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1.  Reduced acquisition and reactivation of human papillomavirus infections among older women treated with cryotherapy: results from a randomized trial in South Africa 
BMC Medicine  2010;8:40.
Treatment of women for high-grade cervical cancer precursors frequently results in clearance of the associated high-risk human papillomavirus (hrHPV) infection but the role of treatment among women without hrHPV is unknown. We investigated whether cervical cryotherapy reduces newly detected hrHPV infections among HIV-positive and HIV-negative women who were hrHPV negative when treated.
The impact of cryotherapy on newly detected hrHPV infections was examined among 612 women of known HIV serostatus, aged 35 to 65 years, who were negative for hrHPV DNA, and randomized to either undergo cryotherapy (n = 309) or not (n = 303). All women underwent repeat hrHPV DNA testing 6, 12, 24, and 36 months later.
Among 540 HIV-negative women, cryotherapy was associated with a significant reduction in newly detected hrHPV infections. Women in the cryotherapy group were 55% less likely to have newly detected hrHPV than women in the control group (95% CI 0.28 to 0.71). This association was independent of the influence of changes in sexual behaviors following therapy (adjusted hazards ratio (HR) = 0.49, 95% CI 0.29 to 0.81). Among 72 HIV-positive women, similar reductions were not observed (HR = 1.10, 95% CI 0.53 to 2.29).
Cervical cryotherapy significantly reduced newly detected hrHPV infections among HIV-negative, but not HIV-positive women. These results raise intriguing questions about immunological responses and biological mechanisms underlying the apparent prophylactic benefits of cryotherapy.
PMCID: PMC2907297  PMID: 20587028
2.  Impact of Vaccination on 14 High-Risk HPV Type Infections: A Mathematical Modelling Approach 
PLoS ONE  2013;8(8):e72088.
The development of high-risk human papillomavirus (hrHPV) infection to cervical cancer is a complicated process. We considered solely hrHPV infections, thus avoiding the confounding effects of disease progression, screening, and treatments. To analyse hrHPV epidemiology and to estimate the overall impact of vaccination against infections with hrHPVs, we developed a dynamic compartmental transmission model for single and multiple infections with 14 hrHPV types. The infection-related parameters were estimated using population-based sexual behaviour and hrHPV prevalence data from Finland. The analysis disclosed the important role of persistent infections in hrHPV epidemiology, provided further evidence for a significant natural immunity, and demonstrated the dependence of transmission probability estimates on the model structure. The model predicted that vaccinating girls at 80% coverage will result in a 55% reduction in the overall hrHPV prevalence and a higher 65% reduction in the prevalence of persistent hrHPV infections in females. In males, the reduction will be 42% in the hrHPV prevalence solely by the herd effect from the 80% coverage in girls. If such high coverage among girls is not reached, it is still possible to reduce the female hrHPV prevalence indirectly by the herd effect if also boys are included in the vaccination program. On the other hand, any herd effects in older unvaccinated cohorts were minor. Limiting the epidemiological model to infection yielded improved understanding of the hrHPV epidemiology and of mechanisms with which vaccination impacts on hrHPV infections.
PMCID: PMC3756967  PMID: 24009669
3.  Primary cervical cancer screening with HPV testing compared with liquid-based cytology: results of round 1 of a randomised controlled trial – the HPV FOCAL Study 
British Journal of Cancer  2012;107(12):1917-1924.
Round 1 data of human papillomavirus (HPV) FOCAL, a three-arm, randomised trial, which aims to establish the efficacy of HPV DNA testing as a primary screen for cervical cancer, are presented.
The three arms are: Control arm – liquid based cytology with atypical squamous cells of unknown significance (ASC-US) triage with hrHPV testing; Intervention Arm – hrHPV at entry with liquid-based cytology (LBC) triage of hrHPV positives, with exit screen at 4 years; Safety check arm – hrHPV at entry with LBC triage of hrHPV positives with exit screen at 2 years.
A total of 6154 women were randomised to the control arm and 12 494 to the HPV arms (intervention and safety check). In the HPV arm, the baseline cervical intraepithelial neoplasia (CIN)2+ and CIN3+ rate was 9.2/1000 (95%CI; 7.4, 10.9) and 4.8/1000 (95%CI; 3.6, 6.1), which increased to 16.1/1000 (95%CI 13.2, 18.9) for CIN2+ and to 8.0/1000 (95%CI; 5.9, 10.0) for CIN3+ after subsequent screening of HPV-DNA-positive/cytology-negative women. Detection rate in the control arm remained unchanged after subsequent screening of ASC-US-positive/hrHPV DNA-negative women at 11.0/1000 for CIN2+ and 5.0/1000 for CIN3+.
After subsequent screening of women who were either hrHPV positive/cytology negative or ASC-US positive/HPV negative, women randomised to the HPV arms had increased CIN2+ detection compared with women randomised to the cytology arm.
PMCID: PMC3516684  PMID: 23169286
HPV; cervical cancer; screening; randomised trial; North America
4.  A Promising DNA Methylation Signature for the Triage of High-Risk Human Papillomavirus DNA-Positive Women 
PLoS ONE  2014;9(3):e91905.
