Enhancer of Zeste Homolog 2 expression is associated with metastasis and adverse clinical outcome in clear cell renal cell carcinoma: a comparative study and review of the literature.
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. (61-63) 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.
MATERIALS AND METHODS
Three tissue microarrays (TMAs) were constructed by using tissues obtained from the surgical pathology archives of the McGill UniversityHealth 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 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 antihuman 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.
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 [chi square] 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 meta-static 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 immuno-labeling 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 ([chi square] 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.
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% [+ or -] 3.6% and 13.3% [+ or -] 1.9% for high and low grade, respectively) and a higher H-score (54.5 [+ or -] 8.0 and 24.1 [+ or -] 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% [+ or -] 4.7%) with higher H-score (74.5 [+ or -] 10.5) than those with lower nuclear grade (percentage of positive labeling: 22.6% [+ or -] 3.3%; H-score: 41.3 [+ or -] 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).
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%) ([chi square] test, P = .02). In addition, low-stage primary tumors were associated with a stronger staining intensity (1.5 6 0.2) (1-way ANOVA, P < .001) than high-stage tumors (0.7 6 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%) ([chi square] 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 ([chi square] test, P = .004). In this cohort, the mean staining intensity of primary and metastatic CRCCs was 0.6 [+ or -] 0.2 and 2.0 [+ or -] 0.2, respectively, and the mean H-scores for both groups were 2.7 [+ or -] 1.0 and 52.7 [+ or -] 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).
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.
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 [+ or -] 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 [+ or -] 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 [+ or -] 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 [+ or -] 33.5 months); and the third included patients who had RCC-related deaths (n = 10; follow-up duration, 13.2 [+ or -] 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 [+ or -] 21.9 months; 17 patients (40%) were alive with residual disease for a follow-up period of 60.4 [+ or -] 15.0 months; and 22 patients (51%) had disease-related death with a follow-up period of 29.3 [+ or -] 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).
The PRC2 is a highly conserved histone methyltransferase that is composed of 3 essential core subunits: EZH2, EED, and SUZ12. (8-10,65) 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. (8-10,65) 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 EZH2induced 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 (RaplGAP), 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. (14,77-79) 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 herpes-virus 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.
At the protein level, numerous reports have confirmed the overexpression of EZH2 in numerous proliferative and neoplastic conditions, including carcinoma, (3,16-21) sarcoma, (22-24) melanoma, (84) primitive pediatric tumor (such as medulloblastoma and nephroblastoma), (75,85,86) lymphoma and leukemia, (25-30) and myeloproliferative disorder. (26,31-34) 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 carcinoma (21,94) (Table 5). The only reported exception of downregulation 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 studies (61-63) 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).
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 al (61) 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 al (63) 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 colleagues (63) 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 al (63) confirming an association between EZH2 and high histo logic 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 breast (87) and prostate cancers, (15) and the result seems to be confirmed by the current study and that of Wagener et al (63) for RCC. It is likely that this effect is mediated by EZH2 epigenetic modulation of key molecules mediating cell invasion and metastasis, including micro-RNAs, 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). (15-17,19,24,35-60) This notwithstanding, conflicting studies exist that report either no prognostic value for EZH2 (21,89-91,93) 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 al (61) reported that the presence of elevated EZH2 mRNA levels is associated with low disease recurrence, Wagener et al (63) 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.
Caption: 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: X100; and magnification of insets: X400.
Caption: 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).
Caption: 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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* References 11, 16, 17, 19,21, 24, 36-47, 49, 51-53, 56-58, 60, 68, 88, 90, 91, 94, 97.
Bin Xu, MD, PhD; Samuel Abourbih, MD; Kanishka Sircar, MD; Wassim Kassouf, MD; Jose Joao Mansure, PhD; Armen Aprikian, MD; Simon Tanguay, MD; Fadi Brimo, MD
Accepted for publication November 19, 2012.
From the Departments of Pathology (Drs Xu and Brimo) and Urology (Drs Abourbih, Kassouf, Mansure, Aprikian, and Tanguay), McGill University Health Centre, Montreal, Quebec, Canada; and the Department of Pathology (Dr Sircar), The University of Texas Maryland Anderson Cancer Center, Houston.
The authors have no relevant financial interest in the products or companies described in this article.
