Departments of 1 Anatomy and Pathology and 2 Medical Oncology, Portuguese Institute of Oncology of Francisco Gentil, Centro Regional do Norte, Porto, Portugal
* Correspondence to: Dr C. Faleiro-Rodrigues or I. Macedo-Pinto, Instituto Português de Oncologia Francisco Gentil, Centro Regional do Norte, Departamento de Anatomia Patológica, Rua Dr António Bernardino de Almeida, 4200-072 Porto, Portugal. Tel: +351-22-5084000 ext. 5111; Fax: +351-22-5084001; Email: cristinafaleiro{at}mail.com
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Abstract |
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Patients and methods: The protein expression of E-cadherin was immunohistochemically evaluated in formalin-fixed, paraffin-embedded samples in 104 patients with primary ovarian carcinomas. The clinicopathological factors studied were age, FIGO staging, histological type, tumour differentiation, the appearance of the ovarian capsule, peritoneal implants and residual tumour after cytoreductive surgery. Overall survival and RFS were evaluated using the KaplanMeier method, and multivariate analysis was completed using the Cox regression model.
Results: Of the 104 carcinomas, negative E-cadherin immunoexpression was observed in seven (7%) cases, and positive immunoexpression in 97 (93%). E-cadherin categorised into negative versus positive expression did not associate with any of the established clinicopathological parameters. However, negative E-cadherin expression significantly predicted a poorer overall survival when compared with positive expression (P=0.006). In the multivariate analyses, negative E-cadherin and the presence of residual tumour after cytoreductive surgery were independent prognostic factors for survival (P=0.014 and P=0.034, respectively).
Conclusions: The presence of residual tumour after primary cytoreductive surgery and negative E-cadherin expression seem to be useful markers in patients with ovarian carcinomas likely to have an unfavourable clinical outcome. The assessment of E-cadherin immunoreactivity may be a useful prognostic indicator in ovarian cancer, complementary to established prognostic factors.
Key words: cell-to-cell adhesion, epithelial cadherin, immunohistochemistry
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Introduction |
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Epithelial ovarian tumours constitute 90% of all cases in women. Because ovarian cancer is often asymptomatic in its early stages, the poor prognosis associated with ovarian carcinomas is related to the extensive dissemination of tumour cells beyond the confines of the ovary at the time of diagnosis. Tumour cells that are released into the abdominal cavity are capable of invasion of the peritoneal surfaces following tumour cell implantation [2]. The molecular mechanisms underlying this process are not well characterised. Nevertheless, it has been realised that the loss of epithelial differentiation in carcinomas, which is accompanied by a higher mobility and invasiveness of the tumour cells, is often a consequence of reduced intercellular cell-to-cell adhesion [3
5
].
Epithelial cadherin (E-cadherin), also known as cell CAM 120/80, belongs to the cadherin family of calcium-dependent adhesion molecules and is mapped to chromosome 16q22.1 [6]. These molecules are transmembrane glycoproteins localised at the adherens junction of epithelial cells and mediate homotypic cell-to-cell adhesions [7
, 8
]. E-cadherin associates with the actin cytoskeleton through a group of membrane-associated proteins,
-catenin, ß-catenin and
-catenin, which are essential for the maintenance of stable E-cadherin-mediated cell adhesion and involved in regulating cell motility [9
11
].
Loss of E-cadherin expression has been regarded as a central event in tumour metastasis, as loss of adhesion between tumour cells facilitates their ability to invade locally and to spread to distant organs [3, 12
]. Studies in vitro have shown that the reduced expression of E-cadherin and the catenins leads to altered phenotypes such as cell morphologic change, loose cell-to-cell contact, cell dissociation and enhanced cell motility, so that the E-cadherincatenin complex is considered to play an important role in the suppression of invasion and metastasis [13
, 14
]. A number of immunohistochemical studies on human carcinomas have demonstrated that the loss and reduced expression of E-cadherin and the catenins is often associated with poor histological differentiation, increased risk of local invasion, metastatic disease and poorer overall survival [4
, 15
27
].
The underlying molecular mechanism for E-cadherin down-regulation is not known; however, mutations and biochemical posttranslational modifications have been observed in several carcinomas [2832
].
