Anchored Multiplex PCR Custom Melanoma Next Generation Sequencing Panel for Analysis of Circulating Tumor DNA

Detection of melanoma mutations using circulating tumor DNA (ctDNA) is a potential alternative to using genomic DNA from invasive tissue biopsies. To date, mutations in the GC-rich <i>TERT</i> promoter region, which is commonly mutated in melanoma, have been technically difficult to detect in ctDNA using next-generation sequencing (NGS) panels. In this study, we developed a custom melanoma NGS panel for detection of ctDNA, which encompasses the top 15 gene mutations in melanoma including the <i>TERT</i> promoter. We analyzed 21 stage III and IV melanoma patient samples who were treatment-naïve or on therapy. The overall detection rate of the custom panel, based on <i>BRAF</i>/<i>NRAS</i>/<i>TERT</i> promoter mutations, was 14/21 (67%) patient samples which included a <i>TERT</i> C250T mutation in one <i>BRAF</i> and <i>NRAS</i> mutation negative sample. A <i>BRAF</i> or <i>NRAS</i> mutation was detected in the ctDNA of 13/21 (62%) patients while <i>TERT</i> promoter mutations were detected in 10/21 (48%) patients. Co-occurrence of <i>TERT</i> promoter mutations with <i>BRAF</i> or <i>NRAS</i> mutations was found in 9/10 (90%) patients. The custom ctDNA panel showed a concordance of 16/21 (76%) with tissue based-detection and included 12 <i>BRAF</i>/<i>NRAS</i> mutation positive and 4 <i>BRAF/NRAS</i> mutation negative patients. The ctDNA mutation detection rate for stage IV was 12/16 (75%) and for stage III was 1/5 (20%). Based on <i>BRAF</i>, <i>NRAS</i> and <i>TERT</i> promoter mutations, the custom melanoma panel displayed a limit of detection of ~0.2% mutant allele frequency and showed significant correlation with droplet digital PCR. For one patient, a novel MAP2K1 H119Y mutation was detected in an <i>NRAS/BRAF/TERT</i> promoter mutation negative background. To increase the detection rate to >90% for stage IV melanoma patients, we plan to expand our custom panel to 50 genes. This study represents one of the first to successfully detect <i>TERT</i> promoter mutations in ctDNA from cutaneous melanoma patients using a targeted NGS panel.