Reprints: Fadi Brimo, MD, Department of Pathology, McGill University Health Centre, Montreal General Hospital, 1650 Cedar Ave, Montreal, QC H3G 1A4, Canada (e-mail: fadi.brimo@muhc. mcgill.ca).
Table 1. Expression of Enhancer of Zeste Homolog 2 (EZH2) in Clear Cell and Papillary Renal Cell Carcinoma (RCC) Highest Stain Intensity (%) N 0 1 2 3 Clear cell RCC 223 55 (25) 44 (20) 43 (19) 81 (36) Primary 91 30 (33) 27 (30) 13 (14) 21 (23) Metastasis 132 25 (19) 17 (13) 30 (23) 60 (45) Primary (matched) 22 12 (55) 5 (23) 2 (9) 3 (14) Metastasis (matched) 22 3 (14) 4 (18) 3 (14) 12 (55) Papillary RCC 21 5 (24) 2 (10) 4 (19) 10 (48) Primary 16 5 (29) 2 (12) 2 (12) 8 (47) Metastasis 5 1 (20) 0 0 4 (80) Overall 244 60 (25) 46 (19) 47 (19) 91 (37) Mean Staining Intensity (a) Clear cell RCC 1.6 [+ or -] 0.1 Primary 1.1 [+ or -] 0.1 (b) Metastasis 1.9 [+ or -] 0.1 (b) Primary (matched) 0.6 [+ or -] 0.2 (b) Metastasis (matched) 2.0 [+ or -] 0.2 (b) Papillary RCC 2.0 [+ or -] 0.3 Primary 1.7 [+ or -] 0.3 Metastasis 2.4 [+ or -] 0.7 Overall 1.6 [+ or -] 0.1 EZH2 Labeling, (a) % Clear cell RCC 19.6 [+ or -] 2.0 Primary 4.3 [+ or -] 1.0 (b) Metastasis 30.1 [+ or -] 2.9 (b) Primary (matched) 1.8 [+ or -] 0.7 (b) Metastasis (matched) 28.2 [+ or -] 6.0 (b) Papillary RCC 24.0 [+ or -] 7.1 Primary 13.7 [+ or -] 6.1 (b) Metastasis 54.0 [+ or -] 19.5 (b) Overall 20.0 [+ or -] 1.9 H-Score (a) Clear cell RCC 37.7 [+ or -] 4.2 Primary 7.5 [+ or -] 2.0 (b) Metastasis 58.5 [+ or -] 6.3 (b) Primary (matched) 2.7 [+ or -] 1.0 (b) Metastasis (matched) 50.0 [+ or -] 11.2 (b) Papillary RCC 45.0 [+ or -] 13.6 Primary 26.7 [+ or -] 12.3% (b) Metastasis 98.0 [+ or -] 36.0% (b) Overall 38.3 [+ or -] 4.0 (a) Data were expressed as mean 6 standard error. (b) Primary tumor versus metastasis, 1-way analysis of variance, P < .05. Table 2. Enhancer of Zeste Homolog 2 (EZH2) Immunoreactivity in Clear Cell Renal Cell Carcinoma (CRCC) Highest Stain Intensity (%) N 0 1 2 3 Nuclear grade of all CRCCs 1 or 2 124 31 (25) 22 (18) 26 (21) 45 (36) 3 or 4 97 24 (25) 22 (23) 17 (18) 34 (35) Nuclear grade of primary CRCCs 1 or 2 61 19 (31) 11 (18) 10 (16) 16 (26) 3 or 4 30 11 (37) 11 (37) 3 (10) 5 (17) Nuclear grade of metastatic CRCCs 1 or 2 63 12 (19) 6 (10) 16 (25) 29 (46) 3 or 4 67 13 (19) 11 (16) 14 (21) 29 (43) Pathologic stage T1 or T2 47 10 (21) 12 (26) 8 (17) 17 (36) T3 or T4 41 182 (44) 15 (37) 4 (10) 4 (10) Staining Intensity EZH2 Labeling, % Nuclear grade of all CRCCs 1 or 2 1.6 [+ or -] 0.1 13.3 [+ or -] 1.9 3 or 4 1.5 [+ or -] 0.1 27.1 [+ or -] 3.6 (a) Nuclear grade of primary CRCCs 1 or 2 1.2 [+ or -] 0.1 3.7 [+ or -] 1.0 3 or 4 0.9 [+ or -] 0.2 5.5 [+ or -] 2.2 Nuclear grade of metastatic CRCCs 1 or 2 1.9 [+ or -] 0.1 22.6 [+ or -] 3.3 3 or 4 1.8 [+ or -] 0.1 36.8 [+ or -] 4.7 (a) Pathologic stage T1 or T2 1.5 [+ or -] 0.22 4.3 [+ or -] 1.0 T3 or T4 0.7 [+ or -] 0.1b 4.5 [+ or -] 1.9 H-Score Nuclear grade of all CRCCs 1 or 2 24.1 [+ or -] 3.7 3 or 4 54.5 [+ or -] 8.0 (a) Nuclear grade of primary CRCCs 1 or 2 6.4 [+ or -] 1.9 3 or 4 9.9 [+ or -] 4.6 Nuclear grade of metastatic CRCCs 1 or 2 41.3 [+ or -] 6.4 3 or 4 74.5 [+ or -] 10.5 (a) Pathologic stage T1 or T2 7.5 [+ or -] 1.8 T3 or T4 8.0 [+ or -] 3.9 (a) Comparison between high and low nuclear grade, P < .05. (b) Comparison between high and low tumor stage, P < .05. Data were expressed as mean 6 standard error. Table 3. Expression of Enhancer of Zeste Homolog 2 (EZH2) in Metastatic Clear Cell Renal Cell Carcinoma Site of Metastasis N Positive Staining Cases, No. (%) Intensity Lymph nodes 26 21 (81) 1.7 [+ or -] 0.04 Bone 20 17 (85) 2.4 [+ or -] 0.1 Brain 19 16 (84) 2.3 [+ or -] 0.1 Lung 17 11 (65) 1.3 [+ or -] 0.1 Adrenal glands 11 9 (82) 1.8 [+ or -] 0.1 Liver 5 5 (100) 2.4 [+ or -] 0.2 Thyroid 2 1 (50) 1.5 [+ or -] 1.5 Others (a) 10 8 (80) 2.0 [+ or -] 0.1 Overall 110 88 (80) 1.9 [+ or -] 0.01 Site of Metastasis EZH2 H-Score Labeling, % Lymph nodes 30.6 [+ or -] 6.9 55.2 [+ or -] 13.9 Bone 33.2 [+ or -] 7.2 63.9 [+ or -] 13.8 Brain 47.5 [+ or -] 8.2 100.2 [+ or -] 20.3 Lung 13.6 [+ or -] 5.7 24.0 [+ or -] 10.8 Adrenal glands 13.8 [+ or -] 5.8 22.8 [+ or -] 10.5 Liver 53.2 [+ or -] 19.9 133.8 [+ or -] 53.0 Thyroid 1.0 [+ or -] 0.7 2.5 [+ or -] 1.8 Others (a) 33.3 [+ or -] 13.5 67.4 [+ or -] 30.1 Overall 30.4 [+ or -] 3.3 60.2 [+ or -] 7.2 (a) This category included 10 metastatic clear cell renal cell carcinomas obtained from stomach (n = 1), ileum (n = 1), colon (n = 1), omentum (n = 1), mesentery (n = 1), retroperitoneum (n = 1), pancreas (n = 1), breast (n = 1), thorax (n = 1), and tongue (n = 1). Data were expressed as mean 6 standard error. Table 4. The Correlation of Clinical Outcomes With the Expression of Enhancer of Zeste Homolog 2 (EZH2) in Clear Cell Renal Cell Carcinoma (CRCC) Clinical N Follow-up Positive Outcome (a) Period, mo Cases, No. (%) (b) Primary CRCC (AJCC stage T1a) NED 37 108.0 [+ or -] 5.1 30 (81) DSD 1 67 0 Primary CRCC (AJCC stage T3 and T4) NED 8 83.6 [+ or -] 29.2 5 (63) AWD 4 47.5 [+ or -] 33.5 3 (75) DSD 10 13.2 [+ or -] 2.6 6 (60) Metastatic CRCC NED 4 83.3 [+ or -] 21.9 4 (100) AWD 17 60.4 [+ or -] 15.0 10 (59) DSD 22 29.3 [+ or -] 5.3 20 (91) Clinical Staining EZH2 Outcome (a) Intensity Labeling, % Primary CRCC (AJCC stage T1a) NED 1.6 [+ or -] 0.2 5.1 [+ or -] 1.2 DSD 0 0 Primary CRCC (AJCC stage T3 and T4) NED 0.6 [+ or -] 0.2 1.6 [+ or -] 1.2 AWD 0.8 [+ or -] 0.3 0.8 [+ or -] 0.3 DSD 1.2 [+ or -] 0.4 13.6 [+ or -] 7.2 (b) Metastatic CRCC NED 2.3 [+ or -] 0.5 12.8 [+ or -] 10.8 AWD 1.1 [+ or -] 0.3 11.5 [+ or -] 5.5 DSD 2.1 [+ or -] 0.2 29.8 [+ or -] 5.8 (b) Clinical H-Score Outcome (a) Primary CRCC (AJCC stage T1a) NED 8.9 [+ or -] 2.3 DSD 0 Primary CRCC (AJCC stage T3 and T4) NED 1.9 [+ or -] 1.5 AWD 0.8 [+ or -] 0.3 DSD 26.0 [+ or -] 14.9 (b) Metastatic CRCC NED 21.3 [+ or -] 18.0 AWD 20.6 [+ or -] 10.6 DSD 55.0 [+ or -] 12.3 (b) Abbreviations: AJCC, American Joint Committee on