Showing papers by "Chris J.L.M. Meijer published in 2014"

Clinical implications of (epi)genetic changes in HPV-induced cervical precancerous lesions

Abstract: Infection of cervical epithelium with high-risk human papilloma virus (hrHPV) might result in productive or transforming cervical intraepithelial neoplasia (CIN) lesions, the morphology of which can overlap. In transforming CIN lesions, aberrations in host cell genes accumulate over time, which is necessary for the ultimate progression to cancer. On the basis of (epi)genetic changes, early and advanced transforming CIN lesions can be distinguished. This paves the way for new molecular tools for cervical screening, diagnosis and management of cervical cancer precursor lesions.

Triage by methylation-marker testing versus cytology in women who test HPV-positive on self-collected cervicovaginal specimens (PROHTECT-3): a randomised controlled non-inferiority trial

Abstract: Summary Background Cytology is a widely used method of triaging women who test positive for human papillomavirus (HPV). However, self-sampled specimens, which can substantially increase participation in screening programmes, are not suitable for accurate cytological assessment. We investigated whether direct DNA methylation-based molecular triage on self-sampled cervicovaginal specimens was non-inferior to cytology triage on additional physician-collected cervical samples in the detection of cervical intraepithelial neoplasia grade 2 (CIN2) or worse in women who did not attend cervical screening programmes. Methods In this randomised controlled non-inferiority trial, we invited women (aged 33–63 years) registered as non-attendees of cervical screening in the Netherlands in 2007 to submit a self-collected cervicovaginal sample for HPV testing. Using a computer-generated sequence, we randomly allocated women who tested positive for high-risk hrHPV on a self-sample to either triage by cytology on an additional physician-taken smear or direct triage on the self-sample by methylation analysis of MAL and miR-124-2 genes (1:1; stratified by age and region, with block sizes by age group). Triage-positive women in either group were referred for colposcopy. The primary endpoint was detection of CIN2 or worse, analysed by intention to treat. The non-inferiority margin was 0·80. This study is registered in the Primary Trial Register of the Netherlands, number NTR6026. Findings We invited 46 001 women to participate, 12 819 of whom returned self-sampled material; 1038 samples tested positive for high-risk HPV. Between Nov 1, 2010, and Dec 31, 2011, after exclusion of women who were ineligible, we enrolled and randomly allocated 515 women to methylation triage and 509 to cytology triage. The detection of CIN2 or worse with methylation triage was non-inferior to that with cytology triage (90 [17%] of 515 women vs 75 [15%] of 509 women; relative risk 1·19, 95% CI 0·90–1·57). Referral for colposcopy was more common in the molecular group (284 [55%] women) than in the cytology group (149 [29%] women; p Interpretation DNA methylation analysis of MAL and miR-124-2 genes on HPV-test-positive self-samples is non-inferior to cytology triage in the detection of CIN2 or worse, opening the way to full molecular screening. Funding Midden-West and Oost Screening Organisations and Stichting Achmea Gezondheidszorg.

Methylation Analysis of the FAM19A4 Gene in Cervical Scrapes Is Highly Efficient in Detecting Cervical Carcinomas and Advanced CIN2/3 Lesions

Abstract: Primary testing for human papillomavirus (HPV) in cervical screening requires triage to differentiate women with transient infection from those with persistent infection who require more intensive management given their risk for cervical (pre)cancer. In this study, the clinical performance of a novel methylation marker FAM19A4 for the triage of high-risk (hr)HPV-positive women was evaluated. Using a training-validation set approach, we analyzed a FAM19A4 quantitative methylation-specific PCR (qMSP). The training set comprised hrHPV-positive cervical scrapes of 43 women with cervical intraepithelial neoplasia grade 3 or worse (CIN3+) and 135 women with ≤CIN1. The validation set comprised hrHPV-positive cervical scrapes of 52 women with CIN2+, including 33 CIN3+, 19 CIN2, and 166 women with ≤CIN1. The methylation threshold of FAM19A4 qMSP that gave rise to CIN3+ specificity of 70% in the training set was applied in the validation set. This resulted in CIN3+ sensitivity of 75.8% [95% confidence interval (CI), 61.1–90.4] at 67.0% (95% CI, 60.3–73.8) specificity. Next, the validated qMSP was applied to an independent series of hrHPV-positive cervical scrapes of 22 women with cervical cancer, 29 with advanced CIN2/3 [i.e., women with a known preceding hrHPV infection (PHI) lasting ≥5 years as proxy of longer duration of lesion existence], and 19 with early CIN2/3 (i.e., PHI <5 years). All carcinomas (22/22) and advanced CIN2/3 lesions (29/29) were FAM19A4 methylation–positive, compared with 42.1% (8/19; 95% CI, 19.9–64.3) of early CIN2/3 lesions. In conclusion, FAM19A4 is an attractive triage marker for hrHPV-positive women, with a high reassurance for the detection of cervical carcinoma and advanced CIN2/3 lesions. Cancer Prev Res; 7(12); 1251–7. ©2014 AACR .

