A complete understanding of the natural history of infection with high-risk human papillomaviruses (HPVs) in cervical cancer requires data from regional and ethnic studies. The prevalence of high-risk HPVs was evaluated retrospectively in 2040 patients with cervicitis, 239 with cervical intraepithelial neoplasia grade 1 (CIN1), 242 with CIN2/3, and 42 patients with invasive squamous cell carcinoma (SCC) based on data from patients who visited our hospital between May 2013 and May 2015. The rates of high-risk HPV infection in patients with cervicitis, CIN1, CIN2/3, and invasive SCC were 40.8%, 74.9%, 70.2%, and 83.3%, respectively. The three most dominant HPV genotypes were HPV16, 58, and 52. HPV16 and HPV58 positivity in cervicitis, CIN1, CIN2/3, and SCC patients were 20.9% and 16.4%, 19.0% and 20.1%, 44.1% and 23.5%, and 60.0% and 31.4%, respectively. Compared to cervicitis, the odds ratios (ORs) for CIN2/3 in HPV16- and HPV58-positive patients were 2.99  and 1.56 (1.11–3.21), respectively; for SCC, the corresponding values were 5.68 (2.31–7.893) and 2.33 (1.41–3.87). Further identifying of carcinogenic HPVs and a fully aware of regional differences in HPV genotype distribution are tasks of top priority for cervical cancer control and prevention. In 2012, the American Cancer Society, American Society for Colposcopy and Cervical Pathology, and American Society for Clinical Pathology jointly published screening guidelines for the prevention and early detection of cervical precancerous lesions and cervical cancer , 4. HoweThus, whether HPV DNA testing alone suffices in the early detection of cervical precancerous lesions and cancer is still uncertain. The question is relevant not only with respect to the adoption of a cervical cancer screening strategy but also points out gaps in the current understanding of the natural history of HPV infection and the role of HPV in oncogenesis. Current opinion is that persistent cervical infection with high-risk HPVs is necessary for the development of cervical cancer; indeed, some epidemiologic data have shown that nearly 100% of patients with cervical cancer test positive for HPV . HPV16 aShanghai is one of the largest cities in China, with a population of more than 25 million. However, the prevalence characteristics of high-risk HPV in its female residents with cervical precancerous lesions and cancer have rarely been evaluated. In this study, high-risk HPV infection rates and HPV genotype distribution were evaluated in 2563 patients living in Shanghai who had been diagnosed with cervicitis, CIN1, CIN2/3, or invasive SCC.th Hospital  approved the study protocols. Written informed consent was obtained from all patients according to the guidelines of the Chinese National Ethics Regulation Committee. All patients were informed of their rights to withdraw consent personally or via kin, caretaker, or guardian.This study was conducted in accordance with the World Medical Association Declaration of Helsinki. The Review Board of the Ethics Committee of Medical Research at Shanghai 7th Hospital, between May 2013 and May 2015 for cervical diseases, were recruited consecutively. The criteria for exclusion in the study were as follows: patients with adenocarcinoma; patients are currently pregnant; patients with history of total uterine or cervical resection; patients with prior chemotherapy or radiation treatment for cervical neoplasia; patients with previous physical treatment of the cervix or hormone treatment for cervical disease, or patients with known human immunodeficiency virus infection. Thus, the study population consisted of 2563 (85.1%) female patients with average age around 40, of these participates, 2040 with cervicitis, 239 with CIN1, 242 with CIN2/3, and 42 with invasive SCC, respectively. The age, clinical gynecological, cytological, and histopathologic data as well as HPV testing results of the study participants were recorded.3011 outpatients and inpatients visiting the Department of Gynecology, Shanghai 7in situ [All participants were screened by clinical gynecological and cytological examinations. For each patient, cervical samples for cytological examination were taken, a sterile cytobrush was inserted into the cervical os and rotated through 360°, the acquired cells were then followed the instruction of the BD SurePath™ liquid-based Pap test . Patients, with epithelial cell abnormalities of atypical squamous cells (ASC) of undetermined significance (US) or higher cytological grade, would undergo colposcopy examination and biopsy. As Wentzensen N. et al. described , four lein situ . Squamouin situ .HPV DNA testing and genotyping are routinely performed in patients visiting the Department of Gynecology of our hospital. Before colposcopy, cervical specimens for HPV testing were also collected using a sterile cytobrush as the cell collecting procedures in cytological examination. The DNA in the specimen was extracted by using a QIAamp DNA Mini Kit , HPV DNA testing and genotyping were done using a commercial detection kit provided by Hybribio . The kit detects and distinguishes among 15 high-risk HPV strains  and 6 low-risk HPV strains . HPV detection by the kit is achieved using polymerase chain reaction followed by HPV genotype-specific DNA microarray analyses. The performance of the HPV DNA testing and genotyping kit was approved by the China Food and Drug Administration. All detection procedures were carried out according to the protocols provided by manufacturers.t test, one-way ANOVA, χ2, or Fisher's exact probability test according to the characteristics of the data distribution. The odds ratio (OR) and relative 95% confidence interval (CI) were calculated using SPSS version 13.0. The significance level α was set at 0.05.Statistical analysis was performed using SPSS version 13.0 . Continuous variables are presented as means ± standard deviations (SDs) and categorical data are presented as numbers (percentages). Differences between groups were examined using the As shown in Table To determine the prevalence of high-risk HPV and of the different viral genotypes in our patients, they were screened for 15 high-risk HPVs. As shown in Table Because HPV16 and HPV58 infections were significantly higher in patients with CIN2/3 and invasive SCC, we calculated the ORs and 95% CIs of such infections (Table To learn a more accurate estimate of HPV prevalence in CIN1, CIN2/3, and SCC patients world widely, we summarized the representative published data. As shown in Table As the genotype distribution of high-risk HPVs differed regionally , we summOur data showed that 40.8% of patients with cervicitis, 74.9% with CIN1, 70.2% with CIN2/3, and 83.3% with invasive SCC were positive for high-risk HPV DNA. HPV infection rates were significantly higher in CIN1, CIN2/3, and invasive SCC patients than in cervicitis patients, but they never reached 100%. Although single-center cross-sectional data do not reflect the real natural history of HPV infection in the population, our results suggest that HPV DNA testing alone might miss patients with cervical cancer or precancerous lesions. In addition, in our population, HPV genotypes 16, 58, and 52 were the dominant high-risk HPVs in patients with cervicitis, CIN1, CIN2/3, and invasive SCC, unlike in other reports , 18 in wIn our group of SCC patients, the 83.3% positivity for HPV DNA was lower than the rates reported in most other studies , 21-23. Because our study was cross-sectional, it was not possible to investigate the pathophysiological process of HPV infection and the development of cervical cancer or precancerous lesions in our patients with cervicitis, CIN1, CIN2/3, and invasive SCC. Therefore, we did not calculate the morbidity associated with the latter three conditions. Nonetheless, the diagnosis of cervical cancer or precancerous lesions was based on standardized histopathology, not cytopathology, which ensured the validity of our data. In this report, we only focused on patients with SCC but excluded patients with adenocarcinoma because the later is very rare in our clinical practice. So we could not know the prevalence characteristics of high-risk HPV in women living in Shanghai with cervical adenocarcinoma, because reports showed that cervical adenocarcinoma displayed specific HPV infection pattern ,23. In oThe distribution of dominant HPV genotypes showed obvious regional differences . HPV18 iCurrent HPV DNA tests are mainly focused on high-risk HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 , 25, 26,


This national online cross-sectional survey in Italy assessed primary care pediatricians’ (PCPs) attitudes and practices regarding Human papillomavirus (HPV) vaccination and the contribution of several characteristics. The questionnaire was distributed from September 2016 to June 2017 to a random sample of 640 PCPs by email via an internet-link leading to a web-based survey platform (Lime Survey). Only 18.4% of PCPs always recommend the HPV vaccine to 11–12 year old boys. PCPs with longer practice activity, working in solo practice, always recommended the HPV vaccine to 11–12 year old girls, and believed that the vaccine was effective for boys were more likely to always recommend the HPV vaccine. PCPs working in a Region where the vaccination was actively recommended and provided free of charge to 11–12 year old boys had higher odds of recommending vaccination. More than two thirds of PCPs (77.4%) always recommend the HPV vaccine to 11–12 year old girls. PCPs who believed that the vaccine was effective for girls and safe in both boys and girls, who always talk with patients of 11–18 years or their parents about HPV infection and vaccination, and who obtain vaccine information from scientific journals were more likely to always recommend the vaccine. PCPs should employ evidence-based educational strategies in order to achieve a better coverage and to reduce the morbidities and mortality of diseases associated with HPV. Human papillomavirus (HPV) infection is still a growing public health threat worldwide due to the morbidity, mortality, and costs related to cervical, vulvar, anal, penile, head, and neck cancers ,2. It isThe online survey was conducted from September 2016 to June 2017 performing a multi-stage sampling. A random sample of Local Health Units (LHUs), which organize, plan, and provide health care services for the community closer to where people live, was selected across Italy. The Head of the selected LHUs received a letter explaining the purpose of the study, and after consent the research team received email-addresses and phone numbers of potential participants. Then, a random sample of 640 PCPs was recruited from the list provided by each selected LHU.The sample size was calculated based on the estimation that 60% of the PCPs recommend HPV vaccination, a 95% confidence interval, and an error of 0.05. Therefore, the minimum number of pediatricians required was estimated at 360. In addition, accounting for a response rate of 55%, the minimum number of participants required was estimated as 640.The study instrument was a questionnaire distributed to the sample by e-mail via an internet-link leading to a web-based survey platform (Lime Survey). The e-mail contained a cover letter with the lead investigator’s name and contact details, and explained, as well as on the web-based surveys platform, the background, the objectives, and the methodology of the study. The participants were invited to self-administer a confidential survey by clicking on a unique URL for each respondent, which remained active for completion until June 30, 2017. The study was absolutely voluntary and the cover letter specified that personal data were collected and analyzed anonymously, and each personal number of the URL link