High-risk human papillomavirus (hrHPV)-DNA testing is frequently performed parallel to cytology for the detection of high-grade dysplasia and cervical cancer particularly in women above 30 years of age. Although highly sensitive, hrHPV testing cannot distinguish between HPV-positive women with or without clinically relevant lesions. However, in principle discrimination is possible on the basis of DNA methylation markers.
In order to identify novel DNA regions which allow an effective triage of hrHPV-positive cases, hypermethylated DNA enriched from cervical cancers was compared with that from cervical scrapes of HPV16-positive cases with no evidence for disease by CpG island microarray hybridization. The most promising marker regions were validated by quantitative methylation-specific PCR (qMSP) using DNA from archived cervical tissues and cervical scrapes. The performance of these markers was then determined in an independent set of 217 hrHPV-positive cervical scrapes from outpatients with histopathological verification.
A methylation signature comprising the 5′ regions of the genes DLX1, ITGA4, RXFP3, SOX17 and ZNF671 specific for CIN3 and cervical cancer (termed CIN3+) was identified and validated. A high detection rate of CIN3+ was obtained if at least 2 of the 5 markers were methylated. In the subsequent cross-sectional study all cervical carcinomas (n = 19) and 56% (13/23) of CIN3 were identified by this algorithm. Only 10% (11/105) of hrHPV-positive women without histological evidence of cervical disease were scored positive by the methylation assay. Of note is that the detection rate of CIN3 differed between age groups. Eight of nine CIN3 were detected among women ≥30 years of age but only five of fourteen among <30 year old group (p = 0.03). The specificity for CIN3+ in the older age group was 76.6% (95% CI 65.6–85.5%). Clinical validation studies are required to determine the usefulness of these novel markers for triage after primary hrHPV testing in a cervical cancer screening setting.
PMCID: PMC3960142  PMID: 24647315
5.  PIK3CA-mediated PI3-kinase signalling is essential for HPV-induced transformation in vitro 
Molecular Cancer  2011;10:71.
High-risk human papillomavirus (hrHPV) infections are causally related to cervical cancer development. The additional (epi)genetic alterations driving malignant transformation of hrHPV-infected cells however, are not yet fully elucidated. In this study we experimentally assessed the role of the PI3-kinase pathway and its regulator PIK3CA, which is frequently altered in cervical cancer, in HPV-induced transformation.
Cervical carcinomas and ectocervical controls were assessed for PIK3CA mRNA and protein expression by quantitative RT-PCR and immunohistochemical staining, respectively. A longitudinal in vitro model system of hrHPV-transfected keratinocytes, representing the immortal and anchorage independent phenotype, was assayed for PI3-kinase activation and function using chemical pathway inhibition i.e. LY294002 treatment, and PIK3CA RNA interference. Phenotypes examined included cellular viability, migration, anchorage independent growth and differentiation. mRNA expression of hTERT and HPV16 E6E7 were studied using quantitative RT-PCR and Northern blotting.
Cervical carcinomas showed significant overexpression of PIK3CA compared to controls. During HPV-induced transformation in vitro, expression of the catalytic subunit PIK3CA as well as activation of downstream effector PKB/AKT progressively increased in parallel. Inhibition of PI3-kinase signalling in HPV16-transfected keratinocytes by chemical interference or siRNA-mediated silencing of PIK3CA resulted in a decreased phosphorylation of PKB/AKT. Moreover, blockage of PI3-kinase resulted in reduced cellular viability, migration, and anchorage independent growth. These properties were accompanied with a downregulation of HPV16E7 and hTERT mRNA expression. In organotypic raft cultures of HPV16- and HPV18-immortalized cells, phosphorylated PKB/AKT was primarily seen in differentiated cells staining positive for cytokeratin 10 (CK10). Upon PI3-kinase signalling inhibition, there was a severe impairment in epithelial tissue development as well as a dramatic reduction in p-PKB/AKT and CK10.
The present data indicate that activation of the PI3-kinase/PKB/AKT pathway through PIK3CA regulates various transformed phenotypes as well as growth and differentiation of HPV-immortalized cells and may therefore play a pivotal role in HPV-induced carcinogenesis.
PMCID: PMC3130697  PMID: 21663621
6.  New Strategies for HPV-based Cervical Screening 
Women's health (London, England)  2013;9(5):10.2217/whe.13.48.
Human papillomavirus (HPV) testing has been shown to be far more sensitive and robust in detecting CIN2+ (and CIN3+) for cervical screening than approaches based on either cytology or visual inspection, but there are a number of issues that need to be overcome if it is to substantially reduce the morbidity and mortality from cervical cancer at a population level. The two main issues are coverage (increasing the numbers of women who participate in screening) and management of women who test high-risk HPV (hrHPV) positive. This article will review the potential for vaginal self-collection to improve coverage and the options for triage of hrHPV-positive women in high-resource and low-resource settings.
