Cervical cancer is the greatest cause of age-weighted years of life lost in the developing world. Human papillomavirus (HPV) infection is associated with a high proportion of cervical cancers, and HPV vaccination may help to reduce the incidence of cancer. The aim of the study was to identify barriers, obstacles, and strategies and to analyze key concerns and lessons learned with respect to the implementation of HPV vaccination program in low- and middle-income countries.The Gardasil Access Program (GAP) is a donation program established to enable organizations and institutions in eligible low-resource countries to gain operational experience designing and implementing HPV vaccination programs. This study used an online survey to capture the experiences and insights of program managers participating in the GAP. Different factors related to HPV vaccination program management were collected. A mixed-method approach enabled the presentation of both quantitative measurements and qualitative insights.Twenty-nine programs implemented by 23 institutions in 19 low- and middle-income countries were included. Twenty programs managers (97.7 %) reported that their institution implemented sensitization strategies about vaccination prior to the launch of vaccination campaign. The most frequently reported obstacles to HPV vaccination by the program managers were erroneous perceptions of population related to the vaccine’s safety and efficacy. Reaching and maintaining follow-up with target populations were identified as challenges. Insufficient infrastructure and human resources financing and the vaccine delivery method were identified as significant health system barriers. Coupling HPV vaccination with other health interventions for mothers of targeted girls helped to increase vaccination and cervical cancer screening. The majority of program managers reported that their programs had a positive impact on national HPV vaccination policy. The majority of institutions had national and international partners that provided support for human resources, technical assistance, and training and financial support for health professionals.Local organizations and institutions can implement successful HPV vaccination campaigns. Adequate and adapted planning and resources that support information sharing, sensitization, and mobilization are essential for such success. These results can inform the development of programs and policies related to HPV vaccination in low- and middle-income countries. Cervical cancer, the third most common cause of cancer in women around the world, is a significant global health challenge, and is the greatest cause of age-weighted years of life lost in the developing world due to its high incidence –3. CerviSince 2006, two prophylactic HPV vaccines have been available, and each has shown 90 % efficacy in preventing HPV type 16- and 18-associated high-grade cervical lesions –12. WhilConcerns about the limited use of HPV vaccine in LMICs led to the implementation of a variety of HPV vaccine demonstration projects designed to generate evidence about effective ways to reach young adolescent girls , 16. WhiDespite the proven safety, efficacy and cost-effectiveness of HPV vaccine, there remain a number of unidentified issues that impede its routine use in LMICs. While a number of HPV vaccination pilot programs have been undertaken in lowest-income countries, published results from these programs do not typically include information related to program management. As more countries consider introducing HPV vaccine, a review of the lessons learned from vaccination programs conducted in LMICs was undertaken in order to identify barriers and difficulties to implementation and sustainability of HPV vaccination programs –19. The Review of the lessons learned from pilot programs undertaken in LMICs is essential for advancing our knowledge of barriers and solutions to implementing sustainable HPV vaccine programs. The aim of this study was to identify barriers, obstacles, and strategies and to analyze key concerns and lessons learned with respect to the implementation of HPV vaccination in institutions that managed HPV vaccination programs in LMICs through the GAP.Donations through the Gardasil Access Program were available to interested government Ministries of Health (MoH) and national and international non-governmental organizations (NGOs), with the approval of national health authorities. Axios Healthcare Development has been responsible for managing, monitoring, and distributing HPV vaccine for the GAP.Organizations and institutions participating in the GAP are responsible for covering all other costs associated with their vaccination campaigns, including importation, storage, cold chain management, distribution of vaccine, data collection, and management of the vaccination campaigns. Program managers were independent from Axios and were employees of the organization at which they helped to manage the GAP. The GAP encourages participating organizations to adhere to WHO guidelines for HPV vaccination, which recommends a target age range of 9 or 10 years of age through 13 years of age . The vacA key method approach of this study was to develop an online survey that could be used to capture the experiences and insights of program managers. The first step in developing this survey was identifying the types of information to be collected. Two health professionals from Axios Healthcare Development  made in-country site visits to 10 institutions representing 15 programs in 6 countries  reviewed all qualitative comments extracted from the questionnaires and placed them into broader categories based on content and thematic saturation. Illustrative quotes have been included in this report along with information about the role that each survey participant played in the vaccination program. Key findings and quotes were compiled in a Microsoft Excel table and coded according to topic or theme. Responses were reported if at least two people gave a similar response. Final decisions on comments categories were discussed with a third author (EA).A total of 29 programs implemented by 23 institutions in 19 countries are included in this study (Table Twenty-two program managers (95.7 %) reported that their institution implemented at least one sensitization strategy about the vaccination program prior to the launch of the campaign; all of the 22 institutions responding implemented sensitization meetings in the communities and organize meetings with the girls (Table “It is important to have good one-on-one education with parents to overcome misinformation.” The integration of the HPV vaccine into the Expanded Program of Immunization (EPI) was also found to be an effective message: “In general parents were open to the HPV vaccine because children received routine immunizations - this helped with acceptance of Gardasil because it was integrated within EPI”.Nineteen program managers 82.6 %) reported that parents had objections to the HPV vaccine, the most common of which were the misconceptions that HPV vaccination is a method for sterilizing girls  required informed consent, including written informed consent , oral informed consent , and informed consent for the parents to refuse vaccination . Three program managers highlighted sociocultural barriers related to the utilization of informed consent: The staff implemented an innovative method of ‘peer tracking’ of girls who needed second and third doses in small communities, some of which did not have cell phone coverage”; “Once the team found one girl who needed a second or third dose, they would ask her to help them locate other girls who needed second or third doses”; “The process of follow up was costly in terms of time, finances, and human resources”.All the institutions were challenged with girls lost to follow-up during the vaccination campaigns. The most frequent obstacles to follow-up were girls changing schools or moving away, and school vacation or examination. One third of the institutions also reported logistical obstacles to follow-up, such as challenges with transportation, and the availability of human and financial resources needed to bring girls to vaccination sites Table . Reachin“Logistics and cold chain management was apparently not a challenge or issue for any of the projects, apart from some isolated and unexpected events”; “Providing supplies and the vaccines in advance (around five days) guarantees the success of the vaccination campaign”; “The main challenge appears to be with the storage capacity at some health facilities.” However, it should be noted that 13 institutions (56.5 %) did report difficulties with HPV vaccine supply chain management, including expiration of vaccine date (46.2 %) and difficulties transporting vaccine to administration sites (38.5 %) . Of those institutions with individual records, seven (31.8 %) recorded the data in an electronic database. Overall, 82.6 % of institutions managed their programs electronically. Sixteen institutions reported difficulties with the data management of vaccination programs, with late or incomplete vaccination reports listed as two of the most common challenges  coupled HPV vaccination with other health interventions for mothers of targeted girls, including screening of cervical cancer in 12 institutions, tetanus vaccination in one institution, and both cervical cancer screening and tetanus vaccination in one institution. The program managers found this approach to be effective: ““The program confirmed that the vaccine was highly acceptable; HPV vaccination was built into the national cervical cancer control strategy”; “The most important result of implementing GAP projects in our country is that we now have the experience of implementing HPV vaccination projects”; “The country benefited from the program: we are now approaching cervical cancer in a comprehensive way, and the GAP led to the establishment of cervical cancer screening centers”; “We had to include Gardasil vaccine issues in the Sexual Reproductive Policy and National Adolescent Health Policy.”Sixteen institutions (69.6 %) reported that their programs had a positive impact on national HPV vaccination policy: “After the GAP project, cervical cancer screening projects started in our country”; “Not exactly a change in national policy, but the National Commission on Immunization Program has recommended undertaking a study on HPV Vaccination activities. The findings from the GAP program will help to inform decision-making related to the vaccination program”; “The start of HPV projects put the issue of cervical cancer on the table for discussion. Because of these discussions and significant political pressure from different groups, a National Prevention Plan against Cervical Cancer was created in the years following the GAP project. This plan included the use of HPV vaccination as an important strategy for cervical cancer management.” At the end of the GAP, ten institutions applied for GAVI Vaccine Alliance Program funding: “The MoH used our experience to apply for GAVI funding.”A positive impact on health policy for cervical cancer screening was also reported: “The level of knowledge related to cervical cancer has increased, in particular in those populations where the vaccination took place”; “It has increased the number of women that do their PAP smears.”Five institutions (21.7 %) also reported a direct impact on cervical cancer awareness: Table This study of 23 key programs managers who were actively involved in the implementation of a variety of HPV programs combines qualitative and quantitative research on the challenges