Expression of CD 20 B-Lymphocyte in oral epithelial dysplasia and oral squamous cell carcinoma: A comparative immunohistochemistry study

Abstract

Background:

As the progressive trends in the field of immunotherapy, it is very favourable to reconsider the role played by B lymphocytes in the tumor microenvironment. Both the protumorogenic and antitumorogenic responses have to be evaluated to formulate an effective immunotherapeutic protocol.

Aim and objective:

The study was primarily conducted to assess the qualitative expression of B lymphocytes in pretumorogenic (oral epithelial dysplasia) and tumorogenic environment (oral squamous cell carcinoma). The differential immunohistochemical staining of CD 20 immune marker was assessed in about 60 cases that included 30 cases of oral epithelial dysplasia and 30 cases of oral squamous cell carcinoma.

Results:

The study found significant correlation between CD 20 IHC immune expression and histopathological diagnosis along with significant correlation between the subject's age group and histopathological diagnosis.

Conclusion:

Modulating the immune response in a precancerous state can be highly beneficial in implementing better immunotherapeutic strategies to treat or prevent malignancy at an early stage.

BACKGROUND

Head and neck squamous cell carcinoma (HNSCC) accounts for more than 90% of all head and neck cancers, with approximately 650,000 cases diagnosed each year worldwide.[1] Despite aggressive multimodal strategies to treat HNSCC using combinations of surgery, radiotherapy, and chemotherapy, the 5-year overall survival of carcinogen-related HNSCC is only 40%-50%. The concept of “immunosurveillance” is the ability of the body to recognize self and nonself. Both natural and acquired immunity have undoubtedly proven their role in check and balance system against tumor progression.[2] In the rapidly evolving field of immunotherapy, identification of biomarkers to predict the immune response can make such a therapy one of the clinically effective treatments of oral squamous cell carcinoma (OSCC). There are many parameters/biomarkers and methods that have been introduced during the last three decades for the assessment of immune response and cluster differentiate 20 (CD 20) is one among them.

The focus of the present study is to determine how the premalignant microenvironment is different from the tumor microenvironment; this might be modulating the immune response during oral carcinogenesis. Thereby, the present study was done to find out presence and distribution of B-lymphocytes in tumoral or peritumoral areas with routine H&E stain and detect B-lymphocytes with CD 20 immunohistochemistry (IHC) staining method.

MATERIALS AND METHODS

The study included archival samples of histopathological diagnosed confirmed cases of oral epithelial dysplasia (OED) (n = 30) and OSCC (n = 30) retrieved from the Department of Oral and Maxillofacial Pathology, V.S Dental College and Hospital, Bangalore and Department of General Pathology, KIMS Hospital, Bangalore. It included demographic data (age, sex), habit history, and type of biopsy technique (incisional or excisional) along with histopathological grading of OSCC and OED were collected and tabulated. (Ethics committee approval was obtained in date of 19-11-2019, KIMS ethical committee).

The 70 cases were studied which included OED cases (n = 30), OSCC (n = 30), breast carcinoma cases (n = 5), and positive control and normal oral mucosa harvested during coronal flap surgery of impacted third molar cases (n = 30) as negative control. The inclusion criteria in this study included only histological diagnosed cases of OED and OSCC with inflammatory infiltrate. Cases of OED without inflammatory infiltrate were not included and histological diagnosed cases of primary intraosseous carcinomas, metastatic carcinomas of oral cavity, and other epithelial tumours were strictly excluded.

The formalin-fixed and paraffin-embedded tissue sections were stained immunohistochemically for the expression of CD 20+ B-lymphocyte using primary antibodies and the antigen-antibody complex was visualized using the DAB detection kit (k3368; Dako). The CD 20 positive cells were counted at the invasive front in OSCC cases and CD 20 positive cells in the OED cases were counted in the subepithelial connective tissue stroma.

A stretch of 10 consecutive high-power fields (40x magnification) at the area of maximum invasion and area with maximum density of inflammatory cells were chosen. For cell counting in mild risk and severe OED cases, a stretch of 10 consecutive high-power fields (40x magnification) at the subepithelial connective tissue stroma with maximum density of inflammatory cells are selected. Immunostaining for CD 20 was interpreted as negative (-); 5% or less of the epithelial cells were positive, weakly positive, or low-grade staining (+); 5% to less than 25% of the epithelial cells were positive, moderately positive, or intermediate-grade staining (++); 25% to less than 50% of the epithelial cells were positive and strongly positive or high-grade staining (+++); and 50% or more of the epithelial cells were positive.