was stored on a secure server to know those who responded. Participants were informed before proceeding to the survey that sending back the anonymous questionnaire, they gave the informed consent to participate, and they were able to exit the survey at any stage. As the survey was completed anonymously, by clicking the “submit” button at the end of the survey, it was not possible to withdraw individual respondents’ data. In an effort to maximize the response rate, eight subsequent additional e-mail reminders at 4-week intervals were sent to the PCPs who had not already responded soliciting them to complete the questionnaire. Finally, non-responders were personally phone called by the research team soliciting their participation. There was no monetary compensation to participate in this study.Prior to the start of the study, the questionnaire was pre-tested and piloted involving a sample of 25 physicians, not included in the final sample, to ensure clarity and ease of administration. The complete questionnaire in Italian and English are found in The Institutional Review Board of the Teaching Hospital of the University of Campania “Luigi Vanvitelli” approved all study procedures.t or chi-square tests, as appropriate, were carried out to evaluate predictors of the different outcomes of interest, after which the candidates for the multivariate regression analysis were selected. Only characteristics with p-values below or equal 0.25 in univariate analyses with the outcomes were included in the regression models. Third, multivariable logistic regression models for dichotomous outcomes were constructed to determine independent associations between different covariates and the following outcomes of interest: profile of PCPs who often/always collect information on the sexual habits of adolescents of 11–18 years (Model 1), profile of PCPs who always recommend the vaccine to 11–12 year old boys (Model 2), and profile of PCPs who always recommend the HPV vaccine to 11–12 year old girls (Model 3). PCP socio-demographic and professional independent variables included in all models were the following: age , gender , number of years since degree (continuous), number of years in practice (continuous), number of hours worked per week (continuous), practice type , always talk about HPV infection and its vaccination with patients of 11–18 years or their parents , sources of information about the HPV vaccination , and need to receive additional information about the HPV vaccination . Moreover, the variable number of patients (continuous) was included in Model 1. The variable believe that the HPV vaccine was safe in both boys and girls (continuous) was included in Models 2 and 3. The following independent variables were also included in Model 2: number of 11–12 year old boys patients (continuous), working in a Region where the HPV vaccination was actively recommended and provided free of charge to 11–12 year old boys , believe that the HPV vaccine was effective for boys (continuous), and having always recommended the HPV vaccine to 11–12 year old girls . Finally, the variables number of 11–12 year old girls patients (continuous), and believe that the HPV vaccine was effective for girls (continuous) were included in Model 3. Stepwise selection was used to determine the final models and the significance level for variables entering in the models was set at 0.2 and for removing at 0.4. Multivariate logistic regression models produced adjusted odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) as measures of association between predictors and outcomes of interest. All statistical tests were two-tailed, and p-value below or equal 0.05 was considered to indicate a statistically significant difference.Statistical analysis of the data (S3 Dataset) was undertaken using the Stata software version 10.1 . First, Of the 640 PCPs selected, a total of 234 participants completed the online questionnaire for a response rate of 36.5%. Regarding the respondents’ attitude towards HPV vaccination, all the three statements received a high value. Indeed, almost all PCPs believed that the vaccine is effective in preventing HPV-related diseases in boys (92.3%) and in girls (97.9%), with an overall mean value of 8.7 out of a maximum score of 10. Moreover, 97.4% of PCPs felt safe the vaccine in both boys and girls, with an overall mean value of 8.8 out of a maximum score of 10.During health maintenance visits, only 10.3% of the PCPs indicated that they collect often/always information on the sexual habits of their patients and a quarter (25.6%) never did it. Only 39.7% and 29.1% talked respectively often and always about HPV infection and its vaccination with patients of 11–18 years or their parents. Findings from the multivariable logistic regression analyses with stepwise elimination procedure of factors associated with the different outcomes of interest are shown in Almost all the sample (98.7%) indicated that they recommend the HPV vaccine with option ranging from rarely to always to 11–12 year old girls, and a total of 77.3% always recommend it. On the basis of univariate findings, the multivariable logistic regression model for the recommendation of the vaccination to this group included eight variables. The final multivariable model predicting this outcome indicated that PCPs who believed that the vaccine was effective for girls  and safe in both boys and girls , those who always talk with patients of 11–18 years or their parents about HPV infection and vaccination  were more likely to recommend the vaccine to 11–12 year old girls. Moreover, compared with PCPs who obtain vaccine information from scientific journals, those who used other sources were less likely  to recommend the vaccine (Model 3). The reasons more frequently reported by PCPs who did not recommend often/always the vaccine for 11–12 year old girls were lack of time to talk about HPV infection and vaccination (60%), concern that vaccination could increase the high-risk sexual behaviors (25%), concern about the efficacy (22.7%) and safety (15%) of the vaccine. Instead, the reasons more frequently reported by respondents who often/always recommend the HPV vaccine were that in their Region it was actively recommended and provided free of charge to girls (75.7%) and that the vaccine can prevent the HPV-related cancers (69.2%).Among survey respondents who recommended the HPV vaccine to girls aged 11–12 years, 71.4% reported that some of them refused to be vaccinated. The most frequent reasons cited by the respondents were concerns regarding the adverse side effects (46.7%), that patients did not believe in the usefulness of vaccination (40.3%), and felt they were not at risk for HPV infection (37%). Furthermore, 51.7% always recommended the vaccine to 13–18 year old girls not previously vaccinated.Overall, 77.4% and 80.8% of PCPs reported having received information about HPV infection and the relative vaccination, respectively. Scientific journals and educational courses/meetings were the most trusted sources by the PCPs in order to obtain information (76.2% and 74.6% for the infection and 79.9% and 83.6% for the vaccination), followed by colleagues (13.2% for the infection and 9.4% for the vaccination), and pharmaceutical representatives (10.6% for the infection and 6.6% for the vaccination). About half (42.3%) of PCPs reported that they felt the need to receive additional information about the HPV infection and more than half (51.3%) about the vaccine.This study characterized the attitudes and practices of a large sample of PCPs in Italy regarding HPV vaccination and determined which factors were associated and it has important implication for the program planning and implementation regarding the vaccination.In this nationally representative sample of PCPs, respectively 77.3% and 18.4% reported that they always recommend the HPV vaccine to girls and boys aged 11–12. In the literature, previous results from the United States highlighted that 57%  and 60% Understanding barriers related with vaccine recommendation are necessary when considering the development of interventions that focus on increasing the vaccination. The most common reason cited by PCPs for not recommending HPV vaccine for boys was that in their Region for this group it was not free of charge, and this finding has important implications for HPV vaccine policy, because there was a policy barrier to recommend the vaccine. Indeed, at the time of the survey, HPV vaccine was provided free of charge to boys in nine Regions . In addition, other reasons affecting HPV vaccine non-recommendation to boys were concerns about vaccine safety and efficacy. This is surprising because the safety and the efficacy, of the HPV vaccine have been determined in multiple studies –34 and tA main objective of the present investigation was to identify factors affecting the behavior of the PCPs regarding HPV vaccination. Indeed, understanding the factors associated with the recommendation of the HPV vaccine to boys and girls is critical because the recommendation of the PCPs is a key factor in their decision to get vaccinated. Findings from the current analysis indicate that the variable working in a Region where HPV immunization was actively recommended and provided free of charge to 11–12 year old boys is a robust predictor of vaccine recommendation by the PCPs to this group. Indeed, as expected, PCPs working in these geographic areas were more likely to recommend the HPV vaccine. The findings of the present study also indicate that personal beliefs about vaccine safety and efficacy were positively associated with vaccination recommendation either for boys or girls aged 11–12 years. Furthermore, not surprisingly and consistent with previous studies, scientific journals and educational courses/meetings were the principal information source that the PCPs trusted –41. AlonIt is important to acknowledge that certain limitations need to be noted in the interpretation of the results of this study. First, since this was a cross-sectional survey, the analysis can only provide evidence of statistical association between explanatory variables and the different outcomes of interest and it is difficult to demonstrate temporal relationship. Second, the intrinsic problem associated with the use of questionnaire with self-reported information and thus the study may be subject to declaration or desirability biases. Social desirability may have influenced the sample and the participants may have responded to questions regarding their practice in a socially desirable way, even though questionnaire was anonymous and the items were carefully framed to avoid a judgmental tone. Third, it is possible that PCPs engaged in HPV vaccination or who had a positive attitude regard vaccination were more likely to respond to the survey than those who did not share these attitudes. These biases would have led to an overestimation of the number of PCPs who recommend HPV vaccination. Fourth, the response rate was modest, but superior to other studies with similar methodology –46 and iIn conclusion, the current results highlight the importance that PCPs address barriers to HPV vaccination promotion and employ evidence-based educational strategies that can facilitate vaccine acceptability for adolescents in order to achieve a better coverage and to reduce the morbidities and mortality of diseases associated with HPV.Preliminary findings from this study have been presented at the Annual Conference of the European Public Health Association, Stockholm, 1–4 November, 2017.S1 FileQuestionnaire in Italian.(DOCX)Click here for additional data file.S2 FileQuestionnaire in English.(DOCX)Click here for additional data file.S3 FileData file.(XLSX)Click here for additional data file.


