Abstract
Human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) is recognized as a distinct disease entity associated with improved survival. DNA hypermethylation profiles differ significantly by HPV status suggesting that a specific subset of methylated CpG loci could give mechanistic insight into HPV-driven OPSCC. We analyzed genome-wide DNA methylation of primary tumor samples and adjacent normal mucosa from 46 OPSCC patients undergoing treatment at Montefiore Medical Center, Bronx, NY using the Illumina HumanMethylation27 beadchip. For each matched tissue set, we measured differentially methylated CpG loci using a change in methylation level (M value). From these analyses, we identified a 22 CpG loci panel for HPV+ OPSCC that included four CDKN2A loci downstream of the p16(INK4A) and p14(ARF) transcription start sites. This panel was significantly associated with overall HPV detection (P < 0.05; ROC area under the curve = 0.96, 95% CI: 0.91–1.0) similar to the subset of four CDKN2A-specific CpG loci (0.90, 95% CI: 0.82–0.99) with equivalence to the full 22 CpG panel. DNA hypermethylation correlated with a significant increase in alternative open reading frame (ARF) expression in HPV+ OPSCC primary tumors, but not to the other transcript variant encoded by the CDKN2A locus. Overall, this study provides evidence of epigenetic changes to the downstream region of the CDKN2A locus in HPV+ oropharyngeal cancer that are associated with changes in expression of the coded protein products.
Highlights
Human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) is recognized as a distinct disease associated with improved survival and response to therapy, and carries distinct pathologic and molecular characteristics, including positive immunostaining for the p16 protein [1]
We showed previously that HPV+ oropharyngeal cancers exhibit significantly higher numbers of differentially methylated CpG loci between tumors and adjacent normal mucosa compared to HPVÀ oropharyngeal, oral cavity, and laryngeal cancers [11] suggesting that a specific subset of CpG loci may be identified that could further stratify these cancers and reflect DNA methylation changes associated with HPV
When mining gene expression data from our head and neck cancer genomics database, HPV+ OPSCC tumors showed a significant increase in the expression of DNA methyltransferase 1 (DNMT1), the major eukaryotic DNA methyltranferase (Fig. 1A) supporting our observation of increased DNA methylation in these tumors [11]
Summary
Human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) is recognized as a distinct disease associated with improved survival and response to therapy, and carries distinct pathologic and molecular characteristics, including positive immunostaining for the p16 protein [1]. Results from molecular array studies support the hypothesis that HPV-positive and negative OPSCC are biologically distinct and may represent different cancer lineages formed through separate etiologic pathways of multistage tumorigenesis [2, 3]. Cancer Medicine published by John Wiley & Sons Ltd. Cancer Medicine published by John Wiley & Sons Ltd HPV-associated Epigenetic Changes in CDKN2A recognized importance of HPV in OPSCC, the pathobiology of HPV in OPSCC tumors is not well understood
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