To increase knowledge about options people have concerning end-of-life-care issues, General Practitioners (GPs) can organise meetings to inform their older patients. We evaluated these meetings, using the following research questions: How did the attendees experience the information meeting? Was there a rise in Advance Care Planning (ACP) behaviour after the information meeting? Was there a change in trust people have that physicians will provide good care at the end of life and that they will follow their end-of-life wishes after the information meetings?Four GPs invited all patients of 75 years and older registered in their GP practices to the meeting via a written letter. Four meetings of 2 h took place in 2016. Meetings started with a presentation on end-of-life topics and ACP by the GP followed by time for questions.0) at the end of the meeting (T1) and 6 months after the meeting (T2).A pre-post evaluation study was done using written questionnaires distributed and filled in at the start of the meeting (T0 and 145 (64%) filled in the questionnaire at T1. After six months, 90 of the 121 people who approved of being sent another questionnaire at T2, returned it (40%). The average age of the respondents was 80 years (T0). The meetings were evaluated positively by the attendees (T1). ACP issues  were discussed with a physician, a relative or both more often in the 6 months after having attended the meeting (T2), compared to before (T0). Compared to before the meeting (T0), trust in the GP providing good end-of-life care and following end-of-life wishes was higher immediately after the meeting (T1), but not after 6 months (T2).In total 225 older people attended a meeting of which 154 (68%) filled in the questionnaire at TInformation meetings on end-of-life care by GPs have a positive influence on the occurrence of ACP, both with the physician and others. Although, this method especially reaches the older people that are already interested in the subject, this seems a relatively easy way to stimulate ACP.The online version contains supplementary material available at 10.1186/s12875-021-01463-3. Many people would like to be cared for at home at the end of life , 2. In tAdvance care planning (ACP) enables individuals to define goals and preferences for future medical treatment and care, to discuss this with their family and healthcare providers, and to record and update these preferences if necessary . AlthougOne barrier to engage in ACP often mentioned by the GP is time –13. FromFour GPs in the Netherlands took the initiative to organize information meetings for their older patient population, because they realized in their daily practices that older people often lacked basic information about palliative care and end-of-life issues. They asked researchers from a university to evaluate these information meetings.How did the attendees experience the information meeting?Was there a rise in ACP behaviour after the information meeting?Was there a change in trust people have that physicians will provide good care at the end of life and that they will follow their end-of-life wishes after the information meetings?In this paper we describe the evaluation of these meetings. The research questions were:0), at the end of the meeting (T1) and 6 months after the meeting (T2) to evaluate the information meetings.A pre- post evaluation study using written questionnaires distributed and filled in at the start of the meeting . The meetings lasted about 2 h, started with a powerpoint presentation on end-of-life topics and ACP by the GP, followed by time for questions. After the first meeting, the presentation was adapted to provide more information on palliative care. In the following three meetings, the GPs in that meeting used the same adapted powerpoint presentation.0, and T1. Attendees were asked by the researchers to fill in the first questionnaire before the meeting started (T0). At the end of the meeting they were asked to fill in a second questionnaire (T1). The researchers collected the T0 and T1 questionnaires when the participants left the meeting.When arriving at the venue, before the meeting started, the researchers handed out the questionnaires for T2). If so, the GP sent the questionnaire after 6 months to the respondents home address and respondents could return the questionnaire to the researchers using a pre-addressed return envelope. With this procedure, the researchers did not have identifying data about the respondents, and GPs did not have the completed questionnaires. The questionnaires had a unique identification number to keep track of the response. Data were stored on a secured network drive of Amsterdam UMC that is only accessible to the researchers.Participants were also asked to fill in a consent form if they consented to the GP sending them a last questionnaire 6 months after the meeting (T0) contained questions on expectations of the meeting and reasons for coming to the meeting, questions on health, illness and quality of life, trust in the GP, previous experience with ACP, and demographics. The questionnaire at the end of the meeting (T1) contained questions evaluating the meeting, ACP related plans after the meeting, and trust in the GP. The questionnaire 6 months after the meeting (T2) had questions on health, illness and quality of life, trust in the GP, experience with ACP, and demographics. All questionnaires were specifically developed for this study. See the The questionnaire at the start of the meeting . Because of small percentages in the categories ‘not much trust’ and ‘no trust’ these were grouped together. Pre- and post-test differences in ACP conversations and Advance Directives were analysed in McNemar-test for paired samples performed on attendees who filled in a questionnaire before and 6 months after the information meeting. In Figs. 0 and 145 (64%) filled in the questionnaire at T1. After six months, 90 of the 121 people who approved of being sent another questionnaire at T2, returned it (40%).In total 225 older people attended a meeting Table  of whichThe majority of attendees were female (61%) and the mean age of attendees was 80 years Table . Althoug1) if the meeting matched their expectations, which was the case for a majority (63%) of participants. They could explain their answer regarding expectations in an open question. A total of 85 comments were given, which were categorised into 113 codes. Most (67%) answers were positive, for instance that the meeting was good or that they thought the information was clear. A total of 18 (9%) comments were less positive, mostly pertaining to inaudibility of the questions asked  or lengthy elaborations of other attendees on personal matters during the discussion. The information on Advance Directives (21%) and the importance of talking to family, friends and care providers (15%) were mentioned most when asked about the most important thing they had heard.More than half of attendees attended the meeting because they wanted to know more about the end-of-life (57%) or because they think about the end-of-life (52%). Most attendees expected information on practices to hasten death  (28%) and possibilities of palliative care (21%). They were asked immediately after the meeting , clarity of information , possibility to ask questions  and answers to questions posed . With regard to clarity of the information and answers to questions meeting B appeared to be evaluated more favourably, compared to meetings A, C and D , discussed with physicians (5% vs 18%) or others 10% vs 36%) and written down (7% vs 18%) preferences regarding treatments under certain circumstances, compared to before (Table 6% and wrn = 56) or plan to put down their wishes in writing (n = 59) have done so six months later in 39% (n = 22) and 36% (n = 21) of the cases. Attendees with plans to discuss wishes with others (n = 58) have acted accordingly in 78% (n = 45) of the cases, and a further 12 out of 14 attendees who did not initially plan to do so did discuss their preferences with others  and to follow end-of-life wishes  after the meeting, compared to before Table . This diThe information meetings were evaluated positively by the attendees and yielded positive outcomes. Six months after the meeting attendees more often had thought about, discussed with physicians or others and written down preferences regarding appointing a proxy, resuscitation, and treatment preferences under certain circumstances, compared to before. Attendees more often had thought about hospitalisation and discussed this with another person (not a physician) after the meeting than before. Whether the attendee could or wanted to stay at home and nursing home admittance were discussed with another person more often after the meeting, compared to before. Euthanasia was discussed with a physician and another person more often after the meeting, compared to before. The percentage of attendees who have acted on plans to discuss wishes with others was higher (78%) than the percentage of attendees who have acted on plans to discuss wishes with physicians (39%) and put preferences in writing (36%). Trust was higher immediately after the meeting, but not after 6 months.The meetings seem to be a time efficient way to stimulate ACP conversations with the GP and others. A recent study on two public health interventions (‘Awareness-Raising’ presentations and ‘How to’ workshops) also yielded positive results with regard to stimulating discussions . The GPsThe data suggest it may be easier to realise plans to discuss wishes with others, compared to talking to physicians or putting preferences in writing. Previous research also indicated that conversations more often were with family or friends than with a physician . TalkingImmediately after the meetings, trust in physicians to provide good care and to follow wishes was higher than before and six months after the meeting. This may indicate that attendees felt that the GP came across as knowledgeable or nuanced  and/or felt that the role of the GP in care delivery and decision making was clearly discussed. The effect disappeared after 6 months. However, when comparing attendees who had or had not spoken to a physician after six months, those who had scored higher on trust than those who had not spoken to a physician. Future studies should explore this rise and fall of trust and the relation with ACP more deeply. Other studies indicate a complex interaction between trust and ACP –15, 22.There are few studies on information meetings , 23, andThe meetings seem to be a time efficient way to stimulate ACP discussions with the GP and others. The meetings were evaluated positively by the attendees and ACP discussions increased after the meetings, especially with friends and family.Additional file 1: Questionnaire at T0. Questionnaire at T1. Questionnaire at T2. Table A.1. Experiences of 145 attendees with information meetings (April – August 2016).


