Application of COLD-PCR for Improved Detection of KRAS Mutations in Clinical Samples

Mod Pathol. 2009 Aug;22(8):1023-31. doi: 10.1038/modpathol.2009.59. Epub 2009 May 8.

Abstract

KRAS mutations have been detected in approximately 30% of all human tumors, and have been shown to predict response to some targeted therapies. The most common KRAS mutation-detection strategy consists of conventional PCR and direct sequencing. This approach has a 10-20% detection sensitivity depending on whether pyrosequencing or Sanger sequencing is used. To improve detection sensitivity, we compared our conventional method with the recently described co-amplification-at-lower denaturation-temperature PCR (COLD-PCR) method, which selectively amplifies minority alleles. In COLD-PCR, the critical denaturation temperature is lowered to 80 degrees C (vs 94 degrees C in conventional PCR). The sensitivity of COLD-PCR was determined by assessing serial dilutions. Fifty clinical samples were used, including 20 fresh bone-marrow aspirate specimens and the formalin-fixed paraffin-embedded (FFPE) tissue of 30 solid tumors. Implementation of COLD-PCR was straightforward and required no additional cost for reagents or instruments. The method was specific and reproducible. COLD-PCR successfully detected mutations in all samples that were positive by conventional PCR, and enhanced the mutant-to-wild-type ratio by >4.74-fold, increasing the mutation detection sensitivity to 1.5%. The enhancement of mutation detection by COLD-PCR inversely correlated with the tumor-cell percentage in a sample. In conclusion, we validated the utility and superior sensitivity of COLD-PCR for detecting KRAS mutations in a variety of hematopoietic and solid tumors using either fresh or fixed, paraffin-embedded tissue.

MeSH terms

  • Adult
  • Aged
  • Aged, 80 and over
  • Cold Temperature
  • DNA Mutational Analysis / methods*
  • Female
  • Humans
  • Male
  • Middle Aged
  • Mutation
  • Neoplasms / genetics*
  • Polymerase Chain Reaction / methods*
  • Proto-Oncogene Proteins / genetics*
  • Proto-Oncogene Proteins p21(ras)
  • Sensitivity and Specificity
  • ras Proteins / genetics*

Substances

  • KRAS protein, human
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins p21(ras)
  • ras Proteins