title: Multiplexed assays for detection of mutations in PIK3CA.
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.
authors: Discovery Medicine, AstraZeneca Pharmaceuticals, Macclesfield, UK. ruth.board@astrazeneca.com
authors: Ruth E
authors: RE
authors: Board
authors: Nicola J
authors: NJ
authors: Thelwell
authors: Paul F
authors: PF
authors: Ravetto
authors: Stephen
authors: S
authors: Little
authors: Malcolm
authors: M
authors: Ranson
authors: Caroline
authors: C
authors: Dive
authors: Andrew
authors: A
authors: Hughes
authors: David
authors: D
authors: Whitcombe
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics*; D001943:Breast Neoplasms / Q000235:genetics; D058534:Class I Phosphatidylinositol 3-Kinases; D015179:Colorectal Neoplasms / Q000235:genetics; D005260:Female; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics; D008545:Melanoma / Q000235:genetics; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
chemical_list: D014408:Biomarkers, Tumor; D019869:Phosphatidylinositol 3-Kinases; D058534:Class I Phosphatidylinositol 3-Kinases; C484760:PIK3CA protein, human