Application of COLD-PCR for Improved Detection of KRAS Mutations in Clinical Samples
Z Zuo, M Soape, S Doan, P Chandra, J Galbincea, B Barkoh, R Luthra. University of Texas MD Anderson Cancer Center, Houston, TX
Background: KRAS mutations, found in 30% of all human tumors, are of prognostic value and can predict response to targeted therapies. Current KRAS mutation detection strategy consists of PCR and direct sequencing. To increase detection sensitivity, we evaluated Co-amplification-at-Lower Denaturation-temperature PCR (COLD-PCR) method which selectively amplifies minority mutant alleles, in various types of clinical samples.
Design: The sensitivity and specificity of KRAS mutation detection by pyrosequencing after COLD-PCR were compared with that using conventional PCR. Both PCR were performed simultaneously with identical set of primers that amplified a 98 bp region from genomic DNA covering codons 12 and 13 of KRAS gene. Based on melting temperature profile, the critical denaturation temperature for COLD-PCR was set to 80 C versus 94 C in conventional PCR. Both PCR reactions comprised of 50 cycles. Sensitivities were determined by serial dilution of a positive patient sample in to a negative control. Clinical specimens included bone marrows from 16 leukemia patients and manually microdissected formalin-fixed paraffin-embedded (FFPE) tissue of 28 solid tumors from colon, rectum, liver, lung and bladder.
Results: COLD-PCR increased the sensitivity of KRAS mutation detection compared to conventional PCR from 1:8 to 1:32 dilution. In subsequent studies with clinical specimens, COLD-PCR successfully detected mutations in all samples that were positive by conventional PCR, and enhanced the mutant to wild-type ratio by 2 to 3 folds. In a leukemia follow up bone marrow, COLD-PCR detected a mutation that was missed by conventional PCR. The enhancement of mutation detection by COLD-PCR inversely correlated with the tumor cell percentage in the samples. Higher enhancement was observed in FFPE samples than in bone marrow specimens. Implementation of the method was straightforward, requiring no additional cost for reagents and instruments. The method maintained high specificity and reproducibility.
Conclusions: Our study showed that COLD-PCR increased the KRAS mutation detection sensitivity from 6% to 1.5% in comparison to conventional PCR methods. This method is especially beneficial for clinical applications in solid tumors due to more prominent enhancement of mutation detection in FFPE samples. COLD-PCR may not only reduce repeat testing due to equivocal results in specimens that have low percentage of tumor cells in high non-neoplastic background but also find utility in monitoring therapy response.
Wednesday, March 11, 2009 1:00 PM
Poster Session VI # 229, Wednesday Afternoon