Head and neck squamous cell carcinoma (HNSCC) incidence has been escalating in the last two decades, particularly in Western Europe and North America. Human papillomavirus (HPV) has been identified as the main culprit for this rise with significant implications for the treatment and outcomes of these patients. The purpose of this retrospective study was to investigate HPV’s impact on HNSCC patient outcomes in the Irish population, which has never been performed before. HPV positivity of HNSCC appears to be associated with improved survival in patients and highlights the importance of surgery, perhaps with less severe chemo-radiation therapy, in HPV-related HNSCC treatment.Human papillomavirus (HPV) infection has been identified as a significant etiological agent in the development of head and neck squamous cell carcinoma (HNSCC). HPV’s involvement has alluded to better survival and prognosis in patients and suggests that different treatment strategies may be appropriate for them. Only some data on the epidemiology of HPV infection in the oropharyngeal, oral cavity, and laryngeal SCC exists in Europe. Thus, this study was carried out to investigate HPV’s impact on HNSCC patient outcomes in the Irish population, one of the largest studies of its kind using consistent HPV testing techniques. A total of 861 primary oropharyngeal, oral cavity, and laryngeal SCC  cases diagnosed between 1994 and 2013, identified through the National Cancer Registry of Ireland (NCRI), were obtained from hospitals across Ireland and tested for HPV DNA using Multiplex PCR Luminex technology based in and sanctioned by the International Agency for Research on Cancer (IARC). Both overall and cancer-specific survival were significantly improved amongst all HPV-positive patients together, though HPV status was only a significant predictor of survival in the oropharynx. Amongst HPV-positive patients in the oropharynx, surgery alone was associated with prolonged survival, alluding to the potential for de-escalation of treatment in HPV-related OPSCC in particular. Cumulatively, these findings highlight the need for continued investigation into treatment pathways for HPV-related OPSCC, the relevance of introducing boys into national HPV vaccination programs, and the relevance of the nona-valent Gardasil-9 vaccine to HNSCC prevention.  Prognosis and survival for head and neck squamous cell carcinoma (HNSCC) are generally poor. Approximately half of all patients with HNSCC have advanced-stage disease at the time of diagnosis, with an expected 5-year survival rate between 10% to 40% .HPV-positive HNSCC, and more specifically oropharyngeal squamous cell carcinoma (OPSCC), has a unique relationship to diagnosis, prognosis, and treatment. These tumors generally present with a more advanced clinical stage, with a higher nodal category ,4, despiDespite more advanced presentation, improved survival, which is consistently higher than 30% , is evidFor most patients with high-risk, resected HNSCC, the standard treatment constitutes adjuvant radiation therapy with high doses of cisplatin. This course of treatment appears to work well for HPV-positive tumors. Adjuvant chemoradiation therapy with one dose of weekly cisplatin had 3-year overall survival rates of 86% and 91% and 3-year recurrence free survival of 82% and 84% in one study, suggesting that cisplatin is a good treatment for HPV-positive OPSCC to preserve survival and minimize toxicity ,25.Given this positive response to therapies ,27,28,29Despite extensive reports in the literature, most studies regarding the differential prognosis and treatment modalities of HPV-related and HPV-unrelated HNSCC analyze fewer than 300 cases ,14,30. Whttp://hpv-ahead.iarc.fr, accessed on 1 July 2022), we collected over 1115 FFPE HNSCC samples from six different hospital sites across Ireland, aiming to determine the relationships between HR HPV status, treatment scheme, and survival. We also standardized the definition of an HPV-positive case, using DNA alone detected by an extremely sensitive Multiplex PCR Luminex technology. In this article, we describe the first results of comprehensive survival analysis of HPV DNA-positive and negative OPSCC, oral cavity SCC (OSCC), and laryngeal SCC (LSCC) diagnosed in Ireland between 1994 and 2013. These patient samples were not routinely analyzed for HPV in this time period.In the context of the worldwide meta-analysis being conducted by the International Agency for Research on Cancer (IARC) on the subject  database, an initial incident population of 5767 OPSCC, OSCC, and LSCC was identified. ICD10 codes used to define these sites were in line with the most up-to-date World Health Organization (WHO) classifications . Strict ® RM2135  [® Bond Plus charged slides  [TM  [FFPE tissue sections were cut in the Trinity College Dublin CERVIVA Molecular Pathology Research Laboratory based at the Coombe Women and Infants’ University Hospital (CWIUH), Dublin, Ireland in accordance with the HPV-AHEAD sectioning protocol  and usinGermany)  instrumeGermany) ) at 5 μmMA, USA) . A new bTwo H+E slides were generated for every block, resulting in 2230 slides from the original 1115 FFPE blocks retrieved. These were all analyzed by a Pathology Review Board (PRB) comprised of 6 pathologists based in St. James’ University Hospital, Dublin, Ireland, and the CWIUH, Dublin, Ireland. A subset of 20% of cases was reviewed a second time by another member of the Pathology Review Board to confirm diagnosis, with little to no divergence in assessment. Only cases with relevant tumor tissue in both associated H+Es were brought forward for molecular testing, resulting in 861 cases ultimately used in the study.At the Trinity College Dublin CERVIVA Molecular Pathology Laboratory at the CWIUH, HPV DNA was extracted from pooled sections S2 to S4 using a 250 μL of digestion buffer  and a 2 h incubation at 56 °C. Water samples were also included to signal contamination at any stage of DNA preparation.HPV DNA was detected by a type-specific multiplex genotyping assay developed previously  and base® technology  [Post PCR, 10 μL of each reaction mixture was analyzed by Multiplex HPV genotyping using Luminex xMAPTX, USA) ,39 as deTX, USA) ,42. The Independent variables, survival, and treatment data were provided for each patient case by the NCRI including all those in Statistics were generated using IBM SPSS Statistics Version 25 , XLSTAT 2019.1.3, and Microsoft Excel Version 16.25. Overall and cancer-specific survival for all oropharyngeal, oral cavity, and laryngeal cancer, and within each sub-site, based on HR HPV status was assessed by Kaplan–Meier curves and log-rank test. Additional Cox proportional hazard statistics were generated to confirm Kaplan–Meier results. The relationship between treatment and HPV status for all cases and for each sub-site was evaluated using chi-square statistics and Fisher’s exact tests in cases where expected counts fell below 5. The cohort of patients assessed for treatment was limited to those who received treatment of any kind within 12 months of diagnosis as these were patients of interest. Predictors of overall and cancer-specific survival were evaluated individually by univariable Cox proportional hazard models. For variables from which more than 10% of data were missing, “missing” was included as a category of its own as is convention in the literature to account for or detect any bias responsible for significance. For those variables with between 0% to 10% missing data, cases with missing data were excluded for univariable and multivariable analyses. Those variables significant in univariable analysis were brought forward for multivariable analysis. All significant variables by univariable models were included in the initial multivariable model. The least significant predictor was then taken out, and the model was run again. The least significant predictor was again taken out, and the model was run again. This continued until all variables remaining in the model proved significantly predictive of survival and risk of death, or until taking another variable out rendered the model as a whole insignificant.To contextualize the study population, its basic demographic characteristics are presented in A basic summary of the number of HPV positive and negative cases by sub-site is provided below in p < 0.0001). Cox proportional hazard model for HPV status and overall survival confirmed the increased risk of death for HPV-negative patients . p = 0.032). Cox proportional hazard model seconded the significantly increased risk of cancer-specific death in the HPV-negative group .p < 0.0001; Cancer-specific-Log-rank: Chi-square 11.902, 1 d.f., p = 0.001). Cox proportional hazard model confirmed this finding .No significant differences in survival by HPV status were noted for LSCC or OSCC sub-sites alone.p < 0.0001). HPV-positive patients were almost twice as likely to be treated more aggressively with all three treatment modalities (surgery/radiotherapy/chemotherapy) than HPV-negative cases. More HPV-negative patients were treated with surgery or radiotherapy alone and almost three times as many HPV-positive patients were treated chemically with radiotherapy/chemotherapy than HPV-negative patients. These patterns were mimicked within the population of oropharyngeal patients alone . No associations were seen within oral cavity cases alone  but were within the laryngeal case population alone . These differences emanated from the disproportionate treatment of HPV-negative cases with radiotherapy alone, and the more frequent treatment of HPV-positive patients with all three modalities.There was significant relationship between HPV status and treatment modality administered  (n = 80) . Surgery alone and all three treatment modalities maximized overall survival for HPV-positive OPSCC patients. Treatment with radiotherapy alone was associated with decreased survival. For cancer-specific survival amongst HPV-positive oropharyngeal cases, there was a significant difference by treatment type . p = 0.039, 0.017, 0.312, 0.600, 0.833).Given the significant relationship between HPV status and treatment modality, and HPV status and survival only for OPSCC, the relationship between all three variables for OPSCC alone is presented. For HPV-positive cases in the oropharynx, there was a significant difference in overall survival by treatment type  . Cox proFor HPV-negative oropharyngeal cases, there was no difference in overall or cancer-specific survival by treatment type, something also reflected by the Cox proportional hazard model.The key finding of the present analysis was that both overall and cancer-specific survival were significantly improved for HPV-positive cases in all oropharyngeal, oral cavity, and laryngeal SCC grouped together. This relationship emanated, however, from the OPSCC population alone. This is reflective of most studies in the literature ,30,43. TThese results highlight the importance of surgical intervention for HPV-positive OPSCC, with treatment approaches not involving surgery seeing very poor survival. The findings are extremely promising in terms of the potential for de-escalation of treatment for these patients. This is indicative of the chance that HPV-related OPSCC presents for drastically improving the quality of life for patients by avoiding the administration of extremely harsh treatments and the long-term side effects that accompany them including difficulties swallowing, breathing, and speaking, chronic pain, osteoradionecrosis, hypertension, pneumonia, dysphagia, weight loss, malnutrition, dental issues, and third-degree burns ,47 alongThe present data thus supports the notion that robotic trans-oral resection (TOR) alone yields extremely good results for HPV-related patients ,55,56 reDespite positive indications of de-escalation potential in HPV-positive OPSCC, the present analysis indicates that HPV-positive HNSCC and OPSCC were more likely to be treated harshly than their HPV-negative counterparts. The population that might have benefited most from less severe treatment schemes was thus the population being treated most severely. The present data and the literature explain that this irony is due to the later stage at which HPV-related OPSCC is diagnosed ,43,65,66To note is that it is increasingly recognized that HPV-negative oropharyngeal cancer represents a treatment-resistant entity and a distinct therapeutic challenge. Comparison among treatment modalities for oropharyngeal