Classical markers alone, such as HPV DNA, p16 and HPV mRNA expression, are not enough to stratify HPV-positive head and neck squamous cell carcinoma (HNSCC) patients, but when combined with serological markers, the latter are strong indicators of prognosis in oropharyngeal squamous cell carcinoma (OPSCC) patients. Specifically, HPV16 E7 oncoprotein in serum at the time of diagnosis, correlates with disease recurrence and patient overall survival. To our knowledge, this is the first study to investigate HPV E7 oncoprotein in patient serum. The E7 oncoprotein detection in serum at the time of diagnosis may be useful as a non-invasive procedure for HPV-positive OPSCC patient stratification and follow-up, helping to identify patients at risk for tumor recurrence and metastasis during follow-up, and ultimately, providing a tool for clinicians to identify patients for de-escalation treatment or those to be kept under close surveillance.n = 67) and controls (n = 58) by qPCR. Subsequently, ELISA tests were used for HPV16 L1 antibody and HPV16 E7 oncoprotein detection in serum at diagnosis and follow-up. All markers were correlated to relapse-free survival (RFS) and overall survival (OS). HPV-DNA was found in HNSCCs (29.85%), HPV16-DNA in 95% of cases, HPV16 E7 mRNA was revealed in 93.75%. p16 was overexpressed in 75% of HPV-positive HNSCC compared to negative samples and controls (p < 0.001). Classical markers correlated with improved OS (p < 0.05). Serological studies showed similar proportions of HPV16 L1 antibodies in all HNSCCs (p > 0.05). Serum E7 oncoprotein was present in 30% HPV-positive patients at diagnosis (p > 0.05) and correlated to HNSCC HPV16 E7 mRNA (p < 0.01), whereas it was associated to worse RFS and OS, especially for oropharyngeal squamous cell carcinoma (OPSCC) (p < 0.01). Detection of circulating HPV16 E7 oncoprotein at diagnosis may be useful for stratifying and monitoring HPV-positive HNSCC patients for worse prognosis, providing clinicians a tool for selecting patients for treatment de-escalation.Despite improved prognosis for many HPV-positive head and neck squamous cell carcinomas (HNSCCs), some cases are still marked by recurrence and metastasis. Our study aimed to identify novel biomarkers for patient stratification. Classical HPV markers: HPV-DNA, p16 and HPV mRNA expression were studied in HNSCC ( Human papillomavirus (HPV)-related head and neck squamous cell carcinomas (HNSCCs) are increasing worldwide . SpecifiHPV plays an important role in OPSCC onset . The maiOptimizing protocols through targeted therapies and personalized treatments is paramount to increase survival rates for all patients . In thisHence, correctly stratifying HPV-positive patients is necessary to select optimized treatment . In an eSerological testing has gained interest in the past few years for HPV-positive OPSCC prognostic studies. The immune response of the host has been studied in association with both HPV-positive tumors and patient prognosis ,19. SeruStudies on cervical cancer have shown that detection of HPV E6 and E7 oncoproteins in cervical scrapings may constitute valuable markers for disease progression ,25. MoreTherefore, we have hypothesized that HPV E6 and E7 oncoproteins could be present in serum from HPV HNSCC patients, and their serum identification could be useful for prognostic purposes. The presence of HPV E6 and E7 oncoproteins in serum from HPV-associated cancer patients is yet to be investigated. Since new kits for testing serum HPV E7 protein are now commercially available new investigations can be carried out.The aim of this study was to identify markers for HPV-positive HNSCC patient stratification. To this purpose, classical tumor markers, such as HPV DNA, p16 expression and HPV E7 mRNA were studied in different HNSCC subtypes, including OPSCC. Sera of HNSCC patients were analyzed for HPV16 L1 antibody titers and, for the first time, HPV16 E7 oncoprotein levels at the time of tumor diagnosis and during follow-up at 3, 6, 12 and 24 months. Finally, results were correlated to patient relapse-free survival (RFS) and overall survival (OS) at 24 months.n = 67) from patients, mean age ± standard deviation [SD] 64.94 ± 10.88 years [y] old, were collected for analyses. Tumor-free tonsillar (TFT) samples (n = 58), from non-oncological patients who had undergone tonsillar surgery, 39 ± 15.17 y old, were used as controls. Samples were collected consecutively from 2016 to 2020 at the Ear, Nose and Throat (ENT) Clinic . Inclusion criteria was the histopathological detection of primary HNSCC in patients 18–95 y old, including hidden or occult SCC with lymph node cervical positive histology. Exclusion criteria were radiotherapy and/or chemotherapy treatment. The 8th edition of AJCC classification was used . DN−ΔΔCt method, as done previously [QPCR was performed for p16 and HPV16 E7 gene expression analyses. Briefly, 50 ng of cDNA were used in 10 μL reactions using 2× SsoAdvanced Universal SYBR Green Supermix (Bio-Rad). A final concentration of 500 nM of each primer was employed . Sampleseviously ,31. Furtn = 20) and HPV-negative (n = 8) HNSCC patients at the time of diagnosis (T0) and during follow-up at 3, 6, 12 and 24 months.Upon collection, blood samples were allowed to clot for 15 min at room temperature and then centrifuged at 1300 g for 15 min. Serum HPV16 L1 IgG antibodies were evaluated in HPV-positive . The test was performed according to the manufacturer’s instructions. The signal intensity was measured as Optical Density (OD) at 450 nm  . The cutn = 20) and HPV-negative (n = 8) HNSCC patients, using the “HPV16 E7 Oncoprotein ELISA Kit” , according to the manufacturer’s instructions. The presence or absence of E7 oncoprotein was determined by considering sample absorbance above or below the cutoff value, respectively, calculated as done previously [Serum HPV16 E7 oncoprotein levels were evaluated in HPV-positive  . The ANO−4 to 4.26 × 102 copies of HPV DNA per cell  of HNSCC samples, consisting of 2/20 (10%) oral squamous cell carcinoma (OSCC), 15/20 (75%) OPSCC, 2/20 (10%) hypopharyngeal cancer and 1/20 (5%) laryngeal cancer . HPV-genper cell A.p < 0.001). HPV-positive patients showed overall p16 gene upregulation compared to controls  with the exception of two samples that harboured p16 downregulated. HPV-negative samples were downregulated compared to control samples . Differences in p16 expression between HPV-positive and -negative were also significant (p < 0.0001)  of HPV16-positive HNSCC and in 5/25 (20%) of HPV-negative HNSCC samples compared to controls ( 0.0001) B.10) of E7 and HPV DNA load . Furthermore, Spearman correlation analyses showed correlation between E7 expression and p16 up-regulation   C. Pearso < 0.05) D. But, Hn = 20) HNSCC patients (n = 8) HNSCC patients, consisting of 4/8 (50%) OPSCCs and 4/8 (50%) OSCCs, were tested for HPV16 L1 IgG antibodies. HPV16 L1 antibodies were found with a similar proportion in 18/20 (90%) HPV–positive HNSCC and 7/8 (87.5%) HPV-negative HNSCC patients at T0 (p > 0.05). HPV DNA-positive HNSCC patients presented higher Optical Density (OD) readings for antibodies anti-HPV16 L1 compared to HPV-negative  (p > 0.05)  A. Antibo > 0.05) B.p > 0.05) (p > 0.05) (HPV16 E7 oncoprotein (ng/mL) amounts were evaluated at the time of diagnosis and during follow-up at 3, 6, 12 and 24 months. At T0, HPV16 E7 oncoprotein was detected in 6/20 (30%) HPV-positive patient serum and no HPV-negative cases ( > 0.05) C. Variat > 0.05) D. Nine op < 0.01), suggesting that circulating E7 protein may be due to release from the tumor site.Finally, HPV16 E7 oncoprotein levels in serum were studied in correlation to the viral mRNA expression in the tumor samples. Results showed correlation between the amount of HPV16 E7 mRNA expressed in the tumors and E7 oncoprotein in serum  (p < 0.01)  and overall survival (OS) were assessed in HPV-positive HNSCC patients compared to HPV-negative cases. Different RFS rates were observed; 72.11% and 48.77% for HPV-positive and -negative, respectively ( > 0.05) A. Furthe < 0.01) B.2 fold change (FC) value  was used as the cut-off criteria. High and low expression were considered when FC was greater than 1 (n = 13) or lower than −1 (n = 17), respectively [p > 0.05) (p < 0.01)  C. OS was < 0.01) D.n = 15) and non (n = 26). Survival proportions indicated that RFS was 64.61% in patients positive for E7 mRNA, and 48.77% in patients HPV mRNA-negative (p > 0.05) (p < 0.05)  E. OS was < 0.05) F.n = 20) at the time of diagnosis and during follow up. RFS was 51.28% and 100% for HPV16 L1 antibody-positive (n = 18), for HPV16 L1 antibody-negative (n = 2) patients, respectively (p > 0.05) (p > 0.05)  A. OS was > 0.05) B. Overalp < 0.0001) and in the OPSCC subgroup (p < 0.001). Statistical analyses on other HNSCC subtypes were not possible due to the small sample size (p < 0.0001) (p < 0.01)  C. OS was < 0.01) D.p > 0.05) (p > 0.05)  E. OS pro > 0.05) F. These p > 0.05) (p > 0.05) (p > 0.05) (p > 0.05) (RFS was 72.9% and 57.14% for patients with T (1–2) and T (3–4), respectively ( > 0.05) A, while  > 0.05) B. Simila > 0.05) C, while  > 0.05) D.p < 0.05) (p > 0.05)  E, while  > 0.05) F.p < 0.05), but not to lymph node involvement in OPSCC (p > 0.05) (p > 0.05).Lastly, we studied the correlation between serum E7 presence and tumor size, lymph node involvement and disease stage. E7 in serum correlated to tumor size ( > 0.05) . Out of The current study aims to find markers for recurrence in HPV-positive patients. For patient stratification, we studied classical HPV markers, such as HPV DNA, p16 mRNA and viral mRNA expression. Once stratified, we studied the presence of potential serological markers, i.e., HPV16 L1 antibodies and, for the first time, the HPV E7 oncoprotein. Serological markers were then correlated to patient prognosis.In an initial screening, we found that 29.85% of HNSCC tumor samples, including 75% OPSCC, harbored HPV-DNA, and 95% tested HPV16-positive. These findings are in accordance to other studies indicating that HPV is found in 25% of all HNSCCs, and in up to 70% of OPSCC tumors ,37,38,39Since HPV status has a great impact on patient prognosis for different HNSCCs, such as OPSCC and OSCC ,44, it iAntibody response against L1 was studied for the prevalence of viral infection in HNSCC patients. HPV L1 antibodies are cumulative markers of past and present infection, although their presence does not imply HPV-driven tumorigenesis . Indeed,We also studied the antibody response during the follow-up to monitor disease status, as proposed by Routman et al. , but no Recently, the study of antibody response against HPV E6/E7 oncoproteins in OPSCC, the major subtype of HNSCC, has been proposed as a marker for disease progression. In spite of good perspectives for both diagnosis and prognosis ,22,23,47To our knowledge, no previous research has been conducted to detect HPV E7 oncoprotein in HNSCC patient serum, while the availability of ELISA kits for oncoprotein detection could rapidly facilitate such study outcomes into clinical use. E7 oncoprotein in serum was specifically found with a frequency of 30% in HPV-positive samples, while the detection of circulating protein at the time of diagnosis strongly correlated to recurrence. Our data are in accordance to other studies showing higher levels of antibodies against HPV oncoproteins at the time of diagnosis in association to a significantly