Cancer; AWD, alive with disease; DSD, disease-specific death; NED, no evidence of disease. (a) Categories of clinical outcome: NED, AWD, and DSD. (b) Comparison between DSD and AWD/NED combined, P < .05. Data were expressed as mean 6 standard error. Table 5. Summary of Published Studies Reporting the Expression of Enhancer of Zeste Homolog 2 in Carcinoma Source, y Carcinoma Tissue (Patient, n) and Methods Breast cancer Bachmann Breast cancer (190) TMA, IHC et al, (16) Endometrial 2006 carcinoma (316) Melanoma (202) Prostate cancer (104) Alford Breast cancer (480) TMA, IHC et al, (36) 2012 Athanassiadou Breast cancer (100) Smear, IHC et al, (37) 2011 Collett et Breast cancer (190) TMA, IHC al, (39) 2006 De Brot et Breast cancer (140) TMA, IHC al, (98) 2012 Gong et Breast cancer (88) TMA, IHC al, (40) 2011 Holm et Breast cancer (428) TMA, IHC, al, (43) 2012 FISH Kleer et Breast cancer (280) TMA, IHC, al, (87) 2003 qRT-PCR Pang et Breast cancer (100) FFPE, IHC al, (88) 2012 Pietersen et Breast cancer (295) TMA, IHC, al, (53) 2008 cRNA oligo- nucleotide array Reijm et Breast cancer (1318) qRT-PCR al, (55) 2011 Gastrointestinal cancer Ha and Esophageal squamous FFPE, IHC Kim, (89) 2012 cell carcinoma (164) He et Esophageal squamous FFPE, IHC al, (41) 2009 cell carcinoma (98) He et Esophageal squamous FFPE, IHC, al, (42) 2010 cell carcinoma (98) FISH Yamada et Esophageal squamous FFPE, IHC al, (24) 2011 cell carcinoma (136) Choi et Gastric cancer (137) TMA, IHC, al, (90) 2010 Western Lee et Gastric cancer (178) TMA, IHC al, (50) 2012 Matsukawa et Gastric cancer (83) FFPE, IHC, al, (52) 2006 Western, RT-PCR Fluge et Colon cancer (290), TMA, IHC al, (96) 2009 rectal cancer (122) Wang et Colorectal cancer TMA, IHC al, (60) 2010 (119) Gynecologic cancer Guo et Ovarian cancer (55) FFPE, IHC, al, (68) 2011 qRT-PCR Li et Ovarian epithelial TMA, IHC, al, (91) 2010 carcinoma (134) Western Rao et Ovarian epithelial TMA, IHC, al, (54) 2010 carcinoma (163) FISH Head and neck cancer (including salivary gland cancer) Cao et Squamous cell qRT-PCR, al, (38) 2012 carcinoma (170) FFPE, IHC Kidani et Squamous cell FFPE, IHC, al, (46) 2009 carcinoma (102) Western Alajez et Nasopharyngeal FFPE, IHC al, (35) 2010 carcinoma (39) Hwang et Nasopharyngeal IHC, RT-PCR al, (45) 2012 carcinoma (89) Vekony et Salivary gland FFPE, IHC al, (59) 2008 adenoid cystic carcinoma (21) Hepatobiliary and pancreatic cancer Cai et Hepatocellular TMA, IHC al, (17) 2011 carcinoma (126) Au et Hepatocellular qRT-PCR al, (11) 2012 carcinoma (59) Sasaki et Hepatocellular IHC, qRT- al, (92) 2008 carcinoma (41) PCR Sudo et Hepatocellular qRT-PCR, al, (93) 2005 carcinoma (66) FFPE, IHC Liu and Gallbladder adeno- FFPE, IHC Yang, (51) 2011 carcinoma (108) Toll et Pancreatic ductal FFPE, IHC al, (57) 2010 adenocarcinoma (54) Lung cancer Takawa et NSCLC (292) TMA, IHC, al, (56) 2011 Colorectal cancer FFPE, IHC, (245) qRT-PCR Bladder cancer (29) Findeis-Hosey Lung NET, NEC, FFPE, IHC et al, (97) 