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cobas 4800 HPV Test, a real-time polymerase chain reaction assay for the detection of human papillomavirus in cervical specimens Expert Rev. Mol. Diagn. 14(1), 5–16 (2014)
Sandra D Isidean*1,2, Franc¸ois Coutle´e2,3 and Eduardo L Franco1,2 1 Department of Epidemiology, Biostatistics, and Occupational Health, McGill University, Montreal, QC, Canada 2 Department of Oncology, McGill University, Montreal, QC, Canada 3 De´partement de MicrobiologieInfectiologie, Hoˆpital Notre-Dame du Centre Hospitalier de l’Universite´ de Montre´al, Montre´al, QC, Canada *Author for correspondence: Tel.: +1 514 398 5519 Fax: +1 514 398 5002 [email protected]
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Cervical cancer screening incorporating high-risk human papillomavirus (HPV) detection has become the preferred screening strategy in some countries and is increasingly more widespread in other countries with organized or opportunistic screening programs. Given knowledge that high-risk HPV genotypes differ in their oncogenic potential, commercial HPV assays with genotyping capabilities have been developed and have garnered attention in the recent literature. The cobas 4800 HPV Test is a qualitative multiplex assay that provides specific genotyping information for HPV types 16 and 18, while concurrently detecting 12 other high-risk HPV genotypes as a pooled result. It is currently the only clinically validated, US FDA-approved assay with this capability. Since HPV types 16 and 18 have been designated as conferring the greatest risk for cervical disease, their detection may prove useful in guiding patient management. KEYWORDS: cervical cancer • cobas 4800 HPV Test • diagnostic test • genotyping • HPV testing • HPV DNA • human papillomavirus • PCR • screening
Although cervical cancer is a preventable disease, it remains the third most common cancer among women worldwide. With an estimated 530,000 new cases and 275,000 deaths annually, it continues to represent a major public health problem [1]. Persistent infection with human papillomavirus (HPV) causes over 99% of cervical cancer cases [2]. Presently, 170 HPV genotypes have been identified, with over 40 infecting the anogenital tract and other mucosal sites [3]. Nonetheless, less than half of these latter 40+ genotypes cause virtually all cervical carcinomas globally (TABLE 1) [4]. HPV types 16 and 18, in particular, have been designated as conferring the greatest risk for cervical disease, as they are associated with over two-thirds of all cervical cancers worldwide [5]. Cervical cancer screening using the Papanicolaou (Pap) cytology test has drastically reduced the incidence of and mortality from cervical 10.1586/14737159.2014.865521
cancer since its introduction in the mid-1900s. Organized and opportunistic screening programs have only been effective, however, among countries able to maintain high-quality and broad coverage screening [6]. Their success has also required the provision of diagnostic and treatment resources for managing cases of disease identified by screening. Accordingly, incidence rates for cervical cancer are lowest in industrialized regions such as Australia, North America and parts of Europe [1]. Given the tremendous infrastructure and personnel costs needed to maintain such programs, however, cervical disease still imparts a heavy burden in the developing world where roughly 85% of cases occur [1]. Consistent clinical evidence has shown cervical cytology to suffer from a high falsenegative rate for the detection of cervical precancerous and cancerous lesions, even in countries with stringent quality assurance systems
2014 Informa UK Ltd
ISSN 1473-7159
5
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´e & Franco Isidean, Coutle
HPV genotype
Percent of cancers caused (%)
Cumulative percent of cancers caused (%)
HPV16
54.4
54.4
HPV18
15.9
70.3
HPV33
4.3
74.6
HPV45
3.7
78.3
HPV31
3.6
81.9
HPV58
3.3
85.2
HPV52
2.5
87.7
HPV35
1.7
89.4
HPV59
1.0
90.4
HPV56
0.7
91.1
HPV51
0.7
91.8
infections will be cleared or suppressed by host immunity within 1–2 years of exposure, and only a small proportion of oncogenic infections may persist for several years and confer a higher risk for future disease [13]. HPV genotyping has been recognized as a potentially useful means for limiting possible harms resulting from over-screening women only transiently HPV-positive. Since it is known that HPV genotypes differ in their oncogenic potential [14], cervical samples of HPV-positive women with normal or equivocal cytology results could be genotyped to detect infections with the highest-risk HPV types (i.e., HPV16 and HPV18). In practice, women with particularly high disease risk could be referred for immediate colposcopy and follow-up, while those with lower disease risk could be followed prospectively and referred for colposcopy only if persistently infected with other oncogenic types. The cobas 4800 HPV Test (Roche Molecular Systems Inc., Pleasanton, CA, USA) is one such HPV test that provides specific genotyping results for HPV types 16 and 18, along with a pooled result for 12 other oncogenic types; it will be the focus of this diagnostic profile.
HPV39
0.6
92.4
Clinical applications & guidelines for HPV testing
HPV73
0.5
92.9
HPV68
0.5
93.4
HPV66
0.3
93.7
HPV70
0.2
93.9
HPV82
0.2
94.1
Not identified
5.9
100.0
Available evidence indicates that HPV testing may be useful in the triage and follow-up of women with equivocal cytology results, as a means of follow-up for women having been treated for cervical disease to assess treatment effectiveness, and as a primary cervical screening method (either alone or in conjunction with cervical cytology) to detect CIN2+ [7,15]. In the USA, recent screening guidelines (2012) have recommended HPV and cytology co-testing as the preferred cervical cancer screening method for women aged 30–65 years [16,17]. Also recommended as a management option for women testing HPV-positive while cytology-negative is HPV genotype-specific testing to detect HPV16 or HPV18, with women testing positive for either/both types being referred for immediate colposcopy. However, these guidelines cited insufficient evidence at the time to recommend HPV testing alone for primary screening in the USA considering the lack of a well-defined and evaluated management strategy for women with positive tests [16]. In Canada, cervical screening programs remain cytologybased. However, HPV testing has been widely used to triage women with a cytology result of atypical squamous cells of undetermined significance (ASC-US). The Canadian Task Force on Preventive Healthcare remains reluctant to recommend the use of HPV testing in national screening guidelines until evidence regarding this screening modality is further developed [18]. Nonetheless, the province of Ontario is expected to begin implementing HPV testing in its provincial screening program in the near-term as a primary screening method for women ‡30 years of age [19]. In Europe, cervical cytology continues to be the cornerstone of screening programs [20]. However, given how rapidly evidence surrounding HPV testing has developed since publication of the second edition of the European Guidelines for Quality Assurance in Cervical Cancer Screening, perspectives regarding the incorporation of HPV
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Table 1. Percentage of invasive cervical cancers attributable to specific HPV genotypes.
Data taken from [4].
in place [7,8]. The low sensitivity of cytology stems from its dependence on optimally collected cervical samples, and the subjective interpretation of cellular morphology in these samples by cytotechnologists. Even among experienced cytotechnologists reviewing adequate cervical samples, nonetheless, there is high inter-observer variability that reduces the diagnostic accuracy of cytology-based screening [9]. Owing to the limitations of cytologic screening and to the understanding of cervical cancer etiology, biotechnology companies have developed molecular assays that detect HPV nucleic acid (DNA or mRNA) for use in diagnostic settings. Over the past 15 years, these molecular assays have consistently demonstrated greater reliability and substantially superior sensitivity for detecting cervical intraepithelial neoplasia grade 2 or worse (CIN2+) than Pap cytology in clinical studies [7,10]. The downside to this improved sensitivity, however, is reduced specificity. Indeed, this raises concern since HPV infections are the most common sexually transmitted infections worldwide, and most sexually active women will acquire an infection at least once in their lifetime [11]. According to a meta-analysis including data from more than 1 million women in 59 countries, the prevalence of genital HPV infection among those with normal cytology ranges from 1.6 to 41.9% [12]. Yet, most (~90%) of these 6
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cobas 4800 HPV Test
testing into screening programs are evolving [7,20]. Consequently, a review and updating of current European guidelines has already been initiated [20]. The Netherlands is the first country soon planning to adopt HPV testing alone into its national screening program for women 30–60 years of age [21]. Nonetheless, primary HPV testing has been introduced as part of pilot programs in countries such as Italy, the UK and Sweden [21,22]. Currently in Europe, HPV testing has been sanctioned for the triage and follow-up of women with equivocal cytology results, and as a test of cure in women following treatment for cervical disease. Exactly how widespread HPV screening is in these countries is somewhat unclear, however. Diagnostic tests for HPV detection
Due to the inability to culture HPV in a laboratory, almost all available methods for detecting the virus involve detection of its nucleic acids in clinical specimens [23]. Over 100 distinct tests for the detection of oncogenic HPV DNA or RNA (mostly DNA) are commercially available [24]. A handful of these tests have been approved for use by the US FDA and Health Canada, and a number more have received CE marking, which allows for broad marketability in the European Economic Area (TABLE 2). It should be noted that the list provided in TABLE 2 is not intended to be comprehensive, but showcases several tests commonly used for HPV detection – along with their main characteristics. For a comprehensive inventory of commercial tests available on the worldwide market, we refer to several recent reviews [24,25]. HPV molecular diagnostic techniques are often broadly categorized by the methodology they employ, namely signal amplification or target amplification. The former is based on an initial hybridization step of nucleic acids in a clinical specimen with target-specific probes, followed by amplification and visualization of this hybridization event [23]. By contrast, target amplification methods duplicate fragments of DNA or RNA from a targeted gene sequence to a level that can be detected using available systems. PCR techniques are based on this latter methodology. The Hybrid Capture 2 (HC2) High-Risk HPV DNA Test (Qiagen, Gaithersburg, MD, USA [previously Digene Corp.]) – which utilizes signal amplification – has been established as the worldwide standard technique for HPV detection, and remains the most evaluated and widely used molecular HPV test on the market. This assay uses a cocktail of reagents to detect any of 13 oncogenic HPV types that may be present either alone or as a co-infection, without distinguishing among the individual genotypes in the sample. In North America and Europe, it has been approved for triage of women with ASC-US cytology to assess the need for referral to colposcopy, and as an adjunct screening test to cytological screening in women ‡30 years of age. Recent guidelines published by Meijer et al. [26] have recommended that new HPV tests aimed for cervical screening purposes demonstrate performance characteristics deemed noninferior to those of the HC2 test (or the EIA kit HPV GP HR Assay [Diassay, Rijswijk, The Netherlands]) prior to use rather www.expert-reviews.com
