Abstract 5033: Sub-0.01% KRAS G12C dPCR variant detection by mutation specific blocker (MSB) cassettes
Authors: Ashley Beams, Kevin Lee, Samantha Addison, Nathan Riccitelli
Journal: Cancer Research (2024)

Abstract Purpose: Kirsten rat sarcoma viral oncogene homologue (KRAS) is one of the most well-known oncogenes and is found commonly mutated in numerous cancer subtypes. Despite its prevalence as a driver of disease state, until recently KRAS-targeting therapeutics have proven difficult to develop due to lack of specificity (Huang 2021). Now, several treatments have been approved for KRAS mutated cancers that are driven specifically by the G12C variant, and additional compounds are under investigation. Enrollment on these therapies is conditional on the G12C mutation presence, and thus accurate, sensitive detection of G12C is critical. Herein, we describe the development of KRAS G12C-targeting MSB dPCR assays capable of detecting G12C variants down to less than 0.01% variant allele frequency in whole blood, FFPE, and cfDNA samples. Methods: Multiple KRAS G12C MSB cassettes were developed, composed of four components: a mutation-specific forward primer with an engineered penultimate nucleotide mismatch, a non-extendable blocking oligo for the wild-type (WT) KRAS G12 sequence, a TaqMan probe, and a standard reverse primer. Initial cassette performance was assessed using synthetic KRAS G12C and KRAS G12 WT sequences inserted into an IDT plasmid backbone and tested directly or spiked into whole blood or extracted genomic DNA obtained from healthy donors prior to testing. Data obtained with the MSB cassettes across multiple matrices was compared to a BioRad off-the-shelf KRAS G12C droplet digital PCR (ddPCR) assay for accuracy. Cassette designs achieving the desired specificity and sensitivity were subsequently run on cell-free DNA samples possessing KRAS G12C, KRAS G12D, and WT sequences to evaluate performance in patient material. Results: The enhanced WT suppression design of the assay allowed the lower limit of detection and limit of blank to remain at 6 copies/reaction (cps/rxn) and 3 cps/rxn, respectively, across 30 ng/rxn to 350 ng/rxn gDNA background. At the theoretical maximum input of 200,000 cps/rxn, the assay can achieve 0.003% frequency detection in a single well, with robust detection down to 5 ng/rxn input, making it ideal for samples with limited availability. The assay demonstrated high accuracy, with a correlation coefficient ≥ 0.95 to known values and the BioRad reference assay, and high precision, with less than 10% coefficient of variation among allele frequencies of the same sample. Conclusion: With the emergence of targeted therapies for KRAS G12C driven cancers, detection of this mutation has become crucial in the diagnosis. While multiple methods are available for variant identification, MSB cassettes represent a powerful approach to achieve a sensitivity of molecular detection not possible with other techniques. This enhanced sensitivity directly impacts patient access to potentially life-saving therapies, ensuring all possible options are available to diagnose the patients sensitively and early. Citation Format: Ashley Beams, Kevin Lee, Samantha Addison, Nathan Riccitelli. Sub-0.01% KRAS G12C dPCR variant detection by mutation specific blocker (MSB) cassettes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5033.