PMCID: PMC3880859  PMID: 24007250
Cancer Prevention; screening for cervical cancer; human papillomavirus testing; triage of hrHPV-positive women; vaginal self sampling
7.  Factors Affecting the Prevalence of Strongly and Weakly Carcinogenic and Lower-Risk Human Papillomaviruses in Anal Specimens in a Cohort of Men Who Have Sex with Men (MSM) 
PLoS ONE  2013;8(11):e79492.
MSM are at higher risk for invasive anal cancer. Twelve human papillomaviruses (HPVs) cause cervical cancer in women (Group 1 high-risk HPVs (hrHPVs)) and 13 HPVs are probable/possible causes (Group 2 hrHPVs) of cervical malignancy. HPVs rarely associated with malignancy are classified as lower-risk HPVs (lrHPVs).
Materials and Methods
Dacron-swab anal-cytology specimens were collected from and data complete for 97% (1262/1296) of Multicenter AIDS Cohort Study (MACS) men tested for HPVs using the Linear Array assay. Multivariate Poisson regression analyses estimated adjusted prevalence ratios for Group 1/2 hrHPVs and lrHPVs, controlling for the effects of age, race, ethnicity, sexual partnerships, smoking; HIV-infection characteristics, treatment, and immune status among HIV-infected men.
HIV-infected men showed 35–90% higher prevalence of Group 1/2 hrHPVs and lrHPVs than HIV-uninfected men, and higher prevalence of multi-Type, and multiple risk-group infections. CD4+ T-cell count was inversely associated with HPV Group 2 prevalence (p<0.0001). The number of receptive anal intercourse (RAI) partners reported in the 24 months preceding HPV testing predicted higher prevalence of Group 1/2 hrHPVs. Men reporting ≥30 lifetime male sex partners before their first MACS visit and men reporting ≥1 RAI partners during the 24 months before HPV testing showed 17–24% and 13–17% higher prevalence of lrHPVs (p-values ≤0.05). Men reporting smoking between MACS visit 1 and 24 months before HPV testing showed 1.2-fold higher prevalence of Group 2 hrHPVs (p = 0.03). Both complete adherence to CART (p = 0.02) and HIV load <50 copies/mL (p = 0.04) were protective for Group 1 hrHPVs among HIV-infected men.
HIV-infected men more often show multi-type and multi-group HPV infections HIV-uninfected men. Long-term mutual monogamy and smoking cessation, generally, and CART-adherence that promotes (HIV) viremia control and prevents immunosuppression, specifically among HIV-infected MSM, are important prevention strategies for HPV infections that are relevant to anal cancer.
PMCID: PMC3835810  PMID: 24278140
8.  Routine cervical screening with primary HPV testing and cytology triage protocol in a randomised setting 
British Journal of Cancer  2005;93(8):862-867.
The role of high-risk human papillomavirus (hrHPV) testing in primary cervical screening has not been established. We generated a randomised evaluation design ultimately to clarify whether primary hrHPV testing implemented into routine screening can bring increase in the programme effectiveness. The aim of the present report on first-year results was to assess the cross-sectional relative validity parameters for routine hrHPV screening, in comparison with conventional screening. An equal number of women invited to routine screening was randomly allocated to primary hrHPV screening (n=7060) and to cytological screening (n=7089). In the hrHPV screening arm, after a single positive hrHPV test result, the need of colposcopy referral was determined by a cytological triage test. Compared with the conventional arm, more colposcopy referrals were made in the hrHPV screening arm (relative risk 1.51, confidence interval 95% 1.03–2.22). Specificity of the primary screening with sole hrHPV test (91.5–92.1%) was much lower than that with the cytology triage (98.7–99.3%), which was not quite as specific as screening with conventional cytology (99.2–99.6%). Compared with conventional cytology, primary screening with hrHPV test results in increased cross-sectional relative sensitivity at the level of all positive lesions at the cost of substantial loss in specificity. With cytology triage, the specificity improves to the level of conventional cytology.
PMCID: PMC2361654  PMID: 16189520
cervical cancer; screening; high-risk HPV; randomised; evaluation; public health
9.  HPV DNA testing in population-based cervical screening (VUSA-Screen study): results and implications 
British Journal of Cancer  2012;106(5):975-981.
Human papillomavirus (HPV) testing is more sensitive than cytology for detecting high-grade cervical intraepithelial neoplasia (CIN). We evaluated the performance of high-risk HPV (hrHPV) testing in routine screening.
In all, 25 871 women (29–61) enrolled in our population-based cohort study were offered both cytology and hrHPV testing. High-risk HPV-positive women with normal cytology and an age-matched subcohort of hrHPV-negative women with normal cytology were invited for repeat testing after 1 and/or 2 years and were referred for colposcopy if they presented with abnormal cytology and/or a positive hrHPV test. The hrHPV-positive women with borderline or mild dyskaryosis (BMD) and all women with moderate dyskaryosis or worse (>BMD) were directly referred for colposcopy. Women with BMD and an hrHPV-negative test were advised to repeat cytology at 6 and 18 months and were referred for colposcopy if the repeat cytology test was abnormal. The main outcome measure was CIN grade 3 or worse (CIN3+). Results were adjusted for non-attendance at repeat testing.
