Our ISH assay detected HR-HPV in 42% of our study population. The majority of ISH positive samples also tested HPV16 positive using sensitive PCR based assays and were more likely to have a history of preceding cytological abnormality. Analysis of subsets of this population revealed HR-HPV to be transcriptionally inactive as there was no evidence of a productive or transforming infection. Critically, the E2 gene was always disrupted in those HPV16 positive cases which were assessed. These findings point to a reservoir of transcriptionally silent, disrupted HPV16 DNA in morphologically normal cervices, re-expression of which could explain the increase in incidence of cervical cancer observed in later life.High-risk human papillomavirus (HR-HPV) causes nearly 100% of cervical carcinoma. However, it remains unclear whether HPV can establish a latent infection, one which may be responsible for the second peak in incidence of cervical carcinoma seen in older women. Therefore, using Ventana  Although the United Kingdom NHS Cervical Screening Programme (NHS CSP) has for many years achieved excellent coverage in women aged between 50 and 64 (~80%), one in five cancers still occur in women aged 65 or older; and 49% of all deaths from cervical cancer are in this age-group1At one time it could be said that the vast majority of these cases occurred in unscreened women. However, a recent NHS CSP audit which included 1341 cervical cancers diagnosed in women aged 65–83 suggested that this is no longer the case. Many of these women exited the screening programme with negative smear tests; 44% had at least three negative smears after the age of 50How do we explain the development of cervical cancer in women who exit the screening programme at 65 with a history of normal smear tests over the preceding fifteen years? It has been argued that incident cervical HPV infections are uncommon in women over the age of 65 and that in any event, the extended natural history of cervical neoplasia makes it improbable that these infections would have sufficient time to progress to invasive cancer. Therefore, we can only conclude that many of these women already had at the time of leaving the screening programme, a cervical HPV infection which was uncoupled from those cytopathic changes associated with virus infection in younger women. One possible explanation for this timeline would be the reactivation of HPV infection from a quiescent or latent state during which productive viral infection is suspended.in vivo studies using the rabbit oral papillomavirus model. These experiments point to a model of virus latency in which papillomaviruses retained under immunological control in the basal epithelial stem cell pool can be induced to reactivate periodicallyAlthough recent epidemiological studies offer circumstantial evidence supporting a natural history of HPV infection which includes such a latent state, the virological characteristics of a latent cervical HPV infection have yet to be defined4in situ hybridisation methods, quantitative PCR assays and viral and cellular biomarkers of productive and transforming viral infection, the prevalence and characteristics of HPV genomes in cervical material taken from a cohort of women undergoing hysterectomy for reasons unrelated to epithelial abnormality. Based on the observations made in animal models we expected to find a low copy number of viral episomes in the basal epithelial cell compartment with evidence of early viral gene expression in the absence of a productive infection. What we found was quite different.Towards this end, we have measured, using sensitive This comprised 106 consecutive patients  who had a hysterectomy performed between 2006 and 2009 at the University Hospital Coventry and Warwickshire NHS Trust for reasons unrelated to uterine malignancy and who were reported to have no histological evidence of epithelial abnormality of the cervix. Hysterectomy samples including both abdominal and vaginal hysterectomies but not sub-total hysterectomies were included in our study. For all samples in our cohort the endocervical canal and cervical transformation zone is represented. Fully informed written consent was obtained from each patient and all experiments were performed in accordance with relevant guidelines and regulations. Approval for this project was given by the National Research Ethics Service, West Midlands - Coventry & Warwickshire (ethics ref no: 04/Q2802/104). Seven patients were excluded from subsequent analyses following further independent histological review undertaken by two of the authors . Cytological histories of women undergoing hysterectomy were obtained from the “Open Exeter National Database System” which holds the records of women attending the NHS Cervical Screening Programme. Cytological histories were recorded as either negative, borderline nuclear changes, mild dyskaryosis or severe dyskariosisin situ analyses with the last of these sections used for HPV DNA testing. To avoid cross contamination, the microtome blades were changed between the cutting of each block. DNA was extracted using AllPrep DNA/RNA FFPE  kit, according to manufacturer’s instructions (Qiagen) and stored at −20 °C.Paraffin embedded blocks were sectioned every 4 μm and one section stained with haematoxylin and eosin; sequential sections were used for routine immunohistochemical staining and The INFORM HPVIII Family 16 Probe(B) which is capable of detecting 12 oncogenic HPV genotypes  was used to stain cervical tissues. Untransfected and HPV18 genome transfected human keratinocytes grown as organotypic rafts were used as negative and positive controlsTM DNA ISH assay; these were optimised on organotypic raft sections of untransfected and HPV18 genome transfected keratinocytes but discarded because of high background staining and low sensitivity after testing on archival FFPE cervical tissue sections.Eleven cases were excluded from this analysis; 6 because of high background staining in non-epithelial and endocervical cells and in the cytoplasm of squamous epithelia; and 5 because of significant squamous epithelial disruption; 88 evaluable cases remained. Two other ISH methods were evaluated during this study, GenPoint DAKO HPV DNA ISH assay, Abbott Vysis Cervical FISH HPV DNA ISH assay and Bond® technology. The assay offers individual typing of 24 HPV types with β-globin as an endogenous control. Cases were classified as invalid when β-globin and HPV were absent. A negative control containing no genomic DNA was included to confirm the absence of contamination.HPV genotyping was performed at the Scottish HPV Reference Laboratory, Edinburgh using the Multimetrix assay (Diamex) which utilises LuminexFifty-five samples were tested for the presence of HPV16 DNA using nested Q-PCR primers for E6. The primer and probe sequences used in this analysis are shown in E2 gene was assessed using overlapping primers that spanned the full length of the HPV16 E2 gene using primers shown in E2 repectively. The water control amplified in the first PCR reaction was carried through to the second PCR reaction to control for PCR product contamination.The integrity of the Ten cases which tested positive for HPV16 using Ventana ISH and either HPV16 nested PCR or Luminex were also tested for HPV16 E4 protein expression using the E4 monoclonal antibody 1D11 by immunohistochemistry® Histology Kit-9511). Organotypic raft tissue of HPV18 transfected keratinocytes and tissue from a patient with koilocytosis were used as positive controls. Sections of organotypic rafts generated from untransfected PHK were used as a negative control. Block-like homogeneous staining involving full thickness of the cervical epithelium  was considered evidence of p16 INK4a protein over-expression, whereas focal staining of clusters or isolated cells was disregardedAll cases were tested for p16 INK4a antigen expression using the monoclonal mouse antibody  tested positive and 51 (58%) tested negative for HPV DNA, with a punctate nuclear staining pattern observed for each HPV positive case . The rem2 = 10.34: 1 df: p < 0.001) : compared to ISH negative samples, ISH positive samples were significantly more likely to test positive for HPV using Luminex (29.6% vs. 0.0% x< 0.001) . Of 55 c< 0.001) . When we< 0.001) . A numbeA cervical smear history prior to hysterectomy was available for 83 women of whom 22 had a history of cytological abnormality [median time from last abnormal smear to hysterectomy = 11.5 years (range 1–22 years)]. Those women who tested positive using ISH were nearly three times as likely to have a history of any cytological abnormality (41.7% vs. 17.5%: RR = 2.8 95% CI 1.4–7.6). When this analysis was repeated exluding borderline nuclear abnormalities and retaining only definite dyskaryosis, the RR increased to 9.1 (95% CI 1.2 to 70.1).To establish if HPV16 DNA positivity was associated with a productive viral infection, 10 tissue sections which tested positive for HPV16 DNA using Ventana and at least one other HPV detection method, were stained for the viral biomarker E4, that is highly expressed in productive HPV infections and that marks the initiation of the vegetative stage of the virus life cycle 17. WhileDeregulation of HPV16 oncoprotein expression is associated with increased levels of the cellular biomarker p16INK4a. No evidence of a block-like homogeneous p16INK4a staining pattern involving the full thickness of the cervical epithelium which is indicative of HPV induced dysplasiaE2 gene was detected in all samples tested; illustrative examples are shown in To determine if the HPV16 DNA detected in the normal cervices was present in episomal forms, the integrity of the E2 gene was investigated by nested PCR using a series of overlapping E2 primers. Nested PCR was performed on all 18 samples which tested positive for HPV using Ventana and Q-PCR . DisruptE2 gene is a strong indicator of viral DNA integration, the assay we used does not identify the viral-cellular junction13202122Our findings point to a reservoir of transcriptionally silent HPV16 in normal cervical epithelium removed from older-women undergoing hysterectomy for reasons unrelated to cervical neoplasia. Our findings offer the first possible scientific explanation for the cause of the second peak of cervical cancer incidence that occurs in older women. As HPV16 could only be detected in these cervices in a disrupted form, the persistence of virus in these samples cannot be attributed to a latent infection as usually definedOur findings help illuminate a number of previously puzzling aspects of the exposure-disease relationship in older women242527de novo methylated following insertion into the host genome, integration-associated changes in the viral epigenome may be a promising line of inquiry1333As to how some HPV integrants are silenced only to be re-expressed in later life is not known. Given that foreign DNA is frequently While our study provides evidence of disrupted forms of high-risk HPV DNA in morphologically normal cervices, it will be important to recapitulate these findings in a validation cohort using both FFPE and fresh tissue collected from women undergoing hysterectomy for reasons unrelated to epithelial abnormality. In addition to replicating our current findings, fresh tissue should be used to characterise the HPV DNA in terms of methylation status, and confirm physical status by identification of specific integration sites in the host genome using a next generation sequencing technology.Our findings point to a reservoir of transcriptionally silent disrupted HPV16 genomes in morphologically normal cervices and re-activation of these “latent” forms of HPV DNA could explain the increase in incidence of cervical cancer observed in later life. A recent report has argued a case for extending cervical screening to women older than 65How to cite this article: Leonard, S. M. et al. Evidence of disrupted high-risk human papillomavirus DNA in morphologically normal cervices of older women. Sci. Rep.6, 20847; doi: 10.1038/srep20847 (2016).


