FAM19A4/miR124-2 methylation analysis as a triage test for HPV-positive women: cross-sectional and longitudinal data from a Dutch screening cohort.

OBJECTIVES: The aim was to evaluate the cross-sectional and long-term triage performance of FAM19A4/miR124-2 methylation analysis in human papillomavirus (HPV)-based cervical screening. METHODS: We conducted a post hoc analysis within a Dutch population-based HPV-positive study cohort of women aged 30-60 years (n = 979). Cross-sectional cervical intraepithelial neoplasia (CIN) 3+ sensitivity, specificity, positive predictive value and negative predictive value as well as cumulative CIN3+ or cervical cancer risks after 9 and 14 years were compared for three baseline triage strategies: (1) cytology, (2) FAM19A4/miR124-2 methylation analysis and (3) combined FAM19A4/miR124-2 methylation with cytology. RESULTS: CIN3+ sensitivity of FAM19A4/miR124-2 methylation analysis was similar to that of cytology (71.3% vs 76.0%, ratio 0.94, 95% CI 0.84 to 1.05), at a lower specificity (78.3% vs 87.0%, ratio 0.90, 95% CI 0.86 to 0.94). Combining FAM19A4/miR124-2 methylation analysis with cytology resulted in a CIN3+ sensitivity of 84.6% (95% CI 78.3 to 90.8) at a specificity of 69.6% (95% CI 66.5 to 72.7). Similar 9- and 14-year CIN3+ risks for baseline cytology-negative women and baseline FAM19A4/miR124-2 methylation-negative women were observed, with risk differences of -0.42% (95% CI -2.1 to 1.4) and -0.07% (95% CI -1.9 to 1.9), respectively. The 14-year cumulative cervical cancer incidence was significantly lower for methylation-negative women compared to cytology-negative women (risk difference 0.98%, 95% CI 0.26 to 2.0). DISCUSSION: FAM19A4/miR124-2 methylation analysis has a good triage performance on baseline screening samples, with a cross-sectional CIN3+ sensitivity and long-term triage-negative CIN3+ risk equalling cytology triage. Therefore, FAM19A4/miR124-2 methylation analysis appears to be a good and objective alternative to cytology in triage scenarios in HPV-based cervical screening.

Measuring vaccine effectiveness against persistent HPV infections: a comparison of different statistical approaches.

BACKGROUND: Persistent high-risk human papillomavirus (HPV) infection is endorsed by the World Health Organization as an intermediate endpoint for evaluating HPV vaccine effectiveness/efficacy. There are different approaches to estimate the vaccine effectiveness/efficacy against persistent HPV infections. METHODS: We performed a systematic literature search in Pubmed to identify statistical approaches that have been used to estimate the vaccine effectiveness/efficacy against persistent HPV infections. We applied these methods to data of a longitudinal observational study to assess their performance and compare the obtained vaccine effectiveness (VE) estimates. RESULTS: Our literature search identified four approaches: the conditional exact test for comparing two independent Poisson rates using a binomial distribution, Generalized Estimating Equations for Poisson regression, Prentice Williams and Peterson total time (PWP-TT) and Cox proportional hazards regression. These approaches differ regarding underlying assumptions and provide different effect measures. However, they provided similar effectiveness estimates against HPV16/18 and HPV31/33/45 persistent infections in a cohort of young women eligible for routine HPV vaccination (range VE 93.7-95.1% and 60.4-67.7%, respectively) and seemed robust to violations of underlying assumptions. CONCLUSIONS: As the rate of subsequent infections increased in our observational cohort, we recommend PWP-TT as the optimal approach to estimate the vaccine effectiveness against persistent HPV infections in young women. Confirmation of our findings should be undertaken by applying these methods after longer follow-up in our study, as well as in different populations.

Grading immunohistochemical markers p16INK4a and HPV E4 identifies productive and transforming lesions caused by low- and high-risk HPV within high-grade anal squamous intraepithelial lesions.

