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Uveal melanoma (UM) development and progression is correlated with specific molecular changes.,Recurrent mutations in GNAQ and GNA11 initiate UM development while tumour progression is correlated with monosomy of chromosome 3 and gain of chromosome 8q.,Hence, molecular analysis of UM is useful for diagnosis and prognosis.,The aim of this study is to evaluate the use of digital PCR (dPCR) for molecular analysis of UM.,A series of 66 UM was analysed with dPCR for three hotspot mutations in GNAQ/GNA11 with mutation specific probes.,The status of chromosomes 3 and 8 were analysed with genomic probes.,The results of dPCR analysis were cross-validated with Sanger sequencing, SNP array analysis, and karyotyping.,Using dPCR, we were able to reconstitute the molecular profile of 66 enucleated UM.,With digital PCR, GNAQ/GNA11 mutations were detected in 60 of the 66 UM.,Sanger sequencing revealed three rare variants, and, combined, these assays revealed GNAQ/GNA11 mutations in 95% of UM.,Monosomy 3 was present in 43 and chromosome 8 aberrations in 52 of the 66 UM.,Survival analysis showed that increasing 8q copy numbers were positively correlated with metastasis risk.,Molecular analysis with dPCR is fast and sensitive.,Just like the recurrent genomic aberrations of chromosome 3 and 8, hotspot mutations in GNAQ and GNA11 are effectively detected in heterogeneous samples.,Increased sensitivity contributes to the number of mutations and chromosomal aberrations detected.,Moreover, quantification of copy number with dPCR validated 8q dosage as a sensitive prognostic tool in UM, of which implementation in disease prediction models will further improve prognostication.

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.

Preclinical data suggest the combination of an anti-programmed death receptor 1 antibody plus dabrafenib and trametinib to have superior antitumor activity compared with dabrafenib plus trametinib alone.,These observations are supported by translational evidence suggesting that immune checkpoint inhibitors plus targeted therapy may improve treatment outcomes in patients with BRAF V600-mutant metastatic melanoma.,COMBI-i is a phase III trial evaluating spartalizumab, an anti-programmed death receptor 1 antibody, in combination with dabrafenib and trametinib (sparta-DabTram), versus placebo plus dabrafenib and trametinib (placebo-DabTram) in patients with BRAF V600-mutant unresectable or metastatic melanoma.,Patients received spartalizumab 400 mg intravenously every 4 weeks plus dabrafenib 150 mg orally twice daily and trametinib 2 mg orally once daily or placebo-DabTram.,Participants were age ≥ 18 years with unresectable or metastatic BRAF V600-mutant melanoma.,The primary end point was investigator-assessed progression-free survival.,Overall survival was a key secondary end point (ClinicalTrials.gov identifier: NCT02967692).,At data cutoff (July 1, 2020), the median progression-free survival was 16.2 months (95% CI, 12.7 to 23.9 months) in the sparta-DabTram arm versus 12.0 months (95% CI, 10.2 to 15.4 months) in the placebo-DabTram arm (hazard ratio, 0.82 [95% CI, 0.66 to 1.03]; P = .042 [one-sided; nonsignificant]).,The objective response rates were 69% (183 of 267 patients) versus 64% (170 of 265 patients), respectively.,Grade ≥ 3 treatment-related adverse events occurred in 55% (146 of 267) of patients in the sparta-DabTram arm and 33% (88 of 264) in the placebo-DabTram arm.,The study did not meet its primary end point; broad first-line use of sparta-DabTram is not supported by these results.,Further biomarker-driven investigation may identify patient subpopulations who could benefit from checkpoint inhibitor plus targeted therapy combinations.

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.

Melanoma represents a significant malignancy in humans and dogs.,Different from genetically engineered models, sporadic canine melanocytic neoplasms share several characteristics with human disease that could make dogs a more relevant preclinical model.,Canine melanomas rarely arise in sun-exposed sites.,Most occur in the oral cavity, with a subset having intra-epithelial malignant melanocytes mimicking the in situ component of human mucosal melanoma.,The spectrum of canine melanocytic neoplasia includes benign lesions with some analogy to nevi, as well as invasive primary melanoma, and widespread metastasis.,Growing evidence of distinct subtypes in humans, differing in somatic and predisposing germ-line genetic alterations, cell of origin, epidemiology, relationship to ultraviolet radiation and progression from benign to malignant tumors, may also exist in dogs.,Canine and human mucosal melanomas appear to harbor BRAF, NRAS, and c-kit mutations uncommonly, compared with human cutaneous melanomas, although both species share AKT and MAPK signaling activation.,We conclude that there is significant overlap in the clinical and histopathological features of canine and human mucosal melanomas.,This represents opportunity to explore canine oral cavity melanoma as a preclinical model.

Recent studies suggested a role for the human endogenous retrovirus (HERV) group HERV-K(HML-2) in melanoma because of upregulated transcription and expression of HERV-K(HML-2)-encoded proteins.,Very little is known about which HML-2 loci are transcribed in melanoma.,We assigned >1,400 HML-2 cDNA sequences generated from various melanoma and related samples to genomic HML-2 loci, identifying a total of 23 loci as transcribed.,Transcription profiles of loci differed significantly between samples.,One locus was found transcribed only in melanoma-derived samples but not in melanocytes and might represent a marker for melanoma.,Several of the transcribed loci harbor ORFs for retroviral Gag and/or Env proteins.,Env-encoding loci were transcribed only in melanoma.,Specific investigation of rec and np9 transcripts indicated transcription of protein encoding loci in melanoma and melanocytes hinting at the relevance of Rec and Np9 in melanoma.,UVB irradiation changed transcription profiles of loci and overall transcript levels decreased in melanoma and melanocytes.,We further identified transcribed HML-2 loci formed by reverse transcription of spliced HML-2 transcripts by L1 machinery or in a retroviral fashion, with loci potentially encoding HML-2-like proteins.,We reveal complex, sample-specific transcription of HML-2 loci in melanoma and related samples.,Identified HML-2 loci and proteins encoded by those loci are particularly relevant for further studying the role of HML-2 in melanoma.,Transcription of HERVs appears as a complex mechanism requiring specific studies to elucidate which HERV loci are transcribed and how transcribed HERVs may be involved in disease.

Reactive oxygen species (ROS/RNS) are produced during inflammation and elicit protein modifications, but the immunological consequences are largely unknown.,Gas plasma technology capable of generating an unmatched variety of ROS/RNS is deployed to mimic inflammation and study the significance of ROS/RNS modifications using the model protein chicken ovalbumin (Ova vs oxOva).,Dynamic light scattering and circular dichroism spectroscopy reveal structural modifications in oxOva compared to Ova.,T cells from Ova‐specific OT‐II but not from C57BL/6 or SKH‐1 wild type mice presents enhanced activation after Ova addition.,OxOva exacerbates this activation when administered ex vivo or in vivo, along with an increased interferon‐gamma production, a known anti‐melanoma agent.,OxOva vaccination of wild type mice followed by inoculation of syngeneic B16F10 Ova‐expressing melanoma cells shows enhanced T cell number and activation, decreased tumor burden, and elevated numbers of antigen‐presenting cells when compared to their Ova‐vaccinated counterparts.,Analysis of oxOva using mass spectrometry identifies three hot spots regions rich in oxidative modifications that are associated with the increased T cell activation.,Using Ova as a model protein, the findings suggest an immunomodulating role of multi‐ROS/RNS modifications that may spur novel research lines in inflammation research and for vaccination strategies in oncology.,Successful vaccination requires increased immunogenicity of antigens.,Cold physical plasmas produce reactive oxygen and nitrogen species capable of eliciting oxidative protein modifications.,Oxidation of ovalbumin (Ova) shows enhanced T cell activation, elevated numbers of antigen‐presenting cells, and decreases tumor burden in vivo upon vaccination.

The Warburg effect in tumour cells is associated with the upregulation of glycolysis to generate ATP, even under normoxic conditions and the presence of fully functioning mitochondria.,However, scientific advances made over the past 15 years have reformed this perspective, demonstrating the importance of oxidative phosphorylation (OXPHOS) as well as glycolysis in malignant cells.,The metabolic phenotypes in melanoma display heterogeneic dynamism (metabolic plasticity) between glycolysis and OXPHOS, conferring a survival advantage to adapt to harsh conditions and pathways of chemoresistance.,Furthermore, the simultaneous upregulation of both OXPHOS and glycolysis (metabolic symbiosis) has been shown to be vital for melanoma progression.,The tumour microenvironment (TME) has an essential supporting role in promoting progression, invasion and metastasis of melanoma.,Mesenchymal stromal cells (MSCs) in the TME show a symbiotic relationship with melanoma, protecting tumour cells from apoptosis and conferring chemoresistance.,With the significant role of OXPHOS in metabolic plasticity and symbiosis, our review outlines how mitochondrial transfer from MSCs to melanoma tumour cells plays a key role in melanoma progression and is the mechanism by which melanoma cells regain OXPHOS capacity even in the presence of mitochondrial mutations.,The studies outlined in this review indicate that targeting mitochondrial trafficking is a potential novel therapeutic approach for this highly refractory disease.

Uveal melanoma (UM) is a lethal intraocular malignancy with an average survival of only 2~8 months in patients with hepatic metastasis.,Currently, there is no effective therapy for metastatic UM.,Here, we reported that niclosamide, an effective repellence of tapeworm that has been approved for use in patients for approximately 50 years, exhibited strong antitumor activity in UM cells in vitro and in vivo.,We showed that niclosamide potently inhibited UM cell proliferation, induced apoptosis and reduced migration and invasion. p-Niclosamide, a water-soluble niclosamide, exerted potent in vivo antitumor activity in a UM xenograft mouse model.,Mechanistically, niclosamide abrogated the activation of the NF-κB pathway induced by tumor necrosis factor α (TNFα) in UM cells, while niclosamide elevated the levels of intracellullar and mitochondrial reactive oxygen species (ROS) in UM cells.,Quenching ROS by N-acetylcysteine (NAC) weakened the ability of niclosamide-mediated apoptosis.,Matrix metalloproteinase 9 (MMP-9) knockdown by shRNA potentiated, while ectopic expression of MMP-9 rescued, the niclosamide-attenuated invasion, implying that MMP-9 is pivotal for invasion blockage by niclosamide in UM cells.,Furthermore, our results showed that niclosamide eliminated cancer stem-like cells (CSCs) as reflected by a decrease in the Aldefluor+ percentage and serial re-plating melanosphere formation, and these phenotypes were associated with the suppressed Wnt/β-catenin pathway by niclosamide in UM.,Niclosamide caused a dose- and time-dependent reduction of β-catenin and the key components [e.g., DVLs, phospho-GSK3β (S9), c-Myc and Cyclin D1] in the canonical Wnt/β-catenin pathway.,Additionally, niclosamide treatment in UM cells reduced ATP and cAMP contents, and decreased PKA-dependent phosphorylation of β-catenin at S552 and S675 which determine the stability of β-catenin protein, suggesting that niclosamide may work as a mitochondrial un-coupler.,Taken together, our results shed light on the mechanism of antitumor action of niclosamide and warrant clinical trial for treatment of UM patients.

Transcription factors regulating the epithelial-to-mesenchymal transition (EMT) program contribute to carcinogenesis and metastasis in many tumors, including cutaneous melanoma.,However, little is known about the role of EMT factors in the growth and metastatic dissemination of uveal melanoma cells.,Here, we analyzed the expression and functions of the EMT factors ZEB1, Twist1, and Snail1 in uveal melanoma cell lines and primary tumors.,ZEB1, Twist1, and Snail1 mRNA levels were measured using qPCR in five uveal melanoma cell lines and in 30 primary tumors.,Gene expression was used to determine class 1 and class 2 signatures in the primary tumors.,Short hairpin RNA was used to downregulate the expressions of the EMT factors; then, growth and transwell invasion assays were performed.,ZEB1, Twist1, and Snail1 were expressed in all five uveal melanoma lines, with ZEB1 having the highest protein levels.,ZEB1 mRNA was significantly elevated in highly metastatic class 2 primary tumors for which survival data were not available, whereas a high gene expression of Twist1 was associated with a worse prognosis in a separate tumor cohort analyzed by expression profiling.,The genetic downregulation of ZEB1 in OCM1, OMM1, and 92.1 resulted in a more than 50% reduction in invasion, but only suppressed growth in OMM1 cells.,Suppression of Twist1 in Mel290 and OMM1 reduced growth and invasion by more than 50%.,The downregulation of Snail1 in the 92.1 cell line reduced invasion by 50%, but did not interfere with growth.,The downregulation of ZEB1, Twist1, and Snail1 reduces the invasive properties of uveal melanoma cells, and the elevated mRNA levels of ZEB1 and Twist1 are associated with a more aggressive clinical phenotype in uveal melanoma samples.,Therefore, these factors could represent new therapeutic targets in patients with ocular melanoma.

Uveal melanoma (UM) typically spreads to the liver, where it is incurable, as there are limited therapeutic interventions available.,This study aimed to standardize laboratory methods for generating three-dimensional (3D) spheroids using UM cell lines and primary UM (PUM) samples for use in drug screening.,Six UM cell lines and nine PUM, of differing genetic characteristics were cultured in two dimensions (2D) and three dimensions.,3D spheroid formation and growth were time monitored, and ImageJ software was used to calculate cross-sectional areas.,PUM spheroids underwent immunohistochemistry for melanoma markers, nuclear BAP1, and cell proliferation.,Chromosomal alterations in patient UM biopsies were compared with the corresponding 3D spheroid.,In vitro drug assays testing doxorubicin and selumetinib assessed drug penetration and toxicity after 48 hours using imaging and the CellTiter-Glo 3D Cell Viability Assay.,All six UM cell lines formed spheroids of varying sizes and compactness; six of the nine PUM samples (67%) also formed spheroids, composed of MelanA+ proliferating melanocytes and admixed macrophages.,PUM spheroids were genetically identical to the original sampled tumor.,In vitro drug assays showed varying penetrations into UM cell line spheroids, with doxorubicin passing into the spheroid core and selumetinib having an effect largely on peripheral cells.,Both drugs caused a dose-dependent reduction in viability of 3D spheroid cells.,UM cell lines and PUM samples can successfully generate uniform 3D spheroids.,PUM spheroids retain histological and genetic characteristics of the primary tumor.,3D spheroids are an important system for use in future high-throughput drug testing.,The use of 3D spheroids allows early-phase drug screening and is an important first step toward treatment personalization for UM patients.

Despite the general focus on an invasive and de-differentiated phenotype as main driver of cancer metastasis, in melanoma patients many metastatic lesions display a high degree of pigmentation, indicative for a differentiated phenotype.,Indeed, studies in mice and fish show that melanoma cells switch to a differentiated phenotype at secondary sites, possibly because in melanoma differentiation is closely linked to proliferation through the lineage-specific transcriptional master regulator MITF.,Importantly, while a lot of effort has gone into identifying factors that induce the de-differentiated/invasive phenotype, it is not well understood how the switch to the differentiated/proliferative phenotype is controlled.,We identify collagen as a contributor to this switch.,We demonstrate that collagen stiffness induces melanoma differentiation through a YAP/PAX3/MITF axis and show that in melanoma patients increased collagen abundance correlates with nuclear YAP localization.,However, the interrogation of large patient datasets revealed that in the context of the tumour microenvironment, YAP function is more complex.,In the absence of fibroblasts, YAP/PAX3-mediated transcription prevails, but in the presence of fibroblasts tumour growth factor-β suppresses YAP/PAX3-mediated MITF expression and induces YAP/TEAD/SMAD-driven transcription and a de-differentiated phenotype.,Intriguingly, while high collagen expression is correlated with poorer patient survival, the worst prognosis is seen in patients with high collagen expression, who also express MITF target genes such as the differentiation markers TRPM1, TYR and TYRP1, as well as CDK4.,In summary, we reveal a distinct lineage-specific route of YAP signalling that contributes to the regulation of melanoma pigmentation and uncovers a set of potential biomarkers predictive for poor survival.

Resistance to targeted cancer therapies is an important clinical problem.,The discovery of anti-resistance drug combinations is challenging as resistance can arise by diverse escape mechanisms.,To address this challenge, we improved and applied the experimental-computational perturbation biology method.,Using statistical inference, we build network models from high-throughput measurements of molecular and phenotypic responses to combinatorial targeted perturbations.,The models are computationally executed to predict the effects of thousands of untested perturbations.,In RAF-inhibitor resistant melanoma cells, we measured 143 proteomic/phenotypic entities under 89 perturbation conditions and predicted c-Myc as an effective therapeutic co-target with BRAF or MEK.,Experiments using the BET bromodomain inhibitor JQ1 affecting the level of c-Myc protein and protein kinase inhibitors targeting the ERK pathway confirmed the prediction.,In conclusion, we propose an anti-cancer strategy of co-targeting a specific upstream alteration and a general downstream point of vulnerability to prevent or overcome resistance to targeted drugs.,DOI:http://dx.doi.org/10.7554/eLife.04640.001,Drugs that target the activity of specific genes could potentially form precise cancer therapies.,Some cancers, including the aggressive skin cancer called melanoma, initially respond well to such treatments.,However, resistance to drugs develops quickly, leading to the rapid regrowth of the tumors.,Resistance can develop in a number of ways.,For example, to prevent the drug from working or to compensate for the effects of a drug, cancer cells can adapt their signaling processes or acquire genetic mutations or other modifications that affect how genes are expressed.,A well-designed combination of drugs that targets multiple molecular pathways can make it harder for cells to resist treatment, as this limits the number of available ‘escape’ pathways that bypass the drug targets.,However, it is difficult to accurately predict how a cell will respond when treated with a particular drug, making it extremely challenging to design effective drug combinations.,In 2013, researchers developed a technique to build predictive models of cellular response pathways based on data collected from perturbation experiments followed by mathematical modeling.,Now, Korkut et al.-including several of the researchers involved in the 2013 work-have refined this technology and applied it to the problem of preventing drug resistance in cancer cells.,Computer simulations that used the mathematical models suggested a particular strategy of ‘upstream-downstream targeting’ in cells that were insensitive to the clinically successful drug vemurafenib (an inhibitor of RAF proteins, which are often mutated in cancers).,In the landscape of signaling pathways, the target of the upstream drug is on or near the mutated RAF protein. c-Myc, the indirect target of the downstream drug helps to express genes that trigger signals that cause the cells to grow.,Inhibiting both targets in parallel may have the dual advantage of blocking the activation of the tumor-specific growth pathway while reducing the cancer cells' attempts to bypass the activation block.,An initial test of the designed drug combination required moving from computer simulations to the laboratory using cell cultures originally derived from melanoma tumors.,When Korkut et al. applied the drug combination, the combined treatment successfully blocked cell growth.,The results suggest that the data-driven computer modeling strategy termed perturbation biology could be a useful tool for identifying effective cancer drug combinations for further preclinical research, possibly followed by clinical trials.,DOI:http://dx.doi.org/10.7554/eLife.04640.002

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.

Talimogene laherparepvec (T-VEC) is an oncolytic immunotherapy designed to induce tumor regression of injected lesions through direct lytic effects, and of uninjected lesions through induction of systemic antitumor immunity.,In this study, we describe the patterns and time course of response to T-VEC from the phase III OPTiM trial of 436 patients with unresected stages IIIB-IV melanoma.,Lesion-level response analyses were performed based on the type of lesion (injected or uninjected cutaneous, subcutaneous, or nodal lesions; or visceral lesions [uninjected]), and the best percentage change from baseline of the sum of products of the longest diameters was calculated.,Patients randomized to T-VEC (n = 295) who experienced a durable response (continuous partial or complete response for ≥6 months) were evaluated for progression prior to response (PPR), defined as the appearance of a new lesion or >25 % increase in total baseline tumor area.,T-VEC resulted in a decrease in size by ≥50 % in 64 % of injected lesions (N = 2116), 34 % of uninjected non-visceral lesions (N = 981), and 15 % of visceral lesions (N = 177).,Complete resolution of lesions occurred in 47 % of injected lesions, 22 % of uninjected non-visceral lesions, and 9 % of visceral lesions.,Of 48 patients with durable responses, 23 (48 %) experienced PPR, including 14 who developed new lesions only.,No difference in overall survival was observed, and median duration of response was not reached in patients with PPR versus those without PPR.,Responses in uninjected lesions provide validation of T-VEC-induced systemic immunotherapeutic effects against melanoma.,PPR did not negatively impact the clinical effectiveness of T-VEC.,The online version of this article (doi:10.1245/s10434-016-5286-0) contains supplementary material, which is available to authorized users.

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

Cutaneous melanoma is epidemiologically linked to ultraviolet radiation (UVR), but the molecular mechanisms by which UVR drives melanomagenesis remain unclear1,2.,The most common somatic mutation in melanoma is a V600E substitution in BRAF, which is an early event3.,To investigate how UVR accelerates oncogenic BRAF-driven melanomagenesis, we used a V600EBRAF mouse model.,In mice expressing V600EBRAF in their melanocytes, a single dose of UVR that mimicked mild sunburn in humans induced clonal expansion of the melanocytes, and repeated doses of UVR increased melanoma burden.,We show that sunscreen (UVA superior: UVB SPF50) delayed the onset of UVR-driven melanoma, but only provided partial protection.,The UVR-exposed tumours presented increased numbers of single nucleotide variants (SNVs) and we observed mutations (H39Y, S124F, R245C, R270C, C272G) in the Trp53 tumour suppressor in ~40% of cases.,TP53 is an accepted UVR target in non-melanoma skin cancer, but is not thought to play a major role in melanoma4.,However, we show that mutant Trp53 accelerated V600EBRAF-driven melanomagenesis and that TP53 mutations are linked to evidence of UVR-induced DNA damage in human melanoma.,Thus, we provide mechanistic insight into epidemiological data linking UVR to acquired naevi in humans5.,We identify TP53/Trp53 as a UVR-target gene that cooperates with V600EBRAF to induce melanoma, providing molecular insight into how UVR accelerates melanomagenesis.,Our study validates public health campaigns that promote sunscreen protection for individuals at risk of melanoma.

Adoptive transfer of large numbers of tumor-reactive CD8+ cytotoxic T lymphocytes (CTLs) expanded and differentiated in vitro has shown promising clinical activity against cancer.,However, such protocols are complicated by extensive ex vivo manipulations of tumor-reactive cells and have largely focused on CD8+ CTLs, with much less emphasis on the role and contribution of CD4+ T cells.,Using a mouse model of advanced melanoma, we found that transfer of small numbers of naive tumor-reactive CD4+ T cells into lymphopenic recipients induces substantial T cell expansion, differentiation, and regression of large established tumors without the need for in vitro manipulation.,Surprisingly, CD4+ T cells developed cytotoxic activity, and tumor rejection was dependent on class II-restricted recognition of tumors by tumor-reactive CD4+ T cells.,Furthermore, blockade of the coinhibitory receptor CTL-associated antigen 4 (CTLA-4) on the transferred CD4+ T cells resulted in greater expansion of effector T cells, diminished accumulation of tumor-reactive regulatory T cells, and superior antitumor activity capable of inducing regression of spontaneous mouse melanoma.,These findings suggest a novel potential therapeutic role for cytotoxic CD4+ T cells and CTLA-4 blockade in cancer immunotherapy, and demonstrate the potential advantages of differentiating tumor-reactive CD4+ cells in vivo over current protocols favoring in vitro expansion and differentiation.

To investigate the efficacy and safety of concurrent stereotactic radiosurgery (SRS) and ipilimumab or nivolumab in patients with untreated melanoma brain metastases.,Eighty consecutive patients with 326 melanoma brain metastases receiving SRS in combination with ipilimumab or nivolumab were identified from an institutional database and retrospectively evaluated.,Patients started systemic treatment with intravenous nivolumab or ipilimumab within one week of receiving SRS.,Nivolumab was given at doses of 3 mg/kg every two weeks.,Ipilimumab was administered up to four doses of 10 mg/kg, one every 3 weeks, then patients had a maintenance dose of 10 mg/kg every 12 weeks, until disease progression or inacceptable toxicity.,Primary endpoint of the study was intracranial progression-free survival (PFS).,Secondary endpoints were extracranial PFS, overall survival (OS), and neurological toxicity.,Eighty patients were analyzed.,Forty-five patients received SRS and ipilimumab, and 35 patients received SRS and nivolumab.,With a median follow-up of 15 months, the 6-month and 12-month intracranial PFS rates were 69% (95%CI,54-87%) and 42% (95%CI,24-65%) for patients receiving SRS and nivolumab and 48% (95%CI,34-64%) and 17% (95%CI,5-31%) for those treated with SRS and ipilimumab (p = 0.02), respectively.,Extracranial PFS and OS were 37 and 78% in SRS and nivolumab group, respectively, and 17 and 68% in SRS and ipilimumab group, respectively, at 12 months.,Sub-group analysis showed significantly better intracranial PFS for patients receiving multi-fraction SRS (3 × 9 Gy) compared to single-fraction SRS (70% versus 46% at 6 months, p = 0.01), especially in combination with nivolumab.,Grade 3 treatment-related adverse events occurred in 11 (24%) patients treated with SRS and ipilimumab and 6 (17%) patients who received SRS and nivolumab.,Radiation-induced brain necrosis (RN) occurred in 15% of patients.,Concurrent SRS and ipilimumab or nivolumab show meaningful intracranial activity in patients with either asymptomatic and symptomatic melanoma brain metastases, although a subset of patients may develop symptomatic RN.,The combination of nivolumab with SRS is associated with better intracranial control.,The online version of this article (10.1186/s40425-019-0588-y) contains supplementary material, which is available to authorized users.

Immune checkpoint inhibitors1 result in impressive clinical responses2-5 but optimal results will require combination with each other6 and other therapies.,This raises fundamental questions about mechanisms of non-redundancy and resistance.,Here, we report major tumor regressions in a subset of patients with metastatic melanoma treated with an anti-CTLA4 antibody (anti-CTLA4) and radiation (RT) and reproduced this effect in mouse models.,Although combined treatment improved responses in irradiated and unirradiated tumors, resistance was common.,Unbiased analyses of mice revealed that resistance was due to upregulation of PD-L1 on melanoma cells and associated with T cell exhaustion.,Accordingly, optimal response in melanoma and other cancer types requires RT, anti-CTLA4, and anti-PD-L1/PD-1.,Anti-CTLA4 predominantly inhibits T regulatory cells (Tregs) to increase the CD8 T cell to Treg (CD8/Treg) ratio.,RT enhances the diversity of the T cell receptor (TCR) repertoire of intratumoral T cells.,Together, anti-CTLA4 promotes expansion of T cells, while RT shapes the TCR repertoire of the expanded peripheral clones.,Addition of PD-L1 blockade reverses T cell exhaustion to mitigate depression in the CD8/Treg ratio and further encourages oligo-clonal T cell expansion.,Similar to results from mice, patients on our clinical trial with melanoma showing high PD-L1 did not respond to RT + anti-CTLA4, demonstrated persistent T cell exhaustion, and rapidly progressed.,Thus, PD-L1 on melanoma cells allows tumors to escape anti-CTLA4-based therapy, and the combination of RT, anti-CTLA4, and anti-PD-L1 promotes response and immunity through distinct mechanisms.

Melanoma, which is usually induced by ultraviolet light exposure and the following DNA damage, is the most dangerous skin cancer.,The purpose of the present study was to screen key molecules involved in melanoma.,Microarray data of E-MTAB-1862 were downloaded from the ArrayExpress database, which included 21 primary melanoma samples and 11 benign nevus samples.,In addition, the RNASeq version 2 and microRNA (miRNA) sequencing data of cutaneous melanoma were downloaded from The Cancer Genome Atlas database.,After identifying the differentially expressed genes (DEGs) using Limma package, enrichment analysis and protein-protein interaction (PPI) network analysis were performed separately for them using DAVID software and Cytoscape software.,In addition, survival analysis and regulatory network analysis were further performed by log-rank test and Cytoscape software, respectively.,Moreover, real-time reverse transcription polymerase chain reaction (RT-PCR) was performed to further verify the expression patterns of several selected DEGs.,A total of 382 DEGs were identified in primary melanoma samples, including 206 upregulated genes and 176 downregulated genes.,Functional enrichment analysis showed that COL17A1 was enriched in epidermis development.,In the PPI network, CXCL8 (degree = 29) and STAT1 (degree = 28) had higher degrees and could interact with each other.,Survival analysis showed that 21 DEGs, 55 long noncoding RNAs (lncRNAs) and 32 miRNAs were found to be associated with prognosis.,Furthermore, several regulatory relationships were found in the lncRNA-gene regulatory network (such as RP11-361L15.4 targeting COL17A1) and the miRNA-gene regulatory network (such as hsa-miR-375 targeting CCL27 and hsa-miR-375 targeting insulin-like growth factor 1 receptor [IGF1R]).,Real-time RT-PCR results showed that the overall direction of differential expression was consistent except COL17A1.,CXCL8 interacted with STAT1, CCL27, and IGF1R targeted by hsa-miR-375, and COL17A1 targeted by RP11-361L15.4 might function in the development and progression of melanoma, which should be verified by more detailed experiments.

