethics approval number nhhaeccltable primers details of rgs and bcl2primeracy1accession nonm_000666ankrd26nm_014915jmjd4nm_001161465ptcd2nm_0247545ppp1r3bnm_024607fbxw9nm_032301nanpnm_1526673plekhm3nm_0010804753tsga10nm_025244nat1nm_001160174ric8bnm_018157gapdhnm_0012897453bcl2nm_0006572sequence ™ ™fw 'cactgacaaccgctatatccgrv 'ctcatgcagccgttcatcgtfw 'tctcggcaagatccacaaagcrv 'aatgtagagccgtcctgttcafw 'gtctgtcaatgtctgtgggagrv 'caggtgtgtgtcgcagagt3'fw 'tatgggacactgcacatcac3'rv 'ggctgaccatcctcttgttta3'fw 'agaacctcgcatttgagaagac3'rv 'tctgaaccggcataagtgtcc3'fw 'tagggcggtgcgatgattc3'rv 'cggattttggcggactgaga3'fw 'ggtccgcctacttctattaacg3'rv 'tctctgctctccacctacaa3'fw 'gatgatatcagcccagccttag3'rv 'ggacttcctggatcccataaac3'fw 'tactcagcgacaccttgctaa3'rv 'ccagatcattgagggttccac3'fw 'gggagggtatgtttacagcac3'rv 'acatctggtatgagcgtccaa3'fw 'atagtgttcaacagtcagatggc3'rv 'gcaagcgcaagtcaaagca3'fw 'tcgacagtcagccgcatcttcttt3'rv 'gcccaatacgaccaaatccgttga3'fw 'ggaggattgtggccttcttt3'rv 'gcccaatacgaccaaatccgttga3'fw forward primer rv reverse primer101371 pone0236338t002amplicon length bptm ˚camplification factor one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genes2017152a subjects with hepatitis bc or hiv and pregnant or lactating women wereexcluded from the studypbmcbmmc peripheral blood mononuclear cells bone marrow mononuclear cellswere separated by layering of blood or bone marrow diluted to with 1x pbs gibco„¢germany above ficollpaque plus histopaque himedia india followed by centrifugation at rcf for mins with brakes off resultant buffy coat was washed twice with 1x pbs andonce with 1x penstrep himedia india before culturing at cell density of to millioncellsml of rpmi himedia india with fbs gibco„¢ germany brazil origin and1x penstrep for subculturing the lymphocyte populationrna cdna and qpcrfrom ffpe formalinfixed paraffinembedded blocks “ curls were deparaffinized inxylene at ˚c followed by proteinase k himedia india treatment prior to rna isolationeither from lymphocytes or from deparaffinized retrospective samples rna was isolatedby trizol„¢ ambion us method and quantified with qubit rna br assay kit thermofisher scientific us before converting to cdna using superscript iv ssiv thermo fisherscientific us as per manufacturers™ instructions with notemplate control ntc qpcrwas performed in triplicates for each sample using kapa sybr green universal reagentssigma aldrich us cdna dilution and primers in a 5μl reaction mix qpcr condition preincubation at ˚c for minutes followed by amplification for cycles“denaturation at ˚c for sec amplification at ˚c for sec and extension at ˚c for sec inroche lightcycler ii machineoptimization of primersprimers were optimized for qpcr as required by the miqe guidelines all primers wereused at four different final concentrations forwardreverse 200nm200nm 200nm100nm100nm200nm and 100nm100nm with pooled cdna template obtained from six normalhealthy volunteers to yield single amplification product primer efficiency was checked using atwofold fivepoint dilution of the template primer efficiency was obtained from standardcurve using the formula amplication factor ¼ 00� table ��slope 00 stability analysis of candidate rgsmean of cq quantification cycle of ntc were subtracted from cq values of each gene inqpcr experiments to obtain δcq cq sampleˆ’mean cq ntc and relative expression aseˆ’δcq for each replicate where e is the amplification factor of corresponding genestability of expression of the candidate rgs was analysed using three independent algorithms“genorm normfinder and bestkeeper and the webbased reffindertool that integrates all three algorithms plus the delta ct method algorithm genorm wasrun using the slqpcr r package whereas authorsupplied r package and excel worksheet were used for normfinder and bestkeeper analysis respectively mean cq values foreach gene for all samples were used as input for bestkeeper and reffinder whereas fenorm and normfinder relative expression values were used since normfinder uses amodelbased approach to quantify inter and intragroup variations the malignant and nonneoplastic or healthynormal samples were used as two groups for normfinder analysiscomprehensive stability rank of each gene was calculated as the geometric mean of stabilityrank given by each method one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesexpression analysis of bcl2rq relative quantification of bcl2 expression was calculated either as ratio of relativeexpression of bcl2 with relative expression of gapdh or the normalization factor which isgeometric mean of relative expression of three candidate rgsrq ðgapdhþ ¼ e 00 dcqðbcl2þe 00 dcqðgapdhþrq ðproposedþ ¼ e 00 dcqðbcl2þgeo mean e 00 dcqðptcd2 ppp1r3b fbxw9þresults and discussionquantification by qpcr could be the choice of pathology laboratories for a quick and costeffective platform for singlegene expression level with appropriate rg towards this effort macrae performed a genome wide search and statistical analysis using rnaseq datafrom leukemia patients in a more recent pancancer study publicly available geneexpression data from microarray studies were analysed to identify a few rg candidates thatshowed minimal variation between malignant and normal samples and were validated in droplet digital pcr on bone marrow samples of all patients we have used types of haematological malignancy samples encompassing bone marrow pbmc and ffpe blocks along with nonneoplastic bone marrow and healthy pbmc samples subsequent to using much wider publiclyavailable data from samples in aml dlbcl and multiple myeloma databases furtherwe have employed an improved statistical analysis including clustering technique described inmethods section instead of an ad hoc approach of selection of top few genes from the clusterswe used important biological considerations to further prune the list of candidate rgssystematic review of commonly used rgs from literaturesystematic review of s yielded rgs used in haematological malignancies througha selection of genes by different analysis methods s4 table and b usage of known rgs inqpcr s1 table fpkm values of all these rgs when examined in public databases showedvaried expression among different types of haematological malignancies s3 and s4 figs withmaybe the exception of pggt1b however since other genes selected in the literatureshowed higher expression and correlated extreme variation we could not depend on the assayand proceeded to select novel rgs with an unbiased approachselection of candidate rgsstatistical analysis stepwise filtration of the number of genes from each dataset is summarized in s6 fig and also in graphical abstract fig shows gene clusters plotted in cv normalized mad and 1pvalue hyperspace for four datasets cluster marked in green in eachfigure represents the cluster with least med value s5 table for the three parametersselected clusters in the four datasets had an overlap of genes indicating large number ofgenes involved in housekeeping processes and hence showing lesser intersample variationacross diverse datasets common genes were pruned further to by go biological processterm filtration disease association and cqr to lead to a final of genes s6 table that weretaken through experimental validation melt curve analysis and efficiency check with pooledcdna from six healthy volunteers narrowed it down to genes with stable median expression and single amplification product of expected size for each table primers for geneswhich did not qualify the efficiency check were eliminated as they failed to show single amplification peak after repeated trials with new experimental conditions and even new primersequences s3 table one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig statistical analysis of candidate genes genes plotted in the cv normalized mad and “pvalue hyperspace for the fourdatasets a tcgalaml b targetaml c gdcdlbc and d mmrfmm cluster shown in green represents the chosencluster with least value of meds101371 pone0236338g001expression of genes with efficient primers were analysed on samples by qpcr usingobserved cq values preliminary stability analysis of the genes were done with online reffinder tool to select top stable genes ptcd2 ppp1r3b fbxw9 nanp ric8b jmjd4plekhm3 nat1 ankrd26 tsga10 as rg candidatessssstability analysis of candidate rgs results of bestkeeper algorithm used independentlyor as part of reffinder were comparable whereas results of genorm or normfinder analysisdiffered as they used different inputs geometric mean of stability ranks assigned in each algorithm was used to create comprehensive stability ranking of all the candidate rgs s7 tableand fig the analysis shows ptcd2 ppp1r3b and fbxw9 to be most stable across all analysed patient samplesptcd2 pentatricopeptide repeatcontaining protein codes for a mitochondrial proteininvolved in rna binding maturation and respiratory chain function though its exact one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig stability rank of candidate reference genes101371 pone0236338g002molecular function is not well understood [ ] ppp1r3b protein phosphatase1 regulatorysubunit3b encodes for a catalytic subunit phosphatase regulatory subunit 3b which isinvolved in hepatic glycogen dysregulation in type diabetes [“] fbxw9 fboxwdrepeatcontaining protein is a cytosolic protein involved in ubiquitination and proteasomedegradation expression analysis of bcl2accurate determination of bcl2 expression among few antiapoptotic markers in patients withhaematological malignancies is emerging as a critical diagnostic test for clinicians to suggest efficacious therapy options fpkm values of rgs common and novel from the publicly availabledatabases when compared fig with bcl2 indicated the novel rgs to be better normalizationcandidate for bcl2 in qpcr assays in pathology labs due to less and stable expressioncomparison of relative expression of gapdh versus the proposed normalization facteometric mean of relative expression of the three rg candidates clearly show a large variation in gapdh expression “ across malignant samples fig 4a s8 table granted itspopularity the expression stability of gapdh has been proven to differ in different conditionsdue to its involvement in apoptotic cell death through ubiquitin ligase membrane trafficking upregulation in aml involvement in nonhodgkin™s bcell lymphomas and inconsistency in several other cancers on the other hand proposed rgs havelesser variation “ and their expressions are consorted with each other making them better candidate as rg compared to gapdh this behaviour is translated to bcl2 expressionrq in malignant samples when normalized with gapdh fig 4b evidently normalizationwith gapdh underestimates relative quantification of bcl2 compared to normalization withproposed rgs with a statistically significant difference in median values p wilcoxonrank sum test between the two schemes bcl2 quantification in haematological malignanciesby qpcr is overtly reliant on rg since availability of œadjacent normal sample is ruled outabove results clearly demonstrate how the quantification may go off limit due to a wrongchoice of rg one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig candidate reference genes in hematological malignancy datasets expression values of candidate genes in four datasets a tcgalamlb targetaml c gdcdlbc and d mmrfmm101371 pone0236338g003broader applicability of proposed reference genesthough primary objective of this study is to discover rg candidates for bcl2 diagnostics in aclinical setting the rgs may have broader utility in other experimental platforms or modelsystems in the systematic review we found a number of research s [“] that haveused taqman probes instead of sybr green whereas our validation experiment was carriedout using sybr green probes however studies in different contexts such as a tropical oilseedplant or measurement of expression of various adenosine receptors in breast cancer tissue and in experiments using human reference rna sybr green pcr assays wereobserved having fair concordance with taqman pcr from these evidences we believe thatstability of proposed rgs is not likely to differ between sybr green and taqman qpcr assaysto assess variation of these stable rgs in cell lines we analyzed rpkm values of proteincoding genes across cell lines of haematopoietic and lymphoid tissue origin frombroad institute cancer cell encyclopedia and found the proposed rgs presenting muchlesser variations in expression compared to the common rgs gapdh abl1 b2m gusband actb in cell lines as well s8 figboth transgenic and wild type and occasional rat models are widely used in leukemia andlymphoma research [ ] usability of rgs common between clinical and animal studies one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig relative expression of chosen reference genes and relative quantification of bcl2 a relative expression of chosen reference genes solidlines and gapdh dashed line across patient samples b relative quantitation of bcl2 expression with respect to the candidate reference genesand gapdh in malignant patient samples101371 pone0236338g004will thus be of immense advantage we find that the proposed rgs“ptcd2 ppp1r3b andfbxw9 have “ sequence similarity and identity with corresponding genes in mice andother commonly used rodent models s9 table suggesting the genes playing similar role incellular function thereby displaying stability similar to that in humans hence normalizationfactor derived from the expression of these rgs may be applicable in murine and other rodentmodels as well with suitable design of primers encompassing conserved regionsbeyond detection of gene expression at mrna level it may be worthwhile to explore theapplicability of protein counterpart of the stable rgs in western blot as control for proteindetection by design we have chosen rgs that are of moderate expression level in middlequartiles of expression among other genes and they may not be detectable by western blotunless a larger amount of sample is loaded which is often not feasible with clinical sampleshowever it may be an interesting proposition to predict stable reference proteins for use inwestern blot by statistical analysis of proteomics data and associated systematic review ofliterature one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesour results indicate that genes ptcd2 ppp1r3b and fbxw9 render more reliability toqpcrbased diagnostic test of bcl2 in haematological malignancies the can beextended to other biomarkers in liquid cancer as well as for research with other model systemssuch as cell lines and rodentssupporting informations1 table list of reference genes in literaturedocxs2 table list of bcl2 primers from literaturedocxs3 table list of unqualified primersdocxs4 table literature explaining analysis and selection of reference genedocxs5 table zscore med valuesdocxs6 table list of selected genesdocxs7 table individual and combined stability rank and scores of candidate reference genesdocxs8 table relative expression of gapdh and the proposed normalization factordocxs9 table sequence similarity and identity with corresponding genes in mice rat andguinea pigdocxs1 fig rgs found in literature with more than one citationtiffs2 fig fpkm values of bcl2 family of antiapoptotic genes in the four datasetstiffs3 fig fpkm values of rgs found in relevant literature with more than one citationtiffs4 fig fpkm values of rgs found in relevant literature with a single citationtiffs5 fig workflow according to prisma guidelines for systematic review for commonlyused reference genestiffs6 fig statistical analysis workflowtiffs7 fig patient samples used in the studytiff one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference geness8 fig variation in stable rgs in cell lines and animal modeltiffs1 graphical abstracttiffacknowledgmentsauthors acknowledge prof joy kuri chair department of electronic science and engineering indian institute of science bangalore for providing the computational resourcesauthor contributionsconceptualization sujan k dhar manjula dasdata curation nehanjali dwivedi sreejeta mondal smitha p k sowmya t kartik sachdeva christopher bathula vishnupriyan kformal analysis sujan k dharfunding acquisition sharat damodar manjula dasinvestigation nehanjali dwivedi sreejeta mondal smitha p k sowmya tmethodology nehanjali dwivedi sreejeta mondal smitha p k sowmya t vishnupriyank manjula dasproject administration manjula dasresources nataraj k s sharat damodarsoftware sujan k dharsupervision manjula dasvalidation nehanjali dwivedi sreejeta mondal smitha p k sowmya t kartik sachdevachristopher bathula vishnupriyan kvisualization manjula daswriting “ original draft sreejeta mondal sujan k dharwriting “ review editing nehanjali dwivedi sreejeta mondal smitha p k sujan kdhar manjula dasreferences perini gf ribeiro gn pinto neto jv campos lt hamerschlak n bcl2 as therapeutic target forhematological malignancies vol of hematology and oncology biomed central ltd gratiotdeans j merino r nuñez g turka la bcl2 expression during tcell development early lossand late return occur at specific stages of commitment to differentiation and survival proc natl acad sciu s a oct “ 101073pnas912210685 pmid merino r ding l veis dj korsmeyer sj nuñez g developmental regulation of the bcl2 protein andsusceptibility to cell death in b lymphocytes embo j feb “ pmid li l li y que x gao x gao q yu m prognostic significances of overexpression myc andorbcl2 in rchoptreated diffuse large bcell lymphoma a systematic review and metaanalysis scirep “ 101038s41598017177655 uchida a isobe y asano j uemura y hoshikawa m takagi m targeting bcl2 with venetoclaxis a promising therapeutic strategy for œdoubleproteinexpression lymphoma with myc and bcl2 one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesrearrangements haematologica jun “ 103324haematol2018 pmid baro´ c espinet b salido m garcı´a m sa´nchez b florensa l cryptic ighbcl2 rearrangementswith variant fish patterns in follicular lymphoma leuk res feb “ 101016jleukres201009011 pmid hofman p heeke s alixpanabières c pantel k liquid biopsy in the era of immunooncology is itready for primetime use for cancer patients suppressed immune microenviron repert brain metastases from patients with resected nsclc “fatani s h mukhtar m h ali a s correlation between serum antiapoptotic bcl2 level and its immunohistochemical expression in relation to apoptosis in gastric cancer j mol biomark diagn albitar m zijun xy wang y manman d tzankov a visco c myc and bcl2 mrna expressionas determined by ngs predicts survival in dlbcl in gcb but not in abc subgroup blood nov 134supplement_15092“ derenzini e rossi a agostinelli c rossi m melle f motta g integration of nanostring profilingand functional characterization of oxidative and replicative stress biomarkers identifies poor prognosis mycbcl2 positive diffuse large bcell lymphoma subsets providing opportunities for precisiontherapies blood nov 132supplement “zhang f yang b zhang k hou ml lu xc li yx ccnd1bcl2 gene network a direct target of amifostine in human acute megakaryocytic leukemia cells chem biol drug des may “101111cbdd12889 pmid patel vm balakrishnan k douglas m tibbitts t xu ey kutok jl duvelisib treatment is associated with altered expression of apoptotic regulators that helps in sensitization of chronic lymphocyticleukemia cells to venetoclax abt199 leukemia sep “ 101038leu2016382 pmid bomben r ferrero s d™agaro t dal bo m re a evangelista a a bcell receptorrelated genesignature predicts survival in mantle cell lymphoma results from the fondazione italiana linfomi mcl trial haematologica apr “ 103324haematol2017184325pmid dheda k huggett jf chang js kim lu
Colon_Cancer
the incidence and death rate of nonsmall cell lung cancer nsclc in china ranks the first among the malignant tumors circular rna circrna was reported to be involved in the progression of nsclc our study aimed to investigate the underlying mechanism of circ_0020123 in nsclc progressionmethods quantitative realtime polymerase chain reaction qrtpcr was used to detect the expression of circ_0020123 mir5905p and thrombospondin thbs2 in nsclc tissues and cells cell proliferation and migration were examined by cell counting kit8 cck8 assay and transwell assay respectively flow cytometry assay was used to detect the apoptosis of nsclc cells the protein levels of ki67 matrix metalloprotein9 mmp9 cleavedcaspase9 cleavedcasp9 and thbs2 were detected by western blot the targets of circ_0020123 and mir5905p were predicted by starbase and targetscan and then confirmed by dualluciferase reporter assay and rna immunoprecipitation rip assay the animal experiment showed the effect of circ_0020123 on tumor growth in vivoresults the expression of circ_0020123 was upregulated in nsclc tissues and cells functionally circ_0020123 downregulation inhibited the proliferation and migration and promoted the apoptosis of nsclc cells interestingly circ_0020123 directly targeted mir5905p and inhibition of mir5905p reversed the knockdown effects of circ_0020123 on nsclc cells more importantly thbs2 was a target of mir5905p and thbs2 overexpression reversed the effects of circ_0020123 knockdown on cell proliferation migration and apoptosis in nsclc cells finally suppression of circ_0020123 inhibited tumor growth in vivo through mir5905pthbs2 axis circular rna circ_0020123 regulated thbs2 by sponging mir5905p to promote cell proliferation and migration and inhibit cell apoptosis in nsclc cellskeywords nsclc circ_0020123 mir5905p thbs2highlights circ_0020123 was upregulated and downregulation of circ_0020123 inhibited cell proliferation migration and promoted cell apoptosis in nsclc cellscorrespondence bskrju163comdepartment of thoracic surgery lianyungang second people™s hospital no hailian east road haizhou district lianyungang jiangsu china circ_0020123 directly targeted mir5905p and mir5905p downregulation reversed the knockdown effects of circ_0020123 on nsclc progression thbs2 acted as a target of mir5905p and overthe effects of expression of thbs2 reversed circ_0020123 knockdown on nsclc progression downregulation of circ_0020123 suppressed tumor growth in vivo through mir5905pthbs2 axis the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the ™s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the ™s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cwang a0et a0al cancer cell int page of lung cancer has the highest incidence of total cases and is the most common cause of cancer death of total cancer deaths in worldwide lung cancer can be divided into several histological subtypes according to the location and the tendency of metastasis small cell lung cancer sclc accounts for about of all lung cancer cases however nonsmall cell lung cancer nsclc accounts for of lung cancer and the a0years overall survival rate os is only about therefore it is important to find the effective treatment and potential molecular targets of nsclc progressioncircular rna circrna is a single stranded rna molecule with a closed circular structure recently amounts of circular dna have been discovered and most of which were thought to be the byproducts of typical splicing [ ] previous reports indicated that the expression of circrna was tissuespecific and the change of its expression intensity was associated with some diseases [“] furthermore circrna was involved in the occurrence and development of the disease and might be used as a potential biomarker in clinical diagnosis prognosis and treatment of diseases [ ] for example circ_0039569 facilitated cell proliferation and migration of renal cell carcinoma by sponging mir34a5p to regulate cc chemokine ligand ccl22 meanwhile hsa_circ_0043256 participated in the progression of nsclc cells by mediating the cinnamaldehyde treatment a previous report suggested that circ_0020123 acted as an oncogene in nsclc and circ_0020123 regulated zincfingerenhancer binding protein zeb1 and enhancer of zeste homolog ezh2 by competitively binding with mir144 to induce cell progression and migration these reports suggested that circ_0020123 was a vital factor in the pathogenesis of nsclc and its function and molecular mechanism need to be further studiedas a small endogenous rna microrna mirna is essential in regulating gene expression and plays a potential role in the exploitation of biomarkers recently some aggregated mirnas have been found in prostate cancer such as mir221222 mir143145 mir23b27b241 and mir1133a which were downregulated and had tumor inhibiting functions a previous study found that circulating mir5905p could be used as routine diagnostic tools for lung cancer and as a potential prognostic marker for liquid biopsy besides overexpression of mir5905p reduced the development of nsclc cells and regulated the expression of epithelialmesenchymal transformation emtrelated proteins by targeting the signal transducers and activators of transcription stat3 however the precise mechanism by which mir5905p affects nsclc needs further investigationthrombospondin thbs2 as a secreted protein was confirmed to be highly expressed in different cancers including cervical cancer colorectal cancer and nsclc a previous report suggested that thbs2 was involved in the proliferation apoptosis and antiautophagy regulation of cervical cancer cells by mir20a tian et a0al found the expression and clinicopathological features of thbs2 in colorectal cancer were significantly correlated with the prognosis of cancer and might be used as a biomarker of prognosis however the molecular function of thbs2 in nsclc remains poorly definedin this study the targeting relationship between circ_0020123 and mir5905p was firstly detected the effects of circ_0020123 on cell proliferation migration apoptosis and tumor growth were performed by gain and lossoffunction experiments and molecular biology techniquesmaterials and a0methodspatients and a0specimensnsclc tissues and the adjacent healthy lung tissues were taken from nsclc patients in the lianyungang second people™s hospital all volunteers signed written informed consents nsclc tissues and the adjacent tissues were immediately frozen in liquid nitrogen and kept at ˆ’ a0 °c for further experiments this research was approved by the ethics committee of lianyungang second people™s hospitalcell culture and a0cell transfectiontwo nsclc cell lines a549 and h1299 and one normal lung cell line imr90 were obtained from the beijing concorde cell library beijing china a549 h1299 and imr90 cells were cultivated in dulbecco™s modified eagle medium dmem hyclone logan ut usa supplementing with fetal bovine serum fbs hyclone and cultured in an incubator at a0„ƒ with co2small interfering rna sirna targeting circ_00201231 sicirc_00201231 and sicirc_00201232 short hairpin rna shrna targeting circ_0020123 shcirc_0020123 mir5905pinhibitors sirna negative control sinc shnc and ncinhibitors were all obtained from biomics biotech jiangsu china full length of thbs2 cdna sangon biotech shanghai china was subcloned into pcdna31 plasmid ekbioscience shanghai china then cell transfection was performed by lipofectamine thermo fisher scientific waltham ma usa 0cwang a0et a0al cancer cell int page of rna isolation and a0quantitative real‘time polymerase chain reaction qrt‘pcrthe trizol reagent invitrogen carlsbad ca usa was used for extracting the total rnas next the reversed transcription was carried out by rtpcr kit invitrogen the qrtpcr was performed using the abi sybr green master mix invitrogen the primers in our study were as follows f5²ttc gga cga ccg tca aac at3² and r5²agg atc cct gca cca caa tg3² for circ_0020123 f5²tga aag acg tga tgg cac ac3² and r5²ctt cca ttt tgg for mir5905p f5²aga agg ggt ttt tgg3 ² ctg ggg ctc att tg3² r5²agg ggc cat cca cag tct tc3² for glyceraldehyde3phosphate dehydrogenase gapdh f5²gcg gct ggg tct att tgt c3² and r5²gca gga ggt gaa gaa cca tc3² for thbs2 f5²att gga acg ata cag aga agatt3² and r5²gga acg ctt cac gaa ttt g3² for u6 gapdh and u6 were the internal parameterscell counting kit‘ cck‘ assaythe proliferation viability of a549 and h1299 cells were detected by the cellcounting kit8 msk wuhan china a549 and h1299 cells were cultivated into a 96well plate with a density of × a0cellswell and incubated in a0°c for or a0h then a0μl fresh medium and cck8 solution was added after incubation at a0°c for a0h the od values were detected by the multiskan ascent microplate reader abcam cambridge ma usatranswell assaytranswell chamber corning life sciences corning ny usa was used to detect cell migration firstly the serumfree dmem thermo fisher scientific was fixed with cell suspension cells and seeded into the upper chamber and the dmem containing serum was put into the lower chamber after incubation for a0h the cells in lower surface of the upper chamber were treated with formaldehyde solution for a0 h and then thoroughly washed finally the migrated cells were stained with crystal violet corning life sciences and observed by using a microscopeflow cytometryfirstly a549 and h1299 cells were cultured and pbs was used for washing cells then the binding buffer was used to resuspend cells and the annexin vfluorescein isothiocyanate vfitcpropidium iodide pi apoptosis detection kit thermo fisher scientific was used to stain cells finally cell apoptosis was detected by flow cytometry thermo fisher scientificwestern blot analysisthe total proteins of nsclc tumors or cells were collected by ripa lysis buffer sangon biotech then the proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis sdspage and transferred to polyvinylidene fluoride pvdf membranes thermo fisher scientific the skimmed milk was added and incubated with primary antigapdh antibody invitrogen carlsbad ca usa antiβactin antibody invitrogen antiki67 antibody invitrogen antimatrix metalloprotein9 mmp9 antibody invitrogen anticleavedcaspase9 cleavedcasp9 antibody invitrogen or antithbs2 antibody invitrogen at a0°c overnight finally the membranes were incubated with the secondary antibody for a0 h at room temperature the results were viewed using kodak film developer fujifilm japandual‘luciferase reporter assaysthe wild type circ_0020123 sequences circ_0020123wt mutant circ_0020123 sequences circ_0020123mut wild type thbs2 ²utr sequences thbs2wt mutant thbs2 ²utr sequences thbs2mut were cloned into pgl3 luciferase reporter plasmid promega madison wi usa then the plasmid and mir5905p or mirnc were cotransfected into a549 and h1299 cells by lipofectamine thermo fisher scientific after transfection for a0h the dualluciferase reporter assay system promega was performed to detect the luciferase activityrna immunoprecipitation ripfirstly the magna rip rnabinding protein immunoprecipitation kit gzscbio guangzhou china was performed to verify the relationship between circ_0020123 and mir5905p in brief the magnetic beads and antiago2 antibody abcam were added into cells and incubated for a0h then the proteinase k and the phenol“chloroformisoamyl alcohol reagent were added for purifying rnas finally qrtpcr was used to measure circ_0020123 enrichmentanimal experimentsthe 4weekold balbc male nude mice vitalriver beijing china were raised in a sterile environment for 0cwang a0et a0al cancer cell int page of experiments then pbs was used to suspend a549 cells × transfected with shcirc_0020123 or shnc next the nude mice were divided into two groups n a549 cells transfected with shcirc_0020123 or shnc were shcirc_0020123 or shnc inoculated into the nude mice the tumor volume was detected every a0 days after a0days the nude mice were euthanatized and the tumor weight was detected besides the tumor tissues from each group were collected to detect the expression of circ_0020123 mir5905p and thbs2 the animal experiment was approved by the animal experimentation ethics committee of lianyungang second people™s hospitalstatistical analysisthe software graphpad prism was performed for statistical analysis the data was displayed as mean ± standard deviation sd the significant difference was calculated by student™s t test and oneway analysis of variance anova p was considered as statistically significantresultscirc_0020123 was a0upregulated in a0nsclc tissues and a0cellsto begin with qrtpcr was used to detect the expression of circ_0020123 the result showed that circ_0020123 was significantly upregulated in nsclc tissues compared with the adjacent healthy tissues fig a0 1a similarly the expression of circ_0020123 in nsclc cells a549 and h1299 was markedly higher than that in normal cells imr90 fig a01b from these data it is speculated that circ_0020123 might be acted as an oncogene in nsclcfig circ_0020123 was upregulated in nsclc tissues and cells a qrtpcr was used to detect the expression of circ_0020123 in adjacent healthy tissues n and tumor tissues n b the expression of circ_0020123 in normal cell line imr90 and nsclc cell lines a549 and h1299 was detected by qrtpcr p downregulation of a0circ_0020123 inhibited the a0proliferation migration and a0induced apoptosis of a0nsclc cellsto investigate the functional effects of circ_0020123 on nsclc cells sicirc_00201231 and sicirc_00201232 were obtained and transfected into a549 and h1299 cells firstly the transfection efficiency was detected by qrtpcr fig a02a next cck8 was used to detect the proliferation and the results showed that the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was reduced fig a0 2b moreover the migration of a549 and h1299 cells was significantly downregulated by circ_0020123 knockdown fig a02c in addition the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was obviously higher than transfected with sinc fig a02d finally the protein levels of cell proliferationrelated protein ki67 and cell migrationrelated protein mmp9 were inhibited while cell apoptosisrelated protein cleavedcasp9 was upregulated in nsclc cells transfected with sicirc_00201231 or sicirc_00201232 fig a02e these data suggested that inhibition of circ_0020123 suppressed cell proliferation migration and promoted apoptosis in nsclc cellscirc_0020123 directly targeted mir‘‘5pby searching in the online software starbase the potential binding sites between circ_0020123 and mir5905p were detected fig a0 3a to confirm that the dualluciferase reporter assay was performed the results showed that the luciferase activity of circ_0020123wt reporter plasmid was reduced by mir5905p mimic while the circ_0020123mut reporter plasmid activity was not changed in a549 and h1299 cells fig a03b furthermore the expression of mir5905p was lower in a549 and h1299 cells compared with that in imr90 cells fig a0 3c in contrast mir5905p expression was elevated in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 fig a0 3d finally the rip assay was also used to confirm the targeting relationship between circ_0020123 and mir5905p and the results showed that circ_0020123 and mir5905p were enriched in antiago2 group fig a03emir‘‘5p downregulation reversed the a0inhibition effects of a0circ_0020123 on a0nsclc cellsto further explore the functional effects between circ_0020123 and mir5905p mir5905pinhibitor was established qrtpcr was used to detect the transfection efficiency fig a0 4a interestingly mir5905p was upregulated in a549 and h1299 cells transfected with sicirc_00201231 while the expression of mir5905p was recovered in cells transfected with 0cwang a0et a0al cancer cell int page of fig downregulation of circ_0020123 inhibited the proliferation and migration and induced the apoptosis of nsclc cells a the transfection efficiency of sicirc_00201231 and sicirc_00201232 in a549 and h1299 cells was detected by qrtpcr b cck8 assay was used to detect the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 c the migration of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was measured by transwell assay d flow cytolysis assay was used to detect the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 e the protein levels of cell proliferation related protein ki67 cell migration related protein mmp9 and cell apoptosis related protein cleavedcasp9 were detected by western blot p fig a0sicirc_00201231 mir5905pinhibitors 4b moreover circ_00201231 knockdown inhibited cell proliferation and migration while the mir5905p inhibitor reversed these effects fig a0 4c d in addition the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 was increased which was abolished by mir5905pinhibitor fig a0 4e similarly mir5905p inhibitors reversed the effects on the protein levels of ki67 mmp9 and cleavedcasp9 in a549 and h1299 cells transfected with sicirc_00201231 fig a0 4f these results that mir5905p downregulation reversed the effects of circ_0020123 downregulation on the proliferation migration and apoptosis of nsclc cellsindicated mir‘‘5p targeted thbs2 in a0nsclc cellsthe thbs2 ²utr was predicted to contain the binding sites of mir5905p through the online software targetscan fig a05a then the dualluciferase reporter assay was used to confirm the targeting relationship the results showed that cotransfection of mir5905p and thbs2wt significantly limited the luciferase activity in both a549 and h1299 cells the luciferase activity was not altered in cells cotransfected with mir5905p and thbs2mut fig a05b importantly the mrna and protein level of thbs2 was enahnced in nsclc cells fig a05c d we further explored whether circ_0020123 affected the functions of thbs2 in nsclc cells the mrna and protein expression of thbs2 were repressed in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 fig a05e f 0cwang a0et a0al cancer cell int page of fig circ_0020123 directly targeted mir5905p a the binding site between circ_0020123 and mir5905p was detected by the online software starbase b the luciferase activity of circ_0020123wt or circ_0020123mut reporter plasmid in a549 and h1299 cells transfected with mirnc or mir5905p was detected by dualluciferase reporter assay c qrtpcr was used to detect the expression of mir5905p in a549 and h1299 cells d the expression of mir5905p in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was detected by qrtpcr e rip assay was used to confirm the relationship between circ_0020123 and mir5905p p thbs2 overexpression reversed the a0effects of a0circ_0020123 knockdown on a0the a0proliferation migration and a0apoptosis of a0nsclc cellsbased on the work ahead of us the pcdna31thbs2 was constructed then the qrtpcr and western blot were used to detect the transfection efficiency and the thbs2 expression was increased in a549 and h1299 cells transfected with pcdna31thbs2 fig a0 6a b in addition the proliferation and migration rates of a549 and h1299 cells transfected with sicirc_00201231 pcdna31thbs2 were higher than that transfected with sicirc_00201231 fig a0 6c d meanwhile a similarly phenomenon was also observed in cell apoptosis the pcdna31thbs2 significantly reversed the promotion effect of circ_0020123 deletion on cell apoptosis fig a0 6e furthermore the effects of circ_0020123 deletion on ki67 mmp9 and cleavedcasp9 protein levels were also reversed by thbs2 overexpression fig a0 6f these data suggested that overexpression of thbs2 could reverse the effects of circ_0020123 downregulation on cell proliferation migration and apoptosisreduction of a0circ_0020123 suppressed tumor growth in a0vivo through a0circ_0020123mir‘‘5pthbs2 axisto further explore the function of circ_0020123 in nsclc cells the shcirc_0020123 was constructed and the xenograft tumor was established then a549 cells transfected with shcirc_0020123 or shnc were injected into the nude mice the xenograft tumor volume was measured every a0 days after injection and the results showed that tumor volume was smaller shcirc_0020123 group than that in shnc group fig a07a moreover tumor weight was inhibited by circ_0020123 knockdown fig a0 7b furthermore the expression circ_0020123 and thbs2 was decreased while the mir5905p was increased in xenograft tumor transfected with shcirc_0020123 fig a0 7c western blot assay also revealed that the protein level of thbs2 was repressed by circ_0020123 knockdown fig a07d finally the digital tomosynthesis dts was used to detect the number of lung metastatic nodules in xenograft tumor and it was reduced in shcirc_0020123 group fig a07e the results suggested that downregulation of circ_0020123 inhibited tumor growth in a0vivodiscussionclinically only a few nsclc patients were diagnosed at an early stage and treated by surgical resection more than of nsclc patients were diagnosed with the advanced stage or metastatic tumors thus finding novel biomarkers and therapeutic targets were necessary for the effective diagnosis and treatment of nsclcrecently circrna was no longer considered as a random product in the rna shearing process and its biological significance and function in malignant tumors 0cwang a0et a0al cancer cell int page of fig mir5905p downregulation reversed circ_0020123 knockdown effects in nsclc cells a qrtpcr was used to detect the expression of mir5905p in a549 and h1299 cells transfected with mir5905pinhibitors b the expression of mir5905p in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors was detected by qrtpcr c the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors was tested by cck8 assay d transwell assay was used to measure the migration of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors e flow cytolysis assay was used to detect the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors f the protein levels of ki67 mmp9 cleavedcasp9 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors were detected by western blot p had received more and more attention previous reports revealed that circ_0020123 was involved in the development of nsclc moreover the level of circ_0020123 was elevated in nsclc cells consistently we found that the expression of circ_0020123 was markedly higher in nsclc tissues and cells moreover this research indicated that inhibition of circ_0020123 suppressed the proliferation migration and induced apoptosis of nsclc cells in a0 vitro besides circ_0020123 promoted tumor growth in a0vivoendogenous circrnas could act as microrna sponges to inhibit their function and some studies linked mirna sponges to human diseases including cancer a previous study indicated that circrna ctransferrin receptor ctfrc regulated tfrc by sponging mir107 to facilitate bladder carcinoma development mir5905p was studied in different cells such as airway smooth muscle cells colon epithelial cells and nsclc cells however the potential relationship between mir5905p and circrna has not been researched in this study circ_0020123 directly targeted mir5905p and mir5905p inhibition reversed the effects of circ_0020123 knockdown on nsclc progression these data provided a clue to the therapeutic strategy for nsclc 0cwang a0et a0al cancer cell int page of fig mir5905p targeted thbs2 in nsclc cells a the potential binding site between thbs2 ²utr and mir5905p was predicted by the online software targetscan b dualluciferase reporter assay was used to measure the luciferase activity of thbs2wt or thbs2mut reporter plasmid in a549 and h1299 cells transfected with mirnc or mir5905p c qrtpcr was used to detect the mrna expression of thbs2 in nsclc cells d the protein level of thbs2 in nsclc cells was tested by western blot e the mrna expression of thbs2 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was detected by qrtpcr f western blot was used to measure the protein level of thbs2 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 p our study also confirmed that mir5905p could target thbs2 directly in nsclc cells thbs2 is a calciumbinding protein that binds to and inactivates matrix metalloproteinase mmp genes involved in tissue formation and repair [ ] a previous document suggested that thbs2 acted as a target of mir2213p and participated in lymph node metastasis in cervical cancer the data in this research showed that the expression of thbs2 in nsclc cells was markedly higher than normal healthy cells furthermore overexpression of thbs2 reversed the effects of circ_0020123 knockdown on proliferation migration and apoptosis of nsclc cells suggesting that circ_0020123 promoted the progression of nsclc cells through mir5905pthbs2 axisin our research showed that the expression of circ_0020123 was higher in nsclc tissues and cells than control and downregulation of circ_0020123 inhibited the proliferation migration and promoted apoptosis of nsclc cells and also suppressed tumor growth in a0 vivo moreover circ_0020123 directly targeted mir5905p while mir5905p inhibition reversed the effects of circ_0020123 knockdown on nsclc cells more importantly circ_0020123 regulated the expression of thbs2 by sponging mir5905p and upregulation of thbs2 reversed the effects of circ_0020123 knockdown on nsclc cells therefore our research demonstrated that circ_0020123 enhanced proliferation migration and inhibited 0cwang a0et a0al cancer cell int page of fig overexpression of thbs2 reversed the effects of circ_0020123 knockdown on proliferation migration and apoptosis of nsclc cells a b the mrna and protein expression of thbs2 in a549 and h1299 cells transfected with pcdna31thbs2 was detected by qrtpcr and western blot c cck8 assay indicated the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 d the migration of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 was measured by transwell assay e the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 was detected by flow cytolysis assay f the protein levels of ki67 mmp9 cleavedcasp9 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 were detected by western blot p apoptosis of nsclc cells by sponging mir5905p to regulate thbs2results and develop the manuscript all authors read and approved the final manuscriptabbreviationsnsclc nonsmall cell lung cancer circrna circular rna qrtpcr quantitative realtime polymerase chain reaction cck8 cell counting kit8 mmp9 matrix metalloprotein9 cleavedcasp9 cleavedcaspase9 cleavedcasp9 cleavedcaspase9 rip rna immunoprecipitation zeb1 zincfingerenhancer binding protein ezh2 zeste homolog stat3 signal transducers and activators of transcription thbs2 thrombospondin acknowledgementsnot applicableauthors™ contributionslw collaborated to design the study lz were responsible for experiments analyzed the data yw wrote the paper all authors collaborated to interpret fundingnoneavailability of data and materialsplease contact corresponding author for data requestsethics approval and consent to participatethis research was approved by the ethics committee of lianyungang second people™s hospital the animal experiment was approved by the animal experimentation ethics committee of lianyungang second people™s hospitalconsent for publicationall listed authors have actively participated in the study and have read and approved the submitted manuscript 0cwang a0et a0al cancer cell int page of fig reduction of circ_0020123 suppressed the tumor growth in vivo through circ_0020123mir5905pthbs2 axis a a total of × a549 cells transfected with shcirc_0020123 or shnc were injected into nude mice to establish the xenograft tumor tumor volume was measured every d after injection b tumor weight was measured on d c the expression of circ_0020123 mir5905p and thbs2 in xenograft tumor was measured by qrtpcr d the protein level of thbs2 in xenograft tumor was evaluated by western blot e the number of lung metastatic nodules in xenograft tumor was detected by digital tomosynthesis dts p competing intereststhe authors declare that they have no competing interestsreceived april accepted july references bray f ferlay j soerjomataram i siegel rl torre la jemal a global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries ca cancer j clin “ abe h takase y sadashima e fukumitsu c murata k ito t kawahara a naito y akiba j insulinomaassociated protein is a novel diagnostic marker of small cell lung cancer in bronchial brushing and cell block cytology from pleural effusions validity and reliability with cutoff value cancer cytopathol “li c zhang l meng g wang q lv x zhang j li j circular rnas pivotal molecular regulators and novel diagnostic and prognostic biomarkers in nonsmall cell lung cancer j cancer res clin oncol “ belousova ea filipenko ml kushlinskii ne circular rna new regulatory molecules bull exp biol med “ zhang z xie q he d ling y li y li j zhang h circular rna new star new hope in cancer bmc cancer li l chen y nie l ding x zhang x zhao w xu x kyei b dai d zhan s guo j zhong t wang l zhang h myodinduced circular rna cdr1as promotes myogenic differentiation of skeletal muscle satellite cells biochim biophys acta gene regul mech “ greco s cardinali b falcone g martelli f circular rnas in muscle function and disease int j mol sci weng xd yan t liu cl circular rna_larp4 inhibits cell migration and invasion of prostate cancer by targeting foxo3a eur rev med pharmacol sci “ deng n lei d huang j yang z fan c wang s circular rna expression profiling identifies hsa_circ_0011460 as a novel molecule in severe preeclampsi
Colon_Cancer
previous studies have shown a strong coexistence of colorectal neoplasia crn and cardiovascular diseases cvd this study was aimed to summarize the available evidence on association of cvd risk with early crn detection in asymptomatic populations pubmed web of science and embase were systematically searched for eligible studies published until dec studies exploring the associations of recommended cvd risk assessment methods eg risk scores carotid artery plaque and coronary artery calcium score [cacs] with risk of crn were included metaanalyses were conducted to determine the overall association of cvd risk with the crn a total of studies were finally included the association of carotid artery plaque with the risk of colorectal adenoma ad was weakest pooled odds ratio [or] confidence interval [ci ] participants with cacs100 had about 2fold increased risk of ad than those with cacs0 the pooled ors were ci and ci for the risk of advanced colorectal neoplasia an and ad respectively in participants with framingham risk score frs20 when compared to participants at low risk frs10 frs might help identify subgroups at increased risk for an but further studies are needed keywords cardiovascular disease risk assessment colorectal neoplasiaintroductionboth colorectal cancer crc and cardiovascular diseases cvd are the leading causes of mortality and morbidity worldwide12 previous studies have shown a strong coexistence of colorectal neoplasia crn and cvd probably due to the shared risk factors eg smoking obesity and metabolic syndrome and pathophysiological mechanisms eg chronic inflammation and oxidative stress3“current guidelines8“ recommend assessing the cvd risk in healthy people using risk estimation scores such as framingham risk score frs1112 procam13 and the pooled cohort equation14 which are based on individuals™ medical history and easily available laboratory data in addition assessment of subclinical atherosclerosis by imaging modalities could be added as risk modifiers to help make clinical decisions for borderline or intermediaterisk adults8“ routine use of imaging modalities is not recommended for cvd risk assessment in clinical practice due to the medical costs or invasiveness but incorporation of imaging data such as the anklebrachial index abi coronary artery calcium score cacs and carotid artery plaques cap could improve the prediction of cvd risk15“clinical epidemiology “ chen this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomtermsphp and incorporate the creative commons attribution “ non commercial unported v30 license httpcreativecommonslicensesbync30 by accessing the work you hereby accept the terms noncommercial uses of the work are permitted without any further permission from dove medical press limited provided the work is properly attributed for permission for commercial use of this work please see paragraphs and of our terms wwwdovepresscomtermsphp 0cchen dovepressvarious risk scores have also been developed for predicting advanced colorectal neoplasia an18“ although several studies2526 have reported that elevated blood lipids the well documented cvd risk factor and history of cvd were associated with increased risk of crc the majority of risk scores developed for an did not include them into the models27 recent studies have reported the associations between cvd risk assessment and risk of1 crn higher frs estimating the 10year risk of developing coronary heart disease chd1112 was significantly associated with the higher risk of an frs vs frs10 odds ratio [or] confidence interval [ci] “ abi was associated with 13fold increased risk of an in a recent study29 cap and cacs were also found to be positively related to the increased risk of adenoma ad and an in several studies30“given a number of shared risk factors and mechanisms between cvd and crc and the emerging epidemiological evidence of association between cvd risk and crc there is a possibility that cvd risk assessment could help trigger crc screening therefore the aim of this review was to provide an overview of the cvd risk assessment methods and their associations with the risk of crn fully understanding of the current knowledge and existing gap might promote better prevention and treatment for cvd and crc circulating and urinary biomarkers have either no or only limited value when added to cvd risk estimation score systems834 thus only score models and imaging methods recommended as risk modifiers abi cacs and cap in the guidelines8“ were included in this reviewmaterials and methodsthis systematic review was conducted following the procedure recommended by the cochrane collaboration35 and was reported according to the preferred reporting items for systematic reviews prisma checklist36 ethical approval and patient informed consent were not necessary since all the data included in the current study were obtained from previously published studiesand metaanalyses remaining publications and reference lists were scrutinized studies that fulfilled the predefined criteria were includedinclusion and exclusion criteriawe required that included studies meet the following criteria published as an original research in a peer reviewed cardiovascular risk has been assessed using either score models or imaging methods recommended as risk modifiers abi cacs and cap in the guidelines3 only included participants who were considered asymptomatic reported the association of cvd risk assessment results with the risk of crn studies were excluded if they were published as conference proceedings dissertations or s only or were not published in english pico eligibility criteria for this review were presented in the supplementary table s1data extractiontwo authors yc and xc independently performed data extraction of all included studies the following information was ed author publication year study period number of participants age number of males outcome ad an and so on data source medical records questionnaires or both cvd risk assessment and association indexdiscriminatory accuracy or hazard ratio [hr] specificity sensitivity or area under the receiver operator characteristic curve values] in case of any disagreement consensus was obtained by discussionquality assessment in eligible studiesrisk of bias and applicability were assessed according to quality assessment of diagnostic accuracy studies2 quadas237 quadas2 evaluates the risk level of bias composed of four basic components patient selection index test reference standard flow and timing clinical applicability is also assessed for the first three components the risk of bias and concerns regarding applicability for each study was then rated as œhigh œlow or œunclearliterature search strategiespubmed embase and web of science were searched up to december to identify the relevant papers the searched items were presented in the appendix which mainly covers expressions for cvd risk score models recommended imaging modalities crn and discriminatory accuracy or strength of association after removal of duplicates titles and s of records were screened according to the inclusion and exclusion criteria full texts of the statistical analysiswe pooled ors for the same cvd risk assessment index using r statistical software version and the r œmeta package version for frs and cacs ors were pooled separately for different levels of scores using the lowest level as reference two kinds of outcomes ad and an were reported in the studies using frs for cvd risk assessment and thus ors were pooled separately for different outcomes heterogeneity across studies was evaluated submit your manuscript wwwdovepresscom dovepress clinical epidemiology 0cdovepress chen using cochrane™s q statistic with p value and the i2 statistic if significant heterogeneity was observed i2 or pqstatistics a randomeffects model was used to calculate pooled estimates otherwise a fixedeffects model was used35 twosided p values of or lower were considered to be statistically significantresultsliterature search resultsa total of records were obtained in the initial search including citations from pubmed citations from embase and citations from web of science after removal of duplicates n1609 and exclusion due to our predefined criteria n5727 records were qualified for fulltext assessment fortyfour records were excluded due to the inclusion and exclusion criteria finally a total of studies28““ including one study which was identified through crossreferences were included the detailed information of the selection process was presented in figure study characteristicstable summarized the basic characteristics of the included studies published between and of the included studies nine were from korea and the other three studies were from japan austria and turkey respectively the study periods stretched from to with sample sizes ranging from to only one was designed as a prospective study41 and the others were crosssectional studies most studies included participants aged older than years and only one study enrolled subjects aged years32 in addition most studies were predominantly in men with proportions of males among participants ranging from to four cvd risk assessment methods abi cap cacs and frs were used in the included studies all studies explored the role of cvd risk assessment method on the detection of ad and some of risk adenoma3032 and an2829384243focused on colorectal high them also figure flowchart of inclusions of studies about relation of cvd risk to crn note adapted from moher d liberati a tetzlaff j preferred reporting items for systematic reviews and metaanalyses the prisma statement plos med creative commons license and disclaimer available from httpcreativecommonslicensesby40legalcode36 abbreviations cvd cardiovascular disease crn colorectal neoplasiaclinical epidemiology submit your manuscript wwwdovepresscom dovepress 0cchen dovepresstable basic characteristics of included studies about relation of cvd risk to colorectal neoplasiastudycountrystudy periodnumber of participantsyamaji y kim j kim h cha jm yun ke choi sh yang mh kim hb lee yj 2019a41lee jy niederseer d basyigit s japankoreakoreakoreakoreakoreakoreakoreakoreakoreaaustriaturkey““““““““““age years mean±sd ± ± 530b median± ± ± ± 526b± ± ±male n outcomec data sourcececum intubation ratedcvd risk assessment ad anad hraadad anad hraadadadadad anad anad anmrqmrqmrqmrmrqmrqmrmrmrqmrqmrqmrqnrnrnrnr‰¥nrabicapcapcapcacscacscacscacscacsfrsfrsfrsnotes ait is a retrospective followup study and all the other studies are crosssectional bsd was not reported cdetected by colonoscopies in all included studies d100 cecum intubation rate participants with failure of cecum intubation were excluded nr not reported studies mentioned that colonoscopies were extended to cecum in the methods section but did not reported the success rate of cecum intubation abbreviations abi anklebrachial index ad colorectal adenoma an advanced colorectal neoplasia cacs coronary artery calcium score cap carotid artery plaque cvd cardiovascular disease frs framingham risk score hra high risk adenoma mr medical records nr not reported q questionnaires sd standard deviationquality assessment of studiesthe results for the quality of included studies using the quadas2 tool are presented in table regarding patient selection one study by kim did not provide detailed information about patient selection31 thus the risk of bias and applicability concerns were rated unclear for this domain in this study otherwise no major risk of bias or applicability concerns were identifiedassociation of cvd risk assessed by different methods with crc risktable described the details of the cvd risk assessment methods in the included studies abi was associated with 13fold ci increased risk of an29 three studies reported the weak association between cap and risk of ad303138 one of them also showed an increased risk of an in the participants with cap but the results were not statistically significant or table risk of bias and applicability judgements in quadas2studyrisk of biasapplicability concernstotalpatient selectionindex testreference standardflow and timingpatient selectionindex testreference standardyamaji y kim j kim h cha jm yun ke choi sh yang mh kim hb lee yj lee jy niederseer d basyigit s totalˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšnotes œ_ high risk œˆš low risk œ unclear riskˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšsubmit your manuscript wwwdovepresscom dovepress clinical epidemiology 0cdovepress chen table details of the cvd risk assessment methods in the included studies about relation of cvd risk to colorectal neoplasiastudycategoriesboutcome or[ ci]yamaji y kim j abnormal abiabnormal abicap yescap yeskim h cap yescha jm yun ke choi sh cap yescap yescacs cacs “cacs cacs cacs “cacs cacs cacs “cacs yang mh201339cacs kim hb cacs “cacs “cacs ‰¥lee yj 2019a41cacs lee jy niederseer d basyigit s frs intermediatefrs highfrs intermediatefrs highfrs intermediatefrs highfrs intermediatefrs highfrs intermediatefrs highfrs intermediatefrs highadanadhraadadanadadadhrahrahraadadadadadadadadadadananadadananadadanan[ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ]hr [ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ]notes ain participants without adenoma cacs0 at baseline compared to cacs0 increased the risk of colorectal adenoma at followup colonoscopy hr ci bthe lowest level was defined as reference abbreviations abi anklebrachial index ad colorectal adenoma an advanced colorectal neoplasia cacs coronary artery calcium score cap carotid artery plaque frs framingham risk score hra high risk adenoma hr hazard ratio or odds ratio ci confidence interval ci in addition the presence of cap was associated with increased risk of colorectal high risk adenoma or ci four studies reported ors for different levels of cacs with cacs0 as reference32333940 highest cacs levels seemed to be associated with the increased risk of ad with or ranging from to the 10year chd risk estimated by frs was categorized as low risk intermediate risk “ and high risk ‰¥ participants with high risk of 10year chd had increased risk of either ad or an in the study by basyigit participants at high chd risk had about 4fold or ci increased risk of an28metaanalyses of available ors for different cvd risk assessment methodsmetaanalyses were performed in the studies that provided ors and their cis for the same cvd risk assessment index the association of cap with the risk of ad was weakest the pooled or ci a medium level of cacs cacs “ was associated with 134fold increased risk of ad when compared to the lowest category of cacs cacs0 participants with cacs100 had an increased risk of ad and the pooled or was ci the pooled ors were ci and ci for the risk of an and ad respectively in participants with high chd risk frs20 when compared to participants at low chd risk frs10 further details were presented in table and in the supplementary figures s1“discussionthis systematic review summarized the associations of recommended cvd risk assessment methods with risk of crn in asymptomatic populations a total of studies including four different methods were identified among these methods frs was most strongly associated with risk of both an and ad participants with frs20 have about 34fold and 23fold increased risk of an and ad respectively when compared to participants at low chd risk frs10 only one study29 reported that abnormal abi greatly increased the risk of an thus it was not included in the metaanalysisboth crc and cvd are thought to develop via a process of insulin resistance inflammation and oxidative clinical epidemiology submit your manuscript wwwdovepresscom dovepress 0cchen dovepresstable metaanalysis of odds ratios for different cvd risk assessment toolsstudycvd risk assessmentcategoriesaoutcomeor cicapcacscacscacsfrsfrsfrsfrsyes vs nocacs vs cacs0cacs “ vs cacs0cacs vs cacs0intermediate vs low riskhigh vs low riskintermediate vs low riskhigh vs low riskadadadadadadanan note athe lowest level was defined as reference abbreviations ad colorectal adenoma an advanced colorectal neoplasia cacs coronary artery calcium score cap carotid artery plaque cvd cardiovascular disease frs framingham risk score or odds ratio ci confidence intervalstress74547 which might partially explain why they share a number of risk factors eg alcohol consumption tobacco use physical activity use of antiinflammatory agents obesity and diabetes mellitus4548 in addition several cellular metabolismrelated pathways eg ampk and pparγ signaling pathways eg wnt signaling pathway and genetic pathways eg lrp6 mutation and tcf7l2 polymorphism are not only associated with accelerated atherosclerosis and an increased risk of cvd but also linked to cancer development and progression7 better understanding of these overlaps might promote shared management of prevention and treatment for both disordersrisk of an in this review the strength of associations between identified cvd risk assessment methods and the risk of crn was generally weak except frs which was modestly associated with frs20 vs frs10 frs was calculated based on age total cholesterol highdensity lipoprotein cholesterol smoking status systolic blood pressure and treatment of blood pressure which are typically available in the medical records44 compared to the more sophisticated risk calculators232449 for predicting an which need variables such as physical activity red meat intake and vegetable consumption frs has relatively higher generalizability and lower recall bias a recent study has recommended the combined preventive screening and research efforts in the prevention of both cvd and cancer50 if participants with highrisk of cvd predicted by frs could be recommended to have a screening for crn which will help increase compliance and uptake of crc screening as persons who are aware of their increased risk are more likely recommendations furthermore it also maximizes the medical values of the comply with to expert information participants obtain from a clinical examination or risk assessment and thus reduces the time and costs for health carehowever there are some issues that merit our attention firstly the included studies are all crosssectional which limits the comparisons between frs and the previously developed risk prediction models for crc secondly frs has its own limitations frs only estimates 10year chd risk for all individuals years or older but not the overall cvd risk in addition it is developed based on the american population while most of study participants are asians in the included studies studies have shown that frs overestimated cvd risk in the asian cohorts51“ at last the included studies tended to yield results with wide ci probably due to the limited number of participants the wider the ci the less the precision in summary higher cvd risk might trigger concurrent crc screening which should be further validated on largescale studies and future studies could consider about using the overall cvd risk score models developed from data of local cohorts to predict the risk of crcas for imaging data the association of cap or cacs with risk of ad is not strong enough that imaging index alone might not be useful for informing early detection of crn similarly routine screening with imaging modalities to predict future cardiovascular events is generally not recommended in clinical practice but use of these imaging techniques has been shown to improve cvd risk assessment and serve as a guide for initiating preventive therapies8“ a high cacs can help modify the predicted risk obtained from frs alone especially among patients in the intermediaterisk category16 up to now only one risk score developed in the multiethnic study of atherosclerosis mesa study used both cacs and submit your manuscript wwwdovepresscom dovepress clinical epidemiology 0cdovepress chen traditional risk factors to predict the 10year chd risk55 inclusion of cacs in the mesa risk score offered significant improvements in risk prediction cstatistic vs p factors in the risk models like smoking behaviors and blood lipids are closely related to the incidence and progression of cvd but they are not direct markers of current status of atherosclerosis this might help explain why the performance of risk models is improved by adding markers with anatomical delineation through imaging technology accounting for the higher performance of the combined use of risk scores and imaging tools on cvd risk assessment further studies could consider about exploring the association of combined form of them with the risk of crcwe also observed that less than half of included studies reported the associations of cvd risk with both risk of an and ad2829384243 colonoscopy is considered to as a valid primary screening tool for crc and is able to detect both ad and an the lower prevalence of an and the limited number of participants in several included studies might limit the power to explore the relation of an with cvd risk which could partly explain why most of studies did not include an as outcome therefore the findings should be carefully interpreted and further validated on largescale studiesour study has some strengths comprehensive search strategies along with welldefined eligibility criteria were used to help identify relevant s in addition two reviewers independently extracted data and assessed the risk of bias in the included studies however several limitations should also be addressed firstly the current meta analysis was based on observational studies there were the possibilities of potential effects of unknown or residual confounding factors on our results secondly as we only considered about established cvd risk models and recommended imaging modalities the potential of other cvd risk assessment index on the detection of crn was not summarized and compared in this study however it is also reasonable to just include these methods since their feasibility and performance for cvd risk prediction have been well approved in the clinical practice thirdly cut off values and group comparisons for the same cvd risk assessment method varied in the included studies which limits the synthesis of results for example the cut off values for cacs are the tertiles of cacs in the study by kim 40 however cacs was categorized into three groups with cut off values at and in the other studies3233 therefore less studies were included in the metaanalysis which might influence the accuracy of the pooled results lastly most of studies were conducted in asian populations which is an inherent limitation of the included studies thus our findings might not be applicable to other populations and needs to be externally validated in racially diverse populationsconclusionsto our knowledge this is the first review that applies metaanalyses to determining the overall association of recommended cv risk assessment methods with the risk of crn in the asymptomatic population frs calculated based on shared risk factors of cvd and crc shows potential to help identify subgroups at increased risk for an whether the combination of frs and imaging index is useful for the optimal evaluation of crn risk remains to be solved in the future studies cvd risk might inform crc screening which needs more research in the future to validate its feasibility and effectivenessabbreviationsabi anklebrachial index ad colorectal adenoma an advanced colorectal neoplasia cacs coronary artery calcium score cap carotid artery plaque chd coronary heart disease ci confidence interval crc colorectal cancer crn colorectal neoplasia cvd cardiovascular disease frs framingham risk score hr hazard ratio hra high risk adenoma mr medical records nr not reported or odds ratio prisma preferred reporting items for systematic reviews and metaanalyses quadas2 quality assessment of diagnostic accuracy studies2 q questionnaires sd standard deviationfundingthis research was funded by national natural science foundation of china grant number disclosurethe authors report no conflicts of interest in this workreferences bray f ferlay j soerjomataram i siegel rl torre la jemal a global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries ca cancer j clin “ doidoi103322caac21492 joseph p leong d mckee m et al reducing the global burden of cardiovascular disease part the epidemiology and risk factors circ res “ doidoi101161circresaha117308903 chan aoo man hj kwok fl et al prevalence of colorectal neoplasm among patients with newly diagnosed coronary artery disease j am med assoc doidoi101001jama29812 clinical epidemiology submit your manuscript wwwdovepresscom dovepress 0cchen dovepress chan aoo lam kf tong t coexistence between colorectal canceradenoma and coronary artery disease results from patients aliment pharmacol ther “ doi doi101111j13652036200602958x wang sc schulmanmarcus j fantauzzi j et al colon cancer laterality is associated with atherosclerosis and coronary artery disease j gastrointest oncol doidoi1021037jgo20180918 kahr pc hammerl s huberschönauer u et al atrial fibrillation a new indicator for advanced colorectal neoplasia in screening colonoscopy j clin med doidoi103390jcm8071083 masoudkabir f sarrafzadegan n krahn a et al cardiovascular disease and cancer evidence for shared disease pathways and pharmacologic prevention cardiovascular disease and cancer evidence for shared disease pathways and pharmacologic prevention hhs public access atherosclerosis “ doidoi101016 jatherosclerosis201706001 piepoli mf hoes aw agewall s european guidelines on cardiovascular disease prevention in clinical practice the sixth joint task force of the european society of cardiology and other societies on cardiovascular disease prevention in clinical practice constituted by representatives of societies and by invited experts developed with the special contribution of the european association for cardiovascular prevention rehabilitation eacpr atherosclerosis “ doidoi101016jatherosclerosis201605037 arnett dk blumenthal rs albert ma et al accaha guideline on the primary prevention of cardiovascular disease a report of the american college of cardiologyamerican heart association task force on clinical practice guidelines circulation 201914011e596“e646 doidoi101161cir0000000000000678 mach f baigent c catapano al esceas guidelines for the management of dyslipidaemias lipid modification to reduce risk eur heart j “ doi cardiovascular doi101093eurheartjehz455 grundy sm becker d clark lt et al detection evaluation and treatment of high blood cholesterol in adults adult treatment panel iii circulation “ doidoi101161circ106 cleeman ji executive summary of the third report of the national cholesterol education program ncep expert panel on detection evaluation and treatment of high blood cholesterol in adults adult treatment panel iii j am med assoc “ doi doi101001jama285192486 assmann g cullen p schulte h simple scoring scheme for calculating the risk of acute coronary events based on the 10year follow up of the prospectiv
Colon_Cancer
" phytolaccaceae species in china are not only ornamental plants but also perennial herbs that areclosely related to human health however both largescale fulllength cdna sequencing and reference genevalidation of phytolaccaceae members are still lacking therefore singlemolecule realtime sequencing technologywas employed to generate fulllength transcriptome in invasive phytolacca americana and noninvasive exotic picosandra based on the transcriptome data rtqpcr was employed to evaluate the gene expression stability in thetwo plant species and another indigenous congener p acinosaresults total of gb and gb clean reads of p americana and p icosandra were generated including and full length nonchimeric flnc reads respectively transcript clustering analysis of flnc readsidentified and consensus isoforms including and highquality ones after removingredundant reads and transcripts were obtained based on structure analysis total and alternative splicing variants and simple sequence repeats ssr as well as and completecoding sequences were detected separately furthermore and lncrna were predicted and and transcripts were annotated respectively subsequently seven reference genes in the two plant species and anative species p acinosa were selected and evaluated by rtqpcr for gene expression analysis when tested indifferent tissues leaves stems roots and flowers 18s rrna showed the highest stability in p americana whetherinfested by spodoptera litura or not ef2 had the most stable expression in p icosandra while ef1α was the mostappropriate one when attacked by s litura ef1α showed the highest stability in pacinosa whereas gapdh wasrecommended when infested by s litura moreover ef1α was the most stable one among the three plant specieswhenever germinating seeds or flowers only were consideredcontinued on next page correspondence yiwangynueducn1yunnan key laboratory of plant reproductive adaption and evolutionaryecology yunnan university kunming china2laboratory of ecology and evolutionary biology state key laboratory forconservation and utilization of bioresources in yunnan yunnan universitykunming chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cliu bmc plant biology page of continued from previous page fulllength transcriptome of p americana and p icosandra were produced individually based on thetranscriptome data the expression stability of seven candidate reference genes under different experimentalconditions was evaluated these results would facilitate further exploration of functional and comparative genomicstudies in phytolaccaceae and provide insights into invasion success of p americanakeywords phytolaccaceae smrt sequencing fulllength transcriptome analysis reference gene evaluation rtqpcr phytolacca americana is a member of the phytolaccaceae family and is native to northeast america becauseof its ornamental and medicinal applications it was introduced into china in unfortunately it hasevolved into an invasive species and spread to mostareas of the country especially in central and southernchina compared to noninvasive exotic congener p icosandra and native congener p acinosa p americana isof interest because it exhibits multiple biological activities such as plant pesticides antimicrobial propertyheavy metal accumulation capacity [“]in order to investigate the mechanisms of various bioactivities of p americana further transcriptomewidestudy is necessary to facilitate reports have showed thatjasmonic acidinduced and cadmiumtreated transcriptome data of p americana have been obtained by illumina hiseq and illumina hiseq platformrespectively [ ] howeverthese data were bothachieved by second generation sequencing sgs whichcould not produce fulllength transcripts genomic dataof p americana was available at the sra under projectprjna544344 but it™s raw reads without coding sequences prediction and functional annotation third generation sequencing tgs is known for itskilobasesized long reads and is an outstanding strategyfor better understanding rna processing for exampleit can be used to analyze different transcript isoformsregulated by alternative splicing which greatly increasesthe repertoire of proteins lead to genetic and functionaldiversity and is prevalent in most eukaryotic anisms the long reads could also provide sequence information on genecoding regions for functional analysis atthe transcriptional level and thus can be applied to refine an assembled genome for better annotation however tgs could not quantify gene expression forthe moment and have a relatively high error rate thansgs the combination of tgs and sgs are able to solvethis problem and are highly recommended by most researchers with the transcriptome and genome data availablefunctional genomics research is being performed whichrelied heavily on gene expression analysis reversetranscription quantitative real time pcr rtqpcr hasbeen reported to be a very sensitive and accurate technique to analyze gene expression level but it requiresappropriate reference gene as an internal control tonormalize mrna levels between different samples foran exact comparison of gene expression [ ] anideal reference gene should be expressed at a constantlevel across various experimental conditions howeverstudies have shown that no single reference gene is universal for all experimental conditions [“] therefore it™s necessary to estimate the stability of referencegenes under particular experimental condition beforeusing them for gene expression analysisin the present study to provide highquality and morecomplete assemblies in genome and transcriptome studies of phytolaccaceae a hybrid sequencing approachcombining the sgs and tgs technologies was carriedout first fulllength transcriptome of the invasive plantspecies of pameracana and an noninvasive exotic conicosandra was generated by singlemoleculegener prealtimesmrtsplicingevents simple sequence repeats ssr coding sequencesprotein annotations and long noncoding sequenceswere analyzed respectively at transcription level furtherthe stability of reference genes was evaluated in two phytolaccaceae species mentioned above and one nativecongener p acinosa by rtqpcr in order to facilitatefuture research on functional gene expressionsequencing alternativeresultsto classify the plant species these three phytolaccaceaemembers p americana p icosandra and pacinosa wereidentified by pcr and followed by sequences alignmentbased on sequences of second internal transcribed spacer its2 and the intergenic spacer of photosystem iiprotein d1 gene and trnahis gene of chloroplast genome psbatrnh table s1 the sequences of its2 andpsbatrnh in p americana that we employed had theidentity of with the sequences reported by chen in p icosandra the sequences of its2 had identity and the sequences of psbatrnh had identity with the results of chen™s in pacinosa 0cliu bmc plant biology page of similarity of its2 and identify of psbatrnh werefound isoforms afterremoving redundantsmrt sequencing data outputusing the pacific biosciences™ smrt sequencing protocol gb clean reads of invasive species p americana were obtained after preprocessing on the basis offull passes and sequence quality circular consensus sequences ccs with fulllength rate were obtained including fulllengthnonchimeric flnc sequences and highqualityconsensussequences from the high quality consensus isoforms transcripts alternative splicing events ssr complete coding sequences lnc rnasand annotated transcripts in p americana wereachieved similarly gb clean reads in p icosandrawere identified and ccs with fulllength rate flnc sequences as well as highquality consensus isoforms were filtered subsequently transcripts and alternative splicingevents were obtained what™s more ssrs complete coding sequences lnc rnas and annotated transcripts were identified in picosandratable transcriptome analysisbased on the structure of achieved transcripts and alternative splicing events were identified in pamericana and p icosandra respectively transcripts of bp in total in p americana wereemployed for ssr analysis based on the sequence lengththat was more than bp including ssrs and ssrcontaining sequences similarly transcripts bp in total in picosandra wereemployed for ssr analysis and ssrs togetherwith ssrcontaining sequences were identifiedtable summary of fulllength transcriptome sequencingclean reads gbccsflncflnc flncccsconsensus isoformhigh quality consensus isoformtranscriptsalternative splicingssrcomplete coding sequenceslncrnaannotated transcriptsp americanap icosandrathe detail information about the number of sequencescontaining more than one ssr the number of ssrspresent in compound formation and the number of different types of ssrs were shown in table in additiontotal of complete coding sequences cds in pamericana and cds in p icosandra were identified by using transdecoder the length distribution ofpredicted proteins was shown in fig s1in pdatabasespecifically nrwith the eight protein databases sequence alignmentswere performed to annotate predicted proteins in total transcripts in p americana and tranicosandra were annotated separatelyscriptstable the number of annotated protein sequencesin p americana was similar with p icosandra under aparticularncbi nonredundant protein analysis revealed that approximately transcripts in p americana and transcripts in picosandra showed the highest sequencesimilarity with beta vulgaris fig go gene ontology assignment also suggested that similar amount ofsequences in the two plant species belonged to the sameterm and many were classified into cell part and cellterm of cellular component catalytic activity and binding of molecular function and metabolic process andcellular process of biological process fig cogclusters of orthologous groups of proteins annotationshowed that a large number of predicted proteins in thetwo plant species were linked to functional class r general function prediction only j translation ribosomalstructure and biogenesis t signal transduction mechanisms g carbohydrate transport and metabolism ando posttranslational modification protein turnoverchaperones fig s2 the result of eggnog evolutionary genealogy of genes nonsupervised orthologousgroup annotation indicated that most of the annotatedproteins in the two plant species were belonging to thefunctional class s function unknown fig s3 kogeukaryotic ortholog groups functional classificationsuggested that r general function prediction only ando posttranslational modification protein turnover andchaperones were the most abundant functional categories in the two plant species fig s4 these results indicated that most of the sequences obtained were trulyfunctional proteins and had a similar functional classification in p americana and its congener picosandraeven though more work is needed to identify sequencesthat regulated or involved in the invasion success of pamericana the annotation of predicted proteins provided necessary information for further studiesbesides the transcripts encoding proteins long noncoding rnas lncrnas were achieved lncrnas arereported to be key regulators in plant biological prolncrna in pameracana andcesses the number ofpicosandra was predicted by cpc coding potential 0cliu bmc plant biology page of table ssrs obtained from transcripts with more than bpsearching itemtotal number of sequences examinedtotal size of examined sequences bptotal number of identified ssrsnumber of ssr containing sequencesnumber of sequences containing more than ssrnumber of ssrs present in compound formationnumber of mono nucleotide ssrnumber of di nucleotide ssrnumber of tri nucleotide ssrnumber of tetra nucleotide ssrnumber of penta nucleotide ssrnumber of hexa nucleotide ssrcalculator cnci codingnoncoding index pfamand cpat coding potential assessment tool respectively in total lncrna in pamericana and lncrna in picosandra were predicted by all these fourmethods fig subsequentlytranscription factorstfs that are key components involved in the transcriptional regulatory system were predicted in p americana tfs of types were filtered and in picosandra tfs of types were predicted thesetwo plant species shared the first types of tfs butthe number of each type tf was not similar especiallyrlkpelle_dlsv c3h snf2 and camk_camklchk1 indicating the particular functions on transcriptregulation fig amplification performance of rtqpcrprimers designed for rtqpcr were evaluated by pcrfirst the primers which produced single ampliconwithout primer dimer were chosen for melting curveanalysis only primers which produced a single fragmentefficiencywereqpcr amplificationchosenforp americanap icosandraassessment the qpcr efficiency of each primer pair wasgenerated from a 10fold serial dilution of pooled cdnaand was shown in table the threshold cycle ct values of each reference genewere employed to evaluate expression level under different experimental conditions fig average ct valuesfor all the seven candidate reference genes ranged from to in which ef1α showed the highest expression level and 28s rrna had the lowest expression levelit was also suggested that ct values of βactin and tubulin fluctuated significantly across all the experimentalsamplesstability of candidate reference genesforto determine the appropriate reference genesnormalization in different experimental conditions theexpression data was analyzed by genorm normfinderand bestkeeper respectively table s2when expression stability of reference genes wereanalyzed in different tissues leaves stems roots andflowers of p americana 18s rrna and ef2 oftable number of proteins annotated via differential protein databasedatabasesp americanaannotated number ‰¤ length length ‰¥ p icosandraannotated number ‰¤ length length ‰¥ coggokeggkogpfamswissproteggnognrall 0cliu bmc plant biology page of fig homologous species distribution of p americana and p icosandra annotated based on the nr database a p americana b p icosandrapamericana were identified as the most suitable reference genes by genorm and normfinder and 18s rrnawas also suggested by bestkeeper pairwise variation valueof v23 was below the cutoff value of which meansthe combination of two reference genes were most suitable for gene expression normalization fig whentested in picosandra ef1α was recommended for normalizing gene expression analysis not only by genorm butalso by normfinder ef2 was also suggested by genormand bestkeeper in pacinosa ef1α was the best reference gene suggested by genorm and bestkeeper but 18srrna was recommended by normfinder the use of tworeference genes was suitable because pairwise variationvalue of v23 was below when pooled the data of different tissues from pamericana and picosandra togetheref2 was shown to be the most stable gene by all the threemethods when investigated the expression stability ofreference genes in different tissues of pamericana andpacinosa 18s rrna showed the best expression stabilityby genorm and normfinder while ef2 was referred asthe most stable one by bestkeeper however the combination of five reference genes was recommended bygenorm for v56 which was less than when thedata of different tissues from picosandra and pacinosawas put together ef1α was identified as the best oneby genorm and normfinder whereas ef2 was suggested to be the best stability reference gene by bestkeeper when set the data of these three plant speciesas a pool ef1α was suggested to be the most stableone by genorm and normfinder while ef2 was alsorecommended by genorm and bestkeeper accordingto these results it is very important to select the appropriate reference gene when analyze the gene expressionlevel among plant species 0cliu bmc plant biology page of fig classification of the transcripts annotated by the gene ontology gowhen analyzed the data among germinating seeds28s rrna and ef1α were identified as the best reference genes by genorm while 18s rrna was recommended by normfinder and gapdh was suggested bybestkeeper three reference genes were sufficient tonormalize gene expression for v34 was below inflowers only of these three plant species ef1α was confirmed by all the three methods the genorm analysisshowed that the value of v45 was below so fourreference genes in combination were suggested thesefig venn diagram of the number of lncrnas predicted by cpc cnci cpat and pfam a p americana b p icosandra 0cliu bmc plant biology page of fig classification of predicted transcription factorsresults indicated that when focusing on particular tissuesof different plant species the selection of reference genewas also very essentialwhen plants were infested by slitura 18s rrnashowed the most expression stability suggested by genorm and normfinder in different tissues of pamericanawhile ef1α wasby bestkeeper therevealedcombination of two reference genes was suggested bygenorm due to the value of v23 was less than 18srrna was also recommended by genorm in s liturainfested picosandra and ef1α was shown to be themost stable one by normfinder and bestkeeper fourreference genes in combination were recommended bygenorm 18s rrna was also identified as the besttable primers for rtqpcr analysisgene nametubulingene descriptiontubulin ef1αelongation factor 1alphaprimer sequence ²²f gtaaggaagccgagaattgr tcaacaacagtgtcagagaf tgaagaaggtcggatacaatr gtagacatcctggagtgggaphdglyceraldehyde3phosphate dehydrogenasef tggtgctaagaaggttattatcef2elongation factor 18s rrna18s rrnaβactinactin728s rrna28s rrnar2 linear regression coefficientr gagtgaacggtggtcataf gtatcaccatcaagtcaactgr acaatcaaccacaacaaggf acttcctcttctcgtatcattr tgttcagcatagactgtgaf atgctatccttcgtctggr tactcttggctgtctctgf tacgattggttacggacatr ttctcatcaacaacagcatatlength bppcr efficiency r2 0cliu bmc plant biology page of together 18s rrna showed the best stability in genormand normfinder while the expression stability of ef1αwas suggested by bestkeeper in pamericana and picosandra 18s rrna was identified as the best referencegene by all the three algorithms in pamericana andpacinosa 18s rrna and βactin were suggested bygenorm in picosandra and pacinosa while gapdhand ef2 were recommended by normfinder and bestkeeper respectively when take all the data of s liturainfested plant species into account 18s rrna exhibitedthe most stable expression suggested by genorm andnormfinder while ef2 was the gene with the most constant expression identified by bestkeeperdiscussionfulllength transcripts are fundamental resources forstructuralfunctional and comparative genomics research [ ] smrt sequencing has been acknowledged by enabling the generation of multikilobasesequences to improve genome and transcriptome assembly the fulllength cdna sequences generated areable to characterize the posttranscriptional processsuch as alternative splicing lncrna prediction and coding sequences for further gene functional studies basedon the fulllength transcriptome data generated about gb of clean data were obtained for pamericana andpicosandra respectively table accordinglythenumber of ccs flnc consensus isoforms highqualityfig rna transcription levels of seven candidate reference genesin p americana picosandra and p acinosa the expression level ofcandidate reference genes in total samples n was presentedas cycle threshold number ctvalue and explained by box andwhisker plots the asterisks represented the minimum and maximumct value the squares indicated the 25th and 75th percentiles andthe median was represented by a bar across the squarereference gene by genorm in plant species pacinosawhile tubulin was suggested by normfinder and 28srrna was recommended by bestkeeper the combination of three reference genes was appropriate by genorm when analyzed the data oftwo plant speciesfig pairwise variation analyzed by genorm to determine the optimal number of reference genes for accurate normalization a threshold valueof was suggested for valid normalization if the value of vnn pairwise variation is less than then n reference genes in combinationare recommended for gene normalization if the value of vnn is more than then vn 1n should be taken into account pam pamericana pic p icosandra pac p acinosa lsrf different tissues of leaves stems roots and flowers gs germinating seeds of these three plantspecies f flowers of these three plant species lsr different tissues of leaves stems and roots i infested by s litura of third instar 0cliu bmc plant biology page of isoforms transcripts alternative splicing events ssrscomplete coding sequeces lncrnas and annotated transcripts were analyzed providing basic transcriptomic information for further studiesreports have showed that fulllength transcriptome ofzea mays have greatly helped in refining gene annotation and revealed the complexity of gene expression inmaize similar analysis has also been conducted inshum bicolor what™s more the world expansioncapability of cydia pomonella has been informed according to its genome information molecularmechanism of rapid growth and invasive adaptation ofan invasive species mikania micrantha has also been investigated according to itsreference genome therefore the fulllength transcriptome data of pamericana and picosandra will contribute to the genomic research and provide insights into invasive mechanism ofpamericana through comparative genomics study inphytolaccaceae speciesgenereliesonanalysisexpressionaccurate relative quantification of rtqpcr for furtherrobustnormalization by stably expressed reference genes tominimize error in the experimental process therefore suitable reference genes for the normalization ofrelative gene expression data in three phytolaccaceaespecies pamericana picosandra and pacinosa weresought under a diverse set of conditions these resultsdemonstrated the importance of validating referencegenes under the relevant experimental conditions forexamplein different tissues leaves stems roots andflowers of pamericana 18s rrna and ef2 were recommended to be the bestsuited reference genes and similar results were found in s liturainfested pamericanahowever even though the appropriate reference genesin picosandra were ranked according to the analyzed results of the three methods all the pairwise variationvalues were above the cutoff value of while thecombination of 18s rrna βactin ef1α and ef2 weremost suitable in s liturainfested picosandra ef2 andef1α have been considered as the ideal reference genesin pacinosa whereas the combination of 18s rrna βactin and gapdh were recommended after s litura infestation researches have also showed that no singlereference gene is stably expressed among different tissues of an anism such as the reference gene selectionin amygdalus persica solanum lycopersicum and glycine max [ ] what™s more our results alsosuggested that reference genes identified based on transcriptome data should be confirmed by experimentalevidence in jainduced transcriptome of p americana28s rrna showed stable expression between exogenousjatreated and control plants ja signal pathway ofplants can be induced by lepidopteran herbivores infestation however 18s rrna and ef2 were identifiedas the most stable expression reference genes in pamericana after s litura infestationin order to conductthe gene expression analysisamong different plant species of phytolaccaceae the dataof the three plant species were also compared togetherwhen compared the data in germinating seeds of threeplant species various genes were recommended by thethree methods the combination of plant species underother experimental conditions showed that the pairwisevalues of almost all the combination were higher thanthe cutoff value of exceptthe combination ofpamericana and pacinosa where five reference geneswere recommended for data normalization as well as thecombination of sliturainfested pamericana and sliturainfested picosandra where three reference geneswere suggested these results indicated that no particular gene was expressed constantly across different plantspecies even though these plants are congeners therefore reference genes should be employed appropriatelyunder the relevant experimental conditionsthe research has provided transcriptomewide fulllength isoforms of pamericana and picosandraproviding insights into invasive success of pamericanaguidelines for selecting appropriate reference genesunder different tissues in one plant species or amongvaried plant species were recommended further no particular gene was expressed constantly under differentexperimental conditions indicating the necessity of reference gene identification these results would facilitatethe exploration of functional and comparative genomicsstudies in phytolaccaceae to better understand plantbiologymethodsplant and insect materialsplants of p americana °²n °²e p icosandra°²n °²e and p acinosa °²n °²eused in this study which was named m k and q firstwere collected in yunnan china sampling was permitted when conducted complying with locallegislationthe formal identification of the samples were conductedby chao chen botany major of laboratory of ecologyand evolutionary biology state key laboratory for conservation and utilization of bioresources in yunnanyunnan university according to flora of china vol5“ flora of north america vol43“ chinese virtual herbarium httpwwwcvhaccn and global plants on jstor httpplantsjstor dna identification was also employed according tothe its2 region of nuclear ribosomal dna one of themost widely used dna fragments in plant molecularsystematics at the generic and species levels and the 0cliu bmc plant biology page of chloroplast psbatrnh intergenic region all voucher specimens were maintained at an experimental fieldof laboratory of ecology and evolutionary biology statekey laboratory for conservation and utilization of bioresources in yunnan yunnan universitytissues of leaves stems roots and flowers from oneindividual plant of p americana or p icosandra werecollected individually from the wild in yunnan provinceand no permission is needed for collecting theses samples each sample was flash frozen in liquid nitrogen andstored at ˆ’ °c for further experimentsshop101732681taobaocomthird instar larvae of spodoptera litura were purchased from henan jiyuan baiyun industry co ltdchinaand then werereared on artificial diet in a climate chamber h at °c with light and h at °c without light for further usefor reference gene evaluation seeds of p americanap icosandra and p acinosa were collected first from thewild in yunnan province and no permission is neededthe seeds were sown separately in agar plates andcultivated in the climate chamber after d five germinating seeds of one plant species were collected togetheras one sample for subsequent experiments each plantspecies have three replications two weeks later othergerminating seeds of each species were transplanted intoplastic pots cm diameter and cm height withsoil jiangsu peilei matrix technology development coltd china and cultivated with adequate water in artificial chambers with same conditions as described abovefour months later leaves stems roots and flowers ofeach plant species were collected individually simultaneously six larvae s litura of third instar were employedto infest on p americana p icosandra or p acinosawith one insect per leaf control treatments were herbivore free after h infestation leaves stems and rootsof these three plant species were harvested individuallyall samples collected were flash frozen in liquid nitrogenand stored in ˆ’ °c for subsequent assays and threereplicates were conducted for each treatmentnucleic acid extraction and assaysgenomic dna was isolated from the leaves of differentplant species following protocols provided by dnaquickplant system tiangen biotech co ltd beijing chinathen it was employed as the pcr template for plantspecies identificationpurekitplanttotal rnas from different tissues was prepared usingrnapreppolysaccharides polyphenolicsrich tiangen biotech co ltd beijingchina according to the manufacturer™s instructionsthe rna quality and purity were measured by using ananophotometer n60 implen germany and the agilent bioanalyzer system agilent technologies causa samples only with a ratio of to a ratio between and and a rin value morethan were chosen for the sequencing library construction an equal amount of total rnas from four different tissues of the same plant species were mixed asone sample for fulllength transcriptome sequencingtotal rnas from the samples collected for referencegene evaluation was also extracted individually as described above for each sample cdna was prepared byusing μg of total rna following the recommendedinstructions of fastquant rt kit with gdnase tiangenbiotech co ltd beijing chinapacbio cdna library preparation and smrt sequencingfulllength cdna was synthesized by using the smarter„¢ pcr cdna synthesis kit clontech ca usathe generated cdna was then reamplified using pcrafter end repairing smrt adaptor with a hairpin loopstructure was ligated to the cdna via exonucleasedigesting the cdna library was constructed after quality measurement of the cdna library smrt sequencingwas performed using the pacific bioscience sequel platform following the provided protocolillumina cdna library construction and secondgenarationsequencingthe extracted mrna was purified using oligo dtattached magnetic beads fragmentation was conducted inthe nebnext first strand synthesis reaction bufferfirststrand cdna was acquired based on the randomhexamers and then the secondstrand cdna was synthesized with dntps rnase h and primestar gxldna polymerase the synthesized cdna was purifiedwith ampure xp beads after end repairing adding polya and adaptor ligation ampure xp beads were used forsize selection the generated cdna was then amplifiedfor building cdna libraries the qualified libraries werepair end sequenced on illumina nova platformquality filtering and error correction of long readsraw smrt sequencing reads were filtered by removingpolymerase reads less than bp and sequence accuracyless than after removing adaptor subreads were obtained clean data was produced with subreads morethan bp ccss were produced from clean data withparameters of full passes and accuracy over after examining the coexistence of ² and ² adaptorsand poly a tail fulllength transcripts were selectedduring the processes of library preparation the chimericsequences formed by the direct linkage of two cdnatemplate strands due to the low concentrations ofadaptor or smrtbell are called artificial chimeric sequences the nonchimeric sequences in the fulllength 0cliu bmc plant biology page of transcripts are the fulllength nonchimeric flncsequencesas smrt sequencing generates a high error rate it isnecessary to perform error correction iterative clustering was used first to obtain consensus isoforms and thefulllength consensus sequences from iterative clusteringfor error correction were refined using quiver [ ]moreover the raw illumina sgs reads were filtered toremove adaptor sequences and low quality reads anderror correction of lowquality isoforms was conductedusing the sgs reads with the software proovread inbriefly the short reads of illumina rnaseq data weremapped to the low quality isoforms and then the basein the low quality isoform was replaced by the particularbase that had the maximum number
Colon_Cancer