The reduction and loss of E-cadherin expression in ovarian tumours have been investigated [3336
]. However, there are only a limited number of studies regarding the prognostic role of E-cadherin [35
, 36
].
Since the loss of E-cadherin has frequently been linked to increased aggressive tumour behaviour and a poor clinical outcome, we evaluated whether loss of E-cadherin expression in our series of patients with primary ovarian carcinomas was associated with any of the classical clinicopathological features, including prognosis.
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Materials and methods |
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The clinicopathological parameters studied were FIGO staging, histological type, tumour differentiation, peritoneal metastasis, residual tumour after surgery, the appearance of the ovarian capsule, peritoneal cytology and lymphatic/vascular invasion.
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, eight endometrioid and two transitional cell carcinomas. These carcinomas were graded into 26 well-differentiated, 27 moderately differentiated and 51 poorly differentiated tumours. In this series, 31 cases were diagnosed with FIGO stage I tumours, seven in FIGO stage II, 47 in FIGO stage III and 19 in FIGO stage IV.
Immunohistochemistry
Section preparation and pre-treatment
Immunohistochemistry was carried out using the avidinbiotin peroxidase complex method. Tissues were sectioned at 3 µm and were mounted on poly-L-lysine treated glass slides (Sigma, St Louis, MO, USA), deparaffinised and rehydrated using xylene, and a series of graded ethanols (100, 90, 80 and 70%). Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 30 min at room temperature. For antigen unmasking, sections were immersed in 0.01 M sodium citrate buffer solution (pH 6.0) and heated in a 600 W microwave on full power for four periods of 2 min, pausing to ensure that there was no fluid lost due to evaporation. The slides were then left to cool for 30 min, and rinsed in phosphate-buffered saline solution (PBS, pH 6.0).
Antibodies and immunohistochemical staining
Non-specific binding was reduced by incubation of the tissue section in normal rabbit serum diluted 1:5 in 12.5% bovine serum albumin (BSA) (Sigma) for 30 min. The slides were then incubated at 4°C overnight with 200 µl of the monoclonal antibody anti-E-cadherin (C20820) 1:2500 (Transduction Laboratories, Lexington, KY, USA). After washing in PBS for 10 min, the slides were incubated in biotinylated rabbit anti-mouse IgG (Dakopatts A/S, Denmark), diluted 1:200 for 30 min, washed in PBS and then incubated for another 30 min with avidinbiotin-complexed labelling antibody (ABC Complex kit, Dako A/S, Denmark) at room temperature. After washing in PBS, chromogen visualisation was achieved by the addition of peroxidase substrate solution [0.05% DAB, 3,3-diaminobenzidine tetrahydrochloride (Sigma) and 0.03% H2O2 in PBS, pH 6.0]. After 7 min, the DAB reaction was stopped under running tap water. The sections were counterstained with modified Mayer's haematoxylin for 30 s and mounted for microscopic examination.
To ensure accurate and reproducible staining, normal skin epithelium was used as a positive control. Strong and homogenous expression of E-cadherin was observed at the cell membrane throughout the epithelium. Normal skin epithelium without the primary antibody was used as a negative control.
Evaluation and quantification of immunostaining
The immunoexpression of the tumours was scored semiquantitatively according to the staining pattern (membranous staining) on a three-point scale of 0 to 3 (0 = complete absence of expression, 1=10%, 2=>10 and
50%, 3=>50%; Table 1).
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The classification of E-cadherin immunoexpression pattern into negative versus positive expression associated significantly with overall survival (OS); therefore, this subdivision was used for further analysis regarding overall and disease-free survival.
As a result of the small number of patients in each category, the patients were grouped into FIGO stage I/II versus FIGO III/IV tumours. Endometrioid and transitional cell carcinomas were grouped into the histological subtype as others. Tumours graded into well and moderately differentiated tumours were grouped versus poorly differentiated tumours. Residual tumour after cytoreductive surgery was categorised into no residual tumour versus presence of residual tumour irrespective of the tumour volume.
The univariate survival analysis was based on the KaplanMeier method. Comparison between the survival curves was analysed using the Log-rank or the Breslow test. The OS was defined as the time between the date of surgery and the last date of follow-up or date of death due to ovarian cancer. Recurrence-free survival (RFS) was defined as the time interval between the date of surgery and the date of recurrence. The prognostic significance of negative E-cadherin expression concerning other pathological variables was assessed using the multivariate Cox's proportional hazard's analysis. A value of P < 0.05 was considered statistically significant.
