There is growing interest in anal cancer screening strategies. However, cytological/molecular evaluation of anal samples is challenging. We aimed to determine the feasibility of detecting, in anal liquid-based cytologies, the expression of biomarkers involved in the cell cycle disturbance elicited by human papillomavirus (HPV). The accuracy of this approach in the identification of high-grade squamous intraepithelial lesions/anal intraepithelial neoplasia grade2–3 (HSIL/AIN2–3) was also evaluated. 215 anal cytologies from men having sex with men living with human immunodeficiency virus were evaluated. Patients showing concordant cytological and anoscopy-directed biopsy diagnosis were selected: 70 with negative cytology and HPV test, 70 with low-grade SIL (LSIL/AIN1) cytology and biopsy, and 75 with cytology and biopsy of HSIL/AIN2–3. CDKN2A/p16, MKI67 and TOP2A mRNA expression was analyzed. HPV detection was performed with Xpert HPV Assay . HSIL/AIN2–3 showed higher expression for the biomarkers than LSIL/AIN1 or negative samples. The specificity for HSIL/AIN2–3 detection for a sensitivity established at 70% was 44.7%  for TOP2A and MKI67 and 54.5% (95%CI 46.0–62.8%) for CDKN2A/p16. mRNA detection of cell biomarkers in anal liquid-based cytology is feasible. Further studies are warranted to confirm if strategies based on mRNA detection have any role in anal cancer screening. Although anal cancer is infrequent in the general population, over the past decade its incidence and mortality have increased by about 2.2% and 2.9% per year, respectively . The incMany analogies have been recognized between anal and cervical cancer. Indeed, the common etiology of these two cancers (HPV) has prompted the use of the screening strategies performed for cervical cancer to prevent anal cancer. However, there are important differences between these two diseases that need to be addressed in the populations at risk ,3,4: theAlthough scant data is available, the screening strategies currently available for the prevention of anal cancer and its precursor, high-grade squamous intraepithelial lesion/anal intraepithelial neoplasia grade 2–3 (HSIL/AIN2–3) are far from ideal. As a result, the proposed screening recommendations are mainly based on expert opinions. Anal cytology has shown a low sensitivity and specificity for HSIL/AIN2–3 detection , whereasThe detection of host-cell mRNA biomarkers involved in DNA replication and cell cycle progression, such as TOP2A, and other HPV-induced cell molecules involved in cell cycle control, such as MKi67 or p16 (CDKN2A), has proved to be feasible in cervical samples preserved in liquid-based cytology media . MoreoveIn the present study we aimed to determine the feasibility of the detection of the mRNA of three HPV-associated biomarkers  in anal samples preserved in liquid-based cytology and to evaluate whether this approach provides valuable information for the detection of HSIL/AIN2–3. For this cross-sectional study, anal cytology samples, collected in the Anal Cancer Prevention Unit from January to December 2016 from men who have sex with men living with HIV, were retrieved from the Department of Pathology of the Hospital Clinic. All patients had been referred to the Anal Cancer Prevention Unit from the HIV Unit of our center. All were under highly active antiretroviral therapy (HAART) and had an undetectable viral load and CD4 counts > 400 cells/mL. Patients with anal sample fulfilling the following inclusion criteria were considered eligible for study: (1) anal liquid-based-cytology sample with adequate material for conventional cytology and HPV testing; (2) in cases with abnormal cytology and/or positive HPV test, a high-resolution anoscopy with at least one directed biopsy .For this validation study we considered the following three diagnostic categories as concordant results: (a) negative (control group): normal anal cytology result and negative HPV test; (b) LSIL: anal cytology result of LSIL and biopsy confirming LSIL/AIN1; and (c) HSIL/AIN: anal cytology result of HSIL and biopsy confirming HSIL/AIN2–3 ,19. The A total of 215 patients who fulfilled the inclusion criteria were included in the study: 70 with a negative result (control group), 70 with LSIL/AIN1 (LSIL group) and 75 with HSIL/AIN2–3 (HSIL group). The study was approved by the institutional ethical review board of Hospital Clínic of Barcelona  and all patients provided signed informed consent.The anal sample was collected using a moistened Dacron swab, which was transferred to PreservCyt solution . In all patients with abnormal cytology a high-resolution anoscopy was performed. Prior to the anoscopy, a digital anorectal examination was carried out to rule out abnormalities suggestive of anal cancer. The high-resolution anoscopy was performed using an Olympus Evis Exera II CV-180 anoscope . Anal evaluation was carefully performed with 5% acetic acid, and iodine-based solution was used to stain the anal canal and to highlight areas of abnormal epithelium. On the detection of suspicious lesions, a high-resolution anoscopy directed biopsy was obtained.The first part of the anal sample was used for cytological analysis. The residual material was used first for HPV testing and thereafter for RNA isolation. Thin-layer cytology slides were prepared using the Thinprep T2000 slide processor (Hologic) and stained using the Papanicolaou method. Cytology slides were evaluated by a cytotechnologist (L.M.) and reviewed by a citopathologist (L.R.) using the revised Bethesda criteria [All histological samples were fixed in 10% formalin-fixed and embedded in paraffin following routine procedures. Four-µm sections were routinely stained with hematoxylin and eosin (HandE). All the histological samples were carefully reviewed by a pathologist (J.O.) with experience in HPV-related lesions to confirm the presence or absence of SIL/AIN and its grade. The histological diagnoses were established using pure morphologic criteria based on the HandE-stained sections, with no knowledge of HPV status or the cytology result . Biopsy HPV detection was performed using the Xpert HPV Assay  in the material collected in liquid-based media (PreservCyt) . The XpeOnce the cytological exam and the HPV testing had been performed, 5mL of the cell suspension was centrifuged at 3000G for 5 min, and the cell pellet was mixed with 700 μL QIAzol lysis reagent . Total RNA was extracted using the RNeasy RNA extraction kit (Qiagen), according to the manufacturer’s protocol. Sample preparation was carried out with the highest measures of quality control to avoid contamination and cross-contamination. RNA concentrations were measured with a Nano-Drop instrument . The total RNA yield varied between 0.5 and 64.3 mg, reflecting the variability of the cell numbers in the specimens.Reverse transcription (RT) was performed starting with 10 μL of total RNA in a 20 μL reaction volume using random hexamers and the high capacity cDNA RT-kit from Applied Biosystems  according to the manufacturer’s protocol. To exclude DNA contamination a reaction without RT was run in parallel with each specimen, as described previously .All PCRs were performed in triplicate at a reaction volume of 25 μL containing 5 μL of cDNA diluted at 1:5 and mixed with Taqman Universal PCR MasterMix (Applied Biosystems). The following protocol was used for all assays: denaturation (10 min at 95 °C) and amplification  repeated for 40 cycles. The expression of CDKN2A/p16, MKI67 and TOP2A, was analyzed. Beta glucuronidase (GUSB) and cGMP-dependent protein kinase 1 (PKG1) were selected as reference genes for quality control of the RNA. This combination of reference genes showed a high stability in expression between groups of normal samples versus HSIL samples. The SiHa cell line was selected as a reference for RNA normalization.The number of cycles required for the signal to cross the threshold  for target genes was set at 35 cycles . For theThe data were analyzed with the SPSS program (Version 24.0). The analysis of variance test was used to compare quantitative variables between the different categories. The χ2 test was used for comparisons between categorical variables. A p ≤ 0.05 was considered statistically significant. All data related to expression are presented as ΔΔCt, which was calculated as  −  ,26. SiHap = 0.419). One-hundred and ninety-nine out of 215 (92.6%) samples were considered adequate for analysis, and only 16 could not be evaluated due to low expression of the reference genes and/or low RNA yield. Of the 199 samples finally included in the study 66 (33.2%) were normal, 66 (33.2%) LSIL, and 67 (33.7%) HSIL. No differences in terms of adequacy for analysis were observed between the three groups of samples , MKI67  and CDKN2A/p16 . CDKN2A/p16 expression in LSIL specimens was also significantly higher compared with negative samples (p = 0.030), but no differences were observed between LSIL and negative samples for the other two biomarkers.The ROC curves and the area under curve for the identification of patients with HSIL/AIN2–3 for the different biomarkers are shown in p = 0.003). Multiple HPV genotypes were found in 61.7% (63/199) of the samples studied. The prevalence of multiple infection was more frequent in the HSIL than in the LSIL group . p = 0.044); 3.22  and 4.12  for MKI67 (p = 0.010) and 0.95  vs. 1.84  for CDKN2A/p16 (p = 0.003). Considering only multiple infections, although the expression of TOP2A, MKI67 and CDKN2A/p16 was higher in patients with multiple infections harboring HPV16 than in those not harboring this genotype, the differences were only statistically significant for CDKN2A/p16.This is the first study evaluating the mRNA expression of host-cell genes involved in anal HPV-associated carcinogenesis in men who have sex with men living with HIV. Our results show that this evaluation may be feasible in anal liquid-based cytology samples in a clinical setting. Thus, molecular assays designed to detect the mRNA of host-cell biomarkers in specimens preserved in liquid-based cytology media may be a promising approach for HSIL/AIN2–3 detection. The current screening options for anal lesions in men who have sex with men living with HIV have shown not only poor reproducibility, but also limited sensitivity and specificity to detect anal HSIL/AIN2–3 ,29. On tIn the present study, we evaluated the feasibility of mRNA detection and its possible usefulness for HSIL/AIN2–3 detection. Due to the technical difficulties of the anal cytology samples, we initially focused on the technical issues. Extraction of high-quality RNA is a prerequisite for the application of RNA transcripts as biomarkers for clinical purposes ,33. Few The biomarkers analyzed in this study have shown good reproducibility, sensitivity, and specificity in other HPV-associated malignancies such as cervical cancer. A previous study performed by our group suggested that the analysis of mRNA expression of the same host-cell genes analyzed in this study  is not only feasible but also useful for the detection of cervical HSIL/CIN2–3 . In our In the present study HPV16 was the most frequent genotype identified in anal precursors, and its prevalence was significantly higher in HSIL/AIN2–3 compared with LSIL/AIN1. These results are in keeping with previous reports showing that HPV16 is the most common genotype as an etiologic factor in HSIL/AIN2–3 and anal cancer ,35. As iIn the present study, we observed that the mRNA expression of host-cell genes was particularly high in HPV16-positive samples. No previous studies have evaluated the relationship between the expression of host-cell genes and different HPV genotypes in cytological anal samples. Thus, this is the first study to evaluate the relation between HPV genotypes and mRNA expression of host-cell genes in anal cytological samples from men having sex with men with HIV and SIL/AIN. Our results suggest that this combination might be an interesting approach in risk-based management anal cancer screening, simulating the approach that has recently been suggested for cervical screening . HoweverThis study has some limitations. The possible changes in the levels of expression of the mRNA of the biomarkers in patients with HPV infection not associated with intraepithelial lesions were not evaluated. In our clinical setting, following the recommendations for anal cancer screening, this particular high-risk population of men who have sex with men living with HIV with a negative cytology does not undergo further examination with high-resolution anoscopy, and consequently, none of these patients had histological confirmation of the negative result. Thus, the control group had to be recruited based only on the cytology result, and we included the additional criteria of a negative HPV test to ensure that the patients were truly negative and not false negative results of the cytology. Nevertheless, the main aim of our study was to determine the feasibility of performing mRNA analysis of host-cells in a standard clinical setting, which was independent of the patients selected. Another limitation is that the presence and effect of low-risk HPV types could not be evaluated with the genotyping test used in our study. However, although it has been suggested that low-risk HPV types may have a role in the appearance of anal lesions, the risk of low-risk HPV infection in the development and progression of anal premalignancies is clearly lower than the risk due to high-risk HPV infection ,45.In conclusion, we have shown that the determination of mRNA expression in anal liquid-based cytology specimens is feasible. Further studies including a larger number of genes and patients representing the complete picture found in an actual screening program are warranted to confirm that strategies based on mRNA detection of cell biomarkers might have a role in the secondary prevention of anal cancer.


