European Journal of Cardiovascular Medicine

Colorectal cancer (CRC) is the third most common cancer in men (663,000 cases, 10.0% of the total cancers) and the second in women (570,000 cases, 9.4% of the total cancers) worldwide. The annual incidence of CRC in India is about 4/100,000 and in Kerala it is about 5.5/100,000.¹ This increased incidence in Kerala is postulated to be due to the increased consumption of red meat in the state which is an established risk factor along with age, environmental factors- diet, smoking, sedentary lifestyle and genetic factors- FAP, Lynch syndrome and related non polyposis associated hereditary conditions. About 6% of all CRC are caused by the inheritance of muted genes with high penetrance.²

Gastrointestinal tumors can be divided into 4 major categories of carcinoma viz, adenocarcinoma, neuroendocrine tumor, neuroendocrine carcinoma and mixed neuroendocrine - non neuroendocrine tumor.

ERβ is the predominant estrogen receptor expressed in normal colonic epithelium, with limited or no expression of ERα observed in the colon. ³ ERβ is thought to have a prominent role in the biological mechanisms of sex steroid action on colorectal tissue.⁴ ERβ is the predominant estrogen receptor expressed in both normal and malignant colonic epithelium. However, during colon cancer progression, ERβ expression is lost, suggesting that estrogen signaling may play a role in disease progression. ERβ is also expressed in CRC although the expression is reduced during colonic tumor genesis as compared to normal tissue. ERβ expression is associated with stage and grade of disease, and an inverse relationship between ERβ expression and tumor progression has been reported in cell lines and clinical samples.^5,6

In post-menopausal women on hormone replacement therapy (HRT), a decreased CRC risk was associated with duration of HRT use specifically in ERβ-positive patients but not in ERβ-negative patients.⁵ It is therefore hypothesized that estrogen-mediated signaling exerts a protective role in CRC and further understanding of this may benefit cancer prevention and also provide additional therapeutic options for ERβ positive tumors.

This study aims to enumerate the loss or decreased expression of CRC and search for any association with clinico-pathological parameters like age, sex, tumor site, histologic type, degree of differentiation and stage.

All colectomy specimens received in the Department of Pathology, Government Medical College Thiruvananthapuram over a period of two years.

Inclusion criteria- All colectomy specimens received in the Department of Pathology which are histopathologically diagnosed as colorectal carcinoma

Exclusion criteria - Post chemotherapy and radiotherapy patients.

Data collection

H&E sections of resected margins of the specimen will be studied and tumor characteristics like histopathological type, invasion, differentiation, metastatic node will be recorded. Formalin fixed paraffin embedded blocks of tumor will be used for IHC staining using primary antibody – ERβ. A three-level scoring system based on Konstantinopoulos et al.⁷ was applied that involved the staining intensity as well as the percentage of positivity in the cancer cell nuclei. The lymphocytes in the lamina propria as well as the cores of adjacent non-neoplastic tissue were used as positive control and standard.

Interpretation of ERβ expression on tumor specimen

• negative for ERβ expression: less than 10% of the cell nuclei showed positive staining.
• moderate expression: staining in 10–50% of the nuclei showed positive staining.
• strong positive: more than 50% of the cell nuclei showed positive staining
• High expression: more than 50% of the cell nuclei showed strong positive staining.

Statistical test of significance - Chi square test for categorical variables and Students t test for quantitative variables. Analysis of data was done using statistical software (SPSS)

Institutional Ethics Committee clearance was obtained, prior to the commencement of study. Patient confidentiality will be ensured and maintained throughout the study.

64 cases were studied for the ERβ status. Of the 64, 48 (71.9%) had loss of IHC expression and 18(28.1%) had no loss of expression.

AGE: The mean age of patients (n=64) was 63.5(S.D=10.03). The mean age of patients (N=46) with   loss of expression of IHC was 63.28 with a standard deviation of 11.069. The mean age of patients (N=18) with no loss of expression of IHC was 64.06 with a standard deviation of 6.949. A Mann Witney U test was employed to look for a significant difference in the mean age of two groups (those with loss of IHC expression vs those without loss of IHC expression).The z score was -0.209 and the p-value (2-tailed) was 0.834 implying no statistically significant difference in mean age between the two groups.

SEX: Of the 64 patients,27(42.19%) were males and 37(57.61%) were females. 17 out of 27 male patients (63%) of male patients and 29 out of 37 female patients(78.4%) had a loss of IHC expression. A chi-square test done to ascertain if there was a significant difference between the two groups showed that there was no significant difference between the two groups as the p value (2 tailed) was 0.260.

HISTOLOGICAL SUBTYPES: 57/64(89.1%) were adenocarcinoma patients and 7/64(10.9%) were mucinous carcinoma patients. 42 out of 57(73.7%) adenocarcinoma patients, and 4 out of 7(57.1%) mucinous carcinoma patients had loss of IHC expression. A fischer's exact test was done to ascertain any significant difference between the two groups of patients for loss of IHC expression which yielded a p value (two tailed) of 0.391 implying no significant difference between the three groups with regards to loss of IHC expression.

Figure 1

SITE OF LESION: 8/10(80%) of left colonic lesions, 10/11(90.9%) of right colonic lesions, 8/12(66.7%) of sigmoid lesions, 1/2(50%) rectosigmoid lesions, 15/25(60%) of rectal lesions and 4/4(100%) of anal canal lesions showed loss IHC expression.

A fischer's exact test done revealed a two tailed p value of 0.283 implying no statistically significant difference between the different sites with regards to loss of expression of IHC.

