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Brief Communications |
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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