ORIGINAL RESEARCH |
https://doi.org/10.5005/jp-journals-10015-2433 |
Immunohistochemical Expression of β-catenin in Different Grades of Oral Squamous Cell Carcinoma
1-3Department of Oral Pathology, Government Dental College and Hospital, Srinagar, Jammu and Kashmir, India
Corresponding Author: Rezhat Abbas, Department of Oral Pathology, Government Dental College and Hospital, Srinagar, Jammu and Kashmir, India, Phone: +91 9797033260, e-mail: writetoempire@gmail.com
Received on: 03 May 2024; Accepted on: 04 June 2024; Published on: 28 June 2024
ABSTRACT
Aim: Oral squamous cell carcinoma (OSCC) is the most common oral malignancy, accounting for up to 80–90% of all oral malignancies. This occurs as a result of the multistep accumulation of heterogeneous genetic changes. β-catenin binds with E-cadherin to form a cell adhesion complex and acts as a tumor suppressor by restricting the Wnt/β-catenin signaling pathway. When β-catenin mutates or loses contact with E-cadherin, it translocates to the nucleus and upregulates transcription factors (TCFs). We analyzed and compared β-catenin expression in various phases of OSCC.
Materials and methods: A retrospective study was conducted on 60 formalin-fixed paraffin-embedded tissue blocks (20 well-differentiated OSCC, 20 moderately differentiated OSCC, and 20 poorly differentiated OSCC). Diagnosis was made using hematoxylin and eosin, and oral mucosa was used as a control.
Results: Statistically substantial downregulation of β-catenin was seen with increasing OSCC grades. As the disease progressed, β-catenin moved from the membrane to the cytoplasm. Nuclear positivity was found in poorly differentiated squamous cell cancer. Nuclear migration activates TCFs, which promote cell proliferation.
Conclusion: The study revealed a good prognostic role of both β-catenin in OSCC.
Clinical significance: The marker can be used for prognostic as well as therapeutic purposes.
How to cite this article: Abbas R, Latoo SH, Dar MS. Immunohistochemical Expression of β-catenin in Different Grades of Oral Squamous Cell Carcinoma. World J Dent 2024;15(5):401-405.
Source of support: Nil
Conflict of interest: None
Keywords: β-catenin, Immunohistochemistry, Oral squamous cell carcinoma, Tumor markers, Wnt pathway.
INTRODUCTION
Oral squamous cell carcinoma (OSCC) is the most common of all malignant neoplasms of the oral cavity. It is defined as ”a malignant epithelial neoplasm exhibiting squamous differentiation as characterized by the formation of keratin and/or the presence of intercellular bridges.”1 The majority of oral cancer cases and one-third of the worldwide burden are found in India.2 The health of nations undergoing economic transformation is seriously threatened by oral cancer. Since oral cancer is one of the more prevalent types of cancer in India, it is the most significant issue for community health.
A series of diverse genetic mutations eventually lead to OSCC. Alcohol usage, tobacco or betel quid use, the human papillomavirus, and poor nutrition are all significant risk factors for OSCC. The tongue, buccal mucosa, and palate are the most often impacted areas by OSCC. It is linked to substantial morbidity, death, and a cumulative 5–7-year survival rate of <50%. This depressing tendency is ascribed to a number of variables, such as late-stage detection, field cancerization, and innate biologic aggressiveness, including a predisposition for invasive growth and lymph node metastatic spread.3
Tumor markers are substances produced by the tumor itself or the body in response to the presence of cancer or certain benign diseases that can help in diagnosing cancer and assessing tumor burden. The amount of production depends on the growth of tumor cells. Molecular markers are associated with the occurrence, progression, and prognosis of cancer.4
β-catenin is a dual-function protein involved in regulation and coordination of cell–cell adhesion and gene transcription. It is produced by the gene CTNNB1, which is found on chromosome 3p22. β-catenin connects E-cadherin to the actin cytoskeleton. Normally, β-catenin is located along the cell membrane. Adenomatous polyposis coli (APC) is a tumor suppressor gene that stops β-catenin from building up in the cytoplasm by generating a macromolecular complex and causing its destruction via the ubiquitin-proteasome pathway. Wnt signaling prevents APC and promotes the movement of β-catenin from the cytoplasm to the nucleus. In the nucleus, β-catenin assembles into a complex transcription factor (TCF) that promotes cell growth. The stratified squamous epithelium’s structural integrity and organization depend on cadherin/catenin health. Transcriptional activity in the nucleus is elevated in cancers as a result of β-catenin stabilization in the cytoplasm.5 With this background, this study was undertaken to evaluate the prognostic role of β-catenin in OSCC.
