| Abstract|| |
Background and Objectives: The product of bcl-2 gene, bcl-2 protein, an anti-apoptotic protein, is known to be over-expressed in potentially malignant disorders and squamous cell carcinoma (SCC) of the oral cavity. The aim of this study is to compare the topographical aspect and degree of bcl-2 over-expression in potentially malignant disorders including leukoplakia, oral submucous fibrosis (OSMF), and oral lichen planus (OLP), with that of the oral squamous cell carcinoma (OSCC), and to determine whether bcl-2 protein can be considered as a tumor marker.
Materials and Methods : A group of 60 histo-pathologically diagnosed, formalin-fixed, paraffin embedded tissue samples was included in the study. The study group was further subdivided into four groups: Group I, consisting of oral leukoplakia; Group II, OSMF; Group III, OLP and Group IV, OSCC. These samples were collected from Government Dental College, Bangalore, and then subjected to immunohistochemical (IHC) staining using indirect immunoenzyme labeled streptavidin biotin (LSAB) method.
Results : Out of 30 cases of OSCC: 11 (36.7%) cases showed greater supra-basal keratinocyte staining; 15 (50%) cases showed greater number of positive cells in the basal cell layer, with relatively less number of supra-basal cells showing positive staining; and, rest of the 4 (13.3%) cases did not show convincing staining. Among the total 30 cases of potentially malignant disorders: 10 each of leukoplakia, OSMF and OLP, 2 (20%), 2 (20%), 4 (40%) of the cases showed greater supra-basal cell layer positive staining and 8 (80%), 6 (60%), 6 (60%) of them showed greater basal cell staining, respectively. Two cases of OSMF did not show convincing staining. In the cases that were bcl-2 positive: 2 (6.67%) of the OSCC, 3 (30%) of leukoplakia, 2 (20%) of OSMF and 1 (10%) of OLP, showed more than 50% of the cells positive. 25-50% cells were positive in 21 (70%) of OSCC, 6 (60%) of leukoplakia, 4 (40%) of OSMF and 6 (60%) of OLP cases. 10-25% of cells were positive in 4 (13.3%) of OSCC, 1(10%) of leukoplakia, 2 (20%) of OSMF and 3 (30%) of OLP cases. Less than 10% of cells were positive in 3 (10%) of OSCC and 2 (20%) of OSMF cases.
Clinical Significance and Conclusion : As definite number of cases showed bcl-2 over expression in our study, the role of bcl-2 in the development and progression of oral neoplasia needs further investigation along with other oncogenes.
Keywords: Bcl-2 protein, immunohistochemistry, leukoplakia, oral lichen planus, oral squamous cell carcinoma, oral submucous fibrosis
|How to cite this article:|
Sudha V M, 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
Oral squamous cell carcinoma (OSCC), the most common oral malignancy, , often presents a clinical diagnostic challenge, particularly in its early stages of development. 
|How to cite this URL:|
Sudha V M, Hemavathy S. Role of bcl-2 oncoprotein in oral potentially malignant disorders and squamous cell carcinoma: An immunohistochemical study. Indian J Dent Res [serial online] 2011 [cited 2019 Nov 16];22:520-5. Available from: http://www.ijdr.in/text.asp?2011/22/4/520/90286
The incidence of OSCC differs widely in various parts of the world, and ranges from 2-10 per 1,00,000 in a year. The South Asian countries such as Srilanka, India, Pakistan and Bangladesh have shown a high incidence. , Despite advances in prevention, diagnosis, and treatment, the 5-year survival rate has not improved over the past 30 years.  Surveys in India have shown that about 80% of oral carcinomas are preceded by oral potentially malignant disorders, most often, persistent leukoplakia (the most common potentially malignant lesion of the oral mucosa  ), or oral submucous fibrosis (OSMF), which predominantly affects the people of South-East origin.  The other premalignant condition seen predominantly is oral lichen planus (OLP). Hence, it would be advantageous if effective diagnostic and prognostic methods of monitoring were available in the premalignant stage.
