Methylation Status of P16Ink4a in Human Papillomavirus-Associated Cancer of Oral Cavity and Oropharynx in Northeastern Thailand

Document Type : Research Articles


1 Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand

2 HPV and EBV and Carcinogenesis Research Group, Khon Kaen University,Khon Kaen, Thailand

3 Department of Otorhinolaryngology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand

4 Department of Obstetrics and Gynecology, Faculty of Medicine, Khon Kaen, University, Thailand

5 Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kae, Thailand

6 Department of Anatomical Pathology, Khon Kaen Central Hospital, Khon Kaen, Khon Kaen, Thailand

7 Department of Epidemiology, Faculty of Public Health, Khon Kaen University

8 Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand


Background: Over-expression of p16INK4a protein is a biomarker for human papillomavirus (HPV)-associated cervical cancer. However, absence of p16INK4a protein expression in HPV-associated cancer of the oral cavity and oropharynx has been reported. Among a number of possible reasons for this is methylation, which is frequently noted in the promoter region of p16INK4a and is associated with silencing of the gene and disease severity. Methods: We investigated the relationships between p16INK4a protein expression, HPV infection and methylation status of the p16INK4a promoter in cancers of the oral cavity and oropharynx. Fifty-three formalin-fixed paraffin-embedded (FFPE) cancer tissue samples from the oral cavity (49 cases) and oropharynx (4 cases) were studied. P16INK4a protein expression was determined using immunohistochemical staining (IHC). Additional oral tissues lacking squamous intraepithelial lesions (SILs), and cervical tissues with high-level SILs, were used as negative and positive controls, respectively. High-risk HPV infection was detected using HPV E6/E7 mRNA in situ hybridization. Methylation status of the p16INK4a promoter was investigated using sodium bisulfite treatment and methylation-specific PCR (MS-PCR).Results: HPV infection was found in 40.8% (20/49) and 50.0% (2/4) of oral cavity and oropharynx cancers, respectively. Promoter methylation of p16INK4a occurred in 73.6 % of all cases and differed significantly in frequency between HPV-positive (90.9%, 20/22) and HPV-negative (61.3%, 19/31) samples. Expression of p16INK4a was found in 35.8% (19/53) and commonly detected in samples with p16INK4a unmethylation (79.5%). Interestingly, the silencing of p16INK4a (64.2%, 34/53) was significantly associated with methylation status (91.2%, 31/34), especially in HPV-infected samples in which the p16INK4a promoter was methylated (52.9%, 18/34). Conclusions: This result demonstrated high frequency of p16INK4a promoter methylation status in HPV-associated HNSCC subsets that could influence the silent p16INK4a expression and might promote disease severity.


Main Subjects

Volume 18, Issue 3
March 2017
Pages 699-705
  • Receive Date: 04 January 2017
  • Revise Date: 11 April 2017
  • Accept Date: 11 April 2017
  • First Publish Date: 11 April 2017