To analyze the clinical performance of p16/Ki-67 dual-stained cytology identifying high-grade cervical intraepithelial neoplasia (CIN2+) in Chinese women.1079 women attending ongoing cervical cancer screening and 211 “enriched” women aged ≥30yrs with biopsy-confirmed CIN2+ from five Chinese hospitals were enrolled during year 2014-2015. Cervical specimens were collected for high-risk human papillomavirus (HR-HPV) DNA analysis, Liquid-based cytology (LBC) and p16/Ki-67 dual staining. Colposcopy and biopsy were performed on women with any abnormal result.p16/Ki-67 positivity increased with histologic severity. It was 18.4%(183/996) in normal histology, 54.0%(34/63) in CIN1, 81.0%(34/42) in CIN2, 93.3%(111/119) in CIN3, 71.4% (5/7) in adenocarcinoma and 95.2%(60/63) in squamous cell carcinoma. Compared with the HR-HPV negatives, p16/Ki-67 expression was significantly higher in the HPV16/18 positive (OR: 35.45(95%CI: 23.35-53.84)) and other 12 HR-HPV types positive group (OR: 8.01(95%CI: 5.81-11.05). The sensitivity and specificity of p16/Ki-67 to detect CIN2+ in the entire population were 90.9% and 79.5%, respectively. In women with ASC-US and LSIL, sensitivity and specificity for detection of CIN2+ were 87.5% and 66.4%, respectively, with a referral rate of 43.8%. In women who tested positive for HR-HPV, sensitivity and specificity of dual-staining for detection of CIN2+ were 92.7% and 52.7%, respectively, and the referral rate was 68.7%.p16/Ki-67 dual-stained cytology provided a high sensitivity and moderate specificity to detect underlying cervical precancer and cancers in various settings, and might be considered as an efficient screening tool in China. As the most populous country in the world, the disease burden of cervical cancer varies greatly in China due to the unbalanced development of economy, uneven distribution of its population and screening coverage. More than 70% of the Chinese population living in rural areas where 90% of incident cervical cancer estimate to occur, however, cervical cancer screening is mainly available in cities, rural Chinese women receive minimal benefit from screening improvement . From 20Papanicolaou (Pap) cytology is widely used for cervical cancer screening since its introduction to China in 1999. However, the positive prediction of atypical squamous cells of undetermined significance (ASC-US) and low-grade intraepithelial lesions (LSIL) cytology results for the presence of high-grade cervical intraepithelial neoplasia (HG CIN) is relatively low, with a 5-year risk for cervical intraepithelial neoplasia grade 3 or worse (CIN3+) of 2.6% and 5.2%, respectively [In resource limited rural areas in China, it is difficult to build the infrastructure necessary for a successful Pap cytology screening system. Alternative screening strategies have been developed, including molecular testing for HR-HPV -the necessary cause of cervical cancer. HR-HPV DNA testing has been evaluated and shown to be more sensitive and reliable than Pap cytology -7. In ApINK4a (p16) is a cyclin-dependent kinase inhibitor that has been proven to be strongly overexpressed in transforming infections with oncogenic types of HPV and is believed to be a surrogate marker for precancerous cervical lesions [p16 lesions , 11. How lesions . In norm lesions -15, but A total of 1,357 Chinese women were enrolled. Of them, 35 (2.6%) women were excluded due to incomplete histology results, 32 (2.4%) women were excluded because of indeterminate cobas HPV Test results or unsatisfactory cytology results. A total of 1,290 women were included in the final analysis, including 1,079 (83.6%) women from the screening group. Among them,481(44.6%) had abnormal test results and received colposcopy, with 63 CIN1, 14 CIN2 and 6 CIN3 cases. There were 211 women in enriched group, i.e. 16.4% of all women. The mean age of the women included in the analysis was 48.3 ± 8.7 years .p < 0.05). It increased significantly with disease severity (trendp < 0.0001). There were 996 (77.2%) women with negative histology, 63 (4.9%) with CIN1, 42 (3.3%) with CIN2, 119 (9.2%) with CIN3, 63 (4.9%) with SCC, and 7 (0.5%) with ADC. The corresponding p16/Ki-67 dual staining positivity was 18.4%, 54.0%, 81.0%, 93.3%, 95.2%, and 71.4%, respectively.As shown in Table p < 0.05).To analyze the associations between HR-HPV infection and p16/Ki-67 dual-stain positivity, the participants were divided into three groups according to HPV status. HPV positivity was strongly associated with p16/Ki-67 dual staining in the whole population Table . Comparep > 0.05); the specificity of p16/Ki-67 to detect CIN2+ was similar with that of HR-HPV(p > 0.05), but slightly higher than that of LBC ;in detection of CIN3+, the specificity of p16/Ki-67 was not significant different with that of LBC (p > 0.05), but a little higher than that of HR-HPV . The similar situation was also observed in the screening population.Sensitivities, specificities, PPVs, NPVs, AUCs, and colposcopy referral rates for all screening methods to detect CIN2+ or CIN3+ are shown in Tables p = 0.727 and 1.000) for both endpoints, but a higher specificity (p = 0.003 and 0.002).There were 256 women diagnosed as ASC-US or LISL in the whole population, and 48 CIN2+ cases were detected in this group; the p16/Ki-67 positivity was 43.8%, indicating that colposcopy referral would be reduced by more than half if p16/Ki-67 dual-stain were used as a triage test  for CIN2+ and CIN3+ but a lower specificity for both endpoints. However, no significant difference was found between LBC and p16/Ki-67. Notably, the positivity of p16/Ki-67 was 68.7%, which means that more than 30% of women would not need referral to colposcopy if p16/Ki-67 dual staining would be used as a triage.Four hundred and sixty-three women were tested positive for HR-HPV in the entire population, and there were 218 CIN2+ cases in this group; the performance of HPV16/18 genotyping, LBC and p16/Ki-67 dual staining were evaluated Table . In contn = 231, including 70 cervical cancer cases) were enrolled from the ongoing cervical cancer screening program or colposcopy clinic. To avoid the verification bias, in the screening group, three screening tests were used in parallel and women who tested positive for any of the tests were refereed to colposcopy and biopsied if needed. Four-quadrant cervical biopsies and ECC were performed to maximize ascertainment of disease if no visible lesions were found under colposcopy. The histopathology of CIN and some of cervical cancer cases were consensus expert-reviewed and p16/PR -supported. In addition, all assays in our study were run in the central laboratory and were evaluated according to the same criteria; all three screening tests were performed by using the same sample.To our knowledge, this is the first study to comprehensively evaluate the performance of p16/Ki-67 dual stained cytology in primary screening and as a tool to triage women with ASCUS/LSIL or positive HR-HPV results in China. To increase the statistical power for the sensitivity estimates of p16/Ki-67, multi-center hospital-based recruitment was undertaken and a large number of women diagnosed with CIN2+ , which is believed to be less preferentially related with HPV infection. Notably, 21 cases of CIN2+ , including 8 cases of CIN2 , 8 cases of CIN3  and 5 cases of cervical cancer  were p16/Ki-67 negative. One of the possible explanations is that not all precancer lesions progress to cervical cancer [In the whole population, the positive rates of p16/Ki-67 increased gradually with disease severity, which correspond with results from previous studies . The ovel cancer , 21; howIn this study, 211 CIN2+ cases were enriched to evaluate the clinical performance of p16/Ki-67 dual staining. We noted that the sensitivity of p16/Ki-67 for the detection of CIN2+ and CIN3+ in the entire population was not significantly different from that of LBC and HR-HPV test (by cobas HPV Test), but the specificity was slightly higher than that of LBC in detection of CIN2+ and HR-HPV in detection of CIN3+. It is inconsistent with previous study . The posAll women with ASC-US and LSIL results were evaluated by biopsy in this study, allowing us to analyze the performance of p16/Ki-67 for triage of these cytology categories. For these women, p16/Ki-67 achieved sensitivity equal to HR-HPV test but with significantly improved specificity, which lead to 50% decrease to colposcopy. Our findings support that p16/Ki-67 can be a viable option for the triage of equivocal and mildly abnormal Pap cytology results, and this is consistent with previous, mostly retrospective studies or studies performed within a colposcopy clinic -26.vs. 71.1%) and the specificity was significantly lower (52.7% vs. 74.7%). Importantly, p16/Ki-67 could cut the referral rate by more than 30% compared with immediately referring of all HR-HPV positive women to colposcopy. Furthermore, there was no significant difference between the performance of p16/Ki-67 and LBC. However, in rural areas of China, where skilled cytopathologists are not available, the broad use of LBC is unrealistic. Interestingly, the recent published studies showed that the interpretation of p16/Ki-67 dual staining could be performed by staff not trained in the morphological interpretation of cytology after a short training phase, and the experimental reproducibility is quite good [Primary HPV DNA testing is believed to be efficient in cervical cancer screening, especially in China, where trained cytopathologists and health care workers are in great shortage. The challenge for HPV DNA testing as primary screening is its lower specificity. Strategies are needed to prioritize women at high cancer risk for immediate intervention. So far, several triage options were considered, including Pap cytology, HPV-genotyping, HR-HPV E6/E7 mRNA or oncoproteins test, or the use of other biomarkers to detect underlying HG CIN, such as p16/Ki-67. Previous studies have been performed to evaluate p16/Ki-67as a triage marker for HPV-positive women with normal cytology , 27. Ourite good , 29, indWith regard to a study weakness, it is important to note that this study was conducted in a mixed population including women who attended cervical cancer screening or referred from colposcopy to enrich for CIN2+ endpoints. Hence, the results are not generalizable to the intended-use screening population. Furthermore, the current study focused on the cross-sectional assessment of the performance of p16/Ki-67 dual-stained cytology, which does not allow the evaluation of the long-term risk of HG CIN in women with negative results in the study. However, two recently published retrospective studies have shown a high long-term NPV of negative p16/Ki-67 in HPV-positive women , 30. FinIn summary, in a large referral and screening combined population with excellent disease ascertainment due to a rigorous colposcopy/biopsy protocol, we found that p16/Ki-67 dual-stained cytology is promising to be used for the efficient detection of cervical precancer and cancers in various settings. Future studies are needed to investigate the management algorithm to meet local needs