Oral squamous cell carcinoma (OSCC) is the most prevalent oral malignancy and a significant oral health problem worldwide [1,2]. It is the most common cause of oral cancer, and it has been linked to high morbidity/mortality. The clinical pattern of OSCC is highly variable in patients and is affected by the size of the tumor, its histological grade, depth of invasion, lymph node involvement, status of surgical margins and other pathological characteristics [3,4]. Surgical resection is the main approach to the treatment of localized disease, but recurrence and progression can occur despite apparently adequate treatment. These differences in the behaviour of these tumors may not be explained by conventional clinicopathological parameters, and there is growing interest in molecular and immunohistochemical markers of tumor progression [5].

Changes in cellular proliferation, DNA damage response, genomic stability and programmed cell death are all involved in the development and progression of OSCC [6]. P53 is a significant tumor suppressor protein, which plays a role in cell-cycle regulation, DNA repair and apoptosis [7]. Abnormal p53 expression, which is often due to changes in the TP53 pathway, has been reported frequently in OSCC and may be related to loss of control of cellular growth and survival [8,9]. The measurement of p53 levels by immunohistochemistry may provide clues to the distribution and accumulation of p53 in the malignant cell and may be associated with poor pathological features [10].

B-cell lymphoma-2 (Bcl-2) is a key regulator of apoptosis, which inhibits programmed cell death thus allowing for cell survival [11]. Overexpression of Bcl-2 could promote the resistance of the malignant cells to apoptosis and be involved in tumor persistence and progression [12]. Anti-apoptotic mechanisms involving Bcl-2 and tumor-suppressive pathways involving p53 may play a role in the biological behavior of OSCC [13].

Although a large body of work has shed light on the role of p53 and Bcl-2 in oral pathologies, their expression patterns in oral cancers and their association with surgical outcomes after treatment have not been reliably determined. Their relationship with recurrence, lymph node involvement, margin status and other factors that influence the surgery prognosis has been limited and inconsistent, especially at the local level.

Research Objective

To determine the immunohistochemical expression of p53 and Bcl-2 in oral squamous cell carcinoma and to assess their correlation with surgical prognosis.

