Expression of Class I Histone Deacetylases Indicates Poor Prognosis

Volume 10 Number 9

September 2008

pp. 1021–1027

1021

www.neoplasia.com

Expression of Class I Histone Deacetylases Indicates Poor Prognosis in Endometrioid Subtypes of Ovarian and Endometrial Carcinomas1

Wilko Weichert*, Carsten Denkert*, Aurelia Noske*, Silvia Darb-Esfahani*, Manfred Dietel*, † † Steve E. Kalloger , David G. Huntsman † and Martin Köbel *Institute of Pathology, Charité Universitätsmedizin, Berlin, Germany; †Genetic Pathology Evaluation Centre of the Prostate Research Centre, Department of Pathology, Vancouver General Hospital and British Columbia Cancer Agency, Vancouver, BC, Canada

Abstract Histone deacetylase (HDAC) inhibitors are an emerging class of targeted cancer therapeutics, and little is known about HDAC expression in gynecologic malignancies. Therefore, we tested the hypothesis whether high-level expression of class 1 HDACs (HDAC1, 2, and 3) is associated with clinically distinct subsets of ovarian and endometrial carcinomas. Expression was assessed by immunohistochemistry in a population-based cohort of 465 ovarian and 149 endometrial carcinomas and correlated with clinicopathologic parameters. Each of the HDACs was expressed at high levels in most ovarian (HDAC1, 61%; HDAC2, 93%; HDAC3, 84%) and endometrial (HDAC1, 61%; HDAC2, 95%; HDAC3, 83%) carcinomas. Further, 55% and 56% of ovarian and endometrial carcinomas, respectively, expressed all three HDACs at high levels. Such cases were less common among endometrioid subtypes of ovarian and endometrial carcinomas (36% and 52% positive cases, respectively) compared with high-grade serous subtypes (64 and 69%, respectively, P < .001). High-level expression of all three HDACs is associated with a poor prognosis in ovarian endometrioid carcinomas (hazard ratio, 6.7; 95% confidence interval, 1.9–23.3). The independent prognostic information and the overall high rate of expression for class I HDACs suggest that these targets should be explored as predictive factors in ovarian and endometrial carcinomas prospectively. Neoplasia (2008) 10, 1021–1027

Introduction Current treatment strategies for ovarian carcinomas with high-risk histology (e.g., high-grade serous and clear cell subtype) are still ineffective [1]. Even in stage I/II, 30% to 40% of women with these neoplasms will die of their disease [2]. For endometrial carcinomas of the serous and clear cell type, the prognosis is comparably grim [3]. Consequently, novel target-based chemotherapeutic strategies are urgently needed. Conversely, endometrioid carcinomas of the ovary and endometrium, which sometimes present synchronously [4], are less-aggressive tumors compared with their high-risk counterparts. The great majority of women (approximately 80%) will be cured by surgery alone because these tumors are frequently organ confined and tend to metastasize late [5–7]. Because there is no good strategy to predict the natural course of these tumors, more than 90% of ovarian endometrioid carcinomas are still treated with platinumbased chemotherapy, and a large percentage of endometrial endometrioid carcinomas are treated with adjuvant radiotherapy and

chemotherapy [8,9]. Along with the opportunity of subtype-specific targeted therapy, there is a need to test for novel markers that will help to classify patient risk independent of currently known risk factors. Histone modifications are known to be centrally involved in the malignant transformation of cells and histone deacetylase (HDAC) inhibitors have proven to be effective as anticancer agents in a broad Abbreviations: HDAC, histone deacetylase; TMA, tissue microarray; HG serous, highgrade serous Address all correspondence to: Martin Köbel, MD, Department of Pathology, Room 1259, 1st Floor JPPN, Vancouver General Hospital, 855 West 12th Ave, Vancouver, BC, Canada V5Z 1M9. E-mail: [email protected] 1 This work was supported by the Cheryl Brown Ovarian Cancer Outcomes Unit of the British Columbia Cancer Agency and by an unrestricted educational grant from sanofiaventis. M.K. received fellowship support from Eli Lilly Canada. Received 10 April 2008; Revised 5 June 2008; Accepted 8 June 2008 Copyright © 2008 Neoplasia Press, Inc. All rights reserved 1522-8002/08/$25.00 DOI 10.1593/neo.08474

1022

HDAC Expression Prognosis Endometrioid Carcinoma

Weichert et al.

