2013;22:43-47 doi: 10.2478^10162-012-0031-2 The Abbott RealTime High Risk HPV test is a clinically validated human papillomavirus assay for triage in the referral population and use in primary cervical cancer screening in women 30 years and older: a review of validation studies Mario Poljak1 Anja Ostrbenk1 Abstract Introduction: Human papillomavirus (HPV) testing has become an essential part of current clinical practice in the management of cervical cancer and precancerous lesions. We reviewed the most important validation studies of a next-generation real-time polymerase chain reaction-based assay, the RealTime High Risk HPV test (RealTime)(Abbott Molecular, Des Plaines, IL, USA), for triage in referral population settings and for use in primary cervical cancer screening in women 30 years and older published in peer-reviewed journals from 2009 to 2013. RealTime is designed to detect 14 high-risk HPV genotypes with concurrent distinction of HPV-16 and HPV-18 from 12 other HPV genotypes. The test was launched on the European market in January 2009 and is currently used in many laboratories worldwide for routine detection of HPV. Methods: We concisely reviewed validation studies of a next-generation real-time polymerase chain reaction (PCR)-based assay: the Abbott RealTime High Risk HPV test. Results: Eight validation studies of RealTime in referral settings showed its consistently high absolute clinical sensitivity for both CIN2+ (range 88.3-100%) and CIN3+ (range 93.0-100%), as well as comparative clinical sensitivity relative to the currently most widely used HPV test: the Qiagen/Digene Hybrid Capture 2 HPV DNA Test (HC2). Due to the significantly different composition of the referral populations, RealTime absolute clinical specificity for CIN2+ and CIN3+ varied greatly across studies, but was comparable relative to HC2. Four validation studies of RealTime performance in cervical cancer screening settings showed its consistently high absolute clinical sensitivity for both CIN2+ and CIN3+, as well as comparative clinical sensitivity and specificity relative to HC2 and GP5+/6+ PCR. Conclusion: RealTime has been extensively evaluated in the last 4 years. RealTime can be considered clinically validated for triage in referral population settings and for use in primary cervical cancer screening in women 30 years and older. Received: 5 May 2013 | Returned for modification: 20 May 2013 | Accepted: 5 June 2013 Introduction Persistent infection with 10 to 15 genotypes of human papillomaviruses (HPV) known as high-risk HPV (hrHPV) is a necessary etiological factor in the development of cervical carcinoma. HPV-16 and HPV-18 together account for approximately 70% of cervical cancers worldwide (1). Consequently, in several countries (including Slovenia) hrHPV testing has become an invaluable part of clinical guidelines for cervical carcinoma screening, triage, and follow-up after treatment. The four major agreed-upon indications for HPV testing in current clinical practice are: 1) triage of women with equivocal cytology results showing the presence of atypical squamous cells of undetermined significance (ASC-US) in order to determine which patients should be referred for col-poscopy; 2) follow-up of women with abnormal screening cytology results that are negative at initial colposcopy/biopsy; 3) prediction of the therapeutic outcome after treatment of CIN2+; and 4) primary screening of women 30 years and older in combination with a Pap smear to detect cervical cancer precursors (2-5). Another potential application of HPV testing is quality control of cervical cytology (6). In a recent comprehensive inventory of commercial tests for detecting alpha-HPV, we identified at least 125 distinct HPV tests and at least 84 variants of the original tests (2). However, our study showed that only a small subset of HPV tests has documented clinical performance for any of the standard HPV testing indications. For