Study Design and Setting To determine the immunohistochemical expression of p53 and Bcl-2 in histologically confirmed oral squamous cell carcinoma (OSCC) and to correlate it with predefined clinicopathological and surgical prognostic parameters, an analytical cross-sectional study was conducted in a hospital setting. The study was carried out in the Islamabad Medical Laboratory (PIMS), Islamabad, a tertiary care teaching hospital which offers specialized diagnostic, histopathological, and surgical services. Any patient who presented for surgical intervention for histologically proven OSCC to the institute was considered for recruitment. The study period was 1 year (from March 25 to December 25). Study Population Patients of OSCC who were histologically confirmed and underwent surgical interventions in the study period at PIMS, Islamabad, were included in the study population. Study population was selected to obtain representative tumour tissue for immunohistochemical evaluation and clinicopathological information relevant to the assessment of the surgical prognosis. Sample Size and Sampling Technique A total of 140 patients with histologically confirmed OSCC were selected for the study. Considering the study protocol, the sample size of 140 was adequate to evaluate p53 and Bcl-2 expression and its relationship to selected clinicopathological and surgical prognostic parameters. The participants were recruited by the use of consecutive sampling. All patients that met the inclusion criteria defined a priori were eligible for inclusion until a sample size of 140 patients was reached. Inclusion and Exclusion Criteria Patients of histologically confirmed oral squamous cell carcinoma who were treated surgically at PIMS, Islamabad with sufficient tumor specimen for histopathological and immunohistochemical analysis were included. Patients with adequate clinical, surgical, and pathological data necessary for analysis of the chosen prognostic factors were also included. Inadequate or unsuitable tissue for an immunohistochemical evaluation, other histopathologic diagnosis than OSCC, extensive necrosis and poor tissue preservation, which made it impossible to obtain reliable immunohistochemical interpretation, or lack of essential clinical, surgical, and pathological data necessary for the study. Histopathological Examination Tumor tissue was collected in surgery and analyzed by conventional histological methods, sampling a representative piece from each tumor. Tissues sections were cut and stained with hematoxylin and eosin for confirmation of oral squamous cell carcinoma and for evaluation of relevant histopathological features. Tumor differentiation, tumor size, depth of invasion (if present), involvement of the lymph nodes, lymphovascular invasion, perineural invasion, pathological tumor stage and other relevant histopathological findings were noted from the pathology reports and, if necessary, confirmed by examination of the tissue sections. The final histopathological evaluation was used to determine the surgical margin status, which was described as involved or uninvolved. Immunohistochemical Assessment of p53 and Bcl-2 Formalin fixed paraffin embedded tumor tissue sections were used for immunohistochemical evaluation for the expression of p53 and Bcl-2. For this, appropriate antigen retrieval and immunostaining procedures were carried out using appropriate validated antibodies for p53 and Bcl-2, with suitable positive and negative controls. Immunohistochemical expression was determined by measuring the percentage of tumor cells staining positive for the tested antigen and by measuring the staining intensity in a uniform manner for all samples using a pre-established scoring system. The resulting immunoreactivity scores were then noted and later divided into pre-established immunoreactivity categories for statistical analysis. To minimize the variability of measurements, the same criteria were used to evaluate the p53 and Bcl-2 in all the specimens studied. Assessment of Surgical Prognostic Parameters The expression of p53 and Bcl-2 were correlated with the pre-defined prognostic parameters of surgery and clinic. They were: tumour size, histological differentiation, depth of invasion (if present), lymph-node involvement, pathological tumour stage, lymphovascular invasion, perineural invasion and surgical margin status. The parameters were chosen for their well-established pathological and surgical prognostic and aggressive characteristics of OSCC. The most important analyses of prognosis included lymph-node involvement, pathological stage, tumor differentiation, and surgical margin status. Study Variables The immunohistochemical levels of p53 and Bcl-2 were selected as the independent variables. Predefined surgical and clinicopathological prognostic parameters, such as lymph-node involvement, pathological tumor stage, surgical margin status, tumor differentiation and other relevant pathological parameters were used as dependent variables. Demographic and clinicopathological characteristics, namely age, sex, tumour site and tumour characteristics, were documented as potential covariates and confounding variables as appropriate. Data Collection Procedure Structured data collection form was used to collect relevant demographic, clinical, surgical, histopathological and immunohistochemical data. A systematic review of the patient records, surgical findings, histopathological and immunohistochemical results were carried out and the appropriate data were entered into the study database. Consistency between the results of immunohistochemistry and the histopathological and surgical records was paid special attention. All respondents had been given a study identification number to ensure anonymity and not to use directly identifiable data while analyzing the data. Statistical Analysis The data were inputted, coded and analyzed using SPSS software. Approximately normally distributed data was summarized using mean and standard deviation, whereas the non-normally distributed data was summarized using median and interquartile ranges; categorical variables were summarized using frequencies and percentages. Continuous variables were assessed for distribution using Shapiro–Wilk test and graphical methods. When frequencies in the cells were small, the correlation between p53 expression with Bcl-2 expression and categorical clinicopathological or prognostic parameters was evaluated by chi-square test. The independent-samples t-test was used for continuous variables for which the following assumptions were met: both variables had normal distributions and the sample sizes were comparable. The Mann–Whitney U test was utilized for continuous variables when the following assumptions were not met: both variables were not normally distributed and sample sizes were not similar. One way analysis of variance (ANOVA) or the Kruskal–Wallis test was used, depending on the distribution and assumptions of the data, for comparisons involving more than two groups. Where appropriate, binary logistic regression analysis was carried out to assess the independent association between p53 and Bcl-2 expression and pre-established adverse prognostic parameters after controlling for relevant clinicopathological and demographic variables. Odds ratios with 95% confidence intervals were reported. A 2-sided p-value of <0.05 was deemed statistically significant.

The study included 140 patients with OSCC (table 1). The majority were male (87, 62.14%), while 53 (37.86%) were female. The largest age group was 51–60 years (39, 27.86%). The tongue was the most frequently involved anatomical site (43, 30.71%), followed by the buccal mucosa (31, 22.14%). Tumors measuring >2–4 cm were most common (61, 43.57%).

Table 1. Baseline demographic and clinical characteristics

Histopathological examination showed that 61 (43.57%) tumors were well differentiated, 56 (40.00%) were moderately differentiated, and 23 (16.43%) were poorly differentiated (table 2). Depth of invasion exceeded 10 mm in 53 (37.86%) cases. Lymphovascular invasion was present in 39 (27.86%) patients, while perineural invasion was identified in 32 (22.86%).

Table 2. Histopathological characteristics

High/positive p53 expression was observed in 82 (58.57%) patients, whereas 58 (41.43%) showed low/negative expression (table 3). For Bcl-2, 64 (45.71%) patients demonstrated high/positive expression and 76 (54.29%) showed low/negative expression.