2011 and small cell carcinoma (68) Huqun et NSCLC (106) FFPE, IHC al, (44) 2012 Kikuchi et NSCLC (157) FFPE, IHC, al, (47) 2010 Western Genitourinary cancer Hinz et Testicular semi- qRT-PCR al, (95) 2010 noma (64) Testicular non- seminoma (36) Varambally Prostate cancer TMA, IHC, et al, (15) 2002 (1023) RT-PCR Laitinen et Prostate cancer (249) TMA, IHC al, (49) 2008 van Leenders Prostate cancer (114) TMA, IHC et al, (58) 2007 Hinz et Urothelial qRT-PCR al, (19) 2008 carcinoma (100) Wang et Urothelial TMA, IHC al, (21) 2012 carcinoma (81) Raman et Urothelial FFPE, IHC, al, (94) 2005 carcinoma (77) RT-PCR Hinz et CRCC (119) qRT-PCR, al, (61) 2009 FFPE, IHC Wagener et RCC (21) qRT-PCR al, (62) 2008 Wagener et RCC (520) including: TMA, IHC al, (63) 2010 CRCC (422) PRCC (45) Chromophobe (23) Collecting duct (3) Unclassified (17) Current study CRCC (223) TMA, IHC PRCC (21) Source, y Over- Association Association expression With With Compared High High to Normal Histologic TNM Tissue Grade Staging Breast cancer Bachmann N/A + (breast, + (prostate et al, (16) endometrial, and breast) 2006 and prostate) Alford N/A + + et al, (36) 2012 Athanassiadou N/A + + et al, (37) 2011 Collett et N/A + + al, (39) 2006 De Brot et N/A N/A N/A al, (98) 2012 Gong et N/A + N/A al, (40) 2011 Holm et N/A + N/A al, (43) 2012 Kleer et + N/A N/A al, (87) 2003 Pang et + + N/A al, (88) 2012 Pietersen et N/A + N/A al, (53) 2008 Reijm et N/A + + al, (55) 2011 Gastrointestinal cancer Ha and + - - Kim, (89) 2012 He et + + + al, (41) 2009 He et N/A + + al, (42) 2010 Yamada et N/A - + al, (24) 2011 Choi et + N/A + al, (90) 2010 Lee et N/A - - al, (50) 2012 Matsukawa et + + + al, (52) 2006 Fluge et N/A - - al, (96) 2009 Wang et + - + al, (60) 2010 Gynecologic cancer Guo et + + + al, (68) 2011 Li et + + - al, (91) 2010 Rao et + N/A N/A al, (54) 2010 Head and neck cancer (including salivary gland cancer) Cao et + + + al, (38) 2012 Kidani et + + + al, (46) 2009 Alajez et N/A N/A N/A al, (35) 2010 Hwang et N/A N/A + al, (45) 2012 Vekony et + N/A N/A al, (59) 2008 Hepatobiliary and pancreatic cancer Cai et + - + al, (17) 2011 Au et N/A + al, (11) 2012 Sasaki et N/A N/A al, (92) 2008 Sudo et - - al, (93) 2005 Liu and Yang, (51) 2011 Toll et - al, (57) 2010 Lung cancer Takawa et + N/A + al, (56) 2011 Findeis-Hosey N/A + N/A et al, (97) 2011 Huqun et N/A - + al, (44) 2012 Kikuchi et N/A + al, (47) 2010 Genitourinary cancer Hinz et (b) N/A - al, (95) 2010 Varambally N/A N/A N/A et al, (15) 2002 Laitinen et N/A + + al, (49) 2008 van Leenders N/A + N/A et al, (58) 2007 Hinz et N/A + + al, (19) 2008 Wang et + + N/A al, (21) 2012 Raman et + + + al, (94) 2005 Hinz et + - - al, (61) 2009 Wagener et + N/A N/A al, (62) 2008 Wagener et + + + al, (63) 2010 Current study N/A + +/- Source, y Over- Adverse expression Prognostic in Metastasis Factor Compared to Primary Tumor Breast cancer Bachmann N/A + et al, (16) 2006 Alford N/A + et al, (36) 2012 Athanassiadou N/A + et al, (37) 