Comparison of TaqMan PCR and Pyrosequencing Assays for Assessment of KRAS Mutations in FFPE Colorectal Cancer Specimens
JA Lefferts, H Chen, A Suriawinata, GJ Tsongalis. Dartmouth-Hitchcock Medical Center, Dartmouth Medical School, Norris Cotton Cancer Center, Lebanon, NH
Background: KRAS mutation detection, specifically in the context of colorectal cancer, is being used with increasing frequency due to findings that KRAS mutations predict poor response to certain drugs targeting the EGFR, upstream of KRAS in signaling pathways. Currently, FDA-approved KRAS mutation assays are not available, requiring laboratories wishing to perform this testing to develop their own assays. 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 DNA samples extracted from FFPE tissue specimens collected during resection of villous adenomas and adenocarcinomas of the colon were subjected to both pyrosequencing and a TaqMan real-time PCR assay. For pyrosequencing the PyroMark 24 platform was used to sequence codons 12 and 13 following PCR amplification of the region surrounding these codons. In the TaqMan PCR analysis each DNA sample was subjected to a set of seven TaqMan PCR assays, each designed to detect one of seven point mutations in codons 12 and 13 of KRAS.
Results: Twelve of the twenty samples tested were found to have KRAS mutations by at least one of the two assays tested. In one of these samples, both assays gave a G13D signal low enough to make distinguishing it from wild-type samples difficult. The detection of the same mutation by two different assays, however, helped confirm the presence of an actual G13D mutation. Additionally, two separate mutations were detected by pyrosequencing in a sample found to be positive for one mutation by the TaqMan assay; another sample found to contain a G12V mutation by the TaqMan assay failed to amplify in the pyrosequencing PCR, resulting in no pyrosequencing results for this sample.
Conclusions: An initial comparison of KRAS mutation detection assays using real-time TaqMan PCR and pyrosequencing indicates a similar ability of both assays to distinguish between wild-type and mutant genomic DNA with respect to KRAS. Anticipated batch size and laboratory instrumentation available may be among the factors to be considered by laboratories implementing KRAS testing.
Monday, March 22, 2010 8:30 AM
Platform Session: Section G 1, Monday Morning