increased risk of recurrence ,49. SimiIt is to be noted that, circulating E7 protein showed correlation with high E7 mRNA expression in the tumor, suggesting that tumor sites may provide the circulating oncoprotein. Circulating E7 protein may be considered a tumorigenesis marker, representing at serological level what occurs at the tumor site. Sources of viral oncoproteins in serum have currently not been established, but some hypotheses could be proposed. Firstly, the transcriptionally active circulating tumor cells may account for the presence of serum viral oncoproteins . Indeed,Moreover, E7 oncoprotein also correlated to tumor size, but not lymph node involvement or disease stage. Overall, 41.66% HNSCC patients with high disease stage III/IV presented E7 oncoprotein in serum, while none of those with low stage I/II did so, in agreement with previous serologic studies ,55, makiThis study demonstrates for the first time, the presence of circulating E7 oncoproteins in serum from HNSCC patients using a direct ELISA assay. Our results indicate that the presence of E7 oncoprotein in OPSCC patient serum at the time of diagnosis is indicative of a higher risk of recurrence. Liquid biopsy for the detection of prognostic markers in HPV-positive OPSCC patients provides valuable information on disease progression and may help stratify and monitor patients over-time; this, can result extremely useful for patients presenting persistent or occult tumors. For future studies, in order to increase the statistical power of the study, a larger sample size for all HNSCC subtypes will be considered. This study takes medicine one step closer to correct patient stratification for therapy de-intensification. The combination of classical markers with serological markers, may be used to plan personalized treatment strategies for HPV-positive patients.Detection of circulating HPV E7 oncoprotein at the time of diagnosis, may be used as non-invasive procedure for patient stratification and follow-up, ultimately providing a tool for clinicians to determine which patients would be good candidates for treatment de-escalation or should be kept under close surveillance.


Complementary and integrative health (CIH) approaches are recommended in national guidelines as viable options for managing chronic pain and de-prescribing opioids. We followed 1,993,455 Veterans with musculoskeletal disorders for two years who were not using opioids at study entry. CIH exposure was ascertained from primary care visits for acupuncture, massage and chiropractic care via natural language processing and structured data. Opioid prescriptions during the 2-year follow-up were abstracted from Veterans Health Administration (VHA) electronic pharmacy records. Propensity score (PS) was used to match CIH recipients with non-recipients with most comparable baseline characteristics. Overall, 140,902 (7.1%) Veterans received CIH, with a prevalence of 2.7% for Veterans aged ≥ 65y, comparing to 6.3% and 10.5% for those aged 50-64y and ≤ 49y, respectively. Among the 1:1 PS-matched sub-cohort , Cox proportional hazard model revealed that time to fill first opioid prescriptions was significantly longer for CIH recipients (mean: 587 days) than non-recipients (mean: 491 days), with adjusted Hazard Ratio of 0.48 : 0.45-0.51) for Veterans ≥ 65y, 0.44 (95% CI: (95% CI: 0.43-0.45) for 50-64y and 0.47 (95% CI: 0.46-0.48) for age ≤ 49y group . Sensitivity analyses among full cohort or modeling total supply of first opioid prescriptions derived consistent results. These findings suggest potential benefit of CIH use in delaying and reducing opioids prescriptions for patients with chronic pain and may have implication for older Veterans ≥ 65y who have been found less likely to seek CIH therapies than their younger counterparts.


To evaluate the clinical performance and utility for risk stratification of DH3 HPV assay in women (≥30 years) with NILM cytology.rd year of follow up, all women that were not biopsy proven CIN2+ would be called back and screened by cytology again. In follow-up period, women with ASC-US and above were referred to colposcopy and biopsied if clinically indicated. CIN2+ was the primary endpoint in analysis. The clinical performance and utility for risk stratification of DH3 HPV assay were assessed by SPSS 22.0 and SAS 9.4.A prospective cohort was established in Central China between November 8 to December 14, 2016 which consisted of 2180 women aging 30-64 years with NILM cytology. At baseline, all women were screened using DH3 HPV assay. HPV 16/18 positive women would be assigned to colposcopy and biopsied if necessary. Then, hr-HPV positive women without CIN2+ lesions would be followed up by cytology every 12 months for two years. In the 3Of 2180 qualified women, the prevalence of hr-HPV was 8.5% (185/2180), 45(2.1%) were HPV 16/18 positive. The clinical performance for HPV16/18 was 91.7% for sensitivity, 98.4% for specificity, respectively against CIN2+ detection at baseline. In four years of study, the corresponding rates of HPV 16/18 were 51.5% and 98.7%, respectively. The cumulative absolute risk for the development of CIN2+ was as high as 37.8% for HPV 16/18 positive women, followed by hr-HPV positive (14.6%), other hr-HPV positive (11.0%) and HPV negative (0.3%) in three years. The relative risk was 125.6 and 3.4 for HPV 16/18 positive group when compared with HPV negative and other hr-HPV positive group, respectively.DH3 HPV assay demonstrated excellent clinical performance against CIN2+ detection in cervical cancer screening and utility of risk stratification by genotyping to promote scientific management of women with NILM cytology. Persistent high-risk human papillomavirus (HPV) infection is the necessary cause for the development of cervical intraepithelial neoplasia (CIN) and cervical cancer , 2. ScreIn general, the strategies used in screening scenario are mainly based on the principle of equal management for equal risk. Although there are no universal thresholds for current strategies, the 3-year and 5-year risk for CIN3+ development which was 4.3% and 5.2%, respectively in related to low-grade squamous intraepithelial lesion (LSIL) cytology, have been identified as the benchmark for referral . Other sIn the light of elimination cervical cancer globally , 12, HPVDH3 HPV assay is a newly developed RNA-DNA hybrid capture-based technique that detects the presence of DNA genome of 14 hr-HPV types with HPV 16/18 genotyping at the same time, which indicated that DH3 HPV assay can be performed in a general laboratory without nucleic acid amplification. Notably, DH3 HPV assay has been reported similarly performance in compared with Cobas4800 HPV in identifying cervical cancer and precursor in cervical cancer screening  for DH3 HPV assay detection after written informed consent obtained. HPV 16/18 positive women would be referred to colposcopy and biopsied if necessary. After then, all hr-HPV positive women would be rescreened by cytology every 12 months if CIN2+ was not reached in next two years. And women with ASC-US and above would undergo colposcopy and biopsied if clinically indicated. In the 3Actually, current study overlapped part of population with another research assessed the clinical performance of DH1 in cervical cancer screening. Therefore, it is possible that CIN2+ cases could be identified by DH1 HPV assay rather than DH3 HPV assay. For such cases, they would be regarded as missing case in analysis.In current study, cytological and HPV samples were collected for screening during baseline and follow up period.In current study, all cytological samples were evaluated according to the TBS (The Bethesda System) system. Each satisfactory cytological sample would be diagnosed by cytologist as following results: negative for intraepithelial lesion or malignancy (NILM), atypical squamous cells undetermined significance (ASC-US), atypical squamous cells‐cannot exclude HSIL (ASC-H), low squamous intraepithelial lesion (LSIL), high squamous intraepithelial lesion (HSIL), squamous of cervical carcinoma (SCC), atypical glandular cells(AGC). Women with ASC-US and above were determined as abnormal cytological results and would be further examinations.All HPV samples would be detected by DH3 HPV assay, a novel domestic high-risk HPV testing based on hybrid capture. Firstly, the denatured samples were mixed with probe A (probe cocktail containing RNA probes for HPV16 and 18) and probe B  in two microplates. The probes targeted the entire HPV genome. The resultant RNA-DNA hybrids were captured by specific antibodies, immobilized on a microplate, and further detected by alkaline phosphatase-conjugated antibodies specific for the RNA-DNA hybrids. As the substrate is cleaved by alkaline phosphatase, light is emitted and measured as relative light unit (RLU) using an illuminometer. The intensity of the emitted light indicates the viral load of the target HPV in the sample. The cut-off value was 1.0 pg/ml for DH3 HPV assay as the threshold indicating HPV-positivity in according to the guidelines of World Health Organization (WHO). In current study, all HPV detection was strictly conducted in accordance with the standard operation protocol.2 tests. Continuous variables were described as mean (SD) or median (IQR). The clinical performance of DH3 HPV assay would be assessed using sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), respectively, based on the HPV result at baseline. The cumulative absolute risk and relative risk for the development of cervical cancer and precursor in three years were also evaluated for different HPV results. And the corresponding 95% confidence interval (95%CI) would also be calculated for clinical performance and risk evaluation. In current study, CIN2+ was used as the clinical endpoint. Statistical significance of all two-tailed tests was set at P ≤ 0.05. The SPSS 22.0 and SAS 9.4. were used for the statistical analysis.In current research, categorical variables were showed as proportions and compared using χIn current study, 2444 enrolled women were invited but 37 were excluded because of hysterectomy, cervix surgery, or cervical cancer treatment, and 227 were dropped in follow up period. Therefore, 2180 were qualified for analysis, and the mean age was 47.3 ± 7.5 years.rd year of follow up were HPV negative at baseline , at the same time, 155 were positive (7.1%) for any other hr-HPV type. At baseline, 5 CIN2 cases and 7 CIN3+ cases were identified. In follow-up period, 12 CIN2 cases and 9 CIN3+ cases were found, respectively, of which 6 CIN2+ cases in the 3baseline Table 1.At baseline, the sensitivity and specificity of DH3 were 100% and 92.0% against CIN2+ detection for hr-HPV positive. In contrast, DH3 showed a sensitivity of 91.7% and a specificity of 98.4% for HPV 16/18 positive. If using CIN3+ as clinical endpoint, they were 100%, 91.8%, and 85.7%, 98.2%, respectively. Considering all cases identified, the overall sensitivity and specificity achieved 81.8% and 92.6% for hr-HPV positive against CIN2+ detection. Similarly, they were 51.5% and 98.7% for HPV 16/18 positive. For CIN3+ detection, the corresponding rates were 81.3%, 92.0%, 50.0% and 98.3%, respectively Table 2.The performance of DH3 for risk prediction was showed in Current study focused on women 30 years or older with NILM cytology. The main findings demonstrated even for NILM women, their absolute