Ramya Bhatia

Papilloplex HR-HPV test has non-inferior clinical performance for detection of human papillomavirus infection: assessment using the VALGENT framework

Aim The Papilloplex high-risk human papillomavirus (hrHPV) test (Genefirst, Oxford, UK) is a single tube real-time HPV test which provides multiplex detection and separate identification of 14 hrHPV types. Here, we present the clinical validation of the test in SurePath samples in comparison to a clinically validated reference test, the GP5+/6+Enzyme ImmunoAssay (GP5+/6+EIA) using the VALGENT (VALidation of HPV GENotyping Tests) framework. Methods Clinical performance was assessed using 998 unselected, cervical screening samples enriched with 297 cytologically abnormal specimens (100 atypical squamous cells of unspecified significance, 100 low-grade squamous intraepithelial lesions, 97 high-grade squamous intraepithelial lesions). Cases were defined as women diagnosed with histologically confirmed cervical intraepithelial neoplasia two or more (≥CIN2, N=119) and controls defined as women with two subsequent negative cytology results (N=834). Results The Papilloplex HR-HPV test has non-inferior sensitivity for detection of cervical precancer (p=0.0001 for ≥CIN2 and p=0.0005 for ≥CIN3) and non-inferior specificity, compared with GP5+/6+EIA (pni=0.0167)). The assay also showed excellent or good agreement for overall hrHPV and nearly all individual HPV types as compared with GP5+/6+EIA/Luminex. Conclusion The Papilloplex HR-HPV applied on cervical specimens stored in SurePath medium fulfils the international clinical accuracy criteria for use in cervical cancer screening.

The challenges of defining sample adequacy in an era of HPV based cervical screening

The implementation of Human Papillomavirus based cervical screening continues apace on a global scale. Understanding the basis and burden of inadequate or invalid samples is important to ensure confidence in high quality laboratory results and inform the development of new technologies. Here we present population based data from Scotland and Denmark which detail the extent of invalid samples for HPV detection in both clinician-taken and self-taken samples. As a comparator we report on the rate of inadequate cytology preparations in both countries. The proportion of samples with an invalid HPV test result was calculated by retrospective analysis of routine laboratory data associated with cervical screening programmes in the two countries. Two assays were in use for the programmes at the time (the Abbott RealTime High Risk HPV assay and the BD Onclarity); both have internal endogenous controls for human genes. In addition, acellular cytology samples were reported through a prospective audit (Scotland) and National quality reporting (Denmark). In total, 89,418 clinician samples and 14,677 self-taken samples were assessed. We observed low rates of invalid HPV tests in clinician taken samples (0.05-0.10 %), irrespective of sample collection media (ThinPrep or SurePath), HPV test system/endogenous control type or clinical indication for testing (primary screening, triage or test of cure). For self-taken samples, the number of invalid samples was 0.18 %. Complete absence of sample material (acellular) in clinician taken samples were observed at a level of 1 in approximately 16.5 thousand. Clinician and self-taken samples appear robust specimens for HPV testing and acellular samples are very rare. Efforts to develop endogenous controls for HPV assays that provide greater insight into true sample adequacy for cervical disease detection, beyond measuring the presence of human cells, will be welcome.

Clinical performance of DNA and RNA based HPV tests for test of cure (TOC) post treatment for cervical intraepithelial neoplasia (CIN) - a retrospective study.