and opportunities associated with implementing the HPV vaccine in LMICs. Our findings document the experiences of various strategies and approaches related to HPV vaccination implementation in LMICs and provide lessons learned and best practices that may be applied to the implementation of other HPV vaccine programs. The most frequently reported obstacles to HPV vaccination were erroneous perceptions that the GAP was using HPV vaccine in the context of a clinical trial, that the HPV vaccine was unsafe, and that the vaccine was being used to sterilize young girls. These misperceptions among parents , in concReaching and maintaining follow-up with adolescent girls in order to deliver HPV vaccines were challenges for program managers. While vaccination programs have traditionally focused on children less than five years of age, a vaccination such as HPV, which is targeted to adolescents, requires different mobilization and delivery infrastructure. There is currently no experience in mass vaccination in adolescent populations, which may explain the reported challenges. Contacts with schoolteachers and administrators were frequently used as a strategy to reach girls lost to follow-up in the 18 programs that utilized school-based delivery models. School-based projects faced additional challenges when vaccination dates occurred outside of scheduled school days, and it was exponentially more difficult to prevent loss to follow-up in these instances. Despite these challenges, school-based vaccination delivery methods were most effective at reaching girls within the WHO-recommended age range, which is likely due to the fact that girls aged 9-13 years are not usual “clients” of the health care system –29. Of tCommunity involvement actions appeared to impact the programs with respect to both sensitization strategies before launch of the HPV vaccination campaign as well as reaching girls lost of follow-up once the campaign was underway , 24, 26.Insufficient infrastructure and human resources financing and the delivery method were identified as significant health system barriers: assistance programs helped to address these factors and support national and international vaccine programs –19. NatiThe data reported here provide lessons for development of public health programs and policies as countries go forward in national decision-making related to HPV vaccination. These results suggest that local organizations and institutions can implement successful HPV vaccination campaigns. Our results also demonstrate that private initiatives to increase access to novel health interventions can have a significant impact on public health issues and foster diverse partnerships among government agencies, NGOs, and private organizations. The diverse HPV vaccine programs included in this study provide concrete examples of how such programs can be adapted to address local and regional issues and concerns, and provide a useful framework in which countries can consider how best to expand their own HPV vaccination programs based on their individual epidemiological, economic, and health system challenges .The results of HPV vaccine experiences so far in LMICs are encouraging, and the continued sharing of outcomes data, successes, and challenges is critical to the reproducibility of successful HPV vaccine interventions. Considerable progress has been made in several LMICs and it is important to identify and understand those factors that are transferable to other settings. Demonstration and pilot projects in several LMICs have shown that a variety of delivery methods, including the use of schools, special campaigns, health centers or combined strategies, can reach a large proportion of eligible girls, and that advocacy and sensitization are essential elements for success , 19, 32.While our results provide practical information about programs for HPV vaccine introduction in low and middle-resource settings, there are some limitations to be noted. A key limitation of this study is that it represents the experiences and opinions of the program managers of the GAP rather than all the HPV vaccination programs in LMICs. Similarly, there are inherent limitations associated with data that are self-reported rather than obtained through objective assessment or observation. However, the design of our study, which utilized both qualitative and quantitative data collection, and utilized a final study questionnaire that was developed following 10 site visits in six countries and was designed to address potential issues related to self-reported data, should help to limit the bias associated with self-reporting. This study is also limited by the design approach, which measured the impact of the GAP in different areas at the program’s end. This approach does not allow us to conduct analyses on a program-by-program or country-by-country basis, or to compare the quality or type of HPV vaccination programs or other type of vaccine delivery provided before and after implementation of the GAP, which could provide additional validation of the program’s impact on these services. However, with the goal of developing a highly informative online survey, we investigated and developed our questionnaire based on site visits, which provides some level of corroboration between reported and observed results. The results of the survey utilized in this study appear to be robust and do provide important insight into the impact of a donation program on a variety of services and health activities at the local, institutional, and national levels. While the current study does not focus on vaccine delivery models, we previously reported that school and health clinic-based models appeared as predictive factors for vaccination coverage and that HPV vaccine campaigns tailored to meet the needs of communities can be effective , 16. TheFor LMICs, it is a challenge to accelerate HPV vaccination programs and also to conduct impact analyses around such programs. Considerable progress has been made in several LMICs and it is important to identify and understand lessons that may be transferable to other settings worldwide , 18. SchRoutine vaccination in adolescents remains challenging. The data reported here from programs focused on vaccinating adolescent girls provides lessons that may be applied to the development of public health programs and policies as countries go forward in national decision-making for HPV vaccination. Our findings in a large sample of HPV vaccination programs demonstrate that continued sharing of evidence, successes, and challenges is critical to the successful scale-up of HPV vaccine interventions worldwide. These findings underscore the need for adequate and adapted planning and resources that support information sharing, sensitization, and mobilization. The GAP provides a model for how private initiatives can have a significant impact on public health issues.The diverse HPV vaccine programs included in this study provide concrete examples of how such programs can be adapted to address local and regional issues and concerns, and provide a useful framework in which countries can consider how to best expand their own HPV vaccination programs based on their individual epidemiological, economic, and health system challenges.


Cervical cancer incidence and mortality rates in Bolivia are among the highest in Latin America. This investigation aims to evaluate the possibility of using simple devices, e.g. a cotton swab and a glass slide, for self-sampling in order to detect human papillomavirus (HPV) DNA by PCR in cervico-vaginal cells.In the first phase of our study we evaluated the use of a glass slide as a transport medium for cervical cells. A physician took paired-cervical samples from 235 women. One sample was transported in Easyfix® solution and the other sample was smeared over a glass slide. Both were further analyzed and compared for human DNA recovery and HPV detection. A kappa value was determined to evaluate the agreement between the HPV DNA detection rates.In the second phase of the study, 222 women from the urban, peri-urban and rural regions of Cochabamba were requested to perform self-sampling using the following devices: a cotton swab combined with a glass slide, and a vaginal tampon. Women gave their opinion about the self-sampling technique.Finally, the agreement for high risk-HPV detection between self- and physician-collected samples was performed in 201 samples in order to evaluate the self-sampling technique.Firstly, the comparison between Easyfix® solution and the glass slide to transport clinical samples gave a good agreement for HPV DNA detection . Secondly, self-sampling, especially with cotton swab combined with glass slide, would generally be preferred over clinician sampling for a screening program based on HPV detection. Finally, we showed a good agreement between self- and physician collected samples for high risk-HPV detection .Simple devices such as a cotton swab and a glass slide can be used to perform self-sampling and HPV DNA detection. Furthermore, most Bolivian women preferred self-sampling over clinician-sampling for cervical cancer screening.The online version of this article (10.1186/s12905-017-0490-z) contains supplementary material, which is available to authorized users. The incidence and mortality rates of cervical cancer in Bolivia are the highest in Latin America: 39.5 and 16.5 per 100,000 women, respectively . FurtherThe current national program for cervical cancer prevention stipulates that every woman should have a Pap test every three years after two consecutive annual negative smears . HoweverIt has been well established that a persistent infection by high risk HPV genotypes (hr-HPV) is the most important risk factor for developing cervical cancer . HPV tesLately, companies have made specialized brushes in order to introduce self-sampling e.g. the Viba-Brush and the Evalyn Brush  , 14. TheIt is noteworthy that in order to be sustainable in a developing country’s health program, such as in Bolivia, new cost-effective techniques for cervical cell transport should be developed and adapted to the socio-economic characteristics of the population. Glass slides are currently used to transport cervical samples for standard cytology tests and could represent an interesting option to economically and conveniently transport cervical cells for hr-HPV DNA detection. However, this device has not yet been tested to transport samples for detection of HPV DNA.Our main objective was to evaluate the possibility of introducing self-sampling for HPV DNA detection using simple devices through an acceptability and feasibility study. The first phase of this study was to evaluate the efficiency of a glass slide as an alternative transport medium for cervico-vaginal cells. Secondly, we evaluated women preference for a self-sampling device between a cotton swab combined with a glass slide and a vaginal tampon, in the urban, peri-urban and rural areas of Cochabamba. Finally, we compared self- and physician-collected samples for hr-HPV detection to assess our self-sampling technique.The Bio-ethical Committee of the “Universidad Mayor de San Simón” approved the study protocol  and each participant signed an inform consent form before enrollment.For the first phase of the study, a group of 235 women attending gynecological services for cytological abnormalities were recruited in the “Hospital Materno Infantil Germán Urquidi” and the gynecological outpatient clinic of the Non-Governmental Organization “Marie Stopes International” in the city of Cochabamba. A physician collected paired-cervical samples from each woman. One