Oral squamous cell carcinoma (OSCC) is one of the most common cancers and accounts for more than 90% of oral malignancies (about 94%). It is the sixth most common cancers in worldwide. ¹ , ² , ³ According to the studies, the age of patient is one of the factors influencing the incidence of cancer. Alcohol and tobacco are known as risk factors for oral and oropharynx cancers. Nutritional factors such as Vitamin A and carotenoid deficiency may also play a role in oral cancers. ⁴ In the last two decades, the prevalence of head and neck cancers including oral and pharynx squamous cell carcinomas (SCCs) had increased. Prevalence increasing especially in young people without the common risk factors suggests the presence of other factors in the etiology of head and neck SCCs. ⁵ Other etiological factors such as genetics, diet, oncogenic viruses, and their effect on the physiological mechanism of cell proliferation control have been recommended. ⁶

Epstein–Barr virus (EBV) is a type of herpes virus that affects more than 90% of the world's population. ⁷ According to the studies, EBV infection is one of the most important cancer-related factors such as Burkitt's lymphoma, Hodgkin's and non-Hodgkin's lymphoma, nasopharyngeal carcinoma, and dendritic cell malignancy. EBV has also been related to esophageal and gastric cancers. ⁸ , ⁹ In addition, a significant association between epithelial and lymphatic cancers such as cancers of the abdomen, lungs, and uterus has been identified with this virus. ⁹ , ¹⁰ , ¹¹ In recent years, the hypothesis of a role of EBV which is an oncogenic virus in the development of OSCC has been proposed. ¹² In the head and neck, EBV infection and genetic alterations of the virus in epithelial cells can cause the development, progression, and invasion of nasopharyngeal carcinoma. Latency-associated proteins, latent membrane protein 1 (LMP-1) and LMP-2 proteins, and EBV nuclear antigens have been identified, including LMP-1 protein as an oncogene protein. ⁷ EBV infection has as important role in delaying cell differentiation. ¹³ , ¹⁴

In She et al.'s study, EBV infection had a direct relationship with increasing risk of OSCC. ¹⁵ Furthermore, in Kikuchi et al.'s study the expression of the latent esophageal variceal bleeding genes in the normal epithelium, inflamed gingiva, dysplastic epithelium, and OSCC has been observed. ¹⁶ Despite the studies on the association of SCCs and EBV, the role of tumorigenesis of EBV in oral epithelial cells is still unclear, therefore more studies in this field are necessary. Due to the high prevalence of SCCs in Iran and its unfavorable prognosis in the most patients and also the lack of studies on EBV in OSCC, the aim of this study was to investigate the EBV expression in OSCCs registered in the Department of Oral Pathology of Isfahan Dental School.

Patients and tissue selection

This cross-sectional study was approve in research and ethics committee of Isfahan (NO: 399247), the samples of 48 patients with a definitive diagnosis of OSCC in the archives of the Department of Oral and Maxillofacial Pathology, Dental School at Isfahan University of Medical Sciences were retrieved. Specimens with definitive diagnosis of OSCC, which were prepared by excisional biopsy and had complete information and tissue block suitable for immunohistochemical (IHC) staining, were included in the study. Demographic information including age, gender, location of lesion, and clinical feature were extracted from the files and recorded in the data collection form. The h and e stained slides of all samples were reviewed by two oral pathologists to confirm the diagnosis and check the quality of samples. Furthermore, the grade of malignancy of the tumor was determined based on the Anneroth et al. classification. ¹⁷

Immunohistochemical staining

For the detection of EBV expression by IHC staining, 3–4 μm sections were cut from the paraffin-embedded tissues. The tissue sections were deparaffinized with xylene and rehydrated with graded ethanol. For antigen retrieval, the sections were heated in a microwave oven at 96°C for 15 min in citrate buffer (0.01 M citrate buffer, pH 6.0), and then cooled at room temperature for 20 min. Endogenous peroxidase activity was blocked by incubation with 3% H2O2 in methanol for 20 min and the sections were washed with phosphate-buffered saline. Slides were treated with Ficin and incubated with the monoclonal mouse EBV antibody (Cell marque clone Epstein-Barr Virus (MRQ-47) Rabbit Monoclonal Antibody) at 1:100 dilution overnight at 2°C–8°C. The slides were then incubated with a biotinylated anti-rabbit secondary antibody (1:100 dilution) for 30 min at room temperature. The slides were parmounted with coverslip and bound antibody was detected for EBV. The tissue sections were considered positive by brown color cytoplasmic or/and nuclear staining of the tumor cells. EBV-infected Hodgkin's lymphoma was included as a positive control.