CADM1, MAL and miR124-2 methylation analysis in cervical scrapes to detect cervical and endometrial cancer.

Abstract: Aims Gene promoter hypermethylation is recognised as an essential early step in carcinogenesis, indicating important application areas for DNA methylation analysis in early cancer detection. The current study was set out to assess the performance of CADM1 , MAL and miR124- 2 methylation analysis in cervical scrapes for detection of cervical and endometrial cancer. Methods A series of cervical scrapes of women with cervical (n=79) or endometrial (n=21) cancer, cervical intraepithelial neoplasia grade 3 (CIN3) (n=16) or CIN2 (n=32), and women without evidence of CIN2 or worse (n=120) were assessed for methylation of CADM1 , MAL and miR124-2 . Methylation analysis was done by the PreCursor-M assay, a multiplex quantitative methylation-specific PCR. Results All samples of women with cervical cancer (79/79, 100%), independent of the histotype, and 76% (16/21; 95% CI 58.0% to 94.4%) of women with endometrial cancer scored positive for DNA methylation for at least one of the three genes. In women without cancer, methylation frequencies increased significantly with severity of disease from 19.2% (23/120; 95% CI 12.1% to 26.2%) in women without CIN2 or worse to 37.5% (12/32; 95% CI 20.7% to 54.3%) and 68.8% (11/16; 95% CI 46.0% to 91.5%) in women with CIN2 and CIN3, respectively. Overall methylation positivity and the number of methylated genes increased proportionally to the lesion severity. Conclusions DNA methylation analysis of CADM1 , MAL and miR124-2 in cervical scrapes consistently detects cervical cancer and the majority of CIN3 lesions, and has the capacity to broaden its use on cervical scrapes through the detection of a substantial subset of endometrial carcinomas.

Primary hrHPV DNA Testing in Cervical Cancer Screening: How to Manage Screen-Positive Women? A POBASCAM Trial Substudy

Abstract: Background: High-risk Human Papillomavirus (hrHPV) testing has higher sensitivity but lower specificity than cytology for cervical (pre)-cancerous lesions. Therefore, triage of hrHPV positive women is needed in cervical cancer screening. Methods: A cohort of 1,100 hrHPV positive women, from a population-based screening trial (POBASCAM: n= 44,938, 29-61 years) was used to evaluate ten triage strategies, involving testing at baseline and six months with combinations of cytology, HPV16/18 genotyping and/or repeat hrHPV testing. Clinical end-point was cervical intra-epithelial neoplasia grade 3 or worse (CIN3+) detected within four years; results were adjusted for women not attending repeat testing. A triage strategy was considered acceptable, when the probability of no CIN3+ after negative triage (negative predictive value, NPV) was at least 98%, and the CIN3+ risk after positive triage (positive predictive value; PPV) was at least 20%. Results : Triage at baseline with cytology only, yielded a NPV of 94.3% (95%CI: 92.0-96.0) and PPV of 39.7% (95%CI: 34.0-45.6). An increase in NPV, against a modest decrease in PPV, was obtained by triaging women with negative baseline cytology by repeat cytology (NPV 98.5%, PPV 34.0%) or by baseline HPV16/18 genotyping (NPV 98.8%, PPV 28.5%). Including both HPV16/18 genotyping at baseline and repeat cytology testing, provided a high NPV (99.6%) and a moderately high PPV (25.6%). Conclusions: Triaging hrHPV positive women by cytology at baseline and after 6-12 months, possibly in combination with baseline HPV16/18 genotyping, seems acceptable for cervical cancer screening. Impact: Implementable triage strategies are provided for primary hrHPV screening in an organized setting.

Methylation marker analysis of self‐sampled cervico‐vaginal lavage specimens to triage high‐risk HPV‐positive women for colposcopy

Abstract: Methylation markers were studied for their suitability to triage human papillomavirus (HPV)-positive women by testing self-collected cervico-vaginal lavage specimens. For this purpose, we analyzed 355 hrHPV-positive self-collected specimens with three methylation markers, that is, CADM1-m18, MAL-m1 and miR-124-2 by quantitative methylation-specific PCR. The areas under the receiver-operating characteristic (ROC) curve for end-point cervical intraepithelial neoplasia grade 3 or worse (CIN3+) were 0.637 for CADM1-m18, 0.767 for MAL-m1 and 0.762 for miR-124-2. This indicates that CADM1-m18 is not suitable as single marker. By varying the thresholds of both markers in the bi-marker panels CADM1-m18/MAL-m1, CADM1-m18/miR-124-2 and MAL-m1/miR-124-2 upper and lower ROC curves were obtained, depicting the maximum and minimum CIN3+ sensitivity, respectively, at given specificity. For all these bi-marker combinations, the upper curves were similar. However, for the MAL-m1/miR-124-2 panel, the distance between upper and lower ROC curves was closest and this panel displayed the highest assay thresholds, indicating that this combination was most robust. At clinical specificities of 50 and 70%, the MAL-m1/miR-124-2 sensitivity for detection of CIN3+ ranged from 77.0 to 87.8% and from 64.9 to 71.6%, respectively. At 70% specificity thresholds no carcinomas were missed. By comparison, the CIN3+ sensitivity of HPV16/18 genotyping on the self-sampled lavage specimens was 58.1% (95%CI: 46.6-68.8) at a specificity of 87.7% (95%CI: 83.2-91.2). In conclusion, methylation analysis is a promising triage tool that in combination with HPV-DNA testing offers feasible, full molecular screening on self-collected cervico-vaginal lavage specimens.

