Comparison of HPV-positive triage strategies combining extended genotyping with cytology or p16/ki67 dual staining in the Italian NTCC2 study.

BACKGROUND: Each high-risk HPV genotype has different oncogenic potential, and the risk of CIN3+ varies according to genotype. We evaluated the performance of different strategies of HPV-positivity triage combining cytology, p16/ki67 dual staining (DS), and extended genotyping. METHODS: Samples from 3180 consecutive women from the NTCC2 study (NCT01837693) positive for HPV DNA at primary screening, were retrospectively analyzed by the BD Onclarity HPV Assay, which allows extended genotyping. Genotypes were divided into three groups based on the risk of CIN3+. HPV DNA-positive women were followed up for 24 months or to clearance. FINDINGS: Combining the three groups of genotypes with cytology or DS results we identify a group of women who need immediate colposcopy (PPV for CIN3+ from 7.8 to 20.1%), a group that can be referred to 1-year HPV retesting (PPV in those HPV-positive at retesting from 2.2 to 3.8), and a group with a very low 24-month CIN3+ risk, i.e. 0.4%, composed by women cytology or DS negative and positive for HPV 56/59/66 or 35/39/68 or negative with the Onclarity test, who can be referred to 3-year retesting. INTERPRETATION: Among the baseline HPV DNA positive/cytology or DS negative women, the extended genotyping allows to stratify for risk of CIN3+, and to identify a group of women with a risk of CIN3+ so low in the next 24 months that they could be referred to a new screening round after 3 years. FUNDING: Italian Ministry of Health (grant number RF-2009-1536040). Hologic-Genprobe, Roche Diagnostics, and Becton & Dickinson provided financial and non-financial support.

Exploring conservative management for cervical intraepithelial neoplasia grade 2 in organised cervical cancer screening programmes: a multicentre study in Italy.

Cervical intraepithelial neoplasia grade 2 (CIN2) lesions may regress spontaneously, offering an alternative to immediate treatment, especially for women of childbearing age (15-45 years).We conducted a prospective multicentre study on conservative CIN2 management, with semiannual follow-up visits over 24 months, biomarkers' investigation and treatment for progression to CIN3+ or CIN2 persistence for more than 12 months. Here, we assess women's willingness to participate and adherence to the study protocol.The study was set in population-based organised cervical cancer screening.From April 2019 to October 2021, 640 CIN2 cases were diagnosed in women aged 25-64 participating in the screening programmes.According to our predefined inclusion and exclusion criteria, 228 (35.6%) women were not eligible; 93 (22.6%) of the 412 eligible refused, and 319 (77.4%) were enrolled. Refusal for personal reasons (ie, desire to become pregnant, anxiety, difficulty in complying with the study protocol) and external barriers (ie, residence elsewhere and language problems) accounted for 71% and 17%, respectively. Only 9% expressed a preference for treatment. The primary ineligibility factor was the upper age limit of 45 years. After enrolment, 12 (4%) women without evidence of progression requested treatment, 125 (39%) were lost to follow-up (mostly after 6-12 months) and 182 (57%) remained compliant. Remarkably, 40% of enrolees did not fully adhere to the protocol, whereas only 5% (20/412) of the eligible women desired treatment.Our study demonstrates a good acceptance of conservative management for CIN2 lesions by the women, supporting its implementation within cervical screening programmes.

Acceptability of deferring the start of cervical cancer screening to age 30 for women vaccinated against human papillomavirus.

HPV vaccination of girls younger than 15 is very effective in reducing their risk of cervical cancer. In Italy, for vaccinated women, the starting age for cervical cancer screening is set to change from 25 to 30. Adherence to a protocol change is crucial to assure efficacy. The aim of our study was to monitor women's reaction to the change and learn about their attitudes. In September 2022, an anonymous online questionnaire was proposed to 3122 women born in 1997, fully vaccinated before 15 years of age and afferent to an organized cervical cancer screening programme in the Veneto region (North-East Italy). The questionnaire included 30 items on knowledge of HPV infection and preventive measures for cervical cancer, gynaecological check-ups and reactions to the deferment of the start of screening. Overall, 147 questionnaires were completed (4.7% participation rate). Almost all women had some information on HPV and HPV vaccination, while one third were unaware of the existence of the screening programme. Over 66% expressed agreement with the rationale for the deferment of screening initiation, but 62% would have preferred to start screening at 25. There was a significant association between having had one or more Pap tests and the willingness to undergo additional testing outside the screening programme before the age of 30. Continued efforts are required to improve the effectiveness of communication to women, especially when implementing existing protocols, together with strategies to promote correct approaches.

Performance of HPV E6/E7 mRNA assay as primary screening test: Results from the NTCC2 trial.