Background and objective: Lipocalin 2 (LCN2) has an oncogenic role in promoting tumorigenesis through enhancing tumor cell proliferation and the metastatic potential. The aim of our study was to determine whether serum LCN2 could serve as a diagnostic marker of cervical cancer (CC) and to evaluate the correlation between its serum concentration, the clinical stage of the cancer and Human Papilloma Virus HPV infections in women. Materials and methods: A total of 33 women with histologically proven cervical cancer (CC), 9 women with high- grade cervical intraepithelial neoplasia (HSIL) and 48 healthy women (NILM) were involved in the study. A concentration of LCN2 was assayed with the Magnetic LuminexR Assay multiplex kit. An HPV genotyping kit was used for the detection and differentiation of 15 high-risk (HR) HPV types in the liquid-based cytology medium (LBCM) and the tissue biopsy. Results: The majority (84.8%) of the women were infected by HPV16 in the CC group, and there was no woman with HPV16 in the control group (P < 0.01). Several types of HR HPV were found more often in the LBCM compared to in the tissue biopsy (P = 0.044). HPV16 was more frequently detected in the tissue biopsy than the LBCM (P < 0.05). The LCN2 level was higher in HPV-positive than in HPV-negative women (P = 0.029). The LCN2 concentration was significantly higher in women with stage IV than those with stage I CC (P = 0.021). Conclusions: Many HR HPV types, together with HPV16/18, can colonize the vagina and cervix, but often HPV16 alone penetrates into the tissue and causes CC. The serum LCN2 concentration was found to be associated not only with HR HPV infection, irrespective of the degree of cervical intraepithelial changes, but also with advanced clinical CC stage. LCN2 could be used to identify patients with advanced disease, who require a more aggressive treatment. The majority of cervical cancer (CC) cases occur as a result of the effect of human papillomavirus (HPV) infection. However, only a small proportion of women with HPV infection and intraepithelial lesions progress to CC development. It is therefore believed that there are different risk factors contributing to the development and progression of this cancer. HPV infection alone is not enough for the complicated processes of cellular change. Tumor formation is a multistage process, the development of which requires other important factors such as genetic damage or mutations, as well as environmental factors . One of Lipocalins (LCNs) are proteins that perform different functions in the regulation of the cell metabolism, immune response, prostaglandin synthesis and others. LCN2 is a glycoprotein released by various cells of the organism, including epithelial cells, macrophages, neutrophils and tumor cells, and this protein can be found in plasma, serum, and urine in different clinical situations, including metastasis of breast cancer or colon cancer . GreaterLCN2, as an acute phase protein, may also be involved in a variety of physiological processes, such as iron transport, prostaglandin synthesis, and the secretion of inflammatory markers, which have been identified in the study on mice and can improve cell survival functions . LCN2 maKim et al. showed a new role for LCN2 as a glycolytic switch in colorectal cancer cells. The results of their study revealed the role of LCN2 major molecular mechanisms in the progression of colorectal cancer, indicating LCN2 as a possible diagnostic marker and a therapeutic target in colorectal cancer . The finThe aim of our study was to determine the serum concentration of LCN2 in CC patients and healthy women with different HPV types as a possible diagnostic marker and to evaluate associations between its serum concentration and the clinical stage of CC.A total of 33 women with histologically proven squamous cells CC and 9 women with high-grade cervical intraepithelial neoplasia, treated in the Department of Obstetrics and Gynaecology, Hospital of Lithuanian University of Health Sciences during 2017–2018, were involved in this study. A revised FIGO staging classification of carcinoma of the cervix uteri was used (2018).The control group consisted of 48 healthy women without cervical intraepithelial lesions or malignancy confirmed by the liquid-based cytology test . The results of the cervicovaginal cytology test were reported by the 2014 Bethesda System: negative for intraepithelial lesions or malignancy (NILM) or for lesions and high-grade squamous intraepithelial lesions (HSIL) .Patients were excluded from the study if they were aged ≤ 18 years, were pregnant, had any autoimmune disease, diabetes and cancer other than CC, or had previously received treatment for carcinoma in situ or HSIL. All patients signed their informed consent before being included in the study. The study was approved by the Kaunas Regional Biomedical Research Ethics Committee (No. BE-2-80/2018).The patients were asked to provide a blood sample before the surgery and cancer treatment. The blood specimens were coded to maintain confidentiality, were frozen and were stored at −80 °C until use. The study population blood was analyzed for LCN2. The concentration of LCN2 was assayed with the Magnetic LuminexR Assay multiplex kit . Magnetic microparticles were coated with test antibodies. The specimens, standards, and microparticles were pipetted into test wells and joined existing antibodies. An antibody mixture specific to the tested markers was added to each well. After washing, a streptavidin-phycoerythrin conjugate was added to each well, and the microparticles were resuspended in buffer and read using the Luminex 100 system . A sample from each well was imaged with a CCD camera, with a set of the filter to differentiate the excitation levels.The HPV DNA detection was performed using a QIAamp DNA mini kit . An HPV genotyping  kit was used for the qualitative and quantitative detection and differentiation of 15 high-risk (HR) HPV types and 3 putative HR types in the liquid-based cytology medium SurePath  using the real-time hybridization-fluorescence detection of amplified products . The hybridization method is based on the simultaneous real-time amplification of DNA fragments of the HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82 and 26, 53, 66, as well as a DNA fragment of the β-globin gene  in one tube. Each genotype was detected in a separate fluorescent channel. A quantitative analysis was performed in the presence of DNA calibrators.U test and Kruskal-Wallis test. The chi-square test and the Fisher exact test  were used to determine whether a relationship exists between the qualitative data. The proportions were compared using the z test. Differences in comparing the groups were considered statistically significant when a P value was under 0.05. The study power analysis was done and determined as 0.8.A statistical data analysis was performed using the statistical package IBM SPSS 23.0. The Kolmogorov-Smirnov test was employed to determine if the quantitative data were normally distributed. All data were found to be non-normally distributed. The non-normally distributed data were compared using the Mann-Whitney P < 0.001)  . We classified HPV16 and HPV18 together (HPV16/18), because they are the types that most often cause cervical lesions; other high-risk HPV types, which were most commonly found in our study population; and HPV16/18 together with other types. The overall prevalence of HPV according to cervical intraepithelial abnormalities is shown in z test.The statistical analysis was performed by a chi-square test and Fisher exact test when the sample sizes were small. The proportions are compared using a P < 0.001). The other HPV types that were most frequently detected in CC and healthy women were the types HPV18, HPV31, and HPV52. HPV39, HPV51, and HPV58 were the most common types among the control women . More than one HR HPV type was found more frequently in LBCM when compared to the tissue biopsy  (P < 0.05). We compared the prevalence of HPV types in the liquid-based cytology medium (LBCM) and in the tissue in the same CC women group. One HR HPV type was detected in 60.6% (n = 20), two in 30.3% (n = 10), and three in 9.1% (n = 3) of the cases in LBCM. We detected a lower number of other HPV types in the tissue samples compared to LBCM . In the = 0.044) . HPV16 aThe statistical analysis was performed by a chi-square test and Fisher exact test when the sample sizes were small.The statistical analysis was performed by a chi-square test and Fisher exact test when the sample sizes were small. P = 0.029), but no difference was observed between HPV16/18 and other HPV types  pg/mL and 15,540  pg/mL, respectively; PV types .U test. The horizontal dashes represent the medians.The statistical analysis was performed by a Mann-Whitney P = 0.087). No significant difference was found in the LCN2 level when comparing the HSIL and CC patients to the healthy HPV-negative women as well. The median LCN2 concentration in the HSIL group was 37,660  pg/mL, and in the CC group it was 32,010  pg/mL.A comparison of the circulating concentrations of LCN2 according to the HPV status and the degree of cervical intraepithelial abnormalities  showed tU test. The horizontal dashes represent the medians.The statistical analysis was performed by a Mann-Whitney P = 0.021), but did not reach a statistical significance between the other CC stages. The median LCN2 concentration was 29,950  pg/mL in women with II stage CC and 20,378  pg/mL in those with III stage CC  and 1044  pg/mL, respectively; stage CC .U test. The horizontal dashes represent the medians.The statistical analysis was performed by a Mann-Whitney In this study, HPV16 was found to be the most prevalent HPV type in the CC women. A study by Félix et al., retrospectively analyzing 1214 tumor samples from 1928 to 2005, reported that the most common HPV16 and HPV18 accounted for almost 70% of CC in the Portuguese female population in all 9 decades that were