It has been suggested that adjustment for incomplete compliance with follow-up in women with positive human papillomavirus (HPV) tests would be appropriate for estimating the true sensitivity of cervical screening with HPV testing. We assessed the compliance and its impact on ⩾CIN3 detection in all eight randomised controlled trials (RCT) with published baseline-round data.We extracted data on recommended follow-up procedures, follow-up compliance, and ⩾CIN3 detection for both arms of each RCT, and assessed their correlation.P=0.33).Compliance with a direct referral for colposcopy was around 90% in all RCTs, whereas compliance with repeated testing among HPV-positive/cytology-negative women was around 60% in three RCTs and 73% in one RCT. Detection of ⩾CIN3 was significantly increased in two out of six RCTs with reported data. The correlation between compliance with follow-up in HPV-positive women and relative ⩾CIN3 detection was 0.48 (There is at present scant evidence to support the view that the measured sensitivity of HPV screening is a simple reflection of compliance with follow-up. Adjustment of measured cervical intraepithelial neoplasia detection on the basis of compliance data may not always be justifiable, and if adjustment is made, it should be used very judiciously. Organised screening programmes using cytological testing are proven to reduce both the incidence of and the mortality from cervical cancer. However, screening with this method is needed every 3–5 years, and even then the disease is not completely eradicated in screened women . Human pvs 84%. Therefore, compliance with follow-up among women with positive screening tests needs to be carefully scrutinised to make an adequate comparison of the sensitivity of HPV testing measured in different trials.However, in recently published data from the ARTISTIC RCT undertaken in the United Kingdom  with abnormal screening tests had a recommended follow-up procedure different from that of older women (35–60 years).Eight RCTs comparing HPV DNA cervical screening with cytology screening had their baseline-round data published by February 2010. Six of these RCTs were undertaken in Europe , one in Canada, and one in India . From thFor both arms of each RCT, we measured compliance with the recommended follow-up by calculating the proportion of screen-positive women who completed the recommended follow-up. For women referred for colposcopy we considered follow-up completed after the first colposcopy. For women not referred for colposcopy, we considered follow-up completed when all recommended repeated tests had been undertaken. Only follow-up with adequate repeated tests completed before the subsequent screening round was considered. We used the relative ⩾CIN3 detection as the indicator of relative sensitivity of HPV screening. The relative compliance and the 95% CI , as wellThe recommended follow-up procedures varied across RCTs, including a direct referral for colposcopy, or repeated testing at 6-, 12- or 18-month intervals with colposcopy recommended only in case of a positive outcome of the repeated testing  and 2. PCompliance with follow-up was dependent on the recommended follow-up procedure. Typically, around 90% of women complied with follow-up if they were immediately referred for colposcopy on the basis of their screening tests alone. In the intervention arms of the RCTs, this was found for women with ⩾ASCUS in Italy phase I, women with HPV+/cyt− tests in Italy phase I , suggesting a modest and non-significant correlation.Could this variation in relative ⩾CIN3 detection be explained by a variation in compliance with follow-up? As explained above, the complete compliance data were available from the Dutch, Italian, Canadian, and Indian RCTs only, whereas data on ⩾CIN3 detection were available from the European RCTs only. Relative compliance and relative ⩾CIN3 detection could therefore only be compared for the Dutch and Italian data  and 2. TP=0.33) in this case, that is, modest and non-significant correlation.Given this paucity of complete data, we additionally considered only the data on the absolute level of compliance among women with HPV+/cyt− or HPV+ tests, which were available for all European RCTs except of the Finnish. Using these data, we calculated the correlation between compliance with follow-up for HPV+/cyt− , and the Netherlands; Although compliance with immediate referral for colposcopy was high (∼90%) in all RCTs with reported data, low compliance with repeated testing was common in all RCTs recommending this procedure. Among HPV+/cyt− women, 55% completed repeated testing in the UK RCT, 58% in the Dutch RCT, 62% in the Italian phase I RCT (25–34 years), and 73% in the Swedish RCT. Furthermore, in the control arm of the Dutch RCT, a comparable proportion, only 66%, of women completed the recommended repeated testing.Our estimates of absolute compliance may have been affected by incomplete reporting of the RCT data. The United Kingdom, Italian, and Dutch RCTs did not systematically report the number of women with inadequate repeated tests. Because we assumed in such cases that all repeated tests were adequate, compliance with follow-up in terms of being ‘properly investigated’ may have been slightly overestimated. For the Dutch RCT, we could not determine, from the original report, whether the number with ‘completed repeat testing’  between compliance with follow-up and measured sensitivity of HPV screening. We used the data from the first reports on baseline data from the Italian RCTs  and 0.87 , respectively. Whereas the relative ⩾CIN3 detection could theoretically be above unity in the Canadian RCT, although not statistically significantly, this seems highly unlikely for the Indian RCT. The available data thus indicate a considerable variation in the relative ⩾CIN3 detection even in RCTs with good compliance with follow-up in both arms.P=0.33).Owing to the lack of completely reported compliance data from all European RCTs, we instead investigated the correlation between the absolute level of completeness of follow-up among HPV+/cyt− or HPV+ women and the relative ⩾CIN3 detection. There was a considerable variation in this compliance—from 94% in Italy phase II to 55% in the United Kingdom—but this was not reflected in a corresponding variation in ⩾CIN3 detection, resulting in a low correlation coefficient of 0.48 (However, in the interpretation of these outcomes, the paucity of the available data should be taken into account. To better elucidate the impact of compliance on the outcome of HPV screening, it is strongly recommended that comprehensive compliance data are published from all RCTs. Taken together, however, at present there is scant evidence from published reports to support the by now widely accepted view that the measured relative sensitivity of HPV screening is a simple reflection of relative compliance with follow-up.If adjustment for incomplete follow-up is applied to RCT data, both arms should undergo adjustment so as to not bias the comparison. However, previously, only adjustment for women in the intervention arm who had an HPV+/cyt− test was applied (Adjustment might, moreover, be problematic even when an unbiased estimate can be made. The European RCTs were undertaken to evaluate HPV screening in real-life settings. In this situation, an upwards correction of CIN detection due to incomplete compliance would be appropriate only if the level of compliance in the RCT was considerably lower than the level of compliance attainable in everyday screening practice. If, on the other hand, compliance with follow-up observed in the RCT is comparable with that attainable in the screening programme, CIN detection in the RCT can be seen as an acceptable estimate for the expected CIN detection. In this case, any further adjustment would lead to an artificial overestimation of ⩾CIN3 detection and would therefore affect the expected outcome of a shift from cytology to HPV screening.Incomplete compliance with recommended follow-up procedures for women with positive screening tests was found in several RCTs comparing HPV screening with cytology screening. Only some of the trials found the detection of ⩾CIN3 cases to be significantly increased in the HPV-screening arm. Follow-up compliance was dependent on the recommended follow-up procedures, but there was scant evidence of an unequivocal relationship between follow-up compliance and ⩾CIN3 detection. Adjustment of measured CIN detection on the basis of compliance data may not always be justifiable, and if adjustment is made, it should be used very judiciously.