Early detection of CIN3 and cervical cancer during long-term follow-up using HPV/Pap smear co-testing and risk-adapted follow-up in a locally organised screening programme

Abstract: We evaluated compliance with human papillomavirus (HPV) testing and risk-adapted patient pathways and monitored changes in high-grade cervical disease during long-term follow-up. Women aged >30 years attending routine screening for cervical cancer were managed according to results from first-round screening tests (cytology and high-risk HPV; Hybrid Capture 2). Between February 2006 and January 2011, 19,795 of 19,947 women agreed to participate, of whom 4,067 proceeded to a second screening round 5 years after recruitment. Predefined endpoints were compliance, grade 3 cervical intraepithelial neoplasia or cancer (CIN3+), new HPV infection, HPV persistence and abnormal smears in round 2. A total of 765 of 19,795 women (3.9%) in round 1 and 41 of 4,067 (1.0%) in round 2 were referred for colposcopy. Compliance rates with colposcopy were 93.1 and 92.7%, respectively, while histological assessment was performed in 680 of 712 (95.5%) and 36 of 38 (94.7%), respectively. CIN3+ rates were 172 of 19,795 (0.87%; 95% confidence intervals: 0.7-1.0) in round 1 and 2 of 4,064 (0.05%; 95% confidence intervals: 0.006-0.2) in round 2; the difference was statistically significant (Fisher's exact test, p<0.001). After 5 years, the incidence of new HPV infection was 124 of 3,906 (3.2%) and HPV persistence was observed in 22 of 161 (13.7%). Locally organised HPV/cytology co-testing is feasible and acceptable to women. Risk-adapted management rapidly detected a high rate of prevalent CIN3+, while the subsequent long-term risk of new high-grade cervical disease was surprisingly low. It remains unclear if this phenomenon is explained by CIN3 mostly occurring early in life or by modifying the natural course of HPV infection with colposcopy and histological assessment.

Triaging borderline/mild dyskaryotic Pap cytology with p16/Ki-67 dual-stained cytology testing: cross-sectional and longitudinal outcome study.

Abstract: Women with borderline/mildly dyskaryotic (BMD) cytology smears are currently followed up with repeat testing at 6 and 18 months. The objective of this study is to analyse the cross-sectional and longitudinal performance of p16/Ki-67 dual-stained cytology for the detection of cervical intraepithelial neoplasia (CIN) grade 3 or worse (CIN3+) and CIN2+ in women with BMD, and to compare the results with baseline human papillomavirus (HPV) testing. Conventional Pap cytology specimens of 256 women with BMD were dual stained for p16/Ki-67 retrospectively, and compared with baseline HPV results and long-term follow-up results. p16/Ki-67 dual-stained cytology showed a sensitivity of 100%, a specificity of 64.4% and a negative predictive value (NPV) of 100.% for CIN3+. Human papillomavirus testing demonstrated similar sensitivity (96.3%), and NPV (99.1%), but a significantly lower specificity (57.6%; P=0.024) for CIN3+. Sensitivity, specificity and NPV for CIN2+ of dual-stained cytology were 89.7%, 73.1% and 95.1%, respectively, which was similar when compared with HPV testing. Dual-stained cytology showed a significant lower referral rate than HPV testing (43.6% vs 49.1%; P=0.043). During long-term follow-up, no CIN3+ lesions developed in HPV-positive, dual-stained negative women. Comparable sensitivity and NPV of dual-stained cytology for CIN3+, combined with a significantly higher specificity, makes p16/Ki-67 dual-stained cytology a viable alternative to HPV testing for triaging BMD.

