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An HPV 16, 18, and 45 genotyping test based on Hybrid Capture® technology
Journal of Clinical Virology 45, S1 (2009) S93 S97
Contents lists available at ScienceDirect
Journal of Clinical Virology j o u r n a l h o m e p a g e : www.elsevier.com/locate/jcv
An HPV 16, 18, and 45 genotyping test based on Hybrid Capture® technology Ha Thai*, Sameera Rangwala, Tanya Gay, Kimberly Keating, Sarah McLeod, Irina Nazarenko, Dominic O’Neil, Dana Pfister, Dirk Loeffert QIAGEN Gaithersburg Inc., Gaithersburg, MD, USA
ARTICLE
INFO
ABSTRACT Background: It has been shown that women positive for HPV 16 and HPV 18 have an increased risk of high-grade cervical intraepithelial neoplasia (CIN) compared with women positive for other high-risk (HR) HPV types. In addition, HPV 18 and HPV 45 have been closely linked to aggressive and difficult to detect adenocarcinomas. Objectives: To develop a test based on the Hybrid Capture® technology capable of specifically detecting the most important carcinogenic HPV types; 16, 18, and 45. Study design: The assay is based on Hybrid Capture technology utilizing a mixture of short type-specific oligoribonucleotides to detect HPV types 16, 18, or 45. The assay utilizes no target amplification and shares workflow and critical reagents with the Digene HC2 HPV screening assay. Studies to evaluate specificity, performance of the test in comparison to HC2, and capability to detect a single genotype in the presence of multiple infections are described. Specificity was evaluated analytically using a panel of HR- and LR-HPV types to illustrate cross-reactivity. Performance in comparison to the HC2 test was evaluated by testing aliquots of the same prepared samples by the genotyping test and HC2. Ability to detect a single genotype during multiple infections was modeled by detecting HPV 16 plasmid in the presence of HPV 6 or HPV 31 at high copy numbers. Results: The proposed genotyping assay specifically detects HPV 16, 18, and 45 with an analytical sensitivity of 5,000 copies per assay. The assay is highly specific and does not detect other tested high-risk or low-risk types at 108 copies per reaction. Utility of the genotyping test was demonstrated using clinical samples collected in Digene Specimen Transport Medium (STM) and results were confirmed by PCR. Conclusions: The target-amplification free assay provides a genotyping method for highly specific detection of HPV 16, 18, and 45 without the complexity of PCR technology. © 2009 Elsevier B.V. All rights reserved.
Keywords: HPV detection HPV genotyping Hybrid Capture Diagnostics
1. Introduction It is widely recognized that persistent infections with highrisk types of human papillomavirus (HR-HPV) are associated with invasive cervical cancer (ICC). Specifically, there are three HR-HPV genotypes, HPV 16, 18, and 45, that are highly prevalent in all regions of the world and frequently identified in women with high-grade cervical intraepithelial neoplasia (CIN 2+) and ICC.1 HPV 16, 18, and 45 have been identified as the primary contributors to ICC accounting for approximately 70% of all cases. HPV 16 is by far the predominant type * Corresponding author. Please add correspondence details as desired? 1590-8658/ $
see front matter © 2009 Elsevier B.V. All rights reserved.
causing greater than 50% of cancers, HPV 18 follows causing 10 14%, and HPV 45 causes approximately 2 8%.2 In addition, HPV 16 and 18 are two of the most common HPV types in women without cancer.3 HPV 45, although an uncommon genotype found amongst women with normal cytology, shows a prevalence of 2.3% in high squamous intraepithelial lesions (HSIL), 4.6% in squamous cervical carcinoma (SCC), and 5.8% in adenocarcinoma (ADC).1 Together, HPV 16, 18, and 45 have been shown to account for nearly 90% of ADC.4 ADC is known to be a much more aggressive and invasive form of cancer as compared to SCC. Technologies available for HPV genotyping vary by method and platform and may offer type-specific characterization
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H. Thai et al. / Journal of Clinical Virology 45 (2009) S93 S97
for a multitude of different high- and low-risk HPV types. For example, the Roche Linear Array HPV Genotyping Test (Roche Molecular Diagnostics, Pleasanton, CA, USA) is designed to detect 37 high- and low-risk HPV types. Innogenetics HPV test, INNO-LiPA HPV Genotyping Extra (Innogenetics NV, Gent, Belgium), detects 28 different HPV types. Greiner Bio-One has a test that utilizes a DNA chip, the Papillo Check® HPV-Screening test (Greiner Bio-One International AG, Kremsmuenster, Austria) for the identification of 24 types of genital HPV. These and many more HPV genotyping solutions involve target amplification in the form of PCR and with it the inherent issues and challenges of PCR-based nucleic acid detection tests, including (1) contamination concerns, (2) high level of technical expertise required to execute, (3) overly sensitive, and (4) non-uniform amplification of multiple targets. In this study, we describe an HPV genotyping assay that is not dependent on target amplification. The genotyping test is highly specific for the detection of HPV 16, 18, and 45 and has an equivalent sensitivity to the digene High-Risk HPV HC2 DNA Test (HC2). The Digene HC2 test is the only FDAapproved assay currently in the US market for the detection of HR HPV. It is widely recognized as the gold standard for HR-HPV screening and is a signal amplification assay utilizing the Hybrid Capture® technology where HPV-DNA targets are hybridized to RNA probes in solution and detected via proprietary hybrid-specific antibodies. The proposed HPV 16, 18, and 45 genotyping test is based on Hybrid Capture technology but utilizes a mixture of short type-specific oligoribonucleotides that can detect HPV types 16, 18, and 45 rather than the long transcribed RNA probes utilized in the HC2 test. In addition, the genotyping assay shares workflow and critical reagents with the Digene HC2 HPV screening assay. 