Published online before print February 7, 2008

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Copyright © 2008 American Society for Investigative Pathology
Journal of Molecular Diagnostics, doi:10.2353/jmoldx.2008.070087

Accepted for publication October 19, 2007.

Article

Detection of Microsatellite Instability in Colorectal Cancer Using an Alternative Multiplex Assay of Quasi-Monomorphic Mononucleotide Markers

Vanessa Deschoolmeester^*@, Marc Baay^*, Wim Wuyts, Eric Van Marck, Nancy Van Damme, Peter Vermeulen^¶, Krzysztof Lukaszuk^||, Filip Lardon^*, and Jan B. Vermorken^*

From the Laboratory of Cancer Research and Clinical Oncology,* Department of Medical Oncology, and the Department of Medical Genetics, University of Antwerp (UA/UZA), Wilrijk, Belgium; the Department of Pathology, University Hospital of Antwerp (UZA), Edegem, Belgium; the Department of Gastroenterology, Ghent University Hospital, Ghent, Belgium; the Translational Cancer Research Group^¶ (Laboratory Pathology, University of Antwerp/University Hospital Antwerp, Oncology Center, St. Augustinus General Hospital), Wilrijk, Belgium; and the Department of Gynaecological Endocrinology,^|| University of Gdansk and INVICTA, Prophylactic Centre, Gdansk, Poland

^@ To whom correspondence should be addressed. E-mail: vanessa.deschoolmeester{at}ua.ac.be.

	Abstract

Colorectal malignancies demonstrating microsatellite instability (MSI) have a very heterogeneous histological appearance, better prognosis, and altered response to therapy. Consequently, identification of the MSI phenotype is both relevant and interesting as a screening and prognostic tool and as a potential predictive factor of chemotherapeutic response. Several groups have argued for the exclusive use of mononucleotide markers for MSI analysis. In this study, an alternative MSI typing multiplex system of mononucleotide microsatellite repeats was developed. This system obviates the need to compare allelic profiles between tumor and matching normal DNA, rendering MSI analysis amenable to high throughput. The quasi-monomorphic allelic distribution of five alternative mononucleotide markers was evaluated in genomic DNA. Only SEC63 and CAT25 were found to be quasi-monomorphic and were thus combined with BAT25 and BAT26 from the Bethesda panel. Consequently, 177 colorectal cancer samples previously analyzed by the Bethesda panel were tested for MSI using this alternative mononucleotide panel. In an attempt to resolve discordant cases, immunohistochemistry of MLH1, MSH2, and MSH6 was performed. The concordance between both panels reached 99.4% when microsatellite stability and MSI-L were grouped together. These new markers were subsequently multiplexed in a single polymerase chain reaction assay. The resulting mononucleotide fluorescent multiplex MSI assay has high accuracy, reliability, and throughput, thus reducing the time and cost involved in MSI testing.