variety of tumors [10–13]. The family of HDACs to date comprises 18 isoenzymes, which, on the basis of structure, could be subclassified into four classes [11], with class I HDACs being the most thoroughly investigated with respect to function and relevance for tumor formation and progression. Lately, research on these proteins has gained momentum because small-molecule inhibitors of HDACs are now available and are currently tested as new antitumor drugs in late-phase clinical studies for different types of solid tumors [12,13], including ovarian cancer [14]. In addition, the HDAC inhibitor vorinostat has recently been approved for therapy in cutaneous T-cell lymphoma [15]. Histone deacetylase inhibitors are known to have profound antitumor effects in both endometrial [16–20] and ovarian carcinoma cell lines [17,21–23] most likely by inducing cell cycle arrest, apoptosis, and differentiation. These drugs act synergistically with paclitaxel[24,25] and platinum-based chemotherapeutics [26,27] in vitro. We have previously found class I HDACs to be highly expressed in different types of human cancers [28–30]. In addition, class I HDAC expression was an unfavorable independent prognostic factor in some of these tumor entities. Currently, little is known about HDAC expression in gynecologic malignancies [23,31]. In our study, we focused on this apparent lack of translational research in this field and addressed the question whether class I HDACs are expressed in gynecologic malignancies and whether expression patterns vary with respect to clinicopathologic and prognostic patient subgroups. Materials and Methods

Study Population From a population-based cohort of 3501 patients with ovarian carcinoma in British Columbia diagnosed between 1984 and 2000, 518 cases were selected on the basis of optimal surgical treatment (i.e., no macroscopic residual tumor after primary surgery) and were eligible for tissue microarray (TMA) construction after complete gynecopathologic review [5]. Two hundred consecutive cases of endometrial carcinoma were retrieved from the archives of the Department of Pathology, Vancouver General Hospital, for the period 1983–1998. All cases underwent gynecopathologic review. Mixed carcinomas were classified according to their highest-grade component and included if the high-grade component was sampled on the TMA. Clinical and pathologic data are shown in Table 1. Both series of cases have been the subject of previous studies [32,33]. A representative area of each tumor was selected and duplicate 0.6-mm tissue cores were punched to construct a TMA (Beecher Instruments, Silver Springs, MD). Table 1. Distribution of Ovarian and Endometrial Carcinomas across Stage.

Neoplasia Vol. 10, No. 9, 2008

Adjuvant Therapy and Follow-up None of the included cases received preoperative radiotherapy or chemotherapy. Approximately 97% of ovarian carcinoma patients were treated according to the provincial treatment guidelines of the British Columbia Cancer Agency [8]. On the other hand, 3% of patients refused the advised chemotherapy and were excluded from survival analysis. All patients with endometrial carcinomas were treated according the BC Cancer Agency treatment recommendation. All women underwent initial simple hysterectomy and bilateral salpingo-oophorectomy. For those with documented widespread disease, debulking surgery as in ovarian carcinomas was performed. Subsequently, radiation was added if the likelihood of local recurrence was >10%, and chemotherapy with different regimens was given if the predicted risk of death was >25%. Systematic lymph node dissection was not routinely performed. Outcomes were tracked through the Cheryl Brown Ovarian Cancer Outcomes Unit at the British Columbia Cancer Agency and were available for all patients. Median follow-up time was 5.1 and 6.2 years for ovarian and endometrial carcinomas, respectively. Approval for the study was obtained from the Research Ethics Board of the University of British Columbia.

Immunohistochemistry Serial 4-μm sections were cut for immunohistochemical analysis and run through an automated system by Ventana, Tuscon, AZ, as per manufacturer’s protocol. Polyclonal rabbit anti-HDAC1 antibody and monoclonal mouse anti-HDAC2 antibody were obtained from Abcam, Cambridge, UK. Monoclonal mouse anti-HDAC3 antibody was obtained from Becton Dickinson, Franklin Lakes, CA. Ample confirmation of antibody specificity has been performed in previous studies [28–30].

