Journal of Dr. NTR University of Health Sciences

ORIGINAL ARTICLE
Year
: 2019  |  Volume : 8  |  Issue : 4  |  Page : 233--237

“A study on association of epstein barr virus in oral squamous cell carcinoma using polymerase chain reaction technique.”


M Mary Prathyusha1, Kiran K Kattappagari2, Deepika Chowdary2, Poosarla C Shekar2, Dasarathi Alivelu2, Baddam V R. Reddy2,  
1 W/O. Dr. Ch. Vijay Prakash, Flat No-401, KNR and SSR Grand, Kannavari Thota, Guntur, Andhra Pradesh, India
2 Department of Oral Pathology, Sibar Institute of Dental Sciences, Guntur, Andhra Pradesh, India

Correspondence Address:
Dr. M Mary Prathyusha
W/O. Dr. Ch. Vijay Prakash, Flat No-401, KNR and SSR Grand, Kannavari Thota, 4th Line, Guntur -522 002, Andhra Pradesh
India

Abstract

Background and Aims: Head and neck cancer is the most common type of cancer in word wide and Oral squamous cell carcinoma (OSCC) accounts for 90% of oral cancers and it represents the sixth most frequent malignant tumour world wide. The main etiological factor causing oral squamous cell carcinoma is tobacco consumption in the form of chewing or smoking, there are few virus such as Human Papilloma virus, Herpes simplex virus and Epistein barr virus are also contributory factor for causing oral squamous cell carcinoma. Among these viruses, Epstein Barr virus shown that increased risk of oral cancer. The present study was conducted to see association between Epstein Barr virus and oral squamous cell carcinoma using polymerase chain reaction technique. Methods: A total of 20 archival tissue blocks which are histopathologically confirmed OSCC and twenty cases of healthy mucosal tissue from retro molar area of the oral cavity. For all these cases DNA extraction was done and subjected to polymerase chain reaction. The results were analysed using statistical package for social sciences (SPSS) version 20.0. Results: Among twenty cases of OSCC twelve were males and eight were females. The mean age ranges from 51.25 ± 2 years in OSCC cases. Out of 20 cases of OSCC, 4 cases (20%) were positive and 16 cases (80%) were negative for EBV. Whereas the mean age ranges from 30.9 ± 2 years, and 11 were males and 9 were females in control group. Among 20 controls, 10 controls (50%) were positive and 10 controls (50%) were negative for EBV. Conclusion: The prevalence of EBV was significantly high in controls than OSCC cases. These observations suggest that location such retro molar area is one of the important factors for considering prevalence of EBV. Further studies with large sample size required to draw the role of location, habits in pathogenesis of EBV causing OSCC.



How to cite this article:
Prathyusha M M, Kattappagari KK, Chowdary D, Shekar PC, Alivelu D, R. Reddy BV. “A study on association of epstein barr virus in oral squamous cell carcinoma using polymerase chain reaction technique.”.J NTR Univ Health Sci 2019;8:233-237


How to cite this URL:
Prathyusha M M, Kattappagari KK, Chowdary D, Shekar PC, Alivelu D, R. Reddy BV. “A study on association of epstein barr virus in oral squamous cell carcinoma using polymerase chain reaction technique.”. J NTR Univ Health Sci [serial online] 2019 [cited 2020 Feb 27 ];8:233-237
Available from: http://www.jdrntruhs.org/text.asp?2019/8/4/233/273128


Full Text



 Introduction



Head and neck cancer is one of the ten most common types of cancer worldwide distressing more than 500,000 individuals every year.[1] Oral squamous cell carcinoma (OSCC) is most frequent accounting for over 90% of oral cancers. It represents the sixth most frequent malignant tumor worldwide[2] Tobacco, smokeless tobacco products such as gutka, pan masala, betel quid and alcohol are the risk factors for the development of OSCC.[3] Viruses such as human papillomavirus (HPV), Epstein-Barr virus and herpes simplex virus-1 (HSV-1) are also implicated to play a role in the development of OSCC.[4] Epstein Barr Viruses (EBVs), member of herpes virus family are small DNA viruses, transmitted by salivary exchange and may initially infect the epithelial cells of the oropharynx, posterior nasopharynx, parotid gland and duct, and possibly tonsillar lymphocytes.[5] There is an increasing evidence of causal association of EBV and OSCC. Several studies have shown that EBV is associated with increased risk of oral cancer, independent of exposure to tobacco and alcohol. The association between EBV and OSCC is because of its detection in oral dysplastic lesions and oral cancer[6] EBV infection is transmitted from host to host via saliva, and the virus pass through the oropharyngeal epithelium to the B lymphocytes. The virus enters the B cell and cause it to proliferate and spread through the B-cell compartment.[7] EBV has been implicated in the development of a wide range of cancers such as Burkitt's Lymphoma, Hodgkin's Disease, Non Hodgkin's Lymphoma in immune competent individuals, nasopharyngeal carcinoma, breast cancer, leiomyo sarcomas, EBV-associated lymphomas in immune compromised individuals, X-linked Lympho proliferative disorders, Posttransplant lympho proliferative disorders, AIDS-related lymphoproliferative disorders.[8] There has been wide range of variation in EBV positivity rates in nasopharyngeal undifferentiated carcinomas and different sites in head and neck region. EBV is most commonly present in the saliva of oral cavity and replicates in the oral epithelial cells.[9] The field of human cancer research has been advanced with the application of highly sensitive molecular biology tools such as Polymerase Chain Reaction which permits virus detection soon after infection and even before the onset of disease. Purpose of the current study is to assess the prevalence of EBV in OSCC. The present was conducted to see the association between EB virus and oral squamous cell carcinoma using polymerase chain reaction.

