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MCL-1 (BCL-2 family anti-apoptotic protein) is responsible for melanoma's resistance to therapy.,Cancer initiating cells also contribute to resistance and relapse from treatments.,Here we examined the effects of the MCL-1 inhibitor SC-2001 in killing non melanoma-initiating-cells (bulk of melanoma), and melanoma-initiating-cells (MICs).,By itself, SC-2001 significantly kills melanoma cells under monolayer conditions in vitro and in a conventional mouse xenograft model.,However, even at high doses (10μM), SC-2001 does not effectively eliminate MICs.,In contrast, the combination of SC-2001 with ABT-737 (a BCL-2/BCL-XL/BCL-W inhibitor) significantly decreases ALDH+ cells, disrupts primary spheres, and inhibits the self-renewability of MICs.,These results were observed in multiple melanomas, including short term cultures of relapsed tumors from current treatments, independent of the mutation status of BRAF or NRAS.,Using a low-cell-number mouse xenograft model, we examined the effects of these treatments on the tumor initiating ability of MIC-enriched cultures.,The combination therapy reduces tumor formation significantly compared to either drug alone.,Mechanistic studies using shRNA and the CRISPR-Cas9 technology demonstrated that the upregulation of pro-apoptotic proteins NOXA and BIM contribute to the combination-induced cell death.,These results indicate that the MCL-1 inhibitor SC-2001 combined with ABT-737 is a promising treatment strategy for targeting melanoma.

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.

Metastatic uveal melanoma is a highly fatal disease; most patients die from their hepatic metastasis within 1 year.,A major drawback in the development of new treatments for metastatic uveal melanoma is the difficulty in obtaining appropriate cell lines and the lack of appropriate animal models.,Patient-derived xenograft (PDX) tumor models, bearing ectopically implanted tumors at a subcutaneous site, have been developed.,However, these ectopically implanted PDX models have obstacles to translational research, including a low engraftment rate, slow tumor growth, and biological changes after multiple passages due to the different microenvironment.,To overcome these limitations, we developed a new method to directly transplant biopsy specimens to the liver of immunocompromised mice.,By using two metastatic uveal melanoma cell lines, we demonstrated that the liver provides a more suitable microenvironment for tumor growth compared to subcutaneous sites and that surgical orthotopic implantation (SOI) of tumor pieces allows the creation of a liver tumor in immunocompromised mice.,Subsequently, 10 of 12 hepatic metastasis specimens from patients were successfully xenografted into the immunocompromised mice (83.3% success rate) using SOI, including 8 of 10 needle biopsy specimens (80%).,Additionally, four cryopreserved PDX tumors were re-implanted to new mice and re-establishment of PDX tumors was confirmed in all four mice.,The serially passaged xenograft tumors as well as the re-implanted tumors after cryopreservation were similar to the original patient tumors in histologic, genomic, and proteomic expression profiles.,CT imaging was effective for detecting and monitoring PDX tumors in the liver of living mice.,The expression of Ki67 in original patient tumors was a predictive factor for implanted tumor growth and the success of serial passages in PDX mice.,Surgical orthotopic implantation of hepatic metastasis from uveal melanoma is highly successful in the establishment of orthotopic PDX models, enhancing their practical utility for research applications.,By using CT scan, tumor growth can be monitored, which is beneficial to evaluate treatment effects in interventional studies.,The online version of this article (doi:10.1186/s12967-017-1247-z) contains supplementary material, which is available to authorized users.

Animal models serve as powerful tools for investigating the pathobiology of cancer, identifying relevant pathways, and developing novel therapeutic agents.,They have facilitated rapid scientific progress in many tumor entities.,However, for establishing a powerful animal model of uveal melanoma fundamental challenges remain.,To date, no animal model offers specific genetic attributes as well as histologic, immunologic, and metastatic features of uveal melanoma.,Syngeneic models with intraocular injection of cutaneous melanoma cells may suit best for investigating immunologic/tumor biology aspects.,However, differences between cutaneous and uveal melanoma regarding genetics and metastasis remain problematic.,Human xenograft models are widely used for evaluating novel therapeutics but require immunosuppression to allow tumor growth.,New approaches aim to establish transgenic mouse models of spontaneous uveal melanoma which recently provided preliminary promising results.,Each model provides certain benefits and may render them suitable for answering a respective scientific question.,However, all existing models also exhibit relevant limitations which may have led to delayed research progress.,Despite refined therapeutic options for the primary ocular tumor, patients' prognosis has not improved since the 1970s.,Basic research needs to further focus on a refinement of a potent animal model which mimics uveal melanoma specific mechanisms of progression and metastasis.,This review will summarise and interpret existing animal models of uveal melanoma including recent advances in the field.

Despite being rarely reported, improved diagnostic and prognostic indicators are necessary for treating malignant melanoma in rabbits.,In this study, two cases of primary skin lesions, on the scrotum and on eyelid, with systemic metastases, were examined.,The tumors formed intra-dermally by sheet-like proliferation of polymorphic cells, with anisocytosis and varying amount of melanin granules.,Tumors had displaced almost 50% of the lung and liver tissue, and tumor metastasis was the cause of early death in both rabbits.,Ki-67-positive population was high in both, and it was found to be useful in assessing the outcome and malignancy.,In addition, Melan-A, HMB-45, PNL2 and S100 established a useful immunohistochemical panel for the diagnosis of melanocytic tumor in rabbits.

Melanoma remains mostly an untreatable fatal disease despite advances in decoding cancer genomics and developing new therapeutic modalities.,Progress in patient care would benefit from additional predictive models germane for human disease mechanisms, tumor heterogeneity, and therapeutic responses.,Toward this aim, this review documents comparative aspects of human and naturally occurring canine melanomas.,Clinical presentation, pathology, therapies, and genetic alterations are highlighted in the context of current basic and translational research in comparative oncology.,Somewhat distinct from sun exposure-related human cutaneous melanomas, there is growing evidence that a variety of gene copy number alterations and protein structure/function mutations play roles in canine melanomas, in circumstances more analogous to human mucosal melanomas and to some extent other melanomas with murine sarcoma viral oncogene homolog B (BRAF), Neuroblastoma RAS Viral (V-Ras) Oncogene Homolog (NRAS), and neurofibromin 1 tumor suppressor NF1 triple wild-type genotype.,Gaps in canine genome annotation, as well as an insufficient number and depth of sequences covered, remain considerable barriers to progress and should be collectively addressed.,Preclinical approaches can be designed to include canine clinical trials addressing immune modulation as well as combined-targeted inhibition of Rat Sarcoma Superfamily/Mitogen-activated protein kinase (RAS/MAPK) and/or Phosphatidylinositol-3-Kinase/Protein Kinase B/Mammalian target of rapamycin (PI3K/AKT/mTOR) signal transduction, pathways frequently activated in both human and canine melanomas.,Future investment should be aimed towards improving understanding of canine melanoma as a predictive preclinical surrogate for human melanoma and for mutually benefiting these uniquely co-dependent species.

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.

Clinical outcomes for advanced malignant melanoma (MM) are often poor due to tumor invasiveness, metastasis, recurrence, and multidrug resistance.,We investigated whether apoptosis, cell cycle regulation, oxidative status, and redox balance were altered by changes in the expression of the long noncoding RNA, growth arrest-specific transcript 5 (GAS5), in MM cells.,Analysis of clinical samples from MM patients showed that the rate of reduced GAS5 expression, relative to that in adjacent noncancerous tissues, was significantly lower for tumors from patients with advanced disease (76.6%, P < 0.001), as evidenced by larger tumor size, higher TNM stage, and higher incidences of ulceration and metastasis (P < 0.001 for all).,Cell culture experiments showed that siRNA-mediated knockdown of GAS5 increased the viability of A375-GAS5si cells.,Flow cytometry and western blotting showed that GAS5 knockdown increased MM cell proliferation by inducing G1/S cell cycle progression through increases in Cyclin D1, CDK4, and p27 expression (P < 0.05 for all) and by inhibiting apoptosis through an increase in Bcl-2 expression (P < 0.001).,Knockdown of GAS5 also increased levels of superoxide anion (P < 0.01), NADP+(P < 0.001), and oxidized glutathiones (P < 0.01) through increases in NOX4 expression (P < 0.001), G6PD expression (P < 0.01), and NOX activity (P < 0.05), and RNA co-immunoprecipitation showed that GAS5 induced these changes through a physical interaction between GAS5 and the G6PD protein.,Our findings show GAS5 contributes to regulation of the apoptosis, cell cycle, homeostasis of reactive oxygen species, and redox balance in MM cells, and suggest that reduced GAS5 expression contributes to disease progression in MM patients.,The online version of this article (10.1007/s00432-018-2820-4) contains supplementary material, which is available to authorized users.

Excessive extracellular matrix (ECM) remodeling and a reactive stroma can affect T-cell infiltration and T-cell activity in the tumor and hereby influence response to immune checkpoint inhibitors (ICI).,In the pursuit of finding biomarkers that predict treatment response, we evaluated the association between serum biomarkers of collagen and vimentin turnover and outcomes in metastatic melanoma patients treated with the anti-CTLA-4 antibody ipilimumab (IPI).,Type III collagen formation (PRO-C3), MMP-degraded type I, type III and type IV collagens (C1M, C3M and C4M), and citrullinated and MMP-degraded vimentin (VICM) were measured with ELISAs in serum from metastatic melanoma patients before (n = 66) and 3 weeks after (n = 52) initiation of IPI treatment.,Biomarker levels were associated with Disease Control Rate (DCR) and survival outcomes.,We found that baseline levels of PRO-C3 (p = 0.011), C1M (p = 0.003), C3M (p = 0.013) and C4M (p = 0.027) were significantly elevated in patients with progressive disease (PD).,Univariate Cox regression analysis identified high PRO-C3 (p = 0.021) and C4M (p = 0.008) as predictors of poor overall survival (OS) and the biomarkers remained significant when evaluated with other covariates (PRO-C3 (p = 0.049) and C4M (p = 0.046)).,Multivariate analysis identified VICM as a predictor of longer OS (p = 0.026).,Similarly, a high C3M/PRO-C3 ratio predicted for increased OS (p = 0.034).,Only C3M (p = 0.003) and VICM (p < 0.0001) increased 3 weeks after treatment.,ECM and tissue remodeling quantified in pre-treatment serum were associated with response and survival outcomes in metastatic melanoma patients treated with IPI.,This highlights the importance of addressing the ECM and stromal component non-invasively in future ICI studies.,The online version of this article (10.1186/s40425-018-0474-z) contains supplementary material, which is available to authorized users.

Melanoma is among the most aggressive tumors, and the occurrence of metastasis leads to a precipitous drop in the patients' survival.,Therefore, identification of metastasis-associated biomarkers and therapeutic targets will contribute a lot to improving melanoma theranostics.,Recently, microRNAs (miRNAs) have been implicated in modulating cancer invasion and metastasis, and are proved as potential non-invasive biomarkers in sera for various tumors.,Here, we reported miR-23a as a novel metastasis-associated miRNA that played a remarkable role in modulating melanoma invasive and metastatic capacity and was of great value in predicting melanoma metastasis and prognosis.,We found that serum miR-23a level was significantly down-regulated in metastatic melanoma patients and highly correlated with poor clinical outcomes.,In addition, miR-23a level was also remarkably decreased in metastatic melanoma tissues and cell lines.,Furthermore, overexpressed miR-23a suppressed the invasive and migratory property of melanoma cells by abrogating autophagy through directly targeting ATG12.,Specially, miR-23a-ATG12 axis attenuated melanoma invasion and migration through autophagy-mediated AMPK-RhoA pathway.,Finally, the overexpression of miR-23a prevented melanoma metastasis in vivo.,Taken together, our findings demonstrate that the metastasis-associated miR-23a is not only a potential biomarker, but also a valuable therapeutic target for melanoma.

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.

Circulating melanoma cells (CMCs) are thought to be valuable in improving measures of prognosis in melanoma patients and may be a useful marker of residual disease to identify non-metastatic patients requiring adjuvant therapy.,We investigated whether immunomagnetic enrichment targeting multiple markers allows more efficient enrichment of CMCs from patient peripheral blood than targeting a single marker.,Furthermore, we aimed to determine whether the number of CMCs in patient blood was associated with disease stage.,We captured CMCs by targeting the melanoma associated markers MCSP and MCAM as well as the melanoma stem cell markers ABCB5 and CD271, both individually and in combination, by immunomagnetic enrichment.,CMCs were enriched and quantified from the peripheral blood of 10 non-metastatic and 13 metastatic melanoma patients.,Targeting all markers in combination resulted in the enrichment of more CMCs than when any individual marker was targeted (p < 0.001-0.028).,Furthermore, when a combination of markers was targeted, a greater number of CMCs were enriched in metastatic patients compared with non-metastatic patients (p = 0.007).,Our results demonstrated that a combination of markers should be targeted for optimal isolation of CMCs.,In addition, there are significantly more CMCs in metastatic patients compared with non-metastatic patients and therefore quantification of CMCs may prove to be a useful marker of disease progression.

Vitiligo is an autoimmune disease in which depigmented skin results from destruction of melanocytes1, with epidemiologic association with other autoimmune diseases2.,In previous linkage and genome-wide association studies (GWAS1, GWAS2), we identified 27 vitiligo susceptibility loci in patients of European (EUR) ancestry.,We carried out a third GWAS (GWAS3) in EUR subjects, with augmented GWAS1 and GWAS2 controls, genome-wide imputation, and meta-analysis of all three GWAS, followed by an independent replication.,The combined analyses, with 4,680 cases and 39,586 controls, identified 23 new loci and 7 suggestive loci, most encoding immune and apoptotic regulators, some also associated with other autoimmune diseases, as well as several melanocyte regulators.,Bioinformatic analyses indicate a predominance of causal regulatory variation, some corresponding to eQTL at these loci.,Together, the identified genes provide a framework for vitiligo genetic architecture and pathobiology, highlight relationships to other autoimmune diseases and melanoma, and offer potential targets for treatment.

Few high penetrance genes are known in Malignant Melanoma (MM), however, the involvement of low-penetrance genes such as MC1R, OCA2, ASIP, SLC45A2 and TYR has been observed.,Lately, genome-wide association studies (GWAS) have been the ideal strategy to identify new common, low-penetrance susceptibility loci.,In this case-control study, we try to validate in our population nine melanoma associated markers selected from published GWAS in melanoma predisposition.,We genotyped the 9 markers corresponding to 8 genes (PARP1, MX2, ATM, CCND1, NADSYN1, CASP8, IRF4 and CYP2R1) in 566 cases and 347 controls from a Spanish population using KASPar probes.,Genotypes were analyzed by logistic regression and adjusted by phenotypic characteristics.,We confirm the protective role in MM of the rs3219090 located on the PARP1 gene (p-value 0.027).,Additionally, this SNP was also associated with eye color (p-value 0.002).,A second polymorphism, rs12203592, located on the IRF4 gene was associated with protection to develop MM for the dominant model (p-value 0.037).,We have also observed an association of this SNP with both lentigines (p-value 0.014) and light eye color (p-value 3.76 × 10-4).,Furthermore, we detected a novel association with rs1485993, located on the CCND1 gene, and dark eye color (p-value 4.96 × 10-4).,Finally, rs1801516, located on the ATM gene, showed a trend towards a protective role in MM similar to the one firstly described in a GWAS study.,To our knowledge, this is the first time that these SNPs have been associated with MM in a Spanish population.,We confirmed the proposed role of rs3219090, located on the PARP1 gene, and rs12203592, located on the IRF4 gene, as protective to MM along the same lines as have previous genome-wide associated works.,Finally, we have seen associations between IRF4, PARP1, and CCND1 and phenotypic characteristics, confirming previous results for the IRF4 gene and presenting novel data for the last two, suggesting that pigmentation characteristics correlated with eye color are potential mediators between PARP1 and MM protection.

NRAS and its effector BRAF are frequently mutated in melanoma.,Paradoxically, CRAF but not BRAF was shown to be critical for various RAS-driven cancers, raising the question of the role of RAF proteins in NRAS-induced melanoma.,Here, using conditional ablation of Raf genes in NRAS-induced mouse melanoma models, we investigate their contribution in tumour progression, from the onset of benign tumours to malignant tumour maintenance.,We show that BRAF expression is required for ERK activation and nevi development, demonstrating a critical role in the early stages of NRAS-driven melanoma.,After melanoma formation, single Braf or Craf ablation is not sufficient to block tumour growth, showing redundant functions for RAF kinases.,Finally, proliferation of resistant cells emerging in the absence of BRAF and CRAF remains dependent on ARAF-mediated ERK activation.,These results reveal specific and compensatory functions for BRAF and CRAF and highlight an addiction to RAF signalling in NRAS-driven melanoma.,The melanoma-driver mutations in NRAS and BRAF are mutually exclusive but the contribution of RAF signalling downstream of NRAS remains to be clarified.,Here, using mouse models, the authors show specific roles of each member of the RAF family at different stages of melanomagenesis.

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.

Patients with metastatic melanoma bearing V600 mutations in BRAF oncogene clinically benefit from the treatment with BRAF inhibitors alone or in combination with MEK inhibitors.,However, a limitation to such treatment is the occurrence of resistance.,Tackling the adaptive changes helping cells survive from drug treatment may offer new therapeutic opportunities.,Very recently the ErbB3 receptor has been shown to act as a central node promoting survival of BRAF mutated melanoma.,In this paper we first demonstrate that ErbB3/AKT hyperphosphorylation occurs in BRAF mutated melanoma cell lines following exposure to BRAF and/or MEK inhibitors.,This strongly correlates with increased transcriptional activation of its ligand neuregulin.,Anti-ErbB3 antibodies impair the establishment of de novo cell resistance to BRAF inhibition in vitro.,In order to more potently ablate ErbB3 activity we used a combination of two anti-ErbB3 antibodies directed against distinct epitopes of its extracellular domain.,These two antibodies in combo with BRAF/MEK inhibitors potently inhibit in vitro cell growth and tumor regrowth after drug withdrawal in an in vivo xenograft model.,Importantly, residual tumor masses from mice treated by the antibodies and BRAF/ERK inhibitors combo are characterized almost exclusively by large necrotic areas with limited residual areas of tumor growth.,Taken together, our findings support the concept that triple therapy directed against BRAF/MEK/ErbB3 may be able to provide durable control of BRAF mutated metastatic melanoma.

Melanoma was again a focus of attention at the 2015 American Society of Clinical Oncology (ASCO) Annual Meeting, in particular the use of combination treatment strategies involving immunotherapies and/or targeted agents.,New data on targeted therapies confirmed previous findings, with combined BRAF inhibitor (vemurafenib) plus MEK inhibitor (cobimetinib) improving progression-free survival (PFS) compared to vemurafenib monotherapy in patients with BRAFV600 mutation-positive tumors (CoBRIM trial).,Positive results were also seen with combined dabrafenib and trametinib in patients with BRAF V600E/K metastatic melanoma and encorafenib plus binimetinib in BRAFV600-mutant cutaneous melanoma.,Even more interesting news centered on the use of combination immunotherapy, in particular the randomized, double-blind CheckMate 067 study in which median PFS with nivolumab plus ipilimumab was 11.5 months, compared to 2.9 months with ipilimumab alone (HR 0.42) and 6.9 months with nivolumab alone (HR 0.57).,Of interest, in patients with ≥5% PD-L1 expression, median PFS was 14 months with the combination or with nivolumab alone compared with 3.9 months in the ipilimumab group, while in the PD-L1 negative cohort, the combination remained superior to both monotherapies.,Given that combination therapy was accompanied by a high occurrence of side-effects, this raises the suggestion that combination therapy might be reserved for PD-L1 negative patients only, with PD-L1 positive patients achieving the same benefit from nivolumab monotherapy.,However, overall survival data are awaited and the equivalence of single agent to the combination remains unconvincing.,Interesting data were also reported on the combination of T-VEC (talimogene laherparepvec) with ipilimumab, and the anti-PD-1 agent MEDI4736 (durvolumab) combined with dabrafenib plus trametinib.,Emerging data also suggested that predictive markers based on immunoprofiling and mismatch repair deficiency may be of clinical use.,In conclusion, the use of combination approaches to treat patients with melanoma, as well as other cancers, is no longer a just a wish for the future but is today a clinical reality with a rapidly growing evidence-base.,Moreover, the most exciting consideration is that this is far from the end of the story, but rather a fantastic introduction.

In order to explore melanoma risk factors through gender-, age-, race-, and site-specific incidence rates, malignant melanoma cases from the Caucasian whites and non-whites were retrieved from the US SEER database.,Age-standardized, age-, and site-specific tumor rates were calculated.,All races and both genders showed positive annual average percentage changes (AAPCs) over the years, but AAPCs varied at different body sites, with men’s trunk exhibiting the fastest increase.,Non-whites were diagnosed at a significantly younger age than whites and showed a trend towards fewer gender differences in the age of diagnosis.,However, non-whites and whites showed a similar pattern of age-specific gender differences in the incidence rate ratios.,A consistent spiked difference (female vs. male, incidence rate ratio (IRR) >2) was observed at or near the age of 20-24 in all race groups and at all body sites.,The highest female vs. male IRR was found in the hip and lower extremities, and the lowest IRR was found in the head and neck region in all races.,These race-, gender-, and site-dependent differences suggest that age-associated cumulative sun exposure weighs significantly more in late-onset melanomas, while genetics and/or pathophysiological factors make important contributions to early-onset melanomas.

Melanoma incidence rates have continued to increase in the United States, and risk behaviors remain high.,Melanoma is responsible for the most skin cancer deaths, with about 9,000 persons dying from it each year.,CDC analyzed current (2011) melanoma incidence and mortality data, and projected melanoma incidence, mortality, and the cost of treating newly diagnosed melanomas through 2030.,Finally, CDC estimated the potential melanoma cases and costs averted through 2030 if a comprehensive skin cancer prevention program was implemented in the United States.,In 2011, the melanoma incidence rate was 19.7 per 100,000, and the death rate was 2.7 per 100,000.,Incidence rates are projected to increase for white males and females through 2019.,Death rates are projected to remain stable.,The annual cost of treating newly diagnosed melanomas was estimated to increase from $457 million in 2011 to $1.6 billion in 2030.,Implementation of a comprehensive skin cancer prevention program was estimated to avert 230,000 melanoma cases and $2.7 billion in initial year treatment costs from 2020 through 2030.,If additional prevention efforts are not undertaken, the number of melanoma cases is projected to increase over the next 15 years, with accompanying increases in health care costs.,Much of this morbidity, mortality, and health care cost can be prevented.,Substantial reductions in melanoma incidence, mortality, and cost can be achieved if evidence-based comprehensive interventions that reduce ultraviolet (UV) radiation exposure and increase sun protection are fully implemented and sustained.

While immune checkpoint blockade has greatly improved clinical outcomes in diseases such as melanoma, there remains a need for predictive biomarkers to determine who will likely benefit most from which therapy.,To date, most biomarkers of response have been identified in the tumors themselves.,Biomarkers that could be assessed from peripheral blood would be even more desirable, because of ease of access and reproducibility of sampling.,We used mass cytometry (CyTOF) to comprehensively profile peripheral blood of melanoma patients, in order to find predictive biomarkers of response to anti-CTLA-4 or anti-PD-1 therapy.,Using a panel of ~ 40 surface and intracellular markers, we performed in-depth phenotypic and functional immune profiling to identify potential predictive biomarker candidates.,Immune profiling of baseline peripheral blood samples using CyTOF revealed that anti-CTLA-4 and anti-PD-1 therapies have distinct sets of candidate biomarkers.,The distribution of CD4+ and CD8+ memory/non-memory cells and other memory subsets was different between responders and non-responders to anti-CTLA-4 therapy.,In anti-PD-1 (but not anti-CTLA-4) treated patients, we discovered differences in CD69 and MIP-1β expressing NK cells between responders and non-responders.,Finally, multivariate analysis was used to develop a model for the prediction of response.,Our results indicate that anti-CTLA-4 and anti-PD-1 have distinct predictive biomarker candidates.,CD4+ and CD8+ memory T cell subsets play an important role in response to anti-CTLA-4, and are potential biomarker candidates.,For anti-PD-1 therapy, NK cell subsets (but not memory T cell subsets) correlated with clinical response to therapy.,These functionally active NK cell subsets likely play a critical role in the anti-tumor response triggered by anti-PD-1.,The online version of this article (10.1186/s40425-018-0328-8) contains supplementary material, which is available to authorized users.

Retreatment with ipilimumab has been shown to re-establish disease control in some patients with disease progression.,Here, we report the efficacy and safety of retreatment with ipilimumab 3 mg kg−1 among patients participating in an expanded access programme in Italy.,Patients who achieved disease control during induction therapy were retreated with ipilimumab upon progression (3 mg kg−1 every 3 weeks for up to four doses), providing they had not experienced toxicity that precluded further dosing.,Tumour assessments were conducted after retreatment, and patients were monitored throughout for adverse events.,Of 855 patients treated with ipilimumab, 51 were retreated upon disease progression.,Of these, 28 (55%) regained disease control upon retreatment and 42% were alive 2 years after the first induction dose of ipilimumab; median overall survival was 21 months.,Eleven patients (22%) had a treatment-related adverse event of any grade during retreatment.,These were generally mild-to-moderate and resolved within a median of 4 days.,No new types of toxicity were reported.,For patients who meet predefined criteria, retreatment with ipilimumab is generally well tolerated and can translate into clinical benefit.,This strategy should be compared with other therapeutic options in randomised controlled trials.

Immune checkpoint inhibitors have improved the prognosis of advanced melanoma.,Although anti-programmed death ligand‐1 (PD‐L1) is a well‐studied biomarker for response to anti-programmed death‐1 PD‐1 therapy in melanoma, its clinical relevance remains unclear.,It has been established that the high expression of indoleamine 2,3‐dioxygenase (IDO) is correlated to a response to anti-CTLA‐4 treatment in melanoma.,However, it is still unknown whether the IDO expression is associated with response to anti-PD‐1 therapy in advanced melanoma.,In addition, acral and mucosal melanomas, which comprise a great proportion of all melanomas in Asians, are genetically different subtypes from cutaneous melanomas; however, they have not been independently analyzed due to their low frequency in Western countries.,To evaluate the association of IDO and PD‐L1 expression with response to anti-PD‐1 antibody in acral and mucosal melanoma patients, we analyzed 32 Japanese patients with acral and mucosal melanomas treated with anti-PD‐1 antibody from the perspective of IDO and PD‐L1 expression levels by immunohistochemistry (IHC).,Multivariate Cox regression models showed that the low expression of IDO in tumors was associated with poor progression‐free survival (HR = 0.33, 95% CI = 0.13‐0.81, P = 0.016), whereas PD‐L1 expression on tumors was not associated with progression‐free survival.,Significantly lower expression of IDO in tumors was found in non-responders compared to responders.,Assessment of the IDO expression could be useful for the identification of suitable candidates for anti-PD‐1 therapy among acral and mucosal melanomas patients.,Further validation study is needed to estimate the clinical utility of our findings.

Treatment with programmed death receptor-1 (PD-1) antibodies is associated with high response rates in patients with advanced melanoma.,Reliable markers for early response and outcome are still sparse.,We evaluated 66 consecutive patients with advanced/metastatic melanoma treated with nivolumab or pembrolizumab between 2013 and 2014.,The main objectives of this study were to investigate whether, first, serum lactate dehydrogenase (LDH) at baseline (normal vs above the upper limit of normal) correlates with overall survival (OS), and, second, whether the change of LDH during treatment predicts response before the first scan and OS in patients with an elevated baseline LDH.,After a median follow-up of 9 months, patients with an elevated baseline LDH (N=34) had a significantly shorter OS compared with patients with normal LDH (N=32; 6-month OS: 60.8% vs 81.6% and 12-month OS: 44.2% vs 71.5% (log-rank P=0.0292).,In those 34 patients with elevated baseline LDH, the relative change during treatment was significantly associated with an objective response on the first scan: the 11 (32%) patients with partial remission had a mean reduction of −27.3% from elevated baseline LDH.,In contrast, patients with progressive disease (N=15) had a mean increase of +39%.,Patients with a relative increase over 10% from elevated baseline LDH had a significantly shorter OS compared with patients with ⩽10% change (4.3 vs 15.7 months, log-rank P<0.00623).,LDH could be a useful marker at baseline and during treatment to predict early response or progression in patients with advanced melanoma who receive anti-PD-1 therapy.