MATERIALS AND METHODS
The study was conducted from January to September 2022 in the Department of Oral Pathology and Microbiology at the Government Dental College and Hospital in Srinagar after obtaining ethical approval from the Institutional Ethics Committee vide number OMFS/GDC-S/11399 dated 24th October 2021. The study was carried out on 65 formalin-fixed paraffin-embedded tissue blocks comprising 20 cases of well-differentiated OSCC, 20 cases of moderately differentiated OSCC, and 20 cases of poorly differentiated OSCC, with five cases of normal oral mucosa (NOM) taken as a control. Tumors were graded according to Broder’s criteria.6
Sections of 3–4 mm thickness were cut from each formalin-fixed and paraffin-embedded tissue block. Immunohistochemical studies were performed using polymer labeling methods. Sections were deparaffinized, rinsed with alcohol, and antigen retrieval was performed in a decloaking chamber using 10 mm Citra solution at 125°C for 30 seconds and then at 90°C for 10 seconds. Slides were naturally cooled and placed at room temperature. The slides were placed into an automatic stainer. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide in methanol for 10 minutes at room temperature. Slides were briefly washed in phosphate-buffered saline (PBS) and incubated with primary antibody against β-catenin (Biocare Medical) for 60 minutes. Sections were washed again with PBS, incubated with polymer for 30 minutes, and then washed again with PBS. Diaminobenzidine (DAB) was used as a chromogen for 10 minutes in hydrogen peroxide. Sections were then counterstained with hematoxylin, mounted, and examined for immunoreactivity by light microscopy.
The presence of a brown-colored end product at the site of the target antigen on the cell membrane, inside the cytoplasm, or in the nucleus was indicative of positive immunoreactivity. In each section, five light microscopic fields (200× magnifications) were randomly selected. Two observers independently recorded the intensity and percentage of β-catenin staining in each field. A pilot study was previously conducted to assess intra- and interobserver reliability. β-catenin expression was assessed as follows.
Staining Intensity
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Score 0 = No staining.
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Score 1 = Weak staining.
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Score 2 = Moderate staining.
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Score 3 = Intense staining.
Percentage Index
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Score 0 = No cell staining in any microscopic field.
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Score 1 = Around <10% of tissue staining positively.
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Score 2 = Around 10–50% of tissue stains positively.
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Score 3 = Around 50–80% of tissue stained positive.
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Score 4 = Around <80% of the tissue stains positively.
Final Immunoreactive Index
The immunoreactivity index was calculated as the product of the percentage index and the intensity index. Final grades were assessed as follows:
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Score 0–1 = Negative.
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Score 2–3 = Mild.
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Score 4–8 = Moderate.
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Score 9–12 = Strongly positive.
The recorded data were compiled and entered into a spreadsheet (Microsoft Excel) and then exported to the data editor of Statistical Package for the Social Sciences (SPSS) Version 20.0 (SPSS Inc., Chicago, Illinois, United States of America).
The variables were compared using the analysis of variance (ANOVA) test, with a p-value of <0.05 indicating statistical significance.
