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Background Primary melanoma ulceration is an unfavourable prognostic factor included in current staging systems. Yet, the immunological and molecular alterations responsible for this adverse outcome have not been fully elucidated. Objectives We aimed to identify immunological differences between ulcerated and non-ulcerated primary melanomas concerning both innate and adaptive immunity and to correlate these with clinical outcome. Methods Formalin-fixed paraffin-embedded primary melanomas from 112 patients (pts) were analysed by immunohistochemistry. The expression of various markers identifying tumour-infiltrating lymphocytes, macrophages and dendritic cells was evaluated semi-quantitatively by three independent investigators. Tumour cell expression of programmed death-ligand 1 (PD-L1), transporter of antigen processing 1 and the MxA protein was also analysed. Results Recurrence occurred in 21/56 pts (37.5%) with ulcerated vs. 14/56 pts (25.0%) with non-ulcerated tumours (P = 0.15). Tumour ulceration was associated with more frequent development of brain metastasis (17.6 vs. 3.6% of pts, P = 0.015). Immunohistochemistry showed an association of ulceration with the presence of intratumoural CD68+ macrophages (P = 0.028) as well as with increased numbers of intratumoural CD11c+ dendritic cells (P = 0.014) and CD163+ macrophages (P = 0.001). PD-L1 positivity (expression in >1% of tumour cells) was more frequent in ulcerated than non-ulcerated tumours [40 (72.7%) vs. 25 (44.6%), P = 0.003]. A positive correlation between intratumoural CD11c+ (Spearman's correlation coefficient ρ: 0.42) and CD163+ (ρ: 0.31) cell count and frequency of tumour cell PD-L1 expression was detected. Conclusions Our results confirm the adverse clinical outcome associated with primary melanoma ulceration, particularly concerning the risk of recurrence and subsequent development of brain metastases. The observed immunological differences suggest a conceivable role of increased intratumoural macrophage and dendritic cell counts associated with enhanced tumour cell PD-L1 expression potentially contributing to the immunosuppressive, growth-promoting microenvironment of ulcerated primary melanomas.

Targeting cyclin-dependent kinases 4/6 (CDK4/6) represents a therapeutic option in combination with BRAF inhibitor and/or MEK inhibitor (MEKi) in melanoma; however, continuous dosing elicits toxicities in patients. Using quantitative and temporal in vivo reporting, we show that continuous MEKi with intermittent CDK4/6 inhibitor (CDK4/6i) led to more complete tumor responses versus other combination schedules. Nevertheless, some tumors acquired resistance that was associated with enhanced phosphorylation of ribosomal S6 protein. These data were supported by phospho-S6 staining of melanoma biopsies from patients treated with CDK4/6i plus targeted inhibitors. Enhanced phospho-S6 in resistant tumors provided a therapeutic window for the mTORC1/2 inhibitor AZD2014. Mechanistically, upregulation or mutation of NRAS was associated with resistance in in vivo models and patient samples, respectively, and mutant NRAS was sufficient to enhance resistance. This study utilizes an in vivo reporter model to optimize schedules and supports targeting mTORC1/2 to overcome MEKi plus CDK4/6i resistance. Significance: Mutant BRAF and NRAS melanomas acquire resistance to combined MEK and CDK4/6 inhibition via upregulation of mTOR pathway signaling. This resistance mechanism provides the preclinical basis to utilize mTORC1/2 inhibitors to improve MEKi plus CDK4/6i drug regimens. Cancer Discov; 8(5); 568–81. ©2018 AACR. See related commentary by Sullivan, p. 532. See related article by Romano et al., p. 556. This article is highlighted in the In This Issue feature, p. 517

A deepened understanding of the cellular and molecular processes in the tumor microenvironment is necessary for the development of precision immunotherapy (IT). We simultaneously investigated CD3, PDL1, and IDO by immunohistochemistry in paired biopsies from various organs of 43 metastatic melanoma patients treated with IT and targeted therapy (TT). Intraindividual biopsies taken after a period of weeks to months demonstrate discordant results in 30% of the cases. Overlap of IDO and PDL1 increased after therapy. IT only marginally impacted PDL1 expression over time in contrast to TT. Standardized repeated assessments of multiple immune markers in repeated biopsies will generate detailed insights in melanoma's immune evolution and adaption during therapies and might be used to support treatment decisions.