in the uk the death toll from severe covid19 is among the highest worldwide1 severe covid19 is characterised by respiratory failure with socalled cytokine storm occurring in some patient subsets2 pathological correlates are required to understand the pathophysiology of covid19 autopsybased histopathological analysis is crucial in this respect in anticipation of the covid19 pandemic our group produced national guidelines for autopsy performance in suspected covid19 cases3covid19 is caused by infection with severe acute respiratory syndrome coronavirus sarscov245 although sarscov2 and its predecessor sarscov causing severe acute respiratory syndrome [sars] are toll similar on a molecular and clinical level covid19 has a lower death rate for covid19 vs for sars and a substantially higher death deaths worldwide from covid19 as of aug vs from sars than sars due to a higher basic reproduction number1 the postmortem findings in patients with sarscov infection included diffuse alveolar damage dad splenic and nodal lymphocyte depletion haemophagocytosis renal acute tubular injury cerebral oedema microthrombosis and adrenalitis with necrosis with intracellular sarscov detected in the lungs kidney brain and haematological ans6 various autopsy series on covid19 have begun to emerge in the literature7“ here we document the major pathological lancet microbe published online august 101016 s2666524720301154department of cellular pathology northwest london pathology b hanley mbbch prof k n naresh md c roufosse phd j weir frcpath prof r goldin md p viola md m osborn frcpath and department of hepatology p manousou phd imperial college london nhs trust london uk centre for haematology b hanley prof k n naresh and centre for inflammatory diseases c roufosse department of immunology and inflammation department of infectious disease prof g s cooke frcp a abdolrasouli phd o c swann mres l baillon bsc r penn msc prof w s barclay phd and department of metabolism prof m thursz md faculty of medicine imperial college london london uk department of histopathology royal brompton and harefield nhs foundation trust and national heart and lung institute imperial college london london uk prof a g nicholson dm renal and transplant centre hammersmith hospital imperial college healthcare nhs london uk r corbett phd department of neuropathology kings college hospital london uk prof s alsarraj frcpath death investigation committee royal college of pathologists london uk m osborn and nightingale nhs hospital london uk m osborn correspondence to dr brian hanley department of cellular pathology northwest london pathology charing cross hospital campus london w6 8na uk bhanleyimperialacukwwwthelancetcommicrobe published online august 101016s2666524720301154 s 0cresearch in contextevidence before this studycovid19 is a new disease and comprehensive descriptions of the histopathological findings at autopsy are scarce we reviewed the literature available on covid19 autopsy findings up to and including may for this we searched pubmed and google scholar databases with no language restrictions using the search terms œcovid19 œsarscov2 œhistology œautopsy and œpostmortemadded value of this studyour series focused on providing a comprehensive description of the histopathological findings in patients with severe fatal covid19 and correlating these findings with data on viral tropism the most prominent findings included diffuse alveolar damage thrombosis haemophagocytosis and immune cell depletion several novel autopsy findings in patients with covid19 were also described including pancreatitis pericarditis adrenal microinfarction secondary disseminated mucormycosis and brain microglial activationimplications of all the available evidenceour study supports the existing clinical and autopsy literature that identified diffuse alveolar damage thrombosis immune cell depletion and macrophage activation as the most prominent pathological features in covid19 other factors including acute kidney injury pancreatitis pericarditis secondary fungal infections and preexisting liver disease require further investigation the presence of ongoing viral replication in late stage covid19 supports the continued use of antiviral therapy even at a point in illness when immunopathology is dominantsee online for appendixfindings of ten postmortem examinations done on patients with clinically confirmed covid19methodspatient selectionfor this study eligible patients were older than years with premortem sarscov2 infection and covid19 listed clinically as the direct cause of death under part on the medical certificate of cause of death [mccd] consent was obtained for all included patients according to the human tissue authority codes of practice by a member of the trust core postmortem consent team consent rate was · ten of patients exclusion criteria included extended postmortem interval before autopsy days and patients with covid19 contributing but not directly leading to death under part of the mccd patients were from imperial college national health service nhs trust nine patients london uk and royal brompton harefield foundation nhs trust one patient london uk premortem sarscov2 infection was identified using the coronavirus typing multiplextandem pcr highplex system aus diagnostics chesham uk ethical approval for this project was provided by the imperial college healthcare tissue bank r20012autopsy proceduresfull autopsies were done on nine patients pm1“ and one patient underwent percutaneous biopsy sampling heart lungs pancreas kidneys and liver using percutaneous biopsy under ultrasound guidance pm10 full postmortem examinations included standard sampling and were done according to royal college of pathologists guidelines3 eight different regions of the brain were sampled for each full neuropathological examination all tissue samples were fixed in formalin for a minimum of h before embedding histochemical stains and immunohistochemistry were applied according to local protocols appendix p ans were reviewed by subspecialist pathologists in lung agn and pv haem ato pathology and immune pathology knn liver rg gastrointestinal mo neuropathology sas and renal pathology cr integrated interpretation was done by a subspecialty autopsy pathologist bh and mo all cases were reviewed independently by at least two pathologistspcr proceduresfresh tissue for quantitative rtpcr qrtpcr analysis was processed within the biosafety level facilities at st mary™s hospital london uk approved by the uk health and safety executive and in accordance with local rules at imperial college london total rna was obtained from fresh tissue samples by use of trizolchloroform extraction followed by precipitation and purification using the rneasy kit qiagen hilden germany qrtpcr against e gene rdrp and subgenomic rna was done as described elsewhere1617 in patient pm5 total fungal genomic dna was extracted from four to five ribbon slices of a formalinfixed paraffinembedded lung tissue block purified dna was amplified with pcr for panfungal and mucoralesspecific targetsstatistical analysisall data was analysed using spss software version and expressed using median iqr and percentagerole of the funding sourcethe funder of the study had no role in study design data collection data analysis data interpretation or writing of the the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publicationresultsbetween march and april ten patients were included in the study the median age at death was years wwwthelancetcommicrobe published online august 101016s2666524720301154s 0ciqr “ seven of ten patients were men three were women and most patients were white or asian nine hypertension four patients and chronic obstructive pulmonary disease three were the most common contributing factors to death according to mccd all ten patients developed fever and had at least two respiratory symptoms or signs cough shortness of breath reduced oxygen saturations or pleuritic chest pain during their early presentation of eight patients assessed for inflammatory markers all had elevated inflammatory markers these features were either apparent upon presentation to hospital eight of ten patients or developed in an inpatient two patients pm8 and pm9 most patients died within weeks of symptom onset seven patients and were not intubated or ventilated six patients four patients were intubated during their presentation pm2 for days pm5 for days pm6 for less than day and pm7 for days the median bodymass index bmi was in the obese range · iqr ·“· and more patients were obese according to bmi at post mortem five of nine than indicated on the mccd one of ten the median interval between death and postmortem examination was days iqr ·“· although the limited post mortem had a shorter interval less than h after death detailed clinical case vignettes are available in the appendix p and clinical data are summarised also in the appendix p all patients had dad six showed purely exudative phase dad and four showed a mixture of exudative and anising dad appendix p figure three of four patients with anisingphase dad had spent a substantial period on a ventilator days days and days florid acute bronchopneumonia and ventilatorassociated pneumonia were not noted in this series although mild interstitial neutrophilic inflammation three of ten patients and patchy acute bronchopneumonia three patients were observed interstitial macrophages were prominent macrophages were accompanied by scattered plasma cells mild or moderate lymphocyte inflammation was present in all ten patients although focal lymphocyte cuffing of small vessels was noted in six patients we noted that lymphocytes in the lung were predominantly cd4positive t cells cd56positive natural killer cells were rarely found occasionally a patient had small aggregates of small b cells chronic bronchiolitis was seen in most patients nine of ten no granulomas or viral inclusion were seen invasive mucormycosis was noted in one patient pm5 figure and confirmed with mucoralesspecific pcr the mucormycosis was vasculocentric and disseminated involving the hilar lymph nodes heart brain and kidney in the same patientmacroscopic two of nine patients and microscopic eight of nine pulmonary thromboemboli were frequent observations appendix p figure both fibrinrich and plateletrich thrombi were identified in smallsized and mediumsized vessels and within the capillaries in alveolar septa figure external examination findings of deep venous thrombosis were not noted very focal lymphocytic vasculitis was identified in one patientthrombotic features were universal in this cohort and all nine patients who underwent a full autopsy had at least one microthrombosis or macrothrombosis in a major an one of nine patients had a macroscopic acute coronary thrombosis in the right coronary artery whereas five patients had thrombi in the microcirculation of the heart on histological analysis coronary artery disease was negligible or mild in most patients seven of nine acute myocardial ischaemic damage h old was noted in the patient with an acute coronary artery thrombus figure 2a pm1 a mottled myocardium and subendocardial contraction band necrosis was noted in a acebdf 03m µm µmfigure pulmonary pathological findings in patients with covid19a macroscopic subpleural petechial haemorrhage in a 24yearold man pm6 b hyaline membranes indicative of exudative phase diffuse alveolar damage in a 79yearold woman pm9 at 20x magnification c cd61 immunohistochemical staining indicating plateletrich microthrombosis in alveolar capillaries pm6 d squamous metaplasia in a 61yearold man pm1 with exudative phase diffuse alveolar damage at × magnification e interstitial multinucleated giant cells in a 79yearold man pm7 with anising phase diffuse alveolar damage at × magnification the top right insert is of multinucleated giant cells showing positive cd68 staining indicative of macrophage lineage the bottom left insert shows absence of staining for cytokeratins f periodic acid schiff staining indicating wide irregular aseptate and ribbonlike hyphae with openangle branching and a vasculocentric pattern indicative of mucormycosis in a 22yearold man pm5 the insert is a grocott silver stain highlighting mucormycosis at 20x magnificationwwwthelancetcommicrobe published online august 101016s2666524720301154 s 0csecond patient pm2 whether the contraction band necrosis was related to ischaemia or inotropic medication received in the intensive care unit is uncertain appendix p pm1 and pm2 were the two patients with the highest active viral load detected in the heart a single patient had a right atrial thrombus pericarditis was acegbdfh µm µm 03m µmfigure thrombotic features identified at autopsy in patients with covid19a macroscopic right coronary artery thrombosis arrow in a 61yearold man pm1 with exudative phase diffuse alveolar damage b macroscopic pulmonary thromboembolism arrow in a 97yearold man pm8 c thrombus in the lung of a 79yearold woman pm9 on haematoxylin and eosin staining at 20x magnification the insert shows cd61 immunohistochemistry indicating moderate staining for platelets d plateletrich thrombus in the mediumsized vessels surrounding the heart in a 61yearold man pm1 the insert shows strong cd61 staining for platelets periodic acid schiff staining showing a glomerular microaneurysm arrow e and microthrombi within glomerular capillary loops arrow f at 40x magnification indicative of thrombotic microangiopathy in a 97yearold man pm8 macroscopic splenic g and hepatic h infarction in a 22year old man pm5identified in two patients one patient showed florid fibrinous pericarditis containing fungal hyphae pm5 while the other showed only microscopic acute pericarditis appendix p figure the median heart weight was high g and four of nine patients had left ventricular hypertrophy nonbacterial thrombotic marantic endocarditis was noted in one patient pm5 with no known history or autopsy findings consistent with malignancy or chronic disorder associated with nonbacterial thrombotic marantic endocarditis appendix p figure pm5 had disseminated mucormycosis and numerous other thrombotic features appendix p cardiac amyloidosis and right atrial thrombosis were identified in one of ten patients pm8 appendix p lymphocyte depletion involving specific compartments and increased phagocytosis were prominent findings appendix p figure increased phagocytosis of other cells was identified in the sinusoidal macrophages of the red pulp of the spleen in four of seven patients sinus histiocytes of hilar lymph nodes in three of six and bone marrow four of eight phagocytosis was identified in at least one of these ans in six of nine patients bone marrow haemophagocytosis was prominent in two patients pm4 and pm8 and focal in two patients pm7 and pm9 depletion of periarteriolar tcell sheaths within the white pulp was observed figure red pulp was generally congested showing reduced numbers of cd8positive t cells plasma cells were variably prominent and sinusoidal histiocytes showed phagocytosis of red blood cells and other cells to varying extents both igmpositive and iggpositive plasma cells were identified and they were polytypic for lightchain expression figure lymph nodes showed preservation of follicles and relative depletion of paracortical areas medullary areas showed prominence of plasma cells and histiocytes were prominent in the sinuses bone marrow samples showed reactive changes with trilineage hyperplasia and prominence of plasma cells and histiocytes were a common finding a necrotising granuloma was noted in a single hilar lymph node in one patient and acidfast bacilli were noted on ziehl neelson staining appendix p all spleen and lymphoid material examined with immuno histochemistry were negative for epsteinbarr virus and cytomegaloviruspancreatitis was noted in two of eight patients pm5 was a 22yearold man with frank necrotichaemorrhagic pancreatitis and secondary mucor mycosis figure no fungal hyphae were noted in the pancreas pm8 was a 97yearold man who showed no substantial macroscopic pancreatitis although micro scopic acute inflammation within the pancreas and periadrenal fat necrosis was noted figure a third of patients three of nine showed patchy areas of infarcttype adrenocortical necrosis with one patient showing anising microthrombi in adrenal vessels figure no wwwthelancetcommicrobe published online august 101016s2666524720301154s 0cadrenalitis was noted two of nine patients showed chronic inflammation in the thyroid with follicular epithelial cell disruption however the significance of this finding is uncertainmedian combined kidney weight was within normal range at g iqr “ salient renal pathology findings were acute tubular injury in all nine patients underlying moderate cortical scarring of uncertain cause in one patient glomerular microaneurysm and thrombi in one patient figure and rare thrombi in interlobular arteries in four patients pm6 24yearold man of arterial intimal thickening than expected for that age appendix p we observed no evidence of focal and segmental glomerulosclerosis diabetic glomerulopathy or glomerulonephritisdegree had a higher large droplet fatty change was seen in most patients seven of eight cirrhosis or bridging hepatic fibrosis were noted in three patients no liver thrombosis was identified histologically but one patient showed macroscopic liver infarction figure the median liver weight was g iqr “ and three of nine patients showed hepatomegaly liver weighing g two patients pm4 and pm7 showed marked autolysis and were not included in analysismoderate to intense microglial activation was the most prominent pathological feature in the cns five of five patients mild tcell infiltration was noted around blood vessels and capillaries in all five patients but b cells were absent we found ischaemic changes of variable extent in the neurons of the cortex and in the white matter detected by bapp β amyloid precursor protein stain however no necrosis of brain tissue or extensive infiltration of inflammatory cells in brain parenchyma or meninges was observed on histological examination although one of nine patients showed macroscopic haemorrhagic transformation in a large recent cerebral infarction in the distribution of the middle cerebral arterytissues from five patients were analysed for presence of viral genomes against e gene and indications of viral replication against subgenomic rna transcripts by qrtpcr viral rna was present in respiratory tract samples including lung of all five cases analysed in addition two of three patients had detectable viral rna in the nasal epithelium and four of five patients in the trachea evidence of viral genomes outside the respiratory tract was found for all five patients but the distribution and viral loads varied case by case figure 5a viral genomes were also detected using a different qrtpcr targeted at rdrp gene and patterns were consistent between the two sets of primers data not shown a third primer set that detected subgenomic rna indicated virus replication in all tissues examined with variation between patients in levels and distribution figure 5bace µmbdf µm µmfigure other notable autopsy findings in patients with covid19a contained aortic dissection green arrow and fibrinous pericarditis red arrow in a 22yearold man pm5 insert is a haematoxylin and eosin stain image of the pericardium showing fibrinous pericarditis 10x magnification b adrenocortical microinfarcts in a 79yearold woman pm9 with reendothelialising thrombus in small adrenal vessels highlighted by cd34 insert bottom left and haematoxylin and eosin insert top right marantic endocarditis c highlight with haematoxylin and eosin staining bottom left at 10x magnification and necrotising haemorrhagic pancreatitis d in a 22yearold man pm5 with covid19 and a secondary fungal lung infection e periodic acid schiff staining showing a granular cast arrow indicative of acute tubular injury in a 24yearold man pm6 20x magnification f microscopic acute pancreatitis on haematoxylin and eosin staining in a 97yearold man pm8 20x magnificationdiscussionin this series we have described the major pathological findings identified at autopsy in ten patients who died of severe covid19 the most consistent findings were dad thrombosis haemophagocytosis and immune cell depletion although unexpected pathologies that are probably related to sarscov2 infection were also identifieddad was the most consistent and prominent feature in our series and others78 the specific phase of dad probably represents the degree and chronicity of the offending insult sarscov2 infection in relation to the time of death this is similar to previous coronavirus epidemics6 the conclusion by copin and colleagues7 that covid19related lung injury œis not diffuse alveolar damage might relate to their sampling strategy and wwwthelancetcommicrobe published online august 101016s2666524720301154 s 0cadbc µm µm µm µm µmef µm µm µmfigure pathological findings in haematological ans in patients with covid19tcell depletion in the spleen of a 79yearold woman pm9 with covid19 haematoxylin and eosin staining of the spleen at 10x magnification a cd20 staining of spleen indicating presence of b cells b 10x magnification with the insert showing the same region at higher power 20x magnification and cd3 staining of spleen indicating depletion of t cells c 10x magnification with the insert showing the same region at higher power 20x magnification bone marrow phagocytosis in a 97yearold man pm8 with covid19 haematoxylin and eosin staining of a well preserved bone marrow with an arrow indicating presence of phagocytosis d 40x magnification and cd68pgm1 staining of bone marrow indicating presence of phagocytosis 20x [e] and 40x [f] magnificationchronicity five patients had spent approximately weeks on a ventilator barton and colleagues8 described prominent acute bronchopneumonia as the major finding in one of two patients although the authors acknowledge that this was probably affected by aspiration in their patient with muscular dystrophy reports of lung histology in early covid19 also suggest a degree of lymphocytic pneumonia although dad is probably superimposed on this over time in the majority of fatal cases7 pulmonary macrophage infiltration and multinucleated giant cell reactions are prominent similar to other series8“ definite evidence of in covid19 will require quantitative analysis comparing tissues from covid19 patients with dad associated with other conditions and unaffected tissues several cases of invasive pulmonary aspergillosis have been reported in patients with severe covid19 pneumonia18 to our knowledge this is the first description of histologically proven mucormycosis in patients with covid19 and suggests that other human fungal pathogens including members of mucoromycotina can complicate covid19associated infectionslymphocyte depletion tissuerelated numerous clinical features including raised serum ddimer concentrations raised procalcitonin concentrations and imaging findings suggest thrombosis is prominent in patients with covid192 thrombotic features were universal among patients who underwent full autopsies all nine patients had thrombi in at least one major an and have been noted to be prominent in other covid19 autopsy series15 in a retrospective study of autopsies in patients with acute respiratory distress syndrome and dad of various causes only showed thrombi within the small vessels of the lung despite sampling of every lobe of the lung19 another study used postmortem angiography and identified thrombi in nearly all cases of acute respiratory distress syndrome from various causes20 whether thrombosis in covid19 is more common than in other causes of dad remains uncertain however our data support thrombosis as being a striking feature in these patients a study suggested endotheliitis as a prominent feature in patients with severe covid1910 but this was not a prominent feature in our patients importantly limited post mortem or postmortem crosssectional imaging are likely to underrepresent the true extent of thrombosis particularly microthrombosis and its impact on patient death the extent of cardiomegaly fibrointimal thickening of renal blood vessels and obesity in our series supports a contribution of hypertension beyond that noted clinically only four patients had hypertension documented on the mccda raised cytokine profile has been documented in a subset of patients with severe covid192 consistent with this haematological ans in our series showed prominent phagocytosis in several patients which has not been documented in previous series21 of the four patients with bone marrow haemophagocytosis one patient pm7 showed mild transaminitis hyperbilirubinaemia elevated serum ferritin concentrations and fever of ·°c however most clinical data were insufficient to assess the presence of haemophagocytic lympho histiocytosis wwwthelancetcommicrobe published online august 101016s2666524720301154s 0ca substantial feature in covid19 is lymphocyte depletion and this is supported in our series by the spleen and lymph node findings when compared with those with mild disease patients with severe covid19 tend to have a higher neutrophil to lymphocyte ratio and higher cd4positive to cd8positive tcell ratio22 additionally a negative correlation exists between peripheral blood lymphocyte count and viral copy number22 we have corroborated this evidence by documenting a low number of t cells especially cd8positive t cells and foxp3positive regulatory t cells in the spleen and lymph nodes in severe fatal covid19 notably normal plasma cell both igm and igg positive response was present in haematological ans in most patientsthe extent to which anspecific pathologies relate to direct viral replication or consequent immunological and cardiovascular complications is of clinical relevance we report here evidence of viral genomic rna outside the respiratory tract this finding is in agreement with several previous studies that have identified viral genomes by qrtpcr in postmortem tissues including the colon14 spleen14 liver1423 skin24 heart23 and brain25 we also report detection of subgenomic rna a product that is only produced in actively infected cells a report identified low viral load in the brain of three of patients with covid19 but could not detect the virus in subsequent immunohistochemistry and concluded that the viral genomes might have been present in the blood25 although we cannot exclude that the rnas detected in our series were similarly carried to the site of sampling in blood the distribution of rna in different tissues varied widely between postmortem casespm3 and pm4 appear to have died earlier in the disease course days after symptom onset and had higher viral loads in the respiratory tract than other patients whereas pm3 and pm4 died after long stays in intensive care units and had either lower overall viral rna pm5 or higher viral rna outside the respiratory tract pm2 pm1 and pm2 were the only patients in whom we detected viral rna and subgenomic rna in the heart and are the only two patients with evidence of acute myocardial injury moreover unlike the previously mentioned study where virus detected in the brain was times less than that detected in the lung the number of viral genomes detected in external tissues in our series was frequently of similar or even higher levels than that found in the respiratory tree one study has detected sarscov2 infection of the endothelium in the vasculature of the skin and lung by immunofluorescent staining for viral antigens24 it is not obvious what determines spread and tropism of sarscov2 outside the respiratory tract several studies have reported ace2 expression levels that were higher in some ans than in others26 the sites of highest expression did not correlate with areas of most severe pathological involvement in our series we should note that cellular receptor status is one of many things that determines the degree of anr latot g 03 rep seipocenege larivpm1pm2pm3pm4pm5abtcbone marrowbrainheartileumkidneyliverspleentonguetrachealungnasal epitheliumfigure tissue tropism of sarscov2 in postmortem samplesfresh tissues were collected from a subset of postmortem examinations and viral load quantified by use of qrtpcr targeting the viral e gene a detection of viral rna was verified by use of qrtpcr against the viral polymerase gene data not shown tissues were additionally tested for subgenomic viral rna transcripts b dotted lines indicate the limit of detection as ascertained by negative control data are included for a 61yearold man pm1 a 64yearold man pm2 a 69yearold woman pm3 a 78yearold man pm4 and a 22yearold man pm5 qrtpcrquantitative rtpcr sarscov2severe acute respiratory syndrome coronavirus pathological involvement other aspects are likely to play a role such as route of transmission acid lability in the stomach coreceptors expression of proteases required to activate entry of virus into the cell eg tmprss2 possibly other yet unidentified host factors and invivo routes of dissemination the evidence of ongoing replication late in disease supports the use of antiviral therapy even at a point in illness when immunopathology is dominantpancreatic pathology was a major unexpected pathology in this series which had not been previously reported in covid19 autopsies or large clinical covid19 series2 a 22yearold man had haemorrhagic necrotic pancreatitis pm5 although this patient also had disseminated mucormycosis he had evidence of viral persistence in the lung and no fungal hyphae were identified in the pancreas on histological sections the other patient with wwwthelancetcommicrobe published online august 101016s2666524720301154 s 0cpancreatic pathology had microscopic evidence of pancreatitis that could be missed at autopsy without adequate sampling the pancreas is a classic site of unexpected pathology identified at autopsy27 serum amylase is not part of the routine care bundle for covid19 in our trust whether the pancreatitis was related to sarscov2 iatrogenic comorbidities or secondary infection is not clear in our study most cases had evidence of hepatic steatosis which is consistent with clinical findings that obesity is a risk factor for poor outcome in covid19 and liver cirrhosis or bridging fibrosis was prominent in this cohort28 from these data nothing suggests direct viral inflammation of the liverinfection or other causes the interval between time of death and autopsy impaired histological interpretation in many ans and affected antigen preservation postmortem endothelial stripping did affect the endothelial interpretation in our study however a subs
Colon_Cancer
" laparoscopic tumorspecific mesorectal excision tsme preserving the left colic artery and superiorrectal artery is still a technically challenging procedure we conducted this study to demonstrate the feasibility ofthis procedure for upper rectal cancermethods a total of patients with upper rectal cancer were retrospectively analyzed in our cancer centerbetween april and april these patients were treated with either laparoscopic tsme n orlaparoscopic total mesorectal excision tme n in the tsme group the left colonic artery and superior rectalartery were preserved while they were not in the tme groupresults the operation time in the tsme group was longer than that in the tme group ± min vs ± min p furthermore the number of resected lymph nodes in the tsme group was greaterthan that in the tme group ± vs ± p the blood loss between the tsme and tmegroups was not significant no mortality occurred in either the tsme or tme groups one patient in the tme groupunderwent conversion to laparotomy the total postoperative complication rates in the tsme and tme groups were and respectively there was no difference in severe complications between the two groupsanastomotic leakage and stenosiss laparoscopic tsme preserving the left colic artery and superior rectal artery can be safely conductedfor upper rectal cancerkeywords laparoscopic surgery rectal cancer tumorspecific mesorectal excision superior rectal artery leftcolonic artery tme correspondence 237721898qqcom 250537471qqcomhuxiang_zc1978sinacom chi zhang haotang wei wenqing hu and yueming sun contributedequally as joint first authors7department of general surgery yizhen people™s hospital clinical medicalcollege yangzhou university yangzhou jiangsu province china3department of gastrointestinal surgery changzhi people™s hospital theaffiliated hospital of changzhi medical college changzhi shanxi provincechina1department of gastrointestinal surgery the first affiliated hospital of dalianmedical university dalian liaoning province chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhang world of surgical oncology page of introductiontotal mesorectal excision tme is an important surgical technique to prevent the local recurrence of rectal cancer on the other hand tme may not besuitable for every case of rectal cancer such as rectosigmoid junction and upper rectal cancers the resection range of tme reaches cm below the inferiorborder of the tumor and has acquired an adequatecure rate reported in previous studies for patientswith rectosigmoid junction and upper rectal cancers this tumorspecific resection according to thetumorsite or t staging is called tumorspecificmesorectal excision tsme itsudeck™s critical point at the rectosigmoid junction isdescribed as the point of origin of the last sigmoid arterial branch originating from the inferior mesenteric artery ima the anastomosis between the lastsigmoidal artery and superior rectal artery sra is absent in some people to avoid the risk of postoperativeischemic necrosis anastomotic leakage colitis and delayed stricturetosudeck™s point for cases where anastomosis may be absent or insufficiently present in addition the rate ofis desirable to ligate proximalabsence of the left colic artery lca is which maybe associated with a risk of anastomotic leakage due toinsufficient vascularization of the proximal colonic conduit this study introduces the procedure and technicalpoints of laparoscopic tsme with preservation of thelca and sra the operation is still a technically challenging procedure we conducted this study to demonstrate the feasibility of this procedure for upper rectalcancer and shortterm prognosismethodspatientslaparoscopic tsme preserving the lca and sra wasperformed on patients with upper rectal cancer fromapril to april in the same period patients with upper rectal cancer underwent standardtme surgery this study was conducted in accordancewith approved guidelines this study was approved bythe institutional review board of the first affiliatedhospital of dalian medical university written informedconsent was obtained from all patientsfig ima 3d cta 0czhang world of surgical oncology page of equipmentangled ° 10mm diameter 3d laparoscope insufflation equipment and bipolar electrosurgical deviceaesculap german harmonic vascular closure systemjohnson usa 10mm and 5mm port trocars teleflexmedical usa laparoscopic linear staplers mm inlength covidien usa hemolock polymer lockingsurgical clips teleflex medical usa and a circularstapler ethicon endosurgery usa were used in thisstudypreoperative preparationinferior mesenteric artery ima 3d cta examinationshould be performed before the operation to assess themesenteric vascular vessel types fig intestinal preparation was performed days before the operation andprophylactic intravenous antibiotics were used beforethe operation for min central venous catheterizationwas performed after general anesthesia the surgicalposture was the starboard lithotomy position with thehead lower and feet higherthe operating surgeon and camera assistant stood onthe patient™s right side and the first assistant stood atthe patient™s left foot side the laparoscopic monitor wasplaced on the patient™s right foot side the trocar for thelaparoscope was inserted from the right paraumbilicalside and four ports were used as working ports fig surgical techniquesthis surgical technique was characterized by thoroughlymph node dissection based on neurovascular preservation and dissection of the left colon and sigmoid andfig position of the trocarupper rectal vessels along the inferior mesenteric vesselsthe region of operation was the superficial layer of thenerve sheath on the vascular surface the left colonicand superior rectal vessels needed to be preserved andthe vascular branch from the sigmoid vessels and theblood vessels from the superior rectal vessels to the intestinal wall were selected and severed according to thetumor positionfirst we adopted a lateral approach by opening themonks™ white line along the descending and sigmoidcolon reaching the splenic flexure as the cephalad dissection point the correct plane of dissection wasachieved by toldt™s fascia we usually used bipolar electrosurgical devices and bipolar scissors to separate thiscorrect plane with gentle blunt and sharp dissectionthe ureter and other retroperitoneal structures weresafely protected by staying in this plane we continuedto dissect along the plane to the root of the ima thehypogastric nerves were visible the nerves were carefully protectedthen the dissection began at the position of the sacralpromontory the junction of the sigmoid mesentery andretroperitoneum from the previous dissection plane in thefirst step ideally we dissected the presacral space belowthe sra from the left side across the midline to the rightside attentively protecting the hypogastric nerves whileusing a bipolar electrosurgical device fig 3a the distaldissection endpoint was approximately “ cm below thetumor we needed to open the peritoneal reflection anddissect the lateral ligament of the rectum by protectingthe neurovascular bundle nvb using a harmonic vascular closure system in some patients we placed the dissected colon and mesocolon to the right celiac side andthoroughly revealed the left side of the mesocolon wecarefully employed dissection in the correct plane on thevessels to avoid tissue damage for the realization of enbloc resection the technique in this step is to identify therelationship between the left colic artery inferior mesenteric vein imv to the ima and sra and the branch ofthe arteriae sigmoideae fig 3b this vascular bundle canbe traced from the origin of the ima to the rectal segmentapproximately “ cm below the inferior border of thetumor fig 3cthe second step was performed using a medial approach this step involved thorough lymph node dissection based on neurovascular preservation the leftcolonic and superior rectal vessels need to be preservedand the sigmoid vessels and vessel branch from the superior rectal vessels to the intestinal wall were selectedand severed according to the tumor positiondissection at the correct presacral space and cephaladdissection to the ima could be employed our generalmedial approach was to begin at the presacral space andobtain a connection with the plane ofthe lateral 0czhang world of surgical oncology page of fig a dissection the presacral space below the superior rectal artery sra approached from the left side across the midline to the right sideattentively protected hypogastric nerves while using a bipolar electrosurgical device b identification of the relationship between left colic arteryimv to the ima and sra and the branch of the arteriae sigmoideae c tracing this vascular bundle from the origin of the ima to the rectumsegment approximately “ cm below the inferior border of the tumor d ligation of arteriae sigmoideae and vascular branch from sra eligation of arteriae sigmoideae and preserving left colonic vasculature f excision of the mesorectum just underneath the rectal wall about “cm and avoiding injury to the rectal wall and sra g tsme preserving left colic artery and superior rectal arteryapproach pelvic dissection was performed from the entrance of the pelvic cavity down to the pelvic floor wecould identify both the hypogastric nerve fibers and pelvicnerve by using highdefinition 3d laparoscopy and preservethem the imvleft colic artery bundle was then carefullytraced to the junction position from the ima and lymphnode no253 was dissected the pelvic nerves and ureterwere already carefully insulated and the circumference ofthe ima could be revealed the mesocolon could be freedfrom the retroperitoneal position by anterior dissection bygently applying a bipolar electrosurgical device we dissected the sra and blood vessels from the sra to the intestinal wall and dissected lymph nodes no252 andno251 at this point we had completed lymph node dissection and completely clarified the relationship betweenthe lca imv ima sra and arteriae sigmoideae finallywe ligated the arteriae sigmoideae and vascular branchfrom the sra into the intestinal wall fig 3d while preserving the left colonic vasculature fig 3e energy devicesand hemolocks were used widely in this step 0czhang world of surgical oncology page of after the above procedure was completed we separated the rectal wall from the mesorectum with an adequate distance from the tumor in accordance with thet stage and position of the tumor using a harmonic vascular closure system in order to provide enough spaceto insert an endoscopic linear stapler we excised themesorectum about “ cm just underneath the rectalwall fig 3f careful surgery was performed to avoid injury to the rectal wall and sra then the endoscopic linear stapler was fixed the rectum was transected andsatisfactory tsme preservation of the left colic and superior rectal arteries was shown fig 3glastly a small 5cm incision was made at the leftlower abdomen and the specimen was taken outside ofthe abdomen and transected intraabdominal presacralanastomosis was performed by double stapling techniques after inserting the anvil head of a 28mm circularstapler into the oral side of the sigmoid colon doubledrains were placed and no diverting stoma wasperformedin the tme group the inferior mesenteric artery wassevered at the root the colon was severed cm awayand digestive tract reconstruction methods were similarto the tsme groupstatisticsspss190 version was used for statistical analysis categorical variables were compared using a χ2 test continuous variables were presented as the mean standarddeviation or median range these variables were compared using a mannwhitney u test p values of were considered statistically significantresultsthe general characteristics of the included patients arelisted in table there were men and women in the tsme group and men and women in the tme group the meanage was ± years and ± years in thetsme and tme groups respectively there were no significant differences in preoperative comorbidity tumorsize depth of invasion and lymph node metastasis between groups the average distance between the tumorand anus of the tsme group was ± cm andthe distal margin was ± cm the pathologicalstages of the patients for the tsme group were as follows stage i stage iia stage iib stage iic stage iiia and stage iiib theproportion of patients with normal preoperative carcinoembryonic antigen cea was approximately of patients had cea levels between and ngml of patients had cea levels between and ngml and only patients had cea levels ngmltable clinicopathological features between the tsme andtme groupsfactorsage yearstme n ± tsme n ± p valuegendermalefemalebmi kgm2comorbidity ± ± cardiovascular disease respiratory diseasediabetes mellitushistological type differentiated typeundifferentiated type tumor size mm ± ± t categoryt1t2t3t4n categoryn0n1n2 conversion to open surgery operation time min ± ± blood loss ml ± ± lymph node dissection ± ± the operation time in the tsme group was longerthan that in the tme group ± vs ± p table furthermore the number ofresected lymph nodes in the tsme group was greaterthan that in the tme group ± vs ± p table the blood loss between groupswas not significantly different table the averagehospital stay in the tsme group was a little shorter thanthat in the tme group ± days vs ± days table no mortality occurred in either group one patient inthe tme group underwent conversion to laparotomy dueto bowel ischemia in the distal colon table the totalpostoperative complication rates in the tsme and tmegroups were and respectively table forsevere complications between the two groups anastomotic leakage and stenosis the severity of complicationswas claviendindo classification grades “ and therewas no significant difference between groups 0czhang world of surgical oncology page of tsme n tme n p value ± table postoperative complicationsfactorspostoperative hospital stay days ± mortalitymorbidityabsentpresentanastomotic leakagebleedingabdominal abscessileuswound infectionanastomotic stenosisurinary tract infectionascitesurinary retentionpneumoniacardiacrelated complications discussionin the british surgeon heald proposed tme for rectalcancer and pointed out that the anatomical level of tmewas clear so that the operative quality can be assessed the main concerns were a higher anastomotic leakage ratelonger operative time and higher blood loss after tme lopezkostner pointed out that tme was the standard operation performed for lower rectal cancers tme isnot necessary for cancers of the upper rectum therefore the tsme technique was introduced to achieve satisfactory local control and low morbidity partial mesorectalexcision is applied in tsme according to willian™s report in only of patients had distal intraluminal diffusion cm pollett and nicholls observed that there were no differencesin the local recurrence rate of rectal cancer between distal margins cm “ cm and cm a randomized prospective study of nsabb the national surgicaladjustburst and bowel project showed that the localrecurrence rate was not significantly different betweendistal rectal margins cm “ cm and cm according to the practice parameters for the management of rectal cancer edition a 2cm distal margin is more acceptable than cm but a 5cm distalmargin is still recommended total mesorectal resectiontme should be used for tumors located in the middleand lowerregardless oftwothirds ofwhether itis performed with low anterior resectionlar or combined abdominal and perineal resectionapr for tumors in the upper onethird of the rectumresection of the mesentery can be carried out accordingto the tumor situation and the distance between thethe rectumdistal margin and tumor should be cm the recommended grade was 1a tme was performed according to the distance between the distal margin of the rectal tumor and anus cm while tsme was performed for patients with adistance between the distal end of the rectal tumor andanus of “ cm in the author™s medical departmentoncological outcomes after surgery can be divided intotwo aspects longterm survival and local recurrence ratelaw reviewed patients the 5year local recurrence rate for tme and partial mesorectal excisionpme for proximal cancer was and respectively the disease stage was associated with a higher riskof local recurrence there was no difference in the localrecurrence rates of tme and pme the 5year cancerspecific survival rates with and without tme were similarat and respectively kim reportedthat the 5year cancerspecific survival rate was andthe local recurrence rate was with cases of rectalcancer after tsme with pathologic stages i“iii the riskfactors affecting cancerspecific survival rate were the ptstage pn stage positive distal resection margin and positive circumferential resection margin the risk factors affecting local recurrence were the pn stage positive distalresection margin and positive circumferential resectionmargin another study from a korean reviewed experience in patients with rectal cancer showed that theoverall local recurrence rate was the 5year local recurrence rates were and in stages i iiand iii respectively the 5year cancerspecific survivalrates were and in stages i ii and iiirespectively the risk factors were the pn stage and circumferential resection margin zakir performed an analysis with years of experience in rectal cancer patients who underwent laparoscopic andopen tsme surgery the 5year local recurrence rate was the overall 5year and cancerspecific survival rateswere and respectively there was no difference in the local recurrence rate between laparoscopic oropen resection the overall and cancerspecific survivalrates were and in the laparoscopic surgerygroup and and in the open surgery group respectively the results showed that laparoscopic surgerywas better than open surgery in overall and cancerspecific survival there was no difference in survival in patients with stage i however the survival rates in patientswith stages ii and iii among the laparoscopic surgerygroup were better than those in the open surgery groupwhich shows the superiority of laparoscopic tsme surgery for the longterm prognosis of rectal cancerkorean scholars conducted a study on the safety andprognosis of tsme after neoadjuvant chemotherapy forrectal cancer patients received 5fu with leucovorinchemotherapy and radiotherapy cgy for cycles 0czhang world of surgical oncology page of leadership was tsme was performed “ weeks later the resultsshowed that the overall complication rate was empiricalinternal construction was the 5year survival rate was and the 5yeardiseasefree survival was at present chinasouth korea and the usa have formulated similarguidelines for preoperative radiotherapy and chemotherapy for middle and low rectal cancer but there is nospecific reference data for preoperative radiotherapy andchemotherapy for upper rectal cancer the purpose ofthis paper is to introduce a new method of tsme anddiscuss the safety of the operation longterm survivaland local recurrence have not been discussedtsme surgery based on tme is now accepted as astandard for rectal cancer surgery and laparoscopic rectal cancer resection is accepted widely in the world eventhough it is a challenging procedure for surgery bloodloss in the laparoscopic group is well shown with anaverage of to ml the average blood loss inour study was ml lower than that reported in the literature we can identify neurovascular lesions usinghighdefinition 3d laparoscopy to preserve them and weuse a bipolar electrosurgical device to reduce injurywhich is beneficial for accurate operationthe overall complication rate in laparoscopic tsmeoperation was lower than that in the open operationgroup the rate of anastomotic leak showed no statistical difference between the two operation methods theaverage leak rate for rectal cancers was zakir reported that the overall complicationrate was in tsme for rectal cancer patients therate of anastomotic leakage was in the open tsmegroup and in the laparoscopic tsme group therewas no statistical difference between groups in our studythe incidence rate of postoperative anastomotic leakagewas three patients had complications after surgeryand the overall complication rate was the threecomplications were wound infection fluid collection andurinary retention with a claviendindo grading of “yoo evaluated the optimal duration of urinarycatheterization after tsme for rectal cancer logistic regression analysis was performed to determine the risk factors for urinary retention the variables including age sexasa grade surgical procedure tnm stage tumor position preoperative radiotherapy duration of urinarycatheterization and time of surgery were not significantrisk factors for urinary retentionat present a 3d laparoscopic system aesculapgerman is used in laparoscopic surgery in our department single and reduced portlaparoscopic surgeryrobot operations and tatme operations are not usedfor tsme the surgeons who performed tsme had morethan years of experience in gastroenterostomy and hadexperience with open tsme the difficulty of the tsmeoperation is the management of the mesorectum seiji has reported on the management of the mesorectumin the narrow pelvis which our treatment method is basedon first the right part of the mesorectum is lifted fromthe right side of the sigmoid mesocolon to expose the inferior mesenteric artery and vein left colonic vessels sigmoid colonic vessels and superior rectum vessels theassistant lifts the left mesentery of the sigmoid colon exposes the above vessels expands the sigmoid mesocolonagain penetrates the mesentery from the right side andexposes the surrounding vessels expansion of the pelviccavity along the vessels is continued and the mesorectumis repaired from the left to the right side “ cm above thetumor according to the location ofthebranches of the severed vessels are determined and “cm of the intestinal wall is repaired the rectum is dissected using an endogia staplerthe tumorlaparoscopic tsme has been used for rectal cancer andcan obtain satisfactory functional results compared to openresection and tme we do not think that the reduction inthe hospital stay is due to the acceleration of the intervention as per enhanced recovery after surgery eras butis due to an increase in the doctors™ confidence in reducingthe risk of postoperative complications after vascular preservation threedimensional cta examination is importantfor the preoperative evaluation of sigmoid colonvascular classification and intraoperative management ofthe sigmoid and left colon vessels however preoperativeexamination could not obtain information on the trafficbranch the biggest advantage of this operation is themaintenance of the blood supply of the proximal and distalintestines and the sufficient length of the intestine so thereis no need for temporary defunctioning stoma temporarydefunctioning stoma only increases the complexity of theoperation and closure of the temporary stoma increasesthe risk of complications in addition the results of the statistical analysis showed that the number of lymph nodes inthe tsme group was greater than that in the tme groupit cannot be concluded that tsme was significantly betterthan tme for lymph node dissection suggesting thattsme was not inferior to tmeslaparoscopic tsme with preserved left colic and superior rectal arteries is a technically challenging procedureintact visceral pelvic fibro is protected with even greateraccuracy than other techniques by 3d laparoscopywhich offers an optimal vision tsme with preserved leftcolic and superior rectum arteries did not increase therisk of operation compared with tme but increased thesurgeon™ s confidence in patient outcomes thereforelaparoscopic tsme with preserved left colic and superior rectal arteries can be safely performed for rectal cancer patients as an alternative to tme 0czhang world of surgical oncology page of abbreviationstme total mesorectal excision tsme tumorspecific mesorectal excisionima inferior mesenteric artery imv inferior mesenteric vein sra superiorrectal artery nvb neurovascular bundle pme partial mesorectal excisionacknowledgementsnoneauthors™ contributionslz yx and xh designed the study cz yx hw qz zd and whcollected and analyzed the data ma lz ys and xh interpreted the datalz and cz drafted the manuscript lz ma yx and xh revised themanuscript the authors read and approved the final manuscriptfundingthis study was supported by the jiangsu natural science foundationbk20180274 this funding supported the collection analysis andinterpretation of the dataavailability of data and materialsall experimental data used to support these findings are included in theethics approval and consent to participatethis study was approved by the institutional review board of the firstaffiliated hospital of dalian medical university written informed consent forpublication was obtained from all patientsconsent for publicationwritten informed consent was obtained from the patients and legalguardian for the publication of these patientscompeting intereststhe authors declare that they have no conflicts of interestauthor details1department of gastrointestinal surgery the first affiliated hospital of dalianmedical university dalian liaoning province china 2department ofgastrointestinal surgery the third affiliated hospital of guangxi medicaluniversity nanning guangxi province china 3department ofgastrointestinal surgery changzhi people™s hospital the affiliated hospital ofchangzhi medical college changzhi shanxi province china 4department ofcolorectal surgery the first affiliated hospital of nanjing medical universitynanjing china 5department of gastrointestinal surgery the first people™shospital of dali city dali yunnan province china 6department ofgastrointestinal surgery graduate school of medicine university of tokyotokyo japan 7department of general surgery yizhen people™s hospitalclinical medical college yangzhou university yangzhou jiangsu provincechinareceived february accepted august heald rj husband em ryall rd the mesorectum in rectal cancer surgerythe clue to pelvic recurrence br j surg “ macfarlane jk ryall rd heald rj mesorectal excision for rectal cancerlancet “lee ky factors influencing oncologic outcomes after tumorspecificmesorectal excision for rectal cancer j korean soc coloproctol “ williams ns dixon mf johnston d reapppraisal of the centimetre rule ofdistal excision for carcinoma of the rectum a study of distal intramuralspread and of patients™ survival br j durg “ pollett wg nicholls rj the relationship between the extent of distalclearance and survival and local recurrence rates after curative anteriorresection for carcinoma of the rectum ann surg “ wolmark n fisher b wieand hs the prognostic value of the modificationsof the dukes™ c class of colorectal cancer an analysis of the nsabp clinicaltrials ann surg “ monson jrt weiser mr buie wd chang gj rafferty jf prepared by thestandards practice task force of the american society of colon and rectalsurgeons practice parameters for the management of rectal cancerrevised dis colon rectum “law wl chu kw anterior resection for rectal cancer with mesorectalexcision a prospective evaluation of patients ann surg “kim sh bae kb kim jm oncologic outcomes and risk factors forrecurrence after tumorspecific mesorectal excision of rectal cancer cases j korean soc coloproctol “kim nk min bs kim js hur h lee ky sohn sk oncologic outcomesand safety after tumorspecific mesorectal excision for resectable rectalcancer a single institution™s experience with patients with rectalcancer j korean soc coloproctol “ zakir k mohamed wai lun law outcome of tumorspecific mesorectalexcision for rectal cancer the impact of laparoscopic resection world jsurg “kim nk baik sh seong js oncologic outcomes after neoadjuvantchemoradiation followed by curative resection with tumorspecificmesorectal excision for fixed locally advanced rectal cancer impact ofpostirradiated pathologic down staging on local recurrence and survivalann surg “ poon jt law wl laparoscopic resection for rectal cancer a review annsurg oncol “ yoo be kye bh kim hj early removal of the urinary catheter aftertotal or tumorspecific mesorectal excision for rectal cancer is safe discolon rectum “seiji o takashi t kazuki s a new laparoscopic surgical procedure toachieve sufficient mesorectal excision in upper rectal cancer int j surgoncol httpsdoi1011552011708439publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsreferencesenker w e thaler h t cranor m l polyak t total mesorectal excision inthe operative treatment of carcinoma of the rectum j am coll surg “lopezkostner i lavery c hool gr rybicki la fazio vw total mesorectalexcision is not necessary for cancer of the upper rectum surgery “zaheer s pemberton jh farouk r dozois rr wolff bg ilstrup d surgicaltreatment of adenocarcinoma of the rectum ann surg “sudeck p ueber die gefässversung des mastdarmes in hinsicht auf dieoperative gangrän muenchen med wschr “van tonder jj boon jm becker jhr anatomical considerations onsudeck™s critical point and its relevance to colorectal surgery clin anat“cirocchi r randolph j cheruiyot i systematic review and metaanalysis of the anatomical variants of the left colic artery color dis httpsdoi101111codi14891 0c"
Colon_Cancer
" the widely recognized anticancer potential of aspirin has created a broad interest to explore theclinical benefits of aspirin in cancer therapy however the current understanding of the molecular mechanismsinvolved in the anticancer potential of aspirin remains limitedmethods cancer stemness assays which contained aldh side population chemoresistance sphere formationand tumorigenesis were performed to validate aspirin function in vitro and in vivo histone modification assay wasperformed to check the effect of aspirin on histone methylation as well as the activity of hdac and kdm6abinhibitor in vivo assay was performed to evaluate therapeutic effects of various inhibitor combination mannersresults in regards to in vitro studies aspirin diminishes cancer cell stemness properties which include reducing thealdh subpopulation side population chemoresistance and sphere formation in three cancer types in regards toin vivo studies aspirin decreases tumor growth and metastasis and prolongs survival in addition our resultsshowed that aspirin inhibits inflammationrelated stemness gene expression especially icam3 identified by a highthroughput sirna platform in regards to the underlying molecular mechanism of action aspirin reduces histonedemethylase kdm6ab expression that mediates histone methylation and suppresses gene expression via a coxindependent manner in regards to therapeutic strategies aspirin combined hdm inhibitors icam3 downstreamsignaling srcpi3k inhibitors or icam3 inhibitor lifitigrast prevents cancer progression in vivos the aforementioned findings suggest a molecular model that explains how aspirin diminishes cancercell stemness properties these findings may provide novel targets for therapeutic strategies involving aspirin in theprevention of cancer progressionkeywords aspirin histone methylation cancer stemness icam3 cox therapeutic strategies correspondence shenwenzhi2011126com1department of pathology and institute of precision medicine jining medicaluniversity hehua road jining chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhang stem cell research therapy page of cancer stem cell csc was found as the chief culprit toinitiate tumor occurrence to enhance tumor malignancyand to cause tumor recurrence whereby the maintenanceof cancer cell stemness mainly depends on the tumormicroenvironment or also called the œniche [“] currently the specific properties of csc were identified likehigh aldh1 activity aldehyde dehydrogenase sidepopulation chemoresistance and other cd moleculescd44 cd133 or markers sox2 oct4 nanoglgr5 positive in cancer clinical therapy which targetedhighly tumorigenic cscs may provide new targets orinsight for cancer therapy however unfortunately cscshad demonstrated a relative resistance to conventionalchemotherapy and radiotherapy moreover the cancer cellstemness and the resultant tumor initiationmalignancycould be maintained by the tumorassociated inflammation factors within the tumor microenvironment niche[ ] our previous work presented a mediumthroughput sirna screen platform to identify inflammation genes that regulate cancer cell stemness and obtainedseveral novel candidates agents that target these genesmay inhibit both inflammation and cancer cell stemnessat the same timeaspirin a nonsteroidal antiinflammatory drugiscommonly used as an antipyretic analgesic antiinflammatory and antithrombotic agent [ ] recentobservational and epidemiological studies have shownthat regular prolonged use of aspirin reduces the riskfor several cancers eg colorectal esophageal breastlung prostate liver and skin cancers [“] althoughthe benefits of aspirin for cancer patients have beenwidely appreciated the mechanism remains unclear previous studies attribute the anticancer potential of aspirin to the inhibition of cyclooxygenase2 cox2which is upregulated in various cancer cells [ ] ofnote an increasing body of evidence suggests that aspirin exhibits anticancer effects in a coxindependentmanner histone modification is a reversible process mediated bythe epigenetic enzymes [ ] histone methylation andacetylation are two important chemical modifications thatact in transcriptional activation or inactivation chromosome packaging and dna damagerepair [ ] histone demethylases hdms and histone deacetylaseshdacs are the key enzymes that remove methyl andacetyl groups respectively to regulate gene transcriptionin this regard aspirin was reported to affect hdacs expression and suppress progression of some cancers like aspirin mediates h3k27 acetylation to prevent coloncarcinogenesis and aspirin cooperates with p300 to activate h3k9 acetylation further to promote colorectal cancer cell apoptosis [ ] however the specific rolesand mechanisms of aspirinmediated histone methylationin cancer stemness remains insufficient thus we studiedthe role of aspirin on histone methylation and the attendantand cancerprogressioneffects on cancercellstemnessthe aldh subpopulationour results indicated that aspirin diminishes various cancer cell stemness properties which include reducingside populationchemoresistance and sphere formation in three cancertypes in vitro aspirin inhibits tumor growth and metastasis as well as prolongs survival in vivo aspirininhibits inflammationrelated stemness genes especiallyicam3aspirin reduces histone demethylasekdm6abexpression which mediates histone methylation respectively with a coxindependent manner and aspirin hdm inhibitors aspirin icam3downstream signaling srcpi3k inhibitors and aspirin icam3 inhibitor lifitigrast all reduce cancer progressionin vivo the abovementioned findings demonstrate apromising mechanism of action and potential therapeutic strategy of aspirin in the prevention of cancerprogressionmethodscell culturemdamb231 breast cancer cell and a549 lung cancercell were purchased from atcc hepg2 was obtainedfrom the chinese academy of sciences mdamb231breast cancer cell and hepg2 liver cancer cell were cultured in dmem medium a549 lung cancer cell was cultured in medium allculture media weresupplemented with fbs gibco and were grown at °c in co2 incubators all cells were passaged forless than months before renewal from frozen earlypassage stocks and tested to ensure thatthey weremycoplasma negativecytotoxicity assayaspirin was purchased in sigma cat a2093 and dissolved in dmso mdamb231 a549 and hepg2 cellswere cultured in 96well plate and treated with variousconcentrations and mm for a549 cells and mm for mdamb231 cells and and mm forhepg2 cells of aspirin for h cell activity was testedby applying cck8 kit dojindo china following themanufacturer™s instructionsaldefluor assaythe aldeflour assay kit stemcell technologies vancouver canada was used to measure aldh enzymaticactivity in three cancer cell lines mdamb231 a549and hepg2 in brief cells were treated with aspirin for h and — cells were suspended in aldeflour 0czhang stem cell research therapy page of assay buffer containing aldh1 substrate and incubated for min at °c cells treated with the specific aldh inhibitor deab served as the negative control stained cellswere analyzed on bd facs calibur flow cytometer bdbiosciences san jose ca data analysis was performedusing flowjo software tree star inc ashland orside population assaymdamb231 a549 and hepg2 cells treated with aspirin for h were harvested and resuspended in prewarmed staining buffer pbs buffer added fbs at adensity of — cellsml hoechst dye wasadded at a final concentration of μgml μgmla549 and μgml hepg2 in the presence or absence of μm fumitremin c ftc the followingsteps were described previously [ ]cell apoptosis assaythe mdamb231 a549 and hepg2 cells were treatedwith aspirin and cisplatin ddp μgml for h simultaneously then harvested and resuspended in prewarmed staining buffer pbs buffer added fbs at adensity of — cellsml apoptotic cells were stainedwith propidiumiodide and annexinvfitc bd biosciences flow cytometry analysis was performed by facscalibur cytometer bd biosciences in which a minimum of events were recordedsphere formation assaycells were collected and rinsed to remove serum thendissociated to singlecellsuspension in serumfreedmemf12 medium supplemented with iumlpenicillin μgml streptomycin ngml human recombinant epidermal growth factor hregf ngmlhuman recombinant basic fibroblast growth factorbfgf and b27 supplement invitrogen cells weresubsequently cultured in ultralow attachment 24wellplates at a density of cells per well then treatedwith aspirin all the time until the spheres were formedanimal studyfemale balbc mice at “ weeks were separated randomly into several groups n ‰¥ — 4t1luci cellswere inoculated sc into each mouse at the right axillafor lung metastasis assay at days after injection micewere treated intraperitoneally with aspirin mgkg aspirin mgkg every days and dmso used as thecontrol for chemoresistance assay at days after injection mice were first treated intraperitoneally with cisplatin mgkg then treated with aspirin mgkgaspirin mgkg every days and dmso used as thecontrolnodscid mice at “ weeks were separated randomly into several groups n ‰¥ — a549luci cellswere inoculated sc into each mouse for inhibitor treatment assay days after tumor cells injection micewere first treated intraperitoneally with aspirin mgkgor kdm6ab inhibitor gsk j1 mgkg or src inhibitor a new specific highefficient src inhibitor [ ] mgkg or pi3k inhibitor ly294002 mgkg orlifitegrast mgkg dmso used as the controltumor volume mm3 was measured with calipers andcalculated by using the standard formulalength —width22 the individual measuring the mice was unaware of the identity of the group measured primarytumor tissues were harvested and separated into threeparts one was formalin fixed paraffin embedded andsectioned for ihc staining the other two were brokenby the tissue homogenizer then used for rna trizoland protein ripa lysis buffer extraction animal usecomplied with nankai university and jining medicaluniversity animal welfare guidelines western blottingthe western blot steps were described previously [ ]the special primary antibodies used in this assay arelisted in supplementary table s1 all western data wasthe representative image of three biologically independent repeats the results were quantified using image jsoftware national institutes of health baltimore mdand analyzed by graphpad prism5 software graphpadsoftware san diego ca usanuclear fractionation analysiscells were harvested and the cytoplasmic and nuclearfractions were separated and extracted with an nepernuclear and cytoplasmic extraction kit thermo fisherscientific inc ma usa h3k3me marker proteinswere detected by western blotimmunofluorescencecells grow on glass slides and tumor tissue slices werefixed in paraformaldehyde and labeled with primaryantibodies overnight at °c followed by incubation withspeciesappropriateroomtemperature for h nuclei were stained with dapi andimages were obtained using a leica dm4000 uprightmicroscope or confocal fluorescence microscopy nikontokyo japan [ ]antibodiessecondaryatchromatin immunoprecipitation assaythe assay was performed with an ezzyme chromatinprep kit millipore according to the manufacturer™sprotocol antihistone modification marker antibodieswere used to precipitate dna crosslinked with histone modification markers respectively and normal rabbitigg was used in parallel as a control enriched dna wasthen used as a template to assess the binding intensity of 0czhang stem cell research therapy page of histone modification markers to putative binding sitesin the icam3 promoter primers used in this assay arelisted in supplementary table s2immunohistochemistryimmunohistochemistry was performed on tumor tissuesections from the mice primary antibodies raise againstthe target proteins at a dilution overnight the expression levels of the proteins were evaluated accordingto the percentage of positive cells in each tumor tissuesections the images were recorded by olympus bx51epifluorescent microscopy under a — or — objective olympus co tokyo japan changes in apoptosis in the three cancer cell lines usingthe cell apoptosis assay after cisplatin ddp treatmentour results indicated that apoptosis increases in theaspirintreated groups versus controls and thereby reduces cisplatin chemo resistance fig 1e f in orderto determine the effects of aspirin on cancer cell stemness we next investigated the changes in cell sphere formation in the three cancer cell lines using the sphereformation assay our results showed that sphere formation decreases in the aspirintreated groups versus controls fig 1g h the abovementioned findings suggestthat aspirin diminishes cancer cell stemness propertiesin vitrostatistical analysisall data were analyzed using graphpad prism5 softwaregraphpad software san diego ca usa values wereexpressed as means ± sem p values were calculatedusing a twotailed student™s t test two groups or oneway anova more than groups unless otherwisenoted a value of p was used as the criterion forstatistical significance an asterisk indicates a significantdifference with p two asterisks indicate a significant difference with p and three asterisks indicatea significant difference with p [ ]resultsaspirin diminishes cancer cell stemness properties in vitroin order to establish the proper working concentrationsof aspirin in various cancer cells we determined theic50 of aspirin in a549 lung cancer cells mdamb231breast cancer cells and hepg2 liver cancer cells using acytotoxicity assay our results showed a 107mm ic50in a549 lung cancer cells a 43mm ic50 in mdamb breast cancer cells and a 97mm ic50 in hepg2liver cancer cells fig s1 based on the ic50 we choseworking concentrations of and mm aspirin fora549 lung cancer cells and mm aspirin formdamb231 breast cancer cells and and mmaspirin for hepg2 liver cancer cells in our studiesthe aldh subpopulation decreasesin order to determine the in vitro effects of aspirin oncancer cell stemness we investigated aldh subpopulation changes in a549 lung cancer cells mdamb231 breast cancer cells and hepg2 liver cancer cellsusing the aldh staining assay our results indicatedthatin theaspirintreated groups versus controls fig 1a b inorder to determine the effects of aspirin on cancer cellstemness we next investigated the changes in the sidepopulation in the three cancer cell lines using the sidepopulation assay our results indicated that the sidepopulation decreases in the aspirintreated groups versuscontrols fig 1c d in order to determine the effects ofaspirin on cancer cell stemness we next investigated theaspirin diminishes cancer cell metastasis and stemnessproperties in vivoin order to determine the effects of aspirin on cancercell metastasis and stemness in vivo we implanted 4t1luciferase cells into the fourth fat pad of female balbcmice seven days after implantation we ip injected themice with mgkg aspirin mgkg aspirin ordmso control group times per week fig 2a ourresults showed thattumor volume decreases in theaspirintreated groups versus the control fig 2b however we found that the body weight did not change inthe aspirintreated groups versus the control fig 2c inaddition we found that the survival time increases in theaspirintreated groups versus control fig 2d with respect to the effect of aspirin on cancer cell metastasiswe found thatlung metastasis decreases in aspirintreated groups versus the control fig 2e“g with respect to the effect of aspirin on cancer cell stemnessproperties we found thatthe immunocytochemicalstaining of sox2 and oct4 stemness markers decreasesin the aspirintreated groups versus dmso controlsfig 2h i the abovementioned findings suggest thataspirin diminishes cancer cell metastasis and stemnessproperties in vivoaspirin reduces cancer cell chemoresistance in vivoin order to determine the effects of aspirin on cancercell chemoresistance in vivo we implanted 4t1luciferase cells into the fourth fat pad of female balbcmice eight days after implantation we ip injected themice with mgkg cisplatin mgkg aspirin mgkgcisplatin mgkg aspirin or dmso control groupevery days fig 2j our results showed that tumor volume decreases in the cisplatinaspirintreated versus thedmso control fig 2k however we found that thebody weight did not change in the cisplatinaspirintreated versus the dmso control fig 2l in additionwe found that the survival time increases in the cisplatinaspirintreated groups versus the dmso controlfig 2m we also found that the rate of tumor growth 0czhang stem cell research therapy page of absllec evitisop hdladeabctrl2mm5mm 4mm10mm ctrleddp10ugml2mm5mm 4mm10mm agpehagpeha549ctrl 5mm 10mm ctrl 2mm 4mm sllec evitisop hdlasllec evitisop hdlahepg2fctrl 5mm 10mm slliec ssotpopaa549 slliec ssotpopactrl 5mm 10mm ctrl 2mm 4mm hepg2 slliec ssotpopactrl 5mm 10mm cblockctrl2mm5mm 4mm10mm agpeha549d noitlaupopeds ia549 noitlaupopeds ictrl 5mm 10mm ctrl 2mm 4mm fig see legend on next pagehepg2h noitlaupopeds ictrl 5mm 10mm rebmun erehpsgctrl2mm5mm 4mm10mm agpehhepg2ctrl 5mm 10mm ctrl 2mm 4mm rebmun erehpsrebmun erehpsctrl 5mm 10mm 0czhang stem cell research therapy page of see figure on previous pagefig aspirin restrains cancer cell stemness properties in vitro a aldh staining assay was performed to check aldh subpopulationpercentage in the three cancer cell lines with or without aspirin treatment b statistic results of aldh subpopulation percentage were shown cside population assay was performed to detect sp percentage in three cancer cell lines with or without aspirin treatment d statistic results of sppercentage were shown e facs was performed to detect cell resistance to cisplatin and the percentage of apoptotic cells was shown f statisticresults of apoptosis cells percentage were shown g sphere formation assay was performed to check the cell sphere formation ability in the threecancer cell lines with or without aspirin treatment scale bars μm h statistic results of sphere amounts were shownwas slower in the cisplatinaspirintreated versus thedmso control fig 2n with respect to the effect of aspirin on cancer cell stemness properties we found thatthe immunocytochemical staining of sox2 and oct4stemness markers decreases in the cisplatinaspirintreated groups versus dmso controls fig 2o p theabovementioned findings suggest that aspirin reducescancer cell chemoresistance in vivoaspirin inhibits the expression of inflammationrelatedstemness genes in vitro and in vivoour previously published report established a mediumthroughput sirna screening platform that identifies inflammation genes that regulate cancer cell stemness specifically we identified several novel candidate genesthat decrease oct4 expression and the aldh subpopulation both of which characterize stemness fig 3ain order to determine whether aspirin decreases theexpression of these novel candidate genes to further diminish cancer cell stemness we investigated the expression of novel candidate genes and stemness markerssox2 and oct4 in a549 lung cancer cells mdamb231 breast cancer cells and hepg2 liver cancer cellsusing western blot our results showed that icam3ccl16 pde3a prtn3 sox2 and oct4 protein expression decreases in the aspirintreated groups versuscontrols fig 3b fig s2a we also found that icam3ccl16 pde3a prtn3traf6 bcar1 il1a il1bnfkb1 ikbkb sox2 and oct4 mrna expression decreasesin the aspirintreated group versus controlfig 3c moreover in icam3 ccl16 pde3a prtn3traf6 bcar1 il1a il1b nfkb1 sox2 and oct4the protein expression decreasesindicated byimmunofluorescencestainingaspirintreatedmbamd231 fig 3d and a549 cells data not shownversus the controlasin thein order to confirm the above in vitro results we theninvestigated mrna and protein expression in tumorsfrom and 46day aspirintreated miceversus control using qpcr and western blot our resultsdemonstrated that icam3 pde3a prtn3 traf6bcar1 il1a il1b nfkb1 ikbkb sox2 and oct4mrna expression decreasesin the aspirintreatedgroups versus control fig 3e in addition we foundthat icam3 pde3a prtn3 traf6 bcar1 il1ail1b nfkb1 sox2 and oct4 protein expressionsimilarly decreases in the aspirintreated groups versuscontrol fig 3f fig s2b the abovementioned findingsexpression ofsuggesttheinflammationrelated stemness genesin vitro andin vivoaspirin decreasesthataspirin mediates histone methylation to regulate targetgenes expressionin order to determine the mechanism underlying the action of aspirin we explored the regulatory effect of aspirin on histone methylation markers in a549 lungcancer cells mdamb231 breast cancer cells andhepg2 liver cancer cells using western blot our resultsindicated thatthe expression of h3 trimethylationmarkersie h3k43me h3k93me h3k273meh3k363me and h3k793me increases in the aspirintreated higher concentration groups versus controlfig 4a s3a we also found that the expression of histone demethylases ie kdm6a and kdm6b decreasesin the aspirintreated higher concentration groups versuscontrol fig 4a in addition we studied the protein expression of h3k43me h3k93me h3k273meh3k363me h3k793me and h3 in a549 lung cancercells mdamb231 breast cancer cells and hepg2 livercancer cells using immunofluorescence ourresultsshowed that the protein expression of the h3 methylation markers within the nucleus increases in the aspirintreated groups versus control fig 4b to further support this we extracted the nuclear proteins of eachgroup and detected these h33me markers the resultsalso showed that the expression of h33me markers wasincreased within the nucleusin the aspirintreatedgroups versus control fig 4c s3bin order to identify the role of h3 methylation in regulating selected inflammationrelated stemness genes wemeasured the amount of icam3 dna fragments in h3modification marker pulldowned dnas in a549 lungcancer cells mdamb231 breast cancer cells andhepg2 liver cancer cells using the chipqpcr assaywe selected icam3 since our previous studies demonstrated that icam mediates cancer cellinflammationand stemness ourtheamount of icam3 dna fragments in the various h3methylation marker pulldowned dnas decreases in alllines fig 4c the abovementionedthree cancer cellfindingssuggesthistoneresults demonstrated thatreducesthataspirin 0cbmcemuovl romutdliavvrus noitcarfctrlasp 50mgkgasp 100mgkgdayscg thgew ydobictrlasp 50mgkgasp 100mgkg days of treatmentectrlasp50 asp100ctrlasp 50mgkgasp100mgkggsedoni ssatsat emgnulfhctrlasp asp ctrlasp asp ——xostcoi sllec xos sllec tcoctrl asp50asp100ctrl asp50 asp100ctrl asp50 asp100zhang stem cell research therapy page of a4t1luci injectionaspirin injectionsurvivalday timej4t1luci injectionaspirin cisplatin 2mgkg injectionsurvivalday kmcemuovl romutctrlcisplatinasp 25cisplatinasp50cisplatin dayslg tihgew ydobctrlcisplatinasp 25cisplatinasp50cisplatin days of treatmentmliavvrus noitcarfncisplatin treatedctrlasp asp50yadyadctrlcisplatinasp 25cisplatinasp50cisplatintimefig see legend on next pageop sllec xoscisplatin treatedctrlasp asp50xostcoctrl asp25 asp50cisplatin treated sllec tcoctrl asp25 asp50cisplatin treated 0czhang stem cell research therapy page of see figure on previous pagefig aspirin suppresses cancer cell metastasis and stemness in vivo a schema of the metastasis model established by subcutaneousimplantation of 4t1luci cells into the 4th pair of mammary fat pad of balbc mice b tumor growth curve of 4t1luci with or without aspirintreatment c the body weight of balbc mice in the course of aspirin treatment d the survival curve of balbc mice inoculated with 4t1luciwith or without aspirin treatment e the representative luciferase images showing the 4t1luci tumors at the primary site and lung metastasissites with or without aspirin treatment f representative he staining images of 4t1luci tumors metastasis to the lung with or without aspirintreatment scale bars lower panel μm g statistic results of metastasis loci of 4t1luci tumors metastasis to the lung with or without aspirintreatment h immunohistochemistry staining of sox2 and oct4 in 4t1luci primary tumors with or without aspirin treatment representativeimages with — magnification were shown scale bars μm i statistic results of sox2 or oct4 positive cells in 4t1luci primary tumors withor without aspirin treatment j schema of the chemoresistance model established by subcutaneous implantation of 4t1luci cells into the 4thpair of mammary fat pad of balbc mice k tumor growth curve of 4t1luci with or without aspirin treatment in the presence of cisplatin l thebody weight of balbc mice in the course of aspirin treatment in the presence of cisplatin m the survival curve of balbc mice inoculated with4t1luci with or without aspirin treatment in the presence of cisplatin n the representative luciferase images showing 4t1luci tumors at theprimary sites with or without aspirin treatment on day before cisplatin administration and day after cisplatin administrationo immunohistochemistry staining of sox2 and oct4 in 4t1luci primary tumors with or without aspirin treatment in the presence of cisplatinrepresentative images with — magnification were shown scale bars μm p statistic results of sox2 or oct4 positive cells in 4t1luciprimary tumors with or without aspirin treatment in the presence of cisplatindemethylase ie kdm6a and kdm6b expressionwhich mediates histone methylation and thereby inhibits gene expression in vitroaspirin mediates h3 methylation to regulate icam3expression in vivoin order to confirm the above in vitro results we nextexamined h3 methylation marker expression in tumorsfrom aspirintreated mice versus control using immunocytochemistry our results demonstrated that the h3methylation marker immunostaining within the nucleusincreases in the aspirintreated group versus controlfig 4d e we also found that the amount of icam3dna fragments in the various h3 methylation markerpulldowned dnas decreasesin the aspirintreatedgroup versus control indicating that icam3 expressionis blocked fig 4f these findings suggest that aspirinmediates h3 methylation and thereby regulates icam3expression in vivoside populationaspirin mediates h3 methylation to regulate icam3expression via a coxindependent mannerin order to determine the role of cox in aspirinmediated h3 methylation and targeted gene expressionwe knocked down cox1 and cox2 expression in a549cells respectively fig 5a s3c and examined thealdh populationand chemoresistance the results showed that the aldh population fig 5b e and side population fig 5c f were decreased in shcox1 or shcox2 cells treated with aspirincompared to shcox1 or shcox2 cells treated withdmso and also the aldh population and side population were decreased in shcox1 or shcox2 cellstreated with aspirin compared to shctrl treated with aspirin moreover the apoptosis was increased in shcox1or shcox2 cells treated with ddp and aspirin comparedto shcox1 or shcox2 cells treated with ddp anddmso ctrl and also the apoptosis was increased inshcox1 or shcox2 cells treated with ddp and aspirincompared to shctrltreated with ddp and aspirinfig 5d g in addition western blot results displayedthat the h3 trimethylation markers were increased andthe histone demethylases ie kdm6a and kdm6bwere decreased in shcox1 or shcox2 cells treated withaspirin compared to shctrl treated with aspirin fig 5hs3d accordingly as the new target genes icam3 expression was decreased in shcox1 or shcox2 cellstreated with aspirin versus shctrl treated with aspirinfig 5i s3e these findings suggest that aspirin mediates h3 methylation and thereby regulates icam3 expression via a coxindependent manneraspirin combined with hdm kdm6ab or icam3signaling inhibitors diminish cancer progression in vivoour previous work proved that icam3 could mediatesrcpi3k signaling to promote cancer cell stemness inorder to investigate the use of aspirin combined withhdm kdm6ab or icam3 signaling inhibitors as thetherapeutic strategies we implanted a549luciferasecells into the fourth fat pad of male nodscid micetwentythree days after implantation we injected ip themice with mgkg aspirin mgkg aspirin mgkg kdm6ab inhibitor gsk j1 mgkg aspirin mgkg src inhibitor mgkg aspirin mgkg pi3kinhibitor ly294002 mgkg aspirin mgkg lifitigrast icam3 inhibitor or dmso control group every days fig 6a our results showed that tumor size andtumor volume decreases in the aspirintreated groupand the aspirin inhibitortreated groups versus dmsocontrol fig 6b c however we found that the bodyweight did not change significantly in the aspirintreatedgroup and the aspirin inhibitortreated groups versusdmso control fig 6din addition we found that the survival time increasesin the aspirintreated group and the aspirin inhibitortreated groups versus dmso control fig 6e these 0czhang stem cell research therapy page of fig see legend on next page 0czhang stem cell research therapy page of see figure on previous pagefig aspirin inhibits the expression of inflammationrelated stemness genes in vitro and in vivo a schematic representation of the sirna screenleft summary of the results from the rnai screen right b western blot examining the expression of inflammatory candidates and stemnessproteins sox2 oct4 in a549 mdamb231 and hepg2 cells with or without aspirin treatment c quantitative pcr examining the mrnaexpression of inflammatory candidates and stemness genes sox2 oct4 in a549 mdamb231 and hepg2 cells with or without aspirintreatment d immunofluorescence staining of inflammatory candidates and stemness genes sox2 oct4 in a549 mdamb231 and hepg2 cellswith or without aspirin treatment scale bars μm e quantitative pcr examining the mrna expression of inflammatory candidates andstemness genes sox2 oct4 in 4t1luci tumors separated from balbc mice treated with aspirin for different survival days f western blotexamining the expression of inflammatory candidates and stemness genes sox2 oct4 in 4t1luci tumors separated from balbc mice treatedwith aspirin for different survival dayssuggestresultsthat aspirin combined with hdmkdm6ab or icam3 signaling inhibitors diminishcancer progression in vivo and may serve as the therapeutic strategiesproposed model of aspirin inhibits cancer cell stemnessand cancer progressionbased on our findings we propose the following modelfig aspirin inhibits histone demethylase hdm expression which then mediates histone methylationh3k43me h3k93me h3k273me h3k363meh3k793me respectively these h3 methylations theninhibit the expression of various inflammationrelatedstemness genes previously identified by highthroughputsirna screening il1a il1b icam3 ccl16 traf6pde3a prtn3 nfkb1 ikbkb bcar1 using theicam3 gene as a representative of the inflammationrelated stemness genes by the aspirinmediated h3modifications restrain icam3 promoter activity andcause icam3 expression is inhibited thus aspirin maydiminish cancer cell stemness properties and cancer progression in vitro and in vivo by inhibiting the expressionof various inflammationrelated stemness genes mostinterestingly the above process was not depending oncox expression as the therapeutic strategies aspirincombined various inhibitors suppressed tumor progression effectivelydiscussionthe widely recognized anticancer potential of aspirin aclassical nonsteroidal and antiinflammatory drug hascreated a broad interest to explore the clinical benefitsof aspirin in cancer therapy [“] previous findingsby many investigators have establishe
Colon_Cancer
" lung carcinoma is a prominent cause of mortality among patients with cancer previous studies have reported the vital role of long noncoding rnas lncrnas in the malignant progression of lung cancer lncrna rp11284f219 was originally identified to be expressed in lung carcinoma but its specific function remains unknown therefore the present study aimed to elucidate the role of lncrna rp11284f219 in lung carcinoma progression the expression of rp11‘284f219 in lung cell lines and tissues was measured using reverse transcription‘quantitative pcr the endogenous expression of rp11284f219 was silenced using rna interference and cell viabilities were measured with a cell counting kit‘ assay the invasion and apoptosis of cells were determined via transwell assays and flow cytometry respectively the protein expression levels were measured by western blotting an increased expression of rp11‘284f219 was identified in both lung carcinoma tissues and cells knockdown of rp11‘284f219 in lung carcinoma cells inhibited cell proliferation and invasion but promoted cell apoptosis the present study identified the existence of a direct interaction between rp11‘284f219 and microrna mirnamir6273p mechanistically it was demonstrated that rp11284f219 promoted the proliferation and invasiveness of lung carcinoma cells in part via the regulation of mir6273p furthermore cell division cycle and apoptosis regulator ccar1 was identified as a target gene of mir6273p the in vivo tumor growth assay also demonstrated that the knockdown of rp11‘284f219 suppressed tumor growth upregulated mir6273p and downregulated correspondence to dr yuan wang department of medical imaging the first affiliated hospital of xi'an jiaotong university west yanta road xi'an shaanxi pr chinaemail wangyuan8003126comabbreviations ccar1 cell division cycle and apoptosis regulator nsclc non‘small cell lung cancer sclc small cell lung cancerkey words rp11284f219 lung carcinoma proliferation invasion microrna6273p ccarccar1 in the xenograft model of nude mice thus the present findings indicated the tumor promoting functions of rp11284f219 in the progression of lung carcinoma and provided a novel lncrnamirna axis as a target for the management of lung cancerintroductionpulmonary malignancies including lung and bronchus cancer rank first and second among different cancer types in terms of mortality and morbidity respectively in both men and women furthermore of lung cancer cases are categorized as nonsmall cell lung cancer nsclc while the remaining are classified as sclc although diagnostic methods and therapeutic strategies based on traditional surgical excision chemotherapy and chest radiotherapy have continuously improved the prognosis of lung carcinoma remains at for an overall 5year survival therefore an increased understanding of the malignant progression and studies on novel therapeutic targets for the improved management of this disease are essentiallong noncoding rnas lncrnas are nucleotides in length and have little or no protein coding capacity the mechanisms via which lncrnas regulate gene expression are diverse and include regulating the transcription of target genes functioning as transcriptional precursors of small rnas generating different splice variants via regulating mrna splicing patterns modulating protein activity and subcellular localization and scaffolding for the assembly of multiple component complexes in recent years previous studies have reported that various human cancer types exhibit lncrnas dysfunction and these lncrnas are involved in different aspects of pathogenesis such as the proliferation metastasis and apoptosis of tumor cells in lung cancer lncrna metastasisassociated lung adenocarcinoma transcript is found to be upregulated in patients with advanced lung adenocarcinoma and may serve as a prognostic marker to predict the survival outcome of patients with cancer lncrna hox transcript antisense rna is also highly expressed in lung cancer and it enhances the aggressiveness of lymph node metastasis and indicates a short diseasefree survival in patients with nsclc furthermore studies have shown that the expression of lncrna urothelial carcinoma‘associated 0cli rp11‘284f219 promotes lung carcinoma proliferation and invasionis significantly upregulated in nsclc and may induce resistance to treatment of egfr‘tyrosine kinase inhibitors by activating the aktmtor pathway lncrna rp11284f219 was primarily discovered in a pancancer transcriptomic analysis lncrna rp11‘284f219 exists as a cluster of three annotated lncrnas rp11284f219107 antisense to brevican which is a proteoglycan linked to invasiveness in glioma but lacks expression in squamous cell lung carcinomas however the specific function and the underlying mechanism of rp11284f219 in lung carcinoma remain unknownto the best of our knowledge the present study demonstrated for the first time that lncrna rp11284f219 was significantly upregulated in lung carcinoma tissues and cell lines and was involved in the carcinogenesis of lung cancer together with microrna mirnamir6273p and cell division cycle and apoptosis regulator ccar1 the regulatory axis of rp11‘284f219mir‘‘3pccar1 exists both in the lung carcinoma cells in vitro and in the tumor growth model in vivo the present study aimed to investigate rp11284f219 function in lung carcinoma and demonstrate the molecular mechanism underlying the regulation process via the rp11‘284f219mir‘‘3pccar1 axismaterials and methodstissue samples and cell lines between may and jan paired tumor and adjacent healthy tissues were isolated from patients with lung carcinoma age range ‘ years nine male patients four female patients who were diagnosed and treated in first affiliated hospital of xi'an jiaotong university the samples were dissected during the surgery and immediately flash‘frozen in liquid nitrogen and transferred to ‘Ëšc storage for further extraction of both rna and protein all the tissue samples were obtained with written informed consent from the patients the protocol was approved by the first affiliated hospital of xi'an jiaotong university approval no a normal lung epithelial cell line beas2b and lung carcinoma cell lines ncih460 ncih1299 and a549 were purchased from american type culture collection atcc and cultured according to the atcc guidelines 293t cells were purchased from procell life sciencetechnology co ltd and cultured in dmem supplemented with fbs cat no ‘ atcc and 1x penicillin‘streptomycin thermo fisher scientific inc beas2b cells were cultured in bronchial epithelial growth medium begm cat no cc‘ clonetics corporation according to the manufacturer's instructions nci‘h460 and nci‘h1299 cells were cultured in rpmi‘ medium cat no ‘ atcc and a549 cells in f12k medium cat no ‘ atcc supplemented with fbs cat no atcc and 1x penicillin‘streptomycin thermo fisher scientific inc all cells were culture at ˚c with co2rna extraction and reverse transcription‘quantitative pcr rt‘qpcr total rna from both tissue samples and cell lines were extracted using trizol® reagent invitrogen thermo fisher scientific inc for each sample ng total rna was reverse transcribed to synthesize the first‘strand cdna using the primescript rt reagent kit takara bio inccdna samples were diluted times to perform the rt‘qpcr using sybr premix ex taq takara bio inc on a cfx96 realtime pcr detection system biorad laboratories inc expression levels of mrnas lncrnas and mirnas were normalized to gapdh the primers used for rtqpcr analyses were as follows gapdh forward '‘aac gac ccc ttc att gac c‘' and reverse '‘tcc acg aca tac tca gca cc‘' rp11‘284f219 forward '‘agg att ggc act cac ttc gg‘' and reverse '‘tct ctc acc acg tct ggt ct‘' and ccar1 forward '‘ctg atg gct agc cct agt atg ga‘' and reverse '‘tgc ctt tca tgc cca cta aaa ‘' the temperature protocol used to perform rt was ˚c for h followed by ˚c for min thermal conditions of pcr reactions were initial denaturation at ˚c for min followed by cycles for sec at ˚c and sec at ˚c the mrna expression levels were determined using the 2δδcq method oligonucleotides and cell transfection the small interfering rna sirna synthetic negative control sinc rp11284f219 sirnas sirp11284f219 mirnc mir‘‘3p mimics and mir‘‘3p inhibitor were purchased from shanghai genepharma co ltdall primer sequence information is presented in table i at a density of 2x105 cellswell the cells were plated in 6well plates h before transfection and were transfected at confluency all of the oligonucleotides were transfected at a final concentration of nm using lipofectamine® reagent invitrogen thermo fisher scientific inc according to the manufacturer's instruction cells were collected at h posttransfection for subsequent experimentscell counting kit cck‘ assay and edu labeling of prolif‘erating cells a cck‘ was used for cell proliferation assay the cells were seeded into ‘well plates 2x103 cellswell and observed for and days or indicated time points following the manufacturer's instructions dojindo molecular technologies inc the optical density was measured at nm using a spectrophotometer thermo fisher scientific incfor the edu assay cells were incubated with µm edu cat no ab219801 abcam for h at ˚c and fixed with formaldehyde at room temperature for min after a brief washing with pbs click reagent was added into each well and incubated in the dark for min at room temperature followed by pbs washing the cells were stained with µgml dapi at room temperature for min images were captured using a fluorescence microscope nikon corporation and measured using adobe photoshop software adobe systems inc the edu labeled cells were analyzed with moflo astrios beckman‘coulter inc magnification x200transwell assay and flow cytometry measurement of cell apoptosis transwell assays were performed with a coating of matrigel bd biosciences mixed with culture medium mixed at ratio at ˚c for h a total of 1x105 cells in µl serum‘free medium were added to the upper layer of the transwell chambers µm pore size corning inc and cultured for h the lower chamber contained the culture medium with fbs the migrated cells were fixed with 0concology reports table i sequence of sirnas and mirna mimics and inhibitorsoligonucleotides si‘nc si‘rp11‘284f219 mir‘nc mir‘‘3p mimics mir‘‘3p inhibitor mir microrna sirna small interfering rna nc negative controlsequence '†’'uucuccgaacgugucacguttuauuggcaccaaggauagcucguuaaucggcuauaauacgcucuuuucuuugagacucacuucuuuucuuugagacucacu paraformaldehyde for min at room temperature stained with crystal violet for min at room temperature and images of six randomly selected fields in each well were captured under a light microscope magnification x200cellular apoptosis was detected using the apoptosis detection kit cat no kgf001 nanjing keygen biotech co ltd according to the manufacturer's instructions cells were stained with fluorescein isothiocyanateconjugated annexin v and pi after incubated for min at ˚c in the dark µl 1x binding buffer was added to each tube and stained cells were analyzed using bd facs canto ii flow cytometry facs calibur bd biosciences data were analyzed using flowjo software version tree star incluciferase reporter assay the rp11284f219 wildtype wt or mutant mut '‘untranslated region '‘utr and ccar1 wt or mut '‘utr sequences were cloned into the pmirglo plasmid youbio httpwwwyoubiocn cat no vt1439 the vectors µgml were co‘transfected with mir‘nc or mir6273p mimic nm and renilla plasmids ngwell used as an internal control into cells seeded in a 48well plate 1x104well using lipofectamine® reagent invitrogen thermo fisher scientific inc cell lysates were collected at h after transfection and the luciferase activities were detected with the dualluciferase reporter assay system promega corporation according to the manufacturer's instructionswestern blotting cell were lysed using ripa lysis buffer sigma‘aldrich merck kgaa and protein concentrations were assessed with the bca protein assay kit according to the manufacturer's instructions beyotime institute of biotechnology shanghai china equal amounts µg of cell protein lysates were loaded and separated by sds‘page transferred to a pvdf membrane and blocked with non‘fat milk at room temperature for h the membranes were then incubated with ccar1 primary antibody cat no ab70243 abcam overnight at ˚c followed by incubation with goat anti‘mouse or goat anti‘rabbit igg‘horseradish peroxidase conjugate secondary antibodies cat no ab205718 abcam at room temperature for h gapdh cat no ab181602 abcam was used as loading control the signals were detected using the ecl system protein simple according to the manufacturer's instructionsin vivo tumorigenicity analysis in mice male balbc nude mice age weeks weight ‘ g were obtained from beijing vital river laboratory animal technology co ltd and housed at a room temperature of ˚c with a h lightdark cycle the mice were maintained in an individually ventilated cage system under specific pathogen‘free conditions temperature ˚c humidity and fed with sterile food and water free access to evaluate the effect of rp11‘284f219 knockdown on the growth of lung carcinoma in vivo 5x106 sinc or sirp11284f219 treated nci‘h1299 cells in µl serum‘free medium were subcutaneously injected into each mouse n5 per group under anesthesia which was induced by isoflurane and maintained by isoflurane flow rate 1lmin the animals were monitored daily and the following criteria for humane endpoint was used severe tumor burden mm in diameter difficulty breathing significant body‘weight loss and clinical signs such as prostration hypothermia and significant abdominal distension tumors were measured on days and and the volumes were calculated using the formula a x b22 [the largest diameter a and the smallest diameter b] then weeks after inoculation the mice were euthanized by co2 inhalation co2 flow rate of cage volume and the death of animals were confirmed by cessation of heartbeat the xenografts were imaged and weighedthe total rna was then extracted from the xenografts as aforementioned animal care and study were approved by the institutional animal care and use committee of the first affiliated hospital of xi'an jiaotong university approval no target prediction potential target mirnas of rp11284f219 were predicted using lncbase v2 httpcarolinaimisathena‘ innovationgrdiana_toolswebindexphprlncbasev2index the target genes of mir‘‘3p were predicted using three bioinformatics algorithms targetscanv72 httpwwwtargetscanorgvert_72 and mirdb httpwwwmirdborgmininghtmlstatistics analysis data were analyzed using the graphpad prism software graphpad software inc and presented as the mean ± sd from ‰¥ independent experiments a two‘tailed unpaired student's t‘test or one‘way anova with tukey's post‘hoc analysis were performed to evaluate the statistical significance p005 was considered to indicate a statistically significant difference 0cli rp11‘284f219 promotes lung carcinoma proliferation and invasionfigure rp11‘284f219 expression is upregulated in lc tissues and cell lines a expression of rp11‘284f219 in lc tissues in comparison with adjacent healthy tissues was analyzed using rtqpcr p0001 vs adjacent tissues n13 b expression of rp11‘284f219 in human lung carcinoma cell lines ncih460 ncih1299 and a549 compared with normal human lung epithelial cell line beas2b was analyzed using rtqpcr p005 p0001 vs beas‘2b n3 lc lung carcinoma rt‘qpcr reverse transcription‘quantitative pcrresultsexpression of rp11‘284f219 is upregulated in lung carci‘noma to investigate the potential role of rp11284f219 in lung carcinoma its expression was analyzed in tissue samples and matched adjacent healthy tissues from patients with lung carcinoma the results demonstrated that the expression of rp11‘284f219 was significantly upregulated in tumor tissues compared with healthy tissues fig 1a the expression of rp11‘284f219 was also analyzed in human lung carcinoma cell lines ncih460 ncih1299 and a549 and normal human lung epithelial cell line beas2b consistent with the findings in the tissue samples the expression of rp11‘284f219 was significantly increased in carcinoma cell lines compared with the normal epithelial cell line fig 1b these results indicated that rp11284f219 may serve an oncogenic role in lung carcinomaknockdown of rp11‘284f219 exerts anti‘oncogenic effects in lung carcinoma cells to study the specific role of rp11284f219 in lung carcinoma cells rp11284f219 sirna was transfected into ncih1299 and ncih460 cells fig 2a after transfection the proliferation of these cells was measured using cck‘ and edu assays fig 2b‘d the results suggested that knocking down rp11‘284f219 significantly reduced the proliferation of lung carcinoma cells compared with the nc group fig 2bd the invasiveness of si‘rp11‘284f219 transfected cells also significantly decreased as indicated by the data from the transwell assay fig 2f to further validate the invasive capability a rt‘qpcr assay was performed to detect the expression levels of invasion‘related genes and the results identified that both mmp2 and mmp9 were significantly decreased when rp11‘284f219 was downregulated fig s1the results of flow cytometry measurement based apoptosis assay suggested that cells transfected with sirp11284f219 had a higher apoptotic rate compared with the sinc transfected group fig 2e these data demonstrated the antitumor effects of rp11‘284f219 knockdown in lung carcinoma cells indicating an oncogenic role of rp11284f219rp11‘284f219 directly interacts with mir‘‘3p based on the prediction of the online tool lncbase v2 from diana prediction module httpcarolinaimisathena‘innovationgrdiana_toolswebindexphprlncbasev2index which was used to identify the downstream mirnas of rp11284f219 the first five mirnas in the output list were tested among the predicted potential targets it was found that mir6273p had the most significant upregulation in ncih1299 cells transfected with sirp11284f219 fig s2using sequence alignment it was identified that mir‘‘3p was partially complementary with the '‘utr of rp11‘284f219 fig 3a subsequently 293t cells were transfected with the pmirglorp11284f219wt or mut vector containing the wt or mut sequence of rp11284f219 '‘utr with or without mir‘‘3p mimics results from the luciferase reporter assay suggested that mir6273p mimics significantly decrease the signal of rp11‘284f219‘wt transfected cells but not the rp11‘284f219‘mut transfected cells indicating a direct interaction between the two non‘coding rnas fig 3a furthermore transfection of sirp11284f219 into ncih1299 and ncih460 cells resulted in the suppression of endogenous rp11‘284f219 leading to a significant increase in mir‘‘3p expression fig 3b thus these findings suggested an inhibitory effect of rp11‘284f219 on the expression of mir‘‘3p in lung carcinoma cellsthe expression of mir‘‘3p was detected in both lung carcinoma tissues and cell lines it was demonstrated that mir‘‘3p was significantly downregulated in carcinoma tissues fig 3c and ncih460 ncih1299 and a549 cells fig 3d compared with healthy tissues and cells collectively these data suggested a direct interaction between rp11284f219 and mir6273p in which rp11284f219 suppresses the expression of mir‘‘3prp11‘284f219 regulates the proliferation and invasiveness of lung carcinoma cells via mir‘‘3p to rescue the antitumor effects of sirp11284f219 in lung carcinoma cells the mir‘‘3p inhibitor which specifically downregulates the expression of mir‘‘3p was transfected into nci‘h1299 and nci‘h460 cells fig 4a the results from the cck‘ and edu assays demonstrated that treatment with si‘rp11‘284f219 0concology reports figure rp11‘284f219 knockdown inhibits lung carcinoma cell proliferation and invasion and promotes cell apoptosis a rp11‘284f219 knockdown was achieved via rp11‘284f219 sirna and the knockdown efficiency was verified using reverse transcription‘quantitative pcr n3 cell counting kit‘ assay was performed to measure the proliferation of b ncih1299 and c ncih460 cells after transfection with sirp11284f219 compared with the si‘nc group n5 d an edu assay was performed to measure the proliferation of nci‘h1299 and nci‘h460 cells after transfection with si‘nc and si‘rp11‘284f219 magnification x200 e flow cytometry analysis was performed to determine the effects of rp11‘284f219 knockdown on apoptotic rates in nci‘h1299 and nci‘h460 cells n3 f transwell assay was performed to determine the effects of rp11‘284f219 knockdown on nci‘h1299 and nci‘h460 cell invasion n3 magnification x200 p005 p001 vs control group nc negative control sirna small interfering rna od optical density and mir‘nc significantly decrease the proliferation of both ncih1299 and ncih460 cells fig 4bd however the administration of mir‘‘3p inhibitor partially reversed the antiproliferative effect of sirp11284f219 indicating that rp11284f219 regulates the proliferation of lung carcinoma cells partially via mir6273p fig 4bd in addition the 0cli rp11‘284f219 promotes lung carcinoma proliferation and invasionfigure rp11‘284f219 directly interacts with mir‘‘3p a binding site between rp11‘284f219 and mir‘‘3p that was identified using the diana tools and a luciferase reporter assay was conducted in pmirglorp11284f219wt or mut treated cells in the presence of mir6273p mimics or mir‘nc n3 p005 vs mir‘nc b expression of mir‘‘3p in nci‘h1299 and nci‘h460 cells transfected with si‘rp11‘284f219 was analyzed using rtqpcr p001 vs si‘nc n3 mir‘‘3p expression in c lc tissues and d nci‘h460 nci‘h1299 and a549 cells compared with adjacent healthy tissues and normal lung epithelial cells was analyzed using rt‘qpcr n3 p005 p001 vs adjacent tissue or beas‘2b cells nc negative control sirna small interfering rna wt wild‘type mut mutant mir microrna lc lung carcinoma mir‘‘3p inhibitor restored the reduction in the number of ncih1299 and ncih460 cells that migrated through the transwell membrane induced by si‘rp11‘284f219 treatment fig 4f these data indicated the participation of mir6273p in the rp11284f219mediated invasive effectthe qpcr assay results identified that both mmp2 and mmp9 expression levels were restored in rp11284f219downregulated cells when mir6273p was inhibited compared with the mir‘nc group fig s3 in addition transfection with mir‘‘3p inhibitor also diminished the pro‘apoptosis effect of si‘rp11‘284f219 in both ncih1299 and ncih460 cells fig 4e therefore it was suggested that rp11284f219 promoted the proliferation and invasion as well as suppressed the apoptosis of lung carcinoma cells by inhibiting the expression of mir‘‘3prp11‘284f219 regulates ccar1 via targeting mir‘‘3p to further evaluate how rp11‘284f219 exerts an oncogenic role via mir‘‘3p the publicly available algorithms of targetscan httpwwwtargetscanorg and mirdb were used which identified ccar1 as a potential target for mir6273p fig 5a in order to validate this prediction mir6273p mimic was transfected into cells and the transfection efficiency was assessed the results demonstrated that transfection of mir6273p mimic increased the expression of mir‘‘3p by times compared with cells transfected with mirnc fig s4after validating the upregulation of mir6273p mimic a ccar1wt vector was constructed which contained the wt binding site between mir‘‘3p and the ccar1 '‘utr and ccar1mut vector containing the mut sequence fig 5a the results from luciferase reporter assays indicated that compared with the mirnc group the mir6273p mimic significantly decreased the luciferase activity of ccar1‘wt treated cells but not the ccar1‘mut treated cells suggesting a direct binding of mir‘‘3p to the '‘utr of ccar1 fig 5b increased expression levels of ccar1 were present in the lung carcinoma tissues compared with the adjacent healthy tissues fig 5c moreover a significant decrease in both mrna and protein expression levels of ccar1 was detected upon transfecting ncih1299 and ncih460 cells with mir6273p mimics fig 5d and e thus ccar1 may be a direct target of mir‘‘3p in lung carcinoma cells and tissuesrp11‘284f219 knockdown inhibits tumor growth and the expression of ccar1 in vivo in order to investigate the effect of rp11284f219 on in vivo tumorigenicity ncih1299 cells were transfected with sinc or sirp11284f219 and injected into the nude mice after weeks a significantly 0concology reports figure rp11‘284f219 regulates proliferation and invasiveness in lung carcinoma cells via mir‘‘3p a expression of mir‘‘3p in nci‘h1299 and nci‘h460 cells transfected with mir‘nc or mir‘‘3p inhibitor was detected using rt‘qpcr analysis n3 p005 vs mir‘nc cell counting kit‘ assay was performed in b nci‘h1299 and c nci‘h460 cells stably transfected with si‘rp11‘284f219 in the presence of mir‘nc or mir‘‘3p inhibitor n5 d edu assay was performed in nci‘h1299 and nci‘h460 cells stably transfected with si‘rp11‘284f219 in the presence of mir‘nc or mir‘‘3p inhibitor magnification x200 e flow cytometry analysis was performed in nci‘h1299 and nci‘h460 cells stably transfected with si‘rp11‘284f219 in the presence of mir‘nc or mir‘‘3p inhibitor n3 f transwell assay was performed in nci‘h1299 and nci‘h460 cells stably transfected with si‘rp11‘284f219 in the presence of mir‘nc or mir‘‘3p inhibitor magnification x200 n3 p005 vs si‘nc nc negative control sirna small interfering rna od optical density mir microrna 0cli rp11‘284f219 promotes lung carcinoma proliferation and invasionfigure mir‘‘3p directly targets ccar1 a bioinformatic analysis of the predicted binding sites between the ccar1 '‘untranslated region and mir‘‘3p b luciferase reporter assay in ccar1‘wt or ccar1‘mut treated cells in the presence of mir‘nc or mir‘‘3p mimic n3 p005 vs mir‘nc c ccar1 expression in lc tissues compared with adjacent healthy tissues was analyzed using rt‘qpcr n13 p001 vs adjacent tissue expression of ccar1 in nci‘h1299 and nci‘h460 cells transfected with mir‘nc or mir‘‘3p mimics was detected using d rt‘qpcr and e western blotting n3 p005 vs mir‘nc mir microrna nc negative control wt wild‘type mut mutant rt‘qpcr reverse transcription‘quantitative pcr ccar1 cell division cycle and apoptosis regulator lc lung carcinoma slower proliferative rate of the tumors was observed in the sirp11284f219 group compared with the sinc group fig 6a and b furthermore the tumor volume and weight were significantly decreased in the si‘rp11‘284f219 group compared with the control group fig 6a and b rtqpcr analysis also demonstrated that compared with the sinc group the tumors in the si‘rp11‘284f219 group expressed higher levels of mir6273p fig 6c and lower levels of ccar1 fig 6d providing further evidence to the existence of the rp11‘284f219mir‘‘3pccar1 regulatory axis in lung carcinoma tumor tissuesdiscussionthe present study investigated the function of rp11284f219 in lung carcinoma it was initially found that rp11284f219 was significantly upregulated in both lung cancer tissues and cell lines following the deduction of a potential oncogenic role of this lncrna sirp11284f219 was transfected into ncih460 and ncih1299 cells and it was demonstrated that knockdown of rp11‘284f219 inhibited the proliferation and invasion while promoting apoptosis of lung carcinoma cells in the mechanistic studies using online prediction tools and in vitro assays the results indicated that mir‘‘3p directly interacts with rp11‘284f219 by binding to its '‘utrthe function of mir627 was initially reported in colorectal cancer crc padi found that when upregulated by calcitriol mir627 targets the histone demethylase jumonji domain containing 1a to increase methylation of histone h3k9 and suppresses the proliferative factors of crc cells thus inhibiting the proliferation of crc both in vitro and in vivo moreover in crc sun discovered the role of mir‘ in vitamin d‘enhanced efficacy of irinotecan via inhibition of the cytochrome p450 enzyme‘mediated intratumoral drug metabolism mir‘ is also reported to be a potential non‘invasive diagnostic marker in gastric and breast cancer types in pulmonary diseases mir627 is downregulated in patients with chronic obstructive pulmonary disease and targets the high‘mobility group box protein to inhibit its expression thus improving transforming growth factorβ1‘induced pulmonary fibrosis the present results demonstrated the inhibitory effect of rp11‘284f219 on the expression of mir‘‘3p in addition it was identified that the mir‘‘3p inhibitor can neutralize the anti‘tumor effects of rp11‘284f219 knockdown indicating that rp11‘284f219 promotes the proliferation and invasiveness of lung carcinoma cells partially by regulating mir‘‘3p this anti‘tumor role of mir6273p under the regulation of rp11284f219 in lung carcinoma tissues and cells is in accordance with the previous aforementioned findings on human crc gastric and breast cancer types 0concology reports figure rp11‘284f219 knockdown inhibits tumor growth in vivo a macroscopic image of xenografted tumors b tumor volume in nude mice injected with nci‘h1299 cells transfected with si‘nc or si‘rp11‘284f219 measured over weeks n5 c weight of tumors in nude mice at weeks after injection of nci‘h1299 cells transfected with si‘nc or si‘rp11‘284f219 n5 expression levels of d mir‘‘3p and e ccar1 in the tumor tissues from nude mice injected with nci‘h1299 cells transfected with si‘nc or si‘rp11‘284f219 for weeks were detected using reverse transcription‘quantitative pcr n5 p005 p001 p0001 vs si‘nc mir microrna nc negative control sh short hairpin rna ccar1 cell division cycle and apoptosis regulator using the publicly available rna interaction prediction algorithms the current study identified that ccar1 which was initially shown as the target gene of mir6273p is also regulated by rp11‘284f219 furthermore the regulatory axis of rp11‘284f219mir‘‘3pccar1 exists in the lung carcinoma cells both in vitro and in vivo in the tumor growth model the interaction between rp11‘284f219 and mir‘‘3p and the interaction between mir‘‘3p and ccar1 were demonstrated by the dual‘luciferase assay although this method has been used to validate rna‘rna interactions in previous studies other assays such as rna pulldown and rna binding protein immunoprecipitation that would provide more direct evidence for the rnarna and rna‘protein interactions should be performedccar1 was initially reported as a protein essential for cancer cell apoptosis induced by retinoids or chemotherapeutics such as adriamycin and etoposide subsequently kim et al revealed that this protein functions as a transcriptional coactivator of nuclear receptors in human breast cancer cells as ccar1 interacts and cooperates with the coactivators of estrogen receptor signaling it promotes the estrogendependent proliferation of cancer cells in crc cells ou reported that ccar1 can be recruited by βcatenin to act as a coactivator for the transcriptional activation of lymphoid enhancer binding factor ccar1 is essential for the expression of wnt target genes as well as the neoplastic transformation of crc cells in gastric cancer cells researchers have revealed the cooperation between ccar1 and βcatenin which leads to the promotion of the proliferation and migration of cancer cells in lung cancer ccar1 was reported to be an effector of doxorubicin‘induced apoptosis moreover muthu demonstrated that certain chemical compounds that bind with ccar1 can increase the expression of ccar1 and induce apoptosis however a contradictory conclusion was reported in a recent study which observed that ccar1 was promoted by serine and arginine rich splicing factor which is activated by glucose intake and further enhanced tumorigenesis by increasing the glucose consumption rate corroborating this finding in the current study via the targeting of mir‘‘3p the expr
Colon_Cancer
