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Breast cancer and melanoma are among the most frequent cancer types leading to brain metastases.,Despite the unquestionable clinical significance, important aspects of the development of secondary tumours of the central nervous system are largely uncharacterized, including extravasation of metastatic cells through the blood‐brain barrier.,By using transmission electron microscopy, here we followed interactions of cancer cells and brain endothelial cells during the adhesion, intercalation/incorporation and transendothelial migration steps.,We observed that brain endothelial cells were actively involved in the initial phases of the extravasation by extending filopodia‐like membrane protrusions towards the tumour cells.,Melanoma cells tended to intercalate between endothelial cells and to transmigrate by utilizing the paracellular route.,On the other hand, breast cancer cells were frequently incorporated into the endothelium and were able to migrate through the transcellular way from the apical to the basolateral side of brain endothelial cells.,When co‐culturing melanoma cells with cerebral endothelial cells, we observed N‐cadherin enrichment at melanoma‐melanoma and melanoma‐endothelial cell borders.,However, for breast cancer cells N‐cadherin proved to be dispensable for the transendothelial migration both in vitro and in vivo.,Our results indicate that breast cancer cells are more effective in the transcellular type of migration than melanoma cells.

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.

Sinonasal mucosal melanoma (SNMM) comprises <1% of all melanomas and lacks well-characterised molecular markers.,Our aim was to determine the frequencies of common mutations and examine their utility as molecular markers in a large series of primary SNMMs.,SNMM patients seen at our institution from August 1991 through July 2016 were identified.,Genomic DNA was extracted from 66 formalin-fixed paraffin-embedded tumours and screened for mutations by direct sequencing.,We investigated the association of mutations with clinicopathological features and survival outcomes.,Overall, 41% (27 out of 66) of the SNMMs harboured mutations.,BRAF and KIT mutations were identified in 8% (five patients) and 5% (three patients) of SNMMs, respectively, whereas NRAS mutations were detected in 30% (20 patients) of SNMMs.,Mutation rates in these oncogenes were similar between SNMMs located in the paranasal sinuses and those in the nasal cavity (30% and 13%, respectively, P=0.09).,In a multivariate analysis, patients with negative margins had significantly better overall survival (hazard ratio 5.43, 95% confidence interval 1.44-21.85, P=0.01) and disease-specific survival (hazard ratio 21.9, 95% confidence interval 3.71-180, P=0.0004).,The mutation status of the tumours showed no association with survival outcomes.,In SNNM, mutation status does not affect survival outcomes, but NRAS mutations are relatively frequent and could be targeted in this disease by MEK inhibitors.

Metastatic melanoma has long been considered to have a very poor prognosis and to be chemo-resistant.,However, a subgroup of patients with metastatic melanoma presents remarkable responses to chemotherapeutic agents, even in the absence of a response to modern targeted therapies and immunotherapies; accordingly, determining predictive biomarkers of the response to chemotherapies for metastatic melanoma remains a priority to guide treatment in these patients.,We report a case study of a patient with B-Raf proto-oncogene serine/threonine kinase-mutated metastatic melanoma harbouring many genetic mutations.,The patient did not respond to prior targeted therapies or immunotherapies but experienced a dramatic objective radiological and clinical response to subsequent dacarbazine-based chemotherapy.,In the era of targeted therapies and immunotherapies for metastatic melanoma, cytotoxic chemotherapies may still represent an interesting therapeutic weapon in a well-defined subgroup of patients presenting with specific genetic and molecular features.

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 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.

Although MAPK pathway inhibitors are becoming a promising anticancer strategy, they are insufficient to fully eliminate cancer cells and their long-term efficacy is strikingly limited in patients with BRAF-mutant melanomas.,It is well established that BRAF inhibitors (BRAFi) hamper glucose uptake before the apparition of cell death.,Here, we show that BRAFi induce an extensive restructuring of mitochondria including an increase in mitochondrial activity and biogenesis associated with mitochondrial network remodeling.,Furthermore, we report a close interaction between ER and mitochondria in melanoma exposed to BRAFi.,This physical connection facilitates mitochondrial Ca2+ uptake after its release from the ER.,Interestingly, Mfn2 silencing disrupts the ER-mitochondria interface, intensifies ER stress and exacerbates ER stress-induced apoptosis in cells exposed to BRAFi in vitro and in vivo.,This mitochondrial control of ER stress-mediated cell death is similar in both BRAF- and NRAS-mutant melanoma cells exposed to MEK inhibitors.,This evidence reinforces the relevance in combining MAPK pathway inhibitors with mitochondriotropic drugs to improve targeted therapies.

Uveal melanoma is a highly aggressive cancer with a strong propensity for metastasis, yet little is known about the biological mechanisms underlying this metastatic potential.,We recently showed that most metastasizing uveal melanomas, which exhibit a class 2 gene expression profile, contain inactivating mutations in the tumor suppressor BAP1.,The aim of this study was to investigate the role of BAP1 in uveal melanoma progression.,Uveal melanoma cells were studied following RNAi-mediated depletion of BAP1 using proliferation, BrdU incorporation, flow cytometry, migration, invasion, differentiation and clonogenic assays, as well as in vivo tumorigenicity experiments in NOD-SCID-Gamma mice.,Depletion of BAP1 in uveal melanoma cells resulted in a loss of differentiation and gain of stem-like properties, including expression of stem cell markers, increased capacity for self-replication, and enhanced ability to grow in stem cell conditions.,BAP1 depletion did not result in increased proliferation, migration, invasion or tumorigenicity.,BAP1 appears to function in the uveal melanocyte lineage primarily as a regulator of differentiation, with cells deficient for BAP1 exhibiting stem-like qualities.,It will be important to elucidate how this effect of BAP1 loss promotes metastasis and how to reverse this effect therapeutically.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play central roles in diverse pathological processes.,In this study, we investigated the effect of microRNA-182 (miR-182) on the development of posterior uveal melanomas.,Initially, we demonstrated that miR-182 expression was dependent on p53 induction in uveal melanoma cells.,Interestingly, transient transfection of miR-182 into cultured uveal melanoma cells led to a significant decrease in cell growth, migration, and invasiveness.,Cells transfected with miR-182 demonstrated cell cycle G1 arrest and increased apoptotic activity.,Using bioinformatics, we identified three potential targets of miR-182, namely MITF, BCL2 and cyclin D2. miR-182 was shown to have activity on mRNA expression by targeting the 3′ untranslated region of MITF, BCL2 and cyclin D2.,Subsequent Western blot analysis confirmed the downregulation of MITF, BCL2 and cyclin D2 protein expression.,The expression of oncogene c-Met and its downstream Akt and ERK1/2 pathways was also downregulated by miR-182.,Concordant with the findings that miR-182 was decreased in uveal melanoma tissue samples, overexpression of miR-182 also suppressed the in vivo growth of uveal melanoma cells.,Our results demonstrated that miR-182, a p53 dependent miRNA, suppressed the expression of MITF, BCL2, cyclin D2 and functioned as a potent tumor suppressor in uveal melanoma cells.

The Microphthalmia associated transcription factor (Mitf) is an important regulator in melanocyte development and has been shown to be involved in melanoma progression.,The current model for the role of Mitf in melanoma assumes that the total activity of the protein is tightly regulated in order to secure cell proliferation.,Previous research has shown that regulation of Mitf is complex and involves regulation of expression, splicing, protein stability and post-translational modifications.,Here we show that microRNAs (miRNAs) are also involved in regulating Mitf in melanoma cells.,Sequence analysis revealed conserved binding sites for several miRNAs in the Mitf 3′UTR sequence.,Furthermore, miR-148 was shown to affect Mitf mRNA expression in melanoma cells through a conserved binding site in the 3′UTR sequence of mouse and human Mitf.,In addition we confirm the previously reported effects of miR-137 on Mitf.,Other miRNAs, miR-27a, miR-32 and miR-124 which all have conserved binding sites in the Mitf 3′UTR sequence did not have effects on Mitf.,Our data show that miR-148 and miR-137 present an additional level of regulating Mitf expression in melanocytes and melanoma cells.,Loss of this regulation, either by mutations or by shortening of the 3′UTR sequence, is therefore a likely factor in melanoma formation and/or progression.

Malignant melanoma displays a high degree of cellular plasticity during disease progression.,Signals in the tumor microenvironment are believed to influence melanoma plasticity through changes in the epigenetic state to guide dynamic differentiation and de-differentiation.,Here we uncover a relationship between geometric features at perimeter regions of melanoma aggregates, and reprogramming to a stem cell-like state through histone marks H3K4Me2 and H3K9Ac.,Using an in vitro tumor microengineering approach, we find spatial enrichment of these histone modifications with concurrent expression of stemness markers.,The epigenetic modifier PRDM14 overlaps with H3K9Ac and shows elevated expression in cells along regions of perimeter curvature. siRNA knockdown of PRDM14 abolishes the MIC phenotype suggesting a role in regulating melanoma heterogeneity.,Our results suggest mechanotransduction at the periphery of melanoma aggregates may orchestrate the activity of epigenetic modifiers to regulate histone state, cellular plasticity, and tumorigenicity.,Junmin Lee et al. study the role of geometric features at the perimeter regions of melanoma aggregates in programming stem cell-like state through histone marks.,They use a tumor microengineering approach in vitro and report a spatial enrichment of histone modifications with stemness markers.,Their work uncovers a mechanotransduction signaling that regulates epigenetic modifiers to regulate tumorigenicity.

Melanoma is a highly aggressive cancer that exhibits metastasis to various critical organs.,Unlike any other cancer cells, melanoma cells can synthesize melanin in large amounts, becoming heavily pigmented.,Until now the role of melanin in melanoma, particularly the effect of melanin presence on the abilities of melanoma cells to spread and metastasize remains unknown.,Recently, we have shown that melanin dramatically modified elastic properties of melanoma cells and inhibited the cells invasive abilities in vitro.,Here, we inoculated human melanoma cells with different melanin content into nude mice and tested the hypothesis that cell elasticity is an important property of cancer cells for their efficient spread in vivo.,The obtained results clearly showed that cells containing melanin were less capable to spread in mice than cells without the pigment.,Our findings indicate that the presence of melanin inhibits melanoma metastasis, emphasizing possible clinical implications of such an inhibitory effect.

Cutaneous squamous cell carcinoma represents the second most common cutaneous malignancy, affecting 7-11% of Caucasians in the United States.,The genetic determinants of susceptibility to cutaneous squamous cell carcinoma remain largely unknown.,Here we report the results of a two-stage genome-wide association study of cutaneous squamous cell carcinoma, totalling 7,404 cases and 292,076 controls.,Eleven loci reached genome-wide significance (P<5 × 10−8) including seven previously confirmed pigmentation-related loci: MC1R, ASIP, TYR, SLC45A2, OCA2, IRF4 and BNC2.,We identify an additional four susceptibility loci: 11q23.3 CADM1, a metastasis suppressor gene involved in modifying tumour interaction with cell-mediated immunity; 2p22.3; 7p21.1 AHR, the dioxin receptor involved in anti-apoptotic pathways and melanoma progression; and 9q34.3 SEC16A, a putative oncogene with roles in secretion and cellular proliferation.,These susceptibility loci provide deeper insight into the pathogenesis of squamous cell carcinoma.,Cutaneous squamous cell carcinoma is the second most common type of skin cancer.,In this genome-wide association study, which includes over 7,000 cases, the authors identify 4 new susceptibility loci for this cancer and also provide independent replication of 9 previously reported susceptibility loci.

The precise mechanisms governing invasion at the leading edge of SCC and its subsequent metastasis are not fully understood.,We aimed to define the cancer related molecular changes that distinguish non-invasive tumor from invasive SCC.,To this end, we combined laser capture microdissection with cDNA microarray analysis.,We defined invasion-associated genes as those differentially regulated only in invasive SCC nests, but not in actinic keratosis or in situ SCC, compared to normal epidermis.,There were 383 up- and 354 down-regulated genes in the “invasion set.”,SCC invasion was characterized by aberrant expression of various proteolytic molecules.,We noted increased expression of MMP7 and IL-24 in invasive SCC.,IL-24 induced the expression of MMP7 in SCC cells in culture.,In addition, blocking of MMP7 by a specific antibody significantly delayed the migration of SCC cells in culture.,These results suggest a possible contribution of IL-24 to SCC invasion via enhancing focal expression of MMP7, though IL-24 has been suggested to have anti-tumor growth effects in other cancer types.,Identification of regional molecular changes that regulate cancer invasion may facilitate the development of new targeted treatments for aggressive cancer.

The mechanisms by which melanoma and other cancer cells evade anti-tumor immunity remain incompletely understood.,Here, we show that the growth of tumors formed by mutant BrafV600E mouse melanoma cells in an immunocompetent host requires their production of prostaglandin E2, which suppresses immunity and fuels tumor-promoting inflammation.,Genetic ablation of cyclooxygenases (COX) or prostaglandin E synthases in BrafV600E mouse melanoma cells, as well as in NrasG12D melanoma or in breast or colorectal cancer cells, renders them susceptible to immune control and provokes a shift in the tumor inflammatory profile toward classic anti-cancer immune pathways.,This mouse COX-dependent inflammatory signature is remarkably conserved in human cutaneous melanoma biopsies, arguing for COX activity as a driver of immune suppression across species.,Pre-clinical data demonstrate that inhibition of COX synergizes with anti-PD-1 blockade in inducing eradication of tumors, implying that COX inhibitors could be useful adjuvants for immune-based therapies in cancer patients.,•Cyclooxygenase in tumors induces PGE2 that subverts myeloid cell function•COX ablation in tumors enables immune control•COX inhibition synergizes with checkpoint blockade therapy•A COX inflammatory signature is conserved across mouse and human cancer biopsies,Cyclooxygenase in tumors induces PGE2 that subverts myeloid cell function,COX ablation in tumors enables immune control,COX inhibition synergizes with checkpoint blockade therapy,A COX inflammatory signature is conserved across mouse and human cancer biopsies,Cyclooxygenase-driven prostaglandin E2, produced by a variety of tumors, drives malignant growth through successful evasion of type I interferon and/or T-cell-dependent tumor elimination.,A remarkable synergy between cyclooxygenase inhibitors and checkpoint blockade immunotherapy results in tumor eradication.

Oncogenic mutations in the serine/threonine kinase B-RAF are found in 50-70% of malignant melanomas1.,Pre-clinical studies have demonstrated that the B-RAFV600E mutation predicts a dependency on the mitogen activated protein kinase (MAPK) signaling cascade in melanoma1-5-an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials6-8.,However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance9-11.,Identification of resistance mechanisms in a manner that elucidates alternative ‘druggable’ targets may inform effective long-term treatment strategies12.,Here, we expressed ~600 kinase and kinase-related open reading frames (ORFs) in parallel to functionally interrogate resistance to a selective RAF kinase inhibitor.,We identified MAP3K8 (COT/TPL2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAFV600E cell lines.,COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signaling.,Moreover, COT expression is associated with de novo resistance in B-RAFV600E cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibition.,We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting.,Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.

Activating mutations in the RAC1 gene have recently been discovered as driver events in malignant melanoma.,Expression of this gene is associated with melanocyte proliferation, and melanoma cells bearing this mutation are insensitive to BRAF inhibitors such as vemurafenib and dabrafenib, and also may evade immune surveillance due to enhanced expression of PD-L1.,Activating mutations in RAC1 are of special interest, as small molecule inhibitors for the RAC effector p21-activated kinase (PAK) are in late-stage clinical development and might impede oncogenic signaling from mutant RAC1.,In this work, we explore the effects of PAK inhibition on RAC1P29S signaling in zebrafish embryonic development, in the proliferation, survival, and motility of RAC1P29S-mutant human melanoma cells, and on tumor formation and progression from such cells in mice.,We report that RAC1P29S evokes a Rasopathy-like phenotype on zebrafish development that can be blocked by inhibitors of PAK or MEK.,We also found and that RAC1 mutant human melanoma cells are resistant to clinical inhibitors of BRAF but are uniquely sensitive to PAK inhibitors.,These data suggest that suppressing the PAK pathway might be of therapeutic benefit in this type of melanoma.

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.

We previously identified PRAME as a biomarker for metastatic risk in Class 1 uveal melanomas.,In this study, we sought to define a threshold value for positive PRAME expression (PRAME+) in a large dataset, identify factors associated with PRAME expression, evaluate the prognostic value of PRAME in Class 2 uveal melanomas, and determine whether PRAME expression is associated with aberrant hypomethylation of the PRAME promoter.,Among 678 samples analyzed by qPCR, 498 (73.5%) were PRAME- and 180 (26.5%) were PRAME+.,Class 1 tumors were more likely to be PRAME-, whereas Class 2 tumors were more likely to be PRAME+ (P < 0.0001).,PRAME expression was associated with shorter time to metastasis and melanoma specific mortality in Class 2 tumors (P = 0.01 and P = 0.02, respectively).,In Class 1 tumors, PRAME expression was directly associated with SF3B1 mutations (P < 0.0001) and inversely associated with EIF1AX mutations (P = 0.004).,PRAME expression was strongly associated with hypomethylation at 12 CpG sites near the PRAME promoter.,Analyses included PRAME mRNA expression, Class 1 versus Class 2 status, chromosomal copy number, mutation status of BAP1, EIF1AX, GNA11, GNAQ and SF3B1, and genomic DNA methylation status.,Analyses were performed on 555 de-identified samples from Castle Biosciences, 123 samples from our center, and 80 samples from the TCGA.,PRAME is aberrantly hypomethylated and activated in Class 1 and Class 2 uveal melanomas and is associated with increased metastatic risk in both classes.,Since PRAME has been successfully targeted for immunotherapy, it may prove to be a companion prognostic biomarker.

Uveal melanoma patients with a poor prognosis can be detected through genetic analysis of the tumor, which has a very high sensitivity.,A large number of patients with uveal melanoma decide to receive information about their individual risk and therefore routine prognostic genetic testing is being carried out on a growing number of patients.,It is obvious that a positive prediction for recidivism in the future will emotionally burden the respective patients, but research on the psychosocial impact of this innovative method is lacking.,The aim of the current study is therefore to investigate the psychosocial impact (psychological distress and quality of life) of prognostic genetic testing in patients with uveal melanoma.,This study is a non-randomized controlled prospective clinical observational trial.,Subjects are patients with uveal melanoma, in whom genetic testing is possible.,Patients who consent to genetic testing are allocated to the intervention group and patients who refuse genetic testing form the observational group.,Both groups receive cancer therapy and psycho-oncological intervention when needed.,The psychosocial impact of prognostic testing is investigated with the following variables: resilience, social support, fear of tumor progression, depression, general distress, cancer-specific and general health-related quality of life, attitude towards genetic testing, estimation of the perceived risk of metastasis, utilization and satisfaction with psycho-oncological crisis intervention, and sociodemographic data.,Data are assessed preoperatively (at initial admission in the clinic) and postoperatively (at discharge from hospital after surgery, 6-12 weeks, 6 and 12 months after initial admission).,Genetic test results are communicated 6-12 weeks after initial admission to the clinic.,We created optimal conditions for investigation of the psychosocial impact of prognostic genetic testing.,This study will provide information on the course of disease and psychosocial outcomes after prognostic genetic testing.,We expect that empirical data from our study will give a scientific basis for medico-ethical considerations.,The online version of this article (doi:10.1186/s12885-016-2479-7) contains supplementary material, which is available to authorized users.

Diversity between metastatic melanoma tumours in individual patients is known; however, the molecular and genetic differences remain unclear.,To examine the molecular and genetic differences between metastatic tumours, we performed gene-expression profiling of 63 melanoma tumours obtained from 28 patients (two or three tumours/patient), followed by analysis of their mutational landscape, using targeted deep sequencing of 1697 cancer genes and DNA copy number analysis.,Gene-expression signatures revealed discordant phenotypes between tumour lesions within a patient in 50% of the cases.,In 18 of 22 patients (where matched normal tissue was available), we found that the multiple lesions within a patient were genetically divergent, with one or more melanoma tumours harbouring 'private' somatic mutations.,In one case, the distant subcutaneous metastasis of one patient occurring 3 months after an earlier regional lymph node metastasis had acquired 37 new coding sequence mutations, including mutations in PTEN and CDH1.,However, BRAF and NRAS mutations, when present in the first metastasis, were always preserved in subsequent metastases.,The patterns of nucleotide substitutions found in this study indicate an influence of UV radiation but possibly also DNA alkylating agents.,Our results clearly demonstrate that metastatic melanoma is a molecularly highly heterogeneous disease that continues to progress throughout its clinical course.,The private aberrations observed on a background of shared aberrations within a patient provide evidence of continued evolution of individual tumours following divergence from a common parental clone, and might have implications for personalized medicine strategies in melanoma treatment.,Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk

Outcomes for melanoma patients with stage III disease differ widely even within the same subcategory.,Molecular signatures that more accurately predict prognosis are needed to stratify patients according to risk.,Proteomic analyses were used to identify differentially abundant proteins in extracts of surgically excised samples from patients with stage IIIc melanoma lymph node metastases.,Analysis of samples from patients with poor (n = 14, <1 yr) and good (n = 19, >4 yr) survival outcomes identified 84 proteins that were differentially abundant between prognostic groups.,Subsequent selected reaction monitoring analysis verified 21 proteins as potential biomarkers for survival.,Poor prognosis patients are characterized by increased levels of proteins involved in protein metabolism, nucleic acid metabolism, angiogenesis, deregulation of cellular energetics and methylation processes, and decreased levels of proteins involved in apoptosis and immune response.,These proteins are able to classify stage IIIc patients into prognostic subgroups (P < 0.02).,This is the first report of potential prognostic markers from stage III melanoma using proteomic analyses.,Validation of these protein markers in larger patient cohorts should define protein signatures that enable better stratification of stage III melanoma patients.

Melanoma is one of the most aggressive cancers with extremely poor prognosis, and the median survival time for stage IV patients is approximately 6 to 8 months.,Unlike cutaneous melanoma, mucosal melanoma is a rare melanoma subtype among Caucasian patients but its incidence remains as high as 22.6% among Chinese patients.,Screening specific genetic variations is the guideline to select targeted drugs for the treatment of advanced melanoma, whereas the genetic variation spectrum and potential therapeutic targets for mucosal melanoma are largely unclear.,It is urgent to identify promising genetic variants for mucosal melanoma so as to develop effective targeted therapies for this disease.,Tumor samples from 213 Chinese mucosal melanoma patients were involved in this study.,P16INK4a/Cyclin D1/CDK4 copy number was examined using the QuantiGene Plex DNA assay and the correlation between abnormal copy number and clinicopathological parameters was analyzed.,Patient-derived xenograft models (PDX) were performed to detect the effects of CDK4/6 inhibitors on the proliferation of mucosal melanoma cells with altered copy number of CDK4 pathway (CDK4, Cyclin D1 and P16INK4a).,The molecular mechanisms of CDK4/6 inhibitors on the proliferation of mucosal melanoma were analyzed by RNAseq.,Among the 213 samples, the amplification rate of CDK4 and CCND1 was 47.0% and 27.7%, respectively, and the deletion rate of P16INK4a was 57.7%.,Patients with more than one genetic abnormality were up to 81.7%.,CDK4 pathway gene copy number variation was not associated with the prognosis of patients with mucosal melanoma (P > 0.05).,Drug sensitivity tests showed that AT7519, a broad-spectrum CDK inhibitor, and PD0332991, a specific CDK4/6 inhibitor, exhibited higher inhibitory effect on CDK4 signaling pathway abnormal mucosal melanoma cells-derived PDX tumors growth than CDK4 signaling pathway normal ones.,RNA-seq analysis showed that CDK4 inhibitors may affect tumor proliferation through multiple signaling pathways.,Abnormal copy number of cell cycle related genes is frequently found in mucosal melanoma.,CDK4/6 inhibitors significantly suppress the PDX tumor growth with abnormal CDK4 pathway.,CDK4 signaling variations predict the effectiveness of CDK4 inhibitors in mucosal melanoma.,The online version of this article (10.1186/s12967-019-1987-z) contains supplementary material, which is available to authorized users.

de novo fatty acid biosynthesis (DNFA) is a hallmark adaptation of many cancers that supports survival, proliferation, and metastasis.,Here we elucidate previously unexplored aspects of transcription regulation and clinical relevance of DNFA in cancers.,We show that elevated expression of DNFA genes is characteristic of many tumor types and correlates with poor prognosis, especially in melanomas.,Elevated DNFA gene expression depends on the SREBP1 transcription factor in multiple melanoma cell lines.,SREBP1 predominantly binds to the transcription start sites of DNFA genes, regulating their expression by recruiting RNA polymerase II to promoters for productive transcription elongation.,We find that SREBP1-regulated DNFA represents a survival trait in melanoma cells, regardless of proliferative state and oncogenic mutation status.,Indeed, malignant melanoma cells exhibit elevated DNFA gene expression after the BRAF/MEK signaling pathway is blocked (e.g. by BRAF inhibitors), and DNFA expression remains higher in melanoma cells resistant to vemurafenib treatment than in untreated cells.,Accordingly, DNFA pathway inhibition, whether by direct targeting of SREBP1 with antisense oligonucleotides, or through combinatorial effects of multiple DNFA enzyme inhibitors, exerts potent cytotoxic effects on both BRAFi-sensitive and -resistant melanoma cells.,Altogether, these results implicate SREBP1 and DNFA enzymes as enticing therapeutic targets in melanomas.

The abnormal expression of long noncoding RNA- (lncRNA-) MEG3 was clearly identified in a number of malignant tumors, but the specific function of MEG3 remains unknown in malignant melanoma until now.,The research attempts to explore the effects of MEG3 on the growth and metastasis of malignant melanoma.,MEG3 and miR-499-5p expression were determined by qRT-PCR method.,Western blotting assay was applied to detect protein expression.,Luciferase reporter assay was used to assess the correlation between MEG3 and miR-499-5p and between CYLD and miR-499-5p.,Cell growth, cell cycle, and cell apoptosis were examined by CCK-8 assay, EdU assay, and flow cytometry assay, respectively.,The invasion ability of melanoma cells was investigated by wound-healing and Transwell assays.,The effect of MEG3 on growth of melanoma in vivo and cell chemosensitivity was detected by xenograft animal model and CCK-8 assay.,As a result, the expression of MEG3 was decreased in melanoma tissues and cell lines.,The level of MEG3 was significantly associated with poor prognosis.,MEG3 could bind to miR-499-5p and CYLD mRNA contained a binding site of miR-499-5p.,The expression of CYLD was reduced and the level of miR-499-5p was elevated in melanoma tissues and cell lines.,Luciferase reporter assay and western blot assay confirmed that MEG3 regulated the expression of CYLD by sponging miR-499-5p.,Functionally, upregulation of MEG3 inhibited melanoma cell proliferation, invasion, and migration, enhanced melanoma cell apoptosis, arrested melanoma cell cycle, and regulated the expression of E-cadherin, N-cadherin, and cyclin D1 by regulating CYLD expression mediated by sponging miR-499-5p.,Importantly, overexpression of MEG3 suppressed the growth of xenograft tumor and improved chemotherapy sensitivity of A375 cells to cisplatin and 5-FU treatment.,In conclusion, MEG3 has a crucial function in the tumorigenesis of melanoma, and MEG3 may be a potential therapeutic target in the treatment of melanoma.