in terms of financial and human resources, infrastructure and capacities, societal norms and level of cancer risk reduction desired.n = 5 centers) study. It was approved by the Institutional Review Board at each participating center ; Shanxi Cancer Hospital; The Second Affiliated Hospital, Sichuan University; Tianjin Central Hospital of Gynecology and Obstetrics, Henan cancer hospital). The methods were carried out in accordance with the approved guidelines. From April 2014 to March 2015, women who were attending cervical cancer screening (screening group) and women who were referred for colposcopy based on one or more prior abnormal Pap test results or a positive HR-HPV test result or other clinical suspicion of cervical cancer with local biopsy-confirmed CIN2+ (enriched group) were enrolled. All participants need to meet the following criteria: aged 30 years and older, were not pregnant, had a cervix, had not been previously diagnosed with cervical cancer and were able to provide informed consent. Exclusion criteria were previous treatment for cervical diseases (including hysterectomy or destructive therapy). Written informed consent was obtained from all participants. In the screening group, cervical cytology was collected using a cytobrush and transferred to PreservCyt solution , stored at 4°C and transported to CICAMS central lab monthly for HPV DNA analysis, Liquid-based cytology (LBC) and p16/Ki-67 dual staining. Women who were positive for any screening test were referred to colposcopy and biopsy. Directed biopsy was taken from all visible cervical lesions, otherwise, the four-quadrant punch biopsy method was indicated; biopsies were taken at positions of 2, 4, 8, and 10 o'clock depending on the quadrant, and endocervical curettage (ECC) was performed. In enriched group, cervical specimens were collected before treatment. Sample collection and processing procedure were the same as for screening group.This is a population based multicenter . The cobas HPV Test features automated sample preparation combined with real-time PCR technology to detect 14 HR-HPV genotypes. PCR amplification and detection occur in a single tube to detect 14 HR-HPV DNA: HPV-16 and HPV-18 individually and the other 12 types pooled . All the procedures were performed according to the recommendations of the manufacturer.Thin-layer cytology slides were prepared with ThinPrep Pap Test  and stained using the Papanicolaou method. Cytology slides were evaluated by senior cytotechnologists and results were reported according to the Bethesda 2001 classification system. Positive LBC cytology results are defined as atypical squamous cells of undetermined significance (ASC-US) or worse (LBC+), which resulted in referral to colposcopy in the cervical cancer screening group.A second cytology slide was prepared from the residual PreservCyt material for p16/Ki-67 which was conducted using the CINtec PLUS Cytology kit  according to the manufacturer's instructions. Samples with one or more cervical epithelial cells that simultaneously showed brownish cytoplasmic immunostaining (p16) and red nuclear immunostaining (Ki-67) were classified as positive regardless of the morphological appearance of the cells. Slides without any double-stained cells were called negative for p16/Ki-67 dual-stain cytology. All the slides were reviewed by a trained cytologist in CICAMS blindly to other tests' results.INK4A immunohistochemistry (IHC) staining  and progestogen receptor (PR) IHC staining  were used as an adjudicator for these selected cases. For the purposes of the study, CIN2, CIN3, squamous cell carcinoma (SCC), and adenocarcinoma (ADC) were referred to as CIN2+ cases, and the other cases were referred to as CIN2- cases.Histopathological diagnosis was made by local pathologists firstly, and then all the CIN, HPV DNA negative cervical cancer and adenocarcinoma cases were selected and reviewed by a panel of expert pathologists from each center. The final diagnosis was based on the results of the panel review. Additionally, p16The study targeted the recruitment of 196 CIN2+ cases, which with a clinical sensitivity of 85% for CIN2+ would result in a 95% confidence interval of approximately ±5%,i.e., 80% to 90%. Chi square of trend for proportion was calculated to test linear associations between screening methods and increasing severity of cytological and histological diagnoses. Associations between p16/Ki-67 expression and HR-HPV positive were examined using logistic regression models. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated for 2 different endpoints, CIN2+ and CIN3+. Estimates were provided with their 95% confidence intervals (95%CI). In addition, area under ROC curve (AUC) and referral rates to colposcopy based on test positivity were calculated. McNemar tests were used to compare paired matching data such as sensitivities, specificities, fractions of positive results and referral rates between different screening methods. All P values less than 0.05 (two-sided) were considered to be statistically significant. SPSS 13.0  was used for the analyses.


Knowledge about cervical cancer (CC) risk factors and benefits of CC prevention motivates women to participate in its screening. However, several studies show that there is a significant knowledge deficit worldwide about human papillomavirus (HPV). The current study explores the level of knowledge about HPV and CC prevention in the context of sociodemographic and behavioral characteristics of women who visited an antenatal clinic in Arkhangelsk, Russia.χ2 test for categorical variables were used in the comparisons of women grouped as having either poor or sufficient knowledge. Linear regression analysis was also employed.This cross-sectional study was conducted in the city of Arkhangelsk, which seats the administrative center of Arkhangelsk County, Northwest Russia. It included women who consulted a gynecologist for any reason between January 1, 2015 and April 30, 2015, were residents of Arkhangelsk, 25 to 65 years of age and sexually active (N = 300). Student’s t-test for continuous variables and Pearson's The level of knowledge about HPV and CC prevention was associated with education, parity, age of initiating of intercourse, and sources of information. After adjustment, women with university education were more likely to have higher knowledge about HPV and CC prevention compared to those with lower education.We observed that most participants had a sufficient level of knowledge. Educational gaps were identified that potentially could be used to tailor interventions in CC prevention. Human papillomavirus (HPV) is one of the most prevalent sexually transmitted infection in both women and men worldwide and more than 100 HPV types are known , 3.CC is the fourth most common cancer affecting women worldwide, with estimates of 527 624 new cases and 265 572 deaths in 2012 .Knowledge about CC risk factors and benefits of its prevention motivate women to participate in screening . HoweverHPV-related knowledge has been explored and described in countries worldwide –14, althSamoylova Clinical Maternity Hospital, which serves as an antenatal clinic for all Arkhangelsk city districts.This cross-sectional study was conducted in the city of Arkhangelsk that seats the administrative center of Arkhangelsk County in Northwest Russia. On January 1, 2015, Arkhangelsk city had a population of 350 982 . EnrolmeA sample size of 300 with an HPV prevalence of 10% was calculated to satisfy the condition (1-β) ≥ 0.80 at α = 0.05. Women who came to a gynecologist for any reason, were sexually active residents of Archangelsk and aged 25 to 65 years were invited to participate in the study. Since there are no national guidelines that regulate the cervical screening age in Russia, we adopted that of the United Kingdom NHC Cervical Screening Programme (NHSCSP). The latter recommends that routine screening be conducted during the 25–65 age interval . Of the The questionnaire sought information about the participants’ knowledge about HPV and CC prevention (through 14 pertinent questions), as well as specific details about their sociodemographic status  and their sexual behavior characteristics  test and related abnormal findings and their management). All the questions were accompanied by several possible answers to choose from. For the questions on knowledge about HPV and CC prevention, only one answer could be keyed in as correct.Participants’ knowledge about HPV and CC prevention was used as both a discrete variable (number of correct answers from 0 to 14) and a binary variable. For the latter, we considered having at least 50% of the questions answered correctly (7–14 out of the 14 questions) as a sufficient level of knowledge, and less than 50% of the questions answered correctly (6 or less out of the 14 questions) as a poor level. Sources of knowledge about HPV and CC prevention included TV/media, physician, or other (including family and friends). The frequency of screening for CC was categorized as once in: six months, a year , and in 3–5 years. The last screening the participants received was categorized into four intervals: less than 3 years ago, more than 3 years ago, never, and do not know.Age as a variable was considered as both continues (years) and categorical (25–44 or ≥ 45 years). Education was categorized as university level or less than university level. Women were divided into three groups based on their marital status: married, cohabiting, or single (including divorced or widowed). Parity was classified as 0, 1, or ≥ 2 deliveries. Smoking was designated as yes or no (ever and never smokers).Age of initiating intercourse was used as a continuous (years) and a categorical variable  and the number of lifetime sexual partners was recorded as three and less or more than three. History of sexually transmitted infections was categorized into ever had or never had, and contraception into four categories .