" phytolaccaceae species in china are not only ornamental plants but also perennial herbs that areclosely related to human health however both largescale fulllength cdna sequencing and reference genevalidation of phytolaccaceae members are still lacking therefore singlemolecule realtime sequencing technologywas employed to generate fulllength transcriptome in invasive phytolacca americana and noninvasive exotic picosandra based on the transcriptome data rtqpcr was employed to evaluate the gene expression stability in thetwo plant species and another indigenous congener p acinosaresults total of gb and gb clean reads of p americana and p icosandra were generated including and full length nonchimeric flnc reads respectively transcript clustering analysis of flnc readsidentified and consensus isoforms including and highquality ones after removingredundant reads and transcripts were obtained based on structure analysis total and alternative splicing variants and simple sequence repeats ssr as well as and completecoding sequences were detected separately furthermore and lncrna were predicted and and transcripts were annotated respectively subsequently seven reference genes in the two plant species and anative species p acinosa were selected and evaluated by rtqpcr for gene expression analysis when tested indifferent tissues leaves stems roots and flowers 18s rrna showed the highest stability in p americana whetherinfested by spodoptera litura or not ef2 had the most stable expression in p icosandra while ef1α was the mostappropriate one when attacked by s litura ef1α showed the highest stability in pacinosa whereas gapdh wasrecommended when infested by s litura moreover ef1α was the most stable one among the three plant specieswhenever germinating seeds or flowers only were consideredcontinued on next page correspondence yiwangynueducn1yunnan key laboratory of plant reproductive adaption and evolutionaryecology yunnan university kunming china2laboratory of ecology and evolutionary biology state key laboratory forconservation and utilization of bioresources in yunnan yunnan universitykunming chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cliu bmc plant biology page of continued from previous page fulllength transcriptome of p americana and p icosandra were produced individually based on thetranscriptome data the expression stability of seven candidate reference genes under different experimentalconditions was evaluated these results would facilitate further exploration of functional and comparative genomicstudies in phytolaccaceae and provide insights into invasion success of p americanakeywords phytolaccaceae smrt sequencing fulllength transcriptome analysis reference gene evaluation rtqpcr phytolacca americana is a member of the phytolaccaceae family and is native to northeast america becauseof its ornamental and medicinal applications it was introduced into china in unfortunately it hasevolved into an invasive species and spread to mostareas of the country especially in central and southernchina compared to noninvasive exotic congener p icosandra and native congener p acinosa p americana isof interest because it exhibits multiple biological activities such as plant pesticides antimicrobial propertyheavy metal accumulation capacity [“]in order to investigate the mechanisms of various bioactivities of p americana further transcriptomewidestudy is necessary to facilitate reports have showed thatjasmonic acidinduced and cadmiumtreated transcriptome data of p americana have been obtained by illumina hiseq and illumina hiseq platformrespectively [ ] howeverthese data were bothachieved by second generation sequencing sgs whichcould not produce fulllength transcripts genomic dataof p americana was available at the sra under projectprjna544344 but it™s raw reads without coding sequences prediction and functional annotation third generation sequencing tgs is known for itskilobasesized long reads and is an outstanding strategyfor better understanding rna processing for exampleit can be used to analyze different transcript isoformsregulated by alternative splicing which greatly increasesthe repertoire of proteins lead to genetic and functionaldiversity and is prevalent in most eukaryotic anisms the long reads could also provide sequence information on genecoding regions for functional analysis atthe transcriptional level and thus can be applied to refine an assembled genome for better annotation however tgs could not quantify gene expression forthe moment and have a relatively high error rate thansgs the combination of tgs and sgs are able to solvethis problem and are highly recommended by most researchers with the transcriptome and genome data availablefunctional genomics research is being performed whichrelied heavily on gene expression analysis reversetranscription quantitative real time pcr rtqpcr hasbeen reported to be a very sensitive and accurate technique to analyze gene expression level but it requiresappropriate reference gene as an internal control tonormalize mrna levels between different samples foran exact comparison of gene expression [ ] anideal reference gene should be expressed at a constantlevel across various experimental conditions howeverstudies have shown that no single reference gene is universal for all experimental conditions [“] therefore it™s necessary to estimate the stability of referencegenes under particular experimental condition beforeusing them for gene expression analysisin the present study to provide highquality and morecomplete assemblies in genome and transcriptome studies of phytolaccaceae a hybrid sequencing approachcombining the sgs and tgs technologies was carriedout first fulllength transcriptome of the invasive plantspecies of pameracana and an noninvasive exotic conicosandra was generated by singlemoleculegener prealtimesmrtsplicingevents simple sequence repeats ssr coding sequencesprotein annotations and long noncoding sequenceswere analyzed respectively at transcription level furtherthe stability of reference genes was evaluated in two phytolaccaceae species mentioned above and one nativecongener p acinosa by rtqpcr in order to facilitatefuture research on functional gene expressionsequencing alternativeresultsto classify the plant species these three phytolaccaceaemembers p americana p icosandra and pacinosa wereidentified by pcr and followed by sequences alignmentbased on sequences of second internal transcribed spacer its2 and the intergenic spacer of photosystem iiprotein d1 gene and trnahis gene of chloroplast genome psbatrnh table s1 the sequences of its2 andpsbatrnh in p americana that we employed had theidentity of with the sequences reported by chen in p icosandra the sequences of its2 had identity and the sequences of psbatrnh had identity with the results of chen™s in pacinosa 0cliu bmc plant biology page of similarity of its2 and identify of psbatrnh werefound isoforms afterremoving redundantsmrt sequencing data outputusing the pacific biosciences™ smrt sequencing protocol gb clean reads of invasive species p americana were obtained after preprocessing on the basis offull passes and sequence quality circular consensus sequences ccs with fulllength rate were obtained including fulllengthnonchimeric flnc sequences and highqualityconsensussequences from the high quality consensus isoforms transcripts alternative splicing events ssr complete coding sequences lnc rnasand annotated transcripts in p americana wereachieved similarly gb clean reads in p icosandrawere identified and ccs with fulllength rate flnc sequences as well as highquality consensus isoforms were filtered subsequently transcripts and alternative splicingevents were obtained what™s more ssrs complete coding sequences lnc rnas and annotated transcripts were identified in picosandratable transcriptome analysisbased on the structure of achieved transcripts and alternative splicing events were identified in pamericana and p icosandra respectively transcripts of bp in total in p americana wereemployed for ssr analysis based on the sequence lengththat was more than bp including ssrs and ssrcontaining sequences similarly transcripts bp in total in picosandra wereemployed for ssr analysis and ssrs togetherwith ssrcontaining sequences were identifiedtable summary of fulllength transcriptome sequencingclean reads gbccsflncflnc flncccsconsensus isoformhigh quality consensus isoformtranscriptsalternative splicingssrcomplete coding sequenceslncrnaannotated transcriptsp americanap icosandrathe detail information about the number of sequencescontaining more than one ssr the number of ssrspresent in compound formation and the number of different types of ssrs were shown in table in additiontotal of complete coding sequences cds in pamericana and cds in p icosandra were identified by using transdecoder the length distribution ofpredicted proteins was shown in fig s1in pdatabasespecifically nrwith the eight protein databases sequence alignmentswere performed to annotate predicted proteins in total transcripts in p americana and tranicosandra were annotated separatelyscriptstable the number of annotated protein sequencesin p americana was similar with p icosandra under aparticularncbi nonredundant protein analysis revealed that approximately transcripts in p americana and transcripts in picosandra showed the highest sequencesimilarity with beta vulgaris fig go gene ontology assignment also suggested that similar amount ofsequences in the two plant species belonged to the sameterm and many were classified into cell part and cellterm of cellular component catalytic activity and binding of molecular function and metabolic process andcellular process of biological process fig cogclusters of orthologous groups of proteins annotationshowed that a large number of predicted proteins in thetwo plant species were linked to functional class r general function prediction only j translation ribosomalstructure and biogenesis t signal transduction mechanisms g carbohydrate transport and metabolism ando posttranslational modification protein turnoverchaperones fig s2 the result of eggnog evolutionary genealogy of genes nonsupervised orthologousgroup annotation indicated that most of the annotatedproteins in the two plant species were belonging to thefunctional class s function unknown fig s3 kogeukaryotic ortholog groups functional classificationsuggested that r general function prediction only ando posttranslational modification protein turnover andchaperones were the most abundant functional categories in the two plant species fig s4 these results indicated that most of the sequences obtained were trulyfunctional proteins and had a similar functional classification in p americana and its congener picosandraeven though more work is needed to identify sequencesthat regulated or involved in the invasion success of pamericana the annotation of predicted proteins provided necessary information for further studiesbesides the transcripts encoding proteins long noncoding rnas lncrnas were achieved lncrnas arereported to be key regulators in plant biological prolncrna in pameracana andcesses the number ofpicosandra was predicted by cpc coding potential 0cliu bmc plant biology page of table ssrs obtained from transcripts with more than bpsearching itemtotal number of sequences examinedtotal size of examined sequences bptotal number of identified ssrsnumber of ssr containing sequencesnumber of sequences containing more than ssrnumber of ssrs present in compound formationnumber of mono nucleotide ssrnumber of di nucleotide ssrnumber of tri nucleotide ssrnumber of tetra nucleotide ssrnumber of penta nucleotide ssrnumber of hexa nucleotide ssrcalculator cnci codingnoncoding index pfamand cpat coding potential assessment tool respectively in total lncrna in pamericana and lncrna in picosandra were predicted by all these fourmethods fig subsequentlytranscription factorstfs that are key components involved in the transcriptional regulatory system were predicted in p americana tfs of types were filtered and in picosandra tfs of types were predicted thesetwo plant species shared the first types of tfs butthe number of each type tf was not similar especiallyrlkpelle_dlsv c3h snf2 and camk_camklchk1 indicating the particular functions on transcriptregulation fig amplification performance of rtqpcrprimers designed for rtqpcr were evaluated by pcrfirst the primers which produced single ampliconwithout primer dimer were chosen for melting curveanalysis only primers which produced a single fragmentefficiencywereqpcr amplificationchosenforp americanap icosandraassessment the qpcr efficiency of each primer pair wasgenerated from a 10fold serial dilution of pooled cdnaand was shown in table the threshold cycle ct values of each reference genewere employed to evaluate expression level under different experimental conditions fig average ct valuesfor all the seven candidate reference genes ranged from to in which ef1α showed the highest expression level and 28s rrna had the lowest expression levelit was also suggested that ct values of βactin and tubulin fluctuated significantly across all the experimentalsamplesstability of candidate reference genesforto determine the appropriate reference genesnormalization in different experimental conditions theexpression data was analyzed by genorm normfinderand bestkeeper respectively table s2when expression stability of reference genes wereanalyzed in different tissues leaves stems roots andflowers of p americana 18s rrna and ef2 oftable number of proteins annotated via differential protein databasedatabasesp americanaannotated number ‰¤ length length ‰¥ p icosandraannotated number ‰¤ length length ‰¥ coggokeggkogpfamswissproteggnognrall 0cliu bmc plant biology page of fig homologous species distribution of p americana and p icosandra annotated based on the nr database a p americana b p icosandrapamericana were identified as the most suitable reference genes by genorm and normfinder and 18s rrnawas also suggested by bestkeeper pairwise variation valueof v23 was below the cutoff value of which meansthe combination of two reference genes were most suitable for gene expression normalization fig whentested in picosandra ef1α was recommended for normalizing gene expression analysis not only by genorm butalso by normfinder ef2 was also suggested by genormand bestkeeper in pacinosa ef1α was the best reference gene suggested by genorm and bestkeeper but 18srrna was recommended by normfinder the use of tworeference genes was suitable because pairwise variationvalue of v23 was below when pooled the data of different tissues from pamericana and picosandra togetheref2 was shown to be the most stable gene by all the threemethods when investigated the expression stability ofreference genes in different tissues of pamericana andpacinosa 18s rrna showed the best expression stabilityby genorm and normfinder while ef2 was referred asthe most stable one by bestkeeper however the combination of five reference genes was recommended bygenorm for v56 which was less than when thedata of different tissues from picosandra and pacinosawas put together ef1α was identified as the best oneby genorm and normfinder whereas ef2 was suggested to be the best stability reference gene by bestkeeper when set the data of these three plant speciesas a pool ef1α was suggested to be the most stableone by genorm and normfinder while ef2 was alsorecommended by genorm and bestkeeper accordingto these results it is very important to select the appropriate reference gene when analyze the gene expressionlevel among plant species 0cliu bmc plant biology page of fig classification of the transcripts annotated by the gene ontology gowhen analyzed the data among germinating seeds28s rrna and ef1α were identified as the best reference genes by genorm while 18s rrna was recommended by normfinder and gapdh was suggested bybestkeeper three reference genes were sufficient tonormalize gene expression for v34 was below inflowers only of these three plant species ef1α was confirmed by all the three methods the genorm analysisshowed that the value of v45 was below so fourreference genes in combination were suggested thesefig venn diagram of the number of lncrnas predicted by cpc cnci cpat and pfam a p americana b p icosandra 0cliu bmc plant biology page of fig classification of predicted transcription factorsresults indicated that when focusing on particular tissuesof different plant species the selection of reference genewas also very essentialwhen plants were infested by slitura 18s rrnashowed the most expression stability suggested by genorm and normfinder in different tissues of pamericanawhile ef1α wasby bestkeeper therevealedcombination of two reference genes was suggested bygenorm due to the value of v23 was less than 18srrna was also recommended by genorm in s liturainfested picosandra and ef1α was shown to be themost stable one by normfinder and bestkeeper fourreference genes in combination were recommended bygenorm 18s rrna was also identified as the besttable primers for rtqpcr analysisgene nametubulingene descriptiontubulin ef1αelongation factor 1alphaprimer sequence ²²f gtaaggaagccgagaattgr tcaacaacagtgtcagagaf tgaagaaggtcggatacaatr gtagacatcctggagtgggaphdglyceraldehyde3phosphate dehydrogenasef tggtgctaagaaggttattatcef2elongation factor 18s rrna18s rrnaβactinactin728s rrna28s rrnar2 linear regression coefficientr gagtgaacggtggtcataf gtatcaccatcaagtcaactgr acaatcaaccacaacaaggf acttcctcttctcgtatcattr tgttcagcatagactgtgaf atgctatccttcgtctggr tactcttggctgtctctgf tacgattggttacggacatr ttctcatcaacaacagcatatlength bppcr efficiency r2 0cliu bmc plant biology page of together 18s rrna showed the best stability in genormand normfinder while the expression stability of ef1αwas suggested by bestkeeper in pamericana and picosandra 18s rrna was identified as the best referencegene by all the three algorithms in pamericana andpacinosa 18s rrna and βactin were suggested bygenorm in picosandra and pacinosa while gapdhand ef2 were recommended by normfinder and bestkeeper respectively when take all the data of s liturainfested plant species into account 18s rrna exhibitedthe most stable expression suggested by genorm andnormfinder while ef2 was the gene with the most constant expression identified by bestkeeperdiscussionfulllength transcripts are fundamental resources forstructuralfunctional and comparative genomics research [ ] smrt sequencing has been acknowledged by enabling the generation of multikilobasesequences to improve genome and transcriptome assembly the fulllength cdna sequences generated areable to characterize the posttranscriptional processsuch as alternative splicing lncrna prediction and coding sequences for further gene functional studies basedon the fulllength transcriptome data generated about gb of clean data were obtained for pamericana andpicosandra respectively table accordinglythenumber of ccs flnc consensus isoforms highqualityfig rna transcription levels of seven candidate reference genesin p americana picosandra and p acinosa the expression level ofcandidate reference genes in total samples n was presentedas cycle threshold number ctvalue and explained by box andwhisker plots the asterisks represented the minimum and maximumct value the squares indicated the 25th and 75th percentiles andthe median was represented by a bar across the squarereference gene by genorm in plant species pacinosawhile tubulin was suggested by normfinder and 28srrna was recommended by bestkeeper the combination of three reference genes was appropriate by genorm when analyzed the data oftwo plant speciesfig pairwise variation analyzed by genorm to determine the optimal number of reference genes for accurate normalization a threshold valueof was suggested for valid normalization if the value of vnn pairwise variation is less than then n reference genes in combinationare recommended for gene normalization if the value of vnn is more than then vn 1n should be taken into account pam pamericana pic p icosandra pac p acinosa lsrf different tissues of leaves stems roots and flowers gs germinating seeds of these three plantspecies f flowers of these three plant species lsr different tissues of leaves stems and roots i infested by s litura of third instar 0cliu bmc plant biology page of isoforms transcripts alternative splicing events ssrscomplete coding sequeces lncrnas and annotated transcripts were analyzed providing basic transcriptomic information for further studiesreports have showed that fulllength transcriptome ofzea mays have greatly helped in refining gene annotation and revealed the complexity of gene expression inmaize similar analysis has also been conducted inshum bicolor what™s more the world expansioncapability of cydia pomonella has been informed according to its genome information molecularmechanism of rapid growth and invasive adaptation ofan invasive species mikania micrantha has also been investigated according to itsreference genome therefore the fulllength transcriptome data of pamericana and picosandra will contribute to the genomic research and provide insights into invasive mechanism ofpamericana through comparative genomics study inphytolaccaceae speciesgenereliesonanalysisexpressionaccurate relative quantification of rtqpcr for furtherrobustnormalization by stably expressed reference genes tominimize error in the experimental process therefore suitable reference genes for the normalization ofrelative gene expression data in three phytolaccaceaespecies pamericana picosandra and pacinosa weresought under a diverse set of conditions these resultsdemonstrated the importance of validating referencegenes under the relevant experimental conditions forexamplein different tissues leaves stems roots andflowers of pamericana 18s rrna and ef2 were recommended to be the bestsuited reference genes and similar results were found in s liturainfested pamericanahowever even though the appropriate reference genesin picosandra were ranked according to the analyzed results of the three methods all the pairwise variationvalues were above the cutoff value of while thecombination of 18s rrna βactin ef1α and ef2 weremost suitable in s liturainfested picosandra ef2 andef1α have been considered as the ideal reference genesin pacinosa whereas the combination of 18s rrna βactin and gapdh were recommended after s litura infestation researches have also showed that no singlereference gene is stably expressed among different tissues of an anism such as the reference gene selectionin amygdalus persica solanum lycopersicum and glycine max [ ] what™s more our results alsosuggested that reference genes identified based on transcriptome data should be confirmed by experimentalevidence in jainduced transcriptome of p americana28s rrna showed stable expression between exogenousjatreated and control plants ja signal pathway ofplants can be induced by lepidopteran herbivores infestation however 18s rrna and ef2 were identifiedas the most stable expression reference genes in pamericana after s litura infestationin order to conductthe gene expression analysisamong different plant species of phytolaccaceae the dataof the three plant species were also compared togetherwhen compared the data in germinating seeds of threeplant species various genes were recommended by thethree methods the combination of plant species underother experimental conditions showed that the pairwisevalues of almost all the combination were higher thanthe cutoff value of exceptthe combination ofpamericana and pacinosa where five reference geneswere recommended for data normalization as well as thecombination of sliturainfested pamericana and sliturainfested picosandra where three reference geneswere suggested these results indicated that no particular gene was expressed constantly across different plantspecies even though these plants are congeners therefore reference genes should be employed appropriatelyunder the relevant experimental conditionsthe research has provided transcriptomewide fulllength isoforms of pamericana and picosandraproviding insights into invasive success of pamericanaguidelines for selecting appropriate reference genesunder different tissues in one plant species or amongvaried plant species were recommended further no particular gene was expressed constantly under differentexperimental conditions indicating the necessity of reference gene identification these results would facilitatethe exploration of functional and comparative genomicsstudies in phytolaccaceae to better understand plantbiologymethodsplant and insect materialsplants of p americana °²n °²e p icosandra°²n °²e and p acinosa °²n °²eused in this study which was named m k and q firstwere collected in yunnan china sampling was permitted when conducted complying with locallegislationthe formal identification of the samples were conductedby chao chen botany major of laboratory of ecologyand evolutionary biology state key laboratory for conservation and utilization of bioresources in yunnanyunnan university according to flora of china vol5“ flora of north america vol43“ chinese virtual herbarium httpwwwcvhaccn and global plants on jstor httpplantsjstor dna identification was also employed according tothe its2 region of nuclear ribosomal dna one of themost widely used dna fragments in plant molecularsystematics at the generic and species levels and the 0cliu bmc plant biology page of chloroplast psbatrnh intergenic region all voucher specimens were maintained at an experimental fieldof laboratory of ecology and evolutionary biology statekey laboratory for conservation and utilization of bioresources in yunnan yunnan universitytissues of leaves stems roots and flowers from oneindividual plant of p americana or p icosandra werecollected individually from the wild in yunnan provinceand no permission is needed for collecting theses samples each sample was flash frozen in liquid nitrogen andstored at ˆ’ °c for further experimentsshop101732681taobaocomthird instar larvae of spodoptera litura were purchased from henan jiyuan baiyun industry co ltdchinaand then werereared on artificial diet in a climate chamber h at °c with light and h at °c without light for further usefor reference gene evaluation seeds of p americanap icosandra and p acinosa were collected first from thewild in yunnan province and no permission is neededthe seeds were sown separately in agar plates andcultivated in the climate chamber after d five germinating seeds of one plant species were collected togetheras one sample for subsequent experiments each plantspecies have three replications two weeks later othergerminating seeds of each species were transplanted intoplastic pots cm diameter and cm height withsoil jiangsu peilei matrix technology development coltd china and cultivated with adequate water in artificial chambers with same conditions as described abovefour months later leaves stems roots and flowers ofeach plant species were collected individually simultaneously six larvae s litura of third instar were employedto infest on p americana p icosandra or p acinosawith one insect per leaf control treatments were herbivore free after h infestation leaves stems and rootsof these three plant species were harvested individuallyall samples collected were flash frozen in liquid nitrogenand stored in ˆ’ °c for subsequent assays and threereplicates were conducted for each treatmentnucleic acid extraction and assaysgenomic dna was isolated from the leaves of differentplant species following protocols provided by dnaquickplant system tiangen biotech co ltd beijing chinathen it was employed as the pcr template for plantspecies identificationpurekitplanttotal rnas from different tissues was prepared usingrnapreppolysaccharides polyphenolicsrich tiangen biotech co ltd beijingchina according to the manufacturer™s instructionsthe rna quality and purity were measured by using ananophotometer n60 implen germany and the agilent bioanalyzer system agilent technologies causa samples only with a ratio of to a ratio between and and a rin value morethan were chosen for the sequencing library construction an equal amount of total rnas from four different tissues of the same plant species were mixed asone sample for fulllength transcriptome sequencingtotal rnas from the samples collected for referencegene evaluation was also extracted individually as described above for each sample cdna was prepared byusing μg of total rna following the recommendedinstructions of fastquant rt kit with gdnase tiangenbiotech co ltd beijing chinapacbio cdna library preparation and smrt sequencingfulllength cdna was synthesized by using the smarter„¢ pcr cdna synthesis kit clontech ca usathe generated cdna was then reamplified using pcrafter end repairing smrt adaptor with a hairpin loopstructure was ligated to the cdna via exonucleasedigesting the cdna library was constructed after quality measurement of the cdna library smrt sequencingwas performed using the pacific bioscience sequel platform following the provided protocolillumina cdna library construction and secondgenarationsequencingthe extracted mrna was purified using oligo dtattached magnetic beads fragmentation was conducted inthe nebnext first strand synthesis reaction bufferfirststrand cdna was acquired based on the randomhexamers and then the secondstrand cdna was synthesized with dntps rnase h and primestar gxldna polymerase the synthesized cdna was purifiedwith ampure xp beads after end repairing adding polya and adaptor ligation ampure xp beads were used forsize selection the generated cdna was then amplifiedfor building cdna libraries the qualified libraries werepair end sequenced on illumina nova platformquality filtering and error correction of long readsraw smrt sequencing reads were filtered by removingpolymerase reads less than bp and sequence accuracyless than after removing adaptor subreads were obtained clean data was produced with subreads morethan bp ccss were produced from clean data withparameters of full passes and accuracy over after examining the coexistence of ² and ² adaptorsand poly a tail fulllength transcripts were selectedduring the processes of library preparation the chimericsequences formed by the direct linkage of two cdnatemplate strands due to the low concentrations ofadaptor or smrtbell are called artificial chimeric sequences the nonchimeric sequences in the fulllength 0cliu bmc plant biology page of transcripts are the fulllength nonchimeric flncsequencesas smrt sequencing generates a high error rate it isnecessary to perform error correction iterative clustering was used first to obtain consensus isoforms and thefulllength consensus sequences from iterative clusteringfor error correction were refined using quiver [ ]moreover the raw illumina sgs reads were filtered toremove adaptor sequences and low quality reads anderror correction of lowquality isoforms was conductedusing the sgs reads with the software proovread inbriefly the short reads of illumina rnaseq data weremapped to the low quality isoforms and then the basein the low quality isoform was replaced by the particularbase that had the maximum number
Colon_Cancer
n6methyladenosine m6a regulators are involved in the progression of various cancers via regulating m6amodification however the potential role and mechanism of the m6a modification in osteosarcoma remains obscurein this study wtap was found to be highly expressed in osteosarcoma tissue and it was an independent prognosticfactor for overall survival in osteosarcoma functionally wtap as an oncogene was involved in the proliferation andmetastasis of osteosarcoma in vitro and vivo mechanistically m6a dot blot rnaseq and meripseq meripqrtpcrand luciferase reporter assays showed that hmbox1 was identified as the target gene of wtap which regulatedhmbox1 stability depending on m6a modification at the ²utr of hmbox1 mrna in addition hmbox1 expressionwas downregulated in osteosarcoma and was an independent prognostic factor for overall survival in osteosarcomapatients silenced hmbox1 evidently attenuated shwtapmediated suppression on osteosarcoma growth andmetastasis in vivo and vitro finally wtaphmbox1 regulated osteosarcoma growth and metastasis via pi3kaktpathway in this study demonstrated the critical role of the wtapmediated m6a modification in theprogression of osteosarcoma which could provide novel insights into osteosarcoma treatmentintroductionosteosarcoma is a primary malignant bone tumor thatis common among childhood and adolescents worldwide1despite the improvements including multiagent chemotherapy with surgery in recent years the 5year survival rate is for localized osteosarcoma and is for recurrent and metastatic osteosarcoma23 thereforea better understanding of molecular mechanism isurgent for developing novel therapeutic strategies forosteosarcomacorrespondence jinglei miao miaojinglei126com orjinsong li jinsongli_csu163com1department of orthopaedics the third xiangya hospital of central southuniversity tongzipo rd changsha hunan china2shanghai key laboratory of regulatory biology institute of biomedicalsciences and school of life sciences east china normal university shanghai chinafull list of author information is available at the end of the edited by a stephanouitn6methyladenosine m6a is the prominent dynamicmrna modification which is involved in various biological process by regulating mrna translocation translation and stability4is catalyzed by m6a writermethyltransferase removed by erasers rna demethylases and recognized by m6a readers involving in variousbiological progression5“ the formation of m6a is catalyzed by a methyltransferase mettl3 and mettl14form a core catalytic complex of methyltransferases that isstabilized by wtap8 recently mettl16910 mettl5zcchc411 and zc3h1312 were showed to play a criticalrole in compositing methyltransferase complex andfacilitating m6a methylation13 the eraser alkbh5 andfto has m6a demethylation activity to remove the m6amodification the reader proteins are from yth familyheterogeneous nuclear ribonucleoprotein hnrp familyand insulinlike growth factor mrnabinding protein the authors open access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate ifchanges were made the images or other third party material in this are included in the ™s creative commons license unless indicated otherwise in a credit line to the material ifmaterial is not included in the ™s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this license visit httpcreativecommonslicensesby40official of the cell death differentiation association 0cchen cell death and disease page of isfamily they recognize the m6a modification to adjustrna metabolisms14dynamic and reversible m6a regulation was reported tobe involved in various physiological and pathologicalprocesses including stem cell differentiation cardiachomeostasis adipogenesis neuronal disorders and spermatogenesis15“ recent years compelling evidence hasrevealed that m6a modification plays an important role inthe tumorigenesis of various cancers20“ for examplemettl3 was reported to play key role in the progressionof colorectal carcinoma25 bladder cancer2627 gastriccancer28 and pancreatic cancer29 ythdf2 was involvedthe progression of acute myeloid leukemia aml viaregulating hematopoietic stem cells hscs activity4 ftowas also reported to play a crucial role in the progressionof melanoma30 breast cancer31 gastric cancer32 andintrahepatic cholangiocarcinoma nevertheless the role ofm6a modification in osteosarcomastill poorlycharacterizedwtap a wilms™ tumor wt1 associated protein33has been reported to be critical in the biological processesincluding g2m transition and premrna splicing34“in addition accumulated studies identified the importantrole of wtap in the progression of various cancers37 forexample wtap function as oncogene in cholangiocarcinoma38 acute myeloid leukemia39 colon cancer40 ovariancancer41 and diffuse large bcelllymphoma42 recentstudies reported that wtap is strictly connected to afunctional m6a methylation complex43 here we revealedthe increased expression of wtap in osteosarcoma tissuewhich was associated with clinicopathological features andpoor prognosis in osteosarcoma patients wtap functionas an oncogenic gene and it promotes the m6a modification and progression of osteosarcoma mechanistically wtap induced the growth and metastasis ofosteosarcoma via downregulating hmbox1 expression inm6adependent manner further investigations demonstrated that wtaphmbox1 regulated osteosarcomagrowth and metastasis via pi3kakt pathway overallthese results imply that wtaphmbox1 may be animportant mechanism of osteosarcoma progression andserve as a novel therapeutically target of osteosarcomamaterials and methodsclinical samples and ethicsonehundredandfour paired fresh osteosarcoma metastatic os sample and nonmetastatic os sampleand normal tissues were obtained from patients withoutradiotherapy andor chemotherapy at the third xiangyahospital of central south university and then immediately frozen in ˆ’ °c for rna and protein extraction orformalxed and paraffinembedded for further usedinformed consent was obtained from each patient or theirguardians and the study were approved by the ethicsofficial of the cell death differentiation associationcommittee of the third xiangya hospital of centralsouth universitycell culture transfection and infectionthe human hfob119 cells and osteosarcoma cell linessjsa1 mg63 hos u2os and 143b were obtainedfrom the cell bank of the chinese academy of sciencesshanghai china the osteosarcoma cells were culturedin dmem with fbs gibco grand island ny usathe hfob119 cells were cultured in dmemf12 df with fbsthe sequences of shrna cloned into plko1 vectorand then shwtapplko1 or shhmbox1plko1 werecotransfected with packing and pax2 plasmids into293tf cells fortyeight hours after transfectionthelentivirus was collected and infected hos and u2os cellsfor h the μgml puromycin was used for screeninginfected cells the primers were listed in table s1western blotproteintechantiythdf2antipi3k ab191606 abcamthe proteins were obtained from cells and tissues beinglysed with ripa buffer and then separated by sdspage the antiwtap ab195380 abcam antihmbox1161231apab220163abcamantippi3kab182651 abcam akt 9272s cell signaling paktser473and antigapdhab181602 abcam were used for pvdf membrane incubating overnight at °c the protein expression was detected by incubating with antirabbit secondary antibodies4051s cellsignalingrnaseq analysis and qrtpcrtotal rnas were extracted from osteosarcoma tissueand cells using trizol thermofisher usa for rnaseqanalysis the library construction and illumina sequencingusing the illumina truseq rna sample prep kit sandiego ca usa for qpcr validation the cdna wasobtained using first strand cdna synthesis kittoyobo finally the mrna expression was detectedusing sybr green biorad california usa all primers were in table s1rna m6a dot blot assay and rna m6a quantificationtotal rnas were extracted from osteosarcoma cells bytrizol thermofisher usa and then nanodrop3000was used for detecting rna quality the m6a content wasdetected by epiquik m6a rna methylation quantification kit colorimetric epigentek usathe polya rnas and ng were spottedonto a nylon membrane ge healthcare for rna m6adot blot assay and then incubated with m6a antibody noabe572 merck millipore usa at °c overnight them6a dots was analyzed using an imaging system biorad usa 0cchen cell death and disease page of meripseq and meripqrtpcrtotal rnas were extracted from osteosarcoma cells bytrizol the dnafree fragmented rnas were incubatedwith magnetic dynabeads bounded antim6a antibodyabcam usa to enrichment the mrna with m6a andthen beads were treated with proteinase k and rna wasextracted for meripseq or validation by qrtpcr theprimers are in table s1cell proliferation assaysthe cck8 cell counting kit8 c0038 beyotimebiotechnology china was used for cell proliferationability44colony formation assaysthe cells were seeded into 6well plates with a density × cells per wells and cultured in dmem medium for weeks and then paraformaldehyde pfa was usedto fix the colonies and crystal violet was used to stain asprevious described45woundhealing assayosteosarcoma cells were cultured for h to reach confluency and then a straight artificial wound wasscraped with a μl pipette tip the cell migration abilitywas measured by photographing the distance at and h45transwell invasion assaysthe transwell chamber bd science bedford mausa was used for invasion assays as previously described1 in brief × osteosarcoma cells were seeded intothe upper chambers and incubated for h the invasivecells were counted and quantified for cellinvasion asprevious described45luciferase reporter assaysthe ²utr of hmbox1 was cloned into pmiglovector promega usa the putative m6a sites bases awere mutated into bases c in ²utr using sitedirectedmutagenesis kit thermo usa the wt and mutplasmids were transfected in osteosarcoma cells and thenthe dualluciferase assay kit promega was used fordetecting the luciferase activity44animal experimentsnude mice weeks male were used for tumor modelall animal care and handling procedures were approvedby the institutional animal care and use committee ofthe third xiangya hospital of central south universitychangsha china for the subcutaneous tumor model ×shwtapshhmbox1 u2os cells seeded into mice via subcutaneous injection tumor volume and tumor weightshhmbox1 orshncshwtap orofficial of the cell death differentiation associationwere measured to analyze tumor growth as previousdescribed46 for orthotopic xenografttumor modelshnc shwtap shhmbox1 or shwtapshhmbox1u2os cells were labeled with a luminescent dye and gfpand injected into the cavity of the tibia of mice thirtydays after injection tumor growth was detected for themetastasis model the cells were injected into the tail veinand the lung metastasis were detected days afterinjection using in vivo bioluminescence imaging systemimmunohistochemistryimmunohistochemistry ihc analysis was performedusing antiwtap ab195380 abcam antihmbox1161231ap proteintech as pervious described26bioinformatics analysisthe geo dataset gse87624 and gse46705 weredownloaded and analyzed by r version httpswwwrproject the different expressed gene wascalculated with edger package and identified by thethreshold criteria of log2 foldchange fc ‰¥ and adjp go and kegg analysis was performed toinvestigate the potential role of genes protein“proteininteraction ppi network was analyzed by the stringdatabasestatistical analysisthe spss spss inc chicago il usa was usedfor the statistical analyses using anova or student™s ttest in this study kaplan“meier analysis was used forsurvival the correlation between wtap and hmbox1were analyzed using pearson analysis statistical significance was defined p resultselevated wtap is associated with poor prognosis ofosteosarcoma patientsto reveal the important role of m6a modification inosteosarcoma we explored the expression levels of m6arelative genes in osteosarcoma tissue and normalbone tissue gse87624 normal and osteosarcomaas shown in figs 1a and s1a we examined the expressionof m6arelated genes in geo database and found thatwtap rbm15 ythdf1 and ythdf2 were differentlyexpressed in tumor tissue compared with control tissuethe m6a writers were reported to play key role on theprogression of various cancers so wtap was selected forfurther analysis next we detected the expression ofwtap in pairs of osteosarcoma tissue and the corresponding paratumor tissues from the third xiangyahospital of central south university txhcsu consistent with the geo results the significant higher mrnaand protein levels of wtap in osteosarcoma was detectedusing the qpcr and wb analysis fig 1b c moreover 0cchen cell death and disease page of fig the expression levels of wtap in osteosarcoma tissue and cell lines a wtap expression in osteosarcoma tissue compared with normalbone tissue from gse87624 b qpcr assay and c western blot assay revealed the wtap expression in osteosarcoma tissue and the correspondingparatumor tissues from txhcsu d the kaplan“meier survival analysis e univariate and multivariate survival analysis f establishment of the overallsurvival nomogram for osteosarcoma patients g timedependent roc analysis h the mrna expression levels of wtap in osteosarcoma cell linesand hfob119 cell line using qpcrthe osteosarcoma patients with high wtap showed pooroverall survival in txhcsu fig 1d and the univariateand multivariate cox analysis showed that wtap andmetastasis were the independent prognostic factor foroverall survival in osteosarcoma patients fig 1e theprognostic nomogram wasthepredictusedtoprobabilities of overall survival rates of osteosarcomapatients and the calibration curves showed has a goodconsistency between the prediction and the actual observation fig 1f in addition the years roc curve aucof wtap in osteosarcoma patients from txhcsu was fig 1g besides the overexpression of wtap wasofficial of the cell death differentiation association 0cchen cell death and disease page of table the association of wtap expression andclinicopathologic characteristics of osteosarcoma patientswtap acts as an oncogene that promoted cell proliferation migration and invasion in osteosarcomacharacteristics case number wtap expressionp valuehmbox1 is potential target of wtap in osteosarcomagendermalefemaleage‰¤tumor size‰¤ cm cmmetastasisyesnotnmiiiiiiivhighn lown p p p p p associated with tumor size metastasis and tnm stagetable and fig s1b finally the higher expression ofwtap was also detected in osteosarcoma cell lines sjsa mg63 hos u2os and 143b compared with that inthe human osteoblast hfob119 cell fig 1h collectively these results demonstrated that wtap is highlyexpressed in osteosarcoma and is correlated with its poorprognosissilenced wtap significantly represses osteosarcomaprogression in vitroto generateto further clarify the role of wtap in osteosarcomashwtap1 andwe next used shrna lentivirusshwtap2stable wtapknockdownosteosarcoma cells and analyzed the role of wtap oncells migrationinvasion and proliferation of osteosarcoma the shwtap1 and shwtap2 significantlyknockdown the expression levels of wtap in osteosarcoma cells fig 2a the cck8 assay and colonyformation assay showed that wtap deficiency significantly reduced proliferative capacity of osteosarcomacells fig 2b c the transwellinvasion and woundhealing assays showed that the invasion and migrationability of the osteosarcoma were significant reduced bysilenced wtap fig 2d e these results determine thatofficial of the cell death differentiation associationto reveal the potential mechanism by which wtapregulates the progression of osteosarcoma we performedrnaseq m6aseq in shwtapshnc u2os cell andclipseq to revealthe potential genes regulated bywtapmediated m6a modification the rnaseq resultsrevealed downregulated degs and upregulateddegs with logfc and adjp in shwtapgroup compared with shnc group fig s2a geneontology go showed that degs were enriched in neutrophil activation cell cycle and so on fig s2b kegganalysis showed that degs were enriched in metabolismrelated signal pathway fig s2c the m6aseq analysisidentified differentially m6a modification genes inwtapsilenced u2os cell compared with normal u2oscells table s2 the clipseq from gse46705 revealed wtaptargeted rna in wtap overexpressed cellusing parclip technology and then we obtained theoverlapped genes in rnaseq m6aseq and clipseq asshown in fig 3a genes were overlapped in three groupsincluding two upregulated gene slc3a2 casp7 andfour downregulated gene abr hs6st1 cd59 andhmbox1 consistent with these results slc3a2 andcasp7 were significantly upregulated and abr hs6st1cd59 and hmbox1 were significantly downregulated inosteosarcoma tissues from geo gse87624 databasefig s3a these results were also confirmed in theosteosarcoma tissues from our txhcsu data fig s3band then we evaluated the regulation of wtap on thesix candidates in osteosarcoma cells using qpcr amongwhich hmbox1 was the most significantly upregulatedgene in shwtap osteosarcoma cells fig 3b and wasselected for further analysis consistent with the mrnaexpressionthe protein level of hmbox1 was alsoremarkably increased by silenced wtap fig 3c wenext evaluated the relationship between wtap andhmbox1 expression in gse87624 and txhcsu as ourspeculation hmbox1 expression was negatively correlated with wtap expression r ˆ’ in gse87624and r ˆ’ in txhcsu fig 3d moreover weanalyzed the relationship between hmbox1 expressionclinicopathological features in osteosarcoma patientsfrom txhcsu hmbox1 expression was significantlyreduced with tumor size and metastasis table and figs3c and the survival analysis results showed that thepatients with low level hmbox1 had poor overall survival in osteosarcoma fig 3e moreover the univariateand multivariate analysis demonstrated hmbox1 as anindependent prognosticsurvivalp in osteosarcoma patients fig 3f we alsorevealedinof hmbox1expressionfactorfor overallthelower 0cchen cell death and disease page of fig silenced wtap significantly repressed osteosarcoma progression in vitro a the shwtap1 and shwtap2 downregulated wtapexpression b cck8 assay revealed that silenced wtap reduced the cell proliferation ability in osteosarcoma c colony formation assay showed thatsilenced wtap decreased the cloning number of osteosarcoma cells d transwell invasion and e woundhealing assay revealed the inhibition ofsilenced wtap on osteosarcoma cell invasion and migrationosteosarcoma cell lines sjsa1 mg63 hos u2os and143b than that in the human osteoblast hfob119 cellline fig 3g in these results indicated thathmbox1 is a potential target of wtap and is related topoor prognosis of osteosarcoma patientswtap regulates hmbox1 expression via m6a modificationin osteosarcomaas meripseq data revealed different m6a modificationof hmbox1 in nc and wtapsilenced cells we nextanalyzed whether wtap regulated the hmbox1official of the cell death differentiation association 0cchen cell death and disease page of fig hmbox1 is a potential target of wtap a the venn diagram was generated from differentially expressed genes in rnaseq m6aseq andclipseq in gse46705 the expression of the overlapped genes in hos and u2os cells with silenced wtap using b qpcr assay and c western blotassay d correlation analysis of wtap and hmbox1 in osteosarcoma from gse87624 and txhcsu e the kaplan“meier survival analysis f univariateand multivariate survival analyses g wtap hmbox1 expression in osteosarcoma cell lines and human osteoblast hfob119 cell lineexpression in an m6adependent manner using m6a dotblot and rna methylation quantification assay asexpected m6a levels were substantially decreased inwtapknockdown osteosarcoma cells compared controlosteosarcoma cells fig 4a moreover meripqpcrassay showed that hmbox1 was effectively enriched bym6aspecific antibody and enriched hmbox1 wasremarkably decreased by in wtapknockdown cells fig4b therefore we supposed that wtap could regulatem6a levels of hmbox1 according to the m6a rnaseqthe ²utr ofresulthmbox1 and the sramp httpwwwcuilabcnsramppredicted three very high confidence m6a sites at the ²utr of hmbox1 fig s4 to further prove the directedthe m6a modification was attarget role of wtap on hmbox1 with m6a modification we cloned the hmbox1 ²utr containing these m6a sites into pmirglo vectors and then we mutant thebases a into bases c in the predicted m6a sites fig3f in fig 3g the luciferase activity of hmbox1 wassignificantly increased by silenced wtap however theluciferase activity of mut hmbox1 did not affected bysilenced wtap in both hos and u2os cells fig 4cythdf2 is a well know m6a reader and plays animportant role in the progression of several cancers viaregulating mrna degradation figure 1s showed thatythdf2 was upregulated in osteosarcoma tissues ingse87624 in fig 4e ythdf2 evidently upregulated inosteosarcoma tissue and cells we next shed light on theofficial of the cell death differentiation association 0cchen cell death and disease page of table the association of hmbox1 expression andclinicopathologic characteristics of osteosarcoma patientscharacteristics case number hmbox1 expressionp valuehighn lown gendermalefemaleage‰¤tumor size‰¤ cm cmmetastasisyesnotnmiiiiiiivp p p p p expression of ythdf2 in osteosarcoma and the role ofythdf2 on hmbox1 expression in osteosarcoma disappointedly silenced ythdf2 showed no effects onhmbox1 expression in osteosarcoma cells fig 4f suggesting that wtap regulated m6amediated hmbox1expression in ythdf2independent mannertogether these data suggested that wtaprepressedhmbox1 expression via regulating m6a modification ofhmbox1 at its ²utrhmbox1 is involved in wtapmediated osteosarcomaproliferation and metastasis in vitrowe next explored whether wtap promoted osteosarcoma progression by regulating hmbox1 expressionas shown in fig 5a hmbox1 expression was evidentlyincreased by wtap knockdown and was decreased byhmbox1 knockdown in osteosarcoma cells the cck8results showed that the proliferation levels were increasedby shhmbox1 in wtapsilenced hos and u2os cellsfig 5b consistent with the cck8 results silencedhmbox1 also alleviated shwtapmediated repressionof cell proliferation in colony formation assay fig 5cthe similar effects of hmbox1 were also observed inwtapsilenced osteosarcoma cell using woundhealingand transwell invasion assays fig 5d and e in additionofficial of the cell death differentiation associationwestern blot results showed that silenced wtap evidently repressed the expression of mesenchymal markerscadherin and vimentin and induced the expression ofepithelial marker ecadherin which was attenuated byshhmbox1 in osteosarcoma cells fig 5f these datasuggested wtap promotes osteosarcoma cells proliferation and metastasis via repressing hmbox1 expressionhmbox1 inhibits osteosarcoma growth and metastasisin vivowe next verified the role of hmbox1 in vivo byinjecting shnc shwtap shhmbox1 and shwtapshhmbox1 u2os cells to induce subcutaneous osteosarcoma mice model orthotopic xenograft tumor modeland tail vein metastasis model the expression levels ofhmbox1 was significantly upregulated by silencedwtap in subcutaneous osteosarcoma tissue figs 6a ands5 and s6 moreover silenced wtap repressed thetumor size and tumor weight in subcutaneous osteosarcoma mice which was rescued by silenced hmbox1fig 6b we next used a luminescent dye and gfplabeled u2os cells to build an orthotopic xenografttumor model the bioluminescence imaging showed thatthe wtap knockdown reduced the proliferation ofu2os cells in situ while silenced hmbox1 alleviatedthis repression fig 6c to further detect the role ofwtap and hmbox1 on the metastatic ability ofosteosarcoma in vivo u2os cells were injected into nudemice via the tail vein the bioluminescence imagingshowed that silenced hmbox1 alleviated the repressionof silenced wtap on osteosarcoma metastasis fig 6dtaken together these results suggest that hmbox1 isinvolved in wtapmediated tumor growth and metastasis of osteosarcomawtaphmbox1 regulates the proliferation and metastasisof osteosarcoma partly by pi3kakt pathwaythe kegg results identified that pi3kakt pathwayscould be regulated by wtap fig s2b pi3kakt signaling pathway promotes the growth migration andinvasion of various cancers including osteosarcoma4748we hypothesized that wtaphmbox1 was involved inthe progression of osteosarcoma via regulating pi3kaktsignaling pathway as shown in fig 7a phosphopi3k andphosphoakt were evidently repressed by shwtap andinduced by shhmbox1 and shhmbox1 significantlyattenuatedandphosphoakt in both hos and u2os cells ly294002 api3kakt pathwaysreducedshhmbox1induced cell proliferation migration andinvasion in both hos and u2os cells fig 7b cthereforethese results imply that wtaphmbox1regulates the proliferation and metastasis of osteosarcomapartly via pi3kakt pathway fig shwtaprepressedphosphopi3kinhibitorremarkably 0cchen cell death and disease page of fig hmbox1 is negative correlation wtap expression and is associated to the poor prognosis of osteosarcoma a the m6a level in hosand u2os cells with silenced wtap b meripqpcr assay followed by qrtpcr revealed the hmbox1 m6a modification c wildtype or m6a sitemutant hmbox1 were cloned in pmirglo d luciferase reporter assays revealed the target role of wtap on the ²utr of hmbox1 e ythdf2expression in osteosarcoma tissue and cells f the effects of silenced ythdf2 on hmbox1 expression in osteosarcoma cellsdiscussionin the past several years m6a modification is considered as a pervasive internal modification of mrna andplays critical roles in the progression of a variety of humandiseases including cancers20 however the underlyinginvolvement of m6a regulators in osteosarcoma progression is still unclear in the present study we focused onthe role and underlying mechanism of wtap and itmediated m6a modification in the progression andmetastasis of osteosarcoma in this study wtap wasfirstly identified to upregulated which was associated withpoor prognosis of osteosarcoma functionally wtappromoted the growth and metastasis of osteosarcomain vitro and vivo mechanistically hmbox1 was identified as the target gene of wtap and it was regulated bywtap with m6a modification at the ²utr finallywtaphmbox1 regulated osteosarcoma growth andmetastasis in a pi3kaktdependent patternactually wtap was reported as an oncogene in various cancers37“ recent studies reported that wtapis strictly connected to a functional m6a methylationcomplex43 howeverfew study demonstrated theimportant role of wtap as a m6a regulator here weconcluded that wtap is not only upregulated but alsoplays key role on the m6a modification in osteosarcomanotably the aberrant of m6a modification is related toofficial of the cell death differentiation association 0cchen cell death and disease page of fig hmbox1 as a tumor suppressor was involved in wtapmediated progression a hmbox1 expression in osteosarcoma cell with shwtapand shhmbox1 knockdown of hmbox1 effectively alleviated wtapdependent b cell proliferation c colony formation d transwell invasion ande woundhealing assay f the protein expression level of emt transition related protein p various biological processes via regulating mrna stability splicing and translation next we shed light on thedownstream mrna that modified by wtapmediatedm6a modification by combining the data from rnaseqm6aseqshowed thathmbox1 is a potential target gene of wtap subsequently meripqpcr western blotand luciferasereporter assay results revealed that wtap repressedand clipseq theresultshmbox1 expressed with wtapdependent m6a modthe ²utr of hmbox1 thus wtapification atinvolved in tumorigenesis depending on its role of m6amodification although ythdf2 is a wellknown m6areader in several cancers we found that ythdf2 showedno effects on hmbox1 expression suggesting thatwtap regulated m6amediated hmbox1 expression inythdf2independent manner and the m6a readerofficial of the cell death differentiation association 0cchen cell death and disease page of fig silenced hmbox1 attenuated shwtaprepressed osteosarcoma growth and metastasis in vivo a the expression levels of hmbox1 inosteosarcoma mice models b knockdown of hmbox1 effectively alleviated shwtaprepressed osteosarcoma growth in mice c the orthotopicxenograft tumor and d lung metastasis were detected using a vivo bioluminescence imaging system representative images and a histogram areshown n each groupwhich was involved in the m6a modification of hmbox1need be further investigatedhmbox1 a homeobox containing protein49 wasreported to be a transcriptional repressor involving in thebiological processes in bone marrowderived stromacells50 nk cells5152 and vascular endothelial cells53recent studies demonstrated the dysregulated hmbox1in various cancers for example high expression ofhmbox1 was observed in gastric cancer and it wasassociated with the poor prognosis of gastric cancer54moreover hmbox1 also observed as tumor suppressorin ovarian cancer55 and cervical cancer56 hmbox1repressed the progression of ovarian cancer via regulatingcell proliferation and apoptosis55 hmbox1 repressedliver cancer progression via regulating autophagy as wellas and immune escape57 consistently hmbox1 isofficial of the cell death differentiation association 0cchen cell death and disease page of fig wtap hmbox1 was involved the progression of osteosarcoma via pi3kakt pathway a western blot analysis for the expression ofppi3k pi3k pakt and akt in osteosarcoma cells b cell proliferation c colony formation transwell invasion and woundhealing assay ofosteosarcoma cell treated with μm akt inhibitor ly294002 abcam ab120243 for hunclear how hmbox1 as a transcriptional repressorregulates pi3kakt pathway nonethelesssilencedhmbox1 only partly alleviated wtapmediated osprogressionthere are other potentialmolecular mechanisms regulated by wtapmediatedepigenetic modulation in osit means thatin summary we identified the elevated wtap inosteosarcoma and which is associated with poor clinicaloutcome and serves as an independent prognostic factorfor osteosarcoma patients wtap dramatically promotedosteosarcoma proliferation and metastasis via regulatinghmbox1 mrna stability in a m6adependent mannerwtaphmbox1 regulated osteosarcoma growth andmetastasis via pi3kakt pathway altogether our resultsdetermined wtap hmbox1 as a potential therapeutictarget for osteosarcomaacknowledgementsthis study was supported by national natural science foundation of chinagrant nos national natural science foundation of china grantnos natural science foundation of hunan province grant nos2018jj2617 natural science foundation of hunan province grant nos2016jj3176 national key research and development program of china no2016yfc1201800 natural science foundation of hunan province no2018sk2090author details1department of orthopaedics the third xiangya hospital of central southuniversity tongzipo rd changsha hunan china 2shanghai keylaboratory of regulatory biology institute of biomedical sciences and schoolof life sciences east china normal university shanghai china 3fourgynecological wards ningbo women and children™s hospital ningbozhejiang china 4the second xiangya hospital of central southuniversity changsha china 5school of basic medical science central southuniversity changsha china 6department of anatomy histology andembryology changsha medical university changsha chinafig a schematic model illustrating wtapmediated m6a regulationin osteosarcomaevidentlydownregulated and closely related to the poor prognosisof osteosarcoma in the present study in addition silencedhmbox1alleviated wtapknockdownmediated repression of osteosarcoma progression whichimplied the import roles of hmbox1 in wtapdrivenosteosarcoma development finally we analyzed thedownstream pathway of wtaphmbox1 in osteosarcoma pi3kakt pathway was reported to an important role in the progression of various cancers includingosteosarcoma58 we found that pi3kakt pathway we
Colon_Cancer
" ethnopharmacological relevance herba patriniae has been used for thousands of years in china as a traditional chinese medicine with heatclearing and detoxicating effects it is applied widly for the treatment of rheumatoid arthritis diarrhea acute hepatitis pelvic inflammatory disease and ulcerative colitis in clinic two species namely patrinia scabiosaefolia fisch ps and patrinia villosa juss pv from the caprifoliaceae family are considered as herba patriniae in the pharmaceutical industry aim of the review this paper aims to comprehensively outline the traditional uses botanical description phytochemistry pharmacology toxicology quality control pharmacokinetics and patents of herba patriniae and elaborate the samedifferent characteristics between ps and pv materials and methods detailed information of herba patriniae was collected from various online databases pubmed web of science google schola china national preproof 0c knowledge infrastructure database national intellectual property administration prc national medical products administration and those published resources msc thesis and books results a total of compounds have been identified in herba patriniae including triterpenoid saponins flavonoids anic acids irids and volatiles a very distinct difference was observed that ps is rich in triterpenoid saponins and volatiles while pv contains more flavonoids two source species of herba patriniae gave similar pharmacological effects on anticancer antiinflammatory antioxidant antimicrobial sedative and hypnotic effects but there were no reports were on antipruritic proangiogenic and antidiarrheal effects for ps and no studies on antidiabetic effects for pv generally herba patriniae showed nontoxic in the clinical dose but mild side effects such as temporary leukopenia dizziness and nausea could be found when large and excessive dosage is used a variety of compounds have been quantified for the quality control of ps and pv the variety growth environment growth time and harvest time not only affected the contents but also the pharmacological activities of the bioactive compounds in the past year patents for compositions containing pv and ps have been filed mainly involving human health hygiene agriculture and animal husbandry unfortunately the research on pharmacokinetics is insufficient only the prototype components and metabolites were repored after intragastric administration of total flavonoids extract from pv in rats herba patriniae has displayed a significant medicinal value in clinic but the differences in phytochemistry pharmacological effects and the content of compounds have been found between two official recorded species about side effects and pharmacokinetic characteristics the differeces between two species have not been well studied for a better clinical use of herba patriniae it is urgent to establish systematic pharmacology quality control pharmacokinetics and clinical researches on the samedifferent characteristics between ps and pv keywords herba patriniae traditional uses phytochemistry pharmacology quality control preproof 0c cells a549 human lung cells cancer aspartate polysaccharide mixture ast list of abbreviations 3t3l1 preadipocytes 5fuhct8 human ileocecal adenocarcinoma cells a2780 human ovarian cancer cells a375s2 human melanoma cells a498 human renal abts carcinoma 'azinobis3ethylbenzothiazoline6sulphonic acid ags human gastric cancer cells akt protein kinase b alt alanine aminotransferase ap acute pancreatitis ap3 aminotransferase bax bcl2associated x protein bcl2 bcell lymphoma2 bclxl b cell lymphoma factor xl bel7402 human hepatoma cells bv2 mouse microglia cells caco2 human colon cancer cells cox2 cyclooxygenase2 crc colorectal cancer dai disease activity index dpph 22diphenyl1picrylhydrazyl ec50 half maximal effective concentration emt epithelialmesenchymal transition fak focal adhesion kinase gcms gas chromatographymass spectrometer glut4 glucose transporter gsh glutathione h2o2 hydrogen peroxide hela human cervical cancer cells hepg2 human hepatoma cells hl60 human promyelocytic leukemia cells ho1 heme oxygenase1 hplc high performance liquid chromatography hsp heat shock proteins hsp heat shock proteins ht1080 human fibrosarcoma cells ht29 human colon carcinoma cells huvecs human umbilical vein endothelial cells ic50 inhibitory concentration icam1 intercellular adhesion molecule icr institute of cancer research il1 interleukin1 beta il6 interleukin il8 interleukin inos inducible nitric oxide synthase irs insulin receptor substrate k562 human malignant myeloid cells ldh lactate dehydrogenase lps lipopolysaccharides mcf7 human breast cancer cells mda malondialdehyde mdamb231 human breast cancer cells mpo myeloperoxidase mrna messenger ribonucleic acid nfκb nuclear factor κb ngf nerve growth factor no nitric oxide nqo1 quinine oxidoreductase nrf2 nuclear factor erythroid 2related factor o2 superoxide anion oh hydroxyl radical pcna proliferating cell nuclear antigen pid pelvic inflammatory disease ps patrinia scabiosaefolia fisch pv patrinia villosa juss raw2647 mouse leukemic monocyte macrophage ros reactive oxygen species rsv respiratory syncytial virus sars severe acute respiratory syndrome sd sprague dawley sgc7901 human gastric cancer cells smmc7721 hepatocellular carcinoma cells stat3 signal transducer and activator of transcription sw480 human colon carcinoma cells tc50 half toxic concentration tcm traditional chinese medicine tgf transforming growth factor beta ti drug treatment index tnfα tumor necrosis factor alpha taoc total antioxidant capacity tsod total superoxide dismutase u14 mice cervical cancer cells u266 human multiple myeloma cancer cells u937 human lymphoma cells uc ulcerative colitis uv ultraviolet preproof 0c table of contents introduction traditional uses botany phytochemistry triterpenoid aglycones and triterpenoid saponins flavonoids anic acids irids volatiles other compounds pharmacology anticancer effect antiinflammatory effect antioxidant effect antimicrobial antiviral and antifungi effects sedative and hypnotic effects others toxicity quality control pharmacokinetics patented formulations and perspectives acknowledgements conflict of interest author contribution references preproof 0c introduction herba patriniae as known as œbai jiang cao in chinese is a traditional chinese medicine tcm originally recorded in œshen nong™s herbal classic as a middle grade medicinal material which has been used for thousands years besides korean ancient pharmacopaea œdonguibogam also record its medical value and it has been used for more than years in korea jeon et al it possesses the tcm properties of pungent and bitter in flavor and slightly cold in nature and has been classified to the stomach large intestine and liver meridians xiao two official species of patrinia scabiosaefolia fisch ps and patrinia villosa juss pv figure were considered as herba patriniae in chinese pharmacopoeia edition and chinese provincial pharmacopoeias these two plants have been widely used for more than years with good biological activities of clearing heat and detoxification eliminating carbuncle and expelling pus dispelling blood stasis and relieving pain through an analysis of ancient and modern literatures herba patriniae was mostly used in intestinal carbuncle lung carbuncle gynecological epigastric pain postpartum blood stasis and eczema in ancient times chen and han modern pharmacological studies have found that it has effects of anticancer antiinflammation antipathogenic microanisms antioxidation sedation and hypnosis wang et al 2019a nowadays herba patriniae is widely used in the respiratory system digestive system genitourinary system gynecology dermatology and other multidisciplinary diseases in clinical practice zhu and jiang and the number of applied patents increases every year httppsssystemcnipagovcn in view of its high content of amino acids vitamins minerals and other nutrients herba patriniae is not only regarded as a potherb with healthy value but also processed into tea products su et al zeng et al zhong et al in the past decades an increasing number of scholars have studied the chemical constituents and pharmacological effects of herba patriniae interestingly based on these studies we found that there are many differentsame characteristics between ps and pv both of them are official species for herba patriniae but differentiated clinical uses of them in different diseases may be better for the clinical outcome unfortunately we cannot found a comprehensive and updated review on the samedifferent characteristics of the two sources of herba patriniae and actually these two species also have not been differentiated in clinical uses therefore this review aims to systematically summarize the similarities and differences from the preproof 0c aspects of the traditional uses botanical description phytochemistry pharmacology and quality control of these two species of herba patriniae as well as being evidences for their clinical application and further research figure two species of herba patriniae a patrinia villosa juss b patrinia scabiosaefolia fisch a httpwwwcvhaccnspmcshcsh0005548 b httpwwwcvhaccnspmsyaufsyauf010108 traditional uses herba patriniae has a wide geographical distribution mainly in east asia and north america he et al some plants such as sonchus arvensis l sonchus asper vill sonchus oleraceus l etc may be confused as herba patriniae lu and hence these adulterants of herba patriniae should be exclude when clinical use traditionally according to records of œshen nong™s herbal classic 神农本草经 œcompendium of materia medica 本 草 纲 目 and œsynopsis of the golden chamber 金匮要略 œtai ping sheng hui fang 太平圣惠方 œpu ji fang 普 济方 œsheng ji zong lu 圣济æ»å½• œqian jin yi fang 千金翼方 and œqian jin fang 千金方 ancient doctors have used the whole herbs and roots of herba patriniae for disease treatment such as the stomach intestine liver gallbladder and gynecological diseases tian and tian zhu and jiang herba patriniae was recorded in chinese pharmacopoeia edition for the treatments of appendicitis dysentery enteritis hepatitis conjunctivitis postpartum blood stasis abdominal pain swollen wellingabscess and clove sores pharmacopoeia committee of the ministry of health of p r china in addition herba patriniae is also preproof 0c recorded in the standards of traditional chinese medicine in many provinces of china table in miao nationality herba patriniae is also called œjia jiang le and used to treat rheumatoid arthritis colds and diarrheal qiu wang in dong medicine lu yi medicine drug control institute of yunnan chuxiong health bureau and dai medicine shi ps is called œnyangt ngeec liongc bail jangl œshe wei long and œpa hong respectively its whole herb is used to treat infantile diarrhea schizophrenia and infantile tinea capitis respectively ps is also called ba gai bao in zhuang medicine and its root is used to treat icteric hepatitis furuncles and snakebites shi pv is called œbitter vegetable by she nationality biological products identification institute of the ministry of health and œpao zi tong by tujia nationality peng and guan its whole herb can be used to treat appendicitis intestinal febrile symptoms constipation mammary abscess blister carbuncle and qi stagnation pv is also called œba gai lan and œhong pa in zhuang medicine biological products identification institute of the ministry of health and dai medicine shi respectively and its root is used to treat jaundice hepatitis furuncle local ulceration caused by snake injury and infantile convulsion moreover in korea people usually use the roots or whole plants of ps and pv as a traditional herbal medicine to treat appendicitis inflammation wound healing edema abscesses endometritis and abdominal pain after childbirth kang et al yang et al in recent years herba patriniae has been extensively applied in clinical practice in china especially in gynecology such as postpartum pain mastitis dysmenorrhea and tubal obstructive infertility liu 2019a it is noteworthy that herba patriniae is one of the most important ingredients in many prescriptions of tcm which is effective in diarrhea he acute hepatitis song pelvic inflammatory disease zhang 1997a typhoid fever paratyphoid fever sun ulcerative colitis liu anal cryptitis shi pelvic endometriosis yan and qiu acute pancreatitis he et al 2019b itching wang and wang gastroesophageal reflux disease benign prostatic hyperplasia rhinosinusitis mumps and phlebitis kong and zhao zhu and jiang a powder composed of coicis semen radix aconiti lateralis preparata and herba patriniae is a classic prescription for treating intestinal carbuncle in the œsynopsis of the golden chamber which is clinically used to treat chronic appendicitis chronic pelvic inflammatory disease and chronic prostatitis ji in addition a powder containing herba preproof 0c patriniae in the prescription is also used to treat sinusitis acute purulent tonsillitis and recurrent upper respiratory tract infection qin and diao zhu and jiang moreover it showed significant efficacy in the treatment of psoriasis vulgaris yan et al keshan disease scientific research cooperation group of herba patriniae in yan'an city for the prevention and control of keshan disease and chronic pelvic inflammation jia in the form of tablets in the chinese pharmacopoeia edition there are chinese herbal medicine prescriptions containing herba patriniae among which kangfu xiaoyan shuan and yifei qinghua gao are used to treat gynecological diseases and respiratory diseases respectively while longqing pian nankang pian niaosaitong pian and qianliexin jiaonang are used to treat genitourinary diseases state pharmacopoeia commission of p r china a summary of the traditional and traditional and clinical preparation of herba patriniae in china is given in table the tender stems and leaves of herba patriniae are rich in nutrients fresh in taste and grow in the mountains without environmental pollution it is a highquality vegetable that urban and rural residents like to eat pv tea is also abundant in hubei province and fujian province jiang 2019a xu et al herba patriniae is not only used in human health but also in agriculture fishery and animal husbandry interplanting herba patriniae in the newly reclaimed tea garden can increase the natural vegetation and reduce soil erosion and surface runoff caused by rainstorm erosion in the rainy season chen the combination of herba patriniae and other medicinal plants can be used to treat poisoned wound of cattle by agkistrodon acutus bitting crawling bee disease liver and skin diseases of turtle and fish and postpartum abdominal pain in cattle chen li shi zhao preproof 0c table the information of herba patriniae in national and local standards in china standards standard of traditional chinese medicine in hunan province standard of traditional chinese medicine in shandong province standard of traditional chinese medicine in heilongjiang province standard of traditional chinese medicine in liaoning province standard of traditional chinese medicine in sichuan province standard of traditional chinese medicine in guizhou province chinese pharmacopoeia edition application acute appendicitis diarrhea enteritis hemorrhagic leucorrhea red eye pterygium postpartum abdominal pain boils and carbuncles appendicitis dysentery enteritis hepatitis conjunctivitis postpartum blood stasis abdominal pain boils and carbuncles acute appendicitis diarrhea hemorrhagic leucorrhea postpartum blood stasis abdominal pain swelling and pain of eye hepatitis boils and carbuncles acute appendicitis diarrhea dysentery postpartum blood stasis abdominal pain conjunctivitis boils and carbuncles acute appendicitis and its abdominal pain postpartum blood stasis abdominal pain boils and carbuncles appendicitis dysentery enteritis hepatitis conjunctivitis postpartum blood stasis abdominal pain boils and carbuncles appendicitis dysentery enteritis hepatitis conjunctivitis postpartum blood stasis abdominal pain boils and carbuncles dosage g standardsetting department hunan food and drug administration g g g g g g shandong medical products administration heilongjiang medical products administration liaoning food and drug administration sichuan food and drug administration guizhou medical products administration pharmacopoeia committee of the ministry of health of p r china preparation name yiyi fuzi baijiang san 薏苡附子败酱散 machixian heji 马齿苋合剂 aiye san 艾叶散 baijiang san 败酱散 baijiang san 败酱散 baijiang tang 败酱汤 baijiang tang 败酱汤 table traditional and clinical preparation of herba patriniae in china formulation main compositions powder coicis semen aconiti lateralis radix praeparata herba patriniae decoction portulacae herba isatidis folium arnebiae radix herba patriniae persicae semen carthami flos paeoniae radix rubar powder powder artemisiae argyi folium angelicae sinensis radix paeoniae radix alba dipsaci radix achyranthis bidentatae radix herba patriniae herba patriniae angelicae sinensis radix chuanxiong rhizoma paeoniae radix alba cinnamomi cortex powder herba patriniae moutan cortex cinnamomi cortex siphonostegiae herba aucklandiae radix decoction herba patriniae notopterygii rhizoma et radix dianthi herba aurantii fructus cinnamomi cortex persicae semen decoction herba patriniae reference jin gui yao lüe ãŠé‡‘匮要略㋠zhang 1997b surgery of chinese medicine ãŠä¸­åŒ»å¤–科学㋠beijing traditional chinese medicine hospital tai ping sheng hui fang ãŠå¤ªå¹³åœ£æƒ æ–¹ã‹ volume wang pu ji fang ãŠæ™®æµŽæ–¹ã‹ volume zhu tai ping sheng hui fang ãŠå¤ªå¹³åœ£æƒ æ–¹ã‹ volume wang sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao qian jin yi fang ãŠåƒé‡‘翼方㋠volume sun preproof 0cpreparation name baijiang tang 败酱汤 baijiang tang 败酱汤 baijiang yin 败酱饮 changyong tang 肠痈汤 decoction chenzhou sheyao pian 郴州蛇药片 chure jili wan 除热蒺藜丸 tablets pills danggui xi tang 当归洗汤 danggui yin 当归饮 ganyan chongji 肝炎冲剂 decoction decoction electuary formulation main compositions decoction herba patriniae rhei radix et rhizoma persicae semen decoction herba patriniae cinnamomi cortex siphonostegiae herba moutan cortex aucklandiae radix decoction herba patriniae angelicae sinensis radix bambusae caulis in taenias rehmanniae radix moutan cortex glycyrrhizae radix et rhizoma herba patriniae zingiberis rhizoma recens poria coicis semen platycodonis radix liriopes radix salviae miltiorrhizae radix et rhizoma paeoniae radix alba rehmanniae radix pv tribuli fructus rhei radix et rhizoma herba patriniae cinnamomi cortex ginseng radix et rhizoma aconiti lateralis radix praeparata coicis semen coptidis rhizoma astragali radix abri herba angelicae sinensis radix aurantii fructus immaturus paeoniae radix alba tetrapanacis medulla angelicae sinensis radix angelicae pubescentis radix angelicae dahuricae radix sanguisorbae radix herba patriniae angelicae sinensis radix herba patriniae dipsaci radix paeoniae radix alba rehmanniae radix bambusae caulis in taenias bupleuri radix angelicae sinensis radix paeoniae radix alba paeoniae radix rubra citri reticulatae pericarpium aurantii fructus curcumae radix cyperi rhizoma salviae miltiorrhizae radix et rhizoma scrophulariae radix artemisiae scopariae herba isatidis radix herba patriniae huangdan tang 黄疸汤 decoction jiedu dihuang wan 解毒地黄丸 pills gualou san powder artemisiae scopariae herba gardeniae fructus lonicerae japonicae flos forsythiae fructus herba patriniae isatidis radix paeoniae radix rubra paeoniae radix alba bupleuri radix perillae caulis platycodonis radix sojae semen germinatum rehmanniae radix astragali radix trichosanthis radix scutellariae radix liriopes radix mantidis ootheca rhei radix et rhizoma ginseng radix et rhizoma gardeniae fructus cistanches herba peucedani radix cimicifugae rhizoma paeoniae radix alba anemarrhenae rhizoma vaccariae semen polygalae radix herba patriniae jujubae fructus trichosanthis semen herba patriniae asari radix et rhizoma zingiberis rhizoma magnoliae officinalis cortex platycodonis radix ginseng radix et rhizoma saposhnikoviae radix reference sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao qian jin fang ãŠåƒé‡‘方㋠volume （sun ） gu jin ming fang ãŠå¤ä»Šåæ–¹ã‹ yan and liu qian jin fang ãŠåƒé‡‘方㋠volume （sun ） qian jin fang ãŠåƒé‡‘方㋠volume （sun ） sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao study on the treatment of common diseases with traditional chinese medicine ãŠå¸¸è§ç—…的中 医 æ²» 疗 研 究 ㋠teaching and research group of traditional chinese medicine the first affiliated hospital of xi'an medical college lin zheng yi an yi fang ãŠä¸´è¯åŒ»æ¡ˆåŒ»æ–¹ã‹sun sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume zhao sheng ji zong lu ãŠåœ£æµŽæ»å½•ã‹ volume preproof 0c preparation name 栝蒌散 lanwei xiaoyan wan 阑尾消炎丸 lanweiyan heji 阑尾炎合剂 formulation main compositions pills lonicerae japonicae flos isatidis folium herba patriniae taraxaci herba spatholobi caulis toosendan fructus rhei radix et rhizoma aucklandiae radix persicae semen paeoniae radix rubra scutellariae radix decoction lonicerae japonicae flos taraxaci herba herba patriniae forsythiae fructus rhei radix et rhizoma paeoniae radix rubra toosendan fructus aucklandiae radix persicae semen lanweiyan tang 阑尾炎汤 lanwei yihao xiaoyan wan 阑尾ä¸å·æ¶ˆç‚Žç‰‡ lenge xiaoji tang 棱莪消积汤 lishi zhiyang pu yao 理湿止痒扑药 lidan tuihuang tang 利胆é黄汤 neibu wuxiang wan 内补五香丸 qianliexian tang 前列腺汤 qumai wan 瞿麦丸 decoction pills decoction powder decoction pills decoction pills yinqiao hongjiang jiedu tang decoction rhei radix et rhizoma moutan cortex persicae semen paeoniae radix alba salviae miltiorrhizae radix et rhizoma bupleuri radix lonicerae japonicae flos forsythiae fructus herba patriniae coicis semen lonicerae japonicae flos isatidis folium herba patriniae taraxaci herba toosendan fructus rhei radix et rhizoma aucklandiae radix persicae semen paeoniae radix rubra scutellariae radix talci pulvis sargentodoxae caulis sparganii rhizoma curcumae rhizoma salviae miltiorrhizae radix et rhizoma paeoniae radix rubra corydalis rhizoma moutan cortex persicae semen coicis semen sargentodoxae caulis herba patriniae kochiae fructus bombyx batryticatus dictamni cortex angelicae dahuricae radix schizonepetae spica artemisiae scopariae herba herba patriniae alumen glycyrrhizae radix et rhizoma talcum cinnabaris artemisiae scopariae herba herba patriniae isatidis radix curcumae radix gardeniae fructus aquilariae lignum resinatum olibanum aucklandiae radix caryophylli flos dipsaci radix rehmanniae radix praeparata paeoniae radix alba magnoliae officinalis cortex herba patriniae ginseng radix et rhizoma poria cervi cornu salviae miltiorrhizae radix et rhizoma lycopi herba paeoniae radix rubra persicae semen carthami flos olibanum myrrha vaccariae semen citri reticulatae pericarpium toosendan fructus foeniculi fructus angelicae dahuricae radix herba patriniae taraxaci herba dianthi herba realgar vaccariae semen rehmanniae radix ephedrae herba imperatae rhizoma herba patriniae saposhnikoviae radix achyranthis bidentatae radix rhei radix et rhizoma lonicerae japonicae flos forsythiae fructus sargentodoxae caulis herba patriniae moutan cortex gardeniae fructus paeoniae radix rubra persicae semen coicis semen corydalis rhizoma toosendan fructus olibanum myrrha reference zhao beijing chinese traditional patent medicine specification ãŠåŒ—京市中成药规范㋠volume beijing municipal bureau of health selected data of acute abdomen treated by integrated traditional chinese and western medicine ãŠä¸­è¥¿åŒ»ç»“合治疗æ¥è…¹ç—‡èµ„æ–™é‰ç¼–ã‹ affiliated hospital of guangzhou college of traditional chinese medicine lin zheng yi an yi fang ãŠä¸´è¯åŒ»æ¡ˆåŒ»æ–¹ã‹ sun compilation of traditional chinese medicine preparations ãŠä¸­è¯åˆ¶å‰‚汇编㋠cao obstetrics and gynecology ㊠妇 产 科 å­¦ ã‹shanghai college of traditional chinese medicine selection of medical prescriptions of cixi and guangxu ãŠæ…ˆç¦§å…‰ç»ªåŒ»æ–¹é‰è®®ã‹ chen gu jin ming fang ãŠå¤ä»Šåæ–¹ã‹ yan and liu tai ping sheng hui fang ãŠå¤ªå¹³åœ£æƒ æ–¹ã‹ volume wang surgery of chinese medicine ãŠä¸­åŒ»å¤–科学㋠beijing traditional chinese medicine hospital qian jin yi fang ãŠåƒé‡‘翼方㋠volume sun obstetrics and gynecology ㊠妇 产 科 å­¦ ã‹shanghai college of traditional chinese preproof 0c formulation main compositions preparation name 银翘红酱解毒汤 danhuang quyu jiaonang 丹黄祛ç˜èƒ¶å›Š qianlieping jiaonang 前列平胶囊 fuping jiaonang 妇平胶囊 fuyan kangfu jujue pian 妇炎康复å’嚼片 fuyan kangfu pian 妇炎康复片 fuyan kangfu jiaonang 妇炎康复胶囊 fuyan kangfu keli 妇炎康复颗粒 fuyanxiao jiaonang 妇炎消胶囊 fuyanqing xiji 妇炎清洗剂 capsules capsules capsules tablets tablets capsules granules capsules lotion xiaoer reke koufuye 小儿热咳口服液 oral liquid kangfu xiaoyan shuan 康复消炎栓 kangfu xiaoyan jiaonang suppository capsules astragali radix salviae miltiorrhizae radix et rhizoma dioscoreae rhizoma smilacis glabrae rhizoma angelicae sinensis radix spatholobi caulis euryales semen houttuyniae herba sparganii rhizoma curcumae rhizoma scorpio herba patriniae cinnamomi cortex atractylodis macrocephalae rhizoma zingiberis rhizoma praepatum eupolyphaga steleophaga corydalis rhizoma toosendan fructus sophorae flavescentis radix herba patriniae salviae miltiorrhizae radix et rhizoma paeoniae radix rubra persicae semen carthami flos lycopi herba pyrrosiae folium olibanum myrrha reference medicine httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa fagopyri dibotryis rhizoma violae herba curcumae rhizoma herba patriniae polygoni perfoliati herba solidaginis herba httpswwwyaozhcom nmpa herba patriniae coicis semen toosendan fructus bupleuri radix scutellariae radix paeoniae radix rubra citri reticulatae pericarpium httpswwwyaozhcom nmpa herba patriniae coicis semen toosendan fructus bupleuri radix scutellariae radix citri reticulatae pericarpium httpswwwyaozhcom nmpa herba patriniae coicis semen toosendan fructus bupleuri radix scutellariae radix paeoniae radix rubra citri reticulatae pericarpium httpswwwyaozhcom nmpa herba patriniae coicis semen toosendan fructus bupleuri radix scutellariae radix paeoniae radix rubra citri reticulatae pericarpium httpswwwyaozhcom nmpa herba patriniae trichosanthis radix rhei radix et rhizoma moutan cortex atractylodis rhizoma linderae radix httpswwwyaozhcom nmpa taraxaci herba herba patriniae coicis semen paeoniae radix rubra atractylodis rhizoma angelicae sinensis radix chuanxiong rhizoma cyperi rhizoma corydalis rhizoma alismatis rhizoma ephedrae herba armeniacae semen amarum forsythiae fructus rhei radix et rhizoma trichosanthis fructus mori cortex herba patriniae carthami flos glycyrrhizae radix et rhizoma sophorae flavescentis radix violae herba herba patriniae andrographis herba suis fellis pulvis taraxaci herba arnebiae radix aloe httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa state pharmacopoeia commission of p r china httpswwwyaozhcom taraxaci herba herba patriniae paeoniae radix rubra coicis semen angelicae sinensis radix atractylodis rhizoma chuanxiong rhizoma cyperi rhizoma alismatis rhizoma corydalis rhizoma httpswwwyaozhcom nmpa preproof 0c formulation main compositions reference preparation name 康妇炎胶囊 manshenning heji 慢肾宁合剂 nankang pian 男康片 decoction tablets astragali radix cinnamomi ramulus epimedii folium rehmanniae radix asini corii colla poria alismatis rhizoma scutellariae radix herba patriniae moutan cortex leonuri herba httpswwwyaozhcom nmpa paeoniae radix rubra rehmanniae radix praeparata cistanches herba glycyrrhizae radix et rhizoma taraxaci herba pyrolae herba phellodendri chinensis cortex carthami flos houttuyniae herba epimedii folium fubi fructus atractylodis macrocephalae rhizoma astragali radix cuscutae semen violae herba herba patriniae chrysanthemi indici flos angelicae sinensis radix sophorae fructus notoginseng radix et rhizoma sophorae flavescentis radix bletillae rhizoma cnidii fructus artemisiae argyi folium herba patriniae lonicerae japonicae flos portulacae herba saposhnikoviae radix alumen borneolum syntheticum glycyrrhizae radix et rhizoma pv sucrose dextrin astragali radix codonopsis radix glehniae radix liriopes radix agrimoniae herba bistortae rhizoma fritillariae cirrhosae bulbus asteris radix et rhizoma platycodonis radix armeniacae semen amarum herba patriniae glycyrrhizae radix et rhizoma codonopsis radix trionycis carapax paridis rhizoma atractylodis macrocephalae rhizoma astragali radix citri reticulatae pericarpium eupolyphaga steleophaga rhei radix et rhizoma persicae semen scutellariae barbatae herba herba patriniae poria coicis semen curcumae radix sappan lignum ostreae concha artemisiae scopariae herba cyperi rhizoma polygoni cuspidati rhizoma et radix ardisiae japonicae herba sedi herba scutellariae barbatae herba magnoliae officinalis cortex herba patriniae arnebiae radix wenyujin rhizoma concisum lonicerae japonicae flos isatidis folium herba patriniae taraxaci herba spatholobi caulis toosendan fructus rhei radix et rhizoma aucklandiae radix persicae semen paeoniae radix rubra scutellariae radix lonicerae japonicae flos isatidis folium herba patriniae taraxaci herba sargentodoxae caulis toosendan fructus rhei radix et rhizoma aucklandiae radix persicae semen paeoniae radix rubra scutellariae radix lonicerae japonicae flos herba patriniae taraxaci herba moutan cortex toosendan fructus paeoniae radix rubra rhei radix et rhizoma persicae semen aucklandiae radix state pharmacopoeia commission of p r china httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa state pharmacopoeia commission of p r china httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa httpswwwyaozhcom nmpa atractylodis rhizoma poria acori tatarinowii rhizoma plantaginis semen indigo naturalis phellodendri amurensis cortex talcum herba patriniae salviae miltiorrhizae radix et rhizoma ht
Colon_Cancer
" the diagnostic gold standard of hirschsprung™s disease hd is based on the histopathological assessmentof colorectal biopsies although data on cholinergic innervation and ganglion cell gc distribution exist only few studieshave examined these two key features together we assessed the pattern of cholinergic innervation and the amount ofgcs in colorectal specimens of hd patientsmethods we established a semiquantitative score for cholinergic innervation using acetylcholinesterase ache enzymehistochemistry and quantitatively analyzed the number of gcs via nadh tetrazolium reductase nadh enzymehistochemistry we examined both the entire length of the resected specimens as well as defined areas of the transitionzone of both pathological and healthy appearing segmentresults high ache score values were associated with absence of gcs and ache scores were inversely correlated withthe number of gcs nevertheless we observed several cases in which one of the two features revealed a normaldistribution pattern whereas the other still displayed pathological featuress our data support the need for transmural colon biopsies to enable the best evaluation of both cholinergicinnervation and gcs for a reliable assessment of hdkeywords hirschsprung™s disease colon rectosigmoid ache aganglionosis hirschsprung™s disease hd is a rare cause of constipation in infants that is microscopically characterized bycongenital absence of gcs in a variable extension mainlyaffecting the rectum and sigmoid colon hd has a prevalence of approximately in newborns male babiesare more frequently affected with a ratio of malefemale a registerbased study has described a slightincrease in incidence over the recent years the clinicalsymptoms of hd comprise severe chronic constipation correspondence michelmittelbronnlnsetatlu anne k braczynski and stefan gfroerer contributed equally to this work4institute of neurology edinger institute goethe university frankfurtgermany13department of oncology donc luxembourg institute of health lihstrassen luxembourgfull list of author information is available at the end of the ongoing defecation problems and occasionally bowel obstruction most newborns with hd present with a delayedmeconium passage in infancy a postnatal passagetime of longer than “ h combined with defecationproblems is clinically suggestive for hd the treatmentof choice for hd is the surgical resection of the affectedaganglionic segment the transition zone which is onlypartially populated by gcs should also be resected theoperation technique should preserve the anorectal function which is usually achieved by connecting the distalfunctional bowel with the sphincter muscle at the area ofthe dentate line despite proper surgical treatmentsthe quality of life may remain negatively affected duringchildhood most cases of hd occur sporadicallynevertheless genetic causes of this disorder have beenidentified [ ] the most frequent mutations affect the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cbraczynski bmc pediatrics page of ret gene that codes for a receptor tyrosine kinase approximately different ret mutations have been reported in hd patients accounting for of familialcases of hd and to of sporadic cases of hd the diagnosis of hd is histopathologically confirmed bythe absence of gcs in the enteric plexus of the distal rectum usually several biopsies ranging between and cmabove the dentate line [“] via either superficial suction or a fullthickness transmural specimen intraoperative diagnostics to detect gcs is most frequentlybased on the hematoxylin and eosin he staining ofcryosections this method can be combined with enzymehistochemistry for i the assessment of cholinergic nerve fibers using acetylcholinesterase ache and ii the detection of ganglion cells via lactate dehydrogenase ldhsuccinate dehydrogenase sdh and nadh tetrazoliumreductase reactions nadh [ ] although averagevalues exist concerning the number and distribution of ganglion cells in normal intestines and intestines affected byhd these values are highly dependent on the stainingmethod histopathologically the diagnosis consists ofan absence of gcs and the presence of hypertrophicachepositive nerve fibers however little is known aboutthe correlation of the gc distribution pattern and the cholinergic innervation hd cases therefore in the currentstudy we aimed at analyzing both the extent and distribution pattern of cholinergic innervation in the mucosal layerin relation to the density of gcs in the myenteric plexus ina cohort of hd patientsmethodspatient datahd patients who were included in this retrospectivemonocentric study underwent transanal pull throughtapt surgeries at the department of pediatric surgery university hospital frankfurtmain germany inall cases the diagnosis of hd was confirmed by transanal rectal biopsies prior to tapt surgeries the specimens for detailed information please see table wereindependently reviewed by at least two experienced neuropathologists akb pnh mm from the institute ofneurology according to the diagnostic criteria that wereproposed by knowles patients with longsegmentdiseases were excluded from this study the ages at thetime of the surgeries ranged from days to monthsmean age months the study cohort consisted of male m and female f patientsethical statementthe parents of the patients gave written informed consent for the surgery the use of patient material wasapproved by the ethical committee ofthe goetheuniversity frankfurt germany gn samplingpartial colectomy specimens were examined and measured n additionally each resected bowel segment was macroscopically analyzed with regard to thechanges in diameter in the zones of bowel obstructionthe length between the proximal resection margin andthe beginning of the dilated colon was documentedfig 1a swiss roll specimens were prepared accordingto the following guidelines first from each of the entirerectosigmoidal specimens a longitudinal “ cm widestrip was excised in paramedian orientation to the antimesocolic tenia afterwards the strip of the intestinalwall was coiled in caudocranial orientation in cryogel tissue tek oct sakura alphen aan den rijntable sample dataidlength of aganglionic segment total length cmswiss rollxtz cmxtz cmxtz cmxtz cmna125abbreviations x specimen available na not availablexxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx 0cbraczynski bmc pediatrics page of fig sampling and study design a intraoperative aspect of rectosigmoid colon during tapt procedure there was a notable change ofdiameter at the beginning of tz tz cm white lines mark the biopsy sites at and cm proximal to the beginning of the transition zonetz cm asterisk indicating aboraldistal end of colon arrowhead the oralproximal resection region b swiss roll specimens stained for heache nadh and ldh asterisk and arrowhead as described in a c first we analyzed the swiss rolls of the fulllength specimens and locatedareas with each of the four grades of the achescore in the same region we counted gcs d second we analyzed the specimen tz and cm for their respective achescore ache and counted the gcmm in the corresponding area nadhnetherlands attached to a cork plate and snap frozensee below swiss rolls were then used to analyze theoverall innervation pattern the œserial biopsies referring to the specimens from the transition zone tz tz cm cm cm and cm were prepared as followsthe beginning of the change in the diameter was definedas the beginning of the transition zone and referred toas tz cm a horizontal incisional antimesocolic biopsyof approximately cm × cm was excised at cm further rostral biopsies were performed at cm cm and cm tz cm tz cm tz cm fig 1a the biopsies were fixed in cryogel on the cryostat specimenholder all specimens were frozen at ˆ’ °c in isopentane which was precooled in liquid nitrogen then μmthick sections were cut with a manual cryostat system leica cm wetzlar germany 0cbraczynski bmc pediatrics page of staining and enzyme histochemistrycryosections were stained with he nadh reactionldh reaction and ache reaction according to standardprotocols fig 1b the incubations were performed at °c for min nadh min ldh and minache stained sections were mounted in entellanmerck millipore darmstadt germanyscoringbefore performing semiquantitative and quantitativeanalyses a joint training was done to reduce interobserver variability akb pnh mm in case of doubtduring quantification a joint analysis was performeduntil agreement was reached a semiquantitative scoring system was applied to evaluate the pathological cholinergic innervation in the mucosal layer fig 1c d thefour grades of the ache score were defined as followsscore no achepositive nerve fibers in the mucosallayer fig 2a e score mild positivity fiber percrypt within the lamina propria mucosae lp fig 2bf score moderate positivity up to fibers per cryptfig 2c g and score strong positivity with or morefibers per crypt fig 2d h in the lp gcs were countedwithin the myenteric plexus using nadh enzyme histochemistry fig based on the ache scoring two different analyseswere performed first we determined the number ofgcsmm in the nadh stainings in areas correspondingto each distinct ache score in the swiss roll fig 1cfor raw values see supplementary table in mostspecimens areas meeting each grade of the ache scorewere present for the second analysis transition zonesamples were analyzed for ache scores as well as forthe number of gcsmm in the corresponding area fig1d for raw values see supplementary table thelength of the myenteric plexus segment was determinedby an open polygon line using image analysis softwareanalysis docu olympus hamburg germany depending on the quality of the specimens the amount gcs ina segment of “ mm were counted absolute gccount was then normalized to the length of the respective myenteric plexus segment to compare gc density ingcmm the imaging and evaluation were performedusing a light microscope olympus bx41 at a magnification of 40x length or 400x cell countstatistical analysisfor statistical analyses we used single case overlay diagrams fig 4b significance levels were calculated byusing an anova and tukey™s test fig 4a c d a significance level of α was determined for all testsstatistical analyses were performed by using the jmp software sas cary nc usaresultsache score negatively correlated with ganglion cell countin areas with no achepositive nerve fibers in the mucosal layer score fig 5ac a median of gcsmm min max was observed in areas withmild ache positivity score a median of gcsmmwas counted min max in areas with moderate ache positivity score a median of gcsmmmin max was counted in areas with severeache positivity score fig 5df a median of gcsfig ache scores of cholinergic innervation in the lamina propria mucosae representative areas of the mucosal layer with cholinergicinnervation score absence of achepositive fibers a e score mild cholinergic innervation b f score moderate cholinergic innervationc g and score severe cholinergic innervation d h scale bar ad μm eh μm 0cbraczynski bmc pediatrics page of fig ganglion cell count in the myenteric plexus a and b healthy bowel areas with normal gc density dashed lines indicating gcs c and dclassic hd pathology with complete absence of gc nadh scale bar a c μm b d μmmm min max was observed there were significant differences in the gc counts when comparingscores vs and vs each p as well asscores vs and vs each p fig 4alow ache score values were associated with a highnumber of ganglion cells but the absence of pathologicalcholinergic innervation was not a reliable indicator for aregular ganglion cell countmoderate or severe pathological cholinergic innervationin the lp was associated with complete absence of ganglion cells in the myenteric plexus fig 4b and fig 5gihowever the absence of or presence of only very fewache positive fibers in the mucosa did not assure aregular gc distribution in all cases in out of a total of resection specimens there were no areas detectedthat could be assigned to an ache score œ furthermore there were two cases with clear aganglionosis atthe aboral resection margin however showing no sign ofpathological cholinergic innervation ache score inthe respective segment exemplarily case id fig 5githe colonic areas with mild cholinergic innervation corresponding to an ache score of œ still exhibited a considerable variation in the gc counts with variationsbetween gcsmm case id and gcsmmcase id fig ache scores did not continuously decrease in thetransition zoneserial biopsies from the defined areas within the transition zone were collected fig 1a and d depending onthe available material and the lengths of the resectedspecimens the sample counts varied tz cm n tz cm n tz cm n and tz cm n the evaluation of the pathological cholinergic innervation in the transition zone specimens revealed a medianache score of at tz cm min max a medianscore of at tz cm min max a median scoreof at tz cm min max and a median scoreof at tz cm min max the decrease between tz cm and tz cm and the increase betweentz cm and tz cm were both significant p fig 4cganglion cell counts increased in the proximal region ofthe transition zone and remained stable rostrallythe gcsmm were counted in the transition zone biopsies at the most distal point of the transition zone tz cm a median of gcsmm min max wasobserved at tz cm a median of gcsmm min max was counted at tz cm a median of gcsmm min max was observed finally at tz cm a median of gcsmm min 0cbraczynski bmc pediatrics page of fig see legend on next page 0cbraczynski bmc pediatrics page of see figure on previous pagefig analysis of the swiss role and ache score and gc distribution in hd transition zone a gcsmm in areas assigned to ache score to inthe full length swiss roll preparations b single case overlay diagram gcsmm per ache score are grouped according to achescore to in ain cases no areas with score were detectable c cholinergic innervation as measured by the ache score and d gcsmm in the tz samples tz cm and cm p p p max was counted there was a significant increase in the the number of gcsmm between cm and cm p and between cm and cm p fig 4d regarding the differences between the othersamples tz cm to tz cm and tz cm to tz cm there was a statistically nonsignificant trend of increasing gc counts with the difference being the smallest between tz cm and tz cm however onlyn for tz cmdiscussionin our cohort of hd patients we assessed the extentof cholinergic innervation in the lamina propria mucosaeby ache scoring and the number of gcs in colonic biopsies over a maximal length of cm extending fromthe proximal area to the beginning of the macroscopically defined transition zonethe amount of gcs was in a similar range to thoseamounts reported by meierruge and brunner byusing ldh stainings “ gcsmm in dysˆ’or hypoganglionic colon segments as well as gcsmm inunaffected colon tissues were reported our slightlyhigher cell numbers may be related by the differentstaining protocol nadh instead of ldh dependingon the staining method as well as the interobservervariability gc counts can vary considerably [ ]fig discrepancy of ganglion cell count in areas with normal appearing ache distribution in general an ache score a corresponded to gcpopulation counts as described in b and c while an ache score d corresponded to the absence of gcs e and f however case id showed no cholinergic innervation ache score g associated with absence of gcs h and i scale bar a d g μm b e h μm c fi μm 0cbraczynski bmc pediatrics page of fig variation of ganglion cell distribution in cases with similarly pathological ache reaction three cases with ache score are shown accase id df case id gi case id while the amount of pathologic cholinergic innervation is comparable gc counts per mm varies from14mm hi case id to 472mm bc case id scale bar a d g μm b e h μm c f i μmswaminathan summarizes the variation of cellcounts depending on the staining method ranging from to gcscm even with laborious counting ofthe entire circumference patienttopatient variationremained very high suggesting that either significant variation in myenteric gc density is normal or variation in technical factors eg stretch might be relevant the question whether the dilatation of the transition zone and the oral part of healthy gut leads to neuronal death or whether a reduced cell count is the resultof dilatation and stretching the same amount of cgsdistributed in a wider circumference remains unsolvedduring tapt surgery longitudinal stretching seems tobe more prominentthan intravital circumferentialstretching which is difficult to assess another factor istissue shrinking during the embedding process which iswell known from ffpe tissue this can be neglected incryoconserved material as in our case current literaturereflectson the disrupted craniocaudal migratorythe gcs “ and a locoregional partialbehaviour ofphenotype which leads to the reduced amount of gcs inthe transition zone rather than technical factors additionally the work by white and langer nicely corroborates the findings on circumferential gc count differences swaminathan and kapur examined thisissue in nonhd cadaveric specimens and found that almost the entire circumference of transverse sectionsfrom a single sample had to be counted to get an accurate estimate of gc density nevertheless all published staining and counting techniques were reportedto have high sensitivity and specificity in an experiencedlaboratory with accuracy rates as high as conventional staining techniques do however not discriminate between gcs and glial bystander cells in the gangliawhich may result in further variation that is caused byinadequate gc identificationour surgical specimens were sampled in a longitudinalorientation from aboral to oral and the beginning of 0cbraczynski bmc pediatrics page of the tz was macroscopically defined based on the end ofthe constricted gut two phenomena may affect gccounts a concerning the circumference some authorshave observed a varying uneven distribution of the gcsin the gut wall see discussion above and b the beginning of the tz œ cm tz biopsies œ cm tz in ourstudy was macroscopically defined thereby still mainlyshowing aganglionic bowel segments in contrast definingthe beginning of the transition zone based on microscopiccriteria a œ cm tz biopsy might be randomly located at“ cm from the aganglionic segment boundary to reduce the impact of these uncertainties further researchstudies should consider an evaluation of intervals that areshorter than cm as expected from normal embryonicdevelopment our samples showed a longitudinal increaseof gcs in oral direction we found significant changes inthe transition zone between tz cm and tz cmfollowed by a trend of increasing gcs in more proximalzones kapur analyzed the relationship between the lengthof the partially aganglionic transition zone compared tothe length of the aganglionic segment in patients andstated that excluding the very long aganglionic segmentsthe hypoganglionic transition zone was consistently lessthan cm in length nevertheless there were significant methodological differences in the study design thecriteria used for the diagnosis of myenteric hypoganglionosis on hestained paraffin sections were descriptiveand designed to detected only moderatetosevere hypoganglionosis despite these formal differences andlimitations regarding to low number of specimens in ourstudy our results suggest a relatively longer transitionzone with still slightly increasing gc counts in areas tz and tz in our study high ache score values score wereonly observed in aganglionic bowel absent or low achescore values score were mostly associated with a highnumber of gcs but the absence of cholinergic innervation did not prove a normal innervation mild cholinergicinnervation has been described in rectal biopsies from patients who did not suffer from hd possibly because mildcholinergic innervation is part of the normal variation little is known about mild persistent cholinergic innervationin the lp in hd patients it is possible that rare achepositive neurites also resolve with the maturation of thechild or persist as a normal variant in older children oradults finding rare achepositive neurites protrudinginto the lp is described to be a relatively common findingin suction biopsies from pediatric patients who do not suffer from hirschsprung disease [“] our findings question that a low amount of remaining ache positive fibersache score has to be considered as a reliable pathological feature in hd patients no significant correlationwas observed between ganglion cell density and absentache score versus ache score which extended upto cm proximally to the aganglionic segment in fourcases we did not identify an area with an ache score of in these cases some degree of cholinergic innervationremained despite a resection margin of up to cm proximal from the tz the lack of correlation between ganglion cell density and low ache score may be based onthe variables discussed above variability in ganglion cellcounts different staining protocolsinterobserver variability tissue stretching and others or alternatively because ache score might be part of the normal variationin our study we exclusively evaluated transmural biopsiesfor both initial diagnosis as well as during definite surgerywhile in other centers mucosal or submucosal biopsiesmay be performed thus resulting in the risk of insufficienttissue being collected for definite diagnostics becauseno definite abnormal cholinergic innervation patternache score was found in the hypoganglionic bowelof most hd patients mucosal ache activity alone shouldnot be used to define the proximal transition zonenevertheless the definition of the end of the transitionzone and the beginning of the normal colon by the use ofgc count cutoff values may not lead to improved surgical patient outcomes with respect to the interobservervariability and methodological differences based on ourresults both mucosal and submucosal tissues should beevaluated however the choice of the resection margins requires further interdisciplinary discussionsin our study we examined the relationship between theamount of cholinergic mucosal innervation and the density of myenteric ganglion cells in resected intestinal specimens from hd patients although we identified anassociation between absent gc counts aganglionosis andmoderate or high ache activity no statistically significantcorrelation between ache activity and ganglion cellcounts in proximal ganglionic intestinal segments couldbe established thus the presence of sparse cholinergicnerves in the mucosa is not a reliable marker of myenterichypoganglionosis and transmural biopsies are essential toquantify ganglion cells to exclude nonacherelated features of transition zone our data support the high validityof transmural colon biopsies for hd diagnostics especiallyfor cases lacking one of the classic features and highlightsthe necessity of a close collaboration between pediatricsurgeons and neuropathologistssupplementary informationsupplementary information accompanies this paper at httpsdoi101186s1288702002299zadditional file table s1 gc count and length of corresponding mpsegment according to ache score swiss role specimen table s2 0cbraczynski bmc pediatrics page of ache score gc count and length of the corresponding mp segment inthe transition zonereceived september accepted august abbreviationsache acetylcholinesterase f female gc ganglion cell he hematoxylinand eosin hd hirschsprung™s disease ldh lactate dehydrogenaselp lamina propria mucosae m male mp myenteric plexus nadh nadhtetrazolium reductase reactions tz transition zone tapt transanal pullthroughacknowledgementswe thank maika dunst tatjana starzetz and cornelia penski for theirexcellent technical assistance this manuscript was language edited byamerican journal experts 475ecb814533a9066fd4authors™ contributionsthe paper is the result of the intellectual collaboration of all listed authors inparticular akb sg pnh ur and mm participated in the conception anddesign of the study drafted the manuscript and performed the neuropathologicalanalysis the statistical methods of this study were reviewed by mm pb followedup the patients rb cosupervised the study and gave critical advice concerningthe stainings sg and ur treated the patients performed the surgery supervisedthe study and helped interpreting the findings all authors read and approved thefinal manuscriptfundingakb received funding through the start and the rotation program of themedical faculty of rwth aachen university the start rotation fund is apersonal grant to akb allowing physicians to do research during thespecialization training mm would like to thank the luxembourg nationalresearch fond fnr for the support fnr pearl p16bm11192868 grantthe pearl fund is a personal grant to mm to develop a research program ofexcellence in the field of clinicaltranslational neuropathologyavailability of data and materialsthe dataset supporting the s of this is included within the and its additional filesethics approval and consent to participatethe use of patient material was approved by the ethical committee of thegoethe university frankfurt germany gn the parents of thepatients gave written informed consent for the surgeryconsent for publicationnot applicablecompeting intereststhe authors declare no conflicts of interestauthor details1department of neurology rwth aachen university hospital aachengermany 2department of physical biology heinrichheine universitydüsseldorf germany 3institute of biological information processing ibi7structural biochemistry forschungszentrum jülich jülich germany 4instituteof neurology edinger institute goethe university frankfurt germany5department of pediatric surgery helios hospital berlinbuch berlingermany 6institute of pathology and neuropathology eberhardkarlsuniversity tuebingen germany 7german cancer consortium dktkheidelberg germany 8german cancer research center dkfz heidelberggermany 9frankfurt cancer institute fci frankfurt am main germany10department of neurosurgery goethe university frankfurt germany11department of pediatric surgery university of frankfurt am main frankfurtgermany 12university children™s hospital goethe university frankfurtgermany 13department of oncology donc luxembourg institute ofhealth lih strassen luxembourg 14luxembourg centre for systemsbiomedicine lcsb university of luxembourg luxembourg cityluxembourg 15national center of pathology ncp laboratoire national desanté lns rue louis rech l3555 dudelange luxembourg16luxembourg center of neuropathology lcnp rue louis rech l3555dudelange luxembourgreferencesborrego s ruizferrer m fernandez rm antinolo g hirschsprung's diseaseas a model of complex genetic etiology histol histopathol “best ke addor mc arriola l balku e barisic i bianchi f hirschsprung'sdisease prevalence in europe a register based study birth defects res aclin mol teratol “friedmacher f puri p classification and diagnostic criteria of variants ofhirschsprung's disease pediatr surg int “gfroerer s rolle u pediatric intestinal motility disorders world jgastroenterol “das k mohanty s hirschsprung disease current diagnosis andmanagement indian j pediatr “collins l collis b trajanovska m khanal r hutson jm teague wj quality of life outcomes in children with hirschsprung disease j pediatrsurg “brooks as oostra ba hofstra rm studying the genetics of hirschsprung'sdisease unraveling an oligogenic disorder clin genet “de lorijn f boeckxstaens ge benninga ma symptomatologypathophysiology diagnostic workup and treatment of hirschsprungdisease in infancy and childhood curr gastroenterol rep “edery p lyonnet s mulligan lm pelet a dow e abel l mutations ofthe ret protooncogene in hirschsprung's disease nature “ meierruge wa bronnimann pb gambazzi f schmid pc schmidt cp stossf histopathological criteria for intestinal neuronal dysplasia of thesubmucosal plexus type b virchows arch “ meierruge w ultrashort segment hirschsprung disease an objective pictureof the disease substantiated by biopsy z kinderchir “ holschneider am puri p hirschsprung's disease and allied disorders berlinheidelberg springer friedmacher f puri p rectal suction biopsy for the diagnosis ofhirschsprung's disease a systematic review of diagnostic accuracy andcomplications pediatr surg int “schappi mg staiano a milla pj smith vv dias ja heuschkel r apractical guide for the diagnosis of primary enteric nervous systemdisorders j pediatr gastroenterol nutr “ meierruge w lutterbeck pm herzog b mer r moser r scharli aacetylcholinesterase activity in suction biopsies of the rectum in thediagnosis of hirschsprung's disease j pediatr surg “ meierruge wa bruder e pathology of chronic constipation in pediatricand adult coloproctology pathobiology ““ meierruge wa brunner la morphometric assessment of hirschsprung'sdisease associated hypoganglionosis of the colonic myenteric plexuspediatr dev pathol “knowles ch veress b kapur rp wedel t farrugia g vanderwinden jm quantitation of cellular components of the enteric nervous system in thenormal human gastrointestinal tractreport on behalf of the gastro international working group neurogastroenterol motil “knowles ch de giio r kapur rp bruder e farrugia g geboes k gastrointestinal neuromuscular pathology guidelines for histologicaltechniques and reporting on behalf of the gastro internationalworking group acta neuropathol “ meierruge wa brunner la engert j heminghaus m holschneider amjordan p a correlative morphometric and clinical investigation ofhypoganglionosis of the colon in children eur j pediatr surg “swaminathan m kapur rp counting myenteric ganglion cells in histologicsections an empirical approach hum pathol “ white fv langer jc circumferential distribution of ganglion cells in thetransition zone of children with hirschsprung disease pediatr dev pathol“ mckeown sj stamp l hao mm young hm hirschsprung disease adevelopmental disorder of the enteric nervous system wiley interdiscip revdev biol “kapur rp histology of the transition zone in hirschsprung disease am jsurg pathol “ 0cbraczynski bmc pediatrics page of schofield de devine w yunis ej acetylcholinesterasestained suction rectalbiopsies in the diagnosis of hirschsprung's disease j pediatr gastroenterolnutr “ moore sw johnson g acetylcholinesterase in hirschsprung's diseasepediatr surg int “ pacheco mc bove ke variability of acetylcholinesterase hyperinnervationpatterns in distal rectal suction biopsy specimens in hirschsprung diseasepediatr dev pathol “ chow cw chan wc yue pc histochemical criteria for the diagnosis ofhirschsprung's disease in rectal suction biopsies by acetylcholinesteraseactivity j pediatr surg “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"
Colon_Cancer