Previous work identified RMEL3 as a lncRNA with enriched expression in melanoma.,Analysis of The Cancer Genome Atlas (TCGA) data confirmed RMEL3 enriched expression in melanoma and demonstrated its association with the presence of BRAFV600E.,RMEL3 siRNA-mediated silencing markedly reduced (95%) colony formation in different BRAFV600E melanoma cell lines.,Multiple genes of the MAPK and PI3K pathways found to be correlated with RMEL3 in TCGA samples were experimentally confirmed.,RMEL3 knockdown led to downregulation of activators or effectors of these pathways, including FGF2, FGF3, DUSP6, ITGB3 and GNG2.,RMEL3 knockdown induces gain of protein levels of tumor suppressor PTEN and the G1/S cyclin-Cdk inhibitors p21 and p27, as well as a decrease of pAKT (T308), BRAF, pRB (S807, S811) and cyclin B1.,Consistently, knockdown resulted in an accumulation of cells in G1 phase and subG0/G1 in an asynchronously growing population.,Thus, TCGA data and functional experiments demonstrate that RMEL3 is required for MAPK and PI3K signaling, and its knockdown decrease BRAFV600E melanoma cell survival and proliferation.

Due to the poor prognosis of advanced metastatic melanoma, it is crucial to find early biomarkers that help identify which melanomas will metastasize.,By comparing the gene expression data from primary and cutaneous melanoma samples from The Cancer Genome Atlas (TCGA), we identified GPC6 among a set of genes whose expression levels can distinguish between primary melanoma and regional cutaneous/subcutaneous metastases.,Glypicans are thought to play a role in tumor growth by regulating the signaling pathways of Wnt, Hedgehogs, fibroblast growth factors (FGFs), and bone morphogenetic proteins (BMPs).,We showed that GPC6 expression was up-regulated in a melanoma cell line compared to normal melanocytes and in metastatic melanoma compared to primary melanoma.,Furthermore, GPC6 expression was positively correlated with genes largely involved in cell adhesion and migration in both melanoma samples and in RNA-seq samples from other TCGA tumors.,Our results suggest that GPC6 may play a role in tumor metastatic progression.,In TCGA melanoma samples, we also showed that GPC6 expression was negatively correlated with miR-509-3p, which has previously been shown to function as a tumor suppressor in various cancer cell lines.,We overexpressed miR-509-3p in A375 melanoma cells and showed that GPC6 expression was significantly suppressed.,This result suggested that GPC6 was a putative target of miR-509-3p in melanoma.,Together, our findings identified GPC6 as an early biomarker for melanoma metastatic progression, one that can be regulated by miR-509-3p.

Metastatic melanoma is the most deadly type of skin cancer.,Despite the success of immunotherapy and targeted agents, the majority of patients experience disease recurrence upon treatment and die due to their disease.,Long non-coding RNAs (lncRNAs) are a new subclass of non-protein coding RNAs involved in (epigenetic) regulation of cell growth, invasion, and other important cellular functions.,Consequently, recent research activities focused on the discovery of these lncRNAs in a broad spectrum of human diseases, especially cancer.,Additional efforts have been undertaken to dissect the underlying molecular mechanisms employed by lncRNAs.,In this review, we will summarize the growing evidence of deregulated lncRNA expression in melanoma, which is linked to tumor growth and progression.,Moreover, we will highlight specific molecular pathways and modes of action for some well-studied lncRNAs and discuss their potential clinical implications.

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

Treatment of early and multiple cutaneous unresectable recurrences is a major therapeutic problem with around 80% of patients relapsing within 5 years.,For lesions refractory to elective treatments, electrochemotherapy (ECT) involving electroporation combined with antineoplastic drug treatment appears to be a new potential option.,This study was undertaken to analyze the short- and long-term responses of lesions treated with ECT with intravenous injection of bleomycin in melanoma patients with in-transit disease or distant cutaneous metastases.,Between June 2007 and September 2012, 60 patients with relapsed and refractory cutaneous melanoma metastases or in-transit disease underwent 100 courses of ECT with intravenous injection of bleomycin.,Response to treatment was evaluated three months after ECT.,A long-lasting response was defined as no cutaneous or in-transit relapse after a minimum of six months.,Three months after ECT, a complete response was observed in 29 patients (48.4%), a partial response in 23 patients (38.3%) and no change or progressive disease in 8 patients (13.3%).,The objective response rate of all treated lesions was 86.6%.,Thirteen patients (44.8% of complete responders) experienced a long-lasting response after one ECT session and were disease-free after a mean duration of follow-up of 27.5 months.,The favorable outcome obtained in the present study demonstrates that ECT is a reliable, and effective procedure that provides long-term benefit in terms of curative and palliative treatment for unresectable cutaneous lesions without adversely impacting the quality of life of patients.

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.

Skin cancer is the most frequently diagnosed cancer in the fair‐skinned population.,In recent years, the incidence of nonmelanoma skin cancer (NMSC) has been increasing worldwide.,However, there is no epidemiological study on skin cancer in the Asian population.,A prospective cohort study including 140 420 participants was initiated in 1990 for cohort Ⅰ and 1993 for cohort Ⅱ at baseline survey from 11 public health center (PHC) areas.,Of these participants, 284 NMSC cases were diagnosed during the follow‐up period (through 2012 in the Osaka PHC area and 2013 in the other PHC areas).,The Cox proportional hazards model was used to estimate hazard ratios and 95% confidence intervals (CI) for NMSC incidence according to occupational type, lifestyle factors (alcohol consumption, coffee consumption, smoking status, physical activity, and body mass index), and family history of cancer.,Among men, compared with indoor workers, outdoor workers were associated with 2.18 (95% CI, 1.17‐4.04) higher risk of squamous cell carcinoma (SCC) but not of basal cell carcinoma (BCC).,Furthermore, men who have a family history of cancer had 1.99 (95% CI, 1.10‐3.62) higher SCC risk.,In women, we did not observe any association between occupational type and the risk of SCC (1.26; 95% CI, 0.68‐2.32) or BCC (0.74; 95% CI, 0.42‐1.28).,In conclusion, men who are outdoor workers or have a family history of cancer had an increased risk of SCC.,We found an increased risk of squamous cell carcinoma skin cancer in men who were outdoor workers and those with a family history of cancer.,As UV exposure in outdoor workers is prevalent and could be avoided to some extent, reducing exposure to sunlight could serve as a prevention strategy to reduce the prevalence of nonmelanoma skin cancer in the Japanese population.

The study aims to evaluate the effects of miR-136 on the proliferation, apoptosis, and epithelial-mesenchymal transition (EMT) of melanoma cells by targetting premelanosome protein (PMEL) through the Wnt signaling pathway.,After establishment of melanoma mouse models, melanoma (model group) and normal tissues (normal group) were collected.,Immunohistochemistry was performed to determine PMEL protein concentration.,Mouse melanoma cells were assigned into control, blank, negative control (NC), miR-136 mimics, miR-136 inhibitors, siRNA-PMEL, and miR-136 inhibitors + siRNA-PMEL, LiC1 (Wnt signaling pathway activator), and siRNA-PMEL+ LiCl groups.,MTT, Scratch test, Transwell assay, and flow cytometry were performed to measure cell proliferation, migration, invasion, and apoptosis.,Quantitative real-time PCR (qRT-PCR) and Western blotting were performed to evaluate miR-136, PMEL, β-catenin, Wnt3a, Bcl-2, Bax, Caspase, E-cadherin, and N-cadherin expressions.,PMEL is highly expressed in melanoma tissues.,MiR-136, Bax, Caspase, and E-cadherin expressions decreased in the model group, whereas PMEL, β-catenin, Bcl-2, Wnt3a, and N-cadherin expressions increased.,Bax, Caspase, and E-cadherin expressions increased in the miR-136 mimics and siRNA-PMEL groups, whereas the expressions decreased in the miR-136 inhibitors group and LiC1 group.,PMEL, β-catenin, Bcl-2, Wnt3a, and N-cadherin expressions, cell proliferation, migration, and invasion decreased, and the apoptosis rate inceased in the miR-136 mimics and siRNA-PMEL groups; whereas the tendencies were opposite to those in the miR-136 inhibitors group and LiC1 group.,In the siRNA-PMEL+ LiCl group, PMEL expression decreased.,These findings indicated that overexpression of miR-136 inhibits melanoma cell EMT, proliferation, migration, invasion, and promotes apoptosis by targetting PMEL through down-regulation of the Wnt signaling pathway.

Aberrations of protein-coding genes are a focus of cancer genomics; however, the impact of oncogenes on expression of the ∼50% of transcripts without protein-coding potential, including long noncoding RNAs (lncRNAs), has been largely uncharacterized.,Activating mutations in the BRAF oncogene are present in >70% of melanomas, 90% of which produce active mutant BRAFV600E protein.,To define the impacts of oncogenic BRAF on the melanocyte transcriptome, massively parallel cDNA sequencing (RNA-seq) was performed on genetically matched normal human melanocytes with and without BRAFV600E expression.,To enhance potential disease relevance by verifying expression of altered genes in BRAF-driven cancer tissue, parallel RNA-seq was also undertaken of two BRAFV600E-mutant human melanomas.,BRAFV600E regulated expression of 1027 protein-coding transcripts and 39 annotated lncRNAs, as well as 70 unannotated, potentially novel, intergenic transcripts.,These transcripts display both tissue-specific and multi-tissue expression profiles and harbor distinctive regulatory chromatin marks and transcription factor binding sites indicative of active transcription.,Coding potential analysis of the 70 unannotated transcripts suggested that most may represent newly identified lncRNAs.,BRAF-regulated lncRNA 1 (BANCR) was identified as a recurrently overexpressed, previously unannotated 693-bp transcript on chromosome 9 with a potential functional role in melanoma cell migration.,BANCR knockdown reduced melanoma cell migration, and this could be rescued by the chemokine CXCL11.,Combining RNA-seq of oncogene-expressing normal cells with RNA-seq of their corresponding human cancers may represent a useful approach to discover new oncogene-regulated RNA transcripts of potential clinical relevance in cancer.

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.

Current prognostic clinical and morphological parameters are insufficient to accurately predict metastasis in individual melanoma patients.,Several studies have described gene expression signatures to predict survival or metastasis of primary melanoma patients, however the reproducibility among these studies is disappointingly low.,We followed extended REMARK/Gould Rothberg criteria to identify gene sets predictive for metastasis in patients with primary cutaneous melanoma.,For class comparison, gene expression data from 116 patients with clinical stage I/II (no metastasis) and 72 with III/IV primary melanoma (with metastasis) at time of first diagnosis were used.,Significance analysis of microarrays identified the top 50 differentially expressed genes.,In an independent data set from a second cohort of 28 primary melanoma patients, these genes were analyzed by multivariate Cox regression analysis and leave-one-out cross validation for association with development of metastatic disease.,In a multivariate Cox regression analysis, expression of the genes Ena/vasodilator-stimulated phosphoprotein-like (EVL) and CD24 antigen gave the best predictive value (p = 0.001; p = 0.017, respectively).,A multivariate Cox proportional hazards model revealed these genes as a potential independent predictor, which may possibly add (both p = 0.01) to the predictive value of the most important morphological indicator, Breslow depth.,Combination of molecular with morphological information may potentially enable an improved prediction of metastasis in primary melanoma patients.,A strength of the gene expression set is the small number of genes, which should allow easy reevaluation in independent data sets and adequately designed clinical trials.

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.

Preclinical studies suggest that treatment with neoadjuvant immune checkpoint blockade is associated with enhanced survival and antigen-specific T cell responses over adjuvant treatment1; however, optimal regimens have not been defined.,Herein, we report results from a randomized phase II study of neoadjuvant nivolumab versus combined ipilimumab with nivolumab in 23 patients with high-risk resectable melanoma (NCT02519322).,RECIST overall response rates (ORR), pathologic complete response rates (pCR), treatment-related adverse events (trAEs), and immune correlates of response were assessed.,Treatment with combined ipilimumab and nivolumab yielded high response rates (RECIST ORR 73%, pCR 45%) but substantial toxicity (73% grade 3 trAEs), whereas treatment with nivolumab monotherapy yielded modest responses (ORR 25%, pCR 25%) and low toxicity (8% grade 3 trAEs).,Immune correlates of response were identified, demonstrating higher lymphoid infiltrates in responders to both therapies and a more clonal and diverse T cell infiltrate in responders to nivolumab monotherapy.,These results are the first to describe the feasibility of neoadjuvant immune checkpoint blockade in melanoma and emphasize the need for additional studies to optimize treatment regimens and to validate putative biomarkers.

Oncogenic mutations in BRAF and NRAS occur in 70% of melanomas.,In this study, we identify a microRNA, miR-146a, that is highly upregulated by oncogenic BRAF and NRAS.,Expression of miR-146a increases the ability of human melanoma cells to proliferate in culture and form tumors in mice, whereas knockdown of miR-146a has the opposite effects.,We show these oncogenic activities are due to miR-146a targeting the NUMB mRNA, a repressor of Notch signaling.,Previous studies have shown that pre-miR-146a contains a single nucleotide polymorphism (C>G rs2910164).,We find that the ability of pre-miR-146a/G to activate Notch signaling and promote oncogenesis is substantially higher than that of pre-miR-146a/C.,Analysis of melanoma cell lines and matched patient samples indicates that during melanoma progression pre-miR-146a/G is enriched relative to pre-miR-146a/C, resulting from a C-to-G somatic mutation in pre-miR-146a/C.,Collectively, our results reveal a central role for miR-146a in the initiation and progression of melanoma.,DOI:http://dx.doi.org/10.7554/eLife.01460.001,Cancer is a leading cause of death worldwide, and although cancer can have many causes, mutations to a small number of genes are often responsible for a large number of cancer cases.,For example, about 70% of cases of the deadliest form of skin cancer have mutations in two specific genes.,Many cancer-causing genes are regulated by small RNA molecules that impede the function of other genes by blocking the machinery that turns a gene into a functional protein.,However, until recently there was a limited understanding of the role of these microRNAs in the skin cancers caused by the most common mutations.,Now, Forloni et al. have looked at all the microRNAs present in cells carrying a mutated form of the one of these genes, and compared these with the microRNAs found in healthy cells.,A microRNA, called miR-146a, was discovered to be much more common in the cancer cells.,Moreover, production of this microRNA was increased by the two cancer-causing mutations.,Forloni et al. found that increasing production of this microRNA caused human skin cancer cells to grow faster, and also caused tumors to develop in mice.,Reducing the level of miR-146a had the opposite effect.,Forloni et al. also looked at cancer cells taken from individuals at different stages of skin cancer and found that, as the disease progresses, an unprocessed form of miR-146a tends to acquire a mutation, which leads to much higher levels of the active, processed form of miR-146a in the cancer cells.,High levels of miR-146a also switched off a gene that encodes for a protein that, in turn, switches off a protein called NOTCH that is linked to skin cancer.,As such, excess miR-146a actually increases the activation of the NOTCH protein.,These results led Forloni et al. to test if delivering drugs that block the production of miR-146a by inhibiting skin cancer-causing genes, along with other drugs that inhibit the Notch signaling pathway, could be more effective than treatment with either drug on its own.,The combined treatment was very effective against human skin cancer cells and could represent a promising development in the treatment of this disease.,DOI:http://dx.doi.org/10.7554/eLife.01460.002

Cancer stem cells have increased resistance against a variety of anti-tumor treatment modalities.,Vasculogenic mimicry (VM) patterns are present in numerous malignant tumor types, represent the formation of perfusion pathways by tumor cells, and their presence in tumors is associated with adverse outcome.,Earlier we have shown that VM-forming tumor cells in three-dimensional (3D) uveal melanoma cultures have increased resistance against cytotoxic agents and oncolytic herpes simplex virus-mediated destruction.,The purpose of the current study was to explore the possibility that this increased resistance of VM-forming tumor cells is due to a cancer stem cell phenotype.,The expression of cancer stem cell marker cluster of differentiation 271 (CD271) was determined in traditional two-dimensional (2D) and 3D cultures of C918 uveal melanoma cells by fluorescent immunocytochemistry.,We found that the VM-forming tumor cell subpopulation in 3D cultures expressed CD271.,In contrast, cells grown in 2D cultures and tumor cell subpopulations not participating in VM formation in 3D cultures were negative for CD271.,These findings suggest that VM-forming uveal melanoma cells acquire a cancer stem cell-like phenotype that may play a role in the increased therapy resistance of these cells.

Melanoma is currently divided on a genetic level according to mutational status.,However, this classification does not optimally predict prognosis.,In prior studies, we have defined gene expression phenotypes (high-immune, pigmentation, proliferative and normal-like), which are predictive of survival outcome as well as informative of biology.,Herein, we employed a population-based metastatic melanoma cohort and external cohorts to determine the prognostic and predictive significance of the gene expression phenotypes.,We performed expression profiling on 214 cutaneous melanoma tumors and found an increased risk of developing distant metastases in the pigmentation (HR, 1.9; 95% CI, 1.05-3.28; P=0.03) and proliferative (HR, 2.8; 95% CI, 1.43-5.57; P=0.003) groups as compared to the high-immune response group.,Further genetic characterization of melanomas using targeted deep-sequencing revealed similar mutational patterns across these phenotypes.,We also used publicly available expression profiling data from melanoma patients treated with targeted or vaccine therapy in order to determine if our signatures predicted therapeutic response.,In patients receiving targeted therapy, melanomas resistant to targeted therapy were enriched in the MITF-low proliferative subtype as compared to pre-treatment biopsies (P=0.02).,In summary, the melanoma gene expression phenotypes are highly predictive of survival outcome and can further help to discriminate patients responding to targeted therapy.

Adoptive cell therapy may be based on isolation of tumor-specific T cells, e.g. autologous tumor infiltrating lymphocytes (TIL), in vitro activation and expansion and the reinfusion of these cells into patients upon chemotherapy induced lymphodepletion.,Together with high-dose interleukin (IL)-2 this treatment has been given to patients with advanced malignant melanoma and impressive response rates but also significant IL-2 associated toxicity have been observed.,Here we present data from a feasibility study at a Danish Translational Research Center using TIL adoptive transfer in combination with low-dose subcutaneous IL-2 injections.,This is a pilot trial (ClinicalTrials.gov identifier: NCT00937625) including patients with metastatic melanoma, PS ≤1, age <70, measurable and progressive disease and no involvement of the central nervous system.,Six patients were treated with lymphodepleting chemotherapy, TIL infusion, and 14 days of subcutaneous low-dose IL-2 injections, 2 MIU/day.,Low-dose IL-2 considerably decreased the treatment related toxicity with no grade 3-4 IL-2 related adverse events.,Objective clinical responses were seen in 2 of 6 treated patients with ongoing complete responses (30+ and 10+ months), 2 patients had stable disease (4 and 5 months) and 2 patients progressed shortly after treatment.,Tumor-reactivity of the infused cells and peripheral lymphocytes before and after therapy were analyzed.,Absolute number of tumor specific T cells in the infusion product tended to correlate with clinical response and also, an induction of peripheral tumor reactive T cells was observed for 1 patient in complete remission.,Complete and durable responses were induced after treatment with adoptive cell therapy in combination with low-dose IL-2 which significantly decreased toxicity of this therapy.

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.

Metformin is the most widely used antidiabetic drug because of its proven efficacy and limited secondary effects.,Interestingly, recent studies have reported that metformin can block the growth of different tumor types.,Here, we show that metformin exerts antiproliferative effects on melanoma cells, whereas normal human melanocytes are resistant to these metformin-induced effects.,To better understand the basis of this antiproliferative effect of metformin in melanoma, we characterized the sequence of events underlying metformin action.,We showed that 24 h metformin treatment induced a cell cycle arrest in G0/G1 phases, while after 72 h, melanoma cells underwent autophagy as demonstrated by electron microscopy, immunochemistry, and by quantification of the autolysosome-associated LC3 and Beclin1 proteins.,In addition, 96 h post metformin treatment we observed robust apoptosis of melanoma cells.,Interestingly, inhibition of autophagy by knocking down LC3 or ATG5 decreased the extent of apoptosis, and suppressed the antiproliferative effect of metformin on melanoma cells, suggesting that apoptosis is a consequence of autophagy.,The relevance of these observations were confirmed in vivo, as we showed that metformin treatment impaired the melanoma tumor growth in mice, and induced autophagy and apoptosis markers.,Taken together, our data suggest that metformin has an important impact on melanoma growth, and may therefore be beneficial in patients with melanoma.

Patients with malignant melanoma often relapse after treatment with BRAF and/or mitogen‐activated protein kinase kinase (MEK) inhibitors (MEKi) owing to development of drug resistance.,To establish the temporal pattern of CD271 regulation during development of resistance by melanoma to trametinib, and determine the association between development of resistance to trametinib and induction of prosurvival autophagy.,Immunohistochemistry for CD271 and p62 was performed on human naevi and primary malignant melanoma tumours.,Western blotting was used to analyse expression of CD271, p62 and LC3 in melanoma subpopulations.,Flow cytometry and immunofluorescence microscopy was used to evaluate trametinib‐induced cell death and CD271 expression.,MTS viability assays and zebrafish xenografts were used to evaluate the effect of CD271 and autophagy modulation on trametinib‐resistant melanoma cell survival and invasion, respectively.,CD271 and autophagic signalling are increased in stage III primary melanomas vs. benign naevi.,In vitro studies demonstrate MEKi of BRAF‐mutant melanoma induced cytotoxic autophagy, followed by the emergence of CD271‐expressing subpopulations.,Trametinib‐induced CD271 reduced autophagic flux, leading to activation of prosurvival autophagy and development of MEKi resistance.,Treatment of CD271‐expressing melanoma subpopulations with RNA interference and small‐molecule inhibitors to CD271 reduced the development of MEKi resistance, while clinically applicable autophagy modulatory agents - including Δ9‐tetrahydrocannabinol and Vps34 - reduced survival of MEKi‐resistant melanoma cells.,Combined MEK/autophagy inhibition also reduced the invasive and metastatic potential of MEKi‐resistant cells in an in vivo zebrafish xenograft.,These results highlight a novel mechanism of MEKi‐induced drug resistance and suggest that targeting autophagy may be a translatable approach to resensitize drug‐resistant melanoma cells to the cytotoxic effects of MEKi.,What's already known about this topic?,The targeted mitogen‐activated protein kinase kinase (MEK) inhibitor (MEKi) trametinib is used for the treatment of patients with BRAF‐mutant metastatic melanoma in combination with BRAF inhibitors, but many tumours rapidly develop resistance to these drugs.Resistance of melanoma to trametinib has been associated with the induction of prosurvival autophagy, but it is unclear how stem‐cell markers, such as CD271 contribute to the transitionary phase of drug resistance.,The targeted mitogen‐activated protein kinase kinase (MEK) inhibitor (MEKi) trametinib is used for the treatment of patients with BRAF‐mutant metastatic melanoma in combination with BRAF inhibitors, but many tumours rapidly develop resistance to these drugs.,Resistance of melanoma to trametinib has been associated with the induction of prosurvival autophagy, but it is unclear how stem‐cell markers, such as CD271 contribute to the transitionary phase of drug resistance.,What does this study add?,Transient induction of the low‐affinity neurotrophin receptor CD271 is a critical regulator of the adaptive drug‐‐response phase of melanoma cells to trametinib.Genetic or chemical inhibition of CD271 prevents emergence of trametinib‐induced drug‐resistant melanoma subpopulations.Progression to trametinib resistance by melanoma subpopulations results in loss of CD271 expression and subsequent resistance to CD271 inhibitors, but not to the cytotoxic effects of autophagy modulation.,Transient induction of the low‐affinity neurotrophin receptor CD271 is a critical regulator of the adaptive drug‐‐response phase of melanoma cells to trametinib.,Genetic or chemical inhibition of CD271 prevents emergence of trametinib‐induced drug‐resistant melanoma subpopulations.,Progression to trametinib resistance by melanoma subpopulations results in loss of CD271 expression and subsequent resistance to CD271 inhibitors, but not to the cytotoxic effects of autophagy modulation.,What is the translational message?,Autophagy modulation either through inhibition of vacuolar protein sorting 34, or activation of cytotoxic autophagy with tetrahydrocannabinol effectively resensitizes drug‐resistant melanoma cells to trametinib in vitro and in vivo.Combined autophagy modulation and MEK inhibition offers a novel personalized therapeutic strategy to overcome MEKi‐induced drug resistance for patients with BRAF‐mutant metastatic melanoma.,Autophagy modulation either through inhibition of vacuolar protein sorting 34, or activation of cytotoxic autophagy with tetrahydrocannabinol effectively resensitizes drug‐resistant melanoma cells to trametinib in vitro and in vivo.,Combined autophagy modulation and MEK inhibition offers a novel personalized therapeutic strategy to overcome MEKi‐induced drug resistance for patients with BRAF‐mutant metastatic melanoma.,Plain language summary available online,Respond to this article

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 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.

EDEM1 is a mannosidase-like protein that recruits misfolded glycoproteins from the calnexin/calreticulin folding cycle to downstream endoplasmic reticulum associated degradation (ERAD) pathway.,Here, we investigate the role of EDEM1 in the processing of tyrosinase, a tumour antigen overexpressed in melanoma cells.,First, we analyzed and modeled EDEM1 major domains.,The homology model raised on the crystal structures of human and Saccharomyces cerevisiae ER class I α1,2-mannosidases reveals that the major mannosidase domain located between aminoacids 121-598 fits with high accuracy.,We have further identified an N-terminal region located between aminoacids 40-119, predicted to be intrinsically disordered (ID) and susceptible to adopt multiple conformations, hence facilitating protein-protein interactions.,To investigate these two domains we have constructed an EDEM1 deletion mutant lacking the ID region and a triple mutant disrupting the glycan-binding domain and analyzed their association with tyrosinase.,Tyrosinase is a glycoprotein partly degraded endogenously by ERAD and the ubiquitin proteasomal system.,We found that the degradation of wild type and misfolded tyrosinase was enhanced when EDEM1 was overexpressed.,Glycosylated and non-glycosylated mutants co-immunoprecipitated with EDEM1 even in the absence of its intact mannosidase-like domain, but not when the ID region was deleted.,In contrast, calnexin and SEL 1L associated with the deletion mutant.,Our data suggest that the ID region identified in the N-terminal end of EDEM1 is involved in the binding of glycosylated and non-glycosylated misfolded proteins.,Accelerating tyrosinase degradation by EDEM1 overexpression may lead to an efficient antigen presentation and enhanced elimination of melanoma cells.

Cutaneous squamous cell carcinoma (cSCC) is a common skin cancer with genetic susceptibility loci identified in recent genome-wide association studies (GWAS).,Transcriptome-wide association studies (TWAS) using imputed gene expression levels can identify additional gene-level associations.,Here we impute gene expression levels in 6891 cSCC cases and 54,566 controls in the Kaiser Permanente Genetic Epidemiology Research in Adult Health and Aging (GERA) cohort and 25,558 self-reported cSCC cases and 673,788 controls from 23andMe.,In a discovery-validation study, we identify 19 loci containing 33 genes whose imputed expression levels are associated with cSCC at false discovery rate < 10% in the GERA cohort and validate 15 of these candidate genes at Bonferroni significance in the 23andMe dataset, including eight genes in five novel susceptibility loci and seven genes in four previously associated loci.,These results suggest genetic mechanisms contributing to cSCC risk and illustrate advantages and disadvantages of TWAS as a supplement to traditional GWAS analyses.,Genetic loci linked to susceptibility for the common skin cancer cutaneous squamous cell carcinoma (cSCC) have been identified by genome wide association studies (GWAS).,Here, the authors impute gene expression levels from GWAS data to perform a transcriptome wide association study (TWAS), identifying five novel genetic loci linked to cSCC susceptibility.