We conducted a study to determine prevalence of HPV types in oropharyngeal cancers in the United States and establish a prevaccine baseline for monitoring the impact of vaccination. HPV DNA was extracted from tumor tissue samples from patients in whom cancer was diagnosed during 1995–2005. The samples were obtained from cancer registries and Residual Tissue Repository Program sites in the United States. HPV was detected and typed by using PCR reverse line blot assays. Among 557 invasive oropharyngeal squamous cell carcinomas, 72% were positive for HPV and 62% for vaccine types HPV16 or 18. Prevalence of HPV-16/18 was lower in women (53%) than in men (66%), and lower in non-Hispanic Black patients (31%) than in other racial/ethnic groups (68%–80%). Results indicate that vaccines could prevent most oropharyngeal cancers in the United States, but their effect may vary by demographic variables. Oropharyngeal cancers include malignancies that occur where the oral cavity and pharynx merge, including in the palatine and lingual tonsils, the posterior 1/3 (base) of the tongue, the soft palate, and the posterior pharyngeal wall. Current worldwide incidence has been estimated at ≈85,000 annually  and 5 additional high-risk types  is in clinical trial. Supported by evidence that existing vaccines effectively reduce oral HPV infections, these formulations may also reduce incidence of oropharyngeal cancers (http://seer.cancer.gov/] ) cancer registry-based residual tissue repositories in Los Angeles County, CA; Hawaii; and Iowa. The following anatomic regions (by ICD-O-3 codes) were included: C01.9 and C02.4 ; C09.0, C09.1, C09.8, C09.9, and c14.2 (tonsil); C14.0, C14.2, C14.8, C02.8, C10.2, C10.8, and C10.9 (other oropharynx) , a systematic review of cases of oropharyngeal cancer diagnosed during 1995–2005 was performed. The cases were selected from 7 participating registries, including 4 central cancer registries in Florida, Kentucky, Louisiana, and Michigan and 3 Surveillance, Epidemiology, and End Results program  by SEER classifications. Differences in prevalence of positive results for high-risk HPV or HPV-16/18, categorized by patient’s age, sex, race/ethnicity, and the anatomic location of cancer, were evaluated by using the χ2 or Fisher exact test whenever possible. Multivariate analysis was performed by using logistic regression with a step-down procedure, adjusting for age, sex, and race/ethnicity as appropriate. All statistical calculations were made by using SAS 9.3 .Statistical analysis was restricted to case-patients that had confirmed invasive OPSCC. Case-patient age at diagnosis was stratified into 4 groups: <50, 50– 59, 60–69, and Of the 1,271 oropharyngeal tumors requested from the participating cancer registries, samples from 588 case-patients were received and successfully tested. Those not received were either unavailable or the remaining tissue was not representative of disease. The demographic characteristics  and cancer stage  (22) of HPV results for 476 (81.0%) samples were from the linear array and 112 (19%) from LiPA. Most tissue was from the primary site (n = 473), but for 15 samples, only metastatic tissue from lymph nodes was available. Most case-patients (77.6%) were from urban areas or counties with a population >250,000. Most (94.4%) diagnoses were made during 2000 or later. Median age at the time of diagnosis was 58 (range 28–97) years. The male-to-female ratio was 3:1 and most of the cases (75.6%) were in non-Hispanic White persons. SCC, the most common histologic type of oropharyngeal cancer, accounted for 557 (94.7%) of all cases, and the main analysis was restricted to these cases .>1 high-risk type (HPV was detected in 403 of the 557 OPSCC cases (72.4%) with valid typing results and 396 (71.1%) were positive for isk type . In 68.4Other high-risk types, including HPV-31, 33, 35, 39, 45, and 52, were found at low frequency . The relProportions of high-risk HPV prevalence and HPV-16/18 were statistically different among the registries and by race/ethnicity, stage, and anatomic subsite . By sex,According to hierarchical assessment, HPV-16 was found in 337 (60.5%) OPSCC cases, HPV-18 in 11 (2.0%), other 9-valent high-risk types in 32 (5.7%), other high-risk types in 16 (2.9%), and low-risk types in the remaining 7 (1.3%) cases . Of the In multivariate analysis for high-risk HPV that included age and sex in the model, only race/ethnicity was a significant independent factor (p = 0.003). Odds for high-risk HPV infections were significantly higher for all other race groups than for non-Hispanic Black persons (p<0.001). When only HPV-16/18 detection was considered, significant differences were found in sex (p = 0.009) and race/ethnicity in (p<0.001), but not age (p = 0.063), between those infected and those who were not .The 31 cases that had histologic results other than SSC included 7 adenocarcinomas  and 2 small cell or neuroendocrine carcinomas (both HPV negative). Twenty-two cases were carcinomas not further specified, of which 7 tested positive for HPV .Our finding of >70% HPV prevalence in a large sample of the oropharyngeal cancer patients from around the United States suggests that a substantially higher faction may be HPV-related than has been reported in many previous investigations  compared with non-Hispanic White persons (73.6%), Hispanic persons (74.4%), or Asian Pacific Islanders (80.0%). Other studies that noted similar differences by race/ethnicity found this to be a recent but ongoing development  or characteristic gene expression profiling, may provide further insights in future assessments and show distinction between actively transforming HPV infections and random, transient occurrences (This study supports a role for oncogenic HPV in high proportions of oropharyngeal cancers. Future assessments are needed to monitor general prevalence and possible type-specific shifts. Data from the present and future studies will provide a baseline for early assessment of vaccine effects. Because the natural history and pre-cancer stages of oropharyngeal cancers are not established as they are for cervical cancer, direct trials with oropharyngeal neoplasia as the endpoint are not feasible. To obtain meaningful, comparable data for this objective, researchers need a universal definition of the anatomic oropharynx and associated malignancies and agreement on laboratory methods.


Basaloid-squamous-carcinomas (BSCC) have been considered as aggressive variants of common squamous-cell-carcinomas (HNSCC). Recent studies demonstrated a different clinical course depending on the tumour site. The aim of the study is to analyze the histopathologic/clinical features of BSCC/HNSCC resolved by the HPV-status.We analysed the histopathologic/clinical features of BSCC (n=59) and HNSCC (n=981), subdivided due to the HPV status. Differences were analysed using Chi square, Fisher exact, and student's t-test. Survival rates were calculated by Kaplan–Meier and log-rank test. Prognostic variables were subsequently evaluated by Cox regression.Our cohort was congruent with the literature regarding sex, age, metastases, and a predilection in the oropharynx. HNSCC/BSCC did not show a different disease-specific-survival. After UICC matching, univariate analysis revealed a better survival of UICC stage IVa BSCC compared to HNSCC  that was associated with a better response to radio-chemotherapy (p = 0.009). These results referred to the high prevalence of HPV+ (86%) oropharyngeal BSCC. Subgroup analysis demonstrated a better survival of HPV+ oropharyngeal BSCC than HPV-BSCC (p=0.017).The clinical outcome in BSCC depends on the tumour site and HPV-status. Prospective studies have to evaluate the beneficial application of postoperative radio-chemotherapy in HPV+ BSCC. Basaloid squamous cell carcinoma (BSCC) has represented a distinctive subtype of squamous cell carcinoma (SCC) since it was first described by Wain et al. in 1986 . BSCC dePatients and tumour characteristics are shown in table Treatment modalities (OP ± R(C)TX or primary R(C)TX) differed within both groups (p < 0.001). BSCC patients underwent more often an adjuvant RCTX . Beside the higher frequency of postoperative positive margins (n = 9) within the BSCC group, other risk factors such as extra-capsular spread (n = 6), angio-invasion (n = 2), lymphangiosis carcinomatosa (n = 5) or a combination (n = 2) led to the prevalent decision towards combined modality adjuvant treatment. Surgery without an adjuvant treatment was done in 19% of the HNSCC and never in the BSCC group. In BSCC 50 patients (85%) completed therapeutic procedures, 874 patients (89%) in the HNSCC group.Recurrence rates and the 5-year RFI were similar in BSCC and HNSCC as well as the oropharyngeal subgroups . After a mean follow-up of 32 months in the BSCC and 33 months in the HNSCC group, there were no significant differences in OS between BSCC and HNSCC  and a trend to a better DSS for BSCC  Fig. . MatchinSixty-two per cent of the BSCC (n = 58) were stained p16-positive. We identified a significant p16 predilection in the oropharynx (86%). Seventy-two per cent of the non-oropharynx BSCC was p16-negative (p < 0.001). p16 positive samples were verified by HPV 16/18 DNA analysis, resulting in a HPV 16 positivity for all cases and one case with a HPV 16 and 18 positivity. There was no difference between p16 negative and positive BSCC regarding sex, age, and UICC stage. Smaller T-stages (T1-2) with loco-regional metastases were frequently observed in p16-positive BSCC (p = 0.017). The 5-year survival rate for all head and neck tumour sites was 79.3% for p16 positive BSCC and 42.0% for p16 negative BSCC (p = 0.015). Subgroup analysis of oropharyngeal BSCC revealed a 5-year survival rate of 71.7% for p16 positive individuals and 50.0% of p16 negative patients (p = 0.017). The 5-year survival rate in HNSCC was 51.3%. Univariate analysis did not indicate age, sex, stage, and localisation as significant prognostic factors. Unadjusted hazard ratios for p16-negative vs. p16-positive BSCC were assessed in the Cox regression model, revealing p16-status as an influential factor on DSS  Fig. . FurtherBSCC was first described 30 years ago, but controversies about the molecular behaviour and patients' prognosis continue. Our BSCC cohort was congruent with the literature regarding sex, age, and a predilection in the oropharynx 70%). Fritsch et al. stated that the increasing incidence of oropharyngeal BSCC seems to reflect the recent epidemic of HPV related oropharyngeal HNSCC and reinforced the hypothesis by sub-analyzing the distribution of the primary site by year. Fritsch et al. showed an increasing proportion of oropharyngeal BSCC from 2000 to 2008 , 17-21. 