15. Suhaib F.doc J Bagh College Dentistry Vol. 27(4), December 2015 P16 protein and Oral Diagnosis 96 P16 Protein and Human Papillomavirus (HPV16, 18) Expressions in Oral Lichen Planus and Squamous Cell Carcinoma Suhaib Raghib Muhsin, B.D.S. (1) Ahlam Hameed Majeed, B.D.S., M.Sc. (2) ABSTRACT Background: Oral carcinogenesis is a molecular and histological multistage process featuring genetic and phenotypic markers for each stage, which involves enhanced function of several oncogenes and/or the deactivation of tumor suppressor genes, resulting in the loss of cell cycle checkpoints. The progression towards malignancy includes sequential histopathological alterations ranging from hyperplasia through dysplasia to carcinoma in situ and invasive carcinoma. The p16 gene produces p16 protein, which in turn inhibits phosphorylation of retinoblastoma, p16 play a significant role in early carcinogenesis. Human papillomavirus is a well established heterogeneous virus and plays an important role in oral cancers. The aims of the study were to evaluate, compare and correlate the immunohistochemical expression of p16 protein and HPV16/18 with each other in oral lichen planus and oral squamous cell carcinoma, and with various clinicopathological findings. Materials and methods: Forty formalin-fixed, paraffin embedded tissue blocks (24 cases of oral lichen planus, and 16 cases of oral squamous cell carcinoma) were included in this study, an immunohistochemical staining was performed using anti p16 monoclonal antibody, and anti HPV16/18 monoclonal antibodies. Results: Positive IHC expression of p16 was found in 11 cases (68.75%) of OSCC, and in 19 cases (79.166%) of OLP. Positive IHC expression of HPV16 was found in 2 cases (12.5%) of OSCC, and in 1 case (4.16%) of OLP. IHC expression of HPV18 showed negative expression in all cases of OSCC, and found only in 1 case (4.16%) of OLP. Conclusions: This study signifies the statistically non significant correlation between p16 and HPV 16/18 in OLP and OSCC. Keywords: OLP, OSCC, P16, HPV. (J Bagh Coll Dentistry 2015; 27(4):96-100). INTRODUCTION Oncogenesis (carcinogenesis) is the progression from a normal healthy cell to a pre- malignant or a potentially malignant cell - characterised by an ability to proliferate autonomously. Oncogenesis involves a series of genetic steps and also epigenetic–outside the gene-changes. These changes include the aberrant expression and function of molecules regulating cell signalling, growth, survival, motility, angiogenesis (blood vessel proliferation), and cell cycle control (1). Cancer of the oral cavity is the sixth most common cancer worldwide and account for nearly 3% of all malignancies (2). P16 is a tumor suppressor protein, that in humans is encoded by the CDKN2A gene (3,4). It regulates the Rb tumor suppressor pathway by keeping Rb in a hypophosphorylated state, which further promotes the binding of E2F to achieve G1 cell- cycle arrest. The disruption of p16 expression has been reported in various human cancers (5-7). Human papillomavirus (HPV) is a well- established heterogeneous virus and is important in human carcinogenesis. It not only causes a vast majority of cervical cancers but also plays an important role in anogenital and oral cancers (8). (1) Master student, Department of Oral Diagnosis, College of Dentistry, University of Baghdad. (2) Professor, Department of Oral Diagnosis, College of Dentistry, University of Baghdad. It has been established that HPVs are exclusively epitheliotropic, meaning that their infection is specifically localised in epithelial cells of the host. In order to complete their life cycle, they rely on