sample was placed in Easyfix® solution and the second sample was smeared on a glass slide. Cervical samples were collected in alternate order to avoid bias.For the second phase of the study, a group of 222 women between 25 and 59 years old from three different regions  tested 2 self-sampling devices: a cotton swab and a vaginal tampon. The recruitment was done through women’s organizations in all areas of the study. The leader of the organization called a meeting and all women who were interested to participate in the study signed an inform consent. No previous cytological results were required to be included in this study. Women were explained how to perform self-sampling with both devices through images  and washed with 300 μl of phosphate-buffered saline solution, centrifuged again (3000 x g for 10 min), and re-suspended in 150 μl of PK-1 buffer . The cell suspension was incubated for 16–20 h. at 56 °C and then incubated at 100 °C for 10 min for proteinase K inactivation. Cell debris was removed by centrifugation . A quality control of DNA extraction was performed by a PCR using the primers PC04 (5’-CAACTTCATCCACGTTCACC-3’) and GH20 (5’-GAAGAGCCAAGGACAGGTAC-3’), amplifying a 260 bp fragment from the human β-globin gene , 28. OnlFor samples transported on a glass slide, DNA was extracted as follows: cells were detached from the glass slide surface with a micropipette tip using 300 μl of Tris-HCl 10 mM pH 8.3 solution. Cells were then centrifuged (3000 x g for 10 min), and DNA was extracted from the cell pellets as described above, in 150 μl of PK-1 buffer.Cervical samples collected with the tampon and transported in 15 ml Easyfix solution were treated as follows: The tampon was taken out from the Falcon tube and squeezed with a syringe piston to recover the cells. The solution was centrifuged as described above and DNA extraction was performed as previously described for the cells stored in Easyfix® solution.2, 0.2 mM dNTP mix, 1 μM of each primer, 1 U of GoTaq® G2 Hot Start Polymerase  and 5 μl of crude DNA extract. PCR was performed in a T100™ Thermal Cycler . Amplified fragments were visualized in a 2% agarose gel  incubated with 0.5 μg/ml ethidium bromide solution (Sigma-Aldrich). Detection of hr-HPV DNA was performed using an enzyme-linked immunoassay as described by Jacobs et al. [HPV DNA was detected with a consensus PCR using the primers GP5+ (5’-TTTGTTACTGTGGTAGATACTAC-3’) and GP6+ (5’-GAAAAATAAACTGTAAATCATATTC-3’), amplifying a 150 bp fragment from the L1 region of the HPV genome . PCR reas et al.  . Oligo-Statistical analyses were performed using SPSS software. Cohen’s kappa factor was calculated to evaluate the agreement between HPV detection results . Kappa vTo evaluate the possibility of using a glass slide to transport cervical cells samples for HPV DNA detection through PCR, we recruited 235 women with cytological abnormalities and compared human β-globin and HPV PCR results from their samples. The PCR results gave similar detection rates of human DNA (94% in Easyfix® solutions vs. 91% on glass slides) and HPV DNA (30% in Easyfix® solutions vs. 33% on glass slides). Comparison of HPV detection rates obtained for the samples with positive β-globin PCR (207 cases) showed a good level of concordance , peri-urban (N = 66) and rural (N = 60). Considerable differences were observed between urban and rural populations in the education level. Most urban women (66%) have been to the university while most rural women (82%) only attended primary school. Furthermore, 76% of women reported to ever had a Pap test (data not shown).This study population included 222 women from three regions of Cochabamba: urban  never used before a vaginal tampon. This implies that women could have misused the device even though 59% of them reported that self-sampling with the tampon was easy. This difference in DNA recovery could also be due to the sampling procedure, i.e. in our study women introduced the vaginal tampons for maximum 30 s, probably allowing little time for the cells to be collected by the vaginal tampon. Indeed, a previous study has shown that tampon samplings with longer cell exposure were equivalent to two swabs for detection of hr-HPV genotypes .The use of a cotton swab is not more common than a vaginal tampon among Bolivian women, however it seems that the procedure for self-collection with the swab is easier to understand for them. It is worth noting that a previous study assessed the possibility of using a cotton swab for self-sampling and concluded that it is not a safe method for sample collection . The difAnother important finding in our study is that most women would prefer self-sampling over standard clinician-sampling for a possible screening program based on HPV detection in all three regions . This is particularly important considering the socio-cultural differences between this regions, specially among urban and rural populations. Furthermore, as 76% of the surveyed women reported having a previous Pap test, they can really compare between sampling procedures. Our results reflect Bolivian women’s commitment to perform self-sampling on a routine basis since more than 80% of them (data not shown) feel confident about the efficiency of the self-collection and therefore would prefer it in a primary screening program for cervical cancer prevention. A recent study about self-sampling acceptability was carried-out in a population with similar socio-cultural characteristics in the northern region of Argentina. This study also found a high percentage of acceptability (86%) .As a final step in our study, we evaluated the efficacy of self-sampling for the detection of hr-HPV detection by comparing it with the standard clinician-sampling. In the design of this part of the study we decided that the physician would use the glass slide to transport the cells samples since we validated its use for that purpose in the first phase of our study. Our results indicate a good agreement between the sampling methods and suggest that the self-collection using a simple cotton swab with a glass slide is a valid method for HPV detection. A previous study has also shown good agreement in hr-HPV detection between clinician and self-sampling with a cotton swab .The results obtained in this study prompted us to design and prepare our own self-sampling kit to offer to the women population. This kit consists in a zipper storage bag containing a cotton swab, a glass slide (properly covered in a cardboard box) and a pair of gloves. The cost of this kit is around half of a dollar, which represents an accessible price for the government considering a future population screening program based on self-sampling.We evaluated the possibility of introducing self-sampling in Bolivia using simple and cheap devices such as cotton swab and glass slide. We showed that these devices can be used to collect and transport cells with acceptable rates for human and viral DNA detection. Moreover, according to the results of our questionnaire, women would prefer self-sampling for a screening program based on HPV DNA detection. Nevertheless, further studies should be performed to prove that our self-sampling method has a sufficient clinical sensitivity for detection of precancerous lesions (CIN2+).


Small cell carcinoma/neuroendocrine carcinoma (SCNEC) of the oropharynx is uncommon. Two cases of SCNEC in an 81-year-old woman and in a 54-year-old man are presented here.We have documented two cases of SCNEC arising in the oropharynx with evidence of high-risk human papillomavirus (HPV) infection. Histologically, both cases were classified as poorly differentiated SCNEC with high nuclear-to-cytoplasmic ratios and nuclear molding. Observations using a transmission electron microscope revealed membrane-bound neuroendocrine granules in some tumor cells. Both tumors expressed high levels of p16, a surrogate marker for high-risk HPV infection. HPV infection was confirmed in both cases using HPV polymerase chain reaction analysis; HPV subtype 16 was identified in one case and HPV subtype 18 in the other.SCNEC of the oropharynx is a rare and novel HPV-associated disease with neuroendocrine granules and aggressive clinical behavior.Herein, we present two cases of SCNEC, focusing on its histologic features and treatment modalities. More studies are required to elucidate the pathophysiology of HPV-associated SCNEC in different organ systems.The online version of this article (doi:10.1186/s40064-016-3501-x) contains supplementary material, which is available to authorized users. Primary malignant tumors of the oropharynx are usually squamous cell carcinomas (SqCCs). Over the past decade, human papillomavirus (HPV) infection has been recognized as a significant etiological factor for a subset of oropharyngeal SqCCs. Primary small cell carcinoma/neuroendocrine carcinoma (SCNEC) of the oropharynx is rare. The larynx is the most commonly involved site, followed by the nasal cavity, paranasal sinuses, salivary glands, and oral cavity Renner . ApproxiAn 81-year-old woman with no history of alcohol consumption or smoking presented with a 3-month history of experiencing a mass in her throat and right-sided neck swelling. On oropharyngoscopy, a tumor was identified in the right anterior wall of the oropharynx  studies were performed for both cases to detect neurosecretory granules. TEM specimens were fixed in 2 % phosphate buffered glutaraldehyde overnight, rinsed in 0.1 mol/L phosphate buffer pH 7.4), post-fixed in 1 % phosphate buffered osmium tetroxide, and dehydrated in epoxy resin. Semi-thin sections from selected areas were stained with uranyl acetate and lead citrate and examined using TEM. TEM evaluation revealed dense-core neurosecretory granules in the cytoplasm of cells in the specimens of both patients , according to the manufacturer’s instructions.HPV status was determined using the HPV Typing Set , a primer set for polymerase chain reaction (PCR) specifically designed to identify HPV genotypes 16, 18, 31, 33, 35, 52, and 58 using genomic DNA. The PCR HPV Typing Set method was performed according to the manufacturer’s instructions. Specimens from both patients tested positively for high-risk HPV. After amplification, HPV typing revealed a 238-bp band for HPV-16 in case 1 and a 268-bp for HPV-18 in case 2 (Fig. The worldwide incidence and prevalence of HPV-associated oropharyngeal cancer have been increasing over time. Among the head and neck regions, SCNEC most commonly arises in the larynx, but it has also been reported in the sinonasal tract and salivary glands Renner . SCNECs Gynecologic extrapulmonary SCNECs most commonly arise in the cervix, and SCNECs of the uterine cervix comprise less than 3 % of cervical cancers (Cohen et al. The diagnosis of SCNEC requires immunohistochemical or ultrastructural studies. With routine hematoxylin and eosin staining, SCNEC exhibits morphology similar to that of nonkeratinizing SqCC. A case with a histopathologic finding of small round cells with scant cytoplasm should be evaluated for synaptophysin, chromogranin A, and CD56 expression, followed by ultrastructural analysis to detect neurosecretory granules in the tumor cells (Mineta et al. Owing to the rarity of these tumors, recommendations for the management of SCNEC of the oropharynx have not been established (Barbeaux et al. In conclusion, SCNEC of the oropharynx is extremely rare and highly aggressive, with a poor prognosis. Herein, we present two cases of SCNEC, focusing on its histologic features and treatment modalities. More studies are required to elucidate the pathophysiology of HPV-associated SCNEC in different organ systems.


