Mutation-enrichment next-generation sequencing for quantitative detection of KRAS mutations in urine cell-free DNA from patients with advanced cancers

Purpose: Tumor-derived cell-free DNA (cfDNA) from urine of patients with cancer offers noninvasive biological material for detection of cancer-related molecular abnormalities such as mutations in Exon 2 of KRAS. Experimental Design: A quantitative, mutation-enrichment next-generation sequencing test for detecting KRAS^G12/G13 mutations in urine cfDNA was developed, and results were compared with clinical testing of archival tumor tissue and plasma cfDNA from patients with advanced cancer. Results: With 90 to 110 mL of urine, the KRAS^G12/G13 cfDNA test had an analytical sensitivity of 0.002% to 0.006% mutant copies in wild-type background. In 71 patients, the concordance between urine cfDNA and tumor was 73% (sensitivity, 63%; specificity, 96%) for all patients and 89% (sensitivity, 80%; specificity, 100%) for patients with urine samples of 90 to 110 mL. Patients had significantly fewer KRAS^G12/G13 copies in urine cfDNA during systemic therapy than at baseline or disease progression (P ¼ 0.002). Compared with no changes or increases in urine cfDNA KRAS^G12/G13 copies during therapy, decreases in these measures were associated with longer median time to treatment failure (P ¼ 0.03). Conclusions: A quantitative, mutation-enrichment next-generation sequencing test for detecting KRAS^G12/G13 mutations in urine cfDNA had good concordance with testing of archival tumor tissue. Changes in mutated urine cfDNA were associated with time to treatment failure.