Methylation of CpG 5962 in L1 of the human papillomavirus 16 genome as a potential predictive marker for viral persistence: A prospective large cohort study using cervical swab samples.

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.

Brd4 inhibition suppresses HPV16 E6 expression and enhances chemoresponse: A potential new target in cervical cancer therapy.

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 HPV-positive 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.

Longitudinal Clinical Performance of the RNA-Based Aptima Human Papillomavirus (AHPV) Assay in Comparison to the DNA-Based Hybrid Capture 2 HPV Test in Two Consecutive Screening Rounds with a 6-Year Interval in Germany.

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. Four hundred eleven test-positive women were followed for up to six years. In addition, 3,295 triple-negative women were screened after a median time of six years. Overall, 28 high-grade cervical intraepithelial neoplasia (CIN3) cases were detected. The absolute risk of developing high-risk HPV-positive CIN3+ over six years among those women that tested negative at baseline was 2.2 (95% confidence interval [95% CI], 1.0 to 4.9) and 3.1 (95% CI, 1.7 to 5.7) per 1,000 women screened by the HC2 and the AHPV tests; the additional risk for those with AHPV-negative compared with HC2-negative results was 0.9 (95% CI, -0.2 to 2.1) per 1,000. In comparison, the absolute risk following a negative LBC test was 9.3 (95% CI, 2.9 to 30.2). The relative sensitivity of AHPV compared to HC2 was 91.5% for CIN3+, and the negative predictive values were 99.8% (95% CI, 99.5 to 99.9%) for HC2 and 99.7% (95% CI, 99.4 to 99.8%) for AHPV. Our data show that the longitudinal performance of the AHPV test over six years is comparable to the performance of the HC2 test and that the absolute risk of CIN3+ over six years following a negative AHPV result in a screening population is low. (This study is registered at ClinicalTrials.gov under registration number NCT02634190.).

HPV16 viral load and physical state measurement as a potential immediate triage strategy for HR-HPV-infected women: a study in 644 women with single HPV16 infections.

High genome copy number (viral load) of human papillomavirus (HPV) is being discussed as a risk factor for high-grade cervical lesions. However, conflicting data about the integration status or viral load of the virus as risk factors for prevalent high-grade squamous intraepithelial lesions (HSIL) are found in the literature. To investigate whether viral load and/or integration status are indicative for prevalent ASCUS/LSIL or HSIL, we determined the HPV16 viral load and the physical state of the genome in 644 women with single HPV16 infections stratified by their cytology results from a large Danish population-based cohort consisting of 40,399 women. Cervical smear samples were tested using a multiplex quantitative real-time PCR (qPCR) with primers specific for HPV16 E2, E6 and beta actin, allowing simultaneous determination of the genome's physical state and the viral copy number per cell. The associations of viral load and physical state with cervical abnormalities were assessed using multinomial logistic regression. We found that a 10-fold increase in viral load was significantly associated with the presence of ASCUS/LSIL (OR=3.91; 95% CI, 2.49-6.13) and HSIL (OR=4.1; 95% CI, 2.45-6.68). A significant association with HSIL was observed for primarily integrated genomes (OR=6.68; 95% CI, 1.45-30.8). Among women with integrated viral genomes, we observed a trend towards increased risk of ASCUS/LSIL (OR=1.32; 95% CI -2.90-3.44) and HSIL (OR=5.10; 95% CI -0.67-38.9) per 10-fold increase in viral load, although not statistically significant. In conclusion, increasing viral load and integrated viral genomes were significantly associated with prevalent HSIL, thus indicating that viral load and physical state may potentially be useful triage markers for HPV16-positive women during cervical screening.

Involvement of Brd4 in different steps of the papillomavirus life cycle.

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.

Study-based evaluation of the Abbott RealTime High Risk HPV test in comparison to the HC2 HR HPV test in women aged ≥30 years using residual LBC ThinPrep specimens.

BACKGROUND: High-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. METHODS: The 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. RESULTS: HR 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 + . CONCLUSION: The RT hrHPV assay is as sensitive and specific in detecting severe cervical lesions in women with abnormal cytology as the HC2 HR HPV test.

TMEM45A, SERPINB5 and p16INK4A transcript levels are predictive for development of high-grade cervical lesions.

Women persistently infected with human papillomavirus (HPV) type 16 are at high risk for development of cervical intraepithelial neoplasia grade 3 or cervical cancer (CIN3+). We aimed to identify biomarkers for progression to CIN3+ in women with persistent HPV16 infection. In this prospective study, 11,088 women aged 20-29 years were enrolled during 1991-1993, and re-invited for a second visit two years later. Cervical cytology samples obtained at both visits were tested for HPV DNA by Hybrid Capture 2 (HC2), and HC2-positive samples were genotyped by INNO-LiPA. The cohort was followed for up to 19 years via a national pathology register. To identify markers for progression to CIN3+, we performed microarray analysis on RNA extracted from cervical swabs of 30 women with persistent HPV16-infection and 11 HPV-negative women. Six genes were selected and validated by quantitative PCR. Three genes were subsequently validated within a different and large group of women from the same cohort. Secondly, Kaplan-Meier and Cox-regression analyses were used to investigate whether expression levels of those three genes predict progression to CIN3+. We found that high transcript levels of TMEM45A, SERPINB5 and p16INK4a at baseline were associated with increased risk of CIN3+ during follow-up. The hazard ratios of CIN3+ per 10-fold increase in baseline expression level were 1.6 (95% CI: 1.1-2.3) for TMEM45A, 1.6 (95% CI: 1.1-2.5) for p16INK4a, and 1.8 (95% CI: 1.2-2.7) for SERPINB5. In conclusion, high mRNA expression levels of TMEM45A, SERPINB5 and p16INK4a were associated with increased risk of CIN3+ in persistently HPV16-infected women.