 Methods



Tissues were collected from clinically suspected patients of OSCC who attended the Department of Oral Pathology and Microbiology. Tissues from retro molar area were collected from patients who underwent surgery for impacted teeth and used as controls after approval from the Institutional Ethics Committee. The study consisted of 40 samples categorized into 2 groups; 20 cases of OSCC and 20 age-matched controls. Part of the tissue was processed and sections were stained and examined for routine haematoxylin and eosin to confirm the histopathological diagnosis of squamous cell carcinoma. From remaining part of the histologically proven tissues, DNA extraction was done and subjected to PCR for the evaluation of EBV-positive samples.

Collection of sample Specimens were collected from both OSCC patients and controls. After obtaining, tissues were kept in a small zip lock bag for immersing into liquid nitrogen then stored at -20° until for further use.

DNA extraction procedure from fresh tissue samples Tissues collected were subjected to dehydration by the addition of 1 ml of alcohol for 30 min then the mixture is centrifuged, and the supernatant is discarded. Later, the pellet was suspended in 500 μl TE buffer and Vortexed. Then centrifuged at 10,000 rpm for 5 min and supernatant was discarded and washed with fresh TE buffer for 2–3 times. Supernatant was discarded and 50 μllysis buffer I was added, Vortexed and kept for 5 min. Later, 50 μlLysis buffer II was added along with 10 μl Proteinase–K (10 mg/ml), vortexed vigorously. Kept in water bath at 60°C for 2 hours. Then, kept in boiling water bath for 10 min for enzyme deactivation. The supernatant containing DNA was taken to fresh tube and stored at −20°C. Amplification was done by conventional PCR using EBV primers [Table 1].[10]{Table 1}

Polymerase chain reaction procedure

The detection of EBV was carried out in 2 separate reactions for each sample. Detection of EBV from tissue done by using biochemical process and evaluation done with a help of PCR. After that a thin-walled PCR tube and is added for each 50 μl reaction. A premixture was prepared and the sample was gently vortexed and spinned down. Then tubes are placed in conventional thermal cycler (Applied Bio systems, USA). The sample was run in polymerase chain reaction with standardized protocol according to Chang Ho Shin et al., in 2003.[11] After the polymerase chain reaction completion the amplified product was run on 2% agarose gel electrophoresis for detection of EBV specific bands viewed using ultra light trans illuminator and recorded using gel documentation system (Major science, USA) Base pair of 326 corresponds to EBV and remain bands was considered as non-specific.

Statistical analysis

The collected data was entered in the excel sheet and statistical analysis was done using software, Statistical Package for Social Sciences (SPSS) version 19.0. Comparison of 2 groups with respect to EBV positivity in cases and controls was done by Chi-square test.

 Results



A total of 20 Oral Squamous Cell Carcinoma cases and 20 controls were included in the study. Distribution of age was done based on less than 40 years and above 40 years of age. 3 cases of oral squamous cell carcinoma were lesser than 40 years, in which no case was positive for EBV, whereas total 17 cases were greater than 40 years, among those only 4 cases of oral squamous cell carcinoma was positive for EBV remaining 14 cases were negative for EBV. When comparison was done between less than 40 and above 40 years results were not statistically significant where as in 20 control subjects 18 were less than 40 years and 2 were above 40 years. Among 8 (80%) control subjects were positive for EBV, where as 2 control were positive for EBV in above 40 years. Results were not statistically significant when compared EBV with Control subjects [Table 2] and [Graph 1].{Table 2}[INLINE:1]

Distribution of gender in terms of positivity for EBV in oral squamous cell carcinoma, 12 cases were males and 8 cases were females. Among these, 3 (75.0%) cases of males were positive for EBV and 1 (25.0%) case was positive for EBV in females. Where as in 20 control subjects, 11 were males and 9 were females. Among males 6 (60.0%) were positive for EBV and 4 (40.0%) were positive for EBV in females. On comparison between squamous cell carcinoma and control subjects, there was no statistically significant result [Table 3] and [Graph 2].{Table 3}[INLINE:2]