P=0.31 for difference). Human papillomavirus was detected in 84% (78 out of 93) of patients. In univariate analysis, 80% (12 out of 15) of lesions without HPV regressed compared to 46% (36 out of 78) of lesions with HPV infection (P=0.016). Women without HPV and those who had a resolution of HPV had a four-fold higher chance of regression than those with persistent HPV . Women with five or fewer lifetime sexual partners had higher rates of regression than women with more than five partners (P=0.003). In multivariate analysis, HPV status and number of sexual partners remained as significant independent predictors of regression. In conclusion, HPV status and number of lifetime sexual partners were strongly predictive of regression of untreated CIN 2 and 3.The aim of this paper was to evaluate the factors that predict regression of untreated CIN 2 and 3. A total of 93 patients with colposcopic persistent CIN 2 and 3 lesions after biopsy were followed for 6 months. Human papillomavirus (HPV) types were determined by polymerase chain reaction at enrolment. We analysed the biologic and demographic predictors of natural regression using univariate and multivariate methods. The overall regression rate was 52% (48 out of 93), including 58% (22 out of 38) of CIN 2 and 47% (26 out of 55) of CIN 3 lesions ( Cervical cancer continues to be the most deadly female cancer in developing countries.  infection has been established as the critical step in the development of most cervical cancers . The cerThe aetiology and risk factors involved with cervical carcinogenesis are amenable to study as invasive cervical cancer typically arises after many years from a morphologically defined precancerous lesion. However, since untreated high-grade lesions can progress to invasive cancer, most women with CIN 2 or 3 are treated promptly. Accordingly, there are few reports on the natural history of high-grade dysplasia. On the other hand, not all CIN lesions progress to invasive cancer. In fact, spontaneous regression of moderate dysplasia has been reported in up to 54% of women . Since mWe evaluated the predictors for natural regression of untreated CIN 2 and 3 such as age, race, parity, smoking history, oral contraceptive (OCP) use, marital status, lesion grade, sexual history, and HPV status on all our patients.β-carotene in the management of women with untreated CIN 2 or 3. Predictors for natural regression, which included age, race, parity, smoking history, OCP use, marital status, lesion grade, sexual history, and HPV status were prospectively analysed. Patients were recruited from the University of California, Irvine, Medical Center, nearby clinics, and colleges. Women greater than 17 years of age with biopsy-proven CIN 2 and 3 were screened. Eligible patients had colposcopic persistence of CIN 2 or 3 one month after initial biopsy and had a negative endocervical curettage. Two pathologists agreed on the cervical histologic diagnosis before enrolment. Based on the guidelines of the Institutional Review Board at University of California, Irvine, written consents were obtained from all patients.We performed an ancillary study to a prospective, double-blinded, randomized, placebo-controlled trial of β-carotene and placebo arms at the end of the study  method was used to determine HPV genotype as described (χ2) and multivariate (logistic regression) analyses performed with Statistical Analysis Systems. All P-values reported are two-tailed.We analysed the age, race, parity, smoking history, OCP use, marital status, lesion grade, sexual history, and HPV status on all our patients. We assessed the impact of various epidemiological risk factors on regression of CIN in all our patients using univariate  patients completed our ancillary study and underwent a 6-month cervical biopsy. The 10 excluded patients did not have a documented persistence of CIN 2 or 3 after initial biopsy. The median age of our patients was 29 years (range 19–55). The majority (59%) presented with CIN 3 lesions and 41% had CIN 2 disease. Approximately one-half (46/93) were Hispanic and the others were either Caucasian (n=45) or Asian (n=2). Fifty (54%) women were married or have been married in the past. Twenty-four (26%) women have completed high school or college. The median parity was one. Forty-two (45%) women had a history of OCP use. Twenty-eight (30%) patients were smokers and nine of these women were smoking more than one pack per day. Of the 75 women who completed the sexual history interview, 54 (72%) women had their first sexual intercourse at 18 years or younger (range: 11–30) with a median of two lifetime sexual partners (range: 1–25). At enrolment, HPV infection was detected in 84% (78 out of 93) of women and most patients had HPV types that were considered high risk for progressing to cancer. The HPV types at baseline included HPV type 16 (42%), type 33 (12%), type 18 (9%), type 31 (8%), type 52 (3%), type 53 (3%), type 35 (1%), type 39 (1%), and mixed/others (22%). Types 16, 18, and 33 were the most prevalent HPV types and 11 patients were infected with multiple HPV types.From 1992 to 1996, 982 patients were screened for our study. We enrolled 124 women into our trial of whom 111 were eligible to be randomized to P=0.31 for difference between grades). Interestingly, 80% (12 out of 15) of lesions without detectable levels of HPV regressed compared to only 46% (36 out of 78) of those infected with HPV (P=0.016). Of the 78 patients who initially tested positive for HPV, 24% (19 out of 78) subsequently developed undetectable levels of HPV. Of the patients who had a resolution of their HPV infection, 63% (12 out of 19) showed regression of their disease compared to a regression rate of only 41% (24 out of 59) in those who had a persistence of their viral infection. After the study, the population was divided into two groups based on the number of lifetime sexual partners: less than or equal to five and more than five. There was a significant inverse relationship (P=0.003) between number of sexual partners and probability of CIN regression , including 58% (22 out of 38) of CIN 2 lesions and 47% (26 out of 55) of CIN 3 lesions (P=0.005), less than or equal to five sexual partners (P=0.003), and Hispanic race (P=0.05) were all significant predictors for regression of high-grade dysplasia. Age, parity, smoking history, OCP use, marital status, lesion grade, and age at first intercourse were not significantly associated with CIN regression (Table 2β-carotene vs placebo) in the multivariate model did not change the estimates of risk of regression associated with HPV infection or number of sexual partners.In univariate analyses, we found that absence of persistent HPV infection (gression . To furtOver the last 30 years, it has been widely accepted that CIN 1, 2, and 3 represent a continuum in the development of invasive cervical cancer . The bioAfter all 93 patients were analysed in our study, we found that the regression rate of CIN 2 and 3 without conventional therapy was unexpectedly high at 52%. Regression included 58% of CIN 2 lesions and 47% of CIN 3 lesions. In contrast, The presence of cervical HPV DNA often is associated with cytologic and histologic changes of CIN. Indeed, up to 90% of women with CIN are HPV DNA positive depending on the diagnostic method used . In our The role of HPV infection provides only a partial explanation for the high rate of spontaneous regression in our patient population. Sexual risk factors also have been recognised as causative determinants for CIN and cervical cancer. In our study, we found an inverse relationship between the number of sexual partners and CIN regression. Multivariate analyses of our study population showed that the effects of sexual risk factors persist even after controlling for HPV infections; suggesting that the presence of other sexually transmitted disease may also play a role in the natural history of CIN 2 or 3.Our data propose that there may be selected patients with CIN who could be followed for short term in anticipation of spontaneous regression; this group includes those with no evidence of an HPV infection and low sexual risk factors. Indeed, the ASCUS-LSIL Triage Study reached this conclusion for patients with ASCUS Pap smears in whom HPV is not present (


HPV infrequently persists and progresses to cervical cancer. We examined host genetic factors hypothesized to play a role in determining which subset of individuals infected with oncogenic human papillomavirus (HPV) have persistent infection and further develop cervical pre-cancer/cancer compared to the majority of infected individuals who will clear infection.OAS3), sulfatase 1 (SULF1), and interferon gamma (IFNG); the DNA repair genes deoxyuridine triphosphate (DUT), dosage suppressor of mck 1 homolog (DMC1), and general transcription factor IIH, polypeptide 3 (GTF2H4); and the EVER1 and EVER2 genes (p<0.01). From each region, the single most significant SNPs associated with CIN3/cancer were OAS3 rs12302655, SULF1 rs4737999, IFNG rs11177074, DUT rs3784621, DMC1 rs5757133, GTF2H4 rs2894054, EVER1/EVER2 rs9893818 (p-trends≤0.001). SNPs for OAS3, SULF1, DUT, and GTF2H4 were associated with HPV persistence whereas IFNG and EVER1/EVER2 SNPs were associated with progression to CIN3/cancer. We note that the associations observed were less than two-fold. We identified variations DNA repair and viral binding and cell entry genes associated with CIN3/cancer. Our results require replication but suggest that different genes may be responsible for modulating risk in the two critical transition steps important for cervical carcinogenesis: HPV persistence and disease progression.We evaluated 7140 tag single nucleotide polymorphisms (SNPs) from 305 candidate genes hypothesized to be involved in DNA repair, viral infection and cell entry in 416 cervical intraepithelial neoplasia 3 (CIN3)/cancer cases, 356 HPV persistent women (median: 25 months), and 425 random controls (RC) from the 10,049 women Guanacaste Costa Rica Natural History study. We used logistic regression to compute odds ratios and p-trend for CIN3/cancer and HPV persistence in relation to SNP genotypes and haplotypes (adjusted for age). We obtained pathway and gene-level summary of associations by computing the adaptive combination of p-values. Genes/regions statistically significantly associated with CIN3/cancer included the viral infection and cell entry genes 2′,5′ oligoadenylate synthetase gene 3 ( HLA) FANCA, associated with both HPV persistence and progression to CIN3/cancer. We also reported a variant in the innate immunity gene IRF3 associated with HPV persistence Persistent infection with one of approximately 15 types of human papillomavirus (HPV) is necessary for the development of cervical cancer and its immediate precursor, cervical intraepithelial neoplasia grade 3 (CIN3). However, HPV infection is not a sufficient cause of cervical cancer/CIN3 and HPV cofactors have been identified, including oral contraceptive use and smoking We evaluated data on 7,140 candidate single nucleotide polymorphisms (SNP) in 305 candidate genes/regions . All known DNA repair genes were included We found the DNA repair pathway as a whole statistically significantly associated with CIN3/cancer (p = 0.0197) and with HPV persistence (p = 0.0472) but not progression (p = 