Multiplexed Assays for Detection of Mutations in PIK3CA

• Published in: Clinical chemistry (Baltimore, Md.) Vol. 54; no. 4; pp. 757 - 760
• Main Authors: Board, Ruth E, Thelwell, Nicola J, Ravetto, Paul F, Little, Stephen, Ranson, Malcolm, Dive, Caroline, Hughes, Andrew, Whitcombe, David
• Format: Journal Article
• Language: English
• Published: Washington, DC Am Assoc Clin Chem 01.04.2008; American Association for Clinical Chemistry; Oxford University Press
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Biomarkers, Tumor - genetics; Breast cancer; Breast Neoplasms - genetics; Class I Phosphatidylinositol 3-Kinases; Colorectal carcinoma; Colorectal Neoplasms - genetics; Deoxyribonucleic acid; DNA; Female; Fundamental and applied biological sciences. Psychology; Genes; Humans; Investigative techniques, diagnostic techniques (general aspects); Kinases; Lung cancer; Lung Neoplasms - genetics; Medical sciences; Melanoma; Melanoma - genetics; Mutation; Ovarian cancer; Phosphatidylinositol 3-Kinases - genetics; Polymerase Chain Reaction - methods; Sensitivity and Specificity; Tissues; Assay; Enzyme; Transferases; Clinical biology; Genetics; Biochemistry; Diagnosis; Mutation; Molecular biology; Detection; 1-Phosphatidylinositol 3-kinase
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1373/clinchem.2007.098376

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. 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). 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. Multiplexed assays, which can easily be applied to clinical samples, have been developed for the detection of PIK3CA mutations.