Evaluation of Immunohistochemistry Tissue microarrays were scored for HDAC1, HDAC2, and HDAC3 by a pathologist (M.K.; scans available online: http://bliss.gpec.ubc.ca/ under OOU ovarian carcinomas, under 02-005 endometrial carcinomas). Only nuclear staining within tumor cells was considered for evaluation. A four-tiered scoring system was used: 0 for negative cases, +1 for weak intensity, +2 for moderate intensity, and +3 for strong intensity (Figure 1). To optimize for disease-specific survival differences, the raw data were binarized as follows: the moderate (+2) and strong (+3) cases were grouped for statistical analysis and assigned the designation 1 and considered as high-level expressors, whereas the completely negative (0) and weak (+1) cases were considered HDAC low-level expressors and designated as 0. In addition, cases were grouped as being high for all three HDACs versus others. In total, 53 (10%) ovarian carcinoma and 51 (26%) endometrial carcinomas were not interpretable at least for one marker, and all analyses were restricted to cases with complete information.

Cell Type

N (%)

Stage I/II (%)

Stage III (%)

Age (Mean ± SE)

Statistical Analysis

Ovarian carcinoma (all) High-grade serous Clear cell Endometrioid Mucinous Other Endometrial carcinoma (all) Endometrioid Serous Clear cell Other

465 180 122 114 24 25 149 127 13 6 3

83 66 93 96 100 88 87 93 46 83 67

17 34 7 4 0 12 13 7 54 17 33

58.0 60.9 56.4 55.8 55.4 60.9 63.5 63.0 70.1 65.5 58.7

Univariable survival analysis was performed by the generation of Kaplan-Meier curves, and differences between the groups were assessed using the log rank statistic. Multivariable survival analysis was performed using the Cox proportional hazards model. Contingency tables and the Pearson χ 2 statistic were used to test the change in the distribution of HDAC expression across primary cell types. The Kruskal-Wallis test was used to compare medians for continuous variables (Ki-67). Spearman rank order correlation was used to determine whether there was a positive or negative correlation between

(39%) (27%) (24%) (5%) (5%) (85%) (9%) (4%) (2%)

± ± ± ± ± ± ± ± ± ± ±

13 12 13 13 13 11 13 13 8 17 12



Weichert et al.

1023

Figure 1. HDAC expression: (A to D) HDAC1 expression in endometrial endometrioid carcinomas score 0 (A), score 1 (B), score 2 (C), score 3 (D); (B) insert normal proliferative endometrium. (E) HDAC2 expression in ovarian clear cell carcinoma, score 3. (F) HDAC3 expression in ovarian high-grade serous carcinoma, score 3.

the levels of expression shown by any two types of immunostaining. All analyses were performed using SPSS v15.0 (SPSS Inc, Chicago, IL). P values < .05 were considered significant.

Results

Expression Patterns of Class I HDAC Isoforms in Normal Tissue HDAC1, HDAC2, and HDAC3 are expressed weakly in nuclei of ovarian surface epithelium of normal ovaries (n = 5). Expression of all class I isoforms in normal endometrium and in ovarian endometriosis was variable but did not exceed moderate staining intensity. Normal endometrial stroma cells and inflammatory cells, if present, partly showed moderate staining intensity as well.

Expression Patterns of Class I HDAC Isoforms in Ovarian Carcinomas Most ovarian carcinomas expressed high levels of HDAC1 (61%), HDAC2 (93%), and HDAC3 (84%) in the nuclei of tumor cells (Figure 1 and Table 2). Cytoplasmic tumor cell expression was not observed. Stromal cells and inflammatory cells were occasionally pos-

itive. Generally, HDAC2 had the highest expression level in all tumor subtypes. Expression of class I HDAC isoforms correlated with each other: HDAC1 with HDAC2 (r = 0.150, P = .001), HDAC1 with HDAC3 (r = 0.300, P < .001), and HDAC2 with HDAC3 (r = 0.265, P = .001), suggesting a shared functional regulation. High-level expression of all three isoforms was detected in 55% of ovarian carcinomas with the mucinous subtype of ovarian carcinoma showing the highest frequency (71%), followed by highgrade serous (64%), clear cell (54%), and endometrioid subtypes (36%). These expression levels are indicative of significant differential expression (P < .001).

Expression Patterns of Class I HDAC Isoforms in Endometrial Carcinomas In parallel to the results reported for ovarian carcinomas, most endometrial carcinomas expressed class I HDAC isoforms in the nuclei of tumor cells (HDAC1, 61%; HDAC2, 95%; HDAC3, 83%; Table 2 and Figure 1). As for ovarian carcinomas, clear cell (83%) and serous subtypes (69%) showed significantly higher expression rates for all three HDACs than endometrioid carcinomas (52%, P < .001; Table 2). In all tumor types, the expression of HDAC2 was highest, followed by HDAC3 and HDAC1 (Table 2). Expression of HDAC

1024


Weichert et al.