0%. FritsThe facts of both a high occurrence of lymph node metastases  that are not associated with a poorer prognosis and the coincident finding of a tremendous prevalence of HPV positive oropharyngeal BSCC underline the impact of HPV in the carcinogenesis of BSCC. The clinical outcome is mainly dependent on the tumour site as oropharyngeal BSCC follow an even better course than oropharyngeal HNSCC that can be traced back on the HPV status.The study included 59 patients with BSCC and 981 with HNSCC that were diagnosed in the ENT department of the University Hospital Rechts der Isar, Munich, during a period of 10 years (01.01.2001–01.09.2011). The diagnosis of BSCC was based on the morphologic criteria defined by Wain  and 32 mDifferences between the BSCC and HNSCC groups were analysed using the Chi square test and Fisher exact test for categorical, and the unpaired student's t-test for continuous variables. As main endpoints the overall survival (OS), disease-specific survival (DSS) and recurrence-free interval (RFI) were assessed measuring the time from treatment to death of any cause, tumour-related death and locoregional recurrence, and/or distant metastasis. Survival rates and curves were calculated and illustrated by the Kaplan–Meier method and further analysed by the log-rank test for univariate analysis. Variables that revealed prognostic or effect modifying potential on the outcome as suggested by univariate analysis were subsequently evaluated by the proportional Cox regression for multivariate analysis. p-values <0.05 were considered statistically significant. Statistical analysis was done using SPSS .FFPE tumour sections (3 μm) were p16  stained and visualized with the Bond Polymer Refine Detection kit . Tissue samples with known p16 expression were used as positive controls. To describe the expression levels we used a scoring system classifying the staining intensity  and the relative proportion of stained cells . A cumulative score (range 0–7 points) was assessed by adding both scores. A positive staining was defined by a cumulative score equal or greater than 3.Small tissue samples that were not suitable for DNA extraction and molecular HPV-16/18 analysis underwent HPV in situ hybridization for high-risk subtypes 16, 18, 31, 33, and 51 (Leica) on a Ventana Benchmark XT automated stainer (Ventana). Tissue samples with an intense nuclear staining of at least 10% tumour cells were defined as positive.DNA was isolated by mixing five 10 μm FFPE sections with 1 ml Xylol and adding 250 μl 70% ethanol. After vortexing, the tubes were centrifuged  and the supernatant was carefully decanted. A vacuum centrifuge was used to dry the probes for 60 min at 45°C. The dried pellet was re-suspended in 600 μl Proteinase K buffer  + 25% Tween . Next 50 μl Proteinase K (Qiagen) was added and incubated over night at 55°C. Enzyme activity was inhibited at 95°C for 10 min.HPV16/18-DNA was detected by PCR amplification. For high-risk genotypes, we used the Abbott Realtime HR HPV assay running on the m2000sp instrument . The analytical sensitivity depends on the subtype: 500 copies per assay for 16, 18, 35, 39, 45, 51, 59, 66, 68; 2000 copies per assay for 31, 33, 52, 56, and 5000 copies per assay for 58, according to the manufacturer. Three forward and two reverse primers target a conserved L1 region. The endogenous human beta globin gene controls for sufficient cell number in the sample, nucleic acid extraction, and amplification efficiency. Tissue samples that were positive for p16 and HPV16/18 DNA were considered HPV positive.A selective literature review in PubMed Central Web addressing BSCC of the head and neck was performed. ‘Basaloid squamous carcinoma’, ‘BSCC’, ‘head and neck’, ‘upper aerodigestive tract’, ‘epidemiology’, ‘survival’, and ‘outcome’ were used as key words. We included all available articles considering more than seven patients and reviewed them for clinic-pathological and survival data .


Vaccination against Human Papillomavirus (HPV) is recommended for adolescent young women prior to sexual debut to reduce cervical cancer related mortality and morbidity. Understanding factors affecting decision-making of HPV vaccination of young women is important so that effective interventions can be developed which address barriers to uptake in population groups less likely to receive the HPV vaccine.We undertook a qualitative systematic review and evidence synthesis to examine decision-making relating to the HPV vaccination of young women in high-income countries. A comprehensive search of databases from inception to March 2012 was undertaken to identify eligible studies reporting the perspectives of key stakeholders including policy makers, professionals involved in programme, parents, and young women. Factors affecting uptake of the vaccine were examined at different levels of the socio-ecological model .Forty-one studies were included. Whether young women receive the HPV vaccine is strongly governed by the decisions of policy makers, healthcare professionals, and parents. These decisions are shaped by: financial considerations; social norms and values relating to sexual activity, and; trust in vaccination programmes and healthcare providers. Financial constraints may be overcome through universal healthcare systems offering the HPV vaccine free at the point of delivery. In the healthcare setting, judgements by healthcare professionals about whether to recommend the vaccine may restrict a young woman’s access to the vaccine irrespective of her own beliefs and preferences. Parents may decide not to allow their daughters to be vaccinated, based on cultural or religious perceptions about sexual activity.Barriers to the uptake of the HPV vaccine have implications for young women’s future sexual, physical and reproductive health. Interventions to address barriers to uptake of the vaccine should target appropriate, and multiple, levels of the socio-ecological model. Issues of trust require clear, accessible, and sometimes culturally appropriate, information about the HPV vaccination programme. Although young women are central to the HPV vaccination programme, their views are underrepresented in the qualitative literature. Future research should consider young women’s perceptions of, and involvement in, consent and decision-making. The World Health Organisation (WHO) recommends a three dose vaccination schedule for females aged between nine and 13 years .The United Nations Convention on the Rights of the Child recognised the rights for all children and young people to participate in decision-making processes which involve them . The legA systematic review and meta-analysis, comprising predominantly studies related to HPV vaccination programmes delivered in healthcare settings, indicated lower initiation by ethnic group and in young women without healthcare coverage insurance. No strong evidence for differences by parental education or family income variables were shown . In the High coverage of HPV vaccination programmes has the potential to reduce substantially cervical cancer incidence and mortality -14. HoweIn order to provide understanding of factors affecting uptake of the HPV vaccination programme, we focused on facilitators and barriers to decision-making by key stakeholders. Additionally, explanations for lower uptake by young women from minority ethnic groups were sought.A protocol for this qualitative systematic review and evidence synthesis was not registered with a database. However, systematic review methods were followed to identify all the relevant qualitative literature pertaining to the research question and are described below.th March 2012: CINAHL; Embase; Medline; PsycINFO, and; ISI Web of Science and ISI Proceedings. All abstracts were saved using Endnote ×3 reference manager software.A comprehensive search strategy for Embase was developed to ensure that all relevant literature was captured. A combination of text words and the following indexing terms (MeSH) was used: “papillomavirus”, “wart virus”, “vaccination”, “immunization”, “immunization programs”, “wart virus vaccines”, “qualitative”, “interviews” and “focus groups”  or open-ended questions in questionnaires were used to explore views and behaviours related to decision-making of HPV vaccination of young women. The populations of interest were: young women; their parents and/or primary care givers; healthcare professionals involved in the delivery of the programme, and; other relevant stakeholders such as policy makers, community leaders, and teachers. Young women were defined as adolescent girls aged between nine and 18 years. Studies which included the views of women over the age of 18 years were included if the views of young women were reported separately.No restriction was imposed by publication date in order to capture views of the HPV vaccine before its general availability (which might influence future delivery or uptake) as well as existing HPV vaccination programmes.Studies not published in English were excluded. Conference abstracts, editorials, letters and books were included only if they presented original qualitative data. Primary studies with adults were excluded if views about HPV vaccination of young women were not reported in the results. Questionnaire studies reporting only closed questions were excluded. For this paper, with a focus on examining factors affecting decision-making in high-income countries, studies were categorised as high-income using the World Bank classifications .Two reviewers (HF and SA) independently assessed the titles and abstracts from the literature searches and the relevance of studies retrieved as full text. Disagreements were resolved by discussion. The reference lists and bibliographies from relevant studies and reviews were hand-searched by one reviewer (HF) for additional primary studies not retrieved by the electronic search.Currently, there is no consensus regarding the assessment of the quality of qualitative research and subsequent exclusion from systematic reviews . For thiData pertaining to the methodology and context, including study and participant characteristics of each primary study, were extracted and entered into an excel spread sheet by one reviewer (HF).a priori aims and objectives designed to directly inform policy and practice. The overall purpose of the synthesis was to ‘pool’ the results from individual primary studies by initially separating the findings, interpreting and then combining them through the identification of key themes across the studies  . Policy er, USA] ,28.The only mandates we currently have for vaccines in our country are for infectious diseases spread in a school setting. It would not be a school issue or a public health issue, but more of an individual issue”     ,31,32: “If HPV vaccine were mandated, I would feel] grateful because we parents want the best for our children, and thank God science has discovered new medicines to prevent diseases”   .There is no way vaccines are cost-effective for us. It costs us an incredible amount of money in terms of time. Because the time we spend, the time the nurses spend…we’re not even close to being reimbursed for the amount of time we spend on vaccines”   .The only reason myself and my colleagues [sic] not able to offer HPV vaccine to our patients is lack of reimbursement [by insurance companies]”   .My gynecologist offers the HPV vaccine but you have to pay $135 up front and it is three shots, so you pay $135 a piece for each shot and insurance does not reimburse you or cover it”  [Insurance don’t pay for everything [provides partial payment] and then a lot of times insurance doesn’t cover everything [some services not covered at all]” [Young woman USA] [Where will I get the money for it [HPV vaccine]? At this price, my family could not support me either”    ;“Insuranman USA] . Where tman USA] :“Where wng Kong] . This costralia] .It is difficult to bring up the question of vaccination to youth if you can see they do not have the money to pay for it, or the family can’t afford to buy the vaccination for them as it is so expensive, it feels a little unethical to take this up with young people”    . Offerin Sweden] .This important barrier to uptake was not evident in studies where the vaccine was offered free at the point of delivery.Social norms and values, particularly those relating to sexual debut and behaviour, shaped the views and actions of healthcare professionals, parents and young women in relation to the HPV vaccine.Well, you know, we try not to be judgmental, but we know there are certain [ethnic minority] populations where sexual activity with multiple partners is more common, more prevalent…we treat