Considering the limited cross protection offered by the current HPV vaccines, understanding the HPV genotype distribution among the different population is essential in predicting the efficacy of current vaccine and devising new vaccine strategy. The present work aimed at investigating the HPV genotypes distribution among women with and without cervical carcinoma in Odisha, Eastern India.n = 210), inflammatory smear (n = 162), normal cytology (n = 68). Statistical analyses were performed by using SPSS version 20.0 software and MediCal version 14.10.2(7). A p-value of ≤ 0.05 was considered statistically significant.A total of 607 participants have been enrolled between January 2014 and June 2016. L1-PCR, sequencing, and E6/E7 nested multiplex type- specific PCR were performed for HPV detection and genotyping. Cytological distribution of 440 cases includes invasive cervical carcinoma or ICC  60.33%. Prevalence of HPV infection was 93.80% (197/210) in invasive cervical cancer (ICC) cases, 54.32% (88/162) in inflammatory smear and 19.11% (13/68) in normal cervical cytology. The most prevalent genotype was HPV16 (87.28%) followed by HPV18 (24.56%) and HPV 51(3.46%). The overall prevalence of single type was 76.58% and highest (78.9%) among ICC cases. The most frequent genotype combination after HPV16 + 18(9.4%) was HPV16 + 66 + 68(2.7%) which was frequently observed in inflammatory cytology. Age > 45years, parity ≥3, low socio-economic condition, rural residential area and post menopause state were significantly associated with HPV infection. Multiple infections did not have a significant association with any of the clinicopathological variables  except tumor size ≥ 2cm in ICC cases. The impact of 2v, 4v, and 9v vaccines in preventing cervical cancer in Odisha were 89.99, 91.65, and 92.16% respectively.This data would help planning an appropriate strategy for disease monitoring and provides baseline data for post-vaccination surveillance in the region. The nonavalent vaccine would be significant in preventing cervical carcinoma in Odisha. Hence an effective vaccination program based on regional HPV epidemiological profile along with the cervical cancer screening is necessary to reduce the cervical cancer burden in India.The online version of this article (doi:10.1186/s12879-016-2136-4) contains supplementary material, which is available to authorized users. Cervical cancer is the fourth most common cancer in women and seventh most common cancer among all the known group of cancers found worldwide . 528,000. Available reports from studies covering few parts of India show a wide variation in the prevalence of HPV infection and genotypes distribution which is attributed to diversified socio-economic and geo-climatic condition [HPV infection plays a central role in causing cervical cancer. Among 184 different HPV genotypes, only 40 diverse types can infect anogenital region which can be classified into 3 classes based on their oncogenic potential. HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82 are included in high-risk group while HPV6, 11, 40, 42, 43, 44, 54, 61, 70, 72 and 81 are included in low-risk group whereas HPV 26, 53 and 66 belong to the group of intermediate risk [Here we determined the genotypes, prevalence and associated risk factors among women in Odisha with and without cervical cancer.Acharya Hari Hara Regional Cancer Center and SCB medical college, Cuttack, Odisha, the two apex referral hospitals of the state were considered to enroll subjects and cervical sample collection. This study was conducted between January 2014 and June 2016. Married women, above 18 years showing any of the symptoms like abnormal vaginal bleeding/discharge, pain during coitus, lower abdominal pain and clinician suspicion of cervical malignancy were included in the study after clinical examination by a gynecologist. Unmarried women, pregnant cases and patients undergoing treatment were excluded. Subjects were enrolled after getting informed written consent from them. This study is approved by the ethics committee of Regional medical research center (ICMR), Bhubaneswar, Odisha, India.Clinical data including signs and symptoms and socio-demographic data such as age, education, economic status, age at marriage, the age of menopause were collected by interviewing the patients with a predesigned questionnaire.Cervical swab specimen was collected using cytobrush and stored inside the viral transport media (Hi-Media) and transported to Virology laboratory, Regional medical research center, Bhubaneswar, Odisha for further analysis. Pap smears were prepared from the collected cervical sample for cytological analysis. Cytological classification was done according to Bethesda system . Part ofDNA was extracted from a 200μl aliquot of exfoliated suspended cell samples using the QIA amp DNA Blood Mini Kit (Qiagen) as per the manufacturer’s instructions. Amplification of the human β-globin gene was performed to test sample sufficiency. To detect HPV, PCR was performed by using PGMY09/PGMY11 primers  targeting a 450-bp region of the HPV L1 gene.http://www.ncbi.nlm.nih.gov/BLAST/).L1 PCR products were isolated from agarose gel and purified. Sequencing was done using Big Dye Terminator sequencing kit   and analPCR followed by sequencing didn’t resolve multiple infections. Hence, E6/E7 type-specific PCRs were performed to confirm multiple types. Type-specific primers were used to detect 18 HPV genotypes that include HPV16, HPV18, HPV31, HPV 33, HPV 35, HPV39, HPV 56, HPV 59, HPV 45, HPV 51, HPV 52, HPV 58, HPV 66, and HPV 68, HPV 6/11, HPV 42, HPV 43, and HPV 44 genotypes. Primers were obtained by following the available literature . The whoBriefly,50 μl PCR reaction mixture containing, 10X Taq buffer with Mgcl2 , 250mM dNTP , 5U Taq DNA polymerase , 20 pmol of each primer and 50ng of DNA was processed for the first PCR cycle. The cycling parameters were composed of 4 min of an initial denaturation step at 94 °C, followed by 35 amplification cycle. Each cycle includes the 30s of denaturation at 94 °C, 30s of annealing at 56 °C, elongation of 72 °C for 45s and a final extension of 4 min at 72 °C. Nested PCR reaction was same as the first PCR except for primers and sample. 2 μl of the PCR product was used for the nested PCRs. 10μl of the amplification products was analyzed by electrophoresis on 2% agarose gels stained with ethidium bromide followed by visualizing under UV light.p-value of ≤ 0.05 was considered as statistically significant.Multivariate logistic regression analysis was performed to determine the risk factors associated with HPV infection. Bivariate analysis was done to determine the variables associated with multiple infections in cervical cancer cases. Statistical analyses were performed by using SPSS version 20.0 software and MediCal version 14.10.2 . A p-valLow and a high estimate of vaccine impact were Calculated by using the following formula:For the quadrivalent vaccine, low estimate was the prevalence of HPV 6/11/16/18 genotypes alone or in association but excluding presence of another HPV type; high estimate was the prevalence of HPV 6/11/16/18 genotypes alone or in association possibly in presence of another HPV type. For the nonavalent vaccine, low estimate was the prevalence of HPV 6/11/16/18/31/33/45/52/58 genotypes alone or in association but excluding presence of another HPV type; high estimate was the prevalence of HPV 6/11/16/18/31/33/45/52/58 genotypes alone or in association possibly in presence of another HPV type . To estiProportion of additional cases potentially prevented by one vaccine compared to the other vaccine was calculated as per the previously published study .n = 68), cervicities (n = 162), invasive carcinoma (n = 210).Enrollment of cases and outcomes were presented in Fig. Socio-demographic features of 595 subjects are presented in Table The common clinical features recorded were abnormal discharge with or without blood stain (55%), postmenopausal bleeding (20%), bleeding and pain during coitus (2%), lower abdominal pain (17%), intermenstrual bleeding (14%), prolapse (6%), and swelling abdomen (1%).n = 9), IIA (n = 8), IIB (=38), IIIB (n = 111), IIIA (n = 2) and IVA (n = 4). The histopathological result of 172 cases showed that 167 cases belonged to Squamous cell carcinoma (SCC) and only 5 cases identified with Adenocarcinoma (ADC). Based on the available data for 89 cases, only 30 cases had tumor size ≥2 cm and 59 cases had <2cm. Similarly, out of 86 available reports 30 cases had lymph node metastasis whereas 56 cases didn’t have lymph node metastasis , ICC cases were classified into different stages which include IB  60.33%. Prevalence of HPV infection was 93.80% 197/210) in invasive cervical cancer (ICC) cases, 54.3% (88/162) in inflammatory smear and 19.11% (13/68) in normal cases  followed by HPV18 (24.56%) , inflammatory smear (n = 88) and ICC (n = 185)  was the most predominant genotype followed by HPV 18(28.4%). Other genotypes detected among this group were HPV 66(6.8%), HPV 68(6.8%), HPV51 (3.4%) and HPV35 (2.27%)  followed by HPV 18(21.08%) and HPV 51(5.4%)  among women aged >55 years , 152(82.6%) and156 (84.32%) respectively. High estimates of genotypes targeted by 2v, 4v, and 9v vaccines were 181(97.83%), 183(98.91%) and 185(100%) respectively. Absolute impact of 2v, 4v and 9v were 89.99, 91.65 and 92.16% respectively. The additional impact of 9v was increased when compared with 4v and 2v (Table vs >50 years), stage at diagnosis (early stage or stage I-II vs late stage or stage III-IV), lymph node metastasis (yes vs no) and histopathology (Squamous cell carcinoma vs Adenocarcinoma). Multiple types were found to be significantly associated with tumor size ≥ 2cm (OR = 4.95 95% CI = 1.68–14.51 p = .0035)  and 1821.08%) were the most predominant genotypes which is quite similar to the studies reported from India and worldwide. In Kolkata, 59–74% of ICC cases are infected with HPV 16 and 2–13.9% cases infected with HPV 18 8% and 18. Other p.08% wereHPV 51 was found to be the 3rd most predominant genotype in the present study, though it is rarely reported in other regions of the country and worldwide. Data on the prevalence and distribution of three most prominent genotypes in the cancer cases from different geographical regions of India shows a great regional variation , 15–17.HPV 66, an intermediate risk genotype, had a prevalence of about 3% and a predominant type in cervicitis cases in the present study is rare in other population. The presence of HPV66 across all age groups and predominantly in cervicitis might indicate its possible role in the development of cervical cancer by triggering inflammation and persistent infection.Low-risk HPV infection is a rare event in this population. Low-risk infection was observed in ICC cases only and always present in association with the high-risk genotypes. It remains unclear whether the association of low-risk genotypes with the high risk induces the progression of lesion or is it the effect of high-risk HPV that makes prone to the infection of other types.The present study showed highest infection rate at the age of ≤35 years among women without cancer. The age-specific trend of HPV infection among noncancer cases showed bimodal shaped infection peak which is similar to many other reports . HPV infAge specific multiple HPV genotype infection curve for ICC cases shows a bimodal peak. Highest peak is at > 55 years and another shift is at 36–45 years  was found in the present work which agrees with the findings of Mungala et al . PrognosThe rate of single genotype infection especially HPV 16, was very high in the abnormal histopathological group as compared to the normal group which is in accordance with Baloch et al . Hence, People staying in a rural area had 20-fold higher risk of acquiring HPV infection. Rural residential area reflects poor socio-economic condition possibly related to lack of access to proper care which facilitates infection and persistence of HPV and an increasing risk of cancer development . In ruraApproximately, more than 70% worldwide ICC could be prevented by the available bivalent HPV vaccines-CervarixTM  and quadrivalent HPV vaccine- Gardasil® (4vHPV)  , 27. TheTo know the impact of a vaccine, estimating the attribution of cases to HPV types is confusing due to multiple genotypes present in the same tumor. In the present study high and low estimate of the impact of vaccines has been estimated to overcome this difficulty. Since the true potential impact lies somewhere between the high and low estimate  the averHPV51, the third most prevalent genotype, and HPV66 another more frequent genotype of this region can’t be cross protected even by the new 9v vaccine for being genetically unrelated. The risk of development of invasive cervical carcinoma associated with these genotypes needs to be estimated.We were unable to assess HIV status or another disease for our participants, which could strongly influence the prevalence of HPV infection.The present study provides first information about the genotype distribution among women with cervical cancer and without cancer cases of the state of Odisha which would help planning an appropriate strategy for disease monitoring. This study also offers the baseline data for future research and post-vaccination surveillance in the region. The nonavalent vaccine would be the most effective vaccine to prevent cervical carcinoma in Odisha. Hence an effective vaccination program based on regional HPV epidemiological profile along with the cervical cancer screening is necessary to reduce the cervical cancer burden in India. Besides, HPV surveillance targeting elderly women and rural people will be useful.


