This single-center, retrospective cohort study sought to estimate the cumulative incidence in HIV-1-infected patients of biopsy-proven high-grade anal intraepithelial neoplasia (HGAIN) recurrence after infrared coagulation (IRC) treatment. The study was based on data from a prospectively compiled database of 665 HIV-1-infected outpatients who attended a hospital Clinical Proctology/HIV Unit between January 2012 and December 2015. Patient records were checked to see which ones had received IRC treatment but later experienced a recurrence of HGAIN. Cytology samples were also checked for the presence of human papilloma virus (HPV). A total of 81 of the 665 patients , of whom 65 were men and 16 women, were diagnosed with HGAIN and again treated with IRC. Of these 81, 20 (25%) experienced recurrent HGAIN, this incidence being true of both men  and women . The median time to recurrence was 6 (2–19) months overall, 6 (2–19) months in men, and 4 (2–6) months in women. HPV infection was detected in all patients except two, with HPV-16 being the most common genotype. This rate of incidence of recurrent HGAIN following IRC treatment is consistent with other reports and highlights the importance of continued post-treatment surveillance, particularly in the first year. Anal cancer (AC) is one of the most common non-AIDS-defining cancers , and itsInterrupting the natural history of AC is a challenge. Infrared coagulation (IRC) has been established as an effective treatment for high-grade squamous intraepithelial lesions (HSIL) . It can In a previous study , our groThe study was a single-center, retrospective cohort study based on data from a prospectively compiled database consisting of the digitized medical records of outpatients who were being attended at the HIV Unit of the Hospital Germans Trias i Pujol in Badalona, Spain. The cohort protocol was approved by the local Institutional Review Board (IRB) and all patients gave their written informed consent.The study population was made up of the 665 HIV-1-infected patients of both genders who had been treated using IRC for biopsy-proven HGAIN between 1 January 2012 and 31 December 2015 and who had yielded positive HIV serology results, were being attended at the hospital’s HIV Unit, and were at least 18 years old. Follow-up of all these patients had ceased by December 2019.An anal canal sample was taken for cytological examination and the Papanicolaou test was performed to check for anal canal cytological changes, as described previously .After topical application of 5% acetic acid in the anal canal for 2 min, HRA was performed using the technique described in [If the result of the histological examination revealed HGAIN , patient consent to undergo IRC ablation was obtained. IRC sessions were usually scheduled between 1 and 2 months after the first HRA. During the IRC process, each lesion was identified, infiltrated with local anesthesia, and then repeatedly coagulated using a Redfield IRC 2100 Infrared Coagulator  in pulses of 1.5 s . All proAfter IRC ablation, patients underwent routine evaluations involving visual inspection, digital rectal examination, and anal canal cytology at 3 to 6-month intervals. Major post-IRC complications were recorded. Patients with abnormal cytology results during the follow-up underwent another examination with HRA. If HRA revealed a new lesion, a biopsy was again performed. Patients with biopsy-proven AIN-2 or AIN-3 were advised to undergo further IRC ablation. Patients with AIN-1 were monitored with anal cytology at 3 to 6-month intervals. Patients with normal cytology results after IRC were monitored using anal cytology at 12-month intervals.In the group of women studied, cervical cytology was assessed  using the Papanicolaou method and results recorded according to the Bethesda Classification System, as follows: normal ; ASCUS ; LSIL ; or HSIL.TM II HPV28 real-time PCR assay . DNA was extracted from cell suspensions using the QiAMP Viral RNA kit .When samples were available from the anal canal or, in the case of women, the cervix, these samples were tested for human papilloma virus (HPV) infection with positive results simultaneously identified for HPV type using the AnyplexBaseline was defined as the moment of the patient’s first anal cytology assessment at which HGAIN was recorded, with recurrent HGAIN defined as biopsy-proven HGAIN subsequent to IRC treatment. The follow-up period was defined as the time between baseline and either biopsy-proven recurrence of HGAIN or last visit available after IRC in case of no recurrence.No formal sample size was calculated. The sample was defined as all HIV-1-infected patients with HGAIN (AIN-2 or AIN-3) as diagnosed by histological examination.The cumulative incidence of biopsy-proven HGAIN recurrence after IRC treatment was estimated, and its 95% confidence interval (95%CI) was calculated. The mean time to recurrence was analyzed using the Kaplan–Meier method. Agreement between the cytological diagnosis (Papanicolaou test) and the histological diagnosis (biopsy) was described. HPV infection and IRC safety was described.p value <0.05 was considered statistically significant. Data were analyzed using SPSS version 20 statistical software .A Of the 665 HIV-1-infected patients who were attended at our Clinical Proctology HIV Section with at least an anal canal cytology performed, 81  patients were diagnosed with HGAIN and were treated with IRC. Of these 81, 65 (80%) were men, of whom 60 (92%) were Men who have Sex with Men (MSM) and 5 (8%) were Men who have Sex with Women (MSW), and 16 (20%) were women. The mean age was 43.1 (SD 11.2) years and the median 44 (range 24–77 years). The cumulative incidence of recurring biopsy-proven HGAIN was 25% : 25%  in men and 25%  in women. The median follow-up period for the study population was 55 months (range 33–71) and the median time to recurrence was 6 months (range 2–19). Broken down by gender, the median was 6 months (range 2–19) in men and 4 months (range 2–6) in women.At baseline, a total of 101 anal lesions in the 81 patients were visually detected by HRA: 30% (19/64) of the men had >1 lesion whereas all the women had only one lesion. At recurrence, 26 lesions in the 20 patients were detected by HRA; here, 6% (1/16) of the men and 50% (2/4) of the women had >1 lesion.n = 81) with abnormal cytological diagnosis  and visualized lesion by HRA had a histological diagnosis of AIN-2  or AIN-3 .Agreement between the cytological and histological diagnosis was described . All patn = 65) were ASCUS 8% (5/65), LSIL 59% (38/65), and HSIL 34% (22/65), while, for the women (n = 16), they were ASCUS 6% (1/16), LSIL 31% (5/16), and HSIL 63% (10/16). The baseline histology results for the men show that 79% (51/65) had AIN 2 and 22% (14/65) had AIN 3, while 63% of the women (10/16) had AIN 2 and 38% (6/16) had AIN 3.The baseline anal canal cytology test results for the men (n = 16) were ASCUS 6% (1/16), LSIL 31% (5/16) and HSIL 63% (10/16), while, for the women (n = 4), they were LSIL 25% (1/4) and HSIL 75% (3/4). The recurrence histology results for the men show that 63% (10/16) had AIN 2 and 38% (6/16) had AIN 3, while 50% of the women (2/4) had AIN 2 and 50% (2/4) had AIN 3.At recurrence, the anal canal cytology test results for the men  and LSIL in 38% (6/16), the remaining 50% women (8/16) yielding normal (negative) results. The four women who presented a recurrence of HGAIN at an anal canal site had at least one historical cervical dysplasia, as revealed in the baseline cytology test.At baseline, 67 out of 81 patients  had samples from an anal canal site for available HPV infection detection. HPV infection was detected in all patients except two . Single HPV infection (for only one HPV genotype) was detected in five patients , and multiple infections (for >1 HPV genotype) in 60 patients . HPV-16 was the most prevalent HPV genotype found at an anal canal site at baseline; this represented 40% of the men  and 50% of the women . Men presented a greater number of HPV genotypes at anal canal sites  relative to women .Of the 20 patients with recurrent HGAIN, 18  had samples from an anal canal site available for the detection of HPV infection. The analysis detected 28 HPV genotypes, of which 13 were high-risk-HPV genotypes , 9 were low-risk-HPV genotypes  and 6 were uncertain-risk-HPV genotypes . HPV infection involving >1 HPV genotype was detected in all patients. HPV-16 was the most prevalent HPV genotype, being present in 47% of the men (95%CI: 25–70%) and 100% of the women (95%CI: 44–100%). The men presented a greater number of HPV genotypes at anal canal sites  relative to the women .No major complications related to IRC were reported. Pain was the main adverse event reported by patients. Likewise, no adverse events related with the obtaining of sampled from the anal canal for cytology and HPV assessment were reported.HGAIN is a prevalent pathology among HIV-1-infected patients, and recurrence of HGAIN in these patients, regardless of treatment, is not uncommon. In the present study of 81 HIV-1-infected patients with a long follow-up (minimum 33 months), we found the same cumulative incidence of biopsy-proven HGAIN recurrence after IRC treatment in both men and women, namely 25%.Though our previous results (published in 2013) on the estimated cumulative recurrence of biopsy-proven HGAIN seem to differ from those reported here, the confidence intervals overlap  ,9,10, thOur study has several limitations. First, the small sample size  means that the results found may be an over- or underrepresentation of the real recurrence rate. Second, our study reports the experience of a single center where a screening program for anal cancer prevention has been active since 2005. However, this program may differ from others , which mIn conclusion, the fact that recurrence of HGAIN in HIV-1-infected patients after IRC ablation is a fairly common occurrence highlights the great importance of continued post-treatment surveillance to prevent new episodes, particularly in the first year.