The hrHPV-positive women with abnormal cytology had a CIN3+ risk of 42.2% (95% confidence interval (CI): 36.4–48.2), whereas the hrHPV-positive women with normal cytology had a much lower risk of 5.22% (95% CI: 3.72–7.91). In hrHPV-positive women with normal cytology, an additional cytology step after 1 year reduced the CIN3+ risk to only 1.6% (95% CI: 0.6–4.9) if the repeat test was normal. The CIN3+ risk in women with hrHPV-positive normal cytology was higher among women invited for the first time (29–33 years of age) (9.1% 95% CI: 5.6–14.3) than among older women (3.0% 95% CI: 1.5–5.5).
Primary hrHPV screening with cytology triage in women aged ⩾30 years is an effective way to stratify women on CIN3+ risk and seems a feasible alternative to cytological screening. Repeat cytology after 1 year for hrHPV-positive women with normal cytology is however necessary before returning women to routine screening.
PMCID: PMC3305964  PMID: 22251922
cytology; human papillomavirus; hybrid capture; cervical intraepithelial neoplasia; early detection of cancer
10.  HPV type-related chromosomal profiles in high-grade cervical intraepithelial neoplasia 
BMC Cancer  2012;12:36.
The development of cervical cancer and its high-grade precursor lesions (Cervical Intraepithelial Neoplasia grade 2/3 [CIN2/3]) result from a persistent infection with high-risk human papillomavirus (hrHPV) types and the accumulation of (epi)genetic host cell aberrations. Epidemiological studies have demonstrated variable CIN2/3 and cancer risks between different hrHPV types. Recent genomic profiling studies revealed substantial heterogeneity in the chromosomal aberrations detected in morphologically indistinguishable CIN2/3 suggestive of varying cancer risk. The current study aimed to investigate whether CIN2/3 with different hrHPV types vary with respect to their chromosomal profiles, both in terms of the number of aberrations and chromosomal loci affected.
Chromosomal profiles were determined of 43 p16INK4a-immunopositive CIN2/3 of women with long-term hrHPV infection (≥ 5 years). Sixteen lesions harboured HPV16, 3 HPV18, 14 HPV31, 1 HPV33, 4 HPV45, 1 HPV51, 2 HPV52 and 2 HPV58.
Unsupervised hierarchical clustering analysis of the chromosomal profiles revealed two major clusters, characterised by either few or multiple chromosomal aberrations, respectively. A majority of 87.5% of lesions with HPV16 were in the cluster with relatively few aberrations, whereas no such unbalanced distribution was seen for lesions harbouring other hrHPV types. Analysis of the two most prevalent types (HPV16 and HPV31) in this data set revealed a three-fold increase in the number of losses in lesions with HPV31 compared to HPV16-positive lesions. In particular, losses at chromosomes 2q, 4p, 4q, 6p, 6q, 8q & 17p and gain at 1p & 1q were significantly more frequent in HPV31-positive lesions (FDR < 0.2).
Chromosomal aberrations in CIN2/3 are at least in part related to the hrHPV type present. The relatively low number of chromosomal aberrations observed in HPV16-positive CIN2/3 suggests that the development of these lesions is less dependent on genetic insult than those caused by other types like HPV31.
PMCID: PMC3305644  PMID: 22273477
Array CGH; Cervical cancer; Chromosomal aberrations; High-grade cervical intraepithelial neoplasia; HPV
11.  Human Papillomavirus (HPV) Upregulates the Cellular Deubiquitinase UCHL1 to Suppress the Keratinocyte's Innate Immune Response 
PLoS Pathogens  2013;9(5):e1003384.
Persistent infection of basal keratinocytes with high-risk human papillomavirus (hrHPV) may cause cancer. Keratinocytes are equipped with different pattern recognition receptors (PRRs) but hrHPV has developed ways to dampen their signals resulting in minimal inflammation and evasion of host immunity for sustained periods of time. To understand the mechanisms underlying hrHPV's capacity to evade immunity, we studied PRR signaling in non, newly, and persistently hrHPV-infected keratinocytes. We found that active infection with hrHPV hampered the relay of signals downstream of the PRRs to the nucleus, thereby affecting the production of type-I interferon and pro-inflammatory cytokines and chemokines. This suppression was shown to depend on hrHPV-induced expression of the cellular protein ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) in keratinocytes. UCHL1 accomplished this by inhibiting tumor necrosis factor receptor-associated factor 3 (TRAF3) K63 poly-ubiquitination which lead to lower levels of TRAF3 bound to TANK-binding kinase 1 and a reduced phosphorylation of interferon regulatory factor 3. Furthermore, UCHL1 mediated the degradation of the NF-kappa-B essential modulator with as result the suppression of p65 phosphorylation and canonical NF-κB signaling. We conclude that hrHPV exploits the cellular protein UCHL1 to evade host innate immunity by suppressing PRR-induced keratinocyte-mediated production of interferons, cytokines and chemokines, which normally results in the attraction and activation of an adaptive immune response. This identifies UCHL1 as a negative regulator of PRR-induced immune responses and consequently its virus-increased expression as a strategy for hrHPV to persist.