Background: Head and neck (H&N) squamous cell carcinoma (SCC) is a significant contributor to worldwide mortality and morbidity. Human papillomavirus (HPV) has been linked with H&N cancer and HPV-positive H&N SCC have been shown to have better survival outcomes.Objective: To evaluate the effect of human papillomavirus (HPV) on laryngeal carcinoma (LSCC) survival outcomes and prognosis.Method: A systematic review and meta-analysis were performed using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. LSCC was confirmed based on histopathology, and HPV status was confirmed by either polymerase chain reaction, immunohistochemistry, and/or in-situ hybridization.Results: There were 1214 studies which were identified, of which 14 studies were eligible for our review. A total of 2,578 cases of LSCC were included in analysis with 413 (16.0%) HPV-positive. Overall survival (OS) was not significant for HPV-positive LSCC in first five years . Disease-free survival (DFS) was similarly not significant for HPV-positive LSCC . When studies are sub-divided into global regions, Chinese studies had better HPV-positive survival compared to North American studies in year five .Conclusion: This is the first study of its kind to evaluate the survival impact of HPV-positive LSCC patients. Unlike oropharyngeal cancer, HPV status does not make a difference to OS or DFS in LSCC. This supports data that HPV is not a prognostic factor in squamous carcinoma of the larynx. Squamous cell carcinoma of the head and neck is a significant contributor to worldwide morbidity and mortality. There are more than 500,000 new cases worldwide reported annually . LaryngeThere is great variation in reporting the incidence of HPV-positive LSCC in literature, however, a recent meta-analysis reports incidence at roughly 25% with minor variation depending on methods of testing . While iThe aim of this study is to conduct a systematic review and meta-analysis to evaluate and quantify the effect of human papillomavirus (HPV) on survival outcome of LSCC and to assess regional variation across the globe.Search strategy and selection criteria:Using the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines, this study was conducted . ElectroUtilising both MeSH terms and keywords, the search was conducted to ensure ideal sensitivity. The terms “larynx”, “squamous cell carcinoma” and “human papilloma virus (HPV)” along with their relevant variations were used. Relevant studies were identified by two independent reviewers (Na.A. and Ni.A.) and were extracted for further evaluations.Inclusion criteria included histologically confirmed LSCC and HPV-DNA or p16 status was confirmed by polymerase chain reaction (PCR), immunohistochemistry (IHC), and/or in-situ hybridization (ISH). Studies were excluded if they did not provide overall survival (OS) or disease-free survival (DFS) comparing HPV-positive and HPV-negative LSCC. Search was limited to papers with minimum length of follow up of one year, English language, and human subjects. Abstracts, case-reports, conference presentation and any papers without original data were excluded.Data extraction and critical appraisal:Two independent investigators (Na.A. and Ni.A.) extracted data, with discrepancies between reviewers resolved by consensus. The senior investigators (N.S. and R.C.) reviewed final results.Statistical analysis:2 statistics to measure percentage variability among summary indices [Meta-analysis was performed with Review Manager  using pooled OS and DFS of HPV-positive and HPV-negative LSCC . Data wa indices -12. StatThrough the search of the nine databases, 1214 studies were identified. After excluding studies with duplicated cohorts, irrelevant studies, and studies with data not extractable, 14 studies were analyzed for statistical analysis. All the studies were case-control with two prospective studies, while the remaining 12 were retrospective. Bias was assessed using Egger’s test for publication bias and no publication bias was identified . PRISMA 2=75%). Majority of LSCC were smokers  and alcohol drinkers . Also, most of LSCC were well differentiated (61.2%) and only the minority were poorly differentiated (15.7%). In terms of tumor classification, there was an even spread between the grades . Most of the LSCC had no regional node involvement (58.6%). Most patients were treated with the combination of radiotherapy and/or chemotherapy (42.4%), while 30.9% had surgery alone and 26.7% had surgery with chemotherapy and/or radiotherapy  cases positive for HPV with 2051 (91.6%) male. HPV rates differed greatly between regions, as is summarised in Table 2=46%, year four: I2=57%, year five: I2=66%). Similarly, there is no difference noted in DFS in the first five years post-diagnosis. There is also significant heterogeneity in third- and fifth-year post-diagnosis . Similarly, there was no significance between DFS in the context of HPV  compared to North America in the same year . This demonstrates a significant difference noted between the regions in fifth-year post-diagnosis (p=0.04). Looking at heterogeneity within regions, there is a significant heterogeneity noted in third- to fifth-year post-diagnosis . The results are summarised in Table The pathogenesis of HPV and its effect on patient treatment and survival outcomes is an area of great importance in head and neck oncology. This study evaluates the current literature with regards to the pathogenesis of HPV as it relates specifically to laryngeal cancer and suggests that unlike in oropharyngeal cancer, HPV in the larynx has little to no effect on survival outcome.HPV consists of double-stranded DNA genomes with more than 100 genotypes, of which more than 24 genotypes are detected in head and neck cancers -17. HPV HPV has a propensity for tumorigenesis in the tonsillar crypts, which are deep recesses with a discontinuous basement membrane, and vasculature, which may explain the early propensity for lymphovascular invasion and early regional nodal spread -22. ThisWhile there is a large body of literature looking at HPV in oropharyngeal cancer, little research has focused on laryngeal pathology with regards to HPV. In this study, we demonstrate that there is no difference in survival in the context of OS and DFS based on HPV status. This suggests that HPV is not a significant driver of LSCC, and as such HPV status has limited clinical relevance in LSCC.Recent meta-analysis by Ndiaye and colleagues showed HPV prevalence of LSCC is 22.1% 95% CI 16.4-28.3) while another meta-analysis demonstrated that the prevalence was 26.8% or 20.3% depending on whether PCR-testing for HPV was utilized or not .4-28.3 w. This isAnother explanation for the different prevalence of HPV is the heterogeneity between different regional populations. The prevalence HPV-positive LSCC of Chinese cohort is 10%, while the prevalence of the rest of the world is 20.3%. While others have shown similar heterogeneity, their reported prevalence is different to our study. Ndiaye, et al. (2014) show the prevalence of HPV is 28.3% in Asia and 18.8% in North America, while Gama, et al. 2016) show point estimate of 0.246 (95%CI: 0.201-0.298) in North America to 0.399 (95%CI: 0.323-0.479) [ show poiThere was no difference noted between HPV-positive and HPV-negative LSCC in terms OS and DFS. However, there was significant heterogeneity noted in studies. As mentioned previously, the AJCC now considers HPV-positive and HPV-negative OSCC different disease entities . This isThere is significant regional variation in LSCC survival. This is most pronounced in the fifth year post-diagnosis, where there is a significant difference between the stratified cohorts Table . As mentLimitationsThis meta-analysis is limited by the number of studies with adequate data to allow for sub-analysis of outcomes and further multivariate analysis. There were also a limited number of studies from various regions of the world, with North American and Chinese studies accounting for most of the cohort of our meta-analysis. Further research is required to better assess survival outcome of LSCC based on HPV status.This is the first meta-analysis to compare HPV-positive LSCC with smoking and alcohol-related LSCC and compare survival outcomes. This is the largest laryngeal meta-analysis looking at survival outcomes in laryngeal SCC with 2578 cases. Even though the study showed heterogeneity across regions in the world, particularly North America and China, the study showed that unlike oropharyngeal squamous cell carcinoma, HPV-positive LSCC does not have a better survival outcome compared to smoking and alcohol-related LSCC.


