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An Official Publication of the Indian Association of Oral and Maxillofacial Pathologists


 
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ORIGINAL ARTICLE  
Year : 2022  |  Volume : 26  |  Issue : 3  |  Page : 316-321
 

To analyse the mitotic and keratinisation correlation with bcl-2 expression in varying grades of oral epithelial dysplasia and squamous cell carcinoma


1 Consultant Oral Pathologist, Paradise Diagnostics, Delhi, India
2 HOD, Department of Oral Pathology, ITS CDSR, Muradnagar, Uttar Pradesh, India
3 HOD, Department of Oral Pathology, Rajas Dental College and Hospital, Kavalkinaru, Tamil Nadu, India
4 Director, Paradise Diagnostics, Delhi, India

Date of Submission11-Sep-2021
Date of Decision10-Mar-2022
Date of Acceptance22-Mar-2022
Date of Web Publication17-Oct-2022

Correspondence Address:
Aparna Pathak
Consultant, Paradise Diagnostics
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jomfp.jomfp_326_21

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   Abstract 


Context: The bcl-2 proto-oncogene was discovered at the chromosomal breakpoint of t (14;18) found in follicular lymphoma. Histological changes in dysplasia are considered the earliest signs preceding the progression into squamous cell carcinoma. Serving as critical regulators of apoptotic pathways, bcl-2 prohibits programmed cell death and subsequently assists in uncontrolled neoplastic growth.
Settings and Design: This study included 48 cases, eight each of epithelial dysplasia and squamous cell carcinoma. Immunohistochemical staining using bcl-2 antibody was performed and different histological parameters were correlated with bcl-2 positive cells in all the cases.
Materials and Methods: All 3 μm thick sections were stained with bcl-2 antibody. After identifying four representative fields at 40x, their images were obtained for assessment of bcl-2 labelled cells and their intensity along with different histological parameters in all the cases.
Statistical Analysis: The differences between different histological parameters were analysed using the Anova test, post hoc test and Bonferroni test. Pearson's Chi-square test was carried out to determine the level of correlation between the bcl-2 positive cells in both epithelial dysplasia and oral squamous cell carcinoma cases.
Conclusion: Sequential increase in the bcl-2 expression was observed in increasing grades of epithelial dysplasia, whereas bcl-2 expression was significantly decreased in ascending stages of squamous cell carcinoma thus, suggesting a possible role of bcl-2 in disease progression from premalignancy to malignancy.


Keywords: Apoptosis, bcl-2, epithelial dysplasia, immunohistochemistry, oral squamous cell carcinoma


How to cite this article:
Pathak A, Shetty DC, Dhanapal R, Kaur G. To analyse the mitotic and keratinisation correlation with bcl-2 expression in varying grades of oral epithelial dysplasia and squamous cell carcinoma. J Oral Maxillofac Pathol 2022;26:316-21

How to cite this URL:
Pathak A, Shetty DC, Dhanapal R, Kaur G. To analyse the mitotic and keratinisation correlation with bcl-2 expression in varying grades of oral epithelial dysplasia and squamous cell carcinoma. J Oral Maxillofac Pathol [serial online] 2022 [cited 2022 Nov 30];26:316-21. Available from: https://www.jomfp.in/text.asp?2022/26/3/316/358736





   Introduction Top


Currently, oral cancer is the sixth most common type of cancer worldwide and is one of the most alarming health problems facing mankind.[1] Sometimes, due to genetic and epigenetic aberrations, certain progressive changes in cellular behaviour from slightly deregulated proliferation to full malignancy are observed.[2] Thus, cells with damaged genomes would not undergo apoptosis, allowing the defective genome to persist and replicate in the offspring cells.[3]

The bcl-2 gene family plays an important role in the regulation of apoptosis and modulation of cell cycle regulating proteins, illustrating the cross-play in mechanisms controlling cell death and proliferation.[4],[5] The founding member of the bcl-2 family of apoptosis-regulating proteins is B-cell lymphoma/leukemia-2 (bcl-2), which gets its name because of its involvement in B-cell lymphomas and leukemias.[6] The bcl-2 proto-oncogene was discovered at the chromosomal breakpoint of t (14;18) found in human follicular lymphoma. The bcl-2 oncoprotein is thought to regulate programmed cell death and facilitate cell survival and is associated with the mitochondrial membrane, nuclear envelope, and endoplasmic reticulum.[7]

