| Abstract|| |
Background: Oral cancer is known to be governed by the antiapototic and loss of cell adhesion properties which dictate its progression. Aim: To study the immunexpression of E-cadherin and Bcl-2 in varying TNM stages and histopathological grades of OSCC. Materials and Methods: 11 cases of well differentiated, 10 cases of moderately differentiated and 11 cases of poorly differentiated OSCC were studied immunohistochemically using archival paraffin embedded tissue specimens. Statistical Analysis: Differences between the different variables were analyzed using ANOVA test, Kruskal–Wallis test and post hoc test followed by Bonferroni test. The resulting data was analyzed using SPSS software version 19. Results: The expression of Bcl-2 and E cadherin immunopositivity was associated positively with tumor grade, high T category and Histopathological grades. Conclusions: The results of this study points to the significance of cell proliferation and invasion as a major determinant of prognosis in OSCC.
Keywords: B-cell lymphoma 2, E-cadherin, oral squamous cell carcinoma, prognosis
|How to cite this article:|
Gulati N, Rathore AS, Juneja S, Rastogi P. Expression of E-cadherin and B-cell lymphoma 2 in oral cancer: A ratio-based planning for targeted therapy. Indian J Dent Res 2017;28:3-9
|How to cite this URL:|
Gulati N, Rathore AS, Juneja S, Rastogi P. Expression of E-cadherin and B-cell lymphoma 2 in oral cancer: A ratio-based planning for targeted therapy. Indian J Dent Res [serial online] 2017 [cited 2019 Jul 19];28:3-9. Available from: http://www.ijdr.in/text.asp?2017/28/1/3/203620
| Introduction|| |
Oral cancer constitutes a significant part of the global burden of cancer. India contributes up to 7.8% of the global cancer burden and 8.33% of global cancer deaths. International Agency for Research on Cancer, an arm of the World Health Organization (WHO), has ranked oral cancer as sixth most common malignancies. The alarmingly high incidence of oral squamous cell carcinoma (OSCC) is indeed a major health issue for developing nations where it accounts for 20 per 1 lakh new cases every year  and the most promising strategy for the treatment of OSCC remains the breakthroughs in the fields of molecular targeted therapy.
E-cadherin acts as a growth suppressor by cyclin-dependent kinase inhibitor p27, dephosphorylates Rb protein, leads to late reduction in cyclin D1 protein and Wnts, upregulates E-cadherin suppressors, i.e., Snail and Twist, and downregulates miR-20014. B-cell lymphoma 2 (Bcl-2) protein is an antiapoptotic gene which is an promoter of invasion, death antagonist which facilitates the permanent acquisition of mutations and malignant transformation.In vitro experiments have shown that intercellular communication may modulate apoptosis both in normal and tumorous tissue through Wnt signaling pathways.
E-cadherin and Bcl-2 have antagonistic role in progression of oral cancer. Thus, the present study is designed to assess and correlate the immunoexpression of E-cadherin and Bcl-2 in various histopathological grades of OSCC, hypothesized E-cadherin and Bcl-2 antagonistic interaction in pathogenesis of OSCC and to deduce a probable E-cadherin/Bcl-2 ratio targeted molecular therapy.
| Materials and Methods|| |
Patients and tumor samples
The biopsy specimens of cases obtained were fixed with 10% neutral buffered formalin processed and embedded in paraffin wax. Three microns section was obtained for hematoxylin and eosin staining and immunohistochemistry (IHC) procedure. The samples comprised of 32 cases of OSCC from the archives of the department. Clinical data of each case such as and tumor, node, metastasis (TNM) staging were collected. Archival specimens were grouped based on the WHO (2005) histopathological grading criteria as follows: Group 1 - Well-differentiated OSCC (n 1 = 11), Group 2 – Moderately differentiated OSCC (n 2 = 10), and Group 3 – Poorly differentiated OSCC (n 3 = 11). Different aspects of the same study were undertaken simultaneously such as clinical parameters, Histopathological grades and pattern of invasion. This study highlights the correlation of expression of markers with TNM stage and histopathological grades of study cases.
