Context.—Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase mediating chromatin condensation and epigenetic modulation, is overexpressed in various human carcinomas and is associated with adverse clinicopathologic characteristics and biologic behavior. The expression of EZH2 in renal cell carcinomas (RCCs) has not been fully characterized yet.

Objective.—To evaluate the prognostic role of EZH2 in RCC by analyzing the immunohistochemical staining pattern of the marker in relation to pathologic features and clinical outcome.

Design.—We correlated the immunolabeling of EZH2 with multiple clinicopathologic features, including Fuhrman nuclear grade, pathologic stage, metastatic status, and clinical outcome in 223 clear cell RCCs (CRCCs) and 21 papillary RCCs, by using tissue microarrays of primary and metastatic cases.

Results.—Most CRCCs (75%) showed positive EZH2 staining, with most primary tumors showing focal staining in comparison to nonfocal staining in metastatic cases. In primary tumors, EZH2 expression was associated with higher nuclear grade and lower pathologic stage. Metastatic tumors showed a higher number of positive cases (81% versus 67%) and a more diffuse and more intense pattern of staining than primary CRCCs. For the 22 locally advanced primary tumors (T3/4) and 43 metastatic RCCs, patients who experienced RCC-related deaths significantly overexpressed the marker in comparison to patients who did not experience RCC-related mortality.

Conclusions.—By showing that EZH2 expression is associated with increased metastatic potential and a worse clinical outcome, this study suggests that EZH2 can serve as a prognostic biomarker for RCC, thus confirming it as a key molecule driving oncogenesis and metastasis.

Epigenetic modulations, such as hypermethylation of areas rich in cytosine and guanine dinucleotides (CpG islands) within the promoters, and acetylation and methylation of histones, are common phenomena observed in a wide variety of human cancers and are believed to contribute to oncogenesis.14  Unlike genetic mutation, such epigenetic changes can potentially be reversed, making enzymes involved in such processes promising therapeutic targets.2,5,6  Enhancer of zeste homolog 2 (EZH2), the catalytic subunit of polycomb repressive complex (PRC2), acts as a histone methyltransferase by adding 3 methyl groups to lysine 27 of histone 3 (H3K27).710  The trimethylation of H3K27 leads to chromatin condensation and mediates epigenetic silencing of a broad range of genes involved in tumoral proliferation, invasion, and angiogenesis.1115  Amplification of the EZH2 gene locus and overexpression of EZH2 protein have been observed in several proliferative and neoplastic conditions, including carcinoma,3,1621  sarcoma,2224  lymphoma, leukemia,2530  and myeloproliferative disorder.26,3134  In addition, recent studies1517,19,24,3560  have shown that EZH2 is an adverse prognostic biomarker that predicts aggressive clinical behavior, including risk of progression and cancer-specific mortality in a variety of carcinoma types.

The significance of EZH2 expression in renal cell carcinoma (RCC) is not fully understood, and the rare studies published on the subject to date have shown contradictory results.6163  In this study, we aimed to evaluate the prognostic role of EZH2 in RCC by analyzing the immunohistochemical staining pattern of the marker in relation to pathologic features and clinical outcome in a large cohort of primary and metastatic clear cell and papillary RCCs.

Case Characteristics

Three tissue microarrays (TMAs) were constructed by using tissues obtained from the surgical pathology archives of the McGill University Health Centre (Montreal, Quebec, Canada). Institutional guidelines regarding experimentation involving human subjects were followed. The first TMA represented primary RCCs obtained from 81 nephrectomy specimens collected from 1985 to 2006, including 68 (84.0%) clear cell RCCs (CRCCs) and 13 (16.0%) papillary RCCs (PRCCs). Each tumor was represented by 2 separate 1-mm cores. The TMA included 30 cases (37.0%) with locally advanced disease defined by an American Joint Committee on Cancer (AJCC)64  pathologic stage of T3 or T4, and 51 cases (63.0%) with organ-confined disease and a pathologic stage of T1. The second TMA contained specimens from primary and matched metastatic tumors of 24 patients. Each patient was represented by at least 2 cores taken from the primary tumor and 2 cores from the metastasis. The 24 primary tumors included 22 CRCCs (91.7%), 1 PRCC (4.2%), and 1 unclassified RCC (4.2%). Sites of metastasis included lymph node (n = 11; 45.8%), adrenal gland (n = 7; 29.2%), lung (n = 2; 8.3%), liver (n = 2; 8.3%), bone (n = 1; 4.2%), brain (n = 1; 4.2%), and omentum (n = 1; 4.2%). Twenty-one metastases (87.5%) were removed at the time of nephrectomy and 3 (12.5%) were removed metachronously. The third TMA consisted of 1-mm cores from 125 metastatic RCCs, including 119 CRCCs (95.2%), 5 PRCCs (4%), and 1 chromophobe RCC (0.8%). The metastatic tumors were obtained from lymph nodes (n = 33; 26.4%), bone (n = 24; 19.2%), lung or pleura (n = 20; 16%), brain (n = 19; 15.2%), adrenal gland (n = 11; 8.8%), liver (n = 6; 4.8%), thyroid (n = 2; 1.6%), and others (n = 10; 8%).