cancer has been limited, and very few studies have evaluated differences based on the HPV subtype. Our findings that HPV-negative cases were treated less aggressively than HPV-positive tumors may in fact just be reflective of clinical practices at that time, or indeed may be related to the patient demographic where smoking and alcohol use may play a role in treatment responses. Further prospective studies into outcomes for HPV-negative tumors are warranted.Two caveats to this study’s support of de-escalation should be noted. First, there were smaller sample sizes available when sub-dividing all 861 cases into their sub-site, HPV status, and treatment groups. Targeted sampling of OPSCC alone is needed for further confirmation of these promising findings. Second, the analysis also emphasizes that in terms of potential de-escalation, it would be unethical to make treatment decisions for these patients, or their negative counterparts, based solely on HPV status. For oropharyngeal, oral cavity, and laryngeal SCC, multivariable predictors of the overall risk of death did include HPV negativity, but HPV status was not confounded by other patient characteristics including older age and current smoker status . For OPSThe findings indicate that the oropharynx is the sub-site in which HPV-related tumors occur and that it is therefore the region for which any HPV-related treatment alterations should be made. They also highlight that though HPV-related tumors are already significantly associated with younger aged patients ,64,65 anThis collection of patient characteristics has recently been recognized in the literature as the only group of oropharyngeal, oral cavity, and laryngeal SCC patients for which de-escalation of treatment is acceptable. In fact, several of the ongoing trials regarding de-escalation only include patients meeting these criteria to assure no jeopardizing of patient safety ,63,64,71With respect to Thus, this study population is further proof of the suggestion that HPV-positive oropharyngeal, oral cavity, and laryngeal SCC, and more specifically, OPSCC alone, is a better-surviving cancer than its HPV-negative counterparts. Furthermore, though more than simply surgery is and will continue to be necessary to treat some late-stage patients, the benefit of treating all HNSCC patients indiscriminately with all three modalities is questionable. The data also indicate that other indicators of clinically relevant HPV infections including patient characteristics will play a significant role in determining treatment options for oropharyngeal, oral cavity, and laryngeal SCC in addition to, and not instead of sensitive HR HPV detection.Importantly, the diverse treatment, survival, and HPV characteristics observed in the present data converge and point to the crucial nature of prevention and early detection in oropharyngeal, oral cavity, and laryngeal SCC if survival, overall and cancer-specific, is to be maximized. All HNSCC are overwhelmingly behaviorally driven cancers, whether by exposure to HPV and/or to smoking .For OPSCC specifically, data are still emerging on the impact of the quadra- and nona-valent Gardasil vaccines on HR HPV prevalence in the oral cavity , though Lastly, the present analysis underlines the urgent need for effective and systematic HNC screening tools. Early detection of those SCC that do go on to develop despite preventative measures is tantamount to prolonging overall survival, no matter how promising or poor cancer-specific survival is and regardless of HPV status. For HPV-unrelated HNSCC in this analysis, especially in the larynx, diagnosis at a later TNM stage was the only predictor of cancer-specific survival after adjustment for other variables . EffortsIn all, pairing early detection with preventative mechanisms and curative approaches suitable to the tumor and patient characteristics will render oropharyngeal, oral cavity, and laryngeal SCC an imminently manageable and rare disease. These public health and clinical measures will ultimately mean huge cost savings, and more importantly, the difference between life and death for potential and current oropharyngeal, oral cavity, and laryngeal SCC patients.


Cervical cancer disproportionately affects women of reproductive age, with 80% of cases occurring in low- and middle-income countries. Persistent infection with high-risk human papillomavirus (HPV) genotypes has been described as the most common non-systemic biological risk factor for the development of cervical cancer. The mucosal immune system plays a significant role in controlling HPV infection by acting as the first line of host defense at the mucosal surface. However, the virus can evade host immunity using various mechanisms, including inhibition of the antiviral immune response necessary for HPV clearance. Pro-inflammatory cytokines and the vaginal microbiome coordinate cell-mediated immune responses and play a pivotal role in modulating immunity. Recently, diverse vaginal microbiome  and genital inflammation have emerged as potential drivers of high-risk HPV positivity and disease severity in women. The potential role of these risk factors on HPV recurrence and persistence remains unclear. This article reviews the role of cellular or cytokine response and vaginal microbiome dysbiosis in the clearance, persistence, and recurrence of HPV infection. Human Papillomavirus (HPV), a primary cause of genital warts and cervical cancer, is the most common sexually transmitted infection . DespiteInnate and adaptive immune responses represent the first line of host defense at the mucosal surface against pathogens such as HPV . SeveralLactobacillus species and high proportions of the Gardnerella, Sneathia, and Atopobium genera are less likely to clear HPV infection -linked bacteria  . Several studies further reported an association between BV-linked microbial communities  and persistent infection with high-risk HPV that may result in the development of CIN  . A Lactolearance , while at of CIN . Furthert of CIN . Diverset of CIN , and devWhile there is evidence of a relationship between the vaginal microbiome, host immune responses, and HPV infection, a link between the vaginal microbiome, host responses, and the progression to HPV-associated cervical cancer remains unclear. Therefore, understanding the concept of vaginal microbiome fluctuations and associated host immune responses during HPV infection stages could shed light on possible mechanisms associated with cervical carcinogenesis. This review summarizes the role of vaginal microbiome fluctuations, cytokine, and cellular immune response during HPV infection, persistent and clearance.Alphapapillomavirus, Gammapapillomavirus, Betapapillomavirus, Deltapapillomavirus, and Mupapillomavirus, with only Alphapapillomavirus, Gammapapillomavirus and Betapapillomavirus affecting humans .HPV is a member of the non-enveloped double-stranded DNA papillomaviridae family that infects squamous epithelium found beneath the foreskin of the penis, the scrotum, vulva, vagina, cervix, skin, and anus . The papg humans . Of the More than 200 genital HPV genotypes have been molecularly characterized, with some categorized into low-risk genotypes  while others are categorized as high-risk genotypes  . High-ri®), quadrivalent (Gardasil®), and nonavalent (Gardasil®9) vaccines  showed that HPV infection peaks in younger women around the age of sexual debut and declines during later decades of life , nucleotide oligomerization domain (NOD)-like receptors (NLRs), and retinoic acid-inducible gene I (RIG-I)-I-like receptors (RLRs) . Antigenvia the expression of inflammatory cytokines such as IL-1α, IL-1β, IL-6, TNF-α, and IL-12 1 cell responses that promote cell-mediated immunity and Th2 cell responses that promote humoral immunity . During nd IL-12 . T lymphnd IL-12 . An antind IL-12 . CTLs, Cnd IL-12 , while tnd IL-12 . Furthernd IL-12 . Some nand IL-12 . In contnd IL-12 . Activatnd IL-12 . Taken tvia downregulation of cytokines (TNF-α and MIP-3α) responsible for activation of these multifunctional cells  demonstrated that HPV-16 downregulated the expression and function of TLR-9 in human epithelial cells  has been associated with suppression of the cyclic guanosine monophosphate-adenosine monophosphate synthase, which plays a role in activating type I interferon genes and production of inflammatory cytokine response . The int+ T cell differentiation and proliferation, thereby modulating the adaptive immune responses  showed a strong association between invasive cervical carcinoma and elevated concentrations of TNF-α, TNF-β, MIP-1α, GMCSF, and IL-10  found that 9 of the 13 cytokines  tested were elevated after the clearance of HPV infection compared to prior visits  (105). Furthermore, L.crispatus abundance in the genital tract was also associated with HPV clearance, suggesting the relative association between clearance and L. crispatus (105).The vagina and ectocervix are dominated by lactic-acid producing bacteria and cervicovaginal fluids that act as a lubricant that traps invading pathogens . A genit and HIV . In contensenii ,. L. galearance . A recenLactobacillus dominant towards more diverse bacteria, including strict and facultative anaerobes such as Gardnerella, Prevotella, and Sneathia, often resulting in vaginitis and discharge  showed that HPV-infected women had lower Lactobacillus species and increased Fusobacteria and Sneathia compared to HPV-uninfected women  are less studied and could be important drivers of HPV risk in young women. There remains a need to conduct more preclinical models to understand how biological risk factors might block efficient HPV clearance from the mucosa and pave the way for cervical cancer.All authors contributed to the article and approved the submitted version.This review was conducted as part of the DST-NRF Centre of Excellence (CoE) in HIV Prevention, supported by the Department of Science and Innovation and the National Research Foundation (grant 96354). LN was funded by DST-NRF CoE in HIV Prevention (grant 96354) and Poliomyelitis Research Foundation Research Bursary (bursary# 21/73). SN was supported by Columbia University-Southern African Fogarty AITRP Programme (grant# D43TW00231), National Research Fund Thuthuka Research Grant (grant# TTK160510164586), and Poliomyelitis Research Foundation Research Grant (grant# 16/17). GM was funded by Poliomyelitis Research Foundation Research Grant (grant# 18/16) and National Research Foundation Grant (grant# SFP180507326699). AM was supported by National Research Foundation Grant (grant# PDG210309589501)The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The Veterans Administration (VA) established pathways to provide VA-issued tablets and increased access to internet for Veterans without these resources. Veterans aged above 65, experience barriers with telemedicine such as access and usability. We sought to improve the usability and experience of telemedicine for rural, older Veterans receiving a VA-issued tablet by modifying materials and qualitatively evaluating their experience with set-up and preparing for their first appointment, guided by user-centered design. We conducted a rapid exploratory evaluation, to understand Veteran and care partner experiences setting up VA-issued tablets and logging into their first appointment using standard 9-page instructions. We interviewed telehealth technicians, and providers to better understand patient barriers from their perspective. Using insights from interviews and evidence-informed guidelines on educational materials for older adults, we created a two-page guidance. A group of Veterans and care partners reviewed the materials and provided feedback. Received feedback provided 17 suggestions, 8 of which were utilized, including enlarging graphics, clarifying abbreviations (or wording), consolidating the instructions further and emphasizing pertinent information further. Modified materials reduced standard written instructions from nine pages to two. Feedback suggests that updated materials are helpful, aesthetically pleasing and preferred over current materials. Utilizing user-centered design methods, addressed barriers experienced by older, rural Veterans with initial telemedicine device and appointment set-up. Veterans, care partners, providers, and telehealth technicians perceived materials adapted for older adults as supportive of video device usability, helping to alleviate barriers that prevent Veterans from initiating telemedicine.


