Board Ruth E, Thelwell Nicola J, Ravetto Paul F, Little Stephen, Ranson Malcolm, Dive Caroline, Hughes Andrew, Whitcombe David
Discovery Medicine, AstraZeneca Pharmaceuticals, Macclesfield, UK.
Clin Chem. 2008 Apr;54(4):757-60. 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.
PIK3CA基因（磷脂酰肌醇-3激酶，催化性，α多肽）的突变最近在多种癌症中被报道，由于传统测序技术灵敏度低，目前对其检测受到限制。
我们将扩增阻滞突变系统（ARMS；阿斯利康）等位基因特异性PCR与蝎形探针（DxS）相结合，开发用于检测肿瘤源性PIK3CA突变的检测方法，并使用实时PCR开发针对最常报道的PIK3CA突变体（H1047L、H1047R、E542K、E545K）的高通量多重检测方法。
这些检测方法比测序更灵敏，能够检测到低至总DNA 0.1%比例中的5份突变DNA。我们检测了从人类肿瘤中提取的DNA，在结直肠癌中检测到PIK3CA突变频率为10.2%，乳腺癌中为38.7%，肺癌中为1.9%，黑色素瘤中为2.9%。相比之下，测序仅检测到我们检测方法所检测到突变的53%。
已开发出可轻松应用于临床样本的多重检测方法，用于检测PIK3CA突变。
