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Role of E-cadherin in differentiation between Squamous and basal cell carcinoma.

Byline: Parvin Rajabi, Mohammad Ali Rajabi, Amin Eftekhari, Mojgan Mokhtari, Ali Asilian and Azar Naimi

Abstract:

Objective: Some type of basal cell carcinoma (ex Keratotic variant) is often misdiagnosed as squamous cell carcinoma and basal cell carinoma. The objective of this study was to determine the role of E-cadherin in differentiation between Squamous and basal cell carcinoma. Methodology: The sampling was performed by simple method. Entry criteria to the study included 2 groups of 34 primary, untreated BCCs and SCCs each from a different patient. They were surgically removed and the diagnosis was confirmed by histopathology. The stained sections were scored by levels of expression and intensity of E-cadherin.

Results: Distribution of E-cadherin staining was more in BCC than SCCs (P=0.006) but the decreased staining intensity of E-cadherin in SCC compared with BCC was not statistically significant (P=0.056). When the results (score of distribution and intensity of E-cadherin) were added together, the acquired E-cadherin index in a statistical analysis was meaningful to differ between SCC and BCC (P=0.002).

Conclusion: Our study showed that E-cadherin distribution and index but not intensity is lower in SCC cells than BCC cells and it can justify the cause of metastasis in SCC. However, this is ambiguous to use it for differentiating of aggressive forms of BCCs from SCCs, practically.

KEY WORDS: Basal cell carcinoma, Squamous cell carcinoma, E-cadherin, Scoring protocol.

Pak J Med Sci April June 2011 (Part-II) Vol. 27 No.3 533-536

How to cite this article:

Rajabi P, Rajabi MA, Eftekhari A, Mokhtari M, Asilian A, Naimi A. Role of E-cadherin in differentiation between Squamous and basal cell carcinoma. Pak J Med Sci 2011;27(3):533-536

INTRODUCTION

The classical cadherins consist of E (epithelial), N (neuronal) and P (placental) cadherin, which are the best studied representatives of this family. The E-cadherin gene is widely regarded as a tumor suppressor gene, and inactivation of this gene is critically involved in tumor formation, invasiveness, and metastatic potentially.1,2

Basal cell carcinomas (BCC) are very common neo-plasms. It mostly occur on the head and neck; but a good number occur on other part of the body, particularly upper trunk and legs. It is estimated that there were about 1.3 million persons suffering from BCC and squamous cell carcinoma (SCC) in the year 2007 and it is about 5 times higher than that of breast or prostate cancer.3

Cutaneous SCC is the second most common type of skin cancer in the United States, after basal cell carcinoma (BCC).4 Most tumours are well differentiated and have a variable degree of pleomorphism. However, the role that loss of E-cadherin function may play in early stages of carcinoma development is not well understood in as much as studies have shown either a decrease5,6 or increase 7, 8 in E-cadherin function during early cancer progression.

In the previous studies there were no meaningful results between the expression of E-cadherin and BCC as well as SCC. Also staging protocol was not used in these studies so we used such a protocol for E-cadherin expression and intensity to compare it with BCC and SCC. We used intensity in addition to distribution and staging them in this study to find a better relationship.

The aim of this study was to define the distribution of E-cadherin in Basal cell carcinoma and Squamous cell carcinoma of skin to determine a scoring protocol according to E-cadherin distribution and intensity for differentiating BCC and SCC.

METHODOLOGY

This is a cross sectional observational study. Purpose of this study was to determine the correlation between epithelial cadherin (E-cadherin) expression in Basal cell carcinoma and squamous cell carcinoma of skin. The sampling was performed by simple method and involved all patients that met the entry criteria to the study. Entry criteria to the study included 2 groups of 34 primary, untreated BCCs and SCCs each from a different patient. They were surgically removed and the diagnosis was confirmed by histopathology in our pathological department (haematoxylin and eosin staining) between 2007-2009. Paraffin blocks sections (3um) were stained using the immunoperoxidase-streptavidin-biotin complex method. Monoclonal antibodies (MAbs) against E-cadherin (Clone NEH-38 Dako Co, California) were used.

Immunohistochemistry was performed using the streptavidin-biotin complex indirect immunoperoxidase method. Sections were dewaxed, rehydrated, and incubated with 3% hydrogen peroxide (H2O2) in water for 5 minutes to block endogenous peroxidase activity. Antigen retrieval was performed using microwave for 20 minutes in 0.01 mol. L-1 citrate buffer (pH 6.0) at 750 W. After rinsing in phosphate-buffered saline (PBS), the sections were incubated with the primary antibody (anti E-cadherin antibody, used at a concentration of 1 microg/mL). After washing with PBS, the sections were incubated with biotinylated Rabit antimouse immunoglobulin (Dako Ltd) for 30 minutes, and washed with PBS, then incubated with streptavidin-peroxidase conjugate (1: 500; Amersham Pharmacia Biotech, Bucks, U.K.) for 30 minutes. The sections were developed with 3, 3'-diaminobenzidine tetrahydrochloride solution (Sigma-Aldrich, Poole, U.K.) and 0.1% H2O2 and counterstained with haematoxylin.