OBJECTIVES: Because current guidelines recognise high-grade anal squamous intraepithelial lesions (HSILs) and low-grade SILs (LSILs), and recommend treatment of all HSILs although not all progress to cancer, this study aims to distinguish transforming and productive HSILs by grading immunohistochemical (IHC) biomarkers p16INK 4a (p16) and E4 in low-risk human papillomavirus (lrHPV) and high-risk (hr)HPV-associated SILs as a potential basis for more selective treatment. METHODS: Immunostaining for p16 and HPV E4 was performed and graded in 183 biopsies from 108 HIV-positive men who have sex with men. The causative HPV genotype of the worst lesion was identified using the HPV SPF10-PCR-DEIA-LiPA25 version 1 system, with laser capture microdissection for multiple infections. The worst lesions were scored for p16 (0-4) to identify activity of the hrHPV E7 gene, and panHPV E4 (0-2) to mark HPV production and life cycle completion. RESULTS: There were 37 normal biopsies, 60 LSILs and 86 HSILs, with 85% of LSILs caused by lrHPV and 93% of HSILs by hrHPV. No normal biopsy showed E4, but 43% of LSILs and 37% of HSILs were E4 positive. No differences in E4 positivity rates were found between lrHPV and hrHPV lesions. Most of the lesions caused by lrHPV (90%) showed very extensive patchy p16 staining; p16 grade in HSILs was variable, with frequency of productive HPV infection dropping with increasing p16 grade. CONCLUSIONS: Combined p16/E4 IHC identifies productive and nonproductive HSILs associated with hrHPV within the group of HSILs defined by the Lower Anogenital Squamous Terminology recommendations. This opens the possibility of investigating selective treatment of advanced transforming HSILs caused by hrHPV, and a 'wait and see' policy for productive HSILs. What's already known about this topic? For preventing anal cancer in high-risk populations, all patients with high-grade squamous intraepithelial lesions (HSILs) are treated, even though this group of lesions is heterogeneous, the histology is variable and regression is frequent. What does this study add? By adding human papillomavirus (HPV) E4 immunohistochemistry to p16 INK4a (p16), and grading expression of both markers, different biomarker expression patterns that reflect the heterogeneity of HSILs can be identified. Moreover, p16/E4 staining can separate high-risk HPV-associated HSILs into productive and more advanced transforming lesions, providing a potential basis for selective treatment.

HPV-based cervical screening: Rationale, expectations and future perspectives of the new Dutch screening programme.

Based on scientific data showing that HPV testing provides better protection against cervical precancer and cancer than cytology, in 2011 the Dutch Health Council advised the Minister of Welfare, Health and Sports to replace cytology by HPV testing in the Dutch population-based screening programme. After a successful evaluation of the feasibility of HPV-based screening in 2014, primary HPV testing for cervical screening was implemented in 2017. The Netherlands has been one of the first countries worldwide to implement nationwide HPV-based screening and its experience with the new programme is therefore followed with great interest. In this manuscript, we present an overview of the studies that were instrumental in the choice of HPV assay and triage strategy, the adjustment of screening starting and exit ages and intervals, and the implementation of HPV self-sampling. Finally, we review the cost-effectiveness of the proposed new screening algorithm and we explore future perspectives. The rationale behind the new Dutch HPV-based screening programme, which is based on risk management, could serve as a guidance to other countries that are planning to implement HPV-based screening in the near future.

High Effectiveness of the Bivalent Human Papillomavirus (HPV) Vaccine Against Incident and Persistent HPV Infections up to 6 Years After Vaccination in Young Dutch Women.

Background: Monitoring vaccine effectiveness (VE) in vaccination programs is of importance for assessing the impact of immunization. This study aimed to estimate the VE of the bivalent human papillomavirus (HPV) vaccine against incident and 12-month persistent infections up to 6 years after vaccination. Methods: In 2009-2010, girls eligible for the vaccination catch-up campaign (ie, those aged 14-16 years) were enrolled into a prospective cohort. Annually, participants completed a questionnaire and submitted a self-collected vaginal swab sample for HPV testing by the SPF10-LiPA25 assay. We compared sociodemographic characteristics and infection rates between vaccinated and unvaccinated girls. The VE was adjusted for characteristics related to HPV vaccination status. We used combined end points for VE estimation. Results: In total, 1635 women, of whom 54% were fully vaccinated, were included for VE estimation. The adjusted VE against HPV16 and 18 persistent infections amounted to 97.7% (95% confidence interval [CI], 83.5%-99.7%). We found a VE against HPV31, 33, and 45 persistent infections of 61.8% (95% CI, 16.7%-82.5%). We found no indications that the protection against vaccine or cross-protective types changes over time. Conclusion: Our findings of nearly full protection against vaccine-type persistent infections and significant cross-protection to nonvaccine types in a population-based cohort study confirm the effectiveness of the bivalent HPV vaccine as estimated in trials. We found no indications for waning protection up to 6 years after vaccination.

