Original Article

Expression of E-cadherin in human colorectal cancer

M.A. Khoursheed T.C. Mathew R.R. Makar S. Louis S.K. Asfar H.M. Al-Sayer H.M. Dashti A. Al-Bader
Department of Surgery, Faculty of Medicine, Kuwait University Health Sciences Centre, Kuwait

Correspondence to: M. A. Koursheed, Department of Surgery, Faculty of Medicine, Kuwait University, P.O. Box 24923, Safat 13110, Kuwait

Introduction Material and methods

Results Discussion References

Keywords: E-cadherin, colorectal cancer, adhesion molecules
Surg J R Coll Surg Edinb Irel., 1 April 2003, 86-91

Aim: To investigate the expression of E-cadherin, a calcium-dependent cell-cell adhesion molecule in colorectal carcinoma. Antibodies to E-Cadherin were used to establish the association of their expression with the clinicopathological characteristics of this disease using immunohistochemical methods. Methods: Immunohistochemical analysis for E-cadherin was carried out in formalin-fixed, paraffin-embedded sections of neoplastic colorectal tissues and non-neoplastic ones adjacent to the lesion from 49 patients who underwent surgery, by the standard peroxidase-antiperoxidase method. Expression of this antigen in normal and malignant epithelium and stromal cells was compared. Results: Both neoplastic and normal tissues showed expression of E-cadherin. There was, however, higher expression of E-cadherin in epithelial cells in both tumour and normal tissues than stromal cells. The percentage of expression in epithelial cells of well-differentiated tumours was significantly higher than moderately differentiated tumours. Loss of normal membranous expression and the presence of cytoplasmic and mixed staining were found frequently in tumour tissues (p = 0.004). This loss of membranous expression, however, did not correlate with Duke’s staging, tumour grade, sex, size or site of the tumour. Conclusion: This study suggests that the lower expression of E-cadherin in less differentiated tumours may explain their aggressive nature, although loss of membranous expression was not significantly correlated to Duke’s staging, tumour grade, sex, size and site of tumour

INTRODUCTION
Tumour metastasis involves two independent processes relevant to cell adhesion: detachment of cells from primary tumours, and reattachment of cells to new sites. Adhesion between normal cells is strong and stable. If the activity of adhesion molecules is suppressed, cell forming tissues tend to dissociate. On the other hand, the reattachment of tumour cells to new sites of metastasis could rely on multiple adhesion molecules such as integrins, selectins and cadherins, in addition to non-specific mechanical trapping of tumour cells in capillaries.¹

Alterations in the function and expression of adhesion receptors which mediate cellcell and cell-substrate interactions have been shown to determine the malignant behaviour of colorectal cancer.² Cadherins constitute a superfamily that share a basic structure (E-cadherin [epithelial or uvomorulin], P-cadherin [placental], N-cadherin [neural or A-CAM], and L-CAM [liver cell adhesion molecule]).^3,4 At least two major subfamilies such as E- and P-cadherin are expressed by tumour cells.⁵ E-cadherin is the prime mediator of epithelial cell-cell adhesion via calcium dependent, homotypic interactions.⁶ It is required for the induction and maintenance of normal epithelial integrity.^3,7,8 It is complexed in the cytoplasm with a-, ß- and y catenin to form a functional unit called the Ecadherincatenin unit (ECCU).^9,10 This unit is then thought to bind to the actin cytoskeleton. Functional regulation of the ECCU may occur, in part, through the APC protein (which competes with E-cadherin for binding sites on ß-catenin) and disruption of any of the components of the ECCU result in loss of E-cadherin-mediated adhesion.^11-16

The levels of cadherins expressed by tumour cells vary. The majority of tumour derived cell lines express high levels of certain cadherins, while, others show down-regulation in expression of these molecules. In general, increased cadherin expression causes tighter association of tumour cells.^1,3,17 Reduction in E-cadherin, on the other hand, may free cancer cells.¹⁸

This study is aimed at determining the immunohistochemical expression of Ecadherin in human colorectal cancer and correlating it with clinicopathological features.