Author Summary
A persistent infection with high-risk human papillomavirus (hrHPV) may cause cancer. Whereas keratinocytes – the cells infected by hrHPV – are equipped with different receptors allowing them to recognize invading pathogens and to activate the immune system, hrHPV has developed ways to evade the host's immune response for sustained periods of time. We showed that hrHPV accomplishes this by interfering with the signaling of the pathogen receptors, thereby hampering the production of cytokines that are known to attract and activate the immune system. HrHPV accomplishes this by upregulating the expression of a cellular protein called ubiquitin carboxyl-terminal hydrolase L1 (UCHL1). This protein suppresses the activation of signals downstream of the pathogen receptor leading to reduced transcription factor activation and downstream gene expression, in particular that of type I interferon and pro-inflammatory cytokines. This lowers the attraction of immune cells and thereby the chance of hrHPV-infected cells to be recognized and eliminated and as such enables hrHPV to persist.
PMCID: PMC3662672  PMID: 23717208
12.  Type-Specific Human Papillomavirus Biological Features: Validated Model-Based Estimates 
PLoS ONE  2013;8(11):e81171.
Infection with high-risk (hr) human papillomavirus (HPV) is considered the necessary cause of cervical cancer. Vaccination against HPV16 and 18 types, which are responsible of about 75% of cervical cancer worldwide, is expected to have a major global impact on cervical cancer occurrence. Valid estimates of the parameters that regulate the natural history of hrHPV infections are crucial to draw reliable projections of the impact of vaccination. We devised a mathematical model to estimate the probability of infection transmission, the rate of clearance, and the patterns of immune response following the clearance of infection of 13 hrHPV types. To test the validity of our estimates, we fitted the same transmission model to two large independent datasets from Italy and Sweden and assessed finding consistency. The two populations, both unvaccinated, differed substantially by sexual behaviour, age distribution, and study setting (screening for cervical cancer or Chlamydia trachomatis infection). Estimated transmission probability of hrHPV types (80% for HPV16, 73%-82% for HPV18, and above 50% for most other types); clearance rates decreasing as a function of time since infection; and partial protection against re-infection with the same hrHPV type (approximately 20% for HPV16 and 50% for the other types) were similar in the two countries. The model could accurately predict the HPV16 prevalence observed in Italy among women who were not infected three years before. In conclusion, our models inform on biological parameters that cannot at the moment be measured directly from any empirical data but are essential to forecast the impact of HPV vaccination programmes.
PMCID: PMC3882251  PMID: 24400036
13.  hTERT promoter activity and CpG methylation in HPV-induced carcinogenesis 
BMC Cancer  2010;10:271.
Activation of telomerase resulting from deregulated hTERT expression is a key event during high-risk human papillomavirus (hrHPV)-induced cervical carcinogenesis. In the present study we examined hTERT promoter activity and its relation to DNA methylation as one of the potential mechanisms underlying deregulated hTERT transcription in hrHPV-transformed cells.
Using luciferase reporter assays we analyzed hTERT promoter activity in primary keratinocytes, HPV16- and HPV18-immortalized keratinocyte cell lines and cervical cancer cell lines. In the same cells as well as cervical specimens we determined hTERT methylation by bisulfite sequencing analysis of the region spanning -442 to +566 (relative to the ATG) and quantitative methylation specific PCR (qMSP) analysis of two regions flanking the hTERT core promoter.
We found that in most telomerase positive cells increased hTERT core promoter activity coincided with increased hTERT mRNA expression. On the other hand basal hTERT promoter activity was also detected in telomerase negative cells with no or strongly reduced hTERT mRNA expression levels. In both telomerase positive and negative cells regulatory sequences flanking both ends of the core promoter markedly repressed exogenous promoter activity.
By extensive bisulfite sequencing a strong increase in CpG methylation was detected in hTERT positive cells compared to cells with no or strongly reduced hTERT expression. Subsequent qMSP analysis of a larger set of cervical tissue specimens revealed methylation of both regions analyzed in 100% of cervical carcinomas and 38% of the high-grade precursor lesions, compared to 9% of low grade precursor lesions and 5% of normal controls.
Methylation of transcriptionally repressive sequences in the hTERT promoter and proximal exonic sequences is correlated to deregulated hTERT transcription in HPV-immortalized cells and cervical cancer cells. The detection of DNA methylation at these repressive regions may provide an attractive biomarker for early detection of cervical cancer.
PMCID: PMC2904279  PMID: 20534141
14.  Implementation of human papillomavirus testing in cervical screening without a concomitant decrease in participation rate 
Journal of Clinical Pathology  2006;59(11):1218-1220.
