Clinical Performance of the APTIMA HPV Assay for Detection of E6/E7 mRNA from High-Risk HPV Types in Liquid Based Cytolo

1. Persistent HPV Infection Initial HPV Infection Host Chromosomal DNA Episomal HPV DNA HPV DNA Integration E6/7 RNA • Low level E6/E7 mRNA expression • Low probability of progression to disease • Increased E6/E7 mRNA expression • Increased probability of progression to disease E6/7 RNA Clinical Performance of the APTIMA HPV Assay for Detection of E6/E7 mRNA from High-Risk HPV Types in Liquid Based Cytology Specimens C. Hill1, J. Dockter1, A. Schroder1, B. Eaton1, J. Monsonego2 1Gen-Probe Incorporated, San Diego, CA 2European Institute of the Cervix, Paris France Abstract Introduction Background: This study evaluated the clinical performance of the APTIMA HPV (AHPV, Gen-Probe Incorporated) assay, a qualitative nucleic acid test to detect the E6/E7 mRNA of 14 high-risk HPV (hrHPV) types. Methods: AHPV was compared to the Hybrid Capture 2 (HC2, Qiagen Incorporated) HPV DNA test. Performance of the assays for detection of hrHPV types was determined by comparison with the Linear Array genotyping test (Roche Molecular Diagnostics). Performance was evaluated using 780 LBC (Cytyc) specimens from women referred to colposcopy. Sensitivity and specificity for detection of disease were calculated based on 753 specimens with histology results defining disease as positive if histology results were CIN2 or greater (CIN2+). Results: The sensitivity of AHPV and HC2 for hrHPV types was 93.0% and 94.1%, respectively. The specificity of the AHPV assay (99.1%) for hrHPV was significantly higher than HC2 (82.4%). The sensitivity for detection of CIN 2+ disease was 90.8% for AHPV and 95.0% for HC2. The clinical specificity of the AHPV assay (55.4%) was significantly higher than HC2 (47.4%). There was no statistical difference in sensitivity between the AHPV and HC2 assays for detection of disease or high risk HPV. Conclusions: The AHPV assay is as sensitive but more specific for detection of hrHPV types and cervical disease compared to the HC2 assay. The increased clinical specificity of AHPV is likely due to the detection of HPV mRNA rather than DNA and the lack of cross-reactivity of AHPV with low-risk HPV genotypes. These results show that the AHPV assay will be useful for the detection of cervical disease in LBC specimens from women. The clinical value of HPV testing for identifying women at increased risk for developing cervical cancer has been established. Detection of HPV infections with DNA molecular tests is more sensitive, but less specific than Pap testing, since most HPV infections and pre-cancerous lesions resolve without treatment. Detection of HPV mRNA, specifically E6/E7, is thought to provide a more specific method to differentiate between transient and persistent HPV infections associated with disease. Figure 1 (1, 2) The APTIMA HPV Assay is a sensitive nucleic acid test that detects HPV mRNA from 14 high-risk HPV types, but does not differentiate them. The assay utilizes Gen-Probe’s magnetic bead-based target capture, transcription-mediated amplification and Duel Kinetic Assay technologies. The assay is CE-Marked and can be performed on the semi-automated DTS and fully-automated TIGRIS DTS systems (Figure 2). • Figure 2. High Throughput Fully Automated TIGRIS DTS SYSTEM • 1,000 results in 14 hours • Time to first result: ~3.5 hrs, ~100 results/hour thereafter • Process controls for all assay steps Figure 1 HPV Infection, DNA Integration and mRNA expression Study Objective The objective of this study was to assess the clinical sensitivity and specificity of the APTIMA HPV Assay for the detection of high-risk HPV and high-grade cervical lesions. APTIMA HPV Assay Clinical Performance Methods Table 1 APTIMA HPV and HC2 Assay Concordance Table 2 Resolution of APTIMA/HC2 Discordant Specimens • Specimens: Residual Thin-Prep liquid-Pap specimens were obtained from 780 subjects referred to colposcopy due to an abnormal Pap and/or HPV infection. Colposcopy/histology results were available for 753 of the subjects. Sensitivity and specificity of the HPV mRNA and DNA tests was calculated using CIN2+ and CIN3+ clinical endpoints. • HPV testing: • A 1 mL aliquot of the Thin-Prep liquid-Pap specimens was transferred to an APTIMA Specimen Transfer tube, containing specimen transfer media. Four hundred microliters of each diluted specimen was tested in the APTIMA HPV Assay (AHPV) according to the instructions for use on the TIGRIS DTS System. • A 4 mL aliquot of the Thin-Prep liquid-Pap specimens was tested in the Hybrid Capture 2 HR HPV DNA test (HC2) according to the manufacturer’s instructions for use. • A subset of the Thin-Prep liquid-Pap specimens were tested in the Linear Array HPV Genotyping test (LA) according to the instructions for use; including 122 AHPV/HC2 discordant and 271 AHPV and HC2 concordant specimens. • Sensitivity/Specificity for detection of HPV: Sensitivity and specificity for detection of high-risk HPV of the AHPV and HC2 tests calculated. High-risk HPV status was based on concordance of 2 of the 3 HPV tests. • Sensitivity/Specificity for detection of high-grade lesions: Sensitivity and specificity for detection of CIN2+ and CIN3+ were calculated. Subjects from whom a biopsy was not obtained (no visible lesion) were considered negative. HR= high-risk HPV DNA LR= low-risk HPV DNA ND= HPV DNA not detected Table 3 Sensitivity and Specificity for Detection of High-risk HPV Conclusions • Good agreement was observed between the APTIMA HPV Assay and the Hybrid Capture 2 HR HPV DNA test in this referral population; kappa value = 0.68. • The APTIMA HPV Assay had similar sensitivity and better specificity than the Hybrid Capture 2 test for detection of high-risk HPV. • The APTIMA HPV Assay had slightly lower sensitivity, although not significantly, but higher specificity for detection of CIN2+ lesions. • The APTIMA HPV Assay had similar sensitivity and significantly higher specificity for detection of CIN3+ lesions. Table 4 Clinical Sensitivity and Specificity for CIN2+ and CIN3+ Clinical Endpoints References • Cushieri, K.S., Whitley, M.J., Cubie, H.A., 2004 Human papillomavirus type specific DNA and RNA persistence-implications for cervical disease progression and monitoring. J. Med. Virol., 73: 65-70. • Wang-Johanning, F., Lu, D.W., Wang, Y., Johnson, M.R., Johanning, G.L., 2002. Quantitation of human papillomavirus 16 E6 and E7 DNA and RNA in residual material from ThinPrep Papanicaolaou tests using real-time polymerase chain reaction analysis. Cancer. 94: 2199-2210. GEN-PROBE, APTIMA, APTIMA COMBO 2, DTS, and TIGRIS are trademarks of Gen-Probe Incorporated CYTYC and PRESERVCYT are trademarks of Cytyc Corporation DIGENE and HYBRID CAPTURE are trademarks of Digene Diagnostics, Inc. LINEAR ARRAY is a trademark of Roche Diagnostics Corporation and ROCHE is a trademark of Hoffmann-La Roche Inc. N= 753 total