Clinical Evaluation of a GP5+/6+-Based Luminex Assay Having Full High-Risk Human Papillomavirus Genotyping Capability and an Internal Control

Abstract: The LMNX genotyping kit HPV GP (LMNX) is based on the clinically validated GP5+/6+ PCR, with a genotyping readout as an alternative for the more established enzyme immunoassay (EIA) detection of 14 targeted high-risk human papillomavirus (HPV) types. LMNX is additionally provided with an internal control probe. Here, we present an analysis of the clinical performance of the LMNX using a sample panel and infrastructure provided by the international VALGENT (Validation of Genotyping Tests) project. This panel consisted of cervical specimens from approximately 1,000 women attending routine screening, "enriched" with 300 women with abnormal cytology. Cases were defined as women classified with cervical intraepithelial neoplasia (CIN) grade 2+ (CIN2+) (n = 102) or CIN3+ (n = 55) within the previous 18 months. Controls were women who had normal cytology results over two subsequent screening rounds at a 3-year interval (n = 746). The GP5+/6+-PCR EIA (EIA) was used as a comparator assay and showed sensitivities of 94.1% and 98.2% for CIN2+ and CIN3+, respectively, with a clinical specificity of 92.4% among women aged ≥ 30 years. The LMNX demonstrated clinical sensitivities of 96.1% for CIN2+ and of 98.2% for CIN3+ and a clinical specificity of 92.6% for women aged ≥ 30 years. The LMNX and EIA were in high agreement (Cohen's kappa = 0.969) for the detection of 14 hrHPVs in aggregate, and no significant difference was observed (McNemar's P = 0.629). The LMNX internal control detected 0.6% inadequate specimens. Based on our study results, we consider the LMNX, similarly to the EIA, useful for HPV-based cervical cancer screening.

Combined CADM1/MAL Methylation and Cytology Testing for Colposcopy Triage of High-Risk HPV-Positive Women

Abstract: Primary screening for high-risk human papillomavirus (hrHPV) requires a triage protocol. Repeat cytology testing at baseline and after 6 to 12 months has emerged as a reasonable triage approach, but carries the risk of loss to follow-up. Repeat cytology testing may be omitted if cytology is supplemented with another, complementary triage test at baseline. In this study, the performance of combined triage by cytology and DNA methylation analysis was assessed. In hrHPV-positive cervical scrapes (n = 250), cytology [threshold: atypical squamous cells of undetermined significance (ASCUS)], bi-marker CADM1/MAL methylation testing (at different assay thresholds), and combinations of both were evaluated for endpoints cervical intraepithelial neoplasia grade 2 or worse (CIN2(+)) and grade 3 or worse (CIN3(+)). At a predefined methylation threshold of 70% specificity for CIN3(+), combined triage revealed a CIN3(+) sensitivity of 86.8% [95% confidence interval (CI), 76.1-97.6] compared with 65.8% (95% CI, 50.7-80.9) for sole cytology triage testing. Corresponding CIN3(+) specificity was 64.8% (95% CI, 58.1-71.5) for combined triage and 78.6% (95% CI, 72.8-84.3) for sole cytology triage testing. For CIN2(+), the sensitivity of combined triage testing was 84.5% (95% CI, 75.2-93.8) compared with 65.5% (95% CI, 53.3-77.7) for sole cytology triage, with corresponding specificities of 69.9% (95% CI, 63.1-76.6) and 83.5% (95% CI, 78.0-89.0), respectively. In conclusion, combined triage reached substantially higher CIN2(+)/3(+) sensitivities compared with sole cytology at a slight drop in specificity. Therefore, it is an attractive triage strategy for colposcopy of hrHPV-positive women with a high reassurance for cervical cancer and advanced CIN lesions.

Methylation-mediated repression of PRDM14 contributes to apoptosis evasion in HPV-positive cancers.

Abstract: Promoter methylation of the transcription factor PRDM14 (PRDI-BF1 and RIZ domain containing 14) represents a highly frequent event in human papillomavirus (HPV)-induced cervical cancers and cancer precursor lesions. Here, we aimed to assess the functional consequences of PRDM14 promoter methylation in HPV-induced carcinogenesis. PRDM14 promoter methylation, expression and consequences of ectopic PRDM14 expression were studied in HPV16-positive cervical and oral cancer cell lines (SiHa, CaSki and 93VU147T), human embryonic kidney 293 (HEK293T) cells and primary human foreskin keratinocytes (HFK). PRDM14 mRNA expression was restricted to HEK293T and HFK cells, and could be upregulated in SiHa cells upon DNA methylation inhibition. Ectopic expression of PRDM14 in SiHa, CaSki and 93VU147T cells resulted in significantly more apoptotic cells, as measured by annexin V labelling, compared to HEK293T and HFK cells. MRNA profiling of 41 apoptosis regulators identified NOXA and PUMA as candidate target genes involved in PRDM14-mediated apoptosis induction. Full-length PRDM14 transactivated both NOXA and PUMA promoters. Transactivation was abolished upon deletion of the PRDM14 DNA binding domain. This suggests that NOXA and PUMA expression is directly regulated by PRDM14, which in case of NOXA was linked to a consensus PRDM14 binding motif in the promoter region. Taken together, these results suggest that PRDM14 acts as a regulator of NOXA and PUMA-mediated apoptosis induction, thereby providing evidence for a tumour suppressive role in HPV-induced carcinogenesis. The contribution of methylation-mediated gene silencing of PRDM14 to apoptosis evasion in HPV-positive cancer cells offers novel therapeutic options for HPV-induced cancers.