Table 2. HDAC Expression across Ovarian and Endometrial Carcinoma Subtypes. Cell Type

N

HDAC1-Positive Cases (%)



All Three HDACs-Positive Cases (%)

Ovarian carcinoma High-grade serous Clear cell Endometrioid Mucinous Other Endometrial carcinoma Endometrioid Serous Clear cell Other

465 180 122 114 24 25 149 127 13 6 3

61 67 64 45 83 64 61 58 69 83 100

93 98 86 94 75 100 95 94 100 100 100

84 92 77 78 83 92 83 81 100 83 100

55 64 54 36 71 64 56 52 69 83 100

Other ovarian carcinomas include nine low-grade serous, six transitional, six undifferentiated, and four adenocarcinoma not otherwise specified. Other endometrial carcinomas include two small cell carcinomas and one large cell carcinoma.

isoforms showed a moderate degree of correlation with one another: HDAC1 with HDAC2 (r = 0.321, P < .001), HDAC1 with HDAC3 (r = 0.512, P < .001), and HDAC2 with HDAC3 (r = 0.339, P < .001). Neither in ovarian nor in endometrial carcinomas a significant association of HDAC1, HDAC2, or HDAC3 expression with stage within histologic subtypes was observed (data not shown).

Correlation of Class I HDAC Expression with Proliferation Because data from cell culture studies suggest a tight interlink between HDACs and cell proliferation [34,35], we aimed to test this potential association for ovarian and endometrial carcinomas in vivo. Those tumors showing higher proliferative capacity as determined by Ki-67 staining usually expressed higher levels of all three class I HDAC isoforms, in both ovarian (P < .001) and endometrial carcinomas (P < .001). This significant association was also observed in most of the major subtypes, with the exception of ovarian endometrioid carcinomas, which showed a borderline significance of (P = .05; Figure 2).

Correlation of Class I HDAC Expression with Patient Prognosis High-level expression of all three class I HDACs was significantly associated with decreased disease-specific survival in endometrioid

ovarian cancer (Figure 3A; P = .007). In this tumor type, 10-year disease-specific survival of women with tumors expressing all three isoforms at high levels was 67% compared with those patients whose tumors expressed no or only some isoforms with 93%. This effect was mainly due to a significant association of HDAC1 expression with prognosis (HDAC1, P = .012; HDAC2, P = .598; HDAC3, P = .391). In multivariable survival analysis performed under inclusion of age, FIGO stage, Silverberg grade, and all three class I HDACs, HDAC expression retained its independent statistical significance (hazard ratio [HR], 6.7, P = .003; Table 3). In serous (Figure 3B; P = .756), mucinous, and clear cell carcinomas (data not shown) of the ovary, none of the HDAC isoforms had significant impact on patient prognosis in univariate survival analysis. In endometrial endometrioid carcinomas, 10-year disease-specific survival of women with tumors expressing all three isoforms at high levels was 63% compared with those patients whose tumors expressed no or only some isoforms with 83%. Although this was not statistically significant (Figure 3C ; P = .123), the survival difference was similar to that observed in their ovarian counterparts (20% vs 26%). In multivariable analysis under inclusion of age, FIGO stage, grade, and grouped HDAC expression, class I HDAC expression showed a trend toward unfavorable prognosis (HR, 2.0, P = .151; Table 4).

Figure 2. Box-percentile plot of Ki-67 labeling index in ovarian carcinoma subtypes. HG serous (indicates high-grade serous), clear cell, and endometrioid ovarian carcinoma and endometrioid subtype of endometrial carcinomas subdivided by low (blue) versus high (red) expression of all three HDACs. P values, Kruskal-Wallis test.



Figure 3. Kaplan-Meier survival analysis: (A) ovarian endometrioid carcinomas (N = 114); (B) ovarian high-grade serous carcinomas (N = 180); (C) endometrial endometrioid carcinomas (N = 127). P values, log rank test.

