The EAT-Lancet universal healthy reference diet recommends an increase in the consumption of healthy foods, among which treenuts and groundnuts. Both are, however, water-intensive products, with a large water footprint (WF) per unit of mass and protein and already today contribute to blue water stress in different parts of the world. The envisaged massive required increase in nut production to feed a global population with this reference diet, needs to occur in a water-sustainable way. In this paper, we identify and quantify where current nut production contributes to local blue water stress and discuss options for water-sustainable nut production. We show that 74% of irrigated nuts are produced under blue water stress (of which 63% under severe water stress), throughout many regions of the world, most notably in India, China, Pakistan, the Middle East, the Mediterranean region and the USA. We critically evaluate which nut types to promote given substantial differences in WFs. We propose sustainable intensification of nut production employing nut-specific WF benchmarks. We also recommend integrated water resources management including maximum sustainable levels of water consumption by setting of WF caps. •The EAT-Lancet reference diet recommends increased nut consumption.•Treenuts and groundnuts are water-intensive products.•74% of irrigated nuts are produced under blue water stress.•63% of irrigated nuts are produced under severe water stress.•Increase in nut production needs to occur in a water-sustainable way. Blue waThe food system is a major cause of both blue water stress (WS) and gree2First, we analyse the quantity of treenut and groundnut production for the current situation as well as the projected EAT-Lancet universal healthy reference diet scenario for the year 2050, based on Third, we use the spatially distributed blue WS data of Blue WS in Following blue WS thresholds are used: values until 1 (low blue WS), 1–1.5 (moderate blue WS), 1.5–2 (significant blue WS) and more than 2 (severe blue WS). In the following discussion we only discuss blue WS as moderate, significant and severe blue WS. We do not include or discuss low blue WS. We assess groundnuts, cashew nuts, chestnuts, almonds, walnuts, pistachios and hazelnuts.33.1Global treenut (shelled) production increased from 4.6 million tons in 2000 to 9.3 million tons in 2017, while global groundnut (shelled) production increased from 24.4 million tons to 33.0 million tons over the same period Fig. 1 a. In 20133.23/y , and the global average WF of groundnut production at 89,256 million m3/y . Groundnuts have the largest blue WF (5107 million m3/y), followed by pistachios (3507 million m3/y) and almonds (3013 million m3/y).Over the period 1996–2005, the global average WF of treenut production has been estimated at 68,267 million m/y green)(SI Table/y green). Among tLarge differences in total, blue and green average unit WFs exist between different nut types, both in terms of litre/kg and litre per gram of protein . Cashew In terms of litre per gram of protein , cashew In terms of litre per kg, almonds, pistachios, hazelnuts and walnuts have unit WFs in the range of red meat from ruminants, whereas groundnuts and chestnuts are in the range of white meat and eggs. In terms of litre per g protein, chestnuts and pistachios have unit WFs in the range of red meat from ruminants, whereas almonds, walnuts and hazelnuts are in the range of other red meat (pigs and sheep) and groundnuts lower than eggs.3.3A significant proportion of irrigated groundnuts is produced under blue WS . Of a toIrrigated production of cashew nuts and chestnuts amounts to each 0.2 million tons (with shell). For casIn total 0.9 million tons of 1.0 million tons irrigated almonds is produced under blue WS, of which 0.8 under severe blue WS . The larIn total 0.59 million tons of irrigated walnut production (0.67 million tons) occurs under blue WS, of which 0.46 under severe blue WS . HotspotPistachios are primarily produced under blue WS in Iran, the USA  and Turkey . Of a toA large proportion of 0.18 million tons of 0.28 million tons irrigated hazelnuts is produced under blue WS, of which 0.13 under severe blue WS . HotspotThese seven nut types account for in total 11.4 million tons of irrigated nuts, of which 8.4 million tons (74%) are produced under blue WS , and 7.3 million tons (63%) under severe blue WS.3.4In the line with these solutions, the most important options for water-sustainable nut production are displayed and discussed in Regarding blue WF unit amounts, especially pistachios are very water demanding both per unit of mass and protein. Groundnuts have generally smaller total and blue WFs both per kg and per gram of protein than tree nuts, but certain authors advocate to produce more perennial crops (treenuts) instead of annual crops (groundnuts) as they promise more sustainable agroecosystems .4Current global nut production contributes to and is affected by different levels of blue water stress, in many regions of the world. The results show for 7 different nut types combined, that 74% of all irrigated nuts are produced under blue water stress  and 63% under severe blue water stress. This was not considered in the recent study of Latter study recommends for a substantial increase in global nut consumption, which requires a substantial increase in global nut production. To achieve such an increased production in a water-sustainable way, we discuss a list of essential options that need to be accounted for. Our study highlights that nuts are water-intensive agricultural commodities that require special attention in order to reach SDG 2 (food security) in harmony with SDG 6 .The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The potential of first-void (FV) urine as a non-invasive method to monitor human papillomavirus (HPV) vaccination has been reported, mainly focusing on urine as a sample to assess HPV DNA. Besides HPV DNA, vaccine-induced HPV antibodies originating from cervicovaginal secretions were recently shown to be detectable in FV urine as well. This presents a novel opportunity for non-invasive sampling to monitor HPV antibody status in women participating in large epidemiological studies and HPV vaccine trials. The simultaneous assessment of both HPV infection and immunogenicity on a non-invasive, readily obtained sample is particularly attractive. The evaluation of immunogenicity in HPV vaccine trials relies largely on serology. In the absence of a correlate of protection, it is generally accepted that the presence of high concentrations of vaccine-induced antibodies in serum—greater than those elicited by natural infection—are the best indicators of long-term protection against HPV infection . NeverthThe presence of HPV-related biomarkers  in the urine of women is based on the fact that discharged mucus and debris from exfoliated cells from the female genital organs (including the cervix) accumulate around the urethra opening, between the small labia, and are washed away with the urine flow. Consequently, the initial flow of urine—defined as first-void urine—contains significantly more human and HPV DNA than random or mid-stream urine –16. CurrStandardized and optimized protocols  have significantly enhanced the sensitivity of urinary HPV detection and demonstrated good concordance with cervical samples , 20, 21.Using urine has several advantages over other more invasive sampling methods. If successful, non-invasive urine sampling could partly replace serum for follow-up of HPV-vaccination and could potentially enhance participation in vaccine trials. Moreover, as urine sampling is non-invasive and does not require trained medical personnel for collection, urine could be of great value for multiple collections at different time points (at home). From the logistical point of view, the possibility to measure both virologic (HPV DNA) and immunological (HPV antibody) end-points in FV urine could ease future surveys and provide major logistical and financial benefits. For example, expansion of HPV vaccination to lower-income countries, particularly with one-or two-dose schedules, may be facilitated by the use of such a tool to assess (early) impact . It is cBesides clear benefits, urine also has drawbacks. Because of inter-individual and intra-individual variability, standardization and normalization is essential. The major variations in urinary antibody concentration are determined by the amount of genital secretions washed away by the initial stream of urine, the volume of urine collected (dilution), and the amount of mucus. We are currently investigating the potential of total IgG concentration, total protein concentration, and other potential normalization markers to develop a normalization algorithm. Besides, more research is necessary to investigate the most appropriate buffer to preserve antibodies in first-void and to determine the maximum time between sample collection and detection.Another challenge using urine consists of generating quantifiable levels of antibodies. Both the amount of antibodies at these mucosal surfaces as well as their presence in urine might be a variable. At the moment we are improving IgG concentration and isolation by pre-treating our urine samples. HPV antibody levels in FV urine were indeed around the detection limit for both the multiplex L1/L2 virus-like particles (VLP)-based ELISA (M4ELISA) and glutathione S-transferase (GST)-L1-based immunoassay (GST-L1-MIA) immunoassay, which complicates the distinction between uronegative and low positive results , 12. As Our current research is focused on HPV, however, the idea and technology could be applied to any infection or disease for which monitoring (vaccine-induced) antibody responses is imperative for disease control. Hence, the use of HPV vaccine-induced antibodies could be a model for wider use of FV urine in immunoassays. It is notable, however, that this simplified sample collection is only applicable in females. Although females are a large part of the population, for most of the vaccination trials, both males and females would need to be surveyed. Hence, further research is necessary to develop accurate and feasible non-invasive sampling that can be performed by men. Oral fluid may be such possible non-invasive alternative to serum for HPV antibody surveillance in men and women –26.It is clear that the successful development of a technology that measures antibodies in female urine as a way to determine HPV vaccination status, besides HPV DNA status, is very promising. Since FV urine collection is less invasive than serum collection, and does not require trained personnel, the validation of such procedure would be of great value for the international HPV research community. In different settings, FV urine could allow vaccine effectiveness and coverage data to be reliably generated post-implementation.The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author/s.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.PV and AV are co-founders and former board members of Novosanis (Belgium), a spin-off company of the University of Antwerp that produces a device for collection and preservation of first void urine. