The overall survival of patients with pancreatic ductal adenocarcinoma is extremely low. Although gemcitabine is the standard used chemotherapy for this disease, clinical outcomes do not reflect significant improvements, not even when combined with adjuvant treatments. There is an urgent need for prognosis markers to be found. The aim of this study was to analyze the potential value of serum cytokines to find a profile that can predict the clinical outcome in patients with pancreatic cancer and to establish a practical prognosis index that significantly predicts patients' outcomes. We have conducted an extensive analysis of serum prognosis biomarkers using an antibody array comprising 507 human cytokines. Overall survival was estimated using the Kaplan-Meier method. Univariate and multivariate Cox's proportional hazard models were used to analyze prognosis factors. To determine the extent that survival could be predicted based on this index, we used the leave-one-out cross-validation model. The multivariate model showed a better performance and it could represent a novel panel of serum cytokines that correlates to poor prognosis in pancreatic cancer. B7-1/CD80, EG-VEGF/PK1, IL-29, NRG1-beta1/HRG1-beta1, and PD-ECGF expressions portend a poor prognosis for patients with pancreatic cancer and these cytokines could represent novel therapeutic targets for this disease. Pancreatic ductal adenocarcinoma (PDAC) accounts for only 2.68% of all cancers, but it represents the fourth leading cancer-related death worldwide just remaining after lung and bronchus, prostate, and colorectum cancers in men and after lung and bronchus, breast, and colorectum cancers in women . The drediagnosis biomarkers); those that predict responses to treatments (predictive biomarkers); and those that forecast the likely course of the disease, including survival and recurrence pattern in the absence of therapy (prognosis biomarkers). Finding a biomarker or a panel of biomarkers that would be able to predict the clinical impact of a chemotherapy regimen even before it is initiated is highly warranted to (1) identify those patients more likely to benefit from aggressive therapies; (2) reduce risks of useless side effects and help to set expectations for doctors and patients; and (3) make attempt to apply new combination of therapies or individualized treatment protocols according to their expected responses. In addition, markers that display prognosis significance also offer the potential to become emergent therapeutic targets and novel strategies in the management of PDAC [As defined by the NIH Biomarker Working Group, a biological marker (biomarker) is a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention . In PDAC of PDAC , 8. In t of PDAC –16.Here we have focused on those inflammatory mediators that may constitute useful prognosis biomarkers for PDAC detection. Altered levels of circulating inflammatory cytokines have been found in cancer patients for nearly every cancer examined, even at early stages of the development, indicating that immune response has an important role during carcinogenesis and that circulating inflammatory markers may be useful cancer biomarkers , 18. CytThe aim of this study was to investigate the prognosis significance of serum cytokines as a reflection of the host response to tumor in PDAC patients. A conditional stepwise algorithm based on likelihood rate analysis according to the Cox's proportional hazard model was used to identify the best combination of significant prognosis factors. An equation was then derived for modeling the survival in our specific cohort. A leave-one-out cross validation was developed to assess the model.n = 14). All information from patients, including gender, age, disease grade, and symptoms was recorded. The mean age of the patients was 66 years  with a male to female ratio of 50 : 50. Clinical staging for patients with pancreatic adenocarcinoma was as follows: stage III (28%) and stage IV (72%)  from 2008 to 2011 (IV (72%) . PDAC pahttp://dx.doi.org/10.1155/2015/518284).Soluble proteins in the sera of PDAC patients were measured using a biotin label-based human antibody array ), according to the recommended protocols. Briefly, all samples were biotinylated. Antibodies were immobilized in specific spot locations on glass slides. Incubation of array membranes with biological samples resulted in the binding of cytokines to corresponding antibodies. Signals were visualized using streptavidin-HRP conjugates and colorimetric. Final spot intensities were measured as the original intensities subtracting the background. Data were normalized to the positive controls in the individual slide. The antibody array data is provided in Supplementary Table 3 . Besides, Wald index was shown to determine the weight of each variable in the global model, both uni- and multivariate. The cytokine levels were introduced in the models as continuous parameters and results were expressed as the hazard ratio (HR) or relative risk ratio for one unit change. By analysis of these variables, a prognosis index (PI) that considers the regression coefficients derived by Cox's model of all significant factors was obtained. This model and the PI calculation were carried out according to the equations provided by Cox [P < 0.05.The overall model fit was considered significant based on chi-squared statistic test  was applied to assess the performance of the prognosis model as the simplest and most widely used method for estimating prediction accuracy . For thiClinical characteristics of the PDAC patients are summarized in P = 0.030 and P = 0.013, resp.). Concerning cytokines, at univariate analysis after feature selection, expression levels of BDNF ); HVEM/TNFRSF14 ); IL-24 ); IL-29 ); leptin R ); LRP-6 ), and ROBO4 ) showed a significant influence on prognosis. Results of the univariate analysis of each cytokine as independent prognosis factors and its beta-coefficients (β), hazard ratios (representing the factor by which the hazard changes for each one-unit increase of the cytokine expression), 95% CI , and P values are shown in P value was provided to show that the differences between the two groups were significant.First, a univariate approach was used in this study to identify relevant and independent measurable factors at prognosis that could be associated to a higher risk of PDAC death. Serum levels of cytokines before treatment and clinicopathologic parameters such as age, gender, stage, and clinical response were analyzed. Amongst the clinicopathologic parameters, age and the clinical response  were asR-squared measurement) of 0.914. These results represent that a 91.4% of the variability in the survival time is accounted for by the statistical model. In the test set, this validation showed an accuracy of 92.3%, sensitivity of 85.57% (true-poor prognosis rate) and specificity of 100% (true-fair prognosis rate) for all the left out samples (test samples). All estimated models are depicted in Supplementary Material .Despite the fact that often only those statistically significant variables in univariate analysis are included in multivariate analysis, some variables not being significant in univariate analysis may appear jointly significant in a multivariate analysis. Thus, in addition to the statistically significant variables related to poor prognosis on the univariate analysis, those also selected by the features selection procedure were also included in the multivariate model. In proteomics studies, the number of samples is usually low compared to the number of variables, due to the limited availability or the cost of measurements. Taking this into account and in order not to introduce bias due to the small sample problem, a wrapper was used as a feature selection method using conditional forward stepwise algorithm based on likelihood rate to reduce the dimensionality of the data . To asseP > 0.05) and were not considered in the global model. Concerning cytokines at multivariate analysis, expression levels of B7-1/CD80 ); EG-VEGF/PK1 ) and IL-29 ) showed a significant influence on prognosis. The significant influence on survival observed in univariate analyses for IL-29 was confirmed in multivariate analyses. Beta-coefficients (β), hazard ratio (HR), 95% CI, and P values for the selected cytokines are shown in P = 0.129), HR 1.020, 95% CI (0.994–1.047)) and PD-ECGF ((P = 0.108) HR 1.302, 95% CI (0.944–1.797)) failed to significantly influence the prognosis as independent factor, the Cox's proportional hazard analyses using conditional forward stepwise algorithm based on likelihood rate did select them as significant variables that influence the overall survival model (see below).The best combination of cytokines selected by the multivariate Cox's proportional hazard analysis is shown in P = 0.026, HR 1.042, 95% CI (1.003–1.023)) and IL-29 ) was returned. The overall model fit was shown to be significant by the chi-squared statistic test (P = 0.0023). So as to establish a prognosis index to determine PDAC patients overall survival, these cytokines β-coefficients were entered in the Cox's model [univariate represents the multivariate model derived from the combination of the underlined markers in the univariate analysis.As combinations of biomarkers are likely to provide more accurate prognosis information, the most accurate subset of variables was sought using the conditional forward stepwise regression approach based on likelihood rate. To illustrate the interrelated effect on OS of the seven markers highlighted by the univariate analysis, the Cox's proportional hazard analysis was employed to select those variables jointly correlated with the survival. As a result of this analysis, a model containing only IL-24 ('s model  and the P = 0.0003) survival model was built and the following PI model was generated:multivariate represents the multivariate model derived from the best of all possible combinations using the cytokines in the multivariate analysis having being previously selected by the wrapper feature selection method.Regarding filtered cytokines obtained by multivariate analysis, a second statistically significant . Indeed, as shown in Supplementary Table 2, the 100% of the PDAC patients were correctly classified as long/short survival according to the previously proposed cut-off in the prognosis index (PI = 17). Prognosis index for univariate model was also depicted and it ranged from 0 to 5. According to this PI, patients were again categorized into two groups: poor (PI > 1.5) and fair prognosis (PI < 1.5). Furthermore, survival curves were compared among these two prognosis groups (P < 0.004) compared with the PI multivariate.Prognosis index for multivariate model with these five cytokines ranged from 0 to 40 in our cohort. Patients were categorized into two groups according to their prognosis index: poor prognosis (PI > 17) and fair prognosis (PI < 17). Survival curves were then compared among these two prognosis groups . The pros groups  and a siIn this work, we have conducted an extensive analysis of serum prognosis biomarkers using an antibody array comprising 507 human proteins including cytokines, chemokines, adipokines, growth factors, angiogenic factors, proteases, soluble receptors, soluble adhesion molecules, and other proteins. The main objective of this analysis was to determine if a specific cytokine panel in patient before Gemcitabine and Erlotinib treatment could influence the survival time after this treatment. This is a powerful tool with great potential in applications for biomarker discovery . To asseIn the course of our evaluation, we first identified 2 cytokines that correlated with patients' prognosis in univariate analysis. Following, a panel of 5 cytokines clearly demonstrated a remarkably better overall performance for modeling OS. Therefore, the multivariate model consisting of B7-1/CD80, EG-VEGF/PK1, IL-29, NRG1-beta1/HRG1-beta1, and PD-ECGF was shown to be more accurate than the univariate model considering the most significant markers. The effectiveness of our model is clearly demonstrated with the evaluation performed by the LOOCV.Notwithstanding proposed roles for B7-1/CD80, EG-VEGF/PK1, and NRG1-beta1/HRG1-beta1 in PDAC, to the best of our knowledge this is the first time that this combination of serum cytokines has been collectively described as prognosis factors for PDAC. An overview of these biomarkers is subsequently given.B7-1/CD80. The B7 system is one of the most important secondary signaling mechanisms and is essential in maintaining the delicate balance between immune potency and suppression of autoimmunity. B7-1 (CD80) and B7-2 (CD86) are ligands expressed on antigen-presenting cells and they are responsible of the costimulatory signaling whereby T cell priming, growth, maturation, and tolerance are regulated. Upon binding to their receptors, T cell activation and survival are promoted. On the other hand, they can also deliver coinhibitory signaling binding to their inhibitory receptors and