and relative risks to develop CIN2+ and CIN3+ were associated closely with hr-HPV infection status, especially whether infected HPV16/18. Therefore, HPV16/18 genotyping could stratify the risk of cervical cancer and precursor, and distinguish high risk population effectively in women with NILM cytology, indicating that the novel hybrid capture HPV test, DH3 HPV assay, had great potential strength in cervical cancer screening, especially in resources limited areas.Here, the prevalence of overall HPV (8.5%) was similar to those findings reported or other FDA-approved HPV assays in women with NILM cytology, including the Onclarity HPV assay  and LA-HThe clinical performance of any hr-HPV and HPV 16/18 was good at baseline because of study design. All CIN2+ cases were hr-HPV positive, but only one CIN3 case was other hr-HPV positive at baseline. Therefore, HPV 16/18 genotyping had a sensitivity of 91.7% for CIN2+ detection, 85.7% for CIN3+ detection. The corresponded specificity was 98.4% and 98.2% respectively. Meanwhile, the overall sensitivity decreased to around 80% for hr-HPV, 50% for HPV 16/18. For specificity, it is about 92% and 98%, correspondingly, if considering all cases found during baseline and follow and period. The performance of DH3 in current research for the detection of CIN2+ and CIN3+ is similarly to those findings of an established, clinically validated HPV assay (HC2) which had a sensitivity of 98.7%, and a specificity of 94.1% for CIN2+ detection . HoweverFor the risk estimation, Uijterwaal MH and colleagues made a five-year risk assessment for the development of CIN2+ or CIN3+ in 2015 . In theiAs other studies found, the risk of women with HPV negative and NILM cytology was very low, in current research, they were 0.3% and 0.2% in three years, indicating that the DH3 HPV assay can effectively predict elevated risk for both CIN2+ and CIN3+ in women at the age of 30 years or older with NILM cytology who were considered to take an extremely low risk before. Finally, these results show that the differential stratification of risk through the detection of individual genotypes (HPV16/18) has the potential to affect patient care pathways in this population. Furthermore, the findings described here are similarly to the established risk thresholds for non–HPV16/18 .Prior cross-sectional and prospective studies have clearly demonstrated that hr-HPV status was an important predictor of the current and future risk for the occurrence of CIN2+ in women with NILM cytology . To makePresently in US clinical practice, acceptable actions following an hr-HPV positive result in the NILM population during screening include either repeat co-testing after 1 year or concurrent HPV genotyping. For the latter step, HPV 16/18 positive women would be directed to colposcopy, whereas HPV 16/18 negative women would undergo repeat co-testing in 1 year. However, it is often underappreciated that HPV positive women with NILM cytology fall below the risk threshold for colposcopy . Current US guidelines regarding co-testing for women ≥30 years of age suggest only HPV 16/18 positive women with NILM be referred based on the risk of cervical cancer and precursor , 26, 27.DH3 HPV assay is a newly developed HPV test based on the hybrid capture technology. Similar to HC2, DH3 HPV assay could detect the presence of 14 hr-HPV by magnifying and detecting the chemical signal without nucleic acid amplification, which make it possible for the assay to be performed in a general laboratory that is more practicable in resource limited areas . But unlIn summary, the performance and risk detection obtained using DH3 HPV assay supports established screening algorithms within the current cervical cancer screening guidelines for co-testing and demonstrates its utility for the triage of women with NILM cytology in cervical cancer screening.The raw data supporting the conclusions of this article will be made available by the authors, upon reasonable request.The studies involving human participants were reviewed and approved by The Institutional Review Board of Affiliated Cancer Hospital of Zhengzhou University. The patients/participants provided their written informed consent to participate in this study.H-FX drafted the manuscript. YL completed the statistical analyses. Y-LL and D-MZ were responsible for the gynecological examination and diagnosis. P-PC, X-A, and M-JL collected the samples and information. M-MJ managed the database. X-BS and S-KZ were supervised the study. S-KZ revised the manuscript substantially. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The combination of human papillomavirus (HPV) vaccination and cervical cancer tests are globally recommended. Although knowledge regarding cervical cancer and HPV and experience of HPV vaccination are reportedly closely associated, the associations between knowledge and frequency of cervical cancer testing are unclear. We conducted a questionnaire survey regarding the knowledge of cervical cancer and HPV and experience of HPV vaccination and frequency of cervical cancer testing including cervical cytology and HPV testing. Among 99 women who visited Tsuruha Festa, most of the 77 non-medical workers who received information on cervical cancer and HPV through the Internet were not more likely to have knowledge about cervical cancer and HPV than were 12 medical workers who had gotten information in vocational school or university curriculum. The rates of HPV vaccination, cervical cytology, and HPV testing were 4.0%, 14.1%, and 4.0%, respectively, among participants and did not differ significantly according to participant job and age. Knowledge about cervical cancer and HPV was associated with experience of HPV vaccination and frequency of cervical cytology and was not associated with frequency of HPV testing. We observed insufficient knowledge about cervical cancer and HPV among non-medical workers as well as low HPV vaccination, cervical cytology, and HPV testing rates, and knowledge about cervical cancer and HPV to which frequency of cervical cancer testing were partially related. Therefore, the government should take measures to enhance public awareness about cervical cancer and HPV through social media such as the Internet. Cervical cancer was the fourth most common cancer in women with an estimated 570,000 new cases in 2018, representing 6.6% of all cancer in women and 311,000 deaths . In Japan, cervical cancer affects about 10,000 and kills about 2,800 women annually. The numbers of both patients and deaths have been gradually increasing since the 1990s . In particular, the incidence is higher in the younger generation (aged between 20 and 40 years), compared to those of other age groups. Long-lasting human papillomavirus (HPV) infection is a main factor contributing to the development of cervical cancer. The HPV vaccine can prevent infection with HPV types 16 and 18, two high-risk HPVs that cause about 70% of cervical cancers. In Japan, funding for HPV vaccination began in 2010 for girls aged 12–16 years, with three-dose coverage initially reaching more than 70%. In June 2013, 2 months after its formal inclusion in the Japanese national immunization program, proactive recommendations for HPV vaccination were suspended. HPV vaccine coverage subsequently dropped to less than 1% and has remained at this level . The proactive recommendation of HPV vaccination remains controversial.Worldwide, the screening modalities for cervical cancer include cervical cytology (Pap test), visual inspection, and HPV deoxyribonucleic acid (DNA) testing (HPV test). The HPV test can be used in combination with cervical cytology to screen for cervical cancer and it is firstly recommended in primary cervical cancer screening . In Japan, cervical cytology is performed for primary screening for cervical cancer . When used with high-risk HPV polymerase chain reaction assays, testing on self-samples was similarly accurate as that for clinician samples . Therefore, HPV tests can be performed with both clinician and self-sampling.Questionnaire surveys on the knowledge of cervical cancer and HPV vaccination coverage have increased in recent years. A meta-analysis of 58 studies, as well as systematic reviews, nationwide interview surveys, and population-based surveys on cervical cancer have been reported . In Japan, only one study has assessed knowledge regarding cervical cancer and HPV and willingness to receive HPV vaccination . The study reported on willingness to receive HPV vaccination but did not describe the actual experience of HPV vaccination, cervical cytology, and HPV testing. The World Health Organization Europe indicates that the combination of HPV vaccination and regular screening is the most effective way for women to protect against cervical cancer . Therefore, we aimed to investigate the association between knowledge about cervical cancer and HPV and experience of HPV vaccination and frequency of cervical cancer testing including cervical cytology and HPV tests.We provided self-collected HPV tests and also conducted a questionnaire survey regarding cervical cancer and HPV on May 18 and 19 and August 24 and 25, 2019, at Tsuruha Festa as part of 2-day general public events related to health and beauty provided by Tsuruha Pharmacies Co., Ltd., a drugstore chain company in Japan. We asked women aged 20–39 years visiting these events to participate in this survey. A total of 99 women participated in the survey. We asked participants to answer the questionnaire and collected them immediately after completion. The questionnaire comprised items relating to the participants’ present knowledge of cervical cancer and HPV as well as their experiences with HPV vaccination and cervical cancer tests including cell cytology and HPV tests (Appendix). After completing the questionnaires, we provided the correct answers and relevant knowledge about HPV and the vaccine. Ethical approval for the study was obtained from the Institutional Review Board of Tsuruha Pharmacies Co., Ltd.Women who responded “I understand” to the question regarding their understanding of cervical cancer and HPV were classified into the “yes” group, while those who answered “I could not understand, but I have heard” or “I did not know” were classified into the “no” group. Women in the “yes” group were considered to have knowledge about the topic of each question on cervical cancer and HPV. We compared the proportions of women with such knowledge according to their job  and age (20–29 years or 30–39 years). Among women with knowledge about cervical cancer and HPV, we also compared the sources of information between medical and non-medical workers. We also compared experience of HPV vaccination and frequency of cervical cancer testing including cervical cytology and HPV testing according to their jobs, ages, and knowledge about cervical cancer and HPV. Fishers’ exact and Cochran-Armitage trend tests were conducted, as appropriate. All statistical analyses were conducted using SAS version 9.4 .Among 99 women, 75 were non-medical workers and 27 were aged 20–29 years. Knowledge about cervical cancer and HPV according to job and age are shown in Experience of HPV vaccination according to participants’ job, age, and knowledge are shown in The frequency of cervical cytology according to participants’ job, age, and knowledge are shown in The frequency of HPV testing according to participants’ job, age, and knowledge are shown in We found that non-medical workers did not have sufficient perceptions about cervical cancer and HPV, that social systems including the government’s financial support for cervical cancer testing were not sufficiently recognized even among medical workers, and that knowledge related to cervical cancer and HPV was associated with experience of HPV vaccination and frequency of cervical cancer testing.Medical workers received information on cervical cancer and HPV as part of their school curriculum, while non-medical workers received such information from the Internet, TV, and other media. The media may be an important tool to provide non-medical workers knowledge of cervical cancer and HPV. Other countries have used the Internet and social media to disseminate health-related information . Therefore, the Japanese government should consider social media in their communication strategies to promote appropriate Web use for the dissemination of medical information on cervical cancer itself to non-medical workers and social systems including the government’s financial support for cervical cancer testing to both non-medical and medical workers.In our study, participants aged 20–29 years with knowledge related to cervical cancer, HPV, and HPV vaccine were significantly more likely to receive HPV vaccination than those without this knowledge. Although the number of participants in the present study was small and the conclusions correspondingly limited, the proactive recommendation for HPV vaccination as was performed until 2013 in Japan may have a great impact on rate of HPV vaccination among participants aged 20–29 years. High levels of knowledge about HPV and the vaccine are related to a greater willingness to receive the vaccine. Studies among undergraduate students reported that the level of knowledge about HPV and HPV vaccine and health education were important predictors of increased vaccination rates  and the main reasons for rejecting the HPV vaccination were insufficient information about the vaccine and possible unknown side effects . A previous study reported that, after educational instruction consisting of a 1-hour group lecture followed by the same questionnaire as that provided after informed consent, vaccine acceptability increased significantly from 77% to 90% among employed women . Therefore, informative group lectures may be effective for the improvement of HPV-related knowledge and HPV vaccine acceptability. However, as the HPV vaccination rate was low even among participants in our study with knowledge about HPV vaccination, the provision of information and education including not only the need for HPV vaccination but also correct knowledge about adverse reactions after vaccination is necessary.In our study, the rates of experience with cervical cytology once every 2 years was similar to the rate of cervical cancer testing within 1 year among women aged 20–49 years in Japan (<30%) . Participants aged 30–39 years with knowledge related to cervical cancer, HPV, and HPV vaccine were significantly more likely to have experienced cervical cytology and no differences were observed in the rates of experience with cervical cytology between participants regardless of their knowledge about cervical cytology. In Japan, cervical cancer screening programs with cervical cytology are conducted once every 2 years and are actively recommended for people over 20 years of age. Therefore, participants with knowledge related to cervical cancer might be willing to receive the screening, resulting in their having experienced cervical cytology. This program might also explain the rate of experience with cervical cytology once every 2 years or irregularly over 60%, regardless of the level of knowledge about cervical cytology in this study. In contrast, the rate of experience with HPV testing was associated with knowledge about HPV testing. One explanation may be that it is necessary to actively acquire to receive an HPV test in Japan . Respondents to this questionnaire survey were participants in self-sampling HPV tests at Tsuruha Festa and, while many had knowledge of HPV testing, the implementation rate was low. The general population may have less knowledge and fewer experiences with HPV testing compared to those in our study participants. Thus, increasing awareness of HPV testing and creating an environment that facilitates HPV testing are required. In Conclusion, the results of this study demonstrated an insufficient knowledge of cervical cancer and HPV including HPV vaccinations and cervical cancer tests in non-medical workers. The HPV vaccination, cervical cytology, and HPV testing rates were very low, and were partially associated with the rates of receiving HPV vaccinations and cervical cancer tests. Our findings suggested that knowledge of cervical cancer and HPV and national programs such as active recommendations to receive HPV vaccination and cervical cancer tests would affect the health behaviors related to cervical cancer, HPV, HPV vaccine, and cervical cancer testing.


Background: To examine completion of the human papillomavirus (HPV) vaccination 3-dose regimen and factors associated with completion among men who have sex with men (MSM) aged ≤ 26 years participating in a time-limited HPV catch-up vaccination program in Victoria, Australia.Methods: MSM who received their first dose of HPV vaccine at Melbourne Sexual Health Centre in 2017 were followed until October 2019. Vaccination completion was defined as those who received three doses. Multivariable logistic regression was performed to examine factors associated with vaccine completion.Results: 931 of 1,947 (47.8%) eligible men received at least one dose of HPV vaccine, 750 (38.5%) received two and 590 (30.3%) received three doses. The median time to receiving the second and third dose was 2.8 (IQR = 2.1–4.8) and 7.2 (IQR = 6.3–10.7) months, respectively. Gay men had higher odds of receiving three doses compared to bisexual men . Compared with HIV-negative MSM not taking PrEP, HIV-positive MSM were more like to complete vaccination  but no difference was found compared to HIV-negative men taking PrEP .Conclusion: Less than one-third of men aged ≤ 26 years completed the three doses of HPV vaccine. Further studies are needed to understand the barriers of men not completing the vaccine. Human papillomavirus (HPV) is one of the most common sexually transmitted viral infections and is associated with genital warts, and cervical, penile and anal cancers. Multiple countries have introduced HPV vaccination programs for girls and women since 2006. A few countries have also introduced gender-neutral programs to include boys –3. Gay, Australia has had a school-based vaccination program for girls aged 12–13 years since 2007 and this program was expanded to include boys aged 12–13 years in 2013. Between April 2017 and October 2019, the Victorian Government funded and implemented a targeted catch-up 3-dose quadrivalent HPV vaccination program targeting MSM aged ≤ 26 years through sexual health and primary care clinics . The recThis was a retrospective cohort study of MSM who received the first dose of HPV vaccine at the MSHC in 2017 (between April and December 2017). MSHC is the largest public HIV/STI clinic in Victoria where men can access HIV/STI services free of charge. HPV vaccines were provided to all MSM aged 16–26 years at MSHC for free between April 2017 and October 2019 during the time-limited HPV vaccination program funded by the Victoria Government.There were 1,947 MSM aged 16–26 years who attended MSHC between April and December 2017 and reported that they did not receive any HPV vaccines from school. These men were eligible for the catch-up program, 830 men (43%) received the first dose of the vaccine at their first consultation during 2017, and the results are described elsewhere  and the corresponding 95% CIs were reported. All data analyses were conducted in Stata (version 17). This study was approved by the Alfred Hospital Ethics Committee, Melbourne, Australia (706/19).We defined vaccination completion as individuals who received three doses of HPV vaccine given at MSHC. We reported the proportion of men who received one, two and three doses of vaccine. The 95% confidence intervals (CI) for the proportion was calculated using the binomial exact method. We calculated the median time from the first to second, and from the first to third doses and presented in months. Demographic characteristics such as age, country of origin, indigenous status, HIV status, use of pre-exposure prophylaxis (PrEP), condoms use and the number of partners in the past 12 months were also extracted from the patient's electronic medical records. Univariable and multivariable logistic regression was performed to examine factors associated with HPV vaccine completion among men who had received at least one dose of vaccine. Variables with n = 1,843, 94.7%) and were born in Australia . A small proportion of men reported having sex with both men and women . There were 1,735 (89.1%) HIV-negative men not taking PrEP, followed by 144 (7.4%) HIV-negative men taking PrEP, and 68 (3.5%) men living with HIV. Of the 1,947 men, 931 (47.8%) received at least one dose of HPV vaccine in 2017 including 590 (30.3%) men who received three doses, 160 men (8.2%) received two doses only and 181 men (9.3%) received one dose only .Only 30% of MSM aged ≤ 26 years received three doses of HPV vaccine via the time-limited catch-up HPV vaccination program for MSM through a sexual health clinic, this is significantly lower than the completion rate of boys who received their HPV vaccines from the school-based program  . The UK There are several factors found to be associated with HPV vaccine completion. First, we found that HIV-positive men are more likely to receive the full course of HPV vaccination compared to HIV-negative men. HIV-positive men are at a higher risk of acquiring anal cancer , and theA few limitations in this study should be noted. This study was conducted at a single urban sexual health clinic in Melbourne, and our findings may not be generalisable to other settings, particularly settings without a free HPV vaccination program for MSM. Additionally, low HPV vaccine completion rate in our study could be due to vaccination was administered opportunistically when men present with STI symptoms or STI testing compared to self-initiated vaccination at general practice clinic. Furthermore, our estimate on vaccine completion may be underestimated as men might have received the HPV vaccine via their general practices or immunisation providers outside MSHC; however, we were unable to justify this. Second, data on sexual practices such as the number of partners and condomless sex are self-reported; and thus, recall bias may have occurred.Although the HPV vaccine completion rate among MSM aged 16–26 years via the time-limited catch-up HPV vaccination program was only 30%, it has been suggested that it may be cost-effective in reducing the burden of HPV diseases in modelling studies . A previeric.chow@monash.edu.The datasets presented in this article are not readily available because data cannot be made publicly available in order to protect patient privacy as per the approved ethics requriment. Requests to access the datasets should be directed to Eric P.F. Chow, The studies involving human participants were reviewed and approved by Alfred Hospital Ethics Committee. Written informed consent from the participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.EC conceived and designed the study. KH and RW performed chart review to verify the vaccination records. KH wrote the first draft of the manuscript. KH and ER performed literature review. KH and EC performed statistical analyses. All authors provided data interpretation, were involved in revising the manuscript for important intellectual content and approved the final version.CF and CB are supported by an Australian National Health and Medical Research Council (NHMRC) Leadership Investigator Grants (GNT1172900 and GNT1173361). EC is supported by an NHMRC Emerging Leadership Investigator Grant (GNT1172873).EC, MC, and CF have received research funding from Merck Sharp & Dohme, outside the submitted work. EC has received an honorarium from Merck Sharp & Dohme, and Roche, outside the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


