AUTHOR=Diefenbach Russell J. , Lee Jenny H. , Stewart Ashleigh , Menzies Alexander M. , Carlino Matteo S. , Saw Robyn P. M. , Stretch Jonathan R. , Long Georgina V. , Scolyer Richard A. , Rizos Helen 

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

JOURNAL=Frontiers in Oncology

VOLUME=12

YEAR=2022

URL=https://www.frontiersin.org/journals/oncology/articles/10.3389/fonc.2022.820510

DOI=10.3389/fonc.2022.820510

ISSN=2234-943X

ABSTRACT=<p>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 <italic>TERT</italic> 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 <italic>TERT</italic> 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 <italic>BRAF</italic>/<italic>NRAS</italic>/<italic>TERT</italic> promoter mutations, was 14/21 (67%) patient samples which included a <italic>TERT</italic> C250T mutation in one <italic>BRAF</italic> and <italic>NRAS</italic> mutation negative sample. A <italic>BRAF</italic> or <italic>NRAS</italic> mutation was detected in the ctDNA of 13/21 (62%) patients while <italic>TERT</italic> promoter mutations were detected in 10/21 (48%) patients. Co-occurrence of <italic>TERT</italic> promoter mutations with <italic>BRAF</italic> or <italic>NRAS</italic> 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 <italic>BRAF</italic>/<italic>NRAS</italic> mutation positive and 4 <italic>BRAF/NRAS</italic> 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 <italic>BRAF</italic>, <italic>NRAS</italic> and <italic>TERT</italic> 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 <italic>NRAS/BRAF/TERT</italic> promoter mutation negative background. To increase the detection rate to &gt;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 <italic>TERT</italic> promoter mutations in ctDNA from cutaneous melanoma patients using a targeted NGS panel.</p>