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than undergo extensive longitudinal validation studies. More specifically, the guidelines state that: the sensitivity and specificity of the candidate test for detecting CIN2+ should be at least 90 and 98%, respectively, of those parameters for the HC2 test; the candidate test should demonstrate intralaboratory reproducibility and inter-laboratory agreement, with a lower confidence bound not less than 87% and samples being evaluated should arise from a population-based screening cohort. According to a recent meta-analysis regarding HPV testing in cervical screening by Arbyn et al. [7], the cobas 4800 HPV Test, RealTime High-Risk HPV PCR assay (Abbott Molecular Inc., Des Plaines, IL, USA) and PapilloCheck HPV-Screening Test (Greiner Bio-One, Frickenhausen, Germany) have all been clinically evaluated for use in primary screening using these criteria. While all three tests fulfilled the sensitivity and specificity criteria, only the first two tests met the reproducibility criteria. The Cervista HPV HR Test (Hologic Inc., Madison, WI, USA [previously Third Wave Technologies]) and APTIMA HPV Assay (Hologic Gen-Probe Inc., San Diego, CA, USA) have not yet been validated for primary screening according to these test requirements, but have received licensing by the FDA, Health Canada, and among countries accepting a CE mark for: triage of women with ASC-US cytology and as an adjunct screening test to cytological screening in women ‡30 years of age. Similarly to the HC2 test, the Cervista HPV HR Test, EIA kit HPV GP HR Assay, and AMPLICOR HPV Test (Roche Molecular Systems Inc.) all detect the presence of high-risk HPV types in aggregate (i.e., without providing information about specific genotypes present in a sample). The APTIMA HPV Assay is also based on the cumulative detection of highrisk HPV types, but targets viral mRNA rather than DNA. It remains the only FDA-approved mRNA-based HPV test available. The PreTect HPV-Proofer Test (NorChip, Klokkarstua, Norway) and NucliSENS EasyQ HPV Test (Biome´rieux, Marcy I’Etoile, France) are also mRNA-based assays; however, they detect transcripts for the five most frequently identified high-risk HPV types in cervical cancers worldwide (HPV types 16, 18, 31, 33 and 45). Tests listed in TABLE 2 providing full genotyping results include the Linear Array HPV Genotyping Test (Roche Molecular Systems Inc.), the INNO-LiPA HPV Genotyping Extra (Innogenetics NV, Gent, Belgium) and PapilloCheck HPV-Screening Test. Although full genotyping assays have been (and remain) a vital component in research settings, their clinical value in population-based cervical screening remains undetermined [27,28]. On the contrary, given compelling evidence demonstrating the exceptional oncogenic potential of HPV types 16 and 18 [4,14], HPV tests with partial concurrent or reflex genotyping capabilities have been developed and include the cobas 4800 HPV Test, the Cervista HPV 16/18 Test (Hologic Inc.), and the RealTime High-Risk HPV PCR assay. In all three assays, individual genotyping results are provided for HPV types 16 and 18 only – explaining why they are termed ‘partial’ genotyping assays. The cobas 4800 HPV Test is the only clinically validated and FDA-approved test that simultaneously provides pooled 7
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Table 2. Summary of common commercially available nucleic acid tests for the detection of HPV in cervical samples of women (not comprehensive). Test
Molecular target
Test methodology
Oncogenic HPV genotypes detected†
Time to results‡
Interpretation of results
Regulatory status
Indications for use§
Hybrid Capture 2 High-Risk HPV DNA Test
HPV DNA
Signal amplification
16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68
Process 352 samples in 8 h
Automated
FDA-approved, CE-marked, HC-approved
ASC-US triage, cervical screening, test of cure
Cervista HPV HR Test
HPV DNA
Signal amplification
16, 35, 52, 66,
Process 96 samples in 6-7 h
Automated
FDA-approved, CE-marked, HC-approved
ASC-US triage, cervical screening, test of cure
Cervista HPV 16/18 Test
HPV DNA
Signal amplification
16, 18
Process 96 samples in 6-7 h
Automated
FDA-approved, CE-marked, HC-approved
ASC-US triage, alongside/ follow-up to Cervista HPV HR Test
cobas 4800 HPV Test
HPV DNA
Target amplification
16, 35, 52, 66,
18, 31, 33, 39, 45, 51, 56, 58, 59, 68
Process 96 samples in 4.5 h
Automated
FDA-approved, CE-marked, HC-approved
ASC-US triage, cervical screening, test of cure
APTIMA HPV Assay
HPV mRNA
Target amplification
16, 35, 52, 66,
18, 31, 33, 39, 45, 51, 56, 58, 59, 68
Process 500 samples in 8.5 h
Automated
FDA-approved, CE-marked
ASC-US triage, cervical screening, test of cure
EIA kit HPV GP HR Assay
HPV DNA
Target amplification
16, 35, 52, 66,
18, 31, 33, 39, 45, 51, 56, 58, 59, 68
Process 96 samples in 9.5 h
Not Automated
CE-marked
ASC-US triage, cervical screening, test of cure
AMPLICOR HPV Test
HPV DNA
Target amplification
16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68
Process 96 samples in 2 working days
Not Automated
CE-marked, HC-approved
ASC-US triage, cervical screening, test of cure
RealTime HighRisk HPV Assay
HPV DNA
Target amplification
16, 35, 52, 66,
18, 31, 33, 39, 45, 51, 56, 58, 59, 68
Process 96 samples in 6 h
Automated
CE-marked, HC-approved
ASC-US triage, cervical screening, test of cure
LINEAR ARRAY HPV Genotyping Test
HPV DNA
Target amplification
16, 33, 51, 58, 68, 82
18, 35, 52, 59, 69,
Process 48 samples in 8 h
Not Automated
CE-marked, HC-approved
HPV genotyping
18, 31, 33, 39, 45, 51, 56, 58, 59, 68
26, 39, 53, 66, 70,
31, 45, 56, 67, 73,
† HPV type 68 is considered a probable carcinogen, and HPV types 26, 53, 66, 67, 69, 70, 73, and 82 are considered possible carcinogens by the International Agency for Research on Cancer (IARC) [61]. ‡ The respective tests vary in their level of automation. Times outlined may not incorporate manual procedure times or PCR amplification times for the various tests. Data shown are from the respective manufacturers. § FDA-approved tests for use in the USA are only indicated for the triage and follow-up of women with ASC-US cytology, and as an adjunct screening test to cytological screening in women ‡30 years of age. Primary, stand-alone screening with these tests is not yet indicated in the USA. In other countries using HPV testing in cervical screening, indications for use may include: triage and follow-up of women with ASC-US cytology; primary screening and follow-up of women after treatment of disease (test of cure).
8
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cobas 4800 HPV Test
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Table 2. Summary of common commercially available nucleic acid tests for the detection of HPV in cervical samples of women (not comprehensive) (cont.). Test
Molecular target
Test methodology
Oncogenic HPV genotypes detected†
Time to results‡
Interpretation of results
Regulatory status
Indications for use§
INNO-LiPA HPV Genotyping Extra
HPV DNA
Target amplification
16, 33, 51, 58, 69,
18, 35, 52, 59, 70,
26, 39, 53, 66, 73,
31, 45, 56, 68, 82
Process 48 samples in 3 h
Automated
CE-marked
HPV genotyping
PapilloCheck HPV-Screening Test
HPV DNA
Target amplification
16, 35, 52, 59, 73,
18, 39, 53, 66, 82
31, 45, 56, 68,
33, 51, 58, 70,
Process 12 samples in 3 h
Automated
CE-marked
ASC-US triage, cervical screening, test of cure
PreTect HPVProofer Test
HPV mRNA
Target amplification
16, 18, 31, 33, 45
Process 30 samples in 2.5 h
Automated
CE-marked
ASC-US triage, cervical screening, test of cure
NucliSENS EasyQ HPV Test
HPV mRNA
Target amplification
16, 18, 31, 33, 45
Process 30 samples in 6.5 h
Automated
CE-marked
ASC-US triage, cervical screening, test of cure
† HPV type 68 is considered a probable carcinogen, and HPV types 26, 53, 66, 67, 69, 70, 73, and 82 are considered possible carcinogens by the International Agency for Research on Cancer (IARC) [61]. ‡ The respective tests vary in their level of automation. Times outlined may not incorporate manual procedure times or PCR amplification times for the various tests. Data shown are from the respective manufacturers. § FDA-approved tests for use in the USA are only indicated for the triage and follow-up of women with ASC-US cytology, and as an adjunct screening test to cytological screening in women ‡30 years of age. Primary, stand-alone screening with these tests is not yet indicated in the USA. In other countries using HPV testing in cervical screening, indications for use may include: triage and follow-up of women with ASC-US cytology; primary screening and follow-up of women after treatment of disease (test of cure).
results for 12 high-risk HPV types, as well as individual results for HPV types 16 and 18 (respectively). The remainder of this article will focus on the specific characteristics of this assay, and will describe its clinical profile through an overview of results from clinical validation studies. cobas 4800 HPV Test: indications for use and assay methodology
The cobas 4800 HPV Test is an in vitro qualitative real-time PCR assay for the detection of high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) in cervical specimens of women undergoing screening. It is performed using the cobas 4800 System, and has been indicated by the US FDA, Health Canada and in countries accepting a CE mark for use as follows: to triage women aged 21 years and older with ASC-US cytology to determine the need for colposcopy referral; to screen women aged 21 years and older with ASC-US cytology to assess the presence or absence of HPV types 16 and 18; to screen women ‡30 years of age adjunctively with cytological screening; to screen women ‡30 years of age to assess the presence or absence of HPV types 16 and 18. In November 2012, Roche Molecular Systems Inc., announced that the test received an expanded indication for use as a primary screening test in countries accepting a CE mark (i.e., cytology screening is no longer required as a pre-test or co-test). www.expert-reviews.com
As indicated in the full package insert by the manufacturer, the cobas 4800 HPV Test has been validated for use with cervical specimens collected in cobas PCR Cell Collection Media (Roche Molecular Systems Inc.), PreservCyt Solution (Hologic Inc.) and SurePath Preservative Fluid (BD DiagnosticsTriPath, Burlington, NC, USA). However, the FDA has only approved use of the test with cervical specimens collected in PreservCyt Solution. While several recent studies have evaluated the use of the cobas 4800 HPV Test with SurePath Preservative Fluid, it is likely that additional evidence will be needed prior to an expanded approval by the FDA [29,30]. Once collected, fully automated specimen preparation for the cobas 4800 HPV Test may be accomplished using the cobas x 480 Instrument – one of two separate technologies in the cobas 4800 System (FIGURE 1). This instrument serves to simultaneously extract, purify and prepare target HPV DNA and b-globin DNA for PCR amplification and detection. The processing of b-globin DNA functions as a control to differentiate HPVnegative specimens from those that fail to exhibit positivity due to a lack of cells or the presence of PCR inhibitors in specimens being tested. With the cobas x 480 Instrument, cervical specimens collected in one of the various media outlined above are processed through cell lysis using a chaotropic reagent. HPV and b-globin DNA released from this process are then purified 9
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A
B
Figure 1. The cobas 4800 System featuring its two component technologies. The cobas x 480 Instrument (A) serves to simultaneously extract, purify, and prepare target HPV DNA and b-globin DNA in collected cervical samples for PCR amplification and detection. Sample preparation using this instrument is fully automated. The cobas z 480 Analyzer (B) serves to then amplify and detect viral nucleic acid in the prepared samples using real-time PCR technology. Manual processing during this procedure is limited to the transfer of a microwell plate containing PCR-ready samples from the cobas x 480 Instrument to the cobas z 480 Analyzer. Technologies are not depicted to scale. Reproduced with permission from Roche Molecular Systems, Inc., Roche Molecular Systems, Inc. (2013).
