Host genetic factors might affect the risk of progression from infection with carcinogenic human papillomavirus (HPV), the etiologic agent for cervical cancer, to persistent HPV infection, and hence to cervical precancer and cancer.PRDX3, and ribosomal protein S19 RPS19. The single most significant SNPs from each gene associated with CIN3/cancer were PRDX3 rs7082598 (Ptrend<0.0001), and RPS19 rs2305809 (Ptrend=0.0007), respectively. Both SNPs were also associated with progression.We assessed 18,310 tag single nucleotide polymorphisms (SNPs) from 1113 genes in 416 cervical intraepithelial neoplasia 3 (CIN3)/cancer cases, 356 women with persistent carcinogenic HPV infection (median persistence of 25 months) and 425 randomly selected women (non-cases and non-HPV persistent) from the 10,049 women from the Guanacaste, Costa Rica HPV natural history cohort. For gene and SNP associations, we computed age-adjusted odds ratio and p-trend. Three comparisons were made: 1) association with CIN3/cancer (compared CIN3/cancer cases to random controls), 2) association with persistence (compared HPV persistence to random controls), and 3) progression (compared CIN3/cancers with HPV-persistent group). Regions statistically significantly associated with CIN3/cancer included genes for peroxiredoxin 3 RSP19 and PRDX3, or other SNPs in linkage disequilibrium, with cervical cancer risk. Further investigation showed that they may be involved in both the persistence and progression transition stages. Our results require replication but, if true, suggest a role for ribosomal dysfunction, mitochondrial processes, and/or oxidative stress, or other unknown function of these genes in cervical carcinogenesis.These data suggest involvement of two genes,  While it is well-known that carcinogenic human papillomaviruses (HPVs) are the causal agents of cervical cancer, HPV infections are extremely common relative to rare cancer incidence, indicating that many infections spontaneously resolve The role of host genetic factors and other co-factors associated with cervical cancer are particularly interesting because the stepwise pathogenesis of the disease has been extensively studied. From its initiation through HPV infection at the cervical transformation zone, and subsequent steps related to viral persistence, progression to precancer, and invasion a priori hypotheses of association with HPV infection and cervical cancer . That effort identified 8 potential genes associated with cervical cancer, including the immune genes 2′,5′ oligoadenylate synthetase gene 3 (OAS3) and sulfatase 1 (SULF1), and the epidermal dysplasia verruciformis (EV)-associated EVER1 and EVER2 genes, TMC6 and TMC8 a priori hypothesized relationship with a wide range of cancers, but not specifically with HPV persistence and cervical cancer. These genes were selected based on the collective effort of numerous cancer researchers and include genes in several immune, cytokine and inflammation response, DNA replication and differentiation, macrophage differentiation, toll-like receptor signaling, and T cell receptor signaling pathways, to name a few, and presumably have lower prior probabilities of association with cervical cancer than those studied in the report of Wang et al. We used data from the well-characterized, longitudinal cohort study on HPV natural history (NHS) in Guanacaste, Costa Rica, and recently reported results from a panel of 7,140 candidate single nucleotide polymorphisms (SNPs). These polymorphisms were chosen to represent variation in 305 genes based on PRDX3 p-value 0.00015; RPS19 p-value 0.00045; DDX1 p-value 0.0006; TELO2 p-value 0.0009; C1RL p-value 0.00165; ILDR1 p-value 0.00285; THRAP4 p-value 0.0037; GDF10 p-value 0.004; and GDF2 p-value 0.004). Two gene regions were identified as statistically significantly associated with disease progression at p-value of ≤0.005 . In addition, 2 gene regions were identified as significantly associated with type-specific HPV persistence . Of the genes identified to be associated with CIN3/cancer RPS19 was also associated with progression to CIN3/cancer . Similarly, of the genes associated with CIN3/cancer, C1RL, GDF10, and GDF2 were also associated with persistence . Only two of the genes - PRDX3 and RPS19, were notable with a FDR≤0.2 comparing CIN3/cancers to random controls. All gene-based results are shown in PRDX3 rs7082598 minor allele variants was associated with statistically significant decreased risk of cervical cancer compared to controls , and this protective effect was observed for both progression (CIN3+ compared with HPV-persistence) and HPV-persistence (persisters compared to controls) . The RPS19 rs2305809 T allele was also associated with statistically significant decreased risk of cervical cancer . Further examination by intermediate steps showed that this effect remained for progression from persistence to CIN3+ . Lastly, the IL2RA rs2476491 T variant was also associated with significantly decreased risk of cervical cancer . This association was strongest for progression from HPV-persistence to CIN3+ .Six SNPs in 6 genes identified by the gene-based analysis were significantly associated with CIN3/cancer, disease progression, or persistent oncogenic HPV infection  in SNP-bTELO2 rs4786772 , C1RL rs12227050 , and TYMS rs2342700  were associated with significantly increased risk of cervical cancer  retained significance in this analysis when comparing CIN3+ vs. random controls .We also carried out a single statistical analysis including all SNPs, and found that the SNP RS7082598  and for HPV-persistence.Effects of putative genetic etiologic factors for cervical cancer may be mapped to specific transition states from HPV infection of the cervical transformation zone, progression of persistently infected cervical cells to precancer and invasive cancer. In this study we had the opportunity to investigate and identify genes which may influence some of these stages of cervical carcinogenesis. We note that the protective effects observed for RPS19 have been associated with Diamond-Blackfan anemia (DBA), which is a constitutional erythroblastopenia characterized by absent or decreased erythroid precursors, in a subset of patients. Patients with DBA have increased risk of osteosarcoma. This association with DBA suggests a possible extra-ribosomal function for this gene in erythropoietic differentiation and proliferation, in addition to its ribosomal function. In some primary colon carcinomas, higher expression levels of this gene have been observed compared to matched normal colon tissues PRDX3 is in the peroxiredoxin family which encodes a protein with antioxidant function, is localized in the mitochondrion, and may function to protect mitochondria from oxidative stress. Sequence comparisons with recently cloned mammalian homologues suggest that these genes consist of a family that is responsible for regulation of cellular proliferation, differentiation, and antioxidant functions Mutations of the ribosomal gene PRDX3 and RPS19) were similarly distributed between NHS and supplemental cases, justifying combining the two cohorts. Additionally, this study was powered on a combined end-point of CIN3 and cancer; 95.5%  of the SNPs were not statistically different between CIN3 and cancer cases. Importantly, none of SNPs associated with the two genes of interest (PRDX3 and RPS19) were statistically significantly different between CIN3 and cancers. Because SNPs were chosen as tagging markers for genetic regions rather than function, the observed associations with SNPs may be due to linkage disequilibrium with other causal unmeasured SNPs. We were underpowered to perform analyses restricted to carcinogenic HPV types due to small sample size as only women enrolled in the original NHS had HPV typing data. Future studies should therefore consider HPV genotypes in analysis. We were also unable to evaluate genetic factors associated with invasive cervical cancer separately. Future studies with large number of cases will be required to address whether certain genes are associated with transition from in situ to invasive cervical cancer. Although we performed haplotype-based analyses (defined by blocks of linkage disequilibrium); results were generally consistent with the gene region- and SNP-based findings. No new regions of interest were identified in haplotype analysis using the sliding window approach of 3 SNPs. The genes evaluated here were also not selected based on their previously reported associations with cervical cancer, but by agnostic analysis, encompassing a global effort to identify genes involved with a range of infection and non-infection-related cancers.There are several study limitations to be considered. We combined the CIN3+ cases from the NHS cohort-based study with the CIN3+ supplemental cases drawn from the community at the same period for increased analytic power. There was a higher proportion of CIN3 (a corollary of detection by screening in NHS rather than symptoms) in the NHS cases than supplemental cases; additionally supplemental cases were older, mainly because of the larger proportion of cancers compared to the NHS cohort-based cases where there were higher proportion of CIN3. Tests of associations between NHS and supplemental populations for 94.2%  of the SNPs were not significant at 5% level, importantly, all the SNPs associated with the two genes of interest , a population-based cohort study in Guanacaste, Costa Rica. Details of the cohort study methods A host genetic sub-study was nested within NHS, as described previously 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.PCR-based HPV DNA testing was conducted using the L1 MY09/MY11 consensus primer methods http://snp500cancer.nci.nih.gov) www.illumina.com). The Infinium included a total of 27,904 tag SNPs. Tag SNPs of the genes were chosen from the designable set of common SNPs  genotyped using the all 3 HapMap populations for tagging dependent on the population of interest for the investigator suggesting the candidate gene  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.A panel was designed as part of an effort of numerous investigators based on their expertise in specific cancers. Genotyping of tag SNPs from 990 candidate gene regions hypothesized to be involved in wide number of cancers such as colon, osteosarcoma, esophageal and stomach, biliary, Fanconi anemia, bladder, breast and other cancers, was conducted at the NCI Core Genotyping Facility , failed validation (no amplification or clustering) and assays that had less than 80% completion or 80% concordance with the 270 HapMap samples used for validation were excluded (n=269). SNPs with low completion rate (<90% of samples) were further excluded (N=482). SNPs with QC discordance among our 100 QC duplicates and among HapMap samples <98% were excluded . We also excluded samples with a completion rate <90% (n=7). Hardy–Weinberg equilibrium was evaluated among controls, 49 SNPs showed evidence of deviation from Hardy–Weinberg proportions. Our QC data did not suggest any obvious genotyping error in these 49, and their results are therefore presented. Of the 20,764 SNPs, 18,310 SNPs from 1113 genes were included in our present analysis.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.19. We considered an FDR value of <0.2 as notable.We obtained a gene-level summary of association 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 their associations for HPV progression and/or persistence by comparing: the group of CIN3/cancer cases (n=415) to HPV persisters (n=356) for evaluation of SNPs relevant to progression and (ii) HPV persisters (n=356) to random controls (n=425) for evaluating SNPs relevant to persistence. We note that persistence does not always precede CIN3, as it may be a result of a CIN3 lesion.Ptrend based on the three-level ordinal variable  of homozygote wildtype, heterozygote, and homozygote variant in a logistic regression model. Because of the small cell size examining some of the SNPs (less than 5%), we also show combined effect of heterozygous and homozygous variant genotypes; thus, results of the two-level models are discussed. All logistic regression models were unconditional and conducted using SAS version 9.1 .We conducted both crude and age-adjusted  analyses. For each outcome, we calculated the Table S1Results for all gene based tests for either an association with (i) cervical precancer/cancer, (ii) progression to cervical precancer/cancer, or (iii) HPV persistence.(XLS)Click here for additional data file.


