KRAS and BRAF Mutation Detection with Multiplex Amplification and Pyrosequencing.
Hidehiro Takei, MaryAnn Szanyi, Federico Monzon. The Methodist Hospital, Houston, TX
Background: Targeted therapies against receptor tyrosine kinase signaling cascades have revolutionized cancer treatment. For metastatic colorectal carcinomas (mCRCs), anti-epidermal growth factor receptor (EGFR) humanized monoclonal antibodies have been shown to improve patient outcomes. However, codon 12/13 mutations in the KRAS gene are associated with resistance to this novel targeted therapy. In addition, mutations in the BRAF gene, a downstream target of the same signaling cascade, have also been shown to confer resistance to anti-EGFR therapy. It is thus desirable to test for both KRAS and BRAF mutations, ideally as a multiplex assay. The aim of study is to design an assay using a multiplex PCR method for both KRAS and BRAF mutations and sequential pyrosequencing on the same amplification reaction.
Design: We designed a multiplex COLD-PCR protocol to amplify segments containing codons 12/13 of the KRAS gene and codon 600 of the BRAF gene. The PCR products were electrophoretically separated to confirm amplicon size. Pyrosequencing to detect KRAS and BRAF amplicon sequences were performed with sequencing primers specific to each gene. A KRAS mutant cell line (HCT116) and a wild-type control (placental DNA) were tested simultaneously.
Results: The electropherogram showed well-resolved peaks at 89 and 219 base pairs, corresponding to KRAS and BRAF amplicons respectively, in both specimens. Pyrosequencing demonstrated mutated (GGC>GAC; G13D) and wild-type sequences of KRAS amplicons, with wild-type sequence of BRAF amplicon, in the KRAS mutant cell line and the control DNAs, respectively. However, peak intensity in the sequencing reaction was reduced, and noise increased when compared to non-multiplexed PCR reactions.
Conclusions: We have demonstrated that both KRAS and BRAF mutations can be tested sequentially on the same multiplex amplification reaction with our assay design. Optimization of the assay and validation with a large number of samples will be performed before possible clinical implementation of this assay. These preliminary results suggest that this protocol should streamline KRAS/BRAF mutation testing by using a single amplification reaction to sequence both genes.
Monday, February 28, 2011 1:00 PM
Poster Session II # 267, Monday Afternoon