Evaluation of the Clinical Performance of the HPV-Risk Assay Using the VALGENT-3 Panel.

Human papillomavirus (HPV) testing is increasingly being incorporated into cervical cancer screening. The Validation of HPV Genotyping Tests (VALGENT) is a framework designed to evaluate the clinical performance of various HPV tests relative to that of the validated and accepted comparator test in a formalized and uniform manner. The aim of this study was to evaluate the clinical performance of the HPV-Risk assay with samples from the VALGENT-3 panel and to compare its performance to that of the clinically validated Hybrid Capture 2 assay (HC2). The VALGENT-3 panel comprises 1,300 consecutive samples from women participating in routine cervical cancer screening and is enriched with 300 samples from women with abnormal cytology. DNA was extracted from original ThinPrep PreservCyt medium aliquots, and HPV testing was performed using the HPV-Risk assay by investigators blind to the clinical data. HPV prevalence was analyzed, and the clinical performance of the HPV-Risk assay for the detection of cervical intraepithelial neoplasia grade 3 or worse (CIN3+) and CIN2 or worse (CIN2+) relative to the performance of HC2 was assessed. The sensitivity of the HPV-Risk assay for the detection of CIN3+ was similar to that of HC2 (relative sensitivity, 1.00; 95% confidence interval [CI], 0.95 to 1.05; P = 1.000), but the specificity of the HPV-Risk assay was significantly higher than that of HC2 (relative specificity, 1.02; 95% CI, 1.01 to 1.04; P < 0.001). For the detection of CIN2+, similar results were obtained, with the relative sensitivity being 0.98 (95% CI, 0.93 to 1.02; P = 0.257) and the relative specificity being 1.02 (95% CI, 1.01 to 1.03; P < 0.001). The performance of the HPV-Risk assay for the detection of CIN3+ and CIN2+ was noninferior to that of HC2, with all P values being ≤0.006. In conclusion, the HPV-Risk assay demonstrated noninferiority to the clinically validated HC2 by the use of samples from the VALGENT-3 panel for test validation and comparison.

Performance of CADM1/MAL-methylation analysis for monitoring of women treated for high-grade CIN.

INTRODUCTION: Recent studies have shown that CADM1/MAL-methylation testing detects high-grade CIN lesions with a high short-term progression risk for cervical cancer. Women treated for CIN2/3 are at risk of post-treatment disease, representing either persistent (incompletely treated) or incident (early onset) lesions. Here, we evaluated CADM1/MAL-methylation analysis as potential tool for detecting recurrent high-grade CIN lesions (rCIN2/3). METHODS AND MATERIALS: A multicenter prospective clinical cohort study was conducted among 364 women treated for CIN2/3. Cervical scrapes were taken prior to treatment, and six and 12months post-treatment and tested for cytology, hrHPV (plus genotype) and CADM1/MAL-methylation. When at six months either of these tests was positive, a colposcopy-directed biopsy was obtained. At 12months, all women underwent an exit-colposcopy with biopsy. In case of rCIN2/3, re-treatment was done. RESULTS: We found 28 rCIN2 (7.7%) and 14 rCIN3 (3.8%), resulting in a total recurrence rate of 11.5%. All 14 women with rCIN3 and 15/28 (54%) with rCIN2 showed hrHPV type-persistence. Of these, 9/14 (64%) rCIN3 and 8/15 (53%) rCIN2 were CADM1/MAL-methylation positive. All incident rCIN2, characterized by hrHPV genotype-switch, were CADM1/MAL-methylation negative. All three carcinomas found after re-treatment were CADM1/MAL-methylation positive. CADM1/MAL-methylation positivity at both baseline and follow-up significantly increased the risk of ≥rCIN3 (from 0.7% to 18.4%), and ≥rCIN2 (from 8.2% to 36.8%), compared to a consistently CADM1/MAL-methylation negative result (p-value: <0.001). CONCLUSION: Post-treatment monitoring by CADM1/MAL-methylation analysis identifies women with an increased risk of rCIN2/3. Our results confirm previous data indicating that CADM1/MAL-methylation analysis provides a high reassurance against cancer.