studied . HR HPV We analyzed the systemic level of LCN2 associated with chronic cervical inflammation during HPV infection as a potential biomarker to identify patients with a different CC stage. Studies have shown that LCN2 plays an important role in the pathogenesis and metastasis of various types of cancer ,16. The Although LCN2 has an oncogenic role in promoting tumorigenesis, it is not a specific marker. High concentrations of LCN2 in serum can be found in various diseases, especially in the case of inflammation developing, infection, or tissue ischemia. LCN2 can enhance the local protective immune barrier against various pathogens . This prIn their study, Sánchez-Zauco et al. evaluated circulating cytokines and chemokines as potential diagnostic biomarkers for gastric cancer (GC). The study included healthy blood donors and found that IL-6, IFN-γ, and IL-10 might be associated with GC and can be used as markers to identify patients at potential risk for GC . In our The role of LCN2 in HR HPV-associated CC was analyzed in a study by Syrjänen et al. . The finThe upregulation of LCN2 in intraepithelial lesions is induced by the E6 protein of HR HPV . In the The study by Chung et al. showed that the increased levels of LCN2 were associated with lymph node involvement in patients with CC. LCN2 can improve invasion abilities in CC cell lines and can activate cancer cell metastasis by increasing cancer cell motility through the activation of mesenchymal transition pathway components . The rolLCN2 can activate the malignant properties of breast cancer cells. The study by Ören et al. reported the tumorigenic potential of LCN2 and provided evidence that LCN2 could be a promising therapeutic goal . In theiOur study data support other authors’ findings that LCN plays an important role in the oncogenesis of cancer, but it is not an early and specific marker of CC. Because of its pro-proliferative, pro-regenerative, and anti-inflammatory properties, LCN2 might be of great importance in the progression of cervical and other cancers, but the diverse biological effects of LCN2 within the tumor-supportive microenvironment, along with other factors besides HR HPV that can affect lipocalin levels, should be further studied.The present study shows that many HR HPV types, together with HPV16/18, can colonize the vagina and the cervix, but that HPV16 often penetrates in the tissue alone and causes CC. The presence of HR HPV significantly increased the LCN2 level, independently of the degree of cervical intraepithelial changes. The significantly elevated LCN2 serum concentration was associated with an advanced clinical CC stage but did not differ between women with early stages (stages I–II) and healthy controls. LCN2 could be used to identify patients with an advanced disease who require a more aggressive treatment.


However, no such study has carried out between Iranian couples yetUrogenital specimen from both males and females of couples were taken and transferred to Nilou laboratory for molecular analysis. HPV DNA extraction and typing were carried out using cobas 4800 platform. Demographic and virological data were analyzed afterwards.P value 0.021). Within the positive samples, 13(5.7%), 8 (7%) and 31(13.5%) were belonged to 16, 18 and other HR genotypes. 59 (51.8%) couples who were negative for HPV showed negative concordance. Of the total positive HPV patients , 9 (16.3%) couples had positive concordance and the rest of 46 (83.7%) couples (either of spouse being negative and the other being positive for HPV) showed neither kinds of concordance.One hundred fourteen couples were enrolled in the study. The mean age of participants were 36 ± 8 and 32 ± 7 for males and females, respectively. 64 (28%) of specimens were positive for at least one HPV genotype. The positive rates within genders were 30.7 and 25.4% for females and males, respectively with a considerable association (Recognition of the dynamics of HPV infection not only in women, but in their sexual partners could impact the implementation of preventive measures like HPV vaccination for cervical cancer and other HPV-related diseases for both sexual partners. Worldwide, HPV infection accounts for an estimated 530,000 cervical cancer cases as well as 270,000 deaths annually occurring substantially in developing countries . HPV alsThe aims of the present survey were to determine HPV prevalence in heterosexual couples and to evaluate HPV type-specific concordance in a predominantly monogamous population.This study was a longitudinal, cross sectional investigation on the prevalence of type-specific HPV concordance in sexually active couples during 2017–2018. To recognize the couples, men files were reviewed firstly. This was due to the fact that women consisting a majority of referrals to Nilou laboratory and finding couples through female files was troublesome. The inclusion criteria were a steady female partner (at least for the last 6 months even if they were not living in the same house) and, if so, whether they would invite their partner to join the couples’ survey. According to the Fig. A written informed consent guaranteeing confidentiality was taken from each patient. The study was approved by the National Ethics Committee for Biomedical Research affiliated to The Ministry of Health and Medical Education. According to HPV testing results, couples categorized into: positive concordance (both partners sharing the same HPV genotype), negative concordance (both being negative for HPV) and neither of discordance (when either of spouse being positive and the other being negative for HPV).thinprep tubes and tested immediately. For women, cervical scrapes were collected by sampling the ectocervix and endocervix with a cytobrush and cervical scrapes were also inserted in a tube containing lysis buffer. Alternatively, cervical samples were taken by a gynecologist from the endocervix and ectocervix areas with a sterile cytobrush and subsequently transferred to Nilou laboratory.The epithelial cells from the urinary meatus, frenulum of prepuce and glance of penis were obtained using a cytobrush, this was cut and inserted in The cobas 4800 HPV test included fully automated sample preparation combined with real-time PCR technology and software that integrates the two modules. One-milliliter aliquots of thinprep fluid were transferred to 13-mL barcoded tubes provided by the manufacturer. The cobas 4800 HPV test was performed according to the manufacturer’s protocol . COBAS generates individual qualitative results for HPV 16, HPV 18 and a pool of other high risk HPV genotypes.P value was considered significant when < 0.05.Statistical analysis was performed using SPSS software version 22 package. Cramer’s V test was used to for correlation intensity between HPV genotypes in couples. This test showed distribution of categorical data in different groups in this study. The concordance analysis was based on the couples’ enrollment data. The proportion of couples who were concordant and discordant was calculated. A couple was classified as having “type-specific positive concordance” if the man and woman had ≥1 HPV genotype in common. A couple was classified as having “negative concordance” if both the man and woman were negative for 14 high risk genotypes. P value, 0.021, Table P value 0.433, Table P value, 0.047, Table Out of 487 men referred to Nilou laboratory, 193 were single without a clear history of sextual partnership and hence excluded from the survey. Finally, 114 couples were eligible and were enrolled in this study Fig. , a and bRegarding the couples, they were classified into four groups. Groups I consisted of 59 (51.8%) couples who were negative for HPV,whereas, groups II, III and IV comprised the total positive HPV patients  (Table HPV infection is very widespread among males and females across all geographical, racial and socio-economic populations worldwide and is the only sexually transmitted disease that is difficult to be handled in both members of a sexual couple. The substantial increased prevalence of genital tract HPV infections in many regions has been ascribed to an early start of sexual activity, poor sexual hygiene, multitude number of sexual partners, and insufficient preventive measures . The objHPV genotypes prevalence and its concordance among heterosexual couples have been studied in many trials although with evident variations in the concordance. However, there are not too many reports on HPV concordance between referral subjects –22 Tabl. GeneralThere are almost no uniform findings between HPV genotypes concordance between sexual partners. They seem to depend upon the type of sexual relationship between male and female and the duration of relationship between couples, immune status differences between them and possibly other environmental factors , 13. ImmThe prevalence of HPV genotypes in couples differs among different surveys, reporting rates of 3.5 to 59% for HPV 16, from 3.5 to 6.7% for HPV 18, and from 3.5% to 8.5% for other HPV genotypes , 23. TypAdmittedly, there are some shortages for this study. Firstly, small sample size prevents generalization of results to the country. Secondly, we did not evaluate the existing risk factors due to unwillingness of participated partners. Evaluation of these factors associated with type-specific positive concordance in healthy heterosexual couples is necessary to increase our understanding of HPV acquisition and transmission dynamics . ThirdlyIn conclusion, it is still important to investigate the HPV genotypes concordance between couples. Recognition of the dynamics of HPV infection not only in women, but in sexual partners could impact the implementation of preventive measures for cervical cancer and other HPV-related diseases. Protective measures like HPV vaccination in men will protect not only them but will also benefit their sexual partners. Therefore, policies to mitigate HPV infections in couples may give rise in public health improvement.