MATERIAL AND METHODS

Patients and tissues
Tissue samples from 49 patients who underwent resection of colorectal carcinoma in Mubarak Al-Kabeer hospital, Kuwait, from 1998 to 2000 were used for this study. A piece of non-necrotic tumour and a piece of unaffected colon mucosa at a distance of 10cm away from the tumour were taken and fixed in 10% formaldehyde. The fixed normal and neoplastic tissues were then processed for paraffin sectioning. Tissue sections of 5µm thickness were taken and stained with haematoxylin and eosin (H and E) stain to confirm the diagnosis and to determine the degree of histological differentiation and Duke’s stage.

Immunohistochemistry
Immunohistochemical analysis was carried out as described previously.¹⁹ Paraffin sections from each case were immunostained with monoclonal antibodies against E-cadherin (Ecad) (DAKO, Denmark). Sections were cut and dried at 37°C, dewaxed in xylene and rehydrated using serial concentrations of ethanol. Sections were washed with Tris buffer and preincubated with normal rabbit serum (Dako, 10%) for 20 minutes. Slides were incubated with primary antibody (Ecad, 1:100 dilution) and exposed to peroxidase antiperoxidase complex. They were then counterstained with haematoxylin and reviewed. For every batch a positive control (reactive lymph node) and negative control, where the primary antibody is replaced with nonimmune immunoglobulin, were run simultaneously.

Immunohistochemical scoring
Expression of E-cadherin, was evaluated by two independent observers. Approximately 500 cells from each slide (50 cells/field) were observed. Where there was a discrepancy, the slides were reviewed by the two observers at a multi-headed microscope to reach a consensus. E-cadherin was evaluated in normal and malignant epithelium and stromal cells (fibroblasts, lymphocytes, macrophages, plasma cells).

Expression of E-cadherin was graded both in terms of percentage and intensity of staining. For percentage the following grades were used: negative - no detectable staining; (+) expression in less than one-third of cells; (++) expression in one-third to two-thirds of cells; (+++) expression in more than two-thirds of cells. Intensity of staining was graded as: negative; weak; moderate or strong. For all positive cases, localisation of staining to the cell membrane, cytoplasm or both (mixed) was assessed. E-cadherin staining in colorectal cancer tissues is shown in Figure 1.

Figure 1: immunohistochemical E-cadherin staining of carcinomatous colorectal tissues in paraffin sections by the peroxidase antiperoxidase method. Note the strong staining of E-cadherin in adenomatous tissue. Majority of neoplastic cells expressed mixed (both membranous and cytoplasmic) staining as compared with normal cells

Statistical Analysis
Results were analysed by using the Statview programme package. Data were compared by using Chi-square test, and the difference between the means of continuous data were compared using the paired t-test. The percentage of expression of E-cadherin in both epithelial and stromal cells was assessed according to Duke’s stage, degree of differentiation, sex, site (colon, rectum) and size of tumour (< or > 4cm).

RESULTS
The mean age of patients was 55.7 years (22 females and 27 males) ranging from 27 to 84 years. In 12 patients the tumour was located in the rectum while in the others the tumour was in the colon (right, transverse and left). All the tumours were adenocarcinomas, 29 were Duke’s B and 20 were Duke’s C. The mean size of the growths was 5.5cm, ranging from 1.5cm to 16cm. Sixteen cancers were well differentiated, while 31 were moderately differentiated and two were poorly differentiated. None of the patients received chemotherapy or radiotherapy prior to surgery.

E-CADHERIN EXPRESSION IN MALIGNANT AND NORMAL TISSUES
The mean percentage of E-cadherin expression in malignant epithelial colorectal tissues was 69.1%, compared with 61.1% in normal tissues (p=0.1). Staining was positive in 89.8% of malignant epithelial tissues and this was found to be similar to normal tissues. There was a lower staining of cells, however, in stromal tissues in both normal and malignant tissues. The mean percentage of expression of E-cadherin in stromal cells of both malignant and normal tissues was 15.7% and 12.5%, respectively. Furthermore, the percentage of strong staining of epithelial cells in normal tissues was significantly higher than in malignant tissues (Table 1).

E-cadherin staining was membranous in 16.3% and mixed in 63.2% of tumour tissues. It was membranous in 42.8% and mixed in 44.9% of normal tissues (Table 2). This difference was found to be highly significant (p=0.004).