in the uk the death toll from severe covid19 is among the highest worldwide1 severe covid19 is characterised by respiratory failure with socalled cytokine storm occurring in some patient subsets2 pathological correlates are required to understand the pathophysiology of covid19 autopsybased histopathological analysis is crucial in this respect in anticipation of the covid19 pandemic our group produced national guidelines for autopsy performance in suspected covid19 cases3covid19 is caused by infection with severe acute respiratory syndrome coronavirus sarscov245 although sarscov2 and its predecessor sarscov causing severe acute respiratory syndrome [sars] are toll similar on a molecular and clinical level covid19 has a lower death rate for covid19 vs for sars and a substantially higher death deaths worldwide from covid19 as of aug vs from sars than sars due to a higher basic reproduction number1 the postmortem findings in patients with sarscov infection included diffuse alveolar damage dad splenic and nodal lymphocyte depletion haemophagocytosis renal acute tubular injury cerebral oedema microthrombosis and adrenalitis with necrosis with intracellular sarscov detected in the lungs kidney brain and haematological ans6 various autopsy series on covid19 have begun to emerge in the literature7“ here we document the major pathological lancet microbe published online august 101016 s2666524720301154department of cellular pathology northwest london pathology b hanley mbbch prof k n naresh md c roufosse phd j weir frcpath prof r goldin md p viola md m osborn frcpath and department of hepatology p manousou phd imperial college london nhs trust london uk centre for haematology b hanley prof k n naresh and centre for inflammatory diseases c roufosse department of immunology and inflammation department of infectious disease prof g s cooke frcp a abdolrasouli phd o c swann mres l baillon bsc r penn msc prof w s barclay phd and department of metabolism prof m thursz md faculty of medicine imperial college london london uk department of histopathology royal brompton and harefield nhs foundation trust and national heart and lung institute imperial college london london uk prof a g nicholson dm renal and transplant centre hammersmith hospital imperial college healthcare nhs london uk r corbett phd department of neuropathology kings college hospital london uk prof s alsarraj frcpath death investigation committee royal college of pathologists london uk m osborn and nightingale nhs hospital london uk m osborn correspondence to dr brian hanley department of cellular pathology northwest london pathology charing cross hospital campus london w6 8na uk bhanleyimperialacukwwwthelancetcommicrobe published online august 101016s2666524720301154 s 0cresearch in contextevidence before this studycovid19 is a new disease and comprehensive descriptions of the histopathological findings at autopsy are scarce we reviewed the literature available on covid19 autopsy findings up to and including may for this we searched pubmed and google scholar databases with no language restrictions using the search terms œcovid19 œsarscov2 œhistology œautopsy and œpostmortemadded value of this studyour series focused on providing a comprehensive description of the histopathological findings in patients with severe fatal covid19 and correlating these findings with data on viral tropism the most prominent findings included diffuse alveolar damage thrombosis haemophagocytosis and immune cell depletion several novel autopsy findings in patients with covid19 were also described including pancreatitis pericarditis adrenal microinfarction secondary disseminated mucormycosis and brain microglial activationimplications of all the available evidenceour study supports the existing clinical and autopsy literature that identified diffuse alveolar damage thrombosis immune cell depletion and macrophage activation as the most prominent pathological features in covid19 other factors including acute kidney injury pancreatitis pericarditis secondary fungal infections and preexisting liver disease require further investigation the presence of ongoing viral replication in late stage covid19 supports the continued use of antiviral therapy even at a point in illness when immunopathology is dominantsee online for appendixfindings of ten postmortem examinations done on patients with clinically confirmed covid19methodspatient selectionfor this study eligible patients were older than years with premortem sarscov2 infection and covid19 listed clinically as the direct cause of death under part on the medical certificate of cause of death [mccd] consent was obtained for all included patients according to the human tissue authority codes of practice by a member of the trust core postmortem consent team consent rate was · ten of patients exclusion criteria included extended postmortem interval before autopsy days and patients with covid19 contributing but not directly leading to death under part of the mccd patients were from imperial college national health service nhs trust nine patients london uk and royal brompton harefield foundation nhs trust one patient london uk premortem sarscov2 infection was identified using the coronavirus typing multiplextandem pcr highplex system aus diagnostics chesham uk ethical approval for this project was provided by the imperial college healthcare tissue bank r20012autopsy proceduresfull autopsies were done on nine patients pm1“ and one patient underwent percutaneous biopsy sampling heart lungs pancreas kidneys and liver using percutaneous biopsy under ultrasound guidance pm10 full postmortem examinations included standard sampling and were done according to royal college of pathologists guidelines3 eight different regions of the brain were sampled for each full neuropathological examination all tissue samples were fixed in formalin for a minimum of h before embedding histochemical stains and immunohistochemistry were applied according to local protocols appendix p ans were reviewed by subspecialist pathologists in lung agn and pv haem ato pathology and immune pathology knn liver rg gastrointestinal mo neuropathology sas and renal pathology cr integrated interpretation was done by a subspecialty autopsy pathologist bh and mo all cases were reviewed independently by at least two pathologistspcr proceduresfresh tissue for quantitative rtpcr qrtpcr analysis was processed within the biosafety level facilities at st mary™s hospital london uk approved by the uk health and safety executive and in accordance with local rules at imperial college london total rna was obtained from fresh tissue samples by use of trizolchloroform extraction followed by precipitation and purification using the rneasy kit qiagen hilden germany qrtpcr against e gene rdrp and subgenomic rna was done as described elsewhere1617 in patient pm5 total fungal genomic dna was extracted from four to five ribbon slices of a formalinfixed paraffinembedded lung tissue block purified dna was amplified with pcr for panfungal and mucoralesspecific targetsstatistical analysisall data was analysed using spss software version and expressed using median iqr and percentagerole of the funding sourcethe funder of the study had no role in study design data collection data analysis data interpretation or writing of the the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publicationresultsbetween march and april ten patients were included in the study the median age at death was years wwwthelancetcommicrobe published online august 101016s2666524720301154s 0ciqr “ seven of ten patients were men three were women and most patients were white or asian nine hypertension four patients and chronic obstructive pulmonary disease three were the most common contributing factors to death according to mccd all ten patients developed fever and had at least two respiratory symptoms or signs cough shortness of breath reduced oxygen saturations or pleuritic chest pain during their early presentation of eight patients assessed for inflammatory markers all had elevated inflammatory markers these features were either apparent upon presentation to hospital eight of ten patients or developed in an inpatient two patients pm8 and pm9 most patients died within weeks of symptom onset seven patients and were not intubated or ventilated six patients four patients were intubated during their presentation pm2 for days pm5 for days pm6 for less than day and pm7 for days the median bodymass index bmi was in the obese range · iqr ·“· and more patients were obese according to bmi at post mortem five of nine than indicated on the mccd one of ten the median interval between death and postmortem examination was days iqr ·“· although the limited post mortem had a shorter interval less than h after death detailed clinical case vignettes are available in the appendix p and clinical data are summarised also in the appendix p all patients had dad six showed purely exudative phase dad and four showed a mixture of exudative and anising dad appendix p figure three of four patients with anisingphase dad had spent a substantial period on a ventilator days days and days florid acute bronchopneumonia and ventilatorassociated pneumonia were not noted in this series although mild interstitial neutrophilic inflammation three of ten patients and patchy acute bronchopneumonia three patients were observed interstitial macrophages were prominent macrophages were accompanied by scattered plasma cells mild or moderate lymphocyte inflammation was present in all ten patients although focal lymphocyte cuffing of small vessels was noted in six patients we noted that lymphocytes in the lung were predominantly cd4positive t cells cd56positive natural killer cells were rarely found occasionally a patient had small aggregates of small b cells chronic bronchiolitis was seen in most patients nine of ten no granulomas or viral inclusion were seen invasive mucormycosis was noted in one patient pm5 figure and confirmed with mucoralesspecific pcr the mucormycosis was vasculocentric and disseminated involving the hilar lymph nodes heart brain and kidney in the same patientmacroscopic two of nine patients and microscopic eight of nine pulmonary thromboemboli were frequent observations appendix p figure both fibrinrich and plateletrich thrombi were identified in smallsized and mediumsized vessels and within the capillaries in alveolar septa figure external examination findings of deep venous thrombosis were not noted very focal lymphocytic vasculitis was identified in one patientthrombotic features were universal in this cohort and all nine patients who underwent a full autopsy had at least one microthrombosis or macrothrombosis in a major an one of nine patients had a macroscopic acute coronary thrombosis in the right coronary artery whereas five patients had thrombi in the microcirculation of the heart on histological analysis coronary artery disease was negligible or mild in most patients seven of nine acute myocardial ischaemic damage h old was noted in the patient with an acute coronary artery thrombus figure 2a pm1 a mottled myocardium and subendocardial contraction band necrosis was noted in a acebdf 03m µm µmfigure pulmonary pathological findings in patients with covid19a macroscopic subpleural petechial haemorrhage in a 24yearold man pm6 b hyaline membranes indicative of exudative phase diffuse alveolar damage in a 79yearold woman pm9 at 20x magnification c cd61 immunohistochemical staining indicating plateletrich microthrombosis in alveolar capillaries pm6 d squamous metaplasia in a 61yearold man pm1 with exudative phase diffuse alveolar damage at × magnification e interstitial multinucleated giant cells in a 79yearold man pm7 with anising phase diffuse alveolar damage at × magnification the top right insert is of multinucleated giant cells showing positive cd68 staining indicative of macrophage lineage the bottom left insert shows absence of staining for cytokeratins f periodic acid schiff staining indicating wide irregular aseptate and ribbonlike hyphae with openangle branching and a vasculocentric pattern indicative of mucormycosis in a 22yearold man pm5 the insert is a grocott silver stain highlighting mucormycosis at 20x magnificationwwwthelancetcommicrobe published online august 101016s2666524720301154 s 0csecond patient pm2 whether the contraction band necrosis was related to ischaemia or inotropic medication received in the intensive care unit is uncertain appendix p pm1 and pm2 were the two patients with the highest active viral load detected in the heart a single patient had a right atrial thrombus pericarditis was acegbdfh µm µm 03m µmfigure thrombotic features identified at autopsy in patients with covid19a macroscopic right coronary artery thrombosis arrow in a 61yearold man pm1 with exudative phase diffuse alveolar damage b macroscopic pulmonary thromboembolism arrow in a 97yearold man pm8 c thrombus in the lung of a 79yearold woman pm9 on haematoxylin and eosin staining at 20x magnification the insert shows cd61 immunohistochemistry indicating moderate staining for platelets d plateletrich thrombus in the mediumsized vessels surrounding the heart in a 61yearold man pm1 the insert shows strong cd61 staining for platelets periodic acid schiff staining showing a glomerular microaneurysm arrow e and microthrombi within glomerular capillary loops arrow f at 40x magnification indicative of thrombotic microangiopathy in a 97yearold man pm8 macroscopic splenic g and hepatic h infarction in a 22year old man pm5identified in two patients one patient showed florid fibrinous pericarditis containing fungal hyphae pm5 while the other showed only microscopic acute pericarditis appendix p figure the median heart weight was high g and four of nine patients had left ventricular hypertrophy nonbacterial thrombotic marantic endocarditis was noted in one patient pm5 with no known history or autopsy findings consistent with malignancy or chronic disorder associated with nonbacterial thrombotic marantic endocarditis appendix p figure pm5 had disseminated mucormycosis and numerous other thrombotic features appendix p cardiac amyloidosis and right atrial thrombosis were identified in one of ten patients pm8 appendix p lymphocyte depletion involving specific compartments and increased phagocytosis were prominent findings appendix p figure increased phagocytosis of other cells was identified in the sinusoidal macrophages of the red pulp of the spleen in four of seven patients sinus histiocytes of hilar lymph nodes in three of six and bone marrow four of eight phagocytosis was identified in at least one of these ans in six of nine patients bone marrow haemophagocytosis was prominent in two patients pm4 and pm8 and focal in two patients pm7 and pm9 depletion of periarteriolar tcell sheaths within the white pulp was observed figure red pulp was generally congested showing reduced numbers of cd8positive t cells plasma cells were variably prominent and sinusoidal histiocytes showed phagocytosis of red blood cells and other cells to varying extents both igmpositive and iggpositive plasma cells were identified and they were polytypic for lightchain expression figure lymph nodes showed preservation of follicles and relative depletion of paracortical areas medullary areas showed prominence of plasma cells and histiocytes were prominent in the sinuses bone marrow samples showed reactive changes with trilineage hyperplasia and prominence of plasma cells and histiocytes were a common finding a necrotising granuloma was noted in a single hilar lymph node in one patient and acidfast bacilli were noted on ziehl neelson staining appendix p all spleen and lymphoid material examined with immuno histochemistry were negative for epsteinbarr virus and cytomegaloviruspancreatitis was noted in two of eight patients pm5 was a 22yearold man with frank necrotichaemorrhagic pancreatitis and secondary mucor mycosis figure no fungal hyphae were noted in the pancreas pm8 was a 97yearold man who showed no substantial macroscopic pancreatitis although micro scopic acute inflammation within the pancreas and periadrenal fat necrosis was noted figure a third of patients three of nine showed patchy areas of infarcttype adrenocortical necrosis with one patient showing anising microthrombi in adrenal vessels figure no wwwthelancetcommicrobe published online august 101016s2666524720301154s 0cadrenalitis was noted two of nine patients showed chronic inflammation in the thyroid with follicular epithelial cell disruption however the significance of this finding is uncertainmedian combined kidney weight was within normal range at g iqr “ salient renal pathology findings were acute tubular injury in all nine patients underlying moderate cortical scarring of uncertain cause in one patient glomerular microaneurysm and thrombi in one patient figure and rare thrombi in interlobular arteries in four patients pm6 24yearold man of arterial intimal thickening than expected for that age appendix p we observed no evidence of focal and segmental glomerulosclerosis diabetic glomerulopathy or glomerulonephritisdegree had a higher large droplet fatty change was seen in most patients seven of eight cirrhosis or bridging hepatic fibrosis were noted in three patients no liver thrombosis was identified histologically but one patient showed macroscopic liver infarction figure the median liver weight was g iqr “ and three of nine patients showed hepatomegaly liver weighing g two patients pm4 and pm7 showed marked autolysis and were not included in analysismoderate to intense microglial activation was the most prominent pathological feature in the cns five of five patients mild tcell infiltration was noted around blood vessels and capillaries in all five patients but b cells were absent we found ischaemic changes of variable extent in the neurons of the cortex and in the white matter detected by bapp β amyloid precursor protein stain however no necrosis of brain tissue or extensive infiltration of inflammatory cells in brain parenchyma or meninges was observed on histological examination although one of nine patients showed macroscopic haemorrhagic transformation in a large recent cerebral infarction in the distribution of the middle cerebral arterytissues from five patients were analysed for presence of viral genomes against e gene and indications of viral replication against subgenomic rna transcripts by qrtpcr viral rna was present in respiratory tract samples including lung of all five cases analysed in addition two of three patients had detectable viral rna in the nasal epithelium and four of five patients in the trachea evidence of viral genomes outside the respiratory tract was found for all five patients but the distribution and viral loads varied case by case figure 5a viral genomes were also detected using a different qrtpcr targeted at rdrp gene and patterns were consistent between the two sets of primers data not shown a third primer set that detected subgenomic rna indicated virus replication in all tissues examined with variation between patients in levels and distribution figure 5bace µmbdf µm µmfigure other notable autopsy findings in patients with covid19a contained aortic dissection green arrow and fibrinous pericarditis red arrow in a 22yearold man pm5 insert is a haematoxylin and eosin stain image of the pericardium showing fibrinous pericarditis 10x magnification b adrenocortical microinfarcts in a 79yearold woman pm9 with reendothelialising thrombus in small adrenal vessels highlighted by cd34 insert bottom left and haematoxylin and eosin insert top right marantic endocarditis c highlight with haematoxylin and eosin staining bottom left at 10x magnification and necrotising haemorrhagic pancreatitis d in a 22yearold man pm5 with covid19 and a secondary fungal lung infection e periodic acid schiff staining showing a granular cast arrow indicative of acute tubular injury in a 24yearold man pm6 20x magnification f microscopic acute pancreatitis on haematoxylin and eosin staining in a 97yearold man pm8 20x magnificationdiscussionin this series we have described the major pathological findings identified at autopsy in ten patients who died of severe covid19 the most consistent findings were dad thrombosis haemophagocytosis and immune cell depletion although unexpected pathologies that are probably related to sarscov2 infection were also identifieddad was the most consistent and prominent feature in our series and others78 the specific phase of dad probably represents the degree and chronicity of the offending insult sarscov2 infection in relation to the time of death this is similar to previous coronavirus epidemics6 the conclusion by copin and colleagues7 that covid19related lung injury œis not diffuse alveolar damage might relate to their sampling strategy and wwwthelancetcommicrobe published online august 101016s2666524720301154 s 0cadbc µm µm µm µm µmef µm µm µmfigure pathological findings in haematological ans in patients with covid19tcell depletion in the spleen of a 79yearold woman pm9 with covid19 haematoxylin and eosin staining of the spleen at 10x magnification a cd20 staining of spleen indicating presence of b cells b 10x magnification with the insert showing the same region at higher power 20x magnification and cd3 staining of spleen indicating depletion of t cells c 10x magnification with the insert showing the same region at higher power 20x magnification bone marrow phagocytosis in a 97yearold man pm8 with covid19 haematoxylin and eosin staining of a well preserved bone marrow with an arrow indicating presence of phagocytosis d 40x magnification and cd68pgm1 staining of bone marrow indicating presence of phagocytosis 20x [e] and 40x [f] magnificationchronicity five patients had spent approximately weeks on a ventilator barton and colleagues8 described prominent acute bronchopneumonia as the major finding in one of two patients although the authors acknowledge that this was probably affected by aspiration in their patient with muscular dystrophy reports of lung histology in early covid19 also suggest a degree of lymphocytic pneumonia although dad is probably superimposed on this over time in the majority of fatal cases7 pulmonary macrophage infiltration and multinucleated giant cell reactions are prominent similar to other series8“ definite evidence of in covid19 will require quantitative analysis comparing tissues from covid19 patients with dad associated with other conditions and unaffected tissues several cases of invasive pulmonary aspergillosis have been reported in patients with severe covid19 pneumonia18 to our knowledge this is the first description of histologically proven mucormycosis in patients with covid19 and suggests that other human fungal pathogens including members of mucoromycotina can complicate covid19associated infectionslymphocyte depletion tissuerelated numerous clinical features including raised serum ddimer concentrations raised procalcitonin concentrations and imaging findings suggest thrombosis is prominent in patients with covid192 thrombotic features were universal among patients who underwent full autopsies all nine patients had thrombi in at least one major an and have been noted to be prominent in other covid19 autopsy series15 in a retrospective study of autopsies in patients with acute respiratory distress syndrome and dad of various causes only showed thrombi within the small vessels of the lung despite sampling of every lobe of the lung19 another study used postmortem angiography and identified thrombi in nearly all cases of acute respiratory distress syndrome from various causes20 whether thrombosis in covid19 is more common than in other causes of dad remains uncertain however our data support thrombosis as being a striking feature in these patients a study suggested endotheliitis as a prominent feature in patients with severe covid1910 but this was not a prominent feature in our patients importantly limited post mortem or postmortem crosssectional imaging are likely to underrepresent the true extent of thrombosis particularly microthrombosis and its impact on patient death the extent of cardiomegaly fibrointimal thickening of renal blood vessels and obesity in our series supports a contribution of hypertension beyond that noted clinically only four patients had hypertension documented on the mccda raised cytokine profile has been documented in a subset of patients with severe covid192 consistent with this haematological ans in our series showed prominent phagocytosis in several patients which has not been documented in previous series21 of the four patients with bone marrow haemophagocytosis one patient pm7 showed mild transaminitis hyperbilirubinaemia elevated serum ferritin concentrations and fever of ·°c however most clinical data were insufficient to assess the presence of haemophagocytic lympho histiocytosis wwwthelancetcommicrobe published online august 101016s2666524720301154s 0ca substantial feature in covid19 is lymphocyte depletion and this is supported in our series by the spleen and lymph node findings when compared with those with mild disease patients with severe covid19 tend to have a higher neutrophil to lymphocyte ratio and higher cd4positive to cd8positive tcell ratio22 additionally a negative correlation exists between peripheral blood lymphocyte count and viral copy number22 we have corroborated this evidence by documenting a low number of t cells especially cd8positive t cells and foxp3positive regulatory t cells in the spleen and lymph nodes in severe fatal covid19 notably normal plasma cell both igm and igg positive response was present in haematological ans in most patientsthe extent to which anspecific pathologies relate to direct viral replication or consequent immunological and cardiovascular complications is of clinical relevance we report here evidence of viral genomic rna outside the respiratory tract this finding is in agreement with several previous studies that have identified viral genomes by qrtpcr in postmortem tissues including the colon14 spleen14 liver1423 skin24 heart23 and brain25 we also report detection of subgenomic rna a product that is only produced in actively infected cells a report identified low viral load in the brain of three of patients with covid19 but could not detect the virus in subsequent immunohistochemistry and concluded that the viral genomes might have been present in the blood25 although we cannot exclude that the rnas detected in our series were similarly carried to the site of sampling in blood the distribution of rna in different tissues varied widely between postmortem casespm3 and pm4 appear to have died earlier in the disease course days after symptom onset and had higher viral loads in the respiratory tract than other patients whereas pm3 and pm4 died after long stays in intensive care units and had either lower overall viral rna pm5 or higher viral rna outside the respiratory tract pm2 pm1 and pm2 were the only patients in whom we detected viral rna and subgenomic rna in the heart and are the only two patients with evidence of acute myocardial injury moreover unlike the previously mentioned study where virus detected in the brain was times less than that detected in the lung the number of viral genomes detected in external tissues in our series was frequently of similar or even higher levels than that found in the respiratory tree one study has detected sarscov2 infection of the endothelium in the vasculature of the skin and lung by immunofluorescent staining for viral antigens24 it is not obvious what determines spread and tropism of sarscov2 outside the respiratory tract several studies have reported ace2 expression levels that were higher in some ans than in others26 the sites of highest expression did not correlate with areas of most severe pathological involvement in our series we should note that cellular receptor status is one of many things that determines the degree of anr latot g 03 rep seipocenege larivpm1pm2pm3pm4pm5abtcbone marrowbrainheartileumkidneyliverspleentonguetrachealungnasal epitheliumfigure tissue tropism of sarscov2 in postmortem samplesfresh tissues were collected from a subset of postmortem examinations and viral load quantified by use of qrtpcr targeting the viral e gene a detection of viral rna was verified by use of qrtpcr against the viral polymerase gene data not shown tissues were additionally tested for subgenomic viral rna transcripts b dotted lines indicate the limit of detection as ascertained by negative control data are included for a 61yearold man pm1 a 64yearold man pm2 a 69yearold woman pm3 a 78yearold man pm4 and a 22yearold man pm5 qrtpcrquantitative rtpcr sarscov2severe acute respiratory syndrome coronavirus pathological involvement other aspects are likely to play a role such as route of transmission acid lability in the stomach coreceptors expression of proteases required to activate entry of virus into the cell eg tmprss2 possibly other yet unidentified host factors and invivo routes of dissemination the evidence of ongoing replication late in disease supports the use of antiviral therapy even at a point in illness when immunopathology is dominantpancreatic pathology was a major unexpected pathology in this series which had not been previously reported in covid19 autopsies or large clinical covid19 series2 a 22yearold man had haemorrhagic necrotic pancreatitis pm5 although this patient also had disseminated mucormycosis he had evidence of viral persistence in the lung and no fungal hyphae were identified in the pancreas on histological sections the other patient with wwwthelancetcommicrobe published online august 101016s2666524720301154 s 0cpancreatic pathology had microscopic evidence of pancreatitis that could be missed at autopsy without adequate sampling the pancreas is a classic site of unexpected pathology identified at autopsy27 serum amylase is not part of the routine care bundle for covid19 in our trust whether the pancreatitis was related to sarscov2 iatrogenic comorbidities or secondary infection is not clear in our study most cases had evidence of hepatic steatosis which is consistent with clinical findings that obesity is a risk factor for poor outcome in covid19 and liver cirrhosis or bridging fibrosis was prominent in this cohort28 from these data nothing suggests direct viral inflammation of the liverinfection or other causes the interval between time of death and autopsy impaired histological interpretation in many ans and affected antigen preservation postmortem endothelial stripping did affect the endothelial interpretation in our study however a subs
Colon_Cancer
" tumor mutational burden tmb has both prognostic value in resected nonsmall cell lung cancernsclc patients and predictive value for immunotherapy response however tmb evaluation by wholeexomesequencing wes is expensive and timeconsuming hampering its application in clinical practice in our study weaimed to construct a mutational burden estimation model with a small set of genes that could precisely estimatewestmb and at the same time has prognostic and predictive value for nsclc patientsmethods tmb estimation model was trained based on genomic data from nsclc samples from the cancergenome atlas tcga validation was performed using three independent cohorts including rizvi cohort and ourown asian cohorts including earlystage and n latestage asian nsclc patients respectively tcga data wereobtained on september the two asian cohort studies were performed from september to march pearson™s correlation coefficient was used to assess the performance of estimated tmb with westmb thekaplanmeier survival analysis was applied to evaluate the association of estimated tmb with diseasefree survivaldfs overall survival os and response to antiprogrammed death1 pd1 and antiprogrammed deathligand pdl1 therapyresults the estimation model consisted of only genes correlated well with westmb both in the training setof tcga cohort and validation set of rizvi cohort and our own asian cohort estimated tmb by the 23gene panelwas significantly associated with dfs and os in patients with earlystage nsclc and could serve as a predictivebiomarker for antipd1 and antipdl1 treatment responsecontinued on next page correspondence zhangli6mailsysueducn jie_969163comzlhuxi163com yanhua tian jiachen xu and qian chu contributed equally to this work5state key laboratory of oncology in south china collaborative innovationcenter for cancer medicine sun yatsen university cancer center eastdong feng road guangzhou guangdong china1state key laboratory of molecular oncology department of medicaloncology national cancer centernational clinical research center forcancercancer hospital chinese academy of medical sciences and pekingunion medical college panjiayuan south lane chaoyang districtbeijing chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ctian bmc medicine page of continued from previous pages the 23gene panel instead of wes or the currently used panelbased methods could be used toassess the westmb with a high relevance this customized targeted sequencing panel could be easily applied intoclinical practice to predict the immunotherapy response and prognosis of nsclckeywords tmb estimation 23gene panel prognostic and predictive value nonsmall cell lung cancertoimmuneinhibitorscheckpoint tumor mutational burden tmb commonly defined asthe number of nonsynonymous mutations has been proposed as a promising predictive biomarker for the responseicisimportantly this metric tightly correlates with overallsurvival os in resected nonsmall celllung cancernsclc patients in rizvi demonstratedthat an increased number of nonsynonymous mutationswere associated with improved objective response durable clinical benefit dcb and progressionfree survivalpfs in nsclc patients who received antiprogrameddeath pd1 therapy clinical studies have also revealed a significant correlation between tmb and objective response rate orr to icis in multiple tumortypes [“] in addition devarakonda recently reported that high tmb was associated with a better survival prognosis in patients with resected nsclc andthe benefit of adjuvant chemotherapy was more pronounced in patients with low tmb the gold standards for tmb calculation are throughwholegenome sequencing wgs or wholeexome sequencing wes however several obstacles such as thehigh demand for quality and quantity of tissue samplesthe cost and time consumption and the unavailabilityfor translation to tmb evaluation by circulating tumordna ctdna in blood btmb hinder the clinicalapplication of these techniques as a result targetednext generation sequencing ngs of cancerrelatedgene panels cgp has been developed serving as surrogates for wes for tmb estimation to date the foodand drug administration fda has approved severalngs panels for tmb estimation eg foundationonecdx f1cdx and memorial sloan kettering cancercenter™s integrated mutation profiling of actionablecancer targets mskimpact which include about“ genes and cover over one megabase of codingdna [ ] recently many new ngs panels consistingof different numbers of genes have been developed andvalidated most of which were designed initially for guiding the use of target therapies these panels mainly include cancerrelated oncogenes and tumor suppressenes many of which do not contribute to or even negatively correlate with tmb thus are not accurate fortmb evaluation besides inclusion of these genes in anngs panel enlarges the panel size used for tmbis importantestimation and can lead to an inferior costeffective consequence itto note that cancer typespecific mutation load estimation models have proven tobe necessary because of the different mutation landscapes among varying tumor types although dnadamage repair ddr genes negatively predictive genesstk11 and keap1 and tmbassociated genes such asmuc16 pole pold1 and ttn have been included inthe ngs panels for tmb evaluation [“] with theburgeoning developments in immunotherapy there is aneed for more specific panels that focus on tmb estimation for nsclcherein by using the cancer genome atlas tcgadatabase as a training set and multiple realworld cohorts as a validation set we constructed an optimizedtmb estimation model with the smallest number ofcarefully selected tmbassociated genes that could beused as both predictive markers for immunotherapy andprognosis biomarkers for resected nsclc patientsmethodspatient cohortsgenomic and clinical data for nsclc samples including lung adenocarcinoma luad and lungsquamous cell carcinoma lusc samples were downloaded from tcga database for the model constructionfor the validation of the model three independent cohorts were used including a previously published studythe rizvi cohort a surgery cohort composing of earlystage nsclc patients who underwent surgicaltreatment and a zs immunotherapy cohort composingof advanced nsclc patients who received ici treatment all the patients in the zs immunotherapy coreceived either antipd1 nivolumab n hortpembrolizumab n shr1210 n or antipdl1 atezolizumab n monotherapy agents there are patients who received durable clinical benefit dcbantipd1 n antipdl1 n and patientswith no durable benefit ndb antipd1 n antipdl1 n all three validation cohorts were used toevaluate the performance of the tmb estimation modeladditionally the surgery cohort was also used for survival validation in resected nsclc patients both therizvi and immunotherapy cohorts were also used forvalidation of ici outcome predictability in advancednsclc patients the clinical details for all enrolled 0ctian bmc medicine page of patients were collected the treatment efficacy for thosetreated with immunotherapy was assessed using response evaluation criteria in solid tumors recistversion with durable clinical benefit dcb definedas partial or stable disease lasting over months allprocedures were approved by the ethics committees ofthe national cancer center all patients provided written informed consentwholeexome sequencing and data processingwe performed wholeexome sequencing of samplesfrom two cohorts in the validation setincluding earlystage nsclc patients who underwent surgicaltreatment and advanced nsclc patients who received ici treatment for those earlystage nsclcpatients both tumor and matched normal samples wereobtained and subjected to wes briefly dna librarieswere prepared using the mgieasy exome capture v4probe set capture kit cat no with a capture region size of mb bgiseq instruments wereused for pairend sequencing × bp the data wereprocessed according to the manufacturer™s protocol the mean coverage was × and × in tumor andnormal samples respectivelyfor those advanced nsclc patients biopsy specimens were available for wes the genomic dna wasextracted using the qiaamp dna ffpe tissue kit andquantified using the dsdna hs assay kit thermofisher scientific usa libraries were constructed withthe kapa hyper prep kit kapa biosystems usa anillumina hiseq4000 platform was used for sequencingwith pe150 sequencing chemistry illumina usa the average coverage depth was ×candidate gene selectiongenomic data for nsclc samples from tcgawere used for candidate gene selection which were usedto construct the mutation load estimation model thecandidate genes were selected based on two criteria mutation frequency higher than or equal to and significant association with mutation load the mutationfrequency of a gene was calculated as the percentage ofpatients with mutation in the gene mutation loadassociated genes were defined as where the westmbwas significantly different between the patients with themutated gene and those with wildtype counterpartsadditional file table s1mutation estimation model constructionthe mutation estimation model construction was basedon tcga data in the training set in detail the first stepwas to build a mutation estimation model using thefewest genes which tightly associated with westmbin our study we constructed the estimation model bysimply randomly selecting a specified number of genesfrom allthe genes or tmbassociated genes andsummed the mutational number as the estimated tmbunder every given number of genes the procedure wasrepeated times resulting in random modelswe then calculated the pearson correlation coefficientr between the estimated and actual mutation load ofwestmb the results allowed us to select the modelwith highest r under the specified number of genes thenext step was to identify which of those best modelsunder the specified number of genes correlated with theclinical outcomes of overall survival os and diseasefree survival dfs the final step was to select a modelusing the fewest genes that tightly associate with thewestmb and have both prognostic value for thoseearlystage nsclc patients and predictive value forthose latestage nsclc patients who received icitreatmentrna expression difference between tmb high and lowgroupsto compare gene expression patterns we downloadedan mrna data set of nsclc patients from tcgadatabase mrna expression was analyzed using gene setenrichment analysis gsea httpsoftwarebroadinstitutegseaindexjsp we divided these patientsinto estimated high ‰¥ mutational counts and lowtmb groups mutational counts and identifiedwhether immunerelated gene signatures associated withtumor mutation status the genes found to be on theleading edge of the enrichment profile were subjected topathway analysis genes with expression over in morethan of the samples were included in the gseathe normalized enrichment score nes is generally theprimary statistic for examining gene set enrichmentresultsstatistical analysisthe mannwhitney u test was used to assess thedifferences in the mutation load between the twogroups the genes with kruskalwalliscorrected pvalues lower than were identified as the mutationloadassociated genes and selected as potential candidate genes survival analysis was performed using thekaplanmeier curves with a p value determined by alogrank test and the statistical tests were twosidedand considered statistically significant at p unless otherwise stated the analyses were performedusing graphpad prism version graphpad prismcorrelations between estimated mutation burden andwholeexome sequencingcalculated tmb were determinedcorrelation coefficient theanalyses were performed using r353by pearson™s 0ctian bmc medicine page of resultscandidate gene selection for model constructionthe flowchart of the construction of estimation model isshown in fig s1 in additional file the somatic mutation data of cases of nsclc were downloaded fromtcga database as the training set tcga cohort including adenocarcinoma and squamous cell carcinoma subtypes of nsclc additional file table s2subsequently a mutation matrix including screened nonsynonymous mutations in genes was generatedfurthermore we identified genetic alterations in genes with mutation frequency ‰¥ in general nsclcpatients and significantly correlating with westmb pvalue range 695eˆ’ to 452eˆ’ these genes werethen used as candidate genes for the construction of thetmb estimation model additional file table s3construction of the tmb estimation modelgenes used for the tmb estimation model were randomly selected from the candidate genes and theserialrandom models theestimated tmb was defined as the sum of all nonsynonymous mutation counts of the selected genes undereach specified number of abstracted genes the procedure was repeated times thus resulting in separatecorrelations ofestimated tmb by these random models and westmbwere evaluated using the pearson correlation coefficientr as expected the correlations between the estimationmodels and westmb increased with the number ofgenes fig 1a b additional file fig s2a b compared with unselected genes in the range of genomicgenes the estimated tmb based on selected geneswas significantly more closely associated with westmb in terms of either the mean or the maximum rfig 1c d additional file fig s2c d the maximumr increased from with one gene included to greaterthan with genes included and then reached aplateau when the included gene number exceeded the r values were comparable though increased slowlyas the number increased fig 1b we asserted that rfig the correlation of westmb and tmb as estimated by different gene panels a b correlation is represented by the pearson correlationcoefficient r genes used for the mutation model construction were either from unselected genes a or from selected genes b that correlatewith westmb c d comparisons of mean c and maximum d r of estimated tmb and westmb using unselected genes or selected genes 0ctian bmc medicine page of greater than in the estimation models was acceptable as such we considered a model with this effectbut including the least number of genes an ideal modelfor clinical applicationin reference to previous reports that tmb is associated with prognosis in patients with resected nsclcsthe optimal tmb estimation model was further evaluated based on the correlation of estimated tmb with osand dfs in models with r over ultimately we constructed an estimated tmb model with only genesand r of p fig 2a additional file which was significantly associated with both os anddfs fig 2b c the cutoff value of the estimated tmbby the 23gene panel was defined as mutational countsthe median value of estimated tmb based on tcgadatabase additional file fig s3a b that were equalor over mutational counts as tmbhigh cases and lessthan mutational counts as tmblow ones these genesincluded unc13c hmcn1 znf536 kmt2d ush2axirp2 pcdh15 ahnak2 adgrl3 reln nf1 ttnadgrg4 cubn cacna1e mrc1 col11a1 nav3csmd1 apob csmd3 col22a1and epha5additional file table s4 the model yielded goodperformances in both subtypes of nsclc with correlations of for luad additional file fig s4aand for lusc additional file fig s4b theaverage cds length of these genes was 12k nucleotides 3k“80k additional file table s4 and the totallength was 028m nucleotides which was considered tobe a great reduction of sequencing cost for mutationload estimation we concluded that the 23gene panel isthe ideal model based on tcga training setanalytic validation of the 23gene panel in asian resectednsclc patientsto validate the performance of the estimation model weconducted wes on chinese stage ia“iiia nsclcpatients after radical pneumonectomy surgery cohortadditional file table s1 the correlation of 23genetmb with wes was r p fig 3a asshown in fig 3b tmbhigh ‰¥ mutational counts according to the 23gene panel associated with a betterdfs compared with those with tmblow logrank p besides a tendency towards improved os wasobserved in the patients with higher estimated tmbthough a statistical difference was not reached due tothe fact that most patients were still alive fig 3cperformance verification by comparing the 23gene panelwith other commercial panelsnext we compared the 23gene panel with two commercial panels based the earlystage nsclc data including f1cdx genes and mskimpact genes there are two overlap genes between the gene panel with f1cdx and mskimpact namelynf1 and epha5 the 23gene tmb has a tight correlation with the tmb estimated by f1cdx f1cdxtmbor mskimpact msktmb r and respectively both p fig 4a b in additionwhen the genes were added to the two commercialpanels the correlation of the incorporated panels withwestmb significantly increased from ci“ to ci “ p for f1cdx fig 4c d and from ci“ to ci “ p for mskimpact fig 4e f to further verify thespecificity of these 23gene panels we compared themwith other randomly selected gene panels from the genes the procedure was repeated timesresulting in the random pearson correlation coefficientsfrom to of f1cdx plus random genesand from to of msk plus random genes the performance of our 23gene model was better than of random models which indicated the irreplaceability of these genesfig tmb estimation model construction based on tcga data in the training set a the correlation of 23gene tmb with westmb is with an empirical p value of r of p b the overall survival is significantly higher in the tmbhigh group ‰¥ mutational counts n than in the tmblow group mutational counts n with logrank test p c the diseasefree survival is significantly higher in thetmbhigh group than in the tmblow group with logrank test p 0ctian bmc medicine page of fig validation of the tmb estimation model based on the earlystage nsclc patients in the validation set a the pearson correlationcoefficient of estimated tmb by the 23gene panel and westmb is with an empirical p value of r of p b the diseasefree survivalis higher in the estimated tmbhigh group ‰¥ mutational counts n than in the tmblow group mutational counts n with logrank test p c the overall survival is comparable in the two groups with logrank test p fig performance evaluation of the 23gene panel against commercially used gene panels a b the pearson correlation coefficient of 23genetmb with f1cdxtmb a and msktmb b c d the pearson correlation coefficient of westmb with f1cdxtmb c and incorporated panel of cancerassociated genes in f1cdx with 23gene panel f1cdx genetmb d e f the pearson correlation coefficient of westmb withmsktmb e and incorporated panel of cancerassociated genes in mskimpact with genepanel msk genetmb f 0ctian bmc medicine page of f1cdxbased on the survival data from our earlystagensclc patients significant correlations were observedbetween survival outcomes dfs and the tmb levelstratified withor mskimpact paneladditional file fig s5a c interestingly the genescould improve the association of these two commercialpanels with dfs additional file fig s5b d if the incorporated panels were used for analysis tmbhigh estimated by both of the two new panels f1cdx 23genepanel or mskimpact 23gene panel demonstratedimproved dfs compared with those of estimated tmblow under the cutoff values indicated in fig s3c and s5of additional file immuneregulatory gene expression signatures stratifiedby tmb level based on the 23gene panelto investigate the difference in immune status betweentmbhigh and tmblow estimated by the 23genepanel we analyzed immuneregulatory gene expressionsignatures based on the rnaseq data of nsclccases from tcga the gsea revealed a prominent enrichment of mrna signatures involved in the inflammainterferonα γ ifnα γtoryresponse tnfαresponse il6jakstat3 signaling and allograft rejection fig immunotherapy response prediction by the established23gene panelfinally we analyzed the performance of tmb estimatedby the 23gene panel in the prediction of response toicis using two independent nsclc cohorts in therizvi cohort the correlation between the tmb estimatedby the 23gene panel and wes was empirical pvalue of r fig 6a the estimated tmb was significantly different between the patients with durableclinical benefit dcb a partial or stable response lastingover months and no durable benefit ndb mannwhitney p fig 6b survival analysis was thenapplied for the comparison of the pfs between the patients n with tmbhigh ‰¥ counts and tmblow counts by the 23gene panel patients withtmbhigh demonstrated significantly improved pfscompared with those with tmblow vs months logrank p fig 6cto further validate the performance of the estimationmodel for response to icis we performed wes of fig gene expression differences between the estimated high tmb and low tmb groups a“f tmbassociated pathways such as inflammatoryresponse tnfα signaling via nfκb interferon α response il6jakstat3 signaling interferon γ response and allograft rejection nes normalizedenrichment score fdr false discovery rate 0ctian bmc medicine page of fig immunotherapy response estimation by the 23gene panel a the correlation of the estimated tmb with westmb using the rizvi datan b estimated tmb in tumors from patients with dcb n or with ndb n mannwhitney p c pfs in tumors withestimated tmbhigh n compared to tumors with tmblow n in patients in the rizvi cohort hr ci to logrank p d the correlation of estimated tmb with westmb using the latestage nsclc patient cohort n e estimated tmb in tumors frompatients with dcb n or with ndb n mannwhitney p f pfs in tumors with estimated tmbhigh n compared totumors with tmblow n in patients in the latestage nsclc patient cohort hr ci to logrank p advanced stage iiib“iv nsclcs in another asian cohort zs immunotherapy cohort all of these patients received with antipd1 or antipdl1 treatmentthe r between the estimated and actual mutation burden was calculated to be empirical p value of r fig 6d the estimated tmb was significantlydifferent between the patients with dcb and ndbmannwhitney p fig 6e the pfs was associated with estimated tmb logrank p fig 6fdemonstrating that the estimated mutation burden derived from caucasian nsclcs from tcga could predict the immunotherapy treatment response quite wellin asian patients we further calculated the hr at different cutoff values in the zs immunotherapy cohort andfound the mutational counts in this cohort resultedthe best hr value additional file fig s6 as a resultwhen applied in clinical practice the cutoff value stillneeds to be further evaluated accordinglycomparison of the 23gene panel with previouslyreported tmbrelated genesmutations in ttn muc16 pole and pold1 havebeen previously reported to correlate with elevated tmblevels [“] the frequencies ofthese genes innsclc based on cases from tcga were and respectively westmb was significantly different between the patients with these mutatedandthose with wildtypegenescounterpartsadditional file fig s7 however only muc16 mutations exhibit significant correlation with os and dfs intcga cohort additional file fig s7ac while theyfailed to confirm the results in our surgery cohortadditional file fig s8 notably none of these genemutations could predict the response or pfs in eitherthe rizvicohortadditional file fig s9cohort or ourimmunotherapydiscussionin the present study we developed a novel and optimaltmb estimation model composed of only geneswhich allowed precise estimation ofthe wesbasedtmb both in earlystage and latestage nsclc patientsimportantly our established 23gene panel can successfully predict the survival outcomes in both resectednsclcs and patients receiving icis in multiple validation cohorts to the best of our knowledge our tmbestimation model is both the first and the smallest paneldescribed to date which can be used as a biomarker tostratify patients not only after radical pneumonectomybut also with advanced nsclc receiving icisthe total cds length of the 23gene panel was 028mnucleotides with an average of 12k 3k“80k the ttnis also included in our panel although it has the longestcds length of 81k the total length was acceptable when 0ctian bmc medicine page of ttn is included besides in a recent study ttn mutation was reported to be associated with tmb in solid tumors including nsclc and correlated with response toicis as a result the 23gene panel was consideredto be a great reduction of sequencing cost for mutationload estimationseveral cancerrelated genes have been previously reported to be associated with westmb in some cancertypes for example melanoma patients with lrp1b mutations exhibited a higher mutationalload than thosewith the wildtype gene li reported that mutations in muc16 are associated with tmb and survivaloutcomes in patients with gastric cancer twoddrrelated genes pole and pold1 were also shownto correlate well with westmb in pancancer types undoubtedly it would be ideal to utilize a singlegene to estimate tmb and effectively predict responseto immunotherapy however we found that singly allthese genes failed to correlate well with the efficacy oficis or survival outcomes after resection the correlationof any individual gene with westmb was moderatemean r “ these results indicate thatusing a single gene to estimate tmb is insufficienttheoretically the larger a ngs gene panel the closerthe estimated tmb is to the actual amount howeverthe costeffective balance for clinical usage must be considered in particular when tmb is detected using peripheral blood super sequencing depth eg “× due to the low abundance of circulating tumordna will significantly drive up the cost to datetwo commercial gene panels f1cdx and mskimpact have been widely used for tmb estimation thesetwo panels demonstrate good performance in correlationwith westmb our established gene panel whichincludes a very limited number of genes demonstratedcomparable correlation coefficients with these two largepanelsindicating the promising reliability of a smallpanel as a surrogate for westmb notably the majority of genes used in our model were not included in thecurrently used commercial gene panels if the genes inour panel were incorporated into the big commercialgene panels the correlation coefficients with westmbincreased these results demonstrate that the geneswe have selected here may be used independently or ascomplement to the currently used gene panels specificfor nsclc inclusion of the genes should be considered in future ngs gene panelsrecently lyu developed a small gene panel with genes to estimate actual tmb derived from luads in tcga database the construction and validation cohorts used for lyu ™s 24gene panel weremainly from caucasian patients however our 23genepanel though also derived from tcga database wassuccessfully validated in multiple asian patient cohortsthese results suggest that our 23gene panel may bemore suitable to nsclc and applicably potent regardless of race and subtypes additional file fig s10similar with the findings of devarakonda weobserved that high tmb associated with improved os inresected nsclc patients in colon cancer patients withresected stage ii mismatch repair deficiency high tmbhas been utilized as a good prognostic biomarker indeed these results possess internal rationality both highneoepitope burden and intense til infiltration have been associated with favorable survival outcomes inearlystage lung cancer high tmb may reflect the immunogenicity in some degree which could mediate theshaping of tumorhost immune interactions taken together these and our findings suggest that quantifyinggenomic instability through tmb estimation can be usedto stratify patients so as to guide adjuvant treatmentowing to the lack of information on hlai it is difficultto judge whether the predictive value of our gene panel isdue to neoantigen generation derived from the includedgene mutations or if the estimated tmb based on the gene panel is simply a representative reflection of genomicinstability as an œaccompanying passenger the otherlimitation of our study is the small number of patientswho received the immunotherapy treatment thus a larger number of cases from a multicenter study are requiredfor the validation of the performance of the treatment response prediction in addition our validation cohorts wereretrospective a prospective study is necessary to translateour estimation model into clinical practice in addition totmb other features such as pdl1 expression microsatellite instability and neoantigen burden have emerged aspotential predictive biomarkers for icis [“] howeverchallenges in defining cutoff valuesintertumoral andintratumoral heterogeneity and test platform uniformitieshave limited their clinical applications therefore future strategies that combine different predictive featuresmay be more effective biomarkers for the accurate prediction of cancer immunotherapy response but need tobe carefully integratedsin summary we have successfully constructed a noveltmb estimation model using only genes that can beused to estimate the westmb and stratify survivalprognosis after radical surgery and clinical outcomes ofici therapy in nsclc patients thus a customized panelfor the targeted sequencing of these selected genes instead of wholeexome sequencing can be designed or utilized as complementary genes included in the currentngs panels consequently by using our model the costeffectiveness may be considerably improved makingrealization of cancer immunotherapy response more accessible in standard clinical settings 0ctian bmc medicine page of supplementary informationsupplementary information accompanies this paper at httpsdoi101186s12916020016948competing interestsno potential conflicts of interest were disclosed by the authorsadditional file table s1 data sets used to calculate westmb forthe study cohorts table s2 characteristics of the patients included inthis study table s3 candidate genes and related information tables4 genes and the corresponding cds length fig s1 flowchart ofthe construction of estimation model fig s2 the correlation of westmb and tmb as estimated by different gene panels fig s3 forestplots of hrs for os and dfs in the tcga and earlystage nsclcpatients study cohort fig s4 the performance of 23gene based tmbestimation model for the luad and lusc subtypes of nsclc tcgadata fig s5 forest plots of hrs for dfs in the earlystage nsclcpatients study cohort fig s6 forest plots of hrs for pfs of the nsclc patients in zs immunotherapy cohort fig s7 westmb is shownbased on muc16 a ttn b and pole c mutation status fig s8 thecorrelation of muc16 mutation status with overall survival a anddiseasefree survival b based on the earlystage nsclc patients figs9 the correlation of muc16 ttn and pold1 mutation status withprogressionfree survival pfs based on the rizvi cohort and ourimmunotherapy cohort fig s10 comparison of predictive performanceof response to icis by our 23gene panel with lyu™s 24gene paneladditional file the correlation of estimation models with genenumber to with os and dfs in the training setacknowledgementswe thank all patients that were involved in this study we also thankguoqiang wang jing zhao and shangli cai the medical department 3dmedicines inc shanghai people™s republic of china for their contribution tothe st
Colon_Cancer
in china esophageal cancer ec is the fourth most frequently diagnosed form of malignant tumor inmales and the fifth most commonly diagnosed form in females approximately and casesoccurred in respectively the incidence of ec in eastern asia is in the top five worldwide including china esophageal squamous cell carcinoma escc is a major histological subtype accountingfor of all ec cases the complex interaction of economical and environmental conditions with individual™s hereditary factors may lead to ec development the etiology and development of ec is notfully understood despite many investigations have payed close attention to the importance of immunity recently it was hypothesized that some important variants in immunerelated genes may influencethe susceptibility of esccreceived november revised july accepted july accepted manuscript online august version of record published august the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20193895101042bsr20193895interleukin4 il4 coded by the il4 gene is an important regulator of the inflammation pathways il4 apleiotropic cytokine may be correlated with survival and growth of lymphocytes il4 is produced by mast cellprecursors and by the tcell thymocyte populations it is important for bcell activation proliferation and differentiation it is reported that il4 is necessary for producing immunoglobulin e and implicated in immune diseasesin the process of innate immune responses il4 may activate m2 macrophage and then play a specific role it hasantiinflammatory effect which is relevant to the development of escc recently a number of studies have focusedon the relationship of il4 with cancer development il4 single nucleotide polymorphisms snps have alsobeen explored for an association with susceptibility to cancer [“] the rs2070874 tc located in the 5cid3utrregion of the il4 gene is an important snp in cancer development some metaanalyses have indicated that il4rs2070874 may be associated with cancer development in asian populations [“] kim reported that il4rs2070874 might affect the role of aspirin in regulating il4 expression rs2243263 gc polymorphism is anintron snp of il4 gene this intron snp might play a role in splicing although the exact role of this intron snpis unknown the associations of il4 rs2243263 gc snp with the human disease have been explored a previousstudy suggested that il4 rs2243263 was associated with the reverse seroconversion of hepatitis b virus hbv this snp was also studied for the relationship of the susceptibility to cancer a previous report investigated the correlation of the il4 rs2243263 locus with colorectal cancer although in this study a null association was identifiedhowever lan in a large simple size study found that the il4 rs2243263 gc snp might increase the susceptibility to nonhodgkin lymphoma currently the associations of il4 the rs2070874 tc and rs2243263 gcpolymorphisms with escc development are unknownthe il10 gene is located in chromosome 1q322 il10 another immune regulator serves as an inhibitor of dendritic cells and macrophages and inhibits the production of many inflammatory cytokines eg tumor necrosis factorα il1 il6 il12 and others il10 is a vital antiinflammatory regulator after il10 combineswith its receptor il10r signal transducer and activator of transcription is triggered which plays a vital role inantiapoptosis and proliferation an investigation found that the upregulated mrna expression of the il10gene and higher serum levels of il10 were found among subjects who carried the rs1800896 gallele thers1800872 snp a promotor variant could influence the level of il10 protein some investigations have suggested that the il10 rs1800896 ag ˆ’ and rs1800872 ac ˆ’ variants may influence thesusceptibility to escc of late a metaanalysis indicated that these il10 snps increased the risk of ec however in this earlier metaanalysis the sample size was very limited ec patients and controls includedthe association of the il10 rs1800896 ag and rs1800872 ac polymorphisms with ec development should befurther studiedherpesvirus entry mediator hvem also known as tnfrsf14 plays a major role in the immune response[“] hvem has been found to be expressed in lymphoid cells as well as in other cells a previous study suggestedthat the hvemb and tlymphocyte attenuatorlymphotoxincd160 network in immune reaction to infection andinflammation could play a bidirectional regulatory role several investigations have focused on the role of hvemin cancer survival [“] zhu reported that higher expression of hvem may promote apoptosis and herald agood prognosis for bladder cancer patients additionally a previous study has indicated that hvem is implicatedin the development of breast cancer bc a snp in the hvem gene the g to a of rs2234167 in the exon regionwas found to influence the development of bc however the association of hvem rs2234167 ga snp withthe expression of hvem is unknown recently migita found that hvem is critical for both tumor survival andthe escape of the host immune system in escc cases thus it could be a useful target for escc therapy to dateinvestigation has not been performed to identify a relationship of the hvem rs2234167 ga polymorphism withescc susceptibilitytherefore in this investigation the hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 andrs1800872 polymorphisms were selected and investigated for their effect on escc development in a chinese hanpopulationmaterials and methodssubjectsour caseˆ’control study was performed in fujian union hospital fuzhou china and the no1 people™s hospital of zhenjiang city zhenjiang china this investigation was approved by jiangsu university registration idk20160036y and fujian medical university registration id 2016zqn25 participants were recruited betweenfebruary and april our study included escc cases and controls these escc patients werehistopathologically confirmed and were from to years old controls were cancerfree individuals from to the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20193895101042bsr20193895 years old the controls were not related to any escc case using a prestructured questionnaire we collectedepidemiological data from participants the escc patients and normal controls signed consent formsdna extraction and genotyping of hvem rs2234167 il4 rs2070874 andrs2243263 and il10 rs1800896 and rs1800872 lociwe collected a blood sample ml from each participant dna was extracted carefully as described in a previousstudy using an snpscan„¢ assay genesky biotechologies inc shanghai china we determined the genotypesof hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 polymorphisms to confirm the accuracy of genotyping samples were selected and retested the genotypes of hvem rs2234167 il4rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 loci were reanalyzed by another technician thegenotypes of hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 snps wereunchangedstatistical analysisthe difference in alcohol consumption body mass index bmi gender cigarette use and age were tested by using χ2 test mean age was calculated by using a student™s t test we used a chisquare test χ2 or fisher™s exact testto determine whether the frequencies of hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896and rs1800872 variants in escc cases and controls were different a multivariate logistic regression analysis methodwas used to calculate the crude and adjusted odds ratios ors and confidence intervals cis sas softwarepackage sas institute inc cary nc usa the relationship of hvem rs2234167 il4 rs2070874 and rs2243263and il10 rs1800896 and rs1800872 polymorphisms with escc development was determined by ors and cisthe statistical significance of all analyses was p005 twosided an internetbased hardy“weinberg equilibriumhwe test httpihggsfdecgibinhwhwa1pl was also harnessed to assess whether the distribution of hvemrs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 genotypes could represent the included populationresultsbaseline characteristicsin total escc cases and controls were recruited table of these escc cases were females and were males average age was ˆ’ years in the control group there were females and maleswith an average age of ˆ’ years there was no difference in terms of mean age p0413 the categoricalvariables age and gender were wellmatched p005 however the distribution of other categorical variables egtobacco use bmi and drinking status were significantly different all p0001 among escc cases there were with lymphatic metastasis the ajcc version criteria was used to determine the escc stage and escc cases were stage iii and were stage iiiiv after genotyping the participants the association ofhvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 genotypes with escc riskwas assessedthe minor allele frequencies mafs of hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896and rs1800872 loci are shown in table they are similar to the data of chinese population as presented in table the hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 genotypes in controlsaccorded with hwerelationship of hvem rs2234167 il4 rs2070874 and rs2243263 andil10 rs1800896 and rs1800872 loci with escctable shows the hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 genotypesthe frequencies of il4 rs2070874 tt tc and cc genotypes were and inescc cases and and in controls when the reference was il4 rs2070874 ttgenotype we found the il4 rs2070874 cc genotype significantly decreased the risk of escc p0023 when thereference was il4 rs2070874 tttc genotype the il4 rs2070874 cc genotype also significantly decreased the riskof escc p0028 adjustment for bmi smoking drinking age and gender the decreased susceptibility was alsoidentified cc vs tt p0008 cc vs tttc p0010hvem rs2234167 il4 rs2243263 and il10 rs1800896 and rs1800872 genotypes are shown in table both crudeand adjusted comparisons indicated that hvem rs2234167 il4 rs2243263 and il10 rs1800896 and rs1800872 lociwere not associated with the risk of escc table the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0ctable distribution of selected demographic variables and risk factors in escc cases and controlsbioscience reports bsr20193895101042bsr20193895p1variableage yearsage yearssex‰¥malefemaletobacco usenevereveralcohol usenevereverbmi kgm2‰¥lymph node statuspositivenegativetmn stageiiiiiiivgradeg1g2g3cases n721n ˆ’ controls n1208n ˆ’ bold values are statistically significant p005 abbreviation tmn tumorlymph nodemetastasis1twosided χ2 test and student™s t testtable primary information for the included snpsgenotyped polymorphismschromosomeposition regionmaf1 in database 1000g chinese hanpopulatonsmaf in our controls n1208pvalue for hwe2 test in our controls genotyping value1maf2hwehvemrs2234167 gail4 rs2070874tcil4 rs2243263gcil10 rs1800872tgil10 rs1800896tc3cid3utr5cid3utrintron variant5cid3flanking5cid3flankingadditionally a subgroup analysis was conducted by escc stage we identified an association between il4rs2070874 tc snp and the decreased susceptibility of escc in stage iii subgroup cc vs tt p0022 cc vstttc p0025 table however this association could not been identified for other snps the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20193895101042bsr20193895table the frequencies of hvem rs2234167 il4 rs2070874 rs2243263 and il10 rs1800896 and rs1800872polymorphisms in different escc subgroupsstage iii patientsstage iiiiv patientsn328n393controls n1208ngenotypehvem rs2234167 gagggaaaa alleleil4 rs2070874 tctttcccc alleleil4 rs2243263 gcgggcccc alleleil10 rs1800872 tgtttgggg alleleil10 rs1800896 tctttcccc alleleoverall casesn721nnnrelationship of hvem rs2234167 il4 rs2070874 and rs2243263 andil10 rs1800896 and rs1800872 loci with escc in stratified analysesin a stratified analysis the il4 rs2070874 genotypes are listed in table after an adjustment we suggested that il4rs2070874 c allele was a protective factor for escc in five subgroups male subgroup cc vs tt p0028 cc vstttc p0031 ‰¥ years old subgroup cc vs tt p0026 cc vs tttc p0029 never smoking subgroupcc vs tt p0041 cctc vs tt p0013 and tc vs tt p0042 drinking subgroup cc vs tt p0025cc vs tttc p0024 and bmi kgm2 subgroup cc vs tt p0010 cc vs tttc p0012 in othersubgroups no association of l4 rs2070874 with escc risk was found table the il4 rs2243263 gc genotypes in the stratified analysis are listed in table after adjustment we identifiedthat il4 rs2243263 gc polymorphism was a risk factor for escc development in the bmi ‰¥ kgm2 subgroupgc vs gg p0030 and gccc vs gg p0018 table in other stratified analyses adjustment comparisons suggested that hvem rs2234167 and il10 rs1800872 andrs1800896 loci did not confer a risk of escc data not shownassociation of hvem rs2234167 il4 rs2070874 and rs2243263 andil10 rs1800896 and rs1800872 loci with lymphatic metastasis in escccasesamong the escc patients patients had lymphatic metastasis as presented in table we found a null association of hvem rs2234167 il4 rs2070874 rs2243263 and il10 rs1800896 and rs1800872 snps with differentlymph node status the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cilcenseccbytheauthorsthisisanopenaccessarticelpublishedbyportlandpressilmitedonbehalfofithebochemcailsocetyianddistributedunderthecreativecommonsattributiontable logistic regression analyses of association of hvem rs2234167 il4 rs2070874 rs2243263 and il10 rs1800896 and rs1800872polymorphisms with risk of esccgenotypehvem rs2234167 gaga vs ggaa vs gggaaa vs ggaa vs gggail4 rs2070874 tctc vs ttcc vs tttccc vs ttcc vs tttcil4 rs2243263 gcgc vs cccc vs gggccc vs ggcc vs gggcil10 rs1800872 tgtg vs ttgg vs ttggtg vs ttgg vs tttgil10 rs1800896 tctc vs ttcc vs tttccc vs ttcc vs tttcpatientsn721vscontrolsoveralln1208crude or cipadjustedor1 ci pstage iii patients n328 vs controlsn1208crude or ciadjustedor1 ci ppstage iiiiv patients n393 vs controlsn1208crude or ciadjustedor1 ci pp “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ 1adjusted for age sex smoking status alcohol use and bmi status bold values are statistically significant p005iiboscencereportsbsrbsr 0cbioscience reports bsr20193895101042bsr20193895table stratified analyses between il4 rs2070874 tc polymorphism and crc risk by sex age bmi smokingstatus and alcohol consumptionil4 rs2070874 tccasecontrol1tttccctttcadjusted or2 ci pcctc cccc vs tcttvariablesexmalefemaleage years‰¥smoking statusnevereveralcohol consumptionnevereverbmi kgm2‰¥ “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p 1for il4 rs2070874 tc the genotyping was successful in crc cases and controls2adjusted for multiple comparisons [age sex bmi smoking status and alcohol consumption besides stratified factors accordingly] in a logisticregression modelbold values are statistically significant p005association of hvem rs2234167 il4 rs2070874 and rs2243263 andil10 rs1800896 and rs1800872 loci with tumor grade of escc casesas presented in table patients had welldifferentiated tumors had moderately differentiated tumors and has poorly differentiated tumors we found an association of the il10 rs1800872 tg snp with a worse differentiation tg vs tt p0048 and ggtg vs tt p0032 table discussionimmunotherapy is altering how we comprehend malignancies and offers new methods to treat them ec is a representative model of immune and inflammationrelated cancer recently some studies indicated that the snps ininflammation and immunerelated genes might influence the risk of ec in this study we explored the role ofimmunerelated gene snps hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872to escc development we observed that il4 rs2070874 tc could decrease a risk to escc even in the stage iiisubgroup however in bmi ‰¥ kgm2 subgroup il4 rs2243263 gc might increase the risk of escc we alsofound an association of the il10 rs1800872 tg snp with a worse differentiationil4 is an important regulator of immune and inflammation pathways some reports have suggested that il4 levelsare higher in untreated escc patients than in controls [“] it is considered that il4 levels may be implicated inthe development of escc the il4 rs2070874 tc polymorphism is a 5cid3utr snp in a highrisk gastric cancergc region a previous study suggested that rs2070874 c allele in the il4 gene might decrease the susceptibilityto gc in a chinese population lu reported that the rs2070874 c allele increased the risk of hcc in amale subgroup however chang and wang found that the il4 rs2070874 polymorphism might notinfluence the susceptibility of cancer in chinese population in this study we included subjects andinvestigated the correlation of this snp to escc susceptibility we found that il4 rs2070874 tc polymorphism the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0ctable stratified analyses between il4 rs2243263 gc polymorphism and crc risk by sex age bmi smokingstatus and alcohol consumptionbioscience reports bsr20193895101042bsr20193895il4 rs2243263 gccasecontrol1gggcccgggcadjusted or2 ci pccgccccc vs gcggvariablesexmalefemaleage‰¥smoking statusnevereveralcohol consumptionnevereverbmi kgm2‰¥ “p “p “p “p p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p p “p “p “p 1for il4 rs2243263 gc the genotyping was successful in crc cases and controls2adjusted for multiple comparisons [age sex bmi smoking status and alcohol consumption besides stratified factors accordingly] in a logisticregression modelbold values are statistically significant p005seemed to be a protective factor for escc development our findings were similar to a previous metaanalysis thatsuggested that the il4 rs2070874 c allele could be associated with a decreased susceptibility of gastrointestinal cancer a functional study indicated that the il4 rs2070874 allele c could promote a higher level of il4 in plasma il4 has an antiinflammatory effect and may decrease the risk of escc by inhibiting the inflammation fitzgerald reported that the il4 rs2070874 allele c could decrease the risk of prostate cancer specific mortality consistent with that report we identified an association between the il4 rs2070874 tc snp and a decreasedsusceptibility to escc in the stage iii subgroup however we did not find an association of il4 rs2070874 tcpolymorphism with lymphatic metastasis this might be due to the limited sample sizes in the future the relationshipof the rs2070874 snp in il4 gene with progress and prognosis should be further exploredrs2243263 gc an intron snp in the il4 gene was studied for the relationship of this snp to some diseasesthis snp might decrease the risk of asthma in the african american children while this relationship was not identified in caucasians hsiao reported that the il4 rs2243263 c allele was a protective factor for hbv surfaceantigen reverse seroconversion in nonhodgkin lymphoma cases undergoing rituximab treatment a previous studyinvestigated the relationship of il4 rs2243263 gc with colon and rectal cancer risk but no association wasfound however in a large simple size study lan found that the il4 rs2243263 gc snp increased the susceptibility to nonhodgkin lymphoma in this study we found that the il4 rs2243263 gc might increase therisk of escc in obese and overweight subjects table it was reported that the il4 level in mothers was inverselylinked to overweight in early childhood and might influence the metabolic profile of childhood in addition thelevel of il4 decreased with antipsychoticinduced weight gain it is suggested that the level of il4 could influence obesity and overweight introns are regulatory sequences that can affect the expression of genes here we foundthat the rs2243263 gc polymorphism a snp in il4 intron region might alter the risk of escc it is presumed the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20193895101042bsr20193895table logistic regression analyses of association between hvem rs2234167 il4 rs2070874 rs2243263 andil10 rs1800896 and rs1800872 polymorphisms and lymph node status in escc patientsgenotypepositive n405negative n316crude or cipadjusted or1 ciphvem rs2234167 gagggaaaga aagggaaaa alleleil4 rs2070874 tctttccccctctttcccc alleleil4 rs2243263 gcgggccccgccgggcccc alleleil10 rs1800872 tgtttgggggtgtttgggg alleleil10 rs1800896 tctttccccctctttcccc allelenn1adjusted for age sex smoking alcohol use and bmi status “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “that the rs2243263 gc polymorphism influences the level of il4 by regulating gene transcription in the future afunctional study should be considered to explore the potential mechanismthe il10 rs1800872 tg is a promotor snp torrespoveda reported that the expression of il10 mrnaand the level of serum il10 were significantly higher in subjects with the il10 rs1800872 t allele a recent studyfound that il10 rs1800872 tg snp promoted the risk of ec a metaanalysis also confirmed this association in our case“control study we did not find the association of il10 rs1800872 tg snp with the developmentof ec even in stratified analyses and reviewing different lymph node status additionally liu reported thatil10 rs1800872 gg genotypes predicted the worse survival of diffuse large bcell lymphoma patients treated withrituximabchop cyclophosphamide doxorubicin vincristine and prednisone in this study we found that the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20193895101042bsr20193895table logistic regression analyses of association between hvem rs2234167 il4 rs2070874 rs2243263 andil10 rs1800896 and rs1800872 polymorphisms and grades of esccgenotypeg2g3 n579hvem rs2234167 gagggaaaga aagggaaaa alleleil4 rs2070874 tctttccccctctttcccc alleleil4 rs2243263 gcgggccccgccgggcccc alleleil10 rs1800872 tgtttgggggtgtttgggg alleleil10 rs1800896 tctttccccctctttcccc allelenng1 n142crude or cipadjusted or1 cip “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “1adjusted for age sex smoking alcohol use and bmi statusbold values are statistically significant p005the il10 rs1800872 g allele was associated with poorly differentiated tumor thus in the future the association ofthe il10 rs1800872 tg snp and the survival of escc cases should be further studiedlimitations in the present study should be acknowledged first in the present study we only included five functional snps and explored the association of the risk to escc second there were other environmental risk factorseg vegetable and fruit intake aspirin and nsaids use and physical exercise which we did not consider for theirinfluence to the development of escc third the number of escc patients was limited and our study may be the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20193895101042bsr20193895underpowered in some subgroups fourth in this investigation the protein expression levels of the suspect factors were not measured finally considering the low penetrance of snp the other functional polymorphisms in thehvem il4 and il10 genes should not be ignoredin summary the present study suggests that the il4 rs2070874 tc polymorphism is a protective factor for esccdevelopment while the il4 rs2243263 gc increases a risk to escc in obese and overweight subjects additionallyit is highlighted that the il10 rs1800872 g allele is associated with poorly differentiated tumorcompeting intereststhe authors declare that there are no competing interests associated with the manuscriptfundingthis work was supported in part by the young and middleaged talent training project of health development planning commission in fujian province [grant number 2016zqn25] the program for new century excellent talents in fujian province university[grant number ncetfj2017b015] and the joint funds for the innovation of science and technology fujian province [grantnumber 2017y9099]author contributionall authors contributed significantly to the present study conceived and designed the experiments wt and mk performed theexperiments sc rc and cl analyzed the data wt and mk contributed reagentsmaterialsanalysis tools mk wrote themanuscript sc and rc other please specify noneacknowledgementswe appreciate all subjects who participated in the present study we wish to thank dr yan liu genesky biotechnologies incshanghai china for technical supportabbreviationsajcc american joint committee on cancer bc breast cancer bmi body mass index ci confidence interval ecesophageal cancer escc esophageal squamous cell carcinoma gc gastric cancer hbv hepatitis b virus hvem herpesvirus entry mediator hwe hardy“weinberg equilibrium il interleukin nsaid nonsteroidal antiinflammatory drug or odds ratio snp single nucleotide polymorphismreferences bray f ferlay j soerjomataram i global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries ca cancer j clin “ 103322caac21492 matejcic m and iqbal parker m geneenvironment interactions in esophageal cancer crit rev clin lab sci “1031091040836320151020358 qin jm yang l chen b interaction of methylenetetrahydrofolate reductase c677t cytochrome p4502e1 polymorphism andenvironment factors in esophageal cancer in kazakh population world j gastroenterol “ pmcid pmc2773864103748wjg146986 lin ew karakasheva ta hicks pd the tumor microenvironment in esophageal cancer oncogene “ pmcidpmc5003768 101038onc201634 park r williamson s kasi a immune therapeutics in the treatment of advanced gastric and esophageal cancer anticancer res “ 1021873anticanres12891 nelms k keegan ad zamorano j the il4 receptor signaling mechanisms and biologic functions annu rev immunol “ 101146annurevimmunol171701 rush js and hodgkin pd b cells activated via cd40 and il4 undergo a division burst but require continued stimulation to maintain divisionsurvival and differentiation eur j immunol “101002152141412001043143c1150aidimmu11503e30co2v suzuki a leland p joshi bh targeting of il4 and il13 receptors for cancer therapy cytokine “101016jcyto201505026 francipane mg alea mp lombardo y crucial role of interleukin4 in the survival of colon cancer stem cells cancer res “ 10115800085472can076874 tan n song j yan m association between il4 tagging single nucleotide polymorphisms and the risk of lung cancer in china molgene genom med e00585 pmcid pmc6465665 101002mgg3585 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc by 0cbioscience reports bsr20193895101042bsr20193895 cardenas dm sanchez ac rosas da preliminary analysis of singlenucleotide polymorphisms in il10 il4 and il4ralpha genesand profile of circulating cytokines in patients with gastric cancer bmc gastroenterol pmcid pmc6288868101186s1287601809139 shamoun l skarstedt m andersson re association study on il4 il4ralpha and il13 genetic polymorphisms in swedish patientswith colorectal cancer clin chim acta “ 101016jcca201809024 jia y xie x shi x associations of common il4 gene polymorphisms with cancer risk a metaanalysis mol med rep “ pmcid pmc5561993 103892mmr20176822 cho ya and kim j association of il4 il13 and il4r polymorphisms with gastrointestinal cancer risk a metaanalysis j epidemiol “ pmcid pmc5394226 101016jje201606002 zhenzhen l xianghua l qingwei w three common polymorphisms in the il4 gene and cancer risk a metaanalysis involving cases and controls tumour biol “ 101007s1327701307618 kim bs park sm uhm tg effect of single nucleotide polymorphisms within the interleukin4 promoter on aspirin intolerance inasthmatics and interleukin4 promoter activity pharmacogenet genomics “ hsiao lt wang hy yang cf human cytokine genetic variants associated with hbsag reverse seroconversion in rituximabtreatednonhodgkin lymphoma patients medicine baltimore e3064 pmcid pmc4839912 101097md0000000000003064 bondurant kl lundgreen a herrick js interleukin genes and associations with colon and rectal cancer risk and overall survival intj cancer “ pmcid pmc3470814 101002ijc27660 lan q wang ss menashe i genetic variation in th1th2 pathway genes and risk of nonhodgkin lymphoma a pooled analysis ofthree populationbased casecontrol studies br j haematol “ pmcid pmc3075370101111j13652141201008424x kwasniak k czarnikkwasniak j maziarz a scientific reports concerning the impact of interleukin interleukin and transforminggrowth factor beta on cancer cells central eur j immunol “ pmcid pmc6745546 105114ceji201876273 d™andrea a asteamezaga m valiante nm interleukin il10 inhibits human lymphocyte interferon gammaproduction bysuppressing natural killer cell stimulatory factoril12 synthesis in accessory cells j exp med “ pmcid pmc2191152101084jem17831041 miteva ld stanilov ns deliysky ts significance of 1082ag polymorphism of il10 gene for progression of colorectal cancer andil10 expression tumour biol “ 101007s1327701425892 pereira apl trugilo kp okuyama ncm il10 c592ca rs1800872 polymorphism is associated with cervical cancer j cancerres clin oncol “ 101007s00432020032560 yang y and fa x role of il10 gene polymorphisms on the susceptibility for esophageal cancer and its association with environmental factorsint j clin exp pathol “ pmcid pmc4583954 sun jm li q gu hy interleukin rs1800872 tg polymorphism was associated with an increased risk of esophageal cancer in achinese population asian pac j cancer prev “ zhao x lu c chu w microrna214 governs lung cancer growth and metastasis by targeting carboxypeptidased dna cell biol “ 101089dna20163398 breloer m hartmann w blankenhaus b cutting edge the btlahvem regulatory pathway interferes with protective immunity tointestinal helmin
Colon_Cancer
" chronic colorectal inflammation has been asso‘ciated with colorectal cancer crc however its exact molecular mechanisms remain unclear the present study aimed to investigate the effect of toll‘like receptor tlr9 on the development of colitis‘associated crc cac through its regulation of the nf‘κb signaling pathway by using a cac mouse model and immunohistochemistry the present study discovered that the protein expression levels of tlr9 were gradually upregulated during the development of crc in addition the expression levels of tlr9 were revealed to be positively correlated with nf‘κb and ki67 expression levels in vitro inhibiting tlr9 expression levels using chloroquine decreased the cell viability proliferation and migration of the crc cell line ht29 and further experiments indicated that this may occur through downregulating the expression levels of nf‘κb proliferating cell nuclear antigen and bcl‘xl in the findings of the present study suggested that tlr9 may serve an important role in the development of cac by regulating nf‘κb signalingcorrespondence to professor youxiang chen or dr chunyan zeng department of gastroenterology the first affiliated hospital of nanchang university yongwaizheng street nanchang jiangxi pr chinae‘mail chenyx102126com e‘mail zcy896163com abbreviations aif acute inflammation aom azoxymethane cac chronic inflammation crc colorectal cancer dai disease activity index dss dextran sodium sulfate ibd inflammatory bowel disease iecs immunohistochemistry pcna proliferating cell nuclear antigen tlr9 toll‘like receptor uc ulcerative colitiskey words colitis‘associated crc aom dss tlr9 nf‘κbintestinal epithelial cells ihc colitis‘associated colorectal cancer cif introductionthe number of patients with colorectal cancer crc world‘wide is increasing annually with an incidence rate of in chronic inflammation is the leading cause of immune cell infiltration and proliferation and it has been suggested to be a high‘risk factor for colitis‘associated crc cac inflammatory bowel disease ibd which encompasses both ulcerative colitis uc and crohn's disease was established as an important precursor to crc for example the incidence of ibd‘associated crc in patients with uc was reported to have a cumulative risk rate of at years at years and at years of disease duration therefore further in vivo studies are required for researchers to gain an improved understanding of the molecular mechanisms of cac which may provide more exact molecular targets for the diagnosis and treatment of cac during the early stages toll‘like receptor tlr9 a member of the tlr family is located in the cytoplasm and intracellular endosomes and can be activated by unmethylated bacterial cpg dna the activation of the tlr9 signaling pathway induces a type t helper cell immune response and stimulates the proliferation of b cells thus protecting the host from external microbial invasion ‘ multiple studies have revealed that abnormal tlr9 expression levels were involved in the pathogenesis and progression of uc in addition abnormal expression levels of tlr9 were also identified during the tumorigenesis and development of crc ‘nf‘κb is an important transcription factor involved in various biological processes including inflammatory reac‘tions immune responses apoptosis and proliferation in fact nf‘κb is regarded as a molecular hub that links inflam‘mation and cancer it was previously suggested that nf‘κb may serve an important role in colorectal carcinogenesis by regulating matrix metalloproteinase‘ expression ‘ previous studies have revealed that tlr9 was related to the biological characteristics of various types of cancer including bladder lung and prostate cancer such as cell proliferation invasion tumor growth and progression ‘ in fact one previous study reported that tlr9 regulated the expression levels of interleukin il‘ through the myeloid differentiation primary response protein myd88 myd88nf‘κb signaling 0cluo tlr9 promotes colorectal carcinogenesispathway in myeloid cells to promote tumor recurrence after irradiation including in melanoma bladder carcinoma and colorectal carcinoma the current study aimed to investigate the effect of tlr9 on the development of cac through its regulation of the nf‘κb signaling pathway owing to the synergistic effects of azoxy‘methane aom a tumor‘inducing agent and dextran sodium sulfate dss a tumor‘promoting agent the present study established cac model mice by co‘administering aom and dss to analyze the expression levels of tlr9 nf‘κb and ki67 in cac tissuesmaterials and methodsreagents and antibodies aom cat no a5486 and chloro‘quine tlr9 inhibitor cat no c6628‘25g were obtained from sigma‘aldrich merck kgaa dss cat no ‘100g was purchased from mp biomedicals llc anti‘tlr9 cat no ab134368 and anti‘myd88 cat no ab135693 primary antibodies were obtained from abcam anti‘nf‘κb nf‘κb p65 cat no 8242s anti‘bcl‘xl cat no and anti‘ki67 cat no 9449s primary antibodies were obtained from cell signaling technology inc the anti‘proliferating cell nuclear antigen pcna cat no sc‘ primary anti‘body was obtained from santa cruz biotechnology inc and the anti‘gapdh cat no ta309157 primary antibody was obtained from origene technologies inc horseradish peroxidase secondary goat anti‘rabbit cat no zb‘ and goat anti‘mouse cat no zb‘ antibodies used for western blotting and goat anti‘mouserabbit antibodies cat no ta130001ta130015 used for immunohistochem‘istry ihc were obtained from origene technologies inccell lines and culture the human crc cell line ht29 was obtained from the american type culture collection and cultured in mccoy's 5a modified medium gibco thermo fisher scientific inc supplemented with fbs gibco thermo fisher scientific inc mgml penicillin and mgml streptomycin maintained in a humidified atmo‘sphere of co2 at ˚cwestern blotting cells were lysed in ripa lysis buffer beijing solarbio science technology co ltd at ˚c for min the protein concentration was measured following centrifugation at x g for min at ˚c and quantified using a bicinchoninic acid protein assay kit beijing solarbio science technology co ltd an equal amount of protein µglane was separated via sds‘page and electrophoretically transferred onto polyvinylidene difluo‘ride membranes emd millipore the membranes were blocked with skimmed milk for h at room temperature and were subsequently incubated overnight at ˚c with the following primary antibodies anti‘tlr9 anti‘nf‘κb anti‘bcl‘xl anti‘pcna anti‘myd88 and anti‘gapdh following the primary antibody incubation the membranes were washed times with pbs for min each and incubated with the corresponding goat anti‘rabbit or goat anti‘mouse secondary antibodies at ˚c for h protein bands were visualized using an ecl reagent thermo fisher scientific inc on a gel doc xr system bio‘rad laboratories inc and analyzed using image lab version software bio‘rad laboratories incwound healing assay a wound healing assay was performed to analyze the cell migratory ability briefly ht29 cells 3x105well were seeded into six‘well plates and cultured to ‘ confluence subsequently a ‘µl pipette tip was used to scratch a wound in the cell monolayer fresh serum‘free mccoy's 5a modified medium containing different concen‘trations of chloroquine or µgml was added to each well and cultured for h in a humidified atmosphere of co2 at ˚c images of each well were captured at and h using a light phase contrast microscope olympus zkx‘ olympus corporation with a magnification of x100 the wound‘healing areas were assessed using imagej 152a software national institutes of health the migratory rate of cells wound area at h‘wound area at harea at hcolony formation assay ht29 cells 12x105well were cultured in ‘well plates for h in medium containing different concentrations of chloroquine or µgml at ˚c and then seeded into ‘cm cell culture dishes cellsdish and incubated in complete medium at ˚c after days the cells were fixed with paraformaldehyde for min and stained with crystal violet for min both at room temperature the number of colonies defined as cellscolony was counted manually using a light microscope with a magnifica‘tion of x100 relative colony number number of colonies in observed groupnumber of colonies in control group all of the experiments were repeated ‰¥ timescell viability assay the viability of ht29 cells was analyzed using an mtt assay briefly ht29 cells were seeded at a density of cellswell in a ‘well plate and treated for or h with chloroquine or µgml at ˚c and then analyzed using an mtt assay as previously described animal studies all animal experiments were approved by the ethics committee of the first affiliated hospital of nanchang university nanchang china a total of female balbc mice weight ‘ g age ‘ weeks were obtained from beijing vital river laboratory animal technology co ltd laboratory animal license no scxk ‘ the animals were maintained with a normal diet and tap water ad libitum at a temperature of ±Ëšc and a relative humidity of ‘ and artificially illuminated on an approximate h lightdark cycle at the animal care facility in the medical college of nanchang university all of the mice experiments were approved by the animal care and use committee of nanchang university the total experiment lasted for weeks the mice were divided into four groups micegroup i group a aom dss which was intraperitoneally injected without anesthesia with mgkg aom once on the first day followed by dss given in the drinking water for week and then weeks of distilled water one cycle which was repeated for two additional cycles ii group b aom which was intraperitoneally injected with mgkg aom once on the first day and provided with distilled drinking water during weeks ‘ iii group c dss which was treated with dss 0concology letters as described for group a but without the aom treatment and iv group d blank control which received neither dss nor aom treatment and was provided with distilled drinking water for the first weeks all the mice were provided with a normal diet and tap water during weeks ‘ fig 1athe disease activity index dai was evaluated at the end of the experiment using the numerical system described by tian the dai parameters included total body weight loss none ‘ ‘ ‘ stool consistency well‘formed pellets loose stool diarrhoea and the presence of fecal occult blood negative positive gross bleedingseveral mice were randomly sacrificed at certain times points following aom injection the 1st 2nd 3rd 6th 9th 12th 18th and 23rd weeks six mice were sacrificed at weeks and one mouse was sacrificed at week and four mice were sacrificed at weeks and in each group additionally six mice in group d were randomly sacrificed at week as control all remaining mice were sacrificed at week after the large bowels were resected and washed with pbs they were carefully examined photographed and fixed in formalin at room temperature for h further histological examinations were subsequently performedhistopathological analysis and immunohistochemistry ihc paraffin‘embedded colorectal sections ‘µm‘thick were stained with hematoxylin for min and eosin for min at room temperature to analyze the degree of inflammation using a light microscope with magnifications of x40 and x100 briefly the severity of inflammation the thickness of inflammation the severity of epithelial damage and the extent of the lesions were each graded from to by two investigators who were blinded to the treatment groups as previously described the severity of inflammation was adapted from the grading system developed by truelove and richards as follows i grade no neutrophil infiltration in the lamina propria ii grade i infiltration of a small number of neutrophils [ cellshigh power field hpf] in the lamina propria involving a few crypts iii grade ii obvious neutrophil infiltration in the lamina propria ‘ cellshpf involving of the crypts iv grade iii infiltration of neutrophils cellshpf in the lamina propria with crypt abscess and v grade iv obvious acute inflammation in the lamina propria with ulcer formation grade i was classi‘fied as mild grade ii was classified as moderate and grades iii and iv were classified as severe the severity of inflammation ranged from to no inflammation mild moderate severe the thickness of inflammation ranged from to no inflammation mucosa mucosa plus submucosa transmural the severity of epithelial damage ranged from to intact epithelium disruption of architectural structure erosion ulceration and the extent of lesions ranged from to no lesions punctuate multifocal diffuseihc was performed as described in our previous study colorectal tissues were fixed in formalin for h at room temperature formalin‘fixed and paraffin‘embedded tissue blocks were cut into ‘µm‘thick sections and mounted on glass slides slides were heated in an oven at ˚c for min and deparaffined in xylene twice for min each at room tempera‘ture rehydrated in a descending ethanol series and ethanol for min each at room temperature and incubated in h2o2 for min at room temperature to block endogenous peroxidase antigen retrieval was performed by heating in a microwave at ˚c in sodium citrate buffer mm ph for min slides were blocked with bovine serum albumin beijing solarbio science technology co ltd for h at room temperature to block non‘specific antibody binding and incubated overnight at ˚c with the following primary anti‘bodies anti‘tlr9 anti‘nf‘κb and anti‘ki67 following the primary antibody incubation the sections were washed three times with pbs and incubated with horse‘radish peroxidase secondary goat anti‘mouserabbit antibodies ready to use at ˚c for min the sections were stained with '‘diaminobenzidine at room temperature the duration of staining was based on the staining observed under a light microscope with a magnification of x100 and the reaction was terminated when the staining was yellowish‘brown the sections were then counterstained with hematoxylin for min at room temperature the slides were observed under a light microscope with a magnification of x100 the widely accepted german semi‘quantitative scoring system was used to determine the staining intensity and area of staining according to the recommendations of remmele and stegner each specimen was assigned a score according to the intensity of the nucleic cytoplasmic andor membrane staining no staining not detected0 weak staining light yellow1 moderate staining yellowish brown2 strong staining brown3 and the extent of stained cells no staining ‘ ‘ ‘ ‘ the final immunoreactive score was determined by multiplying the intensity score with the extent of stained cells score ranging from the minimum to the maximumco‘immunoprecipitation assay ht29 cells were lysed in ripa lysis buffer beijin solarbio science technology co ltd at ˚c for min whole‘cell lysates were pelleted via centrifugation at x g for min at ˚c the super‘natant was incubated with an anti‘tlr9 antibody or goat anti‘mouse igg cat no zb‘ origene technologies inc together with protein ag plus‘agarose beads santa cruz biotechnology at ˚c overnight the beads were washed three times with the non‘lubrol lysis buffer at x g centrifugation for min at ˚c then subjected to sds‘page and subsequent western blotting analysis as aforementioned whole cell lysate was used as a controlstatistical analysis statistical analysis was performed using spss software ibm corp the data are presented as the mean ± sd all experiments were performed at least in triplicate a one‘way anova followed by a tukey's post hoc test was used to determine the statistical differences between groups whereas an unpaired student's t‘test were used to determine the statistical differences between groups a spearman's rank correlation test was used to determine the correlation between the expression levels of tlr9 nf‘κb and ki67 in crc tissues p005 was considered to indicate a statistically significant differenceresultsconstruction of the cac model mice in groups a aom dss and c dss the body weights of the mice decreased with 0cluo tlr9 promotes colorectal carcinogenesisfigure construction of the colitis‘associated‘colorectal cancer model in mice a schematic diagram of the experimental protocol for colitis‘associated colorectal cancer model mice in groups a‘d arrow indicates mgkg azoxymethane administration via intraperitoneal injection black square indicates administration of dextran sodium sulfate in drinking water for week continuous line indicates duration fed a normal diet and distilled water dotted line indicates duration fed a normal diet and tap water and triangle indicates timepoint of sacrifice b body weights of the mice in each group p0001 c disease activity index of groups a and c data are presented as the mean ± sd group a mgkg azoxymethane and dextran sodium sulfate water group b mgkg azoxymethane group c dextran sodium sulfate water group d blank controltime compared with group d blank control group fig 1b the average weight of the mice in group c declined from ± g at the beginning of the experiment to ± g by week while the average weight of the mice in group a decreased from ± g at the beginning of the experiment to ± g by week the differences between groups a and d and groups c and d were statistically significant at week p005 the dai which reflects the severity of colitis was also markedly increased in groups a and c after dss administration on the 1st 3rd and the 6th week fig 1c no weight loss or any signs of inflammation such as loose stool or diarrhea positive fecal occult blood or gross bleeding were observed in groups b aom and d blank control the dai of groups b and d was zero throughout the modeling process and is therefore not shown in fig 1c the lengths of the large bowels in groups a and c were decreased compared with groups b and d fig 2a notably the lengths of the large bowels were significantly decreased in groups a and c from week compared with the control group mice sacrificed in group d at week or with group dp005 fig 2b however the severity of inflamma‘tion was significantly increased in group a compared with in group c at week and the extent of inflammation was signifi‘cantly increased in group a compared with in group c after week p005 while there was no significant differences in the thickness of inflammation and the severity of epithelial damage between groups a and c fig 2cby the 12th week colorectal tumors were only observed in the mice of group a fig 2d pathological results further revealed that the mice in group a presented with acute inflam‘mation aif chronic inflammation cif adenoma and adenocarcinoma of the colorectum at weeks and respectively fig 2d and eexpression levels of tlr9 and nf‘κb are simultaneously upregulated during the development of chronic colitis in crc ihc staining revealed that tlr9 was located in the cytoplasm of the intestinal epithelial cells iecs and inflammatory cells in the lamina propria fig 3a tlr9 expression levels were significantly upregulated in aif cif adenoma and adenocarcinoma tissues compared with the corresponding tissues from control mice group d p00334 p00379 p00437 and p00008 respectively fig 3b furthermore the protein expression levels of tlr9 was significantly upregulated in the adenocarcinoma tissue compared with the aif p00077 cif p00278 and adenoma p00273 tissue fig 3bthe positive nf‘κb region was mainly confined to the cytoplasm of the iecs and inflammatory cells fig 3a the ihc results revealed that the expression levels of nf‘κb were significantly upregulated in the aif cif adenoma and adenocarcinoma tissues compared with the corresponding tissues from control mice p00061 p00043 p00019 and 0concology letters figure colorectal pathological results in mice a length of the large bowels of mice in each group at week representative bowels from ‘ independent animals are shown b mean length of large bowel of mice in each group p005 p001 p0001 vs control mice sacrificed in group d at week or group d bottom graph c histological score of severity thickness damage and extent of lesions of the colorectums from mice in groups a and c p005 d large bowels were retrieved at week and from mice in group a to determine the numbers of tumors formed p005 p001 e histological analysis of various pathological changes in the colons of mice in group a were determined using hematoxylin and eosin staining the control groups represents tissues taken at week from group d scale bars ‘µm upper and ‘µm lower inflammatory cells green arrows and intestinal epithelial cells red arrows are indicated group a mgkg azoxymethane and dextran sodium sulfate water group c dextran sodium sulfate water aif acute inflammation cif chronic inflammationp00001 respectively fig 3d moreover nf‘κb expression levels were significantly upregulated in the adenocarcinoma tissue compared with the aif p00001 and adenoma p00161 tissues fig 3dki67 expression levels were observed in the nuclei of iecs and inflammatory cells fig 3a the ihc results revealed that the ki67 expression levels were gradually upregulated across the intestinal lesions including in the aif cif adenoma and 0cluo tlr9 promotes colorectal carcinogenesisfigure tlr9 and nf‘κb are simultaneously upregulated as cac develops a ihc was used to analyze the expression levels and localization of tlr9 nf‘κb and ki67 in colorectal sections obtained from mice in group a inflammatory cells green arrows and intestinal epithelial cells red arrows are indicated ihc staining scores of b tlr9 c ki67 and d nf‘κb in sections of normal control group d aif cif adenoma and adenocarcinoma tissues which were obtained as described in the methods section scale bar ‘µm p005 p001 and p0001 vs control p005 p001 and p0001 vs adenocarcinoma correlation analysis of e tlr9 and nf‘κb expression levels f ki67 and tlr9 expression levels and g nf‘κb and ki67 expression levels was conducted using spearman's rank correlation group a mgkg azoxymethane and dextran sodium sulfate water aif acute inflammation cif chronic inflammation tlr9 toll‘like receptor ihc immunohistochemistryadenocarcinoma tissues p00331 p00092 p00241 and p00006 respectively compared with the expression levels in the corresponding tissues from the control mice fig 3c interestingly the expression levels of tlr9 and nf‘κb were discovered to be significantly positively correlated with other rho08236 p00001 fig 3e in addition a significant positive correlation was also identified between tlr9 and ki67 expres‘sion levels rho05515 p0001 fig 3f and between nf‘κb and ki67 expression levels rho05103 p001 fig 3gdownregulated tlr9 expression levels reduces the migration viability and colony formation of ht29 cells to further investigate the role of tlr9 in crc the human crc cell line ht29 was treated with chloroquine an inhibitor of tlr9 in vitro the results revealed that suppressing tlr9 with chloroquine at both doses inhibited the migration viability and colony formation ability of ht29 cells in a dose‘dependent manner fig 4a‘e additionally lysates were collected from ht29 cells treated with either different concentrations of chloro‘quine for h or µgml chloroquine for different time periods fig 4f‘i and western blotting was performed the analysis revealed that the expression levels of tlr9 nf‘κb pcna myd88 and bcl‘xl were gradually downregulated in ht29 cells treated with increasing doses of chloroquine compared with the 0concology letters figure chloroquine inhibits the migration viability and colony formation of ht29 cells by inhibiting tlr9 chloroquine and µgml inhibited the migration of ht29 cells in a dose‘dependent manner at h compared with the control group which was determined using a wound healing assay a representative images were photographed magnification x100 and b migration rates were calculated proliferation rate of ht29 cells was reduced by chloroquine treatment and µgml which was determined using a colony formation assay c representative images were photographed and d the relative colony number was analyzed p005 p001 p0001 e viability of ht29 cells was decreased by chloroquine and µgml at and h compared with the control cells as determined using an mtt assay f expression levels of tlr9 nf‘κb pcna myd88 and bcl‘xl in lysates obtained from ht29 cells treated with numerous concentrations of chloroquine or µgml for h were analyzed using western blotting g semi‘quantification of the expression levels of the proteins presented in part f was performed using imagej software gapdh was used as the loading control and for normalization h expression levels of tlr9 nf‘κb pcna myd88 and bcl‘xl in lysates obtained from ht29 cells treated with µgml chloroquine for numerous durations or h were analyzed using western blotting i semi‘quantification of the expression levels of the proteins presented in part h was performed using imagej software gapdh was used as the loading control and for normalization j tlr9 interacted with the nf‘κb protein in ht29 cells which was determined using a co‘immunoprecipitation assay goat anti‘mouse igg antibody was used as the negative control all data are expressed as the mean ± sd of three independent experiments p005 p001 p0001 vs control0 h tlr9 toll‘like receptor pcna proliferating cell nuclear antigen myd88 myeloid differentiation primary response protein myd88 ip immunoprecipitated ib immunoblotting 0cluo tlr9 promotes colorectal carcinogenesiscontrol group fig 4f and g a similar trend was observed in the expression levels of these proteins as the duration of chlo‘roquine treatment increased fig 4h and i thus these results indicated that the expression levels of tlr9 nf‘κb pcna myd88 and bcl‘xl in ht29 cells treated with chloroquine may be downregulated in both a dose‘ and time‘dependent manner fig 4f‘i to verify whether tlr9 affected the colorectal carcinogenesis by interacting with nf‘κb co‘immunoprecipi‘tation assay was used to detect the interaction between tlr9 and nf‘κb in ht‘ cells the results revealed that there was an interaction between tlr9 and nf‘κb fig 4jdiscussioncolorectal carcinogenesis is a multi‘step process starting from normal crypts to aberrant crypt foci then to polyps adenoma and eventually adenocarcinoma it has been reported that individuals with ibd may have an increased risk of developing crc which is directly proportional to the extent and duration of their disease however the exact mechanism and duration required for chronic colitis to develop into adenoma and then adenocarcinoma remains unclear it has been suggested that patients with ibd have an increased risk of crc following the inflammation‘dysplasia‘carcinoma model including dysplasia and crc as primary conse‘quences of chronic inflammation however there are currently still no defined molecular biomarkers or existing monitoring protocols for detecting the occurrence of a malignant tumor except for frequent colonoscopy examinationsin the present study an acute colitis‘chronic colitis‘adenoma‘adenocarcinoma model was successfully constructed via aomdss induction using this model tlr9 expression levels were discovered to be upregulated as the severity of the colorectal lesions increased which indicated that tlr9 protein expression levels may be continuously activated during colitis‘crc development tlr9 is a critical protein associated with innate and acquired immunity and it has been demonstrated to serve a significant role in the development of colitis and sporadic crc however the mechanism by which tlr9 regulates the devel‘opment of crc remains to be elucidatedinterestingly ihc analysis revealed that the expression levels of nf‘κb and ki67 were simultaneously upregulated alongside tlr9 expression levels notably inhibiting tlr9 decreased the migration proliferation and viability of ht29 cells in vitro and tlr9 expression levels in vivo were identi‘fied to be significantly positively correlated with the expression levels of nf‘κb and ki67 a cell proliferation marker during the transition from colitis to crc the present study further revealed that the inhibition of tlr9 in vitro significantly downregulated the expression levels of nf‘κb myd88 pcna and the anti‘apoptotic protein bcl‘xl in a dose‘ and time‘dependent manner notably a previous study reported that tlr9 promoted the tumor‘propagating potential of prostate cancer cells via nf‘κb signaling thus the findings of the present study indicated that tlr9 may promote cac through nf‘κb signaling however these findings may be controver‘sial because other previous studies have revealed that tlr9 agonists exerted an antitumor effect in crc the majority of these studies primarily focused on the role of tlr9 in colitis or sporadic crc whereas the current study focused on the role of tlr9 in the pathogenesis of cac to provide novel targets for the treatment of cac for example altera‘tions in microtubule end‘binding protein were identified as a characteristic of sporadic but not uc‘associated crc and in another previous study the immune profiling patterns of patients with cac were significantly different compared with the patients with sporadic crc chloroquine a non‘specific inhibitor of tlr9 is an old antimalarial drug which has recently attracted significant interest for its potential antitumor properties for example numerous studies have reported that chloroquine directly regu‘lated cancer cells by inducing apoptosis inhibiting autophagy interacting with nucleotides eliminating cancer stem cells and enhancing the growth of cancer cells ‘ chloroquine also inhibited the expression levels of tlr9 by preventing the acidi‘fication and maturation of the endosomes and the trafficking of tlrs due to the multiple effects of chloroquine on tumor cells different concentrations of chloroquine were selected for use in the present study based on the lowest dose according to previous studies in order to obtain the best possible results with low toxicity to the cells in the future investiga‘tions using small interfering rna targeting tlr9 should be performed to determine the effect on the biological processes of crc cell lines to further verify the findings of the present study thus our future studies will focus on investigating the precise molecular mechanism by which tlr9 participates in the early occurrence of colorectal carcinogenesisin the present study developed a cac animal model the findings indicated that tlr9 may be closely asso‘ciated with the process of inflammation‘dysplasia‘carcinoma and may impact carcinogenesis by regulating the nf‘κb signaling pathway these results may provide promising potential to be developed into an early detection protocol or therapeutic molecular target for crcacknowledgementsnot applicablefundingthe present study was supported by grants from the national natural science foundation of china grant nos and the foundation of jiangxi educational committee grant no gjj170016 and the graduate student innovation funding program of nanchang university grant no cx2019119availability of data and materialsall data generated or analyzed during this study are included in this published article the original data are available from the corresponding author on reasonable requestauthors' contributionscz and ql designed the study and drafted the manuscript ql and lz performed the experiments ql ct and zz analyzed the data ql cz and yc revised the manuscript for important 0concology letters intellectual content cz and yc made substantial contribu‘tions to conception design and coordination of the study and gave final approval of the version to be published all authors have read and approved the final manuscriptethics approval and consent to participateall animal experiments were approved by the ethics committee of the first affiliated hospital of nanchang university nanchang china approval no ‘ pa
Colon_Cancer
