Detection of Epstein-Barr Virus in Nasopharyngeal Carcinoma in Sudanese by in Situ Hybridization

Ameera A. M. Adam, Nazik E. Abdullah, Lamyaa A. M. El Hassan, Elwaleed M. Elamin, Muntaser E. Ibrahim, Ahmed M. El Hassan

Department of Molecular Biology, Institute of Endemic Diseases, University of Khartoum, Khartoum, Sudan.
Department of Surgery, Faculty of Medicine, University of Khartoum, Khartoum, Sudan.
Faculty of Medical Laboratory Sciences, Alzaeim Alazhari University, Khartoum, Sudan.
School of Medicine, Ahfad University for Women, Omdurman, Higher Specialization in Histopathology, Khartoum, Sudan.

DOI: 10.4236/jct.2014.56059                

Abstract

Objectives: The aim of this study is to detect Epstein-Barr virus (EBV) in nasopharyngeal carcinoma (NPC) biopsies of Sudanese patients using EBV-encoded RNA (EBER) in Situ hybridization (EBER-ISH). Study Design: This is a descriptive cross-sectional study conducted at the National Center for ENT diseases and Head and Neck Surgery and the Institute of Endemic Diseases, University of Khartoum, Khartoum City, Sudan. Subjects and Methods: Biopsies from 43 patients with nasopharyngeal carcinoma were examined for the presence of Epstein-Barr virus using EBER-ISH. Ten normal samples were used to assess the presence of the virus in non cancer tissues. Results: Fifty three samples were examined for the presence of the virus by EBER-ISH, 43 biopsies were NPC and ten were normal. Histologically the cases were, 20 (46.5%), 20 (46.5%) and 3 (7%) of the biopsies were classified as WHO types II, III and mixed type II and III, respectively; there were no cases of type I NPC. All nasopharyngeal carcinoma biopsies (100%) were positive for EBER1 in almost all carcinoma cells with focal and intense dark-blue staining limited to the nucleus; no hybridization was observed in the cytoplasm. No hybridization was observed in all ten non cancer tissues. Conclusion: All NPC cells are clearly EBV-infected. The virus is located in the nucleus of the tumour cells. The presence of Epstein-Barr virus in normal nasopharyngeal epithelia is not a common event. Objectives: The aim of this study is to detect Epstein-Barr virus (EBV) in nasopharyngeal carcinoma (NPC) biopsies of Sudanese patients using EBV-encoded RNA (EBER) in Situ hybridization (EBER-ISH). Study Design: This is a descriptive cross-sectional study conducted at the National Center for ENT diseases and Head and Neck Surgery and the Institute of Endemic Diseases, University of Khartoum, Khartoum City, Sudan. Subjects and Methods: Biopsies from 43 patients with nasopharyngeal carcinoma were examined for the presence of Epstein-Barr virus using EBER-ISH. Ten normal samples were used to assess the presence of the virus in non cancer tissues. Results: Fifty three samples were examined for the presence of the virus by EBER-ISH, 43 biopsies were NPC and ten were normal. Histologically the cases were, 20 (46.5%), 20 (46.5%) and 3 (7%) of the biopsies were classified as WHO types II, III and mixed type II and III, respectively; there were no cases of type I NPC. All nasopharyngeal carcinoma biopsies (100%) were positive for EBER1 in almost all carcinoma cells with focal and intense dark-blue staining limited to the nucleus; no hybridization was observed in the cytoplasm. No hybridization was observed in all ten non cancer tissues. Conclusion: All NPC cells are clearly EBV-infected. The virus is located in the nucleus of the tumour cells. The presence of Epstein-Barr virus in normal nasopharyngeal epithelia is not a common event.

