In Chile, significant reductions in cervical cancer incidence and mortality have been observed due to implementation of a well-organized screening program. However, it has been suggested that the inclusion of human papillomavirus (HPV) vaccination for young adolescent women may be the best prospect to further reduce the burden of cervical cancer. This cost-effectiveness study comparing two available HPV vaccines in Chile was performed to support decision making on the implementation of universal HPV vaccination.The present analysis used an existing static Markov model to assess the effect of screening and vaccination. This analysis includes the epidemiology of low-risk HPV types allowing for the comparison between the two vaccines (HPV-16/18 AS04-adjuvanted vaccine and the HPV-6/11/16/18 vaccine), latest cross-protection data on HPV vaccines, treatment costs for cervical cancer, vaccine costs and 6% discounting per the health economic guideline for Chile.Projected incremental cost-utility ratio (ICUR) and incremental cost-effectiveness ratio (ICERs) for the HPV-16/18 AS04-adjuvanted vaccine was 116 United States (US) dollars per quality-adjusted life years (QALY) gained or 147 US dollars per life-years (LY) saved, while the projected ICUR/ICER for the HPV-6/11/16/18 vaccine was 541 US dollars per QALY gained or 726 US dollars per LY saved. Introduction of any HPV vaccine to the present cervical cancer prevention program of Chile is estimated to be highly cost-effective . In Chile, the addition of HPV-16/18 AS04-adjuvanted vaccine to the existing screening program dominated the addition of HPV-6/11/16/18 vaccine. In the probabilistic sensitivity analysis results show that the HPV-16/18 AS04-adjuvanted vaccine is expected to be dominant and cost-saving in 69.3% and 77.6% of the replicates respectively.The findings indicate that the addition of any HPV vaccine to the current cervical screening program of Chile will be advantageous. However, this cost-effectiveness model shows that the HPV-16/18 AS04-adjuvanted vaccine dominated the HPV-6/11/16/18 vaccine. Beyond the context of Chile, the data from this modelling exercise may support healthcare policy and decision-making pertaining to introduction of HPV vaccination in similar resource settings in the region.The online version of this article (doi:10.1186/1471-2458-14-1222) contains supplementary material, which is available to authorized users. Cervical cancer is the second most common cancer in women of the Latin America and Caribbean (LAC) region with estimated age-standardized incidence and mortality rates of 23.5 and 10.8 per 100,000 women, respectively , 5. ThisNew opportunities to tackle this high burden of cervical cancer disease are now available. Two different vaccines against human papillomavirus (HPV) have been widely used, both internationally and in the LAC region , 9. It iCost-effectiveness analysis of new interventions, like the introduction of HPV vaccines is a useful tool to assist decision makers in allocating resources. Results reported from these analyses vary widely from one country to another due to epidemiological variation, treatment-related costs and effectiveness of existing secondary prevention programs. The potential impact and cost-effectiveness of introducing the bivalent HPV-16/18 AS04-adjuvanted vaccine to the cervical cancer prevention programs of five Latin American countries was previously evaluated . That anHPV vaccines have now been offered to Latin American countries through the Pan American Health Organization (PAHO) Revolving Fund at low prices per vaccine dose following which some countries in the LAC region have decided its inclusion in their national vaccination programs  . The preThis analysis used a previously published lifetime Markov cohort model with a one year cycle reproducing the natural history of oncogenic HPV in cervical cancer, the effect of screening and of vaccination , 12–15 dMost of the input parameters used to calibrate the model for Chile were previously described  and are The vaccinated cohort assumed in the model included women aged 11 years  . VaccinaIn the present analysis new or modified epidemiological parameters compared with the previous Chilean adaptation of the model are described. The proportion of CIN1 cases under treatment were reduced to 40.0% following the new clinical guidelines developed by the Ministry of Health, Chile . Five-yeThe costs of treatment of precancerous lesions and cervical cancer were obtained from data published by the Ministry of Health, Chile Table  35]. We. We35]. The cost of vaccination was assumed to be 20 US dollars per dose for both vaccines. Although the cost of vaccines is slightly lower through the PAHO Revolving Fund, this value of 20 US dollars per dose was assumed in the analysis since an accurate estimation of the cost of vaccine administration was unavailable. The cost of administration was assumed to be the same for the two vaccines.In the estimation of vaccine effectiveness, HPV type-specific vaccine efficacies and HPV type prevalence in cervical cancer and precancerous lesions for Chile were considered. Vaccine efficacy of 98.0% against cervical cancer, CIN1, CIN grade 2 (CIN2) and CIN grade 3 (CIN3) associated with HPV types 16 and 18 were assumed to be identical for the HPV-16/18 AS04-adjuvanted vaccine and HPV-6/11/16/18 vaccine –27. AddiLifetime sustained protection against HPV types 16 and 18 was considered in the base case analysis. Based on clinical trial data, efficacy against the HPV-16/18 AS04-adjuvanted vaccine after 6.4 years and 9.4 years of follow-up was observed , 26. FurThe present study assessed the incremental difference in health outcomes, total costs and ICER  between scenarios. Future costs and health outcomes were discounted at an annual rate of 6.0% based on the recommendations of the Planning Ministry of Chile  and usedPrevalence of HPV types in cervical cancer cases used for base case analysis was obtained from a recent study  which reDiscount rate of 6.0% was used in base case; however, a discount rate of 3.0% was also tested in-line with international guidelines.Introduction of the HPV-16/18 AS04-adjuvanted vaccine to the screening program of Chile at an increased screening interval as a control was considered of interest for Chile. Therefore, the frequency of screening used in the base case was modified from every three years to five years in the vaccination scenario.Introduction of the HPV-16/18 AS04-adjuvanted vaccine without cross-protection, or with a cross-protective vaccine efficacy of 45.7%  due to the other oncogenic HPV types , 31) wasReduced vaccine prices (both vaccines) according to the PAHO 2012 Revolving Fund (13.48 US dollars per dose for the HPV-16/18 AS04-adjuvanted vaccine and 14.25 US dollars per dose for the HPV-6/11/16/18 vaccine) were considered.The effects of waning of vaccine efficacy against different HPV types used in the base case analysis were assessed in different scenarios. Waning of vaccine efficacy against HPV-18 and the other ten oncogenic HPV types  was assumed to begin 20 years after vaccination and after five years of a linear decrease in vaccine efficacy this value was assumed to be negligible. This scenario of waning vaccine efficacy was evaluated for vaccination with and without a booster vaccine dose 21 years after the first dose (33 years of age) administered to 95% of the cohort and assumed to result in lifetime protection . AlthougA vaccination program based on a two dose schedule was analyzed for the HPV-16/18 AS04-adjuvanted vaccine , 42. TheIn addition to the base case scenarios for both vaccines, deterministic scenario analyses were conducted by varying key input parameters of interest:To test the robustness of model input data and base case assumptions a probabilistic sensitivity analysis (PSA) was performed to quantify the effect of uncertainty surrounding the model input parameters and assumptions on the final ICER estimates. In total, 10000 replicates were generated from the assigned distribution to produce a distribution of the model’s results. The replicates were plotted on the cost-effectiveness plane. The proportion of replicates in each quadrant of the plane was counted and reported as percentage. The PSA was performed by comparing either the HPV-16/18 AS04-adjuvanted vaccine or the HPV-6/11/16/18 vaccine to screening alone or by comparing the HPV-16/18 AS04-adjuvanted vaccine to the HPV-6/11/16/18 vaccine, under different scenarios. The PSA was performed using the @Risk software .The model was validated by comparing modelled outcomes with published epidemiological parameters for Chile which were obtained from the latest local epidemiological data , 35, 36.The most significant health and economic outcomes for the base case analysis are shown in Table The economic analysis projects that the introduction of any HPV vaccine in Chile would be expected to cost 7.0 million US dollars per year Table . HoweverThe PSA on the ICER of the HPV-16/18 AS04-adjuvanted vaccine  of 93.2% is considered the potential impact of the HPV-16/18 AS04-adjuvanted vaccine  or vaccine price was reduced to present 2012 PAHO Revolving Fund vaccine prices or screening interval duration was increased to every five years together with the vaccination program, or a two-dose (versus three dose) vaccination schedule for the HPV-16/18 AS04-adjuvanted vaccine was evaluated, the implementation of cervical cancer vaccination with the HPV-16/18 AS04-adjuvanted vaccine was cost-saving for Chile.One of the many strengths of this analysis is the comparison of the cost-effectiveness profiles of the HPV-16/18 AS04-adjuvanted vaccine and HPV-6/11/16/18 vaccine for the prevention of cervical cancer updated with recent estimates of clinical, economic and epidemiological data for Chile. The ICURs for the HPV-16/18 AS04-adjuvanted vaccine (116 US dollars per QALY gained) were dominant compared with the HPV-6/11/16/18 vaccine (541 US dollars per QALY gained) in the base case analysis. The PSA confirmed that the HPV-16/18 AS04-adjuvanted vaccine remains dominant over the HPV-6/11/16/18 vaccine (base case scenario) in 69.3% of the simulations generated and this difference was strengthened at lower discount rates (87.0% probability at 3.0% discount as used in other developed countries). Since we assumed similar vaccine efficacies against HPV types 16 and 18 for both vaccines, the difference was based mainly on its different cross-protection vaccine efficacy profiles. In addition, greater benefits for the HPV-16/18 AS04-adjuvanted vaccine can be realized if a 93.2% efficacy against CIN3+ (irrespective of HPV type in the lesion) is considered . Under tThe findings from the present analysis are valid and robust mainly due to the inclusion of many plausible scenarios of interest. There is clinical evidence supporting that a two-dose schedule might be possible , 41, howThe present study shows a much better cost effectiveness profile for these vaccines than previously reported . This chA potential limitation of this study is that the real-life effectiveness of these vaccines is unknown or the evidence on the sustainability of vaccine efficacy is limited. While we have attempted to identify publications with the best comparable estimates of cross-protection between vaccines, there is no single trial that directly compares the cross protective efficacy of the HPV-16/18 AS04-adjuvanted and the