RESULTS
Immunolocalization of β-catenin in Various Groups
There was strong homogeneous membranous expression of β-catenin in four (80%) of NOM cases; however, one (20%) of cases showed cytoplasmic staining. No nuclear positivity was seen in normal mucosa. In cases of carcinoma, there was a dramatic shift of expression from membranous to cytoplasmic to nuclear as the grade increased (Table 1).
| Groups | Membranous | Cytoplasmic | Nuclear | Absent | Total |
|---|---|---|---|---|---|
| NOM | 4 (80%) | 1 (20%) | 0 | 00 | 05 |
| WDSCC | 8 (42%) | 6 (28%) | 3 (14%) | 3 (14%) | 20 |
| MDSCC | 6 (28%) | 8 (42%) | 6 (28%) | 00 | 20 |
| PDSCC | 3 (14%) | 3 (14%) | 8 (42%) | 2 (28%) | 20 |
Bold numbers signify total number of cases
Intensity of Staining of β-catenin in Various Groups
Normal mucosa sections showed intensity of tissue staining with a score of 3 in four (80%) of cases and a score of 2 in one (20%) of cases. The staining was profound in basal and spinous layers and least in the superficial layers. Score 1 was found in eight (40%) cases of well-differentiated squamous cell carcinoma (WDSCC), while scores 3, 6, and 0 were detected in six (30%), five (25%), and one (5%) cases, respectively. Moderately differentiated squamous cell carcinoma (MDSCC) presented with a score of 0 in five (25%) cases, score 1 in 10 (50%) cases, a score of 2 in three (15%) cases, and a score of 3 in two (10%) cases. Poorly differentiated squamous cell carcinoma (PDSCC) showed score 0 in 11 (55%) cases and a score of 1 in nine (45%) cases. The differences in intensity of staining among groups were statistically significant with p = 0.00002 (Fig. 1 and Table 2).
| Intensity of staining | NOM | WDSCC | MDSCC | PDSCC |
|---|---|---|---|---|
| Score 0 | 0 | 1 | 5 | 11 |
| Score 1 | 0 | 8 | 10 | 9 |
| Score 2 | 1 | 6 | 3 | 0 |
| Score 3 | 4 | 5 | 2 | 0 |
| Total | 5 | 20 | 20 | 20 |
F-statistic value = 13.1957, p = 0.00002; Bold numbers signify total number of cases
Figs 1A to D: (A) β-catenin expression in NOM (20×); (B) β-catenin expression in WDSCC (20×); (C) β-catenin expression in MDSCC (20×); (D) β-catenin expression in PDSCC (20×)
Percentage of Staining of β-catenin in Various Groups
Normal mucosa sections showed percentage of tissue staining with a score of 3 in two (40%) of cases and a score of 4 in three (60%) of cases. In WDSCC, a score of 0 was found in two (10%) cases, a score of 1 in three (15%) cases, a score of 2 in two (10%) cases, a score of 3 in five (25%) cases, and score 4 in eight (40%) cases. MDSCC presented with a score of 0 in seven (35%) cases, a score of 1 in four (20%) cases, a score of 2 in three (15%) cases, a score of 3 in four (20%) cases, and score of 4 in two (10%) cases. PDSCC showed a score of 0 in 14 (70%) cases, a score of 1 in three (15%) cases, and a score of 2 in three (15%) cases. The differences in percentages of cells with positive staining were statistically significant with p = 0.00001 (Table 3).
| Percentage of staining | NOM | WDSCC | MDSCC | PDSCC |
|---|---|---|---|---|
| Score 0 | 00 | 2 | 7 | 14 |
| Score 1 | 00 | 3 | 4 | 3 |
| Score 2 | 00 | 2 | 3 | 3 |
| Score 3 | 2 | 5 | 4 | 00 |
| Score 4 | 3 | 8 | 2 | 0 |
| Total | 5 | 20 | 20 | 20 |
F-statistic value = 25, p = 0.00001; Bold numbers signify total number of cases
Final Immunoreactive Score of β-catenin in Various Groups
There was strong homogeneous β-catenin staining in NOM with a final immunoreactive score of 12. Among carcinoma cases, WDSCC showed moderate staining with a score of 6, while MDSCC presented with mild staining with a score of 2. Negative staining was observed in PDSCC with a score of 1 (Fig. 2).