0.7451) . These aGTF2H4, DUT, and DMC1 – and the viral infection and cell entry related genes - OAS3, SULF1, and IFNG. The association for GTF2H4 and SULF1 were also associated with HPV persistence (p = 0.005). Three regions were statistically significantly associated with progression to CIN3/cancer at p<0.05: TMC6 (EVER1), TMC8 (EVER2), and FLJ35220. All gene-based results are shown in DUT (rs3784621), GTF2H4 , OAS3 (rs12302655), and SULF1  had significant p-trend<0.05. Of the genes/regions associated with progression to CIN3/cancer, SNPs in IFNG (rs11177074) and between EVER2/TMC8 and EVER1/TMC6 (rs9893818) were statistically significant at p<0.05. Odds ratios and 95% confidence intervals for these SNPs are shown in OAS1, OAS2, and POLN genes  for CIN3/cancer for one or more SNPs in eight of the nine genes/regions with p-trends ≤0.001 . No new regions of interest were identified in haplotype analysis using the sliding window approach of 3 SNPs (data not shown).Results from haplotype-based analyses (defined by blocks of linkage disequilibrium) were generally consistent with the gene/region- and SNP-based findings. Haplotypes statistically significantly associated with CIN3/cancer included SNPs implicated in SNP-based analysis  and three viral infection and cell entry related genes . A unique aspect of our study is the ability to separately evaluate associations with the two important transition steps in the natural history of cervical cancer – viral persistence and progression to precancer/cancer. Of the regions/genes found to be important, the association was primarily with HPV persistence for GTF2H4 and SULF1. IFNG and the epidermodysplasia verruciformis (EV)-associated genes (TMC6-EVER1 and TMC8-EVER2) were primarily associated with progression to CIN3/cancer. All top genes derived from the gene/region- and SNP-based analyses are annotated and briefly described in In this population-based study, we found nine genes/regions associated with CIN3/cancer, 6 of which remained significant at a FDR≤0.2 – three DNA repair genes (GTF2H4) and modulation of nucleotide pools (which includes DUT) to be associated with HPV persistence. The DNA repair family of editing/processing of nucleases was associated with progression to CIN3/cancer and though several genes were statistically significant, none were notable at FDR < 0.2. The association between the Fanconi Anemia family of genes with HPV persistence is also consistent with our previous analyses that reported FANCA variants associated with HPV persistence (5). There have been no reports of polymorphisms in GTF2H4, DUT or DMC with HPV persistence, cervical cancer, or any other cancer to date. However, GTF2H4 is located on chromosome 6p21.3 within the HLA region and is thus of note as a number of studies have investigated HLA Class II and I genes with cervical cancer and have consistently identified alleles  associated with cervical cancer GTF2H4 variants and HPV persistence is due to its biological function and its capacity as a DNA repair gene or to potential linkage disequilibrium with HLA requires further evaluation.Results for our DNA repair pathway-based analyses were consistent with our individual SNP-based results. Specifically, we found that genes within nucleotide excision repair  is induced by interferon. When enzymatically active OAS are bound to viral RNA, RNase L is activated, resulting in the degradation of viral RNA OAS1 and OAS2 SNPs were also associated with HPV persistence in our population (SULF1 (sulfatase 1) is involved in cell signaling and is a coreceptor for heparin-binding growth factors and cytokines. Sulfs potentially play a role in a cellular feed-back mechanism where they edit the sulfation of multiple heparin sulfate proteoglycans Variants in two genes postulated to play a role in viral and HPV binding, pulation . SULF1 , which is characterized by infection with HPV5 EVER1 and EVER2 are also associated with progression to CIN3/cancer may potentially suggest a larger role, though modest, for EVER1 and EVER2 genes in HPV susceptibility and subsequent risk for disease.Three genes were associated primarily with progression to CIN3/cancer including a priori genes and used a FDR of <0.2 to determine which genes were notable, we cannot exclude the possibility of false positives . Importantly, because our population comprises Costa Ricans and genes were tagged based on Caucasian and Yoruban populations where data are available from HapMap, it is plausible that gene coverage in our Costa Rican population is incomplete. Study strengths include our population-based study design. This design approximates a case-cohort design since the proportion of women in the cohort with CIN3 or cancer is small. Another study strength is our ability to evaluate genes relevant for HPV persistence and genes relevant for disease progression. Further, our study had rigorous follow-up, HPV testing and pathology review for case definitions. Our tag-SNP approach also allowed for broader coverage of each a priori gene/region investigation, compared to prior candidate SNP-based genotyping approaches. Our evaluation of the DNA repair pathway was near complete based on most recent literature and allowed for pathway-based analyses. However, our viral binding and cell entry genes were not amenable to similar analyses. We acknowledge that our a priori approach sampled a small subset of genes in the genome and we therefore likely missed other important associations. Increased power and replication of our results are required and larger efforts in genome-wide association studies should shed additional light on genes and regions of significance for cervical cancer. Our study, however, remains the only one designed to delineate between associations with HPV persistence and progression to CIN3/cancer.As described previously In summary, our results require replication but build upon our previous report of potential host genetic variants relevant for HPV persistence and those relevant for progression to CIN3/cancer. If replicated, additional studies to pinpoint the causal SNP(s) and determine their biological relevance should be pursued. Future efforts should include further dissection of long-term persistence that likely leads to progression compared to short-term persistence that is less likely to progress to CIN3/cancer. The role of host gene and HPV methylation and their role on causal genes should also be considered. These data are important for future research in evaluating the interplay between viral and host genetics in determining risk of HPV persistence and for progression to CIN3/cancer.The present study was nested within a population-based cohort study of women in Guanacaste, Costa Rica. Details of the cohort study methods Women selected for the genetic study included: (i) all women in the cohort histologically confirmed with prevalent or incident CIN3 or cancer (n = 184); (ii) all women in the cohort with evidence of HPV persistence, defined as women positive for the same HPV type at two consecutive visits (n = 432) (median length of persistence: 25 months); and (iii) a random selection of controls from the cohort (n = 492) The study was approved by both the US NCI and Costa Rica Institutional Review Boards and all subjects signed informed consent.DNA was extracted from buffy coats with PureGene purification kits/Autopure protocol (Gentra Systems) at SeraCare . For the supplemental cases the DNA extraction was done at the University of Costa Rica using the same kit/protocol.PCR-based HPV DNA testing with L1 MY09/MY11 consensus primer methods http://snp500cancer.nci.nih.gov) www.illumina.com). The Infinium included a total of 27,904 tag SNPs, of which our candidate genes/regions represented 7,765 SNPs. Tag SNPs for the 305 candidate genes were chosen from the designable set of common SNPs >5%) genotyped in the Caucasian (CEU) and Yoruban (YRI) population sample of the HapMap Project  using the software Tagzilla (http://tagzilla.nci.nih.gov/), which implements a tagging algorithm based on the pairwise binning method of Carlson et al. 2>0.8 to define a gene/region. When there were multiple transcripts available for genes, only the primary transcript was assessed.Genotyping of tag SNPs from 305 candidate genes/regions (Genes and SNPs annotated in Tag SNPs that failed manufacturing (ordered but did not convert), failed validation (no amplification or clustering) and assays that had less than 80% completion or 80% concordance with the 90 Hapmap CEU samples used for validation were excluded (n = 104). SNPs with low completion rate (<90% of samples) were further excluded (N = 138). SNPs with QC discordance among our 100 QC duplicates and among HapMap samples <98% were excluded (n = 383). We also excluded samples with a low completion rate (<90%) (N = 7). Hardy–Weinberg equilibrium was evaluated among controls. SNPs showing evidence of deviation from Hardy–Weinberg proportions  are denoted in We evaluated a total of 416 women diagnosed with CIN3 or cancer, 356 women with HPV persistent infection, and 425 random controls for whom validated genotyping results were obtained.We obtained pathway- and gene/region-based summary of associations using the adaptive combination of p-values We calculated odds ratios (OR) and 95% confidence intervals (95% CI) for each genotype with each disease outcome, using the homozygous wild type (WT) genotype as the referent group. We first compared CIN3/cancer cases to random controls. We further evaluated whether the statistically significant associations for CIN3/cancer were consistent for HPV persistence and/or disease progression with the following respective comparisons: (i) HPV persisters compared to random controls and (ii) CIN3/cancer cases compared to HPV persisters.Ptrend based on the three-level ordinal variable  of homozygote WT, heterozygote, and homozygote variant in a logistic regression model. For evaluating associations with HPV persistence, we also conducted analyses where HPV persistence and controls were restricted to any oncogenic-HPV infection . All logistic regression models were unconditional and conducted using SAS version 9.1 .We conducted both crude and analyses adjusted for age . For each outcome, we calculated the http://mayoresearch.mayo.edu/mayo/research/schaid_lab/software.cfm) on all genes. A global score statistic was used to summarize the evidence of association of disease with the haplotypes for each window. Second, we visualized haplotype structures for genes where p<0.01 for gene/region-based analysis using Haploview, version 3.11 We conducted haplotype analyses using two methods. First, we evaluated risk of cancer, progression and HPV persistence associated with haplotypes defined by SNPs within a sliding window of three loci across a gene  for data management and programming support. We are grateful to Amy Hutchinson and Belynda Hicks for their management of this genotyping effort at the NCI Core Genotyping Facility and for their scientific contributions to our research efforts. Results were presented in part at the 25Table S1(0.83 MB XLS)Click here for additional data file.Table S2(0.61 MB DOC)Click here for additional data file.Table S3(0.11 MB DOC)Click here for additional data file.Table S4(5.99 MB XLS)Click here for additional data file.Table S5(0.09 MB DOC)Click here for additional data file.


