Originally published as JCO Early Release 10.1200/JCO.2005.01.5180 on December 5 2005

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EZH2 Expression Is Associated With High Proliferation Rate and Aggressive Tumor Subgroups in Cutaneous Melanoma and Cancers of the Endometrium, Prostate, and Breast

From the the Gade Institute, Section of Pathology; Department of Surgical Sciences, University of Bergen; Department of Clinical Medicine, Section of Obstetrics and Gynecology, Haukeland University Hospital, Bergen, Norway; Department of Biochemistry, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, the Netherlands

Address reprint requests Lars A. Akslen, MD, PhD, Children’s Hospital, Harvard Medical School, Vascular Biology Program, Karp Family Research Labs 12.125, 300 Longwood Ave, Boston, MA 02115-5737; e-mail: lars.akslen{at}childrens.harvard.edu or lars.akslen{at}gades.uib.no

	ABSTRACT

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PURPOSE: EZH2 is a member of the polycomb group of genes and important in cell cycle regulation. Increased expression of EZH2 has been associated previously with invasive growth and aggressive clinical behavior in prostate and breast cancer, but the relationship with tumor cell proliferation has not been examined in human tumors. The purpose of this study was to validate previous findings in a population-based setting, also including tumors that have not been studied previously.

PATIENTS AND METHODS: In our study of nearly 700 patients, we examined EZH2 expression and its association with tumor cell proliferation and other tumor markers, clinical features, and prognosis in cutaneous melanoma and cancers of the endometrium, prostate, and breast.

RESULTS: Strong EZH2 expression was associated with increased tumor cell proliferation in all four cancer types. Associations were also found between EZH2 and important clinicopathologic variables. EZH2 expression showed significant prognostic impact in melanoma, prostate, and endometrial carcinoma in univariate survival analyses, and revealed independent prognostic importance in carcinoma of the endometrium and prostate.

CONCLUSION: Our findings point at EZH2 as a novel and independent prognostic marker in endometrial cancer, and validate previous findings on prostate and breast cancer. Further, EZH2 expression was associated with features of aggressive cutaneous melanoma. The fact that EZH2 might identify increased tumor cell proliferation and aggressive subgroups in several cancers may be of practical interest because the polycomb group proteins have been suggested as candidates for targeted therapy. EZH2 expression should, therefore, be further examined as a possible predictive factor.

	INTRODUCTION

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The enhancer of zeste homolog 2 (EZH2) is a member of the polycomb group of genes (PcG), which are important for transcriptional regulation through nucleosome modification, chromatin remodeling, and interaction with other transcription factors.¹ EZH2 serves as a histone methyl transferase (HMT), and disruption of EZH2 expression may lead to dysregulation of genes critical for the G2-M transition. Further, EZH2 is controlled by E2F transcription factors, which are downstream of the retinoblastoma protein (Rb),² and is also involved in p53-regulated cell cycle control.³ EZH2 was shown previously to be overexpressed in prostate,⁴ and breast cancer,^5,6 and increased expression evaluated by mRNA in situ was found in 34% of human cancers in a recent report.² The aim of our study was to evaluate and validate the expression of EZH2 in a broader range of human cancers, with special attention to key features such as tumor cell proliferation, tumor extent, and patient outcome. This is especially relevant since the PcG proteins have recently been suggested as candidates for targeted therapy,⁷ and EZH2 should be evaluated as a possible predictive factor.