The first parameter that was considered was the intensity of reactivity for E-cadherin. Positivity was scored from 0 to 3 by comparing staining of tumoral cells with normal keratinocytes. E-cadherin was evaluated for each specimen by two simultaneous observers and scaled as follows: 0, no staining; 1, weakly diffuse staining; 2, diffuse staining; 3, membranous staining comparable with that in the normal epidermis. Because, it was common to find areas in the same tumor where tumoral cells didn't express E-cadherin at all, resulting in a patchy distribution of reactivity, a second score was given to each specimen reuecting the proportion of tumor cells that were positive for E-cadherin. We evaluated distribution of E-cadherin expression in tumoral cells by using a semi quantitative scale: score 0, 0%; 1, 1 to 25%; 2, 26% to 50%; 3, greater than 50% positively stained tumor cells for E-cadherin.

Then we summed intensity score with distribution score and calculated E-cadherin index. In this index, we considered negative, 0-2, intermediate, 3-4, and strong, 5-6 and then performed statistical analysis. Non-neoplastic colonic mucosa was used as a positive control. The non-neoplastic adjacent skin in the test sections served as an internal positive control.9

SPSS software (Ver. 16) is used to determine any relation between data with chi-square method.

RESULTS

This study included 68 patients (34 SCCs and 34 BCCs).The BCC growth pattern comprised 4 (11.8%) infiltrative, 25 (73.5%) nodular and 5 (14.7%) superficial tumours. In SCC group about 70% of tumours were moderately differentiated subtype. The number of male and female cases was 37 (54.4%) and 31 (45.6%) respectively. Of the 68 patients included in the study, the mean age of males and females was 49.8 and 47 years old, respectively. The mean age in BCC group was 46.5 +- 18.3 and in SCC group was 50.6 +- 17.6 (T-student test, P greater than 0.05).

In E-cadherin distribution, 94.1% of the BCC cells showed E-cadherin expression but 85% of the SCC cells showed E-cadherin expression in the entire examined specimen. Fig.1 shows the percent of distribution staining of E-cadherin in BCC and SCC. BCC specimens affected to express E-cadherin in greater than 50% of cells. A statistical analysis showed a significant difference between E-cadherin expression in BCC and SCC specimens (Chi square, P=0.006).

In E-cadherin intensity, 16.6% and 44.1% of the 34 BCC samples, stained E-cadherin membranously and strongly, whereas, in SCC samples these were 2.9% and 29.4% respectively. (Fig.2) However, the decreased staining intensity of E-cadherin in SCC compared with the BCC was not statistically significant (Chi-square test, P=0.056).

Summed expression and intensity scores (E-cadherin Index), 58.8% of 34 BCC samples had score 6-7 so this was 17.6% in SCC group. (Fig.3) It shows a significant relationship between it and tumor sub-type (Chi-square test, P=0.002). Interestingly, the P value of it was lower than E-cadherin expression alone.

In 4 infiltrative BCCs, E-cadherin distribution was negative or 1-25%. Also, E-cadherin intensity was negative or weakly positive. In contrast, 5 superficial BCCs showed E-cadherin staining in greater than 50% of cells, and membranously. Although statistical analysis showed a significant difference between subtypes of BCC in E-cadherin distribution, intensity and index but because of specimens loss in subtypes these results are not valid.

We observed that from 10 well differentiated SCCs, in 8, E-cadherin was stained strongly or membranously and only in two, E-cadherin staining was weak. Also, in this group in 5/10, greater than 50% of neoplastic cells and in others 25-50% of cells were stained. But, in moderately differentiated group, 18/24, less than 25% of cells were stained and 21/24 were stained mildly or no. Statistical analysis showed a significant difference for E-cadherin distribution, intensity and index in subtypes of SCC (P less than 0.001, P=0.002 and P less than 0.001 respectively).

DISCUSSION

Tumor progression is responsive to evolving architectural and contextual changes in the tissue microenvironment that may alter tumor cell growth, survival, and differentiation.

