Journal of Clinical Virology Digene HPV Genotyping

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BoÅ¡tjan J. Kocjana, Adriana Vinceb, Mario Poljaka,∗ a Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, ZaloÅ¡ka 4, 1000 ...
Journal of Clinical Virology 46 (2009) 176–179

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Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv

Short communication

Digene HPV Genotyping RH Test RUO: Comparative evaluation with INNO-LiPA HPV Genotyping Extra Test for detection of 18 high-risk and probable high-risk human papillomavirus genotypes ´ Katja Seme a , Snjeˇzana Zˇ idovec Lepej b , Maja M. Lunar a , Janja Iˇscˇ ic-Beˇ s b , Ana Planinic´ b , a b a,∗ Boˇstjan J. Kocjan , Adriana Vince , Mario Poljak a b

Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Zaloˇska 4, 1000 Ljubljana, Slovenia University Hospital for Infectious Diseases, Zagreb, Croatia

a r t i c l e

i n f o

Article history: Received 29 April 2009 Received in revised form 20 July 2009 Accepted 22 July 2009 Keywords: HPV Genotyping Digene INNO-LiPA Reverse hybridization

a b s t r a c t Background: Standardized and validated methods for the specific detection and identification of a spectrum of high-risk (hr) HPV genotypes will be necessary if HPV genotyping gains an important role in the clinical management of HPV-related precancerous lesions and cancers. Objectives: The first comparative evaluation of novel HPV genotyping Digene HPV Genotyping RH Test RUO (Qiagen, Hilden, Germany) with standard INNO-LiPA HPV Genotyping Extra CE assay (Innogenetics, Gent, Belgium). Study design: Seventy hr-HPV positive samples were tested in parallel with both genotyping assays. The results were interpreted taking into account 15 hr-HPV and 3 probable hr-HPV genotypes that can be identified by both assays (assay-common genotypes). Results: Concordant results (a complete match of assay-common genotypes or negative using both assays) and compatible results (at least one genotype in common) were obtained in 42 (60.0%) and 28 (40.0%) samples, respectively. No discordant results for assay-common genotypes were obtained. Of 42 samples with compatible results, the presence of at least one assay-common genotype was detected in 37 samples, while no HPV was detected in two samples by both assays and only a single low-risk HPV was detected by INNO-LiPA in three samples. Conclusions: A novel Digene test is suitable for the detection of hr-HPV genotypes in clinical samples and it provides comparable results to the well established INNO-LiPA assay. Although INNO-LiPA identified significantly more samples with multiple HPV genotypes than the Digene test, the clinical benefit of such a difference is at present unclear. © 2009 Elsevier B.V. All rights reserved.

1. Background Molecular and epidemiologic studies have shown that persistent infection with 15 high-risk human papillomavirus (HPV) genotypes is a necessary but not sufficient etiological factor for the development of cervical carcinoma.1–3 Detection of high-risk HPV (hr-HPV) has consequently become an important part of cervical carcinoma screening and detection algorithms.4–6 For these screening purposes, a range of in-house and commercial molecular tests have been developed in order to distinguish hr-HPV infections from no HPV infection.7,8 The Digene Hybrid Capture II Test (hc2) (Qiagen, Hilden, Germany), is at present the most frequently used

∗ Corresponding author. Tel.: +386 1 543 7453; fax: +386 1 543 7418. E-mail address: [email protected] (M. Poljak). 1386-6532/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2009.07.017

HPV screening test. Although very useful, hc2 does not allow the identification of specific hr-HPV genotypes.9,10 In contrast to hc2, the clinical value of HPV methods that allow an exact determination of HPV genotype(s) has still not been finally determined. Currently, HPV genotyping methods are indispensable for studies of the transmission, natural history, pathogenesis and prevention of HPV. However, it is highly likely that genotyping methods will also have an important role in the clinical management of HPV-related precancerous lesions and cancers in the near future.11,12 Recent studies have thus provided strong evidence that HPV genotyping may be clinically useful for stratifying the risk of women developing a precancerous cervical lesion due to differences in the oncogenic potential of different hr-HPV.13,14 As the use of prophylactic HPV vaccines, becomes more widespread, surveillance for population-level effectiveness will become an increasingly important activity, which will require the use of an HPV genotyping method.12 If HPV genotyping is to be used for diagnostic applications and not just as research tool, it requires

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Table 1 Results of comparative evaluation of Digene and INNO-LiPA HPV genotyping assays. Non assay-common genotypes are bolded. For interpretation criteria see Materials and Methods. Sample no.