Keywords

EBV, NPC, Sudan, ISH, EBER

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Yu, M.C. and Yuan, J. (2006) Nasopharyngeal Cancer. In: Schottenfeld, D. and Fraumeni, J.F., Eds., Cancer Epidemiology and Prevention, Oxford University Press, New York, 620-626. http://dx.doi.org/10.1093/acprof:oso/9780195149616.003.0031
[2]	Abdullah, N.E., Adam, A.A.M., Khalifa, E.H., El Hassan, L.A.M., Elamin, E.M., Hamad, K.M. et al. (2011) Nasopharyngeal Cancer in Sudan: Epidemiology, Clinical and Histological Characteristics. Clinical Medicine Insights: Ear, Nose and Throat, 4, 5-11. http://dx.doi.org/10.4137/CMENT.S5825
[3]	Hidayatalla, A.M.O., Malik, A.E., El Hadi, A.A., Osman, A.A and Hutt, M.S. (1983) Studies on Nasopharyngeal Carcinoma in the Sudan—I Epidemiology and Aetiology. European Journal of Cancer and Clinical Oncology, 19, 705-710. http://dx.doi.org/10.1016/0277-5379(83)90001-9
[4]	Wei, W.I. and Sham, J.S. (2005) Nasopharyngeal Carcinoma. Lancet, 365, 2041-2054. http://dx.doi.org/10.1016/S0140-6736(05)66698-6
[5]	Adam, A.A.M., Abdullah, N.E., Khalifa, E.H., El Hassan, L.A.M., Elamin, E.M., Hamad, K.M., et al. (2011) Pathology of Nasopharyngeal Carcinoma in Sudanese Patients and Its Association with Epstein-Barr Virus: A Report from a Single Center in Khartoum. Advances in Tumor Virology, 2, 1-6. http://dx.doi.org/10.4137/ATV.S5817
[6]	Kieff, E. (1995) Epstein-Barr Virus—Increasing Evidence of a Link to Carcinoma. The New England Journal of Medicine, 333, 724-726. http://dx.doi.org/10.1056/NEJM199509143331110
[7]	Neel, H.B. 3rd, Pearson, G.R. and Taylor, W. (1984) Antibodies to Epstein-Barr Virus in Patients with Nasopharyngeal Carcinoma and in Comparison Groups. Annals of Otology, Rhinology, and Laryngology, 93, 477-482.
[8]	Cohen, J. (2002) Epstein-Barr Virus Infection. The New England Journal of Medicine, 343, 481-492. http://dx.doi.org/10.1056/NEJM200008173430707
[9]	Pagano, J.S., Blaser, M., Buendia, M.A., Damania, B., Khalili, K., Raab-Traub, N. and Roizman, B. (2004) Infectious Agents and Cancer: Criteria for a Causal Relation. Seminars in Cancer Biology, 14, 453-471. http://dx.doi.org/10.1016/j.semcancer.2004.06.009
[10]	Lanier, A.P., Bornkamm, G.W., Henle, W., Henle, G., Bender, T.R., Talbot, M.L., et al. (1981) Association of EpsteinBarr virus with Nasopharyngeal Carcinoma in Alaskan Native Patients: Serum Antibodies and Tissue EBNA and DNA. International Journal of Cancer, 28, 301-305. http://dx.doi.org/10.1002/ijc.2910280308
[11]	Akao, I., Sato, Y., Mukai, K., Uhara, H., Furuya, S., Hoshikawa, T., et al. (1991) Detection of Epstein-Barr Virus DNA in Formalin-Fixed Paraffin-Embedded Tissue of Nasopharyngeal Carcinoma Using Polymerase Chain Reaction and in Situ Hybridization. Laryngoscope, 101, 279-283. http://dx.doi.org/10.1288/00005537-199103000-00010
[12]	Niedobitek, G., Fahraeus, R,. Herbst, H., Latza, U., Ferszt, A., Klein, G., et al. (1992) The Epstein-Barr Virus Encoded Membrane Protein (LMP) Induces Phenotypic Changes in Epithelial Cells. Virchows Archiv B, 62, 55-59. http://dx.doi.org/10.1007/BF02899665
[13]	Weiss, L.M., Movahed, L.A., Butler, A.E., Swanson, S.A., Frierson, H.F.J., Cooper, P.H., et al. (1989) Analysis of Lymphoepithelioma and Lymphoepithelioma-Like Carcinomas for Epstein-Barr Viral Genomes by in Situ Hybridization. American Journal of Surgical Pathology, 13, 625-631. http://dx.doi.org/10.1097/00000478-198908000-00001
[14]	Howe, J.G. and Shu, M.D. (1988) Isolation and Characterization of the Genes for Two Small RNAs of Herpesvirus papio and Their Comparison with Epstein-Barr Virus-Encoded EBER RNAs. Journal of Virology, 62, 2790-2798.
[15]	Tsai, S.T., Jin, Y.T., Mann, R.B. and Ambinder, R.F. (1998) Epstein-Barr Virus Detection in Nasopharyngeal Tissues of Patients with Suspected Nasopharyngeal Carcinoma. Cancer, 82, 1449-1453.