The performance of Dynamic Spectral Imaging colposcopy depends on indication for referrals.

OBJECTIVE. A previous study has shown that Dynamic Spectral Imaging (DSI) colposcopy increases the sensitivity of the colposcopic examination in women referred with abnormal cytology. In this study we have reanalyzed the performance of DSI and conventional colposcopy for new referral conditions and for low-grade cytology referrals versus high-grade cytology referrals. METHOD. Data from a previous validation trial was used to assess the performance of DSI in different cytology groups:Women referred with BMD (borderline and mild dyskaryosis) cytology and women referred with NBMD cytology either hrHPV positive or negative were separately analyzed. Furthermore, we tried to assess the clinical performance by appropriate filtering of patients to replicate two different referral strategies. RESULTS. The sensitivity of DSI and conventional colposcopy to detect CIN2+ lesions in women referred with BMD cytology is 82% and 44% respectively (p= 0.001) and in the NBMD group 77% and 64% respectively (p= 0.24). If the two techniques are combined the sensitivity is 85%.When the conditions of new screening strategies are applied DSI colposcopy has a higher sensitivity to detect CIN2+ than conventional colposcopy. Findings are similar when CIN3+ is used as a threshold. CONCLUSION. We found that in most cases DSI colposcopy has a higher sensitivity than conventional colposcopy, even when referral criteria are changed. Unlike conventional colposcopy, the sensitivity of colposcopy with DSI in low-grade cytology referrals was found similar to the sensitivity in high-grade cytology referrals. This suggests that a baseline colposcopy sensitivity may be possible with the adjunctive use of the DSI map, irrespective of referral cytology.

Which high-risk HPV assays fulfil criteria for use in primary cervical cancer screening?

Several countries are in the process of switching to high-risk human papillomavirus (hrHPV) testing for cervical cancer screening. Given the multitude of available tests, validated assays which assure high-quality screening need to be identified. A systematic review was conducted to answer the question which hrHPV tests fulfil the criteria defined by an international expert team in 2009, based on reproducibility and relative sensitivity and specificity compared to Hybrid Capture-2 or GP5+/6+ PCR-enzyme immunoassay. These latter two hrHPV DNA assays were validated in large randomized trials and cohorts with a follow-up duration of 8 years or more. Eligible studies citing the 2009 guideline were retrieved from Scopus (http://www.scopus.com) and from a meta-analysis assessing the relative accuracy of new hrHPV assays versus the standard comparator tests to detect high-grade cervical intraepithelial neoplasia or cancer in primary screening. The cobas 4800 HPV test and Abbott RealTime High Risk HPV test were consistently validated in two and three studies, respectively, whereas the PapilloCheck HPV-screening test, BD Onclarity HPV assay and the HPV-Risk assay were validated each in one study. Other tests which partially fulfil the 2009 guidelines are the following: Cervista HPV HR Test, GP5+/6+ PCR-LMNX, an in-house E6/E7 RT quantitative PCR and MALDI-TOF (matrix-assisted laser desorption-ionization time-of-flight). The APTIMA HPV assay targeting E6/E7 mRNA of hrHPV was also fully validated. However, the cross-sectional equivalency criteria of the 2009 guidelines were set up for HPV DNA assays. Demonstration of a low risk of CIN3+ after a negative APTIMA test over a longer period is awaited to inform us about its utility in cervical cancer screening at 5-year or longer intervals.

Women's Preferences of Dynamic Spectral Imaging Colposcopy.

BACKGROUND: The focus of testing the dynamic spectral imaging (DSI) colposcope has been on the technical characteristics and clinical performance. However, aspects from a patient's perspective are just as important. METHODS: This study was designed as a substudy of the DSI validation study, a prospective comparative, multicenter clinical trial to assess the clinical performance of DSI colposcopy. All women included in this study were asked to complete two questionnaires: a patient characteristics questionnaire and a patient satisfaction questionnaire. RESULTS: In the initial study a total of 239 women were included in the intention-to-treat cohort. Of these, 230 women (96.2%) completed both questionnaires. When assessing the women's preferences for some of the possible uses of DSI colposcopy, a high level of agreement was noted for all potential implementations. In general, women found the additional time DSI colposcopy took acceptable: just 15 women (6.5%) thought the time DSI colposcopy took made them feel uncomfortable. Furthermore, women ranked test accuracy as the most important characteristic, followed by (more) rapid testing and comfort. Quick notification of the results and costs were considered the least important characteristics. CONCLUSION: Women are willing to accept discomfort in the form of an additional or longer test if there is clinical benefit.

