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

Virginia Alvarez-Garcia,Clare Bartos,Karim Gharbi,Urmi Trivedi,Ieva Keraite,Paul M Brennan,Maïwenn Kersaudy-Kerhoas,Olga Oikonomidou,Nicholas R Leslie

doi:10.1038/s41598-018-22473-9

Abstract

PIK3CA mutations are seemingly the most common driver mutations in breast cancer with H1047R and E545K being the most common of these, accounting together for around 60% of all PIK3CA mutations and have promising therapeutic implications. Given the low sensitivity and the high cost of current genotyping methods we sought to develop fast, simple and inexpensive assays for PIK3CA H1047R and E545K mutation screening in clinical material. The methods we describe are based on a real-time PCR including a mutation specific primer combined with a non-productive oligonucleotide which inhibits wild-type amplification and a parallel internal control reaction. We demonstrate consistent detection of PIK3CA H1047R mutant DNA in genomic DNA extracted from frozen breast cancer biopsies, FFPE material or cancer cell lines with a detection sensitivity of approximately 5% mutant allele fraction and validate these results using both Sanger sequencing and deep next generation sequencing methods. The detection sensitivity for PIK3CA E545K mutation was approximately 10%. We propose these methods as simple, fast and inexpensive diagnostic tools to determine PIK3CA mutation status.

Highlights

PIK3CA mutations are seemingly the most common driver mutations in breast cancer with H1047R and E545K being the most common of these, accounting together for around 60% of all PIK3CA mutations and have promising therapeutic implications
The assays reported rely on the combined use of a set of mutant allele-specific primers with a non-productive phosphate-modified oligonucleotide complementary to the wild-type sequence that blocks the wild-type allele amplification achieving high specificity
A separate internal control reaction allows the quantification of the quantitative PCR (qPCR) product

Summary

Introduction

PIK3CA mutations are seemingly the most common driver mutations in breast cancer with H1047R and E545K being the most common of these, accounting together for around 60% of all PIK3CA mutations and have promising therapeutic implications. The Phosphoinositide 3-Kinase (PI3K) signalling pathway is a key regulator of many essential cellular functions including survival, proliferation and metabolism[1]. It is one of the most commonly altered signal transduction pathways in human cancers[2,3] and its aberrant activation causes mosaic hyper-proliferative syndromes[4,5]. The identified PIK3CA mutations are dominated by a small number of recurrent mutations located within two major hotspots: E542K and E545K in the helical domain (encoded within exon 9) and H1047R in the kinase domain (encoded within exon 20) These missense mutations each produce a constitutively active form of the PI3K protein that promotes enhanced downstream signalling contributing to cellular transformation[7,8]. Due to the complexity and high cost of NGS techniques, their routine implementation into widespread clinical practice seems some way off, in healthcare settings with more limited resources

Methods

Results

Conclusion