Development of integrated PIK3CA mutation enrichment assays

PIK3CA, an oncogene that encodes the p110α catalytic component of PI3K, is one of the two most frequently mutated genes accounting for approximately 30–40% of breast cancer cases. The four most frequent ‘hotspot’ PIK3CA mutations are located within two exons and account for 80–90% of all PIK3CA mutations in human malignancies. Due to the clinical relevance of activating mutations in PI3K, it has become an attractive target for diagnostic purposes and drug development. However, current molecular methods still lack analytical sensitivity to detect mutations of low abundance. Makrigiorgos et al. developed a nuclease-assisted minor-allele enrichment assay with overlapping probes (NaME-PrO), which is practical and cost-effective, and allows detection of very low abundance mutations that could have relevance in the clinic. Thus, we developed PIK3CA mutation specific nuclease based enrichment assays and validated them with clinical samples using crystal digital PCR and our in-house Sybr Green real-time qPCR detection method (Alvarez-Garcia et al. 2018), which allows a quick detection of PIK3CA mutations in circulating cell-free DNA from clinical samples at low-cost. Finally, we have developed a microfluidic device for the integration of this NaME-PrO enrichment step into a complete, continuous sample preparation workflow.