Immunohistochemistry

Immunohistochemistry was performed by using a Ventana automated system and Ventana iVIEW DAB detection kit (Ventana Medical Systems, Inc, Tucson, Arizona) at the immunohistochemistry laboratory of McGill University Health Centre. Mouse anti-human monoclonal EZH2 antibody (BD Transduction Laboratories, San Jose, California) was used. Nuclear staining intensity was evaluated semiquantitatively as follows: 0 = negative, 1 = weak staining, 2 = moderate staining, and 3 = intense staining. The percentages of tumor cells and staining intensity were visually estimated. For each core, the maximal staining intensity, the total percentage of tumor cells with at least 1+ immunostaining per core, and the H-score, defined as the product of the percentage of tumor cells showing EZH2 labeling (0–100) multiplied by the labeling intensity (0–3), were calculated. Averages were taken for statistical purposes if 2 or more cores were available from the same specimen.

Statistical Analysis

All statistical analyses were carried out with Statistica software (Statsoft Inc, Tulsa, Oklahoma) and Statistical Analysis System (SAS Inc, Cary, North Carolina). P values less than .05 were considered to be statistically significant. An immunohistochemistry score of 1 and above was defined as positive for EZH2. The percentages of positive cases were calculated and subjected to χ2  test. A 1-way analysis of variance (ANOVA) test was used to investigate whether the maximal staining intensity, the percentage of positive staining per core, and the H-score differed between different tumor types, histologic grades, AJCC pathologic tumor stages (pT), sites of metastases, or between primary and metastatic tumors. Disease-specific survival was calculated from the date of surgery. The survival end point was the date of last follow-up or disease-specific death. The association between EZH2 immunolabeling and clinical outcome was assessed by the Cox proportional hazards model, using the PHREG procedure, and the hazard ratio and the 95% confidence interval were calculated.

EZH2 Expression in Relation to Tumor Type

Expression of EZH2 in CRCCs and PRCCs is summarized in Table 1. The staining was nuclear with no cytoplasmic staining observed in any case. After excluding cores with insufficient tumor cells, the total number of primary and metastatic cases that were included in the study was 223 CRCCs and 21 PRCCs (14 type I PRCCs and 7 type II PRCCs). Among them, a similar percentage of PRCCs and CRCCs demonstrated EZH2 expression (χ2  test, P = .93) with compatible staining intensity, percentage of positive tumor cells, and H-scores (1-way ANOVA, P = .17, .52, and .61, respectively; Table 1). One hundred sixty-eight cases (75%) of CRCCs and 16 cases (76%) of PRCCs showed at least focal EZH2 immunoreactivity. The TMA also included 1 case of metastatic chromophobe RCC and 1 case of unclassified RCC with matched primary tumor and metastasis, which showed no EZH2 labeling.

Table 1.

Expression of Enhancer of Zeste Homolog 2 (EZH2) in Clear Cell and Papillary Renal Cell Carcinoma (RCC)

Expression of Enhancer of Zeste Homolog 2 (EZH2) in Clear Cell and Papillary Renal Cell Carcinoma (RCC)
Expression of Enhancer of Zeste Homolog 2 (EZH2) in Clear Cell and Papillary Renal Cell Carcinoma (RCC)

