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(American Journal of Pathology. 2002;161:629-634.)
© 2002 American Society for Investigative Pathology

Regular Articles

Establishment and Validation of Real-Time Polymerase Chain Reaction Method for CDH1 Promoter Methylation

Kiyomi O. Toyooka^*, Shinichi Toyooka^*, Anirban Maitra, Qinghua Feng, Nancy C. Kiviat, Alice Smith^*, John D. Minna^*, Raheela Ashfaq and Adi F. Gazdar^*

From the Hamon Center for Therapeutic Oncology Research^*and the Departments of Pathology,Internal Medicine,and Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas; and the Department of Pathology,University of Washington, Seattle, Washington

Aberrant methylation of the promoter region has emerged as the major mechanism for silencing tumor suppressor genes. However, for some genes, such as E-cadherin (CDH1), methylation and protein expression demonstrate considerable heterogeneity, making correlations difficult. We compared methylation and protein expression status of CDH1 in 56 primary breast carcinomas using semiquantitative assays. Aberrant CDH1 methylation was studied by methylation-specific polymerase chain reaction (MSP) and semiquantitative real-time MSP assays. The Cdh1 expression was investigated by immunostaining on archival formalin-fixed sections from 34 primary carcinomas and their accompanying normal epithelium and preinvasive and metastatic lesions. Membrane-specific Cdh1 expression in the neoplastic cells was quantified by image analysis using an automated cellular imaging system and a continuous score. Aberrant promoter methylation of the CDH1 was present in 24 of 56 (43%) breast carcinomas by MSP assay. There was excellent concordance between the standard MSP assay and the real-time assay (91%, P < 0.0001). The concordance between loss of Cdh1 expression and CDH1 methylation by standard MSP was 71% (P = 0.02). Furthermore, there was a strong correlation between the semiquantitative assays for methylation and protein expression (r = 0.47, P = 0.005). We conclude that promoter methylation of CDH1 significantly correlated with the Cdh1 expression level, demonstrating that epigenetic silencing is a valid pathway for silencing of tumor suppressor genes in primary breast carcinomas.

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