Is salience bias—the change in perception of risk due to increased familiarity with the outcome—associated with human papillomavirus (HPV) vaccination decisions?In this cohort study of 757 428 children, there was no major difference in the HPV vaccination rate of children whose mothers had a history of either cervical cancer or a cervical biopsy compared with children whose mothers had neither history.These findings suggest that salience of vaccine-preventable outcomes is not associated with childhood vaccine hesitancy. This cross-sectional study assesses the potential role of salience in influencing vaccination decisions by analyzing human papillomavirus vaccination rates among preadolescent children whose mothers had a history of cervical cancer or cervical biopsy. Barriers to childhood vaccination against vaccine-preventable diseases, such as those due to human papillomavirus (HPV), are well known. However, the role of salience bias—the change in perception of risk due to increased familiarity with the outcome—in decisions to vaccinate children has not been explicitly studied.To assess for salience bias in parental decisions to vaccinate children.This retrospective cohort study used a time-to-event  analysis to compare vaccination rates of children whose mothers had a history of cervical cancer or a cervical biopsy, who have experienced adverse vaccine-preventable outcomes, and for whom vaccination may be more salient, with a control group of children whose mothers had no such history. Participants were accrued from the MarketScan Commercial Database, including US children who turned 11 years old, when HPV vaccination is recommended, from January 1, 2014, to December 31, 2018. Data were analyzed from December 29, 2020, to September 17, 2021.Maternal history of cervical cancer or cervical biopsy.Vaccination against HPV.A total of 757 428 children (370 878 girls [49.0%] and 386 550 boys [51.0%]) were identified, of whom 38 366 had mothers with a history of cervical biopsy alone and 1084 had mothers with a history of cervical cancer. Overall, 54.2% of children (55.7% of girls and 52.7% of boys) received at least 1 vaccination by 16 years of age. In a time-to-event analysis, HPV vaccination did not differ between children whose mothers had cervical cancer vs those whose mothers did not . Maternal history of cervical biopsy was associated with a minimally increased hazard of vaccination . There were no clinically meaningful differences between groups for the tetanus/diphtheria/acellular pertussis and meningococcal vaccinations, which are also recommended at 11 years of age.In this analysis of salience bias in childhood vaccination decisions, mothers’ personal history of cervical cancer or cervical biopsy was not associated with greater vaccination rates among children against HPV. These findings suggest that salience of vaccine-preventable outcomes may not have a major impact on childhood vaccine hesitancy in HPV; the role of salience should be investigated for other vaccines. In 2017, for example, the CDC estimated that only 48.6% of adolescents were up to date on the human papillomavirus (HPV) vaccine, which prevents cervical cancer, although 65.5% had received at least 1 dose of the multidose recommended series .9 Parental decisions on whether to vaccinate their children might reasonably be influenced by how they perceive risk of the disease or risk of a devastating outcome—an outcome with which a parent may have no personal connection or experience. For example, parents who choose not to vaccinate their young children against diseases such as measles perceive their children as less susceptible to infection and perceive the disease as less severe than parents who fully vaccinate their children.10Vaccination, and disease prevention more generally, can be particularly challenging because patients and populations often lack personal or emotional connections to the outcome being targeted.11 This change is likely due to an increase in perceived risk by parents from a combination of an often small increase in absolute risk of the disease and a newfound connection with the disease. This increased perceived risk based solely on personal connection is an example of salience bias, or a tendency to judge the risk of an event by how personally connected one is to the outcome.However, when large outbreaks of a vaccine-preventable disease such as measles occur within a local community, rates of vaccination against that disease increase.12(p1127) Salience bias has been shown to affect decision-making in many areas both inside and outside of medicine, such as automobile pricing, energy conservation while showering, and preferences around cardiopulmonary resuscitation in advance care planning.15 In other areas of medicine, it is possible that a lack of disease salience may lead people to not seek recommended care.In a seminal 1974 study, Tversky and Kahneman framed salience bias in simple terms: “the impact of seeing a house burning on the subjective probability of such accidents is probably greater than reading about a fire in a local paper.”We assessed the potential role of salience in influencing vaccination decisions by analyzing HPV vaccination rates among preadolescent children whose mothers had experienced adverse outcomes that can result from HPV infection  and are now vaccine preventable. We used a nationwide database of insurance claims to select children who turned 11 years old after the HPV vaccine was added to the recommended vaccine schedule and compared HPV vaccination rates between children whose mothers did vs did not have a history of cervical abnormalities . We hypothesized that a large increase in HPV vaccination rates would occur owing to salience  among children whose mothers had experience with cervical abnormalities compared with children whose mothers did not.22 Owing to the use of deidentified data, this study was deemed exempt from review by the Harvard Medical School Institutional Review Board. The study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.We used the MarketScan Commercial Database (IBM Corp), which contains commercial insurance claims of tens of millions of Americans covered by employer-sponsored insurance plans from more than 300 employers. This database has been used in prior studies of vaccinations, including the HPV vaccine, in children, adolescents, and adults.The primary study population was children who turned 11 years old between January 1, 2014, and December 31, 2018, who were continuously enrolled since their 9th birthday, and whose mothers and metropolitan statistical area (MSA) of residence were identifiable in the database. Children were followed up for as long as they were continuously enrolled after their 11th birthday; as such, children contributed variable person-time to the analyses.The HPV vaccine has been recommended by the CDC’s Advisory Committee on Immunization Practices for routine vaccination of girls since 2006 and boys since 2011. We selected children who turned 11 years old starting in 2014 because by then the HPV vaccine would have been a well-established routine vaccination for both girls and boys. Because the Advisory Committee on Immunization Practices allows but does not specifically recommend vaccination as early as 9 years of age, we limited our sample to children who were followed up continuously in the database since that age in case they were vaccinated early, which may have been more likely among children of mothers with a history of cervical abnormalities.Common Procedural Terminology (CPT) codes 90649, 90650, and 90651. The time elapsed was recorded in months from the month of the child’s 9th birthday to the date of vaccine administration. If a child received a second vaccine dose, the timing of that dose was also recorded. Falsification analyses were performed using alternate, non-HPV vaccinations, which were identified by procedure codes for a single dose of the meningococcal vaccine  or the tetanus/diphtheria/acellular pertussis (Tdap) vaccine (CPT code 90715).Human papillomavirus vaccination was identified by the presence of a billing code for an HPV vaccine administration, defined by 23 and the US Census Bureau,24 respectively. The mean number of preventive care visits per year was determined by the number of unique insurance claims for ambulatory encounters with CPT codes 99381 to 99387 or 99391 to 99397, divided by the number of years the participant was included in the database.Children’s month and year of birth were obtained from the database to determine when they turned 11 years of age, when the first HPV vaccination was recommended. Other child demographic characteristics, including sex, MSA of residence, state of residence, and number of insurance dependents in the child’s family, were also obtained from the database. Mean per-capita income and percentage of adults with a bachelor’s degree or higher level of education in 2011 for the primary insurance holder’s MSA of residence were obtained from the US Bureau of Economic AnalysisParticipants’ mothers were identified as the female primary policy holder or the female spouse of a male primary policy holder of the insurance policy under which the child was listed as a dependent. We determined maternal history based on diagnosis codes from mothers’ insurance claims before the first of the following events: the child’s first HPV vaccination, turning 16 years of age, or the child’s departure from the database. Mothers were classified into 3 mutually exclusive groups: (1) history of cervical cancer; (2) history of cervical biopsy without a history of cancer; or (3) controls without a history of cervical biopsy or cervical cancer based on insurance claims.International Classification of Diseases, Ninth Revision, codes 180.X for claims before October 1, 2015, and International Statistical Classification of Diseases and Related Health Problems, Tenth Revision, codes C53.X for subsequent claims. History of cervical biopsy was defined by CPT codes 57452, 57454, 57455, 57456, 57460, and 57461.History of cervical cancer was defined by 25 . We then compared vaccination rates of children in a retrospective cohort study using a cumulative time-to-event  analysis, stratified by both the child’s sex and mother’s history of cervical abnormalities. We hypothesized that mothers with a history of cervical abnormalities would be more likely to have their children vaccinated and more likely to vaccinate them sooner owing to personal history with cervical disease, which would increase salience of HPV vaccination. Although the CDC recommends HPV vaccination among children aged 11 and 12 years, it can be given to children as young as 9 years and to adolescents of any age. A cumulative time-to-event analysis was therefore an ideal approach to detect differences between strata of maternal cervical history during a long period with variable follow-up time among the children studied.Data were analyzed from December 29, 2020, to September 17, 2021. Baseline characteristics between groups of children whose mothers had a history of cervical abnormalities were compared with those of controls using standardized mean differencesIn each time-to-event analysis, participants were followed up from their ninth birthday (the earliest the CDC suggests the vaccine can be given) until the outcome of interest , or until a censoring event, including departure from the database or the month of their 16th birthday at the end of the study period. The study period ended at the 16th birthday to allow any delayed vaccinations to be captured while maximizing the sample size of continuously enrolled patients. We stratified by the child’s sex because we hypothesized a larger effect of salience on vaccination of girls, who are at risk for adverse cervical outcomes. We used Kaplan-Meier methods to generate cumulative event curves and