Human papillomaviruses (HPVs) are oncogenic viruses that cause numerous different cancers as well as benign lesions in the epithelia. To date, there is no effective cure for an ongoing HPV infection. Here, we describe the generation process of a platform for the development of anti-HPV drugs. This system consists of engineered full-length HPV genomes that express reporter genes for evaluation of the viral copy number in all three HPV replication stages. We demonstrate the usefulness of this system by conducting high-throughput screens to identify novel high-risk HPV-specific inhibitors. At least five of the inhibitors block the function of Tdp1 and PARP1, which have been identified as essential cellular proteins for HPV replication and promising candidates for the development of antivirals against HPV and possibly against HPV-related cancers. Human papillomaviruses are causative agents of many different cancers; they are most commonly associated with cervical cancer which leads to about quarter of a million deaths each year. Regardless of extensive studies for decades there is no specific cure against HPV infection. During this research, we have engineered modified HPV marker genomes that express Renilla luciferase reporter gene which expression level correlates directly with viral genome copy number. We have used such modified HPV genome in high-throughput screening of NCI Diversity Set IV chemical library and have identified a number of novel high-risk HPV-specific chemical compounds and drug targets. Such Renilla-expressing marker genomes could be used in various cell systems suitable for HPV replication studies to conduct high-throughput screens and quantify viral genome copy number quickly and effectively. Human papillomaviruses (HPVs) are small, double-stranded DNA viruses that infect the epithelium of the skin and mucosa. To date, at least 202 HPVs have been characterized, but studies suggest that the true number is considerably higher,2. HPVs Regardless of being studied for decades, there is still no effective cure for an ongoing HPV infection. There are approved invasive treatments, such as cryotherapy, larger excision procedures, laser therapy and electrosurgery, which do not eliminate HPV DNA completely, leading to a 40% chance of recurrence of infection,12. ImmuAlthough no effective HPV inhibitors have been developed, several compounds and targets have been analyzed. E1 and E2 are the only two viral proteins necessary for HPV genome replication. The first attempts in the development of HPV inhibitors were focused on E1, specifically targeting its ATPase and DNA helicase activities,19. ThesIn addition to targeting HPV proteins, the viral genome itself could be the target as well. Several sequence-specific DNA binding compounds belonging to the pyrrole-imidazole polyamide class have been developed,25. ThisThe current situation in HPV therapy strongly suggests that there is a need for specific drugs targeting HPV infection, preferably without restriction to specific subtypes.wt HPV11 genome, but it can only be used for a limited number of passages[HPV infects epithelial cells in the cutaneous and mucosal epithelia, and its life cycle is dependent on keratinocyte differentiation. Thus, most of the work regarding the HPV life cycle has been conducted in human primary epithelial keratinocytes; using these cells is relatively time-consuming and expensive–31. This passages. Another passages.U2OS cells, which are derived from moderately differentiated osteosarcoma, have an adherent epithelial morphology and carry wild-type p53 and pRB genes, which have proven to be useful for studying various aspects of the HPV life cycle–38. It hIn this study, we report the generation of various HPV 18, 16 and 5 marker genomes containing reporter genes for rapid and easy quantification of viral copy number. First- and second-generation marker genomes contained reporter genes in various configurations in the late region of the HPV genome. All of these versions had reduced replication ability. Based on the transcription map of HPV18, we nextU2OS cells, which were obtained from the American Type Culture Collection (ATCC no: HTB-96), the modified cell lines U2OS GFP2-Fluc #10.15, U2OS-EBNA1 (Icosagen Cell Factory Ltd) and the HPV18 Rluc-E2-positive U2OS #10.15 subclones #2G10 and #2B3, were grown in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% fetal calf serum (FCS). The U2OS cells were transfected with the indicated amounts of different HPV minicircles or 500 pmol of HPV18-specific siRNAs by electroporation  with a Bio-Rad Gene Pulser II that was supplied with a capacitance extender (Bio-Rad Laboratories). The U2OS GFP2-Fluc #10.15 cell line was generated by transfecting U2OS cells with a linearized expression vector containing both the GFP2 and Firefly luciferase (Fluc) ORFs as well as a puromycin resistance gene. Individual puromycin-resistant clones were picked and analyzed by GFP and Fluc expression. HPV18 Rluc-E2 positive subclones were generated similarly to those described in. The CINThe parental plasmid pMC-HPV18 was constructed for the production of HPV18 genome miniplasmids. A recognition site for the BglII restriction endonuclease was introduced into the HPV18 genome between nt 7473 and nt 7474 . These sites were used previously without observing changes in gene expression compared to unaltered HPV18. The modTdp1 shRNA sequence: GCACGATCTCTCTGAAACAAACTCGAGTTTGTTTCAGAGAGATCGTPARP1 shRNA sequence: GGACTCGCTCCGGATGGCCTTCAAGAGAGGCCATCCGGAGCGAGTCCTdp2-1 shRNA sequence: GTACAGCCCAGATGTGATACGAATATCACATCTGGGCTGTACTdp2-2 shRNA sequence: GAAGGATATTTCACAGCTACGAATAGCTGTGAAATATCCTTCThe U2OS-GFP-Fluc #10.15 cells were transfected with 2 μg of the HPV18-Rluc-E2 minicircle, and the cells were seeded onto 100 mm plates. On the next day, the cells were detached and seeded onto 96-well plates (5000 cells per well). Forty-eight hours after the transfection, the screened compounds were added to the media in 5 μM and 1 μM concentrations. The cells were grown for three days, and both Firefly luciferase (shows cellular viability) and Renilla luciferase (shows HPV copy number) were measured using the Dual-Glo luciferase assay system (Promega) according to manufacturer’s protocol with the GloMAX-96 luminometer (Promega). The results were blotted on a XY-scatter diagram, and HPV-specific hits were chosen.The Diversity set IV and the additional compounds NSC9782, NSC 88915, NSC 82269, NSC 109128 and NSC 305831 were obtained from the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute. Camptothecin (CPT) (sc-200871) and ABT-888 (sc-202901) were purchased from Santa Cruz Biotechnology.HPV). The CtrDNA was detected as a normalization standard from the ribosomal DNA gene sequence in the U2OS genome with the following oligonucleotides (300 nM of each): 5’- GCGGCGTTATTCCCATGACC-3’ and 5’- GGTGCCCTTCCGTCAATTCC-3’. The relative value CN, which reflects the average viral genome copy number per cell, was calculated from the data with the formulas ΔCt = CtHPV − CtrDNA and CN = 2-ΔCt.The viral genome copy number in U2OS cells during replication was analyzed by quantitative real-time PCR (qPCR). Genomic DNA was extracted from U2OS cells, and the samples were linearized by digestion with the appropriate enzyme (see above), and the bacterially produced input DNA was fragmented by digestion with DpnI. For each qPCR reaction, 3 ng of DNA was used, and the reactions were performed with EvaGreen qPCR Mix Rox (Solis BioDyne) according to the manufacturer’s protocol on a 7900 HT Fast Real-Time PCR System (Applied Biosystems). The HPV18 replication signal was amplified with the following oligonucleotides (300 nM of each per reaction): 5’-GCGCTTTGAGGATCCAAC-3’ and 5’-GTTCCGTGCACAGATCAG-3’. For HPV5, the following oligonucleotides were used: 5’-GGTTGCAGGAACTGTGAGGT-3’ and 5’-TCCGCGACAGTCGGGGCACAGG-3’. For HPV16, the oligonucleotides were 5’- CCCACAGCTACAGATACAC-3’ and 5’- GCAGGTGTGGTATCAGTTG-3’. The analysis was performed according to the comparative threshold cycle (ΔCt) method. The results were calculated from the PCR cycle number in which the HPV signal exceeded the threshold value . Specific HPV replication signals were detected by Typhoon TRIO .For first- and second-generation marker genome analyses, episomal DNA was extracted as described in. For SouThe cells were lysed with passive lysis buffer by freezing and thawing. Both Renilla and Firefly luciferase expression was measured with a Glomax 20/20 luminometer using the Dual-Luciferase reporter assay system following the manufacturer’s protocol .The cells were lysed with Laemmli buffer , and 200 mM DTT) and boiled for 10 minutes at 100°C. The samples were resolved in an SDS-PAGE gel and transferred to Immobilon-P PVDF membrane (Millipore). Tdp1 and PARP1 were detected with their specific antibodies from Santa Cruz Biotechnology: sc-365674 and sc-56197, respectively. Tubulin (used as a loading control) was detected using Sigma Aldrich antibody T9026. Anti-mouse peroxidase-conjugated secondary antibody (LabAS) and Amersham ECL Western blotting Detection Kit  were used for visualization. Signals were exposed on an X-ray film (AGFA).The cells were detached and collected by centrifugation 300g 5 minutes. The pellet was resuspended 100μl 1X PBS and 900μl ice-cold 80% ethanol was added drop-wise. The cells were next incubated on ice for at least 30 minutes, collected by centrifugation, resuspended in 1X PBS containing 50μg/ml propidium iodide and 200μg/ml RNase A and incubated at 37°C for 45 minutes. Cell cycle profiles were analyzed by flow cytometry (LSR II from Becton Dickinson) and by FlowJO 10 software using Watson (pragmatic) model.wt, HPV18-RlucE2 and HPV18-RlucE2-K490A genomes. Genomic DNA was extracted 3, 5 and 7 days after the transfection, transient replication was analyzed by Southern blot  and Renilla (from HPV marker genomes) luciferase expression were also measured in the same experiment and are presented as the ratio of Rluc/Fluc, as measured by the dual-luciferase assay described in the materials and methods section. The results in wt and K490A genomes served as negative controls for Renilla luciferase expression. The replication-deficient K490A marker genome shows relatively high Rluc/Fluc ratio the in 3-day timepoint likely because there is still large portion of transfected, transcriptionally active DNA present in the cells.To evaluate cellular viability and the potential toxic effects of the compounds during the HT screen, we generated a monoclonal U2OS cell line stably expressing both the GFP and Firefly luciferase (Fluc) reporter genes, called U2OS-GFP2-Fluc #10.15. Insertion of these reporter genes allows to measure cell growth and viability as well as HPV genome replication . To measern blot  and the ern blot . It is cwt genome, for HPV5, the marker genome replication is 2–3 times more effective than the wt genome. Renilla luciferase expression was reproducibly detectable, the net luminescence from 1 second integration time ranged from 10 to 25 million from approximately 400 000 cells. Using HPV18-specific siRNAs, we showed that the expression level of Renilla luciferase correlated with the HPV18 copy number during initial amplification  based on U2OS-GFP2-Fluc #10.15 that contain an episomal HPV18-RlucE2 genome, as described in. First, The third replication stage of HPVs is late amplification, which is triggered by the initiation of keratinocyte differentiation. This stage could be studied using organotypic raft cultures or by inducing keratinocyte differentiation with high concentrations of calcium,54. It hCollectively, the data shows that cell lines #2B3 and #2G10 can be used in high-throughput screens to identify compounds capable of inhibiting the stable maintenance and/or late amplification of HPVs.wt genome replication, five most effective compounds were chosen for detailed analyses  NCI Diversity set IV using the model system described in the previous sections for compounds inhibiting the initial amplification of the HPV18 genome in U2OS cells. HT screening was performed with all the compounds in this library in 5 μM and 1 μM concentrations, and 80 compounds were selected for further analyses . After validation of the hits on the HPV18 Two of the identified compounds, 88915 and 305831, are known inhibitors of Tdp1,56. To sIt was recently shown that the PARP1 protein activates Tdp1 through PARylation and recruits it to sites of DNA damage. PARP1 iNext, the involvement of Tdp1 and PARP1 in the initial amplification of other HPV types was evaluated by co-transfection of shRNA-s with HPV 31, 33, 11 and 5 minicircle genomes. Genomic DNA was extracted three days post-transfection, analyzed by Southern blot and the signals were quantified . UnexpecEffects of Tdp1 and PARP1 downregulation on cell cycle progression were also evaluated in a similar assay setup. Results in It has been previously shown that instead of Tdp1, Tdp2 is actually necessary for HPV replication . In thisCPT is a Topoisomerase I inhibitor that stabilizes entrapped Top1cc complexes on DNA,60. SincThus far, the experiments for describing the inhibitors were conducted using the high-risk HPV18 genome. Since Tdp1 and PARP1 seem to be necessary for the replication high-risk HPVs but not low-risk or beta HPVs , we expeWhen analyzing chemical compounds, there is always a possibility that the effects may be cell-line specific. In order to ensure that the compounds identified here are true HPV inhibitors, we tested them on HPV31-positive CIN 612E cells (kind gift from Dr. Frank Stubenrauch). The CIN 612E cells were seeded onto 6-well plates and grown in subconfluent conditions in the presence of compounds for 6 days. Genomic DNA was extracted, analyzed by Southern blot and quantified by Phosphoimager. Results in