using magnetic glass particles chemistry, washed and ultimately separated from these particles. Manual processing during this procedure is limited to the transfer of a microwell plate containing PCR-ready samples from the cobas x 480 Instrument to the cobas z 480 Analyzer – the second instrument in the cobas 4800 System (FIGURE 1). This instrument amplifies and detects viral nucleic acid in the prepared samples. A pool of HPV- and b-globin-specific primers present in the Master Mix reagent for the cobas 4800 HPV Test is designed to concurrently amplify HPV DNA from the 14 targeted HPV types and b-globin DNA. This reagent also includes AmpErase (uracil-N-glycosylase) enzyme and deoxyuridine triphosphate (dUTP), allowing for the destruction of contaminating amplicons generated from previous PCR runs. HPV-specific primers target nucleotides within the L1 region of the HPV genome, which encodes the major capsid protein of the virus. Detection of amplified DNA is accomplished using oligonucleotide probes labeled with four fluorescent dyes. Each dye is subsequently visualized according to its characteristic wavelength across one of four detection channels of the cobas z 480 Analyzer. Channel 1 detects the chemiluminescent signal for the 12 non-HPV16/18 genotypes (i.e., 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68), while channels 2, 3 and 4 detect signals for HPV16, HPV18, and b-globin targets (respectively) (FIGURE 2). The cobas 4800 System Software performs all assay and run validations, and automatically displays reportable cobas 4800 HPV Test results as positive, negative or invalid. The software allows the user to select one of two different testing options for samples: the ‘HPV High-Risk Panel’, which provides pooled testing for the 14 targeted high-risk types or the ‘HPV High-Risk Panel Plus Genotyping’, which provides pooled testing for the non-HPV16/18 types and genotype10
specific testing for HPV types 16 and 18 (respectively). Each run of the cobas 4800 System requires one replicate each of a positive and negative control, and valid results for these controls must be obtained for the system software to display testing results. The system is designed to support up to 94 cervical specimens per run (plus the two controls). Notably, a single run can support samples contained in any combination of media described, and can provide results for either of the two testing options listed above for each sample. Up to 384 HPV tests may be processed by the system in a given day, with the first 94 results obtained in 4.5 h. Analytical sensitivity & specificity
As described by Rao et al., analytical studies of the cobas 4800 HPV Test conducted by the manufacturer utilized cut-off values (or cycle thresholds) distinguishing positive from negative samples based on the detection of histologically confirmed CIN2+, so as to optimize clinical sensitivity and specificity (i.e., clinical utility) [31]. Limit of detection (LOD) studies for HPV genotypes 16, 18 and 31 were carried out to assess analytical sensitivity of the test. LOD levels for these studies were defined as the lowest concentration of HPV DNA in a sample resulting in a positive test result at least 95% of the time. Genotypes 16 and 18 were both detected with a 100% positivity rate at 600 copies/ml using PreservCyt Solution, while HPV31 was detected 97% of the time at 300 copies/ml. Using SurePath Preservative Fluid, HPV16 was detected with a 100% positivity rate at 300 copies/ml, and HPV31 was detected with a 95% positivity rate at 150 copies/ml; the LOD level for HPV18 remained the same. To assess analytical specificity, over 100 species of bacteria, fungi, and viruses – to include low-risk HPV genotypes and Expert Rev. Mol. Diagn. 14(1), (2014)
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cobas® 4800 HPV Test: examples of test results HPV HR panel
HPV HR panel plus genotyping
(-)
HR HPV
(-)
HPV16
(-)
HPV18
(-)
HR HPV
(+)
HPV16
(-)
HPV18
(-)
HR HPV
(-)
HPV16
(+)
HPV18
(-)
HR HPV
(-)
HPV16
(-)
HPV18
(+)
HR HPV
(+)
HPV16
(+)
HPV18
(+)
(+) cobas® 4800 HPV Test: assay design (+) 12 HR HPVs
HPV 16
HPV 18
Host DNA control (+)
(+) Channel 1
Channel 2
Channel 3
Channel 4
Figure 2. The cobas 4800 HPV Test: assay design and examples of test results. Assay design (bottom): Channel 1 detects the chemiluminescent signal for the 12 non-HPV16/18 genotypes tested for by the cobas 4800 HPV Test (HPV types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68). Channels 2, 3 and 4 detect signals for HPV16, HPV18 and b-globin targets (respectively). Examples of test results (right): The HPV High-Risk (HR) Panel tests for the presence of all 14 HR HPV types targeted by the assay as a pool. Results for this panel are reported as positive, negative or invalid. The HPV HR Panel Plus Genotyping provides pooled testing for the 12 non-HPV16/ 18 types, and genotype-specific testing for HPV types 16 and 18 (respectively). Test results shown do not represent the total combination of possible results. Adapted with permission from Roche Molecular Systems, Inc., Roche Molecular Systems, Inc. (2013).
other viruses commonly infecting the genitourinary tract – were spiked at high concentrations into two types of specimen: HPV-negative PreservCyt Solution specimen and HPV-negative PreservCyt Solution specimen spiked with HPV types 16, 18 and 31. None of these organisms were found to interfere with the detection of these types, or produce a false-positive result in the HPV-negative specimen. Clinical performance of the cobas 4800 HPV Test
In 2009, Castle et al. published the first report evaluating the prototype of the cobas 4800 HPV Test [32]. Using a convenience sample of 531 cervical specimens, the prototype was compared with the Linear Array HPV Genotyping Test. Percentage of agreement between the two assays was 94.7% (95% CI: 92.5–96.5), and the kappa value was 0.92 (95% CI: 0.89–0.95). In that study, more women without evident cervical abnormalities on histopathology tested positive by the Linear Array HPV Genotyping Test than by the cobas 4800 HPV Test for non-HPV16/18 high-risk types. A conservative clinical threshold of 45 was utilized to determine positivity, as a validated threshold was not available at the time of the study.
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ATHENA HPV Study
The cobas 4800 HPV Test was clinically validated in the ATHENA (Addressing THE Need for Advanced HPV Diagnostics) Study, the largest clinical trial to examine the clinical utility of HPV DNA testing and HPV16/18 genotyping among women attending routine cervical screening in the USA. The study enrolled over 47,000 women ‡21 years of age at one of 61 clinical sites across the country. The test was evaluated in three relevant populations: women aged ‡21 years with ASCUS cytology for the intended use of ASC-US triage to colposcopy; women aged ‡30 years with normal cytology for the intended use as an adjunctive test with cytology and women aged ‡25 years with any cytology result to assess performance of HPV testing with genotyping as a primary screening test [33]. The first evaluation compared performance indices for the cobas 4800 HPV Test with those of the HC2 test for detection of CIN2+ and CIN3+ among 1578 women ‡21 years of age with: valid cytology results demonstrating ASC-US; valid results for HPV testing and valid colposcopic assessments [34]. Performance characteristics of the tests were found to be very similar for all parameters. Sensitivity estimates for the cobas 4800 HPV Test were 90.0% (95% CI: 81.5–94.8) and 93.5% 11
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´e & Franco Isidean, Coutle
(95% CI: 82.5–97.8) for CIN2+ and CIN3+ detection (respectively). These estimates were 87.2% (95% CI: 78.0–92.9) and 91.3% (95% CI: 79.7–96.6) for the HC2 test, respectively, demonstrating the cobas 4800 HPV Test to meet the standard outlined by Meijer et al. for new HPV DNA tests [26]. Specificity of the cobas 4800 HPV Test was 70.5% (95% CI: 68.1–72.7) for CIN2+ and 69.3% (95% CI: 66.9–71.5) for CIN3+, while estimates for HC2 were respectively 71.1% (95% CI: 68.8–73.4) and 70.0% (95% CI: 67.7–72.3). Furthermore, concordance between the assays was high in women with a CIN2+ diagnosis (96.2%; 95% CI: 89.3–98.7). These findings were deemed to clinically validate the use of the cobas 4800 HPV Test for ASC-US triage in women ‡21 years of age. This study also demonstrated how genotyping for HPV16/ 18 identifies women at greatest absolute and relative risk for CIN2+ and CIN3+ [34]. When the cobas 4800 HPV Test was evaluated in women aged ‡30 years with normal cytology, investigators similarly found that both absolute and relative risks for CIN2+ and CIN3 + were not only impacted by HPV status for the 14 targeted types, but were even more influenced by a woman’s HPV16/ 18 status [35]. The relative risk for CIN2+ was 13.7-fold higher (95% CI: 7.3–41.9) with an HPV16- and/or HPV18-positive result on the cobas 4800 HPV Test than with a negative test result, and 35.0-fold higher (95% CI: 13.0–552.8) for CIN3+. Moreover, a positive result for HPV16 and/or HPV18 had a relative risk for CIN2+ that was 2.51-fold higher (95% CI: 1.7–3.6) than a positive result for the 12 remaining high-risk types. Finally, women who were HPV16-positive with normal cytology had an absolute risk of 13.6% (95% CI: 9.5–18.0) for CIN2+; thus, nearly 1 in 7 women testing HPV16-positive had highgrade disease that was missed by cytology [35]. Castle et al. went on to show that in women aged ‡25 years, independent of cytology result, the cobas 4800 HPV Test was more sensitive than liquid-based cytology for the detection of CIN3+ (92.0%; 95% CI: 88.1–94.6 vs 53.3%; 95% CI: 47.4–59.1) [33]. While the addition of cytology to HPV testing increased this sensitivity to 96.7% (95% CI: 93.9–98.3), it also increased the number of positive screening results by 35.2% compared with HPV testing alone. Among HPV-positive women, detection of HPV16 and/or HPV18 alone was equivalent to detection of ASC-US or worse alone in terms of sensitivity and PPV to identify CIN3+. Better sensitivity and similar PPV for CIN3+ was observed via detection of HPV16 and/or HPV18, or low-grade squamous intraepithelial lesions (SIL) or worse cytology than ASC-US or worse cytology alone. This was also shown to be the case when detecting HPV16 and/or HPV18, or high-grade SIL or worse cytology [33]. Final data from the ATHENA Study presented at the Society of Gynecologic Oncology’s 44th Annual Meeting on Women’s Cancer (Los Angeles, CA, USA, 2013) revealed that – among cytology-negative women – HPV testing at enrollment resulted in earlier detection of prevalent CIN3+ disease initially missed by cytology [36]. This allowed for treatment of disease that could have otherwise been delayed or not given due to loss of women 12
to follow-up. HPV16, followed by HPV18, continued to confer the greatest risk for CIN3+. Other comparative studies of the cobas 4800 HPV Test
A study by Heideman et al. showed the cobas 4800 HPV Test to fulfill all criteria by Meijer et al. in their international guidelines for HPV test requirements when assessed relative to the HC2 test [26,37]. Using data from the VUSA-Screen trial in the Netherlands, agreement between the assays was 97.3% (95% CI: 95.9–98.2) for controls and 98.3% (95% CI: 89.1–99.8) for cases. Kappa values for these groups were 0.74 and 0.90, respectively. More recently, Lloveras et al. similarly demonstrated the fulfillment of these criteria by the cobas 4800 HPV Test when compared with HC2 in opportunistic screening in Spain [38]. A third study equated these tests using cervical samples from women enrolled in the HPV PaP Cohort study in the USA [39]. The cobas 4800 HPV Test and Linear Array HPV Genotyping Test were also compared for specific detection of HPV16/18. In 1824 specimens with valid results, the cobas 4800 HPV Test had 85.9% agreement with HC2 and 91.0% agreement with the Linear Array HPV Genotyping Test (kappa values: 0.69 [95% CI: 0.66–0.73] and 0.82 [95% CI: 0.79–0.84], respectively) [39]. The cobas 4800 HPV Test was found more likely to test positive compared with HC2, but the latter test was more likely to cross-react with certain low-risk HPV genotypes. Several other studies also highlighted this cross-reactivity by HC2, despite reporting good to excellent agreement between HC2 and the cobas 4800 HPV Test according to the kappa statistic [40–42]. In a Canadian study using 466 samples from women aged 15 to >60 years, overall agreement between HC2 and the cobas 4800 HPV Test was 93.8% (kappa: 0.87; 95% CI: 0.83–0.92) [43]. In women aged ‡30 years, 92.3% agreement was observed. Another Canadian study by our team compared the performance of the two assays among 396 women aged 24–75 years who were referred to colposcopy following an ASC-US cytology result [44]. Clinical sensitivities and specificities for detecting CIN2+ were respectively 89.7% (95% CI: 72.8–97.2) and 66.7% (95% CI: 61.7–71.3) for the cobas 4800 HPV Test, and 93.1% (95% CI: 77.0–99.2) and 72.2% (95% CI: 67.4–76.5) for HC2. Percent agreement between the tests was 87.9% (95% CI: 84.3–90.8), with a kappa value of 0.74 (95% CI: 0.64–0.83). Agreement between the cobas 4800 HPV Test and Linear Array HPV Genotyping Test to detect the 14 HPV types targeted by the former assay was also reported as 93.2% (95% CI: 90.2–95.3), with a kappa value of 0.86 (95% CI: 0.76–0.96). Several other studies comparing the cobas 4800 HPV Test with other HPV tests, specifically the AMPLICOR HPV Test or RealTime High-Risk HPV PCR assay, have also found good to excellent concordance between the methods [45,46]. In a study population of 1099 women referred for colposcopy in the UK, Szarewski et al. compared the performance of seven different HPV tests – to include the cobas 4800 HPV Test – for identification of CIN2+ and CIN3+ [47]. Cuzick et al. went on to Expert Rev. Mol. Diagn. 14(1), (2014)
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compare the performance of six of these tests in a screening population of 6000 women in the UK [48]. In both studies, the cobas 4800 HPV Test demonstrated high sensitivity to detect highgrade cervical disease, with specificity parameters in line with or greater than those for HC2.
