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Melanoma is characterised by its ability to metastasise at early stages of tumour development.,Current clinico‐pathologic staging based on the American Joint Committee on Cancer criteria is used to guide surveillance and management in early‐stage disease, but its ability to predict clinical outcome has limitations.,Herein we review the genomics of melanoma subtypes including cutaneous, acral, uveal and mucosal, with a focus on the prognostic and predictive significance of key molecular aberrations.,© 2018 The Authors.,The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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Preclinical studies suggest that treatment with neoadjuvant immune checkpoint blockade is associated with enhanced survival and antigen-specific T cell responses over adjuvant treatment1; however, optimal regimens have not been defined.,Herein, we report results from a randomized phase II study of neoadjuvant nivolumab versus combined ipilimumab with nivolumab in 23 patients with high-risk resectable melanoma (NCT02519322).,RECIST overall response rates (ORR), pathologic complete response rates (pCR), treatment-related adverse events (trAEs), and immune correlates of response were assessed.,Treatment with combined ipilimumab and nivolumab yielded high response rates (RECIST ORR 73%, pCR 45%) but substantial toxicity (73% grade 3 trAEs), whereas treatment with nivolumab monotherapy yielded modest responses (ORR 25%, pCR 25%) and low toxicity (8% grade 3 trAEs).,Immune correlates of response were identified, demonstrating higher lymphoid infiltrates in responders to both therapies and a more clonal and diverse T cell infiltrate in responders to nivolumab monotherapy.,These results are the first to describe the feasibility of neoadjuvant immune checkpoint blockade in melanoma and emphasize the need for additional studies to optimize treatment regimens and to validate putative biomarkers.
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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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Oncogenic mutations in the serine/threonine kinase B-RAF are found in 50-70% of malignant melanomas1.,Pre-clinical studies have demonstrated that the B-RAFV600E mutation predicts a dependency on the mitogen activated protein kinase (MAPK) signaling cascade in melanoma1-5-an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials6-8.,However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance9-11.,Identification of resistance mechanisms in a manner that elucidates alternative ‘druggable’ targets may inform effective long-term treatment strategies12.,Here, we expressed ~600 kinase and kinase-related open reading frames (ORFs) in parallel to functionally interrogate resistance to a selective RAF kinase inhibitor.,We identified MAP3K8 (COT/TPL2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAFV600E cell lines.,COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signaling.,Moreover, COT expression is associated with de novo resistance in B-RAFV600E cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibition.,We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting.,Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.
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Side population (SP) is identified as cells capable of excluding the fluorescent Hoechst dye and anticancer drugs, and represents hematopoietic stem cells and chemoresistant cells from several solid tumors.,In this study, we confirmed the presence of SP cells in tumors from melanoma patients.,Melanoma SP cells overexpressed ATP-binding-cassette (ABC) transporters, ABCB1 and ABCB5.,We generated a direct in vivo xenograft model, and demonstrated that SP cells were resistant to paclitaxel, a substrate of ABCB1, both in vitro and in vivo.,However, melanoma SP cells were also resistant to temozolomide, which is not a substrate for ABC transporters, through IL-8 upregulation.,In addition, gene profiling studies identified three signaling pathways (NF- κB, α6-β4-integrin and IL-1) as differentially upregulated in melanoma SP cells, and there was a significant increase of PCDHB11 and decrease of FUK and TBX2 in these cells.,Therefore, we provide evidence that SP is an enriched source of chemoresistant cells in human melanomas, and suggest that the selected genes and signaling pathways of SP cells may be a potential target for effective melanoma therapies.,To our knowledge, this is previously unreported study to isolate SP cells from melanoma patients and to investigate the gene expression profiling of these cells.
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Malignant melanoma, generally described as incurable, is notoriously refractory to chemotherapy.,The mechanisms contributing to this have not yet been defined and the contributions of drug efflux pumps, implicated in chemo-resistance of many other cancer types, have not been extensively investigated in melanoma.,In this study, expression of multi-drug resistant (MDR1/P-gp and MRP-1) proteins was examined, by immunohistochemistry, in archival specimens from 134 melanoma patients.,This included 92 primary tumours and 42 metastases.,On assessing all specimens, MRP-1 and MDR1/P-gp expression was found to be common, with the majority (81%) of melanomas expressing at least one of these efflux pumps.,Although there is significant association between expression of these pumps (P=0.007), MRP-1 was found to be the predominant (67% of cases) form detected. χ2 analysis showed significant associations between expression of MRP-1 and/or MDR1/P-gp and the aggressive nature of this disease specifically increased Breslow's depth, Clark's level and spread to lymph nodes.,This association with aggressiveness and spread is further supported by the observation that a significantly higher percentage of metastases, than primary tumours, express MRP-1 (91% vs 57% P<0.0001) and MDR1/P-gp (74% vs 50% P=0.010).,The predominant expression of these pumps and, in particular, MRP-1 suggests that they may be important contributors to the inherent aggressive and resistant nature of malignant melanoma.
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Melanoma, the most dangerous type of cutaneous neoplasia, contributes to about 75% of all skin cancer-related deaths.,Thus, searching for new melanoma treatment options is an important field of study.,The current study was designed to assess whether the condition of mild and low-dose UVA radiation augments the lomefloxacin-mediated cytotoxic, growth-inhibitory and pro-apoptotic effect of the drug in melanoma cancer cells through excessive oxidative stress generation.,C32 amelanotic and COLO829 melanotic (BRAF-mutant) melanoma cell lines were used as an experimental model system.,The combined exposure of cells to both lomefloxacin and UVA irradiation caused higher alterations of redox signalling pathways, as shown by intracellular reactive oxygen species overproduction and endogenous glutathione depletion when compared to non-irradiated but lomefloxacin-treated melanoma cells.,The obtained results also showed that lomefloxacin decreased both C32 and COLO829 cells’ viability in a concentration-dependent manner.,This effect significantly intensified when melanoma cells were exposed to UVA irradiation and the drug.,For melanoma cells exposed to lomefloxacin or lomefloxacin co-treatment with UVA irradiation, the concentrations of the drug that decreased the cells’ viability by 50% (EC50) were found to be 0.97, 0.17, 1.01, 0.18 mM, respectively.,Moreover, we found that the redox imbalance, mitochondrial membrane potential breakdown, induction of DNA fragmentation, and changes in the melanoma cells’ cell cycle distribution (including G2/M, S as well as Sub-G1-phase blockade) were lomefloxacin in a dose-dependent manner and were significantly augmented by UVA radiation.,This is the first experimental work that assesses the impact of excessive reactive oxygen species generation upon UVA radiation exposure on lomefloxacin-mediated cytotoxic, growth-inhibitory and pro-apoptotic effects towards human melanoma cells, indicating the possibility of the usage of this drug in the photochemotherapy of malignant melanoma as an innovative medical treatment option which could improve the effectiveness of therapy.,The obtained results also revealed that the redox imbalance intensification mediated by the phototoxic potential of fluoroquinolones may be considered as a more efficient treatment model of malignant melanoma and may constitute the basis for the development of new compounds with a high ability to excessive oxidative stress generation upon UVA radiation in cancer cells.
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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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This population-based cohort study examines associations of voriconazole and other antifungal medications with risk of keratinocyte carcinomas in recipients of lung transplants.,What is the association between voriconazole, an antifungal used to treat aspergillosis infections, and the risk of keratinocyte carcinomas among recipients of lung transplants?,In a population-based cohort study of 9599 non-Hispanic white recipients of 9793 lung transplants in the United States (2007-2016) with linkage to pharmacy claims, increasing cumulative voriconazole exposure was associated with an increased risk of cutaneous squamous cell carcinoma.,These findings suggest that physicians caring for lung transplant recipients at high risk for aggressive keratinocyte carcinomas should limit voriconazole exposure when possible and encourage skin protection behaviors and more frequent cancer screenings.,The antifungal medication voriconazole is used to prevent and treat aspergillosis, a major cause of mortality among recipients of lung transplants (hereinafter referred to as lung recipients).,Small studies suggest that voriconazole increases risk of cutaneous squamous cell carcinoma (SCC).,To examine associations of voriconazole and other antifungal medications with risk of keratinocyte carcinomas (SCC and cutaneous basal cell carcinoma [BCC]) in lung recipients.,This population-based cohort study included non-Hispanic white patients (n = 9599) who underwent lung transplant in the United States from January 1, 2007, to December 31, 2016, identified through the national Scientific Registry of Transplant Recipients with data linkable to pharmacy claims.,Data were analyzed from March 1, 2018, to February 13, 2019.,Antifungal medication use, including voriconazole, itraconazole, posaconazole, and other antifungals, was ascertained from pharmacy claims and treated as a time-varying exposure (assessed every 30 days).,Cumulative antifungal exposure was calculated as the total number of exposed months.,Primary outcomes were the first SCC or BCC reported to the transplant registry by transplant centers.,Follow-up began at transplant and ended at SCC or BCC diagnosis, transplant failure or retransplant, death, loss to follow-up, or December 31, 2016.,Cox proportional hazards regression models were used to estimate adjusted hazard ratios (AHRs) for each antifungal medication.,Among the 9793 lung transplants in 9599 recipients included in the analysis, median age at transplant was 59 (interquartile range [IQR], 48-65) years, 5824 (59.5%) were male, and 5721 (58.4%) reported ever smoking.,During a median follow-up of 3.0 (IQR, 1.4-5.0) years after transplant, 1031 SCCs (incidence, 322 per 10 000 person-years) and 347 BCCs (incidence, 101 per 10 000 person-years) were reported.,Compared with lung recipients with no observed voriconazole use, those with 1 to 3 months of voriconazole use experienced increased AHR for SCC of 1.09 (95% CI, 0.90-1.31); 4 to 7 months, 1.42 (95% CI, 1.16-1.73); 8 to 15 months, 2.04 (95% CI, 1.67-2.50); and more than 15 months, 3.05 (95% CI, 2.37-3.91).,Ever itraconazole exposure was associated with increased SCC risk (AHR, 1.20; 95% CI, 1.00-1.45).,For BCC, risk was not associated with voriconazole use but was increased with itraconazole use (AHR, 1.74; 95% CI, 1.27-2.37) or posaconazole use (AHR, 1.55; 95% CI, 1.00-2.41).,In this study, voriconazole use was associated with increased SCC risk among lung recipients, especially after prolonged exposure.,Further research evaluating the risk-benefit ratio of shorter courses or alternative medications in transplant recipients at high risk for SCC should be considered.
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Squamous Cell Carcinoma (SCC) is a type of non-melanoma skin cancer prevalent in immune-suppressed transplant recipients and older individuals with a history of chronic sun-exposure.,SCC itself is believed to be a late-stage manifestation that can develop from premalignant lesions including Intraepidermal Carcinoma (IEC).,Notably, while SCC regression is rare, IEC typically regresses in response to immune modifying topical treatments, however the underlying immunological reasons for these differential responses remain unclear.,This study aimed to define whether IEC and SCC are associated with distinct immune profiles.,We investigated the immune cell infiltrate of photo-damaged skin, IEC, and SCC tissue using 10-colour flow cytometry following fresh lesion digest.,We found that IEC lesions contain higher percentages of CD3+ T-cells than photo-damaged skin, however, the abundance of CD3−CD56+ Natural Killer (NK) cells, CD11c+HLA-DR+ conventional Dendritic Cells (cDC), BDCA-2+HLA-DR+ plasmacytoid DC (pDC), FoxP3+ Regulatory T-cells (T-reg), Vα24+Vβ11+ invariant NKT-cells, and γδ Tcells did not alter with disease stage.,Within the total T-cell population, high percentages of CD4+ T-cells were associated with SCC, yet CD8+ T-cells were less abundant in SCC compared with IEC.,Our study demonstrates that while IEC lesions contain a higher proportion of T-cells than SCC lesions in general, SCC lesions specifically display a lower abundance of CD8+ T-cells than IEC.,We propose that differences in CD8+ T-cell abundance contribute critically to the different capacity of SCC and IEC to regress in response to immune modifying topical treatments.,Our study also suggests that a high ratio of CD4+ T-cells to CD8+ T-cells may be a immunological diagnostic indicator of late-stage SCC development in immune-competent patients.
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Cutaneous melanoma is a complex disorder characterized by an elevated degree of heterogeneity, features that place it among the most aggressive types of cancer.,Although significant progress was recorded in both the understanding of melanoma biology and genetics, and in therapeutic approaches, this malignancy still represents a major problem worldwide due to its high incidence and the lack of a curative treatment for advanced stages.,This review offers a survey of the most recent information available regarding the melanoma epidemiology, etiology, and genetic profile.,Also discussed was the topic of cutaneous melanoma murine models outlining the role of these models in understanding the molecular pathways involved in melanoma initiation, progression, and metastasis.
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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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Merkel cell polyomavirus (MCPyV) is an etiological agent of Merkel cell carcinoma (MCC), a highly aggressive skin cancer.,The MCPyV small tumor antigen (ST) is required for maintenance of MCC and can transform normal cells.,To gain insight into cellular perturbations induced by MCPyV ST, we performed transcriptome analysis of normal human fibroblasts with inducible expression of ST.,MCPyV ST dynamically alters the cellular transcriptome with increased levels of glycolytic genes, including the monocarboxylate lactate transporter SLC16A1 (MCT1).,Extracellular flux analysis revealed increased lactate export reflecting elevated aerobic glycolysis in ST expressing cells.,Inhibition of MCT1 activity suppressed the growth of MCC cell lines and impaired MCPyV-dependent transformation of IMR90 cells.,Both NF-κB and MYC have been shown to regulate MCT1 expression.,While MYC was required for MCT1 induction, MCPyV-induced MCT1 levels decreased following knockdown of the NF-κB subunit RelA, supporting a synergistic activity between MCPyV and MYC in regulating MCT1 levels.,Several MCC lines had high levels of MYCL and MYCN but not MYC.,Increased levels of MYCL was more effective than MYC or MYCN in increasing extracellular acidification in MCC cells.,Our results demonstrate the effects of MCPyV ST on the cellular transcriptome and reveal that transformation is dependent, at least in part, on elevated aerobic glycolysis.
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Merkel cell polyomavirus (MCV) causes the majority of human Merkel cell carcinomas (MCC) and encodes a small T (sT) antigen that transforms immortalized rodent fibroblasts in vitro.,To develop a mouse model for MCV sT-induced carcinogenesis, we generated transgenic mice with a flox-stop-flox MCV sT sequence homologously recombined at the ROSA locus (ROSA sT), allowing Cre-mediated, conditional MCV sT expression.,Standard tamoxifen (TMX) administration to adult Ubc CreERT2; ROSA sT mice, in which Cre is ubiquitously expressed, resulted in MCV sT expression in multiple organs that was uniformly lethal within 5 days.,Conversely, most adult Ubc CreERT2; ROSA sT mice survived low-dose tamoxifen administration but developed ear lobe dermal hyperkeratosis and hypergranulosis.,Simultaneous MCV sT expression and conditional homozygous p53 deletion generated multi-focal, poorly-differentiated, highly anaplastic tumors in the spleens and livers of mice after 60 days of TMX treatment.,Mouse embryonic fibroblasts from these mice induced to express MCV sT exhibited anchorage-independent cell growth.,To examine Merkel cell pathology, MCV sT expression was also induced during mid-embryogenesis in Merkel cells of Atoh1 CreERT2/+; ROSA sT mice, which lead to significantly increased Merkel cell numbers in touch domes at late embryonic ages that normalized postnatally.,Tamoxifen administration to adult Atoh1 CreERT2/+; ROSA sT and Atoh1 CreERT2/+; ROSA sT; p53f lox/flox mice had no effects on Merkel cell numbers and did not induce tumor formation.,Taken together, these results show that MCV sT stimulates progenitor Merkel cell proliferation in embryonic mice and is a bona fide viral oncoprotein that induces full cancer cell transformation in the p53-null setting.
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N-cadherin seems to promote cell migration and invasion in many types of cancers.,The object of this study is recognition of the possible role of N-cadherin and selected downstream protein kinases: PI3K, ERK1/2, and mTOR in cell invasion in malignant melanoma.,Melanoma cells were transfected with the small interfering RNA (siRNA) that targets human N-cadherin gene (CDH2).,Inhibitors LY294002 (PI3K), U0126 (ERK1/2), and everolimus (mTOR) were used to inhibit selected kinases of signalling pathways.,In vitro cell invasion was studied using Matrigel and an analysis of matrix metalloproteinases MMP-2 and MMP-9 activity by gelatinase zymogram assay.,Treatment of melanoma cell with either siRNA against N-cadherin or protein kinase inhibitors led to significantly decreased MMPs expression and activity, as well as diminished invasion.,Both the current and the former results suggest that activation of PI3/AKT, mTOR, and ERK kinase, following N-cadherin expression, contributes not only to increased proliferation but also invasive potential of melanoma cells.,The results also indicate that N-cadherin, as well as the studied kinases, should be considered as a potential target in melanoma therapy.
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Cancer-associated fibroblasts (CAFs) play a central role in the complex process of tumor-stroma interaction and promote tumor growth.,Emerging evidences also suggest that these fibroblasts are involved in the alteration of the anti-tumor immune response by impacting several immune cell populations, especially through their secretion of pro-inflammatory and immunosuppressive factors in the tumor microenvironment.,However, the underlying immuno-modulating mechanisms triggered by these fibroblasts are still only partially defined.,In this study, we provide evidence that melanoma-associated fibroblasts decrease the susceptibility of melanoma tumor cells to NK-mediated lysis through the secretion of active matrix metalloproteinases.,This secretion reduces the expression of the two NKG2D ligands, MICA/B, at the surface of tumor cells and consequently decreases the NKG2D-dependent cytotoxic activity of NK cells against melanoma tumor cells.,Together, our data demonstrate that the modification of tumor cell susceptibility to killer cells is an important determinant of the anti-tumor immune response alteration triggered by CAFs.
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The tumor milieu consists of numerous cell types each existing in a different environment.,However, a characterization of metabolic heterogeneity at single-cell resolution is not established.,Here, we develop a computational pipeline to study metabolic programs in single cells.,In two representative human cancers, melanoma and head and neck, we apply this algorithm to define the intratumor metabolic landscape.,We report an overall discordance between analyses of single cells and those of bulk tumors with higher metabolic activity in malignant cells than previously appreciated.,Variation in mitochondrial programs is found to be the major contributor to metabolic heterogeneity.,Surprisingly, the expression of both glycolytic and mitochondrial programs strongly correlates with hypoxia in all cell types.,Immune and stromal cells could also be distinguished by their metabolic features.,Taken together this analysis establishes a computational framework for characterizing metabolism using single cell expression data and defines principles of the tumor microenvironment.,Each cell type in the tumour microenvironment has unique metabolic demands enabling specific functions.,Here the authors use published single-cell RNA-seq data and develop a computational framework to better understand the heterogeneity of tumour metabolism, highlighting the discordance between results obtained from single cells and bulk tumours.
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Phenotypic plasticity is associated with non-genetic drug tolerance in several cancers.,Such plasticity can arise from chromatin remodeling, transcriptomic reprogramming, and/or protein signaling rewiring, and is characterized as a cell state transition in response to molecular or physical perturbations.,This, in turn, can confound interpretations of drug responses and resistance development.,Using BRAF-mutant melanoma cell lines as the prototype, we report on a joint theoretical and experimental investigation of the cell-state transition dynamics associated with BRAF inhibitor drug tolerance.,Thermodynamically motivated surprisal analysis of transcriptome data was used to treat the cell population as an entropy maximizing system under the influence of time-dependent constraints.,This permits the extraction of an epigenetic potential landscape for drug-induced phenotypic evolution.,Single-cell flow cytometry data of the same system were modeled with a modified Fokker-Planck-type kinetic model.,The two approaches yield a consistent picture that accounts for the phenotypic heterogeneity observed over the course of drug tolerance development.,The results reveal that, in certain plastic cancers, the population heterogeneity and evolution of cell phenotypes may be understood by accounting for the competing interactions of the epigenetic potential landscape and state-dependent cell proliferation.,Accounting for such competition permits accurate, experimentally verifiable predictions that can potentially guide the design of effective treatment strategies.
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Circulating tumour cells (CTCs) can be assessed through a minimally invasive blood sample with potential utility as a predictive, prognostic and pharmacodynamic biomarker.,The large heterogeneity of melanoma CTCs has hindered their detection and clinical application.,Here we compared two microfluidic devices for the recovery of circulating melanoma cells.,The presence of CTCs in 43 blood samples from patients with metastatic melanoma was evaluated using a combination of immunocytochemistry and transcript analyses of five genes by RT-PCR and 19 genes by droplet digital PCR (ddPCR), whereby a CTC score was calculated.,Circulating tumour DNA (ctDNA) from the same patient blood sample, was assessed by ddPCR targeting tumour-specific mutations.,Our analysis revealed an extraordinary heterogeneity amongst melanoma CTCs, with multiple non-overlapping subpopulations.,CTC detection using our multimarker approach was associated with shorter overall and progression-free survival.,Finally, we found that CTC scores correlated with plasma ctDNA concentrations and had similar pharmacodynamic changes upon treatment initiation.,Despite the high phenotypic and molecular heterogeneity of melanoma CTCs, multimarker derived CTC scores could serve as viable tools for prognostication and treatment response monitoring in patients with metastatic melanoma.
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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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An important component of research using animal models is ensuring rigor and reproducibility.,This study was prompted after two experimenters performing virtually identical studies obtained different results when syngeneic B78 murine melanoma cells were implanted into the skin overlying the flank and treated with an in situ vaccine (ISV) immunotherapy.,Although both experimenters thought they were using identical technique, we determined that one was implanting the tumors intradermally (ID) and the other was implanting them subcutaneously (SC).,Though the baseline in vivo immunogenicity of tumors can depend on depth of their implantation, the response to immunotherapy as a function of tumor depth, particularly in immunologically ‘cold’ tumors, has not been well studied.,The goal of this study was to evaluate the difference in growth kinetics and response to immunotherapy between identically sized melanoma tumors following ID versus SC implantation.,We injected C57BL/6 mice with syngeneic B78 melanoma cells either ID or SC in the flank.,When tumors reached 190-230 mm3, they were grouped into a ‘wave’ and treated with our previously published ISV regimen (12 Gy local external beam radiation and intratumoral hu14.18-IL2 immunocytokine).,Physical examination demonstrated that ID-implanted tumors were mobile on palpation, while SC-implanted tumors became fixed to the underlying fascia.,Histologic examination identified a critical fascial layer, the panniculus carnosus, which separated ID and SC tumors.,SC tumors reached the target tumor volume significantly faster compared with ID tumors.,Most ID tumors exhibited either partial or complete response to this immunotherapy, whereas most SC tumors did not.,Further, the ‘mobile’ or ‘fixed’ phenotype of tumors predicted response to therapy, regardless of intended implantation depth.,These findings were then extended to additional immunotherapy regimens in four separate tumor models.,These data indicate that the physical ‘fixed’ versus ‘mobile’ characterization of the tumors may be one simple method of ensuring homogeneity among implanted tumors prior to initiation of treatment.,Overall, this short report demonstrates that small differences in depth of tumor implantation can translate to differences in response to immunotherapy, and proposes a simple physical examination technique to ensure consistent tumor depth when conducting implantable tumor immunotherapy experiments.
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Small aquarium fish models provide useful systems not only for a better understanding of the molecular basis of many human diseases, but also for first-line screening to identify new drug candidates.,For testing new chemical substances, current strategies mostly rely on easy to perform and efficient embryonic screens.,Cancer, however, is a disease that develops mainly during juvenile and adult stage.,Long-term treatment and the challenge to monitor changes in tumor phenotype make testing of large chemical libraries in juvenile and adult animals cost prohibitive.,We hypothesized that changes in the gene expression profile should occur early during anti-tumor treatment, and the disease-associated transcriptional change should provide a reliable readout that can be utilized to evaluate drug-induced effects.,For the current study, we used a previously established medaka melanoma model.,As proof of principle, we showed that exposure of melanoma developing fish to the drugs cisplatin or trametinib, known cancer therapies, for a period of seven days is sufficient to detect treatment-induced changes in gene expression.,By examining whole body transcriptome responses we provide a novel route toward gene panels that recapitulate anti-tumor outcomes thus allowing a screening of thousands of drugs using a whole-body vertebrate model.,Our results suggest that using disease-associated transcriptional change to screen therapeutic molecules in small fish model is viable and may be applied to pre-clinical research and development stages in new drug discovery.
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Elevated levels of cell-free DNA (cfDNA) are frequently observed in tumor patients.,Activating mutations in exon 4 (R183) and exon 5 (Q209) of GNAQ and GNA11 are almost exclusively found in uveal melanoma, thus providing a highly specific marker for the presence of circulating tumor DNA (ctDNA).,To establish a reliable, noninvasive assay that might allow early detection and monitoring of metastatic disease, we determined the proportion of GNAQ or GNA11 mutant reads in cfDNA of uveal melanoma patients by ultradeep sequencing.,Cell-free DNA from 28 uveal melanoma patients with metastases or extraocular growth was isolated and quantified by real-time polymerase chain reaction (PCR) (7-1550 ng DNA/mL plasma).,GNAQ and GNA11 regions of interest were amplified in 22 of 28 patients and ultradeep sequencing of amplicons was performed to detect even low proportions of mutant reads.,We detected Q209 mutations (2-38% mutant reads) in either GNAQ or GNA11 in the plasma of 9 of 22 metastasized patients.,No correlation between the proportion of mutant reads and the concentration of cfDNA could be detected.,Among the nine ctDNA-positive patients, four had metastases in bone, whereas no metastases were detected in the 13 ctDNA-negative patients at this location (P = 0.025).,Furthermore, ctDNA-positive patients tended to be younger at initial diagnosis and show larger metastases.,The results show that ultradeep amplicon sequencing can be used to detect tumor DNA in plasma of metastasized uveal melanoma patients.,It remains to be shown if this approach can be used for early detection of disseminated tumor disease.
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When mutations in two different genes produce the same mutant phenotype, it suggests that the encoded proteins either interact with each other, or act in parallel to fulfill a similar purpose.,Haploinsufficiency of Neurofibromin and over-expression of Endothelin 3 both cause increased numbers of melanocytes to populate the dermis during mouse development, and thus we are interested in how these two signaling pathways might intersect.,Neurofibromin is mutated in the human genetic disease, neurofibromatosis type 1, which is characterized by the development of Schwann cell based tumors and skin hyper-pigmentation.,Neurofibromin is a GTPase activating protein, while the Endothelin 3 ligand activates Endothelin receptor B, a G protein coupled receptor.,In order to study the genetic interactions between endothelin and neurofibromin, we defined the deletion breakpoints of the classical Ednrb piebald lethal allele (Ednrbs-l) and crossed these mice to mice with a loss-of-function mutation in neurofibromin, Dark skin 9 (Dsk9).,We found that Neurofibromin haploinsufficiency requires Endothelin receptor B to darken the tail dermis.,In contrast, Neurofibromin haploinsufficiency increases the area of the coat that is pigmented in Endothelin receptor B null mice.,We also found an oncogenic mutation in the G protein alpha subunit, GNAQ, which couples to Endothelin receptor B, in a uveal melanoma from a patient with neurofibromatosis type 1.,Thus, this data suggests that there is a complex relationship between Neurofibromin and Endothelin receptor B.