more than 75% of HPV tests currently on the market, no single publication in peer-reviewed literature can be identified. We strongly recommended that HPV tests that have not been properly clinically validated and that lack proof of reliability, reproducibility, and accuracy should not be used in clinical management (2). Here we present a concise review of validation studies of a next-generation real-time polymerase chain reaction (PCR)-based assay, the Abbott RealTime High Risk HPV test (RealTime) (Abbott Molecular, Des Plaines, IL, USA), for triage in referral population settings and for use in primary cervical cancer screening in women 30 years and older. RealTime was launched on the European market in January 2009 and is currently used in many laboratories worldwide for routine detection of hrHPV. Together with the cobas® 4800 HPV Test (Roche Molecular Systems Inc., Alameda, CA, USA), RealTime belongs to the group of next-generation real-time PCR-based HPV DNA assays in which detection of 14 HPV genotypes is combined with concurrent genotyping for HPV-16 and HPV-18 (2, 7). The design of such HPV tests was based on the results of clinical studies that demonstrated the exceptionally high oncogenic potential of HPV-16 and HPV-18 compared to other hrHPV types and showed that tests simultaneously 'institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Zaloska 4, SI-1000 Ljubljana, Slovenia. h Corresponding author: mano.poljak@mf.um-lj.si distinguishing HPV genotypes HPV-16 and HPV-18 may identify women at greatest risk of CIN3+ and may permit less aggressive management of women with other hrHPV infections (8). Description of the test The Abbott RealTime High Risk HPV test is a real-time PCR-based assay for concurrent detection and individual genotyping of HPV-16 and HPV-18 and pooled detection of 12 other hrHPV genotypes: HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-66, and HPV-68. The HPV target sequence for RealTime is located in the conserved L1 region of the genome. RealTime is performed on the m2ooort real-time PCR instrument using a modified GP5+/6+ primer mix consisting of three forward primers and two reverse primers designed to hybridize to an approximately 150-base HPV consensus region (9). The internal process control (IC) designed for monitoring sample adequacy and DNA extraction and amplification target sequence is amplified using a primer set targeting a region of 136 bases in the human beta-globin gene. The HPV and internal control probes are single-stranded DNA oligonucleotides modified with a fluorescent moiety covalently linked to one end of the probe and a quenching moiety to the other end (9). Through the distinct labels, signals for HPV-16, HPV-18, non-HPV-16/18 types, and IC can be simultaneously detected and distinguished in a single reaction. The assay also has a proprietary algorithm for amplification curve validation. The RealTime turnaround time is 6 to 8 h for 96 samples, depending on the method used for DNA extraction. The fully automated high-throughput instrument m2ooosp or the smaller m24sp instrument can be used for DNA extraction or, alternatively, DNA can be prepared manually. The assay is officially validated for use with cervical specimens collected with ThinPrep Preserv-Cyt Solution, SurePath Preservative Fluid, and the Abbott Cervi-Collect Specimen Collection Kit; however, in our experience, cervical specimens collected in Digene Specimen Transport Media (STM) are also appropriate (10). In addition to cervical specimens, RealTime can reliably detect and identify targeted HPVs in fresh tissue, fresh-frozen tissue, and tissue fixed with formalin and subsequently embedded in paraffin or paraplast (10-14). Analytical validation studies Probit analysis showed that the analytical sensitivity of RealTime is between 500 and 5,000 copies of HPV DNA per assay, depending on the HPV type (9). An analytical specificity study on a panel of 