Title-loss of βcatenin is an independent prognostic factor in ovarian carcinomas: A multivariate analysis Research Article

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1 Gene Therapy and Molecular Biology Vol 10, page 179 Gene Ther Mol Biol Vol 10, , 2006 Title-loss of βcatenin is an independent prognostic factor in ovarian carcinomas: A multivariate analysis Research Article Cristina Faleiro-Rodrigues 1, *, Isabel Macedo-Pinto 1, Deolinda Pereira 2 1 Department of Anatomy and Pathology 2 Department of Medical Oncology, Portuguese Institute of Oncology of Francisco Gentil, Centro Regional do Norte, Porto, Portugal *Correspondence: Faleiro-Rodrigues C., Instituto Português de Oncologia Francisco Gentil, Centro Regional do Norte, Departamento de Anatomia Patológica, Rua Dr. António Bernardino de Almeida Porto, Portugal; Telephone: Ext 1002; Fax ; cristinafaleiro@mail.com Key words: ovarian cancer, cell adhesion, epithelial cadherin, β-catenin, immunohistochemistry Abbreviations: avidin-biotin peroxidase, (ABC); E-cadherin catenin unit, (ECCU); Epithelial cadherin, (E-cadherin); International Federation of Gynaecology and Obstetrics system, (FIGO); Overall survival, (OS); World Health Organization, (WHO) Received: 10 March 2006; Revised: 26 April 2006 Accepted: 16 May 2006; electronically published: July 2006 Summary In ovarian carcinomas, numerous studies have shown consistent prognostic significance of FIGO stage and residual tumour as independent prognostic factors. However, these prognostic factors alone cannot accurately predict disease outcome since a considerable degree of heterogeneity remains within the various subgroups limiting the predictive value of these factors. Therefore, the identification of new molecular markers that may possibly distinguish patients at a higher risk is of great importance. In two previous studies, the individual loss of E-cadherin and the individual loss of β-catenin were important prognostic factors of poorer overall survival in patients with ovarian carcinomas. Purpose of the present study was to re-analyse the immunohistochemical expression of E- cadherin and β-catenin in 104 patients with ovarian carcinomas, and evaluate whether these two proteins continue to be important independent prognostic factors when assessed together in a multivariate Cox s proportional hazard regression analysis. Results In the multivariate analysis, the most important independent prognostic factors of poorer overall survival were loss of β-catenin expression ([HR], 5.79, 95% CI, 2.38 to 14.10; P=0.0001), FIGO stage IV ([HR], 7.19, 95% CI, 1.02 to 50.8; P=0.04) and residual tumour ([HR], 6.78, 95% CI, 1.41 to 32.56; P=0.034). Conclusion The loss of β-catenin expression is a stronger prognostic factor than E-cadherin. The findings in the present study and previously reported data suggest that β-catenin is a significant prognostic indicator in patients with epithelial ovarian cancer, however, these results should be supported by more and larger studies. I. Introduction In ovarian carcinomas, numerous studies have shown consistent prognostic significance of FIGO tumour stage and size of residual tumour as independent prognostic factors (Rubin et al, 2003). However, these prognostic factors alone cannot accurately predict disease outcome since a considerable degree of heterogeneity remains within the various subgroups limiting the predictive value of these factors. Therefore, the identification of new molecular markers that may possibly distinguish patients at a higher risk is of great importance. Epithelial cadherin (E-cadherin) is a calcium-dependent cell adhesion molecule which plays a key role in cell-cell epithelial adhesion and epithelial tissue integrity. The intracellular domain of E-cadherin is found in a complex linked with the catenins (α-and β-). The association of catenins to cadherins is a key step in the function of intact adhesion complexes. The catenins link the cadherin molecules to the cytoskeleton and mediate signal transduction mechanisms that regulate cell adhesion, growth and differentiation (Frixen et al, 1991; Tsukita et al, 1992; Kemler, 1993; Hinck et al, 1994). Detachment of tumour cells from the primary lesion is considered a main step in the process of invasion and metastases. Increasing evidence points to a role for E-cadherin and the catenins in cancer progression since the loss or reduced expression of E-cadherin and β- 179

