PIK3CA mutation enrichment and quantitation from blood and tissue

• Published in: Scientific reports Vol. 10; no. 1; p. 17082
• Main Authors: Keraite, Ieva, Alvarez-Garcia, Virginia, Garcia-Murillas, Isaac, Beaney, Matthew, Turner, Nicholas C., Bartos, Clare, Oikonomidou, Olga, Kersaudy-Kerhoas, Maïwenn, Leslie, Nicholas R.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 13.10.2020
• Subjects: 631/1647; 631/208; 631/337; 631/67; 692/1807; 692/308; 692/4017; 692/4028; 692/699; 692/700; Biomarkers, Tumor - blood; Biomarkers, Tumor - genetics; Biomarkers, Tumor - metabolism; Breast Neoplasms - diagnosis; Breast Neoplasms - enzymology; Breast Neoplasms - genetics; Cell Line, Tumor; Class I Phosphatidylinositol 3-Kinases - blood; Class I Phosphatidylinositol 3-Kinases - genetics; Class I Phosphatidylinositol 3-Kinases - metabolism; Cohort Studies; DNA Mutational Analysis - methods; DNA, Neoplasm - genetics; DNA, Neoplasm - isolation & purification; Female; Gene Frequency; Humanities and Social Sciences; Humans; MCF-7 Cells; multidisciplinary; Mutation; Real-Time Polymerase Chain Reaction - methods; Science; Science (multidisciplinary)
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-020-74086-w

PIK3CA is one of the two most frequently mutated genes in breast cancers, occurring in 30–40% of cases. Four frequent ‘hotspot’ PIK3CA mutations (E542K, E545K, H1047R and H1047L) account for 80–90% of all PIK3CA mutations in human malignancies and represent predictive biomarkers. Here we describe a PIK3CA mutation specific nuclease-based enrichment assay, which combined with a low-cost real-time qPCR detection method, enhances assay detection sensitivity from 5% for E542K and 10% for E545K to 0.6%, and from 5% for H1047R to 0.3%. Moreover, we present a novel flexible prediction method to calculate initial mutant allele frequency in tissue biopsy and blood samples with low mutant fraction. These advancements demonstrated a quick, accurate and simple detection and quantitation of PIK3CA mutations in two breast cancer cohorts (first cohort n = 22, second cohort n = 25). Hence this simple, versatile and informative workflow could be applicable for routine diagnostic testing where quantitative results are essential, e.g. disease monitoring subject to validation in a substantial future study.