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Patients with primary cutaneous melanomas that are ulcerated and >2 mm in thickness, >4 mm in thickness and those with nodal micrometastases at diagnosis, have few options for adjuvant treatment.,Recent studies have suggested a role for vitamin D to delay melanoma recurrence and improve overall prognosis.,This is a pilot placebo-controlled randomised phase II trial to assess the feasibility, safety and toxicity of an oral loading dose of Vitamin D (500,000 IU) followed by an oral dose of 50,000 IU of Vitamin D monthly for 2 years in patients who have been treated for cutaneous melanoma by wide excision of the primary.,Patients aged 18 - 79 years who have completed primary surgical treatment and have Stage IIb, IIc, IIIa (N1a, N2a) or IIIb (N1a, N2a) disease are eligible for randomisation 2:1 to active treatment or placebo.,The primary endpoints are sufficiency of dose, adherence to study medication and safety of the drug.,The secondary endpoints are participation and progression free survival.,The study has been approved by the Ethics Review Committee (RPAH Zone) of the Sydney Local Health District, protocol number X09-0138.,Effective, non-toxic adjuvant therapy for high risk primary melanoma is not currently available.,Favorable outcomes of this phase II study will form the basis for a multi-centre phase III study to assess whether the addition of oral high-dose vitamin D therapy in patients who have completed primary treatment for melanoma and are at high risk of recurrence will:prolong time to recurrence within 5 yearsimprove overall survival at 5 years andbe both safe and tolerable.,prolong time to recurrence within 5 years,improve overall survival at 5 years and,be both safe and tolerable.,62 patients have been randomised since the study commenced in December 2010.,Target accrual for the study has been met with 75 patients randomised between December 2010 and August 2014.,The Mel-D trial is conducted by the Australia and New Zealand Melanoma Trials Group (ANZMTG 02.09),Australia and New Zealand Clinical Trials Registry (ANZCTR) ACTRN12609000351213
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Melanoma is highly resistant to current modalities of therapy, with the extent of pigmentation playing an important role in therapeutic resistance.,Nuclear factor-κB (NF-κB) is constitutively activated in melanoma and can serve as a molecular target for cancer therapy and steroid/secosteroid action.,Cultured melanoma cells were used for mechanistic studies on NF-κB activity, utilising immunofluorescence, western blotting, EMSA, ELISA, gene reporter, and estimated DNA synthesis assays.,Formalin-fixed, paraffin-embedded specimens from melanoma patients were used for immunocytochemical analysis of NF-κB activity in situ.,Novel 20-hydroxyvitamin (20(OH)D3) and classical 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) secosteroids inhibited melanoma cell proliferation.,Active forms of vitamin D were found to inhibit NF-κB activity in nonpigmented cells, while having no effect on pigmented cells.,Treatment of nonpigmented cells with vitamin D3 derivatives inhibited NF-κB DNA binding and NF-κB-dependent reporter assays, as well as inhibited the nuclear translocation of the p65 NF-κB subunit and its accumulation in the cytoplasm.,Moreover, analysis of biopsies of melanoma patients showed that nonpigmented and slightly pigmented melanomas displayed higher nuclear NF-κB p65 expression than highly pigmented melanomas.,Classical 1,25(OH)2D3 and novel 20(OH)D3 hydroxyderivatives of vitamin D3 can target NF-κB and regulate melanoma progression in nonpigmented melanoma cells.,Melanin pigmentation is associated with the resistance of melanomas to 20(OH)D3 and 1,25(OH)2D3 treatment.
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Talimogene laherparepvec (T-VEC) is a licensed therapy for use in melanoma patients of stage IIIB-IVM1a with injectable, unresectable metastatic lesions in Europe.,Approval was based on the Oncovex Pivotal Trial in Melanoma study, which also included patients with distant metastases and demonstrated an overall response rate (ORR) of 40.5% and a complete response (CR) rate of 16.6%.,The aim of this study was to assess the outcome of melanoma patients treated with T-VEC in a real-life clinical setting.,Based on data from 10 melanoma centers in Austria, Switzerland and southern Germany, we conducted a retrospective chart review, which included 88 patients (44 male, 44 female) with a median age of 72 years (range 36-95 years) treated with T-VEC during the period from May 2016 to January 2020.,88 patients fulfilled the inclusion criteria for analysis.,The ORR was 63.7%. 38 patients (43.2%) showed a CR, 18 (20.5%) had a partial response, 8 (9.1%) had stable disease and 24 (27.3%) patients had a progressive disease.,The median treatment period was 19 weeks (range: 1-65), an average of 11 doses (range: 1-36) were applied.,39 (45.3%) patients developed adverse events, mostly mild, grade I (64.1%).,This real-life cohort treatment with T-VEC showed a high ORR and a large number of durable CRs.
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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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We aimed to estimate the incidence and mortality of uveal melanoma (UM) in Australia from 1982 to 2014.,Deidentified unit data for all cases of ocular melanoma were extracted from the Australian Cancer Database from 1 January 1982 to 31 December 2014.,UM cases were extracted and trends in incidence and disease-specific mortality were calculated.,Incidence rates were age-standardised against the 2001 Australian Standard Population.,Mortality was assessed using Cox regression.,From 1982 to 2014, there were 5087 cases of ocular melanoma in Australia, of which 4617 were classified as UM.,The average age-standardised incidence rate of UM was 7.6 (95% CI 7.3 to 7.9) per million.,There was an increase (p=0.0502) in the incidence of UM from 1982 to 1993 with an annual percent change (APC) of +2.5%, followed by a significant decrease in the incidence of UM from 1993 to 2014 (APC −1.2%).,The average 5-year survival from 1982 to 2011 did not significantly change from an average of 81%, with an average APC (AAPC) of +0.1%.,A multivariate Cox regression revealed that residence in Western Australia (p=0.001) or Tasmania (p=0.05), age ≥60 years (p<0.001) and histological classification as mixed (p<0.001) or epithelioid cells (p<0.001) were significantly associated with reduced survival.,In conclusion, we found that the incidence of UM peaked in the 1990s.,Although treatment for primary UM has improved in the last 30 years, overall survival did not change significantly in the last 30 years.
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Despite outstanding advances in diagnosis and the treatment of primary uveal melanoma (UM), nearly 50% of UM patients develop metastases via hematogenous dissemination, driven by the epithelial-mesenchymal transition (EMT).,Despite the failure in UM to date, a liquid biopsy may offer a feasible non-invasive approach for monitoring metastatic disease progression and addressing protracted dormancy.,To detect circulating tumor cells (CTCs) in UM patients, we evaluated the mRNA expression of EMT-associated transcription factors in CD45-depleted blood fraction, using qRT-PCR. ddPCR was employed to assess UM-specific GNA11, GNAQ, PLCβ4, and CYSLTR2 mutations in plasma DNA.,Moreover, microarray analysis was performed on total RNA isolated from tumor tissues to estimate the prognostic value of EMT-associated gene expression.,In total, 42 primary UM and 11 metastatic patients were enrolled.,All CD45-depleted samples were negative for CTC when compared to the peripheral blood fraction of 60 healthy controls.,Tumor-specific mutations were detected in the plasma of 21.4% patients, merely, in 9.4% of primary UM, while 54.5% in metastatic patients.,Unsupervised hierarchical clustering of differentially expressed EMT genes showed significant differences between monosomy 3 and disomy 3 tumors.,Newly identified genes can serve as non-invasive prognostic biomarkers that can support therapeutic decisions.
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Histone deacetylase inhibitors (HDACi) exert multiple cytotoxic actions on cancer cells.,Currently, different synthetic HDACi are in clinical use or clinical trials; nevertheless, since both pro-invasive and anti-invasive activities have been described, there is some controversy about the effect of HDACi on melanoma cells.,Matrigel and Collagen invasion assays were performed to evaluate the effect of several HDACi (Butyrate, Trichostatin A, Valproic acid and Vorinostat) on two human melanoma cell line invasion (A375 and HT-144).,The expression of N- and E-Cadherin and the activity of the RhoA GTPase were analyzed to elucidate the mechanisms involved in the HDACi activity.,HDACi showed a pro-invasive effect on melanoma cells in vitro.,This effect was accompanied by an up-regulation of N-cadherin expression and an inhibition of RhoA activity.,Moreover, the down-regulation of N-cadherin through blocking antibodies or siRNA abrogated the pro-invasive effect of the HDACi and, additionally, the inhibition of the Rho/ROCK pathway led to an increase of melanoma cell invasion similar to that observed with the HDACi treatments.,These results suggest a role of N-cadherin and RhoA in HDACi induced invasion and call into question the suitability of some HDACi as antitumor agents for melanoma patients.
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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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Locally advanced cutaneous melanoma has marked quality-of-life implications; however, the patient experience of symptom management and subsequent impact on quality of life has not been well described.,This study aims to address the impact on patients of advanced cutaneous melanoma through qualitative interviews.,Adults with stage IIIB, IIIC, or IV (M1a) cutaneous melanoma were recruited from two cancer centers in the USA and one in Australia.,Telephone interviews were conducted to assess how locoregionally advanced cutaneous melanoma impacted everyday life.,Interviews were recorded, transcribed, and coded for qualitative analysis.,Twenty-two melanoma patients were interviewed, mean age 69.7 years (range: 52-83), 64% male.,The study included stage IIIB (36%), stage IIIC (59%), and stage IV M1a (5%) patients.,Emotional health/self-perception issues were the most commonly identified (41% of patient impact expressions), including worry, concern, embarrassment, self-consciousness, fear, and thoughts of death.,Limitations of lifestyle and activities were also identified (28% of expressions) including leisure and social activities, physical functioning, general functioning, and personal care.,Coping strategies such as modified clothing choices, increased use of pain and/or anti-inflammatory medications, and avoidance/protection from the sun represented 20% of all impact expressions.,Ratings of the degree of difficulty patients experienced (using an 11-point numerical rating scale) ranged from 0.0 to 10.0 (mean 5.7, SD 2.9).,Condition-related and treatment-related factors were well characterized in patients with locally advanced cutaneous melanoma.,This provides a strong foundation for assessment of how cutaneous melanoma impacts quality of life.
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In an international, randomized Phase III trial ipilimumab demonstrated a significant overall survival benefit in previously treated advanced melanoma patients.,This report summarizes health-related quality of life (HRQL) outcomes for ipilimumab with/without gp100 vaccine compared to gp100 alone during the clinical trial’s 12 week treatment induction period.,The Phase III clinical trial (MDX010-20) was a double-blind, fixed dose study in 676 previously treated advanced unresectable stage III or IV melanoma patients.,Patients were randomized 3:1:1 to receive either ipilimumab (3 mg/kg q3w x 4 doses) + gp100 (peptide vaccine; 1 mg q3w x 4 doses; ipilimumab plus gp100, n = 403); gp100 vaccine + placebo (gp100 alone, n = 136); or ipilimumab + placebo (ipilimumab alone, n = 137).,The European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30) assessed HRQL.,Baseline to Week 12 changes in EORTC QLQ-C30 function, global health status, and symptom scores were analyzed for ipilimumab with/without gp100 vaccine compared to gp100 alone.,Mean change in scores were categorized “no change” (0-5), “a little” (5-10 points), “moderate” (10-20 points), and “very much” (>20).,In the ipilimumab plus gp100 and ipilimumab alone groups, mean changes from baseline to Week 12 generally indicated “no change” or “a little” impairment across EORTC QLQ-C30 global health status, function, and symptom subscales.,Significant differences in constipation, favoring ipilimumab, were observed (p < 0.05).,For ipilimumab alone arm, subscales with no or a little impairment were physical, emotional, cognitive, social function, global health, nausea, pain, dyspnea, constipation, and diarrhea subscales.,For the gp100 alone group, the observed changes were moderate to large for global health, role function, fatigue, and for pain.,Ipilimumab with/without gp100 vaccine does not have a significant negative HRQL impact during the treatment induction phase relative to gp100 alone in stage III or IV melanoma patients.,Clinicaltrials.gov identification number NCT00094653
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MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play central roles in diverse pathological processes.,In this study, we investigated the effect of microRNA-182 (miR-182) on the development of posterior uveal melanomas.,Initially, we demonstrated that miR-182 expression was dependent on p53 induction in uveal melanoma cells.,Interestingly, transient transfection of miR-182 into cultured uveal melanoma cells led to a significant decrease in cell growth, migration, and invasiveness.,Cells transfected with miR-182 demonstrated cell cycle G1 arrest and increased apoptotic activity.,Using bioinformatics, we identified three potential targets of miR-182, namely MITF, BCL2 and cyclin D2. miR-182 was shown to have activity on mRNA expression by targeting the 3′ untranslated region of MITF, BCL2 and cyclin D2.,Subsequent Western blot analysis confirmed the downregulation of MITF, BCL2 and cyclin D2 protein expression.,The expression of oncogene c-Met and its downstream Akt and ERK1/2 pathways was also downregulated by miR-182.,Concordant with the findings that miR-182 was decreased in uveal melanoma tissue samples, overexpression of miR-182 also suppressed the in vivo growth of uveal melanoma cells.,Our results demonstrated that miR-182, a p53 dependent miRNA, suppressed the expression of MITF, BCL2, cyclin D2 and functioned as a potent tumor suppressor in uveal melanoma cells.
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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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Nivolumab, a monoclonal antibody of immune checkpoint programmed death 1 on T cells (PD-1), combined with ipilimumab, an immune checkpoint cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitor, as combination therapy on the one hand and nivolumab as monotherapy on the other, have both demonstrated improved efficacy compared with ipilimumab alone in the CheckMate 067 study.,However, the combination resulted in a higher frequency of grade 3/4 adverse events (AEs), which could result in diminished health-related quality of life (HRQoL).,Here we report analyses of HRQoL for patients with advanced melanoma in clinical trial CheckMate 067.,HRQoL was assessed at weeks 1 and 5 per 6-week cycle for the first 6 months, once every 6 weeks thereafter, and at two follow-up visits using the European Organization for Research and Treatment of Care Core Quality of Life Questionnaire and the EuroQoL Five Dimensions Questionnaire.,In addition to the randomised population, patient subgroups, including BRAF mutation status, partial or complete response, treatment-related AEs of grade 3/4, and those who discontinued due to any reason and due to an AE, were investigated.,Nivolumab and ipilimumab combination and nivolumab alone both maintained HRQoL, and no clinically meaningful deterioration was observed over time compared with ipilimumab.,In addition, similar results were observed across patient subgroups, and no clinically meaningful changes in HRQoL were observed during follow-up visits for patients who discontinued due to any cause.,These results further support the clinical benefit of nivolumab monotherapy and nivolumab and ipilimumab combination therapy in patients with advanced melanoma.,The finding that the difference in grade 3/4 AEs between the arms did not translate into clinically meaningful differences in the reported HRQoL may be relevant in the clinical setting.,NCT01844505.
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In patients with advanced melanoma in the CheckMate 066 study, baseline health-related quality of life (HRQoL) with nivolumab was maintained over time, with statistically significant and clinically meaningful improvements in some exploratory analyses, and no HRQoL improvements with dacarbazine.,Added to the survival benefit of nivolumab, the benefit-to-risk ratio favors nivolumab over dacarbazine.,Nivolumab has shown significant survival benefit and a favorable safety profile compared with dacarbazine chemotherapy among treatment-naïve patients with metastatic melanoma in the CheckMate 066 phase III study.,Results from the health-related quality of life (HRQoL) analyses from CheckMate 066 are presented.,HRQoL was evaluated at baseline and every 6 weeks while on treatment using the European Organisation for Research and Treatment of Care (EORTC) Core Quality of Life Questionnaire (QLQ-C30) and the EuroQoL Five Dimensions Questionnaire (EQ-5D).,Via a multi-step statistical plan, data were analyzed descriptively, cross-sectionally, and longitudinally, adjusting for baseline covariates, in patients having baseline plus ≥1 post-baseline assessment.,Baseline-adjusted completion rates for all HRQoL questionnaires across treatment arms were 65% and 70% for dacarbazine and nivolumab, respectively, and remained similar throughout treatment.,The mean baseline HRQoL scores were similar for patients treated with nivolumab and dacarbazine.,Baseline HRQoL levels with nivolumab were maintained over time.,This exploratory analysis showed a between-arm difference in favor of nivolumab on the EQ-5D utility index and clinically meaningful EQ-5D improvements from baseline at several time points for patients receiving nivolumab.,Patients treated with nivolumab did not show increased symptom burden as assessed by the EORTC QLQ-C30.,No HRQoL change was noted with dacarbazine patients up to week 43, although the high attrition rate after week 13 did not allow any meaningful analyses.,Patients receiving nivolumab deteriorated significantly later than those receiving dacarbazine on several EORTC QLQ-C30 scales and the EQ-5D utility index.,In addition to prolonged survival, these exploratory HRQoL results show that nivolumab maintains baseline HRQoL levels to provide long-term quality of survival benefit, compared with dacarbazine in patients with advanced melanoma.
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Over the last few years, clinical trials with BRAF and mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitors have shown significant clinical activity in melanoma, but only a fraction of patients respond to these therapies, and development of resistance is frequent.,This has prompted a large set of preclinical studies looking at several new combinatorial approaches of pathway- or target-specific inhibitors.,At least five main drug association strategies have been verified in vitro and in preclinical models.,The most promising include: i) vertical targeting of either MEK or phosphoinositide-3 kinase (PI3K)/mammalian target of rapamycin (mTOR) pathways, or their combined blockade; ii) association of receptor tyrosine kinases (RTKs) inhibitors with other pro-apoptotic strategies; iii) engagement of death receptors in combination with MEK-, mTOR/PI3K-, histone deacetylase (HDAC)-inhibitors, or with anti-apoptotic molecules modulators; iv) strategies aimed at blocking anti-apoptotic proteins belonging to B-cell lymphoma (Bcl-2) or inhibitors of apoptosis (IAP) families associated with MEK/BRAF/p38 inhibition; v) co-inhibition of other molecules important for survival [proteasome, HDAC and Signal transducers and activators of transcription (Stat)3] and the major pathways activated in melanoma; vi) simultaneous targeting of multiple anti-apoptotic molecules.,Here we review the anti-melanoma efficacy and mechanism of action of the above-mentioned combinatorial strategies, together with the potential clinical application of the most promising studies that may eventually lead to therapeutic benefit.
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The sustained clinical activity of the BRAF inhibitor vemurafenib (PLX4032/RG7204) in patients with BRAFV600 mutant melanoma is limited primarily by the development of acquired resistance leading to tumor progression.,Clinical trials are in progress using MEK inhibitors following disease progression in patients receiving BRAF inhibitors.,However, the PI3K/AKT pathway can also induce resistance to the inhibitors of MAPK pathway.,The sensitivity to vemurafenib or the MEK inhibitor AZD6244 was tested in sensitive and resistant human melanoma cell lines exploring differences in activation-associated phosphorylation levels of major signaling molecules, leading to the testing of co-inhibition of the AKT/mTOR pathway genetically and pharmacologically.,There was a high degree of cross-resistance to vemurafenib and AZD6244, except in two vemurafenib-resistant cell lines that acquired a secondary mutation in NRAS.,In other cell lines, acquired resistance to both drugs was associated with persistence or increase in activity of AKT pathway. siRNA-mediated gene silencing and combination therapy with an AKT inhibitor or rapamycin partially or completely reversed the resistance.,Primary and acquired resistance to vemurafenib in these in vitro models results in frequent cross resistance to MEK inhibitors, except when the resistance is the result of a secondary NRAS mutation.,Resistance to BRAF or MEK inhibitors is associated with the induction or persistence of activity within the AKT pathway in the presence of these drugs.,This resistance can be potentially reversed by the combination of a RAF or MEK inhibitor with an AKT or mTOR inhibitor.,These combinations should be available for clinical testing in patients progressing on BRAF inhibitors.
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The clinical use of BRAF inhibitors for treatment of metastatic melanoma is limited by the development of drug resistance.,In this study we investigated whether co-targeting the MAPK and the PI3K-AKT pathway can prevent emergence of resistance or provide additional growth inhibitory effects in vitro.,Anti-tumor effects of the combination of the BRAF inhibitor (BRAFi) dabrafenib and GSK2141795B (AKTi) in a panel of 23 BRAF mutated melanoma cell lines were evaluated on growth inhibition by an ATP-based luminescent assay, on cell cycle and apoptosis by flow cytometry and on cell signaling by western blot.,Moreover, we investigated the possibilities of delaying or reversing resistance or achieving further growth inhibition by combining AKTi with dabrafenib and/or the MEK inhibitor (MEKi) trametinib by using long term cultures.,More than 40% of the cell lines, including PTEN-/- and AKT mutants showed sensitivity to AKTi (IC50 < 1.5 μM).,The combination of dabrafenib and AKTi synergistically potentiated growth inhibition in the majority of cell lines with IC50 > 5 nM dabrafenib.,Combinatorial treatment induced apoptosis only in cell lines sensitive to AKTi.,In long term cultures of a PTEN-/- cell line, combinatorial treatment with the MAPK inhibitors, dabrafenib and trametinib, and AKTi markedly delayed the emergence of drug resistance.,Moreover, combining AKTi with the MAPK inhibitors from the beginning provided superior growth inhibitory effects compared to addition of AKTi upon development of resistance to MAPK inhibitors in this particular cell line.,AKTi combined with BRAFi-based therapy may benefit patients with tumors harboring BRAF mutations and particularly PTEN deletions or AKT mutations.
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Statins are a class of drugs that inhibit the rate-limiting step in the cholesterol biosynthetic pathway and show an anticancer effect, probably through the inhibition of cell proliferation.,To date, the exact mechanism of cancer cell growth arrest induced by statins is not known.,We report that simvastatin is able to induce apoptosis in melanoma cells but not in normal cells and also able to contrast the growth of tumor in an experimental melanoma murine model.,We observed a delay in the tumor development in almost the 50% of the simvastatin administered animals and a strong reduction of the tumor volume with a differences of ∼150% compared to the controls.,Also the survival rate was significantly higher in mice that received the drug with a survival increase of ∼130% compared to the controls.,The tumor growth reduction in mice was supported by the results of cell migration assay, confirming that simvastatin clearly reduced cell migration.,Moreover, simvastatin induced a strong downregulation of NonO gene expression, an important growth factor involved in the splicing regulation.,This result could explain the decrease of melanoma cells proliferation, suggesting a possible action mechanism.,The results derived from our experiments may sustain the many reports on the anticancer inhibitory property of statins and encourage new studies on this drug for a possible use in therapy, probably in combination with conventional chemotherapy.
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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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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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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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YAP and its paralog protein TAZ are downstream effectors of the Hippo pathway.,Both are amplified in many human cancers and promote cell proliferation and epithelial-mesenchymal transition.,Little is known about the status of the Hippo pathway in cutaneous melanoma.,We profiled Hippo pathway component expression in a panel of human melanoma cell lines and melanocytic lesions, and characterized the capacity of YAP and TAZ to control melanoma cell behavior.,YAP and TAZ immuno-staining in human samples revealed mixed cytoplasmic and nuclear staining for both proteins in benign nevi and superficial spreading melanoma.,TAZ was expressed at higher levels than YAP1/2 in all cell lines and in those with high invasive potential.,Stable YAP or TAZ knockdown dramatically reduced the expression of the classical Hippo target CCN2/connective-tissue growth factor (CTGF), as well as anchorage-independent growth, capacity to invade Matrigel, and ability form lung metastases in mice following tail-vein injection.,YAP knockdown also reduced invasion in a model of skin reconstruct.,Inversely, YAP overexpression increased melanoma cell invasiveness, associated with increased TEA domain-dependent transcription and CCN2/CTGF expression.,Together, these results demonstrate that both YAP and TAZ contribute to the invasive and metastatic capacity of melanoma cells and may represent worthy targets for therapeutic intervention.
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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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Unprecedented clinical responses have been reported in advanced stage metastatic melanoma patients treated with targeted inhibitors of constitutively activated mutant BRAF, which is present in approximately half of all melanomas.,We and others have previously observed an association of elevated nuclear β-catenin with improved survival in molecularly-unselected melanoma patients.,This study sought to determine whether levels of Wnt/β-catenin signaling in melanoma tumors prior to treatment might predict patient responses to BRAF inhibitors (BRAFi).,We performed automated quantification of β-catenin immunohistochemical expression in pretreatment BRAF-mutant tumors from 32 BRAFi-treated melanoma patients.,Unexpectedly, patients with higher nuclear β-catenin in their tumors did not exhibit the survival advantage previously observed in molecularly-unselected melanoma patients who did not receive BRAFi.,In cultured melanoma cells treated with long-term BRAFi, activation of Wnt/β-catenin signaling is markedly inhibited, coinciding with a loss of the enhancement of BRAFi-induced apoptosis by WNT3A observed in BRAFi-naïve cells.,Together, these observations suggest that long-term treatment with BRAFi can impact the interaction between BRAF/MAPK and Wnt/β-catenin signaling to affect patient outcomes.,Studies with larger patient cohorts are required to determine whether nuclear β-catenin expression correlates with clinical responses to BRAFi and to specific mechanisms of acquired resistance to BRAFi.,Understanding these pathway interactions will be necessary to facilitate efforts to individualize therapies for melanoma patients.
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Patients with unresectable advanced cutaneous squamous cell carcinoma (cSCC) are generally treated with palliative intent.,Immune checkpoint blockade has significant activity in the palliative setting in patients with recurrent or metastatic cSCC.,This single arm phase 2 prospective study aims to investigate the combination of curative intent chemoradiation and durvalumab (anti-PD-L1 checkpoint inhibitor) for this patient cohort.,Patients with unresectable locally and or regionally advanced pathologically confirmed cSCC (stage III-IVa) deemed fit for CRIO by consensus of the Multidisciplinary meeting will be eligible.,In the first stage of a two-stage minimax design, we aim to recruit a total of 15 patients.,If fewer than 7 patients achieved a complete response in the first stage, we will conclude the treatment is not more effective than standard treatment.,The co-primary endpoints of CRIO are the safety of treatment (acute and late toxicities) and the rate of complete response.,Secondary endpoints would include overall survival, progression free survival, and locoregional control.,Translational research endpoints including biomarkers (CD73, CD39, PD-1, PD-L1) will also be explored utilising multiplex immunohistochemistry on tumour biopsy samples obtained prior to commencing treatment and during treatment (week 2).,In addition, the utility of CXCR-4 PET/CT scan will be explored.,CRIO is a novel trial evaluating the combination of curative intent chemoradiotherapy with concurrent and adjuvant durvalumab for patients with unresectable stage III-IVa cSCC.,Trial registration: Trial registered with the Australian New Zealand Clinical Trial Registry (ACTRN12618001573246)
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A skin field cancerization is a cutaneous area with subclinical changes resultant from chronic sun exposure, with a higher predisposition to development of pre-neoplastic and neoplastic lesions.,So far, there are no well-defined objective parameters that can indicate their degree of activity.,To describe and compare morphometric aspects and expression of factors related to apoptosis and cell proliferation in actinic keratosis (AK), in both photoexposed and photoprotected epidermis.,A cross-sectional study of patients with actinic keratosis in the forearms, biopsied at two points: the actinic keratosis and the axillary region.,The biopsies of the actinic keratosis, perilesional area, and axilla were evaluated through keratinocyte intraepithelial neoplasia (KIN), and immunohistochemistry of p53, survivin, and Ki67.,Nuclear morphometry of basal layer cells was performed through digital image analysis: entropy, area, perimeter, Ra, fractal dimension, circularity, color intensity, and largest diameter.,There were 13 patients included and 38 actinic keratosis biopsied.,In morphometry, 1039 nuclei were analyzed, of which 228 represented axillary skin, 396 demonstrated actinic keratosis, and 415 represented the perilesional area to the actinic keratosis.,There was a significant difference (p < 0.05) in all variables tested for the topographies evaluated.,A significant correlation was identified between nucellar morphometric elements, KIN, proliferation markers, and apoptosis.,Joint patterns of p53, Ki67, and KIN discriminated the topographies sampled.,This was a cross-sectional study with a small number of patients.,There are patterns of proliferation, resistance to apoptosis, and different cellular morphometrics between photoprotected skin and photoexposed skin.,The joint expression of p53, Ki67, and KIN can characterize skin field cancerization activity.
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Tip110, an important regulator of several oncogenic proteins, was significantly downregulated in human metastatic melanoma cells exposed to a hypoxic condition.,Therefore, in this study, we set to determine whether differential expression of Tip110 could be an important indicator for melanoma tumorigenesis and metastasis.,We found that in melanoma, but not in other cancer types, Tip110 knockdown enhanced significant expression and secretion of IL-8 and melanoma cells invasions.,This induction was further potentiated under hypoxia and by inflammatory cytokine and found independent of TNF-α autocrine signaling.,We further showed that Tip110 knockdown-mediated IL-8 induction involved IL-8 mRNA stability.,Furthermore, the transcriptomic profiling data and survival from 455 melanoma patients demonstrated that the correlation between Tip110 expression and the clinical outcomes in melanoma was stage-dependent.,These findings uncover important roles of Tip110 in melanoma tumorigenesis and metastasis through regulation of IL-8 and hope to provide new clues for future therapeutic strategies.,The online version of this article (10.1186/s12943-018-0868-z) contains supplementary material, which is available to authorized users.
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Cutaneous melanoma is an immune-dependent aggressive tumour.,Up to our knowledge, there are no reports regarding immune parameters monitoring in longitudinal followup of melanoma patients.,We report a followup for 36 months of the immune parameters of patients diagnosed in stages I-IV.,The circulatory immune parameters comprised presurgery and postsurgery immune circulating peripheral cells and circulating intercommunicating cytokines.,Based on our analysis, the prototype of the intratumor inflammatory infiltrate in a melanoma with good prognosis is composed of numerous T cells CD3+, few or even absent B cells CD20+, few or absent plasma cells CD138+, and present Langerhans cells CD1a+ or langerin+.,Regarding circulatory immune cells, a marker that correlates with stage is CD4+/CD8+ ratio, and its decrease clearly indicates a worse prognosis of the disease.,Moreover, even in advanced stages, patients that have an increased overall survival rate prove the increase of this ratio.,The decrease in the circulating B lymphocytes with stage is balanced by an increase in circulating NK cells, a phenomenon observed in stage III.,Out of all the tested cytokines in the followup, IL-6 level correlated with the patient's survival, while in our study, IL-8, IL-10, and IL-12 did not correlate statistically in a significant way with overall survival, or relapse-free survival.