Taken together, these data suggest that the inhibitors identified in this study are specific HR-HPV inhibitors, blocking the replication of HPVs that cause more than 99% of all cervical cancer cases worldwide.U2OS cells, an osteosarcoma cell line, are suitable for studying the replication properties of various types of HPVs from different phylogenetic genera,37. Studwt genomes[There are numerous well-characterized chemical compound libraries available, meant for the identification of novel drug candidates via high-throughput screening using appropriate model systems. To date, there was no such system for analyzing the complete HPV genome replication cycle. Thus, the first major goal for this work was to construct a model system for HPV research. It has been shown that HPV genomes that lack the late region replicate similarly to t genomes. Therefowt genomes , which significantly reduces the probability of identifying potential inhibitors. As the model system described here allows to study all three HPV genome replication stages and harbors the expression of all viral proteins, more suitable drug targets and inhibitors could be identified, and new mechanistic insights into HPV pathogenesis could be revealed.Based on the transcriptome analyses of HPV18 in U2OS cells, we next genomes , perhapst genome . Since gt genome . AnalyseAlthough there are currently three vaccines against various types of HPVs that effectively prevent infection with various HPV types, no approved effective cure for an ongoing infection is available. Mostly identification of novel drugs against various targets begins with high-throughput screening of chemical libraries containing thousands of compounds. Thus, uIn almost all HPV-related cancers, the viral genome is integrated into the host genome. Integration of the viral genome usually occurs during persistent infection, and thus to prevent integration and tumor progression, already-established infections must be targeted. Therefore, we next tested these compounds on the stable maintenance and late amplification of HPV18. Four out of five compounds successfully inhibited the stable maintenance phase of viral replication , and thrIt has become clear in recent years that HPVs activate the DNA damage response network during their replication to “invite” cellular replication and DNA repair proteins to their replication foci. MoreoveIt is unclear how Tdp1 is recruited for the HPV replication process and if it is necessary for all HPV types. More than 99% of all cervical cancer cases are caused by five HR-HPV types: 18, 16, 31, 33 and 45. We show here that all five inhibitors successfully block the initial amplification of all of those HR-HPV types Figs . UnexpecIn addition to the involvement in replication, the inhibitors described in this study may  affect HPV gene expression. It is known that Top1 is necessary to relax tensions in DNA during both replication and transcription. Entrapment of Top1cc affects transcription in several ways, perhaps most important of which in the context of HPV life cycle could be the negative impact on splicing–81. SincRegardless, further analyses should be performed to describe the involvement of Tdp1 and PARP1 in HR-HPV replication or gene expression. It would be interesting to identify the specific location(s) in the HPV genome where Top1cc entrapment occurs. Studying the differences in the involvement of Tdp1 between high-risk, low-risk and cutaneous HPV replication would perhaps describe some yet unknown differences between the replication mechanisms of these viruses. It is possible that aside from E6 and E7, differences in replication could be the reason why HR-HPVs, but not LR-HPVs, integrate more readily into the host genome and are therefore causative agents of various cancers.wt genomes and such genomes could be thus used in primary keratinocytes or other suitable cell lines for rapid HPV genome copy number quantification. We used this system in high-throughput screening and have identified several novel HR-HPV-specific inhibitors. Importantly, we have demonstrated that three compounds  inhibit the HPV replication in the cells derived from human cervical intraepithelial neoplasia , replication forks will collide, HPV genome replication is blocked, and eventually, aberrant DNA replication intermediates form.S1 FigA: U2OS GFP2-Fluc #10.15 cell-line is suitable for studying the replication of HPVs. U2OS wt or #10.15 cells were transfected with 2 μg of wt HPV18 minicircle genome. The genomic DNA was extracted 3 and 5 days after the transfection and digested with BglI to linearize the HPV18 genome and with DpnI to eliminate bacterially produced input DNA. Replication signals were quantitated by qPCR and are expressed relative to the U2OS 3-day timepoint, as described in the Materials and Methods section. B: Firefly luciferase expression by U2OS GFP2-Fluc #10.15 cells resembles cell growth. A total of 150 000 U2OS wt or #10.15 cells were seeded onto 6-well plates and grown for 3 and 5 days. The Fluc levels were measured and are expressed relative to the 3-day timepoint of #10.15 cells. U2OS wt served as a negative control. C: Flow cytometric analyses of U2OS wt and #10.15 cells, showing homogenous and clearly detectable GFP signal for U2OS #10.15. U2OS wt served as a negative control. Error bars indicate the standard deviations from two independent experiments. Cell growth and viability could be evaluated by the Firefly luciferase and GFP reporter gene expression.(TIF)Click here for additional data file.S2 Figwt genome in U2OS cells. We therefore generated two different generations of HPV marker genomes that contain reporter genes in the late region. A: Schematic of a first-generation marker genome. Non-HPV regions are marked in black. B: Schematics of the second-generation marker genomes. Non-HPV regions are marked in black. C: U2OS cells were transfected with 1 μg of indicated HPV minicircles, the low-molecular-weight DNA was extracted 48, 72 and 96 hours after the transfection, linearized, and bacterially produced input DNA was digested with DpnI. Southern blot analyses were carried out to measure the replication properties of HPV18 wt (lanes 1–3), the first-generation marker genome (lanes 4–6) and two versions of the second-generation marker genomes (lanes 7–12) Linear replication and DpnI-digested HPV18 DNA is shown. Since both the first- and second-generation marker genome replication levels are very low, the image on panel C is greatly overexposed. Insertion of the reporter gene cassettes into the late region of the HPV18 genome greatly interferes with the gene expression and/or replication properties, suggesting that altering the late region would be very difficult if even possible.It was previously shown that the HPV18 genome that lacks a late region (HPV18 “early” genome) replicates similarly to the (TIF)Click here for additional data file.S3 FigA: The genomic DNA was extracted 2 and 3 days after the transfection, HPV DNA was linearized with BglI, and bacterially produced input DNA was digested with DpnI. Replication signals were quantitated by qPCR. The relative numbers were obtained by normalizing the data points to data from the same timepoint from the HPV18-RlucE2 marker genome and transfection with BPV E1 siRNA. B: Both Renilla (from HPV marker genome) and Firefly (from U2OS genome) luciferase were measured in a dual-luciferase assay and are expressed as the Rluc/Fluc ratio. The relative numbers are obtained by normalizing the data points to data from the same time point HPV18-RlucE2 marker genome and transfection with BPV E1 siRNA. In both panels, the average values with standard deviations from three independent experiments are shown. C: Scheme of HPV18 early region, where positions of the siRNAs are indicated. 83–105 is against the early promoter (p102), 965–987 is against E1 and 3893–3915 is designed against early mRNAs polyadenylated after this sequence. The decrease in the viral copy number is very similar to the reduction of Renilla luciferase expression, and thus it adequately reflects the HPV copy number.To check if the Renilla luciferase levels correlate with the HPV genome copy number, U2OS #10.15 cells were co-transfected with 2 μg of HPV18-Rluc-E2 marker genome minicircle and 500 pmol of different siRNAs as shown. (TIF)Click here for additional data file.S4 FigPolyA+ RNA templates were extracted from U2OS cells that had been transfected with 500 ng of the wt HPV18 genome or with HPV18-RlucE2 (72 h time-point). 500 ng of polyA+ RNA were used as a template for 5'RACE with the HPV18-specific primers Pr1397 (binds to E1 ORF) and Pr904-1 (binds to E7 ORF). The promoter regions from which the detected transcripts arisen, are indicated by arrows on the right.(TIF)Click here for additional data file.S5 FigU2OS cells were transfected with 25 ng of the expression vectors for the HPV18 E1 and E2 proteins together with 500 ng of the HPV18 URR (origin) minicircle plasmid. The cells were grown in the presence of compounds at the indicated concentrations for 24 or 48 hours, with DMSO serving as a vehicle control. Genomic DNA was extracted at the indicated timepoints, HPV18 URR DNA was linearized with BglI, and bacterially produced input DNA was digested with DpnI. HPV URR replication signals were detected by Southern blot analyses. Compound 88915 seems to have a positive effect on HPV18 URR replication. It is possible that this compound has a positive effect on cell growth , or it may stabilize (HPV) replication proteins.(TIF)Click here for additional data file.S6 FigA: U2OS #1.13 cells were grown in sub-confluent conditions in the presence of compounds for 7 days. Genomic DNA was extracted, HPV DNA was linearized with BglI, and the viral replication signal was detected by Southern blot and B: quantified by phosphoimager. C: U2OS #1.13 cells were seeded and grown for 5 days without splitting. A clear rise in the replication level occurred between the 0-day and 5-day timepoints (compare lanes 17 and 18), showing that the switch to late amplification replication occurred. On the 5th day, when late amplification had been turned on, the indicated concentrations of the compounds were added to the media, and the cells were grown for an additional 7 days without splitting. On the 12th day, genomic DNA was extracted, HPV DNA was linearized with BglI, and the viral replication signal was detected by Southern blot and D: quantified by phosphoimager. Error bars represent standard deviations from three independent experiments. Compounds 82269, 88915, 109128 and 305831 (lanes 4–14 in panel A) clearly inhibit the stable maintenance phase of HPV18 replication. Compounds 82269, 109128 and 305831  completely abolish the late amplification of HPV18 genome. Compound 9782 does not inhibit these later stages of HPV18 replication. All the quantified signals are expressed relative to the vehicle control, DMSO, and error bars represent standard deviations from two different experiments. The compound 88915 and 305831 should have identical target (Tdp1) but the 88915 does not inhibit the late amplification of HPV18 in U2OS cells. It may result from the fact that this protein has very low potency and more effective inhibition of Tdp1 is required to inhibit the rapidly ongoing late amplification. The compound 9782 should also inhibit the stable maintenance and late amplification as it acts like a Tdp1 inhibitor by showing synergistic inhibition with CPT Click here for additional data file.S7 FigGiven are name of the compound, its 2D structure, its activities and its expected target molecule.(TIF)Click here for additional data file.S8 FigA) or non-transfected U2OS cells (B) were plated onto 6-well plates and grown in the presence of the compounds for five days. Cell cycle analyses was performed as described in the materials and method section. Results are also shown in C: U2OS cells were transfected with shRNAs specific against Tdp1, Tdp2 or PARP1, grown for three days and cell cycle profile was analyzed. Compounds nor downregulation of Tdp1 and PARP1 showed no significant changes in the cell cycle progression. By contrast, downregulation of Tdp2 with two different shRNAs caused the cell cycle block in G2/M or G1 phases. Results are also shown in HPV18-transfected ((TIF)Click here for additional data file.S9 FigIt is known that high and low risk HPVs differ from each other due to the different oncogenic potential of E6 and E7 proteins. When we saw that all the compounds identified in this study were specific high risk HPV inhibitors , we firs(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


To the Editor;th-7th with more than 3200 participants and over 1300 abstracts. Some studies will draw perspectives for the future and innovate many researchers in new eras.The European Gynecological Oncology Congress, 2017, was held in Vienna between November 4Nowadays, less radical cancer surgery is a topic of discussion to prevent morbidities. On the other hand, molecular and genomic research is increasing tremendously to solve the problem completely.The Lymphadenectomy in Ovarian Neoplasms study by Lorusso et al.  evaluateCervical cancer is generated by human papilloma virus (HPV) infection and the efficacy of HPV vaccines for the prevention of cervical cancer has previously been documented with bivalent and quadrivalent HPV vaccines. The 9-valent HPV vaccine targets HPV type 6, 11, 16, 18 and 31, 33, 45, 52, 58. Joura (Despite efforts to prevent HPV infection before serious high-grade cervical preinvasive lesions, whether high-grade lesions or cervical carcinoma can be treated with vaccines is a matter of research. Park et al.  reportedNew HPV vaccines, with regard to prevention and treatment, will have a role in the limitation and possibly the eradication of cervical cancer.


