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.

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.