As the primary screening test, E6/E7 mRNA has shown similar sensitivity for CIN3+ and lower positivity rate than the HPV DNA test. Nevertheless, the overall mRNA positivity is too high for immediate colposcopy, making a triage test necessary. The aim was to estimate the mRNA performance as a primary test with different triage strategies. All HPV DNA-positives were tested for mRNA, cytology and p16/ki67. A sample of HPV DNA-negatives was also tested for mRNA to estimate test specificity. We included all CIN3+ histologically diagnosed within 24 months since recruitment. Of the 41 127 participants, 7.7% were HPV DNA-positive, of which 66.4% were mRNA-positive. Among the HPV DNA-negatives, 10/1108 (0.9%) were mRNA-positive. Overall, 97 CIN3+ were found. If mRNA was used as the primary test, it would miss about 3% of all CIN3+ with a 22% reduction of positivity compared with HPV DNA. The weighted specificity estimate for

A Retrospective Cohort Study of Young Women Spontaneously Choosing to Be Vaccinated against HPV: Outcomes from Their First Cervical Cancer Screening Test.

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.

Leptomeningeal Carcinomatosis of a Poorly Differentiated Cervical Carcinoma Caused by Human Papillomavirus Type 18.

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.

p16/ki67 and E6/E7 mRNA Accuracy and Prognostic Value in Triaging HPV DNA-Positive Women.

BACKGROUND: The study presents cross-sectional accuracy of E6 and E7 (E6/E7) mRNA detection and p16/ki67 dual staining, alone or in combination with cytology and human papillomavirus (HPV)16/18 genotyping, as a triage test in HPV DNA-positive women and their impact on cervical intraepithelial neoplasia (CIN2+) overdiagnosis. METHODS: Women aged 25-64 years were recruited. HPV DNA-positive women were triaged with cytology and tested for E6/E7 mRNA and p16/ki67. Cytology positive women were referred to colposcopy, and negatives were randomly assigned to immediate colposcopy or to 1-year HPV retesting. Lesions found within 24 months since recruitment were included. All P values were 2-sided. RESULTS: 40 509 women were recruited, and 3147 (7.8%) tested HPV DNA positive; 174 CIN2+ were found: sensitivity was 61.0% (95% confidence interval [CI] = 53.6 to 68.0), 94.4% (95% CI = 89.1 to 97.3), and 75.2% (95% CI = 68.1 to 81.6) for cytology, E6/E7 mRNA, and p16/ki67, respectively. Immediate referral was 25.6%, 66.8%, and 28.3%, respectively. Overall referral was 65.3%, 78.3%, and 63.3%, respectively. Cytology or p16/ki67, when combined with HPV16/18 typing, reached higher sensitivity with a small impact on referral. Among the 2306 HPV DNA-positive and cytology-negative women, relative CIN2+ detection in those randomly assigned at 1-year retesting vs immediate colposcopy suggests a -28% CIN2+ regression (95% CI = -57% to +20%); regression was higher in E6/E7 mRNA-negatives (Pinteraction = .29). HPV clearance at 1 year in E6/E7 mRNA and in p16/ki67 negative women was about 2 times higher than in positive women (Pinteraction < .001 for both). CONCLUSIONS: p16/ki67 showed good performance as a triage test. E6/E7 mRNA showed the highest sensitivity, at the price of too high a positivity rate to be efficient for triage. However, when negative, it showed a good prognostic value for clearance and CIN2+ regression.

Age-independent increasing prevalence of Human Papillomavirus-driven oropharyngeal carcinomas in North-East Italy.

HPV-driven oropharyngeal carcinomas (OPCs) show geographical variations with increasing temporal trends in several areas. We investigated their frequency and clinical outcomes within a prospective multicenter cohort study in North-East Italy. A tumor was defined as HPV-driven by using at least two different biomarkers, usually HPV-DNA positivity and p16INK4A overexpression. Different survival outcomes were compared among patients with HPV-driven and non-HPV-driven tumors. Overall, 42/130 (32.3%) patients with newly diagnosed OPC during the period 2000-2018 resulted HPV-driven; HPV16 was involved in 37 cases (88%), HPV33 in 3 cases (7%), HPV58 and HPV18 in 1 case each. Over time, HPV-driven cases raised from 16.7% (6/36) during 2000-2006 to 46.1% (24/52) during 2013-2018 (p < 0.001). The increase in HPV-driven OPCs was more marked in females than males (p = 0.010), and the frequency of HPV-driven cases was similar in the different age groups. In comparison to cases with non-HPV-driven tumors, a significantly (p < 0.001) better progression-free and overall survival were recorded among patients affected by HPV-driven OPC. The prevalence of HPV-driven OPC cases has been significantly increasing during the last two decades also in North-East Italy and was associated with favorable outcome. OPCs driven by non-HPV16 oncogenic types were restricted to patients older than 68-yrs.

HPV testing for cervical cancer screening: technical improvement of laboratory logistics and good clinical performance of the cobas 6800 in comparison to the 4800 system.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Assessment of viral methylation levels for high risk HPV types by newly designed consensus primers PCR and pyrosequencing.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. IMPACT: Potential application of these methylation protocols in screening settings can be explored to identify women with high probability of progression to high grade lesions.