tested differences between strata using the log-rank test.19), year of birth, number of insurance dependents in the household, number of child preventive care visits, MSA mean per capita income, and MSA percentage of adults with a bachelor’s degree or higher level of education.We conducted a Cox proportional hazards regression analysis to address the possibility that unmeasured confounders might lead to a lack of observed evidence of maternal salience in child HPV vaccinations. Specifically, if confounding factors  are associated with greater likelihood of maternal cervical abnormalities and lower rates of child HPV vaccinations, a failure to account for these factors would lead us to spuriously conclude that a maternal history of cervical abnormalities has no salience for child HPV vaccinations . We used Cox proportional hazards regression to generate models that were adjusted for state of residence, calendar month of birth . Second, we conducted similar time-to-vaccination analyses for non-HPV vaccinations recommended at 11 years of age  under the assumption that any unobserved confounding factors in the HPV analysis would likely lead to differences in non-HPV vaccination rates between children whose mothers had varying cervical abnormality histories, a falsification analysis. Third, to assess for the possibility of bias introduced by static group assignment, we repeated the time to first HPV vaccination analysis excluding children whose mothers’ cervical biopsy or cervical cancer diagnosis occurred after the child turned 11 years of age.P < .05 indicated statistical significance.Analyses were performed using R, version 3.5.2 , and STATA, version 15 (StataCorp LLC). The 95% CIs around estimates reflect an α level of .025 in each tail. Two-sided The primary study population included 757 428 children, of whom 370 878 (49.0%) were girls and 386 550 (51.0%) were boys. Overall, 38 366 children  had mothers with a history of cervical biopsy alone, 1084 children  had mothers with a history of cervical cancer, and 717 978 children  had mothers without a known history of cervical biopsy or cervical cancer. Characteristics of these groups were similar (6Among 281 823 children who were followed up until the month of their 13th birthday or beyond (138 001 girls and 143 822 boys), for whom at least 1 HPV vaccination should have been performed under CDC guidelines, only 54.2% (55.7% of girls and 52.7% of boys) had received at least 1 vaccine by 16 years of age, findings consistent with national survey data.2 = 15.8; P < .001), the differences were clinically small, particularly during the CDC-recommended vaccination window of 11 to 12 years of age (P = .86) and maternal cervical biopsy was associated with only slightly higher hazard of first-dose vaccination  when compared with girls without a maternal history of cervical cancer or cervical biopsy.For both boys and girls, HPV first-dose vaccination patterns were similar regardless of maternal cervical history. For example, unadjusted analysis of time to first dose  for girls showed that although the timing of the first HPV vaccine dose was statistically significantly different between groups of girls based on maternal cervical history  of first HPV vaccine dose for boys with varying maternal cervical history (P = .27) and boys with a maternal history of cervical biopsy had only slightly higher hazard of first-dose HPV vaccination  compared with boys without a maternal history of cervical cancer or cervical biopsy.For boys, unadjusted analysis of time to first dose showed no patterns of statistically or clinically significant difference  and girls  before and after adjustment (Among 281 823 children (138 001 girls and 143 822 boys) who were followed up until or beyond the month of their 13th birthday (the age by which CDC guidelines recommend at least 2 HPV vaccinations should have been administered on the routine schedule), only 38.3% (40.2% of girls and 36.5% of boys) had received 2 vaccines. Findings similar to those for analyses of time to first dose were observed in analyses of time to second dose comparing children whose mothers had a history of cervical cancer or cervical biopsy and those whose mothers did not; findings were similar for both boys  or were diagnosed with cervical cancer (32 girls and 30 boys) after the child turned 11 years of age  may not have been accurately identified in a small number of patients. However, given the high prevalence of HPV and its cervical complications among all maternal populations in the years included in this study, it is unclear why this restriction would lead to selection bias that would affect the study’s conclusions. Finally, this study was performed in a population of commercially insured children, limiting generalizability to other populations.This study has several limitations. First, residual confounding by factors associated with both a mother’s history of cervical cancer or cervical biopsy and the family’s likelihood to vaccinate their child is possible, despite adjustments for family and regional characteristics. Vaccination rates and patterns were similar among groups in the Tdap and meningococcal vaccine analyses, which suggests that confounding factors do not explain the entire lack of observed evidence for major influence of salience in influencing HPV vaccinations specifically. Second, in addition to the assumption of proportional hazards between groups during the study period, the Cox proportional hazards regression models also assumed fixed group assignment, which could introduce bias. Although it is possible that a mother was diagnosed with cervical cancer during the child’s inclusion period—meaning that a child could have been assigned to the group with maternal history of cervical cancer even though until that time they were similar to another group—there is no evidence in our results of any significant bias introduced by these definitions. Third, our analysis was restricted to children whose mothers were on the same insurance policy, which allowed for family linkages; a The findings of this time-to-event cohort study suggest that children whose mothers had a history of cervical cancer or cervical abnormalities requiring biopsy were not more likely to vaccinate their children against HPV, despite their personal experience with vaccine-preventable adverse outcomes. These findings also suggest that mothers with a history of adverse cervical outcomes may be either unaware of the causal link between HPV and cervical disease or that from a behavioral perspective, increased salience of the risks of HPV does not outweigh other factors that contribute to vaccine hesitancy.


INK4a overexpression, suggesting these tumors were HPV+. This was confirmed after PCR, in situ hybridization, and mass spectrometry detected the presence of HPV type 16 (HPV16) DNA, RNA and protein. To further characterize the presence of HPV16, we used a target enrichment strategy on tumor DNA and RNA to isolate the viral sequences from the brain metastases. Analysis by targeted next generation sequencing revealed that both tumors had the HPV genome integrated into the host genome at known hotspots, 8q24.21 and 14q24.1. Applying a similar target enrichment strategy to a larger cohort of HPV+ HNSCC brain metastases could help to identify biomarkers that can predict metastasis and/or identify novel therapeutic options.Human papillomavirus (HPV) positive and negative head and neck squamous cell carcinoma (HNSCC) are known to have differential phenotypes, including the incidence and location of metastases. HPV positive (HPV+) HNSCC are more likely to metastasize to distant sites, such as the lung, brain, and skin. Among these locations, metastasis to the brain is a rare event, and little is known about specific risk factors for this phenotype. In this report, we describe two patients who developed brain metastases from HNSCC. Both patient tumors had p16 Head and neck squamous cell carcinoma (HNSCC) affects more than 50,000 patients in the United States every year and accounts for over 10,000 deaths . The majWhile the presence of HPV in tumors can be used to stratify HNSCC patient populations, the evidence suggests the relationship between HPV and tumor phenotype may be more complex. For example, it has been hypothesized that transcriptional activity of HPV may be more important than HPV status alone in predicting the tumor phenotype. One study reported that transcriptionally inactive HPV+ HNSCC had similar gene expression patterns to HPV− tumors, and both showed worse progression free survival compared to HPV transcriptionally active tumors .HPV integration into the host genome is an important step in HPV-induced tumorigenesis in cervical cancer  and may To this point, limited research has examined the molecular characteristics of HPV in HNSCC brain metastases. Here, we provide a detailed genetic characterization of two cases of HPV16+ OPSCC brain metastases. These tumors were highly aggressive and featured metastases to lymph nodes and other sites. Multiple assays to detect HPV indicated that both patients had HPV16 positive tumors that expressed HPV transcripts. We were able to confirm integration of HPV16 into the human genome at two hotspots for HPV integration, 14q24.1 and 8q24.1. Similar to other studies with primary HNSCC , the HPVAt the time of surgery, tumor tissue was removed and processed by Swedish Medical Center pathology services for paraffin embedding. Excess metastatic tissue (0.5–1 g) was transported to the laboratory on ice, flash frozen in liquid nitrogen, and stored at −80 °C. Paraffin sections were used for immunohistochemistry (IHC) and in situ hybridization (ISH) experiments. All patients were consented to participate in this study under our protocol approved by the WCG IRB (formerly Western IRB).c Receptor Block (Biogenex) and 30 min in 1% normal horse serum (Vector Laboratories). Sections were incubated overnight at 4 °C in 1:200 rabbit IgG anti-CDKN2A/p16INK4a antibody . After washing 3 times in TBST, sections were incubated for 30 min in Vector ImmPRESS HRP Horse Anti-Rabbit IgG Polymer reagent. After washes, slides were developed for 3 min with Vector ImmPACT DAB. Sections were then counterstained with hematoxylin, dehydrated, cleared and mounted using VectaMount (Vector Laboratories). Images were captured using a Leica DM 2000 upright microscope.Paraffin-embedded tissue sections (4 μm) were dewaxed in xylenes 3 times, passed through a series of graded alcohols, and washed in deionized water. For antigen retrieval, 1X CitraPlus AR Buffer (Biogenex) was pre-heated for 20 min in an Oster steamer before adding the slides for an additional 20 min. After cooling for 10 min, slides were washed 3 times in deionized water. Sequential blocking steps were performed—10 min in Bloxall (Vector Laboratories), 30 min in FParaffin-embedded tissue sections were assayed using RNAscope probes from Advanced Cell Diagnostics (ACD) for HPV16/18 E6/E7 mRNA (#311121), PPIB , and dapB . No modifications were made to the described protocol for the Manual RNAscope Assay.Total genomic DNA and RNA were extracted from 10–20 µg of tissue using Qiagen DNeasy and RNeasy kits. PCR for HPV16 E6 and HPV18 L1 was performed on extracted DNA as described in Hashida et al. . To deteA target enrichment high-throughput sequencing based on the SureSelect technology (Agilent) was used to detect HPV encoded DNA and RNA in these tumors. SureSelect RNA baits were custom designed to detect 9 different viruses . To achieve a high sensitivity, a high-resolution tilling design was applied with 5X coverage per region. Our custom designed SureSelect design contains 79,673 RNA biotinylated probes in total (Design ID: S3200262). Target enrichment reactions were performed using SureSelect XT kit. Enriched libraries were sequenced by Illumina sequencing platforms in a pool of 