Abstract 3068: Detection of K-RAS mutations in Korean patients using PNAClampTM KRAS

Abstract

Abstract Mutation of the K-RAS gene located in codons 12 and 13 may play an important role in prognosis of related diseases and responses to certain treatments targeting the pathway. Recent studies suggest that in colon cancer patients, K-RAS mutation status is a strong predictor of success in therapeutics of several tyrosine kinase inhibitors. Therefore, it is important to detect K-RAS mutations fast and accurately. To detect a minority of mutant K-RAS among abundant wild-type alleles, we have developed a highly sensitive and simple method for the detection of K-RAS mutations using PNA-mediated real-time PCR clamping, PNAClampTM. PNAClampTM relies on the following two unique properties of PNA probes: 1) PNA-DNA duplexes generally have greater thermal stability than the corresponding DNA-DNA duplex; 2) PNA oligomers are not recognized by DNA polymerases and consequently can serve as a sequence-selective clamp during PCR amplification. This method is fast and accurate with a detection limit of approximately 0.1% mutant alleles using 10 ng to 25 ng of normal DNA as a template. The turnaround time for performance of this assay is only 2 hrs. The PNAClampTM method facilitates the detection of mutations in more than 1000-fold excess of wild-type DNA samples. K-RAS mutations were detected in 28.3% (36/127) of patients. In detail, 29 cases had mutations in codon 12 and 7 cases had mutations in codon 13. To confirm those results, the same clinical samples were analyzed with a direct sequencing assay. Concordant results were obtained from 124 (97.6%) of the 127 clinical samples in the two assays. The discrepancy was observed in three cases. For comparison, the three clinical samples were also analyzed using DxS K-RAS Mutation Kit (DxS Ltd., UK) to give the same outcome. In this study, we will discuss development of PNAClampTM KRAS and its optimization for higher sensitivity. The PNAClampTM KRAS may serve a rapid and reliable alternative for K-RAS mutation detection in the clinical field. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3068. doi:10.1158/1538-7445.AM2011-3068