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Even though progress has been made, the detection of melanoma still poses a challenge.,In light of this situation, the Nevisense electrical impedance spectroscopy (EIS) system (SciBase AB, Stockholm, Sweden) was designed and shown to have the potential to be used as an adjunct diagnostic tool for melanoma detection.,To assess the effectiveness and safety of the Nevisense system in the distinction of benign lesions of the skin from melanoma with electrical impedance spectroscopy.,This multicentre, prospective, and blinded clinical study was conducted at five American and 17 European investigational sites.,All eligible skin lesions in the study were examined with the EIS-based Nevisense system, photographed, removed by excisional biopsy and subjected to histopathological evaluation.,A postprocedure clinical follow-up was conducted at 7 ± 3 days from the initial measurement.,A total of 1951 patients with 2416 lesions were enrolled into the study; 1943 lesions were eligible and evaluable for the primary efficacy end point, including 265 melanomas - 112 in situ and 153 invasive melanomas with a median Breslow thickness of 0·57 mm [48 basal cell carcinomas (BCCs) and seven squamous cell carcinomas (SCCs)].,The observed sensitivity of Nevisense was 96·6% (256 of 265 melanomas) with an exact one-sided 95% lower confidence bound estimated at 94·2% and an observed specificity of 34·4%, and an exact two-sided 95% confidence bound estimated at 32·0-36·9%.,The positive and negative predictive values of Nevisense were 21·1% and 98·2%, respectively.,The observed sensitivity for nonmelanoma skin cancer was 100% (55 of 48 BCCs and seven SCCs) with an exact two-sided 95% confidence bound estimated at 93·5-100·0%.,Nevisense is an accurate and safe device to support clinicians in the detection of cutaneous melanoma.,Although progress has been made in the detection of melanoma it still poses a challenge.,Electrical impedance spectroscopy (EIS) may potentially be used as a diagnostic aid for the detection of melanoma.,In the largest international prospective study of its kind in melanoma detection, the EIS system Nevisense was shown to be both accurate and safe in the lesion cohort studied.,In the absence of a perfect gold standard, the accuracy of a device should be compared with the consensus diagnosis from multiple experts.
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Dermoscopy is a technique which improves melanoma detection.,Optical dermoscopy uses a handheld optical device to observe the skin lesions without recording the images.,Sequential digital dermoscopy imaging (SDDI) allows storage of the pictures and their comparison over time.,Few studies have compared optical dermoscopy and SDDI from an economic perspective.,The present observational study focused on patients with one-to-three atypical melanocytic lesions, i.e. lesions considered as suspicious by optical dermoscopy.,It aimed to calculate the “extra-costs” related to the process of melanoma detection.,These extra-costs were defined as the costs of excision and pathology of benign lesions and/or the costs of follow-up by SDDI.,The objective was to compare these extra-costs when using optical dermoscopy exclusively versus optical dermoscopy with selective use of SDDI.,In a first group of patients, dermatologists were adequately trained in optical dermoscopy but worked without access to SDDI.,They excised all suspicious lesions to rule out melanoma.,In a second group, the dermatologists were trained in optical and digital dermoscopy.,They had the opportunity of choosing between immediate excision or follow-up by SDDI (with delayed excision if significant change was observed).,The comparison of extra-costs in both groups was made possible by a decision tree model and by the division of the extra-costs by the number of melanomas diagnosed in each group.,Belgian official tariffs and charges were used.,The extra-costs in the first and in the second group were respectively €1,613 and €1,052 per melanoma excised.,The difference was statistically significant.,Using the Belgian official tariffs and charges, we demonstrated that the selective use of SDDI for patients with one-to-three atypical melanocytic lesions resulted in a significant cost reduction.
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Uveal melanoma is a clinically distinct and particularly lethal subtype of melanoma originating from melanocytes in the eye.,Here, we performed multi-region DNA sequencing of primary uveal melanomas and their matched metastases from 35 patients.,We observed novel driver mutations and established the order in which these and known driver mutations undergo selection.,Metastases had genomic alterations distinct from their primary tumors, and metastatic dissemination sometimes occurred early during the development of the primary tumor.,Our study offers new insights into the genetics and evolution of this melanoma subtype, providing potential biomarkers for progression and therapy.
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MITF (microphthalmia-associated transcription factor) is a frequently amplified lineage-specific oncogene in human melanoma, whose role in intrinsic drug resistance has not been systematically investigated.,Utilizing chemical inhibitors for major signaling pathways/cellular processes, we witness MITF as an elicitor of intrinsic drug resistance.,To search kinase(s) targets able to bypass MITF-conferred drug resistance, we employed a multi-kinase inhibitor-directed chemical proteomics-based differential affinity screen in human melanocytes carrying ectopic MITF overexpression.,A subsequent methodical interrogation informed mitotic Ser/Thr kinase Aurora Kinase A (AURKA) as a crucial regulator of melanoma cell proliferation and migration, independent of the underlying molecular alterations, including TP53 functional status and MITF levels.,Crucially, assessing the efficacy of investigational AURKA inhibitor MLN8237, we pre-emptively witness the procurement of a molecular program consistent with acquired drug resistance.,This involved induction of multiple MAPK (mitogen-activated protein kinase) signaling pathway components and their downstream proliferation effectors (Cyclin D1 and c-JUN) and apoptotic regulators (MITF and Bcl-2).,A concomitant AURKA/BRAF and AURKA/MEK targeting overcame MAPK signaling activation-associated resistance signature in BRAF- and NRAS-mutated melanomas, respectively, and elicited heightened anti-proliferative activity and apoptotic cell death.,These findings reveal a previously unreported MAPK signaling-mediated mechanism of immediate resistance to AURKA inhibitors.,These findings could bear significant implications for the application and the success of anti-AURKA approaches that have already entered phase-II clinical trials for human melanoma.
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Solid cancer cells commonly enter the blood and disseminate systemically but are highly inefficient at forming distant metastases for poorly understood reasons.,We studied human melanomas that differed in their metastasis histories in patients and in their capacity to metastasize in NSG mice.,All melanomas had high frequencies of cells that formed subcutaneous tumours, but much lower percentages of cells that formed tumours after intravenous or intrasplenic transplantation, particularly among inefficient metastasizers.,Melanoma cells in the blood and visceral organs experienced oxidative stress not observed in established subcutaneous tumours.,Successfully metastasizing melanomas underwent reversible metabolic changes during metastasis that increased their capacity to withstand oxidative stress, including increased dependence upon NADPH-generating enzymes in the folate pathway.,Anti-oxidants promoted distant metastasis in NSG mice.,Folate pathway inhibition using low-dose methotrexate, ALDH1L2 knockdown, or MTHFD1 knockdown inhibited distant metastasis without significantly affecting the growth of subcutaneous tumors in the same mice.,Oxidative stress thus limits distant metastasis by melanoma cells in vivo.
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Apoptosis-associated speck-like protein containing a CARD (ASC) was originally named because it triggered apoptosis in certain tumors.,More recently, however, ASC was found to be a central adaptor protein of inflammasome which mediates the secretion of pro-tumorigenic inflammation.,Here we examined the role of ASC in tumorigenesis of human melanoma.,Compared with primary melanoma, ASC protein expression was generally downregulated in metastatic melanoma.,While overexpressing ASC in metastatic melanoma showed no effects on cell viability, silencing ASC with short hairpin RNA induced G1 cell cycle arrest, reduced cell viability and suppressed tumorigenesis in metastatic melanoma.,On the other hand, silencing ASC in primary melanoma reduced cell death, increased cell viability and enhanced tumorigenesis.,In primary and metastatic melanoma cells, ASC knockdown inhibited inflammasome-mediated caspase-1 activity and IL-1β secretion.,However, phosphorylated IKKα/β expression and NF-κB activity were suppressed in metastatic melanoma and enhanced in primary melanoma after ASC knockdown.,These findings suggest stage-dependent dual roles of ASC in tumorigenesis.,ASC expression in primary melanoma inhibits tumorigenesis, by reducing IKKα/β phosphorylation and inhibiting NF-κB activity.,In metastatic melanoma, on the other hand, this inhibitory effect is diminished, and ASC induces tumorigenic pathways through enhanced NF-κB activity and inflammasome-mediated IL-1β secretion.
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The present study identified miR-638 as one of the most significantly overexpressed miRNAs in metastatic lesions of melanomas compared with primary melanomas. miR-638 enhanced the tumorigenic properties of melanoma cells in vitro and lung colonization in vivo. mRNA expression profiling identified new candidate genes including TP53INP2 as miR-638 targets, the majority of which are involved in p53 signalling.,Overexpression of TP53INP2 severely attenuated proliferative and invasive capacity of melanoma cells which was reversed by miR-638.,Depletion of miR-638 stimulated expression of p53 and p53 downstream target genes and induced apoptosis and autophagy. miR-638 promoter analysis identified the miR-638 target transcription factor associated protein 2α (TFAP2A/AP-2α) as a direct negative regulator of miR-638, suggestive for a double-negative regulatory feedback loop.,Taken together, miR-638 supports melanoma progression and suppresses p53-mediated apoptosis pathways, autophagy and expression of the transcriptional repressor TFAP2A/AP-2α.
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The prognosis of patients with metastatic melanoma is poor and not influenced by systemic therapy with cytotoxic drugs.,New targeted agents directed against the RAS-RAF-MEK-ERK pathway show promising activity in early clinical development and particular interest is focused on selective inhibitors of mutant BRAF, which is present in one half of the cases of metastatic melanoma.,The majority of patients on early trials of these drugs develop secondary resistance and subsequent disease progression and it is, therefore, critical to understand the underlying escape mechanisms leading to resistance.
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Melanoma is a highly aggressive tumour that can metastasize very early in disease progression.,Notably, melanoma can disseminate using amoeboid invasive strategies.,We show here that high Myosin II activity, high levels of ki-67 and high tumour-initiating abilities are characteristic of invasive amoeboid melanoma cells.,Mechanistically, we find that WNT11-FZD7-DAAM1 activates Rho-ROCK1/2-Myosin II and plays a crucial role in regulating tumour-initiating potential, local invasion and distant metastasis formation.,Importantly, amoeboid melanoma cells express both proliferative and invasive gene signatures.,As such, invasive fronts of human and mouse melanomas are enriched in amoeboid cells that are also ki-67 positive.,This pattern is further enhanced in metastatic lesions.,We propose eradication of amoeboid melanoma cells after surgical removal as a therapeutic strategy.,Amoeboid cells are associated with melanoma invasive capacity.,Here, the authors show that the WNT11-FZD7-DAAM1 pathway regulates tumour-initiating potential, invasion and metastasis lead by amoeboid cells in the invasive front of melanoma tumours.
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ROCK-Myosin II drives fast rounded-amoeboid migration in cancer cells during metastatic dissemination.,Analysis of human melanoma biopsies revealed that amoeboid melanoma cells with high Myosin II activity are predominant in the invasive fronts of primary tumors in proximity to CD206+CD163+ tumor-associated macrophages and vessels.,Proteomic analysis shows that ROCK-Myosin II activity in amoeboid cancer cells controls an immunomodulatory secretome, enabling the recruitment of monocytes and their differentiation into tumor-promoting macrophages.,Both amoeboid cancer cells and their associated macrophages support an abnormal vasculature, which ultimately facilitates tumor progression.,Mechanistically, amoeboid cancer cells perpetuate their behavior via ROCK-Myosin II-driven IL-1α secretion and NF-κB activation.,Using an array of tumor models, we show that high Myosin II activity in tumor cells reprograms the innate immune microenvironment to support tumor growth.,We describe an unexpected role for Myosin II dynamics in cancer cells controlling myeloid function via secreted factors.,•Invasive tumor fronts are enriched in rounded-amoeboid cancer cells high in Myosin II•Myosin II activity in these cells regulates an immunomodulatory secretome•The secreted cytokines and chemokines can induce tumor-promoting macrophages•ROCK-Myosin II and IL-1α/NF-κB cross-talk supports this secretory phenotype,Invasive tumor fronts are enriched in rounded-amoeboid cancer cells high in Myosin II,Myosin II activity in these cells regulates an immunomodulatory secretome,The secreted cytokines and chemokines can induce tumor-promoting macrophages,ROCK-Myosin II and IL-1α/NF-κB cross-talk supports this secretory phenotype,Myosin II activation at the tumor edge promotes invasion and also a secretory phenotype that reshapes the local environment and vasculature to support tumor growth.
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Melanin is major factor that determines skin color as well as one of the defense systems that prevent the UV-induced damage.,In case of abnormal concentration of melanin, skin diseases or problems occur such as albinism, leukoplakia, melasma, freckles, moles, and lentigo.,With the lifespan of humans has been extended, importance of ‘life quality’ has been increased.,White and clean skin is very important part of the satisfaction of appearance, especially for Asia women.,The aim of this study was to find an anti-melanogenesis activity for which the aerial part of Pueraria thunbergiana can be utilized based on the increase in demands for cosmetics, particularly natural products.,We demonstrated anti-pigmentation effects of aerial part of P. thunbergiana by measuring melanin content and through staining in the B16F10 melanoma cell line.,The aerial part of P. thunbergiana decreased tyrosinase activity significantly in B16F10 cell cultures, while there is no direct effect on enzyme in cell-free conditions.,To define the mechanisms, real-time PCR, western blot, glucosidase activity and antioxidant activity assay were implemented.,As results, we demonstrated that aerial part of P. thunbergiana has anti-melanogenesis activity via two mechanisms.,One is downgrading microphthalmia-associated transcription factor by activating Akt/GSK-3β.,Consequently, transcription of tyrosinase and tyrosinase-related protein 1 is decreased.,Another is interrupting maturation of tyrosinase through inhibiting α-glucosidase.,Furthermore, aerial part of P. thunbergiana showed great efficacy on pigmentation in vivo.,These results suggest that aerial part of P. thunbergiana can be used as an anti-melanogenic agent.
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To explore the safety and tolerability of combining two epigenetic drugs: decitabine (a DNA methyltransferase inhibitor) and panobinostat (a histone deacetylase inhibitor), with chemotherapy with temozolomide (an alkylating agent).,The purpose of such combination is to evaluate the use of epigenetic priming to overcome resistance of melanoma to chemotherapy.,A Phase I clinical trial enrolling patients aged 18 years or older, with recurrent or unresectable stage III or IV melanoma of any site.,This trial was conducted with full Institutional Review Board approval and was registered with the National Institutes of Health under the clinicaltrials.gov identifier NCT00925132.,Patients were treated with subcutaneous decitabine 0.1 or 0.2 mg/kg three times weekly for 2 weeks (starting on day 1), in combination with oral panobinostat 10, 20, or 30 mg every 96 h (starting on day 8), and oral temozolomide 150 mg/m2/day on days 9 through 13.,In cycle 2, temozolomide was increased to 200 mg/m2/day if neutropenia or thrombocytopenia had not occurred.,Each cycle lasted 6 weeks, and patients could receive up to six cycles.,Patients who did not demonstrate disease progression were eligible to enter a maintenance protocol with combination of weekly panobinostat and thrice-weekly decitabine until tumor progression, unacceptable toxicity, or withdrawal of consent.,Twenty patients were initially enrolled, with 17 receiving treatment.,The median age was 56 years.,Eleven (65 %) were male, and 6 (35 %) were female.,Eleven (64.7 %) had cutaneous melanoma, 4 (23.5 %) had ocular melanoma, and 2 (11.8 %) had mucosal melanoma.,All patients received at least one treatment cycle and were evaluable for toxicity.,Patients received a median of two 6-week treatment cycles (range 1-6).,None of the patients experienced DLT.,MTD was not reached.,Adverse events attributed to treatment included grade 3 lymphopenia (24 %), anemia (12 %), neutropenia (12 %), and fatigue (12 %), as well as grade 2 leukopenia (30 %), neutropenia (23 %), nausea (23 %), and lymphopenia (18 %).,The most common reason for study discontinuation was disease progression.,This triple agent of dual epigenetic therapy in combination with traditional chemotherapy was generally well tolerated by the cohort and appeared safe to be continued in a Phase II trial.,No DLTs were observed, and MTD was not reached.
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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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Herein, we describe the use of systemic immunotherapy for both locally advanced and metastatic conjunctival melanoma.,Current treatments for advanced conjunctival melanoma typically result in poor local control leading to disfiguring orbital exenteration surgery.,Locoregional spread of conjunctival malignant melanoma typically requires pre-auricular and cervical lymph node dissection with post-operative adjuvant radiation therapy.,In addition, classic systemic chemotherapy has been unsuccessful in the treatment of metastatic disease.,This is a retrospectively analyzed clinical case series of 5 patients with biopsy proven conjunctival melanoma who were treated with checkpoint inhibition therapy.,Of these, 3 patients were treated for residual ocular disease present after failing multiple local therapies and refusing orbital exenteration surgery and two (with local ocular control) for metastatic conjunctival melanoma.,Both those with locally advanced disease and patients with metastatic disease received an anti-PD1 agent in combination with another immunotherapeutic agent.,All 5 were given multiple cycles of systemic anti-PD1 therapy, 1 was initially treated with single agent ipilimumab (3 mg/kg) prior to approval of anti-PD1 agents and two received interferon eye drops.,As part of each ophthalmic examination, photographs of all conjunctival and eyelid surfaces were obtained.,Systemic evaluations involved initial staging scans as well as periodic re-imaging.,All cases have shown responses.,Of the 2 complete responses, 1 was a patient with systemic disease.,No patients developed ocular toxicity or loss of vision.,However, systemic adverse effects included adrenal insufficiency, Grade-III colitis, Grade-II dermatitis, Grade-II hepatotoxicity and Grade-II pneumonitis.,This report suggests that systemic immunotherapy with or without topical interferon is effective in treatment of malignant melanoma of the conjunctiva.,Therefore, it can be considered for patients with advanced local conjunctival melanoma, those who refuse orbital exenteration surgery and those with systemic metastasis.
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Merkel cell polyomavirus (MCV) is the causative agent for the majority of Merkel cell carcinoma (MCC) cases.,Polyomavirus-associated MCC (MCCP) is characterized by the integration of MCV DNA into the tumor genome and a low tumor mutational burden.,In contrast, nonviral MCC (MCCN) is characterized by a high tumor mutational burden induced by UV damage.,Since the discovery of MCV, much work in the field has focused on understanding the molecular mechanisms of oncogenesis driven by the MCV tumor (T) antigens.,Here, we review our current understanding of how the activities of large T (LT) and small T (ST) promote MCC oncogenesis in the absence of genomic instability.,We highlight how both LT and ST inhibit tumor suppressors to evade growth suppression, an important cancer hallmark.,We discuss ST interactions with cellular proteins, with an emphasis on those that contribute to sustaining proliferative signaling.,Finally, we examine active areas of research into open questions in the field, including the origin of MCC and mechanisms of viral integration.
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Approximately one‐third of Merkel cell carcinoma (MCC) patients eventually develop distant metastatic disease.,Little is known about whether the location of the primary lesion is predictive of initial distant metastatic site, or if survival likelihood differs depending on the metastatic site.,Such data could inform imaging/surveillance practices and improve prognostic accuracy.,Multivariate and competing‐risk analyses were performed on a cohort of 215 MCC patients with distant metastases, 31% of whom had two or more initial sites of distant metastasis.,At time of initial distant metastasis in the 215 patients, metastatic sites (n = 305) included non‐regional lymph nodes (present in 41% of patients), skin/body wall (25%), liver (23%), bone (21%), pancreas (8%), lung (7%), and brain (5%).,Among the 194 patients who presented with MCC limited to local or regional sites (stage I‐III) but who ultimately developed distant metastases, distant progression occurred in 49% by 1 year and in 80% by 2 years following initial diagnosis.,Primary MCC locations differed in how likely they were to metastasize to specific organs/sites (P < .001).,For example, liver metastases were far more likely from a head/neck primary (43% of 58 patients) versus a lower limb primary (5% of 39 patients; P < .0001).,Skin‐only distant metastasis was associated with lower MCC‐specific mortality as compared to metastases in multiple organs/sites (HR 2.7; P = .003), in the liver (HR 2.1; P = .05), or in distant lymph nodes (HR 2.0; P = .045).,These data reflect outcomes before PD1‐pathway inhibitor availability, which may positively impact survival.,In conclusion, primary MCC location is associated with a pattern of distant spread, which may assist in optimizing surveillance.,Because it is linked to survival, the site of initial distant metastasis should be considered when assessing prognosis.
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Macrophages are located in essentially all tissues due to their “janitor” function.,Macrophages can exert either anti- or pro-tumor activities depending upon the specific tumor microenvironment they inhabit.,Substantial evidence indicates that macrophages, owing to their plasticity, can be reeducated to adopt a protumoral phenotype within a tumor microenvironment through the help of growth factors in the microenvironment and intercellular interactions.,As the lethality of malignant melanoma is due to its aggressive capacity for metastasis and resistance to therapy, considerable effort has gone toward treatment of metastatic melanoma.,In the present review, we focus on the pro-tumor activities of macrophages in melanoma.,Based upon the information presented in this review it is anticipated that new therapies will soon be developed that target pro-tumor activities of macrophages for use in the treatment of melanoma.
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Anti-CTLA-4 antibody induces selective depletion of T reg cells within tumor lesions in a manner that is dependent on the presence of Fc gamma receptor-expressing macrophages within the tumor microenvironment.,Treatment with monoclonal antibody specific for cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), an inhibitory receptor expressed by T lymphocytes, has emerged as an effective therapy for the treatment of metastatic melanoma.,Although subject to debate, current models favor a mechanism of activity involving blockade of the inhibitory activity of CTLA-4 on both effector (T eff) and regulatory (T reg) T cells, resulting in enhanced antitumor effector T cell activity capable of inducing tumor regression.,We demonstrate, however, that the activity of anti-CTLA-4 antibody on the T reg cell compartment is mediated via selective depletion of T reg cells within tumor lesions.,Importantly, T reg cell depletion is dependent on the presence of Fcγ receptor-expressing macrophages within the tumor microenvironment, indicating that T reg cells are depleted in trans in a context-dependent manner.,Our results reveal further mechanistic insight into the activity of anti-CTLA-4-based cancer immunotherapy, and illustrate the importance of specific features of the local tumor environment on the final outcome of antibody-based immunomodulatory therapies.
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Melanoma is the deadliest form of skin cancer, and its incidence has alarmingly increased in the last few decades, creating a need for novel treatment approaches.,Thus, we evaluated the combinatorial effect of doxorubicin (DOX) and hyperthermia on A375 and MNT-1 human melanoma cell lines.,Cells were treated with DOX for 24, 48, and 72 h and their viabilities were assessed.,The effect of DOX IC10 and IC20 (combined at 43 °C for 30, 60, and 120 min) on cell viability was further analyzed.,Interference on cell cycle dynamics, reactive oxygen species (ROS) production, and apoptosis upon treatment (with 30 min at 43 °C and DOX at the IC20 for 48 h) were analyzed by flow cytometry.,Combined treatment significantly decreased cell viability, but not in all tested conditions, suggesting that the effect depends on the drug concentration and heat treatment duration.,Combined treatment also mediated a G2/M phase arrest in both cell lines, as well as increasing ROS levels.,Additionally, it induced early apoptosis in MNT-1 cells, while in A375 cells this effect was similar to the one caused by hyperthermia alone.,These findings demonstrate that hyperthermia enhances DOX effect through cell cycle arrest, oxidative stress, and apoptotic cell death.
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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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Melanoma is a heterogeneous tumour, but the impact of this heterogeneity upon therapeutic response is not well understood.,Single cell mRNA analysis was used to define the transcriptional heterogeneity of melanoma and its dynamic response to BRAF inhibitor therapy and treatment holidays.,Discrete transcriptional states were defined in cell lines and melanoma patient specimens that predicted initial sensitivity to BRAF inhibition and the potential for effective re-challenge following resistance.,A mathematical model was developed to maintain competition between the drug-sensitive and resistant states, which was validated in vivo.,Our analyses showed melanoma cell lines and patient specimens to be composed of >3 transcriptionally distinct states.,The cell state composition was dynamically regulated in response to BRAF inhibitor therapy and drug holidays.,Transcriptional state composition predicted for therapy response.,The differences in fitness between the different transcriptional states were leveraged to develop a mathematical model that optimized therapy schedules to retain the drug sensitive population.,In vivo validation demonstrated that the personalized adaptive dosing schedules outperformed continuous or fixed intermittent BRAF inhibitor schedules.,Our study provides the first evidence that transcriptional heterogeneity at the single cell level predicts for initial BRAF inhibitor sensitivity.,We further demonstrate that manipulating transcriptional heterogeneity through personalized adaptive therapy schedules can delay the time to resistance.,This work was funded by the National Institutes of Health.,The funder played no role in assembly of the manuscript.
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Efficient treatments against metastatic melanoma dissemination are still lacking.,Here, we report that low-cytotoxic concentrations of 5-aza-2′-deoxycytidine, a DNA demethylating agent, prevent in vitro 3D invasiveness of metastatic melanoma cells and reduce lung metastasis formation in vivo.,We unravelled that this beneficial effect is in part due to MIR-199A2 re-expression by promoter demethylation.,Alone, this miR showed an anti-invasive and anti-metastatic effect.,Throughout integration of micro-RNA target prediction databases with transcriptomic analysis after 5-aza-2′-deoxycytidine treatments, we found that miR-199a-3p downregulates set of genes significantly involved in invasion/migration processes.,In addition, analysis of data from melanoma patients showed a stage- and tissue type-dependent modulation of MIR-199A2 expression by DNA methylation.,Thus, our data suggest that epigenetic- and/or miR-based therapeutic strategies can be relevant to limit metastatic dissemination of melanoma.,The online version of this article (10.1186/s13148-018-0600-2) contains supplementary material, which is available to authorized users.
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Previous analysis of COMBI-d (NCT01584648) demonstrated improved progression-free survival (PFS) and overall survival (OS) with combination dabrafenib and trametinib versus dabrafenib monotherapy in BRAF V600E/K-mutant metastatic melanoma.,This study was continued to assess 3-year landmark efficacy and safety after ≥36-month follow-up for all living patients.,This double-blind, phase 3 study enrolled previously untreated patients with BRAF V600E/K-mutant unresectable stage IIIC or stage IV melanoma.,Patients were randomized to receive dabrafenib (150 mg twice daily) plus trametinib (2 mg once daily) or dabrafenib plus placebo.,The primary endpoint was PFS; secondary endpoints were OS, overall response, duration of response, safety, and pharmacokinetics.,Between 4 May and 30 November 2012, a total of 423 of 947 screened patients were randomly assigned to receive dabrafenib plus trametinib (n = 211) or dabrafenib monotherapy (n = 212).,At data cut-off (15 February 2016), outcomes remained superior with the combination: 3-year PFS was 22% with dabrafenib plus trametinib versus 12% with monotherapy, and 3-year OS was 44% versus 32%, respectively.,Twenty-five patients receiving monotherapy crossed over to combination therapy, with continued follow-up under the monotherapy arm (per intent-to-treat principle).,Of combination-arm patients alive at 3 years, 58% remained on dabrafenib plus trametinib.,Three-year OS with the combination reached 62% in the most favourable subgroup (normal lactate dehydrogenase and <3 organ sites with metastasis) versus only 25% in the unfavourable subgroup (elevated lactate dehydrogenase).,The dabrafenib plus trametinib safety profile was consistent with previous clinical trial observations, and no new safety signals were detected with long-term use.,These data demonstrate that durable (≥3 years) survival is achievable with dabrafenib plus trametinib in patients with BRAF V600-mutant metastatic melanoma and support long-term first-line use of the combination in this setting.
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Advanced melanoma is an incurable disease with complex and expensive treatments.,The best approach to prevent melanoma at advanced stages is an early diagnosis.,A knowledge of factors associated with the process of detecting cutaneous melanomas and the reasons for delays in diagnosis is essential for the improvement of the secondary prevention of the disease.,Identify sociodemographic, individual, and medical aspects related to cutaneous melanoma diagnosis delay.,Interviews evaluated the knowledge of melanoma, signals, symptoms, persons who were suspected, delays in seeking medical attention, physician's deferrals, and related factors of 211 patients.,Melanomas were self-discovered in 41.7% of the patients; healthcare providers detected 29.9% of patients and others detected 27%.,The main component in delay was patient-related.,Only 31.3% of the patients knew that melanoma was a serious skin cancer, and most thought that the pigmented lesion was not important, causing a delay in seeking medical assistance.,Patients (36.4%) reported a wait interval of more than 6 months from the onset of an observed change in a pigmented lesion to the first visit to a physician.,The delay interval from the first physician visit to a histopathological diagnosis was shorter (<1 month) in 55.5% of patients.,Improper treatments without a histopathological confirmation occurred in 14.7% of patients.,A professional delay was related to both inappropriate treatments performed without histopathological confirmation (P = 0.003) and long requirements for medical referrals (P < 0.001).,A deficient knowledge in the population regarding melanoma and physicians’ misdiagnoses regarding suspicious lesions contributed to delays in diagnosis.