The prognosis of oral cancer vastly depends upon the characterisation of the disease. Resultant genomic aberrations could help in the evaluation of high-risk premalignant lesions and their propensity for malignancy as molecular changes occur before histological manifestations.[8] The diagnosis of epithelial dysplasia is a subjective assessment of the discrepancy of epithelial maturation patterns and a variety of cellular changes implying an increased risk of malignant transformation that is relative to the grade of dysplasia.[9]

The present study is aimed to study the expression of bcl-2 in epithelial dysplasia as well as in squamous cell carcinoma to search for the possible role of bcl-2 in modulating the staging and the pathogenesis of squamous cell carcinomas. This study also aims at analysing the link between bcl-2 and the malignant transformation of epithelial dysplasia into squamous cell carcinoma.


   Materials and Methods Top


The study comprised of 48 cases of formalin-fixed, paraffin-embedded blocks, retrieved from the archives of the oral pathology department of I.T.S Centre for Dental Studies and Research, Muradnagar. The selected specimens consisted of 24 cases each of different grades of oral epithelial dysplasia and squamous cell carcinoma which were stained for bcl-2 using the immunohistochemical method. Deparaffinization of 3-μm tissue sections was obtained by dipping in changes of xylene, then rehydrated in decreasing alcohol grades, and washed. The antigen retrieval method was conducted in two cycles further followed by bcl-2 immunohistochemical staining. The lymph node was taken as a positive control for the expression of bcl-2 and immuno-stained in the same manner as other study cases.

All the slides were viewed under a light microscope. The positively stained bcl-2 cells showed the uptake of brown colour. Evaluation of immunohistochemical staining intensity was conducted manually and was further compared in all epithelial dysplasia and squamous cell carcinoma cases. Staining intensity was categorized as a mild or intense expression using the intensity of the positive control as the benchmark.

Selection of field for counting cells

From all positively stained areas, four representative fields at 40x were selected and counted that were not in continuum with each other to minimise any possible errors. The images of each of the four fields were obtained by binocular light microscope (40x) and then transferred to a grid for analysis. The immunoreactive score was then obtained by the assessment of bcl-2 labelled cells and their intensity along with different histological parameters in all the cases.

Statistical analysis

SPSS software was used for statistical analysis and the differences between different variables were analysed using the Anova test and post hoc test, followed by the Bonferroni test. Pearson's Chi-square test was carried out to determine the level of correlation between the study groups. The significance i.e., P value < 0.05, was considered to be significant.


   Results Top


As the epithelial dysplasia grade increased, a significant sequential increase in the bcl-2 expression was observed which was lowest in mild dysplasia, followed by a gradual increase in moderate dysplasia and maximum in severe dysplasia (p < 0.05) [Figure 1]. An increase in differentiating grades of squamous cell carcinoma resulted in a decrease in bcl-2 expression which was statistically significant except for two cases with an absence of bcl-2 expression (p < 0.05) [Table 1].
Figure 1: - (a) Mild epithelial dysplasia showing bcl-2 nuclear and cytoplasmic staining in the lower one-third of the epithelium (×40). (b) Moderate epithelial dysplasia showing nuclear and cytoplasmic bcl-2 staining until two-thirds of the epithelium (×40). (c) Severe epithelial dysplasia showing nuclear and cytoplasmic staining of bcl-2 throughout the epithelium with less immunopositivity in the superficial layer (×40)

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Table 1: Comparative quantitative assessment of bcl-2 expression in varying grades of oral epithelial dysplasia and squamous cell carcinoma

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The qualitative interpretation of the bcl-2 expression in mild, moderate, and severe dysplasia was non-significant (p > 0.05) [Table 2]A. Similarly, the difference in bcl-2 intensity expression in well-differentiated squamous cell carcinoma (WDSCC) moderately differentiated squamous cell carcinoma (MDSCC), and poorly differentiated squamous cell carcinoma (PDSCC) was non-significant (p > 0.05) [Table 2]B.


