Skip to main content

Advertisement

Log in

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

  • Original Article
  • Published:
Virchows Archiv Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Lockhart AC, Berlin JD (2005) The epidermal growth factor receptor as a target for colorectal cancer therapy. Semin Oncol 32:52–60

    Article  PubMed  CAS  Google Scholar 

  2. Ciardiello F, Tortora G (2008) EGFR antagonists in cancer treatment. N Engl J Med 358:1160–1174

    Article  PubMed  CAS  Google Scholar 

  3. Amado RG, Wolf M, Peeters M, van Cutsem E, Siena S, Freeman DJ, Juan T, Sikorski R, Suggs S, Radinsky R, Patterson SD, Chang DD (2008) Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J Clin Oncol 26:1626–1634

    Article  PubMed  CAS  Google Scholar 

  4. Karapetis CS, Khambata-Ford S, Jonker DJ, O'Callaghan CJ, Tu D, Tebbutt NC, Simes RJ, Chalchal H, Shapiro JD, Robitaille S, Price TJ, Shepherd L, Au HJ, Langer C, Moore MJ, Zalcberg JR (2008) K-ras mutations and benefit from cetuximab in advanced colorectal cancer. N Engl J Med 359:1757–1765

    Article  PubMed  CAS  Google Scholar 

  5. Lievre A, Bachet JB, Boige V, Cayre A, Le Corre D, Buc E, Ychou M, Bouche O, Landi B, Louvet C, Andre T, Bibeau F, Diebold MD, Rougier P, Ducreux M, Tomasic G, Emile JF, Penault-Llorca F, Laurent-Puig P (2008) KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab. J Clin Oncol 26:374–379

    Article  PubMed  CAS  Google Scholar 

  6. European Medicines Agency (2008) Committee for Medicinal Product for Human Use, May 2008 plenary meeting monthly report. London

  7. Engstrom PF, Arnoletti JP, Benson AB 3rd, Chen YJ, Choti MA, Cooper HS, Covey A, Dilawari RA, Early DS, Enzinger PC, Fakih MG, Fleshman J Jr, Fuchs C, Grem JL, Kiel L, Knol JA, Leong LA, Lin E, Mulcahy MF, Rao S, Ryan DP, Saltz L, Shibata D, Skibber JM, Sofocleous C, Thomas J, Venook AP, Willett C, National Comprehensive Cancer Network (2009) NCCN clinical practice guidelines in oncology: colon cancer. J Natl Compr Canc Netw 7:778–831

    Google Scholar 

  8. Bellon E, Ligtenberg MJ, Tejpar S, Cox K, de Hertogh G, de Stricker K, Edsjo A, Gorgoulis V, Hofler G, Jung A, Kotsinas A, Laurent-Puig P, Lopez-Rios F, Hansen TP, Rouleau E, Vandenberghe P, van Krieken JJ, Dequeker E (2011) External quality assessment for KRAS testing is needed: setup of a european program and report of the first joined regional quality assessment rounds. Oncologist 16:467–478

    Article  PubMed  CAS  Google Scholar 

  9. van Krieken JH, Jung A, Kirchner T, Carneiro F, Seruca R, Bosman FT, Quirke P, Flejou JF, Plato Hansen T, de Hertogh G, Jares P, Langner C, Hoefler G, Ligtenberg M, Tiniakos D, Tejpar S, Bevilacqua G, Ensari A (2008) KRAS mutation testing for predicting response to anti-EGFR therapy for colorectal carcinoma: proposal for an European quality assurance program. Virchows Arch 453:417–431

    Article  PubMed  Google Scholar 

  10. Querings S, Altmuller J, Ansen S, Zander T, Seidel D, Gabler F, Peifer M, Markert E, Stemshorn K, Timmermann B, Saal B, Klose S, Ernestus K, Scheffler M, Engel-Riedel W, Stoelben E, Brambilla E, Wolf J, Nurnberg P, Thomas RK (2011) Benchmarking of mutation diagnostics in clinical lung cancer specimens. PLoS One 6:e19601

    Article  PubMed  CAS  Google Scholar 

  11. Tsiatis AC, Norris-Kirby A, Rich RG, Hafez MJ, Gocke CD, Eshleman JR, Murphy KM (2010) Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic and clinical implications. J Mol Diagn 12:425–432