There was no oral HPV detected among homosexual women, virgins or among women where sexual exposure was unknown. Never smokers were more likely to be oral HPV+ compared to former smokers (Adjusted Odds Ratio (Adj OR) = 0.1, 95% CI, 0.0–1.1) and there was no difference in risk between never smokers and current smokers . Twenty-five percent (3/12) of oral HPV+ women had a history of HPV and/or genital warts compared to 9% (10/106) of oral HPV-women (p = 0.104). For the women with a history of vaginal sex (n = 110), oral HPV status was statistically significantly different according to oral sex exposure (p = 0.039). A higher proportion of oral HPV-positive women reported that they had no history of oral sex exposure compared to oral HPV-negative women . The prevalence of cervical HPV infection did not vary between heterosexuals and bisexuals (35.7% (25/70) vs. 35.5% (11/31), p-value 0.411) and for all other women the cervical HPV prevalence was significantly lower . Our study suggests that smoking and sexual behavior involving males rather than female partners may be possible predictors of oral HPV infection in women. Further studies with larger sample size are needed to confirm these findings.Human Papillomavirus (HPV) is the main risk factor for cervical cancers and is associated with close to 36% of oropharyngeal cancers. There is increasing evidence that oral HPV transmission is related to sexual behavior but to our knowledge studies that involve women who have sex with women have not been performed. We examined the prevalence of oral HPV according to sexual behavior among a population-based sample of 118 women and have made some inferences of possible predictors of oral HPV infection. Women were categorized as heterosexual (history of vaginal sex and/or oral sex with males only,  Human Papilloma Virus (HPV) is the main risk factor for cervical cancers and is associated with about 25% of head and neck cancers, including close to 36% of oropharyngeal cancers ,2. In caet al. evaluated the prevalence of oral HPV infection in women with cervical HPV infection and performed a meta analysis of the current literature [Despite evidence of HPV as an etiologic risk factor for head and neck cancer, most notably HPV type 16 , there aterature . The pooterature ,18. Maleterature . To our Among women who have sex with women, previous studies have shown cervical HPV prevalence of 13% and 30%, including cases among women with a history of exclusively female sexual partners –22. ThisN = 1043) was contacted by the study coordinator  and invited to participate in this pilot study and if they agree to do so they were consented prior to initiation of the study. All women were called at least once for recruitment into this study and 88 agreed to participate . All study participants were required to be seen at one clinic visit by trained research staff in the Magee Womens Hospital Clinical & Translational Research Center (CTRC). In addition the study population was enriched with 30 African-American participants who were also enrolled during their routine gynecologic clinic visit at the Magee Womens Hospital.This cross-sectional study involves participants who were enrolled in the Epidemiologic Study of HEalth Risk (ESTHER) project . The ESTHER project’s participants were recruited from participants attending women-focused events, news items and advertisements in women-focused news sources throughout the city of Pittsburgh, Pennsylvania. Following approval by the University of Pittsburgh Biomedical Institutional Review Board (IRB), each ESTHER project participant  with a sterile nylon bristle cytology brush. This was followed by the mouthwash collection which required gargling 1–2 min while tilting the head back slightly toward the back of the throat. The goal was to gather exfoliated cells from the tonsil area. The results from each of these sampling techniques were combined to provide the oral HPV status. For the 30 African-American women, only a mouthwash sample was collected.i.e., prior to their enrollment in this study). For the participants who answered no to this question, if the Pap smear collected during this study were abnormal  they were also classified as having a history of abnormal Pap smears.Women who had a Pap smear performed within the previous 12 months prior to the study visit and had the results available were given the option of providing these results at the time of their study visit. Other participants had a Pap smear specimen collected by a study nurse, during their study visit. These Pap smears were read by qualified personnel at Magee Women’s Hospital, Department of Cytology and confirmed by a pathologist. The results of these Pap smears were made available to each study participant at no cost. Women with abnormal Pap test results were referred for follow up with their primary care provider. If the participant did not have their own primary care provider, one was recommended to them. History of abnormal cervical cytology  was determined by combining the history collected from questionnaire data with Pap smear results generated at the time of study enrollment and/or 12 months prior to the time of study enrollment. All participants were asked in the questionnaire whether they had a history of abnormal Pap smears  were obtained for each participant. In this analysis self-reported sexual orientation was not used because it did not correlate with reported sexual behavior patterns. Instead sexual orientation was redefined based on the combination of vaginal and oral sex exposure data and was categorized as heterosexual, bisexual or other . Women with heterosexual exposure were defined as having had vaginal sex and/or oral sex with males only. Women with bisexual exposure had vaginal sex and had oral sex with females and those with homosexual exposure never had vaginal sex and only had oral sex with females.DNA was extracted from all cervical and oral samples using the Puregene DNA purification kit  and HPV genotyping was performed using the Linear Array HPV Genotyping kit (Roche Diagnostics). The details of HPV genotyping and classification of high-risk HPV types has been previously described . HPV staClassification of HPV-risk was based on the epidemiological classification of HPV types associated with cervical cancer . HPV typp-value < 0.05 was considered statistically significant and p > 0.05 and <0.1 was considered marginally statistically significant. Exact logistic regression analyses were performed in order to identify the predictors of oral HPV infection [Statistical analysis was performed using STATA 10 . Fisher’s exact tests were performed to test for group differences between oral HPV-positive and negative groups, with oral HPV status defined by a positive result for either mouthwash or oral brush samples. A nfection . The convs. 12.5% (4/32), p = 0.784). None of the women that were categorized as “other” were positive of oral HPV infection. p = 0.068). A larger proportion of the oral HPV-positive women were never smokers compared to the oral HPV-negative women (75% (9/12) vs. 54% (56/104)). Among the women that were ever smokers, the distribution of former versus current smokers was different according to oral HPV status. The majority of the oral HPV-positive women were current smokers rather than former smokers  while the opposite was observed for the oral HPV-negative women. After adjusting for age, age at first vaginal sex and oral sex exposure, and using never smokers as the reference group, the odds of oral HPV-positivity for current smokers compared to never smokers was not statistically significantly different  while for former smokers, the odds of oral HPV-positivity was marginally statistically significantly lower .There were 118 women included in this study population. Seventy-five (63.6%) were heterosexual , 32 (27.1%) were bisexual  and 11 (9.3%) were categorized as other . The oral HPV prevalence for the entire study population was 10.2% (12/118) and was not different between heterosexuals and bisexuals (10.7% (8/75) vs. 98/104, 94%), the prevalence of oral HPV appears to increase with the number of vaginal sex partners (n = 110), oral HPV status was statistically significantly different according to oral sex exposure (p = 0.039). A higher proportion of oral HPV-positive women reported that they had no history of oral sex exposure compared to oral HPV-negative women . After adjusting for age, age at first vaginal sex and smoking, women who reported a history of vaginal sex but no oral sex exposure were more than six times likely to be oral HPV-positive compared to women who reported a history of vaginal sex and oral sex with males . There were no differences in oral HPV infection according to the total number of oral sex partners (data not shown). A higher proportion of women that were oral HPV-positive were over the age of 21 years old when they had their first oral sex exposure compared to the oral HPV-negative women  but this difference was not statistically significant.Although there was no statistically significant difference in oral HPV infection based on vaginal sex exposure .For all of the women combined, irrespective of sexual exposure, the prevalence of oral HPV infection appears to vary inversely with age when compared to cervical HPV infection . As expeAmong the 12 women found to be infected with oral HPV, five women (42%) had at least one known or probable high risk HPV type, 5 (42%) women had HPV types of unknown risk and 2 (16%) women had low risk HPV infections. Three of the twelve women (25%) with oral HPV infection had more than one HPV types present. One woman was infected with high-risk HPV types 16 and 33, the other with high-risk HPV types 35 and 52 and the third was infected with high-risk HPV66 and low-risk HPV70.vs. 35.5% (11/31), p-value 0.411) and for all other women the cervical HPV prevalence was significantly lower . The frequency of high risk types detected in the cervical mucosa ranked from highest to lowest were 52, 33, 56, 35, 59, 68, 16, 18, 39, 45, 51 and 58. The mean age of women with cervical HPV infection was lower  than the mean age of women with oral HPV infection  but was not statistically significantly different (p = 0.257).In contrast to oral HPV infections, 33.6% (37/110) of the women had detectable cervical HPV infections and among these, more than half of them  were infected with at least one known or probable high-risk HPV type and  were infected with multiple HPV types. The prevalence of cervical HPV infection did not vary between heterosexuals and bisexuals (35.7% (25/70) HPV is found to be associated with cancer in the oropharynx , therefovs. 12.5% (4/32)). In contrast, there was no oral HPV detected among homosexual women, virgins or among women where sexual exposure was unknown. This suggests that oral HPV infection may be more likely associated with sexual exposure to male partners rather than females and further studies are needed to confirm this finding. When oral sex behavior was compared among women who had vaginal sex exposure, there seemed to be an increased prevalence of oral HPV infection for the women that had vaginal sex exposure but no oral sex exposure. This also suggests that that vaginal sex exposure may be a stronger predictor for oral HPV infection. This is supported by results from a recent meta-analysis by Termine, et al. The study involved 100 women and reported that the only significant predictor of oral HPV infection was a younger age at first sexual intercourse and no association was found for other types of sexual behavior such as oral and genital sex and the presence of HPV-related cervical lesions [n = 11), these findings should be interpreted with caution and will need to be confirmed in future studies with a much larger sample size.In our study population, there was no significant difference in the prevalence of HPV infection between heterosexual and bisexual women (10.7% (8/75)  lesions . ConsidePrevious studies have found an association between oral HPV status and number of vaginal sex partners, oral sex partners, and kissing partners ,10. In oet al. reported that the current smoking was associated with the prevalence of cervical HPV infection while there was no association observed for former smokers [We observe a statistically significant difference in smoking status among those women who were oral HPV-positive compared to those who were oral HPV-negative. From our results never smokers as well as current smokers may be more likely to be oral HPV-positive. This observation was marginally statistically significant even after adjusting for age and sexual behavior factors. Vacarella,  smokers  and simi smokers ,31. ThesA recent study of the natural history of oral HPV reported that persons were significantly more likely to be infected with HPV when their spouse had a persistent infections lasting for the 2 year duration of the study . In our Overall our findings suggest that sexual behavior and smoking status may be contributing factors that are related to oral HPV infection in women. It is also possible that in women, sexual exposure to male partners may be more predictive of oral HPV infection rather than sexual exposure to female partners but further studies with larger sample size and with a study design that addresses the limitations and lessons learned from this current study are needed to confirm these findings.