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Results |
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Relationship with clinicopathological parameters
The E-cadherin immunoreactivity categorised into negative versus positive expression did not associate with any of the clinicopathological parameters tested: age, FIGO stage, histological type, tumour differentiation, peritoneal metastasis, residual tumour after primary cytoreductive surgery, peritoneal cytology, appearance of the ovarian capsule and lymphatic/vascular invasion (Table 2).
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In the univariate analyses, FIGO stage, peritoneal cytology, peritoneal metastasis, residual tumour after primary cytoreductive surgery, and lymphatic/vascular invasion, were significant predictors of poor OS (Table 3). In the univariate analyses, negative E-cadherin expression presented a significantly poorer 5-year survival rate (29% versus 66%; P=0.006) than positive expression (Figure 2). Negative expression of E-cadherin observed in seven cases showed the following characteristics: one mucinous stage I tumour, one clear cell stage I tumour, two serous stage III tumours, two serous stage IV tumours and one mucinous stage IV tumour.
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In the RFS analyses, FIGO stage, histological type, tumour differentiation, peritoneal cytology, peritoneal metastasis, residual tumour after primary cytoreductive surgery and lymphatic/vascular invasion were significant predictors of recurrence free survival. The immunoexpression of E-cadherin was not a predictor of RFS (Table 5).
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Discussion |
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To understand the mechanisms of tumour cell dissociation, the role of E-cadherin must be taken into account, as it has a major role in maintaining intercellular junctions in epithelial tissues. In general, adhesion between normal epithelial cells, the origin of carcinomas, is strong and stable. For tumour cells to dissociate, invade and metastasise, cell-to-cell associations must be disrupted. In early mouse development, E-cadherin first functions as an adhesion component during the compaction of blastomeres; at later stages, it is confined to all epithelia originating from ectodermal, mesodermal and endodermal tissue. Thus, E-cadherin plays an important role in the morphogenesis of tissues during embryogenesis, and in mature epithelia [8, 40
]. The critical importance of E-cadherin in normal development and tissue function is demonstrated by the lethality of the E-cadherin gene knockout in mice at a very early stage in embryogenesis [41
, 42
].
Monoclonal antibodies capable of disrupting cell-to-cell adhesion were used to identify E-cadherin in epithelial tissues [43]. In an in vitro model system, Behrens et al. [44
] transformed MadinDarby Canine Kidney epithelial cells with Harvey and Maloney sarcoma viruses and showed that these cells acquired an invasive phenotype when antibodies inhibited intercellular adhesion. Frixen et al. [12
] showed that loss of E-cadherin expression was associated with invasiveness and dedifferentiation of human carcinoma cells, which could be prevented by transfection of E-cadherin cDNA into these poorly differentiated carcinoma cell lines, thereby increasing intercellular cohesion and inhibiting invasion in vitro. As a result, E-cadherin has been recognised as the main calcium-dependent intercellular adhesion molecule that regulates epithelial cell differentiation and acts as an invasion suppressor molecule [13
, 14
]. Since then, many studies have focused on the relationship between loss of E-cadherin expression and the invasive and metastatic process. Immunohistochemical studies have demonstrated that the loss of E-cadherin expression is frequently associated with parameters of enhanced biological aggressiveness such as poor histological differentiation, increased invasiveness, metastatic disease and a poorer survival rate in patients with oral [17
], breast [18
], hepatocellular [19
], bladder [20
], prostate [21
], renal [22
], pancreatic [23
], oesophageal [24
], thyroid [25
], head and neck [26
] and gastric carcinomas [27
].
With regard to the expression of E-cadherin in ovarian tumours, experimental studies in vitro have suggested that the loss and reduced expression of E-cadherin by epithelial cells result in the acquisition of the invasive phenotype [4549
]. Furthermore, in vivo studies have revealed an association between the absence and reduced expression of E-cadherin and poor histological differentiation [33
35
, 48
], increased invasive potential [35
, 45
48
] and poor patient survival [36
].
