Profiling KRAS mutations in whole blood by error-corrected maximum depth sequencing

Clinical use of circulating tumor DNA (ctDNA) sequencing is rapidly increasing, but performance is limited by the sensitivity of next generation sequencing and relies on specialized sample collection. Here we describe sequencing the most frequently mutated oncogene KRAS by adapting error-corrected Maximum Depth Sequencing (K-MDS) for whole blood. K-MDS detected oncogenic KRAS mutations of cancer cells spiked into healthy human blood at a dilution as low as 0.001% with 100% specificity. In a prospectively collected cohort of 190 advanced cancer patients with solid tumors having concurrent commercial ctDNA testing or tumor sequencing, K-MDS detected commercially-identified oncogenic KRAS mutations in 88% of cases. Assay specificity was increased by analyzing both the forward and reverse strands and assigning mutation-specific detection thresholds. Sequencing both strands by K-MDS revealed that the assay differed from the commercial test in two small subsets of the primary cohort. First, multiple oncogenic KRAS mutations were uniquely detected by K-MDS in 7 of 9 patients having prior treatment with EGFR or KRAS inhibitors. Second, in 12 patients with bone metastases, K-MDS detected KRAS mutations more often (10 versus 5) than the commercial ctDNA assay. The sensitivity of the assay suggests potential utility in settings of potentially low ctDNA burden. To this end, when applied to preoperative blood samples collected from a pilot cohort of 18 pancreas adenocarcinoma patients undergoing curative intent resection, K-MDS stratified these patients based on their risk of metastatic disease recurrence. This first-in-human experience with K-MDS suggests the assay has the potential to complement current commercial ctDNA assays for targeted detection of oncogenic mutations in patient whole blood.