Year :
2021
| Month :
July
| Volume :
15
| Issue :
7
| Page :
EC11 - EC14
Full Version
A Study on the Assessment of Galectin-3
Expression in Colorectal Neoplasm and
its Relationship with Tumour Stage, in
Tertiary Care Hospital, Kolkata, India
Published: July 1, 2021 | DOI: https://doi.org/10.7860/JCDR/2021/47792.15092
Nandini Bhaduri Bhattacharyya, Anadi Roy Chowdhury, Susmita Mukhopadhyay, Sneha
1. Assistant Professor, Department of Pathology, RG Kar Medical College, Kolkata, West Bengal, India.
2. Professor, Department of Pathology, RG Kar Medical College, Kolkata, West Bengal, India.
3. Assistant Professor, Department of Pathology, RG Kar Medical College, Kolkata, West Bengal, India.
4. Junior Resident, Department of Pathology, RG Kar Medical College, Kolkata, West Bengal, India.
Correspondence Address :
Anadi Roy Chowdhury,
Kailash 502, Godrej Prakriti, 187f/1 BT Road, Sukhchar, West Bengal, India.
E-mail: dr-anadi@hotmail.com
Abstract
Introduction: Galectin-3 is a β-galactoside-binding lectin found in a considerable number of normal tissues and malignant neoplasms. It was found to be expressed in few thyroid tumours particularly follicular and papillary tumours. Lectins were found to be released in circulation and increased concentration was noted in colorectal cancers especially in metastatic colonic adenocarcinoma. Different types of galectins are expressed in normal colonic and rectal epithelium. Some types do increases in inflammation and cancers of these areas. It was seen that galectin-3 increases in colorectal tumourigenesis and it bears an important role in cancer progression and metastasis. Galectin-3 seems to have an important role in colorectal cancer. Some studies proved that galectin inhibitors could reduce tumour progression and metastasis and it may be a therapeutic target in metastatic colorectal adenocarcinoma.
Aim: To evaluate colon cancer specimens received for biopsy, for galectin-3 expression and its relation with tumour stage, lymphovascular space invasion and tumour differentiation.
Materials and Methods: The study was a cross-sectional observational study conducted in the Department of Pathology of RG Kar Medical College and Hospital from November 2018 to November 2019. It was an immunohistochemistry based assay performed to test the expression levels of galectin-3 in cancer tissues of 62 colorectal neoplasms with the help of galectin-3 primary antibody (mouse monoclonal antibody- clone 9C4). Statistical analysis was done using Statistical Package of Social Sciences (SPSS) version 19.0.
Results: Out of 62 cases, 60 cases were colorectal adenocarcinoma and 2 cases were adenoma with age group between 40-75 years. Total 46 cases had cancer in caecum and ascending colon and rest were in recto-sigmoid colon. A 36/60 cases (60%) of cancer tissues were positive for galectin-3 expression. Strong association of lymphovascular space invasion (p=0.046) and depth of tumour (p=0.0078) with galectin positivity in colon carcinoma was noted.
Conclusion: Evaluation of galectin-3 expression is helpful in the assessment of tumour staging and prognosis in colorectal cancer patients. It may have a therapeutic implication in the management of colon cancer in future.
Keywords
Colon cancer, Galactose binding, Immunohistochemistry, Lectin
Introduction
Galectin-3 is an endogenous carbohydrate binding protein that is implicated in cell growth, differentiation, adhesion, malignant transformation and apoptosis. It is predominantly located in the cytoplasm. Galectin-3 seems to have an important role in colorectal cancer. Dabbs DJ examined the expression of galectin-1 and 3 in a series of few thyroid tumours and reported expression of these lectins in papillary and follicular carcinomas, but not in adenomas, nodular goitre, or normal thyroid tissue. Based on these studies, they concluded that the galectins could be useful in the distinction of benign and malignant thyroid tumours (1).
There is strong in-vitro and in-vivo evidence that tumourigenesis and metastasis can be reduced by galectin inhibitors. Galectin-3-null mice are relatively healthy, indicating that inhibition of galectin-3-mediated actions may present a viable and relatively safe therapeutic approach for cancer treatment (2).
Galectin-3 is also released into the circulation. Concentrations of circulating galectin-3 in the bloodstream of colorectal cancer patients can be increased up to five fold (3). Moreover, patients with metastasis have higher levels of circulating galectin-3 than those with localised tumours (4). The beta subunit of haptoglobin co-precipitates with galectin-3 from the serum of patients with colorectal cancer (4). Recent studies have suggested that the increased circulation of galectin-3 in the bloodstream of cancer patients can be an important promoter of cancer cell metastasis (5),(6).