Proliferation, apoptosis and differentiation are the fundamental aspects of tumor biology.  There is a mounting evidence which suggests that oral carcinogenesis is correlated with progressive accumulation of genetic alterations in molecules that play a crucial role during apoptosis.  Potential biomarkers are needed in order to introduce more objectivity. Some proto-oncogenes are known to prevent apoptosis or programmed cell death, and their altered expression will cause dysregulation of this process.  Of particular interest in this regard is the proto-oncogene bcl-2. The bcl-2 gene was first isolated in cells from leukemia and B cell lymphoma.  Abnormal bcl-2 protein expression is an indicator of blocked apoptosis; thus promoting prolonged cell survival, which facilitates acquisition of mutations and malignant transformation. 
Detection of apoptotic abnormalities before the consequences become clinically or histologically detectable, will greatly enhance the potential for early diagnosis. The detection of these aberrations by immunohistochemistry (IHC) or molecular techniques may serve as an additional diagnostic tool.  This study attempts to study the differential expression of bcl-2 protein in potentially malignant disorders such as leukoplakia, OSMF, OLP and in OSCC for its relevance in development and progression of oral neoplasia, and its potential for allowing early diagnosis and in helping in selection of the appropriate treatment modalities.
| Materials and Methods|| |
10 paraffin blocks of each i.e., leukoplakia, OSMF, OLP, and 30 of OSCC were retrieved from archival paraffin blocks, from the Department of Oral pathology, Government Dental College, Bangalore.
DAKO labeled streptavidin biotin (LSAB)+ system peroxidase (DAKO Corporation, CA, USA) kit was used. Serial sections of 4 μm thickness were taken on silanized slides for IHC. The slides were deparaffinized by heating on the slide warmer at 60°C for 10-20 minutes. The slides were subsequently rehydrated by keeping in two baths each of xylene, absolute alcohol and 95% alcohol for 5, 3 and 3 minutes respectively. Then, the slides were immersed in distilled water for 30 seconds. Antigen retrieval was done using heat induced epitopes retrieval (HIER) microwave oven method. Slides were placed in plastic jar containing 10 mM citrate buffer (pH 6.0) and heated for 20 minutes. They were then allowed to cool for 20 minutes and taken out and washed with wash buffer. All reagents were equilibrated to room temperature (20°-25°C) prior to immunostaining. All the incubations were carried out at room temperature, with a humidifying chamber. At no time, the tissue sections were permitted to dry during the staining procedure.
After tapping off excess buffer, the specimens were covered with 3% hydrogen peroxide for 15 minutes. Then the slides were gently washed with distilled water and placed in a buffer bath for 5 minutes. The excess buffer is tapped off, and primary antibody was used to cover the specimen. For negative control, phosphate buffer (TBS) was used instead of the primary antibody. The slides were incubated for 1 hour at room temperature in a humidifying chamber. Then the slides were rinsed in distilled water and placed in fresh buffer bath.
Excess buffer was tapped off, and the slides were incubated for 15 minutes with link, and then rinsed gently with distilled water, and then placed in a buffer bath for 30 minutes. The excess buffer was tapped off as before, and the specimens were incubated with streptavidin peroxidase for 30 minutes. The slides were washed gently with distilled water and then placed in buffer bath for 5 minutes. Excess buffer was tapped off, and then the sections were covered with substrate chromogen for 2-10 minutes. The slides were then gently rinsed with distilled water and stained lightly with Harris haematoxylin. There were then washed gently under running water for 10 minutes, dehydrated and dipped in xylene and mounted in Di-n-butyl Phthalate in Xylene (DPX), a non-aqueous permanent mounting medium, using coverslips.
Interpretation of staining
Presence of brown colored end product at the site of target antigen was indicative of positive reactivity. The negative control tissue demonstrated absence of specific staining. The positive control used here is in-built, i.e. the sub epithelial lymphocyte infiltration of OLP sections.
Proportions are compared using Chi-square test. P value >0.05 considered as statistically significant.
The topographic patterns of positive keratinocyte staining within the basal cell and prickle cell layers were categorized as follows:
- N - No convincing staining.
- BC ≥ PC - The percentage of keratinocytes stained for each protein in the basal cell layer is greater than or equal to the percentage in prickle cell layer.
- PC > BC - The percentage of keratinocytes stained for each protein is greater in the prickle cell layer than in the basal cell layer.
