RESEARCH ARTICLE Efficient Genotyping of KRASMutant Non- Small Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach

Droplet digital PCR (ddPCR) can be used to detect low frequency mutations in oncogene-driven lung cancer. The range of KRAS point mutations observed in NSCLC necessitates a multiplex approach to efficient mutation detection in circulating DNA. Here we report the design and optimisation of three discriminatory ddPCRmultiplex assays investigating nine different KRASmutations using PrimePCR ™ ddPCR™Mutation Assays and the Bio-Rad QX100 system. Together these mutations account for 95 % of the nucleotide changes found in KRAS in human cancer. Multiplex reactions were optimised on genomic DNA extracted from KRASmutant cell lines and tested on DNA extracted from fixed tumour tissue from a cohort of lung cancer patients without prior knowledge of the specific KRAS genotype. The multiplex ddPCR assays had a limit of detection of better than 1 mutant KRASmolecule in 2,000 wild-type KRASmolecules, which compared favourably with a limit of detection of 1 in 50 for next generation sequencing and 1 in 10 for Sanger sequencing. Multiplex ddPCR assays thus provide a highly efficient methodology to identify KRASmutations in lung adenocarcinoma