Immunohistochemistry with E-cadherin and Bcl-2
Immunohistochemistry procedure was done using standard heat induced epitope retrieval methods. Archival sections on glass slides coated with Poly-L- lysine were used to carry out the immunohistochemistry procedure using heat induced epitope retrieval method. Diaminobenzidine (DAB) was used as chromogen to detect Bcl-2 and E-cadherin positivity.
Five randomly selected fields with minimum 200 cells per field at 40× magnification were selected and the percentage of positive tumor cells out of total number of neoplastic cells present in the same field were scored as mentioned below: Semiquantitative scoring for E-cadherin and Bcl-2 was done as Low, intermediate, and high (<10%, 10%–50%, >50%, respectively)., Similarly, qualitative scoring was done as (+, ++, and +++). The images of five high-power fields (40×) were obtained with a digital camera (Olympus EPL3) Fixed to the research microscope and was transferred to computer system for analysis which was done using a grid. Immunoreactive score was then evaluated by number of positive cells (cytoplasm and nucleus of epithelial cells for Bcl-2 and membranous and cytoplasmic for E-cadherin) per 1000 total tumor cells of all grades of OSCC. Combined scoring was also done and cases divided as low and high grades (Low as 0-2, high as 3-4).
The resulting data were analyzed using SPSS statistics for Windows, Version 19.0(Armonk, NY; IBM Corp). Data have been expressed as mean and standard deviation. Differences between the different variables were analyzed using ANOVA test, Kruskal–Wallis test and post hoc test followed by Bonferroni test. Besides this, area under the curve values was calculated by applying the receiver operating characteristic (ROC) curve analysis for both the molecules. Pearson's Chi-square test was carried out to determine the level of correlation or association between the groups under study. P< 0.05 was considered statistically significant.
| Results|| |
The expression of Bcl-2 and E-cadherin
The qualitative scoring of Bcl-2 immunoexpression increased gradually from well to poorly differentiated OSCC with moderate (++) staining intensity present in 72.7% cases of well-differentiated OSCC in comparison to intense (+++) staining intensity present in 100% cases of poorly differentiated OSCC. Semiquantitative scoring of Bcl-2 immunoexpression increased from well to poorly differentiated OSCC with 81.8% well-differentiated OSCC showing 5%–50% positive cells whereas 90.9% poorly differentiated OSCC with >50% positive cells. The qualitative scoring of E-cadherin immunoexpression decreased in a gradual, incremental manner from well to poorly differentiated OSCC with +++ scoring present in 63.6% cases of well-differentiated OSCC in comparison to + scoring present in 72.7% cases of poorly differentiated OSCC. Semiquantitative scoring of E-cadherin immunoexpression decreased from well to poorly differentiated OSCC with 72.7% well-differentiated OSCC showing >50% positive cells whereas 54.5% poorly differentiated OSCC with <10% positive cells. Results for both the parameters were statistically significant, i.e., P < 0.05 [Table 1] and [Table 2].
The mean score of Bcl-2 immunoexpression quantitatively increased in a gradual gradational manner from well to poorly differentiated OSCC, i.e., well differentiated with 437.55 to poorly differentiated with 926.18 whereas a mean score of E-cadherin immunoexpression quantitatively decreased from well to poorly differentiated OSCC, i.e., well differentiated with 867.45 to poorly differentiated with 301.09. These results were statistically significant when correlated to histopathological grades but not when compared to TNM staging (P < 0.05) [Graph 1] and [Graph 2].
The ratio decreased gradually from well-differentiated OSCC (1.5454) to poorly differentiated OSCC (0.5454), from stage 1 (1.4) to stage 4 (0.72).