The percentages of positive cells were classified as:
- More than 50% of cells positive scored as (+++);
- 25%-50% positive (++);
- 10%-25% (+);
- Fewer than 10% (±), i.e., by counting the number of cells per 1 mm 2 field.
| Results|| |
The topographic expression and degree/percentage of cells expressing bcl-2 in OSCC and in potentially malignant disorders is summarized as below:
Out of 30 cases of OSCC [Figure 1] and [Figure 2], 11 (36.7%) cases showed greater supra-basal keratinocyte staining. 15 (50%) of the cases showed greater number of positive cells in the basal cell layer, with relatively less number of supra-basal cells showing positive staining, and rest of the 4 (13.3%) cases did not show convincing staining. Among the total 30 cases of potentially malignant disorders, 10 each were leukoplakia, OSMF, OLP. In case of leukoplakia, 2 (20%) of the cases showed greater supra-basal cell layer positive staining and 8 (80%) of them showed greater basal cell staining [Figure 3]. Among 10 cases of OSMF, 2 (20%) of the cases showed greater supra-basal cell layer positive staining and 6 (60%) of them showed greater basal cell staining [Figure 4]. Two cases of OSMF did not show convincing staining. In case of OLP, 4 (40%) of the cases showed greater supra-basal cell layer positive staining and 6 (60%) of them showed greater basal cell staining [Figure 5].
|Figure 1: Well differentiated oral squamous cell carcinoma: Epithelial island showing positive reaction at the periphery and greater supra-basal keratinocyte staining|
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|Figure 2: Poorly differentiated carcinoma showing positive reaction for bcl-2 protein|
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|Figure 3: Leukoplakia: The epithelium showing positive reaction in both basal and supra-basal layer for bcl-2 protein|
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|Figure 4: Oral submucous fibrosis: Basal and supra-basal layer of the epithelium showing positive reaction for bcl-2 protein|
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|Figure 5: Lichen planus: Basal and supra-basal cells positive for bcl-2 protein along with lymphocyte infiltrate|
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Statistical analysis of the results of positive topographic expression between OSCC and potentially malignant disorders showed a statistical significance (P value of 0.0144). It was also significant when compared between OSCC and leukoplakia (P value of 0.0139). No statistical significance was observed when compared between OSCC and OSMF, and between OSCC and OLP. Bcl-2 expression in various lesions on the basis of percentage of immunoreactive cells is mentioned in [Table 1]. The majority of the cases are showing 25%-50% (++) of positive cells. No statistical significance was found when compared between OSCC and potentially malignant disorders overall, and between OSCC and each potentially malignant disorder.
|Table 1: Diseases (n) intensity of immunohistochemical staining demonstrating bcl-2 expression in the different lesions of the study|
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| Discussion|| |
The evidence implicating the role of bcl-2 family members in the development of cancer comes from several lines of study. bcl-2 expression appears to be altered to varying degrees in oral mucosal dysplasia. The recent retrospective cross-sectional studies have suggested that up-regulation of bcl-2 may be an early event in epithelial carcinogenesis.  In normal oral mucosa, bcl-2 is not detectable or is expressed only occasionally in the basal cells.  Increased bcl-2 expression is frequently seen adjacent to oral cancers and in dysplastic epithelium. Although there have been no functional studies that explain the role of the bcl-2 protein in oral cancer, one particular study has shown that when oral keratinocytes were transfected with a bcl-2-expression plasmid, their level of differentiation was reduced.  In the current study, the type of control used has been previously validated.  The use of the internal (lymphocyte) positive control suggests that the approach has been optimized for the samples under investigation. 
The proportion of oral cancers in which bcl-2 can be seen by IHC staining has been reported as being between 50% -75% of cases in most studies, although others have found it only rarely. Few other studies employing histologically proven dysplastic tissues for study of bcl-2 expression have reported 16%,  37%  and 81% positivity of IHC.  However, only Singh et al., has attempted to correlate expression levels to grade of dysplasia.  In the study by Singh BB et al.,  it has been clearly demonstrated that bcl-2 immuno-reactivity proportionally paralleled the involved thickness of epithelium in mild, moderate, and severe dysplasia. The other observations by Singh BB et al., were that immunoreactivity for bcl-2 was lower for squamous cell carcinomas than in severe dysplasia.  In the present study, although the cases of OSCC were not categorized as poorly, intermediately or well differentiated, most of the cases (26) were of well-differentiated carcinoma type. In well differentiated carcinomas, more number of peripheral cells showed positivity compared to the central portions of the islands. These observations might be attributed to the down-regulation of bcl-2 expression concomitant with terminal cell differentiation. It also has been reported that bcl-2 expression was significantly more frequent in poorly differentiated, non-keratinizing and early stage carcinomas, in comparison with well- differentiated, keratinizing and advanced stage carcinomas of the esophagus  and oral cavity,  though not of the lung.  This may reflect site specificity, and/or the biological behavior of these tumors. Similarly in the present study, bcl-2 positivity was more and intense in poorly differentiated carcinomas as compared to the well-differentiated ones. The increased bcl-2 expression in poorly differentiated carcinomas may reflect the loss of ability of malignant keratinocytes to differentiate terminally. In an 'in vitro' experiment, it is been shown that bcl-2 actively blocked the differentiation of cultured keratinocytes. 