	PATIENTS AND METHODS

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Patients
For this study, four well-defined patient series from Hordaland County, Norway, were included, and all cases (n = 696) were diagnosed at the Gade Institute, Section of Pathology, University of Bergen (Bergen, Norway). First, 202 consecutive nodular melanomas (diagnosed between 1981 and 1997) were examined (median follow-up time, 89 months; range, 24 to 221 months; 72 melanoma deaths).⁸ For melanomas, the following variables were included: anatomic site of the primary tumor, presence of metastases at diagnosis (local, regional, distant), Breslow’s tumor thickness, Clark’s level of invasion, microscopic ulceration, vascular invasion, and mitotic count (number of mitoses/mm²). In addition, metastatic tumors from 58 patients with recurrent disease were available for analysis. Second, a consecutive series of 104 patients treated with radical prostatectomy for clinically localized prostate cancer from 1988 to 1994 was studied (median follow-up, 104 months; range, 20 to 179 months; 31 patients developed clinical recurrence; 15 patients developed distant [skeletal] metastases; 9 patients died as a result of prostate cancer).⁹ For prostate cancer, these variables were recorded: largest tumor diameter, WHO histologic grade, tumor stage (TNM category), capsular penetration, seminal vesicle invasion, involvement of surgical margins, presence of lymph node metastases, and serum prostate-specific antigen (s-PSA) before and after surgical treatment. Third, a population-based series of 316 endometrial carcinomas (1981 to 1990) was included (median follow-up, 9 years; range, 4 to 16 years; 70 patients died as a result of endometrial cancer).¹⁰ The variables recorded for endometrial carcinomas were histologic type, International Federation of Gynecology and Obstetrics (FIGO) histologic grade, nuclear grade, mitotic count, solid growth, growth pattern, necrosis, vascular invasion, myometrial invasion, and FIGO stage. Finally, 190 breast cancers (95 interval cancers and 95 screen-detected cancers matched by tumor size) were included from the population-based Norwegian Breast Cancer Screening Program (Hordaland County) during two screening rounds and intervals (1996 to 2001; the cases were too recent to provide sufficient number of events during follow-up).¹¹ These variables were recorded for breast cancer: tumor diameter, histologic type, histologic grade,¹² and metastases at diagnosis (lymph node, distant).

Tissue Microarray
The technique of tissue microarray (TMA) has been described¹³ and validated previously.¹⁴ For TMA construction,¹³ representative tumor areas were identified on hematoxylin and eosin–stained slides, and a minimum of three tissue cylinders with a diameter of 0.6 mm were punched from selected areas of the donor block and mounted into a recipient paraffin block using a custom-made precision instrument (Beecher Instruments, Silver Spring, MD). Sections of the resulting TMA blocks (5 µm) were made by standard technique. Sufficient tumor tissue was available and studied in 696 of 812 cases.

Immunohistochemistry
TMA slides were dewaxed with xylene/ethanol before microwave epitope retrieval boiling for 10 minutes at 750 W and 20 minutes at 350 W in tris (hydroxymethyl)aminomethane–EDTA buffer (pH = 9). Sections were incubated for 1 hour with a monoclonal antibody against EZH2 (clone M18)¹⁵ diluted 1:2, using a DAKO Autostainer and the EnVision chain-polymer method (Dako Cytomation, Copenhagen, Denmark) as detection system. A breast carcinoma known to be positive for EZH2 expression was used as a positive control. Staining was recorded using a semiquantitative and subjective grading system, considering the intensity of staining and proportion of tumor cells showing a positive nuclear reaction.

Evaluation of Staining Results
Because of the present lack of standardization of methods and assessment criteria for EZH2 expression, a staining index (SI) was calculated as the product of intensity (0 to 3) and positive tumor cell area (< 10%, 10% to 50%, > 50%).¹⁶ Expression categories were based initially on quartiles of the resulting SI (values 0 to 9), considering also the distribution plots, as well as number of cases and events when determining the final cut points between low and high expression (two categories). In survival analysis, subgroups (based on quartiles) with similar survival were subsequently merged. For each tumor type, the same cutoff point was used for analysis of associations and outcome; median was used for melanoma and breast cancer (SI = 3 for both), and upper quartile was used for prostate and endometrial cancer (SI = 6 for endometrial cancer and SI = 4 for prostate cancer). Thus, categories of high and low expression were consistently defined as groups with staining indices above or below/equal to the cutoff point used for each tumor. Information on other tumor markers was included for comparison; melanoma: Ki-67, p16 and p53,⁸ cyclin D1, Rb and cdk4¹⁷; prostate cancer: Ki-67,⁹ p16 and cdk4¹⁸; and endometrial cancer: Ki-67,¹⁹ p53 and p16.²⁰