2 Faleiro-Rodrigues et al: Loss of β catenin expression in primary ovarian carcinomas catenin correlates with invasive behaviour, increased lymph node metastasis and poor outcome in patients with malignant melanoma and gastric carcinomas (Jawhari et al, 1997; Ramesh et al, 1999; Kageshita et al, 2001; Tanaka et al, 2002). In two previous individual studies, a significant correlation between poor overall survival and the loss of E-cadherin and the loss of β-catenin was observed in patients with ovarian carcinomas. The loss of E-cadherin and β-catenin immunoexpression was also shown to be independent predictors of poorer survival in a multivariate analysis (Faleiro-Rodrigues et al 2004a, b). This study represents a re-analyse of previously published data with the purpose of determining whether these two proteins continue to be important independent prognostic factors when assessed together in a multivariate Cox s proportional hazard regression analysis. II. Material and Methods Routinely formalin-fixed and paraffin-embedded tissue samples from 104 cases of primary ovarian carcinomas were retrieved from the Department of Pathology at the Portuguese Institute of Oncology of Francisco Gentil, Porto, from January 1995 to December The mean age at the time of diagnosis was 56 years (range, 21 to 89 years). None of these patients had undergone neoadjuvant chemotherapy prior to surgery. All tissue specimens were reviewed and re-evaluated by an experienced gynaecological pathologist. Histological classification was performed according to the World Health Organization (WHO) standards. The grading and staging of the tumours were assigned according to the International Federation of Gynaecology and Obstetrics system (FIGO). The mean overall survival duration of the patients was 35 months. At the end of the follow-up period, 65 (62%) patients were without evidence of disease, 35 (34%) patients had died of disease, and 4 (4%) patients were lost for follow up. A. Tissue sections All the tissue sections (stained by haematoxylin and eosin) from each case were observed. Areas of necrosis or deterioration of tissue morphology were avoided. The pathologist selected the best tumour section representing well preserved tissue architecture and cell morphology with approximately 2.0 x 1.0 cm. B. Immunohistochemical staining Archival tissue was fixed in 10% formalin and 3 µm sections were used for both histological and immunohistochemical studies. Immunohistochemistry was performed in all cases using the avidin-biotin peroxidase (ABC) complex with an additional step for microwave antigen retrieval as described (Faleiro-Rodrigues et al, 2004b). The following monoclonal antibodies were used: E-cadherin (C20820), and β- catenin (C19220) (Transduction Laboratories, Lexington, UK). To ensure accurate and reproducible staining, normal skin epithelium was used as a positive control. Staining of E-cadherin and β-catenin was localized on the cell membrane of epithelial cells, particularly at areas of cell-tocell contact. Normal skin epithelium without the primary antibody was used as a negative control. C. Evaluation of E-cadherin and β-catenin immunostaining Membranous immunoreactivity of the catenins was assessed by light microscopy by two independent observers, without previous knowledge of the patients clinicopathological details. E-cadherin and β-catenin immunoexpression in the tumours was scored semi-quantitatively on a scale of 0 to 3 (0=complete absence of expression, 1=10%, 2 >10 and = 50%, 3 >50%). For all the association analyses, the subdivision into negative (0 scale) and positive expression (1-3 scale) was used. D. Statistical analysis The statistical software used was the Statistical Package for the Social Sciences (SPSS version 8.0, SPSS, Chicago). Clinical data was obtained from the Cancer Registry Records of the patients and evaluated by a Medical Oncologist. Overall survival (OS) was defined as the time from diagnosis to death or last clinical control date, and used as a measure of prognosis. Univariate survival curves were estimated using the Kaplan- Meier method and compared using the Log-rank or the Breslow test. Multivariate analysis was performed using the Cox s proportional hazards regression model with overall survival as the outcome measure. Forward stepwise procedure was used to select the independent variables in the multivariate analysis. Forward selection, allows variables to be considered one at a time for entry into the model. After a variable is added to the model, all variables already in the model are examined for removal. The algorithm stops when no more variables meet entry or removal criteria. A value of P<0.05 was regarded as significant. III. Results The present series consisted of 104 carcinomas that were classified into the following histological types, 56 serous carcinomas, 22 mucinous carcinomas, 16 clear cell carcinomas, 8 endometrioid and 2 transitional cell carcinomas. These carcinomas were graded into 26 welldifferentiated, 27 moderately differentiated and 51 poorly differentiated tumours. In this series, 31 cases were diagnosed with FIGO stage I tumours, 7 in FIGO stage II, 47 in FIGO stage III and 19 in FIGO stage IV. The clinicopathological parameters studied were FIGO staging, histological type, tumour differentiation, peritoneal metastasis, and residual tumour after surgery, the appearance of the ovarian capsule, peritoneal cytology and lymphatic/vascular invasion (previously described in Faleiro-Rodrigues et al 2004a). A. Immunoreactivity of E-cadherin and β-catenin in carcinoma tissue Negative E-cadherin expression was observed in 7 (7%) malignant tumours, and positive in 97 (93%). Negative β-catenin expression was observed in 15 (14%) malignant tumours, and positive in 89 (86%). B. Relationship between E-cadherin and β-catenin expression in carcinoma tissue In the 15 carcinomas demonstrating negative expression for β-catenin, 3 carcinomas showed negative expression for E-cadherin, and 12 carcinomas showed positive expression for E-cadherin (Table 1). C. Relationship between the expression of E-cadherin and β-catenin and patient overall survival 180