epithelial differentiation(9). The present study aimed to evaluate, compare and correlate the immunohistochemical expression of p16 protein and HPV16/18 in oral premalignant lesion and oral squamous cell carcinoma, and with each other in various clinicopathological findings. MATERIALS AND METHODS The study samples included 40 formalin-fixed, paraffin embedded tissue blocks (24 OLP, and 16 OSCC) dated from (1975 till 2013), were obtained from the archives of the department of Oral & Maxillofacial Pathology/ College of Dentistry/ University of Baghdad; Al-Shaheed Ghazi Hospital/ Medical City during the period from (1975-2013). Sections of 4μm thickness were mounted on normal glass slides, stained with H&E for histopathologically re-evaluation. Four other 4μm thick sections for each case were cut and mounted on positively charged slides (Fisher scientific and Escho super frost plus, USA) for immunohistochemical staining with monoclonal antibody p16 and HPV16/18 using Abcam expose mouse and rabbit HRP/DAB J Bagh College Dentistry Vol. 27(4), December 2015 P16 protein and Oral Diagnosis 97 immunohistochemical detection kit (Catalog No. ab80436, Cambridge, UK). RESULTS Positive p16 Immunostaining was detected as brown nuclear or (nuclear and cytoplasmic) expression. IHC staining of p16 in OSCC reveals that 5 cases (31.25%) showed negative expression, 5 cases (31.25%) showed weak positive expression, 1 case (6.25%) showed moderate positive expression, and 5 cases (31.25%) showed high positive expression. While in OLP IHC staining of p16 reveals that 19 cases (79.166%) showed negative expression (Cases showing more than 5% of positive cells), and 5 cases (20.833%) showed negative expression (Cases showing less than 5% of positive cells), figures (1,2). Positive HPV16/18 immunostaining was detected as brown nuclear expression. Positive IHC expression of HPV16 was found in 2 cases only (12.5%) of OSCC, and 14 cases (87.5%) showed negative expression. HPV16 positivity was found in 1 case only (4.16%) of OLP, and 23 cases (95.83%) showed negative expression. IHC expression of HPV18 showed negative expression in all cases of OSCC. Positive IHC expression of HPV18 was found in 1 case only (4.16%) of OLP, and 23 cases (95.83%) showed negative expression. Figure (3,4,5). P16 expression were observed in almost cases of OSCC and OLP and according to Chi square test, statistically non significant correlation with clinicopathological findings (age, sex, tumor site, tumor grade) except tumor site of OSCC in p16 was statistically significant, while HPV16/18 expression was detectable in few cases of OSCC and OLP and correlation between the expression of markers (P16, HPV16, HPV18) were non significant statistically in OSCC and OLP (P=0.757, 0.327, 0.874) respectively, as clarified in tables (1,2,3). Figure (1): Positive Expression of p16 in Well Differentiated OSCC (40X). Figure (2): Positive Expression of p16 in OLP (40X). Figure (3): Positive Expression of HPV16 in OSCC (40X). Figure (4): Positive Expression of HPV16 in OLP (40X). J Bagh College Dentistry Vol. 27(4), December 2015 P16 protein and Oral Diagnosis 98 P16 +Ve -VE N X2 Sig OLP 19 5 24 0.096 0.757 OSCC 12 4 16 Total 31 9 40 DISCUSSION The present study is not a large epidemiological one that expressed the incidence and prevalence of different clinicopathological features of OLP and OSCC, however, there was a close correlation between the present data and other published data concerning the incidence of OLP and OSCC in Iraq in the past studies records and studies in other parts in the world (10,11). Assessment of p16 Immunohistochemistry: P16 positivity was found in (68.75%) of OSCC cases. Concerning the correlation between clinicopathological findings of OSCC cases and p16, the present study showed statistically significant correlation between p16 expression and the tumor site, while there was non significant difference between p16 with age, sex, site, and grades of OSCC. The discrepancies in the result of p16 expression in this result and with other studies (12,13) could be attributed to limited sample size of the current study. Concerning OLP cases the results of this study showed that positive expression of p16 was observed in 19 cases (79.166%) and negative expression in 5 cases (20.833%). Montebugnoli et al and Poomsawat et al detected p16 in 64% of OLP samples and reported that p16 expression in 65.2% of OLP cases respectively (14,15). Different cancer-causing agents may lead to p16INK4a gene inactivation as well as altered p53 and pRb tumor suppressive pathways (16,17). The loss of p16 expression as a result of promoter hypermethylation is an early event in oral carcinoma and a useful biomarker for predicting local recurrence in carcinoma of the tongue (18). However, the role of p16 hypermethylation as a predictive risk factor for OSCC or disease recurrence remains unclear and contradictory (19). Assessment of HPV Immunohistochemistry: In OSCC, The present study showed negative HPV16 immunoreactivity in 14 cases (87.5%) and only 2 cases (12.5%) showed positive expression whereas in HPV18, all cases showed negative expression. This finding was in agreement with studies in other part of the world (20-22). In current study, most of OSCC cases that showed negative expression found in cytoplasm. Concerning the correlation between clinicopathological findings of OSCC cases and HPV16 and 18, the present study showed non significant statistical correlation between HPV16, 18 expression with age, sex, site and grade were found (23-28). Regarding OLP, the result of this study showed positive expression of HPV16 only in 1 case and other 23 cases (95.83%) showed negative expression and same result was found in HPV18. Few studies employed immunohistochemical detection of HPV 16 in oral lichen planus, in a Turkish study by Yildirim et al 21% of the cases were positive by immunohistochemistry (29). Concerning correlation between p16 and HPV16/18 in oral squmous cell carcinoma and HPV16 +Ve -VE N X2 Sig OLP 1 23 24 0.096 0.327 OSCC 2 14 16 Total 3 37 40 HPV16 +Ve -VE N X2 Sig OLP 1 23 24 0.684 0.871 OSCC 0 16 16 Total 1 39 40 Figure (5): Positive Expression of HPV18 in OLP (40X). Table (1): Correlation between OSCC and OLP of P16 Expression Table (2): Correlation between OSCC and OLP of HPV16 Table (3): Correlation between OSCC and OLP of HPV18 J Bagh College Dentistry Vol. 27(4), December 2015 P16 protein and Oral Diagnosis 99 oral lichen planus, the present study showed statistically non significant correlation between p16 and HPV16,18 in OSCC and OLP. Nemes et, al., showed that over-expression of p16INK4a proteins in OSCC did not correlate with HR-HPV types (30). In addition, very high p16 expression observed not only in HPV positive groups but also in other groups in the absence of HPVs. Therefore, it revealed unconvincing support for previous claims on the HPV-p16 relationship (31-34). Concerning OLP, non significant correlation was observed in p16 and HPV16 and 18. Montebugnoli et al found p16INK4 expression was detected in 26 specimens, while HPV was found in four lesions: three low-risk HPV, and one high-risk HPV. All HPV-positive lesions also showed p16INK4A overexpression, whereas 22 cases of overexpressed p16INK4A were HPV- negative (35). REFERENCES 1. Surveillance Epidemiology and End Results (SEER). SEER Cancer Statistics Review 1975-2004. National Cancer Institute. 