Immunohistochemical evaluation of E-cadherin in oral epithelial dysplasia and squamous cell carcinoma

Jaishree Sharma; Manish Bhargava; Shivani Aggarwal; Ashim Aggarwal; Anchal Varshney; Disha Chopra

doi:10.4103/ijpm.ijpm_31_21

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ORIGINAL ARTICLE

Year : 2022 | Volume : 65 | Issue : 4 | Page : 755-760

Immunohistochemical evaluation of E-cadherin in oral epithelial dysplasia and squamous cell carcinoma

Jaishree Sharma¹, Manish Bhargava¹, Shivani Aggarwal¹, Ashim Aggarwal², Anchal Varshney¹, Disha Chopra¹
¹ Department of Oral Pathology and Microbiology, Manav Rachna Dental College, Faridabad, Haryana, India
² Department of Oral and Maxillofacial Surgery, Manav Rachna Dental College, Faridabad, Haryana, India

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Date of Submission	11-Jan-2021
Date of Decision	17-Mar-2021
Date of Acceptance	26-Mar-2021
Date of Web Publication	25-May-2022

Abstract

Aim: To evaluate the expression of E-cadherin (E-cad) in oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC). Material and Method: Immunohistochemistry was used to detect E-cad expression in 20 cases each of normal oral mucosa, oral epithelial dysplasia and squamous cell carcinoma. Statistical Analysis Used: Inferential statistical methods for statistical analysis used were Chi-square test for comparison of the frequency between different severity of dysplasia and OSCC. Results: Upon assessing the expression of E-cad in OED and OSCC, increase in E-cad immunoreactivity was seen in early lesions. However, the expression of E-cad decreased significantly as the grade of dysplasia increased. Conclusion: We observed a significant decrease in E-cad expression from dysplasia to poorly differentiated squamous cell carcinoma suggesting that loss of expression of E-cad is closely related to carcinoma.

Keywords: E-cadherin, immunohistochemistry, oral epithelial dysplasia, oral squamous cell carcinoma

How to cite this article:
Sharma J, Bhargava M, Aggarwal S, Aggarwal A, Varshney A, Chopra D. Immunohistochemical evaluation of E-cadherin in oral epithelial dysplasia and squamous cell carcinoma. Indian J Pathol Microbiol 2022;65:755-60

How to cite this URL:
Sharma J, Bhargava M, Aggarwal S, Aggarwal A, Varshney A, Chopra D. Immunohistochemical evaluation of E-cadherin in oral epithelial dysplasia and squamous cell carcinoma. Indian J Pathol Microbiol [serial online] 2022 [cited 2023 Nov 30];65:755-60. Available from: https://www.ijpmonline.org/text.asp?2022/65/4/755/345865

Introduction

Potentially malignant disorders include all clinical presentations that carry a high risk of malignancy when compared to healthy tissue.^[1] Much attention has been focused on oral leukoplakia (OL) due to its high incidence and potential for a malignant transformation.^[2] The malignant transformations are generally associated with the loss of the epithelial phenotype and differentiation.^[3] The epithelial to mesenchymal transition (EMT) features are seen in oral epithelial dysplasia (OED) and in their progression to malignancy.^[1]

Oral squamous cell carcinoma (OSCC) is one of the most prevalent pathological oral cancer, accounting for >80% of all head and neck malignancies. During the progression, there is a loss of cohesion. It is a result of a defect at the cell adhesion molecular level.^[4] Cell adhesion molecules play more than a purely structural role within stratified squamous epithelia. There is a strong relationship between reduced expression of these adhesion molecules, decreased differentiation and increased invasiveness.^[5] E-cadherin (E-cad) is a 120 kDa transmembrane glycoprotein and one of the most important molecules in cell-cell adhesion in epithelial tissues. E-cad, expressed in most epithelial cells, is primarily responsible for the initial adhesion of these cells and also promotes cell polarity and the formation of specialized cell-cell junctions.^[6] Loss of E-cad expression has been noted with poorly differentiated morphology in a large number of malignancies such as cervix and esophagus including head and neck.^[7]

Although some studies have explored the immunoreactivity of E-cad in potentially malignant disorders, non-tumor epithelium adjacent to oral cancer and (OSCC), it remains unclear if E-cad could be used as a biomarker to predict malignant transformation.^[4] Also, the usefulness of E-cadherin for estimating the risk of developing tumors associated with the progression of the dysplasia-carcinoma sequence in the oral cavity is still unknown.