blocking T cell response [response . An inadresponse . The comresponse , 39. AltEG-VEGF/PK1. This molecule was first described as an example of a class of highly specific mitogen that acts to regulate proliferation and differentiation of the vascular endothelium in a tissue-specific manner. Although this protein does not show any structural homology to the VEGF family, they do share multiple regulatory functions related to proliferation and migration [ EG-VEGF/PK1 has been described to be related to multiple cancer types including ovarian [igration . EG-VEGF ovarian , colorec ovarian , prostat ovarian , hepatic ovarian , pancrea ovarian  and neur ovarian . It has  ovarian .IL-29. Also referred as IFN-λ1, it belongs to the type III INF family and it has been described to induce similar biological activities to type I INF family (INT-α and β). Although both are able to induce antiproliferative responses in many cell types, IFN-λ1 appears to be more limited. Signalling via the IFN-λ1 results in activation of STATs, MAPKs and PI3K pathways [λ1 to trigger these alternative pathways could be cell-type specific or altered in cancer cells. Contrasting conclusion has been derived from other study that suggests growth induction in human multiple myeloma cells through MAPK activation [pathways –50. Howetivation . The preNRG1-beta1/HRG1-beta1. Neuregulin-1 or heregulin-1 is an extracellular protein ligand meant to bind to the ErbB receptors family members, ErbB3 and ErbB4. Upon interaction with their receptor, a wide range of biological events including the induction and progression of several epithelial cancers are prompted. The NRG1/HRG1 proteins play essential roles in the nervous system, heart, and breast and are involved in the development of human diseases, including schizophrenia and breast cancer [β mRNA [t cancer , 53. An t cancer . Their pt cancer  which ha [β mRNA . ErbB3 h [β mRNA . It has  [β mRNA .PD-ECGF. It is also known as thymidine phosphorylase; its activity and expression in carcinomas of the esophagus, stomach, colorectum, pancreas, and lung are significantly higher than in the adjacent nonneoplastic tissue and may have an important role in the proliferation of these solid tumours. PD-ECGF is expressed not only in the tumor cells but also in the tumor associated stromal cells [al cells , 60. Regal cells .It may not be possible for one single biomarker to provide the necessary prognosis information about the patient to base treatment options on. For this reason, panels of biomarkers are advisable to accurately predict the stage of the disease and how it will progress. Previous studies have indicated that tumor prognosis is closely associated with immune escape by tumor cells. A dynamic relationship between the host immune system and cancer is emerging . PresentWe are aware of the limitation imposed by population size in this study. However, we have tried to apply a robust statistical analysis and validation. Although PDAC is amongst the less prevalent cancer and studies with large sample size are difficult to be carried out, its aggressiveness and the poor outcome urge to search novel prognosis biomarkers as the basis for rational treatment decisions, analysis of novel therapeutic interventions, and tailored treatment approaches . For thiIn summary, we have identified for the first time a panel of five serum cytokines comprising B7-1/CD80, EG-VEGF/PK1, IL-29, NRG1-beta1/HRG1-beta1, and PD-ECGF with prognosis significance in PDAC. These molecules might not only allow a more accurate prediction of prognosis of patients with PDAC but also represent novel targets for therapeutic agents. Studies in prognosis biomarkers achieving true clinical impact and improving patient management and outcome are a matter of the utmost importance in PDAC. Besides, being able to foresee the prognosis of a PDAC patient may help to avoid futile therapy approaches and to improve quality of life of those whose long-term survival is unpromising.Supplementary Figure 1 shows all estimated models using the different training sets in the Leave-one-out cross-validation (LOOCV). LOOCV was applied as a useful technique for assessing the performance of the model. One sample is removed from the initial 14 sample dataset, leaving a temporary 13 sample training set and one left out-sample. The R-squared measurement indicates the models goodness of fit. Supplementary Table 1 provides the clinicopathological data of the 14 PDAC patients including their overall survival. Supplementary Table 2 compares the real and estimated overall survival for the 14 PDAC patients. Finally, supplementary Table 3 lists the whole normalized data of the cytokine antibody array.


The aim of this study was to analyze the prevalence of HPV and EBV in oral and oropharyngeal squamous cell carcinoma in south-eastern Poland. The correlation between viral infection, OSCC, alcohol use, tobacco smoking, demographic data , anatomic location, pre-treatment staging, evidence of metastases to lymph nodes, and grading was also investigated.The examination samples were collected from paraffin tissue blocks, from 154 patients. Viral DNA was amplified by the nested-PCR method.HPV DNA was detected in 29.2 % of the tested samples . The HPV type 16 was detected in 15.6 % of all samples, and in 53.3 % of HPV-positive group. In HPV-positive samples from oropharyngeal HPV 16 constitute 76.5 %, and in HPV-positive samples from oral cavity HPV 16 constitute 39.3 %. Mixed infection (more than one type of HPV) was observed in 23.5 and 60.7 %, respectively, and in 46.7 % of all HPV-positive samples, and in 12.3 % of the whole study group. EBV DNA was detected in 27.3 % of the cases and HPV-EBV co-infection in 7.8 % of samples.In major patients from Southeastern region of Poland with oropharyngeal cancer HPV type 16 was detected but in oral cavity cancer other mixed infections were observed . HPV 16 was detected more often among patients younger than 50 years of age, whereas the mixed HPV in the group aged 50–59. The pathogenesis of oral squamous cell carcinoma may be connected with EBV infection. Future studies on the mechanisms of HPV/EBV co-infection and/or superinfection and their role in oral squamous cell carcinoma are necessary. Oral squamous cell carcinoma (OSCC) is a serious public health problem in many parts of the world and in Poland too. In Poland oral and oropharyngeal cancer constitutes 3.8 % cancers among men and 1.3 % cancers among women . The etiThe first original observation that implicated HPV as a risk factor in the development of oral cancer was presented by Syrjänen et al. in 1983 . Since tHerpesviridae family, has a double-stranded DNA genome [Epstein-Barr virus, which is also known as human herpesvirus 4 (HHV-4) and which belongs to the A genome . EBV is A genome . EBV wasA genome . EBV is A genome , 13–15. A genome , 16. A lA genome , 18.The aim of this study was to analyze the prevalence of HPV and EBV and their co-infection in oral and oropharyngeal squamous cell carcinoma in south-eastern Poland.Tissue samples were obtained from 154 patients with primary oral and oropharyngeal SCC treated in the Chair and Department of Otolaryngology and Laryngological Oncology of the Medical University of Lublin. Samples were collected in 2006–2009. The group consisted of 131 males and 23 females, aged between 40 and 87 (average 56.8 years). In the examination group there were 92 patients with oral, and 62 with oropharyngeal SCC. It was a retrospective investigation and the examined materials were obtained from formalin-fixed, paraffin-embedded tissues. The samples were collected during surgery, but TNM was calculated during primary diagnosis . One hundred percent of the patients were classified as N0, M0. The patients were treated surgically with or without postoperative radiotherapy, depending on the clinical stage of the disease. The patients did not receive radiotherapy or chemotherapy before. TNM classification was done according to the criteria of the Union Against Cancer (UICC) . HistoloThis research was approved by the Ethics Committee and it was is in accordance with the GCP regulations (no. KE-0254/181/2012).We used 5 × 10-μm sections of formalin-fixed, paraffin-embedded tissues from OSCC samples. DNA was extracted using a protocol as described in the DNeasy Tissue Kit Handbook . Purified DNA was quantified by spectrophotometry .The nested PCR was carried out for detection of EBV DNA. The product size was 54 bp. The primer sequences are:Outer5′ – GTC ATC TAC GGG GAC ACG GA – 3′5′ – AAG AAG AGA TAT GTG GGG GT – 3′Inner5′ – ACC CGG AGC CTG TTT GTG GC– 3′5′ – GGA GAA GGT CTT CTC GGC CTC – 3′2, 0.2 mM dNTPs, 0.2 μM of each forward and reverse primers and 0.5 U of Taq DNA polymerase. During each run the samples were tested together with one negative and one positive control. The negative control consisted of nuclease-free water, positive - EBV-positive cell line, Namalwa, ATCC-CRL-1432. The reaction mixture containing 5 μl of extracted DNA was amplified under the following conditions: 94 °C for 3 min of initial denaturation, then 35 cycles of 94 °C for 30 s, 55 °C for 40 s, 72 °C for 1 min with the final extension at 72 °C for 5 min. The second round amplification was performed with 1 μl of the outer product in the same conditions. The final PCR products were analysed on 3 % agarose gel.All PCR reactions were carried out in the final volume of 25 μl using Taq PCR Core Kit . Concentrations of PCR reaction components were prepared as follows: 2 mM MgClHPV DNA detection; HPV detection and genotyping was performed using the INNO-LiPA HPV Genotyping Extraassay  according to the manufacturer’s protocol. The kit is based on the amplification of a 65 bp fragment from the L1 region of the HPV genome. PCR products are subsequently typed with the reverse hybridization assay. The assay covers all currently known high-risk HPV genotypes and probable high-risk HPV genotypes  as well as a number of low-risk HPV genotypes  and some additional types .p < 0.05.Statistical analysis was performed to investigate the relationship between EBV and HPV presence and clinical and demographical characteristics of patients were determined by means of Pearson’s chi-square test and with Fisher’s exact test for small groups. Statistical significance was defined as Males 85.1 %) with, smoking (85.0 %) and alcohol abuse (64.3 %) problems prevailed in the studied group. The majority of the tumors in the examination group were classified as G2 (81.8 %). T4 (58.4 %) and N2 (50.0 %) traits occurred in the majority of patients. No cases of metastasis were observed (M0 100 %). Characteristics of the study group are shown in Table  % with, p < 0.01). Other mixed infections  were detected in 23.5 and 60.7 % of the cases, respectively, and in 46.7 % of all HPV-positive samples.Of all 154 samples, 29.2 % (45/154) were HPV positive, 27.4 % (17/62) in oropharyngeal and 30.4 % (28/92) in oral cavity cancer. HPV type 16 was detected in 20.9 % (13/62) of oropharyngeal and in 12 % (11/92) of oral cancer. The HPV type 16 was detected in 53.3 % of HPV-positive group; in oropharyngeal more often (76.5 %) than in samples from the oral cavity (39.3 %). This difference is statistically significant (p < 0.05).Of all investigated samples, 27.3 % were positive for EBV, in oropharyngeal 29.1 % and in oral cavity 26.1 %. Co-infection HPV/ EBV was detected in 7.8 % (12/154), in oropharyngeal 9.7 % (6/62) and in the oral cavity 6.5 % (6/92). EBV and HPV 16 co-infection was detected in 6 cases i.e. 50.0 % of co-infected samples. There are no correlation between HPV or EBV infection and age and tobacco smoking  and squamous cell carcinoma of the tongue and oropharyngeal sites , 17, 41.The question remains whether in these cases we have to do with co-infection or superinfection. It is known that chronic virus infection affects the immunological answer of the host. Primary infection with non-oncogenic virus can favour superinfection with oncogenic virus capable of cancer transformation. The oncogenic potential of HPV is related to the expression of E6 and E7, the oncogenic potential of EBV – to the expression of LMP-1 and LMP-2. Toll-like receptors (TLRs) play a critical role in the early innate immune response to the invading pathogens by sensing a microorganism and they are involved in sensing endogenous danger signals , 45. FatIn conclusion, the present study shows that in major patients from Southeastern region of Poland with oropharyngeal cancer HPV type 16 was detected but in oral cavity cancer other mixed infections were observed . HPV 16 was detected more often among patients younger than 50 age of years, whereas the mixed HPV in the group aged 50–59. EBV may play a role in the development of OSCC. Future both epidemiological and studies on the mechanisms of co-infection and/or superinfection and their role in oral squamous cell carcinoma are necessary. The knowledge about these mechanisms may provide targets for therapy and for development of diagnostic methods.