liver cancer the predominant primary malignancy ranks as the fifth most commonly diagnosed cancer in male and the seventh in female around the world in the year it has been estimated thatliver cancer is the fourth leading cause of cancer death with approximated new cases were diagnosed and over patients succumbed to this malignancy the incidence of liver cancer differsamong different geographical regions and the global map of liver cancer revealed that it remains a highestincidence rates in east asia with china accounting for more than of the burden in the world multiple risk factors including chronic infection with hepatitis b virus hbv or hepatitis c virus hcvaflatoxincontaminated eatables intake heavy alcohol consumption smoking corpulence and type diabetes are suggested to be essential for the occurrence of liver cancer of the two infection diseaseshbv is suggested to be implicated in “ of virusassociated liver cancer while hcv is implicated in“ suggesting a predominant role of inflammationimmune response in the process of liver cancer chemokines also named as chemoattractant cytokines are a large subfamily of small heparinbindingproteins and originally characterized by their properties to direct and recruit the movement of variousleukocyte subsets on the basis of the position of the cysteine residues adjacent to the nterminalchemokines were classified into four conserved subfamilies namely cc cxc cx3c and c and theythese authors contributedequally to this workreceived april revised august accepted august accepted manuscript online august version of record published august the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201169101042bsr20201169table sample characteristics in a liver tissue microarraycharacteristiccancer samples casenormal samples casegendermalefemaleage yearrangemedianstageiiiiiivgradeexecute their contributions mainly through interactions with their gproteincoupled receptors identified as ccrcxcr cx3cr and cr it is generally acknowledged that chemokines and their receptors may play multifaceted roles in engendering and mediating inflammation and immune response and therefore be involving inthe pathogenesis and pathological process of various diseases recently expanding evidence highlighted thatchemokinesreceptors were constitutively expressed and responsible for various malignant progression includingtumor cell proliferation migration invasion metastasis and tumor angiogenesis in a variety of human tumor types in the current research we evaluated whether cxcl8 a member of cxc chemokines was constitutively expressedin the tissues with liver cancer and explored the underlying role and mechanism of cxcl8 in regulating malignantprogression of liver cancermaterials and methodscel line cell culture and cell transfectionthe human hepatoblastoma cell line hepg2 were provided by american type culture collection atcc usa andthe cells were cultured in rpmi1640 medium supplemented with fetal bovine serum fbs gibco thermofisher scientific inc containing unitsml penicillin and mgml streptomycin at —¦c with co2 in ahumidified atmosphere a expression plasmid carrying cxcl8 were applied to transfect cells using lipofectamine invitrogen according to the supplier™s instructions and the transfection efficiency was verified using a humancxcl8 elisa kit boster biological technology wuhan china following the manufacturer™s recommendationsimmunohistochemistry staining assaya commercial human tissue microarray containing liver cancer and normal liver tissue samples alenabioxi™an china was used to estimate the expression of cxcl8 sample characteristics were shown in table afterroutine deparaffinization and hydration the microarray was treated with vv hydrogen peroxidemethanolfor min at room temperature for inactivating endogenous peroxidase activity following antigen retrieval with m citrate buffer in a microwave oven for min the microarray was blocked with normal goat serum for min at room temperature and subsequently probed with a primary antibody specific for cxcl8 dilution after rinsing with pbs the microarray was incubated with hrpconjugated secondary antibody boster biologicaltechnology wuhan hubei china at —¦c for min finally dab substrate was utilized for the visualization ofantigen and haematoxylin for a routine nuclear counterstain the expression of cxcl8 at protein level was evaluatedas the following two parameters a the expression intensity negative weak mild strong superstrong staining b the percentage of cells stained “ “ of stainingthe scores for the two parameters are summed to produce a total score a total score of ‰ was referred to as lowexpression whereas ‰¥ as high expression the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201169101042bsr20201169cell proliferation assayhepg2 cells at density of — per well were maintained in 96well plates in μl of rpmi medium containing fbs with or without exogenous cxcl8 following culture for up to or h a cell proliferation assaykit counting kit8 boster biological technology wuhan china was applied to determine the proliferation ability ofcells following the manufacturer™s recommendations the optical density od in individual wells was detected witha microplate reader at nmcell migration assaya total of — hepg2 cells were individually maintained in each chamber of 8μm pore inserts containing μl ofserumfree rpmi medium and the low chamber which contained μl of rpmi medium supplementedwith fbs with or without exogenous cxcl8 was employed as attractant after h incubation the cells that hadpenetrated through the inserts and attached to lower surface of the inserts were fixed and stained using ethanolcontaining viola crystallina finally the migrating cells were photographed and counted under a light microscopeapoptosis assaycell apoptosis was detected using a commercial an apoptotichoechst staining kit beyotime shanghai chinabriefly the cells were fixed with fixative solution and the cells were stained with hoechst followed by washingwith pbs three times a confocal microscope was used to determined cell apoptosis the apoptotic rate was evaluatedby the following formulaapoptotic rate total number of cells ˆ’ number of apoptotic cells— total number of cellswestern blotting assaytotal protein from individual cell pellets was extracted using a commercial ripa lysate buffer boster biologicaltechnology wuhan china following supplier™s instructions about μg of total protein was subjected to electrophoresis in sdspage and subsequently electrotransferred onto a pvdf membrane millipore bedfordma after blocking with nonfat milk at room temperature for h the membrane was respectively incubatedovernight at —¦c with different specific antibodies directed against erk abways perk11000 abwayssurvivin santa cruz bax santa cruz caspase3 affinity and actin hrplinked secondaryantibodies and ecl reagent were used to display protein bands the relative protein levels were normalized againstactinstatistical analysisdata were analyzed using spss software in vivo the pearson correlation coefficient analysis was applied toreveal significant differences among several clinicopathological parameters in vitro data were presented as meansˆ’ standard deviations significant differences between two groups were compared using the student™s ttest p005was considered as statistical significanceresultscxcl8 was overexpressed in tissues with liver cancerthe results from immunohistochemistry assay showed that the expression of cxcl8 at protein level was markedlyincreased in liver cancer tissues figure 1a“c whereas normal liver tissue showed a decreased expression ofcxcl8 protein figure 1d p00246 in addition statistical analysis form liver cancer tissues demonstrated thatupregulated level of cxcl8 was positively concerned with high clinical stage and tumor infiltration p00061 onthe contrary there was no positively association between cxcl8 expression and other clinicopathological parametersincluding pathological grade sex and age table exogenous cxcl8 regulates proliferation migration and apoptosis ofhepg2 cellsto assess the roles of cxcl8 in regulating malignant behavior of liver cancer a classic cell line namely hepg2 wasemployed for following cell experiments cck8 analyses demonstrated that the growth rate of hepg2 cells treatedwith or ngml cxcl8 was significantly elevated as compared with hepg2 cells treated with ngml cxcl8figure 2a coupled with this transwell analyses revealed that migration cells of hepg2 cells treated with the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201169101042bsr20201169figure upregulated expression of cxcl8 in tissues with liver cancer in a human liver tissue microarrayliver cancer tissues with stage ii a iii b and iv c represented strong staining of cxcl3 normal tissue d showed weak stainingof cxcl8table cxcl8 level in a liver tissue microarraycharacteristicnormal liver sampleshcc samplesclinical stagetumorinfiltrationtpathological gradeagesexcase of score‰ case of score‰¥ ii t2iiiivt3t4‰ manwomanχ2por ngml cxcl8 was significantly more than that of hepg2 cells treated with ngml cxcl8 figure 2bc inaddition apoptosis assay showed that treatment of cells with or ngml cxcl8 can significantly inhibit theapoptosis rate of cells figure 2doverexpression of cxcl8 contributes to proliferation migration andapoptosis of hepg2 cellsto reveal the role of cxcl8 in liver cancer gene transfection strategy was used to construct cxcl8overexpressinghepg2 cells and their control fluorescent detection demonstrated that cells exhibited high content of fluorescencefigure 3a moreover elisa analysis showed that the level of cxcl8 in supernatant from cxcl8overexpressioncell medium was remarkably increased as compared with supernatant from their parental and control cell mediumindicating hepg2 cells overexpressing cxcl8 have be established figure 3b cck8 and transwell assays demonstrated that the proliferation figure 3c and migration figure 3de abilities of hepg2 cells overexpressing cxcl8were markedly enhanced as compared with their parental and control cells on the contrary the apoptosis rate ofoverexpression cells was significantly lower than those of parental and control cells figure 3foverexpression of cxcl8 regulates tumorspecific protein expression ofhepg2 cellsfinally we investigated whether overexpression of cxcl8 regulates the expression of tumorrelated genes thedata from western blotting indicated that cxcl8overexpression cells presented significantly increased levelsof erk perk and survivin compared with their control cells inversely the levels of caspase3 and bax incxcl8overexpression cells were predominantly reduced than that in their control cells figure 4ab the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201169101042bsr20201169figure the effects of exogenous cxcl8 in cell proliferation migration and apoptosis of hepg2 cells in vitroproliferation index a the number of migration b and c and apoptosis rate d from hepg2 cells following treatment with exogenous cxcl3 at different concentrations p005 p001 versus ngml of cxcl8discussioncxcl8 also known as il8 is a member of the cxc family of chemokines that is produced by many types of cellsincluding leukocytes fibroblasts endothelial cells and malignant cancer cells and play a various spectrum of biological effects in cell functions through interaction with its g proteincoupled receptors cxcr1 and cxcr2 cxcl8 are best known for their function in the initiation of the inflammatory reaction during the process ofinflammation cxcl8 recruits leukocytes to the site of infection ultimately resulting in increased neutrophil infiltration which is responsible for the damage of endothelial cells this implies that downregulation of cxcl8 is crucialfor the resistance to chronic inflammation in addition to its function in inflammation and immune response increasing evidences suggested that cxcl8 isalso implicated in diseaserelated processes including tissue damage fibronolysis angiogenesis and tumorigenesissuggesting that cxcl8 may be implicated in disease pathologies in which inflammation plays a vital role it is well accepted that inflammation as a predominant regulator is involved in the developmental processes ofnumerous human cancers and is the seventh leading of hallmark of cancer studies showed that many chronicinflammatory states obviously increase the risk of certain cancers and about of cancers are thought to be causedby chronic inflammation or inflammatory states as a proinflammatory chemokine cxcl8 belongs to elrcxc family of chemokines which shares structural homology with cxcl5 to date increasing number of studiesindicated that cxcl8 and its receptors were overexpressed in several types of human cancers including colorectalcancer prostate cancer cervical cancer and nonsmall cell lung cancer in the current investigationwe found that cxcl8 was significantly upregulated in tissues with liver cancer and its overexpression was closelycorrelated with high clinical stage and tumor infiltration in agreement with this it was demonstrated that the expression of cxcl8 was significantly increased in tissues with oesophageal squamous cell carcinoma of which stronger the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201169101042bsr20201169figure the effects of overexpression of cxcl8 in cell proliferation migration and apoptosis of hepg2 cells in vitroa fluorescence images of cxcl8overexpression cells and their control cells b the expression of cxcl in supernatantfrom cxcl8overexpression cells parental cells and control cells was detected by elisa the proliferation index c number ofmigration d and e and apoptosis rate f from hepg2 cells overexpresssing cxcl8 parental cells and control cells p005p001 versus controlexpression of il8 predominantly connected with many advancedstage pathological characteristics including depthof invasion lymph node metastasis pathologic stage lymphatic invasion and venous invasion recently cxcl8was also proposed to be predominately overexpressed in tissues with bladder cancer its overexpression was tightlyassociated with advanced disease and the overall survival rate of patients with increased expression of cxcl8 wasobviously reduced these findings suggest a possible significance of cxcl8 in cancer development and progressionaccumulating studies have revealed that cxcl8 is a critical component that involved in tumor initiation promotion and progression in cervical cancer jia et al reported that the proliferation and migration of hela cervical cancer cells were significantly enhanced after cells treated with different concentrations of exogenous cxcl8 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20201169101042bsr20201169figure the effects of cxcl8 in regulating tumorspecific protein expressiona representative images of western blotting analysis for erk perk survivin caspase3 and bax genes b relative expressionof these genes at protein levels from western blotting analysis p005 p001 versus control accordingly cxcl8 is also responsible for cancer cells malignant behavior in an autocrine fashion for example pc3 prostate cancer cells overexpressing cxcl8 present rapidly tumorigenicity highly proliferation rateremarkably angiogenesis and exhibit incidence of lymph node metastasis this investigation also demonstrated that several genes associated with angiogenesis and metastasis including vggf mmp2 and mmp9 wereupregulated in the clones with high cxcl8 expression on the contrary one study suggested that neutralizing antibodies against cxcl8 exerted a partial inhibition of tumor growth and exhibited antiangiogenesis activity in a nudemouse xenografts model however it is not clear whether cxcl8 stimulates the malignant process of liver cancer in the present studywe demonstrated that exogenous administration of cxcl8 significantly promote hepg2 cells proliferation and migration and overexpression of cxcl8 can also facilitate to these behaviors through an autocrine pathway finallymechanism studies demonstrated that overexpression of cxcl8 in hepg2 cells regulates tumorspecific protein expression including erk12 bax and survivin the involvement of cxcl8 in tumor progression and angiogenesiswere mediated by multifaceted signaling pathways including nfκb jnk pi3kakt p38 mapk and erk [“]in which erk signal pathway is a crucial mediator of a variety of cancer cells fates including proliferation migrationand survival in lung cancer it was suggested that egf stimulated a significant increase of cxcl8 production in lungcancer cells and il8 production from lung cancer cells could be initiated by their own produced factors resulting inthe recruitment of inflammatory cells in the tumor microenvironment as well as the formation of inflammatory microenvironment through pi3kakt and erk pathways cxcl8 signaling has also been proposed to have a vitalrole in promoting tumor progression by regulating apoptosisrelated gene expression for example administrationof the anticancer reagent induced cxcl8 production and the expression of the receptors of cxcl8 cxcr1 andcxcr2 in addition cxcl8mediated chemoresistance to oxaliplatin was demonstrated to be mediated by induction of nfκbtranscription leading to the upregulation of various antiapoptotic genes including bcl2 and survivin conclusionswe showed that cxcl8 expression was upregulated in tissues with liver cancer exogenous administration and overexpression of cxcl8 significantly facilitated to the malignant phenotypes of hepg2 cells by regulating tumorspecificprotein expression including erk12 survivin caspase3 and bax our findings might provide a potential markerand target for the treatment and diagnosis of liver cancerdata availabilitythe supplementary information that accompanies this can be accessed via the corresponding authorcompeting intereststhe authors declare that there are no competing interests associated with the manuscript the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc by 0cbioscience reports bsr20201169101042bsr20201169fundingthe study was financially supported by grant from scientific research project of heilongjiang health and family planning commission [grant number ] and basic scientific research project of provincial colleges and universities in heilongjiangprovince [grant number 2019kyywf1346]author contributionyh designed the study and participated in the revision of the manuscript sy and hw participated in all the experiments andwrote the manuscript cq performed immunohistochemical experiments hs performed cell experiments and data analysisethics approvalthe study was conducted in accordance with the ethical principles for medical research on human beings established by the helsinki protocol and its later amendments or comparable ethical standards and was approved by the institutional ethicscommittee of jiamusi university no jmsu216abbreviationshbv hepatitis b virus hcv hepatitis c virus od optical densityreferences ma x zhuang b and li w microrna2965p downregulated akt2 to inhibit hepatocellular carcinoma cell proliferation migration andinvasion mol med rep “ 103892mmr20176701 bray f ferlay j soerjomataram i siegel rl torre la and jemal a global cancer statistics globocan estimates of incidenceand mortality worldwide for cancers in countries ca cancer j clin “ 103322caac21492 mcglynn ka petrick jl and london wt global epidemiology of hepatocellular carcinoma an emphasis on demographic and regionalvariability clin liver dis “ 101016jcld201501001 zhu rx seto wk lai cl and yuen mf epidemiology of hepatocellular carcinoma in the asiapacific region gut liver “105009gnl15257 hennedige t and venkatesh sk imaging of hepatocellular carcinoma diagnosis staging and treatment monitoring cancer imaging “ 1011021470733020120044 perz jf armstrong gl farrington la hutin yj and bell bp the contributions of hepatitis b virus and hepatitis c virus infections tocirrhosis and primary liver cancer worldwide j hepatol “ 101016jjhep200605013 salazar n castellan m shirodkar ss and lokeshwar bl chemokines and chemokine receptors as promoters of prostate cancer growthand progression crit rev eukaryot gene expr “ 101615critreveukaryotgeneexpr2013006905 karin n chemokines and cancer new immune checkpoints for cancer therapy curr opin immunol “101016jcoi201803004 lazennec g and richmond a chemokines and chemokine receptors new insights into cancerrelated inflammation trends mol med “ 101016jmolmed201001003 chow mt and luster ad chemokines in cancer cancer immunol res “ 10115823266066cir140160 russo rc garcia cc teixeira mm and amaral fa the cxcl8il8 chemokine family and its receptors in inflammatory diseases expertrev clin immunol “ 1015861744666x2014894886 balkwill fr and burke f the cytokine network immunol today “ 1010160167569989900856 balasubramanian a munshi n koziel mj hu z liang tj groopman je et al structural proteins of hepatitis c virus induce interleukin production and apoptosis in human endothelial cells j gen virol “ 101099vir0810560 baggiolini m chemokines and leukocyte traffic nature “ 10103833340 hanahan d and weinberg ra hallmarks of cancer the next generation cell “ 101016jcell201102013 de marzo am deweese tl platz ea meeker ak nakayama m epstein ji et al pathological and molecular mechanisms of prostatecarcinogenesis implications for diagnosis detection prevention and treatment j cell biochem “ 101002jcb10747 li a varney ml and singh rk expression of interleukin and its receptors in human colon carcinoma cells with different metastaticpotentials clin cancer res “ murphy c mcgurk m pettigrew j santinelli a mazzucchelli r johnston pg et al nonapical and cytoplasmic expression ofinterleukin8 cxcr1 and cxcr2 correlates with cell proliferation and microvessel density in prostate cancer clin cancer res “10115810780432ccr041518 jia l li f shao m zhang w zhang c zhao x et al il8 is upregulated in cervical cancer tissues and is associated with the proliferationand migration of hela cervical cancer cells oncol lett “ yuan a yang pc yu cj chen wj lin fy kuo sh et al interleukin8 messenger ribonucleic acid expression correlates with tumorprogression tumor angiogenesis patient survival and timing of relapse in nonsmallcell lung cancer am j respir crit care med “101164ajrccm16252002108 ogura m takeuchi h kawakubo h nishi t fukuda k nakamura r et al clinical significance of cxcl8cxcr2 network in esophagealsquamous cell carcinoma surgery “ 101016jsurg201306013 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc by 0cbioscience reports bsr20201169101042bsr20201169 zhang g gomesgiacoia e dai y lawton a miyake m furuya h et al validation and clinicopathologic associations of a urinebasedbladder cancer biomarker signature diagn pathol 101186s1300001402001 kim sj uehara h karashima t mccarty m shih n and fidler ij expression of interleukin8 correlates with angiogenesistumorigenicity and metastasis of human prostate cancer cells implanted orthotopically in nude mice neoplasia “101038sjneo7900124 moore bb arenberg da stoy k man t addison cl morris sb et al distinct cxc chemokines mediate tumorigenicity of prostatecancer cells am j pathol “ 101016s0002944010654041 bonavia r inda mm vandenberg s cheng sy nagane m hadwiger p et al egfrviii promotes glioma angiogenesis and growth throughthe nfkappab interleukin8 pathway oncogene “ 101038onc2011563 wang y wang w wang l wang x and xia j regulatory mechanisms of interleukin8 production induced by tumour necrosis factoralphain human hepatocellular carcinoma cells j cell mol med “ 101111j15824934201101337x yang ht cohen p and rousseau s il1betastimulated activation of erk12 and p38alpha mapk mediates the transcriptional upregulationof il6 il8 and groalpha in hela cells cell signal “ 101016jcellsig200710025 zhang y wang l zhang m jin m bai c and wang x potential mechanism of interleukin8 production from lung cancer cells aninvolvement of egfegfrpi3kakterk pathway j cell physiol “ 101002jcp22722 wilson c purcell c seaton a oladipo o maxwell pj o™sullivan jm et al chemotherapyinduced cxcchemokinecxcchemokinereceptor signaling in metastatic prostate cancer cells confers resistance to oxaliplatin through potentiation of nuclear factorkappab transcription andevasion of apoptosis j pharmacol exp ther “ 101124jpet108143826 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0c'
Colon_Cancer
" adenovirus serotype ad5 is a commonly used viral vector for transient delivery of transgenes primarily for vaccination against pathogen and tumor antigens however endemic infections with ad5 produce virus specific neutralizing antibodies nabs that limit transgene delivery and constrain target directed immunity following exposure to ad5 based vaccines indeed clinical trials have revealed the limitations that virus specific nabs impose on the efficacy of ad5 based vaccines in that context the emerging focus on immunological approaches targeting cancer self antigens or neoepitopes underscores the unmet therapeutic need for more efficacious vaccine vectorsmethods here we evaluated the ability of a chimeric adenoviral vector ad5f35 derived from the capsid of ad5 and fiber of the rare adenovirus serotype ad35 to induce immune responses to the tumor associated antigen guanylyl cyclase c gucy2cresults in the absence of pre existing immunity to ad5 gucy2c specific t cell responses and antitumor efficacy induced by ad5f35 were comparable to ad5 in a mouse model of metastatic colorectal cancer furthermore like ad5 ad5f35 vector expressing gucy2c was safe and produced no toxicity in tissues with or without gucy2c expression importantly this chimeric vector resisted neutralization in ad5 immunized mice and by sera collected from patients with colorectal cancer naturally exposed to ad5s these data suggest that ad5f35 based vaccines targeting gucy2c or other tumor or pathogen antigens may produce clinically relevant immune responses in more ‰¥ patients compared with ad5 based vaccines inhibitor introductiontherapies immune checkpoint have revolutionized cancer treatment and cancer drug development by engaging the immune system to target various cancers1 despite this success many tumors are immunologically œcold characterized by a dearth of immunogenic neoepitopes3 and lack of tumor infiltrating lymphocytes4 and remain refractory to checkpoint inhibitors6 one emerging strategy to modify a cold tumor into one responsive to immunotherapy is through combination with cancer vaccines8 the goal of this strategy is to use cancer vaccines to create a pool of tumor reactive t cells with antitumor activity alone andor in combination with checkpoint therapies however this approach is significantly limited by the paucity of effective vaccine platforms to safely deliver tumor specificassociated antigens to elicit beneficial antitumor immunitythe ability of adenovirus serotype ad5 to mediate gene transfer and induce potent immune responses has made it a popular vector for experimental vaccines infectious diseases10 against cancer and indeed there have been more than clinical trials using the ad5 vector with most trials focused on developing cancer treatments10 however on natural infection the host immune system develops neutralizing antibodies nabs to the ad5 capsid limiting viral spread and blocking reinfection because ad5 infections are endemic in many human populations pre existing nabs present in of the worldwide population limit ad5 based vaccine strategies12“ these considerations highlight the need for improved vectors for use in vaccines targeting cancer and pathogen associated antigens that can create therapeutic immune responses in the greatest number of patients importantly while the adenovirus capsid is composed of hexon penton and fiber proteins nabs elicited by natural ad5 infection in humans are directed primarily to the ad5 fiber15 suggesting that strategies to flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access circumvent pre existing immunity to this element may improve ad5 based vaccineshere we sought to overcome pre existing ad5 nabs by replacing the ad5 fiber with that of a rare adenovirus serotype ad35 international seroprevalence “ to improve antitumor immunity in mouse models expressing the gastrointestinal gi cancer antigen guanylyl cyclase c gucy2c preclinical models demonstrated that an ad5 based gucy2c directed vaccine ad5 gucy2c s1 elicited cd8 t cell and antibody responses without autoimmunity17 further ad5 gucy2c s1 vaccination of mice induced long term t cell mediated protection against metastatic colorectal cancer in lung and liver19 moreover those results were recapitulated in a recent first in human phase i clinical trial nct01972737 demonstrating that a humanized version of the vaccine ad5 gucy2c padre safely induced gucy2c specific cd8 t cell responses in patients with colorectal cancer following conventional therapies21 however patients possessing high pre existing titers of nabs against ad5 failed to generate gucy2c specific immunity following ad5 gucy2c padre vaccination21 to overcome ad5 nabs we generated a chimeric ad5 vector possessing the fiber of ad35 ad5f35 with equivalent safety and antitumor activity to ad5 and resistance to ad5 nabs in mice and humans this chimeric vaccine can be translated to patients with gi cancer to safely induce gucy2c specific immunity not only in those patients with low ad5 immunity but also in those with high pre existing ad5 nabsmaterials and methodsadenovirus vectorsadenovirus containing mouse extracellular domain gucy2c1429 with the influenza ha107119 cd4 t cell epitope known as site s1 was described previously ad5 gucy2c s120 here gucy2c s1 was cloned into pshuttle and subcloned into the e1 region of previously generated replication deficient chimeric adenovirus ad5f35 in which the ad5 fiber was replaced by the ad35 fiber22 to generate ad5f35 gucy2c s1 all adenovirus vaccines used in this study were produced in hek293 cells and purified by cesium chloride ultracentrifugation under good laboratory practices by the baylor college of medicine in the cell and gene therapy vector development lab and certified to be negative for replication competent adenovirus mycoplasma and host cell dna contamination in vitro gucy2c expression experiments dose“response and time“course were carried out in a549 american type culture collection atcc cells virus was added to the cultures at the indicated doses and culture supernatants were collected at the indicated time points relative gucy2c levels were quantified in supernatants by western blot using μgml ms7 mouse anti gucy2c monoclonal antibody23“ and μgml horseradish peroxidase conjugated goat antimouse secondary antibody jackson immunomice and immunizationseight week old male and female balbcj mice were purchased from the jackson laboratory for experiments animal protocols were approved by the thomas jefferson university institutional animal care and use committee protocol for immunizations mice received or vp of ad5 gucy2c s1 ad5f35 gucy2c s1 or ad5f35 gfp control administered as two μl intramuscular injections one in each hind limb using a ml insulin syringequantifying tcell responses by elispotelispot assays were performed using a mouse interferonÎ ifnÎ single color elispot kit cellular technology according to the manufacturer™s protocol26 briefly well plates were coated with ifnÎ capture antibody overnight at °c the next day plates were washed with phosphate buffered saline pbs and splenocytes from immunized mice were plated at cellswell with no peptide or μgml gucy2c254262 peptide in dimethyl sulfoxide dmso in ctl test medium cellular technology for hours at °c for t cell avidity studies splenocytes were plated at “ cellswell with decreasing concentrations of gucy2c254262 peptide μgml to pgml normalized to cellswell26 after incubation cells were removed and development reagents were added to detect ifnÎproducing spot forming cells the number of spot forming cells per well was determined using the smartcount and autogate functions of an immunospot s6 universal analyzer cellular technology gucy2c specific responses were calculated by subtracting mean spot counts of dmso wells from peptide stimulated wells26 tumor studiesgucy2c expressing mouse balbc ct26 colorectal cancer cells were used for in vivo tumor studies17 luciferase expressing cells were generated by transduction with lentiviral supernatants produced by 293ft cells invitrogen with plenti4 v5 gw luciferase28 for tumor experiments balbcj mice were immunized with vp of ad5 gucy2c s1 ad5f35 gucy2c s1 or pbs control days before delivering × ct26 cells into tail veins tumor burden was quantified weekly by subcutaneous injection of mg of d luciferin potassium salt gold biotechnologies in pbs followed by an min incubation and imaging with a s exposure using a caliper ivis lumina xr imaging station perkinelmer total radiance photonssecond was measured using living image in vivo imaging software perkinelmerantibody neutralization assayserum samples were obtained previously from patients before ad5 gucy2c padre nct01972737 approved by the thomas jefferson university institutional review board21 neutralizing antibody titers against ad5 and ad5f35 vectors were quantified as immunization with flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0cdescribed21 briefly dilutions of heat inactivated serum samples were added to well tissue culture plates containing a549 cells atcc and infected with vp of gfp expressing ad5 or ad5f35 virus ad5 cmv egfp or ad5f35 cmv egfp respectively baylor vector development lab following a hour incubation at °c egfp fluorescence nm excitation nm emission was quantified using a polarstar optimate plate reader bmg labtech sample fluorescence was normalized to control wells containing cells and virus neutralization or wells containing cells alone neutralization titers were quantified using non linear regression as the serum dilution producing neutralization prism v8 graphpad softwaread5 neutralizing immunity studiesto induce anti ad5 immunity mice were exposed intranasally to ad5 gfp once or twice at a week interval thirty days after the last exposure ad5 nabs were quantified in sera as described above and mice were immunized intramuscularly with vp of ad5 gucy2c s1 or ad5f35 gucy2c s1biodistribution and toxicology studybalbcj mice were immunized intramuscularly with a single dose of vp of ad5f35 gucy2c s1 three doses of vp of ad5f35 gucy2c s1 at day intervals or pbs control animals were monitored for adverse events once daily with additional evaluations on the day of dosing min hour and hours after dosing on days and designated animals were sacrificed and brain salivary glands stomach small intestine colon heart lungs kidneys liver and injection site were harvested and weighed for histopathological analysis by a blinded pathologist pathology evaluation was performed by idexx bioanalytics and detection of viral dna by quantitative pcr qpcr using the previously described assay for the gucy2c transgene19 also spleens were collected for histopathological analysis and detection of viral dna as described above as well as quantification of gucy2c specific t cell responses by ifnÎ elispot as described abovestatistical analysisstatistical analyses were conducted using graphpad prism software v8 statistical significance was considered as follows nsp p p p and p cohort sizes were powered based on prior studies with β02 and α005 for multiple comparisons of survival outcomes significance thresholds were corrected using the bonferroni method to identify vaccine induced t cell responders and non responders a previously described21 modified distribution free resampling approach was employed and positive t cell responses were defined as × compared with dmso and specific spots106 cells to determine the impact of gender and number of vaccinations on responses log transformed vaccine response magnitude was compared in mice of different genders cohorts and treatment regimens for up to three way interactions with stepwise backward variable selection by akaike information criterion using r29 package mass30open accessresultsad5gucy2cs1 and ad5f35gucy2cs1 vectorswhile ad5 seroprevalence worldwide exceeds in some regions ad35 is and associated with lower titers figure 1a12 thus we constructed a chimeric adenovirus ad5f35 composed of ad5 in which the fiber was replaced by the ad35 fiber and evaluated its ability to induce gucy2c specific immunity and resist ad5 specific immunity in humans and mice ad5 gucy2c s1 is a replication deficient human ad5 expressing the mouse gucy2c extracellular domain fused to the i ed restricted cd4 epitope known as site at its c terminus20 to generate ad5f35 gucy2c s1 the ad5 fiber l5 was replaced with the ad35 fiber figure 1b replication deficient ad5 gucy2c s1 and ad5f35 gucy2c s1 generated in hek293 cells produced dose dependent figure 1c and time dependent figure 1d expression of gucy2c s1 protein in a549 human alveolar basal epithelial cells in vitroad5f35gucy2cs1 induces gucy2cspecific antitumor immunityfollowing in vitro validation of gucy2c expression by ad5f35 gucy2c s1 we confirmed its ability to induce gucy2c specific immune responses after vaccination in vivo balbc mice immunized intramuscularly with vp of ad5f35 gucy2c s1 produced lower gucy2c specific cd8 t cell responses figure 2a and no gucy2c specific antibody responses figure 2b compared with ad5 gucy2c s1 importantly ad5 and ad5f35 vaccines produced gucy2c specific cd8 t cells of comparable avidity figure 2c a critical determinant of the antitumor efficacy of gucy2c targeted vaccines26 in contrast gucy2c specific antibody responses have no detectable antitumor activity20 similarly ad5 and ad5f35 vaccines produced comparable s1 specific cd4 t cell responses figure 2dluciferase this model previous studies revealed that ad5 gucy2c vaccines induced protective antitumor cd8 t cell responses in murine models of metastatic colorectal cancer17“ thus balbc mice were immunized with ad5 or ad5f35 expressing gucy2c s1 and challenged days later with ct26 colorectal cancer cells expressing gucy2c and firefly specifically emulates secondary prevention of metastatic disease the clinical setting for which the gucy2c vaccine is being developed21 as previously demonstrated ad5 vaccination nearly eliminated metastatic tumor burden figure 3ab delayed disease progression figure 3c and improved survival figure 3d similarly ad5f35 also reduced tumor burden figure 3ab disease progression figure 3c and prolonged survival figure 3d importantly the efficacy of ad5 based and ad5f35 based gucy2c vaccines in flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access figure construction of ad5f35 gucy2c s1 and antigen expression a reported international seroprevalence of ad5 and ad3512 b the l5 gene encoding the fiber protein from ad5 was replaced with the l5 gene from ad35 producing the chimeric adenoviral vector ad5f35 recombinant ad5f35 gucy2c s1 was produced by inserting mouse gucy2c s1 into the e1 region of e1e3 deleted ad5f35 c and d the human alveolar basal epithelial cell line a549 was transduced in duplicate with ad5f35 gucy2c s1 at a multiplicity of infection moi from to for hours c or at an moi of for and hours d supernatants from infected cells were analyzed for gucy2c s1 protein expression by immunoblot protein expression was quantified by densitometry and plotted relative to uninfected cells error bars indicate mean±sem ad5 adenovirus serotype reducing tumor burden opposing disease progression and promoting survival was identical figure 3a“dad5f35 resists ad5directed immunity in mice and humansnabs against ad5 correlated with poor gucy2c specific immune responses in patients receiving ad5 gucy2c padre vaccination and prior exposure of mice to ad5 similarly blunted vaccine induced immunity21 ad5f35 based vaccine resistance to pre existing ad5 immunity was quantified in a model of respiratory pre exposure to ad5 the natural route of infection in patients33 followed by vaccination and quantification of gucy2c specific t cell responses control mice not pre exposed to ad5 naive and those that were pre exposed once × or twice × to intranasal ad5 were vaccinated after weeks with intramuscular ad5 or ad5f35 expressing gucy2c s1 and immune responses were quantified weeks later immunogenicity of ad5 gucy2c s1 and ad5f35 gucy2c s1 a“d balbc mice n4“ micegroup were figure immunized intramuscularly with control or vp of ad5 gucy2c s1 or ad5f35 gucy2c s1 and serum and splenocytes were collected days later gucy2c specific cd8 t cell responses were quantified by interferon gamma ifnÎ elispot a and antibodies were quantified by elisa b c gucy2c specific t cell avidity measurements were analyzed by elispot using non linear regression logagonist versus normalized response with comparisons made using the extra sum of squares f test avidity plots depict the regression line solid with cis dashed d s1 specific cd4 t cell responses were measured by ifnÎ elispot t cell responses in a and d were analyzed by one way analysis of variance values in a b and d indicate individual animals and bars in a and d indicate means tcr t cell receptor ad5 adenovirus serotype flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen accessfigure antitumor efficacy of ad5 gucy2c s1 and ad5f35 gucy2c s1 a“d balbc mice n10 micegroup were immunized intramuscularly with control or vp of ad5 gucy2c s1 or ad5f35 gucy2c s1 and challenged days later with a mouse colorectal cancer cell line ct26 expressing gucy2c and luciferase on days and following challenge mice were injected with d luciferin and imaged a to quantify tumor burden day b mice were weighed twice weekly c and monitored for survival d tumor burden b was analyzed by one way analysis of variance and survival comparisons d were analyzed by the mantel cox log rank test in b and d asterisks indicate comparisons of gucy2c vaccines to the control and brackets ] indicate comparisons between ad5 and ad5f35 vaccines ns not significant ad5 adenovirus serotype figure 4a as expected one ad5 pre exposure induced moderate ad5 nabs online supplementary figure s1 and reduced gucy2c specific t cell responses while two pre exposures induced high ad5 nabs online supplementary figure s1 and reduced gucy2c specific t cell responses following ad5 vaccination figure 4b in contrast gucy2c specific t cell responses were reduced only × pre exposure and × pre exposure following ad5f35 vaccination figure 4b importantly ad5f35 produced t cell responses in a substantially greater fraction of the population cohort responses compared with ad5 cohort responses following serial pre exposures to ad5 figure 4cthese observations in mice were recapitulated using sera from patients with colorectal cancer in the ad5 gucy2c padre phase i trial nct0197273721 here nab titers against ad5 and ad5f35 were quantified using an established ad5ad5f35 reporter virus inhibition bioassay in serum samples collected prior to vaccination with ad5 gucy2c padre21 in these patients ad5f35 specific nab titers were substantially lower than ad5 specific titers figure 4d most importantly of patients possessed low ad5 nabs titers figure 4de which closely correlated with a gucy2c specific response rate21 in striking contrast had low ad5f35 nab titers suggesting that the vast majority of patients immunized with ad5f35 based vaccines could produce gucy2c specific responses figure 4e collectively these observations suggest that pre existing viral immunity induced by repeated environmental exposures which neutralizes ad5 delivery platforms may be overcome by the chimeric ad5f35 vector to enhance fractional population vaccine responsessafety biodistribution and toxicity of ad5f35gucy2cs1food and drug administration ind investigational new drug enabling studies quantified the toxicity biodistribution and immunogenicity of ad5f35 gucy2c s1 in balbc mice employing three schemes to examine acute and chronic effects figure 5a cohorts balanced for sex received ad5f35 gucy2c s1 either as a single intramuscular injection or as three intramuscular injections spaced weeks apart monitored daily and sacrificed on day or for analysis as indicated figure 5a there were no signs of acute or chronic toxicity in the in life phase by observation weight changes or survival figure 5b“d similarly there were no clinically significant differences in organ weights online supplementary figure s2 or histopathology not shown at necropsy small statistical differences in organ weights were considered clinically insignificant and were unrelated to vaccine exposure dose time online supplementary figure s2 biodistribution quantified by qpcr detected ad5f35 gucy2c s1 at the injection site and in the spleen but not appreciably in other organs after acute and chronic exposures online supplementary figure s3 moreover robust cd8 t cell responses were quantified at day that persisted through day in of mice after a single administration figure 5e“g as expected cd8 t cell responses were greater and persisted in more mice at days after three vaccinations figure 5e“gdiscussionthrough decades of gene therapy trials ad5 has remained a popular vector while high ad5 seroprevalence remains a barrier to universal vaccination33 natural respiratory infection can generate long lived antibodies that neutralize ad5 based vaccines eliminating transgene delivery and potential therapeutic benefit in that context ad5 seroprevalence is across multiple countries12 highlighting an unmet need for alternative vectors here we demonstrate that the chimeric ad5f35 resists pre existing ad5 immunity and induces transgene specific antitumor immunity indeed ad5f35 is less susceptible to neutralization associated with ad5 exposure in mice and humans and generates a substantially higher proportion of vaccine responders in mice pre exposed to ad5 these observations support the suggestion that ad5f35 flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access figure ad5f35 resists neutralization associated with pre existing anti ad5 immunity in mice and humans a“c to generate pre existing immunity to ad5 balbc mice n10 micegroup were exposed intranasally once or twice to vp of ad5 gfp at week intervals four weeks after the final ad5 gfp exposure ad5 exposed and naive mice were immunized intramuscularly with vp of ad5 gucy2c s1 or ad5f35 gucy2c s1 b two weeks after immunization gucy2c specific cd8 t cell responses in each group were quantified by interferon gamma ifnÎ elispot and calculated as the of mean responses in naive mice values indicate individual animals and bars indicate means ad5 and ad5f35 were compared by two way analysis of variance c the fraction of animals producing a detectable gucy2c specific cd8 t cell response filled regions in naive × and × ad5 exposed mice was determined from b d and e sera from patients with colorectal cancer collected prior to ad5gucy2c padre vaccination were tested for the ability to neutralize ad5 and ad5f35 vectors and titers were quantified d analyzed by paired t test the dotted line indicates a titer of the threshold for high neutralizing antibody nab titers21 e while subjects had high nab titers against ad5 only had high titers to ad5f35 vector filled regions binomial test ad5 adenovirus serotype will produce a higher proportion of vaccine responders in patient populationsthe extent to which nabs to the ad5 fiber limit reinfection is controversial in some studies replacing the ad5 fiber with that of another serotype circumvents pre existing ad5 immunity34 in contrast other studies suggest that these chimeric adenoviruses do not evade pre existing ad5 nabs suggesting the hexon as the major target of antibody neutralization35 in contrast to those previous studies which generated pre existing ad5 immunity by intramuscular35 or intravenous administration36 here ad5 immunity was induced by intranasal exposure in mice recapitulating natural infection33 moreover natural human respiratory pre existing ad5 nabs in patients with colorectal cancer uniformly produced by repeated respiratory infections33 similarly were overcome by the ad5f35 vector importantly the quality of antibody responses following adenovirus infection is dependent on the route of exposure indeed respiratory infections elicit fiber specific nabs while intramuscular exposure induce capsid specific nabs15 these qualitative differences in nab responses reflecting varying routes of immunization may contribute to observational discrepancies between laboratories the present studies using relevant animal models confirmed and validated with patient samples support the suggestion that ad5f35 based vaccines should produce clinically relevant immune responses in a substantial proportion of patientsflickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen accessfigure safety and immunogenicity of multiple ad5f35 gucy2c s1 administrations a“g balbc mice n10 micegroup were immunized intramuscularly with one or three administrations of vp ad5f35 gucy2c s1 or control at week intervals following immunization body weights b female and c male were recorded weekly and mice were monitored for survival d at days and following first immunization mice were euthanized to quantify organ pathology by weight online supplementary figure s2 biodistribution by quantitative pcr online supplementary figure s3 and gucy2c specific cd8 t cell responses by interferon gamma ifnÎ elispot e“g g pie charts indicate proportion of responding animals ad5 adenovirus serotype recognizing the pervasive limitations imposed by endemic ad5 immunity in global populations12 there is an emerging interest in alternative serotypes and chimeric constructs as a tractable strategy in vaccine development ad26 ad35 and ad48 vectors have been advanced into phase i clinical trials37 in that regard a comparison of ad5 ad26 ad35 and ad48 immunity among healthy patients revealed that endemic ad35 seropositivity was lowest across global populations12 reinforcing chimeric strategies employed herein similarly the first hexon chimeric adenovirus comprising ad5 and ad48 components was safe and immunogenic in patients39 interestingly ad5 ad35 chimeric vectors more efficiently transduce a variety of human cell types in vitro compared with either parental vector40 these observations underscore the future potential of intelligently designed chimeric adenoviruses strategically constructed to deliver transgenes for replacement therapy or vaccination and targeted precisely to the cellular or disease context40while antitumor efficacy was equivalent cd8 t cell responses were lower and antibody responses were absent for ad5f35 gucy2c s1 compared with ad5 gucy2c s1 however the antitumor efficacy of gucy2c directed immunotherapy is driven primarily by t cell avidity rather than effector t cell quantity26 in that context the functional avidity of gucy2c specific cd8 t cells following ad5 and ad5f35 immunizations were equivalent consistent with their comparable antitumor efficacy quantitative differences in transgene specific immunity between vectors may reflect a variety of factors thus the quantity and persistence of gucy2c s1 transgene following ad5f35 immunization is lower compared with ad519 consistent with prior observations that ad5 transduction efficiency in vivo may be several fold higher than ad5f3541 moreover the ad5 fiber binds to cxadr coxsackievirus and adenovirus receptor42 while the ad35 fiber binds to cd4643 suggesting the two viruses may infect distinct cell typesflickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access while checkpoint inhibitors have generated practice shifting results in the clinic and defined immunotherapy as an effective strategy for the treatment of several malignancies they have not been universally successful in that context the dearth of neoepitopes in many cancer types including microsatellite stable colorectal and pancreatic second and third leading causes of cancer mortality respectively makes them insensitive to checkpoint blockade7 indeed examination of neoepitopes presented on the surface of five colorectal cancer specimens revealed a total of three neoepitopes3 thus vaccines targeting cancer associated self antigens have re emerged alone and in combination with checkpoint inhibitors as a strategy to prevent and treat metastases from these cold tumors44 checkpoint inhibitors have become first line therapy in the metastatic setting for some cancers46 while chimeric antigen receptor expressing t cells car t cells are being deployed in patients with metastatic and refractory disease47 in contrast few cancer immunotherapies have been developed for early stage cancer patients with œno evidence of disease ned following conventional surgicalradiochemotherapies who are at significant risk of disease recurrence indeed25 of stage ii and of stage iii patients with colorectal cancer recur following surgery and chemotherapy49 while of patients with resectable pancreatic cancer experience recurrence50 vaccines targeting tumor associated antigens such as ad5f35 gucy2c padre may provide safe and effective immunotherapies for the secondary prevention of metastatic disease in patients with ned who are otherwise ineligible to receive checkpoint inhibitors or car t cellsthe present studies suggest that the chimeric adenoviral vector ad5f35 may be preferable to the widely used ad5 vector and warrants further investigation indeed they suggest that ongoing clinical investigations of gucy2c directed immunotherapy in patients with gucy2c expressing cancers including colorectal pancreatic gastric and esophageal could benefit from using the ad5f35 rather than the ad5 vector in that context an upcoming clinical trial will examine the safety immunogenicity and resistance to pre existing immunity of ad5f35 gucy2c padre in patients with gi cancer nct04111172 safe generation of gucy2c targeted immunity in a high proportion of patients will lead to efficacy trials to establish the ability of ad5f35 gucy2c padre to prevent recurrence following standard therapy in patients with gi cancer who represent of all cancer deaths51 and for whom established immunotherapies are ineffectivetwitter adam e snook adamsnookphdacknowledgements the authors thank adrian p gee phd zhuyong mei md deborah lyon and malcolm brenner md phd center for cell and gene therapy baylor college of medicine for assistance in vaccine manufacturingcontributors jcf js bb saw and aes designed the studies jcf js rc el trb jb ec ap jar and jr carried out the studies tz carried out data analysis and statistical analysis in discussion with aes jcf and aes wrote the manuscript and all authors critically reviewed and approved the final version of the manuscriptfunding this work was supported by the national institutes of health nih r01 ca204881 r01 ca206026 and p30 ca56036 the defense congressionally directed medical research program w81xwh17 prcrp ttsa and targeted diagnostic therapeutics to saw aes received a research starter grant in translational medicine and therapeutics from the phrma foundation a degregorio family foundation award and was supported by the defense congressionally directed medical research programs nos w81xwh1710299 w81xw
Colon_Cancer
posterior reversible encephalopathy syndrome pres is a clinical syndrome that can include headache altered consciousnessvisual disturbances and seizures usually related to autoregulatory cerebral failure and hypertension the neuroimaging isessential to diagnosis showing white matter vasogenic edema in posterior areas we present a case of a 66yearold womanwith severe pneumonia by sarscov2 who developed a posterior reversible encephalopathy syndrome with a typical clinicaland radiological presentation after being treated with antiinterleukin treatment anakinra and tocilizumab following localguidelines we report a case of posterior reversible encephalopathy syndrome in a patient with covid19 disease possiblyrelated to antiil1 or antiil6 suggesting that antiinterleukin treatments may cause this syndrome at least in patients withpredisposing conditions such as infections and hydroelectrolytic disorderskeywords posterior reversible encephalopathy syndrome pres covid19 anakinra tocilizumab immunomodulatorsintroductiona 66yearold woman with covid19 presented with adultrespiratory distress syndrome ards besides bilateralpneumonia she developed multiple complications suchas cardiorespiratory arrest bacterial superinfectionhyponatremia massive hemoptysis requiring embolizationand acute renal injury she was started on lopinavirritonavirhydroxychloroquine and azithromycin after radiological pulmonary progression antiil1 daily anakinra and antiil6single dose of tocilizumab were started following local andhospital guidelines these drugs are recommended incovid19 when there is clinical blood test or radiologicalprogression to avoid an excessive immunological systemicthis is part of the topical collection on covid19 laura llansllansocliniccatxabi urraxurracliniccat department of neurology hospital cl­nic c villarroel barcelona catalonia spain comprehensive stroke center department of neurosciencehospital cl­nic august pi i sunyer biomedical research instituteidibaps barcelona catalonia spainresponse to the virus which is thought to worsen pulmonaryinfiltrates and disease prognosis ten days after the initiationof immunodepressants she developed altered mental statuswithout fever previous headache or visual disturbancescase presentationat the examination the patient opened eyes to painful stimulihad no verbal response and showed withdrawal response topain glasgow coma scale the blood tests showed stablehyponatremia meql and leukocytosis without any other significant findings her vitals were within normal limitsand blood pressure had been mildly increased during the previous h with a maximum systolic pressure of mmhgelectrocardiogram showed sinus rhythm and had not atrioventricular node blocks a ct scan with angiography was performed there was no large vessel occlusion no perfusionalterations and the baseline ct fig showed temporooccipital white matter hypodensity with symmetric obliteration of the sulci in that regionconsidering the infectious the immunomodulatory treatments modest hypertension in the hours before thesymptoms and the distribution of the lesions on the ct scanthe most likely diagnosis is posterior reversible encephalopathy syndrome pres [“] hypertension plays a vital role in 0cfig ct baseline scan showedtemporooccipital symmetricwhite matter hypodensitysn compr clin medthe disease due to a failure in cerebral blood flow autoregulation and in this case rapid rise or fluctuations in blood pressure from baseline may have been harmful despite not beingseverely high the electroencephalogram showed focalslowing and epileptiform discharges in both occipital areasand ruled out nonconvulsive status epilepticus the symptoms improved over the following days after tight controlof blood pressure with labetalol infusion and discontinuinganakinraafter week radiological infiltrates worsened and a bloodtest showed increased acute phase reactants in this context adiffuse alveolar hemorrhage was diagnosed by bronchoalveolar lavage with suspicion of hemophagocytic syndrome sherequired red blood cell transfusions and immunosupressantswere restarted as well as mechanical ventilation the ct pulmonary scan showed worsening of infiltrates and presence ofan intrapulmonary hematoma finally the patient had a torpidevolution to multian failure and deathsthe absence of fever and radiological findings did not suggestan encephalitic cause of the symptoms the serum sodiumlevels were only slightly decreased and had been stable forthe previous days without intravenous infusion of sodiumthus hyponatremia was not assumed to be the cause of thesudden loss of consciousness the cardiorespiratory arresthappened a month before the event while intubated and itwas secondary to a mucus plug after a period of desaturationand bronchospasm it lasted less than min and ended as themucus plug was removed by fibrobronchoscopy the postanoxic cerebral damage was prevented by treating feveravoiding systemic hypotension hypoxemia or glycemicdisbalance and continuing renal replacement therapy withhemodiafiltration instead of hemodialysis to prevent largechanges in volemia one week later sedation was stoppedand a tracheostomy was placed and the patient progressivelyawakened up to a normal state of consciousness without focalneurologic signs the timeline and posterior completerecovery from the respiratory arrest cannot explain the currentepisode as hypoxicischemic encephalopathypres has been associated with immunosuppressive andcytotoxic therapies such as platinumcontaining drugs rchop regimens gemcitabine cyclosporine tacrolimussirolimus and interferon therapies also agents that targetangiogenesis such as bevacizumab antivegf and tyrosinekinase inhibitors tki against vegf receptor pazopanibsorafenib sunitinib have been described as risk factors prior exposure to the predisposing drug does not appearto be protective and patients can develop pres even afterseveral months after exposure furthermore the disorderhas been associated with both acute and chronic renal diseaseas was the case in our patient and medical conditions such ashyponatremia or pulmonary infection could exacerbate theneurological findingsdespite not being described yet the occurrence of pres afew days after antiinterleukin il6 or il1 treatments whichwere given in this patient raises the possibility that these kindsof immunomodulatory agents may also favor prescompliance with ethical standardsconflict of interest the authors declare that they have no conflict ofinterestethical approval and informed consent consent for publication wasobtained from the next of kin daughter approval from the hospital™sirb was provided for this studyreferences fugate je claassen do cloft hj kallmes df kozak osrabinstein aa posterior reversible encephalopathy syndrome associated clinical and radiologic findings mayo clin proc “ gao b lyu c lerner a mckinney am controversy of posteriorreversible encephalopathy syndrome what have we learnt in the last years j neurol neurosurg psychiatry “ 0csn compr clin med allen ja adlakha a bergethon pr reversible posteriorleukoencephalopathy syndrome after bevacizumabfolfiriregimen for metastatic colon cancer arch neurol “ito y arahata y goto y cisplatin neurotoxicity presenting asreversible posterior leukoencephalopathy syndrome ajnr am jneuroradiol schwartz rb jones km kalina p bajakian rl mantello mtgarada b hypertensive encephalopathy findings on ctmr imaging and spect imaging in cases ajr am jroentgenol “publisher™s note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c'
Colon_Cancer
‚ammation is an established risk factor for colorectal cancer we and others have shown that colorectal cancerpatients with elevated cysteinyl leukotriene receptor cyslt2r and 15hydroxyprostaglandin dehydrogenase15pgdh levels exhibit good prognoses however both cyslt2r and 15pgdh which act as tumour suppressorsare often suppressed in colorectal cancer we previously reported that leukotriene c4 ltc4induced differentiationin colon cancer via cyslt2r signalling here we investigated the involvement of hedgehog hh“gli1 signallingwhich is often hyperactivated in colorectal cancer we found that the majority of colorectal cancer patients hadhighgli1 expression which was negatively correlated with cyslt2r 15pgdh and mucin2 and overall survivalcompared with the lowgli1 group ltc4induced 15pgdh downregulated both the mrna and protein expressionof gli1 in a protein kinase a pkadependent manner interestingly the ltc4induced increase in differentiationmarkers and reduction in wnt targets remained unaltered in gli1knockdown cells the restoration of gli1 in15pgdhknockdown cells did not ameliorate the ltc4induced effects indicating the importance of both 15pgdhand gli1 ltc4mediated reduction in the dclk1 and lgr5 stemness markers in colonospheres was abolished incells lacking 15pgdh or gli1 both dclk1 and lgr5 were highly increased in tumour tissue compared with thematched controls reduced mucin2 levels were observed both in zebrafish xenografts with gli1knockdown cellsand in the cysltr2ˆ’ˆ’ colitisassociated colon cancer cac mouse model furthermore gli1 expression waspositively correlated with stemness and negatively correlated with differentiation in crc patients when comparingtumour and mucosal tissues in restoring 15pgdh expression via cyslt2r activation might benefitcolorectal cancer patientsintroductioncolorectal cancer crc one of the most prevalentcancers in the world has a high metastatic efficacy and alow 5year survival rate1 a nontargeted therapeuticapproach combined with late diagnosis leads to poorprognosis and treatment failure more than of crccorrespondence anita sj¶lander anitasjolandermedluse1cell and experimental pathology department of translational medicine lunduniversity sk¥ne university hospital malm¶ sweden2chemical biology therapeutics group department of experimental medicalscience lund university lund swedenfull list of author information is available at the end of the intracellular mechanismscases exhibit anomalous apcwntcatenin signallingwhich regulates the progression of crc by adopting differentthus affecting cancerstem cells and interactions with the tumour microenvironment hedgehog hh signalling which regulatesdifferentiation under physiological conditions has attracted attention because of its emerging role in the promotion and maintenance of crc2“ in the untransformedcolon hh ligands are secreted by epithelial cells targetingmesenchymal cells as a classic paracrine hh signallingpathway to ensuring the proper size and location of thecrypt“villus axis5 as also observed in other tissues6 the authors open access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate ifchanges were made the images or other third party material in this are included in the ™s creative commons license unless indicated otherwise in a credit line to the material ifmaterial is not included in the ™s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this license visit httpcreativecommonslicensesby40oncogenesis 0csatapathy oncogenesis page of in crc abnormal hh signalling functions in a liganddependent manner and is activated in human cc celllines7 and xenograft models4 however the role of hhsignalling and its importance in cell survival in crc arenot well defined although some previous studies havefailed to derive a positive correlation between hh signalling and crc initiation and maintenance89 major bodiesof evidence point to a positive correlation347 moreoverprevious reports have suggested high activity ofthehh“smo“gli axis in crc cell survival and metastasiswhich is coordinated by either canonical signalling viasmo or a noncanonical mode of activation via therasmap kinase pathway47 within these pathways themost prominent factors are gliomaassociated oncogenehomologue gli and and the transcriptional regulators downstream of smo which keep the oncogenicpathway activecrc which is considered to be an ‚ammationassociated cancer is greatly ‚uenced by ‚ammatorymediators such as leukotrienes and prostaglandins whichbelong to the gproteincoupled receptor family thecysteinyl leukotriene receptors cysltrs cyslt1r andcyslt2r are activated by binding with their highaffinityligands leukotriene d4 and c4 ltc4 respectively1011these pro‚ammatory lipid mediators are derived fromthe arachidonic acid pathway via 5lipoxygenase and theyplay crucial roles in pathological ‚ammation such asthat observed in ‚ammatory bowel disease we previously showed that elevated cyslt1r levels were associated with poor prognoses in crc patients whilepatients with high cyslt2r expression had better prognoses12 another important group of eicosanoids areprostaglandins pgs which are produced via the cox2pathway the upregulation of cox2 in crc increasesthe pge2 level which promotes cancer cell proliferationangiogenesis survival migration and invasion these areimportant hallmarks of cancer1314thetumoursuppressor15hydroxyprostaglandindehydrogenase 15pgdh is an enzyme responsible forthe degradation of pge2 into an inactive metabolite15 pgdh is abundantly expressed in normal colon mucosabut its expression is lost in crc cells1617leading todisease progression recently researchers have exploredthe efficacy of 15pgdh as a potential antitumour agentagainst colon cancer18“ in a recent study we established that 15pgdh is induced by ltc4 via cyslt2rsignalling by phosphorylating cjun nterminal kinaseand ap1 to induce 15pgdh promoter activity andfurther guide colon cancer cells toward redifferentiation20however the detailed mechanism underlying this phenomenon remains unclearin this study we elucidated the mechanism by whichltc4induced 15pgdh promotes differentiation incolon cancer cells through cyslt2r activation with theoncogenesisinvolvement of hh“gli signalling we observed thatgli1 was involved in the regulation of the redifferentiation and reduction in stemness induced by ltc4 via pgdh in colon cancer cellsresultsgli1 expression is negatively correlated with cyslt2r pgdh and mucin2 expression in crc patientsto elucidate the regulatory activity of gli1 on theantitumorigenic proteins cyslt2r and 15pgdh and thedifferentiation marker mucin2 in colon cancer we used atissue microarray tma of primary crcs from patients22 after ihc analysis we found only five patientswith negative gli1 staining patients with weak stainingintensity patients with moderate staining intensityand patients with strong staining intensity the mean ±standard deviation sd of the immunoreactive score irsfor gli1 expression was ± then we grouped thepatients with negative and weak staining intensity anddefined them as the lowgli1 expression group n and those with moderate and strong staining intensitywere defined as the highgli1 expression group n fig 1a seventy patients had missing or incomplete coresand were excluded from the final analysisafter ihc evaluation of cyslt2r and 15pgdh weobserved that patients with highgli1 expression hadsignificantly lower levels of cyslt2r irs ± and15pgdh irs ± expression than those with lowgli1 expression fig 1b“d furthermore there was asignificant negative correlation between gli1 and pgdh r ˆ’ p suggesting hyperactivatedhh“gli signalling with suppressed 15pgdh fig 1ehowever no significant correlation was found betweengli1 and cyslt2r expression fig 1eexhibitedhighgli1on the other hand patients with lowgli1 expressionhad a significantly higher irs for cyslt2r ± and15pgdh ± expression fig 1c d which indicates the adverse effects of cyslt2r15pgdh axis activation on gli1 we also noticed that the majority ofpatientspatients and when stratified according to the tumournodemetastasis tnm staging a significantly strongerassociation for the patients with tnm stages iii and ivwas found fig 1e in addition to the above observationstranscriptome data from a public database23 also suggested a significant negative correlation between gli1 andthe tumour suppressors cysltr2 fig 1f and hpgd15pgdh fig 1gexpressionimportantly we observed that patients with lowgli1expression had a lower risk of overall mortalityhazard ratio ci “ than patientswith highgli1 expression after adjusting for age andtnm stage fig 1h the unadjusted survival curve isprovided in supplementary fig s1a the median follow 0csatapathy oncogenesis page of fig see legend on next pageoncogenesis 0csatapathy oncogenesis page of see figure on previous pagefig gli1 expression exhibited a negative correlation with cyslt2r and 15pgdh expression in colorectal cancer patient tissuesa matched pair immunohistochemistry ihc images of gli1 cyslt2r and 15pgdh expression in patients with low and highgli1 expressionshown at — magnification the staining immunoreactivity was quantified by the mean immunoreactive score irs calculated according to thefollowing formula irs staining intensity — of stained cells the mean irs for the groups of patients with low and highgli1 expression b theyaxis represents irs for gli1 in these patients c cyslt2r and d 15pgdh expression according to patients with low and highgli1 expression in crctissue e distribution of tumournodemetastasis tnm stages of crc according to low and highgli1 expression pairwise pearson correlationcoefficient r between the expression of gli1 and that of cyslt2r and 15pgdh p value according to chisquare test xyscatter plots showing mrnalevels of f gli1 and cyslt2r and g gli1 and 15pgdh hpgd gene expression from a public database containing crc patients kaplan“meiercurves for overall survival adjusted for age and tnm stage for patients with h low and highgli1 expression and subgroups of patients with bothgli1 and i cyslt2r or j 15pgdh expression compared by the logrank test the last patient group served as the reference category k western blotanalysis showing the protein expression of cyslt2r 15pgdh and gli1 in matched pairs of six patients with normal n and tumour t areas kdaindicated on the left side of the immunoblots kda represents protein size markers graphical representation of the densitometricanalysis showing the relative protein expression for cyslt2r 15pgdh and gli1 in matched pairs of patient samples n from normal mucosa mwhite and tumour t black areas l qrtpcr analysis of gli1 in matched pairs of normal mucosa m white and tumour t black tissues from crcpatients n scale bar as indicated in the images data represent the mean ± sd p p p mann“whitney testup time was months years with total eventsthisrole of gli1 in coloncarcinogenesissuggests a prominentwe have previously shown that crc patients with lowcyslt2r andor low15pgdh expression have a poorprognosis1220 in this study we noted that patients withhighgli1 expression coupled with either lowcyslt2rexpression orlow15pgdh expression had poorerprognoses than patients with either lowgli1 and low15pgdh expression orlowgli1 and lowcyslt2rexpression fig 1i j western blot analysis of six matchedpairs of crc patients showed significantly higher proteinexpression of cyslt2r and 15pgdh in normal tissuethan in matched tumour tissue fig 1k howevergli1 showed elevated expression in tumour tissue compared with matched normal tissue fig 1kfurthermorethe mrna analysis ofthese pairedtumour tissues with matched mucosa tissues from crcpatients n showed significantly higher mrnaexpression of gli1 in the tumour tissue compared with itsmatched normal mucosa the normal mucosa as referenceset to and tumour tissue ± mean ±sem mann“whitney test p fig 1l furthermore the muc2 mucin2 mrna analysis of thesepaired tumour tissues t and matched normal mucosam showed significantly lower mrna expression ofmuc2 in the tumour tissue compared with its matchednormal mucosa the normal mucosa as reference set to and tumourtissue ± mean ± semmann“whitney test p fig 2a a representativematched pair of high and lowgli1 and the corresponding mucin2 is shown fig 2b we observed thatpatients with highgli1 expression had lower levels ofmucin2 expression than those with lowgli1 expressionfig 2c and by combining these data a significantnegative correlation between elevated expression of gli1and decreased expression of mucin2 in tumour tissueswas found fig 2d moreover grouping the patients intooncogenesismucinous n and nonmucinous n typesrevealed that of patients with nonmucinous status had highgli1 expression suggesting anegative correlation with mucin2expressing cells while of patients in the mucinous category showedhighgli1 expression fig 2d furthermore we found asignificant negative correlation between gli1 and thedifferentiation marker mucin2 expression in these crcpatients n fig 2e we found a better overallsurvival for patients with lowgli1 than those with highgli1 expression regardless of mucin2 expressionfig 2e taken together these results suggest that gli1expression is negatively correlated with cyslt2r pgdh and mucin2 expression but positively with themucinous status of the patientscyslt2r is essential for differentiation in a colitisassociated colon cancer”cac”mouse modelto further validate the role of cyslt2r in promotingdifferentiation in crc we adopted an ‚ammatorymouse model that was induced by azoxymethane aomand dextran sodium sulfate dss briefly c57bl6nwildtype and cysltr2ˆ’ˆ’ mice were subjected to aomand two dss cycles24 fig 2f as described in thematerials and methods section we found that all miceformed polyps in the colon regardless the phenotype butcysltr2ˆ’ˆ’ mice n developed significantlargerpolyps ‰¥ mm p in the colon compared withtheir wildtype wt n littermates supplementaryfig s1b c this result indicates that cysltr2ˆ’ˆ’ micedevelop a more progressive disease supplementary figs1 shows a representative image of colon polypsfig s1d and a likewise representative haemotoxylinand eosinstained imageshowing premalignant areas aberrant cryptfoci and metaplasiaareas from both wt and cysltr2ˆ’ˆ’ mice colonfig s1ecolon tissue sections from wt mice showed abundantmucin2expressing cells compared with sections from 0csatapathy oncogenesis page of fig see legend on next pageoncogenesis 0csatapathy oncogenesis page of see figure on previous pagefig gli1 expression was negatively correlated with differentiation a qrtpcr analysis of mucin2 in matched pairs of normal mucosa mwhite and tumour t black tissues from crc patients n b immunohistochemistry ihc images for gli1 and mucin2 expression in matchedpaired tissue samples from colorectal cancer crc patients with low and highgli1 expression represented at — magnification c quantification ofthe staining immunoreactivity by the mean irs for mucin2 expression according to patients with low and highgli1 expression in crc tissued distribution of tumour type mucinous adenocarcinomas versus nonmucinous adenocarcinomas according to low and highgli1 expressionpairwise pearson correlation coefficient r between the expression of gli1 and mucin2 p value according to chisquare test e kaplan“meier curvesfor overall survival adjusted for age and tnm stage subgroups of patients with both gli1 and mucin2 expression compared by the logrank test thehighgli1 and lowmucin2 patient group was set as the reference category f experimental schematic of the aom“dss mouse model gli1expression exhibited a negative correlation with differentiation in the cysltr2ˆ’ˆ’ aom“dss mouse model immunohistochemical evaluation showingthe protein expression of g mucin2 and h gli1 in wt and cysltr2ˆ’ˆ’ aom“dsschallenged mice graph bars showing the irs scores for mucin2 andgli1 respectively compared between wt and cysltr2ˆ’ˆ’ n micegroup scale bar as indicated in the images data represent the mean ± sdp mann“whitney testcysltr2ˆ’ˆ’ mice ± n fig 2g gli1 exhibited aslightly higher but nonsignificant overall expression incysltr2ˆ’ˆ’ mouse tissue sections compared with wt tissuesections ± n fig 2h taken together theabove observations encouraged us to further investigatehh“gli signalling using both in vitro and in vivo coloncancer model systems and to delineate the involvement ofcyslt2r and 15pgdh in promoting differentiationltc4induced 15pgdh downregulates gli1 in coloncancer cellswe determined whether hh“glisignalling wasinvolved in the ltc4induced 15pgdhpromoted differentiation of cc cells20 interestingly ltc4 stimulationsignificantly downregulated gli1 expression at both themrna and protein levels compared with unstimulatedht29 and caco2 cells fig 3a b in addition immunofluorescence analysis of gli1 expression revealed adecrease in nuclear gli1 upon ltc4 stimulation fig 3cto determine the mechanism of 15pgdhmediateddepletion of gli1 we examined the expression of proteinkinase a pka which is a known gli1 antagonist2526the activation of the pka αδ catalytic subunit incaco2 cells after stimulation by ltc4 was significantlyincreased as indicated by the levels of phosphorylatedpka threonine but remained unchanged in stimulated ht29 cells fig 3b moreover phosphorylation atserine on the catalytic subunit of pka wasincreased after ltc4 stimulation in ht29 cells but wasnot present in caco2 wholecell lysates fig 3b15pgdhspecific shrna shhpgd was employed toinvestigate the involvement of 15pgdh in the ltc4mediated downregulation of gli1 expression comparedwith cells transfected with control shrna shctrl cccells transfected with shhpgd showed no response toltc4 stimulation we observed that shrnamediatedknockdown of hpgd did not affect the expression of theintestinal differentiation markers cdx2 or cdhr2 or thewnt target axin2 supplementary fig s2a“d for ht29cells and unaltered gli1 expression at both the mrnaoncogenesisand protein levels fig 3d“f see supplementary figs3a“f for caco2 cells similarly 15pgdh knockdownreduced the effect of ltc4 stimulation on pka activationin both ht29 and caco2 cells fig 3e supplementaryfig s3fto further confirm the role of pka as an intermediatemolecule in ltc4induced 15pgdhmediated downregulation of gli1 we used h89 a pkaspecific inhibitor nm prior to stimulating the cells with ltc4 wefound that neither ht29 nor caco2 cells treated withthe pka inhibitor affected ltc4induced 15pgdhexpression at either the mrna or protein levels however no significant alteration was observed in either themrna or protein level of gli1 poststimulation withltc4 fig 3g h supplementary fig s4a b which wasalso supported by immunofluorescence analysis fig 3isupplementary fig s4c these data indicate that pkaplays a role in the 15pgdhmediated downregulation ofgli1 in colon cancer cellsgli1 regulates 15pgdhpromoted differentiation in coloncancer cellsnext we determined the mechanism by which ltc4induced 15pgdh promoted differentiation in coloncancer cells20 based on the above evidence we investigated the effects of gli1 knockdown fig 4a“j for ht29cells and supplementary fig s5a“k for caco2 cells onhpgd 15pgdh expression we used shgli1 todetermine possible changes in the expression of pgdh however gli1 knockdown in these cells did notaffect ltc4induced 15pgdh expression at either themrna or protein level compared with the correspondingshctrltransfected cells these data indicate that theeffect of ltc4 signalling on 15pgdh expression isindependent of gli1 suggesting that gli1 is downstreamof 15pgdh we next validated the involvement of gli1in differentiation by testing the mrna expression of sisucrase“isomaltase and muc2 mucin2 which arerepresentative intestinal differentiation markers followingltc4 stimulation the observed increases in the mrna 0csatapathy oncogenesis page of fig 15pgdh regulates the ltc4mediated downregulation of hh“gli signalling in colon cancer cells a qrtpcr analysis of gli1 mrnaexpression in ht29 and caco2 cells with or without ltc4 stimulation for h b western blot analysis of 15pgdh gli1 and phosphopka αγ subunitand subunit levels in ht29 and caco2 cells with or without ltc4 stimulation αtubulin served as the loading control c immunofluorescence analysisof gli1 in ht29 cells with or without ltc4 stimulation for h d qrtpcr analysis of ht29 cells transfected with control shrna shctrl or pgdhspecificshrna shhpgd with or without ltc4 stimulation for h e western blot analysis of ht29 cells transfected with control shrna shctrl or pgdhspecificshrna shhpgd blotted with antibodies against 15pgdh gli1 or phosphopka αγ subunit and subunit with or without ltc4 stimulation for hαtubulin served as the loading control f immunofluorescence analysis of gli1 in ht29 cells transfected with control shrna shctrl or pgdhspecificshrna shhpgd with or without ltc4 stimulation for h g qrtpcr analysis of 15pgdh and gli1 in ht29 cells treated with the pka inhibitor h89 pkainh for h followed by ltc4 for h h western blot analysis showing the expression of 15pgdh gli1 and phosphopka αγ subunit and subunit inht29 cells treated with the pka inhibitor h89 pkainh for h followed by ltc4 for h i immunofluorescence analysis of gli1 in ht29 cells treatedwith the pka inhibitor h89 pkainh for h followed by ltc4 for h hprt1 was used as the housekeeping gene for normalisation of the qrtpcr geneexpression data graphs represent the mean ± sem of data from to independent experiments p p oncogenesis 0csatapathy oncogenesis page of fig 15pgdh promotes differentiation in colon cancer via gli1 qrtpcr validation of gene expression in shctrl control shrna or shgli1gli1specific shrnatransfected ht29 cells with or without ltc4 stimulation for h marker of tumour suppression in a 15pgdh markers ofdifferentiation in b si in c mucin2 and in d cdhr2 marker of differentiation regulation as in e cdx2 markers of wnt activation in f axin2 andg cmyc marker of proliferation in h ccnd1 hprt1 was used as the housekeeping gene for normalization of the gene expression data i western blotanalysis of wholecell lysates for 15pgdh gli1 si cdx2 and cdhr2 expression in shctrl control shrna or shgli1 gli1specific shrnatransfected ht29 cells with or without ltc4 stimulation for h αtubulin served as the loading control j immunofluorescence analysis of gli1 andmucin2 in cells with or without ltc4 stimulation for h and transfected with shctrl or shgli1 graphs represent the mean ± sem of data from threeindependent experiments p p p oncogenesis 0csatapathy oncogenesis page of and protein expression of si after ltc4 stimulation inboth ht29 fig 4b and caco2 cells supplementaryfig s5c were abolished in gli1knockdown cellsfig 4b supplementary fig s5c j furthermore similarresults were observed for the mrna expression of otherdifferentiation markers such as muc2 fig 4c supplementary fig s5d and for both the mrna and proteinexpression of cdhr2 and cdx2 fig 4d e supplementary fig s5e f j ltc4 induced a reduction inaxin2 a potential wnt signalling target fig 4f supplementary fig s5g furthermore myc and ccnd1mrna expression was also abolished in gli1knockdowncells exposed to ltc4 fig 4g h supplementary fig s5hi to further validate the above observations we performed western blot fig 4i and immunofluorescencemicroscopy using double staining for gli1 and mucin2in shctrl and shgli1transfected cells and found thatmucin2 expression was downregulated in cells lackinggli1 fig 4j supplementary fig s5k these findingssupport our hypothesis of a possible regulatory effect ofgli1 in ltc4induced 15pgdhpromoted differentiation in colon cancer cellsgli1 suppresses differentiation in the absence of 15pgdhwe next investigated the ability of gli1 to regulate pgdh by overexpressing gli1 using the pegfphgli1construct in combination with the simultaneous knockdown of hpgd with shhpgd supplementary figs s6ab s7a b we found significant downregulation in theltc4induced increase in si muc2 cdhr2 and cdx2mrna expression levels in ht29 cells fig 5a“d andalso in caco2 cells supplementary fig s7c“f thatoverexpressed gli1 and lacked 15pgdh compared withcells expressing the control vector shctrl we alsofound that the ltc4induced reduction in axin2 mycand ccnd1 mrna was abolished although we observedincreased basal levels of these mrnas fig 5e“g supplementary fig s7g“i we next investigated the proteinexpression of si by western blot fig 5h supplementaryfig s7j and mucin2 by immunofluorescence microscopy fig 5i supplementary fig s7k the ltc4induced increase in si was abolished in hpgdknockdown and gli1overexpressing cells fig 5h supplementary fig s7j a similar pattern was found regardingmucin2 protein expression in these cells which wasobserved using immunofluorescence microscopy fig 5isupplementary fig s7k finallythe ltc4inducedreduction in gli1 was abolished fig 5iltc4induced cyslt2r signalling downregulates gli1 in a15pgdhdependent mannerto determine whether ltc4 acts via cyslt2r as ltc4is the highaffinity ligand of cyslt2r we investigated thespecific involvement of cyslt2r signalling in the ltc4oncogenesisstableofknockdownmediated downregulation of gli1 in colon cancer cellswe first constructed hct116 cells with doxycyclinedoxinduciblecyslt2rshcysltr227 we examined the mrna and proteinexpression levels of cyslt2r 15pgdh and gli1 withand without ltc4 stimulation in this cell line with doxinduction and compared them with the levels in cellsgrown in the absence of dox the mrna expression ofcysltr2 and hpgd wassignificantly upregulatedfig 6a b while gli1 mrna expression was downregulated in cells cultured without dox fig 6c howin the doxinduced cells gli1 gene expressioneverremained unaltered most likely due to the low mrnaexpression levels of cysltr2 and hpgd these observations were also reflected in the levels of proteinexpression fig 6d supplementary fig s8a“cto further study the involvement of cyslt2r we treatedht29 and caco2 cells with the cyslt2rspecificantagonist ap100984 µm followed by ltc4 stimulation for h20 ap100984 treatment efficiently blocked boththe mrna supplementary fig s8d e and proteinexpression supplementary fig s8f of cyslt2r as well asof its downstream signal 15pgdh in ht29 cells as well asin caco2 cells supplementary fig s8g“j ap100984 pretreatment abolished the effect of ltc4 stimulation on gli1at both the mrna and protein levels fig 6e f supplementary fig s8i j taken together the above resultsshowed that cyslt2r signalling plays a role in the ltc4induced 15pgdhmediated downregulation of gli1ltc4induced 15pgdh expression reduces stemness incolonosphereswe extended our study to determine whether ltc4induced 15pgdh affected stemness in colon cancer cellsas well as the possible role of gli1 we created a 3dmodel of multicellular colonospheres derived from coloncancer cells fig 6g we observed that shctrltransfected ht29 or caco2 cellderived colonospheresshowed reduced numbers and sizes with ltc4 stimulation fig unlike the shctrl group shhpgdtransfected cellderived colonospheres showed increases innumber and size howeverthe absence of gli1 inshgli1transfected cellderived colonospheres resulted inno significant alteration in size or number even afterltc4 stimulation the mrna expression levels of thecolon cancerspecific stemness markers dclk1 lgr5and aldh1a1 were elevated in ht29 as well as in caco cellderived colonospheres and were downregulatedafter ltc4 stimulation fig 6i supplementary figs9a“h the mrna and protein levels of the cancer stemcell markers dclk1 and aldh1a1 remained unchangedin both shhpgd and shgli1knockdown ht29 andcaco2 cellderived colonospheres even after ltc4 stimulation compared with their unstimulated counterparts 0csatapathy oncogenesis page of fig gli1 negatively regulates the differentiation and promotes the proliferation of colon cancer cells in the absence of 15pgdh ht29cells were either transfected with shctrl alone or cotransfected with shhpgd and pegfphgli1 followed by ltc4 stimulation for h qrtpcranalysis of the differentiation markers a si b mucin2 and c cdhr2 d the differentiation regulation marker cdx2 e the wnt activation marker axin2f the prooncogene cmyc and g the proliferation marker ccnd1 h western blot analysis of wholecell lysates for 15pgdh gli1 and si expressionαtubulin was used as the loading control i immunofluorescence analysis of gli1 and mucin2 in unstimulated or ltc4stimulated cells transfectedwith shhpgd or shgli1 hprt1 was used as the housekeeping gene for normalization of the qrtpcr gene expression data graphs represent datafrom to independent experiments and represent the mean ± sem p p p fig 6i“k supplementary fig s9a“h gli1 gene andprotein expression in ht29 and caco2derived colonospheres was also downregulated after ltc4 stimulation we next analysed the mrna expression levels intumour and adjacent mucosa samples from pairedcrc patients which also suggested a significantly positivecorrelation between gli1 and the stemness markersdclk1 and lgr5 with elevated expression of all threegenes in tumour tissues figs 1l 6l“n the normalmucosa as reference set to and the tumour tissue fordclk1 ± and lgr5 ± mean± sem respectively mann“whitney test p asexpected the expression level of dclk1 showed a negative correlation with cysltr2 and hpgd expressiononcogenesis 0csatapathy oncogenesis page of fig see legend on next pagefrom our previously published results see ref whilemuc2 had a positive correlation fig 2c20 this suggeststhat a poorly differentiated tumour could occur due toenriched stemnessltc4induced 15pgdh promotes differentiation inzebrafish xenograftsnext we used the zebrafish xenograft model28 to further evaluate and visualise the differentiationpromotingoncogenesis 0csatapathy oncogenesis page of see figure on previous pagefig ltc4induced 15pgdh expression negatively regulates gli1 via cyslt2r graphs showing qrtpcr analysis of a cysltr2 b 15pgdhand c gli1 mrna expression in hct116 cells with stable transfection of doxycyclineregulated shcysltr2 cultured with or without doxycycline µm treatment followed by ltc4 nm stimulation for h d western blot analysis of cyslt2r 15pgdh gli1 and si expression in wholecelllysates αtubulin served as the loading control e qrtpcr analysis showing mrna expression of gli1 and f western blot analysis of gli1 proteinexpression in ht29 cells stimulated with or without ltc4 and with or without ap100984 a cyslt2r antagonist hprt1 was used as the housekeepinggene and αtubulin was used as the loading control in the western blot assay g schematic illustration of colonosphere formation gli1 regulates theeffect of ltc4 on stemness in multicellular colonospheres the cells were cultured in ultralowattachment conditions on matrigel containing serumfree medium for days h representative images of colonospheres from ht29 cells transfected with shctrl shhpgd or shgli1 and stimulated ornot stimulated with ltc4 bar graphs showing the number of colonospheres formed per well and the size of colonospheres with or without ltc4stimulation and comparing the shctrl shhpgd and shgli1transfected groups qrtpcr analysis of the stemness markers i dclk1 j gli1 andm lgr5 in colonospheres derived from shctrl shhpgd or shgli1transfected ht29 cells with or without ltc4 stimulation for h k western blotanalysis showing the expression of dclk1 15pgdh and gli1 in transfected ht29 cells as indicated αtubulin served as the loading control qrtpcr analysis of l dclk1 and n lgr5 in matched pairs of mucosa m and tumour t tissues from crc patients n hprt1 was used as thehousekeeping gene for normalization of the qrtpcr gene expression data data represent the mean ± sem from to independent experimentsp p p role of 15pgdh in colon cancer transgenic zebrafish tgfli1egfp embryos were injected with u
Colon_Cancer
" it is estimated that around “ of patients with early stage colon cancer benefit from adjuvantchemotherapy we are currently not capable of upfront selection of patients who benefit from chemotherapywhich indicates the need for additional predictive markers for response to chemotherapyit has been shown that the consensus molecular subtypes cmss defined by rnaprofiling have prognostic andorpredictive value due to postoperative timing of chemotherapy in current guidelines tumor response tochemotherapy per cms is not known which makes the differentiation between the prognostic and predictive valueimpossible therefore we propose to assess the tumor response per cms in the neoadjuvant chemotherapy settingthis will provide us with clear data on the predictive value for chemotherapy response of the cmsscontinued on next page correspondence jpmedemaamsterdamumcnljhjm van krieken jnm ijzermans jp medema and m koopman havejoint last authorship3laboratory for experimental oncology and radiobiology center forexperimental and molecular medicine cancer center amsterdamamsterdam umc university of amsterdam meibergdreef azamsterdam the netherlands5oncode institute amsterdam umc university of amsterdam amsterdamthe netherlandsfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cberg bmc cancer page of continued from previous pagemethods in this prospective single arm multicenter intervention study patients with resectable microsatellite stablect3“4nxm0 colon cancer will be treated with two courses of neoadjuvant and two courses of adjuvant capecitabine andoxaliplatin the primary endpoint is the pathological tumor response to neoadjuvant chemotherapy per cms secondaryendpoints are radiological tumor response the prognostic value of these responses for recurrence free survival andoverall survival and the differences in cms classification of the same tumor before and after neoadjuvant chemotherapythe study is scheduled to be performed in “ dutch hospitals the first patient was included in february discussion patient selection for adjuvant chemotherapy in early stage colon cancer is far from optimal the cmsclassification is a promising new biomarker but a solid chemotherapy response assessment per subtype is lacking in thisstudy we will investigate whether cms classification can be of added value in clinical decision making by analyzing thepredictive value for chemotherapy response this study can provide the results necessary to proceed to future studies inwhich neo adjuvant chemotherapy may be withhold in patients with a specific cms subtype who show no benefitfrom chemotherapy and for whom possible new treatments can be investigatedtrial registration this study has been registered in the netherlands trial register nl8177 at “ wwwtrialregisternltrial8177 the study has been approved by the medical ethics committee utrecht mec18712keywords colon cancer consensus molecular subtypes neoadjuvant chemotherapy surgery colon cancer is one of the most common types of cancer in the netherlands with an incidence of around patients in approximately of patients present with local disease stage iiii curativesurgery followed by adjuvant systemic chemotherapy isstandard of care in patients with microsatellite stablemss highrisk stage ii and stage iii colon cancer despite this intensive treatment “ of the patients develop metastatic disease these patients do not benefitenough from the current adjuvant systemic therapymoreover it is estimated that will not develop metastases after surgery alone and are therefore overtreated with adjuvant chemotherapy identifying the patients at risk of developing metastases as well as thoseresponding to therapy is a clear unmet need in coloncancer care the development of new prognostic andpredictive markers for chemotherapy response is therefore of utmost importancemany efforts have been undertaken to stratify crc patients into biologically and clinically distinct subtypesone of these led to the development of the consensusmolecular subtypes cmss which is based on rna expression profiling of tumor tissue and which is currentlyconsidered to be the most robust molecular stratificationin crc cms1 is characterized by hypermutationmicrosatellite instability msi and strong immune infiltration cms2 the canonical subtype has marked wntand myc signaling activation cms3 is enriched forkrasmutations and shows evident metabolic deregulation cms4 the mesenchymal subtype is characterizedby prominent tgf activation stromalinvasion andangiogenesis activation subtyping in a large heterogeneous patient cohort n with stage i to iv colorectal cancer showed significant differences in prognosiswith cms4 as the poorprognosis subtype confirmingthe clinical relevance of the intrinsic processes implicated in each cms these results support the idea that the cmss mighthave predictive value for response to chemotherapy dueto the postoperative timing of chemotherapy in currentguidelines the tumor response to chemotherapy is notassessable which makes a distinction between the prognostic value and predictive value of the subtypes impossible only a randomized controlled trialin whichpatients would be randomized in either surgery plus adjuvant chemotherapy or surgery alone would make thisdistinction possible howeverthis causes ethical dilemmas because chemotherapy would be withhold in patients who might actually benefit yet a solid responseassessment per subtype is necessary for implementationin clinical decisionmaking we therefore propose totreat patients with two neoadjuvant and two adjuvantcourses of capox and determine the response in tumorresected specimensapplying neoadjuvant chemotherapy may have severaladvantages the possibility of response monitoring earlyeradication of micrometastases and more complete resections neoadjuvant treatment is already standard ofcare for different gi malignancies including esophagealgastric and rectal cancers [“] the foxtrot collaborative group was the first to set up a neoadjuvant trialin patients with locally advanced resectablecolon cancer and concluded that preoperative chemotherapy is feasible with acceptable toxicity and perioperative morbidity after this pilot studytheyconducted a randomized phase trial investigating theeffect of neoadjuvant chemotherapy in patients with act3“ n02 m0 colon cancer patients were randomized between weeks of neoadjuvant combined with 0cberg bmc cancer page of weeks of adjuvant folfoxcapox or weeks of adjuvant folfoxcapox neoadjuvant chemotherapywas safe with less major surgical complications significant downstaging and a reduced risk of incomplete resection although the primary endpoint of the studyfreedom from recurrent or persistent disease after years was not met the risk of a recurrence after yearswas reduced to with perioperative chemotherapycompared to with adjuvant chemotherapy onlyhr “ p in the proposed study we will investigate the predictivevalue of the cms classification on chemotherapy response in a neoadjuvant setting including pathologicalresponse and radiological response and their correlationwith rfs and os this allows us to determine therapyefficacy in individual patients and per subtypeobjectivethe primary aim of this study is the evaluation of thepathological tumor response to neoadjuvant systemicchemotherapy per cms in patients with mss high riskstage ii and stage iii colon cancermethodsstudy designconnection ii is a prospective multicenter interventional cohort study that will be performed as a substudyofthe prospective dutch colorectal cancer cohortplcrc plcrc is a nationwide cohort study of thedutch colorectal cancer group dccg facilitating scientific research to improve the outcome and quality oflife of patients with colorectal cancer we aim to include patients in “ dutch hospitals that participatein plcrcin connection ii patients with a mss ct3“4nxm0 colon tumor will be treated with two courses ofneoadjuvant and two courses of adjuvant capecitabineand oxaliplatin capox fig and table the cmsclassification will be determined on both the pretreatment biopsies and the resection specimen at least multiregion biopsies will be taken pretreatment toensure a sample with vital tumor and sufficient rnaquality tumor response will be assessed on the resection specimen using the tumor response grading trgsystem as proposed by dworak radiologicalresponse evaluation will be centrally performed by dedicated radiologists on sequential ct scans made at baseline and after two courses of neoadjuvant chemotherapybut before resection pathologists and radiologists will beblinded for the cms classificationoptionally blood samples are taken for circulatingtumor dna ctdna analysis and plasma storage atfour time points at baseline after neoadjuvant treatment after surgery and after completion of the adjuvantchemotherapy followup will be performed until years postsurgery data on local recurrences metastasesand survival will be documentedstudy populationpatients diagnosed with resectable ct3“4nxm0 coloncancer are eligible for the connection ii trial baselinectscans of all patients will be reviewed by dedicated radiologists in the treating hospitals with special focus ontumor staging msi status will be determined on biopsymaterial to exclude patients with an msi tumor patients are eligible when they meet the followingconsent for the connection ii studycriteria 0f able and willing to provide written informed 0f informed consent signed for plcrc components 0f mss based on pretreatment biopsy by immunohis 0f fit to undergo neoadjuvant chemotherapy with˜clinical data™ and ˜future studies™tochemistry ihccapecitabine oxaliplatin and subsequent surgeryjudged by the primary treating physician 0f adequate bone marrow liver and renal functionpatients will be excluded if any of the following criteriaare met 0f any other malignant disease within the preceding years apart from nonmelanotic skin cancerfig flow diagram of clinical study patients with an msi tumor will be excluded from this study at time points blood samples will becollected for ctdna analyses and future biomarker studies 0cweekcheck in and exclusionsign informed consentblood withdrawal for ctdna plasmactscansurgerycapoxrecord medical history xxx axxxcxbx fxgc1d1c2d1xxc3d1exxdc4dxxberg bmc cancer page of table study flowchart of clinical studystudy proceduresinclusion neoadjuvantsurgeryadjuvant chemotherapyfollow upchemotherapy xxxdocument concomitant medicationtherapiesa blood withdrawal may be done at screening or immediately before cycle day b blood withdrawal to be performed after cycle week and before surgeryc blood withdrawal to be performed before cycle day d blood withdrawal to be performed approximately weeks after surgerye cycle day should ideally start within “ weeks after surgery at the latest weeks after surgeryf ct should be performed after completion of cycle and before surgeryg surgery should ideally be performed “ weeks after cycle day but has to be performed weeks after cycle day xcarcinoma in situ and early stage disease with a recurrence risk of less than 0f colonic obstruction that cannot be defunctioned by 0f pregnant or lactating womena stomamain study parameterendpointthe primary endpoint is the pathological tumor responseto neoadjuvant chemotherapy per cms the pathologicalresponse will be centrally scored on hestained slidesfrom the resection specimen using the tumor responsegrading system according to dworak [ ] based on results from the foxtrot study a good response will bedefined as trg2 trg3 or trg4 poor response as trg1or trg0 the cms classification will be determined onthe pretreatment biopsies and on the resection specimens rna will be isolated from ffpe material and analyzed on the ncounter sprint profiler a reliable androbust platform for samples with degraded rna such asffpe samples [ ]secondary study parametersendpoints 0f additional pathological markers to assess the tumorresponse the modified ryan scheme trs andexpression of ki67 and caspase3 and morphological cytostaticcytotoxic effects on hestained tissue slides 0f pathological response per trg and trs categoryseparately for the different cms subtypes 0f radiological tumor response to neoadjuvantchemotherapy 0f recurrence free survival rfs at two and threeyears rfs is defined as the time elapsed betweenthe diagnosis of the primary tumour and either thedate of any recurrence of disease time of death orthe date of the last followup visit at which a patientwas considered to have no recurrence 0f overall survival os at five and ten years 0f therapyinduced cms differences 0f prognostic and predictive value of cytotoxiclymphocytes cytolym and cancerassociated fibroblasts caf infiltration scores 0f diagnostic accuracy of ctdna measurements formonitoring treatment response to neoadjuvanttreatment and detection of residual disease 0f exploration of proteome profiles for monitoringtreatment response to neoadjuvant treatment anddetection of residual disease 0f percentages of pathological complete r0pathological microscopic incomplete r1 andpathologically macroscopic incomplete r2resections 0f surgical complication rate ie wound infections andanastomotic leakstatistical analysisprimary study endpointthe primary study endpoint is the pathological tumorresponse per cms using the trg system according todworak pathologic tumor regression rates withcorresponding confidence intervals will be analyzedper cms subgroup using the wilson method 0cberg bmc cancer page of secondary study endpointscategorical data pathological tumor response accordingto the modified ryan scheme are compared using chisquare analysis or fisher™s exact test and are shown asnumbers relative and absolute rates continuous datacytolym and caf infiltration scoresradiologicaltumor response pathological response by percentage ofki67 and caspase3 positive neoplastic cells are compared using nonparametric ttest or mannwhitney utest where appropriate and are shown as mean andstandard deviation or median and interquartile range“ pvalues are twotailed and results areconsidered significantthe os at and years and rfs at and years willbe calculated and depicted by means of the kaplanmeier technique and will be compared using the stratified logrank test hazard ratios and confidence intervals will be calculated with a stratified coxproportional hazard analysis the rfs will be analyzedper cms subgroup per trg and radiological responseall estimates will be accompanied by confidenceintervalssample size calculationwe based our sample size calculation on the desiredprecision with which we will be able to estimate thepathological response rates to neoadjuvant chemotherapy within each cms subtype this precision is quantified by the margin of error the radius of the confidence interval which we set at a maximum of this margin of error is achieved with patients inthe least prevalent cms subgroup namely cms3 andan anticipated pathologic responses yielding a response rate of with a 95ci of “ based onthe currently observed ratios of subtypes derived fromthe large consensus dataset after exclusion of the msitumors which holds cms1 tumors for most part wewill need a total of mss patients cms2 cms3 cms4 with this sample size we anticipatemaximum margins of error of and forcms2 cms3 and cms4 respectively and overallthe above depends on the assumption that the response rates will not be higher than within eachcms subgroup if response rates will actually be closerto the maximum margin of error will increasethe sample size hence indicates that for the analysis patients will be needed for whom followup andsubtype is known we expect a loss in patients dueto loss of followupinsufficient quality of the biopsymaterial or failure to faithfully assign patients to a subgroup based on the rna expression profiles resulting ina total of patients needed to have sufficient data forboth the primary and secondary outcomesdata collection and data managementdata collection and data management will be performedby the netherlands comprehensive cancer anizationiknl they have broad experience with continuousdata collection based on high quality electronic case reportforms ecrfs which guarantees complete andtimely recording handling and storage of data and documents all local and central data managers are registeredand the electronic database trias is iso certifieddata will be documented in line with ˜good clinicalpractice gcp™ and dutch legal requirements major violations of the protocol will be recordedmonitoringno data and safety monitoring board dsmb will beassigned since patients are subjected to an interventionwith a low postoperative morbidity that is already beingperformed in routine clinical practice no interim analyses will be performedauditingindependent monitoring of the study is performed by aqualified monitor of iknl the monitor plan is basedon the judgement of the irb that study participation isof low to moderate risk monitoring will be performedby investigating the electronic trial database and performing site visits each participating site will be visitedat least once with repeat visits to sites where performance is a concern the quality assessment will focus onthe safety wellbeing and rights of the patients the quality of the documented data in the ecrf and their traceability to source documents and the completeness of theregulatory binder after each monitor visit the trialmonitor reports feedback to the project leader study coordinator and local investigatoradverse eventsthe treatment with capox in this study is standard ofcare therefore ae and sae are not expected to be different as both the treatment with capox and the surgery are part of the standard of care only two specificsaes are defined which are possibly related to the adjusted study schedule information will be collected onpatients who prematurely stop chemotherapy treatmentand of patients who are not able to undergo plannedsurgery due to progressive diseaseobstructionthe following two saes will be reported 0f if the surgery has to be postponed for more than 0f if patients can not complete all the neoadjuvantweeks after the start of cycle of capoxchemotherapy courses 0cberg bmc cancer page of the study coordinator will report these saes to thethat apaccredited institutional review board irbproved the study protocoldiscussioncolon cancer is one of the most common types of cancer in the netherlands the standard of care for patientswith mss high risk stage ii and stage iii colon cancercurrently consists ofsurgery followed by systemicchemotherapy patient selection for adjuvant chemotherapy is still far from optimal approximately wouldnever develop metastases after surgery alone and istherefore overtreated with adjuvant chemotherapymoreover “ still develop metastatic disease despite this intensive treatment leaving merely “ thatin fact benefit from adjuvant chemotherapy this illustrates the evident need for additional predictive markersfor chemotherapy benefitone potential marker is the cms classification whichis based on the integration of six different molecularclassification systems based on rna expression profiling the cms classification divides crc patients intofour subtypes with distinctive biological features guinney showed a clear relapse free survival and overallsurvival advantage for cms1“ compared to cms4 in aheterogeneous patient cohort with stage iiv crc withdivergent treatment schemes besides the prognostic value literature provides somesupport for a predictive value of cms for response tosystemic treatment in a retrospective analysis of thensabp c07 trial on patients n with stage iiicolon cancer only cms2 was associated with benefitfrom oxaliplatin treatment patients with cms4 tumorsdid not benefit from addition of oxaliplatin treatment the mesenchymal subtype showed no benefit from5fu monotherapy compared to no systemic therapy ina nonrandomized retrospective analysis of stage iiicrc patients although being a promising molecular marker a solidchemotherapy response assessment per subtype has notbeen performed and it remains unknown whether thedifference in longterm outcome between cms1“ andcms4 originates from differences in prognosis or response to therapythis makes it impossible to know whether patientswith the poorprognosis subtype cms4 have an impaired survival due to the aggressive nature of the tumoror due to a limited response to chemotherapy therefore it is unknown whether these patients should receivechemotherapy or not this also holds true for the othersubtypes although cms1“ show superior outcomes tocms4 it is unknown whether this is due to a favorabletumor biology or due to a substantial response tochemotherapy wesolidtherefore believethatachemotherapy response assessment per subtype is animportant and essential step to distinguish betweenprognosis and prediction and to incorporate the cmssin clinical decisionmakingadministering neoadjuvant chemotherapy in the suggested study population was proven safe and feasible inthe foxtrot study [ ] importantly the pathological tumor response was evidently associated with recurrence free survival patients with a complete responsetrg4 developed no recurrences after years of followup compared to of patients that showed no regression at all trg0 this illustrates that the responseto chemotherapy of the primary tumor may indeed be areliable measurement for chemotherapy efficiencythe primary endpoint of the proposed study is thepathological tumor response which will be centrallyscored using the trg by dworak a highly reproduciblescoring system which is often used and clinically meaningful evidently tumor response monitoring usinghistology requires invasive procedures as a secondaryendpoint radiological response will be scored by a central board of radiologists and compared to the pathologicaltumor response to evaluate this noninvasivetechnique as a response modality both the histologicaland radiological response will be correlated to rfs andos to assess their prognostic valueinvolvementthe proposed neoadjuvant approach requires reliableclinical tnm staging to minimize the risk of overtreating patients with stage i or low risk stage ii colon cancer a metaanalysis analyzing the accuracy of t and nstaging on ct imaging showed that t1“ can be reliablydistinguished from t3“ sensitivity and specificity while nodalis unreliable with apooled sensitivity and specificity of and respectively therefore only t stage will be used to selectpatients second only patients with an mss status willbe included which will be determined on the biopsiesfollowing the latest recommendations of the update ofthe esmo guideline to refrain from adjuvant chemotherapy in highrisk stage ii msi colon cancer patientsas the possible clinical benefit is too low this wasalso seen in the foxtrot trial where msi status wasassociated with a significantly higher rate of poorno response vs p using the proposed selection of patients with a mss ct3“4nxm0colon tumor up to of patients is estimated to beovertreated [ ]results from this study in which we analyze both thepathological and radiological tumor response per cmswill lead to improved patient stratification and clearerinsight into which patients benefit from chemotherapythis will allow us to identify the group of patients thatreceives chemotherapy appropriately and the group ofpatients that may not benefit from the current treatment 0cberg bmc cancer page of regimen future studiesshould focus on whetherchemotherapy can be withheld in this patient group oron the development of new therapies to improve patientoutcomeabbreviationscaf cancer associated fibroblast capox capecitabin and oxaliplatincms consensus molecular subtype ctdna circulating tumor dnacytolym cytotoxic lymphocytes dccg dutch colorectal cancer groupesmo european society of medical oncology mec medical ethicscommittee msi microsatellite instable mss microsatellite stable os overallsurvival plcrc prospective dutch colorectal cancer cohort rfs recurrencefree survival trg tumor response gradingacknowledgementsnot applicableauthors™ contributionsib sw jr gv rb1 cj se dv mk1 eh wg mo rb2 jk ji jm mk2 authors make substantial contributions toconception and design andor acquisition of data andor analysis andinterpretation of data authors participate in drafting the orrevising it critically for important intellectual content authors give finalapproval of the version to be published served as scientific advisorfundingthe connection ii trial is funded by the dutch cancer society alped™huzes the dutch cancer society is a nonprofit society that funds cancerresearch and has had no direct influence in the structuring of the trial andwill also not benefit financially from the outcomeavailability of data and materialsthe datasets used andor analyzed during the current study are availablefrom the principal investigator on reasonable request results will becommunicated via plcrc presentations at international conferences and viapublications in peer reviewed journalsethics approval and consent to participatethe study has been approved by the medical ethics committee utrechtmec18712 the medical ethics committee utrecht belongs to the umcutrecht and the prinses máxima center reference number slrc19009541the study will be conducted according to the principles of the declarationof helsinki 10th version fortaleza and in concordance with the dutchmedical research improving human subjects act wmo and otherapplicable guidelines regulations and actsauthorships will be defined following the international committee ofmedical journal editors guidelines the patients treating physicians local investigator or research nurse of theparticipating hospitals will follow ichgcp and other applicable regulationsin informing the patient and obtaining consent this includes explaining theconnectionii study to the patient providing himher with informationsuch as the expected efficacy and possible side effects and that refusal toparticipate will not influence further options for therapy before informedconsent may be obtained the investigator should provide the patient ampletime and opportunity to inquire about details of the trial and to decidewhether or not to participate in the trial all questions about the trial shouldbe answered to the satisfaction of the patient only after written informedconsent the patient will be included in this study the inclusion has to takeplace shortly after diagnosis to prevent delay in treatmentpatients are well informed that participation in voluntary and that they maywithdraw at any point during the studyconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interests nielsen t wallden b schaper c ferree s liu s gao d analyticalvalidation of the pam50based prosigna breast cancer prognostic geneauthor details1department of surgery erasmus mc university medical center rotterdamrotterdam the netherlands 2department of medical oncology universitymedical center utrecht utrecht university utrecht the netherlands3laboratory for experimental oncology and radiobiology center forexperimental and molecular medicine cancer center amsterdamamsterdam umc university of amsterdam meibergdreef azamsterdam the netherlands 4department of pathology radboud universitymedical centre nijmegen the netherlands 5oncode institute amsterdamumc university of amsterdam amsterdam the netherlands 6netherlandscomprehensive cancer anisation department of research utrecht thenetherlands 7department of medical oncology amsterdam umc locationvumc amsterdam the netherlands 8julius center for health sciences andprimary care university medical center utrecht utrecht university utrechtthe netherlands 9department of radiology the netherlands cancer instituteamsterdam the netherlands 10department of surgery university medicalcenter utrecht utrecht university utrecht the netherlandsreceived may accepted july referencesnederlandse kankerregistratie nkr iknl retrieved from wwwiknlnlnkrcijfers in may guinney j dienstmann r wang x de reynies a schlicker a soneson c the consensus molecular subtypes of colorectal cancer nat med“van hagen p hulshof mc van lanschot jj steyerberg ew van bergehenegouwen mi wijnhoven bp preoperative chemoradiotherapy foresophageal or junctional cancer n engl j med “bosset jf mercier m triboulet jp conroy t seitz jf surgical resection withand without chemotherapy in oesophageal cancer lancet “author reply cunningham d allum wh stenning sp thompson jn van de velde cjnicolson m perioperative chemotherapy versus surgery alone forresectable gastroesophageal cancer n engl j med “sebagmontefiore d stephens rj steele r monson j grieve r khanna s preoperative radiotherapy versus selective postoperativechemoradiotherapy in patients with rectal cancer mrc cr07 and ncicctgc016 a multicentre randomised trial lancet “roh ms yothers ga connell mjo beart rw pitot hc shields af theimpact of capecitabine and oxaliplatin in the preoperative multimodalitytreatment in patients with carcinoma of the rectum nsabp r04 j clinoncol foxtrot collaborative g feasibility of preoperative chemotherapy for locallyadvanced operable colon cancer the pilot phase of a randomisedcontrolled trial lancet oncol “ matthew t seymour dm and on behalf of the international foxtrot trialinvestigators foxtrot an international randomised controlled trial in patients pts evaluating neoadjuvant chemotherapy nac for colon cancerj clin oncol 20193715_suppl3504 coebergh van den braak rrj van rijssen lb van kleef jj vink gr berbeem van berge henegouwen mi nationwide comprehensive gastrointestinal cancer cohorts the 3p initiative acta oncol “ dworak o keilholz l hoffmann a pathological features of rectal cancerafter preoperative radiochemotherapy int j colorectal dis “ veldmanjones mh brant r rooney c geh c emery h harbron cg evaluating robustness and sensitivity of the nanostring technologiesncounter platform to enable multiplexed gene expression analysis ofclinical samples cancer res “ weissenberg e tnm staging of colorectal carcinoma ajcc 8th ed song n poguegeile kl gavin pg yothers g kim sr johnson nl clinical outcome from oxaliplatin treatment in stage iiiii colon canceraccording to intrinsic subtypes secondary analysis of nsabp c07nrgoncology randomized clinical trial jama oncol “lee j sohn i do ig kim km park sh park jo nanostringbasedmultigene assay to predict recurrence for gastric cancer patients aftersurgery plos one 20149e90133 0cberg bmc cancer page of signature assay and ncounter analysis system using formalinfixed paraffinembedded breast tumor specimens bmc cancer roepman p schlicker a tabernero j majewski i tian s moreno v colorectal cancer intrinsic subtypes predict chemotherapy benefit deficientmismatch repair and epithelialtomesenchymal transition int j cancer“ nerad e lahaye mj maas m nelemans p bakers fc beets gl diagnostic accuracy of ct for local staging of colon cancer a systematicreview and metaanalysis ajr am j roentgenol “ committee eg eupdate early colon cancer treatment recommendations murakami k west n westwood a hemmings g bottomley d davis j the relationship between dna mismatch repair and response to folfoxbased preoperative chemotherapy in the international phase iii foxtrottrial journal of patholog
Colon_Cancer
