VALHUDES: A protocol for validation of human papillomavirus assays and collection devices for HPV testing on self-samples and urine samples

&lt;p&gt;&lt;b&gt;BACK GROUND: &lt;/b&gt;Systematic reviews have concluded that hrHPV DNA testing using target-amplification tests is as accurate on vaginal self-samples as on clinician-taken specimens for the detection of cervical precancer. However, insufficient evidence is available for specific HPV assay/self-sample device combinations.&lt;/p&gt; &lt;p&gt;&lt;b&gt;OBJECTIVES: &lt;/b&gt;The VALHUDES protocol is designed as a diagnostic test accuracy study that aims to compare the clinical sensitivity and specificity of particular hrHPV assay(s) on vaginal self-samples and first-void-urine, collected in agreement with standardized protocols, with hrHPV testing on matched clinician-taken samples.&lt;/p&gt; &lt;p&gt;&lt;b&gt;STUDY DESIGN: &lt;/b&gt;Five hundred enrolled women referred to a colposcopy clinic are invited to collect a first-void urine sample and one or more vaginal self-samples with particular devices before collection of a cervical sample by a clinician. Sample sets are subsequently analysed in a laboratory accredited for HPV testing. Disease verification for all enrolled patients is provided by colposcopy combined with histological assessment of biopsies.&lt;/p&gt; &lt;p&gt;&lt;b&gt;RESULTS: &lt;/b&gt;A first VALHUDES study has started in Belgium in December 2017 with enrolment from four colposcopy centres. The following assays are foreseen to be evaluated: RealTime High Risk HPV assay (Abbott), cobas-4800 and -6800 (Roche), Onclarity (BD), Xpert HPV (Cepheid) and Anyplex II HPV HR (Seegene).&lt;/p&gt; &lt;p&gt;&lt;b&gt;CONCLUSION: &lt;/b&gt;Given empirical evidence that the relative accuracy of HPV-testing on self- vs clinician-samples is robust across clinical settings, the VALHUDES protocol offers a framework for validation of HPV assay/self-sample device combinations that can be translated to a primary screening setting.&lt;/p&gt;</p

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

Comparison of three human papillomavirus DNA assays and one mRNA assay in women with abnormal cytology

AbstractObjectiveTo compare the clinical characteristics of four human papillomavirus (HPV) assays: hybrid capture 2 (HC2), cobas, CLART, and APTIMA in Danish women with abnormal cytology.MethodsSurePath samples from 367 consecutive women from Copenhagen, with atypical squamous cells of undetermined significance or worse, were tested with the four assays. These women were routinely recommended for repeated testing or were referred for colposcopy. Their worst histological diagnosis in 29months from baseline was retrieved from the Danish National Pathology Data Bank.ResultsOf the 367 women, 16 (4%) had no follow-up, 232 (63%) had <CIN2, 35 (10%) had CIN2, 81 (22%) had CIN3, and 3 (1%) had cervical cancer. The sensitivity for ≥CIN3 was 95% (95% CI: 88–99) for HC2, 94% (95% CI: 87–98) for cobas, 93% (95% CI: 85–97) for CLART, and 87% (95% CI: 78–93) for APTIMA. In women of age above 30years, the sensitivities were 98% (95% CI: 87–100), 93% (95% CI: 80–98), 90% (95% CI: 77–97), and 93% (95% CI: 80–98), respectively. One woman with cervical cancer tested negative on CLART and one on cobas; HC2 and APTIMA were positive in all three cancer cases. The specificity for <CIN3 was low for all assays and varied between 22% and 35%. Similar results were seen for ≥CIN2.ConclusionsSmall differences in clinical characteristics were found for the four HPV assays in Danish women with abnormal cytology aged ≥30years. At younger ages, APTIMA was somewhat less sensitive for high-grade CIN than the three HPV DNA assays

Control of pathogenicity and disease specificity of a T-lymphomagenic gammaretrovirus by E-box motifs but not by an overlapping glucocorticoid response element.

Although transcription factors of the basic-helix-loop-helix family have been shown to regulate enhancers of lymphomagenic gammaretroviruses through E-box motifs, overlap of an E-box motif (Egre) with the glucocorticoid response element (GRE) has obscured their function in vivo. We here report that Egre, but not the GRE affects disease induction by the murine T-lymphomagenic SL3-3 virus. Mutating all three copies of Egre prolonged the tumor latency period from 60 to 109 days. Further mutating an E-box motif (Ea/s) outside the enhancer prolonged the latency period to 180 days, suggesting that Ea/s works as a back-up site for Egre. While SL3-3 wt, GRE and Ea/s mutants induced exclusively T-cell lymphomas with wild type latencies, mainly of the CD4+CD8- phenotype, the Egre as well as the Egre plus Ea/s mutants induced B-cell lymphomas and myeloid leukemia in addition to T-cell lymphomas. T-cell lymphomas induced by the two Egre mutants had the same phenotype as those induced by SL3-3 wt, indicating incomplete disruption of T-cell lymphomagenesis in contrast to previous findings for a Runx site mutant of SL3-3. Mutating the Egre site or Egre plus Ea/s triggered several tumor phenotype-associated secondary enhancer changes encompassing neighboring sites, none of which led to regeneration of an E-box motif. Altogether, our results demonstrate a role for the E-box, but not the GRE in T-lymphomagenesis by SL3-3, unveil an inherent broader disease specificity of the virus, and strengthen the notion of selection for more potent enhancer variants of mutated viruses during tumor development

Clinical performance of the HPV-Risk assay on cervical samples in SurePath medium using the VALGENT-4 panel

Background: The VALidation of HPV GENoyping Tests (VALGENT) framework is designed for comparison and clinical validation of HPV assays. Objectives: To evaluate the accuracy of the HPV-Risk assay within VALGENT-4, relative to clinically validated comparator HPV tests. Study design: The VALGENT-4 panel comprises consecutive SurePath cervical samples from routine screening (n=998), of which 51 had abnormal cytology and 13 women had cervical intraepithelial neoplasia (CIN) grade 2 or worse (CIN2+), enriched with SurePath cervical samples from 297 women with abnormal cytology and 109 CIN2+. HPV-Risk assay was performed on DNA extracted panel samples (n=1,295), blinded to clinical data, cytology results, and results from other HPV assays evaluated in VALGENT-4. All assay results were reported to the central VALGENT coordination institute for data and statistical analysis. HPV prevalence was analysed and accuracy for detection of CIN grade 3 or worse (CIN3+) and CIN2+ were assessed relative to GP5+/6+-PCR-EIA and GP5+/6+-PCR-EIA-LMNX. Results: The sensitivity of the HPV-Risk assay for detection of CIN3+ and CIN2+ was similar to that of GP5+/6+-PCR-EIA (relative sensitivity for CIN3+1.01; 95%CI: 0.97-1.06; pMcN=1.000, and for CIN2+1.01; 95%CI: 0.96-1.06; pMcN=1.000) at significantly higher specificity (relative specificity 1.04; 95%CI: 1.02-1.06; pMcN<0.001). The accuracy of the HPV-Risk assay for CIN3+ and CIN2+ was non-inferior compared to GP5+/6+-PCR- EIA and GP5+/6+-PCR-EIA-LMNX, with all p-values ≤0.002. HPV16/18 genotype agreement between HPV-Risk assay and GP5+/6+-PCR-LMNX was high. Conclusions: The HPV-Risk assay demonstrated non-inferiority to clinically validated comparator assays on cervical samples in SurePath medium using the VALGENT-4 panel, and is therefore suitable for cervical cancer screening