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Summary: 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.

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.

Abstract Background Immune checkpoint inhibitors (ICI) are approved for treatment of recurrent or metastatic oropharyngeal head and neck squamous cell carcinoma in the first- and second-line settings. However, only 15–20% of patients benefit from this treatment, a feature increasingly ascribed to the peculiar characteristics of the tumor immune microenvironment (TIME). Methods Immune-related gene expression profiling (GEP) and multiplex immunofluorescence (mIF) including spatial proximity analysis, were used to characterize the TIME of 39 treatment-naïve oropharyngeal squamous cell carcinomas (OPSCC) and the corresponding lymph node metastases. GEP and mIF results were correlated with disease-free survival (DFS). HPV-positive tumors disclosed a stronger activation of several immune signalling pathways, as well as a higher expression of genes related to total tumor-infiltrating lymphocytes, CD8 T cells, cytotoxic cells and exhausted CD8 cells, than HPV-negative patients. Accordingly, mIF revealed that HPV-positive lesions were heavily infiltrated as compared to HPV-negative counterparts, with a higher density of T cells and checkpoint molecules. CD8+ T cells appeared in closer proximity to tumor cells, CD163+ macrophages and FoxP3+ cells in HPV-positive primary tumors, and related metastases. In HPV-positive lesions, PD-L1 expression was increased as compared to HPV-negative samples, and PD-L1+ tumor cells and macrophages were closer to PD-1+ cytotoxic T lymphocytes. Considering the whole cohort, a positive correlation was observed between DFS and higher levels of activating immune signatures and T cell responses, higher density of PD-1+ T cells and their closer proximity to tumor cells or PD-L1+ macrophages. HPV-positive patients with higher infiltration of T cells and macrophages had a longer DFS, while CD163+ macrophages had a negative role in prognosis of HPV-negative patients. Conclusions Our results suggest that checkpoint expression may reflect an ongoing antitumor immune response. Thus, these observations provide the rationale for the incorporation of ICI in the loco-regional therapy strategies for patients with heavily infiltrated treatment-naïve OPSCC, and for the combination of ICI with tumor-specific T cell response inducers or TAM modulators for the “cold” OPSCC counterparts.

Abstract 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.

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 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.

Objectives: To investigate the frequency of Human papillomavirus (HPV)-driven head and neck squamous cell carcinoma (HNSCC) among patients living in North-East Italy, by assessing HPV-DNA positivity in all tumors and additional markers whenever possible. Material and methods: HPV types, viral load, viral RNA, HPV16/18 E6 protein and p16INK4a and pRb expression were determined in primary tumor tissues from 247 HNSCC patients. Tumor-specific HPV seropositivity was analyzed in 102 patients. Results: Tumor HPV-DNA prevalence was 8.5% overall (21/247) and 27% in oropharynx (17/63). HPV16 accounted for 95% of all HPV types found. Among HPV-DNA+ tumors, type-concordant HPV E6*I RNA prevalence was 79%. HPV DNA+ RNA+ tumors showed high viral load, up-regulated p16INK4a, down-regulated pRb and presence of HPV16 E6 protein. Eight cases showed tumor-specific HPV seropositivity, all type-concordant with the tumor. Tumors were defined as HPV-driven when positive for HPV-DNA plus 2 additional HPV transformation-related markers. Conclusion: Relative prevalence of HPV-driven tumors (14 HPV16, 1 HPV58) was 6% overall and 20% among oropharyngeal cancers. In the oropharynx the HPV-driven group showed a trend for better survival versus the HPV-negative group.The relative prevalence of HPV-driven oropharyngeal cancer is low in North-East Italy as compared to Western and Northern Europe. Keywords: Head and neck cancer, Oropharyngeal cancer, HPV-driven, HPV-related markers, HPV antibodies, Prevalence

Offering self-sampling devices improves participation of under-screened women. We evaluated participation in routine screening following the self-sampling intervention in two organized population-based screening programmes located in North-East Italy. Data on participation at 3-years-interval after a randomized clinical trial assessing the response to two strategies offering self-samplers (sent at home or offered free at local pharmacy) with a control action (sending reminders for a cervical specimen taken at the clinic) in 30–64 yr-old women non-respondent to the regular call-recall invitation were analyzed. Up to April 2016, 2300 women out of the 2995 recruited in the trial in 2011 were re-invited to perform a screening test at clinic; overall, 698 women adhered. Participation was similar in the three arms (29–32%), and highest (47–68%) among those who participated in the previous round. Over the two rounds, 44.6%, 32.3% and 30.3% women had at least one test in the self-sampling at home, self-sampling at pharmacy and test at the clinic arms, respectively. Our data indicate that the beneficial effect of offering self-sampling devices to nonparticipating women is maintained over time. Self-samplers are useful to increase overall coverage; their sporadic use does not seem to increase the proportion of women regularly repeating the test.

