Knowledge of the prevalence of HPV infection among adolescent and early adult girls is essential to determining the best age for the introduction of HPV vaccine, monitoring vaccine efficacy, and giving insight into determinants of persistent high-risk HPV infection, a necessary cause of cervical cancer. Yet, there have been limited studies of HPV infection among adolescent and early adult girls in low-and-middle-income countries.10-DEIA/LiPA25 and analyzed data using Stata 14®.In this cross-sectional study, we randomly selected 205 girls, aged 9–20 years, from 10 schools in central Nigeria. We obtained informed consent and assent, collected data, and trained participants to self-collect vaginal samples using swab stick. We genotyped HPV using SPFThe mean (SD) age of the girls was 14.9 (2.3) years. We found HPV in 13.2% of vaginal swabs. The earliest age at which anyHPV and hrHPV infections were detected was 10 and 12 years respectively. The prevalence of any HPV peaked at 16 and 17 years, hrHPV at 16 years, lrHPV at 17 and 18 years and multiple hrHPV 18 years of age. The prevalence of hrHPV infection was 1.5% among the 9–12 years age group, 2.9% among 13–16 years and 3.4% among 17–20 years old. The commonest hrHPV types detected were 52 (3.9%), 18 (1.5%) and 51 (2.4%). The most common lrHPV types was 6 (2.9%).The prevalence of HPV infection in these urbanized young girls in Nigeria is high and commences after 9 years of age. HPV vaccination in this population should start at 9 years of age or younger to prevent the establishment of persistent HPV infection.The online version contains supplementary material available at 10.1186/s12879-022-07215-7. The prevalence of HPV was 13.2% among adolescent girls in Jos, North-Central Nigeria.The earliest age at vaginal HPV infection was 11 years of age.The most predominant hrHPV type in vaginal swab was HPV 52.These findings point to the need for introduction of HPV vaccine starting at 9 years of age among girls in Nigeria to give maximum protection.The online version contains supplementary material available at 10.1186/s12879-022-07215-7. Human papillomavirus (HPV) infection is the commonest oncogenic virus infection in the world. It is associated with cancers of the cervix, anus, vagina, oropharynx, vulva, oral cavity, penis and larynx , 2. In 2HPV vaccination has been shown to be efficacious for prevention of infection and associated diseases  The vaccSubstantial progress has been made in reducing HPV prevalence in developed countries like the USA  and AustWhile there has been several studies of the prevalence of HPV infection among HIV positive and negative women in Nigeria , 19, datWe randomly selected 15 out of 300 registered high schools in Jos, North Central Nigeria for this study and received permission to conduct the study in 10 (66.7%) of the schools. Between May and November 2016, we contacted 700 female students and their parents, of whom 232 girls and their parents agreed to participate in the study giving a 33.1% response rate. We obtained consent from the parents of girls younger than 18 years of age and assent from the girls. Older girls gave consent in their self-cognizance.We conducted face-to-face interview with the girls in company of female nurses or researchers using structured questionnaires that covered socioeconomic factors, family characteristics, gynecologic history, sexual hygiene and practices. Questionnaire designs were guided by the research questions and literature review. The questionnaires were pre-tested on 10 individuals to evaluate their performance and corrected as required.Remove the swab from the reclosable pack.Gently insert the cotton swab into the vagina while squatting or standing with legs apart.Gently turn the swab three times and remove.Replace the swab in the plastic pack.Repeat this procedure with the second swab stick.Swabs were inserted for about 3 cm and NC was always present to guide the girlsPrior to sample collection, we trained study participants on how to self-obtain vaginal samples using sterile swab sticks—a small cotton swab on a wooden handle packaged in an individual reclosable plastic pack. After the training, each girl was given 2 swab sticks and instructed to:The first collected vulvo-vaginal swab was immediately stored on ice pack in a cooler until transported to the Plateau State Human Virology Research Centre (PLASVIREC) sample repository for storage at −80 °C, usually within three hours of collection. The second swab was immediately applied on Hydrion pH test paper   the colour change was compared with the provided standards.Collected samples were transported to the African Collaborative Centre for Microbiome and Genomics Research (ACCME), Institute of Human Virology, Abuja, Nigeria for HPV detection and genotyping. To prepare the vulvo-vaginal swab for DNA extraction, 1 millilitre of PBS was added to the dry swabs inside the cryovials and vortexed to dislodge the cells from the cotton wool.10  which simultaneously detects HPV types 2, 3, 4, 5, 6, 7, 8, 11, 13, 14, 16, 18, 20, 26, 27, 28, 30, 31, 32, 33, 34, 35, 37, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55 (re-classified as a subtype of HPV44), 56, 57, 58, 59, 61, 62, 64 (re-classified as a subtype of HPV34), 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 81, 82, 83, 84, 85, 86, 87, 89, 90, 91, 95, 97, 102, 106, 114 and 115. Positive samples were tested with reverse hybridization line probe assay (LiPA25) which can simultaneously identify 25 HPV types  according to the manufacturer’s  instructions. This is a highly sensitive broad-spectrum PCR-based assay for qualitative detection of HPV and suitable for epidemiological studies.DNA extraction was carried out using Cador® Pathogen 96 QIAcube® HT Kit  on QIAcube HT . The DNA was amplified and HPV detected using SPFThe study protocol was approved by the institutional review board of the Jos University Teaching Hospital (JUTH). Written informed consent was obtained from the parent/guardian of participants who were less than 18 year of age and assent obtained from the participants themselves. For participants who were greater than or equal 18 years of age, they provided written informed consent prior to enrolment.p-value < 0.05 as statistically significant.We entered data into REDCap electronic database and analyzed using Stata version 14 . High-risk HPV types were defined as types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 88, 73 and 82, including the probable hrHPV types and low-risk types were defined as types 6, 11, 40, 42, 43, 44, 54, 61, 68/73, 70, 72, 74, 81 and CP6108 . Univarip-value 0.67). Most of the excluded participants  were from public schools while the remaining ten participants (37.0%) were from private schools. About half of the remaining participants, , were recruited from public schools while 49.3% (101/205) were from private schools . There were no significant differences between girls who were unable to collect their samples and were excluded from the study and those who remained in the study.We excluded 27 participants (11.6%) because they were unable to collect vulvo-vaginal swab leaving 205 participants. The mean age (SD) of the excluded girls was 14.7 (1.97) years while that of the remaining study participants was 14.9 (2.34) years (p = 0.03). Only 9.3% (19/186) of the girls reported positive history of sexual intercourse and this was significantly different (p = 0.01) comparing HPV positive (22.2%) and HPV negative (7.3%) girls. The mean (SD) age at first sexual intercourse for girls who reported positive sexual history was 13.3 (3.58) years, and this was of marginal statistical significance comparing HPV positive (12.0(3.46)) and HPV negative (16.2(1.72) girls (p = 0.07). Most of the girls  who reported sexual intercourse first experienced it at 15 years of age or less and 15.8% (3/19) experienced it before the age of 10 years. Among HPV positive girls, most  of those who have ever had sex had it after 15 years of age