MUTYH and OGG1. The high frequency of C > A substitutions in neuroblastoma contributes to the increased adaptive capacity of these tumors. Thereby, we link basic molecular genetic mutation patterns to clinically significant tumor evolution processes.The collection of large amounts of whole-genome sequencing data allowed for identification of mutational signatures, which are characteristic combinations of substitutions in the context of neighboring bases. The clinical significance of these mutational signatures is still largely unknown. In neuroblastoma, we showed that high levels of cytosine > adenine (C > A) substitutions are associated with poor survival. We identified that these high levels of C > A substitutions result from defects in 8-oxo-guanine repair, specifically from copy number loss of the DNA glycosylases  OGG1 or MUTYH. Both genes encode DNA glycosylases that recognize 8-oxo-guanine (8-oxoG) lesions as a first step of 8-oxoG repair. Tumor organoid models with CNL of OGG1 or MUTYH show increased 8-oxoG levels compared to wild-type cells. We used CRISPR-Cas9 genome editing to create knockout clones of MUTYH and OGG1 in neuroblastoma cells. Whole-genome sequencing of single-cell OGG1 and MUTYH knockout clones identified an increased accumulation of C > A substitutions. Mutational signature analysis of these OGG1 and MUTYH knockout clones revealed enrichment for C > A signatures 18 and 36, respectively. Clustering analysis showed that the knockout clones group together with tumors containing OGG1 or MUTYH CNL. In conclusion, we demonstrate that defects in 8-oxoG repair cause accumulation of C > A substitutions in neuroblastoma, which contributes to mutagenesis and tumor evolution.Neuroblastomas are childhood tumors with frequent fatal relapses after induction treatment, which is related to tumor evolution with additional genomic events. Our whole-genome sequencing data analysis revealed a high frequency of somatic cytosine > adenine (C > A) substitutions in primary neuroblastoma tumors, which was associated with poor survival. We showed that increased levels of C > A substitutions correlate with copy number loss (CNL) of  Neuroblastoma is a childhood tumor of the peripheral sympathetic nervous system that accounts for 15% of childhood cancer mortality . Like otOGG1 and MUTYH when it forms a base pair with a C or A, respectively. 8-oxoG repair is initiated by excision of 8-oxoG by OGG1 or excision of the wrongly inserted A opposite 8-oxoG by MUTYH  of SI Appendix, Fig. S1 A and B).We determined the somatic frequency of each of the six possible base substitutions in our whole-genome sequencing (WGS) data of 86 primary neuroblastoma tumor/normal pairs . C > A sSI Appendix, Fig. S1C). In addition, C > A ratios were not enriched in the control DNA (leukocytes) . Finally, similar levels of C > A substitutions were found in tumors and their corresponding organoid cell lines . Therefore, we conclude that the C > A substitutions found in our tumor cohort were authentic.Since C > A substitutions have been suggested to also occur from sequencing artifacts , we reant test with Welch correction, two-sided, P < 0.0001; P = 2.8 × 10−10) difference in overall survival between patients with a high and a low percentage of C > A substitutions . These results show that the frequency of C > A substitutions is strongly increased in neuroblastoma tumors and that this correlates with a poor prognosis.We next compared the C > A substitution frequencies for the different tumor stages and identified significantly more C > A substitutions in International Neuroblastoma Staging System (INSS) stage 4 tumors compared to the low-stage tumors . OGG1 is located on 3p.25, which is part of the frequently deleted 3p region in neuroblastoma. MUTYH, which is located on 1p.34, was deleted in a subset of tumors with  1p CNL. NUDT1 CNL was identified in two tumors, which also had a MUTYH CNL . CNL of OGG1 and MUTYH was associated with a high frequency of C > A substitutions  or OGG1  and a CNL of the other MUTYH allele . This missense variant in N170T is not reported in the Single Nucleotide Polymorphism Database (dbSNP) (build 153). In addition, this mutation results in an amino acid change from an alanine to a proline, which may affect the protein conformation, although PolyPhen and SIFT do not predict the variant to be damaging. Another tumor (N701T) with a very high frequency (78%) of C > A substitutions carried a germline missense variant in OGG1 (NP_002533.1:p.Gly308Glu) and a CNL of the other OGG1 allele . For this missense variant, it is predicted that it affects function. Overall, five of the seven tumors with the highest C > A substitution frequencies have multiple alterations in genes of the 8-oxoG repair pathway . These results show that C > A substitution frequencies are increased in neuroblastoma tumors with OGG1 or MUTYH CNL.C > A substitutions can result from defects in the 8-oxoG repair pathway . Therefoitutions A. ComparSI Appendix, Fig. S1D). Compared to a wild-type organoid, two organoids with a defect in the 8-oxoG repair pathway show significantly longer comet tails , indicative of higher levels of 8-oxoG . To show that the increased levels of 8-oxoG are indeed caused by the defects in OGG1 and MUTYH, we performed rescue experiments by inducing overexpression of wild-type OGG1 or MUTYH in neuroblastoma cell lines with CNL of OGG1 or MUTYH, respectively. Indeed, we could rescue high levels of 8-oxoG by overexpression (72 h) of wild-type OGG1 or MUTYH, as indicated by a significant decrease in comet tail size . In a neuroblastoma tumor (N701T) with one of the highest C > A mutation frequencies, a germline OGG1 missense variant was identified, as mentioned above. To test whether this mutation in OGG1 is indeed inactivating, we also induced overexpression of OGG1 containing the missense variant (p.G308E) in the neuroblastoma cell line with OGG1 CNL. Since overexpression of this variant increases the tail moment, this shows that this variant does not rescue the levels of 8-oxoG , confirming that this is an inactivating event in OGG1. In addition, we also tested the effect of a germline MUTYH missense variant identified in the tumor with the highest C > A substitution frequency (N170T). Induced overexpression of MUTYH containing this missense variant (p.A425P) in the neuroblastoma cell line with MUTYH CNL, resulted in a lower tail moment compared to the uninduced control . Nevertheless, the tail moment remains higher than for induced overexpression without recombinant OGG1. This indicates that this variant does not rescue the levels of 8-oxoG to a similar extent as wild-type MUTYH and suggests that this variant reduces MUTYH activity. Overall, these results indicate that 8-oxoG levels are increased in neuroblastoma organoids and cell lines with defects in MUTYH and OGG1 and that this can be rescued by overexpression of the affected wild-type gene.To study the relation between 8-oxoG levels and defects in the 8-oxoG repair pathway, we used modified alkaline comet assays . In thisMUTYH, OGG1, or NUDT1 in a neuroblastoma cell line (CHP134). To create these knockout clones, we inserted a puromycin resistance cassette in one of the first exons of these genes to disturb the open reading frame . The selected clones also showed absent expression of the targeted protein . Selected knockout and wild-type clones were passaged for a period between 88 and 126 d to accumulate independent mutations and subsequently single-cell FACS sorted to establish subclones. From both clones and subclones (n = 2 per gene), genomic DNA was isolated and subjected to WGS to study the mutational patterns that accumulate between the two clonal steps . MUTYH and OGG1 knockout clones showed a relative higher contribution of C > A substitutions ranging from 43 to 57% compared to 25 to 30% in control clones . This difference was statistically significant for the MUTYH knockout clones versus the wild-type clones  and the MUTYH knockout clones and OGG1 knockout clones versus the wild-type clones . Also, the total number of base substitutions per genome per day was increased for MUTYH and OGG1 knockout clones compared to the wild-type clones . The substitution frequencies in the NUDT1 knockout clones were similar to the wild-type clones . These results indicate that knockout of OGG1 and MUTYH, but not NUDT1, increases the accumulation of C > A substitutions in individual neuroblastoma cells.To validate the correlation between C > A substitutions and defects in the 8-oxoG pathway, we created CRISPR-Cas9 knockout clones of ng frame A 21). A. AMUTYH,ng frame B. Knocko protein C. Messene clones A. No conMUTYH and OGG1 knockouts are slightly different but cluster close together and separately from the wild-type and NUDT1 knockouts . The mutational spectra were compared using the COSMIC SigProfiler signatures .To further study the mutational patterns of the different knockouts, mutational spectra were analyzed and the relative contributions of mutation signatures were determined. The mutational spectra of the wild-type and knockout clones were visualized by showing the relative contribution of the six possible substitutions in their trinucleotide context B. Cosinegnatures , 22. TheMUTYH knockout clones  with the OGG1, MUTYH, and NUDT1 knockout CHP134 clones. No differential sensitivities compared to the CHP134 mother line were identified . These results indicate that defects in OGG1, MUTYH, and NUDT1 do not render CHP134 cells more sensitive to chemotherapeutics and targeted compounds. The high frequency of C > A substitutions in neuroblastoma might contribute to the adaptive capacity of the tumors. We evaluated a previously published dataset of primary and relapse neuroblastoma tumors   is very rare and, in our cohort, only identified in combination with MUTYH CNL. Strikingly, five of the seven tumors with the highest C > A substitution frequencies have multiple alterations in genes of the 8-oxoG repair pathway. This suggests that multiple alterations in this pathway result in a cumulative effect on the C > A substitution frequency. Other neuroblastoma tumors indicate that partial loss of OGG1 or MUTYH can also be sufficient to induce high C > A substitution levels.CNL of blastoma , 24. On MUTYH and OGG1 knockout clones showed increased accumulation of C > A substitutions. Most neuroblastoma tumors show heterozygous loss instead of homozygous loss of OGG1 or MUTYH, which we expect to result in a similar but more modest phenotype. The NUDT1 knockout clones did not show an increase in C > A substitutions compared to the wild-type clones. NUDT1 prevents the incorporation of 8-oxoG into the DNA. Although 8-oxoG can base pair with both a C and an A, the insertion opposite of an A is expected to be most mutagenic since this will result in A > C (T > G) substitutions, while insertion opposite of a C can be repaired by OGG1. Nevertheless, the mutational spectrum of the NUDT1 knockouts is similar to the wild-type clones, and no increase in T > G substitutions has been detected. This substantiates previous results of NUDT1 knockout in HAP1 cells, which also did not produce detectable mutational signatures compared to the parental clone . These results confirm the correlation between signatures 18 and 36 and defects in the 8-oxoG repair pathway. In concordance, a Nudt1/Ogg1/Mutyh triple knockout mouse model accumulates C > A substitutions with a mutational pattern that is highly similar to signature 18 or 36 -2A-GFP] combined with the corresponding homology arm vector (in pJET1.2/blunt vector) to disrupt the open reading frame with a puromycin resistance cassette (https://www.synapse.org/#!Synapse:syn11967914) were refitted to the obtained mutational profiles for all the clones and tumor samples, and their relative contributions were calculated (excluding low contributing signatures).WGS of 86 neuroblastoma tumors was previously performed , and percassette . From clcassette . To idenSI Appendix, Extended Materials and Methods includes additional experimental details.Supplementary FileSupplementary File