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Expert commentary
Given what is known about the etiology of cervical cancer, and the performance of HPV testing technologies relative to cytological testing, wider utilization of HPV detection methods in cervical screening programs seems inevitable. How to best manage women testing HPV-positive with normal or equivocal cytology results, however, has been a practical concern related to this utilization. Indeed, among high-risk HPV-positive women, only a minority will be found to have meaningful disease. This raises questions regarding how to optimally identify these women needing immediate follow-up. In recent years, HPV genotype-specific detection has been evaluated for its clinical potential to improve screening accuracy and efficiency, and has been shown to identify women at highest risk for cervical disease in numerous studies [14,33–35,49–52]. As a result, novel HPV tests with concurrent or reflex genotyping capabilities to detect high-risk types – and distinguish those types with exceptionally high oncogenic potential (i.e., HPV16/ 18) – have been designed and approved for use in various countries. The cobas 4800 HPV Test is currently the only US FDA-approved test of this kind that provides pooled results for 12 high-risk HPV types concurrently with individual genotyping results for HPV types 16 and 18. As reviewed in this article, the cobas 4800 HPV Test has demonstrated favorable clinical performance in a large, prospective trial (the ATHENA Study), as well as in numerous comparative studies. It has also been evaluated according to the non-inferiority criteria outlined by Meijer et al. for new HPV tests intended for primary screening [26,37,38]. The importance of validating novel HPV tests according to pre-specified validation strategies for sensitivity, specificity and reliability has been strongly emphasized in the recent literature [7,26,27]. Due to deficient health technology regulation in certain parts of the world, however, HPV assays yet to be fully validated are being employed [25]. Alas, lack of proper clinical validation could render a commercial assay more harmful than beneficial. Despite the higher cost of HPV testing relative to cytologic testing for a single round of screening, strategies incorporating HPV testing have been shown to be cost effective over a variety of settings [53–57]. This can be attributed to increases in diagnostic lead-time provided by HPV testing as compared with cytologic testing, which subsequently translates into lower risk following a negative test. As a result, longer screening intervals become permissible. Attainment of such advantages would require a well-organized screening program with optimal compliance, as excessive and unnecessary screening and treatment of women testing HPV-positive – who may only be transiently infected – would obviously render any described advantages extinguished. www.expert-reviews.com
Diagnostic Profile
Five-year view
How will cervical cancer screening be practiced in the future? The answer to this question requires consideration of regionspecific needs. The USA, a country highly risk-averse with respect to medical practice, has recently favored HPV and cytology co-testing – a combination that permits maximal sensitivity for detecting cervical cancer precursors [16,17]. As this review indicates, the main contributor to this high sensitivity is the HPV assay rather than Pap cytology. Nonetheless, because of its superior specificity, the latter is more appropriate as a triage option for women who are HPV-positive. As of this writing, a number of countries have begun to implement screening based on this premise (i.e., primary HPV testing followed by Pap triage in series, not in parallel). Although rapidly gaining in favor, this strategy continues to be formally evaluated in two randomized controlled trials [58,59]. This screening approach will be most attractive to countries with central oversight of the cost–effectiveness of their healthcare systems. Nevertheless, it is not altogether implausible that even the USA may eventually come to revise its guidelines to allow the above serial screening approach to be permitted alongside parallel co-testing. Lowresource countries may adopt simpler screening methodologies, such as visual inspection, with or without a pre-test based on low-cost HPV assays (e.g., the careHPV Test [Qiagen]). A key consideration in the planning of cervical cancer screening should be whether or not a country has succeeded in implementing a public HPV vaccination program of high coverage for pre-adolescent and adolescent girls, with expansion to other young women via catch-up vaccination. As successive cohorts of vaccinated women reach screening age, there will be a gradual decrease in cervical lesion prevalence and – consequently – the positive predictive value of any screening test. Because of its dependence on human interpretation of cytologic abnormalities, Pap cytology is likely to be more affected than molecular HPV assays that are not subjectively interpreted [60]. By screening primarily with a clinically validated HPV test, subsequent triage via cytology will provide cytotechnologists with an enriched caseload of lesions – thereby increasing the positive predictive value of cytologic testing. Furthermore, restricting cytologic assessments to HPV-positive women will reduce the cytology reading workload by more than 10-fold. No country has yet adopted different screening policies for vaccinated and unvaccinated women. For pragmatic and ethical reasons, decisions regarding the optimal age to begin screening, the ideal screening frequency and the most appropriate screening technology cannot be arrived at via randomized controlled trials. Policy decisions stemming from in-depth health technology assessments and cost–effectiveness considerations will prevail. Having a reasonably well-organized screening program and surveillance infrastructure (e.g., vaccination registries) will be necessary for enforcing different policies. Political courage will likely be essential for early adopters, while the role of professional guidelines and the endorsement of national and supranational agencies will be paramount in bringing confidence to providers and the public. 13
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Diagnostic Profile
´e & Franco Isidean, Coutle
Cytology is not the only screening methodology that may be clinically useful as a triage test for women who are HPVpositive. HPV16 or HPV16/18 genotyping, as provided by the cobas 4800 HPV Test, could serve such a role. Indeed, recent guidelines already allow for the use of HPV16/18 genotyping in this capacity [16]. New cytologic/histologic biomarkers, to include p16, Ki-67, MCM2 and MCM7 are also attractive options for an enhanced Pap triage step [28]. However, uncertainty remains as to whether the gains in screening and risk prediction that come from adopting these technologies will be worth the costs and extra labor that they incur. With so many technologies being readied for deployment or already adopted, the horizon for cervical cancer prevention has never been more exciting and dynamic. Primary prevention (via HPV vaccination) and secondary prevention (via screening with molecular HPV tests and other promising technologies) have provided a tremendous potential for reduction in cervical cancer incidence and mortality; no other neoplastic disease can currently rival the magnitude of this potential. Prudent and synergistic use of these prevention fronts will likely be the
hallmark of success stories in the future. Uncoordinated adoption of these technologies in opportunistic and non-universal conditions will only heighten the inequality that has characterized cervical cancer control for so long. Financial & competing interests disclosure
F Coutle´e has received grants through his institution from Merck Sharp & Dohme and Roche Diagnostics, as well as honoraria from Merck and Roche for lectures. EL Franco has served as occasional consultant or advisory board member to Merck and GSK on HPV vaccines, to Qiagen, Roche, GenProbe and Becton & Dickinson on HPV diagnostics, to Cytyc and Ikonisys on cytology and to Innovus and Scimetrika on health economic modeling. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties. No writing assistance was utilized in the production of this manuscript.
Key issues • Persistent infection with high-risk human papillomaviruses (HPVs) causes over 99% of cervical cancers worldwide. • HPV testing has demonstrated greater reliability and sensitivity for detecting cervical disease than cytology-based testing, and has been recommended for use in cervical cancer screening in several industrialized countries. • Over 100 distinct tests for the detection of oncogenic HPVs are now commercially available. • The introduction of HPV testing raises concerns regarding how best to manage women who are HPV-positive while cytologynegative. • Given the exceptional oncogenic potential of HPV types 16 and 18, HPV tests with partial concurrent or reflex genotyping capabilities to detect these types have been developed as a means to further stratify HPV-positive women according to risk, and avoid over-screening and over-treatment. • The cobas 4800 HPV Test is one such HPV test that provides specific genotyping results for HPV types 16 and 18, along with a pooled result for 12 other oncogenic types. • The cobas 4800 HPV Test is an in vitro qualitative real-time PCR assay that is performed using the cobas 4800 System, which includes fully automated specimen preparation, amplification and detection of viral nucleic acid, and automated reportable test results. • The test has demonstrated favorable clinical performance in a large, prospective trial (the ATHENA Study), as well as in numerous comparative studies. It is the first test of its kind to be US FDA-approved. • The benefits of incorporating HPV testing and genotyping into practice will require a well-organized screening program with optimal compliance.