compared to only 23.1% (3/13) of HPV negative girls (p = 0.04). Most of the girls with sexual experience  reported one or two lifetime sexual partners while two girls (10.5%) reported more than two sexual partners. All the sexually experienced HPV negative girls had one or two sexual partners while 66.7% (4/6) of the HPV positive girls had one or two sexual partners and the rest (33.3%) have had three or four sexual partners (p = 0.09). Of the girls who had positive sexual history, 11 (57.9%) reported that the sex was forced and without their consent but the prevalence of this not different comparing HPV positive and HPV negative girls (p = 1.00). Among those girls with positive sexual history who reported the age of their sexual partners, most  were older than 21 years of age, as were the sexual partners of all the HPV positive girls compared to only 33.3% (1/3) of the HPV negative girls (p = 0.11).Some 13.2% (27/205) of the girls had any HPV infection. There was no difference in type of school attended and socio-economic status of HPV positive compared with HPV negative girls. Most  of the girls in this study had attained menarche and there was significant difference in menarcheal status comparing HPV positive (88.9%) and HPV negative (68.0%) girls , history of masturbation, use of toys for masturbation, use of condoms, douching, and pH of the vagina comparing HPV positive and HPV negative girls. The girls in this study discussed sex more with others than with their mothers or fathers. Among the others they discussed sex with, the most common were friends, aunts, and sisters. They had low level of knowledge about HPV infection or HPV vaccination Table .Table 1CThe prevalence of any HPV infection in this study was 13.2% (27/205), while that of hrHPV was 59.3% (16/27), lrHPV was 55.6% (15/27), mixed high and low risk HPV was 14.8% (4/27) and multiple hrHPV was 22.2% (6/27). The age-specific and age group prevalence of any HPV among study participants are shown in Fig. 10LiPA25 in this study population. We detected nine hrHPV types and the commonest hrHPV types were 52 (3.9%), 18 (1.5%) and 51 (2.4%. The other hrHPV types detected in this study were 58 (1.0%), 66 (1.0%), 45 (0.5%), and 31 (0.5%). We did not detect any HPV type 16 in this study sample. The most common lrHPV type we identified was type 6 (2.9%) followed by type 44 (1.0%). Other lrHPV types detected were types 68/73 (0.5%), and 74 (0.5). We did not detect lrHPV type 11 among this cohort of adolescent girls. The distribution of these HPV types in relation to the 27 positive participants is shown in Fig. The prevalence of unknown HPV types found in this study population was 2.4%, suggesting substantial prevalence of HPV types beyond those detected using SPFThe age at first detection of HPV types 18, 45, 66, 6, and 74 was 16 years, while HPV types 51, 52, and 58 was detected at 12 years. HPV types 31, 44 and 68/74 were first detected at 19, 15 and 17 years respectively.p-value = 0.04) was significantly associated with risk of anyHPV. There was no statistically significant associations between low or high risk HPV and any other variable in multivariable analyses : 3.11 (1.04–9.34), es Table .Table 2APrevious studies in Nigeria have reported the prevalence of HPV infection in the general population of women and also among HIV positive women , 19. Up The overall prevalence of HPV found in vaginal swab in this population of adolescent females irrespective of the sample type was 13.2%. Globally, the overall prevalence of HPV infection varies according to geographical regions. Our finding is lower than the 24.5% finding among adolescent girls age 14–19 years in the USA before the introduction of HPV vaccination , 66.7% aWe found significant associations between any HPV infection, menarcheal status and history of ever having sex. Association with age at first sex was marginally significant with most HPV positive girls having first had sex at more than 15 years of age compared with HPV negative girls, most of whom had their first sexual experience at 15 years of age or less. Age is an important determinant of HPV vaccination. The WHO recommends vaccine administration as early as age 9 years targeting girls before the onset of sexual activity. In this study, the earliest age at which anyHPV infections was detected was 11 years. HPV Infection peaked at age 16 years and declined with increasing age. This corroborates with previous findings that adolescent girls have higher exposure to HPV infection  and coulThe high HPV infection prevalence of 13.2% recorded in this study is of public health concern and underscores the importance of introduction of HPV vaccines among adolescents in Nigeria for the prevention of HPV infection and associated malignancies. Although more than 90% of all infection with any HPV resolve without treatment within 2 years approximately 10% of infection fail to resolve, resulting in persistent infection with the virus . PersistHPV vaccination is best administered to HPV naïve individuals and before the onset of sexual activities . The earThe major limitation of the study is the small sample size which reduced the power of the analyses, particularly for exploration of associations between group and type-specific HPV infections and their risk factors. Furthermore, this study was carried out among school attending adolescent and early adult girls. The findings may not be wholly generalizable to girls outside of school settings where other factors are likely to play a role. An important avenue for future research may be to consider including girls who are out of school to give a more generalizable outcome.To the best of our knowledge, this is the first HPV study to be conducted in Nigeria focused on young girls who are the primary target group for HPV vaccine. Knowledge about HPV infection prevalence among adolescent girls had provided an important gateway into HPV vaccine introduction in and vaccine success evaluation. Our findings indicate that HPV vaccination starting at 9 year of age would adequately cover the at-risk population in Nigeria. The study lays the foundation for future monitoring of vaccine efficacy and give insight into the determinants of persistent high-risk HPV infection (hrHPV) among the study population.Additional file 1. HPV among adolescent and young adult grils_dataset.


Human papillomavirus (HPV) was proven to play a significant role in cancer development in the oropharynx. However, its role in the development of laryngeal (LSCC) and hypopharyngeal squamous cell carcinoma (HPSCC) remains to be clarified. High-risk HPV (HR-HPV) viral proteins E6 and E7 are considered to be pertinent to HPV-related carcinogenesis. Hence, our aim was to estimate LSCC and HPSCC for HR-HPV DNA, p16, and E6/E7 oncoprotein status by using molecular virology and immunohistochemistry methods. The prevalence of HPV16 infection was 22/41 (53.7%) and 20/31 (64.5%) for LSCC and HPSCC, accordingly. The majority of HPV16+ tumor samples were stage III or IV. In most samples, the presence of either HPV16 E6 or HPV16 E7 viral protein in dysplastic or tumor cells was confirmed using immunohistochemistry. Our results suggest a high prevalence of HPV16 as a primary HR-HPV type in LSCC and HPSCC. The lack of HPV E6/E7 oncoproteins in some tumor samples may suggest either the absence of viral integration or the presence of other mechanisms of tumorigenesis. The utilization of p16 IHC as a surrogate marker of HR-HPV infection is impractical in LSCC and HPSCC. Laryngeal and hypopharyngeal cancers belong to a large group of squamous cell carcinomas of the head and the neck. The annual incidence of these types of tumors in the world population exceeds 250,000 new registered cases . Among tIn the last three decades, the contribution of HPV to the development of oropharyngeal squamous cell carcinoma (OPSCC) was proven ,4. FurthAccumulating evidence suggests the epithelium of skin and mucosa is infected through superficial defects, and, upon