Table 3. Univariable and Multivariable Analyses of Disease-Specific Survival for Ovarian Endometrioid Carcinomas (N = 114/15 Events). Patient Characteristics

Age Stage Silverberg grade

All three HDACs

Risk Factor

I/II III 1 2 3 0 1

Univariable Analysis

Multivariable Analysis

HR

P

HR

0.423 0.001

*

* 1.0 9.3 1.0 1.7 3.5 1.0 4.9

95% CI

2.5–34.0 0.264 0.5–5.2 0.7–16.7 0.007 1.5–15.7

1.0 16.9 1.0 1.3 3.1 1.0 6.7

CI indicates confidence interval; HR, hazard ratio. *Hazard ratio for age is not available because it is a continuous variable.

95% CI

P 0.246 0.0002

3.8–75.4

Table 4. Univariable and Multivariable Analyses of Disease-Specific Survival for Endometrial Endometrioid Carcinomas (N = 123/20 Events). Patient Characteristics

Age Stage WHO Grade

0.003 1.9–23.3

1025

Discussion In this study, we describe a high-level expression of class I HDAC isoforms in ovarian and endometrial carcinomas. Histone deacetylase expression is especially prominent in high-grade tumors of serous and clear cell subtype. In the endometrioid subtype, we show that expression of the three class I HDACs is an independent prognostic marker. Reports on in vivo expression of single HDAC isoforms in ovarian and endometrioid carcinomas are sparse. In a small set of 10 ovarian carcinomas, high expression levels for class I HDACs were reported [23], which is in line with our results. However, in this cohort, it was not possible to test for prognosis or clinical correlations. Although, in our study, a significant unfavorable prognostic association of class I HDACs for endometrioid tumor subtypes was only observed for ovarian but not for endometrial carcinomas, the same trend and similar differences in 10-year survival rates for class I HDAC expressing groups were evident for tumors arising in both organs. The reason for the differences between ovarian and endometrial carcinomas might be a different assembly of the patient cohorts. The ovarian carcinoma patients were recruited from a populationbased cohort from British Columbia and restricted to patients with completely resectable disease after primary surgery. Patients with endometrial carcinoma were selected from a cohort treated at the Vancouver General Hospital, which is a specialized center where patients with high-risk disease are referred for surgery. This is reflected by a higher number of grade 3 carcinomas in the endometrial endometrioid group (24%) compared to other hospital-based cohorts of endometrioid endometrial carcinoma (6%) [6] and to the ovarian endometrioid group (7%, P = .002, Pearson χ 2). In multivariable analysis, grade and age had a stronger influence on prognosis of endometrial endometrioid than ovarian endometrioid carcinomas. Nevertheless, based on presumably similar biology and given the fact that both tumors occur synchronously in a significant proportion of patients [4], we think that the findings in ovarian endometrioid carcinomas could be transferable to their endometrial counterpart. Larger numbers might be needed to confirm these finding in endometrial endometrioid carcinomas. Previous immunohistochemical studies of HDAC isoform expression have been published for gastric [28], colorectal [29], prostate [30], and breast [34] cancers by our group and others. In colorectal cancers, class I HDAC expression was associated with poor prognosis [29]. Because colorectal carcinomas show pathogenetic pathways (e.g., microsatellite instability, β-catenin mutation, K-Ras mutation) strikingly similar to endometrioid carcinomas [35,36], similar prognostic effects of HDAC expression might be not surprising.

0.408 0.4–1.3 0.6–15.7

Weichert et al.

All three HDACs

Risk Factor

I II/III 1 2 3 0 1

Univariable Analysis

Multivariable Analysis

HR

P

HR

0.0004 0.005

* 1.0 2.1 1.0 0.5 2.5 1.0 2.0

* 1.0 3.6 1.0 1.0 3.1 1.0 2.1

95% CI

1.4–8.9 0.042 0.2–4.4 1.2–7.9 0.123 0.8–5.4

*Hazard ratio for age is not available because it is a continuous variable.

95% CI

P 0.001 0.133

0.8–5.8 0.052 0.1–2.6 0.9–6.8 0.151 0.8–5.4

1026


Weichert et al.