Cervical metaplastic squamous epithelium exhibiting atypia insufficient for a diagnosis of cervical intraepithelial neoplasia (CIN) is usually reported as “atypical squamous metaplasia” (ASM). Stratification impacts treatment since the differential is often between reactive and high grade CIN . Diagnosis with H&E is associated with low intra/interobserver concurrence. P16/Ki-67 immunostains are helpful to assess cervical biopsies for HPV-associated lesions but staining in metaplastic squamous epithelium has received little attention. This study aims to establish staining characteristics of metaplastic squamous epithelium and determine if p16/Ki-67 is useful in ASM stratification. 80 cervical biopsies containing morphologically normal and dysplastic squamous metaplasia were retrieved to determine the staining characteristics of metaplastic epithelium utilizing p16/Ki-67 immunostains. These included 21 benign squamous metaplasia (BSM) from benign cervices, 15 BSM present adjacent to HPV/CIN lesions, and 44 CIN involving squamous metaplasia. Serial sections with controls were stained for p16 and Ki-67 and in-situ hybridization (ISH) for low-risk (LR) and high-risk (HR) HPV was performed. P16 was recorded as negative, spotty, or band-like. Ki-67 was recorded as positive when present in >50% of lesional nuclei. Results were correlated with H&E diagnosis. 95% of the BSMs, whether from normal cervices or adjacent to HPV/CIN were p16/Ki-67 negative. 81% HG CINs involving squamous metaplasia were p16 band/Ki-67 positive. Low grade CIN (CIN I) involving metaplastic epithelium showed a broad distribution of p16/Ki-67 staining patterns. Based on these criteria, 20 ASM were evaluated. 10% of the ASM cases were p16 band/Ki-67 positive indicating HG CIN. 60% of the ASMs were p16/Ki-67 negative indicating reactive change . The remaining 30% of the ASM cases showed variable positivity for p16 and Ki-67 and could not be stratified into the two categories. Thus p16/Ki-67 staining is helpful in stratification of ASM as reactive or CIN. Cervical metaplastic squamous epithelium exhibiting atypia insufficient for a diagnosis of cervical intraepithelial neoplasia (CIN) is usually reported as “atypical squamous metaplasia” (ASM). Stratification of ASM into reactive and CIN groups impacts treatment since the differential is often between reactive and high (rather than low) grade CIN. Small biopsies, suboptimal orientation of sections, coexistent inflammatory/reactive cellular changes, and subjective criteria contribute to low levels of intra- and inter-observer concurrence when the diagnosis of ASM is based on H&E stains alone. Adjuncts that could help stratify ASM would be helpful in patient management.It has been shown that p16/Ki-67 immunostains are helpful in the assessment of cervical  and analThe study was performed in two parts. The first part was designed to determine the p16/Ki-67 staining characteristics of morphologically normal and dysplastic metaplastic epithelium so as to develop algorithms for application to ASM. In the second portion of the study these algorithms were tested on a set of consecutive cases that had been reported as ASM in order to determine if it were possible to stratify cases of ASM into dysplastic or reactive groups.After IRB approval, slides of 80 formalin fixed paraffin embedded cervical biopsies were retrieved from our files and divided into three groups based on examination of routine H&E stained sections: (i) Benign squamous metaplasia (BSM) without morphologic evidence of HPV infection or CIN obtained from benign cervices (21 cases) (ii) uninvolved (benign) squamous metaplasia adjacent to areas with evidence of HPV infection or CIN (15 cases) (iii) metaplastic epithelium involved by HPV or CIN (44 cases). All diagnoses had been rendered by experienced gynecologic pathologists and all were confirmed by the authors  on slide review. Cases with discrepant diagnoses were excluded from the study. No additional exclusion criteria were applied in case selection. For the second portion of the study 20 consecutive cases of ASM were retrieved from the files. These cases had also been reported by the same experienced gynecologic pathologists. An additional 10 ectocervical biopsies with HPV and/or CIN were evaluated as controls. The specimens had been collected from 84 women ranging in age from 10 to 71 years .Serial sections of the retrieved slides and appropriate controls were immunostained for p16  utilizing Envision Plus Mouse Detection  and a DAKO Autostainer and for Ki-67 (CONFIRM Anti-Ki67 (k-2) primary antibody from and as diluted by Ventana Medical Systems, Inc. Tucson, AZ). As positive controls for p16 and Ki-67 immunostains we utilized paraffin sections of a HR HPV positive squamous carcinoma and of a tonsil, respectively. Sections of the cervical biopsies run substituting mouse IgG for p16 and Ki-67 antibody served as negative controls. Slides were treated with 3% hydrogen peroxide to block endogenous staining and the Envision polymer-based system was utilized for antigen detection. P16 antigen retrieval was accomplished by 30 minute incubation with steam and low pH buffer and Ki-67 antigen retrieval was accomplished by pretreatment on the Ventana Benchmark using CC1 buffer. The chromagen for both antibodies was DAB . P16 was recorded as negative , spotty . Nuclear and/or both nuclear and cytoplasmic staining was interpreted as positive for p16; cytoplasmic staining alone was considered nonspecific and recorded as negative. Ki-67 was recorded as positive when present in >50% of lesional cell nuclei. Positive staining for Ki-67 was exclusively nuclear.Serial sections of 88 (88%) of the biopsies  and 10 (100%) of the ectocervical controls were subjected to in-situ hybridization (ISH) for high risk (HR) HPV utilizing HPV-III Family 16 (Ventana). Serial sections of 93 (93%) of the biopsies  of the ectocervical controls were also subjected to in-situ hybridization for low risk (LR) HPV utilizing Inform HPV II-Family 6 and as per the manufacturer’s recommendations. Presence of LR and HR HPV was determined without knowledge of the p16/Ki-67 staining.Immunostaining, ISH, and H&E diagnoses were correlated. Statistical analyses were performed using the Fisher exact test. A 2-sided p value of <0.05 was considered significant.Followup was available for 10 (50%) of the ASMs up to 19 months after the index samples had been obtained.BSM from morphologically normal cervices and BSM adjacent to HPV/CIN showed similar staining and were negative for p16 and Ki-67 . Only twCINs involving metaplastic epithelium showed staining similar to CIN in ectocervical epithelium. Band-like p16 immunostaining and Ki-67 positivity were present in 22 of 27 (81.5%) HG CINs involving metaplastic epithelium. Four of the remaining 5 HG CINs involving metaplastic epithelium showed p16 positivity (2 band and 2 spotty >75%) of which 2 were Ki-67/HR HPV DNA positive, one was Ki-67/HPV DNA negative, and one was Ki-67 negative/HR HPV DNA positive. The fifth case (reported as CIN II) was p16/Ki-67/HPV negative. LG CIN involving metaplastic epithelium showed a broad distribution of p16/Ki-67 immunostaining patterns ranging from negative to positive.Of the 10 ectocervical control cases, four were benign reactive ectocervical biopsies and all were p16/Ki-67/HPV negative, two were HG CIN and were p16 band/Ki-67 positive, one of which was also HR HPV ISH positive. Four were LG CIN and showed variable staining for p16 and Ki-67; two were positive for LR HPV, one was positive for HR HPV, and one was positive for both LR and HR HPV by ISH.Thus to summarize, irrespective of the presence of adjacent HPV/CIN, morphologically normal and dysplastic lesions in metaplastic epithelium show a similar staining pattern to that seen in normal and dysplastic ectocervical epithelium, respectively. HG CIN is p16 band/Ki-67 positive, LG CIN shows a variable staining pattern, and benign squamous/metaplastic epithelium is p16/Ki-67 negative.Based on the above staining patterns, cases with ASM could be further stratified into CIN and benign groups –3. Of thFollowup  for 50% (10) of the ASMs is shown in In this study we have shown that p16/Ki-67 Immunostaining is useful in stratification of ASM into reactive and CIN groups by using the following criteria: (i) p16/Ki-67 negativity is indicative of a reactive change, (ii) band-like staining for p16 and Ki-67 positivity in >50% of lesional nuclei is indicative of HG CIN and has a high correlation with HPV ISH positivity, (iii) p16 band positivity or spotty positivity in >75% of lesional cells by itself is suggestive of HPV infection, (iv) Ki-67 positive staining in >50% of nuclei with negative or spotty staining for p16 in <25% of lesional cells remains a nonspecific finding often observed in reactive epithelium. Our findings are concordant with p16/Ki-67 immunostaining reported by others in cervical biopsies  and by uHuman papillomavirus (HPV) is the most common sexually transmitted infection in the United States with as many as 20 million persons infected . The oveSquamous metaplasia of the cervix is so common that it is virtually regarded as “normal”. Squamous metaplasia can be unifocal but is more frequently multifocal and almost always involves the transformation zone which is also involved in most CINs. Hence it is not surprising that ASM is a frequent diagnosis in surgical pathology. Often a tissue diagnosis of ASM is preceded by a cytological diagnosis of ASC-H   which acIn our study cohort, the majority of biopsies that had been reported as ASM were p16/HPV negative suggesting that the atypia in these epithelia is reactive rather than CIN. Review of the literature indicates that regardless of whether immunostains, ISH, PCR, or followup is used as the gold standard for HPV, the tendency to “overdiagnose” squamous metaplasia as ASM is not unique to our laboratory. In a study utilizing CK17, p16, and p63 immunostains to distinguish atypical immature squamous metaplasia from HG CIN, Regauer  reportedTo date, we are aware of few other studies  that hav