While the majority of veteran suicides involve firearms, no contemporary data describing firearm ownership among US veterans are available. This study uses survey data to describe the prevalence of firearm ownership among a nationally representative sample of veterans, as well as veterans’ reasons for firearm ownership.A cross-sectional, nationally representative web-based survey conducted in 2015.Nearly half of all veterans own one or more firearms , with male veterans more commonly owning firearms than do female veterans . Most veteran firearm owners own both handguns and long guns ; a majority cite protection as a primary reason for firearm ownership .The current study is the first to provide detailed, nationally representative information about firearm ownership among U.S. veterans. Better understanding firearm ownership among veterans can usefully inform ongoing suicide prevention efforts aiming to facilitate lethal means safety among vulnerable veterans during at-risk periods.The online version of this article (10.1186/s40621-017-0130-y) contains supplementary material, which is available to authorized users. Veterans of the United States (U.S.) Armed Forces accounted for nearly 20% of U.S. adult suicides in 2014   was approved by the Institutional Review Board at Northeastern University. The NFS is a cross-sectional, web-based survey conducted by the survey research firm Growth for Knowledge (GfK) in January 2015. The sample was drawn from Knowledge Panel, an online sampling frame of approximately 55,000 U.S. adults selected using address-based sampling methods maintained by GfK. Participants are selected on an ongoing basis using equal probability sampling. For the purposes of this study, veterans and firearm owners were oversampled to ensure reliable national estimates when conducting pre-planned subpopulation analyses. Active duty military personnel were excluded from participation. Of the 7318 panel members invited to complete the survey, 4165 started and 3949 completed the survey . Study-specific analytic weights provided by GfK incorporate base weights for Knowledge Panel that adjust the sample to the most recent U.S. Census Current Population Survey (2010) GfK, , and posInvitations to participate in the study were sent by e-mail. One reminder e-mail was sent to non-responders three days later. GfK has a modest point-based incentive program through which participants accrue points for completing surveys and can later redeem them for cash, merchandise, or participation in sweepstakes.Survey items assessed respondents’ sociodemographic characteristics, political views, history of military service in the U.S. Armed Forces and, for those who served, time period of service, and service branch. Veterans were asked about their use of health care services in the last 12 months and categorized by whether they received no, some, or all of that care from the Veterans Health Administration (VHA). We describe veterans discharged from the military in 2002 or later as “recent veterans”. This group encompasses former military personnel who served in the era of Operations Iraqi Freedom, Enduring Freedom, and New Dawn (OIF/OEF/OND).Firearm ownership status was assessed first by asking “Do you or does anyone you live with currently own any type of gun?” If the answer was affirmative, the question “Do you personally own a gun?” followed. Gun owners were asked about the number and type of gun(s) they own . Firearm owners were asked whether they owned a gun primarily for protection. In addition, for each type of gun that they owned, they were asked to indicate their reasons for ownership (i.e. why they own their handgun(s), why they own their long gun(s)).All analyses were conducted using STATA 14.1 .A total of 1044 veterans of the US Armed Forces completed the 2015 National Firearm Survey and were included in this study. The overall survey completion rate was 54.6%. Further details regarding the overall study population have been previously published   than those who are currently or were previously married . Veterans with children under the age of 18 in their household less commonly own a firearm than those without children . Veterans who grew up without a firearm in their household also less often own a gun than those who grew up with a gun in the home .More than half of veterans living in Southern states own a firearm . In contrast, approximately two in five veterans living in other regions own firearms. Indeed, veterans residing in the South account for nearly half of all veteran firearm owners . Prevalence of firearm ownership does not differ substantially by service branch, service era, or use of VHA health care services. When the analysis was restricted to male veterans only, no important differences were identified , while 22.6% report owning only handguns (95% CI 18.1–27.8%) and 20.6% own only long guns   than do non-veterans (20%) in the general population   and those using VHA services may be important, findings from our survey may nevertheless be informative for the VHA given that veterans engaged in VHA care are those most likely to benefit from its services.By design, the demographic characteristics of veterans responding to our survey closely mirror the distribution of characteristics among the overall veteran population (National Center for Veterans Analysis and Statistics,  Westat, . Though Findings from our survey should be interpreted in light of several limitations. Some gun owners may have chosen not to report their firearm ownership on a survey, particularly if they own firearms illegally. Nevertheless, fewer than 1% of NFS respondents refused to answer the initial question about the presence of a gun in the household, and none refused the subsequent question about whether they personally own a gun. While all data are self-reported, the relatively anonymous nature of the online format mitigates social desirability bias and likely yields more accurate estimates of respondent characteristics than comparable telephone surveys (Chang et al., The current study presents the most comprehensive data available regarding firearm ownership patterns among veterans of the United States Armed Forces. Our finding that a larger proportion of male and female veterans own firearms than do male and female non-veterans is consistent with the fact that the proportion of suicides involving firearms among veterans is considerably higher than that among non-veterans (Office of Suicide Prevention,


Prostatic blue nevus is a rare benign pathologic diagnosis most commonly diagnosed incidentally on many different types of prostate specimens. Blue nevus is the deposition of stromal melanin characterized by spindle cells within the fibromuscular stroma which stains positive for melanin-specific stains Fontana-Masson and S100 and stains negative for CD68, HMB45, and iron stains. We report the case of a multifocal and bilateral blue nevus in a 52-year-old Hispanic male who presented with an elevated prostate-specific antigen of 4.3 and mild obstructive lower urinary tract symptoms, found by transrectal ultrasound-guided prostate needle biopsy. The biopsy also revealed benign prostatic tissue with postatrophic hyperplasia and chronic inflammation. This is the 35th reported case of prostatic blue nevus and the third to show multifocal blue nevus.  Melanocytic lesions are an often-overlooked pathologic process that occurs in the prostate. Of these extremely rare lesions, the most commonly found is the prostatic blue nevus, also known as pigmented melanocytosis or prostatic pigmentary nevohyperplasia . Blue ne3 (PSA density of 0.10 ng/mL/g). Final pathology demonstrated blue nevus in one out of six cores on the right and two out of six cores on the left. On microscopic analysis with hematoxylin-eosin stain, individual heavily pigmented spindle cells distributed in between prostatic stroma and glands were noted  of 4.1 along with mild obstructive lower urinary tract symptoms. There was no family history of prostate cancer. The physical examination including the digital rectal examination was unremarkable. The patient was seen again 3 months later with a PSA of 4.3 and after discussion with the patient he elected to undergo a 12-core TRUS PNBx. The following month when the biopsy was performed, the PSA had slightly decreased to 3.4. The prostate was visualized in the sagittal and transverse planes via ultrasound probe and was unremarkable. Volume was measured to be 33 cmBlue nevus is a rare lesion of dermal melanocytes most commonly found in the skin, but it has been reported in the oral mucosa, sclera, cervix, vagina, and prostate . The appBlue nevus grossly appears as multiple brown to black streaks or nodules that range in size from 0.1 cm to 2.0 cm . The celReview of the literature indicates that blue nevus typically presents as a single focus and is characteristically diagnosed on TURP and prostatectomy specimens. Although no risk factors for blue nevus have been identified, our discovery of just the second case in a Hispanic male may suggest variability in risk among different races/ethnicities . Of the


DcR1 gene promoter hyper-methylation status as well as whether did or not an association between patterns of DNA hypermethylation and high-risk HPV infection, led to risk of cervical cancer.The infection of human papillomavirus (HPV) has been considered as the common cause of cervical cancer, which is the leading cause of cancer death in women, in Vietnam. Recently, hypermethylation at tumor suppressor genes (TSGs) has been also demonstrated to be an early epigenetic event and cofactor in human cancer, including cancer of cervix. This study evaluated the frequency of Methylation-Specific-PCR (MSP) was performed to analyze hypermethylation status from 109 liquid-based Papanicolaou test samples, archived and admitted from the Medic Medical Center and Au Lac Clinic Laboratory, Vietnam, from 2011–2014, a kind of non-invasive samples identified whether HPV/or non-HPV, high-risk/low-risk HPV infection.DcR1 promoter was differentially methylated in 50% cases of high-risk HPV genotype 16 and 18 infected samples. In contrast, a low frequency of hypermethylated DcR1 promoter was found in low risk HPV genotype infected sample (16.0%), and non-HPV infected sample (14.6%). A trend toward positive association was found between hypermethylation of DcR1 gene and HPV exposure was observed (P=0.0005). Moreover, the odds ratio (OR) and relative risk (RR) were found in statistical significant value , RR=3.31 ).DcR1 gene promoter is a significant characteristic of high-risk HPV infected samples in Vietnamese cervical patients. The OR and RR values showed that the strong correlation between DcR1 hypermethylation and high-risk HPV infection, in which increased the risk of cervical cancer. The combination of DcR1 hypermethylation and HPV detection based biomarker could be used in noninvasive samples obtained from high-risk cancer patients, offer significant practical advantages.The hypermethylation of  DcR1 promoter methylation in cervical cancer, especially, in Vietnamese cervical cancer patients. Thus, in order to determine whether promoter hypermethylation profile in Vietnam, the data on DcR1 hypermethylation was evaluated and investigated in a series of clinical HPV infected samples collected from Vietnamese patients were enrolled in current study to develop method for prognosis and early diagnosis of cervical cancer based on the detection of DcR1 methylation status.DcR1 (Decoy receptor 1), also known as TNFRSF10C (Tumor necrosis factor receptor superfamily member 10C), locates on 8p21.3, is homologous of Fas and tumor necrosis factor receptor 1 (TNFR1) trigger apoptosis upon engagement by their cognate death ligands. DcR1 receptor contains an extracellular TRAIL-binding domain and a transmembrane domain, while completely lacks the intracellular death domain, hence is not capable of inducing apoptosis. Additionally, DcR1 has been found to be a p53-regulated DNA damage-inducible gene. The specific sensitivity of cancer cells to the TRIAL apoptosis-inducing activity may be interpreted by the observation of DcR1 expression in many non-cancerous cells , had more advantages and easily developed for non-invasive method applied in Vietnamese cervical cancer patients’ early diagnosis and prognosis based on DNA methylation-specific PCR  and deaths were 265672 cases ASR = 6.8/100000) . All samples were divided into two groups: negative HPV infection group, which consisted of 48 samples; and positive HPV infection group, in which composed of 36 high-risk HPV  infected samples and 25 low-risk HPV infected samples.260 and OD280. The pure preparation of DNA with OD260/OD280 ratio values of 1.8 to 2.0 was used to the bisulfite DNA modification assay. The bisulfite modification was carried out with approximately 2 μg genomic DNA of each sample by DNA modification Kit . The final precipitate was eluted in a volume of 20 μl for MSP assay. Two pairs of primer were used to amplify the regions of interest. One pair recognized a sequence in which CpG sites were methylated (unmodified by bisulfite treatment). Other pair recognized a sequence in which CpG sites were unmethylated (modified to UpG treatment). The primer sequences and X°C for each annealing temperatures were noted in Total of genomic DNA was isolated from PAP samples by phenol/chloroform method. Then, DNA concentration of DNA was quantified by the absorbance at ODMSP assay was carried out in a total of 15 μl containing 3 μl bisulfite-modified template DNA, 0.75 unit iTaq DNA polymerase (Biorad), 0.5 μM each primer, 7.5 μl MyTaqTM Mix (Bioline). Thermal cycling was initiated at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 30 sec, annealing at the X°C for 30 sec, extension at 72 °C for 30 sec, and a final extension at 72 °C for 10 min . The PCR products were run on 2% agarose gel with visualized by ethidium bromide staining. Then, MSP products were sequenced to confirm the specificity of primers, examine the efficiency of bisulfite modification and the hypermethylation status of target gene.DcR1 and HPV infected status were examined by using Chi-square test. The differences in methylation frequencies of DcR1. Moreover, the