Fig. 2: Final immunoreactive score of β-catenin in different grades of OSCC
The difference in the final immunoreactive score among the groups was statistically significant with p = 0.012.
Therefore, it can be inferred from the above-observed results that as the grade of OSCC increases, β-catenin expression translocates from membrane to cytoplasm to nucleus, showing proliferative potential in this group of diseases.
DISCUSSION
The development of oral squamous cell carcinoma is a multistep process involving the accumulation of multiple genetic alterations modulated by genetic predisposition and environmental influences such as tobacco and alcohol use, chronic inflammation, and viral infections. All of these factors can lead to a wide range of genetic and molecular alterations that can be detected using a range of molecular studies. The development of cancer, in part, depends on faults in the structure of the tissue that are caused by deviations from the carefully orchestrated management of cell adhesion. Cell-cell adhesion in epithelial tissues is facilitated by the presence of the E-cadherin/β-catenin complex. A crucial step in the development of cancer is the separation of the E-cadherin/β-catenin complex from the cell membrane. In epithelial malignancies, membrane-bound E-cadherin is frequently lost, while β-catenin dissociates in the cytoplasm and accumulates in the nucleus as a TCF, both of which take place in tandem with the progression of tumors. Membranous E-cadherin and β-catenin down-regulation, as well as cytoplasmic/nuclear accumulation of β-catenin, have all been linked to cancer in the past and show potential as prognostic indicators.7,8
We have attempted to assess the expression of β-catenin in various grades of OSCC. From WDSCC to MDSCC, the expression of β-catenin was found to be significantly lower, and it was observed to be lowest in PDSCC.
With an increase in the grade of the carcinomas, there was a reduction of β-catenin staining. While MDSCC displayed focal heterogeneous staining, WDSCC had focal to decreased homogeneous staining patterns. PDSCC displayed negative staining; however, only a few cases revealed focal heterogeneous staining. The difference in staining pattern and intensity between the grades was statistically significant. The staining intensity was mild to moderate in WDSCC, medium in MDSCC, and mild to absent in PDSCC.
From WDSCC to MDSCC to PDSCC, there was a striking change in the expression from membrane to cytoplasm/nucleus. The prognosis is worsened because the PDSCC displayed severe nuclear positivity, which suggests that β-catenin is migrating within the cells and causing invasion and metastasis. Our findings were consistent with the observations of Bagutti et al.,9 Gasparoni et al.,10 Bankfalvi et al.,11 Tanaka et al.,12 Iwai et al.,13 Rosado et al.,14 Mahomed et al.,15 Wang et al.,16 Cai et al.,17 Liu et al.,18 Chaw et al.,19 Zaid,20 Kumar et al.,21 and Kudo et al.22 Bagutti et al.9 compared the expression of E- and P-cadherin with α-, β-, and γ-catenin. They discovered that cadherins and integrins were less expressed in poorly differentiated cancers; however, catenin expression was reduced in all tumors, regardless of differentiation level.9 Gasparoni et al. analyzed the subcellular location of β-catenin in normal and malignant keratinocyte cultures. Membranous β-catenin localization appeared in normal cells and decreased progressively in malignant cells, consistent with our findings.10 Bankfalvi et al. investigated the role of the E-cadherin/β-catenin complex and epidermal growth factor receptor in the advancement of OSCC. E-cadherin loss was linked to shorter survival, while lower β-catenin staining predicted lymph node metastasis.11 Tanaka et al. studied the expression of E-cadherin, α-catenin, and β-catenin in OSCC. Metastatic patients showed considerably lower expression levels of E-cadherin, α-catenin, and β-catenin compared to nonmetastatic patients.12 Iwai et al. found cytoplasmic accumulation of β-catenin in 90% of their cancer tissue samples.13 The findings were consistent with our study. Rosado studied the expression of E-cadherin and β-catenin in different histological grades