32 barcoded sample batches. FASTQ files were aligned to a reference sequence consisting of all targeted viruses (NC_001526.4) plus human genome GRCh38 by BWA for DNA  and STAR2, and HALT protease inhibitors (Thermo Scientific). Proteins were reduced with 20 mM dithiothreitol (DTT) for 10 min at 95 °C, alkylated with 40 mM iodoacetamide for 30 min at room temperature, and quenched with an additional 20 mM DTT. After alkylation, proteins were then acidified and loaded onto suspension traps (Protifi LLC), where they were digested with 1:25 sequencing grade trypsin (Promega) for 2 h at 47 °C. Resulting peptides were dried with vacuum centrifugation and brought to 1 μg/3 μL in 0.1% formic acid immediately prior to mass spectrometry acquisition.Protein samples were extracted with 5% SDS with 50 mM Triethylammonium bicarbonate, 2 M MgClm/z to 800–900 m/z injections) or 5 μL (900–1000 m/z injections) was loaded and eluted using a two-step gradient: 35 min from 7 to 14% buffer B, followed by 55 min from 14 to 40% buffer B.Tryptic peptides were separated with a Thermo Easy nLC 1000 and emitted into a Thermo Fusion Lumos using a 75 μm inner diameter fused silica capillary with an in-house pulled tip. The column was packed with 3 μm ReproSil-Pur C18 beads (Dr. Maisch) to 30 cm. A Kasil fritted trap column was created from 150 μm inner diameter capillary packed to 4.5 cm with the same C18 beads. Peptide separation was performed using 250 nL/min flow with solvent A as 0.1% formic acid in water and solvent B as 0.1% formic acid in 80% acetonitrile. For each injection, 3 μL  were downloaded from Uniprot on 29 May 2020, containing 1349 total entries. This FASTA database was digested in silico to create all possible +2H and +3H peptides between 7 and 30 amino acids with precursor m/z within 396.43 and 1002.70, assuming up to one missed tryptic cleavage. This peptide sequence and charge state list  was concatenated to a listing of 115,833 unique peptide sequence and charge state pairs that were detected in unpublished experiments of brain tumors and cell lines. Peptide fragmentation and retention time predictions for all peptides were made with the Prosit tool  and collData-independent acquisition (DIA) data were spectrum demultiplexed  with 10 INK4a overexpression  tissue for evidence of HPV in all tumors . Consistent with the presence of HPV, all four tumors displayed p16hemistry I–L. We npression M–P. In apression . HPV16 Epression .To further investigate the HPV molecular signature, we used a custom designed SureSelect kit (Agilent) to enrich for viral nucleic acids followed by next generation sequencing (NGS) on an Illumina Next Seq 500 system. Our kit contained probes covering the whole genomes of HPV16 and HPV18 as well as several herpesviruses. Resulting reads were aligned with BWA to a reference sequence consisting of all targeted viruses (with NC_001526.4 for HPV16) merged with human GRCh38/hg38. We detected HPV16 DNA in both cases, with no evidence of any other viruses above background reads. Case 1 showed coverage across most of the HPV16 genome, except for a 1500 bp deletion (NC_001526.4:643–2122) in the E1/E2 region A. This dWe also sought to determine the extent of viral gene expression in Case 2 using the same SureSelect target enrichment strategy on cDNA libraries prepared from total tumor RNA. As expected from the DNA sequencing data, NGS data showed significant numbers of HPV16 reads and no HPV18 RNA reads. The highest expressed transcripts were the E1^E4, E6 and E7 genes, with comparatively little expression of E1 and E2 A. The seTo detect HPV encoded proteins, we analyzed protein lysates extracted from the metastatic tumor with tandem mass spectrometry. We searched the acquired data with EncyclopeDIA  using a Brain metastases from HNSCC carry a dire prognosis for patients, and the risk factors for their development are poorly understood. In this study, we present two cases of patients with HPV16+ OPSCC. Both patients developed brain metastases at 16 or 20 months after initial disease presentation, and neither patient had prior evidence of other distant metastases at this point. This time frame is within a typical range for HNSCC, as different studies have reported a range of median values between 17 and 36 months ,14,37,38INK4a and ISH for the E6/E7 genes from two common high-risk HPV strains, HPV16 and HPV18. Both assays supported the presence of HPV in both the initial and metastatic tumors, although we could not discriminate between strains with these assays. Subsequent assays were performed on DNA, RNA, and protein extracts from flash frozen metastatic brain tumor tissue. These indicated that only HPV16 was present in both tumors. We also determined that both metastatic tumors expressed HPV transcripts, including two E6 splice forms, E6*I and E6*II. This is important to identify, as active HPV transcription has been shown to have prognostic value in HPV+ OPSCC [We implemented a multifaceted strategy to characterize the molecular phenotype of HPV in these cases. Using FFPE tissue sections from the primary and metastatic tumors, we performed IHC for p16We next supplemented these results with target enrichment sequencing for HPV and human herpesviruses. This approach can be very powerful for molecular profiling of viruses in tumors, and it has already shown promise in identifying HPV sequences from human samples ,40,41. TWe found that Case 1 had HPV integration at 14q24.1 and Case 2 had HPV integration at 8q24.21. Neither one of these directly disrupted human gene expression, and for Case 2, we were also able to show that hybrid human-viral transcripts from this integration were expressed. Understanding these details is important, as evidence suggests that HPV integration into the host genome can influence tumor phenotypes. When hybrid human-HPV transcripts were expressed, this correlated with a worse prognosis . In addiIn summary, we have presented detailed genetic characterizations of HPV16 in two OPSCC brain metastases. We have shown that each tumor was HPV16+ by multiple methods, including PCR, in situ hybridization, next generation sequencing and mass spectrometry. Both cases showed evidence of HPV integration into the host genome at known hotspots, although the significance of these sites is unknown. More comprehensive future studies using target enrichment strategies could help to identify specific commonalities in HPV integration among brain metastases. A more detailed understanding of this relationship could lead to new biomarkers to predict future metastasis and/or new treatment options.


Dear Editor et al. "The Prevalence of HPV Genotypes in Iranian Population: An Update", which pathologically mostly lead to Squamous intraepithelial lesions (9). Our findings suggested which Pap smear report is most associated with each serotype of HPV; however, the "No Inflammation" state is the most common among all serotypes (39.4%). Detailed HPV PCR test and Pap smear cross-tabulation results are shown in . We recommend cytologists report the presence of Anucleated squamous or Keratinized cells and exudates in the positive smears in order to prevent HPV-related Cancers in the population.Thus Type 16 is the most serotype involving in HPV infections among the average Iranian population , 6, 7, 8


There is limited access to effective cervical cancer screening programs in many resource‐limited settings, resulting in continued high cervical cancer burden. Human papillomavirus (HPV) testing is increasingly recognized to be the preferable primary screening approach if affordable due to superior long‐term reassurance when negative and adaptability to self‐sampling. Visual inspection with acetic acid (VIA) is an inexpensive but subjective and inaccurate method widely used in resource‐limited settings, either for primary screening or for triage of HPV‐positive individuals. A deep learning (DL)‐based automated visual evaluation (AVE) of cervical images has been developed to help improve the accuracy and reproducibility of VIA as assistive technology. However, like any new clinical technology, rigorous evaluation and proof of clinical effectiveness are required before AVE is implemented widely. In the current article, we outline essential clinical and technical considerations involved in building a validated DL‐based AVE tool for broad use as a clinical test. What's new?An emerging option for cervical cancer screening is deep learning‐based automated visual evaluation (AVE) of cervical images. Here, the authors lay out parameters for the successful development of deep learning‐based AVE. For instance, an algorithm should be trained on representative images from each of four distinct biological stages: normal cervix; infection with high‐risk HPV; precancer; and invasive cervical cancer. Characteristics that may lead to erroneous classification, such as cervicitis, should also be considered in the training. Introducing deep learning‐based methods prematurely threatens their eventual acceptance and best use. AIartificial intelligenceAISadenocarcinoma in situALTSASCUS‐LSIL Triage StudyASC‐USatypical squamous cells of undetermined significanceAVEautomated visual evaluationCINcervical intraepithelial neoplasiaCNNconvolutional neural networkDLdeep learningDNAdeoxyribose nucleic acidDRdiabetic retinopathyDSLRdigital single lens reflexECCendocervical curettageFGSfemale genital schistosomiasisHIVhuman immunodeficiency virusHPVhuman papillomavirusHRhigh‐riskLFUlosses‐to‐follow‐upLLETZlarge loop excision of the transformation zoneLSILlow‐grade squamous intraepithelial lesionMLmachine learningNHSNatural History StudyROIregion of interestSCJsquamocolumnar junctionSILsquamous intraepithelial lesionTZtransformation zoneVATvisual assessment for treatabilityVIAvisual inspection with acetic acidWHOWorld Health OrganizationWLWHwomen living with HIV1Cervical cancer remains a leading cause of women's morbidity and mortality in resource‐limited settings.Existing cervical cancer screening and triage technologies fall into three categories: visual, microscopic  and molecular .Visual inspection of the cervix after applying acetic acid (VIA), though widely used in low‐resource settings for primary screening or triage, is poorly reproducible across settings and not reliable in discriminating precancers from benign HPV‐related and “look‐alike” changes.Deep learning (DL)‐based automated visual evaluation (AVE) of cervical images is emerging as an alternative novel, low‐cost screening and triage solution. Machine learning (ML) is a type of artificial intelligence (AI) that uses computers to detect patterns in data without being explicitly programmed to do so.General reporting guidelines for clinical trials with AI‐interventions have been reported previously.22.12.1.1Detecting and treating precancer is the main aim of cervical screening.In contrast, among the HPV positives, the prevalence of precancer increases considerably from ~1% to >5%.BOX 1HPV testing for carcinogenic HPV types is the most sensitive method for cervical cancer screening, providing many years of reassurance .Currently, the cost is the prohibitive factor in adopting HPV as a primary screening test in many low‐resource settings. However, based on available tests that cost <5 US dollars and take <1 hour to perform and offer partial HPV genotyping, even lower‐cost, point‐of‐care HPV tests will likely be widely available in a few years.HPV infection is too common to treat all infected women, most of whom do not need treatment, particularly given possible iatrogenic harms. Relying on negative HPV testing to reassure most women against cervical cancer risk permits public health efforts to focus on the triage of HPV‐positive women with newer technologies like HPV typing and AVE.Risk‐informed hierarchical partial genotyping of HPV, if incorporated with minimal additional cost into HPV