association between hypermethylation of DcR1 and risk of cervical cancer was estimated by computing OR, RR and 95% confidence intervals (CI).Statistical analyses were performed by Medcalc® Version 12.7.0.0. The average frequency of methylation was calculated. The association between hypermethylation of DcR1 in clinical samples was evaluated by MSP method. Overall, the promoter frequencies for DcR1 in high-risk HPV, low-risk HPV, and non-HPV infected samples were 50.0% (18 of 36 samples), 16.0% (4 of 25 samples), 14.6% (7 of 48 samples), respectively. Conversely, the promoter unmethylation frequencies were 50.0% (18 of 36 samples), 84.0% (21 of 25 samples) and 85.4% (41 of 48 samples) in high-risk HPV, low-risk HPV, and non-HPV infected samples, respectively. Methylation of DcR1 in high-risk HPV infected samples was found to be significantly higher than other two groups (P=0.0005). The MSP products of samples hypermethylation and/or unmethylation in the promoter of DcR1 were observed in electrophoresis with visualized by ethidium bromide staining and showed in DcR1 in clinical samples were observed in the band of 127 bps and 136 bps length in case of methylation and unmethylation, respectively. The sequencing of samples hypermethylated promoter region of representative sample revealed a conversion of unmethylated Cytosine, but not methylated Cytosine  and 3.31 , respectively.The odds ratio and relative risk values were computed between high-risk HPV infected group and low-risk HPV group combined with non-HPV infected group. The results show that odds ratio, relative risk values were 5.63  test sample by MSP method. Ideally, liquid-based Papanicolaou (Pap) sample, a non-invasive sample, contains exfoliating cells from the transformation zone of the cervix, thus, the presence of DNA in exfoliating cells has offered an opportunity to find out the predictive and prognostic biomarkers for cervical cancer , compared to low-risk HPV infected samples, counting for 16.0% (4 of 25 samples) and non-HPV infected samples, counting for 14.6% (7 of 48 samples). The hypermethylation of DcR1 gene was strongly associated to the HPV infection, particularly, high-risk HPV type infected, here, the infection of HPV 16 and/or 18 high-risk genotype infection were mentioned (P=0.0005). The viral infection has been already proven that it correlated to the epigenetic alteration, especially, the DNA hyper-methylation, in human cervical cancer. The high-risk HPV infection, such as type 16 and 18 infections, is the common cause of cervical cancer development. Additionally, the epigenetic factors, such as hypermethylation, have been suggested as contributing mechanisms to cervical tumorigenesis . Concerning to RR value, high-risk HPV infection significantly increased 3.31 times in the risk of DcR1 hyper-methylation, leading to the inactivation of DcR1 .The hypermethylation of DcR1 gene promoter were significant association and contribution to the risk and development of cervical cancer. Moreover, MSP method for DcR1 hypermethylation detection and HPV typing in Pap test samples, non-invasive clinical samples, could be considered as the promising candidate, non-invasive biomarkers that could be potentially used for diagnosis and prognostic purposes in Vietnam.Therefore, due to our data, identification of HPV high-risk genotype, HPV 16 and HPV 18 geno-type mentioned, and the hypermethylation of DcR1 was commonly found in the high-risk HPV genotype infection, counting for 50.0%, compared to low high-risk HPV infection or non-viral infection. Additionally, the significant association between DcR1 gene hypermethylation and HPV exposure as well as the odds ratio and relative risk were found in the significant correlation, counting for 5.63 and 3.31, respectively were reported. In addition, the screening, which based on the detection of DcR1 promoter hypermethylation, which combined the HPV genotyping, on non-invasive method, will offer the potential method for predictive and prognostic biomarkers for human cervical cancer in Vietnamese population. In further study, the present findings require extension to a larger number of samples and series on many potential genes in order to get the profile of methylated genes related to cancer of cervix.The promoter hypermethylation of Ethical issues  have been completely observed by the authors.


INK4αp16 gene’s promoter and HPV exposure in non-invasive samples (liquid-based papanicolaous samples) in Vietnamese population.Human papillomavirus (HPV) infection has been considered as main cause of cervical cancer. Recently, aberrant DNA methylation at tumor suppressor genes (TSGs), leading to inactivation, has also been an early epigenetic event and cofactor in cervical carcinogenesis. This study was performed to evaluate an association between the hypermethylation of 109 liquid-based papanicolaous test samples were archived and admitted from the Medic Medical Center and Au Lac Clinic Laboratory, Vietnam, from 2011–2014. Methylation-Specific-PCR (MSP) was performed to analyze methylation status from the liquid-based papanicolaous test samples identified whether HPV/or non-HPV, high-risk/low-risk HPV infection.INK4αp16 hypermethylation frequency in high-risk HPV infection, counting for 55.6%, and the low methylation frequency in low-risk and non-HPV infected samples, counting for 22.9%, 8.0%, respectively. The significant correlation between candidate INK4αp16 hypermethylation and HPV exposure was observed (P<0.0001). Moreover, the odds ratio (OR) and relative risk (RR) were found in statistical significant value. .An upward trend was observed concerning the INK4αp16 hypermethylation was the specific characteristic of high-risk HPV infected samples in Vietnamese population. The OR and RR values showed that the strong correlation between INK4αp16 hypermethylation and high-risk HPV infection, in which increased the risk of cervical cancer. The combination of INK4αp16 hyper-methylation and HPV detection based biomarker could be used in non-invasive samples obtained from high-risk cancer patients, offer significant practical advantages.Presence of  Current experimental and clinical data point out high-risk HPV (human papillomavirus) genotype infection, especially HPV 16 and/or 18, play a key role in the etiology of cervical cancer . AlthougINK4αp16 TSGs  is located at 9p21. Its encoded protein functions as a specific inhibitor of cyclin-dependent kinase 4 (cdk4) and cyclin-dependent kinase 6 (cdk6), that initiates D-dependent phosphorylation of the retinoblastoma tumor suppressor protein (Rb)  –18. Hypel cancer , 19–21,.To date, almost limited researches were carried on to provide whether or not an association between patterns of DNA hypermethylation and high-risk HPV infection, led to risk of cervical cancer. A better understanding of those principles will provide the most favorable to prognosis and diagnosis of cervical cancer.INK4αp16 promoter methylation in Vietnamese population and assess the association between INK4αp16 promoter methylation and HPV 16 and/or 18 genotype infection. Notably, the Papanicolaou (Pap) samples, kind of non-invasive samples, collected from Vietnamese cervical cancer patients, enrolled in current study to develop non-invasive method for prognosis and early diagnosis of cervical cancer based on the detection of INK4αp16 methylation status.Here, we performed an MSP (methyl-specific PCR) method to evaluate the frequency of We used anonymized routine specimen surplus to clinical requirements for assay validation, adhering to a governance framework agreed by and with a Medic Medical Center and Au Lac Clinic Laboratory ethics agreement relating to the use of specimen surplus to clinical needs.Totally, 109 Pap test samples, from 2011–2014, were archived and admitted from the Medic Medical Center and Au Lac Clinic Laboratory, Vietnam. All the samples were collected from patient’s diagnosis with HPV infection LightPoweriVA HPV genotype PCR-RDB Kit  was applied in HPV detection and genotyping. Subsequently, all the samples were divided into groups: non-HPV infection consisted of 48 samples; and positive-HPV infection, in which composed of 36 high-risk HPV  infected samples and 25 low-risk HPV infected samples.A total of genomic DNA was isolated from Pap samples by phenol/chloroform method. Cells obtained from Pap samples were lysed in lysis buffer  containing Proteinase K (0.1 mg/ml). Then, total of genomic DNA was isolated and purified by using standard phenol-chloroform and ethanol precipitation. The bisulfite conversion of 2 μg genomic DNA was performed using EpiTect Bisulfite Kits (Qiagen). The final precipitation was eluted in a volume of 20 μl and stored at −20 °C for further studies.Two pairs of primer were used to amplify the regions of interest. One pair recognized a sequence in which CpG sites were methylated (unmodified by bisulfite treatment).Other pair recognized a sequence in which CpG sites were unmethylated (modified to UpG treatment). The primer sequences and X °C for each annealing temperatures were noted in TM Mix (Bioline). Thermal cycling was initiated at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 30 sec, annealing at the X °C for 30 sec, extension at 72 °C for 30 sec, and a final extension at 72 °C for 10 min . The PCR products were run on 2% agarose gel with visualized by ethidium bromide staining. Then, MSP products were sequenced to confirm the specificity of primers, examine the efficiency of bisulfite modification and the hypermethylation status of target gene.MSP assay was carried out in a total of 15 μl containing 3 μl bisulfite-modified template DNA, 0.75 unit iTaq DNA polymerase (Biorad), 0.5 μM each primer, 7.5 μl MyTaqINK4αp16 and HPV infected status were examined by using Chi-square test. The differences of methylation frequencies of INK4αp16 among HPV infection status were considered statistically significant at P≤0.05. Moreover, the association between hypermethylation of INK4αp16 and risk of cervical cancer was estimated by computing OR, RR and 95% confidence intervals (CI).Statistical analyses were performed by Medcalc® ver. 12.7.0.0. The average frequency of methylation was calculated. The association between hypermethylation of INK4αp16 in 48 non-HPV infected samples; 36 high-risk HPV infected samples and 25 low-risks HPV infected samples were examined by MSP. Overall, the promoter frequencies for INK4αp16 in high-risk HPV, low-risk HPV, and non-HPV infected samples were 55.6% (20 of 36 samples), 8.0% (2 of 25 samples), 22.9% (11 of 48 samples), respectively. Conversely, the promoter unmethylation frequencies were 44.4% (16 of 36 samples), 92.0% (23 of 25 samples) and 77.1% (37 of 48 samples) in high-risk HPV, low-risk HPV, and non-HPV infected samples, respectively. Methylation of INK4αp16 in high-risk HPV infected samples was found to be significantly higher than other two groups (P<0.0001) (The frequency of the promoter methylation of <0.0001) .INK4αp16 were observed in electrophoresis with visualized by ethidium bromide staining and showed in INK4αp16 in clinical samples were observed in the band of 149 bps and 150 bps length in case of methylation and unmethylation, respectively. The sequencing of samples hypermethylated promoter region of representative sample revealed a conversion of unmethylated Cytosine, but not methylated Cytosine  and 3.11 , respectively.The odds ratio and relative risk values were computed between high-risk HPV infected group and low-risk HPV group combined with non-HPV infected group. The results show that odds ratio, relative risk values were 5.76  when compared to a previous study, which pointed out the hypermethylation frequency of INK4αp16 reached to 71.4% (55 of 77 cases) (INK4αp16 (P<0.0001). In fact, many studies have been already correlated viral exposure to epigenetic alteration in various human cancers, including cervical cancer. High-risk HPV  infection is necessary to cervical cancer development, only infection is not sufficient to cause of cervical cancer. Recently, growing evidence proved that epigenetic factors, such as hypermethylation, have been suggested as contributing mechanisms to cervical tumorigenesis  . Similarigenesis , 30. Virigenesis . Thus, hINK4αp16 in cervical patients was significantly associated with an approximately 5.76-fold increase in the high-risk HPV infected group than compared to low-risk and/or non-HPV infected group . This result was according to the study, which pointed out the OR=5.82 . Concerning to RR value, high-risk HPV infection significantly increased 3.11 times in the risk of INK4αp16 hypermethylation, leading to the inactivation of INK4αp16 .The hypermethylation of INK4αp16 gene by contributing the risk of cervical cancer development. The MSP method for INK4αp16 hypermethylation detection and HPV typing in Pap test samples could be considered as the promising candidate, non-invasive biomarkers potentially used for diagnosis and prognostic purposes in Vietnam.Therefore, identification of HPV high-risk  and hypermethylation of INK4αp16 promoter hypermethylation in high-risk HPV infected samples, counting for 55.6%. On the contrary, the low frequency of INK4αp16 promoter hypermethylation was found in low-risk and non-HPV infected samples. Additionally, the significant correlation between candidate gene hypermethylation and HPV exposure as well as the odds ratio and relative risk were found in the significant correlation, counting for 5.76 and 3.11, respectively were reported. The screening, which based on the combination of both high-risk HPV detection and INK4αp16 gene’s promoter methylation, will be an auspicious characteristic for early prognosis and diagnosis of cervical cancer. Moreover, MSP assay done in candidate gene on the non-invasive samples (liquid-based Pap test) would provide the potential method, easily applied in clinic, to prognosis and early diagnosis of cervical cancer in Vietnamese population. In further study, the present findings require extension to a larger number of samples and series on many potential genes in order to get the profile of methylated genes related to cancer of cervix.A higher prevalence of Ethical issues  have been completely observed by the authors.


