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Immunotherapy with immune checkpoint inhibitors has radically changed the management of a broad spectrum of tumors.,In contrast, only very limited information is available about the efficacy of these therapies in non-melanoma skin cancers, especially in basal cell carcinoma.,The latter malignancy is often associated with both an impairment of the host immune response and a high mutation burden, suggesting that immune checkpoint inhibitor-based immunotherapy may be effective in the treatment of this tumor.,A 78-year-old woman was diagnosed with a metastatic non-small-cell-lung-cancer.,Following the lack of response to two lines of systemic chemotherapy, she was treated with the anti-PD-1 monoclonal antibody nivolumab, obtaining a prolonged stable disease.,Under nivolumab treatment, the patient developed a basal cell carcinoma of the nose.,The latter was surgically resected.,Immunohistochemical staining of tumor tissue showed a PD-L1 expression < 1% and lack of human leukocyte antigen class I subunit (i.e. heavy and light chain) expression on tumor cells.,In addition, a limited number of T cells (CD3+) was present in the tumor microenvironment, with a higher number of regulatory T cells (Foxp3+) and macrophages (Cd11b+) as compared to a low infiltration of activated cytotoxic T cells (CD8+/ Granzyme B+).,Two months following the surgical removal of the tumor, while still on nivolumab treatment, the patient relapsed with a basal cell carcinoma in the same anatomic site of the previous surgical excision.,The tumor displayed the same pathological characteristics.,Preclinical lines of evidence suggest a potential role of immune checkpoint inhibitors for basal cell carcinoma treatment.,However, limited clinical data is available.,In the patient we have described administration of the immune checkpoint inhibitor nivolumab for the treatment of a responsive non-small cell carcinoma was associated with the development and relapse of a basal cell carcinoma tumor.,This association is likely to reflect the resistance of basal cell carcinoma cells to anti-PD-1 based immunotherapy because of a “cold” tumor microenvironment characterized by lack of human leukocyte antigen class I expression, low PD-L1 expression and high number of immune regulatory cells.
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Patients with locally advanced basal cell carcinoma (laBCC) or metastatic BCC (mBCC), two difficult‐to‐treat populations, have had limited treatment options.,Sonidegib, a hedgehog pathway inhibitor (HPI), was approved in laBCC based on results from the BOLT trial.,To evaluate long‐term efficacy and safety of sonidegib in laBCC and mBCC in the BOLT 18‐ and 30‐month analyses.,BOLT (NCT01327053, ClinicalTrials.gov), a double‐blind phase 2 study, enrolled patients from July 2011 until January 2013.,Eligible HPI‐treatment-naïve patients with laBCC not amenable to curative surgery/radiotherapy or mBCC were randomized 1 : 2 to sonidegib 200 mg (laBCC, n = 66; mBCC, n = 13) or 800 mg (laBCC, n = 128; mBCC, n = 23).,Tumour response was assessed per central and investigator review.,With 30 months of follow‐up, among patients treated with sonidegib 200 mg (approved dose), objective response rates were 56.1% (central) and 71.2% (investigator) in laBCC and 7.7% (central) and 23.1% (investigator) in mBCC.,Tumour responses were durable as follows: median duration of response was 26.1 months (central) and 15.7 months (investigator) in laBCC and 24.0 months (central) and 18.1 months (investigator) in mBCC.,Five patients with laBCC and three with mBCC in the 200‐mg arm died.,Median overall survival was not reached in either population; 2‐year overall survival rates were 93.2% (laBCC) and 69.3% (mBCC).,In laBCC, efficacy was similar regardless of aggressive or non‐aggressive histology.,Sonidegib 200 mg continued to have a better safety profile than 800 mg, with lower rates of grade 3/4 adverse events (43.0% vs.,64.0%) and adverse events leading to discontinuation (30.4% vs.,40.0%).,Sonidegib continued to demonstrate long‐term efficacy and safety in these populations.,These data support the use of sonidegib 200 mg per local treatment guidelines.
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Treatment of BRAF‐mutant melanomas with MAP kinase pathway inhibitors is paradigmatic of the promise of precision cancer therapy but also highlights problems with drug resistance that limit patient benefit.,We use live‐cell imaging, single‐cell analysis, and molecular profiling to show that exposure of tumor cells to RAF/MEK inhibitors elicits a heterogeneous response in which some cells die, some arrest, and the remainder adapt to drug.,Drug‐adapted cells up‐regulate markers of the neural crest (e.g., NGFR), a melanocyte precursor, and grow slowly.,This phenotype is transiently stable, reverting to the drug‐naïve state within 9 days of drug withdrawal.,Transcriptional profiling of cell lines and human tumors implicates a c‐Jun/ECM/FAK/Src cascade in de‐differentiation in about one‐third of cell lines studied; drug‐induced changes in c‐Jun and NGFR levels are also observed in xenograft and human tumors.,Drugs targeting the c‐Jun/ECM/FAK/Src cascade as well as BET bromodomain inhibitors increase the maximum effect (E max) of RAF/MEK kinase inhibitors by promoting cell killing.,Thus, analysis of reversible drug resistance at a single‐cell level identifies signaling pathways and inhibitory drugs missed by assays that focus on cell populations.
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Drug resistance remains a vexing problem in the treatment of cancer patients.,While many studies have focused on cell autonomous mechanisms of drug resistance, we hypothesized that the tumor microenvironment may confer innate resistance to therapy.,Here we developed a co-culture system to systematically assay the ability of 23 stromal cell types to influence the innate resistance of 45 cancer cell lines to 35 anti-cancer drugs.,We found that stroma-mediated resistance is surprisingly common - particularly to targeted agents.,We further characterized the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibition because most of these patients exhibit some degree of innate resistance1-4.,Proteomic analysis showed that stromal secretion of the growth factor hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the MAPK and PI3K/AKT pathways, and immediate resistance to RAF inhibition.,Immunohistochemistry confirmed stromal HGF expression in patients with BRAF-mutant melanoma and a statistically significant correlation between stromal HGF expression and innate resistance to treatment.,Dual inhibition of RAF and MET resulted in reversal of drug resistance, suggesting RAF/MET combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma.,A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines.,More generally, these studies indicate that the systematic dissection of tumor-microenvironment interactions may reveal important mechanisms underlying drug resistance.
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Icaritin (IT) is a flavonoid isolated from Herba Epimedii.,In this study, we evaluated the anti-melanoma activities of IT, and determined its cytotoxic mechanism.,We found that IT exerted cytotoxicity to melanoma cells.,Furthermore, IT induced melanoma cell apoptosis, which was accompanied with PARP cleavage.,Mechanistically, IT suppressed p-STAT3 (tyr705) level in parallel with increases of p-STAT3 (ser727), p-ERK and p-AKT.,IT significantly inhibited STAT3 nuclear translocation and reduced the levels of STAT3 -targeted genes.,IT also inhibited IGF-1-induced STAT3 activation through down-regulation of total IGF-1R level.,No dramatic changes in IGF-1R mRNA levels were observed in IT-treated cells, suggesting that IT acted primarily at a post-transcriptional level.,Using molecular docking analysis, IT was identified as a novel fatty acid synthase (FASN) inhibitor.,We found that IT reduced the level of total IGF-1R via FASN inhibition.,In summary, we reported that IT exerted anti-melanoma activities, and these effects were partially due to inhibition of FASN/IGF-1R/STAT3 signaling.
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Metastatic melanoma is the most aggressive form of skin cancer with a median overall survival of less than one year.,Advancements in our understanding of how melanoma evades the immune system as well as the recognition that melanoma is a molecularly heterogeneous disease have led to major improvements in the treatment of patients with metastatic melanoma.,In 2011, the US Food and Drug Administration (FDA) approved two novel therapies for advanced melanoma: a BRAF inhibitor, vemurafenib, and an immune stimulatory agent, ipilimumab.,The success of these agents has injected excitement and hope into patients and clinicians and, while these therapies have their limitations, they will likely provide excellent building blocks for the next generation of therapies.,In this review we will discuss the advantages and limitations of the two new approved agents, current clinical trials designed to overcome these limitations, and future clinical trials that we feel hold the most promise.
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Patients with advanced metastatic melanoma have poor prognosis and the genetics underlying its pathogenesis are poorly understood.,High throughput sequencing has allowed comprehensive discovery of somatic mutations in cancer samples.,Here, upon analysis of our whole-genome and whole-exome sequencing data of 29 melanoma samples we identified several genes that harbor recurrent non-synonymous mutations.,These included MAP3K5, which in a prevalence screen of 288 melanomas was found to harbor a R256C substitution in 5 cases.,All MAP3K5 mutated samples were wild-type for BRAF, suggesting a mutual exclusivity for these mutations.,Functional analysis of the MAP3K5 R256C mutation revealed attenuation of MKK4 activation through increased binding of the inhibitory protein thioredoxin (TXN/TRX-1/Trx); resulting in increased proliferation and anchorage-independent growth of melanoma cells.,This mutation represents a potential target for the design of new therapies to treat melanoma.
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We sequenced 8 melanoma exomes to identify novel somatic mutations in metastatic melanoma.,Focusing on the MAP3K family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9.,Structural modelling predicts that mutations in the kinase domain may affect the activity and regulation of MAP3K5/9 protein kinases.,The position of the mutations and loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely inactivating.,In vitro kinase assay shows reduction in kinase activity in MAP3K5 I780F and MAP3K9 W333X mutants.,Overexpression of MAP3K5 or MAP3K9 mutant in HEK293T cells reduces phosphorylation of downstream MAP kinases.,Attenuation of MAP3K9 function in melanoma cells using siRNA leads to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.
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Uveal melanoma is the most common primary intraocular malignancy.,A vast majority of metastasizing tumors have mutations in the BAP1 gene.,Here, we investigate the spatiotemporal timing of these mutations.,The size of 177 uveal melanomas and 8.3 million individual tumor cells was measured.,BAP1 sequencing results and BAP1 IHC were available and for 76 (43%) and 101 (57%) of these, respectively.,Tumors with a BAP1 mutation had significantly larger volume (2109 vs. 1552 mm3, p = 0.025).,Similarly, tumor cells with loss of BAP1 protein expression had significantly larger volume (2657 vs. 1593 μm3, p = 0.027).,Using observations of the time elapsed between mitoses, the BAP1 mutation was calculated to occur when the primary tumor had a size of a few malignant cells to 6 mm3, 0.5 to 4.6 years after tumor initiation and at least 9 years before diagnosis.,We conclude that BAP1 mutations occur early in the growth of uveal melanoma, well before the average tumor is diagnosed.,Its timing coincides with the seeding of micrometastases.
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Cancer is thought to arise through the accumulation of genomic aberrations evolving under Darwinian selection.,However, it remains unclear when the aberrations associated with metastasis emerge during tumor evolution.,Uveal melanoma (UM) is the most common primary eye cancer and frequently leads to metastatic death, which is strongly linked to BAP1 mutations.,Accordingly, UM is ideally suited for studying the clonal evolution of metastatic competence.,Here we analyze sequencing data from 151 primary UM samples using a customized bioinformatic pipeline, to improve detection of BAP1 mutations and infer the clonal relationships among genomic aberrations.,Strikingly, we find BAP1 mutations and other canonical genomic aberrations usually arise in an early punctuated burst, followed by neutral evolution extending to the time of clinical detection.,This implies that the metastatic proclivity of UM is “set in stone” early in tumor evolution and may explain why advances in primary treatment have not improved survival.,Uveal melanoma (UM), the most common primary eye cancer, is strongly linked to mutations in the tumor suppressor BAP1.,Here, the authors analyze 151 primary UM samples to find that BAP1 and other canonical genomic aberrations arise in an early punctuated burst followed by neutral tumor evolution.
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In the KEYNOTE-022 study, pembrolizumab with dabrafenib and trametinib (triplet) improved progression-free survival (PFS) versus placebo with dabrafenib and trametinib (doublet) without reaching statistical significance.,Mature results on PFS, duration of response (DOR), and overall survival (OS) are reported.,The double-blind, phase 2 part of KEYNOTE-022 enrolled patients with previously untreated BRAF V600E/K-mutated advanced melanoma from 22 sites in seven countries.,Patients were randomly assigned 1:1 to intravenous pembrolizumab (200 mg every 3 weeks) or placebo plus dabrafenib (150 mg orally two times per day) and trametinib (2 mg orally one time a day).,Primary endpoint was PFS.,Secondary endpoints were objective response rate, DOR, and OS.,Efficacy was assessed in the intention-to-treat population, and safety was assessed in all patients who received at least one dose of study drug.,This analysis was not specified in the protocol.,Between November 30, 2015 and April 24, 2017, 120 patients were randomly assigned to triplet (n=60) or doublet (n=60) therapy.,With 36.6 months of follow-up, median PFS was 16.9 months (95% CI 11.3 to 27.9) with triplet and 10.7 months (95% CI 7.2 to 16.8) with doublet (HR 0.53; 95% CI 0.34 to 0.83).,With triplet and doublet, respectively, PFS at 24 months was 41.0% (95% CI 27.4% to 54.2%) and 16.3% (95% CI 8.1% to 27.1%); median DOR was 25.1 months (95% CI 14.1 to not reached) and 12.1 months (95% CI 6.0 to 15.7), respectively.,Median OS was not reached with triplet and was 26.3 months with doublet (HR 0.64; 95% CI 0.38 to 1.06).,With triplet and doublet, respectively, OS at 24 months was 63.0% (95% CI 49.4% to 73.9%) and 51.7% (95% CI 38.4% to 63.4%).,Grade 3-5 treatment-related adverse events (TRAEs) occurred in 35 patients (58%, including one death) receiving triplet and 15 patients (25%) receiving doublet.,In BRAF V600E/K-mutant advanced melanoma, pembrolizumab plus dabrafenib and trametinib substantially improved PFS, DOR, and OS with a higher incidence of TRAEs.,Interpretation of these results is limited by the post hoc nature of the analysis.
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To increase understanding of the genomic landscape of acral melanoma, a rare form of melanoma occurring on palms, soles or nail beds, whole genome sequencing of 87 tumors with matching transcriptome sequencing for 63 tumors was performed.,Here we report that mutational signature analysis reveals a subset of tumors, mostly subungual, with an ultraviolet radiation signature.,Significantly mutated genes are BRAF, NRAS, NF1, NOTCH2, PTEN and TYRP1.,Mutations and amplification of KIT are also common.,Structural rearrangement and copy number signatures show that whole genome duplication, aneuploidy and complex rearrangements are common.,Complex rearrangements occur recurrently and are associated with amplification of TERT, CDK4, MDM2, CCND1, PAK1 and GAB2, indicating potential therapeutic options.,Acral melanoma occurs on the soles of the feet, palms of the hands and in nail beds.,Here, the authors reports the genomic landscape of 87 acral melanomas and find that some tumors harbor a UV signature and that the tumors are diverse at the levels of mutational signatures, structural aberrations and copy number signatures.
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Around 50% of primary melanomas harbour BRAF mutations, but their prognostic impact has not been clear.,Recently, a BRAF-V600E mutation-specific antibody has become available for immunohistochemistry.,Here, we investigated for the first time the prognostic impact of BRAF-V600E protein expression in primary melanoma.,In a patient series of 248 nodular melanomas, BRAF-V600E and total BRAF expression were assessed by immunohistochemistry using tissue microarray sections of paraffin-embedded archival tissue.,Mutation status was assessed by real-time PCR in cases with sufficient tumour tissue (n=191).,Positive BRAF-V600E expression was present in 86 (35%) of the cases, and was significantly associated with increased tumour thickness, presence of tumour ulceration and reduced survival.,Further, BRAF-V600E expression was an independent prognostic factor by multivariate analysis, whereas BRAF mutation status was not significant.,There was 88% concordance between BRAF-V600E expression and mutation status.,Our findings indicate that BRAF-V600E expression is a novel prognostic marker in primary melanoma.
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The rationale for using small molecule inhibitors of oncogenic proteins as cancer therapies depends, at least in part, on the assumption that metastatic tumors are primarily clonal with respect to mutant oncogene.,With the emergence of BRAFV600E as a therapeutic target, we investigated intra- and inter-tumor heterogeneity in melanoma using detection of the BRAFV600E mutation as a marker of clonality.,BRAF mutant-specific PCR (MS-PCR) and conventional sequencing were performed on 112 tumors from 73 patients, including patients with matched primary and metastatic specimens (n = 18).,Nineteen patients had tissues available from multiple metastatic sites.,Mutations were detected in 36/112 (32%) melanomas using conventional sequencing, and 85/112 (76%) using MS-PCR.,The better sensitivity of the MS-PCR to detect the mutant BRAFV600E allele was not due to the presence of contaminating normal tissue, suggesting that the tumor was comprised of subclones of differing BRAF genotypes.,To determine if tumor subclones were present in individual primary melanomas, we performed laser microdissection and mutation detection via sequencing and BRAFV600E-specific SNaPshot analysis in 9 cases.,Six of these cases demonstrated differing proportions of BRAFV600Eand BRAFwild-type cells in distinct microdissected regions within individual tumors.,Additional analyses of multiple metastatic samples from individual patients using the highly sensitive MS-PCR without microdissection revealed that 5/19 (26%) patients had metastases that were discordant for the BRAFV600E mutation.,In conclusion, we used highly sensitive BRAF mutation detection methods and observed substantial evidence for heterogeneity of the BRAFV600E mutation within individual melanoma tumor specimens, and among multiple specimens from individual patients.,Given the varied clinical responses of patients to BRAF inhibitor therapy, these data suggest that additional studies to determine possible associations between clinical outcomes and intra- and inter-tumor heterogeneity could prove fruitful.
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Inhibitors of RAF inhibit the MAPK pathway that plays an important role in the development and progression of those melanoma carrying the V600E BRAF mutation, but there’s a subset of such patients who do not respond to the therapy.,Various mechanisms of drug resistance have been proposed which include the clonal heterogeneity of the tumor.,We have studied a population of nodular melanoma to investigate the intratumor and intertumor heterogeneity by Laser Capture Microdissection (LCM) analysis.,Our results showed that BRAF and NRAS mutations were detected in 47% and 33% of nodular melanoma, respectively, and that there is a discrepancy in mutational pattern of tumoral sample because in the 36% of patients a different mutation, in at least 1 area of the tumor, was found by LCM analysis, giving evidence of the presence of different clonal cells populations.,Moreover, we found that mutations in BRAF and NRAS are not mutually exclusive because they were simultaneously present in the same tumor specimens and we observed that when the 2 different mutations were present one is a high-frequency mutation and the other is a low-frequency mutation.,This was more evident in lymphonodal metastasis that resulted from wild type to mutational analysis, but showed different mutations following LCM analysis.,Therefore, we believed that, when primary tumoral sample results negative to mutational analysis, if it is possible, metastases should be investigated to verify the presence of mutations.,Generally, it should be searched for other mutations, in addition to BRAF V600E, so as to better understand the mechanism of drug resistance.
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Globally, malignant melanoma shows a steady increase in the incidence among cancer diseases.,Malignant melanoma represents a cancer type where currently no biomarker or diagnostics is available to identify disease stage, progression of disease or personalized medicine treatment.,The aim of this study was to assess the tissue expression of alpha-synuclein, a protein implicated in several disease processes, in metastatic tissues from malignant melanoma patients.,A targeted Selected Reaction Monitoring (SRM) assay was developed and utilized together with stable isotope labeling for the relative quantification of two target peptides of alpha-synuclein.,Analysis of alpha-synuclein protein was then performed in ten metastatic tissue samples from the Lund Melanoma Biobank.,The calibration curve using peak area ratio (heavy/light) versus concentration ratios showed linear regression over three orders of magnitude, for both of the selected target peptide sequences.,In support of the measurements of specific protein expression levels, we also observed significant correlation between the protein and mRNA levels of alpha-synuclein in these tissues.,Investigating levels of tissue alpha-synuclein may add novel aspect to biomarker development in melanoma, help to understand disease mechanisms and ultimately contribute to discriminate melanoma patients with different prognosis.
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Germline CDKN2A mutations occur in 40 % of 3-or-more case melanoma families while mutations of CDK4, BAP1, and genes involved in telomere function (ACD, TERF2IP,POT1), have also been implicated in melanomagenesis.,Mutation of the promoter of the telomerase reverse transcriptase (TERT) gene (c.,−57 T>G variant) has been reported in one family.,We tested for the TERT promoter variant in 675 multicase families wild-type for the known high penetrance familial melanoma genes, 1863 UK population-based melanoma cases and 529 controls.,Germline lymphocyte telomere length was estimated in carriers.,The c.,−57 T>G TERT promoter variant was identified in one 7-case family with multiple primaries and early age of onset (earliest, 15 years) but not among population cases or controls.,One family member had multiple primary melanomas, basal cell carcinomas and a bladder tumour.,The blood leukocyte telomere length of a carrier was similar to wild-type cases.,We provide evidence confirming that a rare promoter variant of TERT (c.,−57 T>G) is associated with high penetrance, early onset melanoma and potentially other cancers, and explains <1 % of UK melanoma multicase families.,The identification of POT1 and TERT germline mutations highlights the importance of telomere integrity in melanoma biology.,The online version of this article (doi:10.1007/s10689-015-9841-9) contains supplementary material, which is available to authorized users.
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Thirteen common susceptibility loci have been reproducibly associated with cutaneous malignant melanoma (CMM).,We report the results of an international two-stage meta-analysis of 11 genome-wide association studies (GWAS, five unpublished) of CMM and Stage two datasets, totaling 15,990 cases and 26,409 controls.,Five loci not previously associated with CMM risk reached genome-wide significance (P < 5×10−8) as did two previously-reported but un-replicated loci and all thirteen established loci.,Novel SNPs fall within putative melanocyte regulatory elements, and bioinformatic and eQTL data highlight candidate genes including one involved in telomere biology.
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Cutaneous melanoma (CM) is a life-threatening form of skin cancer.,Prognostic biomarkers can reliably stratify patients at initial melanoma diagnosis according to risk, and may inform clinical decisions.,Here, we performed a retrospective, cohort-based study analyzing genome-wide DNA methylation of 461 patients with CM from the TCGA database.,Cox regression analyses were conducted to establish a four-DNA methylation signature that was significantly associated with the overall survival (OS) of patients with CM, and that was validated in an independent cohort.,Corresponding Kaplan-Meier analysis displayed a distinct separation in OS.,The ROC analysis confirmed that the predictive signature performed well.,Notably, this signature exhibited much higher predictive accuracy in comparison with known biomarkers.,This signature was significantly correlated with immune checkpoint blockade (ICB) immunotherapy-related signatures, and may have potential as a guide for measures of responsiveness to ICB immunotherapy.
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Immunotherapy is among the most rapidly evolving treatment strategies in oncology.,The therapeutic potential of immune-checkpoint inhibitors is exemplified by the recent hail of Food and Drug Administration (FDA) approvals for their use in various malignancies.,Continued efforts to enhance outcomes with immunotherapy agents have led to the formulation of advanced treatment strategies.,Recent evidence from pre-clinical studies evaluating immune-checkpoint inhibitors in various cancer cell-lines has suggested that combinatorial approaches may have superior survival outcomes compared to single-agent immunotherapy regimens.,Preliminary trials assessing combination therapy with anti-PD-1/PD-L1 plus anti-CTLA-4 immune-checkpoint inhibitors have documented considerable advantages in survival indices over single-agent immunotherapy.,The therapeutic potential of combinatorial approaches is highlighted by the recent FDA approval of nivolumab plus ipilimumab for patients with advanced melanoma.,Presently, dual-immune checkpoint inhibition with anti-programmed death receptor-1/programmed cell death receptor- ligand-1 (anti-PD-1/PD-L1) plus anti-cytotoxic T lymphocyte associated antigen-4 (anti-CTLA-4) monoclonal antibodies (MoAbs) is being evaluated for a wide range of tumor histologies.,Furthermore, several ongoing clinical trials are investigating combination checkpoint inhibition in association with traditional treatment modalities such as chemotherapy, surgery, and radiation.,In this review, we summarize the current landscape of combination therapy with anti-PD-1/PD-L1 plus anti-CTLA-4 MoAbs for patients with melanoma and non-small cell lung cancer (NSCLC).,We present a synopsis of the prospects for expanding the indications of dual immune-checkpoint inhibition therapy to a more diverse set of tumor histologies.
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Germline mutations in BAP1 have been associated with BAP1-Tumor Predisposition Syndrome (BAP1-TPDS), a predisposition to multiple tumors within a family that includes uveal melanoma (UM), cutaneous melanoma, malignant mesothelioma and renal cell carcinoma.,Alternatively, somatic mutations in BAP1 in UM have been associated with high risk for metastasis.,In this study, we compare the risk of metastasis in UM that carry germline versus somatic BAP1 mutations and mutation-negative tumors.,DNA extracted from 142 UM and matched blood samples was sequenced using Sanger or next generation sequencing to identify BAP1 gene mutations.,Eleven of 142 UM (8%) carried germline BAP1 mutations, 43 (30%) had somatic mutations, and 88 (62%) were mutation-negative.,All BAP1 mutations identified in blood samples were also present in the matched UM.,There were 52 unique mutations in 54 tumors.,All were pathogenic or likely pathogenic.,A comparison of tumors carrying somatic vs. germline mutations, or no mutations, showed a higher frequency of metastasis in tumors carrying somatic mutations: 74% vs.,36%, P=0.03 and 74% vs.,26% P<0.001, respectively.,Tumors with a somatic mutation compared to mutation-negative had an older age of diagnosis of (61.8 vs.,52.2 years, P=0.002), and shorter time to metastasis (16 vs. 26 months, P=0.04).,Kaplan-Meier analysis further showed that tumors with somatic (vs. germline) mutations demonstrated a greater metastatic risk (P=0.03).,Cox multivariate analysis showed in addition to chromosome-3 monosomy and larger tumor diameter, the presence of BAP1 somatic, but not germline mutations, was significantly associated with risk of metastasis(P=0.02).,Personal or family history of BAP1-TPDS was available for 79 of the cases.,All eight cases with germline mutations reported a history of BAP1-TPDS, which was significantly greater than what was observed in cases with somatic mutations (10 of 23, P=0.009) or mutation-negative cases (11 of 48, P<0.001).,Defining germline vs. somatic nature of BAP1 mutations in UM can inform the individual about both the risk of metastasis, and the time to metastasis, which are critically important outcomes for the individual.,This information can also change the cascade screening and surveillance of family members.,The online version of this article (10.1186/s12885-018-5079-x) contains supplementary material, which is available to authorized users.
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Common acquired melanocytic nevi are benign neoplasms that are composed of small uniform melanocytes and typically present as flat or slightly elevated, pigmented lesions on the skin.,We describe two families with a new autosomal dominant syndrome characterized by multiple skin-colored, elevated melanocytic tumors.,In contrast to common acquired nevi, the melanocytic neoplasms in affected family members ranged histopathologically from epithelioid nevi to atypical melanocytic proliferations that showed overlapping features with melanoma.,Some affected patients developed uveal or cutaneous melanomas.,Segregating with this phenotype, we found inactivating germline mutations of the BAP1 gene.,The majority of melanocytic neoplasms lost the remaining wild-type allele of BAP1 by various somatic alterations.,In addition, we found BAP1 mutations in a subset of sporadic melanocytic neoplasms showing histologic similarities to the familial tumors.,These findings suggest that loss of BAP1 is associated with a clinically and morphologically distinct type of melanocytic neoplasm.
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The approval of vemurafenib in the US 2011 and in Europe 2012 improved the therapy of not resectable or metastatic melanoma.,Patients carrying a substitution of valine to glutamic acid at codon 600 (p.V600E) or a substitution of valine to leucine (p.V600K) in BRAF show complete or partial response.,Therefore, the precise identification of the underlying somatic mutations is essential.,Herein, we evaluate the sensitivity, specificity and feasibility of six different methods for the detection of BRAF mutations.,Samples harboring p.V600E mutations as well as rare mutations in BRAF exon 15 were compared to wildtype samples.,DNA was extracted from formalin-fixed paraffin-embedded tissues by manual micro-dissection and automated extraction.,BRAF mutational analysis was carried out by high resolution melting (HRM) analysis, pyrosequencing, allele specific PCR, next generation sequencing (NGS) and immunohistochemistry (IHC).,All mutations were independently reassessed by Sanger sequencing.,Due to the limited tumor tissue available different numbers of samples were analyzed with each method (82, 72, 60, 72, 49 and 82 respectively).,There was no difference in sensitivity between the HRM analysis and Sanger sequencing (98%).,All mutations down to 6.6% allele frequency could be detected with 100% specificity.,In contrast, pyrosequencing detected 100% of the mutations down to 5% allele frequency but exhibited only 90% specificity.,The allele specific PCR failed to detect 16.3% of the mutations eligible for therapy with vemurafenib.,NGS could analyze 100% of the cases with 100% specificity but exhibited 97.5% sensitivity.,IHC showed once cross-reactivity with p.V600R but was a good amendment to HRM.,Therefore, at present, a combination of HRM and IHC is recommended to increase sensitivity and specificity for routine diagnostic to fulfill the European requirements concerning vemurafenib therapy of melanoma patients.
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BRAF inhibitors have demonstrated improvement of overall survival in patients with metastatic melanoma and BRAFV600 mutations.,In order to evaluate BRAF tumor heterogeneity between primary and metastatic site, we have evaluated the performance of immunohistochemistry (IHC) with an anti-BRAFV600E antibody in both localization by comparison with high resolution melting analysis followed by Sanger sequencing in a parallel blinded study.,A total of 230 samples distributed as primary melanoma (n = 88) and different types of metastatic samples (n = 142) were studied in 99 patients with advanced or metastatic melanoma (stage III or IV).,The prevalence of each BRAF mutation was c.1799T>A, BRAFV600E (45.2%), c.1799_1800TG>AA, BRAFV600E2 (3.0%), c.1798_1799GT>AA, BRAFV600K (3.0%), c.1801 A>G, BRAFK601E (1.3%), c.1789_1790CT>TC, BRAFL597S (0.4%), c.1780G>A, BRAFD594N (0.9%) respectively.,IHC was positive in 109/112 samples harboring BRAFV600E/E2 mutations and negative in other cases.,The cytoplasmic staining was either strongly positive in tumor cells of BRAFV600E mutated cases.,It appeared strong brown, different from the vesicular grey cytoplasmic pigmentation of melanophages.,Concordance between the two techniques was 96.4%.,Sensitivity of IHC for detecting the BRAFV600E/E2 mutations was 97.3%, while specificity was 100%.,Both our IHC and molecular study demonstrated homogeneity between primary and metastatic sites for BRAF status in melanoma.,This study also provides evidence that IHC may be a cost-effective first-line method for BRAFV600E detection.,Thereafter, molecular techniques should be used in negative, ambiguous or non-contributive cases.
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Talimogene laherparepvec is an oncolytic immunotherapy approved in the US, Europe, Australia and Switzerland.,We report the final planned analysis of OPTiM, a randomized open-label phase III trial in patients with unresectable stage IIIB-IVM1c melanoma.,Patients were randomized 2:1 to receive intratumoral talimogene laherparepvec or subcutaneous recombinant GM-CSF.,In addition to overall survival (OS), durable response rate (DRR), objective response rate (ORR), complete responses (CR), and safety are also reported.,All final analyses are considered to be descriptive and treatment responses were assessed by the investigators.,Of 436 patients in the intent-to-treat population, 295 were allocated to talimogene laherparepvec and 141 to GM-CSF.,Median follow-up in the final OS analysis was 49 months.,Median OS was 23.3 months (95% confidence interval [CI], 19.5-29.6) and 18.9 months (95% CI, 16.0-23.7) in the talimogene laherparepvec and GM-CSF arms, respectively (unstratified hazard ratio, 0.79; 95% CI, 0.62-1.00; p = 0.0494 [descriptive]).,DRR was 19.0 and 1.4% (unadjusted odds ratio, 16.6; 95% CI, 4.0-69.2; p < 0.0001); ORR was 31.5 and 6.4%.,Fifty (16.9%) and 1 (0.7%) patient in the talimogene laherparepvec and GM-CSF arms, respectively, achieved CR.,In talimogene laherparepvec-treated patients, median time to CR was 8.6 months; median CR duration was not reached.,Among patients with a CR, 88.5% were estimated to survive at a 5-year landmark analysis.,Talimogene laherparepvec efficacy was more pronounced in stage IIIB-IVM1a melanoma as already described in the primary analysis.,The safety reporting was consistent with the primary OPTiM analysis.,In this final planned OPTiM analysis, talimogene laherparepvec continued to result in improved longer-term efficacy versus GM-CSF and remained well tolerated.,The final analysis also confirms that talimogene laherparepvec was associated with durable CRs that were associated with prolonged survival.,ClinicalTrials.gov identifier: NCT00769704.,The online version of this article (10.1186/s40425-019-0623-z) contains supplementary material, which is available to authorized users.
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This international, multicenter, single-arm trial assessed efficacy and safety of intralesional rose bengal (PV-10) in 80 patients with refractory cutaneous or subcutaneous metastatic melanoma.,Sixty-two stage III and 18 stage IV melanoma patients with disease refractory to a median of six prior interventions received intralesional PV-10 into up to 20 cutaneous and subcutaneous lesions up to four times over a 16-week period and were followed for 52 weeks.,Objectives were to determine best overall response rate in injected target lesions and uninjected bystander lesions, assess durability of response, and characterize adverse events.,For target lesions, the best overall response rate was 51 %, and the complete response rate was 26 %.,Median time to response was 1.9 months, and median duration of response was 4.0 months, with 8 % of patients having no evidence of disease after 52 weeks.,Response was dependent on untreated disease burden, with complete response achieved in 50 % of patients receiving PV-10 to all of their disease.,Response of target lesions correlated with bystander lesion regression and the occurrence of locoregional blistering.,Adverse events were predominantly mild to moderate and locoregional to the treatment site, with no treatment-associated grade 4 or 5 adverse events.,Intralesional PV-10 yielded durable local control with high rates of complete response.,Toxicity was confined predominantly to the injection site.,Cutaneous bystander tumor regression is consistent with an immunologic response secondary to ablation.,This intralesional approach for local disease control could be complementary to current and investigational treatments for melanoma.