High out-of-pocket (OOP) medical expenses for cervical cancer (CC) can lead to catastrophic health expenditures (CHEs) and medical impoverishment in many low-resource settings. There are 32 million women at risk for CC in Ethiopia, where CC screening is extremely limited. An evaluation of the population health and financial risk protection benefits, and their distributional consequences across socioeconomic groups, from human papillomavirus (HPV) vaccination will be critical to support CC prevention efforts in this setting. We used a static cohort model that captures the main features of HPV vaccines and population demographics to project health and economic outcomes associated with routine HPV vaccination in Ethiopia. Health outcomes included the number of CC cases, and costs included vaccination and operational costs in 2015 US dollars over the years 2019–2118 and CC treatment costs over the lifetimes of cohorts eligible for vaccination in Ethiopia. We estimated the household OOP medical expenditures averted  and cases of CHE averted. A case of CHE was defined as 40% of household consumption expenditures, and the cases of CHE averted depended on wealth quintile, disease incidence, healthcare use and OOP payments. Our analysis shows that, assuming 100% vaccine efficacy against HPV-16/18 and 50% vaccination coverage, routine HPV vaccination could avert up to 970 000 cases of CC between 2019 and 2118, which translates to ∼932 000 lives saved. Additionally, routine HPV vaccination could avert 33 900 cases of CHE. Approximately one-third of health benefits would accrue to the poorest wealth quintile, whereas 50% of financial risk protection benefits would accrue to this quintile. HPV vaccination can reduce disparities in CC incidence, mortality and household health expenditures. This understanding and our findings can help policymakers in decisions regarding targeted CC control efforts and investment in a routine HPV vaccination programme following an initial catch-up programme. Key messagesWe used a static cohort model that captures the main features of HPV vaccines and population demographics to project health and economic outcomes, including financial risk protection and distributional impact, associated with routine HPV vaccination in Ethiopia.Assuming 100% vaccine efficacy against HPV-16/18 and 50% vaccination coverage, our analysis shows that routine two-dose HPV vaccination could avert up to 970 000 cases of cervical cancer between 2019 and 2118, which translates to ∼932 000 lives saved.Approximately one-third of health benefits would accrue to the poorest wealth quintile, whereas 50% of financial risk protection benefits would accrue to this quintile.This analysis can help make decisions regarding routine HPV vaccination, including how to progress towards cervical cancer elimination both efficiently and equitably.Ethiopia is a large (population of 109 million in 2018), low-income country (per capita gross domestic product of $770 in 2018) in East Africa, with 24% of the population living below the national poverty line . EthiopiCC treatment can create a large financial burden on households, with direct medical costs of ∼$330 per patient for consultations, investigations and drugs , withoutBeyond vaccinating the initial cohort of 14-year-old girls (in 2018–19), an evaluation of continued annual routine HPV vaccination in terms of preventing both CC cases and deaths and medical impoverishment from OOP health-related expenditures will be critical to support CC prevention in Ethiopia. Traditional economic evaluations such as cost-effectiveness analysis (CEA) of health interventions often only account for total health impacts and costs incurred , neglectWe conducted an economic evaluation of routine HPV vaccination by quantifying the health, FRP and distributional benefits of rolling out HPV vaccination nationally in Ethiopia, consistent with the dashboard of health and financial outcomes reported in ECEA , we estimate intervention impact along three dimensions: (1) household OOP medical expenditures averted by HPV vaccination, (2) FRP benefits provided through the reduction of OOP medical expenditures by HPV vaccination and (3) distributional impact across the wealth strata (e.g. income quintiles) of the population  two-dose HPV vaccination of 9-year-old girls at an average of 50% coverage. We used the coverage wealth-gradient for diphtheria-tetanus-pertussis third dose (DTP3) coverage from the DHS as a proxy to apportion aggregate 50% coverage by household wealth quintile, resulting in the following coverage levels from poorest to richest quintile: 26%, 48%, 50%, 52% and 74% (i.e. the ‘50% coverage gradient’ scenario) . We assuCC treatment costs included direct medical costs for cancer staging, treatment, palliative care and follow-up associated with stage-specific International Federation of Gynecology and Obstetrics treatment protocols, assuming that cancer treatment costs were not dependent upon vaccination coverage level . We assuWe assumed a vaccine cost of $4.50 per dose for HPV vaccine , which cHousehold medical expenditures related to the treatment of CC cases can be averted with roll-out of HPV vaccination that reduces the incidence of CC cases. Such household OOP direct medical costs averted would then depend on the number of CC cases prevented, probability of seeking care (for CC) and cost of health care. Sixty-eight percent of health expenditures associated with non-communicable diseases are financed by OOP payments in Ethiopia . TherefoBy averting OOP expenditures related to the treatment of CC cases, roll-out of HPV vaccination then provides FRP benefits to the population. Two metrics of (lack of) FRP are commonly used by the World Bank and the World Health Organization in tracking progress towards universal health coverage: catastrophic health expenditures (CHEs), which count the number of occurrences when OOP direct medical expenditures surpass a certain threshold of total consumption expenditures, and impoverishing health expenditures (IHEs), which count the number of occurrences when OOP direct medical expenditures push individuals/households below a defined poverty line  .We analysed six extensions to the base-case scenario: (1) changes in the vaccine price, (2) changes to the level of care-seeking, (3) changes to the level of OOP payments by quintile, (4) changes to the distribution of CC incidence by quintile, (5) changes to the CHE threshold and (6) changes to the distribution of income by quintile. We also examined alternative time horizons of 10, 25 and 50 years in addition to the 100-year base case.In the first scenario analysis, we used a vaccine price of $0.20 per dose, as this is the co-financing level offered by Gavi, to introduce new vaccines in low-income countries such as Ethiopia . In the Four key parameters were identified for probabilistic sensitivity analysis (PSA): HPV-16/18 type distribution, age-specific CC incidence, stage distribution of CC, and stage-specific 5-year survival and treatment access (as a combined parameter). Each parameter was assigned a beta-PERT distribution for probabilistic sampling, with the bounds determined by (1) empirical data for type distribution, (2) confidence intervals estimated from CC cases in Globocan 2020 . Two hunRoutine HPV vaccination assuming a lifelong duration of protection against HPV-16/18 infections could avert 970 000 (95% UI: 740 000–994 000) cases of CC of cohorts vaccinated over 2019–2118 in the base-case scenario (i.e. 50% coverage and 100% vaccine efficacy), compared to a strategy of no HPV vaccination. This translates to ∼932 000 (704 000–965 000) lives saved. Assuming 50% flat coverage, 31% of these health benefits would accrue to the poorest quintile compared with 43% to the richest quintile, whereas, assuming a coverage gradient averaging 50%, 15% of the health benefits would accrue to the poorest quintile compared with 61% in the richest . CurrentThe total vaccine-related costs associated with the base-case scenario of 50% routine coverage were nearly $265 million  between 2019 and 2118, but resulted in long-term cost offsets from future averted CC cases of $93 million ($71–96 million) or 41% of CC treatment costs compared with no vaccination. The vaccination programme of a single cohort of 14-year-old girls at 95% coverage exceeded $16 million but would avert $1.9 million ($1.4–2.0 million) associated with CC treatment costs .Assuming 100% efficacy against HPV-16/18 infections and a lifelong duration of protection, our analysis shows that routine two-dose HPV vaccination could avert $41 200 000  to $46 600 000  in total OOP expenditures over 2019–2118 compared to no vaccination, with the bottom two quintiles accounting for ∼25% of all OOP expenditures averted in the latter . In termThe number of CC deaths and CHE cases averted for the base-case scenario, assuming flat 50% coverage with a vaccine that is 100% efficacious against HPV-16/18 infections, is shown in In the first scenario analysis, if Ethiopia were to introduce the HPV vaccine with Gavi support and face a vaccine price of $0.20 per dose (rather than $4.50 per dose as in the base case), the $265 million in vaccination programme costs would substantially be reduced to $37 million (discounted at 3% per annum), respectively. Given that $93 million ($71–96 million) in CC treatment costs would be averted in the routine programme at 50% coverage, a routine vaccine programme would be cost-saving at this reduced vaccine price. In the second scenario analysis, if our assumed proxy for care-seeking percentage were decreased from 8.5% in the base case to 2.4%, the number of CHE cases averted would decrease, with routine two-dose HPV vaccination