2. Materials and methods 2.1. Sample Transport Media (STM) cervical clinical sample pool A negative pool was created from anonymous de-identified STM (Digene) clinical specimens acquired from Kaiser Permanente (San Rafael, CA, USA). Anonymous individual patient specimens, identified as negative for the thirteen HR-HPV genotypes targeted by the Digene HC2 screening test, were combined to create a larger pool that could be used to mimic clinical background in subsequent experiments. A specimen was considered negative for HR HPV if the HC2 relative light unit/cutoff (RLU/CO) value was <1.0. This pool was primarily utilized for the genotyping to HC2 equivalence study. 2.2. Comparison of the sensitivity of the genotyping test to the sensitivity of HC2 A study was performed to determine if the genotyping assay was able to detect HPV 16, 18, and 45 at a concentration equivalent to that of the HC2 HR-HPV Screening test. In the study, 5,000 copies per assay of either HPV 16, 18, or 45 plasmids were spiked into either clean STM media or a pool of clinical HC2-negative STM specimens. The identical samples were subsequently tested by HC2 and the HPV genotyping test to compare performance in replicates of eight and repeated three times to demonstrate reproducibility. A negative calibrator was included in the experiments
as the baseline and a positive calibrator (1 pg/ml of an HPV 16 standard plasmid) was included in the experiment for determining RLU/CO. The sensitivity for detecting HPV 16, 18, and 45 was determined exclusive from one another in separate experiments. For both the genotyping and HC2 test, a result was considered positive if it had an RLU/CO value of 1.0. 2.3. HR- and LR-HPV cross-reactivity panels for analytical specificity experiments The HR-HPV cross-reactivity panel was made up of 18 selected HPV-DNA plasmids based on homology to HPV 16, 18, and 45. The panel included all 13 of the HR-HPV genotypes contained in the high-risk RNA probe cocktail provided for the Digene HC2 High-Risk HPV DNA test and five other HR-HPV types. The HR cross-reactivity panel contained the following genotypes: HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82. The assay was tested for cross-reactivity against these HR types at 108 copies per assay or 20 ng/ml (except for HPV 18 and HPV 45, which were also tested against each other at 107 copies per assay or 2 ng/ml). The LR-HPV cross-reactivity panel was made up of 19 selected HPV-DNA plasmids based on homology to HPV 16, 18, and 45. The LR panel contained the following genotypes; HPV 1, 2, 3, 4, 5, 6, 8, 11, 13, 30, 34, 40, 42, 43, 44, 67, 69, 70, and 71. The assay was tested for cross-reactivity against these LR types at 108 copies per assay or 20 ng/ml. The individual HPV 16, 18 and 45 probe mixes contained in the genotyping test were evaluated separately and exclusive of each other probe mix. 2.4. HPV DNA testing by HC2 The Digene HC2 High-Risk HPV DNA test is a signal amplification assay utilizing the Hybrid Capture technology where HPVDNA targets are hybridized to transcribed long RNA probes (~8 kb) in solution and detected via proprietary hybrid-specific antibodies. The HC2 high-risk probe set targets 13 HR-HPV genotypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68). The HC2 test was performed manually according to the manufacturer’s instructions and results for each specimen was expressed in relative light unit/cutoff (RLU/CO), which corresponds to the ratio of the specimen luminescence relative to the luminescence of the cutoff, 1.0 pg/ml (5,000 copies per assay) of HPV 16 provided in the kit. Samples considered HC2-HR positive contained an RLU/CO value of 1.0 and samples considered HC2-HR negative contained an RLU/CO value of <1.0. 2.5. HPV 16, 18, and 45 short sequence-specific synthetic oligoribonucleotides A probe mix comprising short sequence-specific synthetic oligoribonucleotides was separately designed for HPV 16, 18, and 45 for use in the HPV genotyping test. The design and development of the probe mixes were performed with both specificity and sensitivity in mind and optimized specifically for the HPV 16, 18, and 45 genotyping tests. Each probe mix for HPV 16, 18, and 45 are comprised of approximately 150 unique oligoribonucleotides of twenty five bases in length. Design of the sequence-specific oligoribonucleotides was performed utilizing two software programs OligoArray 2.1 (Department of Chemical Engineering, University of Michigan, H.H. Dow, Ann Arbor, MI, USA and Department of Mathematical Sciences,
H. Thai et al. / Journal of Clinical Virology 45 (2009) S93 S97
S95
Table 1 Analytical sensitivity of the genotyping test as compared to HC2 a Mean RLU/CO (%CV) HPV 16 HC2
genotyping
HPV 18 HC2
HPV 45
genotyping
HC2
genotyping
1.00 (6.5%)
1.03 (8.0%)
1.49 (6.6%)
1.07 (3.5%)
1.34 (10.8%)
Clean system (5K plasmid in clean STM) 1.09 (6.5%)
1.40 (4.4%)
1.04 (8.1%)
Clinical background (5K plasmid in negative clinical STM pool) 1.02 (8.0%) a
1.21 (5.6%)
1.12 (6.4%)
1.04 (4.9%)
Each parameter was tested with eight replicates. The mean RLU/CO of the replicates is recorded in the table with the %CV. The RLU/CO is the measure of the RLU of the target/RLU of the positive control. The HPV 16, 18, and 45 genotyping test demonstrated equivalent analytical sensitivity to HC2.
Rensselaer Polytechnic Institute, Troy, NY, USA) and the BLASTN program.5
with 5×105 , 5×106 , 5×107 , or 5×108 copies per assay of either HPV 6 or 31.