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Congenital melanocytic naevi (CMN) are a known risk factor for melanoma, with the greatest risk currently thought to be in childhood.,There has been controversy over the years about the incidence of melanoma, and therefore over the clinical management of CMN, due partly to the difficulties of histological diagnosis and partly to publishing bias towards cases of malignancy.,Large cohort studies have demonstrated that melanoma risk in childhood is related to the severity of the congenital phenotype.,New understanding of the genetics of CMN offers the possibility of improvement in diagnosis of melanoma, identification of those at highest risk, and new treatment options.,We review the world literature and our centre's experience over the last 25 years, including the molecular characteristics of melanoma in these patients and new melanoma incidence and outcome data from our prospective cohort.,Management strategies are proposed for presentation of suspected melanoma of the skin and the central nervous system in patients with CMN, including use of oral mitogen‐activated protein kinase kinase inhibitors in NRAS‐mutated tumours.,What's already known about this topic?,Multiple congenital melanocytic naevi (CMN) are the greatest risk factor for paediatric melanoma.Different clinical phenotypes have different risks of malignancy; however, the overall absolute risk for all types of CMN taken together is low.CMN can develop proliferative nodules that can cause diagnostic uncertainty and lead to repeated resections.Histology in patients with CMN is difficult and often requires specialist review.Melanoma in CMN is highly aggressive.,Multiple congenital melanocytic naevi (CMN) are the greatest risk factor for paediatric melanoma.,Different clinical phenotypes have different risks of malignancy; however, the overall absolute risk for all types of CMN taken together is low.,CMN can develop proliferative nodules that can cause diagnostic uncertainty and lead to repeated resections.,Histology in patients with CMN is difficult and often requires specialist review.,Melanoma in CMN is highly aggressive.,What does this study add?,In our prospective cohort, the strongest statistical risk factor for all‐site melanoma in childhood is an abnormal screening MRI of the central nervous system (CNS) in the first months of life, and in this group the incidence is 12%.,Where melanoma does arise in children with multiple CMN, a primary in the CNS is at least as common as in the skin.CNS melanoma currently has 100% mortality, but oral mitogen‐activated protein kinase kinase inhibition in NRAS‐mutation mosaic patients may improve symptom control.Management strategies are proposed for the presentation of a possible malignancy in the skin or CNS.,In our prospective cohort, the strongest statistical risk factor for all‐site melanoma in childhood is an abnormal screening MRI of the central nervous system (CNS) in the first months of life, and in this group the incidence is 12%.,Where melanoma does arise in children with multiple CMN, a primary in the CNS is at least as common as in the skin.,CNS melanoma currently has 100% mortality, but oral mitogen‐activated protein kinase kinase inhibition in NRAS‐mutation mosaic patients may improve symptom control.,Management strategies are proposed for the presentation of a possible malignancy in the skin or CNS.
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Common acquired melanocytic nevi are benign neoplasms that are composed of small uniform melanocytes and typically present as flat or slightly elevated, pigmented lesions on the skin.,We describe two families with a new autosomal dominant syndrome characterized by multiple skin-colored, elevated melanocytic tumors.,In contrast to common acquired nevi, the melanocytic neoplasms in affected family members ranged histopathologically from epithelioid nevi to atypical melanocytic proliferations that showed overlapping features with melanoma.,Some affected patients developed uveal or cutaneous melanomas.,Segregating with this phenotype, we found inactivating germline mutations of the BAP1 gene.,The majority of melanocytic neoplasms lost the remaining wild-type allele of BAP1 by various somatic alterations.,In addition, we found BAP1 mutations in a subset of sporadic melanocytic neoplasms showing histologic similarities to the familial tumors.,These findings suggest that loss of BAP1 is associated with a clinically and morphologically distinct type of melanocytic neoplasm.
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Metastatic melanoma (MM) is a highly aggressive cancer with a median overall survival of 6-9 months, notwithstanding the numerous efforts in development of new therapeutic approaches.,To this aim we tested the clinical applicability of the Ion Torrent Personal Genome Machine to simultaneously screen MM patients in order to individuate new or already known SNPs and mutations able to predict the duration of response to BRAF inhibitors.,An Ampliseq Custom Panel, including 11 crucial full length genes involved in melanoma carcinogenesis and therapy response pathways, was created and used to analyze 25 MM patients.,We reported BRAFV600 and NRASQ61 mutations in 68% and 24% of samples, respectively.,Moreover, we more frequently identified the following alterations related to BRAF status: PIK3CAI391M (44%) and KITD737N (36%) mutations, CTLA4T17A (52%), MC1RV60L (32%) and MITFS473A (60%) polymorphisms.,Considering the progression free survival (PFS), statistical analyses showed that BRAFV600 patients without any of these more frequent alterations had a higher median PFS.,Protein structure changes seem to be due to these variants by in silico analysis.,In conclusion, a Next-Generation Sequencing approach with custom panel may provide new information to evaluate tumor-specific therapeutic susceptibility and individual prognosis to improve the care of MM patients.
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Supplemental Digital Content is available in the text,Treatment of BRAFV600E-mutant melanoma by small molecule inhibitors that target BRAF or MEK kinases is increasingly used in clinical practice and significantly improve patient outcome.,However, patients eventually become resistant and therapeutic improvement is required.,Molecular diversity within individual tumors (intratumor heterogeneity) and between tumors within a single patient (intrapatient heterogeneity) poses a significant challenge to precision medicine.,Using immunohistochemistry, we determined the extent of BRAFV600E intratumor and intrapatient heterogeneity and the influence of morphological heterogeneity in a large series of 171 melanomas of 81 patients.,The BRAFV600E mutation rate found in our melanoma series is 44%, with none of 22 (0%) melanoma in situ, 23 of 56 (41%) primary tumors, 28 of 59 (48%) regional metastases, and 24 of 34 (71%) distant metastases harboring the mutation.,In general, a diffuse homogeneous immunostaining was seen, even in tumors consisting of more than one cell type, that is, epithelioid, spindle, and/or small cell types.,Nevertheless, BRAFV600E-mutant melanomas more often had a purely epithelioid cell population (P = 0.063), that is more evident among distant metastases (P = 0.014).,Only two of 75 (3%) mutated specimens (one primary and one metastasis) displayed heterogeneous BRAFV600E expression.,The primary tumor was also morphologically heterogeneous and exclusively displayed BRAFV600E in the epithelioid component, confirming an association between BRAFV600E and epithelioid cells.,Twenty-eight of 30 patients (93%) had concordant BRAF mutation status between their tumors.,Taken together, BRAFV600E intratumor and intrapatient heterogeneity in melanoma is diminutive, nevertheless, the identified exceptions will have important implications for the clinical management of this disease.
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The effect of apatinib on the formation of vasculogenic mimicry (VM) was studied in a malignant melanoma cell line.,MUM-2B cells cultured in three-dimensional Matrigel were treated with varying concentrations (0, 0.01, 0.05, 0.1, 0.5 μmol/L) of apatinib to test its effect on VM in vitro, followed by MTT proliferation and transwell invasion assays to determine the effect of apatinib on cell proliferation and invasion of MUM-2B cells.,In vivo, we used a melanoma cancer model to test the effect of short-term apatinib (100, 200, 300 mg/kg) treatment on VM.,Western blotting, immunohistochemistry staining, and CD31-PAS dual staining were performed to assess the expression of VEGFR-2, ERK-1/2, PI3K, and MMP-2, and formation of VM.,The results showed apatinib-treated groups formed a lesser number of VM in 3D matrigel, while the cell viability in MTT proliferation assay and the number of migration cells in transwell invasion assay were significantly lower in apatinib-treated groups.,In addition, short-term apatinib treatment inhibited angiogenesis, VM formation, and tumor growth in models of melanoma cancer.,Mice in apatinib-treated groups showed a markedly reduced expression of VEGFR-2, ERK-1/2, PI3K, and MMP-2.,In summary, apatinib could inhibit the expression of VEGFR-2, and downregulate the ERK1/2/PI3K/MMP-2 signaling cascade, which may be one of the underlying mechanisms by which apatinib inhibits angiogenesis and the development of VM in models of melanoma cancer, and restrains the formation of VM by MUM-2B cells.,Apatinib shows inhibitory effects on cell proliferation and invasion of MUM-2B cells, which is a close relationship with the VM.
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Targeted inhibitors elicit heterogeneous clinical responses in genetically stratified groups of patients.,While most studies focus on tumor intrinsic properties, factors in the tumor microenvironment were recently found to modulate the response to inhibitors.,Here, we show that in cutaneous BRAF V600E melanoma, the cytokine TNFα blocks RAF-inhibitor-induced apoptosis via activation of nuclear factor κB (NFκB).,Several NFκB-dependent factors are up-regulated following TNFα and RAF inhibitor treatment.,Of these factors, we show that death receptor inhibitor cellular caspase 8 (FLICE)-like inhibitory protein (c-FLIP) is required for TNFα-induced protection against RAF inhibitor.,Overexpression of c-FLIP_S or c-FLIP_L isoform decreased RAF inhibitor-induced apoptosis in the absence of TNFα.,Importantly, targeting NFκB enhances response to RAF inhibitor in vitro and in vivo.,Together, our results show mechanistic evidence for cytokine-mediated resistance to RAF inhibitor and provide a preclinical rationale for the strategy of co-targeting the RAF-MEK-ERK1/2 pathway and the TNFα/NFκB axis to treat mutant BRAF melanomas.
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Malignant melanoma is one of the most difficult cancers to treat due to its resistance to chemotherapy.,Despite recent successes with BRAF inhibitors and immune checkpoint inhibitors, many patients do not respond or become resistant to these drugs.,Hence, alternative treatments are still required.,Due to the importance of the BCL-2-regulated apoptosis pathway in cancer development and drug resistance, it is of interest to establish which proteins are most important for melanoma cell survival, though the outcomes of previous studies have been conflicting.,To conclusively address this question, we tested a panel of established and early passage patient-derived cell lines against several BH3-mimetic drugs designed to target individual or subsets of pro-survival BCL-2 proteins, alone and in combination, in both 2D and 3D cell cultures.,None of the drugs demonstrated significant activity as single agents, though combinations targeting MCL-1 plus BCL-XL, and to a lesser extent BCL-2, showed considerable synergistic killing activity that was elicited via both BAX and BAK.,Genetic deletion of BFL-1 in cell lines that express it at relatively high levels only had minor impact on BH3-mimetic drug sensitivity, suggesting it is not a critical pro-survival protein in melanoma.,Combinations of MCL-1 inhibitors with BRAF inhibitors also caused only minimal additional melanoma cell killing over each drug alone, whilst combinations with the proteasome inhibitor bortezomib was more effective in multiple cell lines.,Our data show for the first time that therapies targeting specific combinations of BCL-2 pro-survival proteins, namely MCL-1 plus BCL-XL and MCL-1 plus BCL-2, could have significant benefit for the treatment of melanoma.
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In theory, pharmacological inhibition of oncogenic signaling is an effective strategy to halt cellular proliferation, induce apoptosis, and eliminate cancer cells.,In practice, drugs (e.g., PLX-4032) that inhibit oncogenes like B-RAFV600E provide relatively short-term success in patients, due to a combination of incomplete cellular responses and the development of resistance.,To define the relationship between PLX-4032 induced responses and resistance, we interrogated the contributions of anti-apoptotic BCL-2 proteins in determining the fate of B-RAFV600E inhibited melanoma cells.,While PLX-4032 eliminated B-RAFV600E signaling leading to marked cell cycle arrest, only a fraction of cells eventually underwent apoptosis.,These data proposed two hypotheses regarding B-RAFV600E inhibition: (1) only a few cells generate a pro-apoptotic signal, or (2) all the cells generate a pro-apoptotic signal but the majority silences this pathway to ensure survival.,Indeed, the latter hypothesis is supported by our observations as the addition of ABT-737, an inhibitor to anti-apoptotic BCL-2 proteins, revealed massive apoptosis following PLX-4032 exposure.,B-RAFV600E inhibition alone sensitized cells to the mitochondrial pathway of apoptosis characterized by the rapid accumulation of BIM on the outer mitochondrial membrane, which could be functionally revealed by ABT-737 to promote apoptosis and loss of clonogenic survival.,Furthermore, PLX-4032 resistant cells demonstrated collateral resistance to conventional chemotherapy; yet could be re-sensitized to PLX-4032 by BCL-2 family inhibition in vivo and conventional chemotherapies in vitro.,Our data suggest that inhibiting anti-apoptotic BCL-2 proteins will enhance primary responses to PLX-4032, along with reducing the development of resistance to both targeted and conventional therapies.
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Dynamic cellular heterogeneity underlies melanoma progression and therapy resistance.,Advances in single-cell technologies have revealed an increasing number of tumor and microenvironment cell states in melanoma, but little is understood about their function in vivo.,Zebrafish models are a powerful system for discovery, live imaging, and functional investigation of cell states throughout melanoma progression and treatment.,By capturing dynamic melanoma states in living animals, zebrafish have the potential to resolve the complexity of melanoma heterogeneity from a single cell through disease processes within the context of the whole body, revealing novel cancer biology and therapeutic targets.
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Vasculogenic mimicry (VM) describes functional vascular channels composed only of tumor cells and its presence predicts poor prognosis in melanoma patients.,Inhibition of this alternative vascularization pathway might be of clinical importance, especially as several anti-angiogenic therapies targeting endothelial cells are largely ineffective in melanoma.,We show the presence of VM structures histologically in a series of human melanoma lesions and demonstrate that cell cultures derived from these lesions form tubes in 3D cultures ex vivo.,We tested the ability of nicotinamide, the amide form of vitamin B3 (niacin), which acts as an epigenetic gene regulator through unique cellular pathways, to modify VM.,Nicotinamide effectively inhibited the formation of VM structures and destroyed already formed ones, in a dose-dependent manner.,Remarkably, VM formation capacity remained suppressed even one month after the complete withdrawal of Nicotimamid.,The inhibitory effect of nicotinamide on VM formation could be at least partially explained by a nicotinamide-driven downregulation of vascular endothelial cadherin (VE-Cadherin), which is known to have a central role in VM.,Further major changes in the expression profile of hundreds of genes, most of them clustered in biologically-relevant clusters, were observed.,In addition, nicotinamide significantly inhibited melanoma cell proliferation, but had an opposite effect on their invasion capacity.,Cell cycle analysis indicated moderate changes in apoptotic indices.,Therefore, nicotinamide could be further used to unravel new biological mechanisms that drive VM and tumor progression.,Targeting VM, especially in combination with anti-angiogenic strategies, is expected to be synergistic and might yield substantial anti neoplastic effects in a variety of malignancies.
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Uveal melanoma (UM) represents the most common intraocular malignancy in adults and accounts for about 5% of all melanomas.,Primary disease can be effectively controlled by several local therapy options, but UM has a high potential for metastatic spread, especially to the liver.,Despite its clinical and genetic heterogeneity, therapy of metastatic UM has largely been adopted from cutaneous melanoma (CM) with discouraging results until now.,The introduction of antibodies targeting CTLA-4 and PD-1 for immune checkpoint blockade (ICB) has revolutionized the field of cancer therapy and has achieved pioneering results in metastatic CM.,Thus, expectations were high that patients with metastatic UM would also benefit from these new therapy options.,This review provides a comprehensive and up-to-date overview on the role of ICB in UM.,We give a summary of UM biology, its clinical features, and how it differs from CM.,The results of several studies that have been investigating ICB in metastatic UM are presented.,We discuss possible reasons for the lack of efficacy of ICB in UM compared to CM, highlight the pitfalls of ICB in this cancer entity, and explain why other immune-modulating therapies could still be an option for future UM therapies.
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Therapies that target the programmed death-1 (PD-1) receptor have shown unprecedented rates of durable clinical responses in patients with various cancer types.1-5 One mechanism by which cancer tissues limit the host immune response is via upregulation of PD-1 ligand (PD-L1) and its ligation to PD-1 on antigen-specific CD8 T-cells (termed adaptive immune resistance).6,7 Here we show that pre-existing CD8 T-cells distinctly located at the invasive tumour margin are associated with expression of the PD-1/PD-L1 immune inhibitory axis and may predict response to therapy.,We analyzed samples from 46 patients with metastatic melanoma obtained before and during anti-PD1 therapy (pembrolizumab) using quantitative immunohistochemistry, quantitative multiplex immunofluorescence, and next generation sequencing for T-cell receptors (TCR).,In serially sampled tumours, responding patients showed proliferation of intratumoural CD8+ T-cells that directly correlated with radiographic reduction in tumour size.,Pre-treatment samples obtained from responding patients showed higher numbers of CD8, PD1, and PD-L1 expressing cells at the invasive tumour margin and inside tumours, with close proximity between PD-1 and PD-L1, and a more clonal TCR repertoire.,Using multivariate analysis, we established a predictive model based on CD8 expression at the invasive margin and validated the model in an independent cohort of 15 patients.,Our findings indicate that tumour regression following therapeutic PD-1 blockade requires pre-existing CD8+ T cells that are negatively regulated by PD-1/PD-L1 mediated adaptive immune resistance.
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Treatment options are limited for patients with recurrent and/or metastatic (R/M) cutaneous squamous cell carcinoma (cSCC); mortality rates exceed 70% in patients with distant metastases.,Here, we present the first interim analysis of the R/M cSCC cohort from the 2-cohort-locally advanced and R/M-phase II KEYNOTE-629 study.,Patients with R/M cSCC not amenable to surgery or radiation received pembrolizumab 200 mg every 3 weeks.,The primary end point was objective response rate per RECIST v1.1.,Secondary end points were duration of response, disease control rate, progression-free survival, overall survival, and safety.,At data cutoff (April 8, 2019), median follow-up of 105 enrolled patients in the R/M cohort was 11.4 months (range, 0.4 to 16.3 months).,Objective response rate was 34.3% (95% CI, 25.3% to 44.2%; 4 complete responses, 32 partial responses), and disease control rate was 52.4% (95% CI, 42.4% to 62.2%).,Median duration of response was not reached (range, 2.7 to 13.1+ months; ‘+’ refers to ongoing response at data cutoff).,Median progression-free survival was 6.9 months (95% CI, 3.1 months to 8.5 months).,Median overall survival was not reached (95% CI, 10.7 months to not reached).,Treatment-related adverse events occurred in 66.7% of patients (n = 70), the most common of which were pruritus (n = 15; 14.3%), asthenia (n = 14; 13.3%), and fatigue (n = 13; 12.4%).,Grade 3 to 5 treatment-related adverse events occurred in 5.7% (n = 6) of patients.,One patient died of treatment-related cranial nerve neuropathy.,Pembrolizumab demonstrated effective antitumor activity; clinically meaningful, durable responses; and acceptable safety in primarily elderly patients with R/M cSCC, supporting its use in clinical practice.,Pembrolizumab adverse events in this study were consistent with its established safety profile.
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We describe a mechanism of tumorigenesis mediated by kinase-dead BRAF in the presence of oncogenic RAS.,We show that drugs that selectively inhibit BRAF drive RAS-dependent BRAF binding to CRAF, CRAF activation, and MEK-ERK signaling.,This does not occur when oncogenic BRAF is inhibited, demonstrating that BRAF inhibition per se does not drive pathway activation; it only occurs when BRAF is inhibited in the presence of oncogenic RAS.,Kinase-dead BRAF mimics the effects of the BRAF-selective drugs and kinase-dead Braf and oncogenic Ras cooperate to induce melanoma in mice.,Our data reveal another paradigm of BRAF-mediated signaling that promotes tumor progression.,They highlight the importance of understanding pathway signaling in clinical practice and of genotyping tumors prior to administering BRAF-selective drugs, to identify patients who are likely to respond and also to identify patients who may experience adverse effects.,► BRAF activates ERK but in some circumstances BRAF inhibitors can induce tumor growth ► BRAF inhibitors drive BRAF-CRAF binding, activating ERK in cells with oncogenic RAS ► Kinase-dead mutants of BRAF have the same effect as BRAF inhibitors ► Oncogenic RAS and kinase-dead BRAF cooperate to induce melanoma in mice
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Melanoma accounts for the majority of skin cancer deaths.,It has over thirty different subtypes.,Different races have been observed to differ in multiple aspects of melanoma.,SEER (Surveillance, Epidemiology, and End Results) data on six major subtypes, namely melanoma in situ (MIS), superficial spreading melanoma (SSM), nodular melanoma (NM), lentigo maligna melanoma (LMM), acral lentiginous melanoma malignant (ALM), and malignant melanoma NOS (NOS), were analyzed.,The racial groups studied included NHW (non-Hispanic white), HW (Hispanic white), Black, and Asian/PI (Pacific Islanders).,Univariate and multivariate analysis was conducted to quantify racial differences in patients’ characteristics, incidence, treatment, and survival.,Significant racial differences are observed in patients’ characteristics.,For all subtypes except for ALM, NHWs have the highest incidence rates, followed by HWs, while Blacks have the lowest.,For ALM, HWs have the highest rate, followed by NHWs.,In stratified analysis, interaction between gender and race is observed.,For the first five subtypes and localized and regional NOS, the dominating majority of patients had surgery, while for distant NOS, the distribution of treatment is more scattered.,Significant racial differences are observed for distant ALM and NOS.,For MIS, SSM, NM, LMM, and ALM, there is no significant racial difference in survival.,For NOS, significant racial differences in survival are observed for the localized and regional stages, with NHWs having the best and Blacks having the worst five-year survival rates.,Racial differences exist for the six major melanoma subtypes in the U.S.,More data collection and analysis are needed to fully describe and interpret the differences across racial groups and across subtypes.
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Germline CDKN2A mutations occur in 40 % of 3-or-more case melanoma families while mutations of CDK4, BAP1, and genes involved in telomere function (ACD, TERF2IP,POT1), have also been implicated in melanomagenesis.,Mutation of the promoter of the telomerase reverse transcriptase (TERT) gene (c.,−57 T>G variant) has been reported in one family.,We tested for the TERT promoter variant in 675 multicase families wild-type for the known high penetrance familial melanoma genes, 1863 UK population-based melanoma cases and 529 controls.,Germline lymphocyte telomere length was estimated in carriers.,The c.,−57 T>G TERT promoter variant was identified in one 7-case family with multiple primaries and early age of onset (earliest, 15 years) but not among population cases or controls.,One family member had multiple primary melanomas, basal cell carcinomas and a bladder tumour.,The blood leukocyte telomere length of a carrier was similar to wild-type cases.,We provide evidence confirming that a rare promoter variant of TERT (c.,−57 T>G) is associated with high penetrance, early onset melanoma and potentially other cancers, and explains <1 % of UK melanoma multicase families.,The identification of POT1 and TERT germline mutations highlights the importance of telomere integrity in melanoma biology.,The online version of this article (doi:10.1007/s10689-015-9841-9) contains supplementary material, which is available to authorized users.
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Uveal melanoma (UM) is the most common intraocular tumour in adults and despite surgical or radiation treatment of primary tumours, ~50% of patients progress to metastatic disease.,Therapeutic options for metastatic UM are limited, with clinical trials having little impact.,Here we perform whole-genome sequencing (WGS) of 103 UM from all sites of the uveal tract (choroid, ciliary body, iris).,While most UM have low tumour mutation burden (TMB), two subsets with high TMB are seen; one driven by germline MBD4 mutation, and another by ultraviolet radiation (UVR) exposure, which is restricted to iris UM.,All but one tumour have a known UM driver gene mutation (GNAQ, GNA11, BAP1, PLCB4, CYSLTR2, SF3B1, EIF1AX).,We identify three other significantly mutated genes (TP53, RPL5 and CENPE).,Uveal melanoma has a propensity to metastasise.,Here, the authors report the whole genome sequence of 103 uveal melanomas and find that the tumour mutational burden is variable and that two subsets of tumours are characterised by MBD4 mutations and a UV exposure signature.
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B-cell lymphoma and melanoma harbor recurrent mutations in the gene encoding the EZH2 histone methyltransferase, but the carcinogenic role of these mutations is unclear.,Here we describe a mouse model in which the most common somatic EZH2 gain-of-function mutation (Y646F in human, Y641F in the mouse) can be conditionally expressed.,Expression of Ezh2Y641F in mouse B-cells or melanocytes caused high-penetrance lymphoma or melanoma, respectively.,Bcl2 overexpression or p53 loss, but not c-Myc overexpression, further accelerated lymphoma progression, and expression of mutant B-Raf but not mutant N-Ras further accelerated melanoma progression.,Although expression of Ezh2Y641F increased abundance of global H3K27 trimethylation (H3K27me3), it also caused a widespread redistribution of this repressive mark, including a loss of H3K27me3 associated with increased transcription at many loci.,These results suggest that Ezh2Y641F induces lymphoma and melanoma through a vast reorganization of chromatin structure inducing both repression and activation of polycomb-regulated loci.
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The effect of apatinib on the formation of vasculogenic mimicry (VM) was studied in a malignant melanoma cell line.,MUM-2B cells cultured in three-dimensional Matrigel were treated with varying concentrations (0, 0.01, 0.05, 0.1, 0.5 μmol/L) of apatinib to test its effect on VM in vitro, followed by MTT proliferation and transwell invasion assays to determine the effect of apatinib on cell proliferation and invasion of MUM-2B cells.,In vivo, we used a melanoma cancer model to test the effect of short-term apatinib (100, 200, 300 mg/kg) treatment on VM.,Western blotting, immunohistochemistry staining, and CD31-PAS dual staining were performed to assess the expression of VEGFR-2, ERK-1/2, PI3K, and MMP-2, and formation of VM.,The results showed apatinib-treated groups formed a lesser number of VM in 3D matrigel, while the cell viability in MTT proliferation assay and the number of migration cells in transwell invasion assay were significantly lower in apatinib-treated groups.,In addition, short-term apatinib treatment inhibited angiogenesis, VM formation, and tumor growth in models of melanoma cancer.,Mice in apatinib-treated groups showed a markedly reduced expression of VEGFR-2, ERK-1/2, PI3K, and MMP-2.,In summary, apatinib could inhibit the expression of VEGFR-2, and downregulate the ERK1/2/PI3K/MMP-2 signaling cascade, which may be one of the underlying mechanisms by which apatinib inhibits angiogenesis and the development of VM in models of melanoma cancer, and restrains the formation of VM by MUM-2B cells.,Apatinib shows inhibitory effects on cell proliferation and invasion of MUM-2B cells, which is a close relationship with the VM.
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Melanoma is a highly aggressive and drug resistant form of skin cancer.,It arises from melanocytes, the pigment producing cells of the skin.,The formation of these melanocytes is driven by the transcription factor PAX3 early during embryonic development.,As a result of alternative splicing, the PAX3 gene gives rise to eight different transcripts which encode isoforms that have different structures and activate different downstream target genes involved in pathways of cell proliferation, migration, differentiation and survival.,Furthermore, post-translational modifications have also been shown to alter the functions of PAX3.,We previously identified PAX3 downstream target genes in melanocytes and melanoma cells.,Here we assessed the effects of PAX3 down-regulation on this panel of target genes in primary melanocytes versus melanoma cells.,We show that PAX3 differentially regulates various downstream target genes involved in cell proliferation in melanoma cells compared to melanocytes.,To determine mechanisms behind this differential downstream target gene regulation, we performed immunoprecipitation to assess post-translational modifications of the PAX3 protein as well as RNAseq to determine PAX3 transcript expression profiles in melanocytes compared to melanoma cells.,Although PAX3 was found to be post-translationally modified, there was no qualitative difference in phosphorylation and ubiquitination between melanocytes and melanoma cells, while acetylation of PAX3 was reduced in melanoma cells.,Additionally, there were differences in PAX3 transcript expression profiles between melanocytes and melanoma cells.,In particular the PAX3E transcript, responsible for reducing melanocyte proliferation and increasing apoptosis, was found to be down-regulated in melanoma cells compared to melanocytes.,These results suggest that alternate transcript expression profiles activate different downstream target genes leading to the melanoma phenotype.
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Dysregulation of the microRNA (miRNA) network is a typical feature of many cancers.,However, the key specific miRNAs involved in uveal melanoma carcinogenesis are largely unknown.,RT-qPCR was used to detected miR-652 expression in uveal melanoma tissues and cell lines. miR-652 inhibitor was transfected into uveal melanoma cells to decrease miR-652 expression and determine the biological role of miR-652 by CCK-8 and wound healing assays.,Bioinformatic analysis and dual luciferase reporter assay were used to predict and validate the target gene of miR-652.,HOXA9 siRNA was transfected into cells to confirm that miR-652 relies on regulation of HOXA9 to regulate cell proliferation and migration.,RT-qPCR showed that miR-652 was overexpressed in uveal melanoma cell lines (MUM-2B, MEL270) compared with melanocyte cells (ARPE-19).,Overexpression of miR-652 was also observed in uveal melanoma compared to paired non-tumor tissues.,Downregulation of miR-652 inhibited the cell proliferation ability and migration ability of uveal melanoma cells.,Using bioinformatic analysis, HOXA9 was found to be a potential target gene of miR-652.,The direct regulation of HOXA9 by miR-652 was experimentally validated in uveal melanoma cells by dual luciferase assay and Western blotting.,We also observed that miR-652 promoted HIF-1α signaling via repression of HOXA9 in uveal melanoma cells.,Silencing of HOXA9 attenuated the miR-652 inhibitor decreased cell growth rate and decreased migration ability in uveal melanoma cells.,Our data demonstrate an oncogenic role of miR-652 in uveal melanoma, showing that miR-652 may be a useful biomarker for prediction of prognosis for patients with uveal melanoma.
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Simultaneous chemotherapy with vascular endothelial growth factor (VEGF) inhibition has not shown additional benefit over chemotherapy alone in advanced melanoma.,We tested administration of the potent VEGF inhibitor axitinib followed by paclitaxel/carboplatin to determine whether enhanced tumour proliferation during axitinib withdrawal leads to sustained chemosensitivity.,We conducted a prospective phase II trial in metastatic melanoma patients with ECOG performance status 0-1 and normal organ function.,Axitinib 5 mg PO b.i.d. was taken on days 1-14 of each 21-day treatment cycle, and carboplatin (AUC=5) with paclitaxel (175 mg m−2) was administered on day 1 starting with cycle 2.,3′-Deoxy-3′-18F-fluorothymidine (18F-FLT)-PET scans were performed in five patients to assess tumour proliferation on days 1, 14, 17, and 20 of cycle 1.,Molecular profiling for BRAF was performed for all patients with cutaneous, acral, or mucosal melanoma.,The treatment was well tolerated.,The most common grade 3 AEs were hypertension, neutropenia, and anaemia.,Grade 4 non-haematologic AEs were not observed.,Four of five patients completing 18F-FLT-PET scans showed increases (23-92%) in SUV values during the axitinib holiday.,Of 36 evaluable patients, there were 8 confirmed PRs by Response Evaluation Criteria in Solid Tumors.,Overall, 20 patients had SD and 8 had PD as the best response.,The median PFS was 8.7 months and the median overall survival was 14.0 months.,Five BRAFV600E/K patients had significantly worse PFS than patients without these mutations.,Axitinib followed by carboplatin and paclitaxel was well tolerated and effective in BRAF wild-type metastatic melanoma.,3′-Deoxy-3′-18F-fluorothymidine-PET scans showed increased proliferation during axitinib withdrawal.
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To determine if mosaic tuberous sclerosis complex (TSC) can be stratified into subtypes that correspond with prognosis and extent of disease.,Next-generation sequencing of skin tumor and other samples was used to identify patients with mosaic pathogenic variants in TSC1 or TSC2.,Extent of disease, onset age, and family history of TSC were determined through retrospective analysis of patient records.,The median number of disease findings and age at penetrance differed between mosaic patients with asymmetrically distributed facial angiofibromas (4 findings, 24y, n=7), mosaic patients with bilaterally symmetric facial angiofibromas (8 findings, 10y, n=12), and germline TSC patients (10 findings, 4y, n=29).,Cutaneous and internal organ involvement positively correlated in mosaic (R=0.62, p=0.005), but not germline (R=−0.24, p=0.24) TSC.,Variant allele fraction (VAF) in the blood (range: 0-19%) positively correlated with the number of major features (R=0.55, p=0.028).,Five had a TSC2 variant identified in the skin that was below detection in the blood.,One of 12 children from a mosaic parent had TSC.,The phenotype of mosaic TSC ranged from mild to indistinguishable from germline disease.,Patients with mosaicism and asymmetric facial angiofibromas exhibited fewer findings, later onset, and lower VAF in the blood.
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Everolimus does not have sufficient activity to justify its use as single agent in metastatic melanoma.Patients treated with 10 mg per day dose were most likely to require dose reductions.Everolimus appeared to reduce the numbers of regulatory T cells in approximately half of the treated patients; unfortunately, these effects were not correlated with clinical outcomes.,Everolimus does not have sufficient activity to justify its use as single agent in metastatic melanoma.,Patients treated with 10 mg per day dose were most likely to require dose reductions.,Everolimus appeared to reduce the numbers of regulatory T cells in approximately half of the treated patients; unfortunately, these effects were not correlated with clinical outcomes.,Everolimus (RAD‐001) is an orally active rapamycin analogue shown in preclinical data to produce cytostatic cell inhibition, which may be potentially beneficial in treating melanoma.,We conducted a phase II study to evaluate the efficacy and safety of everolimus in patients with unresectable metastatic melanoma (MM).,This study included two cohorts; cohort 1 received 30 mg of everolimus by mouth (PO) weekly, and cohort 2 was dosed with 10 mg of everolimus PO daily.,The endpoints of the study were safety, 16‐week progression‐free survival (PFS), overall survival (OS), and measures of immunomodulatory/antiangiogenic properties with therapy.,Tumor samples before therapy and at week 8 of treatment were analyzed.,Peripheral blood plasma or mononuclear cell isolates collected prior to therapy and at weeks 8 and 16 and at time of tumor progression were analyzed for vascular endothelial growth factor and regulatory T‐cell (Treg) measurements.,A total of 53 patients were enrolled in cohort 1 (n = 24) and cohort 2 (n = 29).,Only 2 patients of the first 20 patients enrolled in cohort 2 had treatment responses (25%; 95% confidence interval, 8.6%-49.1%); this result did not allow full accrual to cohort 2, as the study was terminated for futility.,Median OS was 12.2 months for cohort 1 versus 8.1 months in cohort 2; no PFS advantage was seen in either group (2.1 months vs.,1.8 months).,Dose‐limiting toxicities included grade 4 myocardial ischemia (3.4%); grade 3 fatigue, mucositis, and hyperglycemia (10.3%); and anorexia and anemia (6.9%).,Everolimus significantly reduced the number of Tregs in approximately half of the treated patients; however, these effects were not correlated with clinical outcomes.,Everolimus does not have sufficient single‐agent activity in MM; however, we have identified evidence of biological activity to provide a potential rationale for future combination studies.
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The discovery of the role of the RAS/RAF/MEK/ERK pathway in melanomagenesis and its progression have opened a new era in the treatment of this tumor.,Vemurafenib was the first specific kinase inhibitor approved for therapy of advanced melanomas harboring BRAF-activating mutations, followed by dabrafenib and encorafenib.,However, despite the excellent results of first-generation kinase inhibitors in terms of response rate, the average duration of the response was short, due to the onset of genetic and epigenetic resistance mechanisms.,The combination therapy with MEK inhibitors is an excellent strategy to circumvent drug resistance, with the additional advantage of reducing side effects due to the paradoxical reactivation of the MAPK pathway.,The recent development of RAS and extracellular signal-related kinases (ERK) inhibitors promises to add new players for the ultimate suppression of this signaling pathway and the control of pathway-related drug resistance.,In this review, we analyze the pharmacological, preclinical, and clinical trial data of the various MAPK pathway inhibitors, with a keen interest for their clinical applicability in the management of advanced melanoma.