Cervical cancer and its precursor, high-grade cervical intraepithelial neoplasia (CIN2/3), are associated with persistent high-risk human papillomavirus (HPV) infection. HPV genotype prevalence varies with severity of cervical lesions, patient age and geographical location. The aim of this study was to investigate HPV genotypes prevalence and attribution according to the severity of cervical lesions among Chinese women.A 4-year surveillance study was performed. A total of 1664 female patients were included and their cervical histological diagnosis consisted of cervical intraepithelial neoplasia grade 1 , grade 2 , grade 3  and invasive cervical cancers . HPV genotypes prevalence and attribution to cervical lesions were calculated and analyzed. The 95% confidence interval (CI) for proportion was also calculated.HPV positivity rates increased directly with cervical lesions severity . Infections with multiple HPV types were inversely related to cervical lesions severity. HPV16, 52, 31, 33 and 58 were the most prevalent genotypes in ICC. 49.1% of squamous cell carcinoma, 65.1% of adenocarcinoma and 12.0–43.3% of cervical intraepithelial neoplasia could be attributed to vaccine-covered high-risk genotypes (HPV16/18). Inclusion of HPV52 and HPV31 in future vaccines would provide the highest marginal benefit in protection for individuals residing in this region.These findings provide information about HPV genotypes in this region which may be important to target with future vaccination and screening programs.The online version of this article (doi:10.1186/s12879-017-2223-1) contains supplementary material, which is available to authorized users. Cervical intraepithelial neoplasia (CIN) is a premalignant lesion that is diagnosed by histology as CIN1, CIN2, or CIN3. If left untreated, CIN2 or CIN3 (high-grade CIN) might progress to cervical cancer. There were an estimated 527,600 new cervical cancer cases and 265,700 deaths worldwide in 2012 , 5.For prevention of HPV infection and the potential development of cervical neoplasia, two prophylactic vaccines (Cervarix by GlaxoSmithKline and Gardasil by Merck Sharp and Dohme) are currently available. These two HPV vaccines potentially provide protection against two most common HPV genotypes, HPV16 and 18, which cause approximately 70% of invasive cervical cancers worldwide , 7. In EA recent study performed by our group reported the HPV prevalence among female outpatients from Qingdao , which iFrom January 2012 to December 2015, women who visited gynecologists or oncologists seeking for further management of abnormal cervical cytology results in Qingdao Central Hospital and Qingdao Cancer Hospital, a tertiary hospital attached to Qingdao University with 1600 beds and a tertiary hospital specializing in oncology, were enrolled with informed consents. A participant was eligible if she (a) was sexually active, (b) was a gynecological patient, (c) was no previously miscarriage nor presently pregnant, (d) had not undergone a total hysterectomy, and (e) agreed to receive HPV genotyping and cervical histopathology evaluation. A cervical scrape sample was collected for HPV genotyping. Biopsy-confirmed histopathologic findings were used to define the status of cervical neoplasia. All biopsy samples were taken by a gynecologist according to guidelines from International Agency for Research on Cancer, WHO . All pro°C for 15 min, initial denaturation at 95 °C for 10 min, followed by 10 cycles of 94 °C for 10 s, 42 °C for 90 s and 72 °C for 30 s, followed by 30 cycles of 94 °C for 10 s, 46 °C for 60 s and 72 °C for 20 s, ending with a final extension at 72 °C for 5 min. The PCR product (25 μl) was denatured at 95 °C for 10 min and immediately chilled on ice. Then, the HPV DNA hybridization reaction was performed on an automatic analyzer according to the manufacturer’s instructions. The hybridization procedure was presented in Additional file Cervical scrape samples were stored in a specimen transport medium. DNA extraction, amplification and hybridization were performed using an HPV genotyping kit  according to the manufacturer’s instructions. The HPV DNA chip in the kit contained 23 type-specific probes that recognize 18 high-risk  and 5 low-risk HPV genotypes . PCR amplifications were performed in a T100 Thermal Cycler  with the following parameters: enzyme activation at 50 The biopsy specimens obtained from the subjects were fixed by formalin, embedded in paraffin and stained with hematoxylin-eosin for histopathology in the pathology laboratory at Qingdao Central Hospital. Cervical histological diagnosis of the specimens was reported only when three different pathologists, from Qingdao Central Hospital and Qingdao Cancer Hospital, agreed with each other. Pathology results were classified into cervical intraepithelial neoplasia grade 1 (CIN1), CIN grade 2 (CIN2), CIN grade 3 (CIN3) and invasive cervical cancer (ICC) according to cervical lesions classification criteria .χ2 test and p < 0.05 was considered significant.Frequencies of clinical data were calculated. The prevalence and attribution rates of HPV genotypes were calculated according to a previous method . Crude pp = 6.36 × 10-13). The HPV positivity rate of adenocarcinoma group was significantly lower than squamous cell carcinoma group (p = 2.13 × 10-11). In addition, patients with lower grade cervical lesions  displayed a higher frequency of multiple-type HPV infections compared with those suffering from higher grade cervical lesions  and invasive cancers .According to the inclusive criteria, a total of 1664 women received HPV genotyping and cervical histopathology evaluation. Their cervical pathology status were as follows: 376 cases of CIN1, 408 cases of CIN2, 336 cases of CIN3, 440 cases of squamous cell carcinoma (SCC) and 104 cases of adenocarcinoma. HPV positivity rates and the proportion of single- and multiple-type infection for each cervical pathology status are presented in Table Detailed HPV genotypes distribution according to the severity of cervical lesions was also investigated. Each kind of genotype was counted separately in cases with multiple-type HPV infections. As shown in Fig. The crude prevalence and attribution rates of all HPV genotypes to each cervical pathology status were presented in Table HPV16 also accounted for the majority of high-grade lesions (CIN3 and CIN2) with attribution rate of 43.3 and 30.7%, respectively. HPV52 was the following genotype, which attributed to 11.1 and 14.1% of CIN3 and CIN2, respectively  and age-specific prevalence of cervical lesions and cancers was presented in Fig. Data of HPV prevalence and attribution according to cervical pathology status presented in this study may provide guidance for HPV vaccination program and preventative strategies against cervical cancer in this region of China. Among the involved female patients, HPV positivity rates increase directly with the cervical lesion severity, which is consistent with some previous studies from different regions of China , 13–15. In this study, we found a high rate of young women with high grade cervical lesions (CIN2 and CIN3). 30.6% of patients with high-grade cervical lesions were younger than 35. Notably, 5.0% of patients with high-grade cervical lesions were even younger than 25, which is higher than that from a neighboring area of this region (3.4%) . TherefoFurthermore, an approach adopted from previous studies , 25, whiIn this present work, the “attribution factor” of each HPV genotype was calculated and cumulative attribution rates of prevalent high-risk HPV genotypes were presented. The 95% confidence interval (CI) for proportion was also calculated. These methods have been rarely used to analyze HPV infections in mainland China before. There are still some limitations in this study. The total number of samples involved in this study is not large. Multi-center study, including over three tertiary hospitals, may be more valuable. In addition, interactions between different HPV genotypes still need further investigation.This study presented HPV genotypes prevalence and attribution data among female patients with cervical lesions in this area, where the planning of HPV vaccination should be taken into account the prevalent genotypes of HPV16, HPV52, HPV31, HPV58 and HPV33. These data added new evidence that eastern Asia, including eastern China, exhibited an HPV genotype distribution pattern distinct from other parts of the world. Our results also could provide necessary reference for formulating a regional HPV-based screening strategy in the future.