testing, provides important risk stratification useful for triage of HPV‐positive women.It is pertinent to note that if AVE is used alone for standalone primary screening, “look‐alike” confounding conditions like severe cervicitis could lead to over‐treatmentRecognizing the eventual importance of vaginal HPV testing, we aim to develop a screening strategy using HPV self‐sampling, with risk‐informed partial HPV typingIf found to be effective, this screening strategy is envisioned by our group to be scaled up in a community‐based campaign combining: (a) screen‐and‐treat screening of mid‐adult women , and (b) single‐dose vaccination of multiple birth‐cohorts of girls and younger women to induce herd protection.The prevalence of visual abnormalities and precancer further increase in a colposcopy clinic, where most women have been referred for equivocal or minor cytologic abnormalities such as HPV‐positive atypical squamous cell of undetermined significance (ASC‐US) or squamous intraepithelial lesion (SIL), respectively.In addition, until sufficient supportive evidence accumulates regarding accuracy, reliability and portability of the method to different settings, AVE is best used as an ancillary technology to aid health workers performing VIA to improve their accuracy, rather than a standalone tool.2.1.2Any visual cervical screening methods, including AVE, works best when applied at an appropriate age range .2.1.3Detection of precancer is the main objective of cervical screening. However, based on the natural history, there are four biologically distinct stages in the pathogenesis of cervical cancer, which are: (a) normal cervix; (b) infection with high‐risk (HR)‐HPV (very common); (c) precancer, defined as transforming HR‐HPV infection associated with lesions with a high‐likelihood of invasion if left untreated (uncommon) and (d) invasive cervical cancer .2.1.4AVE needs to be trained on representative cervical images of each of the four natural history categories shown in Figure Invasive cervical cancer is defined histologically unless the clinical picture is so severe that surgical pathology is not obtained.Precancer is defined stringently as a histopathologic CIN3/AIS (adenocarcinoma in situ) since most histopathologic CIN3/AIS cases contain the same HPV types found in invasive cancers.However, histopathology cannot define the normal cervix, as most normal women are never thoroughly biopsied. Since the negative predictive value of the HPV test is very high, the ideal definition of “normal”  will be images from confirmed HR‐HPV negative women.Once cancer and precancer are defined histologically , and the normal cervix is defined visually, the remaining category can be conceived of as “HPV‐related and other equivocal changes.” Histopathology has limitations in defining this category due to subjectivity in microscopic diagnosis and biopsy placement errors .2.22.2.1A typical number of learned parameters in a DL algorithm development tend to be up to millions compared to tens in a traditional multivariate model.2.2.2The provider's training to capture good quality images is a first step for AVE's successful application. However, when an AI‐based image recognition tool is applied in real‐world clinics, variation in the quality of images is inevitable. The image quality is affected, in addition to the user training, by the lighting , image capture device and postcapture processing of images by device‐specific software, anatomic variation, speculums  and so on.Without a quality check, AVE will provide a prediction for any image given to it as an input, including images not even recognizable as cervix and images with a completely obstructed region of interest (ROI)  Figure .20, 5Various parameters define the image quality, such as blur, Gaussian noise, resolution, color, angle and glare/reflections; not all affect the AVE's performance equally. The composite minimal image quality standards needed to obtain a good performance on AVE is an ongoing advanced research topic.2.3Training a DL algorithm is more complex than the simple explanation described previously.In addition, the choice of methods has implications for time and computational speed requirements to run the algorithm. Ideally, a scalable AVE algorithm should be available to run as a standalone app (without internet) on the image‐capture device itself, providing quick (within few seconds), and real‐time predictions for on‐site patient's management to minimize loss‐to‐follow ups.2.42.4.1The essential first parameter in assessing AVE's validity, like any medical test, is reproducibility. Like a thermometer, giving a consistent reading of body temperature on the repeated measurement of the same person, an AVE algorithm should give virtually identical outputs when asked to predict the same image repeatedly. However, in the case of near‐duplicate images , subtle changes in the numerical pixel values of the image due to changes in body or camera position may alter the AVE predictions especially for equivocal images, despite the visual similarity of the images to the human eyes. Clinically, it is confusing to the user if an AVE algorithm were to label one image as a precancer and a near‐duplicate image (or same image in a different run) as normal Figure . Therefo2.4.2To “teach” the algorithm to recognize the target of interest, we provide it with sets of “labeled” cervical images in each target class as a “training” (to learn the features associated with the outcome of interest) and a “validation” set .In addition, it is important to include a realistic set of images in the “test” set on which the performance is finally evaluated. For example, we may observe good case‐control discrimination by AVE on a “restricted” test set, including only the clearest examples of high‐grade cases (CIN3) and HPV‐negative controls. However, it is important to realize that many cervix images will fall into an equivocal intermediate zone, including HPV infection, cervicitis and low‐grade changes. Without examining the discrimination and reproducibility achieved by AVE in this intermediate zone of “not so clear” case or control assigment  where even the expert colposcopists struggle the most, the promising claims about the algorithm's capacity could be misleading for real‐life implementation Figure .A critical aspect to evaluate the algorithm's performance is choosing the appropriate statistical approach. First, AVE class predictions can be compared against the reference standard  in a comprehensive independent test set. Second, the AVE, to be worth adopting, would ideally demonstrate consistently superior performance to the existing standard of care , and at least noninferior performance to the expert clinicians . Of note, AVE is not limited in performance by human factors such as fatigue, mental stress and so on; hence is theorized to have lower intraobserver variability in addition to lower interobserver variability than VIA and colposcopy, leading to higher consistency.2.4.3Verifying an AVE algorithm's performance is a two‐step process. Testing the algorithm's performance (achieving accurate predictions without overfitting) on an independent “test” set of images derived from the same source as the training set  is a crucial first step,2.4.4The AVE algorithm works on a pixel‐level . Therefore, an AVE algorithm trained on images from one type of image capture device tends to be overfitted to the features  of the particular device and works well only on that device.2.4.5Several patient characteristics may contribute to the erroneous classification of a given image by AVE Figure .15 ForThe co‐existence of human immunodeficiency virus (HIV) infection with HPV infection is probably the most important known difficulty in AVE classification. WLWH, due to shared risk factors for acquisition of infection, have 2‐fold increased likelihood of acquiring HPV. Due to HIV‐associated immunosuppression, they have a greatly elevated risk of persistent HPV and precancer, leading to a 6‐fold increased risk of cervical cancer compared to HIV‐negative women.HIV, FGS and cervicitis are highly prevalent in areas with a high burden of cervical cancer as well . Therefore, it is critical to evaluate the need to train the AVE to control for these factors as much as possible and whether to have a subgroup‐specific AVE is needed. Beyond the scope of this discussion, the use of DL algorithms to diagnose FGS is under consideration.2.4.6Before adopting any DL‐based diagnostic tool in clinical practice, the clinician should ask what is the tool measuring  and the limitations of its interpretation for clinical decision‐making. The major goal of AVE, ideally, is to directly predict the risk  of a woman having a precancer today while having some reassurance  for the future.2.4.7AVE algorithms have been commonly trained cross‐sectionally on the woman's present status and images, rather than a longitudinal set of images per woman, and are therefore likely to only predict a prevalent risk of precancer. At present, it is not known for how long a negative AVE test confers reassurance. It is unlikely to be for as long as an HPV test.2.5The considerations described here are mainly focused on the technical efficacy of AVE. When scaled‐up for implementation, outside the research settings, even well‐validated algorithms will have many challenges  as observed in other medical fields.The main important considerations specific for implementation of AVE for cervical cancer screening are human resource capacity‐building to manage screen‐positive women detected by AVE, developing data management systems to support tracking women needing referral and cost‐effectiveness analysis to evaluate AVE's impact in real‐life programs.It is important to emphasize that to prevent cancer we need to detect precancer lesions and treat them adequately. Absence of treatment is a major and unfortunately very common reason for screening program's failure. For women requiring treatment, thermal‐ablation using a battery‐operated mobile device is currently the most portable option given that it is safe, effective, affordable and does not require sophisticated equipment.3DL‐based AVE of the cervical image is a promising but still evolving clinical test. Even though the inner workings of DL remain obscure, DL‐based AVE, in the end, is no different from any other clinical diagnostic test. Since the limitations of the DL described here might not be fully appreciated by end‐users, the onus lies on the developer of an AI‐based device to make the subtle issues explicit, particularly in the less regulated markets. Raising awareness and knowledge of the goodness‐of‐fit and limitations of DL‐based AVE among end users is critical to improve clinical practice. Nonetheless, some AVE‐type products are already being marketed without substantial documentation of effectiveness.DL is the co‐founder of MobileODT. In the last 3 years, he was an executive into company and sat on the board of directors. He is no longer with the company, but still own some stock. He is currently the owner of Imaging and Analytics Consulting, Ltd., a small consulting company based in Israel. His wife is the owner of DL Analytics, LLC, a small business based in California. Other authors have nothing to declare.Kanan T. Desai, Silvia de Sanjosé and Mark Schiffman contributed substantially to the conception and design of the study. Ana‐Cecilia Rodriguez, Brian Befano, David Levitz, Didem Egemen, Helen Kelly, Jose Jeronimo, Julia Gage, Nicole Campos and Paul Pearlman contributed to the interpretation and critical thinking. Sameer Antani and Zhiyun Xue contributed to the DL algorithm development. Kanan T. Desai drafted the manuscript. All authors provided critical revision of the article and provided final approval of the version to publish.Appendix S1 Supporting Information.Click here for additional data file.