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

Tumor cells evade the immune surveillance by up-regulating surface expression of PD-L1, which interacts with PD-1 on T cells to elicit the immune checkpoint response1,2.,Anti-PD-1 antibodies have shown remarkable promise in treating tumors, including metastatic melanoma2-4.,However, patient response rate is low4,5.,A better understanding of PD-L1-mediated immune evasion is needed to predict patient response and improve treatment efficacy.,Here we report that metastatic melanoma releases a high level of extracellular vesicles (EVs), mostly in the form of exosomes, that carry PD-L1 on their surface.,Interferon-γ (IFN-γ) up-regulates PD-L1 on these vesicles, which suppresses the function of CD8 T cells and facilitates tumor growth.,In patients with metastatic melanoma, the level of circulating exosomal PD-L1 positively correlates with that of IFN-γ, and changes during the course of anti-PD-1 therapy.,The magnitudes of the early on-treatment increase in circulating exosomal PD-L1, as an indicator of the adaptive response of the tumor cells to T cell re-invigoration, stratifies clinical responders from non-responders.,Our study unveils a mechanism by which tumor cells systemically suppress the immune system, and provides a rationale for the application of exosomal PD-L1 as a predictor for anti-PD-1 therapy.

Exosomes are extracellular vesicles released by various cell types and play roles in cell-cell communication.,Several studies indicate that cancer cell‐derived exosomes play important pathophysiological roles in tumor progression.,Biodistribution of cancer cell‐derived exosomes in tumor tissue is an important factor for determining their role in tumor proliferation; however, limited studies have assessed the biodistribution of exosomes in tumor tissues.,In the present study, we examined the effect of cancer‐cell derived exosomes on tumor growth by analyzing their biodistribution.,Murine melanoma B16BL6‐derived exosomes increased the proliferation and inhibited the apoptosis of B16BL6 cells, which was associated with an increase and decrease in the levels of proliferation‐ and apoptosis‐related proteins, respectively.,GW4869‐induced inhibition of exosome secretion decreased the proliferation of B16BL6 cells, and treatment of GW4869‐treated cells with B16BL6‐derived exosomes restored their proliferation.,Next, we treated B16BL6 tumors in mice with B16BL6‐derived exosomes and examined the biodistribution and cellular uptake of these exosomes.,After the intratumoral injection of radiolabeled B16BL6‐derived exosomes, most radioactivity was detected within the tumor tissues of mice.,Fractionation of cells present in the tumor tissue showed that fluorescently labeled exosomes were mainly taken up by B16BL6 cells.,Moreover, intratumoral injection of B16BL6‐derived exosomes promoted tumor growth, whereas intratumoral injection of GW4869 suppressed tumor growth.,These results indicate that B16BL6 cells secrete and take up their own exosomes to induce their proliferation and inhibit their apoptosis, which promotes tumor progression.

Most genetic susceptibility to cutaneous melanoma remains to be discovered.,Meta-analysis genome-wide association study (GWAS) of 36,760 melanoma cases (67% newly-genotyped) and 375,188 controls identified 54 significant loci with 68 independent SNPs.,Analysis of risk estimates across geographical regions and host factors suggests the acral melanoma subtype is uniquely unrelated to pigmentation.,Combining this meta-analysis with nevus count and hair color GWAS, and transcriptome association approaches, uncovered 31 potential secondary loci, for a total of 85 cutaneous melanoma susceptibility loci.,These findings provide substantial insights into cutaneous melanoma genetic architecture, reinforcing the importance of nevogenesis, pigmentation, and telomere maintenance together with identifying potential new pathways for cutaneous melanoma pathogenesis.

Melanocytic naevi are an important risk factor for melanoma.,Naevi with distinct dermoscopic patterns can differ in size, distribution and host pigmentation characteristics.,We examined MC1R and 85 other candidate loci in a cohort of children to test the hypothesis that the development and dermoscopic type of naevi are modulated by genetic variants.,Buccal DNAs were obtained from a cohort of 353 fifth graders (mean age 10·4 years).,Polymorphisms were chosen based on a known or anticipated role in naevi and melanoma.,Associations between single-nucleotide polymorphisms (SNPs) and baseline naevus count were determined by multivariate regression adjusting for sex, race/ethnicity and sun sensitivity.,Dermoscopic images were available for 853 naevi from 290 children.,Associations between SNPs and dermoscopic patterns were determined by polytomous regression.,Four SNPs were significantly associated with increasing (IRF4) or decreasing (PARP1, CDK6 and PLA2G6) naevus count in multivariate shrinkage analyses with all SNPs included in the model; IRF4 rs12203952 showed the strongest association with log naevus count (relative risk 1·56, P < 0·001).,Using homogeneous naevi as the reference, IRF4 rs12203952 and four other SNPs in TERT, CDKN1B, MTAP and PARP1 were associated with either globular or reticular dermoscopic patterns (P < 0·05).,Our results provide evidence that subsets of naevi defined by dermoscopic patterns differ in their associations with germline genotypes and support the hypothesis that dermoscopically defined subsets of naevi are biologically distinct.,These results require confirmation in larger cohorts.,If confirmed, these findings will improve the current knowledge of naevogenesis and assist in the identification of individuals with high-risk phenotypes.

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.

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

MiRNAs are increasingly recognized as biomarkers for the diagnosis of cancers where they are profiled from tumor tissue (intracellular miRNAs) or serum/plasma samples (extracellular miRNAs).,To improve detection of reliable biomarkers from blood samples, we first compiled a healthy reference miRNome and established a well-controlled analysis pipeline allowing for standardized quantification of circulating miRNAs.,Using whole miRNome and custom qPCR arrays, miRNA expression profiles were analyzed in 126 serum, whole blood and tissue samples of healthy volunteers and melanoma patients and in primary melanocyte and keratinocyte cell lines.,We found characteristic signatures with excellent prognostic scores only in late stage but not in early stage melanoma patients.,Upon comparison of melanoma tissue miRNomes with matching serum samples, several miRNAs were identified to be exclusively tissue-derived (miR-30b-5p, miR-374a-5p and others) while others had higher expression levels in serum (miR-3201 and miR-122-5p).,Here we have compiled a healthy and widely applicable miRNome from serum samples and we provide strong evidence that levels of cell-free miRNAs only change significantly at later stages of melanoma progression, which has serious implications for miRNA biomarker studies in cancer.

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.

Transcriptomic signatures designed to predict melanoma patient responses to PD-1 blockade have been reported but rarely validated.,We now show that intra-patient heterogeneity of tumor responses to PD-1 inhibition limit the predictive performance of these signatures.,We reasoned that resistance mechanisms will reflect the tumor microenvironment, and thus we examined PD-1 inhibitor resistance relative to T-cell activity in 94 melanoma tumors collected at baseline and at time of PD-1 inhibitor progression.,Tumors were analyzed using RNA sequencing and flow cytometry, and validated functionally.,These analyses confirm that major histocompatibility complex (MHC) class I downregulation is a hallmark of resistance to PD-1 inhibitors and is associated with the MITFlow/AXLhigh de-differentiated phenotype and cancer-associated fibroblast signatures.,We demonstrate that TGFß drives the treatment resistant phenotype (MITFlow/AXLhigh) and contributes to MHC class I downregulation in melanoma.,Combinations of anti-PD-1 with drugs that target the TGFß signaling pathway and/or which reverse melanoma de-differentiation may be effective future therapeutic strategies.,A significant proportion of patients develop innate or acquired resistance to immune checkpoint inhibitors.,Here, the authors show that resistance to anti-PD-1 blockade is associated with TGF-beta driven major histocompatibility complex I (MHCI) down-regulation and a de-differentiated phenotype in melanoma patients.

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.

A high number of nevi is the most significant phenotypic risk factor for melanoma and is in part genetically determined.,The number of nevi decreases from middle age onward but this senescence can be delayed in patients with melanoma.,We investigated the effects of nevus number count on sentinel node status and melanoma survival in a large cohort of melanoma cases.,Out of 2,184 melanoma cases, 684 (31.3%) had a high nevus count (>50).,High nevus counts were associated with favorable prognostic factors such as lower Breslow thickness, less ulceration and lower mitotic rate, despite adjustment for age.,Nevus count was not predictive of sentinel node status.,The crude 5‐ and 10‐year melanoma‐specific survival rate was higher in melanomas cases with a high nevus count compared to those with a low nevus count (91.2 vs.,86.4% and 87.2 vs. 79%, respectively).,The difference in survival remained significant after adjusting for all known melanoma prognostic factors (hazard ratio [HR] = 0.43, confidence interval [CI] = 0.21-0.89).,The favorable prognostic value of a high nevus count was also seen within the positive sentinel node subgroup of patients (HR = 0.22, CI = 0.08-0.60).,High nevus count is associated with a better melanoma survival, even in the subgroup of patients with positive sentinel lymph node.,This suggests a different biological behavior of melanoma tumors in patients with an excess of nevi.,What's new?,Having a lot of pigmented skin lesions known as naevi or moles is a known risk factor for melanoma, but is it also a bad prognostic marker for melanoma patients?,The authors looked at the impact of nevus counts on melanoma survival in more than 2000 patients and found the opposite: high nevus counts were associated with favorable prognostic markers and a higher 5‐ and 10‐year survival rate.,These findings point to biological factor associated with high nevus count that may confer improved survival and may be exploited therapeutically in the future.

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.

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

Autophagy maintains homeostasis and is induced upon stress.,Yet, its mechanistic interaction with oncogenic signaling remains elusive.,Here, we show that in BRAFV600E-melanoma, autophagy is induced by BRAF inhibitor (BRAFi), as part of a transcriptional program coordinating lysosome biogenesis/function, mediated by the TFEB transcription factor.,TFEB is phosphorylated and thus inactivated by BRAFV600E via its downstream ERK independently of mTORC1.,BRAFi disrupts TFEB phosphorylation, allowing its nuclear translocation, which is synergized by increased phosphorylation/inactivation of the ZKSCAN3 transcriptional repressor by JNK2/p38-MAPK.,Blockade of BRAFi-induced transcriptional activation of autophagy-lysosomal function in melanoma xenografts causes enhanced tumor progression, EMT-transdifferentiation, metastatic dissemination, and chemoresistance, which is associated with elevated TGF-β levels and enhanced TGF-β signaling.,Inhibition of TGF-β signaling restores tumor differentiation and drug responsiveness in melanoma cells.,Thus, the “BRAF-TFEB-autophagy-lysosome” axis represents an intrinsic regulatory pathway in BRAF-mutant melanoma, coupling BRAF signaling with TGF-β signaling to drive tumor progression and chemoresistance.,The relationship between autophagy and BRAF signalling is unclear.,Here, the authors describe that BRAF inhibition induces the autophagy-lysosomal function in BRAF-mutant melanomas via modulation of the TFEB and ZKSCAN3 transcriptome, which downregulates TGF-β and suppresses melanoma progression.

Melanoma cells rely on developmental programs during tumor initiation and progression.,Here we show that the embryonic stem cell (ESC) factor Sall4 is re-expressed in the Tyr::NrasQ61K; Cdkn2a−/− melanoma model and that its expression is necessary for primary melanoma formation.,Surprisingly, while Sall4 loss prevents tumor formation, it promotes micrometastases to distant organs in this melanoma-prone mouse model.,Transcriptional profiling and in vitro assays using human melanoma cells demonstrate that SALL4 loss induces a phenotype switch and the acquisition of an invasive phenotype.,We show that SALL4 negatively regulates invasiveness through interaction with the histone deacetylase (HDAC) 2 and direct co-binding to a set of invasiveness genes.,Consequently, SALL4 knock down, as well as HDAC inhibition, promote the expression of an invasive signature, while inhibition of histone acetylation partially reverts the invasiveness program induced by SALL4 loss.,Thus, SALL4 appears to regulate phenotype switching in melanoma through an HDAC2-mediated mechanism.,Melanoma cells can switch between proliferative and invasive phenotypes.,Here the authors show that the embryonic stem cell factor Sall4 is a negative regulator of melanoma phenotype switching where its loss leads to the acquisition of an invasive phenotype, due to derepression of invasiveness genes.

Melanoma is the most fatal skin cancer displaying a high degree of molecular heterogeneity.,Phenotype switching is a mechanism that contributes to melanoma heterogeneity by altering transcription profiles for the transition between states of proliferation/differentiation and invasion/stemness.,As phenotype switching is reversible, epigenetic mechanisms, like DNA methylation, could contribute to the changes in gene expression.,Integrative analysis of methylation and gene expression datasets of five proliferative and five invasion melanoma cell cultures reveal two distinct clusters.,SOX9 is methylated and lowly expressed in the highly proliferative group.,SOX9 overexpression results in decreased proliferation but increased invasion in vitro.,In a B16 mouse model, sox9 overexpression increases the number of lung metastases.,Transcriptional analysis of SOX9-overexpressing melanoma cells reveals enrichment in epithelial to mesenchymal transition (EMT) pathways.,Survival analysis of The Cancer Genome Atlas melanoma dataset shows that metastatic patients with high expression levels of SOX9 have significantly worse survival rates.,Additional survival analysis on the targets of SOX9 reveals that most SOX9 downregulated genes have survival benefit for metastatic patients.,Our genome-wide DNA methylation and gene expression study of 10 early passage melanoma cell cultures reveals two phenotypically distinct groups.,One of the genes regulated by DNA methylation between the two groups is SOX9.,SOX9 induces melanoma cell invasion and metastasis and decreases patient survival.,A number of genes downregulated by SOX9 have a negative impact on patient survival.,In conclusion, SOX9 is an important gene involved in melanoma invasion and negatively impacts melanoma patient survival.,The online version of this article (doi:10.1186/s13059-015-0594-4) contains supplementary material, which is available to authorized users.

Conjunctival melanoma (CjM) is a rare, primary cancer of the ocular region.,Genetic and epigenetic characteristics of conjunctival melanoma have not been completely elucidated yet.,Conjunctival melanoma presents similarities with cutaneous melanoma, with substantial differences in the biological behavior.,We reviewed the genetic and epigenetic insights of CjM involved in invasion and metastatic spread.,CjM is commonly characterized by mutations of v-raf murine sarcoma viral oncogene homolog B1 (BRAF), neurofibromin 1 (NF1) and telomerase reverse transcriptase (TERT), high expression of mammalian target of rapamycin (mTOR) and heat shock protein 90 (HSP90), frequent phosphatase and tensin homolog (PTEN) loss and upregulation of specific miRNAs.,These features should identify CjM as a distinct subset of melanoma with its own profile, which is more similar to cutaneous melanoma than mucosal melanoma and remarkably different from uveal melanoma.

Besides being a preferential site of early metastasis, the sentinel lymph node (SLN) is also a privileged site of T-cell priming, and may thus be an appropriate target for investigating cell types involved in antitumor immune reactions.,In this retrospective study we determined the prevalence of OX40+ activated T lymphocytes, FOXP3+ (forkhead box P3) regulatory T cells, DC-LAMP+ (dendritic cell-lysosomal associated membrane protein) mature dendritic cells (DCs) and CD123+ plasmacytoid DCs by immunohistochemistry in 100 SLNs from 60 melanoma patients.,Density values of each cell type in SLNs were compared to those in non-sentinel nodes obtained from block dissections (n = 37), and analyzed with regard to associations with clinicopathological parameters and disease outcome.,Sentinel nodes showed elevated amount of all cell types studied in comparison to non-sentinel nodes.,Metastatic SLNs had higher density of OX40+ lymphocytes compared to tumor-negative nodes, while no significant difference was observed in the case of the other cell types studied.,In patients with positive sentinel node status, high amount of FOXP3+ cells in SLNs was associated with shorter progression-free (P = 0.0011) and overall survival (P = 0.0014), while no significant correlation was found in the case of sentinel-negative patients.,The density of OX40+, CD123+ or DC-LAMP+ cells did not show significant association with the outcome of the disease.,Taken together, our results are compatible with the hypothesis of functional competence of sentinel lymph nodes based on the prevalence of the studied immune cells.,The density of FOXP3+ lymphocytes showed association with progression and survival in patients with positive SLN status, while the other immune markers studied did not prove of prognostic importance.,These results, together with our previous findings on the prognostic value of activated T cells and mature DCs infiltrating primary melanomas, suggest that immune activation-associated markers in the primary tumor may have a higher impact than those in SLNs on the prognosis of the patients.,On the other hand, FOXP3+ cell density in SLNs, but not in the primary tumor, was found predictive of disease outcome in melanoma patients.

Supplemental Digital Content is available in the text.,The overall survival (OS) of patients with metastatic uveal melanoma is short, the evidence for effectiveness of treatments is limited, and no consensus on the choice of treatment exists.,We aimed to advance interpretation of OS as an outcome by pooling peer-reviewed data.,The design is a systematic review and meta-analysis.,We searched PubMed from 1 January 1980, to 29 March 2017, for articles reporting patient-level survival in Kaplan-Meier or numerical form.,We digitized survival graphs, pooled individual survival times, calculated median OS by treatment modality, and compared each modality by the log-rank test and Cox regression using conventional chemotherapy (CHT) as a reference.,Individual-level data were obtained from 78 articles with 2494 patients.,The median OS across all treatment modalities was 1.07 years (range: 0.59-2.50 years).,Pooled OS reported after isolated hepatic perfusion [median OS: 1.34 years; hazard ratio (HR): 0.92, 95% confidence interval (CI): 0.87-0.97, P = 0.0040], immunoembolization (median OS: 1.63; HR: 0.97, 95% CI: 0.95-1.00, P = 0.0080), and surgery (median OS: 1.43; HR: 0.94, 95% CI: 0.92-0.96, P < 0.0001) was longer, and after checkpoint inhibitor shorter (median OS: 0.59; HR: 1.13, 95% CI: 1.06-1.20, P < 0.0001) than after CHT (median OS: 0.91 years), but subject to identifiable confounding factors.,OS following other modalities did not differ from CHT.,Reported OS was unassociated with the decade of publication, but depended on the percentage of first-line treated patients.,Our results suggest no clinically significant difference in OS by treatment modality or decade.,Most of the difference in reported OS likely is attributable to surveillance, selection, and publication bias rather than treatment-related prolongation.,Our pooled data provide benchmarks for future trials.

To assess clinical characteristics, treatment and survival of patients with uveal melanoma in China.,The retrospective study included all patients with malignant uveal melanoma who were consecutively examined in the study period from January 2005 and June 2015 in the Beijing Tongren hospital.,The mean age of the 582 patients (295(50.7%) women) was 44.6±12.6 years (range:5-77 years).,The tumors were located most often in the superior temporal region (in 117(21.5%) patients) and least common in the inferior region (in 31(5.7%) patients).,In 548(94.2%) patients, the tumors were located in the choroid, in 33(5.7%) patients in the ciliary body, and in one (0.2%) patient in the iris.,Treatment included episcleral brachytherapy (415(71.3%) patients), local tumor resection (48(8.2%) patients) and primary enucleation (119(20.4%) patients).,In 53 individuals out of the 415 patients with primary brachytherapy, episcleral brachytherapy was followed by enucleation, due to an increasing tumor size or due to uncontrolled neovascular glaucoma.,Median follow-up time was of 30 months (range: 1-124 months; mean: 34.8 ± 24.4 months).,Overall survival rate at 5 and 10 years was of 92.7% and 85.1%.,Younger age (P = 0.017), tumor location in the nasal meridian(P = 0.004), smaller tumor size (P<0.001), hemispheric tumor shape (P = 0.025), histological tumor cell type (spindle-cell type versus epitheloid cell type;P = 0.014), and type of treatment (episcleral brachytherapy versus local tumor resection and versus primary enucleation; P<0.001) were significantly associated with the overall survival in univariate analysis, while in multivariate analysis only smaller tumor size was significantly (P<0.001; RR: 4.75; 95% confidence interval:2.11,10.7) associated with better overall survival.,In this study on clinical characteristics of uveal melanoma of a larger group of patients from China, the onset age was considerably younger and survival rate better than in studies from Western countries.,Tumor size was the most significant factor for survival.

Bone metastases occur rarely in patients suffering from malignant melanoma, although their onset severely worsens both prognosis and quality of life.,Extracellular vesicles (EVs) including exosomes (Exos) are active players in melanoma progression involved in the formation of the pre-metastatic niche.,Trans-well assays explored the basal migratory and invasive potential of four melanoma cell lines and investigated their different propensity to be attracted toward the bone.,Exosomes were purified from cell supernatants by ultracentrifugation and explored in their ability to influence the bone tropism of melanoma cells.,The molecular machinery activated during this process was investigated by RT-PCR, droplet digital-PCR, flow-cytometry and Western blot, while loss of function studies with dedicated siRNAs defined the single contribute of CXCR4 and CXCR7 molecules.,Melanoma cells revealed a variable propensity to be attracted toward bone fragments.,Gene profiling of both osteotropic and not-osteotropic cells did not show a different expression of those genes notoriously correlated to chemotaxis and bone metastasis.,However, bone conditioned medium significantly increased CXCR4, CXCR7 and PTHrP expression solely to osteotropic cells, while their Exos were able to revert the original poor bone tropism of not-osteotropic cells through CXCR7 up-regulation.,Silencing experiments also demonstrated that membrane expression of CXCR7 is required by melanoma cells to promote their chemotaxis toward SDF-1 gradients.,Our data correlated the osteotropism of melanoma cells to the activation of the SDF-1/CXCR4/CXCR7 axis following the exposition of tumor cells to bone-derived soluble factors.,Also, we demonstrated in vitro that tumor-derived Exos can reprogram the innate osteotropism of melanoma cells by up-regulating membrane CXCR7.,These results may have a potential translation to future identification of druggable targets for the treatment of skeletal metastases from malignant melanoma.,The online version of this article (10.1186/s12967-019-1982-4) contains supplementary material, which is available to authorized users.

Basal cell carcinoma (BCC) is the most common human cancer and represents a growing public health care problem.,Several tumor suppressor genes and proto-oncogenes have been implicated in BCC pathogenesis, including the key components of the Hedgehog pathway, PTCH1 and SMO, the TP53 tumor suppressor, and members of the RAS proto-oncogene family.,Aberrant activation of the Hedgehog pathway represents the molecular driver in basal cell carcinoma pathogenesis, with the majority of BCCs carrying somatic point mutations, mainly ultraviolet (UV)-induced, and/or copy-loss of heterozygosis in the PTCH1 gene.,Recent advances in sequencing technology allowed genome-scale approaches to mutation discovery, identifying new genes and pathways potentially involved in BCC carcinogenesis.,Mutational and functional analysis suggested PTPN14 and LATS1, both effectors of the Hippo-YAP pathway, and MYCN as new BCC-associated genes.,In addition, emerging reports identified frequent non-coding mutations within the regulatory promoter sequences of the TERT and DPH3-OXNAD1 genes.,Thus, it is clear that a more complex genetic network of cancer-associated genes than previously hypothesized is involved in BCC carcinogenesis, with a potential impact on the development of new molecular targeted therapies.,This article reviews established knowledge and new hypotheses regarding the molecular genetics of BCC pathogenesis.

Metastatic melanoma (mM) and renal cell carcinoma (mRCC) are often treated with anti-PD-1 based therapy, however not all patients respond and further therapies are needed.,High dose interleukin-2 (HD IL-2) can lead to durable responses in a subset of mM and mRCC patients.,The efficacy and toxicity of HD IL-2 therapy following anti-PD-1 or anti-PD-L1 therapy have not yet been explored.,Reports on mM and mRCC patients who had received HD IL-2 after PD-1 or PD-L1 inhibition were queried from the PROCLAIMSM database.,Patient characteristics, toxicity and efficacy were analyzed.,A total of 57 patients (40 mM, 17 mRCC) were treated with high dose IL-2 after PD-1 or PD-L1 inhibition and had data recorded in the PROCLAIM database.,The best overall response rate to HD IL-2 was 22.5% for mM (4 complete response (CR), 5 partial responses (PRs)) and 24% for mRCC (2 CRs, 2 PRs).,The toxicity related to HD IL-2 observed in these patients was similar to that observed in patients treated with HD IL-2 without prior checkpoint blockade.,One patient who had received prior PD-L1 blockade developed drug induced pneumonitis with HD IL-2 requiring steroid therapy.,In this retrospective analysis, HD IL-2 therapy displayed durable antitumor activity in mM and mRCC patients who progressed following treatment with PD-1 and PD-L1 inhibition.,The toxicities were generally manageable and consistent with expectations from HD IL-2 but physicians should watch for immune related toxicities such as pneumonitis.,This analysis supports the development of randomized prospective trials to assess the proper sequencing and combination of immune checkpoint blockade and cytokine therapy.,The online version of this article (10.1186/s40425-019-0522-3) 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.

Recently, a Surveillance Epidemiology and End Results (SEER) survey of melanoma patterns of care by the Mayo Clinic, Scottsdale showed remarkable deviations from best practice patterns throughout the country.,The study, which analyzed the SEER records of 35,126 stage I to III cutaneous malignant melanoma patients treated from 2004 to 2006, showed that adherence to National Comprehensive Cancer Network (NCCN) therapeutic resection margins occurred in less than 36% of patients.,Similarly, considerable variation in the quality of melanoma care in the United States when assessed using 26 quality indicators drawn by a panel of melanoma experts was independently reported.,These observations underscore the significant lack of adherence to published best practice patterns reflected by the NCCN guidelines.,The untoward effects of these variations in practice pattern can have an inordinate impact on the survival of melanoma patients in whom long term outcomes are affected by the adequacy of surgical management.,Thin malignant melanoma is curable; however, thick or node positive melanoma is often incurable.,This outcome is determined not only by the stage at presentation but by the use of best practice patterns as reflected in current NCCN cutaneous melanoma practice guidelines.

To optimise predictive models for sentinal node biopsy (SNB) positivity, relapse and survival, using clinico-pathological characteristics and osteopontin gene expression in primary melanomas.,A comparison of the clinico-pathological characteristics of SNB positive and negative cases was carried out in 561 melanoma patients.,In 199 patients, gene expression in formalin-fixed primary tumours was studied using Illumina's DASL assay.,A cross validation approach was used to test prognostic predictive models and receiver operating characteristic curves were produced.,Independent predictors of SNB positivity were Breslow thickness, mitotic count and tumour site.,Osteopontin expression best predicted SNB positivity (P=2.4 × 10−7), remaining significant in multivariable analysis.,Osteopontin expression, combined with thickness, mitotic count and site, gave the best area under the curve (AUC) to predict SNB positivity (72.6%).,Independent predictors of relapse-free survival were SNB status, thickness, site, ulceration and vessel invasion, whereas only SNB status and thickness predicted overall survival.,Using clinico-pathological features (thickness, mitotic count, ulceration, vessel invasion, site, age and sex) gave a better AUC to predict relapse (71.0%) and survival (70.0%) than SNB status alone (57.0, 55.0%).,In patients with gene expression data, the SNB status combined with the clinico-pathological features produced the best prediction of relapse (72.7%) and survival (69.0%), which was not increased further with osteopontin expression (72.7, 68.0%).,Use of these models should be tested in other data sets in order to improve predictive and prognostic data for patients.

