Iranian Journal of Medical Sciences

Document Type : Original Article(s)


1 Department of Oral and Maxillofacial Pathology, School of Dentistry, Tehran University of Medical Sciences, Tehran, Iran

2 Department of Pathology, Cancer Institute Hospital, IKHC, Tehran University of Medical Sciences, Tehran, Iran

3 Department of Community Oral Health Department, School of Dentistry, Tehran University of Medical Sciences, Tehran, Iran



Background: High-risk Human Papillomavirus (HPV) genotypes are found in malignant oral epithelial lesions, and HPV infection is proposed as a risk factor for initiating Squamous cell carcinoma (SCC) in the head and neck region. This study suggests a practical approach to detect HPV in HPV-associated oral epithelial dysplasia (HAOED).
Methods: Fifty-four oral epithelial dysplasia specimens were examined, comprising twenty-seven cases diagnosed with high-grade dysplasia and twenty-seven cases diagnosed with low-grade dysplasia using a binary grading system. To assess the cases for HPV, the specimens were examined for p16 protein using an immunohistochemical (IHC) study, and then, the Chromatin In Situ Hybridization (CISH) test was performed for all positive cases. Chromatin Immunoprecipitation-Polymerase Chain Reaction (ChIP-PCR) was performed on CISH-positive specimens to assess the outcome. This cross-sectional study was conducted in 2020 at Tehran University of Medical Science. SPSS software version 22.0 was used to perform the Chi square or Fisher’s exact test to examine the relationship between variables (statistically significant level P<0.05).
Results: The expression of p16 protein was not associated with the severity of epithelial dysplasia (81.5% in low-grade and 59.2% in high-grade cases) (P=0.16). Moreover, according to the CISH test result, 9.25% of all specimens were positive (P>0.99), and in the nine cases, undergone the ChIP-PCR study, two cases (22.2%) showed positivity for HPV-16, while one case (11.1%) demonstrated positivity for HPV-51.
Conclusion: Regarding HAOED, here, we proposed a step-by-step combination approach using different diagnostic methods, including IHC for p16 protein, CISH, and ChIP-PCR based on a complementary algorithm.