Infection with high‐risk (HR) genotypes of the human papillomavirus (HPV) is necessary for and causative of almost all cervical cancers and their precursor condition, cervical intraepithelial neoplasia. These conditions have been sharply reduced by cervical cytology screening, and a further decrease is expected because of the recent introduction of prophylactic HPV vaccinations. While significant attention has been given to gynecologic HPV disease, men can be affected by HPV‐related cancers of the anus, penis, and oropharynx. This literature review aims to address disparities in HPV‐related disease in men, and certain HR male subpopulations, compared with women.Overall, immunocompetent men are far less likely than women to develop anogenital HPV‐related cancers, despite harboring HR HPV infections at anogenital sites. On the other hand, men who have sex with men and men living with human immunodeficiency virus infection are at considerably higher risk of HPV‐related disease. Historic rates of prophylactic HPV vaccination in males have trailed those of females due to numerous multilevel factors, although, in recent years, this sex gap in vaccination coverage has been closing. In the absence of routine HPV screening in males, therapeutic vaccinations have emerged as a potential treatment modality for preinvasive neoplasia and are in various phases of clinical testing.Successful reductions in HPV disease morbidity at the population level must acknowledge and target HPV infections in men. In fact, infections predominantly with viruses and some bacteria are responsible for approximately 20% of worldwide cancer cases.While large‐scale screening and vaccination programs have begun to address HPV morbidities in women, men are not spared from potentially cancerous HPV infection. Men can succumb to complications stemming from LR HPV infection  and HR HPV infection , although the course of naturally acquired HPV infections in men is less studied than that of women with cervical infections.2In comparison with women, men are much less likely to incur genital HPV complications.Penile cancer in the United States has an incidence rate of approximately one per 100 000, while the incidence in developing nations can be much higher.A facet of HPV infection that has gained greater appreciation in recent years is the link between HPV infection and head/neck malignancies, most notably, oropharyngeal squamous cell carcinomas (OSCCs). Cancers of the head and neck can be divided into two distinct forms, which vary in epidemiology, etiology, and treatments.infection in men, and HPV‐associated OSCC has been shown to be more common in men, both in the United States and other developed nations.Of particular relevance to this review, oral HPV infection and associated OSCC are far more prevalent in males compared with females. Of 5501 individuals aged 14 to 69 who participated in the National Health and Nutrition Examination Survey (NHANES) in 2009 to 2010, overall prevalence of oral HPV (of any genotype) was approximately 7%. Prevalence of oral HPV infection followed a bimodal distribution in both sexes, with peaks at age 30 to 34 and 60 to 61 years. However, oral HPV infection prevalence was approximately threefold higher in men than women, and the prevalence of HPV 16 was over fivefold higher in men.The causal link between cervical HPV infection and associated cervical dysplasia and carcinoma has a similar parallel in anal HPV infection, anal dysplasia, and its progression to invasive carcinoma. In both men and women, the incidence of anal cancer has increased by approximately 2% per year sinceHPV infections are not systemic but rather localized to the anogenital mucosal sites of viral exposure. As such, anal HPV cases are typically associated with receptive anal sex, a sexual practice common—but not unique—to men who have sex with men (MSM). Numerous studies have shown that anal HPV prevalence  trends higher among MSM, HIV‐positive, and HIV‐positive MSM populations Table . In the While anal HPV has been well characterized in MSM populations, receptive anal intercourse does not appear to be necessary for anal infection. A study comparing 50 HIV‐positive male intravenous drug users with no history of anal intercourse and 67 HIV‐positive MSM showed a 46% and 85% HPV DNA positivity, respectively. This study also reported similar incidences of anal high‐grade squamous intraepithelial lesions (HSIL) in both populations (18%).Because the overwhelming majority of HPV infections resolve within 2 years, an important epidemiological aspect of male HPV infection is seroprevalence, which can provide a measure of viral exposure. Serum antibodies may serve as a surrogate marker for ongoing or previous infections, or even lifetime exposure.Numerous cross‐sectional and longitudinal studies of HPV antibody responses have shown lower HPV seroprevalence in men compared with women. Women disproportionately produce HPV antibody responses to HPV 16 compared with men.3Second to the widespread adoption of cervical cytology screening, the next public health success in the HPV context was the development of prophylactic HPV vaccines. Three vaccines in total have been approved: 4V Gardasil (Merck), bivalent Cervarix (GlaxoSmithKline), and nonavalent Gardasil 9 (Merck). Although genotype coverage varies by vaccine, each of the approved vaccines consists of synthetically made HPV VLPs, which are generated in vitro using a viral capsid protein L1 epitope.Some countries have recognized the public health benefits of prophylactic HPV vaccination and have instituted national government‐funded vaccination programs for adolescents since licensure of the vaccine in their country. Australia, for example, was one of the first countries to institute a national publicly funded school‐based program to vaccinate adolescents, which has resulted in the completion of the prophylactic HPV vaccination series by nearly 80% of Australian girlsProphylactic HPV vaccination policies for males have been influenced by numerous pharmacoeconomic analyses, reviewed in greater detail elsewhere.Other multilevel factors, such as the delayed adoption of sex‐neutral vaccination policies, demographic differences among vaccine‐eligible males, and geographic variation have contributed to low vaccination rates among men. The principal contributing factor to lower vaccination rates among eligible males, particularly in high‐income countries with national HPV vaccination policies, is the delayed adoption of sex‐neutral vaccination policies and publicly funded programs.Newman et al conducted a systematic review and meta‐analysis that identified factors across 23 studies that examined HPV vaccine acceptability among males.4In light of the trailing prophylactic HPV vaccination rates in males, many young men remain at risk of not only contracting new infections and succumbing to HPV‐related anogenital or oropharyngeal complications but also to spreading infection to both female and male sexual partners. Additionally, these risks are also present in all men that are older than that targeted age range for prophylactic vaccination. Thus, new therapeutic modalities are needed to address patients with active HPV infection before oncogenic transformation can occur. Standard‐of‐care excisional procedures exist for cervical dysplasia, such as loop electrical excision procedure (LEEP) or cold knife conization.clinicaltrials.gov identifier: NCT01923116). This vaccine is comprised of nine synthetic HPV 16 E6 peptides and four synthetic HPV 16 E7 peptides and is administered with or without interferon‐α. It is designed to regress HPV 16‐positive AIN 2/3 lesions in HIV‐positive MSMs with recurrent disease or previous unsuccessful treatment, with a follow‐up period of up to 18 months in a phase 1/2 study. Although no data are currently available from this study, the same vaccine has been shown to be efficacious in HPV 16‐positive vulvar intraepithelial neoplasia 3 (VIN3),clinicaltrials.gov identifier NCT02426892) in combination with nivolumab in patients of both sexes with HPV 16‐positive incurable solid tumors of the oropharynx, vulva, vagina, anus, or penis. This phase 2 study has a follow‐up of 11 weeks, and neither of these SLP‐HPV‐01 trials have data currently available. Thus, male therapeutic vaccination for HPV‐related complications may become a viable treatment option in the future.One such therapeutic HPV vaccine currently being tested in male patients is SLP‐HPV‐01, in the Therapeutic HPV‐16 Vaccination for the Treatment of Anal Dysplasia (VACCAIN‐T) study  is comparing 4V Gardasil to normal saline (placebo) in a randomized, double‐blind fashion with a follow‐up of up to 18 months. Secondary outcome measures also include cumulative occurrence of anogenital warts and HPV type‐specific antibody responses. While there are currently no data available from this study, a previous study shows potential for the therapeutic efficacy of 4V Gardasil.Although Gardasil is perhaps the most common prophylactic HPV vaccine, another trial is currently testing 4V Gardasil as a means of preventing recurrence of high‐grade AIN in MSMs who were previously successfully treated. The HPV Vaccination After Treatment of AIN (VACCAIN‐P) study , if these studies in women are successful.6Addressing the HPV disease burden of men themselves and their sexual partners will require a multifaceted prophylactic approach from both biological and public health perspectives. In order to decrease immediate HPV spread, aggressive prophylactic vaccination programs targeting young men and boys  must take place. Mandatory HPV vaccination for school enrollment is one avenue to achieve this, although this remains controversial.The authors have declared that there is no conflict of interest.This work was supported in part by National Institutes of Health  Grant R01 CA143130. The supporting funding source had no involvement in study design; collection, analysis, and interpretation of data; writing of this report; or the decision to submit this report for publication.Conceptualization: Benjamin J. LieblongFunding Acquisition: Mayumi NakagawaProject Administration: Mayumi NakagawaVisualization: Benjamin J. LieblongWriting—Original Draft Preparation: Benjamin J. Lieblong, Brooke E. E. Montgomery, L. Joseph Su, Mayumi NakagawaWriting—Review and Editing: Benjamin J. LieblongAll authors have read and approved the final version of the manuscript. Benjamin J. Lieblong had full access to all of the data in this study and takes complete responsibility for the integrity of the data and the accuracy of the data analysis.