Statistical methods
Analyses were performed using the the Statistical Package for the Social Sciences version 12.0 (SPSS Inc, Chicago, IL). Associations between different categoric variables were assessed by Pearson’s ² test. Distributions of continuous variables were compared between different categories by the Mann-Whitney U test. The Wilcoxon signed rank test was used to compare related samples. Univariate analyses of time to death as a result of cancer, or time to recurrence (recurrence-free survival) were performed using the product-limit procedure (Kaplan-Meier method) and log-rank test. Patients who died as a result of other causes were censored at the date of death. The influence of covariates on patient survival and recurrence-free survival was analyzed by the proportional hazards method, and tested by the likelihood ratio test. The level for inclusion of covariates in Cox multivariate regression analyses was P = .10. Regarding cancer of the prostate, survival analyses were also performed with occurrence of skeletal metastases as end point. Death as a result of causes other than prostate cancer during follow-up was censored at the time of death.

	RESULTS

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EZH2 Expression and Proliferation
Median values for tumor cell proliferation as estimated by Ki-67 staining were 27% (melanoma), 6.7% (prostate), 23% (endometrium) and 12% (breast). Nuclear expression of EZH2 (Fig 1) was significantly associated with proliferation in all tumor series (Table 1). Compared to tumors with low EZH2 expression, those with high expression showed an increase in median tumor cell proliferation rates (estimated by Ki-67 expression) of 38% (melanoma), 103% (prostate), 95% (endometrium) and 250% (breast). Further, mitotic counts were available for melanomas (median, 6.0 mitoses/mm²) and endometrial carcinomas (median, 6.3 mitoses/mm²), showing a significant increase of 241% for melanomas, and 44% for endometrial cancers, comparing subgroups with high and low EZH2 expression (Table 1). For melanomas, median EZH2 expression (SI) was increased 43% in metastatic melanomas, when compared with corresponding primary tumors (P = .041, Wilcoxon signed rank test).

View larger version (135K): [in this window] [in a new window]   Fig 1. Immunohistochemical staining showing low and high levels of nuclear EZH2 expression, in (A) melanoma, (B) endometrial carcinoma, (C) prostate cancer; and (D) breast carcinoma. The corresponding univariate survival curves (Kaplan-Meier method) are shown in the right column; the survival curve for part D represents prostate cancer, not breast carcinoma; follow-up data were not available for the breast cancer patients (see Patients and Methods). Scale bar, 50 µm.

Prostate cancer. High expression of EZH2 (by upper quartile) was associated significantly with moderately/poorly differentiated carcinomas (by WHO histologic grade), as opposed to well-differentiated carcinomas (P = .022), with seminal vesicle invasion (P = .021), and with lymph node infiltration (P = .042). No association between EZH2 and p16 or CDK4 protein expression was noted.

Endometrial cancer. High EZH2 expression was associated significantly with the serous papillary or clear cell histologic subtypes (P = .009), high histologic grade (by FIGO; P = .006), high nuclear grade (P < .0001), high FIGO stage (P = .003), and loss of p16 protein expression (P = .022).

Breast cancer. High EZH2 expression was associated with high histologic grade (P < .001), locally advanced cancers (P = .012), and presence of distant metastatic disease at the time of diagnosis (P = .01).

EZH2 Expression and Patient Survival
Survival data were available for melanoma, prostate cancer, and endometrial cancer. The end point used in survival analyses was death as a result of disease, and other causes of death were censored at time of death. For melanomas, 5-year survival in cases with high EZH2 expression was 48%, compared with 71% among the rest (P = .032; Fig 1). The subgroup with high EZH2 expression (above upper quartile of SI) showed a significant prognostic impact in univariate survival analysis in prostate and endometrial carcinomas. In prostate cancer, 5-year survival regarding time to skeletal metastases, was 97% and 89% for cases with low and high EZH2 expression, respectively (10-year survival, 85% and 29%; P = .0035; Fig 1). Five-year survival regarding time to death of prostate cancer was 99% and 89% (10-year survival, 93% and 53%), for cases with low and high EZH2 expression, respectively (P = .0038). In endometrial carcinomas, 5-year survival was 80% and 56% (low and high expressors), and 10-year survival was 77% and 56% (P < .0001; Fig 1).