The median age for cervical cancer was 45 years old, with an increasing value from 43 to 47 years (p < 0.001). The age classes with greater decreases in hospital admissions for invasive cancers were women aged 35 years or more (APC range from −5.5 to −9.86) and 25–34 years old  for women with cervical carcinoma in situ. After ten years for vaccine introduction and sixteen years for cervical cancer screening availability, a relatively large decrease in hospital admissions for cervical cancer and other HPV-related diseases in Sicily was observed. Some clinical characteristics of hospitalization, such as increasing age, are suggestive clues for the impact of preventive strategies, but further research is needed to confirm this relationship. In light of the implementation of human papillomavirus (HPV) prevention strategies, epidemiological studies in different geographical areas are required in order to assess the impact of HPV-related diseases. The purpose of the present study was to describe the burden of HPV-related hospitalizations in Sicily. A retrospective observational study estimated 43,531 hospitalizations attributable to HPV from 2007 to 2017. During the observed period, there was a decrease for all HPV-related conditions with a higher reduction, among neoplasms, for cervical cancer  = −9.9%,  Human papillomavirus (HPV) is considered the most common causative agent responsible for sexually transmitted infections in the general population . The preCervical cancer remains the fourth most common female cancer globally, with an estimated 570,000 cases and 311,000 deaths yearly . AccordiThe other preventive practice useful to reduce the risk of mortality for cervical cancer is cervical screening. A meta-analysis of 12 case-control studies demonstrated that it is strongly associated with a decreased risk of invasive cervical cancer  . Large-sThe purpose of the present study was to describe the burden of HPV-related hospitalizations in Sicily during 2007–2017, after ten years of vaccine implementation and sixteen years of screening availability.A retrospective observational study was conducted using Sicilian hospital discharge records (HDRs) collected from 2007 to 2017. Sicily is the fourth most populous Italian administrative region, with 5,026,989 inhabitants (2018 Census). The Sicilian Hospital Admission Database is the comprehensive database of all hospital discharges observed in public and private regional health care facilities. It provides information on hospitalized patients, notably demographic  and medical data  . It alsoAccording to several published studies, the following attributable fractions (AFs) were used to quantify the proportion of hospitalizations attributable to HPV: 88% of anal cancers, 50% of penile cancers, 26% of head and neck cancers, 77% of female genitourinary tract cancers, 100% for cervix cancer and warts . In deptAttributable hospitalizations for each HPV-related disease = Specific attributable fraction * Total hospitalizations for each HPV-related disease.Quality control for duplication of hospital admissions and hospitalizations with missing data was performed. The following data were extracted, as de-identified data, for each selected hospital admission: sex, age, days of hospital stay and country of origin. p-value ≤ 0.05 was assumed to indicate statistical significance (two-tailed).Data are described using absolute and relative frequencies for categorical variables, and mean ± standard deviation (SD) or median with interquartile ranges (IQR) for quantitative variables, depending on normal or not normal distribution. The hospitalization rate was calculated by dividing the number of hospital admissions by the total number of at-risk Sicilian population in each observed year . The hosOverall, 73,284 hospitalizations for potential HPV-related diseases were detected among Sicilian people during the entire period. According to the reported attributable fractions, it was estimated that 43,531 hospitalizations could be considered related to HPV in Sicily. As reported in p < 0.001). Among neoplastic diseases, the greatest reduction was detected for cervical cancer, which went down from 166.6 to 63.3 per 100,000 per year , followed by oropharynx cancer from 10.0 to 4.7 per 100,000 per year , and penis cancer with a hospitalization rate decrease from 3.2 to 2.2 per 100,000 per year .p < 0.001). The median age was 45 years old, with an increasing value from 43 to 47 years (p < 0.001). Among the cancers, the most common was cervical cancer with 30,430 hospitalizations occurring in 23,049 women. The demographic and clinical characteristics of hospitalizations with cervical cancer are reported in The number of patients with a hospital admission for invasive cervical cancer was 6,166 (20.3%). Among these, as shown in p < 0.001) followed by 35–44 .On the other hand, people with cervical carcinoma in situ (CIS) had the greatest cervical cancer burden with 24,264 (79.7%) hospital admissions. As reported in This observational study showed the burden of HPV-related disease in Sicily from 2007 to 2017. After ten years for vaccine introduction and sixteen years for cervical cancer screening availability, a relatively large decrease in hospital admissions for cervical cancer and other HPV-related diseases was observed. The reduction in hospitalization rates could be attributable to several factors, including HPV vaccination, which reduces HPV infection and carcinogenesis, cervical screening programs, which are well known to reduce the incidence and mortality of cervical cancer, and level of adherence to preventive practices .Data reported in Sicily can be compared with another Italian study conducted from 2001 to 2012 using a similar methodology [At the Italian administrative region level, there are two different studies that described the burden of HPV-related hospitalization in Tuscany and Veneto in relation with the different adopted vaccination strategies ,23. ThesAmong Sicilian women, there was an increase in age at hospitalization that could be associated with the implementation of HPV vaccination and screening strategies. In this sense, after the implementation of preventive strategies in a target age class (12 years old for vaccination and 25 years old for HPV screening), it was demonstrated that the mean age of the cohort of susceptible people increases year by year . Sicily was the principal entrance for migrant populations from Africa and the Middle East to Europe ,28. SeveThis study highlights a greater hospitalization decrease for CIS among 15–24-year-old women who had access to anti-HPV vaccination. This reduction could be a clue for HPV vaccine effectiveness, as reported by several studies. The most updated data were reported in the meta-analysis of Drolet et al. including 65 studies from 14 high-income countries, pooling data from over 60 million individuals for up to eight years of post-vaccination follow-up . The repIn the Sicilian study, a greater decrease in invasive cervical cancer disease was recorded among women older than 35 years who had access to cervical cancer screening. This result was in accordance with what was reported in Scotland, where vaccination and screening records of nine birth cohorts of 20-year-old women were extracted to evaluate the effectiveness of the HPV vaccine in preventing cervical precancerous lesions . The anaDespite various evidence for anti-HPV vaccine effectiveness, Sicilian vaccination coverage still remains low, with a mean level of 51%, and far from the minimum value required by the Italian Ministry of Health . FurtherConsequently, the Sicilian data could be useful to build up evidence for healthcare authorities to know trends of HPV-related disease . AnotherThis study, however, has to be interpreted in light of certain limitations due to the nature of the source of HDRs. Indeed, errors in diagnosis coding would influence the epidemiological data; however, the errors are expected to be very rare for these well-known diseases. As a second limitation, considering the absence of biological results in the HDR, we could not determine the effective part of HPV-related cancers among identified cases. Another limitation could be related to the decrease in HDRs for some procedures that Italian legislation has shifted from hospitalization to ambulatory care . FinallyHowever, the main objective of the study was to assess the epidemiology and burden of potentially HPV-related diseases during a long-lasting period, where all these limitations can be weakened. HPV-related burden had a constant decrease in Sicily and this decline could be, at least in part, attributed to ten years of HPV vaccine implementation and sixteen years of cervical screening availability. Some clinical characteristics of hospitalizations, such as increasing age, are suggestive clues for the impact of preventive strategies, but further research is needed to confirm the relationship. These data could be useful to local health authorities for improving vaccination coverage and screening adherence among Sicilian populations using an evidence-based approach in order to evaluate their effectiveness.


Background: Substantial declines in genital warts (GW) have been observed in countries with quadrivalent HPV vaccination programmes, with Australia showing the highest reductions due to early commencement and high vaccination coverage. There is a real potential to achieve GW elimination; however, no GW elimination definition exists. Taking Australia as a case study, we aimed to reach expert consensus on a proposed GW elimination definition using a modified Delphi process. Method: We used modelling and epidemiological data to estimate the expected number of new GW cases, from pre-vaccination (baseline) in 2006 to the year 2060 in Australian heterosexuals, men who have sex with men (MSM), and newly arrived international travellers and migrants. We used these data and the literature, to develop a questionnaire containing ten elimination-related items, each with 9-point Likert scales . The survey was completed by 18 experts who participated in a full day face-to-face modified Delphi study, in which individuals and then small groups discussed and scored each item. The process was repeated online for items where consensus (≥70% agreement) was not initially achieved. Median and coefficient of variation (COV) were used to describe the central tendency and variability of responses, respectively. Findings: There was a 95% participation rate in the face-to-face session, and 84% response rate in the final online round. The median item score ranged between 7.0 and 9.0 and the COV was ≤0.30 on all items. Consensus was reached that at ≥80% HPV vaccination coverage, GW will be eliminated as a public health problem in Australia by 2060. During this time period there will be a 95% reduction in population-level incidence compared with baseline, equivalent to <1 GW case per 10,000 population. The reductions will occur most rapidly in Australian heterosexuals, with 73%, 90% and 97% relative reductions by years 2021, 2030 and 2060, respectively. The proportion of new GW cases attributable to importation will increase from 3.6% in 2006 to ~49% in 2060. Interpretation: Our results indicate that the vaccination programme will minimise new GW cases in the Australian population, but importation of cases will continue. This is the first study to define GW elimination at a national level. The framework developed could be used to define GW elimination in other countries, with thresholds particularly valuable for vaccination programme impact evaluation. Funding: LK supported through an Australian Government Research Training Programme Scholarship; unconditional funding from Seqirus to support the Delphi Workshop. In 2018, the qHPV and nHPV vaccine together comprised ~77% of the total vaccine doses delivered globally [Human papillomavirus (HPV) is the most common sexually transmissible infection (STI) globally, and is associated with significant morbidity and mortality due to genital warts (GW), and anogenital and other cancers ,2. Threevaccines . All thrvaccines . The qHP5% of GW , while tqHPV/nHPV vaccination, substantial reductions in GW have been observed in many countries [The primary aim of HPV vaccination is to prevent cervical cancer, which is associated with high global mortality and morbidity . In 2018ountries , suggestountries ,11, has ountries . AustralFirst, there is no standardised definition of GW elimination. The WHO broadly defines elimination of an infection or infectious disease under two categories: (i) interruption of transmission of an infection, defined as reduction to zero of the incidence of infection in a defined geographical area  and (ii)The Delphi method has been widely used in public health to reach consensus among a group of experts in a specific subject area. In situations where available information is contradictory or insufficient, the Delphi method provides an effective tool to make decisions . The metWe designed a modified Delphi study  comprisiWe first conducted a review of the literature (peer-reviewed and grey literature) and held extensive consultations with six Australian experts. Extending our previous work ,15, we eThe epidemiological analyses resulted in the development of a questionnaire containing preliminary GW elimination definitions and thresholds. The questionnaire was