Introduction

Cancer is a term used for identifying a large number of diseases. Perhaps the unique feature of these diseases is the ability for uncontrolled cell proliferation that cannot be checked by normal cell kinetics regulators. A normal cell suddenly turns into a rogue cell and starts dividing continuously without check, leading to the development of solid lumps (tumors) or an abnormal mass with the rise in the number of dispersed cells.[1]

Various factors are responsible for the development of cancer. They are classified into exogenous and endogenous agents. The first group includes nutritional habits, socio-economic status, lifestyle, physical agents (ionizing and non-ionizing radiation), chemical compounds (natural and synthetic), and biological agents (Helicobacter pylori, Epstein Barr virus, human T lymphotropic viruses I and II, human papilloma virus and the hepatitis B virus). Unhealthy lifestyle habits such as excess alcohol consumption; inhalation of tobacco and related products; the ingestion of certain foods and their contamination by mycotoxins are responsible for higher incidences of certain types of neoplasia in several population groups. [2]

Oral squamous cell carcinoma has achieved the status of a disease with a high mortality rate and a high cell turnover which marks the rates of proliferation and a high apoptotic activity. Defective apoptosis plays a major role in the progression of the oral cancer. Recent studies have encouraged some authors to suggest that the underlying etiology and progression of the disease can be reduced for two reasons, mutations that give rise to excessive proliferation and a compensatory disruption of survival signaling pathways that ensures the survival of these hyperproliferative cells. Hence the theory forges the fundamental link between neoplasia and apoptosis.[3]

The Bcl-2 oncogene was first identified as the site of reciprocal translocation of chromosome 18 in follicular lymphoma and encodes a membrane-associated protein present in the endoplasmic reticulum, nuclear, and outer mitochondrial membranes.[4]

Mitochondria play an important role in apoptotic cell death by releasing key effector proteins, including cytochrome c from the mitochondrial intermembrane space (IMS). IMS protein release can result from the remarkable event of mitochondrial outer membrane permeabilization (MOMP), which is thought to be regulated by proteins of the Bcl-2 family. The increase in expression of bcl-2 is not only essential for oral carcinogenesis but influences the progression of the disease. Because it increases the survival rate of neoplastic cells, allowing new genetic mutations to occur and granting their higher resistance to chemotherapy and radiotherapy.[5] Hence the current study was undertaken to evaluate the expression of bcl-2 in various histological grades of OSCC.

Materials and Methods:

The study was done to quantitatively assess the number of bcl2 in OSCC after approval from the institutional ethical committee of Sardar Patel Dental College.

The study comprised 35 patients, who were divided into two groups, Group I contained 30 cases of all grades of OSCC, in which histologically oral squamous cell carcinoma was proven, and Group II consisted of 5 cases of normal mucosa.

The biopsy tissues for Control Group II were retrieved from the department's archival samples. The sample for study group I was collected from the formalin-fixed and paraffin-embedded blocks of the histologically proven OSCC.

Formalin-fixed paraffin-embedded tissues were sectioned at 4μm and3 μm; 4μm tissue sections were subjected to haematoxylin and eosin staining, 3μm sections were stained immunohistochemically with anti-human bcl-2 antibody (Biogenex, USA), on the poly-L-lysine coated slides.

Haematoxylin and eosin-stained tissue sections were first examined to confirm the histological diagnosis and representative areas showing the pathological changes of interest were selected using low power magnification(X10).

The immunohistochemically stained sections were first analyzed for the expression of bcl-2 at low power (X10). The selected fields were used for the quantitative assessment of the bcl-2 positive cells. At least 500 tumor cells were observed and counted in 5 different histological fields using magnification of X40. An eyepiece grid was used to prevent the overlapping of fields.

Fig 1: Well differentiated squamous cell carcinoma.	Fig 2: Bcl2 staining in well differentiated carcinoma
Fig 3: Moderately differentiated squamous cell carcinoma	Fig 4: Bcl2 staining pattern in moderately differentiated squamous carcinoma
Fig 5: Poorly differentiated squamous cell carcinoma	Fig 6: Bcl2 staining pattern in poorly differentiated squamous cell carcinoma

The intensity of staining was graded as light /dark by comparing it with the internal positive control, which was lymphocytes. The pattern of staining was considered as periphery when the expression was seen only in the peripheral cells of the islands, while it was graded as entire when all the tumor cells within the islands expressed bcl-2 reactivity.

The numbers of positively stained and non-stained cells were recorded separately. Bcl-2 overexpression was considered for those cases that demonstrated ≥ 20% positively stained nuclei of tumor cells. A negative result was defined as the absence of stained nuclei or immunoexpression under 20 % of tumor cells. The bcl-2 indices were calculated as the percentage of positively stained cells among the total cells counted. The results were tabulated and subjected to statistical analysis.

Results:

Morphology Parameters in OSCC (Table 1)

Degree of Keratinisation and Nuclear Polymorphism are prominent in 40% of the cases, indicating a significant variation in the differentiation of cells. The number of Mitoses HPF shows that a high mitotic index (36.6%) is present, suggesting rapid cell division, which is often associated with aggressive tumor behavior. Pattern of invasion suggests that 46.6% of the cases have higher stages of invasion, indicating a more aggressive and invasive tumor. Lymphoplasmacytic Infiltration was noted in 20% of the cases, reflecting the body's immune response to the tumor.

Bcl-2 Expression Reliability (Table 2,3)

The t-test and Pearson correlation analysis show high reliability between Observer 1 and Observer 2's cell counts, indicating consistent measurement of Bcl-2 expression across different histological grades.

Bcl-2 Expression in Varying Histological Grades(Table 4)

Mean Bcl-2 expressions show an increase in the number of cells from well-differentiated (WD) to poorly differentiated (PD) grades. This suggests that higher Bcl-2 expression correlates with poorer differentiation and potentially more aggressive tumor behavior.