Efficacy of HPV-based screening for prevention of invasive cervical cancer: Follow-up of four European randomized controlled trials

Abstract: Human papillomavirus (HPV)–based screening is more effective than cytology-based screening for detecting the precursors of invasive cervical carcinoma, cervical intraepithelial neoplasia grade 2 (CIN2), and, particularly, grade 3 (CIN3). Four randomized controlled trials (Swedescreen, POBASCAM, ARTISTIC, and NTCC) compared HPVbased screening for cervical cancer with cytology-based cervical screening. Precursors of this cancer were the end point in each of these trials. In all 4 trials, HPV-based screening detected persistent CIN3 before cytology, increasing the likelihood of treatment before invasion. However, direct estimates are absent in the 4 trials on the relative efficacy of HPVbased versus cytology-based screening for prevention of invasive cancer in women who undergo regular screening and of how efficacy changes according to modifiers of this relative efficacy (age, cancer stage, and morphology) and of the duration of protection against cancer. This extended follow-up study was designed to investigate these outcomes. Data were pooled from the 4 trials. The primary study end point was detected invasive cervical carcinomas in women who have regular screening. A total of 176,464 women with a mean age of 35 to 41 years (range, 20–64 years) were randomly assigned to either HPV-based (experimental arm [HPV group]) or cytology-based (control arm [control group]) screening in the 4 trials. The trials were conducted in Sweden (Swedescreen), the Netherlands (POBASCAM), England (ARTISTIC), and Italy (NTCC). Participants were followed for a median of 6.5 years (1,214,415 person-years). A total of 107 invasive cervical carcinomas were detected by linkage with screening, pathology, and cancer registries and by masked review of histological specimens or from reports. The incidence of invasive cervical carcinoma in the 2 groups was determined by calculating cumulative and study-adjusted rate ratios. The overall rate ratio for detection of invasive cervical carcinoma among all women from recruitment to end of follow-up was 0.60, with a 95% confidence interval (CI) of 0.40 to 0.89; there was no heterogeneity between studies (P = 0.52). During the first 2.5 years of follow-up, the rate ratio for invasive cervical carcinoma was similar between screening methods (0.79; 95% CI, 0.46–1.36) but thereafter was significantly lower in the HIV group (0.45; 95% CI, 0.25–0.81). The rate ratio among women with a negative screening test at entry was 0.30 (95% CI, 0.15–0.60). The cumulative incidence of invasive cervical carcinoma in women with negative entry tests was as follows: HIV group at 3.5 and 5.5 years (4.6 per 10 [95% CI, 1.1–12.1] and 8.7 per 10 [95% CI, 3.3–18.6], respectively) versus control group at 3.5 and 5.5 years (15.4 per 10 [95% CI, 7.9–27.0] and 36.0 per 10 [95% CI, 23.2–53.5], respectively). Rate ratios were lower for adenocarcinoma (0.31; 95% CI, 0.14–0.69) than for squamous cell carcinoma (0.78; 95% CI, 0.49–1.25), but did not differ by cancer stage. The lowest rate ratio (0.36; 95% CI, 0.14–0.94) was found among women aged 30 to 34 years. These data show that HPV-based screening provides 60% to 70% greater protection against invasive cervical cancers compared with cytology. The lower cumulative incidence of cervical cancer 5.5 years after a negative HPV test than 3.5 years after a negative cytology test indicates that 5-year intervals for HPV screening are safer than 3-year intervals for cytology. 472 Obstetrical and Gynecological Survey Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

Six-Month Incidence and Persistence of Oral HPV Infection in HIV-Negative and HIV-Infected Men Who Have Sex with Men

Abstract: Objectives: Our aim was to assess incidence and persistence of oral HPV infection in HIV-negative and HIV-infected men who have sex with men (MSM). Methods: MSM aged $18 years were included in Amsterdam (the Netherlands) in 2010–2011, and followed up 6 months later. Participants completed risk factor questionnaires. HPV DNA was analyzed in oral-rinse and gargle specimens using the SPF10-PCR DEIA/LiPA25 system (version 1). A subset of oral samples was subjected to SPF10 sequencing to identify additional HPV types. Multivariable logistic regression analyses using generalized estimating equations (GEE) were performed to assess determinants for oral high-risk HPV incidence and persistence. Results: 689/795 participant MSM provided both baseline and 6-month data. Baseline prevalence of high-risk HPV was 9.4% in HIV-negative and 23.9% in HIV-infected MSM (P,0.001). 56/689 MSM acquired $1 high-risk HPV infection (6-month incidence 8.1%; 95%CI 6.2–10.4%); incidence was 4.1% in HIV-negative and 14.1% in HIV-infected MSM (P,0.001). HIV infection and recent use of cannabis were both independently associated with high-risk HPV incidence. Persistent high-risk HPV was observed in 48/130 (36.9%) infections. Conclusion: Incidence of oral high-risk HPV infection in MSM is substantial, and is associated with HIV infection. Over a third of HPV infections persisted over a 6-month period.