averting 6930 (5330–7140) cases of CHE (assuming a 50% coverage gradient) or 9550 (7310–9770) cases of CHE (assuming 50% flat coverage). On the other hand, if our assumed proxy for care-seeking percentage were increased from 8.5% in the base case to 50%, the number of CHE cases averted would greatly increase, as expected, with routine two-dose HPV vaccination averting 145 000 (110 000–148 000) cases of CHE (assuming a 50% coverage gradient) or 199 000 (152 000–204 000) cases of CHE (assuming 50% flat coverage) up from 24 600 (18 700–25 200) and 33 900 (25 800–34 700) cases of CHE in the base case, respectively . In the Using a static cohort model that incorporates HPV vaccine characteristics, age-specific CC incidence and HPV type distribution, population demographics, FRP and distributional analysis, we projected that routine two-dose HPV-16/18 vaccination could avert 932 000 CC deaths and 33 900 cases of CHE over 2019–2118 in the simulated base-case scenario (50% coverage and 100% efficacy against HPV-16/18 infections) in Ethiopia. The percentage of CHE cases averted is highly sensitive to the rate of care-seeking for CC that we assumed in this population (8.5%), which is an average across low-income settings as we relied on an estimate of access to radiotherapy, and the 2.4% Ethiopia-specific estimate is likely to be an underestimate of CC care as not all CC case management services are available in tertiary facilities. However, we examined the impact of assuming a 2.4% rate of care-seeking, as well as an aspirational 50% rate of care-seeking in scenario analysis. Assuming 50% flat coverage (which could be reached over the future 2019–2118 time period), ∼30% of the health benefits from HPV vaccination would accrue to the poorest wealth quintile, whereas ∼50% of the FRP benefits would accrue to the same poorest quintile. While the combination of disease incidence and care-seeking by quintile  would yield the greatest health benefits for the richest quintile, the magnitude of the FRP benefits experienced by the poorest quintile  indicates that routine two-dose HPV vaccination could be equity-improving overall. These results underscore the importance of considering perspectives of both total health gains and equity when evaluating public health interventions: in this particular case, we observe that HPV vaccination could be both pro-rich and pro-poor from a health benefit perspective and be pro-poor from a financial protection perspective.Given the SDG focus on poverty reduction  and the There are several important limitations to this analysis. First, we projected intervention impact for only a single cohort of 14-year-old girls in Ethiopia (based on the previously implemented 2018–19 vaccination effort) as a comparison to the roll-out of routine HPV vaccination; even though there are plans in discussion, while there remains a global HPV vaccine shortage, it is currently not feasible for additional cohorts to be vaccinated . If EthiSecond, in order to stratify CC cases by wealth quintile, we assumed that CC incidence and deaths within each quintile would mimic the distribution of STD prevalence across wealth quintiles in Ethiopia . As a reThird, we have only estimated FRP benefits from HPV vaccine using CHE as a metric for lack of FRP . In addiFourth, this analysis captured the costs of the vaccination programme over 100 years; we assumed that CC incidence rates would be stable over this time period. Likewise, we assumed constant expenditures across this time period and no changes to the distribution of income by wealth quintile. The simulation of future outcomes comes with inherent uncertainty, no matter the time horizon, but we attempted to provide a range of scenario analyses to capture how results might be impacted by key assumptions, including alternative time horizons. While we conducted a scenario analysis projecting the Gini coefficient and the associated relative income distribution over time, Gini projections are patchy and inconsistent, which limits our ability to vary the income distribution effectively. This approach also implicitly assumed that mean incomes and OOP costs would evolve relatively similarly over time, which ultimately did not impact the estimation of CHE. Projecting the state of health system financing (as well as the share of OOP vs public financing) in Ethiopia would further rely on political economy factors that are difficult to foresee. These projections also did not take into consideration the impact of the COVID-19 pandemic, which may result in large increases to the world’s poor . VaccineThe results of our work regarding the potential equity-improving impact of HPV vaccination are consistent with previous work on the equity impact of vaccines (In summary, our approach incorporates equity dimensions into the economic evaluation of routine HPV vaccination in Ethiopia, a resource-constrained setting with currently low CC screening coverage. The pro-poor results of this study emphasize the continued priority of routine HPV vaccination once the global vaccine shortage is overcome. This analysis can help make decisions regarding routine HPV vaccination, including how to progress towards CC elimination  both effczab052_SuppClick here for additional data file.


Background: The purpose of this paper is to evaluate the role of health education among young adolescents, regarding their level of knowledge about the HPV and the acceptance of the HPV vaccination, with the aim of increasing vaccination coverage, in Trikala city, mainland of Greece. Methods: This study included high school students from all public and private schools of Trikala city. Questionnaires related to knowledge regarding the HPV infection and HPV vaccination were administered in three phases. In the first phase of the study, the questionnaires were completed by the entire study population. During the second and third phases, the completion of the questionnaires concerned only the population that received the intervention (interactive seminars). The second and third phase questionnaires were completed immediately after the interactive seminar and three months later, respectively. The statistical analysis of the results was performed using IBM SPSS 20.0 statistical program. Results: A total of 434 female students completed the questionnaire (response rate 76.26%). Most participants (66.6%) were females. The questionnaires evaluated the respondents’ awareness of the HPV infection. The results show that the percentage of the participants who stated that they “do not know” what HPV is decreased from 44.4% (first phase), to 1.6% (second phase), and 8.1% (third phase). Similarly, the willingness to accept the HPV vaccine increased from 71% (first phase), to 89.1% (second phase), and 83.5% (third phase). Conclusions: The present study shows that targeted interactive informational interventions in the school environment leads to a statistically significant increase in both the level of knowledge about HPV and the willingness of young adolescent students to be vaccinated against cervical cancer. Most recently in 2014, the nine-valent vaccine against HPV (Gardasil 9®) was released. The HPV vaccine has been incorporated into the Greek national vaccination program since 2008 and since 2017 it is administered for free only until the age of 16 years of age [The Human Papillomavirus (HPV) is currently the leading cause of cervical cancer. The indisputable finding of a causal link between HPV and cervical cancer has led the scientific community to the decision to create HPV vaccines. In 2006, the quadrivalent vaccine . The inclusion criteria consisted of pre-adolescent school students, 11–12 years old, attending first-grade secondary schools in the province of Trikala in Central Greece. The study proposed the participation of all first-grade students from seven school groups in the prefecture of Trikala distributed as follows: four schools were selected from the city of Trikala (urban school complexes), two schools from the province of Kalampaka in the prefecture of Trikala , and the only private school in the prefecture was also selected. The four urban school complexes were categorized into two groups, each of which included two schools: one school was the sample of intervention of the studied population and the other school was the control sample of the studied population. The 1st and 3rd Gymnasiums of Trikala were included in the urban schools, whose students comprised the studied intervention population. The 4th Gymnasium of Trikala and the 9th Gymnasium of Trikala were the control sample of the studied population. Similar to the two rural school complexes in the province of Kalampaka, one  was the sample of intervention and the other school  was the control sample of the studied population. In the only Private Gymnasium of the city of Trikala, half of the students of the 1st grade of Gymnasium were the sample of intervention and the other half the sample of non-intervention—the control of the studied population. The ethical approval to conduct this research was obtained after the submission and approval of the protocol from the Institute of Educational Policy of the Ministry of Education, Research, and Religions (Research and Ethics Committee Decision No. 219048/Δ2/19-12-2018).The anonymity and confidentiality of participants was ensured from the study protocol. Out of 573 questionnaires offered in paper form to the students, 434 completed questionnaires were returned. The largest response was observed in the Private High School (95%), while the 4th Gymnasium of Trikala and the 1st Gymnasium of Kalampaka were those with the lowest participation rates, 68.1% and 68.1%, respectively .The methodological tool of the study was a questionnaire. The questionnaire was chosen after a systematic bibliographic review of studies with a similar research question. The license to use the questionnaire was obtained after contacting the editorial team . Health The survey was conducted during the academic year of 2018–2019. The time period for the study was from January to May 2019. The first phase of the study included the completion of questionnaires by all participants . The intervention group was randomly selected to be included in the study. Two weeks later, an informational lecture was given during allocated school hours that were agreed with the Head of School. The completion of the questionnaire by all the students took place at the end of the seminar (second phase). The informational lectures to the students took place during working days and hours. The duration of the students’ briefing did not exceed 45 min . The conThe collection of the questionnaires, as well as the informative sessions, was carried out by one author (I.T.) of the study. The survey consisted of anonymous questionnaires. Participant pre-adolescents and their parents were shown subscribed assent and consent documents, respectively, informing them of the study’s aims and objectives, as well as the participant’s right to refuse or terminate their participation in the study at any time with no disadvantages.2 statistical test was used to evaluate the possible dependence between questionnaire variables and students’ knowledge status and behavior towards the HPV. The results between the three phases of the study were compared, both with the analysis of the tables obtained from the extraction of descriptive measures