HPV-6/11/16/18 vaccines. In addition, modelling studies based on vaccine efficacy after 6.4 years of follow-up for the HPV-16/18 AS04-adjuvanted vaccine predicted a long-term (≥20 years) persistence of vaccine-induced antibody levels . AlthougUrgent actions are needed in order to reduce cervical cancer mortality. Both interventions (vaccination and screening) must be considered as complementary. Our results show that the inclusion of vaccination against cervical cancer to the current screening program is highly cost-effective or even cost-saving for Chile. The model predicts a substantial impact of the vaccination program not only against cervical cancer cases and deaths but also on pre-cancerous lesions and its associated resource use (diagnostic tests and treatment). However, controversy still exists regarding the best combination of prevention (primary and secondary) programs against cervical cancer and the affordability of such programs. A recent study based on an optimization model have shown that the best combination for Brazil in the prevention of cervical cancer cases under budget constraints was to combine universal HPV vaccination with an increase in screening interval . We haveThe modelling exercise conducted assessed that the implementation of HPV vaccination together with the current screening program of Chile would be cost-effective. These findings may further support or guide the introduction of HPV vaccination in other countries of the LAC region where a higher burden of cervical cancer exists.Details on the cross protection scenario used, on vaccine efficacy waning scenarios analyzed, on the derivation of HPV incidence from HPV prevalence and on the model simulated age distribution of different outcomes.(DOCX 200 KB)Additional file 1:


To investigate the prevalence, genotypes, and prognostic values of Epstein-Barr virus (EBV) and human papillomavirus (HPV) infections in Japanese patients with different types of head and neck cancer (HNC).LMP-1 variants, and HPV mRNA expression were detected by PCR from fresh-frozen HNC samples. HPV genotypes were determined by direct sequencing, and EBV encoded RNA (EBER) was examined by in situ hybridization.HPV and EBV DNA, EBV genotypes and E6/E7 mRNA expression was found in 23 of 60 (38.3%) HPV DNA-positive cases detected. The site of highest prevalence of HPV was the oropharynx (45.9%). Among 146 (69.9%) HNCs in which EBV DNA was identified, 107 (73.3%) and 27 (18.5%) contained types A and B, respectively, and 124 (84.9%) showed the existence of del-LMP-1. However, only 13 (6.2%) HNCs were positive for EBER, 12 (92.3%) of which derived from the nasopharynx. Co-infection of HPV and EBER was found in only 1.0% of HNCs and 10.0% of NPCs. Kaplan-Meier survival analysis showed significantly better disease-specific and overall survival in the HPV DNA+/mRNA+ oropharyngeal squamous cell carcinoma (OPC) patients than in the other OPC patients . Multivariate analysis showed that stage T1–3 (P = 0.002) and HPV mRNA-positive status (P = 0.061) independently predicted better disease-specific survival. No significant difference in disease-specific survival was found between the EBER-positive and -negative NPC patients (P = 0.155).Of the 209 HNC patients, 63 (30.1%) had HPV infection, and HPV-16 was the most common subtype (86.9%). HPV Our findings indicate that co-infection with HPV and EBV is rare in HNC. Oropharyngeal SCC with active HPV infection was related to a highly favorable outcome, while EBV status was not prognostic in the NPC cohort. Head and neck cancer (HNC) is the sixth leading cancer in the world, with more than 600,000 new cases reported each year LMP-1 and LMP-2), and the EBV nuclear antigens (EBNA1–6) EBNA-2, -3A, -3B, and -3CLMP-1 has been found to be an EBV oncogene LMP-1 are required for the maximal activation of nuclear factor-kappa B (NF-kB), the activation of which is linked to the inhibition of apoptosis LMP-1 gene (del-LMP-1) has been reported in NPC in Chinese patients. This deletion can result in loss of amino acids 346–355 in the LMP-1 protein. The deletion protein has a longer half-life and confers enhanced NF-kappa B and JNK/AP1 signaling activity in epithelial cells. These properties of del-LMP-1 variant are localized to the transmembrane-spanning domains and may contribute to a more malignant phenotype of NPC del-LMP-1 to be present in more than 75% of NPC patients in Southeast Asia and North Africa LMP-1 variants in NPC may reflect differences in the biological or molecular properties of LMP-1 variants. Moreover, del-LMP-1 has lower immunogenicity than the non-del variant, which may give rise to tumor development in immunocompetent hosts via escaping immunosurveillance EBER), the gold-standard assay for determining whether a biopsied tumor is EBV-related, is significantly inconsistent in these cancers EBV is an ubiquitous herpes virus that affects more than 90% of the world's adult population and is associated with variety of malignant disorders including Hodgkin's disease, Burkitt's lymphoma, B-cell lymphoma Co-infection with viruses and virus/virus direct or indirect interactions have been found in some malignancies, and a typical example for a malignant tumor due to virus/virus interactions is Kaposi's sarcoma-associated herpesvirus (KSHV)-induced Kaposi sarcoma in AIDS patients del-LMP-1 variants, and HPV genotypes in fresh-frozen samples from patients with HNC in order to analyze the effects of EBV and HPV co-infection on different types of HNC. The findings were then examined for associations with clinical features and prognosis.This prospective study investigated the frequency of EBV genotypes, We recruited 209 patients with HNC pathologically confirmed by the Department of Otorhinolaryngology, Head and Neck Surgery of the University of the Ryukyus, Japan, between December 2006 and March 2014. Written, informed consent was obtained from each patient and the research protocol was approved by the Ethics Committee of the University of the Ryukyus. Tissue samples obtained by biopsy or surgical excision were snap-frozen in liquid nitrogen and stored until further analysis.L1 and E6/E7 genes and the EBV LMP-1 and EBNA-3C genes, respectively, and cultured according to the supplier's instructions.The cell lines CaSki  and Raji  were used as controls for the amplification of the HPV DNA was extracted from the samples and cells using the Gentra Purification Tissue Kit  according to the manufacturer's protocol. The presence and integrity of the DNA in all samples was verified by PCR â-globin gene amplification using primers PC04 and GH20 GP5+/GP6+ and MY09/11GP5+/GP6+ or MY09/11 were re-amplified by (auto-) nested PCR using the GP5+/GP6+ primer pair as previously described The presence of HPV DNA was analyzed by PCR using the general consensus primer sets EBNA-3C gene as previous described EBV-A (153 bp) and EBV-B (246 bp) (EBV DNA presence and typing was carried out by PCR using primers spanning the (246 bp) . As posiLMP-1 gene as previously reported LMP-1. Samples with a shorter PCR product length than that observed with Raji were believed to contain the LMP-1 deletion . In addition, a sample with a 30 bp deletion variant verified by PCR and direct sequencing was also included in each PCR reaction.We also examined the Total RNA extracted from tumor samples using the ToTALLY RNA™ kit  according to the manufacturer's protocols was suspended in 50 µL ultra-high quality diethyl pyrocarbonate-treated water.Before cDNA synthesis, any residual DNA was removed by incubation with 1 U DNase I (Ambion) at room temperature for 25 min. cDNA was then synthesized from DNA-free total RNA using the RETROscript® Kit (Ambion) according to the manufacturer's instructions. To examine for the presence of contaminating DNA in RNA samples, all assays were performed both with and without reverse transcriptase.E6/E7 mRNA transcripts, PCR was performed with cDNA obtained from HPV DNA-positive samples using the TaKaRa PCR Human Papillomavirus Typing Set , which can identify high-risk HPV types 16, 18, 31, 33, 35, 52, and 58. To verify the HPV-16 E6/E7 mRNA transcripts, the HPV-16 DNA-positive samples were also examined using a half-nested PCR approach with cDNA as previously described To detect high-risk EBER. Briefly, 4 µm-thick sections from formalin-fixation paraffin-embedded blocks were deparaffinized, re-hydrated, and predigested with proteinase K. The hybridization solution containing fluorescein isothiocyanate (FITC)-conjugated EBV oligonucleotide probe  was applied for 90 min at 55°C. A Dako ISH detection kit (code No. S5201) with rabbit F(ab′)2 anti-FITC antibody and enzyme substrate (5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium) was used to visualize the hybridization products. Stained slides were counterstained with Nuclear Fast Red . Dark-blue signals appearing within the nucleus were recognized as EBER positive.The presence of EBV in cancer cells was confirmed by ISH for Descriptive statistics were used to characterize patient baseline characteristics. The Mann-Whitney U-test or Kruskal-Wallis test was used for continuous variables, and Pearson's Chi-square test or Fisher's exact test was used for categorical variables.P values less than 0.05 were considered significant. All statistical analyses were performed using SPSS software .Recurrence-free survival was defined as the time from the end of treatment to cancer recurrence or last follow-up. Disease-specific survival was defined as the time from the end of treatment to subsidence of disease or last follow-up. Survival curves were evaluated by the Kaplan-Meier method, and survival distributions were compared using the log-rank test. Location of the primary tumor was the nasopharynx in 20 patients (9.6%), oropharynx in 74 (35.4%), hypopharynx in 50 (23.9%), larynx in 28 (13.4%), and oral cavity in 37 (17.7%). Of the 209 patients, 168 received curative treatment  in 51, surgery alone in 27, and radiation therapy alone in 17), 30 patients received palliative treatment, and 11 had no treatment. Demographic and clinical characteristics are summarized in HPV DNA was detected in 30.1% (63/209) of the 209 patients, and the oropharynx was the site of highest prevalence . Genotyping of the neoplasms showed that most of the HPV DNA-positive specimens contained high-risk HPV-16 . The other HPV types included HPV-33 (4 cases), HPV-35 (2 cases), HPV-56 (1 case), HPV-58 (2 cases), and HPV-67 (1 case). No multiple infection or low-risk HPV types were found .E6/E7 mRNA expression was found in 23 (38.3%) of them. Interestingly, most of the HPV E6/E7 mRNA-positive tumors  derived from the oropharynx. While 60.6% (20/33) of HPV DNA-positive OPCs were positive for viral HPV mRNA expression, HPV E6/E7 mRNA transcripts were found in only 3 of 27 (11.1%) non-oropharyngeal HNCs, deriving from the nasopharynx, larynx, or oral cavity . In addition, significant correlations were observed between HPV status and tumor location (P = 0.002) and between HPV status and tumor differentiation (P<0.001).Analysis of the statistical correlation between HPV infection status and histoclinical features revealed no significant relationships between the presence of HPV DNA and sex, age, TNM stage, or nodal stage. HPV DNA was more likely to be found in nonsmokers or light smokers  of the 209 HNC samples: 107 (73.3%) contained type A, 27 (18.5%) contained type B, and type A and type B were found