Original article Increased EZH2 protein expression is associated with invasive urothelial carcinoma of the bladder Hang Wang, Ph.D. a , Roula Albadine, M.D. b , Ahmed Magheli, M.D. c , Thomas J. Guzzo, M.D., M.P.H. a , Mark W. Ball, M.D. a , Stefan Hinz, M.D. c , Mark P. Schoenberg, M.D. a , George J. Netto, M.D. b , Mark L. Gonzalgo, M.D., Ph.D. d, * a Department of Urology, James Buchanan Brady Urological Institute, Baltimore, MD 21287, USA b Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore, MD 21287, USA c Department of Urology, Universitätsmedizin Berlin, Charité Campus Benjamin Franklin, Berlin, Germany d Department of Urology, Stanford University School of Medicine, Stanford, CA 94305, USA Received 8 February 2010; received in revised form 7 September 2010; accepted 10 September 2010 Abstract Objectives: Elevated polycomb group protein Enhancer of Zest Homolog 2 (EZH2) expression has been associated with progression to more advanced disease in a variety of malignancies. We examined EZH2 protein expression levels in bladder tissue specimens from patients with urothelial carcinoma (UC) and investigated the relationship between EZH2 protein expression and clinical outcomes. Materials and methods: Tissue microarrays (TMAs) were constructed using bladder tissue specimens from radical cystectomies performed for UC at our institution between 1994 and 2002. EZH2 expression was measured by immunohistochemistry and scoring was based on percentage and intensity of positive nuclear staining. A receiver operating curve (ROC) was generated to differentiate cancerous from benign lesions using EZH2 protein scores. Recurrence-free survival was estimated using the Kaplan-Meier approach with log-rank test. A multivariate Cox proportional hazards model was used to assess independent contributions. Results: A total of 454 TMA specimen spots from 81 patients were evaluated. EZH2 protein levels in invasive high grade UC were significantly elevated compared with adjacent benign urothelium, noninvasive low grade UC, and CIS. EZH2 protein levels were also significantly increased in CIS and noninvasive low grade UC compared with adjacent benign urothelium. We found no association between EZH2 protein expression and clinical outcomes following radical cystectomy in our cohort of patients. Conclusion: EZH2 overexpression is a common event in UC of the bladder. Elevated EZH2 protein levels are associated with more aggressive bladder cancer, including invasive UC. EZH2 may therefore serve as a useful biomarker for UC. © 2012 Elsevier Inc. All rights reserved. Keywords: Urinary bladder cancer; Urothelial carcinoma; Tissue microarray; EZH2; Polycomb protein 1. Introduction Bladder cancer is the fourth most common malignancy in men and the eighth most common cancer in women in the United States. The National Comprehensive Cancer Net- work estimates that 68,810 new cases and 14,100 deaths would have occurred in the United States in 2008 [1]. Overall, 70% of bladder tumors present as noninvasive urothelial carcinoma (UC), and the remainder as muscle- invasive disease [2]. Up to 50% of patients initially present- ing with muscle-invasive UC will relapse with metastatic disease. Carcinoma in situ (CIS) is a high grade lesion that is a precursor of invasive UC. The heterogeneity of this disease complicates management decision-making. Precise identification of tumors that will recur and progress vs. tumors that will take a more indolent course remains chal- lenging [3]. EZH2 is a key member of the Polycomb Group (PcG) of proteins. PcG proteins are highly conserved from insects to mammals and play crucial roles in the maintenance of embryonic and adult stem cells and in cell cycle control via maintenance of long-term repressive chromatin states [4,5]. * Corresponding author. Tel.: +1-650-725-5544; fax: +1-650-723-0765. E-mail address: gonzalgo@stanford.edu (M.L. Gonzalgo). Urologic Oncology: Seminars and Original Investigations 30 (2012) 428 – 433 1078-1439/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.urolonc.2010.09.005