There has been no population-based study on human papillomavirus (HPV) prevalence or its genotypes in Bangladesh; a country eligible for GAVI funding for HPV vaccine.We used baseline survey data of a prospective cohort study that was conducted in one urban and one rural area of Bangladesh. A total of 997 urban and 905 rural married women, aged 13 to 64 years, were enrolled in the baseline during July-December, 2011. Information was collected on socio-demographic characteristics and potential risk factors for HPV infection followed by gynecological examination and collection of endocervical samples using the cervical cytobrush . HPV DNA testing was done by Polymerase Chain Reaction (PCR) using a consensus primer set.Prevalence of any HPV infection was 7.7% with no significant difference between urban and rural women. Most common high-risk genotypes were HPV16, HPV66, HPV18, HPV45, HPV31 and HPV53. Urban women working as housemaids or garment workers were at higher risk of any HPV infection  compared to housewives. Rural women whose husband lived overseas were almost two times more likely to have any HPV infection  compared to women whose husbands lived with them.The prevalence of HPV infection among Bangladeshi women is similar to other regions of Asia. However, type-specific patterns are different. The study findings will inform the formulation of HPV vaccination policies in Bangladesh, monitoring the impact of vaccination programmes, and the identification of target populations for screening. Cervical cancer is a major public health problem worldwide. It is the fourth most common cancer in women with an estimated 527,624 new cases and 265,653 deaths in 2012 Human papillomavirus (HPV) is one of the most commonly acquired sexually transmitted infections (STIs) and a significant source of morbidity and mortality The prevalence of HPV infection has shown to vary by region, country and within a country by population sub-groups The literature identifies several risk factors for the acquisition and persistence of HPV infection. Age is a strong predictor, although there are notable differences in age curves of HPV infection across regions While some epidemiological research on HPV has been conducted in the South Asian region The baseline survey was conducted between July and December 2011 and was implemented in two areas of Bangladesh; one rural and one urban . The urbThe rural site of the study is located in Mirzapur, a sub-district of Tangail district, which is 56 kilometers northwest of Dhaka. In Mirzapur, icddr,b has been running a demographic surveillance since 2007 covering a population of approximately 240,000 in eight unions . We purposely selected two of the eight unions  that comUsing systematic random sampling, every third woman in the sampling frame in both urban and rural areas was approached and asked to participate in the study. In the field, the selected women were identified by their name and address recorded in the listing database (urban site) or in the surveillance data base . In both the urban and rural areas, the clusters and villages under study were purposely selected for feasibility issues considering proximity to icddr,b’s field offices, logistics and cost-related issues.The study protocol was approved by the Ethical Review Committee (ERC) of icddr,b. Informed written consent was taken from women aged 18–64 years whereas informed written assent was taken from women aged 13–17 years. For the later group, the assent form was read out to these women in the presence of their parents/legal guardians. If agreed, the assent form was then signed by the participants in the presence of their parents/guardians. The parents/guardians signed the same form.In both study areas, women were first approached at their home and eligibility was determined by trained female research staff. They were briefed about the study background and purpose, and the potential roles they would have in the study. Women who were eligible and willing to participate were sent to a study field office for interview, clinical examination and sample collection. In the case of a refusal, the next eligible woman on the list was approached.Upon arrival at the study field office, a female study physician provided them with detailed information about the study process including the process for collecting the cervical sample. On agreeing to participate in the study, the women were further screened for eligibility criteria by the physician; upon meeting these criteria they were then taken to the next room for interview. Women who were pregnant, or post-partum, or with a history of hysterectomy, cervical cancer or uterine prolapse were excluded from the study. Women with major psychological/psychiatric problems were also excluded.After informed, voluntary, written consent or assent, six trained female research assistants collected information on socio-demographic characteristics , sexual and reproductive history  and other risk factors, such as use of oral contraceptives, tobacco and condom use. Two trained female research physicians performed clinical examinations, including visual examination of the vulva. Following this, the speculum was inserted and the vagina and cervix were examined for any discharge, erosion or lesions. The physician then collected cervical samples using the cervical cytobrush . Following sample collection slides for cervical cytology were prepared The head of the brush was then detached in 10 ml BD SurePath  and stored at 4°C. The specimens (including slides) were then transported to the virology laboratory of icddr,b maintaining appropriate temperature and stored at 4°C until tested. While HPV DNA testing was performed at icddr,b, the cervical slides were sent to the pathology department of Bangabandhu Sheikh Mujib Medical University (BSMMU) for testing. The results of both the HPV DNA test and cervical cytology were provided to the study participants within 2–4 weeks of sample collection. Women presenting the possibility of precancerous or cancerous changes were referred to BSMMU for further assessment and treatment. The details of cervical cytology will be published elsewhere.A 6ml aliquot of cervical swab sample was centrifuged at 5000rpm for 15 min. After decanting the supernatant, the pellet was resuspended in 500 µl phosphate buffer saline. DNA extraction was carried out with a 200 µl aliquot of this concentrated sample using a DNeasy Blood and Tissue Kit  according to the manufacturer’s instructions. The extracted DNA was stored at −20°C until tested.HPV genotyping was carried out in three steps: 1) Detection of HPV L1 gene using PCR; 2) Genotyping using nucleotide sequencing, and 3) Identification of co-infection and genotypes of a small number of HPV positive cases for which typing could not be done in step 2.The first round of PCR amplification was carried out in a 25 µl reaction volume containing 10 µl extracted DNA as a template, 2.5 µl Qiagen 10× PCR buffer, 0.25 µl Hotstar Taq polymerase  (5 U/µl), and 20 pmol papillomavirus specific consensus primers MY09/MY11 HPV positive PCR products either visible in the first or second PCR round were sequenced using respective PCR-product primers with a Big-Dye Xterminator ready reaction kit v3.1  in an ABI 3500xL genetic analyser . Genotyping of HPV was inferred by comparing the resulting sequence for each sample to the GenBank database using BLAST . This method identified HPV types in all samples except six.To identify co-infections, all HPV positive samples were reassessed with a commercially available real time PCR kit . The test specifically identifies HPV16 and HPV18 while concurrently detecting the rest of the 10 high risk types  and two possible high-risk type  in a pooled result (other HR-HPV other than 16/18) and any of the 5 LR-HPV types . The reassessment also assisted us to identify genotypes of six HPV positive cases for which typing could not be done using PCR sequencing in step 2.5′-CAACTTCATCCACGTTCACC-3′), PC04 (5′-GAAGAGCCAAGGACAG GTAC-3′) and HMBB01 (5′-GCGACCCAATGCAAATTGGT-3′) To verify the presence of human genomic DNA in samples, a 268 bp fragment of the β-globin housekeeping gene was amplified using primers GH20  agreed to participate; interviews were completed and samples collected for 1,113 (97%) women. Out of these, 997 women were currently married and having an active sexual relationship with their husband. Similarly, in the rural area, a total of 1,499 eligible women were approached, of which 1,159 (77%) agreed to participate. Interviews were completed and samples collected from 924 (80%) women, of whom 905 women were currently married and having an active sexual relationship with their husband. The rest of the women who were approached but not included in this study either refused to take part , did not show up in the project office on the scheduled day of interview , or were found ineligible after examination by the study physicians . Of the women who were interviewed and from whom samples were collected, some were either divorced, widowed or separated at the time of the interview  and information on their husband’s residence or condom use was not appropriate to collect and therefore, these women were excluded from the analysis.While the majority of women resided with their husbands in both areas , a higher proportion of rural women had a husband living overseas (18%) compared to urban women (6%). About half of the women had three or more children with no difference in the mean number of children between urban and rural women. Most women in both areas reported one lifetime sex partner. While the majority of women reported their husband to have one lifetime sex partner, one in five women reported their husband as having more than one lifetime sex partner, with no difference between urban and rural women. Ever use of oral contraceptives was slightly higher among rural women compared to urban women ; whereas ever use of condoms was significantly higher among urban women compared to rural women .Overall, the prevalence of any HPV infection was 7.7% with no significant difference between urban and rural women . A total of 93 women had single-type HPV infection  and 52 women had multiple HPV infection . HR-HPV infections, single and co-infection combined, were more common (4.2%) than LR-HPV infections (3.4%) with no significant variation between urban and rural women. The most commonly found HR-HPV types in either single or co-infection were HPV16, HPV66, HPV18, HPV45, HPV31 and HPV53. Other HR-HPV types included HPV33, HPV35, HPV52 and HPV58 .Age-specific prevalence of any HPV infection and infection by HR and LR-HPV types are shown in A slightly different HPV infection pattern was observed for rural women. Age-specific prevalence decreased from 9.3% at 24 years or below to 8.3% and 4.4% respectively for the next two age groups of 25–34 and 35–44 years; thereafter, it increased to 7.5% for women aged 45 years and older . When stOccupation had the strongest effect on HPV infection for urban women. Urban women who were working as housemaids or garment workers were at higher risk of any HPV infection  compared to those who reported being housewives. The risk doubled for infection with HR-HPV subtypes  for this group of women.Parity was found to be associated with any HPV infection for rural women only. In particular, having three or more children lowered the risk of any HPV infection for rural women . Similarly, husband’s residency status was found to be associated with any HPV infection for rural women. Rural women whose husband lived overseas were almost twice as likely to have any HPV infection  compared to those women whose husbands lived with them.The number of lifetime sex partners of urban women was significantly associated with detection of any HPV infection; women reporting two or more partners were almost four times more likely to have any HPV infection compared to those reporting only one sex partner . We did not find any association between the numbers of lifetime sex partners of women with HR-HPV infection. We also did not find any association between the number of the husband’s sex partners with the risk of any HPV infection or HR-HPV infection. No effect of contraceptives or tobacco use was found in our analysis. We did not have enough rural samples to investigate the effect