COLD-PCR protocols to enrich KRAS codon 61 and 146 mutations in heterogeneous tumor samples.

Abstract

e22125 Background: About 40% of colorectal cancer patients show mutations on codon 12-13 of KRAS but other, less frequent and mutually exclusive mutations, were described. In Italy, treatments with Cetuximab or Panitumumab for metastatic colorectal cancer are granted only for patients with no mutations on RAS genes and analysis of codon 12,13,59,61,117,146 of KRAS and NRAS are requested before the treatment scheduling. Principal limitation of conventional PCR-based methods for mutation analysis is the inability to selectively amplify low percentages of somatic variants in an elevated background of wild-type sequences. CO-amplification at Lower Denaturation temperature PCR (COLD-PCR) is a simple approach of PCR that selectively enriches minority alleles in a mixture of wild-type and mutant sequences. This method requires the definition of a Critical Temperature (Tc) for each Dna sequence, lower than the melting temperature, at which mismatched Dna heteroduplex (WT-mutated alleles) are preferentially denatured, promoting selective amplification of mutant sequences. Minor allele enrichment by COLD-PCR can be performed in association to different methods of variants detection: Sanger Sequencing, HRM analysis, Pyrosequencing Methods: Comparison of theoretical sensitivity achievable after PCR or COLD-PCR amplification was carried out analyzing serial dilution of mutated cell lines DNA into WT DNA (from 50% to 0.25%). To optimize the enrichment of minority alleles we developed a Full COLD-PCR protocol to analyze the Tm-increasing mutation (183 A>C) on KRAS codon 61 and a Fast COLD-PCR protocol to analyze the Tm-reducing mutation (436 G>A) on KRAS codon 146. Critical temperature was defined experimentally. Results: In KRAS codon 61 and 146 analysis, minimum detectable percentage of mutated allele in WT background was about 10% using conventional PCR followed by both Sanger sequencing and HRM. Conversely limit of sensitivity reached 0.5% using both detection methods after COLD-PCR Conclusions: Our experiments proved as COLD-PCR could be a rapid and economic approach, adaptable to investigate other somatic variants, to enrich very low percentages of mutant alleles in very heterogeneous tumor samples.