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

Background: Evaluation for activating mutations in , , and in colorectal cancer (CRC) and in 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 and 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 , , or 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- dominant gene MAF of 0.34% provided maximum discrimination for predicting SNV detection. Sensitivity for 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 SNVs, 18 assays detected 34 other oncogenic variants, of which 80.6% were not also detected on tissue.

Conclusions: Non- dominant oncogenic mutation MAF ≥1% on cfDNA NGS predicts high sensitivity to detect oncogenic SNVs in CRC and PDAC. MAF £0.34% indicates an assay may not reliably detect SNVs, despite detection on tissue testing. Most variants from assays that did not detect 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.