  1. de Sanjose S, Quint WG, Alemany L, Geraets DT, Klaustermeier JE, Lloveras B, et al. Human papillomavirus genotype attribution in invasive cervical cancer: a retrospective cross-sectional worldwide study. Lancet Oncol. 2010;11:1048-56. doi: 10.1016/S1470-2045(10)70230-8. PubMed PMID: 20952254.
  2. Mazul AL, Taylor JM, Divaris K, Weissler MC, Brennan P, Anantharaman D, et al. Oral health and human papillomavirus-associated head and neck squamous cell carcinoma. Cancer. 2017;123:71-80. doi: 10.1002/cncr.30312. PubMed PMID: 27571516; PubMed Central PMCID: PMCPMC5161679.
  3. Amin MB, Greene FL, Edge SB, Compton CC, Gershenwald JE, Brookland RK, et al. The Eighth Edition AJCC Cancer Staging Manual: Continuing to build a bridge from a population-based to a more “personalized” approach to cancer staging. CA Cancer J Clin. 2017;67:93-9. doi: 10.3322/caac.21388. PubMed PMID: 28094848.
  4. Oliveira MC, Soares RC, Pinto LP, Souza LB, Medeiros SR, Costa Ade L. High-risk human papillomavirus (HPV) is not associated with p53 and bcl-2 expression in oral squamous cell carcinomas. Auris Nasus Larynx. 2009;36:450-6. doi: 10.1016/j.anl.2008.10.011. PubMed PMID: 19124208.
  5. O’Regan EM, Toner ME, Finn SP, Fan CY, Ring M, Hagmar B, et al. p16(INK4A) genetic and epigenetic profiles differ in relation to age and site in head and neck squamous cell carcinomas. Hum Pathol. 2008;39:452-8. doi: 10.1016/j.humpath.2007.08.004. PubMed PMID: 18261630.
  6. Iocca O, Sollecito TP, Alawi F, Weinstein GS, Newman JG, De Virgilio A, et al. Potentially malignant disorders of the oral cavity and oral dysplasia: A systematic review and meta-analysis of malignant transformation rate by subtype. Head Neck. 2020;42:539-55. doi: 10.1002/hed.26006. PubMed PMID: 31803979.
  7. Warnakulasuriya S, Kujan O, Aguirre-Urizar JM, Bagan JV, Gonzalez-Moles MA, Kerr AR, et al. Oral potentially malignant disorders: A consensus report from an international seminar on nomenclature and classification, convened by the WHO Collaborating Centre for Oral Cancer. Oral Dis. 2021;27:1862-80. doi: 10.1111/odi.13704. PubMed PMID: 33128420.
  8. Odell E, Kujan O, Warnakulasuriya S, Sloan P. Oral epithelial dysplasia: Recognition, grading and clinical significance. Oral Dis. 2021;27:1947-76. doi: 10.1111/odi.13993. PubMed PMID: 34418233.
  9. Nalli G, Mastrotta P, Garcia MG, Tatti S, Verdu S. Detection of Oral Human Papillomavirus (HPV) and its Clinical Importance. J Dent (Shiraz). 2022;23:51-7. doi: 10.30476/DENTJODS.2021.88338.1326. PubMed PMID: 35291686; PubMed Central PMCID: PMCPMC8918636.
  10. Woo SB, Cashman EC, Lerman MA. Human papillomavirus-associated oral intraepithelial neoplasia. Mod Pathol. 2013;26:1288-97. doi: 10.1038/modpathol.2013.70. PubMed PMID: 23599160.
  11. Jayaprakash V, Reid M, Hatton E, Merzianu M, Rigual N, Marshall J, et al. Human papillomavirus types 16 and 18 in epithelial dysplasia of oral cavity and oropharynx: a meta-analysis, 1985-2010. Oral Oncol. 2011;47:1048-54. doi: 10.1016/j.oraloncology.2011.07.009. PubMed PMID: 21816661; PubMed Central PMCID: PMCPMC3640331.
  12. Chen X, Zhao Y. Human papillomavirus infection in oral potentially malignant disorders and cancer. Arch Oral Biol. 2017;83:334-9. doi: 10.1016/j.archoralbio.2017.08.011. PubMed PMID: 28886585.
  13. Angiero F, Gatta LB, Seramondi R, Berenzi A, Benetti A, Magistro S, et al. Frequency and role of HPV in the progression of epithelial dysplasia to oral cancer. Anticancer Res. 2010;30:3435-40. PubMed PMID: 20944119.
  14. Armitage P, Berry G, Matthews J. Analysing means and proportions. Statistical methods in medical research. 2002;83:146. doi: 10.1002/9780470773666.ch4.
  15. Kujan O, Oliver RJ, Khattab A, Roberts SA, Thakker N, Sloan P. Evaluation of a new binary system of grading oral epithelial dysplasia for prediction of malignant transformation. Oral Oncol. 2006;42:987-93. doi: 10.1016/j.oraloncology.2005.12.014. PubMed PMID: 16731030.
  16. Thompson L. World Health Organization classification of tumours: pathology and genetics of head and neck tumours. Ear Nose Throat J. 2006;85:74. PubMed PMID: 16579185.
