[1740] KRAS Mutation Detection by a PCR-Based TaqMan Assay Compared to Traditional DNA Sequencing

JA Lefferts, HA Bentley, D Le Corre, P Laurent-Puig, M Gupta, AA Suriawinata, GJ Tsongalis. Dartmouth-Hitchcock Medical Center, Lebanon, NH; Dartmouth Medical School, Hanover, NH; INSERM, Paris, France; Universite Paris Descartes, Paris, France; Hopital Europeen Georges Pompidou, Paris, France; Norris Cotton Cancer Center, Lebanon, NH

Background: Clinical interest has been growing in tests that accurately determine the presence or absence of somatic mutations in the KRAS gene, mainly those found in codons 12 and 13. KRAS mutations have been identified in several cancer types and the presence of these mutations has been shown to indicate poorer prognoses. Many molecular diagnostic laboratories do not currently have DNA sequencing capabilities need for KRAS mutation detection. Additionally, due to the extensive manipulation required of PCR amplified DNA, extreme measures must be taken to avoid PCR contamination which can result in false-postive results. Alternative methods of mutation detection for KRAS have been developed but still tend to be time consuming and do not decrease the possibility PCR contamination. Here, we evaluate a new method of KRAS mutation detection which uses real-time PCR, producing faster results and eliminating the need for post-PCR manipulation.
Design: Twenty-five DNA samples extracted from FFPE sections of resected adenocarcinomas of the colon and seven from mucinous ovarian tumors, which had previously been subjected to KRAS DNA sequencing, were used in a TaqMan real-time PCR assay. For each sample a set of seven primer/probe mixes, each designed to detect one of the seven point mutations commonly seen in KRAS, were used to detect wild-type and/or mutant alleles.
Results: Both DNA sequencing and the TaqMan assay detected codon 12 or 13 mutations in the same ten DNA samples. The remaining 22 samples were found to be negative for KRAS mutations by both methods. Mutations in synthetic and cell line-derived DNA positive controls were also recognized by the TaqMan assay. One primer/probe set which had been designed to detect the G12C mutation was found to correctly identify known G12C sequencing but was also observed to detect other codon 12 mutations.
Conclusions: Real-time PCR using TaqMan probes to determine mutational status of KRAS in tumor specimens is a viable alternative to traditional sequencing. This assay provides results similar to those obtained by sequencing in less time without handling of PCR-amplified DNA and only requires real-time PCR instruments normally found in molecular diagnostic laboratories.
Category: Techniques

Wednesday, March 11, 2009 1:00 PM

Poster Session VI # 230, Wednesday Afternoon