Receiver Operating Characteristic (ROC) curve
The ROC curve shows that area under the graph for Bcl-2 immunoexpression was 20.8% (with standard error as 0.092), and for E-cadherin immunoexpression was 71.9% (with standard error as 0.095), predicting Bcl-2 to be a less sensitive and specific marker as compared to E-cadherin. However, E-cadherin/Bcl-2 ratio had an area under the graph as 78.6% (with standard error as 0.079), indicating it to be the most sensitive and specific parameter to analyze the progression of OSCC cases. The result was statistically significant (P < 0.05) [Graph 3].
| Discussion|| |
Deregulation of oncogenes and tumor suppressor genes involved in apoptosis has been associated with tumor pathogenesis and progression. Bcl-2 protein leads to cellular immortalization, contributing to the formation of the tumor and facilitating the permanent acquisition of mutations. On the contrary, loss or reduction of E-cadherin expression can be caused by smatic mutations of CDH 1 promoter.,,,,,
Bcl-2 expression was seen in the form of granular cytoplasmic to nuclear staining. All well and moderately differentiated OSCC tumor islands showed positive expression except the islands with central keratinization where only peripheral cells were positive and central core with less or no expression suggesting that cells expressing antiapoptotic activity have a stem cell property representing permanent self-renewal which was in contrast to Bcl-2 staining restricted to tumor cells within the centre of tumor islands. Diffuse immunopositivity was seen throughout the tumor cell population in PDSCC suggesting overexpression possibly reflecting the resistance of these tumor cells to apoptosis and increased cell survival  and the loss of ability of malignant keratinocytes to differentiate terminally.
The Bcl-2 mean score was statistically significant (P < 0.05) with 437.55 ± 29.737 in well-differentiated squamous cell carcinoma, 576.10 ± 10.22 in moderately differentiated OSCC, and 926.18 ± 11.28 in poorly differentiated OSCC. Semiquantitative scoring of Bcl-2 immunoexpression also increased from 81.8% WDSCC having 5%–50% positive cells whereas 90.9% PDSCC with >50% positive cells.
It has also been indicated that this oncoprotein may play a role in relatively early events of OSCC , but a decrease in number of Bcl-2 positive cells with a decrease in differentiation has also been reported., Qualitative scoring of Bcl-2 immunoexpression increased, with moderate (++) staining intensity in 72.7% cases of WDSCC while intense (+++) staining intensity in all cases of PDSCC (P < 0.05) [Table 2]. Increase in Bcl-2 staining intensity was seen with progressive histopathological grades of OSCC [Table 3]., Similar results were seen for TNM staging for Bcl-2 qualitative, semi-quantitative, and combined scoring [Table 4] which were similar to studies in oral cavity, laryngeal tumors, and tongue carcinoma but in disagreement to others who noted no correlation of tumor size and TNM staging to Bcl-2 immunoexpression. In our study, the expression of E-cadherin was in the form of membranous to cytoplasmic in WDSCC to cytoplasmic staining in MDSCC, and poorly differentiated OSCC showed almost negligible expression (only two out of 11 cases showed positive cytoplasmic expression) which was in accordance with many other studies., Higher cytoplasmic expression of E-cadherin in OSCC suggested a redistribution of the E-cadherin complex out of tight junctions and increase in its degradation by cytoplasmic endocytosis. The mean E-cadherin count was statistically significant (P < 0.05) with well-differentiated SCC with 867.45 ± 53.655, MDSCC with 756.40 ± 46.867 and PDSCC with 301.09 ± 420.061. This was in accordance with studies of E-cadherin underexpression in carcinoma in situ cases and infiltrative tumors. In the present study, E-cadherin qualitative, semiquantitative, and combined scoring analysis when correlated with TNM staging was nonsignificant like other study  [Table 4] and was in contradiction to studies who noted a significant correlation of decreased E-cadherin immunoexpression to TNM staging. Similarly, the intensity of staining also diminished from well to poorly differentiated OSCC., Qualitative scoring of E-cadherin IHC expression of +++ scoring in 63.6% cases of well-differentiated OSCC and + scoring present in 72.7% cases of poorly differentiated OSCC. Similarly, semiquantitative scoring of E-cadherin IHC expression also decreases from well to poorly differentiated OSCC with 72.7% well-differentiated OSCC showing >50% positive cells whereas 54.5% poorly differentiated OSCC with <10% positive cells [Figure 1],[Figure 2],[Figure 3],[Figure 4].
