Optimized multiplex detection of 7 KRAS Mutations by Taqman Allele-SpecificqPCR

Abstract

Establishing the KRAS mutational status of tumor samples is essential to manage patients with colorectal or lung cancer, since thesemutations preclude treatment with monoclonal anti-epidermalgrowth factor receptor (EGFR) antibodies.We report an inexpensive, rapid multiplex allele-specific qPCR method detecting the 7 most clinically relevant KRAS somaticmutations with concomitant amplification of non-mutated KRAS in tumor cells and tissues from CRC patients. Positive samples evidenced in the multiplex assay were further subjected to individual allele-specific analysis, to define the specificmutation. Reference human cancer DNA harbouring either G12A, G12C, G12D, G12R, G12S, G12V and G13D confirmedassay specificity with ≤1% sensitivity of mutant alleles. KRAS multiplex mutation analysis usefulness was also demonstrated with formalin-fixed paraffin embedded (FFPE) from CRC biopsies. Conclusion. Co-Amplification of non-mutatedDNA avoided false negatives from degraded samples. Moreover, this cost effective assay is compatible withmutation detection by DNA sequencing in FFPE tissues, but with a greater sensitivity when mutant DNA concentrations are limiting.