AZGP1 mRNA levels correlated to improved overall survival (p = 0.023). By showing for the first time that HPV-positive OPSCC patients have increased intratumoral Zn levels and AZGP1 expression, we identify possible positive prognostic biomarkers in HNSCC as well as possible mechanisms of increased sensitivity to chemoradiation in HPV-positive OPSCC.Human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) is biologically distinct from HPV-negative HNSCC. Outside of HPV-status, few tumor-intrinsic variables have been identified that correlate to improved survival. As part of exploratory analysis into the trace elemental composition of oropharyngeal squamous cell carcinoma (OPSCC), we performed elemental quanitification by X-ray fluorescence microscopy (XFM) on a small cohort (n = 32) of patients with HPV-positive and -negative OPSCC and identified in HPV-positive cases increased zinc (Zn) concentrations in tumor tissue relative to normal tissue. Subsequent immunohistochemistry of six Zn-binding proteins—zinc-α2-glycoprotein (AZGP1), Lipocalin-1, Albumin, S100A7, S100A8 and S100A9—revealed that only AZGP1 expression significantly correlated to HPV-status (p < 0.001) and was also increased in tumor relative to normal tissue from HPV-positive OPSCC tumor samples. AZGP1 protein expression in our cohort significantly correlated to a prolonged recurrence-free survival (p = 0.029), similar to HNSCC cases from the TCGA (n = 499), where highest  The continuously rising number of patients presenting with HPV-positive OPSCC in the United States has eclipsed the number of HPV-negative OPSCC3. Increased evidence demonstrates that even within HPV-positive head and neck squamous cell carcinoma (HNSCC), there are divergent clinical outcomes based on factors such as patient smoking history4. Immune-related tumor-extrinsic variables have been identified in HPV-positive OPSCC, including some by our group6, such as the phenotypic and morphologic features of tumor infiltrating lymphocytes. With regards to tumor-intrinsic variables, sex hormone overexpression and gene silencing through promoter methylation8 have recently been correlated to improved survival in HPV-positive OPSCC.Human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) is biologically and clinically distinct from HPV-negative OPSCC and HPV status is a well-established favorable prognostic variable9. Elevated levels of some elements such as Fe, Cu and Zn have also been detected in the serum and tumor tissue from patients with different types of cancers14. At the same time, ample evidence indicates that different types of aggressive hormonally-driven human cancers  have low intratumoral Zn levels relative to normal tissue17. Zn exists largely bound to proteins and one type of Zn-binding proteins are metallothioneins, which are involved in Zn metabolism and have been implicated in chemoradiosensitivity and restoration of mutant p53 in tumor cells23. The analysis of trace elements in HPV-positive OPSCC may assist in better understanding their intrinsic enhanced sensitivity to chemoradiation.As cofactors for multiple enzymes and proteins, essential trace elements—iron (Fe), copper (Cu), cobalt (Co), manganese (Mn) and zinc (Zn)—are required for normal biological functionsIn this study, we utilize elemental mapping of a small cohort of clinically-matched HPV-positive and negative OPSCC cases (n = 32) by X-ray fluorescence microscopy (XFM) and demonstrate Zn is increased in HPV-positive OPSCC tumor tissue relative to matched normal squamous mucosa. By performing a targeted analysis of the expression of six Zn-binding proteins—Lipocalin-1, zinc-α2-glycoprotein (AZGP1), albumin, S100A7, S100A8 and S100A9—in tumors from OPSCC patients (n = 68), we reveal AZGP1 is selectively overexpressed in a subpopulation of HPV-positive OPSCC patients and that its expression is an independent predictor of patient survival in this patient population.A total of 75 cases of OPSCC with sufficient tissue for further testing were identified , using S to normalize the data , the quality of data was high and suitable for analysis for the following eight elements: P, S, K, Cl, Ca, Fe, Cu and Zn. Given that K and Cl measurements are often inaccurate in formalin-fixed paraffin-embedded (FFPE) samplesly) Fig. . We thenly) Fig. . As diffIn order to perform a more in-depth analysis of elemental differences between HPV-positive and -negative samples, we scanned one of the matched HPV-positive and -negative patient pairs with medium-resolution XFM Fig. . Total qFFPE tumor tissues from an HPV-positive and -negative OPSCC patient pair were subjected to exploratory proteomic analysis. Among 276 proteins identified from laser-captured microdissection of tumor tissue, 47 unique proteins were present in the tumor from the HPV-positive patient, 36 were present in the tumor from the HPV-negative patient and the remaining 193 proteins were shared amongst both patient tumor samples  from OPSCC patients by IHC in matched tumor and normal tissue, including: Lipocalin-1 n = 63), AZGP1 (n = 68), albumin (n = 26), S100A7 (n = 53), S100A7 (n = 53), S100A8 (n = 51) and S100A9 (n = 50). We used a graded scoring system for staining intensity  in tumor and matched normal squamous mucosa. Among the six Zn-binding proteins, AZGP1 was the only protein with significant differential expression levels amongst HPV-positive and HPV-negative samples, with higher median expression in tumor relative to normal in HPV-positive cases compared to HPV-negative cases  but not Lipocalin-1 staining intensities; 13 of 14 (93%) of AZGP1-high cases were HPV-positive and among AZGP1-low cases (n = 15), two-third (n = 10) were HPV-negative Fig. . Additio We chose to further focus on Lipocalin-1 and AZGP1 protein expression for patient survival analysis based on our findings of inverse expression trends in our patient cohort. As AZGP1 has been demonstrated to have anti-tumor activity and improved clinical outcome in cancer patients, we hypothesized its overexpression may portend improved patient survival . Among our cohort cases with available survival data  model , similar patterns were evident for both genes with their protein counterparts  correlated to longer overall survival in HNSCC patients, although low AZGP1 mRNA levels  did not portend a worse overall outcome. No significant difference in overall survival was present based on LCN1 expression levels.When survival analysis was extended to The Cancer Genome Atlas (TCGA) to interrogate the relationship of rts Fig. . Overexp28. We performed an analysis of AZGP1 mRNA transcript levels from RNA-seq data available in the databases for HPV-positive and HPV-negative OPSCC cases, including a total of 97 patients with OPSCC from TCGA (n = 80) and GEO portals (n = 17)28. The majority of cases were HPV-positive (n = 83) compared to HPV-negative. While total mean levels were higher in the HPV-negative OPSCC cases compared to HPV-positive OPSCC cases, this was not statistically significant by ANOVA analysis . Among the HPV-positive OPSCC cases, approximately two-thirds had integrated HPV (n = 50) and one-third had episomal HPV (n = 33). HPV-integrated OPSCC cases had significantly higher levels of AZGP1 mRNA transcripts compared to OPSCC cases with episomal HPV  in HNSCC, including OPSCC, from RNA-sequencing (RNA-seq) data in TCGA and Gene Expression Omnibus (GEO)HPV Fig. .28.The elemental and protein-based analysis of OPSCC patients presented here is a coordinated strategy to develop new understandings of the pathogenesis of OPSCC. This is the first attempt to characterize the composition of OPSCC tumor tissue by trace elements using XFM and to demonstrate that Zn-binding protein AZGP1 is a potential biomarker for positive prognosis in HNSCC. Our findings add to other work that also demonstrates that patient outcome in HPV-positive OPSCC is affected by variables such as smoking status, hormonal status, gene expression and methylation patterns and HPV integration status30. In this study we found that the normalized tumor-to-normal P and Zn ratios were elevated in HPV-positive OPSCC patients compared to the ratios for HPV-negative patients. In different aggressive human solid tumor models , intratumoral Zn levels are low relative to normal tissue17. As an antioxidant, Zn has been studied as a tool to interrupt multiple carcinogenesis-related pathways31 and administration of exogenous Zn has been implicated as a strategy to increase chemoradiosensitivity23. In HNSCC, the reduction of systemic Zn levels is correlated to aggressive tumor development—a pilot human longitudinal study of patients with advanced HNSCC revealed that in the immediate timeframe before cancer-related death, Zn levels drop from baseline in the majority of patients32. Subsequently, clinical trials of oral HNSCC have incorporated oral Zn therapy into their protocols as adjuvant therapy33.Studies utilizing XFM have shown distinct patterns in the tumor microenvironment (TME) of different human cancers21. While entirely speculative, our finding of increased intratumoral Zn content in HPV-positive OPSCC cases could provide justification for the intrinsic chemoradiosensitivity of this patient population and offer insight into ways to improve the clinical outcome of patients with HPV-negative HNSCC, who frequently harbor mutant p53.Zn stabilizes the structurally complex DNA-binding domain of the tumor suppressor gene p53 and has also been shown to rescue wild-type p53 activity in mutant p53 by re-establishing chemosensitivity in p53-mutated cell lines34. While we could not conduct a complete survey of all possible Zn binding proteins, we tested for the expression of six Zn-binding proteins in our OPSCC cohort that we initially detected in OPSCC by mass spectroscopy—Lipocalin-1, AZGP1, albumin, S100A7, S100A8 and S100A9—and we found that only AZGP1 expression was significantly higher in HPV-positive OPSCC tumor cells.Unlike P, which is predominantly associated with nucleic acids, the vast majority of intra- and extracellular Zn is bound to proteins, serving either structural or catalytic roles, collectively referred to as the ‘zinc proteome’36. In oral and buccal squamous epithelium, AZGP1 is intracellularly expressed in the mature layers of the stratified squamous epithelium37. AZGP1 expression may be related to HPV infection of human squamous epithelium—transcriptomic analysis of mature keratonicytes infected with HPV-16 demonstrated that AZGP1 mRNA is one of the six most-upregulated genes38. AZGP1 has one strong Zn-binding site and up to 15 weak Zn-binding sites. The strong Zn-binding site is located close to the lipid-binding helical groove that AZGP1 uses as Zn-binding ligands39. Zn, but no other divalent metals, induces the oligomerization of AZGP1 and is required for its functional activity40. While increased Zn and AZGP1 secretion into the serum have been found in different human epithelial malignancies, we found for the first