Assessment of immunohistochemical staining

To analyze IHC staining, all slides were examined by two oral pathologists blindly with light microscopy (Olympus BX41TF, Tokyo, Japan), and cells were counted at ×400 magnification in 10 randomly selected fields. Tissue samples with cytoplasmic and nuclear brown staining of tumor cells were considered positive. The epithelial cells were evaluated using the semi-quantitative scale: 0 (negative: without immunostained cells), +1 (<25% immunostained), +2 (25%–50%), and +3 (>50%). Furthermore, staining intensity was evaluated on the following scores: 0 (without immunostained cells), +1 (very low staining), +2 (low), +3 (moderate), and +4 (high). Staining intensity distribution (SID) score was calculated by multiplying the distribution by staining intensity. ¹⁸

Statistical analysis

The data obtained from clinical, histopathological, and IHC studies were analyzed by the Statistical Package for the Social Sciences, version 24.0 (SPSS Inc., Chicago, IL, USA) using t-test, Chi-square, and Fisher's exact test. The significance level was considered P < 0.05.

In this study, of 48 OSCCs were 23 (48%) negative samples and 25 (52%) positive samples in IHC staining for EBV virus. The mean age of the samples with a negative result was 65.52 ± 14.09 and the mean age of the positive samples was 61.28 ± 17.53. According to t-test, the mean age of the samples did not show a significant difference-based expression of EBV (P = 0.098).

Furthermore, Chi-square test show that no significant relationship was observed between EBV expression and gender in OSCCs (P=0.263) Table 1.{Table 1}

81.8% of buccal mucosa samples and 83.3% of oral floor OSCCs had EBV expression. Based on Fisher's exact test, a significant relationship was observed between the EBV expression and location of OSCCs (P = 0.035). Furthermore, according to Fisher's exact test, no significant relationship was observed between the EBV expression and the clinical presentation (P = 0.772).

Positive staining has been reported in 100% of poorly differentiated OSCCs. Based on Fisher's exact test, a significant difference was not observed between EBV expression and the grading of OSCCs (P = 0.38). The lowest SID index for EBV observed in well-differentiated OSCCs, while SID index has not different significantly between Moderate and poorly differentiated tumors Figure 1. According to Kruskal–Wallis test, the SID index in different grading of tumor was significantly different (P = 0.005). Mann–Whitney statistical test showed the significant difference between well and moderately differentiated tumors based on SID index (P = 0.007), and between well and poorly differentiated tumors (P = 0.024). However, the difference between moderate and poorly differentiated tumors was not significant (P = 0.814) Table 2. Figure 1

(a) Negative result for Epstein–Barr virus, (b) Epstein–Barr virus expression in Oral squamous cell carcinoma (Immunohistochemical staining, ×400).

According to the studies, viruses play a very important role in causing many malignant lesions of the head and neck, especially nasopharyngeal carcinoma, ¹⁹ but the pathogenic role of EBV in oral cancers is still unclear. In the present study, 52% of the samples showed a positive result for EBV in IHC staining. In the studies of Saravani et al. 16.7%, ²⁰ Shamma et al. 81.8%, ¹⁹ Acharya et al. 45.05%, ²¹ Yen et al. 82.5%, ²² Kikuchi et al. ¹⁶ Kis et al. 73.8%, ²³ Began 40%, ²⁴ Sand et al. 9.9%, ²⁵ Broccolo et al. 72.7%, ²⁶ Saleem et al. 22.6%, ¹² Polz-Gruszka et al. 1/26%, ²⁷ and Rahman et al. 59.67% ¹⁴ of the OSCCs were reported positive. The results of these studies indicate the important role of EBV in OSCC. ¹⁹ However, in many studies, the prevalence of EBV has been reported to be higher than in our study, which can be attributed to various diagnostic techniques of the virus including polymerase chain reaction (PCR), Nested PCR, Real-time-quantitative PCR, IHC, in situ hybridization in these studies. ¹⁵ Furthermore, this difference in results can be related to geographical areas and characteristics of people. ²⁰ For example, a significant association between EBV and OSCC has been observed in Europe and the United States, which may be related to individuals' genetics, socioeconomic status, and lifestyle in these areas. ²⁸ According to She et al., EBV infection is directly related to an increased risk of OSCC. ¹⁵ In the Jaloluli's study the incidence of EBV was seen in 55% of the samples from eight different countries. ²⁹ In EBV-associated malignancies, viral proteins have been identified that are involved in regulating proliferation, immune response, and cellular apoptosis. ³⁰ The most of the studies were stated that EBV DNA, mRNA, and EBV proteins are expressed in most of the OSCCs. ¹⁵