χ2 test for categorical variables.Histogram was used to describe the distribution of HPV and CC prevention knowledge (used as a discrete variable) among the study participants. Means and standard deviations for age of the participants and the age of intercourse initiation, and proportions for other sociodemographic and sexual behavior categories were calculated for each of the poor and sufficient levels of knowledge. This statistical comparison between the two levels of knowledge groups was carried out using the independent Student’s t-test for continuous variables and Pearson's We used linear regression analysis to estimate possible associations between the level of knowledge about HPV and CC prevention (when used as a discrete variable) and sociodemographic and sexual behavior characteristics. Crude and mutually adjusted regression coefficients were calculated with 95% confidence intervals. The p-value <0.05 was considered to be statistically significant. The analysis was carried out using SPSS version 24 .All study participants provided written informed consent. The research was approved by the Research Ethics Committee of Northern State Medical University of Arkhangelsk, Northwest Russia Report Number 01/02-17 obtained on 01/03/2017; and the Norwegian Regional Committee for Medical and Health Research Ethics (RECNorth), Tromsø, Norway, Registered Report Number 2014/1670.The mean age of the study participants was 35.8 (9.0) years. Out of 300 participants, 163 (54.3%) had university education, 166 (55.3%) were married and 72 (24%) were cohabiting, 123 (41.0%) had one delivery, and 116 (38.7%) had two or more deliveries; and the prevalence of smoking among the participants was 13%. Almost half of the women (45.3%) reported to ever have had sexually transmitted infection. The mean age of initiating intercourse was 18.3 (2.5) years, and 50.3% reported use of contraception. Condom use was the most common method of contraception (38.3%), with 6.7% indicating the use of combined oral contraceptive pills and 5.3% intrauterine devices.The 14 questions used to estimate women’s knowledge about HPV and CC prevention, the answer keys, and number of women who answered correctly for each of the 14 questions are presented in Of the 300 responders, 74.7% understood the role of having multiple sexual partners as a risk factor of CC, and 67% were generally aware of the prevalence of HPV and sexually active people will likely contract HPV in their lifetime. However, only 35.7% of the women knew about the existence of a vaccine against HPV, and only 9.7% understood that one ought to have follow-up screening after vaccination. Moreover, 79.3% of the study participants answered incorrectly that having had HPV vaccine prevents the development of CC, and most (90%) answered incorrectly that most HPV types clear up on their own.Out of all participants, 37.1% had undertaken screening  less than three years ago; 7.0% more than three years ago; 38.0% never had a Pap test; and 17.7% did not know. Among those who knew that screening can detect CC in its early stages, 48.5% had this test less than three years ago; 8.6% more than 3 years ago; 29.3% never had a Pap test; and 13.6% did not know (p < 0.001).About one third of the study participants reported that their doctor was the main source of information about HPV and CC prevention. Others mentioned TV/media (53.3%) and other sources of information (12.7%).Of the 300 participants, 20.0% had a poor level of knowledge about HPV and CC prevention and 80.0% had sufficient knowledge. Selected characteristics of the study participants stratified by their level of knowledge about HPV and CC prevention are provided in Women in both groups were of comparable age (in their mid-thirties). The level of knowledge about HPV and CC prevention was associated with education (p = 0.049), parity (p = 0.049), age of initiating of intercourse (p = 0.014), and source of information about HPV and CC prevention (p = 0.006) . More spCrude differences in the number of correct answers on the 14 questions about HPV and CC prevention was significant between the educational levels, and were even more pronounced after adjustment. Women with university education were more likely to have higher knowledge about HPV and CC prevention compared to women with lower educational level. Having two or more deliveries was associated with having more questions on HPV and CC prevention answered correctly when compared to nulliparous women. However, this difference was not statistically significant after adjustment. In the crude and adjusted linear regression models age, marital status, smoking, age of initiating of intercourse, number of partners and history of sexually transmitted infections were not associated with the number of correct answers to the 14 questions about HPV and CC prevention.Our study demonstrates that majority of women in Arkhangelsk had sufficient level of knowledge about HPV and CC prevention, and this was associated with level of education, parity, age of initiating of intercourse, and source of information about HPV and CC prevention. After adjustment, women with university level of education were more likely to have a higher score of correct answers on knowledge about HPV and CC prevention compared to less educated women.The level of knowledge and awareness about HPV and CC prevention has been suggested to be important in the development of positive approaches toward CC prevention . Our resWhile many participants in our study were aware of the sexual transmission of HPV, gaps in knowledge about symptoms and treatment of HPV infection were nevertheless evident. Although HPV is one of the most common sexually transmitted infections, it is transient and thus women tend not to seek treatment. Our findings demonstrate that close to 90% of women think that HPV should be treated. This result can be partly explained by the misinformation provided by some health care professionals and pharmaceutical companies that detection of HPV requires antiviral treatment. The HPV test is commercially and widely available in Russia. Another possible explanation is a wide use of colposcopy in Russia, while the number of educational courses and amount of literature on how to perform this procedure correctly is limited , 23. ThiWomen in our study have exhibited good knowledge about CC risk factors. Our study also shows that knowledge about the latter and CC screening is of great importance. Thus those women who were aware that the Pap test is used for CC screening took this test more often than women who did not. However, our analysis also reveals that there was insufficient knowledge about HPV vaccination (in terms of the development of CC and need of Pap test). Although the HPV vaccine is the most effective way to prevent HPV infection, it is not widely available in Russia as it is not yet included in the Russian official vaccination program. These findings highlight a great importance of educational efforts in CC prevention.We found no association between the level of knowledge about HPV and CC prevention and age. An earlier study reported that younger women had a higher level of knowledge of HPV and CC prevention . YoungerWith respect to age, all women in our study were at risk of getting HPV infection and thus developing CC. Clearly, they constitute an age group to whom preventive measures such as CC screening must be directed. However, Tiro et al.  showed tRisky behavior ─ specifically early age of intercourse initiation and high number of sexual partners ─ is generally accepted as increasing the risk of HPV infection . We founWe observed no association between the level of HPV and CC knowledge and history of sexually transmitted infections. Even though women with such history had to seek a physician about their infection, they had not received information about HPV or had forgotten it. This suggests that clinicians need to pay more attention to women`s education about HPV and HPV associated cancers, as well as other sexually transmitted infections. Neither was there an association with the type of contraception. Moreover, every second woman did not use contraception at all. One of possible explanations could be that they are more concerned about being infected with chlamydia or gonorrhea than with HPV. In doing so, they may have underestimated the seriousness of an HPV infection and as a consequence fail to seek help.Interestingly the level of HPV and CC prevention knowledge was associated with source of information. Sufficient level of knowledge was more common among those who reported having a doctor as the primary source of information. Despite the fact that more than half of the study participants received knowledge about CC and its prevention from media and TV, there was no difference in the knowledge level in this context. Since there was a difference for physicians as the information source, we conclude that they could provide more precise and accurate knowledge about CC and its prevention. Consequently for the proper implementation and acceptability of cervical cancer screening, physicians need to provide more education on the importance of routine screening for cervical cancer prevention. In addition, educational campaigns for women are needed, since social surrounding also constitutes a source of information that leads to a poor level of knowledge.Holcomb et al.  also obsSeveral Russian studies and those conducted in former Soviet Union countries have evaluated level of knowledge about HPV and CC among specific groups, such as medical students and health professionals , 12, 13.Our study has several potential limitations. In spite of a relatively high response rate (86%), bias due to non-participation remains a possibility. Taking into account that this is a cross-sectional study and all the information was self-reported, recall bias may have occurred. Moreover, the data were collected at an antenatal health care center, and therefore a built-in selection bias may have occurred. It is possible, for example, that individuals who participated in the survey were more health conscious and curious about HPV than women in the general population. It is also important to mention that our study sample might have over represented frequent users of the health care system, and thus under represented those who live in remote and poorer regions of Arkhangelsk with less access to health care services. If true, the knowledge about HPV and CC prevention would probably be lower than what we assessed. Because of the cross-sectional nature of the study, the associations observed between the level of knowledge about HPV and CC prevention and selected behavioral characteristics cannot be interpreted as causal.To our knowledge, this is the first study in Russia aiming to assess knowledge about HPV and CC prevention and associations between level of this knowledge and a range of sociodemographic and sexual behavioral characteristics among women. Our study population was comprised of women in the age group at risk of HPV infection, and therefore constituted the target group for CC screening and prevention.Our study provides detailed information on knowledge about HPV and CC screening among women of Arkhangelsk, Northwest Russia. We observed that most participants had a sufficient level of knowledge. Women with a university level of education displayed the highest knowledge scores about HPV and CC prevention. Health care professionals as the primary source of information are well positioned to improve women’s knowledge about HPV and CC screening, including promotion of preventive measures. However, a large proportion of participants did not know that most HPV types clear up on their own, that HPV vaccination does not avoid the development of CC nor replaces the need for a Pap test later in life. Consequently, there is need to improve knowledge about the role of CC screening and HPV vaccination in CC prevention among women of Arkhangelsk. These educational gaps might be used to tailor interventions in CC prevention. Consequently, ongoing educational campaigns that emphasize the role of HPV in CC development and importance of regular screening and HPV vaccination are recommended.S1 QuestionnaireIn Russian.(DOC)Click here for additional data file.S2 QuestionnaireIn English.(DOCX)Click here for additional data file.


