Squamous cell carcinoma of the head and neck (SCCHN) comprises a large group of cancers in the oral cavity and nasopharyngeal area that typically arise in older males in association with alcohol/tobacco usage. Within the oral cavity, the mobile tongue is the most common site for tumour development. The incidence of tongue squamous cell carcinoma (TSCC) is increasing in younger people, which has been suggested to associate with a viral aetiology. Two common human oncogenic viruses, human papilloma virus (HPV) and Epstein-Barr virus (EBV) are known causes of certain types of SCCHN, namely the oropharynx and nasopharynx, respectively. EBV infects most adults worldwide through oral transmission and establishes a latent infection, with sporadic productive viral replication and release of virus in the oral cavity throughout life. In view of the prevalence of EBV in the oral cavity and recent data indicating that it infects tongue epithelial cells and establishes latency, we examined 98 cases of primary squamous cell carcinoma of the mobile tongue and 15 cases of tonsillar squamous cell carcinoma for the presence of EBV-encoded RNAs (EBERs), EBV DNA and an EBV-encoded protein, EBNA-1. A commercially available in situ hybridisation kit targeting EBER transcripts (EBER-ISH) showed a positive signal in the cytoplasm and/or nuclei of tumour cells in 43% of TSCCs. However, application of control probes and RNase A digestion using in-house developed EBER-ISH showed identical EBER staining patterns, indicating non-specific signals. PCR analysis of the BamH1 W repeat sequences did not identify EBV genomes in tumour samples. Immunohistochemistry for EBNA-1 was also negative. These data exclude EBV as a potential player in TSCC in both old and young patients and highlight the importance of appropriate controls for EBER-ISH in investigating EBV in human diseases.

2nd University of Naples Multidisciplinary Department of Medical Surgical and Dental Specialties Naples Italy

Department of Clinical Sciences ENT Umeå University Umeå Sweden

Department of Medical Biosciences Umeå University Umeå Sweden

Department of Neuroscience Reproductive and Dentistry Sciences University of Naples Federico 2 Naples Italy

Dipartimento Universitario di Anatomia Patologica Seconda Universita' Degli Studi di Napoli Piazza Miraglia Naples Italy

RECAMO Masaryk Memorial Cancer Institute Zluty kopec 7 Brno Czech Republic

United Arab Emirates University College of Medicine and Health Sciences Dept of Medical Microbiology and Immunology Tawam Hospital Campus Al Ain UAE

University Paris Diderot INSERM UMRS1162 27 rue Juliette Dodu Paris France

Zobrazit více v PubMed

Tsao SW, Tsang CM, To KF, Lo KW. The role of Epstein-Barr virus in epithelial malignancies. J Pathol. 2015;235(2): 323–333. doi: 10.1002/path.4448 PubMed DOI PMC

Young LS, Lau R, Rowe M, Niedobitek G, Packham G, Shanahan F, et al. Differentiation-associated expression of the Epstein-Barr virus BZLF1 transactivator protein in oral hairy leukoplakia. J Virol. 1991;65(6): 2868–2874. PubMed PMC

Gillison ML, Chaturvedi AK, Anderson WF, Fakhry C. Epidemiology of Human Papillomavirus-Positive Head and Neck Squamous Cell Carcinoma. J Clin Oncol. 2015;33(29): 3235–3242. doi: 10.1200/JCO.2015.61.6995 PubMed DOI PMC

Llewellyn CD, Johnson NW, Warnakulasuriya KA. Risk factors for squamous cell carcinoma of the oral cavity in young people—a comprehensive literature review. Oral Oncol. 2001;37(5): 401–418. PubMed

Patel SC, Carpenter WR, Tyree S, Couch ME, Weissler M, Hackman T, et al. Increasing incidence of oral tongue squamous cell carcinoma in young white women, age 18 to 44 years. J Clin Oncol. 2011;29(11): 1488–1494. doi: 10.1200/JCO.2010.31.7883 PubMed DOI

Tota JE, Anderson WF, Coffey C, Califano J, Cozen W, Ferris RL, et al. Rising incidence of oral tongue cancer among white men and women in the United States, 1973–2012. Oral Oncol. 2017;67: 146–152. doi: 10.1016/j.oraloncology.2017.02.019 PubMed DOI

Sgaramella N, Coates PJ, Strindlund K, Loljung L, Colella G, Laurell G, et al. Expression of p16 in squamous cell carcinoma of the mobile tongue is independent of HPV infection despite presence of the HPV-receptor syndecan-1. Br J Cancer. 2015;113(2): 321–326. doi: 10.1038/bjc.2015.207 PubMed DOI PMC

Longnecker R, Kieff E, Cohen JI. Epstein Barr Virus In: Knipe DM, Howley PM, editors. Fields Virology. 6th ed. Philadelphia: Lippincott Williams & Wilkins; 2013. p. 1898–1959.

