Anchored multiplex PCR custom melanoma next generation sequencing panel for analysis of circulating tumor DNA

Abstract

Detection of melanoma mutations using circulating tumor DNA (ctDNA) from plasma is a potential alternative to using genomic DNA from invasive tissue biopsies. In this study, we have developed a custom melanoma next-generation sequencing (NGS) panel which encompasses the top 15 gene mutations in melanoma including the TERT promoter. To date, mutations in the GC-rich TERT promoter region, which is commonly mutated in melanoma, have been technically difficult to detect using NGS panels. We analysed 21 stage III and IV melanoma patients who were treatment-na��ve or on various therapies. A BRAF or NRAS mutation was detected in the ctDNA of 62% of patients while TERT promoter mutations (C250T, C228T or CC242TT) were detected in 48% of patients. Cooccurrence of TERT promoter mutations with BRAF or NRAS mutations was found in 9/10 patients. The detection of a TERT C250T mutation in one BRAF and NRAS mutation negative sample increased the detection rate of the custom panel to 67% (based on BRAF/NRAS/TERTpromoter). The custom ctDNA panel showed a concordance of 76% with tissue based-detection and included 12 BRAF/NRAS mutation positive and 4 BRAF/NRAS mutation negative patients. The ctDNA mutation detection rate for stage IV was 75% and for stage III was 20%. Based on BRAF, NRAS and TERT promoter mutations, the custom melanoma panel displayed a limit of detection of ~0.2% mutant allele frequency (MAF) and showed significant correlation with droplet digital PCR. For one patient, we detected a novel MAP2K1 H119Y mutation in an NRAS/BRAF/ TERT promoter mutation negative background at a MAF of 0.18%. To increase the detection rate to >90% of stage IV melanoma we plan to expand our custom panel to 50 genes. This study represents one of the first to successfully detect TERT promoter mutations in ctDNA from melanoma patients using a targeted NGS panel.