E2. Three HPV-6-positive patients developed recurrent lesions, and HPV-6 copy numbers and mRNA expression decreased after surgical treatment. Among the 96 CpG sites (16/case), 67 (69.8%) were unmethylated, while 23 (30.2%) were heterogeneous (≥ 1 methylated CpG clone). High viral loads and episomal status of HPV-6 were frequently observed in AO-LP; thus, persistent E6/E7 mRNA expression of LR-HPV-6 may be associated with AO-LP recurrences. Hypomethylation and scattered patterns of methylated CpGs at the URR of HPV-6 were identified in AO-LP.The methylation status of HPV-6 upstream regulatory region (URR) in adult-onset laryngeal papillomatosis (AO-LP) remains unclear. The purpose of this study was to investigate the methylation status of URR and the physical status of HPV-6, as well as the dynamic variations of viral load and mRNA expression in AO-LP. We examined 18 specimens from 11 patients with AO-LP by real-time polymerase chain reaction (PCR), bisulfite-sequencing PCR, and amplification of papilloma oncogene transcripts. HPV-6 was identified in 9 of 11 patients (81.8%), and all the 15 specimens derived from 9 HPV-6-positive cases contained only episomal HPV-6 transcripts with intact  Human papillomaviruses (HPVs) are small icosahedral viruses containing 8 or 9 genes on circular double-stranded DNA. More than 150 types have been identified to date. HPV infection can stimulate benign tumors, promote oncogenic changes, or be asymptomatic in epithelial cells , 3. AO-LIt is well known that oncogenic HPVs encode two main viral oncoproteins, E6 and E7, which are responsible for the maintenance of HPV-mediated malignant transformation by their interactions with p53 and retinoblastoma (pRb) tumor suppressor proteins, respectively. Unlike high-risk HPVs, E6 and E7 proteins of LR-HPVs have substantially lower transforming activity and do not induce genomic instability. LR-HPV E6 proteins lack the ability to degrade and inactivate p53 , while lE6 and E7 oncogene expression by impairing the ability of E2 to bind to its specific sites [E6 enhancer region or the E2BS [E6/E7 mRNA expression in SCC of the head and neck in our previous study, suggesting that variations in HPV viral load might reflect differences in the capacity of the viral DNA to replicate and its transcriptional activity [The methylation status of the HPV-16 genome in cancer cell lines and clinical samples of the cervix, head, and neck has been analyzed and identified as an important feature in malignant transformation –16. Previc sites , 17. In the E2BS . Howeverthe E2BS  demonstractivity . With reE6/E7 mRNA in AO-LP, and the dynamic variations of HPV-6 among primary tumors and recurrent tumors after treatment. Moreover, we analyzed the relationship between the methylation pattern of the HPV-6 URR and viral load in AO-LP.In this study, we investigated the methylation status of the URR of LR-HPV-6, the physical status and expression of The clinicopathological characteristics of 18 specimens obtained from 11 patients with laryngeal papilloma are summarized in Table E6 mRNA transcripts for HPV-6 were detected in all cases positive for HPV-6 DNA, and the median mRNA quantity was 0.20 (range 0.08 to 0.43).The pre-operative viral load in 9 primary laryngeal papilloma samples positive for HPV-6 DNA was first evaluated. The relative HPV-6 viral load was normalized by the β-globin standard curve and adjusted to values per nanogram of input DNA for each sample. The viral load of HPV-6 ranged from 2,933 to 2,268,452 copies/ng, with a median of 391,015 copies/ng. E6 mRNA in case 7 slightly decreased from 0.21 to 0.15. Case 5 had 4 recurrences after the primary operation, and the viral load and the expression of E6 mRNA gradually decreased from 178,463 and 0.18, respectively, to 9,550 and 0.10.  that allows differentiation between episome- and integrate-derived  genomes . RegardlBased on a manual comparison of sequence data from the URR as described by Combrinck et al. , HPV-6a,INK4a, p53, and pRb) in 9 of 11 patients with HPV-positive or -negative AO-LP, including cases 1 to 9, was examined by immunohistochemistry  and p53 expression  were expressed in the unique case that developed asynchronous laryngeal papillomatosis and carcinoma.The expression of cell cycle proteins  was consistent with previous studies, in which 75–80% of the papillomas harbored HPV-6 , 23. Regal HPV-6 . A previal HPV-6 , and onlIn a previous study, Huebbers et al. reported a female patient who underwent several hundred revision surgeries for juvenile-onset recurrent respiratory papillomatosis, and eventually developed laryngeal SCC. They identified only the episomal virus in the papilloma and HPV-6 integration in the carcinoma. Loss of the expression of AKR1C3 induced by viral integration may be related to malignant transformation of papillomatosis . In the To our knowledge, this study is the first to demonstrate dynamic variations in HPV-6 viral loads and mRNA expression among patients with AO-LP. Although no relationship between viral load and recurrence could be established due to the limitation of sample size, our results showed a gradual decline in the tendency of HPV-6 copy numbers and mRNA expression over time after surgical treatment. Both the findings of Forslund et al.  and our E6 and E7 oncogenes by adjusting the binding of viral E2 protein to the E2BSs [E6 and E7 expression levels, and the methylation of E2BS3 and E2BS4 in oropharyngeal SCCs is associated with E2 integrity and viral physical status. However, there are few studies on the methylation of LR-HPVs in AO-LP. Ure and Forslund reported a lack of methylation in HPV-6 URR from AO-RRP by analyzing 360 clones derived from both basal/intermediate and superficial cells of 6 HPV-6-positive samples. In their study, no methylated CpG was detected in any of the URRs investigated [E6 and E7 oncogenes in AO-LP. Moreover, the low frequency of p16INK4a overexpression and low expression of p53 in AO-LP were independent of HPV-6 infection in this study. A previous study by Mooren et al. revealed highly variable staining patterns of p16INK4a in head and neck papillomas and laryngeal dysplasia, irrespective of HPV status [INK4A, p53, and pRb. Further studies of large sample sizes should be conducted to investigate associations between the expression of cell-cycle proteins and recurrence.Previous studies have demonstrated that the methylation of high-risk HPV URR is an important feature in cervical neoplasms, and that it regulates the expression of he E2BSs , 17. Reuhe E2BSs  found thstigated . In the V status . We did E6/E7 mRNA expression of LR-HPV-6 may be associated with the recurrence of papillomas. Hypomethylation and the scattered patterns of methylated CpGs at the URR of HPV-6 were identified in AO-LP, but our findings did not provide any evidence of malignant transformation from laryngeal papilloma to carcinoma induced by HPV-6. P16INK4a cannot be recommended as a surrogate biomarker for HPV infection in AO-LP.In summary, high viral loads and episomal HPV-6 were frequently observed in AO-LP, and persistent The study was approved by the Ethics Committee of University of the Ryukyus, and all participants gave written informed consent. All procedures were conducted in accordance with the ethical standards of the Declaration of Helsinki (1975).The study included 16 fresh-frozen tissues and 2 formalin-fixed paraffin-embedded (FFPE) specimens obtained from 11 patients with AO-LP who were diagnosed and treated in the Department of Otorhinolaryngology, Head and Neck Surgery, Graduate School of Medicine, University of the Ryukyus, between September 2009 and February 2017.Tissue samples were snap-frozen in liquid nitrogen during biopsy or surgical excision and stored in liquid nitrogen for further analysis. A Gentra Purification Tissue Kit  was used to isolate DNA from the fresh-frozen samples. The extraction processes were conducted according to the manufacturer’s specifications using the solutions provided in the kit. For the FFPE samples, DNA was extracted by the TaKaRa DEXPAT Easy  following the manufacturer’s instructions.Total RNA was isolated from 5- to 10-mg frozen tumor samples using the ToTALLY RNA kit , according to the manufacturer’s instructions, and suspended in 50 μL ultra-high-quality diethyl pyrocarbonate-treated water. Before cDNA synthesis, any residual DNA was removed through incubation with 1 U DNase I (Ambion) at room temperature for 25 min. cDNA was then synthesized from DNA-free total RNA using the RETROscript Kit (Ambion), according to the manufacturer’s instructions. All assays were performed both with and without reverse transcriptase to examine the presence of contaminating DNA in RNA samples.The presence of HPV-6 DNA was detected by PCR with two general consensus primer sets, GP5+/GP6+ and MY11/MY09, as described previously . PositivE6 open reading frames . Amplification conditions were as follows: 2 min at 50 °C, 10 min at 95 °C, and a two-step cycle of 95 °C for 15 s and 60 °C for 60 s for a total of 50 cycles. A standard curve for the E6 genes was generated by amplification of serial 10-fold dilutions  of a plasmid p1478 carrying the HPV-6b E6 early region . Viral DNA load was determined by calculating E6 copy numbers. Cellular DNA quantification was determined by PCR amplification of β-globin, as described previously [To detect the viral loads of HPV-6, a quantitative real-time PCR (qRT-PCR) was conducted with the ABI Prism 7300 Sequence Detection System  and Taqman PCR Master Mix II . We designed the primers and Taqman probes to target the HPV-6 es Table . The E6 eviously .E6 mRNA was described as each value relative to β-actin mRNA. The β-actin primers and probes were designed as reported previously [A qRT-PCR with the same E6 primers and probes and the same protocol as that described for the detection of viral load in the previous section was used to estimate cDNA viral load as a measurement of mRNA quantity in the samples, and the quantitative value of eviously .The 3′ rapid amplification of cDNA ends (RACE) amplification of papillomavirus oncogene transcripts (APOT) assay described previously by Klaes et al.  was modi2, 10 pmol of each primer, 1.25 units AmpliTaq DNA Polymerase (Applied Biosystems) and 300 ng bisulfite-modified DNA. Amplification was carried out under the following conditions: 95 °C for 5 min, followed by 45 cycles at 95 °C for 1 min, 54 °C for 1 min and 72 °C for 2 min, with a final extension at 72 °C for 7 min.DNA 1–2 μg) extracted from tissue samples was converted with bisulfite using an EpiTect1 Plus Bisulfite Kit , according to the manufacturer’s protocol. The modified DNA was amplified in 3 amplicons with bisulfite-sequencing PCR (BSP) primer assays as BSP-1, BSP-2, and BSP-3 covering the 5′-LCR, and enhancer and promoter regions of the HPV-6 URR, containing 16 CpGs and E2BSs 1, 2, 3, and 4. The primer sequences are shown in Table  μg extraAfter checking in 2% agarose gel, amplified products were gel purified using a Wizard1 SV Gel and PCR Clean-Up System (Promega) and cloned into the pCR™4-TOPO1 vector  for sequencing. For each amplicon, at least 6 individual clones were sequenced using an ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems).INK4a antibody  for p16INK4a staining, primary monoclonal mouse anti-p53 antibody  for p53 staining, and primary monoclonal mouse anti-pRb antibody  for pRb staining. After extensive washing in PBS, the slides were incubated for 30 min at room temperature with a horseradish peroxidase-conjugated goat anti-mouse secondary antibody (MTM Laboratories). Immunolabeling was visualized by incubation in 3-3′-diaminobenzidine and stained slides were counterstained with hematoxylin. Cases were considered pRb-, p53- or p16INK4a-positive if intense nuclear and/or cytoplasmic reactivity was present. Positive expression was defined as pRb and p53 staining in more than 25% [INK4a staining in 40% of 2000 tumor cells [Serial 4-μm-thick sections from FFPE samples were deparaffinized in xylene and hydrated in a graded series of alcohol. Epitope retrieval was performed by heating at 95–99 °C for 10 min in Tris/EDTA buffer (pH 9.0). Endogenous peroxidase activity was quenched by incubating the sections in 3% hydrogen peroxide and 15 mM sodium azide for 5 min. The sections were subsequently incubated overnight at 4 °C with primary monoclonal mouse anti-p16than 25% , or p16Ior cells .