2011 Collett et N/A + al, (39) 2006 De Brot et N/A - al, (98) 2012 Gong et N/A + al, (40) 2011 Holm et N/A + al, (43) 2012 Kleer et + + al, (87) 2003 Pang et N/A N/A al, (88) 2012 Pietersen et N/A + al, (53) 2008 Reijm et N/A + al, (55) 2011 Gastrointestinal cancer Ha and N/A - Kim, (89) 2012 He et N/A + al, (41) 2009 He et N/A + al, (42) 2010 Yamada et N/A + al, (24) 2011 Choi et N/A - al, (90) 2010 Lee et N/A + al, (50) 2012 Matsukawa et N/A + al, (52) 2006 Fluge et N/A (a) al, (96) 2009 Wang et N/A + al, (60) 2010 Gynecologic cancer Guo et N/A N/A al, (68) 2011 Li et N/A - al, (91) 2010 Rao et N/A + al, (54) 2010 Head and neck cancer (including salivary gland cancer) Cao et N/A + al, (38) 2012 Kidani et N/A + al, (46) 2009 Alajez et N/A + al, (35) 2010 Hwang et N/A + al, (45) 2012 Vekony et N/A + al, (59) 2008 Hepatobiliary and pancreatic cancer Cai et N/A + al, (17) 2011 Au et N/A N/A al, (11) 2012 Sasaki et N/A N/A al, (92) 2008 Sudo et N/A - al, (93) 2005 Liu and N/A + Yang, (51) 2011 Toll et N/A + al, (57) 2010 Lung cancer Takawa et N/A + al, (56) 2011 (NSCLC) Findeis-Hosey N/A N/A et al, (97) 2011 Huqun et N/A + al, (44) 2012 Kikuchi et N/A + al, (47) 2010 Genitourinary cancer Hinz et N/A N/A al, (95) 2010 Varambally + + et al, (15) 2002 Laitinen et N/A + al, (49) 2008 van Leenders N/A + et al, (58) 2007 Hinz et N/A + al, (19) 2008 Wang et N/A - al, (21) 2012 Raman et N/A N/A al, (94) 2005 Hinz et N/A (a) al, (61) 2009 Wagener et N/A N/A al, (62) 2008 Wagener et + + al, (63) 2010 Current study + + Abbreviations: CRCC, clear cell renal cell carcinoma; cRNA, complementary RNA; FFPE, formalin fixed, paraffin embedded; FISH, fluorescence in situ hybridization; IHC, immunohistochemistry; N/A, data not available; NEC, neuroendocrine carcinoma; NET, neuroendocrine tumor; NSCLC, non-small cell lung cancer; PRCC, papillary renal cell carcinoma; qRT-PCR, quantitative real-time-polymerase chain reaction; RCC, renal cell carcinoma; RT-PCR, reverse transcriptase-polymerase chain reaction; TMA, tissue microarray; Western, Western blot; +, overexpression of EZH2 is associated with high histologic grade, high American Joint Committee on Cancer (AJCC) stage, or adverse prognosis; +/-, expression of enhancer of zeste homolog 2 (EZH2) is associated with a lower AJCC tumor staging (pT1 or pT2) but a higher AJCC staging for lymph node involvement or distal metastasis (pN1 or pM1); -, no association of EZH2 overexpression with grade, stage, or prognosis. (a) Elevated EZH2 is associated with an improved clinical prognosis. (b) A decreased level of EZH2 is detected in cancer as compared to normal tissue.
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|Title Annotation:||Original Articles|
|Author:||Xu, Bin; Abourbih, Samuel; Sircar, Kanishka; Kassouf, Wassim; Mansure, Jose Joao; Aprikian, Armen; T|
|Publication:||Archives of Pathology & Laboratory Medicine|
|Date:||Oct 1, 2013|
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