together in 12 (8.2%) cases. We also investigated the prevalence of the LMP-1 variant and found del-LMP-1 in 124 (84.9%) of the 146 EBNA-positive cases. Almost all type A EBV cases  contained del-LMP-1, while just over half of type B cases  contained wild-type LMP-1 (P<0.001).As indicated in del-LMP-1, including 6 WHO type I cases, 6 WHO type II cases, and 4 WHO type III cases.The prevalence of EBV DNA by subsite was 95.0% (19/20), 79.7% (59/74), 64.0% (32/50), 50.0% (14/28), and 59.5% (22/37) in tumors from the nasopharynx, oropharynx, hypopharynx, larynx, and oral cavity, respectively. Of the 17 NPC specimens containing type A EBV, 16 (94.1%) showed EBER was also examined by ISH in all HNC samples . EBER was detected in 60.0% (12/20) of patients with NPC, including 2  with WHO type I, 6  with WHO type II, and 4  with WHO type III, but in only 1.4% (1/74) of patients with OPC. None of the EBER-positive cases derived from the hypopharynx, larynx, or oral cavity.To determine the presence of EBV infection in tumor cells,  samples . In contEBER-positive HNCs, only 2 were HPV-16 positive and both derived from the nasopharynx. Since EBV infection in HNC is defined as EBER-positive tumor cells by ISH, the co-infection rates of HPV and EBV in HNC and NPC were only 1.0% and 10.0%, respectively (EBER ISH.Both HPV DNA and EBV DNA were detected by PCR in 45 (21.5%) of the total 209 patients. However, of the 13 ectively . AlthougP = 0.332 and P = 0.378, respectively; P = 0.027 and P = 0.017, respectively). The 3-year disease-specific survival rate was 95.0% (95% CI = 85.5%–100%) in the cohort with HPV DNA+/mRNA+ and 73.8% (95% CI = 61.4%–86.1%) in the cohort with HPV DNA+/mRNA− or HPV DNA−/mRNA−.Since there was insufficient tissue for mRNA detection in one case, we evaluated the treatment outcomes of 73 OPC patients based on HPV status. Fifty-four patients with previously untreated primary OPC had complete remission after receiving primary curative treatment, 5 showed persistent disease after curative treatment, 10 underwent palliative treatment, and 4 had no treatment. Median follow-up for patients whose data were censored was 41 months . At latest follow-up, 18 (24.7%) of the 73 OPC patients had died of the disease and 2 had died of unrelated diseases. Kaplan-Meier analysis showed that no significant differences in disease-specific survival or overall survival between OPC patients with HPV DNA+ and OPC patients with HPV DNA-  = 0.12; 95% confidence interval (CI) = 0.01–0.99) than the other OPC patients  compared with patients categorized as stage T1–3, nodal stage 0–1, and light smokers and non-smokers. The final model of multivariate analysis using a Cox proportional hazards model for identification of fair disease-specific survival of OPCs showed that stage T1–3  and HPV mRNA-positive status  predicted better disease-specific survival  (P = 0.015) (P = 0.032) (P = 0.080)  for OPC patients with HPV DNA− (= 0.002) . Thus, t= 0.015) . Interes= 0.032) , and the= 0.080) .EBER. Among 20 patients with NPC, 18 were treated locally and treatment details and outcome were available from the time of diagnosis for these patients. The median follow-up time was 21 months . Although patients with EBER-positive NPC showed a trend towards improved disease-specific survival compared with patients with EBER-negative NPC, no significant difference was found between these two cohorts (P = 0.155) (EBER-positive NPC and the EBER-negative NPC cohorts (P = 0.381)  . Similar= 0.381) .E6/E7 transcripts are identified in most HPV DNA-positive samples E6/E7 mRNA positivity in the present study. E6/E7 mRNA is considered as a marker of HPV malignant transformation, and demonstrating oncogenic E6/E7 transcription may suggest that HPV-driven carcinogensis is mechanistically possible, whereas HPV DNA-positive/RNA-negative results may simply reflect either low-level transcription or non-transcriptional active, and possibly a “passenger” infection. The fact that approximately 90% of cases with transcriptionally active HPV originated in the oropharynx supported the concept of a close relationship between HPV and OPC. Previous studies have demonstrated that the viral load of HPV DNA exhibits large variation among head and neck regions, and copy numbers of DNA in OPCs were much higher than in non-oropharynx HNCs. The relative low multiplicity of HPV infection in non-oropharynx head and neck sites, including the hypopharynx, larynx, and oral cavity, may correlate to low E6/E7 mRNA expression in tumors at these sites E6/E7 contain a mutated p53 gene, in which the E6 gene is frequently disrupted. The lack of E6/E7 expression might be due to the E6 gene disruption or alterations in transcriptional control The overall HPV prevalence in HNC was slightly higher in the present study (30.1%) than in international studies, which have reported 25.9% HPV-DNA-positive cases for these tumor entities E6/E7 mRNA transcripts, however, the role of HPV in these non-oropharynx HNCs needs further clarification.In previous studies, patients with NPC in Taiwan commonly showed a higher prevalence of HPV (35–51%) del-LMP-1 is common in the Japanese population, findings which agree with data acquired from the same sort of specimens collected in Southeast Asia and North Africa LMP-1 isolates are more prominent than del-LMP-1 variants LMP-1 variants exhibit transformation activity and induce tumorigenic changes, and the deletion protein has a longer half-life and a greater ability to activate NF-kB and JNK/AP1 in epithelial cells, thus contributing to a more malignant NPC phenotype del-LMP-1 in endemic regions of Southeast Asia and North Africa compared with wild-type EBV. However, conflicting findings of no significant differences in the frequency of del-LMP-1 in throat-wash samples between endemic and non-endemic regions in China del-LMP-1 is indeed a NPC phenotype-correlated polymorphism and not a geographical- or ethnicity-related polymorphism.This study using PCR on fresh-frozen samples from NPCs showed that EBV type A is predominant and LMP-1, most of cases with EBV type-A contained del-LMP-1. Our findings are in concordance with a previous study from Korea del-LMP-1 variant and the type B EBV exhibited wild-type LMP-1 more frequently. Interestingly, wild-type del-LMP-1 was not only found in 64.3% of type B EBV but was predominant (64.1%) in type A EBV in peripheral blood mononuclear cells from healthy carriers del-LMP-1 was always detected with type B EBV LMP-1 variants might be due to the intrinsic characteristics of EBV. The variation of LMP-1, together with polymorphisms in other regions of viral genome, may raise the possibility of virus strain-specific disease associations.Although just over half of cases with EBV type-B contained wild-type del-LMP-1 in HNC types other than NPC. However, compared with the high detection rate (60%) of EBER in NPC, all non-nasopharynx HNCs but one were found to be EBER negative by ISH, which suggests that the 30 bp deleted LMP-1 variants might be derived from non-malignant lymphocytes not cancer cells. Moreover, a high frequency of EBV and the del-LMP-1 variant was observed in non-malignant tonsillar tissue in the present and another study EBER detection between NPC and non-NPC HNCs.Our findings showed the same predominance of Although HPV and EBV co-infection has been investigated in a subset of HNC cases in previous studies, the results have been somewhat contradictory. Data from Taiwan and Morocco described such co-infection in NPC in up to 42% of the samples tested by PCR It is well documented that tumors located in specific regions of the head and neck are more or less responsive to radiation and/or chemotherapeutic treatment del-LMP-1 are predominant in Japanese patients with NPC, EBV status was not prognostic in the NPC cohort.In conclusion, our findings indicate that although HPV and EBV are highly prevalent in cancers of the oropharynx and nasopharynx, respectively, HPV and EBV co-infection is rare in HNC, including at these two subsites. Oropharyngeal SCC with active HPV infection is associated with a highly favorable outcome, and an individualized therapeutic regimen based on HPV status and T stage in OPC patients should be evaluated and considered. While EBV type A and


Sub-Saharan countries including Nigeria have the highest burden of Human Papillomavirus (HPV) infection in the world. Most studies on HPV surveillance in Nigeria were done in the southern part of the country. Geographical and socio-cultural diversity of Nigeria makes these data unlikely to be universally representative for the entire country. Northern Nigeria especially the North-East carries a higher prevalence of cervical cancer and many of its risk factors. The region may be harbouring a higher prevalence of HPV infection with a possibility of different genotypic distribution. This study was carried out to determine the burden and confirm the predominant HPV genotypes among women presenting for cervical cancer screening at the Federal Teaching Hospital Gombe (FTHG), North-eastern, Nigeria.The study was an observational hospital based cross sectional study among women who presented for cervical cancer screening in FTHG. A total of 209 consenting women were tested for cervical HPV infection using PCR. DNA sequencing was carried out on positive samples to determine the prevalent HPV genotypes.X2 = 15.897; p = 0.007), age at sexual debut , parity , number of life time sexual partners , age at first pregnancy  and history of other malignancies  were found to have a statistically significant association with HPV infection.The prevalence of cervical HPV infection among the participants with mean age of 39.6 ± 10.4 years was 48.1 %. The five most predominant genotypes were 18, 16, 33, 31 and 35, with prevalence of 44.7 %, 13.2 %, 7.9 %, 5.3 % and 5.3 % respectively. Other genotypes observed were 38, 45, 56, 58, 82 and KC5. Multiple HPV infections were detected among 7.9 % of participants. Risk factors such as level of education  contains supplementary material, which is available to authorized users. Among the approximately 200 known HPV types, about 40 can infect the cervix out of which 13 types  from among the alpha species have been classified as group 1 carcinogens  and GP6+ [5′- GAAAAATAAACTGTAAATCATATTC-3′])  and PGMYt test was used to compare means for continuous variables, while categorical variables were summarized as proportions and further analyzed using Chi square to assess association between them. A logistic regression was performed to ascertain the effects of variables which showed a statistically significant association with HPV testing based on Chi square. Figures and tables were appropriately used in representing the analyzed data . Student’s t test showed no statistically significant difference between the mean ages (P = 0.111).Table More than half, 110 (53.4 %) were educated up to tertiary level with only 32 (15.4 %) having no formal education while 14 (6.7 %) and 51 (24.5 %) had primary and secondary education respectively. Nearly all, 182 (88.0 %) were married as both single and divorced women accounted for only 3.8 % each while 4.3 % were widows. Majority, 128 (64.8 %) of the women were from monogamous family settings. Less than half, 99 (48.1 %) were employed (government and private) with all the remaining 108 (51.9 %) being unemployed among which house wives were predominant. Muslims were slightly more 118 (56.5 %) than Christians 90 (43.5 %).Except for level of education, other socio-demographic characteristics did not show any statistically significant association with HPV infection.HPV DNA was detected in 100 (48.1 %) of the participants while 108 (51.9 %) were negative for the virus. Overall, ten different HPV types were identified from this study. HPV types 18 and 16 were the most predominant with proportions of 44.7 % and 13.2 % respectively. Multiple HPV infections were identified among 7.9 % of the specimens. Other genotypes identified were types 31, 33, 35, 38, 45, 56, 58, 82 and KC5 with corresponding proportions of 5.3 %, 7.9 %, 2.6 %, 2.6 %, 2.6 %, 2.6 %, 2.6 %, 5.3 % and 2.6 % respectively. Of the 209 participants, normal cytological findings were observed among 126 (61.6 %) while 80 (39.0 %) had abnormal features and 3 (1.4 %) had unsatisfactory smears for cytologic studies. Figure X2 = 6.916, P = 0.009). Multiple life time sexual partners also conferred a significant risk for HPV infection  as 26 (68.4 %) out of the 38 respondents with multiple sexual partners were HPV positive compared to 71 (43.3 %) out of 164 with single life time sexual partner. Statistically significant association was also observed between ages at first pregnancy (primigravidity) and HPV infection  as respondents with history of early pregnancy (less than 18 years) showed higher percentage of HPV infection (75.0 %) in comparison to 43.7 % and 42.9 % for normal and late age of primigravidity respectively. Parity of the participants also showed a statistically significant association with HPV infection  with grand multiparous and great grand multiparous women showing a higher percentage of infection; 62.3 % and 80 % respectively. Although women in a polygamous family setting showed higher proportion of HPV infection than those in monogamous setting, the association was not statistically significant . Family history of cervical cancer did not reveal a significant association with HPV infection  but history of other malignancies had a statistically significant association with HPV infection . Among those with history of other malignancies, the type of malignancy (gynaecologic or others) did not show a statistically significant association with HPV infection .Table Other factors as participants’ HIV status, partners’ use of condom and use of hormonal contraceptives did not show statistically significant association with HPV infection. Although not shown in any of the tables, history suggestive of genital tract infection , history of alcohol consumption and cigarette smoking did not show statistical significance in associated with HPV infection. Partners’ circumcision status could not be analyzed as all respondents had circumcised sexual partners.Logistic regression was performed to ascertain the effects of level of education, age at sexual debut, parity, number of sexual partners age at primigravidity and history of other malignancies on the likelihood that participants have HPV infection Table .Table 3LThis study reveals a 48.1 % prevalence of cervical HPV infection among women who presented for cervical cancer screening in Gombe, north-eastern Nigeria. It is one of the highest prevalence rates among one of the oldest studied populations (mean age of 39.6 ± 10.4 years) to be ever reported from any part of Nigeria and the first from North-Eastern Nigeria , 18–20. Several Nigerian studies among diverse population groups had reported lower prevalence rates including 19.6 % among HIV infected patients in Lagos , 14.7 % Studies done in other parts of sub-Saharan Africa among similar population groups have shown similarly high prevalence rates when compared to the value in this study. A prevalence of 66.1 % had been reported among high risk women in Burkina faso  while 49The predominance of HPV types 16 and 18 as observed in this study follows the global trend and is in conformity with some Nigerian studies , 18. HowSeven  out of the eight  global most common cervical HPV types among women were also found in this study . The fivLow prevalence of multiple HPV infections as observed in this study compared to studies that used type specific primers (TS-PCR) in genotyping is not unusual . SequencSeveral variables  observed in this study revealed a statistically significant association with HPV infection. Low socio-economic status, early marriage/coitarche, polygamy and high parity are all well-established risk factors for HPV infection , 38. WomMajority of the studied population fell within the age group 30 to 45 years, hence are suitable for enrolment in screening and treatment programmes in preventing cervical cancer . In addiLogistic regression analysis of the identified risk factors showed that university education, multiple sexual partners, multiparity and grand multiparity are associated with a significantly increased likelihood of having HPV infection among the participants. The apparent significance of university education as a risk factor against our earlier observation and the general trend may not be unconnected with skewed distribution of the respondents in terms of educational qualification. These findings further confirm the need for a larger and community based study to obviate the lopsided distribution of the participants as majority of them are educated and screening is mostly based on referral by healthcare workers.HPV Prevalence of 48.1 % was recorded from this study. Eleven different HPV types were detected with the five most predominant types  being 18, 16, 33, 31 and 35. Other types were 38, 45, 56, 58, 82 and KC5. Low level of education, early age at sexual debut, high parity, multiple life time sexual partners, early age at first pregnancy and positive history of other malignancies were the significant risk factors associated with cervical HPV infection in Gombe.This study has revealed north-eastern Nigeria as having one of the highest HPV prevalence in the country and also a region that will benefit from the currently available HPV vaccines.There is need for a universal, well-coordinated, multi-centred, rural and urban community based study on HPV in addition to a comprehensive national cervical cancer screening programme and improved availability/accessibility of the currently available HPV vaccines in all parts of Nigeria.


Patients with recurrent respiratory papillomatosis (RRP) in Norway treated between 1987 and 2009 were recruited to this cohort study. They were followed from disease onset and data recorded until January 2012. Here, we describe the distribution of human papillomavirus (HPV) genotypes, the prevalence of multiple HPV infections, and the risk of high-grade laryngeal neoplasia and respiratory tract invasive carcinoma in a large cohort of patients with RRP. We also examined whether HPV genotype, gender, age or clinical course are risk factors for this development. Clinical records and histological specimens were reviewed. Using formalin-fixed paraffin-embedded biopsies, HPV genotyping were performed by quantitative polymerase chain reaction assays identifying 15 HPV types. HPV-negative specimens were analyzed by metagenomic sequencing. Paraffin blocks were available in 224/238 patients. The DNA quality was approved in 221/224 cases. HPV DNA was detected in 207/221 patients and all were HPV 6 or HPV 11 positive, comprising HPV 6 in 133/207, HPV 11 in 40/207 cases and HPV 6/11 in 15/207 cases. Co-infection with one or two high-risk HPV types together with HPV 6 or HPV 11 was present in 19/207 patients. Metagenomic sequencing of 14 HPV-negative specimens revealed HPV 8 in one case. In total, 39/221 patients developed high-grade laryngeal neoplasia. 8/221 patients developed carcinoma of the respiratory tract . High-grade laryngeal neoplasias were found more frequently in HPV-negative versus HPV-positive patients, , as well as respiratory tract carcinomas . In summary, the majority of RRP were associated with HPV 6 and/or 11. HPV-negative RRP biopsies occurred more frequently in adult-onset patients, and were associated with an increased risk of laryngeal neoplasia and carcinoma in the respiratory tract. Recurrent respiratory papillomatosis (RRP) is a rare condition characterized by recurrent growth of benign papillomas in the respiratory tract , most coLR-HPV may, in rare cases, be associated with malignancy, including carcinoma in the anogenital region. The study was approved by the regional ethical committee of South Eastern Norway Regional Authority. For the use of clinical records, histological review and tissue samples obtained from prior diagnostics of these patients at Oslo University Hospital and Lovisenberg Diaconal Hospital, ethical committee waived the need for informed consent. Letter to each patient, or the next of kin, was however required for the information of the use of specified data and linkage to the Norwegian Cancer registry. Patients from all regions of Norway who were treated at the departments of otorhinolaryngology at Oslo University Hospital and Lovisenberg Diaconal Hospital during 1987–2009 were recruited to the study. One biopsy specimen from each patient was selected for blinded histological review according to WHO classification 2005 DNA was extracted from formalin-fixed paraffin-embedded tissue using proteinase K treatment  with subsequent clean-up using the Biorobot M48 . Quantitative polymerase chain reaction (qPCR) assay was performed on the Roche Lightcycler 480 II using Roche probe master  reagents according to the manufacturer’s guidelines. Hydrolysis probes labeled with FAM, HEX and Cy-5 distinguished HPV type 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68a and 68b in six multiplex reactions. PCR assays targeting the conserved region E6/E7 in the HPV genome were used. Human genomic DNA was co-amplified and used as internal control. Following whole genome amplification, DNA from HPV-negative samples was sequenced using the Roche GS FLX 454 , as described elsewhere Numerical and categorical data are presented descriptively. Categorical data were analyzed by Pearson’s χ2 test, applying a significance level of 5%. The relative risk with 95% confidential intervals, for developing high-grade dysplasia (including carcinoma) in HPV-negative compared to HPV-positive patients, were calculated using the software package IBM SPSS 18 (USA).In total, clinical records and histological reports from 238 patients with RRP were reviewed, from which 14 patients were excluded from the study because paraffin blocks were not available. Hence, the study population comprised 224 patients . No significant differences were found in gender, age at diagnosis, duration of observation or development of SIN2+ in the 14 excluded patients compared to the study population. The DNA quality was approved in 221/224 cases (98.7%). Observation time, age at onset and number of surgical procedures are shown in SIN2+ developed in the larynx in 39 patients (17.6%) during the observation period, with a median time to SIN2+ diagnosis of 2.7 years. The occurrence of SIN2+ did not differ significantly between adult-onset and juvenile-onset RRP patients, nor did time to diagnosis. There was a preponderance of SIN2+ in men, which was significant among the adult-onset patients (p = 0.028) . A signiIn total, 696,218 metagenomic sequences were obtained, but only 0.12% of these were initially classified as viral sequences (803 sequence-reads) with 688 reads showing a viral