Epidemiologic research also indicates that heterosexual men with hiv, even in the absence of receptive anal intercourse, may have a high prevalence of precancerous anal lesions associated with hpvhpv in heterosexual men may provide important contrasts that will help clarify the picture of anal hpv-related morbidity and mortality in other populations.Although anal cancer is less common than cervical cancer, its incidence in U.S. men is increasing, to 1.6 per 100,000 in 2005 from 0.6 per 100,000 in 1973. During the same period, the incidence of cervical cancer in women decreased hpv types 16 and 18 hpv-induced carcinogenesis leading to cervical cancer is comparable to the process that leads to anal canal cancer. However, although the deployment of a preventive vaccine for hpv infection and cervical dysplasia reflects important progress in preventing cases of cervical cancer hpv vaccine studies in men and from modelling efforts that can provide quantitative insight into the economic and public health effects of the vaccine. However, accurate modeling of vaccine cost-effectiveness with regard to anal hpv in men requires more natural history data hpv in heterosexual men. Indeed, few studies have estimated anal hpv prevalence in heterosexual men.As in cervical cancer, most anal cancers are associated with oncogenic hpv in 222 asymptomatic heterosexual men, we recently estimated anal hpv prevalence at 16.6% for the anal canal and 21.3% for the perianal region. Of men with anal hpv, one third harboured an oncogenic hpv type. Also, men with anal hpv were more than twice as likely as men with no anal hpv to be in their 20s  hpv estimated anal hpv prevalences of 1.2% hpv dna detection.In a community-based study of anal hpv in heterosexual men, but they need to be confirmed in other populations of heterosexual men. We are currently conducting a three-country anal hpv prevalence study with men recruited by a collaborative team led by Dr. Anna Giuliano of the H. Lee Moffitt Cancer Center and Research Institute. Follow-up longitudinal studies are needed, because these prevalence data can blend (and obscure) critical natural history characteristics of anal hpv that are needed to inform prevention strategies.These prevalence data are a first step in understanding anal hpv in men—and especially in heterosexual men. Against this backdrop of scarce data, the incidence of anal cancer has risen considerably. Although knowledge about anal hpv in msm has advanced in the last decade , the much greater number of heterosexual men and the lack of epidemiologic data describing anal hpv in heterosexual men should prompt additional research in this area.Little is known about anal


Squamous cell carcinoma of the vulva is a disease of significant clinical importance, which arises in the presence or absence of human papillomavirus. We used comparative genomic hybridisation to document non-random chromosomal gains and losses within human papillomavirus positive and negative vulvar cancers. Gain of 3q was significantly more common in human papillomavirus-positive cancers compared to human papillomavirus-negative cancers. The smallest area of gain was 3q22–25, a chromosome region which is frequently gained in other human papillomavirus-related cancers. Chromosome 8q was more commonly gained in human papillomavirus-negative compared to human papillomavirus-positive cancers. 8q21 was the smallest region of gain, which has been identified in other, non-human papillomavirus-related cancers. Chromosome arms 3p and 11q were lost in both categories of vulvar cancer. This study has demonstrated chromosome locations important in the development of vulvar squamous cell carcinoma. Additionally, taken together with previous studies of human papillomavirus-positive cancers of other anogenital sites, the data indicate that one or more oncogenes important in the development and progression of human papillomavirus-induced carcinomas are located on 3q. The different genetic changes seen in human papillomavirus-positive and negative vulvar squamous cell carcinomas support the clinicopathological data indicating that these are different cancer types.British Journal of Cancer (2002) 86, 924–928. DOI: 10.1038/sj/bjc/6600112www.bjcancer.comCancer Research UK© 2002  Vulvar cancers affect 1.6 per 100 000 women in Victoria, Australia, 85% of which are squamous cell carcinomas (SCC) . There aThe process of HPV-related carcinogenesis within SCC of the anogenital region depends on functions encoded by the viral E6 and E7 genes. These genes bind with the p53 and Rb genes, respectively, inactivating their function in cell cycle regulation (Comparative genomic hybridization (CGH) is a powerful tool in the study of cancer that has fast gained recognition for its ability to scan an entire genome for DNA copy number changes that are mapped to chromosome regions. HPV-positive and negative vulvar cancers were analysed using CGH in order to determine specific chromosomal regions of DNA copy number gain and loss and to compare these changes with the HPV status of the tumours.The 18 patients with vulvar cancer used in this study were treated surgically between 1982 and 1999 at The Mercy Hospital for Women. Pathologic review was performed and the histologic type confirmed as SCC.2) were microdissected from paraffin-embedded formalin-fixed tissue sections and digested with proteinase K. MCF7 cell-line DNA and reference control DNAs from peripheral blood lymphocytes were extracted using a Progenome kit . DOP–PCR was performed on all reference and tumour samples and MCF-7 control DNA as previously described by CGH was carried out according to methods described in detail elsewhere  but this did not reach statistical significance. The increased number of changes in the HPV-positive group was due to an excess number of losses in these tumours . HPV-positive and negative cancers had a mean of 1.2 and 1.4 gains per case, respectively.The HPV analysis showed that there were 8 HPV-negative tumours and 10 HPV-positive tumours. All HPV-positive tumours contained subtype 16. Figure 1P=0.05 level, in 3q and 8q gain when analysed by HPV status, with 3q gain being more common in HPV-positive cancers , and 8q gain more common in HPV-negative cancers . There was no statistically significant difference by HPV status for 11q and 3p loss. Whilst many tumours showed whole chromosome arm alteration, minimal regions of gain and loss could be determined from some individual tumours. Thus the smallest areas of copy number gain were mapped to 3q22–25 and 8q21 and loss to 3p24, and 11q22-qter.Table 1In this study we have performed CGH analysis of SCC of the vulva, with known HPV status. To our knowledge, this is the first report of a CGH-based, genome-wide analysis of chromosomal gains and losses on this cancer type analysed by HPV status. Vulvar cancer differs from other SCCs of the anogenital region, since a substantial proportion of vulvar cancers are HPV-negative. The different HPV status of these cancers is reflected in their different clinicopathological parameters. We have utilised CGH to identify chromosomal loci important in vulvar oncogenesis in general, as well as those which differ according to the HPV status.Several regions of recurrent chromosomal abnormality were identified in vulvar SCCs. Notably gain of chromosome 3q was a frequent finding in HPV-positive SCCs (50%), but not detected in any of our HPV-negative cases. These data support gain of 3q as an important event in the oncogenesis of HPV-related SCCs since, in a previous study, we have shown that a similar proportion of stage 1b HPV-positive cervical SCCs harbour 3q gain . The smallest area of gain was 8q21, which is centromeric to the location of the proto-oncogene MYC. Gain of 8q, including the 8q21 region, has been previously documented in many different cancer types, the majority of which are not related to HPV infection (MYC. Although our data would suggest that 8q gain is important in the development of HPV-negative vulvar cancer, the number of cases in the present study are too small to clearly indicate whether 8q gain is redundant in the development of HPV-positive vulvar cancers, as is documented for mutations in the p53 gene (Recurrent gain of 8q was also detected in our series of vulvar cancers. Interestingly, this gain was much more frequent in HPV-negative compared to HPV-positive cancers (75% Only one previous study describing CGH analysis of vulvar carcinomas has been published (Our findings indicate that SCC of the vulva harbour several chromosomal alterations in common with other SCCs of the anogenital region. Additionally, there appear to be selected chromosomal regions in which copy number abnormalities are related to HPV status. Identification of candidate genes in these areas of recurrent chromosomal copy number abnormality would give important insights into the development and progression of vulvar and other anogenital cancers.


The study includes 181 HPV-positive controls who did not develop high-grade cervical intraepithelial neoplasia (CIN3) during follow-up, 203 HPV-negative controls, and 199 HPV-positive cases with histologically confirmed CIN3. Detailed information on sexual, reproductive and gynaecological history, oral contraceptive use and smoking was obtained at face-to-face interview. There was a striking division between risk factors for infection and those predictive of disease. Comparing the HPV-positive against the HPV-negative controls, the only risk factors for infection were number of sexual partners , a relatively recent new sexual relationship , and a history of previous miscarriage . The determinants of CIN3 among infected women were, in contrast, early age at first intercourse , a long time since starting a new sexual relationship , and cigarette smoking, with strong evidence for a dose– response . Oral contraceptive use was not significantly associated with either HPV infection or CIN3. © 2000 Cancer Research Campaign


To evaluate the prevalence of human papillomavirus (HPV) types, and risk factors for HPV positivity across cervix, vagina and anus, we conducted a study among 138 women with human immunodeficiency virus (HIV).Compare the prevalence of different HPV types and the risk factors for HPV positivity in three sites.The most frequently detected HPV types in all sites were, in decreasing order, HPV16, 53, 18, 61 and 81. Agreement between the cervix and vagina was good (kappa 0.60 – 0.80) for HPV16 and 53 and excellent (Kappa > 0.80) for HPV18 and 61. HPV positivity was inversely associated with age for all combinations including the anal site.In HIV positive women, HPV18 is the most spread HPV type found in combinations of anal and genital sites. The relationship of anal to genital infection has implications for the development of anal malignancies. Thus, the efficacy of the current HPV vaccine may be considered not only for the cervix, but also for prevention of HPV18 anal infection among immunossuppressed individuals. More than 1.7 million people were living with human immunodeficiency virus (HIV) infection in Latin America at the end of 2004, and Brazilian women made up more than one-third  of this population [Epidemiological and laboratory data have demonstrated that persistent infection with high-risk human papillomavirus (HPV) types  is an essential step in the development of invasive cervical cancer and the majority of anal cancer [In addition, compared to HIV-negative women, HIV-positive women show a higher occurrence of multiple-type HPV infection -10 and aIn this study we have compared the prevalence of different HPV types and the risk factors for HPV positivity in three sites .3 in 24.6% women, between 200–499 cells/mm3 in 37.0%, and < 200 cells/mm3 in 38.4%.Study methods have been reported elsewhere . BrieflyThe same gynecologist applied a standard questionnaire for all patients to elicit information on sociodemographic characteristics, smoking habits, history of drug addiction, sexual behavior and use of condom. The Ethics Committee of the Medical School of the University of São Paulo, Brazil, approved the study protocol, and all participants signed informed consent forms.The same gynecologist collected exfoliated cells from 1) ectocervix, 2) vagina and 3) anal area, in that order, with separate sterile brushes, and stored in separate flasks in phosphate-buffered saline solution (PBS). To