Digene result

INNO-LiPA result

Interpretation

Frequency

Cytology

1–14 15–18 19–20 21 and 22 23 24 and 25 26 and 27 28 29 and 30 31 32 33 34 35 36 37 38 and 39 40 41 and 42 43 and 44 45 and 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70

16 18 31 31 31 33 51 56 59 58 53 16, 33 16, 45 16, 53 18, 53 51, 53 NT NT Negative 16 18 16 16 16 16 16 31 31 33 51 51 51 58 56 56 58 59 66 66 16, 56 16, 56 51, 16 58, 16 16, 56 68, 18

16 18 31 31(31 + 54) 31, 6 33 51 56, 6 59 58 53 16, 33(33 + 54) 16, 45 16, 53 18, 53 51, 53 6 11 Negative 16, 51 18, 51 16, 45 16, 18, 68 16, 33(33 + 54) 16, 66, 68 16, 51, 11, 40, 54 31(31 + 54), 58 31, 51, 66 33(33 + 54), 53 51, 18 51, 53 51, 39, 66, 73, 68 58, 59 56, 52 56, 66 58, 31(31 + 54) 59, 33(33 + 54), 45 66, 16 66, 31(31 + 54) 16, 56, 52 16 51 58 16, 53, 68, 66 68, 31(31 + 54)

Concordant Concordant Concordant Concordant Concordant Concordant Concordant Concordant Concordant Concordant Concordant Concordant Concordant Concordant Concordant Concordant Concordant Concordant Concordant Compatible Compatible Compatible Compatible Compatible Compatible Compatible Compatible Compatible Compatible Compatible Compatible Compatible Compatible Compatible Compatible Compatible Compatible Compatible Compatible Compatible Compatible Compatible Compatible Compatible Compatible

14 4 2 2 1 2 2 1 2 1 1 1 1 1 1 1 2 1 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

7 × HSIL, 2 × ASCUS, 5 × normal 4 × HSIL 2 × ASCUS 1 × HSIL, 1 × normal HSIL 2 × LSIL 1 × LSIL, 1 × normal ASCUS 1 × LSIL, 1 × ASCUS ASCUS ASCUS HSIL ASCUS ASCUS ASCUS ASCUS 1 × ASCUS, 1 × normal ASCUS 1 × HSIL, 1 × LSIL 1 × HSIL, 1 × ASCUS 1 × LSIL, 1 × ASCUS LSIL ASCUS ASCUS ASCUS ASCUS HSIL LSIL HSIL ASCUS HSIL ASCUS LSIL ASCUS ASCUS ASCUS LSIL LSIL ASCUS HSIL HSIL ASCUS HSIL HSIL HSIL

NT: untypable result; HSIL: high-grade squamous intraepithelial lesions; ASCUS: atypical squamous cells of undetermined significance; LSIL: low-grade squamous intraepithelial lesions.

standardized and validated methods for the specific detection and identification of a defined spectrum of HPV genotypes.15 Although DNA sequencing is considered to be the “gold standard” for HPV genotyping, it is costly, time-consuming and difficult to apply in routine diagnostic settings.12 Currently, the most widely used HPV genotyping tests are based on reverse hybridization of amplified HPV products on a membrane strip containing multiple probes immobilized as parallel lines, such as Linear Array HPV Genotyping Test (Roche Molecular Diagnostics, Pleasanton, CA) and INNO-LiPA HPV Assay (Innogenetics, Gent, Belgium).11,16 In December 2008, a novel reverse line-blot based hybridisation assay, Digene HPV Genotyping RH Test RUO (Qiagen, Hilden, Germany), was introduced on the European market. It identifies 15 hrHPV (HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-68, HPV-73 and HPV-82) and 3 probable hr-HPV (HPV-26, HPV-53, and HPV-66). 2. Objectives In this study, we evaluated the performance of the novel HPV genotyping kit Digene HPV Genotyping RH Test RUO in comparison

