Multiplexed assays for detection of mutations in PIK3CA.
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.
Board
Ruth E
RE
Discovery Medicine, AstraZeneca Pharmaceuticals, Macclesfield, UK. ruth.board@astrazeneca.com
Thelwell
Nicola J
NJ
Ravetto
Paul F
PF
Little
Stephen
S
Ranson
Malcolm
M
Dive
Caroline
C
Hughes
Andrew
A
Whitcombe
David
D
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
D014408:Biomarkers, Tumor; D019869:Phosphatidylinositol 3-Kinases; D058534:Class I Phosphatidylinositol 3-Kinases; C484760:PIK3CA protein, human