41 bacteria, viruses, and fungi that can be found in the female anogenital tract revealed no cross-reactivity with any of the organisms tested, including all relevant low-risk HPV genotypes (9). The high analytical specificity of RealTime and virtually no cross-reactivity with non-targeted HPV genotypes in clinical specimens has been confirmed in all studies performed to date, which have focused not only on the clinical performance of RealTime, but also its analytical performance (10, 15-21). Validation of RealTime sensitivity for CIN3 and cervical cancer The clinical sensitivity of the RealTime test was initially evaluated in 593 archived cervical specimens from Amsterdam (the Netherlands) and the presence of hrHPV was detected in 97.2% (246/253) and 98.5% (335/340) of CIN3 and cervical cancer specimens, re- spectively (22). Of 12 hrHPV-negative specimens evaluated further, eight had invalid beta-globin results by the Roche Linear Array HPV Genotyping Test and four contained only low-risk HPVs. In a Slovenian study, HPV was detected by RealTime in 96.4% (245/254) and 98.8% (84/85) of cases of CIN3 and cervical cancer containing targeted hrHPVs, respectively (23). As described below, the high sensitivity of RealTime for CIN3+ lesions, including cervical cancer, has been confirmed in further RealTime validation studies in the referral population and primary cervical cancer screening settings. Clinical validation of RealTime in a referral population setting RealTime clinical performance in a referral population setting has been evaluated in several studies (17-19, 24-28). The results of the most important studies with clinical endpoints (CIN2+ and/ or CIN3+) are presented in Table 1. Although these eight studies differed significantly in the composition of the referral population for colposcopy (the majority enrolling women with abnormal cytology of any grade), the absolute clinical sensitivity of RealTime, as well as its sensitivity relative to the clinically validated comparator test Hybrid Capture® 2 HPV DNA Test (HC2) (Qiagen, Hilden, Germany), were consistently high for both CIN2+ and CIN3+ in all studies performed to date (Table 1). As expected, the absolute clinical specificity for CIN2+/CIN3+ varied greatly across studies due to the heterogeneous nature of the referral populations (Table 1). However, the clinical specificity of RealTime relative to HC2 was above 1.00 in all except one study (Table 1). RealTime also performed well in both multi-HPV assay comparison studies: Predictors 1 (25) and Predictors 2 (28) were among the HPV assays with high clinical sensitivity for cervical high-grade disease and showed comparable clinical specificity with a few other clinically validated HPV DNA assays (25, 28, 29). The high clinical sensitivity and acceptable clinical specificity of RealTime in the referral population was further confirmed in a recent meta-analysis (5). Clinical validation of RealTime for use in primary cervical cancer screening in women 30 years and older RealTime has been clinically validated for use in primary cervical cancer and pre-cancer screening in women 30 years and older in four studies (Table 2) (15, 16, 30, 31). A Slovenian study evaluated RealTime prospectively in comparison with HC2 on 3,129 women (15). Italian (16) and Dutch (30) studies evaluated RealTime following the "Guidelines for HPV DNA test requirements for primary cervical cancer screening in women 30 years and older" (32) in comparison with HC2 and GP5+/6+ PCR on retrospective samples collected from 998 and 927 women, respectively. As shown in Table 3, all three studies showed that RealTime fulfilled the cross-sectional clinical equivalence criteria of the international consensus guidelines (32), which indicates that RealTime can be considered a clinically validated assay for cervical cancer screening purposes. RealTime also performed well in a multi-HPV assay comparison study (Predictors 