of occupation or number of sexual partners on HPV status.To the best of our knowledge, this is the first study to investigate the prevalence of HPV infection and its type-specific distribution in population-based samples of urban and rural women in Bangladesh. The overall prevalence of any HPV infection was found to be 7.7% in this study. This is similar to the prevalence reported in other Asian countries –9% in Nepal HR-HPV was more common than LR-HPV infection in almost all age groups, except among rural women older than 44 years. HPV16 was the most frequently detected type, as found in other parts of the world, including Asia Similar to other countries, we found HPV infection to be most common in younger women with the peak prevalence occurring in women younger than 25 years of age; prevalence started to decline after 30 years of age. We found a secondary increase after age 44 for rural women and ages 35–44 for urban women. In Bangladesh girls marry at an early age and the study was conducted among married women. This may explain high HPV infection in the younger age group. However, the secondary peak seen during 35–44 years in urban women and 45–64 years for rural women requires further explanation.Bangladesh has made remarkable progress in terms of population, economic and health indicators in recent years Occupation was also a significant predictor for HPV infection in the rural study site. In this case however, it was the risk posed by the occupation of women’s husbands. Economic opportunities outside of Bangladesh draw rural men to leave their families for long periods of time for work overseas. This may increase their opportunity for engaging in risky sexual behaviors, thus increasing their wives vulnerability to HPV infection. All these factors may help to explain the secondary peak of HPV infection in our study in women in their late 30′s and 40′s.While we found a similar prevalence of any HPV infection and HR-HPV infections among rural and urban women, we found some important differences in risk factors for infection between rural and urban women. Education had a protective effect for rural women, but not for urban women. Urban women working as garment workers or housemaids were at an increased risk of HPV infection. For urban women, the number of sexual partners was highly associated with HPV infection, a finding consistent with other settings Another common economic opportunity for women in urban areas is to work as domestic servants or housemaids. This occupation may also expose women to increased risk of sexual exploitation as supported by other studies We did not find any association between oral contraceptive use with HPV infection as found in other studies Our study used population-based socio-economically diverse samples from two existing surveillance systems. However, limited participation  may raise questions about the representativeness of the study findings. To address this, we examined the basic socio-demographic characteristics of those participating in the study and those who did not, and we found the two groups to be similar, suggesting limited plausibility for selection bias.Our study adds immense value to the existing knowledge of the burden of HPV infection among the selected urban and rural female population in Bangladesh. The identification of genotypic distribution will help further understanding of the risk of developing cervical cancer in the study population. While we found HPV16 to be the predominant type, HPV66 was the second major HR-HPV type identified in our study population, followed by HPV18. Epidemiological evidence of the carcinogenicity of HPV66 was previously judged sufficient; however, HPV66 has rarely been identified in populations Given that HPV infection is a necessary cause of cervical cancer and preventing it is the most effective way to prevent cervical cancer. HPV vaccines have been proven to be an effective and safe measure to prevent HPV infection and disease related to the vaccine-specific genotypes in women with no evidence of past or current HPV infection This study provides the first population-based HPV prevalence data in Bangladesh. While the findings are similar to studies from neighboring countries, the type-specific distribution, as well as identification of risk factors will provide additional evidence to guide Bangladesh’s response through a cervical cancer prevention programme. The findings are especially timely as the Government of Bangladesh is planning to apply to the Global Alliance for Vaccines and Immunization (GAVI) for funding for pilot testing of the HPV vaccine in a peri-urban setting. The evidence from this study will provide necessary country-specific evidence for this application.


It includes clinically important 1257 integrants and integration sites from different HPV types i.e. 16, 18, 31, 33 and 45 associated with distinct histological conditions. An inclusive HPV integrant and breakpoints browser was designed to provide easy browsing and straightforward analysis. Our study also provides 719 major quantitative HPV DNA methylation observations distributed in 5 distinct HPV genotypes from higher to lower in numbers namely HPV 16 (495), HPV 18 (113), HPV45 (66), HPV 31 (34) and HPV 33 (11). Additionally, we have curated and compiled clinically significant aberrant expression profile of 341 miRNAs including their target genes in distinct carcinomas, which can be utilized for miRNA therapeutics. A user-friendly web interface has been developed for easy data retrieval and analysis. We foresee that HPVbase an integrated and multi-comparative platform would facilitate reliable cancer diagnostics and prognosis.Human papillomaviruses (HPVs) are extremely associated with different carcinomas. Despite consequential accomplishments, there is still need to establish more promising biomarkers to discriminate cancerous progressions. Therefore, we have developed HPVbase ( The human papillomaviruses (HPVs) are circular, double-stranded DNA genome of approximately 8.0 kb in length. It belongs to the papillomaviridae family, which is further taxonomically classified into distinct genera namely alpha, beta, gamma, mupa and nupa12HPVs are well adapted in host differentiating epithelial tissues and prevalent in cervical carcinogenesis. They employ cellular machinery to survive in host. Expression and functions of viral proteins primarily HPV oncoproteins E6 and E7 is very essential for the tumor progression. Upregulation of these oncoproteins in the infected cells have been shown to expedite proliferation, immortalization and malignant transformation. They interact with numerous cellular targets mainly inhibits two essential tumor suppressor proteins, p53 and retinoblastoma (Rb). Interaction of E6 with p53 prohibits proteolytic degradation and abrogates apoptosis5Cervical carcinoma (CC) is the fourth most common cancer and cause of death in women worldwide GLOBOCAN 2012). Series of steps involves in cervical oncogenesis are HPVs infection and transmission, viral persistence, progression of infected epithelium to precancer or high-grade precursor lesion and invasion. Persistence infection with distinct carcinogenic HPVs is a critical factor in the etiology of cervical precursor lesions and cancer012. Seri78HPV infection and associated consequences are valuable determinants of multistage cervical neoplastic progression. In this context, there are alternative biomarkers, which could discriminate latent infection with high-grade precursor lesion and eventually cancer8The infection and integration of HPVs is a well-known factor and event in the development of CC, human genital and head and neck squamous cell carcinoma (HNSCC)1213141516171912In addition, epigenetic mechanisms are also known to influence chromatin conformation and alter gene expression. CpG methylation is a fundamental and well-recognized molecular regulatory mechanism in epigenetics242728Apart from the general concepts of genomic instability govern by carcinogenic HPVs the mechanism of transformation is more complex. The viral oncoproteins E6 & E7) are known to interact with several cellular components and may affect different cellular pathways & E7 are3034HPVbase” is to provide a comprehensive web-based resource of the extensively cross-analyzed different biomarkers.Despite of numerous experimental studies in the field, there is a paucity of computational resources with a unique focus on cancer progression and therapeutically potential biomarkers. Moreover, it is still unclear and less known about the viral mediated processes, such as integration events, methylation pattern in different epidemiological conditions and above all importance of various cellular miRNAs expressions in the etiology of oncogenesis. Hence, it is most important and necessary to have a proper multi-comparative platform of these specific biomarkers. Here, the goal of “The importance and crucial role of HPV integration and human genome integration sites in the progression and etiology of CC were identified and analyzed in several studies. However, complete knowledge and understanding of integration mechanism is still unclear. An inclusive HPV integrant and breakpoints browser were designed to provide analysis and interpretation. In total, 1257 integrants and integration sites were collected which are associated with distinct histological conditions. These integration sites are distributed among several HPV types mainly on HPV16, 18, 45, 33 and 31. It comprises of various clinical information such as viral integrant, host integration sites, gene target region, fragile sites, cancer specimen type, sample size, carcinoma types, detection techniques etc.Using the highly interactive browser user can find and browse distinct HPV integrants. Different color-coded blocks provide corresponding information and existing analysis of HPV integration see . All cliThe most important and critical part of integration events is their frequency to integrate on different chromosomes and genomic region. To facilitate this, we have also analyzed HPV type wise distribution and frequency of integration and breakpoints on different human chromosomes. Moreover, we have also analyzed the complete gene compendium altered/disrupted due to integration events presented in HPVbase. First of all, we have extracted missing gene name information based on human genomic coordinates using UCSC genome browser. Subsequently, from entire set after removing duplicates and other non-genes  we have listed 481 unique genes. Furthermore, disruption frequencies of each unique 481 genes from whole dataset were calculated see Table S3Extensive research in the quantitation of methylation in the carcinogenesis allowed searching methylation patterns in different diseased stages. Our study provides major quantitative HPV DNA methylation observations together and the probable combination of possible CpG methylation sites that could be used to discriminate normal and cancerous progressions. Distinct studies provide different notations for methylation status. To simplify the terminologies, based on methylation levels we have used three categories: hypo or low methylation (HypoM), hyper or high methylation (HyperM) and significantly hyper methylation (HyperM##). Here, we have cataloged 719 methylation entries along with respective important and clinical information. It comprises of CpG methylation sites, gene region, differential methylation patterns, detection techniques, sample size and disease sample type use to identify methylation status see . These a+ve women.Assorted cancer studies have advocated an association between CpG methylation patterns and the carcinogenic development. Integration of episomal HPV genomes in carcinoma usually correlates with elevated DNA methylation. Our resource also provide diverse pattern in methylation outcome in corresponding specimen type, histological conditions and detection techniques used. In HPV positive women, the methylation level at specific CpGs increase with consistent viral infection and even enhance more remarkably in high-grade lesions. Maximum sites from late HPV regions L1 and L2 exhibit conclusive and significant hyper methylation patterns. However, methylation pattern in adjacent long control region (LCR) shows relative inconsistency. It could provide a comprehensive