The spectrum of central nervous system (CNS) abnormalities described in association with congenital melanocytic naevi (CMN) includes congenital, acquired, melanotic and nonmelanotic pathology.,Historically, symptomatic CNS abnormalities were considered to carry a poor prognosis, although studies from large centres have suggested a much wider variation in outcome.,To establish whether routine MRI of the CNS is a clinically relevant investigation in children with multiple CMN (more than one at birth), and to subclassify radiological abnormalities.,Of 376 patients seen between 1991 and 2013, 289 fulfilled our criterion for a single screening CNS MRI, which since 2008 has been more than one CMN at birth, independent of size and site of the largest naevus.,Cutaneous phenotyping and radiological variables were combined in a multiple regression model of long‐term outcome measures (abnormal neurodevelopment, seizures, requirement for neurosurgery).,Twenty‐one per cent of children with multiple CMN had an abnormal MRI.,Abnormal MRI was the most significant predictor of all outcome measures.,Abnormalities were subclassified into group 1 ‘intraparenchymal melanosis alone’ (n = 28) and group 2 ‘all other pathology’ (n = 18).,Group 1 was not associated with malignancy or death during the study period, even when symptomatic with seizures or developmental delay, whereas group 2 showed a much more complex picture, requiring individual assessment.,For screening for congenital neurological lesions a single MRI in multiple CMN is a clinically relevant strategy.,Any child with a stepwise change in neurological/developmental symptoms or signs should have an MRI with contrast of the brain and spine to look for new CNS melanoma.,What's already known about this topic?,Multiple congenital melanocytic naevi (CMN; more than one lesion at birth) can be associated with abnormalities of the central nervous system (CNS).,The spectrum of these abnormalities includes congenital and acquired pathologies, melanotic and nonmelanotic lesions, rendering the term ‘CMN syndrome’ more appropriate than ‘neurocutaneous melanosis’.,Symptomatic CNS abnormalities were previously thought to carry a universally poor prognosis, although cohort data in the last decade have argued against this.,Multiple congenital melanocytic naevi (CMN; more than one lesion at birth) can be associated with abnormalities of the central nervous system (CNS).,The spectrum of these abnormalities includes congenital and acquired pathologies, melanotic and nonmelanotic lesions, rendering the term ‘CMN syndrome’ more appropriate than ‘neurocutaneous melanosis’.,Symptomatic CNS abnormalities were previously thought to carry a universally poor prognosis, although cohort data in the last decade have argued against this.,What does this study add?,A single CNS magnetic resonance imaging scan in multiple CMN, independent of projected adult size or site of the largest naevus, and ideally in the first 6 months of life, is currently an appropriate screening strategy.An abnormal result is a better statistical predictor of clinical outcome than cutaneous phenotype.Clinical management is altered as a result of the radiological result.,A single CNS magnetic resonance imaging scan in multiple CMN, independent of projected adult size or site of the largest naevus, and ideally in the first 6 months of life, is currently an appropriate screening strategy.,An abnormal result is a better statistical predictor of clinical outcome than cutaneous phenotype.,Clinical management is altered as a result of the radiological result.

Despite remarkable advances in the genomic characterization of adult melanoma, the molecular pathogenesis of pediatric melanoma remains largely unknown.,We analyzed 15 conventional melanomas (CMs), 3 melanomas arising in congenital nevi (CNMs), and 5 spitzoid melanomas (SMs), using various platforms, including whole genome or exome sequencing, the molecular inversion probe assay, and/or targeted sequencing.,CMs demonstrated a high burden of somatic single-nucleotide variations (SNVs), with each case containing a TERT promoter (TERT-p) mutation, 13/15 containing an activating BRAF V600 mutation, and >80% of the identified SNVs consistent with UV damage.,In contrast, the three CNMs contained an activating NRAS Q61 mutation and no TERT-p mutations.,SMs were characterized by chromosomal rearrangements resulting in activated kinase signaling in 40%, and an absence of TERT-p mutations, except for the one SM that succumbed to hematogenous metastasis.,We conclude that pediatric CM has a very similar UV-induced mutational spectrum to that found in the adult counterpart, emphasizing the need to promote sun protection practices in early life and to improve access to therapeutic agents being explored in adults in young patients.,In contrast, the pathogenesis of CNM appears to be distinct.,TERT-p mutations may identify the rare subset of spitzoid melanocytic lesions prone to disseminate.

Adiponectin is an insulin-sensitizing and anticarcinogenic hormone that is encoded by a gene on chromosome 3.,Here, we analyzed the expression of adiponectin and its receptor Adipor1 in primary uveal melanoma (UM) with regard to the monosomy-3 status and clinical factors, as well as the physiological response of UM cells to adiponectin.,Immunohistochemistry was performed on the primary UM of 34 patients.,Circulating melanoma cells (CMC) were isolated by immunomagnetic enrichment.,Monosomy-3 was evaluated by Immuno-FISH.,Gene expression was analyzed using the RNAseq data of The Cancer Genome Atlas study.,Cultures of choroidal melanocytes and UM were established from the samples of two patients.,The proliferative potential of the UM cell lines Mel-270 and OMM-2.5 was determined by immunocytochemistry, immunoblotting, cell cycle analysis, nucleolar staining, and adenosine triphosphate (ATP) levels.,UM with monosomy-3 exhibited a lower immunoreactivity for adiponectin and Adipor1, which was associated with monosomy-3-positive CMC and the development of extraocular growth or metastases.,Both proteins were more abundant in the irradiated tumors and present in the cultured cells.,Gene expression profile indicated the impairment of adiponectin-mediated signaling in the monosomy-3 tumors.,Adiponectin induced a significant decline in the ATP levels, Ki-67 expression, cells in the G2/M phase, and nucleolar integrity in UM cultures.,Adiponectin deficiency appears to enhance the metastatic potential of the UM cells with monosomy-3 and the termination of tumor dormancy.,Counteracting insulin resistance and improving the serum adiponectin levels might therefore be a valuable approach to prevent or delay the UM metastases.

Uveal melanoma (UM) is the most common intraocular tumour in adults and despite surgical or radiation treatment of primary tumours, ~50% of patients progress to metastatic disease.,Therapeutic options for metastatic UM are limited, with clinical trials having little impact.,Here we perform whole-genome sequencing (WGS) of 103 UM from all sites of the uveal tract (choroid, ciliary body, iris).,While most UM have low tumour mutation burden (TMB), two subsets with high TMB are seen; one driven by germline MBD4 mutation, and another by ultraviolet radiation (UVR) exposure, which is restricted to iris UM.,All but one tumour have a known UM driver gene mutation (GNAQ, GNA11, BAP1, PLCB4, CYSLTR2, SF3B1, EIF1AX).,We identify three other significantly mutated genes (TP53, RPL5 and CENPE).,Uveal melanoma has a propensity to metastasise.,Here, the authors report the whole genome sequence of 103 uveal melanomas and find that the tumour mutational burden is variable and that two subsets of tumours are characterised by MBD4 mutations and a UV exposure signature.

Over the past decades, melanoma-related mortality has remained nearly stable.,The main reason is treatment failure of metastatic disease and the inherently linked knowledge gap regarding metastasis formation.,In order to elicit invasion, melanoma cells manipulate the tumor microenvironment, gain motility, and adhere to the extracellular matrix and cancer-associated fibroblasts.,Melanoma cells thereby express different cell adhesion molecules like laminins, integrins, N-cadherin, and others.,Epithelial-mesenchymal transition (EMT) is physiological during embryologic development, but reactivated during malignancy.,Despite not being truly epithelial, neural crest-derived malignancies like melanoma share similar biological programs that enable tumorigenesis, invasion, and metastasis.,This complex phenomenon is termed phenotype switching and is intertwined with oncometabolism as well as dormancy escape.,Additionally, it has been shown that primary melanoma shed exosomes that create a favorable premetastatic niche in the microenvironment of secondary organs and lymph nodes.,Although the growing body of literature describes the aforementioned concepts separately, an integrative holistic approach is missing.,Using melanoma as a tumor model, this review will shed light on these complex biological principles in an attempt to clarify the mechanistic metastatic pathways that dictate tumor and patient fate.

Targeted therapies with MAPK inhibitors (MAPKi) are faced with severe problems of resistance in BRAF‐mutant melanoma.,In parallel to the acquisition of genetic mutations, melanoma cells may also adapt to the drugs through phenotype switching.,The ZEB1 transcription factor, a known inducer of EMT and invasiveness, is now considered as a genuine oncogenic factor required for tumor initiation, cancer cell plasticity, and drug resistance in carcinomas.,Here, we show that high levels of ZEB1 expression are associated with inherent resistance to MAPKi in BRAFV 600‐mutated cell lines and tumors.,ZEB1 levels are also elevated in melanoma cells with acquired resistance and in biopsies from patients relapsing while under treatment.,ZEB1 overexpression is sufficient to drive the emergence of resistance to MAPKi by promoting a reversible transition toward a MITF low/p75high stem‐like and tumorigenic phenotype.,ZEB1 inhibition promotes cell differentiation, prevents tumorigenic growth in vivo, sensitizes naive melanoma cells to MAPKi, and induces cell death in resistant cells.,Overall, our results demonstrate that ZEB1 is a major driver of melanoma cell plasticity, driving drug adaptation and phenotypic resistance to MAPKi.

Malignant melanoma is an aggressive tumor of the skin and seems to be resistant to current therapeutic approaches.,Melanocytic transformation is thought to occur by sequential accumulation of genetic and molecular alterations able to activate the Ras/Raf/MEK/ERK (MAPK) and/or the PI3K/AKT (AKT) signalling pathways.,Specifically, mutations of B-RAF activate MAPK pathway resulting in cell cycle progression and apoptosis prevention.,According to these findings, MAPK and AKT pathways may represent promising therapeutic targets for an otherwise devastating disease.,Here we show a computational model able to simulate the main biochemical and metabolic interactions in the PI3K/AKT and MAPK pathways potentially involved in melanoma development.,Overall, this computational approach may accelerate the drug discovery process and encourages the identification of novel pathway activators with consequent development of novel antioncogenic compounds to overcome tumor cell resistance to conventional therapeutic agents.,The source code of the various versions of the model are available as S1 Archive.

Previously we found that terfenadine, an H1 histamine receptor antagonist, acts as a potent apoptosis inducer in melanoma cells through modulation of Ca2+ homeostasis.,In this report, focusing our attention on the apoptotic mechanisms activated by terfenadine, we show that this drug can potentially activate distinct intrinsic signaling pathways depending on culture conditions.,Serum-deprived conditions enhance the cytotoxic effect of terfenadine and caspase-4 and -2 are activated upstream of caspase-9.,Moreover, although we found an increase in ROS levels, the apoptosis was ROS independent.,Conversely, terfenadine treatment in complete medium induced ROS-dependent apoptosis.,Caspase-4, -2, and -9 were simultaneously activated and p73 and Noxa induction were involved.,ROS inhibition prevented p73 and Noxa expression but not p53 and p21 expression, suggesting a role for Noxa in p53-independent apoptosis in melanoma cells.,Finally, we found that terfenadine induced autophagy, that can promote apoptosis.,These findings demonstrate the great potential of terfenadine to kill melanoma cells through different cellular signaling pathways and could contribute to define new therapeutic strategies in melanoma.

The incidence of malignant melanoma has continued to rise during the past decades.,However, in the last few years, treatment protocols have significantly been improved thanks to a better understanding of the key oncogenes and signaling pathways involved in its pathogenesis and progression.,Anticancer therapy would either kill tumor cells by triggering apoptosis or permanently arrest them in the G1 phase of the cell cycle.,Unfortunately, melanoma is often refractory to commonly used anticancer drugs.,More recently, however, some new anticancer strategies have been developed that are “external” to cancer cells, for example stimulating the immune system’s response or inhibiting angiogenesis.,In fact, the increasing knowledge of melanoma pathogenetic mechanisms, in particular the discovery of genetic mutations activating specific oncogenes, stimulated the development of molecularly targeted therapies, a form of treatment in which a drug (chemical or biological) is developed with the goal of exclusively destroying cancer cells by interfering with specific molecules that drive growth and spreading of the tumor.,Again, after the initial exciting results associated with targeted therapy, tumor resistance and/or relapse of the melanoma lesion have been observed.,Hence, very recently, new therapeutic strategies based on the modulation of the immune system function have been developed.,Since cancer cells are known to be capable of evading immune-mediated surveillance, i.e., to block the immune system cell activity, a series of molecular strategies, including monoclonal antibodies, have been developed in order to “release the brakes” on the immune system igniting immune reactivation and hindering metastatic melanoma cell growth.,In this review we analyze the various biological strategies underlying conventional chemotherapy as well as the most recently developed targeted therapies and immunotherapies, pointing at the molecular mechanisms of cell injury and death engaged by the different classes of therapeutic agents.

Melanoma cells can switch their phenotypes in response to microenvironmental insults.,Heterogeneous melanoma populations characterized by long-term growth and a high self-renewal capacity can be obtained in vitro in EGF(+)bFGF(+) medium whilst invasive potential of melanoma cells is increased in serum-containing cultures.,In the present study, we have shown that originally these patient-derived melanoma populations exhibit variable expression of pro-survival genes from the BCL-2 family and inhibitors of apoptosis (IAPs), and differ in the baseline MCL-1 transcript stability as well.,While being transferred to serum-containing medium, melanoma cells are well protected from death.,Immediate adaptive response of melanoma cells selectively involves a temporary MCL-1 increase, both at mRNA and protein levels, and BCL-XL can complement MCL-1, especially in MITFlow populations.,Thus, the extent of MCL-1 and BCL-XL contributions seems to be cell context-dependent.,An increase in MCL-1 level results from a transiently enhanced stability of its transcript, but not from altered protein turnover.,Inhibition of MCL-1 preceding transfer to serum-containing medium caused the induction of cell death in a subset of melanoma cells, which confirms the involvement of MCL-1 in melanoma cell survival during the rapid alteration of growth conditions.,Additionally, immediate response to serum involves the transient increase in MITF expression and inhibition of ERK-1/2 activity.,Uncovering the mechanisms of adaptive response to rapid changes in microenvironment may extend our knowledge on melanoma biology, especially at the stage of dissemination.

Cell migration underlies metastatic dissemination of cancer cells, and fast “amoeboid” migration in the invasive fronts of tumors is controlled by high levels of actomyosin contractility.,How amoeboid migration is regulated by extracellular signals and sustained over time by transcriptional changes is not fully understood.,Transforming growth factor β (TGF-β) is well known to promote epithelial-to-mesenchymal transition (EMT) and contribute to metastasis, but melanocytes are neural crest derivatives that have undergone EMT during embryonic development.,Surprisingly, we find that in melanoma, TGF-β promotes amoeboid features such as cell rounding, membrane blebbing, high levels of contractility, and increased invasion.,Using genome-wide transcriptomics, we find that amoeboid melanoma cells are enriched in a TGF-β-driven signature.,We observe that downstream of TGF-β, SMAD2 and its adaptor CITED1 control amoeboid behavior by regulating the expression of key genes that activate contractile forces.,Moreover, CITED1 is highly upregulated during melanoma progression, and its high expression is associated with poor prognosis.,CITED1 is coupled to a contractile-rounded, amoeboid phenotype in a panel of 16 melanoma cell lines, in mouse melanoma xenografts, and in 47 human melanoma patients.,Its expression is also enriched in the invasive fronts of lesions.,Functionally, we show how the TGF-β-SMAD2-CITED1 axis promotes different steps associated with progression: melanoma detachment from keratinocytes, 2D and 3D migration, attachment to endothelial cells, and in vivo lung metastatic initial colonization and outgrowth.,We propose a novel mechanism by which TGF-β-induced transcription sustains actomyosin force in melanoma cells and thereby promotes melanoma progression independently of EMT.,•TGF-β-SMAD promotes amoeboid migration in melanoma•Downstream of TGF-β, the adaptor CITED1 controls actomyosin contractility•Amoeboid features correlate with CITED1 levels in cell lines, xenografts, and patients•TGF-β-SMAD-CITED1 transcriptional network controls melanoma metastatic ability,TGF-β-SMAD promotes amoeboid migration in melanoma,Downstream of TGF-β, the adaptor CITED1 controls actomyosin contractility,Amoeboid features correlate with CITED1 levels in cell lines, xenografts, and patients,TGF-β-SMAD-CITED1 transcriptional network controls melanoma metastatic ability,Cantelli et al. find that, in melanoma, TGF-β-SMAD-CITED1 controls amoeboid behavior through activation of a transcriptional program.,As a result, melanoma cells detach from keratinocytes, increase their invasive potential, and efficiently colonize the lung.,This is a new function of TGF-β, independent of epithelial-to-mesenchymal transition.

Cutaneous melanoma (CM) is the most lethal skin cancer.,The Fanconi Anemia (FA) pathway involved in DNA crosslinks repair may affect CM susceptibility and prognosis.,Using data derived from published genome-wide association study, we comprehensively analyzed the associations of 2339 common single nucleotide polymorphisms (SNPs) in 14 autosomal FA genes with overall survival (OS) in 858 CM patients.,By performing false-positive report probability corrections and stepwise Cox proportional hazards regression analyses, we identified significant associations between CM OS and four putatively functional SNPs: BRCA2 rs10492396 [AG vs.,GG: adjusted hazard ratio (adjHR)=1.85, 95% confident interval (CI)=1.16-2.95, P=0.010], rs206118 (CC vs.,TT+TC: adjHR=2.44, 95% CI=1.27-4.67, P=0.007), rs3752447 (CC vs.,TT+TC: adjHR=2.10, 95% CI=1.38-3.18, P=0.0005), and FANCA rs62068372 (TT vs.,CC+CT: adjHR=1.85, 95% CI=1.27-2.69, P=0.001).,Moreover, patients with an increasing number of unfavorable genotypes (NUG) of these loci had markedly reduced OS and melanoma-specific survival (MSS).,The final model incorporating with NUG, tumor stage and Breslow thickness showed an improved discriminatory ability to classify both 5-year OS and 5-year MSS.,Additional investigations, preferably prospective studies, are needed to validate our findings.

Inhibitor-kappaB kinase epsilon (IKKε) and TANK-binding kinase 1 (TBK1) are non-canonical IκB kinases, both described as contributors to tumor growth and metastasis in different cancer types.,Several hints indicate that they are also involved in the pathogenesis of melanoma; however, the impact of their inhibition as a potential therapeutic measure in this “difficult-to-treat” cancer type has not been investigated so far.,We assessed IKKε and TBK1 expression in human malignant melanoma cells, primary tumors and the metastasis of melanoma patients.,Both kinases were expressed in the primary tumor and in metastasis and showed a significant overexpression in tumor cells in comparison to melanocytes.,The pharmacological inhibition of IKKε/TBK1 by the approved drug amlexanox reduced cell proliferation, migration and invasion.,Amlexanox did not affect the cell cycle progression nor apoptosis induction but significantly suppressed autophagy in melanoma cells.,The analysis of potential functional downstream targets revealed that NF-кB and ERK pathways might be involved in kinase-mediated effects.,In an in vivo xenograft model in nude mice, amlexanox treatment significantly reduced tumor growth.,In conclusion, amlexanox was able to suppress tumor progression potentially by the inhibition of autophagy as well as NF-кB and MAP kinase pathways and might therefore constitute a promising candidate for melanoma therapy.

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

Melanoma of the uveal tract accounts for approximately 5% of all melanomas and represents the most common primary intraocular malignancy.,Despite improvements in diagnosis and more effective local therapies for primary cancer, the rate of metastatic death has not changed in the past forty years.,In the present study, we made use of bioinformatics to analyze the data obtained from three public available microarray datasets on uveal melanoma in an attempt to identify novel putative chemotherapeutic options for the liver metastatic disease.,We have first carried out a meta-analysis of publicly available whole-genome datasets, that included data from 132 patients, comparing metastatic vs. non metastatic uveal melanomas, in order to identify the most relevant genes characterizing the spreading of tumor to the liver.,Subsequently, the L1000CDS2 web-based utility was used to predict small molecules and drugs targeting the metastatic uveal melanoma gene signature.,The most promising drugs were found to be Cinnarizine, an anti-histaminic drug used for motion sickness, Digitoxigenin, a precursor of cardiac glycosides, and Clofazimine, a fat-soluble iminophenazine used in leprosy.,In vitro and in vivo validation studies will be needed to confirm the efficacy of these molecules for the prevention and treatment of metastatic uveal melanoma.

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

The vascular endothelial growth factor receptor‐1 (VEGFR‐1) is a tyrosine kinase receptor frequently expressed in melanoma.,Its activation by VEGF‐A or placental growth factor (PlGF) promotes tumour cell survival, migration and invasiveness.,Moreover, VEGFR‐1 stimulation contributes to pathological angiogenesis and induces recruitment of tumour‐associated macrophages.,Since melanoma acquired resistance to BRAF inhibitors (BRAFi) has been associated with activation of pro‐angiogenic pathways, we have investigated VEGFR‐1 involvement in vemurafenib resistance.,Results indicate that human melanoma cells rendered resistant to vemurafenib secrete greater amounts of VEGF‐A and express higher VEGFR‐1 levels compared with their BRAFi‐sensitive counterparts.,Transient VEGFR‐1 silencing in susceptible melanoma cells delays resistance development, whereas in resistant cells it increases sensitivity to the BRAFi.,Consistently, enforced VEGFR‐1 expression, by stable gene transfection in receptor‐negative melanoma cells, markedly reduces sensitivity to vemurafenib.,Moreover, melanoma cells expressing VEGFR‐1 are more invasive than VEGFR‐1 deficient cells and receptor blockade by a specific monoclonal antibody (D16F7 mAb) reduces extracellular matrix invasion triggered by VEGF‐A and PlGF.,These data suggest that VEGFR‐1 up‐regulation might contribute to melanoma progression and spreading after acquisition of a drug‐resistant phenotype.,Thus, VEGFR‐1 inhibition with D16F7 mAb might be a suitable adjunct therapy for VEGFR‐1 positive tumours with acquired resistance to vemurafenib.

While multiple mechanisms of BRAFV600-mutant melanoma resistance to targeted MAPK signaling inhibitors (MAPKi) have been reported, the epigenetic regulation of this process remains undetermined.,Here, using a CRISPR-Cas9 screen targeting chromatin regulators, we discover that haploinsufficiency of the histone deacetylase SIRT6 allows melanoma cell persistence in the presence of MAPKi.,Haploinsufficiency, but not complete loss of SIRT6 promotes IGFBP2 expression via increased chromatin accessibility, H3K56 acetylation at the IGFBP2 locus, and consequent activation of the IGF-1 receptor (IGF-1R) and downstream AKT signaling.,Combining a clinically applicable IGF-1Ri with BRAFi overcomes resistance of SIRT6 haploinsufficient melanoma cells in vitro and in vivo.,Using matched melanoma samples derived from patients receiving dabrafenib + trametinib, we identify IGFBP2 as a potential biomarker for MAPKi resistance.,Our study has not only identified an epigenetic mechanism of drug resistance, but also provides insights into a combinatorial therapy that may overcome resistance to standard-of-care therapy for BRAFV600-mutant melanoma patients.,The epigenetic mechanisms of melanoma drug resistance are poorly understood.,Here, the authors develop a CRISPR-Cas9 screen targeting epigenetic regulators and discover that SIRT6 haploinsufficiency induces BRAFV600E melanoma cell resistance to MAPK inhibitors via IGF signalling.

Epigenetic alterations by histone/protein deacetylases (HDACs) are one of the many mechanisms that cancer cells use to alter gene expression and promote growth.,HDAC inhibitors have proven to be effective in the treatment of specific malignancies, particularly in combination with other anticancer agents.,We conducted a phase I trial of panobinostat in patients with unresectable stage III or IV melanoma.,Patients were treated with oral panobinostat at a dose of 30 mg daily on Mondays, Wednesdays, and Fridays (Arm A).,Three of the six patients on this dose experienced clinically significant thrombocytopenia requiring dose interruption.,Due to this, a second treatment arm was opened and the dose was changed to 30 mg oral panobinostat three times a week every other week (Arm B).,Six patients were treated on Arm A and 10 patients were enrolled to Arm B with nine patients treated.,In nine patients treated on Arm B, the response rate was 0% (90% confidence interval [CI]: 0-28%) and the disease‐control rate was 22% (90% CI: 4-55%).,Among all 15 patients treated, the overall response rate was 0% (90% CI: 0-17%) and the disease‐control rate was 27% (90% CI: 10-51%).,There was a high rate of toxicity associated with treatment.,Correlative studies suggest the presence of immune modifications after HDAC inhibition.,Panobinostat is not active as a single agent in the treatment of melanoma.,Further exploration of this agent in combination with other therapies may be warranted.

Malignant melanoma is an aggressive and deadly form of skin cancer, and despite recent advances in available therapies, is still lacking in completely effective treatments.,Rg3, a monomer extracted from ginseng roots, has been attempted for the treatment of many cancers.,It is reported that the expressions of histone deacetylase 3 (HDAC3) and p53 acetylation correlate with tumor cell growth.,However, the antitumor effect of Rg3 on melanoma and the mechanism by which it regulates HDAC3 expression and p53 acetylation remain unknown.,We found high expression of HDAC3 in human melanoma tissues to be significantly correlated to lymph node metastasis and clinical stage of disease (p<0.05).,In melanoma cells, Rg3 inhibited cell proliferation and induced G0/G1 cell cycle arrest.,Rg3 also decreased the expression of HDAC3 and increased the acetylation of p53 on lysine (k373/k382).,Moreover, suppression of HDAC3 by either siRNA or a potent HDAC3 inhibitor (MS-275) inhibited cell proliferation, increased p53 acetylation and transcription activity.,In A375 melanoma xenograft studies, we demonstrated that Rg3 and HDAC3 short hairpin RNA (shHDAC3) inhibited the growth of xenograft tumors with down-regulation of HDAC3 expression and up-regulation of p53 acetylation.,In conclusion, Rg3 has antiproliferative activity against melanoma by decreasing HDAC3 and increasing acetylation of p53 both in vitro and in vivo.,Thus, Rg3 serves as a potential therapeutic agent for the treatment of melanoma.

In general, melanoma can be considered as a UV‐driven disease with an aggressive metastatic course and high mutational load, with only few tumors (acral, mucosal, and uveal melanomas) not induced by sunlight and possessing a lower mutational load.,The most commonly activated pathway in melanoma is the mitogen‐activated protein kinase (MAPK) pathway.,However, the prognostic significance of mutational stratification is unclear and needs further investigation.,Here, in silico we combined mutation data from 162 melanomas subjected to targeted deep sequencing with mutation data from three published studies.,Tumors from 870 patients were grouped according to BRAF,RAS,NF1 mutation or triple‐wild‐type status and correlated with tumor and patient characteristics.,We found that the NF1‐mutated subtype had a higher mutational burden and strongest UV mutation signature.,Searching for co‐occurring mutated genes revealed the RASopathy genes PTPN11 and RASA2, as well as another RAS domain‐containing gene RASSF2 enriched in the NF1 subtype after adjustment for mutational burden.,We found that a larger proportion of the NF1‐mutant tumors were from males and with older age at diagnosis.,Importantly, we found an increased risk of death from melanoma (disease‐specific survival, DSS; HR, 1.9; 95% CI, 1.21-3.10; P = 0.046) and poor overall survival (OS; HR, 2.0; 95% CI, 1.28-2.98; P = 0.01) in the NF1 subtype, which remained significant after adjustment for age, gender, and lesion type (DSS P = 0.03, OS P = 0.06, respectively).,Melanoma genomic subtypes display different biological and clinical characteristics.,The poor outcome observed in the NF1 subtype highlights the need for improved characterization of this group.

Analysis of 501 melanoma exomes revealed RASA2, encoding a RasGAP, as a tumor-suppressor gene mutated in 5% of melanomas.,Recurrent loss-of-function mutations in RASA2 were found to increase RAS activation, melanoma cell growth and migration.,RASA2 expression was lost in ≥30% of human melanomas and was associated with reduced patient survival.,These findings reveal RASA2 inactivation as a melanoma driver and highlight the importance of Ras GAPs in cancer.

In this review, Goding and Arnheiter present the current understanding of MITF's role and regulation in development and disease and highlight key areas where our knowledge of MITF regulation and function is limited.,All transcription factors are equal, but some are more equal than others.,In the 25 yr since the gene encoding the microphthalmia-associated transcription factor (MITF) was first isolated, MITF has emerged as a key coordinator of many aspects of melanocyte and melanoma biology.,Like all transcription factors, MITF binds to specific DNA sequences and up-regulates or down-regulates its target genes.,What marks MITF as being remarkable among its peers is the sheer range of biological processes that it appears to coordinate.,These include cell survival, differentiation, proliferation, invasion, senescence, metabolism, and DNA damage repair.,In this article we present our current understanding of MITF's role and regulation in development and disease, as well as those of the MITF-related factors TFEB and TFE3, and highlight key areas where our knowledge of MITF regulation and function is limited.

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.