The current study aimed to investigate the incidence and survival patterns of HNSCCs arising from different anatomic sites, potentially related (the oropharynx) or unrelated (the oral cavity, the larynx/hypopharynx) to HPV, to provide clues on possible growing impact of HPV in the epidemiology of HNSCC in Italy. Epidemiological data were retrieved from ten long-term Cancer Registries covering a population of 7.8 million inhabitants. Trends were described by means of the estimated annual percent change (APC) stratified by age and gender, and compared between HPV-related and HPV-unrelated anatomical sites. The data regarding 28,295 HNSCCs diagnosed in Italy between 1988 and 2012 were analyzed. In males, the incidence rate (IR) of cancers arising from sites unrelated to HPV infection significantly decreased in all age groups (APC:-3.31 for larynx/hypopharynx; APC:-1.77 for oral cavity), whereas stable IR were observed for cancers arising from sites related to HPV infection. In females, IR for cancers from HPV-related sites increased significantly over the observed period; the largest increment was noted in those over 60 (APC:2.92%) who also showed a significantly lower number of HNSCCs from the larynx/hypopharynx (APC:- 0.84) and a significantly higher number of oral cavity tumors (APC = 2.15). The five-year relative survival remained largely unchanged in the patients with laryngeal/hypopharyngeal SCC and, conversely, significantly improved in the patients with SCC at HPV-related sites. The trends observed suggest a potential increasing impact of HPV infection on the epidemiology of HNSCC in Italy, but to a lesser extent and with a different pattern from that observed in other Western countries.

AIM OF THE STUDY:To assess the feasibility of partial HPV genotyping and methylation analysis of CADM1, MAL, and miR124-2 genes as triage tests in assaying self-collected cervical samples positive for high-risk HPV on primary screening, and to review the literature regarding host cellular gene methylation analysis of self-collected cervical samples. MATERIAL AND METHODS:Women residing in North-East Italy who had failed to respond to the invitation to participate in an organized population-based program were invited to provide a self-sample. Their stored baseline (self-collected) and follow-up (clinician-collected) cervical samples were included in the study. DNA was extracted from HPV-positive (Qiagen's Hybrid Capture 2, HC2) samples. Partial genotyping with separate detection of HPV types 16 and 18 was performed with a hybrid capture-based method and a quantitative PCR assay. Methylation was assayed with a quantitative methylation-specific PCR. RESULTS:High-risk HPV infection was detected in 48% of baseline and 71% of follow-up HC2-positive samples. Methylation was demonstrated respectively in 15% and 23.5% of baseline and follow-up samples and chiefly involved a single gene (miR124-2). Invalid quantitative PCR results were recorded in 5% of self-collected samples. The specificity of miR124-1, MAL, and CADM1 methylation was 84%, 94%, and 98%, respectively, and the specificity of the three markers combined was 84%. Sensitivity was not estimated due to the lack of CIN2+ samples. The systematic review showed that different methylation assays yield different accuracy values. CONCLUSION:Self-collected samples are suitable for methylation assays included in reflex triage testing. The reproducibility and accuracy of the methylation tests described in the literature should be improved.

Although the prevalence of human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) is increasing in developed countries and becoming a relevant health issue, the natural history of oral HPV infection is still unclear. Estimating the infection's prevalence in specific populations and identifying risk factors can widen our understanding of its natural history and help to delineate appropriate prevention strategies. This study sought to (i) determine oral HPV prevalence and genotype distribution in a large series of young Italian adults, (ii) validate an oral rinse sampling/storage protocol, and (iii) pinpoint factors associated with oral HPV infection. Five hundred students, nurses, and technicians (19-35 years-old) studying and working at/for the University of Padua were recruited. Each participant was provided with an oral rinse sampling kit and instructions for use. They were also asked to complete an anonymous questionnaire concerning their demographic characteristics and behaviors. The questionnaires and oral rinse containers were labeled with the same identification code number. The oral rinse samples were tested using a bead-based multiplex BSGP5+/6+-MPG genotyping assay which amplifies the L1 region of 51 mucosal HPV types. The prevalence of oral HPV infection was 4.0% (95% confidence interval (CI), 2.5%-6.1%); those of 14 high-risk HPV types and of HPV-type 16 (HPV16) infection were 2.2% (95% CI, 1.1%-3.9%) and 1.6% (95% CI, 0.6%-3.1%), respectively. HPV16 was the most frequent genotype (40.0% of oral HPV infections). No association was found between oral infection and the co-variables studied (gender, tobacco, alcohol and illegal drug use, number of sex and oral sex partners, HPV vaccination status, history of HPV and sexually transmitted infections, abnormal pap smears, recurrent tonsillitis and tonsillectomy). The oral rinse sampling protocol outlined here proved to be simple, efficient and well tolerated, and the prevalence rate can be considered reliable and thus useful to guide future research. Determinants of oral HPV infection are still unclear and further studies are certainly warranted.

Infection with high-risk (hr) human papillomavirus (HPV) is considered the necessary cause of cervical cancer. Vaccination against HPV16 and 18 types, which are responsible of about 75% of cervical cancer worldwide, is expected to have a major global impact on cervical cancer occurrence. Valid estimates of the parameters that regulate the natural history of hrHPV infections are crucial to draw reliable projections of the impact of vaccination. We devised a mathematical model to estimate the probability of infection transmission, the rate of clearance, and the patterns of immune response following the clearance of infection of 13 hrHPV types. To test the validity of our estimates, we fitted the same transmission model to two large independent datasets from Italy and Sweden and assessed finding consistency. The two populations, both unvaccinated, differed substantially by sexual behaviour, age distribution, and study setting (screening for cervical cancer or Chlamydia trachomatis infection). Estimated transmission probability of hrHPV types (80% for HPV16, 73%-82% for HPV18, and above 50% for most other types); clearance rates decreasing as a function of time since infection; and partial protection against re-infection with the same hrHPV type (approximately 20% for HPV16 and 50% for the other types) were similar in the two countries. The model could accurately predict the HPV16 prevalence observed in Italy among women who were not infected three years before. In conclusion, our models inform on biological parameters that cannot at the moment be measured directly from any empirical data but are essential to forecast the impact of HPV vaccination programmes.