them frequently for various STDs”     . In othean, USA] .They don’t want their child to start talking about sex… or their parents don’t really know how to relay that information to them”     . In one an, USA] . More coan, USA] ,32,44,45an, USA] ,32,44.I haven’t heard it myself, but someone else heard from a girl that had been vaccinated that she was vaccinated against all sexually transmitted disease”   mothers, they were very concerned that by them okaying the needle, that was giving the daughters the green light to go and become sexually active”   . In addi Sweden] ,47: “We Cultures in which a girl must be a virgin at marriage, and so on. Clearly, it becomes very difficult to talk about multiple partners then. And one cannot… it’s like offensive, I think”     . This ap Sweden] -49: “Comher, UK] ,48: “I wI’d say probably [vaccination should happen at age] 11 or 12 definitely before they have sex. I think while they’re still in early middle school. Once they get to the eighth grade and high school they’re already into puberty and thinking. The temptation is out there”     . Others nt, USA] -53: “Eveer, USA] ,48,54: “Because it would encourage my daughter to have sex and I wouldn’t want that” . Parents raised concerns that the HPV vaccine might encourage earlier sexual debut, multiple sexual partners or complacency with regards to safe sexual health practices   [I thought what a fantastic thing [the vaccine], because I actually went to school with a girl who can’t have children because she’s got cervical cancer, and the reason she has cervical cancer is because she was very promiscuous when she was at school with me”   . Other pher, UK] ,54: “I der, USA] . Personaer, USA] ,55,56: “stralia] .So it’s easier to give it to younger children by saying ‘It’s to prevent cancer’ than saying to them ‘you’re having this because when you’re older you’re going to have sex and get all these horrible diseases”   [If my family knows what this [HPV vaccine] is for and if I say I want it, they would think that I am fooling around”   ,58: “We stralia] . Others stralia] -38: “If ng Kong] .Yes, definitely… It’s a very common virus, it causes quite a lot of disease and it can cause some very serious disease, in rare cases, but that can be prevented by the vaccine”   ,43,44 anner, UK] ,30,44.If there is a new vaccine to prevent a disease, we won’t oppose it because we are preventing a disease”   .My first thought was, I am sendin’ my ten-year old to this clinic to put dead HPV cells in her. What if the HPV that they are shooting in her body…what if it comes to life?”    . Such feer, USA] ,49: “I jher, UK] .I have some [parents] that don’t want any vaccination… those I would not even broach the subject with”   ”   . This waan, USA] ,54: “Becstralia] .They [government] may not be telling the full story, which, of course, we found out about syphilis, we found out about AIDS” [African-American participant] [At least [with] the [Centers for Disease Control and Prevention] you know they’ve got some validity behind their name that gives some validity to the drug maker because most drug makers I think are just out there to make a buck”   .There may have been a covering information letter from the school… I suspect I read it…but I guess I must have somewhere made the decision when I saw that it was being offered to the general population that the girls would have it and I don’t think I did any further research at that point”  [If it hadn’t come to school it wouldn’t have crossed my mind to do it… It’s not [a decision you make] on an individual basis, and that [having it as school] makes you more comfortable”   is in our interest so I feel there’s no reason not to get it”   ; “If it stralia] . In one man, UK] .They pump all sorts of things into kids… Do they really know how it will affect them later? I don’t trust the government. Why would they need [the vaccination] when we didn’t get it? What aren’t they telling us? My sister showed me some articles about there being cancer in the vaccine”   .I didn’t realise how ill-informed I am. You just sign off on all these forms…”   .They couldn’t be bothered to read the form or fill it in, you know, motivate themselves enough to do it, but they always wanted the child to have it [the vaccine] as long as somebody bothered to knock on the door and say, ‘just sign on the dotted line’. It was just the process of the filling of the form in was just too much effort”   . Only onrse, UK] .It’s an offer. And it shouldn’t be our job to call and call again and again and invite them. I think that it’s actually the parent’s responsibility, together with the student [aged 12 to 13 years]”   back. I spoke at assembly, and they had year group meetings where the information was distributed and making sure the classes and teachers are informed of what’s happening”   . Targeti Sweden] . This mistralia] .If everybody is given 30 seconds to get in and get out, you can’t reasonably expect a nurse to make a decision in that time. Yes or no? They would have to make some sort of later appointment to speak to the young person and have a serious chat which in itself would then single them out from, you know if there was a mass queue”    ,61,69: “If I know this particular twelve year old girl is a risk of contracting an illness for which there is an injection that will prevent this illness then it is most definitely in her interest to give it to her, even if her parents don’t agree”    . Primaryner, UK] .I didn’t discuss it with the girls, I merely told them of my decision afterwards. I didn’t feel that they were in a position to make a decision for themselves… they are relatively sheltered and therefore it wouldn’t have been relevant to ask them what they thought”     ; “I woulI mean… We didn’t take much notice of the forms, and we handed it to our parents and they make the choice… It’s like your parents are the boss of you, sort of. You don’t choose, ‘oh I’m going to get a cervical cancer vaccination”    [Well she [my daughter] is just needle phobic… Even though I signed the consent forms; she just doesn’t turn up to them. Getting to this age makes it hard, too, because she just says ‘well it’s my body, my choice, and I’m not going’ ”   . Others The studies included in this review illustrate how a young woman’s access to the HPV vaccine is shaped by decisions at different levels of the socio-ecological model. This includes: the policy context; social norms and values, particularly in relation to sexual activity; the views and actions of healthcare professionals, and; parental consent. There is far less qualitative evidence of the role of young women in this important decision affecting their future sexual, physical and reproductive health. The stages at which decisions are made before young women are able to exercise any agency over whether they receive the vaccine are illustrated in Figure Some of the most important decisions affecting access to the HPV vaccine are made at policy level, and relate to whether the vaccine is incorporated into the healthcare system, the financial arrangements for accessing the vaccine, and the arrangements for its delivery to the target population. A systematic review examining predominantly quantitative data from 22 studies (21 of which were conducted in the USA), reported that when cost is a factor in vaccine access it dominates as a barrier . SimilarIn healthcare settings, an important prompt to uptake appeared to be the decision for the healthcare professional to recommend vaccination. This has been reported in a systematic review, comprising predominantly quantitative primary studies, of barriers to HPV vaccination  and a stThe barrier of requiring a healthcare professional’s recommendation to receive the HPV vaccine was largely overcome in school-based programmes where the policy is to offer HPV vaccine universally to eligible young women. However, in this setting the requirement for written parental consent presented the greatest barrier to access. Healthcare professionals delivering the HPV vaccination programme could adapt implementation procedures to reach apparently ‘disengaged’ parents. This could be tested in existing HPV vaccination programmes by introducing strategies to introducing home visiting or chasing up consent forms if not returned ,76.Vaccine safety, misperceptions of need based on sexual activity, and low perceptions of risk of HPV acquisition have been highlighted as barriers to positive HPV vaccine decision-making by young women . HoweverOther combinations of decision-making about HPV vaccine were also identified. Vaccination may be sought by young women when parental consent has been actively refused or when a parental consent form has not been returned. Young women may also exercise their rights to refuse vaccination when parental consent has been granted, although there was little evidence for this within the qualitative studies included in the review. To address gaps in our understanding of this important process, further qualitative research should examine young women’s perceptions of, and involvement in, decision-making about the HPV vaccine. Further, healthcare professionals may require clear guidance and support in understanding how and when a young woman might be given the vaccine without parental consent.A secondary objective of this review was to gain understanding of the factors contributing to inequalities in uptake of the HPV vaccination programme by ethnicity previously identified ,7. CorreMistrust of the motivations of healthcare professionals in relation to the HPV vaccine was a pertinent issue for some minority ethnic groups. In the USA, the historical legacy of the mistreatment of African Americans in the Tuskegee syphilis experiment   with discussions held with the second reviewer (SA) as analysis progressed. The interpretation of the primary study findings, and consequently a different thematic framework, may have emerged if an additional reviewer had undertaken analysis at this stage. The subsequent exclusion of low- and middle-income countries from the study limits applicability of the findings within these settings. Further research to understand barriers and facilitators to HPV vaccination of young women in low- and middle-income countries is recommended. Finally, theoretical frameworks were underutilised in the primary studies. Of the 41 studies included in this review, only eight reported using theoretical models in the study ,60,61,70Our study findings show that access to the HPV vaccine is governed by parental influences, health professional recommendations, social norms and values, organisational factors and policy context. As such, interventions targeted only at young women are likely to be the least effective approach. Although young women are the main participants and beneficiaries of the HPV vaccination programme, their views are underrepresented in the literature. Future research efforts should develop context specific, culturally appropriate strategies that increase equitable access to the HPV vaccine.ACIP: Advisory Committee on Immunisation Practices; HPV: Human Papillomvirus; UK: United Kingdom; USA: United States of America; WHO: World Health Organisation.The author’s declare that they have no competing interests.SA and HF conceived and designed the study. All authors  are responsible for the reported research. HF and SA reviewed titles and abstracts and undertook data extraction. HF analysed the data and prepared the initial manuscript. All authors have made substantial contributions to interpreting the data, revising the manuscript and have given approval of the final version to be submitted.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/14/700/prepubSearch strategy applied to database.Click here for file