obtain samples for anal collection, individual disposable brushes moistened in sterile PBS were inserted into the anal canal and were rotated 360° as they were withdrawn.After collection, cells remaining in the collection devices were washed out into a sterile buffered solution and kept at -20°C for HPV analysis. Beta-globin amplification was used to assess specimen adequacy for HPV DNA detection. Polymerase chain reaction (PCR)-based assays were carried out in the Laboratorio de Patologia e Hemoterapia do Hospital do Cancer, Fundação Antonio Prudente, São Paulo, Brazil .et al. [et al. [HPVX).PCR-positive samples were further typed by restriction fragment length polymorphism according to Bernard et al. , allowin [et al. . Other Hvs vaginal or anal sites. The concordance rate was considered fair to good if Kappa values were between 0.60–0.80, and excellent if greater than 0.80. Agreement for any HPV type is defined as presence of at least one common HPV type in the 2 or 3 combined sites. ORs for detection of HPV infection and corresponding 95% CIs were calculated separately for each anogenital site and for each combination of 2 or 3 sites by means of unconditional regression equations adjusted for age . The analyses were repeated with adjustment for age and CD4 count .The Kappa statistic was used to calculate the agreement between HPV positivity at the cervical The prevalence of HPV infection among HIV-positive women did not differ significantly between the three anogenital sites under study. The prevalence was 61.6% in the cervix, 65.9% in the vagina, and 63.7% in the anal area. The same similarity is seen when high-risk HPV types are considered, in the cervix (36.2%), vagina (42.8%) and the anal area (43.1%). In respect to low-risk HPV types, the prevalence was 34.1% in the cervix, 35.5% in the vagina and 33.3% in the anal area. Multiple HPV infections occurred in 41.2% of HPV-positive cervical samples, 48.4% of HPV-positive vaginal samples and 44.6% of HPV-positive anal samples. The prevalence of HPV types according to the risk category in the studied female population was described elsewhere .In the cervix, the most frequent high-risk HPV types were HPV16 and 53 (n = 13 each type) and HPV18 (n = 12)  (Table vs 2.9% in each other site).In the anal area, the most frequently detected high-risk HPV types were, in decreasing order, HPV53 (n = 13), 18 (n = 12), and 16 (n = 9). The most frequent low-risk HPV types were HPV 61 and 6 (n = 8 for each). In 11.8% of anal samples the HPV type could not be characterized. HPV6, but not HPV11, tended to be more frequent in the anal area than in the cervix or vagina  and varied from 92.8% (HPV16) to 98.6% (HPV61 and HPV6). Kappa values above 0.80 were found for HPV18, 33 and 61. When cervical and anal sites were compared, agreement for positivity for any HPV type was found in 68.6% of women  , but not elsewhere. Nevertheless, condom use did not show associations with HPV positivity at all sites.et al. [Approximately two-thirds of HIV-positive women in our study from Brazil were infected by HPV and the prevalence of HPV infection was similar in the cervix, vagina and anal area. This also held true for the prevalence of the most frequently detected individual HPV types, notably HPV11, 16, 18, 53 and 61, and for the frequency of multiple-type infections. However, the same HPV type(s) were not always present in the three examined anogenital areas and the agreement in the detection of individual types was better between the cervix and the vagina than between the cervix and the anal area. The type most frequently detected at all three sites was HPV18, mainly on account of its high prevalence in the anal area. Recently, Castle et al.  reportedet al. -22.The factors most clearly associated with HPV positivity among HIV-positive women in our study agree with findings from HIV-negative women ,24. AlthTwo characteristics stand out as being more strongly associated with HPV positivity in the anal area than in the cervix and vagina, one being history of anal intercourse. This finding suggests that anal infection is associated among women, as it is among bisexual and homosexual men, to direct HPV exposure ,26. AnalOne of the limitations of this work is its small sample size. As a consequence, many relatively strong associations did not attain statistical significance. Furthermore, the cross-sectional design does not allow for the capture of the dynamic nature of HPV infections. One of the strengths of this study is the wide range of HPV types tested for at different anogenital sites in HIV-positive women.In conclusion, our study showed that high prevalences of HPV infection can be found in the entire lower genital tract in HIV-positive women. In particular, the anal area may represent a reservoir of HPV infection, notably HPV6 and 18. It will thus be important to evaluate the efficacy of the current vaccine against HPV ,30 to pr


INK4a (CDKN2A) and HPV (human papilloma virus) in different grades of conjunctival intraepithelial neoplasia (CIN).The aim of our study was to identify the frequency of expression of p16INK4a and MIB1. The presence of HPV was examined by PCR.Twelve specimens including CIN I (2), II (3), III (5), and CIN with beginning invasion (2), as well as 15 control specimens, were stained with antibodies against p16p16 as well as MIB1 were significantly elevated in CIN compared to control specimens (p<0.01) without correlation with the differentiation grade. Only two cases with CIN grade 3 contained HPV 16.INK4a expression, p16INK4a seems not to be a very reliable marker for the exact determination of CIN. It could serve as an additional diagnostic tool besides the morphological characterization. Our study suggested that p16INK4a elevation is not associated with HPV infection.As few control specimens also showed increased p16 Conjunctival intraepithelial neoplasia (CIN) presents as a spectrum from simple dysplasia to carcinoma in situ. CIN commonly arises in the limbal region and particularly occurs in elderly males who have lived in areas exposed to high levels of UV-B radiation , an assuet al. in 1998 [Recently our immunohistochemical analysis showed a significantly higher expression of p63 in CIN and carcinoma compared to the normal specimens without correlation with the differentiation grade and MIB-1-rate , indicat in 1998 . Here weINK4a  was shown to indicate malignancy and progression into carcinoma [INK4a restrains the progression of the cell cycle by inhibiting the activity of CDK (cyclin dependent kinase) 4, the latter targets pRb for phosphorylation and abolish pRb inhibition of E2F, driving cell cycle progression. In the presence of HPV infection, the HPV E7 protein binds to pRb and displaces E2F with subsequent E2F activation and cell cycle progression from the G1 to S phase and mitosis .In cervical intraepithelial neoplasia, p16arcinoma , 9. p16IDue to pRb inactivation, p16 increases and becomes immunohistochemically detectable -12.INK4a and HPV in different grades of conjunctival intraepithelial neoplasia in order to find out if HPV-vaccination could also be important in order to prevent this disease.The aim of our study was to assess the frequency of expression of p161) excised from the bulbar conjunctiva with the suspicion of conjunctival intraepithelial neoplasia (CIN) and 14 macroscopically normal postmortem conjunctival specimens and 1 conjunctival specimen with slight inflammatory changes  were fixed in 4% formaldehyde in 0.075 M phosphate buffer for 24 h, dehydrated in increasing concentrations of ethanol (70%-99%) and infiltrated with paraffin  at 60°C. Sections of 3 μm thickness were cut and floated on deionized water at 45°C, and single sections were mounted on Superfrost Plus glass slides . Slides were subsequently dried at 60°C for 1h. The haema-toxylin-eosin stained patient slides were diagnosed histologically as follows: 2 CIN grade I (up to 25% of the whole thickness of the specimen shows dysplasia), 3 CIN grade II (25-75% of the whole thickness of the specimen shows dysplasia), 5 CIN grade III , and 2 CIN with beginning invasion . The 15 macroscopically normal conjunctival specimens and 12 CIN specimens were stained immunohistochemically using the avidin biotin method including antigen retrieval using a microwave oven for 10 minutes at pH 6.0, before the mouse monoclonal antibodies p16INK4a Ab-4  and Ki-67  were applied for 2h. The negative controls used only showed occasional and slight cytoplasmic background staining. Cells that were considered positive for either p16INK4a or MIB-1 clearly showed a reddish stained nucleus, and some displayed additional cytoplasmic positive staining.The study was approved by the ethic comitee of the Albert-Ludwig University Freiburg, Germany, written informed consent was obtained from the study participants. 12 conjunctival specimens ; few specimens were almost or completely negative for p16 expression . The proportion of p16-positive cells in the normal postmortem conjunctiva and the conjunctival specimen with inflammatory changes varied between 0 and 51.8%, and the mean was calculated at 16.4%.Most of the normal conjunctival specimens showed stronger p16 expression in the basal layers, compared to the more superficial cell layers .In contrast to observations in normal specimens, many of the CIN specimens stained positive for p16, exhibiting a similar trend to that in normal specimens, with a stronger intensity in the basal layers compared to the superficial layers ). No correlation was found between p16 expression and the differentiation grade (correlation coefficient 0.2).The percentage of p16-positive cells in the CIN specimens was higher than that in normal specimens, and varied between 27.2 and 83.9%, with the mean at 53.6%. Notably, p16 expression was significantly elevated in CIN samples compared to the control specimens .6).Analysis of CIN samples revealed that MIB-1 expression was elevated in the dysplastic specimens, though not to the extent of p16 expression . Neither a correlation between differentiation grade and MIB-1 positive staining (correlation coefficient 0.2) nor between p16 and MIB-1 expression (correlation coefficient 0.4) was found.None of the 15 control specimens were found to contain HPV, while β-actin expression was detected in 14 of the controls. Only 2 of the CIN specimens (case 6 and 8) contained HPV type 16, and both were classified as grade III; eleven of the CIN specimens were found to express β-actin.Cervical intraepithelial neoplasia has been shown to be associated with HPV-infection in more than 99% of the cases , 17. Infet al. [et al. found p16 elevated in conjunctival squamous cell carcinoma [et al. in cervical intraepithelial neoplasia [1)) and subsequent p16 elevation. However, our results indicating the control specimens as HPV negative did not support the above-mentioned hypothesis. Furthermore, only 2 of the 12 CIN specimens turned out to be HPV positive, with type 16 detected. This is in line with the results of Tulvatana et al. who examined 30 CIN specimens and found negative HPV and positive β-globin DNA in 16 cases [et al., who did not find any HPV in 20 malignant epithelial conjunctival tumours [et al. found HPV in 10 CIN cases (5 with HPV 16 and 5 with HPV 18) with negative results in the control specimens. In each of those CIN specimens, 20%–40% of dysplastic cells expressed HPV E6 [Our study shows increased p16 expression in conjunctival intraepithelial neoplasia compared to normal conjunctival specimens. This result is in contrast to that of Kuo et al. , whose Carcinoma , which ceoplasia . This is16 cases , and Eng tumours . In contd HPV E6 . These sIn most of the cases presented here, p16 elevation was not found to be HPV-induced, but seems to be caused by another agent with or without the involvement of pRb status change. p16 did not correlate positively with differentiation grade and MIB-1-positivity, which might indicate that in some cells p16 elevation successfully prevented cell cycle progression beyond the restriction point.In conclusion, p16 seems to be a malignancy indicator in conjunctival epithelial tumours, especially towards CIN, but caution must be applied as normal conjunctiva also occasionally displays high p16 expression. Our study shows that HPV is not the only oncogenic agent in CIN etiology and HPV vaccination seems not to prevent CIN in future.