Changes in attitude were statistically significant in every attitude-based question about HPV (p < 0.05). There were significant differences in knowledge gained and attitudes towards the HPV vaccine when comparing urban and rural locations as well. More research is needed to explore the efficacy of community-based interventions to increase uptake and series completion of HPV vaccination, particularly in rural areas most impacted by HPV-associated cancers. HPV vaccination series completion rates have increased steadily in the USA, yet specific areas continue to be burdened by HPV-related cancers, including rural areas of Virginia. The primary purpose of this study is to compare the impact of an community driven educational film about HPV on intention to vaccinate and knowledge gained in urban and rural areas of Virginia. From October 2016 to September 2019, the CDC-approved documentary “Someone You Love: The HPV Epidemic” was screened and followed by a Q&A session. Intention to vaccinate and knowledge gained after seeing the DVD intervention were measured through a pre-post-survey and analyzed data using chi-squared tests. The sample included males and females of all races and ages 18+ that identified as either student, parent/guardian, and/or healthcare provider from rural and urban geographical areas. Changes in knowledge about HPV were statistically significant in two out of seven questions (The online version contains supplementary material available at 10.1007/s13187-022-02169-5. In the USA, 14 million people are infected with Human Papillomavirus annually, making it the most common sexually transmitted infection . AlthougRural communities, which are defined as areas that contain less than 2,500 people, of which less than 1,500 reside outside institutional group quarters , such asProvider recommendation is a key part of vaccination initiation because it serves as a cue to action for many patients to participate in a healthy behavior. Although this is an important predictor of HPV vaccine series completion, 66% of physicians report that they do not have enough time to educate parents and adolescents about the vaccine . A commuTo date, much of the HPV vaccine-related research has focused on individual interventions, interventions with parents, and interventions with providers, but there is a lack of community-based intervention research. In addition, there are very few studies that compare community-based interventions in urban and rural areas. Although understudied, community-based interventions may be more beneficial in rural areas than urban areas due to limited access to education, fewer providers, and stigma as mentioned previously. It is important to determine whether community-based education interventions are more or less effective in rural areas than urban areas for the sake of being cost-effective and efficient in our effort to influence public health.Although limited, there are a few community-based education intervention research studies that aim to increase HPV vaccine uptake or intention to uptake in rural or urban areas in the past 10 years. Community-based interventions include videos, vaccine fairs, radio or newspaper ads, websites, social media campaigns, school-based reminders, educational sessions, and radionovelas –14. Of tThe literature has shown that utilization of community-based education interventions improves willingness to vaccinate and knowledge gained regarding HPV-related information . StudiesThrough an initiative led by the Indiana Immunization Coalition, the Virginia Department of Health (VDH) conducted multiple screenings of the “Someone You Love: The HPV Epidemic” film in urban and rural areas of Virginia. This film was used as the community-based intervention for this study. The Someone You Love (SYL) documentary follows the lives of 5 women diagnosed with cervical cancer caused by the HPV virus, highlighting the women’s diagnosis and addressing misinformation about and the stigma surrounding HPV . An elecAccording to the Health Belief Models (HBM) theoretical model, disease prevention techniques are used when individuals have an increased perceived susceptibility, an increased perceived severity, are motivated, usually by a cue to action, to change their behavior, and have decreased perceived barriers . The SomBased on the Health Belief Model (HBM), outcomes of this cue to action include changes in knowledge, attitudes, and beliefs regarding the HPV vaccine. Pre-post-surveys included demographics, knowledge, attitudes, and beliefs regarding HPV vaccination to measure these anticipated outcomes.The study population included participant’s ages 18 + , male and female, and all races. Recruitment was solely from those who chose to attend the SYL viewing event. The event was promoted through flyers and email communication. Emails were sent to social, academic, and civic groups to share with their members. The study was conducted in academic institutions throughout Virginia and at the Virginia Department of Health. There were a total of seven geographic areas studied  [Data collection began in October 2016 and ended in the Spring of 2019. This research study consisted of a pre- and post-survey. Before showing the documentary, the study team explained the purpose of the pre- and post-survey and then asked for opt-in consent. The opt-in consent procedure consisted of the researcher reading aloud the consent form to the participants. Participants were asked to use their smartphones to access the provided link projected on the screen which took them to the Qualtrics highly sensitive data portal to begin the pre-survey. After viewing the documentary, the post-survey was administered through the same process. Participants who took the survey through Qualtrics were assigned a numeric code randomly generated through the Qualtrics software which was used to link the pre- and the post-survey. Participants who took the survey on paper did not receive a linking numeric code although all other procedures were conducted the same. This is an important detail to note with regard to how the data was analyzed.The effectiveness of the film intervention was measured by analyzing the differences in the mean responses between the pre/post-survey results in knowledge gained, attitudes towards, and intention towards receiving the HPV vaccine. Although actual uptake of the HPV vaccine would be the best indicator of the effectiveness of the film intervention, intention to vaccinate is significantly associated with uptake .Data analysis was performed using the Statistical Package for the Social Sciences (SPSS). Descriptive statistics were used to analyze the demographics of participants. Chi-squared tests were used to determine the difference in the means between the pre- survey and the post-survey for knowledge-based questions and attitude-based questions in a majority of the data set. A sub-analysis was performed for a small part of the data set that had linking identifiers between the pre- and the post-surveys. Sub-analyses were conducted to compare changes in knowledge and attitudes in part of the data set that had linking IDs, to compare responses between the rural and urban intervention locations, and to compare means between the pre- and post-survey in the data set that excluded participants that identified as healthcare professionals. Due to the nature of the statistical tests used to analyze this data, these sub-analyses were only conducted on the linked data  set rather than the entire data set (paper surveys).n = 127), from a rural geographical area , healthcare providers , and Caucasian   How safe do you think the vaccine is that prevents HPV? (2) How important do you think it is for people between the ages of 9 and 26 to be vaccinated to prevent HPV? 3) If you are a healthcare provider, please answer the following question: How likely are you to talk to your patients about HPV? (4) If you are NOT a healthcare provider, please answer the following question: How likely are you to talk to your/your child’s doctor about HPV? Please refer to Table  If you an = 33), students , and Caucasian . Regarding vaccine initiation/completion rates, 34% (n = 30) participants had received three shots in the HPV vaccine series  Do you plan to get the vaccine in the next 6 months?” and (2) “How important do you think it is for people between the ages of 9 and 26 to be vaccinated to prevent HPV?”  how safe do you think the vaccine is that prevents HPV? and (2) How likely are you to talk to you/your child’s doctor about HPV?  HPV is a rare, sexually transmitted disease, (2) What are some ways to prevent the spread of HPV? (3) How safe do you think the vaccine is that prevents HPV? Attitude-based questions include (1) How safe do you think the vaccine is that prevents HPV? (2) How important do you think it is for people between the ages of 9 and 26 to be vaccinated to prevent HPV? and (3) How likely are you to talk to your patients about the HPV vaccine?  how safe do you think the vaccine is that prevents HPV and (2) how likely you are to talk to your/your child’s doctor about the HPV vaccine? . Participants also indicated that they felt more comfortable talking to their patients about the HPV vaccine if they identified as a healthcare provider. Although previous studies show that community-based interventions have been successful in increasing knowledge about the HPV vaccine and uptake of the vaccine –14, thisThis analysis supports future efforts utilizing videos as community-based interventions to promote HPV vaccination within rural areas. This style of video intervention may be specifically successful if distributed to rural physicians or parent/guardian within schools as a way to increase HPV vaccination education and uptake. This type of video may also be easily incorporated into school-based sexual and reproduction health education curriculum as well. Due to the ease of video creation and distribution, future researchers or healthcare professionals should consider developing an intervention that targets specific populations and addresses cultural barriers in that region to obtaining the HPV vaccine. More research is needed to explore the efficacy of community-based interventions to increase actual uptake and series completion of the HPV vaccine, particularly in rural areas, male, and racial diverse populations. In the context of COVID-19, when HPV vaccination rates have significantly decreased , 22, innSupplementary file1 (DOCX 58 KB)Below is the link to the electronic supplementary material.


