Although melanoma is initiated by acquisition of point mutations and limited focal copy number alterations in melanocytes-of-origin, the nature of genetic changes that characterise lethal metastatic disease is poorly understood. Here, we analyze the evolution of human melanoma progressing from early to late disease in 13 patients by sampling their tumours at multiple sites and times. Whole exome and genome sequencing data from 88 tumour samples reveals only limited gain of point mutations generally, with net mutational loss in some metastases. In contrast, melanoma evolution is dominated by whole genome doubling and large-scale aneuploidy, in which widespread loss of heterozygosity sculpts the burden of point mutations, neoantigens and structural variants even in treatment-naïve and primary cutaneous melanomas in some patients. These results imply that dysregulation of genomic integrity is a key driver of selective clonal advantage during melanoma progression.

The concept of quaternary prevention, resulting from a reflection on the doctor-patient relationship, is presented as a renewal of the age-old ethical requirement: first, a doctor must do no harm; second, the doctor must control himself/herself. The origin of the concept, its endorsement by the World Organization of Family Doctors (WONCA) and the European Union of General Practitioners (UEMO), its dissemination, and the debates to which it has given rise, are presented by a panel of authors from 10 countries. This collective text deals more specifically with: the bioethics of prevention, the importance of teaching Quaternary prevention and factual medicine, the social and political implications of the concept of quaternary prevention, and its anthropological dimensions.

Acquired chemotherapeutic resistance of cancer cells can result from a Darwinistic evolution process in which heterogeneity plays an important role. In order to understand the impact of genetic heterogeneity on acquired resistance and second line therapy selection in metastatic melanoma, we sequenced the exomes of 27 lesions which were collected from 3 metastatic melanoma patients treated with targeted or non-targeted inhibitors. Furthermore, we tested the impact of a second NRAS mutation in 7 BRAF inhibitor resistant early passage cell cultures on the selection of second line therapies.We observed a rapid monophyletic evolution of melanoma subpopulations in response to targeted therapy that was not observed in non-targeted therapy. We observed the acquisition of NRAS mutations in the BRAF mutated patient treated with a BRAF inhibitor in 1 of 5 of his post-resistant samples. In an additional cohort of 5 BRAF-inhibitor treated patients we detected 7 NRAS mutations in 18 post-resistant samples. No NRAS mutations were detected in pre-resistant samples. By sequencing 65 single cell clones we prove that NRAS mutations co-occur with BRAF mutations in single cells. The double mutated cells revealed a heterogeneous response to MEK, ERK, PI3K, AKT and multi RTK -inhibitors.We conclude that BRAF and NRAS co-mutations are not mutually exclusive. However, the sole finding of double mutated cells in a resistant tumor is not sufficient to determine follow-up therapy. In order to target the large pool of heterogeneous cells in a patient, we think combinational therapy targeting different pathways will be necessary.

High-mobility-group-Box1 (HMGB1) is a highly conserved nuclear protein regulating gene expression. Upon cell damage, HMGB1 is actively or passively released into the extracellular space where it displays chemokine and cytokine activities.

Purpose: Cutaneous T-cell lymphomas (CTCL) are a heterogeneous group of malignancies that despite available therapies commonly relapse. The emergence of combination epigenetic therapies in other hematologic malignancies have made investigation of such combinations in CTCL a priority. Here, we explore the synergistic antiproliferative effects of romidepsin, an HDAC inhibitor, and azacitidine, a demethylating agent, combination in CTCL. Experimental Design: The growth inhibition under combination treatment and single agent was explored by the MTT cell viability assay and the Annexin V/propidium iodide (PI) apoptosis assay in different CTCL cell lines and tumor cells derived from Sézary syndrome patients. Quantitative analysis of a dose–effect relationship of romidepsin and azacitidine was done by the CompuSyn software. Investigation of mechanism of action was performed by flow cytometry, immunoblotting, qRT-PCR arrays, and chromatin immunoprecipitation. Global CpG methylation sequencing was utilized to study genome methylation alteration under the treatment modalities. Results: The combination of romidepsin and azacitidine exerts synergistic antiproliferative effects and induction of apoptosis involving activation of the caspase cascade in CTCL cell lines and tumor cells derived from Sézary syndrome patients. We identified genes that were selectively induced by the combination treatment, such as the tumor suppressor gene RhoB that is linked to enhanced histone acetylation at its promoter region in parallel with pronounced expression of p21. Global CpG methylation sequencing in a CTCL cell line and tumor cells demonstrated a subset of genes with a unique change in methylation profile in the combination treatment. Conclusions: The synergistic antiproliferative effects of romidepsin and azacitidine combination treatment justify further exploration in clinical trials for advanced CTCL. Clin Cancer Res; 22(8); 2020–31. ©2015 AACR.