In the present study, an attempt was made to evaluate the expression of E-cad in OED and squamous cell carcinoma, thus, evaluating its significance in the process of oral carcinogenesis and to elucidate its role as a reliable and potential marker in determining the biological behavior of the disease.

Materials and Method

This comparative study comprised of formalin-fixed paraffin-embedded specimen blocks of already diagnosed 20 cases of oral epithelial dysplasia (OED) and 20 cases of OSCC retrieved from the archives of the Department of Oral Pathology and Microbiology. Twenty cases of normal mucosa were taken as controls. Inclusion criteria included histopathologically diagnosed cases of OED and squamous cell carcinoma and exclusion criteria included patients who had undergone chemotherapy and radiotherapy. This study had the ethical clearance of the Institutional Ethics Committee of our college.

Immunohistochemistry procedure

As per standard protocol

Assessment of E-cadherin immunoreactivity

Immunostaining was assessed in each case in three fields that were randomly selected and its average was taken for evaluation for cells expressing E-cad. The distribution and intensity of E-cad immunostaining was assessed semi-quantitatively by two independent observers to eliminate interobserver bias.^[8]

A scale of 0 to ++ was used:

0 - Absence of staining

+/0 - Heterogeneous staining

+ - Weak staining

++ - Strong staining

The pattern of expression of E-cad by tumor cells was also graded by these observers on:

A scale of 1-4 will be used:

Membranous staining
Both membranous and cytoplasmic staining
Cytoplasmic staining
Absence of staining

Histopathological grading of OED: WHO 2005 classification was followed here.

Mild dysplasia
Moderate dysplasia
Severe dysplasia
Carcinoma in situ

Histopathological grading: Broder's (1920) classification is followed here.

Tumors are graded on the basis of the degree of differentiation and keratinization of tumor cells into the following:

Grade I: Well differentiated tumor; 75-100% of the cells are differentiated

Grade II: Moderately differentiated tumor; 50-75% the of cells are differentiated

Grade III: Poorly differentiated tumor; 25-50% of the cells are differentiated

Grade IV: Anaplastic tumor; 0-25% of the cells are differentiated

Statistical analysis

The software used for the statistical analysis was Statistical Package for Social Sciences (SPSS) version 21.0. Chi-square test was used for the comparison of the frequency between different severity of dysplasia and OSCC. The results with P < 0.05 were considered to be statistically significant.

Results

E-cadherin expression in normal oral mucosa

E-cad was immunolocalized at the cell membranes of basal and suprabasal epithelial cells. Staining was weak in the basal aspect of keratinocytes in two cases and was absent in the superficial layers in all cases. Although, the pattern of staining was the same in all cases examined, staining intensity varied in some cases [Figure 1], [Table 1].

Figure 1: Photomicrograph of normal oral mucosa showing strong E-cadherin expression on cell membrane (10x)

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Table 1: Comparison of immunohistochemistry staining intensity between normal oral mucosa (control group) OED and OSCC

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Intergroup comparison of the staining intensity and location of OED

Strong staining was significantly more among mild OED
Weak (+) staining was significantly more among moderate and severe OED
Heterogeneous (+/0) staining was significantly more among severe oral epithelial dysplasia. Membranous location was significantly more among mild and moderate OED in comparison to severe OED.
Both membranous and cytoplasmic was significantly more among OED [Figure 2], [Figure 3], [Figure 4], [Table 2].