There is good evidence that an immune response to melanoma predicts better survival. We report a bioinformatic study designed to identify immune subgroups of primary melanomas in order to better understand the biology of immune cell/tumor interaction, and to identify candidate predictive biomarkers for immunotherapy. We applied an approach described by Angelova et al. to the inference of tumor immune cell infiltrates using differential expression of signature genes, in 703 primary melanoma transcriptomes from the Leeds Melanoma Cohort (LMC). Consensus clustering of immune cell scores identified 3 subgroups in LMC and replication was sought in The Cancer Genome Atlas (TCGA) data set, (primarily metastatic samples). We then used the whole transcriptomic data to explore associated biological pathways: a protein-protein interaction network analysis of genes was performed in Cytoscape plugin Reactome FIVIZ. Nodal genes were identified and the cellular origin of the gene expression signature further explored using immunohistochemistry (IHC) and in patient-derived melanoma cell culture transcriptomes. Clinicopathological and environmental factors, and immune cell scores associated with survival within immune subgroups, were then analysed using a multivariate Cox proportional hazard model. We identified three subgroups with transcriptomic patterns indicative of: low, intermediate and high immune cell infiltration. The groups were concordant with histological evidence of tumor infiltrating lymphocytes. Genes enriched in the low, compared with the high immune group, were in cell cycle, metabolic and Beta-catenin pathways. MYC was the nodal (the most influential) gene of the protein-protein interaction network in this group. In the high immune group, genes were enriched in immune pathways, including antigen processing and presentation, with the nodal gene - NFKB1. Based on the literature and our results, we hypothesised that MYC downregulates HLA-class I, hence we tested MYC correlations in the melanoma cell line transcriptomes (in which immune cells were absent). Indeed, MYC was negatively correlated with many HLA class I genes, with HLA-B having the strongest result: R=-0.57. IHC of primary tumors showed tumor nuclear expression of MYC. Reported smoking independently predicted a worse outcome for patients classified as the high immune group (HR=3.52, P=0.008). The transcriptomic analysis showed that smoking correlated with an increased Th2 cell score (P=0.03) and reduced NK56 bright score (P=0.01) in that group. We report evidence that MYC plays an important role in immune evasion in primary melanoma. The transcriptomic data support in vitro findings that MYC downregulates HLA class I expression. Furthermore, we present important results that smoking status modifies immune response in melanoma and is an independent risk factor for melanoma death. We hypothesise that smoking may impact on immunotherapy efficacy. Citation Format: Joanna Pozniak, Jeremie Nsengimana, Jon Laye, Sally O'Shea, Sabrina Schindler, Mitch Levesque, Tim Bishop, Julia Newton-Bishop. MYC expression and smoking as drivers of failure of immune response to melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5204.

With incidence of melanoma growing worldwide and new therapies prolonging the survival of patients with advanced disease, complex medical care is needed.Best care of complicated melanoma cases is achieved in specialized referral centers. Aims to provide optimized melanoma therapy, best patient-reported treatment outcome, and successful clinical and translational research, necessitate a dedicated interdisciplinary team.We report on critical aspects of the interaction between patients, medical care givers, clinical trial and biobanking teams, and emphasize the importance of interdisciplinary tumor boards. Specialized skin cancer nurses and local patient advocacy groups should be involved in patient care and could be the binding link between the patients and the treatment team.

In the original version of [this article][1] ([1][2]), an error in Fig. 6C was introduced by the compositor during the production of the paper. Specifically, the Raptor Western blot was improperly shifted. The figure has been corrected in the latest online HTML and PDF versions of the article. The

Tumor initiation and metastasis formation in many cancers have been associated with emergence of a gene expression program normally active in embryonic or organ-specific stem cells. In particular, the stem cell transcription factor Sox2 is not only expressed in a variety of tumors, but is also required for their formation. Melanoma, the most aggressive skin tumor, derives from melanocytes that during development originate from neural crest stem cells. While neural crest stem cells do not express Sox2, expression of this transcription factor has been reported in melanoma. However, the role of Sox2 in melanoma is controversial. To study the requirement of Sox2 for melanoma formation, we therefore performed CRISPR-Cas9-mediated gene inactivation in human melanoma cells. In addition, we conditionally inactivated Sox2 in a genetically engineered mouse model, in which melanoma spontaneously develops in the context of an intact stroma and immune system. Surprisingly, in both models, loss of Sox2 did neither affect melanoma initiation, nor growth, nor metastasis formation. The lack of a tumorigenic role of Sox2 in melanoma might reflect a distinct stem cell program active in neural crest stem cells and during melanoma formation.Oncogene advance online publication, 3 April 2017; doi:10.1038/onc.2017.55.

SummaryBackground There is a medical need for new drugs in patients with BRAF wild-type metastatic melanoma. Pazopanib is a multitarget tyrosine kinase inhibitor with antitumour and antiangiogenic activity. Objectives The primary aim was to investigate the metabolic response to pazopanib monotherapy and pazopanib plus paclitaxel in patients with BRAF wild-type melanoma. Secondary end points were the early cytokine and chemokine profiles and histological findings. Methods Pazopanib (400 mg twice daily) was administered orally from days 1 to 10 and from days 14 to 70. An intravenous infusion with paclitaxel (150 mg m−2 body surface) was administered on days 14, 35 and 56. Metabolic response evaluation was performed before treatment, after treatment with pazopanib (day 10) and after treatment with pazopanib and paclitaxel (day 70). Skin biopsy of metastatic tissue for chemokine and cytokine expression analysis and histology and immunohistochemistry (CD68, CD163) evaluation, and blood samples were taken at the same time points. Results Two patients failed screening and 17 were dosed. Of 67 adverse events, nine (13%) were grade 3 or 4. Five of 14 evaluable patients had a partial metabolic response at day 10 under pazopanib monotherapy. The response rate at day 70 under combined pazopanib–paclitaxel treatment was 0%. Immunohistochemistry revealed an increase of M2-like macrophages in nonresponders compared with responders. We observed a significant upregulation of five cytokines (CXCL1, CXCL2, CXCL13, CCL22 and SPP1) in responding vs. nonresponding lesions. Overall, the median progression-free survival was 70 days (range 5–331), which did not differ significantly between responders (148 days) and nonresponders (70 days, P = 0·17). Conclusions In this patient population pazopanib efficacy was limited. Response is associated with low M2-like macrophage density and increased expression of several chemokines.