Hypoxia is a hallmark of cancer that is strongly associated with invasion, metastasis, resistance to therapy and poor clinical outcome. Tumour hypoxia affects immune responses and promotes the accumulation of macrophages in the tumour microenvironment. However, the signals linking tumour hypoxia to tumour-associated macrophage recruitment and tumour promotion are incompletely understood. Here we show that the damage-associated molecular pattern High-Mobility Group Box 1 protein (HMGB1) is released by melanoma tumour cells as a consequence of hypoxia and promotes M2-like tumour-associated macrophage accumulation and an IL-10 rich milieu within the tumour. Furthermore, we demonstrate that HMGB1 drives IL-10 production in M2-like macrophages by selectively signalling through the Receptor for Advanced Glycation End products (RAGE). Finally, we show that HMGB1 has an important role in murine B16 melanoma growth and metastasis, whereas in humans its serum concentration is significantly increased in metastatic melanoma. Collectively, our findings identify a mechanism by which hypoxia affects tumour growth and metastasis in melanoma and depict HMGB1 as a potential therapeutic target.

Fibroblast contamination can make establishing primary melanoma cell cultures from native biopsies a major challenge, due to fibroblasts overgrowing the melanoma cells. Standard protocols therefore enrich for highly proliferative melanoma cells that grow well in vitro but may not represent the full range of in vivo tumor heterogeneity. Here we apply conditional methods that more effectively retrieve melanoma cells by differential trypsinization or by inducing fibroblast senescence through contact inhibition, serum starvation or deprivation of adhesion. Simple mixing experiments of melanoma and fibroblast cells demonstrated the efficacy of the new protocols in retrieving slow-growing melanoma cells. Applying our protocols to 20 cultures that had failed to grow by conventional methods, we could retrieve 12 (60%) validated melanoma cell cultures. Further application of the protocols in the live-cell biobank of 124 early passage cultures significantly improved recovery rates from 13% using standard protocols to 70% overall for the new workflow.

Background: 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.Results: 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.Conclusions: 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.

It has been shown in vitro that melanocyte proliferation and function in palmoplantar skin is regulated by mesenchymal factors derived from fibroblasts. In this study, we investigated in vivo the influence of mesenchymal-epithelial interactions in human tissue-engineered skin substitutes reconstructed from palmar- and nonpalmoplantar-derived fibroblasts. Tissue-engineered dermo-epidermal analogs based on collagen type I hydrogels were populated with either human palmar or nonpalmoplantar fibroblasts and seeded with human nonpalmoplantar-derived melanocytes and keratinocytes. These skin substitutes were transplanted onto full-thickness skin wounds of immunoincompetent rats. Four weeks after transplantation the development of skin color was measured and grafts were excised and analyzed with regard to epidermal characteristics, in particular melanocyte number and function. Skin substitutes containing palmar-derived fibroblasts in comparison to nonpalmoplantar-derived fibroblasts showed (a) a significantly lighter pigmentation; (b) a reduced amount of epidermal melanin granules; and (c) a distinct melanosome expression. However, the number of melanocytes in the basal layer remained similar in both transplantation groups. These findings demonstrate that human palmar fibroblasts regulate the function of melanocytes in human pigmented dermo-epidermal skin substitutes after transplantation, whereas the number of melanocytes remains constant. This underscores the influence of site-specific stromal cells and their importance when constructing skin substitutes for clinical application.

