A p value 0. 05 was considered significant. For further description of the predictive value of EZH2 the concordance probability of the Cox models including EZH2 inhibitor Cabozantinib were calculated and compared to the models excluding EZH2 but retaining all other variables. The statistical analysis system SAS, Version 9. 2 for Windows, the R package, Version 2. 8. 0, and StatXact, Version 8. 0. 0 were used for the analyses. For the calculation of the concordance probability, the CPE package offered by Mo, Goenen and Heller was used with the R programming language. Results In order to identify prognostic markers for RCC in a large cohort of patients, a TMA was constructed which contained RCC tumor tissue and corresponding normal renal tissue samples from 768 patients.
Expression of EZH2 was analyzed by immunohistochemistry using a mouse monoclonal anti EZH2 antibody. Altogether, 520 cases were scored for expression of EZH2. The remaining cases with insufficient tumor tissue or fixation artefacts were excluded from further analyses. Typical examples are depicted in Figure 1. Negative controls showed no immunohistochemical staining. Inhibitors,Modulators,Libraries Non tumorous kidney tissue was mainly negative for EZH2 expression, only spora dically Inhibitors,Modulators,Libraries proximal and distal tubular epithelial cells and infiltrating lymphocytes, if present in the tissue sample, stained positive for EZH2. In contrast, EZH2 protein expression was readily detected in 411 of the cancerous tissues, typically showing nuclear staining, without predominately staining of the central or peripheral zone of tumor.
Ten cases exhibited 50% EZH2 positive nuclei, Inhibitors,Modulators,Libraries 36 25 50% EZH2 positive nuclei, 99 5 25% EZH2 positive nuclei, and 266 1 5% EZH2 positive nuclei. In contrast, 109 samples did not exhibit EZH2 nuclear staining in the tumorous tissue. Next, the survival of patients with RCC was calculated from the time of renal surgery. Until June 2006, 147 patients had died of their disease. The CSS rate at 1 year and at 5 years after surgery for the whole cohort of patients was 88. 4% and 70. 8%, respectively. Clinical and patholo gical features of patients are summarized Inhibitors,Modulators,Libraries in Table 1. Patients with advanced tumor stages, with cancer infiltrated lymph nodes, distant metastases and or a high Fuhrmans grading were found to have significantly more often a higher percentage of EZH2 nuclear staining when compared to patients with localized tumor disease or low Fuhrmans grading.
Moreover, nuclear EZH2 expression and the Mot zer criteria Inhibitors,Modulators,Libraries were found to be independent parameters, whereas a dependency was seen between different histopathological subtypes and nuclear EZH2 expression. As shown in Figures 2A and 2B, Kaplan Meier cancer specific survival curves of the whole cohort of patients and of the non metastatic subgroup revealed a worse prognosis Ivacaftor supplier in patients with more than 25% nuclear EZH2 staining.