Papers of special note have been highlighted as • of interest •• of considerable interest 1
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Diagnostic Profile
cobas 4800 HPV Test, a real-time polymerase chain reaction assay for the detection of human papillomavirus in cervical specimens Expert Rev. Mol. Diagn. 14(1), 5–16 (2014)
Sandra D Isidean*1,2, Franc¸ois Coutle´e2,3 and Eduardo L Franco1,2 1 Department of Epidemiology, Biostatistics, and Occupational Health, McGill University, Montreal, QC, Canada 2 Department of Oncology, McGill University, Montreal, QC, Canada 3 De´partement de MicrobiologieInfectiologie, Hoˆpital Notre-Dame du Centre Hospitalier de l’Universite´ de Montre´al, Montre´al, QC, Canada *Author for correspondence: Tel.: +1 514 398 5519 Fax: +1 514 398 5002 [email protected]
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Cervical cancer screening incorporating high-risk human papillomavirus (HPV) detection has become the preferred screening strategy in some countries and is increasingly more widespread in other countries with organized or opportunistic screening programs. Given knowledge that high-risk HPV genotypes differ in their oncogenic potential, commercial HPV assays with genotyping capabilities have been developed and have garnered attention in the recent literature. The cobas 4800 HPV Test is a qualitative multiplex assay that provides specific genotyping information for HPV types 16 and 18, while concurrently detecting 12 other high-risk HPV genotypes as a pooled result. It is currently the only clinically validated, US FDA-approved assay with this capability. Since HPV types 16 and 18 have been designated as conferring the greatest risk for cervical disease, their detection may prove useful in guiding patient management. KEYWORDS: cervical cancer • cobas 4800 HPV Test • diagnostic test • genotyping • HPV testing • HPV DNA • human papillomavirus • PCR • screening
Although cervical cancer is a preventable disease, it remains the third most common cancer among women worldwide. With an estimated 530,000 new cases and 275,000 deaths annually, it continues to represent a major public health problem [1]. Persistent infection with human papillomavirus (HPV) causes over 99% of cervical cancer cases [2]. Presently, 170 HPV genotypes have been identified, with over 40 infecting the anogenital tract and other mucosal sites [3]. Nonetheless, less than half of these latter 40+ genotypes cause virtually all cervical carcinomas globally (TABLE 1) [4]. HPV types 16 and 18, in particular, have been designated as conferring the greatest risk for cervical disease, as they are associated with over two-thirds of all cervical cancers worldwide [5]. Cervical cancer screening using the Papanicolaou (Pap) cytology test has drastically reduced the incidence of and mortality from cervical 10.1586/14737159.2014.865521
cancer since its introduction in the mid-1900s. Organized and opportunistic screening programs have only been effective, however, among countries able to maintain high-quality and broad coverage screening [6]. Their success has also required the provision of diagnostic and treatment resources for managing cases of disease identified by screening. Accordingly, incidence rates for cervical cancer are lowest in industrialized regions such as Australia, North America and parts of Europe [1]. Given the tremendous infrastructure and personnel costs needed to maintain such programs, however, cervical disease still imparts a heavy burden in the developing world where roughly 85% of cases occur [1]. Consistent clinical evidence has shown cervical cytology to suffer from a high falsenegative rate for the detection of cervical precancerous and cancerous lesions, even in countries with stringent quality assurance systems
2014 Informa UK Ltd
ISSN 1473-7159
5
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HPV genotype
Percent of cancers caused (%)
Cumulative percent of cancers caused (%)
HPV16
54.4
54.4
HPV18
15.9
70.3
HPV33
4.3
74.6
HPV45
3.7
78.3
HPV31
3.6
81.9
HPV58
3.3
85.2
HPV52
2.5
87.7
HPV35
1.7
89.4
HPV59
1.0
90.4
HPV56
0.7
91.1
HPV51
0.7
91.8
infections will be cleared or suppressed by host immunity within 1–2 years of exposure, and only a small proportion of oncogenic infections may persist for several years and confer a higher risk for future disease [13]. HPV genotyping has been recognized as a potentially useful means for limiting possible harms resulting from over-screening women only transiently HPV-positive. Since it is known that HPV genotypes differ in their oncogenic potential [14], cervical samples of HPV-positive women with normal or equivocal cytology results could be genotyped to detect infections with the highest-risk HPV types (i.e., HPV16 and HPV18). In practice, women with particularly high disease risk could be referred for immediate colposcopy and follow-up, while those with lower disease risk could be followed prospectively and referred for colposcopy only if persistently infected with other oncogenic types. The cobas 4800 HPV Test (Roche Molecular Systems Inc., Pleasanton, CA, USA) is one such HPV test that provides specific genotyping results for HPV types 16 and 18, along with a pooled result for 12 other oncogenic types; it will be the focus of this diagnostic profile.
HPV39
0.6
92.4
Clinical applications & guidelines for HPV testing
HPV73
0.5
92.9
HPV68
0.5
93.4
HPV66
0.3
93.7
HPV70
0.2
93.9
HPV82
0.2
94.1
Not identified
5.9
100.0
Available evidence indicates that HPV testing may be useful in the triage and follow-up of women with equivocal cytology results, as a means of follow-up for women having been treated for cervical disease to assess treatment effectiveness, and as a primary cervical screening method (either alone or in conjunction with cervical cytology) to detect CIN2+ [7,15]. In the USA, recent screening guidelines (2012) have recommended HPV and cytology co-testing as the preferred cervical cancer screening method for women aged 30–65 years [16,17]. Also recommended as a management option for women testing HPV-positive while cytology-negative is HPV genotype-specific testing to detect HPV16 or HPV18, with women testing positive for either/both types being referred for immediate colposcopy. However, these guidelines cited insufficient evidence at the time to recommend HPV testing alone for primary screening in the USA considering the lack of a well-defined and evaluated management strategy for women with positive tests [16]. In Canada, cervical screening programs remain cytologybased. However, HPV testing has been widely used to triage women with a cytology result of atypical squamous cells of undetermined significance (ASC-US). The Canadian Task Force on Preventive Healthcare remains reluctant to recommend the use of HPV testing in national screening guidelines until evidence regarding this screening modality is further developed [18]. Nonetheless, the province of Ontario is expected to begin implementing HPV testing in its provincial screening program in the near-term as a primary screening method for women ‡30 years of age [19]. In Europe, cervical cytology continues to be the cornerstone of screening programs [20]. However, given how rapidly evidence surrounding HPV testing has developed since publication of the second edition of the European Guidelines for Quality Assurance in Cervical Cancer Screening, perspectives regarding the incorporation of HPV
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Table 1. Percentage of invasive cervical cancers attributable to specific HPV genotypes.
Data taken from [4].
in place [7,8]. The low sensitivity of cytology stems from its dependence on optimally collected cervical samples, and the subjective interpretation of cellular morphology in these samples by cytotechnologists. Even among experienced cytotechnologists reviewing adequate cervical samples, nonetheless, there is high inter-observer variability that reduces the diagnostic accuracy of cytology-based screening [9]. Owing to the limitations of cytologic screening and to the understanding of cervical cancer etiology, biotechnology companies have developed molecular assays that detect HPV nucleic acid (DNA or mRNA) for use in diagnostic settings. Over the past 15 years, these molecular assays have consistently demonstrated greater reliability and substantially superior sensitivity for detecting cervical intraepithelial neoplasia grade 2 or worse (CIN2+) than Pap cytology in clinical studies [7,10]. The downside to this improved sensitivity, however, is reduced specificity. Indeed, this raises concern since HPV infections are the most common sexually transmitted infections worldwide, and most sexually active women will acquire an infection at least once in their lifetime [11]. According to a meta-analysis including data from more than 1 million women in 59 countries, the prevalence of genital HPV infection among those with normal cytology ranges from 1.6 to 41.9% [12]. Yet, most (~90%) of these 6
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cobas 4800 HPV Test
testing into screening programs are evolving [7,20]. Consequently, a review and updating of current European guidelines has already been initiated [20]. The Netherlands is the first country soon planning to adopt HPV testing alone into its national screening program for women 30–60 years of age [21]. Nonetheless, primary HPV testing has been introduced as part of pilot programs in countries such as Italy, the UK and Sweden [21,22]. Currently in Europe, HPV testing has been sanctioned for the triage and follow-up of women with equivocal cytology results, and as a test of cure in women following treatment for cervical disease. Exactly how widespread HPV screening is in these countries is somewhat unclear, however. Diagnostic tests for HPV detection
Due to the inability to culture HPV in a laboratory, almost all available methods for detecting the virus involve detection of its nucleic acids in clinical specimens [23]. Over 100 distinct tests for the detection of oncogenic HPV DNA or RNA (mostly DNA) are commercially available [24]. A handful of these tests have been approved for use by the US FDA and Health Canada, and a number more have received CE marking, which allows for broad marketability in the European Economic Area (TABLE 2). It should be noted that the list provided in TABLE 2 is not intended to be comprehensive, but showcases several tests commonly used for HPV detection – along with their main characteristics. For a comprehensive inventory of commercial tests available on the worldwide market, we refer to several recent reviews [24,25]. HPV molecular diagnostic techniques are often broadly categorized by the methodology they employ, namely signal amplification or target amplification. The former is based on an initial hybridization step of nucleic acids in a clinical specimen with target-specific probes, followed by amplification and visualization of this hybridization event [23]. By contrast, target amplification methods duplicate fragments of DNA or RNA from a targeted gene sequence to a level that can be detected using available systems. PCR techniques are based on this latter methodology. The Hybrid Capture 2 (HC2) High-Risk HPV DNA Test (Qiagen, Gaithersburg, MD, USA [previously Digene Corp.]) – which utilizes signal amplification – has been established as the worldwide standard technique for HPV detection, and remains the most evaluated and widely used molecular HPV test on the market. This assay uses a cocktail of reagents to detect any of 13 oncogenic HPV types that may be present either alone or as a co-infection, without distinguishing among the individual genotypes in the sample. In North America and Europe, it has been approved for triage of women with ASC-US cytology to assess the need for referral to colposcopy, and as an adjunct screening test to cytological screening in women ‡30 years of age. Recent guidelines published by Meijer et al. [26] have recommended that new HPV tests aimed for cervical screening purposes demonstrate performance characteristics deemed noninferior to those of the HC2 test (or the EIA kit HPV GP HR Assay [Diassay, Rijswijk, The Netherlands]) prior to use rather www.expert-reviews.com