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BRAF and MEK inhibitors are effective in BRAF mutant melanoma, but most patients eventually relapse with acquired resistance, and others present intrinsic resistance to these drugs.,Resistance is often mediated by pathway reactivation through receptor tyrosine kinase (RTK)/SRC-family kinase (SFK) signaling or mutant NRAS, which drive paradoxical reactivation of the pathway.,We describe pan-RAF inhibitors (CCT196969, CCT241161) that also inhibit SFKs.,These compounds do not drive paradoxical pathway activation and inhibit MEK/ERK in BRAF and NRAS mutant melanoma.,They inhibit melanoma cells and patient-derived xenografts that are resistant to BRAF and BRAF/MEK inhibitors.,Thus, paradox-breaking pan-RAF inhibitors that also inhibit SFKs could provide first-line treatment for BRAF and NRAS mutant melanomas and second-line treatment for patients who develop resistance.,•pan-RAF inhibitors also inhibit SRC family kinases•The compounds do not induce paradoxical activation of ERK in RAS mutant cells•The compounds are active in BRAF and NRAS mutant melanomas•The compounds are active in PDXs resistant to BRAF or BRAF plus MEK inhibitors,pan-RAF inhibitors also inhibit SRC family kinases,The compounds do not induce paradoxical activation of ERK in RAS mutant cells,The compounds are active in BRAF and NRAS mutant melanomas,The compounds are active in PDXs resistant to BRAF or BRAF plus MEK inhibitors,Girotti et al. describe two pan-RAF inhibitors that also inhibit SRC-family kinases.,These compounds do not drive paradoxical MEK/ERK activation and can inhibit MEK in NRAS mutant cells.,Moreover, the agents can overcome resistance to clinical BRAF or combination BRAF/MEK inhibitors in patient-derived xenografts.
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Sun-exposure is one of the risk factors associated with the development of a cutaneous neoplasm.,In melanoma, the Ras-Raf-MEK-ERK (MAPK) signaling pathway is constitutively activated through multiple mechanisms, including B-RAF mutation.,It has been hypothesized that B-RAF mutations in melanocytic lesions arise from DNA damage induced by ultraviolet (UV) radiation.,However, it is still discussed if B-RAF mutations are associated with melanoma patients exposed to the sun.,Therefore, in the present study, the known B-RAFV600E mutation was analysed in melanoma samples from 30 indoor and 38 outdoor workers.,B-RAFV600E mutation was detected in 52 and 73% of outdoor workers and indoor workers, respectively.,Of note, this mutation was identified in 12 of 14 (85%) melanoma of the trunk diagnosed in indoor workers and in 9 of 19 (47%) samples from outdoor workers (p=0.03).,By analyzing melanomas of other body sites, no statistical difference in the frequency of B-RAFV600E mutation was identified between the groups of workers.,It appears that the mutation detected among indoor workers may be associated with a recreational or intermittent exposure to the sun, as usually the trunk is a sun-protected body site.,Overall, these data indicate that the B-RAFV600E mutation detected in melanoma is not associated with a chronic exposure to the sun.,Mutations detected in other genes may also contribute to melanoma development in the subset of patients exposed to UV radiation.
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Our understanding of the molecular pathways that underlie melanoma remains incomplete.,Although several published microarray studies of clinical melanomas have provided valuable information, we found only limited concordance between these studies.,Therefore, we took an in vitro functional genomics approach to understand melanoma molecular pathways.,Affymetrix microarray data were generated from A375 melanoma cells treated in vitro with siRNAs against 45 transcription factors and signaling molecules.,Analysis of this data using unsupervised hierarchical clustering and Bayesian gene networks identified proliferation-association RNA clusters, which were co-ordinately expressed across the A375 cells and also across melanomas from patients.,The abundance in metastatic melanomas of these cellular proliferation clusters and their putative upstream regulators was significantly associated with patient prognosis.,An 8-gene classifier derived from gene network hub genes correctly classified the prognosis of 23/26 metastatic melanoma patients in a cross-validation study.,Unlike the RNA clusters associated with cellular proliferation described above, co-ordinately expressed RNA clusters associated with immune response were clearly identified across melanoma tumours from patients but not across the siRNA-treated A375 cells, in which immune responses are not active.,Three uncharacterised genes, which the gene networks predicted to be upstream of apoptosis- or cellular proliferation-associated RNAs, were found to significantly alter apoptosis and cell number when over-expressed in vitro.,This analysis identified co-expression of RNAs that encode functionally-related proteins, in particular, proliferation-associated RNA clusters that are linked to melanoma patient prognosis.,Our analysis suggests that A375 cells in vitro may be valid models in which to study the gene expression modules that underlie some melanoma biological processes (e.g., proliferation) but not others (e.g., immune response).,The gene expression modules identified here, and the RNAs predicted by Bayesian network inference to be upstream of these modules, are potential prognostic biomarkers and drug targets.
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Organ transplant recipients (OTRs) have a 100‐fold increased risk of cutaneous squamous cell carcinoma (cSCC).,We prospectively evaluated the association between β genus human papillomaviruses (βPV) and keratinocyte carcinoma in OTRs.,Two OTR cohorts without cSCC were assembled: cohort 1 was transplanted in 2003‐2006 (n = 274) and cohort 2 was transplanted in 1986‐2002 (n = 352).,Participants were followed until death or cessation of follow‐up in 2016. βPV infection was assessed in eyebrow hair by using polymerase chain reaction-based methods. βPV IgG seroresponses were determined with multiplex serology.,A competing risk model with delayed entry was used to estimate cumulative incidence of histologically proven cSCC and the effect of βPV by using a multivariable Cox regression model.,Results are reported as adjusted hazard ratios (HRs).,OTRs with 5 or more different βPV types in eyebrow hair had 1.7 times the risk of cSCC vs OTRs with 0 to 4 different types (HR 1.7, 95% confidence interval 1.1‐2.6).,A similar risk was seen with high βPV loads (HR 1.8, 95% confidence interval 1.2‐2.8).,No significant associations were seen between serum antibodies and cSCC or between βPV and basal cell carcinoma.,The diversity and load of βPV types in eyebrow hair are associated with cSCC risk in OTRs, providing evidence that βPV is associated with cSCC carcinogenesis and may present a target for future preventive strategies.,In two cohorts of organ transplant recipients, those with five and more different beta‐genus human papillomavirus types and high virus loads in eyebrow hairs subsequently develop significantly more cutaneous squamous cell carcinomas than others, suggesting that these virus types may increase the risk of cutaneous squamous cell carcinoma in these patients.
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Cutaneous beta human papillomavirus (HPV) types are suspected to be involved, together with ultraviolet (UV) radiation, in the development of non-melanoma skin cancer (NMSC).,Studies in in vitro and in vivo experimental models have highlighted the transforming properties of beta HPV E6 and E7 oncoproteins.,However, epidemiological findings indicate that beta HPV types may be required only at an initial stage of carcinogenesis, and may become dispensable after full establishment of NMSC.,Here, we further investigate the potential role of beta HPVs in NMSC using a Cre-loxP-based transgenic (Tg) mouse model that expresses beta HPV38 E6 and E7 oncogenes in the basal layer of the skin epidermis and is highly susceptible to UV-induced carcinogenesis.,Using whole-exome sequencing, we show that, in contrast to WT animals, when exposed to chronic UV irradiation K14 HPV38 E6/E7 Tg mice accumulate a large number of UV-induced DNA mutations, which increase proportionally with the severity of the skin lesions.,The mutation pattern detected in the Tg skin lesions closely resembles that detected in human NMSC, with the highest mutation rate in p53 and Notch genes.,Using the Cre-lox recombination system, we observed that deletion of the viral oncogenes after development of UV-induced skin lesions did not affect the tumour growth.,Together, these findings support the concept that beta HPV types act only at an initial stage of carcinogenesis, by potentiating the deleterious effects of UV radiation.
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Knowledge of key drivers and therapeutic targets in mucosal melanoma is limited due to the paucity of comprehensive mutation data on this rare tumor type.,To better understand the genomic landscape of mucosal melanoma, here we describe whole genome sequencing analysis of 67 tumors and validation of driver gene mutations by exome sequencing of 45 tumors.,Tumors have a low point mutation burden and high numbers of structural variants, including recurrent structural rearrangements targeting TERT, CDK4 and MDM2.,Significantly mutated genes are NRAS, BRAF, NF1, KIT, SF3B1, TP53, SPRED1, ATRX, HLA-A and CHD8.,SF3B1 mutations occur more commonly in female genital and anorectal melanomas and CTNNB1 mutations implicate a role for WNT signaling defects in the genesis of some mucosal melanomas.,TERT aberrations and ATRX mutations are associated with alterations in telomere length.,Mutation profiles of the majority of mucosal melanomas suggest potential susceptibility to CDK4/6 and/or MEK inhibitors.,Mucosal melanomas are challenging to treat partly because so little is known about the genetic drivers underpinning them.,Here, the authors perform a genomic landscape analysis of samples collected from three continents, revealing a potential role for CDK4/6 or MEK inhibition in the treatment of the disease.
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Chimeric antigen receptor (CAR)-engineered T cells have demonstrated promising clinical efficacy in patients with B cell lymphoma.,However, the application of CAR-T cell therapy in the treatment of other solid tumors has been limited.,We incorporated 4-1BB into the anti-GD2 CAR-T cells to test their cytotoxicity in melanoma in vitro and in vivo.,Moreover, we reported the expression of ganglioside GD2 in non-Caucasian melanoma populations for the first time, thus providing a basis for future clinical research.,This study included tumor samples from 288 melanoma patients at the Peking University Cancer Hospital & Institute.,Clinical data were collected.,Immunohistochemical assays using antibodies against ganglioside GD2 were performed on formalin-fixed, paraffin-embedded specimens.,The ability of ganglioside GD2 CAR-T cells to kill ganglioside GD2+ melanoma cells was evaluated in vitro and in a patient-derived xenograft (PDX) model.,Among the 288 samples, 49.3% of cases (142/288) demonstrated positive staining with ganglioside GD2.,The median survival time in patients exhibiting ganglioside GD2 expression was significantly shorter than that in patients without ganglioside GD2 expression (31 vs.,47.1 months, P < 0.001).,In the present study, CAR was constructed using a GD2-specific scFv (14.,G2a), T cell receptor CD3ζ chain, and the CD137 (4-1BB) costimulatory motif.,In addition, the GD2.,BBζ CAR-T cells demonstrated specific lysis of ganglioside GD2-expressing melanoma cells in vitro.,In two PDX models, mice that received intravenous or local intratumor injections of GD2.,BBζ CAR-T cells experienced rapid tumor regression.,These data demonstrate that the rate of GD2 expression in Chinese patients is 49.3%.,GD2.,BBζ CAR-T cells can both efficiently lyse melanoma in a GD2-specific manner and release Th1 cytokines in an antigen-dependent manner in vitro and in vivo.,Anti-GD2/4-1BB CAR-T cells represent a clinically appealing treatment strategy for Chinese melanoma patients exhibiting GD2 expression and provide a basis for future studies of the clinical application of immunotherapy for melanoma.,The online version of this article (10.1186/s13045-017-0548-2) contains supplementary material, which is available to authorized users.
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In an ongoing screen for DNA sequence variants that confer risk of cutaneous basal cell carcinoma (BCC), we conduct a genome-wide association study (GWAS) of 24,988,228 SNPs and small indels detected through whole-genome sequencing of 2,636 Icelanders and imputed into 4,572 BCC patients and 266,358 controls.,Here we show the discovery of four new BCC susceptibility loci: 2p24 MYCN (rs57244888[C], OR=0.76, P=4.7 × 10−12), 2q33 CASP8-ALS2CR12 (rs13014235[C], OR=1.15, P=1.5 × 10−9), 8q21 ZFHX4 (rs28727938[G], OR=0.70, P=3.5 × 10−12) and 10p14 GATA3 (rs73635312[A], OR=0.74, P=2.4 × 10−16).,Fine mapping reveals that two variants correlated with rs73635312[A] occur in conserved binding sites for the GATA3 transcription factor.,In addition, expression microarrays and RNA-seq show that rs13014235[C] and a related SNP rs700635[C] are associated with expression of CASP8 splice variants in which sequences from intron 8 are retained.,Basal cell carcinoma is a common cancer among people of European ancestry, with associated high economic costs to monitor and treat.,Here Stacey et al. conduct a genome-wide association study on Icelandic and other European populations, identifying four novel loci associated with cancer susceptibility.
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Cutaneous squamous cell carcinoma (SCC) is the most prevalent invasive malignancy with metastatic potential.,The epidermis is exposed to a variety of environmental DNA-damaging chemicals, principal among which are polyaromatic hydrocarbons (PAH) ubiquitous in the environment, tobacco smoke, and broiled meats.,Langerhans cells (LC) comprise a network of dendritic cells situated adjacent to basal, suprabasal, and follicular infundibular keratinocytes that when mutated can give rise to SCC, and LC-intact mice are markedly more susceptible than LC-deficient mice to chemical carcinogenesis provoked by initiation with the model PAH, 7,12-dimethylbenz[a]anthracene (DMBA).,LC rapidly internalize and depot DMBA as numerous membrane-independent cytoplasmic foci.,Repopulation of LC-deficient mice using fetal liver LC-precursors restores DMBA-induced tumor susceptibility.,LC expression of p450 enzyme CYP1B1 is required for maximal rapid induction of DNA-damage within adjacent keratinocytes and their efficient neoplastic transformation; however, effects of tumor progression also attributable to the presence of LC were revealed as CYP1B1-independent.,Thus, LC make multifaceted contributions to cutaneous carcinogenesis, including via the handling and metabolism of chemical mutagens.,Such findings suggest a cooperative carcinogenesis role for myeloid-derived cells resident within cancer susceptible epithelial tissues principally by influencing early events in malignant transformation.
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Ocular melanoma consists of posterior uveal melanoma, iris melanoma and conjunctival melanoma.,These malignancies derive from melanocytes in the uveal tract or conjunctiva.,The genetic profiles of these different entities differ from each other.,In uveal melanoma, GNAQ and GNA11 gene mutations are frequently found and prognosis is based on mutation status of BAP1, SF3B1 and EIF1AX genes.,Iris melanoma, also originating from the uvea, has similarities to the genetic makeups of both posterior uveal melanoma (UM) and conjunctival melanoma since mutations in GNAQ and GNA11 are less common and genes involved in conjunctival melanoma such as BRAF have been described.,The genetic spectrum of conjunctival melanoma, however, includes frequent mutations in the BRAF, NRAS and TERT promoter genes, which are found in cutaneous melanoma as well.,The BRAF status of the tumor is not correlated to prognosis, whereas the TERT promoter gene mutations are.,Clinical presentation, histopathological characteristics and copy number alterations are associated with survival in ocular melanoma.,Tissue material is needed to classify ocular melanoma in the different subgroups, which creates a need for the use of noninvasive techniques to prognosticate patients who underwent eye preserving treatment.
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New Zealand and Australia have the highest incidence and mortality rates from cutaneous melanoma in the world.,The predominantly fair-skinned New Zealanders and Australians both enjoy sun, tanned skin and the outdoors, and differences in these activities among generations have been important determinants of trends in melanoma mortality.,We examined whether New Zealand trends in melanoma mortality mirror those in Australia, through detailed comparison of the trends in both countries from 1968 to 2007.,Five-year age-specific and age-standardised mortality rates were calculated for each country for 5-year time periods.,Tests for trends in age-specific rates were performed using the Mantel-Haenszel extension chi-square test.,The age-adjusted mortality rate ratios for New Zealand/Australia were plotted against period of death to show relative changes in mortality over time.,Age-specific mortality rates were plotted against period and the median year of birth to illustrate age-group and birth cohort effects.,To compare the mortality of birth cohorts, age-adjusted melanoma mortality rate ratios were calculated for the birth cohorts in the quin-quennial tables of mortality rates.,The age-standardised mortality rate for melanoma increased in both sexes in New Zealand and Australia from 1968 to 2007, but the increase was greater in New Zealanders and women in particular.,There was evidence of recent significant decreases in mortality in younger Australians and less so in New Zealand women aged under 45 years.,Mortality from melanoma increased in successive generations born from about 1893 to 1918.,In Australia, a decline in mortality started for generations born from about 1958 but in New Zealand there is possibly a decrease only in generations born since 1968.,Mortality trends in New Zealand and Australia are discrepant.,It is too early to know if the pattern in mortality rates in New Zealand is simply a delayed response to melanoma control activities compared with Australia, whereby we can expect the same downward trend in similar age groups in the next few years.,Specific research is needed to better understand and control the increases in mortality and thickness of melanoma in New Zealand.
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The present study aimed to investigate the potential role of the long non-coding RNA (lncRNA) Pvt1 oncogene (non-protein coding) (PVT1) in the progression and metastasis of malignant melanoma, and to reveal its possible molecular mechanisms.,The expression of lncRNA PVT1 in melanoma tissues and adjacent normal skin from patients with melanoma, and in the melanoma A-375 and sk-mel-5 cell lines, was analyzed using reverse transcription-quantitative polymerase chain reaction and western blot analyses.,The effects of PVT1 expression on cell proliferation, the cell cycle, cell migration and cell invasion were analyzed using MTT assay, flow cytometry, Transwell and scratch assays, respectively.,The interaction between PVT1 and enhancer of zeste homolog 2 (EZH2) in melanoma cells was analyzed using RNA immunoprecipitation (RIP) assay.,The effect of PVT1 on microRNA-200c (miR-200c) expression was analyzed by chromatin immunoprecipitation (ChIP) assay.,PVT1 was highly expressed in the melanoma tissues and cells.,Silencing of PVT1 significantly inhibited cell proliferation, migration and invasion, and arrested the cell cycle at the G0/G1 stage.,Additionally, PVT1 silencing significantly decreased the cyclin D1 expression in the melanoma cells.,The expression of E-cadherin was significantly increased and the expression of N-cadherin and vimentin was significantly decreased in the PVT1-silenced group.,The RIP assay found that endogenous PVT1 was highly enriched by EZH2 RIP compared with that of the negative control.,The ChIP assay revealed that the expression of miR-200c was decreased significantly in the PVT1-silenced group compared with the controls.,Overall, the present study demonstrated that the lncRNA PVT1 may contribute to the tumorigenesis and metastasis of melanoma through binding to EZH2 and regulating the expression of miR-200c. lncRNA PVT1 may serve as a potential target for the therapy of melanoma.
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The conversion of melanocytes into cutaneous melanoma is largely dictated by the effects of solar ultraviolet radiation (UVR).,Yet to be described, however, is exactly how these cells are affected by intense solar UVR while residing in their natural microenvironment, and whether their response differs in persons with a history of melanoma when compared to that of healthy individuals.,By using laser capture microdissection (LCM) to isolate a pure population of melanocytes from a small area of skin that had been intermittingly exposed or un-exposed to physiological doses of solar UVR, we can now report for the first time that the majority of UV-responsive microRNAs (miRNAs) in the melanocytes of a group of women with a history of melanoma are down-regulated when compared to those in the melanocytes of healthy controls.,Among the miRNAs that were commonly and significantly down-regulated in each of these women were miR-193b (P<0.003), miR-342-3p (P<0.003), miR186 (P<0.007), miR-130a (P<0.007), and miR-146a (P<0.007).,To identify genes potentially released from inhibition by these repressed UV-miRNAs, we analyzed databases (e.g., DIANA-TarBase) containing experimentally validated microRNA-gene interactions.,In the end, this enabled us to construct UV-miRNA-gene regulatory networks consisting of individual genes with a probable gain-of-function being intersected not by one, but by several down-regulated UV-miRNAs.,Most striking, however, was that these networks typified well-known regulatory modules involved in controlling the epithelial-to-mesenchymal transition and processes associated with the regulation of immune-evasion.,We speculate that these pathways become activated by UVR resulting in miRNA down regulation only in melanocytes susceptible to melanoma, and that these changes could be partially responsible for empowering these cells toward tumor progression.
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Standard-dose pembrolizumab plus alternative-dose ipilimumab (1 mg/kg Q3W for 4 doses) were tolerable and had robust antitumor activity in advanced melanoma in cohort B of the phase 1 KEYNOTE-029 study.,Cohort C evaluated standard-dose pembrolizumab with two other alternative ipilimumab regimens.,Patients with treatment-naive unresectable stage III/IV melanoma were randomly assigned 1:1 to pembrolizumab 200 mg Q3W for ≤24 months plus ipilimumab 50 mg Q6W for 4 doses (PEM200+IPI50), or the same pembrolizumab regimen plus ipilimumab 100 mg Q12W for 4 doses (PEM200+IPI100).,Primary end points were incidence of grade 3-5 treatment-related adverse events (TRAE) and objective response rate (ORR) per RECIST v1.1 by independent central review.,Per protocol-defined thresholds, grade 3-5 TRAE incidence ≤26% indicated meaningful toxicity reduction and ORR ≥48% indicated no decrease in efficacy versus data reported for other PD-1 inhibitor/ipilimumab combinations.,Median follow-up on February 18, 2019, was 16.3 months in PEM200+IPI50 (N = 51) and 16.4 months in PEM200+IPI100 (N = 51).,Grade 3-5 TRAEs occurred in 12 (24%) patients in PEM200+IPI50 and 20 (39%) in PEM200+IPI100.,One patient in PEM200+IPI50 died from treatment-related autoimmune myocarditis.,Immune-mediated AEs or infusion reactions occurred in 21 (42%) patients in PEM200+IPI50 and 28 (55%) in PEM200+IPI100.,ORR was 55% in PEM200+IPI50; 61% in PEM200+IPI100.,Pembrolizumab 200 mg Q3W plus ipilimumab 50 mg Q6W or 100 mg Q12W demonstrated antitumor activity above the predefined threshold; pembrolizumab plus ipilimumab 50 mg Q6W had lower incidence of grade 3-5 TRAEs than the predefined threshold, suggesting a reduction in toxicity.,See related commentary by Jameson-Lee and Luke, p.,5153
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Cancer-secreted exosomal miRNAs regulates the biological processes of many tumours.,The serum level of exosomal miR-106b-5p is significantly increased in melanoma patients.,However, the role and molecular mechanisms of exosomal miR-106b-5p in melanoma remains unclear.,Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-106b-5p and EphA4 in melanoma tissues.,Transmission electron microscopy (TEM) and western blotting were used to identify exosome.,QRT-qPCR and Cy3-labelled miR-106b-5p were used to demonstrated the transmission of melanoma cell-secreted exosomal miR-106b-5p.,Western blotting, Immunofluorescence, adhesion, transwell and scratch wound assay were used to explore the role of exosomal miR-106b-5p in melanocytes.,Luciferase reporter assays and RNA-Chromatin Immunoprecipitation (ChIP) assay were used to confirm whether erythropoietin-producing hepatocellular carcinoma receptor A4 (EphA4) was a direct target of miR-106b-5p.,We found that miR-106b-5p levels were increased in melanoma tissue, and high miR-106b-5p expression is an independent risk factor for the overall survival of patients with melanoma. miR-106b-5p is enriched in melanoma cell-secreted exosomes and transferred to melanocytes.,Exosomal miR-106b-5p promotes the epithelial-to-mesenchymal transition (EMT), migration, invasion and adhesion of melanocytes.,Exosomal miR-106b-5p exerted its role by targeting EphA4 to activate the ERK pathway.,We demonstrated that exosomal miR-106b-5p promoted melanoma metastasis in vivo through pulmonary metastasis assay.,Thus, melanoma cell-secreted exosomal miR-106b-5p may serve as a diagnostic indicator and potential therapeutic target in melanoma patients.,The online version contains supplementary material available at 10.1186/s13046-021-01906-w.
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The MITF transcription factor is a master regulator of melanocyte development and a critical factor in melanomagenesis.,The related transcription factors TFEB and TFE3 regulate lysosomal activity and autophagy processes known to be important in melanoma.,Here we show that MITF binds the CLEAR-box element in the promoters of lysosomal and autophagosomal genes in melanocytes and melanoma cells.,The crystal structure of MITF bound to the CLEAR-box reveals how the palindromic nature of this motif induces symmetric MITF homodimer binding.,In metastatic melanoma tumors and cell lines, MITF positively correlates with the expression of lysosomal and autophagosomal genes, which, interestingly, are different from the lysosomal and autophagosomal genes correlated with TFEB and TFE3.,Depletion of MITF in melanoma cells and melanocytes attenuates the response to starvation-induced autophagy, whereas the overexpression of MITF in melanoma cells increases the number of autophagosomes but is not sufficient to induce autophagic flux.,Our results suggest that MITF and the related factors TFEB and TFE3 have separate roles in regulating a starvation-induced autophagy response in melanoma.,Understanding the normal and pathophysiological roles of MITF and related transcription factors may provide important clinical insights into melanoma therapy.
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Thousands of putative enhancers are characterized in the human genome, yet few have been shown to have a functional role in cancer progression.,Inhibiting oncokinases, such as EGFR, ALK, ERBB2, and BRAF, is a mainstay of current cancer therapy but is hindered by innate drug resistance mediated by up-regulation of the HGF receptor, MET.,The mechanisms mediating such genomic responses to targeted therapy are unknown.,Here, we identify lineage-specific enhancers at the MET locus for multiple common tumor types, including a melanoma lineage-specific enhancer 63 kb downstream from the MET TSS.,This enhancer displays inducible chromatin looping with the MET promoter to up-regulate MET expression upon BRAF inhibition.,Epigenomic analysis demonstrated that the melanocyte-specific transcription factor, MITF, mediates this enhancer function.,Targeted genomic deletion (<7 bp) of the MITF motif within the MET enhancer suppressed inducible chromatin looping and innate drug resistance, while maintaining MITF-dependent, inhibitor-induced melanoma cell differentiation.,Epigenomic analysis can thus guide functional disruption of regulatory DNA to decouple pro- and anti-oncogenic functions of a dominant transcription factor and block innate resistance to oncokinase therapy.
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Melanoma aggressiveness determines its growth and metastatic potential.,This study aimed at identifying new molecular pathways controlling melanoma cell malignancy.,Ten metastatic melanoma cell lines were characterized by their proliferation, migration and invasion capabilities.,The most representative cells were also characterized by spheroid formation assay, gene- and protein- expression profiling as well as cytokines secretion and the most relevant pathways identified through bioinformatic analysis were tested by in silico transcriptomic validation on datasets generated from biopsies specimens of melanoma patients.,Further, matrix metalloproteases (MMPs) activity was tested by zymography assays and TNF-alpha role was validated by anti-TNF cell-treatment.,An aggressiveness score (here named Melanoma AGgressiveness Score: MAGS) was calculated by measuring proliferation, migration, invasion and cell-doubling time in10human melanoma cell lines which were clustered in two distinct groups, according to the corresponding MAGS.,SK-MEL-28 and A375 cell lines were selected as representative models for the less and the most aggressive phenotype, respectively.,Gene-expression and protein expression data were collected for SK-MEL-28 and A375 cells by Illumina-, multiplex x-MAP-and mass-spectrometry technology.,The collected data were subjected to an integrated Ingenuity Pathway Analysis, which highlighted that cytokine/chemokine secretion, as well as Cell-To-Cell Signaling and Interaction functions as well as matrix metalloproteases activity were significantly different in these two cell types.,The key role of these pathways was then confirmed by functional validation.,TNF role was confirmed by exposing cells to the anti-TNF Infliximab antibody.,Upon such treatment melanoma cells aggressiveness was strongly reduced.,Metalloproteases activity was assayed, and their role was confirmed by comparing transcriptomic data from cutaneous melanoma patients (n = 45) and benign nevi (n = 18).,Inflammatory signals such as TNF and MMP-2 activity are key intrinsic players to determine melanoma cells aggressiveness suggesting new venue sin the identification of novel molecular targets with potential therapeutic relevance.,The online version of this article (10.1186/s13046-018-0982-1) contains supplementary material, which is available to authorized users.
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Antibodies against programmed cell death-1 (PD-1) have considerably changed the treatment for melanoma.,However, many patients do not display therapeutic response or eventually relapse.,Moreover, patients treated with anti-PD-1 develop immune-related adverse events that can be cured with anti-tumor necrosis factor α (TNF) antibodies.,Whether anti-TNF antibodies affect the anti-cancer immune response remains unknown.,Our recent work has highlighted that TNFR1-dependent TNF signalling impairs the accumulation of CD8+ tumor-infiltrating T lymphocytes (CD8+ TILs) in mouse melanoma.,Herein, our results indicate that TNF or TNFR1 blockade synergizes with anti-PD-1 on anti-cancer immune responses towards solid cancers.,Mechanistically, TNF blockade prevents anti-PD-1-induced TIL cell death as well as PD-L1 and TIM-3 expression.,TNF expression positively correlates with expression of PD-L1 and TIM-3 in human melanoma specimens.,This study provides a strong rationale to develop a combination therapy based on the use of anti-PD-1 and anti-TNF in cancer patients.,Most melanoma patients do not respond to anti-PD1 therapy.,Here, the authors show that TNFα blockade synergizes with anti-PD-1 by preventing anti-PD-1-induced CD8+ T cell death and TIM-3 expression on such cells.
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Epigenetic events, including covalent post-translational modifications of histones, have been demonstrated to play critical roles in tumor development and progression.,The transcriptional coactivator p300/CBP possesses both histone acetyltransferase (HAT) activity as well as scaffolding properties that directly influence the transcriptional activation of targeted genes.,We have used a potent and specific inhibitor of p300/CBP HAT activity, C646, in order to evaluate the functional contributions of p300/CBP HAT to tumor development and progression.,Here we report that C646 inhibits the growth of human melanoma and other tumor cells and promotes cellular senescence.,Global assessment of the p300 HAT transcriptome in human melanoma identified functional roles in promoting cell cycle progression, chromatin assembly and activation of DNA repair pathways through direct transcriptional regulatory mechanisms.,Additionally, C646 is shown to promote sensitivity to DNA damaging agents, leading to the enhanced apoptosis of melanoma cells following combination treatment with cisplatin.,Together, our data suggest that p300 HAT activity mediates critical growth regulatory pathways in tumor cells and may serve as a potential therapeutic target for melanoma and other malignancies by promoting cellular responses to DNA damaging agents that are currently ineffective against specific cancers.
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Long non-coding RNAs (lncRNAs) have been shown to be implicated in the complex network of cancer including malignant melanoma and play important roles in tumorigenesis and progression.,However, their functions and downstream mechanisms are largely unknown.,This study aimed to investigate whether BRAF-activated non-coding RNA (BANCR), a novel and potential regulator of melanoma cell, participates in the proliferation of malignant melanoma and elucidate the underlying mechanism in this process.,We found that BANCR was abnormally overexpressed in human malignant melanoma cell lines and tissues, and increased with tumor stages by quantitative PCR.,BANCR knockdown induced by shRNA transfection significantly inhibited proliferation of tumor cells and inactivated MAPK pathway, especially by silencing the ERK1/2 and JNK component.,Moreover, combination treatment of BANCR knockdown and suppression ERK1/2 or JNK (induced by specific inhibitors U0126 or SP600125 respectively) produced synergistic inhibitory effects in vitro.,And the inhibitory effects induced by ERK1/2 or JNK could be rescued by BANCR overexpression.,By tumorigenicity assay in BALB/c nude mice, we further found that BANCR knockdown inhibited tumor growth in vivo.,In addition, patients with high expression of BANCR had a lower survival rate.,Taken together, we confirmed the abnormal upregulation of a novel lncRNA, BANCR, in human malignant melanoma.,BANCR was involved in melanoma cell proliferation both in vitro and in vivo.,The linkage between BANCR and MAPK pathway may provide a novel interpretation for the mechanism of proliferation regulation in malignant melanoma.
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Transcriptomic signatures designed to predict melanoma patient responses to PD-1 blockade have been reported but rarely validated.,We now show that intra-patient heterogeneity of tumor responses to PD-1 inhibition limit the predictive performance of these signatures.,We reasoned that resistance mechanisms will reflect the tumor microenvironment, and thus we examined PD-1 inhibitor resistance relative to T-cell activity in 94 melanoma tumors collected at baseline and at time of PD-1 inhibitor progression.,Tumors were analyzed using RNA sequencing and flow cytometry, and validated functionally.,These analyses confirm that major histocompatibility complex (MHC) class I downregulation is a hallmark of resistance to PD-1 inhibitors and is associated with the MITFlow/AXLhigh de-differentiated phenotype and cancer-associated fibroblast signatures.,We demonstrate that TGFß drives the treatment resistant phenotype (MITFlow/AXLhigh) and contributes to MHC class I downregulation in melanoma.,Combinations of anti-PD-1 with drugs that target the TGFß signaling pathway and/or which reverse melanoma de-differentiation may be effective future therapeutic strategies.,A significant proportion of patients develop innate or acquired resistance to immune checkpoint inhibitors.,Here, the authors show that resistance to anti-PD-1 blockade is associated with TGF-beta driven major histocompatibility complex I (MHCI) down-regulation and a de-differentiated phenotype in melanoma patients.
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Stromal fibroblasts are an integral part of the tumor stroma and constantly interact with cancer cells to promote their initiation and progression.,However, the role and function of dermal fibroblasts during the early stage of melanoma development remain poorly understood.,We, therefore, designed a novel genetic approach to deactivate stromal fibroblasts at the onset of melanoma formation by targeted ablation of β‐catenin.,To our surprise, melanoma tumors formed from β‐catenin‐deficient group (B16F10 mixed with β‐catenin‐deficient fibroblasts) appeared earlier than tumors formed from control group (B16F10 mixed with normal dermal fibroblasts).,At the end point when tumors were collected, mutant tumors were bigger and heavier than control tumors.,Further analysis showed that there were fewer amounts of stromal fibroblasts and myofibroblasts inside mutant tumor stroma.,Melanoma tumors from control group showed reduced proliferation, down‐regulated expression of cyclin D1 and increased expression of cyclin‐dependent kinase inhibitor p16, suggesting dermal fibroblasts blocked the onset of melanoma tumor formation by inducing a cell cycle arrest in B16F10 melanoma cells.,Furthermore, we discovered that dermal fibroblasts prevented epithelial‐mesenchymal transition in melanoma cells.,Overall, our findings demonstrated that dermal fibroblasts crosstalk with melanoma cells to regulate in vivo tumor development via multiple mechanisms, and the outcomes of their reciprocal interactions depend on activation states of stromal fibroblasts and stages of melanoma development.
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Melanoma is the most fatal skin cancer displaying a high degree of molecular heterogeneity.,Phenotype switching is a mechanism that contributes to melanoma heterogeneity by altering transcription profiles for the transition between states of proliferation/differentiation and invasion/stemness.,As phenotype switching is reversible, epigenetic mechanisms, like DNA methylation, could contribute to the changes in gene expression.,Integrative analysis of methylation and gene expression datasets of five proliferative and five invasion melanoma cell cultures reveal two distinct clusters.,SOX9 is methylated and lowly expressed in the highly proliferative group.,SOX9 overexpression results in decreased proliferation but increased invasion in vitro.,In a B16 mouse model, sox9 overexpression increases the number of lung metastases.,Transcriptional analysis of SOX9-overexpressing melanoma cells reveals enrichment in epithelial to mesenchymal transition (EMT) pathways.,Survival analysis of The Cancer Genome Atlas melanoma dataset shows that metastatic patients with high expression levels of SOX9 have significantly worse survival rates.,Additional survival analysis on the targets of SOX9 reveals that most SOX9 downregulated genes have survival benefit for metastatic patients.,Our genome-wide DNA methylation and gene expression study of 10 early passage melanoma cell cultures reveals two phenotypically distinct groups.,One of the genes regulated by DNA methylation between the two groups is SOX9.,SOX9 induces melanoma cell invasion and metastasis and decreases patient survival.,A number of genes downregulated by SOX9 have a negative impact on patient survival.,In conclusion, SOX9 is an important gene involved in melanoma invasion and negatively impacts melanoma patient survival.,The online version of this article (doi:10.1186/s13059-015-0594-4) contains supplementary material, which is available to authorized users.