HPV) infection for most of their lives. The duration of protection against HPV‐16/18 from prophylactic vaccination remains unknown. We investigated the 10‐year immune response and long‐term safety profile of the HPV‐16/18 AS04‐adjuvanted vaccine (AS04‐HPV‐16/18 vaccine) in females aged between 15 and 55 years at first vaccination. Females who received primary vaccination with three doses of AS04‐HPV‐16/18 vaccine in the primary phase‐III study (NCT00196937) were invited to attend annual evaluations for long‐term immunogenicity and safety. Anti‐HPV‐16/18 antibodies in serum and cervico‐vaginal secretions (CVS) were measured using enzyme‐linked immunosorbent assay (ELISA). Serious adverse events (SAEs) were recorded throughout the follow‐up period. Seropositivity rates for anti‐HPV‐16 remained high (≥96.3%) in all age groups 10 years after first vaccination. It was found that 99.2% of 15–25‐year olds remained seropositive for anti‐HPV‐18 compared to 93.7% and 83.8% of 26–45‐year olds and 45–55‐year olds, respectively. Geometric mean titers (GMT) remained above natural infection levels in all age groups. Anti‐HPV‐16 and anti‐HPV‐18 titers were at least 5.3‐fold and 3.1‐fold higher than titers observed after natural infection, respectively, and were predicted to persist above natural infection levels for ≥30 years in all age groups. At Year 10, anti‐HPV‐16/18 antibody titers in subjects aged 15–25 years remained above plateau levels observed in previous studies. Correlation coefficients for antibody titers in serum and CVS were 0.64 (anti‐HPV‐16) and 0.38 (anti‐HPV‐18). This study concluded that vaccinated females aged 15–55 years elicited sustained immunogenicity with an acceptable safety profile up to 10 years after primary vaccination, suggesting long‐term protection against HPV.Women remain at risk of human papillomavirus ( Persistent infection with an oncogenic human papillomavirus (HPV) type is a prerequisite for developing cervical cancer (CC) Cervarix, GSK, Belgium) is licensed for administration from the age of 9 years in most countries, with an upper age limit varying from 25 years to no limit The AS04‐HPV‐16/18 vaccine . All subjects who received three doses of the vaccine in the primary study were invited to return for annual immunogenicity and safety evaluations from Year 5 onward. This study was designed to provide 10‐year follow‐up of immunogenicity and safety (NCT00947115) in these subjects. Females who missed an annual visit remained eligible for inclusion at a later time point.Subjects were not enrolled in the study if they had received previous vaccination against HPV other than the AS04‐HPV‐16/18 vaccine, or chronic administration of immunosuppressants, immunoglobulins, or blood products less than 3 months prior to blood sampling.The study was conducted in accordance with the Declaration of Helsinki, principles of Good Clinical Practice (GCP), and all applicable regulatory requirements. Written informed consent was obtained from each subject before conducting any study‐specific procedures.At each annual visit , a 10‐mL sample of whole blood was taken from each subject and CVS samples were also collected in consenting subjects who volunteered for this additional procedure as already described in the primary publication Long‐term safety of the AS04‐HPV‐16/18 vaccine up to 10 years after first vaccination was evaluated. The analysis of safety was based on the TVC comprising all vaccinated subjects who received three doses of the AS04‐HPV‐16/18 vaccine in the primary study and participated in the Year‐10 extension study.A serious adverse event (SAE) was defined as any untoward medical event that resulted in death, was life‐threatening, required hospitalization or prolongation of existing hospitalization, or resulted in disability/incapacity. Medical or scientific judgment was exercised in deciding whether reporting was appropriate in other situations such as important medical events that may not have been immediately life‐threatening or resulted in death or hospitalization but may have jeopardized the subject or may have required medical or surgical intervention. In such cases, these events were also considered serious. Any vaccine‐related, study participation‐related, or GSK concomitant medication‐related SAEs or fatal SAEs were recorded throughout the study period.The primary endpoint was the evaluation of long‐term immunogenicity of the HPV‐16/18 vaccine in serum from all vaccinated subjects by ELISA at each time point  following the first vaccine dose. The according‐to‐protocol (ATP) cohort for analysis of immunogenicity included all evaluable subjects, that is, subjects included in the ATP immunogenicity analysis during the primary study (NCT00196937), who met all eligibility criteria, complied with the study procedures defined in the protocol, had no elimination criteria during the study and in whom serology results were available.μL were excluded from antibody testing as they were considered to be contaminated with blood. The analysis of immunogenicity of CVS samples was based on the total vaccinated cohort (TVC) that was comprised of subjects who volunteered for CVS sampling and for whom CVS results were available. Anti‐HPV‐16/18 antibody titers were determined in both serum and CVS samples. Since antibody levels fluctuate during menstrual cycles, individual anti‐HPV‐16 and anti‐HPV‐18 titers were divided by the total IgG concentration, to evaluate the correlation between vaccine‐induced IgG titers in serum and CVS Seropositivity rates and geometric mean titers (GMTs) were calculated with exact 95% confidence intervals (CIs) for anti‐HPV‐16/18 antibodies. Secondary analyses compared the immune response of subjects to the AS04‐HPV‐16/18 vaccine in this study NCT00947115) with the immunogenicity plateau levels measured in the efficacy study (NCT00689741) and associated long‐term follow‐up studies   with thePost hoc mathematical modeling was conducted to evaluate long‐term immunogenicity against HPV‐16 and HPV‐18 for at least 30 years after vaccination. Two different mixed effects models were used, piecewise and modified power‐law (Fraser) Of 666 subjects from Germany and Poland who received three doses of the AS04‐HPV‐16/18 vaccine in the primary study, 524 participated in the trial and were included in the TVC cohort for safety analysis. Among them, 470 completed the Year 10 assessment, that is, Year 10 TVC, and 451 were included in the Year 10 ATP cohort for immunogenicity Fig. . SubjectSeropositivity rates for anti‐HPV‐16 remained high (≥96.3%) in all age groups 10 years after first vaccination  compared with the 26–45 and 15–25‐year age groups, that is, 334.4 EU/mL  and 965.4 EU/mL , respectively [non‐overlapping CIs]. A similar pattern of change was seen with serum HPV‐18 antibody levels as shown in Table In subjects vaccinated at the age of 15–25, anti‐HPV‐16 antibody titers at Year 10 remained above the plateau levels observed in previous efficacy studies .At Year 10, anti‐HPV‐16 antibody titers in initially seronegative subjects from the Y10 ATP cohort for immunogenicity were 2.31‐fold  higher in the 15–25‐year age group, 0.80‐fold lower  in the 26–45‐year age group and 0.38‐fold lower  in the 46–55‐year age group compared to those measured at the end of the 113‐month (≈ 9.4‐year) follow‐up in subjects vaccinated at the age of 15–25 years in the efficacy and long‐term follow‐up studies. Anti‐HPV‐18 antibody titers were found to be 1.32‐fold higher  in the 15–25‐year age group, 0.48‐fold lower  in the 26–45‐year age group, and 0.29‐fold lower  in the 46–55‐year age group as compared to the same reference. In older age groups, that is, 26–45 and 46–55, antibody titers were similar to or below the plateau levels for HPV‐16 and HPV‐18 antigens , 57.5 (n = 23), and 53.8% (n = 14) of CVS samples of women from the 15–25, 26–45, and 46–55‐year age groups, respectively. Anti‐HPV‐18 antibodies were detected in 39.0, 45.0, and 34.6% of the CVS samples, respectively.At Year 10, 107 women  provided CVS samples with <200 erythrocytes/Overall, correlation coefficients for anti‐HPV‐16 and anti‐HPV‐18 antibody titers between serum and CVS samples were 0.64 and 0.38, respectively.The results of extrapolating anti‐HPV‐16 and anti‐HPV‐18 antibody titers to at least 30 years after the first vaccine dose are shown in Figure The modified power‐law model predicted that anti‐HPV‐16 levels would remain above the natural infection level lifelong in females aged 15–45 years and for up to 31.4 years in those aged 46–55 years. Similarly, this model projected that anti‐HPV‐18 levels would remain above the natural infection level lifelong in females aged 15–25, while the predicted time that 95% of women would still have anti‐HPV‐18 levels above natural infection levels was 25.9 and 3.58 years in the 26–45 and 46–55‐year age groups, respectively  on biopsy indicated the presence of high‐risk HPV although the type causing the lesion could not be identified (PCR mix). The subject was HPV‐16 seropositive and of unknown DNA status at baseline. This event had resolved by the end of the study. Table .This is the first study to examine the 10‐year immune persistence and safety of the AS04‐HPV‐16/18 vaccine administered to women up to 55 years of age.Ten years after receiving the first vaccination, most subjects were still seropositive for HPV‐16/18 in all age groups. Antibody kinetics had similar profiles across all age groups, displaying a decline in GMT values with increasing ages at first vaccination. As previously observed