Metastatic melanoma moves on: translational science in the era of personalized medicine.

Progress in understanding and treating metastatic melanoma is the result of decades of basic and translational research as well as the development of better in vitro tools for modeling the disease. Here, we review the latest therapeutic options for metastatic melanoma and the known genetic and non-genetic mechanisms of resistance to these therapies, as well as the in vitro toolbox that has provided the greatest insights into melanoma progression. These include next-generation sequencing technologies and more complex 2D and 3D cell culture models to functionally test the data generated by genomics approaches. The combination of hypothesis generating and hypothesis testing paradigms reviewed here will be the foundation for the next phase of metastatic melanoma therapies in the coming years.

A new live-cell biobank workflow efficiently recovers heterogeneous melanoma cells from native biopsies.

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.

The EMT Transcription Factor ZEB2 Promotes Proliferation of Primary and Metastatic Melanoma While Suppressing an Invasive, Mesenchymal-Like Phenotype.

Epithelial-to-mesenchymal transition (EMT)-inducing transcription factors (TF) are well known for their ability to induce mesenchymal states associated with increased migratory and invasive properties. Unexpectedly, nuclear expression of the EMT-TF ZEB2 in human primary melanoma has been shown to correlate with reduced invasion. We report here that ZEB2 is required for outgrowth for primary melanomas and metastases at secondary sites. Ablation of Zeb2 hampered outgrowth of primary melanomas in vivo, whereas ectopic expression enhanced proliferation and growth at both primary and secondary sites. Gain of Zeb2 expression in pulmonary-residing melanoma cells promoted the development of macroscopic lesions. In vivo fate mapping made clear that melanoma cells undergo a conversion in state where ZEB2 expression is replaced by ZEB1 expression associated with gain of an invasive phenotype. These findings suggest that reversible switching of the ZEB2/ZEB1 ratio enhances melanoma metastatic dissemination. SIGNIFICANCE: ZEB2 function exerts opposing behaviors in melanoma by promoting proliferation and expansion and conversely inhibiting invasiveness, which could be of future clinical relevance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/14/2983/F1.large.jpg.

Peripheral Blood TCR Repertoire Profiling May Facilitate Patient Stratification for Immunotherapy against Melanoma.

Many metastatic melanoma patients experience durable responses to anti-PD1 and/or anti-CTLA4; however, a significant proportion (over 50%) do not benefit from the therapies. In this study, we sought to assess pretreatment liquid biopsies for biomarkers that may correlate with response to checkpoint blockade. We measured the combinatorial diversity evenness of the T-cell receptor (TCR) repertoire (the DE50, with low values corresponding to more clonality and lack of TCR diversity) in pretreatment peripheral blood mononuclear cells from melanoma patients treated with anti-CTLA4 (n = 42) or anti-PD1 (n = 38) using a multi-N-plex PCR assay on genomic DNA (gDNA). A receiver operating characteristic curve determined the optimal threshold for a dichotomized analysis according to objective responses as defined by RECIST1.1. Correlations between treatment outcome, clinical variables, and DE50 were assessed in multivariate regression models and confirmed with Fisher exact tests. In samples obtained prior to treatment initiation, we showed that low DE50 values were predictive of a longer progression-free survival and good responses to PD-1 blockade, but, on the other hand, predicted a poor response to CTLA4 inhibition. Multivariate logistic regression models identified DE50 as the only independent predictive factor for response to anti-CTLA4 therapy (P = 0.03) and anti-PD1 therapy (P = 0.001). Fisher exact tests confirmed the association of low DE50 with response in the anti-CTLA4 (P = 0.041) and the anti-PD1 cohort (P = 0.0016). Thus, the evaluation of basal TCR repertoire diversity in peripheral blood, using a PCR-based method, could help predict responses to anti-PD1 and anti-CTLA4 therapies.

Co-existence of BRAF and NRAS driver mutations in the same melanoma cells results in heterogeneity of targeted therapy resistance.

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.

Melanoma immunotherapy: historical precedents, recent successes and future prospects.

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.

Hypoxia contributes to melanoma heterogeneity by triggering HIF1α-dependent phenotype switching.

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.