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Combined with cancer screening programs, vaccination against human papillomavirus (HPV) can significantly reduce the high health and economic burden of HPV-related disease in Japan. The objective of this study was to assess the health impact and cost effectiveness of routine and catch-up vaccination of girls and women aged 11–26 years with a 4-valent (4vHPV) or 9-valent HPV (9vHPV) vaccine in Japan compared with no vaccination.We used a mathematical model adapted to the population and healthcare settings in Japan. We compared no vaccination and routine vaccination of 12–16-year old girls with 1) 4vHPV vaccine, 2) 9vHPV vaccine, and 3) 9vHPV vaccine in addition to a temporary catch-up vaccination of 17–26 years old girls and women with 9vHPV. We estimated the expected number of disease cases and deaths, discounted (at 2% per year) future costs (in 2020 ¥) and discounted quality-adjusted life years (QALY), and incremental cost effectiveness ratios (ICER) of each strategy over a time horizon of 100 years. To test the robustness of the conclusions, we conducted scenario and sensitivity analyses.Over 100 years, compared with no vaccination, 9vHPV vaccination was projected to reduce the incidence of 9vHPV-related cervical cancer by 86% . A greater number of cervical cancer cases  and cancer-related deaths  were avoided through the described catch-up vaccination program. Routine HPV vaccination with 4vHPV or 9vHPV vaccine prevented 5,521,000 cases of anogenital warts among women and men. Around 23,520 and 21,400 diagnosed non-cervical cancers are prevented by catch-up vaccination among women and men, respectively. Compared with no vaccination, the ICER of 4vHPV vaccination was ¥975,364/QALY. Compared to 4vHPV, 9vHPV + Catch-up had an ICER of ¥1,534,493/QALY.A vaccination program with a 9-valent vaccine targeting 12 to 16 year-old girls together with a temporary catchup program will avert significant numbers of cases of HPV-related diseases among both men and women. Furthermore, such a program was the most cost effective among the vaccination strategies we considered, with an ICER well below a threshold of ¥5000,000/QALY. A substantial burden of disease results from persistent human papillomavirus (HPV) infections. For example, there were approximately 11,000 new cases of HPV-caused cervical cancer (CC) and 2800 deaths in Japan in 2017 . For exaThere are two types of interventions to prevent HPV-related diseases: secondary prevention through cancer screening  or primary prevention through HPV vaccination. Cervical screening of women aged over 30 years once a year has been practiced in Japan since 1982. The program was modified in 2004 to allow women of age 20 years old and over to receive cervical screening test every other year . HoweverA bivalent human papillomavirus (HPV) vaccine (HPV types 16 and 18) was licensed in Japan in 2009. In 2011, a quadrivalent (4vHPV) vaccine  was approved and indicated for use in girls and women for the prevention of cervical cancer and precursor lesions, vulvar and vaginal intraepithelial neoplasia, and genital warts. However, a 9-valent (9vHPV) HPV vaccine , introduced in many countries since 2014, was recently approved in Japan [Routine vaccination of girls aged 12–16 years against HPV was included in the national immunization program (NIP) in April of 2013. However, following unconfirmed reports of adverse events in the media, the Japanese Ministry of Health, Labour, and Welfare (MHLW) suspended proactive recommendations for routine use of the HPV vaccine in NIP in June 2013 , 11. NotThe Japanese government recently approved the use of the 9vHPV vaccine . PreviouThe cost effectiveness of the 9vHPV vaccine has been evaluated in many countries, particularly those with established 4vHPV vaccine programs . In the In this study, we evaluated population-level health impact and cost effectiveness of routine and catch-up vaccination of girls and women aged 11–26 years with a 4-valent or 9-valent HPV vaccine in Japan compared with no vaccination.We updated and adapted a previously published mathematical platform of HPV disease transmission to the Japanese setting , 25. TheThe demographic model divides the two-sex, heterosexually mixing population into 23 age groups . Same-sex partnerships are not modeled here. The behavioral model defines risk of HPV transmission according to sex, age, sexual activity , and rates of new sex partners per year, all based on sexual activity data from Japan [The epidemiological models simulate the transmission of the nine vaccine-related HPV types  and development of disease using the following 7 groups: cervical, vaginal, vulvar pre-cancers and cancers in women; penile pre-cancer and cancer in men; anal pre-cancer and cancer and head/neck cancer in both men and women; and low-risk cervical intraepithelial neoplasia (CIN) in women, and genital warts (GW) and recurrent respiratory papillomatosis (RRP) in men and women. For simplicity, we developed separate and independent models for each HPV-type/disease group combination. For example, there is a separate model HPV 16 CIN and cervical cancer and a model for HPV 18 anal intraepithelial neoplasia (AIN) and anal cancer. By using this structure, we are assuming that the spread of the various virus types are independent, and that infection by one strain neither increases or decreases the chances of being infected with another strain. In total, the current analysis utilized 44 (7 HPV types × 6 cancers + 2 HPV types X 1 GW/CIN1/RRP) models.Solutions of these epidemiological models for each vaccination strategy consist of numerical functions that describes the number of individuals in each compartment over time. The solved functions are then aggregated into familiar epidemiological variables such as new cases and number of individuals living with cancer over time.The continuous-time economic model uses output from the epidemiological models such as new cases to calculate various types of costs for each vaccination strategy. For example, new cases of cervical cancer at each time are multiplied by lifetime discounted cost per case to obtain healthcare costs of cervical cancer. Similarly, multiplication of number of doses by cost per dose gives total vaccination cost for a given strategy. Numerical functions representing the number of individuals in each compartment, whether living with disease or not, are multiplied by corresponding health utilities and integrated over time to obtain quality-adjusted life years (QALYs). Cost effectiveness of a vaccination strategy relative to a comparator is evaluated using the incremental cost-effectiveness ratio (ICER) obtained by dividing incremental total discounted costs by the incremental total discounted number of QALYs.Cervical cancer screening only and no vaccination (Screen)Cervical cancer screening and routine vaccination of 12–16-year old girls with 4vHPV vaccine (Routine GOV-4vHPV) from 2021 onwardsCervical cancer screening and routine vaccination of 12–16-year old girls with 9vHPV vaccine (Routine GOV-9vHPV) from 2021 onwardsCervical cancer screening and routine vaccination of 12–16-year old girls from 2021 onwards and temporary catch-up vaccination of 17–26 years old girls and women with 9vHPV until 2025 (Routine + catch-up GOV-9vHPV)We considered and assumed no future changes in disease management practices and evaluated the following primary prevention (through HPV vaccination) and secondary prevention  strategies:Due to the recent approval of 9vHPV and given the additional benefit of the 9vHPV vaccine, we do not expect that the routine vaccination plus catchup with 4vHPV to be seriously considered as a viable strategy. We have included routine vaccination with 4vHPV, since it represents the state of the program when the government rescinded its active recommendation.All model input parameters were either calibrated or derived from published sources as described in the supplemental material. Table Sex- and age-specific population size and annual all-cause mortality rates in 2017 were extracted from Japanese Government Statistics  .The base case assumed that up to 70% of all 12-year-old Japanese girls would complete the 3-dose series [Values of health utilities associated with the various health states in the model were derived from published studies  and adjusted to the Japanese population norm using the mean EQ-5D-3L quality of life score .Costs were based on 2020 prices and are evaluated from a public healthcare payer’s perspective. The base-case vaccine cost acquisition cost per dose of 4vHPV was ¥12,000. The vaccine cost acquisition cost per dose of 9vHPV was assumed to be ¥20,000 .Adherence to prescribed number of doses (80 and 100%).Duration of protection of the vaccine .Healthy utility weights .Disease utility weights .Disease costs (±20%).Varying the price of 9vHPV only .Discount Rate Finally, we performed scenario and one-way sensitivity analyses to test sensitivity of outcomes to uncertainty in epidemiology, vaccination coverage, health utilities, discount rates, and cost inputs . We variOver 100 years, the incidence of cervical cancer is expected to drop dramatically, particularly in scenarios where 9vHPV vaccine is used. The estimated public health impact of the various vaccination strategies on other HPV-related diseases is also profound.Given the relatively high incidence of cervical cancer in Japan, HPV vaccination can prevent a large number of cervical cancer cases. Over the 100-year time horizon, there was large drops in the incidence of cancer attributable to the 9 types covered by 9vHPV vaccine, and the overall incidence of cervical cancer  at the top to most effective (GOV-9vHPV + catch-up vaccination) at the bottom, costs, QALYs, and the ICER for each vaccination strategy are shown in Table When using only the diseases as indicated in Japan, the cost-effectiveness results still held, but the ICERs did increase somewhat Table . No vaccThe above results were very robust in a deterministic sensitivity analysis. We varied scenario-related parameters to quantify this robustness. Detailed outcomes of the sensitivity analyses can be found in the technical appendix, and are summarized here, and in Table Results of other scenario or sensitivity analysis are summarized in Table This analysis showed that vaccination against HPV will have a significant public health impact in Japan, resulting substantial reductions in the cases of 9-valent HPV-caused cervical and non-cervical cancers and anogenital warts. At the expected cost of vaccination, HPV vaccination is highly cost effective at a cost-effectiveness threshold of 5000,000 ¥/QALY. We note that there is not a consensus on the local threshold value for Japan, but the threshold we use here is generally used in cost-effectiveness analyses . We noteRoutine vaccination with a 9-valent vaccine with a catch up to age 26 among girls was found to be the most cost-effective strategy, averting many cancers and deaths and improving the quality of life of both men and women in Japan. Even when considering only the diseases for which the HPV vaccine is indicated in Japan, vaccination was still found to be highly cost effective. We also found that strategies that did not include a catch-up program had higher ICERs than more effective strategies (weakly dominated). These results were robust in sensitivity analyses. We can thus conclude that the 9vHPV vaccine with catch up is the most cost-effective strategy, whether we consider all diseases, or only the diseases as indicated for 9vHPV vaccine in Japan.Furthermore, these results are consistent with other work that has been published. Konno et al.  assessedSimilarly, Yamamoto  used a MYamabe et al.  also assSimms et al.  quantifiAs with any modeling study, the results are subject to some limitations. First, we assumed that the system had reached an equilibrium state, and as a result cancer incidence is at a constant state before vaccination. However, there is some evidence to suggest that cervical cancer incidence is increasing in Japan . IncreasWe have also assumed that there is no coverage of the vaccine before 2020, while in actuality some girls were vaccinated in 2013 . HoweverDespite these limitations, our analyses were based on a model that has been extensively validated against existing epidemiological data. The analysis relied heavily on Japanese-specific inputs utilizing data from various sources and published literature. It is reassuring that the model reproduced all baseline incidence of diseases .


