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Table of Contents   
ORIGINAL RESEARCH  
Year : 2022  |  Volume : 33  |  Issue : 3  |  Page : 282-286
Immunohistochemical evaluation of galectin-3 expression in oral squamous cell carcinoma, oral leukoplakia and normal mucosa


Department of Oral Pathology and Microbiology, PMS College of Dental Science and Research, Trivandrum, Kerala, India

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Date of Submission20-Feb-2022
Date of Decision24-Aug-2022
Date of Acceptance01-Sep-2022
Date of Web Publication17-Jan-2023
 

   Abstract 


Background: Galectin 3 (Gal-3) has diverse functions critical in cancer biology including cell proliferation, apoptosis, evasion of immune responses and angiogenesis. The expression of Gal-3 is heterogeneous in normal and neoplastic tissues. In oral squamous cell carcinoma (OSCC) and oral leukoplakia (OL), both increased and decreased expressions of Gal-3 were elicited in numerous studies. Aims: To evaluate, compare and correlate the immunohistochemical expression of Gal-3 in OSCC, OL and normal oral mucosa. Settings and Design: The study was conducted at the Department of Oral Pathology and Microbiology at PMS College of Dental Science and Research, Vattapara, Thiruvananthapuram. This is a retrospective analytical study. Methods and Material: Clinically diagnosed and histopathologically confirmed cases of OSCC (n = 21), OL (n = 21), and normal oral mucosa (n = 21) were included in the study. Paraffin-embedded tissues were subjected to immunohistochemical analysis for Gal-3 expression. Gal-3 staining expression, staining distribution and cellular localisation were evaluated. All sampled categories were compared using immunohistochemical scoring analysis such as the H-score, labelling index (LI), immunoreactive score (IRS) and staining intensity (SI). Statistical Analysis: The results were statistically analysed using multivariate analysis of variance (MANOVA) within and among the groups. Results and Conclusion: The statistical inferences obtained found that the H-score could be used as a guideline for better differentiation between the groups and among the groups. The P value obtained was < 0.0125 and was found to be significant. The observation in our study shows that the immunohistochemical expression of Gal-3 gradually decreased from normal oral mucosa to OL to OSCC.

Keywords: Galectin-3, histo score, immunohistochemistry, oral leukoplakia, oral squamous cell carcinoma

How to cite this article:
Selvaraj FM, Joseph AP, Varun B R, Mony V, Siva Kumar T T. Immunohistochemical evaluation of galectin-3 expression in oral squamous cell carcinoma, oral leukoplakia and normal mucosa. Indian J Dent Res 2022;33:282-6

How to cite this URL:
Selvaraj FM, Joseph AP, Varun B R, Mony V, Siva Kumar T T. Immunohistochemical evaluation of galectin-3 expression in oral squamous cell carcinoma, oral leukoplakia and normal mucosa. Indian J Dent Res [serial online] 2022 [cited 2023 Feb 5];33:282-6. Available from: https://www.ijdr.in/text.asp?2022/33/3/282/367875



   Introduction Top


Oral cancer is a leading cause of death, and oral squamous cell carcinoma (OSCC) accounts for more than 90% of all oral cancers. OSCC is the sixth most common cancer in the world and the third most common in India.[1],[2]

OSCC is preceded in the majority of cases by clinically evident oral potentially malignant disorders, the most common of which is leukoplakia. The prevalence of leukoplakia in India varies from 0.2% to 4.9% with the probability of malignant transformation rate ranging between 5 and 18%.[3]

Oral carcinogenesis is a multistage process which involves numerous molecular alterations. Galectin-3 (Gal-3) is a multifunctional protein, which mediates multiple processes of tumour growth and plays important roles in cell proliferation, angiogenesis, and apoptosis.[4] Galectins are a phylogenetically conserved family of lectins of amino-acid-sequences containing 130 amino acids and the carbohydrate recognition domain (CRD) responsible for β-galactoside binding.[5]

The expression of Gal-3 is heterogeneous in normal and neoplastic tissues. Increased expressions have been shown to be prognostic for poor survival in many cancers including leukaemia, lymphomas, breast cancer, and thyroid cancer. Decreased levels appear to be detrimental to patients with chronic lymphoblastic leukaemia and prostate cancer.[6],[7] A possible explanation for this variance could involve the intracellular localisation of Gal-3 within the cancer cells.[6] In OSCC and oral leukoplakia (OL), both increased and decreased expressions of Gal-3 were elicited in numerous studies.