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When bcl-2 expression was assessed with the histological parameter of mitosis in both epithelial dysplasia and squamous cell carcinoma (SCC) cases, it did not show any significant correlation. Out of eight cases of WDSCC, three showed the presence of mitosis while in five cases it was absent. Thus, the resultant P value was non-significant (p > 0.05). The resultant P value was significant in MDSCC as out of eight cases, seven cases had the presence of mitosis while one was negative. Similarly, all eight cases of poorly differentiated squamous cell carcinoma showed the presence of mitosis and the P value for this grade was statistically significant (p < 0.05). Thus, mitotic activity was maximum in poorly differentiated type followed by moderately differentiated squamous cell carcinoma and least in well-differentiated carcinoma. Out of eight cases of mild dysplasia, four showed the presence of mitosis while in five cases it was absent, whereas in eight cases of moderate dysplasia, four cases had the presence of mitosis while the rest four were negative for it. Similarly, eight cases of severe dysplasia resulted in six cases being positive for mitosis, and two cases showed its absence. The resultant P value in all the three grades of dysplasia was not statistically significant (p > 0.05) [Table 3].
Table 3: Quantitative assessment of the presence of mitosis in different grades of epithelial dysplasia and squamous cell carcinoma

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Out of eight cases, each of mild and moderate dysplasia, individual cell keratinisation, was not observed, whereas out of eight cases of severe dysplasia, only three cases displayed individual cell keratinisation. Thus, the resultant value was statistically non-significant (p > 0.05). Out of eight WDSCC cases, all cases showed the presence of keratin pearls, whereas in eight cases of MDSCC, only six cases had the presence of keratin pearls while eight cases of PDSCC did not show any evidence of keratin pearls. The resultant P value was highly significant (p < 0.05) [Table 4]. The evaluation of the bcl-2 expression in different locations in epithelial dysplasia showed a significant sequential increase in bcl-2 expression from the basal layer to the superficial layer [Table 5]A, whereas, in squamous cell carcinoma, entire islands and peripheral island cells having central keratinisation core showed bcl-2 expression in well and moderately differentiated squamous cell carcinoma [Figure 2]a, [Figure 2]b and PDSCC showed bcl-2 expression throughout the sheet pattern [Figure 2]c. Two cases of WDSCC showed negative bcl-2 expression which was non-significant [Table 5]B. In the present study, the mean bcl-2 count in epithelial dysplasia was comparatively higher (mean-736.42) when compared with OSCC (mean-703.2) [Table 6].
Figure 2: - (a) Tumour islands of WDSCC showing bcl-2 expression at the periphery and diminished expression at the center (×40). b) Tumour islands of MDSCC showing cytoplasmic and nuclear expression of bcl-2 (×40). (c) PDSCC showing sheets of tumour cells having bcl-2 positivity in both cytoplasm and nucleus (×40) [Table 3]: Quantitative assessment of the presence of mitosis in different grades of epithelial dysplasia and squamous cell carcinoma

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Table 4: Quantitative assessment of individual cell keratinization in all grades of epithelial dysplasia and presence of keratin pearls in all grades of squamous cell carcinoma

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Table 6: Quantitative assessment of total bcl-2 expression in epithelial dysplasia and squamous cell carcinoma

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   Discussion Top


In our study, it was observed that as the grade of epithelial dysplasia increased, there was a gradual increase in bcl-2 expression which was significant (p < 0.05-[Table 1]). These findings were also corroborated by Pallavi et al., Singh et al., and Juneja et al. in their respective studies.[9],[10],[11] Similarly, sequential grades of SCC demonstrated an increased expression of bcl-2 which was again significant [Table 1] and was consistent with the findings of Singh et al., Chen et al., and Suri et al.[10],[12],[13] Hence, this concept could be put forth that the bcl-2 oncoprotein expression is inversely proportional to the degree of keratinisation/differentiation as suggested by Jordan et al.[14] and Nitya K et al.[15] that this up-regulation allows a selective growth advantage to the dysplastic cells by prolonging cell survival, thus leading to malignancy. This result is contradictory to the findings by Loro et al.[16] who observed that bcl-2 expression was suppressed in PDSCC and stated that this difference might be topographical or because of different laboratory protocols and antibodies used in various studies.

The qualitative bcl-2 expression evaluation in epithelial dysplasia showed that 16 cases were intensely stained while eight cases stained mildly [Table 2]A. It was evident that an increase in epithelial dysplasia grades resulted in an escalation of bcl-2 staining intensity, thus indicating that this oncoprotein may play a key role in relative early events in the development and progression of oral neoplasia.[10]

Qualitative assessment of all squamous cell carcinoma cases revealed that as the degree of differentiation decreased, there was an increase in the staining intensity except for two cases of well-differentiated squamous cell carcinoma, which did not take up the stain [Table 2]B. The bcl-2 expression in the majority of moderately and poorly differentiated squamous cell carcinoma cases was intense which was confirmed by Nair et al.[17] that this upregulation of bcl-2 may reflect the lost ability of malignant keratinocytes for terminal differentiation and suggested that those cells overexpressing bcl-2 have a stem cell phenotype. The finding of negative bcl-2 staining has also been substantiated by Kannan et al.[18] who demonstrated an inverse correlation between p53 and bcl-2 expression in oral squamous cell carcinoma, suggesting that one of these proteins could substitute the other during carcinogenesis and that the p53 gene can downregulate the bcl-2 gene during the apoptotic process.