The natural history of cutaneous HPV is unclear and in particular, seroprevalence among individuals with different levels of immune function and ethnicity is unknown. As part of a study of cutaneous squamous cell carcinoma (SCC) and HPV among organ transplant recipients (OTR) from London, we investigated the seroprevalence and risk factors for 34 HPV types (detected using Luminex technology) among 409 OTR patients without skin cancer (243 Caucasians and 166 non-Caucasians), 367 individuals with end stage renal failure on dialysis (222 Caucasians and 145 non-Caucasians) and 152 immunocompetent (IC) individuals without skin cancer (102 Caucasians and 50 non-Caucasians) to compare the HPV seroprevalence in patients with differing immune status and ethnicity. In total, seroprevalence data from 928 individuals, all from London, was available.Overall, no difference between HPV seroprevalence by immune status was observed (P = 0.3) among Caucasian or among non-Caucasian individuals, with seroprevalence varying from 87% to 94% across different immune status and ethnic groups. Those individuals seropositive to multiple types of one genus were more likely to be seroreactive to multiple types of another genus, independent of immune status or ethnicity. Lower seroprevalence for gammaHPV 4, and to a lesser extent gammaHPV 48, were observed among OTR compared to IC and dialysis patients. Higher seroprevalence against antibodies to betaHPV 93 were detected more frequently in non-Caucasians than Caucasians whereas muHPV 1 and, to a lesser extent, gammaHPV 4 were found more frequently among Caucasians - these findings were independent of immune status. Within non-Caucasian subgroups, the seroprevalence of 8 HPV  was significantly (P < 0.02) higher in Black compared to Asian patients. HPV16 being sexually transmitted, this might suggest a potential sexual route of transmission for some beta HPV types.We did not observe major disturbance in antibody response between immunocompetent, dialysis and OTR individuals, but significant differences in HPV seroprevalence were identified according to ethnicity. Further research is needed to clarify the natural history of cutaneous HPV, particularly given the growing research interest in its possible role in the pathogenesis of cutaneous SCC. Genital human papillomaviruses (HPV6 and HPV11) are the most common sexually transmitted infectious agents in the UK . A higheHere, we investigate the seroprevalence for 34 HPV types detected using Luminex technology among people in London with different immune status and ethnicity. Information on 409 OTR without skin cancer (243 Caucasian and 166 non-Caucasian) and 152 immunocompetent (IC) individuals (102 Caucasians and 50 non-Caucasians) were included. Patients with end-stage renal disease on dialysis (222 Caucasians and 145 non-Caucasians), at increased risk of infections and cancers ,8, were The present study was conducted as part of research examining the relationship between antibodies against HPV-L1 antigens for 34 HPV types and cutaneous squamous cell carcinoma among OTR. Further details of the study methods can be found elsewhere . BrieflyIn order to assess whether the seroprevalence of HPV was affected by immune status, IC patients were also recruited, and included Caucasians and non-Caucasians without a history of skin cancer enrolled from ophthalmology, plastic surgery or phlebotomy departments. A short questionnaire on basic socio-demographic details  and skin cancer history was completed and a blood sample was obtained. In order to evaluate the influence of renal failure pre-transplantation on HPV seroprevalence, stored sera from Caucasian and non-Caucasian dialysis patients with no history of transplantation, were also included. Skin cancer history was not available for these patients, although it was known that none had previously attended the dermatology department for treatment of skin cancer. Basic socio-demographic details  were provided from the hospital renal database.In total, information on 409 OTR without skin cancer (243 Caucasian and 166 non-Caucasian of whom 5 patients [1%] with a solid organ graft other than kidney), 152 immunocompetent (IC) individuals (102 Caucasians and 50 non-Caucasians) and 367 patients with end-stage renal disease on dialysis (222 Caucasians and 145 non-Caucasians) were included.The study has been approved by the East London and City Health Authority Research Ethics Committee.S-transferase (GST) capture enzyme-linked immunosorbent assay, as previously described [E. coli as double fusion of full-length viral proteins with a N-terminal GST domain and a C-terminal peptide consisting of the last 11 amino acids from the large T antigen of simian virus 40 [2 = 0.97) [HPV antibody detection was performed by multiplex serology, an antibody detection method that is based on a glutathione escribed ,11 in coescribed ,13. All virus 40 . The expvirus 40 ,11,14. Gvirus 40 . The dif = 0.97) ,15. MoreTo assess the relationship between seropositivity to a single HPV type and ethnicity or immune status, logistic regression adjusted for sex, age at recruitment  and, if appropriate, time since transplantation  was applied. Where results are presented in the form of plots, black squares indicate the point estimates and horizontal lines represent 95% confidence intervals (CI). The area of the square is proportional to the amount of statistical information available (inverse of the variance of the logarithm of the estimate). To examine the association between multiple HPV seropositivity and ethnicity or immune status, negative binomial regression adjusted for the same factors was used since over-dispersion was observed when Poisson models were fitted (likelihood ratio test for the null hypothesis of no overdispersion was rejected with P < 0.001). Linear regression was used to compare the means between several groups. Skin type of non-Caucasians was defined using Fitzpatrick classification scale as follows (V) Asian, Middle Eastern and (VI) African/Afro-Caribbean. Detailed ethnicity information was only available for OTR and was defined as Caucasian  and non-Caucasian . Associations between age at recruitment, sex and HPV seropositivity was examined by immune status using logistic regression among Caucasian patients.Sensitivity analysis was performed to compare HPV seroprevalence among IC, dialysis and transplant patients with kidney graft only. To deal with multiple significant tests the level of statistical significance was set to 1% and when a sufficient number of patients was available, agreement of results across population was used to detect genuine associations. Missing value categories were added to adjustment variables with incomplete information in order to retain all the observations in the analyses. Likelihood ratio tests were used to assess heterogeneity tests. All P-values are two-sided. Statistical analyses were carried out using STATA 9 .Table Of the 166 non-Caucasian transplant recipients, 54% identified themselves as Asian, 31% as Black, 8% as Far Eastern and 7% as other ethnic group. In terms of country of birth (excluding missing data for 8% of Caucasians and 33% of non-Caucasians), 96% of Caucasian OTR were born in Europe and 83% of non-Caucasians were born outside Europe .Figure Figure Table In Table The only consistent finding across ethnicity groups was lower HPV4 seroprevalence and, to a lesser extent, gammaHPV 48 among OTR compared to IC or dialysis patients. Among Caucasian patients, seroprevalence of antibodies against betaHPV8 was 24% in OTR compared to 15% among dialysis and 11% among immunocompetent individuals . Otherwise, HPV seroprevalence differed little for most types across the different immune status groups, after adjustment for age and sex. For multiple HPV seropositivity, there was also no statistical difference at the 1% level of significance between the three immune status groups among Caucasian patients whereas seropositivity to multiple mucosal alpha HPV types tended to be higher among dialysis and immunocompetent patients compared to OTR (data not shown).Little is known of the seroepidemiology of HPV, with the exception of those mucosal types associated with cancer of the uterine cervix  or with As expected , mucosalet al., this issue), again suggesting a possible sexual route of transmission for some beta types.To our knowledge, no previously reported studies have investigated the association between the prevalence of cutaneous HPV antibodies by ethnicity. Consistently across immune status, we observed statistically significant differences between Caucasians and non-Caucasians: betaHPV 93 was higher in non-Caucasians, whereas muHPV type 1 and to a lesser extent HPV4 seroprevalence were lower. As most Caucasians were born in Europe and non-Caucasian outside Europe we were not able to distinguish if the observed differences were confounded by birth country. There were some limitations to our analyses on ethnicity as diverse groups  with different birth country were all pooled in the non-Caucasian category. Geographical studies are essential to examine further the HPV seroprevalence among different ethnicities. Studies on sexually transmitted diseases such as gonorrhoea and chlamydia based in London ,18, BirmIn relation to immune status, we observed that seroprevalence of most HPV types did not differ substantially, after controlling for age and sex, between Caucasian IC individuals, dialysis patients without a history of transplantation and OTR, suggesting apparently low disturbance in production of antibodies by immunological status. The only notable exception was a lower HPV4 seroprevalence among OTR compared to dialysis or IC patients independent of ethnicity. Unfortunately, we do not have complete information on the type of dialysis  to explore whether this specifically affected seroprevalence. The observed differences in overall seroprevalence by genera among non-Caucasian patients might be due to the pooling of very heterogeneous groups of non-Caucasians .In summary, we did not find major differences in the prevalence of antibodies against 34 HPV types among people with different immune states. Ethnicity did show significance differences, but results should be interpreted with caution since ethnic groups with different birth country, cultural and socioeconomic backgrounds were pooled together. Further research is needed to clarify the risk factors and the natural history of these viruses.HPV: human papilloma virus; OTR: organ transplant recipients; IC: Immunocompetent; OR: odds ratio; CI: confidence interval.The authors declare that they have no competing interests.CP, CH and RN conceived the study. LM, TW and DC participated in acquisition of all the biological material and data. MP, TW and KM developed the HPV assays and KM analysed the samples. DC analysed the data and drafted the manuscript. All authors read, contributed to and approved the manuscript.