3 Gene Therapy and Molecular Biology Vol 10, page 181 In the univariate survival analysis, patients whose carcinoma tissue demonstrated negative E-cadherin expression had a statistically significant decreased 5-year overall survival rate compared with patients showing positive expression (29% versus 66%, P=0.006), (Faleiro- Rodrigues et al, 2004a). Patients whose carcinoma tissue demonstrated negative β-catenin expression had a statistically significant decreased 5-year overall survival rate compared with patients showing positive expression (44% versus 66%, P=0.022), (Faleiro-Rodrigues et al, 2004b). The parameters that had a significant impact on overall survival as E-cadherin (P=0.006), β-catenin (P=0.022), FIGO stage (P ), peritoneal metastasis (P ), and post-operative residual tumour (P ), peritoneal cytology (P ) and lymphatic/vascular invasion (P=0.008), were then reviewed by a multivariate analysis (Cox s proportional hazards regression model, Table 2). Negative expression of β-catenin (P=0.0001); (Figure 1), FIGO stage IV (P=0.04) and residual postoperative tumour (P=0.01) were shown to associate significantly with poor patient prognosis. IV. Discussion The cause of epithelial ovarian carcinoma is unknown and diagnosis is retarded by the lack of symptoms in early stage disease. Consequently, the poor overall survival and morbidity associated with epithelial ovarian cancer deaths results from the detection of the disease in advanced tumour stages with widespread metastatic disease at the time of diagnosis (Ozols et al, 2000). To date, the molecular mechanisms that allow ovarian cancer cells to detach from the primary tumour and consequently interact with the mesothelium are not fully characterized. Cell adhesion molecules may play an important role in epithelial ovarian carcinogenesis, since cell-to-cell adhesion plays a critical role in a wide variety of biological processes including embryogenesis, maintenance of cell polarity, cell growth, and cell differentiation (Skubitz, 2002). The loss of cell adhesion molecules may lead to changes in cellular adhesion and to increased motility, processes that contribute to the invasive and/or metastatic potential of cells (Vleiminckx et al, 1991; Birchmeier and Behrens, 1993; Mareel et al, 1994; Van Aken et al, 2001). E-cadherin has been identified as an important transmembrane molecule involved in the adhesion of epithelial cells at adherens junctions. Adherens junctions are organized around transmembrane proteins of the cadherin family. While the extracellular domain of the E- cadherin molecule interacts with that of an opposing E- cadherin on a neighbouring cell, the intracellular cytoplasmic domain of E-cadherin associates with ß- catenin, which in turn complexes with α-catenin mediating the connection of E-cadherin to the cytoskeleton (Tsukita et al, 1992). The complex of E-cadherin and the Table 1. Relationship between E-cadherin and β-catenin expression in ovarian carcinomas Table 2. Multivariate analysis for overall survival Immunoexpression Nº β-catenin Negative Positive E-cadherin Negative Positive Total Covariate Hazard ratio (95% CI) P value I 1 II 5.58 ( ) III 2.99 ( ) IV 7.19 ( ) Residual tumour (-) 1 (+) 6.78 ( ) β-catenin (+) 1 (-) 5.79 ( )

4 Faleiro-Rodrigues et al: Loss of β catenin expression in primary ovarian carcinomas Figure 1. Immunoreactivity of β-catenin (A) Negative expression, (B) Mucinous carcinoma showing positive ß-catenin expression. catenins is a functional unit, which is termed here as the E- cadherin-catenin unit (ECCU). Binding to catenins is important for E-cadherin function, rendering the catenins regulatory molecules of E-cadherin. Thus, alterations in E- cadherin or the catenins may lead to loss of cell-cell adhesion, resulting in tumour aggressiveness and invasiveness in neoplastic disease (Ozawa et al, 1990; Frixen et al, 1991; Mareel et al, 1994). In two previous individual studies, the significance of E-cadherin and the catenins α, β- and γ-, as predictors of poorer survival in patients with ovarian carcinomas was assessed. The first study showed that negative E-cadherin immunoexpression significantly predicted a poorer overall survival, and was an independent prognostic factor in the multivariate analyses (Faleiro-Rodrigues et al 2004a). In the second study, although negative immunoexpression of α-catenin and γ-catenin was observed, only negative β- catenin expression was associated with patient poorer overall survival in the univariate analyses. In the multivariate