EZH2 Expression in Relation to Fuhrman Nuclear Grade

Stratifying all CRCCs (primary and metastatic) by Fuhrman nuclear grade, into high grade (grade 3 or 4) (n = 97) and low grade (grade 1 or 2) (n = 124), showed that although the percentage of EZH2-positive cases and the staining intensity did not differ among tumors with different nuclear grades, tumors with higher grade demonstrated more diffuse EZH2 positivity with a significantly higher percentage of positive tumor cells (27.1% ± 3.6% and 13.3% ± 1.9% for high and low grade, respectively) and a higher H-score (54.5 ± 8.0 and 24.1 ± 3.7 for high and low grades, respectively; 1-way ANOVA, P < .001). Interestingly, the observed difference in the overall EZH2 expression between high and low nuclear grade seemed to be related to the metastatic cases only, with cases with higher nuclear grade demonstrating a more diffuse EZH2 staining (36.8% ± 4.7%) with higher H-score (74.5 ± 10.5) than those with lower nuclear grade (percentage of positive labeling: 22.6% ± 3.3%; H-score: 41.3 ± 6.4) (1-way ANOVA, P = .02 for percentage of positive tumor cells, and P = .009 for H-score). In comparison, no difference in EZH2 staining was observed in the primary tumors when they were compared by nuclear grade (1-way ANOVA, P = .41 for percentage and H-score) (Table 2).

Table 2.

Enhancer of Zeste Homolog 2 (EZH2) Immunoreactivity in Clear Cell Renal Cell Carcinoma (CRCC)

Enhancer of Zeste Homolog 2 (EZH2) Immunoreactivity in Clear Cell Renal Cell Carcinoma (CRCC)
Enhancer of Zeste Homolog 2 (EZH2) Immunoreactivity in Clear Cell Renal Cell Carcinoma (CRCC)

EZH2 Expression in Relation to Pathologic Stage

Classifying primary CRCCs into 2 groups, those with high pathologic stage (T3–T4, n = 41) and those with low tumor stage (T1–T2, n = 47), showed that a significantly lower percentage of high-stage primary CRCCs (23 of 41; 56%) overexpressed EZH2 than did low-stage tumors (37 of 47; 79%) (χ2  test, P = .02). In addition, low-stage primary tumors were associated with a stronger staining intensity (1.5 ± 0.2) (1-way ANOVA, P < .001) than high-stage tumors (0.7 ± 0.1) (Table 2). However, such results need to be interpreted with caution given the fact that EZH2 positivity was only observed very focally in less than 5% of tumor cells in both groups.

EZH2 Expression in Primary Versus Metastatic Sites

In comparison with primary CRCCs for which 67% (61 of 91) of cases stained for EZH2, metastatic CRCCs expressed the marker more commonly (107 of 132; 81%) (χ2  test, P = .02). Furthermore, a highly significant difference was detected between primary and metastatic tumors in terms of staining intensity, percentage of positivity per core, and the H-score (1-way ANOVA, P < .001; Figure 1, A through D). These observations were confirmed by analyzing the matched primary and metastatic cases in the second TMA, in which EZH2 staining was observed in 45% (10 of 22) of primary tumors and 86% (19 of 22) of matched metastatic tumors (χ2  test, P = .004). In this cohort, the mean staining intensity of primary and metastatic CRCCs was 0.6 ± 0.2 and 2.0 ± 0.2, respectively, and the mean H-scores for both groups were 2.7 ± 1.0 and 52.7 ± 11.2 (repeated-measures ANOVA, P < .001). Albeit the small number of cases studied for PRCC, comparison between primary and metastatic PRCCs revealed similar findings in which metastatic PRCCs showed more diffuse staining pattern with significantly higher percentage of positive labeling and H-score than did primary PRCCs (1-way ANOVA, P = .01 for percentage, and P = .02 for H-score).

Figure 1. 

Overexpression of enhancer of zeste homolog 2 (EZH2) in metastasis (C and D), while the primary clear cell renal cell carcinoma of the same patient shows no expression of EZH2 (A and B) A and C: hematoxylin-eosin; B and D: EZH2 immunohistochemistry; original magnification: ×100; and magnification of insets: ×400.

Figure 1. 

Overexpression of enhancer of zeste homolog 2 (EZH2) in metastasis (C and D), while the primary clear cell renal cell carcinoma of the same patient shows no expression of EZH2 (A and B) A and C: hematoxylin-eosin; B and D: EZH2 immunohistochemistry; original magnification: ×100; and magnification of insets: ×400.

