Prevalence of salivary epstein-barr virus in potentially malignantoral disorders and oral squamous cell carcinoma

(1)

Med Oral Patol Oral Cir Bucal. 2016 Mar 1;21 (2):e157-60. Salivary Epstein-Barr virus in oral precancer and cancer

e157 Journal section: Oral Medicine and Pathology

Publication Types: Research

Prevalence of salivary epstein-barr virus in potentially malignant oral disorders and oral squamous cell carcinoma

Leticia Bagan ¹, María-Dolores Ocete-Monchon ², Manuel Leopoldo-Rodado ³, Judith Murillo-Cortes ⁴, Jose-M. Díaz-Fernández ⁴, Rafael Medina-Gonzalez ², Concepción Gimeno-Cardona ⁵, Jose-V. Bagan ⁶

1 Associate Professor of Oral Medicine, European University of Valencia, Spain

2 Consultant Microbiology Service. University General Hospital, Valencia

3 Consultant in Oral and Maxillofacial Surgery. Service of Stomatology and Maxillofacial Surgery, Valencia

4 Head Section in Oral and Maxillofacial Surgery. Service of Stomatology and Maxillofacial Surgery, Valencia

5 Professor and Head Service of Microbiology. Valencia University and University General Hospital Valencia

6 Professor and Head Service of Stomatology and Maxillofacial Surgery. Valencia University and University General Hospital Valencia, Spain

Correspondence:

Oral Medicine Department C/ Gasco Oliag 1 46010 Valencia, Spain bagan@uv.es

Received: 18/04/2015 Accepted: 13/01/2016

Abstract

Background: To analyze the presence of salivary Epstein-Barr virus (EBV) DNA in oral squamous cell carcinoma and potentially malignant oral disorders.

Material and Methods: Three groups were studied: Group 1 (12 oral squamous cell carcinomas (OSCC)), Group 2 (12 potentially malignant oral disorders (PMD)) and Group 3 (47 healthy controls). EBV DNA salivary analysis was performed by PCR.

Results: The highest percentage of positive salivary EBV DNA corresponded to the OSCC group (58.3%), followed by the PMD group (41.7%) and the controls (40.4%). The differences between groups were not statistically signifi- cant, however (p>0.05).

Conclusions: Salivary EBV DNA was more prevalent in OSCC than in PMD or the controls.

Key words:EBV DNA, saliva, oral squamous cell carcinoma, oral leukoplakia.

Bagan L, Ocete-Monchon MD, Leopoldo-Rodado M, Murillo-Cortes J, Díaz-Fernández JM, Medina-Gonzalez R, Gimeno-Cardona C, Bagan JV. Prevalence of salivary epstein-barr virus in potentially malignant oral disorders and oral squamous cell carcinoma. Med Oral Patol Oral Cir Bu- cal. 2016 Mar 1;21 (2):e157-60.

http://www.medicinaoral.com/medoralfree01/v21i2/medoralv21i2p157.pdf Article Number: 20785 http://www.medicinaoral.com/

© Medicina Oral S. L. C.I.F. B 96689336 - pISSN 1698-4447 - eISSN: 1698-6946 eMail: medicina@medicinaoral.com

Indexed in:

Science Citation Index Expanded Journal Citation Reports Index Medicus, MEDLINE, PubMed Scopus, Embase and Emcare Indice Médico Español

doi:10.4317/medoral.20785

http://dx.doi.org/doi:10.4317/medoral.20785

(2)

e158

Introduction

Epstein-Barr virus (EBV) is a very well known onco- genic human herpes virus that has been implicated in several malignant tumors affecting epithelial cells and B lymphocytes (1).

The prevalence of EBV in the general population is very high, and there are nearly 200,000 new cases of infection in the world every year. In the first two decades of life EBV infects 90% of all individuals. According to Ueda et al., EBV is a reversible latent infection in B cells (2).