Table 3. p53 and Bcl-2 immunohistochemical expression

High p53 expression was significantly associated with larger tumor size, poorer differentiation, lymph-node involvement, lymphovascular invasion, and perineural invasion (table 4). The strongest association was observed with lymph-node involvement (p<0.001), suggesting that increased p53 expression was associated with more aggressive clinicopathological features.

Table 4. p53 expression versus clinicopathological parameters

High Bcl-2 expression was significantly associated with larger tumor size, poorer differentiation, lymph-node involvement, lymphovascular invasion, and perineural invasion (table 5). The association was strongest for tumor size (p<0.001), with high Bcl-2 expression occurring more frequently among patients with tumors >4 cm.

Table 5. Bcl-2 expression versus clinicopathological parameters

Both markers demonstrated significant associations with pathological stage (table 6). High p53 expression was observed in 36.59% of stage IV cases compared with 12.20% of patients with high expression in stage I. Similarly, high Bcl-2 expression increased from 9.38% in stage I to 39.06% in stage IV, indicating an association between increased marker expression and advanced pathological stage.

Table 6. p53/Bcl-2 expression versus pathological stage

High p53 expression was significantly associated with positive surgical margins (p=0.019) and lymph-node involvement (p<0.001), shown in table 7. Bcl-2 expression was significantly associated with lymph-node involvement (p=0.039), whereas its association with surgical margin status was not statistically significant (p=0.550). These findings suggest a stronger relationship between p53 expression and selected surgical prognostic parameters.

Table 7. p53/Bcl-2 expression versus surgical margin and lymph-node status

In multivariable analysis, high p53 expression remained independently associated with adverse prognostic features (adjusted OR 3.18, 95% CI 1.61–6.29; p=0.001), while high Bcl-2 expression also demonstrated an independent association (adjusted OR 2.07, 95% CI 1.08–3.98; p=0.028), shown in table 8. Tumor size >4 cm and poor differentiation were additional independent predictors, whereas age and sex were not statistically significant.

Table 8. Multivariable logistic regression of p53/Bcl-2 and adverse prognostic parameters