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

Aging and sun exposure are the leading causes of skin cancer.,It has been shown that epigenetic changes, such as DNA methylation, are well established mechanisms for cancer, and also have emerging roles in aging and common disease.,Here, we directly ask whether DNA methylation is altered following skin aging and/or chronic sun exposure in humans.,We compare epidermis and dermis of both sun-protected and sun-exposed skin derived from younger subjects (under 35 years old) and older subjects (over 60 years old), using the Infinium HumanMethylation450 array and whole genome bisulfite sequencing.,We observe large blocks of the genome that are hypomethylated in older, sun-exposed epidermal samples, with the degree of hypomethylation associated with clinical measures of photo-aging.,We replicate these findings using whole genome bisulfite sequencing, comparing epidermis from an additional set of younger and older subjects.,These blocks largely overlap known hypomethylated blocks in colon cancer and we observe that these same regions are similarly hypomethylated in squamous cell carcinoma samples.,These data implicate large scale epigenomic change in mediating the effects of environmental damage with photo-aging.,The online version of this article (doi:10.1186/s13059-015-0644-y) contains supplementary material, which is available to authorized users.

Bacteria within the skin microbiome of some individuals produce an antimetabolite that inhibits tumor growth.,We report the discovery that strains of Staphylococcus epidermidis produce 6-N-hydroxyaminopurine (6-HAP), a molecule that inhibits DNA polymerase activity.,In culture, 6-HAP selectively inhibited proliferation of tumor lines but did not inhibit primary keratinocytes.,Resistance to 6-HAP was associated with the expression of mitochondrial amidoxime reducing components, enzymes that were not observed in cells sensitive to this compound.,Intravenous injection of 6-HAP in mice suppressed the growth of B16F10 melanoma without evidence of systemic toxicity.,Colonization of mice with an S. epidermidis strain producing 6-HAP reduced the incidence of ultraviolet-induced tumors compared to mice colonized by a control strain that did not produce 6-HAP.,S. epidermidis strains producing 6-HAP were found in the metagenome from multiple healthy human subjects, suggesting that the microbiome of some individuals may confer protection against skin cancer.,These findings show a new role for skin commensal bacteria in host defense.

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

Cutaneous melanoma is a complex disorder characterized by an elevated degree of heterogeneity, features that place it among the most aggressive types of cancer.,Although significant progress was recorded in both the understanding of melanoma biology and genetics, and in therapeutic approaches, this malignancy still represents a major problem worldwide due to its high incidence and the lack of a curative treatment for advanced stages.,This review offers a survey of the most recent information available regarding the melanoma epidemiology, etiology, and genetic profile.,Also discussed was the topic of cutaneous melanoma murine models outlining the role of these models in understanding the molecular pathways involved in melanoma initiation, progression, and metastasis.

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.

With the use of a mouse model expressing human Fc-gamma receptors (FcγRs), we demonstrated that antibodies with isotypes equivalent to ipilimumab and tremelimumab mediate intra-tumoral regulatory T (Treg) cell depletion in vivo, increasing the CD8+ to Treg cell ratio and promoting tumor rejection.,Antibodies with improved FcγR binding profiles drove superior anti-tumor responses and survival.,In patients with advanced melanoma, response to ipilimumab was associated with the CD16a-V158F high affinity polymorphism.,Such activity only appeared relevant in the context of inflamed tumors, explaining the modest response rates observed in the clinical setting.,Our data suggest that the activity of anti-CTLA-4 in inflamed tumors may be improved through enhancement of FcγR binding, whereas poorly infiltrated tumors will likely require combination approaches.,•Anti-CTLA-4 of hIgG1 and hIgG2 isotypes promote depletion of intra-tumoral Treg cells•hIgG2 antibodies mediate in vivo depletion of intra-tumoral Treg cells via CD32a•Anti-CTLA-4 with enhanced Fc effector function improves therapeutic outcomes•The CD16-V158F SNP is associated with response to ipilimumab in inflamed tumors,Anti-CTLA-4 of hIgG1 and hIgG2 isotypes promote depletion of intra-tumoral Treg cells,hIgG2 antibodies mediate in vivo depletion of intra-tumoral Treg cells via CD32a,Anti-CTLA-4 with enhanced Fc effector function improves therapeutic outcomes,The CD16-V158F SNP is associated with response to ipilimumab in inflamed tumors,Arce Vargas et al. use a mouse model expressing human FcγRs to show that antibodies with isotypes equivalent to ipilimumab increase the CD8+ to Treg ratio by depleting intra-tumoral Tregs to promote tumor rejection.,In melanoma patients, response to ipilimumab is associated with a high affinity FcγR polymorphism.

CD4+ T cells that express the transcription factor FOXP3 (FOXP3+ T cells) are commonly regarded as immunosuppressive regulatory T cells (Tregs).,FOXP3+ T cells are reported to be increased in tumor-bearing patients or animals and are considered to suppress antitumor immunity, but the evidence is often contradictory.,In addition, accumulating evidence indicates that FOXP3 is induced by antigenic stimulation and that some non-Treg FOXP3+ T cells, especially memory-phenotype FOXP3low cells, produce proinflammatory cytokines.,Accordingly, the subclassification of FOXP3+ T cells is fundamental for revealing the significance of FOXP3+ T cells in tumor immunity, but the arbitrariness and complexity of manual gating have complicated the issue.,In this article, we report a computational method to automatically identify and classify FOXP3+ T cells into subsets using clustering algorithms.,By analyzing flow cytometric data of melanoma patients, the proposed method showed that the FOXP3+ subpopulation that had relatively high FOXP3, CD45RO, and CD25 expressions was increased in melanoma patients, whereas manual gating did not produce significant results on the FOXP3+ subpopulations.,Interestingly, the computationally identified FOXP3+ subpopulation included not only classical FOXP3high Tregs, but also memory-phenotype FOXP3low cells by manual gating.,Furthermore, the proposed method successfully analyzed an independent data set, showing that the same FOXP3+ subpopulation was increased in melanoma patients, validating the method.,Collectively, the proposed method successfully captured an important feature of melanoma without relying on the existing criteria of FOXP3+ T cells, revealing a hidden association between the T cell profile and melanoma, and providing new insights into FOXP3+ T cells and Tregs.

Tremelimumab is an antibody that blocks CTLA-4 and demonstrates clinical efficacy in a subset of advanced melanoma patients.,An unmet clinical need exists for blood-based response-predictive gene signatures to facilitate clinically effective and cost-efficient use of such immunotherapeutic interventions.,Peripheral blood samples were collected in PAXgene® tubes from 210 treatment-naïve melanoma patients receiving tremelimumab in a worldwide, multicenter phase III study (discovery dataset).,A central panel of radiologists determined objective response using RECIST criteria.,Gene expression for 169 mRNA transcripts was measured using quantitative PCR.,A 15-gene pre-treatment response-predictive classifier model was identified.,An independent population (N = 150) of refractory melanoma patients receiving tremelimumab after chemotherapy enrolled in a worldwide phase II study (validation dataset).,The classifier model, using the same genes, coefficients and constants for objective response and one-year survival after treatment, was applied to the validation dataset.,A 15-gene pre-treatment classifier model (containing ADAM17, CDK2, CDKN2A, DPP4, ERBB2, HLA-DRA, ICOS, ITGA4, LARGE, MYC, NAB2, NRAS, RHOC, TGFB1, and TIMP1) achieved an area under the curve (AUC) of 0.86 (95% confidence interval 0.81 to 0.91, p < 0.0001) for objective response and 0.6 (95% confidence interval 0.54 to 0.67, p = 0.0066) for one-year survival in the discovery set.,This model was validated in the validation set with AUCs of 0.62 (95% confidence interval 0.54 to 0.70 p = 0.0455) for objective response and 0.68 for one-year survival (95% confidence interval 0.59 to 0.75 p = 0.0002).,To our knowledge, this is the largest blood-based biomarker study of a checkpoint inhibitor, tremelimumab, which demonstrates a validated pre-treatment mRNA classifier model that predicts clinical response.,The data suggest that the model captures a biological signature representative of genes needed for a robust anti-cancer immune response.,It also identifies non-responders to tremelimumab at baseline prior to treatment.,The online version of this article (doi:10.1186/s40425-017-0272-z) contains supplementary material, which is available to authorized users.

Treatment with ipilimumab, a fully human anti-CTLA-4 antibody approved for the treatment of advanced melanoma, is associated with some immune-related adverse events (irAEs) such as colitis (gastrointestinal irAE, or GI irAE) and skin rash, which are managed by treatment guidelines.,Nevertheless, predictive biomarkers that can help identify patients more likely to develop these irAEs could enhance the management of these toxicities.,To identify candidate predictive biomarkers associated with GI irAEs, gene expression profiling was performed on whole blood samples from 162 advanced melanoma patients at baseline, 3 and 11 weeks after the start of ipilimumab treatment in two phase II clinical trials (CA184004 and CA184007).,Overall, 49 patients developed Grade 2 or higher (grade 2+) GI irAEs during the course of treatment.,A repeated measures analysis of variance (ANOVA) was used to evaluate the differences in mean expression levels between the GI irAE and No-GI irAE groups of patients at the three time points.,In baseline samples, 27 probe sets showed differential mean expression (≥ 1.5 fold, P ≤ 0.05) between the GI irAE and No-GI irAE groups.,Most of these probe sets belonged to three functional categories: immune system, cell cycle, and intracellular trafficking.,Changes in gene expression over time were also characterized.,In the GI irAE group, 58 and 247 probe sets had a ≥ 1.5 fold change in expression from baseline to 3 and 11 weeks after first ipilimumab dose, respectively.,In particular, on-treatment expression increases of CD177 and CEACAM1, two neutrophil-activation markers, were closely associated with GI irAEs, suggesting a possible role of neutrophils in ipilimumab-associated GI irAEs.,In addition, the expression of several immunoglobulin genes increased over time, with greater increases in patients with grade 2+ GI irAEs.,Gene expression profiling of peripheral blood, sampled before or early in the course of treatment with ipilimumab, resulted in the identification of a set of potential biomarkers that were associated with occurrence of GI irAEs.,However, because of the low sensitivity of these biomarkers, they cannot be used alone to predict which patients will develop GI irAEs.,Further investigation of these biomarkers in a larger patient cohort is warranted.

Most skin lesions first present in primary care, where distinguishing rare melanomas from benign lesions can be challenging.,Dermoscopy improves diagnostic accuracy among specialists and is promoted for use by primary care physicians (PCPs).,However, when used by untrained clinicians, accuracy may be no better than visual inspection.,This study aimed to undertake a systematic review of literature reporting use of dermoscopy to triage suspicious skin lesions in primary care settings, and challenges for implementation.,A systematic literature review and narrative synthesis.,We searched MEDLINE, Cochrane Central, EMBASE, Cumulative Index to Nursing and Allied Health Literature, and SCOPUS bibliographic databases from 1 January 1990 to 31 December 2017, without language restrictions.,Studies including assessment of dermoscopy accuracy, acceptability to patients and PCPs, training requirements, and cost-effectiveness of dermoscopy modes in primary care, including trials, diagnostic accuracy and acceptability studies.,23 studies met the review criteria, representing 49 769 lesions and 3708 PCPs, all from high-income countries.,There was a paucity of studies set truly in primary care and the outcomes measured were diverse.,The heterogeneity therefore made meta-analysis unfeasible; the data were synthesised through narrative review.,Dermoscopy, with appropriate training, was associated with improved diagnostic accuracy for melanoma and benign lesions, and reduced unnecessary excisions and referrals.,Teledermoscopy-based referral systems improved triage accuracy.,Only three studies examined cost-effectiveness; hence, there was insufficient evidence to draw conclusions.,Costs, training and time requirements were considered important implementation barriers.,Patient satisfaction was seldom assessed.,Computer-aided dermoscopy and other technological advances have not yet been tested in primary care.,Dermoscopy could help PCPs triage suspicious lesions for biopsy, urgent referral or reassurance.,However, it will be important to establish further evidence on minimum training requirements to reach competence, as well as the cost-effectiveness and patient acceptability of implementing dermoscopy in primary care.,CRD42018091395.

Rising healthcare expenditures places the potential for substitution of hospital care towards primary care high on the political agenda.,As low-risk basal cell carcinoma (BCC) care is one of the potential targets for substitution of hospital care towards primary care the objective of this study is to gain insight in the views of healthcare professionals regarding substitution of skin cancer care, and to identify perceived barriers and potential strategies to facilitate substitution.,A qualitative study was conducted consisting of 40 interviews with dermatologists and GPs and three focus groups with 18 selected GPs with noted willingness regarding substitution of skin cancer care.,The interviews and focus groups focused on general views, perceived barriers and potential strategies to facilitate substitution of skin cancer care, using predefined topic lists.,All sessions were audio-taped, transcribed verbatim and analyzed using the program AtlasTi.,GPs were generally positive regarding substitution of skin care whereas dermatologists expressed more concerns.,Lack of trust in GPs to adequately perform skin cancer care and a preference of patients for dermatologists are reported as barriers by dermatologists.,The main barriers reported by GPs were a lack of confidence in own skills to perform skin cancer care, a lack of trust from both patients and dermatologists and limited time and financial compensation.,Facilitating strategies suggested by both groups mainly focused on improving GPs’ education and improving the collaboration between primary and secondary care.,GPs additionally suggested efforts from dermatologists to increase their own and patients’ trust in GPs, and time and financial compensation.,The selected group of GPs suggested practical solutions to facilitate substitution focusing on changes in organizational structure including horizontal referring, outreach models and practice size reduction.,GPs and, to lesser extent, dermatologists are positive regarding substitution of low-risk BCC care, though report substantial barriers that need to be addressed before substitution can be further implemented.,Aside from essential strategies such as improving GPs’ skin cancer education and time and financial compensation, rearranging the organizational structure in primary care and between primary and secondary care may facilitate effective and safe substitution of low-risk BCC care.

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

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.

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.

Melanoma is notable for its metastatic propensity, lethality in the advanced setting, and association with ultraviolet (UV) exposure early in life1.,To obtain a comprehensive genomic view of melanoma, we sequenced the genomes of 25 metastatic melanomas and matched germline DNA.,A wide range of point mutation rates was observed: lowest in melanomas whose primaries arose on non-UV exposed hairless skin of the extremities (3 and 14 per Mb genome), intermediate in those originating from hair-bearing skin of the trunk (range = 5 to 55 per Mb), and highest in a patient with a documented history of chronic sun exposure (111 per Mb).,Analysis of whole-genome sequence data identified PREX2 - a PTEN-interacting protein and negative regulator of PTEN in breast cancer2 - as a significantly mutated gene with a mutation frequency of approximately 14% in an independent extension cohort of 107 human melanomas.,PREX2 mutations are biologically relevant, as ectopic expression of mutant PREX2 accelerated tumor formation of immortalized human melanocytes in vivo.,Thus, whole-genome sequencing of human melanoma tumors revealed genomic evidence of UV pathogenesis and discovered a new recurrently mutated gene in melanoma.

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

Hotspot mutations in the spliceosome gene SF3B1 are reported in ∼20% of uveal melanomas.,SF3B1 is involved in 3′-splice site (3′ss) recognition during RNA splicing; however, the molecular mechanisms of its mutation have remained unclear.,Here we show, using RNA-Seq analyses of uveal melanoma, that the SF3B1R625/K666 mutation results in deregulated splicing at a subset of junctions, mostly by the use of alternative 3′ss.,Modelling the differential junctions in SF3B1WT and SF3B1R625/K666 cell lines demonstrates that the deregulated splice pattern strictly depends on SF3B1 status and on the 3’ss-sequence context.,SF3B1WT knockdown or overexpression do not reproduce the SF3B1R625/K666 splice pattern, qualifying SF3B1R625/K666 as change-of-function mutants.,Mutagenesis of predicted branchpoints reveals that the SF3B1R625/K666-promoted splice pattern is a direct result of alternative branchpoint usage.,Altogether, this study provides a better understanding of the mechanisms underlying splicing alterations induced by mutant SF3B1 in cancer, and reveals a role for alternative branchpoints in disease.,Mutations in the splicing factor SF3B1 are found in uveal melanoma.,Here, Alsafadi et al. use RNA-sequencing data from these cancers and experimental models, and show that mutant SF3B1 promotes alternative branchpoints in a specific gene subset and that the mutant protein gains a new function.

Supplemental Digital Content is available in the text.,Uveal melanoma is a rare form of melanoma with particularly poor outcomes in the metastatic setting.,In contrast with cutaneous melanoma, uveal melanoma lacks BRAF mutations and demonstrates very low response rates to immune-checkpoint blockade.,Our objectives were to study the transcriptomics of metastatic uveal melanoma with the intent of assessing gene pathways and potential molecular characteristics that might be nominated for further exploration as therapeutic targets.,We initially analyzed transcriptional data from The Cancer Genome Atlas suggesting PI3K/mTOR and glycolysis as well as IL6 associating with poor survival.,From tumor samples collected in a prospective phase II trial (A091201), we performed a transcriptional analysis of human metastatic uveal melanoma observing a novel role for epithelial-mesenchymal transition associating with survival.,Specifically, we nominate and describe initial functional validation of neuropillin-1 from uveal melanoma cells as associated with poor survival and as a mediator of proliferation and migration for uveal melanoma in vitro.,These results immediately nominate potential next steps in clinical research for patients with metastatic uveal melanoma.

Uveal melanoma (UM) represents the most frequent primary tumor of the eye.,Despite the development of new drugs and screening programs, the prognosis of patients with UM remains poor and no effective prognostic biomarkers are yet able to identify high-risk patients.,Therefore, in the present study, microRNA (miRNA or miR) expression data, contained in the TCGA UM (UVM) database, were analyzed in order to identify a set of miRNAs with prognostic significance to be used as biomarkers in clinical practice.,Patients were stratified into 2 groups, including tumor stage (high-grade vs. low-grade) and status (deceased vs. alive); differential analyses of miRNA expression among these groups were performed.,A total of 20 deregulated miRNAs for each group were identified.,In total 7 miRNAs were common between the groups.,The majority of common miRNAs belonged to the miR-506-514 cluster, known to be involved in UM development.,The prognostic value of the 20 selected miRNAs related to tumor stage was assessed.,The deregulation of 12 miRNAs (6 upregulated and 6 downregulated) was associated with a worse prognosis of patients with UM.,Subsequently, miRCancerdb and microRNA Data Integration Portal bioinformatics tools were used to identify a set of genes associated with the 20 miRNAs and to establish their interaction levels.,By this approach, 53 different negatively and positively associated genes were identified.,Finally, DIANA-mirPath prediction pathway and Gene Ontology enrichment analyses were performed on the lists of genes previously generated to establish their functional involvement in biological processes and molecular pathways.,All the miRNAs and genes were involved in molecular pathways usually altered in cancer, including the mitogen-activated protein kinase (MAPK) pathway.,Overall, the findings of the presents study demonstrated that the miRNAs of the miR-506-514 cluster, hsa-miR-592 and hsa-miR-199a-5p were the most deregulated miRNAs in patients with high-grade disease compared to those with low-grade disease and were strictly related to the overall survival (OS) of the patients.,However, further in vitro and translational approaches are required to validate these preliminary findings.

Occupational exposure to ultraviolet radiation is one of the main risk factors for non-melanoma skin cancer (NMSC) development.,The most common variants of NMSC are basal cell carcinomas, squamous cell carcinomas, and actinic keratosis (AK).,The latter is nowadays considered by most authors as an early squamous cell carcinoma rather than a precancerous lesion.,Outdoor workers have a higher risk of developing NMSC because they spend most of the working day outside.,The aim of this descriptive study was to assess the prevalence of skin lesions, especially AK, in a professional category of individuals exposed to ultraviolet (UV) radiation: the Italian Navy.,From January to June 2016, a questionnaire and a total skin examination of 921 military personnel were administered by medical specialists (dermatologists) in seven different Italian Navy centres.,AK was detected in 217 of 921 (23.5%) workers.,Older age, outdoor occupation, longer working life, and fair skin seem to promote the development of AK.,Of the 217 workers with AK, 187 (86.2%) had lesions in chronically sun-exposed skin areas.,Italian Navy personnel have a high AK prevalence.,Further studies are needed to investigate occupational hazards and their health effects among outdoor workers to promote protective behaviour and raise awareness of skin cancer.

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

We have recently demonstrated that invasive melanoma cells are capable of disrupting the brain endothelial barrier integrity.,This was shown using ECIS biosensor technology, which revealed rapid disruption via the paracellular junctions.,In this paper, we demonstrate that melanoma cells secrete factors (e.g., cytokines) that weaken the endothelial barrier integrity.,Through proteome profiling, we attempt to identify the barrier-disrupting cytokines.,Melanoma conditioned media were collected from three New Zealand melanoma lines.,ECIS technology was used to assess if the conditioned media disrupted the endothelial barrier independent of the melanoma cells.,The melanoma cell secretome was assessed using cytometric bead array (CBA), Luminex immunoassay and multiplex Proteome Profilers, to detect the expression of secretory proteins, which may facilitate metastasis.,Finally, ECIS technology was used to assess the direct effects of secreted proteins identified as candidates from the proteome screens.,We show that melanoma-conditioned media significantly disrupted the brain endothelial barrier, however, to a much lesser extent than the cells from which they were collected.,Cytokine and proteome profiling of the conditioned media showed evidence of high concentrations of approximately 15 secreted proteins (including osteopontin, IL-8, GDF-15, MIF and VEGF).,These 15 secreted proteins were expressed variably across the melanoma lines.,Surprisingly, the addition of these individually to the brain endothelial cells did not substantially affect the barrier integrity.,ANGPTL-4 and TGFβ were also produced by the melanoma cells.,Whilst TGFβ-1 had a pronounced effect on the barrier integrity, surprisingly ANGPTL-4 did not.,However, its C-terminal fragment did and within a very similar period to the conditioned media, albeit not to the same extent.,Herein we show that melanoma cells produce a wide-range of soluble factors at high concentrations, which most likely favour support or survival of the cancer cells.,Most of these, except for TGFβ-1 and the C-terminal fragment of ANGPTL-4, did not have an impact on the integrity of the brain endothelial cells.

Matrix metalloproteinase-2 (MMP-2) is a key regulator in the migration of tumor cells. αvβ3 integrin has been reported to play a critical role in cell adhesion and regulate the migration of tumor cells by promoting MMP-2 activation.,However, little is known about the effects of MMP-2 on αvβ3 integrin activity and αvβ3 integrin-mediated adhesion and migration of tumor cells.,Human melanoma cells were seeded using an agarose drop model and/or subjected to in vitro analysis using immunofluorescence, adhesion, migration and invasion assays to investigate the relationship between active MMP-2 and αvβ3 integrin during the adhesion and migration of the tumor cells.,We found that MMP-2 was localized at the leading edge of spreading cells before αvβ3 integrin. αvβ3 integrin-mediated adhesion and migration of the tumor cells were inhibited by a MMP-2 inhibitor.,MMP-2 cleaved fibronectin into small fragments, which promoted the adhesion and migration of the tumor cells.,MMP-2 cleaves fibronectin into small fragments to enhance the adhesion and migration of human melanoma cells mediated by αvβ3 integrin.,These results indicate that MMP-2 may guide the direction of the tumor cell migration.

Statins are a class of drugs that inhibit the rate-limiting step in the cholesterol biosynthetic pathway and show an anticancer effect, probably through the inhibition of cell proliferation.,To date, the exact mechanism of cancer cell growth arrest induced by statins is not known.,We report that simvastatin is able to induce apoptosis in melanoma cells but not in normal cells and also able to contrast the growth of tumor in an experimental melanoma murine model.,We observed a delay in the tumor development in almost the 50% of the simvastatin administered animals and a strong reduction of the tumor volume with a differences of ∼150% compared to the controls.,Also the survival rate was significantly higher in mice that received the drug with a survival increase of ∼130% compared to the controls.,The tumor growth reduction in mice was supported by the results of cell migration assay, confirming that simvastatin clearly reduced cell migration.,Moreover, simvastatin induced a strong downregulation of NonO gene expression, an important growth factor involved in the splicing regulation.,This result could explain the decrease of melanoma cells proliferation, suggesting a possible action mechanism.,The results derived from our experiments may sustain the many reports on the anticancer inhibitory property of statins and encourage new studies on this drug for a possible use in therapy, probably in combination with conventional chemotherapy.

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.

We screen ion channels and transporters throughout the genome to identify those required by human melanoma cells but not by normal human melanocytes.,We discover that Mucolipin-1 (MCOLN1), which encodes the lysosomal cation channel TRPML1, is preferentially required for the survival and proliferation of melanoma cells.,Loss of MCOLN1/TRPML1 function impairs the growth of patient-derived melanomas in culture and in xenografts but does not affect the growth of human melanocytes.,TRPML1 expression and macropinocytosis are elevated in melanoma cells relative to melanocytes.,TRPML1 is required in melanoma cells to negatively regulate MAPK pathway and mTORC1 signaling.,TRPML1-deficient melanoma cells exhibit decreased survival, proliferation, tumor growth, and macropinocytosis, as well as serine depletion and proteotoxic stress.,All of these phenotypes are partially or completely rescued by mTORC1 inhibition.,Melanoma cells thus increase TRPML1 expression relative to melanocytes to attenuate MAPK and mTORC1 signaling, to sustain macropinocytosis, and to avoid proteotoxic stress.

Human melanoma cells express various tumour antigens that are recognized by CD8+ cytotoxic T lymphocytes (CTLs) and elicit tumour-specific responses in vivo.,However, natural and therapeutically enhanced CTL responses in melanoma patients are of limited efficacy.,The mechanisms underlying CTL effector phase failure when facing melanomas are still largely elusive.,Here we show that, on conjugation with CTL, human melanoma cells undergo an active late endosome/lysosome trafficking, which is intensified at the lytic synapse and is paralleled by cathepsin-mediated perforin degradation and deficient granzyme B penetration.,Abortion of SNAP-23-dependent lysosomal trafficking, pH perturbation or impairment of lysosomal proteolytic activity restores susceptibility to CTL attack.,Inside the arsenal of melanoma cell strategies to escape immune surveillance, we identify a self-defence mechanism based on exacerbated lysosome secretion and perforin degradation at the lytic synapse.,Interfering with this synaptic self-defence mechanism might be useful in potentiating CTL-mediated therapies in melanoma patients.,Cytotoxic T lymphocytes recognise and eliminate tumour cells.,Here, the authors show that on contact with these immune cells melanoma cells can resist T cell cytotoxicity by modulating the trafficking of their lysosomal compartment, this results in the degradation of the T cell protein perforin by the protease cathepsin B.

The principles governing evolution of tumors exposed to targeted therapy are poorly understood.,Here we modeled the selection and propagation of BRAF amplification (BRAFamp) in patient-derived tumor xenografts (PDX) treated with a direct ERK inhibitor, alone or in combination with other pathway inhibitors.,Single cell sequencing and multiplex-fluorescence in situ hybridization mapped the emergence of extra-chromosomal amplification in parallel evolutionary tracts, arising in the same tumor shortly after treatment.,The evolutionary selection of BRAFamp is determined by the fitness threshold, the barrier subclonal populations need to overcome to regain fitness in the presence of therapy.,This differed for ERK signaling inhibitors, suggesting that sequential monotherapy is ineffective and selects for a progressively higher BRAF copy number.,Concurrent targeting of RAF, MEK and ERK, however, imposes a sufficiently high fitness threshold to prevent the propagation of subclones with high-level amplification.,Administered on an intermittent schedule, this treatment inhibited tumor growth in 11/11-lung cancer and melanoma PDX without apparent toxicity in mice.,Thus, gene amplification can be acquired and expanded through parallel evolution, enabling tumors to adapt while maintaining their intratumoral heterogeneity.,Treatments that impose the highest fitness threshold will likely prevent the evolution of resistance-causing alterations and merit testing in patients.

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.