It is believed that EBV initially penetrates and multi- plies within epithelial cells, followed by release into saliva, affecting B lymphocytes and spreading throughout the rest of the body. However, most affected individuals are asymptomatic, despite detection of the virus in different body secretions and blood (3).

Saliva plays a significant role in the capacity of EBV to become transmitted to other people. The virus is infec- tive when present in saliva, both in asymptomatic and symptomatic carriers. EBV is released into saliva from the epithelial cells, and it is in this fluid where maxi- mum infectious capacity is observed (1). On the other hand, EBV is found not only in B cells but also in nasopharyngeal carcinomas (4).

Salivary EBV has been analyzed in several oral diseases, particularly in patients with periodontal problems (5-9). In 2007 we already addressed the presence of EBV in potentially malignant disorders (PMD) and in oral squamous cell carcinoma (OSCC), though involving a smaller number of cases (10). However, there have been recent controversial findings in the literature regarding the association between EBV and OSCC. The present study analyzes the frequency of EBV DNA positivity in OSCC and PMD comparing with controls.

Material and Methods

Three groups were studied: Group 1 (12 OSCC), Group 2 (12 PMD) and Group 3 (47 healthy controls).

There were no clinical differences among the three groups regarding age or gender (p>0.05).

Oral squamous cell carcinoma was diagnosed from an incisional biopsy. In the leukoplakia group (Group 2) we also obtained a biopsy to establish the diagnosis follow- ing the criteria of Carrard et al. (11).

The study was approved by the Ethics Committee of the University of Valencia (Spain), and informed consent was obtained from each patient.

Whole non-stimulated saliva was obtained in all cases according to the criteria of Navacek et al. (12). Saliva samples were immediately stored and frozen at -80º un- til EBV DNA analysis.

EBV DNA salivary analysis was performed by PCR following the methodology described elsewhere (3).

Saliva samples were obtained and DNA was extracted

as reported (2). EBV DNA levels were determined by qualitative real-time PCR (qPCR) targeting the EBV Gen LMP1 region.

The present case-control study evaluated the presence and percentage of positive findings regarding EBV DNA and analyzed the association of the virus to the different groups using the χ2 test. Statistical signifi- cance was considered for p < 0.05.

Results

The highest percentage of positive salivary EBV DNA corresponded to the OSCC group, followed by the leukoplakia (PMD) group and the controls (Table 1). The differences between groups were not statistically sig- nificant, however (p>0.05) (Table 2).

Four of the 9 cases of proliferative verrucous leukoplakia (44.4%) presented positive salivary EBV DNA. In those cases with only homogeneous leukoplastic areas, the positivity rate was lower (33.3%).

Discussion

Epstein-Barr virus is very common in normal indivi- duals of the general population. According to Ueda et al. (2), its prevalence may reach 90% in saliva. The virus can penetrate and multiply within the epithelial cells, followed by release into saliva.

The salivary EBV DNA detection rate and consequently shedding of the virus in healthy persons ranges from 22-90% (3). Despite the variability among authors, the detection rate is usually high in healthy controls. In this respect we found 40.4% of our controls to be positive for DNA EBV.

Epstein-Barr virus DNA in saliva has been analyzed in several diseases such as connective tissue disorders (13), adverse drug reactions with eosinophilia and systemic symptoms (DRESS) (14), periimplantitis (15), HIV infection (3,16), periodontal disease (6) and in transplant patients (17).

In cancer patients, EBV in saliva has been described as a useful tool in nasopharyngeal carcinomas. In ad- vances disease stages the EBV DNA levels are higher than in early stages (18).

Epstein-Barr virus has also been studied in OSCC patients, though the results are controversial. According to some investigators, EBV is associated to OSCC and this association seems to be enhanced by betel quid chewing, thus suggesting that EBV may be an impor- tant etiological risk factor for OSCC (19). Furthermore, Jiang et al. (20) described a high prevalence of human papillomavirus (HPV)/EBV infection and coinfection in non-cancerous base of tongue (BOT) lesions and tonsil malignancies, possibly reflecting their origins in lymphoid-rich tissue (20).

