Published online before print October 8, 2009

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Copyright © 2009 American Society for Investigative Pathology
Journal of Molecular Diagnostics, doi:10.2353/jmoldx.2009.090057

Accepted for publication June 10, 2009.

Article

A Multicenter Blinded Study to Evaluate KRAS Mutation Testing Methodologies in the Clinical Setting

Vicki Whitehall^*@, Kayla Tran, Aarti Umapathy^*, Fabienne Grieu, Chelsee Hewitt, Tiffany-Jane Evans^¶, Tuty Ismail^||, Wei Qi Li^**, Peter Collins, Paul Ravetto, Barbara Leggett^*, Manuel Salto-Tellez^||, Richie Soong^**, Stephen Fox, Rodney J. Scott^¶, Alexander Dobrovic, and Barry Iacopetta

From the Conjoint Gastroenterology Laboratory,* Royal Brisbane and Women's Hospital Research Foundation Clinical Research Centre, the Queensland Health Clinical and Statewide Services, Pathology Queensland and Queensland Institute of Medical Research, Herston, Australia; the Department of Tissue Pathology, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Sydney, Australia; the School of Surgery, University of Western Australia, Perth, Australia; the Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, Australia; the Hunter Medical Research Institute,^¶ Newcastle, Australia; the Department of Pathology,^|| National University of Singapore, Singapore; the Cancer Science Institute Singapore,** National University of Singapore, Singapore; DxS Ltd, Manchester, United Kingdom; and the Department of Pathology, University of Melbourne, Parkville, Australia

^@ To whom correspondence should be addressed. E-mail: Vicki.Whitehall{at}qimr.edu.au.

	Abstract

Evidence that activating mutations of the KRAS oncogene abolish the response to anti-epidermal growth factor receptor therapy has revolutionized the treatment of advanced colorectal cancer. This has resulted in the urgent demand for KRAS mutation testing in the clinical setting to aid choice of therapy. The aim of this study was to evaluate six different KRAS mutation detection methodologies on two series of primary colorectal cancer samples. Two series of 80 frozen and 74 formalin-fixed paraffin-embedded tissue samples were sourced and DNA was extracted at a central site before distribution to seven different testing sites. KRAS mutations in codons 12 and 13 were assessed by using single strand conformation polymorphism analysis, pyrosequencing, high resolution melting analysis, dideoxy sequencing, or the commercially available TIB Molbiol (Berlin, Germany) or DxS Diagnostic Innovations (Manchester, UK) kits. In frozen tissue samples, concordance in KRAS status (defined as consensus in at least five assays) was observed in 66/80 (83%) cases. In paraffin tissue, concordance was 46/74 (63%) if all assays were considered or 71/74 (96%) using the five best performing assays. These results demonstrate that a variety of detection methodologies are suitable and provide comparable results for KRAS mutation analysis of clinical samples.