In early breast cancer, the optimal technique for sentinel lymph node biopsy (SLNB) is the combined technique (radioisotope and Patent Blue V) which achieves high identification rates. Despite this, many centres have decided to stop using blue dye due to blue-dye-related complications . We evaluated the SLNB identification rate using the combined technique with and without Patent Blue V and the blue-dye-related complication rates.Clinical and histological data were analysed on patients undergoing SLNB between March 2014 and April 2015. SLNB was performed following standard hospital protocols using the combined technique.n = 158/160), with blue dye alone 92.5% (n = 148/160) and with radioisotope alone 97.5% (n = 156/160). A total of 76.9% (263/342) of nodes were radioactive and blue, 15.5% (53/342) only radioactive and 2.3% (8/342) only blue, 5.3% (18/342) were neither radioactive nor blue. No anaphylactic reactions were reported and blue skin staining was reported in six (3.8%) patients.A total of 208 patients underwent SLNB and 160 patients (342 nodes) with complete operation notes were available for final analysis. The identification rate with the combined technique was 98.8%  has comparable sentinel node identification rates in experienced hands. National guidelines are required to optimise operative documentation. In early breast cancer, sentinel lymph node biopsy (SLNB) is the preferred method for axillary staging –2. SLNB The combined technique using both radioactive TcM99m and blue dye is the current golden standard for SLNB –6. IdentDespite the recognised superiority of the dual technique over radioisotope alone, many centres have decided to stop using blue dye due to blue-dye-related complications  or lack of licensing for blue dyes –13. We eClinical and histological data were evaluated on patients undergoing SLNB between March 2014 and April 2015. This study was approved by the Clinical Review Board (ref 5320).All patients who underwent SLNB for newly diagnosed breast cancer were included. In the case of a bilateral SLNB, only the side with the highest grade, largest primary tumour size or most involved nodes was included to avoid confounding variables.Patients were excluded if there was no procedure description available in operative or pathology notes, if only an injection of radioisotope or Patent Blue V was administered, or if the colour or radioactivity within nodes was not documented.ex vivo. Operation notes included the amount of Patent Blue V injected, amount of nodes excised and if these nodes were blue and/or radioactive. A successful SLNB was defined as a procedure in which at least one true sentinel node was identified to be either blue and/or radioactive.All SLNB procedures were performed using radioisotope, Patent Blue V and a scrubbed consultant surgeon. Patients scheduled to undergo SLNB in the Breast Unit at Guy’s and St. Thomas’ NHS Foundation Trust were injected with radioisotope on the morning of surgery either at the Nuclear Medicine Department or in Day Surgery Unit. Prior to surgery, Patent Blue V was injected peri-areolarly on table followed by a massage of the injection site. During SLNB, the surgeon used a gamma probe to detect the radioisotope and visually identified blue nodes. Any nodes with a count of 10% or more of the highest node count were excised. Beyond four nodes, surgeons excised additional nodes only at their discretion and noted the background count on completion. Palpable nodes felt during the procedure were also removed. For every excised node, the gamma probe was used to determine the radioactivity of nodes All excised SLN’s were sent for histopathological evaluation in accordance with national protocols. Nodes were reported as either normal or involved containing isolated tumour cells (ITC) (≤ 0.2 mm), micrometastases (> 0.2 and ≤ 2.0 mm) or macrometastases (> 2.0 mm). The size of the largest deposit was also reported by the pathologist.p = 0.97, we were prepared to accept non-inferiority with a margin of δ = 0.05. The probability that the addition of radioactivity results in finding new nodes is r0 = kδ with k = 0.2. With 90% power and a 95% one-sided confidence level, zα is the standard normal variate value corresponding to the test size, zα = 1.6449 and zβ the variate corresponding to the test power, zβ = 1.2816. Using the following formula gives a sample size of N = 148 patients.To statistically determine whether SLNB with Patent Blue V provides a non-inferior identification rate compared to the combined technique, a sample size calculation was performed. With an identification rate for the combined technique of p the identification rate of the combined technique. The probability of detecting a node with blue dye or with radioisotope alone should not be lower than p-δ. [A non-inferiority test was performed to define whether the blue dye technique alone is non-inferior to the combined technique using δ = 0.05 and han p-δ. –15t-test. For tumour grade, lymph node status, the presence of lymphovascular invasion, ER/HER2 status, and surgeon’s experience, we performed a crosstab and Fisher’s exact test. We repeated the analysis for sentinel node identification with the combined technique, radioisotope alone, and blue dye alone.To evaluate potential differences in sentinel node identification with different tumour size and volume of blue dye, we performed an independent A total of 208 patients underwent SLNB between March 2014 and April 2015. In the 48 patients who were excluded, operation notes were incomplete in 38 patients (18.3%) and operation notes were missing in eight patients (3.8%). One patient (0.5%) was excluded because she only received Patent Blue V and one patient (0.5%) only received radioisotope. A total of 160 patients in which 342 nodes (range 1–6 nodes) removed were available for final analysis. Patient and tumour characteristics are tabulated in About 78.8% (126/160) procedures were performed by specialised breast surgeons who have over 10 years of experience and 21.2% (34/160) were performed by fellows specialised in breast surgery.In 80 cases (50.0%), SLNB’s were performed on the left and 80 cases (50.0%) on the right. There were 12 cases (7.5%) of bilateral SLNB. SLNB was performed in combination with a mastectomy in 47 cases (29.4%), wide local excision (WLE) in 89 cases (55.7%), and re-excision in five cases (3.1%). In 19 cases (11.9%), an SLNB alone was performed.In 24 cases (15.0%), 1.0 ml of Patent Blue V dye was injected, 2.0 ml was injected in 70 cases (43.8%) and the volume of injected Patent Blue V dye was not documented in 66 cases (41.2%). All 1.0 ml injections were not diluted apart from one which was diluted with 2.0 ml of saline. About 40% (28/70) of 2.0 ml injections were not diluted and 60% (42/70) were diluted with 3.0 ml of saline.n = 158/160), with blue dye alone 92.5% (n = 148/160) and with radioisotope alone 97.5% (n = 156/160) (The sentinel node identification rate with the combined technique was 98.8% (156/160) . In two The extra number of nodes detected by radioisotope was 6.3% ) using the normal approximation of Agresti and Coull . The comt-test found no statistically significant difference in sentinel node identification between patients with different tumour size and volume of blue dye injected. For tumour grade, lymph node status, the presence of lymphovascular invasion, ER/HER2 status and surgeon’s experience, Fisher’s exact test found no statistically significant differences with sentinel node identification.An independent In 158 patients who underwent successful SLNB, a total of 333 sentinel lymph nodes were excised, a mean of 2.1 nodes per patient. One node was excised in 47 patients, two nodes in 68 patients, three nodes in 25 patients, four nodes in 16 patients, five node in one patient and six nodes in one patient. These nodes were both radioactive and blue in 263 nodes (79.0%), only radioactive in 53 nodes (15.9%), only blue in eight nodes (2.4%) and neither radioactive nor blue in nine nodes (2.7%) .The patients with sentinel lymph nodes not detected by blue dye were injected with 1 ml of blue dye , 2 ml , or the amount was not mentioned . Patients with nodes not detected by either blue dye or radioisotope were injected with 1ml of blue dye , 2 ml  or this was not mentioned .The two patients with a failed SLNB had nine nodes excised after injection of 2 ml blue dye  or this was not mentioned .On histopathological examination, 35 patients (16.8%) had involved lymph nodes, 15 patients (9.4%) had micrometastases and 20 patients (12.5%) had macrometastases . In thesOf 18 nodes with micrometastasis, 15 (83.3%) were both radioactive and blue and three (16.7%) were only radioactive. Of 25 nodes with macro-metastases, 18 nodes (72.0%) were both radioactive and blue, six nodes (24.0%) only radioactive and one node (4.0%) only blue . Therefon = 33), haematoma (n = 6), breast oedema (n = 2), infection (n = 2), erythema (n = 1), reduced shoulder movement (n = 1), pain (n = 1), a candida infection (n = 1) and an undocumented complication (n = 1).Post-operative complications were seroma (n = 6/160) of patients which successfully underwent SLNB and no anaphylactic reactions related to blue dye were reported.Blue staining of the skin was reported in 3.8% . Documentation of retrieval of blue or radioactive nodes confirms if the SLNB procedure was successfully completed. There are currently no national  guidelines describing the minimal data set required for the documentation of SLNB procedures. SLNB carries a 6–10% false-negative rate when performed using the standard combined technique, it is likely to be higher if the combined technique is not used with consequently lower identification rates. In this study, the false negative rates for blue dye and radioisotope were 21.4% and 2.9%, respectively. However, no axillary node clearance was performed, to be able to obtain the correct false-negative rate for both techniques. In order to be able to audit SLNB identification rates, it is essential to know the number of nodes removed, their colour and radioactivity count. Guidelines are needed to ensure standardised administration of blue dye and adequate documentation of these procedures, including the type, amount, and dilution of tracers used.Blue dye causes a tattoo at the site of injection and carries a 0.6% risk of anaphylaxis. Complications due to blue dye may be underreported due to inadequate documentation. In this study, a blue tattoo was only reported in 3.8% (six patients) suggesting this is significantly under-reported as it is often asymptomatic. Furthermore, the blue staining was only reported up to three weeks after SLNB, without documentation of resolution. With the increased number of patients suitable for SLNB, it is important to reduce the risk of blue-dye-related complications. In order to understand the impact of the issue, correct documentation is necessary. Therefore, there should be greater emphasis on standardisation of surgical techniques.With respect to nodal involvement the combined technique should be used since if only the radioisotope or blue dye techniques are used alone, involved nodes would have been missed . The limIn the search for new SLNB techniques, it will be difficult to find a technique with an identification rate superior to that of the combined technique in view of its very high identification rate. However, if we can develop a single agent or device that can accomplish the same level of accuracy and rule out the use of radioisotopes, it is likely that the combined technique will be replaced. Currently, several new techniques are under investigation, including the magnetic technique, using superparamagnetic nanoparticles and a hand-held magnetometer, showing an identification rate between 94.4% and 98.5% , 18, 19 The combined technique should continue to be the preferred technique for SLNB and should be standardised. Radioisotope alone  has comparable sentinel node identification rates in experienced hands. National guidelines are required to optimise operative documentation.ALNDAxillary lymph node dissectionSLNBSentinel lymph node biopsyWLEWide local excisionITCIsolated tumour cells“The author(s) declare that they have no conflict of interest.”