Figure 2: Photomicrograph of mild dysplasia showing strong E- cadherin expression on cell membrane (10x)

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Figure 3: Photomicrograph of moderate dysplasia showing weak E-cadherin expression on cell membrane (10x)

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Figure 4: Photomicrograph of severe dysplasia showing heterogeneous E-cadherin expression in both the cytoplasm and on the cell membrane (10x)

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Table 2: Comparison of the staining intensity and location between different grades of oral epithelial dysplasia (OED)

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Intergroup comparison of the staining intensity and locationof OSCC

Weak staining was significantly more among well and moderately differentiated OSCC in comparison to poorly differentiated OSCC
Heterogeneous (+/0) staining was significantly more among poorly differentiated OSCC in comparison to well and moderately differentiated OSCC.
Membranous location was significantly more among well and moderately differentiated OSCC in comparison to poorly differentiated OSCC.
Cytoplasmic location was significantly more among poorly differentiated OSCC in comparison to well and moderately differentiated OSCC [Table 3].

Table 3: Comparison of the staining intensity and location between different grades of oral squamous cell carcinoma

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Comparison of staining intensity and location of OED and OSCC

Out of 20 cases of OED, 14 cases showed membranous localization, 5 cases showed both membranous and cytoplasmic and 1 case showed cytoplasmic localization (Chi-square value = 13.857, P value = 0.008) [Table 2]. Out of 20 cases, 11 cases showed strong intensity, 7 cases weak intensity and 2 cases were heterogeneously stained (Chi-square value = 18.377, P value = 0.001) [Table 4] [Figure 2], [Figure 3], [Figure 4].
Out of 20 cases of squamous cell carcinoma, 11 cases showed membranous localization, 7 cases showed both membranous and cytoplasmic staining and 2 cases showed cytoplasmic staining (Chi-square value = 13.701, P value = 0.008) [Table 2]. The intergroup comparison of distribution of staining intensity was done between well, moderately and poorly differentiated OSCC. Out of 20 cases, 2 cases were strongly stained, 11 cases were weakly stained and 7 cases were heterogeneously stained (Chi-square value = 13.506, P value = 0.009) [Figure 5], [Figure 6], [Figure 7], [Table 5].

Table 4: Comparison of the staining location between different grades of oral epithelial dysplasia and squamous cell carcinoma

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Figure 5: Photomicrograph of well differentiated squamous cell Carcinoma showing strong E-cadherin on cell membrane (10x)

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Figure 6: Photomicrograph of moderately differentiated squamous cell carcinoma showing weak E-cadherin expression on cell membrane (10x)

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Figure 7: Photomicrograph of poorly differentiated squamous cell carcinoma showing heterogeneous E cadherin expression in both the cytoplasm and on the cell membrane (10x)

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Table 5: Comparison of the staining intensity between different grades of oral epithelial dysplasia and squamous cell carcinoma

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Discussion

Head and neck carcinoma constitutes the fifth most frequent human tumor diagnosed worldwide. In India, squamous cell carcinoma constitutes more than 90% of the total of head and neck carcinoma with an incidence rate of 12.6 per lakh of the population.^[8] Recently, various tumor markers have received attention, and among them is cell-to-cell adhesion molecule, E-cad tumor suppressor gene that is expressed in epithelial tissues.^[9]

In the present study, 20 samples of normal oral mucosa (NOM) as the control group was taken, where E-cad was expressed very strongly. These control tissues showed intense continuous membranous staining of E-cad. This observation of our study was similar to the studies conducted by Kaur G et al.^[8] (2009) in 7 samples of NOM and stated that E-cad belongs to the family of cell-cell adhesion molecules essential for maintaining the structural integrity and organization of stratified squamous epithelia. Hence, the immunostaining expression was seen as a strong membranous homogenous staining of the basal/parabasal and superficial cell layers of the epithelium and lost at the upper-third of the epithelium, which may be due to normal desquamation process. In a study by Hung et al.^[10] (2006), E-cad immunoreactivity was observed in the membranous compartments of NOM and positive for all the 10 cases of NOM.