establishment of viral genomes in the nucleus of infected cells as episomes, the early viral genes E1, E2, E6, and E7 are expressed. Furthermore, HPV episomes are maintained in poorly differentiating but actively dividing basal epithelial cells by replicating along with cellular DNA ,12,13. AWhile the viral oncoprotein E6 induces the degradation of p53, leading to the inhibition of elimination by apoptosis in affected epithelial cells, oncoprotein E7 inactivates tumor suppressor proteins of the pRb family, promoting the transcription of p16 ,15,16,17Application of p16 for the assessment of transcriptionally active HR-HPV infection in non-OPSCCs and discussion around it highlight the complexity arising when exploring LSCC and HPSCC ,30,31. CThis study aimed to estimate LSCC and HPSCC for HR-HPV DNA, p16, and E6/E7 oncoprotein status by using molecular virology and immunohistochemistry methods.Seventy-two patients, 68 (94.4%) males (median age 64.9 (range 44.2–83.3)) and 4 (5.6%) females (median age 70.8 (range 53.5–77.5)) with histologically confirmed LSCC and HPSCC, treated at the Latvian Oncology Centre between January 2015 and August 2019, were enrolled in the study.The clinical data of patients included information on TNM stage, smoking and drinking habits, and clinical features of the disease at the time of presentation. Forty-one of 72 patients had LSCC; for 31 patients, the primary tumor site was the hypopharynx. Most patients (88.9%) were smokers; 15 (20.8%) were heavy drinkers . The patThe study was approved by the Ethical Committee of Riga Stradiņš University (Decisions No. 3/24.09.2015.) and conducted according to the Declaration of Helsinki.Total DNA was extracted from either fresh frozen biopsies and surgical materials (34 LSCC and 3 HPSCC) or FFPE tumor tissue blocks (28 HPSCC and 7 LSCC).DNA extraction from the fresh frozen tumor tissue samples was carried out with the phenol/chloroform extraction method.DNA extraction from FFPE tumor samples was carried out with the blackPREP FFPE DNA Kit  following the manufacturer’s protocol. Three to six 10 µm thick sections cut from FFPE samples were used for DNA extraction. Each sample was sectioned separately with a sterile blade to exclude cross-contamination of specimens.The concentration and the quality of the extracted DNA were measured spectrophotometrically . Beta- (β-) globin PCR with appropriate primers was used to determine the quality of isolated DNA . All samInitially, separate regular polymerase chain reactions (PCR) with consensus primers MY9/MY11 and GP5+/6+ were used for the detection of a broad range of HPV types ,40. The Two types of primers were used: the type-specific primers for HPV 16 and 18 (L1) and the Anyplex II HPV28 multiplex real-time-PCR (RT-PCR).Genomic sequences of HPV16 and HPV18 type-specific primers’ are summarized in Anyplex II HPV28 multiplex RT-PCR was performed as recommended by the manufacturers . In total, 5 μL of specimen DNA were added in each of the two sets (wells) with 20 μL PCR reaction mix. Set A consisted of a primer mix for 14 HR-HPV types , and set B consisted of a primer mix for five possible HR-HPV types  and nine LR- HPV types . Both primer sets were designed for the HPV L1 gene and produced 100 and 200 bp amplicons, accordingly. Melting curves allowing the semiquantitative assessment and the differentiation between high (+++), moderate (++), or low (+) viral load were obtained at 30, 40, and 50 cycles and had positive and negative internal controls.The kit had DNA quality control detecting the β-globin gene; all analyzed samples were β-globin positive. The results were analyzed using the Seegene Viewer software .LSCC specimens were obtained during laryngectomy and cordectomy. A small piece of surgically obtained LSCC was further processed for molecular testing, whereas the remaining material as a FFPE tissue was submitted to IHC.In contrast, HPSCC specimens were mostly obtained during a biopsy procedure and further processed as FFPE tissue samples at Latvian Oncology Centre. Only tumor tissue samples confirmed by histopathological examination as HPSCC were used in the study.HPV 16 E6/E7 proteins and p16 were assessed immunohistochemically. Histological sections of 4–5 µm were cut from FFPE tissues and mounted on slides. The consecutive sections were used as negative controls of the immunohistochemical reactions and for hematoxylin and eosin (H&E) staining to confirm the diagnosis. Immunohistochemistry (IHC) was performed manually using sections collected on SuperFrost Plus slides . Immunostaining was carried out following the previously used IHC protocol ,42.The sections were incubated at 4 °C overnight with the following primary antibodies: monoclonal mouse anti-CDKN2A/p16INK4a antibody ; monoclonal mouse anti-HPV16 E6 + HPV18 E6 antibody , which recognize the HPV early antigen E6 of HPV 16 and 18 ,44,45; mThe amplification of the primary antibody and the visualization of reaction products were performed by applying the HiDef Detection HRP Polymer system and diaminobenzidine tetrahydrochloride substrate kit . The sections were counterstained with Mayer’s hematoxylin, washed, mounted, and covered with coverslips. The immunohistochemical controls included the omission of the primary antibody. The assessment of immunostaining was performed by two independent observers blinded to clinicopathological data.The sections were photographed by a Leitz DMRB bright-field microscope using a DFC 450C digital camera or scanned with a Glissando Slide Scanner  with a 10×, 20×, and 40× objective.Cells that were labeled with anti-CDKN2A/p16INK4a, anti- HPV16 E6 + HPV18 E6, and anti-HPV16 E7 antibody and that displayed brown reaction products were considered immunopositive.The assessment of immunostaining of p16 was carried out by determining positive vs. negative structures with a cut-off at 50% tumor cells independently of the reaction proposed by Hong et al., (2013) . The immTo better visualize the cellular distribution and the localization of the HR-HPV16 E7 oncoprotein, the tumor tissue specimens were processed for fluorescence-based immunodetection. The sections immunoreacted with mouse monoclonal anti-HPV16 E7 antibody  overnight at 4 °C were washed in PBS and incubated with goat anti-mouse IgG-FITC: sc-2010  as the secondary antibody. Then, sections were counterstained with 4′,6-diamidino-2phenylindole (DAPI)  and mounted in Prolong Gold with DAPI . Imaging was performed using an Eclipse Ti-E confocal microscope .The workflow of the present study is summarized in p-value of less than 0.05 (p < 0.05) was considered statistically significant.All the statistical analyses were performed using the GraphPad Prism 9 . Anderson–Darling, D’Agostino and Pearson, and Shapiro–Wilk normality tests were applied to assess numerical data distribution. The comparison of means between different groups of numerical variables was performed using one-way ANOVA. For data with a non-Gaussian distribution, Kruskal–Wallis or Friedman’s test (for paired groups) followed by the two-stage step-up method of Benjamini, Krieger, and Yekutieli as a false discovery rate controlling test were used. To compare numerical values between two groups, the Mann–Whitney test or the Wilcoxon test (for pared groups) was applied. The relations between the analyzed groups were investigated using nonparametric Spearman’s correlation analysis . The IHCEleven (15.3%) out of 72 tumor samples were positive for HPV genomic sequences using PCR with MY9/11 consensus primers. In turn, 55 (76.4%) samples demonstrated positivity using PCR with GP5+/6+ primers. By summing up, when tested with consensus PCRs, 61 (84.7%) tumor tissue samples were found positive for HPV DNA—31 HPSCC and 30 LSCC samples.n = 72) were subjected to HPV genotyping using HPV16 and HPV18 L1 primers. A total of 2/72 tumor tissue samples (both LSCC), positive when detected by HPV16 L1 primers, were negative in consensus PCRs. No specific HPV-18 genomic sequence was