ANALYSIS OF IMMUNO HISTOCHEMICAL EXPRESSION OF Bcl2 ANDKi67 INGASTRIC CARCINOMA AND ITS ASSOCIATION WITH HISTOPATHOLOGICAL FEATURES - A CROSS-SECTIONAL STUDY

Gastric cancer remains a major global health problem and is the fifth most commonly diagnosed malignancy and the fourth leading cause of cancer-related mortality worldwide ^[1]. Most cases are diagnosed at an advanced stage due to late clinical presentation, limiting the availability of curative treatment options ^[1]. The five-year survival rate for advanced gastric cancer ranges from 5% to 20%, with a median overall survival of approximately one year ^[2]. The incidence increases with age, peaking between 60 and 80 years, and is rare below 30 years of age ^[2]. Gastric cancer shows a clear male predominance, with men being two to four times more likely to develop the disease than women ^[2]. Dietary factors and Helicobacter pylori infection are major risk factors for distal gastric cancers, while gastroesophageal reflux disease and obesity are more strongly associated with proximal tumors. In India, a higher incidence has been reported in the southern

and northeastern regions ^[2,3].

According to the World Health Organization and Lauren classification, gastric carcinoma is broadly divided into intestinal and diffuse types ^[4]. The intestinal type is more common in older men and is often associated with precursor lesions such as chronic atrophic gastritis and intestinal metaplasia, with H. pylori infection, dietary factors, and obesity playing significant roles ^[5]. This type generally carries a better prognosis than the diffuse variant.

The Bcl-2 family of proteins plays a crucial role in the regulation of apoptosis, comprising both pro-apoptotic and anti-apoptotic members. Dysregulated overexpression of anti-apoptotic Bcl-2 proteins contributes to tumor development and therapeutic resistance ^[6]. In the gastric mucosa, Bcl-2 expression is predominantly observed in proliferative zones, suggesting a role in cellular renewal. During gastric carcinogenesis, Bcl-2 expression has been documented in chronic atrophic gastritis with intestinal metaplasia and epithelial dysplasia, while being infrequent in normal gastric mucosa. Overexpression of Bcl-2 in gastric carcinoma has been associated with resistance to chemotherapy and radiotherapy [6].

Ki-67 is a nuclear protein expressed during the active phases of the cell cycle (G1, S, G2, and M phases) but absent in the resting (G0) phase, making it a reliable marker of cellular proliferation ^[7]. Increased Ki-67 expression has been correlated with aggressive tumor behavior and poor prognosis in several malignancies ^[7].

Evaluation of apoptotic and proliferative markers such as Bcl-2 and Ki-67 may provide valuable insights into tumor biology, aggressiveness, and prognosis in gastric carcinoma. This study aimed to analyze the expression patterns of Bcl-2 and Ki-67 in gastric carcinoma and correlate them with histopathological parameters ^[8].

Table 1.  Interpretation of Bcl2 expression

Ki67 :At least 1000 tumor cells (×400 magnification) from the most immune positive regions of each neoplasm would be visually counted, and the percentage of positive cells (labeling index, LI) will be calculated. Tumors were classified as Ki67-low for scores 0 and 1, and Ki67-high for scores 2 and 3 (Table 2) ^[9]

Table 2. Ki67 immunostaining score

By adhering to these standardized procedures and ethical guidelines, the study aimed to comprehensively analyze the expression patterns of Bcl2 and Ki67 in gastric carcinoma cases.

Statistical Analysis

Comparisons between groups of Ki67 expression levels and Bcl2 expression status were performed using the chi-square test (χ² test) or Fisher’s exact test for categorical variables.

For continuous variables (e.g., age), comparisons were conducted using the independent t-test for normally distributed data and the Mann-Whitney U test for non-normally distributed data.

Normality was assessed using the Shapiro-Wilk test.  

            A p-value < 0.05 was considered statistically significant.

In this study, 42 gastric carcinoma cases, including biopsy and resection specimens were evaluated according to AJCC and WHO guidelines, and subjected to Bcl2 and Ki67 immunohistochemical analysis. Most patients belonged to the 61–70-year age group, with a male predominance. Of the specimens, 19 were biopsies and 23 were gastrectomies, (69% were partial and 30.4% total gastrectomies). Adenocarcinoma NOS was the predominant subtype (54.7%), followed by poorly cohesive (28.5%), mixed (11.9%), and mucinous carcinoma (4.7%). Among the 23 adenocarcinoma NOS cases, 43.4% were Moderately differentiated, 30.4% Poorly differentiated, and 26% were Well differentiated. Lauren classification showed diffuse type in 52.3%, intestinal in 42.8%, and mixed in 4.7% (Table 3). Among resected specimens, T3 was the most frequent pathological stage (69%) and lymph node metastasis was present in 19 cases, with N0 and N1 each comprising 26.09%, N2 21.74%, and N3 17.39%. Lymphovascular invasion and perineural invasion were identified in 82.6% and 73.9% of cases, respectively.

Table 3 Comprehensive overview of Pathological findings in 42 Gastric carcinoma cases

Bcl2 expression showed no significant correlation with histopathological variables as seen in Table 4. Among adenocarcinoma NOS cases, Bcl2 positivity increased with differentiation-50% in poorly, 77.7% in moderately, and 83.3% in well-differentiated tumors (Figure1 and 2). By Lauren classification, Bcl2 was positive in 80% of mixed, 73% of intestinal, and 63.3% of diffuse types (p = 0.381). In resected specimens, Bcl2 positivity occurred in 57.9% of LVI-positive versus 75% of LVI-negative cases (p = 0.524), 58.8% of PNI-positive versus 66.7% of PNI-negative cases (p = 0.735), and 58.8% of node-positive versus 66.7% of node-negative cases (p = 0.735).

