European guidelines for cervical cancer screening now recommend the use of clinically validated assays for high-risk HPV-DNA sequences as primary test in women older than 30 years, performed in centralized laboratories, and run on systems providing automated solutions for all steps. We conducted a comparison study, according to the international guidelines, nested within the organized population-based cervical screening program, between the cobas 4800 and 6800 systems (Roche Diagnostics), to evaluate accuracy and reproducibility of HPV test results and laboratory workflow. In Italy implementation of HPV cervical screening is under way on a regional basis; in Veneto it started in June 2015, following a piloting phase; the assay in use in the three centralized laboratories is the cobas 4800 HPV test, run on the cobas 4800 system. Comparison of HPV results with a new version of the assay (cobas 6800/8800 HPV) run on the cobas 6800 system, and intra- and inter-reproducibility analyses have been conducted in samples collected in PreservCyt medium (Hologic) from women without and with a subsequent diagnosis of high-grade lesion. Samples from women older than 30 years attending organized cervical cancer screening were used. Clinical sensitivity and specificity were evaluated on 60 cases and 925 controls, respectively; intra-laboratory reproducibility and inter-laboratory agreement by the 6800 system were evaluated on 593 and 460 specimens, respectively. Our results showed a very high agreement (> 98%) for overall qualitative results between the two systems; clinical sensitivity and specificity of the HPV assay run on 6800 were non-inferior to those of the HPV assay run on 4800 (p = 0,0157 and p = 0,0056, respectively, at the recommended thresholds of 90 and 98%); kappa values of 0.967 and 0.969 were obtained for intra- and inter-laboratory reproducibility analyses in the 6800 system. The 6800 platform displayed several technological improvements over the 4800 system, with higher throughput and laboratory productivity, and lower operator’s hands-on time. The new cobas 6800/8800 HPV assay run on the 6800 instrument is suitable for use in large centralized laboratories included within population-based cervical cancer screening programs.

Measuring viral DNA methylation in human papillomavirus (HPV) infected women showed promise for accurate detection of high-grade cervical lesions and cancer. Methylation status has been widely investigated for HPV16, sporadically for other HPV types. Objective of this methodological study was to set up molecular methods to test the methylation levels in the twelve oncogenic HPV types by pyrosequencing, minimizing the number of HPV type-specific PCR protocols. Target CpGs were selected on the HPV L1 (two regions, L1 I and L1 II) and L2 genes. Study samples included DNA stored at Turin, Italy, purified by cervical cells collected in Standard Transport Medium or PreservCyt from women who participated in two studies (N = 126 and 140) nested within the regional organized screening programme. PCR consensus primers were designed by PyroMark Assay Design software to be suitable for amplification of many different oncogenic HPV types. Generation of consensus primers was successful for L1 I and II regions, unsuccessful for L2 region, for which HPV type-specific primers remained necessary. The difference between replicated tests on the same sample was ≤4% in 88%, 77% and 91% of cases when targeting the L1 I, L1 II and L2 regions, respectively. The corresponding intra-class correlation coefficients (ICC) were 0.94, 0.87 and 0.97 respectively. When comparing methylation measures based on consensus and type-specific primers, ICC was 0.97 for the L1 I region and 0.99 the for L1 II region. The proposed protocols, applying consensus primers suitable to amplify the oncogenic HPV types and minimize the number of PCR reactions, represent a promising tool to quantify viral methylation in women positive for any high risk HPV type. Potential application of these methylation protocols in screening settings can be explored to identify women with high probability of progression to high grade lesions.