The quadrivalent human papillomavirus (HPV) vaccine has been assumed to give protection against genital warts (GW) as well as cervical cancer. Our main question was whether HPV vaccine has any effects on the prevention of GW reported in randomised controlled clinical trials (RCTs) and time-trend analyses.This meta-analysis was performed according to the PRISMA guidelines using the PICO format. We searched in three electronic databases , and assessed heterogeneity using the Q-test and I-squared statistics, meta-regression was also performed. Odds ratios (OR) and their confidence intervals (CI) were calculated. The sensitivity was tested by leave-one-out method. We evaluated the presence of publication bias using the funnel plot graph and the Copas selection model. The strength of evidence was assessed using the GRADE approach.Eight RCTs (per-protocol populations) and eight time-trend ecological studies were included in this meta-analysis. A significant reduction  of GW in young women was recorded in RCTs, and in time-trend analyses both in young women , and in young men . In subgroup analysis, a significant reduction of the number of GW events was observed especially in women under 21 years . Leave-one-out analysis showed that similar results could be obtained after excluding one study, meta-regression did not show significant difference.Prophylactic, quadrivalent HPV vaccination can prevent GW in healthy women and men, therefore, it should be included in routine immunization programme. Human papillomaviruses (HPV) are sexually transmitted infectious agents and are increasingly common in young people. Three HPV vaccines are currently available worldwide which cover 2, 4, or 9 HPV genotypes . The bivThe World Health Organization (WHO) recognizes the importance of cervical cancer and other HPV-related diseases as global public health problems and reiterates the recommendation that HPV vaccines should be included in national immunization programmes . WHO recAs the quadrivalent vaccine became available in 2006, HPV vaccination has been introduced in an increasing number of high-income countries, while in low- and middle-income countries the bivalent, or both vaccines are funded. There is high certainty evidence that HPV vaccines protect against cervical precancer  in adolescent girls and young women who are vaccinated between aged 15 to 26 , and groAlthough GW are usually not referred to be a serious condition, the loss of quality of life of patients, physical symptoms such as pruritus and pain, and the impact on sexuality can be addressed as significant issues .In the present work our aim was to summarize the available evidence on the efficacy of the quadrivalent HPV vaccine in preventing GW by conducting a meta-analysis. To achieve this, the following PICO  format was applied: P: human population; I: quadrivalent HPV vaccine; C: no vaccine/placebo and O: reduced incidence/prevalence of GW.The present meta-analysis was performed using the Preferred Reporting Items for Systematic Review and Meta-Analyses protocols (PRISMA protocol) . This meWe conducted a systematic search in three electronic databases from inception up to 13th of January, 2020, without language restrictions. The following search queries were used:  OR “condylomata acuminata”[All Fields] OR “condyloma”[All Fields]) OR “condyloma acuminatum”[All Fields]) AND ) AND “humans”[MeSH Terms] for PubMed;  AND (“hpv vaccine” OR “hpv vaccination” OR “human papillomavirus vaccine” OR “human papillomavirus vaccination” OR “gardasil”) AND [humans]/lim AND ([article]/lim OR [article in press]/lim OR [conference abstract]/lim OR [conference paper]/lim OR [conference review]/lim OR [review]/lim) for Embase and  AND (“HPV vaccine” OR “HPV vaccination” OR “human papillomavirus vaccine” OR “human papillomavirus vaccination” OR Gardasil) for Cochrane Trials. We identified relevant studies by reviewing titles and abstracts, and the reference lists of relevant articles were also searched to reveal all relevant studies.We included RCTs and time-trend ecological studies which assessed the effectiveness of the quadrivalent HPV vaccine in the frequency  of GW. In the RCTs, HPV vaccines were administered only to females, except one RCT, where Japanese males were received HPV vaccine or placebo . SimilarWe excluded studies with the following characteristics: just the bivalent vaccine was used; or bivalent and quadrivalent vaccines were used alternatively; or the quadrivalent HPV vaccination was administered in the framework of private care; or the frequency of GW was studied during the post-vaccination period and the comparison of pre- and post-vaccination period was not included. The present analysis included only articles where the absolute number of GW is available. Narrative reviews, mathematical modelling studies, conference abstracts, and conference papers were also excluded.The EndNote X7.4 software package was used for record management. After removing duplicates, the remaining records were screened for eligibility by two authors  based on the title and abstract of the published original papers. The eligibility of full texts of the remaining records was assessed by two reviewers independently. Disagreement between reviewers was resolved by discussion or, if it was necessary, by consulting with a third reviewer (ZSM).The quality of the studies was assessed independently by two authors . We carried out a risk of bias assessment of RCTs using tools proposed by Cochrane for RCTs . This toData extraction was conducted independently by two investigators . In case of disagreement concerning the inclusion of studies and extracted data, a third investigator (ZSM) was involved to resolve discrepancies.In RCTs, the data for primary analysis were collected from a per-protocol susceptible population of women, who received all three doses of vaccination and who had no virological evidence of the relevant HPV types through 7 months after administration of the third dose. In RCTs, we collected information about the number of GW in the vaccinated and placebo groups. Afterwards the number of countries included in the given RCT, the length of the follow-up period, the age of the participants and the lifetime number of their sexual partners were also extracted.In the included ecological trials our primary outcome was the number of GW in the pre- and post-vaccination periods. Secondly, we collected information about the introduction year of the vaccine in the certain country, some details about the vaccination programme, the demographic characteristics of the participants and the duration of the pre- and post-vaccination periods.For data synthesis we used the methods recommended by the working group of the Cochrane Collaboration. In the meta-analysis, odds ratios (ORs) as well as their 95% confidence intervals (CI) were calculated from the original data of the articles and were visualized in forest-plots. Subgroup-analysis was done within ecological studies according to gender and according to age groups (younger or older than 21). In case of gender we applied meta-regression as well. Depending on data quality, the random effect model by DerSimonian and Laird  was usedWe evaluated the presence of publication bias using the funnel plot graph. We also performed Copas selection model to test the publication bias, calculations were performed with R (V. 3.5.2) package metasens (V. 0.4–0).Strength of the overall body of evidence was assessed using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach. Quality of a body of evidence involves consideration of risk of bias, heterogeneity, directness of evidence, precision of effect and risk of publication bias, as described in Section 12.2.2 of the Cochrane Handbook for Systematic Reviews of Interventions . The quaLiterature searches were performed in PubMed, Embase, and Cochrane Library, where altogether 1862 references were identified. After removing duplicates, and reading the titles and abstracts, a total of 106 potentially relevant articles remained. Finally, 16 studies  were eligible for inclusion in the meta-analysis with more than 13,000000 participants. All selected studies corresponded only to the quadrivalent vaccine.The flow chart for identification of relevant studies and the reasons for exclusions are included in Fig. Altogether 8 randomised, double-blind, placebo-controlled studies were included in our final quantitative analysis , 20–26. Additionally, 8 time-trend analyses , 27–33 wp = 0.035).According to the results of RCTs, the quadrivalent HPV vaccine significantly reduced the risk of GW  in healthy women as shown in Fig. p < 0.001) and men .In time-trend analyses Fig. b, the nuWe performed a leave-one-out sensitivity analysis by iteratively removing one study at a time and recalculating the summary OR. The summary ORs remained stable both in RCTs Fig. a and in In subgroup analysis, when the age-groups were separated at 21 years, in women under 21 years of age, the number of GW events decreased significantly by 67% , in women over 21 years of age, GW reduced less substantially (by 50%)  Fig. a. In menBased on the results of leave-one-out sensitivity analysis Fig. a, b, remp = 0.54) among odds ratios of studies . In the analysis of ecological studies (B) publication bias may occur, but the leave-one-out analysis did not show inconsistent result.The results of Copas selection model are included as Additional file Among RCTs only one study  was rateMost of the time-trend analyses showed high risk of confounding factors such as changes in sexual activity and health-seeking behaviour during the long period of the study, which can potentially cause changes in GW. The majority of studies showed low risk of information bias because GW were diagnosed by physicians except in the study of Dominiak-Felden , where aThe GRADE evidence profiles are shown in Table In the present study, we investigated the incidence of GW in healthy women after the administration of quadrivalent HPV vaccination (or placebo) and compared the presence of GW in women and men before and after the administration of the quadrivalent HPV vaccine. Altogether 16 studies (including 8 RCTs) were included in the current meta-analysis. We found that the quadrivalent HPV vaccine is effective in preventing GW in women. According to the results of subgroup analyses, GW reduction was more substantially in women under 21 years of age. Furthermore, a significant herd protection was observed in young men.Leave-one-out analysis showed that similar results could be obtained after excluding one study.RCTs have clearly demonstrated the efficacy of the quadrivalent HPV vaccine in vaccinated persons but not investigated the effects at population level and in men. At population-level, a reduction in the prevalence of GW was reported due to HPV vaccination. In men, we also observed a significant decline in GW, which can most likely be attributed to the indirect protection provided by the vaccination of women by the quadrivalent vaccine. This so called “herd immunity” is the protective effect of the vaccine extending beyond the vaccinated individuals to the unvaccinated ones in the population .Overall, most of the RCTs showed low or unclear risk of bias, therefore, the methodological quality of the trials is acceptable. Ecological studies provide valuable timely information about the population-level effects of HPV vaccination using large populations. However, these studies are vulnerable to carry risk of bias. The possible changes in the clientele and/or in sexual activity or health-seeking behaviour of the clients can modify the diagnosis of GW over time. Despite the unclear/high risk of selection bias and confounding factors, it was important to use the results of time-trend analyses to evaluate the performance of the vaccine under real-life settings to confirm that the efficacy/effectiveness can be seen not exclusively under ideal clinical conditions.Heterogeneity was moderate in RCTs and considerable in time-trend analyses. In the study of Yoshikawa and co-workers only one country was covered in contrast to the other RCTs, in which several countries were involved. In addition, the follow-up period was much shorter in the Japanese study, which could explain the moderate heterogeneity among RCTs. Moreover, the ecological studies were performed in different countries, and the difference in vaccination coverage, sexual behaviour, length of pre- and post-vaccination periods, or source of study data could also be a cause of heterogeneity.Our findings are consistent with previous reviews and meta-analyses, where it was also found that the prevalence of GW decreased significantly in vaccinated girls , 35, 36,Gardasil® has been the vaccine of choice worldwide. It has been chosen by health authorities in the United States, Australia, New Zealand, Canada, Switzerland, Italy, Spain, and Sweden for regional or national vaccination programmes against cervical cancer. The United Kingdom substituted the bivalent vaccine with the quadrivalent one in 2012, since the government clarified that the aim is to protect girls against the types of HPV that cause cervical cancer and those that cause GW . A studyTo the best of our knowledge, our work is the first meta-analysis related to GW which is based on the results of both RCTs and time-trend analyses, and carried out a combined analysis in men and women. The advantage of this paper is the high number of participants involved in ecological studies and consequently in this meta-analysis.However, there are some limitations of this study. First, particularly, the per-protocol population in RCTs is not entirely representative of the general population of women because of the exclusion and inclusion criteria of the trials . Therefore, our results cannot be generalized to other populations with different background risks. Second, in RCTs, many participants from a lot of countries were involved. We tried to come into contact with several authors to get raw data, but the response rate was very low. Thus we cannot state that the actual subsets involved in the different included studies differed totally, but it may not entail extreme distortions. Third, we had to exclude some reports, because we were unable to extract the necessary information from them . Fourth, since our aim was to evaluate the population-level effect of the vaccine, we included time-trend analyses despite the high risk of bias. In addition, this meta-analysis covers time-trend analyses mainly from Australia . Fifth, those articles in which HPV vaccine was administered in the framework of private care, and it was not covered by the National Health Insurance, were excluded, thus the present analysis does not take into account the effect of vaccines that were purchased. We think, that the persons who purchased the HPV vaccine may have different socioeconomic status and sexual habits than those people who were vaccinated in the framework of national/school-based programme, therefore the inclusion of privately vaccinated persons may cause a distortion on the outcome of the study.The results from RCTs and time-trend analyses – representing more than 13,000 000 participants – have shown that the quadrivalent HPV vaccine is highly effective in preventing HPV 6/11 related GW both in women and men which gives an additional value to the application of this type of vaccine. Our meta-analysis provides up-to-date information for the public about the effectiveness of HPV vaccination. Teenagers and their parents should acquire better knowledge about HPV infection and prevention. This is of very high importance, because rumours about vaccine safety have been one of the principal obstacles for the acceptance of HPV vaccination by the public. Despite the early implementation of national vaccination programs, in the majority of developed countries coverage rates remain unsatisfactory .Furthermore, the present work provides reliable information for obstetrician–gynaecologists and other health care providers who should raise the attention of parents and patients for the benefits of HPV vaccination and offer HPV vaccines. Additionally, our results demonstrating strong evidence of quadrivalent HPV vaccine effectiveness can help the governments for making decisions about the implementation of the vaccination. It would be recommended to include the quadrivalent HPV vaccine in routine immunization programme because of its high effectiveness not only against cancer but also against GW.In summary, our results clearly show that the ecological impact of the quadrivalent HPV vaccine is high and its introduction in many countries is strongly suggested.Additional file 1 Graphical output from Copas analysis of 8 RCTs (A) and 8 ecological studies (B) (a): Funnel plot; (b): contour plot; (c): treatment effect plot; (d): P-value plot.


