KRAS mutation analysis on low percentage of colon cancer cells: the importance of quality assurance

Virchows Arch. 2013 Jan;462(1):39-46. doi: 10.1007/s00428-012-1356-2. Epub 2012 Dec 15.


KRAS mutation testing is mandatory for patients with metastatic colorectal cancer who are eligible for treatment with an epidermal growth factor receptor targeting agent, since tumors with a mutation are not sensitive to the drug. Several methods for mutation testing are in use and the need for external quality assurance has been demonstrated. An often little addressed but important issue in external quality assurance schemes is a low percentage of tumor cells in the test samples, where the analytical sensitivity of most tests becomes critical. Using artificial samples based on a mixture of cell lines with known mutation status of the KRAS gene, we assessed the reliability of a series of commonly used methods (Sanger sequencing, high resolution melting, pyrosequencing, and amplification refractory mutation system-polymerase chain reaction) on samples with 0, 2.5, 5, 10, and 15 % mutated cells. Nine laboratories throughout Europe participated and submitted a total of ten data sets. The limit of detection of each method differed, ranging from >15-5 % tumor cells. All methods showed a decreasing correct mutation call rate proportionally with decreasing percentage of tumor cells. Our findings indicate that laboratories and clinicians need to be aware of the decrease in correct mutation call rate proportionally with decreasing percentage of tumor cells and that external quality assurance schemes need to address the issue of low tumor cell percentage in the test samples.

Publication types

  • Comparative Study
  • Multicenter Study

MeSH terms

  • Adenocarcinoma / genetics*
  • Adenocarcinoma / pathology
  • Cell Count
  • Cell Line, Tumor
  • Colorectal Neoplasms / genetics*
  • Colorectal Neoplasms / pathology
  • DNA Mutational Analysis / methods
  • DNA Mutational Analysis / standards
  • DNA, Neoplasm / analysis
  • Genes, ras*
  • Humans
  • Limit of Detection
  • Molecular Diagnostic Techniques / methods
  • Molecular Diagnostic Techniques / standards
  • Mutation*
  • Polymerase Chain Reaction / methods
  • Polymerase Chain Reaction / standards
  • Proto-Oncogene Proteins / genetics*
  • Proto-Oncogene Proteins p21(ras)
  • Quality Assurance, Health Care / methods*
  • Quality Assurance, Health Care / standards
  • Reproducibility of Results
  • ras Proteins / genetics*


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