Following the well-established relationship between human papillomavirus (HPV) and cervical carcinoma, the carcinogenicity of this virus has also been confirmed in subsets of head and neck carcinoma (HNCA), but mainly in the oropharynx. Other subsites of HNCA with less known association to HPV have never been studied in Thailand. Accordingly, the aim of this study was to investigate the prevalence of HPV DNA in hypopharyngeal and laryngeal squamous cell carcinoma in Thai population.This cross-sectional study included hypopharyngeal and laryngeal squamous cell carcinoma patients diagnosed and treated at the Department of Otorhinolaryngology, Siriraj Hospital during the September 2011–December 2013 study period. Presence of HPV genome was confirmed by polymerase chain reaction from pathologically-confirmed fresh specimens. Demographic data and risk factors of HPV infection were evaluated.Eighty patients were included, and 95% of those were male. Only one patient was noted with positive HPV-62 serotype. Most patients consumed tobacco and/or alcohol. Five patients had no risk factors for cancer development. Risk of HPV infection was evaluated by self-reporting questionnaire. The mean age of sexual debut was 20.17 years. Forty-eight patients had multiple sexual partners. Sixteen and seven patients had history of sexually transmitted disease infection and habitual oral sex contact, respectively.There was no oncogenic HPV DNA detected within pathologic specimens of laryngeal and hypopharyngeal cancers in this study. Compared to rates reported from developed countries, the prevalence of HPV-related HNCA in Thailand is very low. The incidence of head and neck cancer (HNCA) is currently declining. In 2012, the World Health Organization (WHO) estimated 686,000 new cases of HNCA [Certain lifestyle behaviors, such as smoking, poor diet, and physical inactivity, are known major risk factors for carcinogenesis. Other possible risk factors for the upper aerodigestive malignancies include chronic mucosal irritation and gastroesophageal reflux disease. Infection has been acknowledged as an etiological factor in certain types of cancer, including stomach, liver, and cervical cancer. In HNCA, human papillomavirus (HPV) infection has been proven to be a major cause of cancer development only at the base of tongue and in the tonsillar regions. Nonetheless, due to the heterogeneity of HNCA, the relationship between HPV infection and other subsites of the head and neck has not been conclusively established.The global impact of HPV-related malignancies has been analyzed in accordance with criteria set forth by the International Agency for Research on Cancer. A strong association has been identified between HPV and HNCA in high Human Development Index (HDI) regions and countries, including Europe, North America, Australia, New Zealand, and Japan . The inc, et al. found 24% prevalence of HPV in more than 1400 laryngeal or hypopharyngeal cancer patients using the polymerase chain reaction (PCR)-based method [Even though HPV infection can develop in almost all parts of the upper aerodigestive tract, the classical manifestation is often located at the larynx. Laryngeal papillomatosis is a benign pathologic lesion that has been recognized for almost a century. The larynx is usually infected by low oncogenicity HPV subtypes that have a malignant transformation rate of less than 2% . A systed method .Subsites of HNCA with less known association to HPV than the oropharynx have never been studied in Thailand. Accordingly, the aim of this study was to investigate the prevalence of HPV DNA in hypopharyngeal and laryngeal squamous cell carcinoma at Thailand’s largest national tertiary referral center.This cross-sectional study included hypopharyngeal and laryngeal squamous cell carcinoma patients diagnosed and treated at the Department of Otorhinolaryngology, Siriraj Hospital, Thailand’s largest national tertiary referral center, during the September 2011 to December 2013 study period. The protocol for this study was approved by the Siriraj Institutional Review Board (SIRB), Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.Patients aged >20 years that were diagnosed with laryngeal or hypopharyngeal squamous cell carcinoma without prior treatment during the study period were included. Patients were divided into one of two study groups according to cancer type. Laryngeal cancer was subclassified into three subsites, including supraglottic, glottic, and subglottic regions. Hypopharyngeal cancer was subdivided into pyriform sinus, posterior pharyngeal wall, and postcricoid regions. Both cancers were staged according to the seventh edition of American Joint Committee on Cancer (AJCC) staging criteria . The samThe case record form used for this study was divided into two parts, as follows: Part 1) Demographic data and clinical information; and, Part 2) Data from the concealed self-reporting Thai-language questionnaire about behavioral risk factors for HPV infection and cancer development, including number of lifetime sexual partners, age of sexual debut, history of habitual oral sex practice, and history of sexually transmitted diseases. Patients were notified of their right to decline answering the sexual behavior questions listed in part 2.A 1.5–2 mm histologically-confirmed fresh specimen of hypopharyngeal and laryngeal squamous cell carcinoma from each included patient was prepared by pathologist. DNA was extracted, and PCR of linear array hybridization for specific HPV test was performed in all 80 specimens. Biotinylated DNA was used as a primer for genotypic testing. The protocol used for HPV identification was previously described by Steinau et al., with 96% sensitivity and 99% specificity .Eighty patients were included, and 95% of those were male. There were 40 patients in the hypopharyngeal group and 40 patients in the laryngeal group. The mean age of patients was 61.5 years (range: 34–89). More than 80% of patients presented with an advanced stage, and almost all of them were smokers (Table More than 70 patients (87.5%) completed the questionnaire that elicited data about risk behaviors for HPV infection Table . There wHPV-positive malignancies represented 5–20% of all HNCA . Data frIn our previous study, only 6% of HPV-related cancer was found in the oropharynx. In this present study, no oncogenic HPV infection was identified in pathologic specimens of laryngeal and hypopharyngeal cancer among Thai study population. This discrepancy in HPV prevalence might be due to a very high rate of tobacco-derived carcinogenesis in our population, which is similar to the result reported from a study in Northern Spain . The patHPV is transmitted sexually through micro-abrasions of skin or mucosa. As a subclinical infection, HPV is the most common sexually transmitted infection worldwide, with an estimated lifetime risk of cervical HPV infection in women of up to 80% . Viral gThe low prevalence of HPV infection in our study population may reflect a conservative culture of Asian countries during the past 50 years. With regard to the sexual behavior questionnaire, it is possible that some of the reported data was inaccurate. For example, the age at first sexual activity and the number of life time partners for some study respondents may have represented only a ‘best guess’ estimate. It should also be noted that detection of HPV genome does not conclusively indicate past high-risk sexual activity. As such, the collection of previous sexual behavior data may not have been beneficially informative in this study.The first reason why HPV may be undetectable in non-oropharyngeal squamous cell cancer is that different mucosal linings have different characteristics. While the palatine and lingual tonsils of the base of the tongue, which are parts of Waldeyer’s ring, are mostly composed of lymphoepithelial tissues, the hypopharynx is lined with columnar epithelium. The other reason is that most of the human papillomavirus could be cleared by intact immunity without causing any clinical manifestations. Only in a small number of people, viral genome can integrate into host tissue and remain in a latent form, and then can progress later into an invasive lesion.The overall rate of high risk HPV infection detected in oral rinsed exfoliated cell among sexually active adults in the US was reported to be 3.7% . There iIn situ hybridization(ISH) is a preferred method for determining the integration of viral genome into a host cell. To cover a broader range of viral subtypes, HPV probe cocktails have recently been invented. This method has higher specificity, lower sensitivity, is more time consuming, and is only available in selected centers . TranscrAs a result of its high positive predictive value, overexpression of p16 protein is proposed as a surrogate marker of HPV activation in oraopharyngeal cancer. Strong staining of p16 in classic HPV-related histomorphology is more practical and cost effective than PCR and ISH . In contLaryngeal papillomatosis is the most common benign neoplasm of the larynx. The HPV genome is integrated at the squamocolumnar junction of the true vocal cord. Most infections are caused by noncarcinogenic viral strains type 6 and 11. No strong evidence has yet been reported that establishes association between HPV infection and laryngeal cancer. Papilloma viral tropism reflects a distinct host-viral interplay among different virus subtypes. A phylogenetic study reported that each variant has its own distinctive potential to induce lesions among different types of mucosal epithelium . ConsistAlphapapillomavirus genus of viruses. HPV-62 is a low-risk HPV that was reported as coinfection in 5.1% of the HPV-positive cervical samples [In our study, the HPV from the patient who was found to be positive for HPV type 62 serotype belongs to the  samples . In Indi samples , 30. Sim samples . RegardlThe patients enrolled in this study were from a single center, Thailand’s largest tertiary referral hospital, which means that we are often referred patients with complicated and intransigent conditions. As such, it is possible that our findings may not be generalizable to patients with the same condition in other settings.The epidemiological trends of head and neck cancer can be classified into two groups, HPV-related and HPV-unrelated HNCA. In recent decades, with the increasing prevalence of HPV-related HNCA, the overall prevalence of HPV-unrelated HNCA has gradually been decreasing due to the reduction of tobacco and alcohol consumption. Hence, HPV status is proved to be an effective prognostic predictor that can potentially affect the outcome of treatments. Particularly, identification of HPV is mandatory in the high prevalence of anatomical subsite.In this study, only 1 of 80 cases (0.0125%) had positive HPV DNA finding. HPV-62 serotype (non-oncogenic) was identified. There was no oncogenic HPV DNA detected within pathologic specimens of laryngeal and hypopharyngeal cancers.Since any subsites other than oropharynx are considered as HPV-unrelated, the detection is thus not routinely recommended in our institute. Moreover, the risks of sexually transmitted diseases in the past do not determine the possible risk of cancer development, especially in countries with less strong association of HPV driven carcinogenesis.


