A simple and robust real-time qPCR method for the detection of PIK3CA H1047R mutations

PIK3CA mutations are seemingly the most common driver mutations in breast cancer and H1047R is the most common of these, accounting for around 40% of all PIK3CA mutations with promising therapeutic implications. Given the low sensitivity and the high cost of current genotyping methods such as Sanger sequencing and/or Next Generation Sequencing, we sought to develop a simple and inexpensive assay for PIK3CA H1047R mutation screening in clinical material. The method we describe for PIK3CA H1047R mutation detection is based on a fast, simple real-time quantitative PCR (qPCR) and can be used in genomic DNA obtained from different types of tissue, including cancer biopsies and formalin-fixed paraffin-embedded (FFPE) samples. We demonstrate consistent detection of PIK3CA H1047R mutant DNA in genomic DNA extracted from frozen tumour biopsies, FFPE material or cancer cell lines with a detection sensitivity of approximately 5% mutant allele fraction and validate these results using Sanger sequencing and deep sequencing methods. Here we describe a robust method for PIK3CA H1047R status in biopsy material based on the widely-used Sybr Green technology for real-time PCR. We propose this method as a simple, fast and inexpensive diagnostic tool to determine PIK3CA H1047R mutation status in clinical samples to inform individualized targeted therapies for cancer patients.