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

Virginia Alvarez-Garcia¹, Clare Bartos^2,3, Ieva Keraite^4,5

¹Institute of Biological Chemistry, Biophysics and Bioengineering, Heriot-Watt University, Edinburgh, EH14 4AS, UK. v.alvarez_garcia@hw.ac.uk.

Scientific Reports

|March 11, 2018

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.

Insights

This study introduces a fast, affordable real-time PCR method for detecting common PIK3CA mutations (H1047R, E545K) in breast cancer. This assay offers a simpler, cost-effective alternative for clinical mutation screening.

Area of Science:

Oncology
Molecular Biology
Genetics

Background:

PIK3CA mutations are frequent in breast cancer, with H1047R and E545K comprising ~60% of cases.
Existing genotyping methods for PIK3CA mutations are costly and lack sensitivity.
Targeting PIK3CA mutations offers significant therapeutic potential in breast cancer treatment.

Purpose of the Study:

To develop rapid, cost-effective assays for screening PIK3CA H1047R and E545K mutations.
To provide a simpler diagnostic tool for PIK3CA mutation status determination in clinical settings.

Main Methods:

Real-time PCR utilizing mutation-specific primers and a wild-type amplification inhibitor.
Incorporation of a parallel internal control reaction for accurate quantification.
Validation using Sanger sequencing and next-generation sequencing (NGS).

Main Results:

Consistent detection of PIK3CA H1047R mutant DNA in various sample types (biopsies, FFPE, cell lines).
Achieved a detection sensitivity of approximately 5% mutant allele fraction for H1047R.
Demonstrated a detection sensitivity of approximately 10% for PIK3CA E545K mutation.

Conclusions:

The developed real-time PCR assays are simple, fast, and inexpensive for PIK3CA mutation screening.
These assays can serve as valuable diagnostic tools for determining PIK3CA mutation status in breast cancer patients.
The methods facilitate wider accessibility to crucial genetic information for personalized cancer therapy.

Read full analysis

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