N = 6051) were randomized (1:1) to receive three doses of HPV‐16/18 vaccine or Al(OH)3 (control) at M0, 1, 6. VE against HPV‐16/18‐associated CIN2+, and cross‐protective VE against infections with nonvaccine oncogenic HPV types, immunogenicity, and safety were assessed. In the according‐to‐protocol efficacy cohort, in initially seronegative/DNA‐negative women , VE against HPV‐16/18‐associated CIN2+ was 87.3% (5.3–99.7); VE against incident infection or against 6‐month persistent infection associated with HPV‐31/33/45 was 50.1% (34.3–62.3) or 52.6% (24.5–70.9), respectively. At least, 99.6% of HPV‐16/18‐vaccines remained seropositive for anti‐HPV‐16/18 antibodies; anti‐HPV‐16 and ‐18 geometric mean titers were 1271.1 EU/mL (1135.8–1422.6) and 710.0 EU/ml (628.6–801.9), respectively. Serious adverse events were infrequent . Of the 1595 reported pregnancies, nine had congenital anomalies  that were unlikely vaccination‐related (blinded data). VE against HPV‐16/18‐associated CIN2+ was demonstrated and evidence of cross‐protective VE against oncogenic HPV types was shown. The vaccine was immunogenic and had an acceptable safety profile.We previously reported the results of a phase II/III, double‐blind, randomized controlled study in Chinese women (NCT00779766) showing a 94.2%  HPV‐16/18 AS04‐adjuvanted vaccine efficacy (VE) against cervical intraepithelial neoplasia grade 1 or higher (CIN1+) and/or 6‐month (M) persistent infection (PI) with a mean follow‐up of <2 years, and immunogenicity until 7 months post‐dose 1. Here, we report efficacy and safety results from an event‐triggered analysis with ~3 years longer follow‐up, and immunogenicity until M24. Healthy 18–25‐year‐old women ( Cervical cancer is the fourth most common cancer among women worldwide and the second most common female cancer in women aged 15–44 years, with nearly 528,000 women diagnosed with cervical cancer and 266,000 deaths in 2012 Persistent infection (PI) with high‐risk (HR) human papillomavirus (HPV) types has been established as the major etiological factor for high‐grade cervical intraepithelial neoplasia (CIN) and cervical cancer In China, cervical cancer is a major public health concern, with an estimated 98,900 cases and 30,500 deaths in 2015 Cervarix™, GSK), is licensed in over 135 countries and was recently approved in China. The vaccine has been shown to be efficacious against high‐grade CIN associated with HPV‐16/18, and irrespective of HPV type in a naïve  population The prophylactic HPV‐16/18 AS04‐adjuvanted vaccine  conducted in four centers in China. Participants were randomized 1:1) to receive three doses of the HPV‐16/18 AS04‐adjuvanted vaccine or aluminium hydroxide ), and HPV‐45 (52.3% [14.4–74.4]), while VE against 6M PI or 12M PI was significant for HPV‐31 (65.6% [27.3–85.0] and 81.2% [34.4–96.5]), but not for HPV‐33 (47.9% [−5.9–75.5] and 10.6% [−161.2–70.0]) or HPV‐45 (11.9% [−178.0–72.8] and −152.3% [−2549.9–58.7]) (Table .2–71.5),The HPV‐16/18 baseline serostatus was similar in both groups. The majority (63.3%) of women were seronegative for both HPV‐16 and HPV‐18 at study start (63.5% in the vaccine group and 63.0% in the control group). In the vaccine group, 11.8% of women were seropositive for both HPV‐16 and HPV‐18 antibodies, 18.7% were seropositive for HPV‐16 alone, and 6.0% were seropositive for HPV‐18 alone. In the control group, 9.2% of women were seropositive for both HPV‐16 and HPV‐18 antibodies, 22.4% were seropositive for HPV‐16 alone, and 5.3% were seropositive for HPV‐18 alone.At M24, all women from the vaccine group remained seropositive for anti‐HPV‐16 antibodies, and 99.6% of women were seropositive for anti‐HPV‐18 antibodies Fig. . In the At M24, in initially seronegative women from the vaccine group, anti‐HPV‐16 and anti‐HPV‐18 GMTs were 1271.1 EU/mL (1135.8–1422.6) and 710.0 EU/mL (628.6–801.9), respectively, and remained above anti‐HPV‐16/18 GMT plateaus associated with sustained protection in a previous efficacy study The safety outcomes are presented in Table A total of 361 women (6.0%) reported at least one medically significant AE  . The majority of pregnancies (74.0%) resulted in live infants with no apparent congenital anomalies, while 17.6% of pregnancies with no apparent congenital anomalies were electively terminated. Two women (0.1%) reported stillbirths with no apparent congenital anomalies. Nine pregnancies (0.6%) resulted in an offspring with congenital anomalies assessed by the investigator as unlikely related to the study vaccine administration; five (0.3%) resulted in live infants, three (0.2%) were electively terminated, and one (0.1%) resulted in a stillbirth. As no pattern in the nature of the congenital anomalies was noticed, the pregnancy data will remain blinded to treatment allocation until the study end. Fifty‐one (3.2%) pregnancies were ongoing at the time of the current analysis.A total of 25 pregnancies were reported around the time of vaccination. Eight pregnancies (0.5%) resulted in birth of a live infant with no congenital anomaly and 14 (0.9%) without any apparent congenital anomaly were electively terminated (Table S2). There were no pregnancies with congenital anomaly around the time of vaccination.In China, cervical cancer is the second most frequent cancer, following breast cancer, among women aged 30–44 years, and the third most common, following thyroid cancer and breast cancer, in women below the age of 30 This study of the HPV‐16/18 AS04‐adjuvanted vaccine is to the best of our knowledge the first large controlled clinical study of vaccination efficacy against HPV conducted in China. We previously reported a high VE (94.2% [62.7–99.9]) against HPV‐16/18‐associated CIN1+ and/or 6M PI within 24 months following last vaccine dose A high VE  against CIN2+ associated with HPV‐16/18 was observed in both ATP‐E and TVC‐E cohorts in women with seronegative status at baseline. One CIN2+ case was observed in the vaccine group; however, the assessment of real causal association was difficult due to the multiple oncogenic HPV infections that were detected in both the lesion and the preceding cytological specimens of this case. A similar phenomenon was also observed in the PATRICIA trial; in 28.6% of the 256 CIN2+ cases associated with HPV‐16 and/or HPV‐18, DNA from nonvaccine HR‐HPV types was also detected in the final event‐driven analysis The overall efficacy results of this study are in line with the previous efficacy trials conducted in similar age populations The anti‐HPV‐16/18 antibody kinetics were also in line with previous observations The proportions of women with SAEs, MSCs, NOCDs, and NOADs were similar in the vaccine and control group, consistent with finding of the previous studies of the HPV‐16/18 AS04‐adjuvanted vaccine in similar age group The study design was similar to other large trials evaluating the efficacy of the HPV‐16/18 AS04‐adjuvanted vaccine in terms of population, vaccination schedule, clinical management for abnormal cytology, colposcopy referral, and HPV DNA testing This study is the first long‐term large‐scale clinical trial of HPV vaccination conducted in China. Additional strengths of the study include the facts that the clinical examination and diagnosis were performed according to a unified standard and the HPV testing was performed in a high‐quality control central laboratory. In this study, local Chinese laboratories were used which were different from the central laboratories in the other efficacy trials. The consistency of the efficacy results between the Chinese and global populations while using different laboratories for diagnosis of the efficacy endpoints demonstrate the robustness of these efficacy findings.A potential limitation of the study is that all women included in this study were sexually active (majority were married) at the time of study entry due to ethical and cultural considerations, and there were more women who were closer to the upper age limit (mean age 23 years at vaccination). Another limitation is inclusion of women from only one of the seven geographic regions in China, which might not fully reflect the overall Chinese population.The results of this follow‐up study in young Chinese women show that the HPV‐16/18 AS04‐adjuvanted vaccine is efficacious against cervical infections, abnormal cytology, and lesions associated with HPV‐16/18. In addition, cross‐protective VE against PI with HPV‐31/33/45 was consistent with that previously reported in the global PATRICIA efficacy trial. The vaccine induced high and sustained anti‐HPV‐16 and anti‐HPV‐18 antibody responses that were comparable with those observed in global trials, and had a clinically acceptable safety profile.http://www.icmje.org/coi_disclosure.pdf and declare the following potential conflicts of interest: The institutions of Fang‐Hui Zhao, Shang‐Ying Hu, Ying Hong, Yue‐Mei Hu, Xun Zhang, Yi‐Ju Zhang, Qin‐Jing Pan, Wen‐Hua Zhang, Cheng‐Fu Zhang, Xiaoping Yang, Jia‐Xi Yu, Jiahong Zhu, Yejiang Zhu, Feng Chen, Qian Zhang, Hong Wang, Changrong Wang, Jun Bi, Shiyin Xue, Lingling Shen, Yan‐Shu Zhang, Feng‐cai Zhu received grants/investigator fees from the GSK group of companies for the conduct of this study. Fang‐Hui Zhao, Feng‐cai Zhu, and Yue‐Mei Hu received support for travel to meetings related to the study from the GSK group of companies. Yunkun He, Haiwen Tang, Naveen Karkada, Pemmaraju Suryakiran, Dan Bi, and Frank Struyf are employees of the GSK group of companies. Haiwen Tang, Dan Bi, and Frank Struyf own stock options and shares in the GSK group of companies.All authors have completed the Unified Competing Interest form at Table S1. Vaccine efficacy against cervical intraepithelial neoplasia and persistent infection associated with HPV‐16 and/or HPV‐18 in women who were HPV DNA‐negative at baseline for the corresponding HPV type, regardless of initial serostatus (ATP‐E and TVC‐E).Table S2. Pregnancy outcomes (TVC).Click here for additional data file.