Clinical evaluation of a GP5+/6+-based luminex assay having full high-risk human papillomavirus genotyping capability and an internal control.

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.

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

BACKGROUND: 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. METHODS: 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. 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. CONCLUSIONS: 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.

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

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.

Cervical cancer screening: on the way to a shift from cytology to full molecular screening.

Cytology-based nation-wide cervical screening has led to a substantial reduction of the incidence of cervical cancer in western countries. However, the sensitivity of cytology for the detection of high-grade precursor lesions or cervical cancer is limited; therefore, repeated testing is necessary to achieve program effectiveness. Additionally, adenocarcinomas and its precursors are often missed by cytology. Consequently, there is a need for a better screening test. The insight that infection with high-risk human papillomavirus (hrHPV) is the causal agent of cervical cancer and its precursors has led to the development of molecular tests for the detection of hrHPV. Strong evidence now supports the use of hrHPV testing in the prevention of cervical cancer. In this review, we will discuss the arguments in favor of, and concerns on aspects of implementation of hrHPV testing in primary cervical cancer screening, such as the age to start hrHPV-based screening, ways to increase screening attendance, requirements for candidate hrHPV tests to be used, and triage algorithms for screen-positive women.

The Aptima HPV assay fulfills the cross-sectional clinical and reproducibility criteria of international guidelines for human papillomavirus test requirements for cervical screening.

The Aptima HPV assay (Hologic Gen-Probe, San Diego, CA) is an FDA-approved assay for detecting human papillomavirus (HPV) E6/E7 mRNA from 14 high-risk HPV types. This study evaluated the clinical performance of the Aptima HPV assay for cervical intraepithelial neoplasia of grade 2 or worse (CIN2+), relative to the high-risk HPV GP5+/GP6+ PCR, in a cross-sectional clinical equivalence analysis using the noninferiority score test with cervical samples from population-based screening, i.e., 69 cervical scraping samples from women with CIN2+ and 843 from women without evidence of CIN2+. In addition, intralaboratory reproducibility over time and interlaboratory agreement of the Aptima HPV assay results were assessed with another set of 548 cervical samples. The Aptima HPV assay showed a clinical sensitivity for CIN2+ of 94.2% (95% confidence interval [CI], 85.5 to 97.8%) and a clinical specificity for CIN2+ of 94.5% (95% CI, 92.8 to 95.9%); by comparison, these figures were 97.1% (95% CI, 89.1 to 99.3%) (67/69 samples) and 93.6% (95% CI, 91.7 to 95.0%) (785/839 samples), respectively, for GP5+/GP6+ PCR. The clinical sensitivity and specificity of the Aptima HPV assay were noninferior to those of GP5+/GP6+ PCR (P = 0.039 and 0.00016, respectively). In addition, high reproducibility of the Aptima HPV assay, as reflected by the intralaboratory reproducibility over time of 96.0% (95% CI, 94.4 to 97.3%) (526/548 samples; kappa = 0.89) and interlaboratory agreement of 96.7% (95% CI, 95.4 to 98.1%) (531/548 samples; kappa = 0.91), was found. Altogether, these data show that the Aptima HPV assay meets the cross-sectional clinical and reproducibility criteria of the international guidelines for HPV test requirements for cervical screening. Longitudinal data are needed to ensure that the long-term negative predictive value of this mRNA assay is similar to those of validated HPV DNA tests.

Clinical validation of the Abbott RealTime High Risk HPV assay according to the guidelines for human papillomavirus DNA test requirements for cervical screening.

This study showed that the Abbott RealTime High Risk HPV assay fulfilled cross-sectional clinical equivalence and reproducibility criteria of international consensus guidelines, which indicates that this assay can be considered clinically validated for cervical cancer screening purposes.

Clinical evaluation of high-risk HPV detection on self-samples using the indicating FTA-elute solid-carrier cartridge.

