Expression of breast cancer gene 2 in oral squamous cell carcinoma and its correlation with the metastatic potential: A retrospective study

Niharika Hitendra Mistry; Suchitra Rajesh Gosavi; Akshay Dhobley; Shubhangi Mishra; Pooja Kherde

doi:10.4103/jpo.jpo_25_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 2 | Issue : 2 | Page : 120-124

Expression of breast cancer gene 2 in oral squamous cell carcinoma and its correlation with the metastatic potential: A retrospective study

Niharika Hitendra Mistry¹, Suchitra Rajesh Gosavi¹, Akshay Dhobley¹, Shubhangi Mishra², Pooja Kherde¹
¹ Department of Oral and Maxillofacial Pathology, Government Dental College and Hospital, Nagpur, Maharashtra, India
² Department of Oral and Maxillofacial Pathology, Bhabha College of Dental Sciences, Bhopal, Madhya Pradesh, India

Date of Submission	30-Oct-2022
Date of Decision	14-Nov-2022
Date of Acceptance	23-Nov-2022
Date of Web Publication	06-Feb-2023

Correspondence Address:
Dr. Niharika Hitendra Mistry
Department of Oral and Maxillofacial Pathology, Government Dental College and Hospital, Nagpur - 440 003, Maharashtra
India

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jpo.jpo_25_22

Abstract

Background: Breast cancer gene 2 (BRCA2), a tumor suppressor gene, has been extensively evaluated in various cancers and correlated with metastatic potentials. However, to the best of our knowledge, the correlation has not been reflected in the literature in patients with oral squamous cell carcinoma (OSCC). Any positive correlation may yield the protein as an exploitable marker for the prediction of metastasis and target for chemotherapy in patients with OSCC.
Materials and Methods: This observational retrospective study was conducted on 54 tissue samples, 18 samples each of normal oral mucosa, and of OSCC with and without cervical lymph node metastasis. The study samples were immunohistochemically stained with BRCA2 antibody. The staining index was calculated as a product of the percentage of positive cells per high-power field (HPF) (A) and the staining intensity (B).
Results: Based on intergroup comparisons, it was found that the percentage of positive cells/HPF, staining intensity, and staining index were higher in OSCCs with cervical lymph node metastasis and lower in normal oral mucosa samples. It was found that a large number of the normal oral mucosa samples (n = 13) did not show any BRCA2 immunoreactivity and a large number of the OSCC samples with cervical lymph node metastasis displayed high BRCA2 immunoreactivity (n = 15).
Conclusion: Overexpression of BRCA2 is linked to the presence of metastases in patients with OSCC and has the potential to be utilised as a marker to predict the outcome of the patient's treatment. The findings may be used in the future to develop targeted therapies, depending on the direction that future research follows.

Keywords: Breast cancer gene 2, metastasis, oral squamous cell carcinoma, tumor suppressor gene

How to cite this article:
Mistry NH, Gosavi SR, Dhobley A, Mishra S, Kherde P. Expression of breast cancer gene 2 in oral squamous cell carcinoma and its correlation with the metastatic potential: A retrospective study. J Precis Oncol 2022;2:120-4

How to cite this URL:
Mistry NH, Gosavi SR, Dhobley A, Mishra S, Kherde P. Expression of breast cancer gene 2 in oral squamous cell carcinoma and its correlation with the metastatic potential: A retrospective study. J Precis Oncol [serial online] 2022 [cited 2023 Mar 28];2:120-4. Available from: https://www.jprecisiononcology.com//text.asp?2022/2/2/120/369219

Introduction

Oral squamous cell carcinoma (OSCC), practically known as oral cancer due to crowning 90% to 95% diagnoses of all the malignant lesions of the oral cavity, is a result of an accumulation of multiple genetic alterations modulated by environmental influences and hereditary predispositions.^[1] Numerous methods have been proposed that can mark and analyze these genetic alterations and aid in the diagnostic and therapeutic approaches toward the life-threatening disease. One of the genetic alterations reported is the breast cancer gene (BRCA) that responds promptly to DNA damage and is involved in recombination-mediated DNA repair, thereby maintaining genetic stability. BRCA1 and BRCA2 are the key tumor suppressor genes and any mutation in them leads to the development of cancers, familial breast, and ovarian cancers, in particular.^[2] More recently, the detection of BRCA1/2 mutations has recently been done in cancers of the maxillofacial region as well.^[3] BRCA1/2 have been revealed to have been designated with their critical role in the development of OSCC. The invasive fronts of the histological sections of OSCC have been seen to exhibit altered subcellular localizations and immunostaining of the genes, suggestive of the genes being an aid in better therapeutic outcomes if detected early for their mutations.^[4]

