Multiplexed assays for detection of mutations in PIK3CA.
Board, Ruth E Thelwell, Nicola J Ravetto, Paul F Little, Stephen Ranson, Malcolm R Dive, Caroline Hughes, Andrew Whitcombe, David
Board, Ruth E
Thelwell, Nicola J
Ravetto, Paul F
Little, Stephen
Ranson, Malcolm R
Dive, Caroline
Hughes, Andrew
Whitcombe, David
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Abstract
BACKGROUND: Mutations in the PIK3CA gene (phosphoinositide-3-kinase, catalytic, alpha polypeptide) have recently been described in a number of cancers, and their detection is currently limited because of the low sensitivity of conventional sequencing techniques. METHODS: We combined Amplification Refractory Mutation System (ARMS; AstraZeneca) allele-specific PCR and Scorpions (DxS) to develop assays for tumor-borne PIK3CA mutations and used real-time PCR to develop high-throughput multiplexed assays for the most commonly reported PIK3CA mutants (H1047L, H1047R, E542K, E545K). RESULTS: These assays were more sensitive than sequencing and could detect 5 copies of mutant DNA in proportions as low as 0.1% of the total DNA. We assayed DNA extracted from human tumors and detected PIK3CA mutation frequencies of 10.2% in colorectal cancer, 38.7% in breast cancer, 1.9% in lung cancer, and 2.9% in melanoma. In contrast, sequencing detected only 53% of the mutations detected by our assay. CONCLUSIONS: Multiplexed assays, which can easily be applied to clinical samples, have been developed for the detection of PIK3CA mutations.
Description
Date
2008-04
Publisher
Keywords
Breast Cancer
Lung Cancer
Colorectal Cancer
PIK3CA
Tumour Markers
Lung Cancer
Colorectal Cancer
PIK3CA
Tumour Markers
Type
Article
Citation
Multiplexed assays for detection of mutations in PIK3CA. 2008, 54 (4):757-60 Clin. Chem.