similarity over their entire length. All of these reads were 99% similar to HPV type 8 and came from a single biopsy sample.To our knowledge, this is the largest cohort study in RRP patients to examine the HPV genotype profile and determine whether RRP is associated with the development of laryngeal neoplasia and SCC in the respiratory tract. Moreover, the follow-up time was relatively long, and the cohort included both juvenile-onset and adult-onset RRP patients.All HPV-positive patients were infected by HPV 6 or HPV 11, which is in line with similar studies with the prevalence of HPV 6 or HPV 11 infection to be in the range of 70–98%. We observed a higher rate of HPV 11 infection in children than adults. Similar prevalence of HPV 11 in children has also been observed in previous studies. et alIn our study, 17.6% of patients acquired SIN2+ in the larynx. It is challenging to compare our results with previous studies due to varying terminology and end-points. This finding is compatible with previous studies in which the prevalence ranged from 3% to 7%. The cumulative risk of laryngeal cancer between 2006 and 2010 in the Norwegian population under the age of 75 was 0.3% in males and 0.1% in females et alWe found that a HPV-negative biopsy to be associated with more than a two-fold increase in the risk of developing SIN2+ and almost a 50-fold increase in the risk of SCC in the respiratory tract, when compared to patients with HPV-positive biopsy. A high risk of malignant transformation in HPV-negative patients has also been reported by Lee Almost 100% of cervical cancers are associated with HPV infection. DNA quality was approved in 98.6% of the cases. Since the paraffin blocks were up to 20 years old, a sensitive and robust qPCR method for detecting HPV in paraffin-embedded tissue was used.This method was validated against a test panel in a HPV reference laboratory. Histology was confirmed in all cases before HPV testing and DNA was only extracted from representative tumor tissue selected by an experienced ear, nose and throat pathologist. Thirteen of the 221 available cases (5.9%) were found to be HPV negative papillomas, which is similar to the rates of HPV negativity reported elsewhere in patients with RRP (4.7%) and anogenital condyloma (5.3%). In each patient the highest grade of dysplasia reported during the time of observation was recorded. Only one biopsy from each patient was revised and used for HPV analysis. 63% of the DNA specimens represented the highest grade of dysplasia found in the patients. Stratifying specimens for whether representing the highest grade of dysplasia or not, revealed similar distribution of HPV profile in these two groups. However, our conclusions regarding association of HPV genotype profile to SIN2+ assume a persistent infection of the same HPV genotype during the clinical course, which is a plausible presumption. Our finding that HPV negativity occurred more frequently in older male RRP patients and with a higher malignancy potential is novel. It is possible that this association may be confounded by other risk factors, such as smoking and alcohol consumption, for which we had only partial or no data. Notably, there was only one daily smoker among the laryngeal carcinoma patients in this RRP cohort. The potentially carcinogenic effect of HPV exposure was, comprehensively assessed in all patients by qPCR and metagenomic sequencing. The impact of selection and misclassification bias was reduced by histologic review and by linking data to the Norwegian cancer registry.Analysis of RRP is challenging. It is a rare disease, and hence larger patient cohorts based on multicenter studies are required to confirm our findings.In the vast majority of this cohort of Norwegian patients, RRP was associated with HPV 6 and/or 11. While co-infection with HR-HPV types was present in 8.6% of patients, follow-up data revealed no significant association with the development of high-grade SIN or carcinoma. HPV-negativity was rare, occurred more frequently with adult-onset versus juvenile onset RRP and was associated with a high relative risk of laryngeal neoplasia or carcinoma in the respiratory tract. There is an increased risk of cancer in the respiratory tract in RPP patients compared to the general population in Norway.


Marital satisfaction is one of the main determinants of a family’s correct function. A large number of veterans have been reported to suffer from depression, anxiety, mood disorders, post-traumatic stress disorder, and physical disorders. The objective of this study is to examine association between Illogical thoughts and Dependence on Others and Marriage Compatibility in the Iranian Veterans Exposed to Chemicals in Iran-Iraq War.The present cross-sectional, analytical study was conducted on 200 veterans exposed to chemicals who were covered by the Foundation of Martyrs and Veterans Affairs, Gilangharb, Kermanshah, Iran. The study sample size was determined according to Krejcie and Morgan formula and the subjects were selected through random sampling. The study data were collected using marriage compatibility questionnaire, illogical thoughts questionnaire, and dependence on others questionnaire. The study data were analyzed using the SPSS statistical software (version18). Pearson correlation coefficient, multiple regression, and t-test were used in order to determine the relationships among the variables and compare the means.The findings of the current study revealed no significant relationship between dependence on others, anxious attention, helplessness, avoiding problems, perfectionism, and autonomy and marriage compatibility. However, a significant relationship was found between failure and marriage compatibility.Overall, the findings of the present study showed that the veterans of Gilangharb did not have disorders, but depended on others, particularly their spouses, due to their abnormal physical status. Sometimes, they cannot even do their personal tasks which results in their dependence on others eventually putting the veterans under pressure and stress. Family is the core of each society and the center for maintaining physical and mental health. It also plays a major role in raising the children or future mothers and fathers . A healtNonetheless, marriage among the healthy individuals is different from the veterans. Veterans have a large number of problems and, consequently, marriage is not quite easy for a veteran. In fact, most of the veterans are opposed to marriage due to their lack of financial and spiritual facilities . VeteranIn psychiatry, thought disorder (TD) or formal thought disorder (FTD) refers to disorganized thinking as evidenced by disorganized speech. Specific thought disorders include poverty of speech, tangentiality, illogicality, perseveration, neologism, and thought blocking . Consequently, veterans experience a lot of problems in their marital life and cannot act as a normal individual in their relationships with their wives. Due to getting hurt during the chemical bombardment, such individuals experience problems in compatibility with their environment and this incompatibility is mostly manifested in their relationships with their family members, particularly their wives . They haThe studies conducted on the handicapped individuals and veterans and their social relationships showed that losing occupation highly affected their mental health and marital relationships. In addition, their children experienced behavioral changes mostly depression and educational failure . In the Furthermore, Bernstein and Goldenberg investigated the behavior of the wives of handicapped individuals and veterans regarding illogical beliefs, cognitive error, and unrealistic beliefs. They mentioned that the couples who continued their life with illogical thoughts and unresolved conflicts experienced considerable critical conflicts. These results can also be generalized to the wives of the veterans exposed to chemicals .Considering the role of the family, Thompson believes that a veteran’srelationship with his family and friends is an inseparable part of his life. Disability mostly affects the whole family, can have negative effects on the family’s integrity, and leads to ambiguities and conflicts in roles because the husband stays at home and the wife has to earn their living .After Iran-Iraq war, a large number of individuals in various parts of Iran underwent a lot of damages due to chemical bombardments. Gilangharb was one of the cities exposed to chemical bombardments during this war. Thus, physical as well as mental injuries were vastly detected in this area.In spite of these individuals’ large number of problems, a limited number of studies have been conducted in this regard. Since most of the war veterans were young and had to live a long life with their new condition, understanding their mental, motivational, psychological, marriage, and familial problems are of utmost importance for enhancing their compatibility. However, due to the small number of researches conducted in this regard in Iran, therapists perform trial and error when providing services for these individuals. Iran - Iraq War occurred between 1980 to1988. Gilangharb is a border town that was vulnerable to enemy attack. Thus, the present study aims to investigate the veterans’ compatibility with their wives and dependence on others and some factors such as anxious attention, helplessness, avoiding problems, perfectionism, failure and autonomy affecting marriage compatibility.The present cross-sectional, analytical study was conducted on 200 veterans exposed to chemicals who were covered by the Foundation of Martyrs and Veterans Affairs, Gilangharb, Kermanshah, Iran. The study sample size was determined according to Krejcie and Morgan formula and the subjects were selected through random sampling . The vetThe study data were collected using marriage compatibility questionnaire, illogical thoughts questionnaire, and dependence on others questionnaire.Singh’s marriage compatibility questionnaire (1958) consists of two forms: for husbands and for wives. This questionnaire includes 10 yes/no questions. In addition, the scores range from 0 to 10. Scores 1 and 0 are assigned to positive and negative responses, respectively. The internal consistency of this questionnaire in a 75-subject sample was shown as 0.80-0.90. Besides, after interviewing 400 couples during more than 1 year, the questionnaire was shown to have desirable face and content validity .Jones’ illogical thoughts questionnaire included 100 closed-ended questions in 10 sections which were scored based on a 5-point Likert scale ranging from completely agree to completely disagree. Various studies have shown the questionnaire as a reliable one with Cranach’s alpha>0.7.The last questionnaire used in the study aimed to determine the dependence on others and reflected the respondents’ capabilities, emotions, and behaviors. This questionnaire was designed by Barret, Hirschfield, Goughand Klerman . The depThe study data were analyzed using the SPSS statistical software (v. 18). The relationship between disability percentage, dependence on others, anxious attention, failure, helplessness, avoiding problems, perfectionism, and autonomy and marriage compatibility was investigated. In addition, Pearson correlation coefficient, multiple regression, and t-test were used in order to determine the relationships among the