and with the use of the statistical paired t-test for dependent samples, in the 95% confidence interval. Through these comparisons, we received the necessary information to enhance students’ awareness about and behavior towards HPV and the vaccination against cervical cancer. Sample with p-value = 0.05 were considered statistically significant.The IBM SPSS  20.0 statistical program , acquired by IBM, was used to analyze the sample data. Using descriptive methods, the sample was analyzed by taking measurements of the frequency and percentages of responses to all questions. The statistical analysis was blinded to the researchers and conducted independently. A Pearson XA total of 434 students who met the criteria for participation in the study filled the questionnaire, 289 (66.6%) of which were girls and 145 (33.4%) were boys. The vast majority of participants (95.6%) were of Greek nationality. Overall, the general demographic characteristics of the study population are summarized in The analysis of the data revealed that no statistically significant difference was observed in the level of knowledge about HPV based on the sex of the participants (Phase II). On the other hand, a statistically significant difference was observed between the levels of students’ annual family income, their nationalities, and the different residential areas of the participants. Similarly, increased levels of knowledge about the virus were noticed among participants of Greek origin, compared to Albanians or other nationalities, as well as in those living in urban centers compared to those living in villages .Analysis of the data from our research  shows thAccording to the final analysis of the results, there was a rapid change regarding the level of knowledge about HPV between the three phases of the study:In the first phase of the study, the percentage of participants who stated that “I don’t know” what HPV is was 44.4% (n = 110), while 52.4% of young adolescent students (n = 130) replied that it was a virus. In addition, 38.3% (n = 95) answered that sexual intercourse is the most common mode of transmission.After the intervention (phase II), participants who stated “I don’t know” what HPV is decreased to 1.6% (n = 4), while 95.6% (n = 237) of those responded it was a virus. At the same time, 95.6% (n = 237) of the participants answered that sexual intercourse is the most common mode of transmission.Three months after the intervention (phase III), participants who replied “I don’t know” what HPV is had a percentage of 8.1% (n = 20), while 88.3% (n = 219) stated that it is a virus. Furthermore, 86.3% (n = 214) of the participants indicated that the most common way of viral transmission is sexual intercourse.Moreover, a change in the responses of the studied population was noticed regarding the willingness to receive the HPV vaccination among young adolescent students. Specifically, in phase 1, 71% (n = 176) of the sample were willing to be vaccinated, in phase 2 (immediately after the informative intervention) this percentage rose to 89.1% (n = 221), and finally, in phase 3 (three months after the informative intervention) the willingness rate for HPV vaccination was 83.5% (n = 207).From the comparative analysis of the data of our sample, we observed that in the third phase of the study the percentage of students (88.3%) who answered that “HPV is a virus” was statistically significantly different from the results of the first phase (52.4%), but slightly reduced compared to the second phase of the study (95.6%).In the second phase of the study, the percentage appears to have increased, but this was without statistical significance in the percentage of those who stated that “they do not know” what HPV is (8.1% in phase III versus 1.6% in phase II). Overall, it is estimated that the level of knowledge of our population in relation to the first phase of sampling is statistically significantly different for the better. When asked about the ways in which the virus is transmitted, 86.3% answered “through sexual intercourse”. This percentage is slightly reduced compared with the second phase of the study , but rapidly increased compared to the first phase of the study .The rate of willingness to be vaccinated against HPV and cervical cancer in young adolescent students in the sample of our study in the third phase remains high (83.5%). This percentage is slightly reduced compared to the second phase of sampling (89.1%), but significantly increased compared to the first phase of the study, which showed only 71.0% of participants were willing to be vaccinated.Collectively, in the above questions examined across the three phases of the study, it was observed that there is a statistically significant difference in the mean values of the sample responses between the first phase and the third phase. At the same time, however, there was a decrease in rates, both in terms of knowledge about HPV and some questions about vaccination, as well as the willingness of individuals to be vaccinated between the second and third phases of the study, but without a statistically significant differentiation.In this study, we recorded young adolescents’ levels of awareness about HPV and their willingness to be vaccinated against it. Vaccination against HPV may be proven to be the best strategic option for the country’s health system. In fact, vaccinating girls at a young age, before the initiation of sexual activity, is the preferred strategy .Our data indicate that the level of knowledge about HPV and the vaccination against cervical cancer is not statistically significant between the two sexes. This finding is opposed to findings from other studies. Durosoy et al. showed that women were better informed about the effects of HPV infection, which was presumed to be related to the campaigns aimed mainly at female population rather than at males . In receThe present study revealed that the residence area of young adolescent students is strongly related to their levels of knowledge about HPV and their willingness to be vaccinated against cervical cancer. It is concluded that students living in urban centers are more aware of HPV and HPV vaccination than students living in rural areas. Students residing in villages tend to be unwilling to be vaccinated against cervical cancer. This is a unique finding, as the only study that comments on the different between areas of the same country is Durusoy et al. That study concluded that participants from Turkey’s western region were more willing to accept and be included in a preventive vaccination program than the residents of the mainland . StudiesAccording to the results of our study, we concluded that annual family income is statistically significantly related to the level of knowledge about HPV and the willingness to undergo vaccination. Families with higher incomes usually offer better education to their children, which resulted in higher levels of awareness about HPV. This is perhaps the case, because these parents are themselves better educated and so are able to educate their children better. Similar outcomes were observed by Durosoy et al. . A recenThe marital status of the parents of the participants in this study was not statistically significantly related to the level of knowledge about HPV and the willingness to be vaccinated against cervical cancer. Similarly, in a previous study, the authors concluded that marital status was not statistically significantly related to the acceptance of the HPV vaccination . In contAnalysis of the data of this study reveals that nationality is statistically significantly related to the level of knowledge about HPV and the willingness to vaccinate. Greek students appear, in our study, to be much more informed about the virus than students of Albanian or other nationalities. Nationality status might, in this instance, be confounded by economic and educational status. However, it is necessary to point out the observed disproportion of nationalities among the participants, 95.6% are of Greek nationality, which does not allow us to statistically validate this result. Similarly, a study from Italy indicated lower vaccination coverage rates against HPV in foreigners compared to Italian citizens . FurtherThe targeted informative intervention showed a statistically significant increase in both the level of knowledge about HPV and the infection as well as in the adolescents’ willingness to be vaccinated against HPV. This was the main result of our research study. Ignorance about the HPV infection and its implications, as well as the benefit of HPV preventive vaccination, is still high among adolescents. The way to improve their knowledge about HPV and the implications of the HPV infection is to provide information through the framework of compulsory schooling, primary health care, and the development of informative interactive interventions. The knowledge about and the perceptible susceptibility to the HPV infection and HPV-related diseases among adolescents demonstrate the need for a well-designed training program to bridge the gap of information about HPV and to encourage acceptance of the HPV vaccine. Implementation of intervention materials, particularly the communication techniques, can improve adolescent HPV vaccination acceptance.The knowledge about and receptive susceptibility to the HPV infection and to HPV-related health issues demonstrate the urgent need for a well-designed training program, in order to bridge the gap between HPV awareness and vaccine acceptance .Previous studies have also indicated that informing young women is an important factor in shaping positive thinking and perception towards the vaccination against HPV ,22,23. OThe main limitations of the present study are the representativeness of the study population. Furthermore, selection bias could have an impact on the participation of school students whose parents were more prone to vaccine compliance. The role of the physician (I.T.) who provided counselling and recommendations for the HPV vaccine target populations may have played a crucial role in the study, biasing the result towards vaccine compliance. The control group was only measured at the baseline and not at other time points, due to the fact that we needed to monitor the intervention . Since few school students refused to participate in the study, the reasons for refusal were not investigated.Young adolescent students are poorly informed about HPV and the HPV vaccination, which can be a significant obstacle to the necessary increase needed in the vaccination coverage of the population. The use of interactive lecturing interventions, provided to young adolescent students in the school environment, could contribute in a statistically significant way to the increase in both the level of knowledge about HPV and the infection caused by it, as well as in the willingness of participants to be vaccinated against HPV.At the same time, the implementation of health education programs in schools, under the responsibility of the State, can be an important measure in an effort to reduce the occurrence of cervical cancer. Sex education of young girls and boys in order to gain a good amount of knowledge concerning issues such as sex life, family planning, proper use and effectiveness of contraceptive methods, and good hygiene, reduces the exposure of the population to predisposing risk factors for developing HPV.