Human Papilloma virus (HPV) is recognized as the etiological agent of benign and malignant ano-genital lesions. The most prevalent genotypes associated with cervical carcinoma are HPV-16 and -18 worldwide. However, recent studies have emphasized the role of other genotypes, such as HPV-51, in the pathogenesis of cervical dysplasia. The aim of the study was to estimate the burden of HPV-51 infection in invasive cervical malignant lesions in Northern Sardinia, Italy.An observational, retrospective, prevalence, mono-center study was carried out to evaluate the presence of HPV genotypes in tissues biopsies of cervical lesions  gathered from 1996 to 2009. Biological samples were collected from women admitted consecutively to a tertiary university hospital situated in Sassari, Italy. Molecular methods were used to identify 28 oncogenic HPV types. A total of 155 formalin-fixed and paraffin-embedded cervical tissue samples were analyzed. Approximately half of the cervical lesions were classified as invasive carcinoma. HPV-DNA was detected in 71% of the samples, with a higher frequency (100%) in those categorized as invasive neoplasia. Mono- or co-infections were demonstrated in 45.8% and 25.8% of the cervical samples, respectively. Overall, the most prevalent HPV types were -16 (49%) and -51 (19.4%), with an increased frequency of detection associated with the severity of the cervical lesions.This survey highlights for the first time the relevant role of HPV-51 infection in the pathogenesis of invasive cervical cancer prior to the introduction of a vaccination program. Although a selection bias could have influenced the results, other recent studies have described the impact of HPV-51. This remarkable epidemiological element should be carefully evaluated, particularly in the view of opting for preventive vaccines, whose cross-protection patterns determine their efficacy in protecting against infection from HPV types that are not included in the vaccine itself. Cervical cancer is deemed the third most frequent neoplasia in women worldwide; in particular, it is the second most prevalent cancer among women aged 15–44 years. Recently, it was estimated an annual number of 529,828 new cases of cervical carcinoma and of 275,128 deaths from cervical carcinoma Human Papilloma viruses (HPVs) have been shown to be associated with cervical cancers since 1976 On this basis, two prophylactic vaccines containing type -16 and -18 specific antigens were recently developed; to date, they have showed a 7–9 year clinical efficacy of about 100% against cervical cytological abnormalities caused by HPV-16 and -18 genotypes, with a variable cross-protective efficacy against cervical lesions associated with other high-risk oncogenic types i.e., HPV types -45, -33, -31, -52, -35, -39, -58, -51, -59, -56, and -66) were associated with more than 20% of cervical carcinomas globally A prevalence study showed that other high-risk HPV genotypes than -16 and -18  and -51 (14.3%) in Northern Sardinia, Italy. Their presence, as single or coinfecting agents, was detected in all kind of cytological lesions; in particular, HPV types -16 and -51 DNA were detected in 12.5 and 6.3% of atypical sqamous cells of undetermined significance; 24.0% and 16.0% of low-grade squamous intraepithelial lesions; 50.0% and 50.0% of high-grade squamous intraepithelial lesions, respectively Since an unexpected proportion of some genotypes could be due to random variability in limited geographical areas, in order to evaluate the epidemiological dynamics of HPV-51, particularly in invasive cervical lesions, we investigated the presence of HPV DNA in embedded tissues samples of cervical lesions collected in the same area of Northern Sardinia, Italy, before and during the above-mentioned prevalence study.Ethical approval and informed consent for this study was unnecessary, according to the Italian legislation concerning the guidelines for the performance of observational studies (G.U. n. 76. 31-3-2008). However, a formal approval of the study protocol was requested in 2007 to the Ethical Committee of the Azienda Sanitaria Locale n°1 of Sassari, Italy .An observational, retrospective, cross-sectional, mono-center study was performed in Northern Sardinia, Italy, to evaluate the prevalence of HPV infection in a subset of embedded cervical lesions , consecutively collected from 1996 to 2009 and belonging to women admitted to a tertiary university hospital located in Sassari, Italy.ad-hoc case report form was created to record hysto-pathological and virological information. Data were anonymized in compliance with the Law Decree No. 196/2003, article 24 .An i.e., 50% in high-grade squamous intraepithelial lesions), an estimated number of three invasive carcinomas per year was needed to obtain a statistically significant difference. Therefore, a sample of 69 formalin-fixed and paraffin-embedded tissues from invasive cervical carcinoma biopsies, collected from 1996 to 2009, was selected. Concomitantly, it was decided to confirm results obtained in our previous prevalence study HPV-51 infection was expected to account for 1% of invasive cervical cancers, on the basis of the estimates of a recent international prevalence study DNA was isolated after extraction and purification using the DNeasy kit  DNA was tested using INNO-LiPA HPV Genotyping Extra, specific for 28 HPV genotypes  When positivity for HR genotypes -16, -18, -31, -45, -51, and -52 was found, confirmation was obtained testing samples with an "in-house" Real-Time quantitative TaqMan PCR assay Cross-protection against cervical carcinoma from preventive vaccines was estimated using statistically significant findings showed in the studies PATRICIA and FUTURE II for the bivalent and tetravalent vaccines, respectively Qualitative variables were summarized with percentages and statistically compared using the z-proportion hypothesis test. Linear trend for proportions was adopted to test the pattern of HPV genotypes in different cervical lesions. 95% confidence intervals were calculated to estimate HPV genotype prevalence.Logistic regression analysis was performed to assess the role of potential variables, including infections by several HPV genotypes, in the development of invasive cervical carcinoma.A p-value <0.05 was considered statistically significant.All the analyses were carried out using the statistical software Stata 11.0 .Cervical tissue samples, obtained during the time period 1996–2009, from 155 women, whose mean (standard deviation) age was 43.71 (14.68) years, were included in the study.The majority of the women were affected by invasive carcinoma , followed by CIN-1 , CIN-2 , and CIN-3 .A total of 110 out of 155 (71%) embedded-tissues were found to be positive for HPV-DNA, ranging from 6 (20%) in CIN-1 to 69 (100%) in invasive cervical neoplasia  .High-risk genotypes were detected in all HPV-DNA positive samples , whereas low-risk genotypes were detected in only 9 cases . A higher percentage of tissue positivity for high-risk HPV oncogenic types was demonstrated in CIN-3 lesions and invasive neoplasias .The prevalence of HPV infection significantly increased alongside with the age: 25% (2/8) in those aged 15–24 years, 52.5% (21/40) in those aged 25–34 years, 68.3% (28/41) in those aged 35–44 years, 82.9% (29/35) in those aged 45–54 years, 93.3% (14/15) in those aged 55–64 years, 100.0% (10/10) in those aged 65–74 years, 100.0% (6/6) in those aged over 75 years . An indistinguishable proportional linear trend was observed for the prevalent high-risk genotypes.Infection by a single HPV genotype was described in about half of the samples ; co-infection with multiple high-risk and/or low-risk genotypes was found in 40 (25.8%) of cervical samples, with an increasing linear trend associated with the severity of the histo-pathological pattern .The most prevalent genotypes were HPV-16  and HPV-51 , which accounted for 64.6% of all detected genotypes. HPV-16 and/or -51 infection was proved in 75.5% (83/110) of all HPV-DNA positive samples. The positivity significantly increased alongside with the severity of the cervical lesions for the genotypes HPV-16 and -51 . Other relevant HPV genotypes were: 45 , 31 , 69 , 71 , and 18 . The observed prevalence of HPV-16/51 co-infection  was almost similar to that estimated on the basis of single HPV-16 and HPV-51 infections . Only a few patients were co-infected by HPV genotypes -16/18 .Among 71 patients infected by a single genotype, the most frequent were: HPV-16 , -51 , -31 , -45 , and -18 .i.e., HPV-16, -18, -31, -33, -45, and -51) is 92.8%.Cervical carcinomas were positive for HPV-16 and -51 in 76.8% and 36.2%, respectively; the proportion of co-infected cases was 20.9%. Therefore, the percentage of cervical carcinomas with at least HPV-16 and/or 51 infection was 81.2% (56/69). The estimated proportion of cases potentially protected by the current vaccines , conferred by the current preventive bivalent and tetravalent vaccines, could be 11.4% and 1.5%, respectively, based on cross-protective efficacy data In particular, cross-protection against HPV-16 and -18 nearest genotypes  responsible of invasive cervical carcinoma did not significantly change during the study period ; however, the analysis of the temporal trend of the only HPV-51 genotype prevalence, in mono- or co-infection, significantly changed due to non-random variability   . The retThe present study confirms the primary role of HPV-16 in the pathogenesis of cervical carcinoma and describes an unusual high prevalence of HPV-51 infection (36.2%) in embedded invasive cervical neoplasias in Northern Sardinia, Italy, in a pre-vaccination period (1996–2007). It confirms that HPV-51, rarely detected in other geographical areas To our knowledge, this is the first study pointing out such a strong association between HPV-51 and invasive cervical cancer .As previously discussed With regards to a potential selection bias, it was already showed that, even if this possibility cannot be excluded, it may not play a role, as no significant differences were detected between HPV positive and negative women in terms of PAP test compliance in the years preceding our survey On the other hand, if the high prevalence demonstrated for HPV-51 is due to a random variability in this restricted geographic area, it should have been lower in the past, in particular in cervical neoplasias occurring several decades after the HPV infection. Therefore, the high prevalence of HPV-51 in embedded tissues from invasive cervical carcinomas, with a significant temporal differences since 1996 up to recent years, strongly indicates that this HPV genotype has been endemic in Northern Sardinia at least for the last thirty years.On this basis, the high HPV-51 prevalence could be deemed a peculiar epidemiological feature of Northern Sardinia. However, the higher diagnostic accuracy of the molecular methods implemented in our study might have positively affected the sensitivity of the survey, explaining, consequently, the differences found comparing our findings with the results of other studies, which used different diagnostic techniques Results from this epidemiological study indirectly indicate that current vaccines may prevent infection by HPV genotypes circulating in Northern Sardinia, particularly as a result of the use of the bivalent vaccine owing to its larger cross-protective effect against HPV genotypes, including HPV-51. Current available vaccines are in fact believed to prevent more than 70% of the cases of cervical carcinoma owing to their cross-protection activity. Nevertheless, it is presently unclear if