"objectives therapeutic radiographers trs are well placed to deliver health behaviour change advice to those living with and beyond cancer lwbc however there is limited research on the opinions of trs around delivering such advice to those lwbc this study aimed to explore trs™ practices and facilitators in delivering advice on physical activity healthy eating alcohol intake smoking and weight managementsetting and participants fifteen uk based trs took part in a telephone interview using a semistructured interview guide data was analysed using the framework analysis methodresults emergent themes highlighted that trs are mainly aware of the benefits of healthy behaviours in managing radiotherapy treatment related side effects with advice provision lowest for healthy eating and physical activity participants identified themselves as well placed to deliver advice on improving behaviours to those lwbc however reported a lack of knowledge as a limiting factor to ng so the trs reported training and knowledge as key facilitators to the delivery of advice with a preference for online trainings there is a need for education resources clear referral pathways and in particular training for trs on delivering physical activity and healthy eating advice to those lwbcintroductionit is estimated that of cancer cases are linked to unhealthy behaviours1 based on evidence from systematic literature reviews and meta analyses the world cancer research fund wcrf recommend that individuals are physically active limit consumption of energy dense foods salty foods red meat and avoid processed meat eat more plant foods maintain a healthy weight limit alcoholic drinks and avoid tobacco to reduce their risk of cancer2 those living with and beyond cancer lwbc are also advised to follow these guidelines due to increasing evidence that healthy behaviours may improve physical strengths and limitations of this study –º this study provides an insight in therapeutic radiographers™ views on all key modifiable health behaviours for those living with and beyond cancer –º the participants worked in different radiotherapy departments offering insight into the practices among therapeutic radiographers in the delivery of healthy behaviour advice from a wide range of hospitals –º whilst data saturation was reached the sample size was small and therefore the findings may not be representative of the views of the wider therapeutic radiography workforce –º the response rate was low therefore the participants might be more interested in the role of health behaviours in cancer survivorship which might bias the responses towards a positive view on the role of therapeutic radiographers in delivering advice within their roleand psychosocial outcomes after a cancer diagnosis2“despite the potential benefits of healthy behaviours few people lwbc are meeting the wcrf recommended health behaviour recommendations9 those lwbc report one key reason for not adopting healthier lifestyle behaviours is lack of advice and support from their healthcare team11 healthcare professionals hcps are well placed to bring about positive health behaviour changes among cancer patients12 a trial of brief advice among breast cancer survivors showed that a simple physical activity recommendation from a hcp doubled the percentage meeting national exercise guidelines12 despite this research to date among both hcps and those lwbc consistently shows that few oncology hcps offer guidance to oncology patients on healthy lifestyle behaviours13“reported barriers in providing health behaviour advice for those among hcps pallin a0nd et a0al bmj open 202010e039909 101136bmjopen2020039909 0copen access lwbc include believing that giving advice was not part of their role lack of time with patients lack of referral programmes lack of resources such as education leaflets for those lwbc and lack of knowledge regarding guidelines and research findings16“ a recent qualitative study with oncology hcps identified that advice on health behaviours provided to those lwbc focussed on general health and controlling side effects with few hcps advising on health behaviours in the context of improving survival outcomes20 while these studies provide useful insight into the practices and barriers among oncology hcps the participants within these studies were primarily oncologists and nurses and focussed on the provision of physical activity and weight management advice there is limited research on the opinions of therapeutic radiographers trs in delivering advice on health behaviours to those lwbc despite at least of cancer patients receiving radiotherapy as part of their cancer treatment22trs are the only health professionals qualified to deliver radiotherapy and play a central role in supporting cancer patients23 in the uk the college of radiographers recognise the importance of trs in providing health behaviour advice to improve patient outcomes24 trs are also seen as an integral part of the health force in driving improvements in well being as outlined in the publication of œahps into action using allied health professionals to transform health care and well being which states that radiographers are key to implementing a preventative healthcare approach and that their expertise should be used to design and deliver health interventions25 trs are ideally placed to deliver health behaviour advice particularly through making every contact count mecc26 mecc is a strategy whereby health professionals use every appropriate opportunity and interaction with patients to promote healthy behaviours and signpost to relevant healthcare services using an ˜ask advise act™ framework27 mecc fits extremely well within the trs™ role in which patient education is a key part of radiotherapy practice with trs providing care to the same patient every day often over a number of weeks23 trs therefore have the potential to make significant contributions in supporting positive health behaviour changes among those lwbchowever despite these opportunities one survey in the uk among trs identified that trs rarely advise patients on the key modifiable health behaviours including smoking alcohol healthy eating and exercise15 the findings also showed lack of knowledge and training as barriers among trs in delivering advice on these topics15 similarly focus group interviews with trs identified that lack of knowledge and training were barriers to the provision of smoking cessation advice28challenges remain in translating behaviour change interventions into existing care pathways and practices in a way that is appropriate for use by health professionals29 understanding trs™ practices and what support they need in delivering advice on the topics of physical activity healthy eating alcohol intake smoking and weight management could inform the development of interventions that will enable trs™ in delivering advice on improving health behaviours to those lwbc qualitative research is appropriate for exploring the beliefs experiences and motivations of individuals on specific matters and allow for more information and clarification30 limited qualitative data exists on trs™ practices and views on delivering advice on these health behaviour topics this study therefore aimed to address this and through a qualitative methodology explore trs™ practices in delivering health behaviour advice in addition to exploring the facilitators in delivering such advice preferences regarding training on delivering this advice were also exploredmethodsparticipants and recruitmentparticipants were trs working in the uk in a clinical role they had provided their contact details on a previous online survey investigating tr™s practices in delivering health behaviour advice agreeing to be invited for a follow up telephone interview an email was sent with an information sheet explaining the research and inviting these trs those who agreed to take part signed a consent form prior to the telephone interviewdata collectionsemi structured individual telephone interviews were carried out between april and may by a lecturer in therapeutic radiography with an msc who had completed qualitative interviewing as part of their training np the interviewer had no previous relationship with the study participants the topic guide see online supplementary material was based on the guide used within a previous study17 which explored oncology hcps views on the provision of lifestyle advice to cancer patients this guide was adapted for use among trs with additional questions added to assess preferences for training on delivering advice the topic guide was piloted with two participants to check for comprehension of the questions this data was included in the analysis because no substantial changes were required the interviews lasted approximately min range to min and were audio recorded anonymised and transcribed verbatim the transcripts were verified by np against each recording to confirm accuracy the aim was to carry out interviews until data saturation was reached it was anticipated that participants would be required to reach data saturation because it was a homogeneous group31 after interviews were carried out they were transcribed verbatim following familiarisation with the data np generated the initial codes and it appeared that saturation was reached after interviews as no new codes occurred in the 10th interview32 a further five interviews were carried out to confirm thispallin a0nd et a0al bmj open 202010e039909 101136bmjopen2020039909 0ctable participant identifier and demographic characteristicsparticipant identifierdemographic characteristicsprofessional gradegendertr tr tr tr tr tr tr tr tr tr tr tr tr tr tr femalefemalefemalefemalefemalefemalefemalemalefemalefemalefemalefemalemalefemalemaleband band band band band band band band band band band band band band band tr therapeutic radiographerpatient and public involvementpatient input was not used in the design of the research methods however the topic guide was piloted with trs in the academic setting additionally the topic guide was piloted with two participants and these were included in the analysisanalysisthe interview transcripts were analysed using the framework analysis method33 this method was chosen because it is an appropriate method for analysing homogeneous data and semi structured interview transcripts it is also appropriate when using inductive qualitative analysis33 a random selection of transcripts n3 were independently coded by af to check for reliability the researchers np af and rjb met and agreed on a final coding list in an iterative process af and rjb are both experienced qualitative researchers and health psychologists these agreed set of codes formed the analytical framework which was then applied to all of the transcripts and the data summarised in a matrix using microsoft excel themes were generated by reviewing the matrix connecting the data between the participants and the codes the completed consolidated criteria for reporting qualitative research checklist is available in the online supplementary material themes are presented in the results with supporting quotes and the participants identifier table resultsparticipantsthe radiotherapy radiography workforce census in the uk only reports the workforce™s professional grade and no other demographics35 in the uk trs™ level of open accessprofessional skills and knowledge are categorised by agenda for change professional band grades to therefore in this study the participants gender and professional grade were collected no other demographic information was collected table the response rate to taking part in the interview was seventy two trs were emailed and invited to take part in an interview returned consent forms and completed the telephone interview fifteen interviews were conducted with women and men the participants came from all regions of the uk including england wales scotland and northern irelandthemesfive main themes were identified trs provide behaviour change advice to manage radiotherapy related side effects trs make judgements about when it is appropriate to deliver health behaviour advice knowledge and training are key facilitators in the delivery of health behaviour advice trs feel patients undergoing radiotherapy treatment seek guidance on health behaviours and trs identify themselves as well placed to give health behaviour advice to patientstrs provide behaviour change advice to manage radiotherapyrelated side effectsmost respondents reported that they only provided advice on health behaviours that they believed would minimise radiotherapy related side effects this meant smoking cessation and alcohol intake were the two health behaviours trs mainly advised onwith head and neck patients we give advice particularly on smoking and drinking obviously get worse side effects tr the only thing we do generally say is about drinking plenty of fluids avoiding alcohol but that™s more to do with prostate side effects bladder reactions and reducing gas tr radiographers are comfortable talking about alcohol when it comes to managing side effects tr no trs reported advising patients on healthy eating some trs mentioned advising patients on dietary intake but this is to patients who are at risk of losing weight for side effect management and potential impact on accuracy of radiotherapy treatment deliveryhealthy eating i don™t tend to discuss too much a lot of patients have difficulty eating and we are encouraging maintaining weight while on treatment tr i™m not very sure if healthy eating is important any patients where we™re treating lower gi or pelvis we would advise them to avoid very high fibre foods spicy foods that might make them have very loose bowels but other than that we say more or less keep on your same diet we wouldn™t generally discuss a healthy diet as a standard for all patients no tr pallin a0nd et a0al bmj open 202010e039909 101136bmjopen2020039909 0copen access some trs mentioned they advise patients to be physically active however this was only in the context of managing radiotherapy and cancer related fatigueso exercise is one of my main ones that i focus on with all patients particularly to help with their fatigue tr exercise i say that™s its quite beneficial to help with fatigue tr i guess when we have patients come in fatigue is one of the side effects so we encourage our patients to remain active tr trs make judgements about when it is appropriate to deliver health behaviour advicetrs explained only discussing health behaviours particularly smoking and alcohol with patients if there were evident indications of a problem trs also often reported making a judgement of whether appropriate to advise a patient on a particular health behaviourso quite often you can tell if a patient is a smoker you can smell it or you can tell by their skin tr i tend to give advice when you make a judgement of when it™s appropriate an example might be if a patient smelt of smoke tr had patients come in and will smell of alcohol and at that time i™ll say to the patient that it can exacerbate side effects tr this meant trs did not provide advice on health behaviours to every patientbut for those patients where it™s not clearly going to benefit them to stop drinking you would just mention it very briefly not every patient will have that information tr knowledge and training are keys facilitator in the delivery of health behaviour advicedelivery of advice matched by knowledgethe reported delivery of advice on health behaviours appeared to be matched by knowledge of the benefits among those lwbcone participant explained how he only appreciated the importance of physical activity in cancer survivorship after attending a talk and being made aware of the evidencemy experience of appreciating the role exercise was from attending a talk i suppose it was really just highlighting in the studies the benefits obviously of a healthy lifestyle and introducing physical activity for patients on treatment tr healthy eating was a topic the participants felt particularly unqualified to deliver advice on and reported lack of knowledge as a barrier to the delivery of advice on healthy eatingit™s a difficult one diet i think it™s more a knowledge thing if you don™t have the knowledge about what you can and can™t say you™re just not going to approach the subject tr a need for continuous postgraduate online trainingall interviewed said they would welcome postgraduate training on delivering health behaviour advice the majority expressed a preference for online training to help overcome the barrier of limited time among trs to attend trainingonline you™re not having to take time out of clinical practice online is more accessible tr participants also mentioned that online training allows for yearly updates and continuous professional developmenti think it™d be good online training because you can do it in your own time because i think that™s sometimes the problem you have this training once and then maybe it never gets brought up again so it would be quite handy to have something small every year alongside all your other mandatory training tr participants did acknowledge that face to face training allows for further questioning that™s not possible with online trainingi think one to one training because you can ask questions that may not be covered within the online training tr to overcome the barrier of not all staff being able attend face to face training participants suggested it would be useful to train some trs through face to face methods that they could then cascade to other trs within the radiotherapy departmentmaybe some face to face with some staff that they could cascade down might be useful as well tr a need for training in the undergraduate settingit was also suggested to incorporate training on delivering lifestyle advice into the undergraduate education programmecertainly get it into the undergraduate course to start with making them aware it is part of the role tr it™s still not something that i can say was primarily covered in the undergraduates™ training about the benefits of healthy lifestyle you know there™s no real formal education that i can see tr trs reported knowledge of resources and referral pathways as facilitators in the delivery of adviceparticipants also felt knowledge of how to refer patients onto further support would enable them to have conversations on improving health behaviours with some pallin a0nd et a0al bmj open 202010e039909 101136bmjopen2020039909 0ctrs reporting that lack of knowledge of resources and referral pathways are barriers to initiating a conversation on behaviour changethere needs to be more information available to professionals of where exactly you can refer patients to whether that be website whether that be an app tr that™s the only reason why they [therapeutic radiographers] don™t want to open these conversations up because they don™t know where to go with it or how to refer on tr they also acknowledge that in the short time they have with patients if they had a resource then would be more inclined to advisehaving something on a piece of paper education and having the resources if you can do it in min you should be able to slip that in tr you don™t always have that information at hand so if it was readily available i think we™d give out a bit more [health behaviour information] if it was just the case of pointing them in the right direction that would be a quick and easy thing to do tr the benefit of incorporating patients™ perspective into trainingparticipants also mentioned that getting patients™ perspectives on receiving advice on improving health behaviours should be incorporated into trainingi think that would be better coming from the patients themselves rather than just feedback from what journals and other literature says tr if there™d even be patients that would be willing to maybe just even be involved with staff training tr trs feel patients undergoing radiotherapy treatment seek guidance on health behavioursmany of the trs also described that patients often ask them for guidance around health behaviour changes particularly on diet and exercise this shows that patients see trs as credible sources of information on health behaviourswe are getting asked the question more and more about weight loss healthy living wanting to exercise more tr it is quite a common thing to be asked at the end of treatment not so much the smoking and alcohol i have to say but diet and exercise is certainly something that people commonly ask tr trs identify themselves as well placed to give health behaviour advice to patientstrs acknowledged that they are a consistent healthcare member for patients undergoing radiotherapy and have many opportunities to deliver lifestyle advice therefore open accesstrs recognised that they are well placed to deliver health behaviour advice to patientswe™re in a unique position because we do see the same patient day after day and you do kind of start to develop a relationship with them tr i think we™re well placed to help influence patients™ behaviours and it™s something we should be seen to encourage and report tr we™re in the best position where we see the patients for a number of weeks every day to encourage any changes tr from the interviews it appeared that many patients undergoing radiotherapy excluding those at risk of malnutrition or significant weight loss are primarily reviewed and assessed by trs this highlights that trs are in an ideal position to deliver advice on health behaviours particularly when asked about nutrition advice deliverythey routinely see the specialist radiographer for the breast patients but they don™t have a dietitian appointment tr prostate and breast are two tumour groups that are fully radiographer led review and about to of our work load they generally wouldn™t be sent to a dietician tr only have a dietitian on board for the head and necks tr discussiontrs in this study saw themselves as well placed to deliver health behaviour advice but also reported that they do not routinely provide advice to all patients trs were particularly unlikely to provide advice on healthy eating and physical activity and were more likely to provide advice on those behaviours they believed would minimise radiotherapy or cancer related side effects this is in line with previous research among trs15 in one qualitative study a key facilitator reported among trs in delivering smoking cessation support to patients was knowledge of the link between smoking and toxicity28 another qualitative study that explored allied health professionals™ views regarding the provision of dietary advice to patients highlighted that trs report giving dietary advice to help counteract the side effects of radiotherapy37 additionally in our study if trs did provide dietary advice this tended to be general advice rather than cancer specific advice on healthy eatingin some studies oncology hcps have reported they do not self identify as the right person to provide lifestyle advice17 however in this study trs identified themselves as being well placed to deliver health behaviour advice and in a unique position as a consistent member of the multidisciplinary team providing care to patients however despite this they do not feel qualified to deliver pallin a0nd et a0al bmj open 202010e039909 101136bmjopen2020039909 0copen access advice particularly on the topic of healthy eating in the uk poor diet has the biggest impact on the national health service budget greater than alcohol consumption smoking and physical inactivity38 it has been noted that there are insufficient dietitians to provide dietary advice to all patients who may need dietary support39 in response to this all hcps are being asked to implement a preventative healthcare approach within their role and the delivery of healthy eating advice is fundamental to this23 key to achieving this is that trs will have the skills knowledge and behaviours to improve the health and well being of individuals24 as with other oncology hcp groups16 this study identifies the need for education and training among trs in delivering health behaviour advice particularly on healthy eating and physical activity this training should also address when and how to refer to other support if necessary as this was identified as a key facilitator in the delivery of advice on health behaviours particularly when time is a barrier to the delivery of this advice15“all interviews demonstrated that trs would welcome training on delivering health behaviour advice and recommended it as a key facilitator in delivering advice in addition to incorporating it into the undergraduate setting the need for postgraduate training among trs in delivering health advice has also recently been reported by charlesworth et al28 in relation to the delivery of smoking cessation advice our findings from this study provide additional insight into trs preferences on the type of training on delivering lifestyle advice to those lwbc with trs demonstrating a preference of online training in the postgraduate setting among hcps online education has been reported to be as effective as face to face education42 additionally the use of online learning enables hcps to carry out training at time that fits in with clinical work43 trs in this study identified this benefit of online learning in overcoming the limited time available for trs to undertake continuous professional development and additional training interestingly trs in this study mentioned having patient input in the training would be helpful while hcps input is key to the development of interventions patient members play key advocacy roles and their input can enhance the outcomes of interventions45 patient input may also help overcome the reported barrier of fear of causing offence to a patient which has been reported as a barrier among oncology hcps in delivery of health behaviour advice17those lwbc wish to receive advice on health behaviours from their healthcare team13 and is of particular importance as the period following a cancer diagnosis has been shown be a teachable moment and an ideal opportunity to motivate patients around the importance of healthy eating and physical activity47 this was made apparent in this study whereby some trs mentioned that healthy eating and exercise were the health behaviours patients ask for advice on more often generally towards the end of their treatment this further highlights the importance of supporting trs in delivering evidence based health behaviour advice to meet patients™ needstrs have a responsibility to educate patients on the importance of following healthy behaviours given the increasing evidence showing implementing healthy behaviours improve a number of physical and psychosocial outcomes after a cancer diagnosis2 among pre menopausal and post menopausal women living with and beyond breast cancer a systematic literature review and meta analysis of follow up studies n213 breast cancer survivors identified that being overweight increases the risk of all cause and breast cancer mortality4 being physically active after a cancer diagnosis is also correlated with improved survival and reduced recurrence5 while data is limited emerging research suggests healthy dietary behaviours after a diagnosis may improve outcomes3 in a prospective observational study of patients with stage iii colon cancer a higher intake of a typical western diet was associated with a threefold increased risk of disease recurrence and a fold increased risk of all cause mortality8 additionally those lwbc are at increased risk for developing cardiovascular disease osteoporosis and diabetes and healthy behaviours can reduce the risk of developing these diseases51 of those interviewed in this study it appeared that those with breast prostate and colorectal cancer are primarily reviewed and assessed by trs therefore it is the responsibility of trs to deliver advice on improving health behaviours to these patients this is also particularly important because the strongest evidence for the benefits of diet and exercise is currently in breast prostate and colorectal cancer survivors53 these are also the most common cancers in the uk and radiotherapy plays a key role in managing these cancers22 therefore with the right skills and knowledge trs could deliver advice on improving health behaviours by supporting self efficacy among patients towards the end of their treatment which very often is in the radiotherapy department can be empowering for patients among those with prostate cancer implementing dietary changes brought psychological benefit as a method of coping and regaining control over their diagnosis46strengths and limitationsthis is the first qualitative study among trs to explore the provision of advice on all key modifiable lifestyle behaviours for those lwbc as per recommendations2 while the aim of qualitative research is not to generalise the findings the sample size was small and therefore the findings may not be representative of the views of the wider therapeutic radiography workforce however data saturation was reached likely due the homogeneous sample of participants additionally the participants worked in different radiotherapy departments and therefore provide insight into the practices among trs in the delivery of healthy behaviour advice from a wide range of hospitals also the participants worked in cancer centres in england wales scotland and northern pallin a0nd et a0al bmj open 202010e039909 101136bmjopen2020039909 0cireland providing insight into the practices across the uk another limitation of this study is the low response rate and that the participants might be more interested in the role of health behaviours in cancer survivorship which might bias the responses towards a positive view on this topic and the role of trs in delivering advice within their role despite this however provision of health behaviour change advice was low suggesting trs may be even less likely to educate patients around the importance of healthy behavioursfuture researchthis study highlights the need for training and education among trs on the delivery of health behaviour advice to cancer patients both in the undergraduate and postgraduate setting particularly on the topics of physical activity healthy eating and weight management higher education institutions have a responsibility in educating the allied health professional workforce on implementing health promotion within their role55 further research among pre registration tr students and lecturers within therapeutic radiography should therefore explore how best to address this need future research among trs should also use purposive sampling to identify the views and health promotion practices among those who may not have a primary interest in the area of health
Colon_Cancer
of hydrophobic fragments into its structure allowed the preparation of waterinsoluble modified dtpa complexes21 the original substance of the modified dtpa dtpamod was synthesized in tomsk polytechnic university preparation of colloid solution dtpamod was produced using the following method a sample of modified dtpa with the mass of a0mg was quantitatively transferred to a volumetric flask of a0ml and dissolved in a0ml of nahco3 solution by heating to a0°c after that the volume was adjusted with the same solvent up to the mark in order to reduce the p size the container with suspension was heated in water to a0°c and treated with ultrasound for a0min 1tomsk polytechnic university lenina avenue tomsk russia 2tomsk national research medical center russian academy of sciences kooperativny street tomsk russia email sadkintpuruscientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0cfigure a0 scheme for determining the sentinel lymph node using nanocolloid radiopharmaceuticals radiopharmaceutical sentinel lymph node detectorfigure a0 the general scheme for the synthesis of 99mtcdtpamodwhich reduced the average p radius up to a0nm the general scheme for the synthesis of 99mtcdtpamod is shown in fig a0the second type of colloids is iron nanops coated with a carbon shell of fec fig a03a these ps were obtained from the institute of metal physics urb ras ekaterinburg russia in order to impart lipophilic properties to ironcarbon ps and to increase their stability in solution in the form of a colloid a technique for preliminary deposition of anic radicals aryldiazonium tosylates adt onto the surface of these ps has been developed an effective method for the synthesis of adt followed by their application onto the carbon surface of ps was developed at the tomsk polytechnic university22 the general scheme for the synthesis of fec ps and their subsequent interaction with 99mtc is shown in fig a03bin the third type of colloids technetium99m was adsorbed on aluminum oxide powder a powder of lowtemperature cubic modification of gammaoxide al2o3 prepared from aluminum hydroxide powder aloh3 by its calcination in a muffle furnace was used the substance was synthesized in tomsk polytechnic universitya reducing agent”tin ii chloride dihydrate was used in order to obtain complexes of 99mtc with colloidsgelatin powdered ph eur uspnf pure pharma grade cas number was purchased from applichem gmbh darmstadt germanymethods method for preparation of 99mtc labeled nanocells the introduction of the radioactive label 99mtc into a colloidal substance was carried out by mixing of the selected substance with the reducing agent sncl2ˆ™2h2o “ a0mgml in different ratios and then adding a a0ml of eluate of 99mtc “ a0mbqml to the mixtures the mixtures were incubated for a0min at a temperature of “ a0°c the preparation is ready for use after cooling at room temperature the reducing agent sncl2ˆ™2h2o was used as a hydrochloric acid solution the amount of a0g of tin chloride ii is added to the vial and a0ml of a0m hydrochloric acid hcl scientific reports 101038s41598020709912vol1234567890wwwnaturecomscientificreports 0cfigure a0 a carbon encapsulated iron nanop b the general scheme for the synthesis of fec psis then added for its preparation after dissolution the volume is adjusted with distilled water to a0ml dissolution was carried out in an inert gas argon mediumdetermination of the size of 99mtc labeled colloidal ps the determination of the size of the labeled nanocolloids was carried out by spectroscopy on a nanophox p size analyzer œsympatec gmbh germany and also by a technique based on measuring the activity of the suspension before and after filtering it successively through filters with predetermined pore sizes and a0nm three samples were taken with a volume of a0μl from each initial solution and filtrates for the subsequent measurement of their activity calculations of the yield of products with different p sizes were determined according to the formulas given belowc220 avc ˆ’ a1avc c100 a1 ˆ’ a2a1 c50 a2 ˆ’ a3a2where avc is the activity of the initial suspension prior to filtration a1 is the activity measured after filtration through a a0nm filter a2 is the activity after filtration through a0nm filter a3 is the activity measured after filtration through a0nm filterin parallel determination of the radiochemical purity rcp of preparations by thin layer chromatography method was carried outthin‘layer chromatography tlc procedure to determine radiochemical purity of 99mtc“nanocolloid a0 µl of prepared sample was spotted on silicagel impregnated strip sorbfil russia — a0 cm to determine scientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0camount of sncl2ˆ™2h2o mga[sn99mtc]a atcviia table change in relative activities of the complex [sn99mtc] and 99mtc viipertechnetate content of the radiopharmaceutical sample first strip was developed using acetone as the mobile phase time of chromatography a0min in this system pertechnetate migrated with the front of the mobile phase rf to determine the colloid content of the preparations the second strip was developed using ethanolwaterammonium hydroxide as the mobile phase time of chromatography a0min in this system the 99mtc“nanocolloid migrated with the front of the mobile phase rf stability the stability of 99mtc“nanocolloid was studied in a0vitro by mixing of a0ml of normal serum and a0ml of 99mtc“nanocolloid following by incubation at a0°c for a0h at different time points a0h a0h and a0h a0ml aliquots of complex were removed and evaluated for radiochemical purity using tlc24determination of the functional suitability of preparations for scintigraphic detection of sln a study to assess the functional suitability of new nanocolloid rps was performed in series of experiments involving white wistar male rats weighing “ a0g injection of rp in a dose of “ a0mbq was performed between the first and second fingers of the rat™s hind paw the animals were anesthetized with ether before the subcutaneous injection and during the scintigraphic study since the introduction the kinetics of radiopharmaceutical distribution throughout ans and tissues was recorded by a framebyframe recording for a0min one frame” a0s in a — pixel matrix static scintigraphy was performed after and a0h in the anterior and posterior projections in a matrix of — with a set of pulses scintigraphy of animals was performed on an ecam signature gamma camera siemens germany the results of scintigraphic studies determined the percentage of accumulation of rp in the lymph node and the injection site the maintenance and participation of the animals in the experiment was carried out in accordance with the rules adopted by the œeuropean convention for the protection of vertebrates used for experiments or other scientific purposes strasbourg the experimental protocols were approved by cancer research institute biomedical ethics committee protocol number all invasive manipulations with animals were performed using inhalation or drug anesthesiastatistical analysis all mean values are expressed as idg ± sd data were analyzed statistically using methods of general statistics with a commercially available software package œstatistics for windows statsoft inc version results and discussionto carry out the labeling of colloids 99mtc extracted from a standard generator in the form of pertechnetate ions contained in a nacl solution was used it has a higher degree of oxidation vii in this chemical form and is not prone to complex formation a reducing agent”tin ii chloride dihydrate widely used for the preparation of various compounds labeled with 99mtc to was used to reduce its valence state in order to obtain complexes with nanoscale ps25 as a result of the reaction of these components the appearance of an untargeted colloid is also possible due to the hydrolysis of excess sncl2·2h2o or the additional formation of a complex of reduced 99mtc with tin26 all this required preliminary experimental studies to establish the necessary and sufficient amount of sncl2·2h2o in the reaction mixtureduring the experiments it was found that the optimal concentration of sn ii in the composition of the reaction mixture when it interacts with 99mtc should be in the range of “ a0mgml table a0it was found that when the eluate with the preliminarily reduced 99mtc vii was added to the nanops the sn ii concentration introduced in the rp was csn a0mgml almost the entire amount of 99mtc has time to enter the composition of the largesize complex with tin even before its mixing with nanops this means that the sequence of the introduction and mixing of the reagents has a great influence on the labeling process especially it concerns the introduction of the sn ii solution into the reaction mixture in this connection the reduction of 99mtc with tin ii was carried out in the presence of the selected substance in this case we can observe a competitive redistribution of the radionuclide between the substance and the tin complex the technique of applying of the 99mtc label to the surface of nanosized ps is given in the previous sectionas a result of the studies reagent compositions and conditions for obtaining three nanocolloid rps were determined table a0 shows their components as well as the radiochemical purity and yield of the target colloid with p sizes of “ a0nmproceeding from the chromatograms it follows that the content of radiochemical impurity of unreduced 99mtc vii in the obtained preparations is “ preliminary tests of these preparations on experimental animals showed that accumulation in lymph nodes is practically not observed although colloids have p sizes in the required range from to a0nm scintigrams of rats obtained after subcutaneous administration of a technetium99m labeled nanocolloid based on aluminum oxide are shown in fig a0scientific reports 101038s41598020709912vol1234567890wwwnaturecomscientificreports 0ccomposition of the preparation per a0mldtpamod a0mg 99mtc “ a0mbq sncl2ˆ™2h2o a0mg n fec a0mg 99mtc “ a0mbq sncl2ˆ™2h2o a0mg n al2o3 a0mg 99mtc “ a0mbq sncl2ˆ™2h2o a0mg n colloid yield “ a0nm rcp ± ± ± table composition of reagents for production of technocium99 a0m nanocolloidsfigure a0 distribution of the preparation in the rat when the preparation is administered [al2o3 99mtc sn ii] a immediately after the administration of the drug b a0min after the administration c a0min after the administrationcomposition of preparations per a0mlal2o3 a0mg 99mtc “ a0mbq sncl2ˆ™2h2o a0mg g a0mg n dtpamod a0mg 99mtc “ a0mbq sncl2ˆ™2h2o a0mg g a0mg n fec a0mg 99mtc “ a0mbq sncl2ˆ™2h2o a0mg g a0mg n yield of colloid “ a0nm rcp ± ± ± table indicators of rcp and the yield of a colloid with a fraction of “ a0nm after the introduction of gelatin in the reagentsscintigrams showed that the drug remains at the injection point for a0h without significant accumulation of 99mtc in the blood of animals which indicates a strong fixation of the radionuclide on the surface of the nanocolloid along with this positive point it should be noted that accumulation of the preparation in the lymph nodes is not observed gelatin g was introduced into the reaction mixture in this connection to increase the œmobility of the labeled ps and increase the speed of their movement through the lymph system matrix systems based on gelatin provide a fairly uniform distribution of the immobilized substance and in this case prevents the formation of a large tin colloid with 99mtc the results of the experiments showed that the addition of gelatin “ a0mgml to the reagent additionally provides an increase in the yield of the target colloid with p sizes of “ a0nm table a0in addition the size of these ps was determined on a nanophox p analyzer the obtained dependence of the change in the density of the distribution of the number of ps on their size constructed from the results of a threedimensional measurement of the preparations is shown in fig a0 a b c the average p size diameter is and a0nm respectivelystability test showed that complex 99mtc“nanocolloid was stable in normal serum at least for a0h radiochemical purity of the tracer at the end of the experiment was ± a study of the functional suitability of the obtained radioactive colloids for the scintigraphic imaging of the sentinel lymph nodes showed that these preparations provide a good level of accumulation in the sentinel lymph nodes fig a0 table a0 displays the al2o3 99mtc dtpamod 99mtc fec 99mtc biodistribution data at different time points postinjectionthe level of accumulation of preparations in the lymph nodes is “ of the total injected activityconclusionas a result of the studies the composition of the reagents and the conditions for the synthesis of three nanocolloid rps were determined an experimental dependence of the change in the content of 99mtc vii impurities on the concentration of tin ii was established and its minimum amount a0mgml was determined to reach a rhp greater than in this case the yield of the target colloid with p sizes of ± a0nm is “ preliminary tests of the developed preparations on experimental animals showed that accumulation of rp in lymph nodes is practically not observed although the sizes of colloidal ps are in the required range increase in the speed of transportation of colloids through the lymphatic system was achieved by the introduction of gelatin in the composition “ a0mgml in addition there was an increase in the yield of the colloid scientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0cfigure a0 change in the density of the distribution of the number of ps from their size in radiopharmaceuticals a œ99mtcal2o3 b œ99mtcfec c œ99mtcdtpamodwith p sizes of “ a0nm to “ with radiochemical purity of the preparations of “ repeated studies in experimental animals have shown that all synthesized nanocolloid preparations provide a good level of scientific reports 101038s41598020709912vol1234567890wwwnaturecomscientificreports 0cstomachtime h99mtcal2o399mtcdtpamod99mtcfec ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± figure a0 distribution of the preparation in the rat with injection of suspension [al2o3 99mtc sn ii gelatin] a immediately after the administration of the preparation b a0min after the administration c a0min after the administration d a0min after the administration the numbers indicate ”lymph node ”site of preparation administrationg ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± liver ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± spleen ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± bloodmlheart ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± lungs ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± table biodistribution data up to a0h after injection of “ a0mbq of 99mtc in healthy male rats data represent the average value n accumulation in the sln thus the level of accumulation of rp œ99mtcdtpamod and rp œ99mtcfecadt in the sln is and respectively at the same time the accumulation level of the preparation based on aluminum oxide is of the total input activityreceived march accepted july references jakobsen j k sentinel node biopsy in urooncology a history of the development of a promising concept urol oncol “ weixler b et al sentinel lymph node mapping with isosulfan blue or indocyanine green in colon cancer shows comparable results and identifies patients with decreased survival a prospective singlecenter trial world j surg 101007s0026 beasley g m et al sentinel lymph node biopsy for recurrent elanoma a multicenter study ann surg oncol moser j et al sentinel node biopsy in melanoma a singlecentre experience with consecutive patients br j dermatol 101245s1043 “ buda a et al optimizing strategies for sentinel lymph node mapping in earlystage cervical and endometrial cancer comparison of realtime fluorescence with indocyanine green and methylene blue int j gynecol cancer “ scientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0c“ sahbai s et al pericervical injection of 99mtcnanocolloid is superior to peritumoral injection for sentinel lymph node detection of endometrial cancer in spectct clin nucl med “ hoogendam j p et al 99mtcnanocolloid spectmri fusion for the selective assessment of nonenlarged sentinel lymph nodes in patients with earlystage cervical cancer j nucl med “ stoffels i leyh j p¶ppel t schadendorf d klode j evaluation of a radioactive and fluorescent hybrid tracer for sentinel lymph node biopsy in head and neck malignancies prospective randomized clinical trial to compare icg99mtcnanocolloid hybrid tracer versus 99mtcnanocolloid eur j nucl med mol imaging “ beisani m et al initial experience in sentinel lymph node detection in pancreatic cancer rev esp med nucl imagen mol schubert t uphoff j henke r p wawroschek f winter a reliability of radioisotopeguided sentinel lymph node biopsy in penile cancer verification in consideration of the european guidelines bmc urol “ jaukovic l et al lymphoscintigraphy and sentinel lymph node biopsy in cutaneous melanoma staging and treatment decisions hell j nucl med “ subramanian s pandey u shah s rangarajan v samuel g an indigenous singlevial kit formulation of human serum albumin nanocolloid for use in sentinel lymph node detection nucl med commun “ ruizdom­nguez j m ibarzservio l garc­ade manuel g calaf peris© o intraoperative injection of 99mtcnanocolloid for localization of nonpalpable intratesticular tumours in ansparing surgery actas urol “ schauer a j specific developments in sentinel node labling using 99mtccolloids in the sentinel lymph node concept springer berlin wang y et al gasphase chemistry of technetium carbonyl complexes chem phys “ o™connor m k et al comparison of tc99m maraciclatide and tc99m sestamibi molecular breast imaging in patients with wang j zhang r evaluation of 99mtcmibi in thyroid gland imaging for the diagnosis of amiodaroneinduced thyrotoxicosis suspected breast cancer ejnmmi res br j radiol costa p et al scintigraphic imaging with technetium99mlabelled ceftizoxime is a reliable technique for the diagnosis of deep sternal wound infection in rats acta cir bras “ vera d r wallace a m hoh c k mattrey r f a synthetic macromolecule for sentinel node detection 99mtcdtpamannosyldextran j nucl med “ hoh c k wallace a m vera d r preclinical studies of [99mtc]dtpamannosyldextran nucl med biol “ skuridin v et al modified dtpa moleculebased nanocolloid radiopharmaceuticals j radioanal nucl chem “ filimonov v d et al unusually stable versatile and pure arenediazonium tosylates their preparation structures and synthetic applicability lett “ lukasz k thin layer chromatography in drug analysis “ crc press london skuridin v et al radiopharmaceutical drug based on aluminum oxide indian j sci technol 1017485 ijst2015v8i36 sazonova s i et al synthesis and experimental study of norfloxacin labeled with technecium99m as a potential agent for infection imaging iran j nucl med “ skuridin v s et al synthesis and biological characterization of 99mtclabeled ciprofloxacin pharm chem j “ acknowledgementsthis work was financially supported by ministry of education and science of the russian federation rfmefi57514x0034author contributionsvs conducting experimental research analysis and interpretation of the data final approval for manuscript publication vs development of the concept and direction of research analysis and interpretation of the data validation of critical intellectual content final approval for manuscript publication en development of the concept and direction of research analysis and interpretation of the data validation of critical intellectual content final approval for manuscript publication es development of the concept and direction of research experimental research development of analytical control methods for the developed kits and radiopharmaceuticals analysis and interpretation of the data validation of critical intellectual content final approval for manuscript publication ar conducting experimental research analysis and interpretation of the data final approval for manuscript publication nv conducting experimental research analysis and interpretation of the data final approval for manuscript publication rz conducting tests of the functional suitability of drugs preparation of the section evaluation of the functional suitability of the preparation by determining its pharmacokinetic characteristics and figures „– final approval of the manuscript for publication of the manuscriptcompeting interests the authors declare no competing interestsadditional informationcorrespondence and requests for materials should be addressed to vsreprints and permissions information is available at wwwnaturecomreprintspublisher™s note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsscientific reports 101038s41598020709912vol1234567890wwwnaturecomscientificreports 0copen access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate if changes were made the images or other third party material in this are included in the ™s creative commons license unless indicated otherwise in a credit line to the material if material is not included in the ™s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this license visit httpcreat iveco mmons licen sesby40 the authors scientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0c'
Colon_Cancer
" lymph node staging of ductal adenocarcinoma of the pancreatic head pdac by crosssectionalimaging is limited the aim of this study was to determine the diagnostic accuracy of expanded criteria in nodalstaging in pdac patientsmethods sixtysix patients with histologically confirmed pdac that underwent primary surgery were included inthis retrospective irbapproved study crosssectional imaging studies ct andor mri were evaluated by aradiologist blinded to histopathology number and size of lymph nodes were measured shortaxis diameter andcharacterized in terms of expanded morphological criteria of border contour spiculated lobulated and indistinctand texture homogeneous or inhomogeneous sensitivities and specificities were calculated with histopathologyas a reference standardresults fortyeight of patients had histologically confirmed lymph node metastases pn sensitivityspecificity and youden™s index for the criterion œsize were and for œinhomogeneous signal intensity and and for œborder contour and respectively there was a significant associationbetween the number of visible lymph nodes on preoperative ct and lymph node involvement pn p lymph node staging in pdac is mainly limited due to low sensitivity for detection of metastatic diseaseusing expanded morphological criteria instead of size did not improve regional nodal staging due to sensitivityremaining low combining specific criteria yields improved sensitivity with specificity and ppv remaining highkeywords ductal adenocarcinoma of the pancreatic head staging lymph nodes computed tomography magneticresonance imaging crosssectional imaging neoadjuvant therapy correspondence florianlochcharitede1charit “ universittsmedizin berlin corporate member of freie universittberlin humboldtuniversitt zu berlin and berlin institute of healthdepartment of surgery campus benjamin franklin hindenburgdamm berlin germanyfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cloch world of surgical oncology page of pancreatic cancer remains one of the most lethal malignancies being the fourth leading cause of cancerdeath in the usa and predicted to be the secondleading cause of cancer death by the overall5year survival after diagnosis is and at thetime of diagnosis the main proportion of patients hasadvancedstage disease leaving only “ qualifiedfor resective surgery pancreatic cancer is locatedin the head of the pancreas in of the cases even after successful resective surgery in patients withcancer of the pancreatic head the 5year survival remains as low as these data underline theimportance of establishing multimodaltherapeuticconcepts for patients with pancreatic cancer as perother entities of abdominal cancerapart from the potential to increase the resectabilityrate of pancreatic cancer by neoadjuvant therapy [ ]there is evidence that patients which are successfullydownstaged from nodepositive disease cn1 to nodenegative disease ypn0 prior to surgery benefit in termsof higher 5year survival rate this would qualifynodalinvolvement as a sufficient basis for indicatingneoadjuvant therapy yet even given advanced imagingtechnologies identifying lymph node metastasis remainschallenging consequently the indication of a potentiallyeffective neoadjuvant therapy cn with side effects inlymph nodepositive patients cn is mainly based onunreliable clinical stagingthe established criterion for lymph node involvement in pancreatic cancer is size using the size underlies the assumption that tumor spread to regionallymph nodes leads to an enlargement of the respective lymph node the usual cutoff value is a shortaxis diameter of mm [ “] it has been shownthough that lymph nodes of ‰¥ mm are not seenmore frequently in patients with histopathologicallymph node involvement pn in various othertumor entities expanded morphological criteria suchas texture and border contour oflymph nodes areused for the assessment oflymph node malignancyon both computed tomography ct and magneticresonance mr imaging this is utilized in order toimprove the accuracy of lymph node staging [“]by applying morphological criteria instead of brown size criterion alone the sensitivity was improvedfrom to and the specificity from to inlymph node staging of rectal cancer thus the aim of this study was to determine thelymph node staging in patients withaccuracy ofductal adenocarcinoma ofthe pancreatic head byboth computed tomography and magnetic resonanceimaging using sizeand expanded morphologicalcriteriamaterial and methodspatientsin this retrospective singlecenter study approved by thelocal ethics committee consecutive patients with histologically proven ductal adenocarcinoma of the pancreatichead that underwent primary surgery between february and november at the department of surgerycampus benjamin franklin charit”university medicine berlin germany were included patients were retrieved from the database of our pancreatic cancercenter certified by the german cancer society n inclusion criteria were primary oncologic tumor resection and the presence of preoperative crosssectional imaging of sufficient quality see below exclusion criteriawere neoadjuvant therapy presence of a potential simultaneous cause of lymphadenopathy of the upper abdominal region eg abdominal lymphoma neuroendocrinetumor and main tumor mass located outside the pancreatic head on histopathology the process of patientselection with the respective reasons for inclusion andexclusion is shown in fig crosssectional imagingall images were retrospectively analyzed for the purposeof this study by a single abdominal radiologist with morethan years of experience in staging of tumors of the visceral ans blinded to the results of histopathologyall crosssectional imaging studies were assessed forsufficient image quality by the radiologist prior to commencement for ct imaging the minimum quality wasdefined as either thinsection ct ‰ mm reconstructedslice thickness or contrastenhanced ct with a slicethickness of ‰ mm for mri minimum quality was defined as availability of an axial t2weighted sequencewith fat suppression slice thickness ‰ mm in combination with a venous phase postcontrast 3d gradientecho sequence slice thickness ‰ mmfor lymph node assessment all visible regional lymphnodes in the field of view were recorded on a score chartand the total number of visible lymph nodes per patientwas calculated then for each patient all lymph nodeswere characterized in terms of size long and shortaxisdiameter in millimeters and the expanded morphological criteria border contour lobulated spiculated indistinct or unaltered and texture homogeneous orinhomogeneous fig based on kim regional lymph nodes of the pancreas are defined asthe following lymph node station numbers 8a8p 11p 11d 12a 12b 12p13a 13b14p14d 17a17b and in all cases in which a lymph node wasnot definitively regional correlation with postoperativecrosssectional imaging was performed to assess whetherthe lymph node was resected or not only resectedlymph nodes were analyzed in this study 0cloch world of surgical oncology page of fig flowchart of patient recruitment the process of patient selection with the respective reasons for inclusion and exclusion is showna second radiologist with more than years of experience in staging of tumors of the visceral ans alsoblinded to the results of histopathology evaluated thect examinations of a representative subgroup of patients for evaluation of interobserver agreementsurgeryall patients underwent primary oncologic pyloruspreserving pancreaticoduodenectomy or whipple procedure with complete lymphadenectomy of the regionallymph nodes mentioned aboveformalinembedded surgicalhistopathologythe original histopathological reportsfor the studyspecimens wereusingreviewed cancer of the pancreatic head was defined as amalignant tumor located within the pancreas to the rightof the superior mesenteric vein and portal vein each patient with histologically proven lymph node metastaseswas classified as nodepositive pn regardless of thenumber of metastatic lymph nodes patients without anymetastatic lymph nodes were classified as nodenegativepn the ratio of metastatic lymph nodes vs the totalnumber of retrieved lymph nodes was documented infig morphological characterization of lymph nodes based on kim the morphological criteria for lymph node assessment used inthis study are shown smooth and homogeneous lymph nodes were considered normal 0cloch world of surgical oncology page of the histopathological report eg or tumorswere classified according to their respective tnm stageusing the 8th edition of tnm classification of malignant tumors table demographic data of patients with ductal adenocarcinomaof the pancreatic head undergoing primary tumor resectionpatientsagen comparison of crosssectional imaging andhistopathologysensitivity specificity and positive predictive value ofthe nodal status using ct and mri with histopathologyas a reference standard were calculated for lymph nodeinvolvement using size and morphological criteria specifically nodal involvement criteria were based on eithersize shortaxis diameter altered border contour lobulated spiculated or indistinct and inhomogeneous signal intensity fig ct and mri examinations wereconsidered nodepositive cnif at least one lymphnode met one of the respective criteria used for involvement if no lymph node with the respective criteria wasseen on ct or mri then the examination was considered nodenegative cnstatistical analysissensitivities specificities and positive predictive valueppv for the size criterion and all morphological criteria were calculated for their respective cutoff valuesan index summarizing the sensitivity and specificity foryouden™s index was calculated sensitivity specificityˆ’ the number of lymph nodes visible on ctand mr images in the group with pn and withoutpn nodal metastases was compared using the mannwhitney u test when calculating the association between ct and mri criteria and lymph node positivitythe χ2test was used interobserver agreement was calculated using cohen™s kappa statistic a p value of ‰ was considered to indicate a statistically significantdifferenceresultspatientssixtysix patients were included in the study fig with the characteristics of the patients presented intable sixty of these patients were staged by preoperative ct twelve of which had additional stagingby mri and six patients were staged by only mri intwo patientsthe mri examinations were excludeddue to insufficient imaging quality both patients hadsufficient staging by ct and were therefore includedin the study of the patients patients receivedpreoperative biliary drainage eight ofthem werestaged by ct only and two by mri onlycomputed tomography ctlymph nodes were detected by ct in ofthe patients the median number of visible lymph nodesmedian age yearsage range yearssexfemalemalecrosssectional imagingct onlyct and mrimri onlyhistopathological stagingpnpnpt1pt2pt3“ was range “ the smallest visible lymph node was mm of size whereas the largest measured mmshortaxis diameter the mean time between ct andsurgery was days with a median of days range “the slice thickness in of the ct examinations was mm or less in five ct examinations slice thickness was mmsize criterion for lymph node involvement onpreoperative ctfigure shows the percentage of patients with pnand without pn lymph node metastases in which alymph node of the respective size was visible “ mmin table sensitivity specificity and youden™s indexare presented for the respective cutoff values lymphnodes of small and medium size “ mm were visiblein patients with “ “ pn and withoutlymph node metastases “ “ pnineven frequency large lymph nodes “ mm wereseen more frequently in the lymph nodepositive group“ “ pnthan in the lymph nodenegative group “ “ pn the maximumvalue of youden™s index for the size criterion was j ci when a cutoff value of mmwas applied yielding a sensitivity of and specificityof additionallylymph nodesgreater than mm on preoperative ct and the histopathological confirmation of a lymph node metastasispn showed a trend towards significance p the presence of 0cloch world of surgical oncology page of fig graph showing lymph node size and morphological criteria of lymph nodepositive and negative patients frequency of regional lymphnodes of the pancreatic head in percent xaxis with different shortaxis diameters and morphological features yaxis in patients with pn redbars or without histologically proven lymph node metastases pn blue bars on preoperative ct imagingexpanded morphological criteria for lymph node involvementon preoperative ctfigure shows the percentage of patients with pnand pn without lymph node metastases in which alymph node of the respective morphological criterionwas visible and table shows the sensitivity specificityand youden™s index of the respective criterionlymph nodes of lobulated border contour were visiblewith a similar frequency in patients with pn and without lymph node metastases pn lymph nodes of spiculated or indistinct bordercontour were only occasionally detected in both groups vs in the lymph nodepositivegroup pn and vs in the lymphnodenegative group pnlymph nodes of inhomogeneous signal intensity were detected in only one patient of the lymph nodenegative group pn and more frequently in patients of the lymphnodepositive group pn resulting in the maximum value of youden™s index for the morphological criteriaj ci consisting of a sensitivity of and a specificity of the ppv was comparison of size with expanded morphological criteriathe maximum value of the youden™s index of the œsizecriterion was j ci cutoff mmwhich is not inferior to the maximum value of the morphological criteria j ci inhomogeneoussignal intensity figure displays respective ct images ofpatients with and without lymph node metastasesfig ct images of patients with and without lymph node metastases a patient with enlarged suspicious lymph node adjacent to the portalvein and hepatic artery who had no lymph node metastases on pathology b patient with enlarged suspicious lymph node adjacent to thehepatic artery who had lymph node metastases on pathology c patient with multiple suspicious lymph nodes based on size and inhomogeneitywho had lymph node metastases on pathology 0cloch world of surgical oncology page of table sensitivity specificity ppv and youden™s index for cutoff values and morphological criteria by ct and mrisensitivity specificity ppvyouden™s indexctsize cutoff value mm mm mm mm mm mm mm mmmorphological criterionlobulatedspiculatedindistinctinhomogeneous mrisize cutoff value mm mm mm mm mm mm mm mm mmmorphological criterionlobulatedspiculatedindistinctnot visibleinhomogeneousnot visible ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ number of visible lymph nodesthere was a significant association between number of visible lymph nodes seen on preoperativect and histopathologicallymph node involvementpn p seven or more lymph nodes were seen on preoperative ct in of patients with lymph nodemetastases pn and in of patients without lymph node metastasis pn p this resulted in a specificity of youden™s index of and ppv of combination of number size and expanded morphologiccriteria on preoperative ctcombining the size criterion and the morphological criterion with the respective highest youden™s index cutoff mm and œinhomogeneous signalintensity andthe criterion œvisible lymph nodes n ‰¥  was significantly associated with nodal metastases pn p for this combined criterion specificity was sensitivity ppv and youden™s index ci interobserver agreementinterobserver agreement was calculated for patientspn pn vs pnfor the criteria sizemorphology and number of visible lymph nodes withthe respective highest youden™s indexinterobserveragreement was substantial for size mm cutoff κ p moderate for the presence of seven ormore lymph nodes κ p and fair forthe morphological criterion inhomogeneous signal intensity κ p magnetic resonance imaging mrilymph nodes were detected in of patientson preoperative mri the median number of visiblelymph nodes was range “ there was no significantassociation between number of visible lymph nodes seenon preoperative mri and histopathological lymph nodeinvolvement pn p the smallest visible lymph node was mm of sizewhereas the largest measured mm shortaxis diameter the mean time between mri and surgery was days with a median of days range “the cutoff values of the highest diagnostic value were mm or mm for the œsize criterion sensitivity specificity youden™s index the presence ofa lymph node of these sizes was not associated with lymphnode metastases pn p lobulated and spiculated lymph nodes were only seen in a few patients n and n and indistinct and inhomogeneous lymphnodes were not seen at all table discussionin this retrospective singlecenter study on lymph nodestaging by ct in ductal adenocarcinoma of the pancreatic head we could show that the morphologic criteriaœinhomogeneous signal intensity and œsize are specificfor regional nodal metastatic disease replacing the sizecriterion by morphologic criteria however did not improve diagnostic accuracy due to sensitivity remaininglow combining specific criteria yields improved sensitivity with specificity remaining highby ct lymph nodes of “ mm in shortaxis diameterwere seen just as often in patients with and without 0cloch world of surgical oncology page of lymph node metastases resulting in poor discriminationlarger lymph nodes mm had a higher prevalence inthe lymph nodepositive group leading to high specificity however these lymph nodes mm or mmwere seen infrequently resulting in a rather low sensitivity the maximum value of the youden™s index for thesize criterion of was achieved when a cutoff valueof mm was applied consisting of a specificity of and sensitivity of yielding a ppv of as for morphologic criteria lymph nodes of lobulatedborder contour were seen in about half of the patients ofboth groups pn and pn and therefore isa criterion that is not suitable to differentiate betweenthe groups lymph nodes of spiculated and indistinctborder contour were seen in few cases in both patientgroups only pn and versus pn and making them poor diagnostic criteria however lymphnodes of inhomogeneous signal intensity were visible in of patients with lymph node metastases pn andonly in of the patients without lymph node metastases pn resulting in a youden™s index of whichwas the maximum value for the morphological criteriaand a ppv of ideally a good discriminator for nodal metastases isnegative in patients without nodal involvement and positive for tumors with lymph node metastases in ourstudy each criterion ie size as well as different morphological features only met one of these prerequisitesthe size criterion mm as well as the presence of alymph node of inhomogeneous signal intensity as morphological criterion turned out to be negative in patientswithout nodal involvement pn and therefore highlyspecific yet lymph nodes of the respective characteristicwere not positive in a sufficiently high number of tumorswith lymph node metastases pn to reach high levelsof sensitivity and consequently did not have a significantdiagnostic valuethe maximum value of the youden™s index for the sizecriterion was when a cutoff value of mm wasapplied and for the morphological criteria whenthe criterion œinhomogeneous signal intensity was usedshowing that morphologic criteria do not yield in higherdiagnostic value than lymph node size in adenocarcinoma of the pancreatic head pdac patients this iscontrary to the findings of brown in rectal cancer one reason might be that brown used mri toassess morphologic criteria which has a higher soft tissuecontrast compared to ct which was used in most patients in our studyinterestingly we could show that with preoperativect the presence of seven or more lymph nodes wasseen more often in patients with lymph node metastasispn than in those without metastasis pn p when applying this as a sole criterion cn for lymphnode metastasis pn this led to a sensitivity of aspecificity of ppv of and youden™s indexof in diagnostic test analysis criteria can be combinedin mainly two ways sensitive criteria can be taken together to improve specificity or specific criteria canbe accumulated to improve sensitivity when combining the highly specific criteria size cutoff value mm inhomogeneous signal intensity and number ofvisible lymph nodes n ‰¥ a highly significant association with nodal metastases pn p wasfound consequently the ct examination was considered nodepositive cn when at least one of thesecriteria was met the application of this criterion improved the sensitivity to with a remaining specificity of and ppv resulting in an alsoimproved youden™s index of the results of the mri examinations must be viewedin a rather descriptive manner since the sample size waslimited n lymph nodes were detected in the majority of examinations generally allowing theevaluation of lymph nodes by mri as well upper abdominal mri generally has a lower spatial resolutionbut a higher soft tissue contrast compared to ct forthe size criterion a cutoff value of mm or mm ledto the best diagnostic results sensitivity specificity youden™s index lymph nodes of abnormal morphological criteria were seen in only veryfew patients table the main limitation of this study is the retrospectivestudy design in which a nodebynode comparison ofcrosssectionalimaging with histopathology was notpossible this was of minor importance though sincelow sensitivity was the main factor that led to compromised diagnostic performance in our study we werealso able to correlate with postoperative crosssectionalimaging in all cases in which it was unclear whether alymph node had been resected during surgery or notalso in our cohort only patients who had subsequentsurgery were included presuming lower tumor stage ascompared to the average patient who undergoes imagingfor presurgical workupthe strength of our singlecenter study is reinforcedby a defined number of surgeons a high standardizationof the ct technique and an experienced radiologist whoperformed the analysisthe results of our study are consistent with recent andinitial data demonstrating that clinical staging by lowsensitivity underestimates histopathological lymph nodeinvolvement pn [ “] however by adding thecriterion œinhomogeneous signal intensity and œnumberof visible lymph nodes to the size criterion we wereable to increase the sensitivity to in comparison toprevious findings roche nanashima 0cloch world of surgical oncology page of and cao with specificity remainingsufficientan additional imaging modality that has shown the potential to improve the sensitivity of detecting metastaticdisease is positron emission tomographycomputed tomography petct however a beneficial role ofpetct in locoregional nodal staging could not be established to date the majority of initial as well as recentstudies show very limited sensitivities for nodal status between and [“] the petpanc study evaluated the incremental diagnostic accuracy and impact ofpetct in addition to multidetector ct in patients withsuspected pancreatic cancer in a prospective multicenterstudy that included patients in this study significantlymore patients with stage iib disease pn were correctlystaged by petct than by multidetector ct p but this only led to a moderate sensitivity of for petct versus for multidetector ct endoscopic ultrasonography eus is a wellestablisheddiagnostic procedure in pancreatic cancer with the benefitof a dynamic diagnostic examination that allows fineneedle aspiration for cytologic diagnosis two metaanalyses evaluating diagnostic accuracy of eus for locoregional nodal staging the pooled sensitivities and specificities were and li studies n patients and and nawaz studiesn patients advanced techniques such ascontrastenhanced eus cheus and eus elastographyare currently in evaluation to date ct remains the standard staging imaging modality recommended by nccn guidelines for locoregional staging of pancreatic cancer neither petctnor eus yields reliable diagnostic accuracy for nodalstagingan advantageous effect on resectability and overallsurvival os in unresectable cases including both borderline resectable and unresectable of pdac by multimodality therapy including neoadjuvanttherapy hasalready been described in several studies the benefit of neoadjuvant therapy in cases of primarily resectable disease at diagnosis is yet less revealedseveral phase ii trials showed that patients who completed neoadjuvant chemoradiation without progressivedisease at restaging had a higher chance of achieving r0resection and consequently higher median and oswhen compared to historical data as seen in othertumor entities a potential benefit of neoadjuvant therapyon the basis of positive nodal status cn is stronglyimplied cao found that the of patients thatwere successfully downstaged from nodepositive diseasecn1 to nodenegative disease ypn0 by neoadjuvanttherapy benefit in terms of higher rates of 5year survivalypn0 vs ypn1 p this is consistent with the findings of portuondo 5yearsurvival ypn0 vs ypn1 p the nccn guidelines for pancreatic adenocarcinomaappreciates these results by stating that considerationcan be given to neoadjuvant therapy for selected patientswith resectable tumor but poor prognostic features suchas large regional lymph nodes markedly elevated ca large primary tumors extreme pain and excessiveweight loss further clarification on this matter isexpected to come from the ongoing neonax trialnct02047513 a phase ii study comparing neoadjuvant plus adjuvant with only adjuvant nabpaclitaxelplus gemcitabine therapy forresectable pancreaticcancer theiii neopa trialphasenct01900327 compares neoadjuvant chemoradiotherapy with upfront surgery of resectable pancreatichead cancer a subgroup analysis in terms of nodalstatus would present reliable dataongoinggiven the suggested benefit of neoadjuvant therapybased on lymph node staging there is an urgent need tofind criteria and modalities to further improve the diagnostic value of lymph node staging by pretherapeuticcrosssectional imaging in patients with ductal adenocarcinoma of the pancreatic head to date none of theexisting modalities and criteria accomplishes reliablenodal staging larger prospective studies are ongoingand necessary to get a more precise idea of the prognostic advantage of neoadjuvant therapy in patients with regionalcn of pdac inpretherapeutic staginglymph node metastasisslymph node staging in pdac patients when using ctmorphological criteria such as border contour or homogeneity compared to diameter cutoff values does notlead to reliable diagnostic value diagnostic accuracy islimited due to low sensitivity for detection of metastaticdisease combining specific criteria yields improved sensitivity with specificity and ppv remaining high theseresults suggest an attentive interpretation of the resultsof pretherapeutic lymph node staging particularly incases in which lymph node metastases are absentabbreviationspdac adenocarcinoma of the pancreatic head ct computed tomographymri magnetic resonance imaging pn histopathologically involved lymphnodes pn histopathologically no involved lymph nodes cn clinicallyinvolved lymph nodes cn clinically no involved lymph nodes ppv positivepredictive value petct positron emission tomographycomputed tomography eus endoscopic ultrasonography cheus contrastenhancedendoscopic ultrasonography os overall survivalacknowledgementswe acknowledge support from the german research foundation dfg andthe open access publication funds of charit “ universittsmedizin berlinauthors™ contributionsall authors were involved in data acquisition and manuscript revision theconception of the design of the study and drafting of the manuscript was 0cloch world of surgical oncology page of done by fn loch c kamphues and p asbach image analysis was performedby p asbach and assisted by fn loch image analysis of the subgroup foranalysis of interobserver agreement was performed by m haas all authorshave approved the submitted version of the manuscript and account fortheir own contribution and the accuracy as well as the integrity of the workpresentedfundingthe study was not fundedavailability of data and materialsthe datasets used during the current study are available from thecorresponding author on reasonable requestethics approval and consent to participatethe study was approved by the local ethics committee of the charituniversity medicine berlin informed consent of participation was waived bythe irb given the retrospective study design irb no ea411418consent for publicationconsent for publication is not applicable for this studycompeting intereststhe authors declare that they have no competing interestsauthor details1charit “ universittsmedizin berlin corporate member of freie universittberlin humboldtuniversitt zu berlin and berlin institute of healthdepartment of surgery campus benjamin franklin hindenburgdamm berlin germany 2charit “ universittsmedizin berlin corporatemember of freie universitt berlin humboldtuniversitt zu berlin and berlininstitute of health department of radiology campus benjamin franklinhindenburgdamm berlin germany 3the johns hopkins universityschool of medicine department of surgery n wolfe street blalock baltimore md usareceived december accepted july zeman rk cooper c zeiberg as kladakis a silverman pm marshall jl tnm staging of pancreatic carcinoma using helical ct am jroentgenol “ m¼ller mf meyenberger c bertschinger p schaer r marincek b pancreatictumors evaluation with endoscopic us ct and mr imaging radiology“ midwinter mj beveridge cj wilsdon jb bennett mk baudouin cj charnleyrm correlation between spiral computed tomography endoscopicultrasonography and findings at operation in pancreatic and ampullarytumours br j surg “ r¶sch t braig c gain t feuerbach s siewert jr schusdziarra v staging of pancreatic and ampullary carcinoma by endoscopicultrasonography comparison with conventional sonography computedtomography and angiography gastroenterology “ prenzel kl h¶lscher ah vallb¶hmer d drebber u gutschow ca m¶nig sp lymph node size and metastatic infiltration in adenocarcinoma of thepancreatic head eur j surg oncol “ rollvn e blomqvist l ¶istm¶ e hjern f csanaky g abrahamnordling mmorphological predictors for lymph node metastases on computedtomography in colon cancer abdom radiol “ brown g richards cj bourne mw newcombe rg radcliffe ag dallimorens morphologic predictors of lymph node status in rectal cancer withuse of highspatialresolution mr imaging with histopathologic comparisonradiology “kim jh beets gl kim mj kessels agh beetstan rgh highresolution mrimaging for nodal staging in rectal cancer are there any criteria in additionto the size eur j radiol “isaji s murata y kishiwada m new japanese classification of pancreaticcancer in neoptolemos j urrutia r abbruzzese j b¼chler mw editorspancreatic cancer new york springer p “ brierley jd gospo
Colon_Cancer
"cellular recognition of microbial dna is an evolutionarily conserved mechanism by which the innate immunesystem detects pathogens cyclic gmpamp synthase cgas and its downstream effector stimulator of interferongenes sting are involved in mediating fundamental innate antimicrobial immunity by promoting the release oftype i interferons ifns and other inflammatory cytokines accumulating evidence suggests that the activation ofthe cgassting axis is critical for antitumor immunity the downstream cytokines regulated by cgasstingespecially type i ifns serve as bridges connecting innate immunity with adaptive immunity accordingly a growingnumber of studies have focused on the synthesis and screening of sting pathway agonists however chronicsting activation may lead to a protumor phenotype in certain malignancies hence the cgassting signalingpathway must be orchestrated properly when sting agonists are used alone or in combination in this review wediscuss the dichotomous roles of the cgassting pathway in tumor development and the latest advances in theuse of sting agonistskeywords cgassting innate immunity type i interferon sting agonists antitumor response cancerdevelopmentintroductionthe discovery of phagocytosis in advanced the understanding of innate immunity the first line of host defenses protection againston patternrecognition receptors prrs which recognize microbialproducts coordinate antimicrobial defenses and activateinfection dependsagainstinfection byvarious pathogens correspondence zqliucsueducn juyan zheng and junluan mo contributed equally to this work1department of clinical pharmacology hunan key laboratory ofpharmacogenetics and national clinical research center for geriatricdisorders xiangya hospital central south university changsha people™s republic of china2institute of clinical pharmacology engineering research center for appliedtechnology of pharmacogenomics of ministry of education central southuniversity changsha people™s republic of chinafull list of author information is available at the end of the adaptive immunity abnormal rna or dna rnadna hybridization and cyclic dinucleotides derived frommicrobes are usually considered pathogenassociated molecular patterns pamps [ ] cells associated with innate immunity recognize different microbial pampsthrough specific prrs thereby playing key roles in hostresistance to microbial infection the pathways governing rna recognition such as retinoid acid induciblegene i rigilike receptors have been reviewed elsewhere and will not be covered herein in the case of dnarecognition one of the best known prrs is tolllike receptor tlr9 which senses extracellular cpg hypomethylated dna that has entered the cytosol through thephagosomelysosome system in addition the aim2like receptor aim2 inflammasome can be triggered afterthe entry of doublestranded dna dsdna into the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czheng molecular cancer page of cytosolic compartment which induces the proteolyticmaturation of proinflammatory cytokines such as il1and il18 and the activation of gasdermin d leading topyroptosis [“] nevertheless the most notable prr iscgas a direct cytosolic dsdna sensor which was identified by dr chen™s group in once cgas bindsto dsdna the cgassting pathway is activated to further induce the expression of type i ifns and other inflammatory cytokinesthus triggering innate immuneresponses mounting evidence suggests that cgassting signaling not only plays pivotal roles in the hostdefense against microbialinfection but also modulatestumorigenesis hence in this review we summarize themechanism of cgassting activation and elaboratefindings regarding its dual effects on tumor developmentcurrent advances in the use of sting agonists as a novelstrategy for antitumor therapy are also reviewedinsights into the cgassting signal transductioncascadecgas is an innate immune sensor that identifies variouscytosolic dsdnaincluding dna with viral bacterialmitochondrial micronuclei and retroelement originswhich can be mainly divided into pathogenderiveddna and selfdna table in the cytoplasm cgas isactivated by interacting with dsdna in a sequence[“]independent butstructural and biochemical analyses have revealed thatthe cterminal lobe of cgas contains a conserved zinclengthdependent mannerionbinding module that mediates dna binding andcgas dimerization [ ] dna ligands promotecgas activation primarily by inducing conformationalchanges around the catalytic site and in the dnabinding structures of cgasthe gscontaining loopundergoes conformational change to maintain stabilitywhich is a major mechanism of cgas activation bydna in addition to the primary dnabinding sitementioned above the secondary site located beside theprimary site is a helix formed between strands 78and several surfaceexposed loops the proximity ofthe two dnabinding sites in cgas leads to a cgasdna complex assembly in which two cgas moleculesembrace two molecules of dsdna [ ] the cgasdimers are anized in œheadtohead alignment nextto the dna and thus form stable œladderlike networks between one long curved dsdna helix or two independent dsdna strands [ ] in this way eachindividual cgasdsdna complex can be cooperativelystabilized and can lead to stronger enzymatic activitywhich may provide a possible explanation for longerdsdna as more likely to activate cgas in additionlong dna is more efficient than short dna in drivingthe liquidliquid phase separation of cgas and the formation ofcriticallydependent on the concentration of cgas and dna inthe cytoplasm cgas and dsdna are spatially concentratedcgasdimerization and activation [“] once cgas andcgas liquidlike dropletsin liquiddropletsistofacilitatetable classification of the cytosolic dsdna that activates the cgassting signaling axisclassificationselfdnasource of dsdnamicronucleipossible mechanismsrupture of the micronuclei membrane leads to exposureof chromatin dna that is recognized by cgas whichactivates the cgassting pathwayreferences mitochondrionnuclear rnapathogenderived dnadna virushsv1 hsv2 kshv adenovirus vacciniavirus cytomegalovirus papillomavirusmurine gammaherpesvirus retrovirushiv siv murine leukemia virusrna viruswest nile virus dengue virus vsvsarscov2bacterialisteria monocytogenes mycobacteriumtuberculosis listeria shigella francisellachlamydia and neisseriamitochondrial stress induces mtdna leakage into thecytosol thus activating the sting pathway and inducingproduction of cytokinesfacilitated by endogenous retroelements nuclear rnacan be reversely transcribed into dna that activatescgassting signaling dna viruses invade host cells and release pathogenderiveddna to induce sting activation[“]dna intermediates generated from reverse transcription maybe recognized by cgas to stimulate downstream stingsignaling infection with rna viruses might cause cellular damage andcell death which results in the release of cellular dna andfurther activation of the cgassting axis sarscov2 bindingto ace2 can lead to excessive angiotensin ii signaling thatactivates the sting pathway in mice[“]bacteria produce cdns such as cyclic digmp and cyclicdiamp which can directly bind to and activate sting[ “]hsv1 herpes simplex virus hsv2 herpes simplex virus kshv kaposi sarcoma“associated herpesvirus hiv human immunodeficiency virus siv simianimmunodeficiency virus vsv vesicular stomatitis virus cdns cyclic dinucleotides and sarscov2 severe acute respiratory syndrome coronavirus 0czheng molecular cancer page of dsdna interacts structural switches rearrange the catalytic pocket to enable cgas to catalyze the synthesis of²²cyclic gmpamp ²²cgamp with atp andgtp as substrates the first step in this process is theformation of a linear dinucleotide ²pppg ²²pawith atp serving as the donor and ²oh on gtp serving as the acceptor then the intermediate product flipsover in the catalytic pocket placing gtp at the donorposition and amp at the acceptor position to form asecond ²² phosphodiester bond [ ] notablyalthough dsrna or singlestrand dna ssdna is ableto bind to cgas neither can rearrange the catalyticpocket which may explain the exclusive activation ofcgas by dsdna ultimately cgamp acts as a secondmessenger to bind to and activate sting a small endoplasmic reticulum erlocated protein kd withfour putative transmembrane domains [ ] normally in a resting state sting is retained in the er byinteracting with the ca2 sensor stromalinteractionmolecule stim1 the cytosolic ligandbindingdomain lbd of sting exists as the most functionalunit capable of integrating with ²² cgamp or cdnscyclic dinucleotides such as cdiamp cdigmp or ²²cgamp from bacteria upon interaction the obviousclosure of the ligand binding pocket in the lbd is observed which is related to the activation of sting next sting transforms into a tetramer through a highorder oligomerization reaction and is translocated fromthe er to the perinuclear area facilitated by cytoplasmiccoat protein complex ii copii and adpribosylationfactor arf gtpases [ ] in the golgi sting ispalmitoylated atcys88 andcys91 a posttranslational modification necessary forsting activation modified sting recruits thekinase tankbinding kinase tbk1 in turn the cterminal domains of sting are phosphorylated bytbk1 and then phosphorylated sting recruits interferon regulatory factor irf3 which is also phosphorylated by tbk1 and dimerizes ultimately dimerizedirf3 enters the nucleus and exerts its function in thetranscription of type i ifns and interferonstimulatedgenes isgs in parallel sting can also bind toand stimulate iκb kinase ikk to mediate the production of nuclear factorκb nfκbdriven inflammatorygenes upon signal transduction termination sting istransferred to endolysosomes for degradation considering that cgamp can be transferred through gapjunctions or delivered in viralexosome packages cgassting signaling may be activated in the cytoplasmwithout dsdna [ ] moreover newly produced typei ifns activate heterodimer interferon receptors ifnar1 and ifnar2 through paracrine signaling and thusinduce the transcription of isgs [ ] in summaryonce virusderived dna and selfdna are located intwo cysteine residuesthe cytoplasm they can be sensed by cgas and a cgasdsdna complex is formed to catalyze the synthesis of ²²cgamp with atp and gtp then ²²cgamp and bacteriaderived cdns induce sting activation and mediate the release of downstream type iifns tnfα and il6 which are prerequisites for antimicrobial defense and antitumor effects the wholeprocess shows that the dsdnacgassting axis canlead to the activation of both innate and adaptive immunity fig the antitumor functions of the cgasstingsignaling pathwayrecent evidence has revealed the close association of thecgassting pathway with cancer development thissignaling pathway is generally regarded as a potent regulator of cancer immunity a stingmediated immunesupportive microenvironment can hamper malignancyoccurrence stressbytumor cell cytosolic dsdna induces sting activationunder normal circumstances dna is strictly unaffiliatedwith the cytoplasm in eukaryotic cells to avoid autoimmunity however dna leaks aberrantly in tumorcells [ ] cancer cells share common features including genome instability tumor suppressor gene mutation or deletion oxidativeand vigorousmetabolism under these intense states nuclear andmitochondrial dna is fragile and easily damaged whichleads to eventual dna leakage in the forms of micronuclei chromatin fragments andor free telomeric dna[ ] chromosomal instability cin is the primary source of cytoplasmic dna in malignant cells andis generally associated with tumor progression distantmetastasis and therapeutic tolerance excessive proliferation of cancer cells results in unstable genomes usuallychromosomal missegregation during mitosis due to defects in segregation lagging chromosomes generate micronuclei in a cellcycledependent manner the vulnerable membraneof micronuclei easily exposes the inner dna to the cytoplasm and activates the cgassting signaling axis exogenous stimuli such as chemotherapy and irradiation can also cause dna damage in addition to leakednuclear dna oxidative stressinduced mitochondrialdna leakage is another crucial initiator of sting pathway activation several anticancer treatments that precisely attack mitochondrial membranes result in effluxand cell death therefore the permeabilization of mitochondria membranes provides a reasonable explanationfor mitochondrial dna escape [ ] other sourcessuch as apoptotic cellderived dna exosomal dnaexodna and transposable elements have also beencharacterized 0czheng molecular cancer page of fig the cgassting dna sensing signaling pathway various dna derived from virus dying tumor cells or nucleus and mitochondria binds toand activates the cytosolic dna sensor cgas cgas catalyzes the synthesis of ²²cgamp in the presence of atp and gtp then ²²cgamp bindsto the er adaptor sting which also can be activated by cdns derived from bacteria upon activation sting translocates from er to golgicompartments where it activates tbk1 and ikk which phosphorylate irf3 and iκbα respectively then irf3 and iκbα dimerize and enter nucleusinitiating the transcription of type i ifn tnf and il6 the primary roles of these cytokines are reflected in host defense inflammation andantitumor immunitydemonstrated to evoke cgas“sting activation intumor cells [ ]type i ifns mediators of sting and adaptive antitumoreffectscgassting signaling exerts antitumor functions incancer cells both in an autonomous and nonautonomousmanner on the one hand dna damage can provokeacute sting signal transduction and induce cellularsenescence an irreversible cell cycle arrest state whichthwarts the aberrant proliferation of tumor cells throughacquisition ofsecretoryphenotype sasp which is associated with the releaseof abundantinflammatory mediators proteases andgrowth factors [ ] in contrast to undergoingsenescence tumor cells also directly propel apoptosisprocesses by upregulating proapoptosis protein bcl2associated x bax and downregulating the bcl2 apoptosis on the other hand stingsenescenceassociatedtheregulatoractivation in tumor cells not only facilitates the transcription of downstream type i ifns to induce dendriticcell maturation but also recruits supportive immunecells for direct nonspontaneous tumor elimination sting activation in nonmalignant cells causes tumorsuppressive effects as well sting signaling protectsagainst colitisassociated carcinomas cacs induced byazoxymethane aom and dextran sulfate sodiumdss which induce dna damage in intestinal epithelialcells and further trigger sting activation downstreamcytokines of sting signaling such as il1 and il18prevent neoplastic transformation by facilitating woundrepair more importantly sting signaling can also provoke cytotoxic t cell responses to control tumorigenesis necrotic cancer cells are commonly engulfed byantigenpresenting cells especially the basic leucine zippertranscription factor atflike batf3drivenlineage of dendritic cells dcs batf3 dcs take intumorassociated antigens and migrate towardsthe 0czheng molecular cancer page of tumordraining lymph node via the lymphatic systemwhere they crossprime tumorspecific cd8 t cellsthen cd8 t cells undergo activation and clonal expansion in the lymph nodes and are trafficked throughblood vessels to kill tumor cells in turn damaged cancercells release more antigens that are further captured bydcs the whole process forms a positive feedback loopcalled the cancerimmunity cycle tumor eradication can be achieved by multiple processes in thecancerimmunity cycle including tumor antigen captureand presentation and t cell priming and activation withtumor antigenspecific t cell priming and activationrelying on dcs and type i ifn release the involvement of type i ifns in innate immune sensing and adaptive immunity provides a reasonable hypothesis forexploring candidate prr pathways as potential immunomodulators mice lacking tlr9 myeloid differentiationprimary response gene myd88 cytosolic rna sensor mavs or the purinergic receptor p2x7r maintainintact antitumor immunity responses whereas mice deficient in sting or irf3 present with impaired cd8 tcell priming and activation [ ] in fact dying tumorcells can release multiple damageassociated molecularpatterns damps to trigger innate immune responsesin dcs among these released stimuli tumor cellderiveddna is a pivotal inducer in general the phagocytosis ofapoptotic cells causesimmune silence because ofdnasebased degradation nevertheless tumor cellreleased dna can be preserved in the dc endolysosomal compartment through an unknown mechanism cgas recognizes dna invading the cytoplasm andinduces the activation of sting cascades excretion oftype i ifns and expression of isgs additionally undersome physiological conditions such as hypoxia andacidic environments nuclear or mitochondrial dnamight be packaged in exosomes exosomal dnaexodna animates sting signaling once it is absorbedby tumorinfiltrating dcs finallytumor cellderived cgamp can also be transferred to host dcs bythe folate transporter slc19a1 and then directly bindsto sting activating it in dcs a recent study moredirectly demonstrated that cellautonomous sting promoted the maintenance of stem celllike cd8 t cellsand augmented antitumor t cell responses and mechanistically cgasstingmediated type i interferon signaling reinforced the stem cell“like cd8 t celldifferentiation program mainly by restraining akt activity immune cellderived type i ifns have crucial functions in antitumor immunity control on the one handtype i ifns boost cross presentation by various mechanisms first they stimulate the maturation of dcs secondthey slow the endosomelysosome acidificationprocess to prevent engulfed tumor antigen clearance andelevate the expression of mhc i molecules on the cellsurface [ ] finally they accelerate dc migrationtowardslymph nodes where they can crossprimetumorspecific cd8 t cells on the other handtype i ifns drive the expression of multiple chemokinessuch as cxcl9 and cxcl10 both of which are necessary for cytotoxic t lymphocyte ctl transfer and infiltration similarly type i ifns restrain the defaultimmune suppressive action of regulatory t treg cellsby downregulating phosphodiesterase pde4 and upregulating cyclic amp camp consequently typei ifns serve as bridges linking the cgassting pathway with cd8 t cellmediated antitumor immunitythe antitumor mechanisms of the cgassting signaling axis are illustrated in fig indeed previous studies revealed that sting activation can stimulate antitumor immune responses inleukemia melanoma glioma and hepatocellular carcinoma [“] additionally sting expression is downregulated in a wide variety of tumor tissues and celllines according to a pancancer analysis with a smallproportion of tumors approximately bearing silent sting expression lower sting expressionwas found in hepatic carcinoma and gastric cancer compared with its level in corresponding normal tissues andthis lower expression level was correlated with highertumor stage and poorer prognosis [ ] consistentlycompared with that in the mcfg10a mammary epithelial cell line lower sting expression was detected inmalignant breast cancer cellincluding mcf7hbl100 and t47d cells as well as human melanomacell lines and colorectal adenocarcinoma lines [ ] collectivelythat cgassting signaling might act as a tumor suppressor in certain types of cancersthese findings suggestlinessting pathway agonists as cancer therapeuticsthe immunostimulatory potential of the cgasstingpathway makes it an attractive pharmacological targetsince its activation in the tumor microenvironmenttme can induce efficient crosspriming oftumorspecific antigens and facilitate the infiltration of effectort cells recent drug research has focused on the development of sting agonists because of their potential inanticancer therapy [ ] to date various kinds ofsting agonists have been discovered and they aremainly divided into the following categories cyclic dinucleotides and their derivates dmxaa and its analogsand small molecular agonists in addition some conventional antitumor therapeutics can also indirectly activatesting such as chemotherapy radiotherapy rt andtargeted therapy in addition sting agonists areable to enhance the efficacy of other anticancer therapeutic agents when used in combination sting 0czheng molecular cancer page of fig the antitumor immunity effect of the cgassting pathway dna damage leads to the formation of dsdna in tumor cells upon itsstimulation sting signaling is activated and promotes the release of type i ifn which is crucial for dc maturation sting signaling activation indcs is the core step of the whole cancerimmunity cycle which can be initiated through engulfment of dyingdamaged tumor cells exosometransfer and cgamp gap junctions then dcs migrate towards the tumordraining lymph node and crossprime tumor specific cd8 t cells withthe help of type i ifns finally t cells undergo clonal expansion and traffic through the blood vessel to conduct tumor killingagonists and their synergistic use with other remedies isfurther explored in detail belowcyclic dinucleotides cdnscdns constitute a main type of sting agonist whichmainly originate from bacteria the known naturalcdns consist of exogenous cyclic digmp cdigmpcdiamp ²²cgamp and endogenous ²²cgampamong these cdigmp cdiamp and ²²cgampare synthesized by bacteria and identified as secondarymessengers that mediate sting signal transduction inprokaryotic cells while ²²cgamp functions as theinitiator of sting in mammalian cells the antitumor potential of these natural dinucleotides was firstproven by the finding that cdigmp could inhibit theproliferation of human colon cancer cells in vitro andbasal cell proliferation of human cecal adenocarcinomah508 cells was inhibited with μm cdigmp intraperitoneal ip injection of highdose cdigmpdirectly activated caspase3 and triggered t1 tumoripcell apoptosis in vitro nmol of cdigmp reduced thegrowth of t1 tumor cells in vitro by and nmreduced it by while lowdose cdigmp nmol accelerated the adaptive t cell response by converting a subgroup of myeloidderived suppressor cellsmdscs into immune stimulatory cells producing il12injection of ²²cgamp consistentlymgkg expedited dramatic leukemic elimination in eltcl1 transgenic mice bearing chronic lymphocyticleukemia cll and promoted tumor shrinkage of multiple myeloma in vivo from the perspective of endogenous cdns ²²cgamp mgkg was alsoshown to restrain tumorigenesis and improve the survival rate of mice bearing ct26 colon adenocarcinomain a dosagedependent manner relying on dc activationand t cell crosspriming more recently ohkurit further demonstrated that intratumoral it injection of ²²cgamp μg25 μldose on and days after the injection of tumor cells significantly mitigated tumor growth and prolonged the survival of breast 0czheng molecular cancer page of cancer t1luc squamous cell carcinoma mscc1colon cancer ct26 and melanoma b16f10 mousemodels notably the it injection of ²²cgampinhibited not only tumor growth but also lung metastases in mice bearing b16f10 cellderived tumors suggesting that cgampinduced cd8 tcell priming can drivesystemic antitumor immunity to control local and distant tumor growth termedvaccinestingvaxconsidering the superior properties of sting signaling in activating adaptive immunityit is rational toutilize sting agonists such as cdns as cancer vaccineadjuvants to increase tumor immunogenicity fu investigated the in vivo therapeutic efficacy of acancercomprisinggranulocytemacrophage colonystimulating factor gmcsf and bacteriaderived or synthetic cdns theyobserved that after it injection of stingvax with μg of cdns per vaccine dose the volume of b16melanoma tumors was dramatically reduced in a dosedependent manner compared to mice receiving gmcsf cancer vaccine alone stingvaxtreated mice hadmore infiltrating cd8 ifnγ t cells in the tumormicroenvironment the in vivo antitumor effect of stingvax was also verified in models of colon carcinomact26 pancreatic carcinoma panc02 and upper aerodigestive squamous cell carcinoma sccfvii although natural cdns are able to produce robust antitumor immunity their chemical features might hindertheir future application in the clinical setting first native cdns are easily degraded by enzymes inside the cellor in the bloodstream second their negatively chargedproperty hydrophilicity and phosphate moieties severelyimpede cdns from penetrating cell membranes to activate cytosolic sting leading to low bioavailability andpoor retention of the cdns in specific cells and tissuesthird unintentional toxicities and narrow therapeuticwindows are also unavoidable thus new strategies toimprove therapeutic efficacy and reduce adverse effectsare urgently needed including drug delivery carrier engineering original structural modification and nonnucleotide agonist screening regarding agonistdelivery smith reported that biopolymer implantscodelivering cdigmp μg and chimeric antigen receptor t cart cells resulted in significant tumor regression in mice bearing pancreatic tumors moreoveriv administration of cdigmpysk05lip equivalent to μg of cdigmp aysk05liposome delivery system encapsulating cdigmp led to a tremendous decrease in metastatic lesionsin a b16f10 mouse melanoma model with nearly ofthe injected mice showing resistance against tumor relapsethe adaptive immune responsememory was successfully induced chen alsofound thatliposomalindicating thatinjection ofintravenousintravenousivnanopdelivered cgamp cgampnp could activate the sting axis more effectively than solublecgamp and converted the immunosuppressive tme toa tumoricidal state in a transplanted b16f10 cell melanoma model and in a genetically engineered triplenegative breast cancer model moreover a recentstudy creatively suggested that modified bacteria mightbe exploited as a selective carrier of sting agonistsintroduction of a dinucleotide cyclasecoding gene intothe escherichia coli nissle strain was an attempt at realizing this effect however advancements to the systemare needed tobysnakeapartdigestionresistancecompoundatoms the modifiedfrom improving delivery methods cdnswith superior properties are currently being synthesized and tested for instance to prevent enzymatichydrolysis of cgamp the nonbridging oxygen atomsin cgamp phosphodiester linkages were replaced by²²sulfurcgsasmp showed resistance against degradation byenpp1 a major ²²cgamp hydrolasetherebyleading to a longer halflife and sustained high affinity for human sting hsting syntheticdithio mixedlinkage cdns with both rp rp r rand rp sp r s dithio diastereomers possessed notonlyvenomphosphodiesterase but also enhanced affinityforsting a novel superior modified product ml rrs2 cda also termed adus100 had the potencyto activate all hsting variants and mouse stingmsting adus100 had higher efficiency in activating sting signaling than endogenous or exogenous cdns mainly because of its enhanced stabilityand lipophilicity its powerful tumor elimination effect was extensively demonstrated in multiple murinemodelsincluding b16 melanoma t1 breast cancer and ct26 colon cancer with all treated animalsshowing significant and durable tumorregressionafter itinjection of adus100 three mg doseswhen tumor volumes reached mm3 theremarkableforhsting laid the foundation for its clinical use related clinicaltrials of adus100 are outlined intable in addition to adus100 some other novelsting agonists have been well designed iacs8779and iacs8803 are two highly potent ²²thiophosphate cdn analogs that induced striking systemicantitumorin a b16 melanoma murineinjection μg on and daysmodel after itposttumor implantation compared with adus100or cgamp the characteristics and preclinicalapplications of all these mentioned cnds are summarized in table because of the structural modification and optimization of delivery strategiestheapplication range and efficacy of cdns have beenand high affinityresponsescurativeeffect 0czheng molecular cancer page of table characteristics and preclinical applications of different sting agonistsclassificationcharacteristicsapplicationmodelsnatural cdnagonistscdigmppoor membrane permeabilitysuitable for various codeliverytechnologiescolon cancer h508cells t1 metastaticbreast cancertreatmentinformation μm nmol ip nmol ip nmol ip²²cgamp²²cgamphigher binding affinity formsting than for hstinghigher affinity for hsting thanits lineage isomers binds tovarious sting nucleotidepolymorphisms observed inhumans easily degraded byphosphodiesteraseimpermeable to the cellmembranechronic lymphocyticleukemia mgkg ipmultiple myeloma mgkg ipct26 colonadenocarcinoma mgkgbreast cancer t1lucsquamous cellcarcinomasmscc1 μg25 μldose it μg25 μldose itcolon cancer ct26 μg25 μldose itmelanoma b16f10 μg25 μldose ittherapeutic effects references[ ] [ ]inhibitsproliferation tumorregression tumorregressionaccelerates tcellresponseleukemiaeliminationsuppressesgrowthrestrainstumorigenesisimproves survivalratedelays tumrowthdelays tumrowthdelays tumrowthdelays tumrowthstingvaxsyntheticcdnagonistspotent in vivo antitumor efficacyin multiple therapeutic modelsof established cancercgampnpsbiopolymer scaffolds cdigmp and car t cellscdigmpysk05lip²²cgsasmpadus100iacs8779iacs8803noncdnagonistsfaaliposomal nanops npsdeliver cgamp intracellularlymore effectively than realizedwith soluble cgamperadicates tumors moreeffectively than systemicdeliveryysk05 is a lipid that can efficientlydeliver cdigmp to the cytosolpossesses high fusogenic activitywhich enhances endosomalescapemore resistant to degradation byenpp1 tenfold more potent atinducing ifn secretion potentialuse as a cancer vaccine adjuvantimproves stability and lipophilicityhigher affinity for hsting thannatural cdn agonists capable toactivate all hsting variants andmstingstimulates a superior systemicantitumor response thanadus100 and cgampcauses hemorrhagic necrosisfailed in a phase i clinical trialdue to species specificity μg cdns itreduces tumorvolume b16 melanomacolon carcinomact26pancreaticcarcinoma panc02b16f10 melanomaivtnbccreates atumoricidal state pancreatic cancer μg cdigmptumor regression b16f10 mousemelanoma μg cdigmp ivdecreasesmetastasisthp1 monocytesb16 melanomathree mg doses it t1 breast cancerthree mg doses itmc26 colon cancerthree mg doses itdurable tumorregressiondurable tumorregressiondurable tumorregression b16 melanoma μg on day and posttumor implantationantitumorresponse murine colontumorsextensive tumorrejection[ ]dmxaafirst discovered as a vascularrat mammary mgkg iphigh anticancer[ 0czheng molecular cancer page of table characteristics and preclinical applications of different sting agonists continuedclassificationcharacteristicsapplicationmodelstreatmentinformationinduces proinflammatory cytokinesin a stingdependent mannerhuman fibroblastsantiviral activity selectively induces stingdependentsynthesis and secretion of bioactiveifns no evidence of binding directlyto stingactivates sting in œopenconformation submicromolarlevels induce sting activationand ifn productionhuman fibroblastsantiviral activity colon tumors mgkg iv of a treatedgroup remainedtumor free faa flavone acetic acid dmxaa 56dimethylxanthenone4acetic acid cma 10carboxymethyl9acrid
Colon_Cancer
"The protocol was approved by the Institutional Review Boards at each participating institution. Informed consent was obtained from each patient. Study design This is a single arm open label multicentre phase II study (ClinicalTrials.gov identifier: NCT00794417). Patients received the three-drug combination intravenously on day 1 of every 21 days with ziv-aflibercept (6?mg?kg?1) first followed by pemetrexed (500?mg?m?2) and cisplatin (75?mg?m?2). Premedications consisted of folic acid vitamin B12 and dexamethasone as a prophylactic measure to reduce pemetrexed-related toxicities and standard anti-emetics. Patients could receive up to six cycles of combination therapy. For patients who completed the combined chemotherapy maintenance ziv-aflibercept every 21 days was to continue until disease progression intolerable toxicity or withdrawal from the study. End points and assessments The two co-primary end points were objective response rate (ORR) and PFS. The ORR was defined as the proportion of patients with complete response plus partial response (CR+PR). The PFS was defined as the time interval from the first dose of combination chemotherapy to tumour progression or death whichever occurred first. Secondary variables were the determination of the adverse events (AEs) pharmacokinetics (PK) and pharmacodynamic profiles (including anti-ziv-aflibercept antibody and hematopoiesis). Pharmacokinetic end points included the area under the concentration curve maximum concentration (Cmax) clearance and terminal half-life (t1/2). Tumour imaging (CT or MRI) was performed at screening on day 21 (±7 days) of every even numbered cycle (every 6 weeks) and when disease progression was suspected. Responses were assessed using RECIST version 1.0 (Therasse et al 2000). Safety and tolerability were assessed at baseline and at least every 21 days as evaluated by AEs and changes in laboratory parameters graded according to the Common Terminology Criteria for Adverse Events (CTCAE) version 3.0 (National Cancer Institute 2006). Ziv-aflibercept (free or bound to VEGF) in plasma samples was quantified using a validated direct enzyme-linked immunosorbent assay. A validated non-quantitative titre-based bridging assay was used to detect anti-ziv-aflibercept antibodies in serum samples. Correlative studies The exploratory objective of the correlative studies was to evaluate changes in erythropoiesis in response to VEGF inhibition. It was hypothesised that VEGF inhibition would result in an increase in haemoglobin via increased hepatic erythropoietin production. Statistical analysis Statistical testing was done to determine whether the ORR was larger than 20% or whether the PFS was greater than 4.5 months. Exact test (one-sided) was used to test the null hypothesis that ORR was ?20% versus the alternative hypothesis that ORR was ?35%. Assuming type I error was not >2.5% a sample of 72 patients would provide >80% power to test the hypothesis using exact binomial test. The calculated sample size of 72 patients would also provide >90% power to test the null hypothesis that median PFS was ?4.5 months versus the alternative hypothesis that PFS was ?6.5 months at one-sided alpha of 2.5% using one sample log-rank test. Safety data were to be summarised. Concentrations of free ziv-aflibercept and adjusted bound ziv-aflibercept: VEGF complex were to be summarised every 21 days over the duration of the study by nominal time point. Noncompartmental parameters were calculated using WinNonlin (version 5.3 Pharsight Corporation Mountain View CA USA) and model 202 (constant infusion) using nominal time points after a single dose of ziv-aflibercept. The noncompartmental analysis was performed over the dosing interval 21 days following the first dose. All analyses used statistical software SAS (version 9.1.3 Cary NC USA). Results Patients This study was closed prematurely because of three confirmed and two suspected but unconfirmed cases of reversible posterior leukoencephalopathy syndrome (RPLS). A total of 42 patients were enrolled from 17 participating sites across the United States and Canada between January 2009 and December 2010. summarises the patient demographics. Median age was 61.5 years; 55% were male 86% were Caucasian and 50% had ECOG PS of 0. Safety evaluation Treatment exposure and dose modifications All 42 patients received at least one dose of each of the three study drugs with a median of 92 days of treatment (range 21“288 days). Twenty-seven (64%) patients completed four or more cycles of the combination treatment. A median of 4.5 (range 1“6) cycles of pemetrexed and 4 (range 1“6) cycles of cisplatin were administered. The median dose intensity was 163.9 (range 110.1“175.5)?mg?m?2 per week for pemetrexed and 24.6 (range 15.3“26.3)?mg?m?2 per week for cisplatin. The delivered dose intensities were 98.3% for pemetrexed and 98.5% for cisplatin. Seventeen (40%) completed six or more cycles of ziv-aflibercept. The median dose intensity of ziv-aflibercept was 1.97?mg?kg?1 per week close to the planned intensity of 2?mg?kg?1 per week. Reasons for treatment discontinuation were disease progression (33%) AEs (33%) and others (34% including withdrawal of consent and investigator request). Seventeen patients had at least one cycle delayed. Eleven patients (26%) had at least 1 dose modification of ziv-aflibercept 6 (14%) of pemetrexed and 11 of cisplatin. Adverse events Thirty-five patients (83%) experienced a treatment-emergent adverse event (TEAE) of grade 3 or 4 (3/4) and 16 patients (38%) experienced a serious TEAE. The most common TEAEs were nausea (69%) fatigue (67%) and hypertension (57%). Hypertension neutropaenia and hypokalaemia were the most common grade 3/4 TEAEs in 36%0.14 and 10% of patients respectively. summarised the most common TEAEs. Thirty-nine patients (93%) experienced at least one haematologic abnormality with grade 3/4 in eight patients (19%) mostly neutropaenia. Every patient experienced at least one abnormal chemistry value with grade 3/4 in 15 (36%) most commonly hyponatraemia. Seven patients (17%) died before clinical cutoff of the study. Five died due to disease progression and two due to TEAEs: 1 pneumonia and 1 sepsis. Neurological toxicities Between 26 September 2010 and 30 December2010 five patients experienced neurological symptoms including altered mental status in four slurred speech in one seizure in two and headache in four patients. Three patients had a brain MRI that was consistent with RPLS (B). Brain MRI was negative for RPLS in two other patients. The three patients diagnosed with RPLS were all Caucasian women aged 3851 and 72 years respectively. One of the three patients with RPLS entered the study with a history of hypertension. All three patients experienced elevated BP and two patients had reduced CrCL during the therapy: one patient's CrCL decreased by 45% from baseline (141 to 78?ml?min?1) after one cycle and the other's decreased by 20% (64 to 51?ml?min?1) after four cycles. They were diagnosed with RPLS after one two and five cycles of ziv-aflibercept respectively. Two patients recovered from RPLS and one died due to disease progression before RPLS resolution. Pharmacokinetic information was available for two of the three RPLS patients and for one suspected case: systemic concentrations of free ziv-aflibercept were within the range of other patients in the treatment cohort. In the phase I study using the same regimen (N=18) five patients experienced a mild neurocognitive disturbance but no RPLS was diagnosed (Diaz-Padilla et al 2012). Rare cases of RPLS have been observed in the ziv-aflibercept clinical development programme but the rate observed in this study (3 out of 42=7%) was much higher than previously reported (i.e. 0.5% of 3795 patients treated with ziv-aflibercept as monotherapy or in combination with chemotherapy ZALTRAP product insert). As a result this study was permanently closed to enrolment. Patients who remained on study were re-consented with updated safety information regarding RPLS in addition to continued close monitoring."