In the KEYNOTE-022 study, pembrolizumab with dabrafenib and trametinib (triplet) improved progression-free survival (PFS) versus placebo with dabrafenib and trametinib (doublet) without reaching statistical significance.,Mature results on PFS, duration of response (DOR), and overall survival (OS) are reported.,The double-blind, phase 2 part of KEYNOTE-022 enrolled patients with previously untreated BRAF V600E/K-mutated advanced melanoma from 22 sites in seven countries.,Patients were randomly assigned 1:1 to intravenous pembrolizumab (200 mg every 3 weeks) or placebo plus dabrafenib (150 mg orally two times per day) and trametinib (2 mg orally one time a day).,Primary endpoint was PFS.,Secondary endpoints were objective response rate, DOR, and OS.,Efficacy was assessed in the intention-to-treat population, and safety was assessed in all patients who received at least one dose of study drug.,This analysis was not specified in the protocol.,Between November 30, 2015 and April 24, 2017, 120 patients were randomly assigned to triplet (n=60) or doublet (n=60) therapy.,With 36.6 months of follow-up, median PFS was 16.9 months (95% CI 11.3 to 27.9) with triplet and 10.7 months (95% CI 7.2 to 16.8) with doublet (HR 0.53; 95% CI 0.34 to 0.83).,With triplet and doublet, respectively, PFS at 24 months was 41.0% (95% CI 27.4% to 54.2%) and 16.3% (95% CI 8.1% to 27.1%); median DOR was 25.1 months (95% CI 14.1 to not reached) and 12.1 months (95% CI 6.0 to 15.7), respectively.,Median OS was not reached with triplet and was 26.3 months with doublet (HR 0.64; 95% CI 0.38 to 1.06).,With triplet and doublet, respectively, OS at 24 months was 63.0% (95% CI 49.4% to 73.9%) and 51.7% (95% CI 38.4% to 63.4%).,Grade 3-5 treatment-related adverse events (TRAEs) occurred in 35 patients (58%, including one death) receiving triplet and 15 patients (25%) receiving doublet.,In BRAF V600E/K-mutant advanced melanoma, pembrolizumab plus dabrafenib and trametinib substantially improved PFS, DOR, and OS with a higher incidence of TRAEs.,Interpretation of these results is limited by the post hoc nature of the analysis.

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.

Merkel cell carcinoma (MCC) is a highly aggressive, neuroendocrine skin cancer that lacks actionable mutations, which could be utilized for targeted therapies.,Epigenetic regulators governing cell identity may represent unexplored therapeutic entry points.,Here, we targeted epigenetic regulators in a pharmacological screen and discovered that the lysine‐specific histone demethylase 1A (LSD1/KDM1A) is required for MCC growth in vitro and in vivo.,We show that LSD1 inhibition in MCC disrupts the LSD1‐CoREST complex leading to displacement and degradation of HMG20B (BRAF35), a poorly characterized complex member that is essential for MCC proliferation.,Inhibition of LSD1 causes derepression of transcriptional master regulators of the neuronal lineage, activates a gene expression signature resembling normal Merkel cells, and induces cell cycle arrest and cell death.,Our study unveils the importance of LSD1 for maintaining cellular plasticity and proliferation in MCC.,There is also growing evidence that cancer cells exploit cellular plasticity and dedifferentiation programs to evade destruction by the immune system.,The combination of LSD1 inhibitors with checkpoint inhibitors may thus represent a promising treatment strategy for MCC patients.,This study identifies that the integrity of the LSD1‐CoREST complex is essential for Merkel cell carcinoma (MCC) proliferation and maintaining cell identity.,LSD1 inhibition causes derepression of transcriptional regulators of the neuronal lineage and is a novel entry point for targeted therapies in MCC.

Merkel cell carcinoma (MCC) is a rare and aggressive, yet highly immunogenic skin cancer.,The latter is due to its viral or UV-associated carcinogenesis.,For tumor progression MCC has to escape the host’s immuno-surveillance, e.g. by loss of HLA class-I expression.,Indeed, a reduced HLA class-I expression was observed in MCC tumor tissues and MCC cell lines.,This reduced HLA class-I surface expression is caused by an impaired expression of key components of the antigen processing machinery (APM), including LMP2 and LMP7 as well as TAP1 and TAP2.,Notably, experimental provisions of HLA class-I binding peptides restored HLA class-I surface expression on MCC cells.,Silencing of the HLA class-I APM is due to histone deacetylation as inhibition of histone deacetylases (HDACs) not only induced acetylation of histones in the respective promoter regions but also re-expression of APM components.,Thus, HDAC inhibition restored HLA class-I surface expression in vitro and in a mouse xenotransplantation model.,In contrast to re-induction of HLA class-I by interferons, HDAC inhibitors did not interfere with the expression of immuno-dominant viral proteins.,In summary, restoration of HLA class-I expression on MCC cells by epigenetic priming is an attractive approach to enhance therapies boosting adaptive immune responses.

Over 100,000 people are diagnosed with cutaneous melanoma each year in the United States.,Despite recent advancements in metastatic melanoma treatment, such as immunotherapy, there are still over 7000 melanoma-related deaths each year.,Melanoma is a highly heterogenous disease, and many underlying genetic drivers have been identified since the introduction of next-generation sequencing.,Despite clinical staging guidelines, the prognosis of metastatic melanoma is variable and difficult to predict.,Bioinformatic and machine learning analyses relying on genetic, clinical, and histopathologic inputs have been increasingly used to risk stratify melanoma patients with high accuracy.,This literature review summarizes the key genetic drivers of melanoma and recent applications of bioinformatic and machine learning models in the risk stratification of melanoma patients.,A robustly validated risk stratification tool can potentially guide the physician management of melanoma patients and ultimately improve patient outcomes.

Melanoma is the most aggressive and dangerous type of skin cancer, but its molecular mechanisms remain largely unclear.,For transcriptomic data of 478 primary and metastatic melanoma, nevi and normal skin samples, we performed high-throughput analysis of intracellular molecular networks including 592 signaling and metabolic pathways.,We showed that at the molecular pathway level, the formation of nevi largely resembles transition from normal skin to primary melanoma.,Using a combination of bioinformatic machine learning algorithms, we identified 44 characteristic signaling and metabolic pathways connected with the formation of nevi, development of primary melanoma, and its metastases.,We created a model describing formation and progression of melanoma at the level of molecular pathway activation.,We discovered six novel associations between activation of metabolic molecular pathways and progression of melanoma: for allopregnanolone biosynthesis, L-carnitine biosynthesis, zymosterol biosynthesis (inhibited in melanoma), fructose 2, 6-bisphosphate synthesis and dephosphorylation, resolvin D biosynthesis (activated in melanoma), D-myo-inositol hexakisphosphate biosynthesis (activated in primary, inhibited in metastatic melanoma).,Finally, we discovered fourteen tightly coordinated functional clusters of molecular pathways.,This study helps to decode molecular mechanisms underlying the development of melanoma.

The non-coding microRNAs (miRNA) have tissue- and disease-specific expression patterns.,They down-regulate target mRNAs, which likely impacts on most fundamental cellular processes.,Differential expression patterns of miRNAs are currently being exploited for identification of biomarkers for early disease diagnosis, prediction of progression for melanoma and other cancers and as promising drug targets, since they can easily be inhibited or replaced in a given cellular context.,Before successfully manipulating miRNAs in clinical settings, their precise expression levels, endogenous functions and thus their target genes have to be determined.,MiR-211, a melanocyte lineage-specific small non-coding miRNA, is located in an intron of TRPM1, a target gene of the microphtalmia-associated transcription factor (MITF).,By transcriptionally up-regulating TRPM1, MITF, which is critical for both melanocyte differentiation and survival and for melanoma progression, indirectly drives the expression of miR-211.,Expression of this miRNA is often reduced in melanoma samples.,Here, we investigated functional roles of miR-211 by identifying and studying new target genes.,We show that MITF-correlated miR-211 expression levels are mostly but not always reduced in a panel of 11 melanoma cell lines and in primary and metastatic melanoma compared to normal melanocytes and nevi, respectively.,MiR-211 itself only marginally impacted on cell invasion and migration, while perturbation of some new miR-211 target genes, such as AP1S2, SOX11, IGFBP5, and SERINC3 significantly increased invasion.,These results and the variable expression levels of miR-211 raise serious doubts on the value of miR-211 as a melanoma tumor-suppressing miRNA and/or as a biomarker for melanoma.

Serum lactate dehydrogenase (LDH) is a prognostic factor for patients with stage IV melanoma.,To gain insights into the biology underlying this prognostic factor, we analyzed total serum LDH, serum LDH isoenzymes, and serum lactate in up to 49 patients with metastatic melanoma.,Our data demonstrate that high serum LDH is associated with a significant increase in LDH isoenzymes 3 and 4, and a decrease in LDH isoenzymes 1 and 2.,Since LDH isoenzymes play a role in both glycolysis and oxidative phosphorylation (OXPHOS), we subsequently determined using tissue microarray (TMA) analysis that the levels of proteins associated with mitochondrial function, lactate metabolism, and regulators of glycolysis were all elevated in advanced melanomas compared with nevic melanocytes.,To investigate whether in advanced melanoma, the glycolysis and OXPHOS pathways might be linked, we determined expression of the monocarboxylate transporters (MCT) 1 and 4.,Analysis of a nevus-to-melanoma progression TMA revealed that MCT4, and to a lesser extend MCT1, were elevated with progression to advanced melanoma.,Further analysis of human melanoma specimens using the Seahorse XF24 extracellular flux analyzer indicated that metastatic melanoma tumors derived a large fraction of energy from OXPHOS.,Taken together, these findings suggest that in stage IV melanomas with normal serum LDH, glycolysis and OXPHOS may provide metabolic symbiosis within the same tumor, whereas in stage IV melanomas with high serum LDH glycolysis is the principle source of energy.

In response to the dynamic intra‐tumor microenvironment, melanoma cells adopt distinct phenotypic states associated with differential expression of the microphthalmia‐associated transcription factor (MITF).,The response to hypoxia is driven by hypoxia‐inducible transcription factors (HIFs) that reprogram metabolism and promote angiogenesis.,HIF1α indirectly represses MITF that can activate HIF1α expression.,Although HIF and MITF share a highly related DNA‐binding specificity, it is unclear whether they co‐regulate subset of target genes.,Moreover, the genomewide impact of hypoxia on melanoma and whether melanoma cell lines representing different phenotypic states exhibit distinct hypoxic responses is unknown.,Here we show that three different melanoma cell lines exhibit widely different hypoxia responses with only a core 23 genes regulated in common after 12 hr in hypoxia.,Surprisingly, under hypoxia MITF is transiently up‐regulated by HIF1α and co‐regulates a subset of HIF targets including VEGFA.,Significantly, we also show that MITF represses itself and also regulates SDHB to control the TCA cycle and suppress pseudo‐hypoxia.,Our results reveal a previously unsuspected role for MITF in metabolism and the network of factors underpinning the hypoxic response in melanoma.

Extensive research has demonstrated a tumor‐promoting role of increased WNT5A expression in malignant melanoma.,However, very little light has been shed upon how WNT5A expression is up‐regulated in melanoma.,A potential regulator of WNT5A expression is the pro‐inflammatory cytokine Interleukin (IL)‐6, which shares the ability of WNT5A to increase melanoma cell invasion.,Here, we investigate whether IL‐6 can promote melanoma cell motility through an increased expression of WNT5A.,We clearly demonstrate that the WNT5A‐antagonistic peptide Box5 could inhibit IL‐6‐induced melanoma cell migration and invasion.,Furthermore, IL‐6 stimulation of the human melanoma cell lines HTB63 and A375 increased the expression of WNT5A in a dose‐dependent manner.,To identify the signaling mechanism responsible for this up‐regulation, we explored the involvement of the three main signals induced by IL‐6; STAT3, Akt and ERK 1/2.,Of these, only STAT3 was activated by IL‐6 in the melanoma cell lines tested.,However, the STAT3 inhibitor S3I‐201 failed to inhibit IL‐6‐induced WNT5A up‐regulation in HTB63 and A375 cells.,Nor did STAT3 siRNA silencing affect the expression of WNT5A.,In search of an alternative signaling mechanism, we detected IL‐6‐induced activation of p38‐MAPK in HTB63 and A375 cells.,The p38‐MAPK inhibitor SB203580 abolished the IL‐6‐induced WNT5A up‐regulation and blocked IL‐6‐induced melanoma cell invasion.,The latter effect could be rescued by the addition of recombinant WNT5A.,Notably, immunoprecipitation analysis revealed that only the p38α‐MAPK isoform was activated by IL‐6, and subsequent siRNA silencing of p38α‐MAPK abolished the IL‐6‐induced up‐regulation of WNT5A.,Taken together, we demonstrate a novel link between the two melanoma pro‐metastatic agents IL‐6 and WNT5A explaining how IL‐6 can increase melanoma cell invasion and thus promote the metastatic process.,This finding provides a basis for future therapeutic intervention of melanoma progression.,We provide a novel link between the melanoma pro‐metastatic agents IL‐6 and WNT5A.WNT5A signaling plays an important role in IL‐6‐induced melanoma cell motility.IL‐6 can increase WNT5A expression by STAT3‐independent signaling in melanoma cells.IL‐6‐induced WNT5A expression is mediated through p38α‐MAPK activation.,We provide a novel link between the melanoma pro‐metastatic agents IL‐6 and WNT5A.,WNT5A signaling plays an important role in IL‐6‐induced melanoma cell motility.,IL‐6 can increase WNT5A expression by STAT3‐independent signaling in melanoma cells.,IL‐6‐induced WNT5A expression is mediated through p38α‐MAPK activation.

Cutaneous squamous cell carcinoma (cuSCC) comprises 15-20% of all skin cancers, accounting for over 700,000 cases in USA annually.,Most cuSCC arise in association with a distinct precancerous lesion, the actinic keratosis (AK).,To identify potential targets for molecularly targeted chemoprevention, here we perform integrated cross-species genomic analysis of cuSCC development through the preneoplastic AK stage using matched human samples and a solar ultraviolet radiation-driven Hairless mouse model.,We identify the major transcriptional drivers of this progression sequence, showing that the key genomic changes in cuSCC development occur in the normal skin to AK transition.,Our data validate the use of this ultraviolet radiation-driven mouse cuSCC model for cross-species analysis and demonstrate that cuSCC bears deep molecular similarities to multiple carcinogen-driven SCCs from diverse sites, suggesting that cuSCC may serve as an effective, accessible model for multiple SCC types and that common treatment and prevention strategies may be feasible.,Cutaneous squamous cell of the skin is a common neoplasm that frequently arises from precancerous actinic keratoses.,Here, the authors carry out genomic analysis on matched sets of human lesions and compare with those in ultraviolet treated mice and identify conserved drivers of tumour development.

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.

Metastasis development represents an important threat for melanoma patients, even when diagnosed at early stages and upon removal of the primary tumor.,In this scenario, determination of prognostic biomarkers would be of great interest.,Serum contains information about the general status of the organism and therefore represents a valuable source for biomarkers.,Thus, we aimed to define serological biomarkers that could be used along with clinical and histopathological features of the disease to predict metastatic events on the early‐stage population of patients.,We previously demonstrated that in stage II melanoma patients, serum levels of dermcidin (DCD) were associated with metastatic progression.,Based on the relevance of the immune response on the cancer progression and the recent association of DCD with local and systemic immune response against cancer cells, serum DCD was analyzed in a new cohort of patients along with interleukin 4 (IL‐4), IL‐6, IL‐10, IL‐17A, interferon γ (IFN‐γ), transforming growth factor‐β (TGF‐ β), and granulocyte-macrophage colony‐stimulating factor (GM‐CSF).,We initially recruited 448 melanoma patients, 323 of whom were diagnosed as stages I‐II according to AJCC.,Levels of selected cytokines were determined by ELISA and Luminex, and obtained data were analyzed employing machine learning and Kaplan-Meier techniques to define an algorithm capable of accurately classifying early‐stage melanoma patients with a high and low risk of developing metastasis.,The results show that in early‐stage melanoma patients, serum levels of the cytokines IL‐4, GM‐CSF, and DCD together with the Breslow thickness are those that best predict melanoma metastasis.,Moreover, resulting algorithm represents a new tool to discriminate subjects with good prognosis from those with high risk for a future metastasis.,Melanoma displays a remarkable capacity for dissemination even when detected at early stages.,We developed a decision rule that considers Breslow thickness of the removed malignant lesion and IL‐4, GM‐CSF, and DCD serum levels in order to foresee the risk for future metastasis development of those stage I‐II patients.,This algorithm may represent a tool to design more personalized follow‐up strategies.

Melanoma is the most aggressive and dangerous type of skin cancer, but its molecular mechanisms remain largely unclear.,For transcriptomic data of 478 primary and metastatic melanoma, nevi and normal skin samples, we performed high-throughput analysis of intracellular molecular networks including 592 signaling and metabolic pathways.,We showed that at the molecular pathway level, the formation of nevi largely resembles transition from normal skin to primary melanoma.,Using a combination of bioinformatic machine learning algorithms, we identified 44 characteristic signaling and metabolic pathways connected with the formation of nevi, development of primary melanoma, and its metastases.,We created a model describing formation and progression of melanoma at the level of molecular pathway activation.,We discovered six novel associations between activation of metabolic molecular pathways and progression of melanoma: for allopregnanolone biosynthesis, L-carnitine biosynthesis, zymosterol biosynthesis (inhibited in melanoma), fructose 2, 6-bisphosphate synthesis and dephosphorylation, resolvin D biosynthesis (activated in melanoma), D-myo-inositol hexakisphosphate biosynthesis (activated in primary, inhibited in metastatic melanoma).,Finally, we discovered fourteen tightly coordinated functional clusters of molecular pathways.,This study helps to decode molecular mechanisms underlying the development of melanoma.

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.

Mucosal melanoma is an aggressive melanoma with poor prognosis.,We assessed efficacy of pembrolizumab in patients with advanced mucosal melanoma in KEYNOTE-001 (NCT01295827), −002 (NCT01704287), and −006 (NCT01866319).,Patients received pembrolizumab 2 mg/kg every 3 weeks (Q3W) or 10 mg/kg Q2W or Q3W.,Response was assessed by independent central review per RECIST v1.1.,1567 patients were treated and 84 (5%) had mucosal melanoma.,Fifty-one of 84 were ipilimumab-naive.,In patients with mucosal melanoma, the objective response rate (ORR) was 19% (95% CI 11-29%), with median duration of response (DOR) of 27.6 months (range 1.1 + to 27.6).,Median progression-free survival (PFS) was 2.8 months (95% CI 2.7-2.8), with median overall survival (OS) of 11.3 months (7.7-16.6).,ORR was 22% (95% CI 11-35%) and 15% (95% CI 5-32%) in ipilimumab-naive and ipilimumab-treated patients.,Pembrolizumab provides durable antitumour activity in patients with advanced mucosal melanoma regardless of prior ipilimumab.

Metastatic melanoma is a malignant cancer with generally poor prognosis, with no targeted chemotherapy.,To identify epigenetic changes related to melanoma, we have determined genome-wide methylated CpG island distributions by next-generation sequencing.,Melanoma chromosomes tend to be differentially methylated over short CpG island tracts.,CpG islands in the upstream regulatory regions of many coding and noncoding RNA genes, including, for example, TERC, which encodes the telomerase RNA, exhibit extensive hypermethylation, whereas several repeated elements, such as LINE 2, and several LTR elements, are hypomethylated in advanced stage melanoma cell lines.,By using CpG island demethylation profiles, and by integrating these data with RNA-seq data obtained from melanoma cells, we have identified a co-expression network of differentially methylated genes with significance for cancer related functions.,Focused assays of melanoma patient tissue samples for CpG island methylation near the noncoding RNA gene SNORD-10 demonstrated high specificity.

The clinical course of cutaneous melanoma (CM) can differ significantly for patients with identical stages of disease, defined clinico-pathologically, and no molecular markers differentiate patients with such a diverse prognosis.,This study aimed to define the prognostic value of whole genome DNA methylation profiles in stage III CM.,Genome-wide methylation profiles were evaluated by the Illumina Human Methylation 27 BeadChip assay in short-term neoplastic cell cultures from 45 stage IIIC CM patients.,Unsupervised K-means partitioning clustering was exploited to sort patients into 2 groups based on their methylation profiles.,Methylation patterns related to the discovered groups were determined using the nearest shrunken centroid classification algorithm.,The impact of genome-wide methylation patterns on overall survival (OS) was assessed using Cox regression and Kaplan-Meier analyses.,Unsupervised K-means partitioning by whole genome methylation profiles identified classes with significantly different OS in stage IIIC CM patients.,Patients with a “favorable” methylation profile had increased OS (P = 0.001, log-rank = 10.2) by Kaplan-Meier analysis.,Median OS of stage IIIC patients with a “favorable” vs. “unfavorable” methylation profile were 31.5 and 10.4 months, respectively.,The 5 year OS for stage IIIC patients with a “favorable” methylation profile was 41.2% as compared to 0% for patients with an “unfavorable” methylation profile.,Among the variables examined by multivariate Cox regression analysis, classification defined by methylation profile was the only predictor of OS (Hazard Ratio = 2.41, for “unfavorable” methylation profile; 95% Confidence Interval: 1.02-5.70; P = 0.045).,A 17 gene methylation signature able to correctly assign prognosis (overall error rate = 0) in stage IIIC patients on the basis of distinct methylation-defined groups was also identified.,A discrete whole-genome methylation signature has been identified as molecular marker of prognosis for stage IIIC CM patients.,Its use in daily practice is foreseeable, and promises to refine the comprehensive clinical management of stage III CM patients.

The focus of the present review is to investigate the role of melanin in the radioprotection of melanoma and attempts to sensitize tumors to radiation by inhibiting melanogenesis.,Early studies showed radical scavenging, oxygen consumption and adsorption as mechanisms of melanin radioprotection.,Experimental models of melanoma in hamsters and in gerbils are described as well as their use in biochemical and radiobiological studies, including a spontaneously metastasizing ocular model.,Some results from in vitro studies on the inhibition of melanogenesis are presented as well as radio-chelation therapy in experimental and clinical settings.,In contrast to cutaneous melanoma, uveal melanoma is very successfully treated with radiation, both using photon and proton beams.,We point out that the presence or lack of melanin pigmentation should be considered, when choosing therapeutic options, and that both the experimental and clinical data suggest that melanin could be a target for radiosensitizing melanoma cells to increase efficacy of radiotherapy against melanoma.

In less than 10 years, melanoma treatment has been revolutionized with the approval of tyrosine kinase inhibitors and immune checkpoint inhibitors, which have been shown to have a significant impact on the prognosis of patients with melanoma.,The early steps of this transformation have taken place in research laboratories.,The mitogen-activated protein kinase (MAPK) pathway, phosphoinositol-3-kinase (PI3K) pathway promote the development of melanoma through numerous genomic alterations on different components of these pathways.,Moreover, melanoma cells deeply interact with the tumor microenvironment and the immune system.,This knowledge has led to the identification of novel therapeutic targets and treatment strategies.,In this review, the epidemiological features of cutaneous melanoma along with the biological mechanisms involved in its development and progression are summarized.,The current state-of-the-art of advanced stage melanoma treatment strategies and the currently available evidence of the use of predictive and prognostic biomarkers are also discussed.

An impressive clinical success has been observed in treating a variety of cancers using immunotherapy with programmed cell death‐1 (PD‐1) checkpoint blockade.,However, limited response in most patients treated with anti‐PD‐1 antibodies remains a challenge, requiring better understanding of molecular mechanisms limiting immunotherapy.,In colorectal cancer (CRC) resistant to immunotherapy, mismatch‐repair‐proficient or microsatellite instability‐low (pMMR‐MSI‐L) tumors have low mutation burden and constitute ~85% of patients.,Here, we show that inhibition of N 6‐methyladenosine (m6A) mRNA modification by depletion of methyltransferases, Mettl3 and Mettl14, enhanced response to anti‐PD‐1 treatment in pMMR‐MSI‐L CRC and melanoma.,Mettl3‐ or Mettl14‐deficient tumors increased cytotoxic tumor‐infiltrating CD8+ T cells and elevated secretion of IFN‐γ, Cxcl9, and Cxcl10 in tumor microenvironment in vivo.,Mechanistically, Mettl3 or Mettl14 loss promoted IFN‐γ‐Stat1‐Irf1 signaling through stabilizing the Stat1 and Irf1 mRNA via Ythdf2.,Finally, we found a negative correlation between METTL3 or METTL14 and STAT1 in 59 patients with pMMR‐MSI‐L CRC tumors.,Altogether, our findings uncover a new awareness of the function of RNA methylation in adaptive immunity and provide METTL3 and METTL14 as potential therapeutic targets in anticancer immunotherapy.,Disruption of m6A methyltransferases leads to enhanced immunotherapy response in colorectal cancer and melanoma cells due to enhanced IFN‐γ‐Stat1‐Irf1 signaling and modulation of the tumor microenvironment.

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.

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.

Uveal melanoma (UM) is characterized by mutually exclusive activating mutations in GNAQ, GNA11, CYSLTR2, and PLCB4, four genes in a linear pathway to activation of PLCβ in almost all tumors and loss of BAP1 in the aggressive subset.,We generated mice with melanocyte-specific expression of GNA11Q209L with and without homozygous Bap1 loss.,The GNA11Q209L mice recapitulated human Gq-associated melanomas, and they developed pigmented neoplastic lesions from melanocytes of the skin and non-cutaneous organs, including the eye and leptomeninges, as well as at atypical sites, including the lymph nodes and lungs.,The addition of Bap1 loss increased tumor proliferation and cutaneous melanoma size.,Integrative transcriptome analysis of human and murine melanomas identified RasGRP3 to be specifically expressed in GNAQ/GNA11-driven melanomas.,In human UM cell lines and murine models, RasGRP3 is specifically required for GNAQ/GNA11-driven Ras activation and tumorigenesis.,This implicates RasGRP3 as a critical node and a potential target in UM.,Moore et al. generate a preclinical mouse model of melanoma that recapitulates features of aggressive uveal melanoma.,By comparing murine and human melanomas, they identify a dependency on RasGRP3 in uveal melanoma.

Malignant melanoma is one of the most metastatic cancer types, and despite recent success with novel treatment strategies, there is still a group of patients who do not respond to any therapies.,Earlier, the prenylation inhibitor hydrophilic bisphosphonate zoledronic acid (ZA) was found to inhibit melanoma growth in vitro, but only a weaker effect was observed in vivo due to its hydrophilic properties.,Recently, lipophilic bisphosphonates (such as BPH1222) were developed.,Accordingly, for the first time, we compared the effect of BPH1222 to ZA in eight melanoma lines using viability, cell-cycle, clonogenic and spheroid assays, videomicroscopy, immunoblot, and xenograft experiments.,Based on 2D and spheroid assays, the majority of cell lines were more sensitive to BPH.,The activation of Akt and S6 proteins, but not Erk, was inhibited by BPH.,Additionally, BPH had a stronger apoptotic effect than ZA, and the changes of Rheb showed a correlation with apoptosis.,In vitro, only M24met cells were more sensitive to ZA than to BPH; however, in vivo growth of M24met was inhibited more strongly by BPH.,Here, we present that lipophilic BPH is more effective on melanoma cells than ZA and identify the PI3K pathway, particularly Rheb as an important mediator of growth inhibition.

Using unique computer-assisted 3D reconstruction software, it was previously demonstrated that tumorigenic cell lines derived from breast tumors, when seeded in a 3D Matrigel model, grew as clonal aggregates which, after approximately 100 hours, underwent coalescence mediated by specialized cells, eventually forming a highly structured large spheroid.,Non-tumorigenic cells did not undergo coalescence.,Because histological sections of melanomas forming in patients suggest that melanoma cells migrate and coalesce to form tumors, we tested whether they also underwent coalescence in a 3D Matrigel model.,Melanoma cells exiting fragments of three independent melanomas or from secondary cultures derived from them, and cells from the melanoma line HTB-66, all underwent coalescence mediated by specialized cells in the 3D model.,Normal melanocytes did not.,However, coalescence of melanoma cells differed from that of breast-derived tumorigenic cell lines in that they 1) coalesced immediately, 2) underwent coalescence as individual cells as well as aggregates, 3) underwent coalescence far faster and 4) ultimately formed long, flat, fenestrated aggregates that were extremely dynamic.,A screen of 51 purified monoclonal antibodies (mAbs) targeting cell surface-associated molecules revealed that two mAbs, anti-beta 1 integrin/(CD29) and anti-CD44, blocked melanoma cell coalescence.,They also blocked coalescence of tumorigenic cells derived from a breast tumor.,These results add weight to the commonality of coalescence as a characteristic of tumorigenic cells, as well as the usefulness of the 3D Matrigel model and software for both investigating the mechanisms regulating tumorigenesis and screening for potential anti-tumorigenesis mAbs.

Solid organ transplantation is associated with increased risk of non-melanoma skin cancer.,Studies with short follow up times have suggested a reduced occurrence of these cancers in recipients treated with mammalian target of rapamycin inhibitors as maintenance immunosuppression.,We aimed to describe the occurrence of skin cancers in renal and liver transplant recipients switched from calcineurin inhibitor to sirolimus-based regimes.,We performed a retrospective study of sirolimus conversion within the Irish national kidney and liver transplant programs.,These data were linked with the National Cancer Registry Ireland to determine the incidence of NMSC among these recipients.,The incidence rate ratio (IRR) for post versus pre-conversion NMSC rates are referred in this study as an effect size with [95% confidence interval].,Of 4,536 kidney transplants and 574 liver transplants functioning on the 1 January 1994 or transplanted between 1 January 1994 and 01 January 1994 and 01 January 2015, 85 kidney and 88 liver transplant recipients were transitioned to sirolimus-based immunosuppression.,In renal transplants, the rate of NMSC was 131 per 1000 patient years pre-switch to sirolimus, and 68 per 1000 patient years post switch, with adjusted effect size of 0.48 [0.31 − 0.74] (p = .001) following the switch.,For liver transplant recipients, the rate of NMSC was 64 per 1,000 patient years pre-switch and 30 per 1,000 patient years post switch, with an adjusted effect size of 0.49 [0.22 − 1.09] (p .081).,Kidney transplant recipients were followed up for a median 3.4 years.,Liver transplants were followed for a median 6.6 years.,In this study, the conversion of maintenance immunosuppression from calcineurin inhibitors to mTOR inhibitors for clinical indications did appear to reduce the incidence of NMSC in kidney and liver transplant recipients.