Figure 2

DIFFERENCIATION IN ADENOCARCINOMA:

8/13 (61.5%) of well differentiated, 33/43(76.7%) of moderately differentiated and 1/1(100%) of poorly differentiated adenocarcinomas exhibited loss of IHC expression.

A fishers exact test was done to ascertain a significant difference between the three grades of adenocarcinoma with regards to loss of IHC expression and yielded a p value (2 tailed) of 0.485 implying no statistically significant difference in IHC expression between the three groups.

Figure 3

IHC EXPRESSION IN DIFFERENT STAGES.

14/24(58.3%) of patients with stage 1, 16/19(84.2%) of patients with stage 2 and 16/21(76.2%) patients with stage 3 malignancy had loss of IHC expression.

A fishers exact test yielded a two tailed p value of 0.170 implying no statistically significant difference in the proportion of patients with loss of IHC expression among the 3 stages of malignancy.

We further compared stage 1and stage 2a patients(disease confined to colon)  with higher stages to look for differential loss of IHC expression.

27/39(69.2%) of stage 1 and 2a patients had loss of IHC expression where as 19/25(76%) of patients with higher stages of malignancy had loss of IHC expression. A chi-square test to ascertain the difference in proportion of patients with IHC expression loss among the two groups yielded a two tailed p value of 0.585 implying no significant difference between the two groups.

Figure 4

Figure 21: ERb negative in well differentiated, moderately and poorly differentiated adenocarcinoma

Figure 24: ERβ 1+ positivity in well differentiated and moderately differentiated adenocarcinoma

Figure 26: ERβ negative in mucinous carcinoma

The present study included 64 cases of patients with colorectal carcinoma who underwent surgical resection in Government Medical College, Thiruvananthapuram over a period of two years.

Of the 64 cases, 48 (71.9%) cases had loss or decreased expression of ERβ in their tumor tissue when compared to adjacent normal tissue and 18 (28.1%) cases showed no loss or decreased expression. This is comparable to the works of Konstantinopoulos et al.⁷, where 76% cases had either no positivity or weak positivity.

AGE: Majority of cases were in the 50 to 75 years age group.  In concordance with literature⁵, mean age of cases were 63.5 years with a standard deviation of 10.03.

SEX: Of the 64 patients, 27(42.19%) were males and 37(57.61%) were females. According to McCashland et al.⁸, Colorectal polyps and tumors have been reported to occur more frequently in men than in women. This disparity could be due to the fewer number of cases studied, which favored the female gender. Or a possible explanation is that older women are at a higher risk of acquiring microsatellite instability tumors, one example being proximal colorectal carcinoma and this study had more women of older age group compared to men.⁹ When comparing the loss of IHC expression, no significant difference was observed between the two sexes.

HISTOPATHOLOGICAL TYPE: 89.1% of cases analysed were adenocarcinomas, 10.9% were mucinous carcinomas, which correlates with the 90% proportion of adenocarcinoma¹⁰ among all the histological types of CRC. No significant difference between the two groups with regards to loss of IHC expression was noted.

DIFFERENTIATION: 22.8% of adenocarcinomas were well differentiated, 75.4% moderately differentiated and 1.75% poorly differentiated. No statistically significant difference in loss of IHC expression between the three groups was observed. This finding contradicts with the works of Konstantinopoulos et al, Jassam et al and Wong et al.^6,7,11 were an inverse relationship between tumour differentiation and ERβ expression was studied. This is further disputed by Hassan et al who further enumerated the stronger intensity if IHC staining in well differentiated adenocarcinomas compared to moderately and poorly differentiated carcinomas. However similar lack of relationship was demonstrated by Rudolph et al.⁵ as in our study. This discrepancy warrants further studies and meta analysis in this regard.

STAGING: 58.3% of patients with stage 1, 84.2% of patients with stage 2 and 76.2% patients with stage 3 malignancy had loss of IHC expression, implying no statistically significant difference in the proportion of patients with loss of  IHC expression among the different stages of malignancy. When carcinomas limited to colon (stage 1 and 2a) were further compared to those with extension to visceral peritoneum, adjacent organs, nodal and distant metastasis (stages 2b,2c,3,4), no significant difference between the two groups was observed. This directly contradicts with the findings of Fang et al and Rudolph et al.^5,12 Such discrepancy could be attributed to the low number of cases studied which could have skewed the results.

In view of our primary objective, 71.9% of cases of CRC were found to have loss of expression of ERβ compared with the 28.1% having retained expression, which closely mimics the works of  Konstantinopoulos et al.⁷ This study however could not identify any significant variables with loss of IHC expression.  Further studies with larger number of cases and including more varied subtypes of CRC are required.

This study investigated the association between the loss of expression of estrogen receptor beta (ERβ) and colorectal carcinoma (CRC) among South Indian population. A significant proportion (71.9%) of CRC cases exhibited a loss or decreased expression of ERβ in tumor tissue compared to adjacent normal tissue. Most patients were between 50 and 75 years old. Female predominance was observed (57.6%). Rectum was the most common site, followed by sigmoid, right, and left colon.

No statistically significant associations were found between ERβ expression and Histological subtypes of CRC, Tumor differentiation or Tumor stage.                                                   

The present study provides evidence that a significant proportion of CRC cases exhibit a loss or decreased expression of ERβ. ERβ expression alone may not be a strong predictor of clinical outcomes such as tumor subtype, differentiation, or stage in CRC. Thus larger sample sizes and inclusion of less common CRC subtypes are necessary to confirm these findings. Further research is crucial to explore the potential role of ER modulation as a therapeutic strategy for CRC.                                     

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