In the present study, 140 patients with OSCC were evaluated, with males comprising 62.14% (87/140) and females 37.86% (53/140). The most common age group was 51-60 years (27.86%) and the most common site of tumor was the tongue (30.71%) and buccal mucosa (22.14%). The largest size category was the Tumors >2–4 cm (43.57%). This distribution is similar to that seen in OSCC, but inter-population variation may be due to variations in tobacco use, lifestyle, referral patterns and distribution of tumor subsites. This is especially noteworthy because the present patient population consisted almost exclusively of clinically larger tumors, a known risk factor for poor prognosis in OSCC [14]. Pathologically, 43.57% of the tumors were well differentiated, 40.00% moderately differentiated and 16.43% poorly differentiated. Pathologically, 43.57% of the tumors were well differentiated, 40.00% moderately differentiated and 16.43% poorly differentiated. LVI was present in 27.86%, PNI in 22.86%, and depth of invasion >10 mm was present in 37.86% of the cases. The results show that although the proportion of well-differentiated tumors is relatively high, there is a significant proportion of pathological features of adverse prognosis. In recent years, there is a growing body of evidence that depth of invasion, lymph-node involvement, and other pathological characteristics are also important in the prognosis of OSCC. Depth of invasion >5 mm was strongly associated with mortality and disease-free survival in a recent systematic review and meta-analysis, which is consistent with the clinical significance of adverse pathological parameters seen in our patient group [15]. The p53 expression was high (positive) in 82 patients (58.57%) and low (negative) in 58 (41.43%). Importantly, an association between high p53 expression and the following was statistically significant: larger tumour size (p<0.001), poorer differentiation (p=0.041), lymph-node involvement (p<0.001), LVI (p=0.021), and PNI (p=0.015). These results are in line with recent studies showing the association between TP53 expression and pathological features of poor prognosis. For instance, Giri et al. (2023) have reported that 45.83% of 48 surgically treated OSCC patients were immunopositive for TP53 and that there was a significant correlation between TP53 expression and surgical margin status (p=0.002), while TP53 expression was more common when LVI was present in tumors [16]. Likewise, Monteiro et al. (2018) identified the relationship between high expression of p53 and a poor cancer-specific survival rate alongside with tumor size, clinical stage, lymph node metastasis and PNI [17]. In the present study we also found that p53 expression gradually increased as the pathological stage progressed (12.20% in stage I and 36.59% in stage IV; (p<0.001). It was also found to be associated with positive surgical margins (31.71% vs. 13.79%, p=0.019) and positive lymph nodes (53.66% vs. 22.41%, p<0.001) in the p53-positive and p53-negative/low-expression groups, respectively. The results support the hypothesis that the accumulation of p53 may represent biologically aggressive OSCC. Liu et al. (2021) conducted a systematic review and meta-analysis which concluded that p53-positive OSCC had a poor 5-year overall survival compared to p53-negative disease and that there was no pooled association between p53 expression and metastasis [18]. Taken together, the finding of a strong nodal association in our study lends credence to the notion that p53 could be used as a prognostic marker, but also shows the importance of considering p53 results in combination with traditional pathological parameters. Sixty-four (45.71%) were high/positive and 76 (54.29%) were low/negative for Bcl-2. Tumor size (p<0.001), differentiation (p=0.032), lymph-node involvement (p=0.039), LVI (p=0.016) and PNI (p=0.026) were significantly associated with high Bcl-2 expression. Moreover, the Bcl-2 expression was raised during the tumor stage from 9.38% at stage I to 39.06% at stage IV (p<0.001). Such results are consistent with the hypothesis that disturbances in the apoptotic process are associated with malignant tumor attributes. The literature is not, however, consistent. In OSCCs, Pallavi et al. (2018) reported that Bcl-2 was positive in 37% of their specimens and that Bcl-2 was found to be variable and decreased in advanced stages of tumour progression [19, 21]. On the other hand, Coutinho-Camillo et al. (2010) found associations between Bcl-2-family proteins and clinicopathological features in 229 OSCC cases, which further underscores the involvement of apoptotic dysregulation in OSCC biology [20, 22]. Lastly, multivariable analysis revealed that high p53 expression was independently associated with unfavorable prognostic factors (adjusted OR=3.18, 95% CI: 1.61–6.29; p=0.001), whereas high Bcl-2 expression was also independently associated (adjusted OR=2.07, 95% CI: 1.08–3.98; p=0.028). Tumor size >4 cm (adjusted OR=2.84, 95% CI: 1.37–5.89; p=0.005) and poor differentiation (adjusted OR=2.61, 95% CI: 1.08–6.32; p=0.034) were additional independent predictors, whereas age and sex were not significant. In general, these results indicate that p53 is more strongly independently associated with poor surgical prognosis, whereas Bcl-2 is more strongly independently associated with additional prognosis information. The findings are similar to those reported in the immunohistochemistry-based studies that included p53, which suggested that p53 is a valuable tool in the identification of high-risk OSCC, with some also highlighting the heterogeneity of p53 evaluation and interpretation in the published literature [17]. Thus, it is justified to standardize the scoring system and to validate it using prospective data prior to using p53 and Bcl-2 routinely in surgical risk stratification. Study Strengths and Limitations An important advantage of this study is the determination of both p53 and Bcl-2 immunohistochemical expressions in a fairly homogeneous group of patients who already have histological diagnosis of OSCC and the determination of its expressions against various known clinicopathological and surgical prognostic parameters. The application of standardized histopathological grading, immunohistochemical analysis and multivariable logistic regression added power to the analysis and enabled studying the independent contribution of the biomarkers to adverse prognostic features, taking important clinical and pathological factors into account. However, there are a number of caveats to keep in mind. The hospital-based cross-sectional design makes it difficult to make temporal or cause-and-effect inferences about the relationship between biomarker expression and clinical outcomes. This study took place in a single tertiary care center and may not be generalizable to other populations and health care facilities. Furthermore, the immunohistochemical expression may be affected by the antibody used for the stain, the staining technique, the scoring system, or the definition of expression positivity; therefore, comparisons with studies using different methodologies are difficult. The study also emphasized mostly pathological and surgical parameters of prognosis (rather than overall survival, disease-free survival or recurrence). Larger multicenter prospective trials with uniform immunohistochemical scoring and long-term follow-up are therefore warranted.

The results revealed that p53 and Bcl-2 immunohistochemical expression was related to various poor clinicopathological and surgical prognostic parameters in OSCC. The notably stronger independent association between p53 and survival suggests that it may be of particular importance in the context of the aggressive nature of the tumour type, while the association between Bcl-2 and survival might be complementary in the context of tumour progression and adverse pathological features. The relationships observed with tumor size, the differentiation grade, lymph-node involvement, pathological stage, lymphovascular invasion, perineural invasion, and some surgical parameters suggest the potential role of these biomarkers in prognostic assessment. However, they should be used as an additional parameter to existing histopathological and surgical criteria rather than as standalone parameters for prognosis. Additional multicenter prospective studies are needed to confirm their prognostic usefulness and to find out if there is a benefit for the combined use of p53/Bcl-2 evaluation in the prognosis and post-operative management of OSCC patients.