Human papillomavirus (HPV) is one of the most widely publicized and researched pathogenic DNA viruses. For decades, HPV research has focused on transforming viral activities in cervical cancer. During the past 15 years, however, HPV has also emerged as a major etiological agent in cancers of the head and neck, in particular squamous cell carcinoma. Even with significant strides achieved towards the screening and treatment of cervical cancer, and preventive vaccines, cervical cancer remains the leading cause of cancer-associated deaths for women in developing countries. Furthermore, routine screens are not available for those at risk of head and neck cancer. The current expectation is that HPV vaccination will prevent not only cervical, but also head and neck cancers. In order to determine if previous cervical cancer models for HPV infection and transformation are directly applicable to head and neck cancer, clinical and molecular disease aspects must be carefully compared. In this review, we briefly discuss the cervical and head and neck cancer literature to highlight clinical and genomic commonalities. Differences in prognosis, staging and treatment, as well as comparisons of mutational profiles, viral integration patterns, and alterations in gene expression will be addressed. Human papillomaviruses (HPVs) comprise a large family of viruses, 170 of which are now sequenced and are extensively referenced on the Papillomavirus Episteme . The hig−) HNCs have been steadily declining in correlation with decreased tobacco use, while HPV positive (HPV+) head and neck cancers are on a steep, upward trajectory especially in younger populations, and are estimated to surpass cervical cancer rates in the U.S. by the year 2020 .,87.86,87possible . Pap tespossible .The peak age of infection for women is under age 25, with the potential for continual infections by either the same or different HPV types over time . ContinuHead and neck squamous cell carcinomas (HNSCCs) account for approximately 50,000 new cancer diagnoses in the United States each year . These tIn general, the prognosis of cervical cancer is excellent at early stages, often with surgical resection alone. Cervical cancer screening in developed countries has led to early detection, thus dramatically reducing cervical cancer mortality. Unfortunately, locally advanced cervical cancer and metastatic disease is much more common in developing countries making it the second leading cause of cancer related death in women worldwide . Women wHNSCC is a much more heterogeneous group of cancers, and therefore, the prognosis varies with site and stage of disease. Much like cervical cancer, however, many localized HNSCCs (stage I and II) are cured with radiation or surgical resection alone. As in cervical cancer, HNSCC survival decreases with advancing stage. Importantly, the prognosis of oropharyngeal HNSCC has greatly improved over the past several decades . In factCervical cancers, whether SCC or adenocarcinoma, have a very high prevalence of HPV. Therefore, it is difficult to determine whether HPV infection per se affects prognosis in these populations. Recently published data suggests HPV is a prognostic factor in anal cancer, predicting both overall and disease-specific survival . HoweverIn general, early stage cervical cancer (Stage IA or IB) is treated with resection alone either by conization for those with early stromal invasion or extrafascial hysterectomy for those with lymphovascular invasion. Patients who desire pregnancy may also be candidates for radical trachelectomy with pelvic lymphadenectomy. Modified radical hysterectomy is often offered for patients with stage IA2 or microscopic IB disease. However, stage IB-IIA with high risk pathologic features  requires radical hysterectomy and adjuvant concurrent radiation and chemotherapy (CRT). Adjuvant radiation is recommended for patients with intermediate risk factors . Radiation therapy alone is also an acceptable option for early stage disease.versus 41% in radiation alone arm at 8 years, Gynecologic Oncology Group (GOG)-120 with 65% in chemotherapy versus 47% in radiation alone, and GOG-123 with 83% OS for chemotherapy versus 74% with radiation alone [In contrast, locally advanced cervical cancer (Stage IIB–IVA), is treated with radiation alone or CRT. Combined treatment has greatly increased survival in locally advanced disease. The addition of cisplatin treatment was shown to increase overall survival (OS) in several studies including Radiation Therapy Oncology Group (RTOG) 90-01 demonstrating 67% OS in chemotherapy arm on alone ,110,111.Patients that develop recurrent disease often undergo either radiation or surgery depending on previous treatments, although some cases may not be amenable to either option. Therefore, these patients and those with metastatic disease are considered incurable and many opt for palliative chemotherapy. In general, chemotherapy is cisplatin-based but many trials have demonstrated increased response rates for combination therapy combining cisplatin and most commonly, paclitaxel. Unfortunately, despite combinations of chemotherapeutics response rates remain low at 25%–35% in recurrent and metastatic disease highlighting a dire need for novel agents ,113.Like cervical cancer, HNSCC is often curable with surgical resection or radiation therapy alone for those with early stage disease. However, many of these patients will develop recurrence and oftentimes present with locally advanced disease. Patients with locally advanced disease may undergo surgery, radiation, or CRT with each treatment carrying certain consequences. Surgical resection can result in disfigurement, poor wound healing and at times loss of voice . However, CRT may result in dysphagia, mucositis, neurotoxicity and renal failure. Despite aggressive treatments, many tumors recur, and patients then undergo the treatment modality not previously offered, or re-resection.Despite improvements of surgical technique, the rate of relapse for both local and metastatic disease after surgery alone is high, particularly in patients with high-risk features such as perineural invasion, multiple lymph node involvement, extracapsular spread and positive surgical margins. Two pivotal studies demonstrated improved progression-free survival (PFS) and overall survival (OS) in patients with high-risk features treated with adjuvant CRT, albeit with greater toxicity over treatment with radiation alone. However, PFS and OS were only 47% and 53%, respectively at 5 years, suggesting that new treatments are necessary in order to improve outcomes in high-risk patients ,115.p = 0.014) [versus 36.4% for radiation alone; thus, cetuximab is an acceptable agent oftentimes associated with less severe toxicities [Combined CRT is frequently offered upfront in locally advanced disease. In 2003, a pivotal phase III trial showed that concurrent chemotherapy and radiation using cisplatin resulted in superior outcomes when compared to radiation alone in unresectable, locally advanced disease. Three year overall survival was 23% in the radiation alone arm compared to 37% in the CRT arm (= 0.014) . As in c= 0.014) ,118. Intxicities . Most ofMany HNSCC relapses occur locally despite successful initial definitive treatment. Upon local relapse, standard of care is often salvage surgical resection which confers local control rates of 33%–50% and a long term overall survival of 20%–40% ,121,122.versus cisplatin and radiation in locally advanced disease. The benefit of cetuximab in cervical cancer is unknown and actively investigated. However, it is clear that not all HPV positive HNSCC tumors behave equally. Positive smoking history, likely resulting in increased genomic mutations, may explain why some HPV positive tumors do not fare as well with therapy. Accordingly, the improved survival of HPV positive patients is negated by tobacco use [Our ability to determine which patients  will recur and which will do well with less intensive therapy and therefore less treatment related toxicity is limited. Active research is ongoing to develop model systems that resemble human HNSCC and that may be useful platforms for the development of alternative treatment strategies ,128. As acco use . As the de novo, or are acquired from exposure to environmental factors. As cancers are quite heterogeneous in nature, with a multitude of mutations, identifying therapies that work on entire tumor cell populations including tumor-initiating or cancer stem cells has been difficult [Cancers often arise due to mutations that are either inherited, occur ifficult . CurrentCervarix®  and Gardasil® . These vaccines cover the most common oncogenic HPV types known to cause cervical cancer, while Gardasil® also includes those responsible for genital warts. Cervarix® is a bivalent vaccine that protects against high risk HPV16 and 18, while Gardasil® is quadrivalent and protects against HPV16 and 18, as well as the low risk HPV6 and 11 types. Additionally, protection for both vaccines has been demonstrated to be ≥5 years [Gardasil®. The current recommendation by the Food and Drug Administration (FDA) is for men and women ages 9–26 years to be vaccinated. This is the age range where HPV infection is most likely to occur. It is imperative to begin the vaccination series at the earliest recommended ages, prior to the start of sexual activity, to offer maximal protection against HPV infection; vaccination after infection does not result in enhanced protection from or clearance of the virus.There are currently two vaccines on the market: ≥5 years ,134,135.Cervarix® and Gardasil® utilize virus-like particles (VLP) formed by self-assembly of the HPV capsid protein L1 to elicit the immune response, as opposed to live or attenuated virus. Antibodies are then produced against the L1-specific to each HPV type included in the vaccine [Cervarix® suggests it may also cross-protect against the oncogenic HPV types 31, 33, 45 and 51 [Both  vaccine . Althoug5 and 51 . Since t5 and 51 ,139. UnlThe advent of whole exome and whole genome sequencing and other “omics” technologies has enabled scientists to identify specific mutations in tumor specimens that may lead to targeted and personalized therapies. This is especially important in cancers such as cervical and head and neck where a diagnosis is often not made until the advanced stages of disease. These late stage tumors are not as treatable and have few targetable molecules identified. Previously, common chromosomal aberrations in both tumor types had been identified in the presence of HPV. These include loss at 13q and 11q22.3–25 and gains at 20q and 3q24–29; albeit unique chromosomal alterations also exist for cervical and HNC . This inet al. [MAPK1, FBXW7, and EP300 [A recent publication by Ojesina et al. , comparend EP300 . AdditioFBXW7 and PIK3CA. The head and neck studies were further segmented into HPV+ and HPV− categories, with far fewer genes mutated in HPV+ as compared to HPV− tumors. Not surprisingly, HPV− tumors harbored higher global mutation frequencies compared to the HPV+ group, presumably due to tobacco use and highlighted by TP53 mutations in almost all HPV− tumors. Similar publications from exome sequencing of HNSCCs were reported in 2011. Only a few somatic mutations match those discovered in the cervical cancers ,143, incHPV integration in cervical cancer can lead to the disruption of E2 as HPV enters common fragile or other sites in the host genome. This is an important event during the progression of cells to a transformed state, albeit not necessarily required, which provides a growth advantage given the control of E6/E7 