found in any of the samples.All the tumor tissue samples  samples were positive for HPV16—10 LSCC and 16 HPSCC samples. In total, 63 HPV+ samples were considered applicable for further analysis; among them, 61 were selected using consensus PCRs, whereas 2 additional samples were selected by HPV16 L1 PCR.All 63 HPV-positive samples confirmed using either consensus primers or HPV16-specific primers were further explored by Anyplex II HPV28 multiplex RT-PCR. All samples were β-globin positive . When assessed by Anyplex II HPV28 multiplex RT-PCR, 28/63 samples were HPV-negative. HPV16 monoinfection was confirmed in 32/63 samples, whereas HPV16 and HPV31 coinfection was confirmed in 2/63 samples, and HPV16 and HPV56 coinfection in 1/63 samples. When HPV+ samples were stratified by the location, 19 LSCC and 13 HPSCC presented as HPV16+, 2 LSCC presented as demonstrating HPV16 and HPV31 coinfection, and 1 HPSCC presented as demonstrating HPV16 and HPV56 coinfection.Interestingly, seven (one LSCC and six HPSCC) HPV16+ tumor tissue samples, confirmed by applying HPV16 L1 PCR, were negative according to Anyplex II HPV28 RT-PCR, thus contributing to a total number of 42/72 (58.3%) HPV16+ samples. The prevalence of HPV16 infection, including multiple infections in a sample, was 22 of 41 (53.7%) for LSCC and 20 of 31 (64.5%) for HPSCC . Most ofTwenty-one HPV16+ samples presented with low, nine with moderate, and two with high viral load, respectively, when detected using the Anyplex II assay. The presence of multiple HPV infections was confirmed in three samples (one HPSCC and two LSCC). A low viral load for both HPV types was established in the HPSCC sample presented with HPV16 and 56 coinfections. Similarly, a low viral load for both HPV types was confirmed in the LSCC sample presented with HPV16 and 31 coinfections. By contrast, a low viral load was confirmed for HPV16, whereas a high viral load was confirmed for HPV31, assessed in the remaining LSCC sample with HPV 16 and 31 coinfections, thus suggesting the dominance of HPV31. The HPV16 RT-PCR data are summarized in n = 6), whereas two were HPSCC —p16+/HPV+, 1/72—p16+/HPV-, 8/72 (11.1%)—p16-/HPV-, and 56/72 (77.8%)—p16-/HPV+. Most of the p16+/HPV+ tumors were LSCC  samples was based on the primary recognition of HPV16 as the main HPV type when applying molecular virology assays.The expression of E6 in HPV16+ LSCC specimens was detected in 21 of 22 cases. The immunoreactive structures were revealed both within the tumor mass and the surface epithelium of the region of interest, demonstrating dysplastic features. One specimen contained only the tumor nest. In 3/22 cases, immunoexpression of HPV16/18 E6 oncoprotein in the tumor mass was strong (>50%); furthermore, among them, 2/22 cases presented with strong positivity in a dysplastic epithelium a,b.In most LSCC samples (12/22), the expression of the E6 oncoprotein in the tumor mass was low a. By conHPV16 E7 protein immunoexpression was confirmed in 20/21 LSCC specimens c. In twoEighteen out of 20 HPSCC samples were HPV16 E6 oncoprotein positive. However, immunohistochemically HPV16 E6 positivity in a tumor mass was confirmed in 13/20 cases e,f. In mAn even smaller number of HPV16 E7 oncoprotein positive HPSCC cases were found when compared to HPV16 E6-positive cases, represented by 13/20 cases g,h. FurtCollectively, the immunoexpression of HPV16 E6/E7 oncoproteins within a tumor mass was not confirmed in 7/42 samples (one LSCC and six HPSCC). Five of these samples were HPV16+ with the Anyplex RT-PCR and two with HPV16 L1 PCR, all of them p16-. Immunohistochemically, only one HPSCC and one LSCC sample were both HPV16 E6 and E7 oncoprotein negative; simultaneously, the HPSCC sample was HPV16+ by HPV16 L1 PCR and the LSCC sample by Anyplex RT-PCR. Matched HPV16 E6/E7 positivity was demonstrated in 2/42 samples within the dysplastic epithelium. No significant differences in tumoral or dysplastic epithelial HPV16 E6/E7 oncoprotein expression were found, except a significant difference demonstrated for E6 oncoprotein positivity in the HPSCC samples e. OveralS = 0.445, p = 0.056) between semiquantitative RT-PCR and HPV16 E7 IHC data was demonstrated in the LSCC tissue samples, particularly in the dysplastic epithelium. Additionally, there were weak-to-moderate positive correlations found in the HPSCC tissue samples; however, they failed to reach statistical significance  detected by molecular biology methods were used. Most HPV16+ samples demonstrated either oncoprotein E6/16 or E7/16 positivity. However, the absence of oncoprotein E6/E7 immunostaining, evidenced in some samples, is likely to suggest other, non-HPV-related mechanisms of tumor development.To the best of our knowledge, few studies previously explored the presence of HPV oncoproteins E6 and E7 in tumor and dysplastic epithelial cells by IHC ,59,60. PThe given study aimed to report on the peculiarities of tumorigenesis in the larynx and the hypopharynx and the likely differences between these two sites, highlighting HR-HPV DNA, p16, and E6/E7 oncoprotein status assessed using molecular virology and IHC methods. Even though most correlations failed to reach statistical significance, weak to moderate positive correlations between the molecular virology and the IHC results may indicate active HPV infection in these samples. However, the data about the activity of HPV infection  could clarify this hypothesis. In this study, PCRs confirmed the presence of HPV DNA in the LSCC and HPSCC samples; still, the molecular virology methods applied failed to distinguish between active and latent infection. On the other hand, the presence of HPV E6/E7 proteins, known as significant contributors to tumor development, suggests active participation of HR-HPV infection in tumorigenesis.Interestingly, in some HPV16+ specimens, tumor cells stained negative for HPV16 E6/E7 oncoproteins, whereas dysplastic epithelium stained positive. Finally, some endothelial cells were found to be positive for HPV16 E6/E7 proteins. These results reflect the limitations of PCR assays, which do not specify the source of the genetic material. In general, the presence of HR-HPV E6 andE7 oncoproteins suggests a possibility of cancerous transformation of these cells. Viral integration and dysregulation of E6 and E7 gene expression is a common tumorigenesis mechanism confirmed in HPV-related cancers in general and in cervical cancer in particular . HoweverSome authors suggested that HR-HPV infection may contribute to laryngeal carcinogenesis via integration of the viral DNA in the host cell genome and a further increase in p16 expression . In thisThe use of broad spectrum of HPV-specific tests such as HPV DNA PCR, detection of HR-HPV and LR-HPV types, along with IHC staining of HPV surrogate marker p16 and viral oncoproteins E6/E7, confirmed by conventional and fluorescence-based immunodetection methods, may be considered as the strength of this study. A few limitations should be considered when interpreting our data. A moderate number of samples were used in this study. The second limitation is related to the absence of HPV mRNA data. These data would be of interest, bringing clarity to the question regarding the activity of HPV infection in analyzed tumors. Finally, some imbalance in gender and tumor stage characteristics, but not affecting the overall results, should be explained by the legal norms and the inclusion criteria used in the given study.In conclusion, this study based on HPV testing assays and a robust platform of IHC methods used to further explore p16 status and the presence of viral oncoproteins E6/E7 confirmed a high prevalence of HPV16 genotype in laryngeal and hypopharyngeal cancers. The absence of the HPV E6/E7 oncoproteins in some tumor samples suggests a mechanism different from the viral integration tumorigenesis mechanism. Unlike in OPSCC, the application of p16 IHC as a surrogate marker of active HR-HPV infection in LSCC and HPSCC appears impractical.