Melanoma is a fatal skin malignant tumor with a poor prognosis.,We found that long noncoding RNA BASP1 ‐AS1 is essential for the development and prognosis of melanoma.,The methylation, RNA sequencing, copy number variation, mutation data, and sample follow‐up information of melanoma from The Cancer Genome Atlas (TCGA) were analyzed using weighted gene co‐expression network analysis and 366 samples common to the three omics were selected for multigroup clustering analysis.,A four‐gene prognostic model (BASP1‐AS1, LOC100506098, ARHGAP27P1, and LINC01532) was constructed in the TCGA cohort and validated using the GSE65904 series.,The expression of BASP1‐AS1 was upregulated in melanoma tissues and various melanoma cell lines.,Functionally, the ectopic expression of BASP1‐AS1 promoted cell proliferation, migration, and invasion in both A375 and SK‐MEL‐2 cells.,Mechanically, BASP1‐AS1 interacted with YBX1 and recruited it to the promoter of NOTCH3, initiating its transcription process.,The activation of the Notch signaling then resulted in the transcription of multiple oncogenes, including c‐MYC, PCNA, and CDK4, which contributed to melanoma progression.,Thus, BASP1‐AS1 could act as a potential biomarker for cutaneous malignant melanoma.,We identified a new type of long noncoding RNA (LncRNA) BASP1 ‐AS1, which can promote melanoma development both in vivo and in vitro.,Detailed studies on the molecular mechanism showed that BASP1‐AS1 promoted the proliferation, invasion, and migration of melanoma cells by regulating YBX1.,In addition, LncRNA BASP1‐AS1 is a poor prognostic indicator and a potential therapeutic target for melanoma.

Cancer cells, including melanoma, often metastasize regionally through lymphatics before metastasizing systemically through the blood1-4; however, the reason for this is unclear.,Here we show that melanoma cells in lymph experience less oxidative stress and form more metastases than melanoma cells in blood.,Immunocompromised mice with patient-derived melanomas and immunocompetent mice with mouse melanomas had more melanoma cells per microliter of tumor-draining lymph than tumor-draining blood.,Cells metastasizing through blood, but not lymph, became dependent on the ferroptosis inhibitor GPX4.,Cells pre-treated with chemical ferroptosis inhibitors formed more metastases than untreated cells after intravenous, but not intralymphatic, injection.,We observed multiple differences between lymph fluid and blood plasma that may contribute to decreased oxidative stress and ferroptosis in lymph, including higher levels of glutathione and oleic acid, and less free iron, in lymph.,Oleic acid protected melanoma cells from ferroptosis in an Acsl3-dependent manner and increased their capacity to form metastatic tumors.,Melanoma cells from lymph nodes were more resistant to ferroptosis and formed more metastases after intravenous injection than melanoma cells from subcutaneous tumors.,Exposure to the lymphatic environment thus protects melanoma cells from ferroptosis and increases their ability to survive during subsequent metastasis through the blood.

In the search for novel, natural melanogenesis inhibitors, a new sesquiterpene, inularin, was isolated from the flowers of Inula britannica, and the structure was determined using spectroscopic and chemical methods.,The antimelanogenic effects of inularin on B16F10 melanoma cells and zebrafish embryos were evaluated.,Inularin dose-dependently reduced melanocyte-stimulating hormone-induced melanin production and L-DOPA oxidation in B16F10 cells.,Zebrafish embryos were used to confirm the antimelanogenic activity.,Inularin significantly decreased the pigmentation of embryos compared with untreated controls.

Genetic variation conferring resistance and susceptibility to carcinogen-induced tumorigenesis is frequently studied in mice.,We have now turned this idea to melanoma using the collaborative cross (CC), a resource of mouse strains designed to discover genes for complex diseases.,We studied melanoma-prone transgenic progeny across seventy CC genetic backgrounds.,We mapped a strong quantitative trait locus for rapid onset spontaneous melanoma onset to Prkdc, a gene involved in detection and repair of DNA damage.,In contrast, rapid onset UVR-induced melanoma was linked to the ribosomal subunit gene Rrp15.,Ribosome biogenesis was upregulated in skin shortly after UVR exposure.,Mechanistically, variation in the ‘usual suspects’ by which UVR may exacerbate melanoma, defective DNA repair, melanocyte proliferation, or inflammatory cell infiltration, did not explain melanoma susceptibility or resistance across the CC.,Instead, events occurring soon after exposure, such as dysregulation of ribosome function, which alters many aspects of cellular metabolism, may be important.,Melanoma is a type of skin cancer.,Melanoma tumors form in the skin’s pigment-producing cells or melanocytes.,Growing evidence points to complex interactions between genetics and environmental exposures that contribute to the risk of developing melanoma.,Ultraviolet (UV) radiation from the sun causes genetic mutations in melanocytes.,This sun exposure interacts with genetic variations that may make people more or less vulnerable to such DNA damage.,For example, genetic variations that control skin color or the cell’s ability to repair DNA, and that influence how easily people develop sunburn, all affect whether UV damage leads to melanoma.,However, some forms of melanoma are not caused by sun exposure at all.,Most of the genetic variations linked to melanoma have a small effect on the risk of developing the disease.,So, it is unlikely that these genes alone cause melanoma.,Few studies have been able to map the complex interactions between genes and the environment that lead to melanoma.,So far, it has been unclear if there are different genetic mechanisms that lead to an increased risk for sun-exposure linked melanoma and non-sun linked melanoma.,Now, Ferguson et al. show that variations in the genes involved in DNA repair during normal cell growth are linked to non-sun linked melanoma.,Sun-linked melanoma, on the other hand, was associated with genes involved in the production of proteins in part of the cell called ribosomes.,In the experiments, the effects of both UV light and various genetic variations were assessed across many different strains of mice.,Mutations that impair the cell’s ability to repair UV-induced DNA damage or that contribute to excessive inflammation in response to sunburn did not increase melanoma susceptibility in these experiments.,Ferguson et al. show that the amount of UV-induced DNA damage alone does not explain melanoma risk, which may not always depend on skin pigmentation.,The experiments also suggest that non-UV linked melanoma is caused by a different mechanism than sun exposure-linked melanoma.,Learning more about different genetic factors that affect the risk of developing different types of melanoma may help scientists develop more specific treatments.

Previous analysis of COMBI-d (NCT01584648) demonstrated improved progression-free survival (PFS) and overall survival (OS) with combination dabrafenib and trametinib versus dabrafenib monotherapy in BRAF V600E/K-mutant metastatic melanoma.,This study was continued to assess 3-year landmark efficacy and safety after ≥36-month follow-up for all living patients.,This double-blind, phase 3 study enrolled previously untreated patients with BRAF V600E/K-mutant unresectable stage IIIC or stage IV melanoma.,Patients were randomized to receive dabrafenib (150 mg twice daily) plus trametinib (2 mg once daily) or dabrafenib plus placebo.,The primary endpoint was PFS; secondary endpoints were OS, overall response, duration of response, safety, and pharmacokinetics.,Between 4 May and 30 November 2012, a total of 423 of 947 screened patients were randomly assigned to receive dabrafenib plus trametinib (n = 211) or dabrafenib monotherapy (n = 212).,At data cut-off (15 February 2016), outcomes remained superior with the combination: 3-year PFS was 22% with dabrafenib plus trametinib versus 12% with monotherapy, and 3-year OS was 44% versus 32%, respectively.,Twenty-five patients receiving monotherapy crossed over to combination therapy, with continued follow-up under the monotherapy arm (per intent-to-treat principle).,Of combination-arm patients alive at 3 years, 58% remained on dabrafenib plus trametinib.,Three-year OS with the combination reached 62% in the most favourable subgroup (normal lactate dehydrogenase and <3 organ sites with metastasis) versus only 25% in the unfavourable subgroup (elevated lactate dehydrogenase).,The dabrafenib plus trametinib safety profile was consistent with previous clinical trial observations, and no new safety signals were detected with long-term use.,These data demonstrate that durable (≥3 years) survival is achievable with dabrafenib plus trametinib in patients with BRAF V600-mutant metastatic melanoma and support long-term first-line use of the combination in this setting.

Molecular diagnostics are increasingly performed routinely in the diagnosis and management of patients with melanoma due to the development of novel therapies that target specific genetic mutations.,The development of next-generation sequencing (NGS) technologies has enabled to sequence multiple cancer-driving genes in a single assay, with improved sensitivity in mutation detection.,The main objective of this study was the design and implementation of a melanoma-specific sequencing panel, and the identification of the spectrum of somatic mutations in a series of primary melanoma samples.,A custom panel was designed to cover the coding regions of 35 melanoma-related genes.,Panel average coverage was 2,575.5 reads per amplicon, with 92,8% of targeted bases covered ≥500×.,Deep coverage enabled sensitive discovery of mutations in as low as 0.5% mutant allele frequency.,Eighty-five percent (85/100) of the melanomas had at least one somatic mutation.,The most prevalent mutated genes were BRAF (50%;50/199), NRAS (15%;15/100), PREX2 (14%;14/100), GRIN2A (13%;13/100), and ERBB4 (12%;12/100).,Turn-around-time and costs for NGS-based analysis was reduced in comparison to conventional molecular approaches.,The results of this study demonstrate the cost-effectiveness and feasibility of a custom-designed targeted NGS panel, and suggest the implementation of targeted NGS into daily routine practice.

Uveal melanoma (UM) is a cancer of eye melanocytes.,Although relatively rare, UM is extremely deadly, as approximately half of all patients develop liver metastases for which there are no approved therapies.,Even therapies that succeed in cutaneous melanoma (CM) treatment have proven ineffectual for UM, highlighting both the distinct nature of these two melanomas and the need to understand the differences between them.,Here, we show that autochthonous UM tumors are rapidly induced by activated YAP and can lack hyperactive ERK, highlighting YAP as a promising therapeutic target.,We further show that MITF functions as a tumor suppressor in UM in contrast to its essential role in CM, establishing that MITF inhibition should not be entertained for UM treatment.,Cutaneous melanoma (CM) and uveal melanoma (UM) both originate from the melanocytic lineage but are primarily driven by distinct oncogenic drivers, BRAF/NRAS or GNAQ/GNA11, respectively.,The melanocytic master transcriptional regulator, MITF, is essential for both CM development and maintenance, but its role in UM is largely unexplored.,Here, we use zebrafish models to dissect the key UM oncogenic signaling events and establish the role of MITF in UM tumors.,Using a melanocytic lineage expression system, we showed that patient-derived mutations of GNAQ (GNAQQ209L) or its upstream CYSLTR2 receptor (CYSLTR2L129Q) both drive UM when combined with a cooperating mutation, tp53M214K/M214K.,The tumor-initiating potential of the major GNAQ/11 effector pathways, YAP, and phospholipase C-β (PLCβ)-ERK was also investigated in this system and thus showed that while activated YAP (YAPAA) induced UM with high potency, the patient-derived PLCβ4 mutation (PLCB4D630Y) very rarely yielded UM tumors in the tp53M214K/M214K context.,Remarkably, mitfa deficiency was profoundly UM promoting, dramatically accelerating the onset and progression of tumors induced by Tg(mitfa:GNAQQ209L);tp53M214K/M214K or Tg(mitfa:CYSLTR2L129Q);tp53M214K/M214K.,Moreover, mitfa loss was sufficient to cooperate with GNAQQ209L to drive tp53-wild type UM development and allowed Tg(mitfa:PLCB4D630Y);tp53M214K/M214K melanocyte lineage cells to readily form tumors.,Notably, all of the mitfa−/− UM tumors, including those arising in Tg(mitfa:PLCB4D630Y);tp53M214K/M214K;mitfa−/− zebrafish, displayed nuclear YAP while lacking hyperactive ERK indicative of PLCβ signaling.,Collectively, these data show that YAP signaling is the major mediator of UM and that MITF acts as a bona fide tumor suppressor in UM in direct opposition to its essential role in CM.

Preclinical studies have suggested that epigenetic therapy could enhance immunogenicity of cancer cells.,We report the results of the PEMDAC phase 2 clinical trial (n = 29; NCT02697630) where the HDAC inhibitor entinostat was combined with the PD-1 inhibitor pembrolizumab in patients with metastatic uveal melanoma (UM).,The primary endpoint was objective response rate (ORR), and was met with an ORR of 14%.,The clinical benefit rate at 18 weeks was 28%, median progression free survival was 2.1 months and the median overall survival was 13.4 months.,Toxicities were manageable, and there were no treatment-related deaths.,Objective responses and/or prolonged survival were seen in patients with BAP1 wildtype tumors, and in one patient with an iris melanoma that exhibited a UV signature.,Longer survival also correlated with low baseline ctDNA levels or LDH.,In conclusion, HDAC inhibition and anti-PD1 immunotherapy results in durable responses in a subset of patients with metastatic UM.,Trial registration ClinicalTrials.gov registration number: NCT02697630 (registered 3 March 2016).,EudraCT registration number: 2016-002114-50.,The authors report the results of the phase II PEMDAC clinical study testing the combination of the HDAC inhibitor entinostat with the anti- PD-1 antibody pembrolizumab in uveal melanoma.,Low tumor burden, a wildtype BAP1 gene in the tumor or iris melanoma correlates with response and longer survival.

Neoangiogenesis is a recognized hallmark of cancer, granting tumor cells to dispose of metabolic substrates through a newly created vascular supply.,Neoangiogenesis was also confirmed in melanoma, where vascular proliferation is associated with increased aggressiveness and poorer prognosis.,Furthermore, melanoma cells show the so-called vascular mimicry, consisting in the assumption of endothelial-like features inducing the expression of pro-angiogenic receptors and ligands, which take part in the interplay with extracellular matrix (ECM) components and are potentiated by the ECM remodeling and the barrier molecule junction alterations that characterize the metastatic phase.,Although neoangiogenesis was biologically proven and clinically associated with worse outcomes in melanoma patients, in the past anti-angiogenic therapies were employed with poor improvement of the already unsatisfactory results associated with chemotherapic agents.,Among the novel therapies of melanoma, immunotherapy has led to previously unexpected outcomes of treatment, yet there is a still strong need for potentiating the results, possibly by new regimens of combination therapies.,Molecular models in many cancer types showed mutual influences between immune responses and vascular normalization.,Recently, clinical trials are investigating the efficacy of the association between anti-angiogenetic agents and immune-checkpoint inhibitors to treat advanced stage melanoma.,This paper reviews the biological bases of angiogenesis in melanoma and summarizes the currently available clinical data on the use of anti-angiogenetic compounds in melanoma.

Acquired melanocytic nevi are commonly found in sun exposed and unexposed human skin, but the potential for their transformation into invasive melanoma is not clear.,Therefore, a mouse model of nevus initiation and progression was developed in C3H/HeN mice using a modified chemical carcinogenesis protocol.,Nevi develop due to DNA damage initiated by dimethylbenz(a) anthracene (DMBA) followed by chronic promotion with 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA).,Dysplastic pigmented skin lesions appeared in 7-9 wk with 100% penetrance.,Nests of melanocytic cells appeared in a subset of skin draining lymph nodes (dLN) by 25 wk, but not in age matched controls.,Immunohistochemistry, real‐time PCR, and flow cytometric analyses confirmed their melanocytic origin.,Transformed cells were present in a subset of nevi and dLNs, which exhibited anchorage‐independent growth, tumor development, and metastasis in nude mice.,Approximately 50% of the cell lines contained H‐Ras mutations and lost tumor suppressor p16Ink4a expression.,While most studies of melanoma focus on tumor progression in transgenic mouse models where the mutations are present from birth, our model permits investigation of acquired mutations at the earliest stages of nevus initiation and promotion of nevus cell transformation.,This robust nevus/melanoma model may prove useful for identifying genetic loci associated with nevus formation, novel oncogenic pathways, tumor targets for immune‐prevention, screening therapeutics, and elucidating mechanisms of immune surveillance and immune evasion.,© 2015 The Authors.,Molecular Carcinogenesis, published by Wiley Periodicals, Inc.

Acral melanoma (AM) tumors arise on the palms, soles, fingers, toes, and nailbeds.,A comprehensive systematic meta‐analysis of AM genomic aberrations has not been conducted to date.,A literature review was carried out to identify studies sequencing AM.,Whole‐genome/exome data from 181 samples were identified.,Targeted panel sequencing data from MSK‐IMPACT were included as a validation cohort (n = 92), and studies using targeted hot spot sequencing were also collated for BRAF (n = 26 studies), NRAS (n = 21), and KIT (n = 32).,Statistical analysis indicated BRAF, NRAS, PTEN, TYRP1, and KIT as significantly mutated genes.,Frequent copy‐number aberrations were also found for important cancer genes, such as CDKN2A, KIT, MDM2, CCND1, CDK4, and PAK1, among others.,Mapping genomic alterations within the context of the hallmarks of cancer identified four components frequently altered, including (i) sustained proliferative signaling and (ii) evading growth suppression, (iii) genome instability and mutation, and (iv) enabling replicative immortality.,This analysis provides the largest analysis of genomic aberrations in AM in the literature to date and highlights pathways that may be therapeutically targetable.

We sequenced 8 melanoma exomes to identify novel somatic mutations in metastatic melanoma.,Focusing on the MAP3K family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9.,Structural modelling predicts that mutations in the kinase domain may affect the activity and regulation of MAP3K5/9 protein kinases.,The position of the mutations and loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely inactivating.,In vitro kinase assay shows reduction in kinase activity in MAP3K5 I780F and MAP3K9 W333X mutants.,Overexpression of MAP3K5 or MAP3K9 mutant in HEK293T cells reduces phosphorylation of downstream MAP kinases.,Attenuation of MAP3K9 function in melanoma cells using siRNA leads to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.

Cutaneous melanoma is an aggressive cancer with a poor prognosis for patients with advanced disease.,The identification of several key molecular pathways implicated in the pathogenesis of melanoma has led to the development of novel therapies for this devastating disease.,In melanoma, both the Ras/Raf/MEK/ERK (MAPK) and the PI3K/AKT (AKT) signalling pathways are constitutively activated through multiple mechanisms.,Targeting various effectors of these pathways with pharmacologic inhibitors may inhibit melanoma cell growth and angiogenesis.,Ongoing clinical trials provide hope to improve progression-free survival of patients with advanced melanoma.,This review summarizes the most relevant studies focused on the specific action of these new molecular targeted agents.,Mechanisms of resistance to therapy are also discussed.

Our understanding of the molecular pathways that underlie melanoma remains incomplete.,Although several published microarray studies of clinical melanomas have provided valuable information, we found only limited concordance between these studies.,Therefore, we took an in vitro functional genomics approach to understand melanoma molecular pathways.,Affymetrix microarray data were generated from A375 melanoma cells treated in vitro with siRNAs against 45 transcription factors and signaling molecules.,Analysis of this data using unsupervised hierarchical clustering and Bayesian gene networks identified proliferation-association RNA clusters, which were co-ordinately expressed across the A375 cells and also across melanomas from patients.,The abundance in metastatic melanomas of these cellular proliferation clusters and their putative upstream regulators was significantly associated with patient prognosis.,An 8-gene classifier derived from gene network hub genes correctly classified the prognosis of 23/26 metastatic melanoma patients in a cross-validation study.,Unlike the RNA clusters associated with cellular proliferation described above, co-ordinately expressed RNA clusters associated with immune response were clearly identified across melanoma tumours from patients but not across the siRNA-treated A375 cells, in which immune responses are not active.,Three uncharacterised genes, which the gene networks predicted to be upstream of apoptosis- or cellular proliferation-associated RNAs, were found to significantly alter apoptosis and cell number when over-expressed in vitro.,This analysis identified co-expression of RNAs that encode functionally-related proteins, in particular, proliferation-associated RNA clusters that are linked to melanoma patient prognosis.,Our analysis suggests that A375 cells in vitro may be valid models in which to study the gene expression modules that underlie some melanoma biological processes (e.g., proliferation) but not others (e.g., immune response).,The gene expression modules identified here, and the RNAs predicted by Bayesian network inference to be upstream of these modules, are potential prognostic biomarkers and drug targets.

Assessment of tumor infiltrating lymphocytes (TILs) as a prognostic variable in melanoma has not seen broad adoption due to lack of standardization.,Automation could represent a solution.,Here, using open source software, we build an algorithm for image-based automated assessment of TILs on hematoxylin-eosin stained sections in melanoma.,Using a retrospective collection of 641 melanoma patients comprising four independent cohorts; one training set (N = 227) and three validation cohorts (N = 137, N = 201, N = 76) from 2 institutions, we show that the automated TIL scoring algorithm separates patients into favorable and poor prognosis cohorts, where higher TILs scores were associated with favorable prognosis.,In multivariable analyses, automated TIL scores show an independent association with disease-specific overall survival.,Therefore, the open source, automated TIL scoring is an independent prognostic marker in melanoma.,With further study, we believe that this algorithm could be useful to define a subset of patients that could potentially be spared immunotherapy.,Histology data exists for many cancer samples and the ability to automatically image this data may provide prognostic information.,Here, the authors generated an algorithm to measure tumour infiltrating lymphocytes in melanoma histology specimens and show that the ratio of these immune cells to tumour cells has prognostic value.

The approval of vemurafenib in the US 2011 and in Europe 2012 improved the therapy of not resectable or metastatic melanoma.,Patients carrying a substitution of valine to glutamic acid at codon 600 (p.V600E) or a substitution of valine to leucine (p.V600K) in BRAF show complete or partial response.,Therefore, the precise identification of the underlying somatic mutations is essential.,Herein, we evaluate the sensitivity, specificity and feasibility of six different methods for the detection of BRAF mutations.,Samples harboring p.V600E mutations as well as rare mutations in BRAF exon 15 were compared to wildtype samples.,DNA was extracted from formalin-fixed paraffin-embedded tissues by manual micro-dissection and automated extraction.,BRAF mutational analysis was carried out by high resolution melting (HRM) analysis, pyrosequencing, allele specific PCR, next generation sequencing (NGS) and immunohistochemistry (IHC).,All mutations were independently reassessed by Sanger sequencing.,Due to the limited tumor tissue available different numbers of samples were analyzed with each method (82, 72, 60, 72, 49 and 82 respectively).,There was no difference in sensitivity between the HRM analysis and Sanger sequencing (98%).,All mutations down to 6.6% allele frequency could be detected with 100% specificity.,In contrast, pyrosequencing detected 100% of the mutations down to 5% allele frequency but exhibited only 90% specificity.,The allele specific PCR failed to detect 16.3% of the mutations eligible for therapy with vemurafenib.,NGS could analyze 100% of the cases with 100% specificity but exhibited 97.5% sensitivity.,IHC showed once cross-reactivity with p.V600R but was a good amendment to HRM.,Therefore, at present, a combination of HRM and IHC is recommended to increase sensitivity and specificity for routine diagnostic to fulfill the European requirements concerning vemurafenib therapy of melanoma patients.

Cutaneous squamous cell carcinoma (cSCC) is the most common type of non-melanoma skin cancer that can metastasize.,The major etiological factor associated with cSCC is Ultraviolet radiation (UVR) with a limited understanding of its molecular mechanism.,It was hypothesized that there is a direct effect of UVR on modulation of microRNAs (miRNAs), a novel class of short noncoding RNAs which affects translation and stability of mRNAs.,To test the hypothesis, the dorsal skin of the SKH1 mice (6-7 week old) was exposed to acute and chronic doses of UVR.,In miRNA array profiling, we found differential expression (log fold change>1) of miR-25-5p between untreated and acute UVR treated (4kJ/m2) SKH1 mice skin.,However, differential expression (>1 log fold) of miR-144-3p, miR-33-5p, miR-32-5p, miR-1983, miR-136-5p, miR-142-3p, miR-376a-3p, miR-142-5p, miR-3968, and miR-29b-3p was observed between untreated and chronically UVR treated mice skin.,Differentially expressed selected miRNAs (miR-32-5p, miR-33-5p, miR-144-3p, and miR-376a-3p) were further validated in real time PCR using miRNA specific primers.,Web based data mining, for the prediction of potential miRNA associated gene pathways in miRBase database revealed a link with important pathways (PI3K-Akt, MAPK, Wnt, transcriptional misregulation, and other oncogenic pathway) associated with cSCC.,Furthermore, findings of PI3K-Akt pathway genes affected due to chronic UVR were confirmed using cDNA array.

Micro RNAs (miRs) have emerged as key regulators during oncogenesis.,They have been found to regulate cell proliferation, differentiation, and apoptosis.,Mir-125b has been identified as an oncomir in various forms of tumours, but we have previously proposed that miR-125b is a suppressor of lymph node metastasis in cutaneous malignant melanoma.,Our goal was therefore to further examine this theory.,We used in-situ-hybridization to visualise miR-125b expression in primary tumours and in lymph node metastasis.,Then using a miRVector plasmid containing a miR-125b-1 insert we transfected melanoma cell line Mel-Juso and then investigated the effect of the presence of a stable overexpression of miR-125b on growth by western blotting, flow cytometry and β-galactosidase staining.,The tumourogenicity of the transfected cells was tested using a murine model and the tumours were further examined with in-situ-hybridization.,In primary human tumours and in lymph node metastases increased expression of miR-125b was found in single, large tumour cells with abundant cytoplasm.,A stable overexpression of miR-125b in human melanoma cell line Mel-Juso resulted in a G0/G1 cell cycle block and emergence of large cells expressing senescence markers: senescence-associated beta-galactosidase, p21, p27 and p53.,Mel-Juso cells overexpressing miR-125b were tumourigenic in mice, but the tumours exhibited higher level of cell senescence and decreased expression of proliferation markers, cyclin D1 and Ki67 than the control tumours.,Our results confirm the theory that miR-125b functions as a tumour supressor in cutaneous malignant melanoma by regulating cellular senescence, which is one of the central mechanisms protecting against the development and progression of malignant melanoma.

A phase II randomised discontinuation trial assessed cabozantinib (XL184), an orally bioavailable inhibitor of tyrosine kinases including VEGF receptors, MET, and AXL, in a cohort of patients with metastatic melanoma.,Patients received cabozantinib 100 mg daily during a 12-week lead-in.,Patients with stable disease (SD) per Response Evaluation Criteria in Solid Tumours (RECIST) at week 12 were randomised to cabozantinib or placebo.,Primary endpoints were objective response rate (ORR) at week 12 and postrandomisation progression-free survival (PFS).,Seventy-seven patients were enroled (62% cutaneous, 30% uveal, and 8% mucosal).,At week 12, the ORR was 5% 39% of patients had SD.,During the lead-in phase, reduction in target lesions from baseline was seen in 55% of evaluable patients overall and in 59% of evaluable patients with uveal melanoma.,Median PFS after randomisation was 4.1 months with cabozantinib and 2.8 months with placebo (hazard ratio of 0.59; P=0.284).,Median PFS from study day 1 was 3.8 months, 6-month PFS was 33%, and median overall survival was 9.4 months.,The most common grade 3/4 adverse events were fatigue (14%), hypertension (10%), and abdominal pain (8%).,One treatment-related death was reported from peritonitis due to diverticular perforation.,Cabozantinib has clinical activity in patients with metastatic melanoma, including uveal melanoma.,Further clinical investigation is warranted.

Numerous tyrosine kinase inhibitors (TKIs) targeting c-Met are currently in clinical trials for several cancers.,Their efficacy is limited due to the development of resistance.,The present study aims to elucidate this mechanism of c-Met TKI resistance by investigating key mTOR and Wnt signaling proteins in melanoma cell lines resistant to SU11274, a c-Met TKI.,Xenografts from RU melanoma cells treated with c-Met TKIs SU11274 and JNJ38877605 showed a 7- and 6-fold reduction in tumor size, respectively.,Resistant cells displayed upregulation of phosphorylated c-Met, mTOR, p70S6Kinase, 4E-BP1, ERK, LRP6, and active β-catenin.,In addition, GATA-6, a Wnt signaling regulator, was upregulated, and Axin, a negative regulator of the Wnt pathway, was downregulated in resistant cells.,Modulation of these mTOR and Wnt pathway proteins was also prevented by combination treatment with SU11274, everolimus, an mTOR inhibitor, and XAV939, a Wnt inhibitor.,Treatment with everolimus, resulted in 56% growth inhibition, and a triple combination of SU11274, everolimus and XAV939, resulted in 95% growth inhibition in RU cells.,The V600E BRAF mutation was found to be positive only in MU cells.,Combination treatment with a c-Met TKI and a BRAF inhibitor displayed a synergistic effect in reducing MU cell viability.,These studies indicate activation of mTOR and Wnt signaling pathways in c-Met TKI resistant melanoma cells and suggest that concurrent targeting of c-Met, mTOR, and Wnt pathways and BRAF may improve efficacy over traditional TKI monotherapy in melanoma patients.

The incidence of malignant melanoma is rising worldwide and survival for metastatic disease is still poor.,Recently, new treatment options have become available.,Still, predictive biomarkers are needed to optimise treatment for this patient group.,In this study, we investigated the predictive value of 60 angiogenic factors in patients with metastatic melanoma treated with the anti‐vascular endothelial growth factor A antibody bevacizumab.,Thirty‐five patients were included in a clinical phase II trial and baseline serum samples were analysed by multiplex protein array.,High‐serum concentration of Activin A was significantly associated with objective response (OR) to treatment (p = 0.014).,Candidate proteins that indicated a borderline association with treatment response were further investigated by immunohistochemistry.,Strong expression of Activin A, interleukin‐1β, and urokinase‐type plasminogen activator receptor in metastases was significantly associated with OR (p = 0.011, p = 0.003, and p = 0.007, respectively), as well as with markers of activated angiogenesis, such as higher number of proliferating vessels and the presence of glomeruloid microvascular proliferations.,Our findings indicate that these proteins may be potential predictive markers for treatment with bevacizumab monotherapy.