To define the cellular composition and architecture of cutaneous squamous cell carcinoma (cSCC), we combined single-cell RNA sequencing with spatial transcriptomics and multiplexed ion beam imaging from a series of human cSCCs and matched normal skin. cSCC exhibited four tumor subpopulations, three recapitulating normal epidermal states, and a tumor-specific keratinocyte (TSK) population unique to cancer, which localized to a fibrovascular niche.,Integration of single-cell and spatial data mapped ligand-receptor networks to specific cell types, revealing TSK cells as a hub for intercellular communication.,Multiple features of potential immunosuppression were observed, including T regulatory cell (Treg) co-localization with CD8 T cells in compartmentalized tumor stroma.,Finally, single-cell characterization of human tumor xenografts and in vivo CRISPR screens identified essential roles for specific tumor subpopulation-enriched gene networks in tumorigenesis.,These data define cSCC tumor and stromal cell subpopulations, the spatial niches where they interact, and the communicating gene networks that they engage in cancer.,•Profiling of 10 human skin SCCs and matched normals via scRNA-seq, ST, and MIBI•Tumor-specific keratinocytes (TSKs) reside within a fibrovascular niche at leading edges•Distinct ligand-receptor and spatial niche associations for tumor and stromal cells.,•Subpopulation essential tumorigenic gene networks defined by in vivo CRISPR screening,Profiling of 10 human skin SCCs and matched normals via scRNA-seq, ST, and MIBI,Tumor-specific keratinocytes (TSKs) reside within a fibrovascular niche at leading edges,Distinct ligand-receptor and spatial niche associations for tumor and stromal cells.,Subpopulation essential tumorigenic gene networks defined by in vivo CRISPR screening,Integration of high-dimensional multi-omics approaches to characterize human cutaneous squamous cell carcinoma identifies a tumor-specific keratinocyte population as well as the immune infiltrates and heterogeneity at tumor leading edges.

Canine malignant melanoma, a significant cause of mortality in domestic dogs, is a powerful comparative model for human melanoma, but little is known about its genetic etiology.,We mapped the genomic landscape of canine melanoma through multi-platform analysis of 37 tumors (31 mucosal, 3 acral, 2 cutaneous, and 1 uveal) and 17 matching constitutional samples including long- and short-insert whole genome sequencing, RNA sequencing, array comparative genomic hybridization, single nucleotide polymorphism array, and targeted Sanger sequencing analyses.,We identified novel predominantly truncating mutations in the putative tumor suppressor gene PTPRJ in 19% of cases.,No BRAF mutations were detected, but activating RAS mutations (24% of cases) occurred in conserved hotspots in all cutaneous and acral and 13% of mucosal subtypes.,MDM2 amplifications (24%) and TP53 mutations (19%) were mutually exclusive.,Additional low-frequency recurrent alterations were observed amidst low point mutation rates, an absence of ultraviolet light mutational signatures, and an abundance of copy number and structural alterations.,Mutations that modulate cell proliferation and cell cycle control were common and highlight therapeutic axes such as MEK and MDM2 inhibition.,This mutational landscape resembles that seen in BRAF wild-type and sun-shielded human melanoma subtypes.,Overall, these data inform biological comparisons between canine and human melanoma while suggesting actionable targets in both species.

Despite being rarely reported, improved diagnostic and prognostic indicators are necessary for treating malignant melanoma in rabbits.,In this study, two cases of primary skin lesions, on the scrotum and on eyelid, with systemic metastases, were examined.,The tumors formed intra-dermally by sheet-like proliferation of polymorphic cells, with anisocytosis and varying amount of melanin granules.,Tumors had displaced almost 50% of the lung and liver tissue, and tumor metastasis was the cause of early death in both rabbits.,Ki-67-positive population was high in both, and it was found to be useful in assessing the outcome and malignancy.,In addition, Melan-A, HMB-45, PNL2 and S100 established a useful immunohistochemical panel for the diagnosis of melanocytic tumor in rabbits.

Previous studies have shown that histone deacetylase 6 (HDAC6) plays critical roles in many cellular processes related to cancer.,However, its biological roles in the development of melanoma remain unexplored.,Our aim was to investigate whether HDAC6 has a biological role in human melanoma development and to understand its underlying mechanism.,In the present study, HDAC6 expression was up-regulated in melanoma tissues and cell lines.,Knockdown of HDAC6 significantly inhibited the proliferation and colony formation ability of A375.,S2 cells, promoted cell arrest at G0/G1 phase and apoptosis.,Additionally, western blotting assay showed that HDAC6 silencing suppressed Bcl-2 level and enhanced Bax level, then activated caspase-9 and caspase-3, and further activated the release of cytochrome c from mitochondria to cytoplasm, finally induced cell apoptosis involving the mitochondrial pathway.,Knockdown of HDAC6 triggered a significant generation of ROS and disruption of mitochondrial membrane potential (MMP).,Furthermore, ROS inhibitor, NAC reduced HDAC6 siRNA-induced ROS production, and blocked HDAC6 siRNA-induced loss of MMP and apoptosis.,NAC also significantly blocked HDAC6 siRNA-induced mtDNA copy number decrease and mitochondrial biogenesis and degradation imbalance.,In conclusion, the results showed that knockdown of HDAC6 induced apoptosis in human melanoma A375.,S2 cells through a ROS-dependent mitochondrial pathway.

Atmospheric gas plasmas (AGPs) up-regulate intracellular ROS levels and induce apoptosis in melanoma cells.,Evidence for TNF-signaling dependence of ASK1-mediated apoptosis suggests possible mechanisms for AGP activation and regulation of apoptosis-signaling pathways in tumor cells.,Atmospheric gas plasmas (AGPs) are able to selectively induce apoptosis in cancer cells, offering a promising alternative to conventional therapies that have unwanted side effects such as drug resistance and toxicity.,However, the mechanism of AGP-induced cancer cell death is unknown.,In this study, AGP is shown to up-regulate intracellular reactive oxygen species (ROS) levels and induce apoptosis in melanoma but not normal melanocyte cells.,By screening genes involved in apoptosis, we identify tumor necrosis factor (TNF)-family members as the most differentially expressed cellular genes upon AGP treatment of melanoma cells.,TNF receptor 1 (TNFR1) antagonist-neutralizing antibody specifically inhibits AGP-induced apoptosis signal, regulating apoptosis signal-regulating kinase 1 (ASK1) activity and subsequent ASK1-dependent apoptosis.,Treatment of cells with intracellular ROS scavenger N-acetyl-l-cysteine also inhibits AGP-induced activation of ASK1, as well as apoptosis.,Moreover, depletion of intracellular ASK1 reduces the level of AGP-induced oxidative stress and apoptosis.,The evidence for TNF-signaling dependence of ASK1-mediated apoptosis suggests possible mechanisms for AGP activation and regulation of apoptosis-signaling pathways in tumor cells.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer.,Although most cSCCs have good prognosis, a subgroup of high-risk cSCC has a higher frequency of recurrence and mortality.,Therefore, the identification of molecular risk factors associated with this aggressive subtype is of major interest.,In this work we carried out a global-scale approach to investigate the DNA-methylation profile in patients at different stages, from premalignant actinic keratosis to low-risk invasive and high-risk non-metastatic and metastatic cSCC.,The results showed massive non-sequential changes in DNA-methylome and identified a minimal methylation signature that discriminates between stages.,Importantly, a direct comparison of low-risk and high-risk stages revealed epigenetic traits characteristic of high-risk tumours.,Finally, a prognostic prediction model in cSCC patients identified a methylation signature able to predict the overall survival of patients.,Thus, the analysis of DNA-methylation in cSCC revealed changes during the evolution of the disease through the different stages that can be of great value not only in the diagnosis but also in the prognosis of the disease.

Melanin possess radioprotective and scavenging properties, and its presence can affect the behavior of melanoma cells, its surrounding environment and susceptibility to the therapy, as showed in vitro experiments.,To determine whether melanin presence in melanoma affects the efficiency of radiotherapy (RTH) we evaluated the survival time after RTH treatment in metastatic melanoma patients (n = 57).,In another cohort of melanoma patients (n = 84), the relationship between melanin level and pT and pN status was determined.,A significantly longer survival time was found in patients with amelanotic metastatic melanomas in comparison to the melanotic ones, who were treated with either RTH or chemotherapy (CHTH) and RTH.,These differences were more significant in a group of melanoma patients treated only with RTH.,A detailed analysis of primary melanomas revealed that melanin levels were significantly higher in melanoma cells invading reticular dermis than the papillary dermis.,A significant reduction of melanin pigmentation in pT3 and pT4 melanomas in comparison to pT1 and T2 tumors was observed.,However, melanin levels measured in pT3-pT4 melanomas developing metastases (pN1-3, pM1) were higher than in pN0 and pM0 cases.,The presence of melanin in metastatic melanoma cells decreases the outcome of radiotherapy, and melanin synthesis is related to higher disease advancement.,Based on our previous cell-based and clinical research and present research we also suggest that inhibition of melanogenesis can improve radiotherapy modalities.,The mechanism of relationship between melanogenesis and efficacy of RTH requires additional studies, including larger melanoma patients population and orthotopic, imageable mouse models of metastatic melanoma.

Mutational signatures have emerged as essential tools in cancer genomics, providing clinically relevant insights as well as accurate background models needed when assessing signals of selection in cancer.,Here, we observe that the mutational signature of ultraviolet (UV) light varies across chromatin states, highlighting a previously unappreciated aspect of mutational signatures.,Our results imply that locally derived, rather than genome-wide or exome-wide, signatures are more accurate, which is of relevance in situations such as cancer driver gene detection, where correct modelling of signatures and expected mutation rates is critical.,We also show that incorporation of longer contextual patterns into the signature further improves modeling of UV mutations.,Mutational signatures can reveal properties of underlying mutational processes and are important when assessing signals of selection in cancer.,Here, we describe the sequence characteristics of mutations induced by ultraviolet (UV) light, a major mutagen in several human cancers, in terms of extended (longer than trinucleotide) patterns as well as variability of the signature across chromatin states.,Promoter regions display a distinct UV signature with reduced TCG > TTG transitions, and genome-wide mapping of UVB-induced DNA photoproducts (pyrimidine dimers) showed that this may be explained by decreased damage formation at hypomethylated promoter CpG sites.,Further, an extended signature model encompassing additional information from longer contextual patterns improves modeling of UV mutations, which may enhance discrimination between drivers and passenger events.,Our study presents a refined picture of the UV signature and underscores that the characteristics of a single mutational process may vary across the genome.

In some organs, adult stem cells are uniquely poised to serve as cancer cells of origin.,It is unclear, however, whether tumorigenesis is influenced by the activation state of the adult stem cell.,Hair follicle stem cells (HFSCs) act as cancer cells of origin for cutaneous squamous cell carcinoma (SCC) and undergo defined cycles of quiescence and activation.,The data presented here show that HFSCs are unable to initiate tumors during the quiescent phase of the hair cycle, indicating that the mechanisms that keep HFSCs dormant are dominant to the gain of oncogenes (Ras) or the loss of tumor suppressors (p53).,Furthermore, Pten activity is necessary for quiescence based tumor suppression, as its deletion alleviates tumor suppression without affecting proliferation.,These data demonstrate that stem cell quiescence is a form of tumor suppression in HFSCs, and that Pten plays a role in maintaining quiescence in the presence of tumorigenic stimuli.

Malignant melanoma is the most aggressive type of skin cancer and is closely associated with the development of brain metastases.,Despite aggressive treatment, the prognosis has traditionally been poor, necessitating improved therapies.,In melanoma, the mitogen activated protein kinase and the phosphoinositide 3-kinase signaling pathways are commonly altered, and therapeutically inhibiting one of the pathways often upregulates the other, leading to resistance.,Thus, combined treatment targeting both pathways is a promising strategy to overcome this.,Here, we studied the in vitro and in vivo effects of the PI3K inhibitor buparlisib and the MEK1/2 inhibitor trametinib, used either as targeted monotherapies or in combination, on patient-derived melanoma brain metastasis cell lines.,Scratch wound and trans-well assays were carried out to assess the migratory capacity of the cells upon drug treatment, whereas flow cytometry, apoptosis array and Western blots were used to study apoptosis.,Finally, an in vivo treatment experiment was carried out on NOD/SCID mice.,We show that combined therapy was more effective than monotherapy.,Combined treatment also more effectively increased apoptosis, and inhibited tumor growth in vivo.,This suggests a clinical potential of combined treatment to overcome ceased treatment activity which is often seen after monotherapies, and strongly encourages the evaluation of the treatment strategy on melanoma patients with brain metastases.

Malignant melanoma has an increasing incidence rate and the metastatic disease is notoriously resistant to standard chemotherapy.,Loss of cell cycle checkpoints is frequently found in many cancer types and makes the cells reliant on compensatory mechanisms to control progression.,This feature may be exploited in therapy, and kinases involved in checkpoint regulation, such as Wee1 and Chk1/2, have thus become attractive therapeutic targets.,In the present study we combined a Wee1 inhibitor (MK1775) with Chk1/2 inhibitor (AZD7762) in malignant melanoma cell lines grown in vitro (2D and 3D cultures) and in xenografts models.,Our in vitro studies showed that combined inhibition of Wee1 and Chk1/2 synergistically decreased viability and increased apoptosis (cleavage of caspase 3 and PARP), which may be explained by accumulation of DNA-damage (increased expression of γ-H2A.X) - and premature mitosis of S-phase cells.,Compared to either inhibitor used as single agents, combined treatment reduced spheroid growth and led to greater tumour growth inhibition in melanoma xenografts.,These data provide a rationale for further evaluation of the combination of Wee1 and Chk1/2 inhibitors in malignant melanoma.,The online version of this article (doi:10.1186/s12885-015-1474-8) contains supplementary material, which is available to authorized users.

Therapies that target the programmed death-1 (PD-1) receptor have shown unprecedented rates of durable clinical responses in patients with various cancer types.1-5 One mechanism by which cancer tissues limit the host immune response is via upregulation of PD-1 ligand (PD-L1) and its ligation to PD-1 on antigen-specific CD8 T-cells (termed adaptive immune resistance).6,7 Here we show that pre-existing CD8 T-cells distinctly located at the invasive tumour margin are associated with expression of the PD-1/PD-L1 immune inhibitory axis and may predict response to therapy.,We analyzed samples from 46 patients with metastatic melanoma obtained before and during anti-PD1 therapy (pembrolizumab) using quantitative immunohistochemistry, quantitative multiplex immunofluorescence, and next generation sequencing for T-cell receptors (TCR).,In serially sampled tumours, responding patients showed proliferation of intratumoural CD8+ T-cells that directly correlated with radiographic reduction in tumour size.,Pre-treatment samples obtained from responding patients showed higher numbers of CD8, PD1, and PD-L1 expressing cells at the invasive tumour margin and inside tumours, with close proximity between PD-1 and PD-L1, and a more clonal TCR repertoire.,Using multivariate analysis, we established a predictive model based on CD8 expression at the invasive margin and validated the model in an independent cohort of 15 patients.,Our findings indicate that tumour regression following therapeutic PD-1 blockade requires pre-existing CD8+ T cells that are negatively regulated by PD-1/PD-L1 mediated adaptive immune resistance.

Sun-exposure is one of the risk factors associated with the development of a cutaneous neoplasm.,In melanoma, the Ras-Raf-MEK-ERK (MAPK) signaling pathway is constitutively activated through multiple mechanisms, including B-RAF mutation.,It has been hypothesized that B-RAF mutations in melanocytic lesions arise from DNA damage induced by ultraviolet (UV) radiation.,However, it is still discussed if B-RAF mutations are associated with melanoma patients exposed to the sun.,Therefore, in the present study, the known B-RAFV600E mutation was analysed in melanoma samples from 30 indoor and 38 outdoor workers.,B-RAFV600E mutation was detected in 52 and 73% of outdoor workers and indoor workers, respectively.,Of note, this mutation was identified in 12 of 14 (85%) melanoma of the trunk diagnosed in indoor workers and in 9 of 19 (47%) samples from outdoor workers (p=0.03).,By analyzing melanomas of other body sites, no statistical difference in the frequency of B-RAFV600E mutation was identified between the groups of workers.,It appears that the mutation detected among indoor workers may be associated with a recreational or intermittent exposure to the sun, as usually the trunk is a sun-protected body site.,Overall, these data indicate that the B-RAFV600E mutation detected in melanoma is not associated with a chronic exposure to the sun.,Mutations detected in other genes may also contribute to melanoma development in the subset of patients exposed to UV radiation.

In this Review, Rambow et al. use melanoma as a model to present a series of theoretical arguments coupled to recent experimental evidence.,The authors discuss key roles for nongenetic state switching at various steps of the evolution of disease progression and therapy resistance.,An incomplete view of the mechanisms that drive metastasis, the primary cause of cancer-related death, has been a major barrier to development of effective therapeutics and prognostic diagnostics.,Increasing evidence indicates that the interplay between microenvironment, genetic lesions, and cellular plasticity drives the metastatic cascade and resistance to therapies.,Here, using melanoma as a model, we outline the diversity and trajectories of cell states during metastatic dissemination and therapy exposure, and highlight how understanding the magnitude and dynamics of nongenetic reprogramming in space and time at single-cell resolution can be exploited to develop therapeutic strategies that capitalize on nongenetic tumor evolution.

Transcriptional reprogramming of proliferative melanoma cells into a phenotypically distinct invasive cell subpopulation is a critical event at the origin of metastatic spreading.,Here we generate transcriptome, open chromatin and histone modification maps of melanoma cultures; and integrate this data with existing transcriptome and DNA methylation profiles from tumour biopsies to gain insight into the mechanisms underlying this key reprogramming event.,This shows thousands of genomic regulatory regions underlying the proliferative and invasive states, identifying SOX10/MITF and AP-1/TEAD as regulators, respectively.,Knockdown of TEADs shows a previously unrecognized role in the invasive gene network and establishes a causative link between these transcription factors, cell invasion and sensitivity to MAPK inhibitors.,Using regulatory landscapes and in silico analysis, we show that transcriptional reprogramming underlies the distinct cellular states present in melanoma.,Furthermore, it reveals an essential role for the TEADs, linking it to clinically relevant mechanisms such as invasion and resistance.,The key regulators that allow transition from proliferative to invasive phenotype in melanoma cells have not been identified yet.,The authors perform chromatin and transcriptome profiling followed by comprehensive bioinformatics analysis identifying new candidate regulators for two distinct cell states of melanoma.

The immune system employs several checkpoint pathways to regulate responses, maintain homeostasis and prevent self-reactivity and autoimmunity.,Tumor cells can hijack these protective mechanisms to enable immune escape, cancer survival and proliferation.,Blocking antibodies, designed to interfere with checkpoint molecules CTLA-4 and PD-1/PD-L1 and counteract these immune suppressive mechanisms, have shown significant success in promoting immune responses against cancer and can result in tumor regression in many patients.,While inhibitors to CTLA-4 and the PD-1/PD-L1 axis are well-established for the clinical management of melanoma, many patients do not respond or develop resistance to these interventions.,Concerted efforts have focused on combinations of approved therapies aiming to further augment positive outcomes and survival.,While CTLA-4 and PD-1 are the most-extensively researched targets, results from pre-clinical studies and clinical trials indicate that novel agents, specific for checkpoints such as A2AR, LAG-3, IDO and others, may further contribute to the improvement of patient outcomes, most likely in combinations with anti-CTLA-4 or anti-PD-1 blockade.,This review discusses the rationale for, and results to date of, the development of inhibitory immune checkpoint blockade combination therapies in melanoma.,The clinical potential of new pipeline therapeutics, and possible future therapy design and directions that hold promise to significantly improve clinical prognosis compared with monotherapy, are discussed.

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.

The CSF-470 cellular vaccine plus BCG and rhGM-CSF increased distant metastases-free survival in cutaneous melanoma patients stages IIB-IIC-III relative to medium dose IFN-α2b (CASVAC-0401 study).,Patient-045 developed a mature vaccination site (VAC-SITE) and a regional cutaneous metastasis (C-MTS), which were excised during the protocol, remaining disease-free 36 months from vaccination start.,CDR3-TCRβ repertoire sequencing in PBMC and tissue samples, along with skin-DTH score and IFN-γ ELISPOT assay, were performed to analyze the T-cell immune response dynamics throughout the immunization protocol.,Histopathological analysis of the VAC-SITE revealed a highly-inflamed granulomatous structure encircled by CD11c+ nested-clusters, brisk CD8+ and scarce FOXP3+, lymphocytes with numerous Langhans multinucleated-giant-cells and macrophages.,A large tumor-regression area fulfilled the C-MTS with brisk lymphocyte infiltration, mainly composed of CD8+PD1+ T-cells, CD20+ B-cells, and scarce FOXP3+ cells.,Increasing DTH score and IFN-γ ELISPOT assay signal against the CSF-470 vaccine-lysate was evidenced throughout immunization.,TCRβ repertoire analysis revealed for the first time the presence of common clonotypes between a VAC-SITE and a C-MTS; most of them persisted in blood by the end of the immunization protocol.,In vitro boost with vaccine-lysate revealed the expansion of persistent clones that infiltrated the VAC-SITE and/or the C-MTS; other persistent clones expanded in the patient's blood as well.,We propose that expansion of such persistent clonotypes might derive from two different although complementary mechanisms: the proliferation of specific clones as well as the expansion of redundant clones, which increased the number of nucleotide rearrangements per clonotype, suggesting a functional antigenic selection.,In this patient, immunization with the CSF-470 vaccine plus BCG and rhGM-CSF induced a T-cell repertoire at the VAC-SITE that was able to infiltrate an emerging C-MTS, which resulted in the expansion of a T-cell repertoire that persisted in blood by the end of the 2-year treatment.

Background.,There is not yet an agreed adjuvant treatment for melanoma patients with American Joint Committee on Cancer stages III B and C.,We report administration of an autologous melanoma vaccine to prevent disease recurrence.,Patients and Methods. 126 patients received eight doses of irradiated autologous melanoma cells conjugated to dinitrophenyl and mixed with BCG.,Delayed type hypersensitivity (DTH) response to unmodified melanoma cells was determined on the vaccine days 5 and 8.,Gene expression analysis was performed on 35 tumors from patients with good or poor survival.,Results.,Median overall survival was 88 months with a 5-year survival of 54%.,Patients attaining a strong DTH response had a significantly better (p = 0.0001) 5-year overall survival of 75% compared with 44% in patients without a strong response.,Gene expression array linked a 50-gene signature to prognosis, including a cluster of four cancer testis antigens: CTAG2 (NY-ESO-2), MAGEA1, SSX1, and SSX4.,Thirty-five patients, who received an autologous vaccine, followed by ipilimumab for progressive disease, had a significantly improved 3-year survival of 46% compared with 19% in nonvaccinated patients treated with ipilimumab alone (p = 0.007).,Conclusion.,Improved survival in patients attaining a strong DTH and increased response rate with subsequent ipilimumab suggests that the autologous vaccine confers protective immunity.

Immune checkpoint inhibitors (ICIs) have significantly improved the outcome in metastatic cutaneous melanoma (CM).,However, therapy response is limited to subgroups of patients and clinically useful predictive biomarkers are lacking.,To discover treatment-related systemic changes in plasma and potential biomarkers associated with treatment outcome, we analyzed serial plasma samples from 24 patients with metastatic CM, collected before and during ICI treatment, with mass-spectrometry-based global proteomics (high-resolution isoelectric focusing liquid chromatography-mass spectrometry (HiRIEF LC-MS/MS)) and targeted proteomics with proximity extension assays (PEAs).,In addition, we analyzed plasma proteomes of 24 patients with metastatic CM treated with mitogen-activated protein kinase inhibitors (MAPKis), to pinpoint changes in protein plasma levels specific to the ICI treatment.,To detect plasma proteins associated with treatment response, we performed stratified analyses in anti-programmed cell death protein 1 (anti-PD-1) responders and non-responders.,In addition, we analyzed the association between protein plasma levels and progression-free survival (PFS) by Cox proportional hazards models.,Unbiased HiRIEF LC-MS/MS-based proteomics showed plasma levels’ alterations related to anti-PD-1 treatment in 80 out of 1160 quantified proteins.,Circulating PD-1 had the highest increase during anti-PD-1 treatment (log2-FC=2.03, p=0.0008) and in anti-PD-1 responders (log2-FC=2.09, p=0.005), but did not change in the MAPKis cohort.,Targeted, antibody-based proteomics by PEA confirmed this observation.,Anti-PD-1 responders had an increase in plasma proteins involved in T-cell response, neutrophil degranulation, inflammation, cell adhesion, and immune suppression.,Furthermore, we discovered new associations between plasma proteins (eg, interleukin 6, interleukin 10, proline-rich acidic protein 1, desmocollin 3, C-C motif chemokine ligands 2, 3 and 4, vascular endothelial growth factor A) and PFS, which may serve as predictive biomarkers.,We detected an increase in circulating PD-1 during anti-PD-1 treatment, as well as diverse immune plasma proteomic signatures in anti-PD-1 responders.,This study demonstrates the potential of plasma proteomics as a liquid biopsy method and in discovery of putative predictive biomarkers for anti-PD-1 treatment in metastatic CM.

The DecisionDx-Melanoma test provides prognostic information for patients with cutaneous melanoma (CM).,Using formalin-fixed paraffin-embedded primary tumor tissue, the RT-PCR-based test classifies patients into a low- (Class 1) or high-risk (Class 2) category for recurrence based on expression of 31 genes.,The current study was designed to assess the analytical validity of this test.,Inter-assay, inter-instrument, and inter-operator studies were performed to evaluate reliability of the 31-gene expression test results, sample stability and reagent stability.,From March 2013 through June 2016, the gene expression test was performed on 8244 CM tumors.,De-identified data from Pathology Reports were used to assess technical success.,Robust sample and reagent stability was observed.,Inter-assay concordance on 168 specimens run on 2 consecutive days was 99% and matched probability scores were significantly correlated (R2 = 0.96).,Inter-instrument concordance was 95%, and probability scores had a correlation R2 of 0.99 (p < 0.001).,From 8244 CM specimens submitted since 2013, 85% (7023) fulfilled pre-specified tumor content parameters.,In these samples with sufficient tumor requirements, the technical success of the test was 98%.,DecisionDx-Melanoma is a robust gene expression profile test that demonstrates strong reproducibility between experiments and has high technical reliability on clinical samples.,The online version of this article (10.1186/s13000-018-0690-3) contains supplementary material, which is available to authorized users.

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.

Loss of expression of surface antigens represents a significant problem for cancer immunotherapy.,Microphthalmia-associated transcription factor (MITF-M) regulates melanocyte fate by driving expression of many differentiation genes, whose protein products can be recognized by cytolytic T lymphocytes.,We previously reported that interleukin-1ß (IL-1ß) can downregulate MITF-M levels.,Here we show that downregulation of MITF-M expression by IL-1ß was paralleled by an upregulation of miR-155 expression in four melanoma lines.,We confirmed that miR-155 was able to target endogenous MITF-M in melanoma cells and demonstrated a role for miR-155 in the IL-1ß-induced repression of MITF-M by using an antagomiR.,Notably, we also observed a strong negative correlation between MITF-M and miR-155 levels in a mouse model of melanoma.,Taken together, our results indicate that MITF-M downregulation by inflammatory stimuli might be partly due to miR-155 upregulation.,This could represent a novel mechanism of melanoma immune escape in an inflammatory microenvironment.

The mitogen-activated protein-kinase pathway consisting of the kinases RAF, MEK, and ERK is central to cell proliferation and survival and is deregulated in more than 90% of melanomas.,MEK inhibitors are currently trialled in the clinic, but despite efficient target inhibition, cytostatic rather than cytotoxic activity limits their efficacy.,We assessed the cytotoxicity to MEK inhibitors (PD184352 and selumetinib) in melanoma cells by toluidine-blue staining, caspase 3 cleavage, and melanoma-sphere growth.,Western blotting and quantitative real-time polymerase chain reaction were applied to determine SMAD-specific E3 ubiquitin protein ligase 2 (SMURF2), PAX3, and MITF expression.,Human melanoma samples (n = 77) from various stages were analyzed for SMURF2 and PAX3 expression.,RNA interference was performed to target SMURF2 during MEK inhibition in vivo in melanoma xenografts in mice and zebrafish.,All statistical tests were two-sided.,Activation of transforming growth factor β (TGF-β) signalling sensitized melanoma cells to the cytotoxic effects of MEK inhibition.,Melanoma cells resistant to the cytotoxic effects of MEK inhibitors counteracted TGF-β signalling through overexpression of the E3 ubiquitin ligase SMURF2, which resulted in increased expression of the transcription factors PAX3 and MITF.,High MITF expression protected melanoma cells against MEK inhibitor cytotoxicity.,Depleting SMURF2 reduced MITF expression and substantially lowered the threshold for MEK inhibitor-induced apoptosis.,Moreover, SMURF2 depletion sensitized melanoma cells to the cytotoxic effects of selumetinib, leading to cell death at concentrations approximately 100-fold lower than the concentration required to induce cell death in SMURF2-expressing cells.,Mice treated with selumetinib alone at a dosage of 10mg/kg body weight once daily produced no response, but in combination with SMURF2 depletion, selumetinib suppressed tumor growth by 97.9% (95% confidence interval = 38.65% to 155.50%, P = .005).,Targeting SMURF2 may be a novel therapeutic approach for increasing the antitumor efficacy of MEK inhibitors.