In the entire cohort and in each subtype, class I HDAC expression is pronounced in highly proliferating tumors as determined by high Ki-67 labeling index. These data fit well with results from functional in vitro studies showing that HDAC inhibitors induce cell cycle arrest in ovarian and endometrial cancer cell lines [17–23]. Our observation that HDAC expression levels vary considerably and have impact on patient survival in ovarian endometrioid carcinoma implicates that expression of these proteins might be useful as a prognostic marker. The absence of or only partial expression of class I HDACs indicates a very good prognosis (a 93% 10-year disease-specific survival). Because 94% of these patients received adjuvant platinum-based chemotherapy, we are not able to compare the outcome with a nontreated group. Anecdotally from seven patients, where adjuvant chemotherapy was not advised, only one woman died of disease, and this tumor expressed a high level of all three HDACs. Further, if we estimate the cure rate of women in the chemotherapy group by using the effectiveness of chemotherapy (33% of patients are cured by adjuvant therapy) described by the authors of the Action trial [37], we calculate that 7% died despite chemotherapy and only 4% of our patients were actually cured by chemotherapy; hence, 89% were treated without benefit. Therefore, we would hypothesize that HDACs’ expression assessment can identify candidates that can be spared from first-line chemotherapy to minimize overtreatment. Future larger studies might clarify whether it is necessary to assess an antibody panel against all three HDACs or whether this prognostic effect is largely based on the expression of one isoform, e.g., HDAC1. An even more interesting aspect of our results is that response of HDAC inhibitors might vary with different expression levels of class I HDACs. Treatment of tumors expressing low levels of HDACs might be ineffective because it has been shown that HDAC-negative tumors caused by the mutation of HDAC genes are resistant to HDAC inhibitors [38]. Hence, we hypothesize that class I HDAC expression in ovarian and endometrial carcinomas might be associated with a positive response to HDAC inhibitors. This would be especially desirable in endometrioid subtypes, because those patients that highly express class I HDACs have a compromised prognosis. Because determination of class I HDAC expression status in tumor tissue is easy, straightforward, and possible on very small tissue samples such as the 0.6-mm cores on a tissue microarray, such analysis is feasible in clinical trials. As we have shown subtype-specific differences in the prognostic value, it is possible that predictive effects will also be restricted to specific pathologic subtypes. References [1] Winter WE III, Maxwell GL, Tian C, Carlson JW, Ozols RF, Rose PG, Markman M, Armstrong DK, Muggia F, McGuire WP, et al. (2007). Prognostic factors for stage III epithelial ovarian cancer: a Gynecologic Oncology Group Study. J Clin Oncol 25, 3621–3627. [2] Kristensen GB, Kildal W, Abeler VM, Kaern J, Vergote I, Tropé CG, and Danielsen HE (2003). Large-scale genomic instability predicts long-term outcome for women with invasive stage I ovarian cancer. Ann Oncol 14, 1494–1500. [3] Soslow RA, Bissonnette JP, Wilton A, Ferguson SE, Alektiar KM, Duska LR, and Oliva E (2007). Clinicopathologic analysis of 187 high-grade endometrial carcinomas of different histologic subtypes: similar outcomes belie distinctive biologic differences. Am J Surg Pathol 31, 979–987. [4] Fujii H, Matsumoto T, Yoshida M, Furugen Y, Takagaki T, Iwabuchi K, Nakata Y, Takagi Y, Moriya T, Ohtsuji N, et al. (2002). Genetics of synchronous uterine and ovarian endometrioid carcinoma: combined analyses of loss of heterozygosity, PTEN mutation, and microsatellite instability. Hum Pathol 33, 421–428. [5] Gilks CB, Ionescu DN, Kalloger SE, Köbel M, and Swenerton K (2008). Tumor cell type can be reproducibly diagnosed and is of independent prognostic sig-

[6]

[7]

[8]

[9]

[10] [11] [12] [13] [14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]