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Melanoma is the most fatal skin cancer, but the etiology of this devastating disease is still poorly understood.,Recently, the transcription factor Sox10 has been shown to promote both melanoma initiation and progression.,Reducing SOX10 expression levels in human melanoma cells and in a genetic melanoma mouse model, efficiently abolishes tumorigenesis by inducing cell cycle exit and apoptosis.,Here, we show that this anti-tumorigenic effect functionally involves SOX9, a factor related to SOX10 and upregulated in melanoma cells upon loss of SOX10.,Unlike SOX10, SOX9 is not required for normal melanocyte stem cell function, the formation of hyperplastic lesions, and melanoma initiation.,To the contrary, SOX9 overexpression results in cell cycle arrest, apoptosis, and a gene expression profile shared by melanoma cells with reduced SOX10 expression.,Moreover, SOX9 binds to the SOX10 promoter and induces downregulation of SOX10 expression, revealing a feedback loop reinforcing the SOX10 low/SOX9 high ant,m/ii-tumorigenic program.,Finally, SOX9 is required in vitro and in vivo for the anti-tumorigenic effect achieved by reducing SOX10 expression.,Thus, SOX10 and SOX9 are functionally antagonistic regulators of melanoma development.
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An impressive clinical success has been observed in treating a variety of cancers using immunotherapy with programmed cell death‐1 (PD‐1) checkpoint blockade.,However, limited response in most patients treated with anti‐PD‐1 antibodies remains a challenge, requiring better understanding of molecular mechanisms limiting immunotherapy.,In colorectal cancer (CRC) resistant to immunotherapy, mismatch‐repair‐proficient or microsatellite instability‐low (pMMR‐MSI‐L) tumors have low mutation burden and constitute ~85% of patients.,Here, we show that inhibition of N 6‐methyladenosine (m6A) mRNA modification by depletion of methyltransferases, Mettl3 and Mettl14, enhanced response to anti‐PD‐1 treatment in pMMR‐MSI‐L CRC and melanoma.,Mettl3‐ or Mettl14‐deficient tumors increased cytotoxic tumor‐infiltrating CD8+ T cells and elevated secretion of IFN‐γ, Cxcl9, and Cxcl10 in tumor microenvironment in vivo.,Mechanistically, Mettl3 or Mettl14 loss promoted IFN‐γ‐Stat1‐Irf1 signaling through stabilizing the Stat1 and Irf1 mRNA via Ythdf2.,Finally, we found a negative correlation between METTL3 or METTL14 and STAT1 in 59 patients with pMMR‐MSI‐L CRC tumors.,Altogether, our findings uncover a new awareness of the function of RNA methylation in adaptive immunity and provide METTL3 and METTL14 as potential therapeutic targets in anticancer immunotherapy.,Disruption of m6A methyltransferases leads to enhanced immunotherapy response in colorectal cancer and melanoma cells due to enhanced IFN‐γ‐Stat1‐Irf1 signaling and modulation of the tumor microenvironment.
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Wnt5a has been implicated in melanoma progression and metastasis, although the exact downstream signaling events that contribute to melanoma metastasis are poorly understood.,Wnt5a signaling results in acyl protein thioesterase 1 (APT1) mediated depalmitoylation of pro-metastatic cell adhesion molecules CD44 and MCAM, resulting in increased melanoma invasion.,The mechanistic details that underlie Wnt5a-mediated regulation of APT1 activity and cellular function remain unknown.,Here, we show Wnt5a signaling regulates APT1 activity through induction of APT1 phosphorylation and we further investigate the functional role of APT1 phosphorylation on its depalmitoylating activity.,We found phosphorylation increased APT1 depalmitoylating activity and reduced APT1 dimerization.,We further determined APT1 phosphorylation increases melanoma invasion in vitro, and also correlated with increased tumor grade and metastasis.,Our results further establish APT1 as an important regulator of melanoma invasion and metastatic behavior.,Inhibition of APT1 may represent a novel way to treat Wnt5a driven cancers.
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BRAFV600E-mutant malignant melanomas depend on RAF/MEK/ERK (MAPK) signaling for tumor cell growth1.,RAF and MEK inhibitors show remarkable clinical efficacy in BRAFV600E melanoma2, 3; however, resistance to these agents remains a formidable challenge2, 4.,Global characterization of resistance mechanisms may inform the development of more effective therapeutic combinations.,Here, we performed systematic gain-of-function resistance studies by expressing >15,500 genes individually in a BRAFV600E melanoma cell line treated with RAF, MEK, ERK, or combined RAF/MEK inhibitors.,These studies revealed a cyclic AMP-dependent melanocytic signaling network not previously associated with drug resistance, including G-protein coupled receptors, adenyl cyclase, protein kinase A and cAMP response element binding protein (CREB).,Preliminary analysis of biopsies from BRAFV600E melanoma patients revealed that phosphorylated (active) CREB was suppressed by RAF/MEK-inhibition but restored in relapsing tumors.,Expression of transcription factors activated downstream of MAP kinase and cAMP pathways also conferred resistance, including c-FOS, NR4A1, NR4A2 and MITF.,Combined treatment with MAP kinase pathway and histone deacetylase inhibitors suppressed MITF expression and cAMP-mediated resistance.,Collectively, these data suggest that oncogenic dysregulation of a melanocyte lineage dependency can cause resistance to RAF/MEK/ERK inhibition, which may be overcome by combining signaling- and chromatin-directed therapeutics.
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We sequenced 8 melanoma exomes to identify novel somatic mutations in metastatic melanoma.,Focusing on the MAP3K family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9.,Structural modelling predicts that mutations in the kinase domain may affect the activity and regulation of MAP3K5/9 protein kinases.,The position of the mutations and loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely inactivating.,In vitro kinase assay shows reduction in kinase activity in MAP3K5 I780F and MAP3K9 W333X mutants.,Overexpression of MAP3K5 or MAP3K9 mutant in HEK293T cells reduces phosphorylation of downstream MAP kinases.,Attenuation of MAP3K9 function in melanoma cells using siRNA leads to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.
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To identify ‘melanoma-specific’ microRNAs (miRNAs) we used an unbiased microRNA profiling approach to comprehensively study cutaneous melanoma in relation to other solid malignancies, which revealed 233 differentially expressed (≥2 fold, p < 0.05) miRNAs.,Among the top 20 most significantly different miRNAs was hsa-miR-514a-3p. miR-514a is a member of a cluster of miRNAs (miR-506-514) involved in initiating melanocyte transformation and promotion of melanoma growth.,We found miR-514a was expressed in 38/55 (69%) melanoma cell lines but in only 1/34 (3%) other solid cancers.,To identify miR-514a regulated targets we conducted a miR-514a-mRNA ‘pull-down’ experiment, which revealed hundreds of genes, including: CTNNB1, CDK2, MC1R, and NF1, previously associated with melanoma.,NF1 was selected for functional validation because of its recent implication in acquired resistance to BRAFV600E-targeted therapy.,Luciferase-reporter assays confirmed NF1 as a direct target of miR-514a and over-expression of miR-514a in melanoma cell lines inhibited NF1 expression, which correlated with increased survival of BRAFV600E cells treated with PLX4032.,These data provide another mechanism for the dysregulation of the MAPK pathway which may contribute to the profound resistance associated with current RAF-targeted therapies.
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Treatment of advanced melanoma has been improved with the advent of the BRAF inhibitors.,However, a limitation to such treatment is the occurrence of resistance.,Several mechanisms have been identified to be responsible for the development of resistance, either MEK-dependent or MEK-independent.,In order to overcome resistance due to reactivation of MEK signaling, MEK inhibitors are being clinically developed with promising results.,However, also in this case resistance inevitably occurs.,It has been recently reported that ErbB3, a member of the EGFR receptor family, may be involved in the establishment of drug resistance.,Three melanoma cell lines were tested: LOX IMVI (BRAF V600E), MST-L (BRAF V600R) and WM266 (BRAF V600D).,Phosphorylation of Receptor Tyrosine Kinases (RTKs) was assessed by an RTK array.,Western blot analysis was performed on total protein extracts using anti-ErbB3, anti-AKT and anti-ERK 1/2 antibodies.,The expression of neuregulin after vemurafenib treatment was assessed by Real Time PCR and Western blotting.,The growth inhibitory effects of vemurafenib, GSK1120212b and/or anti-ErbB3 mAbs were evaluated by in vitro colony formation assays.,In the present study we demonstrate that ErbB3 is the main RTK undergoing rapidly hyperphosphorylation upon either treatment with a BRAF inhibitor or with a MEK inhibitor in a panel of melanoma cell lines harboring a variety of V600BRAF mutations and that this results in a strong activation of phospho-AKT.,Importantly, ErbB3 activation is fully abrogated by the simultaneous use of anti-ErbB3 monoclonal antibodies, which are also shown to potently synergize with BRAF inhibitors in the inactivation of both AKT and ERK pathways and in the inhibition of melanoma cell growth.,We show that upregulation of phospho-ErbB3 is due to an autocrine loop involving increased transcription and production of neuregulin by melanoma cells.,On the basis of these results, we propose that initial co-treatment with BRAF and/or MEK inhibitors and anti-ErbB3 antibodies should be pursued as a strategy to reduce the ErbB3-dependent feedback survival mechanism and enhance duration of clinical response.
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Melanoma patients’ plasma contains exosomes produced by malignant and normal cells.,Plasma exosomes were isolated and separated by immunocapture into two fractions: melanoma cell-derived exosomes (MTEX) and normal cell-derived exosomes (non-MTEX).,Immunosuppressive effects of MTEX on primary human immune cells were evaluated.,Exosomes were isolated from plasma of 12 melanoma patients and six healthy donors (HDs).,Expression levels of 19 immunoregulatory proteins in MTEX, non-MTEX and HDs exosomes were evaluated by on-bead flow cytometry.,Functional/phenotypic changes induced in CD8+ T or natural killer (NK) cells by MTEX or non-MTEX were compared.,Plasma protein levels were higher in patients than HDs (P < 0.0009).,In patients, MTEX accounted for 23-66% of total exosomes.,MTEX were enriched in immunosuppressive proteins (P = 0.03).,MTEX, but not HDs exosomes, inhibited CD69 expression (P ≤ 0.0008), induced apoptosis (P ≤ 0.0009) and suppressed proliferation (P ≤ 0.002) in CD8+ T cells and downregulated NKG2D expression in NK cells (P = 0.001).,Non-MTEX were enriched in immunostimulatory proteins (P = 0.002) and were only weakly immunosuppressive.,Elevated MTEX/total exosome ratios and, surprisingly, non-MTEX ability to induce apoptosis of CD8+ T cells emerged as positive correlates of disease stage.,MTEX emerge as the major mechanism of tumor-induced immune suppression and as an underestimated barrier to successful melanoma immunotherapy.
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Tumor cells evade the immune surveillance by up-regulating surface expression of PD-L1, which interacts with PD-1 on T cells to elicit the immune checkpoint response1,2.,Anti-PD-1 antibodies have shown remarkable promise in treating tumors, including metastatic melanoma2-4.,However, patient response rate is low4,5.,A better understanding of PD-L1-mediated immune evasion is needed to predict patient response and improve treatment efficacy.,Here we report that metastatic melanoma releases a high level of extracellular vesicles (EVs), mostly in the form of exosomes, that carry PD-L1 on their surface.,Interferon-γ (IFN-γ) up-regulates PD-L1 on these vesicles, which suppresses the function of CD8 T cells and facilitates tumor growth.,In patients with metastatic melanoma, the level of circulating exosomal PD-L1 positively correlates with that of IFN-γ, and changes during the course of anti-PD-1 therapy.,The magnitudes of the early on-treatment increase in circulating exosomal PD-L1, as an indicator of the adaptive response of the tumor cells to T cell re-invigoration, stratifies clinical responders from non-responders.,Our study unveils a mechanism by which tumor cells systemically suppress the immune system, and provides a rationale for the application of exosomal PD-L1 as a predictor for anti-PD-1 therapy.
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Uveal melanoma (UM), a rare cancer of the eye, is distinct from cutaneous melanoma by its etiology, the mutation frequency and profile, and its clinical behavior including resistance to targeted therapy and immune checkpoint blockers.,Primary disease is efficiently controlled by surgery or radiation therapy, but about half of UMs develop distant metastasis mostly to the liver.,Survival of patients with metastasis is below 1 year and has not improved in decades.,Recent years have brought a deep understanding of UM biology characterized by initiating mutations in the G proteins GNAQ and GNA11.,Cytogenetic alterations, in particular monosomy of chromosome 3 and amplification of the long arm of chromosome 8, and mutation of the BRCA1-associated protein 1, BAP1, a tumor suppressor gene, or the splicing factor SF3B1 determine UM metastasis.,Cytogenetic and molecular profiling allow for a very precise prognostication that is still not matched by efficacious adjuvant therapies.,G protein signaling has been shown to activate the YAP/TAZ pathway independent of HIPPO, and conventional signaling via the mitogen-activated kinase pathway probably also contributes to UM development and progression.,Several lines of evidence indicate that inflammation and macrophages play a pro-tumor role in UM and in its hepatic metastases.,UM cells benefit from the immune privilege in the eye and may adopt several mechanisms involved in this privilege for tumor escape that act even after leaving the niche.,Here, we review the current knowledge of the biology of UM and discuss recent approaches to UM treatment.
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To assess clinical characteristics, treatment and survival of patients with uveal melanoma in China.,The retrospective study included all patients with malignant uveal melanoma who were consecutively examined in the study period from January 2005 and June 2015 in the Beijing Tongren hospital.,The mean age of the 582 patients (295(50.7%) women) was 44.6±12.6 years (range:5-77 years).,The tumors were located most often in the superior temporal region (in 117(21.5%) patients) and least common in the inferior region (in 31(5.7%) patients).,In 548(94.2%) patients, the tumors were located in the choroid, in 33(5.7%) patients in the ciliary body, and in one (0.2%) patient in the iris.,Treatment included episcleral brachytherapy (415(71.3%) patients), local tumor resection (48(8.2%) patients) and primary enucleation (119(20.4%) patients).,In 53 individuals out of the 415 patients with primary brachytherapy, episcleral brachytherapy was followed by enucleation, due to an increasing tumor size or due to uncontrolled neovascular glaucoma.,Median follow-up time was of 30 months (range: 1-124 months; mean: 34.8 ± 24.4 months).,Overall survival rate at 5 and 10 years was of 92.7% and 85.1%.,Younger age (P = 0.017), tumor location in the nasal meridian(P = 0.004), smaller tumor size (P<0.001), hemispheric tumor shape (P = 0.025), histological tumor cell type (spindle-cell type versus epitheloid cell type;P = 0.014), and type of treatment (episcleral brachytherapy versus local tumor resection and versus primary enucleation; P<0.001) were significantly associated with the overall survival in univariate analysis, while in multivariate analysis only smaller tumor size was significantly (P<0.001; RR: 4.75; 95% confidence interval:2.11,10.7) associated with better overall survival.,In this study on clinical characteristics of uveal melanoma of a larger group of patients from China, the onset age was considerably younger and survival rate better than in studies from Western countries.,Tumor size was the most significant factor for survival.
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Melanoma is the most deadly form of cutaneous malignancy, and its incidence rates are rising worldwide.,In melanoma, constitutive activation of the BRAF/MEK/ERK (MAPK) and PI3K/AKT/mTOR (PI3K) signaling pathways plays a pivotal role in cell proliferation, survival and tumorigenesis.,A combination of compounds that lead to an optimal blockade of these critical signaling pathways may provide an effective strategy for prevention and treatment of melanoma.,The phytochemical fisetin is known to possess anti-proliferative and pro-apoptotic activities.,We found that fisetin treatment inhibited PI3K signaling pathway in melanoma cells.,Therefore, we investigated the effect of fisetin and sorafenib (an RAF inhibitor) alone and in combination on cell proliferation, apoptosis and tumor growth.,Combination treatment (fisetin + sorafenib) more effectively reduced the growth of BRAF-mutated human melanoma cells at lower doses when compared to individual agents.,In addition, combination treatment resulted in enhanced (i) apoptosis, (ii) cleavage of caspase-3 and PARP, (iii) expression of Bax and Bak, (iv) inhibition of Bcl2 and Mcl-1, and (v) inhibition of expression of PI3K, phosphorylation of MEK1/2, ERK1/2, AKT and mTOR.,In athymic nude mice subcutaneously implanted with melanoma cells (A375 and SK-MEL-28), we found that combination therapy resulted in greater reduction of tumor growth when compared to individual agents.,Furthermore, combination therapy was more effective than monotherapy in: (i) inhibition of proliferation and angiogenesis, (ii) induction of apoptosis, and (iii) inhibition of the MAPK and PI3K pathways in xenograft tumors.,These data suggest that simultaneous inhibition of both these signaling pathways using combination of fisetin and sorafenib may serve as a therapeutic option for the management of melanoma.
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BRAFV600E-mutant malignant melanomas depend on RAF/MEK/ERK (MAPK) signaling for tumor cell growth1.,RAF and MEK inhibitors show remarkable clinical efficacy in BRAFV600E melanoma2, 3; however, resistance to these agents remains a formidable challenge2, 4.,Global characterization of resistance mechanisms may inform the development of more effective therapeutic combinations.,Here, we performed systematic gain-of-function resistance studies by expressing >15,500 genes individually in a BRAFV600E melanoma cell line treated with RAF, MEK, ERK, or combined RAF/MEK inhibitors.,These studies revealed a cyclic AMP-dependent melanocytic signaling network not previously associated with drug resistance, including G-protein coupled receptors, adenyl cyclase, protein kinase A and cAMP response element binding protein (CREB).,Preliminary analysis of biopsies from BRAFV600E melanoma patients revealed that phosphorylated (active) CREB was suppressed by RAF/MEK-inhibition but restored in relapsing tumors.,Expression of transcription factors activated downstream of MAP kinase and cAMP pathways also conferred resistance, including c-FOS, NR4A1, NR4A2 and MITF.,Combined treatment with MAP kinase pathway and histone deacetylase inhibitors suppressed MITF expression and cAMP-mediated resistance.,Collectively, these data suggest that oncogenic dysregulation of a melanocyte lineage dependency can cause resistance to RAF/MEK/ERK inhibition, which may be overcome by combining signaling- and chromatin-directed therapeutics.
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microRNAs constitute a complex class of pleiotropic post-transcriptional regulators of gene expression involved in the control of several physiologic and pathologic processes.,Their mechanism of action is primarily based on the imperfect matching of a seed region located at the 5′ end of a 21-23 nt sequence with a partially complementary sequence located in the 3′ untranslated region of target mRNAs.,This leads to inhibition of mRNA translation and eventually to its degradation.,Individual miRNAs are capable of binding to several mRNAs and several miRNAs are capable of influencing the function of the same mRNAs.,In recent years networks of miRNAs are emerging as capable of controlling key signaling pathways responsible for the growth and propagation of cancer cells.,Furthermore several examples have been provided which highlight the involvement of miRNAs in the development of resistance to targeted drug therapies.,In this review we provide an updated overview of the role of miRNAs in the development of melanoma and the identification of the main downstream pathways controlled by these miRNAs.,Furthermore we discuss a group of miRNAs capable to influence through their respective up- or down-modulation the development of resistance to BRAF and MEK inhibitors.
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To identify ‘melanoma-specific’ microRNAs (miRNAs) we used an unbiased microRNA profiling approach to comprehensively study cutaneous melanoma in relation to other solid malignancies, which revealed 233 differentially expressed (≥2 fold, p < 0.05) miRNAs.,Among the top 20 most significantly different miRNAs was hsa-miR-514a-3p. miR-514a is a member of a cluster of miRNAs (miR-506-514) involved in initiating melanocyte transformation and promotion of melanoma growth.,We found miR-514a was expressed in 38/55 (69%) melanoma cell lines but in only 1/34 (3%) other solid cancers.,To identify miR-514a regulated targets we conducted a miR-514a-mRNA ‘pull-down’ experiment, which revealed hundreds of genes, including: CTNNB1, CDK2, MC1R, and NF1, previously associated with melanoma.,NF1 was selected for functional validation because of its recent implication in acquired resistance to BRAFV600E-targeted therapy.,Luciferase-reporter assays confirmed NF1 as a direct target of miR-514a and over-expression of miR-514a in melanoma cell lines inhibited NF1 expression, which correlated with increased survival of BRAFV600E cells treated with PLX4032.,These data provide another mechanism for the dysregulation of the MAPK pathway which may contribute to the profound resistance associated with current RAF-targeted therapies.
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This cohort study explores long noncoding RNA-based immune subtypes associated with survival and response to cancer immunotherapy and presents a novel long noncoding RNA score for immunotherapy prediction using computational algorithms.,Are long noncoding RNAs (lncRNAs) associated with immune molecular classification and clinical outcomes of cancer immunotherapy?,This cohort study of 348 patients with bladder cancer from the IMvigor210 trial and 71 patients with melanoma from The Cancer Genome Analysis identified 4 distinct classes with different immunotherapeutic overall survival and response.,An lncRNA score was developed that also was associated with survival and immunotherapy response.,This study identifies novel lncRNA-based immune classes in cancer immunotherapy and provides an lncRNA score for integration into multiomic panels for precision immunotherapy.,Long noncoding RNAs (lncRNAs) are involved in innate and adaptive immunity in cancer by mediating the functional state of immunologic cells, pathways, and genes.,However, whether lncRNAs are associated with immune molecular classification and clinical outcomes of cancer immunotherapy is largely unknown.,To explore lncRNA-based immune subtypes associated with survival and response to cancer immunotherapy and to present a novel lncRNA score for immunotherapy prediction using computational algorithms.,In this cohort study, an individual patient analysis based on a phase 2, single-arm clinical trial and multicohort was performed from June 25 through September 30, 2019.,Data are from the phase 2 IMvigor210 trial and from The Cancer Genome Atlas (TCGA).,The study analyzed lncRNA and genomic data of 348 patients with bladder cancer from the IMvigor210 trial and 71 patients with melanoma from TCGA who were treated with immunotherapy.,In addition, a pancancer multicohort that included 2951 patients was obtained from TCGA.,The primary end point was overall survival (OS).,Among 348 patients from the IMvigor210 trial (272 [78.2%] male) and 71 patients with melanoma from TCGA (mean [SD] age, 58.3 [13.4] years; 37 [52.1%] female), 4 distinct classes with statistically significant differences in OS (median months, not reached vs 9.6 vs 8.1 vs 6.7 months; P = .002) were identified.,The greatest OS benefit was obtained in the immune-active class, as characterized by the immune-functional lncRNA signature and high CTL infiltration.,Patients with low vs high lncRNA scores had statistically significantly longer OS (hazard ratio, 0.32; 95% CI, 0.24-0.42; P < .001) in the IMvigor210 trial and across various cancer types.,The lncRNA score was associated with immunotherapeutic OS benefit in the IMvigor210 trial cohort (area under the curve [AUC], 0.79 at 12 months and 0.77 at 20 months) and in TCGA melanoma cohort (AUC, 0.87 at 24 months), superior tumor alteration burden, programmed cell death ligand 1 (PD-L1) expression, and cytotoxic T-lymphocyte (CTL) infiltration.,Addition of the lncRNA score to the combination of tumor alteration burden, PD-L1 expression, and CTL infiltration to build a novel multiomics algorithm correlated more strongly with OS in the IMvigor210 trial cohort (AUC, 0.81 at 12 months and 0.80 at 20 months).,This study identifies novel lncRNA-based immune classes in cancer immunotherapy and recommends immunotherapy for patients in the immune-active class.,In addition, the study recommends that the lncRNA score should be integrated into multiomic panels for precision immunotherapy.
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The critical long non-coding RNAs (lncRNAs) involved in the carcinogenesis and progression of malignant melanoma (MM) have not been fully investigated.,In the present study, it was identified that lncRNA activated by transforming growth factor-β (lncRNA-ATB) was upregulated in MM tissues and cells compared with benign nevus cells and human melanocytes, via comparative lncRNA screening from Gene Expression Omnibus datasets and reverse transcription-quantitative polymerase chain reaction analysis.,Furthermore, lncRNA-ATB promoted the cell proliferation, cell migration, and cell invasion of MM cells in vitro, and tumor growth in vivo.,It was additionally identified that lncRNA-ATB attenuated cell cycle arrest and inhibited cellular apoptosis in MM cells.,Finally, it was demonstrated that lncRNA-ATB functions as a competing endogenous RNA (ceRNA) to enhance Yes associated protein 1 expression by competitively sponging microRNA miR-590-5p in MM cells.,In conclusion, the present study revealed the expression and roles of lncRNA-ATB in MM, and indicated that lncRNA-ATB functions as a ceRNA to promote MM proliferation and invasion by sponging miR-590-5p.
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Eosinophils have been identified as a prognostic marker in immunotherapy of melanoma and suggested to contribute to anti-tumor host defense.,However, the influence of immune checkpoint inhibitors (ICI) on the eosinophil population is poorly studied.,Here, we applied routine laboratory tests, multicolor flow cytometry, RNA microarray analysis, and bio-plex assay to analyze circulating eosinophils and related serum inflammatory factors in 32 patients treated with pembrolizumab or the combination of nivolumab and ipilimumab.,We demonstrated that clinical responses to ICI treatment were associated with an eosinophil accumulation in the peripheral blood.,Moreover, immunotherapy led to the alteration of the eosinophil genetic and activation profile.,Elevated serum concentrations of IL-16 during ICI treatment were found to be associated with increased frequencies of eosinophils in the peripheral blood.,Using immunohistochemistry, we observed an enhanced eosinophil degranulation and a positive correlation between eosinophil and CD8+ T cell infiltration of tumor tissues from melanoma patients treated with ICI.,Our findings highlight additional mechanisms of ICI effects and suggest the level of eosinophils as a novel predictive marker for melanoma patients who may benefit from this immunotherapy.
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Neoplastic meningitis is a central nervous system complication that occurs in 3-5% of patients with cancer.,Although most commonly seen in patients with disseminated disease, in a small percentage of patients, it may be the initial manifestation of cancer or even primitive in origin.,In the absence of cancer history, the diagnosis of neoplastic meningitis may be challenging even for expert neurologists.,Prognosis is poor, with a median overall survival of weeks from diagnosis.,In the retrospective study herein, we described three cases of meningeal melanomatosis in patients without previous cancer history.,The patients were diagnosed with significant delay (17 to 47 weeks from symptom onset) mainly due to the deferral in performing the appropriate testing.,Even when the diagnosis was suspected, investigations by MRI, cerebrospinal fluid, or both proved at times unhelpful for confirmation.,Prognosis was dismal, with a median survival of 4 weeks after diagnosis.,Our observations are a cue to analyze the main pitfalls in the diagnosis of neoplastic meningitis in patients without cancer history and emphasize key elements that may facilitate early diagnosis.
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Skin malignancy is a distressing problem for many patients, and clinical management is challenging.,This article describes the protocol for the Calcium Electroporation Response Study (CaEP-R) designed to investigate tumour response to calcium electroporation and is a descriptive guide to calcium electroporation treatment of malignant tumours in the skin.,Calcium electroporation is a local treatment that induces supraphysiological intracellular calcium levels by intratumoural calcium administration and application of electrical pulses.,The pulses create transient membrane pores allowing diffusion of non-permeant calcium ions into target cells.,High calcium levels can kill cancer cells, while normal cells can restore homeostasis.,Prior trials with smaller cohorts have found calcium electroporation to be safe and efficient.,This trial aims to include a larger multiregional cohort of patients with different cancer diagnoses and also to investigate treatment areas using MRI as well as assess impact on quality of life.,This non-randomised phase II multicentre study will investigate response to calcium electroporation in 30 patients with cutaneous or subcutaneous malignancy.,Enrolment of 10 patients is planned at three centres: Zealand University Hospital, University Hospital of Southern Denmark and University Hospital Schleswig-Holstein.,Response after 2 months was chosen as the primary endpoint based on short-term response rates observed in a prior clinical study.,Secondary endpoints include response to treatment using MRI and change in quality of life assessed by questionnaires and qualitative interviews.,The trial is approved by the Danish Medicines Agency and The Danish Regional Committee on Health Research Ethics.,All included patients will receive active treatment (calcium electroporation).,Patients can continue systemic treatment during the study, and side effects are expected to be limited.,Data will be published in a peer-reviewed journal and made available to the public.,NCT04225767 and EudraCT no: 2019-004314-34.
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In the present study, we have aimed to characterize the intrinsic, extrinsic and ER-mediated apoptotic induction by hyperthermia in an in vitro model of human malignant melanoma and furthermore, to evaluate its therapeutic effectiveness in an adjuvant therapeutic setting characterized by combinational treatments with non-targeted (Dacarbazine & Temozolomide) and targeted (Dabrafenib & Vemurafenib) drugs.,Overall, our data showed that both low (43 °C) and high (45 °C) hyperthermic exposures were capable of inducing cell death by activating all apoptotic pathways but in a rather distinct manner.,More specifically, low hyperthermia induced extrinsic and intrinsic apoptotic pathways both of which activated caspase 6 only as opposed to high hyperthermia which was mediated by the combined effects of caspases 3, 7 and 6.,Furthermore, significant involvement of the ER was evident (under both hyperthermic conditions) suggesting its role in regulating apoptosis via activation of CHOP.,Our data revealed that while low hyperthermia activated IRE-1 and ATF6 only, high hyperthermia induced activation of PERK as well suggesting that ultimately these ER stress sensors can lead to the induction of CHOP via different pathways of transmitted signals.,Finally, combinational treatment protocols revealed an effect of hyperthermia in potentiating the therapeutic effectiveness of non-targeted as well as targeted drugs utilized in the clinical setting.,Overall, our findings support evidence into hyperthermia’s therapeutic potential in treating human malignant melanoma by elucidating the underlying mechanisms of its complex apoptotic induction.
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In human mutant BRAF melanoma cells, the stemness transcription factor FOXD3 is rapidly induced by inhibition of ERK1/2 signaling and mediates adaptive resistance to RAF inhibitors.,However, the mechanism underlying ERK signaling control of FOXD3 expression remains unknown.,Here we show that SOX10 is both necessary and sufficient for RAF inhibitor-induced expression of FOXD3 in mutant BRAF melanoma cells.,SOX10 activates the transcription of FOXD3 by binding to a regulatory element in FOXD3 promoter.,Phosphorylation of SOX10 by ERK inhibits its transcription activity toward multiple target genes by interfering with the sumoylation of SOX10 at K55, which is essential for its transcription activity.,Finally, depletion of SOX10 sensitizes mutant BRAF melanoma cells to RAF inhibitors in vitro and in vivo.,Thus, our work discovers a novel phosphorylation-dependent regulatory mechanism of SOX10 transcription activity and completes an ERK1/2/SOX10/FOXD3/ERBB3 axis that mediates adaptive resistance to RAF inhibitors in mutant BRAF melanoma cells.,In BRAF mutant melanoma, inhibition of ERK1/2 induces FOXD3 and mediates RAF inhibitor resistance.,Here, the authors show that ERK1/2 mediated phosphorylation regulates sumoylation of SOX10 which activates FOXD3, and depletion of SOX10 sensitises BRAF mutant melanoma cells to RAF inhibitors.
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Melanoma is the most fatal skin cancer displaying a high degree of molecular heterogeneity.,Phenotype switching is a mechanism that contributes to melanoma heterogeneity by altering transcription profiles for the transition between states of proliferation/differentiation and invasion/stemness.,As phenotype switching is reversible, epigenetic mechanisms, like DNA methylation, could contribute to the changes in gene expression.,Integrative analysis of methylation and gene expression datasets of five proliferative and five invasion melanoma cell cultures reveal two distinct clusters.,SOX9 is methylated and lowly expressed in the highly proliferative group.,SOX9 overexpression results in decreased proliferation but increased invasion in vitro.,In a B16 mouse model, sox9 overexpression increases the number of lung metastases.,Transcriptional analysis of SOX9-overexpressing melanoma cells reveals enrichment in epithelial to mesenchymal transition (EMT) pathways.,Survival analysis of The Cancer Genome Atlas melanoma dataset shows that metastatic patients with high expression levels of SOX9 have significantly worse survival rates.,Additional survival analysis on the targets of SOX9 reveals that most SOX9 downregulated genes have survival benefit for metastatic patients.,Our genome-wide DNA methylation and gene expression study of 10 early passage melanoma cell cultures reveals two phenotypically distinct groups.,One of the genes regulated by DNA methylation between the two groups is SOX9.,SOX9 induces melanoma cell invasion and metastasis and decreases patient survival.,A number of genes downregulated by SOX9 have a negative impact on patient survival.,In conclusion, SOX9 is an important gene involved in melanoma invasion and negatively impacts melanoma patient survival.,The online version of this article (doi:10.1186/s13059-015-0594-4) contains supplementary material, which is available to authorized users.
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We sequenced 8 melanoma exomes to identify novel somatic mutations in metastatic melanoma.,Focusing on the MAP3K family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9.,Structural modelling predicts that mutations in the kinase domain may affect the activity and regulation of MAP3K5/9 protein kinases.,The position of the mutations and loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely inactivating.,In vitro kinase assay shows reduction in kinase activity in MAP3K5 I780F and MAP3K9 W333X mutants.,Overexpression of MAP3K5 or MAP3K9 mutant in HEK293T cells reduces phosphorylation of downstream MAP kinases.,Attenuation of MAP3K9 function in melanoma cells using siRNA leads to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.
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To investigate the feasibility of using bevacizumab to improve the survival of American Joint Committee on Cancer (AJCC) stage III melanoma patients, we investigated how a single bevacizumab treatment affected nodal disease and a panel of biomarkers in clinically fluorodeoxyglucose positron emission tomography (FDG-PET)/computed tomography (CT)-staged, stage III melanoma patients, prior to therapeutic lymph node dissection (TLND).,Four weeks before TLND, nine patients (median age 50, range 28.8-62.1 years; two male, seven female) with palpable lymph node metastases received 7.5 mg/kg bevacizumab.,Before and after this treatment, all patients were assessed by measurements of the maximum standardized uptake value (SUVmax) by FDG-PET scan, and serum S-100B and lactate dehydrogenase (LDH).,After TLND, the dissection specimen was analyzed for number of removed lymph nodes, number of metastatic lymph nodes, and tumor necrosis.,Median follow-up was 15.5 (2.2-32.9) months.,Histopathological analysis revealed tumor necrosis in six patients, of whom five had an S-100B decline and one had an unchanged S-100B level after bevacizumab.,The other three patients showed an S-100B increase and no necrosis.,Tumor necrosis was correlated with S-100B decrease (P = 0.048).,No association was found between necrosis and the markers SUVmax and LDH.,No wound healing disturbances were encountered.,Tumor necrosis in dissection specimens was associated with declining S-100B levels, while elevated S-100B was only found in cases with no necrosis.,Bevacizumab might be useful in treating AJCC stage III melanoma patients prior to TLND, and S100-B appears to be a useful marker for assessment of treatment effects.
