Validation of a Coamplification at Lower Denaturation Temperature PCR (COLD-PCR) Followed by Sanger Sequencing Method for KRAS Mutation Detection in Colon Cancer Specimens
NN Chen, Y El-Gohary, I Ezedi, CN Powers, DS Wilkinson, A Ferreira-Gonzalez, CI Dumur. Virginia Commonwealth University Medical Center, Richmond, VA
Background: KRAS mutations are found in 35% to 45% of patients with colorectal cancer, and it has been shown that tumors harboring wild-type KRAS gene are associated with good response to the EGFR targeted therapies. Recently, ASCO and the National Comprehensive Cancer Network (NCCN) have recommended that patients with metastatic (advanced) colorectal cancer should have KRAS mutation testing done before treatment decision. However, the heterogeneity of clinical specimens may mask the KRAS mutation status, misleading the oncologist in the treatment decision. Here, we employed a novel PCR method combined with direct Sanger sequencing, which resulted in a dramatic improvement of the sensitivity for KRAS mutation detection.
Design: Conventional PCR and COLD-PCR followed by Direct Sanger sequencing were used to amplify a 216-bp DNA fragment in exon 2 of the KRAS gene from genomic DNA extracted from homozygous and heterozygous cell line mixtures ranging from 10% to 100% mutant DNA, to assess the analytical sensitivity of the method. In addition, 17 formalin fixed paraffin embedded (FFPE) tissue specimens with known KRAS mutation status were also tested in a blind experiment. For these samples, KRAS mutation status was previously determined in separate laboratories, by direct sequencing and real-time PCR using Scorpions™ technology.
Results: We were able to improve the limit of detection from 25% using conventional PCR to 10% of mutant DNA using COLD-PCR (positive in 95% of the replicate tested) in cell line mixtures. The clinical sensitivity of KRAS mutation detection was also increased by the use of COLD-PCR compared to Conventional PCR, as well as by the use of tissue macrodissection as shown in Table 1.
Conclusions: COLD-PCR followed by direct Sanger sequencing, combined with macro-dissection of the FFPE tissue sections provides a sensitive and economical method for detecting low-level mutant alleles of KRAS in clinical samples.
Monday, March 22, 2010 9:30 AM
Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 253, Monday Morning