BACKGROUND: High-risk human papillomavirus (hrHPV) testing in cervical screening is usually performed on physician-taken cervical smears in liquid-based medium. However, solid-state specimen carriers allow easy, non-hazardous storage and transportation and might be suitable for self-collection by non-responders in screening and in low-resource settings. OBJECTIVES: We evaluated the adequacy of self-collected cervicovaginal (c/v) samples using a Viba-brush stored on an Indicating FTA-elute cartridge (FTA-based self-sampling) for hrHPV testing in women referred to a gynecology clinic due to an abnormal smear. STUDY DESIGN: 182 women accepted to self-collect a c/v sample. After self-sampling, a physician obtained a conventional liquid-based cervical smear. Finally, women were examined by colposcopy and a biopsy was taken when clinically indicated. Self-samples required only simple DNA elution, and DNA was extracted from physician-obtained samples. Both samples were tested for 14 hrHPVs by GP5+/6+-EIA-LQ Test and SPF(10)-DEIA-LiPA(25). RESULTS: Both assays detected significantly more hrHPV in physician-collected specimens than in self-collected samples (75.3% and 67.6% by SPF(10); 63.3% and 53.3% by GP5+/6+, respectively). The combination of physician-collected specimen and GP5+/6+ testing demonstrated the optimal balance in sensitivity (98.0%) and specificity (48.1%) for CIN2+ detection in this referral population. A test system of FTA-based self-collection and SPF(10) hrHPV detection approached this sensitivity (95.9%) and specificity (42.9%). CONCLUSIONS: These results show that the clinical performance of hrHPV detection is determined by both the sample collection system and the test method. FTA-based self-collection with SPF(10) testing might be valuable when a liquid-based medium cannot be used, but requires further investigation in screening populations.

Increased cytotoxic capacity of tumor antigen specific human T cells after in vitro stimulation with IL21 producing dendritic cells.

Monocyte derived dendritic cells (moDC) electroporated with tumor associated antigen derived mRNA can elicit specific T cells against tumor cells in vivo. IL21 has been shown to enhance activation and cytotoxicity in CD8+ T cells. We therefore investigated in vitro effects on human CD8+ T-cells after stimulation with IL21 mRNA electroporated moDC. Codon modification of the IL21 gene significantly enhanced IL21 production upon electroporation of moDC. Tumor associated antigen specific CTL induction efficiency was significantly enhanced when codon modified IL21 mRNA was co-electroporated with tumor associated antigen mRNA. Tumor associated antigen specific T cells induced by codon modified IL21-DC demonstrated increased cytotoxic capacity and killing compared to control cultures. In conclusion, ectopic expression of codon modified IL21 by moDC enhances the priming efficiency of the DC as well as the cytotoxic potential of the induced CTL.

Altered microRNA expression associated with chromosomal changes contributes to cervical carcinogenesis.

Little is known about the alterations in microRNA (miRNA) expression patterns during the consecutive stages of cervical cancer development and their association with chromosomal instability. In this study, miRNA expression in normal cervical squamous epithelium, high-grade precancerous lesions (cervical intraepithelial neoplasia (CIN2-3)), squamous cell carcinomas (SCCs) and adenocarcinomas (AdCAs) was integrated with previously generated chromosomal profiles of the same samples. Significantly differential expression during the consecutive stages of cervical SCC development was observed for 106 miRNAs. Of these differentially expressed miRNAs, 27 showed early transiently altered expression in CIN2-3 lesions only, 46 miRNAs showed late altered expression in SCCs only and 33 showed continuously altered expression in both CIN2-3 and SCCs. Altered expression of five significantly differentially expressed miRNAs, hsa-miR-9 (1q23.2), hsa-miR-15b (3q25.32), hsa-miR-28-5p (3q27.3), hsa-miR-100 and hsa-miR-125b (both 11q24.1), was directly linked to frequent chromosomal alterations. Functional analyses were performed for hsa-miR-9, representing a potential oncogene with increased expression linked to a chromosomal gain of 1q. Hsa-miR-9 overexpression was found to increase cell viability, anchorage-independent growth and migration in vitro. Upon organic raft culturing, hsa-miR-9 hampered differentiation and induced proliferation in all strata of the epithelial layer. These findings support a potential oncogenic function of hsa-miR-9 in cervical cancer. In summary, differential expression of 106 miRNAs, partly associated with chromosomal alterations, was observed during cervical SCC development. Altered expression of hsa-miR-9 associated with a chromosomal gain of chromosome 1q was shown to be functionally relevant, underlining the importance of deregulated miRNA expression in cervical carcinogenesis.