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

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

doi:10.1038/s41598-018-22473-9

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

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

Research output: Contribution to journal › Article › peer-review

Abstract

PIK3CA mutations are seemingly the most common driver mutations in breast cancer with H1047R and E45K 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.

Original language	English
Pages (from-to)	4290
Journal	Scientific Reports
Volume	8
Issue number	1
Early online date	9 Mar 2018
DOIs	https://doi.org/10.1038/s41598-018-22473-9
Publication status	Published - 9 Mar 2018

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Journal Article

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Olga Oikonomidou
- Deanery of Molecular, Genetic and Population Health Sciences - Senior Clinical Lecturer
- Edinburgh Cancer Research Centre
Person: Academic: Research Active

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title = "A simple and robust real-time qPCR method for the detection of PIK3CA mutations",

abstract = "PIK3CA mutations are seemingly the most common driver mutations in breast cancer with H1047R and E45K 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.",

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AU - Alvarez-Garcia, Virginia

AU - Bartos, Clare

AU - Keraite, Ieva

AU - Trivedi, Urmi

AU - Brennan, Paul

AU - Kersaudy-Kerhoas, Maiwenn

AU - Gharbi, Karim

AU - Oikonomidou, Olga

AU - Leslie, Nicholas R

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AB - PIK3CA mutations are seemingly the most common driver mutations in breast cancer with H1047R and E45K 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.

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

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Olga Oikonomidou

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