The expression of BRCA2 gene has been correlated in a recent study with the different histological grades of OSCC and a positive correlation has been derived.^[4] More recently, the role of BRCA2 has been implicated in the metastatic and recurrent potentials of breast cancers.^[5] Both these recent findings seed relevance in the pursuit of studying the correlation of the gene with the status of metastasis in cases of OSCC. To the best of our knowledge, the expression of BRCA2 has not been lucid or elaborated in cases of metastatic OSCC. Thus, we attempted to evaluate the BRCA2 immunohistochemical expression in cases of OSCC with and without cervical lymph node metastasis and had foreseen the gene to be a marker for predicting metastasis in patients with OSCC.

Materials and Methods

This retrospective study was conducted on biopsy samples of OSCC patients from Central India, reporting to the Department of Oral Pathology and Microbiology, Government Dental College and Hospital, Nagpur. Careful statistical calculation of sample size giving a good external validity to the study dictated us to include 18 samples each of OSCC with and without cervical lymph node metastasis. The biopsy samples of patients with complete clinical data and with no history of any systemic disease or radiotherapy or chemotherapy before the surgical resections of the specimen were included in the study. The study samples were divided into three groups: Group 1, with 18 tissue samples of the normal oral mucosa; Group 2, with 18 tissue samples of OSCC without cervical lymph node metastasis, and Group 3, with 18 tissue samples of OSCC with lymph node metastasis.

The tissue samples were processed for immunohistochemical staining with the BRCA2 antibody following the ideal laboratory protocol. Two independent oral pathologists scored the stained slides. Brown cytoplasmic staining was considered a positive immunoreactivity for BRCA2. The staining index score was calculated as a product of the percentage of positive cells per high-power field (HPF) (A) and staining intensity (B). The percentage of positively stained epithelial cells was scored as 0, for <10% of the cells showing immunoreactivity; as 1, for 10%–40% of the cells; as 2, for 40%–70% of the cells; and as 3, for >70% of the cells. The staining intensity was scored as 0, for no reactivity; as 1, for weak; as 2, for moderate; and as 3, for strong immunoreactivity.

The study was approved by the Institutional Ethical Committee and was in accordance with the Declaration of Helsinki.

Statistical analysis

Data collected were compiled in an MS Office Excel worksheet and subjected to appropriate statistical tests using the IBM SPSS Statistics for Windows, version XX (IBM Corp., Armonk, N.Y., USA). The categorical data, means, and standard deviations of the numerical data in each group were depicted as frequencies and percentages. The values were compared among the different study groups using the Chi-square test. The normality of numerical data was checked using Shapiro–Wilk test or Kolmogorov–Smirnov test. For numerical continuous data following a normal distribution, intergroup comparison (>2 groups) was done using a one-way analysis of variance (ANOVA) test, or else a nonparametric substitute, the Kruskal–Wallis ANOVA test was used. Keeping alpha error at 5%, beta error at 20%, and power at 80%, P < 0.05 was considered statistically significant.

Results

Of the 54 tissue samples included in the study, 21 samples belonged to female patients and 33 to male patients, with 20 samples from the alveolobuccal complex, 16 from the buccal mucosa, 7 from the gingiva, 1 from the lower lip, and 10 from the tongue [Table 1]. Histologically, of the 36 samples of OSCC, 12 samples were diagnosed with well-differentiated SCC, 12 with moderately differentiated SCC, and 12 with poorly differentiated SCC, with six samples being Staged I as per the TNM staging, four samples as Stage II, 10 samples as Stage III, and 16 samples as Stage IV [Table 2].

Table 1: Distribution of the oral squamous cell carcinoma samples according to the site

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Table 2: Distribution of the oral squamous cell carcinoma samples according to the TNM staging

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Of all the 54 samples, 13 samples showed <10% of the cells/HPF to be immunoreactive for BRCA2, 10 samples showed 10%–40% of the cells/HPF to be immunoreactive, 15 samples showed 40%–70% of the cells/HPF to be immunoreactive, and 16 samples showed >70% of the cells/HPF to be immunoreactive. On the evaluation of the staining intensity, 13 samples showed inappreciable staining intensity, seven samples showed weak, 20 samples showed moderate [Figure 1] and [Figure 2], and 14 samples showed strong staining intensity [Figure 3]. Thus, 13 samples each were scored 0, 4, and 9 for the BRCA2 immunoexpression, six samples as 1, five samples as 2, and four samples as 6 [Table 3] and [Graph 1].