tested in a small pilot Delphi with eight early- to mid-career level researchers to ensure the clarity and acceptability of the items. An eight-item questionnaire  and interquartile ranges were used to describe the variability of expert responses on each item. Items were excluded from the subsequent round if ≥70% of participants scored the item ≤4 in the preceding round with a COV of ≤0.5. Items met consensus for inclusion if ≥70% of participants scored the item ≥7 in the preceding round with a COV of ≤0.5. If items failed to meet the inclusion/exclusion criteria in the preceding round, they were modified as per the feedback for the successive round. A response rate of 80% for each round of the study was considered sufficient.Ethical approval was obtained from the UNSW Human Research Ethics Advisory Panel (reference number HC16530). All experts were provided with a participation information sheet. Consent was obtained prior to their participation in the workshop.The sponsor had no role in study design, data collection, analysis, interpretation or writing of the report. The corresponding author has full access to all the data and had full responsibility to submit for publication.We invited 21 experts to participate in the study, of whom 19 agreed. Of these, 18 participated in the workshop (95%), and 16 participated in the online round (84%). The characteristics of the experts are shown in At the end of the workshop, two of four groups of experts scored the concept of interruption of endemic transmission of HPV 6/11 ≤ 4  . Two conSimilarly, for the concept of elimination as a public health problem, two of the four groups of experts agreed on this definition by scoring it ≥ 7  . Some paA review of literature was performed post workshop through which it was determined that the reported prevalence of GW in the pre-vaccination era satisfies the essential criteria for classification of a condition as a public health problem , and theViews were divided on the potential impact of non-vaccine HPV types on the control threshold  and the elimination threshold  at the end of the workshop . Some exeff). Reff denotes the average number of secondary cases per infectious case in a population made up of both susceptible and non-susceptible hosts. Other experts argued that the effects could potentially be less than expected due to growing levels of HPV vaccination globally. Nonetheless, experts suggested that perhaps thresholds needed to be adjusted to account for this. The experts also suggested defining an additional control milestone for the year 2021.Similarly, at the end of the workshop, views were divided regarding the potential impact of ongoing transmission of GW due to importation on the estimated control and elimination thresholds . Some exBased on the experts’ feedback, we conservatively adjusted our proposed control milestone  and control threshold for the years 2021 and 2030, respectively, to account for the effects of chains of transmission due to importation. The relative reductions in new GW diagnoses were changed from 67% to 60% for 2021 and from 82% to 80% for 2030 . The eliqHPV vaccine schedule may be different from two dose coverage (0–6 months apart) for the nHPV vaccine, which replaced the qHPV vaccine in the national HPV vaccination programme in Australia in 2018. At the end of round 2, the reworded item met consensus for inclusion at 75%   for the V: 0.16) .At the end of round 1, most experts disagreed with using sexual health clinics as the main data source for measuring GW elimination and suggested that both general practice and sexual health clinic data should be used, the two out-patient healthcare settings in Australia where GW episodes are seen. The revised item, including both data sources, met consensus for inclusion at 75%  . ExpertsqHPV/nHPV vaccination programme . In the past, elimination targets of other infectious diseases have been presumably developed in consultation with experts, but often very little detail of the process is available ; this isqHPV and nHPV, also provide protection against GW caused by low-risk HPV types 6/11. The choice of vaccine for a national vaccination programme generally depends on affordability, but in some countries, such as the United Kingdom, the decision was influenced by our previous research [qHPV vaccine [qHPV or the nHPV vaccine for their national programme, defining GW elimination thresholds will provide useful additional information to monitor the programmes’ performance. The framework developed through our study for Australia could be replicated for other countries, if comprehensive epidemiological and mathematical modelling information is available in the local context.Cervical cancer is a major HPV-related global public health problem and the aspirational goal of elimination defined by the WHO provides impetus for countries to achieve high HPV vaccination coverage using one of three available HPV vaccines. The primary purpose of HPV vaccination is to prevent cervical cancer, and while all three vaccines are highly effective in providing protection against infections with cancer causing HPV types 16/18, two HPV vaccines, research  on the r vaccine . In the Our HPV vaccination course completion threshold of ≥80% coverage necessary to reach population-level elimination in Australia, is consistent with the WHO target of 90% for female-only vaccination  and the Our results suggest that interruption of endemic transmission of HPV 6/11 is not a suitable elimination definition for GW. For other infections for which elimination thresholds have been set by the WHO for interruption of endemic transmission , case-based surveillance is carried out by conducting genotyping in every outbreak to differentiate endemic versus imported or import-related cases ,33. SuchqHPV vaccination satisfied the essential criteria for classification of a condition as a public health problem [Elimination as a public health problem is a more appropriate approach for defining GW elimination. The burden of GW in Australia prior to the introduction of  problem ,38—namel problem , loss of problem , high fi problem , and the problem . While o problem , transge problem , and mig problem . This wibHPV vaccination programme, with China and India (both currently with no HPV vaccination programme) comprising 24% and 13% of international students, respectively [A key finding from our study is that absolute elimination of GW in Australia is unlikely. Firstly, importation of GW caused by HPV6/11 will continue with over half the remaining cases occurring in international travellers in 2060. Of these, nearly half the cases will be attributed to international students. Currently, a majority (72%) of Australian international students are from countries that either do not have an HPV vaccination programme or only have a ectively . TherefoqHPV vaccination offers significant cross-protection against non-vaccine HPV types, including the majority of those that cause GW [Secondly, a small proportion of GW are caused by HPV types for which current HPV vaccines provide no direct protection. As noted, the control and elimination thresholds defined in this study were not adjusted for the contribution of GW caused by non-vaccine HPV types. The underlying assumption was that their contribution would likely be minimal or insignificant based on new evidence from a study that compared urine samples of vaccinated and unvaccinated cohorts of young girls and suggested that cause GW . In the cause GW , we conccause GW ,46,47. Teff for HPV 6/11 and then use this to estimate the probability that a chain of transmission due to importation will occur. This is outside the scope of this study; however, it is the subject of ongoing work by the authors. We did not adjust the 2060 elimination threshold of 95% relative reduction in GW incidence as the HPV vaccination coverage is likely to increase over time, both in Australia and globally, and the importance of importation is likely to be diminished accordingly. This is particularly important in terms of the anticipated increase in vaccination coverage in low- and middle-income countries where HPV vaccination might become more affordable due to: (a) technology transfers of existing vaccines to manufacturers in middle-income countries [We conservatively adjusted the control milestone and threshold to account for the impact of ongoing chains of transmission in Australia due to importation of HPV types that cause GW. The underlying assumption was that population-level herd protection would not be reached by years 2021 and 2030 and therefore transmission due to importation will continue. One way of addressing this question is to calculate Rountries ,49; and ountries .Our study has some limitations. First, we compromised the anonymity of experts in the workshop as this was necessary to expedite the consensus building process. In doing so, we may have introduced a degree of ‘bandwagon effect’, a phenomenon whereby those with strong opinions may sway the opinions of others . To counOur study also has several strengths and fulfilled important factors that determine the validity of the Delphi technique. These include: (a) selection of a heterogenous panel, with expertise across various areas of the health sector ; (b) achqHPV vaccination programme [In conclusion, we defined thresholds for elimination of GW at a national level, using Australia as the case study, the first country in the world to implement a nationally funded rogramme . On the


Human papillomavirus (HPV)-based cervical screening is now replacing cytology-based screening in several countries and many women in screening programmes will consequently receive HPV-positive results. Because of the sexually transmitted nature of HPV, receiving an HPV-positive result may raise questions about disclosing the infection to a sexual partner.To review the quantitative and qualitative literature exploring women’s concerns about disclosing a high-risk cervical HPV infection to a sexual partner.We searched MEDLINE, PsycINFO, CINAHL Plus, Web of Science and EMBASE for studies reporting at least one disclosure-related outcome among women with high-risk HPV. We also searched the grey literature and carried out forward/backward citation searches. A narrative synthesis for quantitative studies and a thematic synthesis for qualitative studies were conducted.Thirteen articles met the inclusion criteria . In the quantitative study, 60% of HPV-positive women felt disclosing an HPV result was ‘risky’. Concerns about disclosing HPV to a sexual partner were influenced by the stigma that is associated with having an STI and uncertainty about how their partner would respond. Women questioned how, when and to whom they should disclose their HPV-positive status.The studies included in this review provide rich information about the range of concerns women have, the reasons for these concerns, and the questions women have about disclosing HPV to sexual partners. As studies were predominantly qualitative, the prevalence of concerns is unclear. This is the first review to synthesise the literature on women’s concerns about disclosing a high-risk cervical HP Vinfection to a sexual partner.This review identified that concerns about disclosing HPV to a sexual partner are partly because of the stigma associated with having an STI and uncertainty about how a partner might respond.Some women have questions about disclosure including who they should disclose to and how to approach and manage these conversations.Increasing knowledge of the high prevalence of HPV and providing clear information in screening letters and leaflets about disclosing may help women understand their screening result and minimise unnecessary concerns.Virtually all cervical cancers are caused by persistent infection with a high-risk type of human papillomavirus (hrHPV).Until recently, most cervical screening programmes in high-income countries used cytology to detect cervical abnormalities, with HPV testing used as a triage for women with borderline or low-grade cell changes.Research suggests that a key concern among individuals with an STI is disclosing their diagnosis to a sexual partner. In studies with participants with herpes simplex virus (HSV) and chlamydia, disclosure is described as something that is difficult, fear-inducingContact tracing  is important for some STIs so that previous partners can be screened and treated for the infection if necessary. However, there is no treatment for HPV and the World Health Organization (WHO) advise against routine contact tracing for HPV.This review was registered with PROSPERO (CRD42018083969) and followed the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines.www.opengrey.eu) to identify any additional eligible articles.We searched MEDLINE, PsycINFO, CINAHL Plus, Web of Science and EMBASE on 9 January 2019. The search included terms relating to (i) high-risk cervical HPV and (ii) a psychosexual or disclosure-related outcome  and were linked using Boolean operators . Two reviewers (KFB and MR) screened the abstracts of the remaining articles. Articles were included if they mentioned (i) HPV and (ii) a psychosexual or disclosure-related outcome. Reviews, conference abstracts, commentaries, opinion pieces and editorials were excluded. Articles were also excluded if they were not written in English, focused on the psychosexual impact of cervical