Lung_Cancer
"and vimentin was positive in p120ctn cytoplasmic-positive lung cancer cells. .0088064.t001 Correlation between E-cadherin vimentin and lymph node metastasis and p120ctn. p120ctn N membrane cytolymph/nucleolus X2 p E-cadherin negative 56 9 47 30.166 <0.01 positive 22 18 4 Vimentin negative 53 23 30 5.633 0.022 positive 25 4 21 Lymph node metastasis No 41 19 22 5.251 0.032 Yes 37 8 29 Localization of p120ctn is consistent with E-cadherin in lung cancer cells We examined the protein expression levels of p120ctn and E-cadherin in normal HBE cells and nine lung cancer cell lines by Western blot and found that they all expressed mainly isoforms 1A (120 kDa) and 3A (100 kDa) of p120ctn (A). Although the protein expression levels of p120ctn were not related to E-cadherin the localization (membrane or cytoplasm) of p120ctn was always consistent with that of E-cadherin. We then screened cells expressing high levels of p120ctn and E-cadherin in the membrane (H460 cells) or cytoplasm (SPC cells) as well as those expressing low levels of p120ctn and E-cadherin in the membrane (H4299 cells) or cytoplasm (LK2 cells) for further study (B). .0088064.g002 Expression and localization of p120ctn and E-cadherin in H460 SPC H1299 and LK2 cells. (A) Western blot analyses showed expression of p120ctn and E-cadherin in nine lung cancer cell lines and HBE. (B) By immunofluorescence analysis the expression of E-cadherin and p120ctn were observed restricted to the cell membrane at cell-cell adherens junctions in H460 and H1299 cells whereas they both were confined to the cytoplasm in SPC and LK2 cells. Different functions of p120ctn isoform 1A in EMT are dependent on E-cadherin subcellular localization Knockdown of endogenous p120ctn isoform 1A by siRNA-p120ctn-1A resulted in decreased E-cadherin expression and increased N-cadherin snail and vimentin expression in H460 cells (A). However knockdown of endogenous p120ctn-1A by siRNA-p120ctn-1A showed opposite results in SPC cells where we found increased E-cadherin expression and decreased N-cadherin snail and vimentin expression (B). In comparison with the control the ablation of p120ctn isoform 1A also enhanced the H460 cells invasiveness (17.33±1.25 vs. 36.33±1.70 P<0.01) (C) whereas reduced the SPC cells invasiveness (23.0±0.82 vs. 13.0±0.82 P<0.01) (D). These results revealed that the p120ctn isoform 1A plays a different role in EMT and cell invasiveness in different E-cadherin subcellular locations. .0088064.g003 p120ctn isoform 1A plays a different role in regulating EMT in H460 and SPC cells. (A) Ablation of p120ctn isoform 1A decreased E-cadherin expression and increased N-cadherin snail and vimentin expression in H460 cells. (B) SPC cells were treated as in (A) and the opposite results were obtained. (C) Ablation of p120ctn isoform 1A enhanced the invasiveness of H460 cells (**P<0.01). (D) Ablation of p120ctn isoform 1A decreased the invasiveness of SPC cells (**P<0.01). Inhibitory function of p120ctn isoform 3A on EMT is not affected by differences in E-cadherin subcellular localization To verify whether p120ctn isoforms 1A and 3A play different roles in regulating EMT their expression plasmids were transiently transfected into lung cancer cells with low expression of p120ctn (H1299 with membrane E-cadherin expression and LK2 with cytoplasmic E-cadherin expression). The western-blot analysis demonstrated that overexpression of the p120ctn isoform 1A led to increased E-cadherin expression and decreased N-cadherin vimentin and snail expression (A); on the contrary the decreased E-cadherin expression and increased N-cadherin vimentin and snail expression were observed in LK2 cells (B). Overexpression of the p120ctn isoform 1A also reduced the H1299 cell invasiveness (52.0±2.65 vs. 33.33±2.64 P<0.01) (C) while enhanced the LK2 cell invasiveness (18.0±0.82 vs. 39.66±2.05 P<0.01) (D).. Overexpression of p120ctn isoform 3A led to increased E-cadherin expression decreased N-cadherin vimentin and snail expression (A 4B) and reduced cell invasiveness (52.0±2.65 vs. 29.66±1.53 P<0.01; 18.0±0.82 vs. 8.33±expression 0.47 P<0.01) (C 4D) in both of these cell lines. These results further confirmed that the p120ctn isoform 1A had a different effect on EMT depending on the subcellular localization of E-cadherin. They also revealed that the p120ctn isoform 3A maintained an inhibitory role in the EMT of lung cancer cells whether E-cadherin was localized to the membrane or the cytoplasm. .0088064.g004 p120ctn isoform 3A maintains the role of inhibitiing EMT independently of E-cadherin localization. (A B) Both H1299 (E-cadherin membrane localization) and LK2 cells (E-cadherin cytoplasmic localization) transiently transfected with the p120ctn isoform 3A plasmid showed increased E-cadherin expression and decreased N-cadherin vimentin and snail expression. (C D) Transient transfection of p120ctn isoform 3A plasmids into H1299 and LK2 cells resulted in decreased cell invasiveness (**P<0.01). (E) E-cadherin remained localized on the membrane in H1299 cells and in the cytoplasm of LK2 cells after transfection of the p120ctn isoform 3A plasmid. Discussion The phenomenon of EMT in tumor cells often leads to decreased cell adhesion and increased mobility and this transition is accompanied by decreased E-cadherin expression and increased expression of N-cadherin vimentin and other mesenchymal biomarkers [3] [4] [5] [6] [7]. As an important factor for stabilizing E-cadherin p120ctn plays a role in inhibiting or promoting tumor cell proliferation and invasion that is dependent on whether E-cadherin is expressed or not [16] [17]. Furthermore p120ctn isoforms 1A and 3A have shown different effects on E-cadherin expression and tumor cell invasiveness which are based on differences in the localization of E-cadherin [18]. These results strongly suggest that p120ctn most likely regulates the EMT of tumor cells by affecting E-cadherin expression and that p120ctn isoforms 1A and 3A play different roles in EMT expressing E-cadherin in different subcellular locations. We first found that the p120ctn membrane expression was positively correlated with E-cadherin expression and negatively correlated with vimentin expression and lymph node metastasis while the cytoplasmic expression of p120ctn was negatively correlated with E-cadherin expression and positively correlated with vimentin expression and lymph node metastasis by immunohistochemistry. Although these results were consistent with previous studies [13] [14] they further suggested that p120ctn likely affects the EMT by influencing the expression of E-cadherin and vimentin and thereby the cell invasion and metastasis in non-small cell lung cancer (NSCLC). To confirm the different impacts of p120ctn isoforms 1A and 3A on EMT in cells expressing E-cadherin in different locations we selected H460 and H1299 cells with E-cadherin membrane expression and SPC and LK2 cells with E-cadherin cytoplasmic expression for further analysis. Plasmids expressing the p120ctn isoforms 1A and 3A were constructed and the full-length p120ctn siRNA was synthesized for these experiments. Since the sequence beyond amino acids 1“101 of p120ctn isoform 1A is similar to that of p120ctn isoform 3A [24] [25] we could not design an interference sequence specifically for p120ctn isoform 3A. Therefore we had to further study the impact of the two isoforms on EMT and cell invasiveness in lung cancer cells with different E-cadherin locations specifically by knocking down p120ctn isoform 1A in H460 and SPC cells with high p120ctn expression and transfecting cDNA plasmids for exogenous p120ctn isoforms 1A and 3A into H1299 and LK2 cells with low expression of p120ctn. Knockdown of p120ctn isoform 1A in H460 cells destroyed the epithelial cell adhesion complexes. E-cadherin expression was also downregulated due to the loss of its important stabilizing factor p120ctn isoform 1A which was consistent with previous studies [20] [26]. Decreased E-cadherin expression and disrupted cell-cell adhesion may induce EMT [27] [28] [43] [44] which results in increased N-cadherin vimentin and snail expression and enhanced cell invasiveness. On the other hand overexpressed p120ctn isoforms 1A and 3A was shown to bind E-cadherin located on the membrane proactively in tumor cells [29] and then inhibit the degradation of E-cadherin and stabilize its expression contributing to the formation of effective epithelial cell adhesion complexes [30] [31] [32]. As these series of processes maintained the normal cell-cell adhesion connection and inhibited EMT there was increased E-cadherin expression and decreased N-cadherin vimentin and snail expression as well as inhibited cell invasiveness in H1299 cells. Previous studies have shown that although p120ctn isoform 1A could bind E-cadherin in the cytoplasm they could not form effective adhesion complexes on the membrane between epithelial cells [33]. Furthermore the cytoplasmic E-cadherin is likely not to be the full-length E-cadherin but instead cleaved E-cadherin fragments such as E-cad/sE-cad (80 kDa) and E-cad/CTF2 (33 kDa) [34]. The E-cad/CTF2 fragment can bind to p120 in the cytoplasm and then translocate into the nucleus and bind the transcriptional repressor of Kaiso to activate the Wnt/b-catenin pathway [35] [36] finally promoting the EMT of tumor cells and enhancing cell invasion and metastasis [37]. Moreover others have shown that p120ctn-1A is related to abnormal expression of E-cadherin and poor prognosis [38]. These studies illustrated that the cytoplasmic p120ctn isoform 1A can play a role in promoting tumor cell EMT invasion and metastasis. Based on the above we observed on the one hand that the effect of p120ctn isoform 1A to promote tumor cell EMT invasion and metastasis would be lifted by its ablation resulting in increased E-cadherin expression decreased N-cadherin vimentin and snail expression and inhibited invasiveness in SPC cells. On the other hand transfection of the p120ctn isoform 1A plasmid into LK2 cells expressing cytoplasmic E-cadherin resulted in decreased E-cadherin expression increased N-cadherin vimentin and snail expression and enhanced cell invasiveness. Although the precise role of p120ctn during EMT induction is still unclarified previous studies suggested that knockdown of all isoforms of p120ctn could induce EMT indirectly [27] [28] [43] [44]. All inductions were based on decreased E-cadherin expression and intercellular adhesion in previous studies which were also confirmed by our study in H460 cells with E-cadherin membrane localization. Unlike the H460 cells knockdown of the p120ctn isoform 1A in SPC cells with E-cadherin cytoplasmic expression could not decrease E-cadherin expression and intercellular adhesion. Instead we found increased E-cadherin expression and decreased cell invasiveness indicating that the EMT could not be induced by this pathway in SPC cells. It was worth noting that the same result was observed in LK2 and H1299 cells transfected with the p120ctn isoform 3A plasmid both showing increased E-cadherin expression decreased N-cadherin vimentin and snail expression and inhibited cell invasiveness. These results suggested that p120ctn isoform 3A has the function of inhibiting EMT of lung cancer cells and this function is independent of the cellular E-cadherin localization. Past research had also confirmed a shift from p120ctn isoform 3A to p120ctn isoform 1A expression after the induction of EMT [9] [10] [11] which indirectly indicates that p120ctn isoform 3A may inhibit EMT while p120ctn isoform 1A promotes EMT. In addition we also noticed that p120ctn and E-cadherin protein expression levels were significantly increased after transfection of the p120ctn-3A plasmid into LK2 and H1299 cells but p120ctn and E-cadherin were still mainly restricted to the cell membrane at cell-cell adherens junctions in H1299 cells. By contrast E-cadherin and p120ctn were almost exclusively located in the cytoplasm in LK2 cells (E). As a cell adhesion molecule E-cadherin is known to be only located on the cell membrane with the potential to inhibit EMT while in the cytoplasm it is often cleaved into fragments and therefore functions differently from the molecules located on the cell membrane [34]. Thus the cytoplasmic E-cadherin would theoretically not play a role in inhibiting EMT. Based on the above analysis we speculated that there may be some interaction between p120ctn isoform 3A and snail which plays a role in suppressing EMT in lung cancer cells expressing cytoplasmic E-cadherin but this hypothesis requires further study. Importantly we also found that knockdown of p120ctn-1A in SPC cells with cytoplasmic E-cadherin resulted in decreased twist expression (B). Meanwhile transfection of LK2 cells which also showed cytoplasmic localization of E-cadherin with the p120ctn isoform 1A plasmid resulted in increased twist expression (B). However no changes in twist expression were observed in the rest of the experiments (A 4A). As a transcription factor and master gene regulator of EMT [39] [40] twist can downregulate E-cadherin expression [41] and upregulate N-cadherin and other mesenchymal biomarkers [42]. Increased twist expression in LK2 cells transfected with the p120ctn isoform 1A plasmid indicated that transcriptional activation took place and further suggested that the p120ctn isoform 1A may have translocated into the nucleus upon binding of E-cad/CTF2 in the cytoplasm consequently activating the Wnt signaling pathway to promote EMT. Decreased twist expression in SPC cells transfected with p120ctn-1A-siRNA indicated that transcriptional activity was downregulated and suggested that ablation of p120ctn isoform 1A resulted in the inhibtion of EMT by removing the stimulatory effect of the Wnt signaling activity by p120ctn isoform 1A. In the H460 and H1299 cells with E-cadherin localized in the membrane the unchanged twist expression confirmed that p120ctn isoforms 1A and 3A could bind to E-cadherin and maintain effective cell-cell adhesion in order to suppress EMT instead of affecting the Wnt/twist pathway. Intriguingly overexpression of p120ctn isoform 3A did not change twist expression in LK2 cells expressing cytoplasmic E-cadherin indicating that p120ctn isoform 3A did not activate transcription. Therefore we firmly believe in the above hypothesis that p120ctn isoform 3A may interact with snail in some manner to influence E-cadherin expression and suppress EMT in lung cancer cells carrying cytoplasmic E-cadherin. Previous studies have observed that p120ctn-1A restored the cytoplasmic expression of E-cadherin whereas p120ctn-3A could not [20] which seems to be contradictory with the results of this study. However the method in previous studies of knocking down p120ctn expression and then transfecting p120ctn isoforms 1A and 3A plasmids into cells is different from that in the current study in which cells were only transiently transfected with p120ctn isoforms 1A and 3A plasmids. Therefore the different research methods may have led to different effects on E-cadherin. We also noted that in previous studies decreased and almost undetectable levels of E-cadherin by ablation of p120ctn resulted in the failure of exogenous p120ctn-1A to translocate into the nucleus to activate the Wnt/b-catenin pathway and decrease E-cadherin expression due to the deletion of the binding partner E-cad/CTF2. However the LK2 and H1299 cell lines used in these experiments expressed E-cadherin in the present study. E-cadherin binds primarily to unphosphorylated p120ctn isoform 3A whereas tyrosine-phosphorylated p120ctn isoform 1A interacts exclusively with N-cadherin [23]. In the previous studies exogenous p120ctn isoform 3A was prevented from binding and stabilizing E-cadherin after its ablation while in the present study the exogenous p120ctn isoform 3A could stabilize E-cadherin expression directly on the membrane or indirectly by increasing its cytoplasmic expression via regulation of snail expression. Of course all of these findings will need to be further investigated. In conclusion we for the first time found that p120ctn isoforms 1A and 3A to have different functions in EMT of lung cancer cells with E-cadherin expressed in different subcellular locations. When E-cadherin was localized on the cell membrane p120ctn isoforms 1A and 3A both could inhibit EMT and reduce the cell invasiveness phenotype."
Lung_Cancer
"Price of gefitinib was reduced 20% and was fixed in probabilistic sensitivity analysis because it is a brand name drug. b Estimated according to local charges in China. c Varied by ±30%. Health state utility values of the PFS and PS states presented in were derived from the published literature by Nafees et al who assessed quality of life using the visual analogue scale and standard gamble interview in 100 participants on the basis of the health state descriptions which were developed after rounds of in-depth interviews with oncologists oncology specialist nurses and psychometric experts [28]. According to the literature diarrhoea and rash reported in the trial were significantly associated with the utility values of PFS [15] [28]. Therefore we calculated the utility value in PFS based on the published proportion of the adverse events (diarrhoea and rash) [15] and utility values of PFS on oral therapy (0.67) PFS plus rash (0.62) and PFS plus diarrhoea (0.61) [28]. The utility of PS state was 0.47 (range 0.19“0.56) and was used in both arms. Sensitivity Analysis Each key parameter was fitted high/low values and specific pattern of distribution in our model () to reflect substantial uncertainty of the input parameters using one-way and probabilistic sensitivity analyses (PSA). Many ranges were derived from published reports [26]“[28]; price of gefitinib was reduced 20% and fixed in PSA because it is a brand name drug; costs of adverse events were estimated by local charges in China; probabilities of adverse events were varied by ±30%. Lognormal distributions were chosen for all input costs except gefitinib (fixed in PSA); beta distributions were chosen for utility values and probabilities of adverse events; bivariate normal distributions were adopted for the Weibull and Log-logistic parameters; discount rate with high/low values was fixed in PSA in compliance with the request of China Guidelines for Pharmacoeconomic Evaluations (Version 8) [24]. The WTP threshold of 3—per-capita GDP ($16349/QALY) was used. A tornado diagram and an incremental cost-effectiveness scatter plot were developed to depict the results of one-way sensitivity analyses (OSA) and PSA. Results Base Case Model Analysis The Log-logistic and two parameters Weibull model matched the survival curves of the clinical trial by Zhang L et al [15] satisfactorily (). The validity of the simulated survival curves tail beyond the observed time horizon of clinical trial was conducted by comparing the 5 years overall survival rate calculated from the current distribution models for placebo arm (5.7%) to the data from the Surveillance Epidemiology and End Results (SEER) Program which shows that 5-year survival rate of distant lung cancer patients is 3.9% [29] and the site of http://lungcancer.about.com/ which shows that 5-year survival rate of metastatic NSCLC is sadly less than 10%. Base case model analyses in different time horizon are displayed in which suggested that maintenance gefitinib therapy after four chemotherapeutic cycles of stand first-line platinum-based chemotherapy for patients with locally advanced/metastatic NSCLC patients would increase the effectiveness in a 1- 3- 6- or 10-year time horizon with incremental QALYs gained of 0.02330.1462 0.2699 and 0.3496. Incremental costs per QALY for the new therapy compared with placebo were $184828 $19214 $19328 and $21308 respectively at 136 and 10 years. .0088881.g002 Survival curves in patients with maintenance gefitinib group or placebo group after first-line platinum-based combination chemotherapy of four cycles in locally advanced/metastatic non-small-cell lung cancer. The original curves from the clinical trial are shown together with the Weibull and Log-logistic model estimated for progression-free survival and overall survival separately. .0088881.t003 Base-case model analyses of life-years gained (LYGs) quality-adjusted life-years (QALYs) costs and incremental cost per LYG/QALY of maintenance gefitinib therapy arm and placebo arm after four cycles of stand first-line platinum-based chemotherapy for patients with locally advanced/metastatic NSCLC on the basis of 1000 simulation cases. Arm LYGs Gained QALYs Gained Cost ($) Incremental cost ($) Per LYG Per QALY 1-year Placebo arm 0.865 0.465 9082 “ “ Gefitinib arm 0.845 0.488 13396 Dominated 184829 3-year Placebo arm 1.516 0.772 18866 “ “ Gefitinib arm 1.658 0.918 21675 19788 19214 6-year Placebo arm 1.735 0.875 22129 “ “ Gefitinib arm 2.112 1.144 27345 13816 19328 10-year Placebo arm 1.814 0.912 23302 “ “ Gefitinib arm 2.357 1.262 30751 13734 21308 One-way Sensitivity Analysis The results of one-way sensitivity analyses of key populated variables (displayed in ) were depicted in a tornado diagram () to show the influence with regard to the incremental cost-effectiveness ratio (ICER) which means incremental cost per QALY gained in current study. The utility of PFS plus rash impacted utmost on the ICER. The other sensitive variables included utility of PFS plus diarrhoea utility of progressed disease price of gefitinib cost of follow-up treatment in PS state and utility of PFS on oral therapy. All of these variables did not led to an ICER entrancing the WTP threshold of $16349 per QALY (3—per-capita GDP of China). None of the other parameters significantly altered the ICER. .0088881.g003 Tornado diagram for one-way sensitivity analysis revealing variables™ influence on the incremental cost-effectiveness ratio. PFS?=?progression-free survival; GE?=?gefitinib; PL?=?placebo; QALY?=?quality-adjusted life-years. Probabilistic Sensitivity Analysis Incremental cost-effectiveness scatter plot of 1000 simulations () showed a zero probability meeting the WTP threshold of $16349/QALY. If the WTP was >$21323 (per-capita GDP: $7108) more 50% of locally advanced/metastatic NSCLC with maintenance gefitinib therapy after four cycles of first-line platinum-based combination chemotherapy without disease progression could achieve cost-effectiveness. Acceptability curves () suggested that the cost-effectiveness likelihood of maintenance gefitinib therapy increased with increasing thresholds of WTP and about $17700 to $26300 was the sensitivity range. At WTPs >$26300 almost all cases could achieve cost-effectiveness. .0088881.g004 Probabilistic sensitivity analysis of 1000 cases study comparing maintenance gefitinib strategy and placebo strategy. WTP?=?willingness to pay; QALY?=?quality-adjusted life-years. .0088881.g005 Acceptability curves of maintenance gefitinib arm and placebo arm. QALY?=?quality-adjusted life-years. Discussion Maintenance gefitinib therapy has been proven to prolong PFS significantly than placebo for patients with locally advanced/metastatic NSCLC after 4 chemotherapeutic cycles of first-line platinum-based combination chemotherapy without disease progression based on a Chinese phase III trial across 27 centres [15]. However its economic impact is necessary to be considered before it is widely used for the appropriate patients especially for China where the population is >1.3 billion and the health care resources are in serious shortage [30]. Resource allocation decisions in health care are rife based on results from economic assessments. However from Clinical trials it is of difficulty to collect enough financial data for economic evaluation [27]."
Lung_Cancer
" Despite previous investigations it remains unclear how p120-catenin (p120ctn) isoforms 1A and 3A affect the EMT of tumor cells. Here we investigated expression of p120ctn E-cadherin and vimentin in 78 human non-small cell lung cancer (NSCLC) samples by immunohistochemistry and found that p120ctn membrane expression positively correlated with E-cadherin expression (P<0.001) and negatively correlated with vimentin expression and lymph node metastasis (P<0.05). Meanwhile p120ctn cytoplasmic expression negatively correlated with E-cadherin expression (P<0.001) and positively correlated with vimentin expression and lymph node metastasis (P<0.05). Cells expressing high (H460 and SPC) and low (H1299 and LK2) levels of p120ctn were screen to investigate its impact on EMT. E-cadherin was restricted to the cell membrane in H460 and H1299 cells whereas it was expressed in the cytoplasm of SPC and LK2 cells. Ablation of endogenous p120ctn isoform 1A in cells expressing high levels of the protein resulted in decreased E-cadherin expression increased N-cadherin vimentin and snail expression and enhanced invasiveness in H460 cells. Meanwhile completely opposite results were observed in SPC cells. Furthermore transfection of in H1299 cells expressing low p120ctn levels with the p120ctn isoform 1A plasmid resulted in increased E-cadherin expression decreased N-cadherin vimentin and snail expression and weakened invasiveness while LK2 cells showed completely opposite results. Both cell lines expressing low p120ctn levels and transfected with the p120ctn isoform 3A plasmid appeared to have increased E-cadherin expression decreased N-cadherin vimentin and snail expression and weakened invasiveness. In in cells with membrane E-cadherin both p120ctn isoforms 1A and 3A inhibited EMT and decreased cell invasiveness. In cells with cytoplasmic E-cadherin p120ctn isoform 1A promoted EMT and increased cell invasiveness while p120ctn isoform 3A inhibited the EMT and decreased cell invasiveness. This work was supported by grants from the National Natural Science Foundation of China (grants 81071905 and 81272606 to E.-H. W. grants 81101780 to Y.Z.). The funders had no role in study design data collection and analysis decision to publish or preparation of the manuscript. Introduction The epithelial mesenchymal transition (EMT) is a rapid and often reversible change of cell phenotype and plays a particularly important role in tumor development. In the process of EMT epithelial cells undergo a phenotypic switch to form mesenchymal cells that are similar in appearance to fibroblasts [1] [2]. Such phenotypic changes cause epithelial cells to lose their characteristic cell-cell adhesion structures alter their polarity modulate the anization of their cytoskeletal systems switch expression from keratin- to vimentin-type intermediate filaments as well as become isolated motile and resistant to anoikis [3] [4]. Typically cells undergoing EMT show decreased E-cadherin expression [5] [6] and decreased expression of mesenchymal biomarkers such as N-cadherin vimentin snail slug and twist [7] [8]. Previous studies on the relationship between p120-catenin (p120ctn) and EMT have been confined to the switch from short to long p120ctn isoforms during the EMT induced by expression of SIP1/ZEB2 [9] twist [10] or Zeppo1 [11]. However the mechanism by which p120-catenin isoforms 1A and 3A affect EMT of tumor cells remains unknown. The p120ctn protein has four isoforms (1 to 4) resulting from four transcriptional start sites and each isoform has a full central Armadillo repeat domain that can interact with the juxtamembrane domain of E-cadherin in order to participate in the formation of an adhesion complex on the cell membrane [12]. These observations suggest that the subcellular localization and function of p120ctn can be affected by the localization of E-cadherin. Previous studies have shown that p120ctn may play opposing roles depending on whether it is located on the membrane or in the cytoplasm of cells [13] [14]. Others have also found that p120ctn isoforms 1A and 3A have different regulatory functions on tumor cell proliferation invasion and metastasis [15] [16] . These studies indicate that if p120ctn has an impact on the EMT it is likely to be different between p120ctn isoforms 1A and 3A. Some studies have shown that p120ctn may promote or inhibit tumor growth and invasiveness depending on whether E-cadherin expressed or not [18] [19]. Yu and colleagues also found different effects of p120ctn isoforms 1A and 3A on proliferation and invasion in tumor cells exhibiting different localizations of E-cadherin [20]. Thus whether p120ctn isoforms 1A and 3A also play different roles in regulating EMT in tumor cells with E-cadherin at different locations remains unknown. The aim of this study was to determine the potential effects and regulatory mechanisms of p120ctn isoforms 1A and 3A on EMT in lung cancer cells. We first revealed that the membrane or cytoplasmic expression of p120ctn correlated with expression of E-cadherin and vimentin or lymph node metastasis by immunohistochemistry. We further detected the expression levels of p120ctn E-cadherin and vimentin in lung cancer cells by Western blot and screened cell lines expressing both low and high levels of p120ctn and with E-cadherin in the membrane or cytoplasm. Changes in expression of EMT-related molecules and cell invasion were also investigated by knockdown of endogenous p120ctn-1A or overexpression by transfection of p120ctn-1A and 3A plasmids into cells. Materials and Methods Materials This study was conducted with the approval of the institutional review board at China Medical University. Written consent was given by the participants for their information to be stored in the hospital database and for their specimens to be used in this study. All clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. Samples were collected from 78 cases of squamous cell lung cancer and lung adenocarcinoma diagnosed at the First Affiliated Hospital of China Medical University (Sheny-ang China). The samples were from 46 male and 32 female patients with an average age of 57 years. The samples were classified according to lung tumor histological criteria (2004) of the World Health anization (WHO) [21] as squamous cell lung carcinoma (32 cases) or lung adeno-carcinoma (46 cases). Thirty cases were highly differentiated and forty-eight were moderately or poorly differentiated. Lymph node metastases were present in 43 cases but not in the other 35. We selected cases with lymph node metastases to compare the metastatic nodules with the primary tumor. Tumor staging was performed according to the tumor-node-metastasis (TNM) staging system of the International Union against Cancer (UICC) [22]. There were 39 cases at stage I“II and 39 cases at stage IIIa“IIIb. None of the patients had received radiotherapy or chemotherapy before the operation and were given the standard treatment following the surgery. All samples were fixed in formalin embedded in paraffin and stained with hematoxylin and eosin for pathological analysis and diagnosis. Cell culture Normal human bronchial epithelial (HBE) cells and A549 H1299 H460 and H157 cell lines were obtained from the American Type Culture Collection (Manassas VA USA). The SPC-A-1 LTEP-A-2 and LK2 cell lines were purchased from the Shanghai Cell Bank of Chinese Academy of Science. The human lung ADC Anip973 and AGZY83a cell lines were purchased from Shanghai Bioleaf Biotech Co. Ltd (http://www.bioleaf.com) and stored in the Department of Pathology Harbin Medical University. Cells were cultured in RPMI 1640 (Invitrogen Carlsbad CA USA) containing 10% fetal calf serum (Invitrogen) 100 IU/ml penicillin (Sigma St. Louis MO USA) and 100 mg/ml streptomycin (Sigma). Plasmid construction and transfection Expression plasmids for p120ctn isoforms 1A and 3A (donated by Dr. Albert B. Reynolds Department of Cancer Biology Vanderbilt University School of Medicine TN USA) have been described previously [16]. Sequences of p120ctn-1A-siRNA (Guangzhou Ruibo Co. Ltd Guangzhou China) used in the experiments were as follows: si-h-CTNND1: 5?-CACAAGAUGCCAACCCACU dTdT-3? 3?-dTdT GUGUUCUACGGUUGGGUGA-5?. The cells were transiently transfected with p120ctn-1A-siRNA and plasmids expressing p120ctn isoforms 1A and 3A using Lipofectamine 2000 (Invitrogen Carlsbad CA) or Attractene Transfection Reagent (QIAGEN GmbH Hilden Germany) according to the manufacturer's instructions. Immunohistochemistry The paraffin embedded samples were cut serially into 4-?m thick sections. Normal bronchial epithelium present in the tumor slides was used as an internal positive control."
Lung_Cancer
"of erythrocytes in systemic lupus erythematosus: analysis of the stability of the defect and of a restriction fragment length polymorphism of the CR1 gene J Immunol 1987 138 2708 2710 2881967 Xiang L Rundles JR Hamilton DR Wilson JG Quantitative alleles of CR1: coding sequence analysis and comparison of haplotypes in two ethnic groups J Immunol 1999 163 4939 4945 10528197 Birmingham DJ Chen W Liang G Schmitt HC Gavit K Nagaraja HN A CR1 polymorphism associated with constitutive erythrocyte CR1 levels affects binding to C4b but not C3b Immunology 2003 108 531 538 10.1046/j.1365-2567.2003.01579.x 12667215 Holers VM Chaplin DD Leykam JF Gruner BA Kumar V Atkinson JP Human complement C3b/C4b receptor (CR1) mRNA polymorphism that correlates with the CR1 allelic molecular weight polymorphism Proc Natl Acad Sci U S A 1987 84 2459 2463 10.1073/pnas.84.8.2459 3031685 Zhang Q Yu JT Zhu QX Zhang W Wu ZC Miao D Tan L Complement receptor 1 polymorphisms and risk of late-onset Alzheimer™s disease Brain Res 2010 1348 216 221 20558149 He JR Xi J Ren ZF Qin H Zhang Y Zeng YX Mo HY Jia WH Complement receptor 1 expression in peripheral blood mononuclear cells and the association with clinicopathological features and prognosis of nasopharyngeal carcinoma Asian Pac J Cancer Prev 2012 13 6527 6531 10.7314/APJCP.2012.13.12.6527 23464487 Srivastava A Mittal B Complement receptor 1 (A3650G RsaI and intron 27 HindIII) polymorphisms and risk of gallbladder cancer in north Indian population Scand J Immunol 2009 70 614 620 10.1111/j.1365-3083.2009.02329.x 19906204 Ferreira CG Lung cancer in developing countries Am Soc Clin Oncol Educ Book 2013 327 331 PMID:23714537 23714537 Amos CI Wu X Broderick P Gorlov IP Gu J Eisen T Dong Q Zhang Q Gu X Vijayakrishnan J Sullivan K Matakidou A Wang Y Mills G Doheny K Tsai YY Chen WV Shete S Spitz MR Houlston RS Genome-wide association scan of tag SNPs identifies a susceptibility locus for lung cancer at 15q25.1 Nat Genet 2008 40 616 622 10.1038/ng.109 18385676 Hung RJ McKay JD Gaborieau V Boffetta P Hashibe M Zaridze D Mukeria A Szeszenia-Dabrowska N Lissowska J Rudnai P Fabianova E Mates D Bencko V Foretova L Janout V Chen C Goodman G Field JK Liloglou T Xinarianos G Cassidy A McLaughlin J Liu G Narod S Krokan HE Skorpen F Elvestad MB Hveem K Vatten L Linseisen J A susceptibility locus for lung cancer maps to nicotinic acetylcholine receptor subunit genes on 15q25 Nature 2008 452 633 637 10.1038/nature06885 18385738 Wang Y Broderick P Webb E Wu X Vijayakrishnan J Matakidou A Qureshi M Dong Q Gu X Chen WV Spitz MR Eisen T Amos CI Houlston RS Common 5p15.33 and 6p21.33 variants influence lung cancer risk Nat Genet 2008 40 1407 1409 10.1038/ng.273 18978787 McKay JD Hung RJ Gaborieau V Boffetta P Chabrier A Byrnes G Zaridze D Mukeria A Szeszenia-Dabrowska N Lissowska J Rudnai P Fabianova E Mates D Bencko V Foretova L Janout V McLaughlin J Shepherd F Montpetit A Narod S Krokan HE Skorpen F Elvestad MB Vatten L Nj¸lstad I Axelsson T Chen C Goodman G Barnett M Loomis MM Lung cancer susceptibility locus at 5p15.33 Nat Genet 2008 40 1404 1406 10.1038/ng.254 18978790 Markiewski MM Lambris JD The role of complement in inflammatory diseases from behind the scenes into the spotlight Am J Pathol 2007 171 715 727 10.2353/ajpath.2007.070166 17640961 Ricklin D Lambris JD Complement-targeted therapeutics Nat Biotechnol 2007 25 1265 1275 10.1038/nbt1342 17989689 Hugli TE Biochemistry and biology of anaphylatoxins Complement 1986 3 111 127 3542363 Ostrand-Rosenberg S Cancer and complement Nat Biotechnol 2008 26 1348 1349 10.1038/nbt1208-1348 19060872 Markiewski MM DeAngelis RA Benencia F Ricklin-Lichtsteiner SK Koutoulaki A Gerard C Coukos G Lambris JD Modulation of the antitumor immune response by complement Nat Immunol 2008 9 1225 1235 10.1038/ni.1655 18820683 Markiewski MM Lambris JD Is complement good or bad for cancer patients? A new perspective on an old dilemma Trends Immunol 2009 30 286 292 10.1016/j.it.2009.04.002 19428302 Fan Q He JF Wang QR Cai HB Sun XG Zhou XX Qin HD Shugart YY Jia WH Functional polymorphism in the 5?-UTR of CR2 is associated with susceptibility to nasopharyngeal carcinoma Oncol Rep 2013 30 11 16 23612877 Cerhan JR Novak AJ Fredericksen ZS Wang AH Liebow M Call TG Dogan A Witzig TE Ansell SM Habermann TM Kay NE Slager SL Risk of non-Hodgkin lymphoma in association with germline variation in complement genes Br J Haematol 2009 145 614 623 10.1111/j.1365-2141.2009.07675.x 19344414 Zhang Z Yu D Yuan J Guo Y Wang H Zhang X Cigarette smoking strongly modifies the association of complement factor H variant and the risk of lung cancer Cancer Epidemiol 2012 36 e111 e115 10.1016/j.canep.2011.11.004 22197220 Shen M Vermeulen R Rajaraman P Menashe I He X Chapman RS Yeager M Thomas G Burdett L Hutchinson A Yuenger J Chanock S Lan Q Polymorphisms in innate immunity genes and lung cancer risk in Xuanwei China Environ Mol Mutagen 2009 50 285 290 10.1002/em.20452 19170196 Fearon DT Identification of the membrane glycoprotein that is the C3b receptor of the human erythrocyte polymorphonuclear leukocyte B lymphocyte and monocyte J Exp Med 1980 152 20 30 10.1084/jem.152.1.20 6967510 Besaratinia A Pfeifer GP Second-hand smoke and human lung cancer Lancet Oncol 2008 9 657 666 10.1016/S1470-2045(08)70172-4 18598930 Coyle YM Minahjuddin AT Hynan LS Minna JD An ecological study of the association of metal air pollutants with lung cancer incidence in Texas J Thorac Oncol 2006 1 654 661 10.1097/01243894-200609000-00009 17409932 Garshick E Laden F Hart JE Rosner B Smith TJ Dockery DW Speizer FE Lung cancer in railroad workers exposed to diesel exhaust Environ Health Perspect 2004 112 1539 1543 10.1289/ehp.7195 15531439 Brennan P Buffler PA Reynolds P Wu AH Wichmann HE Agudo A Pershagen G Jockel KH Benhamou S Greenberg RS Merletti F Winck C Fontham ET Kreuzer M Darby SC Forastiere F Simonato L Boffetta P Secondhand smoke exposure in adulthood and risk of lung cancer among never smokers: a pooled analysis of two large studies Int J Cancer 2004 109 125 131 10.1002/ijc.11682 14735478 Lee G Walser TC Dubinett SM Chronic inflammation chronic obstructive pulmonary disease and lung cancer Curr Opin Pulm Med 2009 15 303 307 10.1097/MCP.0b013e32832c975a 19417670 Engels EA Inflammation in the development of lung cancer: epidemiological evidence Expert Rev Anticancer Ther 2008 8 605 615 10.1586/14737140.8.4.605 18402527 Kullo IJ Ding K Shameer K McCarty CA Jarvik GP Denny JC Ritchie MD Ye Z Crosslin DR Chisholm RL Manolio TA Chute CG Complement receptor 1 gene variants are associated with erythrocyte sedimentation rate Am J Hum Genet 2011 89 131 138 10.1016/j.ajhg.2011.05.019 21700265 Teeranaipong P Ohashi J Patarapotikul J Kimura R Nuchnoi P Hananantachai H Naka I Putaporntip C Jongwutiwes S Tokunaga K A functional single-nucleotide polymorphism in the CR1 promoter region contributes to protection against cerebral malaria J Infect Dis 2008 198 1880 1891 10.1086/593338 18954261 Chen GB Xu Y Xu HM Li MD Zhu J Lou XY Practical and theoretical considerations in study design for detecting gene-gene interactions using MDR and GMDR approaches PLoS One 2011 6 e16981 .0016981 21386969 Lou XY Chen GB Yan L Ma JZ Zhu J Elston RC Li MD A generalized combinatorial approach for detecting gene-by-gene and gene-by-environment interactions with application to nicotine dependence Am J Hum Genet 2007 80 1125 1137 10.1086/518312 17503330 PLoS One one 1932-6203 Public Library of Science San Francisco USA 24416392 3887046 PONE-D-13-33822 .0085329 Research Mathematics Statistics Biostatistics Statistical Methods Medicine Clinical Research Design Meta-Analyses Oncology Basic Cancer Research Metastasis Cancers and Neoplasms Lung and Intrathoracic Tumors Pulmonology Surgery Thoracic Surgery Evaluation of Video-Assisted Thoracoscopic Surgery for Pulmonary Metastases: A Meta-Analysis VATS for Pulmonary Metastases Dong Siyuan Zhang Lin * Li Wenya Du Jiang Liu Xiangli Chen Xitao Department of Thoracic Surgery First Hospital of China Medical University Shenyang Liaoning Province People's Republic of China Arnold Paul Editor University of Kansas United States of America * E-mail: [email protected] Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: SYD LZ. Performed the experiments: SYD LZ WYL JD XLL XTC. Analyzed the data: SYD LZ WYL JD XLL XTC. Contributed reagents/materials/analysis tools: SYD LZ WYL JD XLL XTC. Wrote the paper: SYD LZ. 2014 9 1 2014 9 1 e85329 16 8 2013 25 11 2013 2014 Dong et al This is an open-access distributed under the terms of the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited. Background To evaluate the evidence comparing video-assisted thoracic surgery (VATS) and open thoracotomy in the treatment of metastatic lung cancer using meta-analytical techniques. Methods A literature search was undertaken until July 2013 to identify the comparative studies evaluating disease-free survival rates and survival rates. The pooled odds ratios (OR) and the 95% confidence intervals (95% CI) were calculated with the fixed or random effect models. Results Six retrospective studies were included in our meta-analysis. These studies included a total of 546 patients: 235 patients were treated with VATS and 311 patients were treated with open thoracotomy. The VATS and the thoracotomy did not demonstrate a significant difference in the 1-3-5-year survival rates and the 1-year disease-free survival rate. There were significant statistical differences between the 3-year disease free survival rate (p?=?0.04) which favored open thoracotomy. Conclusions The VATS approach is a safe and feasible treatment in terms of the survival rate for metastatic lung cancer compared with the thoracotomy. The 3-year disease-free survival rate in the VATS group is inferior to that of open thoracotomy. The VATS approach could not completely replace open thoracotomy. The authors have no support or funding to report. Introduction Metastasectomy is considered a beneficial treatment for a patient with metastatic lung cancer whose primary tumor has been well controlled[1].After surgery 5-year survival rates of 30% to 50% could be achieved depending on the underlying primary cancer[2]“[4].In practice the surgical approaches to pulmonary metastases are variable. Video-assisted thoracoscopic surgery (VATS) is an emerging technique; many procedures that had previously required a thoracotomy have been performed with the minimally invasive VATS. VATS has been used for the treatment of pulmonary metastases. The routine use of VATS for the treatment of respectable metastatic lung cancer remains controversial. Critics of the VATS approach have argued that it might not be an equivalent oncological operation[5] [6]. A prospective study by Cerfolio[7]found that 22% of the nodules that could be detected by thoracotomy were missing by VATS.Whether the VATS approach can provide a satisfactory outcome is unknown. An evidenced-based investigation of the VATS approach is needed we undertook this meta-analysis to achieve a more objective assessment of the published studies and to provide a more accurate comparison between VATS and thoracotomy for metastatic lung cancer. Methods Search Strategy Electronic searches were of the MEDLINECochrane Controlled Trial Register (CENTRAL) Ovid MEDILINE PubMed and Embase databases were performed until July 2013.The following MeSH search headings were used: œmetastatic lung cancer œpulmonary metastases œvideo-assisted thoracic surgery œthoracotomy and œcomparative study.We searched the reference lists of relevant studies reviews editorials lettersand meeting abstracts. We used the Science Citation Index to cross-reference for further studies that met our criteria. Study Selection The studies included in this meta-analysis were based on our predetermined criteria as follows: (1) clinical trials that include the full text of the paper published in peer-reviewed English journals or reports of presentations at major thoracic surgery meetings; (2) comparison of the efficacy of VATS to that of thoracotomy in patients with metastatic lung cancer; and (3) similarity in the patients' baseline characteristics. Data extraction and quality assessment Two independent reviewers (Siyuan and Wenya) assessed the quality and the risk of bias of the included trials as follows: (1) the studies that did not include a comparative group with surgery as a form of intervention were excluded; (2) the trials focusing on patients undergoing surgery for primary lung cancer were excluded; (3) the studies on robotic video-assisted thoracic surgery were excluded; (4) if there was an overlap between authors centers or patient cohorts evaluated in the published literature only the most recent report was included; (5) studies published more than 20 years ago were excluded because of the significant technological changes that has occurred. The s were evaluated with the Downs and Black quality assessment method[8]. Discrepancies between the two investigators were resolved by discussion and consensus with a senior investigator. The final results were reviewed by two senior investigators (Lin and Jiang).The disease-free survival was defined as the date of the initial metastasectomy until the date of a recurrence. Statistical and sensitivity analyses The meta-analysis was performed using the RevMan 5.1.0. software package. The odds ratio (OR) or the mean difference with 95% confidence intervals (95% CI) was calculated for the dichotomous outcomes and the continuous outcomes respectively. A P value<0.05 was considered a significant difference in the value between the two groups. We used the I2 statistic to investigate the heterogeneity among the studies.The heterogeneity was explored by X2 and I2; I2<25% and I2>50% reflect a small and large inconsistency respectively. P<0.05 was considered significant. If there were a statistical difference in terms of the heterogeneity (P?0.05) a random-effect model was selected to pool the data. Otherwise a fixed-effect model was used. Taking into account the presence of different sample sizes of the included studies a sensitivity analysis was performed to compare the of 1-year survival rate and the 3-year disease free survival rate between VATS and open thoracotomy. Publication bias A funnel plot was used to explore bias. Asymmetry in the funnel plot of trial size against treatment effect was used to assess the risk of bias. Results Description of the studies Six retrospective cohort studies the met our criteria were included in this meta-analysis. A total of 546 patients were included in the six studies;235 patients were allocated to the VATS group whereas 311 were allocated to the open thoracotomy group to evaluate their survival rate.The search algorithm results of the search strategies and selection criteria are shown in Fig 1. The patient characteristics and evaluation index are shown in Table 1. .0085329.g001 Figure 1 Identification of studies for inclusion. .0085329.t001 Table 1 Study Design Country NO(V/O) Gender (M/F) Mean age (years) Assessment score Nakajima2001[28] OC Japan 45/55 V59/41 O34/21 V55±15 O55±14 13 Mutsaerts2002[29] OC Netherlands 8/12 NR NR 19 Nakas2009[30] OC UK 25/27 V16/9 O 19/8 V69 O66 16 Carballo2009[31] OC USA 36/135 V18/18 O82/53 V58.5 O49 15 Gossot2009[32] OC France 31/29 V21/10 O13/16 V43 O40 18 Chao2012[33] OC Taiwan 90/53 V49/41 O35/18 NR 13 V VATS; O Open thoracotomy; NR Not reported; OC observational cohort. Assessment of Recurrence and Survival Six studies documented the 1-year survival rateand there was no significant heterogeneity among the six studies (x2?=?3.79 P?=?0.58I2?=?0%).A fixed effect model was used.The combined result is shown in Fig 2(OR?=?1.15; 95%CI 0.72“1.84; p?=?0.58). Because of the heterogeneity in sample size the sensitivity analyses were conducted using larger sample sizes. There was no difference between the two surgical methods with an OR of 1.00(95%CI 0.55“1.79) and with heterogeneity(?2?=?3.23P?=?0.07 I2?=?69%). Five studies reported the 3-year survival rate and heterogeneity was identified through the five studies (x2?=?11.32P?=?0.02I2?=?65%); and a random effect model was adopted (OR?=?1.07; 95%CI 0.50“2.27; p?=?0.86) (Fig 3). Three studies compared the 5-year survival rate (OR?=?0.96; 95%CI 0.34“2.71; p?=?0.93) with certain heterogeneity(x2?=?8.86P?=?0.01I2?=?77%) (Fig 4). .0085329.g002 Figure 2 1-year survival rate. Forest plot of the Odds Ratio(OR) of the 1-year survival rate following VATS versus open thoracotomy for metastatic lung cancer.The estimate of the OR of each individual trial corresponds to the middle of the squares and horizontal line gives the 95% CI.On each linethe numbers of events as a fraction of the total number randomized are shown for both treatment groups.For each subgroupthe sum of the statistics along with the summary OR is represented by the middle of the solid diamonds.A test of heterogeneity between the trials within a subgroup is given below the summary statistics. .0085329.g003 Figure 3 3-year survival rate. Forest plot of the Odds Ratio(OR) of the 3-year survival rate following VATS versus open thoracotomy for metastatic lung cancer."
Lung_Cancer
"As differential LysoTracker retention may reflect differences in basal lysosomal mass and/or lysosomal pH we therefore next examined phenothiazine-induced changes in LysoTracker retention (?LysoTracker) as an indicator of perturbation in lysosomal pH. Exposure to TFP for 24?h was associated with varying degrees of loss in LysoTracker retention in almost all of the LC cell lines tested (b). Furthermore there was a highly significant positive correlation between the extent of TFP-induced loss in LysoTracker retention and the magnitude of cytotoxicity (b left). Importantly the loss of LysoTracker retention became irreversible in SCLC at lower TFP concentrations than in NSCLC cells (10??M in H82 and 20??M in U-1810) (c) and the afflicted cells underwent clear mitochondrial depolarization (d). By contrast cells exposed to non-cytotoxic concentrations of TFP recovered LysoTracker Green staining to various degrees over time (c). These data showed that prolonged disruption of lysosomal functions was detrimental to LC cell viability and that SCLC were more sensitive to phenothiazines as result of lower intrinsic lysosomal buffer capacity. In line with these results TFP-induced cell death was significantly antagonized by the protease inhibitor E-64d (e). Moreover preventing the accumulation of TFP within lysosomes by BafA1 pre-treatment also largely abolished its negative impact on cell viability (f). Thus our data suggest that the more prominent cytotoxic effect of phenothiazines in SCLC is related to a higher degree of lysosomal pH neutralization (?LysoTracker) that is more persistent in SCLC than in NSCLC. Moreover although phenothiazines initially induced LC3-II in both NSCLC and SCLC only SCLC failed to adapt to phenothiazine-induced lysosomal dysfunction and this led to increased cell death preferentially in SCLC. Together these data reinforce the notion that persistent lysosomal perturbation induced upon single-agent phenothiazine treatment is a major mediator of cytotoxicity in SCLC cells. SCLC cells show increased sensitivity toward lysosome-disrupting agents On the basis of the findings presented above we reasoned that the increased susceptibility of SCLC cells to phenothiazines may be due to lower basal lysosomal mass and/or lysosomal pH buffer capacity leading to intrinsically lower tolerance toward compounds that disrupt lysosomal functions. Consistent with this idea we found that SCLC cells were typically more sensitive than NSCLC counterparts toward a number of lysosomotropic agents (Supplementary Figure S3) including the clinically approved drugs tamoxifen (TMX) and chloroquine (CQ) in addition to the phenothiazine compounds TFP and CPZ. Notably TFP CPZ TMX and CQ all dissipated LysoTracker staining to a larger extent in SCLC (H69 H82 H592 and U-1285) than in NSCLC (A549 and U-1810) cell lines (Figures 6a and b). Moreover the increased sensitivity of SCLC cells toward lysosomotropic agents was not recapitulated with the CaM antagonist W7 suggesting that phenothiazine-induced lysosomal perturbation does not involve modulation of Ca2+/CaM signaling a well-known process inhibited by phenothiazines (c Supplementary Figure S3A). Overall our findings highlight not only the potential for treating SCLC with FDA-approved drugs that target lysosomal acidification in general but also the use of phenothiazines against this tumor type in particular. Discussion Phenothiazines are most well-known for their dopamine antagonistic activity in the central nervous system (CNS) which has been utilized clinically for the management of psychiatric disorders such as schizophrenia.1 2 In addition these compounds affect the fate of non-CNS cells in a variety of ways and depending on the experimental context may result in cellular differentiation cell death or protection from toxic injuries.34 9 One well-recognized property of phenothiazines is their ability to induce apoptosis in certain cell types.34 9 The mechanism(s) behind this is not clearly understood although inhibition of CaM-regulated processes and increases in membrane fluidity/permeability are thought to be at least partially responsible. However the cell-intrinsic determinants that govern which of these effects that will be elicited in a particular model system are not known and the underlining molecular pathways remain poorly defined. Elucidating the contextual utilities of phenothiazines in in vitro system response in tumor xenografts and animal models will enable them to be harnessed appropriately for treating different human ailments including tumors as illustrated here with SCLC. Although targeted agents have been introduced for the clinical management of LC cases the majority of SCLC and NSCLC patients with advanced disseminated diseases are still treated with conventional CT agents such as cisplatin. For SCLC the initial response is often good but most cases relapse and present high degrees of chemoresistance while for NSCLC a less CT-sensitive phenotype is usually found already at start of treatment.14 Consequently there is an urgent need for the development of new treatment regimen to combat both subtypes of LC. In this study we evaluated a novel strategy involving the use of phenothiazines as single treatment agents in LC. Our data demonstrate that phenothiazines are generally more cytotoxic in SCLC than in NSCLC cell lines despite comparable responses to cisplatin etoposide and gemcitabine which are standard chemotherapeutic agents for the treatment of LC. Importantly we show that normal lung fibroblasts are less affected by phenothiazines at concentrations which were toxic for SCLC indicating a favorable therapeutic window that would allow its use in SCLC without incurring significant adverse effects on healthy tissues. Although it needs to be confirmed by further in vivo toxicity analysis; several earlier reports have shown that phenothiazines are in general well-tolerated by cancer patients.10 15 To uncover responsible mechanisms for the preferential susceptibility of SCLC to phenothiazines we dissected the molecular details of phenothiazine-induced cell death using multiple SCLC and NSCLC cell lines and with TFP as a model compound. We found that TFP treatment led to a rapid neutralization of lysosomal pH as judged by decreased retention of the lysosomotropic dye LysoTracker Green accompanied by accumulation of LC3-II in SCLC cells. Our data thus corroborated a previous study that identified TFP as an inducer of autophagy at low doses in H4 human neuroglioma cells.16 However we found that TFP at cytotoxic concentrations irreversibly disrupted lysosomal homeostasis especially in SCLC cells driving LC3 conversion while blocking its degradation through autophagy. This was logical given that protonation of the weakly basic phenothiazines within lysosomes is expected to increase lumenal pH and could thereby adversely affect the activities of acid hydrolases.13 17 Prolonged exposure to cytotoxic concentrations of TFP (10??M in H82 and 20??M in U-1810) was associated with lysosomal membrane permeabilization followed by mitochondrial depolarization."
Lung_Cancer
"We demonstrated in this study that the MSLN-targeted fusion protein elicited significant tumor-specific CD8+ T-cell immune responses in ovarian cancer-bearing mice and this adaptive antitumor response has an absolute requirement for tumor-specific CD8+ T cells. Although at the dosing schedule used in these studies tumor-specific T-cell responses did not eventually lead to rejection of the established tumors they significantly prolonged survival time in tumor-bearing mice. DCs are believed to play a pivotal role in the initiation and programming of tumor-specific T-cell responses and are becoming an essential target in efforts to generate therapeutic immunity against cancer [33]. Two main approaches are currently under consideration for providing DCs with tumor-specific antigens. One approach is to culture patient-derived DCs ex vivo with an adjuvant that induces DC maturation in the presence of tumor specific antigens followed by adoptive transfer into the patient [33]. This approach is fraught with technical and practical difficulties such as selection of a suitable antigenic target inappropriate maturation state of selected DCs and the difficulty of generating a sufficient number of DCs ex vivo. In addition a number of investigators have recently reported that ex vivo-derived DC vaccines have an insignificant role in the direct priming of T cells in vivo[33-35]. An alternative approach to generate tumor-specific antigen bearing DCs is to induce them to take up tumor-specific antigens in vivo. It has been shown that in vivo specific targeting of tumor antigens to DCs improves the induction of antigen-specific CD4+ and CD8+ T-cell immunity. In these studies an agonistic anti-CD40 monoclonal antibody was used to mature DCs and eliminate antigen-specific tolerance [36-39]. MTBHsp70 has also been shown to stimulate inflammation and DC maturation via an interaction with CD40 receptors on both DCs and monocytes thus acting as an alternative ligand to CD40L [2940]. In our study we showed the fusion protein up-regulates surface expression of phenotypic markers of DC maturation. Interestingly in addition to CD80 CD86 and MHC class II molecules the expression of CD40 is also enhanced indicating a possible positive feedback loop involving CD40 signaling components. Beyond promoting DC maturation the scFvMTBHsp70 fusion protein also targets tumor cells towards the matured DCs. We propose that binding of the fusion protein with both tumor cells and DCs improves phagocytosis of parts of tumor cells by DCs and therefore any tumor antigen can be processed and loaded on both MHC class II and MHC class I molecules and presented to CD4+ and CD8+ T cells. This could explain the observed augmentation of tumor antigen presentation and cross-presentation brought about by the fusion protein in vitro. This may also explain the observed increased anti-Her2/neu CD8+ T-cell responses in the scFvMTBHsp70-treated ovarian tumor bearing mice although Her2/neu is not directly targeted. We recapitulated these in vitro findings in an in vivo tumor cell immunogenicity study. We used the fusion protein to activate and mature DCs in the skin such as Langerhans cells. These DCs then captured tumor cells or tumor cell fragments through the connection established by the fusion protein and migrated to the draining lymphoid ans where they presented tumor antigens to na¯ve T cells. T cells recovered from the draining lymph node showed significantly enhanced responses to stimulation with a range of tumor antigens. Conclusion Our study provides preclinical evidence that supports a protein-based immunotherapy that induces anti-tumor immune responses which normally require dendritic cell-based approaches. The MSLN-targeted MTBHsp70 fusion protein binds MSLN on tumor cells recruits and activates APCs including DCs loads DCs in vivo with the broadest profile of naturally processed tumor antigens promotes tumor antigen presentation and cross-presentation and enhances tumor specific CD4+ and CD8+ T-cell responses (Figure 5). Our study supports the continued exploration of this novel fusion protein alone or in combination with immune checkpoint inhibitors following conventional surgical reduction and chemotherapy for MSLN-expressing cancers. This new approach could significantly increase time to recurrence and survival in humans with ovarian cancer and mesothelioma where effective second line treatment options are very limited. A schematic model showing that the scFvMTBHsp70 fusion protein binds with MSLN on tumor cells and activates antigen presenting cells (APCs) thus promoting uptake of tumor cells or tumor cell fragments and promoting tumor antigen presentation and cross-presentation as well as adjuvanting tumor specific CD4 + and CD8 + T-cell responses. Methods Production of proteins The plasmid pQE30-MTBhsp70 that encodes full length MTBHsp70 was a generous gift from Dr. Peter Sveshnikov (Moscow Medical Academy Russia). The plasmid pTOR2-scFv that encodes an scFv fragment specific to MSLN and the recombinant P4 scFv protein [13] generated and purified from yeast were generous gifts from Dr. Nathalie Scholler (Penn Ovarian Cancer Research Center University of Pennsylvania). The DNA fragment corresponding to a 15 amino acid linker (GGGGSGGGGSGGGGS) was connected to the scFv at its C-terminal using an overlap PCR approach. The PCR product scFv-linker was subcloned into pQE30-MTBhsp70 at the N-terminal of MTBhsp70. The DNA fragment for scFvMTBhsp70 was PCR amplified and cloned into pPMY5 (Promab) downstream of a human IgG1 Fc domain and separated from the Fc region by the signal cleavage sequence for Tobacco Etch Virus protease (TEV enzyme). scFvMTBHsp70 the MSLN-targeted fusion protein was generated from HEK293 cells and purified using Protein G resin (Pierce). The Fc region of the Protein G eluted protein was then cleaved from the fusion protein by TEV enzyme (Promab) digestion. MTBHsp70 was generated using the same expression system. The production and purification of these two proteins was accomplished by Promab Biotechnologies Inc. at Richmond CA. After purification and hIgG-Fc tag removal the integrity of scFv-MTBHsp70 and MTBHsp70 were determined by SDS-PAGE followed by staining with RAPIDstain (G-Bioscience). Endotoxin contamination levels in scFvMTBHsp70 and MTBHsp70 were determined by Limulus Amebocyte Lysate Assay (LAL-assay Cambrex). Cells The BR5FVB1 ovarian cancer cells a kind gift from Dr. Orsulic in Women™s Cancer Research Institute at Cedars-Sinai Medical Center [41] or 40L mesothelioma cells a kind gift from Dr. Kane in Department of Pathology and Laboratory Medicine at Brown University [42] were maintained at 37°C in DMEM with 2 mmol/L L-glutamine 10 units/ml penicillin 10 ?g/ml streptomycin and 10% fetal bovine serum in humidified atmosphere with 5% CO2. Cells were cultured until 80% confluent and harvested with enzyme-free cell-dissociation buffer (Gibco) for in vitro tumor cell binding assays and cross-presentation studies or harvested with Trypsin EDTA (Mediatech) for animal injections. Mouse PBLs were obtained from FVB mice via tail vein bleeds after lysis of erythrocytes using M-lyse buffer (R&D systems). Small pieces of parietal peritoneal membrane were taken from the mice and digested in enzyme-free cell-dissociation buffer to obtain mouse peritoneal mesothelial cells. To test whether scFvMTBHsp70 or MTBHsp70 binds to the MSLN-expressing tumor cells or non-cancerous cells we incubated BR5FVB1 ovarian tumor cells 40L mesothelioma cells or normal cells from FVB mice including PBLs splenocytes and peritoneal mesothelial cells with 40 ?g/ml scFvMTBHsp70 or 26 ?g/ml MTBHsp70 followed by anti-MTBHsp70 (IgG2a) (Biodesign International) biotinylated anti-IgG2a (BD Bioscience) and Streptavidin-APC (BioLegend) and then analyzed the tumor cells by flow cytometry. As controls cells were incubated with the reagents described above except scFvMTBHsp70 or MTBHsp70. To confirm that scFv portion of the fusion protein binds to MSLN on the surface of tumor cells scFvMTBHsp70 or MTBHsp70 was preincubated with 12 ?g/ml of recombinant human MSLN (R&D Systems) for 30 min before adding to the cells. For fluorescence microscopy cells were cultured on coverslips until 50% confluent stained with 10 ?g/ml scFvMTBHsp70 or 6.5 ?g/ml MTBHsp70 followed by mouse anti-MTBHsp70 (1:500 dilution) and Donkey anti-mouse Alexa Fluor 594 (Invitrogen 1:500 dilution). Cells were observed using a Nikon Eclipse TiE fluorescence microscope. In some experiments tumor cells were treated with 20 ?g/ml mitomycin C at a concentration of 5 — 106/ml for 1 h in a 37°C water bath and washed with complete medium at least 3 times before use. Animal models and tumor treatment Ovarian cancer was established by i.p. injection of syngeneic cancer cells BR5FVB1 (107 cells per mouse) into 6-week-old female FVB/NJ mice as previously described [25]. All mice were purchased from Jackson laboratories. Intraperitoneal mesotheliomas were established by i.p. injection of syngeneic 40L cells (2?—?106 per mouse) into 6-week-old male C57BL/6 mice as previously described [42]. Mice with ovarian tumors were treated 7 days after BR5FVB1 tumor cell inoculation with i.p. injections of scFvMTBHsp70 (2 ?g per mouse) normal saline or an equimolar mixture of MTBHsp70 plus P4 scFv. This was followed by 3 further treatments at 4-day intervals. In the mesothelioma model C57BL/6 mice were treated 5 days after 40L tumor cell inoculation and injected i.p. with scFvMTBHsp70 (2 ?g per mouse) normal saline or an equimolar mixture of MTBHsp70 plus P4 scFv. Three subsequent doses were administered at 3-day intervals thereafter. For survival studies we observed the mice daily 3 weeks after inoculation of BR5FVB1 cells or 1 week after inoculation of 40L cells. Tumor generations were consistently first evident via abdominal distension secondary to malignant ascites and tumor-bearing mice were euthanized at the endpoint when there were signs of distress including fur ruffling rapid respiratory rate hunched posture reduced activity and progressive ascites formation as previously described [25]. For the investigation of anti-tumor T-cell responses all ovarian tumor-bearing mice were sacrificed 7 days after the final scheduled treatment. All studies were performed in a manner that was blinded to the observer under protocols that were approved by the Massachusetts General Hospital Subcommittee on Research Animal Care (SRAC). Treatment of na¯ve mice with experimental or control protein 6-week-old male C57BL/6 mice were injected i.p. with scFvMTBHsp70 (2 ?g per mouse) normal saline or an equimolar mixture of MTBHsp70 plus P4 scFv. Three subsequent doses were administered at 3-day intervals thereafter. Seven days post the administration of the final treatment mice were sacrificed and abdominal wall and intestine were retrieved for histopathological studies of mesothelial tissues. Ex vivo assessment of tumor specific T-cell functions Single cell suspensions were prepared from spleens. Cells were plated in round-bottomed 96-well plates pulsed with a validated CD8+ T-cell Her2/neu peptide (PDSLRDLSVF 1 ?g/ml; EZBiolab) [2543] an in-house designed H2d-restricted MSLN Ld1 peptide (IPLSYLCDF 1 ?g/ml; EZBiolab) that did not induce ovarian cancer specific T-cell response in H-2q FVB mice or medium alone for 72 hours when Golgi Plug (BD Bioscience) was added for the last 5 hours as previously described [44] and then stained with fluorophore-conjugated anti-CD3 anti-CD4 anti-CD8 anti-IFN? (BD Pharmingen) and anti-Granzyme B (eBioscience) antibodies. Cells were then analyzed on a LSRII 4 laser (BD Biosciences). Depletion of CD8+ T cells in vivo FVB/NJ mice were injected i.p. with 200 ?g of anti-CD8 monoclonal antibody (mAb)(53“6.72 Bio X Cell) or an isotype-matched irrelevant rat IgG2a (2A3 Bio X Cell) 2 days before 1 day before and 1 day after i.p. inoculation with BR5FVB1 ovarian tumor cells. Depletion was continued once every week until 29 days after tumor inoculation. The mice were treated with scFvMTBHsp70 or saline as described above. All the mice were bled from the tail vein and the depletion of CD8+ cells was examined by flow cytometry analysis of peripheral blood cells stained with fluorophore-conjugated anti-CD8 on days 7 and 28 after tumor inoculation. Generation and purification of bone marrow-derived DCs (BMDCs) CD11c+ DCs were generated from bone marrow cells of FVB/NJ mice as described [45-47] with minor modifications. Briefly erythrocyte-depleted mouse bone marrow cells from flushed marrow cavities were cultured in complete RPMI 1640 with 10 ng/ml GM-CSF and 1 ng/ml IL-4 at 1 — 106 cells/ml. Medium was changed on day 3. On day 7 DCs were harvested by gentle pipetting and purified with magnetic microbeads conjugated to a monoclonal antibody against CD11c (MiltenyiBiotec) as described [4648] according to the manufacturer™s recommended protocol. In vitro activation of BMDCs CD11c+ BMDCs were plated in a 24-well plate at a density of 2?—?106 cells/ml and incubated with 2 ?g/ml scFvMTBHsp70 (105 kDa) 1.3 ?g/ml MTBHsp70 (70 kDa) 1 ?g/ml LPS equivalent to 103 EU/ml endotoxin (InvivoGen San Diego CA) or 0.1 ng/ml (0.1 EU/ml) LPS equivalent to endotoxin found in 2 ?g/ml of proteins (since LPS level is less than 50 EU per mg of protein) for 24 h at 37°C in humidified atmosphere with 5% CO2. Cells were then placed on ice collected by vigorous pipetting washed and stained with the following fluorophore-conjugated antibodies: anti-CD11c and anti-CD40 (eBioscience) anti-CD80 (BD Horizon) anti-CD86 and anti-MHC class II (I-Aq) (BD Pharmingen). Afterwards the cells were analyzed on an LSRII 4 laser (BD Biosciences). In vitro tumor antigen presentation assay BR5FVB1 cells were harvested and treated with mitomycin C and plated in a 96-well round-bottomed plate with 20 ?g/ml scFvMTBHsp70 or 13 ?g/ml MTBHsp70. After pre-incubation at 4°C for 1 h CD11c+ BMDCs (ratio of tumor cells: DCs = 3: 1) were added to the wells and the plate was incubated at 37°C for 24 h. For generation of BR5FVB1 cell-primed T cells we inoculated FVB/NJ mice by i.p. injection with 107mitomycin C-treated BR5FVB1 cells and sacrificed the mice 60 days after the immunization according to the approved animal protocol. Splenocytes were then harvested and T cells were isolated using the Pan T-Cell Isolation Kit II (MiltenyiBiotec). BR5FVB1 cell-primed T cells were then added to the wells at a DC/T-cell ratio of 1:20. After a 24-hour co-culture of BR5FVB1 cell-pulsed DCs with BR5FVB1 cell-primed T cells the cells were harvested washed and resuspended in PBS with 5% FBS stained for CD3 CD4 CD8 and IFN? and analyzed on a LSRII 4 laser (BD Biosciences). In vivo immunization with mitomycin C-treated ovarian tumor cells BR5FVB1 ovarian tumor cells were harvested with enzyme-free cell-dissociation buffer and treated with mitomycin C as described above. Cells were then pre-incubated with scFvMTBHsp70 (10 ?g/106 cells) MTBHsp70 (6.5 ?g/106 cells) or PBS alone at 4°C for 1 h. 6-week-old FVB mice were shaved and depilated on both left and right flanks and then injected i.d. with 50 ?l of PBS or 1?—?106 tumor cells in 50 ?l of PBS with or without a pre-incubation with scFvMTBHsp70 or MTBHsp70 at both flanks. Histopathology Abdominal walls and intestines from mice were fixed for at least 24 h in PBS-buffered 10% formalin. Tissues were routinely embedded in paraffin. 5 ?m thick sections were stained routinely with H&E. For staining tumor-infiltrating T cells mice were perfused with 4% paraformaldehyde (PFA) in PBS and tumor nodules were fixed in 4% PFA/PBS for additional 2 hours washed and infiltrated with 30% sucrose/PBS at 4°C. 6 ?m thick frozen sections were stained with rat anti-mouse CD8 (BD Biosciences 1:100 dilution) or rat anti-mouse Foxp3 (eBioscience 1:12 dilution) followed by polyclonal rabbit anti-rat immunoglobulin/HRP (Dako 1:750 dilution). Signal was developed with diaminobenzidine (DAB Dako). Images were acquired on a Zeiss Axio A1 microscope. All histopathological and immunohistochemical samples were reviewed and the quantitation of the cellular infiltrate was performed in a blinded manner to the observer. Statistical analysis Statistical differences between three or more experimental groups were analyzed using One-Way ANOVA followed by Turkey™s multiple comparison tests when mean of each group is compared with that of every other group or followed by Dunnett™s multiple comparison tests when mean of each group is compared with that of a control group. Statistical differences between two experimental groups were analyzed using Student™s t-test. Survival was analyzed with the Log-rank test. Prism 6.0 software (GraphPad Software) was used for all the statistical analysis. Abbreviations DC: Dendritic cell; scFv: Single-chain antibody variable fragment; MSLN: Mesothelin; MTB: Mycobacterium tuberculosis; Hsp: Heat shock protein; i.p.: Intraperitoneal; i.d.: Intradermal; BMDCs: Bone marrow-derived dendritic cells; APCs: Antigen-presenting cells; PBMCs: Peripheral blood mononuclear cells; PBLs: Peripheral blood leukocytes; LPS: Lipopolysaccharide; H&E: Haematoxylin and eosin; PFA: Paraformaldehyde; DAB: Diaminobenzidine; mAb: monoclonal antibody. Competing interests The authors declare that they have no competing interests. Authors™ contributions JY played a role in the design of the experiments acquisition analysis and interpretation of the data and writing the manuscript. PR JN YY NHA MN GJ-M XT SK HC PU BF TC and PL participated in the performance of experiments. SK and TB were involved in design of the experiments. RB was involved in data analysis. ER was involved in setting up murine ovarian cancer model. SO provided the murine ovarian cancer model. NS provided the plasmid that encodes an scFv fragment specific to MSLN and the recombinant P4 scFv protein. GD NS and SO gave constructive input on experimental design and data analysis. JG played a role in conception and design of the fusion protein. MP and JG were involved in the conceptualization and design of the study analysis and interpretation of datasets and in writing the manuscript. All authors read and approved the final manuscript. Supplementary Material Additional file 1: Figure S1 scFvMTBHsp70 binds to 40L mesothelioma cells. 40L cells were stained with scFvMTBHsp70 or MTBHsp70 followed by mouse anti-MTBHsp70 and Donkey anti-mouse Alexa Fluor 594. Cells were observed using a Nikon Eclipse TiE fluorescence microscope. A Representative pictures from three independent experiments. Scale bar 10 ?m. B Images were analyzed using the NIS-Elements AR Microscope Imaging Software. Mean Fluorescence Intensity was analyzed using ImageJ. P values were determined using One-Way ANOVA followed by Turkey™s multiple comparison tests. ****p?<?0.0001. Click here for file Additional file 2: Figure S2 scFvMTBHsp70 or MTBHsp70 plus P4 scFv treatment does not lead to infiltration of inflammatory cells into abdominal or intestinal mesothelial tissues. Samples of abdominal wall and intestine were prepared from C57BL/6 mice that had previously received multiple i.p. injections of scFvMTBHsp70 MTBHsp70 plus P4 scFv or saline as described in the Methods section. Sections of these tissues were stained with H&E and images were acquired on a Zeiss Axio A1 microscope. Representative images from 3 animals per treatment group are shown. No detectable level of mononuclear cell or granulocyte infiltrate within mesothelial tissues was seen in any sampled tissues. Scale bar 20 ?m. Click here for file Additional file 3: Figure S3 scFvMTBHsp70 treatment does not affect numbers of tumor-infiltrating CD8+ or Foxp3+ T cells. (A) Representative images of intratumoral CD8+ and Foxp3+ T cells from saline (n?=?3) scFvMTBHsp70 (n?=?3) or MTBHsp70 plus P4 scFv (n?=?3) -treated mice. Mouse spleen sections were used as positive controls: CD8+ and Foxp3+ T cells are clearly evident in the sections. Scale bar 20 ?m. (B) Numbers of CD8+ and Foxp3+ cells were quantified from 3“5 randomized fields. Click here for file Additional file 4: Figure S4 Validation of in vivo depletion of CD8+ cells in FVB/NJ mice. Mice were injected i.p. with 200 ?g of anti-CD8 mAb or an isotype-matched irrelevant rat IgG2a as described in Methods. All the mice were bled from the tail vein and the depletion of CD8+ cells was examined by flow cytometry analysis of peripheral blood cells stained with fluorophore-conjugated anti-CD8 on days 7 and 28 after tumor inoculation. (A) Representative results of flow analyses on 10 mice per group and reported as the percentage of CD8+ cells in lymphocytes. (B) CD8+ cells in the mice treated with isotype IgG2a or anti-CD8 mAb were compared. ***p< 0.001. Click here for file Acknowledgments This manuscript is dedicated to the memory of Janet Gelfand a victim of ovarian cancer. The authors gratefully acknowledge the continuing support for this work from the Edmund C. Lynch Jr. Cancer Fund Arthur Luxenberg Esq. Perry Weitz Esq. and the VIC Mesothelioma Research and Resource Program at MGH and the Friends of VIC Fund. PU and NHA were supported by the Prof. Dulcie V. Coleman Studentship at Imperial College London. We thank Oliver Mitchell John Cao Lujia Zhou Rumbidzai Mushavi and Sayinthen Vivekanantham for their technical assistances Dr. Yuhui Huang for his useful comments Michael Waring Dr. Michael Santuosuosso and Dr. Ravi Mylvaganam for their technical advice Dr. Musie Ghebremichael for his advice in statistical analysis and Mahnoor Valibhoy for her assistance with the schematic figure. Banchereau J Palucka AK Dendritic cells as therapeutic vaccines against cancer Nature reviews Immunology 2005 5 296 306 10.1038/nri1592 15803149 Mellman I Coukos G Dranoff G Cancer immunotherapy comes of age Nature 2011 480 480 489 10.1038/nature10673 22193102 Topalian SL Weiner GJ Pardoll DM Cancer immunotherapy comes of age J Clin Oncol 2011 29 4828 4836 10.1200/JCO.2011.38.0899 22042955 Kantoff PW Higano CS Shore ND Berger ER Small EJ Penson DF Redfern CH Ferrari AC Dreicer R Sims RB Xu Y Frohlich MW Schellhammer PF IMPACT Study Investigators Sipuleucel-T immunotherapy for castration-resistant prostate cancer The New England journal of medicine 2010 363 411 422"
Lung_Cancer
"trial in patients with advanced non-small-cell lung cancer. J Clin Oncol 2010 28 3076 3083 20479403 29. Miller VA Hirsh V Cadranel J Afatinib versus placebo for patients with advanced metastatic non-small-cell lung cancer after failure of erlotinib gefitinib or both and one or two lines of chemotherapy (LUX-Lung 1): a phase 2b/3 randomised trial. Lancet Oncol 2012 13 528 538 22452896 Clin Orthop Relat Res Clin. Orthop. Relat. Res Clinical Orthopaedics and Related Research 0009-921X 1528-1132 Springer US Boston 24249538 3971232 3385 10.1007/s11999-013-3385-9 Clinical Research Does Intensity of Surveillance Affect Survival After Surgery for Sarcomas? Results of a Randomized Noninferiority Trial Puri Ajay MS docpurigmail.com Gulia Ashish MS Hawaldar Rohini PhD Ranganathan Priya MD Badwe Rajendra A. MS Orthopaedic Oncology Tata Memorial Hospital Room No. 45 E Bes Road Mumbai India Tata Memorial Hospital Mumbai India Anaesthesiology Critical Care and Pain Tata Memorial Hospital Mumbai India Surgical Oncology Tata Memorial Hospital Mumbai India 19 11 2013 5 2014 472 5 1568 1575 30 7 2013 8 11 2013 The Association of Bone and Joint Surgeons® 2013 Background Whether current postoperative surveillance regimes result in improved overall survival (OS) of patients with extremity sarcomas is unknown. Questions/purposes We hypothesized that a less intensive followup protocol would not be inferior to the conventional followup protocol in terms of OS. We (1) assessed OS of patients to determine if less intensive followup regimens led to worsened survival and asked (2) whether chest radiograph followup group was inferior to CT scan followup group in detecting pulmonary metastasis; and (3) whether less frequent (6-monthly) followup interval was inferior to more frequent (3-monthly) followup in detecting pulmonary metastasis and local recurrence. Methods A prospective randomized single-center noninferiority trial was conducted between January 2006 and June 2010. On the basis of 3-year survival of 60% with intensive more frequent followup 500 nonmetastatic patients were randomized to demonstrate noninferiority by a margin (delta) of 10% (hazard ratio [HR] 1.36). The primary end point was OS at 3 years. The secondary objective was to compare disease-free survival (DFS) (time to recurrence) at 3 years. At minimum followup of 30 months (median 42 months; range 30“81 months) 178 deaths were documented. Results Three-year OS and DFS for all patients was 67% and 52% respectively. Three-year OS was 67% and 66% in chest radiography and CT groups respectively (HR 0.9; upper 90% confidence interval [CI] 1.13). DFS rate was 54% and 49% in chest radiography and CT groups respectively (HR 0.82; upper 90% CI 0.97). Three-year OS was 64% and 69% in 6-monthly and 3-monthly groups respectively (HR 1.2; upper 90% CI 1.47). DFS was 51% and 52% in 6-monthly and 3-monthly groups respectively (HR 1.01; upper 90% CI 1.2). Almost 90% of local recurrences were identified by patients themselves. Conclusions Inexpensive imaging detects the vast majority of recurrent disease in patients with sarcoma without deleterious effects on eventual outcomes. Patient education regarding self-examination will detect most instances of local recurrence although this was not directly assessed in this study. Although less frequent visits adequately detected metastasis and local recurrence this trial could not conclusively demonstrate noninferiority in OS for a 6-monthly interval of followup visits against 3-monthly visits."
