Identification of an optimal mutant allele frequency to detect activating KRAS , NRAS , and BRAF mutations in a commercial cell-free DNA next-generation sequencing assay in colorectal and pancreatic adenocarcinomas.

Background: Evaluation for activating mutations in KRAS , NRAS , and BRAF in colorectal cancer (CRC) and in KRAS in pancreatic ductal adenocarcinoma (PDAC) is essential for clinical care. Plasma cell-free DNA (cfDNA) next-generation sequencing (NGS) allows convenient assessment of a tumor's molecular profile, however low tumor DNA shedding limits sensitivity. We investigated mutant allele frequency (MAF) of other oncogenic dominant genes to identify a threshold for accurate detection of KRAS, NRAS, and BRAF (RAS/RAF) mutations in cfDNA.
Methods: Molecular and clinical data were obtained from the Duke Molecular Registry of Tumors and the SCRUM-Japan GOZILA study. Patients with CRC or PDAC and a KRAS , NRAS , or BRAF activating single nucleotide variant (SNV) present on tissue NGS and with available cfDNA assays were included. Recursive partitioning and Wilcoxon-rank statistics methods identified potential cut-points for discriminative MAF values.
Results: One hundred and thirty-five CRC and 30 PDAC cases with 198 total cfDNA assays met criteria. Greatest non- RAS/RAF dominant gene MAF of 0.34% provided maximum discrimination for predicting RAS/RAF SNV detection. Sensitivity for RAS/RAF SNVs increased with dominant gene MAF, with MAF ≥1% predicting sensitivity >98%, MAF between 0.34 and 1% predicting sensitivity of 84.0%, and MAF £0.34% predicting sensitivity of 50%. For 43 cfDNA assays that did not detect RAS/RAF SNVs, 18 assays detected 34 other oncogenic variants, of which 80.6% were not also detected on tissue.
Conclusions: Non- RAS/RAF dominant oncogenic mutation MAF ≥1% on cfDNA NGS predicts high sensitivity to detect RAS/RAF oncogenic SNVs in CRC and PDAC. MAF £0.34% indicates an assay may not reliably detect RAS/RAF SNVs, despite detection on tissue testing. Most variants from assays that did not detect RAS/RAF had MAF <1% and were not detected on tissue, suggesting potential confounding. These data suggest a practical approach to determining cfDNA assay adequacy, with implications for guiding clinical decisions in CRC and PDAC.
Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-23-114/coif). KU reports honoraria from Chugai Pharmaceutical, Taiho Pharmaceutical and Yakult Honsha. MG reports royalties from SciMed Solutions as part of a software licensing agreement. MI reports grants/research funding from Eisai, Merck biopharma, Eli Lilly Japan, Yakult, Ono, ASLAN, J-Pharma, AstraZeneca, Pfizer, Merus N.V., Nihon Servier, Delta-Fly Pharma, Chiome Bioscience, Bristol-Myers Squibb, Chugai Pharmaceutical, Novartis, Bayer, Takeda, MSD, Syneos Health, as well as honoraria from Eisai, MSD, Eli Lilly Japan, Yakult, Teijin Pharma, Astellas, Sumitomo Dainippon, Otsuka, Nihon Servier, Taiho, Chugai Pharmaceutical, Bristol-Myers Squibb, Novartis, Bayer, Takeda, EA Pharma, AstraZeneca, AbbVie, Abbott Japan, Fujifilm Toyama Chemical, and Incyte Biosciences Japan. MU reports grants/research fundings from Taiho Pharmaceutical, AstraZeneca, Merck, MSD, Astellas Pharma, Eisai, Ono Pharmaceutical, Incyte, Chugai Pharmaceutical, DFP, Daiichi Sankyo, Novartis, Boehringer Ingelheim, and J-pharma, and also honoraria from Taiho Pharmaceutical, AstraZeneca, Yakult Honsha, MSD, Nihon Servier, Ono Pharmaceutical, Uncyte, Chugai Pharmaceutical, Boehringer