cancer, or treatment for cervical cancer or colposcopy. We decided not to include articles that focused exclusively on low-risk types of HPV  because (i) primary HPV testing will be for high-risk types of HPV and (ii) feelings about disclosing low-risk HPV were expected to be distinct because of its symptomatic, visible nature. Full-texts were obtained where an article could not be assessed from the abstract. Disagreements were resolved by discussion.Data were extracted from each article using a standardised data extraction form see . Extract10.1136/bmjsrh-2019-200503.supp2Supplementary dataA quality assessment was carried out for each article using modified versions of the National Institute for Health and Care Excellence (NICE) quality appraisal checklists for quantitative and qualitative studies see . One rev10.1136/bmjsrh-2019-200503.supp3Supplementary data10.1136/bmjsrh-2019-200503.supp4Supplementary dataFor qualitative studies we conducted a thematic synthesis, following three stages outlined by Thomas and Harden:The initial search returned 7336 articles, which reduced to 4801 after the removal of duplicates. Of these, 4465 were excluded on the basis of their title, leaving 336 abstracts to be reviewed. Following exclusions, 41 full-texts were reviewed. Thirteen articles were excluded during the full-text review and an additional two articles were identified following backward and forward citation searches, resulting in 30 papers see . ThirteeStudies were conducted in the US (n=7), UK (n=2), Australia (n=2), Taiwan (n=1) and Brazil (n=1) and were published between 2005 and 2016. Studies were predominantly qualitative (n=12),All qualitative studies were judged to be well conducted. The single quantitative study was judged to have been designed or conducted in such a way as to minimise the risk of bias and had good internal and external validity  Anticipated psychological impact of disclosure, (ii) When is disclosure necessary? and (iii) Managing disclosure. The first theme describes the thoughts, feelings and concerns women had prior to disclosing HPV to a sexual partner. In addition to expressing general concerns, women were concerned about the stigma that was attached to having an STI and how their partner would respond.While some women were not worried about disclosing the infection, others felt that the prospect of disclosure was challenging, complicated and something they wished to avoid. Women were often anxious, worried, fearful and stressed about discussing HPV with their sexual partners:"The thought of having it, deciding when to do it and how and what to say - it was extremely stressful" [P = participant comment]."Women repeatedly described feeling highly anxious about informing their partner, with descriptions of 'bursting into tears' and feeling intensely 'guilty' and worried that they may have infected their partner with the virus" [A = author comment].Their concerns about disclosing were partly due to the stigma and shame associated with having an STI and how others would respond. Concern that they may have transmitted the infection to their partner and perceptions that partners had a poor understanding of HPV also enhanced anxiety around disclosure:Women’s concerns about anticipated disclosure were partly due to the stigma of having an STI. Women were apprehensive that they might be viewed as being promiscuous.I was more worried about my partner reading it and saying 'aha'. I was worried about him thinking it was sexually transmitted and that I picked it up before I met him which would have concerned him a lot as we had only been together about 4 or 5 months at that stage… I was worried that it might change his opinion of me and being early in a relationship [it was a] bit of a concern" [P]."If I told men that I had it they might not want to have sex with me" [P].Concerns about how others would respond and react to HPV disclosure seemed to be influenced by the negative connotations of having an STI. Women were concerned that their partner would perceive them differently:Should I be telling sexual partners that I have this? And one person would say yes of course you must and another would say don’t be silly almost all the population’s been exposed to it … I couldn’t get to the truth … they were giving me conflicting advice … I found that very distressing that I couldn’t actually get real information that I could trust" [P].The second theme related to women’s views on whether it was necessary to disclose their HPV-positive status to a sexual partner. While some women felt obligated to disclose the infection to current and prospective partners,It's always in the back of your head. You know, 'Is he going to ask me back to his place? If he does, should I tell him?' It was just, 'When do I tell him?'… So, it was very much like 'What's the best timing?'… It was a lot of planning and stressing out and asking my friends, 'Do you think I need to tell him?'" [P].motivated by women’s desire to minimise their own anxiety during an already stressful period and to avoid dealing with a difficult issue of which they had only limited understanding" [A].The third theme related to managing disclosure. Some women reported that they were uncertain about how to approach disclosureOnly one quantitative study reported outcomes relating to disclosure of HPV to a sexual partner.To our knowledge, this is the first review to synthesise the literature on women’s concerns about disclosing a high-risk cervical HPV infection to a sexual partner. The qualitative literature identified a range of concerns about disclosing HPV to a sexual partner. These concerns were partly because of the stigma associated with having an STI and the ways in which women anticipated their partners might respond. Some HPV-positive women used strategies to manage disclosure of their HPV diagnosis to a sexual partner, for example, focusing on having an abnormal screening result rather than HPV per se. The qualitative literature also found that women questioned how, when and to whom they should disclose their result. While quantitative and qualitative articles were included in the review, only one quantitative article was identified which found that over half of HPV-positive participants felt that disclosing their HPV-positive result was ‘risky’.The results of this review suggest that some women feel anxious, worried and fearful about disclosing HPV to a sexual partner and described it as something they wished to avoid. These feelings were partly related to the stigma of having an STI and concerns about how others would respond to the disclosure of an HPV diagnosis. These findings are consistent with previous research with individuals diagnosed with other STIs such as HSV and chlamydia, where disclosure has been described as something that is difficult, fear-inducingDuring disclosure some women deliberately avoided mentioning HPV, focusing instead on their abnormal cytology or other aspects of their screening results. Managing the psychological implications of disclosure may be more challenging for women undergoing primary HPV testing who are told they are HPV-positive with normal cytology, given that HPV will be the only abnormal result they receive. They could, however, choose to focus on the normal cytology result. Following the introduction of primary HPV testing, it may be necessary to have additional support available for women. Healthcare professionals, particularly those carrying out cervical screening, are ideally placed to give brief information during screening which could help to mitigate the psychological impact of an HPV-positive result.There are several advantages of primary HPV testing over cytology including increased sensitivity for detecting high-grade cervical abnormalities;Some women had questions about disclosing the infection to sexual partners, including whether disclosing was necessary. Disclosure is important for some STIs so that previous partners can be screened and treated for the infection if necessary, and future transmission of the infection can be prevented. However, while HPV is classified as an STI, it differs from other infections in that it does not usually need any treatment or cause any long-term problems. In addition, because most people will be infected with HPV at some point in their life,A strength of this review is that it was systematic and followed PRISMA guidelines. In addition, a broad search strategy was used with no date restrictions. It is possible that because of the range of terms that can be used to describe disclosure, some eligible studies may not have been identified in our search; however, we conducted forward and backward citation searching for all included studies to reduce the likelihood of this. Data were extracted by one author, with a second reviewer independently extracting data for 30% of the studies. It is possible that if the second reviewer extracted data from all the studies the results of the review could have changed; however, we feel this is unlikely as the agreement rate between reviewers was very good.Only one quantitative paper was identified that reported disclosure-related outcomes, compared with six exploring the broader psychosexual impact of HPV, as identified by our related review.This review synthesises the literature on women’s concerns about disclosing a high-risk cervical HPV infection to a sexual partner. The studies included in the review provide rich information about the range of concerns women have, the reasons for these concerns, and the questions women have about disclosing HPV to sexual partners. Increasing knowledge of HPV and providing clear information in screening information letters and leaflets about disclosing HPV to sexual partners may help women understand their screening result and minimise any unnecessary concern surrounding disclosure.https://www.nhs.uk/conditions/human-papilloma-virus-hpv/Human papillomavirus: https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/827426/Cervical_screening_helping_you_decide_HPV.pdfNHS cervical screening - helping you decide: https://www.jostrust.org.uk/professionalsScreening, colposcopy, and cervical cancer:


We detected disproportionate reports of premature ovarian insufficiency (POI) and related events, including amenorrhea, menstruation irregular, FSH increased, and premature menopause, following human papillomavirus (HPV) vaccine from FDA Vaccine Adverse Event Reporting System (VAERS). The signal was detected by the methods of Bayesian Confidence Propagation Neural Network (BCPNN) and Multi-item Gamma Poisson Shrinker (MGPS). When both methods detected a positive result, a signal was generated. Besides, time-scan map is drawn based on the IC value and 95%CI of BCPNN, if the IC curve showed a steady upward trend and the 95%CI narrowed, the signal was stable and strong association.The results showed that there were not POI reports of HPV vaccine, but VAERS received a total of 2, 389, 27 POI related events for HPV2, HPV4, HPV9 respectively from the year of marketed to 2018. No signal was detected for HPV2. HPV4-POI ralated events were all detected as signals by two methods. There was only one signal of menstruation irregular for HPV9. Time scan of HPV4-POI ralated events showed those signals were stability and strong association, but not for HPV9. Our results only represent statistical association between HPV vaccine and POI related events, causal relationship needs further investigation. Continuous infection of high-risk HPV types is highly associated with the development of cervical cancer, and administration HPV vaccine can effectively prevent cervical cancer. Three prophylactic HPV vaccines are currently available worldwide, including HPV bivalent recombinant vaccine (HPV2), HPV quadrivalent recombinant vaccine (HPV4), HPV 9-valent recombinant vaccine (HPV9).Human papillomavirus (HPV) is the most common viral infection of the reproductive tract and is the cause of a range of conditions in both men and women and cervical cancer is caused by certain types of HPV. It was estimated that 569,847 new cervical cancer in 2018, of which 311,365 deaths around the world2, is a clinical syndrome defined by loss of ovarian activity before the age of 40, which is characterized by menstrual disturbance (amenorrhea or oligomenorrhea) with raised gonadotrophins and low estradiol 3. Women with POI have a little chance of spontaneous pregnancy, and no interventions could increase ovarian activity and natural conception rates 3. With the widespread of HPV vaccine, there were an increasing number of reports and studies on HPV vaccine-POI combination. For example, case reports and case series study reported a possible link about adolescent POI following HPV4 4, but a population-based retrospective cohort study found no elevated risk of POI after HPV4 administration 5. Although these observations were not identified the possible risk for HPV vaccine-POI association, we connot exclude HPV vaccination has a role in pathogenesis of POI 6. So far, there are no reports and studies of HPV2 and HPV9-POI combination, and whether various HPV vaccines associated with POI is still inconclusive.Premature ovarian insufficiency (POI), also known as premature menopause or premature ovarian failure7, the World Health Organization Uppsala Montoring Centre (WHO-UMC) 8, the Netherlands Pharmacovigilance Centre Lareb. 