|Table 3: Correlation of histopathological grades with mean scores of qualitative, semi-quantitative and combined assessment of Bcl-2 and E-cadherin in study cases|
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|Table 4: Correlation of TNM stages with mean scores of qualitative, semi-quantitative and combined assessment of Bcl-2 and E-cadherin in study cases|
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|Figure 1: Well-differentiated oral squamous cell carcinoma showing immunopositivity of Bcl-2. Periphery cells of tumor islands shows Bcl-2 positivity (×10, inset ×40)|
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|Figure 2: Well-differentiated squamous cell carcinoma showing immunopositivity of E-cadherin. All cells in tumor islands showing positive expression of E-cadherin (×10, inset ×40)|
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|Figure 3: Poorly differentiated squamous cell carcinoma showing diffuse and intense immunopositivity of Bcl-2 in tumor cells (×10, inset ×40)|
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|Figure 4: Poorly differentiated oral squamous cell carcinoma showing weak immunopositivity of E-cadherin in tumor cells (×10, inset ×40)|
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The E-cadherin/Bcl-2 ratio decreased overall from well-differentiated OSCC (1.5454 ± 0.522) to poorly differentiated OSCC (0.5454 ± 0.1507) and from stage 1 (1.4 ± 0.54) to stage 4 (0.72 ± 0.609). Approximately, 40% cases of well-differentiated OSCC showed increase in ratio from TNM stage 1 (1.4 ± 0.54) to TNM stage 3 (1.75 ± 0.5) while among poorly differentiated OSCC, 30% cases showed a gradual increase in ratio from TNM stage 1 (0) to TNM stage 4 (0.508 ± 0) [Table 5].
|Table 5: Combined score E-cadherin/Bcl-2 ratio correlation to histopathological grades and TNM stages|
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Evidence to support the antagonistic role of E-cadherin and Bcl-2, have been studied in other tumors such as breast, colon, prostate, and pancreas. Bcl-2 acts to antagonize the action of E – cadherin as has been shown by previous studies., Certain other studies also suggested that cadherin catenin is associated with increased activity of Bcl-2. Bcl-2 expression increases in response to estrogen and is correlated with estrogen receptor positivity in breast carcinomas.
| Conclusions|| |
Collectively, the present work has provided new insights on how elevated expression of antiapoptotic Bcl-2 not only increases tumorigenicity but, through induction of an epithelial-mesenchymal transition-like phenotype, triggers an invasion-metastatic profile. It remains unclear exactly the means by which Bcl-2 induce such a diverse set of crucial signaling events culminating in enhanced metastatic potential. Thus, a combined ratio of both molecules is a helpful guide to categorize OSCC cases based on their prognostic implications. Moreover, loss of E-cadherin in invasive tumor cells may lead to increased Bcl-2 expression and resistance to chemotherapeutic drugs.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Landis SH, Murray T, Bolden S, Wingo PA. Cancer statistics, 1999. CA Cancer J Clin 1999;49:8-31.
St Croix B, Sheehan C, Rak JW, Flørenes VA, Slingerland JM, Kerbel RS. E-Cadherin-dependent growth suppression is mediated by the cyclin-dependent kinase inhibitor p27(KIP1). J Cell Biol 1998;142:557-71.
Saydam O, Shen Y, Würdinger T, Senol O, Boke E, James MF, et al.
Downregulated microRNA-200a in meningiomas promotes tumor growth by reducing E-cadherin and activating the Wnt/beta-catenin signaling pathway. Mol Cell Biol 2009;29:5923-40.
Hung KF, Chang CS, Liu CJ, Lui MT, Cheng CY, Kao SY. Differential expression of E-cadherin in metastatic lesions comparing to primary oral squamous cell carcinoma. J Oral Pathol Med 2006;35:589-94.