Young LS, Yap LF, Murray PG. Epstein-Barr virus: more than 50 years old and still providing surprises. Nat Rev Cancer. 2016;16(12): 789–802. doi: 10.1038/nrc.2016.92 PubMed DOI

Hutt-Fletcher LM. The Long and Complicated Relationship between Epstein-Barr Virus and Epithelial Cells. J Virol. 2017;91(1). doi: 10.1128/JVI.01677-16 PubMed DOI PMC

Vockerodt M, Yap LF, Shannon-Lowe C, Curley H, Wei W, Vrzalikova K, et al. The Epstein-Barr virus and the pathogenesis of lymphoma. J Pathol. 2015;235(2): 312–322. doi: 10.1002/path.4459 PubMed DOI

Niedobitek G, Young LS, Lau R, Brooks L, Greenspan D, Greenspan JS, et al. Epstein-Barr virus infection in oral hairy leukoplakia: virus replication in the absence of a detectable latent phase. J Gen Virol. 1991;72 (Pt 12): 3035–3046. doi: 10.1099/0022-1317-72-12-3035 PubMed DOI

Khammissa RA, Fourie J, Chandran R, Lemmer J, Feller L. Epstein-Barr Virus and Its Association with Oral Hairy Leukoplakia: A Short Review. Int J Dent. 2016;2016: 4941783 doi: 10.1155/2016/4941783 PubMed DOI PMC

Nawandar DM, Wang A, Makielski K, Lee D, Ma S, Barlow E, et al. Differentiation-Dependent KLF4 Expression Promotes Lytic Epstein-Barr Virus Infection in Epithelial Cells. PLoS Pathog. 2015;11(10): e1005195 doi: 10.1371/journal.ppat.1005195 PubMed DOI PMC

Shimakage M, Horii K, Tempaku A, Kakudo K, Shirasaka T, Sasagawa T. Association of Epstein-Barr virus with oral cancers. Hum Pathol. 2002;33(6): 608–614. PubMed

Kikuchi K, Noguchi Y, de Rivera MW, Hoshino M, Sakashita H, Yamada T, et al. Detection of Epstein-Barr virus genome and latent infection gene expression in normal epithelia, epithelial dysplasia, and squamous cell carcinoma of the oral cavity. Tumour Biol. 2016;37(3): 3389–3404. doi: 10.1007/s13277-015-4167-7 PubMed DOI

Shamaa AA, Zyada MM, Wagner M, Awad SS, Osman MM, Abdel Azeem AA. The significance of Epstein Barr virus (EBV) & DNA topoisomerase II alpha (DNA-Topo II alpha) immunoreactivity in normal oral mucosa, oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC). Diagn Pathol. 2008;3: 45 doi: 10.1186/1746-1596-3-45 PubMed DOI PMC

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(4): 252–257. doi: 10.1111/jop.12231 PubMed DOI

Gonzalez-Moles MA, Gutierrez J, Rodriguez MJ, Ruiz-Avila I, Rodriguez-Archilla A. Epstein-Barr virus latent membrane protein-1 (LMP-1) expression in oral squamous cell carcinoma. Laryngoscope. 2002;112(3): 482–487. doi: 10.1097/00005537-200203000-00014 PubMed DOI

Kis A, Feher E, Gall T, Tar I, Boda R, Toth ED, et al. Epstein-Barr virus prevalence in oral squamous cell cancer and in potentially malignant oral disorders in an eastern Hungarian population. Eur J Oral Sci. 2009;117(5): 536–540. doi: 10.1111/j.1600-0722.2009.00660.x PubMed DOI

Kobayashi I, Shima K, Saito I, Kiyoshima T, Matsuo K, Ozeki S, et al. Prevalence of Epstein-Barr virus in oral squamous cell carcinoma. J Pathol. 1999;189(1): 34–39. doi: 10.1002/(SICI)1096-9896(199909)189:1<34::AID-PATH391>3.0.CO;2-4 PubMed DOI

Cruz I, Van Den Brule AJ, Brink AA, Snijders PJ, Walboomers JM, Van Der Waal I, et al. No direct role for Epstein-Barr virus in oral carcinogenesis: a study at the DNA, RNA and protein levels. Int J Cancer. 2000;86(3): 356–361. PubMed

Cruz I, Van den Brule AJ, Steenbergen RD, Snijders PJ, Meijer CJ, Walboomers JM, et al. Prevalence of Epstein-Barr virus in oral squamous cell carcinomas, premalignant lesions and normal mucosa—a study using the polymerase chain reaction. Oral Oncol. 1997;33(3): 182–188. PubMed

Khan G, Coates PJ, Gupta RK, Kangro HO, Slavin G. Presence of Epstein-Barr virus in Hodgkin's disease is not exclusive to Reed-Sternberg cells. Am J Pathol. 1992;140(4): 757–762. PubMed PMC

Daskalogianni C, Pyndiah S, Apcher S, Mazars A, Manoury B, Ammari N, et al. Epstein-Barr virus-encoded EBNA1 and ZEBRA: targets for therapeutic strategies against EBV-carrying cancers. J Pathol. 2015;235(2): 334–341. doi: 10.1002/path.4431 PubMed DOI