In contrast, other authors have found no significant OSCC risk in subjects with EBV infection (21,22). Like-

(3)

e159

Case Group Age Gender Type of lesion EBV + DNA

1 Cancer 60 2 Ulceration -

3 Cancer 79 1 Ulceration +

13 PMD 55 1 Verrucous leukoplakia -

15 PMD 86 1 Verrucous leukoplakia +

22 PMD 69 2 Homogeneous leukoplakia -

23 PMD 48 1 Homogeneous leukoplakia +

24 PMD 56 1 Homogeneous leukoplakia -

25 Control 58 2 No lesions -

29 Control 43 2 No lesions +

Table 1. Epstein-Barr virus DNA findings among the three groups.

PMD: Potentially malignant disorder Gender: 1 Female, 2 Male

Cancer: Group 1 PMD: Group 2

(4)

e160 wise, the data published in 2014 by Saravani et al. (23) neither supported the hypothesis that EBV and HHV-6 are directly involved in OSCC nor ruled out the possi- bility that these viruses might play an indirect carcino- genic role in this area.

Considering the above discrepancies, we tried to analyze the presence of EBV DNA in the saliva of patients with potentially malignant disorders and oral squamous cell carcinoma. No significant differences were observed among OSCC, PMD and the controls (p>0.05). Howev- er, EBV DNA positivity was greater in the OSCC group than in the PMD group or controls (Table 2). Studies with a larger number of cases are required to determine whether such higher percentage EBV DNA positivity in OSCC is also found in other larger populations.

References

1. Tugizov SM, Herrera R, Palefsky JM. Epstein-Barr virus transcyto- sis through polarized oral epithelial cells. J Virol. 2013;87:8179-94.

2. Ueda S, Uchiyama S, Azzi T, Gysin C, Berger C, Bernasconi M, et al. Oropharyngeal group A streptococcal colonization disrupts latent Epstein-Barr virus infection. J Infect Dis. 2014;209:255-64.

3. de França TR, de Albuquerque Tavares Carvalho A, Gomes VB, Gueiros LA, Porter SR, Leao JC. Salivary shedding of Epstein-Barr virus and cytomegalovirus in people infected or not by human im- munodeficiency virus 1. Clin Oral Investig. 2012;16:659-64.

4. Shan J, Pow EH, Tsang PC, Perera RA, Kwong DL. Comparison of two laboratory extraction techniques for the detection of Epstein- Barr virus in the saliva of nasopharyngeal carcinoma patients. J In- vestig Clin Dent. 2014;5:104-8.

5. Grande SR, Imbronito AV, Okuda OS, Pannuti CM, Nunes FD, Lima LA. Relationship between herpesviruses and periodon- topathogens in patients with HIV and periodontitis. J Periodontol.

2011;82:1442-52.

6. Sahin S, Saygun I, Kubar A, Slots J. Periodontitis lesions are the main source of salivary cytomegalovirus. Oral Microbiol Immunol.

2009;24:340-2.

7. Dawson DR, Wang C, Danaher RJ, Lin Y, Kryscio RJ, Jacob RJ, et al. Salivary levels of Epstein-Barr virus DNA correlate with subgin- gival levels, not severity of periodontitis. Oral Dis. 2009;15:554-9.

8. Slots J, Saygun I, Sabeti M, Kubar A. Epstein-Barr virus in oral diseases. J Periodontal Res. 2006;41:235-44.

9. Bilder L, Elimelech R, Szwarcwort-Cohen M, Kra-Oz Z, Machtei EE. The prevalence of human herpes viruses in the saliva of chronic periodontitis patients compared to oral health providers and healthy controls. Arch Virol. 2013;158:1221-6.

