Abstract 5059: Detection of K-RAS mutation in clinical samples by one-step PNA-mediated real-time PCR clamping techniques

Abstract

Abstract Mutations in the K-RAS oncogene are frequently found in human cancers. K-RAS mutations may indicate prognosis and drug response, and many new cancer therapies are being targeted to the K-RAS pathway. Many researchers suggest that in colon cancer patients the K-RAS mutation status is strong predictor of resistance to therapy with tyrosine kinase inhibitors such as Erbitux® (cetuximab/ Imclone system Inc) or Vectibix® (panitumumab/ Amgen Inc). Therefore, it is important that detection of K-RAS mutations is fast and simple with high accuracy and high sensitivity for predict drug response and prognosis. To detect a minority of mutant K-RAS among abundant wild-type alleles, we developed a high sensitivity and simple method for detection of K-RAS mutations using PNA-mediated real-time PCR clamping. PNA mediated real-time PCR clamping 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 and 2) PNA oligomers are not recognized by DNA polymerases and consequently can serve as sequence selective clamp during PCR amplification. One-step PNA-mediated real-time PCR clamping method was optimized for detection of K-RAS mutations in exon 12 and 13 with high sensitivity. This method was simple and correct with detection limit approximately 0.1% mutant alleles using 10ng to 50ng of normal DNA as the template. The turnaround time for performing this assay was only 2.5hrs. K-RAS mutations were detected in 24.4% (22/90) of clinical samples, of which 18 had mutations in codon 12 and 4 had these in codon 13. To confirm these results, same clinical samples were analyzed with sequencing assay. The concordant results were obtained from 88 (98%) of the 90 clinical samples by the two assays. The PNA-mediated real-time PCR clamping is a rapid and reliable tool for K-RAS mutation detection and allowed a minority of mutant in clinical field. Keywords: K-RAS, Mutation, PNA, cancer, PCR clamping This work was supported be the Small and Medium Business Administration funded by the Korean Government. (S1060400) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5059.