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Activating NRAS mutations are found in 15% to 20% of melanomas. Immune therapies have become a mainstay in advanced melanoma treatment. We sought to evaluate whether tumor genotype (e.g., NRAS mutations) correlates with benefit from immune therapy in melanoma. We identified 229 patients with melanoma treated with immune therapies [IL2, ipilimumab, or anti-programmed cell death-1/ligand-1 (PD-1/PD-L1)] at three centers and compared clinical outcomes following immune therapy for patients with or without NRAS mutations. Of the 229 patients with melanoma, 60 had NRAS mutation, 53 had BRAF mutation, and 116 had NRAS/BRAF wild type. The NRAS-mutant cohort had superior or a trend to superior outcomes compared with the other cohorts in terms of response to first-line immune therapy (28% vs. 16%, P = 0.04), response to any line of immune therapy (32% vs. 20%, P = 0.07), clinical benefit (response + stable disease lasting ≥24 weeks; 50% vs. 31%, P < 0.01), and progression-free survival (median, 4.1 vs. 2.9 months, P = 0.09). Benefit from anti–PD-1/PD-L1 was particularly marked in the NRAS cohort (clinical benefit rate 73% vs. 35%). In an independent group of patient samples, NRAS-mutant melanoma had higher PD-L1 expression (although not statistically significant) compared with other genotypes (8/12 vs. 9/20 samples with ≥1% expression; 6/12 vs. 6/20 samples with ≥5% expression), suggesting a potential mechanism for the clinical results. This retrospective study suggests that NRAS mutations in advanced melanoma correlate with increased benefit from immune-based therapies compared with other genetic subtypes. If confirmed by prospective studies, this may be explained in part by high rates of PD-L1 expression. Cancer Immunol Res; 3(3); 288–95. ©2015 AACR.

Purpose: Knowledge of tumor mutation status is becoming increasingly important for the treatment of cancer, as mutationspecific 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. Methods: 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. Results: 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. Conclusion: 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.