The prevalence of peripheral artery disease (PAD) increases exponentially with age and is associated with heightened risk of functional impairment. We assessed baseline function and compared changes in mobility among older Veterans with PAD participating in Gerofit, a clinical exercise program. A total of 545 Veterans (mean age: 79 years) completed baseline, 3-, 6-, and 12-month mobility assessments and were divided into the following cohorts: PAD (n=99) and no PAD (n=446). Assessments included 10-m walk speed, 6-min walk distance, 30-s chair stands, and 8-ft up-and-go time. Veterans with PAD performed worse on all measures than non-PAD Veterans at baseline. Veterans with PAD demonstrated significant improvements (p < 0.05) in all measures from baseline at 3, 6 and 12 months. There were no significant differences in change scores between PAD and non PAD across all time points, controlling for age, except for 8-ft up-and go. PAD Veterans benefit from function-based exercise.


Results: HPV16 was the dominant genotype in 53% (9/17) of patients with confirmed precancerous lesions . In specimens with confirmed cytological abnormalities and hrHPV infection, we detected a wide diversity of microbes, while the most common species were Gardnerella vaginalis, Atopobium vaginae, Prevotella bivia, Ureaplasma parvum, Ureaplasma urealyticum, Leptotrichia amnionii, Bacteroides ureolyticus, and Sneathia sanguinegens. The presence of pathogens did not differ, depending on the degree of precancerous lesions or HPV type. Conclusion: In our work, HPV16 dominated in patients with cervical precancerous lesions. We also suggest an increased bacterial diversity of the vaginal microbiome in patients with cervical lesions, for which the HPV virus is largely responsible. Background: Cervical cancer is the fourth most common malignancy and fourth leading cause of cancer death in women worldwide. More than 99.7% of cases are caused by human papillomavirus (HPV), while HPV types 16 and 18 cause over 70% of all cervical cancer cases. In this preliminary study, we aimed to investigate the presence of HPV infection and diversity of bacteria associated with bacterial vaginosis. Methods: Cervical swabs ( Cervical cancer is the fourth most common malignancy and fourth leading cause of cancer death in women worldwide . More thThe HPV virus affects up to 80% of women during their lifetimes . DespiteLactobacillus crispatus; in group II, Lactobacillus gasseri dominated; in group III, Lactobacillus iners was dominant; and Lactobacillus jensenii was dominant in group V. Vaginal swab examinations in the remaining 27% patients showed the dominance of strict anaerobes, including Gardnerella, Prevotella, Megasphaera, and Sneathia. The lowest pH was found in group I, while the highest pH was observed in group IV, corresponding to a higher risk of BV development [The human vaginal microbiome, in the right composition, contributes to the prevention of urological tract infections , urogenielopment . The vaginal microbiome associated with bacterial vaginosis is a predisposing factor for sexually transmitted infections ,15 and tThe aim of our work is to investigate the possible association of vaginal dysbiosis with hrHPV infection and subsequent development of precancerous lesions of the cervix. We also sought to determine the representation of HPV genotypes in the studied cohort, according to the types of precancerous lesions.Sampling was performed during the expert colposcopic examination of patients with cytological abnormalities. Control samples from women negative for intraepithelial lesions of the cervix were taken during a routine gynecological examination. In both cases, all participants were fully informed that anonymity is assured, why the research is being conducted, how their data will be used, and the associated risks. All participants expressed clear consent for the collection of biological material. During the expert colposcopic examination, the samples of the vaginal microbiome were taken from the posterior vaginal fornix with, and from, the area of the cervical external orificium using cytobrush. The optical examination of the cervix consisted of native and extended colposcopy, using a solution of 3% acetic acid and iodine solution (Schiller’s test), which also served to describe the cervical lesions in the case of tissue biopsy.Cervical swabs were immersed in RNA stabilization and storage solution , transported to Biomedical Centre in Martin at room temperature, and stored at −20 °C until further analysis.Cervical swabs were unfrozen, vortexed, and rinsed with phosphate buffered saline (PBS). A total of 100 μL of cervical cell suspension was employed for DNA extraction using a MasterPure Complete DNA and RNA Isolation kit , according to the manufacturer’s instructions. Extracted DNA was diluted in 35 μL of TE buffer, and the quantity was measured using a Qubit dsDNS HS assay kit . All samples were stored at −20 °C before subsequent analysis.2, 50 nM of each primer, and 1 U of Hot StartTaq DNA polymerase . PCR amplicons were checked on 1.75% agarose gel stained with gel red nucleic acid gel stain  and purified by NucleoSpin Gel and PCR Clean-Up kit . Reactions for the cycle sequencing of 420 bp fragments  were purified on gel matrix . The HPV genotypes were distinguished via sequencing on a 3500 genetic analyzer  and compared to other known sequences in an online database .HPV genotyping was performed using PCR targeting the L1  conserveIn total, 250 ng of DNA was tested via a real-time PCR to identify of microbial strains associated with bacterial vaginosis, according to the manufacturer’s instructions . The microbial qPCR array contained assays for 42 pathogenic bacterial species, as well as control human genes (GAPDH and HBB1) ,24, bactHere, we present partial results for 21 patients, from a set of 152 collected samples, as sampling is still ongoing. The presented patients were between 21 and 47 years of age, with cytological abnormalities demonstrated in 17 patients (ASCUS (5), LSIL (9), and HSIL (3)). The other four women had negative cytology smears.HPV genotyping was performed according to the previously published method , thus alOverall, HPV 16 was the dominant genotype identified in three patients with ASCUS cytological smear abnormalities; four patients with low-grade lesions, two patients with high-grade lesions, and one patient negative for an intraepithelial lesion or malignancy (NILM) were infected with this type. One patient with a low-grade lesion was infected simultaneously with two types, HPV18 and HPV66. Among the remaining samples, in the ASCUS lesion category, one patient was infected with HPV33, and one patient was infected with HPV66. One patient with an LSIL lesion was infected with HPV66, and another LSIL patient was infected with HPV70. The remaining patient with an HSIL lesion was infected with HPV52. Three NILM participants and two LSIL patients were HPV negative. The results are summarized in Gardnerella vaginalis, Atopobium vaginae, Leptotrichia amnionii, Sneathia sanguinegens, Parvimonas micra, Prevotella melaninogenica, Prevotella disiens, Prevotella intermedica, Prevotella nigrescens, Prevotella bivia, Streptococcus mitis, Mycoplasma hominis, Aerococcus christensenii, Fusobacterium nucleatum, Corynebacterium aurimucosum, Streptococcus intermedius, Ureaplasma parvum, Ureaplasma urealyticum, Campylobacter gracilis, Treponema denticola, Bacteroides ureolyticus, Mobiluncus curtisii, and Candida albicans.In all patients with confirmed cytological smear abnormalities, pathogens disrupting the physiological composition of the vaginal microflora were present in the vaginal swabs. These pathogens comprised a spectrum of various microorganisms, such as Gardnerella vaginalis was confirmed in 75% of HPV-positive patients, but it was confirmed in patients with every type of precancerous lesion detected in our cohort. Gardnerella vaginalis was also confirmed in 80% HPV-negative women. Prevotella bivia and Atopobium vaginae were detected, respectively, in 44% and in 50% of HPV-positive patients, while Prevotella bivia was confirmed in of 40% HPV-negative participants. Ureaplasma parvum/urealyticum infection was observed in 56% of HPV positive cases and 60% of HPV-negative women, followed by Leptotrichia amnionii in 38%, Bacteroides ureolyticus in 25%, and Sneathia sanguinegens in 19% of HPV positive cases . The abudance of genus Gardnerella was almost nine-fold higher in the HPV positive group, compared to that among HPV negative women (6.42% vs. 0.72%), while settlement of the genus Prevotella was more than five times higher (2.44% vs. 0.44%). Other microbes associated with CST IV, such as Megasphaera and Atopobium, were also found in a higher quantity in the vaginal microbiomes of HPV-positive women, compared to the HPV negative group [Prevotella bivia, Enterococcus durans, and Porphyromonas uenonis in the vaginal microbiome related to persistent HPV infection [Gardnerella in the cervicovaginal swabs at the first visit as a biomarker for hrHPV progression, while the abundance of L. iners was associated with the clearance of the HPV infection [Wei et al.  comparedve group . This renfection  reportednfection . Anaerococcus vaginae, Garderella vaginalis, and L. iners identified in the vaginal microbiome, alongside a simultaneous decrease in the representation of L. crispatus, compared to that in hrHPV-negative group [Garderella vaginalis in hrHP- positive patients. Brotman et al. [L. gasseri and appears to be a potentially therapeutic species that can improve the clearance of HPV infection [Community state types IV and III are often associated with the presence of HPV infection in women, as well as the development of precancerous lesions. A study of 70 women with cervical intraepithelial lesions (CINs) and 50 healthy women showed a higher diversity of the vaginal microbiome in women with CINs, compared to that in controls. There was an increased risk of developing CIN observed in hrHPV-positive women with ve group . Our worn et al.  examinednfection .In vitro, after treatment of HPV16-positive Caski cells with Lactobacillus supernatants, a reduction in the number of cells at the G2/M phase transition was observed. Additionally, a lower expression of mRNA E6 and E7 oncogenes was demonstrated and detected a more than six-fold reduction