Abstract 2119: Very high sensitivity detection of K-RAS exon 2 mutations using fast COLD-PCR
Authors: Candau Reyes , Shandilya Harini , Han Ying , Eastlake Phil , Kaldjian Eric , Makrigiorgos G. Mike , Gerard Gary F. 
Published: February 2011

Abstract
Therapeutic agents such as cetuximab and panitumumab are epidermal growth factor receptor (EGFR) antagonists that can be effective in the treatment of colorectal cancer. However some 40% of these tumors have activating K-RAS exon 2 codon 12 and 13 mutations that have a strong association with poor response to EGFR antagonists. Attempts have been made to detect the presence of K-RAS exon 2 mutations in free circulating DNA from plasma/serum from colorectal cancer patients with K-RAS exon 2 mutations in their tumors. The small amount of circulating DNA in serum and the low sensitivity of the detection techniques used have resulted in sporadic success in establishing this correlation.
Co-amplification at Lower Denaturation Temperature-PCR (COLD-PCR) is a novel form of PCR that preferentially amplifies mutant alleles in a mutant/wild-type mixture with wild-type alleles in vast excess. Fast COLD-PCR, one of two forms of the method, is applied to point mutations or insertion/deletions that lower the melting temperature (Tm) of the mutant allele. In Fast COLD-PCR, preferential amplification of a mutant allele is achieved by setting the PCR denaturation temperature during each cycle at the critical temperature (Tc) at which mutant DNA duplexes but not wild-type duplexes are denatured. COLD-PCR is normally carried out in a thermocycler with independent temperature control of each well, ensuring that Tc is precisely achieved. Well-to-well temperature variation of standard, bench thermocyclers is often not within the narrow range required to achieve efficient COLD-PCR mutation enrichment. We have developed a variation of the Fast COLD-PCR method that reduces the need for precise well-to-well temperature control. In this study we have taken advantage of the Tm lowering of most of the K-RAS exon 2 mutations in codons 12 and 13 (G&gt;A or G&gt;T), and have successfully applied our variation of Fast COLD-PCR to their enrichment.
A plasmid-based model system was established to measure the Limit of Detection (LOD) of the K-RAS exon 2 codon 12 mutation G12S (GGT&gt;GAT). Mixtures of two plasmids, one bearing a wild-type copy of exon 2 and the other the G12S mutation were prepared at ratios of mutant/wild-type ranging from 1:10 to 1:1,000 (0.1%). A total of 10,000 plasmid copies alone or combined with purified human serum DNA were enriched. Two rounds of PCR were performed: standard PCR producing a 218-bp amplicon followed by enrichment using nested Fast COLD-PCR at a Tc of 80.9 °C producing a 161-bp amplicon. SURVEYOR® Nuclease/WAVE® HPLC and Sanger DNA sequencing were used to identify the G&gt;A mutation in the enriched PCR product. The LOD for the G12S mutation before enrichment was 1% by SURVEYOR/WAVE and 5% by sequencing. After Fast COLD-PCR enrichment, the LOD was &lt;0.1% in the absence and &lt;0.4% in the presence of serum with both detection methods. This represents detection of less than 1 mutant in 1000 wild-type K-RAS copies.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2119.