2.6. HPV DNA testing by HPV 16, 18, and 45 genotyping test
3. Results
The HPV genotyping test is a non-target amplification assay for the specific detection of HPV 16, 18, and 45 at 1 pg/ml (5,000 copies per assay). Each genotype is detected individually in separate wells. The method is based on the specific hybridization of short sequence-specific synthetic oligoribonucleotide probes in solution to the corresponding DNA target. The DNA RNA hybrids are subsequently captured onto HC2 capture plates and detected via proprietary hybridspecific antibodies conjugated with alkaline phosphatase which provides additional sensitivity via signal amplification. The determination of a positive and negative result is identical to the method used by the HC2 test where an HPV 16-positive standard (1 pg/ml of HPV 16 DNA plasmid) is utilized as the cutoff and the ratio of the specimen luminescence relative to the luminescence of the cutoff is the measurement by which samples are evaluated. A positive HPV genotyping test result produces an RLU/CO value of 1 and a negative HPV genotyping test result produces an RLU/CO value of <1.0. The generic reagents and assay flow of the HPV genotyping test are the same as for the Digene HC2 screening test. An evaluation was also performed using HPV 18 and HPV 45 standards (calibrators) for the detection these two HPV types respectively rather than using HPV 16 as the universal calibrator. The evaluation demonstrated that the results were comparable if we used HPV 16 as the universal calibrator or if we used HPV 16, 18, or 45 as individual calibrators to detect only its own specific HPV type.
3.1. Equivalent sensitivity of the HPV 16, 18, and 45 genotyping test with HC2
2.7. Detecting HPV 16 target in the presence of HPV 6 or 31 at high copy numbers To demonstrate the ability of the HPV genotyping test to detect a single HPV target in the presence of other HPV targets at high concentrations, a study was performed where 5,000 copies per assay of HPV 16 plasmid DNA was spiked into fresh STM media either by itself (baseline) or in the presence of up to 5×108 copies per assay of a competing HPV-DNA plasmid, HPV 31 or HPV 6. The study encompassed three separate experiments and replicates of eight for each condition to demonstrate reproducibility of the results. Conditions tested in each experiment included 5,000 copies per assay of HPV 16 plasmid alone and 5,000 copies per assay of HPV 16 plasmid
On average the genotyping test produced a slightly higher RLU/CO when detecting HPV 16 or 45 (1.40 and 1.49, respectively) as compared to HC2 (1.09 and 1.03, respectively) and HC2 produces a slightly higher RLU/CO (1.04) when detecting HPV 18 as compared to the HPV genotyping test (1.00). The results demonstrate the equivalent performance of the genotyping test to HC2 for each of the three targets (HPV 16, 18, and 45) detected in either a clean system or a system with clinical background (Table 1). In addition, the reproducibility of each test was very good with HC2 generating %CV 8.1% and the genotyping test generating %CV 10.8%. 3.2. Demonstrating high specificity against a panel of HRand LR-HPV genotypes Assay specificity studies demonstrated that the HPV 16, 18, and 45 probe mixes do not cross-react (RLU/CO 1) with any HPV genotypes in the HR and LR panels at concentrations equal to or less than 108 copies per assay. When the HR panel was tested, the RLU/CO ratios ranged from 0.24 to 0.17 for HVP 16, from 0.28 to 0.16 for HPV 18 and from 0.72 to 0.16 for HPV 45 (Table 2). The highest RLU/CO ratio (0.72) was for HPV 45 when tested against HPV 18, a highly similar genotype. However, this RLU/CO ratio was still below the positive cut off value of 1.0. HPV 45 and HPV 18 cross-reactivity was shown to be significantly less at 107 copies per assay (2 ng/ml) with a RLU/CO ratio of 0.21 (Table 2). The RLU/CO ratios ranged from 0.29 to 0.14 for the HPV 16, 18, and 45 assays when tested with the LR panel (Table 3). 3.3. Detection of a specific HPV genotype during multiple infections The results demonstrated that 5,000 copies of HPV 16 were detected when only HPV 16 was in the sample or when HPV 16 was present with HPV 31 or HPV 6 at high concentrations. The RLU/CO ratios when detecting HPV 16 alone or in the presence of HPV 31 or HPV 6 were comparable. The same experiment, repeated in triplicate, yielded an average signal to noise (S/N) ratio for HPV 16 alone of 4.8, while the average S/N ratio of
H. Thai et al. / Journal of Clinical Virology 45 (2009) S93 S97
S96
Table 2 Analytical specificity of the genotyping test against a panel of HR-HPV types a genotype
Pos Con b
16
18 @ 107
18 @ 108
26
31
33
16
2.06 (3%)
n/a
n/a
0.22 (8%)
0.17 (6%)
0.21 (10%)
0.19 (4%)
18
1.43 (8%)
0.2 (13%)
n/a
n/a
0.18 (14%)
0.18 (3%)
0.19 (13%)
45
1.54 (5%)
0.16 (8%)
0.21 (8%)
0.50 (8%)
0.15 (5%)
0.17 (7%)
0.17 (10%)
genotype
35
39
45 @ 107
45 @ 108
51
52
53
16
0.2 (7%)
0.2 (18%)
n/a
0.2 (16%)
0.19 (11%)
0.2 (9%)
0.2 (7%)
18
0.2 (13%)
0.17 (8%)
0.2 (7%)
0.28 (14%)
0.18 (5%)
0.19 (5%)
0.18 (9%)
45
0.17 (7%)
0.16 (8%)
n/a
n/a
0.16 (8%)
0.16 (3%)
0.15 (9%)
genotype
56
58
59
66
68
73
82
16
0.19 (11%)
0.2 (24%)
0.2 (9%)
0.21 (22%)
0.21 (19%)
0.2 (17%)
0.24 (16%)
18
0.2 (11%)
0.2 (9%)
0.17 (3%)
0.16 (8%)
0.19 (7%)
0.18 (11%)
0.17 (10%)
45
0.16 (10%)
0.45 (12%)
0.15 (5%)
0.16 (6%)
0.16 (7%)
0.14 (5%)
0.16 (12%)
a
8
b