3), being among HPV assays with the highest clinical sensitivity for cervical high-grade disease in women 30 years and older and showing comparable clinical specificity with other clinically validated HPV DNA assays (31). This was further confirmed in a recent meta-analysis (5), in which RealTime was listed as one of four currently available HPV assays that can be considered clinically validated for use in primary screening. £ S a ÔE £ .c *u M ÏC m -Q ™ < jfr JÏ > £ "3 U u '= • « C (fc ¡^i « S £ u ™ (0 = « SL u CC is -o . > c W (0 (0 M *> "S tH -s > 5 S (A JW CO o re o ® <2 s ■¡3 is is £ n to « g £ a) S c 15 E ¡3 "o ib o) m ® -c .Q oc -M < -a S a. in Q) J3 . O to .Q CO t= « 3« CC OJ U"! •S ^ *o /—) Œ 3 . O to .Q co to ^ m < o < to 5 o CD oô 9 00 00 sS s? ro oo 'l o fN \û C3 m CO 10 (N O | 00 ■ th O LO cn (N o o ^ ? ^ 7 fs rH CO ™ vo ~ Sp ^ \0 sp vP o^ nv o^ nv /—) ^ °° T 9 T ^ ^ 9 K © CD LO K S 0\ CN • CN 00 CN 00 CN O < m < m 1 ^ H (N 00 LO 00 O 'R 'iR CN \0 fN rs "S Wl CL c 1= C 0 0 M— 3 ro 3 ^ X X O X — CC U d O ta /_) CO CO CN _: eu V < Wl ~s to § (U c ce QJ N > U") V tj H 15 u > <0 V .c 1b W- ■a S ¡S « 13 = 12 o 10 .n != o ^ o ? = -c n H tn . ^ ^ .■i^ c o ÎC w J0 •n M "îr w g bo o S» ^ îs -Ji w o ^ iË "¡5 s " M ce T5 Z -i u <» & •= o O ë * ^ I ^^ „ + IB H lO S * ^ > .ç a) E ¿g £ s ^ ^ -a in S JS V E -IB 10 (P (P c 3 ; JS v "S « ¡2 ^ -s ^ ^ iû i— co M— V CL X ^ 'u CJ i_ ^ « îfi ^ ^ ^ 13 '> u ^ '¡^ S® m « ï ui (P > c E -n — o 5S fN tn 00 S N fN t» n V-; ^ ^ CD O 00 O + \0 CN CN C£ CN O O + O O X X CL X CL CD fN nP ^ ^ ^ vP m o o o 00 o 00 o vH 00 0\ (N ^ (P œ (P Œ tu ^ M w M w ^ ^ O ÎJ O lu LO -- Table 3 | Validation of intra-laboratory reproducibility and inter-laboratory agreement of the Abbott RealTime High Risk HPV test. Study (reference) Type of study Number of samples RealTime overall reproducibility (95% CI) Kappa (95% CI) Carozzi FM et al. (16) Intra-laboratory reproducibility 521 98.5% (97-99) 0.97 (0.95-0.99) Poljak M et al. (15) Intra-laboratory reproducibility 500 100.0% (99-100) 1.00 (0.90-1.00) Poljak M et al. (15) Inter-laboratory agreement (first round) 500 100.0% (99-100) 1.00 (0.98-1.00) Poljak M et al. (15) Inter-laboratory agreement (second round) 500 99.8% (98.7-99.9) 0.99 (0.98-1.00) Hesselink AT et al. (30) Intra-laboratory reproducibility 504 98.4% (97.2-99.2) 0.96 Hesselink AT et al. (30) Inter-laboratory agreement 500 99.8% (99.1-99.9) 0.99 Validation of RealTime intra-laboratory reproducibility and inter-laboratory agreement Intra-laboratory reproducibility and inter-laboratory agreement of RealTime have been extensively validated following the "Guidelines for HPV DNA test requirements for primary cervical cancer screening in women 30 years and older" (32) in three and two studies, respectively (15, 16, 30). As shown in Table 3, both intra-laboratory reproducibility and inter-laboratory agreement of RealTime were well above confidence levels in all studies (> 87%, with kappa values > 0.5), confirming the exceptional reproducibil-ity and reliability of RealTime for detecting targeted hrHPV, even in samples stored for more than 3 years. Conclusions RealTime has been extensively evaluated in the last 4 years and can be considered clinically validated for triage in referral population settings and for use in primary cervical cancer screening in women 30 years and older. Disclosures The Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana has received research funding from Abbott Molecular. Mario Poljak has received travel grants and honoraria from Abbott Molecular for speaking and for participating in conferences and sitting on an advisory board. Figure 1 | Example of an Abbott RealTime High Risk HPV test result in a cervical sample in which both HPV-16 and HPV-18 were detected. The assay uses four channels for detecting fluorescent signals: one for detecting an internal process control for sample adequacy and DNA extraction and amplification, a second for