[16]	Nakado, K., Mochiki, M., Nibu, K., Sugasaywa, M. and Uozaki, H. (2006) Analysis of Prognostic Factors of Nasopharyngeal Carcinoma: Impact of in Situ Hybridization for Epstein-Barr Virus Encoded Small RNA 1. Otolaryngology—Head and Neck Surgery, 134, 639-645. http://dx.doi.org/10.1016/j.otohns.2005.11.022
[17]	Kengjian, K., Haiyun, W., Zichen, S., Liping, D., Dabo, L. and Jin, Y. (2014) Epstein-Barr Virus-Encoded RNAs as a Survival Predictor in Nasopharyngeal Carcinoma. Chinese Medical Journal, 2, 294-299.
[18]	zur Hausen, H., Schulte-Holthausen, H., Klein, G., Henle, W., Henle, G., Clifford, P., et al. (1970) EBV DNA in Biopsies of Burkitt’s Tumours and Anaplastic Carcinomas of the Nasopharynx. Nature, 228, 1056-1058. http://dx.doi.org/10.1038/2281056a0
[19]	Chang, K.L., Chen, Y.Y., Shibata, D. and Weiss, L.M. (1992) Description of an in Situ Hybridization Methodology for Detection of Epstein-Barr Virus RNA in Paraffin-Embedded Tissues, with a Survey of Normal and Neoplastic Tissues. Diagnostic Molecular Pathology, 1, 246-255. http://dx.doi.org/10.1097/00019606-199203000-00037
[20]	Chan, A.T.C., Teo, P.M.L and Johnson, P.J. (2002) Nasopharyngeal Carcinoma. Annals of Oncology, 13, 1007-1015. http://dx.doi.org/10.1093/annonc/mdf179
[21]	Collins, S.L. (1996) Squamous Cell Carcinoma of the Oral Cavity and Oropharynx. In: Ballenger, J. and Snow, J., Eds., Otorhinolaryngology: Head and Neck Surgery, Media, Williams & Wilkins, 249-368.
[22]	Inoue, H., Sato, Y., Tsuchiya, B., Nagai, H., Takahashi, H. and Kameya, T. (2002) Expression of Epstein-Barr Virus Encoded Small Nuclear RNA1 in Japanese Nasopharyngeal Carcinomas. Acta Oto-Laryngologica, 547, 113-117. http://dx.doi.org/10.1080/000164802760057725
[23]	Pathmanathan, R., Prasad, U., Chandrika, G., Sadler, R., Flynn, K. and Traub, R.N. (1995) Undifferentiated, Nonkeratinizing, and Squamous Cell Carcinoma of the Nasopharynx: Variants of Epstein Barr Virus-Infected Neoplasia. American Journal of Pathology, 146, 1355-1367.
[24]	Gabusi, E., Lattes, C., Fiorentino, M., D’Errico, A. and Grigioni, W.F. (2001) Expression of Epstein-Barr Virus-Encoded RNA and Biological Markers in Italian Nasopharyngeal Carcinomas. Journal of Experimental & Clinical Cancer Research, 20, 371-376.
[25]	Plaza, G., Manzanal, A.I., Fogué, L., Santón, A., Martínez-Montero, J.C. and Bellas, C. (2002) Association of Epstein-Barr Virus and Nasopharyngeal Carcinoma in Caucasian Patients. Annals of Otology, Rhinology, and Laryngology, 111, 210-216.
[26]	Lin, Z., Khong, B., Kwok, S., Cao, H., West, R.B., Le, Q. and Kong C. (2014) Human Papiloma Virus 16 Detected in Nasopharyngeal Carcinomas in Caucasian American But Not in Endemic Southern Chinese Patients. Head & Neck, 36, 709-714. http://dx.doi.org/10.1002/hed.23362
[27]	Laantri, N., Attaleb, M., Kandil, M., Naji, F., Mouttaki, T., Dardari, R., et al. (20011) Human Papillomavirus Detection in Moroccan Patients with Nasopharyngeal Carcinoma. Infect Agent Cancer, 6, 3.
[28]	Mirzamani, N., Salehian, P., Farhadi, M. and Tehran, E.A. (2006) Detection of EBV and HPV in Nasopharyngeal Carcinoma by in Situ Hybridization. Experimental and Molecular Pathology, 81, 231-234 http://dx.doi.org/10.1016/j.yexmp.2006.04.006
[29]	Ertan, Y., Hekimgil, M., Karaarslan, S. and Soydan, S. (2008) Expression of Epstein-Barr-Virus-Encoded Small Nuclear RNA in Nasopharyngeal Carcinomas of Aegean Turkish Patients. Virchows Arch, 452, 411-414. http://dx.doi.org/10.1007/s00428-008-0589-6
[30]	Sam, C.K., Boorks, L.A., Niedoitek, G., Young, L.S., Prasad, U. and Riskinson, A.B. (1993) Analysis of Epstein-Barr Virus Infection in Nasopharyngeal Carcinoma from a Group at High Risk of Nasopharyngeal Carcinoma. International Journal of Cancer, 53, 957-962. http://dx.doi.org/10.1002/ijc.2910530616
[31]	Dickens, P., Srivastava, G., Loke, S.L., Chan, C.W. and Liu, Y.T. (1992) Epstein-Barr Virus DNA in Nasopharyngeal Carcinomas from Chinese Patients in Hong Kong. Journal of Clinical Pathology, 45, 396-397. http://dx.doi.org/10.1136/jcp.45.5.396