Abstract 4226: PIK3CA mutation detection in clinical samples using PNA mediated real-time PCR clamping assay..

Abstract

Abstract Somatic mutations in the PIK3CA gene have been frequently found in various human cancers, and their presence correlates to therapy response. These somatic mutations within the PIK3CA gene are localized in exon 9 and 20. We have developed a highly sensitive and simple method to detect 13 mutations in exons 9 and 20 of PIK3CA using PNA-mediated real-time PCR clamping, PNAClampTM. PNAClampTM is based on unique properties of PNA probes that PNA oligomers are not recognized by DNA serving as sequence-selective clamps during PCR amplification. This method can also detect 13 mutations through only three tube reactions each with 10 ng of the target DNA. PNAClampTM is simple and reliable with a detection limit of approximately 3% mutant alleles using 10 to 50 ng of normal DNA as a template. The turnaround time for performing this assay was only 2 hours. Furthermore, while direct sequencing method will detect most of PIK3CA samples as having E545A mutation, due to homology in exon 9 in PIK3CA gene, highly specific PNAClampTM Method have overcome this perceived problem. In clinical studies with breast cancer patients in Korea, PIK3CA mutations were detected utilizing PNAClampTM in 60 (35.7 %) of clinical samples, and the results were compared with direct sequencing method. Among 168 patients, 6 (3.6 %) had mutations in codon 542, 23 (13.7 %) had mutations in codon 545 and 31 (18.5 %) had mutations in codon 1047. To confirm these results, the same clinical samples were analyzed with a sequencing assay. Consistent results were obtained from 137 (81.5 %) of the 168 clinical samples between PNAClampTM and the sequencing assay. The results not in agreement with the sequencing numbered 21 (12.5 %) samples. Ten samples did not react by direct sequencing due to quality problems with the target DNA. The remaining samples were initially determined as the wild type by direct sequencing, but they were found to be PIK3CA mutant using PNAClampTM. This result was likely due to the high sensitivity of PNAClampTM. PNAClampTM is a rapid, sensitive and easy to perform tool that can be used for the detection of PIK3CA mutations in the clinical field. [j1]Check this - original was unclear. Is this what you mean? Citation Format: Hysun Kim, Jae Jin Choi, Heekyung Park. PIK3CA mutation detection in clinical samples using PNA mediated real-time PCR clamping assay. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4226. doi:10.1158/1538-7445.AM2013-4226