Targeted DNA Sequencing of Cutaneous Melanoma Identifies Prognostic and Predictive Alterations
Authors: Haugh Alexandra M. , Osorio Robert C. , Francois Rony A. , Tawil Michael E. , Tsai Katy K. , Tetzlaff Michael , Daud Adil , Vasudevan Harish N. 
Published: January 2024

AbstractImportanceCutaneous melanoma (CM) can be molecularly classified into four groups:BRAFmutant,NRASmutant,NF1loss, 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.ObjectiveTo explore how molecular features and tumor mutational burden (TMB) impact outcomes in patients with cutaneous melanoma.DesignThis 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.SettingTertiary referral, comprehensive NCI cancer center from 2013 - 2023.ParticipantsA total of 254 patients with CM who had a CLIA certified targeted sequencing assay performed on their tumor tissue were includedExposureA CLIA certified targeted sequencing assay was performed as standard of care on 254 patients with CM treated at a single institution.Main OutcomeNRASmutation 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).ResultsOf 254 patients with cutaneous melanoma, 77 wereBRAFmutant (30.3%), 77 wereNRASmutant (30.3%), 47 wereNF1mutant (18.5%), 33 were TWT (13.0%) and the remaining 20 (7.9%) carried mutations in multiple driver genes (BRAF/NRAS/NF1co-mutated). The majority of this co-mutation group carried mutations inNF1(n=19 or 90%) with co-occurring mutations inBRAForNRAS,often with a weaker oncogenic variant. Consistently,NF1mutant tumors harbored numerous significantly co-altered genes compared toBRAForNRASmutant 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).NRASmutant 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 RelevanceNRASmutation 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.