of E6 expression after 24 h of treatment with the dominant CST I microbe, L. crispatus [Bifidobacterium adolescentis [As previously mentioned, it is also possible to describe the relationship between the physiological lactobacillus settlement of the vagina and regression of HPV infection. rispatus . Anotherescentis . In our pilot study, the collection of samples is still ongoing; and we are increasing the number of participants to determine specific bacterial species and HPV genotype. Our study’s main limitations are the limited number of samples, differences in age, and phase of the menstrual cycle of the examined women, which can also affect the composition of the vaginal microbiome. On the other hand, the main strength of our work is that the microbial qPCR array that we used is able to identify 42 pathogenic bacterial species. In the next step, we are planning to investigate the colonization of the vagina with lactobacilli, which would allow us to evaluate bacterial vaginosis in the examined samples. The goal of our work is to point out the vaginal microbiome as a possible auxiliary tool in the diagnosis of cervical precancerosis. Some works also suggest that physiological correction of the composition of the vaginal microbiome may play an important role in the regression of the HPV infection of the cervix.Gardnerella, Atopobium, Prevotella, Ureaplasma, Leptotrichia, Bacteroides, and Sneathia. Cervical cancer is easily avoidable with the available methods for both primary and secondary prevention; nevertheless, the prevalence of this cancer among the population is still high. We suggest that support for the physiological composition of vaginal microflora could become a widely available tool for preventing the development of cervical precancerous lesions.The HPV virus is responsible for almost all cases of cervical cancer. In our work, HPV16 dominated in patients with cervical precancerous lesions. We believe that there is an increased bacterial diversity of the vaginal microbiome in patients with cervical lesions, for which the HPV virus is largely responsible. In our study, the most common genera of bacteria were


IntroductionCervical cancer is the fourth most frequent cancer in women worldwide, and it continues to be a big issue in developing countries. The current case-control study sought to determine the presence of high-risk human papillomaviruses (hr-HPV) in the development of cervical cancer, as well as their relationship with the cell cycle inhibitor gene p16INK4A in cervical cancer.MethodsThe association between p16INK4A protein and the presence of hr-HPV DNA in cervical lesions was explored in this study, which included 150 cervical cancer patients and 100 normal cervix samples. The immunohistochemistry approach was used to identify the expression of the p16INK4A protein, while the semi-quantitative polymerized chain reaction (PCR) method was used to identify the genomic identity of hr-HPV.ResultsAbout 90.67% (n=136) of the 150 case samples were found to be hr-HPV positive. Within the 136 HPV-positive samples, 45 (33.08%) show moderate expression of the p16INK4A protein, whereas 91 (66.91%) show overexpression, which is statistically significant (0.05). Among the 136 HPV-positive samples, 22.08% (N=30) were classified as having cervical intraepithelial neoplasia (CIN), with 56.66% (n=17) having CIN3, 36.66% (n=11) having CIN2, and 6.67% (n=2) having CIN1.ConclusionBased on the semi-quantitative immune staining scoring method of p16INK4A protein, genomic expression of HPV demonstrates that the expression of p16INK4A protein increases with the infectious load of the hr-HPV genome in the host cell. The result directly shows that immunostaining of the p16INK4A protein, in conjunction with the assessment of high-risk HPV in the host genome, will aid in the identification of cervical cancer in the cervix. Uterine cervical cancer remains the most common cause of death for women, ranks fourth among all cancers worldwide in females, and is the first major human cancer reported with a single infectious etiology . It is tBiomarker-based test systems have been developed and clinically evaluated on the basis of a clarified understanding of the molecular pathogenesis of how HPVs contribute to the neoplastic transformation of cervical squamous epithelial cells . In partThough previous studies mentioned that immunohistochemical (IHC) identification of p16INK4a could be a biomarker to predict the outcomes of cervical cancer lesions, the predominance of comparing as a biomarker along with HPV genotyping was not fully elucidated. Therefore, in the present study, we aimed to identify the existence of the HPV genome along with the immunoexpression of p16INK4a protein in the uterine cervix as an effective biomarker to identify the role of high-risk HPV in the development of cervical cancer and also to find an association between the HPV genome and the cell cycle inhibitor protein p16INK4a. Doing so, we could provide a strong inference from India to strengthen the association between p16INK4a and HPV for the early detection of high-grade cervical lesions and cervical cancer tissue toward better prophylaxis of the scenario.Study population and specimen collectionThe Institutional Ethics Committee, MNJ Institute of Oncology and Regional Cancer Center, Hyderabad, India, reviewed and approved the protocol on July 12th, 2018 (Regd No. ECR/227/Inst/AP/2013/RR-16). The prospective study consists of 250 samples, 150 cases and 100 controls. A total of 150 histopathologically diagnosed specimens were included for cervical tumors. Both the cervical samples and normal tissues were obtained in normal saline and formalin and were processed based on the methodology demands. Cervical biopsy positive for dysplasia or malignancy in epithelial cells was included, and the sections that were negative for dysplasia or malignancy were excluded from the study.Histopathological analysisSample biopsies were fixed in 10% neutral buffered formalin, dehydrated and paraffin-embedded. Formalin-fixed paraffin-embedded blocks were sectioned on a microtome (5 μm in thickness) for both hematoxylin and eosin (H&E) staining and immunostaining. The H&E stained slides were examined for histopathological diagnosis by an experienced pathologist to record morphological alterations .Immunohistochemical staining and semi-quantitative scoringAll the cervix carcinoma samples were diagnosed histologically and were subjected to immunohistochemical analysis for p16INK4A expression as described elsewhere . The antSemi-quantitative scoring of immunostained cervical cancer sections was performed by an experienced pathologist. Normal cervical tissue blocks were used as controls. Strong, diffuse nuclear, as well as cytoplasmic staining in the tumor cells of the squamous epithelium, is considered p16INK4A positive. First, the points were given based on the intensity of staining and as follows: a grade of 0 is given for no staining; 1 for weak staining; 2 for moderate staining; and 3 for strong staining. Secondly, the points were given according to the proportion of cells stained for p16INK4A as follows: 0 is given for no staining; 1 for <1%; 2 for 1-10%; 3 for 11-33%; 4 for 34-66%; and 5 for >66% of cell staining. The final score was tabulated by adding the points of intensity and the proportion of cells stained for the p16INK4A primary antibody. The maximum score was given 8 and the minimum of 0. A score of 0 to 2 was taken as low expression, in-between 3 and 5 as moderate expression, and a score calculated in-between 6 and 8 was taken as overexpression .Identification of HPV viral genome expression by semi-quantitative PCR methodThe procedure for the identification of high-risk HPV viral genome expression from fresh tissue samples was followed as described elsewhere . In brieStatistical analysisStatistical analysis was performed using SPSS version 20.0 . A descriptive statistics test was used to determine the statistical significance. The p-value of <0.05 was considered statistically significant.This study includes 250 samples, of which 150 were reported with squamous cell carcinoma (cases) and 100 without squamous cell carcinoma (controls). The expression of the high-risk HPV genome was analyzed using the semi-quantitative PCR method. The data show that among 150 case samples, 90.67% n=136) of the cases were identified with the presence of the HPV genome and are categorized as HPV-positive samples. Only 9.33% (n=14) were found to be negative for high-risk HPV in their host genome. Hematoxylin and eosin staining was performed to identify the changes in the cellular morphology  were grouped under cervical intraepithelial neoplasia (CIN). Within the CIN identified, 56.66% (n=17) were noticed with CIN3, 36.66% (n=11) were with CIN2 and 6.67% (n=2) were with CIN1. We noticed that the two individuals grouped under CIN were found to be negative for HPV but they expressed p16INK4A protein with a low score.Categorization of high-risk HPV genome expression was performed based on the immunostaining scoring of the p16INK4A protein. Figure HPV is a definite etiological agent and a sensitive marker for the identification of the risk of progression of cervical neoplasia in cervical cancer patients . The tumCervical carcinogenesis is a complex mechanism due to the integration of the HPV gene into the host cell transcription machinery, which results in the destruction of the immune response of host cells . The rolThe immunohistochemical identification of p16INK4A protein in cervical intraepithelial neoplasia has been reported to be a useful diagnostic biomarker for the identification of cervical cancer among women ,19. SomeLimitation of the studyDue to financial constrictions and the limited duration of the study, the sample size is less, and more sample study is required to be more authentic. We have done an hr-HPV PCR study representing combined genotypes of hr-HPV. We have not used specific genotype markers like HPV 16, 18, or 33 separately.We conclude that there is a strong association between genome expression of high-risk HPV and immune expression of p16, both in cervical intraepithelial lesions as well as cervical malignancies, highlighting the significance of immunohistochemistry with p16 in cases of cervical cancer, and making p16, a surrogate marker for HPV in low resource settings.