The development of acquired drug resistance hampers the long-term success of B-RAF inhibitor (B-RAFi) therapy for melanoma patients.,Here we show V600EB-RAF copy number gain as a mechanism of acquired B-RAFi resistance in four out of twenty (20%) patients treated with B-RAFi.,In cell lines, V600EB-RAF over-expression and knockdown conferred B-RAFi resistance and sensitivity, respectively.,In V600EB-RAF amplification-driven (vs. mutant N-RAS-driven) B-RAFi resistance, ERK reactivation is saturable, with higher doses of vemurafenib down-regulating pERK and re-sensitizing melanoma cells to B-RAFi.,These two mechanisms of ERK reactivation are sensitive to the MEK1/2 inhibitor AZD6244/selumetinib or its combination with the B-RAFi vemurafenib.,In contrast to mutant N-RAS-mediated V600EB-RAF bypass, which is sensitive to C-RAF knockdown, V600EB-RAF amplification-mediated resistance functions largely independently of C-RAF.,Thus, alternative clinical strategies may potentially overcome distinct modes of ERK reactivation underlying acquired B-RAFi resistance in melanoma.

Immunologic responses to anti-PD-1 therapy in melanoma patients occur rapidly with pharmacodynamic T cell responses detectable in blood by 3 weeks.,It is unclear, however, whether these early blood-based observations translate to the tumor microenvironment.,We conducted a study of neoadjuvant/adjuvant anti-PD-1 therapy in stage III/IV melanoma.,We hypothesized that immune reinvigoration in the tumor would be detectable at 3 weeks and this response would correlate with disease-free survival.,We identified a rapid and potent anti-tumor response, with 8/27 patients experiencing a complete or major pathological response after a single dose of anti-PD-1, all of whom remain disease-free.,These rapid pathologic and clinical responses were associated with accumulation of exhausted CD8 T cells in the tumor at 3 weeks with reinvigoration in the blood observed as early as 1 week.,Transcriptional analysis demonstrated a pre-treatment immune signature (Neoadjuvant Response Signature) that was associated with clinical benefit.,In contrast, patients with disease recurrence displayed mechanisms of resistance including immune suppression, mutational escape, and/or tumor evolution.,Neoadjuvant anti-PD-1 treatment is effective in high-risk resectable stage III/IV melanoma.,Pathological response and immunological analyses after a single neoadjuvant dose can be used to predict clinical outcome and to dissect underlying mechanisms in checkpoint blockade.

Anti-PD-1 therapy has shown significant clinical activity in advanced melanoma.,We developed and validated a clinical prediction scale for response to anti- PD-1 monotherapy.,A total of 315 patients with advanced melanoma treated with pembrolizumab (2 or 10 mg kg−1 Q2W or Q3W) or nivolumab (3 mg kg−1 Q2W) at four cancer centres between 2011 to 2013 served as the setting for the present cohort study.,Variables with significant association to response on a univariate analysis were entered into a forward stepwise logistic regression model and were given a score based on ORs to calculate a clinical prediction scale.,The developed clinical prediction scale included elevated LDH (1 point), age <65 years (1 point), female sex (1 point), history of ipilimumab treatment (2 points) and the presence of liver metastasis (2 points).,The scale had an area under the receiver-operating curve (AUC) of 0.73 (95% CI 0.67, 0.80) in predicting response to therapy.,The predictive performance of the score was maintained in the validation cohort (AUC 0.70 (95% CI 0.58, 0.81)) and the goodness-to-fit model demonstrated good calibration.,Based on a large cohort of patients, we developed and validated a simple five-factor prediction scale for the clinical activity of PD-1 antibodies in advanced melanoma patients.,This scale can be used to stratify patients participating in clinical trials.

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.

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5).,Mouse models have been broadly utilized to study tissue morphogenesis in vivo.,However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans.,Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis.,The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types.,The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis.,Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction.,The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin.,As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes.,Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo.,Recently, dermal stem cells have been identified in the human dermis (6).,These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

Melanoma is the deadliest form of skin cancer.,In the early stages, melanoma can be treated successfully with surgery alone and survival rates are high, but after metastasis survival rates drop significantly.,Therefore, early and correct diagnosis is key for ensuring patients have the best possible prognosis.,Melanoma misdiagnosis accounts for more pathology and dermatology malpractice claims than any cancer other than breast cancer, as an early misdiagnosis can significantly reduce a patient’s chances of survival.,As far as treatment for metastatic melanoma goes, there have been several new drugs developed over the last 10 years that have greatly improved the prognosis of patients with metastatic melanoma, however, a majority of patients do not show a lasting response to these treatments.,Thus, new biomarkers and drug targets are needed to improve the accuracy of melanoma diagnosis and treatment.,This article will discuss the major advancements of melanoma diagnosis and treatment from antiquity to the present day.

BRAF is a serine/threonine protein kinase activating the MAP kinase/ERK-signaling pathway.,About 50 % of melanomas harbors activating BRAF mutations (over 90 % V600E).,BRAFV600E has been implicated in different mechanisms underlying melanomagenesis, most of which due to the deregulated activation of the downstream MEK/ERK effectors.,The first selective inhibitor of mutant BRAF, vemurafenib, after highly encouraging results of the phase I and II trial, was compared to dacarbazine in a phase III trial in treatment-naïve patients (BRIM-3).,The study results showed a relative reduction of 63 % in risk of death and 74 % in risk of tumor progression.,Considering all trials so far completed, median overall survival reached approximately 16 months for vemurafenib compared to less than 10 months for dacarbazine treatment.,Vemurafenib has been extensively tested on melanoma patients expressing the BRAFV600E mutated form; it has been demonstrated to be also effective in inhibiting melanomas carrying the V600K mutation.,In 2011, both FDA and EMA therefore approved vemurafenib for metastatic melanoma carrying BRAFV600 mutations.,Some findings suggest that continuation of vemurafenib treatment is potentially beneficial after local therapy in a subset of patients with disease progression (PD).,Among who continued vemurafenib >30 days after local therapy of PD lesion(s), a median overall survival was not reached, with a median follow-up of 15.5 months from initiation of BRAF inhibitor therapy.,For patients who did not continue treatment, median overall survival from the time of disease progression was 1.4 months.,A clinical phase I/II trial is evaluating the safety, tolerability and efficacy of vemurafenib in combination with the CTLA-4 inhibitor mAb ipilimumab.,In the BRIM-7 trial vemurafenib is tested in association with GDC-0973, a potent and highly selective inhibitor of MEK1/2.,Preliminary data seem to indicate that an additional inhibitor of mutated BRAF, GSK2118436, might be also active on a wider range of BRAF mutations (V600E-K-D-R); actually, treatment with such a compound is under evaluation in a phase III study among stage III-IV melanoma patients positive for BRAF mutations.,Overall, BRAF inhibitors were well tolerated; common adverse events are arthralgia, rash, fatigue, alopecia, keratoacanthoma or cutaneous squamous-cell carcinoma, photosensitivity, nausea, and diarrhea, with some variants between different inhibitors.

Uveal melanoma represents ∼85% of all ocular melanomas and up to 50% of patients develop metastatic disease.,Metastases are most frequently localised to the liver and, as few patients are candidates for potentially curative surgery, this is associated with a poor prognosis.,There is currently little published evidence for the optimal management and treatment of metastatic uveal melanoma and the lack of effective therapies in this setting has led to the widespread use of systemic treatments for patients with cutaneous melanoma.,Uveal and cutaneous melanomas are intrinsically different diseases and so dedicated management strategies and therapies for uveal melanoma are much needed.,This review explores the biology of uveal melanoma and how this relates to ongoing trials of targeted therapies in the metastatic disease setting.,In addition, we consider the options to optimise patient management and care.

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

Melanomas can switch to a dedifferentiated cell state upon exposure to cytotoxic T cells.,However, it is unclear whether such tumor cells pre-exist in patients and whether they can be resensitized to immunotherapy.,Here, we chronically expose (patient-derived) melanoma cell lines to differentiation antigen-specific cytotoxic T cells and observe strong enrichment of a pre-existing NGFRhi population.,These fractions are refractory also to T cells recognizing non-differentiation antigens, as well as to BRAF + MEK inhibitors.,NGFRhi cells induce the neurotrophic factor BDNF, which contributes to T cell resistance, as does NGFR.,In melanoma patients, a tumor-intrinsic NGFR signature predicts anti-PD-1 therapy resistance, and NGFRhi tumor fractions are associated with immune exclusion.,Lastly, pharmacologic NGFR inhibition restores tumor sensitivity to T cell attack in vitro and in melanoma xenografts.,These findings demonstrate the existence of a stable and pre-existing NGFRhi multitherapy-refractory melanoma subpopulation, which ought to be eliminated to revert intrinsic resistance to immunotherapeutic intervention.,Dedifferentiation state has been associated with therapy resistance in melanoma.,Here, the authors uncover a pre-existing NGFR-expressing, targetable subpopulation that is resistant to immunotherapy and other treatments in melanoma cells and preclinical models.

The blossom of immunotherapy in melanoma highlights the need to delineate mechanisms of immune resistance.,Recently, we have demonstrated that the RNA editing protein, adenosine deaminase acting on RNA-1 (ADAR1) is down-regulated during metastatic transition of melanoma, which enhances melanoma cell proliferation and tumorigenicity.,Here we investigate the role of ADAR1 in melanoma immune resistance.,Importantly, knockdown of ADAR1 in human melanoma cells induces resistance to tumor infiltrating lymphocytes in a cell contact-dependent mechanism.,We show that ADAR1, in an editing-independent manner, regulates the biogenesis of miR-222 at the transcription level and thereby Intercellular Adhesion Molecule 1 (ICAM1) expression, which consequently affects melanoma immune resistance.,ADAR1 thus has a novel, pivotal, role in cancer immune resistance.,Corroborating with these results, the expression of miR-222 in melanoma tissue specimens was significantly higher in patients who had no clinical benefit from treatment with ipilimumab as compared to patients that responded clinically, suggesting that miR-222 could function as a biomarker for the prediction of response to ipilimumab.,These results provide not only novel insights on melanoma immune resistance, but also pave the way to the development of innovative personalized tools to enable optimal drug selection and treatment.

The total number of acquired melanocytic nevi on the skin is strongly correlated with melanoma risk.,Here we report a meta-analysis of 11 nevus GWAS from Australia, Netherlands, UK, and USA comprising 52,506 individuals.,We confirm known loci including MTAP, PLA2G6, and IRF4, and detect novel SNPs in KITLG and a region of 9q32.,In a bivariate analysis combining the nevus results with a recent melanoma GWAS meta-analysis (12,874 cases, 23,203 controls), SNPs near GPRC5A, CYP1B1, PPARGC1B, HDAC4, FAM208B, DOCK8, and SYNE2 reached global significance, and other loci, including MIR146A and OBFC1, reached a suggestive level.,Overall, we conclude that most nevus genes affect melanoma risk (KITLG an exception), while many melanoma risk loci do not alter nevus count.,For example, variants in TERC and OBFC1 affect both traits, but other telomere length maintenance genes seem to affect melanoma risk only.,Our findings implicate multiple pathways in nevogenesis.,Melanocytic nevus count is associated with melanoma risk.,In this study, a meta-analysis of 11 nevus GWAS studies identifies novel SNPs in KITLG and 9q32, and bivariate analysis with melanoma GWAS meta-analysis reveals that most nevus genes affect melanoma risk, while melanoma risk loci do not alter the nevus count.

Genome wide association studies (GWAS) have mapped multiple independent cancer susceptibility loci to chr5p15.33.,Here, we show that fine-mapping of pancreatic and testicular cancer GWAS within one of these loci (Region 2 in CLPTM1L) focuses the signal to nine highly correlated SNPs.,Of these, rs36115365-C associated with increased pancreatic and testicular but decreased lung cancer and melanoma risk, and exhibited preferred protein-binding and enhanced regulatory activity.,Transcriptional gene silencing of this regulatory element repressed TERT expression in an allele-specific manner.,Proteomic analysis identifies allele-preferred binding of Zinc finger protein 148 (ZNF148) to rs36115365-C, further supported by binding of purified recombinant ZNF148.,Knockdown of ZNF148 results in reduced TERT expression, telomerase activity and telomere length.,Our results indicate that the association with chr5p15.33-Region 2 may be explained by rs36115365, a variant influencing TERT expression via ZNF148 in a manner consistent with elevated TERT in carriers of the C allele.,Genetic variants at multiple loci of chr5p15.33 have been associated with susceptibility to numerous cancers.,Here the authors show that the association of one of these loci may be explained by a variant, rs36115365, influencing telomerase reverse transcriptase (TERT) expression via ZNF148.

Melanoma accounts for 1.7% of global cancer diagnoses and is the fifth most common cancer in the US.,Melanoma incidence is rising in developed, predominantly fair-skinned countries, growing over 320% in the US since 1975.,However, US mortality has fallen almost 30% over the past decade with the approval of 10 new targeted or immunotherapy agents since 2011.,Mutations in the signaling-protein BRAF, present in half of cases, are targeted with oral BRAF/MEK inhibitor combinations, while checkpoint inhibitors are used to restore immunosurveillance likely inactivated by UV radiation.,Although the overall 5-year survival has risen to 93.3% in the US, survival for stage IV disease remains only 29.8%.,Melanoma is most common in white, older men, with an average age of diagnosis of 65.,Outdoor UV exposure without protection is the main risk factor, although indoor tanning beds, immunosuppression, family history and rare congenital diseases, moles, and obesity contribute to the disease.,Primary prevention initiatives in Australia implemented since 1988, such as education on sun-protection, have increased sun-screen usage and curbed melanoma incidence, which peaked in Australia in 2005.,In the US, melanoma incidence is not projected to peak until 2022-2026.,Fewer than 40% of Americans report practicing adequate protection (sun avoidance from 10 a.m.-4 p.m. and regular application of broad-spectrum sunscreen with an SPF > 30).,A 2-4-fold return on investment is predicted for a US sun-protection education initiative.,Lesion-directed skin screening programs, especially for those at risk, have also cost-efficiently reduced melanoma mortality.

(1) Cisplatin (CDDP) is used in melanoma chemotherapy, but it has many side effects.,Hence, the search for natural substances that can reduce the dose of CDDP, and CDDP-related toxicity, is highly desired.,Coumarins have many biological properties, including anticancer and antiproliferative effects.,(2) An in vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay on two human melanoma cell lines (FM55P and FM55M2) examined the antitumor properties of CDDP and five naturally occurring coumarins (osthole, xanthotoxin, xanthotoxol, isopimpinellin, and imperatorin).,The antiproliferative effects produced by combinations of CDDP with the coumarins were assessed using type I isobolographic analysis.,(3) The most potent anticancer properties of coumarins were presented by osthole and xanthotoxol.,These compounds were characterized by the lowest median inhibitory concentration (IC50) values relative to the FM55P and FM55M2 melanoma cells.,Isobolographic analysis showed that for both melanoma cell lines, the combination of CDDP and osthole exerted synergistic and additive interactions, while the combination of CDDP and xanthotoxol exerted additive interactions.,Combinations of CDDP with xanthotoxin, isopimpinellin, and imperatorin showed antagonistic and additive interactions in two melanoma cell lines.,(4) The combination of CDDP and osthole was characterized by the most desirable synergistic interaction.,Isobolographic analysis allows the selection of potential candidates for cancer drugs among natural substances.

Despite intensive research and novel adjuvant therapies, there is currently no cure for metastatic melanoma.,The chemokine receptor CXCR4 controls metastasis to sites such as the liver; however, the therapeutic blockade with the existing agents has proven difficult.,AMD11070, a novel orally bioavailable inhibitor of CXCR4, was tested for its ability to inhibit the migration of melanoma cells compared with the commonly described antagonist AMD3100.,AMD11070 abrogated melanoma cell migration and was significantly more effective than AMD3100.,Importantly for the clinical context, the expression of B-RAF-V600E did not the affect the sensitivity of AMD11070.,Liver-resident myofibroblasts excrete CXCL12, which is able to promote the migration of CXCR4-expressing tumour cells from the blood into the liver.,Blockade of this axis by AMD11070 thus represents a novel therapeutic strategy for both B-RAF wild-type and mutated melanomas.

We have interrogated a 12-chemokine gene expression signature (GES) on genomic arrays of 14,492 distinct solid tumors and show broad distribution across different histologies.,We hypothesized that this 12-chemokine GES might accurately predict a unique intratumoral immune reaction in stage IV (non-locoregional) melanoma metastases.,The 12-chemokine GES predicted the presence of unique, lymph node-like structures, containing CD20+ B cell follicles with prominent areas of CD3+ T cells (both CD4+ and CD8+ subsets).,CD86+, but not FoxP3+, cells were present within these unique structures as well.,The direct correlation between the 12-chemokine GES score and the presence of unique, lymph nodal structures was also associated with better overall survival of the subset of melanoma patients.,The use of this novel 12-chemokine GES may reveal basic information on in situ mechanisms of the anti-tumor immune response, potentially leading to improvements in the identification and selection of melanoma patients most suitable for immunotherapy.

While the role of genetic risk factors in the etiology of uveal melanoma (UM) has been strongly suggested, the genetic susceptibility to UM is currently vastly unexplored.,Due to shared epidemiological risk factors between cutaneous melanoma (CM) and UM, in this study we have selected 28 SNPs identified as risk variants in previous genome-wide association studies on CM or CM-related host phenotypes (such as pigmentation and eye color) and tested them for association with UM risk.,By logistic regression analysis of 272 UM cases and 1782 controls using an additive model, we identified five variants significantly associated with UM risk, all passing adjustment for multiple testing.,The three most significantly associated variants rs12913832 (OR = 0.529, 95% CI 0.415-0.673; p = 8.47E-08), rs1129038 (OR = 0.533, 95% CI 0.419-0.678; p = 1.19E-07) and rs916977 (OR = 0.465, 95% CI 0.339-0.637; p = 3.04E-07) are correlated (r2 > 0.5) and map at 15q12 in the region of HERC2/OCA2, which determines eye-color in the human population.,Our data provides first evidence that the genetic factors associated with pigmentation traits are risk loci of UM susceptibility.

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.

Sézary syndrome (SS) is an aggressive leukemic variant of cutaneous T-cell lymphoma (L-CTCL) that arises from malignant clonally derived skin-homing CD4+ T cells.,Based on advancements in our understanding of the mechanisms underlying L-CTCL, boosting the suppressed immune response emerges as a promising strategy in SS management.,Immune checkpoint inhibitory molecules have already demonstrated efficacy in a wide spectrum of malignancies.,Currently, agents targeting the programmed death-1 (PD-1) axis are under evaluation in L-CTCL.,Here we investigated the expression of PD-1 and its ligands, PD-L1 and PD-L2 in blood and skin from patients with L-CTCL.,We demonstrate that PD-1 expression is markedly increased on tumor T cells compared to non-tumor CD4+ T cells from SS patients and to CD4+ cells from healthy individuals.,In contrast, PD-L1 shows decreased expression on tumor T cells, while PD-L2 expression is low without significant differences between these groups.,Functional PD-1 blockade in vitro resulted in reduced Th2 phenotype of non-tumor T lymphocytes, but enhanced the proliferation of tumor T cells from SS patients.,Our study sheds some light on the PD-1 axis in both peripheral blood and skin compartments in SS patients, which may be relevant for the treatment of L-CTCL with immune checkpoint inhibitor.

In patients with cutaneous T-cell lymphoma (CTCL) bacterial infections constitute a major clinical problem caused by compromised skin barrier and a progressive immunodeficiency.,Indeed, the majority of patients with advanced disease die from infections with bacteria, e.g., Staphylococcus aureus.,Bacterial toxins such as staphylococcal enterotoxins (SE) have long been suspected to be involved in the pathogenesis in CTCL.,Here, we review links between bacterial infections and CTCL with focus on earlier studies addressing a direct role of SE on malignant T cells and recent data indicating novel indirect mechanisms involving SE- and cytokine-driven cross-talk between malignant- and non-malignant T cells.

The Bateson-Dobzhansky-Muller (BDM) model describes negative epistatic interactions that occur between genes with a different evolutionary history to account for hybrid incompatibility and is a central theory explaining genetic mechanisms underlying speciation.,Since the early 1900 s when the BDM model was forwarded examples of BDM incompatibility have been described in only a few nonvertebrate cases.,This study reports the only vertebrate system, with clearly defined interacting loci, that supports the BDM model.,In addition, this study also poses that tumorigenesis serves as a novel mechanism that accounts for postzygotic isolation.,Mixing genomes of different species by hybridization can disrupt species-specific genetic interactions that were adapted and fixed within each species population.,Such disruption can predispose the hybrids to abnormalities and disease that decrease the overall fitness of the hybrids and is therefore named as hybrid incompatibility.,Interspecies hybridization between southern platyfish and green swordtails leads to lethal melanocyte tumorigenesis.,This occurs in hybrids with tumor incidence following progeny ratio that is consistent with two-locus interaction, suggesting melanoma development is a result of negative epistasis.,Such observations make Xiphophorus one of the only two vertebrate hybrid incompatibility examples in which interacting genes have been identified.,One of the two interacting loci has been characterized as a mutant epidermal growth factor receptor.,However, the other locus has not been identified despite over five decades of active research.,Here we report the localization of the melanoma regulatory locus to a single gene, rab3d, which shows all expected features of the long-sought oncogene interacting locus.,Our findings provide insights into the role of egfr regulation in regard to cancer etiology.,Finally, they provide a molecular explainable example of hybrid incompatibility.

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential.,Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors.,Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity.,In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models.,Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions.,The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells.,The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo.,Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Once melanomas have progressed with acquired resistance to mitogen-activated protein kinase (MAPK)-targeted therapy, mutational heterogeneity presents a major challenge.,We therefore examined the therapy phase before acquired resistance had developed and discovered the melanoma survival oncogene MITF as a driver of an early non-mutational and reversible drug-tolerance state, which is induced by PAX3-mediated upregulation of MITF.,A drug-repositioning screen identified the HIV1-protease inhibitor nelfinavir as potent suppressor of PAX3 and MITF expression.,Nelfinavir profoundly sensitizes BRAF and NRAS mutant melanoma cells to MAPK-pathway inhibitors.,Moreover, nelfinavir is effective in BRAF and NRAS mutant melanoma cells isolated from patients progressed on MAPK inhibitor (MAPKi) therapy and in BRAF/NRAS/PTEN mutant tumors.,We demonstrate that inhibiting a driver of MAPKi-induced drug tolerance could improve current approaches of targeted melanoma therapy.,•MITF is a driver of a reversible non-mutational drug-tolerance phase in melanoma•Drug repositioning identifies nelfinavir mesylate as a suppressor of MITF expression•Nelfinavir sensitizes BRAF and NRAS mutant melanoma to MAPK inhibitor treatment•A nelfinavir combination therapy overcomes NRAS-driven acquired resistance,MITF is a driver of a reversible non-mutational drug-tolerance phase in melanoma,Drug repositioning identifies nelfinavir mesylate as a suppressor of MITF expression,Nelfinavir sensitizes BRAF and NRAS mutant melanoma to MAPK inhibitor treatment,A nelfinavir combination therapy overcomes NRAS-driven acquired resistance,Smith et al. discover PAX3-mediated overexpression of MITF as a reversible resistance mechanism to MAPK-pathway inhibition in BRAF mutant melanomas and identify nelfinavir, which inhibits this mechanism and sensitizes not only BRAF mutant but also BRAF and NRAS mutant melanoma cells to MAPK-pathway inhibitors.

The most common mutation in melanoma, BRAF(V600E), activates the BRAF serine/threonine kinase and causes excessive MAPK pathway activity1,2.,BRAF(V600E)mutations are also present in benign melanocytic nevi3, highlighting the importance of additional genetic alterations in the genesis of malignant tumors.,Such changes include recurrent copy number variations that result in the amplification of oncogenes4,5.,For certain amplifications, the large number of genes in the interval has precluded an understanding of cooperating oncogenic events.,Here, we have used a zebrafish melanoma model to test genes in a recurrently amplified region on chromosome 1 for the ability to cooperate with BRAF(V600E) and accelerate melanoma.,SETDB1, an enzyme that methylates histone H3 on lysine 9 (H3K9), was found to significantly accelerate melanoma formation in the zebrafish.,Chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-Seq) and gene expression analyses revealed target genes, including Hox genes, that are transcriptionally dysregulated in response to elevated SETDB1.,Our studies establish SETDB1 as an oncogene in melanoma and underscore the role of chromatin factors in regulating tumorigenesis.

Cutaneous melanoma is a lethal disease, even when diagnosed in advanced stages.,Although recent progress in biology and treatment has dramatically improved survival rates, new therapeutic approaches are still needed.,Deregulation of epigenetics, which mainly controls DNA methylation status and chromatin remodeling, is implied not only in cancer initiation and progression, but also in resistance to antitumor drugs.,Epigenetics in melanoma has been studied recently in both melanoma preclinical models and patient samples, highlighting its potential role in different phases of melanomagenesis, as well as in resistance to approved drugs such as immune checkpoint inhibitors and MAPK inhibitors.,This review summarizes what is currently known about epigenetics in melanoma and dwells on the recognized and potential new targets for testing epigenetic drugs, alone or together with other agents, in advanced melanoma patients.

The presence of immune cells in the tumor microenvironment has been associated with response to immunotherapies across several cancer types, including melanoma.,Despite its therapeutic relevance, characterization of the melanoma immune microenvironments remains insufficiently explored.,To distinguish the immune microenvironment in a cohort of 180 metastatic melanoma clinical specimens, we developed a method using promoter CpG methylation of immune cell type‐specific genes extracted from genome‐wide methylation arrays.,Unsupervised clustering identified three immune methylation clusters with varying levels of immune CpG methylation that are related to patient survival.,Matching protein and gene expression data further corroborated the identified epigenetic characterization.,Exploration of the possible immune exclusion mechanisms at play revealed likely dependency on MITF protein level and PTEN loss‐of‐function events for melanomas unresponsive to immunotherapies (immune‐low).,To understand whether melanoma tumors resemble other solid tumors in terms of immune methylation characteristics, we explored 15 different solid tumor cohorts from TCGA.,Low‐dimensional projection based on immune cell type‐specific methylation revealed grouping of the solid tumors in line with melanoma immune methylation clusters rather than tumor types.,Association of survival outcome with immune cell type‐specific methylation differed across tumor and cell types.,However, in melanomas immune cell type‐specific methylation was associated with inferior patient survival.,Exploration of the immune methylation patterns in a pan‐cancer context suggested that specific immune microenvironments might occur across the cancer spectrum.,Together, our findings underscore the existence of diverse immune microenvironments, which may be informative for future immunotherapeutic applications.,The importance of the tumor immune microenvironment has been highlighted in multiple cancers, but information regarding the contribution of individual immune cell types is lacking for melanoma.,In this study, we have sought to fill the gap by identifying distinct DNA methylation patterns for specifc immune cells.,These were corroborated by other molecular correlates and found to be shared across other types of solid tomors.

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.

Skin cancer, which includes melanoma and squamous cell carcinoma, represents the most common type of cutaneous malignancy worldwide, and its incidence is expected to rise in the near future.,This condition derives from acquired genetic dysregulation of signaling pathways involved in the proliferation and apoptosis of skin cells.,The development of animal models has allowed a better understanding of these pathomechanisms, with the possibility of carrying out toxicological screening and drug development.,In particular, the zebrafish (Danio rerio) has been established as one of the most important model organisms for cancer research.,This model is particularly suitable for live cell imaging and high-throughput drug screening in a large-scale fashion.,Thanks to the recent advances in genome editing, such as the clustered regularly-interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) methodologies, the mechanisms associated with cancer development and progression, as well as drug resistance can be investigated and comprehended.,With these unique tools, the zebrafish represents a powerful platform for skin cancer research in the development of target therapies.,Here, we will review the advantages of using the zebrafish model for drug discovery and toxicological and phenotypical screening.,We will focus in detail on the most recent progress in the field of zebrafish model generation for the study of melanoma and squamous cell carcinoma (SCC), including cancer cell injection and transgenic animal development.,Moreover, we will report the latest compounds and small molecules under investigation in melanoma zebrafish models.