The human papilloma virus (HPV) is responsible for different pathological manifestations in humans. This agent gives rise to lesions of different types and in different areas of the organism, including the oral cavity. The aim of this study is to show which are the main diseases for which HPV is responsible and to bring to light some of the interceptive and therapeutic strategies. The analysis was conducted by consulting the major scientific databases with the aim of obtaining information on the characteristics of oral HPV and its management; furthermore, the literature was supported by some clinical cases proposed by the authors. The role of dentistry is essential in the early diagnosis of this type of pathologies and above all in knowing how to direct patients towards a path that can lead to patient management, especially in the event that these lesions have a malignant potential. Enhancing the knowledge and role of dentistry can lead to early diagnosis of this type of injury, intercepting a pathology that could have multiorgan implications. Papilloma viruses (from the Latin “papilla” meaning pustule and from the Greek suffix “oma”) are classified in the Papillomaviridae family, officially recognized by the International Committee on Taxonomy of Viruses (ICTV). The human papilloma virus (HPV) is a small-sized DNA virus (diameter 50-55 nm) without envelope, which is resistant to heat, acids, and ether. Currently, over 120 types of HPV have been identified, identifiable in 16 different genera based on biological properties and the organization of the genome .Papilloma viruses have a high tropism for stratified squamous epithelial cells and replicate only in differentiating epithelial cells of the skin and mucous .The infection begins with the entry, following small wounds or superficial abrasions of the host's mucosa, of a viral particle into a cell of the basal epithelial layer called keratinocyte characterized by a marked proliferative activity. Once the virus has penetrated into the keratinocyte, it could remain in a latent state or undergo active replication. This virus is responsible for causing lesions of the affected tissues –6.Cancer of the uterine cervix, whose incidence is about 600,000 new cases every year with rates of 40 cases per 100,000 women in some geographic areas at higher risk such as Central America, South America, and Africa, is the first tumor recognized by the World Health Organization with infectious etiology. The etiological agent is the human papillomavirus responsible for most sexually transmitted diseases. It is estimated that about 75% of women will contract an HPV infection in their lifetime, but only a small percentage of these infections will become persistent and may lead to cell atypia up to the development of carcinoma. The role of the virus in oral carcinogenesis is highly controversial, but it is now believed that HPV infection, in particular HPV 16, is important in the pathogenesis of oropharyngeal cancer; oral carcinoma accounts for more than 90% of all malignant neoplasms of the mouth. It most frequently affects individuals over fifty years of age and males to a greater extent than females in a ratio of about 2 : 1 .–26.11–26The narrative review of the literature was also supported by some clinical cases managed by the authors with the aim of giving better visibility to the scientific manuscript, with a particular case also of negative PCR and histological positivity. The analysis of the literature provided a large number of results that were screened and read independently by the authors. The individual manuscripts were evaluated on the basis of the title and abstract, to evaluate a first inclusion , and subsequently, they were subjected to reading of the full-text. In this way, it was possible to analyze in detail the individual results and include only those that contained sufficient data to conduct a narrative review.Group 1: carcinogenic to humans Group 2A: probably carcinogenic to humans (HPV 68)Group 2B: possibly carcinogenic to humans Group 3: not classifiable as to its carcinogenicity to humans (HPV 6 and 11) .The IARC  in its latest epidemiological classification divides HPV based on their oncogenicity in mankind into the following:Based on their tropism, papillomaviruses are divided into two broad categories:Cutaneous HPV is responsible for skin lesions such as common or vulgar warts (these are the most common forms) which are frequently localized in the hands and feet and which manifest themselves in the form of white-greyish or brown flat or raised papules. The genotypes most frequently associated with warts are types 1, 2, 3, 4, and 7. Cutaneous HPV genotypes 5, 8, 9, 12, 14, 15, 17, 19, 20, 47, and 49 are associated with epidermodysplasia Lewandowsky-Lutz verruciform. This is a rare familiar dermatosis, with chronic evolution, characterized by a dissemination of numerous papular and macular elements, which usually appears in childhood and which within 20-30 years of onset can encounter a neoplastic transformation (first bowenoid carcinoma in situ and subsequently squamous cell epithelioma) which manifests itself in the form of flat warts and reddish macules and which can also degenerate into squamous cell carcinomas , 28.Mucosal HPV is responsible for benign mucous lesions that mainly include sharp and flat warts that arise in the female and male genitalia, the urethra, the perianal area, and the anus. The genotypes associated with these infections are genotype 6 and genotype 11.Malignant mucosal lesions were associated with high-risk genotypes and characterized by low-grade (LSIL) or high-grade (HSIL) squamous intraepithelial lesions (SIL) which on the Pap test show characteristic cells with nuclear atypia and perinuclear halo called koilocytes –32.The tests available for the laboratory diagnosis of HPV infections, given the lack of effective culture methods, are numerous and differ in applicability on biological material, sensitivity, and specificity. They can be divided into cytological, immunohistochemical, and molecular methods.ASC  with the subcategory ASC-US  which includes cellular alterations suggestive of SIL  and the ASC-subcategory H  which does not exclude H-SIL LGSIL  and low-grade intraepithelial squamous cell lesionsHGSIL  and high-grade squamous cell lesionsSquamous cell carcinoma , 33, 34Cytology uses the Papanicolaou stain of smears of samples taken from the uterine cervix (Pap test). This technique consists of evaluating any alterations present in HPV-infected cells. Squamous cell abnormalities are divided into four categories:Immunohistochemical methods use fresh or fixed cytological material that is incubated with polyclonal or monoclonal antibodies, directed against major and minor capsid antigens, capable of recognizing different subtypes. Subsequently, biotinylated antibodies linked to the avidin-peroxidase complex and capable of binding to the primary antibody are used. The final phase of the revelation involves the addition of specific chromogens and countercoloring with hematoxylin. The nuclei of infected cells show a red or brown color depending on the chromogen used. The limits of this method are related to low sensitivity, especially in cases where the capsid antigens are not expressed , 36.Molecular methods allow the identification of viral DNA with relative genotyping in preparations obtained from different biological samples. In women, superficial cells of the cervical mucosa, fresh cervical biopsies or those fixed in formalin or paraffin, and vulvar papillary biopsies are used as biological samples. In humans, the superficial cells of the glans, urine, and seminal fluid are used .Except for the paraffinized samples which must be treated with xylene and washed with ethanol, all the others are first treated with proteinase K at different concentrations based on the type and quantity of cellular material to be digested and subsequently lysed with isopropanol. For the extraction of viral DNA to chemical methods, which use phenol and chloroform, those on the column are preferred today, which involve a lysis step, a binding of the nucleic acid to the column membrane, repeated washing, and a final elution of the acid. Nucleic acid is purified with a vacuum system. Once the purified DNA has been obtained, the three main methods used are direct hybridization, signal amplification, and gene amplification (PCR). Direct hybridization uses appropriately labeled probes that bind specifically to homologous DNA sequences contained in the sample to be analyzed. The probe-DNA binding of the sample is then revealed either by using radioactive tracers or by using streptavidin labeled with alkaline phosphatase which is made to act on a suitable substrate –40. The Gene amplification (polymerase chain reaction) is a PCR technology invented in April 1983 by Kary B. Mullis who received the Nobel Prize in Chemistry for this in 1993. It is a biomolecular technique that allows the exponential amplification in vitro of a given DNA sequence defined as target DNA , 43.Denaturation (separation): the target dsDNA (double-stranded DNA) is denatured at about 95°C and is converted into single-stranded DNAPairing : the complementary oligonucleotide “primers” at the two ends 3′ of the sequence to be amplified hybridize with the two denatured filaments; their sequence is oriented so as to be able to guide DNA polymerization in the stretch between the two regions to which they associateExtension: the oligonucleotide “primers”, in the presence of the four triphosphate deoxynucleotides and a DNA polymerase, are each extended towards the other but on two different complementary chains leading to the synthesis of two molecules of ds-DNA copies of the delimited target region from the primers. The cycle can also be repeated up to 60 times leading to the exponential amplification of the “target” DNA (target DNA) The essential components of the PCR reaction mixture are a thermostable DNA polymerase, the “primers” which are single-stranded DNA oligonucleotides complementary to the two ends of the target segment, the deoxyribonucleotide triphosphates (dNTPs), the magnesium ions, and the target DNA. The amplification reaction has three phases:RFLP (restriction fragment length polymorphism) which uses restriction enzymes that generate genotype-specific bands. Interpretation is somewhat difficult in the case of packagingELISA (enzyme-linked immunoabsorbent assay) which uses type-specific oligonucleotide probes in microplates and a colorimetric reaction to highlight the hybridizationLIPA (line probe assay) which identifies amplified DNA by selective hybridization with membrane-immobilized oligonucleotides. The method is sensitive and also has a good level of discrimination in the packagingSequencing that uses terminator nucleotides in which the DNA sequence is determined by enzymatic synthesis of polynucleotide chains interrupted with the addition of dideoxy terminator nucleotides. It allows the identification of the viral genotype by comparing sequences using an automatic sequencer Different types of primers designed to amplify