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than undergo extensive longitudinal validation studies. More specifically, the guidelines state that: the sensitivity and specificity of the candidate test for detecting CIN2+ should be at least 90 and 98%, respectively, of those parameters for the HC2 test; the candidate test should demonstrate intralaboratory reproducibility and inter-laboratory agreement, with a lower confidence bound not less than 87% and samples being evaluated should arise from a population-based screening cohort. According to a recent meta-analysis regarding HPV testing in cervical screening by Arbyn et al. [7], the cobas 4800 HPV Test, RealTime High-Risk HPV PCR assay (Abbott Molecular Inc., Des Plaines, IL, USA) and PapilloCheck HPV-Screening Test (Greiner Bio-One, Frickenhausen, Germany) have all been clinically evaluated for use in primary screening using these criteria. While all three tests fulfilled the sensitivity and specificity criteria, only the first two tests met the reproducibility criteria. The Cervista HPV HR Test (Hologic Inc., Madison, WI, USA [previously Third Wave Technologies]) and APTIMA HPV Assay (Hologic Gen-Probe Inc., San Diego, CA, USA) have not yet been validated for primary screening according to these test requirements, but have received licensing by the FDA, Health Canada, and among countries accepting a CE mark for: triage of women with ASC-US cytology and as an adjunct screening test to cytological screening in women ‡30 years of age. Similarly to the HC2 test, the Cervista HPV HR Test, EIA kit HPV GP HR Assay, and AMPLICOR HPV Test (Roche Molecular Systems Inc.) all detect the presence of high-risk HPV types in aggregate (i.e., without providing information about specific genotypes present in a sample). The APTIMA HPV Assay is also based on the cumulative detection of highrisk HPV types, but targets viral mRNA rather than DNA. It remains the only FDA-approved mRNA-based HPV test available. The PreTect HPV-Proofer Test (NorChip, Klokkarstua, Norway) and NucliSENS EasyQ HPV Test (Biome´rieux, Marcy I’Etoile, France) are also mRNA-based assays; however, they detect transcripts for the five most frequently identified high-risk HPV types in cervical cancers worldwide (HPV types 16, 18, 31, 33 and 45). Tests listed in TABLE 2 providing full genotyping results include the Linear Array HPV Genotyping Test (Roche Molecular Systems Inc.), the INNO-LiPA HPV Genotyping Extra (Innogenetics NV, Gent, Belgium) and PapilloCheck HPV-Screening Test. Although full genotyping assays have been (and remain) a vital component in research settings, their clinical value in population-based cervical screening remains undetermined [27,28]. On the contrary, given compelling evidence demonstrating the exceptional oncogenic potential of HPV types 16 and 18 [4,14], HPV tests with partial concurrent or reflex genotyping capabilities have been developed and include the cobas 4800 HPV Test, the Cervista HPV 16/18 Test (Hologic Inc.), and the RealTime High-Risk HPV PCR assay. In all three assays, individual genotyping results are provided for HPV types 16 and 18 only – explaining why they are termed ‘partial’ genotyping assays. The cobas 4800 HPV Test is the only clinically validated and FDA-approved test that simultaneously provides pooled 7
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Table 2. Summary of common commercially available nucleic acid tests for the detection of HPV in cervical samples of women (not comprehensive). Test
Molecular target
Test methodology
Oncogenic HPV genotypes detected†
Time to results‡
Interpretation of results
Regulatory status
Indications for use§
Hybrid Capture 2 High-Risk HPV DNA Test
HPV DNA
Signal amplification
16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68
Process 352 samples in 8 h
Automated
FDA-approved, CE-marked, HC-approved
ASC-US triage, cervical screening, test of cure
Cervista HPV HR Test
HPV DNA
Signal amplification
16, 35, 52, 66,
Process 96 samples in 6-7 h
Automated
FDA-approved, CE-marked, HC-approved
ASC-US triage, cervical screening, test of cure
Cervista HPV 16/18 Test
HPV DNA
Signal amplification
16, 18
Process 96 samples in 6-7 h
Automated
FDA-approved, CE-marked, HC-approved
ASC-US triage, alongside/ follow-up to Cervista HPV HR Test
cobas 4800 HPV Test
HPV DNA
Target amplification
16, 35, 52, 66,
18, 31, 33, 39, 45, 51, 56, 58, 59, 68
Process 96 samples in 4.5 h
Automated
FDA-approved, CE-marked, HC-approved
ASC-US triage, cervical screening, test of cure
APTIMA HPV Assay
HPV mRNA
Target amplification
16, 35, 52, 66,
18, 31, 33, 39, 45, 51, 56, 58, 59, 68
Process 500 samples in 8.5 h
Automated
FDA-approved, CE-marked
ASC-US triage, cervical screening, test of cure
EIA kit HPV GP HR Assay
HPV DNA
Target amplification
16, 35, 52, 66,
18, 31, 33, 39, 45, 51, 56, 58, 59, 68
Process 96 samples in 9.5 h
Not Automated
CE-marked
ASC-US triage, cervical screening, test of cure
AMPLICOR HPV Test
HPV DNA
Target amplification
16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68
Process 96 samples in 2 working days
Not Automated
CE-marked, HC-approved
ASC-US triage, cervical screening, test of cure
RealTime HighRisk HPV Assay
HPV DNA
Target amplification
16, 35, 52, 66,
18, 31, 33, 39, 45, 51, 56, 58, 59, 68
Process 96 samples in 6 h
Automated
CE-marked, HC-approved
ASC-US triage, cervical screening, test of cure
LINEAR ARRAY HPV Genotyping Test
HPV DNA
Target amplification
16, 33, 51, 58, 68, 82
18, 35, 52, 59, 69,
Process 48 samples in 8 h
Not Automated
CE-marked, HC-approved
HPV genotyping
18, 31, 33, 39, 45, 51, 56, 58, 59, 68
26, 39, 53, 66, 70,
31, 45, 56, 67, 73,
† HPV type 68 is considered a probable carcinogen, and HPV types 26, 53, 66, 67, 69, 70, 73, and 82 are considered possible carcinogens by the International Agency for Research on Cancer (IARC) [61]. ‡ The respective tests vary in their level of automation. Times outlined may not incorporate manual procedure times or PCR amplification times for the various tests. Data shown are from the respective manufacturers. § FDA-approved tests for use in the USA are only indicated for the triage and follow-up of women with ASC-US cytology, and as an adjunct screening test to cytological screening in women ‡30 years of age. Primary, stand-alone screening with these tests is not yet indicated in the USA. In other countries using HPV testing in cervical screening, indications for use may include: triage and follow-up of women with ASC-US cytology; primary screening and follow-up of women after treatment of disease (test of cure).
8
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cobas 4800 HPV Test
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Table 2. Summary of common commercially available nucleic acid tests for the detection of HPV in cervical samples of women (not comprehensive) (cont.). Test
Molecular target
Test methodology
Oncogenic HPV genotypes detected†
Time to results‡
Interpretation of results
Regulatory status
Indications for use§
INNO-LiPA HPV Genotyping Extra
HPV DNA
Target amplification
16, 33, 51, 58, 69,
18, 35, 52, 59, 70,
26, 39, 53, 66, 73,
31, 45, 56, 68, 82
Process 48 samples in 3 h
Automated
CE-marked
HPV genotyping
PapilloCheck HPV-Screening Test
HPV DNA
Target amplification
16, 35, 52, 59, 73,
18, 39, 53, 66, 82
31, 45, 56, 68,
33, 51, 58, 70,
Process 12 samples in 3 h
Automated
CE-marked
ASC-US triage, cervical screening, test of cure
PreTect HPVProofer Test
HPV mRNA
Target amplification
16, 18, 31, 33, 45
Process 30 samples in 2.5 h
Automated
CE-marked
ASC-US triage, cervical screening, test of cure
NucliSENS EasyQ HPV Test
HPV mRNA
Target amplification
16, 18, 31, 33, 45
Process 30 samples in 6.5 h
Automated
CE-marked
ASC-US triage, cervical screening, test of cure
† HPV type 68 is considered a probable carcinogen, and HPV types 26, 53, 66, 67, 69, 70, 73, and 82 are considered possible carcinogens by the International Agency for Research on Cancer (IARC) [61]. ‡ The respective tests vary in their level of automation. Times outlined may not incorporate manual procedure times or PCR amplification times for the various tests. Data shown are from the respective manufacturers. § FDA-approved tests for use in the USA are only indicated for the triage and follow-up of women with ASC-US cytology, and as an adjunct screening test to cytological screening in women ‡30 years of age. Primary, stand-alone screening with these tests is not yet indicated in the USA. In other countries using HPV testing in cervical screening, indications for use may include: triage and follow-up of women with ASC-US cytology; primary screening and follow-up of women after treatment of disease (test of cure).
results for 12 high-risk HPV types, as well as individual results for HPV types 16 and 18 (respectively). The remainder of this article will focus on the specific characteristics of this assay, and will describe its clinical profile through an overview of results from clinical validation studies. cobas 4800 HPV Test: indications for use and assay methodology
The cobas 4800 HPV Test is an in vitro qualitative real-time PCR assay for the detection of high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) in cervical specimens of women undergoing screening. It is performed using the cobas 4800 System, and has been indicated by the US FDA, Health Canada and in countries accepting a CE mark for use as follows: to triage women aged 21 years and older with ASC-US cytology to determine the need for colposcopy referral; to screen women aged 21 years and older with ASC-US cytology to assess the presence or absence of HPV types 16 and 18; to screen women ‡30 years of age adjunctively with cytological screening; to screen women ‡30 years of age to assess the presence or absence of HPV types 16 and 18. In November 2012, Roche Molecular Systems Inc., announced that the test received an expanded indication for use as a primary screening test in countries accepting a CE mark (i.e., cytology screening is no longer required as a pre-test or co-test). www.expert-reviews.com
As indicated in the full package insert by the manufacturer, the cobas 4800 HPV Test has been validated for use with cervical specimens collected in cobas PCR Cell Collection Media (Roche Molecular Systems Inc.), PreservCyt Solution (Hologic Inc.) and SurePath Preservative Fluid (BD DiagnosticsTriPath, Burlington, NC, USA). However, the FDA has only approved use of the test with cervical specimens collected in PreservCyt Solution. While several recent studies have evaluated the use of the cobas 4800 HPV Test with SurePath Preservative Fluid, it is likely that additional evidence will be needed prior to an expanded approval by the FDA [29,30]. Once collected, fully automated specimen preparation for the cobas 4800 HPV Test may be accomplished using the cobas x 480 Instrument – one of two separate technologies in the cobas 4800 System (FIGURE 1). This instrument serves to simultaneously extract, purify and prepare target HPV DNA and b-globin DNA for PCR amplification and detection. The processing of b-globin DNA functions as a control to differentiate HPVnegative specimens from those that fail to exhibit positivity due to a lack of cells or the presence of PCR inhibitors in specimens being tested. With the cobas x 480 Instrument, cervical specimens collected in one of the various media outlined above are processed through cell lysis using a chaotropic reagent. HPV and b-globin DNA released from this process are then purified 9
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A
B
Figure 1. The cobas 4800 System featuring its two component technologies. The cobas x 480 Instrument (A) serves to simultaneously extract, purify, and prepare target HPV DNA and b-globin DNA in collected cervical samples for PCR amplification and detection. Sample preparation using this instrument is fully automated. The cobas z 480 Analyzer (B) serves to then amplify and detect viral nucleic acid in the prepared samples using real-time PCR technology. Manual processing during this procedure is limited to the transfer of a microwell plate containing PCR-ready samples from the cobas x 480 Instrument to the cobas z 480 Analyzer. Technologies are not depicted to scale. Reproduced with permission from Roche Molecular Systems, Inc., Roche Molecular Systems, Inc. (2013).
