The present study defines EZH2 as a powerful and independent negative prognostic marker of CSS in patients with metastasized and non-metastasized RCC. Thus, assessment of EZH2 expression may allow improved patient selection for systemic therapies. Moreover, integration of the EZH2 status into current prognostic models could result in more accurate survival prediction and may also be useful for individualizing follow-up and selecting patients for clinical trials.
RCC is commonly characterized by a poor response towards current treatment options. Even after complete resection of the primary tumor, relapse occurs in 20-30% of cases. The overall 5-year survival rate is 60%; in patients with metastases, the median survival is only about 13 months, with a 5-year survival of less than 10% [23, 24]. Cytokine therapy and novel targeted therapies, i.e. tyrosine kinase inhibitors, have been of limited benefit [23, 25–29]. Progression and treatment response of the disease are still not sufficiently predictable. Suitable molecular markers could help to refine individual risk stratification and treatment plans . However, for RCC, as for other cancers in general, only few markers have been validated for clinical practice. This may be partly due to the fact that many studies have been small and poorly designed, used inappropriate statistical analysis, and employed different assay methods and outcome measures .
The present study, defining EZH2 as a novel prognostic marker in RCC, has been conducted according to the REMARK criteria . These include a large sample size, a long prospective follow-up of patients, and a description of the predictive value of the marker. Possible limitations of our study include the lack of external validation using a second cohort of patients with RCC. For analyzing RCC patients without metastases, our Cox regression model-which included tumor stage, grading, Karnofsky performance index, age, sex, histopathological subtype, and EZH2 expression-revealed a concordance probability of 73.4% compared to 71.8% excluding EZH2 expression. In RCC patients with metastatic disease, the concordance probability including EZH2 expression was 68.4%, compared to 63.0% excluding EZH2 expression. These values are in a similar range as obtained by the University of California Los Angeles Integrated Staging System (UISS) , a well-accepted prognostic factor model for RCC with good predictive accuracy, which exhibits a concordance index of 76% . Including molecular markers to the clinical models increased the concordance probability, in our model as well as in the UISS model [33, 34]. Therefore, EZH2 should represent a powerful new marker for predicting prognosis in RCC and could be integrated in established prognostic models to provide an even better consultation for patients regarding diagnosis, treatment, and follow-up.
Our findings concerning the prognostic value of EZH2 in RCC, are in contrast to a recent study of 119 clear cell carcinoma patients, who reported that high tumor EZH2 levels indicate less aggressive tumor phenotypes with a favorable prognosis, as assessed by real-time RT-PCR . We do not know whether this discrepancy is related to the different detection method for EZH2 expression. Our results are in line with studies in several other tumor forms, including malignant melanoma and cancers of the breast, prostate, endometrium, stomach, and liver, where increased EZH2 expression has been linked to more aggressive tumor behaviour and poor prognosis [7, 9, 17, 36, 37]. EZH2 expression showed significant prognostic impact in melanoma, prostate, and endometrial carcinoma in univariate survival analyses, but revealed independent multivariate prognostic importance only in carcinoma of the endometrium and prostate . In breast cancer, high EZH2 expression was a strong independent predictive parameter of outcome, providing a better information about CSS than other independent prognostic features . Thus, EZH2 may be an interesting novel prognostic marker for a large panel of different cancer types.
RCCs exhibited significantly higher EZH2 expression levels than histologically normal kidney, indicating that an increase in EZH2 expression is acquired during RCC tumorigenesis. Increased EZH2 expression in tumorous versus corresponding normal tissue has been also reported for other cancers as well, including malignant melanoma, prostate carcinoma, breast cancer and hepatocellular carcinoma [7, 9, 17, 37]. However, it should be noted that infiltrating lymphocytes and, sporadically, proximal and distal tubule epithelial cells stained positive for EZH2 in normal renal tissue. This indicates that detectable EZH2 expression is not stringently restricted to tumor cells. In line, EZH2 expression could be detected in the proliferating parabasal cell layer in normal cervical epithelium  and in proliferating cells of normal mammary gland tissue . Interestingly, the latter study raised the possibility that EZH2 is expressed in mammary stem cells, in line with studies indicating a dual role of the PcG proteins in self-renewal of stem cells and oncogenesis [39, 40].
Apart form serving as a novel prognostic marker in RCC, EZH2 expression may also have therapeutic and diagnostic implications. Mechanistically, EZH2 is likely to contribute to the growth of RCC cells, since silencing of EZH2 expression exerts profound anti-proliferative effects in RCC lines . These findings indicate that EZH2 may represent a novel therapeutic target for RCC treatment in that specific EZH2 inhibitors should repress tumor growth. Under diagnostic aspects, it is noteworthy that upregulation of EZH2 expression can be detected very early in breast cancer development, even before atypic cells are histologically evident [15, 16]. Thus, the determination of EZH2 expression may be an important new tool to identify patients at risk for developing breast cancer [15, 16]. In view of the substantial portion of RCCs expressing EZH2, it will be interesting to investigate in future studies whether EZH2 expression is an early event in RCC development and may also have diagnostic potential for RCC detection.