Bcl-2 Staining Location Frequency (Table 5)

Staining Locations show that the entire island staining is more prevalent in well and moderately differentiated grades, whereas poorly differentiated grades predominantly show staining in sheets. This might indicate a change in the spatial distribution of Bcl-2 expression with the progression of tumor grade.

Discussion

Apoptosis occurs in several pathological situations in multicellular organisms and constitutes part of a common mechanism of cell replacement, tissue remodelling, and the removal of damaged cells. Bcl2 is the most common oncoprotein investigated for apoptosis. Bcl-2 represents the founding member of the new and growing class of cell death-inhibiting oncoproteins. Ensuing analysis of experimental retrovirus-mediated Bcl-2 overexpression in mouse bone marrow cells indicated, however, that Bcl-2 overexpression on its own would not suffice for tumorigenesis. Rather, high levels of the Bcl-2 protein may ensure the persistence of a cell clone with the overactive bcl-2 gene until further tumorigenic mutations come into effect.[5]

In our study, bcl-2 expression was detected in 100 % of cases of Oral squamous cell carcinoma. The epithelial and the connective tissue components were analyzed to find out the pattern of invasion, the number of mitoses, nuclear and cellular pleomorphism, lymphoplasmacytosis, and the degree of keratinization as followed by Anneroth’s et al (1987) multifactorial grading system.[6,7]

We found a higher nuclear (40%) and cellular pleomorphism (50%) in the 30 cases of OSCC. The no of mitoses (4- 5) per high power field was about 43.3%. Twelve cases showed high keratinisation (40%) whereas 3 cases showed no keratinisation (10%).

All the 30 cases of oral squamous cell carcinoma showed positive bcl2 immunostaining and none were negative. The immunohistochemical staining of the tumor cells showed granular cytoplasm and the nuclear envelope staining was positive. The sites of expression (periphery/entire island) in tumor islands/sheets were noted and the intensity of the expression of bcl-2 was also graded as light or dark when compared to the internal positive control (Lymphocytes)

The overall positivity was expressed as between 7% and 60% in different studies. Jordan et al (1996) reported a positivity of 60%.[8] Ravi et al (1999) found 87% positivity in their study on OSCC.[9] Teni T, Pawar S, Sanghvi V, and Saranath D (2002) have mentioned that the overall positivity ranges from 60%[10] whereas Suri C (2009), Koshy V (2012) has found that the overall positivity of bcl-2 immunoreactivity in OSCC was 100%.[11] Solomon MC, Carnelio S, Gudattu V (2010) – Reported 63% positivity for bcl-2. Our study has reported a 100% positivity of bcl-2 immunoreactivity.[12]

On the contrary, a few cases have reported all the cases to be negative for bcl2 expression. Leyva-Huerta ER, Ledesma-Montes C, Rojo-Botello RE, and Vega-Memije E (2012) have reported 0% immunoreactivity of bcl2 in OSCC[13] and S Nafarzadeh (2013) expressed a mild positivity of Bcl-2 in OSCC.[14]

Lee et al., Thongsuksai et al., and Ito et al. indicated in their study that Bcl-2 expression is not related to aggressiveness and prognosis in head and neck cancers.[15-17] Other studies contradict the results where the positive or negative expression of Bcl-2 was considered an important prognostic factor [18-21] This disparity in bcl-2 expression in OSCC in different studies may reflect subtle inherent differences in upstream genetic events among different populations, gender and age of the patients, anatomical location of the studies lesions, and possibly differences in the environmental, hygienic and dietary conditions. [22]

Ravi et al (2009) in their study on squamous cell carcinoma found the expression of bcl-2 in the form of granular cytoplasmic staining with an accentuation around the nuclear membrane. This is due to the cellular localization of bcl- 2 in the outer membrane of the mitochondria, endoplasmic reticulum, and the nuclear membrane. In our study, the staining was observed more in moderate and poorly differentiated squamous cell carcinoma.[9]

Suri C (2009) found the expression of the staining pattern of the oncoprotein bcl-2 was more in poorly differentiated SCC than in well-differentiated SCC. The staining was noted in the cytoplasm and the periphery of the nuclear membrane. The moderate and poorly differentiated OSCCs showed a prominent pattern of staining. [11,23]

In a study conducted by Namita Bhutani, Pooja Poswal, Shilpi Moga, and Sunil Arora bcl-2 scoring was done taking into consideration the number (percentage) of positive cells in the tumor parenchyma. No correlation was seen between the expression of bcl-2 and the grade of the tumor but 89% of the cases revealed bcl-2 expression in tumor cells. They state that the role of bcl-2 immunoexpression has been extensively studied; however, the thresholds for staining intensity and distribution are not consistently standardized, and various authors have applied different criteria for the same.[22]

In our study, we also noticed the expression of bcl2 oncoprotein in the endothelial cells near the tumor islands. This is attributed to the induction of VEGF in the endothelial cells. Kaneko et al also found that Bcl-2 is the orchestrator of a cross-talk between neovascular endothelial cells and tumor cells, which has a direct effect on tumor growth. Bcl-2 induces vascular endothelial growth factor (VEGF) expression in neovascular endothelial cells through a signal transducer and activator of transcription 3 (STAT3)-mediated pathway.[24]

Conclusion

On the basis of our study, we conclude that the anti-apoptotic protein bcl2 is expressed more in moderately to poorly differentiated oral squamous cell carcinomas. The results emphasize the role of apoptosis in oral carcinogenesis. The more the expression in poorly differentiated carcinomas the worse the prognosis. Further research is still needed with larger study groups and by comparing it with more recent techniques like mRNA analysis of bcl2.

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