The role that alterations in E-cadherin-mediated adhesion may play in the development of incipient invasive carcinoma has remained unclear, as previous studies have shown conflicting findings regarding how loss of E-cadherin alters the early pre-invasive stages of cancer progression in stratified squamous epithelium. The expression of E-cadherin, in both SCC and on BCC cells was investigated at light microscopic level in the current study.

Reduced expression of E-cadherin on invasive neoplastic cells has been demonstrated for cancers of the stomach, liver, breast, and several other organs. This reduced expression of E-cadherin is observed in squamous cell carcinoma and Paget's disease.10 In BCC and SCC, but not in premalignant lesions, cell-surface expression of E-cadherin is reduced. It is in agreement with the observation that the loss of E-cadherin is associated with tumor invasion.11

Ultrastructural studies show that E-cadherin on the cytoplasmic membrane of the keratinocytes is shifted to desmosomes under physiological conditions and therein expresses an adhesion function in association with other desmosomal cadherins.10 We observed that E-cadherin was strongly expressed membranously in all normal keratinocytes and specialized epithelial structures. Fuller et al11 obtained the same results; they also showed strong surface expression of E-cadherin, similar to normal skin in the Bowen's disease. Alt-Holland et al12 could show that loss of E-cadherin-mediated adhesion led to the acquisition of phenotypic properties that augmented cell motility and directed the transition from the precancer to cancer in skin-like tissues. It can justify local invasion and no metastasis in BCC rather than SCC.

We observed that E-cadherin expression in SCC (distribution and intensity) correlated with cell differentiation. The previous studies also showed this correlation.13-16 Kaur et al14 in their study on oral SCC showed that increase in E-cadherin immunoreactivity was seen in early lesions, that is, in well differentiated and moderately differentiated SCC. Further-more, E-cadherin was negative in majority of metastatic lymph nodes. They showed that loss of the cell adhesion and E-cadherin plays an important role in progression of SCC, that is, downward regulation of its expression is associated with de-differentiation and metastasis. Koseki et al17 also obtained the same results on SCC of skin. They showed that decreased expression of E-cadherin in the primary lesions is correlated with regional lymph node metastasis in SCC and that it is more frequently correlated with well-differentiated than poorly differentiated SCC.

A heterogeneous distribution of cells ( less than 25% of cells) with different immunostaining intensity (weakly or no staining) was more frequently observed in specimens of infiltrative BCCs. These results show that E-cadherin has a major role in the growth pattern and the local aggressive behavior of BCC. The same results were also obtained in previous studies.18, 19

We have shown that loss of distribution of E-cadherin expression but not intensity occurred more in SCC tumor cells than BCC tumor cells. In previous studies, this difference in intensity of E-cadherin was shown.11,20 Interestingly, E-cadherin index that was a semi-quantitative scale of both intensity and distribution was less in SCCs than BCCs; and its P value was less than E-cadherin distribution. Although, these results made us hopeful that E-cadherin can be used for differentiating BCCs from SCCs, but practically, it was seen that aggressive forms of BCC (infiltrative) expressed E-cadherin weakly (both in distribution and intensity). Therefore, it needs more studies with bigger data (especially aggressive forms of BCC) for recommending E cadherin scaling in differentiation of BCCs from SCCs. But in future studies we can recommend the use from this scaling system.

In conclusion, our study showed that E-cadherin distribution and index but not intensity is lower in SCC cells than BCC cells and it can justify the cause of metastasis in SCC. However, this is ambiguous to use it for differentiating of aggressive forms of BCCs from SCCs, practically.

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Parvin Rajabi, Mohammad Ali Rajabi, Amin Eftekhari, Mojgan Mokhtari, Ali Asilian and Azar Naimi

1. Parvin Rajabi, MD, Department of Pathology,

2. Mohammad Ali Rajabi, MD, Department of Surgery,

3. Amin Eftekhari, MD, Department Pathology,

4. Mojgan Mokhtari, MD, Department of Pathology,

5. Ali Asilian, MD, Department of Dermatology,

6. Azar Naimi, MD, Department of Pathology,

1-6: Isfahan University of Medical Science, Isfahan, Iran.

Correspondence: Amin Eftekhari, 46, Mehrang Alley, Nazar Gharbi Street, Postal code: 817575455, Esfahan, Iran. E-mail: surgicalpathology@gmail.com

Received For Publication: December 2, 2010

Revision Received: March 31, 2011

Revision Accepted: April 1, 2011
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Author:Rajabi, Parvin; Rajabi, Mohammad Ali; Eftekhari, Amin; Mokhtari, Mojgan; Asilian, Ali; Naimi, Azar
Publication:Pakistan Journal of Medical Sciences
Article Type:Report
Geographic Code:7IRAN
Date:Sep 30, 2011
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