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Metastatic mucosal melanoma responds poorly to anti-programmed cell death-1 (PD-1) monotherapy.,Vascular endothelial growth factor (VEGF) has been shown to play an important immunosuppressive role in the tumor microenvironment.,The combination of VEGF inhibition and PD-1 blockade provides therapeutic opportunities for patients refractory to either therapy alone.,We conducted a single-center, phase IB trial evaluating the safety and preliminary efficacy of toripalimab, a humanized immunoglobulin G4 monoclonal antibody against PD-1 in combination with the VEGF receptor inhibitor axitinib in patients with advanced melanoma, including patients with chemotherapy-naïve mucosal melanomas (88%).,Patients received toripalimab at 1 or 3 mg/kg via intravenous infusion every 2 weeks, in combination with axitinib 5 mg orally twice a day, in a dose-escalation and cohort-expansion study until confirmed disease progression, unacceptable toxicity, or voluntary withdrawal.,The primary objective was safety.,Secondary objectives included efficacy, pharmacokinetics, pharmacodynamics, immunogenicity, and tumor tissue biomarkers.,Thirty-three patients were enrolled.,No dose-limiting toxicities were observed.,Ninety-seven percent of patients experienced treatment-related adverse events (TRAEs).,The most common TRAEs were mild (grade 1 or 2) and included diarrhea, proteinuria, hand and foot syndrome, fatigue, AST or ALT elevation, hypertension, hypo- or hyperthyroidism, and rash.,Grade 3 or greater TRAEs occurred in 39.4% of patients.,By the cutoff date, among 29 patients with chemotherapy-naïve mucosal melanoma, 14 patients (48.3%; 95% CI, 29.4% to 67.5%) achieved objective response, and the median progression-free survival time was 7.5 months (95% CI, 3.7 months to not reached) per Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1.,The combination of toripalimab plus axitinib was tolerable and showed promising antitumor activity in patients with treatment-naïve metastatic mucosal melanoma.,Patients enrolled in this study were all Asian, and this combination therapy must be validated in a randomized phase III trial that includes a non-Asian population before it can become a standard of care.
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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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This study identified sex differences in progression of cutaneous melanoma.,Of 7,338 patients who were diagnosed as an invasive primary CM without clinically detectable metastases from 1976 to 2008 at the University of Tuebingen in Germany, 1,078 developed subsequent metastases during follow up.,The metastatic pathways were defined in these patients and analyzed using the Kaplan-Meier method.,Multivariate survival analysis was performed using Cox modeling.,In 18.7% of men and 29.2% of women (P<0.001) the first metastasis following diagnosis of primary tumor was locoregional as satellite/in-transit metastasis.,The majority of men (54.0%) and women (47.6%, P = 0.035) exhibited direct regional lymph node metastasis.,Direct distant metastasis from the stage of the primary tumor was observed in 27.3% of men and 23.2% of women (P = 0.13).,Site of first metastasis was the most important prognostic factor of survival after recurrence in multivariate analysis (HR:1.3; 95% CI: 1.0-1.6 for metastasis to the regional lymph nodes vs. satellite/in-transit recurrence, and HR:5.5; 95% CI: 4.2-7.1 for distant metastasis vs. satellite/in-transit recurrence, P<0.001).,Median time to distant metastasis was 40.5 months (IQR, 58.75) in women and 33 months (IQR, 44.25) in men (P = 0.002).,Five-year survival after distant recurrence probability was 5.2% (95% CI: 1.4-2.5) for men compared with 15.3% (95% CI: 11.1-19.5; P = 0.008) for women.,Both, the pattern of metastatic spread with more locoregional metastasis in women, and the time course with retracted metastasis in women contributed to the more favorable outcome of women.,Furthermore, the total rate of metastasis is increased in men.,Interestingly, there is also a much more favorable long term survival of women after development of distant metastasis.,It remains a matter of debate and of future research, whether hormonal or immunologic factors may be responsible for these sex differences.
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Activating mutations in BRAF kinase are common in melanomas.,Clinical trials with PLX4032, the mutant-BRAF inhibitor, show promising preliminary results in patients selected for the presence of V600E mutation.,However, activating V600K mutation is the other most common mutation, yet patients with this variant are currently excluded from the PLX4032 trials.,Here we present evidence that a patient bearing the BRAF V600K mutation responded remarkably to PLX4032, suggesting that clinical trials should include all patients with activating BRAF V600E/K mutations.
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Skin cancer is the most frequently diagnosed cancer in the fair‐skinned population.,In recent years, the incidence of nonmelanoma skin cancer (NMSC) has been increasing worldwide.,However, there is no epidemiological study on skin cancer in the Asian population.,A prospective cohort study including 140 420 participants was initiated in 1990 for cohort Ⅰ and 1993 for cohort Ⅱ at baseline survey from 11 public health center (PHC) areas.,Of these participants, 284 NMSC cases were diagnosed during the follow‐up period (through 2012 in the Osaka PHC area and 2013 in the other PHC areas).,The Cox proportional hazards model was used to estimate hazard ratios and 95% confidence intervals (CI) for NMSC incidence according to occupational type, lifestyle factors (alcohol consumption, coffee consumption, smoking status, physical activity, and body mass index), and family history of cancer.,Among men, compared with indoor workers, outdoor workers were associated with 2.18 (95% CI, 1.17‐4.04) higher risk of squamous cell carcinoma (SCC) but not of basal cell carcinoma (BCC).,Furthermore, men who have a family history of cancer had 1.99 (95% CI, 1.10‐3.62) higher SCC risk.,In women, we did not observe any association between occupational type and the risk of SCC (1.26; 95% CI, 0.68‐2.32) or BCC (0.74; 95% CI, 0.42‐1.28).,In conclusion, men who are outdoor workers or have a family history of cancer had an increased risk of SCC.,We found an increased risk of squamous cell carcinoma skin cancer in men who were outdoor workers and those with a family history of cancer.,As UV exposure in outdoor workers is prevalent and could be avoided to some extent, reducing exposure to sunlight could serve as a prevention strategy to reduce the prevalence of nonmelanoma skin cancer in the Japanese population.
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Supplemental Digital Content is available in the text,The metabolic hormone leptin has been implicated in the pathogenesis of various malignancies and may contribute to the high rate of cancer in obese individuals.,We reported that leptin and its receptor are expressed by melanoma tumors and cell lines, and that leptin stimulates proliferation of cultured melanoma cells.,Here, we tested the hypothesis that leptin contributes to early melanoma progression by assessing its association with sentinel node positivity in cutaneous melanoma patients.,The study enrolled 72 patients who were scheduled to undergo lymphatic mapping and sentinel node biopsy.,Fasting blood was obtained before surgery, and serum leptin levels were measured by enzyme-linked immunosorbent assay (ELISA) with a “raw” (assay value) and an “adjusted” value (raw value divided by body mass index).,Leptin levels and other clinicopathologic parameters were compared between sentinel node positive and negative groups.,Logistic regression models were used to predict sentinel node status using leptin and other relevant clinical parameters.,The raw and adjusted leptin levels were significantly higher in the 15 patients with positive sentinel nodes.,These findings could not be attributed to differences in body mass indices.,Univariate models revealed raw leptin, adjusted leptin, Breslow thickness, and mitotic rate as significant predictors of sentinel node status.,Leptin levels and Breslow thickness remained significant in multivariate models.,Survival and follow-up analysis revealed more aggressive disease in diabetic patients.,Elevated serum leptin levels predict sentinel node metastasis in melanoma.,Validation of this finding in larger cohorts should enable better stratification of early stage melanoma patients.
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Uveal melanoma (UM) is the most common primary tumor of the eye diagnosed in adults, associated with a high risk of metastasis and thereby, poor prognosis.,Among known risk factors for the development of metastatic disease is the loss of BAP1 expression and chromosome 3 monosomy in the primary tumor.,However, the expression levels of specific micro RNAs (miRNA) in tumor tissue may also serve as a valuable marker for determining the risk of metastatic disease in patients with primary uveal melanoma.,In our study, we analyzed the miRNA expression data of cases selected from The Cancer Genome Atlas study on uveal melanoma, and determined a panel of 15 miRNAs differentially expressed between patients with primary and metastatic disease.,Next, 6 miRNAs were validated on a group of 46 tumor samples from primary and metastatic patients.,We have shown, that expression of hsa-miR-592, hsa-miR-346, and hsa-miR-1247 was significantly increased, while hsa-miR-506 and hsa-miR-513c were decreased in the tumors of patients with metastatic disease.,Hsa-miR-196b expression did not differ between the two subgroups, however, we showed significant correlation with BAP1 expression.,Moreover, hsa-miR-592 also showed correlation with monosomy 3 tumors.,Gene ontology analysis revealed involvement of those miRNAs with cellular processes mediating the metastatic process.,Our results showed that miRNAs play an important role in the deregulation of several oncogenic pathways in UM and can, thereby, promote metastatic spread to distant organs.,Moreover, differentially expressed miRNAs may be used as an interesting biomarker for the assessment of metastatic risk in uveal melanoma patients.
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MicroRNAs (miRNAs) are a group endogenous small non-coding RNAs that inhibit protein translation through binding to specific target mRNAs.,Recent studies have demonstrated that miRNAs are implicated in the development of cancer.,However, the role of miR-144 in uveal melanoma metastasis remains largely unknown.,MiR-144 was downregulated in both uveal melanoma cells and tissues.,Transfection of miR-144 mimic into uveal melanoma cells led to a decrease in cell growth and invasion.,After identification of two putative miR-144 binding sites within the 3' UTR of the human c-Met mRNA, miR-144 was proved to inhibit the luciferase activity inMUM-2B cells with a luciferase reporter construct containing the binding sites.,In addition, the expression of c-Met protein was inhibited by miR-144.,Furthermore, c-Met-mediated cell proliferation and invasion were inhibited by restoration of miR-144 in uveal melanoma cells.,In conclusion, miR-144 acts as a tumor suppressor in uveal melanoma, through inhibiting cell proliferation and migration. miR-144 might serve as a potential therapeutic target in uveal melanoma patients.
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Most melanomas occur on the skin, but a small percentage of these life-threatening cancers affect other parts of the body, such as the eye and mucous membranes, including the mouth.,Given that most melanomas are caused by ultraviolet radiation (UV) exposure, close attention has been paid to the impact of oxidative stress on these tumors.,The possibility that key epigenetic enzymes cannot act on a DNA altered by oxidative stress has opened new perspectives.,Therefore, much attention has been paid to the alteration of DNA methylation by oxidative stress.,We review the current evidence about (i) the role of oxidative stress in melanoma initiation and progression; (ii) the mechanisms by which ROS influence the DNA methylation pattern of transformed melanocytes; (iii) the transformative potential of oxidative stress-induced changes in global and/or local gene methylation and expression; (iv) the employment of this epimutation as a biomarker for melanoma diagnosis, prognosis, and drug resistance evaluation; (v) the impact of this new knowledge in clinical practice for melanoma treatment.
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Melanoma is the most aggressive type of skin cancer and has very high rates of mortality.,An early stage melanoma can be surgically removed, with a survival rate of 99%.,However, metastasized melanoma is difficult to cure.,The 5-year survival rates for patients with metastasized melanoma are still below 20%.,Metastasized melanoma is currently treated by chemotherapy, targeted therapy, immunotherapy and radiotherapy.,The outcome of most of the current therapies is far from optimistic.,Although melanoma patients with a mutation in the oncogene v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) have an initially higher positive response rate to targeted therapy, the majority develop acquired drug resistance after 6 months of the therapy.,To increase treatment efficacy, early diagnosis, more potent pharmacological agents, and more effective delivery systems are urgently needed.,Nanotechnology has been extensively studied for melanoma treatment and diagnosis, to decrease drug resistance, increase therapeutic efficacy, and reduce side effects.,In this review, we summarize the recent progress on the development of various nanoparticles for melanoma treatment and diagnosis.,Several common nanoparticles, including liposome, polymersomes, dendrimers, carbon-based nanoparticles, and human albumin, have been used to deliver chemotherapeutic agents, and small interfering ribonucleic acids (siRNAs) against signaling molecules have also been tested for the treatment of melanoma.,Indeed, several nanoparticle-delivered drugs have been approved by the US Food and Drug Administration and are currently in clinical trials.,The application of nanoparticles could produce side effects, which will need to be reduced so that nanoparticle-delivered drugs can be safely applied in the clinical setting.
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Oncogene addiction describes how cancer cells exhibit dependence on single oncogenes to escape apoptosis and senescence.,While oncogene addiction constitutes the basis for new cancer treatment strategies targeting individual kinases and pathways activated by oncogenic mutations, the biochemical basis for this addiction is largely unknown.,Here we provide evidence for a metabolic rationale behind the addiction to V600EBRAF in two malignant melanoma cell lines.,Both cell lines display a striking addiction to glycolysis due to underlying dysfunction of oxidative phosphorylation (OXPHOS).,Notably, even minor reductions in glycolytic activity lead to increased OXPHOS activity (reversed Warburg effect), however the mitochondria are unable to sustain ATP production.,We show that V600EBRAF upholds the activity of glycolysis and therefore the addiction to glycolysis de facto becomes an addiction to V600EBRAF.,Finally, the senescence response associated with inhibition of V600EBRAF is rescued by overexpression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), providing direct evidence that oncogene addiction rests on a metabolic foundation.
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Antiangiogenic agents that disrupt the vascular endothelial growth factor pathway have been demonstrated to normalize tumor vasculature and improve tumor oxygenation in some studies and to induce hypoxia in others.,The aim of this preclinical study was to investigate the effect of sunitinib treatment on the morphology and function of tumor vasculature and on tumor oxygenation.,A-07-GFP and R-18-GFP human melanoma xenografts grown in dorsal window chambers were used as preclinical tumor models.,Morphologic parameters of tumor vascular networks were assessed from high-resolution transillumination images, and tumor blood supply time was assessed from first-pass imaging movies recorded after a bolus of 155 kDa tetramethylrhodamine isothiocyanate-labeled dextran had been administered intravenously.,Tumor hypoxia was assessed from immunohistochemical preparations of the imaged tissue by use of pimonidazole as a hypoxia marker.,Sunitinib treatment reduced vessel densities, increased vessel segment lengths, did not affect blood supply times, and increased hypoxic area fractions.,Sunitinib treatment did not improve vascular function but induced hypoxia in A-07-GFP and R-18-GFP tumors.
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MicroRNAs-221 and -222 are highly upregulated in several solid tumors, including melanomas.,We demonstrate that the proto-oncogene ETS-1, involved in the pathogenesis of cancers of different origin, is a transcriptional regulator of miR-222 by direct binding to its promoter region.,Differently from 293FT cells or early stage melanomas, where unphosphorylated ETS-1 represses miR-222 transcription, in metastatic melanoma the constitutively Thr-38 phosphorylated fraction of ETS-1 induces miR-222.,Despite its stepwise decreased expression along with melanoma progression, the oncogenic activity of ETS-1 relies on its RAS/RAF/ERK-dependent phosphorylation status more than on its total amount.,To close the loop, we demonstrate ETS-1 as a direct target of miR-222, but not miR-221, showing the novel option of their uncoupled functions.,In addition, a spatial redistribution of ETS-1 protein from the nucleus to the cytoplasm is also evidenced in advanced melanoma cells.,Finally, in vivo studies confirmed the contribution of miR-222 to the increased invasive potential obtained by ETS- silencing.
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The search for melanoma biomarkers is crucial, as the incidence of melanoma continues to rise.,We have previously demonstrated that serum CEACAM1 (sCEACAM1) is secreted from melanoma cells and correlates with disease progression in metastatic melanoma patients.,Here, we have used a different cohort of melanoma patients with regional or metastatic disease (N = 49), treated with autologous vaccination.,By monitoring sCEACAM1 in serum samples obtained prior to and after vaccination, we show that sCEACAM1 correlates with disease state, overall survival, and S100B.,The trend of change in sCEACAM1 following vaccination (increase/decrease) inversely correlates with overall survival.,DTH skin test is used to evaluate patients' anti-melanoma immune response and to predict response to vaccination.,Importantly, sCEACAM1 had a stronger prognostic value than that of DTH, and when sCEACAM1 decreased following treatment, this was the dominant predictor of increased survival.,Collectively, our results point out the relevance of sCEACAM1 in monitoring melanoma patients.
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Our understanding of how checkpoint inhibitors (CPI) affect T cell evolution is incomplete, limiting our ability to achieve full clinical benefit from these drugs.,Here we analyzed peripheral T cell populations after one cycle of CPI and identified a dynamic awakening of the immune system revealed by T cell evolution in response to treatment.,We sequenced T cell receptors (TCR) in plasma cell-free DNA (cfDNA) and peripheral blood mononuclear cells (PBMC) and performed phenotypic analysis of peripheral T cell subsets from metastatic melanoma patients treated with CPI.,We found that early peripheral T cell turnover and TCR repertoire dynamics identified which patients would respond to treatment.,Additionally, the expansion of a subset of immune-effector peripheral T cells we call TIE cells correlated with response.,These events are prognostic and occur within 3 weeks of starting immunotherapy, raising the potential for monitoring patients responses using minimally invasive liquid biopsies.”
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In vitro differentiated CD8+ T cells have been the primary focus of immunotherapy of cancer with little focus on CD4+ T cells.,Immunotherapy involving in vitro differentiated T cells given after lymphodepleting regimens significantly augments antitumor immunity in animals and human patients with cancer.,However, the mechanisms by which lymphopenia augments adoptive cell therapy and the means of properly differentiating T cells in vitro are still emerging.,We demonstrate that naive tumor/self-specific CD4+ T cells naturally differentiated into T helper type 1 cytotoxic T cells in vivo and caused the regression of established tumors and depigmentation in lymphopenic hosts.,Therapy was independent of vaccination, exogenous cytokine support, CD8+, B, natural killer (NK), and NKT cells.,Proper activation of CD4+ T cells in vivo was important for tumor clearance, as naive tumor-specific CD4+ T cells could not completely treat tumor in lymphopenic common gamma chain (γc)-deficient hosts. γc signaling in the tumor-bearing host was important for survival and proper differentiation of adoptively transferred tumor-specific CD4+ T cells.,Thus, these data provide a platform for designing immunotherapies that incorporate tumor/self-reactive CD4+ T cells.
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Uveal melanoma (UM) development and progression is correlated with specific molecular changes.,Recurrent mutations in GNAQ and GNA11 initiate UM development while tumour progression is correlated with monosomy of chromosome 3 and gain of chromosome 8q.,Hence, molecular analysis of UM is useful for diagnosis and prognosis.,The aim of this study is to evaluate the use of digital PCR (dPCR) for molecular analysis of UM.,A series of 66 UM was analysed with dPCR for three hotspot mutations in GNAQ/GNA11 with mutation specific probes.,The status of chromosomes 3 and 8 were analysed with genomic probes.,The results of dPCR analysis were cross-validated with Sanger sequencing, SNP array analysis, and karyotyping.,Using dPCR, we were able to reconstitute the molecular profile of 66 enucleated UM.,With digital PCR, GNAQ/GNA11 mutations were detected in 60 of the 66 UM.,Sanger sequencing revealed three rare variants, and, combined, these assays revealed GNAQ/GNA11 mutations in 95% of UM.,Monosomy 3 was present in 43 and chromosome 8 aberrations in 52 of the 66 UM.,Survival analysis showed that increasing 8q copy numbers were positively correlated with metastasis risk.,Molecular analysis with dPCR is fast and sensitive.,Just like the recurrent genomic aberrations of chromosome 3 and 8, hotspot mutations in GNAQ and GNA11 are effectively detected in heterogeneous samples.,Increased sensitivity contributes to the number of mutations and chromosomal aberrations detected.,Moreover, quantification of copy number with dPCR validated 8q dosage as a sensitive prognostic tool in UM, of which implementation in disease prediction models will further improve prognostication.
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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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Melanoma is the deadliest form of skin cancer and remains a diagnostic challenge in the dermatology clinic.,Several non-invasive imaging techniques have been developed to identify melanoma.,The signal source in each of these modalities is based on the alteration of physical characteristics of the tissue from healthy/benign to melanoma.,However, as these characteristics are not always sufficiently specific, the current imaging techniques are not adequate for use in the clinical setting.,A more robust way of melanoma diagnosis is to “stain” or selectively target the suspect tissue with a melanoma biomarker attached to a contrast enhancer of one imaging modality.,Here, we categorize and review known melanoma diagnostic biomarkers with the goal of guiding skin imaging experts to design an appropriate diagnostic tool for differentiating between melanoma and benign lesions with a high specificity and sensitivity.
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Malignant cancer may contain highly heterogeneous populations of cells, including stem-like cells which were resistant to chemotherapy agents, radiation, mechanical stress, and immune surveillance.,The characterization of these specific subpopulations might be critical to develop novel strategy to remove malignant tumors.,We selected and enriched small population of human melanoma A2058 cells by repetitive selection cycles (selection, restoration, and amplification).,These subpopulation of melanoma cells persisted the characteristics of slower cell proliferation, enhanced drug-resistance, elevated percentage of side population as analyzed by Hoechst33342 exclusion, in vitro sphere formation, and in vivo xenograft tumor formation by small amount of tumor cells.,The selected populations would be melanoma stem-like cells with high expression of stem cell markers and altered kinase activation.,Microarray and bioinformatics analysis highlighted the high expression of angiopoietin-like 4 protein in drug-selected melanoma stem-like cells.,Further validation by specific shRNA demonstrated the role of angiopoietin-like 4 protein in drug-selected subpopulation associated with enhanced drug-resistance, sphere formation, reduced kinase activation, in vitro tube-forming ability correlated with heparan-sulfate proteoglycans.,Our finding would be applicable to explore the mechanism of melanoma stemness and use angiopoietin-like 4 as potential biomarkers to identify melanoma stem-like cells.
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The increasing incidence and mortality associated with advanced stages of melanoma are cause for concern.,Few treatment options are available for advanced melanoma and the 5-year survival rate is less than 15%.,Targeted therapies may revolutionize melanoma treatment by providing less toxic and more effective strategies.,However, maximizing effectiveness requires further understanding of the molecular alterations that drive tumor formation, progression, and maintenance, as well as elucidating the mechanisms of resistance.,Several different genetic alterations identified in human melanoma have been recapitulated in mice.,This review outlines recent progress made in the development of mouse models of melanoma and summarizes what these findings reveal about the human disease.,We begin with a discussion of traditional models and conclude with the recently developed RCAS/TVA somatic cell gene delivery mouse model of melanoma.
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Melanocytic proliferations are common in horses but the diagnosis of malignancy is not always straightforward.,To improve diagnosis and prognosis, markers of malignancy are needed.,Receptor for activated C kinase 1 (RACK1) protein may be such a marker.,RACK1 was originally found to characterize malignant melanocytic lesions in the Melanoblastoma-bearing Libechov minipig (MeLiM) and, later, in human patients.,Our purpose was to investigate the value of RACK1 in the classification of cutaneous melanocytic proliferations in horses.,Using immunofluorescence, we report here that both MITF (Microphthalmia-associated transcription factor) and PAX3 (Paired box 3) allow the identification of melanocytic cells in horse skin samples.,Importantly, RACK1 was detected in melanocytic lesions but not in healthy skin melanocytes.,Finally, we found that RACK1 labeling can be used in horses to distinguish benign melanocytic tumors from melanomas.,Indeed, RACK1 labeling appeared more informative to assess malignancy than individual histomorphological features.,This study confirms that horses provide an interesting model for melanoma genesis studies.,It establishes MITF and PAX3 as markers of horse melanocytic cells.,RACK1 emerges as an important marker of malignancy which may contribute to progress in the diagnosis of melanomas in both human and veterinary medicine.
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Over the past decades, melanoma-related mortality has remained nearly stable.,The main reason is treatment failure of metastatic disease and the inherently linked knowledge gap regarding metastasis formation.,In order to elicit invasion, melanoma cells manipulate the tumor microenvironment, gain motility, and adhere to the extracellular matrix and cancer-associated fibroblasts.,Melanoma cells thereby express different cell adhesion molecules like laminins, integrins, N-cadherin, and others.,Epithelial-mesenchymal transition (EMT) is physiological during embryologic development, but reactivated during malignancy.,Despite not being truly epithelial, neural crest-derived malignancies like melanoma share similar biological programs that enable tumorigenesis, invasion, and metastasis.,This complex phenomenon is termed phenotype switching and is intertwined with oncometabolism as well as dormancy escape.,Additionally, it has been shown that primary melanoma shed exosomes that create a favorable premetastatic niche in the microenvironment of secondary organs and lymph nodes.,Although the growing body of literature describes the aforementioned concepts separately, an integrative holistic approach is missing.,Using melanoma as a tumor model, this review will shed light on these complex biological principles in an attempt to clarify the mechanistic metastatic pathways that dictate tumor and patient fate.
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TP53 has been proved to be associated with cytotoxic T-cell induced apoptosis, however, the association between TP53 and the benefit of immunotherapy in melanoma has not been studied.,In the present study, we examined the relationship between TP53 mutation and response to CTLA-4 blockade in metastatic melanoma by analyzing the data from one public cohort consisting of 110 patients with metastatic melanoma.,The sequencing, mRNA and survival data of 368 patients with skin melanoma from The Cancer Genome Atlas (TCGA) was used to explore the underlying mechanism.,TP53 mutation was associated with significant poorer progression-free survival (HR, 2.25; 95% CI, 1.15-4.37; P = 0.014), poorer overall survival (HR, 2.05; 95% CI, 1.02-4.13; P = 0.040) and trend of poorer response (OR, 0.20; 95% CI, 0.02-1.62; P = 0.131).,The correlations were significant in multivariate analysis including lactate dehydrogenase, tumor mutational burden and tumor stage (P < 0.05).,In TCGA, no association was observed between TP53 mutation and survival (P = 0.55).,The mRNA expression of FAS was lower in patients with TP53 mutation than TP53 wild-type.,Our findings suggest that TP53 mutation is a potential negative predictor of metastatic melanoma treated with CTLA-4 blockade.,•TP53 mutation is associated with poorer outcomes in patients with metastatic melanoma receiving anti-CTLA-4 treatment.,•The mRNA expression of FAS was lower in patients with TP53 mutation than TP53 wild-type.,•TP53 is a potential negative predictor of metastatic melanoma treated with CTLA-4 blockade.,TP53 mutation is associated with poorer outcomes in patients with metastatic melanoma receiving anti-CTLA-4 treatment.,The mRNA expression of FAS was lower in patients with TP53 mutation than TP53 wild-type.,TP53 is a potential negative predictor of metastatic melanoma treated with CTLA-4 blockade.,Immune checkpoint blockades by enhancing the anti-tumor activity of immune system have been standard treatment for several advanced tumors, however, only a subset of patients can benefit from them.,In this study, we find patients with melanoma harboring TP53 mutation show poorer survival outcome and response of anti-CTLA-4 treatment.,The mRNA expression of FAS is lower in patients with TP53 mutation, suggesting that TP53 down-regulates the expression of FAS and impede cytotoxic T-cell induced apoptosis in melanoma.,These results suggest that TP53 mutation may serve as a negative predictor of anti-CTLA-4 treatment in melanoma.
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Data on the KIT mutation rate in melanoma in the central European region is missing.,Accordingly, in a cohort of 79 BRAF/NRAS double wild type cutaneous melanoma and 17 mucosal melanoma KIT mutation was assessed by Sanger sequencing of exons 9,11,13,17 and 18.,In this cutaneous melanoma cohort KIT mutation frequency was found to be 34/79 (43.04%) with a significantly higher rate in acrolentiginous melanoma (ALM) as compared to UV-induced common variants (20/34, 58.8% versus 14/45, 31.1%, p = 0.014).,In the double wild type mucosal melanoma cohort the KIT mutation frequency was found to be comparable (41.2%).,The actual frequency of KIT mutation in the original 227 patient cutaneous melanoma cohort was 34/227, 14.9%.,Exon 11 was the most frequent mutation site (44.7%) followed by exon 9 (21.1%) equally characterizing UV-induced common histotypes and ALM tumors.,In mucosal melanoma exon 9 was the most frequently involved exon followed by exon 13 and 17.,KIT mutation hotspots were identified in exon 9 (c482/491/492), in exon 11 (c559,c572, c570), in exon 13 (c642), in exon 17 (c822) and in exon 18 (c853).,The relatively high KIT mutation rate in cutaneous melanoma in this central-European cohort justifies regular testing of this molecular target in this entity, not only in mucosal variants.