In a study by Teni, 35/65 oral squamous cell carcinomas were bcl-2 positive, 23% cases were 3+, 26% cases were 2+, and 51% cases were 1+ (classification was similar to ours as mentioned above).  In the present study on 30 cases of OSCC: 6.67% cases were 3+, 70% cases were 2+, and 13.3% cases were 1+ and rest 10% cases did not show convincing staining. The reason for different results may be related to the longevity of the disease and also other factors like age, causative factors and number of cases under study.
In human cancers, over-expression of bcl-2 protein has also been observed in several tumor types, including cancer of the lung,  ovary,  colon,  prostate,  esophagi  and breast.  Additionally, over-expression has been observed in potentially malignant disorders of the colorectal system, oral cavity, stomach and esophagus. ,, This finding suggests that the bcl-2 may be associated with early oncogenesis in these organs.  Singh BB et al., demonstrated expression of bcl-2 in non-dysplastic basal and parabasal cells contiguous to neoplastic epithelium.  This raises the possibility that bcl-2 alterations precede the overt appearance of oral potentially malignant disorders and neoplastic lesions.
Tanda et al., in 2000, reported that the percentage of supra-basal keratinocytes expressing bcl-2 and MDM2 (negative regulators of apoptosis and p53) in leukoplakia lesions was higher than that in OLP and normal mucosa, i.e. five (38.5%) of leukoplakia and 2 (20.0%) of lichen planus lesions showed greater number of prickle cell layer expressing bcl-2.  In the present study, 2 (20%) of leukoplakia and 4 (40%) of lichen planus lesions showed greater suprabasal cell layer positivity for bcl-2. In lichen planus, the increased supra-basal cell postivity may also be correlated to the basal cell degeneration along with its risk of carcinoma development. In a study by Teni et al., 2002, bcl-2 expression was detected in superficial layers of oral lesions such as OSMF (14) and leukoplakia (17)  and 16% of the oral lesions showed bcl-2 positivity. In the present study, the results are almost at par, with 20% of the lesions (both OSMF and leukoplakia) being bcl-2 positive in the suprabasal cell layer.
Alterations in the expression of proteins related to cell proliferation and apoptosis are a strong indicator of the malignant transformation potential of certain lesions.  As the oncoprotein bcl-2 regulates programmed cell death by allowing the tumor cells to escape apoptosis, it promotes their survival, thereby facilitating the acquisition of further mutations.  Hence, increased expression in leukoplakia and OSMF may be attributed to their potential risk of transforming into malignant lesions.These studies indicate that alterations in bcl-2 may play a role in potential for malignant transformation, probably by allowing escape from apoptosis. In addition, over-expression of the proteins in potentially malignant disorders suggests early stages of oral carcinogenesis.
| Conclusion|| |
In this study, we demonstrated with IHC that there is an increased bcl-2 expression in oral potentially malignant disorders including leukoplakia, OSMF, lichen planus and OSCC. It is clearly demonstrated that the bcl-2 expression is more in OSCC, than potentially malignant disorders in the study. The degree of cells showing positivity is closely related. Among the potentially malignant disorders, there is a difference seen in topographical expression, and also in the degree of expression of bcl-2; however, this can be better appreciated with investigation of more number of cases. Clearly, the role of bcl-2 along with other oncogenes, in the context of multiple interconnected changes in the gene expression during the development and progression of oral neoplasia, needs further investigation.
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V M Sudha
Department of Oral Pathology, Government Dental College, Bangalore
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]