Urine self-sampling for human papillomavirus (HPV)-based cervical cancer screening is a non-invasive method that offers several logistical advantages and high acceptability, reducing barriers related to low screening coverage. This study developed and evaluated the performance of a low-cost urine self-sampling method for HPV-testing and explored the acceptability and feasibility of potential implementation of this alternative in routine screening.A series of sequential laboratory assays examined the impact of several pre-analytical conditions for obtaining DNA from urine and subsequent HPV detection. Initially, we assessed the effect of ethylaminediaminetetraacetic acid (EDTA) as a DNA preservative examining several variables including EDTA concentration, specimen storage temperature, time between urine collection and DNA extraction, and first-morning micturition versus convenience sample collection. We further evaluated the agreement of HPV-testing between urine and clinician-collected cervical samples among 95 women. Finally, we explored the costs of self-sampling supplies as well as the acceptability and feasibility of urine self-sampling among women and healthcare workers.Our results revealed higher DNA concentrations were obtained when using a 40mM EDTA solution, storing specimens at 25°C and extracting DNA within 72 hrs. of urine collection, regardless of using first-morning micturition or a convenience sampling. We observed good agreement (Kappa = 0.72) between urine and clinician-collected cervical samples for HPV detection. Furthermore, urine self-sampling was an affordable method (USD 1.10), well accepted among cervical cancer screening users, healthcare workers, and decision-makers.These results suggest urine self-sampling is feasible and appropriate alternative for HPV-testing in HPV-based screening programs in lower-resource contexts. Cervical cancer is the second leading cause of cancer deaths in low- and middle-income countries (LMICs), with almost 285,000 new cases every year . CurrentUrine self-sampling has been proposed as an alternative primary screening method for detecting high-risk human papillomavirus (hrHPV), given self-sampling may increase women’s willingness to participate in screening –5. PreviThere are several challenges to obtaining proper urine DNA preservation in order to achieve an easy and affordable method applicable to real-world conditions. Among the main barriers are: 1) time between sampling and processing, as studies have shown that urine DNA nucleases reduce concentrations of DNA in urine over time ,9; 2) anIt is key to have simple and low-cost urine collection and preservation procedures able to overcome previously described difficulties that may allow use of self-collected urine samples for the detection of hrHPV as a primary test for cervical cancer screening ,14. hrHPWe aimed to evaluate a low-cost method for urine self-sampling for HPV-testing that could be potentially translated into clinical practice and public policy, requiring minimum training among healthcare providers. Additionally, cost, acceptability and feasibility were evaluated as core elements for implementation.The study was conducted in two phases. During the first phase, we conducted a series of sequential laboratory evaluations to examine the impact of different pre-analytical conditions on the DNA concentration of stored urine samples. All these evaluations were performed in a set of urine samples donated by five volunteer women, aged 25 to 45, postgraduate students from Mexico City. The second phase was conducted to evaluate the agreement between hrHPV-testing in urine samples and clinician-collected cervical samples. Finally, we explored the acceptability, perception of feasibility and costs of urine self-sampling for hrHPV-testing in cervical cancer screening. The IRB of the National Public Health Institute (INSP) (CI:1417 CB: 1408–2016) approved the first phase protocol. The second phase was conducted within the FASTER study, reviewed and approved by the IRB of the INSP (Num: 1322–2015 and 1417–2016), the Mexican regulatory agency Federal Committee for Protection from Sanitary Risks (COFEPRIS) (163300410A0160/2016), and the Mexico City Ministry of Health (211/001/003/16); and registered in ClinicalTrials.gov, number: NCT03105856. The objective of the study, procedures, and possible consequences were explained to potential participants before obtaining written informed consent  compared to a micturition collected at any time of the day  (p = 0.11 for Wilcoxon test) when stored at room temperature with 40mM EDTA.We evaluated the agreement of specific hrHPV detection on 95 paired urine samples compared to clinician-collected cervical samples. We used urine samples preserved with the optimal collection method based on the results of our initial studies to achieve optimization. The general characteristics of the participants in this paired comparison are presented in Among the 95 paired samples, all urine and cervical samples had the amplification control target of the β-globin gene detected. In cervical samples, the prevalence of hrHPV was 13.7%, of HPV16 1.1% and 0% for HPV18. Urine samples had a prevalence of hrHPV of 22.1%, 3.2% for HPV16, and 0% positive samples for HPV18.We observed that all 13 hrHPV positives identified by the HPV testing in cervical samples were successfully detected in urine samples. The specific hrHPV types detected in cervical samples, were also detected in urine samples. In addition, we detected a case with HPV 45 and another with HPV 52 in urine that were not detected in cervical samples. Among these paired samples we observed an acceptable agreement of hrHPV detection in paired urine and cervical samples as shown by a Kappa value of >0.6 .The majority (64%) had completed basic or middle-level education; half of them lived with a partner and had at least one child. All women stated that urine self-sampling was a more comfortable and acceptable method of cervical cancer screening compared to clinician-directed cervical sampling. They felt that collecting a urine sample was an easy and common procedure, and women were familiar with routine urinalysis. Eliminating the shame associated with clinician-directed cervical sampling, with the added advantages of a substantially faster and more comfortable process along with the possibility of collecting urine at home made the future use of urine samples for the detection of cervical cancer a very attractive alternative.We also interviewed five physicians, three nurses and one social worker, ages 34 to 62, of which 89% were women. All interviewed agreed that urine collection was a more straightforward strategy for cervical cancer screening than sampling collection requiring a pelvic examination. Furthermore, they felt that urine analysis was an excellent opportunity for women to be able to obtain the sample in their own homes and deliver it to their healthcare centers. Moreover, 78% of physicians considered that urine collection may be implemented as a cervical cancer screening strategy given the convenience and simplicity of the procedure.Of nine policy decision-makers interviewed, 40% were women, 44% had specialty or postgraduate studies, with several years of experience in the field of preventive medicine. Six of them were heads of departments . All considered urine self-sampling as an alternative that could overcome barriers such as the discomfort associated with pelvic examination and cervical sampling, with a good possibility for its implementation in healthcare centers at no additional cost.The preliminary cost of a urine sample was estimated at USD $1.10, the details of prices are listed in Our results suggest that DNA for HPV-testing can be obtained from urine samples with only a simple solution of 40mM EDTA and can be stored at room temperature up to 72 hours before processing. Urine HPV-testing, employing this low-cost sampling procedure, offers good agreement with paired clinician-collected cervical samples. Furthermore, urine self-sampling seems to be highly acceptable and is an affordable method with good potential for implementation within the cervical cancer screening program in Mexico.Vorsters et al. previously tried a new in-house buffer for urine samples destined for HPV detection that contained bovine serum albumin, a microbicide and a fungicide in addition to the chelating agent . AlthougRoom temperature was not suitable for storing urine samples in the absence of EDTA. In contrast, studies by Bosschieter et al., Cannas et al. and Augustus et al. found that appropriate DNA concentrations could be extracted from urine stored at room temperature for several days as long as EDTA was added ,10,22. IRegarding time from collection to processing, we found that processing within the first 72 hours yielded DNA concentrations appropriate for HPV testing. These results are consistent with previous findings documenting that DNA concentration decreases over time ,9. We diThe cost estimate of a urine sample in our study ~ USD $1.10, was much less than currently available commercial kits ,25. WithhrHPV detection in urine samples has previously been reported with acceptable agreement compared to clinician-directed cervical samples –14,23,27We defined the degree of agreement according to the guidelines outlined by Landis and Koch in 1977: < 0 no agreement, 0.01 to 0.2 slight agreement, 0.21 to 0.40 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.80 substantial agreement and 0.8 to 1.0 almost perfect agreement . In our Our qualitative analysis reveals that women consider acceptable urine self-sampling and preferred this because of characteristics such as comfort, simplicity and speed of collection. Our participants consider it could be a feasible strategy for implementation at their healthcare centers. These findings are similar to those reported in previous studies, highlighting the advantages of urine collection as a method for HPV DNA detection ,30–32. SIn accordance with earlier reports, we believe that the urine self-sampling method has the potential to impact screening coverage ,34. In hOne of the strengths of our study was our focus on real-world applicability. Phase one focused on pre-analytical conditions geared towards techniques most likely to be applied to our environment with favorable results, such as room temperature storage, processing within 72 hours, and use of a simple EDTA-based solution for preservation. Furthermore, the second phase, albeit with a small number of participants, was executed applying the lessons learned during phase one, with valuable input on acceptability and feasibility from both patients and healthcare professionals actively involved in cervical cancer early detection programs in Mexico.However, as mentioned above, an important limitation of this study was the small sample size of the second phase which was descriptive to support future appropriately powered investigations. Our main objective was to provide early evidence about whether urine sample using a new low-cost method could be successful in subsequent diagnostic trials.Nevertheless, acknowledging that the second phase of our study was to evaluate the agreement between hrHPV-testing in urine samples and regular cervical samples, we performed a post-hoc power calculation to detect differences in HPV detection for paired samples. All HPV positives cases identifying in cervical samples were successfully detected in urine samples with a power of 81.79%, with an alpha of 0.05 using paired samples from 95 participants.This study showed only the concordance between urine and cervical sample for HPV detection. For HPV tests using self-collected urine to be clinically applied, further investigation will be required to measure the clinical performance of HPV tests with evaluation of CIN2+ detection.Overall, we found urine-self sampling to be less expensive than cervical samples, was able to overcome technical and cultural barriers, and had an acceptable agreement with clinician-collected cervical sampling in the detection of HPV. Our findings and those of previous studies, support proposing urine as promising alternative for increasing coverage of cervical cancer screening programs worth further evaluation. Our findings also suggest that a urine sampling strategy is worth further evaluation as it is also seen as feasible by healthcare professionals and accepted by patients for implementation as part of the cervical cancer screening program in Mexico.S1 FileInterview guides applied for women, healthcare professionals and decision-makers. The Interview guides were focused on attitudes, acceptability, and feasibility of urine sampling for primary hrHPV-testing in cervical cancer screening.(PDF)Click here for additional data file.S2 FileDNA quantification and HPV results.(XLSX)Click here for additional data file.S1 TableDemographic and clinical characteristics of the 95 participants in the study.(PDF)Click here for additional data file.