9, to evaluate possible association between vaccine/drug and event combination, which including detection of novel signal, strengthening or weaking of existing signal and providing evidence for or against exicting safety concern not evaluated by data mining.Published adverse reactions about HPV vaccines were mainly available form clinical trials, which might not reflect the full safety characteristics of HPV vaccines because of strict trial design, relatively small sample size and short duration of follow-up. Large-scale adverse event spontaneous reporting system is an important data source for identifying and discovering new or rare adverse reactions and after marketing. Signal detection from spontaneous reporting system (SRS) is carried out by many regulatorty authorities, such as the US Food and Drug Administration (FDA) Published researches have supported or opposed the possible association between HPV4 vaccination and POI. We designed this study to evaluate the statistical association and detect the signal of POI and related events of three HPV vaccines, and to provide evidence for or against exicting HPV4-associated POI concern based on the data mining and signal detection method. The research data was obtained from the Vaccine Adverse Event Reporting System (VAERS).Between 2009 and 2018, VAERS received a total of 275,595 vaccinated alone reports, including 171 for HPV2; from 2006 to 2018, there were 322,932 single use reports, of which 27,386 were HPV4; 143,909 were extracted from 2015 to 2018 and 7,255 for HPV9 , suggesting that the report was authentic and professional. The healthcare providers include physician, physician assistant, medical assistant, certified medical assistant, nurse, registered nurse, licensed practical nurse, nurse practitioner, health professional, healthcare worker, doctor of pharmacy, pharmacist, pharmacy technician, physical therapist.The age distribution was mainly concentrated on 9–18 years old , and the proportion of less than 40 years old is 100.00%, 77.12%, and 70.37%, respectively for HPV2, HPV4 and HPV9. For serious adverse event (SAE), there are 0, 55 and 1 reports for HPV2, HPV4 and HPV9 respectively. The general characteristics and composition of each HPV adverse event reports are shown in Table Signal detection was performed on POI related events of three HPVs. The results are shown in Table 10. The results are shown in Figs. A time-scan map is drawn for the HPV-associated POI related events to show the change tendency of the signal over time. When the lower 95% confidence interval (CI) limit of the IC for the HPV vacccine-AE combinations changes from a negative a positive value, the signal appears. When the curve of IC shows a steady upward trend and the 95%CI narrowed, it indicates that the signal tends to be stable and strong association The time scan of HPV4-associated amenorrhea and menstruation irregular shown in Fig. Figure For the HPV4-associated premature menopause Fig. d, becausThe time scans of HPV9-associated AEs are shown in Fig3. SAE reports were few in this database, mainly in disability . Although two methods, BCPNN and MGPS, were used to improve the signal detection, some positive signals might have been missed due to the low sensitivity of Bayesian method.To our knowledge, this is the first signal detection research in which POI and related events were evaluated as a potential HPV vaccine adverse event. For the general characteristics of the reports, the reporter of healthcare providers accounted for 54.76% in HPV4, for 29.63% in HPV9, indicating that reports mainly accessed by professional staff. The patient age composition mainly concentrated on adolescent of 9–18 years old  and reproductive-age women between 19 and 26 years old , within the age for ovarian development and fertility. In addition, more than 70% of patients were younger than 40 years old, consistent with the epidemiological characteristics and diagnostic criteria of POI 11. Amenorrhea, FSH increased, and menstruation irregular are the symptoms and examination indicators of POI that serve as early warning on the development of POI for healthcare providers to identify POI early. Although POI is often used as synonyms of menopause, they are not equivalent. Thus, we conducted signal detection for amenorrhea, FSH increased, menstruation irregular, and premature menopause to assessing the sataistical risk of POI caused by HPV vaccines.The American Society of Reproductive Medicine classified POI into occultation period, biochemical abnormality period and clinical abnormal period according to FSH level, menstrual status and fertility. The fertility is reduced with the increase of FSH as early manifestation, and finally develops into irregular menstruation or amenorrhea The signal detection results showed: (a) HPV2 did not yield any signal; (b) HPV4 was associated with amenorrhea, FSH increased, menstrual irregularities and premature menopause, indicating HPV4 with a sataistical risk of POI and related events; (c) irregular menstruation was detected as signal for HPV9 only. The diagrams of time scan showed: (a) because there was no signal for HPV2, no time scan figure was done for it; (b) the time scan of HPV4-associated POI related events showed that the signals were stable and strong association; (c) time scan of HPV9-POI related events indicated that the value of IC increasing but with a large 95% CI bars, suggesting the unstable signals. It was worth noting that the signals only represented a statistical relationship between HPV vaccines and adverse events, did not represent a causal relationship.12 and the adjuvant adverse effect of vaccine 13. A possible relationship between POI and HPV vaccination has been proposed in view of the temporal vaccine-event association 12. POI is a clinical disorder with complicated aetiology, and 5–30% of POI cases have an autoimmune mechanism 14. But the pathogenesis of HPV vaccine as a tigger factor for autoimmune disease is difficult to assess and did not found conclusive evidence 12. In immunology, according to defined by the National Cancer Institute, an adjuvant is an agent which might stimulate and increase the immune response to a vaccine, the aluminum salt was the most common adjuvant in human vaccines 15, and aluminum salt is the adjuvant in HPV2, HPV4 and HPV9. The adjuvant of aluminum salt could enhance or prolong antigen-specific immune response, but it may have potential adverse effect. According to animal experiments, aluminum could accumulate in the ovary, damage the structure of the ovary and decrease ovarian weight, which suppressed the concentrations of estrogen, progestogen, FSH, luteinizing hormone and resulted in abnormal development of ovarian follicles, and the reproductive function of female rats was inhibited by aluminum exposure 17.The possible pathogenesis between POI and HPV vaccines were multifactorial, such as autoimmune response prompted by HPV vaccines 5 and Pellegrino 6, which did not obversed an elevated risk of POI after HPV4 administration but they suspected presenting of a rare risk factor of determining the adverse event 6, our results provided a signal evidence in the view of pharmacovigilance. Furthermore, long-term reproductive safety studies are surely needed. HPV4 and HPV9 have the same preparation process and adjuvants except for the different viral subtypes. Short-term of marketed and lack of long-term safety data may be attribute to the difference in signals between the both. In addition, no studies have shown that the difference in adverse events due to different virus subtypes.Our study detected the signals of HPV4-associated POI related events and the signal of menstruation irregular for HPV9. Although our results were different from other reseauches, such as Naleway 18, this study only includes the reports of vaccination alone and excluding data from co-vaccination. But it resulted in a reduction in sample size and it is also a bias in our study. According to the clinical trials for HPV4 and HPV9 co-vaccination with other vaccines, they found that it is safety in vaccination combination, but lack of the data of post-marketed 20.However, data mining and signal detection of VAERS has several limitations. First, the major limitations of our study are that it is unclear whether these AEs caused by disease, and lack of the data of medical history, medication history and menstrual history. Therefore, disease-oriented adverse events could be detected as signals. Second, VAERS is subject to various biased such as missing data, over-reporting, under-reporting. Third, because there is no specific value of FSH in VAERS and each report is only one FSH test result without continuous result, the FSH increase signal may bias to POI. Fourth, since combined vaccination may inhibit or enhance the immune response AE reports in VAERS were reviewed to analysis the safety profiles of HPV vaccine-associated POI and related events. From the results of signal detection, it was suggesting that HPV4 have statistically signaificant association with POI, including amenorrhea, menstruation irregular, FSH increased and premature menopause, and HPV9 have potential statistical risk in menstruation irregular. However, it should be noted that our research data is accessed from VAERS and results are affected by the quantity and quality of AE reports, and studying POI as an HPV vaccine adverse event is challenging for many reasons. Finally, further research and cauaslity investigation between HPV vaccine and POI are strongly suggested in the future.https://vaers.hhs.gov/data/datasets.html, or using the CDC WONDER online search tool. The raw data in VAERS is presented by calendar year and updated monthly. The downloaded data include 3 separated data files, which are VAERSDATA, VAERSSYMTOMS, VAERSVAX, and each file have a unique ID for one report 21. In our study, we downloaded raw data from VAERS wepsite and extract data using ID.VAERS is a passive surveillance system created by the Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC) to receive adverse event (AE) reports that may be possible associated with vaccines. The primary purpose of VAERS is to find early signal and generate hypotheses about possible new vaccine adverse events that do not be found during pre-market trails. VAERS is a free and open database, and VAERS data is accessible by two ways: downloading raw data in comma-separated value (CSV) files from In VAERSVAX file, HPV vaccine was encoded as HPVX, HPV2, HPV4, HPV9, so the search terms of this study were defined as HPV2, HPV4 and HPV9. The search time was set to the year of vaccine launch until 2018, with HPV2 from January 2009 to December 2018, HPV4 from January 2006 to December 2018, and HPV9 from January 2015 to December 2018. In order to avoid the impact of co-vaccination, this study only included adverse event reports of HPV vaccination alone. We used EXCEL 2016 to screen and extract data on target vaccine-adverse event reports, including: number of reports, age, the type of reporter, and serious adverse events.3, which are oligomenorrhea/amenorrhea for at least 4 months, an elevated follicle stimulating hormone (FSH) level more than 25 IU/L on two occasion more than 4 weeks apart. We defined 3 related events: amenorrhea, irregular menstruation (including oligomenorrhea and delayed menstruation), and FSH increased to evaluate the association between HPV and POI. Considering that the two terms of POI and premature menopause are often used interchangeably 5, we also included the event of premature menopause into the study.Medical Dictionary for Regulatory Activities (MedDRA) is a collection of international medical terminology prepared by the International Council for Harmonization (ICH) for the standardization and unification of adverse event report. MedDRA contains 5 hierarchical structures, which are Lowest Level Term (LLT), Preferred Term (PT), High Level Terms (HLT), High Group Terms (HLGT), System Organ Classes (SOC). Adverse events in VAERS reports are coded using the MedDRA of PT. We searched adverse events in MedDRA 19.0. Since MedDRA 19.0 did not include the term of POI, we redefined POI and related events according to definition and diagnostic criteria from ESHRE Guideline 22 and Multi-item Gamma Poisson Shrinker (MGPS)24. The basis of those methods is based on two-by-two contingency table le Table .Table 3TThe theory is to organize the reports of HPV-associated POI and related events in VAERS into the four analysis units of the vaccine-AE combination, including target vaccine-target AE, target vaccine-other AEs, other vaccine-target AE, other vaccines-other AEs. Then, we conducted statistical and disproportionalily analysis. In order to reduce the generation of false positive signals, our study used both BCPNN and MGPS methods to detect signals. When the results of both methods were positive, a signal was detected. The signal detection standards of each method are shown in Table In addition, this study will plot the time scan for HPV-associated POI and related events of information component(IC) and 95% CI, in order to reflect the change tendency of the target vaccine-target adverse event association in the database as the number of reports increasing over time, and verify the signal stability and association strength. When the IC curve is steady upward trend and the 95% CI narrowed, the signal is stable and strong association.