detecting HPV-16, a third for detecting HPV-18, and a fourth for aggregate detection of the 12 HPV types. A positive result for HPV-16 is visible as dark blue curve (positive in cycle number 14.37) and a positive result for HPV-18 as a pale blue curve (positive in cycle number 13.45). Internal process control based on amplification of the housekeeping beta-globin gene gave a positive signal in cycle number 19.51 (purple curve). The result for the aggregate detection of the 12 HPV types is negative (no specific curve present). References Arbyn M, Castellsague X, de Sanjose S, Bruni L, Saraiya M, Bray F, et al. Worldwide burden of cervical cancer in 2008. Ann Oncol. 2011;22:2675-86. Poljak M, Cuzick J, Kocjan BJ, Iftner T, Dillner J, Arbyn M. Nucleic acid tests for the detection of alpha human papillomaviruses. Vaccine. 20i2;30(Suppl 5):Fioo-6. Cox JT. History of the use of HPV testing in cervical screening and in the management of abnormal cervical screening results. J Clin Virol. 2009;45(Suppl i):S3-12. Cuschieri KS, Cubie HA. The role of human papillomavirus testing in cervical screening. J Clin Virol. 2005;32(Suppl 1)^34-42. Arbyn M, Ronco G, Anttila A, Meijer CJ, Poljak M, Ogilvie G, et al. Evidence regarding human papillomavirus testing in secondary prevention of cervical cancer. Vaccine. 2012;30(Suppl 5):F88-99. Arbyn M, Ronco G, Cuzick J, Wentzensen N, Castle PE. How to evaluate emerging technologies in cervical cancer screening? Int J Cancer. 2009;125:2489-96. Poljak M, Kocjan BJ. Commercially available assays for multiplex detection of alpha human papillomaviruses. Exp Rev Anti Infect Ther. 2010;8:1139-62. Khan MJ, Castle PE, Lorincz AT, Wacholder S, Sherman M, Scott DR, et al. The elevated 10-year risk of cervical precancer and cancer in women with human papillomavirus (HPV) type 16 or 18 and the possible utility of type-specific HPV testing in clinical practice. J Natl Cancer Inst. 2005;97:1072-9. Huang S, Tang N, Mak WB, Erickson B, Salituro J, Li Y, et al. Principles and analytical performance of Abbott RealTime High Risk HPV test. J Clin Virol. 2009;45(Suppl 1):S13-7. Poljak M, Kovanda A, Kocjan BJ, Seme K, Jancar N, Vrtacnik-Bokal E. The Abbott RealTime High Risk HPV test: comparative evaluation of analytical specificity and clinical sensitivity for cervical carcinoma and CIN 3 lesions with the Hybrid Capture 2 HPV DNA test. Acta Dermatovenerol Alp Panonica Adriat. 2009^8:94-103. 7. 8. 10. 11. Kocjan BJ, Maver PJ, Hosnjak L, Zidar N, Odar K, Gale N, et al. Comparative evaluation of the Abbott RealTime High Risk HPV test and INNO-LiPA HPV Genotyping Extra test for detecting and identifying human papillomaviruses in archival tissue specimens of head and neck cancers. Acta Dermatovenerol Alp Panonica Adriat. 2012;21:73-5. 12. Kocjan BJ, Seme K, Poljak M. Comparison of the Abbott RealTime High Risk HPV test and INNO-LiPA HPV Genotyping Extra test for the detection of human papillomaviruses in formalin-fixed, paraffin-embedded cervical cancer specimens. J Virol Methods. 2011;175:117-9. 13. Snietura M, Jaworska M, Pigtowski W, Goraj-Zajac A, Wozniak G, Lange D. High risk HPV DNA status and p16 (INK4a) expression as prognostic markers in patients with squamous cell cancer of oral cavity and oropharynx. Pol J Pathol. 2010;61:133-9. 14. Snietura M, Piglowski W, Jaworska M, Mucha-Malecka A, Wozniak G, Lange D, et al. Impact of HPV infection on the clinical outcome of p-CAIR trial in head and neck cancer. Eur Arch Otorhinolaryngol. 2011;268:721-6. 15. Poljak M, Ostrbenk A, Seme K, Ucakar V, Hillemanns P, Bokal EV, et al. Comparison of clinical and analytical performance of the Abbott Realtime High Risk HPV test to the performance of Hybrid Capture 2 in population-based cervical cancer screening. J Clin Microbiol. 2011;49:1721-9. 16. Carozzi FM, Burroni E, Bisanzi S, Puliti D, Confortini M, Giorgi Rossi P, et al. Comparison of clinical performance of Abbott RealTime High Risk HPV test with that of Hybrid Capture 2 assay in a screening setting. J Clin Microbiol. 