expression by E2 under normal circumstances. Transformation is continuously dependent upon E6/E7 expression and can be reversed by the reintroduction of E2 ,145,146.TP63 as a gene affected by integration events found in both cervical and HNCs. Schmitz et al. defined the chromosomal hotspot 3q28, where the TP63 gene is located, and demonstrated TP63 was disrupted by HPV integration in multiple cervical tumors. Likewise, Akagi et al. identified HPV integration within the TP63 gene, resulting in the loss of normal TP63 gene expression and the creation of a novel protein isoform, along with viral-host fusion transcripts. Furthermore, c-myc was also amplified and overexpressed in both cancer types in the presence of integrated HPV [in vitro, the accumulation of oncogenic mutations in cellular tumor drivers or suppressors is necessary to achieve neoplastic transformation and cancer development [Even with some disparate findings between the above reports, both identify ated HPV ,152. Altelopment . In dept+ cervical with HPV+ HNCs. Given the large number of specimens already banked for both cancer types, clear trends in gene expression should delineate common molecular and genomic occurrences that are due to HPV activities.It is clear that HPV infection and transformation de-regulates gene expression patterns in keratinocytes. For many years, studies of HPV-driven host gene expression involved either the introduction of the HPV genome or of the HPV E6 and E7 oncogenes in existing cell lines. More recently, studies of primary tumors compared to control specimens have provided additional insights in clinically relevant model systems. To create a database of reliable genomics and transcriptomics information for many cancer types, the National Cancer Institute (NCI) along with the National Human Genome Research Institute (NHGRI) collaborated on the ambitious “The Cancer Genome Atlas” (TCGA) project . The goaCDKN2A (p16), TOP2A, E2F1, FOXM1, MCM2/4/5, and PTGES, with a high prevalence of genes involved in cell cycle regulation. Down-regulated genes including TGFβ1, TGFα, and KRT16, were clustered in differentiation and tumor suppressor pathways [In the absence of published TCGA based comparisons, smaller studies that profile HPV-related gene expression are publically available. One of the first papers published comparing cervical cancer (SCC and adenocarcinoma) to normal cervical keratinocytes identified approximately 500 genes up- or down-regulated in the presence of HPV . Up-regupathways . This repathways .FOXM1, TOP2A, and E2F1 were not only overexpressed, but where their respective expression levels gradually increased as cervical lesions progressed from low-grade to high-grade lesions and eventually to carcinomas. Other de-regulated genes included CDC25A, TP73, and CCNE2. Conversely, genes that demonstrated reduced expression in the progression from dysplastic tissue to carcinoma included PTGS2, BCL2, and FOS [Analyses in 2013 utilized a retrospective approach to identify novel genes involved in dysplastic and neoplastic cervical lesions, based on targets previously published in the literature . The aut and FOS .+ to HPV− HNSCCs are available. One study compared HPV+versus HPV−versus normal oral mucosa and analyzed these specimens in a number of ways [CDKN2A, TOP2A, and MCM2/3 in HPV-infected HNCs compared to normal mucosa. Other up-regulated genes reflected pathways such as DNA replication, cell cycle progression, DNA repair and differentiation, while down-regulated genes reflected processes such as proteolysis and chemotaxis. Additional comparisons between HPV+ and HPV− HNCs identified up-regulated genes involved in nuclear structure, mitosis, DNA repair and methylation, and down-regulated genes involved in signal transduction and proteolysis [+ cervical and head and neck cancers and discovered similar trends where cell cycle-related genes are highly up-regulated [A multitude of publications comparing the gene expression profiles of HPV of ways . The autteolysis . Finallyegulated . Other degulated ,159,160.CDKN2A [CDKN2A locus [+ cancers that it is used clinically as a surrogate marker for HPV positivity during cancer diagnosis.p53 and Rb-dependent functions are regulated by p14ARF and p16, respectively, which are encoded by the same gene, CDKN2A ,162. Thi2A locus . p16 is Epigenetics is another mechanism whereby gene expression patterns can be altered in normal and transformed cells. This is a complex field that investigates DNA changes that do not affect the inherent nucleotide sequence, but rather processes such as methylation patterns and histone modifications. The presence of HPV greatly affects methylation signatures across the genome, promoting hyper- and hypomethylation. For example, tumor suppressor genes, such as TERT, exhibit promoter hypermethlyation and thus gene silencing in CIN lesions and cervical cancers ,165. TheInitial studies comparing cervical and HNCs to normal controls suggested HPV elicits similar gene expression alterations between tumor types. Publication and analysis of the TCGA data will likely confirm this trend, but novel details are also expected which should reflect differential tumor origins in various tumor types, data which may be exploited for the targeting of tumor cells by novel agents. The large number of samples combined with thorough bioinformatics analyses and ultimately confirmatory studies will help validate genes of interest shared or unique to cervical and HNCs. Further separation of these groups based on clinical, environmental and demographic data is bound to enrich and surprise the HPV community.Human papillomavirus is the most common sexually transmitted viral pathogen and causes approximately 5% of the global cancer burden . Even af


Human papillomavirus (HPV) genotyping assays are becoming increasingly attractive for use in mass screening, as they offer a possibility to integrate HPV screening with HPV vaccine monitoring, thereby generating a synergy between the two main modes of cervical cancer prevention. The Genomica CLART HPV2 assay is a semi-automated PCR-based microarray assay detecting 35 high-risk and low-risk HPV genotypes. However, few reports have described this assay in cervical screening.An aim of the present study, Horizon, was to assess the prevalence of high-risk HPV infections in Copenhagen, Denmark, an area with a high background risk of cervical cancer where women aged 23-65 years are targeted for organized screening.Material from 5,068 SurePath samples of women participating in routine screening and clinical follow-up of cervical abnormalities was tested using liquid based cytology, CLART HPV2 and Hybrid Capture 2 (HC2).At least one of the 35 defined genotypes was detected by CLART in 1,896 (37%) samples. The most frequent high-risk genotypes were HPV 16 (7%), HPV 52 (5%), and HPV 31 (4%). The most frequent low-risk genotypes were HPV 53 (5%), HPV 61 (4%), and HPV 66 (3%). Among 4,793 women targeted by the screening program (23-65 years), 1,166 (24%) tested positive for one or more of the 13 high-risk genotypes. This proportion decreased from 40% at age 23-29 years to 10% at age 60-65 years. On HC2, 1,035 (20%) samples were positive for any high-risk and thus CLART showed a higher analytical sensitivity for 13 high-risk HPV genotypes than HC2, and this was found in all age-groups and in women normal cytology.CLART performed well with a positive reproducibility for high-risk genotypes of 86%, and a negative reproducibility of 97%. This report furthermore updates the genotype distribution in Denmark prior to the inclusion of the HPV-vaccinated cohorts into the screening program, and as such represents a valuable baseline for future vaccine impact assessment.The online version of this article (doi:10.1186/1471-2334-14-413) contains supplementary material, which is available to authorized users. Human Papillomavirus (HPV) infection is a necessary cause of cervical cancer . At presSeveral HPV assays are now commercially available. The most frequently used assay, the Hybrid Capture 2 (HC2), detects a combination of 13 genotypes without individual genotyping. However, inclusion of HPV16 and 18 in the present vaccines, and a higher number of genotypes in next generation vaccines call for more detailed monitoring of genotypes. For this purpose, HPV assays with genotype resolution will be necessary. Commercially available full-genotyping assays, e.g. Linear Array  detecting 37 high- and low-risk genotypes, or INNO-LiPA  detecting 28 genotypes, have been extensively used in research. However, their results are most often read manually, and the tests are therefore prone to inter-observer variability making quality assurance for use in routine screening a challenge.The CLART HPV2  assay offers an alternative for genotype specific screening covering 35 individual HPV genotypes, including all 13 high-risk or probably high-risk genotypes. A set of primers to amplify a fragment of the human CFTR gene 892 bp) is included as a DNA control for sample adequacy, while an additional set of primers to amplify a spiked plasmid (1202 bp) is included as a PCR process control. The resulting visualized microarray is analysed on a computer guided reader with automated reading software. This reduces inter-observer variation in reporting the results, and is useful for high-throughput settings. Furthermore, CLART is a versatile high-throughput assay that can be applied to several sample types for HPV diagnostics, including formalin fixed paraffin embedded specimens –8. Versa92 bp is Earlier studies reported mixed results when the detection of HPV and cervical lesions by CLART was compared to that of HC2 –13. All The Department of Pathology of Copenhagen University Hospital in Hvidovre is the largest cervical screening laboratory in Denmark. While the study was on-going, it annually evaluated 66,000 cervical SurePath samples from women living in the municipalities of Copenhagen and Frederiksberg (denoted as Copenhagen in the remainder of the manuscript). The laboratory handles all cervical cytology regardless of the reason for sample-taking. Since the 1960’s, Copenhagen has been covered by an organized cervical screening program. At present, women aged 23-49 years are targeted for screening every three years and women aged 50-65 years every five years. In 2012, 75% of women had been screened at least once within the recommended interval .The Horizon study was nested into routine laboratory practice. Upon arrival in the laboratory, SurePath samples were sequentially arranged into racks of 48. Samples from the first one to four racks were collected for the study, equally from Monday to Friday, between 10 June and 25 August 2011. This collection method mimicked a cohort of unselected consecutive liquid-based cytology (LBC) samples, assuming that the time the sample arrived in the laboratory was not associated with the woman’s characteristics. Residual material was collected after completion of routine LBC which included HC2 triage of women aged ≥30 years with atypical squamous cells of undetermined significance (ASCUS). The volume of the collected residual material was approximately 2 ml per sample, and it was diluted with 2 ml of SurePath (dilution factor approximately 1:1) to obtain enough volume for additional testing reported elsewhere –20. All Cytological evaluation of SurePath samples was undertaken by cytoscreeners, though in collaboration with a pathologist in case of abnormal findings. Reading was assisted by FocalPoint GS Imaging System . The outcomes were reported using the Bethesda 2001 system. They were classified as negative for intraepithelial lesion or malignancy, ASCUS, low-grade squamous