Lung_Cancer
"mediation with focus on breath - Attention for breath - Awareness of pleasant events - Attention for routine activity 3. Observing limits - Yoga while lying down - Seeing exercise to demonstrate difference between observation and interpretation - Bodyscan or yoga - 3-min breathing space - Sitting meditation - Awareness of unpleasant events - 3-min breathing space 4. Opening up to distress - Sitting mediation with focus on breath body and sound - Interrelatedness of feelings thoughts and bodily sensations - Bodyscan or yoga - Sitting meditation - 3-min breathing space - Psychoeducation about grief - Awareness of stress reactions - 3-min breathing space 5. Responding to distress - Sitting mediation with focus on breath body sound thoughts difficulty - Reacting versus responding - Meditation by choice - Coping with grief - Awareness of reaction in difficult situation - Walking meditation - Awareness of communication difficulties - 3-min breathing space - 3-min breathing space 6. Mindful communication - Yoga in standing position - Mindful communication exercise about effect of lung cancer with their own partner - Sitting meditation or yoga - 3-min breathing space - Awareness of communication - 3-min breathing space during stress Silent day - Varying meditation exercises - Silent lunch and tea break 7. Taking care of yourself - Sitting meditation ending in choiceless awareness - Exercise on taking care of yourself by examining how to improve balance in life - Meditation without CD - Yoga or walking meditation - Reflect on training - 3-min breathing space 8. The rest of your life - Bodyscan - Reflection on training - Further sources of information - Short sitting meditation - Maintaining practice Outcome measures Primary outcome measure Psychological distress The primary outcome measure is the total score on the HADS [39-41] which is developed to measure psychological distress in somatic patient populations. It consists of a 7-item anxiety (HADS-A) and 7-item depression (HADS-D) subscale. The HADS shows good psychometric properties in the general medical population including oncology patients [42]. Internal consistency as measured with Cronbach™s ? varied from .84 to .90 [4042].Test-retest reliability was good as Pearson™s r > .80 were obtained [4043]. Though the cut-off scores of the HADS vary among populations [44] in lung cancer patients they have found to be <8 versus ?8 on the HADS-A or HADS-D [45]. The HADS has been shown to be highly correlated with the Beck Depression Inventory [42]. It has previously been used in intervention studies of mindfulness and shown to be sensitive to change (e.g. [46]). Secondary outcome measures Quality of life (only for patients) The European anisation for Research and Treatment of Cancer (EORTC) Core Quality of Life Questionnaire (QLQ-C30) [47] is included along with the supplemental Lung Cancer questionnaire module (QLQ-LC13) [48]. The QLQ-C30 is designed to use in clinical trials on physical treatments for cancer patients. It incorporates five functional scales (physical role cognitive emotional social) three symptom scales (fatigue pain nausea and vomiting) a global health and quality of life scale and an array of single-item symptom measures. After revisions in the role functioning global health and physical functioning scale internal consistency of the subscales varied between .65 and .94 except for the cognitive functioning scale with ? varying from .56 to .63 [474950]. Test-retest reliability varied from .63 to .86 [51]. The lung cancer questionnaire module is designed to supplement the core questionnaire and comprises specific symptoms associated with lung cancer (coughing haemoptysis dyspnoea pain) and side-effects from conventional chemo- and radiotherapy (hair loss neuropathy sore mouth dysphagia). While the multi-item dyspnoea scale showed high internal consistency the pain subscale did not. When combined with the dyspnoea and pain items of the core questionnaire both the dyspnoea (? = .86) and pain (? = .71) subscale showed high internal consistency. Since the QLQ-C30 and QLQ-LC13 are mainly focused on physical symptoms we added the items Social Interaction and Alertness Behavior of the Sickness Impact Profile (SIP) [52]. Internal consistency was .94 and test-retest reliability was .92. The SIP correlated with self-assessed sickness and dysfunction [52]. Caregiver appraisal (only for partners) We use the 9-item Self-Perceived Pressure from Informal Care (SPPIC) [53] to assess the extent to which caregiving is experienced as burdensome. To also measure positive aspects of caregiving the 9-item subscale Care-Derived Self-Esteem of the Caregiver Reaction Assessment (CRA-SE) [54] is included. Internal consistency of the SPPIC was .79 and of the CRA-SE was .73. The SPPIC and CRA-SE were unrelated to each other [55]. Relationship quality To measure relationship satisfaction we included the 10-item Satisfaction subscale of the Investment Model Scale (IMS-S) [56]. The IMS-S starts with 5 items that measure concrete examplars of satisfaction to enhance the comprehensibility of the global items which are utilized to form the construct. Internal consistency varied from .79 to .95 and the IMS-S was related to the Dyadic Adjustment Scale. Also the Mutual Interpersonal Sensitivity scale (MIS) [57] is included to measure communication between partners about the cancer. It contains 18 items and is divided into two scales: open communication and avoiding negative thoughts about the cancer. Spirituality is measured with the Spiritual Attitude and Involvement List (SAIL) [58] and consists of 26 items divided into the subscales meaningfulness trust acceptance caring for others connectedness with nature transcendent experiences and spiritual activities. The internal consistency varied from .74 to .88 and test-retest reliability varied from .77 to .92. All subscales except for connectedness with nature were related with the Functional Assessment of Chronic Illness Therapy “ Spiritual Well-Being Scale. Costs (only for patients) The cost-effectiveness evaluation is carried out from a societal perspective considering direct as well as indirect health costs. Data on costs are collected prospectively using a diary in which participants register a) health care utilization: the type of care and its duration and b) cancer-related absence from work. Unit cost estimates are derived from the national manual for cost prices in the health care sector [59]. Costs of reduced ability to work are estimated using the friction costs method which results in a more realistic estimate than the human capital approach [60]. Treatment costs of MBSR are calculated using activity-based-costing methods thus measuring actual resources (time of therapist time of patients facilities) used. All unit cost prices are adjusted to 2013 prices. Unit cost estimates are combined with resource utilization data to obtain a net cost per patient over the entire follow-up period. Process measures Mindfulness skills are examined with the 39-item Five Facet Mindfulness Questionnaire (FFMQ) [6162]. The FFMQ is based on an exploratory factor analysis of five mindfulness measures which allowed items from different instruments to form factors providing an empirical integration of these independent attempts to operationalize mindfulness. This led to the following five subscales: observing describing acting with awareness non-judging of inner experience and non-reactivity to inner experience. Internal consistency varied from .72 to .93 among the different subscales. Most subscales were related to meditation experience Psychological Well-Being scales and psychological symptoms including the Brief Symptom Inventory [61]. FFMQ is sensitive to change in mindfulness-based interventions and is found to mediate the relationship between mindfulness practice and improvements in psychological symptoms (e.g. [63]). Self-compassion is assessed with the Self Compassion Scale (SCS) [6465] which has 26 items and is divided into six subscales: self-kindness versus self-judgment common humanity versus isolation and mindfulness versus over-identification. Internal consistency of the different subscales varied from .75 to .81 and test-retest reliability varied from .80 to .93. SCS correlated moderately with self-esteem measures including the Rosenberg Self-Esteem Scale. Furthermore whereas the self-esteem measures correlated significantly with the Narcissistic Personality Inventory the SCS was unrelated to narcissism [64]. SCS is sensitive to change through mindfulness-based interventions and is found to mediate MBCT™s treatment effects [66]. To measure rumination we administered the extended version of the Ruminative Response Scale (RRS-EXT) [67] Raes and Hermans: The revised version of the Dutch Ruminative Response Scale unpublished instrument]. The RRS-EXT contains 26 items in which a more adaptive thinking style (i.e. reflection) is distinguished from a more maladaptive one (i.e. brooding). Internal consistency varied from .72 to .77 and test-retest reliability varied from .60 to .62 for the brooding and reflection subscales. The concept of rumination seems to be sensitive to change through mindfulness-based interventions and has been shown to mediate the effect of MBSR on depressive symptoms in oncology patients [68]. The psychological stress reaction is measured with the 15-item Impact of Event Scale (IES) [6970] which assesses two categories of responses: intrusive experiences and avoidance of thoughts and images associated with the event. Internal consistency varied from .65 to .92 [71] and test-retest reliability varied from .79 to .87 among the subscales [69]. IES correlated with anxiety and depression subscales of the General Health Questionaire. Adherence to MBSR is assessed during the entire study period with a calendar on which participants in the MBSR condition fill out on a daily basis whether they adhere to the mindfulness exercises: either formal practice (e.g. meditation exercise like the bodyscan) informal practice (e.g. activity with awareness) or no exercise. Adherence to MBSR has been shown to mediate the effects of MBCT on depressive symptoms [72]. Statistical analysis plan Sample size To determine the required sample size first the sample size was calculated that would be needed for a simple t-test and subsequently it was corrected for clustering repeated measurements and baseline. A two-sided t-test on the total HADS score [3940] (i.e. our primary outcome measure examining psychological distress (HADS-total) anxiety symptoms (HADS-A) and depressive symptoms (HADS-D)) would require 64 participants in each group to have 80% power to detect a medium-sized difference (effect size = 0.5) with alpha = 0.05. To correct for clustering we multiplied this sample size of 64 with the design factor (1 + (n ? 1) * ICC) where n denotes the cluster size and where ICC denotes the intra-cluster correlation. In our study the treatment groups will consist of 14 people of whom about 7 will be patients. With n = 7 and an estimated ICC = 0.01. [72] the correction factor equals 1.06. To correct for repeated measurements and the use of the baseline measurement as a covariate we multiplied the required sample size by the design factor 1+?/2??02 where ? denotes the correlation between the post-treatment HADS measurements and ?0 denotes the correlation between the baseline HADS with the post-treatment HADS measurements. With ? = 0.8 and ? = 0.5 as conservative estimates the second design factor equals 0.65. Consequently after correction for clustering and covariates we arrived at a required sample size of 0.65 * 1.06 * 64 = 44 patients per arm. So 88 patients with lung cancer would be required for the study. Based on our pilot study [van den Hurk Schellekens Molema Speckens and van der Drift in preparation] we expect a 20% drop-out rate. Therefore we intend to include 110 patients and 110 partners. Primary analyses The samples of lung cancer patients and partners will be analyzed separately. Baseline characteristics of the population will be compared between MBSR and control group to ensure that key variables were evenly distributed by randomization. First analyses will be based on the intention-to-treat approach. Next we will perform per-protocol analyses with the treatment-adherent sample (i.e. in the MBSR condition participants have to attend at least four of the eight MBSR sessions [73] and in the TAU condition participants do not attend a mindfulness-based programme). We will use linear mixed models to analyze all outcome variables (i.e. psychological distress quality of life (only for patient) caregiver appraisal (only for partner) relationship quality and spirituality) with treatment as fixed factor baseline measurement as covariate and a random intercept based on MBSR group. This procedure will use all observed data in our analyses. In addition Cohen™s d effect size [74] will be reported based on the difference between the group means on baseline and follow-up scores divided by the pooled standard deviation at baseline and follow-up. Secondary analyses Cost effectiveness The quality of life measures (i.e. QLQ-C30; QLQ-LC13) will be used to calculate Quality of Adjusted Life Years (QALYs) for each individual. Costs and effects (in terms of QALYs) will be combined in the incremental cost-effectiveness ratio (ICER). The ICER expresses cost-effectiveness in terms of incremental costs per QALY gained. To estimate confidence intervals for the mean of the ICER a non-parametric bootstrapping method will be used performing 1000 replications of the original data. In order to express the implications of the cost-effectiveness results more clearly a cost-acceptability curve will be constructed. In case of dominance a full cost analysis will be conducted to estimate the mean savings per patient per year. Mediation analyses To examine the possible underlying mechanisms of change in MBSR mediation analyses will be conducted. Only the data of the treatment-adherent sample will be included in these analyses. By means of a multiple mediation model suggested by Preacher and Hayes [75] we will test the mediating effect of mindfulness skills self-compassion rumination and adherence to MBSR on psychological distress quality of life (only in patients) caregiver appraisal (only in partners) relationship quality and spirituality. Discussion In the last ten years MBSR has not only proven to be a feasible and acceptable intervention in cancer patients [76] but it also seems to be effective in reducing psychological distress [30]. However the generalization of these results is limited because most participants were female patients with breast cancer. A large part of lung cancer patients already have advanced cancer at time of diagnosis and are confronted with a poor prognosis and low health status. Consequently they more often report psychological distress than patients with other diagnoses of cancer [89]. Hence it is not yet clear whether MBSR is a feasible acceptable and effective intervention in patients with lung cancer. Moreover little is known about the effectiveness of MBSR in partners of cancer patients [30] though they also often report psychological distress. Our pilot study of 19 lung cancer patients and 16 partners participating in an MBSR course provides preliminary evidence that MBSR is feasible and acceptable in this population (van den Hurk Schellekens Molema Speckens and van der Drift in preparation). The current trial will answer the question whether MBSR is effective in patients with lung cancer and their partners. We started enrolment of participants in February 2012. At the moment we think recruiting a sufficient number of patients and partners will be a challenge due to rapidly fluctuating health status and sudden changes in cancer treatment [77]. The main reasons for declining participation in patients is ˜being too ill™ or that it is ˜too much of a burden during chemo and/or radiotherapy™. Furthermore no perceived need or motivation for the training is commonly mentioned. Among partners participation is highly depending on whether the patient is willing to participate. Although partners can take part separately partners who are interested do often not participate when the patients decline participation. Considering the difficulty of studying lung cancer patients and their partners [77] our trial will offer valuable information on whether MBSR as one of the few available psychosocial care programmes contributes to the alleviation of their psychological distress. Abbreviations MBSR: Mindfulness-based stress reduction; RCT: Randomized controlled trial; RUNMC: Radboud University Nijmegen Medical Centre; MBCT: Mindfulness-based cognitive therapy; MMSE: Mini mental state examination; DT: Distress thermometer; HADS: Hospital anxiety and depression scale; QLQ-C30: Quality of life “ cancer; QLQ-LC13: Quality of life “ lung cancer; SIP: Sickness impact profile; SPPIC: Self-perceived pressure from informal care; CRA-SE: Caregiver reaction assessment “ care-derived self-esteem; IMS-S: Investment model scale-satisfaction; MIS: Mutuality and interpersonal sensitivity; SAIL: Spiritual attitude and involvement list; FFMQ: Five facet mindfulness questionnaire; SCS: Self-compassion scale; RRS-EXT: Rumination response scale “ extended version; IES: Impact of event scale. Competing interests The authors declare that they have no competing interests. Authors™ contributions All authors contributed to the design of the study. AS MD and JP are the principal investigators of the study. MS drafted the paper which was modified and supplemented by all other authors. DH MS and MD are involved in recruiting participants while MS and DH take care of the logistics of the study and data collection. RD contributed specifically to the statistical analysis plan and WW contributed specifically to the design of the cost-effectiveness evaluation. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2407/14/3/prepub Acknowledgements This research is funded by Foundation Alpe d™HuZes and the Dutch Cancer Society (Grant number KUN 2011“5077 awarded to Prof. dr. Anne E. M. Speckens Dr. Miep A. van der Drift and Prof. dr. Judith B. Prins). Jemal A Bray F Center MM Ferlay J Ward E Forman D Global cancer statistics CA Cancer J Clin 2011 61 2 69 90 10.3322/caac.20107 21296855 Akechi T Okamura H Nishiwaki Y Uchitomi Y Psychiatric disorders and associated and predictive factors in patients with unresectable nonsmall cell lung carcinoma: a longitudinal study Cancer 2001 92 10 2609 2622 10.1002/1097-0142(20011115)92:10<2609::AID-CNCR1614>3.0.CO;2-K 11745196 Uchitomi Y Mikami I Kugaya A Akizuki N Nagai K Nishiwaki Y Akechi T Okamura H Depression after successful treatment for nonsmall cell lung carcinoma: a 3-month follow-up study Cancer 2000 89 5 1172 1179 10.1002/1097-0142(20000901)89:5<1172::AID-CNCR27>3.0.CO;2-U 10964348 Montazeri A Milroy R Hole D McEwen J Gillis CR Anxiety and depression in patients with lung cancer before and after diagnosis: findings from a population in Glasgow Scotland J Epidemiol Community Health 1998 52 3 203 204 10.1136/jech.52.3.203 9616429 Hyodo I Eguchi K Takigawa N Segawa Y Hosokawa Y Kamejima K Inoue R Psychological impact of informed consent in hospitalized cancer patients: a sequential study of anxiety and depression using the Hospital Anxiety and Depression scale Support Care Cancer 1999 7 6 396 399 10.1007/s005200050299 10541981 Turner NJ Muers MF Haward RA Mulley GP Psychological distress and concerns of elderly patients treated with palliative radiotherapy for lung cancer Psychooncology 2007 16 8 707 713 10.1002/pon.1109 17115458 Hopwood P Stephens RJ Depression in patients with lung cancer: prevalence and risk factors derived from quality-of-life data J Clin Oncol 2000 18 4 893 903 10673533 Zabora J Brintzenhofeszoc K Curbow B Hooker C Piantadosi S The prevalence of psychological distress by cancer site Psychooncology 2001 10 1 19 28 10.1002/1099-1611(200101/02)10:1<19::AID-PON501>3.0.CO;2-6 11180574 Carlson LE Angen M Cullum J Goodey E Koopmans J Lamont L MacRae JH Martin M Pelletier G Robinson J High levels of untreated distress and fatigue in cancer patients Br J Cancer 2004 90 12 2297 2304 15162149 Temel JS Greer JA Muzikansky A Gallagher ER Admane S Jackson VA Dahlin CM Blinderman CD Jacobsen J Pirl WF Early Palliative Care for Patients with Metastatic Non-Small-Cell Lung Cancer N Engl J Med 2010 363 8 733 742 10.1056/NEJMoa1000678 20818875 Abernethy AD Chang HT Seidlitz L Evinger JS Duberstein PR Religious coping and depression among spouses of people with lung cancer Psychosomatics 2002 43 6 456 463 10.1176/appi.psy.43.6.456 12444228 Thielemann PA Conner NE Social support as a mediator of depression in caregivers of patients with end-stage disease J Hosp Palliat Nurs 2009 11 2 82 90 10.1097/NJH.0b013e31819974f9 Pinquart M Duberstein PR Optimism pessimism and depressive symptoms in spouses of lung cancer patients Psychol Health 2005 20 5 565 578 10.1080/08870440412331337101 Kim Y Duberstein PR Sorensen S Larson MR Levels of depressive symptoms in spouses of people with lung cancer: effects of personality social support and caregiving burden Psychosomatics 2005 46 2 123 130 10.1176/appi.psy.46.2.123 15774950 Mosher CE Jaynes HA Hanna N Ostroff JS Distressed family caregivers of lung cancer patients: an examination of psychosocial and practical challenges Support Care Cancer 2013 21 2 431 437 10.1007/s00520-012-1532-6 22797839 Mosher CE Bakas T Champion VL Physical health mental health and life changes among family caregivers of patients with lung cancer Oncol Nurs Forum 2013 40 1 53 61 10.1188/13.ONF.53-61 23269770 Ostlund U Wennman-Larsen A Persson C Gustavsson P Wengstrom Y Mental health in significant others of patients dying from lung cancer Psychooncology 2010 19 1 29 37 10.1002/pon.1433 19253315 Wennman-Larsen A Persson C Ostlund U Wengstrom Y Gustavsson JP Development in quality of relationship between the significant other and the lung cancer patient as perceived by the significant other Eur J Oncol Nurs 2008 12 5 430 435 10.1016/j.ejon.2008.07.004 18845476 Siminoff LA Wilson-Genderson M Baker S Jr Depressive symptoms in lung cancer patients and their family caregivers and the influence of family environment Psychooncology 2010 19 12 1285 1293 10.1002/pon.1696 20119935 Manne S Badr H Intimacy processes and psychological distress among couples coping with head and neck or lung cancers Psychooncology 2010 19 9 941 954 10.1002/pon.1645 19885852 Badr H Taylor CLC Effects of relationship maintenance on psychological distress and dyadic adjustment among couples coping with lung cancer Health Psychol 2008 27 5 616 627 18823188 Buccheri G Depressive reactions to lung cancer are common and often followed by a poor outcome Eur Respir J 1998 11 1 173 178 10.1183/09031936.98.11010173 9543289 Walker J Sawhney A Hansen CH Symeonides S Martin P Murray G Sharpe M Treatment of depression in people with lung cancer: a systematic review Lung Cancer 2013 79 1 46 53 10.1016/j.lungcan.2012.09.014 23102652 Follwell M Burman D Le LW Wakimoto K Seccareccia D Bryson J Rodin G Zimmermann C Phase II study of an outpatient palliative care intervention in patients with metastatic cancer J Clin Oncol 2009 27 2 206 213 10.1200/JCO.2008.17.7568 19064979 Jordhoy MS Fayers P Loge JH Ahlner-Elmqvist M Kaasa S Quality of life in palliative cancer care: results from a cluster randomized trial J Clin Oncol 2001 19 18 3884 3894 11559726 Gustafson DH DuBenske LL Namkoong K Hawkins R Chih M-Y Atwood AK Johnson R Bhattacharya A Carmack CL Traynor AM An eHealth system supporting palliative care for patients with non“small cell lung cancer Cancer 2013 119 9 1744 1751 10.1002/cncr.27939 23355273 Greer JA Pirl WF Jackson VA Muzikansky A Lennes IT Heist RS Gallagher ER Temel JS Effect of early palliative care on chemotherapy use and end-of-life care in patients with metastatic non-small-cell lung cancer J Clin Oncol 2012 30 4 394 400 10.1200/JCO.2011.35.7996 22203758 Kabat-zinn J Full catastrophe living: using the wisdom of your body and mind to face stress pain and illness 1990 New York: Delacourt Segal ZV Williams JMG Teasdale JD Mindfulness-Based Cognitive Therapy for depression: a new approach to preventing relapse 2002 New York: Guilford Press Piet J Wurtzen H Zachariae R The effect of Mindfulness-Based Therapy on symptomps of anxiety and depression in adult cancer patients and survivors: a systematic review and meta-analysis J Consult Clin Psychol 2012 80 6 1007 1020 22563637 Birnie K Garland SN Carlson LE Psychological benefits for cancer patients and their partners participating in Mindfulness-Based Stress Reduction (MBSR) Psychooncology 2010 19 9 1004 1009 10.1002/pon.1651 19918956 Hagedoorn M Sanderman R Bolks HN Tuinstra J Coyne JC Distress in couples coping with cancer: a meta-analysis and critical review of role and gender effects Psychol Bull 2008 134 1 1 30 18193993 Folstein MF Folstein SE McHugh PR Mini-mental state: practical method for grading cognitive state of patiens for clinician J Psychiatr Res 1975 12 3 189 198 10.1016/0022-3956(75)90026-6 1202204 Roth AJ Kornblith AB Batel-Copel L Peabody E Scher HI Holland JC Rapid screening for psychologic distress in men with prostate carcinoma Cancer 1998 82 10 1904 1908 10.1002/(SICI)1097-0142(19980515)82:10<1904::AID-CNCR13>3.0.CO;2-X 9587123 Tuinman MA Gazendam-Donofrio SM Hoekstra-Weebers JE Screening and referral for psychosocial distress in oncologic practice Cancer 2008 113 4 870 878 10.1002/cncr.23622 18618581 Kübler-Ross E On death and dying 1969 New York: Macmillan MBSR teacher certification pathway: complete checklist"
Lung_Cancer
"was connected to the scFv at its C-terminal using an overlap PCR approach. The PCR product scFv-linker was subcloned into pQE30-MTBhsp70 at the N-terminal of MTBhsp70. The DNA fragment for scFvMTBhsp70 was PCR amplified and cloned into pPMY5 (Promab) downstream of a human IgG1 Fc domain and separated from the Fc region by the signal cleavage sequence for Tobacco Etch Virus protease (TEV enzyme). scFvMTBHsp70 the MSLN-targeted fusion protein was generated from HEK293 cells and purified using Protein G resin (Pierce). The Fc region of the Protein G eluted protein was then cleaved from the fusion protein by TEV enzyme (Promab) digestion. MTBHsp70 was generated using the same expression system. The production and purification of these two proteins was accomplished by Promab Biotechnologies Inc. at Richmond CA. After purification and hIgG-Fc tag removal the integrity of scFv-MTBHsp70 and MTBHsp70 were determined by SDS-PAGE followed by staining with RAPIDstain (G-Bioscience). Endotoxin contamination levels in scFvMTBHsp70 and MTBHsp70 were determined by Limulus Amebocyte Lysate Assay (LAL-assay Cambrex). Cells The BR5FVB1 ovarian cancer cells a kind gift from Dr. Orsulic in Women™s Cancer Research Institute at Cedars-Sinai Medical Center [41] or 40L mesothelioma cells a kind gift from Dr. Kane in Department of Pathology and Laboratory Medicine at Brown University [42] were maintained at 37°C in DMEM with 2 mmol/L L-glutamine 10 units/ml penicillin 10 ?g/ml streptomycin and 10% fetal bovine serum in humidified atmosphere with 5% CO2. Cells were cultured until 80% confluent and harvested with enzyme-free cell-dissociation buffer (Gibco) for in vitro tumor cell binding assays and cross-presentation studies or harvested with Trypsin EDTA (Mediatech) for animal injections. Mouse PBLs were obtained from FVB mice via tail vein bleeds after lysis of erythrocytes using M-lyse buffer (R&D systems). Small pieces of parietal peritoneal membrane were taken from the mice and digested in enzyme-free cell-dissociation buffer to obtain mouse peritoneal mesothelial cells. To test whether scFvMTBHsp70 or MTBHsp70 binds to the MSLN-expressing tumor cells or non-cancerous cells we incubated BR5FVB1 ovarian tumor cells 40L mesothelioma cells or normal cells from FVB mice including PBLs splenocytes and peritoneal mesothelial cells with 40 ?g/ml scFvMTBHsp70 or 26 ?g/ml MTBHsp70 followed by anti-MTBHsp70 (IgG2a) (Biodesign International) biotinylated anti-IgG2a (BD Bioscience) and Streptavidin-APC (BioLegend) and then analyzed the tumor cells by flow cytometry. As controls cells were incubated with the reagents described above except scFvMTBHsp70 or MTBHsp70. To confirm that scFv portion of the fusion protein binds to MSLN on the surface of tumor cells scFvMTBHsp70 or MTBHsp70 was preincubated with 12 ?g/ml of recombinant human MSLN (R&D Systems) for 30 min before adding to the cells. For fluorescence microscopy cells were cultured on coverslips until 50% confluent stained with 10 ?g/ml scFvMTBHsp70 or 6.5 ?g/ml MTBHsp70 followed by mouse anti-MTBHsp70 (1:500 dilution) and Donkey anti-mouse Alexa Fluor 594 (Invitrogen 1:500 dilution). Cells were observed using a Nikon Eclipse TiE fluorescence microscope. In some experiments tumor cells were treated with 20 ?g/ml mitomycin C at a concentration of 5 — 106/ml for 1 h in a 37°C water bath and washed with complete medium at least 3 times before use. Animal models and tumor treatment Ovarian cancer was established by i.p. injection of syngeneic cancer cells BR5FVB1 (107 cells per mouse) into 6-week-old female FVB/NJ mice as previously described [25]. All mice were purchased from Jackson laboratories. Intraperitoneal mesotheliomas were established by i.p. injection of syngeneic 40L cells (2?—?106 per mouse) into 6-week-old male C57BL/6 mice as previously described [42]. Mice with ovarian tumors were treated 7 days after BR5FVB1 tumor cell inoculation with i.p. injections of scFvMTBHsp70 (2 ?g per mouse) normal saline or an equimolar mixture of MTBHsp70 plus P4 scFv. This was followed by 3 further treatments at 4-day intervals. In the mesothelioma model C57BL/6 mice were treated 5 days after 40L tumor cell inoculation and injected i.p. with scFvMTBHsp70 (2 ?g per mouse) normal saline or an equimolar mixture of MTBHsp70 plus P4 scFv. Three subsequent doses were administered at 3-day intervals thereafter. For survival studies we observed the mice daily 3 weeks after inoculation of BR5FVB1 cells or 1 week after inoculation of 40L cells. Tumor generations were consistently first evident via abdominal distension secondary to malignant ascites and tumor-bearing mice were euthanized at the endpoint when there were signs of distress including fur ruffling rapid respiratory rate hunched posture reduced activity and progressive ascites formation as previously described [25]. For the investigation of anti-tumor T-cell responses all ovarian tumor-bearing mice were sacrificed 7 days after the final scheduled treatment. All studies were performed in a manner that was blinded to the observer under protocols that were approved by the Massachusetts General Hospital Subcommittee on Research Animal Care (SRAC). Treatment of na¯ve mice with experimental or control protein 6-week-old male C57BL/6 mice were injected i.p. with scFvMTBHsp70 (2 ?g per mouse) normal saline or an equimolar mixture of MTBHsp70 plus P4 scFv. Three subsequent doses were administered at 3-day intervals thereafter. Seven days post the administration of the final treatment mice were sacrificed and abdominal wall and intestine were retrieved for histopathological studies of mesothelial tissues. Ex vivo assessment of tumor specific T-cell functions Single cell suspensions were prepared from spleens. Cells were plated in round-bottomed 96-well plates pulsed with a validated CD8+ T-cell Her2/neu peptide (PDSLRDLSVF 1 ?g/ml; EZBiolab) [2543] an in-house designed H2d-restricted MSLN Ld1 peptide (IPLSYLCDF 1 ?g/ml; EZBiolab) that did not induce ovarian cancer specific T-cell response in H-2q FVB mice or medium alone for 72 hours when Golgi Plug (BD Bioscience) was added for the last 5 hours as previously described [44] and then stained with fluorophore-conjugated anti-CD3 anti-CD4 anti-CD8 anti-IFN? (BD Pharmingen) and anti-Granzyme B (eBioscience) antibodies. Cells were then analyzed on a LSRII 4 laser (BD Biosciences). Depletion of CD8+ T cells in vivo FVB/NJ mice were injected i.p. with 200 ?g of anti-CD8 monoclonal antibody (mAb)(53“6.72 Bio X Cell) or an isotype-matched irrelevant rat IgG2a (2A3 Bio X Cell) 2 days before 1 day before and 1 day after i.p. inoculation with BR5FVB1 ovarian tumor cells. Depletion was continued once every week until 29 days after tumor inoculation. The mice were treated with scFvMTBHsp70 or saline as described above. All the mice were bled from the tail vein and the depletion of CD8+ cells was examined by flow cytometry analysis of peripheral blood cells stained with fluorophore-conjugated anti-CD8 on days 7 and 28 after tumor inoculation. Generation and purification of bone marrow-derived DCs (BMDCs) CD11c+ DCs were generated from bone marrow cells of FVB/NJ mice as described [45-47] with minor modifications. Briefly erythrocyte-depleted mouse bone marrow cells from flushed marrow cavities were cultured in complete RPMI 1640 with 10 ng/ml GM-CSF and 1 ng/ml IL-4 at 1 — 106 cells/ml. Medium was changed on day 3. On day 7 DCs were harvested by gentle pipetting and purified with magnetic microbeads conjugated to a monoclonal antibody against CD11c (MiltenyiBiotec) as described [4648] according to the manufacturer™s recommended protocol. In vitro activation of BMDCs CD11c+ BMDCs were plated in a 24-well plate at a density of 2?—?106 cells/ml and incubated with 2 ?g/ml scFvMTBHsp70 (105 kDa) 1.3 ?g/ml MTBHsp70 (70 kDa) 1 ?g/ml LPS equivalent to 103 EU/ml endotoxin (InvivoGen San Diego CA) or 0.1 ng/ml (0.1 EU/ml) LPS equivalent to endotoxin found in 2 ?g/ml of proteins (since LPS level is less than 50 EU per mg of protein) for 24 h at 37°C in humidified atmosphere with 5% CO2. Cells were then placed on ice collected by vigorous pipetting washed and stained with the following fluorophore-conjugated antibodies: anti-CD11c and anti-CD40 (eBioscience) anti-CD80 (BD Horizon) anti-CD86 and anti-MHC class II (I-Aq) (BD Pharmingen). Afterwards the cells were analyzed on an LSRII 4 laser (BD Biosciences). In vitro tumor antigen presentation assay BR5FVB1 cells were harvested and treated with mitomycin C and plated in a 96-well round-bottomed plate with 20 ?g/ml scFvMTBHsp70 or 13 ?g/ml MTBHsp70. After pre-incubation at 4°C for 1 h CD11c+ BMDCs (ratio of tumor cells: DCs = 3: 1) were added to the wells and the plate was incubated at 37°C for 24 h. For generation of BR5FVB1 cell-primed T cells we inoculated FVB/NJ mice by i.p. injection with 107mitomycin C-treated BR5FVB1 cells and sacrificed the mice 60 days after the immunization according to the approved animal protocol. Splenocytes were then harvested and T cells were isolated using the Pan T-Cell Isolation Kit II (MiltenyiBiotec). BR5FVB1 cell-primed T cells were then added to the wells at a DC/T-cell ratio of 1:20. After a 24-hour co-culture of BR5FVB1 cell-pulsed DCs with BR5FVB1 cell-primed T cells the cells were harvested washed and resuspended in PBS with 5% FBS stained for CD3 CD4 CD8 and IFN? and analyzed on a LSRII 4 laser (BD Biosciences). In vivo immunization with mitomycin C-treated ovarian tumor cells BR5FVB1 ovarian tumor cells were harvested with enzyme-free cell-dissociation buffer and treated with mitomycin C as described above. Cells were then pre-incubated with scFvMTBHsp70 (10 ?g/106 cells) MTBHsp70 (6.5 ?g/106 cells) or PBS alone at 4°C for 1 h. 6-week-old FVB mice were shaved and depilated on both left and right flanks and then injected i.d. with 50 ?l of PBS or 1?—?106 tumor cells in 50 ?l of PBS with or without a pre-incubation with scFvMTBHsp70 or MTBHsp70 at both flanks. Histopathology Abdominal walls and intestines from mice were fixed for at least 24 h in PBS-buffered 10% formalin. Tissues were routinely embedded in paraffin. 5 ?m thick sections were stained routinely with H&E. For staining tumor-infiltrating T cells mice were perfused with 4% paraformaldehyde (PFA) in PBS and tumor nodules were fixed in 4% PFA/PBS for additional 2 hours washed and infiltrated with 30% sucrose/PBS at 4°C. 6 ?m thick frozen sections were stained with rat anti-mouse CD8 (BD Biosciences 1:100 dilution) or rat anti-mouse Foxp3 (eBioscience 1:12 dilution) followed by polyclonal rabbit anti-rat immunoglobulin/HRP (Dako 1:750 dilution). Signal was developed with diaminobenzidine (DAB Dako). Images were acquired on a Zeiss Axio A1 microscope. All histopathological and immunohistochemical samples were reviewed and the quantitation of the cellular infiltrate was performed in a blinded manner to the observer. Statistical analysis Statistical differences between three or more experimental groups were analyzed using One-Way ANOVA followed by Turkey™s multiple comparison tests when mean of each group is compared with that of every other group or followed by Dunnett™s multiple comparison tests when mean of each group is compared with that of a control group. Statistical differences between two experimental groups were analyzed using Student™s t-test. Survival was analyzed with the Log-rank test. Prism 6.0 software (GraphPad Software) was used for all the statistical analysis. Abbreviations DC: Dendritic cell; scFv: Single-chain antibody variable fragment; MSLN: "
Lung_Cancer
"inhibitors by causing immediate and nearly complete intracellular and extracellular thiamine deprivation [6]. In previous studies we have shown that thiaminase has both in vitro and in vivo cytotoxicity activity further supporting the concept that TDEs could represent new targets for novel therapies [6] [7] [8]. We have also previously reported that rapamycin has antagonistic effect on thiaminase-mediated growth inhibition of leukemia cells [7] a surprising finding since rapamycin generally acts as a sensitizing agent in combination with cytotoxic drugs. We now present metabolic and metabolomic observations regarding the anticancer activities and metabolic effects of thiaminase in leukemia and breast cancer cells. We chose to focus on breast and leukemia models because these were the models in which we observed the most promising activity of thiaminase in xenografts. These studies help define thiaminase metabolic effects that may be responsible for its cytotoxic activity. These studies also further elucidate the role of mTOR as an inhibitor of thiaminase-mediated alterations in cellular metabolism and demonstrate the role of mTOR in regulating expression of enzymes involved in thiamine-dependent metabolism. Methods Ethics statement All animal studies were approved by the University of Kentucky Institutional Animal Care and Use Committee. Cell Lines The human breast cancer cell line MCF-7 and the non-malignant breast cell line MCF-10A were obtained from ATCC; human leukemia cell lines Reh and RS4 were originally obtained from ATCC and generously provided by Dr. Terzah Horton Baylor College of Medicine. Cell line authentication was performed by PCR amplification of nine short tandem repeat (STR) loci (Research Animal Diagnostic Laboratory St. Louis MO) and comparing the profile to the ATCC STR database. The STR profile of MCF-7 and RS4 cell lines were identical to the ATCC profile. The Reh cell line matched all alleles in the ATCC Reh profile plus one extra allele at two loci. Cytotoxicity assays Human leukemia cell lines Reh or RS4 were plated in triplicate in 96-well microtiter plates in RPMI-1640 (with 25 mM HEPES) medium containing 10% fetal bovine serum at final densities of 8—104 cells/well. MCF-7 cells were plated in the same medium at the final density of 1000cells/well. Medium containing native thiaminase at a concentration range of 1—10?6 to 4 units/ml was added to cells and incubated for four days. Following incubation an MTT Cell Proliferation Assay (ATCC) was performed according to the ATCC protocol. The IC50 was calculated from the dose response curve as the concentration of drug producing a 50% decrease in the mean absorbance compared to the untreated wells using Prism GraphPad software. The cytotoxicity experiments were repeated a minimum of three times in triplicate. Analysis of primary human ALL specimens was performed by plating cells at a density of 1—106/ml and treating for 24 hours with the indicated concentration of thiaminase. Viability was evaluated by dead cell exclusion labeling with trypan blue dye as well as flow cytometric analyses using Annexin-V labeling as previously described [9]. Xenograft studies RS4 tumor xenografts were established by subcutaneously inoculating 1—107 cells into the right flank of five-week-old female Crl:NU-Foxn1 nude mice (Charles River Laboratories Wilmington MA). For the establishment of MCF-7 xenograft a 17?-estradiol pellet (0.72 mg 60 days release; Innovative Research of America Sarasota FL) was implanted subcutaneously into the neck to facilitate optimal tumor growth for the estrogen receptor“positive MCF-7 cells. The xenografts were initiated by subcutaneously injecting 5—106 MCF-7 cells into the right flank of five-week-old female Crl:NU-Foxn1 nude mice. When palpable tumors had formed mice were treated with native thiaminase at its MTD (2000 units/kg body weight) twice a week at a site distant from the formed tumor for three weeks. Mice were treated with a single dose of thiaminase enzyme conjugated with 1k linear chain PEG (1K-LCPTE) at its MTD (50 U/kg) at a site distant from the formed tumor. Mice treated with N3-pyridyl thiamine (N3PT) received an intraperitoneal (i.p.) dosage of 80 mg/kg daily for five days. Mice treated with both 1K-LCPTE and N3PT received a single dose of 1K-LCPTE at 50 U/kg first then after 5five days mice received N3PT at 80 mg/kg daily for five days. Tumors were measured twice a week in a blinded manner by measuring perpendicular diameters with a digital caliper and tumor volumes (mm3) were calculated using the following formula: volume ?=? width — width — length — ?/6. The predetermined endpoint was a tumor volume of 1500 mm3. The control mice were injected with MCF-7 or RS4 cells and left untreated and results combined with a previous control group of the same xenograft [7]. Kaplan-Meier survival curves and statistical analysis was performed with GraphPad Prism software. Primary lymphoblastic leukemia xenograft methods A primary lymphoblastic leukemia specimen was transplanted by IV injection into sub-lethally irradiated immune deficient NOD/SCID/IL2Rg mice. When the tumor burden was established in the bone marrow (day 17) animals received treatment with thiaminase 2000 units/kg on days 17 20 and 24 administered by subcutaneous injection (the longer interval between treatments in these mice in comparison to the Crl:NU-Foxn1 nude mice was due to tolerability). The animals were sacrificed on day 33 and marrow cells were isolated and examined by flow cytometry using human-specific antibodies for CD45 and CD19 to determine the level of human leukemia cell engraftment as previously described [9]. Mitochondrial bioenergetics measurements Oxygen consumption was determined using the Seahorse Extracellular Flux (XF-96) analyzer (Seahorse Bioscience Chicopee MA). The XF-96 measures the concentration of oxygen and free protons in the medium above a monolayer of cells in real-time. Thus the rates of oxygen consumption and proton production can be measured across several samples at a time. To allow comparison between experiments data are presented as oxygen consumption rate (OCR) in pMoles/min/104 cells and the extracellular acidification rate (ECAR) in mpH/min/104 cells. RS4 and Reh leukemia cells were seeded at 125000 cells/well into gelatin-coated Seahorse Bioscience XF microplates cultured in the presence or absence of 2 g/L D-glucose and then centrifuged to adhere to the bottom of the wells while for the MCF-7 and MCF-10A about 45000 cells were plated and allowed to adhere overnight. OCR was measured four times and plotted as a function of cells under the basal condition followed by the sequential addition of oligomycin (1 µg/ml) FCCP (1 µM) and rotonone (1 µM). The ATP-linked OCR was calculated as the basal OCR minus the OCR measured after the addition of oligomycin. The OCR maximal capacity was the direct rate measured after the addition of FCCP. The reserve capacity is the FCCP OCR minus the basal OCR. For the ECAR measurements cells were washed following overnight incubation and changed to assay media lacking glucose. Basal ECAR were measured four times and plotted as a function of cells under the basal condition followed by the sequential addition of glucose (25 mM) oligomycin (1 µg/ml) and 2-deoxyglucose (25 mM). The rate of glycolysis was determined by subtracting the basal ECAR from the ECAR after the addition of glucose. Glycolytic reserve was determined by subtracting the ECAR following the addition of oligomycin from the ECAR following the addition of glucose. Differences between treatment groups were calculated using the Kruskal Wallis test. Metabolomic studies RS4 leukemia cells and MCF-7 breast cancer cells were analyzed under six conditions: control for 24 hrs (C-24); incubation in thiaminase for 24 hrs (T-24); control for 48 hours (C-48); thiaminase for 48 hrs (T-48); rapamycin for 48 hrs (R-48); and both rapamycin and thiaminase for 48 hrs (R+T-48). Four independent samples were produced for each time point for each cell line and cell pellets were stored at ?80°C until the separate experiments were all completed. Metabolomic studies were performed at Metabolon Inc. (Durham NC). The non-targeted metabolic profiling platform consisted of three independent instrumental methods: ultrahigh performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS2) optimized for basic species; UHPLC/MS/MS2 optimized for acidic species; and gas chromatography/mass spectrometry (GC/MS). The detailed process of the platform; including sample processing instrument configuration data acquisition as well as metabolite identification and quantitation were published previously [10] [11]. Three hundred and forty two metabolites were identified by automated comparison of the ion features in the experimental samples to a reference library of chemical standard entries that included retention time molecular weight (m/z) preferred adducts in-source fragments and associated MS spectra [12]. Instrument variability was determined by calculating the median relative standard deviation (RSD) for the internal standards that were added to each sample prior to injection into the mass spectrometers. Overall process variability was determined by calculating the median RSD for all endogenous metabolites (i.e. non-instrument standards) present in 100% of a set of technical replicates of pooled samples. Values for instrument and process variability meet Metabolon™s acceptance criteria with instrument variability of 3% and overall process variability of 12%. Following normalization to total protein (Bradford assay) log transformation and imputation with minimum observed values for each compound Welch™s two-sample t-tests were used to identify biochemicals that differed significantly between experimental groups. The entire metabolomic data sets for RS4 and MCF-7 cells with statistical results are included in data tables (Table S1). Immunoblot analysis Cells were treated with thiaminase (0.001 U/ml) and/or rapamycin (0.1 µM) for 96 hours. Cells were lysed with a triple-detergent lysis buffer (50 mM Tris pH8.0 150 mM NaCl 1% NP-40 0.5% DOC 0.1% SDS 0.02% sodium azide 100 µg/ml PMSF protease inhibitors (Roche) and phosphatase inhibitors (Thermo Scientific)). Equal amounts of protein were loaded into each well and separated by SDS-PAGE gel followed by transfer onto nitrocellulose membranes. The membranes were blocked incubated with the indicated primary antibodies at 4°C overnight and the appropriate horseradish peroxidase“conjugated secondary antibody was added for 1 hour at room temperature. Immunoblots were developed by use of SuperSignal West Pico chemiluminescent substrate (Thermo Scientific) according to the manufacturer™s protocol and analyzed by FujiFilm LAS-4000 luminescent image analyzer (Multigauge software). The primary and secondary antibodies used in this study are listed as follows. Anti- PKM1; anti-BCAT2; anti-TPK1 and anti-THTPA antibodies were purchased from Proteintech Group Inc. (Chicago IL). Anti- PKM2 and anti-CPT1A antibodies were from Cell Signaling Technology (Danvers MA). Anti-BCKDE1 and anti-phospho-BCKDE1 antibodies were obtained from Bethyl laboratories (Montgomery TX). Anti-BCAT1 anti-?-actin and all secondary antibodies were obtained from Sigma-Aldrich (St. Louis MO). Results Evidence of antitumor activity of thiaminase in leukemia and breast cancer tumor models is shown in Figure 1. Figure 1A is a Kaplan-Meier plot of MCF-7 subcutaneous xenografts treated with thiaminase showing a prolongation in the time to endpoint (pre-defined tumor volume) (TTE) from 41 days in the mock treated cohort to 59 days in the treated cohort (p?=?0.03). In Figure 1B RS4 subcutaneous xenografts show an increase in median TTE from 16.5 days from the start of treatment to undefined TTE after 60 days (p<0.001). We have also previously shown evidence of thiaminase activity against MDA231 breast cancer [8]. In Figure 1C the activity of thiaminase is shown against primary human leukemia cells. The most sensitive primary leukemia specimens appear to be lymphoblastic specimens with MLL-gene re-arrangements. This was of interest as the RS4 cell line is also an MLL-rearranged cell line. Figure 1D shows flow cytometric analysis of bone marrow of a primary MLL-rearranged leukemia cell xenograft treated with thiaminase demonstrating a decrease in leukemia cell proportion after treatment. These studies along with previous studies [7] [8] provided the rationale for performing further detailed examination of the metabolic effects of thiaminase in the breast cancer cell line MCF-7 and in the RS4 leukemia cell line. In addition for further points of comparison we included selected studies in two additional cell lines Reh leukemia cells another lymphoblastic leukemia cell line and MCF-10A a non-malignant breast cell line. 10.1371/journal.pone.0085702.g001 Figure 1 In vivo evidence of thiaminase anticancer activity. A. A Kaplan-Meyer plot of time to pre-defined tumor volume endpoint for subcutaneous MCF-7 breast cancer xenografts treated with thiaminase 2000 units SC QOD or buffer control. The median time to endpoint was 41 "
Lung_Cancer
"The Ventana assay using the D5F3 antibody gave the most intense cytoplasmic signal but this was accompanied by higher background staining which was especially noticeable in macrophages. The ALK1 and 5A4 antibody assays produced weaker staining but with less background. Of the two 5A4 had marginally more background staining especially in macrophages. The value of both intensity and proportion scores was assessed. Cutoffs for positivity were set using ROC analysis to optimize correct classification of ALK status using FISH results as the standard (). The intensity score alone was seen to outperform both proportion and the aggregate score. Therefore intensity scoring using the optimized cutoff was used in subsequent analyses. TABLE 3. Optimised Cutoff Values for Immunohistochemical Tests Concordance between FISH and IHC is shown in . No cases with negative FISH results and positive IHC were identified (100% specificity of IHC in these data). Sensitivity was the same (83%) for all three assays in excision specimens. In small biopsies and cytological specimens however the D5F3 antibody was the most sensitive. The ALK1 and 5A4 assays failed to identify a further one or two FISH-positive cases respectively. All three assays failed to stain the same two cases which contain rearranged ALK genes detectable by FISH. TABLE 4. Concordance of Immunohistochemistry (IHC) and Fluorescence In Situ Hybridization (FISH) Assays Response to Therapy Seven cases with ALK rearrangements detected by FISH went on to receive crizotinib therapy. All but one showed at least a partial response. This crizotinib-refractory case showed no detectable ALK expression by all three IHC assays. DISCUSSION We have compared three different antibody assays for the ALK kinase domain to the current standard FISH assay in a set of archival tumors including 15 FISH-positive cases. We found all three assays to be specific (100%) and sensitive (up to 86%) especially when a signal amplification technique is employed. Furthermore data on response to crizotinib therapy in seven treated cases showed all but one case responded. The case that failed to respond to therapy was negative by all three IHC assays. Of the three antibody methods compared the D5F3 antibody using a Ventana proprietary assay performed the best especially in scanty samples which is likely to be a consequence of the tyramide signal amplification step incorporated into the Ventana assay. It is possible that the other two antibodies would perform as well if a suitable signal amplification step were introduced. However an assay using the 5A4 without tyramide amplification has been successfully applied by To et al in a recent comparable assessment of IHC as a test for ALK rearrangement.5 In a set of 373 tumors that included 20 ALK rearrangements as detected by FISH their IHC assay was 99% specific and 100% sensitive. In contrast to this we find 100% specificity and (at best) 86% sensitivity; that is to say we identified rare FISH-positive IHC-negative cases whereas To et al found occasional FISH-negative IHC-positive cases which were proved to harbor EML4-ALK rearrangements by reverse transcription polymerase chain reaction (RT-PCR).5 It is unsurprising that we do not identify FISH-negative IHC-positive cases as we only examined 17 FISH-negative or indeterminate cases in comparison to the 356 examined by To et al.5 It is more notable that To et al.5 do not identify FISH-positive IHC-negative cases. This might be explained by their use of tissue microarrays for FISH which is even more technically demanding and hard to interpret than FISH using whole sections. This possible shortcoming of tissue microarray methods might be apparent in two other recent studies using tissue microarrays for a comparison of FISH with ALK1 5A4 and D5F3 antibodies.1314 Selinger et al.13 describe 100% sensitivity for all three antibody assays. Conklin et al.14 also find 100% sensitivity and a maximum specificity of 88% (again using the 5A4 antibody). Again it may be that in both these additional studies the approach used hampered the identification of FISH-positive IHC-negative cases because of the difficulty of applying FISH to TMAs especially when the primary test has been IHC and the FISH test is not œblind to the IHC result. Other recent studies compare various immunohistochemical assays and FISH for the detection of ALK rearrangements.1516 As in this study Sholl et al.15 identify occasional FISH-positive IHC-negative cases. They explain two cases by identifying co-existent mutations in other driving oncogenes (presumably thereby relieving the tumor œaddiction to ALK) and one by insufficient tumor material for accurate IHC assessment. Savic et al.16 compared an immunocytochemical assay using the 5A4 antibody to FISH in cytological specimens and achieved a sensitivity of 93% and specificity of 96% which is comparable with our findings in cytological and small biopsy cases (sensitivity 88% specificity 100%). In the current study we detected two œfalse-negative cases which were positive by FISH and negative by all three IHC assays. This combination has two possible explanations. Firstly they might represent tumors which are not expressing ALK protein at detectable levels because of a false-positive FISH result or an absence of addiction to rearranged ALK protein despite presence of recombined ALK DNA. These tumors are unlikely to respond to crizotinib. Secondly they might represent a failure of the IHC assay because of poor preservation of antigen insufficient material or another technical error. In this case crizotinib therapy would still be likely to be effective. The study includes seven cases with positive FISH results with data on response to crizotinib. Six showed at least a partial response to crizotinib therapy as assessed by Response Evaluation Criteria in Solid Tumors criteria. A single case showed no response and this was one of the two œfalse negatives. Thus in this one case the IHC test would have correctly predicted response. This was a scanty cytological specimen in which FISH interpretation was difficult and only 20% of 515 cells showed rearranged ALK signals (fusion plus split red/green probes and fusion plus isolated red signal). Therefore it seems possible that this represents a technical failure of the FISH assay. The other case an excision specimen showed 39% of 626 cells with rearranged signals. It was also shown to harbor a driving mutation in a second gene; PCR testing demonstrated the loss of exon 19 of endothelial growth factor receptor. This has been described in another study that characterized two such œfalse-negative cases.15 Thus these tumors may well escape œoncogene addiction to the ALK kinase activity which would be consistent with indetectable ALK protein expression. Again IHC would be expected to be the best predictor of response to tyrosine kinase inhibitor therapy in such cases. It is essential to identify and molecularly characterize other œfalse-negative cases that have received crizotinib therapy. In addition it seems likely that IHC should guide treatments in œfalse-positive cases that express high levels of ALK from genetic lesions that are invisible to the current FISH assay. Although we identified no œfalse positives i.e. FISH-negative IHC-positive cases our sensitivity may be an overestimate (as judged by FISH) because of the small number of FISH-negative cases under study. Several studies have identified rare cases with rearrangements that are indetectable by FISH but detectable by IHC and confirmed by reverse-transcriptase PCR.5910 Such cases would be expected to respond to crizotinib and a recent study shows that at least one novel œFISH-indetectable rearrangement does indeed drive the malignant phenotype.5"
Lung_Cancer
"The remaining trusts formed either the control group (if they had agreed to participate) or the non-participant group and had no further contact with the study team but continued to submit data to the NLCA as usual. Intervention The study timeline is shown in . Following introductory workshops the multidisciplinary teams within each pair undertook facilitated reciprocal site visits. The visits consisted of observation of the host team's multidisciplinary team meeting three discussion sessions focusing on the functioning of the mulitdisciplinary team meeting the host team's NLCA data and patient experience questionnaire results. The final session aimed to identify the focus of improvement work to be undertaken by the host team. The quality improvement facilitator introduced a structured template for the quality improvement plans and provided a short introduction to using the model of improvement to guide implementation of the plans. Over the next 12 months the quality improvement facilitator provided support via electronic mail telephone and follow-up visits where required. Teams within the intervention group supported each other via mini-collaboratives in the form of web-based teleconferences and two face-to-face workshops. Outcomes Changes in process and outcome were assessed using data from local quality-improving plans and the following indicators from the NLCA: the proportion of patients discussed at a multidisciplinary team meeting histological confirmation rate active treatment rate surgical resection rate the proportion of patients with small-cell lung cancer receiving chemotherapy and the proportion of patients seen by a lung cancer nurse specialist. Patient experience was assessed in the intervention group using a new lung cancer-specific patient experience questionnaire designed in collaboration with the Roy Castle Lung Cancer Foundation. The questionnaire included 11 questions selected with permission from the previously validated 2004 national cancer patient survey. The questions covered the following domains: communication privacy respect and dignity and three free text questions (see Appendix I). Participating teams were asked to distribute 30 questionnaires to patients recently seen in their services. The clinical nurse specialists distributed the questionnaires to patients who anonymously returned them to the Royal College of Physicians. An independent qualitative ethnographic evaluation of the study was undertaken by the Social Science Applied to Healthcare Improvement Research Group at the University of Leicester. Statistical methods Data were tested for normality using the Shapiro“Wilk test. Baseline NLCA indicators were taken from the 2009 NLCA report and the intervention control and non-participant groups were compared using a ?2- test. The changes in NLCA indicators from 2009 to 2011 were compared using an independent t-test. Patient experience questionnaire responses for each question were labelled and re-coded to separate them into the worst patient experience category (score 1) vs all other responses (score 0). These scores were then summated to create a domain and a total patient experience score with a possible range of 0“11 whereby a higher score indicates a worse patient experience. Analyses were performed using the statistical software package SPSS (International Business Machines Corp. Armonk NY USA). Funding and ethics The study was funded by a ˜Closing the Gap' grant from the Health Foundation. The National Research Ethics Service confirmed that the study was service evaluation and quality improvement and did not require ethical review. Results One hundred trusts (66%) replied to the invitation to participate and 91 (61%) agreed to participate in the study. Eighty-one trusts had 2008 NLCA data of sufficient quality to allow pairing. Two trusts provided a joint multidisciplinary team allowing 40 pairs of multidisciplinary teams to be created. One pair agreed to act as a pilot and was excluded from further analysis. Of the remaining 39 pairs 15 pairs (31 trusts) were randomised to the intervention group. The remaining 24 pairs formed the control group. During the study two trusts in the control group amalgamated to form one trust so the total number of trusts in the control group was 47 (). Quality improvement plans Two hundred and thirty medical professionals from 31 trusts participated in the review visits. Twenty-nine teams submitted a total of 67 quality improvement plans. The issues identified in the quality improvement plans are shown in . Eighteen teams collected local data to measure impact. An example of such data is shown in . This trust identified small-cell lung cancer chemotherapy as an area for improvement. They introduced a number of changes to their diagnostic and treatment pathways including prioritisation of small-cell pathology reporting faxing of the results to the multidisciplinary team coordinator and lung nurse specialist to allow early booking of oncology appointments. These changes were monitored using a run chart that demonstrated a reduction in the time from multidisciplinary team meeting to chemotherapy treatment and an increase in the proportion of small-cell lung cancer patients receiving chemotherapy from 60% in 2009 to 71% in 2011. National lung cancer audit indicators Baseline (2009) NLCA indicators for the intervention control and non-participant groups were similar (). The mean change for each NLCA indicator from baseline to 2011 in the intervention and control group is shown in . The proportion of patients receiving active anti-cancer treatment in the intervention group increased by 5.2% compared with 1.2% in the controls (mean difference 4.1% 95% CI ?0.1 to 8.2% P=0.055). The remaining NLCA indicators improved similarly both in the intervention and control groups. Patient experience In the intervention group patient experience questionnaires were returned by 438 patients from 30 multidisciplinary teams at baseline (return rate 49%) and 372 patients from 27 trusts following the intervention (return rate 41%). Baseline total scores were low (0“1.31) indicating high levels of patient satisfaction with the care received although there was a statistically significant (P<0.001) variation in results by the multidisciplinary team (). In particular the proportion of patients responding yes to the question ˜did you find that the person who told you about your diagnosis did so with sufficient sensitivity/care?' varied significantly by 57%“100% (P<0.001). The total questionnaire scores did not change significantly during the study (0.22“0.17 P=0.377) however the variation by the multidisciplinary team reduced (). Given that the study aimed to bring the standard of the lower performing trusts to that of the best we performed a post hoc analysis for the five trusts with the worst baseline patient experience scores. This demonstrated that the mean total score improved significantly for these trusts from 0.86 to 0.22 P<0.001. The biggest improvement in this group was seen in the proportion of patients responding yes to the question ˜did you find that the person who told you about your diagnosis did so with sufficient sensitivity/care?' which increased from 75% to 90% (P=0.05). One multidisciplinary team in this group achieved this improvement by using their baseline questionnaire results as a lever to encourage attendance at an advanced communications skills course. The questionnaire domain-specific scores did not change significantly during the study. Of the individual questions a significant improvement was seen in the rating of the quality of information provided as excellent which rose from 53%“59% P<0.05. Qualitative evaluation Participants' experiences were overwhelmingly positive. The reciprocal peer-to-peer visits with supported quality improvement were seen as a strong driver to change. The method of pairing multidisciplinary teams was important. In particular pairing teams with different results not just ˜good' with ˜bad' and allowing teams to visit each other's sites to ensure a two-way sharing of best practice. The independent quality improvement facilitator role was seen as crucial to ensure the visits remained focussed and that the engagement with quality improvement plans was maintained. Finally the involvement of senior managers was crucial to the successful implementation of the quality improvement plans. The detailed findings from the independent evaluation of this project have been reported elsewhere (Aveling et al 2012). Discussion Lung cancer outcomes remain relatively poor and reducing unexplained variation is an attractive proposition to promote improvement. There are a number of ways that clinical teams may share best practice and innovative service delivery models however studies formally evaluating their impact are limited. To our knowledge this is the first study to formally test a national quality improvement strategy which aimed to bring the standard of all lung cancer teams to that of the best. We have demonstrated that reciprocal peer-to-peer review with supported quality improvement is both feasible and effective at stimulating local quality improvement activity but had a relatively modest and somewhat disappointing impact on process and outcome measures as measured by NLCA indicators and a new lung cancer patient experience questionnaire. The facilitated reciprocal visits represented a new and unique opportunity for all members of a lung cancer team to exchange ideas in a supported environment and to formally design then implement quality improvement plans. Nearly two-thirds of lung cancer multidisciplinary teams in England agreed to take part in the study and reassuringly baseline NLCA indicators did not differ significantly between participants and non-participants suggesting that the willingness to participate in quality improvement activity is not related to baseline performance. There were a wide range of areas identified for improvement but nearly half of the teams identified multidisciplinary team meeting effectiveness as a key issue. This is not surprising given that these meetings are pivotal in the lung cancer pathway. Live observation of each multidisciplinary team meeting followed by facilitated feedback proved to be a strong driver to improve on problems such as ensuring weekly presence of all the treatment specialists as well as more simple issues such as room layout. The need to streamline diagnostic and treatment pathways was also identified as a common problem. Recent NICE guidance on the management of lung cancer (National Institute for Health and Care Excellence 2011) recommended a paradigm shift in the diagnostic algorithm from performing multiple diagnostic and staging investigations to performing a single test that will provide both diagnostic and staging information. A number of teams within our study were able to introduce such pathways and demonstrate impressive reductions in diagnostic times and more prompt treatment. This together with more effective multidisciplinary team working may have led to the small increase in the active anti-cancer treatment rates seen within the intervention group. However an alternative explanation for the improvement is regression to the mean given that treatment rates in the intervention group were lower at baseline and overall the lack of significant improvement across the range of NLCA indicators in the intervention group was disappointing. One possible explanation for this is the challenge that some participating teams encountered converting enthusiastic quality improvement plans into tangible improvements for patients over a relatively short time period. The qualitative evaluation confirmed that participants often underestimated the time and energy required to implement and sustain change and highlighted the importance of early engagement with hospital managers to maintain momentum (Aveling et al 2012). Alternatively other national lung cancer initiatives implemented at the time of the study may have driven coexistent improvements in the control group. For example the drive to encourage all lung cancer patients to be referred for clinical nurse specialist support has subsequently been shown to increase the probability that a lung cancer patient receives active treatment. Although even small improvements in lung cancer treatment rates are very welcome it is recognised that undergoing investigation for suspected lung cancer generates high levels of patient anxiety and many patients will remain too unwell to benefit from currently available drugs. The assessment of patient experience is therefore of particular importance in lung cancer. This has proved challenging in detailed national cancer surveys owing to the advance in age poor health and short median survival of lung cancer patients. The response rate to our short questionnaire was relatively high at 41“49% compared with the 2011 national survey in which only 7% of lung cancer patients responded (Department of Health 2012) but still represents the views of less than half of lung cancer patients and is a relative limitation in terms of generalisability of the results. It was reassuring to note that at entry to the study patients in the intervention group generally rated their experience as highly satisfactory. This may explain the low number of teams who specifically identified patient experience as an area for quality improvement. In terms of assessing the impact of the reciprocal peer-to-peer review visits and supported quality improvement on patient experience it is likely that this high-baseline satisfaction and the lack of patient experience data for the control group limited our ability to detect a significant change. However our results suggest that those teams with poor scores may be able to use patient experience data to promote significant improvements particularly in areas such as communication skills. Further work is required to develop a lung cancer patient experience measure that is both acceptable to patients and able to detect small but clinically important changes in experience. Although similar in name to the national cancer peer review process there are a number of important differences between the reciprocal peer-to-peer review and supported quality improvement process employed in the current study and national cancer peer review. The latter predominantly performs a quality assurance role ensuring that cancer teams meet a minimum standard via compliance with a number of process measures. Support with quality improvement is not provided and site visits are now rarely performed. The qualitative evaluation of our study highlighted the importance of an independent quality improvement facilitator to the success of the peer review visits and the subsequent implementation of the quality improvement plans. Integration of facilitated reciprocal peer-to-peer review and supported quality improvement into national cancer peer review both for lung cancer and other tumour sites is an attractive proposition and requires further study. However our results suggest that this strategy alone is unlikely to have a major impact on lung cancer treatment rates. This phenomenon is not new in lung cancer for example the introduction and NICE approval of gefitinib treatment for the first-line treatment of lung cancer in 2010 was associated with only a 1% increase in active anti-cancer treatment rates over the following year (Health and Social Care Information Centre 2012). Achieving a stepwise increase in lung cancer treatment rates and survival is likely to require a multi-targeted approach including earlier diagnosis streamlined lung cancer pathways new treatments and a reduction in unexplained variation via supported quality improvement programmes. This project was funded by a Health Foundation Closing the Gap award. (grant number: 7797/5557). Appendix I Improving lung cancer outcomes project: patient experience questionnaireWhat is this survey about? This questionnaire asks about your experience of lung cancer treatment and care at the hospital. It was developed in 2010 and it has been used by Lung Cancer Nurse Specialists in 30 hospital across participating in the ˜Improving Lung Cancer Outcomes Project' led by the Royal College of Physicians and several other organisations. The project aims to improve the quality of services and care for people affected by lung cancer. Why should I complete the survey? We need to know your opinion of the current services and care to help improve these for people affected by lung cancer. Your participation in this survey is voluntary and your answers will be treated in "
Lung_Cancer
"Anchorage of tissue cells to their physical environment is an obligate requirement for survival which is lost in mature hematopoietic and in transformed epithelial cells. Here we find that a lymphocyte lineage-restricted transcription factor Aiolos is frequently expressed in lung cancers and predicts markedly reduced patient survival. Aiolos decreases expression of a large set of adhesion-related genes disrupting cell-cell and cell-matrix interactions. Aiolos also reconfigures chromatin structure within the SHC1 gene causing isoform-specific silencing of the anchorage reporter p66Shc and blocking anoikis in vitro and in vivo. In lung cancer tissues and single cells p66Shc expression inversely correlates with that of Aiolos. Together these findings suggest that Aiolos functions as an epigenetic driver of lymphocyte mimicry in metastatic epithelial cancers. Int J Clin Exp Pathol Int J Clin Exp Pathol ijcep International Journal of Clinical and Experimental Pathology 1936-2625 e-Century Publishing Corporation 24427333 3885467 Original Activation of AKT/ERK confers non-small cell lung cancer cells resistance to vinorelbine Fan Da-Ping Zhang Yi-Mei Hu Xiao-Chen Li Jing-Jing Zhang Wei Department of Respiratory Medicine First Clinical Medical College Affiliated to Harbin Medical University Harbin China Address correspondence to: Dr. Wei Zhang Department of Respiratory Medicine First Clinical Medical College Affiliated to Harbin Medical University No. 23 Youzheng Street Harbin Heilongjiang Province China. Tel: 0451-85552560; 86-13030052121; E-mail: zhangwei_harbinyeah.net 2014 15 12 2013 7 1 134 143 30 10 2013 10 12 2013 IJCEP Copyright 2014 2014 Vinorelbine is a semi-synthetic vinca-alkaloid approved for the treatment of non-small cell lung cancer (NSCLC). However the lower objective response rate and higher adverse effects of vinorelbine hinder its wide use in treatment of advanced NSCLC. Therefore it is of great interest to uncover the biomarkers for sensitivity of NSCLC cells to vinorelbine to allow the identification of patients most likely to benefit from vinorelbine-based chemotherapy and to improve the therapy. In present work four NSCLC cell lines were divided into vinorelbine-sensitive (VS) group and vinorelbine-resistant (VR) group according to their sensitivities to vinorelbine. And then the gene expression profiles of these two groups was compared the differentially expressed genes (expression difference higher than 100% and p<0.05 totally 496 genes) were applied to Ingenuity Pathway Analysis (IPA). IPA results showed that NF-?B and PTEN signaling were predicted to be inactivated in VR cell lines which was partially validated by quantitative PCR or western blotting experiments. The higher expression of RAF1 mRNA and the activation of AKT/ERK proteins in VR NSCLC cell lines may confer resistance to vinorelbine. Our work may provide potential pathway signature for vinorelbine sensitivity and some therapeutic targets for combined therapy. Non-small cell lung cancer vinorelbine NF-?B signaling PTEN signaling AKT ERK Proc Natl Acad Sci U S A Proc. Natl. Acad. Sci. U.S.A pnas pnas PNAS Proceedings of the National Academy of Sciences of the United States of America 0027-8424 1091-6490 National Academy of Sciences 24550494 3948305 201319911 10.1073/pnas.1319911111 Biological Sciences Cell Biology Analysis of the tumor-initiating and metastatic capacity of PDX1-positive cells from the adult pancreas PDX1-positive cells from adult pancreas Ischenko Irene a Petrenko Oleksi b 1 Hayman Michael J. a 1 Departments of aMolecular Genetics and Microbiology and bPathology Stony Brook University Stony Brook NY 11794 1To whom correspondence may be addressed. E-mail: alexei.petrenkostonybrook.edu or michael.haymanstonybrook.edu. Edited by Douglas R. Lowy National Cancer Institute Bethesda MD and approved January 22 2014 (received for review October 22 2013) Author contributions: O.P. and M.J.H. designed research; I.I. and O.P. performed research; I.I. and O.P. contributed new reagents/analytic tools; O.P. and M.J.H. analyzed data; and O.P. and M.J.H. wrote the paper. 4 3 2014 18 2 2014 111 9 3466 3471 Significance Pancreatic cancer is characterized by aggressive growth and a high propensity for metastatic spread. Despite growing understanding of the genetic causes of pancreatic cancer the mechanism and timing of cancer metastasis the main cause of deaths in pancreatic cancer patients remain relatively unexplored. In this study we used experimental mouse models of pancreatic carcinogenesis to show that hyperactivation of the Ras/MAPK/ERK pathway and stabilization of the MYC protein are the two main driving forces behind the development of pancreatic cancer cells with high metastatic potential. Our results suggest that pancreatic cells bearing Kras mutation can be induced to differentiate into quasi-normal cells with suppressed tumorigenicity by selective inhibition of the MAPK/ERK/MYC signaling cascade. These findings may have important therapeutic implications. Pancreatic cancer is one of the deadliest human malignancies. A striking feature of pancreatic cancer is that activating Kras mutations are found in ?90% of cases. However apart from a restricted population of cells expressing pancreatic and duodenal homeobox 1 (PDX1) most pancreatic cells are refractory to Kras-driven transformation. In the present study we sought to determine which subsets of PDX1+ cells may be responsible for tumor growth. Using the Lox-Stop-Lox“KrasG12D genetic mouse model of pancreatic carcinogenesis we isolated a population of KrasG12D-expressing PDX1+ cells with an inherent capacity to metastasize. This population of cells bears the surface phenotype of EpCAM+CD24+CD44+CD133“SCA1? and is closer in its properties to stem-like cells than to more mature cell types. We further demonstrate that the tumorigenic capacity of PDX1+ cells is limited becoming progressively lost as the cells acquire a mature phenotype. These data are consistent with the hypothesis that the adult pancreas harbors a dormant progenitor cell population that is capable of initiating tumor growth under conditions of oncogenic stimulation. We present evidence that constitutive activation of the mitogen-activated protein kinase (MAPK/ERK) signaling and stabilization of the MYC protein "
Lung_Cancer
"PAX6 expression was obviously weakened in A549 PAX6 KD and H1299 PAX6 KD cells. To elucidate whether PAX6 expression has any effect on the growth of lung cancer cells RNAi was used to generate pax6 knock-down (PAX6 KD) cell lines. We selected two target cell lines: H1299 which showed high levels of PAX6 expression and A549 which showed low levels of expression. In the present study pGCSIL-pax6 shRNA-GFP was infected into H1299 and A549 cells. Cells were also infected with pGCSIL-NS shRNA-GFP (PAX6 NS) as a negative control (NC). To determine the function of PAX6 H1299 H1299NC A549 or A549NC cells were used as controls in all assays. The PAX6 mRNA level in H1299 PAX6 KD and A549 PAX6 KD cells was determined by real-time PCR to confirm whether PAX6 expression was specifically inhibited through RNAi in A549 and H1299 cells. As shown in B PAX6 expression in A549 PAX6 KD cells was inhibited by 80“90% compared to cells infected with lentivirus-mediated NS-shRNA. We found similar results in H1299 PAX6 KD cells. PAX6 mRNA expression in these cells was also inhibited by 90“95% as compared to NC cells (**P<0.01; C). PAX6 protein expression in these cells was detected by Western blotting. As shown in D PAX6 protein in H1299 PAX6 KD and A549 PAX6 KD cells was not readily detected whereas a clear PAX6 protein band was evident in the control cells. Inhibition of PAX6 expression leads to a decline in cell proliferation PAX6 is a critical transcription factor that plays an important role in regulating proliferation and differentiation during human embryonic development [3]. A cell proliferation assay was performed to determine whether PAX6 plays a role in cellular growth. A549 PAX6 KD H1299 PAX6 KD and control cells were seeded in 96-well plates and cell proliferation activity was measured using a Cell Proliferation Assay kit. A549 and H1299 cell growth was obviously suppressed when PAX6 expression was inhibited by RNAi (A and B). As shown in A and B the decrease in cell growth caused by the inhibition of PAX6 expression in H1299 was much stronger than that in A549 cells. These different results may be attributable to the different PAX6 expression levels between H1299 and A549 cells displayed in A and D. .0085738.g002 The lentivirus-mediated pax6-shRNA knockdown of PAX6 expression could suppress lung cancer cell growth. A -B A549 PAX6 KD H1299 PAX6 KD cells and control cells were seeded in 96-well plates and an MTS assay was performed. The absorbance at 490 nm (y -axis) was measured at 24-h intervals up to 120 h. C -D Colony formation efficiency in A549 PAX6 KD H1299 PAX6 KD cells and control cells. The y -axis represents the normalized colony formation rate relative to A549 (C) or H1299 (D) cells. E -F A soft -agar assay was performed to investigate the effects of PAX6 on tumorigenesis in vitro. The y -axis represents the normalized soft -agar colony formation rate relative to A549 (E) or H1299 (F) cells. The data are expressed as the means ± SEM from three separate experiments. Times of the experiments are listed above the graph.*P <0.05 **P <0.01. Reduced colony formation and soft-agar colony formation in PAX6 KD cells Colony formation represents a loss of contact inhibition or the ability to maintain cell growth and movement despite contact with surrounding cells. To clarify whether PAX6 could confer a loss of contact inhibition cells infected with pGCSIL-pax6 shRNA-GFP as well as their control cells were seeded into 6-well plates and cultured for 7 days. After 2% gentian violet staining colonies containing more than 50 cells were counted under a light microscope. As displayed in C and D the inhibition of PAX6 expression in A549 and H1299 cells led to an obvious decrease in the number of foci generated compared to the control cells (**P<0.01). To further study the function of PAX6 soft-agar colony formation was analyzed to determine whether PAX6 contributed to anchorage-independent colony formation in lung cancer cells. The rate of soft-agar colony formation declined in A549 PAX6 KD and H1299 PAX6 KD cells compared to NC cells (**P<0.01 *P<0.05; E and F). PAX6 expression increased cell growth by promoting faster progression into S phase of the cell cycle To detect the effect of PAX6 on cell cycle progression the cell cycle progression of A549 PAX6 KD H1299 PAX6 KD A549 PAX6 NC H1299 PAX6 NC A549 and H1299 cells was analyzed by flow cytometry. As displayed in A the percentage of cells entering S phase was decreased in the A549 PAX6 KD cell line along with an increase in the population of G0-G1 phase cells. A similar result was observed in H1299 PAX6 KD H1299 PAX6 NC and H1299 cells (B). In these experiments PAX6 expression led to cell growth by inducing cell cycle progression. .0085738.g003 PAX6 expression promoted cell cycle progression. A B Cell cycle analysis. Cells were stained with propidium iodide (PI) and analyzed for cell cycle phase distribution. The histogram was the statistical data from three independent experimental replicates. *P <0.05 **P <0.01. In this study the expression level of cyclin D1 a relevant cyclin regulating G1-S progression [15] [16] was detected in A549 PAX6 KD and H1299 PAX6 KD cells. As indicated in A cyclin D1 expression was decreased in A549 PAX6 KD cell lines compared to control cells."
Lung_Cancer
"Because the meQTL SNPs affecting CpG sites in gene body/non-CGI regions were mostly enriched for SQ risk (Fig. 5d) we performed further analysis in this category by integrating the ENCODE SAEC data. We chose SAEC data because this cell type may be involved in SQ development. We restricted enrichment analysis to the œregulatory meQTL SNPs which localized in the CTCF binding regions DNaseI hypersensitive sites or histone marks (H3K27me3 H3K4me3 and H3K36me3) or had at least one LD SNP (r2? 0.95) residing in these regions. The strong enrichment in SQ was driven by SNPs overlapping with CTCF binding sites (P<10?4 based on 10000 permutations) or the repressive mark H3K27me3 (P<10?4 based on 10000 permutations) (Fig. 5e). The enrichment test was not significant after excluding the SNPs overlapping with these regulatory regions (P=0.14 based on 10000 permutations). Replication of meQTLs in TCGA breast and kidney tissues To explore the tissue-specificity of the genetic effects on DNA methylation we examined whether the meQTLs detected in EAGLE lung tissue data could be replicated in TCGA breast (n=87) or kidney (n=142) histologically normal tissue samples the only two ans to date with data available for a large number of normal tissues of European ancestry. Results are in and Supplementary Fig. 8. For both cis- and trans- meQTLs a large proportion of associations had the same direction of EAGLE meQTLs in both breast and kidney samples. For cis-associations 54.7% and 70.0% were replicated with FDR=5% based on single-sided P-values in two data sets respectively. For the strong cis associations with P<10?10 in EAGLE the replication rates increased to 82.7% and 89.2% in the two data sets. For trans- associations 83.4% and 86.4% were replicated in breast and kidney samples respectively. The detected master regulator (Fig 2a) was strongly replicated in both data sets (Supplementary ). Interestingly some cis-meQTLs but not trans-meQTLs had an opposite but very strong association (P<10?6) in breast (n=7) or kidney (n=58) compared with the EAGLE lung data a phenomenon previously reported in a cell-type specific eQTL study40. Discussion We found that inherited genetic variation profoundly and extensively impacts DNA methylation in target ans. Based on high-density methylation arrays in a large sample size we identified 34304 cis-meQTLs and 585 trans-meQTLs one to two orders of magnitude larger compared to previous studies3“57. meQTLs involved nearly half of the autosomal genes of which 9330 in cis and 373 in trans with 9525 unique genes in total. We show that approximately 10% of the cis-meQTLs were affected by at least two SNPs independently. Moreover we detected a master regulator SNP associated with the methylation levels of five CpG probes on different chromosomes demonstrating the existence of regulatory hotspots for DNA methylation as previously shown for eQTL4142. Most meQTLs were replicated in independent histologically normal lung tissue samples from TCGA. We also showed a high similarity of genetic control on DNA methylation across different tissues. Our findings show that genetic effects on DNA methylation are extensive in scale and complex in structure across the whole genome and suggest a series of important biological implications. First our results show that the genomic architecture surrounding cis- and trans-meQTLs is distinct. cis-meQTLs are very large in number impact predominantly the CpG sites mapping to non-gene regions and when they occur in genes are mostly in non-promoter and non-CpG island regions. In contrast trans-meQTLs are rarer mainly affect promoter CGI regions and may be associated with distal CpG sites through the mediation effect of proximal CpG sites. We found preliminary evidence that the cis-CpG sites mediating the trans-meQTL associations were enriched for genes involved in methylation regulation such as genes encoding for GTPase or proteins involved in genetic imprinting. GTPase-related gene pathways appear to modulate expression of DNA methyltransferases43. Methylation-induced expression changes of these genes may result in further methylation changes of other genes (i.e. in trans). Moreover a noncoding RNA within the intron of KCNQ1 a key gene regulating genetic imprinting can influence chromatin 3-D structure via a protein complex including DNA methyltransferase proteins4445. These findings suggest intricate mechanisms for trans-regulating effects through proximal methylation. cis-meQTLs may affect cancer risk. To understand the functional consequences of GWAS loci is challenging and multiple principles for post-GWAS™ functional characterization of genetic loci have been proposed including the exploration of epigenetic mechanisms46. In our study the top GWAS lung cancer loci were strongly associated with methylation levels of CpG sites in nearby gene bodies through cis-regulation and adjusting for smoking status or intensity did not change the results. Furthermore SNPs affecting the DNA methylation of gene bodies (which are typically methylated) were also collectively associated with risk for squamous cell carcinoma after excluding the established GWAS loci and were enriched for genes in cancer pathways. In contrast no enrichment was observed for SNPs affecting the methylation of gene promoters or CGI regions which are typically not methylated in normal tissues. This suggests a potential novel mechanism for genetic effects on cancer risk. In fact gene body-enriched cis-meQTLs outside CGI regions may increase the risk for germline and somatic mutations due to their increased propensity to become mutated1112. Upon spontaneous hydrolytic deamination methylated cytosine residues turn into thymine which are less likely to be efficiently repaired than the uracils that result from deamination of unmethylated cytosine residues. For example about 25% of mutations in TP53 in cancers are thought to be due to epigenetic effects47. Indeed analyses of comprehensive human catalogues of lung tumors have identified frequent G>T mutations enriched for CpG dinucleotides outside CGI regions suggesting a role for methylated cytosine since CGI as we confirmed are usually unmethylated48. A similar signature was recently observed in other tumors14. Thus inherited genetic variation may have a profound impact on carcinogenesis by regulating the human methylome. We observed a high similarity of genetic control on DNA methylation across tissues. Since tissue of origin determines cancer-associated CpG island promoter hypermethylation patterns49 a natural question is whether the genetic regulation of methylation is tissue specific. While the tissue-specificity of eQTLs has been investigated for a few tissues50 for cis-meQTL only a recent investigation was conducted6 showing that 35.7% of 88751 cis meQTLs detected in 662 adipose samples were replicated in ~200 whole blood samples. We found that a large proportion of meQTLs in EAGLE lung samples particularly those with large effect sizes were robustly replicated in breast and kidney tissue samples from TCGA suggesting a high similarity of genetic regulation of methylation across these tissues and related impact on somatic mutation rates1448. "
Lung_Cancer
"The lung tissues were fixed in 10% neutral formalin embedded in paraffin and cut into 5 µm thick slices after we took photographs to record staining on the lung surface. We made 4 axial slices that covered the center of the staining. The slices were subjected to hematoxylin-eosin (H-E) stain to the evaluate lung parenchymal change. We evaluated the presence or absence of neutrophil infiltration vasculitis necrosis hemorrhage and foam cell in alveolus. The extent of each histopathologic finding was estimated using visual grading scores as 0 (no) 1 (focal) or 2 (diffuse). Localized parenchymal change (<50% of total area) surrounded by normal lung was defined as focal. Extensive lung parenchymal change (?50% of total area) that replaced normal lung was defined as diffuse. An experienced pathologist with eight years of experience reviewed all slices. The overall severity of the lung parenchymal change was defined as a total score by adding visual grading scores for each histopathologic finding. We compared the overall severity score between MLM and methylene blue as well as between Group A and Group B. Statistical analysis All data are expressed as mean±standard deviation (SD) unless otherwise stated. Comparisons of the average scores were performed by two-tailed unpaired Student's t-test or Mann-Whitney test. We used a Fisher's exact test to compare the number of subjects in the subgroups. Linear by linear association evaluated the association of the extent of lung parenchymal change and materials or groups. Null hypotheses of no difference were rejected if the P values were less than 0.05. The statistical analysis was performed with commercially available statistical software IBM SPSS Statistics version 20.0 (IBM Corp. in Armonk NY USA). RESULTS Subject characteristics procedural records time interval of injection and examinations Among the 24 subjects included in our study successful CT-guided percutaneous injections into the desired location of the lung were achieved in 21 subjects (11 in Group A and 10 in Group B). Three subjects died during anesthesia. Mean weight was 3.2±0.2 kg for Group A and 3.3±0.2 kg for Group B. Injection depth from visceral pleura to needle tip was 0.4±0.1 cm (range: 0.3-0.6 cm) for MLM and 0.4±0.1 cm (range: 0.3-0.7 cm) for methylene blue (P=0.43). Distance from skin to needle tip was 2.8±0.6 cm (range: 2.1-5.0 cm) for MLM and 2.8±0.3 cm (range: 2.2-3.5 cm) for methylene blue (P=0.83). Of 42 CT-guided percutaneous injections total number of procedure related complications was 10 (24%) including 7 leakage (all in MLM) and 3 pneumothorax (2 in MLM 1 in methylene blue). The complication rate in MLM was significantly higher than methylene blue (43% vs 5%) (P=0.004). On post-procedural CT images the extent of the radio-opacity of MLM was 1.3±0.4 cm (range: 0.7-2.0 cm) for Group A and 0.6±0.3 cm (range: 0.3-1.1 cm) for Group B. Discrete compact nodular opacity was achieved in 15 subjects (72%) scattered nodular opacities in 3 (14%) and small faint opacity in 3 (14%) (Fig. 4). The average value of radio-opacity of MLM was 1415±856 HU (range: 307-2768 HU). The interval between injection and sacrifice was 7.9±0.1 hr (range: 7.8-8.0 hr) for Group A and 23.5±0.1 hr (range: 23.4-23.7 hr) for Group B. Time from injection to initial and follow up fluoroscopy was 3.4±0.5 hr (range: 2.5-4.2 hr) and 6.8±0.4 hr (range: 6.3-7.7 hr) for Group A and 1.5±0.4 hr (range: 0.9-2.1 hr) and 22.6±0.4 hr (range: 21.9-23.2 hr) for Group B respectively. Scores and extent of staining and radio-opacity demonstrates the staining extent and localization ability of MLM and methylene blue. In total groups the staining extent of MLM was significant smaller than methylene blue (0.6 cm vs 1.0 cm P<0.001). MLM showed a significantly higher staining ability score than methylene blue (2.8 vs 2.2 P=0.010). Radio-opacity in the initial fluoroscopy was not significantly different from the follow up (2.0 vs 1.9 P=0.49). showed the number of subjects in each score of localization ability of staining or radio-opacity. In Group A appropriate staining was 100% for both MLM and methylene blue. In Group B appropriate staining was 90% for MLM and 70% for methylene blue. Appropriate staining of MLM was not significantly different from that of methylene blue (95% vs 86% P=0.61); however excellent staining in MLM was significantly higher than methylene blue (81% vs 38% P=0.011) (). shows the localization ability of MLM regarding both staining ability and radio-opacity. There was no subject with a score of 0 or 1 in both radio-opacity and staining. MLM achieved appropriate staining or radio-opacity in 21 subjects (100%) with a dual localization feature. Histopathologic findings demonstrates the results of the histopathologic findings. In all lung specimens both methylene blue and MLM showed acute lung parenchymal change that included neutrophil infiltration hemorrhage and foam cell in alveolus (Fig. 4). Comparing the two materials the number of specimen having neutrophil infiltration vasculitis necrosis hemorrhage and foam cell in alveolus was similar in each extent. In terms of all features the number of specimen that showed diffuse extent was more in Group B than Group A for both MLM and methylene blue. The extent of the histopathologic findings was not significantly associated with the materials for all histopathologic features (). Among the histopathologic findings the extent of vasculitis was significantly associated with Group for both MLM and methylene blue (P=0.002 for both MLM and methylene blue). Focal or diffuse extent of vasculitis was more frequently found in Group A than Group B (P=0.001 for both MLM and methylene blue). The overall severity of lung parenchymal change was not different between MLM and methylene blue (5.6±1.6 vs 5.7±1.5 P=0.839); in addition Group B showed a significantly higher overall severity score of lung parenchymal change than Group A (6.6±1.6 vs 4.7±0.9 P=0.005). DISCUSSION The results of this study show that MLM is a useful percutaneous injection material for a successful localization in the lung. The average staining score of MLM was significantly higher than methylene blue (2.8±0.5 vs 2.2±0.7 P=0.010). In terms of staining the appropriate localization rate (acceptable or excellent staining) in our study was 95% using MLM. The result was in close agreement with previous studies that showed a high success performance rate of lipiodol localization (99%-100%) (21-23). An appropriate localization rate (acceptable or excellent staining) of methylene blue injection was 86% in our study. This is lower than the results found in previous studies where the success rate of methylene blue injection was 96%-100% (18 20). We found that an acceptable (or excellent staining rate) of MLM and methylene blue was not significantly different (95% vs 86% P=0.610). However MLM showed excellent staining for localization in 17 (81%) of 21 subjects and was significantly higher than methylene blue (38%) (P=0.011). The results indicate that lipiodol reduced the spread of methylene blue. This is the first study to indicate that MLM is an available percutaneous injection material for localization with superior staining ability compared to methylene blue. The complication rate was 43% in MLM and 5% in the methylene blue (P=0.004). Possible complications after percutaneous injection for pulmonary localization include pneumothorax leakage hemorrhage pain hemoptysis hemothorax and embolism. Previous studies reported that the complication rate was 17-29% for lipiodol and 33% for methylene blue (2023 24). The complication rate of MLM in the current study was higher than the results of previous studies mainly due to the leakage of MLM into the pleural cavity (n=9). This difference was probably because the distance from the pleura to the injecting needle tip (0.4±0.1 cm for MLM) was inadequate to avoid leakage into the pleural cavity. In the previous studies of lipiodol marking for localization the mean distance from the pleura to the target nodule was 1.0-1.9 cm (22-24) more than twice our study. The results indicate that the high complication rate of our study is associated with the inserting procedure of the needle rather than MLM itself. The dispersion of methylene blue throughout the lung parenchyma may lead to unnecessarily large wedge resections; in addition some have reported instances of the dispersion of methylene blue throughout the entire pleural surface or intraoperative identification failure due to severe anthracosis of the visceral pleura. The failure rate was reported to be 0%-13% with the use of methylene blue (1819 25). The results are similar to our study and indicate that inappropriate staining on the lung surface was 14% in methylene blue. In this study we found that the dispersion of methylene blue in MLM through the lung parenchyma was significantly smaller than methylene blue (0.6±0.3 cm vs 1.0±0.4 cm P<0.05). The result implies that lipiodol reduces the spread of methylene blue in lung parenchyma. Regarding the score of radio-opacity 38% of MLM showed non-visualization or minimally increased opacity on the fluoroscopic examinations. It means the proportion of lipiodol in MLM at the time of the percutaneous injection was too small to be detected. Post-procedural CT images also revealed that 3 subjects had small faint radio-opacity after the injection of MLM. It suggests that the uneven blending of lipiodol and methylene blue occurred during the preparation of MLM. Water-insolubility of lipiodol would result in the uneven mixing of water soluble methylene blue after mechanical blending of the two materials. Further research is required to reduce non-homogeneity of MLM at the time of injection. Previous studies reported the availability of a mixture of methylene blue with other materials such as collagen or autologous blood (15 16). They performed VATS resection on the same day as localization. In our study we evaluated the localization ability of MLM on the same day of localization (6 hr) as well as 24 hr after injection. Localization is usually performed on the day of surgery. This requires the simultaneous use of the CT and the operating room which is not always available. Surgeries on the next day of localization were reported in several published articles (26 27). MLM shows a prolonged localization ability of up to 24 hr in terms of staining ability and radio-opacity. Stable localization ability is the advantage of MLM in our study. Due to uneven blending of MLM one subject (10%) showed inappropriate staining and appropriate radio-opacity and required an intraoperative fluoroscopic examination to detect MLM. Possible radiation exposure is a drawback of MLM. We would like to justify the use of intraoperative fluoroscopy because the operator can avoid radiation exposure with a lead apron. In regards to the risk-benefit for patients lowering the risk of detection failure is thought to be more important than radiation exposure. Histopathologic examinations showed lung parenchymal changes in all specimens. Both methylene blue and MLM induced acute lung injury that included neutrophil infiltration vasculitis necrosis hemorrhage and foam cell in alveolus (). The results of our study are similar to those of a previous study by Kwon et al. (28) that showed that lipiodol led to acute lung injury. They described that lipiodol creates the histopathologic feature of acute lung injury such as peripheral endothelial cell damage neutrophil infiltration necrosis hemorrhage alveolar wall destruction vasculitis emboli (or thrombi in arteriole) and macrophages in the alveolar space (28). In our results the extent of lung parenchymal change was not associated with the materials for all histopathologic features. In addition the overall severity score of lung parenchymal change in MLM was not different from methylene blue (5.6 and 5.7 P=0.839). This suggests that MLM shows similar histopathologic effects in the lung parenchyma to methylene blue. The overall severity score of parenchymal change was higher in Group B (follow up interval of 24 hr) than Group A (follow up interval of 6 hr) (6.6 vs 4.7 P=0.005). The extent of lung parenchymal change depends on the time interval. Acute lung injury after the percutaneous injection of lipiodol or methylene blue was reported in animal studies (28 29); however there are no clinical results that show the adverse effect of acute lung injury in human lungs. Injection material (such as barium) can potentially complicate the pathologic diagnosis of the target lesion due to acute inflammation (29 30). To our knowledge no study has indicated that lipiodol or methylene blue hinders the histopathologic diagnosis of target lesions in human lungs. The small amount of material injection in human lungs might not create a significant parenchymal change or disrupt underlying lung disease. It is necessary to avoid directly injecting materials into the target lesion in human lungs in order to avoid the adverse effect of injection materials on underlying lung disease (especially ground glass opacity nodule or potential benign lesion). There were several limitations in our study. First we included only a small number of subjects. Second the overall localization success rate was low and the complication rate was high (compared to the results of previous studies) due to the difficulty in an accurate percutaneous injection at the desired location and depth in the small sized rabbit lung. Third we used a 1 mL syringe with manual administration to inject materials in the lung parenchyma and there were possible individual difference in the administering volume of materials. Fourth we could not evaluate complications such as intractable pain material related anaphylaxis or embolism. Fifth we could not evaluate if the histopathologic changes had any effect on underlying lung disease because the lung parenchyma of the experimental rabbits were normal. Finally we did not evaluate a successful localization for the true target lesion in lung parenchyma. The criteria for appropriate staining and radio-opacity were subjective. We expect that further clinical studies might provide an answer to if MLM can be a useful percutaneous injection material for localization in the human lung. In conclusion MLM is available for percutaneous injection for the pulmonary localization. The results of this study showed that MLM provides superior ability for appropriate localization than that of methylene blue. Further research on human lungs can clarify the availability of MLM as a CT guided percutaneous injection material. This study was supported by grant from the Seoul National University College of Medicine Research Fund 2012 (800-20120036). We have no potential conflicts of interest or commercial involvement to disclose. 1 Nakashima S Watanabe A Obama T Yamada G Takahashi H Higami T Need for preoperative computed tomography-guided localization in video-assisted thoracoscopic surgery pulmonary resections of metastatic pulmonary nodules Ann Thorac Surg 2010 89 212 218 20103238 2 Chen S Zhou J Zhang J Hu H Luo X Zhang Y Chen H Video-assisted thoracoscopic solitary pulmonary nodule resection after CT-guided hookwire localization: 43 cases report and literature review Surg Endosc 2011 25 1723 1729 21181200 3 Ciriaco P Negri G Puglisi A Nicoletti R Del Maschio A Zannini P Video-assisted thoracoscopic surgery for pulmonary nodules: rationale for preoperative computed tomography-guided hookwire localization Eur J Cardiothorac Surg 2004 25 429 433 15019673 4 Suzuki K Nagai K Yoshida J Ohmatsu H Takahashi K Nishimura M Nishiwaki Y Video-assisted thoracoscopic surgery for small indeterminate pulmonary nodules: indications for preoperative marking Chest 1999 115 563 568 10027460 5 Seo JM Lee HY Kim HK Choi YS Kim J Shim YM Lee KS Factors determining successful computed tomography-guided localization of lung nodules J Thorac Cardiovasc Surg 2012 143 809 814 22104686 6 Gossot D Miaux Y Guermazi A Celerier M Friga J The hook-wire technique for localization of pulmonary nodules during thoracoscopic resection Chest 1994 105 1467 1469 8181339 7 Pittet O Christodoulou M Pezzetta E Schmidt S Schnyder P Ris HB Video-assisted thoracoscopic resection of a small pulmonary nodule after computed tomography-guided localization with a hook-wire system: experience in 45 consecutive patients World J Surg 2007 31 575 578 17318707 8 Chen W Chen L Yang S Chen Z Qian G Zhang S Jing J A novel technique for localization of small pulmonary nodules Chest 2007 131 1526 1531 17494801 9 Bernard A Resection of pulmonary nodules using video-assisted thoracic surgery: the Thorax Group Ann Thorac Surg 1996 61 202 204 8561553 10 Martin AE Chen JY Muratore CS Mayo-Smith WW Luks FI Dual localization technique for thoracoscopic resection of lung lesions in children J Laparoendosc Adv Surg Tech A 2009 19 S161 S164 18999984 11 Kawanaka K Nomori H Mori T Ikeda K Ikeda O Tomiguchi S Yamashita Y Marking of small pulmonary nodules before thoracoscopic resection: injection of lipiodol under CT-fluoroscopic guidance Acad Radiol 2009 16 39 45 19064210 12 Yamagami T Miura H Yoshimatsu R Tanaka O Ono S Iehara T Hosoi H Nishimura T Experience of fluoroscopy-aided thoracoscopic resection of pulmonary nodule localised with Lipiodol in a child J Med Imaging Radiat Oncol 2011 55 401 403 21843175 13 Iwasaki Y Nagata K Yuba T Hosogi S Kohno K Ohsugi S Kuwahara H Takemura Y Yokomura I Fluoroscopy-guided barium marking for localizing small pulmonary lesions before video-assisted thoracic surgery Respir Med 2005 99 285 289 15733503 14 Yoshida J Nagai K Nishimura M Takahashi K Computed tomography-fluoroscopy guided injection of cyanoacrylate to mark a pulmonary nodule for thoracoscopic resection Jpn J Thorac Cardiovasc Surg 1999 47 210 213 10402768 15 Nomori H Horio H Colored collagen is a long-lasting point marker for small pulmonary nodules in thoracoscopic operations Ann Thorac Surg 1996 61 1070 1073 8607658 16 McConnell PI Feola GP Meyers RL Methylene blue-stained autologous blood for needle localization and thoracoscopic resection of deep pulmonary nodules J Pediatr Surg 2002 37 1729 1731 12483642 17 Hu J Zhang C Sun L Localization of small pulmonary nodules for videothoracoscopic surgery ANZ J Surg 2006 76 649 651 16813634 18 Wicky S Mayor B Cuttat JF Schnyder P CT-guided localizations of pulmonary nodules with methylene blue injections for thoracoscopic resections Chest 1994 106 1326 1328 7956378 19 Vandoni RE Cuttat JF Wicky S Suter M CT-guided methylene-blue labelling before thoracoscopic resection of pulmonary nodules Eur J Cardiothorac Surg 1998 14 265 270 9761435 20 Lenglinger FX Schwarz CD Artmann W Localization of pulmonary nodules before thoracoscopic surgery: value of percutaneous staining with methylene blue AJR Am J Roentg"
Lung_Cancer
"This phenomenon which we did not observe in our previous cohorts might relate to the average quality of chimeras as for this experiment we induced tumors in chimeric mice with a wide range (5“95%) of coat-color chimerism (supplementary Fig S8B). Monitoring of Luciferase expression in individual mice from the F1 cohort revealed that all Mycl1 expressing tumors initiated around the same time point and showed identical growth leftacteristics making this model very suitable for tumor intervention studies (Fig 4E). To proof that Mycl1 is the driver gene for the 4qD2.2 amplicon we verified Mycl1 amplification status in genomic DNA of tumors from control Rb1F/F ;Trp53F/F mice or from mice with either the invCAG-Luc or the invCAG-Mycl1-Luc2 transgene (both chimeras and F1) by low-coverage DNA sequencing and real-time PCR (Fig 4F). Mycl1 was amplified in 37.5“66.7% of the control tumors whereas it was only amplified in 6% of the Mycl1 transgenic tumors clearly validating Mycl1 as a driver for SCLC development. Discussion The GEMM-ESC procedure as presented here can be divided in two separate phases: a resource phase and an experimental phase (Fig 5). The resource phase starting from the selection of the original GEMM until cryogenic storage of quality controlled Col1a1-frt targeted GEMM-ESC clones is fairly laborious taking up to 6 months. The success rate is high and independent of the strain background. This can be largely attributed to the optimized ESC culture conditions which not only simplify procedures but also allow for better quality ESCs as compared to previous protocols. The 2i culture protocol also enables derivation of ESCs from mouse strains that were previously considered refractory (Ying et al 2008; Reinholdt et al 2012). In this study we derived 47 ESC clones from 364 embryos representing 13% derivation efficiency. Although this seems low most of our ESC derivation attempts were successful. The derivation efficiency could be further increased by expanding all early ESC clones instead of selecting them on the basis of their morphology and growth rate (). Efficiency of the GEMM-ESC approach.Schematic representation of the GEMM-ESC approach including the performance of the individual steps. The approach is divided in two phases: a resource phase and an experimental phase. The resource phase includes ESC's derivation and targeting with the Col1a1-frt vector performed once per GEMM and takes ˜6 months including the necessary quality controls. The experimental phase is mainly focused on introducing a transgene-coding plasmid in a validated GEMM-ESC clone using the Flp-in method that allows for consecutive manipulations and takes ˜4 months to obtain a chimeric cohort. Alternatively GEMM-ESC clones are also suitable for direct targeting of a specific gene or the introduction of mutant alleles using gene editing (arrows with dotted lines). The experimental phase also includes the option to follow an F1 route as almost all GEMM-ESC clones showed germline transmission (GLT). In practice we advise that for each model (i) multiple Col1a1-frt targeted GEMM-ESC clones are screened for their ability to efficiently generate high quality chimeras (ii) two of the best-performing clones are selected for the Flp-in procedure and (iii) at least two transgene-coding GEMM-ESC clones are used to generate cohorts. The final clones should originate from different Col1a1-frt targeted parental clones to minimize the chance of miss-interpreting phenotypes due to possible unwanted genetic alterations introduced by long-term culture. The selection of best-performing Col1a1-frt targeted GEMM-ESC clones is crucial for the efficiency to later generate experimental cohorts as the number of chimeras born per injected embryo is likely to decline after additional manipulations and propagation in culture. One issue we noted is that different genetic backgrounds of the original GEMMs require fine-tuning of the ESC injection procedure in order to achieve an optimal balance between quality and yield of the resulting chimeras. This balance is affected by several factors including the strain background and the developmental stage of the host embryos. Injection of ESCs into morulae instead of blastocysts (Plagge et al 2000) leads to very high quality chimeras but also causes an increase in birth problems still-born animals and pups with growth retardation in particular for FVB/n and FVB/n;129/Ola ESC clones (Fig 1D and supplementary Table S1). We therefore optimized the injection procedure for each ESC background. In practice ESCs derived from C57BL/6J strains were injected into FVB/n morulae whereas ESCs from FVB/n;129/Ola or FVB/n strains were injected into C57BL/6N blastocysts. Also other strategies are available to shift the balance between the quality and the yield of chimeras. For instance fully ESC-derived chimeras can be produced using tetraploid complementation techniques (Eakin & Hadjantonakis 2006). Alternatively lower numbers of ESCs can be injected per morula or blastocyst in order to improve the yield of life-born chimeras. The quality of GEMM-ESC clones remains stable over time even after genetic manipulation by targeting or Flp-mediated integration as 70“78% of newly modified ESC clones gave good chimeras and GLT (Fig 5). The genetic stability of ESCs cultured in 2i medium is comparable to ESCs cultured under classic conditions (Fig 3B; Liang et al 2008). The occurrence of CNVs highlights the need for thorough genetic screening of the Col1a1-frt targeted GEMM-ESC clones by e.g. aCGH deep sequencing or spectral karyotyping especially because a fraction of these CNVs will be transmitted from the chimeras to the F1 offspring (Fig 3C). The development of CNVs might be related to targeting or Flp-mediated integration; however we have also observed CNVs in freshly subcloned ESCs (supplementary Table S4). It is therefore more likely that CNVs arise spontaneously when cells are stressed for instance by single-cell cloning or antibiotic selection regimens and not by specific recombination or integration events. Occasionally we observed identical CNVs in independent subclones from the same GEMM-ESC implying that some CNVs are already present in a subpopulation of the derived GEMM-ESC clone (supplementary Table S4). This places the occurrence of some CNVs very early in the ESC derivation process. Thus far we did not observe any phenotypic changes in our chimeras or their offspring that could be attributed to a particular CNV; however this remains a possibility. Also aCGH screening may not allow for full identification of all aberrations that occur during in vitro culture as some apparently normal clones failed to give rise to chimeras indicating an underlying genetic or epigenetic defect. It should be noted that these issues are not specific for the approach described here but plays a role in any experiment using cloned ESCs (Liang et al 2008); in a conventional ESC approach these CNVs are likely bred out of the cohort which is not the case when chimeras are used directly although use of independent ESC clones can further reduce this risk. Further refinements in the ESC culture conditions might improve the (epi)genetic stability of the GEMM-ESC clones. The experimental phase of the GEMM-ESC procedure is based on the ˜plug-and-play™ principle which ensures optimal flexibility and short time frames. It takes typically <4 months to obtain an experimental cohort. Another advantage of the plug-and-play system is that it is compatible with a wide array of vectors for gain-of-function or loss-of-function studies. To this end we are developing additional Flp-in vector backbones for inducible expression of shRNAs to allow for specific knockdown of target genes. In the near future one only has to clone a cDNA or validated shRNA construct into one of these vectors and introduce that into the GEMM-ESC clone of choice after which an experimental cohort can be produced. This will permit the swift validation of candidate cancer genes identified in genomic sequencing efforts and functional genetic screens in a suitable mouse model as we have shown for Mycl1. Moreover in the case where full target gene silencing is required to observe a phenotype the highly efficient gene editing tools TALENS and CRISPR/Cas can be applied on GEMM-ESCs to generate a homozygous null allele that can be directly evaluated in chimeras (Fig 5). By using a GEMM instead of a xenograft model for in vivo validation also the effects of the target gene on tumor initiation tumor progression and tumor maintenance can be monitored including interactions between tumor cells and immune cells. The GEMM-ESC procedure allows for the direct use of chimeras for experimentation. We show that the tumor leftacteristics of chimeric mice are very similar to those of conventional mice (Fig 2) though the tumor latency can differ depending on the model and level of chimerism (Fig 4C supplementary Fig S2). Typically the level of chimerism is estimated on basis of coat-color contribution although this consistently results in an overestimation of the true chimerism in the various tissues (Fig 2C and D). In our experience 70“100% chimeras give a quite consistent reproducible tumor phenotype when the penetrance is high. In GEMMs with low penetrant phenotypes it is advisable to backcross the chimeras to the parental strain and use the F1 cohort. We feel that a more quantitative analysis on a particular tissue e.g. tail does not provide a substantial advantage above estimating chimerism on the basis of coat color as variations can also be found among different tissues. The chimeric approach is particularly useful for side-by-side comparison of multiple allelic variants of the same gene or for in vivo screening of multiple candidate cancer genes in one GEMM in a semi-high throughput fashion. Direct comparison of tumorigenesis in chimeras that differ only on basis of the expression of one candidate cancer gene will accelerate the identification of true driver genes within a large group of candidate cancer genes for instance Mycl1 in the SCLC model. Furthermore all tumors will have an identical genetic background excluding any variation caused by modifier alleles introduced via breeding. This simplifies subsequent deep sequencing efforts as all tumors can be compared to the same GEMM-ESC derived reference DNA. It is however advisable to use two independent ESC lines as also recombinase-mediated introduction of constructs into the ESCs can give rise to chromosomal changes that could influence the outcome. We realize that in most cases the chimeras will be crossed to the original GEMM to establish stable mouse strains. Indeed a single cross of the chimeras to the original strain will result in F1 mice with or without the introduced construct. These mice can be directly used as experimental and control cohorts respectively (Fig 4D). The feasibility of this approach depends on the efficiency of GLT of the chimeras. Based on two GEMMs we observed that twelve out of fourteen chimeras gave GLT in their first litter and one chimera in the second (supplementary Table S1)."
Lung_Cancer
"Although there is no report about SUV in primary or metastatic lung tumors in dogs a previous human study investigated the role of FDG PET in the differentiation between lung metastasis and multiple primary lung tumors [5] because the FDG uptake reflects metabolic activity depending on the tumor types. Based on the SUV of pulmonary nodules in this case there is possibility that all pulmonary nodules were identical tumors showing similar metabolic activity and one of them was the primary tumor and the others were metastasis. Also the degree of FDG uptake provides important information useful in predicting the patient™s prognosis [17] even though TNM staging is used as a reliable prognostic factor in patients with tumors. For example in clinical stage IA lung adenocarcinoma with high FDG uptake lymph node metastasis in people more frequently occurred than did low FDG uptake [17]. The criterion for FDG uptake SUV 3.3 was proposed to be a poor prognosis in humans [17]. In a similar study a significant correlation was identified between the FDG uptake and the aggressiveness of the adenocarcinoma. That is the mean SUV in aggressive pulmonary adenocarcinoma (4.36 ± 1.94) was reported significantly higher than that of non-aggressive ones (1.53 ± 0.88) [8]. In this dog the maximum value of SUV would have been considered to be high taking into account the same criterion in human. However circumstantial assessment of the pulmonary nodule on the basis of the SUV value was impossible because to authors™ knowledge there has been no previous study related to FDG PET in canine adenocarcinoma. Primary pulmonary tumor tends to develop in the peripheral regions in the caudal lung lobe [12 18]. Thus transthoracic fine needle aspiration or biopsy can be considered in pulmonary adenocarcinoma. A transthoracic biopsy under ultrasonography was performed in this case. However pulmonary adenocarcionoma could not be confirmed histopathologically due to the extent of necrosis. Extensive necrosis was considered as the cause of leukocytosis. This study described the diagnostic features of radiographs ultrasonography CT and PET-CT of primary adenocarcinoma in a dog. In this case the primary pulmonary tumor was tentatively diagnosed with CT and PET-CT by excluding an undetected primary tumor and pulmonary nodules could be determined to be malignancies based on the SUV of PET-CT. Although this report is a single case of pulmonary adenocarcinoma in which CT and PET-CT examinations were applied the procedure of this case can be used as fundamental information for diagnosis of canines with pulmonary adenocarcinoma"
Lung_Cancer
"In current study a semi-Markov model along with two-parametric Weibull and Log-logistic distribution were used for measuring the time-dependency transition probabilities and calculating the direct medical costs LYGs and QALYs gained of the practice presented in the trial [15]. A cost-effectiveness evaluation was performed to analysis the economic impact of maintenance gefitinib therapy for patients with locally advanced/metastatic NSCLC with unknown EGFR mutations. Base case analyses of 1- 3- 6- and 10-year time horizon showed an unfavorable ICER of $184829 $19214 $19328 and $21308 per QALY gained respectively. OSA and PSA all revealed that the model we applied was robust to the results. Monte Carlo simulations of 1000 cases suggested that all ICERs for maintenance gefitinib therapy were higher than the recommended WTP threshold (3—per-capita GDP) of cost-effectiveness guidelines from Word Health anization (WHO). There are 31 province-level administrative units in Chinese mainland the per-capita GDP of which differs significantly. In 2011 for example it ranged from $2495 in Guizhou province to $13392 in Tianjin city [31]. According to the recommended threshold of WHO [25] the WTP threshold of different province-level administrative units extended from $7485 (3—$2495) to $40176 (3—$13392) per QALY gained which exceeded the sensitivity range of the WTP (about $17700 to $26300) obtained from PSA of the current study. Obviously local government could take fully into account covering maintenance gefitinib treatment following first-line platinum-based chemotherapy for locally advanced/metastatic NSCLC with unknown EGFR mutations in accordance with local economic development level. Cost-effective probability for different economic level provinces displayed in could supply available information for local governments when gefitinib is approved by local governments™ finance before it has access to the directory of drugs for national basic medical insurance in China. .0088881.t004 The cost-effective probabilities of gefitinib arm for 31 provinces of Chinese mainland. Region Per-capita GDP ($) WTP (3—Per-capita GDP $) Cost-effective Probability Mainland China 5449.71 16349 0 More affluent regionsa >8767 >26300 1.00 Guangdong 7819 23457 0.932 Liaoning 7795 23385 0.926 Fujian 7344 22032 0.717 Shandong 7273 21819 0.655 Less affluent regionsb <5900 <17700 0 a Consist of Tianjin Shanghai Beijing Jiangsu Zhejiang and Inner Mongolia. b Consist of Jilin Chongqing Hubei Hebei Shanxi Ningxia Heilongjiang Shangxi Xinjiang Hunan Qinghai Henan Hainan Jiangxi Sichuan Guangxi Anhui Tibet Gansu Yuannan and Guizhou. A number of different survival models such as Weibull Exponential Log-logistic Gompertz et al can be used to perform extrapolation according to the observed trial data [32]. It is therefore very vital to choose the justifiable extrapolation approach to ensure the associated results of economic analysis confident to decision makers. In the current study after the deviance information criterion test (reported by Jackson et al [33] to alternative models introduced by Latimer [32] we chose Weibull and Log-logistic for PFS and OS respectively instead of Weibull for extrapolating both PFS and OS curves like the previous study undertaken by Zhu J et al [23]. In addition a hazard ration (HR) of PFS was applied to derive the PFS curve for the gefitinib strategy in the previous study [23]. Latimer however in the resent published paper pointed out that the HR used may cause bias because of the requirement of the assumptions“that is the HR was from a related model and was constant over time [34]. Obviously the bias should be considered especially if the HR impacts the results markedly. Unfortunately the HR of PFS was one of the two most influential parameters on the basis of one-way sensitivity analyses performed by Zhu J et al [23]. In view of the above cases independent parametric models were fitted to both control and experimental groups in our study. Utility of PFS played a great role in the results not only in the resent study [23] but also in the current study. Nafees et al [28] reviewed that all toxicities (diarrhoea rash nausea and vomiting neutropenia fatigue and hair loss) were related to pulling utility down significantly. Of the toxicities rash and diarrhoea were associated with maintenance gefitinib strategy as reported the clinical trial [15]. For higher accuracy we weighted the utility of PFS according to the risks of the rash and diarrhoea which were displayed in . In particularly one point revealed by one-way sensitivity analysis () should be highlighted that the price of gefitinib would be the most significant parameter that could reduce the ICER. With the gefitinib price reduction of 20% discount the ICER decreased to $16731 per QALY gained which is very close to the WTP threshold of $16349 per QALY. Therefore if the price of gefitinib decreases >20% maintenance gefitinib therapy after the standard chemotherapy in patients with locally advanced/metastatic NSCLC may be a cost-effectiveness strategy. There are some limitations in the present study. First using Weibull and Log-logistic distribution to extrapolate the survival curves beyond the time scope of the trial was an unavoidable limitation of this process. There is not enough survival data provided by the short follow-ups of the clinical trial to compare the long-term outcomes estimated by the model. Our results should be updated when long-term survival data are available. Another important limitation is that the utility weight parameters originated from the published literature that may not reflect Chinese patients™ trait. It is an inevitable limitation of the current analysis because utilities data are not yet available for China. Fortunately opinions from Chinese oncologists suggested that quality of life of locally advanced or metastatic NSCLC patients in China should not be of significant difference from abroad patients. Finally because there is no head-to-head clinical trial comparing maintenance gefitinib with other maintenance drugs (eg erlotinib) after the standard chemotherapy of four chemotherapeutic cycles we have not conducted a cost-effectiveness analysis of gefitinib in comparison with other maintenance therapies. Although the current estimates were derived from just one study which is also the only phase III trial compared maintenance gefitinib treatment in patients with locally advanced/metastatic NSCLC according to our literature search we believe that the analysis of our study based on a current Chinese phase III trial and the justifiable extrapolation approach can provide important reference information for decision makers in China. First of all the clinical study itself is a multicentre double-blind randomized controlled-trial (RCT) which represents the best evidence available and is deemed to be the most accepted scientific method of determining the benefit of a drug or a therapeutic procedure. Second the analysis method applied in our study was reliable and widely used in economic evaluations especially in the field of medical and health care. In addition the Log-logistic and two parameters Weibull model matched the survival curves of the clinical trial satisfactorily () which shows that the model we constructed can mirror the effectiveness data of the trial commendably. And then direct medical costs related to each strategy were estimated including maintenance gefitinib therapy treatment of major adverse events routine follow-up treatment for patients without progression follow-up treatment in PS state and terminal-phase cost. Although the costs originated from our previous study [26] the published literature [27] or estimates according to local charges based on expert opinion all of them stemmed from a Chinese health care system perspective as well as in view of patients with advanced NSCLC which echoed the purpose of the current study. Last but not least to reflect substantial uncertainty of the input parameters the sensitivity analyses (including OSA and PSA) were conducted for each key parameter and all sensitivity analyses revealed that the model we applied was robust to the results. In conclusion according to the recommended WTP threshold (3—per-capita GDP) of cost-effectiveness guidelines from WHO maintenance gefitinib therapy after the standard chemotherapy of four chemotherapeutic cycles in locally advanced/metastatic NSCLC patients with unknown EGFR mutations is likely to be not cost-effective for Chinese mainland from the Chinese health care system perspective. Local governments with different economic level however could take fully into account covering maintenance gefitinib treatment. Because for rich regions (the per-capita GDP> $8767) the new strategy seems to be a reasonable option and if the per-capita GDP ranges from $5900 to $8767 the maintenance therapy may be favourable in terms of the different cost-effective probabilities. Decreasing the price of gefitinib the most significant parameter that could reduce the ICER should be considered to as a preferential factor for meeting widely treatment demands in China. Prof. L.B. Peng and J.H. Li are the guarantors for the overall content. The authors greatly thank many clinicians and the data managers who have recorded the initial data diligently of medicines over the years. In particular they thank Ouyang Lihui Wang Siying Zhao Ziying and Qiu Zhenhua for their help in the data collection and valuable discussions and advices. References 1 JemalA BrayF (2011) Center MM Ferlay J Ward E et al (2011) Global cancer statistics. CA Cancer J Clin61: 69“9021296855 2 FathiAT BrahmerJR (2008) Chemotherapy for advanced stage non-small cell lung cancer. Semin Thorac Cardiovasc Surg20: 210“21619038730 3 GovindanR PageN MenszternD ReadW TierneyR et al (2006) Changing epidemiology of small-cell lung cancer in the United States over the last 30 years: analysis of the surveillance epidemiologic and end results database. J Clin Oncol24: 4539“454417008692 4 Nation Comprehensive Cancer Network (2013) Non“small cell lung cancer (version 2.2014). Available: http://www.nccn./professionals/physician_gls/pdf/nscl.pdf Accessed 21 January 2014. 5 AzzoliCG BakerJS TeminS PaoW AliffT et al (2009) American Society of Clinical Oncology Clinical Practice Guideline update on chemotherapy for stage IV non-small-cell lung cancer. J Clin Oncol27: 6251“626619917871 6 D™Addario G Felip E (2009) Non-small-cell lung cancer: ESMO clinical recommendations for diagnosis treatment and follow-up. Ann Oncol (Suppl 4): 68“70. 7 BareschinoMA SchettinoC RossiA MaioneP SaccoPC et al (2011) Treatment of advanced non small cell lung cancer. J Thorac Dis3: 122“13322263075 8 BrodowiczT KrzakowskiM ZwitterM TzekovaV RamlauR et al (2006) Cisplatin and gemcitabine first-line chemotherapy followed by maintenance gemcitabine or best supportive care in advanced non-small cell lung cancer: a phase III trial. Lung Cancer52: 155“16316569462 9 FidiasPM DakhilSR LyssAP LoeschDM WaterhouseDM et al (2009) Phase III study of immediate compared with delayed docetaxel after front-line therapy with gemcitabine plus carboplatin in advanced non-small-cell lung cancer. J Clin Oncol27: 591“59819075278 10 CiuleanuT BrodowiczT ZielinskiC KimJH KrzakowskiM et al (2009) Maintenance pemetrexed plus best supportive care versus placebo plus best supportive care for non-small-cell lung cancer: a randomised double-blind phase 3 study. Lancet374: 1432“144019767093 11 CappuzzoF CiuleanuT StelmakhL CicenasS Szcz©snaA et al (2010) Erlotinib as maintenance treatment in advanced non-small-cell lung cancer: a multicentre randomised placebo-controlled phase 3 study. Lancet Oncol11: 521“52920493771 12 Paz-AresL de MarinisF DediuM ThomasM PujolJL et al (2012) Maintenance therapy with pemetrexed plus best supportive care versus placebo plus best supportive care after induction therapy with pemetrexed plus cisplatin for advanced non-squamous non-small-cell lung cancer (PARAMOUNT): a double-blind phase 3 randomized controlled trial. Lancet Oncol13: 247“25522341744 13 CohenMH JohnsonJR ChattopadhyayS TangS JusticeR et al (2010) Approval summary: erlotinib maintenance therapy of advanced/metastatic non-small cell lung cancer (NSCLC). Oncologist15: 1344“135121148614 14 CohenMH CortazarP JusticeR PazdurR (2010) Approval summary: pemetrexed maintenance therapy of advanced/metastatic nonsquamous non-small cell lung cancer (NSCLC). Oncologist15: 1352“135821148615 15 ZhangL MaS SongX HanB ChengY et al (2012) Gefitinib versus placebo as maintenance therapy in patients with locally advanced or metastatic non-small-cell lung cancer (INFORM; C-TONG 0804): a multicentre double-blind randomised phase 3 trial. Lancet Oncol13: 466“47522512843 16 WalleserS RayJ BischoffH Vergnen¨greA RoseryH et al (2012) Maintenance erlotinib in advanced non-small cell lung cancer: cost-effectiveness in EGFR wild-type across Europe. Clinicoecon Outcomes Res4: 269“27523028234 17 Vergnen¨greA RayJA ChouaidC GrossiF BischoffHG et al (2012) Cross-market cost-effectiveness analysis of erlotinib as first-line maintenance treatment for patients with stable non-small cell lung cancer. Clinicoecon Outcomes Res4: 31“3722347803 18 GreenhalghJ McLeodC BagustA BolandA FleemanN et al (2010) Pemetrexed for the maintenance treatment of locally advanced or metastatic non-small cell lung cancer. Health Technol Assess14: 33“3921047489 19 KleinR WielageR MuehlenbeinC LiepaAM BabineauxS et al (2010) Cost-effectiveness of pemetrexed as first-line maintenance therapy for advanced nonsquamous non-small cell lung cancer. J Thor Oncol5: 1263“1272 20 TsuchiyaT FukudaT FuruiyeM KawabuchiK (2011) Pharmacoeconomic analysis of consolidation therapy with pemetrexed after first-line chemotherapy for non-small cell lung cancer. Lung Cancer74: 521“52921570734 21 Matter-WalstraK JoergerM K¼hnelU SzucsT PestalozziB et al (2012) Cost-Effectiveness of Maintenance Pemetrexed in Patients with Advanced Nonsquamous-Cell Lung Cancer from the Perspective of the Swiss Health Care System. Value Health15: 65“7122264973 22 ZengXH PengLB LiJH ChenGN TanCQ et al (2013) Cost-Effectiveness of Continuation Maintenance Pemetrexed after cisplatin and pemetrexed chemotherapy for Advanced Non-squamous Non-small-cell Lung Cancer: estimates from the Chinese Perspective of Health Care System. Clin Ther35: 54“6523328269 23 ZhuJ LiT WangXH YeM CaiJ et al (2013) Gene-guided Gefitinib switch maintenance therapy for patients with advanced EGFR mutation-positive Non-small cell lung cancer: an economic analysis. BMC Cancer13: 3923360224 24 China Center for Health Economic Research. China Guidelines for Pharmacoeconomic Evaluations (Version 8) [in Chinese] (2010) Available: http://www.cpa..cn/Article/UploadFiles/201011/2010112509052247.pdfAccessed 21 January 2014. 25 WHO. Cost-effectiveness thresholds. Available: http://www.who.int/choice/costs/CER_thresholds/en/ Accessed 21 January 2014. 26 ZengXH KarnonJ WangSY WuB WanXM et al (2012) The cost of treating advanced non-small cell lung cancer: estimates from the Chinese experience. PLoS ONE7: e4832323118985 27 WuB ChenH ShenJ YeM (2011) Cost-effectiveness of adding rh-endostatin to first-line chemotherapy in patients with advanced non-small-cell lung cancer in China. Clin Ther33: 1446“145521992806 28 NafeesB StaffordM GavrielS BhallaS WatkinsJ (2008) Health state utilities for non small cell lung cancer. Health Qual Life Out6: 84 29 National Cancer Institute (2013) SEER Stat Fact Sheets: Lung and Bronchus Cancer. Available: http://seer.cancer.gov/statfacts/html/lungb.html Accessed 21 January 2014. 30 LiuQ WangB KongY ChengKK (2011) China™s primary health-care reform. Lancet377: 2064“206621453962 31 National Bureau of Statistics of China (2012) China statistical yearbook 2012. Available: http://www.stats.gov.cn/english/ Accessed 21 January 2014. 32 Latimer N (2011) NICE DSU Technical Support Document 14: Undertaking survival analysis for economic evaluations alongside clinical trials“extrapolation with patient-level data. Available: http://www.nicedsu..uk Accessed 21 January 2014. 33 JacksonCH SharplesLD ThompsonSG (2010) Survival models in health economic evaluations: Balancing fit and parsimony to improve prediction. Int J Biostat6: 34 34 LatimerNR (2013) Survival analysis for economic evaluations alongside clinical trials“extrapolation with patient-level data: inconsistencies limitations and a practical guide. Med Decis Making33: 743“75423341049 0374236 2771 Cancer Cancer Cancer 0008-543X 1097-0142 24711210 4219619 10.1002/cncr.28683 NIHMS637693 Article Guideline-Concordant Cancer Care and Survival Among American Indian/Alaskan Native Patients Javid Sara H. MD 1 Varghese Thomas K. MD MS 1 Morris Arden M. MD 2 Porter Michael P. MD MS 3 He Hao PhD 1 Buchwald Dedra MD 4 Flum David R. MD MPH 1 for the Collaborative to Improve Native Cancer Outcomes (CINCO) 1Department of Surgery Surgical Outcomes Research Center School of Medicine University of Washington Seattle Washington 2Department of Surgery School of Medicine University of Michigan Ann 3Department of Urology Surgical Outcomes Research Center School of Medicine University of Washington Seattle Washington 4Division of General Internal Medicine School of Medicine University of Washington Seattle Washington Corresponding author: Sara H. Javid MD University of Washington 1959 NE Pacific Street Box 356410 Seattle WA 98195; Fax: (206) 543-8136; [email protected] 27 10 2014 07 4 2014 15 7 2014 04 11 2014 120 14 2183 2190 © 2014 American Cancer Society. 2014 BACKGROUND American Indians/Alaskan Natives (AI/ANs) have the worst 5-year cancer survival of all racial/ethnic groups in the United States. Causes for this disparity are unknown. The authors of this report examined the receipt of cancer treatment among AI/AN patients compared with white patients. METHODS This was a retrospective cohort study of 338204 patients who were diagnosed at age ?65 years with breast colon lung or prostate cancer between 1996 and 2005 in the Surveillance Epidemiology and End Results-Medicare database. Nationally accepted guidelines for surgical and adjuvant therapy and surveillance were selected as metrics of optimal guideline-concordant care. Treatment analyses compared AI/ANs with matched whites. RESULTS Across cancer types AI/ANs were less likely to receive optimal cancer treatment and were less likely to undergo surgery (P ? .025 for all cancers). Adjuvant therapy rates were significantly lower for AI/AN patients with breast cancer (P <.001) and colon cancer (P = .001). Rates of post-treatment surveillance also were lower among AI/ANs and were statistically significantly lower for AI/AN patients with breast cancer (P = .002) and prostate cancer (P <.001). Nonreceipt of optimal cancer treatment was associated with significantly worse survival across cancer types. Disease-specific survival for those who did not undergo surgery was significantly lower for patients with breast cancer (hazard ratio [HR] 0.62) colon cancer (HR 0.74) prostate cancer (HR 0.52) and lung cancer (HR 0.36). Survival rates also were significantly lower for those patients who did not receive adjuvant therapy for breast cancer (HR 0.56) colon cancer (HR 0.59) or prostate cancer (HR 0.81; all 95% confidence intervals were <1.0). "
Lung_Cancer