We also observed that when epithelial dysplasia was correlated with the presence of mitosis in both basal and suprabasal layers, a greater number of mitotic figures were seen as the grade of dysplasia increased from mild to severe, which was significant (p < 0.05-[Table 3]). It was suggested that loss of bcl-2 in oral keratinocytes may be involved in dysplastic and malignant progression of oral epithelium by making oral keratinocytes more responsive to mitotic stimuli.[19] In squamous cell carcinoma cases, fewer abnormal mitoses were evident as the grade of differentiation increased [Table 3]. Another observation in our study was, as the number of mitoses increased due to the progression of the disease, that there was a simultaneous increase in the intensity of bcl-2 expression. The significant increase in numbers of mitoses from normal to dysplasia to carcinomas possibly represents increased stem cell turnover, thus leading to early invasive tumour development.[4],[20]

Out of the many dysplastic features, one essential component for malignant transformation is the presence of individual cell keratinisation.[9],[21] As the disease progressed from mild to moderate dysplasia, individual cell keratinisation was absent except in three cases of individual cell keratinisation evident in severe dysplasia [Table 4], which suggested that as the dysplastic grade increases, it leads to increased bcl-2 expression, thus enhancing its further malignant transformation potential. It was also evident that as the degree of differentiation in squamous cell carcinoma increased, there was a simultaneous increase in the number of keratin pearls which was significant [Table 4].

Our findings suggest that as the grade of dysplasia increased, it resulted in a sequential increase in the level of bcl-2 expression from the lowermost basal layer up till the superficial layer which was significant [Table 5]A. The expression of bcl-2 in superficial layers proposed a modulation of its expression due to the binding of bcl-2 to other cellular proteins as the bcl-2 expression is only confined to the basal cell layer.[13] The present study noted that the entire islands devoid of central keratinised core showed bcl-2 positivity, whereas the tumour islands having central keratinisation stained only in the peripheral cells with very less or no immunopositivity in the centre. Cases of poorly differentiated squamous cell carcinoma showed immunopositivity throughout in the pattern of sheets [Table 5]B. Similar results were found by Suri et al.[13] and Sudha et al.[22] that this overexpression possibly reflected the resistance of these tumour cells to apoptosis and the loss of ability of malignant keratinocytes to differentiate terminally. In contrast to our study, Teni et al.[23] reported that in well and moderately differentiated cases, bcl-2 staining was restricted to the cells within the centre of tumour islands.

In our study, it was reflected that the mean bcl-2 expression was higher in epithelial dysplasia than in squamous cell carcinoma [Table 6]. Pallavi et al.[9] showed similar findings that bcl-2 immunoreactivity was more in epithelial dysplasia and had diminished activity in squamous cell carcinoma cases. Positive bcl-2 expression was also perceived in the endothelial cells lining the blood vessels that might be due to increased production of interleukin-8 (IL-8) resulting in enhanced trans-membrane endothelial cell permeability, thus promoting tumour angiogenesis.[24] Our study revealed that the expression of bcl-2 was directly proportional to the degree of dysplasia, whereas an inverse correlation was found between bcl-2 positivity and degree of differentiation in cases of squamous cell carcinoma, thus probably contemplating that bcl-2 may play an important role in early events of tumour progression. In previous studies, only quantitative and qualitative analysis of bcl-2 has been done in epithelial dysplasia and squamous cell carcinoma cases, but this is the first study that has attempted to correlate the histopathological parameters along with quantitative and qualitative analysis of bcl-2 in varying grades of both the lesions.


   Conclusion Top


Additional follow-up studies are the need of the hour to extract the intricate molecular mechanisms substantiated with other molecular markers in predicting the sequential progression from premalignancy to malignancy which might be instrumental in advocating the use of bcl-2 judiciously and could further pinpoint and emphasize the role of bcl-2 in the pathogenesis and progression model of oral squamous cell carcinoma.

Acknowledgements

We acknowledge I.T.S Dental College, Oral Pathology Department, and various staff members who helped in conducting this original study on the role of bcl-2 in varying grades of epithelial dysplasia and squamous cell carcinoma.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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