Gao S, Eiberg H, Krogdahl A, Liu CJ, Sørensen JA. Cytoplasmic expression of E-cadherin and beta-Catenin correlated with LOH and hypermethylation of the APC gene in oral squamous cell carcinomas. J Oral Pathol Med 2005;34:116-9.
Jäckel MC, Dorudian MA, Marx D, Brinck U, Schauer A, Steiner W. Spontaneous apoptosis in laryngeal squamous cell carcinoma is independent of bcl-2 and bax protein expression. Cancer 1999;85:591-9.
Jameson JL. Principles of Molecular Medicine. Totowa, New Jersey: Humana Press; 1998. P. 89-95.
Tsujimoto Y, Cossman J, Jaffe E, Croce CM. Involvement of the bcl-2 gene in human follicular lymphoma. Science 1985;228:1440-3.
Hirohashi S. Inactivation of the E-cadherin-mediated cell adhesion system in human cancers. Am J Pathol 1998;153:333-9.
Strathdee G. Epigenetic versus genetic alterations in the inactivation of E-cadherin. Semin Cancer Biol 2002;12:373-9.
Maretzky T, Reiss K, Ludwig A, Buchholz J, Scholz F, Proksch E, et al.
ADAM10 mediates E-cadherin shedding and regulates epithelial cell-cell adhesion, migration, and beta-catenin translocation. Proc Natl Acad Sci U S A 2005;102:9182-7.
Berx G, Becker KF, Höfler H, van Roy F. Mutations of the human E-cadherin (CDH1) gene. Hum Mutat 1998;12:226-37.
Yoshiura K, Kanai Y, Ochiai A, Shimoyama Y, Sugimura T, Hirohashi S. Silencing of the E-cadherin invasion-suppressor gene by CpG methylation in human carcinomas. Proc Natl Acad Sci U S A 1995;92:7416-9.
Bolós V, Peinado H, Pérez-Moreno MA, Fraga MF, Esteller M, Cano A. The transcription factor Slug represses E-cadherin expression and induces epithelial to mesenchymal transitions: A comparison with Snail and E47 repressors. J Cell Sci 2003;116(Pt 3):499-511.
Teni T, Pawar S, Sanghvi V, Saranath D. Expression of bcl-2 and bax in chewing tobacco-induced oral cancers and oral lesions from India. Pathol Oncol Res 2002;8:109-14.
Suri C. The immunohistochemical evaluation of the expression of bcl-2 in different histological grades of squamous cell carcinoma. J Clin Diagn Res 2009;3:1891-9.
Sudha VM, Hemavathy S. Role of bcl-2 oncoprotein in oral potentially malignant disorders and squamous cell carcinoma: An immunohistochemical study. Indian J Dent Res 2011;22:520-5.
] [Full text]
Arul AS, Solomon RD, Arul AS, Santhi VS. Immunohistochemical evaluation of bcl-2 and Ki-67 in varying grades of oral squamous cell carcinoma. J Sci Ind Res 2011;70:923-8.
Chen Y, Kayano T, Takagi M. Dysregulated expression of bcl-2 and bax in oral carcinomas: Evidence of post-transcriptional control. J Oral Pathol Med 2000;29:63-9.
Muzio M, Pannone T, Rosa N. Interaction between bcl2 and p53 in neoplastic progression of basal cell carcinoma of head and neck. Anticancer Res 2001;21:3757-64.
Nair RG, Shameena PM, Varghese I, Sudha S. Immunohistochemical evaluation of bcl-2 oncoprotein in oral dysplasia and carcinoma. J Oral Maxillofac Pathol 2011;2:83-8.
Jordan RC, Catzavelos GC, Barrett AW, Speight PM. Differential expression of bcl-2 and bax in squamous cell carcinomas of the oral cavity. Eur J Cancer B Oral Oncol 1996;32B: 394-400.
