Abstract LB-122: Single copy detection of KRAS mutations in colorectal cancer tissue and blood by qPCR using primers with enhanced analytical selectivity

Abstract

Abstract The development of highly sensitive genotyping assays that are suitable for clinical diagnostics opens new opportunities for the detection, assessment and management of cancer. Anticipated uses for these assays include profiling tissue biopsies and circulating tumor cells (CTCs), and detecting mutations in circulating cell-free nucleic acids. Swift Biosciences has developed myT™ Primers which have unique thermodynamic properties that make them highly sensitive to mismatch discrimination. myT™ Primer qPCR assays have been developed for the seven most frequent KRAS mutations in codons 12 and 13. These assays demonstrated single copy sensitivity for all seven KRAS mutations when used to genotype formalin fixed paraffin embedded (FFPE) specimens from Stage III colorectal tumors. When compared to a leading commercially available KRAS qPCR mutation test kit, myT™ Primer KRAS assays demonstrated several orders of improved specificity. A myT™ Primer assay was also developed for BRAF V600E/K mutations; results from FFPE melanoma specimens indicate high sensitivity and specificity. As a proof of concept for CTC detection, limiting dilutions of cells harboring the KRAS G12V mutation were added to whole human blood. Single copy detection of G12V was observed from an equivalent of a few microliters of blood. Because CTCs and cell-free cancer DNA can exist in blood at extremely low frequencies, we tested assay performance at even lower target concentrations. Mutant KRAS plasmid templates were titrated with wt KRAS plasmids such that the total KRAS copy number was 1×108. Under these conditions, the G12V-specific assay was able to detect the presence of ten mutant templates per 100 million (108) wt templates. The extreme selectivity of the myT™ Primer assays may make them especially useful for detection of mutations present at extremely low copy number and for genotyping difficult samples such as needle biopsies, CTCs and serum. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-122. doi:10.1158/1538-7445.AM2011-LB-122