    Article  PubMed  CAS  Google Scholar 

  12. Angulo B, Garcia-Garcia E, Martinez R, Suarez-Gauthier A, Conde E, Hidalgo M, Lopez-Rios F (2010) A commercial real-time PCR kit provides greater sensitivity than direct sequencing to detect KRAS mutations: a morphology-based approach in colorectal carcinoma. J Mol Diagn 12:292–299

    Article  PubMed  CAS  Google Scholar 

  13. Feigelson HS, Goddard KA, Johnson MA, Funk KC, Rahm AK, Kauffman TL, Chitale DA, Le Marchand L, Richards CS (2012) Reliability of KRAS mutation testing in metastatic colorectal cancer patients across five laboratories. BMC Res Notes 5:196

    Article  PubMed  CAS  Google Scholar 

  14. Oliner K, Juan T, Suggs S, Wolf M, Sarosi I, Freeman DJ, Gyuris T, Baron W, Bakker A, Parker A, Patterson SD (2010) A comparability study of 5 commercial KRAS tests. Diagn Pathol 5:23

    Article  PubMed  Google Scholar 

  15. Weichert W, Schewe C, Lehmann A, Sers C, Denkert C, Budczies J, Stenzinger A, Joos H, Landt O, Heiser V, Rocken C, Dietel M (2010) KRAS genotyping of paraffin-embedded colorectal cancer tissue in routine diagnostics: comparison of methods and impact of histology. J Mol Diagn 12:35–42

    Article  PubMed  CAS  Google Scholar 

  16. Whitehall V, Tran K, Umapathy A, Grieu F, Hewitt C, Evans TJ, Ismail T, Li WQ, Collins P, Ravetto P, Leggett B, Salto-Tellez M, Soong R, Fox S, Scott RJ, Dobrovic A, Iacopetta B (2009) A multicenter blinded study to evaluate KRAS mutation testing methodologies in the clinical setting. J Mol Diagn 11:543–552

    Article  PubMed  CAS  Google Scholar 

  17. Dijkstra JR, Opdam FJ, Boonyaratanakornkit J, Schonbrunner ER, Shahbazian M, Edsjo A, Hoefler G, Jung A, Kotsinas A, Gorgoulis VG, Lopez-Rios F, de Stricker K, Rouleau E, Biesmans B, van Krieken JH (2012) Implementation of formalin-fixed, paraffin-embedded cell line pellets as high-quality process controls in quality assessment programs for KRAS mutation analysis. J Mol Diagn 14:187–191

    Article  PubMed  Google Scholar 

  18. Richman SD, Seymour MT, Chambers P, Elliott F, Daly CL, Meade AM, Taylor G, Barrett JH, Quirke P (2009) KRAS and BRAF mutations in advanced colorectal cancer are associated with poor prognosis but do not preclude benefit from oxaliplatin or irinotecan: results from the MRC FOCUS trial. J Clin Oncol 27:5931–5937

    Article  PubMed  CAS  Google Scholar 

  19. Zuo Z, Chen SS, Chandra PK, Galbincea JM, Soape M, Doan S, Barkoh BA, Koeppen H, Medeiros LJ, Luthra R (2009) Application of COLD-PCR for improved detection of KRAS mutations in clinical samples. Mod Pathol 22:1023–1031

    Article  PubMed  CAS  Google Scholar 

  20. Dijkstra JR, Mekenkamp LJ, Teerenstra S, De Krijger I, Nagtegaal ID (2011) MicroRNA expression in formalin-fixed paraffin embedded tissue using real time quantitative PCR; the strengths and pitfalls. J Cell Mol Med 16:683–90

    Google Scholar 

  21. Kramer D, Thunnissen FB, Gallegos-Ruiz MI, Smit EF, Postmus PE, Meijer CJ, Snijders PJ, Heideman DA (2009) A fast, sensitive and accurate high resolution melting (HRM) technology-based assay to screen for common K-ras mutations. Cell Oncol 31:161–167

    PubMed  CAS  Google Scholar 

  22. Lewis F, Maughan NJ, Smith V, Hillan K, Quirke P (2001) Unlocking the archive—gene expression in paraffin-embedded tissue. J Pathol 195:66–71

    Article  PubMed  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to J. R. Dijkstra.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOC 97 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Dijkstra, J.R., Heideman, D.A.M., Meijer, G.A. et al. KRAS mutation analysis on low percentage of colon cancer cells: the importance of quality assurance. Virchows Arch 462, 39–46 (2013). https://doi.org/10.1007/s00428-012-1356-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00428-012-1356-2

Keywords

Navigation