of OSCC and noted a loss of the abovementioned markers with histological grades.14 Mahomed et al. discovered a strong link between E-cadherin/β-catenin and tumor differentiation. Regardless of nodal status, 93% of E-cadherin and 73% of β-catenin patients showed expression reduction in the invasive tumor front.15 Wang et al. investigated the correlation between the expression of desmoglein 3, desmocollin 3, and β-catenin in OSCC and lymph node metastasis/cell proliferation. When compared to normal oral epithelium, they discovered that all indicators were expressed less or not at all.16 Cai et al. examined the expression of β-catenin in oral squamous cell cancer. A higher grade of malignancy was associated with lower expression of β-catenin in the cell membrane and increased expression in the cytoplasm and nucleus.17 Our results also show reduced expression of β-catenin in OSCC as the grade worsens. Furthermore, there was a substantial shift of expression from membranous to nuclear in increasing grades of OSCC. Liu et al. investigated the expression of vimentin, E-cadherin, and β-catenin in OSCC using immunohistochemistry. In recurring tumors, they discovered increased vimentin expression and decreased E-cadherin expression. Invasive tumors showed reduced β-catenin expression.18 Chaw et al. investigated biomarkers for epithelial to mesenchymal transition in OSCC, including E-cadherin, β-catenin, APC, and vimentin. As OSCC’s histopathological grade increased, β-catenin expression shifted from membranous to cytoplasmic/nuclear staining.19 Zaid found a significant correlation between E-cadherin and β-catenin expressions in different histological grades of OSCC. Expression of β-catenin shifted from membrane to cytoplasm and nucleus as histological grades advanced.20 Kumar et al. found a significant difference in the expression of β-catenin between normal mucosa, WDSCC, and MDSCC.21 Wang suggested abnormal β-catenin expression in the progression of oral carcinomas, lymph node metastasis, and cell proliferation in OSCCs.16 Kudo et al. discovered that E-cadherin downregulation and membrane β-catenin degradation are necessary for oral cancer cell invasion and metastasis. Down-regulation of E-cadherin and β-catenin accumulation in the cytoplasm/nucleus may promote malignant transformation.22 A study by Balasundaram23 revealed the downregulation of molecular markers like β-catenin and E-cadherin in OSCC along with aberrant expression of vimentin.23
β-catenin is a multifunctional protein that maintains cell-cell adhesion by building complexes with the adhesion molecule E-cadherin. These complexes are responsible for the preservation of squamous epithelial homeostasis. The loss of β-catenin expression in the cell membrane, failure of cytoplasmic degradation mechanisms (related to activation of the Wnt canonical oncogenic pathway), and/or its translocation to the nucleus (acting as a TCF of oncogenes) are aberrant mechanisms with oncogenic implications in oral carcinogenesis.
Loss of β-catenin expression is an early event in oral tumorigenesis; its association with aggressive tumor behavior and disease recurrence underscore its potential as a prognostic marker. Reduction in intensity of β-catenin can be used as a reliable marker for the progression of potentially malignant disorders toward invasive malignancy. It may also aid in providing new strategies for early diagnosis, prevention, and treatment planning of oral cancer to bring down the mortality rate.
Given the small sample size, we propose that additional studies be conducted on a larger scale using multiple panels of markers or better methods, which may provide more insights to better clarify the molecular nature of tumor spread and aid in predicting the treatment protocol for the patient’s survival.
CONCLUSION
Reducing β-catenin expression and shifting its location from the cell membrane to the cytoplasm and nucleus may suggest malignant transformation in tissues. We conclude that both expressions of β-catenin can be employed as prognostic indicators in oral squamous cell cancer.
ORCID
Rezhat Abbas https://orcid.org/0000-0003-1413-8337
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