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

Bennett A Caughey; Kumiko Umemoto; Michelle F Green; Masafumi Ikeda; Melissa E Lowe; Makoto Ueno; Donna Niedzwiecki; Hiroya Taniguchi; Daniel J Walden; Yoshito Komatsu; Rachel D'Anna; Taito Esaki; Tadamichi Denda; Michael B Datto; Hideaki Bando; Tanios Bekaii-Saab; Takayuki Yoshino; John H Strickler; Yoshiaki Nakamura

doi:10.21037/jgo-23-114

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

J Gastrointest Oncol. 2023 Oct 31;14(5):2083-2096. doi: 10.21037/jgo-23-114. Epub 2023 Sep 19.

Authors

Bennett A Caughey¹, Kumiko Umemoto², Michelle F Green³, Masafumi Ikeda⁴, Melissa E Lowe⁵, Makoto Ueno⁶, Donna Niedzwiecki⁵, Hiroya Taniguchi⁷, Daniel J Walden⁸, Yoshito Komatsu⁹, Rachel D'Anna¹⁰, Taito Esaki¹¹, Tadamichi Denda¹², Michael B Datto³, Hideaki Bando¹³, Tanios Bekaii-Saab⁸, Takayuki Yoshino¹³, John H Strickler¹, Yoshiaki Nakamura¹³

Affiliations

¹ Division of Medical Oncology, Department of Medicine, Duke University Medical Center, Durham, NC, USA.
² Department of Clinical Oncology, St. Marianna University School of Medicine, Kawasaki, Japan.
³ Department of Pathology, Duke University Medical Center, Durham, NC, USA.
⁴ Department of Hepatobiliary and Pancreatic Oncology, National Cancer Center Hospital East, Kashiwa, Japan.
⁵ Duke Cancer Institute-Biostatistics Shared Resource, Durham, NC, USA.
⁶ Department of Gastroenterology, Hepatobiliary and Pancreatic Medical Oncology, Kanagawa Cancer Center, Yokohama, Japan.
⁷ Department of Clinical Oncology, Aichi Cancer Center Hospital, Nagoya, Japan.
⁸ Division of Hematology and Oncology, Mayo Clinic, Phoenix, Arizona, USA.
⁹ Department of Cancer Center, Hokkaido University Hospital, Sapporo, Japan.
¹⁰ Duke Cancer Institute-Biostatistics Shared Resource and Department of Biostatistics and Bioinformatics, Duke University, Durham, NC, USA.
¹¹ Department of Gastrointestinal and Medical Oncology, National Hospital Organization Kyushu Cancer Center, Fukuoka, Japan.
¹² Division of Gastroenterology, Chiba Cancer Center, Chiba, Japan.
¹³ Department of Gastroenterology and Gastrointestinal Oncology, National Cancer Center Hospital East, Kashiwa, Japan.

Abstract

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.

Keywords: Cell-free DNA (cfDNA); colorectal cancer (CRC); pancreatic cancer.