In describing the perils of modelling, the statistician George Box famously claimed that ‘All models are wrong, but some are useful’. Over the last few decades, several useful conceptual models of tumor progression have been proposed to describe cancer initiation, metastasis and therapeutic resistance. These include the clonal evolution (CE), cancer stem cell (CSC) and phenotype switching (PS) models. Recently, the PS model has become increasingly invoked by melanoma researchers and clinicians as it seems to best explain how a dynamic and adaptive reprogramming of melanoma cells, independent of hierarchical tumor organization, can drive tumor progression. When developing this model, we and others incorporated aspects of CSC and CE to explain how plastic phenotypic states could allow melanoma cells to respond to microenvironmental cues by downregulating a proliferative, melanocytic program, and by activating a neural crest stem cell module more conducive to invasion and metastasis (Fig. 1). Despite the popularity of PS, each of the other models still has useful explanatory power. For instance, according to a selection-based model such as CE, microenvironmental forces act on genetic subclonal populations, which then evolve according to their differential responses to a selective pressure. This may be particularly relevant in a therapeutic setting where targeted inhibitors might select for subclones that remain proliferative in the presence of the drug, and where less-fit subclones cannot compete. In contrast, the CSC model was the preferred model in melanoma until it was shown that most cells in a tumor are capable of acting as tumor initiating cells, ruling out the possibility that just a few stem cells were necessary 1. However, the reactivation of neural crest, stem cell-like developmental programs during tumor progression in invasive phenotype melanoma cells may be seen as a variation on the CSC theme 2. A recently published study provides further support for the PS model 3. Marconi and colleagues used Reflectance Confocal Microscopy to group 12 melanoma biopsies into three subtypes. All samples were stained by immunohistochemistry and analysed for the expression of HIF1alpha and CD271. They observed an anticorrelation between HIF1alpha and CD271 expression. A correlation of the stainings with phases of progression led them to conclude that HIF1alpha is expressed in more aggressive melanoma subtypes, whereas CD271 was expressed in the slow growing, superficial melanoma and was progressively lost during melanoma progression 3. The two proteins that Marconi et al. investigated are well-known in melanoma biology. Both have been reported to be important players in PS whereby proliferative melanoma cells acquire mesenchymal, invasive characteristics 4, 5. HIF1alpha is a mediator of the hypoxic response, which we and others have shown can induce a switch from proliferative to invasive phenotype cells, thereby increasing melanoma invasion 6 (S1,S2). CD271 on the other hand is a marker of stem-like cells for which the data are contradictory (S3,S4,S5). However, their postulation that CD271 is progressively lost during melanoma progression does not agree with previous reports that CD271 expression in melanoma is associated with invasiveness (S4,S6). Hypoxia is not the only factor able to induce a phenotype switch. Many studies have observed an (epithelial to mesenchymal transition) EMT-like transformation of melanoma cells from a melanocytic, differentiated state to a more dedifferentiated, mesenchymal state. During this process, the typical melanocytic markers are downregulated and mesenchymal markers are upregulated. In addition to hypoxia, TGFbeta has been shown to induce an EMT equivalent in melanoma, thereby pushing the cells towards an invasive phenotype 7. These processes are probably not so much involved in the initial melanoma formation but seem to play a key role in tumor progression and even therapeutic resistance 8. Generally, tumors are composed of numerous genetic and epigenetic subclones which show a high transcriptional diversity and adaptability. Tissue-dependent microenvironments further complicate the situation which makes targeting melanoma a difficult endeavour. Some studies comparing tumors within the same patients have found multiple different mechanisms pointing towards the presence of a high number of genetic subclones. Newly developed single-agent drugs are likely to target only one subset of melanoma cells explaining the common development of resistance. Evidence suggests that the majority of genetic resistance mechanisms lead to a reactivation of the MAPK pathway (S7,S8). Future therapies will be composed of multiple drugs in order to find a way to cope with this heterogeneity in melanoma. Despite an enormous effort and thousands of sequenced tumors in hundreds of patients, there is still a large proportion of cases where no mutation could be found to explain treatment resistance. In addition to a long tail of potential oncogenic drivers, among which PPP6C, RAC1 and MAP2K1 may be the most prominent ones (S9), the modulation of epigenetic and transcriptional states may also play an important role in progression and resistance mechanisms, as predicted by the PS model (S10). We have just started to understand the different factors driving melanoma, but the accumulating evidence suggests that PS contributes to the strategies that melanoma cells use to escape microenvironmental and therapeutic pressures 8 (S11,S12). The systematic application of increasingly more powerful panomics technologies on better curated biobank collections will improve our ability to more accurately study tumor biology in vitro and in vivo and will contribute to more sophisticated and useful conceptual models 9. Ultimately, of course, the usefulness of any tumor model will be measured by how well it can generate hypotheses and melanoma-targeting treatment strategies that directly improve the well-being of cancer patients. Daniel S. Widmer and Mitchell P. Levesque wrote the manuscript and created the illustration. Ossia M. Eichhoff contributed to creating the illustration and helped in writing the manuscript. Reinhard Dummer helped in writing the manuscript. The authors of this study do not have any conflict of interests to declare. Data S1. References. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