to the standard HPV genotyping assay, INNO-LiPA HPV Genotyping Extra CE (INNO-LiPA), in a routine diagnostic laboratory setting. The comparator assay identifies the same 15 hr-HPV and 3 probable hr-HPV genotypes as Digene assay and, in addition, 7 low-risk HPV genotypes (HPV-6, HPV-11, HPV-40, HPV-43, HPV-44, HPV-54 and HPV-70). To the best of our knowledge, this study is first to evaluate the performance of Digene HPV Genotyping RH Test RUO. 3. Study design The study enrolled 1615 women who attended the Outpatient STD Clinics at the University Hospital for Infectious Diseases, Zagreb, Croatia for HPV DNA testing and treatment follow-up. DNA of hr-HPV genotypes was detected in cervical swab specimens in 618/1615 women (38.3%) using a high-risk hc2 cocktail (Qiagen). For this study, 70 hr-HPV hc2-positive samples were randomly selected and genotyped with Digene HPV Genotyping RH Test RUO and INNO-LiPA HPV Genotyping Extra CE, following the manufacturers’ instructions. For both genotyping assays, DNA was isolated using QIAamp DNA Mini Kit (Qiagen) from the same 200 ␮l aliquot of cervical specimens that had been deliberately removed from

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Digene Specimen Transport Medium® (Qiagen) before the addition of a denaturating reagent (the first step of hc2 testing). DNA was eluted in a final volume of 50 ␮l and 10 ␮l was used for each genotyping assay. The performance of the two assays was compared by taking into account 15 hr-HPV and 3 probable hr-HPV genotypes that can be identified by both genotyping assays (assay-common genotypes). The results of the comparison were considered concordant if a complete match of assay-common genotypes or negative result were obtained by both assays, compatible if at least one genotype in common was detected and discordant if different assay-common HPV genotypes were detected by the two assays.

concordant results for HPV-16 and HPV-18 were obtained in 13/16 (81%) and 3/6 (50%) samples, respectively. INNO-LiPA and Digene missed HPV-16 and HPV-18 in three samples each. The clinical relevance of undetected HPV-16 and HPV-18 infections in six samples with discordant results is unclear since cytological abnormalities found in these samples might be caused by other hr-HPV present in these samples rather then by HPV-16 and HPV-18 which were missed by one of two assays. In conclusion, although performed on the relatively small sample size, our study showed that the Digene test is a reliable new diagnostic tool for the detection and identification of hr-HPV genotypes in clinical samples.

4. Results

Conflict of interest

As shown in Table 1, 8 women had normal cytology, 27 atypical squamous cells of undetermined significance (ASCUS), 11 lowgrade squamous intraepithelial lesions (LSIL) and 24 high-grade squamous intraepithelial lesions (HSIL). Concordant and compatible results for assay-common genotypes were obtained in 42 (60.0%) and 28 (40.0%) samples, respectively. No discordant results for assay-common genotypes were obtained. Of 42 samples with concordant results, the presence of at least one assay-common genotype was detected in 37 (88.0%) samples, while no HPV was detected by both assays in two samples (samples 41 and 42) and only a single low-risk HPV was detected by INNO-LiPA in three samples (samples 38–40). The absence of HPV DNA in samples 41 and 42 was additionally confirmed using in-house PCRs, using 3 different HPV consensus primers: CPI/IIg, MY09/11 and GP5+/6+.17–19 The presence of low-risk-HPV only in samples 38–40 was additionally confirmed using Linear Array HPV Genotyping Test (Roche Molecular Diagnostics, Pleasanton, CA). These results are probably explained by previously described findings,9–10 hc2 false positivity due to cross-reactivity of the hr-hc2 probe cocktail with untargeted HPV genotypes (samples 38–40) or may be due to unknown reasons (samples 41 and 42). In 32 of 37 (86.5%) samples with concordant results, a single assay-common HPV genotype was detected. Co-infection with two hr-HPV genotypes was detected in 5 of 37 (13.5%) samples with concordant results. Of 28 samples with compatible results, INNO-LiPA detected additional assay-common genotypes in 23 samples (1, 2 and 4 additional genotype in samples 18, 4 and 1, respectively). The Digene test detected one additional assay-common genotype in three samples. In two samples, (samples 69 and 70) both tests detected additional assay-common genotypes.

Mario Poljak has served as consultant and received lecture, advisory board and consultancy fees from Abbott Molecular and Roche. Snjeˇzana Zˇ idovec Lepej has received travel grants from Digene and Roche.

