PIK3CA mutation enrichment and detection in clinical samples

The PI3K/AKT/mTOR signalling pathway is one of the most frequently mutated pathways in human cancers, including breast cancer. Given the current drive for more personalized approaches, there is an increasing need for a rigorous list of biomarkers and detection methods using less invasive, reliable and low-cost technologies. According to limited clinical results to date, the detection of PIK3CA mutations shows great potential to contribute in cancer patient management in the future at many different levels, including diagnosis, treatment choice and monitoring, and identification of drug resistance. However, current molecular methods still lack analytical sensitivity to detect mutations of low abundance in high wild type DNA background typically found in patient samples. Makrigiorgos et al. recently developed a nuclease-assisted minor-allele enrichment assay with overlapping probes (NaME-PrO). This assay is practical and cost-effective, and allows detection of very low abundance mutations that would have relevance in the clinic. Here we develop PIK3CA mutation specific NaME-PrO assay and validate it with clinical tissue and circulating cell-free DNA samples using digital droplet PCR (Stilla technologies). A key aspect of our used ddPCR technique is sample partitioning into thousands of individual PCR reactions in a microfluidic Sapphire chip using reagents similar to TaqMan probe-based assays, thus massively improving sensitivity for target detection. We then also combined our validated enrichment assay with a rapid and low-cost in-house SYBR Green real-time qPCR detection method (Alvarez-Garcia et al. 2018) for detection of PIK3CA mutations from low mutant allele tissue biopsy samples.