in the same study, a more or less potent correlation was observed between the anti‐HPV‐16/18 antibody levels in the serum and CVS samples, further supporting the previous observation of long‐lasting transudation of serum antibodies across the cervical epithelium, although this association was not as strong as that seen at the Year 6 time point and was quite weak for HPV‐18, presumably as a result of a smaller number of evaluable samples at the Year 10 time point The long‐term safety profile of the vaccine was clinically acceptable. The occurrence of cervical dysplasia was of unclear clinical significance as the subject had already been infected by HPV‐16 before vaccination and no baseline DNA testing was performed at study entry. It is not known if the lesion was the result of a reactivation or a new infection by a high‐risk HPV type.This study confirmed, for the first time, the long‐term persistence of anti‐HPV‐16/18 antibodies in women vaccinated up to the age of 55. The study was further strengthened by the collection of serum and CVS samples that allowed for correlation of antibody titers, although few CVS samples were collected and evaluable at study end. It was limited by the lack of cervical screening at enrolment and the absence of clinical or virological endpoints.Ten years after first vaccination, the AS04‐HPV‐16/18 vaccine showed sustained immunogenicity in all age groups. Antibody titers at Year 10 were still at least three times higher than those observed after natural infection across all age groups, and in the older age categories (≥25 years), titers were similar to or just below the plateau levels associated with long‐term efficacy Mathematical modeling predicted detectable anti‐HPV‐16 and anti‐HPV‐18 antibody responses for at least 30 years after the first vaccination. Since women are potentially at risk of exposure to HPV for a long period of time throughout their lives, these findings suggest that the duration of protection against CC conferred by HPV vaccination may be close to lifelong.This study confirmed the sustained immunogenicity of the AS04‐HPV‐16/18 vaccine in women aged 15–55 years, with an acceptable safety profile for at least 10 years after primary vaccination. Mathematical modeling predicted that anti‐HPV‐16/18 antibody titers, 30 years post vaccination would still be above natural infection levels in all age groups using a modified power‐law model, and in the 15–25 years and 26–45‐year age groups using a piecewise model. Considering the time required to develop cervical cancer, this suggests that protection may be close to lifelong. Women in age groups not targeted by pediatric or adolescent immunization and catch‐up programs may still individually benefit from HPV vaccination.Cervarix is a trademark owned by the GSK group of companies.GlaxoSmithKline Biologicals SA took in charge all costs associated with developing and publishing this manuscript. TF Schwarz received personal fees from the GSK group of companies outside the submitted work. L Lin was previously insourced to the GSK group of companies as a consultant and is now an employee of the GSK group of companies. S Poncelet, PV Suryakiran, N Folschweiller, and F Struyf are employees of the GSK group of companies. S Poncelet, L Lin, and F Struyf hold shares in the GSK group of companies as part of their employee remuneration.Table S1. List of related or fatal SAEs during the entire study Click here for additional data file.Figure S1. Study design flowchart showing subject disposition.Click here for additional data file.


E6 and E7 proteins encoded by high-risk HPV play a key role in cellular transformation. HPV-33 and HPV-58 types are highly prevalent among Chinese women. To study the gene intratypic variations, polymorphisms and positive selections of HPV-33 and HPV-58 E6/E7 in southwest China, HPV-33  and HPV-58  E6 and E7 genes were sequenced and compared to others submitted to GenBank. Phylogenetic trees were constructed by Maximum-likelihood and the Kimura 2-parameters methods by MEGA 6 . The diversity of secondary structure was analyzed by PSIPred software. The selection pressures acting on the E6/E7 genes were estimated by PAML 4.8  software. The positive sites of HPV-33 and HPV-58 E6/E7 were contrasted by ClustalX 2.1. Among 216 HPV-33 E6 sequences, 8 single nucleotide mutations were observed with 6/8 non-synonymous and 2/8 synonymous mutations. The 216 HPV-33 E7 sequences showed 3 single nucleotide mutations that were non-synonymous. The 405 HPV-58 E6 sequences revealed 8 single nucleotide mutations with 4/8 non-synonymous and 4/8 synonymous mutations. Among 405 HPV-58 E7 sequences, 13 single nucleotide mutations were observed with 10/13 non-synonymous mutations and 3/13 synonymous mutations. The selective pressure analysis showed that all HPV-33 and 4/6 HPV-58 E6/E7 major non-synonymous mutations were sites of positive selection. All variations were observed in sites belonging to major histocompatibility complex and/or B-cell predicted epitopes. K93N and R145 (I/N) were observed in both HPV-33 and HPV-58 E6.Cancer of the cervix is associated with infection by certain types of human papillomavirus (HPV). The gene variants differ in immune responses and oncogenic potential. The  Cervical cancer is the third most common cancer in women worldwide, and a persistent infection of the high-risk human papillomavirus (HPV) is a major risk factor for cervical cancer ,2. ApproE6 and E7 of high-risk HPV are the primary oncoproteins involved in human epithelial cell immortalization and transformation, and act through their interactions with numerous host proteins [E6 is the p53 tumor suppressor protein. Additionally, E6 activates telomerase expression and modulates the activities of PDZ domain-containing proteins and tumor necrosis factor receptors [E7 proteins have evolved as a primary target of the retinoblastoma (Rb) family of proteins that control the activity of E2F transcription factors for degradation, resulting in the increase in the expression of E2F-responsive genes [E6 and E7 expression in cervical cancer cells successfully suppresses cell growth and induces apoptosis [E6/E7 can form an ideal target for diagnostic detection and therapeutic vaccine design.The early expressing proteins proteins . A primaeceptors ,10. The ve genes . Moreovepoptosis . Thus, Hin situ and cervical cancer caused by HPV-58 were more than the cases caused by HPV-18, were rated second; while HPV-33 was ranked third or fourth [Worldwide, HPV-33 and HPV-58 account for approximately 5% and 2% of cervical cancer cases, respectively; nevertheless, these HPV types show remarkably high infection rates in East Asia –16. The r fourth –19. Furtr fourth . TherefoE6/E7 might affect carcinogenic potential, host immune responses, and therapeutic effects of the vaccine. Among different types of HPV, there are types and variants that can acquire biological advantages through fixed mutations in their genomes, and even small variations could result in small adaptive improvements, possibly altering the composition of a HPV population [E6/E7 are still limited, especially in China. Therefore, the present study aimed to investigate the HPV-33 and HPV-58 E6/E7 gene polymorphisms, intratypic variations and positive selections in southwest China by examining a large series of samples covering the Chinese population. This study can provide essential data for future research on viral prevention and therapeutics. Above all, our findings may offer critical information for developing diagnostic probes and vaccines, specifically designed based on HPV-33 and HPV-58 E6/E7.The distribution and variation of HPV types show some degree of ethnic and geographical differences; consequently, the diagnosis, treatment, and ideal vaccine constructs necessarily need to be local ,22. Aminpulation . This stThe study was approved by the Education and Research Committee and the Ethics Committee of Sichuan University, Sichuan, China. Before the samples were collected, a written informed consent was obtained from all the patients or their guardians, and patient/study subject privacy was carefully protected.6H5CO2Na 10g, H₂O 1L) at -20°C.Between January 1, 2009 and December 31, 2015, 16793  cervical specimens were collected from women who underwent cervical screenings, histology and cytology evaluations for cervical diseases at Sichuan Reproductive Health Research Center Affiliated Hospital, The Angel Women’s and Children’s Hospital, The Chengdu Western Hospital Maternity Unit, The People’s Hospital of Pengzhou, and Chongqing The Fourth Hospital. Women over 14 years of age and with visible cervical lesions and/or HPV-related diseases  were eligible for inclusion [HPV-DNA was extracted and examined using the Human Papillomavirus Genotyping Kit For 23 Types  according to the manufacturer’s instructions. This kit enabled the classification of the 23 HPV types . 