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Differential Methylation Hybridization Array of Endometrial Cancers Reveals Two Novel Cancer-Specific Methylation Markers

Authors' Affiliations: ¹ Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, ² Endocrine and Oncologic Surgery, Department of Surgery, Washington University School of Medicine and Siteman Cancer Center, St. Louis, Missouri, and ³ Department of Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer Center, Ohio State University, Columbus, Ohio

Requests for reprints: Israel Zighelboim, Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Washington University School of Medicine, 4911 Barnes Jewish Plaza, Box 8064, St. Louis, MO 63110. Phone: 314-362-3181; Fax: 314-362-2893; E-mail: zighelboimi{at}wustl.edu.

Purpose: To identify novel endometrial cancer-specific methylation markers and to determine their association with clinicopathologic variables and survival outcomes.

Experimental Design: Differential methylation hybridization analysis (DMH) was done for 20 endometrioid endometrial cancers using normal endometrial DNA as a reference control. Combined bisulfite restriction analysis (COBRA) was used to verify methylation of sequences identified by DMH. Bisulfite sequencing was undertaken to further define CpG island methylation and to confirm the reliability of the COBRA. The methylation status of newly identified markers and the MLH1 promoter was evaluated by COBRA in a large series of endometrioid (n = 361) and non-endometrioid uterine cancers (n = 23).

Results: DMH and COBRA identified two CpG islands methylated in tumors but not in normal DNAs: SESN3 (PY2B4) and TITF1 (SC77F6/154). Bisulfite sequencing showed dense methylation of the CpG islands and confirmed the COBRA assays. SESN3 and TITF1 were methylated in 20% and 70% of endometrioid tumors, respectively. MLH1 methylation was seen in 28% of the tumors. TITF1 and SESN3 methylation was highly associated with MLH1 methylation (P < 0.0001). SESN3 and TITF1 were methylated in endometrioid and non-endometrioid tumors, whereas MLH1 methylation was restricted to endometrioid tumors. Methylation at these markers was not associated with survival outcomes.

Conclusions: The 5' CpG islands for SESN3 and TITF1 are novel cancer-specific methylation markers. Methylation at these loci is strongly associated with aberrant MLH1 methylation in endometrial cancers. SESN3, TITF1 and MLH1 methylation did not predict overall survival or disease-free survival in this large cohort of patients with endometrioid endometrial cancer.