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EZH2 Expression in Relation to Metastatic Sites

When analyzing EZH2 labeling in metastatic tumors taken from a variety of sites, H-scores and percentage of positive cases were significantly different across all sites of metastasis (1-way ANOVA, P = .03 for percentage and P = .009 for H-score; Table 3). Among all the sites studied, metastatic tumors to the liver and brain had the strongest and most diffuse labeling, while lung metastasis showed the lowest percentage of positive cases, and a more focal staining pattern.

Table 3.

Expression of Enhancer of Zeste Homolog 2 (EZH2) in Metastatic Clear Cell Renal Cell Carcinoma

Expression of Enhancer of Zeste Homolog 2 (EZH2) in Metastatic Clear Cell Renal Cell Carcinoma
Expression of Enhancer of Zeste Homolog 2 (EZH2) in Metastatic Clear Cell Renal Cell Carcinoma

EZH2 Expression in CRCC in Relation to Outcome

For the 223 included CRCCs, clinical outcomes were available for 103 patients, with cases including 38 primary T1a tumors, 22 primary T3 and T4 tumors, and 43 metastatic tumors. All patients except one (37 of 38; 97%) with an initial diagnosis of stage T1a CRCC showed no evidence of disease for an average follow-up period of 108.0 ± 5.1 months (Table 4). Twenty-two patients with locally advanced primary CRCCs (pT3/4) had available clinical follow-up with an average follow-up duration of 45.0 ± 13.8 months. These patients were classified into 3 categories by their clinical outcomes: the first group were patients showing no evidence of disease (n = 8; follow-up duration of 83.6 ± 29.2 months); the second were patients who were alive with evidence of recurrent or metastatic disease (n = 4; follow-up duration of 47.5 ± 33.5 months); and the third included patients who had RCC-related deaths (n = 10; follow-up duration, 13.2 ± 2.6 months; Table 4). While the percentage of EZH2-positive cases did not differ between the 3 groups (Fisher exact test, P = .44; Table 4), regression analysis using the Cox proportional hazards model showed that patients with RCC-related deaths had a higher H-score (hazard ratio [HR] = 1.02; 95% confidence interval [CI] = 1.00–1.03, P = .03) and a higher percentage of positive tumor cells (HR = 1.04, 95% CI = 1.00–1.07, P = .03) than those patients who were still alive (Figure 2, A and C; Table 4). Among the 43 patients with metastatic CRCC and available clinical data, 4 (9%) showed no further evidence of disease with a follow-up period of 83.3 ± 21.9 months; 17 patients (40%) were alive with residual disease for a follow-up period of 60.4 ± 15.0 months; and 22 patients (51%) had disease-related death with a follow-up period of 29.3 ± 5.3 months. Similar to what was observed in the primary CRCCs, regression analysis demonstrated that RCC-related deaths were also significantly associated with EZH2 overexpression in the metastatic setting. The H-score (HR = 1.01, 95% CI = 1.00–1.01, P = .049) and the percentage of positive tumor cells (HR = 1.02, 95% CI = 1.00–1.03, P = .022) were significantly higher in patients who died of disease (Figure 2, B and D; Table 4).

Table 4.

The Correlation of Clinical Outcomes With the Expression of Enhancer of Zeste Homolog 2 (EZH2) in Clear Cell Renal Cell Carcinoma (CRCC)

The Correlation of Clinical Outcomes With the Expression of Enhancer of Zeste Homolog 2 (EZH2) in Clear Cell Renal Cell Carcinoma (CRCC)
The Correlation of Clinical Outcomes With the Expression of Enhancer of Zeste Homolog 2 (EZH2) in Clear Cell Renal Cell Carcinoma (CRCC)
Figure 2. 

The expression of enhancer of zeste homolog 2 (EZH2) relative to the clinical outcomes in locally advanced AJCC (American Joint Committee on Cancer) pathologic tumor stage T3 and above (A and C) and metastatic renal cell carcinoma (RCC) (B and D). A and B, The association of H-score with follow-up period in 3 groups with different clinical outcomes (NED: no evidence of disease, triangle; AWD: alive with disease, diamond; and DOD: death from disease, square). C and D, Kaplan-Meier survival curves for overall survival. EZH2 expression is divided into 2 groups (high H-score and low H-score) by using the median H-score (H-score = 0.5 for primary T3 and T4 clear cell RCC; H-score = 10 for metastatic clear cell RCC).