In the present study, the mean age of samples with a negative result of EBV (65.52 ± 14.09) was higher than positive samples (61.28 ± 17.53), but there was no statistically significant difference between the mean age and EBV expression that consistent with the studies of Saleem et al., ¹² Sand et al., ²⁵ She et al., ¹⁵ Shamma et al. ¹⁹ and Saravani et al. ²⁰ In Yen et al.'s study, the mean age of the negative cases was significantly higher than the positive cases. ²² In the Polz-Gruszka et al.'s study, most of the samples in the age group of 50–59 years had the EBV genome. ²⁷ The virus enters saliva, squamous epithelium, lymphoid organs, and B cells and can remain latent in the human body. The virus can reactivate without causing symptoms and can be detected in the saliva of infected people. ³¹

In the present study, most of OSCCs in men (58.1%) had EBV expression, but most of the female samples (58.8%) had not EBV expression, although there was no statistically significant difference between gender and EBV expression, which is consistent with the study of Saravani et al. ²⁰ In the Saleem et al.'s study, 97% of cases with EBV expression are men and only 3% are females. ¹² In the Shamma et al.'s study, a significant relationship was reported between gender and the incidence of EBV, which in this study also showed a higher expression of EBV in men. ¹⁹ One of the reasons for these results is related to the risk factors in men associated with OSCC. Various studies have examined the role of alcohol consumption and various forms of tobacco and its relationship with EBV in OSCCs. In the Acharya et al.'s study, Betel quid chewing was associated with EBV, but alcohol and smoking were not associated with the virus. ²¹ The study of Saleem et al., ¹² Polz-Gruszka et al., ²⁷ and Drop et al. ³¹ also reported an EBV association with oral cancer in tobacco users.

In the present study, a significant relationship was found between the EBV expression and location of the lesion. OSCCs in buccal mucosa and floor of the mouth have more frequently of EBV expression, which is consistent with the Saleem et al. ¹² and Deng et al. ⁷ studies. In Mao and Smith study, the tongue was the main location for EBV expression. ³² In addition, in Yen et al. ²² and Shamma et al. ¹⁹ study, no relationship was observed between the location of the lesion and EBV expression. In some OSCC studies, tonsils and the base of the tongue have been associated with a higher incidence of EBV. One of the reasons for the result may be the expression of a specific C3D receptor on the surface of the keratinized squamous epithelium of the buccal mucosa. This receptor has been evaluated in previous studies of nasopharyngeal carcinoma and its role in virus binding has been demonstrated. ¹² , ¹³

In the present study, the most exophytic lesions had EBV expression, although the relationship between the EBV expression and the clinical appearance of the lesion did not show a statistically significant difference. In Yen et al.'s study, a higher EBV expression was reported significantly in samples with exophytic appearance and ulcer lesions. The lesions with invading deeper tissues also showed a higher expression of EBV. ²²

In the present study, there was no significant difference between the grading of tumor malignancy and EBV expression, although SID index increased significantly with increasing tumor malignancy, which is consistent with the Saleem et al.'s ¹² and Broccolo et al.'s ²⁶ studies. In some studies, a significant relationship has been found between EBV expression and histological types of nasopharyngeal carcinoma. ¹² , ³³ In Broccolo et al.'s study, ²⁶ EBV expression was higher in nonkeratinized SCC than in keratinized SCC. The Polz-Gruszka et al.'s study ²⁷ also showed the more EBV expression in invasive types of oral tumors. Although the presence of EBV was not influential in the prognosis of OSCC in Deng et al. ⁷ study, but in Shamma et al. ¹⁹ study, the EBV expression was significantly higher in less differentiation tumors and the tumors with lymph node metastasis than well and moderate differentiation tumors. Thus, EBV infection delays epithelial cell differentiation and increases epithelial cells. ³¹

Restrictions in choosing more sample size and low number of OSCC with moderate and poorly differentiation of OSCC were the current research limitations.

EBV expression was observed in the most of OSCCs and especially in poorly differentiated tumors. The pathogenesis of OSCC may be related with EBV. OSCCs in buccal mucosa and floor of the mouth have more frequently EBV expression. Future studies on the mechanisms of EBV and their role in OSCC are required with larger sample sizes.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.