We report a genome-wide association study of melanoma, conducted by GenoMEL, of 2,981 cases, of European ancestry, and 1,982 study-specific controls, plus a further 6,426 French and UK population controls, all genotyped for 317,000 or 610,000 SNPs.,The analysis confirmed previously known melanoma susceptibility loci.,The 7 novel regions with at least one SNP with p<10−5 and further local imputed or genotyped support were selected for replication using two other genome-wide studies (from Australia and Houston, Texas).,Additional replication came from UK and Dutch case-control series.,Three of the 7 regions replicated at p<10−3: an ATM missense polymorphism (rs1801516, overall p=3.4×10−9); a polymorphism within MX2 (rs45430, p=2.9×10−9) and a SNP adjacent to CASP8 (rs13016963, p=8.6×10−10).,A fourth region near CCND1 remains of potential interest, showing suggestive but inconclusive evidence of replication.,Unlike the previously known regions, the novel loci showed no association with nevus or pigmentation phenotypes in a large UK case-control series.

Recently, it has been described that programmed cell death protein 1 (PD-1) overexpressing melanoma cells are highly aggressive.,However, until now it has not been defined which factors lead to the generation of PD-1 overexpressing subpopulations.,Here, we present that melanoma-derived exosomes, conveying oncogenic molecular reprogramming, induce the formation of a melanoma-like, PD-1 overexpressing cell population (mMSCPD-1+) from naïve mesenchymal stem cells (MSCs).,Exosomes and mMSCPD-1+ cells induce tumor progression and expression of oncogenic factors in vivo.,Finally, we revealed a characteristic, tumorigenic signaling network combining the upregulated molecules (e.g., PD-1, MET, RAF1, BCL2, MTOR) and their upstream exosomal regulating proteins and miRNAs.,Our study highlights the complexity of exosomal communication during tumor progression and contributes to the detailed understanding of metastatic processes.

The melanocortin-1 receptor (MC1R), a G protein-coupled receptor, plays a crucial role in human and mouse pigmentation1-8.,Activation of MC1R in melanocytes by α-melanocyte-stimulating hormone (α-MSH)9 stimulates cAMP signaling and melanin production and enhances DNA repair after UV irradiation (UVR)10-16.,Individuals carrying MC1R variants, especially those associated with red hair color, fair skin and poor tanning ability (RHC-variants), are associated with higher risk of melanoma5,17,18,19,20.,However, how MC1R activity might be modulated by UV irradiation, why redheads are more prone to developing melanoma, and whether the activity of RHC variants might be restored for therapeutic benefit remain unresolved questions.,Here we demonstrate a potential MC1R-targeted intervention strategy to rescue loss-of-function MC1R in MC1R RHC-variants for therapeutic benefit based on activating MC1R protein palmitoylation.,Specifically, MC1R palmitoylation, primarily mediated by the protein-acyl transferase (PAT) ZDHHC13, is essential for activating MC1R signaling that triggers increased pigmentation, UVB-induced G1-like cell cycle arrest and control of senescence and melanomagenesis in vitro and in vivo.,Using C57BL/6J-MC1Re/eJ mice expressing MC1R RHC-variants we show that pharmacological activation of palmitoylation rescues the defects of MC1R RHC-variants and prevents melanomagenesis.,The results highlight a central role for MC1R palmitoylation in pigmentation and protection against melanoma.

Podoplanin (PDPN), a small transmembrane mucin-like glycoprotein, is ectopically expressed.,It is also known to be linked with several aspects of tumor malignancy in some types of human tumors, including invasion, metastasis, and cancer stemness.,However, there are few reports on the expression of dog PDPN (dPDPN) in canine tumors, and the association between dPDPN and tumor malignancy has not been elucidated.,We identified that 11 out of 18 types of canine tumors expressed dPDPN.,Furthermore, 80% of canine malignant melanoma (MM), squamous cell carcinoma, and meningioma expressed dPDPN.,Moreover, the expression density of dPDPN was positively associated with the expression of the Ki67 proliferation marker.,The silencing of dPDPN by siRNAs resulted in the suppression of cell migration, invasion, stem cell-like characteristics, and cell viability in canine MM cell lines.,The suppression of cell viability was caused by the induction of apoptosis and G2/M phase cell cycle arrest.,Overall, this study demonstrates that dPDPN is expressed in various types of canine tumors and that dPDPN silencing suppresses cell viability through apoptosis and cell cycle arrest, thus providing a novel biological role for PDPN in tumor progression.

Podoplanin (PDPN), a transmembrane O-glycoprotein, is up-regulated in many tumors and is involved in tumor metastasis and malignant progression.,In previous studies, we generated a functional blocking monoclonal antibody (mAb, SZ168) against the extracellular domain of human PDPN.,This study is aimed to investigate whether blocking PDPN by SZ168 inhibits tumor growth and metastasis.,Malignant melanoma xenograft model by inoculating subcutaneously human malignant melanoma cell line C8161 into the back of BALB/c nude mice was used.,Endogenous PDPN expression in C8161 cells and nasopharyngeal cancer cell line CNE-2 was detected using western blot and flow cytometry.,SZ168 significantly inhibited C8161 or CNE-2 cell-induced platelet aggregation in a dose-dependent manner with a maximal inhibition of 73.9 ± 3.0% in C8161 cells or 77.1 ± 2.7% in CNE-2 cells.,Moreover, SZ168 inhibited the growth and pulmonary metastasis of C8161cells in vivo.,The number of lung metastatic foci in the SZ168-treated group was significantly decreased compared with that in the control mouse IgG group (1.61 ± 0.44 vs.3.83 ± 0.60, P < 0.01).,Subcutaneous tumor volume, weight, and incidence were also significantly reduced in the SZ168-treated group compared to the control group (P < 0.05).,Additionally, SZ168 recognized PDPN in immunohistochemical analyses of tumor tissue sections.,SZ168 blocks growth and pulmonary metastasis of human malignant melanoma by inhibiting the interaction between tumor PDPN and platelet CLEC-2 and therefore is a promising antibody for therapeutic development against malignant melanoma.

Melanoma is an aggressive malignancy with a poor prognosis.,Current studies show that imatinib treatment is a promising approach in treating advanced melanoma patients harboring c-Kit mutations or amplifications.,We retrospectively analyzed the clinical medical records of 78 patients with metastatic melanoma harboring c-Kit mutations or amplifications.,These patients were treated with imatinib at a dose of 400 mg/day continuously unless intolerable toxicities or disease progression occurred.,Endpoints for exploration included overall survival (OS), progression-free survival (PFS), objective response rate (ORR), and disease of control rate (DCR).,The median OS and PFS of all patients were 13.1 and 4.2 months, respectively.,ORR and DCR were 21.8% and 60.3%, respectively.,The survival time of patients who achieved partial response or stable disease was significantly superior to those with disease progression.,Cox regression analysis showed that patients with M1c stage, subtype of cutaneous melanoma, or elevated LDH level (>upper limit of normal) had higher hazard ratios for overall survival.,Our study, combined with those studies targeting patients with a c-Kit alteration, validates the role of imatinib as an important and promising therapeutic agent in the treatment of patients with advanced melanoma.

Immune checkpoint inhibitors have improved patient survival in melanoma, but the innate resistance of many patients necessitates the investigation of alternative immune targets.,Many immune checkpoint proteins lack proper characterization, including V-domain Ig suppressor of T cell activation (VISTA).,VISTA expression on immune cells can suppress T cell activity; however, few studies have investigated its expression and regulation in cancer cells.,In this study, we observe that VISTA is expressed in melanoma patient samples and cell lines.,Tumor cell-specific expression of VISTA promotes tumor onset in vivo, associated with increased intratumoral T regulatory cells, and enhanced PDL-1 expression on tumor-infiltrating macrophages.,VISTA transcript levels are regulated by the stemness factor Forkhead box D3 (FOXD3).,BRAF inhibition upregulates FOXD3 and reduces VISTA expression.,Overall, this study demonstrates melanoma cell expression of VISTA and its regulation by FOXD3, contributing to the rationale for therapeutic strategies that combine targeted inhibitors with immune checkpoint blockade.

Pembrolizumab demonstrated robust antitumor activity and safety in the phase Ib KEYNOTE-001 study (NCT01295827) of advanced melanoma.,Five-year outcomes in all patients and treatment-naive patients are reported herein.,Patients whose disease progressed following initial response and who received a second course of pembrolizumab were also analyzed.,Patients aged ≥18 years with previously treated or treatment-naive advanced/metastatic melanoma received pembrolizumab 2 mg/kg every 3 weeks, 10 mg/kg every 3 weeks, or 10 mg/kg every 2 weeks until disease progression, intolerable toxicity, or patient/investigator decision to withdraw.,Kaplan-Meier estimates of overall survival (OS) and progression-free survival (PFS) were calculated.,Objective response rate and PFS were based on immune-related response criteria by investigator assessment (data cut-off, September 1, 2017).,KEYNOTE-001 enrolled 655 patients with melanoma; median follow-up was 55 months.,Estimated 5-year OS was 34% in all patients and 41% in treatment-naive patients; median OS was 23.8 months (95% CI, 20.2-30.4) and 38.6 months (95% CI, 27.2-not reached), respectively.,Estimated 5-year PFS rates were 21% in all patients and 29% in treatment-naive patients; median PFS was 8.3 months (95% CI, 5.8-11.1) and 16.9 months (95% CI, 9.3-35.5), respectively.,Median response duration was not reached; 73% of all responses and 82% of treatment-naive responses were ongoing at data cut-off; the longest response was ongoing at 66 months.,Four patients [all with prior response of complete response (CR)] whose disease progressed during observation subsequently received second-course pembrolizumab.,One patient each achieved CR and partial response (after data cut-off).,Treatment-related AEs (TRAEs) occurred in 86% of patients and resulted in study discontinuation in 7.8%; 17% experienced grade 3/4 TRAE.,This 5-year analysis of KEYNOTE-001 represents the longest follow-up for pembrolizumab to date and confirms the durable antitumor activity and tolerability of pembrolizumab in advanced melanoma.,ClinicalTrials.gov, NCT01295827.

The programmed death-1 inhibitor pembrolizumab has demonstrated efficacy and safety in clinical trials for treating advanced (unresectable/metastatic) melanoma.,We investigated the real-world utilization of pembrolizumab and associated patient outcomes for advanced melanoma in US community oncology practices.,This retrospective, observational study used deidentified data from electronic health records for adult patients with advanced melanoma who received pembrolizumab at The US Oncology Network sites from September 2014 through December 2015, with follow-up through September 2016.,Patients enrolled in clinical trials were excluded.,Overall survival (OS) and physician-stated progression-free survival (PFS) were analyzed from pembrolizumab initiation using Kaplan-Meier, and associations between pembrolizumab therapy and OS/PFS, using multivariable Cox regression.,Of 168 patients studied, 110 (65%) were male; the median age was 66 years (range, 26-over 90).,Pembrolizumab was prescribed as first-line, second-line, and third-line/later for 39 (23%), 87 (52%), and 42 (25%) patients, respectively.,In total, 41 patients (24%) had brain metastases.,At pembrolizumab initiation, 21/129 (16%) had Eastern Cooperative Oncology Group performance status (ECOG PS) >1; 51/116 (44%) had elevated lactate dehydrogenase.,Median follow-up was 10.5 months (range, 0-25.1); median OS was 19.4 months (95% confidence interval, 14.0-not reached); median PFS was 4.2 months (95% confidence interval, 2.9-5.3).,Brain metastases, ECOG PS>1, elevated lactate dehydrogenase, and third-line/later (vs. first-line) pembrolizumab were significant predictors (P<0.01) of decreased survival.,Treatment-related toxicity was a discontinuation reason for 25% (29/117) of patients, and for 10 of these 29 patients (6% of the full-study cohort) treatment-related toxicity was the only reported reason.,The real-world effectiveness and safety of pembrolizumab for advanced melanoma are consistent with clinical trial findings.

The current study defines a fibroblast-derived niche that facilitates the therapeutic escape of melanoma cells from BRAF inhibition.,Vemurafenib treatment led to the release of TGF-β from the melanoma cells that increased the differentiation state of the fibroblasts; an affect associated with fibronectin deposition, increase in α-smooth muscle actin (α-SMA) expression and the release of neuregulin (NRG).,At the same time, vemurafenib directly activated the fibroblasts through paradoxical stimulation of the MAPK pathway, causing them to secrete hepatocyte growth factor (HGF).,Treatment with the BRAF/MEK inhibitor combination reversed the release of HGF.,Adhesion of melanoma cells to fibronectin was critical in amplifying the fibroblast-derived NRG and HGF-mediated PI3K/AKT survival signaling in the melanoma cells following BRAF inhibition.,In co-culture studies, combination treatment with inhibitors of BRAF/MET/HER kinase was ineffective at reversing the fibroblast-mediated therapeutic escape from BRAF inhibition.,Instead, it was noted that combined BRAF/PI3K inhibition overcame fibroblast-mediated drug resistance in vitro and was associated with enhanced anti-tumor effects in an in vivo xenograft model.,Thus, we show melanoma cells and fibroblasts remodel their microenvironment in response to BRAF inhibition and that these adaptations allow tumor cells to evade therapy through increased PI3K/AKT survival signaling.

The recurrent BRAF driver mutation V600E (BRAFV600E) is currently one of the most clinically relevant mutations in melanoma.,However, the genome-wide transcriptional and epigenetic dysregulations induced by BRAFV600E are still unclear.,The investigation of this driver mutation’s functional consequences is critical to the understanding of tumorigenesis and the development of therapeutic strategies.,We performed an integrative analysis of transcriptomic and epigenomic changes disturbed by BRAFV600E by comparing the gene expression and methylation profiles of 34 primary cutaneous melanoma tumors harboring BRAFV600E with those of 27 BRAFWT samples available from The Cancer Genome Atlas (TCGA).,A total of 711 significantly differentially expressed genes were identified as putative BRAFV600E target genes.,Functional enrichment analyses revealed the transcription factor MITF (p < 3.6 × 10−16) and growth factor TGFB1 (p < 3.1 × 10−9) were the most significantly enriched up-regulators, with MITF being significantly up-regulated, whereas TGFB1 was significantly down-regulated in BRAFV600E, suggesting that they may mediate tumorigenesis driven by BRAFV600E.,Further investigation using the MITF ChIP-Seq data confirmed that BRAFV600E led to an overall increased level of gene expression for the MITF targets.,Furthermore, DNA methylation analysis revealed a global DNA methylation loss in BRAFV600E relative to BRAFWT.,This might be due to BRAF dysregulation of DNMT3A, which was identified as a potential target with significant down-regulation in BRAFV600E.,Finally, we demonstrated that BRAFV600E targets may play essential functional roles in cell growth and proliferation, measured by their effects on melanoma tumor growth using a short hairpin RNA silencing experimental dataset.,Our integrative analysis identified a set of BRAFV600E target genes.,Further analyses suggested a complex mechanism driven by mutation BRAFV600E on melanoma tumorigenesis that disturbs specific cancer-related genes, pathways, and methylation modifications.,The online version of this article (doi:10.1186/s12943-015-0328-y) contains supplementary material, which is available to authorized users.

The treatment of metastatic melanoma has been revolutionized in the past decade because of the development of immunotherapies and targeted therapies.,Despite these developments, there is still an unmet clinical need for more advanced combination therapies for the subset of patients who remain resistant to immunotherapy or targeted therapy alone.,To our knowledge, no reports have been published on combinations of PD-1 blockades and c-KIT inhibitors in melanoma patients.,Furthermore, data are limited regarding the safety and efficacy of this combination in patients harboring KIT mutations.,We report a case of an 82-year-old female with metastatic melanoma who was found to have double KIT mutations at V559 and N822I.,She was treated with a combination of c-KIT inhibitor and PD-1 blockade after being resistant to anti-PD-1 monotherapy.,Patient developed two episodes of grade 2 liver toxicity requiring treatment breaks followed by a dose reduction.,Her transaminitis eventually resolved and patient remained on combination treatment for almost two years with good control of her disease prior to progression.,Treatment options for patients who progress after PD-1 inhibitors are very limited; therefore, there is a high unmet clinical need for this patient population.,Combining Imatinib with checkpoint inhibitors may be efficacious in patients with metastatic melanoma and KIT mutations.,This novel combination can cause additional toxicities which seem to be overall manageable.

Melanoma of the skin is the sixth most common type of cancer in Europe and accounts for 3.4% of all diagnosed cancers.,More alarming is the degree of recurrence that occurs with approximately 20% of patients lethally relapsing following treatment.,Malignant melanoma is a highly aggressive skin cancer and metastases rapidly extend to the regional lymph nodes (stage 3) and to distal organs (stage 4).,Targeted oncotherapy is one of the standard treatment for progressive stage 4 melanoma, and BRAF inhibitors (e.g. vemurafenib, dabrafenib) combined with MEK inhibitor (e.g. trametinib) can effectively counter BRAFV600E-mutated melanomas.,Compared to conventional chemotherapy, targeted BRAFV600E inhibition achieves a significantly higher response rate.,After a period of cancer control, however, most responsive patients develop resistance to the therapy and lethal progression.,The many underlying factors potentially causing resistance to BRAF inhibitors have been extensively studied.,Nevertheless, the remaining unsolved clinical questions necessitate alternative research approaches to address the molecular mechanisms underlying metastatic and treatment-resistant melanoma.,In broader terms, proteomics can address clinical questions far beyond the reach of genomics, by measuring, i.e. the relative abundance of protein products, post-translational modifications (PTMs), protein localisation, turnover, protein interactions and protein function.,More specifically, proteomic analysis of body fluids and tissues in a given medical and clinical setting can aid in the identification of cancer biomarkers and novel therapeutic targets.,Achieving this goal requires the development of a robust and reproducible clinical proteomic platform that encompasses automated biobanking of patient samples, tissue sectioning and histological examination, efficient protein extraction, enzymatic digestion, mass spectrometry-based quantitative protein analysis by label-free or labelling technologies and/or enrichment of peptides with specific PTMs.,By combining data from, e.g. phosphoproteomics and acetylomics, the protein expression profiles of different melanoma stages can provide a solid framework for understanding the biology and progression of the disease.,When complemented by proteogenomics, customised protein sequence databases generated from patient-specific genomic and transcriptomic data aid in interpreting clinical proteomic biomarker data to provide a deeper and more comprehensive molecular characterisation of cellular functions underlying disease progression.,In parallel to a streamlined, patient-centric, clinical proteomic pipeline, mass spectrometry-based imaging can aid in interrogating the spatial distribution of drugs and drug metabolites within tissues at single-cell resolution.,These developments are an important advancement in studying drug action and efficacy in vivo and will aid in the development of more effective and safer strategies for the treatment of melanoma.,A collaborative effort of gargantuan proportions between academia and healthcare professionals has led to the initiation, establishment and development of a cutting-edge cancer research centre with a specialisation in melanoma and lung cancer.,The primary research focus of the European Cancer Moonshot Lund Center is to understand the impact that drugs have on cancer at an individualised and personalised level.,Simultaneously, the centre increases awareness of the relentless battle against cancer and attracts global interest in the exceptional research performed at the centre.

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.

The clinical use of BRAF inhibitors for treatment of metastatic melanoma is limited by the development of drug resistance.,In this study we investigated whether co-targeting the MAPK and the PI3K-AKT pathway can prevent emergence of resistance or provide additional growth inhibitory effects in vitro.,Anti-tumor effects of the combination of the BRAF inhibitor (BRAFi) dabrafenib and GSK2141795B (AKTi) in a panel of 23 BRAF mutated melanoma cell lines were evaluated on growth inhibition by an ATP-based luminescent assay, on cell cycle and apoptosis by flow cytometry and on cell signaling by western blot.,Moreover, we investigated the possibilities of delaying or reversing resistance or achieving further growth inhibition by combining AKTi with dabrafenib and/or the MEK inhibitor (MEKi) trametinib by using long term cultures.,More than 40% of the cell lines, including PTEN-/- and AKT mutants showed sensitivity to AKTi (IC50 < 1.5 μM).,The combination of dabrafenib and AKTi synergistically potentiated growth inhibition in the majority of cell lines with IC50 > 5 nM dabrafenib.,Combinatorial treatment induced apoptosis only in cell lines sensitive to AKTi.,In long term cultures of a PTEN-/- cell line, combinatorial treatment with the MAPK inhibitors, dabrafenib and trametinib, and AKTi markedly delayed the emergence of drug resistance.,Moreover, combining AKTi with the MAPK inhibitors from the beginning provided superior growth inhibitory effects compared to addition of AKTi upon development of resistance to MAPK inhibitors in this particular cell line.,AKTi combined with BRAFi-based therapy may benefit patients with tumors harboring BRAF mutations and particularly PTEN deletions or AKT mutations.

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.

Magnolol, a hydroxylated biphenol compound isolated from the bark of Magnolia officinalis, has been shown to exhibit anti-proliferative effect in various cancer cells, including skin cancer cells.,Methoxylation of magnolol appears to improve its anti-inflammatory activity, yet the effect of this modification on the agent's antitumor activity remains unknown.,In this work, we report that 2-O-methylmagnolol (MM1) displays improved antitumor activity against skin cancer cells compared to magnolol both in vitro and in vivo.,The increased antitumor activity of MM1 appears to correlate with its increased ability to induce apoptosis.,DNA microarray and network pathway analyses suggest that MM1 affects certain key factors involved in regulating apoptosis and programmed cell death.,Interestingly, the level of the long non-coding (lnc) RNA of growth arrest-specific 5 (GAS5) was increased in MM1-treated cells, and inhibition of lncRNA GAS5 inhibited MM1-induced apoptosis.,Conversely, overexpression of lncRNA GAS5 inhibited cell proliferation and promoted cell apoptosis in skin cancer cells.,The expression of lncRNA GAS5 in the skin cancer tissues was found to be lower than that in the adjacent normal tissues in a majority of patients.,Taken together, our findings suggest that MM1 has improved antitumor activity in skin cancer cells, and that this is due, at least in part, to the upregulation of lncRNA GAS5 and the enhancement of apoptosis.

Preclinical studies suggest that treatment with neoadjuvant immune checkpoint blockade is associated with enhanced survival and antigen-specific T cell responses over adjuvant treatment1; however, optimal regimens have not been defined.,Herein, we report results from a randomized phase II study of neoadjuvant nivolumab versus combined ipilimumab with nivolumab in 23 patients with high-risk resectable melanoma (NCT02519322).,RECIST overall response rates (ORR), pathologic complete response rates (pCR), treatment-related adverse events (trAEs), and immune correlates of response were assessed.,Treatment with combined ipilimumab and nivolumab yielded high response rates (RECIST ORR 73%, pCR 45%) but substantial toxicity (73% grade 3 trAEs), whereas treatment with nivolumab monotherapy yielded modest responses (ORR 25%, pCR 25%) and low toxicity (8% grade 3 trAEs).,Immune correlates of response were identified, demonstrating higher lymphoid infiltrates in responders to both therapies and a more clonal and diverse T cell infiltrate in responders to nivolumab monotherapy.,These results are the first to describe the feasibility of neoadjuvant immune checkpoint blockade in melanoma and emphasize the need for additional studies to optimize treatment regimens and to validate putative biomarkers.

Neoadjuvant immunotherapy utilizing novel combinations has the potential to transform the standard of care for locally/regionally advanced melanoma.,We hypothesized that neoadjuvant ipilimumab in combination with high dose IFNα2b (HDI) is safe and associated with durable pathologic complete responses (pCR).,Patients with locally/regionally advanced melanoma were randomized to ipilimumab 3 or 10 mg/kg × 4 doses bracketing definitive surgery, then every 12 weeks × 4.,HDI was given concurrently.,We evaluated the safety and efficacy of the combination with ipilimumab 3 or 10 mg/kg.,The impact on T-cell fraction and clonality were investigated in tumor and blood.,Thirty patients (age 37-76), 15 each at 3 and 10 mg/kg, 18 male and 12 female were treated.,Considering immune related adverse events (irAEs) of interest, more grade 3/4 irAEs were seen with ipilimumab 10 mg/kg versus 3 mg/kg (p = 0.042).,Among 28 evaluable patients, 11 relapsed, of whom 5 died.,Median follow-up for 17 patients who have not relapsed was 32 months.,The radiologic preoperative response rate was 36% (95% CI, 21-54); 4 patients at ipilimumab 3 mg/kg and 6 at 10 mg/kg and 2 (at 10 mg/kg) later relapsed.,The pCR was 32% (95% CI, 18-51); 5 patients at ipilimumab 3 mg/kg and 4 at 10 mg/kg and one (at 3 mg/kg) had a late relapse.,In patients with pCR, T-cell fraction was significantly higher when measured in primary melanoma tumors (p = 0.033).,Higher tumor T-cell clonality in primary tumor and more so following neoadjuvant therapy was significantly associated with improved relapse free survival.,Neoadjuvant ipilimumab-HDI was relatively safe and exhibited promising tumor response rates with an associated measurable impact on T-cell fraction and clonality.,Most pCRs were durable supporting the value of pCR as a primary endpoint in neoadjuvant immunotherapy trials.,ClinicalTrials.gov, NCT01608594.,Registered 31 May 2012.,The online version of this article (10.1186/s40425-018-0428-5) contains supplementary material, which is available to authorized users.

Autophagy is a resistance mechanism to BRAF/MEK inhibition in BRAFV600-mutant melanoma.,Here we used hydroxychloroquine (HCQ) to inhibit autophagy in combination with dabrafenib 150 mg twice daily and trametinib 2 mg every day (D+T).,We conducted a phase I/II clinical trial in four centers of HCQ + D+T in patients with advanced BRAFV600-mutant melanoma.,The primary objectives were the recommended phase II dose (RP2D) and the one-year progression-free survival (PFS) rate of >53%.,Thirty-four patients were evaluable for one-year PFS rate.,Patient demographics were as follows: elevated lactate dehydrogenase: 47%; stage IV M1c/M1d: 52%; prior immunotherapy: 50%.,In phase I, there was no dose-limiting toxicity.,HCQ 600 mg orally twice daily with D+T was the RP2D.,The one-year PFS rate was 48.2% [95% confidence interval (CI), 31.0%-65.5%], median PFS was 11.2 months (95% CI, 5.4-16.9 months), and response rate (RR) was 85% (95% CI, 64%-95%).,The complete RR was 41% and median overall survival (OS) was 26.5 months.,In a patient with elevated LDH (n = 16), the RR was 88% and median PFS and OS were 7.3 and 22 months, respectively.,HCQ + D+T was well tolerated and produced a high RR but did not meet criteria for success for the one-year PFS rate.,There was a high proportion of patients with pretreated and elevated LDH, an increasingly common demographic in patients receiving targeted therapy.,In this difficult-to-treat population, the RR and PFS were encouraging.,A randomized trial of D+T + HCQ or placebo in patients with BRAFV600-mutant melanoma with elevated LDH and previous immunotherapy is being conducted.