Figure 2. 

The expression of enhancer of zeste homolog 2 (EZH2) relative to the clinical outcomes in locally advanced AJCC (American Joint Committee on Cancer) pathologic tumor stage T3 and above (A and C) and metastatic renal cell carcinoma (RCC) (B and D). A and B, The association of H-score with follow-up period in 3 groups with different clinical outcomes (NED: no evidence of disease, triangle; AWD: alive with disease, diamond; and DOD: death from disease, square). C and D, Kaplan-Meier survival curves for overall survival. EZH2 expression is divided into 2 groups (high H-score and low H-score) by using the median H-score (H-score = 0.5 for primary T3 and T4 clear cell RCC; H-score = 10 for metastatic clear cell RCC).

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The PRC2 is a highly conserved histone methyltransferase that is composed of 3 essential core subunits: EZH2, EED, and SUZ12.810,65  EZH2, the catalytic subunit of PRC2, catalyzes trimethylation of lysine 27 of histone 3, leading to chromatin condensation and the mediation of epigenetic silencing of multiple transcription factors and signaling components.810,65  Since the discovery of EZH2 as an epigenetic modification enzyme, numerous studies have reported the abundance of EZH2 in cancerous tissue and its oncogenic-like effects in tumorigenesis. The regulatory role of EZH2 has been demonstrated in several vital cellular processes, including cell proliferation, cell mobilization, metastasis, and angiogenesis (Figure 3). By silencing multiple signaling pathways, EZH2 inhibits apoptosis and triggers cell proliferation via epigenetic effects that affect several well-characterized tumor suppressor genes and oncogenes. As an example, the tumor suppressor Runt-related transcription factor 3 (RUNX3) is subject to EZH2-induced methylation, which gene leads to down-regulation of RUNX3 messenger RNA (mRNA), promoting proliferation in multiple cancer cell lines.66,67 In ovarian cancer cells, EZH2 suppresses another anti-oncogene, P57 (also known as cyclin-dependent kinase inhibitor 1C), which is a crucial cell cycle inhibitor causing G1 phase arrest.68  In addition, EZH2 hypermethylates the promoter region of Rap1 GTPase-activating protein gene (Rap1GAP), a critical tumor suppressor gene in head and neck squamous cell carcinoma, thyroid cancer, pancreatic cancer, and melanoma, which subsequently down-regulates Rap1 (also called Ras-proximate-1 or Ras-related protein 1) and results in cell proliferation.69,70  In prostate cancer, EZH2 has been shown to inhibit the transcription of MSMB, the coding gene of prostatic secretory protein of 94 amino acids (PSP94), a suppressor of tumor growth.71  Furthermore, EZH2 mediates epigenetic silencing of the promoters of DAB2IP, a crucial inhibitory modulator of oncogenic Ras-mediated signaling, which ultimately promotes tumor growth and induces metastasis in prostate cancer cell lines.72–75 In anaplastic thyroid cancer cell lines, EZH2 directly prevents cell growth inhibition and differentiation by silencing the thyroid-specific transcription factor paired box gene 8 (PAX8).76  EZH2 also plays a key role in tumor invasiveness and metastasis. Together with histone deacetylases, EZH2 acts as an essential inhibitory mediator, resulting in transcription silencing of E-cadherin, consequently promoting cell migration and invasion.14,7779  In breast cancer cell lines, EZH2 was shown to trigger cells' migration and invasion by repressing the expression of forkhead box C1 (FOXC1), a member of forkhead box transcription factors.80  One study has also recently proved that EZH2 promotes metastasis in breast and prostate cancer cells via transcriptional repression of Raf-1 kinase inhibitor protein (RKIP).81  Lastly, EZH2 is an important mediator for tumoral angiogenesis, a key step allowing tumor cells to obtain sufficient oxygen and be spread hematogenously. Kaposi sarcoma–associated herpesvirus induces upregulation of EZH2 in endothelial cells, which serves as an essential induction signal for the proangiogenic molecule Ephrin-B2.82  EZH2-mediated epigenetic methylation also silences the expression of vasohibin 1, a soluble inhibitor of neovascularization,83  thus promoting angiogenesis.

Figure 3. 

