Methylation status of HPV16 E2-binding sites classifies subtypes of HPV-associated oropharyngeal cancers.

The human papillomavirus (HPV) E2 protein is a transcriptional repressor of the oncogenes E6/E7 and loss of E2 function is considered a key step in carcinogenesis. Integration of HPV into the host genome may disrupt the E2 gene. Furthermore, methylation of CpG dinucleotides in E2-binding sites (E2BSs) in the HPV upstream regulatory region may interfere with transcriptional repression of E6 and E7 by E2. The authors hypothesized that the CpG methylation status of E2BS identifies subtypes of HPV type 16 (HPV16)-associated oropharyngeal squamous cell cancers (OPSCC) in association with E2 gene integrity and viral integration. Methylation of 10 CpG dinucleotides within the upstream regulatory region, encompassing E2BSs 1, 2, 3, and 4, was quantitatively analyzed by bisulfite pyrosequencing in 57 HPV16-associated OPSCC cases. E2 status was analyzed by gene amplification and quantitative real-time reverse transcriptase-polymerase chain reaction. Viral integration was determined by integration-specific polymerase chain reaction methods. Three subgroups with differential methylation at E2BS3 and E2BS 4 were identified: 1) complete methylation (>80%) associated with the presence of integrated HPV genomes with an intact E2 gene; 2) intermediate methylation levels (20%-80%) with predominantly episomal HPV genomes with intact E2; and 3) no methylation (<20%) with a disrupted E2 gene. Patients with high methylation levels tended to have a worse 5-year overall survival compared with patients with intermediate methylation (hazard ratio, 3.23; 95% confidence interval, 1.13-9.24 [P = .06]). Methylation of E2BS3 and E2BS4 in OPSCC is associated with E2 integrity and viral physical status. It might explain deregulated viral oncogene expression in the presence of E2. The prognostic significance of E2BS methylation for patients with HPV-associated OPSCC needs to be analyzed further.

Reuschenbach M ，Huebbers CU ，Prigge ES ，Bermejo JL ，Kalteis MS ，Preuss SF ，Seuthe IM ，Kolligs J ，Speel EJ ，Olthof N ，Kremer B ，Wagner S ，Klussmann JP ，Vinokurova S ，von Knebel Doeberitz M ... -  《-》

Virological characteristics of cervical cancers carrying pure episomal form of HPV16 genome.

Many studies on integration have reported conflicting results regarding the role of HPV integration in cervical cancer. We hypothesized that high viral load and disruption of E2 gene associated with integration of HPV were not the only pathway leading to cancer development. This study analysed the viral load and integration status of HPV16, measured the HPV16 E6/E7 mRNA transcript levels, delineated the E2 and LCR sequence variation, and determined the methylation status of two E2 binding sites. The results showed that viral load was not associated with the physical status of HPV genome. Levels of the three E6/E7 mRNA transcripts in invasive cervical cancers containing purely episomal viral genome were found to be similar to those containing integrated viral genome, suggesting that cancers containing episomal viral genome were also mediated by an up-regulated E6/E7 mRNA expression, and more importantly, did not depend on integration and disruption of the E2 gene. The alternative mechanism that up-regulated the expression of E6 and E7 in invasive cancers harbouring episomal viral genome was likely to be a consequence of methylation of the two E2 binding sites located at the promoter region of HPV16. These observations are in line with the hypothesis that HPV integration was not the only mechanism leading to the development of cervical cancer.

Cheung JL ，Cheung TH ，Yu MY ，Chan PK ... -  《-》

HPV 16 E2 binding sites 1 and 2 become more methylated than E2 binding site 4 during cervical carcinogenesis.

E2 protein binding to the four E2 binding sites (E2BSs) at the long control region of Human Papillomavirus (HPV) 16/18 genome may exert either transcriptional activation/repression on E6 and E7 oncoproteins. Methylation status at the E2BSs may affect the relative binding of E2 protein to them. In this study, methylation percentage at E2BS 1, 2 (promoter-proximal), and 4 (promoter-distal) were assessed by pyrosequencing and compared among HPV 16/18-positive cervical cancer, high-grade, and low-grade Cervical Intraepithelial Neoplasia, Atypical Squamous Cells of Undetermined Significance, and normal cervical epithelium. HPV 16 E2BS1&2 were more methylated than HPV 16 E2BS4 in cervical cancer whereas in cervical premalignant lesions and normal epithelium, HPV 16 E2BS1&2 were less methylated than HPV 16 E2BS4. HPV 18 E2BS1&2 remained more methylated than E2BS4 in all histological groups. HPV 16 E2BS1&2 methylation increased from high-grade lesions to cervical cancer (P < 0.001). HPV 16 E2BS4 methylation increased from low-grade to high-grade premalignant lesions (P = 0.041). Both HPV 18 E2BS1&2 and E2BS4 methylation increased from low-grade to high-grade Cervical Intraepithelial Neoplasia (P = 0.019 and 0.001 respectively) and further increased form high-grade lesions to cervical cancer (P < 0.001 and 0.005 respectively). Conclusively, HPV 16 E2BS1&2 (for transcriptional repression of E6/E7 oncoproteins) became more heavily methylated than E2BS4 (for transcriptional activation of E6/E7) in cervical cancer, favouring the differential binding of E2 protein to E2BS4. Increasing methylation at HPV 16/18 E2BSs are potentially useful adjunctive molecular markers for predicting progression from low-grade to high-grade cervical premalignant lesions and from high-grade lesions to cervical cancer.