Singh BB, Chandler FW Jr., Whitaker SB, Forbes-Nelson AE. Immunohistochemical evaluation of bcl-2 oncoprotein in oral dysplasia and carcinoma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:692-8.
Popovic B, Jekic B, Novakovic I, Milasin J. Analysis of the anti-apoptotic protein bcl-2 in oral squamous cell carcinoma. Serbian Dent J 2007;54:153-60.
Yao L, Iwai M, Furuta I. Correlations of bcl-2 and p53 expression with the clinicopathological features in tongue squamous cell carcinomas. Oral Oncol 1999;35:56-62.
Yuen AP, Lam KY, Choy JT, Ho WK, Wong LY, Wei WI. Clinicopathologic significance of bcl-2 expression in the surgical treatment of oral tongue carcinoma. Eur J Surg Oncol 2002;28:667-72.
Kaur G, Carnelio S, Rao N, Rao L. Expression of E-cadherin in primary oral squamous cell carcinoma and metastatic lymph nodes: An immunohistochemical study. Indian J Dent Res 2009;20:71-6.
] [Full text]
Janabi A, Sarkis S. Immunohistochemical expression of E-cadherin and CD44 adhesion molecules in oral squamous cell carcinoma. J Baghdad Coll Dent 2013;25:36-42.
Williams HK, Sanders DS, Jankowski JA, Landini G, Brown AM. Expression of cadherins and catenins in oral epithelial dysplasia and squamous cell carcinoma. J Oral Pathol Med 1998;27:308-17.
Hanemann JA, Oliveira DT, Nonogaki S, Nishimoto IN, de Carli ML, Landman G, et al.
Expression of E-cadherin and ß-catenin in basaloid and conventional squamous cell carcinoma of the oral cavity: Are potential prognostic markers? BMC Cancer 2014;14:395.
Braakhuis BJ, Bloemena E, Leemans CR, Brakenhoff RH. Molecular analysis of surgical margins in head and neck cancer: More than a marginal issue. Oral Oncol 2010;46:485-91.
Ahmad M, Khadim M, Bukhari N. E-cadherin expression, a valuable predictor of invasiveness in histological grades of oral squamous cell carcinoma. Pak Dent J 2013;33:222-34.
Narayan TV, Shreedhar B, Shashidhara R, Mohanty L. Expression of E-cadherin and cathepsin -D in normal oral mucosa, oral epithelial dysplasia and oral squamous cell carcinoma: A comparative analysis between immunohistochemistry and routine histopathology. J Oral Maxillofac Pathol 2011;15:288-94. [Full text]
Karch I, Schipper E, Christgen H, Kreipe H, Lehmann U, Christgen M. Is upregulation of BCL2 a determinant of tumor development driven by inactivation of CDH1/E-cadherin? PLoS One 2013;8:e73062.
Ke H, Zhang JY, Akiyama SK, French JE. BCL2 interaction with actin in vitro
may inhibit cell motility by enhancing actin polymerization. Cell Adh Migr 2011;5:6-10.
Sasaki CY, Lin HC, Passaniti A. Expression of E-cadherin reduces bcl-2 expression and increases sensitivity to etoposide-induced apoptosis. Int J Cancer 2000;86:660-6.
Tran NL, Adams DG, Vaillancourt RR, Heimark RL. Signal transduction from N-cadherin increases Bcl-2. Regulation of the phosphatidylinositol 3-kinase/Akt pathway by homophilic adhesion and actin cytoskeletal organization. J Biol Chem 2002;277:32905-14.
Teixeira C, Reed JC, Pratt MA. Estrogen promotes chemotherapeutic drug resistance by a mechanism involving Bcl-2 proto-oncogene expression in human breast cancer cells. Cancer Res 1995;55:3902-7.
Dr. Nikita Gulati
Department of Oral and Maxiilofacial Pathology and Microbiology, ITS Dental Collge, Murad Nagar, Ghaziabad, Uttar Pradesh - 201 206
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]