data on cross-protective efficacy, obtained from clinical trials, indicates long-lasting immunity However, the introduction of HPV vaccines in the Italian market, followed by the implementation of a specific public health program, could modify the epidemiological scenario, favoring the spread of HPV genotypes whose antigens are not included in the vaccines. This has been described for other bacterial infections following the introduction of vaccination programmes Data obtained from this prevalence survey will allow to evaluate the possible epidemiological impact of HPV vaccine on the etiological dynamics of cervical cancer; in particular, to determine whether the implementation of a vaccination program based on the bivalent  vaccine in Northern Sardinia can reduce the burden of HPV-51 infection as a consequence of its cross protective efficacy against HPV-31, -33, -45, and -51 Several limitations can be indentified in the survey carried out in Northern Sardinia: firstly, the cross-sectional nature of this study could not permit to adequately evaluate the dynamics of the HPV genotypes circulation in North Sardinia, being an observational, prospective design more suitable to recognize temporal changes. Nevertheless, the 10-year period selected for this study is deemed to be sufficient to give an overview of the HPV genotype prevalence in the pre-vaccination era, when, theoretically, no selective pressure for ecological changes occurred in this geographical area.The confidence intervals of the estimates of the prevalence data show a wide range for some of the less frequent HPV genotypes, underlying an issue of poor statistical power; therefore, an under-reporting of cervical lesions, particularly CIN lesions, could be hypothesized, although samples of all invasive cervical carcinomas, notified in the study period to the official Northern Sardinian registry, were collected to undergo molecular analysis for HPV-DNA. Moreover, we consecutively enrolled samples of individuals complaining of clinical symptoms and, consequently, a selection bias could not be excluded, though our previous prevalence study In conclusion, as previously reported, this study has confirmed the high prevalence of HPV infection in women with cervical dysplasia and invasive disease in our region; moreover, it highlighted the role of HPV-51 in determining invasive disease, emphasizing the importance of orienting immunoprophylaxis towards this HPV type in the prevention of cervical cancer in our geographical area.


Cervical cancer arises from cells localized in the ectoendocervical squamocolumnar junction of the cervix persistently infected with one of about 13 human papillomavirus (HPV) genotypes. The majority of HPV infections induces low grade squamous epithelial lesions that in more than 90% of cases spontaneously regress and in about 10% eventually progress to high grade lesions and even less frequently evolve to invasive cancer. Tumor progression is characterized by (1) increased expression of E6 and E7 genes of high risk HPVs, known to bind to and inactivate p53 and pRb oncosuppressors, respectively; (2) integration of viral DNA into host genome, with disruption of E2 viral genes and host chromosomal loci; and (3) molecular alterations of key regulators of cell cycle. Molecular markers with high sensitivity and specificity in differentiating viral infections associated with cellular abnormalities with high risk of progression are strongly needed for cervical cancer screening and triage. This review will focus on the analysis of clinical validated or candidate biomarkers, such as HPV DNA, HPV E6/E7 mRNA, HPV proteins, p16(INK4a) and Ki67, TOP2A and MCM2 cellular factors, and DNA methylation profiles, which will likely improve the identification of premalignant lesions that have a high risk to evolve into invasive cervical cancer. Cervical cancer is the third most common tumour in women worldwide with more than 85% of the cases occurring in low-to-medium-resource countries . The intThe role of human papillomaviruses (HPVs) in the aetiology of invasive cervical carcinoma has been well established. At least 13 genotypes of the alpha genus  have been found to be associated with the risk to develop cervical cancer and defined as “carcinogenic” viral types . HPV16 iThe HPV lifecycle is characterized by infection of undifferentiated proliferating cells of the basal epithelial layer that become exposed through microwounds. HPV DNA episomes are maintained at low copy number in the nucleus and only early proteins are expressed from the viral genome. Differentiation of HPV-positive epithelial cells is accompanied by viral DNA replication and activation of the productive phase of the viral life cycle . In partIntegration of high risk HPV DNA into the host genome is also a crucial event in cervical carcinogenesis as it is found almost exclusively in high-grade lesions and invasive cancer often in association with progression and invasiveness . NotablySeveral biomarkers have been recognized which roughly identify specific stages in the natural history of HPV infection and cervical cancer progression. They include the presence of viral nucleic acids, viral proteins, or alteration of cellular factors induced by viral oncoproteins . The floNowadays, numerous assays have been developed to detect nucleic acids of oncogenic and nononcogenic HPVs in cervical samples. The main advantage of using HPV testing is the high sensitivity, with a consequent high negative predictive value, since the absence of carcinogenic HPV indicates an extremely low risk of cervical intraepithelial neoplasia grade 3 (CIN3) or cancer –22, and There are a wide range of commercially available HPV detection assays which are based on different techniques such as target amplification (mainly PCR), signal amplification, and probe amplification . They caFour HPV DNA assays have been approved by the Food and Drug Administration (FDA): (1) the Hybrid Capture 2 (Qiagen), detecting 13 high-risk HPVs; (2) Cervista HPV HR (Hologic), targeting 14 high-risk HPVs; (3) Cervista HPV 16/18 (Hologic), specifically designed to identify HPV16 and 18; and (4) Cobas 4800 HPV (Roche Diagnostics), targeting 14 high-risk HPVs . Hybrid In respect to the accuracy of Hybrid Capture 2 to find CIN2 or worse in the triage of women with low SIL, the meta-analysis of 24 studies showed that the pooled absolute sensitivity was 95.4% (95% CI: 94.0–96.5%) and 96.4% (95% CI: 90.5–98.7%), whereas the pooled specificity was 27.8% (95% CI: 23.8–32.1%) and 23.7% (95% CI: 19.4–28.7%) for the outcomes of CIN2 and CIN3 or worse, respectively. Similar to Hybrid Capture 2, the sensitivity of most of the other assays targeting the DNA of all 13 high risk HPV DNAs was high (>90% for CIN2 and CIN3), whereas the specificity was low in low SIL triage . HPV DNASeveral other HPV tests targeting at minimum the 13 high risk HPVs have been clinically evaluated, following the strategy reported by Meijer et al. , and theThe U.S. Preventive Services Task Force (USPSTF) and a multidisciplinary partnership among the American Cancer Society/American Society for Colposcopy and Cervical Pathology/American Society for Clinical Pathology (ACS/ASCCP/ASCP), as well as several health authorities in Europe  , 34, recHPV RNA assays are designed to detect viral mRNAs encoding for the E6 and E7 proteins which are the most critical factors for the development of cervical cancer. Overexpression of HPV E6 and E7 mRNAs has been evaluated as a marker for the transition from a productive infection to an abortive infection that eventually promotes cell transformation. Four assays are currently available for detection of HPV E6/E7 mRNA in cervical samples. The APTIMA (GenProbe) and OncoTect (IncellDX) assays are based on reverse transcriptase (RT) and PCR technique and detect E6/E7 mRNA from 14 and 13 high risk HPV genotypes, respectively. The PreTect HPV-Proofer (Norchip) and NucliSENS EasyQ (Biomerieux), both relying on nucleic acid sequence-based amplification (NASBA), are able to detect E6/E7 transcripts from the five most common high-risk viral types in cervical carcinoma . APTIMA was approved by FDA in 2011 for the triage of women with ASCUS cytology and older than the age of 21 years or for screening of women of 30 years of age and older in conjunction with cytology. Several studies have compared the APTIMA to Hybrid Capture 2 and cytology-based tests for both primary cervical cancer screening as well as triage of ASCUS or low SIL. A recent meta-analysis showed that testing for viral RNA with APTIMA assay in the triage of women with ASCUS was as sensitive but more specific for identifying CIN2 or worse compared with Hybrid Capture 2 (specificity ratio of 1.19 (95% CI: 1.08–1.37)) . HoweverTwo HPV protein assays are available to detect protein levels in cervical cell exfoliates, namely, the OncoE6 (Arbor Vita Corporation) and Cytoactiv (Cytoimmun Diagnostics). The OncoE6 is able to identify the E6 protein encoded by HPV 16, 18, and 45. A pilot study performed with OncoE6assay showed that the expression of E6 from HPV 16, 18, and/or 45 may be more specific for the detection of CIN3 or worse compared with HPV-DNA tests . ClinicaThe functional inactivation of p53 and pRb oncosuppressors by E6 and E7 oncoproteins determines the alteration of several cellular pathways relevant for cell transformation and cancer development. The expression of E7 determines the inactivation of pRb with a consequent increase of free E2F in the cell, leading to both an increase of cyclin-dependent kinase inhibitor p16 (p16INK4a) and aberrant proliferation (marked by increased levels of Ki-67 expression) , 41. TheThe use of p16INK4a immunohistochemistry is also a very important tool for the improvement of the diagnostic accuracy, reliability and quality in histopathology of cervical lesions . The accThe proliferation antigen K1-67, which is expressed during the G2 and mitotic phases of the cell cycle, has been demonstrated in many studies to be a reliable indicator of the growth fraction of a tumor. Reuschenbach et al. analyzed a series of 138 cervical cone biopsies and showed that p16INK4a and Ki-67 were coexpressed in dysplastic lesions only . A dual Recent studies have shown that minichromosome maintenance protein 2 (MCM 2) and topoisomerase II alpha (TOP2A) proteins are expressed in cells with aberrant S-phases and including HPV-transformed cells in association with elevated expression of the HPV E6/E7 proteins , 52. TheThe E6 proteins of oncogenic HPVs have been shown to promote the transcription of telomerase reverse transcriptase (TERT) which stabilizes and repairs the repeated DNA sequences at the telomere end of chromosomes . Gain ofEmerging evidence suggests that microRNAs (miRNAs), small noncoding single-stranded RNAs that regulate cell gene expression, might be involved