highly conserved sequences of the L1, E1, or E6/E7 region of the viral genome can be used for DNA detection of papillomavirus. The most commonly used are the consensus primers directed towards the L1 region of the virus . MY09/MY11 identifies 25 HPV genotypes and generate a 450 bp fragment, while GP5+/GP6+ generates a 140 bp fragment identifying an HPV spectrum similar but not identical to MY09/MY11. The primers directed towards the E6/E7 region that identify only the high-risk genotypes are the pU-IM/pU2R which generate a fragment of 233-268 bp on the E6 region. Once the amplification reaction is completed, the type-specific identification is carried out which can be carried out using various methods:The viral load is determined using real-time amplification techniques based on the use of highly specific probes which emit a fluorescent signal when probe/DNA hybridization occurs and which allow an accurate quantitative analysis to be performed, also detecting a low number of DNA copies. The fluorescent signal is read in real time at each amplification cycle, generating curves which, compared to a standard, allow for a quantitative evaluation of the viral load. Several studies have shown a statistically valid correlation between viral load and risk of developing cervical cancer and high sensitivity, reproducibility, and speed of execution .The determination of the mRNA transcripts of E6/E7 oncoproteins, a useful prognostic indicator of the progression of lesions towards cervicocarcinoma, uses NASBA (nucleic acid sequence-based amplification) technology based on isothermal amplification (conducted at 41°C) of the target RNA and on the use of particular probes defined as molecular beacons capable of emitting fluorescence at two different wavelengths when a complementary sequence is present , 45–47.Direct horizontal contact : oral sexual contact is certainly one of the most frequent ways of transmission of the papilloma virus; a relationship has been demonstrated between the presence of HPV in the oral cavity and the age of onset of sexual activity in the young; the presence of oral HPV is also more evident in couples in which oral sex is practiced, compared to couples in which only genital sex is practiced; a 10 times higher risk of oral HPV infection was then calculated in patients who have had more than 20 sexual partners in their lifetime compared to individuals with fewer than 3 partners Indirect contact Vertical maternal-fetal transmission  , 50Transmission of the infection occurs with three different methods of contact:Subclinical infection: the presence of HPV in a clinically normal mucosa is found in about 10% of subjects, which represent a potential reservoir for the transmission of infectionSquamous papilloma: it is associated with HPV 6 and 11, and it is the most common manifestation of oral HPV infection; it is localized on the nonkeratinized mucosa  or keratinized  and appears as an exophytic neoformation with a cauliflower surface. From the histological point of view, it is composed of a connective axis with a villous and papillary morphology (arborescent appearance) covered by a paving epithelium which presents a notable hyperkeratosis and acanthosis (verrucous hyperplasia); at the cellular level, there is a clear vacuolisation of the cells of the spinous layer (koilocytosis) characteristic of HPV infectionsVerruca vulgaris: it is associated with HPV 2 and 4, is rare in the oral mucosa, and is localized at the level of the keratinized mucosa ; it is found more frequently in children, as a consequence of self-inoculation, and is generally self-limited, resolving in less than 2 yearsCondyloma acuminata: it is associated with HPV 2, 6, and 11, and it is extremely contagious and is often found associated with genital warts. It is localized on the nonkeratinized mucosa  and has a wider implant base than papillomas and warts and a more pinkish color as it is often devoid of hyperkeratosisSquamous cell carcinoma: the role of the virus in carcinogenesis of the oral cavity is highly controversial, but it is now believed that HPV infection, in particular HPV 16, is important in the pathogenesis of oropharyngeal cancer (cancer of the back of the mouth); in fact, oropharyngeal tumors are 70% HPV positive (HPV 16), and compared to the healthy mucosa, HPV infection in these tumors is almost 13 times more frequent –53. TherIn Figures Contrary to other viral infectious conditions that respond to drug therapy, for HPV infection, there are no active ingredients available that can eradicate the infection or induce regression of clinical lesions where present. Some drugs have been tested for topical and/or systemic use for the treatment of genital lesions, but to date there are no available treatment for genital lesions. For oral subclinical infection, similar to genital infection, no treatment is provided; none of the antiviral drugs tested  proved effective in eliminating the infection. The management provides for the patient's follow-up and the repetition of the HPV test 8-12 months after the first detection and periodically to verify the eradication of the infection by the immune system. In the presence of HPV-related lesions, their treatment is mainly surgical as they are not responsive to topical application or systemic administration of cytotoxic or immunomodulating drugs. The recommended surgical treatment is the excisional one using a cold blade scalpel, quantum resonance scalpel, or laser, which allows performing the histological examination of the piece. The application of low-level laser therapy (LLLT) protocols to the oral manifestations of viral infections and in particular to the relapses of cold sores (herpes simplex virus 1)  is justiThe new therapeutic perspectives concern the possibility of using “therapeutic vaccines,” i.e., real molecules capable of arresting tumor growth linked to vectors directed against some viral antigens and therefore directed against positive HPV CSCs –67.Monovalent anti-HPV 16 vaccineDirect quadrivalent vaccine against HPV 6, 11, 16, and 18Bivalent anti-HPV vaccine 16 and 18 , 69There are currently three prophylactic vaccines already evaluated by randomized clinical trials, but only the last two have been released:The HPV vaccine also protects against oral infection caused by the virus, which causes more than 30% of oropharyngeal cancers. So much so as to lower the prevalence of infection by 88% in those who have carried out at least one dose of vaccine compared to those who have decided not to get vaccinated, the HPV vaccine is indicated for the prevention of cancer 5of the cervix, vulva, vagina, anus, and penis because it has been shown to be able to prevent infection of the most aggressive HPV strains, those that cause warts or cancer.The epithelial tropism of the virusThe confirmed etiological role of HPV-HR in almost all squamous cell carcinomas of the uterine cervix and in about 40% of cases of other anogenital squamous cell carcinomasThe histological similarities between the lining epithelia of the two mucous districtsDespite the histological similarities of the two epithelia, it should however be reiterated that the environment of the uterine cervix is much more receptive to the permanence and entry of the virus than any other anatomical siteThe role of the virus in oral carcinogenesis is highly controversial The most common malignant neoplasm of the oral cavity is squamous cell carcinoma. According to recent estimates, both the annual incidence rates of the disease and the mortality rates have not decreased in recent years, still representing a serious public health problem. The disease is in fact characterized by considerable local aggression and an often-poor prognosis, with a five-year survival ranging from 59.4% to 67%. The initial hypotheses about a possible etiological role of the virus in oral and oropharyngeal carcinogenesis, dating back to the early 1980s, were subsequently supported by the following evidence:HPV-associated cancer, on the other hand, occurs in young subjects, mainly men, is independent of traditional risk factors, and is instead correlated with sexual behavior , mainly involving oropharynx   70]..70].In some cases, a combined protocol may be required for diagnosis. The biopsy intervention can be split and stored in a different way so as to perform a double analysis with a single surgical intervention on the patient. Keeping part of the excised tissue in formalin and part in physiological can allow performing both a histological examination  and a PCR test to identify traces of DNA of certain viral strains. However, there are reported cases where PCR is negative and histology is positive Figures  and 5 7. This maThe virus is normally found in the genital and perianal area. If these come into contact with the mouth, the passage into the oropharyngeal mucosa is quite obvious. In most cases, the infection resolves spontaneously and the virus disappears. In other cases, however, it lurks and can give rise to pretumor lesions. The cigarette itself is a risk factor for various forms of airway cancer, including the oropharynx. Smoking aggravates the state of inflammation and could facilitate the evolution of tissues towards cancer. It is possible that smoking also acts on the defensive capacities of the immune system at the level of the mucous membranes: marijuana smoke, for example, has been associated with a greater risk of oral HPV infection, and it is believed precisely because of its immunodepressive activity. It should be checked whether the same also applies to the cigarette one. Limiting the number of oral sex partners, not smoking, and getting vaccinated against HPV are three strategies for reducing risk , 72.Patients at risk or those already affected by the disease must undergo periodic checks by both ENT /dentist (ENT/D) and gynecologist. The ENT/D and the gynecologist, in synergy, could schedule follow-ups for these patients at risk, even for couples. The sensation of an oral-pharyngeal foreign body, the pain or burning of the oral mucosa, and the bleeding (even if rare) immediately require a specialist visit and therefore the appropriate care of the case. In recent decades, the amount of information regarding HPV infection and its oncogenic potential has grown considerably, hand in hand with the progress of molecular biology techniques applied to the diagnostic, preventive, and therapeutic management of HPV-induced lesions. Understanding the interactions between the viral agent and the host has provided new targets for the control of the infection and laid the foundation for the formulation of new targeted therapeutic protocols. It is essential to emphasize that the general population has little knowledge about the methods of transmission and clinical presentation of HPV infection, an aspect mostly due to the scarcity of information campaigns, the implementation of which is desirable in order to achieve better infection control. While waiting for these information and prevention programs to be implemented, the role of the dentist is of great importance, for head and neck cancer detection too.


