High sensitivity detection and robust enrichment for PIK3CA somatic mutations using ice COLD-PCR technology.

Abstract

e13546 Background: High sensitivity detection of cancer biomarkers facilitates early diagnosis and provides insight for intervention including stratification for drug treatment and monitoring changes in tumor mutation profiles by analyzing DNA isolated from serum/plasma or circulating tumor cells. PIK3CA, encoding the PI3K p110α catalytic subunit, is mutated frequently in common cancers, including breast and colon carcinomas. 80% of these mutations are represented by three amino acid substitutions in the enzyme’s helical (E542K and E545K) or kinase (H1047R) domains. Methods: In this study, we have applied improved and complete enrichment CO-amplification at lower denaturation temperature PCR (Ice COLD-PCR) technology for high sensitivity detection and robust enrichment of any mutations present in a DNA sample. Ice-COLD-PCR preferentially amplifies low levels of any mutant DNA in a sample containing a vast excess of wild-type DNA. The use of a reference sequence oligonucleotide complimentary to one of the wild-type DNA strands results in linear amplification of one of the wild type strands while both mutant strands amplify exponentially during ice COLD- PCR. After Ice COLD-PCR, there is sufficient mutant allele enrichment to allow Sanger sequencing confirmation of the mutation. Results: The use of Ice COLD-PCR allows for the enrichment of mutations any of the key mutations in the PIK3CA gene that cannot be detected by standard PCR followed by Sanger sequencing methods. Initial limit of detection experiments indicate that the detection and sequence confirmation of mutations in PIK3CA more sensitive than standard PCR amplification or allele specific probe methodologies. Conclusions: After Ice COLD-PCR, low level PIK3CA mutant alleles are enriched over the wild-type allele such that simple sequence confirmation on multiple downstream systems such as Sanger Sequencing or allele-specific probe platforms is possible. Ice COLD-PCR amplification of PIK3CA and other genes requires only a standard thermocycler coupled to any downstream sequence analysis platform and therefore can be developed as a powerful, highly sensitive diagnostic assay.