Uveal melanoma (UM) is a highly metastatic cancer that, in contrast to cutaneous melanoma, is largely unresponsive to checkpoint immunotherapy.,Here, we interrogate the tumor microenvironment at single-cell resolution using scRNA-seq of 59,915 tumor and non-neoplastic cells from 8 primary and 3 metastatic samples.,Tumor cells reveal novel subclonal genomic complexity and transcriptional states.,Tumor-infiltrating immune cells comprise a previously unrecognized diversity of cell types, including CD8+ T cells predominantly expressing the checkpoint marker LAG3, rather than PD1 or CTLA4.,V(D)J analysis shows clonally expanded T cells, indicating that they are capable of mounting an immune response.,An indolent liver metastasis from a class 1B UM is infiltrated with clonally expanded plasma cells, indicative of antibody-mediated immunity.,This complex ecosystem of tumor and immune cells provides new insights into UM biology, and LAG3 is identified as a potential candidate for immune checkpoint blockade in patients with high risk UM.,Uveal melanoma is highly metastatic and unresponsive to checkpoint immunotherapy.,Here, the authors present single-cell transcriptomics of 59,915 cells in 8 primary and 3 metastatic samples, highlighting the diversity of the tumour microenvironment.

PD‐1 inhibitors are routinely used for the treatment of advanced melanoma.,This study sought to determine whether PD‐L1 expression on circulating tumor cells (CTCs) can serve as a predictive biomarker of clinical benefit and response to treatment with the PD‐1 inhibitor pembrolizumab.,Blood samples were collected from patients with metastatic melanoma receiving pembrolizumab, prior to treatment and 6-12 weeks after initiation of therapy.,Multiparametric flow cytometry was used to identify CTCs and evaluate the expression of PD‐L1.,CTCs were detected in 25 of 40 patients (63%).,Patients with detectable PD‐L1+ CTCs (14/25, 64%) had significantly longer progression‐free survival (PFS) compared with patients with PD‐L1− CTCs (26.6 months vs.,5.5 months; p = .018).,The 12‐month PFS rates were 76% versus 22% in the PD‐L1+ versus PD‐L1− CTCs groups (p = .012), respectively.,A multivariate linear regression analysis confirmed that PD‐L1+ CTC is an independent predictive biomarker of PFS (hazard ratio, 0.229; 95% confidence interval, 0.052-1.012; p = .026).,Our results reveal the potential of CTCs as a noninvasive real‐time biopsy to evaluate PD‐L1 expression in patients with melanoma.,PD‐L1 expression on CTCs may be predictive of response to pembrolizumab and longer PFS.,The present data suggest that PD‐L1 expression on circulating tumor cells may predict response to pembrolizumab in advanced melanoma.,This needs further validation in a larger trial and, if proven, might be a useful liquid biopsy tool that could be used to stratify patients into groups more likely to respond to immunotherapy, hence leading to health cost savings.,The objective of this study was to determine whether PD‐L1 expression on circulating tumor cells can serve as a predictive biomarker of clinical benefit and response to treatment with the PD‐1 inhibitor pembrolizumab.

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.

Personalized cancer vaccines hold promises for future cancer therapy.,Targeting neoantigens is perceived as more beneficial compared to germline, non-mutated antigens.,However, it is a practical challenge to identify and vaccinate patients with neoantigens.,Here we asked whether two neoantigens are sufficient, and whether the addition of germline antigens would enhance the therapeutic efficacy.,We developed and used a personalized cancer nano-vaccine platform based on virus-like particles loaded with toll-like receptor ligands.,We generated three sets of multi-target vaccines (MTV) to immunize against the aggressive B16F10 murine melanoma: one set based on germline epitopes (GL-MTV) identified by immunopeptidomics, another set based on mutated epitopes (Mutated-MTV) predicted by whole exome sequencing and a last set combines both germline and mutated epitopes (Mix-MTV).,Our results demonstrate that both germline and mutated epitopes induced protection but the best therapeutic effect was achieved with the combination of both.,Our platform is based on Cu-free click chemistry used for peptide-VLP coupling, thus enabling bedside production of a personalized cancer vaccine, ready for clinical translation.

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.

There is growing evidence that tripartite motif-containing protein 44 (TRIM44) plays crucial role in tumor development.,However, the underlying mechanism of this deubiquitinating enzyme remains unclear.,Large clinical samples were used to detect TRIM44 expression and its associations with clinicopathological features and prognosis.,Gain- and loss-of-function experiments in cell lines and mouse xenograft models were performed to elucidate the function and underlying mechanisms of TRIM44 induced tumor progression.,Co-immunoprecipitation (Co-IP) assays and mass spectrometric analyses were applied to verify the interacting proteins of TRIM44.,We found that TRIM44 was commonly amplified in melanoma tissues compared with paratumoral tissues.,TRIM44 expression also positively correlated with more aggressive clinicopathological features, such as Breslow depth (p = 0.025), distant metastasis (p = 0.012), and TNM stage (p = 0.002).,Importantly, we found that TRIM44 was an independent indicator of prognosis for melanoma patients.,Functionally, overexpression of TRIM44 facilitated cell invasion, migration, apoptosis resistance and proliferation in vitro, and promoted lung metastasis and tumorigenic ability in vivo.,Importantly, high level of TRIM44 induced melanoma cell epithelial-mesenchymal transition (EMT), which is one of the most important mechanisms for the promotion of tumor metastasis.,Mechanistically, high levels of TRIM44 increased the levels of p-AKT (T308) and p-mTOR (S2448), and a specific AKT inhibitor inhibited TRIM44-induced tumor progression.,Co-IP assays and mass spectrometric analyses indicated that TRIM44 overexpression induces cell EMT through activating AKT/mTOR pathway via directly binding and stabilizing TOLL-like receptor 4 (TLR4), and TLR4 interference impeded TRIM44 induced tumor progression.,Moreover, we demonstrated that TRIM44 is the target of miR-26b-5p, which is significantly downregulated in melanoma tissues and may be responsible for the overexpression of TRIM44.,TRIM44, regulated by miR-26b-5p, promotes melanoma progression by stabilizing TLR4, which then activates the AKT/mTOR pathway.,TRIM44 shows promise as a prognostic predictor and a therapeutic target for melanoma patients.,The online version of this article (10.1186/s13046-019-1138-7) contains supplementary material, which is available to authorized users.

DNA methylation, an epigenetic alteration typically occurring early in cancer development, could aid in the molecular diagnosis of melanoma.,We determined technical feasibility for high-throughput DNA-methylation array-based profiling using formalin-fixed paraffin-embedded tissues for selection of candidate DNA-methylation differences between melanomas and nevi.,Promoter methylation was evaluated in 27 common benign nevi and 22 primary invasive melanomas using a 1505 CpG site microarray.,Unsupervised hierarchical clustering distinguished melanomas from nevi; 26 CpG sites in 22 genes were identified with significantly different methylation levels between melanomas and nevi after adjustment for age, sex, and multiple comparisons and with β-value differences of ≥0.2.,Prediction analysis for microarrays identified 12 CpG loci that were highly predictive of melanoma, with area under the receiver operating characteristic curves of >0.95.,Of our panel of 22 genes, 14 were statistically significant in an independent sample set of 29 nevi (including dysplastic nevi) and 25 primary invasive melanomas after adjustment for age, sex, and multiple comparisons.,This first report of a DNA-methylation signature discriminating melanomas from nevi indicates that DNA methylation appears promising as an additional tool for enhancing melanoma diagnosis.

Treatment with immune checkpoint blockade (CPB) therapies often leads to prolonged responses in patients with metastatic melanoma, but the common mechanisms of primary and acquired resistance to these agents remain incompletely characterized and have yet to be validated in large cohorts.,By analyzing longitudinal tumor biopsies from 17 metastatic melanoma patients treated with CPB therapies, we observed point mutations, deletions or loss of heterozygosity (LOH) in beta-2-microglobulin (B2M), an essential component of MHC class I antigen presentation, in 29.4% of patients with progressing disease.,In two independent cohorts of melanoma patients treated with anti-CTLA4 and anti-PD1, respectively, we find that B2M LOH is enriched threefold in non-responders (~30%) compared to responders (~10%) and associated with poorer overall survival.,Loss of both copies of B2M is found only in non-responders.,B2M loss is likely a common mechanism of resistance to therapies targeting CTLA4 or PD1.,Resistance to immune-checkpoint blockade often occurs in treated patients.,Here, the authors demonstrate that B2M loss is a mechanism of primary and acquired resistance to therapies targeting CTLA4 or PD-1 in melanoma patients.

As researchers begin probing deep coverage sequencing data for increasingly rare mutations and subclonal events, the fidelity of next generation sequencing (NGS) laboratory methods will become increasingly critical.,Although error rates for sequencing and polymerase chain reaction (PCR) are well documented, the effects that DNA extraction and other library preparation steps could have on downstream sequence integrity have not been thoroughly evaluated.,Here, we describe the discovery of novel C > A/G > T transversion artifacts found at low allelic fractions in targeted capture data.,Characteristics such as sequencer read orientation and presence in both tumor and normal samples strongly indicated a non-biological mechanism.,We identified the source as oxidation of DNA during acoustic shearing in samples containing reactive contaminants from the extraction process.,We show generation of 8-oxoguanine (8-oxoG) lesions during DNA shearing, present analysis tools to detect oxidation in sequencing data and suggest methods to reduce DNA oxidation through the introduction of antioxidants.,Further, informatics methods are presented to confidently filter these artifacts from sequencing data sets.,Though only seen in a low percentage of reads in affected samples, such artifacts could have profoundly deleterious effects on the ability to confidently call rare mutations, and eliminating other possible sources of artifacts should become a priority for the research community.

Melanoma accounts for nearly 80% of all deaths associated with skin cancer.CD147 plays a very important role in melanoma progression and the expression level may correlate with tumor malignancy.,RING1 can bind DNA and act as a transcriptional repressor, play an important role in the aggressive phenotype in melanoma.,The interactions between CD147 and RING1 were identified with a yeast two-hybrid and RING1 interacted with CD147 through the transmembrane domain.,RING1 inhibits CD147’s capability promoting melanoma cell migration.,In conclusion, the study identified novel interactions between CD147 and RING1, recovered CD147 nuclear envelope distribution in melanoma cells, and suggested a new mechanism underlying how cytoplasmic CD147 promotes melanoma development.

Melanoma is the most lethal form of skin cancer and successful treatment of metastatic melanoma remains challenging.,BRAF/MEK inhibitors only show a temporary benefit due to rapid occurrence of resistance, whereas immunotherapy is mainly effective in selected subsets of patients.,Thus, there is a need to identify new targets to improve treatment of metastatic melanoma.,To this extent, we searched for markers that are elevated in melanoma and are under regulation of potentially druggable enzymes.,Here, we show that the pro-proliferative transcription factor FOXM1 is elevated and activated in malignant melanoma.,FOXM1 activity correlated with expression of the enzyme Pin1, which we found to be indicative of a poor prognosis.,In functional experiments, Pin1 proved to be a main regulator of FOXM1 activity through MEK-dependent physical regulation during the cell cycle.,The Pin1-FOXM1 interaction was enhanced by BRAFV600E, the driver oncogene in the majority of melanomas, and in extrapolation of the correlation data, interference with\ Pin1 in BRAFV600E-driven metastatic melanoma cells impaired both FOXM1 activity and cell survival.,Importantly, cell-permeable Pin1-FOXM1-blocking peptides repressed the proliferation of melanoma cells in freshly isolated human metastatic melanoma ex vivo and in three-dimensional-cultured patient-derived melanoids.,When combined with the BRAFV600E-inhibitor PLX4032 a robust repression in melanoid viability was obtained, establishing preclinical value of patient-derived melanoids for prognostic use of drug sensitivity and further underscoring the beneficial effect of Pin1-FOXM1 inhibitory peptides as anti-melanoma drugs.,These proof-of-concept results provide a starting point for development of therapeutic Pin1-FOXM1 inhibitors to target metastatic melanoma.

Small aquarium fish models provide useful systems not only for a better understanding of the molecular basis of many human diseases, but also for first-line screening to identify new drug candidates.,For testing new chemical substances, current strategies mostly rely on easy to perform and efficient embryonic screens.,Cancer, however, is a disease that develops mainly during juvenile and adult stage.,Long-term treatment and the challenge to monitor changes in tumor phenotype make testing of large chemical libraries in juvenile and adult animals cost prohibitive.,We hypothesized that changes in the gene expression profile should occur early during anti-tumor treatment, and the disease-associated transcriptional change should provide a reliable readout that can be utilized to evaluate drug-induced effects.,For the current study, we used a previously established medaka melanoma model.,As proof of principle, we showed that exposure of melanoma developing fish to the drugs cisplatin or trametinib, known cancer therapies, for a period of seven days is sufficient to detect treatment-induced changes in gene expression.,By examining whole body transcriptome responses we provide a novel route toward gene panels that recapitulate anti-tumor outcomes thus allowing a screening of thousands of drugs using a whole-body vertebrate model.,Our results suggest that using disease-associated transcriptional change to screen therapeutic molecules in small fish model is viable and may be applied to pre-clinical research and development stages in new drug discovery.

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.

Despite several therapeutic advances, malignant melanoma still remains a fatal disease for which novel and long-term curative treatments are needed.,The successful development of innovative therapies strongly depends on the availability of appropriate pre-clinical models.,For this purpose, several mouse models holding the promise to provide insight into molecular biology and clinical behavior of melanoma have been generated.,The most relevant ones and their contribution for the advancement of therapeutic approaches for the treatment of human melanoma patients will be here summarized.,However, as models, mice do not recapitulate all the features of human melanoma, thus their strengths and weaknesses need to be carefully identified and considered for the translation of the results into the human clinics.,In this panorama, the concept of comparative oncology acquires a priceless value.,The revolutionary importance of spontaneous canine melanoma as a translational model for the pre-clinical investigation of melanoma progression and treatment will be here discussed, with a special consideration to the development of innovative immunotherapeutic approaches.

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.

Immune-checkpoint blockade (ICB) has demonstrated efficacy in many tumor types, but predictors of responsiveness to anti-PD1 ICB are incompletely characterized.,In this study, we analyzed a clinically annotated cohort of patients with melanoma (n = 144) treated with anti-PD1 ICB, with whole-exome and whole-transcriptome sequencing of pre-treatment tumors.,We found that tumor mutational burden as a predictor of response was confounded by melanoma subtype, whereas multiple novel genomic and transcriptomic features predicted selective response, including features associated with MHC-I and MHC-II antigen presentation.,Furthermore, previous anti-CTLA4 ICB exposure was associated with different predictors of response compared to tumors that were naive to ICB, suggesting selective immune effects of previous exposure to anti-CTLA4 ICB.,Finally, we developed parsimonious models integrating clinical, genomic and transcriptomic features to predict intrinsic resistance to anti-PD1 ICB in individual tumors, with validation in smaller independent cohorts limited by the availability of comprehensive data.,Broadly, we present a framework to discover predictive features and build models of ICB therapeutic response.,Analysis of fully clinically annotated and sequenced melanoma tumor samples collected before anti-PD1 treatment suggests that determinants of response differ on the basis of previous anti-CTLA4 therapy, and that tumor mutational burden may not be a strong predictor of response across melanoma subtypes.

Treatment with immune checkpoint blockade (CPB) therapies often leads to prolonged responses in patients with metastatic melanoma, but the common mechanisms of primary and acquired resistance to these agents remain incompletely characterized and have yet to be validated in large cohorts.,By analyzing longitudinal tumor biopsies from 17 metastatic melanoma patients treated with CPB therapies, we observed point mutations, deletions or loss of heterozygosity (LOH) in beta-2-microglobulin (B2M), an essential component of MHC class I antigen presentation, in 29.4% of patients with progressing disease.,In two independent cohorts of melanoma patients treated with anti-CTLA4 and anti-PD1, respectively, we find that B2M LOH is enriched threefold in non-responders (~30%) compared to responders (~10%) and associated with poorer overall survival.,Loss of both copies of B2M is found only in non-responders.,B2M loss is likely a common mechanism of resistance to therapies targeting CTLA4 or PD1.,Resistance to immune-checkpoint blockade often occurs in treated patients.,Here, the authors demonstrate that B2M loss is a mechanism of primary and acquired resistance to therapies targeting CTLA4 or PD-1 in melanoma patients.

Treatment options are limited for patients with recurrent and/or metastatic (R/M) cutaneous squamous cell carcinoma (cSCC); mortality rates exceed 70% in patients with distant metastases.,Here, we present the first interim analysis of the R/M cSCC cohort from the 2-cohort-locally advanced and R/M-phase II KEYNOTE-629 study.,Patients with R/M cSCC not amenable to surgery or radiation received pembrolizumab 200 mg every 3 weeks.,The primary end point was objective response rate per RECIST v1.1.,Secondary end points were duration of response, disease control rate, progression-free survival, overall survival, and safety.,At data cutoff (April 8, 2019), median follow-up of 105 enrolled patients in the R/M cohort was 11.4 months (range, 0.4 to 16.3 months).,Objective response rate was 34.3% (95% CI, 25.3% to 44.2%; 4 complete responses, 32 partial responses), and disease control rate was 52.4% (95% CI, 42.4% to 62.2%).,Median duration of response was not reached (range, 2.7 to 13.1+ months; ‘+’ refers to ongoing response at data cutoff).,Median progression-free survival was 6.9 months (95% CI, 3.1 months to 8.5 months).,Median overall survival was not reached (95% CI, 10.7 months to not reached).,Treatment-related adverse events occurred in 66.7% of patients (n = 70), the most common of which were pruritus (n = 15; 14.3%), asthenia (n = 14; 13.3%), and fatigue (n = 13; 12.4%).,Grade 3 to 5 treatment-related adverse events occurred in 5.7% (n = 6) of patients.,One patient died of treatment-related cranial nerve neuropathy.,Pembrolizumab demonstrated effective antitumor activity; clinically meaningful, durable responses; and acceptable safety in primarily elderly patients with R/M cSCC, supporting its use in clinical practice.,Pembrolizumab adverse events in this study were consistent with its established safety profile.

Long noncoding RNAs (LncRNAs), including MALAT1, are critical regulators of tumor development.,However, the roles and molecular mechanisms of LncRNAs in cutaneous squamous cell carcinoma (cSCC) remain underexplored.,In this study, functional studies using in vitro cellular and in vivo xenograft models confirmed the pro-carcinogenic roles of MALAT1 in cSCC.,Further, MALAT1 was identified to regulate epidermal growth factor receptor (EGFR) protein expression but did not affect EGFR mRNA expression.,Transcriptomic sequencing identified kinectin 1 (KTN1) as the key mediator for MALAT1 regulation of EGFR.,Mechanistic study revealed that MALAT1 interacts with c-MYC to form a complex and directly binds to the promoter region of KTN1 gene and enhances its transactivation to positively regulate EGFR protein expression.,Our findings, therefore, establish a novel c-MYC-assisted MALAT1-KTN1-EGFR axis, which contributes to cSCC development and may serve as novel target for therapeutic intervention.

Patients with newly resected stage II melanoma (n = 104) were randomized to receive adjuvant vitamin D3 (100,000 IU every 50 days) or placebo for 3 years to investigate vitamin D3 protective effects on developing a recurrent disease.,Median age at diagnosis was 50 years, and 43% of the patients were female.,Median serum 25-hydroxy vitamin D (25OHD) level at baseline was 18 ng/mL, interquartile range (IQ) was 13-24 ng/mL, and 80% of the patients had insufficient vitamin D levels.,We observed pronounced increases in 25OHD levels after 4 months in the active arm (median 32.9 ng/mL; IQ range 25.9-38.4) against placebo (median 19.05 ng/mL; IQ range 13.0-25.9), constantly rising during treatment.,Remarkably, patients with low Breslow score (<3 mm) had a double increase in 25OHD levels from baseline, whereas patients with Breslow score ≥3 mm had a significantly lower increase over time.,After 12 months, subjects with low 25OHD levels and Breslow score ≥3 mm had shorter disease-free survival (p = 0.02) compared to those with Breslow score <3 mm and/or high levels of 25OHD.,Adjusting for age and treatment arm, the hazard ratio for relapse was 4.81 (95% CI: 1.44-16.09, p = 0.011).,Despite the evidence of a role of 25OHD in melanoma prognosis, larger trials with vitamin D supplementation involving subjects with melanoma are needed.

Objective To synthesise the literature on indoor tanning and non-melanoma skin cancer.,Design Systematic review and meta-analysis.,Data sources PubMed (1966 to present), Embase (1974 to present), and Web of Science (1898 to present).,Study selection All articles that reported an original effect statistic for indoor tanning and non-melanoma skin cancer were included.,Articles that presented no data, such as review articles and editorials, were excluded, as were articles in languages other than English.,Data extraction Two investigators independently extracted data.,Random effects meta-analysis was used to summarise the relative risk of ever use versus never use of indoor tanning.,Dose-response effects and exposure to indoor tanning during early life were also examined.,The population attributable risk fraction for the United States population was calculated.,Results 12 studies with 9328 cases of non-melanoma skin cancer were included.,Among people who reported ever using indoor tanning compared with those who never used indoor tanning, the summary relative risk for squamous cell carcinoma was 1.67 (95% confidence interval 1.29 to 2.17) and that for basal cell carcinoma was 1.29 (1.08 to 1.53).,No significant heterogeneity existed between studies.,The population attributable risk fraction for the United States was estimated to be 8.2% for squamous cell carcinoma and 3.7% for basal cell carcinoma.,This corresponds to more than 170 000 cases of non-melanoma skin cancer each year attributable to indoor tanning.,On the basis of data from three studies, use of indoor tanning before age 25 was more strongly associated with both squamous cell carcinoma (relative risk 2.02, 0.70 to 5.86) and basal cell carcinoma (1.40, 1.29 to 1.52).,Conclusions Indoor tanning is associated with a significantly increased risk of both basal and squamous cell skin cancer.,The risk is higher with use in early life (<25 years).,This modifiable risk factor may account for hundreds of thousands of cases of non-melanoma skin cancer each year in the United States alone and many more worldwide.,These findings contribute to the growing body of evidence on the harms of indoor tanning and support public health campaigns and regulation to reduce exposure to this carcinogen.

No standard treatment has been defined for metastatic uveal melanoma (mUM).,Although clinical trials testing Nivolumab/Pembrolizumab for cutaneous melanoma did not include mUM, anti PD-1 agents are commonly used for this disease.,In this prospective observational cohort single arm study, we investigated efficacy and safety of Pembrolizumab as first-line therapy for mUM.,The efficacy was evaluated in terms of progression-free survival (PFS), response rate and overall survival (OS).,Toxicity was also assessed.,Seventeen patients were enrolled.,A median of 8 cycles were administered (range 2-28).,Two patients achieved partial response (11.7%), 6 a disease stabilization (35.3%), whereas 9 (53%) had a progression.,No complete response was observed.,PFS of the overall population was 3.8 months.,PFS was 9.7 months for patients with an interval higher than 5 years from diagnosis of primary tumor to metastatic disease and 2.6 months for patients with an interval lower than 5 years [p = 0.039, HR 0.2865 (95% CI 0.0869-0.9443)].,Median OS was not reached.,The two responding patients were still on treatment with Pembrolizumab at the time of data analysis.,Survival was 12.8 months for patients with clinical benefit, while OS for progressive patients was 3.1 months.,PD-L1 expression and genomic abnormalities predictive of relapse after diagnosis of primary tumor were not associated with PFS.,Toxicity was mild, without grade 3-4 side effects.,The efficacy of Pembrolizumab does not seem particularly different when compared to other agents for mUM, but responding patients had a remarkable disease control.

Metastatic uveal melanoma is a deadly disease with no proven standard of care.,Here we present a metastatic uveal melanoma patient with an exceptional high sensitivity to a PD-1 inhibitor associated with outlier CpG>TpG mutation burden, MBD4 germline deleterious mutation, and somatic MBD4 inactivation in the tumor.,We identify additional tumors in The Cancer Genome Atlas (TCGA) cohorts with similar hypermutator profiles in patients carrying germline deleterious MBD4 mutations and somatic loss of heterozygosity.,This MBD4-related hypermutator phenotype may explain unexpected responses to immune checkpoint inhibitors.,Hypermutated tumors respond more favorably to checkpoint inhibitor-based immune therapy.,Here, the authors describe a new hypermutated phenotype due to germline mutations and subsequent somatic loss of heterozygosity of MBD4, and a dramatic response to the PD-1 inhibitor pembrolizumab in a patient with a MBD4-inactivated hypermutated uveal melanoma.

Molecular signatures specific to particular tumor types are required to design treatments for resistant tumors.,However, it remains unclear whether tumors and corresponding cell lines used for drug development share such signatures.,We developed similarity core analysis (SCA), a universal and unsupervised computational framework for extracting core molecular features common to tumors and cell lines.,We applied SCA to mRNA/miRNA expression data from various sources, comparing melanoma cell lines and metastases.,The signature obtained was associated with phenotypic characteristics in vitro, and the core genes CAPN3 and TRIM63 were implicated in melanoma cell migration/invasion.,About 90% of the melanoma signature genes belong to an intrinsic network of transcription factors governing neural development (TFAP2A, DLX2, ALX1, MITF, PAX3, SOX10, LEF1, and GAS7) and miRNAs (211-5p, 221-3p, and 10a-5p).,The SCA signature effectively discriminated between two subpopulations of melanoma patients differing in overall survival, and classified MEKi/BRAFi-resistant and -sensitive melanoma cell lines.,Cancer cell lines are at the forefront of drug discovery but are often limited in representing the tumor of origin due to the artificial culture conditions.,Rambow et al. develop a computational approach for identifying tumor cell lineage expression cores.,These core genes reveal relevant molecular dependencies linking aggressiveness, patient survival, and drug sensitivity.

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.

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.

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.

Actinic Keratosis (AK), Intraepidermal Carcinoma (IEC), and Squamous Cell Carcinoma (SCC) are generally considered to be advancing stages of the same disease spectrum.,However, while AK often regress spontaneously, and IEC often regress in response to immune-activating treatments, SCC typically do not regress.,Therefore, it is vital to define whether fundamental immunological changes occur during progression to SCC.,Here we show that proinflammatory cytokine expression, chemokine expression, and immune cell infiltration density change during progression to SCC.,Our findings suggest a switch from predominantly proinflammatory cytokine production to chemokine production is a key feature of progression from precancer to cancer.,Together, these observations propose a model that can underpin current research and open new avenues of exploration into the clinical significance of these profiles with respect to immunotherapeutic or other treatment outcomes.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer.,In immunosuppressed populations it is a source of considerable morbidity and mortality due to its enhanced recurrence and metastatic potential.,In common with many malignancies, leucocyte populations are both protective against cancer development and also play a role in ‘sculpting’ the nascent tumor, leading to loss of immunogenicity and tumor progression.,UV radiation and chronic viral carriage may represent unique risk factors for cSCC development, and the immune system plays a key role in modulating the response to both.,In this review, we discuss the lessons learned from animal and ex vivo human studies of the role of individual leucocyte subpopulations in the development of cutaneous SCC.,We then discuss the insights into cSCC immunity gleaned from studies in humans, particularly in populations receiving pharmacological immunosuppression such as transplant recipients.,Similar insights in other malignancies have led to exciting and novel immune therapies, which are beginning to emerge into the cSCC clinical arena.

