Abstract 2115: Increased sensitivity of KRAS mutation detection by High-Resolution Melting analysis of COLD-PCR products

Abstract

Abstract Much effort has been put into the development of sophisticated technologies enabling the detection of clinically significant low-level tumor specific KRAS mutations. The constitutively active KRAS oncogene harbors a mutation hotspot in codon 12 and 13 of exon 2. These mutations are predictors of response to treatment with the monoclonal antibodies panitumumab and cetuximab targeting the epidermal growth factor receptor (EGFR). Therefore, it is crucial to develop robust and sensitive assays targeting these mutations. Coamplification at lower denaturation temperature-PCR (COLD-PCR) is a new form of PCR that selectively amplifies mutation containing amplicons based on the lower melting temperature of heteroduplexes versus homoduplexes. We have developed an assay based on real-time COLD-PCR and high-resolution melting (HRM) targeting codon 12 and 13 for simultaneous detection of the various mutations found in this mutation hotspot. By using mutation containing cell-lines serially diluted into wild-type DNA we have compared the sensitivity of HRM after standard PCR with the sensitivity of HRM after COLD-PCR. We have also analyzed 60 colorectal cancer samples for which KRAS mutation status previously has been evaluated by the DxS kit which is approved by the U.S. Food and Drug Administration. Replacing standard PCR by COLD-PCR increased the sensitivity of the assay by up to 50 fold depending on the specific mutation. Furthermore, we have obtained very similar results when using our COLD-PCR assay as we obtained when using the DxS kit and confirmed the HRM results with Sanger sequencing. In conclusion, applying COLD-PCR to HRM analysis is a simple way of increasing the sensitivity of KRAS mutation detection without adding to the complexity and cost of the experiments. 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 2115.