Basal cell carcinomas (BCCs) exhibit aberrant activation of the hedgehog pathway.,Sonidegib is a hedgehog pathway inhibitor approved for the treatment of locally advanced BCC (laBCC) and metastatic BCC (mBCC) based on primary results of the BOLT study [Basal Cell Carcinoma Outcomes with LDE225 (sonidegib) Treatment].,This is the final 42‐month analysis of the BOLT study, evaluating the efficacy and safety of sonidegib.,Adults with no prior hedgehog pathway inhibitor therapy were randomized in a 1 : 2 ratio to sonidegib 200 mg or 800 mg once daily.,Treatment continued for up to 42 months or until disease progression, unacceptable toxicity, death, study termination or withdrawal of consent.,The primary efficacy end point was the objective response rate (ORR) by central review, assessed at baseline; weeks 5, 9 and 17; then subsequently every 8 or 12 weeks during years 1 or 2, respectively.,Safety end points included adverse event monitoring and reporting.,The study enrolled 230 patients, 79 and 151 in the 200‐mg and 800‐mg groups, respectively, of whom 8% and 3.3% remained on treatment by the 42‐month cutoff, respectively.,The ORRs by central review were 56% [95% confidence interval (CI) 43-68] for laBCC and 8% (95% CI 0·2-36) for mBCC in the 200‐mg group and 46·1% (95% CI 37·2-55·1) for laBCC and 17% (95% CI 5-39) for mBCC in the 800‐mg group.,No new safety concerns emerged.,Sonidegib demonstrated sustained efficacy and a manageable safety profile.,The final BOLT results support sonidegib as a viable treatment option for laBCC and mBCC.,What's already known about this topic?,Basal cell carcinoma (BCC) is usually treatable with surgery or radiation therapy, but there are limited treatment options for patients with advanced BCC.Sonidegib, a hedgehog pathway inhibitor approved for the treatment of advanced BCC, demonstrated clinically relevant efficacy and manageable safety in prior analyses of the phase II randomized, double‐blind BOLT study [Basal Cell Carcinoma Outcomes with LDE225 (sonidegib) Treatment].,Basal cell carcinoma (BCC) is usually treatable with surgery or radiation therapy, but there are limited treatment options for patients with advanced BCC.,Sonidegib, a hedgehog pathway inhibitor approved for the treatment of advanced BCC, demonstrated clinically relevant efficacy and manageable safety in prior analyses of the phase II randomized, double‐blind BOLT study [Basal Cell Carcinoma Outcomes with LDE225 (sonidegib) Treatment].,What does this study add?,This final 42‐month analysis of BOLT is the longest follow‐up available for a hedgehog pathway inhibitor.Clinically relevant efficacy results were sustained from prior analyses, with objective response rates by central review of the approved 200‐mg daily dose of 56% in locally advanced BCC and 8% in metastatic BCC.No new safety concerns were raised.The results confirmed sonidegib as a viable long‐term treatment option for patients with advanced BCC.,This final 42‐month analysis of BOLT is the longest follow‐up available for a hedgehog pathway inhibitor.,Clinically relevant efficacy results were sustained from prior analyses, with objective response rates by central review of the approved 200‐mg daily dose of 56% in locally advanced BCC and 8% in metastatic BCC.,No new safety concerns were raised.,The results confirmed sonidegib as a viable long‐term treatment option for patients with advanced BCC.,Linked Comment:Fife.,Br J Dermatol 2020; 182:1322-1323.

In the primary analysis of the ERIVANCE BCC trial, vismodegib, the first US Food and Drug Administration-approved Hedgehog pathway inhibitor, showed objective response rates (ORRs) by independent review facility (IRF) of 30% and 43% in metastatic basal cell carcinoma (mBCC) and locally advanced BCC (laBCC), respectively.,ORRs by investigator review were 45% (mBCC) and 60% (laBCC).,Herein, we present long-term safety and final investigator-assessed efficacy results in patients with mBCC or laBCC.,One hundred four patients with measurable advanced BCC received oral vismodegib 150 mg once daily until disease progression or intolerable toxicity.,The primary end point was IRF-assessed ORR.,Secondary end points included ORR, duration of response (DOR), progression-free survival, overall survival (OS), and safety.,At data cutoff (39 months after completion of accrual), 8 patients were receiving the study drug (69 patients in survival follow-up).,Investigator-assessed ORR was 48.5% in the mBCC group (all partial responses) and 60.3% in the laBCC group (20 patients had complete response and 18 patients had partial response).,ORRs were comparable across patient subgroups, including aggressive histologic subtypes (eg, infiltrative BCC).,Median DOR was 14.8 months (mBCC) and 26.2 months (laBCC).,Median OS was 33.4 months in the mBCC cohort and not estimable in the laBCC cohort.,Adverse events remained consistent with clinical experience.,Thirty-three deaths (31.7%) were reported; none were related to vismodegib.,This long-term update of the ERIVANCE BCC trial demonstrated durability of response, efficacy across patient subgroups, and manageable long-term safety of vismodegib in patients with advanced BCC.,This study was registered prospectively with Clinicaltrials.gov, number NCT00833417 on January 30, 2009.,The online version of this article (doi:10.1186/s12885-017-3286-5) contains supplementary material, which is available to authorized users.

Melanoma affects about 6000 patients a year in Spain.,A group of medical oncologists from Spanish Society of Medical Oncology (SEOM) and Spanish Multidisciplinary Melanoma Group (GEM) has designed these guidelines to homogenize the management of these patients.,The diagnosis must be histological and determination of BRAF status has to be performed in patients with stage ≥ III.,Stage I-III resectable melanomas will be treated surgically.,In patients with stage III melanoma, adjuvant treatment with immunotherapy or targeted therapy is also recommended.,Patients with unresectable or metastatic melanoma will receive treatment with immunotherapy or targeted therapy, the optimal sequence of these treatments remains unclear.,Brain metastases require a separate consideration, since, in addition to systemic treatment, they may require local treatment.,Patients must be followed up closely to receive or change treatment as soon as their previous clinical condition changes, since multiple therapeutic options are available.

Drug tolerance brought about by reversible adaptive responses precedes the emergence of irreversible mutation-driven drug resistance and sustains tumor cells when at their most vulnerable.,Young et al. delineate a signaling relay incorporating IL-1 and CXCR2 ligands emanating from melanoma-associated macrophages and fibroblasts, respectively, that confer tolerance to MAPK inhibitors.,Mitogen-activated protein kinase (MAPK) pathway antagonists induce profound clinical responses in advanced cutaneous melanoma, but complete remissions are frustrated by the development of acquired resistance.,Before resistance emerges, adaptive responses establish a mutation-independent drug tolerance.,Antagonizing these adaptive responses could improve drug effects, thereby thwarting the emergence of acquired resistance.,In this study, we reveal that inflammatory niches consisting of tumor-associated macrophages and fibroblasts contribute to treatment tolerance through a cytokine-signaling network that involves macrophage-derived IL-1β and fibroblast-derived CXCR2 ligands.,Fibroblasts require IL-1β to produce CXCR2 ligands, and loss of host IL-1R signaling in vivo reduces melanoma growth.,In tumors from patients on treatment, signaling from inflammatory niches is amplified in the presence of MAPK inhibitors.,Signaling from inflammatory niches counteracts combined BRAF/MEK (MAPK/extracellular signal-regulated kinase kinase) inhibitor treatment, and consequently, inhibiting IL-1R or CXCR2 signaling in vivo enhanced the efficacy of MAPK inhibitors.,We conclude that melanoma inflammatory niches adapt to and confer drug tolerance toward BRAF and MEK inhibitors early during treatment.

The current development of BRAF inhibitors has revolutionized the treatment of unresectable melanoma.,As the potential heterogeneity of BRAF mutations in melanoma has been reported, accurate detection of BRAF mutations are important.,However, the genetic heterogeneity of acral melanoma-a distinct type of melanoma with a unique genetic background-has not fully been investigated.,We conducted a retrospective review of our acral melanoma patients.,Of the 196 patients with acral melanoma, we retrieved 31 pairs of primary and matched metastatic melanomas.,We immunostained the 31 pairs with VE1, a BRAFV600E-mutation-specific monoclonal antibody.,Immunohistochemistry with VE1 showed a high degree of sensitivity and specificity for detecting BRAFV600E mutations compared with the real-time polymerase chain reaction method.,A total of nine primary (29.0%) and eight metastatic (25.8%) acral melanomas were positive for VE1.,In three patients (9.7%), we observed a discordance of VE1 staining between the primary and metastatic lesions.,Of note, VE1 immunohistochemical staining revealed a remarkable degree of intra-tumor genetic heterogeneity in acral melanoma.,Our study reveals that VE1 immunostaining is a useful ancillary method for detecting BRAFV600E mutations in acral melanoma and allows for a clear visualization of intra- and inter-tumor BRAF heterogeneity.

Selective BRAF inhibitors, vemurafenib and dabrafenib, and the MEK inhibitor, trametinib, have been approved for treatment of metastatic melanomas with a BRAF p.V600E mutation.,The clinical significance of non-codon 600 mutations remains unclear, in part, due to variation of kinase activity for different mutants.,In this study, we categorized BRAF mutations according to the reported mutant kinase activity.,A total of 1027 lung cancer, colorectal cancer or melanoma specimens were submitted for clinical mutation detection by next generation sequencing.,Non-codon 600 mutations were observed in 37 % of BRAF-mutated tumors.,Of all BRAF mutants, 75 % were kinase-activated, 15 % kinase-impaired and 10 % kinase-unknown.,The most common kinase-impaired mutant involves codon 594, specifically, p.D594G (c.1781A > G) and p.D594N (c.1780G > A).,Lung cancers showed significantly higher incidences of kinase-impaired or kinase-unknown mutants.,Kinase-impaired BRAF mutants showed a significant association with concomitant activating KRAS or NRAS mutations, but not PIK3CA mutations, supporting the reported interaction of these mutations.,BRAF mutants with impaired or unknown kinase activity as well as concomitant kinase-impaired BRAF mutations and RAS mutations were detected in lung cancers, colorectal cancers and melanomas.,Different therapeutic strategies based on the BRAF mutant kinase activity and the concomitant mutations may be worthwhile.

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.

Female sex and history of prior pregnancies are associated with favorable melanoma outcomes.,Here, we show that much of the melanoma protective effect likely results from estrogen signaling through the G protein-coupled estrogen receptor (GPER) on melanocytes.,Selective GPER activation in primary melanocytes and melanoma cells induced long-term changes that maintained a more differentiated cell state as defined by increased expression of well-established melanocyte differentiation antigens, increased pigment production, decreased proliferative capacity, and decreased expression of the oncodriver and stem cell marker c-Myc.,GPER signaling also rendered melanoma cells more vulnerable to immunotherapy.,Systemically delivered GPER agonist was well tolerated, and cooperated with immune checkpoint blockade in melanoma-bearing mice to dramatically extend survival, with up to half of mice clearing their tumor.,Complete responses were associated with immune memory that protected against tumor rechallenge.,GPER may be a useful, pharmacologically accessible target for melanoma.

Single-cell RNA-seq's (scRNA-seq) unprecedented cellular resolution at a genome-wide scale enables us to address questions about cellular heterogeneity that are inaccessible using methods that average over bulk tissue extracts.,However, scRNA-seq data sets also present additional challenges such as high transcript dropout rates, stochastic transcription events, and complex population substructures.,Here, we present a single-cell RNA-seq analysis and klustering evaluation (SAKE), a robust method for scRNA-seq analysis that provides quantitative statistical metrics at each step of the analysis pipeline.,Comparing SAKE to multiple single-cell analysis methods shows that most methods perform similarly across a wide range of cellular contexts, with SAKE outperforming these methods in the case of large complex populations.,We next applied the SAKE algorithms to identify drug-resistant cellular populations as human melanoma cells respond to targeted BRAF inhibitors (BRAFi).,Single-cell RNA-seq data from both the Fluidigm C1 and 10x Genomics platforms were analyzed with SAKE to dissect this problem at multiple scales.,Data from both platforms indicate that BRAF inhibitor-resistant cells can emerge from rare populations already present before drug application, with SAKE identifying both novel and known markers of resistance.,These experimentally validated markers of BRAFi resistance share overlap with previous analyses in different melanoma cell lines, demonstrating the generality of these findings and highlighting the utility of single-cell analysis to elucidate mechanisms of BRAFi resistance.

Effective management of melanoma depends heavily on early diagnosis.,When detected in early non-metastatic stages, melanoma is almost 100% curable by surgical resection, however when detected in late metastatic stages III and IV, 5-year survival rates drop to ~50% and 10-25%, respectively, due to limited efficacy of current treatment options.,This presents a pressing need to identify biomarkers that can detect patients at high risk of recurrence and progression to metastatic disease, which will allow for early intervention and survival benefit.,Accumulating evidence over the past few decades has highlighted the potential use of circulating molecular biomarkers for melanoma diagnosis and prognosis, including lactate dehydrogenase (LDH), S100 calcium-binding protein B (S100B) and circulating tumor DNA (ctDNA) fragments.,Since 2010, circulating microRNAs (miRNAs) have been increasingly recognised as more robust non-invasive biomarkers for melanoma due to their structural stability under the harsh conditions of the blood and different conditions of sample processing and isolation.,Several pre-analytical and analytical variables challenge the accurate quantification of relative miRNA levels between serum samples or plasma samples, leading to conflicting findings between studies on circulating miRNA biomarkers for melanoma.,In this review, we provide a critical summary of the circulating miRNA biomarkers for melanoma published to date.

Increasing evidence highlights the association of occupational exposure and cutaneous malignant melanoma (CMM).,We estimated the burden of CMM and total skin cancer burden in Britain due to occupational solar radiation exposure.,Attributable fractions (AF) and numbers were estimated for CMM mortality and incidence using risk estimates from the published literature and national data sources for proportions exposed.,We extended existing methods to account for the exposed population age structure.,The estimated total AF for CMM is 2.0% (95% CI: 1.4-2.7%), giving 48 (95% CI: 33-64) deaths in (2012) and 241 (95% CI: 168-325) registrations (in 2011) attributable to occupational exposure to solar radiation.,Higher exposure and larger numbers exposed led to much higher numbers for men than women.,Industries of concern are construction, agriculture, public administration and defence, and land transport.,These results emphasise the urgent need to develop appropriate strategies to reduce this burden.

Postal delivery workers spend a large proportion of their work time outdoors, placing them at increased risk of skin cancer.,To date, no studies have examined occupational sun safety knowledge and practice within this group in the UK.,To describe the occupational sun safety knowledge and practice of UK postal delivery workers and to investigate the association of demographic, personal and occupational factors with knowledge and practice in order to identify potential strategies for improving sun safety in this occupational group.,Postal delivery workers completed a questionnaire that collected data on occupational sun safety knowledge and practice in addition to demographic, personal and workplace characteristics.,One-way analysis of variances were applied to assess differences in knowledge and practice by these characteristics.,A total of 1153 postal delivery workers completed the questionnaire, a 60% response rate.,Thirty-three per cent reported receiving sun safety training within the previous 12 months.,The majority of respondents reported correct knowledge on three of the six domains and good practice on four of the six behavioural domains.,However, only one-fifth of respondents reported wearing sunglasses and ensuring a plentiful intake of water.,Knowledge and practice differed significantly according to demographic, personal and workplace characteristics.,There is a need to raise the profile of occupational skin cancer in this occupational group and to increase the priority given to occupational sun safety policies alongside targeted and tailored interventions, the effect of which can be evaluated.

Drug resistance remains as one of the major challenges in melanoma therapy.,It is well known that tumour cells undergo phenotypic switching during melanoma progression, increasing melanoma plasticity and resistance to mitogen-activated protein kinase inhibitors (MAPKi).,We investigated the melanoma phenotype switching using a partial reprogramming model to de-differentiate murine melanoma cells and target melanoma therapy adaptation against MAPKi.,Here, we show that partially reprogrammed cells are a less proliferative and more de-differentiated cell population, expressing a gene signature for stemness and suppressing melanocyte-specific markers.,To investigate adaptation to MAPKi, cells were exposed to B-Raf Proto-Oncogene (BRAF) and mitogen-activated protein kinase kinase (MEK) inhibitors.,De-differentiated cells became less sensitive to MAPKi, showed increased cell viability and decreased apoptosis.,Furthermore, T-type calcium channels expression increased in adaptive murine cells and in human adaptive melanoma cells.,Treatment with the calcium channel blocker mibefradil induced cell death, differentiation and susceptibility to MAPKi in vitro and in vivo.,In summary, we show that partial reprogramming of melanoma cells induces de-differentiation and adaptation to MAPKi.,Moreover, we postulated a calcium channel blocker such as mibefradil, as a potential candidate to restore sensitivity to MAPKi in adaptive melanoma cells.

Melanoma is one of the most lethal cancers when it reaches a metastatic stage.,Despite the spectacular achievements of targeted therapies (BRAF inhibitors) or immuno-therapies (anti-CTLA4 or anti-PD1), most patients with melanoma will need additional treatments.,Here we used a photoactive NADPH analogue called NS1 to induce cell death by inhibition of NADPH oxidases NOX in melanoma cells, including melanoma cells isolated from patients.,In contrast, healthy melanocytes growth was unaffected by NS1 treatment.,NS1 established an early Endoplasmic Reticulum stress by the early release of calcium mediated by (a) calcium-dependent redox-sensitive ion channel(s).,These events initiated autophagy and apoptosis in all tested melanoma cells independently of their mutational status.,The autophagy promoted by NS1 was incomplete.,The autophagic flux was blocked at late stage events, consistent with the accumulation of p62, and a close localization of LC3 with NS1 associated with NS1 inhibition of NOX1 in autophagosomes.,This hypothesis of a specific incomplete autophagy and apoptosis driven by NS1 was comforted by the use of siRNAs and pharmacological inhibitors blocking different processes.,This study highlights the potential therapeutic interest of NS1 inducing cell death by triggering a selective ER stress and incomplete autophagy in melanoma cells harbouring wt and BRAF mutation.

Drugs that inhibit RAF/MEK signaling, such as vemurafenib, elicit profound but often temporary anti-tumor responses in patients with BRAFV600E melanoma.,Adaptive responses to RAF/MEK inhibition occur on a timescale of hours to days, involve homeostatic responses that reactivate MAP kinase signaling and compensatory mitogenic pathways, and attenuate the anti-tumor effects of RAF/MEK inhibitors.,We profile adaptive responses across a panel of melanoma cell lines using multiplex biochemical measurement, single-cell assays, and statistical modeling and show that adaptation involves at least six signaling cascades that act to reduce drug potency (IC50) and maximal effect (i.e., Emax ≪ 1).,Among these cascades, we identify a role for JNK/c-Jun signaling in vemurafenib adaptation and show that RAF and JNK inhibitors synergize in cell killing.,This arises because JNK inhibition prevents a subset of cells in a cycling population from becoming quiescent upon vemurafenib treatment, thereby reducing drug Emax.,Our findings demonstrate the breadth and diversity of adaptive responses to RAF/MEK inhibition and a means to identify which steps in a signaling cascade are most predictive of phenotypic response.

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.

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.

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.

Effective management of melanoma depends heavily on early diagnosis.,When detected in early non-metastatic stages, melanoma is almost 100% curable by surgical resection, however when detected in late metastatic stages III and IV, 5-year survival rates drop to ~50% and 10-25%, respectively, due to limited efficacy of current treatment options.,This presents a pressing need to identify biomarkers that can detect patients at high risk of recurrence and progression to metastatic disease, which will allow for early intervention and survival benefit.,Accumulating evidence over the past few decades has highlighted the potential use of circulating molecular biomarkers for melanoma diagnosis and prognosis, including lactate dehydrogenase (LDH), S100 calcium-binding protein B (S100B) and circulating tumor DNA (ctDNA) fragments.,Since 2010, circulating microRNAs (miRNAs) have been increasingly recognised as more robust non-invasive biomarkers for melanoma due to their structural stability under the harsh conditions of the blood and different conditions of sample processing and isolation.,Several pre-analytical and analytical variables challenge the accurate quantification of relative miRNA levels between serum samples or plasma samples, leading to conflicting findings between studies on circulating miRNA biomarkers for melanoma.,In this review, we provide a critical summary of the circulating miRNA biomarkers for melanoma published to date.

microRNAs constitute a complex class of pleiotropic post-transcriptional regulators of gene expression involved in the control of several physiologic and pathologic processes.,Their mechanism of action is primarily based on the imperfect matching of a seed region located at the 5′ end of a 21-23 nt sequence with a partially complementary sequence located in the 3′ untranslated region of target mRNAs.,This leads to inhibition of mRNA translation and eventually to its degradation.,Individual miRNAs are capable of binding to several mRNAs and several miRNAs are capable of influencing the function of the same mRNAs.,In recent years networks of miRNAs are emerging as capable of controlling key signaling pathways responsible for the growth and propagation of cancer cells.,Furthermore several examples have been provided which highlight the involvement of miRNAs in the development of resistance to targeted drug therapies.,In this review we provide an updated overview of the role of miRNAs in the development of melanoma and the identification of the main downstream pathways controlled by these miRNAs.,Furthermore we discuss a group of miRNAs capable to influence through their respective up- or down-modulation the development of resistance to BRAF and MEK inhibitors.

Melanoma is a malignant tumor deriving from neoplastic transformation of melanocytes.,The incidence of melanoma has increased dramatically over the last 50 years.,It accounts for most cases of skin cancer deaths.,Early diagnosis leads to remission in 90% of cases of melanoma; conversely, for melanoma at more advanced stages, prognosis becomes more unfavorable also because dvanced melanoma is often resistant to pharmacological and radiological therapies due to genetic plasticity, presence of cancer stem cells that regenerate the tumor, and efficient elimination of drugs.,This review illustrates the role of autophagy in tumor progression and resistance to therapy, focusing on molecular targets for future drugs.

BCL2 19 kD protein-interacting protein 3 (BNIP3) is a BH3-containing protein of the BCL-2 family; it can regulate cell death, autophagy, and cytoprotection.,The upregulation of BNIP3 has been reported to relate to progression and poor prognosis in different cancer types.,However, the clinical significance of BNIP3 in uveal melanoma (UM) is still unknown.,In our study, 47 patients with UM were enrolled; the expression of BNIP3 was detected with immunohistochemistry.,According to BNIP3 immunohistochemical scores, the patients were divided into BNIP3 high- and low-expression subgroups.,The correlation between the expression of BNIP3 and clinicopathological factors was evaluated with Fisher’s test; the associations with survival rates were analyzed with log-rank test.,The independent prognostic factors were identified with the Cox-regression model.,BNIP3 was mainly localized in the cytoplasm, and high expression of BNIP3 accounted for 31.9% (15/47) of the patients in our study.,High expression of BNIP3 was demonstrated to be significantly associated with more pigment (P=0.018) and deeper scleral invasion (P=0.013).,High expression of BNIP3 was also correlated with lower overall survival rate (P=0.006).,Multivariate analysis confirmed positive ciliary body involvement and lymphatic infiltration as independent prognostic factors.,High expression of BNIP3 was significantly associated with poor prognosis of patients with UM, indicating that BNIP3 detection could help stratify high-risk patients and identify new therapies targeting BNIP3 as a promising approach to treat UM.

V600BRAF mutations drive approximately 50% of metastatic melanoma which can be therapeutically targeted by BRAF inhibitors (BRAFi) and, based on resistance mechanisms, the combination of BRAF and MEK inhibitors (BRAFi + MEKi).,Although the combination therapy has been shown to provide superior clinical benefits, acquired resistance is still prevalent and limits the overall survival benefits.,Recent work has shown that oncogenic changes can lead to alterations in tumor cell metabolism rendering cells addicted to nutrients, such as the amino acid glutamine.,Here, we evaluated whether melanoma cells with acquired resistance display glutamine dependence and whether glutamine metabolism can be a potential molecular target to treat resistant cells.,Isogenic BRAFi sensitive parental V600BRAF mutant melanoma cell lines and resistant (derived by chronic treatment with vemurafenib) sub-lines were used to assess differences in the glutamine uptake and sensitivity to glutamine deprivation.,To evaluate a broader range of resistance mechanisms, isogenic pairs where the sub-lines were resistant to BRAFi + MEKi were also studied.,Since resistant cells demonstrated increased sensitivity to glutamine deficiency, we used glutaminase inhibitors BPTES [bis-2-(5 phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide] and L-L-DON (6-Diazo-5-oxo-l-norleucine) to treat MAPK pathway inhibitor (MAPKi) resistant cell populations both in vitro and in vivo.,We demonstrated that MAPKi-acquired resistant cells uptook greater amounts of glutamine and have increased sensitivity to glutamine deprivation than their MAPKi-sensitive counterparts.,In addition, it was found that both BPTES and L-DON were more effective at decreasing cell survival of MAPKi-resistant sub-lines than parental cell populations in vitro.,We also showed that mutant NRAS was critical for glutamine addiction in mutant NRAS driven resistance.,When tested in vivo, we found that xenografts derived from resistant cells were more sensitive to BPTES or L-DON treatment than those derived from parental cells.,Our study is a proof-of-concept for the potential of targeting glutamine metabolism as an alternative strategy to suppress acquired MAPKi-resistance in melanoma.

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.

SPRED1 is a negative regulator of the MAPK pathway frequently deleted in human melanoma.,Through in vivo modeling in zebrafish and mechanistic analyses in human cell lines, Ablain et al. demonstrate that SPRED1 inactivation confers resistance to targeted inhibition of V600 mutant BRAF, the most common driver of melanoma.,Functional evaluation of genetic lesions can discover a role in cancer initiation and progression and help develop novel therapeutic strategies.,We previously identified the negative MAPK regulator SPRED1 as a novel tumor suppressor in KIT-driven melanoma.,Here, we show that SPRED1 is also frequently deleted in human melanoma driven by mutant BRAF.,We found that SPRED1 inactivation in human melanoma cell lines and primary zebrafish melanoma conferred resistance to BRAFV600E inhibition in vitro and in vivo.,Mechanistically, SPRED1 loss promoted melanoma cell proliferation under mutant BRAF inhibition by reactivating MAPK activity.,Consistently, biallelic deletion of SPRED1 was observed in a patient whose melanoma acquired resistance to MAPK-targeted therapy.,These studies combining work in human cells and in vivo modeling in zebrafish demonstrate a new mechanism of resistance to BRAFV600E inhibition in melanoma.

Vanicosides A and B are the esters of hydroxycinnamic acids with sucrose, occurring in a few plant species from the Polygonaceae family.,So far, vanicosides A and B have not been evaluated for anticancer activity against human malignant melanoma.,In this study, we tested these two natural products, isolated from Reynoutria sachalinensis rhizomes, against two human melanoma cell lines (amelanotic C32 cell line and melanotic A375 cell line, both bearing endogenous BRAFV600E mutation) and two normal human cell lines-keratinocytes (HaCaT) and the primary fibroblast line.,Additionally, a molecular docking of vanicoside A and vanicoside B with selected targets involved in melanoma progression was performed.,Cell viability was studied using an MTT assay.,A RealTime-Glo™ Annexin V Apoptosis and Necrosis assay was used for monitoring programmed cell death (PCD).,Vanicoside A demonstrated strong cytotoxicity against the amelanotic C32 cell line (viability of the C32 cell line was decreased to 55% after 72 h incubation with 5.0 µM of vanicoside A), significantly stronger than vanicoside B.,This stronger cytotoxic activity can be attributed to an additional acetyl group in vanicoside A.,No significant differences in the cytotoxicity of vanicosides were observed against the less sensitive A375 cell line.,Moreover, vanicosides caused the death of melanoma cells at concentrations from 2.5 to 50 µM, without harming the primary fibroblast line.,The keratinocyte cell line (HaCaT) was more sensitive to vanicosides than fibroblasts, showing a clear decrease in viability after incubation with 25 µM of vanicoside A as well as a significant phosphatidylserine (PS) exposure, but without a measurable cell death-associated fluorescence.,Vanicosides induced an apoptotic death pathway in melanoma cell lines, but because of the initial loss of cell membrane integrity, an additional cell death mechanism might be involved like permeability transition pore (PTP)-mediated necrosis that needs to be explored in the future.,Molecular docking indicated that both compounds bind to the active site of the BRAFV600E kinase and MEK-1 kinase; further experiments on their specific inhibitory activity of these targets should be considered.

Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer associated with a high risk of metastasis.,In 2017, avelumab (anti-programmed death-ligand 1 (PD-L1)) became the first approved treatment for patients with metastatic MCC (mMCC), based on the occurrence of durable responses in a subset of patients.,Here, we report long-term efficacy and safety data and exploratory biomarker analyses in patients with mMCC treated with avelumab.,In a cohort of this single-arm, phase 2 trial (JAVELIN Merkel 200), patients with mMCC and disease progression after prior chemotherapy received avelumab 10 mg/kg intravenously every 2 weeks.,The primary endpoint was confirmed objective response rate (ORR) by independent review per Response Evaluation Criteria in Solid Tumors V.1.1.,Other assessments included duration of response, progression-free survival, overall survival (OS), safety and biomarker analyses.,As of 14 September 2018, 88 patients had been followed up for a median of 40.8 months (range 36.4-49.7 months).,The ORR was 33.0% (95% CI 23.3% to 43.8%), including a complete response in 11.4% (10 patients), and the median duration of response was 40.5 months (95% CI 18.0 months to not estimable).,As of 2 May 2019 (≥44 months of follow-up), the median OS was 12.6 months (95% CI 7.5 to 17.1 months) and the 42-month OS rate was 31% (95% CI 22% to 41%).,Of long-term survivors (OS >36 months) evaluable for PD-L1 expression status (n=22), 81.8% had PD-L1+ tumors.,In exploratory biomarker analyses, high tumor mutational burden (≥2 non-synonymous somatic variants per megabase) and high major histocompatibility complex class I expression (30% of tumors with highest expression) were associated with trends for improved ORR and OS.,In long-term safety assessments (≥36 months of follow-up), no new or unexpected adverse events were reported, and no treatment-related deaths occurred.,Avelumab showed continued durable responses and meaningful long-term survival outcomes in patients with mMCC, reinforcing avelumab as a standard-of-care treatment option for this disease.,NCT02155647

Case series,Patients: Male, 58 • Male, 65 • Male, 75 • Male, 61 • Male, 69,Final Diagnosis: Merkel cell carcinoma,Symptoms: Metastatic disease,Medication: Pazopanib • Cabozantinib,Clinical Procedure: Systemic therapy,Specialty: Oncology,Rare disease,Merkel cell carcinoma (MCC) is a rare but aggressive neuroendocrine skin cancer.,The estimated 5-year survival of patients with metastatic disease is approximately 14%.,Cytotoxic chemotherapy is associated with a modest median progression-free survival (PFS) of only 3 months.,In recent studies, immunotherapy with anti-PD-1/anti-PD-L1 antibodies has demonstrated a high response rate in immunocompetent patients (>50% in chemotherapy-naïve patients) and responses are typically durable.,However, approximately 50% of immunocompetent patients do not respond to immunotherapy.,In addition, immunosuppressed patients have limited therapeutic options.,Hence, there is a significant unmet need for effective treatments in these subpopulations.,We describe 5 patients (out of 24 total) with metastatic MCC who were treated with a vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor (TKI), either pazopanib (n=4) or cabozantinib (n=1), with clinical benefit.,One patient had a complete response to pazopanib after 3 months of therapy.,Four patients had stabilization of disease that lasted from 5 months to 3.5 years.,In an immunosuppressed patient with psoriatic arthritis, stabilization of MCC was also associated with improvement in his arthritis that allowed cessation of immunosuppression.,Patients did not develop any unusual toxicities from VEGFR-TKIs.,Treatment with VEGFR-TKIs demonstrated clinical benefit in this selected small group of patients with metastatic MCC.,Prospective investigation of VEGFR-TKIs is warranted in this population, especially in patients with disease refractory to immunotherapy.

Cancer is a leading cause of death worldwide, and while great advances have been made particularly in chemotherapy, many types of cancer still present a dismal prognosis.,In the case of glioma, temozolomide (TMZ) is the main option for treatment, but it has limited success due to drug resistance.,While this resistance is usually associated to DNA repair mechanisms, in this work we demonstrate that oxidative stress plays an important role.,We showed that upon TMZ treatment there is an induction of the nuclear factor erythroid 2-related factor 2 (NRF2), which is the main antioxidant transcription factor regulator in human cells.,This is accompanied by an enhancement of glutathione (GSH) concentration in the tumor cells.,The effectiveness of this pathway was proven by silencing NFR2, which greatly enhanced cell death upon TMZ treatment both in vitro and in vivo.,Also, higher DNA damage and induced cell death was observed by combining BSO - a GSH inhibitor - with TMZ.,Similar effects were also observed using in vitro and in vivo models of melanoma, thus possibly indicating that GSH has a decisive role in TMZ resistance in a wider range of tumors.,Thus, a combined regimen of BSO and TMZ configures an interesting therapeutic alternative for fighting both glioma and melanoma.

BRAFV600E-mutant malignant melanomas depend on RAF/MEK/ERK (MAPK) signaling for tumor cell growth1.,RAF and MEK inhibitors show remarkable clinical efficacy in BRAFV600E melanoma2, 3; however, resistance to these agents remains a formidable challenge2, 4.,Global characterization of resistance mechanisms may inform the development of more effective therapeutic combinations.,Here, we performed systematic gain-of-function resistance studies by expressing >15,500 genes individually in a BRAFV600E melanoma cell line treated with RAF, MEK, ERK, or combined RAF/MEK inhibitors.,These studies revealed a cyclic AMP-dependent melanocytic signaling network not previously associated with drug resistance, including G-protein coupled receptors, adenyl cyclase, protein kinase A and cAMP response element binding protein (CREB).,Preliminary analysis of biopsies from BRAFV600E melanoma patients revealed that phosphorylated (active) CREB was suppressed by RAF/MEK-inhibition but restored in relapsing tumors.,Expression of transcription factors activated downstream of MAP kinase and cAMP pathways also conferred resistance, including c-FOS, NR4A1, NR4A2 and MITF.,Combined treatment with MAP kinase pathway and histone deacetylase inhibitors suppressed MITF expression and cAMP-mediated resistance.,Collectively, these data suggest that oncogenic dysregulation of a melanocyte lineage dependency can cause resistance to RAF/MEK/ERK inhibition, which may be overcome by combining signaling- and chromatin-directed therapeutics.

WNT-signaling controls important cellular processes throughout embryonic development and adult life, so any deregulation of this signaling can result in a wide range of pathologies, including cancer.,WNT-signaling is classified into two categories: β-catenin-dependent signaling (canonical pathway) and β-catenin-independent signaling (non-canonical pathway), the latter can be further divided into WNT/planar cell polarity (PCP) and calcium pathways.,WNT ligands are considered as unique directional growth factors that contribute to both cell proliferation and polarity.,Origin of cancer can be diverse and therefore tissue-specific differences can be found in WNT-signaling between cancers, including specific mutations contributing to cancer development.,This review focuses on the role of the WNT-signaling pathway in melanoma.,The current view on the role of WNT-signaling in cancer immunity as well as a short summary of WNT pathway-related drugs under investigation are also provided.

Hepatocyte growth factor (HGF)/ mesenchymal-epithelial transition factor (c-MET) signaling is involved in complex cellular programs that are important for embryonic development and tissue regeneration, but its activity is also utilized by cancer cells during tumor progression.,HGF and c-MET usually mediate heterotypic cell-cell interactions, such as epithelial-mesenchymal, including tumor-stroma interactions.,In the skin, dermal fibroblasts are the main source of HGF.,The presence of c-MET on keratinocytes is crucial for wound healing in the skin.,HGF is not released by normal melanocytes, but as melanocytes express c-MET, they are receptive to HGF, which protects them from apoptosis and stimulates their proliferation and motility.,Dissimilar to melanocytes, melanoma cells not only express c-MET, but also release HGF, thus activating c-MET in an autocrine manner.,Stimulation of the HGF/c-MET pathways contributes to several processes that are crucial for melanoma development, such as proliferation, survival, motility, and invasiveness, including distant metastatic niche formation.,HGF might be a factor in the innate and acquired resistance of melanoma to oncoprotein-targeted drugs.,It is not entirely clear whether elevated serum HGF level is associated with low progression-free survival and overall survival after treatment with targeted therapies.,This review focuses on the role of HGF/c-MET signaling in melanoma with some introductory information on its function in skin and melanocytes.

The dominant phenotype of greying with age in horses, caused by a 4.6-kb duplication in intron 6 of STX17, is associated with a high incidence of melanoma and vitiligo-like skin depigmentation.,However, the progressive greying and the incidence of melanoma, vitiligo-like depigmentation, and amount of speckling in these horses do not follow a simple inheritance pattern.,To understand their inheritance, we analysed the melanoma grade, grey level, vitiligo grade, and speckling grade of 1,119 Grey horses (7,146 measurements) measured in six countries over a 9-year period.,We estimated narrow sense heritability (h2), and we decomposed this parameter into polygenic heritability (h2 POLY), heritability due to the Grey (STX17) mutation (h2 STX17), and heritability due to agouti (ASIP) locus (h2 ASIP).,A high heritability was found for greying (h2 = 0.79), vitiligo (h2 = 0.63), and speckling (h2 = 0.66), while a moderate heritability was estimated for melanoma (h2 = 0.37).,The additive component of ASIP was significantly different from zero only for melanoma (h2 ASIP = 0.02).,STX17 controlled large proportions of phenotypic variance (h2 STX17 = 0.18-0.55) and overall heritability (h2 STX17/h2 = 0.28-0.83) for all traits.,Genetic correlations among traits were estimated as moderate to high, primarily due to the effects of the STX17 locus.,Nevertheless, the correlation between progressive greying and vitiligo-like depigmentation remained large even after taking into account the effects of STX17.,We presented a model where four traits with complex inheritance patterns are strongly influenced by a single mutation.,This is in line with evidence of recent studies in domestic animals indicating that some complex traits are, in addition to the large number of genes with small additive effects, influenced by genes of moderate-to-large effect.,Furthermore, we demonstrated that the STX17 mutation explains to a large extent the moderate to high genetic correlations among traits, providing an example of strong pleiotropic effects caused by a single gene.

Aberrations in gene expression are a hallmark of cancer cells.,Differential tumor-specific transcript levels of single genes or whole sets of genes may be critical for the neoplastic phenotype and important for therapeutic considerations or useful as biomarkers.,As an approach to filter out such relevant expression differences from the plethora of changes noted in global expression profiling studies, we searched for changes of gene expression levels that are conserved.,Transcriptomes from massive parallel sequencing of different types of melanoma from medaka were generated and compared to microarray datasets from zebrafish and human melanoma.,This revealed molecular conservation at various levels between fish models and human tumors providing a useful strategy for identifying expression signatures strongly associated with disease phenotypes and uncovering new melanoma molecules.

Malignant melanoma is often used as a model tumor for the establishment of novel therapies.,It is known that two-dimensional (2D) culture methods are not sufficient to elucidate the various processes during cancer development and progression.,Therefore, it is of major interest to establish defined biofabricated three-dimensional (3D) models, which help to decipher complex cellular interactions.,To get an impression of their printability and subsequent behavior, we printed fluorescently labeled melanoma cell lines with Matrigel and two different types of commercially available bioinks, without or with modification (RGD (Arginine-Glycine-Aspartate)-sequence/laminin-mixture) for increased cell-matrix communication.,In general, we demonstrated the printability of melanoma cells in all tested biomaterials and survival of the printed cells throughout 14 days of cultivation.,Melanoma cell lines revealed specific differential behavior in the respective inks.,Whereas in Matrigel, the cells were able to spread, proliferate and form dense networks throughout the construct, the cells showed no proliferation at all in alginate-based bioink.,In gelatin methacrylate-based bioink, the cells proliferated in clusters.,Surprisingly, the modifications of the bioinks with RGD or the laminin blend did not affect the analyzed cellular behavior.,Our results underline the importance of precisely adapting extracellular matrices to individual requirements of specific 3D bioprinting applications.

The novel nitrobenzoxadiazole (NBD) derivative MC3181 is endowed with remarkable therapeutic activity in mice bearing both sensitive and vemurafenib-resistant human melanoma xenografts.,Here, we report that subtoxic concentrations of this compound significantly reduced invasiveness of BRAF-V600D mutated WM115 and WM266.4 melanoma cell lines derived from the primary lesion and related skin metastasis of the same patient, respectively.,The strong antimetastatic activity of MC3181 was observed in both 2D monolayer cultures and 3D multicellular tumor spheroids, and confirmed in vivo by the significant decrease in the number of B16-F10 melanoma lung metastases in drug-treated mice.,Our data also show that MC3181 affects the lactate production in the high glycolytic WM266.4 cell line.,To unveil the MC3181 mechanism of action, we analyzed the ability of MC3181 to affect the degree of activation of different MAPK pathways, as well as the expression/activity levels of several proteins involved in angiogenesis, invasion, and survival (i.e.,AP2, MCAM/MUC18, N-cadherin, VEGF and MMP-2).,Our data disclosed both a decrease of the phospho-active form of JNK and an increased expression of the transcription factor AP2, events that occur in the very early phase of drug treatment and may be responsible of the antimetastatic effects of MC3181.

Targeted cancer therapies have shown some progress in treating BRAF‐mutant melanoma, but not against NRAS‐mutant and treatment‐resistant melanoma.,In this issue of EMBO Molecular Medicine, Echevarría‐Vargas et al (2018) report that cotargeting BET and MEK pathways efficiently kills immune therapy‐resistant and NRAS‐mutant melanoma tumor cells.

Although the number of new cases of Basal Cell Carcinoma (BCC) has increased rapidly in the last few decades, the molecular basis of its pathogenesis is not completely understood.,Activation of Hedgehog (Hh) signaling pathway has been shown to be a key factor driving the development of BCC.,The Wnt/β-catenin signaling pathway was also shown to be activated in BCCs and to perhaps modulate the activity of Hh pathway.,We have previously identified a novel mechanism by which Wnt signaling regulates the transcriptional outcome of Hh signaling pathway.,We demonstrated that CRD-BP, a direct target of the Wnt/β-catenin signaling, binds to GLI1 mRNA, stabilizes it, and consequently upregulates its levels (mRNA and protein) and activities.,We hypothesized that Wnt-induced and CRD-BP-dependent regulation of GLI1 expression and activities is important to the development of BCC.,In this study, we show that CRD-BP is over-expressed in BCC and that its expression positively correlates with the activation of both Wnt and Hh signaling pathways.,We also describe the generation and characterization of a human BCC cell line.,This cell line was utilized to demonstrate the importance of CRD-BP-dependent regulation of GLI1 expression and activities in the development of BCC.

Trials dedicated to metastatic uveal melanoma are needed because of the poor prognosis of this rare cancer and because its biology is distinct from that of cutaneous melanoma.Agents targeting the MEK/ERK/MAP kinase pathways are being tested.,Trials dedicated to metastatic uveal melanoma are needed because of the poor prognosis of this rare cancer and because its biology is distinct from that of cutaneous melanoma.,Agents targeting the MEK/ERK/MAP kinase pathways are being tested.,In experimental models, bevacizumab suppressed in vitro growth and in vivo hepatic metastasis of ocular melanoma cells.,Additional preclinical data suggested a potential benefit when combining bevacizumab with dacarbazine.,This noncomparative phase II study evaluated a combination of bevacizumab (10 mg/kg on days 8 and 22) with temozolomide (150 mg/m2 on days 1-7 and 15-21) in 36 patients with metastatic uveal melanoma (MUM).,The primary endpoint was the progression-free rate (PFR) at 6 months.,Using a modified 2-step Fleming plan, at least 10 of 35 patients were required to support a predefined PFR at 6 months of 40%.,Secondary objectives were progression-free survival (PFS), overall survival (OS), and safety; liver perfusion computed tomography (CT) for response imaging; and impact of VEGF-A gene polymorphisms on bevacizumab pharmacodynamics.,First- and second-step analyses revealed nonprogression at 6 months in 3 of 17 and 8 of 35 patients, respectively.,Finally, the 6-month PFR was 23% (95% confidence interval [CI]: 10-39), with long-lasting stable disease in 5 patients (14%).,Median PFS and OS were 12 weeks and 10 months, respectively.,No unexpected toxicity occurred.,Liver perfusion CT imaging was not useful in assessing tumor response, and VEGF-A gene polymorphisms were not correlated with toxicity or survival.,In patients with MUM, a combination of bevacizumab plus temozolomide achieved a 6-month PFR of 23%.

Uveal melanoma (UM) is the most common intraocular malignancy in adults.,Despite improvements in surgical, radiation and chemotherapy treatments, the overall survival of UM and prognosis remain poor.,In the present study, we hypothesized that Sirtuin 1 and 2 (SIRT1/2), class III histone deacetylases (HDACs), were critical in controlling the destiny of bulk tumor cells and cancer stem cells (CSCs) of UM.,We testified this hypothesis in four lines of UM cells (92.1, Mel 270, Omm 1 and Omm 2.3).,Our results showed that inhibition of SIRT1/2 by Tenovin-6 induced apoptosis in UM cells by activating the expression of tumor suppressor genes such as p53 and elevating reactive oxygen species (ROS).,Tenovin-6 inhibited the growth of UM cells.,Tenovin-6 and vinblastine was synergistic in inducing apoptosis of UM cell line 92.1 and Mel 270.,Furthermore, Tenovin-6 eliminated cancer stem cells in 92.1 and Mel 270 cells.,In conclusion, our findings suggest that Tenovin-6 may be a promising agent to kill UM bulk tumor cells and CSCs.

The mechanisms by which some melanoma cells adapt to BRAF inhibitor therapy are incompletely understood.,In the present study, we used mass spectrometry-based phosphoproteomics to determine how BRAF inhibition remodeled the signaling network of melanoma cell lines that were BRAF-mutant and PTEN-null.,Short-term BRAF inhibition was associated with marked changes in fibronectin-based adhesion signaling that were PTEN-dependent.,These effects were recapitulated through BRAF siRNA knockdown and following treatment with chemotherapeutic drugs.,Increased fibronectin expression was also observed in mouse xenograft models as well as specimens from melanoma patients undergoing BRAF inhibitor treatment.,Analysis of a melanoma TMA showed loss of PTEN expression to predict for a lower overall survival, with a trend for even lower survival being seen when loss of fibronectin was included in the analysis.,Mechanistically, the induction of fibronectin limited the responses of these PTEN-null melanoma cell lines to vemurafenib, with enhanced cytotoxicity observed following the knockdown of either fibronectin or its receptor α5β1 integrin.,This in turn abrogated the cytotoxic response to BRAF inhibition via increased AKT signaling, which prevented the induction of cell death by maintaining the expression of the pro-survival protein Mcl-1.,The protection conveyed by the induction of fibronectin expression could be overcome through combined treatment with a BRAF and PI3K inhibitor.

Despite remarkable efforts, metastatic melanoma (MM) still presents with significant mortality.,Recently, mono-chemotherapies are increasingly replenished by more cancer-specific combination therapies involving death ligands and drugs interfering with cell signaling.,Still, MM remains a fatal disease because tumors rapidly develop resistance to novel therapies thereby regaining tumorigenic capacity.,Although genetically engineered mouse models for MM have been developed, at present no model is available that reliably mimics the human disease and is suitable for studying mechanisms of therapeutic obstacles including cell death resistance.,To improve the increasing requests on new therapeutic alternatives, reliable human screening models are demanded that translate the findings from basic cellular research into clinical applications.,By developing an organotypic full skin equivalent, harboring melanoma tumor spheroids of defined sizes we have invented a cell-based model that recapitulates both the 3D organization and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.,By extending our previous findings on melanoma cell sensitization toward TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) by co-application of sublethal doses of ultraviolet-B radiation (UVB) or cisplatin, we show significant differences in the therapeutical outcome to exist between regular two-dimensional (2D) and complex in vivo-like 3D models.,Of note, while both treatment combinations killed the same cancer cell lines in 2D culture, skin equivalent-embedded melanoma spheroids are potently killed by TRAIL+cisplatin treatment but remain almost unaffected by the TRAIL+UVB combination.,Consequently, we have established an organotypic human skin-melanoma model that will facilitate efforts to improve therapeutic outcomes for malignant melanoma by providing a platform for the investigation of cytotoxic treatments and tailored therapies in a more physiological setting.

Despite the remarkable benefits associated with the interventional treatment of melanomas (and other solid cancers) with immune checkpoint and Braf inhibitors (Brafi), most patients ultimately progress on therapy.,The presence of multifocal/disseminated disease in patients increases their mortality risk.,Hence, the development of novel strategies to effectively treat patients with melanomas that are resistant to anti-PD1 mAb (αPD1) and/or Brafi, particularly those with multifocal/disseminated disease remains a major unmet clinical need.,Mice developing induced/spontaneous BrafV600E/Pten−/− melanomas were treated by cutaneous immunization with a DNA vaccine encoding the melanoma-associated antigen TRP2, with Brafi or αPD1 alone, or with a combination of these treatments.,Tumor progression, tumor-infiltration by CD4+ and CD8+ T cells, and the development of TRP2-specific CD8+ T cells were then monitored over time.,Vaccination led to durable antitumor immunity against PD1/Brafi-resistant melanomas in both single lesion and multifocal disease models, and it sensitized PD1-resistant melanomas to salvage therapy with αPD1.,The therapeutic efficacy of the vaccine was associated with host skin-resident cells, the induction of a systemic, broadly reactive IFNγ+CD8+ T cell repertoire, increased frequencies of CD8+ TIL and reduced levels of PD1hi/intCD8+ T cells.,Extended survival was associated with improved TIL functionality, exemplified by the presence of enhanced levels of IFNγ+CD8+ TIL and IL2+CD4+ TIL.,These data support the use of a novel genetic vaccine for the effective treatment of localized or multifocal melanoma refractory to conventional αPD1-based and/or Brafi-based (immune)therapy.

The development of brain metastases in patients with advanced stage melanoma is common, but the molecular mechanisms responsible for their development are poorly understood.,Melanoma brain metastases cause significant morbidity and mortality and confer a poor prognosis; traditional therapies including whole brain radiation, stereotactic radiotherapy, or chemotherapy yield only modest increases in overall survival (OS) for these patients.,While recently approved therapies have significantly improved OS in melanoma patients, only a small number of studies have investigated their efficacy in patients with brain metastases.,Preliminary data suggest that some responses have been observed in intracranial lesions, which has sparked new clinical trials designed to evaluate the efficacy in melanoma patients with brain metastases.,Simultaneously, recent advances in our understanding of the mechanisms of melanoma cell dissemination to the brain have revealed novel and potentially therapeutic targets.,In this review, we provide an overview of newly discovered mechanisms of melanoma spread to the brain, discuss preclinical models that are being used to further our understanding of this deadly disease and provide an update of the current clinical trials for melanoma patients with brain metastases.

Metastatic melanoma (MM) is a highly aggressive cancer with a median overall survival of 6-9 months, notwithstanding the numerous efforts in development of new therapeutic approaches.,To this aim we tested the clinical applicability of the Ion Torrent Personal Genome Machine to simultaneously screen MM patients in order to individuate new or already known SNPs and mutations able to predict the duration of response to BRAF inhibitors.,An Ampliseq Custom Panel, including 11 crucial full length genes involved in melanoma carcinogenesis and therapy response pathways, was created and used to analyze 25 MM patients.,We reported BRAFV600 and NRASQ61 mutations in 68% and 24% of samples, respectively.,Moreover, we more frequently identified the following alterations related to BRAF status: PIK3CAI391M (44%) and KITD737N (36%) mutations, CTLA4T17A (52%), MC1RV60L (32%) and MITFS473A (60%) polymorphisms.,Considering the progression free survival (PFS), statistical analyses showed that BRAFV600 patients without any of these more frequent alterations had a higher median PFS.,Protein structure changes seem to be due to these variants by in silico analysis.,In conclusion, a Next-Generation Sequencing approach with custom panel may provide new information to evaluate tumor-specific therapeutic susceptibility and individual prognosis to improve the care of MM patients.

The detection of V600E BRAF mutations has fundamental clinical consequences as the treatment option with BRAF inhibitors such as vemurafenib or dabrafenib yields response rates of ∼48%.,Heterogeneity with respect to BRAF mutation in different metastases has been described in single cases.,As this has important implications for the determination of BRAF status and treatment of patients, it is essential to acquire more data.,A total of 300 tumour samples from 187 melanoma patients were analysed for BRAF mutations by pyrosequencing.,Equivocal results were confirmed by capillary sequencing.,Clinical data with respect to melanoma type, tumour site and survival were summarised for 53 patients with multiple analysed tumour samples (2-13 per patient).,BRAF mutations were found in 84 patients (44.9%) and 144 tumour samples (48%) with BRAF mutations in 45.5% of primary tumours and 51.3% of metastases, respectively.,In 10 out of 53 patients (18.9%) where multiple samples were analysed results were discordant with respect to mutation findings with wild-type and mutated tumours in the same patient.,Mutations did not appear more frequently over the course of disease nor was its occurrence associated with a specific localisation of metastases.,As heterogeneity with respect to BRAF mutation status is detected in melanoma patients, subsequent testing of initially wild-type patients can yield different results and thus make BRAF inhibitor therapy accessible.,The role of heterogeneity in testing and for clinical response to therapy with a BRAF inhibitor needs to be further investigated.

Standard-dose pembrolizumab plus alternative-dose ipilimumab (1 mg/kg Q3W for 4 doses) were tolerable and had robust antitumor activity in advanced melanoma in cohort B of the phase 1 KEYNOTE-029 study.,Cohort C evaluated standard-dose pembrolizumab with two other alternative ipilimumab regimens.,Patients with treatment-naive unresectable stage III/IV melanoma were randomly assigned 1:1 to pembrolizumab 200 mg Q3W for ≤24 months plus ipilimumab 50 mg Q6W for 4 doses (PEM200+IPI50), or the same pembrolizumab regimen plus ipilimumab 100 mg Q12W for 4 doses (PEM200+IPI100).,Primary end points were incidence of grade 3-5 treatment-related adverse events (TRAE) and objective response rate (ORR) per RECIST v1.1 by independent central review.,Per protocol-defined thresholds, grade 3-5 TRAE incidence ≤26% indicated meaningful toxicity reduction and ORR ≥48% indicated no decrease in efficacy versus data reported for other PD-1 inhibitor/ipilimumab combinations.,Median follow-up on February 18, 2019, was 16.3 months in PEM200+IPI50 (N = 51) and 16.4 months in PEM200+IPI100 (N = 51).,Grade 3-5 TRAEs occurred in 12 (24%) patients in PEM200+IPI50 and 20 (39%) in PEM200+IPI100.,One patient in PEM200+IPI50 died from treatment-related autoimmune myocarditis.,Immune-mediated AEs or infusion reactions occurred in 21 (42%) patients in PEM200+IPI50 and 28 (55%) in PEM200+IPI100.,ORR was 55% in PEM200+IPI50; 61% in PEM200+IPI100.,Pembrolizumab 200 mg Q3W plus ipilimumab 50 mg Q6W or 100 mg Q12W demonstrated antitumor activity above the predefined threshold; pembrolizumab plus ipilimumab 50 mg Q6W had lower incidence of grade 3-5 TRAEs than the predefined threshold, suggesting a reduction in toxicity.,See related commentary by Jameson-Lee and Luke, p.,5153

Wnt5a has been implicated in melanoma progression and metastasis, although the exact downstream signaling events that contribute to melanoma metastasis are poorly understood.,Wnt5a signaling results in acyl protein thioesterase 1 (APT1) mediated depalmitoylation of pro-metastatic cell adhesion molecules CD44 and MCAM, resulting in increased melanoma invasion.,The mechanistic details that underlie Wnt5a-mediated regulation of APT1 activity and cellular function remain unknown.,Here, we show Wnt5a signaling regulates APT1 activity through induction of APT1 phosphorylation and we further investigate the functional role of APT1 phosphorylation on its depalmitoylating activity.,We found phosphorylation increased APT1 depalmitoylating activity and reduced APT1 dimerization.,We further determined APT1 phosphorylation increases melanoma invasion in vitro, and also correlated with increased tumor grade and metastasis.,Our results further establish APT1 as an important regulator of melanoma invasion and metastatic behavior.,Inhibition of APT1 may represent a novel way to treat Wnt5a driven cancers.

Detachment of cells from the primary tumour precedes metastatic progression by facilitating cell release into the tissue.,Solid tumours exhibit altered pH homeostasis with extracellular acidification.,In human melanoma, the Na+/H+ exchanger NHE1 is an important modifier of the tumour nanoenvironment.,Here we tested the modulation of cell-cell-adhesion by extracellular pH and NHE1.,MV3 tumour spheroids embedded in a collagen matrix unravelled the efficacy of cell-cell contact loosening and 3D emigration into an environment mimicking physiological confinement.,Adhesive interaction strength between individual MV3 cells was quantified using atomic force microscopy and validated by multicellular aggregation assays.,Extracellular acidification from pHe7.4 to 6.4 decreases cell migration and invasion but increases single cell detachment from the spheroids.,Acidification and NHE1 overexpression both reduce cell-cell adhesion strength, indicated by reduced maximum pulling forces and adhesion energies.,Multicellular aggregation and spheroid formation are strongly impaired under acidification or NHE1 overexpression.,We show a clear dependence of melanoma cell-cell adhesion on pHe and NHE1 as a modulator.,These effects are opposite to cell-matrix interactions that are strengthened by protons extruded via NHE1.,We conclude that these opposite effects of NHE1 act synergistically during the metastatic cascade.

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.

Resistance to immune checkpoint blockade and targeted therapy in melanoma patients is currently one of the major clinical challenges.,With the approval of talimogene laherparepvec (T-VEC), oncolytic viruses are now in clinical practice for locally advanced or non-resectable melanoma.,Here, we describe the usage of T-VEC in stage IVM1b-M1c melanoma patients, who achieved complete remission or stable disease upon systemic treatment but suffered from a loco-regional recurrence.,To our knowledge, there are no case reports so far describing T-VEC as a means to overcome acquired resistance to immune checkpoint blockade or targeted therapy.,All melanoma patients in our department treated with T-VEC in the period of 2016-2018 were evaluated retrospectively.,Data on clinicopathological characteristics, treatment response, and toxicity were analyzed.,Fourteen melanoma patients were treated with T-VEC in our center.,Six patients (43%) received T-VEC first-line.,In eight patients (57%), T-VEC followed a prior systemic therapy.,Three patients with M1b stage and one patient with M1c stage melanoma were treated with T-VEC.,These patients suffered from loco-regional progress, whilst distant metastases had regressed during prior systemic treatment. 64% of patients showed a benefit from therapy with T-VEC.,The durable response rate was 36%.,T-VEC represents an effective and tolerable treatment option.,This is true not only for loco-regionally advanced melanoma patients, but also for patients with stable or regressive systemic metastases who develop loco-regionally acquired resistance upon treatment with immune checkpoint blockade or targeted therapy.,A sensible selection of suitable patients seems to be crucial.