EBV DNA was detected in 4 out of 20 cases constituting 20% positivity and 10 out of 20 controls constituting 50% positivity. EBV was absent in 16 cases constituting 80%. EBV DNA was detected indicating higher percentage of EBV presence among 10 controls (50%) than in cases of oral squamous cell carcinoma. Statistical analysis was performed using chi square test and the value was 3.956. The difference was statistically significant (p 0.0470) [Table 4] and [Graph 3].{Table 4}[INLINE:3]

Pertaining to the grade of differentiation in oral squamous cell carcinoma, 8 cases were detected in moderately differentiated SCC, which constitutes 40%, 12 cases were detected in well differentiated SCC which constitutes 60%. Out of 20 cases, EBV positivity was present in 4, in which 3 out of 4 cases constituting 75% were well differentiated SCC, and 1 out of 4 cases constituting 25% were moderately differentiated SCC. The result was not statistically significant (p 0.49) [Table 5].{Table 5}

Pertaining to habits and EBV, 75% of cases with habits, were positive for EBV. None of the controls had habits, but EBV positivity was seen in 50% of the controls. P-0.02 was statistically significant [Table 6].{Table 6}

 Discussion



Squamous cell carcinoma (SCC) is the most frequent oral cavity malignancy accounting for over 90% of oral cancers. It represents the sixth most frequent malignant tumour worldwide.[12] Patients with chewing or smoking tobacco or consuming alcohol will cause prototypic changes and lead to oral squamous cell carcinoma.[3] Squamous cell carcinoma (OSCC) emphasizes the role of other risk factors such as genetic susceptibility and oncogenic viruses such as HPV, EBV. Some viruses are strongly associated with OSCC while the association of others is less frequent and may depend on cofactors for their carcinogenic effects. Therefore, the exact role of EBV viruses must be evaluated with care in order to improve the diagnosis and treatment of OSCC[6] EBV was the first human virus to be directly implicated in carcinogenesis and it infects >90% of the world's population. Although most humans coexist with the virus without serious sequelae, but small percentage of population may develop neoplasm.[7] Epstein-Barr virus is known to cause nasopharyngeal carcinoma and oral squamous cell carcinoma.[13] Prevalence of EBV is highest in United Kingdom This high prevalence of EBV in United Kingdom with oral cancers indicate that viral infection may be an important etiological agent along with dietary habits and a probable genetic predisposition can cause additional mutations leading to malignancy. The lowest prevalence of EBV-positive HNSCC was in Yeman.[14] The genetic susceptibility, endemic environment factors, and Epstein-Barr virus (EBV) infection are believed to be the major etiologic factors of OSCC. Once metastasis occurs, prognosis is very poor.[15]

According to Udeabor et al. in 2012 in their study the mean age was under 40 years and 78.9% were males and 21.1% were females.[16] Another study done by Polz-Gruszka et al. in 2015 found that 14.3% in the age group of 40-49 years and 27.3% of the cases EBV was detected. In the present study, the age was more than 40 years of age group in both control and cases of oral squamous cell carcinoma and detection of positive for EBV was observed less than 40 years. These observations are accordance with above study.

In the present study, when compared with gender and prevalence of EBV, it was observed that 4 cases which were positive for EBV among 4 cases oral squamous cell carcinoma 3 cases was positive for EBV and 1 was positive in females where as 10 control subjects 6 were males and 4 were females, this observation was accordance with Polz-Gruszkaet al. in 2015[17] According to Kis A et al. in 2009 observed that prevalence of EBV in OSCC patients was significantly higher than in controls[18] The present study was shown that EBV prevalence was high in oral squamous cell carcinoma than healthy individuals and the results showed statistically significant. (P ≤ 0.05) Kobayashi I et al. in 1999 said that, 46 samples of oral squamous cell carcinoma (OSCC) were evaluated for the prevalence of Epstein-Barr virus (EBV) infection by the polymerase chain reaction (PCR), Southern blot hybridization, and in situ hybridization (ISH). EBV DNA was detected in 15% combination of PCR and Southern blot hybridization methods. When comparison of different grading of squamous cell carcinoma cases, 7 were positive for EBV in well-differentiated carcinoma, thus suggesting a possible relationship between EBV infection and the degree of differentiation of carcinoma tissue.[19] In the present study, 4 cases of well differentiated oral squamous cell carcinoma showed positive for EBV, thus showing that correlation of EBV with the grade of differentiation which is in accordance with Kobayashi I et al. study.

 Conclusion



EBV is commonly found in normal oral mucosa. But presence of habits along with the EBV might play a role in the causation of Oral Squamous Cell Carcinoma. Hence further studies on larger samples using sensitive detecting techniques like RT-PCR provide conclusive results.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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