Repeat tumor biopsies to study genomic changes during therapy are difficult, invasive and data are confounded by tumoral heterogeneity.,The analysis of circulating tumor DNA (ctDNA) can provide a non-invasive approach to assess prognosis and the genetic evolution of tumors in response to therapy.,Mutation-specific droplet digital PCR was used to measure plasma concentrations of oncogenic BRAF and NRAS variants in 48 patients with advanced metastatic melanoma prior to treatment with targeted therapies (vemurafenib, dabrafenib or dabrafenib/trametinib combination) or immunotherapies (ipilimumab, nivolumab or pembrolizumab).,Baseline ctDNA levels were evaluated relative to treatment response and progression-free survival (PFS).,Tumor-associated ctDNA was detected in the plasma of 35/48 (73%) patients prior to treatment and lower ctDNA levels at this time point were significantly associated with response to treatment and prolonged PFS, irrespective of therapy type.,Levels of ctDNA decreased significantly in patients treated with MAPK inhibitors (p < 0.001) in accordance with response to therapy, but this was not apparent in patients receiving immunotherapies.,We show that circulating NRAS mutations, known to confer resistance to BRAF inhibitors, were detected in 3 of 7 (43%) patients progressing on kinase inhibitor therapy.,Significantly, ctDNA rebound and circulating mutant NRAS preceded radiological detection of progressive disease.,Our data demonstrate that ctDNA is a useful biomarker of response to kinase inhibitor therapy and can be used to monitor tumor evolution and detect the early appearance of resistance effectors.

Caveolin-1 (CAV1) is a scaffolding protein that plays a dual role in cancer.,In advanced stages of this disease, CAV1 expression in tumor cells is associated with enhanced metastatic potential, while, at earlier stages, CAV1 functions as a tumor suppressor.,We recently implicated CAV1 phosphorylation on tyrosine 14 (Y14) in CAV1-enhanced cell migration.,However, the contribution of this modification to the dual role of CAV1 in cancer remained unexplored.,Here, we used in vitro [2D and transendothelial cell migration (TEM), invasion] and in vivo (metastasis) assays, as well as genetic and biochemical approaches to address this question in B16F10 murine melanoma cells.,CAV1 promoted directional migration on fibronectin or laminin, two abundant lung extracellular matrix (ECM) components, which correlated with enhanced Y14 phosphorylation during spreading.,Moreover, CAV1-driven migration, invasion, TEM and metastasis were ablated by expression of the phosphorylation null CAV1(Y14F), but not the phosphorylation mimicking CAV1(Y14E) mutation.,Finally, CAV1-enhanced focal adhesion dynamics and surface expression of beta1 integrin were required for CAV1-driven TEM.,Importantly, CAV1 function as a tumor suppressor in tumor formation assays was not altered by the Y14F mutation.,In conclusion, our results provide critical insight to the mechanisms of CAV1 action during cancer development.,Specific ECM-integrin interactions and Y14 phosphorylation are required for CAV1-enhanced melanoma cell migration, invasion and metastasis to the lung.,Because Y14F mutation diminishes metastasis without inhibiting the tumor suppressor function of CAV1, Y14 phosphorylation emerges as an attractive therapeutic target to prevent metastasis without altering beneficial traits of CAV1.

Melanomas are highly lethal skin tumours that are frequently treated by surgical resection.,However, the efficacy of such procedures is often limited by tumour recurrence and metastasis.,Caveolin-1 (CAV1) has been attributed roles as a tumour suppressor, although in late-stage tumours, its presence is associated with enhanced metastasis.,The expression of this protein in human melanoma development and particularly how the presence of CAV1 affects metastasis after surgery has not been defined.,CAV1 expression in human melanocytes and melanomas increases with disease progression and is highest in metastatic melanomas.,The effect of increased CAV1 expression can then be evaluated using B16F10 murine melanoma cells injected into syngenic immunocompetent C57BL/6 mice or human A375 melanoma cells injected into immunodeficient B6Rag1−/− mice.,Augmented CAV1 expression suppresses tumour formation upon a subcutaneous injection, but enhances lung metastasis of cells injected into the tail vein in both models.,A procedure was initially developed using B16F10 melanoma cells in C57BL/6 mice to mimic better the situation in patients undergoing surgery.,Subcutaneous tumours of a defined size were removed surgically and local tumour recurrence and lung metastasis were evaluated after another 14 days.,In this postsurgery setting, CAV1 presence in B16F10 melanomas favoured metastasis to the lung, although tumour suppression at the initial site was still evident.,Similar results were obtained when evaluating A375 cells in B6Rag1−/− mice.,These results implicate CAV1 expression in melanomas as a marker of poor prognosis for patients undergoing surgery as CAV1 expression promotes experimental lung metastasis in two different preclinical models.

An anti-programmed cell death protein 1 monoclonal antibody, nivolumab, is one of the most effective drugs for advanced melanoma.,Tumor cell-derived or immune cell-derived markers and clinical predictors such as serum lactate dehydrogenase (LDH) and cutaneous adverse events, have already been described as prognostic factors for advanced melanoma treated with nivolumab.,We sought to identify further clinical predictors that can be determined in routine clinical practice.,We retrospectively analyzed clinical findings of 98 consecutive patients with unresectable stage III or IV melanoma treated with nivolumab, at the National Cancer Center Hospital or at Keio University Hospital, in Tokyo, Japan, between July 2014 and July 2016.,These patients had been administered nivolumab at a dose of 2mg/kg every 3 weeks.,As for pretreatment prognostic factors, ECOG performance status (PS) ≥1, maximum tumor diameters of ≥30mm, elevated LDH and elevated C-reactive protein were significantly associated with poor overall survival (OS) (hazard ratio [HR] 0.29 [P<0.001], HR 0.40 [p=0.003], HR 0.29 [P<0.001], HR 0.42 [P=0.004], respectively) on univariate analysis.,Among these factors, PS and LDH were identified as independent variables by multivariate analysis.,As for early markers examined during therapy, patients with absolute lymphocyte count (ALC) ≥ 1000/μl (Week3: HR 0.40 [P=0.004], Week6: HR 0.33 [P=0.001]) and absolute neutrophil count (ANC) <4000/μl (Week3: HR 0.46 [P=0.014], Week6: HR 0.51 [P=0.046]) had significantly better OS.,ALC≥1000/μl and ANC<4000/μl during treatment appear to be early markers associated with OS.,Nivolumab might have minimal efficacy in patients with a massive tumor burden.

Anti‐cytotoxic T lymphocyte‐associated antigen‐4 (CTLA‐4) and anti‐programmed cell death‐1 (PD‐1) inhibitors have been shown to significantly improve survival in patients with metastatic cutaneous melanoma.,However, there was some heterogeneity as well as some variation in the degree of benefit across studies.,We reviewed randomized trials and performed a meta‐analysis to determine the efficacy and safety of immune checkpoint inhibitors in comparison with conventional regimens.,Eligible studies were limited to randomized controlled trials comparing anti‐CTLA‐4 or anti‐PD‐1 inhibitors to chemotherapy or vaccination treatment in adult patients with unresectable cutaneous metastatic melanoma.,Progression‐free survival (PFS) rate at 6 months was 28.5% versus 17.7% (RR: 0.84, 95% CI: 0.76-0.93), overall survival (OS) rate at 1 year was 51.2% versus 38.8% (RR: 0.72, 95% CI: 0.59-0.88), and overall response rate (ORR) at 6 months was 29.6% versus 17.7% (RR: 0.85, 95% CI: 0.76-0.95) favoring immune check point inhibitors over chemotherapies or vaccination.,Immune check point inhibitors were associated with more frequent immune‐related adverse events at 13.7% versus 2.4% of treated patients (RR: 6.74, 95% CI: 4.65-9.75).,Subgroup analyses demonstrated significant PFS (RR: 0.92 vs.,0.74, P < 0.00001) and ORR (RR: 0.95 vs.,0.76, P = 0.0004) improvement with anti‐PD‐1 treatment compared to anti‐CTLA‐4 when each of them was compared to control treatments.,Collectively, these results demonstrate that immune checkpoint inhibitors have superior outcomes compared to conventional chemotherapies or vaccination, and support the results of recent randomized trials that showed superior outcomes with anti‐PD‐1 agents over ipilimumab in unresectable metastatic cutaneous melanoma patients.

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

HEDGEHOG (HH) signaling is a key regulator of tissue development and its aberrant activation is involved in several cancer types, including melanoma.,We and others have shown a reciprocal cross talk between HH signaling and p53, whose function is often impaired in melanoma.,Here we present evidence that both GLI1 and GLI2, the final effectors of HH signaling, regulate the transcription factor E2F1 in melanoma cells, by binding to a functional non-canonical GLI consensus sequence.,Consistently, we find a significant correlation between E2F1 and PATCHED1 (PTCH1), GLI1 and GLI2 expression in human melanomas.,Functionally, we find that E2F1 is a crucial mediator of HH signaling and it is required for melanoma cell proliferation and xenograft growth induced by activation of the HH pathway.,Interestingly, we present evidence that the HH/GLI-E2F1 axis positively modulates the inhibitor of apoptosis-stimulating protein of p53 (iASPP) at multiple levels.,HH activation induces iASPP expression through E2F1, which directly binds to iASPP promoter.,HH pathway also contributes to iASPP function, by the induction of Cyclin B1 and by the E2F1-dependent regulation of CDK1, which are both involved in iASPP activation.,Our data show that activation of HH signaling enhances proliferation in presence of E2F1 and promotes apoptosis in its absence or upon CDK1 inhibition, suggesting that E2F1/iASPP dictates the outcome of HH signaling in melanoma.,Together, these findings identify a novel HH/GLI-E2F1-iASPP axis that regulates melanoma cell growth and survival, providing an additional mechanism through which HH signaling restrains p53 proapoptotic function.

The treatment of immunotherapy relapsed cutaneous melanoma constitutes a challenge in both research and clinical practice fields given the lack of effective therapeutic options.,Homologous recombination deficiency (HRD) has been identified in several solid cancers including cutaneous melanoma.,However, the utility of medications targeting HRD cancer cells is an uncharted territory in melanoma.,Moreover, preclinical evidence suggests a synergistic role of combining immune checkpoint blockade (ICB) with drugs targeting HRD cancer cells such as PARP inhibitors.,Here, we present a case study of a patient with immunotherapy relapsed melanoma who was found to have detected HRD and was treated with nivolumab (ICB) and olaparib (PARP inhibitors).

PARP inhibitors niraparib and talazoparib are FDA approved for special cases of breast cancer.,PARP is an interesting repair protein which is frequently affected in cancer cells.,We studied the combined action of talazoparib or niraparib with ionizing radiation in melanoma cells and healthy fibroblasts.,Homologous recombination (HR) status in six different melanoma cell lines and healthy fibroblasts was assessed.,Cell cultures were treated with PARP inhibitors talazoparib or niraparib and ionizing radiation (IR).,Apoptosis, necrosis and cell cycle distribution was analyzed via flow cytometry.,Cell migration was studied by scratch assays.,Studied melanoma cell cultures are HR deficient.,Studied healthy fibroblasts are HR proficient.,Talazoparib and niraparib have congruent effects within the same cell cultures.,In all cell cultures, combined treatment increases cell death and G2/M arrest compared to IR.,Combined treatment in melanoma cells distinctly increases G2/M arrest.,Healthy fibroblasts are less affected by G2/M arrest.,Treatment predominantly decelerates or does not modify migration.,In two cell cultures migration is enhanced under the inhibitors.,Although the two PARP inhibitors talazoparib and niraparib appear to be suitable for a combination treatment with ionizing radiation in our in vitro studies, a combination treatment cannot generally be recommended.,There are clear interindividual differences in the effect of the inhibitors on different melanoma cells.,Therefore, the effect on the cancer cells should be studied prior to a combination therapy.,Since melanoma cells increase more strongly than fibroblasts in G2/M arrest, the fractional application of combined treatment should be further investigated.

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.

Previous analysis of COMBI-d (NCT01584648) demonstrated improved progression-free survival (PFS) and overall survival (OS) with combination dabrafenib and trametinib versus dabrafenib monotherapy in BRAF V600E/K-mutant metastatic melanoma.,This study was continued to assess 3-year landmark efficacy and safety after ≥36-month follow-up for all living patients.,This double-blind, phase 3 study enrolled previously untreated patients with BRAF V600E/K-mutant unresectable stage IIIC or stage IV melanoma.,Patients were randomized to receive dabrafenib (150 mg twice daily) plus trametinib (2 mg once daily) or dabrafenib plus placebo.,The primary endpoint was PFS; secondary endpoints were OS, overall response, duration of response, safety, and pharmacokinetics.,Between 4 May and 30 November 2012, a total of 423 of 947 screened patients were randomly assigned to receive dabrafenib plus trametinib (n = 211) or dabrafenib monotherapy (n = 212).,At data cut-off (15 February 2016), outcomes remained superior with the combination: 3-year PFS was 22% with dabrafenib plus trametinib versus 12% with monotherapy, and 3-year OS was 44% versus 32%, respectively.,Twenty-five patients receiving monotherapy crossed over to combination therapy, with continued follow-up under the monotherapy arm (per intent-to-treat principle).,Of combination-arm patients alive at 3 years, 58% remained on dabrafenib plus trametinib.,Three-year OS with the combination reached 62% in the most favourable subgroup (normal lactate dehydrogenase and <3 organ sites with metastasis) versus only 25% in the unfavourable subgroup (elevated lactate dehydrogenase).,The dabrafenib plus trametinib safety profile was consistent with previous clinical trial observations, and no new safety signals were detected with long-term use.,These data demonstrate that durable (≥3 years) survival is achievable with dabrafenib plus trametinib in patients with BRAF V600-mutant metastatic melanoma and support long-term first-line use of the combination in this setting.

NME1 is a metastasis-suppressor gene (MSG), capable of suppressing metastatic activity in cell lines of melanoma, breast carcinoma and other cancer origins without affecting their growth in culture or as primary tumours.,Herein, we selectively ablated the tandemly arranged Nme1 and Nme2 genes to assess their individual impacts on metastatic activity in a mouse model (HGF:p16−/−) of ultraviolet radiation (UVR)-induced melanoma.,Metastatic activity was strongly enhanced in both genders of Nme1- and Nme2-null mice, with stronger activity in females across all genotypes.,The study ascribes MSG activity to Nme2 for the first time in an in vivo model of spontaneous cancer, as well as a novel metastasis-suppressor function to Nme1 in the specific context of UVR-induced melanoma.

Fascin is a F-actin bundling protein and its overexpression is correlated with poor prognosis and increases metastatic potential in a number of cancers.,But underlying function and mechanism of fascin on tumorigenesis in melanoma remain elusive.,The melanoma cell lines WM793 and WM39 were employed for the soft agar and sphere formation assay.,Quantitative RT-PCR and Western blot were performed for identifying the gene expression at mRNA and protein levels, respectively.,Co-IP and in vitro GST pulldown experiments were used to test the interaction between fascin and MST2.,Fascin regulates tumorigenesis and cancer cell stemness in melanoma through inhibition of the Hippo pathway kinase MST2 and the activation of transcription factor TAZ.,Our data showed that fascin interacts with the kinase domain of MST2 to inhibit its homodimer formation and kinase activity.,Depletion of fascin led to increase of p-LATS level and decrease of TAZ, but not YAP.,We also demonstrated that fascin regulates melanoma tumorigenesis independent of its actin-bundling activity.,Fascin is a new regulator of the MST2-LATS-TAZ pathway and plays a critical role in melanoma tumorigenesis.,Inhibition of fascin reduces melanoma tumorigenesis and stemness, and thus fascin could be a potential therapeutic target for this malignancy.,The online version of this article (10.1186/s12964-018-0250-1) contains supplementary material, which is available to authorized users.

Recently, the targeting of ERK with ATP-competitive inhibitors has emerged as a potential clinical strategy to overcome acquired resistance to BRAF and MEK inhibitor combination therapies.,In this study, we investigate an alternative strategy of targeting the D-recruitment site (DRS) of ERK.,The DRS is a conserved region that lies distal to the active site and mediates ERK-protein interactions.,We demonstrate that the small molecule BI-78D3 binds to the DRS of ERK2 and forms a covalent adduct with a conserved cysteine residue (C159) within the pocket and disrupts signaling in vivo.,BI-78D3 does not covalently modify p38MAPK, JNK or ERK5.,BI-78D3 promotes apoptosis in BRAF inhibitor-naive and resistant melanoma cells containing a BRAF V600E mutation.,These studies provide the basis for designing modulators of protein-protein interactions involving ERK, with the potential to impact ERK signaling dynamics and to induce cell cycle arrest and apoptosis in ERK-dependent cancers.,The ERK signalling pathway is activated in many cancers, however ERK1 and ERK2 are difficult to target pharmacologically.,Here, the authors identify a small molecule inhibitor that binds covalently to the D-recruitment site of ERK and induces cell death and reduces tumour growth in mice.

Solid cancer cells commonly enter the blood and disseminate systemically but are highly inefficient at forming distant metastases for poorly understood reasons.,We studied human melanomas that differed in their metastasis histories in patients and in their capacity to metastasize in NSG mice.,All melanomas had high frequencies of cells that formed subcutaneous tumours, but much lower percentages of cells that formed tumours after intravenous or intrasplenic transplantation, particularly among inefficient metastasizers.,Melanoma cells in the blood and visceral organs experienced oxidative stress not observed in established subcutaneous tumours.,Successfully metastasizing melanomas underwent reversible metabolic changes during metastasis that increased their capacity to withstand oxidative stress, including increased dependence upon NADPH-generating enzymes in the folate pathway.,Anti-oxidants promoted distant metastasis in NSG mice.,Folate pathway inhibition using low-dose methotrexate, ALDH1L2 knockdown, or MTHFD1 knockdown inhibited distant metastasis without significantly affecting the growth of subcutaneous tumors in the same mice.,Oxidative stress thus limits distant metastasis by melanoma cells in vivo.

Intratumoral heterogeneity is a major obstacle for the treatment of cancer, as the presence of even minor populations that are insensitive to therapy can lead to disease relapse.,Increased clonal diversity has been correlated with a poor prognosis for cancer patients, and we therefore examined genetic, transcriptional, and functional diversity in metastatic melanoma.,Amplicon sequencing and SNP microarrays were used to profile somatic mutations and DNA copy number changes in multiple regions from metastatic lesions.,Clonal genetic and transcriptional heterogeneity was also assessed in single cell clones from early passage cell lines, which were then subjected to clonogenicity and drug sensitivity assays.,MAPK pathway and tumor suppressor mutations were identified in all regions of the melanoma metastases analyzed.,In contrast, we identified copy number abnormalities present in only some regions in addition to homogeneously present changes, suggesting ongoing genetic evolution following metastatic spread.,Copy number heterogeneity from a tumor was represented in matched cell line clones, which also varied in their clonogenicity and drug sensitivity.,Minor clones were identified based on dissimilarity to the parental cell line, and these clones were the most clonogenic and least sensitive to drugs.,Finally, treatment of a polyclonal cell line with paclitaxel to enrich for drug-resistant cells resulted in the adoption of a gene expression profile with features of one of the minor clones, supporting the idea that these populations can mediate disease relapse.,Our results support the hypothesis that minor clones might have major consequences for patient outcomes in melanoma.

Metastasis is characterized by spreading of neoplastic cells to an organ other than where they originated and is the predominant cause of death among cancer patients.,This holds true for melanoma, whose incidence is increasing more rapidly than any other cancer and once disseminated has few therapeutic options.,Here we performed whole exome sequencing of two sets of matched normal and metastatic tumor DNAs.,Using stringent criteria, we evaluated the similarities and differences between the lesions.,We find that in both cases, 96% of the single nucleotide variants are shared between the two metastases indicating that clonal populations gave rise to the distant metastases.,Analysis of copy number variation patterns of both metastatic sets revealed a trend similar to that seen with our single nucleotide variants.,Analysis of pathway enrichment on tumor sets shows commonly mutated pathways enriched between individual sets of metastases and all metastases combined.,These data provide a proof-of-concept suggesting that individual metastases may have sufficient similarity for successful targeting of driver mutations.

The development and progression of melanoma have been attributed to independent or combined genetic and epigenetic events.,There has been remarkable progress in understanding melanoma pathogenesis in terms of genetic alterations.,However, recent studies have revealed a complex involvement of epigenetic mechanisms in the regulation of gene expression, including methylation, chromatin modification and remodeling, and the diverse activities of non-coding RNAs.,The roles of gene methylation and miRNAs have been relatively well studied in melanoma, but other studies have shown that changes in chromatin status and in the differential expression of long non-coding RNAs can lead to altered regulation of key genes.,Taken together, they affect the functioning of signaling pathways that influence each other, intersect, and form networks in which local perturbations disturb the activity of the whole system.,Here, we focus on how epigenetic events intertwine with these pathways and contribute to the molecular pathogenesis of melanoma.

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.

Anti-PD1 treatment has improved the survival of metastatic melanoma patients, yet it is unknown which patients benefit from the treatment.,In this exploratory study, we aimed to understand the effects of anti-PD1 therapy on the patients’ immune system and discover the characteristics that would result in successful treatment.,We collected peripheral blood (PB) samples from 17 immuno-oncology-naïve metastatic melanoma patients before and after 1 and 3 months of anti-PD1 therapy.,In addition, matching tumor biopsies at the time of diagnosis were collected for tissue microarray.,The complete blood counts, PB immunophenotype, serum cytokine profiles, and tumor-infiltrating lymphocytes were analyzed and correlated with the clinical data.,Patients were categorized based on their disease control into responders (complete response, partial response, stable disease > 6 months, N = 11) and non-responders (progressive disease, stable disease ≤ 6 months, N = 6).,During therapy, the PB natural killer T (NKT) cell frequency, expression of CD25 and CD45RO on cytotoxic natural killer (NK) cells, and serum CXC chemokine levels were significantly increased in responders.,Furthermore, higher age together with age-associated characteristics from PB, lower frequency of PB-naïve CD8+ T cells, and elevated levels of serum MCP-4 and OPG were discovered as baseline predictors of treatment response.,We therefore propose that in addition to T cells, anti-PD1 treatment is associated with NK- and NKT-cell population dynamics, and that the age-associated characteristics from PB together with older age may contribute to prolonged PFS in anti-PD1-treated melanoma patients.,The online version of this article (10.1007/s00262-020-02497-9) contains supplementary material, which is available to authorized users.

PD-1 inhibitors are approved for multiple malignancies and function by stimulating T cells.,However, the role of B cells in the anti-tumor activity of these drugs is unknown, as is their activity in patients who have received B cell depleting drugs or with immunoglobulin deficiencies.,We studied B cell content in 40 melanomas from patients treated with pembrolizumab or nivolumab and assessed the association with response to therapy.,Murine MC38 colon cancer and YUMMER1.7 melanoma models were used to determine whether concomitant anti-CD20 antibody injections diminish the anti-tumor effects of anti-PD-1.,Results were validated in muMT mice, which lack B cells.,B cells were sparse in most melanomas and B cell content was not associated with response to anti-PD-1 or overall survival.,Employing MC38 and YUMMER1.7 models, we demonstrated that anti-CD20 antibodies reduce tumor-infiltrating B cells yet had no effect on tumor growth, response to PD-1 inhibition, or survival.,In muMT mice, T-cell dependent tumor rejection and anti-PD-1 responses were no different than in wildtype C57BL/6 J mice.,The degree of tumor infiltrating B cell content is not associated with response to anti-PD-1 inhibitors in melanoma.,PD-1 inhibitors cause tumor shrinkage in murine cancer models even when B cells are absent or are depleted.,PD-1 inhibitors are likely to be active in patients with impaired B cell function, such as patients undergoing B cell depletion with drugs including rituximab for conditions such as B cell malignancies or autoimmune disorders.,The online version of this article (10.1186/s40425-019-0613-1) contains supplementary material, which is available to authorized users.

Shikonin, a natural product isolated from the roots of Lithospermum erythrorhizon, exhibits pharmacological effects against inflammation, ulcers, infections, and tumors.,In the present study, we aimed to investigate the antitumor effects of shikonin on the human melanoma cell line, A375SM, and in an in vivo mouse xenograft model.,We examined the anticancer effects of shikonin by in vitro experiments (MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, 4′,6-diamidino-2-phenylindole (DAPI) stain, annexin V/ propidium iodide (PI) stain, and protein analysis of apoptosis and mitogen-activated protein kinase (MAPK) pathways).,Further, the anticancer effect in vivo was confirmed through a xenograft model.,Our results showed that shikonin inhibited the proliferation of melanoma cells in a dose-dependent manner.,In addition, shikonin significantly increased nucleus and chromatin condensation and early/late apoptosis.,Shikonin also increased the pro-apoptotic proteins and decreased the anti-apoptotic proteins.,Additionally, shikonin was overexpressed in MAPK pathways.,Investigation of the effects of shikonin in a mouse xenograft model not only showed decreased A375SM tumor volume but also increased apoptosis as determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay.,Furthermore, pathologic changes were not observed in the liver and kidney of mice.,Collectively, the study indicated that shikonin inhibited the proliferation of the human melanoma cells by inducing apoptosis, mediated by MAPK pathway and that it is a potential candidate for an anticancer drug against melanoma cancer.

Immune checkpoint inhibitors and targeted therapies are approved for adjuvant treatment of patients with resected melanoma; however, they have not been compared in randomized controlled trials (RCTs).,We compared the efficacy and safety of adjuvant nivolumab with other approved treatments using available evidence from RCTs in a Bayesian network meta-analysis (NMA).,A systematic literature review was conducted through May 2019 to identify relevant RCTs evaluating approved adjuvant treatments.,Outcomes of interest included recurrence-free survival (RFS)/disease-free survival (DFS), distant metastasis-free survival (DMFS), all-cause grade 3/4 adverse events (AEs), discontinuations, and discontinuations due to AEs.,Time-to-event outcomes (RFS/DFS and DMFS) were analyzed both assuming that hazard ratios (HRs) are constant over time and that they vary.,Of 26 identified RCTs, 19 were included in the NMA following a feasibility assessment.,Based on HRs for RFS/DFS, the risk of recurrence with nivolumab was similar to that of pembrolizumab and lower than that of ipilimumab 3 mg/kg, ipilimumab 10 mg/kg, or interferon.,Risk of recurrence with nivolumab was similar to that of dabrafenib plus trametinib at 12 months, however, was lower beyond 12 months (HR [95% credible interval] at 24 months, 0.46 [0.27-0.78]; at 36 months, 0.28 [0.14-0.59]).,Based on HRs for DMFS, the risk of developing distant metastases was lower with nivolumab than with ipilimumab 10 mg/kg or interferon and was similar to dabrafenib plus trametinib.,Adjuvant therapy with nivolumab provides an effective treatment option with a promising risk-benefit profile.,Supplementary information accompanies this paper at 10.1186/s12885-020-07538-1.

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

Melanoma is the most highly malignant skin cancer, and nucleotide excision repair (NER) is involved in melanoma susceptibility.,In this analysis of 1042 melanoma patients, we evaluated whether genetic variants of NER genes may predict survival outcome of melanoma patients.,We used genotyping data of 74 tagging single nucleotide polymorphisms (tagSNPs) in eight core NER genes from our genome-wide association study (including 2 in XPA, 14 in XPC, 3 in XPE, 4 in ERCC1, 10 in ERCC2, 8 in ERCC3, 14 in ERCC4, and 19 in ERCC5) and evaluated their associations with prognosis of melanoma patients.,Using the Cox proportional hazards model and Kaplan-Meier analysis, we found a predictive role of XPE rs28720291, ERCC5 rs4150314, XPC rs2470458 and ERCC2 rs50871 SNPs in prognosis of melanoma patients (rs28720291: AG vs.,GG, adjusted hazard ratio [adjHR] = 11.2, 95% confidence interval [CI] 3.04-40.9, P = 0.0003; rs4150314: AG vs.,GG, adjHR = 4.76, 95% CI 1.09-20.8, P = 0.038; rs2470458: AA vs.,AG/GG, adjHR = 2.11, 95% CI 1.03-4.33, P = 0.040; and rs50871: AA vs.,AC/CC adjHR =2.27, 95% CI 1.18-4.35, P = 0.015).,Patients with an increasing number of unfavorable genotypes had dramatically increased death risk.,Genetic variants of NER genes, particularly XPE rs28720291, ERCC5 rs4150314, XPC rs2470458 and ERCC2 rs50871, may independently or jointly modulate survival outcome of melanoma patients.,Because our results were based on a median follow-up of 3 years without multiple test corrections, additional large prospective studies are needed to confirm our findings.