Aims. To investigate the possible associationof human papillomavirus (HPV) with endometrial hyperplasias andneoplasia. Does HPV play any role in the initiation orprognosis of endometrial adenocarcinomas?Methods. Twenty-five endometrial adenocarcinomasof the endometrioid cell type, with and without squamousdifferentiation, and twenty-four endometrial hyperplasias of variousforms  were analyzed for thepresence of type 16 and 18 HPV by the polymerase chainreaction (PCR). The results were related to histopathologicalfeatures of the tumour, and the patients' age, andprognosis. Results. Six of 25 endometrialadenocarcinomas were HPV 16-positive(Conclusion. It appears that thepresence of HPV in the endometrium, as detected by PCR, doesnot play any role in the initiation or prognosis ofendometrial adenocarcinoma. Human papillomavirus(HPV) infection in the female genital tract has been connected with specificsites: vulva, vagina, cervix, and specific epithelium—the stratified squamousepithelium, leading to epithelial cell proliferation and often malignancy. Theinfection, most reliably detected by polymerase chain reaction (PCR), is recognized by distinct histological changes in epithelial cells consisting of multinucleation and koilocytosis , 2. TypiA number of reports havedemonstrated the presence of HPV in endometrial adenocarcinomas –8, a tisIn view of this paucity ofinformation, this study was designed to evaluate the presence of HPV in theendometrium by PCR and its potential role in the genesis of endometrialadenocarcinoma.The tissues used for this study were drawn from the files of theDepartment of Pathology, Democritus University of Thrace Medical School . They were all hysterectomy specimens and had been routinely fixed in10% formol-saline. The original haematoxylin and eosin- (H and E-) stainedsections consisted of 24 endometrial hyperplasias of various forms: simple,Democritus University of Thrace Medical School .Paraffin sections were cut at 7  aguide . Normal 2, 1 U Platinum Taq Polymerase , and 50 pmol each of the GP5+(5'-TTTGTTACTGTGGTAGATACTAC-3') and GP6+(5'-GAAAAATAAACTGTAAATCATATTC-3') primers.Polymerase chain reaction amplification conditions were 96°C (1 minute), 45°C (1.5 minutes) and 72°C(1 minute) for 40 cycles, followed by final extension at 72°C for 10 minutes.Isolated DNA was subjected to GP5+/GP6+ PCR .Briefly'-CCCAGCTGTAATCATGCATGGAGA-3' andReverse: 3'-CACACGGGTAATTCAGAAGGT-5'generating a 253 bps PCR product. TheHPV 18 specific primers were as follows: Forward: 5'-CGACAGGAACGACTCCAACGA-3' and Reverse:3'-TCAATTTAGTAGTTGTAAATGGTCG-5' generating a 201 bps PCR product. PCRamplification was carried out in 25 2, 200 Taq Polymerase . Polymerase chain reactionamplification conditions were 96°C (30 seconds), 60°C (30 seconds), and 72°C(30 seconds) for 35 cycles, followed by final extension at 72°C for 10 minutes.Single PCR was carried outusing type-specific primers to investigate the incidence of both HPV 16 and HPV18 sequences in GP5+/GP6+ samples. The HPV 16 specific primers were as follows: Forward: 5The amplifications werecarried out in a Mastercycler gradient  thermal cycler and the PCRproducts were visualized in ethidium bromide stained agarose gels (2%).In all PCR assays, appropriatepositive controls for HPV 16 (human Caski cell line DNA), HPV 18 (human HeLAcell line DNA), and cervical squamous cell carcinomas were used and identified.In addition, the commercially provided positive controls for the identificationof HPV 16 and HPV 18 by Maxim Biotech, Inc  were applied successfully. Human lung and liver tissues were tested asnegative controls and were consistently negative. Allreactions were performed in a “blinded” manner by DP.Statistical analysis of the data was performed using Fisher’s exact test.Humanpapillomavirus (HPV) 16 was a somewhat more common inhabitant than HPV 18 inendometrial neoplasms and in all forms of endometrial hyperplasia. It wasdetected in 24% (versus 20% of HPV 18) of the endometrial adenocarcinomasstudied, and in 16.6% (versus 4% of HPV 18) of the endometrial hyperplasias. Both tyDetectionof HPV 16/18 DNA sequences in primary endometrial adenocarcinomas by PCRamplification products in representative specimens are shown in Figures None of the HPV 16 and 18-positive cases, whether hyperplastic or neoplastic,demonstrated cellular evidence of viral infection, that is, koilocytotic atypiaor multinucleation. There was no statistical correlation between HPV 16/18 andsquamous differentiation, depth of myometrial invasion, lymphatic involvement,lymphocytic response, patients’ age, or survival (data not shown).The detection ofhuman papillomavirus (HPV) in the human endometrium, whether hyperplastic orneoplastic, is fraught with curiosities. HPV was thought as being site- andtissue-specific, infecting the stratified squamous epithelium of the lowerfemale genital tract: vulva, vagina, and exocervix , most coDetectingHPV DNA sequences in tissues originally fixed in formaldehyde and embedded in paraffin wax may provedifficult by PCR methods , and theGiventhat HPV infection precedes the development of cancer , it is aInterestingly, the presenceof HPV in endometrial neoplasms was unrelated to histopathological features,patients’ age, or patients’ survival. This is also the experience of otherinvestigators . It is rSince HPV 16/18 infectionis, by and large, site- and tissue-specific , the endometrium, as indeed the glandular lesions of the endocervix,may not be a suitable host for HPV replication and maturation. This is furthersupported by the absence of relevant epithelial changes, lack of correlationwith histological features or prognosis, and the low incidence rates withprecancerous endometrial lesions. There is, of course, some evidence thatkoilocytotic-like changes may occur in the squamoid component of someendometrial adenocarcinomas with squamous differentiation , 10 and


Senescence                        is blocked in human cervical keratinocytes infected with high risk human                        papillomavirus (e.g. HPV type16). Viral oncoproteins HPV E6 and HPV E7                        access the cell cycle via cellular p53 and retinoblastoma proteins                        respectively. Previously we have shown that HPV E7, not HPV E6, is also                        responsible for cervical cancer cell survival . We                        now present evidence that SIRT1, an aging-related NAD-dependent                        deacetylase, mediates HPV E7 survival function in SiHa cervical cancer                        cells. Moreover, HPV E7 up-regulates SIRT1 protein when expressed in                        primary human keratinocytes. Conversely, SIRT1 levels decrease following                        RNAi-mediated silencing of HPV E7 in SiHa cells. Silencing HPV E6 has no                        effect on SIRT1 but, as expected, causes marked accumulation of p53 protein                        accompanied by p53-mediated up-regulation of p21. However, p53 acetylation                        (K382Ac) was barely detectable. Since p53 is a known SIRT1 substrate we                        propose that elevated SIRT1 levels (induced by HPV E7) attenuate p53                        pro-apoptotic capacity via its de-acetylation. Our discovery that HPV E7 up-regulates                        SIRT1 links a clinically important oncogenic virus with the                        multi-functional SIRT1 protein. This link may open the way for a more                        in-depth understanding of the process of HPV-induced malignant                        transformation and also of the inter-relationships between aging and                        cancer.                 The processes of aging and cancer share                        many characteristics despite their opposing phenotypes of senescence versus                        immortalisation . In bothThe yeast model provided                        the initial evidence linking SIRT1 with aging. Thus Sir2, the ancestral                        homologue of SIRT1, is a yeast NAD-dependent deacetylase and longevity factor ,17. In Much of our understanding                        of the molecular biology of cancer derives from early studies in which DNA                        tumour viruses were employed and the mechanisms by which they induce cell                        transformation from normal to cancerous growth were elucidated. Indeed studies                        with DNA tumour viruses led to the initial discovery of p53, the major tumour                        suppressor in humans . One ofin vivo).                                HPV E6 and E7 are expressed                        as bicistronic mRNA. In this current work we have exploited a previous                        unexpected observation, namely that in HPV16-positive SiHa cells it is possible                        to selectively and individually silence E6 and E7 by RNA interference (RNAi)                        despite the contiguous nature of their mRNAs . Here weIt is already established that HPV16 E6                        targets the p53 protein for rapid degradation with consequential loss of p53                        tumour suppressor functions, including maintenance of chromosomal integrity. We                        have now discovered that the second HPV viral oncogene, HPV16 E7, targets                        SIRT1. Specifically we demonstrate (i) that exogenous expression of HPV E7 (but                        not HPV E6) in primary human keratinocytes induces abnormally high levels of                        the SIRT1 protein, similar to those observed in human cervical cancer cells,                        and (ii) that HPV E7 (but not HPV E6) is required to maintain the abnormally                        high levels of SIRT1 protein expressed in cervical cancer cells. The ability of                        HPV E7 to up-regulate SIRT1 appears to be linked with HPV E7-mediated                        suppression of apoptosis in cervical cancer cells  since ouOur discovery that HPV E7                        influences the expression of SIRT1 provides the first link between an oncogenic                        virus and the aging-related SIRT1 protein, and enables access to diverse                        SIRT1-dependent cellular regulatory systems. This link may open the way for a                        more in-depth understanding of the inter-relationships between aging and                        cancer. It may also provide insight into the mechanism of chromosomal                        re-distribution during the switch from latent to malignant HPV viral infection,                        and also for the maintenance of HPV-induced malignancy.                                For                            this study we exploited the ability of RNAi to separately knockdown the viral                            oncogenes HPV16 E6 and HPV16 E7 in HPV16-positive SiHa cervical cancer cells.                            Individual knockdown of HPV E6 and E7 by their respective siRNAs was verified                            by quantitative RT-PCR .                            Treatment with trichostatin A, a class I and II histone deacetylase inhibitor,                            induced a dramatic increase in both K9Ac and K14Ac of histone H3 (data not                            shown) indicating high deacetylase activity at these sites in SiHa cells.                            Neither E6 nor E7 significantly affected K9 methylation of histone H3 in SiHa                            cells Figure .       To explore how E7 silencing might decrease S10P we examined levels of aurora B and                            survivin. Aurora B is the principal histone H3 S10 kinase in human cells  and itsIn                            the mouse model for BRCA1 tumor suppressor function there is evidence that wild                            type BRCA1 suppresses survivin expression via SIRT1-dependent epigenetic                            modification of histone H3 . Our ownE6                            silencing caused a delayed reduction in survivin protein levels Figure  39-41].-41.39-41HPV E7 silencing also resulted in reduced mRNA levels of survivin Figure         1 cells from ~60% to ~11% was                            observed 72h post-transfection  were unaffected by HPV E7 knock-down (data not shown)                            demonstrating that the reduction in SIRT1 protein levels reflects a selective effect                            of HPV E7 depletion. In contrast to HPV E7 depletion, the depletion of HPV E6                            had no effect on levels of SIRT1 protein . The                            HPV E7 effect on SIRT1 could be a late event in the process of malignant cell                            transformation by the HPV virus. Alternatively HPV E7-mediated up-regulation of                            SIRT1 might play a causative role in malignant cell transformation. In favour                            of the latter alternative we show that SIRT1 suppresses apoptosis in SiHa human                            cervical cancer cells . Primary human keratinocytes                            were transfected with HPV E6 or HPV E7 expression vectors and exogenously                            expressed E6 and E7 proteins were detected by immunoblotting  formulated into liposomes as described [                escribed ,30,45,46escribed .       escribed ,30,45,46E6                                and E7 cDNA cloning and exogenous expression.                 Mammalian                        expression vectors for HPV16 E6 and HPV16 E7 were generated by subcloning HPV16                        E6 cDNA or E7 cDNA, freshly cloned from low passage SiHa cells, into the                        multiple cloning site of pcDNA3.1. Generated constructs were verified by                        sequencing. DNA transfection was performed using lipofectamine (Invitrogen)                        according to the manufacturer's instructions as previously described [                escribed .       mRNA                                quantification.                 Total cellular RNA was isolated using RNeasy kit                        (Qiagen) [0C                        for 30min; 950C for 15 min; 30 cycles of 940C for 30 sec,                        580C for 45 sec, 720C for 1 min; followed by 720C                        for 5 min. For aurora B, primers 5'-ACAGACGGCTCCATCTGGCCT -3' and 5'-TCAGGCGACAGATTGAAGGGCA-3' were                        used in the thermal cycle: 500C for 30min; 950C for 15                        min; 50 cycles of 940C for 30 sec, 580C for 45 sec, 720C                        for 1 min; and then 720C for 5 min. Cycle parameters and primers for                        HPV E6, HPV E7, SIRT1 and lamin A/C mRNAs were as previously described [                (Qiagen) . 100ng RImmunoblotting.                 Total cell                        extracts were prepared from transfected cells [                ed cells  and    ed cells . ComparaCell                                cycle analysis and quantitation of apoptosis.                 Cell cycle                        analysis, and cell aggregate discrimination and histogram deconvolution using                        Cylchred software, were as previously described [                escribed ,20,45,46escribed .