nificance in patients with maximally debulked ovarian carcinoma. Hum Pathol 39, 1239–1251. Köbel M, Langhammer T, Hüttelmaier S, Schmitt WD, Kriese K, Dittmer J, Strauss HG, Thomssen C, and Hauptmann S (2006). Ezrin expression is related to poor prognosis in FIGO stage I endometrioid carcinomas. Mod Pathol 19, 581–587. Creutzberg CL, van Putten WL, Koper PC, Lybeert ML, Jobsen JJ, WárlámRodenhuis CC, De Winter KA, Lutgens LC, van den Bergh AC, van de SteenBanasik E, et al. (2000). Surgery and postoperative radiotherapy versus surgery alone for patients with stage-1 endometrial carcinoma: multicentre randomised trial. PORTEC Study Group. Post Operative Radiation Therapy in Endometrial Carcinoma. Lancet 355, 1404–1411. Swenerton KD (1992). Prognostic indices in ovarian cancer. Their significance in treatment planning. Acta Obstet Gynecol Scand Suppl 155, 67–74, and updated ovarian carcinoma treatment guidelines in British Columbia (http://www. bccancer.bc.ca/PPI/TypesofCancer/Ovary/default.htm). Maggi R, Lissoni A, Spina F, Melpignano M, Zola P, Favalli G, Colombo A, and Fossati R (2006). Adjuvant chemotherapy vs radiotherapy in high-risk endometrial carcinoma: results of a randomised trial. Br J Cancer 95, 266–271. Yoo CB and Jones PA (2006). Epigenetic therapy of cancer: past, present and future. Nat Rev Drug Discov 5, 37–50. Minucci S and Pelicci PG (2006). Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat Rev Cancer 6, 8–51. Xu WS, Parmigiani RB, and Marks PA (2007). Histone deacetylase inhibitors: molecular mechanisms of action. Oncogene 26, 5541–5552. Bolden JE, Peart MJ, and Johnstone RW (2006). Anticancer activities of histone deacetylase inhibitors. Nat Rev Drug Discov 5, 769–784. Candelaria M, Gallardo-Rincón D, Arce C, Cetina L, Aguilar-Ponce JL, Arrieta O, González-Fierro A, Chávez-Blanco A, de la Cruz-Hernández E, Camargo MF, et al. (2007). A phase II study of epigenetic therapy with hydralazine and magnesium valproate to overcome chemotherapy resistance in refractory solid tumors. Ann Oncol 18, 1529–1538. Marks PA and Breslow R (2007). Dimethyl sulfoxide to vorinostat: development of this histone deacetylase inhibitor as an anticancer drug. Nat Biotechnol 25, 84–90. Zhou XC, Dowdy SC, Podratz KC, and Jiang SW (2007). Epigenetic considerations for endometrial cancer prevention, diagnosis and treatment. Gynecol Oncol 107, 143–153. Takai N and Narahara H (2007). Human endometrial and ovarian cancer cells: histone deacetylase inhibitors exhibit antiproliferative activity, potently induce cell cycle arrest, and stimulate apoptosis. Curr Med Chem 14, 2548–2553. Ahn MY, Jung JH, Na YJ, and Kim HS (2008). A natural histone deacetylase inhibitor, Psammaplin A, induces cell cycle arrest and apoptosis in human endometrial cancer cells. Gynecol Oncol 108, 27–33. Takai N, Desmond JC, Kumagai T, Gui D, Said JW, Whittaker S, Miyakawa I, and Koeffler HP (2004). Histone deacetylase inhibitors have a profound antigrowth activity in endometrial cancer cells. Clin Cancer Res 10, 1141–1149. Jiang S, Dowdy SC, Meng XW, Wang Z, Jones MB, Podratz KC, and Jiang SW (2007). Histone deacetylase inhibitors induce apoptosis in both type I and type II endometrial cancer cells. Gynecol Oncol 105, 493–500. Takai N, Kawamata N, Gui D, Said JW, Miyakawa I, and Koeffler HP (2004). Human ovarian carcinoma cells: histone deacetylase inhibitors exhibit antiproliferative activity and potently induce apoptosis. Cancer 101, 2760–2770. Strait KA, Dabbas B, Hammond EH, Warnick CT, Iistrup SJ, and Ford CD (2002). Cell cycle blockade and differentiation of ovarian cancer cells by the histone deacetylase inhibitor trichostatin A are associated with changes in p21, Rb, and Id proteins. Mol Cancer Ther 1, 1181–1190. Khabele D, Son DS, Parl AK, Goldberg GL, Augenlicht LH, Mariadason JM, and Rice VM (2007). Drug-induced inactivation or gene silencing of class I histone deacetylases suppresses ovarian cancer cell growth: implications for therapy. Cancer Biol Ther 6, 795–801. Cooper AL, Greenberg VL, Lancaster PS, van Nagell JR Jr, Zimmer SG, and Modesitt SC (2007). In vitro and in vivo histone deacetylase inhibitor therapy with suberoylanilide hydroxamic acid (SAHA) and paclitaxel in ovarian cancer. Gynecol Oncol 104, 596–601. Sonnemann J, Gänge J, Pilz S, Stötzer C, Ohlinger R, Belau A, Lorenz G, and Beck JF (2006). Comparative evaluation of the treatment efficacy of suberoylanilide hydroxamic acid (SAHA) and paclitaxel in ovarian cancer cell lines and primary ovarian cancer cells from patients. BMC Cancer 6, 183. Strait KA, Warnick CT, Ford CD, Dabbas B, Hammond EH, and Ilstrup SJ (2005). Histone deacetylase inhibitors induce G2-checkpoint arrest and apoptosis


