Targeted DNA Sequencing of Cutaneous Melanoma Identifies Prognostic and Predictive Alterations

Importance Cutaneous melanoma (CM) can be molecularly classified into four groups: BRAF mutant, NRAS mutant, NF1 loss, and triple wild type (TWT) tumors lacking any of these three alterations. In the era of immune checkpoint inhibition (ICI) and targeted molecular therapy, the clinical significance of these groups remains unclear. Here, we integrate targeted DNA sequencing with comprehensive clinical follow-up in CM patients. Objective To explore how molecular features and tumor mutational burden (TMB) impact outcomes in patients with cutaneous melanoma. Design This was a retrospective cohort study that assessed clinical and molecular features from patients with localized or metastatic CM who underwent targeted next-generation sequencing as part of routine clinical care. Setting Tertiary referral, comprehensive NCI cancer center from 2013 - 2023. Participants A total of 254 patients with CM who had a CLIA certified targeted sequencing assay performed on their tumor tissue were included Exposure A CLIA certified targeted sequencing assay was performed as standard of care on 254 patients with CM treated at a single institution. Main Outcome NRAS mutation correlated with significantly worse overall survival compared to other TCGA driver groups. Elevated TMB correlated with improved progression-free survival on combination checkpoint inhibition (anti-PD1 plus anti-CTLA4). Results Of 254 patients with cutaneous melanoma, 77 were BRAF mutant (30.3%), 77 were NRAS mutant (30.3%), 47 were NF1 mutant (18.5%), 33 were TWT (13.0%) and the remaining 20 (7.9%) carried mutations in multiple driver genes (BRAF/NRAS/NF1 co-mutated). The majority of this co-mutation group carried mutations in NF1 (n=19 or 90%) with co-occurring mutations in BRAF or NRAS, often with a weaker oncogenic variant. Consistently, NF1 mutant tumors harbored numerous significantly co-altered genes compared to BRAF or NRAS mutant tumors. The majority of TWT tumors (n=29, 87.9%) harbored a pathogenic mutation within a known Ras/MAPK signaling pathway component. Of the 154 cases with available TMB data, the median TMB was 20 (range 0.7 – 266 mutations/Mb). A total of 14 cases (9.1%) were classified as TMB low (<5 mutations/Mb), 64 of 154 (41.6%) were TMB intermediate (>5 and <20 mutations/Mb), 40 of 154 (26.0%) were TMB high (>20 and <50 mutations/Mb) and 36 of 154 (23.4%) were classified as TMB very high (>50 mutations/Mb). NRAS mutant melanoma demonstrated significantly decreased overall survival on multivariable analysis (HR for death 2.95, 95% CI 1.13 – 7.69, p = 0.027, log rank test) compared with other TCGA molecular subgroups. Other factors correlated with decreased overall survival included age and ECOG score. Of the 116 patients in our cohort with available treatment data, 36 received combination dual ICI with anti-CTLA4 and anti-PD1 inhibition as first line therapy. Elevated TMB was associated with significantly longer progression-free survival following dual agent ICI (HR 0.26, 95% CI 0.07 – 0.90, p =0.033, log rank test). Conclusions and Relevance NRAS mutation in CMs correlated with significantly worse overall survival. Elevated TMB was associated with increased progression-free survival for patients treated with combination dual ICI, supporting the potential utility of TMB as a predictive biomarker for ICI response in melanoma.