Rationale

To evaluate, compare and correlate the immuno-histochemical expression of Gal-3 in OSCC, OL and normal oral mucosa.


   Methods Top


Sample size

This study was designed as a retrospective analytical study. The sample size was calculated by using the following formula by keeping the power of study at 80%.



As per the equation, the minimum sample size calculated was 21 for each group (OSCC, OL and normal mucosa).

Sample collection

Clinically diagnosed and histopathologically confirmed cases of OSCC (n = 21), OL (n = 21), and normal oral mucosa (n = 21) were obtained from the archives of the Department of Oral Pathology and Microbiology, PMS College of Dental Science and Research, Vattappara, Thiruvananthapuram. For normal oral mucosa, non-inflamed buccal mucosa was taken during the removal of the impacted third molar. Most of them were collected from the archives of the Department of Oral Pathology and Microbiology and few from the Department of Oral and Maxillofacial surgery. All specimens were obtained with prior informed consent, which was reviewed and approved by the Institutional Ethics Committee of PMS College of Dental Science and Research, Thiruvananthapuram (IEC no. PMS/IEC/2018-19/02). OL was graded according to the WHO 2017 criteria as mild, moderate and severe dysplasias. According to the degree of differentiation, the tumours were graded histologically as well, moderately (Mod) and poorly differentiated OSCC.[8]

The immunohistochemistry (IHC) procedure was used for the detection of Gal-3 using the monoclonal mouse galectin 3 antibody (9C4) (PM211-6 ml RTU). Poly Excel Poly horseradish peroxidase (HRP) (pre-diluted, PEH002, 6 ml) was used as the secondary antibody. Papillary carcinoma thyroid (PCT) was taken as the positive control. For negative control, the primary antibody was replaced with serum.

Standard HRP IHC was performed to detect the expression of Gal-3. Tissue sections of 4 μm thickness were made in a rotary semiautomatic microtome. The ribbons of the tissue section were transferred onto the APES (3-aminopropyltriethoxysilane)-coated slide. The tissues were deparaffinised with xylene and rehydrated by decreasing the concentrations of ethanol in water. Slides were then placed in a jar containing Tris-EDTA buffer (PathnSitu Cat # PS009) at a pH of 9 and subjected to heat-induced antigen retrieval using PathnSitu's MERS (Multi Epitope Retrieval System) in a 5L pressure cooker at 1000°C until 2 whistles were heard. It was then allowed to cool for 10 minutes and then transferred into distilled water. Endogenous peroxidases were quenched for 10 min with incubation in 3% hydrogen peroxide (PathnSitu). Slides were incubated overnight with primary antibody at room temperature in a humidified chamber, followed by 30-min incubation with biotinylated goat anti-rabbit IgG (PathnSitu). The expression was visualised using a diaminobenzine tetrahydrochloride (DAB) solution detection system (VKAN Life Care, Chennai, India). The duration of DAB incubation was determined through pilot experiments and was made constant for 35 min for all slides. Between each step, slides were washed with immuno wash buffer (VKAN Life Care). The sections were counterstained with hematoxylin, dehydrated with ethanol, and cover-slipped using a xylene-based mounting medium

Immunohistochemical scoring system

Oral squamous cell carcinoma

Cases that showed specific staining of more than 5% of the tumour cells, regardless of SI, were scored as positive for Gal-3.[9] For analysis of the staining results, a semi-quantitative score, the immunoreactive score (IRS) was used. The IRS gives a product of multiplication between the score of optical SI (grades: 0 = none, 1 = weak, 2 = moderate and 3 = strong staining) and the score of percentage of positively stained cells (0 = no staining, 1 ≤10% of the cells, 2 = 11–50% of the cells, 3 = 51–80% of the cells and 4 ≥81% of the cells).[10] The percentage of positive cells is calculated as the Labelling Index (LI).[11]

To get more precision, the results were evaluated by a semi-quantitative approach using the histo score (H-score). Gal-3 expression was assessed on the nuclear and cytoplasmic staining of tumour cells, and the number (%) of tumour cells was counted. The intensity score is given by

0 -negative (non-stained cells)

1 -staining expression weaker than PCT: weak

2 -staining expression equivalent to PCT: moderate

3 -staining expression stronger than PCT: strong

The percentage of cells at each SI level was assigned using the following formula: {[1× % cells 1+] + [2× % cells 2+] + [3× % cells 3+]}. The H-score was obtained from the calculation with a range of 0–300. An H-score >0 is considered positive staining, and an H-score <0 is considered complete negative staining.[12]