The aim of this study was to evaluate (1) the female medical students’ knowledge about HPV infection; (2) the associations between the HPV vaccination intent and coping strategies, health locus of control (HLOC), and sense of coherence; and (3) the specific differences between preclinical and clinical students in terms of the vaccination intent. Participants included 1243 female medicine students , who completed The Multidimensional Health Locus of Control (MHLC)—Form A, the Brief COPE Scale, the Sense of Coherence Scale (SOC-13), and two questionnaires measuring the knowledge about the HPV infection and the HPV vaccination intent. Results show a good knowledge about HPV, which progressively increased during the study cycles. Still, the main contributors to vaccination intent are represented by coping strategies and health locus of control. Refusal of vaccination is associated to behavioral disengagement and the use of religion, precontemplation and contemplation to denial, and preparation to planning, positive reframing, and the powerful others component of HLOC. Sense of coherence did not predict vaccination intent. In clinical years, active coping outweighs HLOC in making the decision to get vaccinated. These results could be helpful in designing personalized strategies for addressing vaccine hesitancy in academic communities. Human papillomavirus (HPV) is the most commonly sexually transmitted infection (STI) in the world  and has The administration of the HPV vaccine has been reported to decrease the incidence of HPV infections and cervical cancer in general population ,7,8. AltIn Romania, the age-standardized incidence of cervical cancer is 20 new cases/100,000 women , with th-Health locus of control (HLOC): low levels of internal HLOC may lead to decreased compliance to vaccinations . In cont-Coping strategies: avoidance-focused coping is reported in literature to have a negative impact on the vaccination intent, while more active, problem-focused strategies are related to preventive behavior and a higher propensity to seek vaccination ,41;-Sense of coherence (SOC): has a moderating and a mediating effect on health . People -Stages of change: people in the precontemplation stage have no intention to change their behavior  and therWhile exploring the psychological factors associated with vaccination opposition and vaccine hesitancy, a series of variables appear to be important:Beside the aforementioned variables, a considerable barrier in vaccination campaigns is represented by the lack of awareness and knowledge about HPV infection and vaccination ,46. As rIn the academic communities, in particularly in medical universities, not many female students talk freely about the health risks associated with their sexual behavior or about their HPV vaccine hesitancy  even in The aim of this study was (1) to evaluate the female medical students’ knowledge about HPV infection; (2) to assess the specific differences and the comparative predictive value of coping strategies, health locus of control (HLOC), and sense of coherence in regards to the HPV vaccination intent; and (3) to evaluate the specific differences between vaccination intent in students having an exclusive theoretical knowledge of HPV and those students with clinical experience with HPV infection and consequences, i.e., cervical cancer.The design of the study was cross-sectional, with a single administration of a series of standardized psychometric instruments.In order to ensure representativity of the study sample, we included in the study 1243 medical students  from 9 medical universities in three different regions of Romania and Bucharest. We recruited a convenience sample of medical students through posted announcements on their institutional online groups. This process was based on the participants’ availability until the time limit was reached. All participants met the inclusion criteria, set as (1) being female, at least 19 years of age, and having the status of current undergraduate students in the abovementioned institutions and (2) having offered the informed consent to participate in the study. Exclusion criteria were represented by (1) students already vaccinated with one or more doses of the HPV vaccine, (2) students displaying current self-reported somatic or psychiatric morbidity, (3) students with cognitive deficits or any other impairments that would render the understanding and completion of the study questionnaires difficult, and (4) lack of completion of one or more study instruments.® Inc., Chicago, IL, USA) database.Data were collected in May–November 2020 through the administering of an online set of questions containing the study instruments. Before taking part in this research, students who expressed interest in participating received an explanatory statement about the study and completed informed consent forms. Subsequently, they were sent the web link for answering the psychometric instruments and the survey. Procedures in the study were designed in accordance with the World Medical Association Declaration of Helsinki and with the ethical guidelines published by the Committee of Ethics at the University of Medicine and Pharmacy Carol Davila—Bucharest . A researcher (TC) was available by phone or email in case the participants had questions concerning the filling of the questionnaires. All responses were processed anonymously, and a numerical code was assigned for each participant. The collected data were accessible exclusively to study researchers , while regular didactic staff had no access to the nominal distribution, collection, or interpretation of questionnaires. The interpretation of the questionnaires was performed independently by two researchers  and cross-checked for congruence afterwards. Final results were included in a SPSS 21 —Form A  compriseBrief COPE Scale contains 28 items, which measure 14 distinct coping strategies . The ansThe Sense of Coherence Scale—the shorter version with 13 questions (SOC-13)  measuresThe questionnaire of knowledge about HPV infection, extracted from previous published research on this theme , contain-The refusal of vaccination ;-Precontemplation ;-Contemplation ;-Preparation  in the next 6 months, but I have not tried yet to schedule an appointment”);-Action .The questionnaire about the HPV vaccination intent, adapted from an instrument used previously  and consThe questionnaire was reported to display a good content validity .Descriptive analyses were realized for socio-demographic and psychological variables.® Inc., Chicago, IL, USA). It comprised one-way ANOVA (followed by Tukey’s post-hoc tests) to compare the differences among study groups and multiple logistic regression to identify the predictors of the vaccination intent for each distinct phase of the TTM model. Additionally, separate t-tests for independent samples to compare the results of students in the sixth study year (who have been exposed to both theory and practice regarding HPV and its consequences) to those in the second study year .Statistical analysis was performed with SPSS Statistics 21 ;Group 1: no vaccination intent: -n = 175 (n = 14.1%)) or about the personal benefit from getting vaccinated (contemplation: n = 546 (43.9%)); andGroup 2: the vaccination intent is only theoretical and consists in thoughts about the utility of the vaccine (precontemplation: -n = 326 (26.2%)).Group 3: the vaccination intent is not only theoretical, but it has also a practical component (preparation: The study sample was subdivided into three distinct categories according to their responses at the questionnaire about the HPV intent :-Group 1The vast majority of the respondents (79.001%) originated in urban areas. A majority of them studied in the Northwest regions of the country (54.625%). Most of the participants were enrolled in the clinical study years (3–6) (63.878%).Across the whole sample, the most preferred coping strategies were planning, active coping, and acceptance, while the least preferred were substance use and behavioral disengagement. Several differences emerged between groups, with a more substantial use of behavioral disengagement and religion in group 1, denial and seek for instrumental support in group 2, and planning and positive reframing in group 3.The HPV knowledge was satisfactory in all study groups and the whole sample (range 12.214–12.269 out of 16).The sense of coherence had a high dispersion of results (range 23–83), with the sample mean (49.134) placed among the low scores reported in literature for academic populations ,67,68.In terms of health locus of control, most students placed its main source internally, followed by the powerful others and by chance. This hierarchy was maintained irrespective of the participants’ vaccination intent.Students refusing vaccination (group 1) had significantly higher scores in behavioral disengagement and lower scores in positive reinterpretation compared to students preparing for getting vaccinated (group 3). Moreover, they had a significantly higher use of religion compared to students only thinking about vaccination (group 2).Students in group 3 had significantly lower scores of denial and marginally higher