Synaptogyrin-3 (SYNGR3) is robustly expressed in immune cells of HPV(+) squamous cancers. Multiplex immunostaining and single-cell RNA sequencing analyses confirmed SYNGR3 expression in T cells, but also unexpectedly in B cells of HPV(+) tumors. ROC curve analyses revealed that combining SYNGR3 and p16 provides more sensitivity and specificity for HPV detection compared with p16 IHC alone. Patients with SYNGR3-high HNSC have significantly better prognosis with 5-year OS and DSS rates of 60% and 71%, respectively. Moreover, combining p16 localization and SYNGR3 expression can further risk stratify HPV(+) patients such that high cytoplasmic, low nuclear p16 do significantly worse  compared with patients with high cytoplasmic, high nuclear p16. SYNGR3 expression in T and B cells is associated with HPV status and enhanced survival outcomes of patients with HNSC.Over 70% of oropharyngeal head and neck squamous cell carcinoma (HNSC) cases in the United States are positive for human papillomavirus (HPV) yet biomarkers for stratifying oropharyngeal HNSC patient risk are limited. We used immunogenomics to identify differentially expressed genes in immune cells of HPV(+) and HPV(−) squamous carcinomas. Candidate genes were tested in clinical specimens using both qRT-PCR and IHC and validated by IHC using the Carolina Head and Neck Cancer Study tissue microarray of HNSC cases. We performed multiplex immunofluorescent staining to confirm expression within the immune cells of HPV(+) tumors, ROC curve analyses, and assessed survival outcomes. The neuronal gene These findings indicate that codetection of SYNGR3 in immune cells and p16 in tumor cells by IHC can more reliably identify the HPV(+) subgroup of patients with low-risk head and neck cancer that may be appropriate for clinical trials involving treatment deescalation. Head and neck squamous cell carcinomas (HNSC) are the sixth most common cancer worldwide . PatientAmong the currently available HPV detection assays commonly used in clinical settings are those involving PCR of the viral oncogenes E6/E7, IHC of p16 protein, and ISH of either DNA or RNA for E6/E7. These assays have varying reliability and availability depending upon sample requirements, test specificity and sensitivity, as well as presence of specific equipment and technical expertise . While P+TCF-1+CD45RO+ stem-like CD8 T cells suggesting that these patients with HNSC retain the ability to respond to PD-1 checkpoint blockade  respond to these therapies . Recent blockade . These iblockade  to bioinblockade , as a ton = 11) were from the following anatomic sites: larynx (n = 2), oral cavity (n = 7), and oropharynx (n = 2). Histopathologic assessments were made by a pathologist and presented in a nonquantitative, binary format (either negative or positive) for both HPV ISH (high-risk HPV strains) and p16 IHC.All research involving human tumor tissues was reviewed and approved by The University of North Carolina at Chapel Hill Institutional Review Board (IRB) under IRB protocols 15-1604 and 17-2947 and the studies were performed in accordance with recognized U.S. Common Rule ethical guidelines. We obtained a waiver of written informed consent from all subjects for the use of their biological specimens. Fresh-frozen HNSC human tumor specimens with affiliated HPV assay clinical diagnoses were obtained through NC Cancer Hospital and UNC Lineberger Comprehensive Cancer Center's Pathology Services Core. Samples , whereas HPV(−) samples were required to have a negative HPV status as determined by both ISH and by p16 IHC testing (n = 56). Normal samples were omitted from the analysis, according to TCGA records (clin.merged file). Similarly, the cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) tumor data from TCGA was also used to perform differential expression analysis between HPV(+) (n = 281) and HPV(−) (n = 22) subjects. HPV status was taken from the available patient HPV test results column in the available merged clinical data. Normal samples were also omitted. Differential expression analysis on both individual bulk RNA-seq datasets was performed using the DESeq2 package in R , and transcript abundance was normalized using quantile normalization. Abundance differences were assessed using Wilcoxon signed-rank test.TCGA RNA-seq datasets used in this study were downloaded from The Broad Institute TCGA GDAC Firehose (gdac.broadinsitue.org), which provides TCGA level 3 data and level 4 analyses packaged in a form amenable to immediate algorithmic analysis. Specifically, publicly available HNSC tumor data from TCGA were used to evaluate the differential expression of genes between HPV(+) and HPV(−) subjects. HPV(+) samples were defined as having a gene expression–based ratio The analyzed immune-related genes were previously identified as belonging to the modified “Immunome” signature’, which can be found in Porrello and colleagues . In partSYNGR3 expression level in various immune cell types across HPV-positive (n = 8) and HPV-negative (n = 18) samples . Single-cell RNA-seq analysis was performed using the Seurat v4 package in R  tissue microarray (TMA) used for these studies includes distinct anatomic locations of the oropharynx, hypopharynx, oral cavity, and larynx and has been thoroughly characterized previously (78). Slides used were reviewed for presence of evaluable tumor. Cores lacking evaluable tumor or with fewer than 500 cells detected by analysis algorithm were excluded. Data presented here include 190 evaluable tumor cores taken from 98 separate tumors (1–3 cores/tumor block). Patient details including sex, race/ethnicity, smoking status, pack years, alcohol use, and diagnosis age are described in Ventana Benchmark XT autostainer was used for HPV ISH according to manufacturer's protocol as described previously (79). INFORM HPV III Family 16 Probe  was used for staining of HPV strains 16, 18, 31, 33, 35, 39, 45, 51,52, 56, 58, and 66. Either punctate or diffuse signal pattern in tumor nuclei was scored as positive staining. HPV ISH positive was defined as nuclear score 1–3 at any percent.Anti-SYNGR3 rabbit polyclonal antibody (referenced as Antibody #1) was purchased from Invitrogen . Anti-SYNGR3 (E-11) mouse mAb (referenced as Antibody #2) was purchased from Santa Cruz Biotechnology . Anti-p16 mouse mAb (D-25) used for individual stain was Sigma-Aldrich ; for multiplex staining anti-p16 mouse mAb by Ventana  was used. pan-Cytokeratin rabbit polyclonal antibody used was from Dako . A mouse mAb to CD3  and a CD45 mouse mAb  from Leica (Leica Microsystems Inc.) were used for multiplex staining.Chromogenic IHC was performed on paraffin-embedded tissues that were sectioned at 5 μm. All IHC was carried out in the Bond III Autostainer (Leica Microsystems Inc.). Slides were dewaxed in Bond Dewax solution (AR9222) and hydrated in Bond Wash solution (AR9590). Antigen retrieval was performed for 20 minutes at 100°C in Bond-Epitope Retrieval solution 1, pH-6.0 (AR9961).For the SYNGR3 Invitrogen antibody, slides were incubated for at 1:500 for 1 hour then ImmPress horseradish peroxidase (HRP) anti-rabbit IgG secondary . For the SYNGR3 Santa Cruz antibody, slides were incubated at 1:50 for 4 hours followed by Novocastra Post Primary  and Novolink Polymer  secondary antibodies for 8 minutes each. Antibody detection with 3,3′-diaminobenzidine (DAB) was performed using the Bond Intense R detection system (DS9263) with ImmPress HRP anti-rabbit IgG .Slides were incubated with SYNGR3 Invitrogen antibody at 1:300 for 1 hour and were detected with ImmPress HRP anti-rabbit IgG and TSA Cy5 (SAT705A001EA). After completion of SYNGR3 staining, a second round of denaturation  was followed by incubation in either anti- pan-Cytokeratin  or CD45  and detection with ImmPress HRP anti-rabbit IgG and TSA Cy3 . Following pan-Cytokeratin/CD45 staining, a third denaturation step was performed for 10 minutes in Bond-Epitope Retrieval solution 2  followed by incubation with either anti-CD3  or p16  then detection with Bond Polymer (DS9455) and TSA Alexa-488 .Stained slides were dehydrated and coverslipped with either Cytoseal 60  or Prolong gold . Positive and negative controls (no primary antibody) were included during staining runs. The slides were digitally scanned at 20× magnification using Aperio AT2 (Aperio Technologies) and uploaded to the Aperio eSlideManager database (Leica Biosystems Inc) at the Pathology Services Core at UNC.In addition to the digital image analysis described below, p16 IHC was also previously scored by pathologists for protein expression (79). Each core was scored for cytoplasmic intensity staining and nuclear intensity staining on a 0–3 scale. The percent of positive staining tumor cells was quantified using 10 microscopic fields of 100 cells each. p16 positive was defined as a cytoplasmic or nuclear score of 1–3 in at least 70% cells.DAB-stained slides for SYNGR3 were digitally scanned using the Aperio ScanScope-XT . DAB-stained slides for p16 were digitally scanned using Aperio ScanScope CS . Multiplex immunofluorescent slides were scanned using the Aperio ScanScope FL . All images were scanned at an apparent 20× magnification and uploaded to the Aperio eSlideManager database  at the Translational Pathology Laboratory at UNC.TMA images stained for p16 or SYNGR3 were digitally segmented into individual cores using TMALab (Aperio Technologies). Whole tissue sections stained were annotated using Aperio ImageScope to remove staining artifacts and tissue folds before they were analyzed. The Cytoplasmic v2 algorithm (Aperio Technologies) was used to analyze p16 and both the Cytoplasmic v2 and the Membrane v9 algorithms (Aperio Technologies) were used to analyze SYNGR3 staining. Using these algorithms, cells were analyzed for DAB signal and the number and percentage of cells with light (1+), medium (2+), and strong (3+) cell