The aim of this study was to carry out a case control study comparing the HPV genome in patients with oral cavity squamous cell carcinoma (OC-SCC) to normal patients using metagenomic shotgun sequencing. We recruited 50 OC-SCC cases which were then matched with a control patient by age, gender, race, smoking status and alcohol status. DNA was extracted from oral wash samples from all patients and whole genome shotgun sequencing performed. The raw sequence data was cleaned, reads aligned with the human genome (GRCH38), nonhuman reads identified and then HPV genotypes identified using HPViewer. In the 50 patients with OC-SCC, the most common subsite was tongue in 26 (52%). All patients were treated with primary resection and neck dissection. All but 2 tumors were negative on p16 immunohistochemistry. There were no statistically significant differences between the cases and controls in terms of gender, age, race/ethnicity, alcohol drinking, and cigarette smoking. There was no statistically significant difference between the cancer samples and control samples in the nonhuman DNA reads . HPV was detected in 5 cases (10%) of OC-SCC  but only 1 tumor sample (genotype 16) yielded a high number of reads to suggest a role in the etiology of OC-SCC. HPV was detected in 4 control patients  but all had only 1–2 HPV reads per human genome. Genotypes of HPV are rarely found in patients with oral cancer. More than 95% of these cases will be squamous cell carcinoma (SCC)2. Although approximately 50% of these patients might be alive 5 years from now, about 20% will die from the disease within 1 year2. The 5-year survival of oral cancer has not significantly improved over the past several decades3. Although tobacco use is among the strongest risk factors for oral cancer, this factor does not completely explain the incidence of all oral cancer as the drastic decline in the prevalence of cigarette smoking since 1975 (from ~ 40 to 20%) has caused only a moderate change in the incidence of oral cancer5. This indicates a paradigm shift in the cause of oral cancer and the need to search for other risk factors.The American Cancer Society estimates about 53,260 people will be diagnosed and 10,750 people will die of oral cancer and oropharyngeal cancer in the United States in 202011. This has led to the hypothesis that some genotypes of HPV may also be responsible for the epidemiology change in the etiology of oral cancer as well. Although the prevalence of the high risk genotypes HPV 16, 18 and 33 varies greatly across multiple studies on oral cancer12 it is now accepted that these high risk genotypes are unlikely to be responsible. With regards to other genotypes of HPV, over the past 10 years an increasing number of HPV types have being found in oral samples14. The currently available HPV detection kits only detect a limited number of HPV genotypes. Since there are over 200 different genotypes of HPV, we hypothesized these may be responsible for some cases of oral cancer. The aim of our study was therefore to carry out a case control study in 50 oral cancer patients and 50 matched control patients to detect all 200 genotypes of HPV using next generation sequencing.High risk genotypes of Human papilloma virus (HPV), genotypes 16, 18 and 33, now account for 60–80% of all oropharyngeal squamous cell cancersTo examine the hypothesis that oral HPV is associated with OC-SCC, we performed a case control study with mouthwash samples from 50 patients with OC-SCC and 50 subjects with no oral lesions. Total genomic DNA was extracted from cell pellets of the mouthwash samples. Subject recruitment, sample collection, data generation and analysis are detailed below.A case–control study was approved by the Institutional Review Board of Memorial Sloan Kettering Cancer Center (IRB 15-256) and New York University School of Medicine (i15-00389). All methods were performed in accordance with the relevant guidelines and regulations. Informed consent was obtained from all patients in the study population. From Memorial Sloan Kettering Cancer Center (MSKCC), we recruited 50 oral cavity squamous cell carcinoma (OC-SCC) cases. These cases were then matched with a control patient by age, gender, race, smoking status and alcohol status. Overall, 100 subjects (50 cases and 50 controls) were enrolled in this study. The controls comprised patients with thyroid nodules . These patients had complete head and neck examination including flexible laryngoscopy and were found to have no evidence or oral cancer. In patients with oral cancer, OC-SCC was confirmed by histological examination of biopsy specimens. Pathological grade and stage of OC-SCC were determined by histopathological examination at the time of surgical resection. Demographic and clinical information was collected for each patient.15. This is cheaper and easier to carry out than ISH and PCR and therefore immunostaining of tumor sections for p16INK4a is now used as an indirect marker for HPV status in clinical pathology laboratories around the world16. In prospective randomized trials on treatment of patients with HPV related oropharyngeal cancer, p16 immunohistochemistry is now used as the surrogate marker for HPV positivity in the USA. Rarely some p16 positive tumors may not be HPV related. The addition of HPV PCR to the detection methodology would increase specificity as described by Prigge et al.17. In our study, all pathology specimens were examined by a single pathologist specialized in head and neck pathology (NK). p16 immunohistochemistry was performed as follows: four-micrometer tumor sections were deparaffinized, and after heat-induced epitope retrieval, immunohistochemistry for p16INK4a was performed with the primary antibody dilution of 1:7 as per manufacturer’s protocol . Cases with nuclear and cytoplasmic immunolabeling in at least 70% of the tumor cells were considered positive for p16. In patients with available tissue, HR HPV PCR was done to confirm results of the p16 immunohistochemistry.High risk (HR) HPV infection was determined by Tissue HR HPV PCR and p16 immunohistochemistry on tumor tissue of OC-SCC patients. Ang et al. has reported that the expression of p16INK4a by immunohistochemistry correlated well  with the presence of HPV DNA in tumors25. This technique has been reported as a potential screening method for HPV oropharynx cancer22 and also for the detection of persistent disease or recurrent disease following treatment in patients with HPV related oropharyngeal cancer25. We therefore used oral rinse specimens from patients with oral cavity cancer and patients with no head and neck cancer for the detection of HPV genotypes. The workflow for the collection and processing of samples and detection of HPV by DNA sequencing is shown in Fig. The detection of HPV in oral rinse samples and saliva samples using PCR has been reported as being a sensitive and specific method for the detection of HPV related oropharyngeal cancer×g for 20 min, supernatants were decanted and then the cell pellets were transferred into a 2-ml Eppendorf tube and stored at − 80 °C freezer for further study. All oral rinse specimens were taken prior to surgical resection of the OC-SCC. These samples were de-identified and coded. Using the MoBio method, we successfully extracted DNA from all 100 oral wash samples. DNA yield was measured by the Nanodrop method.The participants rinsed their mouth vigorously with 10 ml sterile saline for 30 s and then mouthwash collected in a 50 cc falcon canical flask container. After centrifugation at 3120The DNA fragmentation and shotgun metagenomic library construction and sequencing was carried out at the BGI Americas Corp  using Kapa kit and Illumina HISeq X Ten, with 100 samples pooled into 8 lanes.The Illumina sequencing process supplied raw sequence reads in fastq format and assigned a quality score called ‘Phred scores’ to describe the base accuracy. The raw sequence quality was reviewed using FASTQC software and any adapters and low-quality reads were removed using Trimmomatic. Low quality reads were defined as leading low quality or N bases, quality < 25; trailing low quality or N bases, quality < 25, scanning the read with a 4-base wide sliding window and cutting when the average quality per base drops below 25 and drops reads below 50 bases long. On average, each sample yielded 70,984,784 ± 7,145,632 raw reads, ranging 54,314,162 to 88,936,078. After trimming, 43,400,676 ± 5,586,559 reads left, ranging 32,127,260 to 58,483,416. The average percentage of clean to raw reads was 61.16%  . Those with ≥ 90% similarity were considered as human reads. On average, each sample yielded 36,184,844 ± 7,255,847 human reads, ranging 18,446,834 to 56,005,990. The sequencing depth was estimated by averaging the coverage across all 22 human autosomal chromosomes. There was no statistically significant difference between the cancer samples and control samples in the sequencing depth  . HPViewer detected HPV reads in all tumor samples [288–4229 HPV (type 6 or 11) reads per tumor sample] while in the six matched oral wash samples, only two were positive for the concordant HPV types with only 2 HPV reads/sample. Because the 2 HPV reads in the oral wash samples shared the same SNPs with the HPV reads in the corresponding tumor samples, it was concluded that the oral HPV reads represent the HPV released from the tumors in the larynx. In various oral cavity sites in healthy people, HPV prevalence ranged 2.9 to 7.1% and type 16 occasionally detected27. Thus, the low level HPV reads detected in the oral washes samples most likely represent HPV released from the tumors or from infection site in the oral cavity.We compared HPV prevalence and abundance between the 50 patients with OC-SCC and 50 subjects with no oral lesions. The nonhuman DNA reads were searched for HPV DNA using HPViewer  for the cases and 54.3 ng/µl (range 12.6–106.5) for the controls.Nonhuman reads averaged 7,215,832 ± 6,165,570, ranging 601,022 to 27,624,778. On average, nonhuman reads accounted for 10.06 ± 8.36% of the total reads, ranging 1.01% to 37.37%  yielded a considerable number of HPV reads suggesting a role in the etiology of OC-SCC in this patient. This was genotype 16. This patient was also positive on tissue HPV by PCR and positive p16 immunohistochemistry. The location of the tumor was the anterior 2/3rds of the oral tongue. The other 4 tumor cases yielded only about 1–2 HPV reads per human genome. These were in serotypes HPV 10, 16 (2 cases) and 98. All 4 patients were negative on p16 tissue immunohistochemistry and tissue HPV-PCR. Of the 4 control patients, all had only 1–2 HPV reads per human genome. These were in genotypes HPV 16, 22, 76, and 200.Using HPViewer, HPV was detected in five cases (10%) of OC-SCC and four controls (8%) Table . The raw3. There has been a steady decline in the use of cigarettes and alcohol in the population4. Despite this, the incidence of oral cancer has failed to decline. New studies show that there is an increasing number of patients who do not smoke or drink alcohol excessively but still develop oral cancer5. These patients tend to be younger with an increased frequency in females. The cause of oral cancer in these patients remains an enigma.Over the past 20 years the epidemiology of oral cancer has been changing. Traditionally, oral cancer has been caused by smoking and heavy alcohol consumption12.This change in epidemiology has occurred