Population- and Type-Specific Clustering of Multiple HPV Types Across Diverse Risk Populations in the Netherlands

Abstract: In view of possible type replacement upon introduction of human papillomavirus (HPV) vaccination, we aimed to explore patterns of type-specific clustering across populations with various background infection risks. A total of 3,874 women from 3 cross-sectional studies in the Netherlands (in 2007-2009) provided vaginal self-samples, which were tested for 25 HPV genotypes by a sensitive molecular assay (SPF10 line probe assay, DDL Diagnostic Laboratory, Voorburg, the Netherlands). The number of concurrent HPV infections per woman was studied by Poisson regression. Associations between HPV types were investigated by generalized estimating equation analyses. The prevalence of any HPV type was 14% in a population-based study, 54% in a chlamydia screening intervention study, and 73% in a study among attendees of sexually transmitted infection clinics. Overall, multiple HPV infections were detected in 26% of the women. The number of concurrent HPV infections conformed to an overdispersed Poisson distribution, even after correction for known risk factors. Types differed significantly in their tendencies to be involved in coinfections, but no evidence for particular type-type interactions was found. Moreover, the strongest associations were observed in the lowest-risk population and vice versa.We found no indications of pairwise interactions, but our findings do suggest that clustering differs among HPV types and varies across risk groups.

Differential In Vitro Immortalization Capacity of Eleven, Probable High-Risk Human Papillomavirus Types

Abstract: Epidemiological studies identified 12 high-risk HPV (hrHPV) types and 8 probable/possible hrHPV types that display different cancer risks. Functional studies on transforming properties of hrHPV are mainly limited to HPV16 and -18, which induce immortalization of human foreskin keratinocytes (HFKs) by successive bypass of two proliferative life span barriers, senescence and crisis. Here, we systematically compared the in vitro immortalization capacities, as well as influences on p53, pRb, hTERT, growth behavior, and differentiation capacity, of nine hrHPV types (HPV16, -18, -31, -33, -35, -45, -51, -52, and -59), and two probable hrHPV types (HPV66 and -70). By retroviral transduction, the respective E6/E7 coding sequences were expressed in HFKs from two or three independent donors. Reduced p53 levels and low-level hTERT expression in early-passage cells, as seen in HPV16-, -31-, -33-, and -35-, and to a lesser extent HPV18-transduced HFKs, was associated with continuous growth and an increased immortalization capacity. Less frequent immortalization by HPV45 and -51 and immortalization by HPV66 and -70 was preceded by an intervening period of strongly reduced growth (crisis) without prior increase in hTERT expression. Immortalization by HPV59 was also preceded by a period crisis, despite the onset of low hTERT expression at early passage. HPV52 triggered an extended life span but failed to induce immortality. Variations in p53 and pRb levels were not correlated with differences in alternative E6/E7 mRNA splicing in all hrHPV-transduced HFKs. On collagen rafts, transductants showed disturbed differentiation reminiscent of precancerous lesions. In conclusion, in vitro oncogenic capacities differ between the established hrHPV types, and both some established and probable hrHPV types display weak or moderate immortalization potential.

Twelve-month incidence and clearance of oral HPV infection in HIV-negative and HIV-infected men who have sex with men: the H2M cohort study

Abstract: Our aim was to compare the 12-month incidence and clearance of oral high-risk HPV infection between HIV-infected men who have sex with men (MSM) and HIV-negative MSM. MSM aged 18 years or older were recruited in Amsterdam, the Netherlands. Questionnaire data and oral-rinse and gargle samples were collected at baseline, and after 6 and 12 months. HPV DNA was genotyped using the SPF10-PCR & DEIA-LiPA25 system (version 1). Determinants of oral HPV incidence and clearance were explored using Cox and logistic regression analyses respectively. 433 HIV-negative and 290 HIV-infected MSM were included in these analyses. The median follow-up time per participant was 12 months (range 3–15). During follow-up, 114 incident oral high-risk HPV infections were observed. The incidence rate of HPV-16 was 3.5/1000 person-months (PM) in HIV-infected and 0.9/1000 PM in HIV-negative MSM (IRR 4.1; 95% CI 1.3-13.2). The incidence rates of other high-risk HPV types ranged between 1.3-3.5/1000 PM in HIV-infected and 0.0-1.1/1000 PM in HIV-negative MSM. In multivariable analyses, HIV infection (adjusted hazard ratio [aHR] 3.8; 95% CI 2.3-6.2) and a higher number of recent oral sex partners (aHR 2.4 for ≥8 partners compared to ≤2; 95% CI 1.4-4.2) were associated with HPV incidence. Of the 111 baseline oral high-risk infections, 59 (53.2%) were cleared. In multivariable analyses, only a higher number of recent oral sex partners was associated with HPV clearance (adjusted odds ratio 3.4 for ≥8 compared to ≤2 partners; 95% CI 1.3-9.0). The incidence rate of oral high-risk HPV infection was higher in HIV-infected MSM and in those with a higher number of recent oral sex partners. Just over half of the oral high-risk HPV infections at baseline were cleared after 12 months, with a higher likelihood of clearance among MSM reporting higher numbers of recent oral sex partners, but no difference by HIV status.