staining was determined. H scores were calculated using the following formula: 3 × percentage of strongly staining cells + 2 × percentage of moderately staining cells + the percentage of weakly staining cells, giving a range of 0 to 300. The average DAB intensities in cells, cytoplasm, and nuclei were also determined.TMA images were digitally segmented into individual cores using Tissue Studio in IF TMA mode . Cellular Coexpression analysis algorithms were used to quantify the number of cells expressing individual markers, two of three markers, and all three markers, and cells that were negative for all markers. Tumor microenvironment analysis algorithm was used to segment cores into regions of interest (ROI) based on designated epithelial marker  and quantify cell number expressing/coexpressing each marker. The average cytoplasmic intensity was also determined for all ROIs. Tumor stroma was defined as 25 μm on either side of the border of the tumor core.Cores were binned into categories DN, SP-p16 (SP1), SP-ISH (SP2), and DP as described in For survival analysis, one core per patient tumor block was selected and cores were binned by p16 or SYNGR3 expression from digital image analysis. High p16 cytoplasmic expression was defined as an H-score of 70 or higher; high nuclear expression as a H-score of 100 or higher. High SYNGR3 expression was defined as a cytoplasmic H-score of 70 or higher. For SYNGR3 expression based on p16 staining groups in the HPV(+) TMA, all DN and HPV(−) by droplet digital PCR (ddPCR) cores were excluded; any SP1 core that also had a DN core in its same patient block were also excluded.ROC curve analysis was carried out to examine and compare the classification accuracy on HNSC tumor HPV status among several measures, including p16 and SYNGR3 IHC percentage of tumor staining and H-score. For each of the classifications measured, the AUC was calculated using the pROC package in R  from whiSummary statistics, univariate and bivariate methods including Kaplan–Meier survival analyses and bivariate tests with a significance level alpha set to <0.05, were used to describe the distribution of our patient sample and present their demographic  and clinicopathologic (tumor site) characteristics overall and by p16 cytoplasmic/nuclear expression status. Crude 5- and 10-year survival rates by p16 status were also calculated.Multivariable Cox regression models adjusted for age, sex, race, smoking status, alcohol status, and tumor site were created to estimate the associations of p16 status with OS and DSS. To evaluate these associations, we used HRs and corresponding 95% confidence intervals (CI). All statistical analyses were performed with Stata 16.1  and the same program was also used for figure production.ESR1) gene. Specific primers and hydrolysis probes were designed to amplify a portion of E6 and E7 genes encoded by HPV16 while only a portion of E7 gene was amplified in case of other high risk HPV strains namely, 18, 31, 33, and 35. Two TMA cores which were not included in ddPCR evaluation but were positive by ISH E6/E7 were included as “true positives” due to the high specificity of HPV ISH. Appropriate HPV plasmid controls were used as positive control for each of the digital PCR assays.HPV was detected by ddPCR on the QX-200 platform (Bio-Rad) using QuantaSoft software v1.7.4.0917 (Bio-Rad). The assay was performed as described previously ; detailst test, one-way ANOVA or two-way ANOVA where applicable. Data are presented as mean ± SD or mean ± SEM as indicated in the figure legends.Statistical analyses were performed with GraphPad Prism  using Student https://www.ncbi.nlm.nih.gov/geo/). The raw IHC data analyzed for this study were generated at the UNC Pathology Services Core. Derived data supporting the findings of this study are available from the corresponding author upon request.The bioinformatics data analyzed in this study were obtained from TCGA and the NCBI GEO at (E6/E7 ISH status which resulted in 53 HPV(+) samples  and 56 HPV(−) samples . Unsupervised hierarchical clustering of these data confirmed previous studies (SYNGR3 was fourth most significantly (P = 9.40E-79) upregulated gene identified in HPV(+) tumors  tumors (SYNGR3 expression is high in HPV(+)OPSCC and low in HPV(−) OPSCC  and HPV(−) tumors using TCGA HNSC dataset. Specifically, we examined gene expression in tumors for which clinical information was available. Of the original 279 published HNSC tumors analyzed ( studies  demonstr studies . SYNGR3 ) tumors . Moreove−) OPSCC , suggestSYNGR3 expression in both HPV(+) HNSC  and HPV(+) CESC  compared with HPV(−) tumors , double positive , and DN . There were no available samples positive for only HPV ISH in this tumor cohort. While mRNA levels of CDKN2A/p16 unexpectedly did not correlate with HPV status (SYNGR3 mRNA (P < 0.01) in DP tumors  in the DN group compared with the DP group (P < 0.05) in DP samples than in SP1 or DN samples  Hospitals . We performed qPCR on fresh-frozen specimens to determine levels of the canonical HPV biomarker p16, as well as other candidate genes identified in V status , we obseP tumors . AlternaDP group . To vali samples . Thus, aGiven the sample size limitations of our initial validation cohort, we sought to extend these findings to a larger panel of HNSC tumors. Thus, we next performed SYNGR3 IHC on a TMA that we previously generated for which the results of HPV clinical assays (p16 IHC and HPV ISH) were available . These HWe next evaluated the performance of p16 IHC and SYNGR3 IHC in comparison with a “gold standard” assay for HPV16 DNA that we recently validated in our ongoing multi-institutional prospective phase II clinical trials for patients with HPV-associated HNSC . SpecifiWe performed ROC analyses to determine the optimal cut-off points for the two different antibodies examined in this study for SYNGR3 IHC interpretation in comparison with p16 IHC ; Table 1SYNGR3 expression in HPV(+) HNSCs indicated that it is highly expressed within the Th1 subset of tumor-infiltrating T cells , and CK  and found that the number of coexpressed SYNGR3+/ CD3+ cells was significantly higher  in DP (p16 IHC+/HPV ISH+) tumors compared with DN (p16 IHC-/HPV ISH−) tumors , SP1 = 2,291 cells (23.4%), SP2 = 4,088 cells (47.6%), and DP = 5,054 cells  tumors . The ave+/ CD45+ cells was significantly higher  in DP (p16 IHC+/HPV ISH+) tumors compared with DN (p16 IHC−/HPV ISH−) tumors  compared with the cancer cell islet (pan-CK and p16 positive) regions  for SYNGR3 expression in HPV(+) HNSCs  HNSCs , and p16, and found that the number of coexpressed SYNGR3) tumors . Similar+) HNSCs . Unsuper+) HNSCs . Notably+) HNSCs .SYNGR3 expression quartiles revealed a marked separation in the survival curves such that patients with high SYNGR3 expression show significantly better OS (P = 0.0242) and DSS (P = 0.02607) compared with patients with low SYNGR3 expression  HNSC, we used the UCSC Xenabrowser to analyze publicly available RNA-seq data for TCGA head and neck cancer dataset . Stratifpression .n = 25); low cytoplasmic, low nuclear ; and high cytoplasmic, low nuclear  and LC/LN (P < 0.0001) groups and multivariate analysis demonstrates that this group is associated with significantly improved DSS , source , and measurement method . In the SYNGR3 expression in HPV(+) HNSC and CESC, is also associated with improved response to immunotherapy therapy  tumors have increased tumor immune infiltrates, which have also been shown to be positive for prognosis . With th therapy . Interes therapy  as well  therapy , suggestignature . Probingignature , as wellhigh SYNGR3 may indicate better prognosis. SYNGR3 was also identified as being downregulated in a subgroup of HNSCC with high beta-adrenergic signaling, which was also associated with HPV negativity  and HPV(−) tumors . SYNGR3 Several prognostic biomarkers of HNSC have been identified over the past decade including expression of the estrogen receptor α (ERα) and TRAF3/CYLD . While tDespite HPV infection being a well-established indicator of improved HNSCC outcomes, the reliability of currently available diagnostic assays can be improved. Similarly, increased interest in the tumor immune microenvironment in recent years has revealed its importance in evaluating patient prognosis, as well. This, combined with recent studies detailing disparate genetic signatures and tumor-immune landscapes depending on HPV infection status, motivated our immunogenomic approach to identifying an improved biomarker for HPV diagnosis. Further evaluation of SYNGR3 biology and clinical validation as a biomarker in HNSCC could directly influence patient care.Figure S1HPV(+) and HPV(-) HNSC are characterized by distinct immunogenomic signatures.Click here for additional data file.Figure S2Validation of upregulated and downregulated genes in HPV(+) HNSC patient tumors.Click here for additional data file.Figure S3Additional multiplex staining coexpression and ROI quantification.Click here for additional data file.Figure S4Association of SYNGR3 expression patient survival.Click here for additional data file.Supplementary Table ST1List of Primers.Click here for additional data file.Supplementary Table ST2Demographic and clinical characteristics of the study cases.Click here for additional data file.Supplementary Table ST3Differentially expressed genes from the TCGA Head and Neck Squamous Cell Carcinoma (HNSC).Click here for additional data file.Supplementary Table ST4Differentially expressed genes from the TCGA Cervical Squamous Cell Carcinoma (CESC).Click here for additional data file.Supplementary Table ST5Descriptive statistics for HNSC patients from the CHANCE study by p16 cytoplasmic/nuclear status.Click here for additional data file.Supplementary Table ST6Crude 5- and 10-year survival rates by p16 cytoplasmic/nuclear status.Click here for additional data file.