variables and compare the means.The present study was conducted on 200 veterans exposed to chemicals including 102 males (51%) and 98 females (49%). The subjects’ age ranged from 25 to over 50 years and their mean age was 42 years.By t-test determined that the mean difference between the male and female in terms of Dependence on others, Helplessness, Avoiding problems, Perfectionism and failure statistically were significant . Due to As the table depicts, the highest means in both males and females were related to autonomy . On the other hand, the lowest mean was related to avoiding problems in males (17.37) and dependence on others in females (17.58). By t-test determined that the mean difference between the high age and low age in terms of Dependence on others, anxious attention, helplessness, Avoiding problems, Perfectionism and Autonomy statistically were significant. In this study, ages above 40 were considered as high ages and the rest were considered as low ages. The highest means in both high and low age groups were related to autonomy . On the other hand, the lowest mean was related to avoiding problems in high age groups (17.47) and dependence on others in low age groups (17.58) .In this study, more than 40% disability was considered as high disability percentage and the rest were considered as low disability percentage. The highest means were 34.23 and 32.77 in high and low disability percentage groups, respectively. On the other hand, the lowest means were 16.41 and 17.53 in high and low disability percentage groups, respectively . Given tThe findings of the current study revealed no significant relationship between dependence on others, anxious attention, helplessness, avoiding problems, perfectionism, and autonomy and marriage compatibility. However, a significant relationship was found between failure and marriage compatibility by Pearson correlation coefficient (P=0.03) .Age, sex, disability percentage, illogical changes, and dependence on others are independent variables and marriage compatibility is dependent variable.Sex explained 6.1% of marriage compatibility variance which was statistically significant (P=0.001). After entering the emotional dependence variable into the model, it explained 8.3% of marriage compatibility variance. .The most significant effect was related to sex (β=0.26) followed by dependence on others (β=-0.148). According to the results, female gender increased marriage compatibility. In addition, as the emotional dependence increased, marriage compatibility significantly decreased. The study findings revealed a significant correlation among the veterans’ and their wives’ anxiety, hostility, and phobic anxiety. However, no significant relationship was observed between age and disability percentage and marriage compatibility.Considering the problems in various dimensions, 30%, 34%, and 36% of the veterans with above 70% disability were faced with the aforementioned problems at low, average, and high levels, respectively. Yet, after categorizing the problems, 26% of the veterans had little treatment problems, while 44% were faced with a lot of treatment problems. Besides, 46% of the veterans had serious social and cultural problems and 36.2% had a large number of economic problems.The findings of the present study showed that the type of injury was effective in social problems; as the veterans suffered from more types of disabilities; they were faced with a larger number of social problems. Moreover, the number of years the veterans were covered by the Foundation of Martyrs and Veterans Affairs affected their personal and familial problems; the longer the veterans were supported by the organization, the less they were encountered with the above-mentioned problems. Residence status was also effective in the veterans’ problems. In case the veterans lived in their own houses rather than renting a house or living in their father’s house, they were faced with fewer economic problems.The present study aimed to investigate the relationship between marriage compatibility and illogical thoughts and dependence on others among the male and female veterans exposed to chemicals in Gilangharb, Iran. The study results revealed no significant relationship between marriage compatibility and dependence on others. This is in contrast with the study by Thompson showing the veterans’ relationship with their spouses and families as an inseparable part of their lives . Due to In this study, no significant relationship was observed between marriage compatibility and anxious attention, which is on the contrary to the results of the studies by Bernstein and Goldenberg . They asIn this study, no significant association was found between marriage compatibility and helplessness for change, which is in contrast with the results of the study by Ellis . He beliThe findings of the present study showed no significant relationship between avoiding problems and marriage compatibility, which is not in agreement with any of the studies conducted on the issue. This reveals the sufficient satisfaction in the study population. They receive their income from the government and do not expect much. Therefore, they try to avoid problems and not to interfere in others’ affaires. Thus, it can be concluded that the couples solve their own problems which leads to increase in marriage compatibility.In the current study, no significant relationship was observed between marriage compatibility and perfectionism. It should be noted that perfectionism in the society under investigation has its roots in religious beliefs and reveals adherence to traditions. Therefore, perfectionism strengthens marriage compatibility because marriage compatibility and respect to spouse are highly admired in religious beliefs.The present study results revealed a significant relationship between marriage compatibility and failure. The veterans under the present study avoided problems and rejected any change which was against their tradition. Otherwise, they would experience failure which has negative impacts on the couples’ relationships.In this study, no significant relationship was observed between marriage compatibility and autonomy. Similar results were observed in another study . In the In one study conducted on the relationship between illogical thinking and dependence on others and marriage compatibility among non-veteran couples in Mashhad, marriage compatibility was similar in the two sexes. However, the findings of the present study revealed a significant difference between the two sexes regarding marriage compatibility .The studies performed on the veterans’ marriage compatibility in Iran have shown losing occupation as one of the main factors affecting marriage compatibility as well as social and psychological problem (Overall, the findings of the present study showed that the veterans of Gilangharb did not have disorders, but depended on others, particularly their spouses, due to their abnormal physical status. Sometimes, they cannot even do their personal tasks which results in their dependence on others eventually putting the veterans under pressure and stress.Some information could not be accessed because of being secret and most of the information was collected based on the memories of war. These are cross sectional data and the measures used were dated. Iran- Iraq war began about 34 years ago. Moreover, due to the small number of chemical wars compared to other types of wars, a limited number of studies have been conducted on this issue and we faced a great difficulty in reviewing the literature.Supporting the veterans both emotionally and psychologicallyProviding the veterans with occupations according to their disability percentageSupplying facilities including transferAppreciating the veterans’ efforts and considering facilities and rewards for themTimely hospitalization of the veterans and providing them with medicationSince the veterans have been forgotten in some dimensions of the society, it is necessary to provide a condition through advertisement and culturalization so that the veterans would not feel unsupported.


Head and neck squamous cell carcinoma (HNSCC) has a variety of causes. Recently, the human papilloma virus (HPV) has been implicated in the rising incidence of oropharyngeal cancer and has led to variety of studies exploring the differences between HPV-positive and HPV-negative HNSCC. The calcium-activated chloride channel TMEM16A is overexpressed in a variety of cancers, including HNSCC, but whether or not it plays different roles in HPV-positive and HPV-negative HNSCC is unknown. Here, we demonstrate that TMEM16A is preferentially overexpressed in HPV-negative HNSCC and that this overexpression of TMEM16A is associated with decreased patient survival. We also show that TMEM16A expression is decreased in HPV-positive HNSCC at the DNA, RNA, and protein levels in patient samples as well as cell lines. We demonstrate that the lower levels of TMEM16A expression in HPV-positive tumors can be attributed to both a combination of copy number alteration and promoter methylation at the DNA level. Additionally, our cellular data show that HPV-negative cell lines are more dependent on TMEM16A for survival than HPV-positive cell lines. Therefore, we suspect that the down-regulation of TMEM16A in HPV-positive HNSCC makes TMEM16A a poor therapeutic target in HPV-positive HNSCC, but a potentially useful target in HPV-negative HNSCC. Head and neck squamous cell carcinoma (HNSCC) represents the sixth most common cancer in the world and has an overall five-year survival of approximately 50%While HPV-positive and HPV-negative HNSCC are indistinguishable upon initial presentation, there are important distinctions to be made between these two classifications of tumors. For example, HPV-negative HNSCC occurs at various sites in the head and neck, but HPV-positive HNSCC generally arises in the oropharynxTMEM16A gene is located in the chromosomal band 11q13, which is frequently amplified in breast, bladder, esophageal, and head and neck cancersTMEM16A , a calcium-activated chloride channelWhile TMEM16A has been found to be a poor prognostic indicator and a possible therapeutic target in HNSCC, no study has compared the role of TMEM16A in HPV-positive and HPV-negative HNSCC. Here, we show that TMEM16A is overexpressed in HPV-negative HNSCC, but not HPV-positive HNSCC. We demonstrate that overexpression of TMEM16A is associated with poor outcome in patients with HPV-negative tumors, but not necessarily in those with HPV-positive cancer. Additionally, we show that TMEM16A expression is in part governed by methylation in both HPV-negative and HPV-positive tumors, although HPV-positive tumors demonstrate higher levels of promoter methylation. Lastly, we demonstrate that cancer cell lines derived from HPV-negative tumors are more reliant on TMEM16A for survival than HPV-positive cell lines. Taken together, these results demonstrate that TMEM16A may prove to be a novel therapeutic target for HPV-negative, but not HPV-positive, HNSCC.in situ hybridization (FISH) analysis  and fluorescent analysis . In our analysis .TMEM16A gene amplification by FISH. This was done by taking the ratio of TMEM16A signal intensity  to CEP11 intensity (centromere-to-nucleus ratio). A total of 44 HPV-positive and 20 HPV-negative tumors had this data available. HPV-negative tumors had a significantly higher TMEM16A/CEP11 ratio