over the same time period as the change in epidemiology of oropharyngeal cancer. In oropharyngeal cancer it is now recognized that the agent causing cancer is high risk genotypes of the Human Papilloma virus, notably HPV 16, 18, and 3312. However, it is now generally accepted that the high risk genotypes HPV16, 18, and 33 are unlikely to be responsible for the change in epidemiology or oral cancer. There are 200 different genotypes of HPV. The traditional HPV detection kits/methods cover only a limited number of high/low risk (HLR) HPV genotypes32. Most of these traditional HPV detection methods are PCR-based and detect 14 genotypes . Since these detection kits do not cover all 200 different genotypes of HPV, it is possible that these detection methods may be failing to identify other genotypes of HPV which may be responsible for causing oral cancer.This has led several studies to be carried out on oral cancer patients to identify high risk HPV genotypes, either in saliva or in tissue tumor specimens. The results of these studies have been highly variable with some studies showing little association whereas others have reported a strong association38. In particular, a study that involved condyloma samples shown to be negative for HPV by traditional PCR revealed the ability of MSS to identify many putative HPV sequences33. Using MSS, we surveyed HPV distribution in various body sites of 103 healthy human subjects and found that the majority of the 109 HPV types detected could not be detected using the widely used commercial kits and do not belong to the HLR HPV types26. Interestingly, the HPV types detected have strong organ tropism and the oral HPV community is different from that of the vagina. These findings raised the possibility that the oral HPV types that are invisible to the traditional detection methods contribute to the etiology of human diseases, such as OC-SCC. The aim of our study was to carry out a case control study in 50 oral cancer patients and 50 matched control patients to detect all 200 genotypes of HPV by MSS.The limited ability of current commercial HPV detection kits can be overcome by metagenomic shotgun sequencing (MSS). This is a non-selective approach that, in theory, permits the identification of all HPV sequences. Recently, MSS has been used to detect HPV in some human samples and to identify several novel HPV types22 as well as the detection of persistent or recurrent cancer in patients who have completed treatment25. Our sequencing methodology using MSS was also sound with comparable reads in both tumor and control samples. Through our comprehensive detailed sequencing analysis we have shown that only 1 patient had a tumor sample (2%) that could be directly related to HPV. In this case, it was HPV16 which is the main genotype responsible for oropharyngeal cancer. Importantly, no other genotype of HPV was able to be detected at a high copy number indicating that these other genotypes are not responsible for oral cancer in these patients. These observations are highly relevant because they now provide evidence that HPV is rarely associated with oral cancer. There are 2 other studies which support our findings. A study by Bragelman using RNASeq to sequence the tumor mRNA in 7 patients with oral tongue cancer who did not smoke or drink alcohol failed to show any HPV sequences39. Another study by Li et al. examined the tumor transcriptomes of 20 patients with oral tongue cancer and failed to identify any HPV viral transcriptome40. A recent metanalysis by Sahovaler reported that in oral cavity locations, overall survival was not significantly associated with HPV positivity 41.In our study our cancer patients and control patients were well matched in terms of age, gender, and smoking and alcohol status. We used oral rinse samples from each patient and extracted DNA from cell pellets prior to sequencing. DNA extracted from oral rinse samples has been reported to be a sensitive and specific method for the identification of patients with oropharyngeal cancer40. Even more research is ongoing to identify bacteria which may be responsible. Studies on the oral microbiome have recently been published suggesting specific bacteria may be responsible43. Our own group recently identified that the periodontal pathogens Fusobacterium, Prevotella, Alloprevotella were enriched while commensal Streptococcus depleted in OC-SCC in nonsmoking patients with premalignant oral cavity lesions as well as oral cancer19. Clearly this is an area which requires much research effort to try to provide new insight into this devastating disease.There is much interest in identifying factors responsible for oral cancer in these patients. It is possible other viruses such as herpes simplex, herpes zoster, Epstein Barr virus may be responsible though research on these common viruses have not shown any association to dateIn conclusion, our study suggests no role in the aetiology of HPV in oral cavity cancer. However, our population of patients is fairly homogenous population (88% white ethnicity) and all from the USA. It is possible that there may be geographic or ethnic differences in the role of HPV across populations and we therefore cannot extrapolate from a single 50 patient study”. Further research in different geographic and ethnic populations is needed. New research is also needed to explore other infectious agents such as bacteria or viruses that may be responsible for oral cancer. Although we saw few HPV reads in our metagenomic data, it is important to analyse the metagenomic sequences for other non human reads from other viruses, bacteria or even fungi. It is possible these other microbes may have an association with OSCC pathogenesis. To carry out such a comprehensive analysis requires complex bioinformatics as well as validation studies. These studies are currently underway.Supplementary Figures.Supplementary Table.


Lesbian, gay, and bisexual (LGB) Veterans report stress  and mental health conditions  that may increase risk for neurocognitive changes like dementia. Subjective cognitive decline (SCD) can be an early indicator of neurocognitive change – yet no known studies have examined SCD in LGB Veterans. Cross-sectional data from the Vietnam Era Health Retrospective Observational Study (VE-HEROeS) were examined for 260 LGB and 17,796 heterosexual Veterans. VE-HEROeS is the latest probability-based survey of Vietnam Era Veterans (1961–1975) as older adults (2016-2017). SCD was assessed using two subscales of the Functional Assessment of Cancer Therapy-Cognitive Instrument Version-3 (FACT-Cog). Good reliability was observed in this sample: Cronbach’s alpha =.94 for the 7-item Perceived Cognitive Abilities subscale and .88 for the 4-item Comments from Others. Analyses were weighted to account for the complex survey design. LGB Veterans were slightly younger  than heterosexual Veterans ; were more likely to be female ; and had fewer people living in the household . LGB Veterans were also more likely than heterosexual Veterans to report feeling depressed most or all of the time over the past 30 days  on a single 5-point Likert-scale. SCD indicators did not vary by Veteran sexual orientation  and were elevated compared to published studies in healthy adult samples. More work is needed to examine neurocognitive risk factors in aging LGB Veterans.


INK4a, HPV-DNA, and HPV RNA biomarkers were used to assess the HPV status in head and neck cancer in a retrospective cohort of approximately 700 patients. In our study, HPV prevalence in oropharyngeal cancers was much higher than in oral and laryngeal cancers, and HPV positivity conferred better prognosis only in oropharyngeal cancers. Importantly, we have observed an increase of the prevalence of HPV positivity in oropharyngeal cancers in the most recent calendar periods, suggesting that this disease is increasing in Italy, as has happened before in other developed regions.This is the largest and most comprehensive assessment of the role of human papillomavirus (HPV) in head and neck cancer (HNC) in Italy, which is a region currently considered bearing a low burden of HPV-driven HNC. p16INK4a staining. Multivariate models were used to assess for factors associated with HPV positivity and proportional hazards for survival and recurrence. The percentage of HPV-driven cases (considering HPV-E6*I mRNA positivity) was 1.8, 2.2, and 40.4% for oral cavity (OC), laryngeal (LC), and oropharyngeal (OPC) cases, respectively. The estimates were similar for HPV-DNA/p16INK4a double positivity. Being a non-smoker or former smoker or diagnosed at more recent calendar periods were associated with HPV-E6*I mRNA positivity only in OPC. Being younger was associated with HPV-E6*I mRNA positivity in LC. HPV-driven OPC, but not HPV-driven OC and LC, showed better 5 year overall and disease-free survival. Our data show that HPV prevalence in OPC was much higher than in OC and LC and observed to increase in most recent years. Moreover, HPV positivity conferred better prognosis only in OPC. Novel insights on the role of HPV in HNC in Italy are provided, with possible implications in the clinical management of these patients.Literature on the role of human papillomavirus (HPV) in head and neck cancer (HNC) in Italy is limited, especially for non-oropharyngeal tumours. Within the context of the HPV-AHEAD study, we aimed to assess the prognostic value of different tests or test algorithms judging HPV carcinogenicity in HNC and factors related to HPV positivity at the European Institute of Oncology. We conducted a retrospective cohort study (2000–2010) on a total of 696 primary HNC patients. Formalin-fixed, paraffin-embedded cancer tissues were studied. All HPV-DNA-positive and a random sample of HPV-DNA-negative cases were subjected to HPV-E6*I mRNA detection and p16 Head and neck carcinoma (HNC) is a heterogeneous group of tumours located at the nasopharynx, oropharynx, hypopharynx, larynx, and oral cavity. Over 90% of HNC are squamous cell carcinomas (HNSCC) and are caused mainly by environmental factors such as smoking, alcohol consumption, human papillomavirus (HPV), and Epstein Barr (EBV) infections. According to the Surveillance, Epidemiology, and End Results (SEER), approximately 4% of all worldwide cancers are in the head and neck region, with more than 430,000 cases per year .Globocan future estimations of HNC in Italy are projected to increase: specifically, a boost of more than 10% of new cases is estimated in all head and neck (HN) areas for both sexes, at all ages, per 100,000 people , within The HPV distribution in HNSCC largely differs by anatomical site: while the prevalence of HPV-driven OPC ranges between 30–40%, much lower are the estimated rates for the other areas, specifically 2.1–4.4% for OC and 2.7–3.5% for LC and HPC ,7,8,9.Italian data on HPV status in HNSCC were reported from three other studies with HPV-DNA/HPV-E6*I mRNA double positivity with variable ranges such as 37.9% in OPC 1992–2015) , and 32. , and 32INK4a staining) has been considered for the stage classification of the tumour [INK4a immunohistochemistry (IHC) [INK4a high expression detected by IHC is the most widely implemented technique in clinical settings for HPV-driven OPC diagnosis [INK4a-positive OPCs are HPV-DNA-negative with no prognostic advantage with respect to HPV-DNA/p16INK4a double-negative tumours, as they might not be related to HPV [INK4a IHC is starting