The nuclear Gal-3 expression was scored 0 (<5% positive tumour nuclei) or 1 (nuclear staining >5%) independently of the galectin-3 scoring in the tumour cell cytoplasm. The distribution pattern of stroma was classified as focal (≤30% of the epithelium) and diffuse (>30% of the epithelium).[13]

Oral leukoplakia

The staining distribution of Gal-3 was classified into focal (30% of the epithelium) and diffuse (>30% of the epithelium). Epithelial localisation, that is, the predominant site of expression in the epithelium, was divided into upper, middle and lower third.[14] Cellular localisation, corresponding to the site of expression in the cell, was classified as membrane, membrane/cytoplasmic and nuclear/cytoplasmic staining and was adapted from Sobel et al.[15] The percentage of positive cells was calculated as the LI. The intensity and distribution pattern of the specific immunohistochemical staining was evaluated by using a semi-quantitative method, IRS. Thus, the SI is graded as no staining = 0, weak staining = 1, moderate staining = 2 or strong staining = 3 and the percentage of positively stained cells is defined as no staining = 0, <10% of stained cells = 1, 10–50% of stained cells = 2, 51–80% stained cells = 3 and >80% of stained cells = 4. For calculating the IRS score, SI and percentage of positively stained cells were multiplied.[16] To get more precision, results were also evaluated using the H-score.

Statistical analysis

Multivariate analysis of variance (MANOVA) was used to assess the staining intensities within and among the groups. MANOVA compares multivariate sample means and reduces type 1 error of ANOVA by considering more uncorrelated dependent variables at a time by losing a degree of freedom for each addition of a dependent variable. The differences between the groups were determined by Wilks' lambda (Λ) test. The significance of the change in Λ is measured by an F-test. These data were analysed using the Excel solver add-on.


   Results Top


The biopsy specimens of OSCC and OL were taken from anatomic sites including the buccal mucosa, tongue, gingiva and alveolar mucosa within the oral cavity. A summary of demographic details of patients, including their age- and sex-matched cohorts is provided in [Table 1].
Table 1: Demographic data of patients

Click here to view


All sampled categories such as OSCC, OL and normal mucosa were compared using the immunohistochemical scoring analysis such as H-score, LI, IRS and staining intensity (SI). The statistical inferences obtained found that the H-score could be used as a guideline for better differentiation between the groups and among the groups. The P value obtained was <0.0125 and was found to be significant. The average H-score of OSCC, OL and normal mucosa was 99.492, 107.606, and 135.21 respectively [Graph 1]. The highest H-score was seen with normal mucosa which was decreasing towards OL and OSCC. In the OL group, the H-score was 118, 109.66, and 54.375 for mild, moderate and severe dysplasia, respectively. The expression of Gal-3 was found to decrease as the severity of dysplasia increased.



Staining distribution of Gal-3 was seen in a more diffuse pattern than focal in OL [Graph 2]. In moderate dysplasia, no focal patterns were noted. The diffuse pattern of distribution decreased as the severity of dysplasia increased, whereas the focal pattern of distribution remained the same. In early invasive (EI) OSCC, only focal patterns of distribution of Gal-3 were noted. Well- and moderately differentiated OSCC showed a more diffuse pattern than the focal pattern of staining distribution of Gal-3. Normal mucosa showed a more diffuse pattern of staining distribution than the focal pattern.



Cellular localisation was seen more in cytoplasm as the severity of dysplasia increases [Graph 3]. Combinations of nuclear, cytoplasm and membrane staining were seen in normal mucosa. Combination stains of the nucleus, cytoplasm and membrane and the individual stains of these were seen in both OL and normal mucosa. Among the OSCC groups, the nuclear stains decreased as the grade of OSCC increased, whereas it was the reverse for cytoplasmic expressions. In the OL group, the combination stains of the cytoplasm, nucleus and membrane stains were seen only in mild dysplasia. In mild dysplasia, more cytoplasm and nuclear stains were seen when compared to severe dysplasias. Also, individual nuclear stains were seen only in severe dysplasias.



The observation in our study shows that the immunohistochemical expression of Gal-3 was gradually decreasing from normal oral mucosa to OL to OSCC [Figure 1].
Figure 1: (a) Gal-3 expression in normal mucosa. (b) Gal-3 expression in OL (40 × magnification). (c) Gal-3 expression membrane staining in OL (40 × magnification). (d) Gal-3 expression in OSCC (10 × magnification). (e) Gal-3 expression in OSCC (40 × magnification). (f) Gal-3 expression membrane staining in OSCC (40 × magnification)

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   Discussion Top


Gal-3 is expressed widely in epithelial and immune cells and has been reported to be involved in various biological phenomena. Several studies have demonstrated the involvement of Gal-3 in tumourigenesis and have emphasised their diagnostic and prognostic value, particularly for squamous cell carcinoma of the head and neck regions.