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MicroRNAs (miRNAs) are small non-coding RNAs with regulatory roles, which are involved in a broad spectrum of physiological and pathological processes, including cancer.,A common strategy for identification of miRNAs involved in cell transformation is to compare malignant cells to normal cells.,Here we focus on identification of miRNAs that regulate the aggressive phenotype of melanoma cells.,To avoid differences due to genetic background, a comparative high-throughput miRNA profiling was performed on two isogenic human melanoma cell lines that display major differences in their net proliferation, invasion and tube formation activities.,This screening revealed two major cohorts of differentially expressed miRNAs.,We speculated that miRNAs up-regulated in the more-aggressive cell line contribute oncogenic features, while the down-regulated miRNAs are tumor suppressive.,This assumption was further tested experimentally on five candidate tumor suppressive miRNAs (miR-31, -34a, -184, -185 and -204) and on one candidate oncogenic miRNA (miR-17-5p), all of which have never been reported before in cutaneous melanoma.,Remarkably, all candidate Suppressive-miRNAs inhibited net proliferation, invasion or tube formation, while miR-17-5p enhanced cell proliferation. miR-34a and miR-185 were further shown to inhibit the growth of melanoma xenografts when implanted in SCID-NOD mice.,Finally, all six candidate miRNAs were detected in 15 different metastatic melanoma specimens, attesting for the physiological relevance of our findings.,Collectively, these findings may prove instrumental for understanding mechanisms of disease and for development of novel therapeutic and staging technologies for melanoma.
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The major challenge in antigen‐specific immunotherapy of cancer is to select the most relevant tumor antigens to target.,To this aim, understanding their mode of expression by tumor cells is critical.,We previously identified a melanoma‐specific antigen, melanoma‐overexpressed antigen 1 (MELOE‐1)-coded for by a long noncoding RNA-whose internal ribosomal entry sequence (IRES)‐dependent translation is restricted to tumor cells.,This restricted expression is associated with the presence of a broad‐specific T‐cell repertoire that is involved in tumor immunosurveillance in melanoma patients.,In the present work, we explored the translation control of MELOE‐1 and provide evidence that heterogeneous nuclear ribonucleoprotein A1 (hnRNP‐A1) binds to the MELOE‐1 IRES and acts as an IRES trans‐activating factor (ITAF) to promote the translation of MELOE‐1 in melanoma cells.,In addition, we showed that endoplasmic reticulum (ER) stress induced by thapsigargin, which promotes hnRNP‐A1 cytoplasmic translocation, enhances MELOE‐1 translation and recognition of melanoma cells by a MELOE‐1‐specific T‐cell clone.,These findings suggest that pharmacological stimulation of stress pathways may enhance the efficacy of immunotherapies targeting stress‐induced tumor antigens such as MELOE‐1.,Our study demonstrates that reticular stress in melanoma cells promoted the translocation of the IRES transacting factor hnRPN‐A1 to the cytoplasm.,By binding to the IRES site of meloe mRNA, hnRPN‐A1 promoted the translation of the melanoma‐specific antigen MELOE‐1 and led to the expression of a new immunogenic HLA/peptide complex at the cell surface.,Altogether, our data suggest that pharmacological stimulation of stress pathways may enhance the efficacy of T‐cell based immunotherapies in targeting stress‐induced tumor antigens, such as MELOE‐1 in patients with melanoma.
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Despite major advances in targeted melanoma therapies, drug resistance limits their efficacy.,Long noncoding RNAs (lncRNAs) are transcriptome elements that do not encode proteins but are important regulatory molecules.,LncRNAs have been implicated in cancer development and response to different therapeutics and are thus potential treatment targets; however, the majority of their functions and molecular interactions remain unexplored.,In this study, we identify a novel cytoplasmic intergenic lincRNA (MIRAT), which is upregulated following prolonged MAPK inhibition in NRAS mutant melanoma and modulates MAPK signaling by binding to the MEK scaffold protein IQGAP1.,Collectively, our results present MIRAT’s direct modulatory effect on the MAPK pathway and highlight the relevance of cytoplasmic lncRNAs as potential targets in drug resistant cancer.
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Combining PD-L1 blockade with inhibition of oncogenic mitogen-activated protein kinase (MAPK) signaling may result in long-lasting responses in patients with advanced melanoma.,This phase 1, open-label, dose-escalation and -expansion study (NCT02027961) investigated safety, tolerability and preliminary efficacy of durvalumab (anti-PD-L1) combined with dabrafenib (BRAF inhibitor) and trametinib (MEK inhibitor) for patients with BRAF-mutated melanoma (cohort A, n = 26), or durvalumab and trametinib given concomitantly (cohort B, n = 20) or sequentially (cohort C, n = 22) for patients with BRAF-wild type melanoma.,Adverse events and treatment discontinuation rates were more common than previously reported for these agents given as monotherapy.,Objective responses were observed in 69.2% (cohort A), 20.0% (cohort B) and 31.8% (cohort C) of patients, with evidence of improved tumor immune infiltration and durable responses in a subset of patients with available biopsy samples.,In conclusion, combined MAPK inhibition and anti-PD-L1 therapy may provide treatment options for patients with advanced melanoma.,Immune checkpoints inhibitors or MAPK inhibitors are currently used as standard of care therapies for patients with advanced melanoma.,Here the authors report a phase 1 clinical trial testing the anti-PD-L1 antibody durvalumab in combination with the BRAF inhibitor dafrafenib and the MEK inhibitor trametinib in patients with BRAFV600-mutant melanoma.
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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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Establishing basal cell carcinoma (BCC) subtype is sometimes challenging for pathologists.,Deep-learning (DL) algorithms are an emerging approach in image classification due to their performance, accompanied by a new concept - transfer learning, which implies replacing the final layers of a trained network and retraining it for a new task, while keeping the weights from the imported layers.,A DL convolution-based software, capable of classifying 10 subtypes of BCC, was designed.,Transfer learning from three general-purpose image classification networks (AlexNet, GoogLeNet, and ResNet-18) was used.,Three pathologists independently labeled 2249 patches.,Ninety percent of data was used for training and 10% for testing on 100 independent training sequences.,Each of the resulted networks independently labeled the whole dataset.,Mean and standard deviation (SD) accuracy (ACC) [%]/sensitivity (SN) [%]/specificity (SP) [%]/area under the curve (AUC) for all the networks was 82.53±2.63/72.52±3.63/97.94±0.3/0.99.,The software was validated on another 50-image dataset, and its results are comparable with the result of three pathologists in terms of agreement.,All networks had similar classification accuracies, which demonstrated that they reached a maximum classification rate on the dataset.,The software shows promising results, and with further development can be successfully used on histological images, assisting pathologists’ diagnosis and teaching.
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The treatment of basal cell carcinoma depends on its histological subtype.,Therefore, a biopsy should be performed before definitive treatment.,However, as the biopsy is only a sample of the tumor, it does not always shows every histological subtype present in the neoplasm.,Few studies have compared the histological findings of biopsies with the findings of Mohs micrographic surgery.,By evaluating the totality of the peripheral margins, in addition to sampling large tumor areas, this technique provides a more representative amount of tissue than preoperative biopsy.,a) Determine the agreement between the histological subtype of basal cell carcinoma from punch biopsy and the findings of Mohs surgery; b) To assess, among the discordant cases, the prevalence of non-aggressive tumors in the preoperative biopsy that were reclassified as aggressive by Mohs surgery.,Retrospective analysis of 79 cases of basal cell carcinomas submitted to punch biopsy and subsequent Mohs surgery.,The agreement between the classification of the subtypes in the biopsy and in Mohs surgery was 40.5%.,Punch biopsy was able to predict the most aggressive basal cell carcinoma growth pattern in 83% of cases.,Retrospective nature, sample size, and biopsies performed by different professionals.,The agreement between the histopathological subtypes of basal cell carcinoma as seen in preoperative biopsy and Mohs surgery was low.,However, preoperative biopsy presented good accuracy (83%) in detecting aggressive histopathological subtypes.
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Talimogene Laherparepvec (T-VEC) is an oncolytic virus approved as an intratumoral therapy for treating unresectable stage IIIB-IV metastatic melanoma.,The mechanisms of action for T-VEC and checkpoint inhibitor are highly complementary.,Recent studies have shown that combining checkpoint inhibitor therapy with T-VEC injection can lead to improved response rates for stage IIIB-IV melanoma patients.,We reviewed 10 consecutive cases of stage IIIC to stage IVM1b melanoma patients that received T-VEC plus checkpoint inhibitor(s) therapy (pembrolizumab, ipilimumab/nivolumab, or nivolumab) treated between June 2016 and August 2017 at the Cleveland Clinic with a median follow-up of 7 months (range: 4 to 13 months).,Responses of injected (on-target) and uninjected (off-target) lesions were evaluated according to RECIST 2.0.,The overall response rate for on-target lesions was 90%, with 6 patients experiencing a complete response in injected lesions.,Two patients had off-target lesions, which were completely resolved after treatment.,Blood samples were tested for 3 complete responders and 2 partial responders.,CD4:CD8 ratio and frequencies of circulating PD1+ CD4 and CD8 T cells were elevated in complete responders but not partial responders.,One patient died due to causes unrelated to melanoma and one patient died of progression of the disease.,Our data suggest that combining checkpoint inhibitor(s) with T-VEC injection may provide a synergistic efficacy for patients with unresectable melanoma.,We observed a better overall response rate and complete response rate compared to published studies on similar therapeutic regimens.
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Immunotherapy plays a key role in the treatment of metastatic melanoma.,Patients with autoimmune conditions and/or on immunosuppressive therapy due to orthotropic transplants, however, are systematically excluded from clinical trials.,Talimogene laherparepvec (T-VEC) is the first oncolytic virus to be approved by the FDA for cancer therapy.,To our knowledge, this is the first report of T-VEC being administered in the setting of an organ transplant recipient.,Here we present the case of a patient with recurrent locally advanced cutaneous melanoma receiving salvage T-VEC therapy in the setting of orthotropic heart transplantation.,After 5 cycles of therapy, no evidence of graft rejection has been observed to date, and the patient achieved a complete remission, and is currently off therapy.,This case advocates for further investigation on the safety and efficacy of immunotherapeutic approaches, such as T-VEC, in solid organ transplant recipients.
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The p.E318K variant of the Melanocyte Inducing Transcription Factor (MITF) has been implicated in genetic predisposition to melanoma as an intermediate penetrance allele.,However, the impact of this variant on clinico-phenotypic, as well as on dermoscopic patterns features of affected patients is not entirely defined.,The purpose of our study was to assess the association between the p.E318K germline variant and clinic-phenotypical features of MITF+ compared to non-carriers (MITF−), including dermoscopic findings of melanomas and dysplastic nevi.,we retrospectively analyzed a consecutive series of 1386 patients recruited between 2000 and 2017 who underwent genetic testing for CDKN2A, CDK4, MC1R and MITF germline variants in our laboratory for diagnostic/research purposes.,The patients were probands of melanoma-prone families and apparently sporadic single or multiple primary melanoma patients.,For all, we collected clinical, pathological information and dermoscopic images of the histopathologically diagnosed melanomas and dysplastic nevi, when available.,After excluding patients positive for CDKN2A/CDK4 pathogenic variants and those affected by non-cutaneous melanomas, our study cohort comprised 984 cutaneous melanoma patients, 22 MITF+ and 962 MITF−.,MITF+ were more likely to develop dysplastic nevi and multiple primary melanomas.,Nodular melanoma was more common in MITF+ patients (32% compared to 19% in MITF−).,MITF+ patients showed more frequently dysplastic nevi and melanomas with uncommon dermoscopic patterns (unspecific), as opposed to MITF− patients, whose most prevalent pattern was the multicomponent.,MITF+ patients tend to develop melanomas and dysplastic nevi with histopathological features, frequency and dermoscopic patterns often different from those prevalent in MITF− patients.,Our results emphasize the importance of melanoma prevention programs for MITF+ patients, including dermatologic surveillance with digital follow-up.
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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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Uveal melanoma arises in the eye, and it spreads to distant organs in almost half of patients, leading to a fatal outcome.,To metastasize, uveal melanoma cells must transmigrate into and out of the microvasculature, crossing the monolayer of endothelial cells that separates the vessel lumen from surrounding tissues.,We investigated how human uveal melanoma cells cross the endothelial cell monolayer, using a cultured cell system with primary human endothelial cell monolayers on hydrogel substrates.,We found that uveal melanoma cells transmigrate by a novel and unexpected mechanism.,Uveal melanoma cells intercalate into the endothelial cell monolayer and flatten out, assuming a shape and geometry similar to those of endothelial cells in the monolayer.,After an extended period of time in the intercalated state, the uveal melanoma cells round up and migrate underneath the monolayer.,VCAM is present on endothelial cells, and anti-VCAM antibodies slowed the process of intercalation.,Depletion of BAP1, a known suppressor of metastasis in patients, increased the amount of transmigration of uveal melanoma cells in transwell assays; but BAP1 depletion did not affect the rate of intercalation, based on movies of living cells.,Our results reveal a novel route of transendothelial migration for uveal melanoma cells, and they provide insight into the mechanism by which loss of BAP1 promotes metastasis.
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Pigment epithelium-derived factor (PEDF), a member of the serine protease inhibitor (SERPIN) superfamily, displays a potent antiangiogenic and antimetastatic activity in a broad range of tumor types.,Melanocytes and low aggressive melanoma cells secrete high levels of PEDF, while its expression is lost in highly aggressive melanomas.,PEDF efficiently abrogates a number of functional properties critical for the acquisition of metastatic ability by melanoma cells, such as neovascularization, proliferation, migration, invasiveness and extravasation.,In this study, we identify hypoxia as a relevant negative regulator of PEDF in melanocytes and low aggressive melanoma cells.,PEDF was regulated at the protein level.,Importantly, although downregulation of PEDF was induced by inhibition of 2-oxoglutarate-dependent dioxygenases, it was independent of the hypoxia inducible factor (HIF), a key mediator of the adaptation to hypoxia.,Decreased PEDF protein was not mediated by inhibition of translation through untranslated regions (UTRs) in melanoma cells.,Degradation by metalloproteinases, implicated on PEDF degradation in retinal pigment epithelial cells, or by the proteasome, was also excluded as regulatory mechanism in melanoma cells.,Instead, we found that degradation by autophagy was critical for PEDF downregulation under hypoxia in human melanoma cells.,Our findings show that hypoxic conditions encountered during primary melanoma growth downregulate antiangiogenic and antimetastasic PEDF by a posttranslational mechanism involving degradation by autophagy and could therefore contribute to the acquisition of highly metastatic potential characteristic of aggressive melanoma cells.
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Stromal fibroblast senescence has been linked to aging-associated cancer risk.,However, density and proliferation of cancer-associated fibroblasts (CAF) are frequently increased.,Loss or down-modulation of the Notch effector CSL/RBP-Jκ in dermal fibroblasts is sufficient for CAF activation and ensuing keratinocyte-derived tumors.,We report that CSL silencing induces senescence of primary fibroblasts from dermis, oral mucosa, breast and lung.,CSL functions in these cells as direct repressor of multiple senescence- and CAF-effector genes.,It also physically interacts with p53, repressing its activity.,CSL is down-modulated in stromal fibroblasts of premalignant skin actinic keratosis lesions and squamous cell carcinomas (SCC), while p53 expression and function is down-modulated only in the latter, with paracrine FGF signaling as likely culprit.,Concomitant loss of CSL and p53 overcomes fibroblast senescence, enhances expression of CAF effectors and promotes stromal and cancer cell expansion.,The findings support a CAF activation/stromal co-evolution model under convergent CSL/p53 control.
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Calcineurin inhibitors such as cyclosporin A (CsA) are the mainstay of immunosuppressive treatment for organ transplant recipients.,Squamous cell carcinoma (SCC) of the skin is a major complication of treatment with these drugs, with a 65-100 fold higher risk than in the normal population1.,By contrast, the incidence of basal cell carcinoma (BCC), the other major keratinocyte-derived tumour of the skin, of melanoma and of internal malignancies increases to a significantly lesser extent 1.,Here we report that genetic and pharmacological suppression of calcineurin/NFAT function promotes tumour formation in mouse skin and in xenografts, in immune compromised mice, of H-rasV12 expressing primary human keratinocytes or keratinocyte-derived SCC cells.,Calcineurin/NFAT inhibition counteracts p53-dependent cancer cell senescence thereby increasing tumourigenic potential.,ATF3, a member of the “enlarged” AP-1 family, is selectively induced by calcineurin/NFAT inhibition, both under experimental conditions and in clinically occurring tumours, and increased ATF3 expression accounts for suppression of p53-dependent senescence and enhanced tumourigenic potential.,Thus, intact calcineurin/NFAT signalling is critically required for p53 and senescence-associated mechanisms that protect against skin squamous cancer development.
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PD-L1 and PD-L2 are ligands for the PD-1 immune inhibiting checkpoint that can be induced in tumors by interferon exposure, leading to immune evasion.,This process is important for immunotherapy based on PD-1 blockade.,We examined the specific molecules involved in interferon-induced signaling that regulates PD-L1 and PD-L2 expression in melanoma cells.,These studies revealed that the interferon-gamma-JAK1/JAK2-STAT1/STAT2/STAT3-IRF1 axis primarily regulates PD-L1 expression, with IRF1 binding to its promoter.,PD-L2 responded equally to interferon beta and gamma and is regulated through both IRF1 and STAT3, which bind to the PD-L2 promoter.,Analysis of biopsy specimens from patients with melanoma confirmed interferon signature enrichment and upregulation of gene targets for STAT1/STAT2/STAT3 and IRF1 in anti-PD-1-responding tumors.,Therefore, these studies map the signaling pathway of interferon-gamma-inducible PD-1 ligand expression.,Garcia-Diaz et al. performed a small hairpin RNA screen and genetic and functional studies to map the signaling pathways that result in reactive PD-L1 and PD-L2 on melanoma cells upon interferon gamma exposure.,The authors highlight the importance of the JAK1/JAK2-STAT1/STAT2/STAT3-IRF1 axis for clinical responses to PD-1 blockade therapy.
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To inform the benefit-risk assessment of nivolumab in patients with advanced melanoma, analyses of efficacy and safety exposure-response (E-R) relationships were conducted with data from patients with advanced melanoma enrolled in two clinical studies (phase I and phase III) who received nivolumab 0.1-10.0 mg/kg every 2 weeks.,E‐R efficacy analyses were performed by relating the nivolumab time‐averaged concentration after the first dose (Cavg1) to two endpoints: RECIST objective response (OR) and overall survival (OS).,E-R safety analyses characterized the relationship between nivolumab Cavg1 and the hazard of all‐causality adverse events leading to discontinuation or death (AE‐DC/D).,Nivolumab exposure represented by Cavg1 was not a significant predictor of OR, OS, or the hazard of AE‐DC/D.,E-R efficacy and safety relationships were relatively flat over the exposure range.
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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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BRAF inhibitors can extend progression‐free and overall survival in melanoma patients whose tumors harbor mutations in BRAF.,However, the majority of patients eventually develop resistance to these drugs.,Here we show that BRAF mutant melanoma cells that have developed acquired resistance to BRAF inhibitors display increased oxidative metabolism and increased dependency on mitochondria for survival.,Intriguingly, the increased oxidative metabolism is associated with a switch from glucose to glutamine metabolism and an increased dependence on glutamine over glucose for proliferation.,We show that the resistant cells are more sensitive to mitochondrial poisons and to inhibitors of glutaminolysis, suggesting that targeting specific metabolic pathways may offer exciting therapeutic opportunities to treat resistant tumors, or to delay emergence of resistance in the first‐line setting.,We examine the metabolic requirements in melanoma cell lines resistant to BRAF inhibitor PLX4720.,Cells that are resistant to BRAF inhibitors are more dependent on glutamine than glucose as their carbon source.Combined glutaminolysis and BRAF inhibition was effective to promote a delay in the onset of resistance.,We examine the metabolic requirements in melanoma cell lines resistant to BRAF inhibitor PLX4720.,Cells that are resistant to BRAF inhibitors are more dependent on glutamine than glucose as their carbon source.,Combined glutaminolysis and BRAF inhibition was effective to promote a delay in the onset of resistance.
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The pocket protein (PP) family consists of the three members RB1, p107 and p130 all possessing tumor suppressive properties.,Indeed, the PPs jointly control the G1/S transition mainly by inhibiting E2F transcription factors.,Notably, several viral oncoproteins are capable of binding and inhibiting PPs.,Merkel cell polyomavirus (MCPyV) is considered as etiological factor for Merkel cell carcinoma (MCC) with expression of the viral Large T antigen (LT) harboring an intact PP binding domain being required for proliferation of most MCC cells.,Therefore, we analyzed the interaction of MCPyV-LT with the PPs.,Co-IP experiments indicate that MCPyV-LT binds potently only to RB1.,Moreover, MCPyV-LT knockdown-induced growth arrest in MCC cells can be rescued by knockdown of RB1, but not by p107 or p130 knockdown.,Accordingly, cell cycle arrest and E2F target gene repression mediated by the single PPs can only in the case of RB1 be significantly reverted by MCPyV-LT expression.,Moreover, data from an MCC patient indicate that loss of RB1 rendered the MCPyV-positive MCC cells LT independent.,Thus, our results suggest that RB1 is the dominant tumor suppressor PP in MCC, and that inactivation of RB1 by MCPyV-LT is largely sufficient for its growth supporting function in established MCPyV-positive MCC cells.
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Merkel Cell Polyomavirus (MCPyV) is associated with Merkel Cell carcinoma (MCC), a rare, aggressive skin cancer with neuroendocrine features.,The causal role of MCPyV is highly suggested by monoclonal integration of its genome and expression of the viral large T (LT) antigen in MCC cells.,We investigated and characterized MCPyV molecular features in MCC, respiratory, urine and blood samples from 33 patients by quantitative PCR, sequencing and detection of integrated viral DNA.,We examined associations between either MCPyV viral load in primary MCC or MCPyV DNAemia and survival.,Results were interpreted with respect to the viral molecular signature in each compartment.,Patients with MCC containing more than 1 viral genome copy per cell had a longer period in complete remission than patients with less than 1 copy per cell (34 vs 10 months, P = 0.037).,Peripheral blood mononuclear cells (PBMC) contained MCPyV more frequently in patients sampled with disease than in patients in complete remission (60% vs 11%, P = 0.00083).,Moreover, the detection of MCPyV in at least one PBMC sample during follow-up was associated with a shorter overall survival (P = 0.003).,Sequencing of viral DNA from MCC and non MCC samples characterized common single nucleotide polymorphisms defining 8 patient specific strains.,However, specific molecular signatures truncating MCPyV LT were observed in 8/12 MCC cases but not in respiratory and urinary samples from 15 patients.,New integration sites were identified in 4 MCC cases.,Finally, mutated-integrated forms of MCPyV were detected in PBMC of two patients with disseminated MCC disease, indicating circulation of metastatic cells.,We conclude that MCPyV molecular features in primary MCC tumour and PBMC may help to predict the course of the disease.
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RNA modifications, including RNA methylation, are widely existed in cutaneous melanoma (CM).,Among epigenetic modifications, N7-methylguanosine (m7G) is a kind of modification at 5’ cap of RNA which participate in maintaining the stability of mRNA and various cell biological processes.,However, there is still no study concerning the relationship between CM and m7G methylation complexes, METTL1 and WDR4.,Here, long non-coding RNA (lncRNAs) and gene expression data of CM from the Cancer Genome Atlas (TCGA) database were retrieved to identify differentially expressed m7G-related lncRNAs connected with overall survival of CM.,Then, Cox regression analyses was applied to construct a lncRNA risk signature, the prognostic value of identified signature was further evaluated.,As a result, 6 m7G-associated lncRNAs that were significantly related to CM prognosis were incorporated into our prognostic signature.,The functional analyses indicated that the prognostic model was correlated with patient survival, cancer metastasis, and growth.,Meanwhile, its diagnostic accuracy was better than conventional clinicopathological characteristics.,The pathway enrichment analysis showed that the risk model was enriched in several immunity-associated pathways.,Moreover, the signature model was significantly connected with the immune subtypes, infiltration of immune cells, immune microenvironment, as well as several m6A-related genes and tumor stem cells.,Finally, a nomogram based on the calculated risk score was established.,Overall, a risk signature based on 6 m7G-associated lncRNAs was generated which presented predictive value for the prognosis of CM patients and can be further used in the development of novel therapeutic strategies for CM.
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Despite major advances in targeted melanoma therapies, drug resistance limits their efficacy.,Long noncoding RNAs (lncRNAs) are transcriptome elements that do not encode proteins but are important regulatory molecules.,LncRNAs have been implicated in cancer development and response to different therapeutics and are thus potential treatment targets; however, the majority of their functions and molecular interactions remain unexplored.,In this study, we identify a novel cytoplasmic intergenic lincRNA (MIRAT), which is upregulated following prolonged MAPK inhibition in NRAS mutant melanoma and modulates MAPK signaling by binding to the MEK scaffold protein IQGAP1.,Collectively, our results present MIRAT’s direct modulatory effect on the MAPK pathway and highlight the relevance of cytoplasmic lncRNAs as potential targets in drug resistant cancer.
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Acral lentiginous melanoma is associated with worse survival than other subtypes of melanoma.,Understanding prognostic factors for survival and recurrence can help better inform follow-up care.,To analyze the clinicopathologic features, melanoma-specific survival, and recurrence-free survival by substage in a large, multi-institutional cohort of primary acral lentiginous melanoma patients.,Retrospective review of the United States Melanoma Consortium database, a multi-center prospectively collected database of acral lentiginous melanoma patients treated between January 2000 and December 2017.,Of the 433 primary acral lentiginous melanoma patients identified (median [range] age: 66 [8-97] years; 53% female, 83% white), 66% presented with stage 0-2 disease and the median time of follow-up for the 392 patients included in the survival analysis was 32.5 months (range: 0-259).,The 5-year melanoma-specific survivals by stage were 0 = 100%, I = 93.8%, II = 76.2%, III = 63.4%, IIIA = 80.8%, and IV = 0%.,Thicker Breslow depth ((HR) = 1.13; 95% CI = 1.05-1.21; P < .001)) and positive nodal status ((HR) = 1.79; 95% CI = 1.00-3.22; P = .050)) were independent prognostic factors for melanoma-specific survival.,Breslow depth ((HR = 1.13; 95% CI = 1.07-1.20; P < .001), and positive nodal status (HR = 2.12; 95% CI = 1.38-3.80; P = .001) were also prognostic factors for recurrence-free survival.,In this cohort of patients, acral lentiginous melanoma was associated with poor outcomes even in early stage disease, consistent with prior reports.,Stage IIB and IIC disease were associated with particularly low melanoma-specific and recurrence-free survival.,This suggests that studies investigating adjuvant therapies in stage II patients may be especially valuable in acral lentiginous melanoma patients.
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Metastasized or unresectable melanoma has been the first malignant tumor to be successfully treated with checkpoint inhibitors.,Nevertheless, about 40-50% of the patients do not respond to these treatments and severe side effects are observed in up to 60%.,Therefore, there is a high need to identify reliable biomarkers predicting response.,Tumor Mutation Burden (TMB) is a debated predictor for response to checkpoint inhibitors and early measurement of ctDNA can help to detect treatment failure to immunotherapy in selected melanoma patients.,However, it has not yet been clarified how TMB and ctDNA can be used to estimate response to combined CTLA-4 and PD-1 antibody therapy in metastatic melanoma.,In this prospective biomarker study, we included 35 melanoma patients with ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) therapy.,In all patients, a tumor panel of 710 tumor-associated genes was applied (tumor vs. reference tissue comparison), followed by repetitive liquid biopsies.,Cell-free DNA was extracted and at least one driver mutation was monitored.,Treatment response was evaluated after about three months of therapy.,TMB was significantly higher in responders than in nonresponders and TMB > 23.1 Mut/Mb (TMB-high) was associated with a survival benefit compared to TMB ≤ 23.1 Mut/Mb (TMB-low or TMB-intermediate).,Furthermore, a > 50% decrease of cell-free DNA concentration or undetectable circulating tumor DNA (ctDNA), measured by tumor-specific variant copies/ml of plasma at first follow-up three weeks after treatment initiation were significantly associated with response to combined immunotherapy and improved overall survival, respectively.,It is noticeable that no patient with TMB ≤ 23.1 Mut/Mb and detectable or increasing ctDNA at first follow-up responded to immunotherapy.,High TMB, > 50% decrease of cell-free DNA concentration, and undetectable ctDNA at first follow-up seem to be associated with response and overall survival under combined immunotherapy.,The evaluation of ctDNA and cell-free DNA three weeks after treatment initiation may be suitable for early assessment of efficacy of immunotherapy.,The online version of this article (10.1186/s40425-019-0659-0) contains supplementary material, which is available to authorized users.