We recently described 17 patients with a new subtype of congenital melanocytic naevi (CMN), designated as naevus spilus-type (1). The affected patients present with a large cafe-au-lait (CAL) background macule and dozens of superimposed small and medium-sized CMN. Here we report an additional case with a novel NRAS mutation (Q61L). CMN are categorized by their largest diameter (projected adult size, PAS) (2). Small CMN (SCMN, <1.5 cm PAS) and medium-sized CMN (MCMN, 1.5 - 20 cm PAS) represent rather common, solitary, slightly thickened plaques with prominent terminal hairs. This article is protected by copyright. All rights reserved.

In our laboratory, we have been using human pigmented dermo‐epidermal skin substitutes for short‐term experiments since several years. Little is known, however, about the long‐term biology of such constructs after transplantation. We constructed human, melanocyte‐containing dermo‐epidermal skin substitutes of different (light and dark) pigmentation types and studied them in a long‐term animal experiment. Developmental and maturational stages of the epidermal and dermal compartment as well as signs of homoeostasis were analysed 15 weeks after transplantation. Keratinocytes, melanocytes and fibroblasts from human skin biopsies were isolated and assembled into dermo‐epidermal skin substitutes. These were transplanted onto immuno‐incompetent rats and investigated 15 weeks after transplantation. Chromameter evaluation showed a consistent skin colour between 3 and 4 months after transplantation. Melanocytes resided in the epidermal basal layer in physiological numbers and melanin accumulated in keratinocytes in a supranuclear position. Skin substitutes showed a mature epidermis in a homoeostatic state and the presence of dermal components such as Fibrillin and Tropoelastin suggested advanced maturation. Overall, pigmented dermo‐epidermal skin substitutes show a promising development towards achieving near‐normal skin characteristics and epidermal and dermal tissue homoeostasis. In particular, melanocytes function correctly over several months whilst remaining in a physiological, epidermal position and yield a pigmentation resembling original donor skin colour.

The identification of targetable mutations has revolutionized the therapy of metastatic melanoma. In particular, BRAF and MEK inhibitors have a well-documented impact on overall survival in metastatic disease. However, therapeutic success is highly dependent on the correct identification of these mutations. We discuss the impact of molecular heterogeneity in this context.

Epithelial to mesenchymal transition (EMT) is a programme defined in epithelial cells and recognized as playing a critical role in cancer progression. Although melanoma is not a cancer of epithelial cells, hallmarks of EMT have been described to play a critical role in melanoma progression. Here, we demonstrate that long‐term TGFβ exposure can induce a dedifferentiated EMT‐like state resembling a previously described invasive phenotype (EMT‐like). TGFβ‐induced EMT‐like is marked by the downregulation of melanocyte differentiation markers, such as MITF, and the upregulation of mesenchymal markers, such as N‐cadherin, and an increase in melanoma cell migration and cell invasion. Pharmacological interference shows the dependency of TGFβ‐induced EMT‐like on the activation of the PDGF signalling pathway and the subsequent activation of PI3K in human melanoma cells. Together, the data provide novel insights into the transcriptional plasticity of melanoma cells that might contribute to tumor progression in patients and propose avenues to therapeutic interventions.