Ingelheim, and J-Pharma. HT reports grants/research funding from Daiichi Sankyo and Takeda, and also honoraria from Guardant Health AMEA, Chugai Pharmaceutical, Ono, Eli Lilly, Takeda, and Merck. YK reports scholarly/research support or grant funding from Ono, Chugai Pharmaceutical, Taiho, Shionogi, Nippon Zoki, Asahi Kasei, Nippon Kayaku, Daiichi Sankyo, IQVIA Services Japan, MSD, Astellas Pharma, Incyte, Eisai, National Cancer Center Japan, Syneos Health, ShiftZero, Parexel International, Japan Clinical Cancer Research Organization, EPS, Sysemx Corporation, and the Public Health Research Foundation; and also honoraria from Ono, Taiho, Astellas Pharma, EA Pharma, Daiichi Sankyo, Nippon Kayaku, Pfizer, Nippon Zoki, Sanofi, Nipro, Asahi, Chugai Pharmaceutical, MSD, Zeria, Eli Lilly, Bayer, Yakult, Sumitomo Dainippon Pharma, Incyte, and Merck. TE reports research grants and honoraria from MSD, Daiichi Sankyo, and, Chugai, grants from Pfizer, Astellas, Quintiles, Syneos Health, Amgen, Ono, Novartis, Astellas, Amgen Biopharma, Asahi Kasei Pharma, and IQVIA, and honoraria from Taiho, Eli Lilly, and Bristol Myers Squib, outside the submitted work. TD reports research funds from MSD, Amgen and Ono Pharmaceutical, and honoraria from Sysmex, Ono Pharmaceutical, SAWAI Pharmaceutical Co and Daiichi Sankyo, outside the submitted work. HB reports research funding from Ono Pharmaceutical and honoraria from Ono Pharmaceutical, Taiho, and Eli Lilly. TBS reports research funding/grants from Agio, Arys, Arcua, Atreca, Boston Biomedical, Bayer, Eisai, Celgene, Lilly, Ipsen, Clovis, Seattle Genetics, Genentech, Novartic, Mirati, Merus, Abgenomics, Incyte, Pfizer, and BMS; consulting fees from Ipsen, Arcus, Pfizer, Seattle Genetics, Bayer, Genentech, Incyte, Eisai, Merus, Merck, Stemline, Abbvie, Boehringer Ingelheim, Janssen, Daiichi Sankyo, Natera, TreosBio, Celularity, Caladrius Biosciences, Exact Science, Sobi, Beigene, Kapaph, AstraZeneca, Deciphera, Zai Labs, Exelixis, MJH Life Sciences, Aptitude Health, Illumina, Foundation Medicine, and Sanofi; patents currently licensed to Imugene and Recursionl; and participation in DSMB or Scientific Advistory Boards for The Valley Hospital, Fibrogen, Suzhou Kintor, AstraZeneca, Exelixis, Merck, Eisai, PanCan, 1GLOBE, Imugene, Immuneering, Xilis, Replimune, Artiva, and Sun Biopharma. TY reports research support/grants from Amgen, Chugai Pharmaceutical, Daiichi Sankyo, Eisai, FALCO, Genomedia, Molecular Health GmbH, MSD, Boehringer Ingelheim, Ono, Pfizer, Roche Diagnostics, Sanofi, Sysmex Corporation, and Taiho as well as consulting fees from Sumitomo Corporation and honoraria from Bayer, Chugai Pharmaceutical, Merck, MSD, Ono, and Takeda. JS reports grant/research support from Abbvie, Amgen, AStar D3, Bayer, Beigene, Curegenix, Daiichi-Sankyo, Eli Lilly, Erasca, Gossamer Bio, Leap Therapeutics, Nektar, Roche/Genentech, Seagen, and Silverback Therapeutics; and consulting fees from or serving in an advisory role to Abbvie, Amgen, AstraZeneca, Bayer, Beigene, Daiichi-Sankyo, Eli Lilly, G3 Therapeutics, GSK, Natera, Pfizer, Pionyr Immunotherapeutics, Roche/Genentech, Seagen, Silverback Therapeutics, Takeda, and Viatris. YN reports grants and honoraria from Chugai, grants from Taiho, Guardant Health, Genomedia, Daiichi-Sankyo, Seagen, and Roche Diagnostics, and honoraria from Guardant Health AMEA and Merck. The other authors have no conflicts of interest to declare.
(2023 Journal of Gastrointestinal Oncology. All rights reserved.)