Clinical Chemistry
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Clinical Chemistry 54: 757-760, 2008; 10.1373/clinchem.2007.098376
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(Clinical Chemistry. 2008;54:757-760.)
© 2008 American Association for Clinical Chemistry, Inc.

Brief Communications

Multiplexed Assays for Detection of Mutations in PIK3CA

Ruth E. Board1,2,3,a, Nicola J. Thelwell4, Paul F. Ravetto4, Stephen Little4, Malcolm Ranson2, Caroline Dive3, Andrew Hughes1 and David Whitcombe4

1 Discovery Medicine, AstraZeneca Pharmaceuticals, Macclesfield, UK; 2 CRUK Department of Medical Oncology, Christie Hospital NHS Trust, Manchester, UK; 3 Clinical and Experimental Pharmacology Group, Paterson Institute of Cancer Research, University of Manchester, Manchester, UK; 4 DxS Ltd, Manchester, UK;

aaddress correspondence to this author at: Discovery Medicine, AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire, UK SK10 4TG. Fax 01625-230824; e-mail ruth.board{at}astrazeneca.com.


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 (ARMSTM; AstraZeneca) allele-specific PCR and ScorpionsTM (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.




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