[27]

[28]

[29]

[30]

[31]

[32]


in cisplatinum-resistant ovarian cancer cells associated with overexpression of the Bcl-2–related protein Bad. Mol Cancer Ther 4, 603–611. Ozaki KI, Kishikawa F, Tanaka M, Sakamoto T, Tanimura S, and Kohno M (2008). Histone deacetylase inhibitors enhance the chemosensitivity of tumor cells with cross-resistance to a wide range of DNA-damaging drugs. Cancer Sci 99, 376–384. Weichert W, Röske A, Gekeler V, Beckers T, Ebert MP, Pross M, Dietel M, Denkert C, and Röcken C (2008). Association of patterns of class I histone deacetylase expression with patient prognosis in gastric cancer: a retrospective analysis. Lancet Oncol 9, 139–148. Weichert W, Roeske A, Niesporek S, Noske A, Buckendahl AC, Dietel M, Gekeler V, Boehm M, Beckers T, and Denkert C (2008). Class I histone deacetylase expression has independent prognostic impact in human colorectal cancer—specific role of class I HDACs in vitro and in vivo. Clin Cancer Res 14, 1669–1677. Weichert W, Röske A, Gekeler V, Beckers T, Stephan C, Jung K, Fritzsche FR, Niesporek S, Denkert C, Dietel M, et al. (2008). Histone deacetylases 1, 2 and 3 are highly expressed in prostate cancer and HDAC2 expression is associated with shorter PSA relapse time after radical prostatectomy. Br J Cancer 98, 604–610. Krusche CA, Vloet AJ, Classen-Linke I, von Rango U, Beier HM, and Alfer J (2007). Class I histone deacetylase expression in the human cyclic endometrium and endometrial adenocarcinomas. Hum Reprod 22, 2956–2966. Prentice LM, Klausen C, Kalloger S, Köbel M, McKinney S, Santos JL, Kenney C, Mehl E, Gilks CB, Leung P, et al. (2007). Kisspeptin and GPR54 immuno-

[33]

[34]

[35]

[36]

[37]

[38]

Weichert et al.

1027

reactivity in a cohort of 518 patients defines favourable prognosis and clear cell subtype in ovarian carcinoma. BMC Med 5, 33. Alkushi A, Clarke BA, Akbari M, Makretsov N, Lim P, Miller D, Magliocco A, Coldman A, van de Rijn M, Huntsman D, et al. (2007). Identification of prognostically relevant and reproducible subsets of endometrial adenocarcinoma based on clustering analysis of immunostaining data. Mod Pathol 20, 1156–1165. Krusche CA, Wülfing P, Kersting C, Vloet A, Böcker W, Kiesel L, Beier HM, and Alfer J (2005). Histone deacetylase-1 and -3 protein expression in human breast cancer: a tissue microarray analysis. Breast Cancer Res Treat 90, 15–23. Matias-Guiu X, Catasus L, Bussaglia E, Lagarda H, Garcia A, Pons C, Munoz J, Arguelles R, Machin P, and Prat J (2001). Molecular pathology of endometrial hyperplasia and carcinoma. Hum Pathol 32, 569–577. Shen L, Toyota M, Kondo Y, Lin E, Zhang L, Guo Y, Hernandez NS, Chen X, Ahmed S, Konishi K, et al. (2007). Integrated genetic and epigenetic analysis identifies three different subclasses of colon cancer. Proc Natl Acad Sci USA 104, 18654–18659. Trimbos JB, Vergote I, Bolis G, Vermorken JB, Mangioni C, Madronal C, Franchi M, Tateo S, Zanetta G, Scarfone G, et al. (2003). Impact of adjuvant chemotherapy and surgical staging in early-stage ovarian carcinoma: European Organisation for Research and Treatment of Cancer-Adjuvant ChemoTherapy in Ovarian Neoplasm trial. J Natl Cancer Inst 95, 113–125. Ropero S, Fraga MF, Ballestar E, Hamelin R, Yamamoto H, Boix-Chornet M, Caballero R, Alaminos M, Setien F, Paz MF, et al. (2006). A truncating mutation of HDAC2 in human cancers confers resistance to histone deacetylase inhibition. Nat Genet 38, 566–569.