scores of acceptance compared to those in group 2.All the three groups differed significantly in terms of the role played by the powerful others, this getting progressively more important from group 1 to group 3.Multinominal regression analysis was used to identify the predictors of the vaccination intent. Results are displayed in p < 0.341).The goodness-of-fit of the model was satisfactory , positive reframing (p < 0.010), religion (p < 0.013), and health locus of control (the powerful others component) (p < 0.001).Across the whole TTM stages, the most important predictors were denial (p < 0.008) and religion  and lower scores in positive reframing . In addition, those in group 1 counted less on powerful others .Participants in group 1 were characterized, compared to group 3, by higher scores in behavioral disengagement .Similarly, participants in group 2 were characterized, compared to group 3, by lower scores on the powerful others component of the HLOC scale  and students in the clinical cycle . The significant results of this analysis are displayed in As expected, students in the sixth study year, irrespective of their vaccination intent, had statistically more HPV knowledge than those in the second year.Still, the students in the preparation phase had two additional particularities: they tended to follow, in terms of health, an authoritative figure  or were significantly more active .This study focused on assessing the female medical students’ knowledge about HPV infection; the specific differences and the comparative predictive value of coping strategies, health locus of control (HLOC), and sense of coherence in regards to the HPV vaccination intent; and the specific differences between vaccination intent in students in the preclinical and clinical study cycles after having been exposed to HPV and its consequences.The descriptive data illustrated a satisfactory overall level of knowledge about HPV (12.25 out of 16). This finding was to a certain point predictable given that medical students are in direct and frequent contact with health information about HPV and associated risk factors.The most used coping strategies were generally the adaptive ones , while the least used were the avoidant ones . Generally, the use of this kind of strategies is associated, in medical students, with better health outcomes . In the Still, significant differences were met between the three study groups in terms of coping strategies preferences. Participants who did not express the intention to get the HPV vaccine (group 1) were characterized by behavioral disengagement and the use of religion, reflecting a higher orientation towards emotion-focused coping in handling difficult, uncertain, or risky circumstances. In contrast, the respondents in the precontemplation and contemplation stages (group 2) used more often denial. In terms of long-term evolution, this could be potentially dangerous, as they may lack awareness about the risk to get the HPV infection and/or cervical cancer and may stagnate in these stages for a long time or even return to the previous one (no vaccination intent). Still, the regular use of instrumental support by these participants could offer reasons of hope, as this could be associated with a higher tendency to ask for help and support if needed. Individuals belonging to the preparation stage (group 3) preferred planning and positive reframing as tools to better handle stressful emotions (such as those related to the risk of HPV infection or to fear of the vaccine’s side effects). These could have helped them to see the HPV-associated risks in more positive and resolvable terms.In terms of the sense of coherence, this was assessed as lower than normal in all three study groups. Furthermore, the sense of coherence did not prove in our study to be predictive for the vaccination intent; in other words, understanding the link between vaccination and the lower risk of cervical cancer could have not played a significant role in adopting this preventive behavior. These findings come in contrast with other literature studies that illustrate the importance of the sense of coherence for developing the intention to get vaccinated . This coRegarding the health locus of control, most of the students had a high level of internal HLC, followed by the powerful others HLC and chance HLC. This evidence is encouraging, as the predominance of the internal HLC could reflect the will and the ability to perform preventive behaviors, such as vaccination. Literature data using standardized instruments also confirm that high internal HLC correlates positively with preventive behaviors ,74,75,76In what concerned the contribution brought by the powerful others HLC, this could have played in the studied group an additional role in the decision to actually perform HPV vaccination. This effect, in relation to the specific authority brought by the physicians’ expertise, has been also reported in literature . HoweverIn terms of the predictors of the vaccination intent, the students in group 1 differed from those in group 3 essentially through their higher scores in behavioral disengagement and religion. They seem also to be characterized by a certain psychological rigidity (as they display a low ability of positive reframing) and higher autonomy . In contrast, groups 2 and 3 differed essentially only in what concerned the higher importance of powerful others in the decision of getting vaccinated, met by the students from group 3. From this point of view, the personal example of the persons invested with authority  seems to be essential for making the transition from precontemplation/contemplation to preparation.Although the level of knowledge about HPV was satisfactory, it did not represent, as such, a predictor of the vaccination intent. The high level of knowledge about HPV infection seems to be a necessary but not sufficient condition for determining the young, academic population to adopt a preventive HPV vaccine behavior. This result does not replicate the findings of previous studies on the same theme, which illustrate the positive predictive value of the level of HPV knowledge on vaccination intent ,82.The specific comparisons realized between students in the preclinical cycle (second study year) and those in the clinical cycle (sixth study year) revealed that medical knowledge does not predict alone the active preparation for vaccination. Important additional elements are represented by the role of the powerful others  and active coping . These characteristics could be very informative for the key players in the field of education in designing effective personalized programs in favor of vaccination.Our study has several limitations. Despite including students from all study years, it realized only a cross-evaluation of their vaccination intent. The study included only psychological variables and not social ones. Some variables, such as the powerful others HLOC, may have had an ambivalent influence in time on the vaccination intent. To further investigate the predictors of HPV vaccination, future research could include additional psychosocial factors relevant for personality or social conformity as well as social parameters reported in literature to influence the vaccination intent .This study offers evidence about the HPV knowledge and the factors influencing HPV vaccination intent among female medical students. Among these factors, the most important are HLOC (the powerful others component), coping strategies, and the position in the academic cycle.Consequently, a series of practical aspects emerge as important for ensuring a higher chance of success of HPV vaccination programs in academic settings. This kind of programs should not rely only on information  but also on psychological variables . They should be doable and flexible and always consider the placement of the individual in the stages-of-change continuum. For example, for vaccine skeptics and deniers, the most suitable strategies seem to be those focused on emotional coping in the sense of including positive cues for vaccination in their preferred ways of facing reality. For individuals in the precontemplation and contemplation phases, a particular importance should be given to the ambivalent use of denial and to their real (and not imaginary) ability to ask for and use instrumental support. In the case of those in the stage of preparation, they could be stimulated into action via strategies supporting planning, positive reframing, and external influencers.Furthermore, the transition from preclinical to clinical teaching in medical universities should not only be considered as a simple change of the study curriculum but as an opportunity for many students to commute from a more external-oriented thinking style to more autonomy and goal-oriented behavior. This in turn could be reflected in the attitude towards medical interventions and should be addressed in programs supporting HPV vaccination.As a whole, these findings can be useful in designing more effective strategies, whether they be health policies or informal actions from influencers or other opinion leaders, in order to increase HPV vaccine acceptance.