5. Discussion The first evaluation of the novel Digene HPV Genotyping RH Test RUO showed that this test provides comparable results to the well established INNO-LiPA assay. Although INNO-LiPA identified significantly more samples with multiple HPV genotypes than the Digene test, the clinical benefit of such a difference is at present unclear due to the lack of evidence that the presence of multiple hr-HPV rather than a single hr-HPV is connected with different risk for cervical cancer. The most possible reason for increased detection rate of multiple HPV infection by INNO-LiPA is considerably smaller amplicon size in comparison to Digene test (65-bp versus 150-bp). Recent studies showed that HPV-16 and HPV-18 are remarkably powerful human carcinogens that are different from other hr-HPV genotypes in a way that merits separate laboratory and clinical consideration.13,14 Thus we performed an additional analysis to measure assays concordance for those two genotypes in 21 samples containing HPV-16 and/or HPV-18. As shown in Table 1,

Acknowledgements This study was partially sponsored by the Croatian Ministry of Science, Education and Sports through grants 143-1080116-0097 and 143-0000000-0117, by the Slovenian Research Agency through grant Z3-0220-0381-08 and through Slovenian–Croatian bilateral grant BI-HR/09-10-017. References [1]. de Villiers EM, Fauquet C, Broker TR, Bernard HU, zur Hausen H. Classification of papillomaviruses. Virology 2004;324:17–27. [2]. Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003;348:518–27. [3]. Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999;189:12–9. [4]. Cuschieri KS, Cubie HA. The role of human papillomavirus testing in cervical screening. J Clin Virol 2005;32(Suppl. 1):S34–42. [5]. Iftner T, Villa LL. Human papillomavirus technologies. J Natl Cancer Inst Monogr 2003;31:80–8. [6]. Smith RA, Cokkinides V, Eyre HJ. American Cancer Society guidelines for the early detection of cancer, 2003. CA Cancer J Clin 2003;53:27–43. [7]. Wright Jr TC, Cox JT, Massad LS, Twiggs LB, Wilkinson EJ, ASCCP-Sponsored Consensus Conference, 2002. 2001 Consensus Guidelines for the management of women with cervical cytological abnormalities. JAMA 2001;287: 2120–9. [8]. Solomon D, Schiffman M, Tarone R, The ALTS Study Group. Human papillomavirus testing for triage of women with cytologic evidence of low-grade squamous intraepithelial lesions: baseline data from a randomized trial. J Natl Cancer Inst 2000;92:397–402. [9]. Poljak M, Marin IJ, Seme K, Vince A. Hybrid Capture II HPV test detects at least 15 human papillomavirus genotypes not included in its current high risk cocktail. J Clin Virol 2002;25:S89–97. [10]. Castle PE, Solomon D, Wheeler CM, Gravitt PE, Wacholder S, Schiffman M. Human papillomavirus genotype specificity of hybrid capture 2. J Clin Microbiol 2008;46:2595–604. [11]. van Hamont D, van Ham MA, Bakkers JM, Massuger LF, Melchers WJ. Evaluation of the SPF10-INNO LiPA human papillomavirus (HPV) genotyping test and the Roche linear array HPV genotyping test. J Clin Microbiol 2006;449: 3122–9. [12]. Feng Q, Cherne S, Winer RL, Balasubramanian A, Lee SK, Hawes SE, et al. Development and evaluation of a liquid bead microarray assay for genotyping genital human papillomaviruses. J Clin Microbiol 2009;47:547–53. [13]. Castle PE, Solomon D, Schiffman M, Wheeler CM. Human papillomavirus type 16 infections and 2-year absolute risk of cervical precancer in women with equivocal or mild cytologic abnormalities. J Natl Cancer Inst 2005;97: 1066–71. [14]. Khan MJ, Castle PE, Lorincz AT, Wacholder S, Sherman M, Scott DR, et al. The elevated 10-year risk of cervical precancer and cancer in women with human papillomavirus (HPV) type 16 or 18 and the possible utility of type-specific HPV testing in clinical practice. J Natl Cancer Inst 2005;97:1072–9. [15]. Dalstein V, Merlin S, Bali C, Saunier M, Dachez R, Ronsin C. Analytical evaluation of the PapilloCheck test, a new commercial DNA chip for detection and genotyping of human papillomavirus. J Virol Methods 2009;156:77–83.

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