NaME-PrO-assisted ARMS (NAPA): a novel approach for the improvement of ARMS-PCR specificity in liquid biopsies

Allele-specific polymerase chain reaction (PCR) (amplification-refractory mutation system, ARMS) is one of the most commonly used methods for mutation detection. However, a main limitation of ARMS-PCR is the false positive results obtained due to nonspecific priming that can take place with wild-type (WT) DNA, which often precludes detection of low-level mutations. To improve the analytical specificity of ARMS, we present here a new technology, NAPA: NaME-PrO-assisted ARMS, that overcomes the ARMS deficiency by adding a brief enzymatic step that reduces wild-type alleles just prior to ARMS. We performed this technology for the simultaneous detection of two hot-spot PIK3CA mutations (E545 K and H1047R) in circulating tumor cells (CTCs) and cell free DNA (cfDNA). The developed protocol could simultaneously detect mutation-allelic-frequency of 0.5% for PIK3CA exon 9 (E545 K) and 0.1% for PIK3CA exon 20 (H1047R) with high specificity. We further compared the developed NAPA assay with (a) ddPCR considered as t...