Analytical specificity of genotyping test against a panel of HR-HPV genotypes tested at 10 copies per assay (unless noted). HPV 18 was tested against HPV 45 at both 107 and 108 and HPV 45 was tested against HPV 18 at both 107 and 108 . Pos Con is equal to 5000 copies/assay of either HPV 16, HPV 18, or HPV 45. Values presented represent mean RLU/CO (%CV) generated by HPV genotyping test
Table 3 Analytical specificity of the genotyping test against a panel of LR-HPV types a genotype
Pos Con b
1
2
3
4
5
6
16
2.37 (22%)
0.16 (10%)
0.16 (11%)
0.17 (16%)
0.16 (9%)
0.15 (8%)
0.17 (10%)
18
1.36 (12%)
0.23 (12%)
0.23 (11%)
0.21 (10%)
0.21 (10%)
0.21 (10%)
0.24 (8%)
45
1.51 (10%)
0.30 (11%)
0.18 (9%)
0.16 (7%)
0.17 (3%)
0.24 (26%)
0.17 (12%)
genotype
8
11
13
30
34
40
42
16
0.17 (15%)
0.19 (25%)
0.15 (7%)
0.18 (15%)
0.17 (8%)
0.15 (8%)
0.15 (6%)
18
0.25 (7%)
0.24 (7%)
0.25 (23%)
0.19 (4%)
0.2 (6%)
0.21 (9%)
0.2 (9%)
45
0.16 (7%)
0.17 (6%)
0.18 (6%)
0.21 (16%)
0.17 (12%)
0.23 (17%)
0.19 (7%)
genotype
43
44
67
69
70
71
16
0.15 (7%)
0.14 (3%)
0.15 (10%)
0.14 (4%)
0.14 (11%)
0.15 (10%)
18
0.19 (3%)
0.2 (5%)
0.21 (8%)
0.19 (5%)
0.21 (12%)
0.22 (16%)
45
0.21 (23%)
0.24 (13%)
0.18 (13%)
0.17 (11%)
0.19 (9%)
0.16 (12%)
a b
LR-HPV types were tested at a concentration of 108 copies per assay. Pos Con is equal to 5000 copies/assay of either HPV 16, HPV 18, or HPV 45. Values presented represent mean RLU/CO (%CV) generated by HPV genotyping test
HPV 16 in the presence of 108 copies per assay of HPV 31 and HPV 6, respectively, was 4.7 and 4.3. 4. Discussion This preliminary study presents the analytical performance of a hybrid capture-based detection strategy for the specific identification of HPV 16, 18, and 45 DNA targets. The HPV genotyping test does not require target amplification and utilizes the well established Hybrid Capture technology and digene HPV-HC2 test to create a highly-specific assay. The test incorporates a mix of short sequence-specific RNA probes which provide the high analytical specificity for the detection of HPV 16, 18, and 45. High analytical specificity and the ability to detect specific HR-HPV types in a background of multiple HRor LR-HPV types is especially important for an HPV genotyping test used for clinical diagnostics since it has been shown that among women diagnosed with CIN 2+ nearly 80% of HPV clinical samples may have multiple infections.6 Determining with confidence the HPV genotype present could impact patient management. The extremely homologous genotypes
HPV 18 and 45, when tested with the assay did not show crossreactivity against each other at 107 copies per assay and only minimal cross-reactivity at 108 copies per assay. In addition, the results also show that the genotyping test is capable of detecting its designated target in an environment where another HPV genotype is present at high copy numbers. The HPV genotyping test has equivalent sensitivity to the digene HC2 screening test for the detection of HPV 16, 18, and 45. The described genotyping test shares the same standard cutoff as the well established HC2 test giving it the same clinically validated assay sensitivity of 1 pg/ml or 5,000 copies per assay. It has been demonstrated by Snijders et al. that for the population of women with abnormal cytology the detection threshold of 1 pg/ml has predictive value and is above the clinically irrelevant level for HPV viral load.7 In summary, the Digene genotyping test is a simple, reproducible, and analytically accurate assay for the specific detection of HPV 16, 18, and 45. The genotyping test was shown to detect HPV 16, 18, and 45 in cervical specimens that contained both single and multiple HPV genotype infections. The clinical assessment of this assay, using well characterized
H. Thai et al. / Journal of Clinical Virology 45 (2009) S93 S97
patient samples, is essential to fully validate the assay’s performance. Acknowledgements: We would like to personally thank Kim McPartland for her contributions to the project. We are honored and thankful that you choose to spend your brief summer and winter breaks toiling in the laboratory with us. Competing interests: All authors are employees of Qiagen. References 1. Bosch FX, Burchell AN, Schiffman M, Giuliano AR, de Sanjose S, Bruni L, et al. Epidemiology and natural history of human papillomavirus infections and typespecific implications in cervical neoplasia. Vaccine 2008;26(Suppl 10):K1 16.
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2. Clifford GM, Smith JS, Plummer M, Munoz N, Franceschi S. Human papillomavirus types in invasive cervical cancer worldwide: a meta-analysis. Br J Cancer 2003;88:63 73. 3. Herrero R, Hildesheim A, Bratti C, Sherman ME, Hutchinson M, Morales J, et al. Population-based study of human papillomavirus infection and cervical neoplasia in rural Costa Rica. J Natl Cancer Inst 2000;92:464 74. 4. Bosch FX, de Sanjose S. Chapter 1: Human papillomavirus and cervical cancerburden and assessment of causality. J Natl Cancer Inst Monogr 2003;31:3 13. 5. Zhang Z, Schwartz S, Wagner L, Miller W. A greedy algorithm for aligning DNA sequences. J Comput Biol 2000;7(1 2):203 14. 6. Spinillo A, Dal Bello B, Gardella B, Roccio M, Dacc` o MD, Silini EM. Multiple human papillomavirus infection and high grade cervical intraepithelial neoplasia among women with cytological diagnosis of atypical squamous cells of undetermined significance or low grade squamous intraepithelial lesions. Gynecol Oncol 2009 Jan 30 [Epub ahead of print]. 7. Snijders P, van den Brule A, Meijer C. The clinical relevance of human papillomavirus testing: relationship between analytical and clinical sensitivity. J Pathol 2003;201:1 6.
