Source of Data
This prospective study was conducted on colectomy, low anterior resection and abdominoperineal resection specimens of colorectal carcinoma, received in the Department of Pathology, for routine histopathological evaluation from the Departments of Surgery and Surgical Oncology, M S Ramaiah Medical College and Hospitals, Bengaluru, over a period of 2 years (from June 2015 to May 2017). Cases where only a biopsy, endoscopic mucosal resection or polypectomy has been performed and cases where there was extensive tumor necrosis without  sufficient viable tumor cells were excluded from the study.
The specimens were received in 10% formalin. In every case, the standard protocol for surgical grossing of resected specimens was followed. After a detailed gross specimen examination, multiple representative tissue bits were taken from the tumor, surgical margins, mesentery, and all the lymph nodes. The latter were processed as per standard protocol and paraffin embedded tissue blocks were cut and stained by hematoxylin and eosin (H & E). The H & E stained slides were studied for the tumor histology and grade. The tumor was staged according to AJCC cancer staging system (6).

Processing for Immunohistochemistry
Immunohistochemical detection of Bcl-2 was done on 4µm thick sections, cut from a paraffin block of tumor tissue. The technique for IHC includes antigen retrieval in citrate buffer in a microwave oven, blocking endogenous peroxidase with 3% hydrogen peroxide, incubating with primary mouse anticlonal antibody against Bcl-2 (Anti-bcl-2 oncoprot), linking with rabbit anti mouse secondary antibody (Biogenex), enzyme labelling with streptavidin- horseradish peroxidase, developing chromogen with deaminobenzidine (DAB) and counterstaining with hematoxylin (7). Staining was defined as positive for Bcl-2 protein whenever any specific cytoplasmic staining was detected. In each case, the percentage of positive staining tumor cells (the number of positive tumor cells over the total number of tumor cells) was evaluated. A semi-quantitative assessment of staining was done as follows:

• Negative (0) – No Bcl-2 immunoreactivity detectable,

• Weak positive (1+) – less than 5% of tumor cells showing Bcl-2 positivity.

• Moderate positive (2+) - 5-50% of tumor cells showing Bcl-2 positivity.

• Strong positive (3+) - More than 50% of tumor cells positive for Bcl-2 (8).
   

Statistical Analysis of Data
Descriptive statistics were employed to express quantitative parameters such as age, duration of the disease etc. and were summarized in terms of percentage with 95% confidence interval. Differences in the proportion of expression between different grades, types, etc. were tested for statistical significance by Chi-square test significance/ Fisher`s Exact test. Differences in mean values were tested by appropriate students` t`/ Mann-Whitney test.

During the study period, 58 resected specimens of colorectal carcinoma were received in the department of pathology. The Mean patient age was 47.07 years (age range: 25 to 75 years). Males (65.5%) were found to be highly susceptible to CRC when compared to females (34.5%) with a male to female ratio of 1.89:1.  The most common site for colorectal carcinoma was sigmoid colon (37.9 %), followed by cecum (20.7%), descending colon (20.7%), ascending colon (6.9%) , hepatic flexure (6.9%), splenic flexure (3.4%), transverse colon (1.7%), and rectum (1.7%). The ulceroinfiltrative pattern was the dominant pattern seen in the gross specimen, accounting for 58.6%, followed by ulceroproliferative pattern (31.0%), annular constricting (5.2%) and diffuse infiltrating pattern (5.2%). Adenocarcinoma –NOS (ACa-NOS) (75.8%) was the predominant histologic type of colorectal carcinoma, followed by mucinous adenocarcinoma (MACa; 19.0%), and signet ring cell carcinoma (SCa; 5.2%). Majority of the tumors were grade 2 (moderately differentiated, 69%) followed by grade 1 (well differentiated; 17.2%), and grade 3 (poorly differentiated; 13.8%) morphology. Stage II (43.1%) was the most common presentation, followed by stage III (39.7%), stage I (13.8%), and stage IV (3.4%). Overall, 32.7% of CRC showed Bcl-2 positivity, out of which 20.6% of Bcl-2 expression was noted in left colonic tumors, 12.1% of Bcl-2 positivity was seen in right colonic tumors. Weak positivity, moderately intense positivity and strong positivity were observed in 12.1%, 12.1% and 8.5% of CRC cases respectively (Figures 1, 2, and 3 respectively & Table 1). Well and moderately differentiated CRC were associated with greater expression of Bcl-2 compared to poorly differentiated CRC (Table 2). Early stage (stages I and II) tumors showed greater expression of Bcl-2 in contrast to advanced stage tumors (stages III and IV) (Table 3). However, there was no statistically significant association between the Bcl-2 expression and tumor stage (P=0.5) and grade (P=0.58).