Cutaneous malignant melanoma is a highly aggressive and frequently chemoresistant cancer, whose incidence continues to rise.,Epidemiological studies reveal that the major etiological melanoma risk factor is ultraviolet (UV) solar radiation, with the highest risk associated with intermittent burning doses, especially during childhood1,2.,We have experimentally validated these epidemiological findings using the hepatocyte growth factor/scatter factor (HGF/SF) transgenic mouse model, which develops lesions in stages highly reminiscent of human melanoma with respect to biological, genetic and etiologic criteria, but only when irradiated as neonatal pups with UVB, not UVA3,4.,However, mechanisms underlying UVB-initiated, neonatal-specific melanomagenesis remain largely unknown.,Here we introduce a mouse model permitting fluorescence-aided melanocyte imaging and isolation following in vivo UV irradiation.,We use expression profiling to show that activated neonatal skin melanocytes isolated following a melanomagenic UVB dose bear a distinct, persistent interferon response signature, including genes associated with immunoevasion.,UVB-induced melanocyte activation, characterized by aberrant growth and migration, was abolished by antibody-mediated systemic blockade of interferon-γ (IFN-γ), but not type-I interferons.,IFN-γ was produced by macrophages recruited to neonatal skin by UVB-induced ligands to the chemokine receptor Ccr2.,Admixed recruited skin macrophages enhanced transplanted melanoma growth by inhibiting apoptosis; notably, IFN-γ blockade abolished macrophage-enhanced melanoma growth and survival.,IFN-γ-producing macrophages were also identified in 70% of human melanomas examined.,Our data reveal an unanticipated role for IFN-γ in promoting melanocytic cell survival/immunoevasion, and suggest that IFN-γ-R signaling represents a novel therapeutic melanoma target.

Recent advances in cancer treatment have emerged from new immunotherapies targeting T-cell inhibitory receptors, including cytotoxic T-lymphocyte associated antigen (CTLA)-4 and programmed cell death (PD)-1.,In this context, anti-CTLA-4 and anti-PD-1 monoclonal antibodies have demonstrated survival benefits in numerous cancers, including melanoma and non-small-cell lung carcinoma.,PD-1-expressing CD8+ T lymphocytes appear to play a major role in the response to these immune checkpoint inhibitors (ICI).,Cytotoxic T lymphocytes (CTL) eliminate malignant cells through recognition by the T-cell receptor (TCR) of specific antigenic peptides presented on the surface of cancer cells by major histocompatibility complex class I/beta-2-microglobulin complexes, and through killing of target cells, mainly by releasing the content of secretory lysosomes containing perforin and granzyme B.,T-cell adhesion molecules and, in particular, lymphocyte-function-associated antigen-1 and CD103 integrins, and their cognate ligands, respectively, intercellular adhesion molecule 1 and E-cadherin, on target cells, are involved in strengthening the interaction between CTL and tumor cells.,Tumor-specific CTL have been isolated from tumor-infiltrating lymphocytes and peripheral blood lymphocytes (PBL) of patients with varied cancers.,TCRβ-chain gene usage indicated that CTL identified in vitro selectively expanded in vivo at the tumor site compared to autologous PBL.,Moreover, functional studies indicated that these CTL mediate human leukocyte antigen class I-restricted cytotoxic activity toward autologous tumor cells.,Several of them recognize truly tumor-specific antigens encoded by mutated genes, also known as neoantigens, which likely play a key role in antitumor CD8 T-cell immunity.,Accordingly, it has been shown that the presence of T lymphocytes directed toward tumor neoantigens is associated with patient response to immunotherapies, including ICI, adoptive cell transfer, and dendritic cell-based vaccines.,These tumor-specific mutation-derived antigens open up new perspectives for development of effective second-generation therapeutic cancer vaccines.

This international, multicenter, single-arm trial assessed efficacy and safety of intralesional rose bengal (PV-10) in 80 patients with refractory cutaneous or subcutaneous metastatic melanoma.,Sixty-two stage III and 18 stage IV melanoma patients with disease refractory to a median of six prior interventions received intralesional PV-10 into up to 20 cutaneous and subcutaneous lesions up to four times over a 16-week period and were followed for 52 weeks.,Objectives were to determine best overall response rate in injected target lesions and uninjected bystander lesions, assess durability of response, and characterize adverse events.,For target lesions, the best overall response rate was 51 %, and the complete response rate was 26 %.,Median time to response was 1.9 months, and median duration of response was 4.0 months, with 8 % of patients having no evidence of disease after 52 weeks.,Response was dependent on untreated disease burden, with complete response achieved in 50 % of patients receiving PV-10 to all of their disease.,Response of target lesions correlated with bystander lesion regression and the occurrence of locoregional blistering.,Adverse events were predominantly mild to moderate and locoregional to the treatment site, with no treatment-associated grade 4 or 5 adverse events.,Intralesional PV-10 yielded durable local control with high rates of complete response.,Toxicity was confined predominantly to the injection site.,Cutaneous bystander tumor regression is consistent with an immunologic response secondary to ablation.,This intralesional approach for local disease control could be complementary to current and investigational treatments for melanoma.

BRAFV600E-mutant malignant melanomas depend on RAF/MEK/ERK (MAPK) signaling for tumor cell growth1.,RAF and MEK inhibitors show remarkable clinical efficacy in BRAFV600E melanoma2, 3; however, resistance to these agents remains a formidable challenge2, 4.,Global characterization of resistance mechanisms may inform the development of more effective therapeutic combinations.,Here, we performed systematic gain-of-function resistance studies by expressing >15,500 genes individually in a BRAFV600E melanoma cell line treated with RAF, MEK, ERK, or combined RAF/MEK inhibitors.,These studies revealed a cyclic AMP-dependent melanocytic signaling network not previously associated with drug resistance, including G-protein coupled receptors, adenyl cyclase, protein kinase A and cAMP response element binding protein (CREB).,Preliminary analysis of biopsies from BRAFV600E melanoma patients revealed that phosphorylated (active) CREB was suppressed by RAF/MEK-inhibition but restored in relapsing tumors.,Expression of transcription factors activated downstream of MAP kinase and cAMP pathways also conferred resistance, including c-FOS, NR4A1, NR4A2 and MITF.,Combined treatment with MAP kinase pathway and histone deacetylase inhibitors suppressed MITF expression and cAMP-mediated resistance.,Collectively, these data suggest that oncogenic dysregulation of a melanocyte lineage dependency can cause resistance to RAF/MEK/ERK inhibition, which may be overcome by combining signaling- and chromatin-directed therapeutics.

A molecular linkage between the MAPK and the LKB1-AMPK energy sensor pathways suggests that combined MAPK oncogene inhibition and metabolic modulation of AMPK would be more effective than either manipulation alone in melanoma cell lines.,The combination of the BRAF inhibitor vemurafenib (formerly PLX4032) and metformin were tested against a panel of human melanoma cell lines with defined BRAF and NRAS mutations for effects on viability, cell cycle and apoptosis.,Signaling molecules in the MAPK, PI3K-AKT and LKB1-AMPK pathways were studied by Western blot.,Single agent metformin inhibited proliferation in 12 out of 19 cell lines irrespective of the BRAF mutation status, but in one NRASQ61K mutant cell line it powerfully stimulated cell growth.,Synergistic anti-proliferative effects of the combination of metformin with vemurafenib were observed in 6 out of 11 BRAFV600E mutants, including highly synergistic effects in two BRAFV600E mutant melanoma cell lines.,Antagonistic effects were noted in some cell lines, in particular in BRAFV600E mutant cell lines resistant to single agent vemurafenib.,Seven out of 8 BRAF wild type cell lines showed marginally synergistic anti-proliferative effects with the combination, and one cell line had highly antagonistic effects with the combination.,The differential effects were not dependent on the sensitivity to each drug alone, effects on cell cycle or signaling pathways.,The combination of vemurafenib and metformin tended to have stronger anti-proliferative effects on BRAFV600E mutant cell lines.,However, determinants of vemurafenib and metformin synergism or antagonism need to be understood with greater detail before any potential clinical utility of this combination.

Malignant cutaneous epithelial tumors comprise various skin malignancies originating from the cutaneous epithelium, including cutaneous squamous cell carcinoma, basal cell carcinoma, and malignant cutaneous adnexal tumors.,Treatment options are limited, as the rarity of these tumors, especially among Asians, renders well-controlled clinical trials extremely challenging to conduct.,Thus, we designed a clinical trial to evaluate the efficacy and safety of the anti-programmed cell death-1 (PD-1) monoclonal antibody nivolumab in patients with metastatic cutaneous squamous cell carcinomas and other rare metastatic cutaneous epithelial tumors.,This is an open-label, single-arm, multicenter, phase 2 clinical trial involving patients with metastatic malignant cutaneous epithelial tumors.,Nivolumab (480 mg) will be administered intravenously every 4 weeks for a maximum of 26 doses.,The primary outcome of the study will be the response rate based on response evaluation criteria in solid tumors, version 1.1.,Assuming a null hypothesis of a response rate ≤5% and an alternative hypothesis of a 25% response rate, a minimum of 26 patients are required to achieve a 5% two-sided type I error and 80% power based on the exact binomial distribution.,Finally, a target cohort size of 30 patients was determined as some patient dropout will be expected.,This is the first phase 2 clinical trial evaluating the efficacy and safety of the PD-1 inhibitor nivolumab in Asian patients with metastatic malignant cutaneous epithelial tumors.,The findings of the study will contribute to the development of novel treatment approaches for patients with rare cutaneous malignancies, which remains an unmet clinical need.,Registry number: jRCT 2031190048

Cutaneous basal cell carcinoma (BCC) is the commonest cancer worldwide.,BCC is locally invasive and the surrounding stromal microenvironment is pivotal for tumourigenesis.,Cancer associated fibroblasts (CAFs) in the microenvironment are essential for tumour growth in a variety of neoplasms but their role in BCC is poorly understood.,Material included facial BCC and control skin from the peritumoural area and from the buttocks.,With next-generation sequencing (NGS) we compared mRNA expression between BCC and peritumoural skin. qRT-PCR, immunohistochemical and immunofluorescent staining were performed to validate the NGS results and to investigate CAF-related cyto-and chemokines.,NGS revealed upregulation of 65 genes in BCC coding for extracellular matrix components pointing at CAF-related matrix remodeling. qRT-PCR showed increased mRNA expression of CAF markers FAP-α, PDGFR-β and prolyl-4-hydroxylase in BCC.,Peritumoural skin (but not buttock skin) also exhibited high expression of PDGFR-β and prolyl-4-hydroxylase but not FAP-α.,We found a similar pattern for the CAF-associated chemokines CCL17, CCL18, CCL22, CCL25, CXCL12 and IL6 with high expression in BCC and peritumoural skin but absence in buttock skin.,Immunofluorescence revealed correlation between FAP-α and PDGFR-β and CXCL12 and CCL17.,Matrix remodeling is the most prominent molecular feature of BCC.,CAFs are present within BCC stroma and associated with increased expression of chemokines involved in tumour progression and immunosuppression (CXCL12, CCL17).,Fibroblasts from chronically sun-exposed skin near tumours show gene expression patterns resembling that of CAFs, indicating that stromal fibroblasts in cancer-free surgical BCC margins exhibit a tumour promoting phenotype.,The online version of this article (doi: 10.1186/s12885-017-3663-0) contains supplementary material, which is available to authorized users.

While invasion and metastasis of tumour cells are the principle factor responsible for cancer related deaths, the mechanisms governing the process remain poorly defined.,Moreover, phenotypic divergence of sub-populations of tumour cells is known to underpin alternative behaviors linked to tumour progression such as proliferation, survival and invasion.,In the context of melanoma, heterogeneity between two transcription factors, BRN2 and MITF, has been associated with phenotypic switching between predominantly invasive and proliferative behaviors respectively.,Epigenetic changes, in response to external cues, have been proposed to underpin this process, however the mechanism by which the phenotypic switch occurs is unclear.,Here we report the identification of the NFIB transcription factor as a novel downstream effector of BRN2 function in melanoma cells linked to the migratory and invasive characteristics of these cells.,Furthermore, the function of NFIB appears to drive an invasive phenotype through an epigenetic mechanism achieved via the upregulation of the polycomb group protein EZH2.,A notable target of NFIB mediated up-regulation of EZH2 is decreased MITF expression, which further promotes a less proliferative, more invasive phenotype.,Together our data reveal that NFIB has the ability to promote dynamic changes in the chromatin state of melanoma cells to facilitate migration, invasion and metastasis.,•NFIB mediates a slow cycling, highly invasive/migratory melanoma cell phenotype downstream of BRN2.,•NFIB increases EZH2 expression downstream of BRN2, which further decreases MITF levels.,•NFIB expression is defined by an invasive gene signature and colocalises with BRN2 in primary and metastatic human melanoma tumours.,NFIB mediates a slow cycling, highly invasive/migratory melanoma cell phenotype downstream of BRN2.,NFIB increases EZH2 expression downstream of BRN2, which further decreases MITF levels.,NFIB expression is defined by an invasive gene signature and colocalises with BRN2 in primary and metastatic human melanoma tumours.,Melanoma is a heterogeneous cancer, made up of many cellular populations that differ in their ability to induce tumour growth or invasion throughout the body (metastasis).,These populations have been found to switch back and forth to drive invasion and progression.,This process appears to be controlled by an inverse axis between two genes, MITF and BRN2.,BRN2 drives metastatic spread, but the process by which it acts is not well characterized and cannot be targeted clinically.,This study has uncovered a role for the gene NFIB in driving invasion downstream of BRN2.,Importantly, it appears to drive this process through EZH2, which can be targeted therapeutically to reduce metastasis.

In cancer, Wnt/β-catenin signaling is ubiquitously referred to as an “oncogenic” pathway that promotes tumor progression.,This review examines how the regulation and downstream effects of Wnt/β-catenin signaling in cancer varies depending on cellular context, with a focus on malignant melanoma.,We emphasize that the cellular homeostasis of Wnt/β-catenin signaling may represent a more appropriate concept than the simplified view of the Wnt/β-catenin pathway as either oncogenic or tumor-suppressing.,Ultimately, a more refined understanding of the contextual regulation of Wnt/β-catenin signaling will be essential for addressing if and how therapeutic targeting of this pathway could be leveraged for patient benefit.

Immunodeficient mice injected with human cancer cell lines have been used for human oncology studies and anti‐cancer drug trials for several decades.,However, rodents are not ideal species for modelling human cancer because rodents are physiologically dissimilar to humans.,Therefore, anti‐tumour drugs tested effective in rodents have a failure rate of 90% or higher in phase III clinical trials.,Pigs are similar to humans in size, anatomy, physiology and drug metabolism rate, rendering them a desirable pre‐clinical animal model for assessing anti‐cancer drugs.,However, xenogeneic immune rejection is a major barrier to the use of pigs as hosts for human tumours.,Interleukin (IL)‐2 receptor γ (IL2RG), a common signalling subunit for multiple immune cytokines including IL‐2, IL‐4, IL‐7, IL‐9, IL‐15 and IL‐21, is required for proper lymphoid development.,IL2RG−/Y pigs were generated by CRISPR/Cas9 technology, and examined for immunodeficiency and ability to support human oncogenesis.,Compared to age‐matched wild‐type pigs, IL2RG −/Y pigs exhibited a severely impaired immune system as shown by lymphopenia, lymphoid organ atrophy, poor immunoglobulin function, and T‐ and NK‐cell deficiency.,Human melanoma Mel888 cells generated tumours in IL2RG −/Y pigs but not in wild‐type littermates.,The human tumours grew faster in IL2RG −/Y pigs than in nude mice.,Our results indicate that these pigs are promising hosts for modelling human cancer in vivo, which may aid in the discovery and development of anti‐cancer drugs.

Fourier transform infrared (FTIR) microspectroscopy was used to evaluate the growth of human melanoma cells (SK-MEL-2) in two-dimensional (2D) versus three-dimensional (3D) spheroid culture systems.,FTIR microspectroscopy, coupled with multivariate analysis, could be used to monitor the variability of spheroid morphologies prepared from different cell densities.,The characteristic shift in absorbance bands of the 2D cells were different from the spectra of cells from 3D spheroids.,FTIR microspectroscopy can also be used to monitor cell death similar to fluorescence cell staining in 3D spheroids.,A change in the secondary structure of protein was observed in cells from the 3D spheroid versus the 2D culture system.,FTIR microspectroscopy can detect specific alterations in the biological components inside the spheroid, which cannot be detected using fluorescence cell death staining.,In the cells from 3D spheroids, the respective lipid, DNA, and RNA region content represent specific markers directly proportional to the spheroid size and central area of necrotic cell death, which can be confirmed using unsupervised PCA and hierarchical cluster analysis.,FTIR microspectroscopy could be used as an alternative tool for spheroid cell culture discrimination, and validation of the usual biochemical technique.

Herbert et al. show that BRN2 is associated with DNA damage response proteins and suppresses an apoptosis-associated gene expression program to protect against UVB-, chemotherapy-, and vemurafenib-induced apoptosis.,Whether cell types exposed to a high level of environmental insults possess cell type-specific prosurvival mechanisms or enhanced DNA damage repair capacity is not well understood.,BRN2 is a tissue-restricted POU domain transcription factor implicated in neural development and several cancers.,In melanoma, BRN2 plays a key role in promoting invasion and regulating proliferation.,Here we found, surprisingly, that rather than interacting with transcription cofactors, BRN2 is instead associated with DNA damage response proteins and directly binds PARP1 and Ku70/Ku80.,Rapid PARP1-dependent BRN2 association with sites of DNA damage facilitates recruitment of Ku80 and reprograms DNA damage repair by promoting Ku-dependent nonhomologous end-joining (NHEJ) at the expense of homologous recombination.,BRN2 also suppresses an apoptosis-associated gene expression program to protect against UVB-, chemotherapy- and vemurafenib-induced apoptosis.,Remarkably, BRN2 expression also correlates with a high single-nucleotide variation prevalence in human melanomas.,By promoting error-prone DNA damage repair via NHEJ and suppressing apoptosis of damaged cells, our results suggest that BRN2 contributes to the generation of melanomas with a high mutation burden.,Our findings highlight a novel role for a key transcription factor in reprogramming DNA damage repair and suggest that BRN2 may impact the response to DNA-damaging agents in BRN2-expressing cancers.

Malignant melanoma is a neoplasm of melanocytes, and the microphthalmia‐associated transcription factor (MITF) is essential for the existence of melanocytes.,MITF's relevance for this cell lineage is maintained in melanoma, where it is an important regulator of survival and balances melanoma cell proliferation with terminal differentiation (pigmentation).,The MITF gene is amplified in ~20% of melanomas and MITF mutation can predispose to melanoma development.,Furthermore, the regulation of MITF expression and function is strongly linked to the BRAF/MEK/ERK/MAP‐kinase (MAPK) pathway, which is deregulated in >90% of melanomas and central target of current therapies.,MITF expression in melanoma is heterogeneous, and recent findings highlight the relevance of this heterogeneity for the response of melanoma to MAPK pathway targeting drugs, as well as for MITF's role in melanoma progression.,This review aims to provide an updated overview on the regulation of MITF function and plasticity in melanoma with a focus on its link to MAPK signaling.

Inflammation promotes phenotypic plasticity in melanoma, a source of non-genetic heterogeneity, but the molecular framework is poorly understood.,Here we use functional genomic approaches and identify a reciprocal antagonism between the melanocyte lineage transcription factor MITF and c-Jun, which interconnects inflammation-induced dedifferentiation with pro-inflammatory cytokine responsiveness of melanoma cells favouring myeloid cell recruitment.,We show that pro-inflammatory cytokines such as TNF-α instigate gradual suppression of MITF expression through c-Jun.,MITF itself binds to the c-Jun regulatory genomic region and its reduction increases c-Jun expression that in turn amplifies TNF-stimulated cytokine expression with further MITF suppression.,This feed-forward mechanism turns poor peak-like transcriptional responses to TNF-α into progressive and persistent cytokine and chemokine induction.,Consistently, inflammatory MITFlow/c-Junhigh syngeneic mouse melanomas recruit myeloid immune cells into the tumour microenvironment as recapitulated by their human counterparts.,Our study suggests myeloid cell-directed therapies may be useful for MITFlow/c-Junhigh melanomas to counteract their growth-promoting and immunosuppressive functions.,The c-Jun transcription factor can mediate a cell's response to TNFa.,Here, Riesenberg et al. show in melanoma cells that c-Jun has an inverse relationship with the key melanocyte transcription factor MITF and that high c-Jun levels contribute to melanoma heterogeneity and an inflammatory microenvironment.

Transcriptional reprogramming of proliferative melanoma cells into a phenotypically distinct invasive cell subpopulation is a critical event at the origin of metastatic spreading.,Here we generate transcriptome, open chromatin and histone modification maps of melanoma cultures; and integrate this data with existing transcriptome and DNA methylation profiles from tumour biopsies to gain insight into the mechanisms underlying this key reprogramming event.,This shows thousands of genomic regulatory regions underlying the proliferative and invasive states, identifying SOX10/MITF and AP-1/TEAD as regulators, respectively.,Knockdown of TEADs shows a previously unrecognized role in the invasive gene network and establishes a causative link between these transcription factors, cell invasion and sensitivity to MAPK inhibitors.,Using regulatory landscapes and in silico analysis, we show that transcriptional reprogramming underlies the distinct cellular states present in melanoma.,Furthermore, it reveals an essential role for the TEADs, linking it to clinically relevant mechanisms such as invasion and resistance.,The key regulators that allow transition from proliferative to invasive phenotype in melanoma cells have not been identified yet.,The authors perform chromatin and transcriptome profiling followed by comprehensive bioinformatics analysis identifying new candidate regulators for two distinct cell states of melanoma.

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.

Despite the clinical success of RAF inhibitors in BRAF-mutated melanomas, attempts to target RAF kinases in the context of RAS-driven or otherwise RAF wild-type tumours have not only been ineffective, but RAF inhibitors appear to aggravate tumorigenesis in these settings.,Subsequent preclinical investigation has revealed several regulatory mechanisms, feedback pathways and unexpected enzymatic quirks in the MAPK pathway, which may explain this paradox.,In this review, we cover the various proposed molecular mechanisms for the RAF paradox, the clinical consequences and strategies to overcome it.

Major limitations of current melanoma treatments are for instances of relapse and the lack of therapeutic options for BRAF wild-type patients who do not respond to immunotherapy.,Many studies therefore focus on killing resistant subpopulations, such as Melanoma Initiating Cells (MICs) to prevent relapse.,Here we examined whether combining a GSI (γ-Secretase Inhibitor) with ABT-737 (a small molecule BCL-2/BCL-XL/BCL-W inhibitor) can kill both the non-MICs (bulk of melanoma) and MICs.,To address the limitations of melanoma therapies, we included multiple tumor samples of patients relapsed from current treatments, with a diverse genetic background (with or without the common BRAF, NRAS or NF1 mutations) in these studies.,Excitingly, the combination treatment reduced cell viability and induced apoptosis of the non-MICs; disrupted primary spheres, decreased the ALDH+ cells, and inhibited the self-renewability of the MICs in multiple melanoma cell lines and relapsed patient samples.,Using a low-cell-number mouse xenograft model, we demonstrated that the combination significantly reduced the tumor initiating ability of MIC-enriched cultures from relapsed patient samples.,Mechanistic studies also indicate that cell death is NOXA-dependent.,In summary, this combination may be a promising strategy to address treatment relapse and for triple wild-type patients who do not respond to immunotherapy.

While response rates to BRAF inhibitiors (BRAFi) are high, disease progression emerges quickly.,One strategy to delay the onset of resistance is to target anti-apoptotic proteins such as BCL-2, known to be associated with a poor prognosis.,We analyzed BCL-2 family member expression levels of 34 samples from 17 patients collected before and 10 to 14 days after treatment initiation with either vemurafenib or dabrafenib/trametinib combination.,The observed changes in mRNA and protein levels with BRAFi treatment led us to hypothesize that combining BRAFi with a BCL-2 inhibitor (the BH3-mimetic navitoclax) would improve outcome.,We tested this hypothesis in cell lines and in mice.,Pretreatment mRNA levels of BCL-2 negatively correlated with maximal tumor regression.,Early increases in mRNA levels were seen in BIM, BCL-XL, BID and BCL2-W, as were decreases in MCL-1 and BCL2A.,No significant changes were observed with BCL-2.,Using reverse phase protein array (RPPA), significant increases in protein levels were found in BIM and BID.,No changes in mRNA or protein correlated with response.,Concurrent BRAF (PLX4720) and BCL2 (navitoclax) inhibition synergistically reduced viability in BRAF mutant cell lines and correlated with down-modulation of MCL-1 and BIM induction after PLX4720 treatment.,In xenograft models, navitoclax enhanced the efficacy of PLX4720.,The combination of a selective BRAF inhibitor with a BH3-mimetic promises to be an important therapeutic strategy capable of enhancing the clinical efficacy of BRAF inhibition in many patients that might otherwise succumb quickly to de novo resistance.,Trial Registrations: ClinicalTrials.gov NCT01006980;,ClinicalTrials.gov NCT01107418;,ClinicalTrials.gov NCT01264380;,ClinicalTrials.gov NCT01248936;,ClinicalTrials.gov NCT00949702;,ClinicalTrials.gov NCT01072175

Let-7 microRNAs (miRNAs) are highly conserved well-established promoters of terminal differentiation that are expressed in healthy adult tissues and frequently repressed in cancer cells.,The tumor suppressive role of let-7 in a variety of cancers in vitro and in vivo has been widely documented and prompted these miRNAs to be candidate genes for miRNA replacement therapy.,In this study we described a new role of let-7a in reprogramming cancer metabolism, recently identified as a new hallmark of cancer.,We show that let-7a down-regulates key anabolic enzymes and increases both oxidative phosphorylation and glycolysis in triple-negative breast cancer and metastatic melanoma cell lines.,Strikingly, the accelerated glycolysis coexists with drastically reduced cancer features.,Moreover, let-7a causes mitochondrial ROS production concomitant with the up-regulation of oxidative stress responsive genes.,To exploit these increased ROS levels for therapeutic purposes, we combined let-7a transfection with the chemotherapeutic drug doxorubicin.,In both cancer types let-7a increased cell sensitivity to doxorubicin.,Pre-treatment with N-acetyl cysteine (NAC) totally abolished this effect, indicating that the increased doxorubicin sensitivity of let-7a cells depends on the redox pathway.,We thus have demonstrated that let-7a plays a prominent role in regulating energy metabolism in cancer cells, further expanding its therapeutic potential.

Almost 50% of metastatic melanoma patients harbor a BRAFV600 mutation andthe introduction of BRAF inhibitors has improved their treatment options.,BRAF inhibitors vemurafenib and dabrafenib achieved improved overall survival over chemotherapy and have been approved for the treatment of BRAF-mutated metastatic melanoma.,However, most patients develop mechanisms of acquired resistance and about 15% of them do not achieve tumor regression at all, due to intrinsic resistance to therapy.,Moreover, early adaptive responses limit the initial efficacy of BRAF inhibition, leading mostly to incomplete responses that may favor the selection of a sub-population of resistant clones and the acquisition of alterations that cause tumor regrowth and progressive disease.,The purpose of this paper is to review the mechanisms of resistance to therapy with BRAF inhibitors and to discuss the strategies to overcome them based on pre-clinical and clinical evidences.

The prognosis of metastatic melanoma is very poor, due to the development of drug resistance.,Cancer stem cells (CSCs) may play a crucial role in this mechanism, contributing to disease relapse.,We first characterized CSCs in melanoma cell lines.,We observed that A375 (but not BLM) cells are able to form melanospheres and show CSCs traits: expression of the pluripotency markers SOX2 and KLF4, higher invasiveness and tumor formation capability in vivo with respect to parental adherent cells.,We also showed that a subpopulation of autofluorescent cells expressing the ABCG2 stem cell marker is present in the A375 spheroid culture.,Based on these data, we investigated whether δ-TT might target melanoma CSCs.,We demonstrated that melanoma cells escaping the antitumor activity of δ-TT are completely devoid of the ability to form melanospheres.,In contrast, cells that escaped vemurafenib treatment show a higher ability to form melanospheres than control cells. δ-TT also induced disaggregation of A375 melanospheres and reduced the spheroidogenic ability of sphere-derived cells, reducing the expression of the ABCG2 marker.,These data demonstrate that δ-TT exerts its antitumor activity by targeting the CSC subpopulation of A375 melanoma cells and might represent a novel chemopreventive/therapeutic strategy against melanoma.