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

Pro-apoptotic BCL2 associated X (BAX) is traditionally thought to be regulated by anti-apoptotic BCL-2 family members, like BCL2-like 1 (BCL-XL), at the protein level.,However, the posttranscriptional regulation of BAX is under explored.,In this study, we identified BAX as the novel downstream target of miR-365, which is supported by gain- and loss-of-function studies of onco-miR-365.,Loss of BAX by either RNA interference or highly-expressed miR-365 in cells of cutaneous squamous cell carcinoma (CSCC) enhanced the tumor resistance against apoptosis, while repressing cell proliferation, migration, and invasiveness.,In vivo experiment confirmed that BAX knockdown promotes the growth of CSCC xenografts.,Collectively, our results find a miR-365-BAX axis for alleviating the pro-apoptotic effects of BAX, which promotes CSCC development and may facilitate the generation of novel therapeutic regimens to the clinical treatment of CSCC.

Melanoma is often caused by mutations due to exposure to ultraviolet radiation.,This study reports a recurrent somatic C > T change causing a P131L mutation in the RQCD1 (Required for Cell Differentiation1 Homolog) gene identified through whole exome sequencing of 20 metastatic melanomas.,Screening in 715 additional primary melanomas revealed a prevalence of ~4%.,This represents the first reported recurrent mutation in a member of the CCR4-NOT complex in cancer.,Compared to tumors without the mutation, the P131L mutant positive tumors were associated with increased thickness (p = 0.02), head and neck (p = 0.009) and upper limb (p = 0.03) location, lentigo maligna melanoma subtype (p = 0.02) and BRAF V600K (p = 0.04) but not V600E or NRAS codon 61 mutations.,There was no association with nodal disease (p = 0.3).,Mutually exclusive mutations of other members of the CCR4-NOT complex were found in ~20% of the TCGA melanoma dataset suggesting the complex may play an important role in melanoma biology.,Mutant RQCD1 was predicted to bind strongly to HLA-A0201 and HLA-Cw3 MHC1 complexes.,From thirteen patients with mutant RQCD1, an anti-tumor CD8+ T cell response was observed from a single patient's peripheral blood mononuclear cell population stimulated with mutated peptide compared to wildtype indicating a neoantigen may be formed.

We sequenced 8 melanoma exomes to identify novel somatic mutations in metastatic melanoma.,Focusing on the MAP3K family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9.,Structural modelling predicts that mutations in the kinase domain may affect the activity and regulation of MAP3K5/9 protein kinases.,The position of the mutations and loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely inactivating.,In vitro kinase assay shows reduction in kinase activity in MAP3K5 I780F and MAP3K9 W333X mutants.,Overexpression of MAP3K5 or MAP3K9 mutant in HEK293T cells reduces phosphorylation of downstream MAP kinases.,Attenuation of MAP3K9 function in melanoma cells using siRNA leads to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.

Strategies to battle malignant tumors have always been a dynamic research endeavour.,Although various vehicles (e.g., chemotherapeutic therapy, radiotherapy, surgical resection, etc.) are used for skin cancer management, they mostly remain unsatisfactory due to the complex mechanism of carcinogenesis.,Increasing evidence indicates that redox imbalance and aberrant reactive oxygen species (ROS) are closely implicated in the oncogenesis of skin cancer.,When ROS production goes beyond their clearance, excessive or accumulated ROS could disrupt redox balance, induce oxidative stress, and activate the altered ROS signals.,These would damage cellular DNA, proteins, and lipids, further leading to gene mutation, cell hyperproliferation, and fatal lesions in cells that contribute to carcinogenesis in the skin.,It has been known that ROS-mediated skin carcinogenesis involves multiple ways, including modulating related signaling pathways, changing cell metabolism, and causing the instability of the genome and epigenome.,Nevertheless, the exact role of ROS in skin cancer has not been thoroughly elucidated.,In spite of ROS inducing skin carcinogenesis, toxic-dose ROS could trigger cell death/apoptosis and, therefore, may be an efficient therapeutic tool to battle skin cancer.,Considering the dual role of ROS in the carcinogenesis and treatment of skin cancer, it would be essential to clarify the relationship between ROS and skin cancer.,Thus, in this review, we get the related data together to seek the connection between ROS and skin carcinogenesis.,Besides, strategies basing on ROS to fight skin cancer are discussed.

Skin cancer is currently diagnosed as one in every three cancers.,Melanoma, the most aggressive form of skin cancer, is responsible for 79% of skin cancer deaths and the incidence is rising faster than in any other solid tumor type.,Previously, we have demonstrated that dimethylacrylshikonin (DMAS), isolated from the roots of Onosma paniculata (Boraginaceae), exhibited the lowest IC50 values against different tumor types out of several isolated shikonin derivatives.,DMAS was especially cytotoxic towards melanoma cells and led to apoptosis and cell cycle arrest.,In this study, we performed a comprehensive gene expression study to investigate the mechanism of action in more detail.,Gene expression signature was compared to vehicle-treated WM164 control cells after 24 h of DMAS treatment; where 1192 distinct mRNAs could be identified as expressed in all replicates and 89 were at least 2-fold differentially expressed.,DMAS favored catabolic processes and led in particular to p62 increase which is involved in cell growth, survival, and autophagy.,More in-depth experiments revealed that DMAS led to autophagy, ROS generation, and loss of mitochondrial membrane potential in different melanoma cells.,It has been reported that the induction of an autophagic cell death represents a highly effective approach in melanoma therapy.

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.

Knowledge of key drivers and therapeutic targets in mucosal melanoma is limited due to the paucity of comprehensive mutation data on this rare tumor type.,To better understand the genomic landscape of mucosal melanoma, here we describe whole genome sequencing analysis of 67 tumors and validation of driver gene mutations by exome sequencing of 45 tumors.,Tumors have a low point mutation burden and high numbers of structural variants, including recurrent structural rearrangements targeting TERT, CDK4 and MDM2.,Significantly mutated genes are NRAS, BRAF, NF1, KIT, SF3B1, TP53, SPRED1, ATRX, HLA-A and CHD8.,SF3B1 mutations occur more commonly in female genital and anorectal melanomas and CTNNB1 mutations implicate a role for WNT signaling defects in the genesis of some mucosal melanomas.,TERT aberrations and ATRX mutations are associated with alterations in telomere length.,Mutation profiles of the majority of mucosal melanomas suggest potential susceptibility to CDK4/6 and/or MEK inhibitors.,Mucosal melanomas are challenging to treat partly because so little is known about the genetic drivers underpinning them.,Here, the authors perform a genomic landscape analysis of samples collected from three continents, revealing a potential role for CDK4/6 or MEK inhibition in the treatment of the disease.

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.

The effects of epigallocatechingallate (EGCG) on the migration and expression of MMP-2 of uveal melanoma cells have not been reported.,We studied this effect and relevant signaling pathways in a human uveal melanoma cell line (M17).,MTT study found that EGCG did not affect the cell viability of M17 cells up to 100 µM.,Wound-healing assay showed that EGCG significantly reduced the migration of melanoma cells in a dose-dependent manner from 20 to 100 µM.,Gelatin zymography showed that secreted MMP-2 activity was dose-dependently inhibited by EGCG, whereas the MMP-2 expression at protein and mRNA levels was not affected as determined by western blot and RT-PCR analysis.,EGCG significantly increased the expressions of MMP-2 endogenous inhibitors (TIMP-2 and RECK) in M17 cells.,Western blot analysis of MAPK signal pathways showed that EGCG significantly decreased phosphorylated ERK1/2 levels, but not p38 and JNK levels, in melanoma cells.,ERK1/2 inhibitors also reduced the migration and activity of MMP-2 in M17 cells.,The present study suggested EGCG at nontoxic levels could inhibit migration of melanoma cells via downregulation of activities of secreted MMP-2 through the inhibition of the ERK1/2 phosphorylation.,Therefore, EGCG may be a promising agent to be explored for the prevention of metastasis of uveal melanoma.

A series of imidazothiazole derivatives possessing potential activity against melanoma cells were investigated for molecular mechanism of action.,The target compounds were tested against V600E-B-RAF and RAF1 kinases.,Compound 1zb is the most potent against both kinases with IC50 values 0.978 and 8.2 nM, respectively.,It showed relative selectivity against V600E mutant B-RAF kinase.,Compound 1zb was also tested against four melanoma cell lines and exerted superior potency (IC50 0.18-0.59 µM) compared to the reference standard drug, sorafenib (IC50 1.95-5.45 µM).,Compound 1zb demonstrated also prominent selectivity towards melanoma cells than normal skin cells.,It was further tested in whole-cell kinase assay and showed in-cell V600E-B-RAF kinase inhibition with IC50 of 0.19 µM.,Compound 1zb induces apoptosis not necrosis in the most sensitive melanoma cell line, UACC-62.,Furthermore, molecular dynamic and 3D-QSAR studies were done to investigate the binding mode and understand the pharmacophoric features of this series of compounds.

New therapies are urgently needed in melanoma particularly in late-stage patients not responsive to immunotherapies and kinase inhibitors.,Drug screening, IC50 determinations as well as synergy assays were detected by the MTT assay.,Apoptosis using Annexin V and 7AAD staining was assessed using flow cytometry.,TUNEL staining was performed using immunocytochemistry.,Changes in phosphorylation of key molecules in PI3K/Akt/mTOR and other relevant pathways were detected by western blot as well as immunocytochemistry.,To assess in vivo anti-tumor activity of Tegaserod, syngeneic intravenous and subcutaneous melanoma xenografts were used.,Immunocytochemical staining was performed to detect expression of active Caspase-3, cleaved Caspase 8 and p-S6 in tumors.,Evaluation of immune infiltrates was carried out by flow cytometry.,Using a screen of 770 pharmacologically active and/or FDA approved drugs, we identified Tegaserod (Zelnorm, Zelmac) as a compound with novel anti-cancer activity which induced apoptosis in murine and human malignant melanoma cell lines.,Tegaserod (TM) is a serotonin receptor 4 agonist (HTR4) used in the treatment of irritable bowel syndrome (IBS).,TM’s anti-melanoma apoptosis-inducing effects were uncoupled from serotonin signaling and attributed to PI3K/Akt/mTOR signaling inhibition.,Specifically, TM blunted S6 phosphorylation in both BRAFV600E and BRAF wildtype (WT) melanoma cell lines.,TM decreased tumor growth and metastases as well as increased survival in an in vivo syngeneic immune-competent model.,In vivo, TM also caused tumor cell apoptosis, blunted PI3K/Akt/mTOR signaling and decreased S6 phosphorylation.,Furthermore TM decreased the infiltration of immune suppressive regulatory CD4+CD25+ T cells and FOXP3 and ROR-γt positive CD4+ T cells.,Importantly, TM synergized with Vemurafenib, the standard of care drug used in patients with late stage disease harboring the BRAFV600E mutation and could be additively or synergistically combined with Cobimetinib in both BRAFV600E and BRAF WT melanoma cell lines in inducing anti-cancer effects.,Taken together, we have identified a drug with anti-melanoma activity in vitro and in vivo that has the potential to be combined with the standard of care agent Vemurafenib and Cobimetinib in both BRAFV600E and BRAF WT melanoma.

Certain cutaneous human papillomaviruses (HPVs), which are ubiquitous and acquired early during childhood, can cause a variety of skin tumors and are likely involved in the development of non-melanoma skin cancer, especially in immunosuppressed patients.,Hence, the burden of these clinical manifestations demands for a prophylactic approach.,To evaluate whether protective efficacy of a vaccine is potentially translatable to patients, we used the rodent Mastomys coucha that is naturally infected with Mastomys natalensis papillomavirus (MnPV).,This skin type papillomavirus induces not only benign skin tumours, such as papillomas and keratoacanthomas, but also squamous cell carcinomas, thereby allowing a straightforward read-out for successful vaccination in a small immunocompetent laboratory animal.,Here, we examined the efficacy of a virus-like particle (VLP)-based vaccine on either previously or newly established infections.,VLPs raise a strong and long-lasting neutralizing antibody response that confers protection even under systemic long-term cyclosporine A treatment.,Remarkably, the vaccine completely prevents the appearance of benign as well as malignant skin tumors.,Protection involves the maintenance of a low viral load in the skin by an antibody-dependent prevention of virus spread.,Our results provide first evidence that VLPs elicit an effective immune response in the skin under immunocompetent and immunosuppressed conditions in an outbred animal model, irrespective of the infection status at the time of vaccination.,These findings provide the basis for the clinical development of potent vaccination strategies against cutaneous HPV infections and HPV-induced tumors, especially in patients awaiting organ transplantation.

Cutaneous beta human papillomavirus (HPV) types appear to be involved in the development of non-melanoma skin cancer (NMSC); however, it is not entirely clear whether they play a direct role.,We have previously shown that E6 and E7 oncoproteins from the beta HPV type 38 display transforming activities in several experimental models.,To evaluate the possible contribution of HPV38 in a proliferative tissue compartment during carcinogenesis, we generated a new transgenic mouse model (Tg) where HPV38 E6 and E7 are expressed in the undifferentiated basal layer of epithelia under the control of the Keratin 14 (K14) promoter.,Viral oncogene expression led to increased cellular proliferation in the epidermis of the Tg animals in comparison to the wild-type littermates.,Although no spontaneous formation of tumours was observed during the lifespan of the K14 HPV38 E6/E7-Tg mice, they were highly susceptible to 7,12-dimethylbenz(a)anthracene (DMBA)/12-0-tetradecanoylphorbol-13-acetate (TPA) two-stage chemical carcinogenesis.,In addition, when animals were exposed to ultraviolet light (UV) irradiation, we observed that accumulation of p21WAF1 and cell-cycle arrest were significantly alleviated in the skin of Tg mice as compared to wild-type controls.,Most importantly, chronic UV irradiation of Tg mice induced the development of actinic keratosis-like lesions, which are considered in humans as precursors of squamous cell carcinomas (SCC), and subsequently of SCC in a significant proportion of the animals.,In contrast, wild-type animals subjected to identical treatments did not develop any type of skin lesions.,Thus, the oncoproteins E6 and E7 from beta HPV38 significantly contribute to SCC development in the skin rendering keratinocytes more susceptible to UV-induced carcinogenesis.

Recently, activating mutations in the TERT promoter were identified in cutaneous melanoma.,We tested a cohort of ocular melanoma samples for similar mutations.,The TERT promoter region was analysed by Sanger sequencing in 47 uveal (ciliary body or choroidal) melanomas and 38 conjunctival melanomas.,Mutations of the TERT promoter were not identified in uveal melanomas, but were detected in 12 (32%) conjunctival melanomas.,Mutations had a UV signature and were identical to those found in cutaneous melanoma.,Mutations of TERT promoter with UV signatures are frequent in conjunctival melanomas and favour a pathogenetic kinship with cutaneous melanomas.,Absence of these mutations in uveal melanomas emphasises their genetic distinction from cutaneous and conjunctival melanomas.

Besides being a preferential site of early metastasis, the sentinel lymph node (SLN) is also a privileged site of T-cell priming, and may thus be an appropriate target for investigating cell types involved in antitumor immune reactions.,In this retrospective study we determined the prevalence of OX40+ activated T lymphocytes, FOXP3+ (forkhead box P3) regulatory T cells, DC-LAMP+ (dendritic cell-lysosomal associated membrane protein) mature dendritic cells (DCs) and CD123+ plasmacytoid DCs by immunohistochemistry in 100 SLNs from 60 melanoma patients.,Density values of each cell type in SLNs were compared to those in non-sentinel nodes obtained from block dissections (n = 37), and analyzed with regard to associations with clinicopathological parameters and disease outcome.,Sentinel nodes showed elevated amount of all cell types studied in comparison to non-sentinel nodes.,Metastatic SLNs had higher density of OX40+ lymphocytes compared to tumor-negative nodes, while no significant difference was observed in the case of the other cell types studied.,In patients with positive sentinel node status, high amount of FOXP3+ cells in SLNs was associated with shorter progression-free (P = 0.0011) and overall survival (P = 0.0014), while no significant correlation was found in the case of sentinel-negative patients.,The density of OX40+, CD123+ or DC-LAMP+ cells did not show significant association with the outcome of the disease.,Taken together, our results are compatible with the hypothesis of functional competence of sentinel lymph nodes based on the prevalence of the studied immune cells.,The density of FOXP3+ lymphocytes showed association with progression and survival in patients with positive SLN status, while the other immune markers studied did not prove of prognostic importance.,These results, together with our previous findings on the prognostic value of activated T cells and mature DCs infiltrating primary melanomas, suggest that immune activation-associated markers in the primary tumor may have a higher impact than those in SLNs on the prognosis of the patients.,On the other hand, FOXP3+ cell density in SLNs, but not in the primary tumor, was found predictive of disease outcome in melanoma patients.

Both targeted therapy and immunotherapy have been used successfully to treat melanoma, but the development of resistance and poor response rates to the individual therapies has limited their success.,Designing rational combinations of targeted therapy and immunotherapy may overcome these obstacles, but requires assessment in preclinical models with the capacity to respond to both therapeutic classes.,Herein, we describe the development and characterization of a novel, immunogenic variant of the BrafV600ECdkn2a−/−Pten−/− YUMM1.1 tumor model that expresses the immunogen, ovalbumin (YOVAL1.1).,We demonstrate that, unlike parental tumors, YOVAL1.1 tumors are immunogenic in vivo and can be controlled by immunotherapy.,Importantly, YOVAL1.1 tumors are sensitive to targeted inhibitors of BRAFV600E and MEK, responding in a manner consistent with human BRAFV600E melanoma.,The YOVAL1.1 melanoma model is transplantable, immunogenic and sensitive to clinical therapies, making it a valuable platform to guide strategic development of combined targeted therapy and immunotherapy approaches in BRAFV600E melanoma.

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

In this study, Falletta et al. show that microenvironmental cues, including inflammation-mediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B.,Their results suggest that translation reprogramming, an evolutionarily conserved starvation response, has been hijacked by microenvironmental stress signals in melanoma to drive phenotypic plasticity and invasion and determine therapeutic outcome.,The intratumor microenvironment generates phenotypically distinct but interconvertible malignant cell subpopulations that fuel metastatic spread and therapeutic resistance.,Whether different microenvironmental cues impose invasive or therapy-resistant phenotypes via a common mechanism is unknown.,In melanoma, low expression of the lineage survival oncogene microphthalmia-associated transcription factor (MITF) correlates with invasion, senescence, and drug resistance.,However, how MITF is suppressed in vivo and how MITF-low cells in tumors escape senescence are poorly understood.,Here we show that microenvironmental cues, including inflammation-mediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B.,ATF4, a key transcription mediator of the integrated stress response, also activates AXL and suppresses senescence to impose the MITF-low/AXL-high drug-resistant phenotype observed in human tumors.,However, unexpectedly, without translation reprogramming an ATF4-high/MITF-low state is insufficient to drive invasion.,Importantly, translation reprogramming dramatically enhances tumorigenesis and is linked to a previously unexplained gene expression program associated with anti-PD-1 immunotherapy resistance.,Since we show that inhibition of eIF2B also drives neural crest migration and yeast invasiveness, our results suggest that translation reprogramming, an evolutionarily conserved starvation response, has been hijacked by microenvironmental stress signals in melanoma to drive phenotypic plasticity and invasion and determine therapeutic outcome.

BRAF inhibitors can extend progression‐free and overall survival in melanoma patients whose tumors harbor mutations in BRAF.,However, the majority of patients eventually develop resistance to these drugs.,Here we show that BRAF mutant melanoma cells that have developed acquired resistance to BRAF inhibitors display increased oxidative metabolism and increased dependency on mitochondria for survival.,Intriguingly, the increased oxidative metabolism is associated with a switch from glucose to glutamine metabolism and an increased dependence on glutamine over glucose for proliferation.,We show that the resistant cells are more sensitive to mitochondrial poisons and to inhibitors of glutaminolysis, suggesting that targeting specific metabolic pathways may offer exciting therapeutic opportunities to treat resistant tumors, or to delay emergence of resistance in the first‐line setting.,We examine the metabolic requirements in melanoma cell lines resistant to BRAF inhibitor PLX4720.,Cells that are resistant to BRAF inhibitors are more dependent on glutamine than glucose as their carbon source.Combined glutaminolysis and BRAF inhibition was effective to promote a delay in the onset of resistance.,We examine the metabolic requirements in melanoma cell lines resistant to BRAF inhibitor PLX4720.,Cells that are resistant to BRAF inhibitors are more dependent on glutamine than glucose as their carbon source.,Combined glutaminolysis and BRAF inhibition was effective to promote a delay in the onset of resistance.

PD-L1 is expressed by a subset of patients with metastatic melanoma (MM) with an unfavorable outcome.,Its expression is increased in cells resistant to BRAF or MEK inhibitors (BRAFi or MEKi).,However, the function and regulation of expression of PD-L1 remain incompletely understood.,After generating BRAFi- and MEKi-resistant cell lines, we observed marked up-regulation of PD-L1 expression.,These cells were characterized by a common gene expression profile with up-regulation of genes involved in cell movement.,Consistently, in vitro they showed significantly increased invasive properties.,This phenotype was controlled in part by PD-L1, as determined after silencing the molecule.,Up-regulation of PD-L1 was due to post-transcriptional events controlled by miR-17-5p, which showed an inverse correlation with PD-L1 mRNA.,Direct binding between miR-17-5p and the 3’-UTR of PD-L1 mRNA was demonstrated using luciferase reporter assays.,In a cohort of 80 BRAF-mutated MM patients treated with BRAFi or MEKi, constitutive expression of PD-L1 in the absence of immune infiltrate, defined the patient subset with the worst prognosis.,Furthermore, PD-L1 expression increased in tissue biopsies after the metastatic lesions became resistant to BRAFi or MEKi.,Lastly, plasmatic miR-17-5p levels were higher in patients with PD-L1+ than PD-L1- lesions.,In conclusion, our findings indicate that PD-L1 expression induces a more aggressive behavior in melanoma cells.,We also show that PD-L1 up-regulation in BRAFi or MEKi-resistant cells is partly due to post-transcriptional mechanisms that involve miR-17-5p, suggesting that miR-17-5p may be used as a marker of PD-L1 expression by metastatic lesions and ultimately a predictor of responses to BRAFi or MEKi.

Micro RNAs (miRs) have emerged as key regulators during oncogenesis.,They have been found to regulate cell proliferation, differentiation, and apoptosis.,Mir-125b has been identified as an oncomir in various forms of tumours, but we have previously proposed that miR-125b is a suppressor of lymph node metastasis in cutaneous malignant melanoma.,Our goal was therefore to further examine this theory.,We used in-situ-hybridization to visualise miR-125b expression in primary tumours and in lymph node metastasis.,Then using a miRVector plasmid containing a miR-125b-1 insert we transfected melanoma cell line Mel-Juso and then investigated the effect of the presence of a stable overexpression of miR-125b on growth by western blotting, flow cytometry and β-galactosidase staining.,The tumourogenicity of the transfected cells was tested using a murine model and the tumours were further examined with in-situ-hybridization.,In primary human tumours and in lymph node metastases increased expression of miR-125b was found in single, large tumour cells with abundant cytoplasm.,A stable overexpression of miR-125b in human melanoma cell line Mel-Juso resulted in a G0/G1 cell cycle block and emergence of large cells expressing senescence markers: senescence-associated beta-galactosidase, p21, p27 and p53.,Mel-Juso cells overexpressing miR-125b were tumourigenic in mice, but the tumours exhibited higher level of cell senescence and decreased expression of proliferation markers, cyclin D1 and Ki67 than the control tumours.,Our results confirm the theory that miR-125b functions as a tumour supressor in cutaneous malignant melanoma by regulating cellular senescence, which is one of the central mechanisms protecting against the development and progression of malignant melanoma.

Melanoma is a heterogeneous tumour, but the impact of this heterogeneity upon therapeutic response is not well understood.,Single cell mRNA analysis was used to define the transcriptional heterogeneity of melanoma and its dynamic response to BRAF inhibitor therapy and treatment holidays.,Discrete transcriptional states were defined in cell lines and melanoma patient specimens that predicted initial sensitivity to BRAF inhibition and the potential for effective re-challenge following resistance.,A mathematical model was developed to maintain competition between the drug-sensitive and resistant states, which was validated in vivo.,Our analyses showed melanoma cell lines and patient specimens to be composed of >3 transcriptionally distinct states.,The cell state composition was dynamically regulated in response to BRAF inhibitor therapy and drug holidays.,Transcriptional state composition predicted for therapy response.,The differences in fitness between the different transcriptional states were leveraged to develop a mathematical model that optimized therapy schedules to retain the drug sensitive population.,In vivo validation demonstrated that the personalized adaptive dosing schedules outperformed continuous or fixed intermittent BRAF inhibitor schedules.,Our study provides the first evidence that transcriptional heterogeneity at the single cell level predicts for initial BRAF inhibitor sensitivity.,We further demonstrate that manipulating transcriptional heterogeneity through personalized adaptive therapy schedules can delay the time to resistance.,This work was funded by the National Institutes of Health.,The funder played no role in assembly of the manuscript.

Development of resistance to inhibitors of BRAF (BRAFi) and MEK (MEKi) remains a great challenge for targeted therapy in patients with BRAF-mutant melanoma.,Here, we explored the role of miRNAs in melanoma acquired resistance to BRAFi.,miRNA expression in two BRAF-mutant melanoma cell lines and their dabrafenib-resistant sublines was determined using Affymetrix GeneChip® miRNA 3.1 microarrays and/or qRT-PCR.,The effects of miR-126-3p re-expression on proliferation, apoptosis, cell cycle, ERK1/2 and AKT phosphorylation, dabrafenib sensitivity, invasiveness and VEGF-A secretion were evaluated in the dabrafenib-resistant sublines using MTT assays, flow cytometry, immunoblotting, invasion assays in Boyden chambers and ELISA.,ADAM9, PIK3R2, MMP7 and CXCR4 expression in the sensitive and dabrafenib-resistant cells was determined by immunoblotting.,Small RNA interference was performed to investigate the consequence of VEGFA or ADAM9 silencing on proliferation, invasiveness or dabrafenib sensitivity of the resistant sublines.,Long-term proliferation assays were carried out in dabrafenib-sensitive cells to assess the effects of enforced miR-126-3p expression or ADAM9 silencing on resistance development.,VEGF-A serum levels in melanoma patients treated with BRAFi or BRAFi+MEKi were evaluated at baseline (T0), after two months of treatment (T2) and at progression (TP) by ELISA.,miR-126-3p was significantly down-regulated in the dabrafenib-resistant sublines as compared with their parental counterparts. miR-126-3p replacement in the drug-resistant cells inhibited proliferation, cell cycle progression, phosphorylation of ERK1/2 and/or AKT, invasiveness, VEGF-A and ADAM9 expression, and increased dabrafenib sensitivity.,VEGFA or ADAM9 silencing impaired proliferation and invasiveness of the drug-resistant sublines.,ADAM9 knock-down in the resistant cells increased dabrafenib sensitivity, whereas miR-126-3p enforced expression or ADAM9 silencing in the drug-sensitive cells delayed the development of resistance.,At T0 and T2, statistically significant differences were observed in VEGF-A serum levels between patients who responded to therapy and patients who did not.,In responder patients, a significant increase of VEGF-A levels was observed at TP versus T2.,Strategies restoring miR-126-3p expression or targeting VEGF-A or ADAM9 could restrain growth and metastasis of dabrafenib-resistant melanomas and increase their drug sensitivity.,Circulating VEGF-A is a promising biomarker for predicting patients’ response to BRAFi or BRAFi+MEKi and for monitoring the onset of resistance.,The online version of this article (10.1186/s13046-019-1238-4) contains supplementary material, which is available to authorized users.

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

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.

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.