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A novel pathway of vitamin D3 (D3) metabolism, initiated by C20-hydroxylation of D3 by CYP11A1, has been confirmed to operate in vivo.,Its major product, 20(OH)D3, exhibits antiproliferative activity in vitro comparable to that of 1,25(OH)2D3, but is noncalcemic in mice and rats.,To further characterize the antimelanoma activity of 20(OH)D3, we tested its effect on colony formation of human melanoma cells in monolayer culture and anchorage-independent growth in soft agar.,The migratory capabilities of the cells and cell-cell and cell-extracellular matrix interactions were also evaluated using transwell cell migration and spheroid toxicity assays.,To assess the antimelanoma activity of 20(OH)D3 in vivo, age-matched immunocompromised mice were subcutaneously implanted with luciferase-labelled SKMel-188 cells and were randomly assigned to be treated with either 20(OH)D3 or vehicle (n=10 per group).,Tumor size was measured with caliper and live bioimaging methods, and overall health condition expressed as a total body score scale.,The following results were observed: (i) 20(OH)D3 inhibited colony formation both in monolayer and soft agar conditions, (ii) 20(OH)D3 inhibited melanoma cells in both transwell migration and spheroid toxicity assays, and (iii) 20(OH)D3 inhibited melanoma tumor growth in immunocompromised mice without visible signs of toxicity.,However, although the survival rate was 90% in both groups, the total body score was higher in the treatment group compared to control group (2.8 vs.,2.55).,In conclusion, 20(OH)D3, an endogenously produced secosteroid, is an excellent candidate for further preclinical testing as an antimelanoma agent.
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The retinoic acid-related orphan receptors (RORs) regulate several physiological and pathological processes, including immune functions, development and cancer.,To study the potential role of RORs in melanoma progression, we analysed RORα and RORγ expression in nevi and primary melanomas and non-lesional skin and metastases in relation to melanoma clinico-pathomorphological features.,The expression of RORα and RORγ was lower in melanomas than in nevi and decreased during melanoma progression, with lowest levels found in primary melanomas at stages III and IV and in melanoma metastases.,Their expression correlated with pathomorphological pTNM parameters being low in aggressive tumors and being high in tumors showing histological markers of good prognosis.,Higher nuclear levels of RORα and RORγ and of cytoplasmic RORγ correlated with significantly longer overall and disease free survival time.,Highly pigmented melanomas showed significantly lower level of nuclear RORs.,This study shows that human melanoma development and aggressiveness is associated with decreased expression of RORα and RORγ, suggesting that RORs could be important in melanoma progression and host responses against the tumor.,Furthermore, it suggests that RORα and RORγ might constitute a novel druggable target in anti-melanoma management using tumor suppressor gene therapy restoring their normal functions.
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RAC1 P29 is the third most commonly mutated codon in human cutaneous melanoma, after BRAF V600 and NRAS Q61.,Here, we study the role of RAC1P29S in melanoma development and reveal that RAC1P29S activates PAK, AKT, and a gene expression program initiated by the SRF/MRTF transcriptional pathway, which results in a melanocytic to mesenchymal phenotypic switch.,Mice with ubiquitous expression of RAC1P29S from the endogenous locus develop lymphoma.,When expressed only in melanocytes, RAC1P29S cooperates with oncogenic BRAF or with NF1-loss to promote tumorigenesis.,RAC1P29S also drives resistance to BRAF inhibitors, which is reversed by SRF/MRTF inhibitors.,These findings establish RAC1P29S as a promoter of melanoma initiation and mediator of therapy resistance, while identifying SRF/MRTF as a potential therapeutic target.,•RAC1P29S activates PAK, AKT, and the SRF/MRTF transcription program in melanocytes•RAC1P29S induces a melanocytic to mesenchymal transition through SRF/MRTF and PAK•RAC1P29S cooperates with BRAF mutation or NF1 deletion to promote melanomagenesis•RAC1P29S induces resistance to BRAF inhibitors through SRF/MRTF,RAC1P29S activates PAK, AKT, and the SRF/MRTF transcription program in melanocytes,RAC1P29S induces a melanocytic to mesenchymal transition through SRF/MRTF and PAK,RAC1P29S cooperates with BRAF mutation or NF1 deletion to promote melanomagenesis,RAC1P29S induces resistance to BRAF inhibitors through SRF/MRTF,RAC1P29S is a common mutation in human cutaneous melanoma.,Lionarons et al. show that RAC1P29S induces a melanocytic to mesenchymal switch via an SRF/MRTF-mediated gene expression program, cooperates with BRAF in melanomagenesis, and drives BRAF inhibitor resistance, which is reversed by SRF/MRTF inhibition.
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Cancer immunotherapy restores and/or enhances effector function of CD8+ T cells in the tumor microenvironment1,2.,CD8+ T cells activated by cancer immunotherapy execute tumor clearance mainly by inducing cell death through perforin-granzyme- and Fas/Fas ligand-pathways3,4.,Ferroptosis is a form of cell death that differs from apoptosis and results from iron-dependent lipid peroxide accumulation5,6.,Although it was mechanistically illuminated in vitro7,8, emerging evidence has shown that ferroptosis may be implicated in a variety of pathological scenarios9,10.,However, the involvement of ferroptosis in T cell immunity and cancer immunotherapy is unknown.,Here, we find that immunotherapy-activated CD8+ T cells enhance ferroptosis-specific lipid peroxidation in tumor cells, and in turn, increased ferroptosis contributes to the anti-tumor efficacy of immunotherapy.,Mechanistically, interferon gamma (IFNγ) released from CD8+ T cells downregulates expression of SLC3A2 and SLC7A11, two subunits of glutamate-cystine antiporter system xc-, restrains tumor cell cystine uptake, and as a consequence, promotes tumor cell lipid peroxidation and ferroptosis.,In preclinical models, depletion of cyst(e)ine by cyst(e)inase in combination with checkpoint blockade synergistically enhances T cell-mediated anti-tumor immunity and induces tumor cell ferroptosis.,Expression of system xc- is negatively associated with CD8+ T cell signature, IFNγ expression, and cancer patient outcome.,Transcriptome analyses before and during nivolumab therapy reveal that clinical benefits correlate with reduced expression of SLC3A2 and increased IFNγ and CD8.,Thus, T cell-promoted tumor ferroptosis is a novel anti-tumor mechanism.,Targeting tumor ferroptosis pathway constitutes a therapeutic approach in combination with checkpoint blockade.
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Mitogen activated-protein kinase pathway inhibitors (MAPKis) improve treatment outcome in patients with disseminated BRAFV600 mutant cutaneous malignant melanoma (CMM) but responses are of limited duration due to emerging resistance.,Although extensive research in mechanisms of resistance is being performed, predictive biomarkers for durable responses are still lacking.,We used miRNA qPCR to investigate if different levels of extracellular microvesicle microRNA (EV miRNA) in matched plasma samples collected from patients with metastatic IV BRAFV600 mutated CMM before, during and after therapy with MAPKis could serve as predictive biomarkers.,EV miRNAs were extracted from plasma samples from 28 patients collected before and during therapy, measured by quantitative PCR-array and correlated to therapy outcome.,Increased levels of EV let-7g-5p during treatment compared to before treatment (EV let-7g-5p_delta) were associated with better disease control with MAPKis (odds ratio 8568.4, 95% CI = 4.8-1.5e+07, P = 0.000036).,Elevated levels of EV miR-497-5p during therapy were associated with prolonged progression free survival (PFS) (hazard ratio = 0.27, 95% CI = 0.13-0.52, P <0.000061).,EV miRNAs let-7g-5p and miR-497-5p were identified as putative novel predictive biomarkers of MAPKi treatment benefit in metastatic CMM patients highlighting the potential relevance of assessing EV miRNA during and after treatment to unravel novel mechanisms of resistance.
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Specifically targeted drug delivery systems with low immunogenicity and toxicity are deemed to increase efficacy of cancer chemotherapy.,Acridine Orange (AO) is an acidophilic dye with a strong tumoricidal action following excitation with a light source at 466 nm.,However, to date the clinical use of AO is limited by the potential side effects elicited by systemic administration.,The endogenous nanocarrier exosomes have been recently introduced as a natural delivery system for therapeutic molecules.,In this article, we show the outcome of the administration to human melanoma cells of AO charged Exosomes (Exo-AO), in both monolayer and spheroid models.,The results showed an extended drug delivery time of Exo-AO to melanoma cells as compared to the free AO, improving the cytotoxicity of AO.,This study shows that Exo-AO have a great potential for a real exploitation as a new theranostic approach against tumors based on AO delivered through the exosomes.
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Large-scale genomic analyses of cutaneous melanoma have revealed insights into the aetiology and heterogeneity of this disease, as well as opportunities to further personalise treatment for patients with targeted and immune therapies.,Herein, we review the proposed genomic classification of cutaneous melanoma from large-scale next-generation sequencing studies, including the largest integrative analysis of melanoma from The Cancer Genome Atlas (TCGA) Network.,We examine studies that have identified molecular features of melanomas linked to immune checkpoint inhibitor response.,In addition, we draw attention to low-frequency actionable mutations and highlight frequent non-coding mutations in melanoma where little is known about their biological function that may provide novel avenues for the development of treatment strategies for melanoma patients.
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Melanoma is highly resistant to current modalities of therapy, with the extent of pigmentation playing an important role in therapeutic resistance.,Nuclear factor-κB (NF-κB) is constitutively activated in melanoma and can serve as a molecular target for cancer therapy and steroid/secosteroid action.,Cultured melanoma cells were used for mechanistic studies on NF-κB activity, utilising immunofluorescence, western blotting, EMSA, ELISA, gene reporter, and estimated DNA synthesis assays.,Formalin-fixed, paraffin-embedded specimens from melanoma patients were used for immunocytochemical analysis of NF-κB activity in situ.,Novel 20-hydroxyvitamin (20(OH)D3) and classical 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) secosteroids inhibited melanoma cell proliferation.,Active forms of vitamin D were found to inhibit NF-κB activity in nonpigmented cells, while having no effect on pigmented cells.,Treatment of nonpigmented cells with vitamin D3 derivatives inhibited NF-κB DNA binding and NF-κB-dependent reporter assays, as well as inhibited the nuclear translocation of the p65 NF-κB subunit and its accumulation in the cytoplasm.,Moreover, analysis of biopsies of melanoma patients showed that nonpigmented and slightly pigmented melanomas displayed higher nuclear NF-κB p65 expression than highly pigmented melanomas.,Classical 1,25(OH)2D3 and novel 20(OH)D3 hydroxyderivatives of vitamin D3 can target NF-κB and regulate melanoma progression in nonpigmented melanoma cells.,Melanin pigmentation is associated with the resistance of melanomas to 20(OH)D3 and 1,25(OH)2D3 treatment.
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Melanoma is an aggressive malignancy of melanocytes and most commonly arises in the skin.,In 2002, BRAF gene mutations were identified in melanoma, and this finding resulted in the development of several small-molecule molecular inhibitors that specifically targeted the BRAF V600E mutation.,The development of targeted therapies for advanced-stage melanoma, including tyrosine kinase inhibitors (TKIs) of the BRAF (V600E) kinase, vemurafenib and dabrafenib, have been approved for the treatment of advanced melanoma leading to improved clinical outcomes.,However, the development of BRAF inhibitor (BRAFi) resistance has significantly reduced the therapeutic efficacy after prolonged treatment.,Recent studies have identified the molecular mechanisms for BRAFi resistance.,This review aims to describe the impact of BRAFi resistance on the pathogenesis of melanoma, the current status of molecular pathways involved in BRAFi resistance, including intrinsic resistance, adaptive resistance, and acquired resistance.,This review will discuss how an understanding of the mechanisms associated with BRAFi resistance may aid the identification of useful strategies for overcoming the resistance to BRAF-targeted therapy in patients with advanced-stage melanoma.
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Knowledge of tumor mutation status is becoming increasingly important for the treatment of cancer, as mutation-specific inhibitors are being developed for clinical use that target only sub-populations of patients with particular tumor genotypes.,Melanoma provides a recent example of this paradigm.,We report here development, validation, and implementation of an assay designed to simultaneously detect 43 common somatic point mutations in 6 genes (BRAF, NRAS, KIT, GNAQ, GNA11, and CTNNB1) potentially relevant to existing and emerging targeted therapies specifically in melanoma.,The test utilizes the SNaPshot method (multiplex PCR, multiplex primer extension, and capillary electrophoresis) and can be performed rapidly with high sensitivity (requiring 5-10% mutant allele frequency) and minimal amounts of DNA (10-20 nanograms).,The assay was validated using cell lines, fresh-frozen tissue, and formalin-fixed paraffin embedded tissue.,Clinical characteristics and the impact on clinical trial enrollment were then assessed for the first 150 melanoma patients whose tumors were genotyped in the Vanderbilt molecular diagnostics lab.,Directing this test to a single disease, 90 of 150 (60%) melanomas from sites throughout the body harbored a mutation tested, including 57, 23, 6, 3, and 2 mutations in BRAF, NRAS, GNAQ, KIT, and CTNNB1, respectively.,Among BRAF V600 mutations, 79%, 12%, 5%, and 4% were V600E, V600K, V600R, and V600M, respectively. 23 of 54 (43%) patients with mutation harboring metastatic disease were subsequently enrolled in genotype-driven trials.,We present development of a simple mutational profiling screen for clinically relevant mutations in melanoma.,Adoption of this genetically-informed approach to the treatment of melanoma has already had an impact on clinical trial enrollment and prioritization of therapy for patients with the disease.
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Vemurafenib/PLX4032, a selective inhibitor of mutant BRAFV600E, constitutes a paradigm shift in melanoma therapy.,Unfortunately, acquired resistance, which unavoidably occurs, represents one major limitation to clinical responses.,Recent studies have highlighted that vemurafenib activated oxidative metabolism in BRAFV600E melanomas expressing PGC1α.,However, the oxidative state of melanoma resistant to BRAF inhibitors is unknown.,We established representative in vitro and in vivo models of human melanoma resistant to vemurafenib including primary specimens derived from melanoma patients.,Firstly, our study reveals that vemurafenib increased mitochondrial respiration and ROS production in BRAFV600E melanoma cell lines regardless the expression of PGC1α.,Secondly, melanoma cells that have acquired resistance to vemurafenib displayed intrinsically high rates of mitochondrial respiration associated with elevated mitochondrial oxidative stress irrespective of the presence of vemurafenib.,Thirdly, the elevated ROS level rendered vemurafenib-resistant melanoma cells prone to cell death induced by pro-oxidants including the clinical trial drug, elesclomol.,Based on these observations, we propose that the mitochondrial oxidative signature of resistant melanoma constitutes a novel opportunity to overcome resistance to BRAF inhibition.
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Recent insights into the genetic and somatic aberrations have initiated a new era of rapidly evolving targeted and immune-based treatments for melanoma.,After decades of unsuccessful attempts to finding a more effective cure in the treatment of melanoma now we have several drugs active in melanoma.,The possibility to use these drugs in combination to improve responses to overcome the resistance, to potentiate the action of immune system with the new immunomodulating antibodies, and identification of biomarkers that can predict the response to a particular therapy represent new concepts and approaches in the clinical management of melanoma.,The third “Melanoma Research: “A bridge from Naples to the World” meeting, shortened as “Bridge Melanoma Meeting” took place in Naples, December 2 to 4th, 2012.,The four topics of discussion at this meeting were: advances in molecular profiling and novel biomarkers, combination therapies, novel concepts toward integrating biomarkers and therapies into contemporary clinical management of patients with melanoma across the entire spectrum of disease stage, and the knowledge gained from the biology of tumor microenvironment across different tumors as a bridge to impact on prognosis and response to therapy in melanoma.,This international congress gathered more than 30 international faculty members who in an interactive atmosphere which stimulated discussion and exchange of their experience regarding the most recent advances in research and clinical management of melanoma patients.
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To assess the incidence and to review experience with the treatment of mucosal melanoma of the head and neck (MMHN) in Slovenia between 1985 and 2013.,The National Cancer Registry database and clinical records with outcome data of identified patients treated during the period 1985-2013 in Slovenia were reviewed.,In a 29-year period, 61 patients with MMHN were identified, representing 0.5 % of all head and neck malignant tumors and 42 % of all mucosal melanomas in Slovenia. 72 % originated in the sinonasal tract and were predominantly (78 %) diagnosed as a local disease.,Regional metastases at diagnosis were more frequent in patients with oral/oropharyngeal primary (44 %; sinonasal MMHN 11 %, p = 0.006).,Curative intent treatment was given to 48 (79 %) patients.,The overall survival (OS) rates at 2 and 5 years for the whole cohort were 43 % and 18 %, respectively, and for the curative intent group 53 % and 24 %, respectively.,In the latter group, multivariate analyses showed postoperative radiotherapy (PORT) to be predictive for locoregional control (LRC) (hazard ratios [HR] for surgery with PORT vs. surgery alone: 1.0 vs.,3.9, p = 0.037), whereas only the World Health Organization performance status (HR for grade 0 vs. grade 1 vs. grade >1: 1.0 (p = 0.022) vs.,1.2 (p = 0.640) vs.,7.7 (p = 0.008)) significantly influenced OS.,MMHN is a rare tumor with a poor prognosis.,Combination of surgery and PORT offers the best prospects for LRC but without improvement of OS.,Due to potential toxicity of high-dose RT such treatment is indicated in patients in whom LRC outweighs the risks of serious adverse effects.
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Carbon ion radiotherapy (C-ion RT) is an advanced modality for treating malignant melanoma.,After we treated our first case of gynecological melanoma using C-ion RT in November 2004, we decided to conduct a clinical trial to evaluate its usefulness for the treatment of gynecological melanoma.,The eligibility criteria for enrollment in this study were histologically proven malignant melanoma of the gynecological regions with lymph node metastasis remaining in the inguinal and pelvic regions.,The small pelvic space, including the GTV and the metastatic lymph node, was irradiated with up to a total dose of 36 GyE followed by a GTV boost of up to a total dose of 57.6 GyE or 64 GyE in 16 fractions.,A series of 23 patients were treated between November 2004 and October 2012.,Patient age ranged from 51-80 with a median of 71.,Of the tumor sites, 14 were located in the vagina, 6 in the vulva, and 3 in the cervix uteri.,Of the 23 patients, 22 were irradiated with up to a total dose of 57.6 GyE, and 1 patient was irradiated with up to a total dose of 64 GyE.,Chemotherapy and interferon-β were also used to treat 11 of the patients.,Acute and late toxicities of Grade 3 or higher were observed in 1 patient treated with concurrent interferon-β.,The median follow-up time was 17 months (range, 6-53 months).,There was recurrence in 14 patients, and the 3-year local control and overall survival rates were 49.9% and 53.0%, respectively.,C-ion RT may become a non-invasive treatment option for gynecological melanoma.
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Melanoma is the deadliest form of skin cancer, and little is known about the impact of deregulated expression of long noncoding RNAs (lncRNAs) in the progression of this cancer.,In this study, we explored RNA-Seq data to search for lncRNAs associated with melanoma progression.,We found distinct lncRNA gene expression patterns across melanocytes, primary and metastatic melanoma cells.,Also, we observed upregulation of the lncRNA ZEB1-AS1 (ZEB1 antisense RNA 1) in melanoma cell lines.,Data analysis from The Cancer Genome Atlas (TCGA) confirmed higher ZEB1-AS1 expression in metastatic melanoma and its association with hotspot mutations in BRAF (B-Raf proto-oncogene, serine/threonine kinase) gene and RAS family genes.,In addition, a positive correlation between ZEB1-AS1 and ZEB1 (zinc finger E-box binding homeobox 1) gene expression was verified in primary and metastatic melanomas.,Using gene expression signatures indicative of invasive or proliferative phenotypes, we found an association between ZEB1-AS1 upregulation and a transcriptional profile for invasiveness.,Enrichment analysis of correlated genes demonstrated cancer genes and pathways associated with ZEB1-AS1.,We suggest that the lncRNA ZEB1-AS1 could function by activating ZEB1 gene expression, thereby influencing invasiveness and phenotype switching in melanoma, an epithelial-to-mesenchymal transition (EMT)-like process, which the ZEB1 gene has an essential role.
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The incidence of cutaneous melanoma (CM) has increased in the past few decades.,The biology of melanoma is characterized by a complex interaction between genetic, environmental and phenotypic factors.,A greater understanding of the molecular mechanisms that promote melanoma cell growth and dissemination is crucial to improve diagnosis, prognostication, and treatment of CM.,Both small and long non‐coding RNAs (lncRNAs) have been identified to play a role in melanoma biology; microRNA and lncRNA expression is altered in transformed melanocytes and this in turn has functional effects on cell proliferation, apoptosis, invasion, metastasis, and immune response.,Moreover, specific dysregulated ncRNAs were shown to have a diagnostic or prognostic role in melanoma and to drive the establishment of drug resistance.,Here, we review the current literature on small and lncRNAs with a role in melanoma, with the aim of putting into some order this complex jigsaw puzzle.
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To establish the maximum tolerated dose (MTD), dose‐limiting toxicities (DLT), safety profile, and anti‐tumor efficacy of RAF265.,We conducted a multicenter, open‐label, phase‐I, dose‐escalation trial of RAF265, an orally available RAF kinase/VEGFR‐2 inhibitor, in patients with advanced or metastatic melanoma.,Pharmacokinetic (PK) analysis, pharmacodynamics (PD) and tumor response assessment were conducted.,We evaluated metabolic tumor response by 18[F]‐fluorodeoxyglucose‐positron‐emission tomography (FDG‐PET), tissue biomarkers using immunohistochemistry (IHC), and modulators of angiogenesis.,RAF265 has a serum half‐life of approximately 200 h.,The MTD was 48 mg once daily given continuously.,Among 77 patients, most common treatment‐related adverse effects were fatigue (52%), diarrhea (34%), weight loss (31%) and vitreous floaters (27%).,Eight of 66 evaluable patients (12.1%) had an objective response, including seven partial and one complete response.,Responses occurred in BRAF‐mutant and BRAF wild‐type (WT) patients.,Twelve of 58 (20.7%) evaluable patients had a partial metabolic response.,On‐treatment versus pretreatment IHC staining in 23 patients showed dose‐dependent p‐ERK inhibition.,We observed a significant temporal increase in placental growth factor levels and decrease in soluble vascular endothelial growth factor receptor 2 (sVEGFR‐2) levels in all dose levels.,RAF265 is an oral RAF/VEGFR‐2 inhibitor that produced antitumor responses, metabolic responses, and modulated angiogenic growth factor levels.,Antitumor activity occurred in patients with BRAF‐mutant and BRAF‐WT disease.,Despite low activity at tolerable doses, this study provides a framework for the development of pan‐RAF inhibitors and modulators of angiogenesis for the treatment of 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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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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There is currently no curative treatment for melanoma once the disease spreads beyond the original site.,Although activation of the PI3K/Akt pathway resulting from genetic mutations and epigenetic deregulation of its major regulators is known to cause resistance of melanoma to therapeutic agents, including the conventional chemotherapeutic drug dacarbazine and the Food and Drug Administration-approved mutant BRAF inhibitors vemurafenib and dabrafenib, the role of extracellular stimuli of the pathway, such as insulin, in drug resistance of melanoma remains less understood.,To investigate the effect of insulin on the response of melanoma cells to dacarbazine, and in particular, the effect of insulin on the response of melanoma cells carrying the BRAFV600E mutation to mutant BRAF inhibitors.,An additional aim was to define the role of the PI3K/Akt pathway in the insulin-triggered drug resistance.,The effect of insulin on cytotoxicity induced by dacarbazine or the mutant BRAF inhibitor PLX4720 was tested by pre-incubation of melanoma cells with insulin.,Cytotoxicity was determined by the MTS assay.,The role of the PI3K/Akt pathway in the insulin-triggered drug resistance was examined using the PI3K inhibitor LY294002 and the PI3K and mammalian target of rapamycin dual inhibitor BEZ-235.,Activation of the PI3K/Akt pathway was monitored by Western blot analysis of phosphorylated levels of Akt.,Recombinant insulin attenuated dacarbazine-induced cytotoxicity in both wild-type BRAF and BRAFV600E melanoma cells, whereas it also reduced killing of BRAFV600E melanoma cells by PLX4720.,Nevertheless, the protective effect of insulin was abolished by the PI3K and mTOR dual inhibitor BEZ-235 or the PI3K inhibitor LY294002.,Insulin attenuates the therapeutic efficacy of dacarbazine and PLX4720 in melanoma cells, which is mediated by activation of the PI3K/Akt pathway and can be overcome by PI3K inhibitors.
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Next generation sequencing of uveal melanoma (UM) samples has identified a number of recurrent oncogenic or loss-of-function mutations in key driver genes including: GNAQ, GNA11, EIF1AX, SF3B1 and BAP1.,To search for additional driver mutations in this tumor type we carried out whole-genome or whole-exome sequencing of 28 tumors or primary cell lines.,These samples have a low mutation burden, with a mean of 10.6 protein changing mutations per sample (range 0 to 53).,As expected for these sun-shielded melanomas the mutation spectrum was not consistent with an ultraviolet radiation signature, instead, a BRCA mutation signature predominated.,In addition to mutations in the known UM driver genes, we found a recurrent mutation in PLCB4 (c.G1888T, p.D630Y, NM_000933), which was validated using Sanger sequencing.,The identical mutation was also found in published UM sequence data (1 of 56 tumors), supporting its role as a novel driver mutation in UM.,PLCB4 p.D630Y mutations are mutually exclusive with mutations in GNA11 and GNAQ, consistent with PLCB4 being the canonical downstream target of the former gene products.,Taken together these data suggest that the PLCB4 hotspot mutation is similarly a gain-of-function mutation leading to activation of the same signaling pathway, promoting UM tumorigenesis.
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Uveal melanoma is the most common primary cancer of the eye and often results in fatal metastasis.,Here, we describe mutations occurring exclusively at arginine-625 in splicing factor 3B subunit 1 (SF3B1) in low-grade uveal melanomas with good prognosis.,Thus, uveal melanoma is among a small group of cancers associated with SF3B1 mutation, and these mutations denote a distinct molecular subset of uveal melanomas.
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The aim of this study was to compare the expressions of mRNA for metalloproteinases (MMP-2 and MMP-9) and type IV collagen in two different histological types of basal-cell carcinoma (BCCs; nodular and infiltrative) and in normal tissues from the tumor interface.,The study included biopsy specimens of the skin involved with BCC and normal skin adjacent the lesion.,The expressions of mRNA for MMP-2, MMP-9 and type IV collagen were determined by means of RT-PCR (Reverse transcription polymerase chain reaction).,The level of type IV collagen mRNA in nodular and infiltrative BCCs turned out to be significantly lower, and the expressions of MMP-2 and MMP-9 mRNA significantly higher than in normal tissues adjacent to these tumors.,The expression of mRNA for MMP-9 but not for MMP-2 was significantly higher in infiltrative BCCs than in the nodular BCCs.,In turn, normal tissues adjacent to nodular BCCs showed significantly higher levels of mRNA for MMP-2 and significantly lower levels of type IV collagen mRNA than the normal tissues from the interface of infiltrative BCCs.,The findings suggest that MMP-2 and MMP-9 could be used as prognostic factors of BCCs.
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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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The steadily increasing incidence of malignant melanoma (MM) and its aggressive behaviour makes this tumour an attractive cancer research topic.,The tumour microenvironment is being increasingly recognised as a key factor in cancer biology, with an impact on proliferation, invasion, angiogenesis and metastatic spread, as well as acquired therapy resistance.,Multiple bioactive molecules playing cooperative roles promote the chronic inflammatory milieu in tumours, making inflammation a hallmark of cancer.,This specific inflammatory setting is evident in the affected tissue.,However, certain mediators can leak into the systemic circulation and affect the whole organism.,The present study analysed the complex inflammatory response in the sera of patients with MM of various stages.,Multiplexed proteomic analysis (Luminex Corporation) of 31 serum proteins was employed.,These targets were observed in immunohistochemical profiles of primary tumours from the same patients.,Furthermore, these proteins were analysed in MM cell lines and the principal cell population of the melanoma microenvironment, cancer-associated fibroblasts.,Growth factors such as hepatocyte growth factor, granulocyte-colony stimulating factor and vascular endothelial growth factor, chemokines RANTES and interleukin (IL)-8, and cytokines IL-6, interferon-α and IL-1 receptor antagonist significantly differed in these patients compared with the healthy controls.,Taken together, the results presented here depict the inflammatory landscape that is altered in melanoma patients, and highlight potentially relevant targets for therapy improvement.
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Blocking CCR8 inhibits entry of metastases from the collecting lymphatic vessel into the lymph node.,Lymphatic vessels are thought to contribute to metastasis primarily by serving as a transportation system.,It is widely believed that tumor cells enter lymph nodes passively by the flow of lymph.,We demonstrate that lymph node lymphatic sinuses control tumor cell entry into the lymph node, which requires active tumor cell migration.,In human and mouse tissues, CCL1 protein is detected in lymph node lymphatic sinuses but not in the peripheral lymphatics.,CCR8, the receptor for CCL1, is strongly expressed by human malignant melanoma.,Tumor cell migration to lymphatic endothelial cells (LECs) in vitro is inhibited by blocking CCR8 or CCL1, and recombinant CCL1 promotes migration of CCR8+ tumor cells.,The proinflammatory mediators TNF, IL-1β, and LPS increase CCL1 production by LECs and tumor cell migration to LECs.,In a mouse model, blocking CCR8 with the soluble antagonist or knockdown with shRNA significantly decreased lymph node metastasis.,Notably, inhibition of CCR8 led to the arrest of tumor cells in the collecting lymphatic vessels at the junction with the lymph node subcapsular sinus.,These data identify a novel function for CCL1-CCR8 in metastasis and lymph node LECs as a critical checkpoint for the entry of metastases into the lymph nodes.
