Optimization of routine KRAS mutation PCR-based testing procedure for rational individualized first-line-targeted therapy selection in metastatic colorectal cancer

Cancer Med. 2013 Feb;2(1):11-20. doi: 10.1002/cam4.47. Epub 2013 Feb 3.


KRAS mutation detection represents a crucial issue in metastatic colorectal cancer (mCRC). The optimization of KRAS mutation detection delay enabling rational prescription of first-line treatment in mCRC including anti-EGFR-targeted therapy requires robust and rapid molecular biology techniques. Routine analysis of mutations in codons 12 and 13 on 674 paraffin-embedded tissue specimens of mCRC has been performed for KRAS mutations detection using three molecular biology techniques, that is, high-resolution melting (HRM), polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP), and allelic discrimination PCR (TaqMan PCR). Discordant cases were assessed with COBAS 4800 KRAS CE-IVD assay. Among the 674 tumor specimens, 1.5% (10/674) had excessive DNA degradation and could not be analyzed. KRAS mutations were detected in 38.0% (256/674) of the analysable specimens (82.4% in codon 12 and 17.6% in codon 13). Among 613 specimens in whom all three techniques were used, 12 (2.0%) cases of discordance between the three techniques were observed. 83.3% (10/12) of the discordances were due to PCR-RFLP as confirmed by COBAS 4800 retrospective analysis. The three techniques were statistically comparable (κ > 0.9; P < 0.001). From these results, optimization of the routine procedure consisted of proceeding to systematic KRAS detection using HRM and TaqMan and PCR-RFLP in case of discordance and allowed significant decrease in delays. The results showed an excellent correlation between the three techniques. Using HRM and TaqMan warrants high-quality and rapid-routine KRAS mutation detection in paraffin-embedded tumor specimens. The new procedure allowed a significant decrease in delays for reporting results, enabling rational prescription of first-line-targeted therapy in mCRC.

Keywords: Colorectal cancer; HRM; KRAS; PCR-RFLP; TaqMan PCR.

MeSH terms

  • Adult
  • Aged
  • Aged, 80 and over
  • Colorectal Neoplasms / drug therapy
  • Colorectal Neoplasms / genetics*
  • DNA Mutational Analysis / methods
  • DNA, Neoplasm / genetics
  • Female
  • Humans
  • Male
  • Middle Aged
  • Molecular Targeted Therapy
  • Mutation*
  • Neoplasm Metastasis
  • Nucleic Acid Denaturation
  • Patient Selection
  • Polymerase Chain Reaction / methods
  • Polymorphism, Restriction Fragment Length
  • Proto-Oncogene Proteins / genetics*
  • Proto-Oncogene Proteins p21(ras)
  • Sensitivity and Specificity
  • Young Adult
  • ras Proteins / genetics*


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