intraepithelial lesions (LSIL), or high-grade squamous intraepithelial lesions or worse (≥HSIL) including atypical squamous cells - cannot exclude HSIL (ASC-H), atypical glandular cells (AGC), adenocarcinoma in situ (AIS), and squamous cell carcinoma. Cytoscreeners and pathologists were blinded to the outcomes of HPV testing.CLART HPV2  is a low-density microarray assay based on PCR amplification of genotype specific HPV L1 fragments from 35 individual HPV genotypes , with analytical sensitivity calibrated against known copies of cloned plasmids.One ml of the diluted SurePath sample was spun down , with the supernatant removed and cell pellet resuspended in a mix of 180 μl phosphate buffered saline  and 20 μl Proteinase K . Samples were then vortexed and incubated for one hour at 56°C and one hour at 90°C. HPV DNA was purified using the MagNa Pure LC 96 and MagNA Pure LC 32 instruments with the MagNA Pure LC Total Nucleic Acid Isolation Kit . Five μl of purified DNA were used for the PCR amplification. During amplification the PCR products were labeled with biotin. Prior to visualization, the PCR products were denatured at 95°C for 10 minutes. Visualization was performed on the CLART microarray, using 10 μl of the denatured PCR products. Hybridization between the amplicons and their specific probes on CLART results in formation of an insoluble precipitate of peroxidase when adding a Streptavidin conjugate that bind to the biotin-labeled PCR products. Precipitate is analyzed on the Clinical Array Reader . All samples returning an invalid outcome were retested, and the second result was considered definitive.Samples for evaluation of CLART’s intra-laboratory reproducibility were collected, in part, within the Horizon study. To obtain sufficient numbers of positive and negative samples for the analysis, Horizon samples were supplemented with other routine screening samples. Extracted DNA was used for this purpose, in order to minimize any influence of the MagNA Pure step.® System  using scripts depending on pretreatment. A small number of samples from women with ASCUS aged ≥30 years, in whom HPV triage was undertaken routinely, were denatured and tested manually.HC2 is a hybridization assay detecting a combination of 13 high-risk genotypes, without an internal control for sufficiency of sample material. SurePath samples were either pretreated manually with DNA denatured prior to testing according to the manufacturer’s protocol, or DNA was isolated and purified using the DSP AXpH DNA kit on the QIASymphony SP platform . Testing of these samples was performed on automated Rapid CaptureThe study protocol and assay testing protocols were agreed upon with all manufacturers prior to the study. All instrumentation and software were used as supplied and maintained by the manufacturers.Women’s screening history from 1 January 2000 onwards was retrieved from the Danish Pathology Data Bank , where tχ2 test. Trends in HPV positivity by age were tested with the Mantel-Haenszel χ2 test for trend.CLART was assigned a positive result if it detected at least one of the 13 high-risk HPV genotypes that are included in HC2; HC2 if relative light unit to cut-off (rlu/co) value was ≥1, and cytology when ≥ ASCUS. Differences in the distributions of age, screening history, cytology and HC2 outcomes between included and excluded samples were tested with the Horizon was designed as a quality development study, utilizing only residual material that would otherwise have been discarded. According to Danish regulations of biomedical research, published on 5 May 2011 in the Guidelines about Notification etc. of a Biomedical Research Project to the Committee System on Biomedical Research Ethics No. 9154 section 2.5, quality development studies do not require ethical approval. The study was notified to the Danish Data Protection Agency in concordance with the current guidelines .Of the 12,138 consecutive samples received in the laboratory during the collection period, 6,258 (52%) were selected for the study. All selected samples were tested with HC2 except 23 (0.4%), and with LBC. In total, 1,190 (19%) samples were not tested with CLART owing to insufficient residual volume  or to human error (n = 25), and were excluded. Excluded samples were not significantly different from the remaining 5,068 samples in terms of age (P = 0.09), cytology (P = 0.55), and HC2 outcome (P = 0.31). There was a small statistically significant difference in the women’s screening history, with 13% of the included and 10% of the excluded samples being follow-up samples (P = 0.01). Five thousand and nine women contributed one sample , 28 contributed two, and one woman three samples. The mean age of the women was 37.3 years . The mean number of days between the receipt of the sample in the laboratory and storing was 2 (range: 1-5).At least one of 35 genotypes was detected by CLART in 1,896 (37%) samples , 1,166 (24%) tested positive for high-risk genotypes  of samples. The proportion of CLART positive samples increased by cytological grade; 1,001 (21%) samples with normal cytology were positive on CLART; 72 (59%) with ASCUS; 103 (73%) with LSIL; and 97 (92%) with ≥ HSIL (P < 0.0001). The small proportion of samples with only low-risk genotypes showed no trend by cytological grade (P = 0.28). Among women aged 23-65 years, HPV 16 was found in 235 (5%), and HPV 18 in 88 (2%) of 4,435 samples with normal cytology  samples were positive on HC2, HC2 being positive in 874 (69%) CLART-positive samples, and in 160 (4%) CLART-negative samples. The majority, 111 (69%), of CLART-negative/HC2 positive samples tested positive on CLART low-risk genotypes, indicating some HC2 cross-reactivity to low-risk HPV genotypes. HC2 showed slightly lower prevalence of high-risk HPV infections than CLART in all age groups; the prevalence decreased from 57% (92/162) at age 16-22 years, to 33%  at age 23-29 years, 17%  at age 30-39 years, 11% (112/991) at age 40-49 years, 7% (37/506) at age 50–59 years, 6% (14/234) at age 60-65 years, and 6% (7/112) at age >65 years. Compared to CLART, HC2 tested positive in fewer samples with normal cytology, 16% , but in more samples with low-grade abnormal cytology, 64% (79/123) in ASCUS, and 90% (128/142) in LSIL. Both assays tested positive on 92% (97/106) of samples diagnosed as ≥ HSIL; on 94 of these 97 samples both CLART and HC2 returned a positive result, whereas three samples tested positive on CLART and negative on HC2, and additional three samples tested negative on CLART and positive on HC2.When only the 4,410 primary screening samples without a recent abnormality were considered, 1,024 (23%) tested positive on CLART; 822 (19%) on HC2; and 241 (5%) had ≥ ASCUS  could be reproduced. Among the 17 (6%) non-reproducible samples, 8 showed high-risk infections in the second run. Hence, when only high-risk infections were considered as a positive result in the second run, the negative reproducibility was 97% (264/273).Among the 371 samples in which at least one of the 35 HPV genotypes was detected by CLART in the first run, 329 (89%) still had at least one genotype detectable in the second run. No genotype was detected in the second run among the remaining 42 (11%) samples, of which 28 initially showed infections with at least one high-risk genotype. Restricting the evaluation to the 283 samples with high-risk genotypes detected in the first run, 243 (86%) reproduced a high-risk genotype. An HPV 16 result from the first run (n = 71) was reproduced in 67 (94%) samples, and for HPV 18 (n = 30) this was the case in 25 (83%) samples.Assuming that in routine work, 25% had high-risk HPV on CLART Table , and 95%The Horizon study is the first population-based study to quantify cervical HPV infections using CLART HPV2 genotyping assay with cross comparison to HC2 on unselected screening samples stored in SurePath. Denmark has a high-risk, but well screened, population with respect to cervical cancer, and CLART detected at least one high-risk genotype in 25% of the samples, compared to 20% by HC2. The proportion testing positive decreased strongly with women’s age. In primary screening of women aged ≥30 years, 16% tested positive on CLART, and 12% on HC2; and most of these women had normal cytology. HPV 16 causing more than half of cervical cancers , was fouWe also evaluated CLART’s laboratory performance. Here, focus was on intra-laboratory reproducibility, as a lack thereof may pose problems for laboratories, e.g. in terms of their ability to verify the initial findings. In total, 94% of samples initially negative on all 35 genotypes were negative at retesting. When only high-risk genotypes were considered, this was 97%. Similarly, 89% of samples initially testing positive for at least one of the 35 genotypes, were positive at retesting. When only high-risk genotypes were considered, this was 86%. Taking the proportions of HPV positive and negative samples into account, overall 94% of samples were reproducible. This was similar to the reproducibility evaluated in the Horizon study of two other newly available commercial assays, Roche’s cobas  and HoloWe had to exclude 19% of the samples owing to insufficient volume of available residual material. However, there was no significant difference between the included and the excluded samples in terms of the women’s age, cytology, and the HC2 outcome. All samples were tested in one laboratory by the same staff, and the instrumentation was used as supplied and maintained by the manufacturers. Although there may be some differences between automated and manual DNA extraction, we used the former method in our study. This allowed us to evaluate CLART in a relatively high throughput fashion. This is important as manual extraction in a laboratory routinely running >10,000 HPV tests per year would be feasible neither from a patient safety nor from a quality control/quality assurance point of view. Although SurePath is a widely used screening medium for sample collection, no previous study has addressed the use of CLART technology for HPV detection on this media type, using automated extraction and testing.In 2004-2005, a total of 11,617 consecutive (screening) SurePath samples evaluated in the same laboratory were tested with HC2 . The medSo far CLART’s clinical performance has only been evaluated in a handful of studies, and several of these did not compare with that of HC2 , 23–26. Experts largely discourage use of HPV DNA testing in routine screening of women below age 30 years . In our me HPV test, BD Onclarity and APTIMA, was found in 3% (157/5,068; Table Several marketed HPV assays have partial or full genotyping. The added value for screening of detailed genotyping is widely debated, yet it has been suggested to use genotyping in triage of HPV-positive women to colposcopy . HoweverIn monitoring the effect of HPV vaccination, the use of genotyping assays will be very important. HPV vaccination is expected to change the dynamics of the screening programs by decreasing the background incidence of cervical cancer and hence also the need for treatment of CIN . Until nIn summary, this is the first investigation of performance of the CLART HPV2 genotyping assay in a population-based study using unselected screening samples collected in SurePath. CLART performed well with positive and negative reproducibility comparable to that of other commercially available assays. Moreover, CLART showed a higher positivity rate for 13 high-risk HPV genotypes than HC2. As HPV-vaccinated birth cohorts are soon to enter screening age, our study maps the last pre-vaccination HPV genotype distribution in Danish women, and can as such serve as reference for the monitoring vaccine impact.