In multivariate survival analyses (Cox proportional hazards method), EZH2 expression did not reach significance (P = .25) in the melanoma series when included along with strong prognostic factors such as tumor thickness (Breslow thickness), level of invasion (Clark level), vascular invasion, and tumor ulceration. The presence of tumor cell proliferation (Ki-67) did not influence the impact of EZH2 expression in the multivariate model. In prostate cancer, including standard prognostic variables such as histologic grade (by WHO) and pathologic stage as covariates, EZH2 expression (cutpoint upper quartile) independently predicted clinical recurrence (hazard ratio [HR] = 3.4; P = .037), together with histologic grade (clinical recurrence was used to ensure sufficient number of events; n = 31). EZH2 was also significant in multivariate analyses when using time to skeletal metastases (Table 2) or time to prostate cancer deaths (not shown). In the series of endometrial carcinomas, EZH2 expression had strong and independent prognostic influence (HR = 2.2; P = .02), when included together with multiple known prognostic variables such as histologic type, histologic grade (FIGO), vascular invasion, depth of myometrial infiltration, and FIGO stage (Table 2). In the presence of tumor cell proliferation (Ki-67), EZH2 expression was still an independent prognostic factor (not shown).

	DISCUSSION

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Although EZH2 expression has been promoted as a marker of invasion and aggressive tumors,^4-6 experimental data indicate a role in cell cycle regulation and proliferation.^1,21 Recently published cDNA microarray data show that EZH2 is specifically downregulated in senescent fibroblasts, and that disruption of EZH2 expression retards cell proliferation and induces cell cycle arrest at the G2-M transition.³ Further, overexpression of EZH2 in cultured mouse embryonic fibroblasts was found to shorten the G1 phase of the cell cycle and lead to accumulation of cells in the S phase.² Thus, our data from several human tumors support an influence of EZH2 on proliferation because EZH2 was consistently associated with increased Ki-67 expression in all tumor types. In addition to the strong associations between increased EZH2 expression and proliferation, our findings indicate a connection with local tumor invasion, advanced disease, and patient outcome.

Notably, we found a significant association between strong EZH2 expression and loss of the cell cycle suppressor protein p16 in melanomas and endometrial carcinomas. Low or absent p16 protein is known to be associated with increased Rb expression,^17,22 which could explain our findings in these subgroups. In melanomas, high levels of EZH2 were also associated with increased expression of cyclin D1, which is involved in the same regulatory pathway.

In conclusion, our findings point at EZH2 as a novel marker of aggressive tumors in malignant melanoma and endometrial cancer, and these data validate and extend previous findings on prostate and breast cancer. We found strong associations between EZH2 expression and increased tumor cell proliferation, and correlations with indicators of local tumor invasion and advanced disease. The fact that EZH2 might be a marker of aggressive subgroups in several cancers may be of significant practical interest, since the PcG proteins have been proposed recently as candidates for targeted therapy. EZH2 should, therefore, be further studied as a possible predictive factor.

	Authors’ Disclosures of Potential Conflicts of Interest

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors’ Disclosures of... Author Contributions GLOSSARY REFERENCES

 
The authors indicated no potential conflicts of interest.

	Author Contributions

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors’ Disclosures of... Author Contributions GLOSSARY REFERENCES

 

Conception and design: Ingeborg M. Bachmann, Lars A. Akslen Financial support: Lars A. Akslen Administrative support: Lars A. Akslen Provision of study materials or patients: Ole J. Halvorsen, Oddbjørn Straume, Svein A. Haukaas, Helga B. Salvesen, Arie P. Otte, Lars A. Akslen, Karin Collett, Ingunn M. Stefansson Collection and assembly of data: Ingeborg M. Bachmann, Ole J. Halvorsen, Karin Collett, Ingunn M. Stefansson, Oddbjørn Straume, Helga B. Salvesen, Lars A. Akslen Data analysis and interpretation: Ingeborg M. Bachmann, Ole J. Halvorsen, Karin Collett, Ingunn M. Stefansson, Lars A. Akslen Manuscript writing: Ingeborg M. Bachmann, Ole J. Halvorsen, Lars A. Akslen Final approval of manuscript: Ingeborg M. Bachmann, Ole J. Halvorsen, Karin Collett, Ingunn M. Stefansson, Oddbjørn Straume, Svein A. Haukaas, Helga B. Salvesen, Arie P. Otte, Lars A. Akslen

