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Published on: April 11, 2016
PIK3CA mutation enrichment and quantitation from blood and tissue
Ieva Keraite1,2, Virginia Alvarez-Garcia1,3, Isaac Garcia-Murillas4
1Institute of Biological Chemistry, Biophysics and Bioengineering, Heriot-Watt University, Edinburgh, EH14 4AS, UK.
Abstract:
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.
Insights
A new assay significantly improves the detection of PIK3CA mutations in breast cancer, enhancing diagnostic accuracy for targeted therapies. This method offers a simple, versatile, and informative workflow for routine testing and disease monitoring.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- PIK3CA gene mutations are prevalent in 30-40% of breast cancers.
- Four hotspot mutations (E542K, E545K, H1047R, H1047L) are common and serve as predictive biomarkers.
Purpose of the Study:
- To develop a highly sensitive assay for detecting and quantifying PIK3CA mutations.
- To establish a flexible prediction method for calculating mutant allele frequency in various sample types.
Main Methods:
- A nuclease-based enrichment assay was combined with real-time qPCR.
- A novel prediction method was developed for mutant allele frequency calculation.
Main Results:
- Assay sensitivity was enhanced to 0.3-0.6% for specific PIK3CA mutations.
- Accurate detection and quantitation were demonstrated in two breast cancer cohorts.
- The method proved effective for samples with low mutant fractions.
Conclusions:
- The developed workflow offers a quick, accurate, and simple method for PIK3CA mutation detection.
- This versatile assay is suitable for routine diagnostic testing and disease monitoring.
- Further validation in larger studies is recommended for clinical implementation.