Enhancer of zeste homolog 2 (EZH2)–mediated epigenetic regulation and EZH2-dependent oncogenesis. Black line: inhibitory signals; red arrow: excitatory signals. Abbreviations: FOXC1, forkhead box C1; H3K27me3, H3K27 trimethylation; miRNAs, microRNAs; PAX8, paired box gene 8; PRC2, polycomb repressive complex 2; PSP94, prostate secretory protein of 94 amino acids; Rap1, Ras-proximate-1 or Ras-related protein 1; Rap1GAP, Rap1 GTPase-activating protein; RKIP, Raf-1 kinase inhibitor protein; RUNX3, Runt-related transcription factor 3 gene.

Figure 3. 

Enhancer of zeste homolog 2 (EZH2)–mediated epigenetic regulation and EZH2-dependent oncogenesis. Black line: inhibitory signals; red arrow: excitatory signals. Abbreviations: FOXC1, forkhead box C1; H3K27me3, H3K27 trimethylation; miRNAs, microRNAs; PAX8, paired box gene 8; PRC2, polycomb repressive complex 2; PSP94, prostate secretory protein of 94 amino acids; Rap1, Ras-proximate-1 or Ras-related protein 1; Rap1GAP, Rap1 GTPase-activating protein; RKIP, Raf-1 kinase inhibitor protein; RUNX3, Runt-related transcription factor 3 gene.

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At the protein level, numerous reports have confirmed the overexpression of EZH2 in numerous proliferative and neoplastic conditions, including carcinoma,3,1621  sarcoma,2224  melanoma,84  primitive pediatric tumor (such as medulloblastoma and nephroblastoma),75,85,86  lymphoma and leukemia,2530  and myeloproliferative disorder.26,3134  Among carcinomas, EZH2 mRNA and/or protein levels were shown to be elevated, compared to normal tissue, in breast cancer,87,88  esophageal squamous cell carcinoma,41,89  gastric cancer,52,90  colorectal cancer,60  ovarian cancer,54,68,91  head and neck cancer,38,46,59  hepatobiliary and pancreatic cancer,11,17,51,57,92,93  lung cancer,56  and urothelial carcinoma21,94  (Table 5). The only reported exception of down-regulation of EZH2 in human carcinoma was observed in testicular germ cell tumors, in which a decreased EZH2 mRNA was detected in seminoma and nonseminoma tumor compared to normal testicular parenchyma.95  Using quantitative real-time polymerase chain reaction (qRT-PCR) or immunohistochemistry, only 3 previous studies6163  have reported a similar increase of EZH2 in RCC. Table 5 shows a detailed summary of all published studies reporting the association of EZH2 at the protein level with the tumor's clinicopathologic characteristics and outcome in carcinomas of different organs. With a few exceptions,50,89,93,96  most published reports show that EZH2 overexpression is associated with higher histologic grade, more advanced clinicopathologic stage, and/or a higher risk of lymph node or distant metastasis.* Taking AJCC TNM staging as an example, a large proportion of the published literature (26 of 33 reports) demonstrated that EZH2 overexpression was correlated with more locally advanced disease and/or higher risk of lymph node or distal metastasis, while the remaining 7 reports showed no difference of EZH2 labeling in tumors with different TNM staging (as summarized in Table 5, column 6).

Table 5.

Summary of Published Studies Reporting the Expression of Enhancer of Zeste Homolog 2 in Carcinoma

Summary of Published Studies Reporting the Expression of Enhancer of Zeste Homolog 2 in Carcinoma
Summary of Published Studies Reporting the Expression of Enhancer of Zeste Homolog 2 in Carcinoma

To date, only 3 published studies have reported on the significance of EZH2 expression in RCC, and these have shown conflicting results. Using qRT-PCR technique, Hinz et al61  investigated EZH2 mRNA level in 119 cases of CRCC and found that cases with detectable EZH2 mRNA (100 of 119; 84%) had no association with clinicopathologic tumor features, such as Fuhrman nuclear grade, pathologic stage, and presence of metastasis at time of surgery. By applying EZH2 immunohistochemical staining using TMA, Wagener et al63  studied 520 RCCs including 423 CRCCs, 45 PRCCs, 23 chromophobe RCCs, 3 collecting duct carcinomas, and 17 unclassified carcinomas. In their hands, expression of EZH2 was detected in 411 cases (79%), compatible with the positivity rates observed by Hinz et al. 61  However, Wagener and colleagues63  were able to demonstrate a strong association between EZH2 expression on the one hand and high Fuhrman grade, advanced tumor stage, regional lymph node metastasis, and distant metastasis on the other. Our results are in line with the findings of Wagener et al63  confirming an association between EZH2 and high histologic grade of the tumor, but also go against their findings in that an advanced pathologic stage was associated with lower rather than higher expression of EZH2. This being said, since most primary tumors in our cohort expressed the marker in very focal fashion (≍5% of cells staining), the significance of the marker's expression in different subgroups of the primary tumors (by nuclear grade or pathologic stage) should be interpreted with caution. In comparison, in the study of Wagener et al,63  51% of the included RCCs expressed the marker in a very focal fashion as well (1%–5% of positive cells).