base for comparative examination between distinctive features and cancers, which in turn facilitate advancement in clinical screening tests. Furthermore, it could be used as a cost-effective molecular tool for the triage of HPVIn various studies, miRNAs are suggested being involved in pathogenesis of cancers usually in CC and HNSCC including other cancer types i.e. breast cancer, thyroid papillary malignancy, ovarian cancer, prostate cancer etc3737To facilitate this, we developed an integrated source for the cellular miRNAs with description, which could be explored for greater insight between miRNAs and HPV related tumorogenesis. Regulation of cellular miRNAs is key to prognosis. We have curated and compiled clinically significant aberrant expression profile of 341 miRNAs from different carcinomas. It includes 142 miRNAs, which comprises of 80 upregulated and 62 downregulated in CC, 176 miRNAs among which 85 and 91 are up and downregulated respectively from HNSCCs and 22 miRNAs includes 9 upregulated and 13 downregulated from vulvar carcinoma. Beside this, 1 downregulated miRNA is related to penile carcinoma. It includes miRNAs and their mirbase id, host chromosomal location, genome coordinates, their potential target genes as shown in We have cross-analyzed chromosomal distribution of these aberrantly (up and down) regulated miRNAs in different carcinomas i.e. CC, HNSCC and vulvar carcinoma. The vital role of HPV integrations and human genome chromosomal sites were analyzed and illustrated in various studies. HR-HPVs integration is a key incident in HPV-mediated carcinogenesis, which leads to increase expression and stability of viral oncogenes (E6/E7) as well as disruption of various key cellular proteins. It is usually considered as one of the main events takes place during the transition from low grade to high-grade lesions. Additionally, it was also observed that these are frequently allied with FS normally play a role in the structural alterations, translocations and large deletions. Thus, it is one of the important mechanisms that enhances and contributes in aberrant proliferation, genomic instability and cellular immortalization, which enhance malignant progression. It plays an acute function not only to alter viral genome as well as host genomic regions mainly associated with cancer related genes.From our analysis, we have identify and reported certain specific chromosomal loci regions such as 8q24.21, 3q28, 13q22.1, 9q22.33, 14q24.1 and many more which shows higher level of viral integration indicating preferential target in diverse carcinomas see . In thisFor example, TP63 is a tumor protein 63 belongs to family of transcription factors. It mainly involves in complex events and regulates cellular proliferation and neoplastic progression. MYC, it is a v-myc avian myelocytomatosis viral oncogene homolog usually plays a role in cellular transformation, apoptosis and cell cycle. ETS2, v-ets avian erythroblastosis virus E26 oncogene homolog 2 is a tumor suppressor gene mainly regulates genes involved in development and apoptosis. It also found to be involved in regulation of telomerase. FHIT, belongs to histidine triad protein family is a tumor suppressor gene. It encloses the common fragile site FRA3B and highly susceptible to the translocations and aberrant expression which leads to the various carcinomas. RAD51B  is a member of the RAD51 family. It is essential element of the DNA repair pathway and found to be associated with cell cycle delay and cell apoptosis. Hence, integration at unstable regions and at certain genes concomitant with DNA repair mechanism also signifies its relation with various chromosomal machineries.These interpretations from existing knowledge suggest that prevalence of certain viral integration sites on specific genomic locations could play an essential role to determine eventual cancer phenotype. Therefore, revealing these imperative events in the transforming lesions from low grade to invasive carcinomas might be useful and precious to be used as potential cancer biomarker.Apart, from genetic changes due to integration, HPVs are also known to cause epigenetic alterations mainly modification in DNA methylation pattern and miRNAs aberrant expression. These mechanisms contribute to the development of cancer either reducing expression of tumor suppressor genes or the enhancing oncogene expression supporting cancer progression. With the advent of high throughput technologies, researchers are now focusing on the application of these alterations to explore their prognostic and diagnostic value to determine disease progression3941Additionally, differential expression of oncogenic and tumor suppressive miRNAs has confounding affect on cancer paradigm. They generally post transcriptionally modulate or regulate expression of target genes and play significant role in cell cycle progression, apoptosis, angiogenesis etc. the observation of different circulatory miRNA signatures might be useful as diagnostic tool as well as could serve as therapeutic target in HPV induced oncogenesis. Furthermore, distinct characteristics of miRNAs such as resistant to temperature, degradation and non-immunogenic nature to utilize host regulatory machinery make them more appropriate to serve as prognostic indicator of cancer aggressiveness.Some approaches like restoring miRNA level or blocking its function can be used in drugs development. Concluding same, certain miRNA based cancer-targeted drugs such as miR-34-mimic (MRX34); a liposome-based drug is in clinical trial for the treatment of hepatocellular carcinoma. Likewise, “miravirsen”, antisense oligonucleotides based an antiviral compound known as locked nucleic acids (LNA anti-miRs) is in clinical evaluation for the treatment of hepatitis c virus (HCV)43HPVs play a cardinal role in the etiology of distinct carcinomas including CC, HNSCC etc. CC is still account as fourth most common cancer globally and remained as leading cause of cancer mortalities. However, regardless of substantial efforts in the field to develop screening and prevention methods, there is no effectual solution is available to combat against these lethal diseases. The general progression of HPV mediated cancer is associated with the various events and changes with other risk factors. These events can be used as biomarkers. Here, the main aim and feature of HPVbase is to deliver integrative and multi comparative platform for HPV integrations, methylation patterns and cellular miRNAs expression to facilitate identification and analysis of valuable cancer biomarkers. These are very essential factors especially in cervical progression see .The HPVbase is the first web knowledgebase to provide highly interactive and manually curated resource for the clinically significant viral and cellular biomarkers. Nevertheless, we have also searched and compared existing sources related to these events and papillomaviruses. For viral integration sites, DR. VIS45http://pave.niaid.nih.gov)http://cvc.dfci.harvard.edu/hpv/)HPVbase” for the extensive comparative analysis of different biomarkers and genomic information. It will provide systematic compilation of knowledge and user-friendly visualization of clinical content. It can be exploited for assessment of clinically significant biomarker data and will provide a comprehensive base for examination between distinct features and cancers, which in turn facilitate advancement in clinical screening tests. Finally, combined impact of multiple events can be utilized for the future prevention and therapeutics. Thus, in turn this exhaustive platform promises to be precious to facilitate and accelerate reliable cancer diagnostics and prognosis.Some sources also developed which provide HPV genomic and proteomic information. The Papillomavirus Episteme (PaVE) contains 303 genomes, 3126 gene region, 2823 protein sequences and 48 protein structures  the integration and breakpoint events in HPV associated carcinomas (2) HPVs methylation events relevant to the cancer clinical outcomes and (3) the HPV mediated aberrant expression of distinct host miRNAs that are crucial in cancer etiology were included and analyzed.Collection of integration and breakpoints were derived from scientific literatures searched using set of keywords “((((((HPVs) OR human papillomaviruses) OR human papillomavirus) OR HPV*)) AND (((cancer) OR carcinoma))) AND ((integration*) OR breakpoint*)” as query. Overall, 755 articles were found, out of which 117 review articles were excluded from study and 638 research papers were screened for extensive data mining. Furthermore, maximum studies only state HPV status or the information related to presence or absence of HPV genes such studies does not provide integration sites, coordinates and further clinical details were filtered and removed. After vigilant reading, comprehensive knowledge associated with integration events were extracted such as HPV type , HPV region , viral integrant/breakpoints , human chromosome number  and coordinates , cytoband , target genes , fragile sites , detection technique or methods , histological conditions , specimen or sample type  etc. The collected data were manually checked and rectified for errors and inconsistencies.“(((((((HPVs) OR human papillomaviruses) OR human papillomavirus) OR HPV*))) AND (((cancer) OR carcinoma))) AND methylation”. In total, 320 articles excluding 31 review articles were screened. Most of the literature covers host genome or gene methylations, which are not in the scope of this study and hence excluded. All-inclusive information related to HPV methylation i.e. HPV type , its genomic region , methylation status , technique used for detecting methylation , sample type , associated cancer type  or stage ) etc. were mined and compiled.The HPV DNA methylation studies were extensively searched using combination of keywords All literature information were collected using keywords query “((((((HPVs) OR human papillomaviruses) OR human papillomavirus) OR HPV*)) AND ((cancer) OR carcinoma)) AND ((((microRNA) OR miRNA) OR microRNAs) OR miRNAs)” from PubMed. Overall, 99 articles excluding 24 review articles were searched. We have extracted information comprises of miRNAs, their expression profile, mirbase id, host chromosomal location and genome coordinates from distinct human carcinomas. Apart from manually curated expression profiles, these are further integrated and linked with the external resources. Along with this, we also explored and obtained known target genes of different miRNAs using mirtarbaseThe web interface (front and back end) is developed using the open source LAMP (Linux-Apache-MySQL-PHP) solution stack reinforced by combination of programming and scripting languages i.e. HTML, javascript and PERL. We have also utilized the JBrowseHPVbase is a first web-based, HPV knowledgebase for the effective analysis and assessment of viral and cellular biomarkers involves in pathogenicity of different HPV mediated carcinomas. It has well-designed user interface for the interactive visualization and evaluation. In HPVbase, data is classified and organized in three different sections- integration sites and breakpoints, methylation patterns and miRNAs expression profiles see . SubsequAlongside, HPVbase also facilitate user affable data searching and retrieval options. The resulting two search tools (a) integration search and (b) advance search have been implemented at website.User can extensively search integration sites based on various keywords with exact or containing sub-options from number of provided fields including integration site, HPV type, chromosomal locations, cancer type etc.This tool permits user to perform systematic search using combination of logical operators (OR/AND). It allows user to build a query using different combination of keywords to narrow down search.We have incorporated the latest and highly curated data in HPVbase. To cope with this increasing amount of valuable clinical data, we would like to further update our resource in every six-month.How to cite this article: Kumar Gupta, A. and Kumar, M. HPVbase- a knowledgebase of viral integrations, methylation patterns and microRNAs aberrant expression: As potential biomarkers for Human papillomaviruses mediated carcinomas. Sci. Rep.5, 12522; doi: 10.1038/srep12522 (2015).