Of the 20 cases of OED, the expression pattern was similar to NOM in all mild dysplasia but slight loss of expression pattern was noted as the severity of dysplasia increased. This observation was similar to the study conducted by Yogesh et al.^[7] (2011) a where majority of cases (70% of 10 cases) scored 1+ (weak) and 2+ (mild to moderate) expression. There is a variation in the expression of E-cad in the dysplastic epithelium with varying degrees of dysplasia or severity of dysplasia and location of tissue. This would suggest that these alterations are the result of the progression of dysplasia and could be a late event, which is suggestive of change toward a cell phenotype with the ability to invade. There was a significant difference in the distribution of expression of E-cad staining intensity between mild, moderate and severe OED , i.e., intensity decreases from strong to weak as the severity of dysplasia increased. A similar observation was made in the studies done by Chaw et al.^[11] (2012) and Yuwanati B et al.^[6] (2011). Out of the 20 cases of epithelial dysplasia, 14 cases showed membranous localization, 5 cases showed both membranous and cytoplasmic and 1 case showed cytoplasmic localization There was a significant difference in expression of E-cad as dysplasia increased in severity and expanded higher within the epithelium. Loss of expression of E-cad was associated with inflammatory infiltrate in the epithelium. A similar observation was made by Hung et al.^[10] (2006) and Kaur J et al.^[12] (2013). Out of 20 cases of OSCC, 11 cases showed membranous localization, 7 cases showed both membranous and cytoplasmic staining and 2 cases showed cytoplasmic staining. There was a significant difference in the distribution of staining location, a decrease of membranous staining and an increase in cytoplasmic staining of E-cad was seen more frequently in poorly differentiated tumors than in well and moderately differentiated tumor. A similar observation was made in a study by Kaur G et al.^[8] (2009). Similar observation was found in various studies by Williams HK et al.^[13] (1998) and Hung et al.^[10] (2006), who concluded that immunoreactivity of E-cad was progressively reduced from normal mucosa followed by oral precancerous lesions and significantly decreased in primary OSCC (58%), which was also coinciding with our findings of E-cad expression.

Out of 20 cases of OSCC, 2 cases were strongly stained, 11 cases were weakly stained and 7 cases were heterogeneously stained. There was a significant difference in the distribution of staining intensity between well, moderately and poorly differentiated squamous cell carcinoma. Weak staining intensity was significantly more among well and moderately differentiated OSCC in comparison to poorly differentiated OSCC. Heterogeneous staining was significantly more among poorly differentiated OSCC in comparison to well and moderately differentiated OSCC. Kaur G^[8] (2009) observed that well differentiated OSCC expressed E-cad often as strongly as normal stratified squamous epithelium, while in poorly differentiated OSCC expression of E-cad was lost or cytoplasmic, and in moderately differentiated tumors, it was expressed in a heterogeneous fashion. Similar were the observations of Tanaka N, Odajima T, Ogi K, Ikeda T, Satoh M^[14] (2003), Caballero T, López J, Rey J and Gracia A^[15](2006).

Freitas et al.^[16] (2006) and Shinohara M^[17] (1998) stated that there is no significant association between the degree of differentiation of tumor and expression of E-cad. Tumors were examined for intensity of staining for the adhesion molecule E-cad. Reduction in E-cad staining was correlated with the mode of invasion and with reduction in desmosomal staining, but not with poor differentiation as indicated by cytokeratin staining.

In our study (20 cases of OED and OSCC each), dysplastic epithelium showed slight loss of expression of E-cad as the grade of dysplasia increased. The expression of E-cad decreased significantly in advanced cases of OSCC. Hence, E-cad immunoreactivity was found to inversely correlate with the loss of cell differentiation. Our study gives an insight into the expression of E-cad in OED and squamous cell carcinoma. We believe further additional studies with larger sample size and trials are required to establish whether immunoexpression of this tumor marker in OSCC correlates with higher stage and grade of disease; lymph node metastasis, tumor invasive behavior and carcinogenesis can be used in estimating the invasiveness and aggressiveness of the disease. Hence, in future, it may help us to understand the mechanism of metastatic behavior, and aid in the design of new strategies for diagnosis and treatment of oral carcinoma.

Conclusion

In the present study, we observed a significant decrease in E-cad expression from dysplasia to poorly differentiated squamous cell carcinoma, suggesting that loss of expression of E-cad is closely related to the carcinoma. We believe further additional studies with much larger sample size are required to establish whether immunoexpression of this tumor marker in OSCC correlates with a higher stage and grade of disease.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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