In this study, we evaluated the immunohistochemical expression of E-cadherin in formalin-fixed, paraffin-embedded tissue specimens from primary ovarian cancer tissue. E-cadherin immunoreactivity categorised into negative versus positive expression did not associate with any of the standard clinicopathological parameters tested. However, in the univariate analyses, the 5-year survival rate of patients with negative E-cadherin expression was significantly lower than for patients with positive E-cadherin expression. These results are in agreement with those of Darai et al. [36] and reinforce the observation that negative E-cadherin protein expression may be of value as a prognostic factor in ovarian cancer. Furthermore, in the multivariate regression analyses, the residual tumour after cytoreductive surgery and the negative expression of E-cadherin were shown to be significant independent predictors of poorer OS. Although the prognostic value of E-cadherin needs to be confirmed in a larger number of patients, our results indicate that the immunohistochemical assessment of E-cadherin into negative versus positive expression on primary ovarian carcinomas could prove to be a useful marker for selecting a small group of patients with a high risk of suffering an unfavourable clinical outcome. Whether this information can be used to stratify patients better for therapeutic strategies also needs to be explored in future clinical studies.
In this study, immunohistochemical expression of E-cadherin scored into negative versus positive expression was limited to the assessment of the protein expression level, but it does not provide information about its functional status. Recent studies have offered strong evidence that loss of E-cadherin expression plays a causal role in cancer. Germline-inactivating mutations in the E-cadherin gene (CDH1) have been found in families with inherited predisposition to gastric carcinomas, particularly those with diffuse-type histology [50]. Somatic mutations in CDH1 are the most prevalent in lobular breast carcinomas and in diffuse gastric carcinomas [51
55
]. However, somatic mutations in the E-cadherin gene are rare in ovarian carcinomas, since only one missense mutation in 63 cases was found, suggesting that other mechanisms of inactivation may be involved [56
]. Therefore, we propose that the loss of E-cadherin expression observed in our study may be due to epigenetic events observed in several human carcinomas such as CpG methylation of the E-cadherin promoter region, rearrangements in chromatin structure, aberrant tyrosine phosphorylation and alterations of specific transcription factor pathways regulating E-cadherin gene expression, rather than inactivating mutations of CDH1 [28
31
, 57
59
].
In the literature, a limited number of studies on E-cadherin inactivating mechanisms in ovarian cancers have been carried out [60, 61
]. One recent study examined the DNA methylation profiles in primary sporadic ovarian cancers and ovarian tissues from high-risk women. Here, Rathi and colleagues [60
] demonstrated that the methylation rate in the E-cadherin gene was significantly higher in tumours compared with non-malignant ovarian tissues, suggesting that aberrant methylation of CDH1 may be important in ovarian cancer pathogenesis. Also, the biological function of E-cadherin may be modified by a number of different growth factors, such as the epidermal growth factor receptor (EGFR). Alper et al. [61
] demonstrated that the loss and reduced EGFR expression in the human ovarian carcinoma cell line NIH: OVCAR-8 led to the reduced expression of E-cadherin and the catenin proteins that form the E-cadherin complex, suggesting that EGFR may regulate the expression of cell adhesion molecules that influence cell growth and invasiveness. Furthermore, the E-cadherin molecule is only part of a complex cell adhesion system in which the cytoplasmic domain of E-cadherin interacts with the actin filament of the cytoskeleton through the catenins. Thus, further biomolecular studies are necessary to obtain deeper insights into the dynamics of E-cadherin expression and its regulation that might bring about the observed loss of E-cadherin in ovarian carcinomas. Nevertheless, irrespective of the exact mechanism, our retrospective study suggests that loss of E-cadherin expression may be a useful marker in patients with epithelial ovarian carcinoma likely to show a less favourable disease outcome. Our study may also encourage further investigations to confirm the prognostic value of E-cadherin under prospective conditions, so as to validate the use of E-cadherin as a prognostic marker in ovarian cancer.
In conclusion, residual tumour and the examination of E-cadherin immunoreactivity in ovarian carcinomas might be beneficial in evaluating patient prognosis, since a strong correlation was observed between negative E-cadherin expression and poor clinical outcome.
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Acknowledgements |
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Received for publication November 21, 2003. Revision received March 29, 2004. Accepted for publication May 17, 2004.
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