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.

Circulating cell-free(cf) microRNAs (miRNAs) have been reported to exist in plasma.,MicroRNA-210(miR-210) is known to play important roles in the tumor hypoxic state.,We hypothesized that the expression levels of cf-miR-210 in plasma would predict early clinical recurrence in melanoma patients.,A direct miRNA assay on plasma (RT-qPCR-DP) was developed to improve cf-miRNA assay logistics, eliminate RNA extraction, and reduce specimen amount required.,RNA was extracted from formalin-fixed paraffin-embedded (FFPE) melanoma tissues (n = 108) and assessed by RT-qPCR.,Plasma (10 μl; n = 264) was procured from AJCC Stage III/IV patients in phase III clinical trials.,A RT-qPCR-DP was performed to detect cf-miR-210.,MiR-210 was significantly higher in metastatic tumors compared to primary tumors.,Cf-miR-210 was significantly higher in melanoma patients versus healthy donor controls.,In serial bloods within individual patients, cf-miR-210 < 3 months prior to disease recurrence significantly increased compared to baseline levels (p = 0.012).,ROC curve analysis demonstrated that patients with elevated cf-miR-210 were more likely to have disease recurrence.,Moreover, cf-miR-210 increase significantly correlated with poorer prognosis (p < 0.001).,Lactate dehydrogenase (LDH) level was also assessed within patients, and the AIC values for proportional hazards regression models of cf-miR-210(120.01) and LDH (122.91) demonstrated that cf-miR-210 is a better recurrence indicator.,We concluded enhanced cf-miR-210 provides identification of early systemic melanoma recurrence.

Chemotherapeutic refractoriness of advanced cutaneous melanoma may be linked with melanoma-initiating cells, also known as melanoma stem cells.,This study aimed to determine relative risk of clonal dominance of the CD133+ phenotype in tissues from melanoma patients with different clinical outcomes that could be applied to early diagnosis, prognosis or disease monitoring.,Significant overexpression of CD133 (p<0.02) was observed by immunohistochemical staining in tissues from patients with recurrent disease versus those without disease recurrence.,Relative risk analysis between these two groups suggested that the patients with recurrence or metastatic lesion had a greater than 2-fold overexpression of CD133.,In addition, immunodetectable CD133 corroborated with upregulation of CD133 RNA levels (14- to 30-fold) as assessed by quantitative real-time reverse transcription-PCR (qRT-PCR) comparison of melanoma cell lines derived from patients with poor clinical outcomes and short overall survival (<10 months), vs. those derived from patients with good clinical outcomes and longer overall survival (>24 months).,Further, cells derived from patients, and MACS-sorted according to their CD133 status retained their CD133-positivity (>95%) or CD133-negativity (>95%) for more than 8 passages in culture.,CD133+ cells could repopulate and form tumors (p<0.03) in athymic NCr-nu/nu mice within 8 weeks while no tumors were observed with CD133− phenotype (up to 200,000 cells).,Taken together, the study demonstrates, for the first time, that there exists a clonal dominance of a CD133+ population within the hierarchy of cells in cutaneous tissues from patients that have undergone successive progressive stages of melanoma, from primary to metastatic lesions.,CD133, thus, provides a predictive marker of disease as well as a potential therapeutic target of high-risk melanoma.

Metastatic melanoma is the most aggressive skin cancer.,Recently, phenotypically distinct subpopulations of tumor cells were identified.,Among them, ABCB5-expressing cells were proposed to display an enhanced tumorigenicity with stem cell-like properties.,In addition, ABCB5+ cells are thought to participate to chemoresistance through a potential efflux function of ABCB5.,Nevertheless, the fate of these cells upon drugs that are used in melanoma chemotherapy remains to be clarified.,Here we explored the effect of anti-melanoma treatments on the ABCB5-expressing cells.,Using a melanoma xenograft model (WM266-4), we observed in vivo that ABCB5-expressing cells are enriched after a temozolomide treatment that induces a significant tumor regression.,These results were further confirmed in a preliminary study conducted on clinical samples from patients that received dacarbazine.,In vitro, we showed that ABCB5-expressing cells selectively survive when exposed to dacarbazine, the reference treatment of metastatic melanoma, but also to vemurafenib, a new inhibitor of the mutated kinase V600E BRAF and other various chemotherapeutic drugs.,Our results show that anti-melanoma chemotherapy might participate to the chemoresistance acquisition by selecting tumor cell subpopulations expressing ABCB5.,This is of particular importance in understanding the relapses observed after anti-melanoma treatments and reinforces the interest of ABCB5 and ABCB5-expressing cells as potential therapeutic targets in melanoma.

Merkel cell carcinoma (MCC) is a rare but clinically aggressive cancer with a high mortality rate.,In recent years, antibodies blocking the interactions among PD-1 and its ligands have generated durable tumor regressions in patients with advanced MCC.,However, there is a paucity of data regarding effective therapy for patients whose disease is refractory to PD-1 pathway blockade.,This retrospective case series describes a heterogeneous group of patients treated with additional immune checkpoint blocking therapy after MCC progression through anti-PD-1.,Among 13 patients treated with anti-CTLA-4, alone or in combination with anti-PD-1, objective responses were seen in 4 (31%).,Additionally, one patient with MCC refractory to anti-PD-1 and anti-CTLA-4 experienced tumor regression with anti-PD-L1.,Our report - the largest case series to date describing this patient population - provides evidence that sequentially-administered salvage immune checkpoint blocking therapy can potentially activate anti-tumor immunity in patients with advanced anti-PD-1-refractory MCC and provides a strong rationale for formally testing these agents in multicenter clinical trials.,Additionally, to the best of our knowledge, our report is the first to demonstrate possible anti-tumor activity of second-line treatment with a PD-L1 antibody in a patient with anti-PD-1-refractory disease.,The online version of this article (10.1186/s40425-019-0661-6) contains supplementary material, which is available to authorized users.

Currently, there is no established standard of care for patients with metastatic CSCC.,Based on the mechanisms of CSCC carcinogenesis has been postulated that these tumors may be amenable to PD-1/PD-L1 blockade.,This case illustrates a patient with CSCC with nodal involvement and pulmonary metastases, refractory to two lines of platinum-based regimens and salvage surgery, for whom treatment with nivolumab was recommended.,His clinical course was marked by an atypical pattern of response, with initial reduction of soft tissue/visceral lesions, yet development of new bone findings, followed by overall improvement in subsequent scans and sustained disease control upon treatment continuation.,The case of patient with metastatic CSCC, refractory, received immunotherapy and evolved with atypical pattern of response.

Melanoma is an extremely aggressive malignant skin tumor with a high mortality.,Various long noncoding RNAs (lncRNAs) have been reported to be associated with the oncogenesis of melanoma.,The purposes of this study were to investigate the potential role of lncRNA PVT1 in melanoma progression and to explore its possible mechanisms.,A total of 35 patients who were diagnosed with malignant melanoma were enrolled in this study.,Expression of PVT1 was significantly upregulated in melanoma tissue and was associated with a poor prognosis.,Loss-of-function experiments showed that PVT1 knockdown markedly suppressed the proliferation activity, induced cell cycle arrest at the G0/G1 phase, and enhanced the apoptosis of melanoma cell lines.,Bioinformatics analysis and dual-luciferase reporter assay revealed that PVT1 directly bound to miR-26b, which had been verified to be a tumor suppressor in melanoma.,Moreover, further functional rescue experiments revealed that PVT1 knockdown could observably reverse the tumor-promoting role of the miR-26b inhibitor.,Overall, our study demonstrates the oncogenic role of PVT1 as a miR-26b sponge, possibly providing a novel therapeutic target for melanoma.

Long intergenic nonprotein coding RNA 518 (LINC00518) has been shown to promote cancer cell growth and metastasis in some human tumors.,Although it has been reported that LINC00518 is dysregulated in melanoma, its exact role and molecular mechanism in melanoma remain unclear.,RNA-seq analysis and qRT-PCR was used to detect the expression of LINC00518 in melanoma tissues.,Melanoma cases from The Cancer Genome Atlas (TCGA), GEO#GSE15605 and GEO#GSE24469 were included in this study.,3D migration, transwell and scratch wound assay were used to explore the role of LINC00518 in melanoma cells.,Bioinformatics, luciferase reporter assays, MS2-RIP assay, RNA pull-down assay and RNA-ChIP assay were used to demonstrate the mechanism of LINC00518 in melanoma.,We found that LICN00518 was significantly upregulated in melanoma tissue, and high LICN00518 level was an independent risk factor for melanoma patients.,LICN00518 promoted the invasion and migration of melanoma cells.,LICN00518 exerted its role by decoying miR-204-5p to upregulate Adaptor Related Protein Complex 1 Sigma 2 Subunit (AP1S2) expression.,We also demonstrated that LICN00518 promoted melanoma metastasis in vivo through pulmonary metastasis assay.,This result elucidates a new mechanism for LICN00518 in the metastasis of melanoma.,LICN00518 may serve as a survival indicator and potential therapeutic target in melanoma patients.

Cutaneous melanoma is the deadliest type of skin cancer, characterized by a high molecular and metabolic heterogeneity which contributes to therapy resistance.,Despite advances in treatment, more efficient therapies are needed.,Olive oil compounds have been described as having anti-cancer properties.,Here, we clarified the cytotoxic potential of oleic acid, homovanillyl alcohol, and hydroxytyrosol on melanoma cells.,Metabolic viability was determined 48 h post treatment of A375 and MNT1 cells.,Metabolic gene expression was assessed by qRT-PCR and Mitogen-Activated Protein Kinase (MAPK) activation by Western blot.,Hydroxytyrosol treatment (100 and 200 µM) significantly reduced A375 cell viability (p = 0.0249; p < 0.0001) which, based on the expression analysis performed, is more compatible with a predominant glycolytic profile and c-Jun N-terminal kinase (JNK) activation.,By contrast, hydroxytyrosol had no effect on MNT1 cell viability, which demonstrates an enhanced oxidative metabolism and extracellular signal-regulated kinase (ERK) activation.,This compound triggered cell detoxification and the use of alternative energy sources in A375 cells, inhibiting JNK and ERK pathways.,Despite oleic acid and homovanillyl alcohol demonstrating no effect on melanoma cell viability, they influenced the MNT1 glycolytic rate and A375 detoxification mechanisms, respectively.,Both compounds suppressed ERK activation in MNT1 cells.,The distinct cell responses to olive oil compounds depend on the metabolic and molecular mechanisms preferentially activated.,Hydroxytyrosol may have a cytotoxic potential in melanoma cells with predominant glycolytic metabolism and JNK activation.

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.

Melanoma is a molecularly heterogeneous disease with many genetic mutations and altered signaling pathways.,Activating mutations in the BRAF oncogene are observed in approximately 50% of cutaneous melanomas and the use of BRAF inhibitor (BRAFi) compounds has been reported to improve the outcome of patients with BRAF-mutated metastatic melanoma.,However, the majority of these patients develop resistance within 6-8 months following the initiation of BRAFi treatment.,In this study, we examined the possible use of the poly(ADP-ribose) polymerase 1 (PARP1) inhibitor, ABT-888 (veliparib), as a novel molecule that may be successfully employed in the treatment of BRAFi-resistant melanoma cells.,Sensitive and resistant to BRAFi dabrafenib A375 cells were exposed to increasing concentrations of ABT-888.,Cell viability and apoptosis were assessed by MTT assay and Annexin V-FITC analysis, respectively.,The cell migratory and invasive ability was investigated using the xCELLigence technology and Boyden chamber assays, respectively.,ABT-888 was found to reduce cell viability and exhibited pro-apoptotic activity in melanoma cell lines, independently from the BRAF/NRAS mutation status, in a dose-dependent manner, with the maximal effect being reached in the 25-50 µM concentration range.,Moreover, ABT-888 promoted apoptosis in both the sensitive and resistant A375 cells, suggesting that ABT-888 may be useful in the treatment of BRAFi-resistant subsets of melanoma cells.,Finally, in accordance with the involvement of PARP1 in actin cytoskeletal machinery, we found that the cytoskeletal organization, motility and invasive capability of both the A375 and A375R cells decreased upon exposure to 5 µM ABT-888 for 24 h.,On the whole, the findings of this study highlight the pivotal role of PARP1 in the migration and invasion of melanoma cells, suggesting that ABT-888 may indeed be effective, not only as a pro-apoptotic drug for use in the treatment of BRAFi-resistant melanoma cells, but also in suppressing their migratory and invasive activities.

Notoriously resistant malignant melanoma is one of the most increasing forms of cancer worldwide; there is thus a precarious need for new treatment options.,The Wee1 kinase is a major regulator of the G2/M checkpoint, and halts the cell cycle by adding a negative phosphorylation on CDK1 (Tyr15).,Additionally, Wee1 has a function in safeguarding the genome integrity during DNA synthesis.,To assess the role of Wee1 in development and progression of malignant melanoma we examined its expression in a panel of paraffin-embedded patient derived tissue of benign nevi and primary- and metastatic melanomas, as well as in agarose-embedded cultured melanocytes.,We found that Wee1 expression increased in the direction of malignancy, and showed a strong, positive correlation with known biomarkers involved in cell cycle regulation: Cyclin A (p<0.0001), Ki67 (p<0.0001), Cyclin D3 (p = 0.001), p21Cip1/WAF1 (p = 0.003), p53 (p = 0.025).,Furthermore, high Wee1 expression was associated with thicker primary tumors (p = 0.001), ulceration (p = 0.005) and poor disease-free survival (p = 0.008).,Transfections using siWee1 in metastatic melanoma cell lines; WM239WTp53, WM45.1MUTp53 and LOXWTp53, further support our hypothesis of a tumor promoting role of Wee1 in melanomas.,Whereas no effect was observed in LOX cells, transfection with siWee1 led to accumulation of cells in G1/S and S phase of the cell cycle in WM239 and WM45.1 cells, respectively.,Both latter cell lines displayed DNA damage and induction of apoptosis, in the absence of Wee1, indicating that the effect of silencing Wee1 may not be solely dependent of the p53 status of the cells.,Together these results reveal the importance of Wee1 as a prognostic biomarker in melanomas, and indicate a potential role for targeted therapy, alone or in combination with other agents.

Patients with primary cutaneous melanomas that are ulcerated and >2 mm in thickness, >4 mm in thickness and those with nodal micrometastases at diagnosis, have few options for adjuvant treatment.,Recent studies have suggested a role for vitamin D to delay melanoma recurrence and improve overall prognosis.,This is a pilot placebo-controlled randomised phase II trial to assess the feasibility, safety and toxicity of an oral loading dose of Vitamin D (500,000 IU) followed by an oral dose of 50,000 IU of Vitamin D monthly for 2 years in patients who have been treated for cutaneous melanoma by wide excision of the primary.,Patients aged 18 - 79 years who have completed primary surgical treatment and have Stage IIb, IIc, IIIa (N1a, N2a) or IIIb (N1a, N2a) disease are eligible for randomisation 2:1 to active treatment or placebo.,The primary endpoints are sufficiency of dose, adherence to study medication and safety of the drug.,The secondary endpoints are participation and progression free survival.,The study has been approved by the Ethics Review Committee (RPAH Zone) of the Sydney Local Health District, protocol number X09-0138.,Effective, non-toxic adjuvant therapy for high risk primary melanoma is not currently available.,Favorable outcomes of this phase II study will form the basis for a multi-centre phase III study to assess whether the addition of oral high-dose vitamin D therapy in patients who have completed primary treatment for melanoma and are at high risk of recurrence will:prolong time to recurrence within 5 yearsimprove overall survival at 5 years andbe both safe and tolerable.,prolong time to recurrence within 5 years,improve overall survival at 5 years and,be both safe and tolerable.,62 patients have been randomised since the study commenced in December 2010.,Target accrual for the study has been met with 75 patients randomised between December 2010 and August 2014.,The Mel-D trial is conducted by the Australia and New Zealand Melanoma Trials Group (ANZMTG 02.09),Australia and New Zealand Clinical Trials Registry (ANZCTR) ACTRN12609000351213

The interplay between a non-lethal autophagic response and apoptotic cell death is still a matter of debate in cancer cell biology.,In the present study performed on human melanoma cells, we investigate the role of basal or stimulated autophagy in cisplatin-induced cytotoxicity, as well as the contribution of cisplatin-induced activation of caspases 3/7 and conventional calpains.,The results show that, while down-regulating Beclin-1, Atg14 and LC3-II, cisplatin treatment inhibits the basal autophagic response, impairing a physiological pro-survival response.,Consistently, exogenously stimulated autophagy, obtained with trehalose or calpains inhibitors (MDL-28170 and calpeptin), protects from cisplatin-induced apoptosis, and such a protection is reverted by inhibiting autophagy with 3-methyladenine or ATG5 silencing.,In addition, during trehalose-stimulated autophagy, the cisplatin-induced activation of calpains is abrogated, suggesting the existence of a feedback loop between the autophagic process and calpains.,On the whole, our results demonstrate that in human melanoma cells autophagy may function as a beneficial stress response, hindered by cisplatin-induced death mechanisms.,In a therapeutic perspective, these findings suggest that the efficacy of cisplatin-based polychemotherapies for melanoma could be potentiated by inhibitors of autophagy.

Recent reports have confirmed highest levels of growth hormone (GH) receptor (GHR) transcripts in melanoma, one of the most aggressive forms of human cancer.,Yet the mechanism of GH action in melanoma remains mostly unknown.,Here, using human malignant melanoma cells, we examined the effects of GH excess or siRNA mediated GHR knock-down (GHRKD) on tumor proliferation, migration and invasion.,GH promoted melanoma progression while GHRKD attenuated the same.,Western blot analysis revealed drastic modulation of multiple oncogenic signaling pathways (JAK2, STAT1, STAT3, STAT5, AKT, mTOR, SRC and ERK1/2) following addition of GH or GHRKD.,Further, we show that GH excess upregulates expression of markers of epithelial mesenchymal transition in human melanoma, while the effects were reversed by GHRKD.,Interestingly, we observed consistent expression of GH transcript in the melanoma cells as well as marked modulation of the IGF receptors and binding proteins (IGF1R, IGF2R, IR, IGFBP2, IGFBP3) and the oncogenic HGF-MET mRNA, in response to excess GH or GHRKD.,Our study thus identifies the mechanistic model of GH-GHR action in human melanoma and validates it as an important pharmacological target of intervention.

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.

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.

Icaritin (IT) is a flavonoid isolated from Herba Epimedii.,In this study, we evaluated the anti-melanoma activities of IT, and determined its cytotoxic mechanism.,We found that IT exerted cytotoxicity to melanoma cells.,Furthermore, IT induced melanoma cell apoptosis, which was accompanied with PARP cleavage.,Mechanistically, IT suppressed p-STAT3 (tyr705) level in parallel with increases of p-STAT3 (ser727), p-ERK and p-AKT.,IT significantly inhibited STAT3 nuclear translocation and reduced the levels of STAT3 -targeted genes.,IT also inhibited IGF-1-induced STAT3 activation through down-regulation of total IGF-1R level.,No dramatic changes in IGF-1R mRNA levels were observed in IT-treated cells, suggesting that IT acted primarily at a post-transcriptional level.,Using molecular docking analysis, IT was identified as a novel fatty acid synthase (FASN) inhibitor.,We found that IT reduced the level of total IGF-1R via FASN inhibition.,In summary, we reported that IT exerted anti-melanoma activities, and these effects were partially due to inhibition of FASN/IGF-1R/STAT3 signaling.

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.

The optimal treatment sequence for patients with advanced BRAF V600 mutant melanoma is unknown.,BRAF/MEK inhibition (BRAF/MEKi), single agent anti‐PD‐1 (aPD‐1) antibodies and combination immune checkpoint inhibition with nivolumab and ipilimumab (niv/ipi) are all approved; however, they have not been prospectively compared.,Therefore, we sought to compare overall survival of patients with advanced BRAF mutant melanoma treated with either front‐line BRAF/MEKi, aPD‐1, or niv/ipi.,Patients with advanced BRAF mutant melanoma who had received BRAF/MEKi, niv/ipi, or aPD‐1 in the front‐line setting were identified from a nationwide database comprising de‐identified patient‐level structured and unstructured data derived from electronic health records.,Survival was compared using Kaplan‐Meier curves and log‐rank analysis.,Univariate and multivariate Cox regression models were used to measure the effect of front‐line treatment, age (>64 or not), LDH (elevated or not), and Eastern Cooperative Oncology Group (ECOG) performance status (>1 or not) on survival.,Five hundred and sixty seven patients with advanced disease and treated with front‐line aPD‐1 (n = 162), BRAF/MEKi (n = 297) or niv/ipi (n = 108) were identified.,With a median follow‐up of 22.4 months, median overall survival (OS) for patients treated with front‐line niv/ipi was not reached (NR) while median OS for patients treated with aPD‐1 or BRAF/MEKi was 39.5 months and 13.2 months, respectively.,Front‐line treatment with PD‐1 and niv/ipi were associated with statistically longer survival than BRAF/MEKi in multivariate analyses.,In our real‐world retrospective analysis, patients with advanced BRAF mutant melanoma treated with front‐line niv/ipi or aPD‐1 had longer survival compared to those treated with front‐line BRAF/MEKi.,Real‐world overall survival of patients with advanced BRAF mutant melanoma treated with front‐line BRAF/MEK inhibitors, anti‐PD‐1 antibodies, or nivolumab/ipilimumab.

Clinical benefit from programmed cell death 1 receptor (PD-1) inhibitors relies on reinvigoration of endogenous antitumor immunity.,Nonetheless, robust immunological markers, based on circulating immune cell subsets associated with therapeutic efficacy are yet to be validated.,We isolated peripheral blood mononuclear cell from three independent cohorts of melanoma and Merkel cell carcinoma patients treated with PD-1 inhibitor, at baseline and longitudinally after therapy.,Using multiparameter flow cytometry and cell sorting, we isolated four subsets of CD8+ T cells, based on PD-1 and TIGIT expression profiles.,We performed phenotypic characterization, T cell receptor sequencing, targeted transcriptomic analysis and antitumor reactivity assays to thoroughly characterize each of these subsets.,We documented that the frequency of circulating PD-1+TIGIT+ (DPOS) CD8+ T-cells after 1 month of anti-PD-1 therapy was associated with clinical response and overall survival.,This DPOS T-cell population was enriched in highly activated T-cells, tumor-specific and emerging T-cell clonotypes and T lymphocytes overexpressing CXCR5, a key marker of the CD8 cytotoxic follicular T cell population.,Additionally, transcriptomic profiling defined a specific gene signature for this population as well as the overexpression of specific pathways associated with the therapeutic response.,Our results provide a convincing rationale for monitoring this PD-1+TIGIT+ circulating population as an early cellular-based marker of therapeutic response to anti-PD-1 therapy.

Among Immunotherapeutic approaches for cancer treatment, the adoptive transfer of antigen specific T cells is still a relevant approach, that could have higher efficacy when further combined with immune check-point blockade.,A high number of adoptive transfer trials have been performed in metastatic melanoma, due to its high immunogenic potential, either with polyclonal TIL or antigen-specific polyclonal populations.,In this setting, the extensive characterization of T cell functions and receptor diversity of infused polyclonal T cells is required, notably for monitoring purposes.,We developed a clinical grade procedure for the selection and amplification of polyclonal CD8 T cells, specific for two shared and widely expressed melanoma antigens: Melan-A and MELOE-1.,This procedure is currently used in a clinical trial for HLA-A2 metastatic melanoma patients.,In this study, we characterized the T-cell diversity (T-cell repertoire) of such T cell populations using a new RNAseq strategy.,We first assessed the added-value of TCR receptor sequencing, in terms of sensitivity and specificity, by direct comparison with cytometry analysis of the T cell populations labeled with anti-Vß-specific antibodies.,Results from these analyzes also confirmed specific features already reported for Melan-A and MELOE-1 specific T cell repertoires in terms of V-alpha recurrence usage, on a very high number of T cell clonotypes.,Furthermore, these analyses also revealed undescribed features, such as the recurrence of a specific motif in the CDR3α region for MELOE-1 specific T cell repertoire.,Finally, the analysis of a large number of T cell clonotypes originating from various patients revealed the existence of public CDR3α and ß clonotypes for Melan-A and MELOE-1 specific T cells.,In conclusion, this method of high throughput TCR sequencing is a reliable and powerful approach to deeply characterize polyclonal T cell repertoires, and to reveal specific features of a given TCR repertoire, that would be useful for immune follow-up of cancer patients treated by immunotherapeutic approaches.

Taking into consideration that the immune system plays a very important role in the development of melanoma and non-melanoma skin cancers, which have a high prevalence in immunosuppressed patients and after prolonged ultraviolet radiation, the interest in developing novel therapies, in particular targeting the inflammation in cancer, has increased in the past years.,The latest data suggest that therapies such as imiquimod (IMQ), ingenol mebutate (IM), 5-fluorouracil (5-FU), retinoids, and nonsteroidal anti-inflammatory drugs (NSAIDs) have been used with success in the topical treatment of some cancers.,Herein, we review the topical treatment targeting the inflammation in skin cancer and the mechanisms involved in these processes.,Currently, various associations have shown a superior success rate than monotherapy, such as systemic acitretin and topical IMQ, topical 5-FU with tretinoin cream, or IMQ with checkpoint inhibitor cytotoxic T lymphocyte antigen 4.,Novel therapies targeting Toll-like receptor-7 (TLR-7) with higher selectivity than IMQ are also of great interest.

Photodynamic therapy (PDT) is a widely used technique for epithelial skin cancer treatment. 5-aminolevulinic acid (5-ALA) is a drug currently used for PDT and is a hydrophilic molecule at its physiological pH, and this limits its capacity to cross the stratum corneum of skin.,Since skin penetration is a key factor in the efficacy of topical 5-ALA-mediated PDT, numerous strategies have been proposed to improve skin penetration.,Yet this problem is still ongoing.,The results of a previous study showed a low rate of 5-ALA encapsulated in liposomes (5.7%) that were 400 nm in size.,In the present study, we used 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes as vehicles and tested their delivery efficacy of 5-ALA-medicated PDT both in vitro and in vivo.,Our data shows that 5-ALA encapsulated in 0.1 or 0.5% DPPC liposomes (5-ALA/DPPC) had a better encapsulated rate (15~16%) and were smaller in size (84~89 nm).,We found the 5-ALA/DPPC formulation reduced cell viability, mitochondria membrane potential, and enhanced intracellular ROS accumulation as compared to 5-ALA alone in melanoma cells.,Furthermore, the 5-ALA/DPPC formulation also had better skin penetration ability as compared to the 5-ALA in our ex vivo data by assaying 5-ALA converted into protoporphyrin IX (PpIX) in the skin of the mice that were experimented on.,In melanoma xenograft models, 5-ALA/DPPC enhanced PpIX accumulation only in tumor tissue but not normal skin.,In conclusion, we found DPPC liposomes to be good carriers for 5-ALA delivery and believe that they may prove useful in 5-ALA-mediated PDT in the future.