  17. Koo CL, Kok LF, Lee MY, Wu TS, Cheng YW, Hsu JD, et al. Scoring mechanisms of p16INK4a immunohistochemistry based on either independent nucleic stain or mixed cytoplasmic with nucleic expression can significantly signal to distinguish between endocervical and endometrial adenocarcinomas in a tissue microarray study. J Transl Med. 2009;7:25. doi: 10.1186/1479-5876-7-25. PubMed PMID: 19366452; PubMed Central PMCID: PMCPMC2672079.
  18. Dost F, Le Cao K, Ford PJ, Ades C, Farah CS. Malignant transformation of oral epithelial dysplasia: a real-world evaluation of histopathologic grading. Oral Surg Oral Med Oral Pathol Oral Radiol. 2014;117:343-52. doi: 10.1016/j.oooo.2013.09.017. PubMed PMID: 24388536.
  19. Fonseca-Silva T, Farias LC, Cardoso CM, Souza LR, Carvalho Fraga CA, Oliveira MV, et al. Analysis of p16(CDKN2A) methylation and HPV-16 infection in oral mucosal dysplasia. Pathobiology. 2012;79:94-100. doi: 10.1159/000334926. PubMed PMID: 22285991.
  20. Antonsson A, Neale RE, Boros S, Lampe G, Coman WB, Pryor DI, et al. Human papillomavirus status and p16(INK4A) expression in patients with mucosal squamous cell carcinoma of the head and neck in Queensland, Australia. Cancer Epidemiol. 2015;39:174-81. doi: 10.1016/j.canep.2015.01.010. PubMed PMID: 25677091.
  21. Szalmas A, Konya J. Epigenetic alterations in cervical carcinogenesis. Semin Cancer Biol. 2009;19:144-52. doi: 10.1016/j.semcancer.2009.02.011. PubMed PMID: 19429477.
  22. Rahrotaban S, Mahdavi N, Abdollahi A, Yazdani F, Kaghazloo A, Derakhshan S. Carcinoma-associated Fibroblasts are a Common Finding in the Microenvironment of HPV-positive Oropharyngeal Squamous Cell Carcinoma. Appl Immunohistochem Mol Morphol. 2019;27:683-8. doi: 10.1097/PAI.0000000000000687. PubMed PMID: 31593979.
  23. Dalakoti P, Ramaswamy B, Bhandarkar AM, Nayak DR, Sabeena S, Arunkumar G. Prevalence of HPV in Oral Squamous Cell Carcinoma in South West India. Indian J Otolaryngol Head Neck Surg. 2019;71:657-64. doi: 10.1007/s12070-018-1470-9. PubMed PMID: 31742038; PubMed Central PMCID: PMCPMC6848302.
  24. Thankappan P, Ramadoss MN, Joseph TI, Augustine PI, Shaga IB, Thilak J. Human Papilloma Virus and Cancer Stem Cell markers in Oral Epithelial Dysplasia-An Immunohistochemical Study. Rambam Maimonides Med J. 2021;12. doi: 10.5041/RMMJ.10451. PubMed PMID: 34709167; PubMed Central PMCID: PMCPMC8549839
  25. Nemes JA, Deli L, Nemes Z, Marton IJ. Expression of p16(INK4A), p53, and Rb proteins are independent from the presence of human papillomavirus genes in oral squamous cell carcinoma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006;102:344-52. doi: 10.1016/j.tripleo.2005.10.069. PubMed PMID: 16920543.
  26. Zhang L, Lewis JS, Jr., El-Mofty SK, Gandhi M, Chernock RD. Nonkeratinizing Squamous Cell Carcinoma In Situ of the Upper Aerodigestive Tract: An HPV-Related Entity. Head Neck Pathol. 2017;11:152-61. doi: 10.1007/s12105-016-0749-y. PubMed PMID: 27492445; PubMed Central PMCID: PMCPMC5429272.
  27. Venkatesh A, Elengkumaran S, Ravindran C, Malathi N. Association of Human Papilloma Virus in Oral Squamous Cell Carcinoma: An Alarming Need for Human Papillomavirus 16 Screening in Cancer Patients. J Pharm Bioallied Sci. 2021;13:S1224-S7. doi: 10.4103/jpbs.jpbs_370_21. PubMed PMID: 35017960; PubMed Central PMCID: PMCPMC8686889.
  28. Cunningham LL, Jr., Pagano GM, Li M, Tandon R, Holm SW, White DK, et al. Overexpression of p16INK4 is a reliable marker of human papillomavirus-induced oral high-grade squamous dysplasia. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006;102:77-81. doi: 10.1016/j.tripleo.2005.11.028. PubMed PMID: 16831676.
  29. Husain N, Neyaz A. Human papillomavirus associated head and neck squamous cell carcinoma: Controversies and new concepts. J Oral Biol Craniofac Res. 2017;7:198-205. doi: 10.1016/j.jobcr.2017.08.003. PubMed PMID: 29124000; PubMed Central PMCID: PMCPMC5670302.
  30. Fauzi FH, Hamzan NI, Rahman NA, Suraiya S, Mohamad S. Detection of human papillomavirus in oropharyngeal squamous cell carcinoma. J Zhejiang Univ Sci B. 2020;21:961-76. doi: 10.1631/jzus.B2000161. PubMed PMID: 33843162; PubMed Central PMCID: PMCPMC7759454.