The development of effective strategies against cervical cancer in Africa requires accurate type specific data on human papillomavirus (HPV) prevalence, including determination of DNA sequences in order to maximise local vaccine efficacy. We have investigated cervical HPV infection and squamous intraepithelial lesions (SIL) in an unselected cohort of 1061 women in a rural Gambian community. Squamous intraepithelial lesions was diagnosed using cytology and histology, HPV was typed by PCR-ELISA of DNA extracts, which were also DNA sequenced. The prevalence of cervical HPV infection was 13% and SIL were observed in 7% of subjects. Human papillomavirus-16 was most prevalent and most strongly associated with SIL. Also common were HPV-18, -33, -58 and, notably, -35. Human papillomavirus DNA sequencing revealed HPV-16 samples to be exclusively African type 1 (Af1). Subjects of the Wolof ethnic group had a lower prevalence of HPV infection while subjects aged 25–44 years had a higher prevalence of cervical precancer than older or younger subjects. This first report of HPV prevalence in an unselected, unscreened rural population confirms high rates of SIL and HPV infection in West Africa. This study has implications for the vaccination of Gambian and other African populations in the prevention of cervical cancer. It is well established, on a cellular and epidemiological basis, that the sexual transmission of high oncogenic risk (HR) human papillomavirus (HPV) types is the essential prerequisite for cervical carcinogenesis . ConsequSamples were collected during a large reproductive morbidity survey in rural Gambia between January and July 1999 as described elsewhere  or knife cone biopsy. Histology and cytology results were subjected to clinical review to decide on a final diagnosis viz low- or high-grade squamous intraepithelial lesions (LSIL or HSIL) , microinβ-globin gene in order to verify the presence of an adequate DNA sample and the absence of PCR inhibitors. The presence of HPV and β-globin DNA was visualised on an agarose gel. Human papillomavirus typing was preformed by enzyme linked immuno-sorbent assay (ELISA) using streptavidin coated microtitre plates to capture PCR product  (http://www.ncbi.nlm.nih.gov/BLAST/) search was carried out for each of the consensus strands produced by sequencing at least twice in each direction. Alignment analysis was carried out using the Lasergene suit of software . The sequences most homologous to the Gambia samples are listed in Samples that could not be identified by ELISA were characterised by DNA sequencing. Selected HPV positive samples were also sequenced to confirm typing accuracy and to detect any relevant sequence variations in known HPV types. Sequencing was carried out on a Beckman Coulter CEQ2000 Automated Sequencer, using protocols and reagents supplied by the manufacturer. Human papillomavirus DNA sequencing utilised the MY09/MY11 consensus primer pair as the product of these (≈455 bp) is a more suitable target for sequencing than that of GP5+/6+ (≈140 bp). Furthermore, MY09/11 primers amplify a region containing highly conserved amino acids, which simplifies sequence alignment in HPV classification . Frequency distributions of HPV infection and different HPV types were calculated. Socio-demographic characteristics and other morbidities were examined as risk factors for HPV infection by cross tabulation and c groups . An adjuβ-globin human housekeeping gene was amplified in 710 out of 934 (76%) samples; consequently, only these samples were suitable for HPV analysis. The β-globin negative samples may have been incorrectly stored or damaged during transfer to the UK, prevalence figures are therefore based upon the 710 β-globin positive samples. Of the three main ethnic groups, Wolof women were slightly under-represented (38.7% of eligible population vs 32.1% of those with adequate cytology and 34.1% of those with adequate HPV samples) and Mandinka women were slightly over-represented (43.9% of eligible population vs 49.5% of those with adequate cytology and 46.2% of those with adequate HPV samples). Younger women aged 15–24 years were under-represented .Of 1871 eligible women, 1348 (72%) participated in the study  and 1061Human papillomavirus infection was present in 95 of 710 adequate samples ). Adjusting HPV prevalence for under-representation of Wolof subjects reduced prevalence slightly to 13.0%. Human papillomavirus typing by PCR-ELISA was successful in 84 samples revealing 6 different LR and 13 different HR types . Dual ceHuman papillomavirus DNA was amplified with the GP5+/6+ primers in 11 samples but were negative by ELISA, and therefore contained HPV types not included in the probe cocktails. These samples were sequenced with the MY 09/11 primers; five had either degenerated in storage or would not amplify, two contained multiple templates and were impossible to sequence, while four were successfully sequenced and identified but none were infected with novel types, merely types not included in the ELISA probe cocktail .DNA sequencing was conducted on samples successfully identified by ELISA as HPV types associated with HSIL. All HPV-16 samples sequenced showed DNA homology in the amplified region with a variant of HPV-16 African type 1  . Both HPn=22; 16 confirmed by histology), LSIL were found in 3.3%  and atypical squamous cells of uncertain significance (ASCUS) in 1.1% (n=10). A single case of invasive cervical cancer was diagnosed by clinical and cytological criteria but the woman sadly died before the histology could be confirmed; it was included in the HSIL analysis.The overall crude prevalence of cervical precancer was 6.7% (63/946 adequate smears (95% CI: 5.2–8.4%)), or 6.5% after adjusting for the under-representation of Wolof women Cytological abnormalities were confirmed by histology in 55.6% (35/63) of cases with HSIL present in 2.3% (χ2P=0.03) and cervical HPV infection. Rates of SIL were highest at ages 25–34 years (22 out of 265 (8%)) and 35–44 years (24/268 (9%)), intermediate at ages 45–54 (9/150 (6%)) and lowest at ages 15–24 (8/263 (3%)). In subjects where both adequate cytology/histology and HPV analysis were available, 574/612 (93%) had normal cytology, 38 out of 612 (6%) had SIL and seven out of 612 (1%) had ASCUS. Of the subjects with normal cytology 525/574 (91%) were negative for HPV while in those with SIL/ASCUS 20/38 (53%) had HR HPV types. Of subjects with HSIL, 13/15 (87%) had HR HPV types, although the other two were negative for HPV. Among subjects with HSIL, HPV-16, HPV-33 and HPV-58 were most common, each found in 3/15 (20%). HPV-16 was found in the single invasive cancer case. HPV-18, -31, -35, -39 and -45 occurred once each among subjects with HSIL. Of 30 subjects with LSIL/ASCUS, seven were infected with HR HPV types; of subjects with normal cytology 66/576 (12%) had HPV infection.Risk factors for cervical precancer included age  . Moderatet al was urban  and the higher HPV prevalence may be explained by differing sexual behaviour as well as the much higher prevalence of LR HPV types in the Nigerian study than in our study.This first unselected study of cervical HPV and SIL in an unscreened, rural West-African population of its kind shows HPV infection to be common at 13.4% (adjusted=13.0%) with the majority HR types. Although younger subjects were under-represented, such an age profile is comparable with other studies. The high cervical HPV infection prevalence and SIL are in agreement with Gambian Cancer Registry data  in this study and meta-analysis of worldwide HPV prevalence studies shows low overall prevalence of HPV-35 (≈2%), even in Africa . No HPV samples in this study were found to be novel types after identification by sequencing, although some were unusual subtypes or variants . The DNAHistologically confirmed cervical neoplasia prevalence in our study was 6.7% (adjusted=6.5%). Diagnostic accuracy was improved by classifying subjects with abnormal cytology but normal histology as ‘normal cervix’. The prevalence of abnormal cytology alone was slightly higher at 7.4% (70/946 adequate smears). Other investigators have used a cytological screening to identify cervical disease providing a less accurate prevalence of SIL (In this study, HPV infection was not associated with age, parity or concurrent STI. The even distribution of HPV prevalence at different ages is unusual though observed in other developing countries such as India  and ArgeThe ideal treatment strategy for this rural Gambian population would involve the use of a prophylactic vaccine to prevent cellular viral entry. Current prophylactic vaccine formulations require three needle injections, cold storage and offer only type specific protection (The ethnic groups in our study population have not previously been studied using these methods. Other studies in African populations using the same methods to those used in our study (