HPV testing as a "test of cure" (TOC) of women treated for cervical high-grade lesions can support and inform appropriate clinical management pathways. However, there is a lack of studies that report the discrete performance of different HPV assays in this context, including HPV mRNA based assays. To address this, we performed an analysis of the clinical performance of two hrHPV assays in the (TOC) setting; the recently launched DNA based Alinity m HR HPV (Abbott Molecular) and RNA based Aptima HPV assay (Hologic). Using a retrospective case-control design, two panels of archived cervical liquid based cytology samples, originally taken as per routine TOC protocols in Scotland were assessed. Each panel contained 63 cases, where cervical intraepithelial neoplasia 2 or worse (CIN2+) was detected and 160 controls (women with no CIN2+ and two subsequent cytology negative results (minimum) 3 years apart or women who had histologically confirmed ≤CIN1). All samples were previously tested using the RealTime High Risk HPV assay (Abbott Molecular) as per national TOC protocol. Panel A and Panel B were tested using Alinity and Aptima assay respectively. Both assays showed similar performance to the original RealTime assay. Aptima had sensitivity for CIN2+ of 96.8% (95% CI: 89.0- 99.6) compared to RealTime (93.7% (95% CI: 84.5 - 98.2)). Alinity had sensitivity for CIN2+ of 92.1% (95% CI: 82.4- 97.4) compared to RealTime (98.4% (95% CI: 91.5- 99.95)). Both mRNA based and DNA based HPV tests show robust performance for the monitoring of residual disease post-treatment.

Agreement between L1 and E6/E7-based assays for detection of high-risk HPV in cervical, oropharyngeal and penile cancers

Aims Human papillomavirus (HPV) molecular testing targets either the late gene L1 or early genes E6 and/or E7. Loss of L1 during integration is suggested to compromise sensitivity in samples associated with cancer, however, clear evidence for this is lacking. Our aim is to address this by performing a head-to-head comparison between assays targeting L1 vs E6/E7, using a series of high-grade and invasive disease samples within different biological matrices and anatomical sites. Methods We obtained 298 samples comprising of liquid-based cytology and biopsies of cervical cancer and cervical intraepithelial neoplasia grade 3, in addition to biopsies of penile and oropharyngeal cancers. Two commercially available HPV primary screening assays and two assays with extended genotyping were applied to the sample set targeting L1 (Abbott RealTime HR HPV Assay and Optiplex HPV Genotyping Test) and E6/E7 genes (Xpert HPV Test and EuroArray HPV Test). Results Agreement for high-risk HPV (hrHPV) for all samples types between the screening assays is over 88% and over 96% for the two genotyping assays. For HPV 16 agreement is over 90% for both screening and genotyping assays. Kappa statistics show good to very good agreement between the screening and genotyping assays for hrHPV and HPV 16. Conclusions Analysis of the valid results from our data indicates that L1 and E6/E7 targeting assays show similar performance for detection of hrHPV in high grade cervical lesions and cancers of cervix, penis and oropharynx.

Clinical performance of the novel full‐genotyping OncoPredict HPV Quantitative Typing assay using the VALGENT framework

AbstractClinical validation of human papillomavirus (HPV) assays according to international criteria is prerequisite for their implementation in cervical cancer screening. OncoPredict HPV Quantitative Typing (QT) assay (Hiantis Srl, Milan, Italy) is a novel full‐genotyping multiplex real‐time PCR quantitative assay targeting E6/E7 genes, allowing individual viral load determination of 12 high‐risk (HR) HPV types. Quality controls for sample adequacy, efficiency of nucleic acid extraction and PCR inhibition are included in the assay. Clinical performance of OncoPredict HPV QT test was assessed as part of the “Validation of HPV Genotyping Tests” (VALGENT‐2) framework, consisting of 1300 cervical liquid‐based cytology (LBC) samples of women aged between 20 and 60 years who had originally attended for routine cervical screening in Scotland. The clinical accuracy of the OncoPredict HPV QT (index test) for the detection of CIN2+ was assessed relative to the GP5+/6+ Enzyme ImmunoAssay (GP5+/6+ EIA) (comparator test), using noninferiority criteria. Intra‐ and interlaboratory reproducibility of the assay was assessed on a subpopulation, comprising 526 samples. The relative sensitivity and specificity for OncoPredict HPV QT vs GP5+/6+‐PCR‐EIA were 1.01 (95% CI: 0.99‐1.03) and 1.03 (95% CI: 1.0‐1.06) respectively. The P‐values for noninferiority were ≤0.001. The intra‐ and inter‐laboratory reproducibility demonstrated a high concordance (>98.7%) with kappas for individual types ranging from 0.66 to 1.00. OncoPredict HPV QT fulfills the international validation criteria for the use of HPV tests in cervical cancer screening.