Multivariable logistic regression models were performed to address the study objectives.Data from 2008–2012 National Immunization Survey - Teen (NIS - Teen) were examined in this study. Parents with “no-intent” to vaccinate their daughters were asked to identify reasons for their decision. All responses were categorized into five domains identified as barriers to receive the HPV vaccine series: Number of people in the household, household income, mother’s age, education, health insurance, recommendation of a health care provider, and the survey year were significantly associated with parental “no-intent”. Race/ethnicity, mother’s education, marital status, recommendation of a health care provider, household income, age of the unvaccinated daughter, and the survey year, were significantly associated with one or more domains identified as barriers to receive the HPV vaccine.This study identified sub-groups of parents across different socio-demographic factors with “no-intent” for their adolescent daughters to receive the HPV vaccine. Developing strategies that target educational tools towards the identified sub-groups of parents about the purpose, safety, and efficacy of the HPV vaccine, and HPV infection, may help increase HPV vaccine acceptance, initiation and completion rates. In the United States (U.S.), human papillomavirus (HPV) is the most prevalent sexually transmitted infection (STI) with up to 24.5% HPV infection among females aged 14–19 years . HPV typA considerable number of studies have focused on the factors associated with initiation and completion of the HPV vaccine series –30. For In a recent study among unvaccinated females aged 15–25 years, Liddon and colleagues described the correlates of the main reasons for foregoing vaccination in females . HoweverIn this study, we used a sub-sample of unvaccinated females aged 13–17 years from the National Immunization Survey - Teen (NIS - Teen), 2008–2012, to address three specific objectives: 1) To identify socio-demographic factors associated with parental “no-intent” for their 13–17 year old unvaccinated daughter to receive the human papillomavirus (HPV) vaccine series in the next twelve months, 2) to describe patterns in “no-intent” by socio-demographic factors, and 3) to identify socio-demographic factors associated with parental reasons for “no-intent”.Data from 2008–2012 NIS-Teen surveys were used for the analyses . The NIShow likely is it the teen will receive HPV vaccinations in the next 12 months?”. Of the five possible responses, parents who responded “very likely” or “somewhat likely” were considered as having “intent” and those who responded “not too likely”, “not likely at all”, or “not sure/do not know” were considered as having “no-intent” for their daughter to receive the HPV vaccine series within the next 12 months. The percentage of parents who responded “not sure/do not know” was very small (<3%). Parents with “no-intent” were then asked the question, “What is the main reason [your teen] will not receive HPV shots in the next 12 months? “ Parents were allowed to report one or more reasons from a list which CDC created. For the present analyses, we created the following five domains for the reasons parents reported: i) Safety and Effectiveness Concerns, ii) Systemic Barriers, iii) Vaccine Misinformation, iv) Lack of Knowledge about the Vaccine, and v) Socio-cultural Barriers. Table Parent (or guardian) of a female adolescent with no history of HPV vaccination at the time of the interview was asked the question “n = 5,126; 2009: n = 4,901; 2010: n = 4,614; 2011: n = 5,231; and 2012: n = 3,850), 21,467 had information on all the covariates of interest. Of these, parents of 12,716 females were in the “no-intent” group, of which 12,274 of them reported at least one reason for their decision.Of the 23,722 unvaccinated females  were computed to describe the socio-demographic characteristics of unvaccinated female adolescents. Univariate analyses were performed to test for associations between socio-demographic factors and parental “intent” vs. “no-intent” for their daughters to receive the HPV vaccine series within the next 12 months. Multivariable logistic regression analysis was performed to determine the factors associated with “no-intent”. To describe how the socio-demographic factors associated with parental “no-intent” changed over time, we computed the unadjusted weighted prevalence estimates with 95% CIs for each survey year. The sample size for these analyses was 21,467. To determine how the socio-demographic factors associated with parental reasons for “no-intent”, we performed multivariable logistic regression analysis treating the five domains as binary outcomes (Yes/No) correspondingly. The sample size for these analyses was 12,274.Sampling weights provided in the 2008–2012 NIS - Teen public use data were used to obtain population-based estimates. All analyses were performed using SAS® version 9.3  survey procedures  to account for the complex sampling design of the NIS-Teen data. All analyses were conducted in 2014.Overall, 51.1% (95% CI: 50.3%–52.0%) of the female adolescents were completely unvaccinated for HPV . The mean age of these adolescents was 14.3 years. Table During 2008–2012, 59.9% (95% CI: 58.7%–61.1%) of parents of unvaccinated adolescent females indicated that they did not intend to have their daughters vaccinated against HPV within the next 12 months. Table Table accine misinformation, 18.0% safety and effective concerns, 17.0% lack of knowledge about the vaccine, 16.0% systemic barriers, 5.5% socio-cultural reasons.Of the 12,274 parents who reported “no-intent” for their daughters to receive the HPV vaccine series within the next 12 months, 92.3% of parents cited one reason, 6.6% cited two, and 1.1% cited three or more reasons with the maximum being seven reasons. The proportion of parents who reported one or more reasons which fall in one of more of the five domains are as follows: 43.8% vSafety and Effectiveness Concerns”.Socio-demographic factors associated with parental reasons for “no-intent” for their daughters to receive the HPV vaccine series within the next 12 months is presented in Table Systemic Barriers” (AOR: 6.84 [95% CI: 5.25–8.91]) and/or “Vaccine Misinformation” (AOR: 1.18 [95% CI: 1.02–1.35]) compared to those parents of female adolescents with a provider recommendation. An increasing number of people in the household was also associated with greater odds to report vaccine misinformation (AOR: 1.08 [95% CI: 1.03–1.14]).Parents who did not receive a health care provider recommendation for their daughter to receive the HPV vaccine series had greater odds to report “Socio-cultural Barriers” were significantly greater among female adolescents 15 years of age (AOR: 1.57 [95% CI: 1.09–2.26]) and, 17 years of age (AOR: 1.52 [95% CI: 1.01–2.30]) compared to 13 years of age; among Black non-Hispanic parents (AOR: 1.63 [95% CI: 1.15–2.32]) compared to White non-Hispanic parents; among parents who reported their annual household income as > $75,000 (AOR: 1.91 [95% CI: 1.14–3.19]), and $35,001–$75,000 (AOR: 1.62 [95% CI: 1.04–2.55]) compared to parents who reported their annual household income as ≤ $35,000. Parents in survey years 2009–2012 were more likely to report safety and effectiveness concerns compared to parents in the survey year 2008. Additionally, parents in the survey year 2012 (AOR: 1.57 [95% CI: 1.17–2.09]) had higher odds of reporting systemic barriers compared to parents in the survey year 2008.The odds of reporting “This study is one of the first to identify sub-groups of parents across different socio-demographic factors who reported “no-intent” and reasons for “no-intent” for their daughters to receive the HPV vaccine series within the next 12 months in a nationally representative sample of unvaccinated female adolescents 13–17 years of age. Though the proportion of HPV vaccine uptake among female adolescents has significantly increased over time, this study found that three out of five parents of unvaccinated females reported “no-intent” to vaccinate their daughters within the next 12 months . In addition, this study describes the patterns over time in socio-demographic factors of parents who reported “no-intent”. The study findings provide useful information to develop strategies for outreach and education geared toward health care providers whose lack of recommendation for the HPV vaccine significantly influenced parental “intent” for their daughters to receive the HPV vaccine series, to address parents’ concerns about the HPV vaccine across different socio-demographic characteristics. This may help improve the vaccine uptake among females 13–17 years of age.Consistent with previous reports , 39–41, Lack of Knowledge about the Vaccine” as a reason for “no-intent”. Consistent with our finding, recent studies in Latino parents’ reported low HPV vaccine knowledge as one of the main barriers to vaccine receipt [Socio-cultural Barriers”. These findings highlight the importance of disseminating health communication about HPV across various demographics.In this study, mothers with a high school education or higher were more likely to report “no-intent” for their daughters to receive the HPV vaccine series within the next 12 months. Other studies have reported similar findings , 35, 46. receipt , 49. FurOverall, this study found that the patterns in socio-demographic factors between proportions of parents reporting “no-intent” were similar across years 2008–2012. These patterns highlight that HPV vaccine acceptance beliefs among parents have not significantly changed over time, thereby impacting vaccine uptake among certain sub-groups of adolescents identified in this study. Considering that the HPV vaccine is intended to protect against HPV infection and pre-cancerous lesions caused by high-risk HPV types, delaying the initiation or refusing the vaccine altogether by these sub-groups of parents for their daughters is a matter of public health concern.We acknowledge that the analysis of this study has some limitations. In the years 2008–2010, during which cell-phone-only households were increasing, the NIS-Teen sampling frame included only households that had landline telephones. For the first time in 2011, NIS-Teen utilized a dual-frame (both landline and cell-phone sampling frames) but the cellular household response rate was low compared to the landline household response rate in both 2011 and 2012. As a result, non-response and non-coverage bias may exist, even after adjusting sampling weights. Geographic region in the US, which we did not consider in our study, may be helpful to better understand and address parents’ reported reasons for “no-intent” to vaccinate their adolescent daughters . Also, iThis study identified sub-groups of parents across different socio-demographic factors with “no-intent” for their daughters of 13–17 years of age to receive the HPV vaccine series within the next 12 months, and their reasons for such a decision. Developing strategies that target educational tools towards the identified sub-groups of parents about the purpose, safety, and efficacy of the HPV vaccine, and HPV infection, may help increase HPV vaccination acceptance, and initiation and completion rates among female adolescents. In addition, strategies to improve vaccine uptake should focus on developing communication tools for health care providers to better address parents’ reasons for “no-intent” in support of making recommendations for vaccinating the adolescents during their health-care encounter.This study identified subgroups of parents across different socio-demographics and the reported reasons for no-intent to vaccinate their daughters with the HPV vaccine. The socio-demographic characteristics of such parents with no-intent as identified in this study have implications for developing strategies to address the various concerns (reasons) reported by the parents with “no-intent” to vaccinate their adolescent daughters with HPV vaccine. Also, communication tools focused on health care providers would address the significant systemic failure due to lack of provider recommendation. These efforts may help increase HPV vaccine acceptance. Future studies should focus on the impact of strategies developed on HPV vaccine uptake among the subgroups of parents identified in this study.