Common acquired melanocytic nevi are benign neoplasms that are composed of small uniform melanocytes and typically present as flat or slightly elevated, pigmented lesions on the skin.,We describe two families with a new autosomal dominant syndrome characterized by multiple skin-colored, elevated melanocytic tumors.,In contrast to common acquired nevi, the melanocytic neoplasms in affected family members ranged histopathologically from epithelioid nevi to atypical melanocytic proliferations that showed overlapping features with melanoma.,Some affected patients developed uveal or cutaneous melanomas.,Segregating with this phenotype, we found inactivating germline mutations of the BAP1 gene.,The majority of melanocytic neoplasms lost the remaining wild-type allele of BAP1 by various somatic alterations.,In addition, we found BAP1 mutations in a subset of sporadic melanocytic neoplasms showing histologic similarities to the familial tumors.,These findings suggest that loss of BAP1 is associated with a clinically and morphologically distinct type of melanocytic neoplasm.

Uveal melanoma is an aggressive cancer that metastasizes to the liver in about half of the patients, with a high lethality rate.,Identification of patients at high risk of metastases may provide indication for a frequent follow-up for early detection of metastases and treatment.,The analysis of the gene expression profiles of primary human uveal melanomas showed high expression of SDCBP gene (encoding for syndecan-binding protein-1 or mda-9/syntenin), which appeared higher in patients with recurrence, whereas expression of syndecans was lower and unrelated to progression.,Moreover, we found that high expression of SDCBP gene was related to metastatic progression in two additional independent datasets of uveal melanoma patients.,More importantly, immunohistochemistry showed that high expression of mda-9/syntenin protein in primary tumors was significantly related to metastatic recurrence in our cohort of patients.,Mda-9/syntenin expression was confirmed by RT-PCR, immunofluorescence and immunohistochemistry in cultured uveal melanoma cells or primary tumors.,Interestingly, mda-9/syntenin showed both cytoplasmic and nuclear localization in cell lines and in a fraction of patients, suggesting its possible involvement in nuclear functions.,A pseudo-metastatic model of uveal melanoma to the liver was developed in NOD/SCID/IL2Rγ null mice and the study of mda-9/syntenin expression in primary and metastatic lesions revealed higher mda-9/syntenin in metastases.,The inhibition of SDCBP expression by siRNA impaired the ability of uveal melanoma cells to migrate in a wound-healing assay.,Moreover, silencing of SDCBP in mda-9/syntenin-high uveal melanoma cells inhibited the hepatocyte growth factor (HGF)-triggered invasion of matrigel membranes and inhibited the activation of FAK, AKT and Src.,Conversely syntenin overexpression in mda-9/syntenin-low uveal melanoma cells mediated opposite effects.,These results suggest that mda-9/syntenin is involved in uveal melanoma progression and that it warrants further investigation as a candidate molecular marker of metastases and a potential therapeutic target.

Tumor-associated macrophages (TAMs) have been detected in most skin cancers.,TAMs produce various chemokines and angiogenic factors that promote tumor development, along with other immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and tumor-associated neutrophils.,TAMs generated from monocytes develop into functional, fully activated macrophages, and TAMs obtain various immunosuppressive functions to maintain the tumor microenvironment.,Since TAMs express PD1 to maintain the immunosuppressive M2 phenotype by PD1/PD-L1 signaling from tumor cells, and the blockade of PD1/PD-L1 signaling by anti-PD1 antibodies (Abs) activate and re-polarize TAMs into immunoreactive M1 phenotypes, TAMs represent a potential target for anti-PD1 Abs.,The main population of TAMs comprises CD163+ M2 macrophages, and CD163+ TAMs release soluble (s)CD163 and several proinflammatory chemokines (CXCL5, CXCL10, CCL19, etc.) as a result of TAM activation to induce an immunosuppressive tumor microenvironment together with other immunosuppressive cells.,Since direct blockade of PD1/PD-L1 signaling between tumor cells and tumor-infiltrating T cells (both effector T cells and Tregs) is mandatory for inducing an anti-immune response by anti-PD1 Abs, anti-PD1 Abs need to reach the tumor microenvironment to induce anti-immune responses in the tumor-bearing host.,Taken together, TAM-related factors could offer a biomarker for anti-PD1 Ab-based immunotherapy.,Understanding the crosstalk between TAMs and immunosuppressive cells is important for optimizing PD1 Ab-based immunotherapy.

The culture supernatant from macrophages overexpressing TRIM59 has a cytotoxic effect on melanoma, but the mechanism remains unclear.,To investigate whether deletion of TRIM59 in macrophages affects the metastatic potential of melanoma cells, we polarized control and TRIM59-deficient bone marrow-derived macrophages to the M2 phenotype and collected the respective conditioned media (CM).,Exposure to CM from TRIM59-/--M2 cultures significantly promoted migration and invasion by B16-F0 and B16-F10 cells.,Cytokine profiling indicated a ~15-fold increase in TNF-α production in CM from TRIM59-/--M2 cultures, and neutralizing TNF-α activity abrogated the referred stimulatory effects on cell motility.,Transcriptome analysis revealed significant upregulation of MMP-9 and Madcam1 in melanoma cells exposed to TRIM59-/--M2 CM.,Inhibitory experiments determined that these changes were also TNF-α-dependent and mediated by activation of ERK signaling.,Independent knockdown of MMP9 and Madcam1 in B16-F10 cells impeded epithelial-mesenchymal transition and inhibited subcutaneous tumor growth and formation of metastatic lung nodules in vivo.,These data suggest TRIM59 expression attenuates the tumor-promoting effect of tumor-associated macrophages, most of which resemble the M2 phenotype.,Moreover, they highlight the relevance of TRIM59 in macrophages as a potential regulator of tumor metastasis and suggest TRIM59 could serve as a novel target for cancer immunotherapy.

We characterized the mutational landscape of melanoma, the form of skin cancer with the highest mortality rate, by sequencing the exomes of 147 melanomas.,Sun-exposed melanomas had markedly more ultraviolet (UV)-like C>T somatic mutations compared to sun-shielded acral, mucosal and uveal melanomas.,Among the newly identified cancer genes was PPP6C, encoding a serine/threonine phosphatase, which harbored mutations that clustered in the active site in 12% of sun-exposed melanomas, exclusively in tumors with mutations in BRAF or NRAS.,Notably, we identified a recurrent UV-signature, an activating mutation in RAC1 in 9.2% of sun-exposed melanomas.,This activating mutation, the third most frequent in our cohort of sun-exposed melanoma after those of BRAF and NRAS, changes Pro29 to serine (RAC1P29S) in the highly conserved switch I domain.,Crystal structures, and biochemical and functional studies of RAC1P29S showed that the alteration releases the conformational restraint conferred by the conserved proline, causes an increased binding of the protein to downstream effectors, and promotes melanocyte proliferation and migration.,These findings raise the possibility that pharmacological inhibition of downstream effectors of RAC1 signaling could be of therapeutic benefit.

The paradoxical coexistence of spontaneous tumor antigen-specific immune responses with progressive disease in cancer patients furthers the need to dissect the molecular pathways involved in tumor-induced T cell dysfunction.,In patients with advanced melanoma, we have previously shown that the cancer-germline antigen NY-ESO-1 stimulates spontaneous NY-ESO-1-specific CD8+ T cells that up-regulate PD-1 expression.,We also observed that PD-1 regulates NY-ESO-1-specific CD8+ T cell expansion upon chronic antigen stimulation.,In the present study, we show that a fraction of PD-1+ NY-ESO-1-specific CD8+ T cells in patients with advanced melanoma up-regulates Tim-3 expression and that Tim-3+PD-1+ NY-ESO-1-specific CD8+ T cells are more dysfunctional than Tim-3−PD-1+ and Tim-3−PD-1− NY-ESO-1-specific CD8+ T cells, producing less IFN-γ, TNF, and IL-2.,Tim-3-Tim-3L blockade enhanced cytokine production by NY-ESO-1-specific CD8+ T cells upon short ex vivo stimulation with cognate peptide, thus enhancing their functional capacity.,In addition, Tim-3-Tim-3L blockade enhanced cytokine production and proliferation of NY-ESO-1-specific CD8+ T cells upon prolonged antigen stimulation and acted in synergy with PD-1-PD-L1 blockade.,Collectively, our findings support the use of Tim-3-Tim-3L blockade together with PD-1-PD-L1 blockade to reverse tumor-induced T cell exhaustion/dysfunction in patients with advanced melanoma.

Brain metastases are common in patients with melanoma, and optimal management is not well defined.,As melanoma has traditionally been thought of as “radioresistant,” the role of whole brain radiation therapy (WBRT) in particular is unclear.,We conducted this retrospective study to identify prognostic factors for patients treated with stereotactic radiosurgery (SRS) for melanoma brain metastases and to investigate the role of additional up-front treatment with whole brain radiation therapy (WBRT).,We reviewed records of 147 patients who received SRS as part of initial management of their melanoma brain metastases from January 2000 through June 2010.,Overall survival (OS) and time to distant intracranial progression were calculated using the Kaplan-Meier method.,Prognostic factors were evaluated using the Cox proportional hazards model.,WBRT was employed with SRS in 27% of patients and as salvage in an additional 22%.,Age at SRS > 60 years (hazard ratio [HR] 0.64, p = 0.05), multiple brain metastases (HR 1.90, p = 0.008), and omission of up-front WBRT (HR 2.24, p = 0.005) were associated with distant intracranial progression on multivariate analysis.,Extensive extracranial metastases (HR 1.86, p = 0.0006), Karnofsky Performance Status (KPS) ≤ 80% (HR 1.58, p = 0.01), and multiple brain metastases (HR 1.40, p = 0.06) were associated with worse OS on univariate analysis.,Extensive extracranial metastases (HR 1.78, p = 0.001) and KPS (HR 1.52, p = 0.02) remained significantly associated with OS on multivariate analysis.,In patients with absent or stable extracranial disease, multiple brain metastases were associated with worse OS (multivariate HR 5.89, p = 0.004), and there was a trend toward an association with worse OS when up-front WBRT was omitted (multivariate HR 2.56, p = 0.08).,Multiple brain metastases and omission of up-front WBRT (particularly in combination) are associated with distant intracranial progression.,Improvement in intracranial disease control may be especially important in the subset of patients with absent or stable extracranial disease, where the competing risk of death from extracranial disease is low.,These results are hypothesis generating and require confirmation from ongoing randomized trials.

Brain metastases (BMs) are common in melanoma patients.,Adjuvant whole brain radiotherapy (WBRT) following local treatment of intracranial melanoma metastases with neurosurgery and/or stereotactic radiosurgery is controversial.,A randomised trial is needed.,However, accrual to WBRT trials has been problematic.,A pilot study by Australia and New Zealand Melanoma Trials Group (ANZMTG) was conducted to see if accrual was feasible.,Sites canvassed for interest included those who treat melanoma patients, had a proven accrual in previous melanoma trials and who had the relevant infrastructure support.,Feasibility forecasts from interested sites were sought.,These were compared to the patient numbers documented in the research contracts.,A target accrual of 60 patients in 2 years was set.,Funding was sought for the pilot study.,Basic demographics of the pilot study cohort were collected.,The first centre opened December 2008; the first patient was randomised in April 2009.,The pilot accruing period concluded in September, 2011.,In 30 months, 54 patients from 10 of a total of 17 activated sites in Australia (39, 72%) and in Norway (15, 28%) had been accrued.,Feasibility forecasts predicted 133 trial eligible patients per year (including 108 Australian + 25 International patients).,Site estimates generally overestimated accrual with 4 of 17 active sites estimating within 50% of target numbers.,Sites with patient estimates calculated from records were more accurate than those estimated from memory.,The overall recruitment target was lower in the research contracts when compared to the feasibility evaluation.,Basic demographics of the pilot study revealed 62% of patients were males; 58% had a single metastasis, 28% had two and 14% had three metastases. 12-month overall survival was 50%.,Despite only 54 patients and not the full 60 patient target being accrued in two years the Trial Management Committee and Data Safely Monitoring Committee approved the continuation of the pilot study to the main trial.,On the basis of this successful pilot study, funding was achieved for the full study. 143 patients of a target of 200 have been randomised by June 2014.

Cutaneous squamous cell carcinoma (cSCC) has a high tumour mutational burden (50 mutations per megabase DNA pair).,Here, we combine whole-exome analyses from 40 primary cSCC tumours, comprising 20 well-differentiated and 20 moderately/poorly differentiated tumours, with accompanying clinical data from a longitudinal study of immunosuppressed and immunocompetent patients and integrate this analysis with independent gene expression studies.,We identify commonly mutated genes, copy number changes and altered pathways and processes.,Comparisons with tumour differentiation status suggest events which may drive disease progression.,Mutational signature analysis reveals the presence of a novel signature (signature 32), whose incidence correlates with chronic exposure to the immunosuppressive drug azathioprine.,Characterisation of a panel of 15 cSCC tumour-derived cell lines reveals that they accurately reflect the mutational signatures and genomic alterations of primary tumours and provide a valuable resource for the validation of tumour drivers and therapeutic targets.,It is known cutaneous squamous cell carcinoma (cSCC) involves a high tumour mutation burden.,Here the authors identify common cSCC mutated genes, copy number changes, altered pathways and report the presence of a novel mutation signature associated with chronic exposure to the immunosuppressive drug azathioprine.

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.

Immunotherapy targeting immune checkpoint molecules, programmed cell death 1 (PD-1) and PD-ligand 1 (PD-L1), using therapeutic antibodies has been widely used for some human malignancies in the last 5 years.,A costimulatory receptor, PD-1, is expressed on T cells and suppresses effector functions when it binds to its ligand, PD-L1.,Aberrant PD-L1 expression is reported in various human cancers and is considered an immune escape mechanism.,Antibodies blocking the PD-1/PD-L1 axis induce antitumour responses in patients with malignant melanoma and other cancers.,In dogs, no such clinical studies have been performed to date because of the lack of therapeutic antibodies that can be used in dogs.,In this study, the immunomodulatory effects of c4G12, a canine-chimerised anti-PD-L1 monoclonal antibody, were evaluated in vitro, demonstrating significantly enhanced cytokine production and proliferation of dog peripheral blood mononuclear cells.,A pilot clinical study was performed on seven dogs with oral malignant melanoma (OMM) and two with undifferentiated sarcoma.,Objective antitumour responses were observed in one dog with OMM (14.3%, 1/7) and one with undifferentiated sarcoma (50.0%, 1/2) when c4G12 was given at 2 or 5 mg/kg, every 2 weeks. c4G12 could be a safe and effective treatment option for canine cancers.

A xenogeneic DNA vaccination has been licensed for use in dogs with locally controlled stage II and III oral malignant melanoma (OMM).,At present, there are limited outcome data for dogs with OMM treated with surgery and immunotherapy.,The aim of this study is to retrospectively review the outcome and survival of 32 dogs affected by OMM that were treated with a combination of surgery and the xenogeneic DNA vaccination (with the addition of radiotherapy in some cases) and to determine the influence of surgical margins and delay in receiving vaccination.,The overall median survival time (MST) was 335 days (95% CI: 301-540 days), and the overall median progression-free survival (PFS) was 160 days (mean 182 days, 95% CI: 132-232 days).,Stage, completeness of surgical margins and delay in administration of the vaccine did not appear to statistically influence survival or PFS, although these results may reflect the low statistical power of the study due to small numbers.,Further studies are required to assess whether the addition of any adjuvant treatment to surgery, including immunotherapy, is able to significantly prolong survival in cases of canine oral melanoma.

Circulating tumor DNA (ctDNA) may serve as a surrogate to tissue biopsy for noninvasive identification of mutations across multiple genetic loci and for disease monitoring in melanoma.,In this study, we compared the mutation profiles of tumor biopsies and plasma ctDNA from metastatic melanoma patients using custom sequencing panels targeting 30 melanoma‐associated genes.,Somatic mutations were identified in 20 of 24 melanoma biopsies, and 16 of 20 (70%) matched‐patient plasmas had detectable ctDNA.,In a subgroup of seven patients for whom matching tumor tissue and plasma were sequenced, 80% of the mutations found in tumor tissue were also detected in ctDNA.,However, TERT promoter mutations were only detected by ddPCR, and promoter mutations were consistently found at lower concentrations than other driver mutations in longitudinal samples.,In vitro experiments revealed that mutations in promoter regions of TERT and DPH3 are underrepresented in ctDNA.,While the results underscore the utility of using ctDNA as an alternative to tissue biopsy for genetic profiling and surveillance of the disease, our study highlights the underrepresentation of promoter mutations in ctDNA and its potential impact on quantitative liquid biopsy applications.

Metastatic melanoma is a highly heterogeneous tumor; thus, methods to analyze tumor-derived cells circulating in blood should address this diversity.,Taking this into account, we analyzed, using multiparametric flow cytometry, the co-expression of the melanoma markers melanoma cell adhesion molecule and melanoma-associated chondroitin sulphate proteoglycan and the tumor-initiating markers ATP-binding cassette sub-family B member 5 (ABCB5), CD271, and receptor activator of NF-κβ (RANK) in individual circulating tumor cells (CTCs) from 40 late-stage (III-IV) and 16 early-stage (I-II) melanoma patients.,CTCs were heterogeneous within and between patients, with limited co-expression between the five markers analyzed.,Analysis of patient matched blood and metastatic tumors revealed that ABCB5 and RANK subpopulations are more common among CTCs than in the solid tumors, suggesting a preferential selection for these cells in circulation.,Pairwise comparison of CTC subpopulations longitudinally before and 6-13 weeks after treatment initiation showed that the percentage of RANK+ CTCs significantly increased in the patients undergoing targeted therapy (N=16, P<0.01).,Moreover, the presence of ⩾5 RANK+ CTCs in the blood of patients undergoing targeted therapies was prognostic of shorter progression-free survival (hazards ratio 8.73, 95% confidence interval 1.82-41.75, P<0.01).,Taken together, our results provide evidence of the heterogeneity among CTC subpopulations in melanoma and the differential response of these subpopulations to targeted therapy.

Metastatic melanoma is the most aggressive form of this cancer.,It is important to understand factors that increase or decrease metastatic activity in order to more effectively research and implement treatments for melanoma.,Increased cell invasion through the extracellular matrix is required for metastasis and is enhanced by matrix metalloproteinases (MMPs).,Tissue inhibitor of metalloproteinases 3 (TIMP3) inhibits MMP activity.,It was previously shown by our group that miR-21, a potential regulator of TIMP3, is over-expressed in cutaneous melanoma.,It was therefore hypothesized that increased levels of miR-21 expression would lead to decreased expression of TIMP3 and thereby enhance the invasiveness of melanoma cells. miR-21 over-expression in the melanoma cell lines WM1552c, WM793b, A375 and MEL 39 was accomplished via transfection with pre-miR-21.,Immunoblot analysis of miR-21-overexpressing cell lines revealed reduced expression of TIMP3 as compared to controls.,This in turn led to a significant increase in the invasiveness of the radial growth phase cell line WM1552c and the vertical growth phase cell line WM793b (p < 0.05), but not in the metastatic cell lines A375 or MEL 39.,The proliferation and migration of miR-21 over-expressing cell lines was not affected.,Reduced expression of TIMP3 was achieved by siRNA knockdown and significantly enhanced invasion of melanoma cell lines, mimicking the effects of miR-21 over-expression.,Treatment of tumor cells with a linked nucleic acid antagomir to miR-21 inhibited tumor growth and increased tumor expression of TIMP3 in vivo in 01B74 Athymic NCr-nu/nu mice.,Intra-tumoral injections of anti-miR-21 produced similar effects.,This data shows that increased expression of miR-21 enhanced the invasive potential of melanoma cell lines through TIMP3 inhibition.,Therefore, inhibition of miR-21 in melanoma may reduce melanoma invasiveness.

The Microphthalmia associated transcription factor (Mitf) is an important regulator in melanocyte development and has been shown to be involved in melanoma progression.,The current model for the role of Mitf in melanoma assumes that the total activity of the protein is tightly regulated in order to secure cell proliferation.,Previous research has shown that regulation of Mitf is complex and involves regulation of expression, splicing, protein stability and post-translational modifications.,Here we show that microRNAs (miRNAs) are also involved in regulating Mitf in melanoma cells.,Sequence analysis revealed conserved binding sites for several miRNAs in the Mitf 3′UTR sequence.,Furthermore, miR-148 was shown to affect Mitf mRNA expression in melanoma cells through a conserved binding site in the 3′UTR sequence of mouse and human Mitf.,In addition we confirm the previously reported effects of miR-137 on Mitf.,Other miRNAs, miR-27a, miR-32 and miR-124 which all have conserved binding sites in the Mitf 3′UTR sequence did not have effects on Mitf.,Our data show that miR-148 and miR-137 present an additional level of regulating Mitf expression in melanocytes and melanoma cells.,Loss of this regulation, either by mutations or by shortening of the 3′UTR sequence, is therefore a likely factor in melanoma formation and/or progression.

Excessive UV exposure is considered the major environmental factor in melanoma progression.,Human skin is constantly exposed to selected tryptophan-derived aryl hydrocarbon receptor (AhR) ligands, including kynurenine (KYN) and kynurenic acid (KYNA), as they are endogenously produced and present in various tissues and body fluids.,Importantly, recent studies confirmed the biological activity of KYN and KYNA toward melanoma cells in vitro.,Thus, in this study, the potential biological interactions between UVB and tryptophan metabolites KYN and KYNA were studied in melanoma A375, SK-MEL-3, and RPMI-7951 cells.,It was shown that UVB enhanced the antiproliferative activity of KYN and KYNA in melanoma cells.,Importantly, selected tryptophan-derived AhR ligands did not affect the invasiveness of A375 and RPMI-7951 cells; however, the stimulatory effect was observed in SK-MEL-3 cells exposed to UVB.,Thus, the effect of tryptophan metabolites on metabolic activity, cell cycle regulation, and cell death in SK-MEL-3 cells exposed to UVB was assessed.,In conclusion, taking into account that both UVB radiation and tryptophan-derived AhR ligands may have a crucial effect on skin cancer formation and progression, these results may have a significant impact, revealing the potential biological interactions in melanoma cells in vitro.

Background and objectives: Melanin, which has a confirmed role in melanoma cell behaviour, is formed in the process of melanogenesis and is synthesized from tryptophan, L-tyrosine and their metabolites.,All these metabolites are easily detectable by chromatography in urine.,Materials and Methods: Urine samples of 133 individuals (82 malignant melanoma patients and 51 healthy controls) were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC).,The diagnosis of malignant melanoma was confirmed histologically.,Results: Chromatograms of melanoma patients showed increased levels of 5,6-dihydroxyindole-2-carboxylic acid, vanilmandelic acid, homovanilic acid, tryptophan, 5-hydroxyindole-3-acetic acid, and indoxyl sulphate compared to healthy controls.,Concentration of indoxyl sulphate, homovanilic acid and tryptophan were significantly increased even in the low clinical stage 0 of the disease (indoxyl sulphate, homovanilic acid and tryptophan in patients with clinical stage 0 vs. controls expressed as medium/ interquartile range in µmol/mmol creatinine: 28.37/15.30 vs.,5.00/6.91; 47.97/33.08 vs.,7.33/21.25; and 16.38/15.98 vs.,3.46/6.22, respectively).,Conclusions: HPLC detection of metabolites of L-tyrosine and tryptophan in the urine of melanoma patients may play a significant role in diagnostics as well as a therapeutic strategy of melanoma cancer.

Different types of tumor‐infiltrating immune cells not only augment but also dampen antitumor immunity in the microenvironment of melanoma.,Therefore, it is critical to provide an overview of tumor‐infiltrating immune cells in melanoma and explore a novel strategy for immunotherapies.,We analyzed the immune states of different stages in melanoma patients by the immune, stromal, and estimation of stromal and immune cells in malignant tumor tissues using expression data (ESTIMATE) scores.,Immune cell types were identified by the estimating relative subsets of RNA transcripts (CIBERSORTx) algorithm in 471 melanoma and 324 healthy tissues.,Moreover, we performed a gene set variation analysis (GSVA) to determine the differentially regulated pathways in the tumor microenvironment.,In melanoma cohorts, we found that ESTIMATE and immune scores were involved in survival or tumor clinical stage.,Among the 22 immune cells, CD8+ T cells, M2 macrophages, and regulatory T cells (Tregs) showed significant differences using the CIBERSORTx algorithm.,Furthermore, GSVA identified the immune cell‐related pathways; the primary immunodeficiency pathway, intestinal immune network for IgA, and TGF‐β pathways were identified as participants of the crosstalk in CD8+ T cells, Tregs, and M2 macrophages in the melanoma microenvironment.,These results reveal the cellular and molecular characteristics of immune cells in melanoma, providing a method for selecting targets of immunotherapies and promoting the efficacy of therapies for the treatment of melanoma.,Immunotherapy has shown excellent responses in melanoma, while the reaction is low.,However, the molecular mechanisms of tumor infiltrated immune cells have not been explored.,In this study, we found that higher ESTIMATE and immune scores were associated with a clinical‐stage in melanoma patients and also filtered the crosstalks between cells that immune cells.,Our work revealed the immune cellular and molecular characteristics of melanoma, providing a method for selecting targets promoting immunotherapy efficacy.

Cisplatin is a potent antitumor agent.,However, toxicity and primary and secondary resistance are major limitations of cisplatin-based chemotherapy, leading to therapeutic failure.,We have previously reported that mono-sulfonamide platinum complexes have good antitumor activity against different tumoral cell lines and with a different and better cytotoxic profile than cisplatin.,Besides, N-sulfonamides have been used extensively in medicinal chemistry as bactericides, anticonvulsant, inhibitors of the carbonic anhydrase, inhibitors of histone deacetylases, and inhibitors of microtubule polymerization, among others.,We aimed to compare the cytotoxic effects of cisplatin and a trans-sulfonamide-platinum-complex (TSPC), in two human melanoma cell lines that differ in their TP53 status: SK-MEL-5, TP53 wild type, and SK-MEL-28, TP53 mutated.,We performed cytotoxicity assays with both drugs, alone and in combination, cell cycle analyses, western blotting and immunoprecipitation, and fluorescence immunocytochemistry.,TSPC had similar antiproliferative activity than cisplatin against SK-MEL-5 (3.24 ± 1.08 vs 2.89 ± 1.12 μM) and higher against SK-MEL-28 cells (5.83 ± 1.06 vs 10.17 ± 1.29 μM).,Combination of both drugs inhibited proliferation in both cell lines, being especially important in SK-MEL-28, and showing a synergistic effect.,In contrast to cisplatin, TSPC caused G1 instead G2/M arrest in both cell lines.,Our present findings indicate that the G1 arrest is associated with the induction of CDKN1A and CDKN1B proteins, and that this response is also present in melanoma cells containing TP53 mutated.,Also, strong accumulation of CDKN1A and CDKN1B in cells nuclei was seen upon TSPC treatment in both cell lines.,Overall, these findings provide a new promising TSPC compound with in vitro antitumor activity against melanoma cell lines, and with a different mechanism of action from that of cisplatin.,Besides, TSPC synergism with cisplatin facilitates its potential use for co-treatment to reduce toxicity and resistance against cisplatin.,TSPC remains a promising lead compound for the generation of novel antineoplastic agent and to explore its synergism with other DNA damaging agents.

