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

Markou, Athina; Tzanikou, Elena; Ladas, Ioannis; Makrigiorgos, G Mike; Lianidou, Evi S

Allele-specific PCR (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 non-specific priming that can take place with 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 (E545K and H1047R) in CTCs and cfDNA. The developed protocol could simultaneously detect mutation-allelic-frequency of 0.5% for PIK3CA exon 9 (E545K) and 0.1% for PIK3CA exon 20 (H1047R), with high specificity. We further compared the developed NAPA assay with a) ddPCR considered as the gold standard and b) our previous assay based on the combination of allele-specific, asymmetric rapid PCR and melting analysis. Our data show that the newly developed NAPA assay gives consistent results with both these assays (p=0.001). The developed assay resolves the false positive signals issue derived through classic ARMS-PCR and provides an ideal combination of speed, accuracy and versatility, and should be easily applicable in routine diagnostic labs.