In our study, Gal-3 expression was found higher in normal mucosa when compared to OL and OSCC in the decreasing order. Among the OL groups, Gal-3 expression was seen high in mild dysplasias when compared to moderate and severe dysplasias, which shows a decrease in the expression of Gal-3 as the epithelium acquires dysplastic characteristics. This apparently reduced expression may indicate early biomolecular alterations in dysplastic tissue. Among the OSCC groups, the expressions were found more in well-differentiated OSCCs when compared to the moderately differentiated OSCCs, which also shows that as the grade of OSCCs increases, the staining expression of Gal-3 decreases. This apparently decreased expression may indicate early biomolecular alterations in altered tissues such as OSCCs. Similar results of reduced expression of Gal-3 in OSCCs were shown in other studies also.[13],[17],[18],[19] Studies have also shown a reduction in the expression of Gal-3 in intraepithelial and cervical carcinoma, ovarian carcinoma, and uterine adenocarcinomas when compared to normal tissues. However, overexpression of Gal-3 has been observed in other tumours such as pancreatic, gastric, thyroid, renal, and head and neck squamous cell carcinoma. A possible explanation for this variance could involve the intracellular localisation of Gal-3 within the cancer cells as the molecule's function will vary depending on whether it is in the nucleus, cytoplasm, nucleus, or extracellular spaces for that matter. Hence, our study also suggests that the Gal-3 expression could be related to cell differentiation rather than tumour progression and prognosis.

A shift in the site of expression of Gal-3 from the membrane to the intracellular medium is observed when the epithelium becomes dysplastic. In our study, both cytoplasmic and nuclear expressions were seen in most of the cases. In addition to these cellular localisations, membrane staining was also detected in few cases. In OSCC, a shift in the cytoplasm to nuclear staining was seen as the severity increased. Nuclear stains were seen more in normal mucosa and also seen more when the severity decreased. Ectopic expression of Gal-3 occurs during the early stage of phenotypic transformation of the tumour, and increased cytoplasmic expression is related to tumour progression. The intracellular (mainly cytoplasmic) localisation of Gal-3 is probably related to its antiapoptotic action. Alterations in the expression of apoptosis-related proteins are important indicators of the malignant transformation of a lesion.[20]

In our study, even though there was an overall decrease in the immunoexpression of galectin-3 in OSCC, few specimens showed higher expressions when compared to OL and normal mucosa. This differential expression of Gal-3 may be due to the involvement of galectin-3 in various biological phenomena and the change in localisation from the nucleus to the cytoplasm and to extracellular spaces. For more accurate results to be obtained to assess the staining expressions of Gal-3, we can increase the sample sizes of OSCC, OL and the normal mucosa. Further research on specific antigen recognition sites of Gal-3 antibodies with larger samples and clinical follow-ups may provide additional insight into the role of Gal-3 as a potential biomarker of oral carcinogenesis.


   Conclusion Top


OL is the most important indicator for determining the risk of the development of oral cancer in premalignant lesions. Evaluation of Gal-3 may provide additional information beyond histopathological features in oral dysplastic lesions. Due to its involvement in diverse biological functions, many attempts have been made to use Gal-3 as a therapeutic target. Gal-3 suppresses apoptosis and increases the resistance of tumour cells to chemotherapeutic drugs; it has been used as a target to resensitise cancer cells to chemotherapeutic drugs also. Galectin-3C is also an effective adjuvant therapy in advanced, refractory multiple myeloma and ovarian cancer. With further research on the different roles of Gal-3, it can be used as a promising target for tumour therapy in OSCC. Based on the results of our study, it could be concluded that Gal-3 may be a valuable marker for evaluating the malignant potential of OL and tumour progression of OSCC.

Acknowledgements

The authors acknowledge PMS College of Dental Science and Research for facilitating the infrastructure. The authors also acknowledge Dr. Calwin Parthibaraj S, PhD, for the statistical analysis works to complete the research work.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient (s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Dr. Freeda M Selvaraj
PMS College of Dental Science and Research, Vattapara, Trivandrum - 695 028, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.ijdr_157_22

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