First-void urine usually contains exfoliated cells of the debris and mucus from the female genital organs and cervix, i.e., high concentration of human papillomavirus deoxyribonucleic acid (HPV DNA). We conducted a meta-analysis of published data and determined an accuracy of HPV detection in first-void urine compared to the women’s cervix. According to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, we carried out a comprehensive literature search. Eligible articles published from 2011 until 2021 were gathered by searching Embase, PubMed and Cochrane Library Central databases. The patient selection, index test, standard test, and patient flow were the factors involved in quality evaluation. A meta-analysis of 15 studies (3412 women) based on 5054 potential records was conducted. Pooled sensitivity for high-risk HPV detection in urine of 78% (70–84%) and specificity of 89% (81–94%) were calculated. Any HPV detection in urine of 87% (74–94%) and 91% (83–96%) were pooled sensitivity and specificity, respectively. HPV 16 and 18 had a pooled sensitivity of 77% (76–77%) and specificity of 98% (98–98%). Meta-analysis indicated variations between the pooled specificities and sensitivities. In meta-regression analysis, a heterogeneity in accuracy by using covariates  were not detected. Our meta-analysis demonstrates the accuracy of detection of HPV in urine for the presence of cervical HPV. Although progress is continuously made in urinary HPV detection, further studies are needed to evaluate and to improve the accuracy of the first-void urine test in order to be comparable with other screening methods. Is widely known that HPV is the primary cause of cervical cancer . CervicaA major impediment to controlling cervical cancer is lack of attendance for screening, i.e., in those countries without well-developed screening programs, from 50% to more than 80% of women are not screened .There has been a drastic decline in the incidence, as well as the mortality, of cervical cancer worldwide since the introduction of the Pap test ,11. HoweThere has been a great deal of interest lately in using urine as a liquid biopsy for HPV DNA testing, and this has increased due to observation of high correlations between urine and cervical HPV infections ,18,19,20Urine voiding in the first part (first-void urine) usually contains exfoliated cells of the debris and mucus from the female genital organs and cervix, i.e., the first-void urine contains higher concentrations of HPV DNA than midstream urine. According to this theory, the identification of biomarkers in first-void urine, as well as HPV DNA, can be used to screen for (pre)cervical cancer .Therefore, we conducted a systematic review and meta-analysis to determine the accuracy of detection of HPV in first-void urine compared with the cervix in women.According to recommended methods, a meta-analysis and systematic review was conducted in compliance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines .A literature review for the past 10 years (from January 2011 up to May 2021) in the three databases: Embase and Cochrane library (Title/Keywords/Abstracts) and PubMed (Title/Abstracts) was conducted. In each database, using Boolean logic, we searched for the following terms: (HPV or hrHPV or human papillomavir *) OR (HPV or hrHPV or human papillomavir *) AND (test * or assay * or genotyping or typing or detection or amplification) OR (HPV or hrHPV or human papillomavir *) AND (deoxyribonucleic or ribonucleic or nucleic or DNA or RNA or mRNA) OR  AND  OR  AND (urin *). We manually searched the relevant publications.The eligibility criteria included any test-of-accuracy study comparing HPV DNA detection in urine and cervix samples, in women with concern about infection with HPV or development of cervical cancer. If the reference standard was different or not available, we excluded the study. Meta-analysis included studies with data that could be converted into 2 × 2 table. A test’s diagnostic value can be overestimated by certain factors. Therefore, we excluded case-control studies, i.e., studies testing only cervical cancer patients or non-infected patients from the meta-analysis.For relevant studies, we screened all titles and abstracts. Two reviewers (P.B. and J.S.) independently performed a systematic literature search. In addition, P.B. screened the full texts of the included papers and extracted the subsequent data: characteristics of the study , characteristics of the patients , index test characteristics . To all studies the quality assessment of diagnostic accuracy studies-2 (QUADAS-2) was applied . The patUpon the detection of any HPV, high-risk HPV, HPV 16 and 18, the 2 × 2 table was made. If the study included more than one method for testing urine HPV, we selected the one with methods closest to those used by other studies. From the estimates, we derived a summary receiver operating characteristic (SROC) curve and the summary accuracy measures with 95% confidence interval (CI) ). The shape of a receiver operating characteristic (ROC) curve and the area under the curve (AUC) can help us get a sense of a test’s discriminative power, i.e., AUC presents the measure of diagnostic accuracy. If the curve is located as close as possible to the upper-lefthand corner, and the larger the area under curve, then the test will discriminate better between diseased and healthy individuals. A good indicator of the quality of the test is the area under the curve, which can range from 0 to 1. In a perfect diagnostic test, the AUC is 1, whereas in a nondiscriminating one, the AUC is 0.5 . The forhttps://crsu.shinyapps.io/dta_ma/ (Accessed date: 13 December 2021)). The MetaDTA statistical tool pools the sensitivity and specificity estimates for bivariate random-effects models. This model was fitted as a generalized linear mixed-effect model using the glmer function from the package lme4 of the statistical software R with shiny [A meta-analysis of diagnostic test accuracy was conducted using an online, freely available interactive web-based tool: MetaDTA, version 2.01 . To explore sources of heterogeneity in the studies, we used the Moses–Shapiro–Littenberg method by adding covariates to the model . Meta-rep value < 0.05 was considered statistically significant. Publication bias was conducted using R Studio (version 1.3.959) with “metafor” package. A Identifying and selecting studies is summarized in The characteristics of included studies in this review and meta-analysis are shown in Results of cytological analysis were recorded for 15 populations, i.e., 51% (1706/3360) women had normal conditions, 25% (848/3360) had atypical squamous cells of undetermined significance (ASCUS), 16% (542/3360) had low-grade squamous intraepithelial lesion (LSIL), 0.42% (14/3360) had atypical squamous cells, possible high-grade lesion (ASC-H), and 7.4% (250/3360) had high-grade squamous intraepithelial lesion (HSIL). From the 9 populations with reported histology results, 33.3% (304/912) of women had normal conditions, 25% (229/912) had CIN1, 14.6% (133/912) had CIN2, 1.2% (11/912) had CIN2+, 25% (229/912) had CIN3, and 0.66% (6/912) had histology proved cervical cancer. Conventional PCR was used in most studies, but the testing methods used were not uniform. Five of the 15 studies used real-time PCR ,34,40,41A quality evaluation of the studies is shown in The heterogeneity of sensitivity and specificity between individual urine detection studies of any HPV (10 studies), high-risk HPV (12 studies), and HPV 16 and 18 (7 studies) is shown in A SROC plot for pooled sensitivity and specificity for the three groups, (a) any HPV, (b) high-risk HPV and (c) HPV 16 and 18 is shown in A meta-regression with the following covariates  was conducted to identify the possible sources of heterogeneity. Using the Cochran’s Q test, likelihood ratios and diagnostic odds ratios were tested for homogeneity between studies. Heterogeneity and variation between studies were not confirmed using the covariates listed above .p = 0.19).We investigated the potential publication bias by using Deek´s funnel plot asymmetry test, as shown in The purpose of diagnostic tests in healthcare settings is to confirm or exclude diagnoses. Assessment of accuracy is determined by comparing the diagnostic test results with the “gold standard” according to which individuals’ true diagnosis can be determined. In our study, the HPV DNA in cervix samples represented the gold standard test, to compare with the HPV DNA in first-void urine samples. In Pathak’s review, accuracy of urinary HPV testing for cervical human papillomavirus was investigated through meta-analysis. There was only one source of heterogeneity identified, which was urine sampling, i.e., the accuracy of samples collected as random or midstream, as opposed to first-void samples, decreased by more than 22 times . The firTo evaluate the performance of a diagnostic test, we synthesized sensitivity and specificity from a meta-analysis of diagnostic test accuracy studies. In our meta-analysis, a heterogeneity between the pooled sensitivities and specificities was detected, i.e., pooled sensitivity for high-risk HPV detection in urine was 78% (70% to 84%) and specificity was 89% (81% to 94%). For any HPV detection in urine of 87% (74% to 94%) and 91% (83% to 96%), we pooled sensitivity and specificity, respectively. HPV 16 and 18 had a pooled sensitivity of 77% (76% to 77%) and a specificity of 98% (98% to 98%). The bivariate model has been shown to be mathematically identical to the HSROC model when covariates are not included. The HSROC parameters were estimated using parameters of the bivariate model and the equivalence equations of Harbord et al. The SROC plot was drawn using the resulting HSROC parameters , and it + and LR-. In our study, higher values of the positive likelihood ratio were detected, i.e., for any HPV detection, the LR+ was 15.62 (95% CI 4.60 to 53.05) and the LR- was 0.14 (95% CI 0.08 to 0.24). For high-risk HPV detection, the LR+ was 6.81 (4.07 to 11.41) and the LR- was 0.25 (0.18 to 0.34). For HPV 16 and 18 detection, the LR+ was 39.73 (39.33 to 40.14) and the LR- was 0.24 (0.24 to 0.24).The estimates of logit sensitivity and logit specificity were used to calculate LRQUADAS-2 was used as a revised tool for the quality assessment of diagnostic accuracy studies . The patTo determine whether these differences in testing methods influenced results, a meta-regression was used. In the meta-regression analysis, the variation in accuracy was not seen by using covariates . However, a heterogeneity between the pooled sensitivities and specificities, and higher values of the positive likelihood ratio were detected. These factors could have a significant impact on the probability of infection in HPV-positive women. Therefore, the false positive results could lead to unnecessary invasive examination and costs, which is the advantage of the urine-testing method. However, the high specificity of our test suggests that this scenario is less likely to occur. For these reasons, our results should be interpreted cautiously because there is always the risk of over- or underestimating data. Testing methods need to be more consistent and reproducible if the test is to be successfully implemented in current practice. Therefore, we recommend standardizing urine testing methods, i.e., before incorporating urine testing for HPV into cervical cancer screening guidelines, it is important to minimise variation.Based on the above-mentioned facts, it is necessary to optimise the HPV DNA detection in first-void urine in order to minimise variation of the first-void urine test (sensitivity and specificity) for the presence cervical HPV in women. Optimised HPV DNA detection in urine should include the following: (1) use of the first-void urine (morning or later during the day) captured with a urine collection device ; (2) immOur meta-analysis demonstrates the accuracy of detection of HPV in urine for the presence of cervical HPV. Although progress is continuously made in urinary HPV detection, further studies are needed to evaluate and to improve the accuracy of the first-void urine test in order to be comparable with other screening methods. Different testing platforms and conditions were used in these studies. Therefore, all results should be interpreted carefully, as they may have been over- or underestimated.


