Results of first proficiency test for KRAS testing with formalin-fixed, paraffin-embedded cell lines in China

Rui Zhang; Yanxi Han; Jie Huang; Liang Ma; Yulong Li; Jinming Li

doi:10.1515/cclm-2014-0227

Article

Results of first proficiency test for KRAS testing with formalin-fixed, paraffin-embedded cell lines in China

Rui Zhang , Yanxi Han , Jie Huang , Liang Ma , Yulong Li and Jinming Li

Published/Copyright: June 19, 2014

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From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 52 Issue 12

Abstract

Background: Laboratory testing for KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) mutations in metastatic colorectal cancer (mCRC) is performed by various methods in China, but there is no standardized system for proficiency testing or assay performance evaluations. The aim of this study was to evaluate assay and laboratory performance with artificial samples derived from formalin-fixed, paraffin-embedded (FFPE) cell lines.

Methods: Artificial FFPE samples were prepared from cultured cell lines to construct a proficiency panel of 10 samples covering eight KRAS mutations and two wild-type samples. The samples were validated by Sanger sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The panel was distributed to participating laboratories and their reported results were compared to the reference sequences.

Results: The percentages of mutant KRAS alleles in each mutant sample were more than 50% by MALDI-TOF-MS. Sixty-three laboratories reported results, including 41 hospital laboratories and 22 commercial laboratories and reagent manufacturers. Only 55.6% (35/63) of the laboratories correctly identified the mutations in all samples and 33.3% (21/63) reported at least one false-positive result. The false-positive ratio was 7.1% (45/630) and the false-negative ratio was 3.0% (15/504).

Conclusions:KRAS mutations can be missed even by the most sensitive methods if the procedures are not performed correctly. False-positive results are a substantial problem in KRAS testing; laboratories must use sufficient negative controls to identify cross-contamination from PCR-amplified products or between samples during handling and DNA extraction.

Keywords: colorectal cancer; KRAS; proficiency testing; quality assurance

Corresponding author: Jinming Li, MD, PhD., National Center for Clinical Laboratories, Beijing Hospital of the Ministry of Health, 1 Dahua Road, Dongdan, Beijing 100730, P.R. China, Phone: +86 10 58115053, Fax: +86 10 65212064, E-mail: jmli63hn@gmail.com

a Rui Zhang and Yanxi Han contributed equally to the work.

References

1. Brink M, de Goeij AF, Weijenberg MP, Roemen GM, Lentjes MH, Pachen MM, et al. K-ras oncogene mutations in sporadic colorectal cancer in the Netherlands Cohort Study. Carcinogenesis 2003;24:703–10.10.1093/carcin/bgg009Search in Google Scholar PubMed

2. Ogino S, Kawasaki T, Kirkner G, Suemoto Y, Meyerhardt J, Fuchs CS. Molecular correlates with MGMT promoter methylation and silencing support CpG island methylator phenotype-low (CIMP-low) in colorectal cancer. Gut 2007;56:1564–71.10.1136/gut.2007.119750Search in Google Scholar PubMed PubMed Central

3. Kim ST, Park KH, Kim JS, Shin SW, Kim YH. Impact of KRAS mutation status on outcomes in metastatic colon cancer patients without anti-epidermal growth factor receptor therapy. Cancer Res Treat 2013;45:56–62.10.4143/crt.2013.45.1.55Search in Google Scholar PubMed PubMed Central

4. Peeters M, Douillard JY, Van Cutsem E, Siena S, Zhang K, Williams R, et al. Mutant KRAS codon 12 and 13 alleles in patients with metastatic colorectal cancer: assessment as prognostic and predictive biomarkers of response to panitumumab. J Clin Oncol 2013;31:759–65.10.1200/JCO.2012.45.1492Search in Google Scholar PubMed

5. Bos JL. Ras oncogenes in human cancer: a review. Cancer Res 1989;49:4682–9.Search in Google Scholar

6. Rouleau E, Spyratos F, Dieumegard B, Guinebretière JM, Lidereau R, Bièche I. KRAS mutation status in colorectal cancer to predict response to EGFR targeted therapies: the need for a more precise definition. Br J Cancer 2008;99:2100.10.1038/sj.bjc.6604815Search in Google Scholar PubMed PubMed Central

7. Lièvre A, Bachet JB, Le Corre D, Boige V, Landi B, Emile JF, et al. KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer. Cancer Res 2006;66:3992–5.10.1158/0008-5472.CAN-06-0191Search in Google Scholar PubMed

8. Ciardiello F, Tejpar S, Normanno N, Mercadante D, Teague T, Wohlschlegel B, et al. Uptake of KRAS mutation testing in patients with metastatic colorectal cancer in Europe, Latin America and Asia. Target Oncol 2011;6:133–45.10.1007/s11523-011-0181-xSearch in Google Scholar PubMed

9. Bando H, Yoshino T, Tsuchihara K, Ogasawara N, Fuse N, Kojima T, et al. KRAS mutations detected by the amplification refractory mutation system-Scorpion assays strongly correlate with therapeutic effect of cetuximab. Br J Cancer 2011;105:403–6.10.1038/bjc.2011.247Search in Google Scholar PubMed PubMed Central