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NME1 is a metastasis-suppressor gene (MSG), capable of suppressing metastatic activity in cell lines of melanoma, breast carcinoma and other cancer origins without affecting their growth in culture or as primary tumours.,Herein, we selectively ablated the tandemly arranged Nme1 and Nme2 genes to assess their individual impacts on metastatic activity in a mouse model (HGF:p16−/−) of ultraviolet radiation (UVR)-induced melanoma.,Metastatic activity was strongly enhanced in both genders of Nme1- and Nme2-null mice, with stronger activity in females across all genotypes.,The study ascribes MSG activity to Nme2 for the first time in an in vivo model of spontaneous cancer, as well as a novel metastasis-suppressor function to Nme1 in the specific context of UVR-induced melanoma.
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To identify ‘melanoma-specific’ microRNAs (miRNAs) we used an unbiased microRNA profiling approach to comprehensively study cutaneous melanoma in relation to other solid malignancies, which revealed 233 differentially expressed (≥2 fold, p < 0.05) miRNAs.,Among the top 20 most significantly different miRNAs was hsa-miR-514a-3p. miR-514a is a member of a cluster of miRNAs (miR-506-514) involved in initiating melanocyte transformation and promotion of melanoma growth.,We found miR-514a was expressed in 38/55 (69%) melanoma cell lines but in only 1/34 (3%) other solid cancers.,To identify miR-514a regulated targets we conducted a miR-514a-mRNA ‘pull-down’ experiment, which revealed hundreds of genes, including: CTNNB1, CDK2, MC1R, and NF1, previously associated with melanoma.,NF1 was selected for functional validation because of its recent implication in acquired resistance to BRAFV600E-targeted therapy.,Luciferase-reporter assays confirmed NF1 as a direct target of miR-514a and over-expression of miR-514a in melanoma cell lines inhibited NF1 expression, which correlated with increased survival of BRAFV600E cells treated with PLX4032.,These data provide another mechanism for the dysregulation of the MAPK pathway which may contribute to the profound resistance associated with current RAF-targeted therapies.
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Uveal melanoma is a malignant tumor of the eye that often metastasizes to the liver conferring poor prognosis.,When comparing immune profiles in peripheral blood of untreated patients with uveal melanoma liver metastasis and healthy blood donors, it was observed that immune cells of uveal melanoma patients carried immunosuppressive features.,Patient blood contained an increased content of CD14+HLA-DR−/low M-MDSCs and inflammatory CD16+ monocytes, while their dendritic cells expressed lower levels of activation markers.,Melanoma patients also harbored an enhanced fraction of CD4+Foxp3+ regulatory T cells, while their effector T cells expressed lower levels of the activation marker HLA-DR.,Biopsies from liver metastases were obtained from patients with uveal melanoma that subsequently underwent hyperthermic isolated hepatic perfusion (IHP) with melphalan.,There were trends indicating a positive correlation between a high infiltration of CD8+ T cells in metastases and an activated immune cell profile in blood.,High metastatic infiltration of CD8+ T cells and CD68+ macrophages, but not of immunosuppressive CD163+ macrophages, correlated to a longer overall survival in patients treated with IHP.,Hence, while the immune system of patients with uveal melanoma shows signs of immunosuppression, the presence of activated immune cells may correlate to a longer survival, at least following IHP treatment.
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Uveal melanoma (UM), a rare cancer of the eye, is distinct from cutaneous melanoma by its etiology, the mutation frequency and profile, and its clinical behavior including resistance to targeted therapy and immune checkpoint blockers.,Primary disease is efficiently controlled by surgery or radiation therapy, but about half of UMs develop distant metastasis mostly to the liver.,Survival of patients with metastasis is below 1 year and has not improved in decades.,Recent years have brought a deep understanding of UM biology characterized by initiating mutations in the G proteins GNAQ and GNA11.,Cytogenetic alterations, in particular monosomy of chromosome 3 and amplification of the long arm of chromosome 8, and mutation of the BRCA1-associated protein 1, BAP1, a tumor suppressor gene, or the splicing factor SF3B1 determine UM metastasis.,Cytogenetic and molecular profiling allow for a very precise prognostication that is still not matched by efficacious adjuvant therapies.,G protein signaling has been shown to activate the YAP/TAZ pathway independent of HIPPO, and conventional signaling via the mitogen-activated kinase pathway probably also contributes to UM development and progression.,Several lines of evidence indicate that inflammation and macrophages play a pro-tumor role in UM and in its hepatic metastases.,UM cells benefit from the immune privilege in the eye and may adopt several mechanisms involved in this privilege for tumor escape that act even after leaving the niche.,Here, we review the current knowledge of the biology of UM and discuss recent approaches to UM treatment.
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Metastatic melanoma is a highly heterogeneous tumor; thus, methods to analyze tumor-derived cells circulating in blood should address this diversity.,Taking this into account, we analyzed, using multiparametric flow cytometry, the co-expression of the melanoma markers melanoma cell adhesion molecule and melanoma-associated chondroitin sulphate proteoglycan and the tumor-initiating markers ATP-binding cassette sub-family B member 5 (ABCB5), CD271, and receptor activator of NF-κβ (RANK) in individual circulating tumor cells (CTCs) from 40 late-stage (III-IV) and 16 early-stage (I-II) melanoma patients.,CTCs were heterogeneous within and between patients, with limited co-expression between the five markers analyzed.,Analysis of patient matched blood and metastatic tumors revealed that ABCB5 and RANK subpopulations are more common among CTCs than in the solid tumors, suggesting a preferential selection for these cells in circulation.,Pairwise comparison of CTC subpopulations longitudinally before and 6-13 weeks after treatment initiation showed that the percentage of RANK+ CTCs significantly increased in the patients undergoing targeted therapy (N=16, P<0.01).,Moreover, the presence of ⩾5 RANK+ CTCs in the blood of patients undergoing targeted therapies was prognostic of shorter progression-free survival (hazards ratio 8.73, 95% confidence interval 1.82-41.75, P<0.01).,Taken together, our results provide evidence of the heterogeneity among CTC subpopulations in melanoma and the differential response of these subpopulations to targeted therapy.
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A fundamental problem in cancer research is identifying the cell type that is capable of sustaining neoplastic growth and its origin from normal tissue cells.,Recent investigations of a variety of tumor types have shown that phenotypically identifiable and isolable subfractions of cells possess the tumor-forming ability.,In the present paper, using two lineage-related human melanoma cell lines, primary melanoma line IGR39 and its metastatic derivative line IGR37, two main observations are reported.,The first one is the first phenotypic evidence to support the origin of melanoma cancer stem cells (CSCs) from mutated tissue-specific stem cells; and the second one is the identification of a more aggressive subpopulation of CSCs in melanoma that are CXCR6+.,We defined CXCR6 as a new biomarker for tissue-specific stem cell asymmetric self-renewal.,Thus, the relationship between melanoma formation and ABCG2 and CXCR6 expression was investigated.,Consistent with their non-metastatic character, unsorted IGR39 cells formed significantly smaller tumors than unsorted IGR37 cells.,In addition, ABCG2+ cells produced tumors that had a 2-fold greater mass than tumors produced by unsorted cells or ABCG2- cells.,CXCR6+ cells produced more aggressive tumors.,CXCR6 identifies a more discrete subpopulation of cultured human melanoma cells with a more aggressive MCSC phenotype than cells selected on the basis of the ABCG2+ phenotype alone.,The association of a more aggressive tumor phenotype with asymmetric self-renewal phenotype reveals a previously unrecognized aspect of tumor cell physiology.,Namely, the retention of some tissue-specific stem cell attributes, like the ability to asymmetrically self-renew, impacts the natural history of human tumor development.,Knowledge of this new aspect of tumor development and progression may provide new targets for cancer prevention and treatment.
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The tumor microenvironment (TME) plays a critical role in tumorigenesis, development, and therapeutic efficacy.,Major advances have been achieved in the treatment of various cancers through immunotherapy.,Nevertheless, only a minority of patients have positive responses to immunotherapy, which is partly due to conditions of the immunosuppressive microenvironment.,Therefore, it is essential to identify prognostic biomarkers that reflect heterogeneous landscapes of the TME.,Based upon the ESTIMATE algorithm, we evaluated the infiltrating levels of immune and stromal components derived from patients afflicted by various types of cancer from The Cancer Genome Atlas database (TCGA).,According to respective patient immune and stromal scores, we categorized cases into high‐ and low‐scoring subgroups for each cancer type to explore associations between TME and patient prognosis.,Gene Set Enrichment Analyses (GSEA) were conducted and genes enriched in IFNγ response signaling pathway were selected to facilitate establishment of a risk model for predicting overall survival (OS).,Furthermore, we investigated the associations between the prognostic signature and tumor immune infiltration landscape by using CIBERSORT algorithm and TIMER database.,Among the cancers assessed, the immune scores for skin cutaneous melanoma (SKCM) were the most significantly correlated with patients' survival time (P < .0001).,We identified and validated a five‐IFNγ response‐related gene signature (UBE2L6, PARP14, IFIH1, IRF2, and GBP4), which was closely correlated with the prognosis for SKCM afflicted patients.,Multivariate Cox regression analysis indicated that this risk model was an independent prognostic factor for SKCM.,Tumor‐infiltrating lymphocytes and specific immune checkpoint molecules had notably differential levels of expression in high‐ compared to low‐risk samples.,In this study, we established a novel five‐IFNγ response‐related gene signature that provided a better and increasingly comprehensive understanding of tumor immune landscape, and which demonstrated good performance in predicting outcomes for patients afflicted by SKCM.,We established an effective five‐IFNγ response‐related gene‐based risk score, which has potential to be a novel prognostic signature and may provide insight into tumor immune microenvironment of cutaneous melanoma.
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An impressive clinical success has been observed in treating a variety of cancers using immunotherapy with programmed cell death‐1 (PD‐1) checkpoint blockade.,However, limited response in most patients treated with anti‐PD‐1 antibodies remains a challenge, requiring better understanding of molecular mechanisms limiting immunotherapy.,In colorectal cancer (CRC) resistant to immunotherapy, mismatch‐repair‐proficient or microsatellite instability‐low (pMMR‐MSI‐L) tumors have low mutation burden and constitute ~85% of patients.,Here, we show that inhibition of N 6‐methyladenosine (m6A) mRNA modification by depletion of methyltransferases, Mettl3 and Mettl14, enhanced response to anti‐PD‐1 treatment in pMMR‐MSI‐L CRC and melanoma.,Mettl3‐ or Mettl14‐deficient tumors increased cytotoxic tumor‐infiltrating CD8+ T cells and elevated secretion of IFN‐γ, Cxcl9, and Cxcl10 in tumor microenvironment in vivo.,Mechanistically, Mettl3 or Mettl14 loss promoted IFN‐γ‐Stat1‐Irf1 signaling through stabilizing the Stat1 and Irf1 mRNA via Ythdf2.,Finally, we found a negative correlation between METTL3 or METTL14 and STAT1 in 59 patients with pMMR‐MSI‐L CRC tumors.,Altogether, our findings uncover a new awareness of the function of RNA methylation in adaptive immunity and provide METTL3 and METTL14 as potential therapeutic targets in anticancer immunotherapy.,Disruption of m6A methyltransferases leads to enhanced immunotherapy response in colorectal cancer and melanoma cells due to enhanced IFN‐γ‐Stat1‐Irf1 signaling and modulation of the tumor microenvironment.
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Effective treatment options are limited for patients with advanced (metastatic or unresectable) melanoma who progress after immune checkpoint inhibitors and targeted therapies.,Adoptive cell therapy using tumor-infiltrating lymphocytes has demonstrated efficacy in advanced melanoma.,Lifileucel is an autologous, centrally manufactured tumor-infiltrating lymphocyte product.,We conducted a phase II open-label, single-arm, multicenter study in patients with advanced melanoma who had been previously treated with checkpoint inhibitor(s) and BRAF ± MEK targeted agents.,Lifileucel was produced from harvested tumor specimens in central Good Manufacturing Practice facilities using a streamlined 22-day process.,Patients received a nonmyeloablative lymphodepletion regimen, a single infusion of lifileucel, and up to six doses of high-dose interleukin-2.,The primary end point was investigator-assessed objective response rate (ORR) per RECIST, version 1.1.,Sixty-six patients received a mean of 3.3 prior therapies (anti-programmed death 1 [PD-1] or programmed death ligand 1 [PD-L1]: 100%; anticytotoxic T-lymphocyte-associated protein-4: 80%; BRAF ± MEK inhibitor: 23%).,The ORR was 36% (95% CI, 25 to 49), with two complete responses and 22 partial responses.,Disease control rate was 80% (95% CI, 69 to 89).,Median duration of response was not reached after 18.7-month median study follow-up (range, 0.2-34.1 months).,In the primary refractory to anti-PD-1 or PD-L1 therapy subset, the ORR and disease control rate were 41% (95% CI, 26 to 57) and 81% (95% CI, 66 to 91), respectively.,Safety profile was consistent with known adverse events associated with nonmyeloablative lymphodepletion and interleukin-2.,Lifileucel demonstrated durable responses and addresses a major unmet need in patients with metastatic melanoma with limited treatment options after approved therapy, including the primary refractory to anti-PD-1 or PD-L1 therapy subset.
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The PI 3-kinase (PI3K) pathway has been implicated as a target for melanoma therapy.,Given the high degree of genetic heterogeneity in melanoma, we sought to understand the breadth of variation in PI3K signalling in the large NZM panel of early passage cell lines developed from metastatic melanomas.,We find the vast majority of lines show upregulation of this pathway, and this upregulation is achieved by a wide range of mechanisms.,Expression of all class-IA PI3K isoforms was readily detected in these cell lines.,A range of genetic changes in different components of the PI3K pathway was seen in different lines.,Coding variants or amplification were identified in the PIK3CA gene, and amplification of the PK3CG gene was common.,Deletions in the PIK3R1 and PIK3R2 regulatory subunits were also relatively common.,Notably, no genetic variants were seen in the PIK3CD gene despite p110δ being expressed in many of the lines.,Genetic variants were detected in a number of genes that encode phosphatases regulating the PI3K signalling, with reductions in copy number common in PTEN, INPP4B, INPP5J, PHLLP1 and PHLLP2 genes.,While the pan-PI3K inhibitor ZSTK474 attenuated cell growth in all the lines tested, isoform-selective inhibition of p110α and p110δ inhibited cell growth in only a subset of the lines and the inhibition was only partial.,This suggests that functional redundancy exists between PI3K isoforms.,Furthermore, while ZSTK474 was initially effective in melanoma cells with induced resistance to vemurafenib, a subset of these cell lines concurrently developed partial resistance to PI3K inhibition.,Importantly, mTOR-selective or mTOR/PI3K dual inhibitors effectively inhibited cell growth in all the lines, including those already resistant to BRAF inhibitors and ZSTK474.,Overall, this indicates a high degree of diversity in the way the PI3K pathway is activated in different melanoma cell lines and that mTOR is the most effective point for targeting the growth via the PI3K pathway across all of these cell lines.,The online version contains supplementary material available at 10.1186/s12885-021-07826-4.
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The effect of apatinib on the formation of vasculogenic mimicry (VM) was studied in a malignant melanoma cell line.,MUM-2B cells cultured in three-dimensional Matrigel were treated with varying concentrations (0, 0.01, 0.05, 0.1, 0.5 μmol/L) of apatinib to test its effect on VM in vitro, followed by MTT proliferation and transwell invasion assays to determine the effect of apatinib on cell proliferation and invasion of MUM-2B cells.,In vivo, we used a melanoma cancer model to test the effect of short-term apatinib (100, 200, 300 mg/kg) treatment on VM.,Western blotting, immunohistochemistry staining, and CD31-PAS dual staining were performed to assess the expression of VEGFR-2, ERK-1/2, PI3K, and MMP-2, and formation of VM.,The results showed apatinib-treated groups formed a lesser number of VM in 3D matrigel, while the cell viability in MTT proliferation assay and the number of migration cells in transwell invasion assay were significantly lower in apatinib-treated groups.,In addition, short-term apatinib treatment inhibited angiogenesis, VM formation, and tumor growth in models of melanoma cancer.,Mice in apatinib-treated groups showed a markedly reduced expression of VEGFR-2, ERK-1/2, PI3K, and MMP-2.,In summary, apatinib could inhibit the expression of VEGFR-2, and downregulate the ERK1/2/PI3K/MMP-2 signaling cascade, which may be one of the underlying mechanisms by which apatinib inhibits angiogenesis and the development of VM in models of melanoma cancer, and restrains the formation of VM by MUM-2B cells.,Apatinib shows inhibitory effects on cell proliferation and invasion of MUM-2B cells, which is a close relationship with the VM.
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Cutaneous melanoma is a metastatic disease characterized by high resistance to treatment, the incidence of which has alarmingly increased worldwide over the past years.,A thorough characterization of tumor onset, progression and metastasis is compulsory to overcome the gaps existent in melanoma biology.,The present study suggests a well-established protocol and a detailed histological description of human melanoma models in ovo and in vivo obtained by the inoculation of A375 cells to chick embryo chorioallantoic membrane (CAM) and Balb/c nude mice.,The inoculation of A375 cells on CAM led to the formation of compact primary and secondary tumors on day 4 post-inoculation, with mean surface area values of 2.2±0.4 mm2 and 1.5±0.3 mm2, respectively.,Moreover, the vessels around the tumors presented a spike wheel pattern, indicating a strong angiogenic reaction.,All the injected mice, apart from one, developed solid polypoid primary tumors with lobulated surfaces and intense vascularization, and achromic epithelioid malignant melanocytes with vesiculous nuclei and necrosis area were detected.,Metastasis was histologically confirmed in only 30% of the mice with the tumor xenografts.,These data indicate that the standardization protocols proposed are complex and reproducible, and can be further employed for the therapeutic surveillance of antiangiogenic and anticancer agents.
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Up to 50% of patients with uveal melanoma (UM) develop metastatic disease with limited treatment options.,The immunomodulating agent ipilimumab has shown an overall survival (OS) benefit in patients with cutaneous metastatic melanoma in two phase III trials.,As patients with UM were excluded in these studies, the Dermatologic Cooperative Oncology Group (DeCOG) conducted a phase II to assess the efficacy and safety of ipilimumab in patients with metastatic UM.,We undertook a multicenter phase II study in patients with different subtypes of metastatic melanoma.,Here we present data on patients with metastatic UM (pretreated and treatment-naïve) who received up to four cycles of ipilimumab administered at a dose of 3 mg/kg in 3 week intervals.,Tumor assessments were conducted at baseline, weeks 12, 24, 36 and 48 according to RECIST 1.1 criteria.,Adverse events (AEs), including immune-related AEs were graded according to National Cancer Institute Common Toxicity Criteria (CTC) v.4.0.,Primary endpoint was the OS rate at 12 months.,Forty five pretreated (85%) and eight treatment-naïve (15%) patients received at least one dose of ipilimumab. 1-year and 2-year OS rates were 22% and 7%, respectively.,Median OS was 6.8 months (95% CI 3.7-8.1), median progression-free survival 2.8 months (95% CI 2.5-2.9).,The disease control rate at weeks 12 and 24 was 47% and 21%, respectively.,Sixteen patients had stable disease (47%), none experienced partial or complete response.,Treatment-related AEs were observed in 35 patients (66%), including 19 grade 3-4 events (36%).,One drug-related death due to pancytopenia was observed.,Ipilimumab has very limited clinical activity in patients with metastatic UM.,Toxicity was manageable when treated as per protocol-specific guidelines.,ClinicalTrials.gov NCT01355120
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Arginine-depleting therapy with pegylated arginine deiminase (ADI-PEG20) was reported to have activity in advanced melanoma in early phase I-II trial, and clinical trials are currently underway in other cancers.,However, the optimal patient population who benefit from this treatment is unknown.,Advanced melanoma patients with accessible tumours had biopsy performed before the start of treatment with ADI-PEG20 and at the time of progression or relapse when amenable to determine whether argininosuccinate synthetase (ASS) expression in tumour was predictive of response to ADI-PEG20.,Twenty-seven of thirty-eight patients treated had melanoma tumours assessable for ASS staining before treatment.,Clinical benefit rate (CBR) and longer time to progression were associated with negative expression of tumour ASS.,Only 1 of 10 patients with ASS-positive tumours (ASS+) had stable disease, whereas 4 of 17 (24%) had partial response and 5 had stable disease, when ASS expression was negative (ASS−), giving CBR rates of 52.9 vs 10%, P=0.041.,Two responding patients with negative ASS expression before therapy had rebiopsy after tumour progression and the ASS expression became positive.,The survival of ASS− patients receiving at least four doses at 320 IU m−2 was significantly better than the ASS+ group at 26.5 vs 8.5 months, P=0.024.,ADI-PEG20 is safe and the drug is only efficacious in melanoma patients whose tumour has negative ASS expression.,Argininosuccinate synthetase tumour positivity is associated with drug resistance and tumour progression.
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Alterations in transcriptional programs promote tumor development and progression and are targetable by bromodomain and extraterminal (BET) protein inhibitors.,However, in a multi‐site clinical trial testing the novel BET inhibitor, PLX51107, in solid cancer patients, liver metastases of uveal melanoma (UM) patients progressed rapidly following treatment.,Mechanisms of resistance to BET inhibitors in UM are unknown.,We show that fibroblast growth factor 2 (FGF2) rescued UM cells from growth inhibition by BET inhibitors, and FGF2 effects were reversible by FGF receptor (FGFR) inhibitors.,BET inhibitors also increased FGFR protein expression in UM cell lines and in patient tumor samples.,Hepatic stellate cells (HSCs) secrete FGF2, and HSC‐conditioned medium provided resistance of UM cells to BET inhibitors.,PLX51107 was ineffective in vivo, but the combination of a FGFR inhibitor, AZD4547, and PLX51107 significantly suppressed the growth of xenograft UM tumors formed from subcutaneous inoculation of UM cells with HSCs and orthotopically in the liver.,These results suggest that co‐targeting of FGFR signaling is required to increase the responses of metastatic UM to BET inhibitors.
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Activated RAS promotes dimerization of members of the RAF kinase family1-3.,ATP-competitive RAF inhibitors activate ERK signaling4-7 by transactivating RAF dimers4.,In melanomas with mutant BRAF(V600E), levels of RAS activation are low and these drugs bind to BRAF(V600E) monomers and inhibit their activity.,This tumor-specific inhibition of ERK signaling results in a broad therapeutic index and RAF inhibitors have remarkable clinical activity in patients with melanomas that harbor mutant BRAF(V600E)8.,However, resistance invariably develops.,Here, we identify a novel resistance mechanism.,We find that a subset of cells resistant to vemurafenib (PLX4032, RG7204) express a 61kd variant form of BRAF(V600E) that lacks exons 4-8, a region that encompasses the RAS-binding domain. p61BRAF(V600E) exhibits enhanced dimerization in cells with low levels of RAS activation, as compared to full length BRAF(V600E).,In cells in which p61BRAF(V600E) is expressed endogenously or ectopically, ERK signaling is resistant to the RAF inhibitor.,Moreover, a mutation that abolishes the dimerization of p61BRAF(V600E) restores its sensitivity to vemurafenib.,Finally, we identified BRAF(V600E) splicing variants lacking the RAS-binding domain in the tumors of six of 19 patients with acquired resistance to vemurafenib.,These data support the model that inhibition of ERK signaling by RAF inhibitors is dependent on levels of RAS-GTP too low to support RAF dimerization and identify a novel mechanism of acquired resistance in patients: expression of splicing isoforms of BRAF(V600E) that dimerize in a RAS-independent manner.
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The melanoma-specific graded prognostic assessment (msGPA) assigns patients with brain metastases from malignant melanoma to 1 of 4 prognostic groups.,It was largely derived using clinical data from patients treated in the era that preceded the development of newer therapies such as BRAF, MEK and immune checkpoint inhibitors.,Therefore, its current relevance to patients diagnosed with brain metastases from malignant melanoma is unclear.,This study is an external validation of the msGPA in two temporally distinct British populations.,Performance of the msGPA was assessed in Cohort I (1997-2008, n=231) and Cohort II (2008-2013, n=162) using Kaplan-Meier methods and Harrell's c-index of concordance.,Cox regression was used to explore additional factors that may have prognostic relevance.,The msGPA does not perform well as a prognostic score outside of the derivation cohort, with suboptimal statistical calibration and discrimination, particularly in those patients with an intermediate prognosis.,Extra-cerebral metastases, leptomeningeal disease, age and potential use of novel targeted agents after brain metastases are diagnosed, should be incorporated into future prognostic models.,An improved prognostic score is required to underpin high-quality randomised controlled trials in an area with a wide disparity in clinical care.
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To retrospectively access outcome and prognostic parameters of linear accelerator-based stereotactic radiosurgery in brain metastases from malignant melanoma.,Between 1990 and 2011 140 brain metastases in 84 patients with malignant melanoma (median age 56 years) were treated with stereotactic radiosurgery.,At initial stereotactic radiosurgery 48 % of patients showed extracerebral control.,The median count of brain metastases in a single patient was 1, the median diameter was 12 mm.,The median dose applied was 20 Gy/80 % isodose enclosing.,The median follow-up was 7 months and the median overall survival 9 months.,The 6-, 12- and 24 month overall survival rates were 71 %, 39 % and 25 % respectively.,Cerebral follow-up imaging showed complete remission in 20 brain metastases, partial remission in 39 brain metastases, stable disease in 54 brain metastases, progressive disease in 24 brain metastases and pseudo-progression in 3 brain metastases.,Median intracerebral control was 5.3 months and the 6- and 12-month intracerebral progression-free survival rates 48 % and 38 %, respectively.,Upon univariate analysis, extracerebral control (log-rank, p < 0.001), the response to stereotactic radiosurgery (log-rank, p < 0.001), the number of brain metastases (log-rank, p = 0.007), the recursive partitioning analysis class (log-rank, p = 0.027) and the diagnosis-specific graded prognostic assessment score (log-rank, p = 0.011) were prognostic for overall survival.,The most common clinical side effect was headache common toxicity criteria grade I.,The most common radiological finding during follow-up was localized edema within the stereotactic radiosurgery high dose region.,Stereotactic radiosurgery is a well-tolerated and effective treatment option for brain metastases in malignant melanoma and was able to achieve local remissions in several cases.,Furthermore, especially patients with controlled extracerebral disease and a low count of brain metastases seem to benefit from this treatment modality.,Prospective trials analysing the effects of combined stereotactic radiosurgery and new systemic agents are warranted.
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The application of immune-based therapies has revolutionized cancer treatment.,Yet how the immune system responds to phenotypically heterogeneous populations within tumors is poorly understood.,In melanoma, one of the major determinants of phenotypic identity is the lineage survival oncogene MITF that integrates diverse microenvironmental cues to coordinate melanoma survival, senescence bypass, differentiation, proliferation, invasion, metabolism and DNA damage repair.,Whether MITF also controls the immune response is unknown.,By using several mouse melanoma models, we examine the potential role of MITF to modulate the anti-melanoma immune response.,ChIP-seq data analysis, ChIP-qPCR, CRISPR-Cas9 genome editing, and luciferase reporter assays were utilized to identify ADAM10 as a direct MITF target gene.,Western blotting, confocal microscopy, flow cytometry, and natural killer (NK) cytotoxicity assays were used to determine the underlying mechanisms by which MITF-driven phenotypic plasticity modulates melanoma NK cell-mediated killing.,Here we show that MITF regulates expression of ADAM10, a key sheddase that cleaves the MICA/B family of ligands for NK cells.,By controlling melanoma recognition by NK-cells MITF thereby controls the melanoma response to the innate immune system.,Consequently, while melanoma MITFLow cells can be effectively suppressed by NK-mediated killing, MITF-expressing cells escape NK cell surveillance.,Our results reveal how modulation of MITF activity can impact the anti-melanoma immune response with implications for the application of anti-melanoma immunotherapies.,The online version contains supplementary material available at 10.1186/s13046-021-01916-8.
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How targeted therapies and immunotherapies shape tumours and thereby influence subsequent therapeutic responses is poorly understood.,Here, we show in melanoma patients and mouse models that when tumours relapse following targeted therapy with MAPK pathway inhibitors, they are cross-resistant to immunotherapies, despite the different modes of action of these therapies.,We find that cross-resistance is mediated by a cancer cell-instructed, immunosuppressive tumour microenvironment that lacks functional CD103+ dendritic cells, precluding an effective T cell response.,Restoring the numbers and functionality of CD103+ dendritic cells can re-sensitize cross-resistant tumours to immunotherapy.,Cross-resistance does not arise from selective pressure of an immune response during evolution of resistance, but from the MAPK pathway, which is not only reactivated, but also exhibits an increased transcriptional output that drives immune evasion.,Our work provides mechanistic evidence for cross-resistance between two unrelated therapies, and a scientific rationale for treating patients with immunotherapy before they acquire resistance to targeted therapy.
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Patients with high-risk stage II/III resected melanoma commonly develop distant metastases.,At present, we cannot differentiate between patients who will recur or those who are cured by surgery.,We investigated if circulating tumor DNA (ctDNA) can predict relapse and survival in patients with resected melanoma.,We carried out droplet digital polymerase chain reaction to detect BRAF and NRAS mutations in plasma taken after surgery from 161 stage II/III high-risk melanoma patients enrolled in the AVAST-M adjuvant trial.,Mutant BRAF or NRAS ctDNA was detected (≥1 copy of mutant ctDNA) in 15/132 (11%) BRAF mutant patient samples and 4/29 (14%) NRAS mutant patient samples.,Patients with detectable ctDNA had a decreased disease-free interval [DFI; hazard ratio (HR) 3.12; 95% confidence interval (CI) 1.79-5.47; P < 0.0001] and distant metastasis-free interval (DMFI; HR 3.22; 95% CI 1.80-5.79; P < 0.0001) versus those with undetectable ctDNA.,Detectable ctDNA remained a significant predictor after adjustment for performance status and disease stage (DFI: HR 3.26, 95% CI 1.83-5.83, P < 0.0001; DMFI: HR 3.45, 95% CI 1.88-6.34, P < 0.0001).,Five-year overall survival rate for patients with detectable ctDNA was 33% (95% CI 14%-55%) versus 65% (95% CI 56%-72%) for those with undetectable ctDNA.,Overall survival was significantly worse for patients with detectable ctDNA (HR 2.63; 95% CI 1.40-4.96); P = 0.003) and remained significant after adjustment for performance status (HR 2.50, 95% CI 1.32-4.74, P = 0.005).,ctDNA predicts for relapse and survival in high-risk resected melanoma and could aid selection of patients for adjuvant therapy.,ISRCTN 81261306
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There is limited data on the efficacy of anti-programmed death 1 (PD-1) antibodies in patients (pts) with melanoma brain metastasis (BM), particularly those which are symptomatic.,We retrospectively assessed pts with melanoma BM treated with PD-1 antibodies, nivolumab and pembrolizumab.,Clinicopathologic and treatment parameters were collected and outcomes determined for intracranial (IC) response rate (RR) using a modified RECIST criteria, with up to five IC target lesions used to determine IC response, disease control rate (DCR) and progression-free survival (PFS).,A total of 66 pts were identified with a median follow up of 7.0 months (range 0.8-24.5 months).,A total of 68% were male and 45% BRAF V600 mutation positive.,At PD-1 antibody commencement, 50% had an elevated LDH; 64% had local therapy to BM prior to commencing anti-PD1, of which 5% had surgical resection, 14% stereotactic radiosurgery (SRS), 18% whole-brain radiotherapy (WBRT), 27% had surgery and radiotherapy.,Twenty-one per cent started anti-PD-1 as first line systemic therapy.,No pt had prior anti-PD-1 treatment.,The IC overall RR was 21 and DCR 56%.,Responses occurred in 21% of pts with symptomatic BM.,The median OS was 9.9 months (95% CI 6.93-17.74).,Pts with symptomatic BM had shorter PFS than those without symptoms (2.7 vs 7.4 months, P=0.035) and numerically shorter OS (5.7 vs 13.0 months, P=0.068).,Pts requiring corticosteroids also had a numerically shorter PFS (3.2 vs 7.4 months, P=0.081) and OS (4.8 vs 13.1 months, P=0.039).,IC responses to anti-PD-1 antibodies occur in pts with BM, including those with symptomatic BM requiring corticosteroids.,Prospective trials evaluating anti-PD-1 therapy in pts with BM are underway.
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Dendritic cell (DC)-based vaccination effectively induces anti-tumor immunity, although in the majority of cases this does not translate into a durable clinical response.,However, DC vaccination is characterized by a robust safety profile, making this treatment a potential candidate for effective combination cancer immunotherapy.,To explore this possibility, understanding changes occurring in the tumor microenvironment (TME) upon DC vaccination is required.,In this line, quantitative and qualitative changes in tumor-infiltrating T lymphocytes (TILs) induced by vaccination with autologous tumor lysate/homogenate loaded DCs were investigated in a series of 16 patients with metastatic melanoma.,Immunohistochemistry for CD4, CD8, Foxp3, Granzyme B (GZMB), PDL1, and HLA class I was performed in tumor biopsies collected before and after DC vaccination.,The density of each marker was quantified by automated digital pathology analysis on whole slide images.,Co-expression of markers defining functional phenotypes, i.e., Foxp3+ regulatory CD4+ T cells (Treg) and GZMB+ cytotoxic CD8+ T cells, was assessed with sequential immunohistochemistry.,A significant increase of CD8+ TILs was found in post-vaccine biopsies of patients who were not previously treated with immune-modulating cytokines or Ipilimumab.,Interestingly, along with a maintained tumoral HLA class I expression, after DC vaccination we observed a significant increase of PDL1+ tumor cells, which significantly correlated with intratumoral CD8+ T cell density.,This observation might explain the lack of a significant concurrent cytotoxic reactivation of CD8+ T cell, as measured by the numbers of GZMB+ T cells.,Altogether these findings indicate that DC vaccination exerts an important role in sustaining or de novo inducing a T cell inflamed TME.,However, the strength of the intratumoral T cell activation detected in post-DC therapy lesions is lessened by an occurring phenomenon of adaptive immune resistance, yet the concomitant PDL1 up-regulation.,Overall, this study sheds light on DC immunotherapy-induced TME changes, lending the rationale for the design of smarter immune-combination therapies.