OBJECTIVES/GOALS: Cervical cancer is the fourth most common cancer among women worldwide, with approximately 570,000 newly diagnosed cases and 311,000 related deaths among women in 2018.In the United States, approximately 13,000 new cases of cervical cancer are diagnosed annually with approximately 4,000 women dying each year from cervical cancer. Nearly all cervical cancer is caused by oncogenic strains of the human papillomavirus (HPV). Prevention strategies to reduce cervical cancer after HPV exposure entail treatment of cervical dysplasia at the premalignant state, specifically low-grade squamous intraepithelial lesions (LSIL) and high-grade squamous intraepithelial lesions (HSIL), along with the eventual clearance of HPV.The most common treatment for persistent LSIL or HSIL is the loop electrical excisional procedure (LEEP). This procedure is unfortunately not widely available in resource-limited countries and is associated with potential significant morbidities, including decreased fertility, preterm birth, premature rupture of membranes and cervical incompetence. Despite undergoing standard of care treatment, in certain high-risk populations, specifically HIV-infected women, there is a higher rate of premalignant HPV-related cervical disease persistence, progression and recurrence.There is a paramount need for novel nonsurgical treatments to stabilize or treat precancerous lesions of the cervix and to decrease the persistence of HPV infection. Medical treatment with the natural herb curcumin may allow subjects to receive treatment of cervical lesions without undergoing a surgical procedure.Curcumin is a major active component extracted from turmeric with anti-inflammatory, anti-infectious, and anti-cancer properties.Topical intravaginal curcumin has the promise of delivering this drug directly to the site of disease, ensuring adequate concentrations at the site of disease while avoiding systemic side effects. This proposed study will determine if there are higher rates of HPV clearance after curcumin administration among women with and without HIV who have premalignant HPV-related cervical disease, specifically LSIL or recently treated (with a LEEP) HSIL. METHODS/STUDY POPULATION: We are proposing a prospective double-blind randomized control trial to investigate the utility of topical intra-vaginal curcumin in increasing rates of HPV clearance and mitigating the high rates of disease recurrence in women with and without HIV. A sample of approximately 200 women with and without HIV who have biopsy-proven LSIL or recently treated HSIL will be randomized to one of two arms: 2000 mg of curcumin powder in capsules or placebo inserted intravaginally at bedtime once weekly for 20 weeks (excluding the time of menses). Cervical cytology and HPV testing will be performed at baseline and 6 months post-randomization. If a participant has abnormal cytology or a positive high-risk HPV test 6 months post-randomization, they will be scheduled to undergo a colposcopy with biopsies of all suspicious cervical lesions. If biopsy results are HSIL or greater, subjects will be referred back to routine clinical treatment, which may include a LEEP. Clinicians performing the colposcopies and the study participants will be blinded since the placebo has the same appearance as the curcumin capsules. At the end of the study, study participants will be offered the opportunity to participate in 2-3 hour focus groups to discuss acceptability of the product as a treatment for premalignant HPV-related cervical disease until data saturation is achieved. Power and sample size calculations are based on the primary outcome of interest, which is clearance of HPV at 6 months. Basu et al (2013) documented HPV clearance in as many as 80% of subjects with topical curcumin. To account for an expected lower success rate in HIV-infected women, who will also be included in the study, we intend to power our study to determine a more conservative 20% improvement in clearance rate at 6 months with curcumin treatment, assuming an expected clearance rate of HPV in HIV-infected women of 25%. In order to detect a 20% difference of HPV clearance among those treated with intravaginal curcumin vs. placebo at 6 months, about 80 patients per arm would achieve 80% power at the 5% significance level. To account for up to 20% loss to follow-up or discontinuation, the total sample size in each arm would be 100 subjects with a total of approximately 200 subjects enrolled in both arms. RESULTS/ANTICIPATED RESULTS: We are currently in the process of collecting data for this study. We hypothesize that intravaginal curcumin will have a 20% higher rate of HPV clearance at 6 months as compared to placebo. Primary outcome measures will include clearance of HPV at 6 months in curcumin vs. placebo. Secondary outcomes measures will include recurrence of disease by either cytologic or histologic abnormality requiring further surveillance or treatment at 6 months. We also hypothesize that intravaginal curcumin administered once weekly at bedtime for 20 weeks will be safe, acceptable, and well tolerated. This is based off of previous findings from the Phase 1 trial of intravaginal curcumin that we performed. During this Phase 1 trial, we explored daily intravaginal administration of 2000 mg of curcumin to further understand curcumin’s tolerability. Our focus group participants displayed an overwhelming consensus that daily administration affected quality of life, specifically due to the yellow-colored vaginal discharge from the medication. Study participants expressed that once or twice weekly administration was more tolerable and feasible. Our proposed study would therefore test the tolerability and effects of weekly curcumin administration and its ability to clear HPV infection. The primary outcome measure will be the proportion of study participants who discontinue treatment for any reason (acceptability) and the proportion of study participants who discontinue treatment due to adverse effects (tolerability). DISCUSSION/SIGNIFICANCE OF IMPACT: Non-surgical treatments that decrease the morbidity and risk of progression of premalignant HPV-related cervical disease are greatly needed, especially in low-resource settings and among women experiencing barriers to care and/or at high risk for disease progression. Medical treatment with the natural herb curcumin is an emerging strategy that may allow subjects to receive treatment of cervical lesions without undergoing a surgical procedure.Several preclinical and clinical studies have shown curcumin’s ability to reduce tumors and precancerous lesions in animal and human cancer cells. Curcumin can suppress the activation of transcription factor NF-κB and the expression and activity of VEGF and p16INK4a, biomarkers known to be elevated in premalignant HPV-related cervical disease.Studies have also shown that curcumin alters HPV-associated molecular pathways in cancer cells, suppressing cervical cancer growth by inhibiting the transcription of oncoproteins HPV16 and HPV18 (designated as E6 and E7) and restoring p53 and retinoblastoma function. Our proposed study would therefore test the tolerability and effects of weekly curcumin administration and its ability to clear HPV infection. Our results will generate novel data as to what is an acceptable and well-tolerated dosing regimen of intravaginal curcumin, which would be crucial in designing further curcumin intervention studies. The results of our proposal will explore the effect of intravaginal curcumin as a standalone and adjuvant therapy to a LEEP among women with premalignant HPV-related cervical disease. The potential to not just excise diseased tissue, but to directly augment the clearance of the causative agent HPV, would have profound long-term ramifications in resource-limited settings and among women experiencing barriers to care and/or at high risk for disease progression.


Oral cavity and oropharynx cancer associated with human papil loma virus infection, particularly in young people who are continuously exposed to this virus, is a serious public health problem worldwide, especially for high-risk strains that are most associated with premalignant lesions and tumors. These neoplasms remain asymptomatic for a long time and, when they occur, they are already at an advanced stage. If diagnosed and treated early, oral cancer induced by human papilloma virus allows for high survival, as it often has a more favorable prognosis than oral cancers not directly related to viral infection. In this study, the presence of different high-risk and low-risk HPV strains was investigated to assess the epidemiological status in a population of healthy individuals. Two types of samples, one from the tonsils and one from the base of the tongue, were collected from 2015 healthy individuals of different sex and age. A total of 1885 DNA samples belonging to 1285 patients were tested for the presence of 11 high-risk HPV types plus 4 low-risk HPV types using real-time PCR. Of the patients’ DNA samples screened for 15 HPV types, only four samples were positive, all of which were taken from male smokers. These results indicate that newly acquired oral oncogenic HPV infections in the healthy population are rare and, in many cases, controversial. Therefore, more studies are needed to ensure fewer variations in outcomes and a greater clarification of HPV infection and its prevalence in the oropharynx of the healthy population, and to guide efforts to prevent the development of this infection which, if undiagnosed, can lead to the onset of malignant tumors in the oral cavity. The etiological role of human papillomavirus (HPV) infection in the development of squamous cell carcinoma of the uterine cervix has been extensively demonstrated ,2. In alDue to the high tropism of the virus in the epithelium and similarities between genital and oropharyngeal epithelia, HPV is responsible not only for cervical tumors but also for 25% of head and neck squamous cell carcinoma (HNSCC) cases, particularly in the oropharyngeal region, with an incidence of HPV in up to 70% of cases . The etiTo date, more than 200 HPV strains have been identified, which have been divided into two categories based on their neoplastic potential. Low-risk HPV genotypes, which include six types, 11, 40–44 and 54, are responsible for the development of benign lesions such as genital warts or respiratory papillomatosis. High-risk HPV genotypes, including types 16, 18, 26, 31, 33, 35, 39, 45, 51–53, 56, 58, 59, 66, 68, 70, 73, and 82, are related to the development of tumors in the oral cavity of the oropharynx, and in genitals and the anal area [Several studies have evaluated the prevalence of human papillomavirus (HPV) in HNSCC, highlighting a wide variability of results linked to both the type of population and the location of the tumor, as well as the type of specimen and the method of virus detection ,11,12,13HPV is more frequently found in tumors of the tonsillar crypts and the base of the tongue than in other head and neck districts, and HPV16 is reported as the predominant type detected ,15,16.Generally, HNSCC related to the presence of HPV arise at a relatively early age. Since the presence of HPV remains asymptomatic for a long time before generating a neoplasm, it is assumed that the infection occurs at a very early age .Studies show that sexual contact is the most important risk factor, especially among young adults, and it has been shown that the risk increases linearly with an increasing number of sexual partners. Consequently, preventive considerations related to sex education could reduce the incidence of this cancer ,19,20,21There has been increasing discussion on whether HPV-related HNSCC may be positively influenced by planned prevention and vaccination programs for cervical cancer-related HPV ,23.A secondary