Epithelial-to-mesenchymal transition (EMT), in which epithelial cells loose their polarity and become motile mesenchymal cells, is a determinant of melanoma metastasis.,We compared gene expression signatures of mesenchymal-like melanoma cells with those of epithelial-like melanoma cells, and identified Thrombospondin 1 (THBS1) as highly up-regulated in the mesenchymal phenotype.,This study investigated whether THBS1, a major physiological activator of transforming growth factor (TGF)-beta, is involved in melanoma EMT-like process.,We sought to examine expression patterns in distinct melanoma phenotypes including invasive, de-differentiated, label-retaining and drug resistant populations that are putatively associated with an EMT-like process.,Here we show that THBS1 expression and secretion was elevated in melanoma cells exhibiting invasive, drug resistant, label retaining and mesenchymal phenotypes and correlated with reduced expression of genes involved in pigmentation.,Elevated THBS1 levels were detected in Vemurafenib resistant melanoma cells and inhibition of THBS1 led to significantly reduced chemoresistance in melanoma cells.,Notably, siRNA-mediated silencing of THBS1 and neutralizing antibody to THBS1 reduced invasion in mesenchymal-like melanoma cells, while ectopic THBS1 expression in epithelial-like melanoma cells enhanced invasion.,Furthermore, the loss of THBS1 inhibited in vivo motility of melanoma cells within the embryonic chicken neural tube.,In addition, we found aberrant THBS1 protein expression in metastatic melanoma tumor biopsies.,These results implicate a role for THBS1 in EMT, and hence THBS1 may serve as a novel target for strategies aimed at the treatment of melanoma invasion and drug resistance.

A functional role of microRNAs (miRNAs or miRs) in neoplasia and metastasis is becoming clear, and the miR-200 family has received much attention for potentially regulating tumor progression.,The miRNAs of this family have been shown to suppress epithelial-mesenchymal transition, and their down-regulation in some tumors promotes invasion and metastasis.,Interestingly, while miR-200 is down-regulated in some cancers, it is up-regulated in others.,We show that levels of miR-200 are increased in melanoma cell lines compared to normal melanocytes and that miR-200 family members play a role in determining modes of tumor cell migration.,Individual tumor cells can invade in either elongated, “mesenchymal-type” or rounded, “amoeboid-like” modes and these two modes of invasion are inter-convertible [1].,In melanoma cell lines, expression of miR-200 members does not suppress invasion but rather leads to a switch between modes of invasion.,MicroRNA-200c results in a higher proportion of cells adopting the rounded, amoeboid-like mode of invasion, while miR-200a results in a protrusion-associated elongated mode of invasion.,Functional target identification studies suggest that the morphological effects of miR-200c may be mediated by reduced expression of MARCKS, which has been linked to formation of cell protrusions.,In contrast miR-200a reduces actomyosin contractility, a feature of rounded morphology.,Overall our findings call into question the general role of miR-200 in suppressing invasion and metastasis, and highlight novel distinguishing characteristics of individual miR-200 family members.

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.

UV radiation (UV) is classified as a “complete carcinogen” because it is both a mutagen and a non-specific damaging agent and has properties of both a tumor initiator and a tumor promoter.,In environmental abundance, UV is the most important modifiable risk factor for skin cancer and many other environmentally-influenced skin disorders.,However, UV also benefits human health by mediating natural synthesis of vitamin D and endorphins in the skin, therefore UV has complex and mixed effects on human health.,Nonetheless, excessive exposure to UV carries profound health risks, including atrophy, pigmentary changes, wrinkling and malignancy.,UV is epidemiologically and molecularly linked to the three most common types of skin cancer, basal cell carcinoma, squamous cell carcinoma and malignant melanoma, which together affect more than a million Americans annually.,Genetic factors also influence risk of UV-mediated skin disease.,Polymorphisms of the melanocortin 1 receptor (MC1R) gene, in particular, correlate with fairness of skin, UV sensitivity, and enhanced cancer risk.,We are interested in developing UV-protective approaches based on a detailed understanding of molecular events that occur after UV exposure, focusing particularly on epidermal melanization and the role of the MC1R in genome maintenance.

The microphthalmia-associated transcription factor (MITF) is the “master melanocyte transcription factor” with a complex role in melanoma.,MITF protein levels vary between and within clinical specimens, and amplifications and gain- and loss-of-function mutations have been identified in melanoma.,How MITF functions in melanoma development and the effects of targeting MITF in vivo are unknown because MITF levels have not been directly tested in a genetic animal model.,Here, we use a temperature-sensitive mitf zebrafish mutant to conditionally control endogenous MITF activity.,We show that low levels of endogenous MITF activity are oncogenic with BRAFV600E to promote melanoma that reflects the pathology of the human disease.,Remarkably, abrogating MITF activity in BRAFV600Emitf melanoma leads to dramatic tumor regression marked by melanophage infiltration and increased apoptosis.,These studies are significant because they show that targeting MITF activity is a potent antitumor mechanism, but also show that caution is required because low levels of wild-type MITF activity are oncogenic.

Melanoma is the most aggressive and lethal form of skin cancer.,Because of the increasing incidence and high lethality of melanoma, animal models for continuously observing melanoma formation and progression as well as for testing pharmacological agents are needed.,Using the combinatorial Gal4 -UAS system, we have developed a zebrafish transgenic line that expresses oncogenic HRAS under the kita promoter.,Already at 3 days transgenic kita-GFP-RAS larvae show a hyper-pigmentation phenotype as earliest evidence of abnormal melanocyte growth.,By 2-4 weeks, masses of transformed melanocytes form in the tail stalk of the majority of kita-GFP-RAS transgenic fish.,The adult tumors evident between 1-3 months of age faithfully reproduce the immunological, histological and molecular phenotypes of human melanoma, but on a condensed time-line.,Furthermore, they show transplantability, dependence on mitfa expression and do not require additional mutations in tumor suppressors.,In contrast to kita expressing melanocyte progenitors that efficiently develop melanoma, mitfa expressing progenitors in a second Gal4-driver line were 4 times less efficient in developing melanoma during the three months observation period.,This indicates that zebrafish kita promoter is a powerful tool for driving oncogene expression in the right cells and at the right level to induce early onset melanoma in the presence of tumor suppressors.,Thus our zebrafish model provides a link between kita expressing melanocyte progenitors and melanoma and offers the advantage of a larval phenotype suitable for large scale drug and genetic modifier screens.

Drug resistance remains a vexing problem in the treatment of cancer patients.,While many studies have focused on cell autonomous mechanisms of drug resistance, we hypothesized that the tumor microenvironment may confer innate resistance to therapy.,Here we developed a co-culture system to systematically assay the ability of 23 stromal cell types to influence the innate resistance of 45 cancer cell lines to 35 anti-cancer drugs.,We found that stroma-mediated resistance is surprisingly common - particularly to targeted agents.,We further characterized the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibition because most of these patients exhibit some degree of innate resistance1-4.,Proteomic analysis showed that stromal secretion of the growth factor hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the MAPK and PI3K/AKT pathways, and immediate resistance to RAF inhibition.,Immunohistochemistry confirmed stromal HGF expression in patients with BRAF-mutant melanoma and a statistically significant correlation between stromal HGF expression and innate resistance to treatment.,Dual inhibition of RAF and MET resulted in reversal of drug resistance, suggesting RAF/MET combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma.,A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines.,More generally, these studies indicate that the systematic dissection of tumor-microenvironment interactions may reveal important mechanisms underlying drug resistance.

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.

Interleukin-2 (IL-2) is a component of most protocols of adoptive cell transfer (ACT) therapy for cancer, but is limited by short exposure and high toxicities.,NKTR-214 is a kinetically-engineered IL-2 receptor βγ (IL-2Rβγ)-biased agonist consisting of IL-2 conjugated to multiple releasable polyethylene glycol chains resulting in sustained signaling through IL-2Rβγ.,We report that ACT supported by NKTR-214 increases the proliferation, homing and persistence of anti-tumor T cells compared to ACT with IL-2, resulting in superior antitumor activity in a B16-F10 murine melanoma model.,The use of NKTR-214 increases the number of polyfunctional T cells in murine spleens and tumors compared to IL-2, and enhances the polyfunctionality of T and NK cells in the peripheral blood of patients receiving NKTR-214 in a phase 1 trial.,In conclusion, NKTR-214 may have the potential to improve the antitumor activity of ACT in humans through increased in vivo expansion and polyfunctionality of the adoptively transferred T cells.,Adoptive cell transfer (ACT) of T cells for tumor treatment often requires IL-2 administration.,Here, the authors show that a modified IL-2 cytokine (NKTR-214) can outperform IL-2 in a melanoma mouse model.

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.

Adoptive therapy with tumour-infiltrating lymphocytes (TILs) induces durable complete responses (CR) in ∼20% of patients with metastatic melanoma.,The recruitment of T cells through CXCR3/CCR5 chemokine ligands is critical for immune-mediated rejection.,We postulated that polymorphisms and/or expression of CXCR3/CCR5 in TILs and the expression of their ligands in tumour influence the migration of TILs to tumours and tumour regression.,Tumour-infiltrating lymphocytes from 142 metastatic melanoma patients enrolled in adoptive therapy trials were genotyped for CXCR3 rs2280964 and CCR5-Δ32 deletion, which encodes a protein not expressed on the cell surface.,Expression of CXCR3/CCR5 in TILs and CXCR3/CCR5 and ligand genes in 113 available parental tumours was also assessed.,Tumour-infiltrating lymphocyte data were validated by flow cytometry (N=50).,The full gene expression/polymorphism model, which includes CXCR3 and CCR5 expression data, CCR5-Δ32 polymorphism data and their interaction, was significantly associated with both CR and overall response (OR; P=0.0009, and P=0.007, respectively).,More in detail, the predicted underexpression of both CXCR3 and CCR5 according to gene expression and polymorphism data (protein prediction model, PPM) was associated with response to therapy (odds ratio=6.16 and 2.32, for CR and OR, respectively).,Flow cytometric analysis confirmed the PPM.,Coordinate upregulation of CXCL9, CXCL10, CXCL11, and CCL5 in pretreatment tumour biopsies was associated with OR.,Coordinate overexpression of CXCL9, CXCL10, CXCL11, and CCL5 in pretreatment tumours was associated with responsiveness to treatment.,Conversely, CCR5-Δ32 polymorphism and CXCR3/CCR5 underexpression influence downregulation of the corresponding receptors in TILs and were associated with likelihood and degree of response.

Several lines of evidence suggest a dichotomy between immune active and quiescent cancers, with the former associated with a good prognostic phenotype and better responsiveness to immunotherapy.,Central to such dichotomy is the master regulator of the acute inflammatory process interferon regulatory factor (IRF)-1.,However, it remains unknown whether the responsiveness of IRF-1 to cytokines is able to differentiate cancer immune phenotypes.,IRF-1 activation was measured in 15 melanoma cell lines at basal level and after treatment with IFN-γ, TNF-α and a combination of both.,Microarray analysis was used to compare transcriptional patterns between cell lines characterised by high or low IRF-1 activation.,We observed a strong positive correlation between IRF-1 activation at basal level and after IFN-γ and TNF-α treatment.,Microarray demonstrated that three cell lines with low and three with high IRF-1 inducible translocation scores differed in the expression of 597 transcripts.,Functional interpretation analysis showed mTOR and Wnt/β-cathenin as the top downregulated pathways in the cell lines with low inducible IRF-1 activation, suggesting that a low IRF-1 inducibility recapitulates a cancer phenotype already described in literature characterised by poor prognosis.,Our findings support the central role of IRF-1 in influencing different tumour phenotypes.

BRAF and MEK inhibitors are effective in BRAF mutant melanoma, but most patients eventually relapse with acquired resistance, and others present intrinsic resistance to these drugs.,Resistance is often mediated by pathway reactivation through receptor tyrosine kinase (RTK)/SRC-family kinase (SFK) signaling or mutant NRAS, which drive paradoxical reactivation of the pathway.,We describe pan-RAF inhibitors (CCT196969, CCT241161) that also inhibit SFKs.,These compounds do not drive paradoxical pathway activation and inhibit MEK/ERK in BRAF and NRAS mutant melanoma.,They inhibit melanoma cells and patient-derived xenografts that are resistant to BRAF and BRAF/MEK inhibitors.,Thus, paradox-breaking pan-RAF inhibitors that also inhibit SFKs could provide first-line treatment for BRAF and NRAS mutant melanomas and second-line treatment for patients who develop resistance.,•pan-RAF inhibitors also inhibit SRC family kinases•The compounds do not induce paradoxical activation of ERK in RAS mutant cells•The compounds are active in BRAF and NRAS mutant melanomas•The compounds are active in PDXs resistant to BRAF or BRAF plus MEK inhibitors,pan-RAF inhibitors also inhibit SRC family kinases,The compounds do not induce paradoxical activation of ERK in RAS mutant cells,The compounds are active in BRAF and NRAS mutant melanomas,The compounds are active in PDXs resistant to BRAF or BRAF plus MEK inhibitors,Girotti et al. describe two pan-RAF inhibitors that also inhibit SRC-family kinases.,These compounds do not drive paradoxical MEK/ERK activation and can inhibit MEK in NRAS mutant cells.,Moreover, the agents can overcome resistance to clinical BRAF or combination BRAF/MEK inhibitors in patient-derived xenografts.

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.

The overall 5-year survival for melanoma is 91%.,However, if distant metastasis occurs (stage IV), cure rates are < 15%.,Hence, melanoma detection in earlier stages (stages I-III) maximises the chances of patient survival.,We measured the expression of a panel of 17 microRNAs (miRNAs) (MELmiR-17) in melanoma tissues (stage III; n = 76 and IV; n = 10) and serum samples (collected from controls with no melanoma, n = 130; and patients with melanoma (stages I/II, n = 86; III, n = 50; and IV, n = 119)) obtained from biobanks in Australia and Germany.,In melanoma tissues, members of the ‘MELmiR-17’ panel were found to be predictors of stage, recurrence, and survival.,Additionally, in a minimally-invasive blood test, a seven-miRNA panel (MELmiR-7) detected the presence of melanoma (relative to controls) with high sensitivity (93%) and specificity (≥ 82%) when ≥ 4 miRNAs were expressed.,Moreover, the ‘MELmiR-7’ panel characterised overall survival of melanoma patients better than both serum LDH and S100B (delta log likelihood = 11, p < 0.001).,This panel was found to be superior to currently used serological markers for melanoma progression, recurrence, and survival; and would be ideally suited to monitor tumour progression in patients diagnosed with early metastatic disease (stages IIIa-c/IV M1a-b) to detect relapse following surgical or adjuvant treatment.,•A seven-miRNA panel (MELmiR-7) detected the presence of melanoma with high sensitivity (93%) and specificity (≥ 82%).,•In serially collected stage IV specimens, members of the ‘MELmiR-7’ panel confirmed tumour progression in 100% of cases.,•The ‘MELmiR-7’ panel is superior to currently used serological markers for melanoma progression, recurrence, and survival.,A seven-miRNA panel (MELmiR-7) detected the presence of melanoma with high sensitivity (93%) and specificity (≥ 82%).,In serially collected stage IV specimens, members of the ‘MELmiR-7’ panel confirmed tumour progression in 100% of cases.,The ‘MELmiR-7’ panel is superior to currently used serological markers for melanoma progression, recurrence, and survival.

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.

We conducted the phase III double-blind European Organisation for Research and Treatment of Cancer (EORTC) 1325/KEYNOTE-054 trial to evaluate pembrolizumab versus placebo in patients with resected high-risk stage III melanoma.,On the basis of 351 recurrence-free survival (RFS) events at a 1.25-year median follow-up, pembrolizumab prolonged RFS (hazard ratio [HR], 0.57; P < .0001) compared with placebo.,This led to the approval of pembrolizumab adjuvant treatment by the European Medicines Agency and US Food and Drug Administration.,Here, we report an updated RFS analysis at the 3.05-year median follow-up.,A total of 1,019 patients with complete lymph node dissection of American Joint Committee on Cancer Staging Manual (seventh edition; AJCC-7), stage IIIA (at least one lymph node metastasis > 1 mm), IIIB, or IIIC (without in-transit metastasis) cutaneous melanoma were randomly assigned to receive pembrolizumab at a flat dose of 200 mg (n = 514) or placebo (n = 505) every 3 weeks for 1 year or until disease recurrence or unacceptable toxicity.,The two coprimary end points were RFS in the overall population and in those with programmed death-ligand 1 (PD-L1)-positive tumors.,Pembrolizumab (190 RFS events) compared with placebo (283 RFS events) resulted in prolonged RFS in the overall population (3-year RFS rate, 63.7% v 44.1% for pembrolizumab v placebo, respectively; HR, 0.56; 95% CI, 0.47 to 0.68) and in the PD-L1-positive tumor subgroup (HR, 0.57; 99% CI, 0.43 to 0.74).,The impact of pembrolizumab on RFS was similar in subgroups, in particular according to AJCC-7 and AJCC-8 staging, and BRAF mutation status (HR, 0.51 [99% CI, 0.36 to 0.73] v 0.66 [99% CI, 0.46 to 0.95] for V600E/K v wild type).,In resected high-risk stage III melanoma, pembrolizumab adjuvant therapy provided a sustained and clinically meaningful improvement in RFS at 3-year median follow-up.,This improvement was consistent across subgroups.

Over the past 5 years, adjuvant treatment options for surgically resected stage III melanoma have expanded with the introduction of several novel immune checkpoint inhibitors and targeted therapies.,Pembrolizumab, a programmed cell death protein 1 inhibitor, received US Food and Drug Administration approval in 2019 for resected high-risk stage III melanoma based on significantly longer recurrence-free survival versus placebo.,This study evaluated the cost-effectiveness of pembrolizumab versus other adjuvant treatment strategies for resected high-risk stage III melanoma from a US health system perspective.,A Markov cohort-level model with four states (recurrence-free, locoregional recurrence, distant metastases, death) estimated costs and quality-adjusted life-years (QALYs) for pembrolizumab versus routine observation and other adjuvant comparators: ipilimumab in the overall population; and dabrafenib + trametinib in the BRAF-mutation positive (BRAF+) subgroup.,Transition probabilities starting from recurrence-free were estimated through parametric multi-state modeling based on phase 3 KEYNOTE-054 (NCT02362594) trial data for pembrolizumab and observation, and network meta-analyses for other comparators.,Post-recurrence transitions were modeled based on electronic medical records data and trials in advanced/metastatic melanoma.,Utilities were derived using quality-of-life data from KEYNOTE-054 and literature.,Costs of treatment, adverse events, disease management, and terminal care were included.,Over a lifetime, pembrolizumab, ipilimumab, and observation were associated with QALYs of 9.24, 7.09, and 5.95 and total costs of $511,290, $992,721, and $461,422, respectively (2019 US dollars).,Pembrolizumab was thus dominant (less costly, more effective) versus ipilimumab, with an incremental cost-effectiveness ratio of $15,155/QALY versus observation.,In the BRAF+ subgroup, pembrolizumab dominated dabrafenib + trametinib and observation, decreasing costs by $62,776 and $11,250 and increasing QALYs by 0.93 and 3.10 versus these comparators, respectively.,Results were robust in deterministic and probabilistic sensitivity analyses.,As adjuvant treatment for resected stage III melanoma, pembrolizumab was found to be dominant and therefore cost-effective compared with the active comparators ipilimumab and dabrafenib + trametinib.,Pembrolizumab increased costs relative to observation in the overall population, with sufficient incremental benefit to be considered cost-effective based on typical willingness-to-pay thresholds.,The online version of this article (10.1007/s40261-020-00922-6) contains supplementary material, which is available to authorized users.

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.

Although CDKN2A is the most frequent high-risk melanoma susceptibility gene, the underlying genetic factors for most melanoma-prone families remain unknown.,Using whole exome sequencing, we identified a rare variant that arose as a founder mutation in the telomere shelterin POT1 gene (g.7:124493086 C>T, Ser270Asn) in five unrelated melanoma-prone families from Romagna, Italy.,Carriers of this variant had increased telomere length and elevated fragile telomeres suggesting that this variant perturbs telomere maintenance.,Two additional rare POT1 variants were identified in all cases sequenced in two other Italian families, yielding a frequency of POT1 variants comparable to that of CDKN2A mutations in this population.,These variants were not found in public databases or in 2,038 genotyped Italian controls.,We also identified two rare recurrent POT1 variants in American and French familial melanoma cases.,Our findings suggest that POT1 is a major susceptibility gene for familial melanoma in several populations.

Genome-wide association studies (GWAS) have identified ~20 melanoma susceptibility loci, most of which are not functionally characterized.,Here we report an approach integrating massively-parallel reporter assays (MPRA) with cell-type-specific epigenome and expression quantitative trait loci (eQTL) to identify susceptibility genes/variants from multiple GWAS loci.,From 832 high-LD variants, we identify 39 candidate functional variants from 14 loci displaying allelic transcriptional activity, a subset of which corroborates four colocalizing melanocyte cis-eQTL genes.,Among these, we further characterize the locus encompassing the HIV-1 restriction gene, MX2 (Chr21q22.3), and validate a functional intronic variant, rs398206. rs398206 mediates the binding of the transcription factor, YY1, to increase MX2 levels, consistent with the cis-eQTL of MX2 in primary human melanocytes.,Melanocyte-specific expression of human MX2 in a zebrafish model demonstrates accelerated melanoma formation in a BRAFV600E background.,Our integrative approach streamlines GWAS follow-up studies and highlights a pleiotropic function of MX2 in melanoma susceptibility.,There are more than 20 known melanoma susceptibility genes.,Here, using a massively parallel reporter assay, the authors identify risk-associated variants that alter gene transcription, and demonstrate that expression of one such gene, MX2, leads to the promotion of melanoma in a zebrafish model.

Deleterious germline variants in CDKN2A account for around 40% of familial melanoma cases1, while rare variants in CDK4, BRCA2, BAP1, and the promoter of TERT, have also been linked to the disease2-5.,Here we set out to identify novel high-penetrance susceptibility genes in unexplained cases by sequencing 184 melanoma patients from 105 pedigrees recruited in the United Kingdom, the Netherlands, and Australia that were negative for variants in known predisposition genes.,We identify families where melanoma co-segregates with loss-of-function variants in the protection of telomeres 1 (POT1) gene, a proportion of members presenting with an early age of onset and multiple primaries.,We show that these variants either affect POT1 mRNA splicing or alter key residues in the highly conserved oligonucleotide-/oligosaccharide-binding (OB) domains of POT1, disrupting protein-telomere binding, leading to increased telomere length.,Thus, POT1 variants predispose to melanoma formation via a direct effect on telomeres.

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.

Finding the best technique to identify BRAF mutations with a high sensitivity and specificity is mandatory for accurate patient selection for target therapy.,BRAF mutation frequency ranges from 40 to 60% depending on melanoma clinical characteristics and detection technique used.,Intertumoral heterogeneity could lead to misinterpretation of BRAF mutational status; this is especially important if testing is performed on primary specimens, when metastatic lesions are unavailable.,Aim of this study was to identify the best combination of methods for detecting BRAF mutations (among peptide nucleic acid - PNA-clamping real-time PCR, immunohistochemistry and capillary sequencing) and investigate BRAF mutation heterogeneity in a series of 100 primary melanomas and a subset of 25 matched metastatic samples.,Overall, we obtained a BRAF mutation frequency of 62%, based on the combination of at least two techniques.,Concordance between mutation status in primary and metastatic tumor was good but not complete (67%), when agreement of at least two techniques were considered.,Next generation sequencing was used to quantify the threshold of detected mutant alleles in discordant samples.,Combining different methods excludes that the observed heterogeneity is technique-based.,We propose an algorithm for BRAF mutation testing based on agreement between immunohistochemistry and PNA; a third molecular method could be added in case of discordance of the results.,Testing the primary tumor when the metastatic sample is unavailable is a good option if at least two methods of detection are used, however the presence of intertumoral heterogeneity or the occurrence of additional primaries should be carefully considered.

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.

Long non-coding RNAs (lncRNAs) are involved in tumorigenesis.,Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), an lncRNAs, is associated with the growth and metastasis of many human tumors, but its biological roles in malignant melanoma remain unclear.,In this study, the aberrant up-regulation of MALAT1 was detected in melanoma.,We determined that MALAT1 promotes melanoma cells proliferation, invasion and migration by sponging miR-22.,MiR-22 was decreased and acted as a tumor suppressor in melanoma, and MMP14 and Snail were the functional targets of miR-22.,Furthermore, MALAT1 could modulate MMP14 and Snail by operating as a competing endogenous RNA (ceRNA) for miR-22.,The effects of MALAT1 in malignant melanoma is verified using a xenograft model.,This finding elucidates a new mechanism for MALAT1 in melanoma development and provides a potential target for melanoma therapeutic intervention.

Anti-PD-1 monoclonal antibodies, nivolumab and pembrolizumab, and anti-CTLA-4 antibody ipilimumab are being in clinic trials to treat melanoma.,Here, we performed a meta-analysis to evaluate the efficacy and toxicity of them against advanced melanoma.,Eleven reports from 6 randomized control trials on treating metastatic melanoma, which were divided into 3 subgroups, nivolumab/pembrolizumab versus chemotherapy, nivolumab versus ipilimumab, and nivolumab-plus-ipilimumab versus ipilimumab, were included and the meta-analysis was performed for each subgroup.,The outcome measures were objective response rates (ORR), median progression free survival (PFS), 1-year overall survival rates (OS), and toxicity estimated by grade 3 to 4 adverse events.,For nivolumab/pembrolizumab versus chemotherapy, nivolumab versus ipilimumab, and nivolumab-plus-ipilimumab versus ipilimumab, the pooled risk ratios (RR) of the ORR were 3.43 (95% CI: 2.57-4.58), 2.51 (95% CI: 2.03-3.09), and 3.28 (95% CI: 2.58-4.17), respectively.,The pooled HR of PFS were 0.42 (95% CI: 0.36-0.49), 0.58 (95% CI: 0.50-0.66), and 0.41 (95% CI: 0.30-0.52), respectively.,The pooled RR of 1-year OS was 1.37 (95% CI: 1.08-1.74) and 1.54 (95% CI: 0.90-2.63) for nivolumab versus ipilimumab and nivolumab-plus-ipilimumab versus ipilimumab.,These results suggested that anti-PD-1 monotherapy and nivolumab-plus-ipilimumab therapy had ORR and PFS benefit compared with the control group.,Anti-PD-1 treatment increased 1-year OS for patients compared with ipililumab treatment.,But there is no significantly difference on 1-year OS between the nivolumab-plus-ipilimumab treatment and the ipilimumab treatment group.,The toxicity analysis showed that there is less risk of adverse events in the anti-PD-1 treatment group compared with the chemotherapy and ipilimumab group.,Combining nivolumab with ipilimumab increased the risk for high-grade adverse events compared with ipilimumab alone but the adverse events were generally manageable.,Anti-PD-1 monotherapy and nivolumab-plus-ipilimumab therapy improved ORR and prolonged PFS of patients with advanced melanoma and the adverse events are generally manageable.,The therapy is indeed a promising approach for treatment of advanced melanoma.