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Epacadostat is a potent inhibitor of the immunosuppressive indoleamine 2,3-dioxygenase 1 (IDO1) enzyme.,We present phase 1 results from a phase 1/2 clinical study of epacadostat in combination with ipilimumab, an anti-cytotoxic T-lymphocyte-associated protein 4 antibody, in advanced melanoma (NCT01604889).,Only the phase 1, open-label portion of the study was conducted, per the sponsor’s decision to terminate the study early based on the changing melanoma treatment landscape favoring exploration of programmed cell death protein 1 (PD-1)/PD-ligand 1 inhibitor-based combination strategies.,Such decision was not related to the safety of epacadostat plus ipilimumab.,Patients received oral epacadostat (25, 50, 100, or 300 mg twice daily [BID]; 75 mg daily [50 mg am, 25 mg pm]; or 50 mg BID intermittent [2 weeks on/1 week off]) plus intravenous ipilimumab 3 mg/kg every 3 weeks.,Fifty patients received ≥1 dose of epacadostat.,As of January 20, 2017, 2 patients completed treatment and 48 discontinued, primarily because of adverse events (AEs) and disease progression (n = 20 each).,Dose-limiting toxicities occurred in 11 patients (n = 1 each with epacadostat 25 mg BID, 50 mg BID intermittent, 75 mg daily; n = 4 each with epacadostat 50 mg BID, 300 mg BID).,The most common immune-related treatment-emergent AEs included rash (50%), alanine aminotransferase elevation (28%), pruritus (28%), aspartate aminotransferase elevation (24%), and hypothyroidism (10%).,Among immunotherapy-naive patients (n = 39), the objective response rate was 26% by immune-related response criteria and 23% by Response Evaluation Criteria in Solid Tumors version 1.1.,No objective response was seen in the 11 patients who received prior immunotherapy.,Epacadostat exposure was dose proportional, with clinically significant IDO1 inhibition at doses ≥25 mg BID.,When combined with ipilimumab, epacadostat ≤50 mg BID demonstrated clinical and pharmacologic activity and was generally well tolerated in patients with advanced melanoma.,ClinicalTrials.gov identifier, NCT01604889.,Registration date, May 9, 2012, retrospectively registered.,The online version of this article (10.1186/s40425-019-0562-8) contains supplementary material, which is available to authorized users.
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Immunologic responses to anti-PD-1 therapy in melanoma patients occur rapidly with pharmacodynamic T cell responses detectable in blood by 3 weeks.,It is unclear, however, whether these early blood-based observations translate to the tumor microenvironment.,We conducted a study of neoadjuvant/adjuvant anti-PD-1 therapy in stage III/IV melanoma.,We hypothesized that immune reinvigoration in the tumor would be detectable at 3 weeks and this response would correlate with disease-free survival.,We identified a rapid and potent anti-tumor response, with 8/27 patients experiencing a complete or major pathological response after a single dose of anti-PD-1, all of whom remain disease-free.,These rapid pathologic and clinical responses were associated with accumulation of exhausted CD8 T cells in the tumor at 3 weeks with reinvigoration in the blood observed as early as 1 week.,Transcriptional analysis demonstrated a pre-treatment immune signature (Neoadjuvant Response Signature) that was associated with clinical benefit.,In contrast, patients with disease recurrence displayed mechanisms of resistance including immune suppression, mutational escape, and/or tumor evolution.,Neoadjuvant anti-PD-1 treatment is effective in high-risk resectable stage III/IV melanoma.,Pathological response and immunological analyses after a single neoadjuvant dose can be used to predict clinical outcome and to dissect underlying mechanisms in checkpoint blockade.
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Neoadjuvant immunotherapy utilizing novel combinations has the potential to transform the standard of care for locally/regionally advanced melanoma.,We hypothesized that neoadjuvant ipilimumab in combination with high dose IFNα2b (HDI) is safe and associated with durable pathologic complete responses (pCR).,Patients with locally/regionally advanced melanoma were randomized to ipilimumab 3 or 10 mg/kg × 4 doses bracketing definitive surgery, then every 12 weeks × 4.,HDI was given concurrently.,We evaluated the safety and efficacy of the combination with ipilimumab 3 or 10 mg/kg.,The impact on T-cell fraction and clonality were investigated in tumor and blood.,Thirty patients (age 37-76), 15 each at 3 and 10 mg/kg, 18 male and 12 female were treated.,Considering immune related adverse events (irAEs) of interest, more grade 3/4 irAEs were seen with ipilimumab 10 mg/kg versus 3 mg/kg (p = 0.042).,Among 28 evaluable patients, 11 relapsed, of whom 5 died.,Median follow-up for 17 patients who have not relapsed was 32 months.,The radiologic preoperative response rate was 36% (95% CI, 21-54); 4 patients at ipilimumab 3 mg/kg and 6 at 10 mg/kg and 2 (at 10 mg/kg) later relapsed.,The pCR was 32% (95% CI, 18-51); 5 patients at ipilimumab 3 mg/kg and 4 at 10 mg/kg and one (at 3 mg/kg) had a late relapse.,In patients with pCR, T-cell fraction was significantly higher when measured in primary melanoma tumors (p = 0.033).,Higher tumor T-cell clonality in primary tumor and more so following neoadjuvant therapy was significantly associated with improved relapse free survival.,Neoadjuvant ipilimumab-HDI was relatively safe and exhibited promising tumor response rates with an associated measurable impact on T-cell fraction and clonality.,Most pCRs were durable supporting the value of pCR as a primary endpoint in neoadjuvant immunotherapy trials.,ClinicalTrials.gov, NCT01608594.,Registered 31 May 2012.,The online version of this article (10.1186/s40425-018-0428-5) contains supplementary material, which is available to authorized users.
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Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer and frequently progresses from an actinic keratosis (AK), a sun-induced keratinocyte intraepithelial neoplasia (KIN).,Epigenetic mechanisms involved in the phenomenon of progression from AK to cSCC remain to be elicited.,Expression of microRNAs in sun-exposed skin, AK and cSCC was analysed by Agilent microarrays.,DNA methylation of miR-204 promoter was determined by bisulphite treatment and pyrosequencing.,Identification of miR-204 targets and pathways was accomplished in HaCat cells.,Immunofluorescence and immunohistochemistry were used to analyze STAT3 activation and PTPN11 expression in human biopsies.,cSCCs display a marked downregulation of miR-204 expression when compared to AK.,DNA methylation of miR-204 promoter was identified as one of the repressive mechanisms that accounts for miR-204 silencing in cSCC.,In HaCaT cells miR-204 inhibits STAT3 and favours the MAPK signaling pathway, likely acting through PTPN11, a nuclear tyrosine phosphatase that is a direct miR-204 target.,In non-peritumoral AK lesions, activated STAT3, as detected by pY705-STAT3 immunofluorescence, is retained in the membrane and cytoplasm compartments, whereas AK lesions adjacent to cSCCs display activated STAT3 in the nuclei.,Our data suggest that miR-204 may act as a “rheostat” that controls the signalling towards the MAPK pathway or the STAT3 pathway in the progression from AK to cSCC.,The online version of this article (doi:10.1186/s12943-016-0537-z) contains supplementary material, which is available to authorized users.
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Zhang et al. show that miR-125b is abundantly expressed, particularly at early stages of malignant progression to squamous cell carcinoma.,When elevated in normal murine epidermis, miR-125b promotes tumor initiation and contributes to malignant progression. mir-125b directly represses stress-responsive MAPK genes and indirectly prolongs activated EGFR signaling by repressing Vps4B, encoding a protein implicated in negatively regulating the endosomal sorting complexes that are necessary for the recycling of active EGFR.,Previously, we identified miR-125b as a key regulator of the undifferentiated state of hair follicle stem cells.,Here, we show that in both mice and humans, miR-125b is abundantly expressed, particularly at early stages of malignant progression to squamous cell carcinoma (SCC), the second most prevalent cancer worldwide.,Moreover, when elevated in normal murine epidermis, miR-125b promotes tumor initiation and contributes to malignant progression.,We further show that miR-125b can confer “oncomiR addiction” in early stage malignant progenitors by delaying their differentiation and favoring an SCC cancer stem cell (CSC)-like transcriptional program.,To understand how, we systematically identified and validated miR125b targets that are specifically associated with tumors that are dependent on miR-125b.,Through molecular and genetic analysis of these targets, we uncovered new insights underlying miR-125b’s oncogenic function.,Specifically, we show that, on the one hand, mir-125b directly represses stress-responsive MAP kinase genes and associated signaling.,On the other hand, it indirectly prolongs activated (phosphorylated) EGFR signaling by repressing Vps4b (vacuolar protein-sorting 4 homolog B), encoding a protein implicated in negatively regulating the endosomal sorting complexes that are necessary for the recycling of active EGFR.,Together, these findings illuminate miR-125b as an important microRNA regulator that is shared between normal skin progenitors and their early malignant counterparts.
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The introduction of a newly developed target therapy for metastatic melanomas poses the challenge to have a good molecular stratification of those patients who may benefit from this therapeutic option.,Practically, BRAF mutation status (V600E) is commonly screened although other non-V600E mutations (i.e., K-R-M-D) could be found in some patients who respond to therapy equally to the patients harboring V600E mutations.,Furthermore, other mutations, namely, N-RAS, KIT, and GNAQ, should be sequenced according to distinct melanoma specific subtypes and clinical aspects.,In our report, a practical flow chart is described along with our experience in this field.
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According to the prevailing multistep model of melanoma development, oncogenic BRAF or NRAS mutations are crucial initial events in melanoma development.,It is not known whether melanocytic nevi that are found in association with a melanoma are more likely to carry BRAF or NRAS mutations than uninvolved nevi.,By laser microdissection we were able to selectively dissect and genotype cells either from the nevus or from the melanoma part of 46 melanomas that developed in association with a nevus.,In 25 cases we also genotyped a control nevus of the same patients.,Available tissue was also immunostained using the BRAFV600E-mutation specific antibody VE1.,The BRAFV600E mutation was found in 63.0% of melanomas, 65.2% of associated nevi and 50.0% of control nevi.,No significant differences in the distribution of BRAF or NRAS mutations could be found between melanoma and associated nevi or between melanoma associated nevi and control nevi.,In concordant cases immunohistochemistry showed a higher expression (intensity of immunohistochemistry) of the mutated BRAFV600E-protein in melanomas compared to their associated nevi.,In this series the presence of a BRAF- or NRAS mutation in a nevus was not associated with the risk of malignant transformation.,Our findings do not support the current traditional model of stepwise tumor progression.
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The MITF(E318K) variant confers moderate risk for cutaneous melanoma.,While there are small studies suggesting that this risk is associated with other malignancies (e.g. renal cell carcinoma), little is known about the role of this variant in specifying risk for other cancers.,In this study, we perform a systematic review and meta-analysis of the published data as a backdrop to a whole-exome sequence(WES)-based characterization of MITF(E318K) risk for various cancers in sporadic samples from the TCGA and several genetically-enriched patient cohorts.,We found minimal evidence of MITF(E318K)’s contribution to non-melanoma cancer risk among individuals with low inherited risks of melanoma (OR 1.168; 95% CI 0.78-1.74; p = 0.454), suggesting that earlier reports of an association between this variant and other malignancies may be related to shared environmental or polygenic risk factors rather than MITF(E318K).,Interestingly, an association was observed with uterine carcinosarcoma, (OR 9.24; 95% CI 2.08-37.17; p = 0.024), which was not previously described.,While more research needs to be completed, this study will help update cancer screening recommendations for patients with the MITF(E318K) variant.
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The mitogen-activated protein-kinase pathway consisting of the kinases RAF, MEK, and ERK is central to cell proliferation and survival and is deregulated in more than 90% of melanomas.,MEK inhibitors are currently trialled in the clinic, but despite efficient target inhibition, cytostatic rather than cytotoxic activity limits their efficacy.,We assessed the cytotoxicity to MEK inhibitors (PD184352 and selumetinib) in melanoma cells by toluidine-blue staining, caspase 3 cleavage, and melanoma-sphere growth.,Western blotting and quantitative real-time polymerase chain reaction were applied to determine SMAD-specific E3 ubiquitin protein ligase 2 (SMURF2), PAX3, and MITF expression.,Human melanoma samples (n = 77) from various stages were analyzed for SMURF2 and PAX3 expression.,RNA interference was performed to target SMURF2 during MEK inhibition in vivo in melanoma xenografts in mice and zebrafish.,All statistical tests were two-sided.,Activation of transforming growth factor β (TGF-β) signalling sensitized melanoma cells to the cytotoxic effects of MEK inhibition.,Melanoma cells resistant to the cytotoxic effects of MEK inhibitors counteracted TGF-β signalling through overexpression of the E3 ubiquitin ligase SMURF2, which resulted in increased expression of the transcription factors PAX3 and MITF.,High MITF expression protected melanoma cells against MEK inhibitor cytotoxicity.,Depleting SMURF2 reduced MITF expression and substantially lowered the threshold for MEK inhibitor-induced apoptosis.,Moreover, SMURF2 depletion sensitized melanoma cells to the cytotoxic effects of selumetinib, leading to cell death at concentrations approximately 100-fold lower than the concentration required to induce cell death in SMURF2-expressing cells.,Mice treated with selumetinib alone at a dosage of 10mg/kg body weight once daily produced no response, but in combination with SMURF2 depletion, selumetinib suppressed tumor growth by 97.9% (95% confidence interval = 38.65% to 155.50%, P = .005).,Targeting SMURF2 may be a novel therapeutic approach for increasing the antitumor efficacy of MEK inhibitors.
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Cutaneous melanoma is one of the most aggressive human cancers due to its high invasiveness.,Germline mutations in high-risk melanoma susceptibility genes have been associated with development hereditary melanoma; however, most genetic culprits remain elusive.,To unravel novel susceptibility genes for hereditary melanoma, we performed whole exome sequencing (WES) on eight patients with multiple primary melanomas, high number of nevi, and negative for high and intermediate-risk germline mutations.,Thirteen new potentially pathogenic variants were identified after bioinformatics analysis and validation.,CDH23, ARHGEF40, and BRD9 were identified as the most promising susceptibility genes in hereditary melanoma.,In silico analysis of CDH23 and ARHGEF40 variants provided clues for altered protein structure and function associated with the identified mutations.,Then, we also evaluated the clinical value of CDH23, ARHGEF40, and BRD9 expression in sporadic melanoma by using the TCGA dataset (n = 461).,No differences were observed in BRD9 expression between melanoma and normal skin samples, nor with melanoma stage, whereas ARHGEF40 was found overexpressed, and CDH23 was downregulated and its loss was associated with worse survival.,Altogether, these results reveal three novel genes with clinical relevance in hereditary and sporadic melanoma.
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Germline mutations within melanoma susceptibility genes are present only in minority of melanoma patients and it is expected that additional genes will be discovered with next generation sequence technology and whole-exome sequencing (WES).,Herein we performed WES on a cohort of 96 unrelated Polish patients with melanoma diagnosed under the age of 40 years who all screened negative for the presence of CDKN2A variants.,A replication study using a set of 1,200 melanoma patient DNA samples and similarly large series of healthy controls was undertaken.,We selected 21 potentially deleterious variants in 20 genes (VRK1, MYCT1, DNAH14, CASC3, MS4A12, PRC1, WWOX, CARD6, EXO5, CASC3, CASP8AP2, STK33, SAMD11, CNDP2, CPNE1, EFCAB6, CABLES1, LEKR1, NUDT17, and RRP15), which were identified by WES and confirmed by Sanger sequencing for an association study.,Evaluation of the allele distribution among carriers and their relatives in available family trios revealed that these variants were unlikely to account for many familial cases of melanoma.,Replication study revealed no statistically significant differences between cases and controls.,Although most of the changes seemed to be neutral we could not exclude an association between variants in VRK1, CREB3L3, EXO5, and STK33 with melanoma risk.
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In the phase III CheckMate 067 trial, durable clinical benefit was demonstrated previously with nivolumab plus ipilimumab and nivolumab alone versus ipilimumab.,Here, we report 6.5-year efficacy and safety outcomes.,Patients with previously untreated unresectable stage III or stage IV melanoma were randomly assigned 1:1:1 to receive nivolumab 1 mg/kg plus ipilimumab 3 mg/kg once every 3 weeks (four doses) followed by nivolumab 3 mg/kg once every 2 weeks (n = 314), nivolumab 3 mg/kg once every 2 weeks (n = 316), or ipilimumab 3 mg/kg once every 3 weeks (four doses; n = 315).,Coprimary end points were progression-free survival and overall survival (OS) with nivolumab plus ipilimumab or nivolumab versus ipilimumab.,Secondary end points included objective response rate, descriptive efficacy assessments of nivolumab plus ipilimumab versus nivolumab alone, and safety.,Melanoma-specific survival (MSS; descriptive analysis), which excludes deaths unrelated to melanoma, was also evaluated.,Median OS (minimum follow-up, 6.5 years) was 72.1, 36.9, and 19.9 months in the combination, nivolumab, and ipilimumab groups, respectively.,Median MSS was not reached, 58.7, and 21.9 months, respectively; 6.5-year OS rates were 57%, 43%, and 25% in patients with BRAF-mutant tumors and 46%, 42%, and 22% in those with BRAF-wild-type tumors, respectively.,In patients who discontinued treatment, the median treatment-free interval was 27.6, 2.3, and 1.9 months, respectively.,Since the 5-year analysis, no new safety signals were observed.,These 6.5-year CheckMate 067 results, which include the longest median OS in a phase III melanoma trial reported to date and the first report of MSS, showed durable, improved clinical outcomes with nivolumab plus ipilimumab or nivolumab versus ipilimumab in patients with advanced melanoma and, in descriptive analyses, with the combination over nivolumab monotherapy.
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Malignant melanoma is the most aggressive type of skin cancer and is closely associated with the development of brain metastases.,Despite aggressive treatment, the prognosis has traditionally been poor, necessitating improved therapies.,In melanoma, the mitogen activated protein kinase and the phosphoinositide 3-kinase signaling pathways are commonly altered, and therapeutically inhibiting one of the pathways often upregulates the other, leading to resistance.,Thus, combined treatment targeting both pathways is a promising strategy to overcome this.,Here, we studied the in vitro and in vivo effects of the PI3K inhibitor buparlisib and the MEK1/2 inhibitor trametinib, used either as targeted monotherapies or in combination, on patient-derived melanoma brain metastasis cell lines.,Scratch wound and trans-well assays were carried out to assess the migratory capacity of the cells upon drug treatment, whereas flow cytometry, apoptosis array and Western blots were used to study apoptosis.,Finally, an in vivo treatment experiment was carried out on NOD/SCID mice.,We show that combined therapy was more effective than monotherapy.,Combined treatment also more effectively increased apoptosis, and inhibited tumor growth in vivo.,This suggests a clinical potential of combined treatment to overcome ceased treatment activity which is often seen after monotherapies, and strongly encourages the evaluation of the treatment strategy on melanoma patients with brain metastases.
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Ocular melanoma is the most common primary intraocular malignancy and has a very poor prognosis once liver metastases occur.,The aim of this study was to prospectively assess the efficacy and safety of percutaneous hepatic perfusion with melphalan (M-PHP) using the new second-generation (GEN 2) hemofiltration system in patients with ocular melanoma metastases confined to the liver.,Prospective, single-center, single-arm, phase II study including patients with unresectable ocular melanoma metastases confined to the liver.,Treatment consisted of two M-PHP procedures at 6-8 weeks interval.,Procedures were performed using the CHEMOSAT (GEN 2) system with 3 mg/kg melphalan.,Primary endpoints were overall response rate (ORR) and best overall response (BOR).,Secondary endpoints included overall survival (OS), progression-free survival (PFS), hepatic PFS (hPFS), and safety.,Sixty-four M-PHP procedures were performed in 35 patients between February 2014 and June 2017.,The ORR was 72%.,BOR was as follows: complete response in 3%, partial response in 69%, stable disease in 13%, and progressive disease in 16%.,There was no treatment-related mortality.,Fourteen serious adverse events occurred.,At a median follow-up of 19.1 months (range 5.6-69.5), median OS was 19.1 months and was significantly longer in responders than in nonresponders (27.5 vs.,11.9 months, p < 0.001).,The 1- and 2-year OS was 77% and 43%, respectively.,PFS and hPFS were 7.6 and 11.2 months, respectively.,M-PHP using the GEN 2 filter can achieve a high ORR and prolonged survival in patients with liver-only ocular melanoma metastases.
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Chemosaturation with percutaneous hepatic perfusion (CS-PHP; Hepatic CHEMOSAT® Delivery System; Delcath Systems Inc, USA) is a novel interventional procedure, which delivers high doses of melphalan directly to the liver in patients with liver tumors while limiting systemic toxicity through hemofiltration of the hepatic venous blood.,We have previously shown promising efficacy for patients with ocular melanoma (OM) and cholangiocarcinoma (CCA) within our single-center and multi-center experiences.,The aim of this study was to analyze the safety and efficacy of CS-PHP after 141 treatments at Hannover Medical School, Germany.,Overall response rates (ORR) were assessed according to Response Evaluation Criteria In Solid Tumors (RECIST1.1).,Median Overall survival (mOS), median progression-free survival (mPFS), and median hepatic PFS (mhPFS) were analyzed using the Kaplan-Meier estimation.,Overall, 60 patients were treated with CS-PHP in the salvage setting from October 2014 until January 2019 at Hannover Medical School with a total of 141 procedures.,Half of the patients were patients with hepatic metastases of ocular melanoma (OM) (n = 30), 14 patients had CCA (23.3%), 6 patients had hepatocellular carcinoma (10%), and 10 patients were treated for other secondary liver malignancies (16.7%).,In total, ORR and disease stabilization rate were 33.3% and 70.3% (n = 25), respectively.,ORR was highest for patients with OM (42.3%), followed by patients with CCA (30.8%).,Independent response-associated factors were normal levels of lactate dehydrogenase (odds ratio (OR) 13.7; p = 0.015) and diagnosis with OM (OR 9.3; p = 0.028).,Overall, mOS was 9 months, mPFS was 4 months, and mhPFS was 5 months.,Patients with OM had the longest mOS, mPFS, and mhPFS with 12, 6, and 6 months, respectively.,Adverse events included most frequently significant, but transient, hematologic toxicities (80% of grade 3/4 thrombopenia), less frequently hepatic injury up to liver failure (3.3%) and cardiovascular events including two cases of ischemic insults (5%).,Salvage treatment with CS-PHP is safe and effective particularly in patients OM and CCA.,Careful attention should be paid to possible, serious hepatic, and cardiovascular complications.,The online version of this article (10.1007/s00432-020-03289-5) contains supplementary material, which is available to authorized users.
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Treatment of BRAF‐mutant melanomas with MAP kinase pathway inhibitors is paradigmatic of the promise of precision cancer therapy but also highlights problems with drug resistance that limit patient benefit.,We use live‐cell imaging, single‐cell analysis, and molecular profiling to show that exposure of tumor cells to RAF/MEK inhibitors elicits a heterogeneous response in which some cells die, some arrest, and the remainder adapt to drug.,Drug‐adapted cells up‐regulate markers of the neural crest (e.g., NGFR), a melanocyte precursor, and grow slowly.,This phenotype is transiently stable, reverting to the drug‐naïve state within 9 days of drug withdrawal.,Transcriptional profiling of cell lines and human tumors implicates a c‐Jun/ECM/FAK/Src cascade in de‐differentiation in about one‐third of cell lines studied; drug‐induced changes in c‐Jun and NGFR levels are also observed in xenograft and human tumors.,Drugs targeting the c‐Jun/ECM/FAK/Src cascade as well as BET bromodomain inhibitors increase the maximum effect (E max) of RAF/MEK kinase inhibitors by promoting cell killing.,Thus, analysis of reversible drug resistance at a single‐cell level identifies signaling pathways and inhibitory drugs missed by assays that focus on cell populations.
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Transcriptional reprogramming of proliferative melanoma cells into a phenotypically distinct invasive cell subpopulation is a critical event at the origin of metastatic spreading.,Here we generate transcriptome, open chromatin and histone modification maps of melanoma cultures; and integrate this data with existing transcriptome and DNA methylation profiles from tumour biopsies to gain insight into the mechanisms underlying this key reprogramming event.,This shows thousands of genomic regulatory regions underlying the proliferative and invasive states, identifying SOX10/MITF and AP-1/TEAD as regulators, respectively.,Knockdown of TEADs shows a previously unrecognized role in the invasive gene network and establishes a causative link between these transcription factors, cell invasion and sensitivity to MAPK inhibitors.,Using regulatory landscapes and in silico analysis, we show that transcriptional reprogramming underlies the distinct cellular states present in melanoma.,Furthermore, it reveals an essential role for the TEADs, linking it to clinically relevant mechanisms such as invasion and resistance.,The key regulators that allow transition from proliferative to invasive phenotype in melanoma cells have not been identified yet.,The authors perform chromatin and transcriptome profiling followed by comprehensive bioinformatics analysis identifying new candidate regulators for two distinct cell states of melanoma.
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We conducted the phase III double-blind European Organisation for Research and Treatment of Cancer (EORTC) 1325/KEYNOTE-054 trial to evaluate pembrolizumab versus placebo in patients with resected high-risk stage III melanoma.,On the basis of 351 recurrence-free survival (RFS) events at a 1.25-year median follow-up, pembrolizumab prolonged RFS (hazard ratio [HR], 0.57; P < .0001) compared with placebo.,This led to the approval of pembrolizumab adjuvant treatment by the European Medicines Agency and US Food and Drug Administration.,Here, we report an updated RFS analysis at the 3.05-year median follow-up.,A total of 1,019 patients with complete lymph node dissection of American Joint Committee on Cancer Staging Manual (seventh edition; AJCC-7), stage IIIA (at least one lymph node metastasis > 1 mm), IIIB, or IIIC (without in-transit metastasis) cutaneous melanoma were randomly assigned to receive pembrolizumab at a flat dose of 200 mg (n = 514) or placebo (n = 505) every 3 weeks for 1 year or until disease recurrence or unacceptable toxicity.,The two coprimary end points were RFS in the overall population and in those with programmed death-ligand 1 (PD-L1)-positive tumors.,Pembrolizumab (190 RFS events) compared with placebo (283 RFS events) resulted in prolonged RFS in the overall population (3-year RFS rate, 63.7% v 44.1% for pembrolizumab v placebo, respectively; HR, 0.56; 95% CI, 0.47 to 0.68) and in the PD-L1-positive tumor subgroup (HR, 0.57; 99% CI, 0.43 to 0.74).,The impact of pembrolizumab on RFS was similar in subgroups, in particular according to AJCC-7 and AJCC-8 staging, and BRAF mutation status (HR, 0.51 [99% CI, 0.36 to 0.73] v 0.66 [99% CI, 0.46 to 0.95] for V600E/K v wild type).,In resected high-risk stage III melanoma, pembrolizumab adjuvant therapy provided a sustained and clinically meaningful improvement in RFS at 3-year median follow-up.,This improvement was consistent across subgroups.
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There is growing evidence that tripartite motif-containing protein 44 (TRIM44) plays crucial role in tumor development.,However, the underlying mechanism of this deubiquitinating enzyme remains unclear.,Large clinical samples were used to detect TRIM44 expression and its associations with clinicopathological features and prognosis.,Gain- and loss-of-function experiments in cell lines and mouse xenograft models were performed to elucidate the function and underlying mechanisms of TRIM44 induced tumor progression.,Co-immunoprecipitation (Co-IP) assays and mass spectrometric analyses were applied to verify the interacting proteins of TRIM44.,We found that TRIM44 was commonly amplified in melanoma tissues compared with paratumoral tissues.,TRIM44 expression also positively correlated with more aggressive clinicopathological features, such as Breslow depth (p = 0.025), distant metastasis (p = 0.012), and TNM stage (p = 0.002).,Importantly, we found that TRIM44 was an independent indicator of prognosis for melanoma patients.,Functionally, overexpression of TRIM44 facilitated cell invasion, migration, apoptosis resistance and proliferation in vitro, and promoted lung metastasis and tumorigenic ability in vivo.,Importantly, high level of TRIM44 induced melanoma cell epithelial-mesenchymal transition (EMT), which is one of the most important mechanisms for the promotion of tumor metastasis.,Mechanistically, high levels of TRIM44 increased the levels of p-AKT (T308) and p-mTOR (S2448), and a specific AKT inhibitor inhibited TRIM44-induced tumor progression.,Co-IP assays and mass spectrometric analyses indicated that TRIM44 overexpression induces cell EMT through activating AKT/mTOR pathway via directly binding and stabilizing TOLL-like receptor 4 (TLR4), and TLR4 interference impeded TRIM44 induced tumor progression.,Moreover, we demonstrated that TRIM44 is the target of miR-26b-5p, which is significantly downregulated in melanoma tissues and may be responsible for the overexpression of TRIM44.,TRIM44, regulated by miR-26b-5p, promotes melanoma progression by stabilizing TLR4, which then activates the AKT/mTOR pathway.,TRIM44 shows promise as a prognostic predictor and a therapeutic target for melanoma patients.,The online version of this article (10.1186/s13046-019-1138-7) contains supplementary material, which is available to authorized users.
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Advanced and undifferentiated skin squamous cell carcinomas (SCCs) exhibit aggressive growth and enhanced metastasis capability, which is associated in mice with an expansion of the cancer stem-like cell (CSC) population and with changes in the regulatory mechanisms that control the proliferation and invasion of these cells.,Indeed, autocrine activation of PDGFRα induces CSC invasion and promotes distant metastasis in advanced SCCs.,However, the mechanisms involved in this process were unclear.,Here, we show that CSCs of mouse advanced SCCs (L-CSCs) express CXCR4 and CXCR7, both receptors of SDF-1.,PDGFRα signaling induces SDF-1 expression and secretion, and the autocrine activation of this pathway in L-CSCs.,Autocrine SDF-1/CXCR4 signaling induces L-CSC proliferation and survival, and mediates PDGFRα-induced invasion, promoting in vivo lung metastasis.,Validation of these findings in patient samples of skin SCCs shows a strong correlation between the expression of SDF1, PDGFRA, and PDGFRB, which is upregulated, along CXCR4 in tumor cells of advanced SCCs.,Furthermore, PDGFR regulates SDF-1 expression and inhibition of SDF-1/CXCR4 and PDGFR pathways blocks distant metastasis of human PD/S-SCCs.,Our results indicate that functional crosstalk between PDGFR/SDF-1 signaling regulates tumor cell invasion and metastasis in human and mouse advanced SCCs, and suggest that CXCR4 and/or PDGFR inhibitors could be used to block metastasis of these aggressive tumors.
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Cutaneous SCC (cSCC) is the most frequent skin cancer with metastatic potential and can manifest rapidly as a common side effect in patients receiving systemic kinase inhibitors.,Here we use massively parallel exome and targeted level sequencing 132 sporadic cSCC, 39 squamoproliferative lesions and cSCC arising in patients receiving the BRAF inhibitor vemurafenib, as well as 10 normal skin samples to identify significant NOTCH1 mutation as an early event in squamous cell carcinogenesis.,Bisected vemurafenib induced lesions revealed surprising heterogeneity with different activating HRAS and NOTCH1 mutations identified in two halves of the same cSCC suggesting polyclonal origin.,Immunohistochemical analysis using an antibody specific to nuclear NOTCH1 correlates with mutation status in sporadic cSCC and regions of NOTCH1 loss or down-regulation are frequently observed in normal looking skin.,Our data indicate that NOTCH1 acts as a gatekeeper in human cSCC.
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Fluid-conducting extracellular matrix patterns known as vasculogenic mimicry (VM) have been associated with poor prognosis in uveal melanoma and other cancers.,We investigate the correlations between VM, presenting symptoms, mortality, and the area density of periodic acid-Schiff positive histological patterns (PAS density).,Sixty-nine patients that underwent enucleation for uveal melanoma between 2000 and 2007 were included.,Clinicopathological parameters presenting symptoms and outcomes were collected.,Histological tumor sections were evaluated for VM and PAS density was quantified with digital image analysis.,Thirty-four patients (49%) presented with blurred vision. 18 (26%) with a shadow in the visual field, 7 (10%) with photopsia and/or floaters, and 2 (3%) with metamorphopsia.,Nine patients (13%) had no symptoms at all.,Median follow-up was 16.7 years (SD 2.6).,A shadow in the visual field, but no other symptom, was positively correlated with the presence of VM (φ 0.70, p < 0.001) and greater PAS density (p < 0.001).,In multivariate regression, retinal detachment (RD), presence of VM, and PAS density ≥ median were independent predictors of a shadow, but not tumor distance to the macula, tumor apical thickness, tumor diameter, or ciliary body engagement.,The presence of VM was associated with significantly shorter cumulative disease-specific survival (Wilcoxon p = 0.04), but not PAS density ≥ median, presenting symptoms or RD (p > 0.28).,Tumors from uveal melanoma patients that report a visual field shadow are likely to display VM and greater PAS density, likely explaining the previously reported association between this symptom and poor prognosis.
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Uveal melanoma (UM) is the most common intraocular tumour in adults and despite surgical or radiation treatment of primary tumours, ~50% of patients progress to metastatic disease.,Therapeutic options for metastatic UM are limited, with clinical trials having little impact.,Here we perform whole-genome sequencing (WGS) of 103 UM from all sites of the uveal tract (choroid, ciliary body, iris).,While most UM have low tumour mutation burden (TMB), two subsets with high TMB are seen; one driven by germline MBD4 mutation, and another by ultraviolet radiation (UVR) exposure, which is restricted to iris UM.,All but one tumour have a known UM driver gene mutation (GNAQ, GNA11, BAP1, PLCB4, CYSLTR2, SF3B1, EIF1AX).,We identify three other significantly mutated genes (TP53, RPL5 and CENPE).,Uveal melanoma has a propensity to metastasise.,Here, the authors report the whole genome sequence of 103 uveal melanomas and find that the tumour mutational burden is variable and that two subsets of tumours are characterised by MBD4 mutations and a UV exposure signature.
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Drug resistance remains a vexing problem in the treatment of cancer patients.,While many studies have focused on cell autonomous mechanisms of drug resistance, we hypothesized that the tumor microenvironment may confer innate resistance to therapy.,Here we developed a co-culture system to systematically assay the ability of 23 stromal cell types to influence the innate resistance of 45 cancer cell lines to 35 anti-cancer drugs.,We found that stroma-mediated resistance is surprisingly common - particularly to targeted agents.,We further characterized the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibition because most of these patients exhibit some degree of innate resistance1-4.,Proteomic analysis showed that stromal secretion of the growth factor hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the MAPK and PI3K/AKT pathways, and immediate resistance to RAF inhibition.,Immunohistochemistry confirmed stromal HGF expression in patients with BRAF-mutant melanoma and a statistically significant correlation between stromal HGF expression and innate resistance to treatment.,Dual inhibition of RAF and MET resulted in reversal of drug resistance, suggesting RAF/MET combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma.,A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines.,More generally, these studies indicate that the systematic dissection of tumor-microenvironment interactions may reveal important mechanisms underlying drug resistance.
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Activated RAS promotes dimerization of members of the RAF kinase family1-3.,ATP-competitive RAF inhibitors activate ERK signaling4-7 by transactivating RAF dimers4.,In melanomas with mutant BRAF(V600E), levels of RAS activation are low and these drugs bind to BRAF(V600E) monomers and inhibit their activity.,This tumor-specific inhibition of ERK signaling results in a broad therapeutic index and RAF inhibitors have remarkable clinical activity in patients with melanomas that harbor mutant BRAF(V600E)8.,However, resistance invariably develops.,Here, we identify a novel resistance mechanism.,We find that a subset of cells resistant to vemurafenib (PLX4032, RG7204) express a 61kd variant form of BRAF(V600E) that lacks exons 4-8, a region that encompasses the RAS-binding domain. p61BRAF(V600E) exhibits enhanced dimerization in cells with low levels of RAS activation, as compared to full length BRAF(V600E).,In cells in which p61BRAF(V600E) is expressed endogenously or ectopically, ERK signaling is resistant to the RAF inhibitor.,Moreover, a mutation that abolishes the dimerization of p61BRAF(V600E) restores its sensitivity to vemurafenib.,Finally, we identified BRAF(V600E) splicing variants lacking the RAS-binding domain in the tumors of six of 19 patients with acquired resistance to vemurafenib.,These data support the model that inhibition of ERK signaling by RAF inhibitors is dependent on levels of RAS-GTP too low to support RAF dimerization and identify a novel mechanism of acquired resistance in patients: expression of splicing isoforms of BRAF(V600E) that dimerize in a RAS-independent manner.