effect of cervical cancer screening and HPV vaccination could, therefore, be to reduce in parallel the number of cases of HNSCC related to the presence of HPV .Although a direct relationship between the HPV vaccine and HNSCC prevention has not yet been demonstrated, there has been a significative reduction, up to 90%, of oropharyngeal HPV infections among vaccinated young adults in recent years ,25.This study aims to test the presence of multiple types of HPV (low and high risk) in a group of healthy male and female patients, over 18 years of age, in two specific anatomical areas, such as the base of the tongue and the tonsil, to investigate the prevalence of the virus in the oral cavity in a healthy population.A total of 2015 males and females (median age 30 years) who had given their consent were included in the study. Patients were recruited from the University of Tor Vergata  and from the University of L’Aquila . This study was approved by the L’Aquila ethical committee .The inclusion criteria were absence of systemic diseases , absence of facial trauma, good oral hygiene, and acceptance of the proposed follow-up protocols.Patients who did not give their consent to conduct this study were excluded. Patients who could not guarantee adequate compliance, those with pathologies inducing systemic immunosuppression, patients who had undergone radio and/or chemotherapy, those with a positive history of taking immunomodulatory or immunosuppressive drugs, and patients with poor oral hygiene were also excluded.Two samples were taken from each patient, one at the base of the tongue and one from the tonsil, using an oral swab that collects the desquamation epithelial cells. Swabs were immediately placed in a test tube containing silica gel capsules, which rapidly drIed the sample and preserved the integrity of the biological material.The samples were stored at 4 °C until processing.Total DNA was extracted from each swab (tonsil and base tongue) with an automated procedure using a QIAcube HT extractor  and the dedicated QIAamp 96 DNA QIAcube HT Kit .The samples were initially digested in a lysis buffer containing 20 mg/mL of proteinase K and then purified on silica membranes capable of simultaneously binding both genomic and viral DNA. The DNA was washed and then eluted in 150 µL of TE .Total DNA concentration and quality were measured using a NanoDrop 2000 spectrophotometer .Each PCR oligonucleotide set was designed with the Primer-BLAST tool in order to identify primers that are specific to the intended HPV type (PMID: 22708584). This tool combines Primer3 software to design PCR primers and the BLAST and global alignment algorithms to screen primers against a user-selected database to avoid non-specific amplifications. For this investigation, the Papillomavirus Episteme (PaVE) database of curated human papillomavirus genomic sequences was used (accessed on 1 April 2019) .Primer quality was further checked with MFEprimer v3.0 software, which also helped to set multiplex PCR assays . This alFive multiplex real-time PCRs were performed for each sample. The primer and probe sequences used in the reactions are shown in Human papillomaviruses 18 and 45 have been identified and quantified with a single probe marked with cyanine 5. Human papillomaviruses 33 and 58 have been identified and quantified with a single probe marked with FAM.Absolute quantification assays were performed using ABI PRISM 7500 . Each reaction was performed in 20 µL containing 10 µL of 2X qPCRBIO Probe Mix Lo-ROX, 100 nM of DNA purified from samples, 200nM of each primer and 100 nM probe of each probe.The amplification profile consists of an initial denaturation at 95 °C for 10 min, followed by a two-step amplification of 15″ at 95 °C and 60″ at 60 °C for 40 cycles. All PCR plates included non-template controls to ensure no contamination of reagents. Each experiment plate included non-template controls to ensure no contamination of reagents, and serial dilutions of the specific synthetic template. These positive controls were used to plot standard curves, i.e., threshold cycle values against the log of the copy number, which were used to check amplification efficiency and for quantification of targets in each sample.The purified DNA obtained from each specimen was evaluated by real-time PCR using a unique human genome sequence as a target to quantify the number of copies analyzed in each reaction.1 to 107 copies. Serial dilutions of DNA extracted from blood, with amounts ranging from 101 to 105 copies, were used as the standard to quantify the human genome copy number in each reaction.Standard curves for the human papillomavirus types were constructed in a multiplex reaction by using a mix of the same amount of different plasmids containing the target synthetic DNAs, in serial dilutions ranging from 10Descriptive statistics and data analysis was performed using SPSS software v.25 .A total of 2015 volunteers who signed an informed consent form were recruited for this study. The clinicians performed two oral swabs, the first at the base of the tongue and the second at the tonsil. The specimens were sent to the laboratory by express courier. The laboratory received and processed 1882 specimens from the base of the tongue and 1844 from the tonsil. Missing specimens were due to a number of reasons, including patients refusing to give consent for sampling at the chair, clinician decisions, inadequate conservation of samples, or lost shipment. About half of all samples did not pass the minimal amount threshold of 1000 human cell genomes; this threshold was set to reduce the chance of false negatives of the HPV assay due to the low DNA concentration. The samples over the threshold were 971 from the base of the tongue and 914 from the tonsil, with median amounts of DNA of 6573 (IQR: 3946–11148) and 4381 (IQR: 2240–8652) human genome equivalents, respectively. These samples were then tested for the presence of 11 high-risk HPV types plus 4 low-risk HPV types. The screening involved 1285 patients, of which 600 were tested with two specimens from base of the tongue and tonsil, 371 were tested with a specimen from the base of the tongue only, and 314 were tested with a specimen from the tonsil only. The pyramid plot shows the age and gender distributions of patients  and reveAmong the 1885 DNA samples from 1285 patients that were screened for 15 types of HPV, only four samples returned positive results . All of The calculated prevalence of any of the tested HPV types (4 out of 1285 patients) was 0.31% (95% C.I. 0.12–0.80), and prevalence for high-risk HPV (3 out of 1285 patients) was 0.23% (95% C.I. 0.08–0.68). The prevalence values among the 971 samples from the tongue were 0.42% (95% C.I. 0.16–1.05) and 0.31% (I.C. 95%: 0.11–0. 90) for any HPV and high-risk HPV, respectively.https://www.salute.gov.it/portale/documentazione/p6_2_8_3_1.jsp?lingua=italiano&id=27 (accessed on 1 December 2021), which reports the percentage of the population who are fully vaccinated, according to age, date of birth, and gender in the area of recruitment  have been associated with high-risk HPV serotypes .The high spread of this virus has meant that HPV-associated OPSCC is now the most common cancer in the United States, surpassing cervical cancer .The neoplasm caused by HPV remains asymptomatic for a long time and when it manifests it is already in an advanced stage. If diagnosed and treated early, HPV-induced cancer allows for 85–90% survival, and HPV-positive OPSCCs have been shown to have a better prognosis than HPV-negative ones ,34.HPV infection occurs mainly by sexual transmission, and it is estimated that over 60% of adults have contracted the virus at least once in their lifetime ,35,36. FOnly in 10% of cases, especially in cases of HPV 16 and 18 infections, does it turn into persistent infection and cause precancerous lesions. Precancerous lesions in turn usually take a few years to develop into infiltrating carcinoma ,38.Since oral and pharyngeal cancer is diagnosed in 70% of cases at an advanced stage, surgery is the only therapeutic tool that can be used with a consequent large waste of economic resources ,40.For these reasons, it is important to have an overview of the epidemiological situation in the healthy population. Although members of this population may be asymptomatic, they could be HPV positive with the potential to develop a precancerous lesion over many years.In this study, the presence of different high-risk and low-risk HPV strains was examined to assess the epidemiological situation in a study sample consisting of healthy individuals of different age groups.The screening involved 1285 patients, of which 600 were tested with two specimens from the base of the tongue and tonsil, 371 tested with a specimen from the base of the tongue only, and 314 tested with a specimen from the tonsil only.The gender distribution of age class was between 20 and 30 years and the majority of participants were female.These samples were tested for the presence of 11 high-risk HPV types plus 4 low-risk HPV types.Among the 1885 DNA samples from 1285 patients that were screened for 15 types of HPV, only four samples returned positive results. All of them were collected from the base of the tongue of middle-aged, male smokers. Only one tonsil sample was available from these four individuals, this tested negative for HPV.Low rates of infection in tonsil tissue are consistent with higher immunological exposure in the epithelium of the tonsils than in cervical epithelia. This site contains IgA and proteolytic enzymes that protect against HPV infection .It was also found that, in the young population, most HPV infections are effectively eliminated by the immune system within 6–12 months ,43.There are numerous conflicting results described in the literature on the presence of HPV in the oral cavity and its correlation with any infections at the genital level or with sexual habits. In fact, the prevalence percentage varies from 0% to 50% ,10,12,13This high heterogeneity in HPV prevalence across countries and cultures could be due to a number of factors that may be intrinsic to the type of study performed, such as differences in sample collection, processing, and testing, as well as the small sample size which represents a limitation for the correct assessment of prevalence .Kreimer et al. , in a stThe same authors, in a systematic review of the literature, showed that the prevalence of oral HPV16 was 1.3% among healthy individuals and appeared to differ according to the geographic region of origin .Castro et al.  conducteAnother study reported that oral HPV16 infection is present in only 1% of cancer-free individuals .A bimodal age distribution in HPV infection has been described, with the prevalence peaking at 30–34 and 60–64 years. Some studies suggest that the higher prevalence of oral HPV in old age could be caused by a longer duration of infections, rather than a higher incidence ,48.Our study, based on a large sample of healthy young individuals, demonstrates that the oral cavity is not a reservoir of HPV in healthy people. Therefore, it is worth understanding the vaccination status of study participants to assess what roles HPV vaccines may have in the protection against oral HPV infections and, ultimately, against OPSCC-HPV.More studies are needed which have fewer variables on outcomes and greater clarification of HPV infection and its prevalence in the oropharynx of the healthy population, to guide efforts to prevent the development of this infection which, if undiagnosed, can lead to the onset of malignant neoplasms of the oral cavity.Oral healthcare professionals are in an excellent position to screen for and detect oropharynx cancers thorough intra- and extra-oral examinations, which should be emphasized for the early detection of these types of cancers.