Adding high‐risk human papillomavirus (hrHPV) testing to screening increases the efficacy of cervical screening programmes. However, hrHPV testing may result in a lower participation rate because of the perceived association with sexually transmitted infections. We describe how testing for hrHPV was added to cervical screening in the POpulation‐BAsed SCreening study AMsterdam (POBASCAM) trial. Participation rates of the screening programme before and after hrHPV implementation were evaluated in the region where the POBASCAM trial was carried out. The participation rate was 58.7% before and 61.4% after the addition of hrHPV testing to screening (p<0.001). An inventory of frequently asked questions is presented. Thus, hrHPV testing can be added to cervical screening by cytology without a decrease in participation rate.
PMCID: PMC1860522  PMID: 16943223
15.  Clinical Validation of a Type-Specific Real-Time Quantitative Human Papillomavirus PCR against the Performance of Hybrid Capture 2 for the Purpose of Cervical Cancer Screening 
Journal of Clinical Microbiology  2012;50(12):4073-4077.
To be acceptable for use in cervical cancer screening, a new assay that detects DNA of high-risk human papillomavirus (hrHPV) types must demonstrate high reproducibility and performance not inferior to that of a clinically validated HPV test. In the present study, a real-time quantitative PCR (qPCR) assay targeting the E6 and E7 genes of hrHPV was compared with Hybrid Capture 2 (hc2) in a Belgian cervical cancer screening setting. In women >30 years old, the sensitivity and specificity for intraepithelial neoplasias of grade 2 or worse (93 cases of cervical intraepithelial neoplasias of grade 2 or worse (CIN2+) and 1,207 cases of no CIN or CIN1) were 93.6% and 95.6%, respectively, and those of hc2 were 83.9% and 94.5%, respectively {relative sensitivity of qPCR/hc2 = 1.12 [95% confidence interval (CI), 1.01 to 1.23]; relative specificity = 1.01 [95% CI, 0.99 to 1.03]}. A score test showed that the sensitivity (P < 0.0001) and specificity (P < 0.0001) of the qPCR assay were not inferior to those of hc2 at the required thresholds of 90% and 98%, respectively. The overall agreement of hrHPV positivity between the two runs of the qPCR tests was 98.7% (95% CI, 97.5 to 99.4%), with a kappa value of 0.96 (95% CI, 0.83 to 1.00). The qPCR assay used in this study can be considered a reliable HPV assay that fulfills the clinical validation criteria defined for use in cervical cancer screening.
PMCID: PMC3503006  PMID: 23052314
16.  Consistent condom use increases spontaneous regression in high-risk non-HPV16 but not in HPV16 CIN2-3 lesions, a prospective population-based cohort study 
The major cause of cervical intraepithelial neoplasia (CIN) is persistent infection with human papillomavirus (HPV). Most CIN grade 2 and 3 lesions are treated with cone excision, although a substantial proportion (6-50%) of CIN2-3 lesions will regresses spontaneously. Predictors for regression of CIN2-3 are desirable in order to reduce this overtreatment.
In this prospective cohort study, 145 consecutive women with first-time onset CIN2-3 in colposcopy-directed biopsies and standardized biopsy-cone excision interval were included. The genotype of the high-risk human papillomaviruses (=hrHPV) and clinical factors including sexual behaviour, parity, contraception and smoking were assessed. Patients were divided into two groups according to lesions containing HPV16 (hrHPV16+) and high-risk non-HPV16 (hrHPV16-) genotypes.
Women whose partners consistently used condoms showed a significantly higher regression rate than women using other types of contraception (53% versus 13%, p<0.0001). However, this effect was only seen in hrHPV16- patients (73% regression rate versus 13%, p<0.0001). HrHPV16+ patients had a significantly higher number of sexual partners and more current smokers compared to hrHPV16- patients. The regression rate was not significantly different in CIN2-3 lesions containing HPV16 (hrHPV16+) versus hrHPV16- genotypes.
Heterogeneity among hrHPV genotypes excists. HPV-genotype analyses can identify women who significantly increase their chance of regression by consistent condom use.
PMCID: PMC3523032  PMID: 23126423
CIN2-3; High-risk HPV; HPV16; Regression; Condom use; Clinical factors
17.  HPV Detection Methods 
Disease Markers  2007;23(4):273-281.
Given the causal relation between a persistent high-risk human papillomavirus (hrHPV) infection and the development of high-grade cervical intraepithelial neoplasia (CIN) and cervical cancer, hrHPV testing has been advocated in addition to cytology for the detection of clinically relevant cervical lesions. HrHPV testing is thought to improve cervical screening algorithms, the management of women with cytologically equivocal smears, and the management of women treated for high grade CIN. In this chapter we discuss different methods for HPV detection and genotyping and their respective applications.
PMCID: PMC3851620  PMID: 17627062
Human papillomavirus; polymerase chain reaction; reverse hybridization; hybrid capture; real-time PCR; cervical carcinoma; NASBA; DNA probes; epidemiology
18.  Limitations of widely used high-risk human papillomavirus laboratory-developed testing in cervical cancer screening 
To increase awareness of the limitations of high-risk human papillomavirus (hrHPV) laboratory-developed testing (LDT) widely used in US cervical cancer screening.