In the normal human colon and rectum, four galectins, galectin(gal)-1, 3, 4 and 8, are expressed (7),(8),(9),(10). Galectin-1 is expressed weakly in normal colonic epithelium but its expression is increased in inflammation and cancer. The results on gal-1 and gal-3 clearly show that galectins are involved in the malignant progression of colon cancer and their migration properties (11).
Differential expression of galectin expression is very much common in cancers of the human gastrointestinal tract. Accumulating evidences support an active role of these endogenous carbohydrates binding protein in the regulation of colorectal cancer development, progression and distant metastasis (12).
Galectin-3 expression is greater in advanced cancer (13) and metastases express higher levels of galectin-3 than the primary tumours from which they arise (14). There is a general change in galectin-3 sub-cellular localisation from nucleus to the cytoplasm in colorectal cancer during progression from colorectal adenoma to carcinoma (8),(15). As cytoplasmic galectin-3 is known to be an apoptosis inhibitor, it is very likely that this change in localisation may contribute to cancer cell survival.
Galectin-4 is expressed in the human intestinal and colonic mucosa and its expression is generally lower in cancer than in normal mucosa (9),(16),(17).
Galectin-8 is expressed widely in the gastrointestinal tract. Low basal levels of galectin-8 are observed in the human intestine (18),(19). Hence, the present study aimed to evaluate the colon cancer specimens for galectin-3 expression and its relation with tumour stage, lymphovascular space invasion and tumour differentiation.
Material and Methods
The present study was an observational cross-sectional study carried out in the Department of Pathology, RG Kar Medical College, Kolkata, India from November 2018 to November 2019. Total 62 colorectal specimen with growth were collected during this period. Out of 62 cases, 40 cases were from male patients and 22 cases were from female patients. Institutional Ethics Committee permission (IEC No RKC/47) was taken.
Study Procedure
Surgically resected colorectal cancer tissues were fixed in 10% formalin and embedded in paraffin for routine pathological diagnosis. Paraffin blocks containing representative cancer tissue were selected and used for immunohistochemical study to see galectin 3 expression. The final result was achieved by multiplying the scores from the 2 categories: 0 to 3 points is negative expression; 4 to 6 points weakly positive expression (+), 7 to 9 points, fairly strong positive expression (++), 9 to 12 points, strong positive expression (+++) (13).
Tumour staging was done from assessment of depth of tumour penetration, lymphovascular invasion and lymph node metastasis. The IHC-2 sections of 2-3 μm thickness were prepared, one for H&E staining and one for IHC with galectin-3 primary antibody (mouse monoclonal antibody- clone number 9C4). The samples were evaluated by cell staining with the following scores: brown- 3 points; pale brown- 2 points; light brown- 1 point; and no colouration- 0 points. The quantity of stained cells was evaluated with the following scores: quantity of stained cells in one visual field of >75%- 4 points; 51% to 75%- 3 points; 11% to 50%- 2 points; less than 10%- 1 point, and negative- 0 points (13). (Table/Fig 1)a: Poorly differentiated colorectal carcinoma with Lymphovascular Space Invasion (LVSI) in surrounding area (100X magnification, H&E). (Table/Fig 1)b: Tumour deposit in the surrounding adipose tissue with LVSI. (100X magnification, H&E). (Table/Fig 1)c: strong positive galectin stain of 3×4=12 score (IHC) and (Table/Fig 1)d: weakly positive galectin stain of 2×2=4 score (IHC) in this study.
Statistical Analysis
Statistical analysis was done using 2×2 contingency table from each category with the help of SPSS version 19.0 and Chi-square test was done to assess level of significance.
Results
In this study the expression of galectin-3 in 62 tissue specimens were assessed, which included 2 adenomas (one rectal villous adenoma and one was traditional serrated adenoma in ascending colon) and 60 cases of colon carcinomas of known Tumour Node Metastasis (TNM) stage. Therefore, the result analysis was done on 60 cases only, excluding the adenomas. The average age of 62 patients in this study was between 40 years to 75 years. There were 40 males and 22 females. A total of 46 cases had tumour in ascending colon and caecum and 16 had tumour in recto-sigmoid region. Total 36 out of 60 (60%) of the tumours showed galectin-3 expression. The various parameters studied for galectin-3 expression are: 1) Tumour stage; 2) LVSI; and 3) Tumour differentiation.
Clinicopathologic characteristics of all the cases are shown in (Table/Fig 2). There is strong association (p-value <0.05) of Lymphovascular space invasion and galectin positivity in colon carcinoma which is depicted in (Table/Fig 3). Strong association was also noted in cases of depth of tumour and galectin positivity (p-value=0.014 i.e., p<0.05) noted in (Table/Fig 4).
