Detection of High-Risk Papillomavirus DNA with Commercial Invader-Technology-Based Analyte-Specific Reagents following Automated Extraction of DNA from Cervical Brushings in ThinPrep Media

Abstract

We compared the performances of the Third Wave Technology Invader method and the Digene Hybrid Capture 2 assay to detect high-risk human papillomaviruses in 87 cervical brushing specimens submitted in Cytyc ThinPrep media. Two different methods for the extraction of DNA from squamous epithelial cells were also evaluated.The human papillomavirus (HPV) family consists of more than 100 types. Those shown to be causative agents of cervical cancer are referred to as "high-risk" strains. Current guidelines recommend the testing of all women with a cytological diagnosis of atypical squamous cells of undetermined significance (ASCUS) for infection by high-risk types of HPV (16,23). HPV testing has been found to have a higher sensitivity than standard or liquid-based Pap testing for detecting high-grade cervical intraepithelial neoplasia (1,(17)(18)(19)(20). Only the Digene Hybrid Capture 2 (HC2) assay (Digene, Gaithersburg, MD) has been approved by the FDA for the detection of high-risk strains of HPV as an ASCUS triage for referral to colposcopy, and, in conjunction with cervical cytology, as a primary screen for cervical cancer. The purpose of this study was to compare a laboratory-developed test utilizing analyte-specific reagents (ASRs) developed by Third Wave Technologies Inc. (TWT, Madison, WI) with the Digene HC2 assay for the detection of high-risk HPV DNA in cervical brushings. The TWT method used in this study, based on proprietary Invader technology, employed isothermal signal amplification to detect 13 high-risk HPV types, utilizing three probe pools (A5/A6 [types 51 and 56], A7 [types 18, 39, 45, 59, and 68], and A9 [types 16, 31, 33, 35, 52, and 58]) based on phylogenetic relatedness. The assay also incorporated an internal control for human alpha-actin to control for DNA quality and quantity in each reaction.The 87 specimens used in this study were cervical brushings collected in ThinPrep fluid (Cytyc Corp., Marlborough, MA) submitted to the cytology laboratory at CompuNet Clinical Laboratories for routine Pap testing. Those samples that received a cytological diagnosis of ASCUS were tested for highrisk HPV by using the Digene HC2 high-risk HPV test according to the manufacturer's instructions. Cells were collected from 2 ml of ThinPrep fluid by centrifugation at 14,000 ϫ g for 10 min and suspended in 0.4 ml of phosphate-buffered saline.DNA was extracted using the Qiagen BioRobot M48 and the MagAttract virus DNA minikit (Qiagen Inc., Valencia, CA) and eluted into 100 l of RNase-free distilled water. Duplicate samples were prepared from 2 ml of each cytology sample by using Gentra Systems Puregene spin columns (Qiagen Inc., Valencia, CA) by following the manufacturer's instructions. All extracted samples were stored at Ϫ20°C until tested. The extracted DNA was tested for the presence of high-risk HPV by a laboratory-developed assay utilizing Invader HPV ASRs. The TWT ASRs consisted of three separate oligonucleotide pools with 6-carboxyfluorescein (FAM)-labeled probes, one Redmond red (RED)-labeled oligonu...

Clinical Laboratory Experience with Cervista HPV HR as a Function of Cytological Classification: Comparison with Retrospective digene HC2 High-Risk HPV DNA Test Data

“…Other studies published from a laboratory (2)(3)(4)(5)(6) or clinical (7) perspective do not reflect this opinion. In this letter, we compare a large data set generated from consecutive 9-month intervals of HC2 and Cervista screening, stratified by age and cytological classification.…”

Commercially available assays for multiplex detection of alpha human papillomaviruses

“…Interference or inhibition was not observed with any of the tested substances that may be present in cervical specimens [40]. In four studies, which included a total of 1248 samples, different prototypic Review versions of Cervista showed 81.6-90.8% agreement with hc2 [33,[41][42][43]45]. After testing 12,490 specimens using Cervista assay prototype Invader V2.0, Harvey et al recently assessed the clinical performance of this prototype assay by comparison of obtained HPV results with corresponding biopsy results for a total of 1931 cases [44].…”

“…A genotyping study of 3179 specimens of women participating in an ALTS trial (a clinical trial to evaluate management strategies for women with ASC-US or low-grade squamous intraepithelial lesions) showed that 7.8% of all hc2-positive results in this population were false-positive due to the cross-reactivity of hc2 with untargeted, noncarcinogenic HPV types [32]. Some recent studies have also shown that hc2 has an additional 5% false-positive rate due to positive hr-HPV results when no HPV DNA is present in the clinical specimen based on the use of different usually highly sensitive and broad-range PCR tests [32,33]. The relatively high false-positivity rate of the current hc2 hr-HPV cocktail has important clinical consequences as the 2006 American Society for Colposcopy and Cervical Pathology (ASCCP) guidelines state that it is 'unacceptable' for an HPV assay to test for nononcogenic HPV types if used in cervical cancer screening programs because false-positive results may result in unnecessary colposcopy procedures [34].…”

“…To the best of our knowledge, eight evaluations have been published to date: one analytical [40] and seven evaluations comparing the performance of different prototypic versions of the current assay with other HPV assays, mainly hc2 [33,[42][43][44][45][46][47]. A study that characterized the analytical performance of an investigational use only version of Cervista showed that analytical sensitivity for the targeted 14 HPV types ranged from 1250 to 7500 copies per reaction, depending on the HPV type [40].…”

Clinical Experience with the Cervista HPV HR Assay

“…This assumption is supported by previous studies that reported occasional false-negative results for hc2 when comparing hc2 and Invader-based assays. 11,14,15 In addition, results from nearly one-third of the cytologically unsatisfactory samples in the Cervista population could not be reported because of low internal control signal; however, all samples of this type from the hc2 population that had sufficient volume for testing were reported as being definitively positive or definitively negative for HR-HPV. The clinical usefulness of HR-HPV testing largely relies on its high sensitivity and strong negative predictive value; thus, false-negative results are of particular concern because they fail to identify women who require further testing.…”