There are >200 different types of human papilloma virus (HPV) of which >51 infect genital epithelium, with ~14 of these classed as high-risk being more commonly associated with cervical cancer. During development of the disease, high-risk types have an increased tendency to develop a truncated non-replicative life cycle, whereas low-risk, non-cancer-associated HPV types are either asymptomatic or cause benign lesions completing their full replicative life cycle. HPVs can also be present as non-replicative so-called “latent” infections and they can also show superinfection exclusion, where cells with pre-existing infections with one type cannot be infected with a different HPV type. Thus, the HPV repertoire and replication status present in an individual can form a complex dynamic meta-community which changes with respect to both time and exposure to different HPV types. In light of these considerations, it is not clear how current prophylactic HPV vaccines will affect this system and the potential for iatrogenic outcomes is discussed in light of recent outcome data. Although published work overwhelmingly supports the efficacy of the current prophylactic HPV vaccines, there has been a great deal of publicity and numerous studies carried out on potential vaccine-related adverse events ,2,3,4. TAlthough there are ~200 distinct types of HPV, there is substantial evidence that many others are yet to be identified ,7 as exeOf these, ~14 are classed as high risk and are associated with cervical cancer although some are more carcinogenic than others ,9,10. FoThe unique life cycle of HPV is intimately associated with how the virus manages to evade the immune system. At its simplest, the virus hides by not penetrating below the basement membrane where the majority of immune effector cells reside. Furthermore, it does not produce secreted proteins, inflammation, viremia or cell death ,15. TherHowever, during the development of HPV-related high-grade cervical neoplasia, the production of mature virions is reduced since the virus downregulates its full replicative life cycle and expression of the L1 protein is also reduced , Gardasil targets L1 from HPV16/18/6/11  and Gardasil 9 targets L1 from 16/18/31/33/45/52/58/6/11  and their prophylactic use is currently advocated for women prior to infection with HPV. They all induce HPV type-specific L1 antibodies which provide humoral immunity against HPV infections in HPV-negative women by blocking the virus from interacting with its cellular receptors and many studies have demonstrated their effectiveness at protecting against CIN lesions caused by infection with vaccine-covered HPV types. However, for >15 years, there has been considerable debate on the ability of Cervarix and Gardasil to protect both HPV-positive and -negative women from HPV-associated cervical cancer. For example, it is well known that HPV-negative women vaccinated with either the bivalent or quadrivalent vaccines can still develop CIN lesions positive for HPV types not covered by these products although it is anticipated that the more recently introduced Gardasil 9 should improve this ,30. FurtIn women who are HPV positive at the time of vaccination, additional concerns are based on the mode of action of the L1 targeted vaccines. Since expression of the L1 protein is downregulated as a consequence of the truncated HPV life cycle that is a common feature of high-grade type 3 cervical intraepithelial neoplasia (CIN3)  19], it, it19], As previously discussed, many women are known to acquire cervical infections with multiple HPV types of which only some are associated with cervical cancer  6,9,10],9,106,9,As previously mentioned, it is known that HPV can exist at very low copy numbers, where the virus is present as a non-productive so-called latent infection  which haThese observations illustrate there is a distinct lack of knowledge concerning potential interactions within HPV meta-communities which may be present at various stages of HPV infection and how these may contribute to either progression or regression of disease . In thisTo date, long-term efficacy studies with Gardasil 9 are not available so only Cervarix and Gardasil will be considered further. Prior to, and immediately following approval, reports on the efficacy of Gardasil were produced by the Food and Drug Administration  and the Analysis of the combined results from protocols p007, 013 and 15 showed that Gardasil was 98–100% effective at preventing HPV6/11/16/18-related CIN2/3 in PPE and MITT-2 women who were sexually naive and HPV negative with normal smears at baseline. However, this dropped to 36.3% in the MITT-3 cohort which represents a real-world population of 16–26-year-old women who were positive for either vaccine or non-vaccine HPV types and also included women who had an abnormal pap test at baseline . NotablThe Future 1 p013 study was also analysed for Gardasil efficacy against all grades of CIN related to any HPV type in the baseline HPV-negative RMITT-2 population which includes women who were either sexually naïve or experienced. This demonstrated 31% efficacy where 6.4% (92/1429) of vaccinated and 9.1% (132/1441) of placebo-treated women developed CIN lesions due to any HPV type including types 16/18 . Thus, A combined analysis of both Future 1 and 2 trials (p013 and p015) was also carried out for CIN2/3 lesions related to any HPV type including types 16/18. This showed a vaccination efficacy of 46% in RMITT-2 and 27% in MITT-2 women against CIN2/3 . SignifExtent of vaccine efficacy in HPV-positive women.Extent of HPV cross-type protection.Extent of HPV type replacement and the potential role played by latent infections.The previously discussed FDA and EMA reports on Gardasil were made between 2006 and 2008 which both indicate that the vaccine provides virtually 100% protection against all grades of CIN related to HPV infection with vaccine-covered types in HPV PCR-negative women and many additional studies have since confirmed this observation. However, important questions still remain such as:Attempts to address these issues have relied on monitoring pre- and post-vaccination levels of both vaccine-covered and non-vaccine-covered HPV types in various populations. In this regard, a meta-analysis of 9 studies involving 13,886 girls and women ≤19 years of age and 23,340 women 20–24 years of age was carried out by Mesher et al. in 2016 . The resThe issue of HPV type replacement is still a subject of significant controversy and a study by Gray et al. evaluated pre- and post-vaccination HPV type prevalence in Finnish women . It was Furthermore, it was shown that a substantial proportion of this increase was due to increased prevalence of additional HPV types covered by Gardasil 9. However, there was also an overall increase of ~12% in the prevalence of CIN2 in presumably catch-up vaccinated women aged between 30 and 35 years which was attributed to changes in screening practices between 2008 and 2016.A similar study was carried out in New Zealand which analysed changes in HPV types associated with CIN2 between 2013 and 2016 in 392 women under the age of 25 . New ZeaAlthough HPV16/18 lesions decreased substantially, there was a 33% increase in lesions associated with other high-risk types and it was noted there was a significant increase in the number of women diagnosed with high-grade cytology over the same period . IntereMore recently, Innes at al evaluated the effect of vaccination on the incidence of abnormal cervical cytology and histology in 104,313 New Zealand women aged 20–24 years between 2010 and 2015 . CompareDrolet et al. carried a systematic review and meta-analysis on the population level impact and herd effects related to HPV vaccination derived from 65 articles covering 60 million individuals over a maximum period of 8 years in order to compare pre- and post-vaccination relative risk of HPV-related end points . This stUnfortunately, there are currently no studies with sufficient follow up and suitable efficacy end points to definitively answer this question although it is known that the bivalent vaccine provides 91% efficacy at protecting women older than 25 which lasts for 7 years . FurtherVaccinate prior to onset of sexual activity and begin assessment of end points at age of usual cervical screening once sexually active.Make all clinical study reports, including anonymised individual patient data, publicly available.Separate trials to assess benefits in women already exposed to HPV without restrictions based on risk factors.Analyse data by country and study site.Ensure the testing interval is in line with usual cervical screening protocols.Continue follow up for a minimum of 20 years from time of sexual debut.Power trials for primary composite outcomes CIN3/AIS/cervical cancer due to oncogenic HPV types.Define secondary outcome of persistent HPV16/18 infection at a minimum of 12 months.Use standardised methods for HPV testing.Undertake a saline placebo-controlled efficacy trial of Gardasil 9 in previously unvaccinated populations as it is difficult to draw conclusions on efficacy and risk of harms based on comparing Gardasil 9 to Gardasil.It is very clear that the previously discussed trial results raise many questions and do not provide conclusive evidence that any of the prophylactic vaccines will actually prevent cervical cancer. Indeed a very recent review by Rees et al. critically assessed the available published data in relation to this goal and drew the same conclusion . These aAlthough implementation of these suggestions would obviously clarify some of the issues raised, it is very clear that such trials would also benefit from improved HPV testing in relation to vaccine efficacy. For example, analysis of the prevalence of all 51 genital HPV types  in womenSelection of the most appropriate outcome is critical for assessing vaccine efficacy and CIN3 was judged to be preferable to either HPV status or low-grade disease since these have much higher rates of spontaneous regression . HoweverIn the UK the overall incidence of cervical cancer decreased by ~35% from 1990 to 2005 and vaccination was started in 2008.The overall incidence of cervical cancer remained at a constant level until 2017 A. HoweveAustralia started Gardasil vaccination in 2007, achieving a coverage of >80%. The age-stratified five-year average cervical cancer incidence in New Zealand from 1987 to 2016 is shown in Sweden introduced cervical screening in the mid-1960s and by 2011 the incidence of cervical cancer had declined 3 fold. Vaccination started in Sweden in 2008 firstly with Cervarix and then Gardasil from 2012 with a coverage of 10% in the period 2008–2010 rising to >80% in 2018. The conclusion from this study was that the increase may be due to insufficient detection of precursor lesions prior to 2009 and the possibility that vaccination may have played a role was dismissed since <0.5% of women in the study cohorts were vaccinated. Indeed a more recent study of vaccination and the risk of cervical cancer in Sweden concluded that vaccinated women aged 30 years or less had a significantly lower risk than unvaccinated women . HoweverNorway started single cohort vaccination of 12-year-old girls with Gardasil in 2009 achieving a coverage of 89% but, unlike most other nations, did not start catch-up vaccinations until 2016 . It is nIt is significant that the UK, Australia, New Zealand, Sweden and Norway all have vaccination coverage rates of 50–86%, whereas France started vaccination in 2007 and only achieved coverage of 19% in 16-year-old girls. Indeed France has the lowest coverage in Western Europe which begs the question, how does the incidence of cervical cancer in France compare to nations with higher vaccine coverage over the same period? In 2007, the age-standardised incidence of cervical cancer was ~10 per 100,000 women, whereas in 2018 it had declined to 6.7 . Thus, uUse of high-sensitivity methods of HPV analysis in order to detect latent infections.Determination of the impact of vaccination over time on the prevalence of all 51 HPV types known to infect genital mucosa in all age groups and combine this with vaccination status, age at vaccination, vaccine type and sexual history.Use of this to discriminate between direct and indirect (herd) effects of vaccination on the incidence of different HPV types in unvaccinated women.Carry out these analyses on normal, CIN1, 2 and 3 and cervical cancers.Do not assume that unvaccinated women residing in a vaccinated population will experience only beneficial herd effects since increased transmission of non-vaccine-covered high-risk HPV types combined with decreased transmission of low-risk types has been shown to occur .Conclusions on vaccine efficacy should not be based on comparing vaccinated and unvaccinated women over time at the same geographical location since herd-effect transmission may confound this.Ideally, the incidence and disease association of different HPV types should be evaluated in any population before and after implementing vaccination.Clearly the preceding observations do not prove any causal relationship between the current prophylactic vaccines and iatrogenic outcomes with respect to cervical cancer. However, it is also very clear that there is a distinct lack of knowledge concerning the complex biology of mucosal HPV communities and the purpose of this review was to draw attention to how this could be affected by L1 targeting prophylactic vaccines. With regard to assessing vaccine efficacy, the recommendations of Rees et al.  are cleaWhilst it is very clear that the current HPV vaccines have substantial efficacy against disease related to vaccine-covered HPV types, there are many questions left unanswered concerning the full spectrum of their effects on HPV biology. As discussed, HPVs of the genital mucosa can represent a dynamic meta-community which changes over time with respect to many factors such as age, promiscuity and type of contraception used which means it is difficult to predict outcomes. In this regard, it is notable that the French PAPCLEAR trial  has someChanges in sexual practice.Changes in screening practice.Problems with assay sensitivity.Influx of migrant communities.Finally, as discussed, the recent increase in the incidence of cervical cancer in several countries with high vaccine coverage does not in any way establish cause and effect and there have been a variety of explanations put forward to explain this, such as:Indeed it has been speculated that this may be due to relaxed screening intervals which were adopted by some countries in order to cover the cost of vaccination and HPV testing. However, it is very unlikely that these same factors will be present in all the countries cited and it is very clear it will be of crucial importance to analysed the incidence of HPV types, CINs 1–3 and cervical cancers in women of all age groups in countries with high and low vaccine coverage in the next decade.