in the pathogenesis of several human cancers, including cervical carcinoma . SeveralDNA methylation is one of the epigenetic mechanisms that influence gene transcription, chromatin structure, genomic stability, and the inactivation of imprinted genes and X chromosome . AbnormaDNA methylation of several human genes has been shown to be also a relevant event for cervical carcinoma development. The treatment of HPV-positive cervical cancer cell lines with demethylating agents, coupled to expression microarrays, has allowed the identification of genes encoding for SPARC and TFPI2 as highly methylated in invasive cervical cancer . AnotherTesting for nucleic acids of high risk HPVs or for cellular surrogate markers of HPV transforming processes will most probably be the primary cervical cancer screening method in many countries. HPV-based tests offer a more sensitive way to identify women with high-grade cervical disease compared with cytology-based methods and in the future specific HPV testing will be very important for screening, for triage of women with abnormal cytology, and for viral surveillance and monitoring of vaccine efficacy. Many commercially available assays have been already approved by FDA or have been clinically validated in accordance with international approved validation guidelines. In general the large majority of available assay have high sensitivity and specificity for the identification of CIN2. But the majority of the CIN2 and many CIN3 lesions regress and it would be very relevant to identify biomarkers that identify the minority of high grade CIN lesions that will not regress. Unfortunately, CIN2 and CIN3 are the endpoint of all the available studies and the target of treatment; consequently we can have only indirect evidence for prognostic biomarkers able to identify high grade lesions that could be not treated.New biomarkers such as viral and cellular methylation profiles could represent the most accurate markers for cancer progression. Nevertheless, results on the novel promising biomarkers are in general based on small sample size, and additional clinical trials are needed to determine the true clinical value of these new assays.


Investigation of HPV infection in men remains important due to its association with genital warts and anorectal cancer, as well as to the role men play in HPV transmission to their female sexual partners. Asymptomatic men (n = 43), whose sexual partners had presented cervical HPV infection, were enrolled in this study. Among the 43 men, 23 had their female partner included and tested for HPV-DNA, totaling 23 couples. HPV-DNA was detected by PCR. Type specific PCR to detect HPV 16, 18, 31, 33, 45 and 6/11 was performed. At least one type of HPV was detected in 86.0% (37/43) of the male patients and more than one HPV type was identified in 39.5% (17/43) of the samples, including high and low risk HPV. HPV-16 proved to be the most prevalent viral type in both male and female samples. Concordance of at least one viral type was observed in 56.5% (13/23) of the couples. Among couples that have shown concordance of viral types, 84.6% (11/13) of the men had the same high risk viral type presented by the female sexual partner. These data suggest that HPV infected men is an important reservoir, contributing to a higher transmission to women and maintenance of infection, and consequently, a higher risk of developing cervical cancer. HPV vaccination in men will protect not only them but will also have implications for their sexual partners. Genital human papillomavirus  infection is one of the most common sexually transmitted diseases. It is estimated that the prevalence of HPV-infection is 20% of all men, reaching 70% in some age groups, especially among individuals between 15 and 24 years of age. Although HPV infection in men may be associated with low mortality and morbidity, investigation remains important due to its association with genital warts, penile cancer, anorectal cancer and oropharyngeal cancer as well as to the role men play in HPV transmission to their female sexual partners Transmission can occur easily between sexual partners, and in many cases, multiple transmission events may take place with a couple without being detected Compared to cervical cancer, penile cancer is a relatively rare disease and generally occurs late in life. Approximately 40% of penile carcinomas can be attributed to infection with high risk HPV, whose presence is related mainly to basaloid and warty histological subtypes The HPV type concordance between sex partners has been addressed in previous studies Several lines of evidence have suggested that the sexual behavior of males can contribute to the risk of cervical cancer in their sexual partners The prevalence of HPV infection detected in male partners of women who have received a positive diagnosis for this virus, and/or cervical intraepithelial neoplasia or squamous carcinoma, varies between 23% and 73% Therefore, this study aimed to detect the prevalence of DNA-HPV in the male partners of HPV-infected women and to assess the concordance of viral types in infected couples.Based on the cytological or histopathological diagnosis of cervical squamous intraepithelial lesions associated to HPV infection of the female partner, couples were invited to participate of this study. Of the invited couples, all the 43 male partners attended to diagnostic evaluation and were included, while only 23 women attended for a new sample collection and had their cervical samples tested for HPV-DNA, totaling 23 couples . BesidesThe study protocol was approved by the Ethics Committee of Research of Federal University of Minas Gerais (protocol 0088.0410203-09). Information concerning the research project was provided to all participants, and all signed a free and informed consent form approved by the institutional ethics committee of the Federal University of Minas Gerais (UFMG).The penile samples were collected using the DNA Citoliq system . Material was brushed from the glans and prepuce internal surfaces, including the sulcus and the corona, and placed in transport tubes. Cells were then suspended in a preservative liquid, which allowed them to be used for both liquid-based cytology and DNA extraction . The cytopathological changes observed in these samples had been previously reported , 33, 45, and 6/11, using primers and conditions presented in 2, and the specific oligonucleotides in a final volume of 10.0 µL. PCR were performed in MJ PT100 thermocyclers  and consisted of 35 cycles of 1 min. at 94°C for denaturing, 1 min. at 55°C for annealing, and 1 min. at 72°C for extension to amplify the human β-globulin gene fragment. To amplify the HPV-DNA, a “touch-down” PCR condition was used, which consisted of 5 initial cycles of 1 min. at 95°C, 1 min. at 45°C, and 1 min. at 72°C, followed by 35 cycles of 1 min. at 95°, 1 min. at 45°C, and 1 min. at 72°C. Negative and positive controls were included within the settings for each reaction. PCR products were analyzed in silver stained 6% polyacrylamide gel electrophoresis. Oligonucleotides and PCR conditions for genotyping HPV through PCR are listed in DNA extraction was carried out in aliquots obtained from the 1 mL liquid medium samples . The molecular analysis was performed in DNA obtained using proteinase K, phenol:chloroform extraction, and isopropanol DNA precipitation. All samples were submitted to PCR, using the oligonucleotides PC03 and PC04, which amplified a 120 pb sequence of a human β-globulin gene, so as to create an internal control to verify the integrity and quality of the extracted DNA Statistical significance for prevalence of different HPV types between age groups or gender was tested by using Proportion Test Z and considered significant when p<0.05.The prevalence of DNA-HPV among the 43 penile samples, as detected by PCR, is shown on Cervical and penile samples collected in 23 couples were also submitted to HPV genotyping. The prevalence of viral types in these samples is shown in The concordance of viral types between couples is shown in HPV infection was prevalent in 86% of the men in the present study. Previous data of other Brazilian studies At least one HPV type appeared in 37 men (86%), although in only 22 of these (51.2%) DNA-HPV was detected by means of GP5+/GP6+ oligonucleotides. By contrast, the detection of HPV by GP5+/GP6+ oligonucleotides in female samples, as compared to male samples, was higher, suggesting that a higher cellular representation may be present in cervical samples, in turn improving the chances of detecting HPV. In men, several factors may affect the detection of DNA-HPV, such as the absence of a delimited lesion, the excess of cornea squamous, and lower viral loads. Negative results when consensus oligonucleotides are used in positive samples, as detected by specific oligonucleotides, have been reported in prior studies In the present study, among the 43 male subjects, 33% presented only high risk HPV. Moreover, multiple infections of high risk and low risk HPV (39.5%) were also frequently present, which is in agreement with prior reports By comparing the concordance of viral types between couples, it has been observed that 56.5% (13/23) of the couples shared at least one viral type, while 47.8% (11/23) of the male subjects shared at least one high risk viral type with their partners. Studies evaluating the HPV infection in men who are sexual partners of women with intraepithelial squamous lesions and/or HPV have shown that 13% to 63.2% of partners are infected by the same HPV viral type In addition, the lack of concordance of HPV types in 43.5% of couples may be explained by the fact that the transmission of HPV infection occurs mainly at the beginning of sexual life and is associated with immunity The prevalence of viral types in partners of infected women varies in different studies, presenting rates of 3.5% to 59% for HPV 16, from 3.5% to 6.7% for HPV 18, and from 3.5% to 8.5% for HPV 31 The low number of patients and couples studied, as well as an insufficient monitoring of the duration and clearance of the HPV infection among couples, creates certain limitations to the present study. It would be also interesting to genotype all HPV positive samples for additional viral types. Thus, further investigation is needed to better elucidate these facts. Although similar studies have been conducted in different countries, it is still important to investigate the different epidemiological aspects regarding HPV infection in men, attempting to reproduce and confirm results. Moreover, these findings, including those reported herein, may help to better comprehend men’s role in the transmission of this infection, to guide adoption and to follow up of control measures, such as vaccination and the alterations in epidemiological patterns of this significant STD.