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Metastatic melanoma carries a poor prognosis despite modern systemic therapies.,Understanding the evolution of the disease could help inform patient management.,Through whole-genome sequencing of 13 melanoma metastases sampled at autopsy from a treatment naïve patient and by leveraging the analytical power of multi-sample analyses, we reveal evidence of diversification among metastatic lineages.,UV-induced mutations dominate the trunk, whereas APOBEC-associated mutations are found in the branches of the evolutionary tree.,Multi-sample analyses from a further seven patients confirmed that lineage diversification was pervasive, representing an important mode of melanoma dissemination.,Our analyses demonstrate that joint analysis of cancer cell fraction estimates across multiple metastases can uncover previously unrecognised levels of tumour heterogeneity and highlight the limitations of inferring heterogeneity from a single biopsy.,Metastatic melanoma is associated with a poor prognosis and understanding the genetic features of metastases may enable better treatment strategies.,Here, the authors analyse multiple metastases from individual patients finding high levels of heterogeneity in metastases from different organs.
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Clonal selection and transcriptional reprogramming (e.g., epithelial-mesenchymal transition or phenotype switching) are the predominant theories thought to underlie tumor progression.,However, a “division of labor” leading to cooperation among tumor-cell subpopulations could be an additional catalyst of progression.,Using a zebrafish-melanoma xenograft model, we found that in a heterogeneous setting, inherently invasive cells, which possess protease activity and deposit extracellular matrix (ECM), co-invade with subpopulations of poorly invasive cells, a phenomenon we term “cooperative invasion”.,Whereas the poorly invasive cells benefit from heterogeneity, the invasive cells switch from protease-independent to an MT1-MMP-dependent mode of invasion.,We did not observe changes in expression of the melanoma phenotype determinant MITF during cooperative invasion, thus ruling out the necessity for phenotype switching for invasion.,Altogether, our data suggest that cooperation can drive melanoma progression without the need for clonal selection or phenotype switching and can account for the preservation of heterogeneity seen throughout tumor progression.,•Inherently invasive and poorly invasive melanoma subpopulations co-invade•Leader cells are inherently invasive and provide MT1-MMP and deposit ECM•Follower cells affect the mode of invasion and thereby elicit protease dependency•Cooperative invasion bypasses phenotype switching and maintains tumor heterogeneity,Inherently invasive and poorly invasive melanoma subpopulations co-invade,Leader cells are inherently invasive and provide MT1-MMP and deposit ECM,Follower cells affect the mode of invasion and thereby elicit protease dependency,Cooperative invasion bypasses phenotype switching and maintains tumor heterogeneity,Tumor heterogeneity has been viewed as the outcome of adaptive competition or differential transcriptional reprogramming (phenotype switching).,Alternatively, heterogeneity may reflect a division of labor between tumor cell subpopulations with complementary acquired characteristics.,Chapman et al. now show reciprocal interactions between heterogeneous melanoma cell subpopulations that lead to cooperative invasion without competition and without phenotype switching.,Thus, cooperative behavior emerges as a property of heterogeneity relevant for tumor biology and worth targeting therapeutically.
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We report the performance of a gene expression profile test to classify the recurrence risk of cutaneous melanoma tumors of the head and neck as low‐risk Class 1 or high‐risk Class 2.,Of note, 157 primary head and neck cutaneous melanoma tumors were identified.,Survival analyses were performed using Kaplan‐Meier and Cox methods.,Gene expression profile class and node status stratified tumors into significantly different 5‐year survival groups by Kaplan‐Meier method (P < .0001 for all end points), and both were independent predictors of recurrence in multivariate analysis.,Overall, 74% of distant metastases and 88% of melanoma‐specific deaths had Class 2 risk.,The gene expression profile test identifies cases at increased risk for metastasis and death independent of a clinically or pathologically negative nodal status, suggesting that incorporation of this molecular tool could improve clinical management of patients with head and neck cutaneous melanoma, especially in those with a negative sentinel lymph node biopsy.
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Matrix metalloproteinase-23 (MMP-23) can block the voltage-gated potassium channel Kv1.3, whose function is important for sustained Ca2+ signaling during T cell activation.,MMP-23 may also alter T cell activity and phenotype through cleavage of proteins affecting cytokine and chemokine signaling.,We therefore tested the hypothesis that MMP-23 can negatively regulate the anti-tumor T cell response in human melanoma.,We characterized MMP-23 expression in primary melanoma patients who received adjuvant immunotherapy.,We examined the association of MMP-23 with the anti-tumor immune response - as assessed by the prevalence of tumor-infiltrating lymphocytes and Foxp3+ regulatory T cells.,Further, we examined the association between MMP-23 expression and response to immunotherapy.,Considering also an in trans mechanism, we examined the association of melanoma MMP-23 and melanoma Kv1.3 expression.,Our data revealed an inverse association between primary melanoma MMP-23 expression and the anti-tumor T cell response, as demonstrated by decreased tumor-infiltrating lymphocytes (TIL) (P = 0.05), in particular brisk TILs (P = 0.04), and a trend towards an increased proportion of immunosuppressive Foxp3+ regulatory T cells (P = 0.07).,High melanoma MMP-23 expression is also associated with recurrence in patients treated with immune biologics (P = 0.037) but not in those treated with vaccines (P = 0.64).,Further, high melanoma MMP-23 expression is associated with shorter periods of progression-free survival for patients receiving immune biologics (P = 0.025).,On the other hand, there is no relationship between melanoma MMP-23 and melanoma Kv1.3 expression (P = 0.27).,Our data support a role for MMP-23 as a potential immunosuppressive target in melanoma, as well as a possible biomarker for informing melanoma immunotherapies.,The online version of this article (doi:10.1186/s12967-014-0342-7) contains supplementary material, which is available to authorized users.
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Basal cell carcinoma (BCC) represents the most commonly diagnosed human cancer among persons of European ancestry with etiology mainly attributed to sun-exposure.,In this study we investigated mutations in coding and flanking regions of PTCH1 and TP53 and noncoding alterations in the TERT and DPH3 promoters in 191 BCC tumors.,In addition, we measured CpG methylation within the TERT hypermethylated oncological region (THOR) and transcription levels of the reverse transcriptase subunit.,We observed mutations in PTCH1 in 58.6% and TP53 in 31.4% of the tumors.,Noncoding mutations in TERT and DPH3 promoters were detected in 59.2% and 38.2% of the tumors, respectively.,We observed a statistically significant co-occurrence of mutations at the four investigated loci.,While PTCH1 mutations tended to associate with decreased patient age at diagnosis; TP53 mutations were associated with light skin color and increased number of nevi; TERT and DPH3 promoter with history of cutaneous neoplasms in BCC patients.,Increased reverse transcriptase subunit expression was observed in tumors with TERT promoter mutations and not with THOR methylation.,Our study signifies, in addition to the protein altering mutations in the PTCH1 and TP53 genes, the importance of noncoding mutations in BCC, particularly functional alterations in the TERT promoter.
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An association between low serum vitamin D levels and poorer melanoma survival has been reported.,We have studied inheritance of a polymorphism of the GC gene, rs2282679, coding for the vitamin D-binding protein, which is associated with lower serum levels of vitamin D, in a meta-analysis of 3137 melanoma patients.,The aim was to investigate evidence for a causal relationship between vitamin D and outcome (Mendelian randomization).,The variant was not associated with reduced overall survival (OS) in the UK cohort, per-allele hazard ratio (HR) for death 1.23 (95% confidence interval (CI) 0.93, 1.64).,In the smaller cohorts, HR in OS analysis was 1.07 (95% CI 0.88, 1.3) and for all cohorts combined, HR for OS was 1.09 (95% CI 0.93, 1.29).,There was evidence of increased melanoma-specific deaths in the seven cohorts for which these data were available.,The lack of unequivocal findings despite the large sample size illustrates the difficulties of implementing Mendelian randomization.
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The skin’s tendency to sunburn rather than tan is a major risk factor for skin cancer.,Here we report a large genome-wide association study of ease of skin tanning in 176,678 subjects of European ancestry.,We identify significant association with tanning ability at 20 loci.,We confirm previously identified associations at six of these loci, and report 14 novel loci, of which ten have never been associated with pigmentation-related phenotypes.,Our results also suggest that variants at the AHR/AGR3 locus, previously associated with cutaneous malignant melanoma the underlying mechanism of which is poorly understood, might act on disease risk through modulation of tanning ability.,The skin’s tanning response to sun exposure shows great interindividual variability.,Here, Visconti et al. perform a genome-wide association study for ease of skin tanning and identify 20 genetic loci, ten of which had not previously been associated with pigmentation-related traits.
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Whole-genome sequencing identifies a novel germline variant in the oncogene MITF, which is associated with the development of melanoma.,The online version of this article (doi:10.1038/nature10630) contains supplementary material, which is available to authorized users.,Two papers in this issue of Nature demonstrate that missense substitutions in the gene encoding for microphthalmia-associated transcription factor (MITF) are associated with susceptibility to melanoma and renal cell carcinoma.,Functional analysis shows that the variant has impaired sumoylation that leads to differential regulation of several MITF targets, and promotes tumour cell clonogenicity, migration and invasion.,The online version of this article (doi:10.1038/nature10630) contains supplementary material, which is available to authorized users.,So far, two genes associated with familial melanoma have been identified, accounting for a minority of genetic risk in families.,Mutations in CDKN2A account for approximately 40% of familial cases1, and predisposing mutations in CDK4 have been reported in a very small number of melanoma kindreds2.,Here we report the whole-genome sequencing of probands from several melanoma families, which we performed in order to identify other genes associated with familial melanoma.,We identify one individual carrying a novel germline variant (coding DNA sequence c.G1075A; protein sequence p.E318K; rs149617956) in the melanoma-lineage-specific oncogene microphthalmia-associated transcription factor (MITF).,Although the variant co-segregated with melanoma in some but not all cases in the family, linkage analysis of 31 families subsequently identified to carry the variant generated a log of odds (lod) score of 2.7 under a dominant model, indicating E318K as a possible intermediate risk variant.,Consistent with this, the E318K variant was significantly associated with melanoma in a large Australian case-control sample.,Likewise, it was similarly associated in an independent case-control sample from the United Kingdom.,In the Australian sample, the variant allele was significantly over-represented in cases with a family history of melanoma, multiple primary melanomas, or both.,The variant allele was also associated with increased naevus count and non-blue eye colour.,Functional analysis of E318K showed that MITF encoded by the variant allele had impaired sumoylation and differentially regulated several MITF targets.,These data indicate that MITF is a melanoma-predisposition gene and highlight the utility of whole-genome sequencing to identify novel rare variants associated with disease susceptibility.,The online version of this article (doi:10.1038/nature10630) contains supplementary material, which is available to authorized users.
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Uveal melanoma (UM) is a highly metastatic cancer that, in contrast to cutaneous melanoma, is largely unresponsive to checkpoint immunotherapy.,Here, we interrogate the tumor microenvironment at single-cell resolution using scRNA-seq of 59,915 tumor and non-neoplastic cells from 8 primary and 3 metastatic samples.,Tumor cells reveal novel subclonal genomic complexity and transcriptional states.,Tumor-infiltrating immune cells comprise a previously unrecognized diversity of cell types, including CD8+ T cells predominantly expressing the checkpoint marker LAG3, rather than PD1 or CTLA4.,V(D)J analysis shows clonally expanded T cells, indicating that they are capable of mounting an immune response.,An indolent liver metastasis from a class 1B UM is infiltrated with clonally expanded plasma cells, indicative of antibody-mediated immunity.,This complex ecosystem of tumor and immune cells provides new insights into UM biology, and LAG3 is identified as a potential candidate for immune checkpoint blockade in patients with high risk UM.,Uveal melanoma is highly metastatic and unresponsive to checkpoint immunotherapy.,Here, the authors present single-cell transcriptomics of 59,915 cells in 8 primary and 3 metastatic samples, highlighting the diversity of the tumour microenvironment.
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B-RAF is the most frequently mutated protein kinase in human cancers.1 The finding that oncogenic mutations in BRAF are common in melanoma2 followed by the demonstration that these tumors are dependent on the RAF/MEK/ERK pathway3 offered hope that inhibition of B-RAF kinase activity could benefit melanoma patients.,Herein, we describe the structure-guided discovery of PLX4032 (RG7204), a potent inhibitor of oncogenic B-RAF kinase activity.,Preclinical experiments demonstrated that PLX4032 selectively blocked the RAF/MEK/ERK pathway in BRAF mutant cells and caused regression of BRAF mutant xenografts.4 Toxicology studies confirmed a wide safety margin consistent with the high degree of selectivity, enabling Phase 1 clinical trials using a crystalline formulation of PLX4032.5 In a subset of melanoma patients, pathway inhibition was monitored in paired biopsy specimens collected before treatment initiation and following two weeks of treatment.,This analysis revealed substantial inhibition of ERK phosphorylation, yet clinical evaluation did not show tumor regressions.,At higher drug exposures afforded by a new amorphous drug formulation,4,5 greater than 80% inhibition of ERK phosphorylation in the tumors of patients correlated with clinical response.,Indeed, the Phase 1 clinical data revealed a remarkably high 81% response rate in metastatic melanoma patients treated at an oral dose of 960 mg twice daily.5 These data demonstrate that BRAF-mutant melanomas are highly dependent on B-RAF kinase activity.
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Despite recent improvements in prevention, diagnosis and treatment, vast differences in melanoma burden still exist between populations.,Comparative data can highlight these differences and lead to focused efforts to reduce the burden of melanoma.,To assess global, regional and national melanoma incidence, mortality and disability‐adjusted life year (DALY) estimates from the Global Burden of Disease Study 2015.,Vital registration system and cancer registry data were used for melanoma mortality modelling.,Incidence and prevalence were estimated using separately modelled mortality‐to‐incidence ratios.,Total prevalence was divided into four disease phases and multiplied by disability weights to generate years lived with disability (YLDs).,Deaths in each age group were multiplied by the reference life expectancy to generate years of life lost (YLLs).,YLDs and YLLs were added to estimate DALYs.,The five world regions with the greatest melanoma incidence, DALY and mortality rates were Australasia, North America, Eastern Europe, Western Europe and Central Europe.,With the exception of regions in sub‐Saharan Africa, DALY and mortality rates were greater in men than in women.,DALY rate by age was highest in those aged 75-79 years, 70-74 years and ≥ 80 years.,The greatest burden from melanoma falls on Australasian, North American, European, elderly and male populations, which is consistent with previous investigations.,These substantial disparities in melanoma burden worldwide highlight the need for aggressive prevention efforts.,The Global Burden of Disease Study results can help shape melanoma research and public policy.,What's already known about this topic?,Melanoma incidence and mortality has been assessed in the past for individual countries or world regions.,Melanoma incidence and mortality has been assessed in the past for individual countries or world regions.,What does this study add?,As part of the Global Burden of Disease Study, melanoma burden was estimated at the global, regional and country level for incidence, mortality, prevalence, years lived with disability, years of life lost and disability‐adjusted life years.These estimates can be used to guide prevention and treatment strategies, as well as resource allocation.,As part of the Global Burden of Disease Study, melanoma burden was estimated at the global, regional and country level for incidence, mortality, prevalence, years lived with disability, years of life lost and disability‐adjusted life years.,These estimates can be used to guide prevention and treatment strategies, as well as resource allocation.,Respond to this article,Plain language summary available online
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The development and progression of melanoma have been attributed to independent or combined genetic and epigenetic events.,There has been remarkable progress in understanding melanoma pathogenesis in terms of genetic alterations.,However, recent studies have revealed a complex involvement of epigenetic mechanisms in the regulation of gene expression, including methylation, chromatin modification and remodeling, and the diverse activities of non-coding RNAs.,The roles of gene methylation and miRNAs have been relatively well studied in melanoma, but other studies have shown that changes in chromatin status and in the differential expression of long non-coding RNAs can lead to altered regulation of key genes.,Taken together, they affect the functioning of signaling pathways that influence each other, intersect, and form networks in which local perturbations disturb the activity of the whole system.,Here, we focus on how epigenetic events intertwine with these pathways and contribute to the molecular pathogenesis of melanoma.
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The common mutation BRAFV600 in primary melanomas activates the mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) pathway and the introduction of proto-oncogene B-Raf (BRAF) and mitogen-activated protein kinase kinase (MEK) inhibitors (BRAFi and MEKi) was a breakthrough in the treatment of these cancers.,However, 15-20% of tumors harbor primary resistance to this therapy, and moreover, patients develop acquired resistance to treatment.,Understanding the molecular phenomena behind resistance to BRAFi/MEKis is indispensable in order to develop novel targeted therapies.,Most often, resistance develops due to either the reactivation of the MAPK/ERK pathway or the activation of alternative kinase signaling pathways including phosphatase and tensin homolog (PTEN), neurofibromin 1 (NF-1) or RAS signaling.,The hyperactivation of tyrosine kinase receptors, such as the receptor of the platelet-derived growth factor β (PDFRβ), insulin-like growth factor 1 receptor (IGF-1R) and the receptor for hepatocyte growth factor (HGF), lead to the induction of the AKT/3-phosphoinositol kinase (PI3K) pathway.,Another pathway resulting in BRAFi/MEKi resistance is the hyperactivation of epidermal growth factor receptor (EGFR) signaling or the deregulation of microphthalmia-associated transcription factor (MITF).
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Metastatic melanoma is an aggressive form of skin cancer with a high mortality rate and the fastest growing global incidence rate of all malignancies.,The introduction of BRAF/MEK inhibitor combinations has yielded significant increases in PFS and OS for melanoma.,However, at present, no direct comparisons between different BRAF/MEK combinations have been conducted.,In light of this, an indirect treatment comparison was performed between two BRAF/MEK inhibitor combination therapies for metastatic melanoma, dabrafenib plus trametinib and vemurafenib plus cobimetinib, in order to understand the relative efficacy and toxicity profiles of these therapies.,A systematic literature search identified two randomized trials as suitable for indirect comparison: the coBRIM trial of vemurafenib plus cobimetinib versus vemurafenib and the COMBI-v trial of dabrafenib plus trametinib versus vemurafenib.,The comparison followed the method of Bucher et al. and analyzed both efficacy (overall survival [OS], progression-free survival [PFS], and overall response rate [ORR]) and safety outcomes (adverse events [AEs]).,The indirect comparison revealed similar efficacy outcomes between both therapies, with no statistically significant difference between therapies for OS (hazard ratio [HR] 0.94, 95% confidence interval [CI] 0.68 − 1.30), PFS (HR 1.05, 95% CI 0.79 − 1.40), or ORR (risk ratio [RR] 0.90, 95% CI 0.74 − 1.10).,Dabrafenib plus trametinib differed significantly from vemurafenib plus cobimetinib with regard to the incidence of treatment-related AE (RR 0.92, 95% CI 0.87 − 0.97), any AE grade ≥3 (RR 0.71, 95% CI 0.60 − 0.85) or dose interruption/modification (RR 0.77, 95% CI 0.60 − 0.99).,Several categories of AEs occurred significantly more frequently with vemurafenib plus cobimetinib, while some occurred significantly more frequently with dabrafenib plus trametinib.,For severe AEs (grade 3 or above), four occurred significantly more frequently with vemurafenib plus cobimetinib and no severe AE occurred significantly more frequently with dabrafenib plus trametinib.,This indirect treatment comparison suggested that dabrafenib plus trametinib had comparable efficacy to vemurafenib plus cobimetinib but was associated with reduced adverse events.
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Cancer cell phenotype largely depends on oxygen availability.,The atmospheric oxygen concentration (21%) used in in vitro studies is much higher than in any human tissue.,Using well-characterized patient-derived melanoma cell lines, we compared: (i) activities of several signaling pathways, and (ii) the effects of vemurafenib and trametinib in hyperoxia (21% O2), normoxia (6% O2) and hypoxia (1% O2).,A high plasticity of melanoma cells in response to changes in oxygen supplementation and drug treatment was observed, and the transcriptional reprograming and phenotypic changes varied between cell lines.,Normoxia enhanced the expression of vascular endothelial growth factor (VEGF), glucose metabolism/transport-related genes, and changed percentages of NGFR- and MITF-positive cells in cell line-dependent manner.,Increased protein stability might be responsible for high PGC1α level in MITFlow melanoma cells.,Vemurafenib and trametinib while targeting the activity of MAPK/ERK pathway irrespective of oxygen concentration, were less effective in normoxia than hyperoxia in reducing levels of VEGF, PGC1α, SLC7A11 and Ki-67-positive cells in cell line-dependent manner.,In conclusion, in vitro studies performed in atmospheric oxygen concentration provide different information on melanoma cell phenotype and response to drugs than performed in normoxia, which might partially explain the discrepancies between results obtained in vitro and in clinical settings.
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The neurotrophin Neuritin1 (NRN1; cpg15) belongs to the candidate plasticity gene (CPG) family and is expressed in postmitotic-differentiating neurons of the developmental nervous system and neuronal structures associated with plasticity in the brain of human adult.,Our newest findings document that NRN1 deregulation could contribute also to disease development and have impact on malignant melanoma.,Our analyses displayed the over-expression of NRN1 in melanoma in vitro and in vivo, shown by immunohistochemistry and qRT-PCR on microdissected melanoma tissue; furthermore, soluble NRN1 was detectable in tissue culture supernatant and serum of melanoma patients.,To investigate the role of NRN1 in melanoma we performed knockdown, over-expression and recombinant-NRN1-treatment experiments affiliated by functional assays.,Our results show that migration, attachment independent growth and vasculogenesis were affected after manipulation of NRN1 on endogenous and extrinsic level.,Interestingly, high NRN1 serum levels correlate with low MIA serum levels (< 10ng/ml).,Therefore, we speculate that NRN1 could be a marker for early melanoma stages, in particular.,In summary, we detected an overexpression of NRN1 in melanoma patient.,In functional cell culture experiments we found a correlation between NRN1 expression and the cancerous behavior of melanoma cells.
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New therapies, including the anti‐cytotoxic T lymphocyte antigen (CTLA)‐4 antibody, ipilimumab, is approved for metastatic melanoma.,Prognostic biomarkers need to be identified, because the treatment has serious side effects.,Serum samples were obtained before and during treatment from 56 patients with metastatic or unresectable malignant melanoma, receiving treatment with ipilimumab in a national Phase IV study (NCT0268196).,Expression of a panel of 17 inflammatory‐related markers reflecting different pathways including extracellular matrix remodeling and fibrosis, vascular inflammation and monocyte/macrophage activation were measured at baseline and the second and/or third course of treatment with ipilimumab.,Six candidate proteins [endostatin, osteoprotegerin (OPG), C‐reactive protein (CRP), pulmonary and activation‐regulated chemokine (PARC), growth differentiation factor 15 (GDF15) and galectin‐3 binding‐protein (Gal3BP)] were persistently higher in non‐survivors.,In particular, high Gal3BP and endostatin levels were also independently associated with poor 2‐year survival after adjusting for lactate dehydrogenase, M‐stage and number of organs affected.,A 1 standard deviation increase in endostatin gave 1·74 times [95% confidence interval (CI) = 1·10-2·78, P = 0·019] and for Gal3BP 1·52 times (95% CI = 1·01-2·29, P = 0·047) higher risk of death in the adjusted model.,Endostatin and Gal3BP may represent prognostic biomarkers for patients on ipilimumab treatment in metastatic melanoma and should be further evaluated.,Owing to the non‐placebo design, we could only relate our findings to prognosis during ipilimumab treatment.
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Tremelimumab is an antibody that blocks CTLA-4 and demonstrates clinical efficacy in a subset of advanced melanoma patients.,An unmet clinical need exists for blood-based response-predictive gene signatures to facilitate clinically effective and cost-efficient use of such immunotherapeutic interventions.,Peripheral blood samples were collected in PAXgene® tubes from 210 treatment-naïve melanoma patients receiving tremelimumab in a worldwide, multicenter phase III study (discovery dataset).,A central panel of radiologists determined objective response using RECIST criteria.,Gene expression for 169 mRNA transcripts was measured using quantitative PCR.,A 15-gene pre-treatment response-predictive classifier model was identified.,An independent population (N = 150) of refractory melanoma patients receiving tremelimumab after chemotherapy enrolled in a worldwide phase II study (validation dataset).,The classifier model, using the same genes, coefficients and constants for objective response and one-year survival after treatment, was applied to the validation dataset.,A 15-gene pre-treatment classifier model (containing ADAM17, CDK2, CDKN2A, DPP4, ERBB2, HLA-DRA, ICOS, ITGA4, LARGE, MYC, NAB2, NRAS, RHOC, TGFB1, and TIMP1) achieved an area under the curve (AUC) of 0.86 (95% confidence interval 0.81 to 0.91, p < 0.0001) for objective response and 0.6 (95% confidence interval 0.54 to 0.67, p = 0.0066) for one-year survival in the discovery set.,This model was validated in the validation set with AUCs of 0.62 (95% confidence interval 0.54 to 0.70 p = 0.0455) for objective response and 0.68 for one-year survival (95% confidence interval 0.59 to 0.75 p = 0.0002).,To our knowledge, this is the largest blood-based biomarker study of a checkpoint inhibitor, tremelimumab, which demonstrates a validated pre-treatment mRNA classifier model that predicts clinical response.,The data suggest that the model captures a biological signature representative of genes needed for a robust anti-cancer immune response.,It also identifies non-responders to tremelimumab at baseline prior to treatment.,The online version of this article (doi:10.1186/s40425-017-0272-z) contains supplementary material, which is available to authorized users.
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Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer.,Although most cSCCs have good prognosis, a subgroup of high-risk cSCC has a higher frequency of recurrence and mortality.,Therefore, the identification of molecular risk factors associated with this aggressive subtype is of major interest.,In this work we carried out a global-scale approach to investigate the DNA-methylation profile in patients at different stages, from premalignant actinic keratosis to low-risk invasive and high-risk non-metastatic and metastatic cSCC.,The results showed massive non-sequential changes in DNA-methylome and identified a minimal methylation signature that discriminates between stages.,Importantly, a direct comparison of low-risk and high-risk stages revealed epigenetic traits characteristic of high-risk tumours.,Finally, a prognostic prediction model in cSCC patients identified a methylation signature able to predict the overall survival of patients.,Thus, the analysis of DNA-methylation in cSCC revealed changes during the evolution of the disease through the different stages that can be of great value not only in the diagnosis but also in the prognosis of the disease.
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The expression levels of miR-365 vary in different malignancies.,Herein, we found that miR-365 was overexpressed in both cells and clinical specimens of cutaneous squamous cell carcinoma (SCC).,We demonstrated that the HaCaTpre-miR-365-2 cell line, which overexpressed miR-365, could induce subcutaneous tumors in vivo.,Antagomir-365, an anti-miR-365 oligonucleotide, inhibited cutaneous tumor formation in vivo, along with G1 phase arrest and apoptosis of cancer cells.,These findings suggest that miR-365 may act as an onco-miR in cutaneous SCC both in vitro and in vivo.,The present study provides valuable insight into the role of miR-365 in cutaneous SCC formation, which can help develop new drug and miR-365 target-based therapies for cutaneous SCC.
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In less than 10 years, melanoma treatment has been revolutionized with the approval of tyrosine kinase inhibitors and immune checkpoint inhibitors, which have been shown to have a significant impact on the prognosis of patients with melanoma.,The early steps of this transformation have taken place in research laboratories.,The mitogen-activated protein kinase (MAPK) pathway, phosphoinositol-3-kinase (PI3K) pathway promote the development of melanoma through numerous genomic alterations on different components of these pathways.,Moreover, melanoma cells deeply interact with the tumor microenvironment and the immune system.,This knowledge has led to the identification of novel therapeutic targets and treatment strategies.,In this review, the epidemiological features of cutaneous melanoma along with the biological mechanisms involved in its development and progression are summarized.,The current state-of-the-art of advanced stage melanoma treatment strategies and the currently available evidence of the use of predictive and prognostic biomarkers are also discussed.
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In addition to alterations concerning the expression of oncogenes and onco-suppressors, melanoma is characterized by the presence of distinctive gangliosides (sialic acid carrying glycosphingolipids).,Gangliosides strongly control cell surface dynamics and signaling; therefore, it could be assumed that these alterations are linked to modifications of cell behavior acquired by the tumor.,On these bases, this work investigated the correlations between melanoma cell ganglioside metabolism profiles and the biological features of the tumor and the survival of patients.,Melanoma cell lines were established from surgical specimens of AJCC stage III and IV melanoma patients.,Sphingolipid analysis was carried out on melanoma cell lines and melanocytes through cell metabolic labeling employing [3-3H]sphingosine and by FACS.,N-glycolyl GM3 was identified employing the 14 F7 antibody.,Gene expression was assayed by Real Time PCR.,Cell invasiveness was assayed through a Matrigel invasion assay; cell proliferation was determined through the soft agar assay, MTT, and [3H] thymidine incorporation.,Statistical analysis was performed using XLSTAT software for melanoma hierarchical clustering based on ganglioside profile, the Kaplan-Meier method, the log-rank (Mantel-Cox) test, and the Mantel-Haenszel test for survival analysis.,Based on the ganglioside profiles, through a hierarchical clustering, we classified melanoma cells isolated from patients into three clusters: 1) cluster 1, characterized by high content of GM3, mainly in the form of N-glycolyl GM3, and GD3; 2) cluster 2, characterized by the appearance of complex gangliosides and by a low content of GM3; 3) cluster 3, which showed an intermediate phenotype between cluster 1 and cluster 3.,Moreover, our data demonstrated that: a) a correlation could be traced between patients’ survival and clusters based on ganglioside profiles, with cluster 1 showing the worst survival; b) the expression of several enzymes (sialidase NEU3, GM2 and GM1 synthases) involved in ganglioside metabolism was associated with patients’ survival; c) melanoma clusters showed different malignant features such as growth in soft agar, invasiveness, expression of anti-apoptotic proteins.,Ganglioside profile and metabolism is strictly interconnected with melanoma aggressiveness.,Therefore, the profiling of melanoma gangliosides and enzymes involved in their metabolism could represent a useful prognostic and diagnostic tool.,The online version of this article (doi:10.1186/1471-2407-14-560) contains supplementary material, which is available to authorized users.
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Supplemental Digital Content is available in the text.,The overall survival (OS) of patients with metastatic uveal melanoma is short, the evidence for effectiveness of treatments is limited, and no consensus on the choice of treatment exists.,We aimed to advance interpretation of OS as an outcome by pooling peer-reviewed data.,The design is a systematic review and meta-analysis.,We searched PubMed from 1 January 1980, to 29 March 2017, for articles reporting patient-level survival in Kaplan-Meier or numerical form.,We digitized survival graphs, pooled individual survival times, calculated median OS by treatment modality, and compared each modality by the log-rank test and Cox regression using conventional chemotherapy (CHT) as a reference.,Individual-level data were obtained from 78 articles with 2494 patients.,The median OS across all treatment modalities was 1.07 years (range: 0.59-2.50 years).,Pooled OS reported after isolated hepatic perfusion [median OS: 1.34 years; hazard ratio (HR): 0.92, 95% confidence interval (CI): 0.87-0.97, P = 0.0040], immunoembolization (median OS: 1.63; HR: 0.97, 95% CI: 0.95-1.00, P = 0.0080), and surgery (median OS: 1.43; HR: 0.94, 95% CI: 0.92-0.96, P < 0.0001) was longer, and after checkpoint inhibitor shorter (median OS: 0.59; HR: 1.13, 95% CI: 1.06-1.20, P < 0.0001) than after CHT (median OS: 0.91 years), but subject to identifiable confounding factors.,OS following other modalities did not differ from CHT.,Reported OS was unassociated with the decade of publication, but depended on the percentage of first-line treated patients.,Our results suggest no clinically significant difference in OS by treatment modality or decade.,Most of the difference in reported OS likely is attributable to surveillance, selection, and publication bias rather than treatment-related prolongation.,Our pooled data provide benchmarks for future trials.
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Mutations in GNAQ and GNA11, encoding the oncogenic G-protein alpha subunit q and 11, respectively, occur frequently in the majority of uveal melanomas.,Exons 4 and 5 from GNAQ and GNA11 were amplified and sequenced from 92 ciliary body and choroidal melanomas.,The mutation status was correlated with disease-free survival (DFS) and other parameters.,None of the tumours harboured a GNAQ exon 4 mutation.,A GNAQ mutation in exon 5 codon 209 was found in 46 out of 92 (50.0%) of the tumours.,Only 1 out of 92 (1.1%) melanomas showed a mutation in GNA11 exon 4 codon 183, whereas 39 out of 92 (42.4%) harboured a mutation in exon 5 of GNA11 codon 209.,Six tumours did not show any mutations in exons 4 and 5 of these genes.,Univariate analyses showed no correlation between DFS and the mutation status.,GNAQ and GNA11 mutations are, in equal matter, not associated with patient outcome.
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Emerging evidence suggests that a diagnosis of cutaneous melanoma (CM) may be associated with prostate cancer (PC) incidence.,We examined if the incidence of CM was associated with an increased subsequent risk of PC.,We used data from the New South Wales Cancer Registry for all CM and PC cases diagnosed between January 1972 and December 2008.,We calculated the age standardized incidence ratio (SIR) and 95% confidence intervals (95% CI) for PC incidence following a CM diagnosis, applying age- and calendar- specific rates to the appropriate person years at risk.,We determined rate ratio (RR) and 95% CI of PC incidence according to specified socio-demographic categories and disease related characteristics, using a negative binomial model.,There were 143,594 men diagnosed with PC or CM in the study period and of these 101,198 and 42,396 were diagnosed with PC and CM, respectively, as first primary cancers.,Risk of PC incidence increased following CM diagnosis (n = 2,114; SIR = 1.25; 95% CI:1.20.8-1.31: p < 0.0001), with the increased risk apparent in men diagnosed with localised CM (n = 1,862;SIR = 1.26; 95% CI:1.20-1.32).,CM diagnosis increased the subsequent risk of PC incidence.,This raises the potential for future PC risk to be discussed with newly diagnosed males with CM.