To assess the knowledge of students from public high schools in poor communities about HPV and Sexually Transmitted Infections and their attitude towards and prevention of such diseases.Cross-sectional study with adolescents from public schools of São Paulo – Brazil. Participants were selected for an interview by a randomization program. A questionnaire about knowledge, attitudes, and preventive practices regarding STIs, including HPV, according to sex was administered and answers were analyzed by the Poisson regression model with robust variance.Median age of the 269 participants was 16 years. The majority was of African descent , most  were religious and the vast majority  lived with their parents. The Poisson regression revealed statistically significant sex-related differences regarding the following questions: “Do you know how it is prevented?” ; “Have you ever been concerned with HPV?” ; “Have you ever sought health care due to concerns about HPV?” ; “Do you know what a Pap Smear is?” ; “Do you know what the cervix is?” ; “Do you know what cervical cancer is?” .The present results show that adolescents from public schools in poor communities in São Paulo City know little about HPV and cervical cancer. Male adolescents know less than female adolescents and are less concerned with health care. Adolescence is the period when sexual life might initiate. Currently, most adolescents engage in sexual intercourse at an increasingly early age.The prevalence of HPV in Brazil is 40% to 60%, and in the state of São Paulo, it is over 56%.,Distribution of male condoms and HPV vaccination are strategies to reduce transmission of HPV and other STIs and prevent precursor lesions of cervical cancer among teenagers. Adherence to condom use and to vaccination are behaviors dependent on one's knowledge of these infections, and they may be influenced by sex, socioeconomic level, and educational, cultural, and religious backgrounds.,A school is a place of learning for adolescents. It not only plays an important role in education, but it is also a setting for the promotion of reproductive and sexual health among youth.,HPV infection is associated with age, for it is young people who are most prone to taking risks. In Brazil, the quadrivalent HPV vaccine is freely distributed to girls from 9 to 14 years old, boys from 11 to 14 years old, and immunosuppressed men and women, aged 9 to 45 years, living with HIV/AIDS, transplanted solid organs or bone marrow, and cancer patients.,However, the authors don't know if public high schools in underprivileged areas are being able to transmit necessary information about HPV and STIs and to develop the interest of adolescents from low-income families in their sexual health.This cross-sectional study assessing students from three public high schools was conducted by the Discipline of Gynecology of the Universidade de São Paulo (USP) between 2018 and 2019. The project was approved by the Ethics Committee of the “Faculdade de Medicina da Universidade de São Paulo” (FMUSP). The protocol number is 38719314.2.00000068.Of the 2588 students from three state high schools located in the city of São Paulo, 375 were selected for an interview according to the following inclusion criteria: proper enrollment in a public school and 19 years of age or less. The sample size was estimated at 253 students, given a confidence level of 85% and power test of 80% in line with the Kops et al. (2019)The students were selected for an interview by a computer-generated randomization list. If a student on the list was unwilling to participate, no substitution was made . Prior to data collection, all interviewers were trained and certified.The questionnaire was designed by the authors in accordance with the scientific literature. It addresses the student's knowledge of the prevention, transmission, and consequences of STIs, including HPV, as well as their attitude toward the diseases and their preventive practices. The questionnaire contains 13 items grouped into four categories: knowledge, attitudes, health practices, and HPV vaccination.Programa Institucional de Bolsas de Iniciação Científica (PIBIC) , which is a program of prescientific initiation and technology innovation at USP. Before the questionnaires were handed out, the participants were informed about the research objectives, and they signed an informed consent form. No student identification was required on the questionnaire. After the students completed the questionnaires, any questions they had were answered for further clarification.The questionnaires were administered by students from the A pilot study was conducted in 2018 (n = 50) to evaluate and improve the questions and ensure comprehension. Validation of the questionnaire was based on experts’ analyses , semantic analyses, and pretesting. After completion, each questionnaire was analyzed and then tabulated. Thirty pilot interviews were recorded, and three interviewers listened to each interview. The questionnaire was then improved further, and the interviewers received instructions for administering an interview and training as well as shown in Supplementary Material.Bolsa Família), sexual intercourse, and family nucleus.In addition to the questionnaire above, a sociodemographic questionnaire was drawn up with questions about birthplace, age, ethnicity, sex, religion, family income, the social welfare program  software. Answers to questions 1, 2, 3, 6, 7, 8, 9 10, 11, 12, and 13 were dichotomized into “presence of knowledge” when the answer was “I know a little” or “I know”, and “lack of knowledge”, when the answer was “I don't know”, “I heard of” or “I know partially”.On the other hand, answers to questions 4 and 5 were dichotomized into “I was worried/I sought health care”, “when the answer was “frequently”, “I'm aware”, or “I worried a lot”, and into ‘I wasn't worried/I didn't seek health care’ when the answer was “never” or “sometimes”.https://doi.org/10.7910/DVN/OCNLUF.The datasetParticipants were divided into 2 groups according to their sex: male or female. Qualitative variables were reported as absolute and relative frequencies, and the quantitative variable ‘age’ was expressed by the median and confidence interval. The Chi-Square and the Mann-Whitney tests were used to analyze homogeneity between the two groups in relation to socioeconomic variables.The Poisson regression model with robust variance was used to analyze the answers to questions about students’ knowledge, attitudes, and preventive practices with respect to STIs, including HPV, according to sex. The level of statistical significance for the sample was set at 5%. The model was adjusted according to income, age, ethnicity, grade repetition, cohabitation with parents, and religion.The internal consistency of the questionnaire, expressed by Cronbach's alpha coefficient, 0.78 in this case, was substantial.The total study population consisted of 269 high school students in which themedian age was 16 years . The majority  lived in the city of São Paulo, and most of these  resided in neighborhoods near their schools. A considerable number of the students  were of African descent.Bolsa Família  declared they had already engaged in sexual intercourse; most  had religious beliefs; a vast majority  lived with their parents; and over two-thirds  had never failed a grade in school. Despite the fact that most  received less than the minimum salary, a very large number  were not enrolled in the country's social welfare program, the so-called  Família .Table 1SThe comparison of sociodemographic characteristics between the female and the male students yielded a significant statistical difference only in terms of the variable “has religious beliefs” (p = 0.037).A sex-based analysis of the student's responses to questions about their knowledge, attitudes, and preventive practices with respect to HPV and STIs, along with the 95% CI, prevalence ratio, and p results, is shown in The use of the adjusted Poisson regression model with robust variance revealed a difference in the answers of boys and girls. The following questions had statistically different answers when comparing the female and the male adolescents: “Do you know what HPV is?” ; “Do you know how it is prevented?” ; “Have you ever been concerned with HPV?” ; “Have you ever sought health care due to concerns about HPV?” ; “Do you know what a Pap Smear is?” ; “Do you know what the cervix is?” ; “Do you know what cervical cancer is?” . A statistical difference was also found in the crude model, which also exhibited a difference in relation to the question “Do you know how it is transmitted?”  .In adolescence, the desire for sexual experience frequently predisposes young people to risky sexual habits due to their immaturity and inexperience. Access to information and health professionals can assist with guidance and the acquisition of healthy habits.The present study indicates that adolescents know little about STIs, that less than 40% show some concern about HPV, and that only 12.6% have sought health care due to worries about HPV. The male sex was less knowledgeable and concerned than the female sex.,Studies of knowledge about HPV/STI are important guidelines in public health, as they show that gaps in understanding can be barriers to the self-care process and to adherence to vaccination programs. The consequences of HPV are not immediate since cancer precursor lesions and HPV-induced lesions take a while to be detected and treated, with repercussions that include high mortality from these cancers, mainly in developing countries.The sample was characterized by a majority population of Afro-descendants. This epidemiological profile is similar to that of other studies with parallel topics. The predominance of students of African descent in public schools should be highlighted. Racial inequality among low-income adolescents in Brazil is a factor that may have a negative psychological influence, especially when one feels discriminated against by their ethnicity.The assessment of homogeneity between characteristics of male and female adolescents shows that the variable ‘has religious beliefs’ is answered positively more often by the female population. The studied country is predominantly Catholic, and the Church provides information on reproductive and sexual health,An important point in the present study is the difference in knowledge about STIs, including HPV, between the male and the female sexes: the latter know more about the subject than the former. Many factors may be associated with the discrepancy between the sexes. The study by Genz et al. (2017)Besides, in Brazil, the implementation of the government's HPV vaccine program in schools, initially was limited to girls and their parents, which may have contributed to the differences in knowledge between the sexes.In the study by Sousa et al. (2018),The results of this study reinforce the need for health promotion actions with adolescents – especially male students ‒ in order to improve their knowledge about HPV and/or STIs beyond the dissemination of such knowledge in schools,This shows a clear need to address the male population; even if male adolescents have not been strongly affected by HPV, they may have transmitted the virus and spread it among the female population.Cervical cancer has HPV as its main etiological viral factor and other STIs as associated factors, being highly prevalent in Brazil.Thus, there is a need to intensify campaigns, which should aim mostly at public school male teenagers. Not only chemical and biological processes, but also intense and unstructured emotions occur in this phase, possibly triggering affective relationships that become the gateway to erroneous, premature, and unprotected sex predisposing adolescents to contract STIs.One of the limitations of this cross-sectional study is that the present study's sample consisted of adolescents from public schools. This narrow selection of subjects may have biased the variable ‘knowledge and perception’ because it does not necessarily express the reality of other teenagers with the same socioeconomic level or access to education.Another limiting factor is the use of quantitatively analyzed objective questionnaires, where students were asked whether or not they had knowledge about the subjects in question, but these answers were not confirmed. In this way, the knowledge investigated is referred/perceived. In addition, the questionnaires do not allow the exploration of socioeconomic aspects, such as religious beliefs. nor the prevalence of STI/HPV and vaccination. Another shortcoming is the fact that this study was not designed to assess the real causes of male disinterest in their health. Therefore, other studies are needed.The highlights of this study conducted in a poor community include an elucidation of the divergences of sexual health knowledge among adolescents and a comparison of boys’ and girls’ knowledge. Such information can be used to underpin sex education, reproductive health programs, and specific health promotion actions. Public health education should be the focus of pediatric and adolescent gynecologists.The presentresults show that about half of the adolescents from public schools in the city of São Paulo do not know what HPV is, nor do they know what cervical cancer is. Indeed, a large part never worried about HPV and the vast majority did not seek a health service due to HPV-related issues. In addition, male adolescents not only know less than female adolescents but are also less concerned about health care.The project was approved by the Ethics Committee of the Faculdade de Medicina da USP. The protocol number is 38719314.2.00000068.The participants were informed about the research objectives, and they signed an informed consent form.J.M.S.J.: He made substantial contributions to the concept, study design, and definition of intellectual content; he was involved in literature search, data analysis, statistical analysis, manuscript preparation, and manuscript writing; he drafted the article or revised it critically for important intellectual content, and he approved the final version to be published.H.M.C.O.: She made substantial contributions to the concept, study design, and definition of intellectual content; she was involved in literature search, data analysis, statistical analysis, manuscript preparation, and manuscript writing; she drafted the article or revised it critically for important intellectual content; and she approved the final version to be published.C.M.L.: She was involved in literature search, data analysis, statistical analysis, manuscript preparation, and manuscript writing; she drafted the article or revised it critically for important intellectual content.L.T.S.Z.: She was involved in literature search, data analysis, and statistical analysis; and she approved the final version to be published.E.C.A.V: He was involved in literature search, data analysis, and statistical analysis; and he approved the final version to be published.J.Z.R.: She was involved in literature search, data analysis, statistical analysis; and she approved the final version to be published.F.W.S.F.: He was involved in literature search, data analysis, and statistical analysis; and he approved the final version to be published.M.S.A.: She was involved in literature search, manuscript preparation, and manuscript writing; and she approved the final version to be published.I.C.E.S.: She made substantial contributions to the concept, study design, and definition of intellectual content; she was involved in literature search, data analysis, statistical analysis, manuscript preparation, and manuscript writing; she drafted the article or revised it critically for important intellectual content; and she approved the final version to be published.E.C.B: He was involved in literature search, data analysis, and statistical analysis; and he approved the final version to be published.FAPESP (#2016/16847-0 and #2017/07276-2), CNPq (#301725/2017-9), and CAPES .This study was supported by The authors declare no conflicts of interest.