RESULTS

The statistical analysis performed on various parameters of the present study includes age, sex, habit history of tobacco, type of biopsy, histopathological diagnosis, and IHC staining grade. Correlation tests were done to determine the significance between the two study groups of OSCC and OED. All statistical procedures were performed using Statistical Package for Social Sciences (SPSS) 20.0. All quantitative variables like age and gender were expressed in mean and standard deviation. Qualitative variables were expressed in percentages especially of staining of CD 20 IHC marker. Chi-square test was used for association between variables and correlation between different groups. Probability value (P < .05) was considered statistically significant.

Table 1 shows IHC grading of CD 20 distribution between the range of 80% (high) and 8.3% (low), with an intermediate grade of about 11.7.

Table 1.

Shows the type of IHC grading were 80.0% of the subjects high grading, 11.7% intermediate, and 8.3% with low grade

	n	%
High	48	80.0
Intermediate	7	11.7
Low	5	8.3

Table 2 whereas presents the correlation between expression of CD 20 IHC expression and histopathological diagnosis. It has about 70% high-grade expression in well differentiated squamous cell carcinoma (WDSCC), 20% intermediate, and 10% low. The moderately differentiated squamous cell carcinoma (MDSCC) and CD 20 IHC expression is about 80% high and 20% with intermediate grade. However, the correlation established between poorly differentiated carcinoma (PDSCC) is about 100% with high-grade expression of CD 20 IHC marker. Whereas in cases of OED in mild epithelial dysplasia is about 60% with high grade, 10% is intermediate, and about 30% as low grade. In moderate cases of epithelial dysplasia, there is about 80% high-grade IHC, 10% as intermediate IHC grading, and 10% low grade. However, in cases of severe epithelial dysplasia there is about 90% correlation with high grade and 10% correlation with intermediate IHC grading.

Table 2.

Shows the correlation between expression of CD 20 IHC expression and histopathological diagnosis

	High n (%)	Intermediate n (%)	Low n (%)	P
WDSCC	7 (70)	2 (20)	1 (10)
MDSCC	8 (80)	2 (20)	0	.03
PDSSC	10 (100)	0	0
Mild ED	6 (60)	1 (10)	3 (30)
Moderate ED	8 (80)	1 (10)	1 (10)
Severe ED	9 (90)	1 (10)	0

Table 3 demonstrates association between age and H/P diagnosis that there is a significant association between age and their histopathological diagnosis with significant P value of. 01. This is statistically highly significant as the P value was less than. 05.

Table 3.

Shows the correlation between different age groups and their histopathological diagnosis. This is highly significant as the P value was less than .01

	WDSCC n (%)	MDSCC n (%)	PDSSC n (%)	Mild ED n (%)	Moderate ED n (%)	Severe ED n (%)	P
25-35	1 (12.5)	0	1 (12.5)	4 (50.0)	1 (12.5)	1 (12.5)
36-45	3 (16.7)	3 (16.7)	4 (22.2)	1 (5.6)	4 (22.2)	2 (11.1)	.01*
46-55	0	5 (35.7)	3 (21.4)	3 (21.4)	3 (21.4)	0
56-65	3 (33.3)	1 (11.1)	0	1 (11.1)	0	4 (44.4)
>65	3 (27.3)	1 (9.1)	2 (18.2)	1 (9.1)	2 (18.2)	3 (27.3)

*: P value is highly significant. Chi-square test; P value < .05 is statistically significant

The H&E-stained sections observed under the light microscope shows CD 20 expression in OSCC and OED. The prominent areas assessed were peritumoral areas, tumoral areas, and inflammatory cells in the subepithelial connective tissue.

DISCUSSION

OSCC consists of more than 90% of oral cancer. The average incidence of new cases of oral cancer worldwide is around 354,864 (2% of all cancers). Among them, 227,906 (64.22%) cases are reported from Asia, where India accounted for 107,424 (30.27%) incidences as per 2018 data.[3]

The application of T cell as an immunotherapeutic tool has already gained acceptance especially in the cases of HNSCC. The role of humoral response, especially the role played by B lymphocytes is still considered controversial for their protumorogenic and antitumorogenic role in tumor microenvironment.

Mature B lymphocytes constitute about 10%-20% of circulating peripheral lymphocyte population. The function of B lymphocytes in humoral immune system includes producing antibodies, secreting cytokines, acting as antigen presenting cells, and as regulatory molecules. B cells recognize antigens through the B cell receptors complex on their cell membrane and evokes an antigen-antibody interaction.[4] B lymphocytes identify tumor-associated antigens and secrete specific antitumor antibodies as an immune reaction against tumor progression.

The present study highlights the importance of age group, as there is an increase in rise of oral cancer cases in younger individuals. The mean age group that as the maximum subjects is from 36-45 years, that was about 30%. The results of the study are similar to Abdulla R et al. (2018)[5] study conducted across South India between 1996 and 2012. The present study had similar results to the study conducted by Shruthi Singh et al. (2020),[6] on prevalence of OSCC and OED.