10. Bagan JV, Jiménez Y, Murillo J, Poveda R, Díaz JM, Gavaldá C, et al. Epstein-Barr virus in oral proliferative verrucous leukoplakia and squamous cell carcinoma: A preliminary study. Med Oral Patol Oral Cir Bucal. 2008;13:E110-3.

11. Carrard VC, Brouns ER, van der Waal I. Proliferative verrucous leukoplakia; a critical appraisal of the diagnostic criteria. Med Oral Patol Oral Cir Bucal. 2013;18:e411-3.

12. Navazesh M. Methods for collecting saliva. Ann N Y Acad Sci.

1993;694:72-7.

13. Kuhara T, Watanabe D, Ishida N, Tamada Y, Matsumoto Y, Ihira M, et al. Quantitative analysis of shedding of Epstein-Barr virus in saliva from patients with connective tissue diseases: a pilot study. Int J Dermatol. 2013;52:887-90.

14. Descamps V, Avenel-Audran M, Valeyrie-Allanore L, Bensaid B, Barbaud A, AlJawhari M, et al. French Study Group of Cutaneous Drug Adverse Reactions. Saliva polymerase chain reaction assay for detection and follow-up of herpesvirus reactivation in patients with drug reaction with eosinophilia and systemic symptoms (DRESS).

JAMA Dermatol. 2013;149:565-9.

15. Verdugo F, Castillo A, Castillo F, Uribarri A. Epstein-Barr virus associated peri-implantitis: a split-mouth study. Clin Oral Investig.

2015;19:535-43.

16. Amornthatree K, Sriplung H, Mitarnun W, Nittayananta W. Ef- fects of long-term use of antiretroviral therapy on the prevalence of oral Epstein-Barr virus. J Oral Pathol Med. 2012;41:249-54.

17. Nikoobakht M, Beitollahi J, Nikoobakht N, Aloosh M, Saheb- jamee M, Rezaeidanesh M, et al. Evaluation of Epstein-Barr virus load in saliva before and after renal transplantation. Transplant Proc.

2011;43:540-2.

18. Pow EH, Law MY, Tsang PC, Perera RA, Kwong DL. Salivary Epstein-Barr virus DNA level in patients with nasopharyngeal carcinoma following radiotherapy. Oral Oncol. 2011;47:879-82.

19. Acharya S, Ekalaksananan T, Vatanasapt P, Loyha K, Phusingha P, Promthet S, et al. Association of Epstein-Barr virus infection with oral squamous cell carcinoma in a case-control study. J Oral Pathol Med. 2015;44:252-7.

20. Jiang R, Ekshyyan O, Moore-Medlin T, Rong X, Nathan S, Gu X, et al. Association between human papilloma virus/Epstein- Barr virus coinfection and oral carcinogenesis. J Oral Pathol Med.

2015;44:28-36.

21. Sand L, Jalouli J. Viruses and oral cancer. Is there a link? Mi- crobes Infect. 2014;16:371-8.

22. Nasher AT, Al-Hebshi NN, Al-Moayad EE, Suleiman AM. Viral infection and oral habits as risk factors for oral squamous cell carcinoma in Yemen: a case-control study. Oral Surg Oral Med Oral Pathol Oral Radiol. 2014;118:566-72.

23. Saravani S, Miri-Moghaddam E, Sanadgol N, Kadeh H, Nazeri MR. Human herpesvirus-6 and epstein-barr virus infections at different histopathological grades of oral squamous cell carcinomas. Int J Prev Med. 2014;5:1231-8.

Conflict of Interest

Authors declare no conflicts of interest

Value Significance

Chi square of Pearson 1.270 0.530

Saliva DNA Epstein-Barr virus

Negative Positive Total

Groups Controls 28 (59.6%) 19 (40.4%) 47

OSCC 5 (41.7%) 7 (58.3%) 12

PMD 7 (58.3%) 5 (41.7%) 12

Table 2. Summary of DNA EBV detection in saliva of the three groups.

OSCC: Group 1.

PMD: Group 2.