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The immune response to melanoma improves the survival in untreated patients and predicts the response to immune checkpoint blockade. Here, we report genetic and environmental predictors of the immune response in a large primary cutaneous melanoma cohort. Bioinformatic analysis of 703 tumor transcriptomes was used to infer immune cell infiltration and to categorize tumors into immune subgroups, which were then investigated for association with biological pathways, clinicopathologic factors, and copy number alterations. Three subgroups, with “low”, “intermediate”, and “high” immune signals, were identified in primary tumors and replicated in metastatic tumors. Genes in the low subgroup were enriched for cell-cycle and metabolic pathways, whereas genes in the high subgroup were enriched for IFN and NF-κB signaling. We identified high MYC expression partially driven by amplification, HLA-B downregulation, and deletion of IFNγ and NF-κB pathway genes as the regulators of immune suppression. Furthermore, we showed that cigarette smoking, a globally detrimental environmental factor, modulates immunity, reducing the survival primarily in patients with a strong immune response. Together, these analyses identify a set of factors that can be easily assessed that may serve as predictors of response to immunotherapy in patients with melanoma.Significance:These findings identify novel genetic and environmental modulators of the immune response against primary cutaneous melanoma and predict their impact on patient survival.See related commentary by Anichini, p. 2457

Gene lists per immune cell type after successive filtration steps.

Supplementary Tables 1-8

Immunohistochemistry of melanoma sections from LMC for VDR expression

Supplementary methods

Legend for Supplementary tables

1α,25-Dihydroxyvitamin D3 signals via the vitamin D receptor (VDR). Higher serum vitamin D is associated with thinner primary melanoma and better outcome, although a causal mechanism has not been established. As patients with melanoma commonly avoid sun exposure, and consequent vitamin D deficiency might worsen outcomes, we interrogated 703 primary melanoma transcriptomes to understand the role of vitamin D–VDR signaling and replicated the findings in The Cancer Genome Atlas metastases. VDR expression was independently protective for melanoma-related death in both primary and metastatic disease. High tumor VDR expression was associated with upregulation of pathways mediating antitumor immunity and corresponding with higher imputed immune cell scores and histologically detected tumor-infiltrating lymphocytes. High VDR–expressing tumors had downregulation of proliferative pathways, notably Wnt/β-catenin signaling. Deleterious low VDR levels resulted from promoter methylation and gene deletion in metastases. Vitamin D deficiency (VDR-dependent manner. In vitro functional validation studies showed that elevated vitamin D–VDR signaling inhibited Wnt/β-catenin signaling genes. Murine melanoma cells overexpressing VDR produced fewer pulmonary metastases than controls in tail-vein metastasis assays. In summary, vitamin D–VDR signaling contributes to controlling pro-proliferative/immunosuppressive Wnt/β-catenin signaling in melanoma and this is associated with less metastatic disease and stronger host immune responses. This is evidence of a causal relationship between vitamin D–VDR signaling and melanoma survival, which should be explored as a therapeutic target in primary resistance to checkpoint blockade.Significance:VDR expression could potentially be used as a biomarker to stratify patients with melanoma that may respond better to immunotherapy.

Estimation of VDR expression in murine melanoma cells using Western blot and qRT-PCR

Additional information on methods used and supplementary figures and tables. Figure S1: Schematic summary of consensus clustering; Figure S2: Comparison of the newly obtained immune subgroups with two published melanoma molecular signatures; Figure S3: Whole transcriptome differences among the three immune subgroups; Figure S4: Association of GPR15 gene expression, smoking and the three immune subgroups; Table S1: Survival analysis for the immune cell scores in our data (LMC) and TCGA datasets; Table S2: Association between clinico-pathological characteristics and the three immune subgroups in the LMC; Table S3: Differences in the immune cell scores between ever and never smokers in the High Immune Subgroup; Table S4: Association between smoking and tumor histological features in the High Immune Subgroup.

List of 70 genes shared between the signature used to generate the three immune subgroups in this study and the TCGA signature as well as tables of additional results from the analysis of the 3 immune subgroups: Genes and pathways upregulated in the High and Low Immune Subgroups; List of genes significantly correlating with MYC in melanoma cell lines; Results of copy number data analysis for MYC, NF-kappa B and IFN-gamma pathways; Results of the survival analyses of environmental and clinico-pathological factors with alternative definitions of smoking variable.

Comparison of CD3 positive immune infiltrate in murine lungs

Multiple BAP1 negative melanocytic neoplasms are a hallmark of familial cancer susceptibility syndrome caused by BAP1 germline mutation. The syndrome is characterized by increased incidence of renal cell carcinoma, mesothelioma, cholangiocarcinoma, cutaneous and uveal melanoma and some other neoplasms.We report histomorphologic characteristics of six cutaneous melanocytic neoplasms with loss of BAP1 expression in two members of a family with BAP1-associated cancer susceptibility syndrome.The neoplasms were dermal melanocytic nevi characterized by a proliferation of large epithelioid (spitzoid) melanocytes, and adipocytic metaplasia. Nuclear pseudoinclusions and multinucleated melanocytes were present in most neoplasms. In two of the cases, a nodular melanoma was found associated with a dermal nevus. None of the melanomas recurred or metastasized after 6 and 3 years of follow up.We report two new cases of melanoma arising in a BAP1-deficient melanocytic nevus in the setting of familial tumor predisposition syndrome. Adipocytic metaplasia and nuclear pseudoinclusions may be additional morphologic clues to a BAP1-deficient nevus. It remains to be seen whether these features are more common in familial than sporadic lesions.