Over two-thirds of melanomas have activating mutations in B-Raf, leading to constitutive activation of the B-Raf/MKK/ERK signaling pathway.,The most prevalent mutation, B-RafV600E, promotes cancer cell behavior through mechanisms that are still incompletely defined.,Here, we used a sensitive microarray profiling platform to compare microRNA (miRNA) expression levels between primary melanocytes and B-RafV600E-positive melanoma cell lines, and between melanoma cells treated in the presence and absence of an MKK1/2 inhibitor.,We identified a network of >20 miRNAs deregulated by B-Raf/MKK/ERK in melanoma cells, the majority of which modulate the expression of key cancer regulatory genes and functions.,Importantly, miRNAs within the network converge on protein regulation and cancer phenotypes, suggesting that these miRNAs might function combinatorially.,We show that miRNAs augment effects on protein repression and cell invasion when co-expressed, and gene-specific latency and interference effects between miRNAs were also observed.,Thus, B-Raf/MKK/ERK controls key aspects of cancer cell behavior and gene expression by modulating a network of miRNAs with cross-regulatory functions.,The findings highlight the potential for complex interactions between coordinately regulated miRNAs within a network.

Skin cutaneous melanoma (SKCM) is one of the most deadly malignancies.,Although immunotherapies showed the potential to improve the prognosis for metastatic melanoma patients, only a small group of patients can benefit from it.,Therefore, it is urgent to investigate the tumor microenvironment in melanoma as well as to identify efficient biomarkers in the diagnosis and treatments of SKCM patients.,A comprehensive analysis was performed based on metastatic melanoma samples from the Cancer Genome Atlas (TCGA) database and ESTIMATE algorithm, including gene expression, immune and stromal scores, prognostic immune‐related genes, infiltrating immune cells analysis and immune subtype identification.,Then, the differentially expressed genes (DEGs) were obtained based on the immune and stromal scores, and a list of prognostic immune‐related genes was identified.,Functional analysis and the protein-protein interaction network revealed that these genes enriched in multiple immune-related biological processes.,Furthermore, prognostic genes were verified in the Gene Expression Omnibus (GEO) databases and used to predict immune infiltrating cells component.,Our study revealed seven immune subtypes with different risk values and identified T cells as the most abundant cells in the immune microenvironment and closely associated with prognostic outcomes.,In conclusion, the present study thoroughly analyzed the tumor microenvironment and identified prognostic immune‐related biomarkers for metastatic melanoma.

The combination of liquid biomarkers from a single blood tube can provide more comprehensive information on tumor development and progression in cancer patients compared to single analysis.,Here, we evaluated whether a combined analysis of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and circulating cell‐free microRNA (miRNA) in total plasma and extracellular vesicles (EV) from the same blood sample is feasible and how the results are influenced by the choice of different blood tubes.,Peripheral blood from 20 stage IV melanoma patients and five healthy donors (HD) was collected in EDTA, Streck, and Transfix tubes.,Peripheral blood mononuclear cell fraction was used for CTC analysis, whereas plasma and EV fractions were used for ctDNA mutation and miRNA analysis.,Mutations in cell‐free circulating DNA were detected in 67% of patients, with no significant difference between the tubes.,CTC was detected in only EDTA blood and only in 15% of patients. miRNA NGS (next‐generation sequencing) results were highly influenced by the collection tubes and could only be performed from EDTA and Streck tubes due to hemolysis in Transfix tubes.,No overlap of significantly differentially expressed miRNA (patients versus HD) could be found between the tubes in total plasma, whereas eight miRNA were commonly differentially regulated in the EV fraction.,In summary, high‐quality CTCs, ctDNA, and miRNA data from a single blood tube can be obtained.,However, the choice of blood collection tubes is a critical pre‐analytical variable.,We assessed whether a combined analysis of circulating tumor cells, circulating tumor DNA, and microRNA in total plasma or extracellular vesicles from one blood sample is feasible.,We show that high‐quality data from a single blood tube can be obtained in melanoma patients.,However, the choice of the tube affects the outcome of the analysis, underlining the importance of pre‐analytical factors.

Melanoma is the most aggressive skin-cancer, showing high mortality at advanced stages.,Platelet Derived Growth Factor Receptor-alpha (PDGFR-alpha) potently inhibits melanoma- and endothelium-proliferation and its expression is significantly reduced in melanoma-biopsies, suggesting that melanoma progression eliminates cells expressing PDGFR-alpha.,In the present study transient overexpression of PDGFR-alpha in endothelial (HUVEC) and melanoma (SKMel-28, A375, Preyer) human-cells shows strong anti-proliferative effects, with profound transcriptome and miRNome deregulation.,PDGFR-alpha overexpression strongly affects expression of 82 genes in HUVEC (41 up-, 41 down-regulated), and 52 genes in SKMel-28 (43 up-, 9 down-regulated).,CXCL10/IP-10 transcript showed up to 20 fold-increase, with similar changes detectable at the protein level. miRNA expression profiling in cells overexpressing PDGFR-alpha identified 14 miRNAs up- and 40 down-regulated, with miR-503 being the most down-regulated (6.4 fold-reduction). miR-503, miR-630 and miR-424 deregulation was confirmed by qRT-PCR.,Interestingly, the most upregulated transcript (i.e., CXCL10/IP-10) was a validated miR-503 target and CXCL10/IP-10 neutralization significantly reverted the anti-proliferative action of PDGFR-alpha, and PDGFR-alpha inhibition by Dasatinb totally reverted the CXCL10/IP10 induction, further supporting a functional interplay of these factors.,Finally, integration of transcriptomics and miRNomics data highlighted several pathways affected by PDGFR-alpha.,This study demonstrates for the first time that PDGFR-alpha strongly inhibits endothelial and melanoma cells proliferation in a CXCL10/IP-10 dependent way, via miR-503 down-regulation.

MicroRNAs (miRNAs) are attractive therapeutic targets for various therapy-resistant tumors.,However, the association between miRNA and BRAF inhibitor resistance in melanoma remains to be elucidated.,We used microarray analysis to comprehensively study the miRNA expression profiling of vemurafenib resistant (VemR) A375 melanoma cells in relation to parental A375 melanoma cells.,MicroRNA-7 (miR-7) was identified to be the most significantly down-regulated miRNA in VemR A375 melanoma cells.,We also found that miR-7 was down-regulated in Mel-CVR cells (vemurafenib resistant Mel-CV melanoma cells).,Reestablishment of miR-7 expression could reverse the resistance of both cells to vemurafenib.,We showed that epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF-1R) and CRAF were over-expressed in VemR A375 melanoma cells.,Introduction of miR-7 mimics could markedly decrease the expressions of EGFR, IGF-1R and CRAF and further suppressed the activation of MAPK and PI3K/AKT pathway in VemR A375 melanoma cells.,Furthermore, tumor growth was inhibited in an in vivo murine VemR A375 melanoma tumor model transfected with miR-7 mimics.,Collectively, our study demonstrated that miR-7 could reverse the resistance to BRAF inhibitors in certain vemurafenib resistant melanoma cell lines.,It could advance the field and provide the basis for further studies in BRAF inhibitor resistance in melanoma.

Melanoma cells rely on developmental programs during tumor initiation and progression.,Here we show that the embryonic stem cell (ESC) factor Sall4 is re-expressed in the Tyr::NrasQ61K; Cdkn2a−/− melanoma model and that its expression is necessary for primary melanoma formation.,Surprisingly, while Sall4 loss prevents tumor formation, it promotes micrometastases to distant organs in this melanoma-prone mouse model.,Transcriptional profiling and in vitro assays using human melanoma cells demonstrate that SALL4 loss induces a phenotype switch and the acquisition of an invasive phenotype.,We show that SALL4 negatively regulates invasiveness through interaction with the histone deacetylase (HDAC) 2 and direct co-binding to a set of invasiveness genes.,Consequently, SALL4 knock down, as well as HDAC inhibition, promote the expression of an invasive signature, while inhibition of histone acetylation partially reverts the invasiveness program induced by SALL4 loss.,Thus, SALL4 appears to regulate phenotype switching in melanoma through an HDAC2-mediated mechanism.,Melanoma cells can switch between proliferative and invasive phenotypes.,Here the authors show that the embryonic stem cell factor Sall4 is a negative regulator of melanoma phenotype switching where its loss leads to the acquisition of an invasive phenotype, due to derepression of invasiveness genes.

The PI 3-kinase (PI3K) pathway has been implicated as a target for melanoma therapy.,Given the high degree of genetic heterogeneity in melanoma, we sought to understand the breadth of variation in PI3K signalling in the large NZM panel of early passage cell lines developed from metastatic melanomas.,We find the vast majority of lines show upregulation of this pathway, and this upregulation is achieved by a wide range of mechanisms.,Expression of all class-IA PI3K isoforms was readily detected in these cell lines.,A range of genetic changes in different components of the PI3K pathway was seen in different lines.,Coding variants or amplification were identified in the PIK3CA gene, and amplification of the PK3CG gene was common.,Deletions in the PIK3R1 and PIK3R2 regulatory subunits were also relatively common.,Notably, no genetic variants were seen in the PIK3CD gene despite p110δ being expressed in many of the lines.,Genetic variants were detected in a number of genes that encode phosphatases regulating the PI3K signalling, with reductions in copy number common in PTEN, INPP4B, INPP5J, PHLLP1 and PHLLP2 genes.,While the pan-PI3K inhibitor ZSTK474 attenuated cell growth in all the lines tested, isoform-selective inhibition of p110α and p110δ inhibited cell growth in only a subset of the lines and the inhibition was only partial.,This suggests that functional redundancy exists between PI3K isoforms.,Furthermore, while ZSTK474 was initially effective in melanoma cells with induced resistance to vemurafenib, a subset of these cell lines concurrently developed partial resistance to PI3K inhibition.,Importantly, mTOR-selective or mTOR/PI3K dual inhibitors effectively inhibited cell growth in all the lines, including those already resistant to BRAF inhibitors and ZSTK474.,Overall, this indicates a high degree of diversity in the way the PI3K pathway is activated in different melanoma cell lines and that mTOR is the most effective point for targeting the growth via the PI3K pathway across all of these cell lines.,The online version contains supplementary material available at 10.1186/s12885-021-07826-4.

Cutaneous melanoma represents the most fatal skin cancer due to its high metastatic capacity.,According to the “phenotype switching” model, the aggressive nature of melanoma cells results from their intrinsic potential to dynamically switch from a high-proliferative/low-invasive to a low-proliferative/high-invasive state.,Here we identify the low affinity neurotrophin receptor CD271 as a key effector of phenotype switching in melanoma.,CD271 plays a dual role in this process by decreasing proliferation, while simultaneously promoting invasiveness.,Dynamic modification of CD271 expression allows tumor cells to grow at low levels of CD271, to reduce growth and invade when CD271 expression is high, and to re-expand at a distant site upon decrease of CD271 expression.,Mechanistically, the cleaved intracellular domain of CD271 controls proliferation, while the interaction of CD271 with the neurotrophin receptor Trk-A modulates cell adhesiveness through dynamic regulation of a set of cholesterol synthesis genes relevant for patient survival.,The aggressive nature of melanoma cells relies on their ability to switch from a high-proliferative/low-invasive to a low-proliferative/high-invasive state; however, the mechanisms governing this switch are unclear.,Here, using in vivo models of human melanoma, the authors show that CD271 is a key regulator of phenotype switching and metastasis formation.

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 the deadliest of all skin cancers due to its high metastatic potential.,In recent years, advances in targeted therapy and immunotherapy have contributed to a remarkable progress in the treatment of metastatic disease.,However, intrinsic or acquired resistance to such therapies remains a major obstacle in melanoma treatment.,Melanoma disease progression, beginning from tumor initiation and growth to acquisition of invasive phenotypes and metastatic spread and acquisition of treatment resistance, has been associated with cellular dedifferentiation and the hijacking of gene regulatory networks reminiscent of the neural crest (NC)-the developmental structure which gives rise to melanocytes and hence melanoma.,This review summarizes the experimental evidence for the involvement of NC stem cell (NCSC)‐like cell states during melanoma progression and addresses novel approaches to combat the emergence of stemness characteristics that have shown to be linked with aggressive disease outcome and drug resistance.

Cutaneous melanoma represents the most fatal skin cancer due to its high metastatic capacity.,According to the “phenotype switching” model, the aggressive nature of melanoma cells results from their intrinsic potential to dynamically switch from a high-proliferative/low-invasive to a low-proliferative/high-invasive state.,Here we identify the low affinity neurotrophin receptor CD271 as a key effector of phenotype switching in melanoma.,CD271 plays a dual role in this process by decreasing proliferation, while simultaneously promoting invasiveness.,Dynamic modification of CD271 expression allows tumor cells to grow at low levels of CD271, to reduce growth and invade when CD271 expression is high, and to re-expand at a distant site upon decrease of CD271 expression.,Mechanistically, the cleaved intracellular domain of CD271 controls proliferation, while the interaction of CD271 with the neurotrophin receptor Trk-A modulates cell adhesiveness through dynamic regulation of a set of cholesterol synthesis genes relevant for patient survival.,The aggressive nature of melanoma cells relies on their ability to switch from a high-proliferative/low-invasive to a low-proliferative/high-invasive state; however, the mechanisms governing this switch are unclear.,Here, using in vivo models of human melanoma, the authors show that CD271 is a key regulator of phenotype switching and metastasis formation.

Transcriptomic signatures designed to predict melanoma patient responses to PD-1 blockade have been reported but rarely validated.,We now show that intra-patient heterogeneity of tumor responses to PD-1 inhibition limit the predictive performance of these signatures.,We reasoned that resistance mechanisms will reflect the tumor microenvironment, and thus we examined PD-1 inhibitor resistance relative to T-cell activity in 94 melanoma tumors collected at baseline and at time of PD-1 inhibitor progression.,Tumors were analyzed using RNA sequencing and flow cytometry, and validated functionally.,These analyses confirm that major histocompatibility complex (MHC) class I downregulation is a hallmark of resistance to PD-1 inhibitors and is associated with the MITFlow/AXLhigh de-differentiated phenotype and cancer-associated fibroblast signatures.,We demonstrate that TGFß drives the treatment resistant phenotype (MITFlow/AXLhigh) and contributes to MHC class I downregulation in melanoma.,Combinations of anti-PD-1 with drugs that target the TGFß signaling pathway and/or which reverse melanoma de-differentiation may be effective future therapeutic strategies.,A significant proportion of patients develop innate or acquired resistance to immune checkpoint inhibitors.,Here, the authors show that resistance to anti-PD-1 blockade is associated with TGF-beta driven major histocompatibility complex I (MHCI) down-regulation and a de-differentiated phenotype in melanoma patients.

Understanding mechanisms of late/acquired cancer immunotherapy resistance is critical to improve outcomes; cellular immunotherapy trials offer a means to probe complex tumor-immune interfaces through defined T cell/antigen interactions.,We treated two patients with metastatic Merkel cell carcinoma with autologous Merkel cell polyomavirus specific CD8+ T cells and immune-checkpoint inhibitors.,In both cases, dramatic remissions were associated with dense infiltration of activated CD8+s into the regressing tumors.,However, late relapses developed at 22 and 18 months, respectively.,Here we report single cell RNA sequencing identified dynamic transcriptional suppression of the specific HLA genes presenting the targeted viral epitope in the resistant tumor as a consequence of intense CD8-mediated immunologic pressure; this is distinguished from genetic HLA-loss by its reversibility with drugs.,Transcriptional suppression of Class I loci may underlie resistance to other immunotherapies, including checkpoint inhibitors, and have implications for the design of improved immunotherapy treatments.,Acquired resistance is a major problem in cancer immunotherapy.,Here the authors report a study of two patients with Merkel cell carcinoma under immunotherapy treatment who develop resistance after deep responses for >1 year and identified a novel mechanism of acquired, gene-specific transcriptional suppression of HLAs.

Although melanoma is one of the most immunogenic tumors, it has an ability to evade anti-tumor immune responses by exploiting tolerance mechanisms, including negative immune checkpoint molecules.,The most extensively studied checkpoints represent cytotoxic T lymphocyte-associated protein-4 (CTLA-4) and programmed cell death protein 1 (PD-1).,Immune checkpoint inhibitors (ICI), which were broadly applied for melanoma treatment in the past decade, can unleash anti-tumor immune responses and result in melanoma regression.,Patients responding to the ICI treatment showed long-lasting remission or disease control status.,However, a large group of patients failed to respond to this therapy, indicating the development of resistance mechanisms.,Among them are intrinsic tumor properties, the dysfunction of effector cells, and the generation of immunosuppressive tumor microenvironment (TME).,This review discusses achievements of ICI treatment in melanoma, reasons for its failure, and promising approaches for overcoming the resistance.,These methods include combinations of different ICI with each other, strategies for neutralizing the immunosuppressive TME and combining ICI with other anti-cancer therapies such as radiation, oncolytic viral, or targeted therapy.,New therapeutic approaches targeting other immune checkpoint molecules are also discussed.

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.

Vasculogenic mimicry (VM) is a functional microcirculation formed by tumor cells.,Matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9, promote VM formation.,Another specific MMP, collagenase-3 (MMP-13), has broad substrate specificity and potentially affects tumor metastasis and invasion.,Here we found that MMP-13 was associated with metastasis and poor survival in 79 patients with melanoma.,MMP-13 expression was inversely correlated with VM.,These results were confirmed in human and mouse melanoma cell lines.,We found that MMP-13 cleaves laminin-5 (Ln-5) into small fragments to accelerate tumor metastasis.,Degradation of Ln-5 and VE-cadherin by MMP-13 inhibited VM formation.,In conclusion, MMP-13 has a dual effect in melanoma, as it promotes invasion and metastasis but disrupts VM formation.

The aggressiveness of melanoma tumors is likely to rely on their well-recognized heterogeneity and plasticity.,Melanoma comprises multi-subpopulations of cancer cells some of which may possess stem cell-like properties.,Although useful, the sphere-formation assay to identify stem cell-like or tumor initiating cell subpopulations in melanoma has been challenged, and it is unclear if this model can predict a functional phenotype associated with aggressive tumor cells.,We analyzed the molecular and functional phenotypes of melanoma spheroids formed in neural crest cell medium.,Whether from metastatic or advanced primary tumors, spheroid cells expressed melanoma-associated markers.,They displayed higher capacity to differentiate along mesenchymal lineages and enhanced expression of SOX2, NANOG, KLF4, and/or OCT4 transcription factors, but not enhanced self-renewal or tumorigenicity when compared to their adherent counterparts.,Gene expression profiling attributed a neural crest cell signature to these spheroids and indicated that a migratory/invasive and immune-function modulating program could be associated with these cells.,In vitro assays confirmed that spheroids display enhanced migratory/invasive capacities.,In immune activation assays, spheroid cells elicited a poorer allogenic response from immune cells and inhibited mitogen-dependent T cells activation and proliferation more efficiently than their adherent counterparts.,Our findings reveal a novel immune-modulator function of melanoma spheroids and suggest specific roles for spheroids in invasion and in evasion of antitumor immunity.,The association of a more plastic, invasive and evasive, thus a more aggressive tumor phenotype with melanoma spheroids reveals a previously unrecognized aspect of tumor cells expanded as spheroid cultures.,While of limited efficiency for melanoma initiating cell identification, our melanoma spheroid model predicted aggressive phenotype and suggested that aggressiveness and heterogeneity of melanoma tumors can be supported by subpopulations other than cancer stem cells.,Therefore, it could be constructive to investigate melanoma aggressiveness, relevant to patients and clinical transferability.

Gene expression profiles of cutaneous melanoma were analyzed to identify critical genes associated with metastasis.,Two gene expression datasets were downloaded from Gene Expression Omnibus (GEO) and another dataset was obtained from The Cancer Genome Atlas (TCGA).,Differentially expression genes (DEGs) between metastatic and non-metastatic melanoma were identified by meta-analysis.,A protein-protein interaction (PPI) network was constructed for the DEGs using information from BioGRID, HPRD and DIP.,Betweenness centrality (BC) was calculated for each node in the network and the top feature genes ranked by BC were selected to construct the support vector machine (SVM) classifier using the training set.,The SVM classifier was then validated in another independent dataset.,Pathway enrichment analysis was performed for the feature genes using Fisher's exact test.,A total of 798 DEGs were identified and a PPI network including 337 nodes and 466 edges was then constructed.,Top 110 feature genes ranked by BC were included in the SVM classifier.,The prediction accuracies for the three datasets were 96.8, 100 and 94.4%, respectively.,A total of 11 KEGG pathways and 13 GO biological pathways were significantly over-represented in the 110 feature genes, including endometrial cancer, regulation of actin cytoskeleton, focal adhesion, ubiquitin mediated proteolysis, regulation of apoptosis and regulation of cell proliferation.,A SVM classifier of high prediction accuracy was acquired.,Several critical genes implicated in melanoms metastasis were also revealed.,These results may advance understanding of the molecular mechanisms underlying metastasis, and also provide potential therapeutic targets.

In the recent past years, many discoveries in the tumor microenvironment have led to changes in the management of melanoma and it is rising up hopes, specially, to those in advanced stages.,FDA approved seven new drugs from 2011 to 2014.,They are: Vemurafenib, Dabrafenib and Trametinib, kinases inhibitors used for patients that have BRAFV600E mutation; Ipilimumab (anti-CTLA4), Pembrolizumab (anti-PD-1) and Nivolumab (anti-PD-1), monoclonal antibodies that stimulate the immune system; and Peginterferon alfa-2b, an anti-proliferative cytokine used as adjuvant therapy.,In this article, we will review the molecular bases for these new metastatic melanoma therapeutic agents cited above and also analyze new molecular discoveries in melanoma study, as Cancer-Testis antigens (CT).,They are capable of induce humoral and cellular immune responses in cancer patients and because of this immunogenicity and their restrict expression in normal tissues, they are considered an ideal candidate for vaccine development against cancer.,Among CT antigens, NY-ESO-1 is the best characterized in terms of expression patterns and immunogenicity.,It is expressed in 20-40% of all melanomas, more in metastatic lesions than in primary ones, and it is very heterogeneous inter and intratumoral.,Breslow index is associate with NY-ESO-1 expression in primary cutaneous melanomas, but its relation to patient survival remains controversial.

CTCs provide prognostic information and their application is under investigation in multiple tumor types.,Of the multiple variables inherent in any such process, none is more important to outcome than the appropriateness of the sample source.,To address this question, we investigated CTCs in paired peripheral venous and arterial blood specimens obtained from stage IV uveal melanoma patients.,Blood specimens were obtained from both common femoral arteries and antecubital veins in 17 uveal melanoma patients with multiple hepatic metastases for CTC measurements.,CTCs were detectable with greater frequency (100%) and in larger numbers (median 5, range 1 to 168) in all arterial blood specimens than in venous samples (52.9%; median 1, range 0 to 8).,Patients with hepatic as well as extra-hepatic metastasis showed higher number of arterial CTCs, compared to patients with liver-only metastasis (p = 0.003).,There was no significant association between the number of arterial CTCs and the tumor burden within the liver in patients who had liver-only metastases.,Our data indicate that arterial blood specimens might be a better source of circulating uveal melanoma cells.,Although less conveniently processed, perhaps arterial blood should be evaluated as sample source for measurement of CTCs.,•CTCs were detectable in 100% of arterial blood obtained from metastatic uveal melanoma patients, while only 53% of venous blood was positive for CTCs.,CTCs were detectable in 100% of arterial blood obtained from metastatic uveal melanoma patients, while only 53% of venous blood was positive for CTCs.,CTCs have been investigated to provide prognostic information in multiple tumor types.,Of the multiple variables, none is more important than the appropriateness of the sample source.,Blood specimens were obtained from both femoral arteries and antecubital veins in 17 uveal melanoma patients with multiple hepatic metastases.,CTCs were detectable with greater frequency (100%) and in larger numbers in all arterial blood specimens than in venous samples (52.9%).,Our data indicate that arterial blood specimens might be a better source of circulating uveal melanoma cells.,Although less convenient, arterial blood should be evaluated as sample source for measurement of CTCs.

The transcriptional response promoted by hypoxia-inducible factors has been associated with metastatic spread of uveal melanoma.,We found expression of hypoxia-inducible factor 1α (HIF-1α) protein in well-vascularized tumor regions as well as in four cell lines grown in normoxia, thus this pathway may be important even in well-oxygenated uveal melanoma cells.,HIF-1α protein accumulation in normoxia was inhibited by rapamycin.,As expected, hypoxia (1% pO2) further induced HIF-1α protein levels along with its target genes VEGF and LOX.,Growth in hypoxia significantly increased cellular invasion of all 5 uveal melanoma lines tested, as did the introduction of an oxygen-insensitive HIF-1α mutant into Mel285 cells with low HIF-1α baseline levels.,In contrast, HIF-1α knockdown using shRNA significantly decreased growth in hypoxia, and reduced by more than 50% tumor invasion in four lines with high HIF-1α baseline levels.,Pharmacologic blockade of HIF-1α protein expression using digoxin dramatically suppressed cellular invasion both in normoxia and in hypoxia.,We found that Notch pathway components, including Jag1-2 ligands, Hes1-Hey1 targets and the intracellular domain of Notch1, were increased in hypoxia, as well as the phosphorylation levels of Erk1-2 and Akt.,Pharmacologic and genetic inhibition of Notch largely blocked the hypoxic induction of invasion as did the pharmacologic suppression of Erk1-2 activity.,In addition, the increase in Erk1-2 and Akt phosphorylation by hypoxia was partially reduced by inhibiting Notch signaling.,Our findings support the functional importance of HIF-1α signaling in promoting the invasive capacity of uveal melanoma cells in both hypoxia and normoxia, and suggest that pharmacologically targeting HIF-1α pathway directly or through blockade of Notch or Erk1-2 pathways can slow tumor spread.

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.

A fundamental problem in cancer research is identifying the cell type that is capable of sustaining neoplastic growth and its origin from normal tissue cells.,Recent investigations of a variety of tumor types have shown that phenotypically identifiable and isolable subfractions of cells possess the tumor-forming ability.,In the present paper, using two lineage-related human melanoma cell lines, primary melanoma line IGR39 and its metastatic derivative line IGR37, two main observations are reported.,The first one is the first phenotypic evidence to support the origin of melanoma cancer stem cells (CSCs) from mutated tissue-specific stem cells; and the second one is the identification of a more aggressive subpopulation of CSCs in melanoma that are CXCR6+.,We defined CXCR6 as a new biomarker for tissue-specific stem cell asymmetric self-renewal.,Thus, the relationship between melanoma formation and ABCG2 and CXCR6 expression was investigated.,Consistent with their non-metastatic character, unsorted IGR39 cells formed significantly smaller tumors than unsorted IGR37 cells.,In addition, ABCG2+ cells produced tumors that had a 2-fold greater mass than tumors produced by unsorted cells or ABCG2- cells.,CXCR6+ cells produced more aggressive tumors.,CXCR6 identifies a more discrete subpopulation of cultured human melanoma cells with a more aggressive MCSC phenotype than cells selected on the basis of the ABCG2+ phenotype alone.,The association of a more aggressive tumor phenotype with asymmetric self-renewal phenotype reveals a previously unrecognized aspect of tumor cell physiology.,Namely, the retention of some tissue-specific stem cell attributes, like the ability to asymmetrically self-renew, impacts the natural history of human tumor development.,Knowledge of this new aspect of tumor development and progression may provide new targets for cancer prevention and treatment.

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

Melanoma progression is associated with increased invasion and, often, decreased levels of microphthalmia-associated transcription factor (MITF).,Accordingly, downregulation of MITF induces invasion in melanoma cells, however little is known about the underlying mechanisms.,Here, we report for the first time that depletion of MITF results in elevation of intracellular GTP levels and increased amounts of active (GTP-bound) RAC1, RHO-A and RHO-C.,Concomitantly, MITF-depleted cells display larger number of invadopodia and increased invasion.,We further demonstrate that the gene for guanosine monophosphate reductase (GMPR) is a direct MITF target, and that the partial repression of GMPR accounts mostly for the above phenotypes in MITF-depleted cells.,Reciprocally, transactivation of GMPR is required for MITF-dependent suppression of melanoma cell invasion, tumorigenicity, and lung colonization.,Moreover, loss of GMPR accompanies downregulation of MITF in vemurafenib-resistant BRAFV600E-melanoma cells and underlies the increased invasion in these cells.,Our data uncover novel mechanisms linking MITF-dependent inhibition of invasion to suppression of guanylate metabolism.