time that both Zn and AZGP1 levels were increased in the cytoplasmic compartment of HPV-positive OPSCC tumor cells. Further work is needed to elucidate the co-localization of Zn with AZGP1 in the cytoplasm of tumor cells.AZGP1 is a soluble 40-kDa protein and major histocompatibility complex homolog and adipokine located on human chromosome 7q22.1. It is secreted by epithelial ductal cells of the salivary glands, breast, prostate and gastrointestinal tract45. In the TME, data supports the conclusion that AZGP1 could function as a tumor suppressor gene through inhibition of ERK signaling27.Abundant evidence demonstrates that the loss of expression of AZGP1—just like Zn—in tumor cells is a negative prognostic biomarker for different solid tumors, including breast, gastric, esophageal, soft tissue and prostate cancers. In these cancers, loss of AZGP1 protein was also associated with an increased likelihood of lymph node metastasis and advanced clinical stage36. This is in-keeping with the typical morphologic features of HPV-associated HNSCC and reflects an important distinction in AZGP1 staining patterns in distinct anatomic locations of the head and neck.From our exploratory protein expression analysis by IHC, we found that a subset of all OPSCC patients overexpressed AZGP1 protein and had a significantly longer RFS compared to patients with absent or low AZGP1 expression, which we corroborated with survival analysis of HNSCC patients in the TCGA. In comparison to previous literature that reports increased AZGP1 staining in oral cavity squamous cell carcinoma with histologic evidence of maturation (keratinization), the vast majority  of AZGP1-high OPSCC tumors in our cohort showed undifferentiated morphology (data not shown)AZGP1 mRNA levels in HPV-positive and HPV-negative OPSCC patients. Our analysis of TCGA and GEO RNA-seq data for AZGP1 mRNA levels revealed that total mean AZGP1 mRNA transcrips were higher in HPV-negative OPSCC, though it is impossible to make conclusions from this based on the small patient size of TCGA HPV-negative OPSCC cases. If AZGP1 mRNA levels are indeed higher in HPV-negative OPSCC, our findings of its reduced expression at the protein level in our HPV-negative OPSCC, albeit also small in size and difficult to permit general conclusions, could be due to AZGP1 post-translational and/or epigenetic silencing through histone deacetylase (HDAC) activity, which is an established epigenetic silencing mechanism of AZGP1in different human cancers46.In light of these protein expression results, we wanted to explore corresponding AZGP1 mRNA levels compared to those cases with episomal HPV. What these findings from RNA-seq mean for the protein expression, however, remains to be established. The HPV oncoproteins E6 and E7 encode Zn finger transcription activation domains comprised of cysteine residues that are required for their function49. Additionally, the HPV16 E5 protein blocks the negative regulation of Zn redistribution into host cell nucleoli through interactions with the Zn transporter complex made of proteins EVER1 and 2 and ZnT-150, resulting in increased cell proliferation. Further investigation is warranted to determine if HPV-related proteins such as EVER2 contribute to our finding that Zn is elevated in HPV-positive tumor cells. We speculate other Zn-binding proteins that are not encoded by the HPV genome, such as AZGP1, also contribute to increased detectable Zn levels in OPSCC cases.While our methods did not allow us to segregate between episomal and integrated HPV-status in our OPSS cohort, we show that TCGA/GEEO cases with integrated HPV had significantly higher This study is intended to draw previously unrecognized correlations between HPV-status in OPSCC with intratumoral trace element content and the expression of metal-binding proteins and not to establish mechanisms for which one finding may be related to another. We believe that both Zn and AZGP1 are promising therapeutic targets in HNSCC, though their role in patient sensitivity to chemoradiation needs to be further studied in a larger population. Major limitations of this study include the relatively small cohort size and the robustness of the correlation of overall tumor Zn levels with specific Zn binding proteins. With respect to the former, the number of patients in this cohort is not of adequate size to correlate to the general population. We attempted to remedy this by extending our survival analysis of AZGP1 to the larger HNSCC population database available in TCGA. Also, due to lack of tissue in some cases, we were not able to achieve statistical significance for a correlation between AZGP1 expression and Zn levels on a per-case basis.All archived patient specimens for retrospective analysis were collected from patients diagnosed with OPSCC at Northwestern Memorial Hospital (NMH), Department of Pathology under a Northwestern University (NU) IRB-approved protocol. All experiments were performed in accordance with relevant guidelines and regulations. The need for informed consent was waived by the NU IRB. Cases were selected over a six-year period (2010–2016) based on the following criteria: a primary diagnosis of squamous cell carcinoma of the oropharynx with available p16 immunohistochemistry (IHC) results and adequate tissue for testing. Among all patients, 16 HPV-positive and HPV-negative patient pairs were created (A.R.) for XFM imaging using the following matching variables: age, smoking, alcohol use, tumor differentiation, tumor location, tumor stage and gender.FFPE tissues selected for this study were used for construction of TMAs as three 1.5 mm cores per case, comprised of two tumor cores and one core of adjacent histologically normal squamous mucosa, verified by a pathologist (K.P.). Tonsil cores from healthy donors were included in selection for the TMA as staining controls. Five μm sections were cut from tissue and TMA blocks for staining with hematoxylin and eosin (H&E) and for IHC. Tissue sections for XFM were placed on Ultralene membranes .51—and interpreted as positive when >70% strong tumor nuclear and cytoplasmic staining was present. We also performed high-risk (HR) HPV testing on all cases using DNA and mRNA in situ hybridization (ISH). DNA ISH: ZytoFast® HPV High-Risk (HR) Types Probe specific for oncogenic E6/E7 from 15 HPV types, Cat. T-1140-400 combined with the Zytofast PLUS Implementation Kit, Cat. T-1061-40). mRNA ISH: RNAscope 2.0 HD Detection Kit . RNAscope testing used a probe against 18 high-risk (HR) HPV genotypes E6/E7 mRNA . Slides were processed according to the manufacturer’s instructions; the bacterial gene DapB was used as a negative control and housekeeping gene POLR2A served as a positive control.At the time of diagnostic work-up, all cases were stained for p16—the HPV surrogate marker53. At the 8BMB beamline, the beam was focused to a 30 μm spot (low resolution) and at the beamline 2-ID-E the beam was focused to a 0.5 μm spot (medium resolution). Spectra were collected with a SII Vortex ME4 4-element silicon drift detector . Tissue sections were scanned with step sizes equal or larger than beam spot sizes and dwell times of 100–500 milliseconds; their elemental content was quantified and mapped. Elemental concentrations were calibrated using thin film AXO standards  and the data were calibrated using MAPS software54. Per pixel counts were converted to elemental concentrations (μg/cm2).XFM was done (T.P.) on FFPE whole-sections of tumor from patients with OPSCC , sulfur (S), chlorine (Cl), potassium (K), calcium (Ca), chromium (Cr), nickel (Ni), arsenic (As), selenium (Se), bromine (Br) and mercury (Hg). Concentrations of some of the elements  fell within the error range and those elements were not used for final analysis. Final measurement of elemental concentrations was done as follows: for each selected region of interest (ROI), mean per pixel value background area concentration for each element was subtracted from elemental concentrations for tumor and adjacent histologically benign squamous mucosa. Finally, these values were normalized relative to sulfur. Sulfur was chosen for this purpose due to the fact that it has a uniform signal in proteins from FFPE tissue, serving as a proxy for tissue density2  covering tumor were collected in adhesive caps 200 . Tissue fragments were stored at −80 °C until use. Protein digestion with trypsin was done, followed by purification with C18 spin columns prior to analysis by mass spectrometry. Mass spectrometry data acquisition was done at NU Proteomics core facility. In brief, peptides were analyzed by LC-MS/MS using a Dionex UltiMate 3000 Rapid Separation nanoLC and a Q Exactive™ HF Hybrid Quadrupole-Orbitrap™ Mass Spectrometer . Peptides were separated on a 120-min analytical gradient from 5% ACN/0.1% FA to 30% ACN/0.1% FA. The top 15 most abundant precursor ions in each MS1 scan were selected for fragmentation. Proteins were identified from the tandem mass spectra extracted by Xcalibur version 4.0. MS/MS spectra were searched against the SwissProt Homo sapiens database using Mascot search engine . The search result was visualized by Scaffold .For two patients, FFPE tumor sections eight μm in thickness were mounted on membrane slides  and were deparaffinized and stained for 3–5 seconds in 0.1% Toluidine Blue Solution . Laser capture microdissection (LCM) was performed using a type P-MB device (Carl Zeiss MicroImaging). From each section, areas of approximately 500,000 μmTMAs were cut at 5-um thickness and deparaffinized. Antigen retrieval was performed at 95 °C for 30 minutes followed by blocking with HRP solution. Slides were then incubated with antibodies for: AZGP1 , Lipocalin-1 , Albumin , S100A7 , S100A8  and S100A9  followed by HRP-conjugated secondary antibody and DAB staining. IHC staining in tumor for AZGP1 (cytoplasmic), Lipocalin-1 (cytoplasmic), Albumin (cytoplasmic), S100A7 (cytoplasmic and nuclear), S100A8 (cytoplasmic and nuclear) and S100A9 (cytoplasmic) were scored by a pathologist (KP)  and the GEO (https://www.ncbi.nlm.nih.gov/geo/). The TCGA cohort was comprised of 515 patients with clinical and genomic data. For some analysis, RNA-seq data was extracted from HNSCC patients from the following coded sites: ‘oropharynx,’ ‘tonsil’ and ‘base of tongue’. Preprocessed and normalized htseq count data for AZGP1 and LCN1 gene expression was performed by aligning reads with STAR and Tophat256. Detection of HPV status and integration sites was performed by realigning data to the HPV genome as previously described57. AZGP1 RNAseq data for integrated and episomal HPV-positive OPSCC cases were obtained from: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE74956. All downloaded data was subsequently normalized using the DESeq2R package58.We evaluated HNSCC patient clinical and RNA-seq data extracted from TCGA Research Network  starting at the date of diagnosis and defined as the absence of locoregional or distant recurrence or disease-related death. In HNSCC TCGA survival analysis, the primary endpoint was overall survival. Patient survival trends were tested by log-rank. Multivariable parametric a AFT models were performed to estimate associations between protein expression levels and survival outcomes. Covariate adjustments included: smoking status, age, sex and treatment with surgery only, radiation only, chemoradiation only, surgery followed by radiation or surgery followed by chemoradiation. P values ≤ 0.05 were considered statistically significant. All statistical analyses were performed in Prism (GraphPad Software) and R (v3.4).Comparisons between elemental concentrations were calculated using paired T tests. The relationship between HPV status and AZGP1/Lipocalin-1/Albumin/S100A7/S100A8/ S100A9 IHC scores were determined by linear regression and Pearson correlation. The relationship between logSupplemental Information