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Local acidification of stroma is proposed to favour pre-metastatic niche formation but the mechanism of initiation is unclear.,We investigated whether Human Melanoma-derived exosomes (HMEX) could reprogram human adult dermal fibroblasts (HADF) and cause extracellular acidification.,HMEX were isolated from supernatants of six melanoma cell lines (3 BRAF V600E mutant cell lines and 3 BRAF wild-type cell lines) using ultracentrifugation or Size Exclusion Chromatography (SEC).,Rapid uptake of exosomes by HADF was demonstrated following 18 hours co-incubation.,Exposure of HDAF to HMEX leads to an increase in aerobic glycolysis and decrease in oxidative phosphorylation (OXPHOS) in HADF, consequently increasing extracellular acidification.,Using a novel immuno-biochip, exosomal miR-155 and miR-210 were detected in HMEX.,These miRNAs were present in HMEX from all six melanoma cell lines and were instrumental in promoting glycolysis and inhibiting OXPHOS in tumour cells.,Inhibition of miR-155 and miR-210 activity by transfection of miRNA inhibitors into HMEX reversed the exosome-induced metabolic reprogramming of HADF.,The data indicate that melanoma-derived exosomes modulate stromal cell metabolism and may contribute to the creation of a pre-metastatic niche that promotes the development of metastasis.
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Growing evidence is showing that metastatic cell populations are able to transfer their characteristics to less malignant cells.,Exosomes (EXOs) are membrane vesicles of endocytic origin able to convey their cargo of mRNAs, microRNAs (miRs), proteins and lipids from donors to proximal as well as distant acceptor cells.,Our previous results indicated that miR-221&222 are key factors for melanoma development and dissemination.,The aim of this study was to verify whether the tumorigenic properties associated with miR-222 overexpression can be also propagated by miR-222-containing EXOs.,EXOs were isolated by UltraCentrifugation or Exoquick-TC® methods.,Preparations of melanoma-derived vesicles were characterized by using the Nanosight™ technology and the expression of exosome markers analyzed by western blot.,The expression levels of endogenous and exosomal miRNAs were examined by real time PCR.,Confocal microscopy was used to evaluate transfer and uptake of microvesicles from donor to recipient cells.,The functional significance of exosomal miR-222 was estimated by analyzing the vessel-like process formation, as well as cell cycle rates, invasive and chemotactic capabilities.,Besides microvesicle marker characterization, we evidenced that miR-222 exosomal expression mostly reflected its abundance in the cells of origin, correctly paralleled by repression of its target genes, such as p27Kip1, and induction of the PI3K/AKT pathway, thus confirming its functional implication in cancer.,The possible differential significance of PI3K/AKT blockade was assessed by using the BKM120 inhibitor in miR-222-transduced cell lines.,In addition, in vitro cultures showed that vesicles released by miR-222-overexpressing cells were able to transfer miR-222-dependent malignancy when taken-up by recipient primary melanomas.,Results were confirmed by antagomiR-221&222 treatments and by functional observations after internalization of EXOs devoid of these miRs.,All together these data, besides generally confirming the role of miR-222 in melanoma tumorigenesis, supported its responsibility in the exosome-associated melanoma properties, thus further indicating this miR as potential diagnostic and prognostic biomarker and its abrogation as a future therapeutic option.,The online version of this article (doi:10.1186/s12967-016-0811-2) contains supplementary material, which is available to authorized users.
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Immunotherapy has revolutionised the treatment of advanced cutaneous squamous cell carcinoma (cSCC).,It is important to understand both safety and efficacy in a real-world and trial-ineligible cSCC population.,We aimed to evaluate safety, efficacy and molecular insights among a broader cSCC population, including immunosuppressed patients, treated with immune checkpoint inhibitors (CPI).,We present a cohort of advanced cSCC patients (n = 61) treated from 2015 to 2020 evaluating the best overall response (BOR) (RECISTv1.1) to CPI therapy, immune-related adverse events (irAEs) and tumour mutational burden (TMB) to correlate with outcomes.,A validated geriatric scoring index (CIRS-G) was utilised to assess comorbidities among patients ≥75.,These data were compared with published clinical trial results among the broader cSCC population.,BOR to CPI was lower among the entire cohort when compared with trial data (31.5 vs.,48%, P < 0.01), with higher rates of progression (59 vs.,16.5%, P < 0.01), regardless of immunosuppression history or age.,Grade 3+ irAEs were more common among responders (P = 0.02), while pre-treatment lymphocyte count and TMB predicted response (P = 0.02).,We demonstrate comparatively lower response rates to CPI among real-world cSCC patients not explained by older age or immunosuppression history alone.,Immune-related toxicity, absolute lymphocyte count and TMB predicted CPI response.
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Prior studies of conventional chemotherapy or epidermal growth factor receptor inhibitors for advanced (ie, locally advanced cutaneous squamous cell carcinoma [laCSCC] or metastatic [mCSCC]) cutaneous squamous cell cancer enrolled ≤ 40 patients.,This retrospective, observational study assessed real‐world treatment patterns and clinical outcomes in patients with unresectable laCSCC or mCSCC using electronic health records of patients who initiated first‐line (1L) systemic treatment from 1 January 2008 to 31 December 2015, with follow‐up to 30 September 2017.,The median duration of follow‐up from 1L treatment was 10.1 months (range 0.03‐67.6 months).,Duration of therapy (DOT) and overall survival (OS) were assessed using Kaplan‐Meier analysis.,Response rate was calculated as the proportion of patients who achieved physician‐assessed‐response.,Eighty‐two patients were identified (17 laCSCC and 65 mCSCC).,Median age at 1L treatment initiation was 75 years; 85% were male, 88% had an Eastern Cooperative Oncology Group performance status of 1, and 84% had received radiotherapy.,The most common 1L regimens were carboplatin + paclitaxel (27%) and cetuximab monotherapy (24%).,The median 1L DOT was 4.1 months for laCSCC and 2.3 months for mCSCC.,The physician‐assessed response rate for 1L therapy was 17.6% for laCSCC, and 18.5% for mCSCC.,The median OS from 1L treatment initiation was 16.2 months for laCSCC, and 15.3 months for mCSCC.,Only 24 patients (29%) received second‐line therapy.,This is the largest retrospective data set regarding patients with advanced CSCC treated with anticancer systemic therapy prior to approval of the anti‐programmed cell death‐1 antibody, cemiplimab.,Efficacy was low in both laCSCC and mCSCC.,These data provide historic benchmarks for outcomes in patients with advanced CSCC prior to Food and Drug Administration approval of cemiplimab‐rwlc.,This retrospective study describes the limited efficacy of systemic therapy in a real‐world population of patients with locally advanced and metastatic cutaneous squamous cell carcinoma.,The data provide historic benchmarks for outcomes of treatment for these patients prior to the approval of cemiplimab.
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Microenvironment cues involved in melanoma progression are largely unknown.,Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host.,A role of exosomes in driving melanoma progression under microenvironmental acidity was never described.,We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0-6.7.,To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis.,Functional roles of exosomes were tested in migration and invasion tests.,Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression.,We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive.,We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes.,In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis.,A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases.,A crucial step of melanoma progression does occur at melanoma intermediate -stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes.,Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement.,The online version of this article (10.1186/s13046-018-0915-z) contains supplementary material, which is available to authorized users.
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Despite the general focus on an invasive and de-differentiated phenotype as main driver of cancer metastasis, in melanoma patients many metastatic lesions display a high degree of pigmentation, indicative for a differentiated phenotype.,Indeed, studies in mice and fish show that melanoma cells switch to a differentiated phenotype at secondary sites, possibly because in melanoma differentiation is closely linked to proliferation through the lineage-specific transcriptional master regulator MITF.,Importantly, while a lot of effort has gone into identifying factors that induce the de-differentiated/invasive phenotype, it is not well understood how the switch to the differentiated/proliferative phenotype is controlled.,We identify collagen as a contributor to this switch.,We demonstrate that collagen stiffness induces melanoma differentiation through a YAP/PAX3/MITF axis and show that in melanoma patients increased collagen abundance correlates with nuclear YAP localization.,However, the interrogation of large patient datasets revealed that in the context of the tumour microenvironment, YAP function is more complex.,In the absence of fibroblasts, YAP/PAX3-mediated transcription prevails, but in the presence of fibroblasts tumour growth factor-β suppresses YAP/PAX3-mediated MITF expression and induces YAP/TEAD/SMAD-driven transcription and a de-differentiated phenotype.,Intriguingly, while high collagen expression is correlated with poorer patient survival, the worst prognosis is seen in patients with high collagen expression, who also express MITF target genes such as the differentiation markers TRPM1, TYR and TYRP1, as well as CDK4.,In summary, we reveal a distinct lineage-specific route of YAP signalling that contributes to the regulation of melanoma pigmentation and uncovers a set of potential biomarkers predictive for poor survival.
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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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Aberrations of protein-coding genes are a focus of cancer genomics; however, the impact of oncogenes on expression of the ∼50% of transcripts without protein-coding potential, including long noncoding RNAs (lncRNAs), has been largely uncharacterized.,Activating mutations in the BRAF oncogene are present in >70% of melanomas, 90% of which produce active mutant BRAFV600E protein.,To define the impacts of oncogenic BRAF on the melanocyte transcriptome, massively parallel cDNA sequencing (RNA-seq) was performed on genetically matched normal human melanocytes with and without BRAFV600E expression.,To enhance potential disease relevance by verifying expression of altered genes in BRAF-driven cancer tissue, parallel RNA-seq was also undertaken of two BRAFV600E-mutant human melanomas.,BRAFV600E regulated expression of 1027 protein-coding transcripts and 39 annotated lncRNAs, as well as 70 unannotated, potentially novel, intergenic transcripts.,These transcripts display both tissue-specific and multi-tissue expression profiles and harbor distinctive regulatory chromatin marks and transcription factor binding sites indicative of active transcription.,Coding potential analysis of the 70 unannotated transcripts suggested that most may represent newly identified lncRNAs.,BRAF-regulated lncRNA 1 (BANCR) was identified as a recurrently overexpressed, previously unannotated 693-bp transcript on chromosome 9 with a potential functional role in melanoma cell migration.,BANCR knockdown reduced melanoma cell migration, and this could be rescued by the chemokine CXCL11.,Combining RNA-seq of oncogene-expressing normal cells with RNA-seq of their corresponding human cancers may represent a useful approach to discover new oncogene-regulated RNA transcripts of potential clinical relevance in cancer.
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The development of targeted inhibitors, like vemurafenib, has greatly improved the clinical outcome of BRAFV600E metastatic melanoma.,However, resistance to such compounds represents a formidable problem.,Using whole-exome sequencing and functional analyses, we have investigated the nature and pleiotropy of vemurafenib resistance in a melanoma patient carrying multiple drug-resistant metastases.,Resistance was caused by a plethora of mechanisms, all of which reactivated the MAPK pathway.,In addition to three independent amplifications and an aberrant form of BRAFV600E, we identified a new activating insertion in MEK1.,This MEK1T55delinsRT mutation could be traced back to a fraction of the pre-treatment lesion and not only provided protection against vemurafenib but also promoted local invasion of transplanted melanomas.,Analysis of patient-derived xenografts (PDX) from therapy-refractory metastases revealed that multiple resistance mechanisms were present within one metastasis.,This heterogeneity, both inter- and intra-tumorally, caused an incomplete capture in the PDX of the resistance mechanisms observed in the patient.,In conclusion, vemurafenib resistance in a single patient can be established through distinct events, which may be preexisting.,Furthermore, our results indicate that PDX may not harbor the full genetic heterogeneity seen in the patient’s melanoma.
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Blocking oncogenic signaling induced by the BRAFV600E mutation is a promising approach for melanoma treatment.,We tested the anti-tumor effects of a specific inhibitor of Raf protein kinases, PLX4032/RG7204, in melanoma cell lines.,PLX4032 decreased signaling through the MAPK pathway only in cell lines with the BRAFV600E mutation.,Seven out of 10 BRAFV600E mutant cell lines displayed sensitivity based on cell viability assays and three were resistant at concentrations up to 10 μM.,Among the sensitive cell lines, four were highly sensitive with IC50 values below 1 μM, and three were moderately sensitive with IC50 values between 1 and 10 μM.,There was evidence of MAPK pathway inhibition and cell cycle arrest in both sensitive and resistant cell lines.,Genomic analysis by sequencing, genotyping of close to 400 oncogeninc mutations by mass spectrometry, and SNP arrays demonstrated no major differences in BRAF locus amplification or in other oncogenic events between sensitive and resistant cell lines.,However, metabolic tracer uptake studies demonstrated that sensitive cell lines had a more profound inhibition of FDG uptake upon exposure to PLX4032 than resistant cell lines.,In conclusion, BRAFV600E mutant melanoma cell lines displayed a range of sensitivities to PLX4032 and metabolic imaging using PET probes can be used to assess sensitivity.
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Up to 60% of patients with metastatic melanoma develop melanoma brain metastasis (MBM) during the course of their disease.,Surgery, radiosurgery (SRS), stereotactic radiotherapy (SRT), and whole‐brain radiation therapy (WBRT) or combinations of these are commonly used local treatment modalities.,Inhibitory monoclonal antibodies against the CTLA‐4 and PD‐1 immune checkpoint receptors significantly improved the survival of metastatic melanoma patients, including patients with MBM.,This prolonged survival, and potentially also the immunostimulatory mechanisms, may expose patients to a higher risk for long‐term complications such as focal postradiation necrosis of the brain (RNB).,We analyzed the incidence of pseudotumoral RNB in a single institution cohort of 142 melanoma patients that were prospectively followed after starting treatment with pembrolizumab in an expanded access program.,Of the 142 patients, 43 (30.7%) patients had MBM at initiation pembrolizumab.,Of these, 31 (72.1%) were treated with SRS, 8 (18.6%) with WBRT while 4 (9.3%) had no prior local therapy.,Of patients treated with RT, 28 (71.1%) received RT before the initiation of pembrolizumab.,5 (12.8%) patients developed a new symptomatic pseudotumoral lesion at a median time of 11.15 months (range 8‐46) after the RT.,In all patients, the diagnosis of RNB was radiologically confirmed.,The RNB was treated with corticosteroids in two patients, bevacizumab in two patients, and surgery in three symptomatic patients.,The diagnosis was histologically confirmed in the patients treated with surgery.,Melanoma patients with MBM treated with radiosurgery and showing a beneficial response to pembrolizumab are at risk for late RNB.,In case of suspected isolated progression at the site of a previously irradiated MBM, the diagnosis of RNB should be considered.
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Combination concepts of radiotherapy and immune checkpoint inhibition are currently of high interest.,We examined imaging findings, acute toxicity, and local control in patients with melanoma brain metastases receiving programmed death 1 (PD-1) inhibitors and/or robotic stereotactic radiosurgery (SRS).,Twenty-six patients treated with SRS alone (n = 13; 20 lesions) or in combination with anti-PD-1 therapy (n = 13; 28 lesions) were analyzed.,Lesion size was evaluated three and six months after SRS using a volumetric assessment based on cranial magnetic resonance imaging (cMRI) and acute toxicity after 12 weeks according to the Common Terminology Criteria for Adverse Events (CTCAE).,Local control after six months was comparable (86%, SRS + anti-PD-1, and 80%, SRS).,All toxicities reported were less than or equal to grade 2.,One metastasis (5%) in the SRS group and six (21%) in the SRS + anti-PD-1 group increased after three months, whereas four (14%) of the six regressed during further follow-ups.,This was rated as pseudoprogression (PsP).,Three patients (23%) in the SRS + anti-PD-1 group showed characteristics of PsP.,Treatment with SRS and anti-PD-1 antibodies can be combined safely in melanoma patients with cerebral metastases.,Early volumetric progression of lesions under simultaneous treatment may be related to PsP; thus, the evaluation of combined radioimmunotherapy remains challenging and requires experienced teams.
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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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Brain metastases are common in patients with melanoma, and optimal management is not well defined.,As melanoma has traditionally been thought of as “radioresistant,” the role of whole brain radiation therapy (WBRT) in particular is unclear.,We conducted this retrospective study to identify prognostic factors for patients treated with stereotactic radiosurgery (SRS) for melanoma brain metastases and to investigate the role of additional up-front treatment with whole brain radiation therapy (WBRT).,We reviewed records of 147 patients who received SRS as part of initial management of their melanoma brain metastases from January 2000 through June 2010.,Overall survival (OS) and time to distant intracranial progression were calculated using the Kaplan-Meier method.,Prognostic factors were evaluated using the Cox proportional hazards model.,WBRT was employed with SRS in 27% of patients and as salvage in an additional 22%.,Age at SRS > 60 years (hazard ratio [HR] 0.64, p = 0.05), multiple brain metastases (HR 1.90, p = 0.008), and omission of up-front WBRT (HR 2.24, p = 0.005) were associated with distant intracranial progression on multivariate analysis.,Extensive extracranial metastases (HR 1.86, p = 0.0006), Karnofsky Performance Status (KPS) ≤ 80% (HR 1.58, p = 0.01), and multiple brain metastases (HR 1.40, p = 0.06) were associated with worse OS on univariate analysis.,Extensive extracranial metastases (HR 1.78, p = 0.001) and KPS (HR 1.52, p = 0.02) remained significantly associated with OS on multivariate analysis.,In patients with absent or stable extracranial disease, multiple brain metastases were associated with worse OS (multivariate HR 5.89, p = 0.004), and there was a trend toward an association with worse OS when up-front WBRT was omitted (multivariate HR 2.56, p = 0.08).,Multiple brain metastases and omission of up-front WBRT (particularly in combination) are associated with distant intracranial progression.,Improvement in intracranial disease control may be especially important in the subset of patients with absent or stable extracranial disease, where the competing risk of death from extracranial disease is low.,These results are hypothesis generating and require confirmation from ongoing randomized trials.
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Identifying predictive biomarkers of therapeutic response for melanoma patients treated with immune checkpoint inhibitors is a major challenge.,By combining microfluidic enrichment for melanoma circulating tumor cells (CTCs) together with RNA-based droplet digital PCR quantitation, we have established a highly sensitive and robust platform for noninvasive, blood-based monitoring of tumor burden.,Serial monitoring of melanoma patients treated with immune checkpoint inhibitors shows rapid changes in CTC score, which precede standard clinical assessment and are highly predictive of long-term clinical outcome.,Early on-treatment digital monitoring of CTC dynamics may thus help identify patients likely to benefit from immune checkpoint inhibition therapy.,A subset of patients with metastatic melanoma have sustained remissions following treatment with immune checkpoint inhibitors.,However, analyses of pretreatment tumor biopsies for markers predictive of response, including PD-1 ligand (PD-L1) expression and mutational burden, are insufficiently precise to guide treatment selection, and clinical radiographic evidence of response on therapy may be delayed, leading to some patients receiving potentially ineffective but toxic therapy.,Here, we developed a molecular signature of melanoma circulating tumor cells (CTCs) to quantify early tumor response using blood-based monitoring.,A quantitative 19-gene digital RNA signature (CTC score) applied to microfluidically enriched CTCs robustly distinguishes melanoma cells, within a background of blood cells in reconstituted and in patient-derived (n = 42) blood specimens.,In a prospective cohort of 49 patients treated with immune checkpoint inhibitors, a decrease in CTC score within 7 weeks of therapy correlates with marked improvement in progression-free survival [hazard ratio (HR), 0.17; P = 0.008] and overall survival (HR, 0.12; P = 0.04).,Thus, digital quantitation of melanoma CTC-derived transcripts enables serial noninvasive monitoring of tumor burden, supporting the rational application of immune checkpoint inhibition therapies.
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Repeat tumor biopsies to study genomic changes during therapy are difficult, invasive and data are confounded by tumoral heterogeneity.,The analysis of circulating tumor DNA (ctDNA) can provide a non-invasive approach to assess prognosis and the genetic evolution of tumors in response to therapy.,Mutation-specific droplet digital PCR was used to measure plasma concentrations of oncogenic BRAF and NRAS variants in 48 patients with advanced metastatic melanoma prior to treatment with targeted therapies (vemurafenib, dabrafenib or dabrafenib/trametinib combination) or immunotherapies (ipilimumab, nivolumab or pembrolizumab).,Baseline ctDNA levels were evaluated relative to treatment response and progression-free survival (PFS).,Tumor-associated ctDNA was detected in the plasma of 35/48 (73%) patients prior to treatment and lower ctDNA levels at this time point were significantly associated with response to treatment and prolonged PFS, irrespective of therapy type.,Levels of ctDNA decreased significantly in patients treated with MAPK inhibitors (p < 0.001) in accordance with response to therapy, but this was not apparent in patients receiving immunotherapies.,We show that circulating NRAS mutations, known to confer resistance to BRAF inhibitors, were detected in 3 of 7 (43%) patients progressing on kinase inhibitor therapy.,Significantly, ctDNA rebound and circulating mutant NRAS preceded radiological detection of progressive disease.,Our data demonstrate that ctDNA is a useful biomarker of response to kinase inhibitor therapy and can be used to monitor tumor evolution and detect the early appearance of resistance effectors.
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Basal cell carcinomas are the most frequent skin cancers in the fair-skinned adult population over 50 years of age.,Their incidence is increasing throughout the world.,Ultraviolet (UV) exposure is the major carcinogenic factor.,Some genodermatosis can predispose to formation of basal cell carcinomas at an earlier age.,Basal cell carcinomas are heterogeneous, from superficial or nodular lesions of good prognosis to very extensive difficult-to-treat lesions that must be discussed in multidisciplinary committees.,Recent guidelines have updated the management of basal cell carcinoma.,The prognosis is linked to the risk of recurrence of basal cell carcinoma or its local destructive capacity.,Characteristic molecular events in these tumours are: (i) activation of the hedgehog pathway, which has allowed the development of hedgehog inhibitors for difficult-to-treat lesions that are not accessible to surgery or radiotherapy; (ii) high mutational burden, which suggests that hedgehog inhibitor refractory tumours could be offered immunotherapy; some trials are ongoing.,The standard treatment for most basal cell carcinomas is surgery, as it allows excision margin control and shows a low risk of recurrence.,Superficial lesions can be treated by non-surgical methods with significant efficacy.
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Microvascular networks of human basal cell carcinomas (BCC) and surrounding skin were assessed with optical coherence angiography (OCA) in conjunction with photodynamic therapy (PDT).,OCA images were collected and analyzed in 31 lesions pre-treatment, and immediately/24 hours/3-12 months post-treatment.,Pre-treatment OCA enabled differentiation between prevalent subtypes of BCC (nodular and superficial) and nodular-with-necrotic-core BCC subtypes with a diagnostic accuracy of 78%; this can facilitate more accurate biopsy reducing sampling error and better therapy regimen selection.,Post-treatment OCA images at 24 hours were 98% predictive of eventual outcome.,Additional findings highlight the importance of pre-treatment necrotic core, vascular metrics associated with hypertrophic scar formation, and early microvascular changes necessary in both tumorous and peri-tumorous regions to ensure treatment success.
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Vemurafenib/PLX4032, a selective inhibitor of mutant BRAFV600E, constitutes a paradigm shift in melanoma therapy.,Unfortunately, acquired resistance, which unavoidably occurs, represents one major limitation to clinical responses.,Recent studies have highlighted that vemurafenib activated oxidative metabolism in BRAFV600E melanomas expressing PGC1α.,However, the oxidative state of melanoma resistant to BRAF inhibitors is unknown.,We established representative in vitro and in vivo models of human melanoma resistant to vemurafenib including primary specimens derived from melanoma patients.,Firstly, our study reveals that vemurafenib increased mitochondrial respiration and ROS production in BRAFV600E melanoma cell lines regardless the expression of PGC1α.,Secondly, melanoma cells that have acquired resistance to vemurafenib displayed intrinsically high rates of mitochondrial respiration associated with elevated mitochondrial oxidative stress irrespective of the presence of vemurafenib.,Thirdly, the elevated ROS level rendered vemurafenib-resistant melanoma cells prone to cell death induced by pro-oxidants including the clinical trial drug, elesclomol.,Based on these observations, we propose that the mitochondrial oxidative signature of resistant melanoma constitutes a novel opportunity to overcome resistance to BRAF inhibition.
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Oncogenic mutations in the serine/threonine kinase B-RAF are found in 50-70% of malignant melanomas1.,Pre-clinical studies have demonstrated that the B-RAFV600E mutation predicts a dependency on the mitogen activated protein kinase (MAPK) signaling cascade in melanoma1-5-an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials6-8.,However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance9-11.,Identification of resistance mechanisms in a manner that elucidates alternative ‘druggable’ targets may inform effective long-term treatment strategies12.,Here, we expressed ~600 kinase and kinase-related open reading frames (ORFs) in parallel to functionally interrogate resistance to a selective RAF kinase inhibitor.,We identified MAP3K8 (COT/TPL2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAFV600E cell lines.,COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signaling.,Moreover, COT expression is associated with de novo resistance in B-RAFV600E cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibition.,We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting.,Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.
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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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To reveal the clonal architecture of melanoma and associated driver mutations, whole genome sequencing (WGS) and targeted extension sequencing were used to characterize 124 melanoma cases.,Significantly mutated gene analysis using 13 WGS cases and 15 additional paired extension cases identified known melanoma genes such as BRAF, NRAS, and CDKN2A, as well as a novel gene EPHA3, previously implicated in other cancer types.,Extension studies using tumors from another 96 patients discovered a large number of truncation mutations in tumor suppressors (TP53 and RB1), protein phosphatases (e.g., PTEN, PTPRB, PTPRD, and PTPRT), as well as chromatin remodeling genes (e.g., ASXL3, MLL2, and ARID2).,Deep sequencing of mutations revealed subclones in the majority of metastatic tumors from 13 WGS cases.,Validated mutations from 12 out of 13 WGS patients exhibited a predominant UV signature characterized by a high frequency of C->T transitions occurring at the 3′ base of dipyrimidine sequences while one patient (MEL9) with a hypermutator phenotype lacked this signature.,Strikingly, a subclonal mutation signature analysis revealed that the founding clone in MEL9 exhibited UV signature but the secondary clone did not, suggesting different mutational mechanisms for two clonal populations from the same tumor.,Further analysis of four metastases from different geographic locations in 2 melanoma cases revealed phylogenetic relationships and highlighted the genetic alterations responsible for differential drug resistance among metastatic tumors.,Our study suggests that clonal evaluation is crucial for understanding tumor etiology and drug resistance in melanoma.
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This study aimed to investigate the effect and underlying mechanism of lncRNA CASC2 in malignant melanoma (MM).,Expression of CASC2 in MM tissues and cells was detected.,A375 cells were transfected with pc-CASC2, si-CASC2, miR-18a-5p inhibitor, or corresponding controls, and then cell proliferation, migration, and invasion were detected using MTT assay, colony formation assay, and Transwell analysis, respectively.,The relationship of miR-18a-5p and CASC2 or RUNX1 was detected by luciferase reporter assay.,The levels of CASC2 and RUNX1 were significantly reduced in MM tissues compared with normal skin tissues or cells, while the miR-18a-5p level was obviously increased (all p < 0.01).,Cell viability, colony number, migration, and invasion were significantly decreased in cells with pc-CASC2 compared with cells transfected with pcDNA3.1 (all p < 0.05).,These effects were consistent with the cells transfected with miR-18a-5p inhibitor.,The luciferase reporter assay revealed that CASC2 acted as a molecular sponge for miR-18a-5p, and RUNX1 was a target gene of miR-18a-5p.,Moreover, CASC2 overexpression promoted the expression of RUNX1, while upregulated miR-18a-5p significantly reversed the effect of CASC2 on the RUNX1 level (all p < 0.05).,Upregulated CASC2 may inhibit cell proliferation, migration, and invasion through regulating miR-18a-5p and its target gene RUNX1 in MM.