Methods and results:
A young woman in her 30s was diagnosed and treated for stage 1B1 cervical squamous cell carcinoma in which HPV 16 DNA was detected using polymerase chain reaction testing. Both 1 month before and 42 months before cervical cancer diagnosis, the patient had highly abnormal cytology findings; however, residual SurePath™ (Becton, Dickson and Company, Franklin Lakes, NJ) vial fluid yielded negative Hybrid Capture 2 (HC2; Qiagen NV, Hilden, Germany) hrHPV LDT results from each of the two specimens. This prompted questions to be asked concerning the performance characteristics of hrHPV LDT. A review of the available data indicates that (1) purification of DNA from SurePath specimens requires complex sample preparation due to formaldehyde crosslinking of proteins and nucleic acids, (2) HC2–SurePath hrHPV testing had not been Food and Drug Administration-approved after multiple premarket approval submissions, (3) detectible hrHPV DNA in the SurePath vial decreases over time, and (4) US laboratories performing HC2–SurePath hrHPV LDT testing are not using a standardized manufacturer-endorsed procedure.
Recently updated cervical screening guidelines in the US recommend against the use of hrHPV LDT in cervical screening, including widely used HC2 testing from the SurePath vial. The manufacturer recently issued a technical bulletin specifically warning that use of SurePath samples with the HC2 hrHPV test may provide false negative results and potentially compromise patient safety. Co-collection using a Food and Drug Administration-approved hrHPV test medium is recommended for HPV testing of patients undergoing cervical screening using SurePath samples.
PMCID: PMC3496968  PMID: 23152707
HPV; SurePath; Hybrid Capture 2; LDT; cervical screening
19.  Human Papillomavirus Type 16 E7 Oncoprotein Associates with the Cullin 2 Ubiquitin Ligase Complex, Which Contributes to Degradation of the Retinoblastoma Tumor Suppressor▿  
Journal of Virology  2007;81(18):9737-9747.
Human papillomavirus type 16 (HPV16) and other high-risk HPVs are etiologically linked to the development of cervical carcinomas and contribute to a number of other tumors of the anogenital tract, as well as oral cancers. The high-risk HPV E6 and E7 oncoproteins are consistently expressed in cervical cancer cells and are necessary for the induction and maintenance of the transformed phenotype. An important aspect of HPV16 E7's oncogenic activities is destabilization of the retinoblastoma tumor suppressor (pRB) through a ubiquitin/proteasome-dependent mechanism, although the exact molecular mechanism is unknown. Here, we report that HPV16 E7 is associated with an enzymatically active cullin 2 ubiquitin ligase complex and that the HPV16 E7/pRB complex contains cullin 2. Depletion of cullin 2 by RNA interference causes increased steady-state levels and stability of pRB in HPV16 E7-expressing cells, and ectopic expression of HPV16 E7 and the cullin 2 complex leads to pRB ubiquitination in vivo. Hence, we propose that the HPV16 E7-associated cullin 2 ubiquitin ligase complex contributes to aberrant degradation of the pRB tumor suppressor in HPV16 E7-expressing cells.
PMCID: PMC2045412  PMID: 17609271
20.  HPV-DNA testing for cervical cancer precursors: from evidence to clinical practice 
ecancermedicalscience  2012;6:258.
The large amount of literature published over the last two decades on human papillomavirus (HPV)-DNA testing has definitely demonstrated the association between high-risk viral genotypes (hrHPV) and cervical cancer. Moreover, hrHPV-DNA testing has shown excellent performance in several clinical applications, from screening settings to the follow-up of treated patients, compared to conventional cytology or colposcopy options. On the other hand, when a huge number of reports are published on the same subject in a relatively short period of time, with many variations in settings, study designs and applications, the result is often confusion and decreased comprehension by readers. In daily office practice, several different situations (in symptomatic or asymptomatic women) can be positively managed by the correct use of hrHPV-DNA testing. Validated hrHPV-DNA testing and, specifically, the HC2® assay, due to its excellent sensitivity and negative predictive value together with optimal reproducibility, currently represent a powerful tool in the clinician’s hands to optimally manage several situations related to HPV infection and the potential development of cervical cancer.
PMCID: PMC3388143  PMID: 22778786
cervical cancer; human papillomavirus (HPV); HPV-DNA; screening
21.  Preventive and Therapeutic Vaccines against Human Papillomaviruses Associated Cervical Cancers 
Cervical cancer is, globally known to be, one of the most common cancers among women especially in developing countries. More than 90% of cervical cancers are associated with high-risk human papillomaviruses (HPVs) particularly HPV types 16 and 18. Two major strategies have been developed for prevention and treatment of cervical cancer and other HPV-associated malignancies; the first one is based on HPV virus-like particles (VLPs) containing HPV structural proteins. VLP based vaccines can induce genotype specific virus neutralizing antibodies for preventing HPV infections. The other strategy is based on HPV early genes especially E6 and E7 for eliminating the established HPV infections; therefore they are classified as HPV therapeutic vaccines. This article reviews the preventive and therapeutic vaccines against HPV infections and cervical cancer.