CLINICAL AND HISTOPATHOLOGICAL FEATURES
The percentage of expression of E-cadherin in the well differentiated adenocarcinoma was significantly higher than that of the moderately differentiated adenocarcinoma (p=0.02). This, however, was not found to be significant with respect to Duke’s staging, sex, size and site of the tumour (Table 3). Furthermore, loss of membranous expression did not correlate significantly with Duke’s staging, tumour grade, sex, size and site of tumour.

DISCUSSION
In vitro studies have shown that loss of E-cadherin is associated with invasive and poorly differentiated phenotype of colon carcinoma cells.^20-23 Furthermore, transfection of E-cadherin cDNA into poorly differentiated human colon carcinoma cell lines increases cell polarity and intercellular cohesion, and inhibits invasion in vitro.²⁴ Reduction of E-cadherin expression in a noninvasive clone resulted in the acquisition of invasive behaviour.²⁵ In this study, we have investigated the expression of E-cadherin in colorectal cancers and adjacent normal tissues in formalinfixed, paraffin-embedded tissue sections, as previously described.^26-28 Expression of E-cadherin in the epithelial cells and stromal cells of colorectal cancer tissues was similar to the adjacent normal tissues. There was, however, a higher expression of E-cadherin in epithelial cells than in stromal cells of both tumour and normal tissues, as reported by others.^7,8 This study did not support the reports that E-cadherin expression in cancer cells is reduced, when compared with normal epithelium since the expression was similar.²⁰ This can be due to perturbation of the cadherin cell adhesion system without the loss of cadherin. This mechanism is most likely connected to the cytoplasmic control of cadherin function, where the expression site of cadherins in cells is an important factor despite the strong expression.^1,4,29

Normal colorectal epithelial cells show more membranous expression of E-cadherin protein at the cell-cell borders. This reflects the normal localisation of an intercellular adhesion molecule to permit homotypic adhesion since cytoplasmic E-cadherin is by definition non-functional.²⁶ Our study has demonstrated that staining of tumour tissue epithelial cells was mixed (cytoplasmic and membranous) in 63.2% of patients. The staining of the normal adjacent tissues, however, was membranous in 42.8% of patients and mixed in 44.9% (p = 0.004). These findings are in agreement with what has been reported in previous studies.^{19,20,28,30-33} The abnormal Ecadherin staining pattern is likely to reflect reductions or loss of E-cadherinmediated adhesion. The increased detection of E-cadherin in the cytoplasm of tumour tissue could be related to increased production rate, failure to translocate or to anchor to the membrane. This may be due to alternation in a-, ß- and y-catenins which link the cadherin molecule to the actin cytoskeleton.^34,35

  Loss of E-cadherin expression is associated with high grade and advanced stage breast, prostatic and bladder tumours.^1,36 It has been reported that reduced cadherin expression is correlated with invasiveness rather than metastasis.³⁷ In colorectal cancer, however, this has not been consistently the case.^19,28,32-35 Nevertheless, a number of studies have shown that a reduced or negative expression of E-cadherin is associated with undifferentiated tumour histology and an advanced clinical tumour stage.^16,38,39 Furthermore, some studies, in colon cancer, have shown that reduced expression of E-cadherin is significantly associated with poor prognosis.^20,28 It was also demonstrated that there was a significant association between Jass classification and tumour recurrence.^24,40 There was no significant difference in E-cadherin expression, however, between non-recurrent and recurrent cases in moderately differentiated Duke’s B colon cancers, as reported earlier.²⁷

We found that the percentage of expression of E-cadherin in epithelial cells of well differentiated carcinomas was significantly higher than moderately differentiated carcinomas as shown in early reports.^26,28 Tumour grade, Duke’s classification, size, site and sex, however, were not found to have significant correlation with the tumour staining site. Furthermore, investigation of expression intensity show that strong staining of adjacent normal tissue was higher than the tumour tissues, which may reflect the reduction or loss of Ecadherin in these transformed tissues.

Our study has shown that there was loss of membranous expression of E-cadherin in tumour tissues. The percentage of expression was significantly higher in well differentiated than moderately differentiated cancers, which may explain the more aggressive nature of the less differentiated tumours. This may be indicative of tumour progression and metastasis in colorectal cancer. Hence, loss of E-cadherin expression could be utilised in selecting candidates for aggressive adjuvant therapy.

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Copyright: 12 March 2003