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Here, we report that the long noncoding RNA (lncRNA) ovarian adenocarcinoma-amplified lncRNA (OVAAL) is a mediator of cancer cell resistance, counteracting the effects of apoptosis-inducing agents acting through both the extrinsic and intrinsic pathways.,Building upon previous reports associating OVAAL amplification with ovarian and endometrial cancers, we now show that OVAAL overexpression occurs during the pathogenesis of colorectal cancer and melanoma.,Mechanistically, our findings also establish that OVAAL expression more generally contributes a prosurvival role to cancer cells under steady-state conditions.,OVAAL accomplishes these actions utilizing distinct functional modalities: one promoting activation of RAF/MEK/ERK signaling and the other blocking cell entry into senescence.,Our study demonstrates that expression of a single OVAAL in cancer cells drives two distinct but coordinated actions contributing to cancer pathology.,Long noncoding RNAs (lncRNAs) function through a diverse array of mechanisms that are not presently fully understood.,Here, we sought to find lncRNAs differentially regulated in cancer cells resistant to either TNF-related apoptosis-inducing ligand (TRAIL) or the Mcl-1 inhibitor UMI-77, agents that act through the extrinsic and intrinsic apoptotic pathways, respectively.,This work identified a commonly up-regulated lncRNA, ovarian adenocarcinoma-amplified lncRNA (OVAAL), that conferred apoptotic resistance in multiple cancer types.,Analysis of clinical samples revealed OVAAL expression was significantly increased in colorectal cancers and melanoma in comparison to the corresponding normal tissues.,Functional investigations showed that OVAAL depletion significantly inhibited cancer cell proliferation and retarded tumor xenograft growth.,Mechanically, OVAAL physically interacted with serine/threonine-protein kinase 3 (STK3), which, in turn, enhanced the binding between STK3 and Raf-1.,The ternary complex OVAAL/STK3/Raf-1 enhanced the activation of the RAF protooncogene serine/threonine-protein kinase (RAF)/mitogen-activated protein kinase kinase 1 (MEK)/ERK signaling cascade, thus promoting c-Myc-mediated cell proliferation and Mcl-1-mediated cell survival.,On the other hand, depletion of OVAAL triggered cellular senescence through polypyrimidine tract-binding protein 1 (PTBP1)-mediated p27 expression, which was regulated by competitive binding between OVAAL and p27 mRNA to PTBP1.,Additionally, c-Myc was demonstrated to drive OVAAL transcription, indicating a positive feedback loop between c-Myc and OVAAL in controlling tumor growth.,Taken together, these results reveal that OVAAL contributes to the survival of cancer cells through dual mechanisms controlling RAF/MEK/ERK signaling and p27-mediated cell senescence.
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In this study, Falletta et al. show that microenvironmental cues, including inflammation-mediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B.,Their results suggest that translation reprogramming, an evolutionarily conserved starvation response, has been hijacked by microenvironmental stress signals in melanoma to drive phenotypic plasticity and invasion and determine therapeutic outcome.,The intratumor microenvironment generates phenotypically distinct but interconvertible malignant cell subpopulations that fuel metastatic spread and therapeutic resistance.,Whether different microenvironmental cues impose invasive or therapy-resistant phenotypes via a common mechanism is unknown.,In melanoma, low expression of the lineage survival oncogene microphthalmia-associated transcription factor (MITF) correlates with invasion, senescence, and drug resistance.,However, how MITF is suppressed in vivo and how MITF-low cells in tumors escape senescence are poorly understood.,Here we show that microenvironmental cues, including inflammation-mediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B.,ATF4, a key transcription mediator of the integrated stress response, also activates AXL and suppresses senescence to impose the MITF-low/AXL-high drug-resistant phenotype observed in human tumors.,However, unexpectedly, without translation reprogramming an ATF4-high/MITF-low state is insufficient to drive invasion.,Importantly, translation reprogramming dramatically enhances tumorigenesis and is linked to a previously unexplained gene expression program associated with anti-PD-1 immunotherapy resistance.,Since we show that inhibition of eIF2B also drives neural crest migration and yeast invasiveness, our results suggest that translation reprogramming, an evolutionarily conserved starvation response, has been hijacked by microenvironmental stress signals in melanoma to drive phenotypic plasticity and invasion and determine therapeutic outcome.
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The unfolded protein response (UPR) regulates cell fate following exposure of cells to endoplasmic reticulum stresses.,PERK, a UPR protein kinase, regulates protein synthesis and while linked with cell survival, exhibits activities associated with both tumor progression and tumor suppression.,For example, while cells lacking PERK are sensitive to UPR-dependent cell death, acute activation of PERK triggers both apoptosis and cell cycle arrest, which would be expected to contribute tumor suppressive activity.,We have evaluated these activities in the BRAF-dependent melanoma and provide evidence revealing a complex role for PERK in melanoma where a 50% reduction is permissive for BrafV600E-dependent transformation, while complete inhibition is tumor suppressive.,Consistently, PERK mutants identified in human melanoma are hypomorphic with dominant inhibitory function.,Strikingly, we demonstrate that small molecule PERK inhibitors exhibit single agent efficacy against BrafV600E-dependent tumors highlighting the clinical value of targeting PERK.
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RAC1 P29 is the third most commonly mutated codon in human cutaneous melanoma, after BRAF V600 and NRAS Q61.,Here, we study the role of RAC1P29S in melanoma development and reveal that RAC1P29S activates PAK, AKT, and a gene expression program initiated by the SRF/MRTF transcriptional pathway, which results in a melanocytic to mesenchymal phenotypic switch.,Mice with ubiquitous expression of RAC1P29S from the endogenous locus develop lymphoma.,When expressed only in melanocytes, RAC1P29S cooperates with oncogenic BRAF or with NF1-loss to promote tumorigenesis.,RAC1P29S also drives resistance to BRAF inhibitors, which is reversed by SRF/MRTF inhibitors.,These findings establish RAC1P29S as a promoter of melanoma initiation and mediator of therapy resistance, while identifying SRF/MRTF as a potential therapeutic target.,•RAC1P29S activates PAK, AKT, and the SRF/MRTF transcription program in melanocytes•RAC1P29S induces a melanocytic to mesenchymal transition through SRF/MRTF and PAK•RAC1P29S cooperates with BRAF mutation or NF1 deletion to promote melanomagenesis•RAC1P29S induces resistance to BRAF inhibitors through SRF/MRTF,RAC1P29S activates PAK, AKT, and the SRF/MRTF transcription program in melanocytes,RAC1P29S induces a melanocytic to mesenchymal transition through SRF/MRTF and PAK,RAC1P29S cooperates with BRAF mutation or NF1 deletion to promote melanomagenesis,RAC1P29S induces resistance to BRAF inhibitors through SRF/MRTF,RAC1P29S is a common mutation in human cutaneous melanoma.,Lionarons et al. show that RAC1P29S induces a melanocytic to mesenchymal switch via an SRF/MRTF-mediated gene expression program, cooperates with BRAF in melanomagenesis, and drives BRAF inhibitor resistance, which is reversed by SRF/MRTF inhibition.
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The melanocortin-1 receptor (MC1R), a G protein-coupled receptor, plays a crucial role in human and mouse pigmentation1-8.,Activation of MC1R in melanocytes by α-melanocyte-stimulating hormone (α-MSH)9 stimulates cAMP signaling and melanin production and enhances DNA repair after UV irradiation (UVR)10-16.,Individuals carrying MC1R variants, especially those associated with red hair color, fair skin and poor tanning ability (RHC-variants), are associated with higher risk of melanoma5,17,18,19,20.,However, how MC1R activity might be modulated by UV irradiation, why redheads are more prone to developing melanoma, and whether the activity of RHC variants might be restored for therapeutic benefit remain unresolved questions.,Here we demonstrate a potential MC1R-targeted intervention strategy to rescue loss-of-function MC1R in MC1R RHC-variants for therapeutic benefit based on activating MC1R protein palmitoylation.,Specifically, MC1R palmitoylation, primarily mediated by the protein-acyl transferase (PAT) ZDHHC13, is essential for activating MC1R signaling that triggers increased pigmentation, UVB-induced G1-like cell cycle arrest and control of senescence and melanomagenesis in vitro and in vivo.,Using C57BL/6J-MC1Re/eJ mice expressing MC1R RHC-variants we show that pharmacological activation of palmitoylation rescues the defects of MC1R RHC-variants and prevents melanomagenesis.,The results highlight a central role for MC1R palmitoylation in pigmentation and protection against melanoma.
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The LATS1 kinase has been described as a tumor suppressor in various cancers.,However, its role in melanoma has not been fully elucidated.,There are several processes involved in tumorigenesis, including melanin production.,Melanin content positively correlates with the level of reactive oxygen species (ROS) inside the cell.,Accordingly, the purpose of the study was to assess the role of LATS1 in melanogenesis and oxidative stress and its influence on tumor growth.,We have knocked down LATS1 in primary melanocytes and melanoma cells and found that its expression is crucial for melanin synthesis, ROS production, and oxidative stress response.,We showed that LATS1 ablation significantly decreased the melanogenesis markers’ expression and melanin synthesis in melanocyte and melanoma cell lines.,Moreover, silencing LATS1 resulted in enhanced oxidative stress.,Reduced melanin content in LATS1 knocked down tumors was associated with increased tumor growth, pointing to melanin’s protective role in this process.,The study demonstrated that LATS1 is highly engaged in melanogenesis and oxidative stress control and affects melanoma growth.,Our results may find the implications in the diagnosis and treatment of pigmentation disorders, including melanoma.
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Malignant melanoma is the most aggressive form of skin carcinoma, which possesses fast propagating and highly invasive characteristics.,Curcumin is a natural phenol compound that has various biological activities, such as anti-proliferative and apoptosis-accelerating impacts on tumor cells.,Unfortunately, the therapeutical activities of Cur are severely hindered due to its extremely low bioavailability.,In this study, a cooperative therapy of low concentration Cur combined with red united blue light irradiation was performed to inspect the synergistic effects on the apoptosis, proliferation and autophagy in human melanoma A375 cell.,The results showed that red united blue light irradiation efficaciously synergized with Cur to trigger oxidative stress-mediated cell death, induce apoptosis and inhibit cell proliferation.,Meanwhile, Western blotting revealed that combined disposure induced the formation of autophagosomes.,Conversely, inhibition of the autophagy enhanced apoptosis, obstructed cell cycle arrest and induced reversible proliferation arrest to senescence.,These findings suggest that Cur combined with red united blue light irradiation could generate photochemo-preventive effects via enhancing apoptosis and triggering autophagy, and pharmacological inhibition of autophagy convert reversible arrested cells to senescence, therefore reducing the possibility that damaged cells might escape programmed death.
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A subset of basal cell carcinomas (BCCs) are directly derived from hair follicles (HFs).,In some respects, HFs can be defined as ‘ordered’ skin appendage growths, while BCCs can be regarded as ‘disordered’ skin appendage growths.,The aim of the present study was to examine HFs and BCCs to define the expression of common and unique signaling pathways in each skin appendage.,Human nodular BCCs, along with HFs and non-follicular skin epithelium from normal individuals, were examined using microarrays, qPCR, and immunohistochemistry.,Subsequently, BCC cells and root sheath keratinocyte cells from HFs were cultured and treated with Notch signaling peptide Jagged1 (JAG1).,Gene expression, protein levels, and cell apoptosis susceptibility were assessed using qPCR, immunoblotting, and flow cytometry, respectively.,Specific molecular mechanisms were found to be involved in the process of cell self-renewal in the HFs and BCCs, including Notch and Hedgehog signaling pathways.,However, several key Notch signaling factors showed significant differential expression in BCCs compared with HFs.,Stimulating Notch signaling with JAG1 induced apoptosis of BCC cells by increasing Fas ligand expression and downstream caspase-8 activation.,The present study showed that Notch signaling pathway activity is suppressed in BCCs, and is highly expressed in HFs.,Elements of the Notch pathway could, therefore, represent targets for the treatment of BCCs and potentially in hair follicle engineering.
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Activating mutations in the TERT promoter were recently identified in up to 71% of cutaneous melanoma.,Subsequent studies found TERT promoter mutations in a wide array of other major human cancers.,TERT promoter mutations lead to increased expression of telomerase, which maintains telomere length and genomic stability, thereby allowing cancer cells to continuously divide, avoiding senescence or apoptosis.,TERT promoter mutations in cutaneous melanoma often show UV-signatures.,Non-melanoma skin cancer, including basal cell carcinoma and squamous cell carcinoma, are very frequent malignancies in individuals of European descent.,We investigated the presence of TERT promoter mutations in 32 basal cell carcinomas and 34 cutaneous squamous cell carcinomas using conventional Sanger sequencing.,TERT promoter mutations were identified in 18 (56%) basal cell carcinomas and in 17 (50%) cutaneous squamous cell carcinomas.,The recurrent mutations identified in our cohort were identical to those previously described in cutaneous melanoma, and showed a UV-signature (C>T or CC>TT) in line with a causative role for UV exposure in these common cutaneous malignancies.,Our study shows that TERT promoter mutations with UV-signatures are frequent in non-melanoma skin cancer, being present in around 50% of basal and squamous cell carcinomas and suggests that increased expression of telomerase plays an important role in the pathogenesis of these tumors.
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Melanoma, as for many other cancers, undergoes a selection process during progression that limits many innate and adaptive tumor control mechanisms.,Immunotherapy with immune checkpoint blockade overcomes one of the escape mechanisms but if the tumor is not eliminated other escape mechanisms evolve that require new approaches for tumor control.,Some of the innate mechanisms that have evolved against infections with microorganisms and viruses are proving to be active against cancer cells but require better understanding of how they are activated and what inhibitory mechanisms may need to be targeted.,This is particularly so for inflammasomes which have evolved against many different organisms and which recruit a number of cytotoxic mechanisms that remain poorly understood.,Equally important is understanding of where these mechanisms will fit into existing treatment strategies and whether existing strategies already involve the innate killing mechanisms.
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Inhibition of complex I (CI) of the mitochondrial respiratory chain by BAY 87-2243 (‘BAY') triggers death of BRAFV600E melanoma cell lines and inhibits in vivo tumor growth.,Here we studied the mechanism by which this inhibition induces melanoma cell death.,BAY treatment depolarized the mitochondrial membrane potential (Δψ), increased cellular ROS levels, stimulated lipid peroxidation and reduced glutathione levels.,These effects were paralleled by increased opening of the mitochondrial permeability transition pore (mPTP) and stimulation of autophagosome formation and mitophagy.,BAY-induced cell death was not due to glucose shortage and inhibited by the antioxidant α-tocopherol and the mPTP inhibitor cyclosporin A.,Tumor necrosis factor receptor-associated protein 1 (TRAP1) overexpression in BAY-treated cells lowered ROS levels and inhibited mPTP opening and cell death, whereas the latter was potentiated by TRAP1 knockdown.,Knockdown of autophagy-related 5 (ATG5) inhibited the BAY-stimulated autophagosome formation, cellular ROS increase and cell death.,Knockdown of phosphatase and tensin homolog-induced putative kinase 1 (PINK1) inhibited the BAY-induced Δψ depolarization, mitophagy stimulation, ROS increase and cell death.,Dynamin-related protein 1 (Drp1) knockdown induced mitochondrial filamentation and inhibited BAY-induced cell death.,The latter was insensitive to the pancaspase inhibitor z-VAD-FMK, but reduced by necroptosis inhibitors (necrostatin-1, necrostatin-1s)) and knockdown of key necroptosis proteins (receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and mixed lineage kinase domain-like (MLKL)).,BAY-induced cell death was also reduced by the ferroptosis inhibitor ferrostatin-1 and overexpression of the ferroptosis-inhibiting protein glutathione peroxidase 4 (GPX4).,This overexpression also inhibited the BAY-induced ROS increase and lipid peroxidation.,Conversely, GPX4 knockdown potentiated BAY-induced cell death.,We propose a chain of events in which: (i) CI inhibition induces mPTP opening and Δψ depolarization, that (ii) stimulate autophagosome formation, mitophagy and an associated ROS increase, leading to (iii) activation of combined necroptotic/ferroptotic cell death.
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A considerably high number of outdoor workers worldwide are constantly exposed for the majority of their working life to solar radiation (SR); this exposure is known to induce various adverse health effects, mainly related to its ultraviolet (UV) component.,The skin and the eye are the principal target organs for both acute and long-term exposure.,Actinic keratosis, non-melanoma skin cancers, and malignant melanoma are the main long-term adverse skin effects, whereas in the eye pterygium, cataracts, and according to an increasing body of evidence, macular degeneration may be induced.,Despite this, SR exposure risk is currently undervalued, if not neglected, as an occupational risk factor for outdoor workers.,SR exposure is influenced by various environmental and individual factors, and occupation is one of the most relevant.,For a better understanding of this risk and for the development of more effective prevention strategies, one of the main problems is the lack of available and adequate methods to estimate SR worker exposure, especially long-term exposure.,The main aims of this review were to provide a comprehensive overview of SR exposure risk of outdoor workers, including the UV exposure levels and the main methods recently proposed for short-term and cumulative exposure, and to provide an update of knowledge on the main adverse eye and skin effects.,Finally, we also outline here preventive interventions to reduce occupational risk.
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The biological clock has received increasing interest due to its key role in regulating body homeostasis in a time-dependent manner.,Cancer development and progression has been linked to a disrupted molecular clock; however, in melanoma, the role of the biological clock is largely unknown.,We investigated the effects of the tumor on its micro- (TME) and macro-environments (TMaE) in a non-metastatic melanoma model.,C57BL/6J mice were inoculated with murine B16-F10 melanoma cells and 2 weeks later the animals were euthanized every 6 h during 24 h.,The presence of a localized tumor significantly impaired the biological clock of tumor-adjacent skin and affected the oscillatory expression of genes involved in light- and thermo-reception, proliferation, melanogenesis, and DNA repair.,The expression of tumor molecular clock was significantly reduced compared to healthy skin but still displayed an oscillatory profile.,We were able to cluster the affected genes using a human database and distinguish between primary melanoma and healthy skin.,The molecular clocks of lungs and liver (common sites of metastasis), and the suprachiasmatic nucleus (SCN) were significantly affected by tumor presence, leading to chronodisruption in each organ.,Taken altogether, the presence of non-metastatic melanoma significantly impairs the organism’s biological clocks.,We suggest that the clock alterations found in TME and TMaE could impact development, progression, and metastasis of melanoma; thus, making the molecular clock an interesting pharmacological target.
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Merkel cell carcinoma (MCC) is a rare but lethal cancer with the highest case-by-case fatality rate among all skin cancers. 80% of cancers are associated with the Merkel cell polyomavirus (MCPyV). 20% of MCCs are virus negative.,Recent epidemiological data suggest that there are important, clinically relevant differences between these two subtypes of MCC.,Recent studies in cancer genomics, mouse genetics, and virology experiments have transformed our understanding of MCC pathophysiology.,Importantly, dramatic differences in the genetics of these two MCC subtypes suggest fundamental differences in their pathophysiology.,We review these recent works and find that they provocatively suggest that MCPyV-positive and MCPyV-negative MCCs arise from two different cells of origin: the MCPyV-negative MCC from epidermal keratinocytes and the MCPyV-positive MCC from dermal fibroblasts.,If true, this would represent the first cancer that we are aware of that evolves from cells of origin from two distinct germ layers: MCPyV-negative MCCs from ectodermal keratinocytes and MCPyV-positive MCCs from mesodermal fibroblasts.,Future epigenetic experiments may prove valuable in confirming these distinct lineages for these MCC subtypes, especially for the clinical importance the cell of origin has on MCC treatment and prevention.
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Merkel cell carcinomas (MCC) are rare but highly malignant skin cancers associated with a novel polyomavirus.,MCC tumors were infiltrated by T cells, including effector, central memory and regulatory T cells.,Infiltrating T cells showed markedly reduced activation as evidenced by reduced expression of CD69 and CD25.,Treatment of MCC tumors in vitro with IL-2 and IL-15 led to T cell activation, proliferation, enhanced cytokine production and loss of viable tumor cells from cultures.,Expanded tumor-infiltrating lymphocytes showed TCR repertoire skewing and upregulation of CD137.,MCC tumors implanted into immunodeficient mice failed to grow unless human T cells in the tumor grafts were depleted with denileukin diftitox, suggesting tumor-specific T cells capable of controlling tumor growth were present in MCC.,Both CD4+ and CD8+ FOXP3+ regulatory T cells were frequent in MCC. 50% of non-activated T cells in MCC expressed PD-1, a marker of T-cell exhaustion, and PD-L1 and PD-L2 were expressed by a subset of tumor dendritic cells and macrophages.,In summary, we observed tumor-specific T cells with suppressed activity in MCC tumors.,Agents that stimulate T cell activity, block Treg function or inhibit PD-1 signaling may be effective in the treatment of this highly malignant skin cancer.
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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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It is unknown whether melanoma patients achieving complete response (CR) with targeted therapy can safely discontinue treatment.,All patients treated with BRAF/MEK inhibitors achieving CR and ceasing treatment before progression were identified.,Clinical data at treatment initiation, cessation and progression were examined.,A total of 12 eligible patients were identified, with median follow-up of 16 months, of whom 6 (50%) recurred at a median of 6.6 months after treatment cessation.,One patient lost to follow-up until presentation with symptomatic recurrence was the only relapser to die.,At relapse, the remaining five patients had an LDH <1.2 times ULN, four were ECOG 0 and one ECOG 1.,Baseline characteristics and time to CR and to discontinuation did not influence the rate of relapse.,A large proportion of patients achieving CR with BRAF/MEK inhibitors relapse after treatment cessation.,The optimal treatment duration in such patients is unclear, particularly where alternative treatments are available.
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Spatial profiling of cutaneous melanoma at the single-cell level provided molecular features of progression and immunosuppression, which include paracrine cytokine signaling and direct immune cell-cell contact.,Cutaneous melanoma is a highly immunogenic malignancy that is surgically curable at early stages but life-threatening when metastatic.,Here we integrate high-plex imaging, 3D high-resolution microscopy, and spatially resolved microregion transcriptomics to study immune evasion and immunoediting in primary melanoma.,We find that recurrent cellular neighborhoods involving tumor, immune, and stromal cells change significantly along a progression axis involving precursor states, melanoma in situ, and invasive tumor.,Hallmarks of immunosuppression are already detectable in precursor regions.,When tumors become locally invasive, a consolidated and spatially restricted suppressive environment forms along the tumor-stromal boundary.,This environment is established by cytokine gradients that promote expression of MHC-II and IDO1, and by PD1-PDL1-mediated cell contacts involving macrophages, dendritic cells, and T cells.,A few millimeters away, cytotoxic T cells synapse with melanoma cells in fields of tumor regression.,Thus, invasion and immunoediting can coexist within a few millimeters of each other in a single specimen.,The reorganization of the tumor ecosystem in primary melanoma is an excellent setting in which to study immunoediting and immune evasion.,Guided by classic histopathology, spatial profiling of proteins and mRNA reveals recurrent morphologic and molecular features of tumor evolution that involve localized paracrine cytokine signaling and direct cell-cell contact.,This article is highlighted in the In This Issue feature, p.,1397
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Anti-PD-1 therapy yields objective clinical responses in 30-40% of advanced melanoma patients.,Since most patients do not respond, predictive biomarkers to guide treatment selection are needed.,We hypothesize that MHC-I/II expression is required for tumour antigen presentation and may predict anti-PD-1 therapy response.,In this study, across 60 melanoma cell lines, we find bimodal expression patterns of MHC-II, while MHC-I expression was ubiquitous.,A unique subset of melanomas are capable of expressing MHC-II under basal or IFNγ-stimulated conditions.,Using pathway analysis, we show that MHC-II(+) cell lines demonstrate signatures of ‘PD-1 signalling', ‘allograft rejection' and ‘T-cell receptor signalling', among others.,In two independent cohorts of anti-PD-1-treated melanoma patients, MHC-II positivity on tumour cells is associated with therapeutic response, progression-free and overall survival, as well as CD4+ and CD8+ tumour infiltrate.,MHC-II+ tumours can be identified by melanoma-specific immunohistochemistry using commercially available antibodies for HLA-DR to improve anti-PD-1 patient selection.,Immunotherapy is used to treat melanoma, however patient responses vary widely highlighting the need for factors that can predict therapeutic success.,Here, the authors show that MHC-II molecules expressed by tumour cells are positively correlated with a good response to therapy and overall patient survival.
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Cutaneous melanoma is a very aggressive neoplasia of melanocytic origin with constantly growing incidence and mortality rates world-wide.,Epigenetic modifications (i.e., alterations of genomic DNA methylation patterns, of post-translational modifications of histones, and of microRNA profiles) have been recently identified as playing an important role in melanoma development and progression by affecting key cellular pathways such as cell cycle regulation, cell signalling, differentiation, DNA repair, apoptosis, invasion and immune recognition.,In this scenario, pharmacologic inhibition of DNA methyltransferases and/or of histone deacetylases were demonstrated to efficiently restore the expression of aberrantly-silenced genes, thus re-establishing pathway functions.,In light of the pleiotropic activities of epigenetic drugs, their use alone or in combination therapies is being strongly suggested, and a particular clinical benefit might be expected from their synergistic activities with chemo-, radio-, and immuno-therapeutic approaches in melanoma patients.,On this path, an important improvement would possibly derive from the development of new generation epigenetic drugs characterized by much reduced systemic toxicities, higher bioavailability, and more specific epigenetic effects.
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In addition to modulating the function and stability of cellular mRNAs, microRNAs can profoundly affect the life cycles of viruses bearing sequence complementary targets, a finding recently exploited to ameliorate toxicities of vaccines and oncolytic viruses.,To elucidate the mechanisms underlying microRNA-mediated antiviral activity, we modified the 3′ untranslated region (3′UTR) of Coxsackievirus A21 to incorporate targets with varying degrees of homology to endogenous microRNAs.,We show that microRNAs can interrupt the picornavirus life-cycle at multiple levels, including catalytic degradation of the viral RNA genome, suppression of cap-independent mRNA translation, and interference with genome encapsidation.,In addition, we have examined the extent to which endogenous microRNAs can suppress viral replication in vivo and how viruses can overcome this inhibition by microRNA saturation in mouse cancer models.
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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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In general, melanoma can be considered as a UV‐driven disease with an aggressive metastatic course and high mutational load, with only few tumors (acral, mucosal, and uveal melanomas) not induced by sunlight and possessing a lower mutational load.,The most commonly activated pathway in melanoma is the mitogen‐activated protein kinase (MAPK) pathway.,However, the prognostic significance of mutational stratification is unclear and needs further investigation.,Here, in silico we combined mutation data from 162 melanomas subjected to targeted deep sequencing with mutation data from three published studies.,Tumors from 870 patients were grouped according to BRAF,RAS,NF1 mutation or triple‐wild‐type status and correlated with tumor and patient characteristics.,We found that the NF1‐mutated subtype had a higher mutational burden and strongest UV mutation signature.,Searching for co‐occurring mutated genes revealed the RASopathy genes PTPN11 and RASA2, as well as another RAS domain‐containing gene RASSF2 enriched in the NF1 subtype after adjustment for mutational burden.,We found that a larger proportion of the NF1‐mutant tumors were from males and with older age at diagnosis.,Importantly, we found an increased risk of death from melanoma (disease‐specific survival, DSS; HR, 1.9; 95% CI, 1.21-3.10; P = 0.046) and poor overall survival (OS; HR, 2.0; 95% CI, 1.28-2.98; P = 0.01) in the NF1 subtype, which remained significant after adjustment for age, gender, and lesion type (DSS P = 0.03, OS P = 0.06, respectively).,Melanoma genomic subtypes display different biological and clinical characteristics.,The poor outcome observed in the NF1 subtype highlights the need for improved characterization of this group.
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Most anti-cancer drugs are derived from natural resources such as marine, microbial and botanical sources.,Cutaneous malignant melanoma is the most aggressive form of skin cancer, with a high mortality rate.,Various treatments for malignant melanoma are available, but due to the development of multi-drug resistance, current or emerging chemotherapies have a relatively low success rates.,This emphasizes the importance of discovering new compounds that are both safe and effective against melanoma.,In vitro testing of melanoma cell lines and murine melanoma models offers the opportunity for identifying mechanisms of action of plant derived compounds and extracts.,Common anti-melanoma effects of natural compounds include potentiating apoptosis, inhibiting cell proliferation and inhibiting metastasis.,There are different mechanisms and pathways responsible for anti-melanoma actions of medicinal compounds such as promotion of caspase activity, inhibition of angiogenesis and inhibition of the effects of tumor promoting proteins such as PI3-K, Bcl-2, STAT3 and MMPs.,This review thus aims at providing an overview of anti-cancer compounds, derived from natural sources, that are currently used in cancer chemotherapies, or that have been reported to show anti-melanoma, or anti-skin cancer activities.,Phytochemicals that are discussed in this review include flavonoids, carotenoids, terpenoids, vitamins, sulforaphane, some polyphenols and crude plant extracts.
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Cutaneous melanoma is a challenge to treat.,Over the last 30 years, no drug or combination of drugs demonstrated significant impact to improve patient survival.,From 1995 to 2000, the use of cytokines such as interferon and interleukin become treatment options.,In 2011, new drugs were approved by the U.S.,Food and Drug Administration, including peginterferon alfa-2b for patients with stage III disease, vemurafenib for patients with metastatic melanoma with the BRAF V600E mutation, and ipilimumab, a monoclonal antibody directed to the CTLA-4 T lymphocyte receptor, to combat metastatic melanoma in patients who do not have the BRAF V600E mutation.,Both ipilimumab and vemurafenib showed results in terms of overall survival.,Other trials with inhibitors of other genes, such as the KIT gene and MEK, are underway in the search for new discoveries.,The discovery of new treatments for advanced or metastatic disease aims to relieve symptoms and improve patient quality of life.
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