In the last decade, genome-wide transcriptome analyses have been routinely used to monitor tissue-, disease- and cell type-specific gene expression, but it has been technically challenging to generate expression profiles from single cells.,Here we describe a novel and robust mRNA-Seq protocol (Smart-Seq) that is applicable down to single cell levels.,Compared with existing methods, Smart-Seq has improved read coverage across transcripts, which significantly enhances detailed analyses of alternative transcript isoforms and identification of SNPs.,We have determined the sensitivity and quantitative accuracy of Smart-Seq for single-cell transcriptomics by evaluating it on total RNA dilution series.,Applying Smart-Seq to circulating tumor cells from melanomas, we identified distinct gene expression patterns, including new candidate biomarkers for melanoma circulating tumor cells.,Importantly, our protocol can easily be utilized for addressing fundamental biological problems requiring genome-wide transcriptome profiling in rare cells.

Epigenetic changes influence various physiological and pathological conditions in the human body.,Recent advances in epigenetic studies of the skin have led to an appreciation of the importance of epigenetic modifications in skin diseases.,Cutaneous sarcomas are intractable skin cancers, and there are no curative therapeutic options for the advanced forms of cutaneous sarcomas.,In this review, we discuss the detailed molecular effects of epigenetic modifications on skin sarcomas, such as dermatofibrosarcoma protuberans, angiosarcoma, Kaposi’s sarcoma, leiomyosarcoma, and liposarcoma.,We also discuss the application of epigenetic-targeted therapy for skin sarcomas.

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.

Solid tumors are complex systems characterized by dynamic interactions between neoplastic cells, non-tumoral cells, and extracellular components.,Among all the stromal cells that populate tumor microenvironment, fibroblasts are the most abundant elements and are critically involved in disease progression.,Cancer-associated fibroblasts (CAFs) have pleiotropic functions in tumor growth and extracellular matrix remodeling implicated in local invasion and distant metastasis.,CAFs additionally participate in the inflammatory response of the tumor site by releasing a variety of chemokines and cytokines.,It is becoming clear that understanding the dynamic, mutual melanoma-fibroblast relationship would enable treatment options to be amplified.,To better characterize melanoma-associated fibroblasts, here we analyzed low-passage primary CAFs derived from advanced-stage primary skin melanomas, focusing on the immuno-phenotype.,Furthermore, we assessed the expression of several CAF markers and the production of growth factors.,To deepen the study of CAF-melanoma cell crosstalk, we employed CAF-derived supernatants and trans-well co-culture systems to evaluate the influences of CAFs on (i) the motogenic ability of melanoma cells, (ii) the chemotherapy-induced cytotoxicity, and (iii) the release of mediators active in modulating tumor growth and spread.

Malignant melanoma is a highly metastatic type of cancer, which arises frequently from transformed pigment cells and melanocytes as a result of long-term UV radiation exposure.,In recent years, the incidence of newly diagnosed melanoma patients reached 5% of all cancer cases.,Despite the development of novel targeted therapies directed against melanoma-specific markers, patients’ response to treatment is often weak or short-term due to a rapid acquisition of drug resistance.,Among the factors affecting therapy effectiveness, elements of the tumor microenvironment play a major role.,Melanoma niche encompasses adjacent cells, such as keratinocytes, cancer-associated fibroblasts (CAFs), adipocytes, and immune cells, as well as components of the extracellular matrix and tumor-specific physicochemical properties.,In this review, we summarize the current knowledge concerning the influence of cancer-associated cells (keratinocytes, CAFs, adipocytes) on the process of melanomagenesis, tumor progression, invasiveness, and the emergence of drug resistance in melanoma.,We also address how melanoma can alter the differentiation and activation status of cells present in the tumor microenvironment.,Understanding these complex interactions between malignant and cancer-associated cells could improve the development of effective antitumor therapeutic strategies.

Melanoma is the most aggressive and dangerous form of skin cancer that develops from transformed melanocytes.,It is crucial to identify melanoma at its early stages, in situ, as it is “curable” at this stage.,However, after metastasis, it is difficult to treat and the five-year survival is only 25%.,In recent years, a better understanding of the etiology of melanoma and its progression has made it possible for the development of targeted therapeutics, such as vemurafenib and immunotherapies, to treat advanced melanomas.,In this review, we focus on the molecular mechanisms that mediate melanoma development and progression, with a special focus on the immune evasion strategies utilized by melanomas, to evade host immune surveillances.,The proposed mechanism of action and the roles of immunotherapeutic agents, ipilimumab, nivolumab, pembrolizumab, and atezolizumab, adoptive T- cell therapy plus T-VEC in the treatment of advanced melanoma are discussed.,In this review, we implore that a better understanding of the steps that mediate melanoma onset and progression, immune evasion strategies exploited by these tumor cells, and the identification of biomarkers to predict treatment response are critical in the design of improved strategies to improve clinical outcomes for patients with this deadly disease.

Cutaneous melanoma is a metastatic disease characterized by high resistance to treatment, the incidence of which has alarmingly increased worldwide over the past years.,A thorough characterization of tumor onset, progression and metastasis is compulsory to overcome the gaps existent in melanoma biology.,The present study suggests a well-established protocol and a detailed histological description of human melanoma models in ovo and in vivo obtained by the inoculation of A375 cells to chick embryo chorioallantoic membrane (CAM) and Balb/c nude mice.,The inoculation of A375 cells on CAM led to the formation of compact primary and secondary tumors on day 4 post-inoculation, with mean surface area values of 2.2±0.4 mm2 and 1.5±0.3 mm2, respectively.,Moreover, the vessels around the tumors presented a spike wheel pattern, indicating a strong angiogenic reaction.,All the injected mice, apart from one, developed solid polypoid primary tumors with lobulated surfaces and intense vascularization, and achromic epithelioid malignant melanocytes with vesiculous nuclei and necrosis area were detected.,Metastasis was histologically confirmed in only 30% of the mice with the tumor xenografts.,These data indicate that the standardization protocols proposed are complex and reproducible, and can be further employed for the therapeutic surveillance of antiangiogenic and anticancer agents.

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.

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.

Despite recent advances in therapy, liver metastasis from melanoma is still associated with poor prognosis.,Although targeting the mTOR signaling pathway exerts potent anti-tumor activity, little is known about specific mTORC2 inhibition regarding liver metastasis.,Using the novel mTORC2 specific inhibitor JR-AB2-011, we show significantly reduced migration and invasion capacity by impaired activation of MMP2 in melanoma cells.,In addition, blockade of mTORC2 induces cell death by non-apoptotic pathways and reduces tumor cell proliferation rate dose-dependently.,Furthermore, a significant reduction of liver metastasis was detected in a syngeneic murine metastasis model upon therapy with JR-AB2-011 as determined by in vivo imaging and necropsy.,Hence, our study for the first time highlights the impact of the pharmacological blockade of mTORC2 as a potent novel anti-cancer approach for liver metastasis from melanoma.

Understanding the underlying molecular mechanisms involved in the formation of cutaneous malignant melanoma is critical for improved diagnosis and treatment.,Keratinocytic nuclear receptor Retinoid X Receptor α (RXRα) has a protective role against melanomagenesis and is involved in the regulation of keratinocyte and melanocyte homeostasis subsequent acute ultraviolet (UV) irradiation.,We generated a trigenic mouse model system (RXRα ep−/−| Tyr-NRAS Q61K | CDK4 R24C/R24C ) harboring an epidermal knockout of Retinoid X Receptor α (RXRα ep−/−), combined with oncogenic NRAS Q61K (constitutively active RAS) and activated CDK4 R24C/R24C (constitutively active CDK4).,Those mice were subjected to a single neonatal dose of UVB treatment and the role of RXR α was evaluated by characterizing the molecular and cellular changes that took place in the untreated and UVB treated trigenic RXRα ep−/− mice compared to the control mice with functional RXRα.,Here we report that the trigenic mice develops spontaneous melanoma and exposure to a single neonatal UVB treatment reduces the tumor latency in those mice compared to control mice with functional RXRα.,Melanomas from the trigenic RXRα ep−/− mice are substantial in size, show increased proliferation, exhibit increased expression of malignant melanoma markers and exhibit enhanced vascularization.,Altered expression of several biomarkers including increased expression of activated AKT, p21 and cyclin D1 and reduced expression of pro-apoptotic marker BAX was observed in the tumor adjacent normal (TAN) skin of acute ultraviolet B treated trigenic RXRα ep−/− mice.,Interestingly, we observed a significant increase in p21 and Cyclin D1 in the TAN skin of un-irradiated trigenic RXRα ep−/− mice, suggesting that those changes might be consequences of loss of functional RXRα in the melanoma microenvironment.,Loss of RXRα in the epidermal keratinocytes in combination with oncogenic NRAS Q61K and CDK4 R24C/R24C mutations in trigenic mice led to significant melanoma invasion into the draining lymph nodes as compared to controls with functional RXRα.,Our study demonstrates the protective role of keratinocytic RxRα in (1) suppressing spontaneous and acute UVB-induced melanoma, and (2) preventing progression of the melanoma to malignancy in the presence of driver mutations like activated CDK4 R24C/R24C and oncogenic NRAS Q61K .,The online version of this article (10.1186/s12885-017-3714-6) contains supplementary material, which is available to authorized users.

A 60-year-old Caucasian female was referred for biopsy-proven amelanotic orbito-conjunctival melanoma.,Map biopsies revealed residual invasive melanoma on the deep tarsal margin at the site of previous surgery.,Repeat excisions were required after recurrence was detected following 3 months and 7 months.,Positron emission tomography scan detected liver metastasis and additional orbito-conjunctival melanoma recurrence.,Biomarker testing showed NRAS mutation without BRAF or c-KIT mutations and without PD-L1 expression.,Systemic checkpoint inhibitor therapy was initiated with regression of both the orbito-conjunctival melanoma and liver metastasis.,Invasive, non resectable orbito-conjunctival melanoma with liver metastasis can demonstrate a response to systemic checkpoint inhibitor therapy.

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.

Ferroptosis is a regulated form of cell death driven by small molecules or conditions that induce lipid-based reactive oxygen species (ROS) accumulation.,This form of iron-dependent cell death is morphologically and genetically distinct from apoptosis, necroptosis, and autophagy. miRNAs are known to play crucial roles in diverse fundamental biological processes.,However, to date no study has reported miRNA-mediated regulation of ferroptosis.,Here we show that miR-137 negatively regulates ferroptosis by directly targeting glutamine transporter SLC1A5 in melanoma cells.,Ectopic expression of miR-137 suppressed SLC1A5, resulting in decreased glutamine uptake and malondialdehyde (MDA) accumulation.,Meanwhile, antagomir-mediated inactivation of endogenous miR-137 increased the sensitivity of melanoma cells to erastin- and RSL3-induced ferroptosis.,Importantly, knockdown of miR-137 increased the antitumor activity of erastin by enhancing ferroptosis both in vitro and in vivo.,Collectively, these data indicate that miR-137 plays a novel and indispensable role in ferroptosis by inhibiting glutaminolysis and suggest a potential therapeutic approach for melanoma.

Blocking oncogenic signaling induced by the BRAFV600E mutation is a promising approach for melanoma treatment.,We tested the anti-tumor effects of a specific inhibitor of Raf protein kinases, PLX4032/RG7204, in melanoma cell lines.,PLX4032 decreased signaling through the MAPK pathway only in cell lines with the BRAFV600E mutation.,Seven out of 10 BRAFV600E mutant cell lines displayed sensitivity based on cell viability assays and three were resistant at concentrations up to 10 μM.,Among the sensitive cell lines, four were highly sensitive with IC50 values below 1 μM, and three were moderately sensitive with IC50 values between 1 and 10 μM.,There was evidence of MAPK pathway inhibition and cell cycle arrest in both sensitive and resistant cell lines.,Genomic analysis by sequencing, genotyping of close to 400 oncogeninc mutations by mass spectrometry, and SNP arrays demonstrated no major differences in BRAF locus amplification or in other oncogenic events between sensitive and resistant cell lines.,However, metabolic tracer uptake studies demonstrated that sensitive cell lines had a more profound inhibition of FDG uptake upon exposure to PLX4032 than resistant cell lines.,In conclusion, BRAFV600E mutant melanoma cell lines displayed a range of sensitivities to PLX4032 and metabolic imaging using PET probes can be used to assess sensitivity.

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.

The precise mechanisms governing invasion at the leading edge of SCC and its subsequent metastasis are not fully understood.,We aimed to define the cancer related molecular changes that distinguish non-invasive tumor from invasive SCC.,To this end, we combined laser capture microdissection with cDNA microarray analysis.,We defined invasion-associated genes as those differentially regulated only in invasive SCC nests, but not in actinic keratosis or in situ SCC, compared to normal epidermis.,There were 383 up- and 354 down-regulated genes in the “invasion set.”,SCC invasion was characterized by aberrant expression of various proteolytic molecules.,We noted increased expression of MMP7 and IL-24 in invasive SCC.,IL-24 induced the expression of MMP7 in SCC cells in culture.,In addition, blocking of MMP7 by a specific antibody significantly delayed the migration of SCC cells in culture.,These results suggest a possible contribution of IL-24 to SCC invasion via enhancing focal expression of MMP7, though IL-24 has been suggested to have anti-tumor growth effects in other cancer types.,Identification of regional molecular changes that regulate cancer invasion may facilitate the development of new targeted treatments for aggressive cancer.

Genes, proteins, or cells influence each other and consequently create patterns, which can be increasingly better observed by experimental biology and medicine.,Thereby, descriptive methods of statistics and bioinformatics sharpen and structure our perception.,However, additionally considering the interconnectivity between biological elements promises a deeper and more coherent understanding of melanoma.,For instance, integrative network-based tools and well-grounded inductive in silico research reveal disease mechanisms, stratify patients, and support treatment individualization.,This review gives an overview of different modeling techniques beyond statistics, shows how different strategies align with the respective medical biology, and identifies possible areas of new computational melanoma research.

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.

ROCK-Myosin II drives fast rounded-amoeboid migration in cancer cells during metastatic dissemination.,Analysis of human melanoma biopsies revealed that amoeboid melanoma cells with high Myosin II activity are predominant in the invasive fronts of primary tumors in proximity to CD206+CD163+ tumor-associated macrophages and vessels.,Proteomic analysis shows that ROCK-Myosin II activity in amoeboid cancer cells controls an immunomodulatory secretome, enabling the recruitment of monocytes and their differentiation into tumor-promoting macrophages.,Both amoeboid cancer cells and their associated macrophages support an abnormal vasculature, which ultimately facilitates tumor progression.,Mechanistically, amoeboid cancer cells perpetuate their behavior via ROCK-Myosin II-driven IL-1α secretion and NF-κB activation.,Using an array of tumor models, we show that high Myosin II activity in tumor cells reprograms the innate immune microenvironment to support tumor growth.,We describe an unexpected role for Myosin II dynamics in cancer cells controlling myeloid function via secreted factors.,•Invasive tumor fronts are enriched in rounded-amoeboid cancer cells high in Myosin II•Myosin II activity in these cells regulates an immunomodulatory secretome•The secreted cytokines and chemokines can induce tumor-promoting macrophages•ROCK-Myosin II and IL-1α/NF-κB cross-talk supports this secretory phenotype,Invasive tumor fronts are enriched in rounded-amoeboid cancer cells high in Myosin II,Myosin II activity in these cells regulates an immunomodulatory secretome,The secreted cytokines and chemokines can induce tumor-promoting macrophages,ROCK-Myosin II and IL-1α/NF-κB cross-talk supports this secretory phenotype,Myosin II activation at the tumor edge promotes invasion and also a secretory phenotype that reshapes the local environment and vasculature to support tumor growth.

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.

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.

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.

RAC1 P29 is the third most commonly mutated codon in human cutaneous melanoma, after BRAF V600 and NRAS Q61.,Here, we study the role of RAC1P29S in melanoma development and reveal that RAC1P29S activates PAK, AKT, and a gene expression program initiated by the SRF/MRTF transcriptional pathway, which results in a melanocytic to mesenchymal phenotypic switch.,Mice with ubiquitous expression of RAC1P29S from the endogenous locus develop lymphoma.,When expressed only in melanocytes, RAC1P29S cooperates with oncogenic BRAF or with NF1-loss to promote tumorigenesis.,RAC1P29S also drives resistance to BRAF inhibitors, which is reversed by SRF/MRTF inhibitors.,These findings establish RAC1P29S as a promoter of melanoma initiation and mediator of therapy resistance, while identifying SRF/MRTF as a potential therapeutic target.,•RAC1P29S activates PAK, AKT, and the SRF/MRTF transcription program in melanocytes•RAC1P29S induces a melanocytic to mesenchymal transition through SRF/MRTF and PAK•RAC1P29S cooperates with BRAF mutation or NF1 deletion to promote melanomagenesis•RAC1P29S induces resistance to BRAF inhibitors through SRF/MRTF,RAC1P29S activates PAK, AKT, and the SRF/MRTF transcription program in melanocytes,RAC1P29S induces a melanocytic to mesenchymal transition through SRF/MRTF and PAK,RAC1P29S cooperates with BRAF mutation or NF1 deletion to promote melanomagenesis,RAC1P29S induces resistance to BRAF inhibitors through SRF/MRTF,RAC1P29S is a common mutation in human cutaneous melanoma.,Lionarons et al. show that RAC1P29S induces a melanocytic to mesenchymal switch via an SRF/MRTF-mediated gene expression program, cooperates with BRAF in melanomagenesis, and drives BRAF inhibitor resistance, which is reversed by SRF/MRTF inhibition.

Autophagy is a dynamic cell survival mechanism by which a double-membrane vesicle, or autophagosome, sequesters portions of the cytosol for delivery to the lysosome for recycling.,This process can be inhibited using the antimalarial agent chloroquine (CQ), which impairs lysosomal function and prevents autophagosome turnover.,Despite its activity, CQ is a relatively inadequate inhibitor that requires high concentrations to disrupt autophagy, highlighting the need for improved small molecules.,To address this, we screened a panel of antimalarial agents for autophagy inhibition and chemically synthesized a novel series of acridine and tetrahydroacridine derivatives.,Structure-activity relationship studies of the acridine ring led to the discovery of VATG-027 as a potent autophagy inhibitor with a high cytotoxicity profile.,In contrast, the tetrahydroacridine VATG-032 showed remarkably little cytotoxicity while still maintaining autophagy inhibition activity, suggesting that both compounds act as autophagy inhibitors with differential effects on cell viability.,Further, knockdown of autophagy-related genes showed no effect on cell viability, demonstrating that the ability to inhibit autophagy is separate from the compound cytotoxicity profiles.,Next, we determined that both inhibitors function through lysosomal deacidification mechanisms and ultimately disrupt autophagosome turnover.,To evaluate the genetic context in which these lysosomotropic inhibitors may be effective, they were tested in patient-derived melanoma cell lines driven by oncogenic BRAF (v-raf murine sarcoma viral oncogene homolog B).,We discovered that both inhibitors sensitized melanoma cells to the BRAF V600E inhibitor vemurafenib.,Overall, these autophagy inhibitors provide a means to effectively block autophagy and have the potential to sensitize mutant BRAF melanomas to first-line therapies.

Tissue-resident memory CD8+ T (Trm) cells mediate potent local innate and adaptive immune responses and play a central role against solid tumors.,However, whether Trm cells cross-talk with dendritic cells (DCs) to support anti-tumor immunity remains unclear.,Here we show that antigen-specific activation of skin Trm cells leads to maturation and migration to draining lymph nodes of cross-presenting dermal DCs.,Tumor rejection mediated by Trm cells triggers the spread of cytotoxic CD8+ T cell responses against tumor-derived neo- and self-antigens via dermal DCs.,These responses suppress the growth of intradermal tumors and disseminated melanoma lacking the Trm cell-targeted epitope.,Moreover, analysis of RNA sequencing data from human melanoma tumors reveals that enrichment of a Trm cell gene signature associates with DC activation and improved survival.,This work unveils the ability of Trm cells to amplify the breath of cytotoxic CD8+ T cell responses through DCs, thereby strengthening anti-tumor immunity.,Immunotherapy can induce antigen spreading of antitumor T cell response, which correlates with better outcomes.,Here the authors show that tissue-resident memory CD8 T cells promote antigen spreading via lysing tumor cells and promoting their uptake and cross-presentation by dendritic cells, thereby eliciting de novo T cell responses.

The infiltration of melanoma tumors by macrophages is often correlated with poor prognosis.,However, the molecular signals that regulate the dialogue between malignant cells and the inflammatory microenvironment remain poorly understood.,We previously reported an increased expression of sphingosine kinase-1 (SK1), which produces the bioactive lipid sphingosine 1-phosphate (S1P), in melanoma.,The present study aimed at defining the role of tumor SK1 in the recruitment and differentiation of macrophages in melanoma.,Herein, we show that downregulation of SK1 in melanoma cells causes a reduction in the percentage of CD206highMHCIIlow M2 macrophages in favor of an increased proportion of CD206lowMHCIIhigh M1 macrophages into the tumor.,This macrophage differentiation orchestrates T lymphocyte recruitment as well as tumor rejection through the expression of Th1 cytokines and chemokines.,In vitro experiments indicated that macrophage migration is triggered by the binding of tumor S1P to S1PR1 receptors present on macrophages whereas macrophage differentiation is stimulated by SK1-induced secretion of TGF-β1.,Finally, RNA-seq analysis of human melanoma tumors revealed a positive correlation between SK1 and TGF-β1 expression.,Altogether, our findings demonstrate that melanoma SK1 plays a key role in the recruitment and phenotypic shift of the tumor macrophages that promote melanoma growth.

With the goal of diagnosing skin cancer in an early and noninvasive way, an extended near infrared multispectral imaging system based on an InGaAs sensor with sensitivity from 995 nm to 1613 nm was built to evaluate deeper skin layers thanks to the higher penetration of photons at these wavelengths.,The outcomes of this device were combined with those of a previously developed multispectral system that works in the visible and near infrared range (414 nm-995 nm).,Both provide spectral and spatial information from skin lesions.,A classification method to discriminate between melanomas and nevi was developed based on the analysis of first-order statistics descriptors, principal component analysis, and support vector machine tools.,The system provided a sensitivity of 78.6% and a specificity of 84.6%, the latter one being improved with respect to that offered by silicon sensors.

Early and accurate diagnosis of melanoma, the deadliest type of skin cancer, has the potential to reduce morbidity and mortality rate.,However, early diagnosis of melanoma is not trivial even for experienced dermatologists, as it needs sampling and laboratory tests which can be extremely complex and subjective.,The accuracy of clinical diagnosis of melanoma is also an issue especially in distinguishing between melanoma and mole.,To solve these problems, this paper presents an approach that makes non-subjective judgements based on quantitative measures for automatic diagnosis of melanoma.,Our approach involves image acquisition, image processing, feature extraction, and classification. 187 images (19 malignant melanoma and 168 benign lesions) were collected in a clinic by a spectroscopic device that combines single-scattered, polarized light spectroscopy with multiple-scattered, un-polarized light spectroscopy.,After noise reduction and image normalization, features were extracted based on statistical measurements (i.e. mean, standard deviation, mean absolute deviation, L1 norm, and L2 norm) of image pixel intensities to characterize the pattern of melanoma.,Finally, these features were fed into certain classifiers to train learning models for classification.,We adopted three classifiers - artificial neural network, naïve bayes, and k-nearest neighbour to evaluate our approach separately.,The naive bayes classifier achieved the best performance - 89% accuracy, 89% sensitivity and 89% specificity, which was integrated with our approach in a desktop application running on the spectroscopic system for diagnosis of melanoma.,Our work has two strengths.,(1) We have used single scattered polarized light spectroscopy and multiple scattered unpolarized light spectroscopy to decipher the multilayered characteristics of human skin.,(2) Our approach does not need image segmentation, as we directly probe tiny spots in the lesion skin and the image scans do not involve background skin.,The desktop application for automatic diagnosis of melanoma can help dermatologists get a non-subjective second opinion for their diagnosis decision.

A newer generation of anti-cancer drugs targeting underlying somatic genetic driver events have resulted in high single-agent or single-pathway response rates in selected patients, but few patients achieve complete responses and a sizeable fraction of patients relapse within a year.,Thus, there is a pressing need for identification of combinations of targeted agents which induce more complete responses and prevent disease progression.,We describe the results of a combination screen of an unprecedented scale in mammalian cells performed using a collection of targeted, clinically tractable agents across a large panel of melanoma cell lines.,We find that even the most synergistic drug pairs are effective only in a discrete number of cell lines, underlying a strong context dependency for synergy, with strong, widespread synergies often corresponding to non-specific or off-target drug effects such as multidrug resistance protein 1 (MDR1) transporter inhibition.,We identified drugs sensitizing cell lines that are BRAFV600E mutant but intrinsically resistant to BRAF inhibitor PLX4720, including the vascular endothelial growth factor receptor/kinase insert domain receptor (VEGFR/KDR) and platelet derived growth factor receptor (PDGFR) family inhibitor cediranib.,The combination of cediranib and PLX4720 induced apoptosis in vitro and tumor regression in animal models.,This synergistic interaction is likely due to engagement of multiple receptor tyrosine kinases (RTKs), demonstrating the potential of drug- rather than gene-specific combination discovery approaches.,Patients with elevated biopsy KDR expression showed decreased progression free survival in trials of mitogen-activated protein kinase (MAPK) kinase pathway inhibitors.,Thus, high-throughput unbiased screening of targeted drug combinations, with appropriate library selection and mechanistic follow-up, can yield clinically-actionable drug combinations.

Knowledge of tumor mutation status is becoming increasingly important for the treatment of cancer, as mutation-specific inhibitors are being developed for clinical use that target only sub-populations of patients with particular tumor genotypes.,Melanoma provides a recent example of this paradigm.,We report here development, validation, and implementation of an assay designed to simultaneously detect 43 common somatic point mutations in 6 genes (BRAF, NRAS, KIT, GNAQ, GNA11, and CTNNB1) potentially relevant to existing and emerging targeted therapies specifically in melanoma.,The test utilizes the SNaPshot method (multiplex PCR, multiplex primer extension, and capillary electrophoresis) and can be performed rapidly with high sensitivity (requiring 5-10% mutant allele frequency) and minimal amounts of DNA (10-20 nanograms).,The assay was validated using cell lines, fresh-frozen tissue, and formalin-fixed paraffin embedded tissue.,Clinical characteristics and the impact on clinical trial enrollment were then assessed for the first 150 melanoma patients whose tumors were genotyped in the Vanderbilt molecular diagnostics lab.,Directing this test to a single disease, 90 of 150 (60%) melanomas from sites throughout the body harbored a mutation tested, including 57, 23, 6, 3, and 2 mutations in BRAF, NRAS, GNAQ, KIT, and CTNNB1, respectively.,Among BRAF V600 mutations, 79%, 12%, 5%, and 4% were V600E, V600K, V600R, and V600M, respectively. 23 of 54 (43%) patients with mutation harboring metastatic disease were subsequently enrolled in genotype-driven trials.,We present development of a simple mutational profiling screen for clinically relevant mutations in melanoma.,Adoption of this genetically-informed approach to the treatment of melanoma has already had an impact on clinical trial enrollment and prioritization of therapy for patients with the disease.