Detection of
melanoma-associated mutations using
circulating tumor DNA (ctDNA) from plasma is a potential alternative to using genomic
DNA from invasive tissue biopsies. In this study, we developed a custom
melanoma next-generation sequencing (NGS) panel which includes 123 amplicons in 30 genes covering driver and targetable mutations and alterations associated with treatment resistance. Analysis of a cohort of 74 stage III and IV treatment-naïve
melanoma patients revealed that sensitivity of ctDNA detection was influenced by the amount of circulating-free
DNA (
cfDNA) input and stage of
melanoma. At the recommended
cfDNA input quantity of 20 ng (available in 28/74 patients), at least one
cancer-associated mutation was detected in the ctDNA of 84% of stage IV patients and 47% of stage III patients with a limit of detection for mutant allele frequency (MAF) of 0.2%. This custom
melanoma panel showed significant correlation with droplet digital PCR (ddPCR) and provided a more comprehensive
melanoma mutation profile. Our custom panel could be further optimized by replacing amplicons spanning the TERT promoter, which did not perform well due to the high GC content. To increase the detection rate to 90% of stage IV
melanoma and decrease the sensitivity to 0.1% MAF, we recommend increasing the volume of plasma to 8 mL to achieve minimal recommended
cfDNA input and the refinement of poorly performing amplicons. Our panel can also be expanded to include new targetable and treatment resistance mutations to improve the tracking of treatment response and resistance in
melanoma patients treated with systemic
drug therapies.