326 HPV-33 and 592 HPV-58 positive samples were collected.E6/E7 genes of HPV-33 were first amplified by polymerase chain reaction (PCR) in the thermal cycler  using the primers: 5'-AAAAAAGTAGGGTGTAACCGA-3' and 5'-TGCCACTGTCATCTGCTGT-3'; this step was followed by a second round of amplification using the inner primers: 5'-ACGGTGCATATATAAAGCAAACATT-3' and 5'-CTTCTACCTCAAACCAACC-AGTACA-3', when needed. The HPV-58 E6/E7 fragment was amplified using specific primers described previously [The complete eviously . Each 50The sequences were subsequently analyzed by NCBI Blast and DNAMAN version5.2.2 . HPV-33 and HPV-58 DNA nucleotide positions were numbered according to their reference sequences M12732.1 and D90400, respectively. The E6/E7 sequences of 33HE01-33HE15 in HPV-33 and 58HE01-58HE17 in HPV-58 were published with the GenBank accession codes KX354744-KX354775.http://bioinf.cs.ucl.ac.uk/psipred/) with the default parameters, which provided a simple and accurate secondary structure prediction method. Using a very stringent cross validation technique to evaluate the method’s performance, PSIPRED 3.2 achieves an average Q3 score of 81.6% [The secondary structures of the reference proteins were predicted by PSIPred server  [http://www.imtech.res.in/raghava/propred/) [http://www.imtech.res.in/raghava/abcpred/ABC_submission.html), which uses an artificial neural network, was used for the prediction of B-cell epitope(s) in the E6 and E7 sequences at the default settings  [ProPred-I server (opred1/)  was usedropred/)  was usedter: no) .E6/E7 gene sequences, the codeml program in the PAML 4.8 was used. This program performed the likelihood ratio tests (LRTs) to infer nonsynonymous and synonymous nucleotide divergence for coding regions employing the method described by Nei and Gojobor [E6/E7 protein sequences were aligned by ClustalX 2.1  [To estimate for positive selection at particular sites of the HPV-33 and HPV-58  Gojobor –42. HPV-stalw2/) .E6/E7 gene and 216 sequences of HPV-33 E6/E7 gene were obtained owing to the small number of copies of infected HPV in some women and limited amplicons obtained for sequencing.Among all the HPV-58 and HPV-33 samples, 405 sequences of HPV-58 E6/E7 sequence homology with the reference and the remaining 112 (51.85%) isolates showed nucleotide variation.Compared with the HPV-33 reference sequence (GenBank: M12732.1), 104 of the 216 HPV-33 isolates (48.15%) showed complete E6/E7 fragment showing sequence polymorphisms were identified among the 216 isolates. 8 biallelic mutations occurred in the 450-bp E6 Open Reading Frame (ORF); more specifically, 6/8 were non-synonymous substitutions and 2/8 were synonymous mutations. In addition, two biallelic mutations and one triallelic (C706A/T) mutation were found over the 294-bp E7 ORF, resulting in 4 amino acid changes of S29T, A45V, A45E, and Q97L  occurred in the HPV-33 Among the 216 determinable samples of HPV-33, the A1 and A3 variants were found in 199 (92.13%) and 17 (7.87%) samples, respectively . All of E6/E7 sequence homology with the reference, and the nucleotide variation rate of HPV-58 E6/E7 was 88.15% (357/405) among the 405 HPV-58 isolates. Overall, 8 nucleotide sequence variations were detected in the 450-bp E6 ORF; specifically, 4 mutations were non-synonymous and 4 mutations were synonymous. 13 single nucleotide changes were identified in the 297-bp E7 ORF, out of which 10 substitutions were non-synonymous and 3 substitutions were synonymous mutations (E7 than for E6. The average probability of a nucleotide sequence deviation from prototype was 20.63 substitutions/10000 bp for E6 and 78.90 substitutions/10000 bp for E7. There were no mutations generating a frame shift or a premature stop codon.When compared with the HPV-58 reference sequences (GenBank: D90400), 11.85% (48/405) of the isolates showed complete utations . SequencE6 sequences encoding the alpha helix or the beta sheet; while three non-synonymous mutations  occurred in HPV-58 E7 sequences encoding the alpha helix.In addition, there were no non-synonymous mutations in the HPV-58 Among 405 isolates of HPV-58, the A and B variants were found in 404 (99.75%) and 1 (0.25%) isolates, respectively. The nonprototype-like variant (lineage B) of HPV-58 was rare in our study. The sub-lineage A1, A2, and A3 variants were found in 168 (41.48%), 163 (40.25%), and 73 (18.02%) HPV-58 isolates, respectively . LineageE6 and E7 genes  have been summarized in Tables E6 were K35N, K93N, and R145I; for HPV-33 E7 were S29T, A45V, A45E, and Q97L; for HPV-58 E6 were K93N, R145K; and for HPV-58 E7 were T20I, G41R, G63S, and G63D. The homology comparison results were shown in E6 were observed at the same two positions.The variable dN/dS ratios were tested among various lineages using the PAML 4.8 software. The results of the selective pressure analysis of HPV-58 and HPV-33 E6, 5 for HPV-33 E7, 11 for HPV-58 E6 and 5 for HPV-58 E7, respectively; For MHC II, 6 good epitopes were obtained for HPV-33 E6, 6 for HPV-33 E7, 9 for HPV-58 E6 and 6 for HPV-58 E7, respectively; For B-cell, 1 high score epitope was obtained for HPV-33 E6, 3 for HPV-33 E7, 2 for HPV-58 E6 and 3 for HPV-58 E7, respectively. Amino acids change may influence the epitope’s binding ability, decrease or increase the binding ability, or even lead to disappearance of epitopes or appearance of new epitopes (The results of major histocompatibility complex (MHC) epitopes  and B-cell epitopes (only score ≥ 0.85 were shown), as predicted, were summarized in epitopes  Tables.E6 and E7 can offer useful information for the distinction and identification of known or new HPV types [E6/E7 variations than HPV-33 E6/E7. Furthermore, HPV-33 E7 was observed to be steadier than E6; therefore, E7 was selected as a more suitable target for diagnostic detection of HPV-33 than E6. On the contrary, HPV-58 E6 was steadier than E7 ; and was chosen as a more appropriate target for diagnostic detection of HPV-58 than E7.HPV types are divided into different genus according to their biological characteristics. HPV-33 and 58 are known to be closely related to each other, and belong to the α-9 species, which is the principal species consisting of almost all carcinogenic types . The intPV types ,46. The E6/E7 segment of 213 HPV-33 samples, the A2 sub-lineage (59.72%) was the main type in the Asia and Oceania region [Previous studies revealed two main groups—prototype-like group (lineage A) and nonprototype-like group (lineage B) among HPV-33 variants , 29. HowIn the current research, the prototype-like group of HPV-58 was the dominating variant; this classification of lineages was in agreement with the results of previous studies ,47. NeveE6 were A231C  and A387C ; in HPV-33 E7 were G658C  and C706A ; in HPV-58 E6 were C307T (237/405) and A388C (K93N 111/405); while the most common non-synonymous mutations in HPV-58 E7 were T744G (319/405) and G761A . These mutations may be considered important when E6/E7 are chosen as targets for primer design or diagnostic detection.The most common mutations observed in HPV-33 E6/E7 sites under positive selection. The key characteristic of positive selection is that it causes an unusually rapid rise in allele frequency, under positive selection, the positive mutation(s) may rise to high frequency rapidly, help species to adapt to the environments [E6, they may have evolutionary significance in making HPV-33 and HPV-58 adaptive to their environments. The HPV-58 E6 mutations T20I and G63S have been reported to increase the risk of developing cervical cancer [E6, D658C  for HPV-33 E7 as well as A259G and T395C for HPV-58 E6 [To the best of our knowledge, the present study is the first to contrast the HPV-33 and HPV-58 ronments . The selronments ; and reml cancer ; interesPV-58 E6 ,47,49–51E6 protein; amino acid positions 21–29 form short linear motif responsible for Rb binding in E7 protein; whereas the positions 58, 61, 91, and 94 act as Zn binding sites in the E7 protein [E6 and G543A  were found at residues 145–149; G658  in HPV-33 E7 was found at residues 21–29, G632T  in HPV-58 E7 was found beside residues 21–29; C755A (T61N) in HPV-58 E7 was found at residues involved in Zn binding for E7 protein. Until now, there is no published data to prove that immunity to one variant can prevent another variant in HPV infections [Amino acid positions 145–149 form the PDZ binding domain in the  protein . In the fections .E6 K93N, HPV-33 E7 Q97L and HPV-58 E7 T20I were observed in sites belonging to ideal B-cell and/or MHC predicted epitopes. Some variations, like HPV-58 E6 R145K, occurred at the sites belonging to both B-cell and MHC epitopes; Amino acids change may influence the epitopes, and then the immune recognition of HPV-infected cells. For example, the score of HPV-58 E6 B-cell epitope 81-96YSLYGDTLEQTLKKCL was 0.90, because of the mutation D86E, the score of epitope 81-96YSLYGETLEQTLNKCL decreased to 0.86; the HPV-58 E7 predicted epitope 77-85VRTLQQLLM disappeared because of the mutation V77A. However, further experimentation is required to validate the prediction through immunoinformatics.Modern immunoinformatics provide new strategies for the design and identification of antigen-specific epitopic sites that could be used as vaccine targets. The prediction of MHC and B-cell epitope in this study can be potentially used for the vaccine development against specific HPV variants in the Chinese population. In the present study, variations other than HPV-33 E6/E7 epitopes of HPV-58 variants in southwest China and to report the variants of HPV-33 E6/E7 in China. The data presented in this study may have significant implications in understanding the intrinsic geographical relatedness and biological differences between HPV-33 and HPV-58 E6/E7, and may also contribute to the design of clinical diagnostic probes and second-generation therapeutic vaccines based on HPV-33 and HPV-58 E6/E7.This is the first study to examine the changes in S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S1 FigNote: The residues conserved across HPV types were shown in capital letters, whereas the nonconserved residues are given in lowercase letters.(TIF)Click here for additional data file.