The aim of the survey was to introduce knowledge of HPV's role in head and neck pathologies to general physicians (GPs), otorhinolaryngologists (ENTs) and newly graduated doctors, as well as to promote HPV-related diseases prevention.Cross-sectional study.Self-designed questionnaire was sent to 2100 doctors. A total of 404 doctors, including 144 ENTs, 192 GPs and 68 trainees, responded.The majority of ENTs (86.8%) had contact with recurrent respiratory papillomatosis (RRP) and oropharyngeal cancers (OPCs) patients; in contrast, the majority of GPs (55.7%) did not (p = 0.00). The knowledge of HPV aetiology of cervical cancer versus OPCs and RRP was statistically higher. 7% of ENTs, 20% of GPs and 10% of trainees had not heard about HPV in oropharyngeal diseases. Women had greater knowledge than men. Both in the group of GPs and ENTs, 100% of respondents had heard about the impact of vaccination on the reduction of cervical cancer incidence. Only 39.11% of respondents had heard about the possibility of using vaccination against HPV in RRP—ENT doctors significantly more often than GPs and trainees (p = 0.00). Only 28.96% of physicians had heard about the potential value of HPV vaccination in preventing OPCs, including 44.44% of ENT doctors, 23.44% of GPs and 11.76% of trainees (p = 0.00). The doctors from district hospitals showed lower level of knowledge compared with clinicians (p = 0.04).The different levels of knowledge and awareness of HPV issues highlight the need for targeted awareness strategies in Poland with implementation of HPV testing and vaccination. The information should be accessible especially to those with lower education levels: ENTs from small, provincial wards, GPs from cities of < 200 000 inhabitants and older physicians. The incorporation of HPV issues into the studies curriculum would be fruitful in terms of improving the knowledge of trainees. Human papillomavirus (HPV) is a common sexually transmitted infection, and a well-documented agent in the development of cervical, vulvar, anal, penile and head and neck carcinomas. The high-risk HPV types 16 and 18 are implicated in over 70% of cervical cancer cases. The presence of HPV in oropharyngeal cancers (OPCs) is slightly lower: 60% in the USA, 40% in Europe (EUROCARE project) and 33% in other parts of the world, but it was documented that until 2000 the share of HPV infections was 41%, until 2004–72% and after 2004 there was an increase of up to 96% [Testing for the high-risk types of HPV has been an established method in the cervical screening context around the world. The European Guidelines for Quality Assurance in Cervical Cancer Screening created the principles of organized population-based screening . HoweverIn contrast to cervical cancer in the region of Central and Eastern Europe and Central Asia (CEECA), there is scarce data concerning the evaluation of the incidence and mortality from other HPV-related cancers and diseases . Head anThe overall knowledge of HPV infections with special regard to the head and neck has never been examined in the influential milieu of Polish physicians. The aim of this survey was to introduce knowledge of HPV’s role in head and neck pathologies to general physicians (GPs), otorhinolaryngologists (ENTs) and newly graduated doctors. One purpose of the study was also to determine which demographic factors influenced this knowledge. The second aim was to test the hypothesis that the need for vaccination should be higher among people who have heard of HPV.The survey was conducted among otorhinolaryngologists , general practitioners—GPs and trainees. For ENT doctors, GPs and trainees a questionnaire was constructed based on eleven slightly different questions . The surIn the group of 144 ENT doctors the following age categories were analyzed: below 30 years old (n = 6), 31–40 years old (n = 26), 41–50 years old (n = 46), 51–60 years old (n = 51) and 15 respondents over 60 years old; 62 men and 82 women. The analysis of the workplace included: 48, 40 and 56 ENT doctors from clinical centres, district hospitals and private practices, respectively. 127 specialists treated adult patients and 17 treated children.192 responses were obtained from family doctors, including 134 from women and 58 from men; the age group 41–50 years old was the most numerous (40%). Our respondents in a similar percentage  came from population areas of up to 10 thousand, 10–200 thousand, and more than 200 thousand residents; 81% of them treated mostly adults.The study analyzed the responses from 68 doctors who had just completed their studies and had not yet taken up a post. They constituted a homogeneous age group of people living in a city of > 200 thousand residents. The group of trainees included 44 women and 24 men.Due to the fact that the studied traits are not quantitative  but qualitative, the analysis of differences between the groups of ENTs, GPs and trainees can be carried out only through the study of relationships rather than differences.Two questions in the survey for ENTs and GPs related to the contact/treatment/care of patients with head and neck pathology of HPV aetiology: oropharyngeal cancer patients  and with recurrent respiratory papillomatosis (RRP). The following categories were examined: I do not treat such patients; I treat 1–5 patients with this disease; I treat 5–10 patients with this disease; the group with OPCs, which is a much more widespread disease than RRP, additionally included the category: I treat more than 10 patients with this disease. In the group of surveyed ENTs 99/144 treated patients from these groups. 38 ENT doctors treated 1–5 patients with RRP and 61 treated more than 5 such patients. 125 ENTs took care of OPCs patients, 48 treated 1–5 patients a year, 19 treated 5–10 patients a year and 58 treated more than 10 patients a year. The vast majority of GPs (77%) did not take care of patients with RRP but almost 45% had patients with OPCs. Differences between the two professional groups in terms of contact with patients with RRP and OPCs were significant ; ENT doctors took care of these patients significantly more often .Respondents from the three groups: GPs, ENT doctors and trainees answered how important, in their opinion, the impact of HPV was on the development of genital and upper respiratory tract pathology. Knowledge of the importance of HPV to the carcinogenesis of cervical carcinoma was widespread; awareness of HPV’s role was 100%, 85% and 98.5% of respondents in the group of ENTs, GPs and trainees, respectively. The importance of HPV to the development of upper respiratory tract pathology was not as common. It was tested in the following categories: the role of HPV is definitely large; I know about the problem; the significance is overestimated; I have not heard about the problem . A relatBoth in the group of GPs and ENTs, 100% of respondents had heard about the impact of vaccination on the reduction of cervical cancer incidence.In the group of ENT doctors and GPs, 47% had a daughter. To the question: Has your daughter been vaccinated against HPV? respondents gave the following answers: ENT doctors: I have an adult, unvaccinated daughter—22,9%; she has been vaccinated—17,4%; I will vaccinate my daughter—15,9%; I will not vaccinate my daughter—3,5%; whereas in the group of GPs: 20,8% had an adult daughter vaccinated against HPV, 12,5% had an unvaccinated daughter, 13% planned to vaccinate a daughter, and 1,6% did not plan to vaccinate .A total of 158/404 (39.11%) physicians had heard about the possibility of using vaccination against HPV in laryngeal papilloma: 88/144 (61.11%) ENT doctors, 59/192 GPs (30.73%) and 11/68 (16.18%) trainees. Knowledge of the problem differed significantly between the groups of ENT doctors and GPs (p = 0.00) and the group of ENTs and trainees (p = 0.00).A total of 117/404 (28.96%) physicians had heard about the potential value of HPV vaccination in preventing OPC, including 64/144 (44.44%) ENT doctors, 45/192 (23.44%) GPs and 8/68 (11.76%) trainees. Knowledge of the problem differed significantly between the groups of ENTs and GPs (p = 0.00) and the group of ENTs and trainees (p = 0.00).The analysis of the data characterizing the group of ENTs and the data describing the state of their knowledge of HPV indicates that the age of respondents did not matter in terms of the knowledge about HPV or the possibility of using vaccination against HPV in laryngeal papilloma or precancerous conditions of the oral cavity and pharynx. The age of respondents, however, had a significant influence on the decision whether to vaccinate daughters: the younger the age of respondents, the greater their willingness to vaccinate previously unvaccinated adolescent girls (p = 0.00006).In the group of ENT doctors gender was important in assessing the role of HPV in the development of upper respiratory tract pathology: HPV was considered a crucial pathological factor by 20% of men and 39% of women (p = 0.009). This predilection, however, did not affect the state of knowledge about the role of vaccines in RRP or precancerous conditions of the oral cavity and pharynx or the decision to vaccinate daughters.The workplace of ENT doctors was important in the assessment of the significance of HPV in the development of upper airway pathology (p = 0.00025): 28% of employees of clinical centres assessed the importance as very high, whereas only 11% of the staff of district hospitals and 18% of employees of private practices shared this view; the last two groups assessed the role of HPV as significant (11% and 18%). The place of work did not affect the attitude to vaccination, both in terms of using it in own daughters as well as the option for patients with RRP. As for the potential importance of vaccination to protect against the development of OPC, doctors working in clinical centres (19%) and private practices (16%) gave significantly higher percentages of affirmative responses compared to the staff of district hospitals (9%) (p = 0.04).The analysis of the data characterizing the group of GPs and the data describing the state of their knowledge of HPV showed that the age of respondents did not significantly affect the way they perceived the importance of HPV in the development of upper respiratory tract pathology, knowledge of the potential role of vaccination against HPV in laryngeal papilloma, and the prevention of OPCs. Age, however, was crucial in the decision whether to vaccinate daughters. The cohort of 41–50 year-olds was the most enthusiastic to take this action compared to older doctors (p = 0.00). The gender of the surveyed physicians did not affect any of the studied parameters.There were no significant differences in terms of the workplace of GPs (size of the city) that would influence the assessment of the importance of HPV in pathology of the cervix, genitals and upper respiratory tract, the possibility of vaccination against HPV in laryngeal papilloma, and the role in OPC prevention. In contrast, a desire to vaccinate daughters was declared mostly by respondents of cities of over 200 thousand inhabitants (p = 0.02).The analysis of the data characterising the group of trainees and the data describing the state of their knowledge showed that the gender of respondents did not affect the assessment of HPV’s significance in the development of pathology of the cervix, genitals and upper respiratory tract. 