A novel KRAS Exon 2 drop-off Digital PCR Assay for Mutation Detection in Cell- free DNA of Cancer Patients

Background: KRAS exon 2 mutations are highly prevalent in human malignancies, making them attractive targets for detection and monitoring in cell-free DNA (cfDNA) of cancer patients. Drop-off assays designed for digital polymerase chain reaction (ddPCR drop-off) span entire mutational hotspots and detect any mutated allele within the covered region, overcoming a major limitation of mutation-specific ddPCR assays. We therefore set out to develop a novel KRAS codon 12/13 ddPCR drop-off assay for the robust, highly sensitive and specific detection of KRAS exon 2 hotspot mutations in cfDNA. Methods: We designed, optimized and extensively validated a KRAS codon 12/13 ddPCR drop-off ssay. We compared assay performance to a commercially available KRAS multiplex assay. For clinical validation, we analyzed plasma samples collected from patients with KRAS- mutated gastrointestinal malignancies. Results: Limit of detection of the newly established ddPCR drop-off assay was 0.57 copies/µL, limit of blank was 0.13 copies/µ. The inter-assay precision (r ² ) was 0.9096. Our newly developed KRAS ddPCR drop-off assay accurately identified single nucleotide variants in 35/36 (97.2%) of circulating tumor-positive samples from the patient validation cohort. Assay cross-validation showed that the newly established KRAS codon 12/13 ddPCR drop-off assay outperformed a commercially available KRAS multiplex ddPCR assay in terms of specificity . Moreover, the newly developed assay proved to be suitable for multiplexing with mutation-specific probes. Conclusion: We developed and clinically validated a highly accurate ddPCR drop-off assay for KRAS exon 2 hot-spot detection in cfDNA with broad applicability for clinic and research.