to be recommended to diagnose HPV-related OPCs [INK4a IHC is not recommended for the diagnosis of HPV association; however, there is limited information about the accuracy and prognostic value of dual HPV-DNA and p16INK4a testing in non-oropharyngeal HNSCC.The distinction between HPV-driven and non-HPV-driven OPC is underscored also in the 8th edition of the TNM, where the HPV status  . The ideiagnosis . Howeverd to HPV . The comted OPCs . OutsideMoreover, country-specific estimates of HPV-attributable fractions in OPC and non-oropharyngeal HNC are warranted in order to evaluate the possible protective effect of HPV vaccination.INK4a double positivity.In this study, we assessed the prognostic value of HPV positivity (as defined by HPV-E6*I mRNA positivity) in a sample of OPC and non-OPC Italian patients and compared the results with those from HPV-DNA positivity and HPV-DNA/p16We also studied factors related to HPV positivity (as defined by different HPV-relatedness definitions) and the overall proportion and type distribution of HPV-positive at different anatomical sites, as well as the trend of the proportion of HPV-positive HNSCC in more recent years, in Italy. Finally, we highlight the differences between HPV-positive versus negative cancers at three different anatomical regions in terms of prognosis and survival in an Italian setting.INK4a double positivity was 2.4, 1.8, and 43.9% for OC, LC, and OPC cases, respectively. All OPC HPV-DNA-negative cases tested for p16INK4a were also p16INK4a-negative, but three (15.0%) OC and one (3.8%) LC HPV-DNA-negative cases were p16INK4a-positive.A total of 1594 cases consecutively diagnosed with a primary HNSCC at European Institute of Oncology, IRCCS (IEO) in 2000–2010 were identified, of which 835 (52%) had unavailable formalin-fixed, paraffin-embedded (FFPE) tissue blocks at diagnosis. Some differences were observed between cases with and without available FFPE tissue blocks: specimens from younger patients, cases diagnosed with stage IV a-b (7th TNM edition) or located at sites distal to the oropharynx were over-represented among OC cases with available FFPE tissue blocks. Cases diagnosed with stage III (7th TNM edition) were over-represented among OPC cases with available FFPE tissue blocks, whereas OPC cases located at the base of the tongue were under-represented. Smokers and drinkers, as well as cases diagnosed at earlier periods and located at anatomical subsites proximal to the oropharynx, were over-represented among LC cases with available FFPE tissue blocks. A total of 696 primary cases had a valid HPV result and 675 were finally included in the analyses: 165 OC, 109 OPC, and 401 LC cases . The percentage of HPV-driven cases (considering HPV-E6*I mRNA positivity) was 1.8, 2.2, and 40.4% for OC, LC, and OPC cases, respectively . The perINK4a, the prevalence of HPV16 increased in LC and OPC but not in OC. When only considering HPV-E6*I mRNA-positive cases, the prevalence of HPV16 increased in OC and OPC, but not in LC. Differences in HPV type distribution by HPV relatedness definitions were statistically significant. INK4a double positivity and HPV-E6*I mRNA positivity after adjusting for significant covariates such as tobacco use. Being younger was associated with HPV-DNA/p16INK4a double positivity and HPV-E6*I mRNA positivity in LC. Being a non-smoker was associated with HPV-E6*I mRNA positivity in LC. None of these associations neither others were observed for OC after adjusting for significant covariates, with the exception of an anatomical location proximal to the oropharynx for HPV-DNA positivity. This association was not observed for HPV-DNA/p16INK4a double positivity and HPV- E6*I mRNA positivity.HPV16 was the most common type among HPV-DNA-positive cases for all HN sites, although with lower proportions in LC (59.3%) than in OC (90.0%) and OPC (96.4%) . The nexp < 0.001), as compared to HPV-non-driven OPC (p = 0.004), as compared to HPV-non-driven OPC   [A previous study of 248 HNC cases diagnosed in 2003–2012 in Northern Italy found HPV prevalence of 1.6% in OC, 20% in OPC, and 1% in LC, when considering HPV-DNA/HPV-E6*I RNA double positivity . Our est = 0.06) . We alsoA cancer registry-based Italian study assessing the incidence and survival patterns of HNC diagnosed in Italy between 1988 and 2012  found inOur study was conducted in the context of the international HPV-AHEAD study ,32,33, wHPV16 was the most common type among HPV-positive cases across all HN sites and HPV-relatedness definitions. However, its predominance was far higher in OC and OPC than in LC. Moreover, when considering only cases were HPV was the truly triggering carcinogenic agent  the percentage of HPV16-positive cases increased for OC and OPC but decreased for LC. These results, if confirmed in other larger studies, may have implications on the estimation of HPV vaccination effects.Being a non-smoker or former smoker was associated with HPV positivity in OPC, consistently with what is reported in the literature regardless of geographical region ,34,35. HRegarding non-oropharyngeal HNSCC cases, being younger and a non-smoker were associated with HPV positivity in LC. As compared to OPC, fewer studies have analysed the factors associated with HPV positivity in non-oropharyngeal cancer sites. The ICO international study also observed that HPV-positive LC patients, as well as OC ones, showed younger ages at diagnosis than HPV-negative ones . HoweverINK4a). HPV-positive LC and OC cases did not show better OS and PFS than HPV-negative cases. Noteworthy, HPV-DNA/p16INK4a-positive and HPV-E6*I mRNA double-positive OC cases showed a marginally non-significant better PFS than the rest of cases. The prognostic value of HPV in non-oropharyngeal HNC is still unclear. While some studies have observed a better outcome for HPV-positive HNC, others have not [As expected, HPV-positive OPC patients showed better OS and PFS than HPV-negative OPC cases regardless of the HPV-relatedness definition herein considered. However, as it has been already observed ,36 the phave not . The moshave not . In thisINK4a, in accordance with the protocol established within the HPV-AHEAD consortium. For an important number of primary HNC cases consecutively diagnosed at IEO during the study period and targeted to be included in the study, no FFPE tissue blocks were available. Moreover, some differences were observed between cases with and without tissue sample, as it has been noted in other studies [INK4a positivity was established before the publication of the guidelines for HPV testing in HNC by the College of American Pathologists [INK4a-positive cases, which are less than 70%. A total of 15 out of 68 cases classified as p16INK4a-positive did not reach the 70% nuclear and cytoplasmic staining threshold.We acknowledge that our study has several limitations. The retrospective nature of the cohort may have limited the accuracy of data related to some risk factors such as tobacco/alcohol use. Only 11% of HPV-DNA-negative cases were further tested for HPV-E6*I mRNA and p16 studies . We had ologists , where aA retrospective cohort of patients consecutively diagnosed with a primary HNC at the IEO in Milan from 2000 to 2010 was conducted within the HPV-AHEAD project ,32,33. HInformation on demographics, smoking and alcohol consumption, and clinical and follow-up data up to 2019 was extracted from electronic medical records. The definition of a drinker was consumption of three or more drinks per week. We only considered FFPE tumour samples from the diagnosis previous to treatment.Ethical clearance was obtained from IEO Ethical Committee (code IEO N101), Milan, Italy as well as IARC, Lyon, France. The study did not involve any contact with the patients. All clinical and pathological data were collected using well-designed case report forms (CRF) according to good clinical practice guidelines.INK4a staining at Roche mtm laboratories.Adequate measures to ensure data protection, confidentiality, patients’ privacy, and anonymization were taken into account. FFPE tissue blocks were used to perform several laboratory assays, as described below. Each assay was performed in a single laboratory: (i) HPV-DNA assay at IARC, (ii) HPV RNA assay at DKFZ, and (iii) p16Cancer samples having tested negative for both HPV-DNA and beta-globin DNA were excluded from the analyses.FFPE tissues were in part processed at the International Agency for Research on Cancer, Lyon, France and at IEO, Milan, Italy, following the HPV-AHEAD sectioning protocol .All sections were evaluated by the HPV-AHEAD histopathology review panel six pathologists . An online pathology form was used. Each pathologist evaluated tissues from approximately 80 patients. All sections were re-evaluated by a second panel of pathologists. Only FFPE tissue blocks for which the first and last haematoxylin/eosin stained sections reflected tumour tissue were included in the study.DNA was purified from three consecutive FFPE sections (S6–8), as previously described . HPV-DNAINK4a staining.All HPV-DNA-positive FFPE specimens and a random subgroup of approximately 11% of HPV-DNA-negative cases were subjected to HPV-E6*I mRNA detection and p16An ultra-short amplicon, E6*I mRNA RT-PCR assay was chosen for HPV-mRNA detection for its applicability to FFPE material and absolute RNA specificity by using a splice-site as identification target . Total RINK4a within the cancer area of the tissue sections was considered as positive, while a focal staining or no staining was considered negative. IHC slides were analysed without knowledge of any other clinical information (including HPV-DNA and RNA status) by R.R. and F.M. Discordant cases were analysed by O.C. and consensus was reached.Expression of p16 was evaluated manually by IHC in FFPE sections using the CINtec p16 Histology Kit  according to the manufacturer’s instructions and as previously described . A contip < 0.05) and the Akaike information criterion were applied to exclude non-significant factors. Histological variables were not considered in multivariate analyses as they were considered to be intermediate variables in the carcinogenic process, as previously described [Differences in the covariate distribution  between cases with and without available FFPE block were assessed by Pearson’s chi2 test. We used HPV-E6*I mRNA positivity as the reference test for viral carcinogenic activity. We assumed that HPV-DNA-negative cases not tested for HPV-E6*I mRNA were HPV-E6*I mRNA-negative and HPV-DNA/HPV-E6*I mRNA double-positive cases were considered as HPV-E6*I mRNA-positive cases. HPV-prevalence and HPV-type distribution were assessed. Unconditional logistic regression analyses were performed to identify independent factors  associated with HPV etiological involvement in each HNSCC site according to the three different HPV-relatedness definitions using backward selection of significant covariates. The likelihood ratio test (LR escribed . Crude aOur findings from a large cohort of unselected HNSCC patients provide a comprehensive picture on the role of HPV in OPC and non-oropharyngeal cancer in a setting of Southern Europe, which is a region currently considered to bear a low burden of HPV-driven HNC. We have observed an increase of the prevalence of HPV positivity in OPC with most recent calendar periods. These results, together from previous findings of increasing incidence of OPC in Italy , suggest