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Virchows Archiv

, Volume 462, Issue 1, pp 39–46 | Cite as

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

  • J. R. Dijkstra
  • D. A. M. Heideman
  • G. A. Meijer
  • J. E. Boers
  • N. A. ‘t Hart
  • J. Diebold
  • A. Hirschmann
  • G. Hoefler
  • G. Winter
  • G. Miltenberger-Miltenyi
  • S. V. Pereira
  • S. D. Richman
  • P. Quirke
  • E. L. Rouleau
  • J. M. Guinebretiere
  • S. Tejpar
  • B. Biesmans
  • J. H. J. M. van Krieken
Original Article

Abstract

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.

Keywords

KRAS Mutation Colon cancer EQA 

Notes

Conflict of interest

At the time of the study, Roche Molecular had an exclusive distribution agreement with DxS. In 2010, the distribution agreement was terminated with the acquisition of DxS by Qiagen. Roche provided aid in specimen preparation, data, and statistical analysis. Roche has declined authorship in this manuscript. All other authors declare no conflict of interest.

Supplementary material

428_2012_1356_MOESM1_ESM.doc (97 kb)
ESM 1 (DOC 97 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • J. R. Dijkstra
    • 1
  • D. A. M. Heideman
    • 2
  • G. A. Meijer
    • 2
  • J. E. Boers
    • 3
  • N. A. ‘t Hart
    • 3
  • J. Diebold
    • 4
  • A. Hirschmann
    • 4
  • G. Hoefler
    • 5
  • G. Winter
    • 5
  • G. Miltenberger-Miltenyi
    • 6
  • S. V. Pereira
    • 6
  • S. D. Richman
    • 7
  • P. Quirke
    • 7
  • E. L. Rouleau
    • 8
  • J. M. Guinebretiere
    • 8
  • S. Tejpar
    • 9
  • B. Biesmans
    • 9
  • J. H. J. M. van Krieken
    • 1
  1. 1.Department of PathologyRadboud University Nijmegen Medical CentreNijmegenThe Netherlands
  2. 2.Department of PathologyVU University Medical CenterAmsterdamThe Netherlands
  3. 3.Department of PathologyIsala KliniekenZwolleThe Netherlands
  4. 4.Institute of PathologyLuzerner KantonsspitalLucerne 16Switzerland
  5. 5.Institute of PathologyMedical University GrazGrazAustria
  6. 6.Instituto de Medicina MolecularFaculdade de Medicina da Universidade de LisboaLisboaPortugal
  7. 7.The Sections of Pathology and Tumour Biology and Oncology, Leeds Institute of Molecular Medicine, Wellcome Trust Brenner BuildingSt James University HospitalLeedsUK
  8. 8.Institut Curie, Oncogenetic LaboratoryHôpital René HugeninSaint-CloudFrance
  9. 9.Center for Human Genetics O&N1Katholieke Universiteit LeuvenLeuvenBelgium

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