2. Shah JP, Gil Z. Current Concepts in Management of Oral Cancer-Surgery. Oral Oncol 2009; 45(4): 394- 401. 3. Stone S, Jiang P, Dayananth P, Tavtigian SV, Katcher H, Parry D, Peters G, Kamb A, Complex structure and regulation of the P16 (MTS1) locus. Cancer Res 1995; 55(14): 2988–94. 4. Nobori T, Miura K, Wu DJ, Lois A, Takabayashi K, Carson DA, Deletions of the cyclin-dependent kinase- 4 inhibitor gene in multiple human cancers. Nature 1994; 368(6473): 753–6. 5. Maruschke M, Thur S, Kundt G, Nizze H, Hakenberg OW, Immunohistochemical expression of retinoblastoma protein and p16 in renal cell carcinoma. Urol Int. 2011; 86(1): 60–7. 6. Koh VM, Shi YX, Tang QH, P16 and retinoblastoma protein expression in endometrial carcinoma and clinical significance. Eur J Gynaecol Oncol 2011; 32 (3): 309–315. 7. Arima Y, Hayashi N, Hayashi H, Sasaki M, Kai K, Sugihara E, et al, Loss of p16 expression is associated with the stem cell characteristics of surface markers and therapeutic resistance in estrogen receptor- negative breast cancer. Int J Cancer 2012; 130(11): 2568-79. 8. Chaudhary AK, Pandya S, Singh M, Singh M, Mehrotra R, Identification of high-risk human papillomavirus-16 and -18 infections by multiplex PCR and their expression in oral submucous fibrosis and oral squamous cell carcinoma. Head Neck Oncol 2013; 5(1): 4. 9. Doorbar J, The papillomavirus life cycle. J Clin Virol 2005; 32: 7-15. 10. Regezi. Oral Pathology, Clinical Pathologic Correlations. 5th ed. St. Louis, Missouri: Elisver Saunders; 2008. 11. Al-Bayati SAAF. Oral Lichen planus: A clinical study of 123 patients attending an Oral Medicine Clinic, Baghdad University, Iraq, Gulf Medical J 2012; 1(1): 10-4. 12. Abrahao AC, Bonelli BV, Nunes FD, Dias EP, Cabral MG, Immunohistochemical expression of p53, p16 and hTERT in oral squamous cell carcinoma and potentially malignant disorders. Braz Oral Res 2011; 25(1): 34-41. 13. Chen YW, Kao SY, Yang MH, Analysis of p16INK4A expression of oral squamous cell carcinomas in Taiwan: Prognostic correlation without relevance to betel quid consumption. J Surg Oncol 2012; 106(2): 149–54. 14. Montebugnoli L, Gissi DB, Scapoli L, Palmieri A, Morandi L, Manelli I, Foschini MP. p16INK4 expression is not associated with human papilloma virus in oral lichen planus. Oral Surg Oral Med Oral Pathol Oral Radiol 2014; 118: 694-702. 15. Poomsawat S, Buajeeb W, Khovidhunkit SO, Punyasingh J, Overexpression of cdk4 and p16 in oral lichen planus supports the concept of premalignancy. J Oral Pathol Med 2011; 40: 294-9. 16. De Oliveira LR, Ribeiro-Silva A, Zucoloto S, Prognostic impact of p53 and p63 immunoexpression in oral squamous cell carcinoma. J Oral Pathol Med 2007; 36(4): 191-7. 17. Karsai S, Abel U, Roesch-Ely M, Affolter A, Hofele C, Joos S, et al, Comparison of p16(INK4a) expression with p53 alterations in head and neck cancer by tissue microarray analysis. J Pathol 2007; 211(3): 314-22. 18. Sinha P, Bahadur S, Thakar A, Matta A, Macha M, Ralhan R. and Gupta S D, Significance of promoter hypermethylation of p16 gene for margin assessment in carcinoma tongue. Head Neck 2009; 31(11): 1423- 30. 19. Ruesga M T, Acha-Sagredo A, Rodríguez M J, Aguirregaviria J I, Videgain J, Rodríguez C, de Pancorbo M D L A M, Aguirre J M, p16INK4a promoter hypermethylation in oral scrapings of oral squamous cell carcinoma risk patients. Cancer Lett 2007; 250(1): 140-5. 20. Tsuchiya H, Y Tomita, H Shirasawa, H Tanzawa, K. Sato, and B Simizu, Detection of human papillomavirus in head and neck tumors with DNA hybridization and immunohistochemical analysis, Oral Surgery Oral Medicine and Oral Pathology 1991; 71 (6): 721–5. 