Human papillomavirus (HPV) bivalent and quadrivalent vaccines have been widely implemented in worldwide organized immunization programs. A nonavalent HPV vaccine is now available in several countries. The objective was to describe the fraction of squamous non-invasive high-grade cervical intraepithelial lesions attributable to genotypes targeted by bi-quadrivalent vaccines and by nonavalent vaccine according to age and diagnosis in women living in the city of Vigo .n = 145) and grade 3-carcinoma in situ  were tested with Linear Array HPV Genotyping test . Hierarchical estimation of the fraction attributable to HPV 16/18 or HPV 31/33/45/52/58 detected alone or in combination was calculated. Absolute additional fraction attributable to genotypes targeted by nonavalent vaccine compared to genotypes targeted by bi-quadrivalent vaccines was calculated as the increment of attributable cases with respect to all studied cases. Age group 1, 2 and 3 included women 18 to 34, 35–44 and ≥45 years old, respectively. EPIDAT 3.1 was used.Cervical scrapings (2009–2014) of women with histological diagnosis of cervical intraepithelial neoplasia grade 2 . It was 63/51/50% of CIN2 and 78/66/45% of CIN3-CIS in age group 1, 2, 3, respectively. Fraction attributable to genotypes targeted by nonavalent vaccine was 86% CIN2 and 86% CIN3-CIS. It was 87/91/75% of CIN2 and 90/86/76% of CIN3-CIS in age group 1, 2, 3, respectively. Fraction attributable to genotypes targeted by these vaccines tended to decrease as age increased (p-trend <0.05). Globally, absolute additional attributable fraction was 16%, 26% and 29% in age group 1, 2 and 3, respectively (p < 0.005).Fraction attributable to genotypes targeted by bi-quadrivalent vaccines was 59% CIN2 vs. 69% CIN3-CIS (Absolute additional fraction of CIN2 and CIN3-CIS attributable to genotypes targeted by nonavalent vaccine was observed in women of any age, especially in those over 35 years old. Human papillomavirus (HPV) vaccines have demonstrated their preventive potential for different HPV-related diseases , 2. InvaNo HPV vaccine protects against all HR HPV genotypes responsible for ICC. HPV 16 and HPV 18 cause the majority -around 70%- of ICC. HPV genotypes targeted by 9-valent vaccine are responsible for approximately 90% of ICC. The process for making a decision about introducing HPV vaccine into an immunization programme or about changing to a second generation vaccine has to be systematic and transparent  and theyThe objective of this study was to describe the fraction of squamous non-invasive high-grade cervical intraepithelial lesions attributable to HPV genotypes targeted by a 9-valent vaccine and by 2/4-valent vaccines according to age and diagnosis in women living in the city of Vigo .n = 123) or grade 3-carcinoma in situ  were prospectively recruited between the years 2011–2014 in the University Hospital of Vigo, Spain. Women with CIN2 (n = 22) and CIN3-CIS (n = 51) lesions histologically diagnosed from 2009 to 2010 in the same hospital were also included in this study. Characteristics of these 2009–2010 cases were previously described in a retrospective study  or 2.7 of these 3 multiple type infected lesions would be attributed to HPV 16 and [3 × 1/ , only if there were not detected HPV 16 and/or HPV 18 . For example, a lesion was attributed to HPV 35/39/51/56/59  only if there were not detected HPV 16/18/31/33/45/52/58.Absolute additional fraction of cervical lesions attributable to genotypes targeted by 9-valent vaccine compared to 2/4-valent vaccines was calculated as the increment of attributable cases with respect to all studied cases. Three age groups were considered: Group 1 (18–34 years), 2 (35–44 years) and 3 (≥45 years).p-value <0.05 or p-trend value <0.05 were considered statistically significant (EPIDAT software version 3.1) [For means, standard deviation (SD) and 95% confidence interval (95%CI) were calculated. Quantitative values were compared by Chi-square test. A two-sided ion 3.1) .Ethics Committee of Clinical Investigation of Galicia approved this study (CEIC reference number 2008/190). Women signed a consent form before their participation.In this study, 389 cases  were included. Global mean age was 35.3 years . Mean age of CIN2 cases was 33.5 years . Mean age of CIN3-CIS cases was 36.4 years  (Table HR HPV was detected in 386/389 lesions. Single genotype infection was detected in 66 CIN2 and 135 CIN3-CIS lesions. Multiple genotype infection was detected in 77 CIN2 and 108 CIN3-CIS lesions.n = 201). The most frequently detected genotypes were HPV 16 among 2/4-valent vaccine genotypes, HPV 31/33 among 9-valent vaccine genotypes other than HPV 16/18, HPV 35/51 among HR non vaccine genotypes and HPV 82 among probable/possible HR genotypes. A linear trend was found for HPV 16 prevalence  in age group 1 , 2  and 3  (p = 0.01). Prevalence of HPV 31 was 15.1% (10/66) in CIN2 vs 3.0% (4/135) in CIN3-CIS (p = 0.003).Prevalence of individual HPV genotypes is shown in Fig. n = 386). Data of 2/4-valent vaccine genotypes converged for all estimate methods except for hierarchical attribution and crude prevalence in CIN2 (6% difference with respect to minimum estimate). Fraction attributable to 2/4-valent vaccine genotypes was 53.0% (35/66) of CIN2 vs. 65.9% (89/135) of CIN3-CIS (p < 0.001) (minimum estimate) and 58.6% (85/145) of CIN2 vs. 69.3% (169/244) of CIN3-CIS (p < 0.001) . Data of 9-valent vaccine genotypes converged for all estimate methods. Fraction attributable to 9-valent vaccine genotypes was 84.8% (56/66) of CIN2 vs. 81.5% (110/135) of CIN3-CIS (minimum estimate) and 86.2% (125/145) of CIN2 vs. 86.5% (211/244) of CIN3-CIS .Figure p-trend <0.05). Considering hierarchical attribution, fraction of CIN2–3 attributable to 9-valent vaccine genotypes also tended to decrease with increasing age (p-trend <0.05).Age specific attributable fraction according to lesion grade is shown in Table p < 0.05) (Table p < 0.005), in comparison with 2/4-valent vaccine genotypes.For 9-valent vaccine genotypes, absolute additional attributable fraction of CIN2–3 was observed for all age groups , Joste et al.  and Riethmuller et al. 90%) [0% [9, 16The lowest oncogenic HPV genotypes were found mainly in CIN2–3 lesions diagnosed in the oldest women. This influenced the analysis of the vaccine genotype attributable fraction in each specific age group. First, vaccine genotype attributable fraction decreased as age increased. Secondly, the greatest absolute additional protective effect would be observed when vaccinated preadolescent women were more than 35 years old.Half cases were infected by only one genotype. These single infections could be useful for testing if global results obtained after mathematical attribution of HPV genotypes were reasonable. Among these single genotype lesions, HPV 16 was more prevalent in the youngest than in the oldest women, as previously reported , 11; HPVThe introduction of 2/4-valent vaccines has demonstrated to reduce cervical abnormalities, genital warts and HPV prevalence as well as the appearance of herd immunity after immunization programmes –20. TherLimitations: Quadrivalent and bivalent vaccine genotype attributable fraction of lesions was considered to be the same. The possible oncogenic effect of HPV 6/11  has not been taken into account. Cross protection against other related genotypes was not considered. Nonavalent vaccine antigens might probably produce a much stronger and longer lasting immunogenic response than cross protection. It would not be simple to establish whether the immunity directly induced by a vaccine and the cross-protection should be quantified in the same way. In this study, influence of vaccination coverage and vaccine efficacy were not taken into account. Current Spanish vaccine coverage is high , exceediIn comparison with bivalent and quadrivalent HPV vaccine, additional fraction of CIN2 and CIN3-CIS attributable to genotypes targeted by nonavalent HPV vaccine was observed in women of any age, especially on women more than 35 years old. Age-specific potential impact of nonavalent HPV vaccine should be taken into account in cost-effectiveness evaluations of HPV immunization programmes and in the organization of screening of vaccinated populations.


The replacement of cytology with human papillomavirus testing as the primary cervical screening test in England is imminent. In light of newly available evidence, we revised our previous estimates of the likely impact of primary human papillomavirus testing on incidence of cervical cancer.Using screening data on women aged 25–64 diagnosed with cervical cancer in England between 1988 and 2012, we previously reported that 38.8% had a negative test six months to six years prior to diagnosis. However, not all of these cancers would be prevented by human papillomavirus testing: for 1.0% the human papillomavirus positive test would come too late (within 18 months of diagnosis) to make a difference; 7.6% will have a negative human papillomavirus test (based on 79.9% sensitivity of human papillomavirus testing in cytology negative women); and 2.0% will develop cancer despite a positive human papillomavirus test. Additionally, we estimate that some women  whose cancers are currently prevented by cytology-based screening will have a false-negative human papillomavirus test.Overall, we estimate that 23.9% (95% CI: 19.3–27.6%) of current cases in women invited for screening could be prevented. Based on 2013 cancer incidence statistics, absolute numbers could be reduced by 487 (95% CI 394 to 563) or 3.4 (95% CI 2.8 to 4.0) per 100,000 women per year. Since then Ronco et al.3 have published a pooled analysis of randomised controlled trials with cervical cancer as an outcome, comparing HPV testing with cytology. They found that among women with a negative cytology screening test at entry, the rate ratio for invasive cervical carcinoma was 0.30 : 0.15–0.60). Here, we update our previous estimates of the impact of primary HPV testing on incidence of cervical cancer in England, in light of this new evidence, and taking into account cancers that will be diagnosed despite a previous positive HPV test.The intention to introduce primary human papillomavirus (HPV) testing in the United Kingdom was recently announced by the UK National Screening Committee.5We used a population-based case–control study of prospectively recorded cervical screening data (from 1988 onwards) for women with cervical cancer (ICD-10 C53) diagnosed aged 25 to 64 in England between April 2007 and March 2012. Data on screening were abstracted from routinely recorded cervical cytology records held on the National Cervical Screening Call/Recall System. After linking, the data were pseudonymised before being transferred to us for analysis. Details of the National Audit of Cervical Cancer have been published previously.Previously, we found that 38.8% (95% CI 37.8% to 39.8%) of women with cervical cancer had a negative cytology test resulting in routine recall  between six months and six years prior to their diagnosis. Here, we excluded the 1.0% of women whose only negative test within six years of diagnosis was within 18 months of diagnosis, on the grounds that a positive HPV test, together with a negative cytology test, would result in a repeat test a year later, and would probably not have led to the prevention (or even earlier diagnosis) of that cancer.6; (ii) there will be 70% (95% CI 40–85%) fewer cancers in HPV negative women compared with cytology negative women3; (iii) in women who are cytology positive, the sensitivity of HPV to pre-cancer will be 97.0% (95% CI 95.1–98.9%).7 These values result in a 93.0% (95% CI 88.0–98.0%) overall sensitivity of HPV testing for identifying women who have or would develop cervical cancer over the next six years, compared with an overall sensitivity of 96.5% (95% CI 93.0–100.0%) in the previous publication (see We modelled the likely results of HPV testing using the following scenario: (i) The overall sensitivity of English cytology to “pre-cancer” (i.e. disease that would lead to a cancer diagnosis over the next six years) is 76.6% (95% CI 65.1–85.1%)tion see .Table 1Of the women with negative cytology from 18 months to 6 years before diagnosis (37.8% of 8774 cases in the audit), 79.9% (95% CI 74.8–85.0%) would have been positive on the HPV test  cancer, despite a positive screen between 18 months and 6 years prior to diagnosis. From As HPV testing is to become the sole primary screening test in the UK, it is necessary to take into account the proportion of cancers currently prevented by cytology-based screening which would be missed by HPV testing. We estimate this number to be equal to 4.3% of the current total number of cancers .Taking all of these adjustments into account, we estimate that with HPV primary screening, the number of cervical cancers in women aged 25–64 in England might be reduced by 23.9% (corresponding to 38.8−(1.0 + 7. 6 + 2.0 + 4.3)). Taking into account the uncertainty in the figures combined to reach this percentage, we estimate the 95% confidence interval to be: 19.3% to 27.6% .8 for 2010 to estimate absolute risks. The rate then in women aged 25–64 was 13.0 per 100,000 women, with a total of 1801 cancers. The most recent data are for 20139 show 2039 cancers and a rate of 14.4 per 100,000 women. Applying a reduction of 23.9% to 2013 cancer incidence data yields a reduction of 487 (95% CI 394 to 563) cancers  in women aged 25–64 by introducing HPV primary testing.In our original publication, we estimated a maximum reduction of 587 cancers , using cancer statistics for EnglandWe believe that these updated estimates provide a more realistic estimate of the impact of switching to HPV testing in England. Nevertheless, other factors, such as vaccination (as of September 2015 the first vaccinated catch-up cohorts entered the screening programme) and falling screening coverage (particularly among older women) are not considered here and are likely to have an impact on the effectiveness of HPV primary screening.