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Cutaneous malignant melanoma, whose incidence is increasing steadily worldwide, is the result of complex interactions between individual genetic factors and environmental risk factors.,Ultraviolet radiation represents the most important environmental risk factor for the development of skin cancers, including melanoma.,Sun exposure and early sunburn during childhood are the principal causes of cutaneous melanoma insurgence in adults, with double the risk relative to a nonexposed population.,Consequently, ultraviolet protection has long been recognized as an important measure to prevent such a malignancy.,Biological and epidemiological data suggest that vitamin D status could affect the risk of cancer and play a role in cancer prevention by exerting antiproliferative effects.,Solar radiations are critical for vitamin D synthesis in humans; however, uncontrolled and intensive sun exposure is dangerous to skin health and may contribute toward the development of cutaneous malignant melanoma.,An optimum balance between sun protection and exposure is thus advocated.,Additional research is required to confirm the preventive role of vitamin D in melanoma incidence or a positive influence on patient outcome.
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Most genetic susceptibility to cutaneous melanoma remains to be discovered.,Meta-analysis genome-wide association study (GWAS) of 36,760 melanoma cases (67% newly-genotyped) and 375,188 controls identified 54 significant loci with 68 independent SNPs.,Analysis of risk estimates across geographical regions and host factors suggests the acral melanoma subtype is uniquely unrelated to pigmentation.,Combining this meta-analysis with nevus count and hair color GWAS, and transcriptome association approaches, uncovered 31 potential secondary loci, for a total of 85 cutaneous melanoma susceptibility loci.,These findings provide substantial insights into cutaneous melanoma genetic architecture, reinforcing the importance of nevogenesis, pigmentation, and telomere maintenance together with identifying potential new pathways for cutaneous melanoma pathogenesis.
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We sequenced 8 melanoma exomes to identify novel somatic mutations in metastatic melanoma.,Focusing on the MAP3K family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9.,Structural modelling predicts that mutations in the kinase domain may affect the activity and regulation of MAP3K5/9 protein kinases.,The position of the mutations and loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely inactivating.,In vitro kinase assay shows reduction in kinase activity in MAP3K5 I780F and MAP3K9 W333X mutants.,Overexpression of MAP3K5 or MAP3K9 mutant in HEK293T cells reduces phosphorylation of downstream MAP kinases.,Attenuation of MAP3K9 function in melanoma cells using siRNA leads to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.
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Despite several therapeutic advances, malignant melanoma still remains a fatal disease for which novel and long-term curative treatments are needed.,The successful development of innovative therapies strongly depends on the availability of appropriate pre-clinical models.,For this purpose, several mouse models holding the promise to provide insight into molecular biology and clinical behavior of melanoma have been generated.,The most relevant ones and their contribution for the advancement of therapeutic approaches for the treatment of human melanoma patients will be here summarized.,However, as models, mice do not recapitulate all the features of human melanoma, thus their strengths and weaknesses need to be carefully identified and considered for the translation of the results into the human clinics.,In this panorama, the concept of comparative oncology acquires a priceless value.,The revolutionary importance of spontaneous canine melanoma as a translational model for the pre-clinical investigation of melanoma progression and treatment will be here discussed, with a special consideration to the development of innovative immunotherapeutic approaches.
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Therapies that target the programmed death-1 (PD-1) receptor have shown unprecedented rates of durable clinical responses in patients with various cancer types.1-5 One mechanism by which cancer tissues limit the host immune response is via upregulation of PD-1 ligand (PD-L1) and its ligation to PD-1 on antigen-specific CD8 T-cells (termed adaptive immune resistance).6,7 Here we show that pre-existing CD8 T-cells distinctly located at the invasive tumour margin are associated with expression of the PD-1/PD-L1 immune inhibitory axis and may predict response to therapy.,We analyzed samples from 46 patients with metastatic melanoma obtained before and during anti-PD1 therapy (pembrolizumab) using quantitative immunohistochemistry, quantitative multiplex immunofluorescence, and next generation sequencing for T-cell receptors (TCR).,In serially sampled tumours, responding patients showed proliferation of intratumoural CD8+ T-cells that directly correlated with radiographic reduction in tumour size.,Pre-treatment samples obtained from responding patients showed higher numbers of CD8, PD1, and PD-L1 expressing cells at the invasive tumour margin and inside tumours, with close proximity between PD-1 and PD-L1, and a more clonal TCR repertoire.,Using multivariate analysis, we established a predictive model based on CD8 expression at the invasive margin and validated the model in an independent cohort of 15 patients.,Our findings indicate that tumour regression following therapeutic PD-1 blockade requires pre-existing CD8+ T cells that are negatively regulated by PD-1/PD-L1 mediated adaptive immune resistance.
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The incidence of melanoma is increasing worldwide, and the prognosis for patients with high-risk or advanced metastatic melanoma remains poor despite advances in the field.,A systematic literature review of treatments for advanced, metastatic disease was conducted to present the success of current treatments and the promise of those still in clinical development that may yield incremental improvements in the treatment of advanced, metastatic melanoma.,Advances in the understanding of the mechanism of chemotherapy resistance offer the hope for improved results with chemotherapy, and the triumvirate of more effective chemotherapy, immunotherapy, and targeted therapy are likely to be combined with one another for significant advances in melanoma over the coming few years.,The incidence of melanoma is increasing worldwide, and the prognosis for patients with high-risk or advanced metastatic melanoma remains poor despite advances in the field.,Standard treatment for patients with thick (≥2.0 mm) primary melanoma with or without regional metastases to lymph nodes is surgery followed by adjuvant therapy or clinical trial enrollment.,Adjuvant therapy with interferon-α and cancer vaccines is discussed in detail.,Patients who progress to stage IV metastatic melanoma have a median survival of ≤1 year.,Standard treatment with chemotherapy yields low response rates, of which few are durable.,Cytokine therapy with IL-2 achieves durable benefits in a greater fraction, but it is accompanied by severe toxicities that require the patient to be hospitalized for support during treatment.,A systematic literature review of treatments for advanced, metastatic disease was conducted to present the success of current treatments and the promise of those still in clinical development that may yield incremental improvements in the treatment of advanced, metastatic melanoma.
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B-RAF is the most frequently mutated protein kinase in human cancers.1 The finding that oncogenic mutations in BRAF are common in melanoma2 followed by the demonstration that these tumors are dependent on the RAF/MEK/ERK pathway3 offered hope that inhibition of B-RAF kinase activity could benefit melanoma patients.,Herein, we describe the structure-guided discovery of PLX4032 (RG7204), a potent inhibitor of oncogenic B-RAF kinase activity.,Preclinical experiments demonstrated that PLX4032 selectively blocked the RAF/MEK/ERK pathway in BRAF mutant cells and caused regression of BRAF mutant xenografts.4 Toxicology studies confirmed a wide safety margin consistent with the high degree of selectivity, enabling Phase 1 clinical trials using a crystalline formulation of PLX4032.5 In a subset of melanoma patients, pathway inhibition was monitored in paired biopsy specimens collected before treatment initiation and following two weeks of treatment.,This analysis revealed substantial inhibition of ERK phosphorylation, yet clinical evaluation did not show tumor regressions.,At higher drug exposures afforded by a new amorphous drug formulation,4,5 greater than 80% inhibition of ERK phosphorylation in the tumors of patients correlated with clinical response.,Indeed, the Phase 1 clinical data revealed a remarkably high 81% response rate in metastatic melanoma patients treated at an oral dose of 960 mg twice daily.5 These data demonstrate that BRAF-mutant melanomas are highly dependent on B-RAF kinase activity.
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Wnt proteins are important for developmental processes and certain diseases.,WNT5A is a non-canonical Wnt protein that previously has been shown to play a role in the progression of malignant melanoma.,High expression of WNT5A in melanoma tumors correlates to formation of distant metastasis and poor prognosis.,This has partly been described by the findings that WNT5A expression in melanoma cell lines increases migration and invasion.,Malignant melanoma cell lines were treated with rWNT5A or WNT5A siRNA, and mRNA versus protein levels of soluble mediators were measured using RT-PCR, cytokine bead array and ELISA.,The induced signaling pathways were analyzed using inhibitors, Rho-GTPase pull down assays and western blot.,Ultracentrifugation and electron microscopy was used to analyze microvesicles.,Gene expression microarray data obtained from primary malignant melanomas was used to verify our data.,We show that WNT5A signaling induces a Ca2+-dependent release of exosomes containing the immunomodulatory and pro-angiogenic proteins IL-6, VEGF and MMP2 in melanoma cells.,The process was independent of the transcriptional machinery and depletion of WNT5A reduced the levels of the exosome-derived proteins.,The WNT5A induced exosomal secretion was neither affected by Tetanus toxin nor Brefeldin A, but was blocked by the calcium chelator Bapta, inhibited by a dominant negative version of the small Rho-GTPase Cdc42 and was accompanied by cytoskeletal reorganization.,Co-cultures of melanoma/endothelial cells showed that depletion of WNT5A in melanoma cells decreased endothelial cell branching, while stimulation of endothelial cells with isolated rWNT5A-induced melanoma exosomes increased endothelial cell branching in vitro.,Finally, gene expression data analysis of primary malignant melanomas revealed a correlation between WNT5A expression and the angiogenesis marker ESAM.,These data indicate that WNT5A has a broader function on tumor progression and metastatic spread than previously known; by inducing exosome-release of immunomodulatory and pro-angiogenic factors that enhance the immunosuppressive and angiogenic capacity of the tumors thus rendering them more aggressive and more prone to metastasize.
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Conventional calpains are ubiquitous cysteine proteases whose activity is promoted by calcium signaling and specifically limited by calpastatin.,Calpain expression has been shown to be increased in human malignant cells, but the contribution of the calpain/calpastatin system in tumorigenesis remains unclear.,It may play an important role in tumor cells themselves (cell growth, migration, and a contrario cell death) and/or in tumor niche (tissue infiltration by immune cells, neo-angiogenesis).,In this study, we have used a mouse model of melanoma as a tool to gain further understanding of the role of calpains in tumor progression.,To determine the respective importance of each target, we overexpressed calpastatin in tumor and/or host in isolation.,Our data demonstrate that calpain inhibition in both tumor and host blunts tumor growth, while paradoxically increasing metastatic dissemination to regional lymph nodes.,Specifically, calpain inhibition in melanoma cells limits tumor growth in vitro and in vivo but increases dissemination by amplifying cell resistance to apoptosis and accelerating migration process.,Meanwhile, calpain inhibition restricted to host cells blunts tumor infiltration by immune cells and angiogenesis required for antitumor immunity, allowing tumor cells to escape tumor niche and disseminate.,The development of highly specific calpain inhibitors with potential medical applications in cancer should take into account the opposing roles of the calpain/calpastatin system in initial tumor growth and subsequent metastatic dissemination.
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Melanomas can switch to a dedifferentiated cell state upon exposure to cytotoxic T cells.,However, it is unclear whether such tumor cells pre-exist in patients and whether they can be resensitized to immunotherapy.,Here, we chronically expose (patient-derived) melanoma cell lines to differentiation antigen-specific cytotoxic T cells and observe strong enrichment of a pre-existing NGFRhi population.,These fractions are refractory also to T cells recognizing non-differentiation antigens, as well as to BRAF + MEK inhibitors.,NGFRhi cells induce the neurotrophic factor BDNF, which contributes to T cell resistance, as does NGFR.,In melanoma patients, a tumor-intrinsic NGFR signature predicts anti-PD-1 therapy resistance, and NGFRhi tumor fractions are associated with immune exclusion.,Lastly, pharmacologic NGFR inhibition restores tumor sensitivity to T cell attack in vitro and in melanoma xenografts.,These findings demonstrate the existence of a stable and pre-existing NGFRhi multitherapy-refractory melanoma subpopulation, which ought to be eliminated to revert intrinsic resistance to immunotherapeutic intervention.,Dedifferentiation state has been associated with therapy resistance in melanoma.,Here, the authors uncover a pre-existing NGFR-expressing, targetable subpopulation that is resistant to immunotherapy and other treatments in melanoma cells and preclinical models.
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Identifying cancer biomarkers is of great interest for patient prognosis and monitoring.,However, tumor-associated factors are diluted in blood and challenging to detect.,Broggi et al. show that lymphatic exudate, accessible at the time of lymphadenectomy, is highly enriched in tumor-associated factors and therefore a promising source for biomarker discovery.,Liquid biopsies allow monitoring of cancer progression and detection of relapse, but reliable biomarkers in melanoma are lacking.,Because secreted factors preferentially drain to lymphatic vessels before dilution in the blood, we hypothesized that lymph should be vastly enriched in cancer biomarkers.,We characterized postoperative lymphatic exudate and plasma of metastatic melanoma patients after lymphadenectomy and found a dramatic enrichment in lymphatic exudate of tumor-derived factors and especially extracellular vesicles containing melanoma-associated proteins and miRNAs, with unique protein signatures reflecting early versus advanced metastatic spread.,Furthermore, lymphatic exudate was enriched in memory T cells, including tumor-reactive CD137+ and stem cell-like types.,In mice, lymph vessels were the major route of extracellular vesicle transport from tumors to the systemic circulation.,We suggest that lymphatic exudate provides a rich source of tumor-derived factors for enabling the discovery of novel biomarkers that may reflect disease stage and therapeutic response.
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BRAF V600 mutant circulating cell-free tumor DNA (BRAF V600mut ctDNA) could serve as a specific biomarker in patients with BRAF V600 mutant melanoma.,We analyzed the value of BRAF V600mut ctDNA from plasma as a monitoring tool for advanced melanoma patients treated with BRAF/MEK inhibitors.,Allele-specific quantitative PCR analysis for BRAF V600 E/E2/D/K/R/M mutations was performed on DNA extracted from plasma of patients with known BRAF V600 mutant melanoma who were treated with dabrafenib and trametinib.,245 plasma samples from 36 patients were analyzed.,In 16 patients the first plasma sample was obtained before the first dosing of dabrafenib/trametinib.,At baseline, BRAF V600mut ctDNA was detected in 75 % of patients (n = 12/16).,BRAF V600mut ctDNA decreased rapidly upon initiation of targeted therapy (p < 0.001) and became undetectable in 60 % of patients (n = 7/12) after 6 weeks of treatment.,During treatment, disease progression (PD) was diagnosed in 27 of 36 patients.,An increase of the BRAF V600mut ctDNA copy number and fraction, identified PD with a sensitivity of 70 % (n = 19/27) and a specificity of 100 %.,An increase in the BRAF V600mut ctDNA fraction was detected prior to clinical PD in 44 % of cases (n = 12/27) and simultaneously with PD in 26 % of patients (n = 7/27).,Quantitative analysis of BRAF V600mut ctDNA in plasma has unique features as a monitoring tool during treatment with BRAF/MEK inhibitors.,Its potential as an early predictor of acquired resistance deserves further evaluation.
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Circulating tumor cells have emerged as prognostic biomarkers in the treatment of metastatic cancers of epithelial origins viz., breast, colorectal and prostate.,These tumors express Epithelial Cell Adhesion Molecule (EpCAM) on their cell surface which is used as an antigen for immunoaffinity capture.,However, EpCAM capture technologies are of limited utility for non-epithelial cancers such as melanoma.,We report a method to enrich Circulating Melanoma Cells (CMCs) that does not presuppose malignant cell characteristics.,CMCs were enriched by centrifugation of blood samples from healthy (N = 10) and patient (N = 11) donors, followed by RBC lysis and immunomagnetic depletion of CD45-positive leukocytes in a specialized magnetic separator.,CMCs were identified by immunocytochemistry using Melan-A or S100B as melanoma markers and enumerated using automated microscopy image analyses.,Separation was optimized for maximum sensitivity and recovery of CMCs.,Our results indicate large number of CMCs in Stage IV melanoma patients.,Analysis of survival suggested a trend toward decreased survival with increased number of CMCs.,Moreover, melanoma-associated miRs were found to be higher in CMC-enriched fractions in two patients when compared with the unseparated samples, validating this method as applicable for molecular analyses.,Negative selection is a promising approach for isolation of CMCs and other EpCAM -negative CTCs, and is amenable to molecular analysis of CMCs.,Further studies are required to validate its efficacy at capturing specific circulating cells for genomic analysis, and xenograft studies.
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Tumor cells evade the immune surveillance by up-regulating surface expression of PD-L1, which interacts with PD-1 on T cells to elicit the immune checkpoint response1,2.,Anti-PD-1 antibodies have shown remarkable promise in treating tumors, including metastatic melanoma2-4.,However, patient response rate is low4,5.,A better understanding of PD-L1-mediated immune evasion is needed to predict patient response and improve treatment efficacy.,Here we report that metastatic melanoma releases a high level of extracellular vesicles (EVs), mostly in the form of exosomes, that carry PD-L1 on their surface.,Interferon-γ (IFN-γ) up-regulates PD-L1 on these vesicles, which suppresses the function of CD8 T cells and facilitates tumor growth.,In patients with metastatic melanoma, the level of circulating exosomal PD-L1 positively correlates with that of IFN-γ, and changes during the course of anti-PD-1 therapy.,The magnitudes of the early on-treatment increase in circulating exosomal PD-L1, as an indicator of the adaptive response of the tumor cells to T cell re-invigoration, stratifies clinical responders from non-responders.,Our study unveils a mechanism by which tumor cells systemically suppress the immune system, and provides a rationale for the application of exosomal PD-L1 as a predictor for anti-PD-1 therapy.
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Vitamin D is a lipid soluble steroid hormone with pleiotropic biological properties, including regulation of cell proliferation, differentiation and apoptosis.,As to these desirable anticancer actions, 1,25-dihydroxyvitamins D and analogs have been reported to inhibit the proliferation and to induce differentiation of a wide variety of cancer cell types, including human malignant melanoma.,However, there is a need for novel and more efficacious vitamin D analogs, and how best to design such is still an open issue.,A series of double point modified (DPM) analogs of 1,25-dihydroxyvitamin D2 (1,25(OH)2D2) induced differentiation of the vitamin D receptor (VDR) positive A375 and VDR negative SK-MEL 188b human malignant melanoma cell lines.,Surprisingly, the dose of 1,25(OH)2D2 required to inhibit the proliferation of the A375 melanoma cell line by was several fold lower than that required in the case of 1,25(OH)2D3.,To evaluate the impact of the modification in the side chain (additional 22-hydroxyl) and in the A-ring (5,6-trans modification), the regular side-chain of vitamin D2 or D3 was retained in the structure of our analogs.,As expected, 5,6-trans modification was advantageous to enhancing the anti-proliferative activity of analogs, but not as a single point modification (SPM).,Very unexpectedly, the additional 22-hydroxyl in the side-chain reduced significantly the anti-proliferative activity of both the natural and 5,6-trans series analogs.,Finally, an induction of pigmentation in melanoma SK-MEL 188b cells was observed to sensitized cells to the effect of vitamin D analogs.
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The microphthalmia-associated transcription factor (MITF) is the “master melanocyte transcription factor” with a complex role in melanoma.,MITF protein levels vary between and within clinical specimens, and amplifications and gain- and loss-of-function mutations have been identified in melanoma.,How MITF functions in melanoma development and the effects of targeting MITF in vivo are unknown because MITF levels have not been directly tested in a genetic animal model.,Here, we use a temperature-sensitive mitf zebrafish mutant to conditionally control endogenous MITF activity.,We show that low levels of endogenous MITF activity are oncogenic with BRAFV600E to promote melanoma that reflects the pathology of the human disease.,Remarkably, abrogating MITF activity in BRAFV600Emitf melanoma leads to dramatic tumor regression marked by melanophage infiltration and increased apoptosis.,These studies are significant because they show that targeting MITF activity is a potent antitumor mechanism, but also show that caution is required because low levels of wild-type MITF activity are oncogenic.
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Melanoma is the most aggressive and lethal form of skin cancer.,Because of the increasing incidence and high lethality of melanoma, animal models for continuously observing melanoma formation and progression as well as for testing pharmacological agents are needed.,Using the combinatorial Gal4 -UAS system, we have developed a zebrafish transgenic line that expresses oncogenic HRAS under the kita promoter.,Already at 3 days transgenic kita-GFP-RAS larvae show a hyper-pigmentation phenotype as earliest evidence of abnormal melanocyte growth.,By 2-4 weeks, masses of transformed melanocytes form in the tail stalk of the majority of kita-GFP-RAS transgenic fish.,The adult tumors evident between 1-3 months of age faithfully reproduce the immunological, histological and molecular phenotypes of human melanoma, but on a condensed time-line.,Furthermore, they show transplantability, dependence on mitfa expression and do not require additional mutations in tumor suppressors.,In contrast to kita expressing melanocyte progenitors that efficiently develop melanoma, mitfa expressing progenitors in a second Gal4-driver line were 4 times less efficient in developing melanoma during the three months observation period.,This indicates that zebrafish kita promoter is a powerful tool for driving oncogene expression in the right cells and at the right level to induce early onset melanoma in the presence of tumor suppressors.,Thus our zebrafish model provides a link between kita expressing melanocyte progenitors and melanoma and offers the advantage of a larval phenotype suitable for large scale drug and genetic modifier screens.
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Before licensing, ipilimumab was first made available to previously treated advanced melanoma patients through an expanded access programme (EAP) across Europe.,We interrogated data from UK EAP patients to inform future clinical practice.,Clinicians registered in the UK EAP provided anonymized patient data using a prespecified variable fields datasheet.,Data collected were baseline patient characteristics, treatment delivered, toxicity, response, progression-free survival and overall survival (OS).,Data were received for 193 previously treated metastatic melanoma patients, whose primary sites were cutaneous (82%), uveal (8%), mucosal (2%), acral (3%) or unknown (5%).,At baseline, 88% of patients had a performance status (PS) of 0-1 and 20% had brain metastases.,Of the patients, 53% received all four planned cycles of ipilimumab; the most common reason for stopping early was disease progression, including death from melanoma.,Toxicity was recorded for 171 patients, 30% of whom experienced an adverse event of grade 3 or higher, the most common being diarrhoea (13%) and fatigue (9%).,At a median follow-up of 23 months, the median progression-free survival and OS were 2.8 and 6.1 months, respectively; the 1-year and 2-year OS rates were 31 and 14.8%, respectively.,The 2-year OS was significantly lower for patients with poorer PS (P<0.0001), low albumin concentrations (P<0.0001), the presence of brain metastases (P=0.007) and lactate dehydrogenase levels more than two times the upper limit of normal (P<0.0001) at baseline.,These baseline characteristics are negative predictors of benefit from ipilimumab and should be taken into consideration before prescription.
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Ipilimumab, a fully human, recombinant, monoclonal antibody to cytotoxic T-lymphocyte antigen 4 improves overall survival (OS) in previously treated and untreated metastatic melanoma.,This retrospective analysis reports data gathered by a questionnaire on the demographics, outcomes, and toxicity of ipilimumab administered through an Expanded Access Program (EAP).,Ipilimumab 3 mg/kg was administered intravenously every 3 weeks for four cycles to adults with metastatic melanoma.,Efficacy outcomes included complete response, partial response (PR), progressive disease, stabilized disease, and OS.,EAP data were collected from EAP physicians.,A subgroup analysis examined efficacy in elderly patients (≥70 years) and factors predictive of survival were identified.,Of 355 requests for ipilimumab, resulting in 288 treatments, completed questionnaires were received for 153 ipilimumab recipients (median age 58 years, 57.2% men).,Efficacy was evaluated in 144 patients: complete response in 1.3%, PR in 9.6%, PR with previous progression 8.4%, stabilized disease in 14.5%, and progressive disease in 66.2%.,The median OS was 6.5 months (199 days); 1-year survival was 32.9%.,Predictive survival factors included lymphocytes over 1000/ml (P=0.0008) and lactate dehydrogenase more than 1.5×upper limit of normal (P=0.003).,Cutaneous, hepatic, and gastrointestinal toxicities were mild.,In 30 patients aged more than 70 years, ipilimumab efficacy and tolerability was similar to that of the overall population.,In the clinical practice setting, ipilimumab is effective and well tolerated in patients with advanced melanoma, including elderly patients, when administered at the recommended dosage.,Ipilimumab improves treatment options for patients who, until recently, have had little hope of an improved prognosis.
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The BRIM-3 trial showed improved progression-free survival (PFS) and overall survival (OS) for vemurafenib compared with dacarbazine in treatment-naive patients with BRAFV600 mutation-positive metastatic melanoma.,We present final OS data from BRIM-3.,Patients were randomly assigned in a 1 : 1 ratio to receive vemurafenib (960 mg twice daily) or dacarbazine (1000 mg/m2 every 3 weeks).,OS and PFS were co-primary end points.,OS was assessed in the intention-to-treat population, with and without censoring of data for dacarbazine patients who crossed over to vemurafenib.,Between 4 January 2010 and 16 December 2010, a total of 675 patients were randomized to vemurafenib (n = 337) or dacarbazine (n = 338, of whom 84 crossed over to vemurafenib).,At the time of database lock (14 August 2015), median OS, censored at crossover, was significantly longer for vemurafenib than for dacarbazine {13.6 months [95% confidence interval (CI) 12.0-15.4] versus 9.7 months [95% CI 7.9-12.8; hazard ratio (HR) 0.81 [95% CI 0.67-0.98]; P = 0.03}, as was median OS without censoring at crossover [13.6 months (95% CI 12.0-15.4) versus 10.3 months (95% CI 9.1-12.8); HR 0.81 (95% CI 0.68-0.96); P = 0.01].,Kaplan-Meier estimates of OS rates for vemurafenib versus dacarbazine were 56% versus 46%, 30% versus 24%, 21% versus 19% and 17% versus 16% at 1, 2, 3 and 4 years, respectively.,Overall, 173 of the 338 patients (51%) in the dacarbazine arm and 175 of the 337 (52%) of those in the vemurafenib arm received subsequent anticancer therapies, most commonly ipilimumab.,Safety data were consistent with the primary analysis.,Vemurafenib continues to be associated with improved median OS in the BRIM-3 trial after extended follow-up.,OS curves converged after ≈3 years, likely as a result of crossover from dacarbazine to vemurafenib and receipt of subsequent anticancer therapies.,NCT01006980.
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The initial use of BRAF targeted therapeutics in clinical trials has demonstrated encouraging responses in melanoma patients, although a rise in drug-resistant cells capable of advancing malignant disease has been described.,The current study uses BRAFV600E expressing WM793 melanoma cells to derive data aimed at investigating the molecular determinant of cell invasion following treatment with clinical BRAF inhibitors.,Small-molecule inhibitors targeting BRAF reduced MEK1/2-ERK1/2 pathway activation and cell survival; yet, viable cell subpopulations persisted.,The residual cells exhibited an elongated cell shape, prominent actin stress fibers and retained the ability to invade 3-D dermal-like microenvironments.,BRAF inhibitor treatments were associated with reduced expression of RND3, an antagonist of RHOA activation, and elevated RHOA-dependent signaling.,Restoration of RND3 expression or RHOA knockdown attenuated the migratory ability of residual cells without affecting overall cell survival.,The invasive ability of BRAF inhibitor treated cells embedded in collagen gels was diminished following RND3 re-expression or RHOA depletion.,Conversely, melanoma cell movement in the absence of BRAF inhibition was unaffected by RND3 expression or RHOA depletion.,These data reveal a novel switch in the requirement for RND3 and RHOA in coordinating the movement of residual WM793 cells that are initially refractive to BRAF inhibitor therapy.,These results have important clinical implications because they suggest that combining BRAF inhibitors with therapies that target the invasion of drug-resistant cells could aid in controlling disease relapse.
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Melanoma is often caused by mutations due to exposure to ultraviolet radiation.,This study reports a recurrent somatic C > T change causing a P131L mutation in the RQCD1 (Required for Cell Differentiation1 Homolog) gene identified through whole exome sequencing of 20 metastatic melanomas.,Screening in 715 additional primary melanomas revealed a prevalence of ~4%.,This represents the first reported recurrent mutation in a member of the CCR4-NOT complex in cancer.,Compared to tumors without the mutation, the P131L mutant positive tumors were associated with increased thickness (p = 0.02), head and neck (p = 0.009) and upper limb (p = 0.03) location, lentigo maligna melanoma subtype (p = 0.02) and BRAF V600K (p = 0.04) but not V600E or NRAS codon 61 mutations.,There was no association with nodal disease (p = 0.3).,Mutually exclusive mutations of other members of the CCR4-NOT complex were found in ~20% of the TCGA melanoma dataset suggesting the complex may play an important role in melanoma biology.,Mutant RQCD1 was predicted to bind strongly to HLA-A0201 and HLA-Cw3 MHC1 complexes.,From thirteen patients with mutant RQCD1, an anti-tumor CD8+ T cell response was observed from a single patient's peripheral blood mononuclear cell population stimulated with mutated peptide compared to wildtype indicating a neoantigen may be formed.
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We characterized the mutational landscape of human skin cutaneous melanoma (SKCM) using data obtained from The Cancer Genome Atlas (TCGA) project.,We analyzed next-generation sequencing data of somatic copy number alterations and somatic mutations in 303 metastatic melanomas.,We were able to confirm preeminent drivers of melanoma as well as identify new melanoma genes.,The TCGA SKCM study confirmed a dominance of somatic BRAF mutations in 50% of patients.,The mutational burden of melanoma patients is an order of magnitude higher than of other TCGA cohorts.,A multi-step filter enriched somatic mutations while accounting for recurrence, conservation, and basal rate.,Thus, this filter can serve as a paradigm for analysis of genome-wide next-generation sequencing data of large cohorts with a high mutational burden.,Analysis of TCGA melanoma data using such a multi-step filter discovered novel and statistically significant potential melanoma driver genes.,In the context of the Pan-Cancer study we report a detailed analysis of the mutational landscape of BRAF and other drivers across cancer tissues.,Integrated analysis of somatic mutations, somatic copy number alterations, low pass copy numbers, and gene expression of the melanogenesis pathway shows coordination of proliferative events by Gs-protein and cyclin signaling at a systems level.
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Pembrolizumab demonstrated robust antitumor activity and safety in the phase Ib KEYNOTE-001 study (NCT01295827) of advanced melanoma.,Five-year outcomes in all patients and treatment-naive patients are reported herein.,Patients whose disease progressed following initial response and who received a second course of pembrolizumab were also analyzed.,Patients aged ≥18 years with previously treated or treatment-naive advanced/metastatic melanoma received pembrolizumab 2 mg/kg every 3 weeks, 10 mg/kg every 3 weeks, or 10 mg/kg every 2 weeks until disease progression, intolerable toxicity, or patient/investigator decision to withdraw.,Kaplan-Meier estimates of overall survival (OS) and progression-free survival (PFS) were calculated.,Objective response rate and PFS were based on immune-related response criteria by investigator assessment (data cut-off, September 1, 2017).,KEYNOTE-001 enrolled 655 patients with melanoma; median follow-up was 55 months.,Estimated 5-year OS was 34% in all patients and 41% in treatment-naive patients; median OS was 23.8 months (95% CI, 20.2-30.4) and 38.6 months (95% CI, 27.2-not reached), respectively.,Estimated 5-year PFS rates were 21% in all patients and 29% in treatment-naive patients; median PFS was 8.3 months (95% CI, 5.8-11.1) and 16.9 months (95% CI, 9.3-35.5), respectively.,Median response duration was not reached; 73% of all responses and 82% of treatment-naive responses were ongoing at data cut-off; the longest response was ongoing at 66 months.,Four patients [all with prior response of complete response (CR)] whose disease progressed during observation subsequently received second-course pembrolizumab.,One patient each achieved CR and partial response (after data cut-off).,Treatment-related AEs (TRAEs) occurred in 86% of patients and resulted in study discontinuation in 7.8%; 17% experienced grade 3/4 TRAE.,This 5-year analysis of KEYNOTE-001 represents the longest follow-up for pembrolizumab to date and confirms the durable antitumor activity and tolerability of pembrolizumab in advanced melanoma.,ClinicalTrials.gov, NCT01295827.
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Metastatic melanoma is a highly aggressive disease.,Recent progress in immunotherapy (IT) and targeted therapy (TT) has led to significant improvements in response and survival rates in metastatic melanoma patients.,The current project aims to determine the benefit of the introduction of these new therapies in advanced melanoma across several regions of Switzerland.,This is a retrospective multicenter analysis of 395 advanced melanoma patients treated with standard chemotherapy, checkpoint inhibitors, and kinase inhibitors from January 2008 until December 2014.,The 1-year survival was 69% (n=121) in patients treated with checkpoint inhibitors (IT), 50% in patients treated with TTs (n=113), 85% in the IT+TT group (n=66), and 38% in patients treated with standard chemotherapy (n=95).,The median overall survival (mOS) from first systemic treatment in the entire study cohort was 16.9 months. mOS of patients treated either with checkpoint or kinase inhibitors (n=300, 14.6 months) between 2008 and 2014 was significantly improved (P<0.0001) compared with patients treated with standard chemotherapy in 2008-2009 (n=95, 7.4 months). mOS of 61 patients with brain metastases at stage IV was 8.1 versus 12.5 months for patients without at stage IV (n=334), therefore being significantly different (P=0.00065).,Furthermore, a significant reduction in hospitalization duration compared with chemotherapy was noted.,Treatment with checkpoint and kinase inhibitors beyond clinical trials significantly improves the mOS in real life and the results are consistent with published prospective trial data.
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The MITF transcription factor is a master regulator of melanocyte development and a critical factor in melanomagenesis.,The related transcription factors TFEB and TFE3 regulate lysosomal activity and autophagy processes known to be important in melanoma.,Here we show that MITF binds the CLEAR-box element in the promoters of lysosomal and autophagosomal genes in melanocytes and melanoma cells.,The crystal structure of MITF bound to the CLEAR-box reveals how the palindromic nature of this motif induces symmetric MITF homodimer binding.,In metastatic melanoma tumors and cell lines, MITF positively correlates with the expression of lysosomal and autophagosomal genes, which, interestingly, are different from the lysosomal and autophagosomal genes correlated with TFEB and TFE3.,Depletion of MITF in melanoma cells and melanocytes attenuates the response to starvation-induced autophagy, whereas the overexpression of MITF in melanoma cells increases the number of autophagosomes but is not sufficient to induce autophagic flux.,Our results suggest that MITF and the related factors TFEB and TFE3 have separate roles in regulating a starvation-induced autophagy response in melanoma.,Understanding the normal and pathophysiological roles of MITF and related transcription factors may provide important clinical insights into melanoma therapy.
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MITF (microphthalmia-associated transcription factor) represents a melanocytic lineage-specific transcription factor whose role is profoundly extended in malignant melanoma.,Over the last few years, the function of MITF has been tightly connected to plasticity of melanoma cells.,MITF participates in executing diverse melanoma phenotypes defined by distinct gene expression profiles.,Mutation-dependent alterations in MITF expression and activity have been found in a relatively small subset of melanomas.,MITF activity is rather modulated by its upstream activators and suppressors operating on transcriptional, post-transcriptional and post-translational levels.,These regulatory mechanisms also include epigenetic and microenvironmental signals.,Several transcription factors and signaling pathways involved in the regulation of MITF expression and/or activity such as the Wnt/β-catenin pathway are broadly utilized by various types of tumors, whereas others, e.g., BRAFV600E/ERK1/2 are more specific for melanoma.,Furthermore, the MITF activity can be affected by the availability of transcriptional co-partners that are often redirected by MITF from their own canonical signaling pathways.,In this review, we discuss the complexity of a multilevel regulation of MITF expression and activity that underlies distinct context-related phenotypes of melanoma and might explain diverse responses of melanoma patients to currently used therapeutics.
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