Figure 1: IHC stained photomicrograph shows low-to-moderate BRCA2 staining in tumor islands. (×10). IHC: Immunohistochemical, BRCA2: Breast cancer gene 2

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Figure 2: IHC stained photomicrograph shows low-to-moderate BRCA2 membranous staining in tumor cells (×40). IHC: Immunohistochemical, BRCA2: Breast cancer gene 2

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Figure 3: IHC stained photomicrograph shows moderate BRCA2 membranous staining in tumor cells (×40). IHC: Immunohistochemical, BRCA2: Breast cancer gene 2

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Table 3: Intergroup comparison of frequencies of the percentage of positive cells/high-power field (A), staining intensity (B), and staining index (A × B)

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Intergroup comparisons revealed that the percentage of positive cells/HPF, staining intensity, and thereby the staining index was highest in the samples of OSCC with cervical lymph node metastasis and lowest in normal oral mucosa, with most tissue samples of the normal oral mucosa (n = 13) showing absence of any BRCA2 immunoreactivity [Figure 4], [Figure 5] and [Figure 6] and of OSCC with cervical lymph node metastasis showing high BRCA2 immunoreactivity (n = 15).

Figure 4: IHC stained photomicrograph shows high BRCA2 membranous staining in tumor islands (×10). IHC: Immunohistochemical, BRCA2: Breast cancer gene 2

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Figure 5: IHC stained photomicrograph shows high BRCA2 staining in tumor islands (×40). IHC: Immunohistochemical, BRCA2: Breast cancer gene 2

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Figure 6: IHC stained photomicrograph shows high BRCA2 membranous and cytoplasmic staining in tumor cells (×40). IHC: Immunohistochemical, BRCA2: Breast cancer gene 2

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Discussion

The BRCA2, a tumor suppressor gene, has been known for its involvement in the development of epithelial malignancies, namely, breast and ovarian cancers, upon undergoing mutations. The involvement has so strongly been evidenced that in the Jewish population, who exhibit a higher likelihood of BRCA2 mutations, even the males have been seen to develop breast cancers.^[6] Integral parts of healthy control of cell division are the tumor suppressor genes that limit abnormal proliferation. Mutations may inactivate the genes relieving the cells of the growth restraint. The BRCA2 has widely been studied in the cancers of the breast, ovaries, prostate, colon, and pancreas, and increased surveillance for patients with this gene mutation has been advocated. Furthermore, personalized treatment protocols are devised for cancer patients with BRCA1/2 mutations.^[6]

With this background, we attempted to evaluate the expression of BRCA2 in OSCC patients and if it can be used as a marker for predicting the status of metastasis in the patients. We found that BRCA2 expression was higher in OSCC patients as compared to individuals with normal oral mucosa. These results are in accordance with several studies on BRCA2 exhibiting higher expressions in epithelial malignancies as well as with the study done on different histological grades of OSCC, with BRCA2 expression being evidently correlated with the invasive front of the tumors.^[4] Furthermore, the BRCA2 expression was higher in OSCC patients exhibiting cervical lymph node metastasis than in the ones with no metastasis. These results have no comparable equivalent studies with regard to the findings in OSCC patients; however, the results can be seen to be in accordance with the finding of higher BRCA2 expression being related to higher metastatic and recurrent potentials in breast cancer cell lines.^[5] Similarly, a case report on BRCA2-associated breast cancer patients extensively reviewed the literature to conclude similar facts about BRCA2 and also reported the manifestation of the orbital metastasis in the patient.^[7] Another case report added a case of metastasis of BRCA2 pathogenic variant of breast cancer to the fimbrial part of the Fallopian tube ^{More Details}, stressing the role of the genetic mutation in metastasis.^[8] Furthermore, the latest research has again emphasized the higher likelihood of brain metastasis in patients with BRCA1/2 invasive breast cancer.^[9]

Apart from the literature on the association of BRCA2 in the mechanism of metastasis of BRCA2-associated cancers and its diagnostic and prognostic potential of being exploited as a marker for the prediction of metastasis, the research also houses the clinical implication of this association. In a study conducted on oral cancer cell lines, it was observed that the downregulation or knockdown of BRCA2 helped in sensitizing the tumor cells to increased sensitivity to 5-fluorouracil chemotherapy.^[10]

Conclusion

BRCA2 being a tumor suppressor gene can be exploited as a marker for diagnosis and prognosis in OSCC patients using appropriate genetic tests. The higher BRCA2 immunoexpression in OSCC patients with cervical lymph node metastasis is clearly indicative of its potential in metastasis. This further stresses the future scope of its exploitation in the therapeutic management of patients with OSCC and the prevention of metastasis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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