Objectives: To estimate years of potential life lost (YPLL) and present value of future lost productivity (PVFLP) associated with premature mortality due to HPV-attributable cancers, specifically those targeted by nonavalent HPV (9vHPV) vaccination, in the United States (US) before vaccine use.Methods: YPLL was estimated from the reported number of deaths in 2017 due to HPV-related cancers, the proportion attributable to 9vHPV-targeted types, and age- and sex-specific US life expectancy. PVFLP was estimated as the product of YPLL by age- and sex-specific probability of labor force participation, annual wage, value of non-market labor, and fringe benefits markup factor.Results: An estimated 7,085 HPV-attributable cancer deaths occurred in 2017 accounting for 154,954 YPLL, with 6,482 deaths (91%) and 141,019 YPLL (91%) attributable to 9vHPV-targeted types. The estimated PVFLP was $3.8 billion for cancer deaths attributable to 9vHPV-targeted types (84% from women). The highest productivity burden was associated with cervical cancer in women and anal and oropharyngeal cancers in men.Conclusions: HPV-attributable cancer deaths are associated with a substantial economic burden in the US, much of which could be vaccine preventable. Human papillomavirus (HPV) can cause cervical, vulvar, vaginal, penile, anal, and oropharyngeal cancers . The medThe quadrivalent HPV vaccine 4vHPV), which targets high-risk HPV 16 and 18 as well as low-risk HPV 6 and 11, was introduced in the US in 2006 and was the predominant vaccine administered through 2015 vHPV, whi. HoweverAn estimated 32,100 HPV-attributable cancers 92%) per year are attributable to HPV 16, 18, 31, 33, 45, 52, and 58, the high-risk types targeted by the 9vHPV vaccine 2% per ye, 12 and Cancer-related mortality has a wide impact on society and individuals, including the grief of bereaved family members and friends , 15. EcoUnderstanding the economic burden of HPV-attributable cancers, including work-related and non-work productivity loss, can help provide decision-makers in the US and other countries around the world with a more complete picture of the disease's impact on society , 21. TheThe objective of this analysis was to estimate the years of potential life lost (YPLL) and productivity loss associated with an expected loss of life years due to HPV-attributable cancers in the US in 2017, stratified by cancers attributable to vaccine-preventable high-risk HPV types 16/18 and 31/33/45/52/58.We estimated the number of deaths due to HPV-attributable cervical, vulvar, vaginal, penile, anal, and oropharyngeal cancers in the US in the year 2017. From these data, we calculated the YPLL and total 2017 present value of future lifetime productivity (PVFLP) due to HPV-attributable cancer deaths, stratified by cancer type, HPV type, and sex.International Statistical Classification of Diseases, Tenth Revision (ICD-10) codes: C53 , C51 (vulvar), C52 , C60 (penile), C21 , and C00-C14 . The WONDER-reported US cancer deaths by age and sex in the year 2017 are shown in To estimate the expected number of deaths caused by HPV, the number of deaths by age and sex in each cancer site reported in the year 2017 was multiplied by either the proportion of those cancers attributable to any HPV or the proportions attributable to the high-risk HPV types targeted in the 4vHPV (HPV 16/18) or 9vHPV vaccine (HPV 31/33/45/52/58). The number of cancer-related deaths by age and sex for individuals aged 15 years and older was obtained from the CDC's Wide-ranging Online Data for Epidemiologic Research (WONDER) , a publiYPLL was calculated for each death using the sex-specific life expectancy at the age of death taken from the US life table from 2016 . SpecifiTo estimate the PVFLP for each death, the economic value of market and non-market labor for each future year of lost life was estimated and discounted to 2017 US dollars. Given an individual's sex and age at death, his or her lost market earnings were calculated as the age- and sex-specific probability of labor force participation  multipliThis analysis estimated that a total of 7,085 HPV-attributable cancer deaths occurred in the United States in 2017; of these, 6,482 (91%) deaths were attributable to the high-risk types targeted by 9vHPV  and $626 million (15%), respectively. The average PVFLP per death among men and women were $529,248 and $608,906, respectively.The estimated deaths, PVFLP, and PVFLP per death for all cancers in these sites, regardless of HPV attribution, are presented in This study provides estimates of the YPLL and PVFLP due to premature HPV-attributable cancer deaths in the US in the year 2017, stratified by HPV types 16/18 and 31/33/45/52/58. These estimates are based on the latest data available for the US population, labor force participation, and wages.ICD codes C01-02, C09, C14]) without regard to whether any of the cases were attributable to HPV. If we were to assume that 100% of each cancer is due to HPV, our estimated YPLL would be 182,391, on par with the Ekwueme et al. study. The difference in estimated PVFLP in our study is due to the attribution of HPV-related cancers to our calculation as well as general inflation costs between the years 2003 and 2017. Assuming 100% HPV attribution to the cancer deaths, our total PVFLP estimate would be $4.9 billion in 2017 US dollars; adjusted for inflation, this value corresponds to $3.7 billion in 2003 US dollars, which is the same as the value estimated by Ekwueme et al.Previously, Ekwueme et al.  estimateIt is not surprising that our estimate of 7,085 HPV-attributable cancer deaths in 2017 is fairly similar to the Ekwueme et al. estimate of 8,302 HPV-associated cancer deaths in 2003. The ongoing HPV vaccination program is not expected to impact US cancer deaths yet because of the long lead times between HPV infection and cancer death. In a natural history study of data from women diagnosed with cervical intraepithelial neoplasia 3 (CIN3) from 1955 through 1976 in New Zealand, the median time from CIN3 diagnosis to cervical or vaginal cancer diagnosis was 27 years . In the Although the incidence of cervical cancer attributable to 9vHPV-targeted types has decreased in the years since the Ekwueme et al. study, the incidence of head and neck cancers and other HPV-associated cancers has increased . OropharOverall, HPV-attributable cancer deaths in women accounted for the highest number of YPLL in our analysis and represented 86% of the total YPLL. HPV-attributable cervical cancer deaths were associated with the highest total YPLL and YPLL per death in our analysis, contributing to the higher burden in women as compared to men. Similar results were estimated by Ekwueme et al.: HPV-associated cancer deaths in women accounted for 71% of total YPLL in their analysis, and HPV-associated cervical cancer was associated with the highest YPLL and YPLL per death . The higAmong men, the average YPLL per death in our analysis was the highest among those who died of HPV-attributable anal cancer, and the total YPLL was the highest among those who died of HPV-attributable oropharyngeal cancer; the same rankings were observed in the Ekwueme et al. study of HPV-associated cancer deaths. The higher average YPLL per death seen with HPV-attributable anal cancer may be due to the younger median age at diagnosis (59 years) resulting in more YPLL, whereas the higher total YPLL observed with HPV-attributable oropharyngeal cancer may be attributable to the higher overall volume of oropharyngeal cancer deaths .Our results show that the economic burden of HPV-attributable cancer in men is not trivial. HPV-attributable cancer deaths among men in 2017 caused a total future productivity loss of $655 million. Although the average PVFLP per HPV-attributable cancer death overall was higher among women  than men , the PVFLP of HPV-attributable anal and oropharyngeal cancers in our analysis was higher for men compared to women. For example, there were 19 YPLL per anal cancer death for both men and women, but the PVFLP per death was much higher for men: ~$600,000 vs. $431,000 for women. For oropharyngeal cancer, the YPLL per death is slightly lower among men than women , but again, the PVFLP is higher for men . These differences are driven by higher labor force participation and higher wages for men compared to women.In addition to the estimation of YPLL and PVFLP due to any HPV, we estimated these outcomes for the high-risk 9vHPV-targeted types (HPV 16/18/31/33/45/52/58). Cancer deaths caused by high-risk 9vHPV-targeted types accounted for 91% of the total YPLL and total PVFLP. These results highlight that more than three-quarters of HPV-related cancer deaths in 2017 are vaccine preventable and are associated with a significant economic burden of mortality and associated productivity loss among men and women in the US.Our analysis was specific to the US, but the YPLL and PVFLP due to premature HPV-attributable cancer deaths could be estimated in the same manner for any country if sufficient data are available. For example, nearly 90% of cervical cancer deaths worldwide in 2018 occurred in low- and middle-income countries, and more than 60% of women with cervical cancer in these countries die from the disease, which is more than twice the proportion in high-income countries (~30%) . Thus, aIn August 2020, the World Health Assembly adopted a global strategy to accelerate cervical cancer elimination by focusing on prevention through HPV vaccination, screening and treatment of precancerous lesions, and treatment and palliative care for invasive cervical cancer . Althougafter mortality. The direct medical, non-direct medical, and productivity costs to patients and their caregivers during the course of the disease can also be substantial  in the US and the first conducted since the initiation of routine vaccination against HPV. Vaccination with 9vHPV may prevent HPV-attributable cancers and the associated losses (http://engagezone.msd.com/ds_documentation.php. Requests for access to the study data can be submitted through the EngageZone site or via email to dataaccess@merck.com.Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA's data sharing policy, including restrictions, is available at All authors were involved in the conception or design of the work, the acquisition, analysis, or interpretation of data, drafting the manuscript, revised and/or reviewed the manuscript for important intellectual content, provided final approval of the version to be published, and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.VP, BK, CN, and DC are employees of Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA, who may own stock and/or hold stock options in Merck & Co., Inc., Kenilworth, NJ, USA. MP, SS, and SC are employees of Pharmerit – an OPEN Health Company, paid consultants to Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