Cervical cancer in 2013: Screening comes of age and treatment progress continues

Abstract: In 2013, studies confirmed that HPV infection of target cells predisposes to cervical (pre)cancer. In developed countries, HPV screening revealed superior protection than cytology screening. In India, visual inspection of the cervix after acetic acid application significantly reduced cervical cancer mortality after 12 years. Improved survival for women with advanced disease was observed after adjuvant bevacizumab.

Posttreatment assessment of women at risk of developing high-grade cervical disease: proposal for new guidelines based on data from the Netherlands

Abstract: OBJECTIVE: Women treated for high-grade cervical disease (cervical intraepithelial neoplasia grade 2 or grade 3 [CIN2/3]) face a significant risk of developing post-treatment disease. Therefore, in most European countries, they are monitored by cytologic testing at 6, 12, and 24 months after treatment. Although testing for high-risk types of the human papillomavirus (hrHPV) in the follow-up seems to be a valuable supplementary method, its use is not yet fully explored. METHODS: Besides reviewing the literature, we completed a long-term follow-up study describing the cumulative risk for CIN2/3 or cancer (CIN2+) of different hrHPV and cytology test results after treatment. CONCLUSIONS: High-risk HPV testing improves the sensitivity to detect posttreatment CIN2/3 (relative sensitivity=1.15, 95% confidence interval [CI]=1.06-1.25), but the highest sensitivity (95%, 95% CI=91%-98%) is reached by performing cotesting (both cytology and hrHPV). The CIN2+ risk after a single negative cotesting result taken 6 months after treatments was similar to the risk after 3 consecutive negative cytologic test results (5-y CIN2+ risk being 3.0% [95% CI=1.5%-6.1%] and 2.9% [95% CI=1.2%-7.1%], respectively). Women who test negative for cotesting at both 6 and 24 months after treatment have a minimal risk of developing CIN3+ in the next 5 years (0.0%, 95% CI=0.0%-3.0%). RECOMMENDATIONS: We propose a new posttreatment surveillance protocol, consisting of combined testing with both cytology and hrHPV at 6 and 24 months after treatment. After 2 negative cotesting results, women should be retested after 5 years.

Prevalence of HPV infection and other risk factors in a Fijian population

Abstract: Background: Cancer is among the leading contributors to morbidity and mortality in the Pacific, but the magnitude of the problem and the potential for prevention have not been comprehensively studied. Over the past decade, cervical cancer has been the most common cancer among women in Fiji with an age standardised cervical cancer incidence rate of 51 per 100,000. This rate is among the highest in the South Pacific region and in the world. This high cervical cancer incidence rate is likely linked to the low cervical screening rate, but it points also to the possibility of a high burden of human papillomavirus (HPV) infection. Methods: We conducted a population-based survey in Fiji to provide information on human papillomavirus (HPV) prevalence, and the distribution of individual HPV types in a Fijian health-sub-district. We included 1,261 women aged between 16 and 64 years. A general primer GP5+/6+mediatedpolymerase chain reaction (PCR) assay was used for HPV testing of 44 HPV types. Results: The crude HPV prevalence in 1,244 women with an adequate HPV sample was 24.0% (95% confidence interval (CI), 21.7-26.4%) and the corresponding age standardised prevalence was 25.5% (95% CI, 23.1-28.1%). The prevalence of high-risk HPV types was 13.6% (95% CI, 11.8-15.6%). Among 1,192 women with adequate cytological results, 13 (1.1%) showed cervical abnormalities, the majority of which were high-grade intraepithelial lesions or worse. HPV prevalence declined from 35.8% in women aged <25 years to 18.6% in those aged 55–64 years of age. After adjustment, the only variables significantly associated with HPV-positivity were age (ranging from odds ratio (OR) 0.57 (95% CI, 0.36-0.89) for 25–34 year-old-women to OR 0.43 (95% CI, 0.20-0.89) for 55–64 year-old-women) and ‘husband’s extramarital sexual relationships’ (OR 1.69; 95% CI, 1.17-2.34). Conclusion: These findings on HPV provide key information for future policy decisions on the most appropriate methods of cervical cancer prevention in Fiji and in the Pacific region.

Risk of HIV acquisition among circumcised and uncircumcised young men with penile human papillomavirus infection.