98% had heard about the impact of vaccination on the reduction of cancers of the cervix. However, when asked the questions: Have you been vaccinated? Has our partner been vaccinated?, as many as 82% of respondents answered that they had not: 15% of the surveyed women, and fewer than 3% of the partners of the surveyed men were vaccinated.The increasing knowledge about HPV’s significance in head and neck pathology may help to minimize any negative psychological consequences associated with HPV testing and vaccination. Medical recommendations for cancer screening and prevention are connected with the compliance of the patient . Thus, tIn contrast with the USA, where HPV vaccination has been presented through public health initiatives, with an overriding sense of community obligation to be vaccinated for whatever herd immunity may occur –11, in PIn the presented analysis 404 Polish physicians responded to the survey. Taking into consideration the everyday contact with RRP and OPCs patients, the majority of ENTs (125/144) have contact with these entities; in contrast, the majority of GPs (107/192) do not. The knowledge of HPV aetiology of cervical cancer versus OPCs and RRP was statistically higher. Around two thirds of those who had heard of HPV in ano-genital pathologies had also heard of HPV in oropharyngeal diseases. However, 7% of ENTs, 20% of GPs and 10% of trainees had not heard about the problem. The differences were significant and indicate that GPs should be a target for the intensification of education. Women had greater knowledge than men, which could have been expected, since HPV is commonly associated with cervical cancer, so it is women who are currently referred for HPV testing.In the group of ENT doctors the workplace was important in the assessment of HPV’s significance in the development of upper respiratory tract pathology: 28% of employees of clinical centres assessed this significance as very high, whereas only 11% of the staff of district hospitals and 18% of employees of private practices shared this view. As for the potential importance of vaccination to protect against the development of OPCs, doctors working in clinical centres (19%) and private practices (16%) gave significantly higher percentages of affirmative responses compared to the staff of district hospitals (9%) (p = 0.04). In both categories doctors from district hospitals showed the lowest level of knowledge compared with clinicians. The high level of self-education in doctors running their own practices is noteworthy.The high impact of medical recommendations for vaccination has been proven –19. Of nThere are some limitations of the study. Participants were recruited from online panels and so may not be representative of the whole population of doctors but they are probably the most open and questing group. To summarize, the different levels of knowledge and awareness of HPV issues highlight the need for targeted awareness strategies in Poland where we would like to implement HPV testing and vaccination. Knowledge and awareness could be improved through ensuring that information is accessible to those with lower education levels: ENTs from small, provincial wards, GPs from cities of < 200 000 inhabitants and older physicians. Thus, it may be helpful to encourage the discussion about HPV testing and vaccination in the media and to incorporate information about HPV and the implications of HPV test results in community outreach initiatives. The incorporation of HPV issues into the studies curriculum would be fruitful in terms of improving the knowledge of trainees.


While prophylactic human papilloma virus (HPV) vaccination is considered effective in young girls, it is unclear whether a catch-up vaccination of older girls would be beneficial. We, therefore, aimed to examine the potential health impact of a HPV catch-up vaccination of girls who were too old at the time of vaccine introduction, hence aged 16 and older.We systematically searched the literature for randomized clinical trials (RCTs) that examined the effect of HPV vaccines on overall mortality, cancer mortality and incidence, high-grade cervical intraepithelial neoplasia grade 2 and higher (CIN2+), vulvar intraepithelial neoplasia (VIN) and vaginal intraepithelial neoplasia (VaIN) grade 2 and higher lesions  genital warts (condyloma). We considered all lesions and those associated with HPV type(s) included in the vaccines. RCTs reporting on serious adverse events were also eligible. Selected publications were assessed for potential risk of bias, and we ascertained the overall quality of the evidence for each outcome using Grading of Recommendations Assessment, Development and Evaluation (GRADE). Meta-analyses were performed, assuming both random and fixed effects, to estimate risk ratios (RR) and corresponding 95% confidence intervals (CI), using intention-to-treat and per-protocol populations.We included 46 publications reporting on 13 RCTs. Most of the RCTs had a maximum follow-up period of four years. We identified no RCT reporting on the effect of HPV catch vaccination on overall and cancer related mortality, and on cervical cancer incidence. We found a borderline protective effect of a HPV catch-up vaccination on all CIN2+, with a pooled RR of 0.80 (95% CI: 0.62-1.02) for a follow-up period of 4 years. A HPV catch-up vaccination was associated with a reduction in VIN2+ and VaIN2+ lesions, and condyloma. No difference in risk of serious adverse events was seen in vaccinated participants versus unvaccinated women (pooled RR of 0.99 (0.91-1.08)).This systematic review indicates that a HPV catch-up vaccination could be beneficial, however the long-term effect of such a vaccination, and its effect on cervical cancer incidence and mortality is still unclear.The online version of this article (doi:10.1186/1471-2458-14-867) contains supplementary material, which is available to authorized users. Human papillomavirus (HPV) is considered the most common sexually transmitted agent worldwide , and mosEfficient prophylactic vaccines could have an important public health impact . Since 2To the best of our knowledge, three meta-analyses have been published to date and they reported results on the effect of HPV vaccination of older girls on persistent HPV infection , and onlIn this article, we present a systematic review of the international literature to investigate the health impact of a HPV catch-up vaccination of girls who were too old at the time of vaccine introduction. Taking into account the age range covered by HPV vaccines licensing, and the age at introduction of HPV vaccination in different countries, we have therefore examined the health impact of HPV vaccination of girls aged 16 and older.We included randomised clinical trials (RCT) that examined the efficacy of a HPV catch-up vaccination of young women aged 16 and older. Eligible RCTs examined the effect of HPV vaccines on overall mortality, cancer related mortality, cervical cancer, high-grade cervical intraepithelial neoplasia grades 2 and higher (CIN2+), vulvar intraepithelial neoplasia (VIN) and vaginal intraepithelial neoplasia (VaIN) grade 2 and higher lesions , and genital warts (condyloma). RCTs investigating HPV vaccination safety and reporting on serious adverse events were also eligible. RCTs that used the following comparison groups were included: HPV vaccine against placebo, HPV vaccine against placebo with in addition another vaccine (such as hepatitis B vaccine) used in the intervention and in the placebo groups, or RCTs comparing two different HPV vaccines. No language restriction was applied during the literature search.We systematically searched several databases from 1999 up to October 2012 (Ovid MEDLINE(R) In-Process & Other Non-Indexed Citations, Ovid MEDLINE(R), Embase, Cochrane Central Register of Controlled Trials, ISI web of Science, PubMed, and Google scholar). Details of the search strategy are provided in the Additional file To assess the potential health impact of a HPV catch-up vaccination, we have examined several outcomes. We planned to investigate the effect of a HPV catch-up vaccination on overall and cancer mortality, and on cervical cancer incidence. Furthermore, we aimed to examine several female genital HPV related diseases and investigate the association between HPV vaccination and these outcomes. These outcomes were CIN2+, VIN2+, VaIN2+ and genital warts (condyloma acuminata). We also examined the association between HPV vaccination and these lesions considering only HPV related lesions (i.e. HPV type(s) found in the lesion is/are the HPV type(s) covered by the examined vaccine).We examined also adverse events possibly linked to HPV vaccination. We considered only adverse events reported as serious adverse events in the included publications.The selection of articles was carried out by two of the review authors . All titles and abstracts from the reference lists of articles were screened, and full-text articles were retrieved for publications judged potentially relevant. Each full-length article was assessed for possible inclusion according to the predefined eligibility criteria. Potential disagreements were resolved by discussion with a review author.Selected publications were assessed for potential risk of bias according to the Cochrane risk of bias tool . All assOne review author extracted the data and another verified the information. When data were reported in several publications, we used the publication with the longest follow-up period. If a publication included several trials, preference was given to the publication that included the most trials. We extracted detailed data on characteristics of the clinical trial and participants, on administration of vaccine and control, and on relevant outcomes.Two review authors assessed the overall quality of the evidence for each selected outcome using GRADE  .When possible, we carried out meta-analyses to investigate the association between HPV vaccination and outcomes described above, comparing vaccine and control groups. Extracted data were pooled together by performing meta-analyses using the Review Manager software (RevMan). When the outcome data could not be pooled in meta-analyses, we described the results in a narrative manner.Random and fixed effect models were used to calculate pooled risk ratios (RR) and corresponding 95% confidence intervals (CI). If we identified fewer than three studies reporting data on the same clinical outcome, we report pooled estimates using fixed effect models. Otherwise, pooled estimates obtained with random effect models are presented.We performed intention-to-treat (ITT) analyses. However, none of the included studies used an intention-to-treat population including only randomized subjects. We, therefore, used the modified intention-to-treat populations as defined in the included publications. The modified ITT population was most commonly defined as participants who received at least one vaccine or control dose and had at least one follow-up visit post-dose 1. When possible, we conducted also analyses according to per-protocol-population (PPP). The PPP typically included participants who received the three vaccine or control doses. We pooled results published for the safety population, using estimates reported for the longest follow-up period in each study. In most trials, the safety population was similar to the modified ITT population.The study selection process is presented in Figure Characteristics from included studies –40 are pVaccines used in the trials were the bivalent vaccine containing HPV 16 and 18 virus-like particles (VLP) from GlaxoSmithKline, or the monovalent vaccine containing HPV 16 VLP and the quadrivalent vaccine containing HPV 6, 11, 16 and 18, both from Sanofi Pasteur MSD. All trials used placebo as comparator except for two studies: one used hepatitis B vaccine in both the intervention and the control groups , and oneSome of the included studies had unclear allocation concealment and unclear blinding. However, all studies were assessed as having low risk of bias.While, one of our aims was to investigate the effect of a HPV catch-up vaccination on overall and cancer related mortality, and