High Ki67 expression was observed in 54.7% of cases, with no significant association with age (p = 0.241). In adenocarcinoma NOS, high Ki67 expression tended to increase with poorer differentiation 71.4% in poorly, 66.8% in moderately, and 57.1% in well-differentiated tumors (p = 0.800) (Figure 3 and 4). Ki67 distribution was similar across Lauren types, with high expression in 54% of diffuse, 53% of intestinal, and 40% of mixed tumors (p = 0.801). Among resected specimens, high Ki67 expression was found in 52.6% of LVI-positive cases, while none of the LVI-negative cases showed high Ki67. High expression was present in 41.2% of PNI-positive and 50% of PNI-negative cases (p = 0.708) and in 41.2% of node-positive and 50% of node-negative cases (p = 0.708).

Bcl2 positivity was noted in 28 cases, of which 57.1% showed high Ki67 expression, compared with 50% among Bcl2-negative cases. This association was not statistically significant (p = 0.661).

Table 4 Association of Bcl2 and Ki67 Immunohistochemical Expression with Histological Grade, Lauren Classification, and Tumor Invasion Features

Figure. 1 Immunohistochemical Bcl-2 cytoplasmic positivity in well-

differentiated gastric adenocarcinoma (200×)

Figure. 2 Photomicrograph of IHC Bcl2 cytoplasmic staining showing positive in moderately differentiated adenocarcinoma, grade 2 (200x)

Figure. 3 Photomicrograph of IHC Ki67 nuclear staining showing positive

with score 2 (400x)

Figure. 4 Photomicrograph of IHC Ki67 nuclear staining

showing positive with score 3 (400x)

Gastric carcinoma is the fifth most commonly diagnosed cancer worldwide and shows a clear male predominance [1]. Diagnosis is primarily based on endoscopy and histopathological examination of biopsy specimens, with tumor grade, stage, and growth pattern being important prognostic indicators. However, as histological grading may be subjective, immunohistochemistry serves as a valuable adjunct in tumor assessment [10]. Bcl2 is an anti-apoptotic protein localized to the mitochondrial, nuclear, and endoplasmic reticulum membranes, where it regulates caspase activation and cytochrome-c release [11]. Ki67 is a well-established proliferation marker expressed during the G1, S, G2, and M phases of the cell cycle but absent in the G0 phase [12]. The present study evaluated the expression of Bcl2 and Ki67 in gastric carcinoma and correlated their expression with histopathological parameters.

The mean age of patients in this study was 62 years (range 34–75 years), which is comparable to previous studies reporting peak incidence in the sixth to seventh decades of life [13]. A male predominance with a male-to-female ratio of 2.2:1 was observed, consistent with earlier reports. According to Lauren classification, diffuse-type carcinoma was the most frequent subtype (52%), similar to findings by Qiu et al. [14], but in contrast to studies by Park et al. [1], Lee et al. [15], Crețu et al. [16], Chen et al. [17], and where intestinal-type carcinoma predominated. Moderately differentiated adenocarcinoma constituted the largest proportion of cases (43%), comparable to the observations of Lee et al. [15].

Bcl2 expression was noted in 66.7% of cases, which is in agreement with Tsamandas et al. [18] and comparable to findings by Zhou et al. [19], and although lower expression rates have been reported in some studies. A slightly higher frequency of Bcl2 positivity was observed in older patients, however this association was not statistically significant, consistent with study by Chakrabarthi et al. [20]. Although Bcl2 expression appeared to increase with better tumor differentiation, no statistically significant association was observed. Similarly, no significant differences in Bcl2 expression were found across Lauren subtypes or in relation to lymphovascular invasion and lymph node metastasis [21].

High Ki67 expression was observed in 54.7% of cases, comparable to previously published studies by Chatpalli Pranjali et al. [22]. Ki67 expression tended to be higher in older patients and in poorly differentiated tumors; however, these associations did not reach statistical significance, consistent with most published literature. Except for studies by Lazăr et al. [23,24,25,26], the majority of reports have demonstrated no significant association between tumor grade and Ki67 expression. In this study, Ki-67 expression did not show any significant association with lymphovascular or perineural invasion, which is similar to what has been reported in earlier studies.

 Associations between Ki67 expression and lymph node metastasis have been variable across studies. No significant correlation was observed between Ki67 and Bcl2 expression (p = 0.661), consistent with the findings of Zhou et al. [19], although some studies have reported contrasting results done by Kyueng-Whan Min et. [14]

Overall, the findings of this study demonstrate demographic characteristics, a predominance of diffuse-type and moderately differentiated tumors, and heterogeneous expression patterns of Bcl2 and Ki67. The absence of statistically significant associations with most clinicopathological parameters underscores the biological heterogeneity of gastric carcinoma and suggests that the prognostic significance of these biomarkers remains uncertain. These observations highlight the need for larger, multicentric studies to more clearly define the role of Bcl2 and Ki67 in gastric carcinoma.

This study assessed the association of Bcl2 and Ki67 with histopathological features of gastric carcinoma. Ki67 expression showed a heterogeneous pattern, with score 2 being most frequent (35.7%), and increased expression observed with higher tumor grade. Bcl2 was positive in 66.7% of cases, with higher expression in well-differentiated tumors. These patterns indicate that tumor progression involves dynamic changes in apoptotic and proliferative pathways. The observed expression trends suggest a potential role of Bcl2 and Ki67 in tumor differentiation and progression, contributing to a better understanding of the biological behavior of gastric carcinoma.

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