Abstract: Objectives There are very few data from men on the risk of HIV acquisition associated with penile human papillomavirus (HPV) infection and no data on the potential modifying effect of male circumcision. Therefore, this study evaluated whether HPV is independently associated with risk of HIV. Design A cohort study of HPV natural history nested within a randomized control trial of male circumcision to reduce HIV incidence in Kisumu, Kenya. Methods Prospective data from 2519 men were analyzed using 6-month discrete-time Cox models to determine if HIV acquisition was higher among circumcised or uncircumcised men with HPV compared to HPV-uninfected men. Results Risk of HIV acquisition was nonsignificantly increased among men with any HPV [adjusted hazard ratio (aHR) 1.72; 95% confidence interval (CI) 0.94-3.15] and high-risk HPV (aHR 1.92; 95% CI 0.96-3.87) compared to HPV-uninfected men, and estimates did not differ by circumcision status. Risk of HIV increased 27% with each additional HPV genotype infection (aHR 1.27; 95% CI 1.09-1.48). Men with persistent (aHR 3.27; 95% CI 1.59-6.72) or recently cleared (aHR 3.05; 95% CI 1.34-6.97) HPV had a higher risk of HIV acquisition than HPV-uninfected men. Conclusions Consistent with the findings in women, HPV infection, clearance, and persistence were associated with an increased risk of HIV acquisition in men. Given the high prevalence of HPV in populations at risk of HIV, consideration of HPV in future HIV-prevention studies and investigation into mechanisms through which HPV might facilitate HIV acquisition are needed.

DNA copy number aberrations in endobronchial lesions: a validated predictor for cancer

Abstract: We recently identified a DNA copy number aberration (CNA)-based classifier, including changes at 3p26.3-p11.1, 3q26.2-29, and 6p25.3-24.3, as a risk predictor for cancer in individuals presenting with endobronchial squamous metaplasia. The current study was set out to validate the prediction accuracy of this classifier in an independent series of endobronchial squamous metaplastic and dysplastic lesions. The study included 36 high-risk subjects who had endobronchial lesions of various histological grades that were identified and biopsied by autofluorescence bronchoscopy and were subjected to arrayCGH in a nested case-control design. Of the 36 patients, 12 had a carcinoma in situ or invasive carcinoma at the same site at follow-up (median 11 months, range 4-24), while 24 controls remained cancer free (78 months, range 21-142). The previously defined CNA-based classifier demonstrated 92% (95% CI 77% to 98%) accuracy for cancer (in situ) prediction. All nine subjects with CNA-based classifier-positive endobronchial lesions at baseline experienced cancer outcome, whereas all 24 controls and 3 cases were classified as being low risk. In conclusion, CNAs prove to be a highly accurate biomarker for assessing the progression risk of endobronchial squamous metaplastic and dysplastic lesions. This classifier could assist in selecting subjects with endobronchial lesions who might benefit from more aggressive therapeutic intervention or surveillance.

Arguments in favor of HPV testing for cervical screening and post-treatment CIN2+ monitoring

Abstract: Several studies have shown that the human papilloma virus (HPV) test is a more sensitive and objective primary cervical cancer screening tool than cytology. Therefore, conversion of cytology into HPV screening (as is planned in The Netherlands and some other European regions) will result in a better protection against cervical cancer and high-grade precursor lesions. Moreover, offering self-sampling for HPV testing will increase screening attendance by re-attracting former non-attendees. However, triage of HPV positive women is necessary because the specificity of HPV testing is 2–4% lower than of cytology. Several triage strategies have been evaluated, of which two, with cytology testing included, are feasible and were recently recommended. As an alternative for cytology triage, objective, non-morphological disease markers are upcoming and so far have shown promising results. Finally, HPV testing can also contribute to a more efficient monitoring of women treated for high-grade cervical precursor lesions...

Clinical validation of the PapilloCheck assay according to international guidelines for HPV test requirements for cervical screening

Abstract: Objective To compare the clinical performance of the PapilloCheck assay (Greiner Bio-One, Germany) with that of the clinically validated high-risk HPV GP5+/6+-PCR according to the international guidelines for HPV test requirements for cervical screening (Meijer et al., IJC., 2009). Methods 1,437 cervical scrapings of women without evidence CIN2+ and 192 of women with CIN3+, originating from a population-based screening cohort (POBASCAM) were tested with both PapilloCheck and the GP5+/6+-PCR. In addition, intra-laboratory reproducibility over time and inter-laboratory agreement of the PapilloCheck assay were assessed on another set of 550 cervical samples. Clinical sensitivity and specificity values of the PapilloCheck assay were compared with those of GP5+/6+-PCR by non-inferiority score testing using previously defined thresholds for non-inferiority, i.e., relative sensitivity for CIN2+ >90% and relative specificity for CIN2+ >98%. For intraand inter-laboratory agreement a lower confidence bound not less than 87 % was used as threshold. Conclusions When restricting PapilloCheck analysis to the 14 hrHPV types targeted by GP5+/6+-PCR the clinical sensitivity and specificity were 95.8 % (95 % CI 92.8–98.8) and 96.7 % (95 % CI 95.7–97.7), respectively. By comparison, these figures were 96.4 % (95 % CI : 93.9–98.9) and 97.7 % (95 % CI : 96.9–98.5), respectively, for GP5+6+-PCR. Both the clinical sensitivity and specificity of PapilloCheck were non-inferior to that of GP5+/6+-PCR (non-inferiority score test ; P