than HPV-positive tumors , chi-squared analysis showed that 55% of HPV-negative tumors showed TMEM16A amplification, while only 7% of HPV-positive tumors showed it . Stratification of TCGA data revealed 36 HPV-positive tumors and 243 HPV-negative tumors. The mRNA Z-Score is defined as the relative mRNA expression of an individual gene to the gene’s expression distribution in a reference population. In this case, the reference population consists of tumors of the same type (i.e. HNSCC) in TCGA that are known to be diploid for TMEM16A. When comparing mRNA Z-Scores by t-test, HPV-negative tumors were found to express significantly more TMEM16A mRNA than HPV+ tumors  in TCGA are calculated using log2 ratios of a tumor’s DNA to a reference normal DNA. A log2 ratio of 0 indicates no up-regulation (i.e. diploid), and a positive log2 ratio is considered gain, with a value of 1 representing a full doubling of copy number. When comparing log2 ratios, HPV-negative tumors showed significantly more CNA for TMEM16A than HPV-positive tumors , we found that 28.3% of HPV-negative tumors showed TMEM16A amplification, while only 5.5% of HPV-positive tumors showed it  of a single CPG island within the promoter region of the We found similar results in our own institution’s tumor samples, in which 18 of the HPV-positive and seven of the HPV-negative tumors could be analyzed by qMSP as described below. These samples were also compared to 12 normal pharynx specimens. Pairwise comparison revealed that the level of methylation in HPV-negative tumors (mean = 0.2243 a.u.) was significantly less than that of HPV-positive tumors  for 93-VU-147T  data clearly indicates that TMEM16A is significantly more highly expressed in HPV-negative HNSCC than in HPV-positive. Previous studies have shown that overexpression of TMEM16A is associated with decreased survival in HNSCCTMEM16A gene has significantly less promoter methylation in HPV-negative tumors in both TCGA and our institution’s TMA. This difference in methylation, in combination with the difference in gene amplification, helps to explain why TMEM16A is more highly expressed in HPV-negative HNSCC.TMEM16A expression has been shown to be regulated by promoter methylationin vitro, we saw the importance of TMEM16A for survival in HPV-negative cell lines. We found concurring results when inhibiting TMEM16A either with CaCCinh, a small molecule inhibitor, or by knockdown of TMEM16A with either doxycycline-induced shRNA or siRNA. The HPV-negative cell lines, FaDu and PE/CA-PJ34, were much more sensitive to CaCCinh than the HPV-positive cell lines, SCC90 and 93-VU-147T. Using colony formation assays, we found that knockdown of TMEM16A did not alter colony formation in the HPV-positive 93-VU-147T cells, but did decrease colony formation in HPV-negative FaDu and PE/CA-PJ34 cells. Similarly, overexpression of TMEM16A in HPV-positive SCC90 cells did not confer a survival benefit, showing that this is not a protein that promotes survival in HPV-positive tumors.When working with HPV-negative and HPV-positive cell lines TMEM16A compared to HPV-positive ones; 4) HPV-negative cell lines are more sensitive to pharmacologic inhibition of TMEM16A than HPV-positive cell lines; 5) Knockdown of TMEM16A decreases colony formation in HPV-negative cell lines but not HPV-positive cell lines; and 6) TMEM16A overexpression does not confer an increase in colony formation in HPV-positive cell lines. Taken together, these results suggest that TMEM16A may be a viable target for therapy in HPV-negative tumors, but not necessarily for head and neck squamous cell carcinoma that has been caused by HPV.In conclusion, we have shown that TMEM16A plays a more important role in HPV-negative HNSCC than in HPV-positive HNSCC. We have determined that 1) HPV-negative tumors express more TMEM16A at the DNA, mRNA, and protein level; 2) only patients with HPV-negative HNSCC have decreased survival when TMEM16A is overexpressed; 3) HPV-negative tumors have decreased promoter methylation of et al.All experiments were carried out after obtaining approval from the University of Pittsburgh Institutional Review Board, and all experiments were performed in accordance with guidelines set out by that institution. Before obtaining primary tissue samples, informed consent was obtained from each subject. As described by Shiwarski in situ hybridization (FISH) studies were carried out on the TMA samples using a probe for the centromere of chromosome 11 (CEP11) labeled with SpectrumGreen (Abbott Molecular) and a probe prepared from a BAC clone  and labeled by nick translation with SpectrumOrangeFluorescence http://cancergenome.nih.gov/), parameters of 279 HNSCC patient samples and associated clinical data were analyzed. The data were accessed using the cBioPortal14Using the Head and Neck Squamous Cell Carcinoma in Revision database from The Cancer Genome Atlas (TCGA)  to generate completely methylated DNA. Serial dilutions (30–0.003 ng) of this bisulfite-treated methylated DNA were used to construct a calibration curve. All data points were within the range of sensitivity and reproducibility of the assay based on the calibration curve. The methylation levels in each sample were determined as a ratio of qMSP-amplified gene to β-actin (reference gene) and then multiplied by 1000 for easier tabulation .Leukocyte DNA from a healthy individual was methylated 2. Survival analysis was performed using the Kaplan-Meier method and log-rank testing. For cell viability assays, IC50 was calculated by constraining maximal response to 100% and minimal response to 0%. For colony formation assays, all areas are reported with the control group normalized to 1.Statistical analysis was performed using GraphPad Prism 5. All data are reported as mean ± SEM unless stated otherwise. For continuous variables, t-tests and ANOVA were performed where appropriate. For categorical data, chi-squared test was used to determine signifance. Correlations were calculated using Pearson’s rHow to cite this article: Dixit, R. et al. TMEM16A/ANO1 is differentially expressed in HPV-negative versus HPV-positive head and neck squamous cell carcinoma through promoter methylation. Sci. Rep.5, 16657; doi: 10.1038/srep16657 (2015).


We observed that the HPV16 integration sites were broadly located across the whole viral genome. In addition, either single or multiple integration events could occur frequently for HPV16, ranging from 1 to 19 per sample. The viral integration sites were distributed across almost all the chromosomes, except chromosome 22. All the cervical cancer cases harboring more than four HPV16 integration sites showed clinical diagnosis of stage III carcinoma. A significant enrichment of overlapping nucleotides shared between the human genome and HPV genome at integration breakpoints was observed, indicating that it may play an important role in the HPV integration process. The results expand on knowledge from previous findings on HPV16 and HPV18 integration sites and allow a better understanding of the molecular basis of the pathogenesis of cervical carcinoma.Integration of human papillomavirus (HPV) DNA into the host genome can be a driver mutation in cervical carcinoma. Identification of HPV integration at base resolution has been a longstanding technical challenge, largely due to sensitivity masking by HPV in episomes or concatenated forms. The aim was to enhance the understanding of the precise localization of HPV integration sites using an innovative strategy. Using HPV capture technology combined with next generation sequencing, HPV prevalence and the exact integration sites of the HPV DNA in 47 primary cervical cancer samples and 2 cell lines were investigated. A total of 117 unique HPV integration sites were identified, including HPV16 ( Cervical carcinoma is one of the most commonly occurring cancers in women worldwide , 2, whilStudies have shown a significant correlation between integration and progression of cervical dysplastic lesions to invasive carcinomas. In pre-invasive lesions, HPV16 integration has already been found to occur in cervical intraepithelial neoplasia (CIN) –10. AlthDetermining the DNA sequence of viral-cellular junctions would therefore indicate most directly HPV integration, as attempted in a number of previous studies where genomic HPV integration sites had been analyzed via many different assays. However, the precise identification of tiny stretches against a background of massive amount of episomal forms is an ongoing technical challenge. In addition, due to the nature of integration, which may be incorporated either as a single or multiple numbers of concatenated form, the latter can actually hinder integration detection . Thus, dHere, in order to contribute to the understanding of the presence and the precise localization of HPV integration sites in cervical cancer, we studied a large series of cases using base-resolution genome-wide HPV capture technology with the next generation sequencing. More specifically, we simultaneously evaluate HPV prevalence and detailed characterization of the exact integration sites of the HPV DNA into the human genome in 47 primary cervical cancer samples and 2 cell lines.In this study, 49 DNA samples including 47 from fresh-frozen cervical carcinomas and 2 cervical cancer cell lines (SiHa and HeLa) were detected using the method of HPV capture combined with next generation sequencing .n = 4), 58 (n = 5), 45 (n = 1), 31 (n = 1). Two samples in this analysis harbored both types of HPV, HPV16 and HPV18 in T35, HPV18 and HPV58 in T9, respectively. However, in T35, the sequencing depth of HPV16 is 34-fold greater than that of HPV18, and in T9, the sequencing depth of HPV18 is 13 fold greater than that of HPV58. Therefore, only HPV16 in T35 and HPV18 in T9 were analyzed in subsequent HPV assay. SiHa was positive for HPV16, and HeLa was positive for HPV18 . Of the 47 cancer samples, we were able to capture HPV DNA in 39 samples. Of these 39 samples, 30 were positive for HPV16. In addition to HPV 16, we also detected HPV18 , with frequencies ranging from 1 to 59 per sample . The pren = 101), HPV18 (n = 7), and HPV58 (n = 9) , 47.8% (22/46), 44.4% (4/9), and 83.3% (85/102), respectively . The seq9) Table  and S2; SiHa and HeLa are cell lines with defined integration sites, which provide an invaluable standard for validation of diagnostic assays . Consistn = 5), E7 (n = 6), E1 (n = 31), E1/E2 (n = 2), E2 (n = 20), E2/E4 (n = 7), E5 (n = 1), L2 (n = 15), L2/L1 (n = 4), L1 (n = 3), and LCR (n = 7)  Table . Due to 7) Table , the mosn = 99 sites)  Figure . Altogetn = 2), E2 (n = 3), L1 (n = 1) and LCR (n = 1) region of HPV genome , E7 (n = 1), E1 (n = 3), E2 (n = 1), L1 (n = 2), and LCR (n = 1)  Table .n = 4), 9p22.3 in T38 (n = 6), 11p15.4 in T34 (n = 4), 20p12.1 in T38 (n = 6), 20q11.22 in T23 (n = 6), and 20q13.2 in T11 (n = 4). In the second group, some integrant clusters arose from at least three individual cases, and they may represent authentic hot spots, such as 13q22.1 in SiHa (n = 2), T15 (n = 1), and T43 (n = 2). In the last group, the clusters showed both of the aforementioned properties, such as 8q24.21 in HeLa (n = 4), T34 (n = 7), and T35 (n = 3) , including overlapping, inserting some unaligned nucleotides, and direct ligation . OverlapAll integration loci were checked for the presence of fragile sites. Of the 117 integration loci identified, 69 (59%) were located in or close