KRAS and HRAS mutations were detected in 18 (35%) and 17 (33%) SCPs, respectively. Notably, KRAS mutations, HRAS mutations and HPV infection were mutually exclusive. The larynx and trachea  were more preferentially infected by HPV than the other sites  and HPV was associated with multifocal development . In contrast, KRAS and HRAS mutations in SCPs were evenly distributed across the anatomical sites and found only in single SCPs. In conclusion, this study demonstrated that HPV was not frequently involved in SCPs and that RAS mutations were more common alterations. In contrast to inverted sinonasal papillomas and oncocytic sinonasal papillomas, SCP may not be a precursor lesion of carcinoma, because these aetiological events in SCP are distinct from squamous cell carcinoma in the same sites.Squamous cell papilloma (SCP) is a benign neoplasm of the head and neck. Human papillomavirus (HPV) has been reported to be a tumourigenic factor for SCP. However, not all SCPs are positive for HPV, suggesting that other possible mechanisms are involved in their development. In this study, we examined the mutational status of 51 SCPs using targeted panel sequencing in addition to HPV status using GP5+/GP6+ PCR. HPV DNA was detected in 6 (12%) SCPs, while  Squamous cell papilloma (SCP) is a relatively common benign lesion, showing papillary proliferation of squamous epithelium. SCPs develop widely in the mucosa of the upper aerodigestive system, including the oral cavity, pharynx, larynx, oesophagus, and trachea. The involvement of low‐risk human papillomavirus (HPV) in a subset of SCPs, particularly recurrent respiratory papillomatosis, is well known et alEGFR mutations in inverted sinonasal papillomas KRAS mutational status, resulting in the detection of the mutation in 38% of SCPs. Few reports have focused on oncogenic genetic alterations in SCPs. Therefore, we attempted in this study to address the relationship between HPV status and possible recurrent oncogenic drivers.Consistent with the first study by Udager We selected 51 SCPs of the head and neck  that were resected or biopsied in 51 patients from the database of the Department of Pathology and Molecular Diagnostics at Aichi Cancer Center Hospital, Nagoya, Japan. Twenty‐three of the SCP cases in this cohort were reported in a previous study Tumour areas were marked on haematoxylin and eosin (H&E) stained sections. DNA was extracted from tumour areas on each unstained paraffin section while referring to the marks on the H&E‐stained sections. We confirmed that isolated tumour areas contained a minimum of 20% tumour cell nuclei. Targeted panel sequencing was performed on extracted DNA. These methods of detection have been described in detail elsewhere et alHPV status was examined using GP5+/GP6+ consensus primers for the L1 region (150 bp product) P value less than 0.05 was considered statistically significant.The chi‐squared test, the Fisher's exact test for independence, and the Kruskal–Wallis test were used to compare the frequencies of the clinicopathological variables. Statistical analysis was performed using StatView . A The patients were 40 men and 11 women with a median age of 63 years . The majority (75%) had a history of smoking. The average tumour size was 5.4 mm . Five patients had multiple tumours; all five had synchronous multiple tumours, and two had local recurrences. None of the patients had malignant transformation and therefore there were no disease‐associated deaths.KRAS and HRAS mutations were detected in 35% (18/51) and 33% (17/51) of tumours, respectively , followed by KRAS G12D . In 10 of 51 (20%) tumours, neither RAS mutations nor HPV were detected. Interestingly, none of the tumours harboured two or more alterations of RAS mutations and/or HPV, suggesting a mutually exclusive nature of these alterations. In two tumours, FGFR3 S249C mutations were detected, and both tumours were negative for RAS mutations and HPV infection.In a total of 51 SCPs from 51 patients, HPV DNA was detected in 12% (6/51) of tumours. The predominant virotype was HPV6, a low‐risk HPV type . y Figure  and S3. RAS mutations or positive‐HPV were detected in 67% (8/12) of oral SCPs, 90% (18/20) of pharyngeal SCPs, 75% (9/12) of oesophageal SCPs, and 86% (6/7) of laryngeal/tracheal SCPs. The distributions of the alterations were significantly different according to the anatomic sites . Moreover, the frequency of KRAS mutations was significantly higher in the pharynx  than in the oral cavity  (p = 0.0107). In contrast, there was no significant difference in the distribution of HRAS mutations according to the anatomic sites. It was also different between single and multiple occurrence; HPV‐positive SCPs were frequently multiple , while RAS‐mutated SCPs were always single .Either in 67% 8/2 of oralp < 0.0001)  of HPV‐positive SCPs but absent in HPV‐negative SCPs  Figure . The hisRAS mutations accounted for the major alterations  in the SCPs, and were mutually exclusive of HPV positivity in a manner independent of the anatomic sites.In this study, we found that HPV was associated with laryngeal/tracheal and multicentric development. In contrast, RAS genes RAS mutations and low‐risk HPV lead to activation of the same RAS‐MAPK pathway, suggesting a crucial role in molecular pathogenesis. Mutually exclusive involvement of HPV and RAS also supports the importance of this pathway.The oncogenic mechanism of HPV infection has been shown to be related to the intrinsic proteins of HPV. The E5 protein of HPV activates the mitogen activated protein kinase (MAPK) pathways, a downstream target of Specific involvement of HPV in the larynx and trachea might be associated with the histological characteristics of the sites. Recurrent respiratory papilloma, a representative HPV‐related papilloma, tends to occur in the transition zone of the larynx between stratified squamous epithelium and ciliated epithelium KRAS and HRAS mutations were the major genetic alterations in SCPs. These findings in SCPs are in line with those of previous studies. Frequent KRAS mutations are found in oncocytic sinonasal papilloma HRAS mutations are frequent in inverted urothelial papilloma Hras and Kras, but not Nras, activation induces papillomas in the skin and oral mucosa Ras mutations in mice is different among cancers; Hras induced papillomas and haematopoietic tumours, whereas Kras elicited gastric tumours and lung lesions RAS signal activation are explained by a narrow window of RAS mutations for tumourigenesis. Too little signalling leads cells to fail to proliferate, while too much signalling leads to abortive processes, such as growth arrest, that may result in a benign lesion incapable of further progression to carcinoma. In addition, the magnitude of the optimal signal varies according to the intrinsic cellular context Our results first revealed that KRAS mutations in head and neck cancers are extremely rare (less than 1%), whereas HRAS Q61L mutations, the most common variant in SCPs, are found in a small proportion RAS mutations supports the suggestion that papilloma is not a precursor lesion of squamous cell carcinoma of the head and neck. We speculate that features of the tumour are closely associated with the anatomical site, which defines susceptibility, and alterations, including the type of genes involved and the expression level of the mutated gene, according to the intrinsic cellular context.Interestingly, the mutational spectrum is distinct between the papillomas and carcinomas that arise from the same sites. There are several other types of papilloma in the head and neck. Malignant transformation has been reported to occur in 2–27% and 4–17% of cases of inverted sinonasal papilloma and oncocytic sinonasal papilloma, respectively RAS mutations and HPV infection. FGFR3 S249C mutations were detected in two SCPs without RAS mutations or HPV infection. In line with the previous discussion, this mutation can activate the MAPK pathway RAS activation in SCPs.In this study, 20% (10/51) of all SCPs were negative for RAS/HPV status and smoking history irrespective of anatomic site (data not shown). However, this may be masked due to the high percentage  of patients with a history of smoking.The association of SCP tumourigenesis with smoking has remained unknown. The mucosa in the upper aerodigestive tract is directly exposed to smoking, and head and neck cancer is closely related to smoking RAS mutations and low‐risk HPV infection. These early aetiological events may have crucial implications for developing the benign nature of SCPs.In conclusion, our study shows frequent involvement of the RAS‐MAPK pathway in SCPs, including ES and YY conceived and designed the study. ES, NH and YY contributed to the materials and patients. ES and YY provided pathology review. ES, KM and SF carried out experiments. ES, KM, SF and YY analysed and interpreted the data. ES and YY generated tables and figures. All authors were involved in writing the paper and had final approval of the submitted manuscript.Table S1. Hotspot panel of 23 cancer‐related genesClick here for additional data file.Table S2. Oncogenic mutations related to the MAPK pathway and HPV status for 51 SCPsClick here for additional data file.Table S3. Average read depth for the targeted KRAS, HRAS, and FGFR3 ampliconsClick here for additional data file.