Abstract
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.
Highlights
The analysis of circulating tumor DNA is progressively being integrated into routine clinical care to monitor cancer progression, response to therapy, emergence of resistance and to direct therapy [1,2,3,4,5,6]
Seventeen patients were excluded from analysis as they were subsequently confirmed to have a non-melanoma malignancy (n = 3) or the liquid biopsy sample was taken after surgical resection (n = 14)
We developed a custom melanoma next-generation sequencing (NGS) panel
Summary
The analysis of circulating tumor DNA (ctDNA) is progressively being integrated into routine clinical care to monitor cancer progression, response to therapy, emergence of resistance and to direct therapy [1,2,3,4,5,6]. The use of customized gene mutation panels in NGS of ctDNA can produce significantly lower levels of mutation detection [22], and these panels typically monitor common melanoma driver mutations in BRAF, NRAS and KIT, along with mutations in tumor suppressor genes, such as TP53. Such gene panels do not include many established mutations associated with treatment resistance and do not allow for the discovery and tracking of novel resistance mutations [22]. The UltraSEEK panel is based on mutation detection by mass spectrometry, and, it showed similar sensitivity to ddPCR in Stage IV melanoma known to carry mutations in plasma, the concordance in the mutations detected was only 88% between these two platforms [24]
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