analysis, β-catenin immunoexpression and residual tumour were shown to be independent prognostic factors for survival (Faleiro-Rodrigues et al 2004b). In the present study, when E-cadherin and β-catenin were assessed together in a Cox s multivariate regression analysis to determine whether the immunoexpression of these two proteins continued to be independent prognostic factors, only β-catenin continued to be an independent prognostic factor of poor survival. The loss of β-catenin expression, FIGO stage IV and residual tumour, when considered with parameters that had a significant impact on overall survival as peritoneal metastasis, peritoneal cytology, lymphatic/vascular invasion and E-cadherin expression, were shown to be the strongest independent predictors of poor survival. The results of this study suggest that when β-catenin and E-cadherin are assessed in a multivariate analysis, the loss of β-catenin proves to be a more important prognostic marker than the loss of E- cadherin in patients with ovarian carcinomas. A study by the group of Akimoto et al, showed that the expression of E-cadherin in murine adenocarcinomas correlated well with the expression of β-catenin. They also showed that reduced expression of β-catenin in these tumours correlated with enhanced metastasis formation (Akimoto et al, 1999). Whether β-catenin alone could have affected the propensity of these tumour cells to metastasise is unclear. However, some recent studies show an independent role of the catenins in tumour invasion and metastases (Kawanishi et al, 1995; Vermeulen et al, 1995). Studies on the molecular organization of the ECCU using recombinant proteins have demonstrated β-catenin to play a central role in the formation of the E-cadherin complex (Oyama et al, 1994; Kawanishi et al, 1995; Vermeulen et al, 1995; Harington and Syrigos, 2000). In general, adhesion between normal epithelial cells is strong and stable. For tumour cells to dissociate, invade and metastasize, cell-to-cell associations must be disrupted. In our series of ovarian carcinomas, despite the small number of tumours showing loss of expression for β-catenin the observation that a) 15 carcinomas demonstrated negative expression for β-catenin, of which 3 were negative and 12 were positive for E-cadherin, respectively, and b) β- catenin expression was shown to be an independent prognostic factor in a previous (Faleiro-Rodrigues et al 2004b) and in the present study, reinforces the viewpoint that β-catenin is a crucial and indispensable component in the formation of the ECCU, and that loss of β-catenin alone may be sufficient to disassemble the adherent junction, leading to loss of intercellular adhesion. Thus, loss of β-catenin expression may be an important step in the development of a malignant tumour, by this approach, enable the dissociation of cells from the primary tumour, and thus possibly contribute to tumour cell invasion and tumour peritoneal implantation in ovarian cancer patients. Although the prognostic value of β-catenin needs to be supported by more studies and a larger number of patients, this retrospective study, suggests that the immunohistochemical assessment of β-catenin into negative versus positive expression on primary ovarian carcinomas may prove to be a useful marker for selecting 182

5 Gene Therapy and Molecular Biology Vol 10, page 183 a small group of patients with a high risk of suffering an unfavourable clinical outcome. Whether this information can be used to stratify patients for therapeutic strategies also needs to be explored in future clinical studies. In several carcinomas, loss of β-catenin expression by immunohistochemistry has been associated with malignant transformation as increased invasiveness, disease progression, and poor prognosis (Takayama et al, 1996; Jawhari et al, 1997; Muzio et al, 1999; Ramesh et al, 1999; Garcia del Muro et al, 2000; Kageshita et al, 2001; Tanaka et al, 2002). However, the molecular mechanisms that bring about the loss of β-catenin in these tumours have not been characterized and yet to be investigated. The causal mechanism for the loss of β-catenin protein expression in our series of ovarian carcinomas is not clear. Several mechanisms may impair β-catenin from being expressed, such as hypermethylation of the β-catenin gene (CTNNB1) promoter, CTNNB1 mutations and deletions (Ebert et al, 2003; Ueda et al, 2001). It is now of interest to evaluate further the molecular mechanisms that underlie the observed loss of β-catenin observed in the present study. Future investigations on the regulation of the expression of β-catenin may elucidate possible mechanisms resulting in the loss of this protein. Nevertheless, irrespective of the mechanism that impairs the expression of β-catenin, based on the above results, it seems that tumour cells may become increasingly invasive and show an aggressive cellular phenotype upon the loss of β-catenin, which may be an important step in the progression of ovarian carcinomas. 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