using magnetic glass particles chemistry, washed and ultimately separated from these particles. Manual processing during this procedure is limited to the transfer of a microwell plate containing PCR-ready samples from the cobas x 480 Instrument to the cobas z 480 Analyzer – the second instrument in the cobas 4800 System (FIGURE 1). This instrument amplifies and detects viral nucleic acid in the prepared samples. A pool of HPV- and b-globin-specific primers present in the Master Mix reagent for the cobas 4800 HPV Test is designed to concurrently amplify HPV DNA from the 14 targeted HPV types and b-globin DNA. This reagent also includes AmpErase (uracil-N-glycosylase) enzyme and deoxyuridine triphosphate (dUTP), allowing for the destruction of contaminating amplicons generated from previous PCR runs. HPV-specific primers target nucleotides within the L1 region of the HPV genome, which encodes the major capsid protein of the virus. Detection of amplified DNA is accomplished using oligonucleotide probes labeled with four fluorescent dyes. Each dye is subsequently visualized according to its characteristic wavelength across one of four detection channels of the cobas z 480 Analyzer. Channel 1 detects the chemiluminescent signal for the 12 non-HPV16/18 genotypes (i.e., 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68), while channels 2, 3 and 4 detect signals for HPV16, HPV18, and b-globin targets (respectively) (FIGURE 2). The cobas 4800 System Software performs all assay and run validations, and automatically displays reportable cobas 4800 HPV Test results as positive, negative or invalid. The software allows the user to select one of two different testing options for samples: the ‘HPV High-Risk Panel’, which provides pooled testing for the 14 targeted high-risk types or the ‘HPV High-Risk Panel Plus Genotyping’, which provides pooled testing for the non-HPV16/18 types and genotype10
specific testing for HPV types 16 and 18 (respectively). Each run of the cobas 4800 System requires one replicate each of a positive and negative control, and valid results for these controls must be obtained for the system software to display testing results. The system is designed to support up to 94 cervical specimens per run (plus the two controls). Notably, a single run can support samples contained in any combination of media described, and can provide results for either of the two testing options listed above for each sample. Up to 384 HPV tests may be processed by the system in a given day, with the first 94 results obtained in 4.5 h. Analytical sensitivity & specificity
As described by Rao et al., analytical studies of the cobas 4800 HPV Test conducted by the manufacturer utilized cut-off values (or cycle thresholds) distinguishing positive from negative samples based on the detection of histologically confirmed CIN2+, so as to optimize clinical sensitivity and specificity (i.e., clinical utility) [31]. Limit of detection (LOD) studies for HPV genotypes 16, 18 and 31 were carried out to assess analytical sensitivity of the test. LOD levels for these studies were defined as the lowest concentration of HPV DNA in a sample resulting in a positive test result at least 95% of the time. Genotypes 16 and 18 were both detected with a 100% positivity rate at 600 copies/ml using PreservCyt Solution, while HPV31 was detected 97% of the time at 300 copies/ml. Using SurePath Preservative Fluid, HPV16 was detected with a 100% positivity rate at 300 copies/ml, and HPV31 was detected with a 95% positivity rate at 150 copies/ml; the LOD level for HPV18 remained the same. To assess analytical specificity, over 100 species of bacteria, fungi, and viruses – to include low-risk HPV genotypes and Expert Rev. Mol. Diagn. 14(1), (2014)
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cobas® 4800 HPV Test: examples of test results HPV HR panel
HPV HR panel plus genotyping
(-)
HR HPV
(-)
HPV16
(-)
HPV18
(-)
HR HPV
(+)
HPV16
(-)
HPV18
(-)
HR HPV
(-)
HPV16
(+)
HPV18
(-)
HR HPV
(-)
HPV16
(-)
HPV18
(+)
HR HPV
(+)
HPV16
(+)
HPV18
(+)
(+) cobas® 4800 HPV Test: assay design (+) 12 HR HPVs
HPV 16
HPV 18
Host DNA control (+)
(+) Channel 1
Channel 2
Channel 3
Channel 4
Figure 2. The cobas 4800 HPV Test: assay design and examples of test results. Assay design (bottom): Channel 1 detects the chemiluminescent signal for the 12 non-HPV16/18 genotypes tested for by the cobas 4800 HPV Test (HPV types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68). Channels 2, 3 and 4 detect signals for HPV16, HPV18 and b-globin targets (respectively). Examples of test results (right): The HPV High-Risk (HR) Panel tests for the presence of all 14 HR HPV types targeted by the assay as a pool. Results for this panel are reported as positive, negative or invalid. The HPV HR Panel Plus Genotyping provides pooled testing for the 12 non-HPV16/ 18 types, and genotype-specific testing for HPV types 16 and 18 (respectively). Test results shown do not represent the total combination of possible results. Adapted with permission from Roche Molecular Systems, Inc., Roche Molecular Systems, Inc. (2013).
other viruses commonly infecting the genitourinary tract – were spiked at high concentrations into two types of specimen: HPV-negative PreservCyt Solution specimen and HPV-negative PreservCyt Solution specimen spiked with HPV types 16, 18 and 31. None of these organisms were found to interfere with the detection of these types, or produce a false-positive result in the HPV-negative specimen. Clinical performance of the cobas 4800 HPV Test
In 2009, Castle et al. published the first report evaluating the prototype of the cobas 4800 HPV Test [32]. Using a convenience sample of 531 cervical specimens, the prototype was compared with the Linear Array HPV Genotyping Test. Percentage of agreement between the two assays was 94.7% (95% CI: 92.5–96.5), and the kappa value was 0.92 (95% CI: 0.89–0.95). In that study, more women without evident cervical abnormalities on histopathology tested positive by the Linear Array HPV Genotyping Test than by the cobas 4800 HPV Test for non-HPV16/18 high-risk types. A conservative clinical threshold of 45 was utilized to determine positivity, as a validated threshold was not available at the time of the study.
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ATHENA HPV Study
The cobas 4800 HPV Test was clinically validated in the ATHENA (Addressing THE Need for Advanced HPV Diagnostics) Study, the largest clinical trial to examine the clinical utility of HPV DNA testing and HPV16/18 genotyping among women attending routine cervical screening in the USA. The study enrolled over 47,000 women ‡21 years of age at one of 61 clinical sites across the country. The test was evaluated in three relevant populations: women aged ‡21 years with ASCUS cytology for the intended use of ASC-US triage to colposcopy; women aged ‡30 years with normal cytology for the intended use as an adjunctive test with cytology and women aged ‡25 years with any cytology result to assess performance of HPV testing with genotyping as a primary screening test [33]. The first evaluation compared performance indices for the cobas 4800 HPV Test with those of the HC2 test for detection of CIN2+ and CIN3+ among 1578 women ‡21 years of age with: valid cytology results demonstrating ASC-US; valid results for HPV testing and valid colposcopic assessments [34]. Performance characteristics of the tests were found to be very similar for all parameters. Sensitivity estimates for the cobas 4800 HPV Test were 90.0% (95% CI: 81.5–94.8) and 93.5% 11
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´e & Franco Isidean, Coutle
(95% CI: 82.5–97.8) for CIN2+ and CIN3+ detection (respectively). These estimates were 87.2% (95% CI: 78.0–92.9) and 91.3% (95% CI: 79.7–96.6) for the HC2 test, respectively, demonstrating the cobas 4800 HPV Test to meet the standard outlined by Meijer et al. for new HPV DNA tests [26]. Specificity of the cobas 4800 HPV Test was 70.5% (95% CI: 68.1–72.7) for CIN2+ and 69.3% (95% CI: 66.9–71.5) for CIN3+, while estimates for HC2 were respectively 71.1% (95% CI: 68.8–73.4) and 70.0% (95% CI: 67.7–72.3). Furthermore, concordance between the assays was high in women with a CIN2+ diagnosis (96.2%; 95% CI: 89.3–98.7). These findings were deemed to clinically validate the use of the cobas 4800 HPV Test for ASC-US triage in women ‡21 years of age. This study also demonstrated how genotyping for HPV16/ 18 identifies women at greatest absolute and relative risk for CIN2+ and CIN3+ [34]. When the cobas 4800 HPV Test was evaluated in women aged ‡30 years with normal cytology, investigators similarly found that both absolute and relative risks for CIN2+ and CIN3 + were not only impacted by HPV status for the 14 targeted types, but were even more influenced by a woman’s HPV16/ 18 status [35]. The relative risk for CIN2+ was 13.7-fold higher (95% CI: 7.3–41.9) with an HPV16- and/or HPV18-positive result on the cobas 4800 HPV Test than with a negative test result, and 35.0-fold higher (95% CI: 13.0–552.8) for CIN3+. Moreover, a positive result for HPV16 and/or HPV18 had a relative risk for CIN2+ that was 2.51-fold higher (95% CI: 1.7–3.6) than a positive result for the 12 remaining high-risk types. Finally, women who were HPV16-positive with normal cytology had an absolute risk of 13.6% (95% CI: 9.5–18.0) for CIN2+; thus, nearly 1 in 7 women testing HPV16-positive had highgrade disease that was missed by cytology [35]. Castle et al. went on to show that in women aged ‡25 years, independent of cytology result, the cobas 4800 HPV Test was more sensitive than liquid-based cytology for the detection of CIN3+ (92.0%; 95% CI: 88.1–94.6 vs 53.3%; 95% CI: 47.4–59.1) [33]. While the addition of cytology to HPV testing increased this sensitivity to 96.7% (95% CI: 93.9–98.3), it also increased the number of positive screening results by 35.2% compared with HPV testing alone. Among HPV-positive women, detection of HPV16 and/or HPV18 alone was equivalent to detection of ASC-US or worse alone in terms of sensitivity and PPV to identify CIN3+. Better sensitivity and similar PPV for CIN3+ was observed via detection of HPV16 and/or HPV18, or low-grade squamous intraepithelial lesions (SIL) or worse cytology than ASC-US or worse cytology alone. This was also shown to be the case when detecting HPV16 and/or HPV18, or high-grade SIL or worse cytology [33]. Final data from the ATHENA Study presented at the Society of Gynecologic Oncology’s 44th Annual Meeting on Women’s Cancer (Los Angeles, CA, USA, 2013) revealed that – among cytology-negative women – HPV testing at enrollment resulted in earlier detection of prevalent CIN3+ disease initially missed by cytology [36]. This allowed for treatment of disease that could have otherwise been delayed or not given due to loss of women 12
to follow-up. HPV16, followed by HPV18, continued to confer the greatest risk for CIN3+. Other comparative studies of the cobas 4800 HPV Test
A study by Heideman et al. showed the cobas 4800 HPV Test to fulfill all criteria by Meijer et al. in their international guidelines for HPV test requirements when assessed relative to the HC2 test [26,37]. Using data from the VUSA-Screen trial in the Netherlands, agreement between the assays was 97.3% (95% CI: 95.9–98.2) for controls and 98.3% (95% CI: 89.1–99.8) for cases. Kappa values for these groups were 0.74 and 0.90, respectively. More recently, Lloveras et al. similarly demonstrated the fulfillment of these criteria by the cobas 4800 HPV Test when compared with HC2 in opportunistic screening in Spain [38]. A third study equated these tests using cervical samples from women enrolled in the HPV PaP Cohort study in the USA [39]. The cobas 4800 HPV Test and Linear Array HPV Genotyping Test were also compared for specific detection of HPV16/18. In 1824 specimens with valid results, the cobas 4800 HPV Test had 85.9% agreement with HC2 and 91.0% agreement with the Linear Array HPV Genotyping Test (kappa values: 0.69 [95% CI: 0.66–0.73] and 0.82 [95% CI: 0.79–0.84], respectively) [39]. The cobas 4800 HPV Test was found more likely to test positive compared with HC2, but the latter test was more likely to cross-react with certain low-risk HPV genotypes. Several other studies also highlighted this cross-reactivity by HC2, despite reporting good to excellent agreement between HC2 and the cobas 4800 HPV Test according to the kappa statistic [40–42]. In a Canadian study using 466 samples from women aged 15 to >60 years, overall agreement between HC2 and the cobas 4800 HPV Test was 93.8% (kappa: 0.87; 95% CI: 0.83–0.92) [43]. In women aged ‡30 years, 92.3% agreement was observed. Another Canadian study by our team compared the performance of the two assays among 396 women aged 24–75 years who were referred to colposcopy following an ASC-US cytology result [44]. Clinical sensitivities and specificities for detecting CIN2+ were respectively 89.7% (95% CI: 72.8–97.2) and 66.7% (95% CI: 61.7–71.3) for the cobas 4800 HPV Test, and 93.1% (95% CI: 77.0–99.2) and 72.2% (95% CI: 67.4–76.5) for HC2. Percent agreement between the tests was 87.9% (95% CI: 84.3–90.8), with a kappa value of 0.74 (95% CI: 0.64–0.83). Agreement between the cobas 4800 HPV Test and Linear Array HPV Genotyping Test to detect the 14 HPV types targeted by the former assay was also reported as 93.2% (95% CI: 90.2–95.3), with a kappa value of 0.86 (95% CI: 0.76–0.96). Several other studies comparing the cobas 4800 HPV Test with other HPV tests, specifically the AMPLICOR HPV Test or RealTime High-Risk HPV PCR assay, have also found good to excellent concordance between the methods [45,46]. In a study population of 1099 women referred for colposcopy in the UK, Szarewski et al. compared the performance of seven different HPV tests – to include the cobas 4800 HPV Test – for identification of CIN2+ and CIN3+ [47]. Cuzick et al. went on to Expert Rev. Mol. Diagn. 14(1), (2014)
cobas 4800 HPV Test
compare the performance of six of these tests in a screening population of 6000 women in the UK [48]. In both studies, the cobas 4800 HPV Test demonstrated high sensitivity to detect highgrade cervical disease, with specificity parameters in line with or greater than those for HC2.