In the recent years, dramatic increases in HIV transmission among men who have sex with men (MSM) have been observed in China. Human papillomavirus (HPV) infection related anal cancer is more common among HIV-infected MSM as compared to the general population. However, HPV infection and anal cytology has been rarely studied in HIV-infected MSM in China.HIV-infected MSM in Beijing, China were invited to participate in this study between January and April 2011. Anal swabs were collected for examining cytology and HPV genotypes.Ninety-five eligible participants with complete questionnaire and laboratory data were included in the analyses. Thirty six of them (37.9%) showed abnormal anal cytology as follows: atypical squamous cells of undetermined significance (ASC-US) in 19 (20.0%), atypical squamous cells but cannot exclude HSIL (ASC-H) in 1 (1.1%), low-grade squamous intraepithelial lesion (LSIL) in 15 (15.8%), and high-grade squamous intraepithelial lesion (HSIL) in 1 (1.1%). HPV6 (20.0%), HPV16 (10.9%), HPV56 (10.9%), HPV52 (9.1%) and HPV39 (9.1%) were observed most frequently among those with normal anal cytology, while different distribution was found in the ones with abnormal anal cytology as HPV6 (19.4%), HPV16 (19.4%), HPV45 (16.7%), HPV52 (16.7%) and HPV18 (11.1%). In addition, HPV16, HPV45, HPV52 and HPV18 were the most frequent high-risk types in patients with abnormal anal cytology. HPV multiplicity was found to be significantly related to the prevalence of abnormal anal cytology (p for trend = 0.04).High prevalence of HPV infection and abnormal anal cytology was observed among HIV-infected MSM in China. Infection of multiple HPV types or high-risk types was found to be associated with an increased risk of abnormal anal cytology. While anal carcinoma is rare in the general population Human papillomavirus (HPV) infection, especially high-risk HPV (HR-HPV) viral types such as HPV 16 and HPV 18, is a significant risk factor for development of anal intraepithelial neoplasia (AIN) and anal cancer A total of 98 eligible participants gave consent to be included into the study. Three of them were exclude in the final data analysis because of unsatisfactory cytological evaluation. Of the remaining participants, about three quarters (75.1%) were aged 20–39 years . The majAs shown in As shown in To our knowledge, this pilot study was the first investigation into the prevalence of HPV genotypes in HIV-infected MSM with abnormal anal cytology in China. Based on a cross-sectional study design, a total of 95 HIV-infected MSM were included in the study, 36 of them (37.9%) showed abnormal anal cytology including 17 with ASIL/ASC-H (17.9%). Different HPV genotype distributions were found between patients with normal and abnormal anal cytology. Infection of multiple HPV types and high-risk types were found to be associated with an increased risk of abnormal anal cytology.Invasive anal cancer has well-documented precursors, known as HSIL (cytology) or AIN2/3 (histology) In the past decade, multiple studies have reported that anal HPV infection is common among MSM especially in HIV-infected MSM Some limitations of this study should be considered. First, HRA needs more advanced medical recourses especially for HIV-infected patients In conclusion, the high prevalence of HPV infection and abnormal anal cytology was observed among HIV-infected MSM in China. Infection of multiple HPV types and high-risk types were found to be associated with an increased risk of abnormal anal cytology. Considering the high prevalence of HIV infection in this high-risk population in China, more studies addressing anal HPV infection and related diseases in HIV-infected MSM are needed.The study was approved by the Ethics Committee of the Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College. Written informed consent was obtained from each study participant before the interview, sample collection and testing.Study participants were outpatients of the Capital City Dermatology Hospital and were recruited through local MSM nongovernment organization (Beijing Rainbow Volunteers Workstation) between January and April 2011. To maximize outreach of study information to HIV-infected MSM community, multiple methods were used for recruitment including MSM website advertisement, distributing flyers with study-related information at MSM-frequented venues , and participants were also encouraged to refer their friends and acquaintances to attend the study. Those eligible to participate were males, 18 years or older, HIV positive, ever had homosexual behaviors, willing to take tests for anal HPV infection and anal cytology, and provision of written informed consent. The presence or absence of any symptoms, including anal symptoms, did not influence eligibility. Study participants who were positive for suspicious symptoms related to STD  and abnormal cytology were informed by study personnel in private and referred to the Capital City Dermatology Hospital or the Beijing Cancer Hospital for further diagnosis and treatment.Sociodemographic and sexual behavior data were collected using one-to-one interviews in a separate room and a standardized questionnaire. The content of the questionnaire includes self-reported sociodemographic characteristics , regular homosexual behavior, sexual behaviors in the past 6 months, status of HIV infection and treatment, knowledge on STD, history of STD, and lifestyle factors . As explained to the participants, regular homosexual behavior was defined as at least 50% of sexual activities occurring with males. Each study participant was assigned a unique code that was used to link the questionnaire and specimens. Personal contact information, which was blinded to researchers, was kept by the Beijing Rainbow Volunteers Workstation for test results feedback and data validation.Trained personnel collected anal samples for cytological analysis and HPV typing by rotating a saline water moistened nylon flocked swab in the anal canal for about 2 minutes. The cytological specimens were then placed on a glass slide and stained with a Papanicolaou's stain. The cytology slides were read by a cytologist from Hospital of Cancer, Chinese Academy of Medical Sciences and Peking Union Medical College (Dr. Zhihui Zhang). Using the Bethesda system criteria for evaluation of cervical cytological results Double entered questionnaires and laboratory results were analyzed using Statistical Analysis System . The study population was described by age, ethnicity, education, marriage status, age at the first homosexual act, number of homosexual partners ever had, status of syphilis, ever had STD other than HIV, had receipted HAART, latest CD4 cell count (tested in the recent three months), time since HIV diagnosis, regular sex behavior , number of partners in the past six months and condom use during these activities. Differences between groups were assessed using Pearson chi-square or Fisher exact test.The associations of potential risk factors with HPV infection and anal cytological results were estimated by means of OR and 95% confidence intervals (CI) using logistic regression analysis. Covariates with a significant association in the univariate analyses were included the following multivariate analysis using a significance level of 0.05. In order to investigate the combined effect of HPV types, the risk of anal cytological results was then assessed with respect to the HPV subgroups according to carcinogenicity and the number of HPV types in each sample. Tests for trend by were performed by treating categories of the number as continuous variable in the logistic regression analysis.


Vulvar cancer is a relatively rare malignancy, which occurs most often in postmenopausal women. We have previously identified a geographic cluster of vulvar cancer in young Indigenous women living in remote communities in the Arnhem Land region of Australia. In this population, we investigated the prevalence of oncogenic human papillomavirus (HPV) infection in anogenital samples  and compared the overall, type-specific and multiple infection prevalence between sites.A cross-sectional survey of 551 Indigenous women aged 18–60 years was undertaken in 9 Arnhem Land communities. Women were consented for HPV detection and genotyping collected by a combined vulvar/vaginal/perianal (VVP) sweep swab and a separate PreservCyt endocervical sample collected during Pap cytology screening. HPV DNA testing was undertaken using PCR with broad spectrum L1 consensus PGMY09/11 primers with genotyping of positive samples by Roche Linear Array. The primary outcomes were the prevalence of cervical and VVP high-risk (HR) HPV.The prevalence of VVP HR-HPV was 39%, which was significantly higher than the cervical HR-HPV prevalence . HPV-16 was the most common genotype detected in both sites . HPV-16 infection peaked in women aged <20 years; however, there was a marked decline in cervical HPV-16 prevalence with age (p=0.007), whereas following an initial decline, the prevalence of VVP HPV-16 remained constant in subsequent age-groups (p=0.835).In this population experiencing a cluster of vulvar cancer, the prevalence of cervical oncogenic HPV infection was similar to that reported by studies of other Australian women; however there was a significantly higher prevalence of vulvar/vaginal/perianal infection to cervical. The large discrepancy in HPV prevalence between anogenital sites in this population may represent more persistent infection at the vulva. This needs further investigation, including the presence of possible environmental and/or genetic factors that may impair host immunity. Vulvar cancer is a relatively rare malignancy, accounting for approximately 3% of all gynecological cancers worldwide . Two indWorldwide, the incidence of vulvar cancer ranges from 0.0 to 3.5 per 100,000 . AlthougBetween 1996–2005, in the Arnhem Land region, the incidence of vulvar cancer among Indigenous women aged less than 50 years was 31.1 per 100,000  13.1-49.1), over 50 times higher than the rate for the total Australian population of the same age-group . An exceThe cause of this very high incidence is unclear. The cases are almost entirely restricted to the under 50 age-group, suggesting that excess oncogenic HPV infection is a key etiological factor. The prevalence of vulvar HPV infection in this population and in other Australian women is unknown. We undertook a cross-sectional study to assess prevalence of oncogenic anogenital HPV infection in Indigenous women residing in the Arnhem Land region. Samples were collected from the cervix and vulvar/vaginal/perianal area. The aims of this study were two-fold. First, to compare the vulvar/vaginal/perianal and cervical HPV prevalence in Arnhem Land women. Second, to compare the prevalence of cervical HPV in this population to that of other Australian women, to provide an indirect indication of whether oncogenic vulvar HPV infection is more common in the Arnhem land region.A cross-sectional survey was undertaken in 2007–2009 in 11 primary health centres providing services to 9 Indigenous communities and a number of smaller outstations in the Arnhem Land region. The characteristics of this region have been described previously . BrieflyCommunities were identified based on our earlier work  and reprWomen were eligible to participate if they met the following criteria: Aboriginal and/or Torres Strait Islander, aged between 18 and 60 years, and their usual place of residence was in a community in the