There was also increased negativity of galectin in relation to tumour differentiation in colorectal carcinoma shown in (Table/Fig 5). Poorly differentiated carcinoma show 13/19 (68.4%) negative expression compared to well differentiated carcinoma where only 5 out of 13 cases (38.4%) showed negative expression.
Discussion
Galectin-3 is an endogenous carbohydrate binding protein implicated in cell growth, differentiation, adhesion, malignant transformation and apoptosis. Being predominantly located in the cytoplasm, it seems to have an important role in colorectal carcinogenesis.
In the present study, patients were in stage 2 in 17 cases and in stage 3 and stage 4 combined in 43 cases. 6 cases of stage 2 tumour, were galectin-3 positive and 30 cases in stages 3 and 4 were galectin-3 positive. The p-value was 0.014 and significant (p<0.05).
A total of 39 samples were LVSI positive and 27 of them were galectin-3 positive. Twenty one cases were negative for LVSI. Out of LVSI negative cases 9 were galectin-3 positive. The p-value was calculated to be 0.046, result is significant at p<0.05.
Similar study was done by Schoeppner HL et al., on galectin-3 expression in 153 tissue specimens. They found galectin-3 expression was significantly higher in high grade dysplasia and early invasive cancers compared with the adenomatous tissue. A linear relationship noted between galectin-3 expression in invasive cancers with advancing stage (p=0.008). Decreased long term patient survival (p=0.021) correlated with enhanced expression. Higher level of galectin-3 expression noted in metastasis compared with the primary cancers (p<0.005) (14).
A similar study using immunohistochemistry assay was used to test the expression status of galectin-3 in cancer tissues of 61 colorectal cancer (61 cases) and in normal intestinal mucosa adjacent to the cancer (23 cases). Clinicopathological features, such as age, sex, pathological type, lymphatic metastasis, and prognosis were also analysed with the galectin-3 level. Rate of positivity of galectin-3 in cancer tissues was significantly higher than that of normal epithelium adjacent to cancer 62.5% (38/61) versus 13.0% (3/23) (p<0.05) respectively. Positive correlation was noted between the protein expression of galectin-3 and the tumour size (p<0.05), tumour differentiation (p<0.05) and duke’s stage (p<0.05) (20).
Another similar study conducted in Kyushu University, Fukuoka, Japan on 121 colorectal cancer patients, showed positive expression in 65% patients. The incidence of lymph node metastasis was significantly high in galectin-3 positive cases with significance level p<0.05 (p=0.0007). Deeper invasion into wall and lymphatic permeation was higher in positive cases (p=0.01 and p=0.041, respectively). Larger tumour size had higher expression (p=0.016) (21).
Another study conducted in Department of General Surgical Science of Gunma University Graduate School of Medicine, Maebashi, Japan on 108 patients with colorectal cancer was investigated using immunohistochemical analysis. Galectin-3 expression at the surface of the tumour was correlated with the depth of invasion (p=0.02) and blood vessel invasion (p<0.01) in this study (22).
A study from a chinese article showed that galectin 3 was expressed in 158 cases including 30 normal mucosa, 25 adenomas, 65 carcinomas and 38 metastatic tumour specimens. Normal tissues showed different expression than adenomas (p<0.001). Poorly differentiated and colorectal cancer with metastasis showed higher expression than well and moderately differentiated tumours (p=0.03 and p<0.001, respectively). Invasive tumours had higher expression than non invasive tumours (p<0.001) (23).
Limitation(s)
Not all parameters were satisfactorily described in the present study because of low sample size. It can be made more statistically significant if a large sample size would be taken.
Conclusion
Immunohistochemical detection of elevated expression of galectin-3 is a potent prognostic marker in colorectal cancer, but its biological function is still to be explored. Elucidation of this function may contribute to our search for a new therapeutic regime against cancer progression and metastasis in future. The higher expression of galectin-3 may contribute as a metastasis predictor for colorectal carcinoma. This study is restricted in this regard due to low sample volume. Further studies are needed.
Acknowledgement
Authors would like to thank Prof. (Dr.) Tushar Kanti Das, Head of the Department, Department of Pathology, and all technical staff of the department.
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10.7860/JCDR/2021/47792.15092
Date of Submission: Nov 15, 2020
Date of Peer Review: Dec 31, 2020
Date of Acceptance: Feb 17, 2021
Date of Publishing: Jul 01, 2021
AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? NA
• For any images presented appropriate consent has been obtained from the subjects. NA
PLAGIARISM CHECKING METHODS:
• Plagiarism X-checker: Nov 17, 2020
• Manual Googling: Feb 13, 2021
• iThenticate Software: Mar 15, 2021 (19%)
ETYMOLOGY: Author Origin
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