2011;49:1446-51. 17. Halfon P, Benmoura D, Agostini A, Khiri H, Penaranda G, Martineau A, et al. Evaluation of the clinical performance of the Abbott RealTime High-Risk HPV for carcinogenic HPV detection. J Clin Virol. 2010;48:246-50. 18. Venturoli S, Leo E, Nocera M, Barbieri D, Cricca M, Costa S, et al. Comparison of Abbott RealTime High Risk HPV and Hybrid Capture 2 for the detection of high-risk HPV DNA in a referral population setting. J Clin Virol. 2012;53:121-4. 19. Jentschke M, Soergel P, Lange V, Kocjan B, Doerk T, Luyten A, et al. Evaluation of a new multiplex real-time polymerase chain reaction assay for the detection of human papillomavirus infections in a referral population. Int J Gynecol Cancer. 2012;22:1050-6. 20. Park Y, Lee E, Choi J, Jeong S, Kim HS. Comparison of the Abbott RealTime High-Risk Human Papillomavirus (HPV), Roche Cobas HPV, and Hybrid Capture 2 assays to direct sequencing and genotyping of HPV DNA. J Clin Microbiol. 2012;50:2359-65. 21. Hwang Y, Lee M. Comparison of the AdvanSure human papillomavirus screening real-time PCR, the Abbott RealTime High Risk human papillomavirus test, and the Hybrid Capture human papillomavirus DNA test for the detection of human papillomavirus. Ann Lab Med. 2012;32:201-5. 22. Tang N, Huang S, Erickson B, Mak WB, Salituro J, Robinson J, et al. High-risk HPV detection and concurrent HPV 16 and 18 typing with Abbott RealTime High Risk HPV test. J Clin Virol. 2009;45(Suppl i):S25-8. 23. Kovanda A, Juvan U, Sterbenc A, Kocjan BJ, Seme K, Jancar N, et al. Pre-vaccination distribution of human papillomavirus (HPV) genotypes in women with cervical intraepithelial neoplasia grade 3 (CIN 3) lesions in Slovenia. Acta Dermatovenerol Alp Panonica Adriat. 2009;18:47-52. 24. Huang S, Erickson B, Tang N, Mak WB, Salituro J, Robinson J, et al. Clinical performance of Abbott RealTime High Risk HPV test for detection of high-grade cervical intraepithelial neoplasia in women with abnormal cytology. J Clin Virol. 2009;45(Suppl i):Si9-23. 25. Cuzick J, Ambroisine L, Cadman L, Austin J, Ho L, Terry G, et al. Performance of the Abbott RealTime high-risk HPV test in women with abnormal cervical cytology smears. J Med Virol. 2010;82:1186-91. 26. Halfon P, Benmoura D, Agostini A, Khiri H, Penaranda G, Martineau A, et al. Stepwise algorithm combining HPV high-risk DNA-based assays and RNA-based assay for high grade CIN in women with abnormal smears referred to colposcopy. Cancer Biomark. 2010;7:133-9. 27. Wong OG, Lo CK, Szeto E, Cheung AN. Efficacy of Abbott RealTime High Risk HPV test in evaluation of atypical squamous cells of undetermined significance from an Asian screening population. J Clin Virol. 2011;51:136-8. 28. Szarewski A, Mesher D, Cadman L, Austin J, Ashdown-Barr L, Ho L, et al. Comparison of seven tests for high-grade cervical intraepithelial neoplasia in women with abnormal smears: the Predictors 2 study. J Clin Microbiol. 2012;50:1867-73. 29. Mesher D, Szarewski A, Cadman L, Austin J, Ashdown-Barr L, Ho L, et al. Comparison of human papillomavirus testing strategies for triage of women referred with low-grade cytological abnormalities. Eur J Cancer. 2013 (in press). 30. Hesselink AT, Meijer CJ, Poljak M, Berkhof J, van Kemenade FJ, van der Salm ML, et al. Clinical validation of the Abbott RealTime High Risk HPV assay according to the guidelines for HPV DNA test requirements for cervical screening. J Clin Microbiol. 2013 (in press). 31. Cuzick J, Cadman L, Mesher D, Austin J, Ashdown-Barr L, Ho L, et al. Comparing the performance of six human papillomavirus tests in a screening population. Br J Cancer. 2013;108:908-13. 32. Meijer CJ, Berkhof J, Castle PE, Hesselink AT, Franco EL, Ronco G, et al. Guidelines for human papillomavirus DNA test requirements for primary cervical cancer screening in women 30 years and older. Int J Cancer. 2009;124:516-20.