Arnhem Land region.There were four methods of recruitment: (1) approaching women due for Pap screening based on health centre recall systems; (2) holding community forums; (3) holding “women’s health weeks” which had a focus on health education and screening; and (4) approaching women who presented to the health centre when the research team were present.All participants gave written informed consent, and the study procedures conformed to the principles of the Declaration of Helsinki. Consenting women were invited to have a Pap test, specimen collection for combined vulvar/vaginal/perianal (VVP) and separate cervical HPV detection and genotyping, swabs and collection of blood (where indicated) for assessment of other sexually transmissible infections (STIs), and clinical examination for vulvar lesions. Samples for VVP genotyping were collected by clinical staff performing a single sweep swab of the vulva, vagina and peri-anal region using an established protocol . The VVPWomen with clinical signs of anogenital disease were referred to a gynecologist for colposcopy and biopsy for definitive histological diagnosis and treatment. Abnormal Pap and STI test results were managed by the health centre in accordance with local guidelines. Women testing positive for high-risk (HR) HPV but with a normal Pap test result were offered a repeat Pap test and HPV genotyping at 12 months.For cervical and VVP specimens, cellular and viral DNA was extracted using the MagNA Pure LC (MP) isolation and purification system  with a modified protocol . For cerAny samples negative for HR-HPV on the Amplicor HPV test were further assessed for the presence of HR- and low risk (LR)-HPV. Briefly, 20 uL of extracted DNA was amplified for 40 cycles using 50 rmol of each of the Ll consensus primers PGMY09/11 . AmplifiAny sample positive for HR- or LR-HPV DNA was genotyped on the Roche Linear Array (LA) HPV genotyping test (Roche Molecular Systems). This test directs amplification of a 450 bp region of the HPV L1 gene and allows identification of 37 anogenital HPV genotypes  as well as amplification of 265 bp region of the human ß-globin gene, serving as an internal control. Samples were denatured and detected using a modified method as previously described ,16. Due At the completion of the study any cervical or VVP sample that was positive on any test had the corresponding cervical or VVP sample genotyped to ensure correct comparison of differing samples. There were 10 cervical samples and 5 VVP samples that were positive on initial HPV DNA testing, but no HPV genotype was detected on LA.The primary outcomes were the cervical and VVP prevalence of HR-HPV, defined as detection of any one of the following HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 or 59). Secondary outcomes included the cervical and VVP prevalence of: probable HR-HPV (type 68), possible HR-HPV , any-HPV (presence of any of the 37 types detectable on LA), LR-HPV , multiple HR types, multiple LR types, individual type-specific HPV and vaccine preventable types . All HPV prevalence figures were based on HPV as detected on LA. HPV types were classified into carcinogenic groups according to the International Agency for Research on Cancer [Proportions and means/medians were calculated to summarize the data as appropriate. As there is no data on the prevalence of vulvar HPV infection in Australian women, we compared the prevalence of cervical HR-HPV infection in Arnhem Land women to that of other Australian women (Indigenous and non-Indigenous) based on the results of the Women's HPV prevalence Indigenous Non-Indigenous Urban Rural Study (WHINURS) . This isThe sample size was estimated using preliminary data in 2006 from the WHINURS . To deteThis study was approved by the Human Research Ethics Committee of the Menzies School of Health Research and the Northern Territory Department of Health and Community Services, and its Aboriginal subcommittee.Five-hundred and sixty-two women consented to participate. Eleven women 1.9%) were found to be ineligible after enrolment due to age and were excluded. The characteristics of participants are summarised in Table .9% were All women consented to a vulvar examination and VVP swab collection. However, VVP samples were not adequate for HPV testing for 30 women, due to samples leaking during transportation. Five-hundred and nine women consented to Pap screening, and of these, cervical samples were not adequate for HPV testing in two women. A vault smear was undertaken in six women with a hysterectomy, and these samples were excluded from the cervical samples. As a result, a total of 521 VVP swabs and 501 cervical swabs were adequate for analysis.On examination, one new case of vulvar cancer and two new cases of high-grade vulvar lesion were found; six definitive high-grade cervical lesions were detected on Pap cytology screening Table . No new The prevalence of VVP HR-HPV was 39% compared with the cervical HR-HPV prevalence of 26%  adequate for HPV analysis. The prevalence of HR-HPV was significantly higher in the VVP samples than the cervical samples , as was the presence of multiple HR-HPV types  and possible HR-HPV types .Type-specific HPV prevalence was significantly higher in the VVP samples for the following HR-HPV types: 16 , 51 , 52 , 56  and 35 ; and possible HR-HPV types: 53 , 70  and 73 .For both VVP and cervical infections, HR-HPV prevalence peaked in the age-group <20 years and declined thereafter Figure . There wIn this community-based study of Indigenous women in the Arnhem Land region, where there is a very high incidence of vulvar neoplasia , genitalJust over a quarter of all women in this study had a cervical HR-HPV infection, which is very similar to the prevalence found in the WHINURS, the largest examination of cervical HPV genotype prevalence in Australian women  . In our A higher rate of HPV detection in vulvar/vaginal samples than cervical samples has been reported previously ,21: howeThe higher prevalence of VVP infection may reflect increased persistence of infection or continuing re-infection with different genotypes. Our finding that VVP HPV-16 prevalence did not vary significantly by age, whereas there was a marked and significant decline in cervical HPV-16 prevalence, suggests that increased persistence of HR types on the vulva alone may be occurring. This may explain why the cervical cancer incidence in this population is no higher than in other Indigenous women in the Northern Territory . We are The higher prevalence of infection in the VVP area relative to the cervix could also reflect incident infections that have not yet ascended to the cervix. Winer et al.  demonstrLongitudinal data are needed to examine persistence of VVP HPV infection in this population. However, while speculative, neither a 1.5-fold higher prevalence nor increased persistence of HR-HPV types is likely to be sufficient to explain the fifty-times higher incidence of vulvar cancer in this population compared to other Australian women. Other factors that alter the balance between HPV infection and host immunity are likely to be present. Such factors could be genetic, affecting the susceptibility to or inability to clear HPV, or environmental, possibly altering the natural history of infection of the vulvar epithelium. An interaction between a combination of risk factors  is also possible; genetic variation in Th1 cytokines has been shown to modify the risk of vulvar cancer among smokers . A very There are several limitations of this study. There is no data on HPV prevalence in the VVP area in other Australian women, and so a direct comparison of VVP HPV prevalence was not possible. The study design was cross-sectional, therefore persistence of HPV infection could not be adequately assessed. Similarly, detection of HPV DNA only reflects current infection or carriage status, hence there is no information about cumulative lifetime exposure to HPV. Some women (7%) did not consent to have a Pap smear, which reduced the number of women with both samples available for analysis. There was no difference in demographic characteristics or history of anogenital lesions between women who consented to a Pap smear and those who did not, suggesting that this was not a major source of bias. Finally, our results will not generalize to other Indigenous or non-Indigenous women, as the study was purposely designed to examine the prevalence of HPV in a population with, to our knowledge, the highest incidence of vulvar cancer reported worldwide.In young Indigenous women in the Arnhem Land region of Australia, a population subject to a cluster of vulvar cancer, we have shown that the prevalence of cervical HPV infection is similar to other Australian women, both Indigenous and non-Indigenous. We found a significantly higher prevalence of vulvar/vaginal/perianal infection to cervical in Arnhem Land Indigenous women. Although in other Australian women the prevalence of HPV infection in the vulvar/vaginal/perianal area is unknown, in our study population, the higher prevalence of infection in this area relative to cervical is unlikely to completely explain this cancer cluster. Further investigation of possible genetic and/or environmental risk factors that may impair host immunity is required.BMI: Body mass index; DNA: Deoxyribonucleic acid; HGSIL: High–grade squamous intraepithelial lesion; HIV: Human immunodeficiency virus; HPV: Human papillomavirus; HR: High-risk; HR-HPV: High-risk human papillomavirus; IARC: International agency for research on cancer; IQR: Interquartile range; LGSIL: Low-grade squamous intraepithelial lesion; LR: Low-risk; SCC: Squamous cell carcinoma; SD: Standard deviation; STI: Sexually transmissible infection; VIN: Vulvar intraepithelial neoplasia; VVP: Vulvar/vaginal/perianal; WHINURS: Women's HPV prevalence indigenous non-indigenous urban rural study.SMG has received advisory board fees and grant support from CSL and GSK; lecture fees from Merck, GSK and Sanofi Pasteur; funding (through her employing institution) to conduct HPV vaccine studies for MSD and GSK; and is a member of the Merck Global Advisory Board and the Merck Scientific Advisory Committee for HPV. SET is the recipient of a GlaxoSmithKline Australian Postgraduate Support Grant for work outside of this submitted manuscript. CSL Biotherapies have provided funding to the authorship group to support a workshop on genetic susceptibility to vulvar cancer.JRC, ARR, SNT and SMG had a primary role in developing the study design; DT and MN were responsible for participant recruitment, sample collection and data management; SET undertook the HPV genotyping of samples and all other associated laboratory work under the supervision of SNT. ARR conducted the analyses and drafted the manuscript; JRC, SET, DT, MN, MJD, MMO, CMC, IZ, JM and SMG contributed to the interpretation of analyses and assisted with preparation and editing of the manuscript. All authors read and approved the final manuscript.This work was supported by the National Health and Medical Research Council (NHMRC) in Australia, Grant ID: 436013. The views expressed in this publication are those of the authors and do not reflect the views of NHMRC. Alice Rumbold is supported by the Jean B Reid Fellowship from the University of Adelaide Medical Endowment Funds. Sarah Tan is supported by the Royal Women’s Hospital Post Graduate Degree Scholarship from the Royal Women’s Hospital, Melbourne Australia.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/243/prepub