To validate the only study reporting overexpression of EZH2 in metastatic tumors in comparison to primary tumors, we compared the staining patterns of the 2 groups in 2 different cohorts: (1) nonmatched primary RCCs versus metastatic RCCs overall, and (2) matched primary and metastatic tumors. In our hands, significant EZH2 overexpression was detected in metastatic compared to primary RCCs. These findings were also confirmed in the TMA, which included 22 cases of matched primary and metastatic tumors, with a significant increase of EZH2 positivity and staining intensity observed in the metastatic cases. The current study is the first to test EZH2 expression in matched primary and metastatic tumors, which allowed us to avoid confounders and selection bias that could have been introduced if the primary and metastatic cases had been derived from different patients. Therefore, our findings provide compelling evidence for a functional link between EZH2 dysregulation and metastasis in RCC. A similar association between metastasis and EZH2 was previously reported in breast87  and prostate cancers,15  and the result seems to be confirmed by the current study and that of Wagener et al63  for RCC. It is likely that this effect is mediated by EZH2 epigenetic modulation of key molecules mediating cell invasion and metastasis, including microRNAs, CDH1, FOXC1, and RK1P. However, this has not been confirmed yet at the molecular level in RCC.

In terms of clinical outcome, numerous investigators have reported that EZH2 overexpression is an adverse prognostic biomarker in different types of carcinomas in which it is associated, with decreased overall survival, progression-free survival, and disease-free survival (Table 5).1517,19,24,3560  This notwithstanding, conflicting studies exist that report either no prognostic value for EZH221,89–91,93 or even an association with improved disease-free survival,96  albeit to a more limited extent. Similarly, contradictory results have been reported in RCC: whereas Hinz et al61  reported that the presence of elevated EZH2 mRNA levels is associated with low disease recurrence, Wagener et al63  showed that patients with overexpression of EZH2 had decreased disease-specific survival. In the current study, EZH2 upregulation was associated with disease-specific death in both primary and metastatic CRCCs, confirming what has been reported by Wagener et al.63 

One potential limitation of the current study is that TMAs were used. A well-documented disadvantage of TMA is that each core represents only a fraction of the tumor. In tumors with only focal expression of certain proteins, TMAs may underestimate the prevalence of protein expression as compared with whole sections. Such a drawback is overcome by a large sample size or the use of multiple cores per sample, allowing results to be generated that are compatible with those of studies using whole sections. Although concordance between TMA and whole section was not directly addressed in the current study, the large number of tumors included and the fact that at least 2 cores were taken for the first and second TMAs theoretically balance the potential errors associated with intratumor heterogeneity. Storage of the paraffin blocks for a protracted period may result in decreased intensity of staining for certain antigens. Such effect should be taken into consideration, since the current study included cases dating back to 1985. To evaluate the effects of storage time on the EZH2 labeling, we compared the H-score among cases that were stored for less than 10 years, between 10 to 20 years, and for more than 20 years by using multivariate analysis of variance. A compatible average H-score was observed among the 3 groups, indicating that duration of storage did not affect the staining intensity for EZH2 (data not shown, P = .76).

In summary, this study evaluating the relation between RCC and EZH2 confirms that EZH2 expression is associated with increased metastatic potential and worse clinical outcome. Our study, together with emerging evidence derived from other human carcinomas, indicates that EZH2 is a key molecule driving oncogenesis, tumor invasiveness, and metastasis.

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References 11, 16, 17, 19, 21, 24, 36–47, 49, 51–53, 56–58, 60, 68, 88, 90, 91, 94, 97.

Author notes

The authors have no relevant financial interest in the products or companies described in this article.