on the incidence of cervical cancer, no RCT examining these outcomes were identified.Table Overall mortality was seldom reported, and primarily only in the text. Overall mortality was reported in 7 RCTs , 41, 45.The intention-to-treat (ITT) analysis based on five studies showed a borderline statistically significant reduction in CIN2+ lesions associated with HPV vaccination with a pooled RR of 0.80 (95% CI: 0.62-1.02) for a follow-up period of 4 years  showed a non statistically significant reduction for all CIN2+ lesions after a four year follow-up period   based on two RCTs reported in one publication . The results also indicate a protective effect against all CIN2 lesions (HPV associated and not). No difference in the occurrence of serious adverse events was found in vaccinated participants compared to control groups.High grade cervical lesions (CIN2+) were proposed by the WHO as an appropriate surrogate outcome for RCTs examining the effect of HPV vaccination . A reasoHPV vaccines were shown to be highly efficacious to prevent persistent infections with HPV , 49, 50.While HPV types 16 and 18 are the two most common HPV types worldwide, prevalence of HPV types differs geographically [While prophylactic HPV vaccination is considered effective and cost-effective in young girls, it is unclear whether a catch-up vaccination of older girls would have beneficial effect on health outcomes. This is primarily due to differences of HPV status between young and older girls, with younger more likely to be free of HPV infection. In the population included in this systematic review, the HPV status of participants varies, because of the age-range examined in this review, and differences in studies inclusion criteria: Some studies included only participants with no history of HPV infection and negative HPV tests at entry into the study , and othThe conducted RCTs on the efficacy of HPV vaccination have a relatively short follow-up period with most results published up to date based on follow-up periods of approximately four years. Two included trials published results after longer follow-up periods (6 and 8 years) but the results were based on few participants and are inconclusive , 36. TheIn most developed countries, national cervical cancer screening programs have been implemented, with a reported reduction in cervical cancer incidence probably partly due to these programs –56. A puThe evidence, to date, shows that a HPV catch-up vaccination can be considered safe, with no differences seen in serious complications between the vaccination and the control groups. However, the number of cases of published clinical studies may not be sufficient to determine the occurrence of rarely occurring (severe) adverse events in a reliable way. Furthermore, due to the relatively short follow-up period since HPV vaccination implementation in different countries, the long-term safety of the vaccines is unclear and needs to be further monitored in future studies. While different adverse events have been reported, as we examined only events reported as serious this ensures a better homogeneity of the ascertained outcome across studies.This systematic review indicates that HVP catch-up vaccination could be a valuable primary prevention against cervical cancer and national catch up vaccination programs have already been implemented in several countries. However, the long term effect of such a vaccination strategy and its impact on cervical cancer prevention remains to be determined.Appendix.(DOCX 14 KB)Additional file 1:


Countries must decide whether or not to replace primary cytology-based screening with primary human papillomavirus (HPV)-based screening. We aimed to assess how primary screening for an HPV infection, a sexually transmitted infection (STI), and the type of information included in the invitation letter, will affect screening intention.We randomized a representative sample of Norwegian women to one of three invitation letters: 1) Pap smear, 2) HPV testing or 3) HPV testing with additional information about the nature of the infection. Intention to participate, anxiety level and whether women intend to follow-up abnormal results were measured between groups using chi-squared and nonparametric Kruskal-Wallis tests. Determinants of intention were explored using logistic regression.Responses from 3540 women were representative of the Norwegian population with respect to age, civil status and geographic location. No significant difference across invitation letters was found in women’s stated intention to participate (range: 91.8-92.3%), anxiety (39-42% were either quite or very worried) or to follow-up after an abnormal result (range: 97.1-97.6%). Strength of intention to participate was only marginally lower for HPV-based invitation letters, albeit significant , when measured on a scale. Only 36–40% of respondents given the HPV invitations correctly understood that they likely had an STI.We found that switching to primary HPV screening, independent of additional information about HPV infections, is not likely to reduce screening participation rates or increase anxiety; however, women lacked the ability to interpret the meaning of an HPV-test result. High participation and follow-up rates of population-based cervical cancer screening programs using cytology-based methods (Pap smear) have been effective in reducing the incidence and mortality of invasive cervical cancer in many developed countries . Since 1Due to the causal link between cervical cancer and human papillomavirus (HPV), a common sexually transmitted infection (STI), diagnostic tests, which explicitly detect the presence of HPV, have been developed. Large, randomized controlled trials have shown that primary HPV testing can detect more high-grade precancerous lesions than conventional or liquid-based cytology (LBC) . While tWhether or not explicitly testing for an STI will affect screening adherence in Norway is not known. As in other countries, public understanding of HPV and its role in cervical cancer development is poor . The mosOne potential concern is that some specific subgroups may respond differently to primary HPV testing than others. In a qualitative study performed in the United States , women >Intention to perform an action is often cited as the most important predictor of behavioral performance or one’s true actions  and can In 2011, we conducted a randomized web-based survey of a representative sample of Norwegian women using TNS Gallup’s active internet panel of more than 50,000 individuals. Baseline demographic characteristics, knowledge of cervical cancer and HPV infection, perceived risk of cervical cancer and anxiety towards developing STIs were obtained. To elicit the intent to participate in screening, women were randomized to one of three versions of cervical cancer screening invitation letters  would be needed for 80% power with a significance level of 0.05, to detect a 3.5% difference  between expressed participation rates. Based on published estimates of the prevalence of hysterectomized women in Norway , we expeWe calculated proportions for binary responses and means for the scale responses. We conducted Chi-squared tests of statistical significance for the difference between proportions and nonparametric analysis of variance  to test the differences between groups for questions measured on a continuous scale. Odds ratios (ORs) were derived from univariable and multivariable logistic regression models for the binary dependent variable of intent to participate. The dependent variable was set to equal 1 if the respondent intended to participate, and to zero if they did not intend to participate. Independent variables were selected to reflect author hypotheses and factors, which have been associated with screening participation in previous studies. In addition to the main effects, we studied whether invitation letter type interacts with age  and civil status (single vs. cohabiting/married) with regard to participation rate. The α-level was set at 0.05. All data was analyzed in STATA, release 12 .The mean age of the final sample of 3540 women was 45.1 years. The baseline demographic characteristics were evenly distributed among the randomized groups  and thus, the variables were not included in the models presented in Table Socio-demographic factors were significantly associated with intention to participate in the univariable models, though effects did not remain significant in the multivariable model  factors that influences participation. One possible explanation would be if the rationale to attend screening in Norway stems from a sense of duty. This "obligation factor” has been tied to screening previously, and is often documented in welfare countries where state-run health care systems dominate . In neigPrevious knowledge of HPV was higher than what has been previously reported in 2005 , though Confounders and statistical variation between letter versions are likely minimized due to randomization and our large sample size; however, generalizing our results to the Norwegian population may be mitigated by 1) not allowing all women adequate time to respond to the survey (due to the target number of women reached within two weeks) and 2) the over representativeness of women with a university education when compared to the Norwegian population (Table It is important for policy-makers to gauge the impact primary HPV testing will have on attendance prior to implementation in Norway. Our findings are similar to results from a previous study conducted within a large randomized trial setting for primary screening  and to aResults indicate that, in Norway, switching to primary HPV screening, independent of additional information on HPV infections, is neither likely to reduce screening participation rates nor increase anxiety. However, women lacked the proper interpretation of their HPV-test result and it is uncertain whether increased awareness of HPV and its implications will impact future participation rates in primary HPV screening programs.EAB, DGH and TAM have no interests to declare. MN and ISK are members of the Norwegian Directorate of Health’s national advisory board for cervical cancer screening.EAB, MN, DGH and ISK designed the study. EAB and TAM analyzed the data. EAB, MN, DGH, ISK interpreted results. EAB drafted the manuscript and all authors aided revision and accepted the final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/14/360/prepubTranslated survey invitations letters and additional results.Click here for file


We benchmarked pT356RB1 to total RB1, Ki67, pT202/Y204ERK1/2, and TP53, as quantified by automatic quantitative analysis (AQUA), and correlated protein expression with tumor stage and grade. High expression of pT356RB1 but not total RB1 predicted reduced overall survival , indicating the potential relevance of post-translational phosphorylation. Paired analysis of The Cancer Genome Atlas (TCGA) data for regulators of this RB1 phosphorylation identified loss or truncating mutation of negative regulator CDKN2A (p16) and elevated expression of the CDK4/6 activator CCND1 (cyclin D) as also predicting poor survival. Given that CDK4/6 inhibitors have been most effective in the context of functional RB1 and low expression or deletion of p16 in other tumor types, these data suggest such agents may merit evaluation in HPV-negative SCCHN, specifically in cases associated with high pT356RB1.Locally advanced squamous cell carcinoma of the head and neck (SCCHN) that is not associated with human papillomavirus (HPV) has a poor prognosis in contrast to HPV-positive disease. To better understand the importance of RB1 activity in HPV-negative SCCHN, we investigated the prognostic value of inhibitory CDK4/6 phosphorylation of RB1 on threonine 356 (T Da