PMCID: PMC3586871  PMID: 23493151
Cervical cancer; Human papillomavirus; Preventive vaccine; Therapeutic vaccine
22.  Growth Suppression Induced by Downregulation of E6-AP Expression in Human Papillomavirus-Positive Cancer Cell Lines Depends on p53 
Journal of Virology  2005;79(14):9296-9300.
The ubiquitin-protein ligase E6-AP is utilized by the E6 oncoprotein of human papillomaviruses (HPVs) associated with cervical cancer to target the tumor suppressor p53 for degradation. Here, we report that downregulation of E6-AP expression by RNA interference results in both the accumulation of p53 and growth suppression of the HPV-positive cervical cancer cell lines HeLa and SiHa. In addition, HeLa cells, in which p53 expression was suppressed by RNA interference, are significantly less sensitive to the downregulation of E6-AP expression with respect to growth suppression than parental HeLa cells. These data indicate that the anti-growth-suppressive properties of E6-AP in HPV-positive cells depend on its ability to induce p53 degradation.
PMCID: PMC1168765  PMID: 15994823
23.  Distribution of High-Risk Human Papillomavirus Genotypes among HIV-Negative Women with and without Cervical Intraepithelial Neoplasia in South Africa 
PLoS ONE  2012;7(9):e44332.
Large studies describing the profile of high-risk Human papillomavirus (hrHPV) genotypes among women in sub-Saharan Africa are lacking. Here we describe the prevalence and distribution of hrHPV genotypes among HIV-negative women in South Africa, with and without cervical intraepithelial neoplasia (CIN).
We report data on 8,050 HIV-negative women, aged 17–65 years, recruited into three sequential studies undertaken in Cape Town, South Africa. Women had no history of previous cervical cancer screening. Cervical samples were tested for hrHPV DNA using the Hybrid Capture 2 (HC2) assay and all positive samples were genotyped using a PCR-based assay (Line Blot). Women underwent colposcopy and biopsy/endocervical curettage to determine CIN status. The prevalence and distribution of specific hrHPV genotypes were examined by age and CIN status.
Overall, 20.7% (95% CI, 19.9–21.6%) of women were hrHPV-positive by HC2, with women with CIN having the highest rates of positivity. Prevalence decreased with increasing age among women without CIN; but, a bimodal age curve was observed among women with CIN. HPV 16 and 35 were the most common hrHPV genotypes in all age and CIN groups. HPV 45 became more frequent among older women with CIN grade 2 or 3 (CIN2,3). Younger women (17–29 years) had more multiple hrHPV genotypes overall and in each cervical disease group than older women (40–65 years).
HPV 16, 35, and 45 were the leading contributors to CIN 2,3. The current HPV vaccines could significantly reduce HPV-related cervical disease; however, next generation vaccines that include HPV 35 and 45 would further reduce cervical disease in this population.
PMCID: PMC3435398  PMID: 22970201
24.  Human Papillomavirus and Cervical Cancer 
Clinical Microbiology Reviews  2003;16(1):1-17.
Of the many types of human papillomavirus (HPV), more than 30 infect the genital tract. The association between certain oncogenic (high-risk) strains of HPV and cervical cancer is well established. Although HPV is essential to the transformation of cervical epithelial cells, it is not sufficient, and a variety of cofactors and molecular events influence whether cervical cancer will develop. Early detection and treatment of precancerous lesions can prevent progression to cervical cancer. Identification of precancerous lesions has been primarily by cytologic screening of cervical cells. Cellular abnormalities, however, may be missed or may not be sufficiently distinct, and a portion of patients with borderline or mildly dyskaryotic cytomorphology will have higher-grade disease identified by subsequent colposcopy and biopsy. Sensitive and specific molecular techniques that detect HPV DNA and distinguish high-risk HPV types from low-risk HPV types have been introduced as an adjunct to cytology. Earlier detection of high-risk HPV types may improve triage, treatment, and follow-up in infected patients. Currently, the clearest role for HPV DNA testing is to improve diagnostic accuracy and limit unnecessary colposcopy in patients with borderline or mildly abnormal cytologic test results.
PMCID: PMC145302  PMID: 12525422
25.  Long-term protective effect of high-risk human papillomavirus testing in population-based cervical screening 
British Journal of Cancer  2005;92(9):1800-1802.
We prospectively evaluated the 5-year predictive values of adding high-risk human papillomavirus (hrHPV) testing to cytology for the detection of ⩾cervical intraepithelial neoplasia (CIN)3 lesions in a population-based cohort of 2810 women. At baseline, nine (0.3%) women had prevalent lesions ⩾CIN3, all being hrHPV positive. After 5 years of follow-up, four (6.5%) of the 62 hrHPV-positive women with normal cytology developed lesions ⩾CIN3, vs only one (0.05%) of the 2175 hrHPV-negative women with normal cytology. High-risk human papillomavirus testing or combined screening revealed a much higher sensitivity, at the cost of a small decrease in specificity, and a higher negative predictive value for the detection of lesions ⩾CIN3 till the next screening round (5 years) than cytology alone.
PMCID: PMC2362030  PMID: 15827553
human papillomavirus; cervical cancer; screening; long-term

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