21. Young S K, K. W, Min, In situ DNA hybridization analysis of oral papillomas, leukoplakias, and carcinomas for human papillomavirus, Oral Surgery Oral Medicine and Oral Pathology 1991; 71(6): 726– 9. 22. Ringström E, Peters E, Hasegawa M, et al, Human Papillomavirus Type 16 and Squamous Cell Carcinoma of the Head and Neck, Department of Cancer Cell Biology, Harvard School of Public Health, Boston, Massachusetts 02115; Department of Oral Epidemiology and Department of Oral Medicine, Harvard School of Dental Medicine, Boston, Massachusetts; and Department of Head and Neck Oncology, Dana Farber Cancer Institute, Boston, Massachusetts, Clin Cancer Res 2002; 8: 3187-92. 23. Kurose K, M Terai, N Soedarsono et al, Low prevalence of HPV infection and its natural history in normal oral mucosa among volunteers on Miyako Island, Japan. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004; 98(1): 91–6. J Bagh College Dentistry Vol. 27(4), December 2015 P16 protein and Oral Diagnosis 100 24. Agrawal GP, Joshi PS, Agrawal A. Role of HPV-16 in pathogenesis of oral epithelial dysplasia and oral squamous cell carcinoma and correlation of p16ink4a expression in HPV-16 positive cases: An immunohistochemical study. ISRN Pathology 2013: Article ID 807095. Pages 7. 25. Cruz IBF, Snijders PJF, Steenbergen RDM, et al, Agedependence of human papillomavirus DNA presence in oral squamous cell carcinomas, European J Cancer B, 1996; 32(1): 55–62. 26. Sanders JHCM, Prime SS, Scully C, Maitland NJ. Detection of human pappiloma virus type 16 DNA in oral squames from normal young adults, Journal of Oral Pathology and Medicine 1992; 21(10): 465–70. 27. Schlecht NF, Burk RD, Adrien L, et al. Gene expression profiles in HPV-infected head and neck cancer. J Pathol 2007; 213(3): 283–93. 28. Badaracco G, Venuti A, Bartolazzi A, Morello R, Marzetti F, Marcante ML. Overexpression of p53 and bcl-2 proteins and the presence of HPV infection are independent events in head and neck cancer, Journal of Oral Pathology and Medicine 2000; 29(4): 173–9. 29. Yildirim B, Sengüven B, Demir C, Prevalence of herpes simplex, Epstein Barr and human papilloma viruses in oral lichen planus. Med Oral Patol Oral Cir Bucal 2011; 16(2): 170–4. 30. Nemes JA, Deli L, Nemes Z, Márton IJ. Expression of p16INK4A, p53, and Rb proteins are independent from the presence of human papillomavirus genes in oral squamous cell carcinoma. Oral Surg Oral Med Oral Rad Oral Endod 2006; 102(3): 344-52. 31. Aulmann S, Schleibaum J, Penzel R, Schirmacher P, Gebauer G, Sinn H P, Gains of chromosome region 3q26 in intraepithelial neoplasia and invasive squamous cell carcinoma of the vulva are frequent and independent of HPV status. J Clin Pathol 2008; 61(9): 1034-7. 32. Bohn OL, Navarro L, Saldivar J, Sanchez-Sosa S. Identification of human papillomavirus in esophageal squamous papillomas. World J Gastroenterol 2008; 14(46): 7107-11. 33. Samama B, Schaeffer C, Boehm N, p16 expression in relation to human papillomavirus in liquid-based cervical smears. Gynecol Oncol 2008; 109(2): 285-90. 34. Smeets SJ, Hesselink AT, Speel EM, Haesevoets A, Snijders PJF, Pawlita M, Meijer CJ LM, Braakhuis BJ M, Leemans CR, Brakenhoff RH. A novel algorithm for reliable detection of human papillomavirus in paraffin embedded head and neck cancer specimen. Int J Cancer 2007; 121(1): 2465-72. 35. Montebugnoli L, Gissi DB, Scapoli L, Palmieri A, Morandi L, Manelli I, Foschini MP. p16INK4 expression is not associated with human papilloma virus in oral lichen planus. Oral Surg Oral Med Oral Pathol Oral Radiol 2014; 118: 694-702.