The skin represents a physical and chemical barrier against invading pathogens, which is additionally supported by restriction factors that provide intrinsic cellular immunity. These factors detect viruses to block their replication cycle. Here, we uncover the Myb-related transcription factor, partner of profilin (MYPOP) as a novel antiviral protein. It is highly expressed in the epithelium and binds to the minor capsid protein L2 and the DNA of human papillomaviruses (HPV), which are the primary causative agents of cervical cancer and other tumors. The early promoter activity and early gene expression of the oncogenic HPV types 16 and 18 is potently silenced by MYPOP. Cellular MYPOP-depletion relieves the restriction of HPV16 infection, demonstrating that MYPOP acts as a restriction factor. Interestingly, we found that MYPOP protein levels are significantly reduced in diverse HPV-transformed cell lines and in HPV-induced cervical cancer. Decades ago it became clear that the early oncoproteins E6 and E7 cooperate to immortalize keratinocytes by promoting degradation of tumor suppressor proteins. Our findings suggest that E7 stimulates MYPOP degradation. Moreover, overexpression of MYPOP blocks colony formation of HPV and non-virally transformed keratinocytes, suggesting that MYPOP exhibits tumor suppressor properties. The physical and chemical barrier of the skin mediates the first line of defense against invading pathogens. This is additionally supported by the cellular innate immunity . Here, sHuman papillomaviruses are small DNA viruses associated with a wide range of benign and malignant epithelial tumors. These viruses account for 5.2% of the worldwide cancer burden . PersistThe viral replication cycle starts with virus entry via endocytosis –18. AftePOP is able to repress the consensus Myb recognition element (MRE) when introduced into the minimal herpesvirus thymidine kinase promoter [In this study, we identified the so far unknown human Myb-related transcription factor (TF), MYPOP, as a novel interaction partner of the minor capsid protein L2 and the viral DNA. It was shown that MYPOP’s orthologous murine protein p42promoter . We uncoDuring HPV entry, the minor capsid protein L2 accompanies the viral DNA into the host cell nucleus and is accessible to cytoplasmic and nuclear proteins , 23, 24.Chromatin immunoprecipitation (ChIP) studies in HeLa cells, which are transformed by oncogenic HPV and harbor the integrated viral genome within the host cell genome  revealedPOP had showed that the MYPOP’s orthologous murine protein represses transcription [Next, we investigated whether MYPOP directly regulates the activity of the HPV16 LCR by performing promoter-reporter gene assays. Non-virally transformed HaCaT keratinocytes were co-transfected with the promoter-reporter plasmid pGL4.20 containing the HPV16 long control region (pGL4.20 HPV16 LCR), MYPOP and/or L2 expression vectors Fig. . Measurecription , suggestDirect binding of Myb TFs to viral and cellular promoters is a well-characterized interaction of the DNA-binding domain (DBD) with the MRE –42. Our Next, we uncovered that the MYPOP-mediated silencing of the LCR is not limited to HPV16, but was also observed for the high-risk HPV18 LCR, which comprises three MREs Fig. . By contThe repressive effect of MYPOP on the transfected HPV LCR was extended to incoming histone associated HPV-transduced DNA. For this study, we replaced the viral genome by the pGL4.20 HPV16 LCR plasmid, thereby generating HPV16 LCR PsV. Next, we reduced the endogenous MYPOP level in HaCaT cells by RNA interference. These experiments provided deeper insight into the nature of the TF Fig. . First, To verify the negative correlation of the MYPOP expression level and HPV16 PsV infection in different cells, we tested total cellular MYPOP amounts and infection efficiencies of HaCaT cells and primary keratinocytes (NHEK) Fig. . These rAs our MYPOP-based ChIP and promoter studies demonstrated binding to the HPV LCR and repression of the LCR activity, we controlled the effect of MYPOP in the whole HPV genome context on the expression of viral early genes. We co-transfected squamous cell carcinoma cells SCC-13  with HPVIt has been described that early viral oncoproteins E6 and E7, which are expressed in HPV-transformed cells, eliminate detectable tumor suppressors p53 and pRb, respectively . InteresThe high-risk HPV E6 or E7 oncoproteins are potential candidates to induce MYPOP downregulation. These proteins are highly expressed in HPV-induced cancers and possess the ability to target tumor suppressors for degradation , 50–52. The maintenance of early gene expression is a precondition for proliferation and survival of HPV-induced cancer cells –55 and sMYPOP’s repressive activity might be explained by competition of MYPOP with activating TFs. For c-Myb, it has been shown earlier that it binds to one MRE in the HPV16 LCR and activates early gene expression . TherefoOverall, our study provides first indications for the so far unknown roles of MYPOP by demonstrating that this Myb-related protein senses incoming viruses and represses viral gene transcription. Thereby, MYPOP acts as a restriction factor and limits cells’ permissiveness to infection with oncogenic HPV viruses. Mechanistically, we propose a model in which MYPOP senses incoming oncogenic HPV by interaction with the accessible cytoplasmic part of the L2 protein until release of the viral DNA within the nucleus. Subsequently, MYPOP binds to the LCR via its DBD. This results in silencing of HPV early/oncogene expression and subsequently, suppression of cancer. During cell transformation, the HPV16 onco-protein E7 mediates degradation of MYPOP by a mechanism that is yet to be determined, which results in increased expression of the early viral genes, cell proliferation, and finally oncogenesis. A detailed investigation and elucidation of this transcriptional repressor will be crucial for better understanding of infections by oncogenic papillomaviruses and tumor suppression in general.Supplementary figuresSupplementary methods


KR-20 coefficient of 0.74 for the second part and a Cronbach’s alpha of 0.85 for the third part). Furthermore, the present study confirmed the importance of health-care providers in recommending the immunization practice. Parents that had been strongly advised by health-care providers to vaccinate their children accepted immunization for their girls (odds ratio (OR) 1.09 (95% CI 1.04–1.14)) and boys (OR 1.06 (95% CI 1.02–1.10)), had a lower probability of deciding to postpone the immunization appointment (OR 0.81 (95% CI 0.66–0.98)), had fewer doubts and concerns about the vaccine (OR 0.69 (95% CI 0.54–0.89)), and had a lower probability of refusing the vaccination (OR 0.93 (95% CI 0.86–0.99)). Interestingly, the use of new, emerging tools such as ad hoc websites, applications, and other interactive devices reduced vaccine hesitancy (OR 0.90 (95% CI 0.82–0.99)) and concerns about the side-effects of the vaccine (OR 0.92 (95% CI 0.86–0.99)). However, among Israeli health-care workers, knowledge was generally moderate, with updated information lacking in about 30% of surveyed health-care providers and approximately 20% of them not recommending the anti-HPV vaccine among boys. This study has practical implications for policy- and decision-makers in that they should be aware of the overall level of knowledge among health-care workers and should implement ad hoc educational interventions to address gaps in knowledge and help medical providers routinely recommend the anti-HPV vaccine both to male and female children and adolescents.Human papillomavirus (HPV) is a highly widespread virus which is responsible for one of the most common sexually transmitted infections. Two main preventative strategies exist: anti-HPV vaccination and cervical screening. Health-care workers play a key role in promoting public health campaigns; however, vaccine hesitancy is an often under-recognized challenge. To investigate the overall knowledge of HPV and HPV-related issues, as well as the attitudes and practices of health professionals towards recommending the anti-HPV vaccine, an ad hoc knowledge, attitudes, and practices (KAP) questionnaire was developed and validated in a sample of 139 Israeli pediatricians, gynecologists, and internal medicine doctors. The KAP questionnaire was found to be psychometrically valid and sound (with an r Human papillomavirus (HPV), a nonenveloped DNA virus, is a highly widespread and common pathogen, which is responsible for one of the most common sexually transmitted infections . It geneGlobally, approximately 4.5% of all malignancies can be attributed to HPV (8.6% among women and 0.8% among men), with HPV being the main etiopathogenetic factor of up to 83% of all cervical cancers ,8. p-value for trend = 0.0001). Similarly, the annual age-adjusted incidence rate of CIN3 ranged from 3.9 per 100,000 in 1986 to 40.4 per 100,000 in 2010  [Specifically in Israel, HPV generates a dramatic burden, even though this is partially mitigated by cultural and religious factors  . In thisAnti-HPV vaccination and gynecological screening (the Papanicolaou smear or Pap test) represent two major preventative strategies to counteract or, at least, mitigate such a burden, the former being an approach of primary prevention, and the latter being a strategy of secondary prevention ,12. ©, against HPV types 16 and 18), a quadrivalent one , and finally, a nonavalent one  [Currently, three types of anti-HPV vaccine exist: a bivalent preparation (traded as Cervarix and 58) ,13. While being generally administered among females, recently, the American Academy of Pediatrics (AAP) has recommended the use of anti-HPV vaccines also among males . Various© vaccine was added to the health basket, that is, the basket of drugs and medical treatments that are subsidized by the government. This enabled achieving an overall coverage rate of approximately 60%, being higher among secular Jews and Arabs and lower among Orthodox Jews. In 2015, the