Despite existing vaccination strategies targeting TRP-2, its function is not yet fully understood. TRP-2 is an enzyme involved in melanin biosynthesis and therefore discussed as a differentiation antigen. However, in mice Trp-2 was shown to be expressed in melanocyte stem cells of the hair follicle and therefore also considered as an indicator of stemness. A proper understanding of the TRP-2 function is crucial, considering a vaccination targeting cells with stemness properties would be highly effective in contrast to a therapy targeting differentiated melanoma cells. Analysing over 200 melanomas including primaries, partly matched metastases and patients’ cell cultures we show that TRP-2 is correlated with Melan A expression and decreases with tumor progression. In mice it is expressed in differentiated melanocytes as well as in stem cells. Furthermore, we identify a TRP-2 negative, proliferative, hypoxia related cell subpopulation which is significantly associated with tumor thickness and diseases progression. Patients with a higher percentage of those cells have a less favourable tumor specific survival. Our findings underline that TRP-2 is a differentiation antigen, highlighting the importance to combine TRP-2 vaccination with other strategies targeting the aggressive undifferentiated hypoxia related subpopulation.

The idea of cancer immunotherapy has been around for more than a century; however, the first immunotherapeutic ipilimumab, an anti-CTLA-4 antibody, has only recently been approved by the US FDA for melanoma. With an increasing understanding of the immune response, it is expected that more therapies will follow. This review aims to provide a general overview of immunotherapy in melanoma. We first explain the development of cancer immunotherapy more than a century ago and the general opinions about it over time. This is followed by a general overview of the immune reaction in order to give insight into the possible targets for therapy. Finally, we will discuss the current therapies for melanoma, their shortcomings and why it is important to develop patient stratification criteria. We conclude with an overview of recent discoveries and possible future therapies.

We have previously reported a model for melanoma progression in which oscillation between melanoma cell phenotypes characterized by invasion or proliferation is fundamental to tumor heterogeneity and disease progression. In this study we examine the possible role of hypoxia as one of the microenvironmental influences driving metastatic progression by promoting a switch from a proliferative to an invasive phenotype. Immunohistochemistry on primary human cutaneous melanoma biopsies showed intratumoral heterogeneity for cells expressing melanocytic markers, and a loss of these markers correlated with hypoxic regions. Furthermore, we show that the downregulation of melanocytic markers is dependent on hypoxia inducible factor 1α (HIF1α), a known regulator of the hypoxic response. In vitro invasion assays showed that a hypoxic environment increases the invasiveness of proliferative melanoma cell cultures in a HIF1α-dependent manner. In contrast, invasive phenotype melanoma cells showed no increase in invasive potential upon exposure to hypoxia. Thus, exposure of proliferative melanoma cells to hypoxic microenvironments is sufficient, in a HIF1α-dependent manner, to downregulate melanocytic marker expression and increase their invasive potential.

There is growing evidence that the metastatic spread of melanoma is driven not by a linear increase in tumorigenic aggressiveness, but rather by switching back and forth between two different phenotypes of metastatic potential. In vitro these phenotypes are respectively defined by the characteristics of strong proliferation/weak invasiveness and weak proliferation/strong invasiveness. Melanoma cell phenotype is tightly linked to gene expression. Taking advantage of this, we have developed a gene expression-based tool for predicting phenotype called Heuristic Online Phenotype Prediction. We demonstrate the predictive utility of this tool by comparing phenotype-specific signatures with measurements of characteristics of melanoma phenotype-specific biology in different melanoma cell lines and short-term cultures. We further show that 86% of 536 tested melanoma lines and short-term cultures are significantly associated with the phenotypes we describe. These findings reinforce the concept that a two-state system, as described by the phenotype switching model, underlies melanoma progression.