Multiplexed Detection of Mutant Circulating Tumor DNA Using Peptide Nucleic Acid Clamping Asymmetric Polymerase Chain Reaction and Liquidchip

: Circulating tumor DNA (ctDNA) in blood has been investigated as a feasible substitute for genetic alterations in tumor tissues to predict and assess drug responses, but current techniques of screening clinical relevant mutations still have great limitations in sensitivity, specificity, or multiplexed detection because of highly fragmented ctDNA and its low concentration in a high background of normal DNA. In this study, we developed PNA-aPCR-Liquidchip (PAPL), a novel method that aims to detect multiple mutant ctDNA. In order to demonstrate its utility, we analyzed three high frequent epidermal growth factor receptor (EGFR) mutations (exon 19 deletion, L858R, and T790M) in non-small cell lung cancer (NSCLC). Multiplexed analyses indicated that this method has high specificity and sensitivity which could detect down to 2∼5 copies of mutant EGFR in a background of 10,000 copies of wild-type genomic DNA, achieving the mutant abundance of 0.02%∼0.05%. Furthermore, PAPL had no significant differences with droplet digital PCR (ddPCR) in plasma cell-free DNA (cfDNA) detection. Thus, PAPL can be used to detect EGFR mutations in NSCLC patients' plasma, indicating that this method has great potential for application in the context of precision medicine based on mutant ctDNA detection.