Contents lists available at ScienceDirect
Journal of Clinical Virology j o u r n a l h o m e p a g e : www.elsevier.com/locate/jcv
An HPV 16, 18, and 45 genotyping test based on Hybrid Capture® technology Ha Thai*, Sameera Rangwala, Tanya Gay, Kimberly Keating, Sarah McLeod, Irina Nazarenko, Dominic O’Neil, Dana Pfister, Dirk Loeffert QIAGEN Gaithersburg Inc., Gaithersburg, MD, USA
ARTICLE
INFO
ABSTRACT Background: It has been shown that women positive for HPV 16 and HPV 18 have an increased risk of high-grade cervical intraepithelial neoplasia (CIN) compared with women positive for other high-risk (HR) HPV types. In addition, HPV 18 and HPV 45 have been closely linked to aggressive and difficult to detect adenocarcinomas. Objectives: To develop a test based on the Hybrid Capture® technology capable of specifically detecting the most important carcinogenic HPV types; 16, 18, and 45. Study design: The assay is based on Hybrid Capture technology utilizing a mixture of short type-specific oligoribonucleotides to detect HPV types 16, 18, or 45. The assay utilizes no target amplification and shares workflow and critical reagents with the Digene HC2 HPV screening assay. Studies to evaluate specificity, performance of the test in comparison to HC2, and capability to detect a single genotype in the presence of multiple infections are described. Specificity was evaluated analytically using a panel of HR- and LR-HPV types to illustrate cross-reactivity. Performance in comparison to the HC2 test was evaluated by testing aliquots of the same prepared samples by the genotyping test and HC2. Ability to detect a single genotype during multiple infections was modeled by detecting HPV 16 plasmid in the presence of HPV 6 or HPV 31 at high copy numbers. Results: The proposed genotyping assay specifically detects HPV 16, 18, and 45 with an analytical sensitivity of 5,000 copies per assay. The assay is highly specific and does not detect other tested high-risk or low-risk types at 108 copies per reaction. Utility of the genotyping test was demonstrated using clinical samples collected in Digene Specimen Transport Medium (STM) and results were confirmed by PCR. Conclusions: The target-amplification free assay provides a genotyping method for highly specific detection of HPV 16, 18, and 45 without the complexity of PCR technology. © 2009 Elsevier B.V. All rights reserved.
Keywords: HPV detection HPV genotyping Hybrid Capture Diagnostics
1. Introduction It is widely recognized that persistent infections with highrisk types of human papillomavirus (HR-HPV) are associated with invasive cervical cancer (ICC). Specifically, there are three HR-HPV genotypes, HPV 16, 18, and 45, that are highly prevalent in all regions of the world and frequently identified in women with high-grade cervical intraepithelial neoplasia (CIN 2+) and ICC.1 HPV 16, 18, and 45 have been identified as the primary contributors to ICC accounting for approximately 70% of all cases. HPV 16 is by far the predominant type * Corresponding author. Please add correspondence details as desired? 1590-8658/ $
see front matter © 2009 Elsevier B.V. All rights reserved.
causing greater than 50% of cancers, HPV 18 follows causing 10 14%, and HPV 45 causes approximately 2 8%.2 In addition, HPV 16 and 18 are two of the most common HPV types in women without cancer.3 HPV 45, although an uncommon genotype found amongst women with normal cytology, shows a prevalence of 2.3% in high squamous intraepithelial lesions (HSIL), 4.6% in squamous cervical carcinoma (SCC), and 5.8% in adenocarcinoma (ADC).1 Together, HPV 16, 18, and 45 have been shown to account for nearly 90% of ADC.4 ADC is known to be a much more aggressive and invasive form of cancer as compared to SCC. Technologies available for HPV genotyping vary by method and platform and may offer type-specific characterization
S94
H. Thai et al. / Journal of Clinical Virology 45 (2009) S93 S97
for a multitude of different high- and low-risk HPV types. For example, the Roche Linear Array HPV Genotyping Test (Roche Molecular Diagnostics, Pleasanton, CA, USA) is designed to detect 37 high- and low-risk HPV types. Innogenetics HPV test, INNO-LiPA HPV Genotyping Extra (Innogenetics NV, Gent, Belgium), detects 28 different HPV types. Greiner Bio-One has a test that utilizes a DNA chip, the Papillo Check® HPV-Screening test (Greiner Bio-One International AG, Kremsmuenster, Austria) for the identification of 24 types of genital HPV. These and many more HPV genotyping solutions involve target amplification in the form of PCR and with it the inherent issues and challenges of PCR-based nucleic acid detection tests, including (1) contamination concerns, (2) high level of technical expertise required to execute, (3) overly sensitive, and (4) non-uniform amplification of multiple targets. In this study, we describe an HPV genotyping assay that is not dependent on target amplification. The genotyping test is highly specific for the detection of HPV 16, 18, and 45 and has an equivalent sensitivity to the digene High-Risk HPV HC2 DNA Test (HC2). The Digene HC2 test is the only FDAapproved assay currently in the US market for the detection of HR HPV. It is widely recognized as the gold standard for HR-HPV screening and is a signal amplification assay utilizing the Hybrid Capture® technology where HPV-DNA targets are hybridized to RNA probes in solution and detected via proprietary hybrid-specific antibodies. The proposed HPV 16, 18, and 45 genotyping test is based on Hybrid Capture technology but utilizes a mixture of short type-specific oligoribonucleotides that can detect HPV types 16, 18, and 45 rather than the long transcribed RNA probes utilized in the HC2 test. In addition, the genotyping assay shares workflow and critical reagents with the Digene HC2 HPV screening assay. 