Bromodomain-containing protein 4 (Brd4) is a cellular chromatin-binding factor and transcriptional regulator that recruits sequence-specific transcription factors and chromatin modulators to control target gene transcription. Papillomaviruses (PVs) have evolved to hijack Brd4’s activity in order to create a facilitating environment for the viral life cycle. Brd4, in association with the major viral regulatory protein E2, is involved in multiple steps of the PV life cycle including replication initiation, viral gene transcription, and viral genome segregation and maintenance. Phosphorylation of Brd4, regulated by casein kinase II (CK2) and protein phosphatase 2A (PP2A), is critical for viral gene transcription as well as E1- and E2-dependent origin replication. Thus, pharmacological agents regulating Brd4 phosphorylation and inhibitors blocking phospho-Brd4 functions are promising candidates for therapeutic intervention in treating human papillomavirus (HPV) infections as well as associated disease.

Longitudinal data on the E6/E7 mRNA-based Aptima human papillomavirus (AHPV) assay exceeding three years in comparison to the gold standard Digene Hybrid Capture 2 (HC2) test are not available. We previously reported the cross-sectional data of the German AHPV Screening Trial (GAST) in which 10,040 women were recruited and tested by liquid-based cytology, the HC2 assay, and the AHPV assay.

Although a vast amount of research underlines the roles of the HR HPV E6 and E7 oncogenes in HPV-induced carcinogenesis of cervical cancer, it remains unclear whether these oncogenes are also involved in the resistance of the cancer against chemotherapy.We examined the role of the HPV16 E6 oncogene in cisplatin resistance by analyzing its expression in newly established cisplatin-sensitive versus -resistant cervical cancer cell lines (CC7, CC10). Resistant variants were obtained by interval exposure treatment with 1-2 μM cisplatin for 8-9 months. Our results demonstrate that the expression level of HPV16 E6 directly correlates with the extent of cisplatin resistance in novel as well as established (SiHa) drug resistant cervical cancer cell lines. Overexpression of HPV16 E6 in cisplatin-naïve cells rendered these cells more resistant to cisplatin. Reducing E6 expression by JQ1 treatment reversed the drug resistant phenotype and strongly enhanced chemoresponse only in HPVpositive cisplatin-resistant variants and not in HPV-negative C33A cervical cancer cells. The level of E6 directly correlated with the extent of cisplatin sensitivity and was shown to be increased in newly established drug-resistant cell line variants, while reducing E6 expression using Brd4-inhibitors enhanced chemoresponse when co-delivered with cisplatin. Inhibition of Brd4 could represent a new therapeutic option by increasing treatment response in cervical cancer cells and might allow lower cisplatin dosages, thus reducing negative side effects.

Several studies have demonstrated that the viral genome can be methylated by the host cell during progression from persistent infection to cervical cancer. The aim of this study was to investigate whether methylation at a specific site could predict the development of viral persistence and whether viral load shows a correlation with specific methylation patterns. HPV16-positive samples from women aged 20-29 years (n = 99) with a follow-up time of 13 years, were included from a Danish cohort comprising 11 088 women. Viral load was measured by real-time PCR and methylation status was determined for 39 CpG sites in the upstream regulatory region (URR), E6/E7, and L1 region of HPV16 by next-generation sequencing. Participants were divided into two groups according to whether they were persistently (≥ 24 months) or transiently HPV16 infected. The general methylation status was significantly different between women with a persistent and women with a transient infection outcome (P = .025). One site located in L1 (nt. 5962) was statistically significantly (P = .00048) different in the methylation status after correction using the Holm-Sidak method (alpha = 0.05). Correlation analyses of samples from HPV16 persistently infected women suggest that methylation is higher although viral load is lower.This study indicates that methylation at position 5962 of the HPV16 genome within the L1 gene might be a predictive marker for the development of a persistent HPV16 infection. K E Y W O R D SBiomarker, DNA Methylation, HPV16, Persistence

BackgroundHigh-risk human papillomavirus (HR HPV) testing is already part of cervical cancer screening programs in a number of countries. New tests need to be validated not only in clinical studies but also in routine screening settings with regard to their clinical performance.MethodsThe Abbott RealTime High Risk HPV Test (RT hrHPV test) was evaluated in a random sample of 1,456 patients from a German routine screening population of 13,372 women ≥30 years of age screened primarily by liquid-based cytology (LBC) that was complemented by 48 CIN3+ cases. Clinical sensitivities, relative specificities and positive predictive values (PPV) for both HPV tests were determined based on histologically confirmed high-grade cervical disease (CIN3+) as clinical outcome.ResultsHR HPV prevalence in residual LBC samples was found to be 5.4 % by the RT hrHPV test and 5.6 % by the HR HC2 test, respectively. The Kappa-value for overall agreement between the RT hrHPV test and the HC2 assay for detection of HR HPV was 0.87. Relative sensitivities for detection of CIN3+ in patients with abnormal cytology was 93.8 % for the RT hrHPV assay and 97.9 % for HC2 (p-value = 0.5). Relative specificities and PPVs were comparable for both tests. The highest PPV was calculated for the specific detection of HPV16 by the RT hrHPV test (84.2 %). The RT hrHPV test showed a reduced sensitivity for detection of HVP31-positive CIN3 + .ConclusionThe RT hrHPV assay is as sensitive and specific in detecting severe cervical lesions in women with abnormal cytology as the HC2 HR HPV test.