to a fragile site : 20  of the 117 confirmed HPV integration sites were located within cellular genes, and 54 cellular DNA breakpoints were located in introns and one breakpoint was located in exon . MoreoveP = 0.435). In consideration of HPV integration status, we found that in 13 HPV DNA-positive patients treated with radiotherapy and chemotherapy before surgery, HPV integrations were not detected in 10 patients, including 8 patients positive for HPV16 and 1 patient positive for HPV45 and 1 patient positive for HPV58. Altogether, in 17 patients with cervical cancer who had received radiotherapy and chemotherapy before surgery, 82.4% (14/17) showed HPV negative or no HPV integration sites  in discriminating HPV mRNAs derived from integrated and episomal viral genomes , 20, ResHPV capture in this study was designed based on probes from full-length HPV genome of 17 types. Thus, the HPV subtypes and HPV DNA integration sites were simultaneously detected in 49 cervical cancer samples (47 primary tumors and 2 cell lines). Out of which 82.9% of the 47 cervical cancer cases was HPV DNA positive, including HPV16(63.8%), HPV18(8.5%), HPV58(10.6%), HPV45 (2.1%), and HPV31 (2.1%). These percentages are relatively well aligned with existing findings –26, thatThe sensitivity of the HPV capture combined with sequencing can be estimated from the results obtained from the well characterized cervical carcinoma cell lines SiHa and HeLa. SiHa, a HPV16-positive cervical cell line, harbors only one HPV16 DNA copy per cell , 28. TwoWith this strategy, 117 unique and validated HPV integration sites and high-quality nucleotide sequences of the viral-cellular junctions were obtained. Of the 30 HPV16 DNA-positive cervical cancer samples, 63% (19/30) harbored HPV integration sites, similar to previously reported percentages , 29, indIt is worthy to note that our data, obtained from 101 validated HPV16 integration sites including 2 integration sites from SiHa cell line, indicated that all the regions of HPV genome can be targeted for the linkage to cellular sequences, even E6 and E7. Doubt arises on the feasibility of the widely used E2:E6/E7 ratio by real-time PCR amplification to determine the physical status of HPV infection, which is based on the assumption that the viral E2 gene is frequently disrupted upon HPV integration, and the HPV E6 and E7 open reading frames are almost always retained, whereas other portions of the HPV genome may be deleted . In contin vitro and its reported clinical association with more aggressive cervical cancers [Of the 4 HPV18 DNA-positive cervical carcinomas including HeLa, 100% (4/4) harbored HPV integration sites. This percentage conforms to the estimation of 100% of HPV18-positive cervical carcinomas harboring integrated viral DNA by Southern analysis . The hig cancers .PCR and Sanger sequencing were used to verify the HPV integration breakpoints based on one or more discordant paired-end reads. Although the low validation rate for one to three discordant paired-end reads may arise from technical error or integration occurred in a small subset of cancer cells, practically, the criterion using more than three discordant read pairs can cover most breakpoints identified (85/117). It should be noted that no explanation could sufficiently capture the failure of the remaining 16.7% of integration sites for validation based on more than three discordant reads, such as the number of discordant paired-end reads, and the proportion of repetitive sequence in the human genome. Additional research is warranted to reduce the false positive rate.Viral integration within fragile sites has previously been reported in several studies , 35. FraIn total, 55 (47%) of the 117 confirmed HPV integration sites were located within cellular genes, most frequently in intronic regions. Moreover, several cellular genes existed in two or more unique HPV integration sites in the current study, including CACNG7, CAPG, HDAC4, TP63, CAGE1, PVT1, MACROD2, and NFIB. HPV integration into an intron will probably disturb expression of the targeted cellular gene. However, only few examples exist, because a direct link between HPV integration and gene alteration must be verified by functional data. TP63, a member of the p53 protein family, acts as a tumor suppressor protein and aberrant expression was noted for several cancer entities including cervical cancer , 37, supIn our data, overlapping was the most prevalent pattern accounting for 67% of the 117 validated viral-cellular sequences. A significant enrichment of overlapping nucleotides shared between the human genome and HPV genome at integration breakpoints was observed, indicating that it may play a vital role in the HPV integration process.Additionally, we observed that the prevalence of HPV DNA in 17 patients who had been treated with radiotherapy and chemotherapy before surgery was lower than that in the group of 30 patients without the pre-operative procedures, but no statistically significant difference was found. While considering the status of HPV integration, we found that 82.4% showed negative for HPV or an absence of HPV integration sites in 17 patients with cervical cancer who had received pre-operative radiotherapy and chemotherapy. These results might indicate that radiotherapy and chemotherapy before surgery had some impact on HPV in cervical carcinoma, especially on HPV integration. A plausible explanation for the decrease of HPV integration is that these cells have been more effectively cleared by treatment. Additionally, we found that all the cervical cancer cases harboring more than four HPV16 integration sites showed clinical features and diagnosis of stage III carcinoma, indicating that the complexity of HPV integration might be associated with advanced cervical cancers.Taken together, the method of HPV capture combined with sequencing allow a better analysis of multiple HPV subtypes at the same time and to identify the precise nucleotide sequences at the viral-cellular junctions as well as the affected cellular genes. The high number of 117 validated unique HPV integration sites deduced in the study demonstrates the effective performance of this strategy at single-nucleotide resolution. HPV integration is a special pattern of cancer mutation, which has the well-established potential to affect the cellular genes involved in cancer carcinogenesis. Therefore, identification of HPV integration in cervical cancers can provide new insight into the understanding of the cancer genome projects and its role in the pathogenesis of cervical carcinoma. Furthermore, because of the intrinsic random distribution of HPV integration sites, the efficient determination of HPV integration sites would allow them to be served as individualized markers in cervical cancer screening and in the follow-up of patients for the early detection of recurrent disease and metastasis in the future.A total of 47 fresh tissue specimens were collected from patients with cervical cancers who had undergone surgeries at Anyang Cancer Hospital, Henan province, China, between 2009 and 2010. Median age of these patients was 51 years with range 29 to 81 years. Among whom 42 had squamous cell carcinoma, 3 had adenocarcinoma and 2 adenosquamous carcinoma. The HPV16- and HPV18-positive human cervical carcinoma cell lines SiHa and HeLa with defined integration sites served as positive control. SiHa and HeLa cell lines had been obtained from AACT many years ago, and were often used for research purposes. Before undergoing experimentation in this study, these two cell lines had been identified using a PCR-based short tandem repeat (STR) genotyping method.Individual informed consents had been collected from all study participants. This study received ethical approval from the Institutional Review Board of the hospital.DNA was extracted using DNeasy Blood & Tissue kit  according to the manufacturer's protocol. The DNA concentration was determined via a Nano-Drop .In order to establish the whole genomic library with the targeted gene specifically, a stretch of 3~5 μg genomic DNA was sheared to about 150 bp fragments by Covaris S2. These fragments were subsequently end blunted, “A” tailed, adaptor ligated and amplified 10 cycles by PCR. Out of which 1 μl of the prepared library samples was quantified using Nanodrop 2000, and 3 μl identified in 2% agarose gel and the final size of the electrophoresis fragment was around 300bp~500 bp.HPV probes were designed according to full- length genome of 17 HPV types  by MyGenostics . The overall experiment was conducted according to the manufacturer's protocol. In brief, the whole-genomic libraries were hybridized with HPV probes (MyGenostics GenCap Technology), adsorbed onto the beads via biotin and streptavidin magnetic beads, and the uncaptured DNA fragments were removed by washing. Then the eluted fragments containing the targeted gene were enriched by 18 cycles of PCR to generate libraries for sequencing. Libraries were quantified and sequenced for paired-end 100bp using the Illumina HiSeq 2000 sequencer .The 100 bp paired-end reads were preceded into bioinformatics analysis. For quality control, we first filtered low quality reads using the Trim Galore program. Then, 3′/5′ adaptors were trimmed using the Cutadapt program implemented in Trim Galore, thereby rendering high quality clean reads, whose quality value exceeds 20 and read length greater than 80 bp. The high quality reads were obtained for subsequent analysis.Illumina clean reads were mapped to human genome (GRCh37/hg19) and HPV genome of 17 types  using the BWA program. The quality scores were recalibrated and realigned to reference sequences using the GATK software package. For the potential HPV integration sites in the human genome, we aligned the Illumina clean reads to the reference human genome hg19 using the BWA program, and simultaneously performing recalibration and realignment with GATK. The paired-end read, uniquely mapped with one end to a human chromosome and the other to the HPV reference genome, is identified as a discordant read pair. If a specific position has one or more discordant read pairs, it would be considered as a potential HPV integration site and the breakpoints of which were then identified using the BreakDancer program. The integration sites were annotated according to human genome (GRCh37/hg19) from the UCSC database and HPV genome.The sample was considered HPV DNA positive when the average sequencing depth was more than 10 and more than 50% of the virus genome's total length was covered by at least 4x.The average sequencing depth was calculated based on sequencing tags containing HPV sequences and according to the following formula: Sequencing Depth = Total HPV bases/genome size. Genome size here means the size of the specific HPV genome.To validate the potential HPV integration sites, we designed primers to amplify regions of interest by PCR. PCR products were sequenced directly from both directions using conventional Sanger sequencing. If unsuccessful, the integration sites were identified by TA cloning of PCR products and sequencing. All sequences of the fusion genes were characterized by the NCBI human mega Blast database alignment tool and the UCSC Blat database.P-value of less than 0.05 was considered as statistically significant. All the P-values presented are two-sided.Fisher's exact test was used for statistical analysis in the present study.