Cervical intraepithelial neoplasia grade 2 (CIN2) can spontaneously regress in a sizable proportion of cases. The aim of this prospective multicenter cohort study was to identify the biomarkers associated with a high probability of regression. A total of 319 women aged 25–45 years fulfilling predefined inclusion and exclusion criteria (full visibility of transformation zone and lesion; no previous history of CIN2+ or immune impairment) were enrolled and subjected to active surveillance for 24 months. HPV genotyping, p16/ki67 expression, methylation status of FAM19A4/miR124‐2 genes, and cytology were evaluated at baseline. The probability of CIN2 regression according to the different biomarkers was evaluated through binomial logistic regression. At follow‐up, regression, persistence, and progression (evaluated on 294 women) were recorded in 165 (56%), 68 (23%), and 61 (21%) cases, respectively; no association with age was observed. Overall, 110 women underwent excisional treatment during follow‐up; 53 CIN2 and 50 CIN3+ were diagnosed. The probability of CIN2 regression significantly increased with early HPV negativity (odds ratio [OR] 6.45, 95% confidence intervals [CI] 1.68–42.6), no p16/ki67 expression (OR 2.49, 95%CI 1.38–4.52), and unmethylated status (OR 2.12, 95%CI 1.09–4.20). Our results indicate that active CIN2 surveillance could be implemented for women up to 45 years, after selection according to anatomo‐clinical criteria and biomarker status. To improve feasibility, the biomarkers can be used sequentially, taking advantage of the HPV genotyping available in primary screening tests, and eventually refining the selection by using the other biomarkers in selected subgroups.

Cervical cancer is caused by a persistent infection with high-risk types of Papillomaviruses (hrHPV); HPV16 and HPV18 are associated with about 70% of the cases. In the last decades the introduction of a cervical cancer screening has allowed a decrease in cervical cancer incidence and mortality; regular adhesion to the screening procedures, by pap test or HPV test, and colposcopy, according to the international guidelines, prevents cancer development and allows for diagnosis at the early stages. Nowadays, in industrialized countries, it is not common to diagnose this pathology in advanced stages, and this occurrence is frequently associated with patient’s unattendance of cervical screening programs. We describe a case of delayed diagnosis of cervical cancer, posed only after the onset of the neurological symptoms caused by leptomeningeal metastases, despite a two-year history of abnormal cytology. The endocervical mass was analyzed by immunohistochemistry, and search and typing of HPV sequences was performed by PCR in the meningeal carcinomatous cells. A poorly differentiated squamous cell carcinoma was diagnosed, and HPV18 sequences were detected. This rapidly fatal case highlights the importance of following the evidence-based recommended protocols and the preventive role of the population-based cervical cancer screening programs.

Background: Efficacy for cervical cancer prevention of opportunistic HPV vaccination in post-pubertal girls is lower than in 11-year-olds. Methods: Women born between 1986 and 1992 vaccinated at 15–25 years of age (at least one dose of 4-valent HPV vaccine) and screened at 24–27 years of age were included. Frequency of opportunistic vaccination, overall and by birth cohort, was calculated; screening outcomes were compared between vaccinated and unvaccinated women. Results: Overall, 4718 (4.9%) HPV-vaccinated, and 91,512 unvaccinated, women were studied. The frequency of vaccination increased by birth cohort, ranging between 1.8% and 9.8%; age at vaccination decreased progressively by birth cohort (p < 0.0001). Participation in screening was 60.8% among vaccinated, and 56.6% among unvaccinated, women (p < 0.0001). Detection rates (DR) for high-grade lesions were lower in vaccinated women (2.11‰ vs. 3.85‰ in unvaccinated, for CIN3+, p = 0.24; 0.0‰ vs. 0.22‰ for cancer). The DR of CIN3+ increased with age at vaccination, scoring respectively 0.0‰, 0.83‰, and 4.68‰ for women vaccinated when they were 15–16, 17–20, and 21–25 years old (p = 0.17). Conclusions: In comparison to unvaccinated women, higher compliance with cervical cancer screening invitation and lower CIN3+ DR among vaccinated women was observed. Age at vaccination was inversely correlated to vaccination efficacy.

• Cervical cancer risk is lower in HPV vaccinated than unvaccinated women. • Women vaccinated before age 15 will start screening at 30 instead of 25 years. • The change is being communicated with a personal letter. • An on-line questionnaire was proposed together with the letter to 25 year-old women. • The reactions were positive but intention to undergo spontaneous screening emerged.