The HPV16 upstream regulatory region (URR) undergoes shifts in methylation during cervical carcinogenesis. Four binding sites for E2 (E2BS), a key regulatory protein in HPV infection, are located in this region. Methylation of E2BS 3 and 4 is thought to promote uncontrolled expression of the HPV oncogenes E6 and E7 which drive carcinogenesis.
It has been shown previously that methylation at HPV E2BS3/4 allows for classification of subpopulations of oropharyngeal squamous cell cancer (OPSCC). In order to gain additional insight into the epigenetic regulation of HPV oncogene expression, we analyzed a series of tonsillar and base of the tongue carcinomas along with matched lymph node metastases and correlated E2BS3 and 4 methylation levels with clinical data.
We hypothesized that there might be distinct shifts in the binding sites' methylation during formation of metastases, possibly due to the selection and subsequent metastatic expansion of cell populations. Additionally, there might be subgroups of OPSCC with discernible differences in E2BS3 and 4 methylation and associated patient survival.
FFPE tissue from 67 HPV16 DNA+ and p16INK4a+ neoplastic lesions consisting of 42 OPSCC primaries with 25 matched lymph node metastases was obtained from St. Gertrauden-Krankenhaus, Berlin, Germany. DNA was bisulfite-converted and analyzed for methylation in five CpG sites in E2BS3/4 (positions 31-58) of the HPV16 URR by pyrosequencing. Differences in methylation levels among groups defined by clinical characteristics were assessed using ANOVA and Tukey's post-hoc tests. A cut-off level for assorting samples into groups of low versus high methylation was established using hierarchical cluster analysis. Kaplan-Meier-curves and log-rank-tests were used to examine different overall (OS) and progression-free survival (PFS) among these groups.
There was a trend towards lower methylation levels in E2BS CpGs in lymph node metastases compared to OPSCC primaries, which reached significance for CpG 43 in E2BS3 (p=.022).
Tumor histology was significantly associated with methylation: OPSCC with basaloid histology were significantly higher methylated than non-basaloid OPSCC considering confounding clinical factors.
High methylation levels (> 52% as established by cluster analysis) were associated with reduced OS and PFS.
OPSCC histological subtypes apparently harbor differences in HPV16 E2BS3/4 methylation levels which could be responsible for changes in the specific regulation of HPV E6/E7 oncogene expression, thereby potentially modifying the course of the disease.
Differences in URR methylation could be a marker for predicting survival in specific OPSCC subpopulations.