To characterize the differences between the primary and metastatic melanoma cell lines grown in 2D cultures and 3D cultures.,Primary melanoma cells (WM115) and metastatic melanoma cells (WM266) extracted from a single donor was cultured in 2D as well as 3D cultures.,These cells were characterized using proton NMR spectrometry, and the qualitative chemical shifts markers were identified and discussed.,In monolayer culture (2D), we observed one qualitative chemical shift marker for primary melanoma cells.,In spheroid cultures (3D), we observed nine significant chemical shifts, of which eight markers were specific for primary melanoma spheroids, whereas the other one marker was specific to metastatic melanoma spheroids.,This study suggests that the glucose accumulation and phospholipid composition vary significantly between the primary and metastatic cells lines that are obtained from a single donor and also with the cell culturing methods. 14 qualitative chemical shift markers were obtained in the comparison between monolayer culture and spheroids cultures irrespective of the differences in the cell lines.,Among which 4 were unique to monolayer cultures whereas 10 chemical shifts were unique to the spheroid cultures.,This study also shows that the method of cell culture would drastically affect the phospholipid composition of the cells and also depicts that the cells in spheroid culture closely resembles the cells in vivo.,This study shows the high specificity of proton NMR spectrometry in characterizing cancer cell lines and also shows the variations in the glucose accumulation and phospholipid composition between the primary and metastatic melanoma cell lines from the same donor.,Differences in the cell culture method does plays an important role in phospholipid composition of the cells.,The online version of this article (doi:10.1186/s40659-017-0117-8) contains supplementary material, which is available to authorized users.

Cutaneous basal cell carcinoma (cBCC) is the most common malignancy diagnosed in the human population. cBCC presents an increasing incidence which, in the near future, will be higher than all other cancers combined.,The majority of cBCC are located in the head and the neck.,A diversity of management modalities is currently available; nonetheless, surgical excision remains the main modality of treatment. cBCC rarely metastasises and presents a low mortality rate. cBCC morbidity is influenced by local invasion and destruction, especially in the face, where function and aesthetics are major issues.,Easy accessibility to the face and skin on the neck makes cBCC an important issue for otorhinolaryngology head and neck surgeons who must be aware and committed in its management, as the main modality of treatment continues to be surgical.,The aim of this review is to present a brief and practical overview of head and neck cBCC management for ear, nose and throat (ENT) surgeons, discussing key issues about its epidemiology, risk factors, diagnosis and pathogenesis.

Metastasis causes as many as 90% of cancer-related deaths, especially for the deadliest skin cancer, melanoma.,Since hematogenous dissemination of circulating tumor cells is the major route of metastasis, detection and destruction of circulating tumor cells are vital for impeding metastasis and improving patient prognosis.,Exploiting the exquisite intrinsic optical absorption contrast of circulating melanoma cells, we developed dual-wavelength photoacoustic flow cytography coupled with a nanosecond-pulsed melanoma-specific laser therapy mechanism.,We have successfully achieved in vivo label-free imaging of rare single circulating melanoma cells in both arteries and veins of mice.,Further, the photoacoustic signal from a circulating melanoma cell immediately hardware-triggers a lethal pinpoint laser irradiation to kill it on the spot in a thermally confined manner without causing collateral damage.,A pseudo-therapy study including both in vivo and in vitro experiments demonstrated the performance and the potential clinical value of our method, which can facilitate early treatment of metastasis by clearing circulating tumor cells from vasculature.

Cellular plasticity of cancer cells is often associated with phenotypic heterogeneity and drug resistance and thus remains a major challenge for the treatment of melanoma and other types of cancer.,Melanoma cells have the capacity to switch their phenotype during tumor progression, from a proliferative and differentiated phenotype to a more invasive and dedifferentiated phenotype.,However, the molecular mechanisms driving this phenotype switch are not yet fully understood.,Considering that cellular heterogeneity within the tumor contributes to the high plasticity typically observed in melanoma, it is crucial to generate suitable models to investigate this phenomenon in detail.,Here, we discuss the use of complete and partial reprogramming into induced pluripotent cancer (iPC) cells as a tool to obtain new insights into melanoma cellular plasticity.,We consider this a relevant topic due to the high plasticity of melanoma cells and its association with a strong resistance to standard anticancer treatments.

The development of acquired drug resistance hampers the long-term success of B-RAF inhibitor (B-RAFi) therapy for melanoma patients.,Here we show V600EB-RAF copy number gain as a mechanism of acquired B-RAFi resistance in four out of twenty (20%) patients treated with B-RAFi.,In cell lines, V600EB-RAF over-expression and knockdown conferred B-RAFi resistance and sensitivity, respectively.,In V600EB-RAF amplification-driven (vs. mutant N-RAS-driven) B-RAFi resistance, ERK reactivation is saturable, with higher doses of vemurafenib down-regulating pERK and re-sensitizing melanoma cells to B-RAFi.,These two mechanisms of ERK reactivation are sensitive to the MEK1/2 inhibitor AZD6244/selumetinib or its combination with the B-RAFi vemurafenib.,In contrast to mutant N-RAS-mediated V600EB-RAF bypass, which is sensitive to C-RAF knockdown, V600EB-RAF amplification-mediated resistance functions largely independently of C-RAF.,Thus, alternative clinical strategies may potentially overcome distinct modes of ERK reactivation underlying acquired B-RAFi resistance in melanoma.

Metastasis requires cancer cells to undergo poorly-understood metabolic changes1-3.,We found that metabolic differences among melanoma cells confer differences in metastatic potential as a result of differences in Monocarboxylate Transporter 1 (MCT1) function.,In vivo isotope tracing in patient-derived xenografts revealed differences in nutrient handling between efficiently and inefficiently metastasizing melanomas, with circulating lactate being a more prominent source of tumor lactate in efficient metastasizers.,Efficient metastasizers had higher MCT1 levels and MCT1 inhibition reduced lactate uptake.,MCT1 inhibition had little effect on primary subcutaneous tumor growth but depleted circulating melanoma cells and reduced metastatic disease burden in patient-derived xenografts and in mouse melanomas.,MCT1 inhibition suppressed the oxidative pentose phosphate pathway and increased ROS levels.,Anti-oxidants blocked the effect of MCT1 inhibition on metastasis.,MCT1high and MCT1−/low cells from the same melanomas had similar capacities to form subcutaneous tumors, but MCT1high cells formed more metastases after intravenous injection.,Metabolic differences among cancer cells thus confer differences in metastatic potential as metastasizing cells depend upon MCT1 to manage oxidative stress.

The mechanisms by which some melanoma cells adapt to BRAF inhibitor therapy are incompletely understood.,In the present study, we used mass spectrometry-based phosphoproteomics to determine how BRAF inhibition remodeled the signaling network of melanoma cell lines that were BRAF-mutant and PTEN-null.,Short-term BRAF inhibition was associated with marked changes in fibronectin-based adhesion signaling that were PTEN-dependent.,These effects were recapitulated through BRAF siRNA knockdown and following treatment with chemotherapeutic drugs.,Increased fibronectin expression was also observed in mouse xenograft models as well as specimens from melanoma patients undergoing BRAF inhibitor treatment.,Analysis of a melanoma TMA showed loss of PTEN expression to predict for a lower overall survival, with a trend for even lower survival being seen when loss of fibronectin was included in the analysis.,Mechanistically, the induction of fibronectin limited the responses of these PTEN-null melanoma cell lines to vemurafenib, with enhanced cytotoxicity observed following the knockdown of either fibronectin or its receptor α5β1 integrin.,This in turn abrogated the cytotoxic response to BRAF inhibition via increased AKT signaling, which prevented the induction of cell death by maintaining the expression of the pro-survival protein Mcl-1.,The protection conveyed by the induction of fibronectin expression could be overcome through combined treatment with a BRAF and PI3K inhibitor.

Clinical outcomes for advanced malignant melanoma (MM) are often poor due to tumor invasiveness, metastasis, recurrence, and multidrug resistance.,We investigated whether apoptosis, cell cycle regulation, oxidative status, and redox balance were altered by changes in the expression of the long noncoding RNA, growth arrest-specific transcript 5 (GAS5), in MM cells.,Analysis of clinical samples from MM patients showed that the rate of reduced GAS5 expression, relative to that in adjacent noncancerous tissues, was significantly lower for tumors from patients with advanced disease (76.6%, P < 0.001), as evidenced by larger tumor size, higher TNM stage, and higher incidences of ulceration and metastasis (P < 0.001 for all).,Cell culture experiments showed that siRNA-mediated knockdown of GAS5 increased the viability of A375-GAS5si cells.,Flow cytometry and western blotting showed that GAS5 knockdown increased MM cell proliferation by inducing G1/S cell cycle progression through increases in Cyclin D1, CDK4, and p27 expression (P < 0.05 for all) and by inhibiting apoptosis through an increase in Bcl-2 expression (P < 0.001).,Knockdown of GAS5 also increased levels of superoxide anion (P < 0.01), NADP+(P < 0.001), and oxidized glutathiones (P < 0.01) through increases in NOX4 expression (P < 0.001), G6PD expression (P < 0.01), and NOX activity (P < 0.05), and RNA co-immunoprecipitation showed that GAS5 induced these changes through a physical interaction between GAS5 and the G6PD protein.,Our findings show GAS5 contributes to regulation of the apoptosis, cell cycle, homeostasis of reactive oxygen species, and redox balance in MM cells, and suggest that reduced GAS5 expression contributes to disease progression in MM patients.,The online version of this article (10.1007/s00432-018-2820-4) contains supplementary material, which is available to authorized users.

Conventional clinico-pathological features in melanoma patients should be integrated with new molecular diagnostic, predictive, and prognostic factors coming from the expanding genomic profiles.,Cutaneous melanoma (CM), even differing in biological behavior according to sun-exposure levels on the skin areas where it arises, is molecularly heterogeneous.,The next-generation sequencing (NGS) approaches are providing data on mutation landscapes in driver genes that may account for distinct pathogenetic mechanisms and pathways.,The purpose was to group and classify all somatic driver mutations observed in the main NGS-based studies.,Whole exome and whole genome sequencing approaches have provided data on spectrum and distribution of genetic and genomic alterations as well as allowed to discover new cancer genes underlying CM pathogenesis.,After evaluating the mutational status in a cohort of 686 CM cases from the most representative NGS studies, three molecular CM subtypes were proposed: BRAFmut, RASmut, and non-BRAFmut/non-RASmut.

The molecular background of a significant proportion of spitzoid neoplasms is still unknown.,Recently, activating mutations in MAP2K1 have been described in a few spitzoid lesions, but not in benign Spitz nevi.,We report four cases of melanocytic tumors with spitzoid features in which a MAP2K1 mutation was detected.,The lesions did not show a single distinct phenotype and ranged from benign to malignant.,Two cases resembled desmoplastic Spitz nevi.,Based on the combination of morphological, immunohistochemical, and molecular findings, one case was classified as benign, one as probably benign, possibly intermediate low-grade (MELTUMP-melanocytic tumor of unknown malignant potential), one case was classified as intermediate (MELTUMP), and one case was considered a superficial spreading melanoma with spitzoid features.,Based on this, we conclude that MAP2K1 mutations can indicate a spitzoid genetic signature and can be found in both benign and malignant spitzoid neoplasms.,The online version of this article (10.1007/s00428-020-02940-3) contains supplementary material, which is available to authorized users.

Spitzoid neoplasms constitute a morphologically distinct category of melanocytic tumors, encompassing Spitz nevus (benign), atypical Spitz tumor (intermediate malignant potential), and spitzoid melanoma (fully malignant).,Currently, no reliable histopathological criteria or molecular marker is known to distinguish borderline from overtly malignant neoplasms.,Because TERT promoter (TERT-p) mutations are common in inherently aggressive cutaneous conventional melanoma, we sought to evaluate their prognostic significance in spitzoid neoplasms.,We analyzed tumors labeled as atypical Spitz tumor or spitzoid melanoma from 56 patients with available follow-up data for the association of TERT-p mutations, biallelic CDKN2A deletion, biallelic PTEN deletion, kinase fusions, BRAF/NRAS mutations, nodal status, and histopathological parameters with risk of hematogenous metastasis.,Four patients died of disseminated disease and 52 patients were alive and disease free without extranodal metastasis (median follow-up, 32.5 months).,We found TERT-p mutations in samples from the 4 patients who developed hematogenous metastasis but in none of tumors from patients who had favorable outcomes.,Presence of TERT-p mutations was the most significant predictor of haematogenous dissemination (P < 0.0001) among variables analyzed.,We conclude that TERT-p mutations identify a clinically high-risk subset of patients with spitzoid tumors.,Application of TERT-p mutational assays for risk stratification in the clinic requires large-scale validation.

Talimogene laherparepvec is an oncolytic immunotherapy approved in the US, Europe, Australia and Switzerland.,We report the final planned analysis of OPTiM, a randomized open-label phase III trial in patients with unresectable stage IIIB-IVM1c melanoma.,Patients were randomized 2:1 to receive intratumoral talimogene laherparepvec or subcutaneous recombinant GM-CSF.,In addition to overall survival (OS), durable response rate (DRR), objective response rate (ORR), complete responses (CR), and safety are also reported.,All final analyses are considered to be descriptive and treatment responses were assessed by the investigators.,Of 436 patients in the intent-to-treat population, 295 were allocated to talimogene laherparepvec and 141 to GM-CSF.,Median follow-up in the final OS analysis was 49 months.,Median OS was 23.3 months (95% confidence interval [CI], 19.5-29.6) and 18.9 months (95% CI, 16.0-23.7) in the talimogene laherparepvec and GM-CSF arms, respectively (unstratified hazard ratio, 0.79; 95% CI, 0.62-1.00; p = 0.0494 [descriptive]).,DRR was 19.0 and 1.4% (unadjusted odds ratio, 16.6; 95% CI, 4.0-69.2; p < 0.0001); ORR was 31.5 and 6.4%.,Fifty (16.9%) and 1 (0.7%) patient in the talimogene laherparepvec and GM-CSF arms, respectively, achieved CR.,In talimogene laherparepvec-treated patients, median time to CR was 8.6 months; median CR duration was not reached.,Among patients with a CR, 88.5% were estimated to survive at a 5-year landmark analysis.,Talimogene laherparepvec efficacy was more pronounced in stage IIIB-IVM1a melanoma as already described in the primary analysis.,The safety reporting was consistent with the primary OPTiM analysis.,In this final planned OPTiM analysis, talimogene laherparepvec continued to result in improved longer-term efficacy versus GM-CSF and remained well tolerated.,The final analysis also confirms that talimogene laherparepvec was associated with durable CRs that were associated with prolonged survival.,ClinicalTrials.gov identifier: NCT00769704.,The online version of this article (10.1186/s40425-019-0623-z) contains supplementary material, which is available to authorized users.

A clinical oncolytic herpes simplex virus (HSV) encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), talimogene laherparepvec, causes regression of injected and non-injected melanoma lesions in patients and is now licensed for clinical use in advanced melanoma.,To date, limited data are available regarding the mechanisms of human anti-tumor immune priming, an improved understanding of which could inform the development of future combination strategies with improved efficacy.,This study addressed direct oncolysis and innate and adaptive human immune-mediated effects of a closely related HSV encoding GM-CSF (HSVGM-CSF) alone and in combination with histone deacetylase inhibition.,We found that HSVGM-CSF supported activation of anti-melanoma immunity via monocyte-mediated type I interferon production, which activates NK cells, and viral maturation of immature dendritic cells (iDCs) into potent antigen-presenting cells for cytotoxic T lymphocyte (CTL) priming.,Addition of the histone deacetylase inhibitor valproic acid (VPA) to HSVGM-CSF treatment of tumor cells increased viral replication, viral GM-CSF production, and oncolysis and augmented the development of anti-tumor immunity.,Mechanistically, VPA increased expression of activating ligands for NK cell recognition and induced expression of tumor-associated antigens, supporting innate NK cell killing and CTL priming.,These data support the clinical combination of talimogene laherparepvec with histone deacetylase inhibition to enhance oncolysis and anti-tumor immunity.,Jennings et al., demonstrate that oncolytic HSVGM-CSF activates NK cells and matures immature dendritic cells to promote cytotoxic T lymphocyte (CTL) priming.,Moreover, they report that histone deacetylase inhibition can augment innate and adaptive anti-tumor immune responses through increased expression of NK cell-activating ligands and tumor-associated antigens.

We report phase 1b data from patients enrolled in the JAVELIN Solid Tumor clinical trial (NCT01772004) with unresectable stage IIIC or IV melanoma that had progressed after ≥1 line of therapy for metastatic disease.,Patients received avelumab (10 mg/kg)-a human anti-PD-L1 antibody.,Assessments included objective response rate (ORR), progression-free survival (PFS), overall survival (OS), and safety.,As of December 31, 2016, 51 patients were treated and followed for a median of 24.2 months (range, 16.1-31.5).,Most patients had cutaneous (n = 28 [54.9%]) or ocular (n = 16 [31.4%]) melanoma and had received a median of 2 prior lines of therapy (range, 0-4), including ipilimumab (n = 26 [51.0%]).,The confirmed ORR was 21.6% (95% CI, 11.3-35.3; complete response, 7.8%; partial response, 13.7%).,The median duration of response was not estimable (95% CI, 2.6 months-not estimable).,Median PFS and OS were 3.1 months (95% CI, 1.4-6.3) and 17.2 months (95% CI, 6.6-not estimable), respectively.,Subgroup analyses suggested meaningful clinical activity (ORR [95% CI]) in patients with non-ocular melanoma (31.4% [16.9-49.3]), PD-L1-positive tumors (42.1% [20.3-66.5]), or prior ipilimumab therapy (30.8% [14.3-51.8]).,Thirty-nine patients (76.5%) had a treatment-related adverse event (TRAE), most commonly infusion-related reaction (29.4%), fatigue (17.6%), and chills (11.8%); 4 patients (7.8%) had a grade 3 TRAE.,Five patients (9.8%) had an immune-related TRAE (all were grade 1/2).,No grade 4 TRAEs or treatment-related deaths were reported.,Avelumab showed durable responses, promising survival outcomes, and an acceptable safety profile in patients with previously treated metastatic melanoma.,ClinicalTrials.gov identifier: NCT01772004.,The online version of this article (10.1186/s40425-018-0459-y) contains supplementary material, which is available to authorized users.

Immunotherapy with PD-1 antibodies has greatly increased prognosis of patients with advanced melanoma.,Identifying biomarkers that predict overall survival (OS) and response to immunotherapy is important.,OS and best overall response according to RECIST version 1.1 were analysed, and S100B and lactate dehydrogenase (LDH) serum levels were assessed retrospectively in 152 patients treated with anti-PD-1, and in 86 patients treated with anti-PD-1 plus anti-CTLA-4 antibodies at University Hospital Tuebingen, Germany.,In the pembrolizumab group, patients with elevated baseline S100B or LDH exhibited significantly impaired OS compared with patients with normal S100B (1-year OS: 51.1% vs 83.1%, log-rank P < .0001) and normal LDH (1-year OS: 44.4% vs 80.8%, P = .00022), respectively.,LDH increases of >25% and S100B increases of >145% compared to baseline were significantly associated with impaired OS (both P < .0001).,In patients treated with ipilimumab and nivolumab, baseline S100B and increasing S100B levels of >145% as well as baseline LDH were associated with impaired OS (P < .0001, P = .00060, and P = .0050, respectively), whereas increasing LDH of >25% was not (P = .64).,S100B could serve as a strong baseline marker for OS in melanoma patients receiving anti-PD-1 therapy.,Rising S100B levels during the first weeks of therapy could help guide treatment decisions.

Cancer is thought to arise through the accumulation of genomic aberrations evolving under Darwinian selection.,However, it remains unclear when the aberrations associated with metastasis emerge during tumor evolution.,Uveal melanoma (UM) is the most common primary eye cancer and frequently leads to metastatic death, which is strongly linked to BAP1 mutations.,Accordingly, UM is ideally suited for studying the clonal evolution of metastatic competence.,Here we analyze sequencing data from 151 primary UM samples using a customized bioinformatic pipeline, to improve detection of BAP1 mutations and infer the clonal relationships among genomic aberrations.,Strikingly, we find BAP1 mutations and other canonical genomic aberrations usually arise in an early punctuated burst, followed by neutral evolution extending to the time of clinical detection.,This implies that the metastatic proclivity of UM is “set in stone” early in tumor evolution and may explain why advances in primary treatment have not improved survival.,Uveal melanoma (UM), the most common primary eye cancer, is strongly linked to mutations in the tumor suppressor BAP1.,Here, the authors analyze 151 primary UM samples to find that BAP1 and other canonical genomic aberrations arise in an early punctuated burst followed by neutral tumor evolution.

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.

Treatment of BRAF‐mutant melanomas with MAP kinase pathway inhibitors is paradigmatic of the promise of precision cancer therapy but also highlights problems with drug resistance that limit patient benefit.,We use live‐cell imaging, single‐cell analysis, and molecular profiling to show that exposure of tumor cells to RAF/MEK inhibitors elicits a heterogeneous response in which some cells die, some arrest, and the remainder adapt to drug.,Drug‐adapted cells up‐regulate markers of the neural crest (e.g., NGFR), a melanocyte precursor, and grow slowly.,This phenotype is transiently stable, reverting to the drug‐naïve state within 9 days of drug withdrawal.,Transcriptional profiling of cell lines and human tumors implicates a c‐Jun/ECM/FAK/Src cascade in de‐differentiation in about one‐third of cell lines studied; drug‐induced changes in c‐Jun and NGFR levels are also observed in xenograft and human tumors.,Drugs targeting the c‐Jun/ECM/FAK/Src cascade as well as BET bromodomain inhibitors increase the maximum effect (E max) of RAF/MEK kinase inhibitors by promoting cell killing.,Thus, analysis of reversible drug resistance at a single‐cell level identifies signaling pathways and inhibitory drugs missed by assays that focus on cell populations.

The expansion of cell colonies is driven by a delicate balance of several mechanisms including cell motility, cell-to-cell adhesion and cell proliferation.,New approaches that can be used to independently identify and quantify the role of each mechanism will help us understand how each mechanism contributes to the expansion process.,Standard mathematical modelling approaches to describe such cell colony expansion typically neglect cell-to-cell adhesion, despite the fact that cell-to-cell adhesion is thought to play an important role.,We use a combined experimental and mathematical modelling approach to determine the cell diffusivity, D, cell-to-cell adhesion strength, q, and cell proliferation rate, λ, in an expanding colony of MM127 melanoma cells.,Using a circular barrier assay, we extract several types of experimental data and use a mathematical model to independently estimate D, q and λ.,In our first set of experiments, we suppress cell proliferation and analyse three different types of data to estimate D and q.,We find that standard types of data, such as the area enclosed by the leading edge of the expanding colony and more detailed cell density profiles throughout the expanding colony, does not provide sufficient information to uniquely identify D and q.,We find that additional data relating to the degree of cell-to-cell clustering is required to provide independent estimates of q, and in turn D.,In our second set of experiments, where proliferation is not suppressed, we use data describing temporal changes in cell density to determine the cell proliferation rate.,In summary, we find that our experiments are best described using the range D=161−243μm2hour−1, q=0.3−0.5 (low to moderate strength) and λ=0.0305−0.0398hour−1, and with these parameters we can accurately predict the temporal variations in the spatial extent and cell density profile throughout the expanding melanoma cell colony.,Our systematic approach to identify the cell diffusivity, cell-to-cell adhesion strength and cell proliferation rate highlights the importance of integrating multiple types of data to accurately quantify the factors influencing the spatial expansion of melanoma cell colonies.

Melanoma is a highly metastatic disease with an increasing rate of incidence worldwide.,It is treatment refractory and has poor clinical prognosis; therefore, the development of new therapeutic agents for metastatic melanoma are urgently required.,In this study, we created a lung-seeking A375LM5IF4g/Luc BRAFV600E mutant melanoma cell clone and investigated the bioefficacy of a plant sesquiterpene lactone deoxyelephantopin (DET) and its novel semi-synthetic derivative, DETD-35, in suppressing metastatic A375LM5IF4g/Luc melanoma growth in vitro and in a xenograft mouse model.,DET and DETD-35 treatment inhibited A375LM5IF4g/Luc cell proliferation, and induced G2/M cell-cycle arrest and apoptosis.,Furthermore, A375LM5IF4g/Luc exhibited clonogenic, metastatic and invasive abilities, and several A375LM5IF4g/Luc metastasis markers, N-cadherin, MMP2, vimentin and integrin α4 were significantly suppressed by treatment with either compound.,Interestingly, DET- and DETD-35-induced Reactive Oxygen Species (ROS) generation and glutathione (GSH) depletion were found to be upstream events important for the in vitro activities, because exogenous GSH supplementation blunted DET and DETD-35 effects on A375LM5IF4g/Luc cells.,DET and DETD-35 also induced mitochondrial DNA mutation, superoxide production, mitochondrial bioenergetics dysfunction, and mitochondrial protein deregulation.,Most importantly, DET and DETD-35 inhibited lung metastasis of A375LM5IF4g/Luc in NOD/SCID mice through inhibiting pulmonary vascular permeability and melanoma cell (Mel-A+) proliferation, angiogenesis (VEGF+, CD31+) and EMT (N-cadherin) in the tumor microenvironment in the lungs.,These findings indicate that DET and DETD-35 may be useful in the intervention of lung metastatic BRAFV600E mutant melanoma.

FKBP51 immunophilin is abundantly expressed by immune cells.,Co-inhibitory immune receptor signalling generates the splicing isoform FKBP51s.,Tregs stained by FKBP51s are increased in melanoma patients and their counts are associated with anti-CTLA-4 response.,An expansion of FKBP51s+PD-L1+ monocytes was measured in a group of non-responding patients to anti-CTLA-4.,The aim of this work was to confirm the predictive value of response of FKBP51s+Tregs in a cohort of patients undergoing anti-PD1 treatment and shed light on a monocyte subset co-expressing PD-L1/FKBP51s.,Co-cultures of organoids and autologous lymphocytes were used to confirm that tumour T-cell interaction can induce FKBP51s.,PBMC immunophenotype and flow cytometry served to assess and monitor FKBP51s+Treg and FKBP51s+PD-L1+ monocytes in 22 advanced melanoma patients treated with anti-PD1.,Silencing and overexpression of FKBP51s in human macrophages served to address the protein role in the tolerant macrophages’ behaviour.,FKBP51s+Tregs count was increased in responders and had a prognostic value.,Non-responders showed an early increase in FKBP51s+ PD-L1+ monocytes during anti-PD1 treatment.,Manipulation of FKBP51s modulated the macrophage-phenotype, with forced protein expression promoting aspects associated with tolerance.,FKBP51s may guide in the selection and monitoring of melanoma patient candidates to immune-checkpoint-targeted therapy.,Manipulation of FKBP51s may overcome resistance.

Melanoma is an aggressive skin carcinoma with poor prognosis, and is prevalent worldwide.,It was demonstrated that microRNA (miR)-21 and mitogen-activated protein kinase kinase 3 (MKK3) both participated in the occurrence and development of various tumors; however, their detailed roles in the progression of melanoma remain unclear.,Reverse transcription-quantitative PCR (RT-qPCR) and western blot analyses were conducted to examine the expression levels of miR-21 and MKK3 in clinical specimens of patients with melanoma and melanoma cell lines.,A dual-luciferase reporter assay was performed to verify the target interaction between miR-21 and MKK3.,The mRNA and protein expressions of MKK3 were measured using RT-qPCR and western blot analysis, respectively, following transfection with miR-21 mimics and inhibitor.,Subsequently, Cell Counting Kit-8 and colony formation assays, and flow cytometry were conducted to assess the effects of miR-21 and MKK3 on the cell growth of melanoma.,Cell migration and invasion experiments were performed to evaluate the effects of miR-21 and MKK3 on the cell metastasis of melanoma.,It was revealed that MKK3 was upregulated, and miR-21 was downregulated in patients with melanoma and melanoma cell lines.,MKK3 was demonstrated to be a direct target of miR-21.,Furthermore, it was demonstrated that upregulated miR-21 expression and downregulated MKK3 expression suppressed cell proliferation and colony formation, promoted apoptosis, delayed the cell cycle, and inhibited cell migration and invasion.,The present findings suggested that miR-21 could inhibit the cell growth and metastasis of melanoma by negatively regulating MKK3.

Conventional clinico-pathological features in melanoma patients should be integrated with new molecular diagnostic, predictive, and prognostic factors coming from the expanding genomic profiles.,Cutaneous melanoma (CM), even differing in biological behavior according to sun-exposure levels on the skin areas where it arises, is molecularly heterogeneous.,The next-generation sequencing (NGS) approaches are providing data on mutation landscapes in driver genes that may account for distinct pathogenetic mechanisms and pathways.,The purpose was to group and classify all somatic driver mutations observed in the main NGS-based studies.,Whole exome and whole genome sequencing approaches have provided data on spectrum and distribution of genetic and genomic alterations as well as allowed to discover new cancer genes underlying CM pathogenesis.,After evaluating the mutational status in a cohort of 686 CM cases from the most representative NGS studies, three molecular CM subtypes were proposed: BRAFmut, RASmut, and non-BRAFmut/non-RASmut.