 

	GLOSSARY

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Breslow’s tumor thickness:

The microstage of malignant melanoma is defined by Breslow’s and Clark’s classifications. The Breslow classification defines the absolute vertical thickness in mm of the primary tumor in the skin. This microstaging more accurately predicts subsequent behavior of cutaneous melanoma.

Clark’s level of invasion:

The microstage of malignant melanoma is defined by Breslow’s and Clark’s classifications. The Clark classification defines the anatomical level of primary tumor invasion in the skin and is reported as level I (involves only the epidermis and considered as noninvasive/in situ lesions), level II (involves invasion of the papillary der-mis, not reaching the papillary-reticular dermal interface), level III (involves complete invasion and expansion into the papillary dermis, not penetrating the reticular dermis), level IV (involves invasion into the reticular dermis, not subcutane ous tissue), and level V (involves invasion into the subcutaneous tissue via the reticular dermis). The revised American Joint Committee on Cancer (AJCC) staging includes Clark’s level of invasion when defining subcategories of T1 melanomas, but it is not used for thicker melanomas (ie, T2-T4).

Cyclin-dependent kinase 4 (cdk4):

A member of the serine/threonine protein kinase family, ckd4 is the catalytic subunit of the kinase complex (Cyclin D/cdk4) important in the G1 phase of the cell cycle. Regulatory subunits of this complex are Cyclin D (positive) and cdk inhibitors (negative). Alternately spliced forms of cdk4 are known and some phosphorylation targets (eg, Rb) are also known to modulate events that are necessary for cell cycle progression.

EZH2:

An acronym for Enhancer of Zeste Homolog 2, EZH2 is a transcriptional repressor

FIGO histologic grading:

A grading system for endometrial carcinoma defined by the International Federation of Gynecology and Obstetrics (FIGO); a three-grade system primarily defined by tumor architecture and modified in the presence of "notable nuclear atypia." The FIGO grade has prognostic value.

FIGO staging:

A tumor staging system established and revised by the International Federation of Gynecology and Obstetrics (FIGO) that takes into account the postoperative histopathologic evaluation of the specimen. The FIGO stage classification has prognostic value.

Ki-67:

A marker of proliferation, Ki67 is a protein that is expressed in the nucleus of proliferating cells. Absent only in resting cells, cells in the G1, S, G2, and M phase of the cell cycle express this marker.

p16:

Also known as p16INK4, p16 binds to cyclin-dependent kinase 4 and 6, thereby preventing their interaction with cyclin D. It thus behaves as a negative regulator of proliferation and arrests cells in the G0/G1 phase of the cell cycle.

PSA (prostate-specific antigen):

A protein produced by cells of the prostate gland, the blood level of PSA is used as a tumor marker for men who may be suspected of having prostate cancer. Most physicians consider 0 to 4.0 ng/mL as the normal range. Levels of 4 to 10 and 10 to 20 ng/mL are considered slightly and moderately elevated, respectively. PSA levels have to be complemented with other tests to make a firm diagnosis of prostate cancer.

RB:

The first tumor suppressor gene identified in children with hereditary retinoblastomas, its phosphorylation state has important implications for cell cycle progression. Hypophosphorylated RB tightly binds the transcriptional factor E2F (also important for cell cycle regulation), thus preventing E2F-mediated cell cycle entry.

	Acknowledgment

 
The authors want to thank Gerd Lillian Hallseth, Karien Hamer, and Bendik Nordanger for excellent technical assistance.

	NOTES

 
Supported by Norwegian Cancer Society, Norwegian Research Council, Meltzer Research Fund, and Helse Vest Research Fund.

Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org.

Authors’ disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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Submitted February 7, 2005; accepted August 8, 2005.

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