2. Materials and methods 2.1. Sample Transport Media (STM) cervical clinical sample pool A negative pool was created from anonymous de-identified STM (Digene) clinical specimens acquired from Kaiser Permanente (San Rafael, CA, USA). Anonymous individual patient specimens, identified as negative for the thirteen HR-HPV genotypes targeted by the Digene HC2 screening test, were combined to create a larger pool that could be used to mimic clinical background in subsequent experiments. A specimen was considered negative for HR HPV if the HC2 relative light unit/cutoff (RLU/CO) value was <1.0. This pool was primarily utilized for the genotyping to HC2 equivalence study. 2.2. Comparison of the sensitivity of the genotyping test to the sensitivity of HC2 A study was performed to determine if the genotyping assay was able to detect HPV 16, 18, and 45 at a concentration equivalent to that of the HC2 HR-HPV Screening test. In the study, 5,000 copies per assay of either HPV 16, 18, or 45 plasmids were spiked into either clean STM media or a pool of clinical HC2-negative STM specimens. The identical samples were subsequently tested by HC2 and the HPV genotyping test to compare performance in replicates of eight and repeated three times to demonstrate reproducibility. A negative calibrator was included in the experiments
as the baseline and a positive calibrator (1 pg/ml of an HPV 16 standard plasmid) was included in the experiment for determining RLU/CO. The sensitivity for detecting HPV 16, 18, and 45 was determined exclusive from one another in separate experiments. For both the genotyping and HC2 test, a result was considered positive if it had an RLU/CO value of 1.0. 2.3. HR- and LR-HPV cross-reactivity panels for analytical specificity experiments The HR-HPV cross-reactivity panel was made up of 18 selected HPV-DNA plasmids based on homology to HPV 16, 18, and 45. The panel included all 13 of the HR-HPV genotypes contained in the high-risk RNA probe cocktail provided for the Digene HC2 High-Risk HPV DNA test and five other HR-HPV types. The HR cross-reactivity panel contained the following genotypes: HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82. The assay was tested for cross-reactivity against these HR types at 108 copies per assay or 20 ng/ml (except for HPV 18 and HPV 45, which were also tested against each other at 107 copies per assay or 2 ng/ml). The LR-HPV cross-reactivity panel was made up of 19 selected HPV-DNA plasmids based on homology to HPV 16, 18, and 45. The LR panel contained the following genotypes; HPV 1, 2, 3, 4, 5, 6, 8, 11, 13, 30, 34, 40, 42, 43, 44, 67, 69, 70, and 71. The assay was tested for cross-reactivity against these LR types at 108 copies per assay or 20 ng/ml. The individual HPV 16, 18 and 45 probe mixes contained in the genotyping test were evaluated separately and exclusive of each other probe mix. 2.4. HPV DNA testing by HC2 The Digene HC2 High-Risk HPV DNA test is a signal amplification assay utilizing the Hybrid Capture technology where HPVDNA targets are hybridized to transcribed long RNA probes (~8 kb) in solution and detected via proprietary hybrid-specific antibodies. The HC2 high-risk probe set targets 13 HR-HPV genotypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68). The HC2 test was performed manually according to the manufacturer’s instructions and results for each specimen was expressed in relative light unit/cutoff (RLU/CO), which corresponds to the ratio of the specimen luminescence relative to the luminescence of the cutoff, 1.0 pg/ml (5,000 copies per assay) of HPV 16 provided in the kit. Samples considered HC2-HR positive contained an RLU/CO value of 1.0 and samples considered HC2-HR negative contained an RLU/CO value of <1.0. 2.5. HPV 16, 18, and 45 short sequence-specific synthetic oligoribonucleotides A probe mix comprising short sequence-specific synthetic oligoribonucleotides was separately designed for HPV 16, 18, and 45 for use in the HPV genotyping test. The design and development of the probe mixes were performed with both specificity and sensitivity in mind and optimized specifically for the HPV 16, 18, and 45 genotyping tests. Each probe mix for HPV 16, 18, and 45 are comprised of approximately 150 unique oligoribonucleotides of twenty five bases in length. Design of the sequence-specific oligoribonucleotides was performed utilizing two software programs OligoArray 2.1 (Department of Chemical Engineering, University of Michigan, H.H. Dow, Ann Arbor, MI, USA and Department of Mathematical Sciences,
H. Thai et al. / Journal of Clinical Virology 45 (2009) S93 S97
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Table 1 Analytical sensitivity of the genotyping test as compared to HC2 a Mean RLU/CO (%CV) HPV 16 HC2
genotyping
HPV 18 HC2
HPV 45
genotyping
HC2
genotyping
1.00 (6.5%)
1.03 (8.0%)
1.49 (6.6%)
1.07 (3.5%)
1.34 (10.8%)
Clean system (5K plasmid in clean STM) 1.09 (6.5%)
1.40 (4.4%)
1.04 (8.1%)
Clinical background (5K plasmid in negative clinical STM pool) 1.02 (8.0%) a
1.21 (5.6%)
1.12 (6.4%)
1.04 (4.9%)
Each parameter was tested with eight replicates. The mean RLU/CO of the replicates is recorded in the table with the %CV. The RLU/CO is the measure of the RLU of the target/RLU of the positive control. The HPV 16, 18, and 45 genotyping test demonstrated equivalent analytical sensitivity to HC2.
Rensselaer Polytechnic Institute, Troy, NY, USA) and the BLASTN program.5
with 5×105 , 5×106 , 5×107 , or 5×108 copies per assay of either HPV 6 or 31.