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Expert commentary
Given what is known about the etiology of cervical cancer, and the performance of HPV testing technologies relative to cytological testing, wider utilization of HPV detection methods in cervical screening programs seems inevitable. How to best manage women testing HPV-positive with normal or equivocal cytology results, however, has been a practical concern related to this utilization. Indeed, among high-risk HPV-positive women, only a minority will be found to have meaningful disease. This raises questions regarding how to optimally identify these women needing immediate follow-up. In recent years, HPV genotype-specific detection has been evaluated for its clinical potential to improve screening accuracy and efficiency, and has been shown to identify women at highest risk for cervical disease in numerous studies [14,33–35,49–52]. As a result, novel HPV tests with concurrent or reflex genotyping capabilities to detect high-risk types – and distinguish those types with exceptionally high oncogenic potential (i.e., HPV16/ 18) – have been designed and approved for use in various countries. The cobas 4800 HPV Test is currently the only US FDA-approved test of this kind that provides pooled results for 12 high-risk HPV types concurrently with individual genotyping results for HPV types 16 and 18. As reviewed in this article, the cobas 4800 HPV Test has demonstrated favorable clinical performance in a large, prospective trial (the ATHENA Study), as well as in numerous comparative studies. It has also been evaluated according to the non-inferiority criteria outlined by Meijer et al. for new HPV tests intended for primary screening [26,37,38]. The importance of validating novel HPV tests according to pre-specified validation strategies for sensitivity, specificity and reliability has been strongly emphasized in the recent literature [7,26,27]. Due to deficient health technology regulation in certain parts of the world, however, HPV assays yet to be fully validated are being employed [25]. Alas, lack of proper clinical validation could render a commercial assay more harmful than beneficial. Despite the higher cost of HPV testing relative to cytologic testing for a single round of screening, strategies incorporating HPV testing have been shown to be cost effective over a variety of settings [53–57]. This can be attributed to increases in diagnostic lead-time provided by HPV testing as compared with cytologic testing, which subsequently translates into lower risk following a negative test. As a result, longer screening intervals become permissible. Attainment of such advantages would require a well-organized screening program with optimal compliance, as excessive and unnecessary screening and treatment of women testing HPV-positive – who may only be transiently infected – would obviously render any described advantages extinguished. www.expert-reviews.com
Diagnostic Profile
Five-year view
How will cervical cancer screening be practiced in the future? The answer to this question requires consideration of regionspecific needs. The USA, a country highly risk-averse with respect to medical practice, has recently favored HPV and cytology co-testing – a combination that permits maximal sensitivity for detecting cervical cancer precursors [16,17]. As this review indicates, the main contributor to this high sensitivity is the HPV assay rather than Pap cytology. Nonetheless, because of its superior specificity, the latter is more appropriate as a triage option for women who are HPV-positive. As of this writing, a number of countries have begun to implement screening based on this premise (i.e., primary HPV testing followed by Pap triage in series, not in parallel). Although rapidly gaining in favor, this strategy continues to be formally evaluated in two randomized controlled trials [58,59]. This screening approach will be most attractive to countries with central oversight of the cost–effectiveness of their healthcare systems. Nevertheless, it is not altogether implausible that even the USA may eventually come to revise its guidelines to allow the above serial screening approach to be permitted alongside parallel co-testing. Lowresource countries may adopt simpler screening methodologies, such as visual inspection, with or without a pre-test based on low-cost HPV assays (e.g., the careHPV Test [Qiagen]). A key consideration in the planning of cervical cancer screening should be whether or not a country has succeeded in implementing a public HPV vaccination program of high coverage for pre-adolescent and adolescent girls, with expansion to other young women via catch-up vaccination. As successive cohorts of vaccinated women reach screening age, there will be a gradual decrease in cervical lesion prevalence and – consequently – the positive predictive value of any screening test. Because of its dependence on human interpretation of cytologic abnormalities, Pap cytology is likely to be more affected than molecular HPV assays that are not subjectively interpreted [60]. By screening primarily with a clinically validated HPV test, subsequent triage via cytology will provide cytotechnologists with an enriched caseload of lesions – thereby increasing the positive predictive value of cytologic testing. Furthermore, restricting cytologic assessments to HPV-positive women will reduce the cytology reading workload by more than 10-fold. No country has yet adopted different screening policies for vaccinated and unvaccinated women. For pragmatic and ethical reasons, decisions regarding the optimal age to begin screening, the ideal screening frequency and the most appropriate screening technology cannot be arrived at via randomized controlled trials. Policy decisions stemming from in-depth health technology assessments and cost–effectiveness considerations will prevail. Having a reasonably well-organized screening program and surveillance infrastructure (e.g., vaccination registries) will be necessary for enforcing different policies. Political courage will likely be essential for early adopters, while the role of professional guidelines and the endorsement of national and supranational agencies will be paramount in bringing confidence to providers and the public. 13
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Diagnostic Profile
´e & Franco Isidean, Coutle
Cytology is not the only screening methodology that may be clinically useful as a triage test for women who are HPVpositive. HPV16 or HPV16/18 genotyping, as provided by the cobas 4800 HPV Test, could serve such a role. Indeed, recent guidelines already allow for the use of HPV16/18 genotyping in this capacity [16]. New cytologic/histologic biomarkers, to include p16, Ki-67, MCM2 and MCM7 are also attractive options for an enhanced Pap triage step [28]. However, uncertainty remains as to whether the gains in screening and risk prediction that come from adopting these technologies will be worth the costs and extra labor that they incur. With so many technologies being readied for deployment or already adopted, the horizon for cervical cancer prevention has never been more exciting and dynamic. Primary prevention (via HPV vaccination) and secondary prevention (via screening with molecular HPV tests and other promising technologies) have provided a tremendous potential for reduction in cervical cancer incidence and mortality; no other neoplastic disease can currently rival the magnitude of this potential. Prudent and synergistic use of these prevention fronts will likely be the
hallmark of success stories in the future. Uncoordinated adoption of these technologies in opportunistic and non-universal conditions will only heighten the inequality that has characterized cervical cancer control for so long. Financial & competing interests disclosure
F Coutle´e has received grants through his institution from Merck Sharp & Dohme and Roche Diagnostics, as well as honoraria from Merck and Roche for lectures. EL Franco has served as occasional consultant or advisory board member to Merck and GSK on HPV vaccines, to Qiagen, Roche, GenProbe and Becton & Dickinson on HPV diagnostics, to Cytyc and Ikonisys on cytology and to Innovus and Scimetrika on health economic modeling. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties. No writing assistance was utilized in the production of this manuscript.
Key issues • Persistent infection with high-risk human papillomaviruses (HPVs) causes over 99% of cervical cancers worldwide. • HPV testing has demonstrated greater reliability and sensitivity for detecting cervical disease than cytology-based testing, and has been recommended for use in cervical cancer screening in several industrialized countries. • Over 100 distinct tests for the detection of oncogenic HPVs are now commercially available. • The introduction of HPV testing raises concerns regarding how best to manage women who are HPV-positive while cytologynegative. • Given the exceptional oncogenic potential of HPV types 16 and 18, HPV tests with partial concurrent or reflex genotyping capabilities to detect these types have been developed as a means to further stratify HPV-positive women according to risk, and avoid over-screening and over-treatment. • The cobas 4800 HPV Test is one such HPV test that provides specific genotyping results for HPV types 16 and 18, along with a pooled result for 12 other oncogenic types. • The cobas 4800 HPV Test is an in vitro qualitative real-time PCR assay that is performed using the cobas 4800 System, which includes fully automated specimen preparation, amplification and detection of viral nucleic acid, and automated reportable test results. • The test has demonstrated favorable clinical performance in a large, prospective trial (the ATHENA Study), as well as in numerous comparative studies. It is the first test of its kind to be US FDA-approved. • The benefits of incorporating HPV testing and genotyping into practice will require a well-organized screening program with optimal compliance.
Papers of special note have been highlighted as • of interest •• of considerable interest 1
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