Leung TW ，Liu SS ，Leung RC ，Chu MM ，Cheung AN ，Ngan HY ... -  《-》

Methylation at 3'LCR of HPV16 can be affected by patient age and disruption of E1 or E2 genes.

CpG methylation at early promoter of HPV16 DNA, in the 3' end of the Long Control Region (3'LCR), has been associated to the presence of episomal forms of viral genome and, consequently, intact E1 and E2 ORFs. The DNA methylation would block the access of E2 viral protein to the E2 binding sites at early-promoter. However, is still unclear if methylation at 3'LCR of HPV16 DNA can also vary depending of other tumor characteristics in addition to viral DNA physical state. In this study, we evaluate whether the methylation level at the five CpG located at 3'LCR of HPV16 is associated to patient age and E1 and/or E2 ORFs integrity. DNA pyrosequencing was used to measure the methylation level in 69 invasive cervical cancer samples obtained from biopsies of patients attended at Brazilian National Institute of Cancer (INCA). PCR amplifications were performed to assess disruption status of E1 and E2 genes of HPV16. The methylation average per sample ranged widely, from <1 to 88.00%. Presence of intact E1/E2 genes and patient age were positively associated with average methylation in both bivariate analyses (p=0.003 and p=0.006, respectively), and multivariate analysis (p=0.002 and p=0.021, respectively), adjusted for tumor type (squamous cell carcinomas or adenocarcinomas) and HPV16 lineage. These findings showed that presence of intact E1/E2 open reading frames was associated with high levels of DNA methylation, and older patients showed higher levels of methylation than younger ones independently of viral genome disruption.

Filho SMA ，Bertoni N ，Brant AC ，Vidal JPCB ，Felix SP ，Cavalcanti SMB ，Carestiato FN ，Martins LFL ，Almeida LM ，Moreira MAM ... -  《-》

Differential methylation of E2 binding sites in episomal and integrated HPV 16 genomes in preinvasive and invasive cervical lesions.

Enhanced expression of the HPV 16 E6-E7 oncogenes may trigger neoplastic transformation of the squamous epithelial cells at the uterine cervix. The HPV E2 protein is a key transcriptional regulator of the E6-E7 genes. It binds to four E2 binding sites (E2BSs 1-4) in the viral upstream regulatory region (URR). Modification of E2 functions, for example, by methylation of E2BSs is hypothesized to trigger enhanced expression of the viral E6-E7 oncogenes. In the majority of HPV-transformed premalignant lesions and about half of cervical carcinomas HPV genomes persist in an extra-chromosomal, episomal state, whereas they are integrated into host cells chromosomes in the remaining lesions. Here we compared the methylation profile of E2BSs 1-4 of the HPV 16 URR in a series of 18 HPV16-positive premalignant lesions and 33 invasive cervical cancers. CpGs within the E2BSs 1, 3, and 4 were higher methylated in all lesions with only episomal HPV16 genomes compared with lesions displaying single integrated copies. Samples with multiple HPV16 integrated copies displayed high methylation levels for all CpGs suggesting that the majority of multiple copies were silenced by extensive methylation. These data support the hypothesis that differential methylation of the E2BSs 1, 3 and 4 is related to the activation of viral oncogene expression in cervical lesions as long as the viral genome remains in the episomal state. Once the virus becomes integrated into host cell chromosomes these methylation patterns may be substantially altered due to complex epigenetic changes of integrated HPV genomes.

Chaiwongkot A ，Vinokurova S ，Pientong C ，Ekalaksananan T ，Kongyingyoes B ，Kleebkaow P ，Chumworathayi B ，Patarapadungkit N ，Reuschenbach M ，von Knebel Doeberitz M ... -  《-》