2.6. HPV DNA testing by HPV 16, 18, and 45 genotyping test
3. Results
The HPV genotyping test is a non-target amplification assay for the specific detection of HPV 16, 18, and 45 at 1 pg/ml (5,000 copies per assay). Each genotype is detected individually in separate wells. The method is based on the specific hybridization of short sequence-specific synthetic oligoribonucleotide probes in solution to the corresponding DNA target. The DNA RNA hybrids are subsequently captured onto HC2 capture plates and detected via proprietary hybridspecific antibodies conjugated with alkaline phosphatase which provides additional sensitivity via signal amplification. The determination of a positive and negative result is identical to the method used by the HC2 test where an HPV 16-positive standard (1 pg/ml of HPV 16 DNA plasmid) is utilized as the cutoff and the ratio of the specimen luminescence relative to the luminescence of the cutoff is the measurement by which samples are evaluated. A positive HPV genotyping test result produces an RLU/CO value of 1 and a negative HPV genotyping test result produces an RLU/CO value of <1.0. The generic reagents and assay flow of the HPV genotyping test are the same as for the Digene HC2 screening test. An evaluation was also performed using HPV 18 and HPV 45 standards (calibrators) for the detection these two HPV types respectively rather than using HPV 16 as the universal calibrator. The evaluation demonstrated that the results were comparable if we used HPV 16 as the universal calibrator or if we used HPV 16, 18, or 45 as individual calibrators to detect only its own specific HPV type.
3.1. Equivalent sensitivity of the HPV 16, 18, and 45 genotyping test with HC2
2.7. Detecting HPV 16 target in the presence of HPV 6 or 31 at high copy numbers To demonstrate the ability of the HPV genotyping test to detect a single HPV target in the presence of other HPV targets at high concentrations, a study was performed where 5,000 copies per assay of HPV 16 plasmid DNA was spiked into fresh STM media either by itself (baseline) or in the presence of up to 5×108 copies per assay of a competing HPV-DNA plasmid, HPV 31 or HPV 6. The study encompassed three separate experiments and replicates of eight for each condition to demonstrate reproducibility of the results. Conditions tested in each experiment included 5,000 copies per assay of HPV 16 plasmid alone and 5,000 copies per assay of HPV 16 plasmid
On average the genotyping test produced a slightly higher RLU/CO when detecting HPV 16 or 45 (1.40 and 1.49, respectively) as compared to HC2 (1.09 and 1.03, respectively) and HC2 produces a slightly higher RLU/CO (1.04) when detecting HPV 18 as compared to the HPV genotyping test (1.00). The results demonstrate the equivalent performance of the genotyping test to HC2 for each of the three targets (HPV 16, 18, and 45) detected in either a clean system or a system with clinical background (Table 1). In addition, the reproducibility of each test was very good with HC2 generating %CV 8.1% and the genotyping test generating %CV 10.8%. 3.2. Demonstrating high specificity against a panel of HRand LR-HPV genotypes Assay specificity studies demonstrated that the HPV 16, 18, and 45 probe mixes do not cross-react (RLU/CO 1) with any HPV genotypes in the HR and LR panels at concentrations equal to or less than 108 copies per assay. When the HR panel was tested, the RLU/CO ratios ranged from 0.24 to 0.17 for HVP 16, from 0.28 to 0.16 for HPV 18 and from 0.72 to 0.16 for HPV 45 (Table 2). The highest RLU/CO ratio (0.72) was for HPV 45 when tested against HPV 18, a highly similar genotype. However, this RLU/CO ratio was still below the positive cut off value of 1.0. HPV 45 and HPV 18 cross-reactivity was shown to be significantly less at 107 copies per assay (2 ng/ml) with a RLU/CO ratio of 0.21 (Table 2). The RLU/CO ratios ranged from 0.29 to 0.14 for the HPV 16, 18, and 45 assays when tested with the LR panel (Table 3). 3.3. Detection of a specific HPV genotype during multiple infections The results demonstrated that 5,000 copies of HPV 16 were detected when only HPV 16 was in the sample or when HPV 16 was present with HPV 31 or HPV 6 at high concentrations. The RLU/CO ratios when detecting HPV 16 alone or in the presence of HPV 31 or HPV 6 were comparable. The same experiment, repeated in triplicate, yielded an average signal to noise (S/N) ratio for HPV 16 alone of 4.8, while the average S/N ratio of
H. Thai et al. / Journal of Clinical Virology 45 (2009) S93 S97
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Table 2 Analytical specificity of the genotyping test against a panel of HR-HPV types a genotype
Pos Con b
16
18 @ 107
18 @ 108
26
31
33
16
2.06 (3%)
n/a
n/a
0.22 (8%)
0.17 (6%)
0.21 (10%)
0.19 (4%)
18
1.43 (8%)
0.2 (13%)
n/a
n/a
0.18 (14%)
0.18 (3%)
0.19 (13%)
45
1.54 (5%)
0.16 (8%)
0.21 (8%)
0.50 (8%)
0.15 (5%)
0.17 (7%)
0.17 (10%)
genotype
35
39
45 @ 107
45 @ 108
51
52
53
16
0.2 (7%)
0.2 (18%)
n/a
0.2 (16%)
0.19 (11%)
0.2 (9%)
0.2 (7%)
18
0.2 (13%)
0.17 (8%)
0.2 (7%)
0.28 (14%)
0.18 (5%)
0.19 (5%)
0.18 (9%)
45
0.17 (7%)
0.16 (8%)
n/a
n/a
0.16 (8%)
0.16 (3%)
0.15 (9%)
genotype
56
58
59
66
68
73
82
16
0.19 (11%)
0.2 (24%)
0.2 (9%)
0.21 (22%)
0.21 (19%)
0.2 (17%)
0.24 (16%)
18
0.2 (11%)
0.2 (9%)
0.17 (3%)
0.16 (8%)
0.19 (7%)
0.18 (11%)
0.17 (10%)
45
0.16 (10%)
0.45 (12%)
0.15 (5%)
0.16 (6%)
0.16 (7%)
0.14 (5%)
0.16 (12%)
a
8
b
