This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bettstetter, M. Articles by Dietmaier, W. Search for Related Content PubMed PubMed Citation Articles by Bettstetter, M. Articles by Dietmaier, W.

Distinction of Hereditary Nonpolyposis Colorectal Cancer and Sporadic Microsatellite-Unstable Colorectal Cancer through Quantification of MLH1 Methylation by Real-time PCR

Authors' Affiliations: ¹ Institute of Pathology and Molecular Diagnostics and ² Department of Surgery, University of Regensburg, Regensburg, Germany; ³ Institute of Human Genetics, Ludwig-Maximilian-University; and ⁴ Institute of Pathology, University of Polytechniques, Munich, Germany

Requests for reprints: Wolfgang Dietmaier, Department of Pathology, University of Regensburg, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, Germany. Phone: 49-941-944-6624; Fax: 49-941-944-6602; E-mail: Wolfgang.dietmaier{at}klinik.uni-regensburg.de.

Purpose: Promoter hypermethylation occurs frequently in tumors and leads to silencing of tumor-relevant genes like tumor suppressor genes. In a subset of sporadic colorectal cancers (CRC), inactivation of the mismatch repair gene MLH1 due to promoter methylation causes high level of microsatellite instability (MSI-H). MSI-H is also a hallmark of hereditary nonpolyposis colorectal cancer (HNPCC) in which mismatch repair inactivation results from germ-line mutations. For differentiation of sporadic and hereditary MSI-H tumor patients, MLH1 promoter methylation analysis is a promising tool but is not yet used in daily diagnostics because only qualitative techniques without standardization are available. The aim of this study is to establish a reliable and quantitative MLH1 methylation analysis technique and to define valid MLH1 methylation cutoff values for HNPCC diagnostics.

Experimental Design: We developed a new real-time PCR–based technique to detect and quantify methylation of both proximal and distal hMLH1 promoter regions. We established and validated this technique in a cohort of 108 CRCs [94 MSI-H and 16 microsatellite stable (MSS) cases] comprising a reference (n = 58) and a tester tumor group (n = 50).

Results: The reference tumor group contained 28 HNPCC with proven germ-line mutations or positive Amsterdam I criteria (median age, 37 years) and loss of MLH1 expression, 14 sporadic MSI-H CRC tumors with loss of MLH1 expression and BRAF V600E mutation (median age, 80.5 years), and 16 sporadic MSS CRC (median age, 76.5 years). No MLH1 promoter methylation could be found in any MSS tumors. HNPCC patients showed no or low level of MLH1 promoter methylation. A cutoff value of 18% methylation extent could be determined in this study to define MLH1 hypermethylation specific for sporadic MSI-H cases. Methylation could also be verified qualitatively by melting point analysis. BRAF V600E mutations were not detected in any HNPCC patients (n = 22 informative cases).

Conclusion: According to the present data, quantitative MLH1 methylation analysis in MSI-H CRC is a valuable molecular tool to distinguish between HNPCC and sporadic MSI-H CRC. The detection of a BRAF V600E mutation further supports the exclusion of HNPCC.

This article has been cited by other articles:

				K. E. Varley, D. G. Mutch, T. B. Edmonston, P. J. Goodfellow, and R. D. Mitra Intra-tumor heterogeneity of MLH1 promoter methylation revealed by deep single molecule bisulfite sequencing Nucleic Acids Res., June 3, 2009; (2009) gkp457v1. [Abstract] [Full Text] [PDF]

				K. Nosho, K. Shima, N. Irahara, S. Kure, Y. Baba, G. J. Kirkner, L. Chen, S. Gokhale, A. Hazra, D. Spiegelman, et al. DNMT3B Expression Might Contribute to CpG Island Methylator Phenotype in Colorectal Cancer Clin. Cancer Res., June 1, 2009; 15(11): 3663 - 3671. [Abstract] [Full Text] [PDF]

				A. Paya, C. Alenda, L. Perez-Carbonell, E. Rojas, J.-L. Soto, C. Guillen, A. Castillejo, V. M. Barbera, A. Carrato, A. Castells, et al. Utility of p16 Immunohistochemistry for the Identification of Lynch Syndrome Clin. Cancer Res., May 1, 2009; 15(9): 3156 - 3162. [Abstract] [Full Text] [PDF]

				R. C. Green, P. S. Parfrey, M. O. Woods, and H. B. Younghusband Prediction of Lynch Syndrome in Consecutive Patients With Colorectal Cancer J Natl Cancer Inst, March 4, 2009; 101(5): 331 - 340. [Abstract] [Full Text] [PDF]

				E. I. Joensuu, W. M. Abdel-Rahman, M. Ollikainen, S. Ruosaari, S. Knuutila, and P. Peltomaki Epigenetic Signatures of Familial Cancer Are Characteristic of Tumor Type and Family Category Cancer Res., June 15, 2008; 68(12): 4597 - 4605. [Abstract] [Full Text] [PDF]

				K. Imai and H. Yamamoto Carcinogenesis and microsatellite instability: the interrelationship between genetics and epigenetics Carcinogenesis, April 1, 2008; 29(4): 673 - 680. [Abstract] [Full Text] [PDF]