Ahmed W, Khan G. The labyrinth of interactions of Epstein-Barr virus-encoded small RNAs. Rev Med Virol. 2014;24(1): 3–14. doi: 10.1002/rmv.1763 PubMed DOI

Lee N, Yario TA, Gao JS, Steitz JA. EBV noncoding RNA EBER2 interacts with host RNA-binding proteins to regulate viral gene expression. Proc Natl Acad Sci U S A. 2016;113(12): 3221–3226. doi: 10.1073/pnas.1601773113 PubMed DOI PMC

Loizou C, Laurell G, Lindquist D, Ofverman C, Stefansson K, Nylander K, et al. Incidence of tonsillar cancer in northern Sweden: Impact of human papilloma virus. Oncol Lett. 2015;10(6): 3565–3572. doi: 10.3892/ol.2015.3775 PubMed DOI PMC

Khan G. Screening for Epstein-Barr virus in Hodgkin's lymphoma. Methods Mol Biol. 2009;511: 311–322. doi: 10.1007/978-1-59745-447-6_13 PubMed DOI

Khan G, Coates PJ, Kangro HO, Slavin G. Epstein Barr virus (EBV) encoded small RNAs: targets for detection by in situ hybridisation with oligonucleotide probes. J Clin Pathol. 1992;45(7): 616–620. PubMed PMC

Coates PJ, d'Ardenne AJ, Khan G, Kangro HO, Slavin G. Simplified procedures for applying the polymerase chain reaction to routinely fixed paraffin wax sections. J Clin Pathol. 1991;44(2): 115–118. PubMed PMC

Hassani A, Khan G. A simple procedure for the extraction of DNA from long-term formalin-preserved brain tissues for the detection of EBV by PCR. Exp Mol Pathol. 2015;99(3): 558–563. doi: 10.1016/j.yexmp.2015.09.013 PubMed DOI

Khan G, Ahmed W, Philip PS, Ali MH, Adem A. Healthy rabbits are susceptible to Epstein-Barr virus infection and infected cells proliferate in immunosuppressed animals. Virol J. 2015;12: 28 doi: 10.1186/s12985-015-0260-1 PubMed DOI PMC

Cheung A, Kieff E. Long internal direct repeat in Epstein-Barr virus DNA. J Virol. 1982;44(1): 286–294. PubMed PMC

Sand L, Jalouli J. Viruses and oral cancer. Is there a link? Microbes Infect. 2014;16(5): 371–378. doi: 10.1016/j.micinf.2014.02.009 PubMed DOI

Metgud R, Astekar M, Verma M, Sharma A. Role of viruses in oral squamous cell carcinoma. Oncol Rev. 2012;6(2): e21 doi: 10.4081/oncol.2012.e21 PubMed DOI PMC

Gondivkar SM, Parikh RV, Gadbail AR, Solanke V, Chole R, Mankar M, et al. Involvement of viral factors with head and neck cancers. Oral Oncol. 2012;48(3): 195–199. doi: 10.1016/j.oraloncology.2011.10.007 PubMed DOI

Prabhu SR, Wilson DF. Evidence of Epstein-Barr Virus Association with Head and Neck Cancers: A Review. J Can Dent Assoc. 2016;82: g2 PubMed

Fok V, Friend K, Steitz JA. Epstein-Barr virus noncoding RNAs are confined to the nucleus, whereas their partner, the human La protein, undergoes nucleocytoplasmic shuttling. J Cell Biol. 2006;173(3): 319–325. doi: 10.1083/jcb.200601026 PubMed DOI PMC

Higa M, Kinjo T, Kamiyama K, Chinen K, Iwamasa T, Arasaki A, et al. Epstein-Barr virus (EBV)-related oral squamous cell carcinoma in Okinawa, a subtropical island, in southern Japan—simultaneously infected with human papillomavirus (HPV). Oral Oncol. 2003;39(4): 405–414. PubMed

Higa M, Kinjo T, Kamiyama K, Iwamasa T, Hamada T, Iyama K. Epstein-Barr virus (EBV) subtype in EBV related oral squamous cell carcinoma in Okinawa, a subtropical island in southern Japan, compared with Kitakyushu and Kumamoto in mainland Japan. J Clin Pathol. 2002;55(6): 414–423. PubMed PMC

Jiang R, Gu X, Moore-Medlin TN, Nathan CA, Hutt-Fletcher LM. Oral dysplasia and squamous cell carcinoma: correlation between increased expression of CD21, Epstein-Barr virus and CK19. Oral Oncol. 2012;48(9): 836–841. doi: 10.1016/j.oraloncology.2012.03.017 PubMed DOI PMC

Hermann RM, Fuzesi L, Pradier O, Christiansen H, Schmidberger H. Presence of human papillomavirus-18 and Epstein-Barr virus in a squamous cell carcinoma of the tongue in a 20-year-old patient. Case report and review of the current literature. Cancer Radiother. 2004;8(4): 262–265. doi: 10.1016/j.canrad.2004.06.003 PubMed DOI

High immune cytolytic activity in tumor-free tongue tissue confers better prognosis in patients with squamous cell carcinoma of the oral tongue