The risk of malignant tumors increases with age.[7,8,9,10,11,12,13] The present study shows a correlation with age and histopathological diagnosis, that is consistent with the concept of immunosenescence with age progression. Studies conducted by various author like on immune system and age like Kline GH et al. (1999),[14] Caruso C et al. (2013),[15] and Cepeda S et al. (2018)[16] also proves the same especially age changes related to B lymphocytes.

The present study is at a preliminary attempt to show the immune response especially B lymphocytes in epithelial dysplasia and OSCC. The role of B lymphocytes is critically expressed in pretumor environment or premalignancy state. The present study on the cases of epithelial dysplasia shows similar study results as Ellis and Auclair.[17] (1996) done in HLA/DR-positive cells. The increase in immune cell infiltration with progression of oral epithelium from hyperkeratosis to dysplasia and carcinoma is noted by G Gannot et al. (2002).[18] The study done on human tongue tissues (n = 53) diagnosed as hyperkeratosis (11 cases), mild dysplasia (nine cases), moderate and severe dysplasia (14 cases), and squamous cell carcinoma (19 cases) The results revealed that in the tongue lesions, the changes in the epithelium from normal appearance to transformed were accompanied by a corresponding increase in the infiltration of CD4, CD8, CD14, CD19+20, and HLA/DR-positive cells. The most significant change was an increase in B lymphocytes in tongue lesions and was in accordance with the transformation level.

The humoral response in the tumor microenvironment especially lymphocytic infiltration into the tumor microenvironment (TME) is generally considered to represent host immunity against tumors. The CD 20 being a surface immune marker that acts as an independent prognostic marker in early-stage oral-tongue cancer, which emphasizes the biological impact of clustered B-cells and supports their critical role in antitumor responses in OSCC. Tsou P et al. (2016),[19] Lao X M et al. (2016),[20] and Wirsing A M et al. (2018)[21] study results suggested the antitumor response of B lymphocytes in the tumor microenvironment. This includes secretion of antibodies, presentation of tumor antigen to adjacent T-cells, and production of immune-potentiating cytokines, such as IFNγ and IL12.[22,23,24,25,26] On the other hand, B-cells may mediate a protumorogenic effect through induction of neovascularization, becoming regulatory B-cells (Bregs). This was suggested from the results of the study Zhou X et al. (2016).[27] The other studies conducted by de Visser KE et al. (2005)[28] and Ammirante et al. (2010)[29] suggested the production of immune-suppressive cytokines, such as IL10, IL-35, and TGFβ would promote the protumorogenic response in the tumor microenvironment.

The results presented illustrated in the present study shows an increased expression of CD 20 B lymphocyte in advanced stages of OSCC. In PDSCC there was 100% expression of CD 20 IHC marker that suggested an increased humoral response of B lymphocytes in the tumor microenvironment, subsequently associated as in MDSCC and WDSCC. The study results were consistent with findings of C. Phanthunane et al. (2021)[30] study, using multiplex in situ immunofluorescence and computational image analyses of 138 patients with T1-T2 primary oral-tongue squamous cell carcinoma, observed a high density of CD 20 cells that clustered together in the IM-S regions. The study concluded that the so-called CD 20 Cluster Score acts as a strong independent prognostic factor in early-stage oral-tongue cancer. However, the study results of Wouters MCA et al. (2018)[31] and Gentles AJ et al. (2015)[32] contradicting pointing out that tumor infiltrated B-cells and B-cells–associated genes expressed in cancers tend to give a negative prognosis or even no effect on patients' survival.

The CD 20 immune marker provides adequate evidence of B lymphocytic infiltration into the peritumoral, tumoral, and connective tissue of dysplastic lesions. The study ascertained the expression of B lymphocytes in the tumor microenvironment through the differential staining of CD 20 IHC marker.

The future perspectives to validate the results of the present study can include to assess the B lymphocytes by quantitative measures to signify the B cell population and composition. Additionally, larger sample size and advanced molecular techniques could also aid in the study.

CONCLUSION

This study assessed the clinical parameters and the expression of CD 20 in OSCC (n = 30) and OED (n = 30) cases using IHC. This study revealed that there is significant variation in expression of CD 20 B lymphocytes in both malignant and potentially malignant cases. Age as a clinical parameter of the present study correlated with the histopathological diagnosis of OSCC and OED cases. The association with age and immune response was ascertained and found similar to previous studies done in the same field. The probable humoral immune response through B lymphocytes is expressed both in pretumor and tumor microenvironment. However, the dual roles played by B lymphocytes are to be critically analyzed and assessed through advanced research modalities. Possibilities of B-lymphocyte associated with immune therapy can be fairly considered as a treatment option or as an adjuvant therapeutic aid in future.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.