Characteristics of Co-Amplification at Lower Denaturation Temperature-PCR (COLD-PCR) for KRAS Mutant Detection in Colorectal Carcinoma
Shengle Zhang, Jamie Tull. SUNY Upstate Medical University, Syracuse, NY
Background: KRAS mutation analysis at codon 12 or 13 is important to predict response of targeted therapy against epidermal growth factor receptor (EGFR) on metastatic colorectal cancer with Erbitux. Clinical utility of regular PCR with Sanger's sequencing, a commonly used method, is limited by its low test sensitivity, i.e. 20% detection sensitivity of a mutant allele in a wild-type background. COLD-PCR provides a new approach of selective amplification on minority mutated alleles in a background of wild-type DNA. However, the characteristics of COLD-PCR plus Sanger's sequencing for KRAS mutation detection remains unclear in clinical samples of formalin fixed paraffin embedded tissue (FFPE).
Design: Eighteen FFPE samples of colorectal carcinoma with 10-80% tumor cellularity and known KRAS mutation were tested by regular PCR and COLD-PCR followed by Sanger's sequencing. The mutant to wild-type ratio between regular PCR and COLD-PCR were compared, and their correlation with tumor cellularity and variants of mutation were evaluated.
Results: The mutant/wild type ratios were generally increased in the group of COLD-PCR. However, the degree of increase was associated with mutant to wild-type ratio seen in regular PCR. When the ratios in regular PCR were ≤ 50% (8 cases), the ratios in COLD-PCR were increased by 82% on average. When the ratios in regular PCR were 50 ∼ 130% (8 cases), the ratios in COLD-PCR were increased by only 19% on average. Interestingly, when the ratios in regular PCR were > 140% (2 cases), no increase or even reverse effect was observed in COLD-PCR group. Mutant/wild-type ratio was associated with tumor % cellularity only in a lower cellular group, and not related to types of KRAS mutation.
Conclusions: Improved KRAS mutant detection with COLD-PCR was demonstrated predominately in those with lower mutant/wild-type ratios, typically < 50%, in regular PCR. Through the COLD-PCR approach with Sanger's sequencing, test sensitivity for KRAS mutation can be elevated from 20% to 10% in general.
Monday, March 19, 2012 1:00 PM
Poster Session II # 293, Monday Afternoon