10. Karim B, Florence C, Kamel R, Nadia K, Ines O, Raja M, et al. KRAS mutation detection in Tunisian sporadic coloractal cancer patients with direct sequencing, high resolution melting and denaturating high performance liquid chromatography. Cancer Biomark 2010–2011;8:331–40.10.3233/CBM-2011-0222Search in Google Scholar PubMed

11. Hsieh LL, Er TK, Chen CC, Hsieh JS, Chang JG, Liu TC. Characteristics and prevalence of KRAS, BRAF, and PIK3CA mutations in colorectal cancer by high-resolution melting analysis in Taiwanese population. Clin Chim Acta 2012;413:1605–11.10.1016/j.cca.2012.04.029Search in Google Scholar PubMed

12. Blons H, Laurent-Puig P. Technical considerations for KRAS testing in colorectal cancer. The biologist’s point of view. Bull Cancer 2009;96(Suppl):S47–56.10.1684/bdc.2009.0996Search in Google Scholar PubMed

13. Dijkstra JR, Opdam FJ, Boonyaratanakornkit J, Schönbrunner ER, Shahbazian M, Edsjö A, et al. 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 2012;14:187–91.10.1016/j.jmoldx.2012.01.002Search in Google Scholar PubMed

14. Deans ZC, Tull J, Beighton G, Abbs S, Robinson DO, Butler R. Molecular genetics external quality assessment pilot scheme for KRAS analysis in metastatic colorectal cancer. Genet Test Mol Biomark 2011;15:777–83.10.1089/gtmb.2010.0239Search in Google Scholar PubMed

15. Dijkstra JR, Heideman DA, Meijer GA, Boers JE,’t Hart NA, Diebold J, et al. KRAS mutation analysis on low percentage of colon cancer cells: the importance of quality assurance. Virchows Archiv 2013;462:39–46.10.1007/s00428-012-1356-2Search in Google Scholar PubMed

16. Kamel-Reid S, Zhang T, Persons DL, Nikiforova MN, Halling KC. Molecular Oncology Resource Committee of the College of American Pathologists. Validation of KRAS testing for anti-EGFR therapeutic decisions for patients with metastatic colorectal carcinoma. Arch Pathol Lab Med 2012;136:26–3.10.5858/arpa.2011-0220-OASearch in Google Scholar PubMed

17. Bando H, Tsuchihara K, Yoshino T, Kojima M, Ogasawara N, Fukushima H, et al. Biased discordance of KRAS mutation detection in archived colorectal cancer specimens between the ARMS-Scorpion method and direct sequencing. Jpn J Clin Oncol 2011;41:239–44.10.1093/jjco/hyq216Search in Google Scholar PubMed

18. Bellon E, Ligtenberg MJ, Tejpar S, Cox K, de Hertogh G, de Stricker K, et al. External quality assessment for KRAS testing is needed: setup of a European program and report of the first joined regional quality assessment rounds. Oncologist 2011;16:467–78.10.1634/theoncologist.2010-0429Search in Google Scholar PubMed PubMed Central

19. Thunnissen E, Bovée JV, Bruinsma H, van den Brule AJ, Dinjens W, Heideman DA, et al. EGFR and KRAS quality assurance schemes in pathology: generating normative data for molecular predictive marker analysis in targeted therapy. J Clin Pathol 2011;64:884–92.10.1136/jclinpath-2011-200163Search in Google Scholar PubMed

20. Dufort S, Richard MJ, de Fraipont F. Pyrosequencing method to detect KRAS mutation in formalin-fixed and paraffin-embedded tumor tissues. Anal Biochem 2009;391:166–8.10.1016/j.ab.2009.05.027Search in Google Scholar PubMed

21. Whitehall V, Tran K, Umapathy A, Grieu F, Hewitt C, Evans TJ, et al. A multicenter blinded study to evaluate KRAS mutation testing methodologies in the clinical setting. J Mol Diagn 2009;11:543–52.10.2353/jmoldx.2009.090057Search in Google Scholar PubMed PubMed Central

22. Normanno N, Pinto C, Taddei G, Gambacorta M, Castiglione F, Barberis M, et al. Results of the First Italian External Quality Assurance Scheme for somatic EGFR mutation testing in non-small-cell lung cancer. J Thorac Oncol 2013;8:773–8.10.1097/JTO.0b013e31828c2b08Search in Google Scholar PubMed

23. Normanno N, Pinto C, Castiglione F, Bardelli A, Gambacorta M, Botti G, et al. KRAS mutations testing in colorectal carcinoma patients in Italy: from guidelines to external quality assessment. PLoS One 2011;6:e29146.10.1371/journal.pone.0029146Search in Google Scholar PubMed PubMed Central

Received: 2014-2-28

Accepted: 2014-5-28

Published Online: 2014-6-19

Published in Print: 2014-12-1

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Articles in the same Issue

https://doi.org/10.1515/cclm-2014-0227

Keywords for this article

colorectal cancer; KRAS; proficiency testing; quality assurance