Analytical specificity of genotyping test against a panel of HR-HPV genotypes tested at 10 copies per assay (unless noted). HPV 18 was tested against HPV 45 at both 107 and 108 and HPV 45 was tested against HPV 18 at both 107 and 108 . Pos Con is equal to 5000 copies/assay of either HPV 16, HPV 18, or HPV 45. Values presented represent mean RLU/CO (%CV) generated by HPV genotyping test
Table 3 Analytical specificity of the genotyping test against a panel of LR-HPV types a genotype
Pos Con b
1
2
3
4
5
6
16
2.37 (22%)
0.16 (10%)
0.16 (11%)
0.17 (16%)
0.16 (9%)
0.15 (8%)
0.17 (10%)
18
1.36 (12%)
0.23 (12%)
0.23 (11%)
0.21 (10%)
0.21 (10%)
0.21 (10%)
0.24 (8%)
45
1.51 (10%)
0.30 (11%)
0.18 (9%)
0.16 (7%)
0.17 (3%)
0.24 (26%)
0.17 (12%)
genotype
8
11
13
30
34
40
42
16
0.17 (15%)
0.19 (25%)
0.15 (7%)
0.18 (15%)
0.17 (8%)
0.15 (8%)
0.15 (6%)
18
0.25 (7%)
0.24 (7%)
0.25 (23%)
0.19 (4%)
0.2 (6%)
0.21 (9%)
0.2 (9%)
45
0.16 (7%)
0.17 (6%)
0.18 (6%)
0.21 (16%)
0.17 (12%)
0.23 (17%)
0.19 (7%)
genotype
43
44
67
69
70
71
16
0.15 (7%)
0.14 (3%)
0.15 (10%)
0.14 (4%)
0.14 (11%)
0.15 (10%)
18
0.19 (3%)
0.2 (5%)
0.21 (8%)
0.19 (5%)
0.21 (12%)
0.22 (16%)
45
0.21 (23%)
0.24 (13%)
0.18 (13%)
0.17 (11%)
0.19 (9%)
0.16 (12%)
a b
LR-HPV types were tested at a concentration of 108 copies per assay. Pos Con is equal to 5000 copies/assay of either HPV 16, HPV 18, or HPV 45. Values presented represent mean RLU/CO (%CV) generated by HPV genotyping test
HPV 16 in the presence of 108 copies per assay of HPV 31 and HPV 6, respectively, was 4.7 and 4.3. 4. Discussion This preliminary study presents the analytical performance of a hybrid capture-based detection strategy for the specific identification of HPV 16, 18, and 45 DNA targets. The HPV genotyping test does not require target amplification and utilizes the well established Hybrid Capture technology and digene HPV-HC2 test to create a highly-specific assay. The test incorporates a mix of short sequence-specific RNA probes which provide the high analytical specificity for the detection of HPV 16, 18, and 45. High analytical specificity and the ability to detect specific HR-HPV types in a background of multiple HRor LR-HPV types is especially important for an HPV genotyping test used for clinical diagnostics since it has been shown that among women diagnosed with CIN 2+ nearly 80% of HPV clinical samples may have multiple infections.6 Determining with confidence the HPV genotype present could impact patient management. The extremely homologous genotypes
HPV 18 and 45, when tested with the assay did not show crossreactivity against each other at 107 copies per assay and only minimal cross-reactivity at 108 copies per assay. In addition, the results also show that the genotyping test is capable of detecting its designated target in an environment where another HPV genotype is present at high copy numbers. The HPV genotyping test has equivalent sensitivity to the digene HC2 screening test for the detection of HPV 16, 18, and 45. The described genotyping test shares the same standard cutoff as the well established HC2 test giving it the same clinically validated assay sensitivity of 1 pg/ml or 5,000 copies per assay. It has been demonstrated by Snijders et al. that for the population of women with abnormal cytology the detection threshold of 1 pg/ml has predictive value and is above the clinically irrelevant level for HPV viral load.7 In summary, the Digene genotyping test is a simple, reproducible, and analytically accurate assay for the specific detection of HPV 16, 18, and 45. The genotyping test was shown to detect HPV 16, 18, and 45 in cervical specimens that contained both single and multiple HPV genotype infections. The clinical assessment of this assay, using well characterized
H. Thai et al. / Journal of Clinical Virology 45 (2009) S93 S97
patient samples, is essential to fully validate the assay’s performance. Acknowledgements: We would like to personally thank Kim McPartland for her contributions to the project. We are honored and thankful that you choose to spend your brief summer and winter breaks toiling in the laboratory with us. Competing interests: All authors are employees of Qiagen. References 1. Bosch FX, Burchell AN, Schiffman M, Giuliano AR, de Sanjose S, Bruni L, et al. Epidemiology and natural history of human papillomavirus infections and typespecific implications in cervical neoplasia. Vaccine 2008;26(Suppl 10):K1 16.
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2. Clifford GM, Smith JS, Plummer M, Munoz N, Franceschi S. Human papillomavirus types in invasive cervical cancer worldwide: a meta-analysis. Br J Cancer 2003;88:63 73. 3. Herrero R, Hildesheim A, Bratti C, Sherman ME, Hutchinson M, Morales J, et al. Population-based study of human papillomavirus infection and cervical neoplasia in rural Costa Rica. J Natl Cancer Inst 2000;92:464 74. 4. Bosch FX, de Sanjose S. Chapter 1: Human papillomavirus and cervical cancerburden and assessment of causality. J Natl Cancer Inst Monogr 2003;31:3 13. 5. Zhang Z, Schwartz S, Wagner L, Miller W. A greedy algorithm for aligning DNA sequences. J Comput Biol 2000;7(1 2):203 14. 6. Spinillo A, Dal Bello B, Gardella B, Roccio M, Dacc` o MD, Silini EM. Multiple human papillomavirus infection and high grade cervical intraepithelial neoplasia among women with cytological diagnosis of atypical squamous cells of undetermined significance or low grade squamous intraepithelial lesions. Gynecol Oncol 2009 Jan 30 [Epub ahead of print]. 7. Snijders P, van den Brule A, Meijer C. The clinical relevance of human papillomavirus testing: relationship between analytical and clinical sensitivity. J Pathol 2003;201:1 6.