We conclude that EZH2 is a powerful and independent predictor of RCC related death, which can add to the development of a modified risk stratification system. Methods Patients Clinical data of patients with RCC who under went radical nephrectomy or nephron sparing surgery at the all targets Department of Urology, University of Heidelberg, between 1990 and 2005 and had no other malignant tumor before or within Inhibitors,Modulators,Libraries one month after surgery were entered into a prospective database. Tumor stage was classified according to the tumor node metastasis sta ging system of 2002, tumors were graded on the basis Inhibitors,Modulators,Libraries the Fuhrman four tiered nuclear Inhibitors,Modulators,Libraries grading system. In patients with metastases, the Motzer criteria were evaluated and patients categorized to one of the following risk groups low, intermediate or high risk.

Patients Inhibitors,Modulators,Libraries were prospectively evaluated every 3 months for the first 2 years after surgery, every 6 months for the next 3 years, and yearly thereafter. No adjuvant therapy was administered after radical surgery. Patients with metastases, a Karnofsky severity rating 80, and with no medical contraindications received palliative Interferon alpha and Interleukin 2 based immunother apy. No tyrosine kinase inhibitors have been given. The work was covered by a votum of the ethical com mittee of the University of Heidelberg No. 206 2005. Informed and or written consent was obtained from each patient. Tissue micro arrays Tissue samples of all 768 patients included in the pro spective clinical database were obtained from the Tissue Bank of the National Center for Tumor Diseases Heidelberg and included in a TMA, containing 768 primary tumor and corresponding normal tissue samples, as previously described.

Briefly, representative tissue blocks were selected as donor blocks for the TMA. Sec tions were Inhibitors,Modulators,Libraries cut from each donor block and stained with H E. Then, a morphologically representative region was chosen from each of the RCC and normal renal tissue samples. Two cylindrical core tissue specimen per tumor following block were punched from these regions and arrayed into the recipient paraffin block using a semiautomatic system. In total, 16 tissue array blocks were generated, each containing up to 200 core tissue specimens, matching 50 patients per array. Immunohistochemistry The TMA slides were dewaxed and rehydrated using xylene and a series of graded alcohols, followed by heat induced antigen retrieval using a target retrieval solution in a pressure cooker for 10 min. Immunohistochemical staining was performed on an automated staining sys tem with a mouse monoclonal anti EZH2 antibody for 45 min. An avidin biotin complex peroxidase technique using aminoethylcarbazole for visualisation and hema toxylin for counterstaining was applied.