immunization program was further expanded, introducing the anti-HPV vaccination (Gardasil©) also for men and boys aged between 9 and 26 years. For the year 2019–2020, the Gardasil-9© vaccine is being provided both to eighth-grade girls and boys at schools. According to the Israeli Ministry of Health’s guidelines [In 2013, in Israel, a universal program for HPV vaccination was introduced for eighth-grade girls at middle schools and for ninth-grade girls at health bureaus . In greaidelines , cervicaHealth-care providers are essential in disseminating high-quality and scientific-evidence-based information on HPV and the HPV-related burden. A recent state-wide survey carried out in Minnesota, United States, found that anti-HPV vaccine hesitancy among health-care professionals may discourage vaccine uptake among children and adolescents . HoweverTherefore, the present cross-sectional survey-based study was designed to address this gap in knowledge.The study protocol of the present investigation was reviewed in depth and, being questionnaire based, received an exemption from the Helsinki Commission of the Ziv Medical Center, Safed, Israel. A KAP questionnaire concerning knowledge of HPV, as well as attitudes and practices towards anti-HPV vaccination, was developed based on (i) an extensive literature search; (ii) a focus group with a selected number of Israeli pediatricians, gynecologists, and internal medicine doctors; (iii) a revision from a panel of experts and scholars; and, finally, (iv) a pilot test in a sample of 20–30 subjects. The final form of the questionnaire was administered to pediatricians, gynecologists, and internal medicine doctors of the Galilee Medical Center in Nahariya and of the Ziv Hospital in Safed, Israel. The questionnaire consisted of three parts: a sociodemographic part with questions concerning age, gender, marital status, religion, profession, clinical ward attended, and years of experience; a part regarding knowledge of HPV and HPV-related burden; and a part related to attitudes and practices towards anti-HPV vaccination. More in detail, the third part of the questionnaire was theoretically built to consist of two subsets of items: the first subset concerned the awareness of the health-care provider about the importance, safety, and effectiveness of the anti-HPV vaccination and his/her attitude towards strongly recommending it by establishing a relationship and alliance with the child and his/her family. The second subset of items regarded vaccine counseling and the use of new, emerging tools, such as ad hoc vignettes, cartoons, websites, applications, and other interactive devices.Before proceeding with data handling and statistical processing, figures were visually inspected to capture potential outliers. Continuous variables were expressed as mean ± standard deviation, whereas categorical parameters were computed as percentages, where appropriate. KR-20) [K is the number of items, i, i, and n is the sample size. The KR-20 represents a particular case of the Cronbach’s alpha for items that imply a dichotomous reply . Unlike the Kuder–Richardson Formula 21 (KR-21), the KR-20 makes the more realistic assumption that the test items differ in terms of difficulty . Therefore, different variables, such as the spread among the scores, the presence of some particularly challenging questions, and the length of the questionnaire, could impact on the rKR-20.The validity of the second part was computed by calculating the Kuder–Richardson Formula 20 (KR-20) coefficient (rKR-20) ,26,27,28i for the current sample of subjects.The validity of the third part was computed calculating the Cronbach’s alpha coefficient, using the following formula :(3)α=KK−The magnitude of the Cronbach’s alpha coefficient was interpreted based on the following rule of thumb: unacceptable if less than 0.5, poor in the range of 0.5–0.6, questionable in the range of 0.6–0.7, acceptable in the range of 0.7–0.8, good in the range of 0.8–0.9, and excellent if equal to or greater than 0.9.KR-20 and the Cronbach’s alpha coefficient can be standardized as follows:Both the rp-value less than 0.25 at the univariate analysis were entered in the models. Gender and age, being potential confounders, were included into the regression model. The goodness of fit of the model was verified performing the Hosmer–Lemeshow test. A logistic regression model was conducted to shed light on the determinants of overall knowledge of HPV and HPV-related issues, the attitudes and practices towards recommending the anti-HPV vaccination, as well as their impact on parents’ intention to vaccinate their children. According to the Hosmer–Lemeshow procedure, only covariates with a Finally, the impact on parents’ intention to vaccinate or not their children was assessed by means of odds ratios (ORs) computed together with their 95% confidence interval (CI).p-values less than 0.05 were considered statistically significant. All statistical analyses were performed by means of the commercial “Statistical Package for Social Sciences” (SPSS) version 24.0 for Windows . For all statistical analyses, figures with A sample of 139 health-care workers took part in the present study (average age 31 years (95% inter-quartile range (IQR) 29–47), 61.2% females and 38.8% males, 53.2% married, 68.3% specialists/doctors and 31.7% residents, 64.0% with less than 5 years of experience, and 37.4% of Jewish creed). Further characteristics of the sample are shown in KR-20 coefficient of 0.74, standardized coefficient adjusted for the number of items of 0.75). Dropping one item per time generally resulted in a decreased internal consistency coefficient (except for item one), confirming the validity of the questionnaire. The third part displayed an overall good internal consistency . For further information, the reader is referred to The items of the questionnaire are presented in Specifically concerning the third part of the questionnaire, the first subset of items had a satisfactory internal consistency of 0.776, whereas the second subset had a validity of 0.841 .Overall, HPV-related knowledge resulted in a mean score of 7 (95% IQR 5–8), with the percentage of right replies ranging from 35.3%  to 87.1% , as reported in p < 0.0001). The mean score of the third part of the questionnaire was 3.34 ± 0.41. Average scores for the first and second subsets were 3.32 ± 0.37 and 3.39 ± 0.75, respectively. Correlation between the first and the second subsets was 0.47 ), whereas no significant predictor was associated with the score of the second subset .Most health-care workers recommended anti-HPV vaccination among girls (98.6%), whereas only 79.9% of them recommended the immunization practice among boys. Parents that had been strongly advised by health-care providers to vaccinate their children accepted immunization for their girls ) and boys (OR 1.06 (95% CI 1.02–1.10)), had a lower probability of deciding to postpone the immunization appointment (OR 0.81 (95% CI 0.66–0.98)), had fewer doubts and concerns about the vaccine (OR 0.69 (95% CI 0.54–0.89)), and had a lower probability of refusing the vaccination (OR 0.93 (95% CI 0.86–0.99)). Interestingly, the use of new, emerging tools such as ad hoc websites, applications, and other interactive devices reduced vaccine hesitancy (OR 0.90 (95% CI 0.82–0.99)) and concerns about side-effects of the vaccine (OR 0.92 (95% CI 0.86–0.99)). Anti-HPV vaccination and HPV testing, such as the Pap test, represent, respectively, a safe, effective immunization practice and a major secondary preventative strategy that can reduce HPV-related burden. Health-care providers play a key role in advising parents to vaccinate their children in terms of trust and being perceived of as authoritative, highly reliable sources of information. Indeed, some studies have shown that parents who had been very strongly recommended to have their son immunized had higher odds of vaccinating their children compared with parents who were not very strongly recommended . It is tOur findings indicate that knowledge of HPV and HPV-generated clinical burden is moderate among health-care workers in Israel. These results are in line with the existing scholarly literature. In Greece, several gaps in knowledge among female health-care providers could be detected, especially in terms of updated information on cervical cancer prevention through HPV testing and vaccination, with only 80% of them being aware of the existence of cervical screenings . In SpaiHowever, in our study, we could not find differences in terms of level of HPV-related knowledge between residents and specialists/doctors as well as among gynecologists, pediatricians, and internal medicine doctors (except for the first subset of items of the third part of the questionnaire), whereas some scholars have reported differences, with the highest level of knowledge of and belief in the anti-HPV vaccine, its effectiveness, and its safety being observed among pediatricians and school medicine specialists . On the Furthermore, in Israel, the decision to also include boys in the immunization program ignited a rather intense debate, given the relationship between the vaccine and sexual behaviors ,36. ConcIn Israel, several parents are hesitant to vaccinate their sons ,40 due tVaccine hesitancy among health-care professionals is an under-recognized public health concern that may jeopardize the success of immunization programs and needs, as such, to be properly addressed . Health However, the present investigation is not without limitations, including the small sample size employed and its cross-sectional study design. Longitudinal, prospective studies verifying, for instance, the effect of programs of continuing education and utilizing random stratified sampling techniques, in order to ensure the generalizability of the findings, are warranted. The KAP questionnaire was found to be psychometrically valid and reliable and can be used, as such, by other scholars to investigate knowledge of HPV, as well as attitudes and practices towards anti-HPV vaccination. Among Israeli pediatricians, gynecologists, and internal medicine doctors, knowledge was generally moderate, with updated information lacking in about 30% of surveyed health-care providers and approximately 20% of them not recommending the anti-HPV vaccine among boys. On the other hand, a good level of knowledge and positive attitudes towards recommending the anti-HPV vaccine significantly impacted on parents’ intention to vaccinate their children. This study has practical implications for policy- and decision-makers in that they should be aware of the overall level of knowledge among health-care workers and their attitudes towards recommending the anti-HPV vaccine. Continuing medical education and other educational interventions could be an effective strategy to keep health professionals constantly updated and to potentially counteract vaccine hesitancy among health-care workers.