Oropharyngeal squamous cell carcinoma (OPSCC) is an uncommon malignancy worldwide. Remarkably, the rising incidence of OPSCC has been observed in many developed countries over the past few decades. On top of tobacco smoking and alcohol consumption, human papillomavirus (HPV) infection has become a major etiologic factor for OPSCC. The radiotherapy-based or surgery-based systemic therapies are recommended equally as first-line treatment, while chemotherapy-based strategy is applied to advanced diseases. Immunotherapy in head and neck squamous cell carcinoma (HNSCC) is currently under the spotlight, especially for patients with advanced diseases. Numerous researches on programmed death-1/programmed death-ligand 1 checkpoint inhibitors have proven beneficial to patients with metastatic HNSCC. In 2016, nivolumab and pembrolizumab were approved as the second-line treatment for advanced metastatic HNSCC by the USA Food and Drug Administration. Soon after, in 2019, the USA Food and Drug Administration approved pembrolizumab as the first-line treatment for patients with unresectable, recurrent, and metastatic HNSCC. It has been reported that HPV-positive HNSCC patients were associated with increased programmed death-ligand 1 expression; however, whether HPV status indicates different treatment outcomes among HNSCC patients treated with immunotherapy has contradicted. Notably, HPV-positive OPSCC exhibits a significantly better clinical response to primary treatment  and a more desirable prognosis compared to the HPV-negative OPSCC. This review summarizes the current publications on immunotherapy in HNSCC/OPSCC patients and discusses the impact of HPV infection in immunotherapeutic efficacy, providing an update on the immune landscape and future perspectives in OPSCC. Oropharyngeal squamous cell carcinoma (OPSCC) is one of the head and neck squamous cell carcinoma (HNSCC), developing in the following areas: soft palate, base of the tongue, palatoglossal folds, palatine tonsils, valleculae, and posterior pharyngeal wall . AccordiThe standard of care (SoC) for OPSCC, which includes surgery, radiotherapy, and chemotherapy, has continuously improved. However, the prognosis of OPSCC patients remains poor due to late diagnosis, high rates of primary-site recurrence, and lymphatic metastasis , 11. RecThis review intends to update the current evidence in immunotherapy among OPSCC patients and the impact of HPV infection on the treatment efficacy of immunotherapy, providing future perspectives in OPSCC treatment.via a variety of molecules/pathways  , followed by PIK3CA (17%) and KMT2D (10.6%); and TP53 was more commonly mutated in the HPV-negative group   . BesidesP<0.001) –34. In gP<0.001) , a high e HNSCCs . A curre 0.0005) .th edition of the Union for International Cancer Control and American Joint Committee on Cancer (UICC/AJCC) staging system failed to differentiate the impact of HPV infection on survival (hazard consistency) between stages and lost the capability in predicting features of any stage  has been observed and related to differentiated survival . Based on these two landmark trials, the FDA approved pembrolizumab and nivolumab as the second-line treatment for R/M HNSCC in 2016. Soon after, the phase II, single-arm study, KEYNOTE-055, demonstrated that 16%  of the 171 R/M HNSCC patients (22% were HPV+ and 77% were HPV-) refractory to platinum and cetuximab who received pembrolizumab achieved confirmed response, with 8 months median duration of response  . The phase Ib trial published in 2016, KEYNOTE-012 (NCT01848834), was the first study investigating PD-1 blockade therapy in 104 recurrent/metastasis (R/M) HNSCC patients expressing PD-L1 (38% were HPV-positive and 62% were HPV-negative) . The ove months) –45. Arou months) –45. In 2 in 2019 –45.SoC of HNSCC, including surgery, radiotherapy, chemotherapy, and targeted therapy, have been well-studied and widely applied in clinics with a proven impact . NeverthLenvatinib is a tyrosine kinase inhibitor of several VEGF receptors and could modulate immune suppression in the tumor micro-environment by angiogenesis inhibition. The effectiveness of pembrolizumab in combination with lenvatinib in patients with HNSCC has been supported by a phase II trial (NCT02501096)  towards P=0.0034), while pembrolizumab monotherapy was non-inferior to cetuximab with chemotherapy (11.6 vs. 10.7 months). When stratifying the patient population with CPS score, both arms show survival benefits compared to cetuximab with chemotherapy. However, 85% of the R/M HNSCC patients in the pembrolizumab with chemotherapy group suffered grade 3 or worse all-cause adverse events (AEs), while 55% of the patients in the pembrolizumab monotherapy group endured AEs. It indicates that when clinicians decide on monotherapy or combined therapy, the toxicity should be considered in the clinical settings.Besides, the impact of ICI therapy combined with chemotherapy was investigated in a wide range of solid tumors. Some of these trials  observe statistically significant survival benefits  in patients with corresponding cancers –78, suggThe desirable effect of single-agent immunotherapy sparks research into the combination of anti-CTLA-4 and anti-PD-1 therapies . IpilimuIn addition, a phase III trial, EAGLE (NCT02369874), aimed to assess the combination of durvalumab plus tremelimumab  . This trApart from immune-combined therapy and a combination of anti-CTLA4 and anti-PD-1/PD-L1 therapy, there are other immunotherapies toward HNSCCs using anti-PD-L1 drugs  , 87. BesP = 0.03)  agonists therapy through clinical trials. The results of the phase Ib study (NCT02471846) were disappointing that the combination of navoximod (IDO1 inhibitors) and atezolizumab (anti-PD-L1 agent) failed to improve clinical benefit among patients with various solid tumors, including HNSCC . However = 0.03) .In all, immunotherapy provides a promising future, but the application in the treatment of OPSCC is still lacking. Researchers should take steps to discover more information in this field.HPV is a solid causative agent in the formation and progression of OPSCCs, making viral neoantigens an attractive target for therapeutic immunization. Tumor vaccine aims to reduce tumor burden and control tumor recurrence by stimulating both humoral and cellular immune response, offering an immune activation strategy . CurrentGiven the essential roles E6 and E7 play in HPV-positive cancers, they are usually selected as targets for a therapeutic vaccine. A phase I study (ACTRN12618000140257) assessed the safety, tolerability, and immunogenicity of an HPV E6/E7 vaccine (AMV002) in patients with HPV-positive OPSCC . The vacOn top of various vaccines, Ramireddy et al. showed that tumor membrane vesicle (TMV) vaccine comprises glycolipid-anchored immuno-stimulatory molecules GPI-B7-1 and GPI-IL-12, magnified the efficacy of anti-PD1 antibodies and inhibited tumor growth, and thus improved the survival of mice with stage VII SCC . In addiHowever, those HPV therapeutic vaccines were neither successful in achieving desired clinical outcomes nor applied to human study. Researchers put forward that one reason might be that patients obtained immune tolerance to antigens due to chronic exposure to the virus for years . TherefoCurrently, there are few studies on immunotherapy towards HPV-negative OPSCC. DURTRERAD is a randomized phase II trial evaluating feasibility and efficacy of durvalumab (D arm) versus durvalumab and tremelimumab (DT arm) in combination with radiotherapy as primary treatment for locally advanced HPV-negative HNSCC, more than a half being OPSCC . In thisP = 0.06) , progressive disease (PD), or OS when patients were classified by HPV status. However, ORR was approaching significance in HPV-positive patients compared to HPV-negative ones  .It should be pointed out that the accuracy of existing detection methods would influence the result of these trials. A study held by Miren Taberna et al. Proves that the ICON-S model in 8th AJCC predicts overall survival assessment better in HPV-related OPC patients when using at least two biomarkers to define HPV-causality (HPV-DNA and (p16INK4a or HPV-mRNA) or double positivity for HPV-DNA/p16INK4a) . These rCancer biomarkers help evaluate treatment effects, monitor tumor recurrence and predict survival. The application of biomarkers will influence patient outcomes; hence, exploring and discovering novel biomarkers have significant research value.The prediction value of PD-(L)1 expression measured by immunohistochemistry for prognosis in patients with HNSCC was analytically validated . PreviouTP53 and CDK2NA and the newly discovered mutations in FBXW7, TP63, IRF6, and NOTCH1 (NCAN, NRXN1, COL19A1, SYCP2, RPA2, and SMC1B, related to HPV infection among HNSCCs regardless of the anatomic site (Recent efforts to excavate molecular biomarkers through next-generation sequencing provided deeper and broader insights. Genetic and epidemic alterations are involved in the initiation of HNSCC formation. Genetic alterations include the classic mutations in d NOTCH1 . NOTCH sd NOTCH1 –103. Giad NOTCH1 . Besidesmic site . Moreovemic site .Most clinical trials evaluating the RR to ICIs in OPSCC patients indicate that less than 15% of patients receiving ICI therapy could achieve durable responses , 15, 107ex vivo platform, CANscript system, has been proven helpful to for profiling the response of immunotherapy combining chemotherapy (In the clinical practice of immunotherapy, evaluating patients’ immune status could be challenging. As discussed above, feasible biomarkers could guide researchers to identify potential beneficial patients and monitor adverse events. However, it is insufficient to select patients for ICI immunotherapy based on a single parameter without considering other factors given the highly heterogeneous microenvironment in OPSCC. An otherapy . Besidesotherapy , 116. ItIn summary, classified by HPV status, OPSCC is a heterogeneous disease. The unique TME shaping by HPV status calls for distinct therapeutic approaches. Immunotherapy offers a wide range of therapeutic strategies which will be especially useful in meeting this need. Monotherapy of novel agents has proved effective, while combinations of immunotherapy with conventional therapies and dual immunotherapy are undergoing clinical investigation. Notably, there is an urgent need for a feasible treatment stratification by HPV status. Immunosuppression and lack of desirable biomarkers for personalized therapy are the two significant issues in immunotherapy. More clinical trials are warranted to assess the efficacy of novel immunotherapies based on HPV status.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.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.