Results showed that HPV vaccination was associated with being knowledgeable . Vaccination against HPV did not predict perception of sexual risk. Knowledge was low (only 87/385 or 22.6% of vaccinated girls were knowledgeable), but predicted perception of a high sexual risk . HPV vaccination, knowledge and perceived sexual risk did not predict sexual behaviour intentions. High parental communication was associated with adolescent attitudes that support postponement of sexual debut in both bivariate and multiple regression analyses. In conclusion, findings of this study suggest that HPV vaccination is not likely to encourage adolescent sexual activity. Influence of knowledge on sexual behaviour intentions was not definitively explained. Prospective cohort studies were proposed to address the emerging questions.The purpose of the study was to investigate the influence of human papillomavirus (HPV) vaccination on adolescent girls’ knowledge of HPV and HPV vaccine, perception of sexual risk and intentions for sexual debut. This cross-sectional comparative study was conducted in Ibanda and Mbarara districts. Data was collected using a standardized self-administered questionnaire and analyzed using the Statistical Package for the Social Sciences computer software. Univariate, bivariate, and logistic regression analyses were conducted with significance level set at  Globally, about 500,000 new cases of cervical cancer and about 274,000 deaths due to the disease occur annually. More than 80% of these deaths occur in developing countries, where cervical cancer is the leading cause of cancer deaths among adult women. This is projected to increase to 90% by 2020 . The humFrom 2008 to 2011, cohorts of adolescent girls in primary schools in Ibanda and Nakasongola districts in Uganda were vaccinated against the HPV using the bivalent vaccine . This was part of a demonstration project by the Government of Uganda and the Program for Appropriate Technology for Health (PATH) to evaluate different HPV vaccine delivery strategies. A school-based HPV vaccine delivery strategy was adopted in Ibanda targeting girls enrolled in primary grade five (P5). In Nakasongola, the HPV vaccine was delivered during the routine Child Days Plus (CDP) program, targeting girls of at least 10 years. Each eligible girl was to receive three doses of the vaccine administered in month 1, month 2 and month 6 . Prior tThis comparative cross-sectional study was conducted between November and December 2011. Girls vaccinated against HPV were drawn from Ibanda district while unvaccinated participants for comparison were selected from Mbarara district.Using multi-stage sampling, 3 rural sub-counties were randomly selected in Ibanda and 5 in Mbarara. Also, one urban sub-county was purposively selected in each district. Sixteen out of 120 schools were then randomly selected in Ibanda and 16 out of 66 schools in Mbarara, stratified by rural-urban location.In Ibanda, all assenting and consenting primary five and primary six girls present on the day of the survey who had received at least one dose of the HPV vaccine and whose parents or guardians gave written consent for their participation were recruited in the survey. Similar assent/consent criteria were followed for the unvaccinated comparison group in Mbarara. Using a formula for sample size calculation in studies comparing two proportions in terms of risk , a samplA pre-tested and translated self-administered questionnaire was used to collect data. Selected girls were assembled in a room on the day of the survey. Each girl filled the questionnaire under close supervision of the first author and research assistants who also explained to the girls the purpose and procedure of the survey. The questionnaire excluded respondents’ names to ensure their anonymity in the collected data. Data was collected first in Ibanda before Mbarara district.Independent variables were: HPV vaccination status, socio-demographic characteristics , parental communication and perceived peer norms about delayed sexual debut. Dependent variables were: knowledge about HPV and HPV vaccine; perceived sexual risk, and intentions for sexual debut. Questions on HPV vaccination status and socio-demographic characteristics required yes/no answers. Each of the other variables was assessed through responses to a set of questions or statements, scored on a Likert scale. Scores for all questions in a variable were added to generate a subscale for the variable. The subscales were analyzed as dichotomous variables.Parental communication was assessed by asking respondents how often their parents or guardians talked to them about their future and the dangers of pre-marital sex. Peer norms were assessed by asking each adolescent if most of her friends; would like to have sex at their current age, thought that one had to be older before having sex, thought she should not have sexual intercourse, and had been encouraging her to have sexual intercourse. Each adolescent was also asked if she believed sexual intercourse made a girl popular and if she believed sexual intercourse at her age was a “great” thing for a girl.Knowledge was assessed through responses to 14 factual statements, some correct and others incorrect. These were mainly based on information about HPV and HPV vaccine contained in two leaflets, “Your guide to prevent cancer of the cervix” and “What you need to know about cancer of the cervix and its prevention”. The leaflets produced by the Ministry of Health and partners were used for sensitization prior to HPV vaccination. The statements were: HPV is the primary cause of cervical cancer, HPV is usually sexually transmitted, men cannot get the virus that causes cervical cancer, HPV can be transmitted through skin-to-skin genital contact without sexual intercourse, use of condoms during sex can completely prevent HPV, life-long sexual abstinence prevents HPV, and two doses of HPV vaccine can effectively protect a girl against HPV infection. Others were: HPV vaccine can cure individuals who are already infected with HPV, HPV vaccine can cause infertility, HPV vaccine protects against HIV, HPV vaccine protects against other STDs, young girls who are infected with HPV can develop cervical cancer, Uganda is the first country in the world to use the HPV vaccine, and it is possible for a healthy-looking person to have the HPV. Each statement had three answer choices . The scale used to assess knowledge had good internal consistency (Cronbach’s α = .75).Perceived sexual risk was assessed by questions on currently perceived chance of acquiring HIV and other sexually transmitted infections (STIs), separately. Each question had five answer choices . Scores for HIV and other STIs were combined during analysis; the resultant two-item sexual risk subscale had inadequate internal consistency (Cronbach’s α = .61). Intentions for sexual debut was assessed using a 12-item subscale adapted from a study by Khan et al (2004); it had Data was analyzed using the Statistical Package for the Social Sciences (SPSS) version 16.0 . UnivariThe research, “Human Papillomavirus (HPV) Vaccination and Adolescent Girls’ Knowledge and Sexuality in Western Uganda: A Comparative Cross-Sectional Study” was approved by the Higher Degrees Research and Ethics Committee of the then Makerere University Medical School  (REC REF 2011–200) and the Uganda National Council for Science and Technology (REF SS 2478). Additional approvals for this research were obtained from relevant local government and school authorities. Prior to the interviews in each selected school, parents or guardians gave written informed consent by filling and returning a form sent from school through their respective targeted children a day before the survey date. In addition, on the day of the survey written informed assent was obtained from the targeted minors (below 18 years of age) while older girls gave written informed consent.p = 0.02).The overall mean age of girls was 13.3 years (SD = 1.39). The mean age of vaccinated girls  did not differ significantly with the mean age of unvaccinated girls . The results in p = 0.000).Knowledge was low; 102/670 or 17.6% of the adolescents overall were knowledgeable. A bivariate analysis to identify association of the girls’ background characteristics with their knowledge showed that significantly more vaccinated girls (87/385 or 22.6%) than unvaccinated (15/285 or 5.3%) were knowledgeable . Being a younger adolescent was significantly associated with perception of a low sexual risk . Knowledge was significantly associated with perception of a high sexual risk .Perception of sexual risk (risk for HIV and other STIs infection) was low; 17.9% of the girls overall perceived a high sexual risk. p = 0.008). Knowledge was significantly associated with perception of a high sexual risk even after controlling for other background variables (p = 0.008).Logistic regression analysis was conducted with perceived sexual risk as the dependent variable. Predictor variables were the significant socio-demographic variables in bivariate analysis, HPV vaccination status, and knowledge. As shown in p = 0.68). As expected, knowledgeable girls were more likely to support delayed sexual debut (60/100 or 61.0%) than the unknowledgeable (297/546 or 54.4%) although the difference was not statistically significant . Girls who perceived a high sexual risk were more likely to support delayed sexual debut (65/111 or 59.8%) than those who perceived a low risk (292/535 or 54.6%) but the difference was not significant .We expected that being vaccinated against HPV, being knowledgeable and perceiving a high sexual risk would be associated with adolescents’ support for PSD. Consistent with expectation, vaccinated girls (208/370 or 56.2%) were more likely to support delayed sexual debut compared to the unvaccinated (149/276 or 54.0%) but the difference was not statistically significant .Variables entered in the model of logistic regression to predict attitudes/beliefs about PSD were; parental communication that was significantly associated with intentions for sexual debut in bivariate analysis and the study hypothesized predictors: HPV vaccination, knowledge, and perceived sexual risk. The results are presented in It was anticipated that HPV-vaccinated girls were more knowledgeable, perceived a higher sexual risk, and were more likely to support delayed sexual debut owing to their exposure to health information during the HPV vaccination. This was premised on a hypothesis that vaccination against an STI could provide an opportunity to increase the girls’ awareness about their sexual risk when they become sexually active, and the need for prevention , 19.As expected in this study, HPV vaccination was associated with being knowledgeable. Notably, although significantly more vaccinated than unvaccinated girls were knowledgeable it is a minority (23%) of vaccinated girls that were knowledgeable. Low knowledge has been documented in other post-vaccination studies –26. HoweIn this study, vaccination against HPV was associated with knowledge and knowledge predicted perception of a high sexual risk. Contrary to expectation however, HPV vaccination did not predict perception of sexual risk. This could be attributed to the low level of knowledge even among vaccinated girls. Previous studies of risk perception after HPV vaccination reported that higher knowledge was associated with a higher awareness of the risk of sexual contact  and percWe expected HPV-vaccinated girls to be more likely to intend to delay sexual debut, but results showed that HPV vaccination, knowledge, and perceived sexual risk did not predict intentions for sexual debut. This was consistent with previous studies that showed no association of HPV vaccination with adolescent and young women’s sexual initiation and activity , 32–34. In this study, high parental communication irrespective of vaccination status predicted adolescent attitudes that were likely to support PSD. This was consistent with previous adolescent studies, not necessarily related to HPV vaccination, which reported PSD to be positively associated with parental communication –43. Our This study has some methodological limitations to be considered while interpreting the findings. The research design was cross-sectional, rendering it difficult to make definitive inferences about the direction of the observed relationships between dependent and independent variables. Adolescents’ self-reported perceptions of sexual risk and sexual behaviour intentions may change with time. The vaccinated P6 study participants had completed their vaccination about one year before while their P5 colleagues completed vaccination about one month prior to the date of data collection. It is therefore possible that long term socio-behavioural effects of the vaccination were yet to be evident. Since the P6 vaccinated girls were interviewed about one year after their vaccination, some of their responses could have been compromised due to recall bias. There was also a risk of contamination arising from comparing bordering districts. This and the risk of recall bias were minimised by limiting our assessment of knowledge to the information in leaflets that were distributed in the targeted schools. In all, the sample size was relatively large, thereby increasing the power of the study. Data that was collected and its subsequent interpretation are reasonably valid and reliable for programming and policy purposes.Levels of knowledge and perceived sexual risk were low among vaccinated girls despite the pre-vaccine sensitization. Yet, knowledge predicted perception of a high sexual risk. Sensitization strategies should be revised to increase their effectiveness, improve adolescents’ awareness of HPV sub-types not prevented by the currently available vaccines, HIV and other STIs as pertinent risks of sexual contact. This would likely pave the way for adoption of safer sexual behaviours which remain necessary even after individuals have been vaccinated against HPV. Our results showing parental communication as a steady predictor of attitudes that support PSD suggest that involvement of parents and promotion of parental communication would likely enhance effectiveness of strategies to promote safer sexual behaviours of adolescents after vaccination. HPV vaccination, knowledge and perceptions of sexual risk did not predict adolescent girls’ intentions for sexual debut. Therefore, our study adds to the body of evidence that vaccination against HPV is unlikely to encourage adolescent sexual activity.HPV vaccination was associated with being knowledgeable. HPV vaccination did not influence perception of sexual risk but knowledge did. HPV vaccination, knowledge, and sexual risk perception did not influence sexual behaviour intentions. This implies that HPV vaccination is not likely to encourage adolescent sexual activity. However, this study did not decisively explain the influence of knowledge on adolescents’ sexual behaviour intentions due to the low levels of knowledge even among vaccinated girls. Further prospective cohort studies are needed to get more definitive results on the possible behavioral effects of HPV vaccination and its accompanying knowledge. Future research would be more helpful if it could be interventional.