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Comparison of the Abbott RealTime High Risk HPV test and INNO-LiPA HPV Genotyping Extra test for the detection of human papillomaviruses in formalin-fixed, paraffin-embedded cervical cancer specimens
Journal of Virological Methods 175 (2011) 117–119
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
Comparison of the Abbott RealTime High Risk HPV test and INNO-LiPA HPV Genotyping Extra test for the detection of human papillomaviruses in formalin-fixed, paraffin-embedded cervical cancer specimens Boˇstjan J. Kocjan, Katja Seme, Mario Poljak ∗ Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Zaloˇska 4, 1105 Ljubljana, Slovenia
a b s t r a c t Article history: Received 2 February 2011 Received in revised form 29 March 2011 Accepted 5 April 2011 Available online 12 April 2011 Keywords: HPV Genotyping INNO-LiPA Abbott RealTime Archival specimens
Comparative evaluation of Abbott RealTime and Innogenetics INNO-LiPA on alternately processed formalin-fixed, paraffin-embedded specimens of 31 cervical cancers and 31 uterine myomas showed complete agreement in the detection of 14 assay-common HPV genotypes and partial genotyping of HPV-16 and HPV-18. The tissue preparation protocol was shown to be sample-to-sample contamination safe. © 2011 Elsevier B.V. All rights reserved.
Cervical cancer is the second most frequent cancer in women worldwide. Persistent infection with high-risk alpha-human papillomaviruses (hr-HPV) is a necessary although not sufficient etiological factor for the development of cervical cancer and its immediate precursors (Bouvard et al., 2009; Schiffman et al., 2009). In addition to cervical cancer, hr-HPVs, the most frequent being HPV-16, play the leading etiological role in the development of anal cancer and a substantial proportion of vaginal, penile, vulvar and oropharyngeal cancers (zur Hausen, 2009). Formalin-fixation and subsequent paraffin/paraplast embedding (FFPE) is a standard method for long-term preservation of tissue specimens in pathological departments worldwide. These specimens, used routinely for histopathological diagnoses, also represent an invaluable resource for etiological and epidemiological studies of HPV and other viral infections in cases in which fresh or frozen tissue is not available (Janˇcar et al., 2009; Poljak et al., 1998; Tan et al., 2010). However, the detection of viruses in FFPE specimens is often challenging, due to substantial degradation of viral DNA/RNA as a result of excessive fixation or tissue ageing or the presence of substances that inhibit PCR amplification ˜ or proteinase K (Gilbert et al., 2007; Gillio-Tos et al., 2007; MunozCadavid et al., 2010; Tan et al., 2010). Since the fragmentation of DNA influences significantly the efficiency of PCR amplification,
∗ Corresponding author. Tel.: +386 1 543 7453; fax: +386 1 543 7418. E-mail address: [email protected] (M. Poljak). 0166-0934/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2011.04.006
HPV methods that amplify a relatively small portion of the viral genome are the most suitable when working with archival specimens (Cuschieri and Cubie, 2005; Greer et al., 1991; Poljak et al., 1998, 2000; Poljak and Kocjan, 2010; Snijders et al., 2010). SPF10 and GP5+/GP6+ primers amplifying the 65-bp and 150-bp portions of the HPV L1 gene, respectively, have been used most frequently for detection of HPV in FFPE specimens (Chen et al., 2009; Hannisdal et al., 2010; Janˇcar et al., 2009; Kleter et al., 1999; Odida et al., 2010; Rubin et al., 2001). The Abbott RealTime High Risk HPV test (RealTime) (Abbott, Wiesbaden, Germany) is a novel real-time PCR-based assay based on concurrent individual genotyping for HPV-16 and HPV-18 and pooled detection of 12 HPV genotypes: HPV-31, HPV-33, HPV35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-66 and HPV-68. The assay was launched on the European market in January 2009. RealTime is performed on a fully automated nucleic acid preparation instrument m2000sp and the real-time PCR instrument m2000rt using a modified GP5+/GP6+ primer mix; co-amplification of a 136-bp region of human beta-globin is used as an internal process control for sample adequacy, DNA extraction and amplification (Huang et al., 2009a). The assay is validated for use with cervical specimens collected with ThinPrep PreservCyt solution (Hologic, Madison, WI), SurePath Preservative Fluid (BD, Frankli Lakes, NJ) and Cervi-Collect Specimen Collection Kit (Abbott Molecular) (Huang et al., 2009a) but, in our experience, cervical specimens collected in Digene Specimen Transport Medium (Qiagen, Hilden, Germany) are also appropriate (Poljak et al., 2009).
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B.J. Kocjan et al. / Journal of Virological Methods 175 (2011) 117–119
Table 1 Results of comparative evaluation of RealTime and INNO-LiPA assays on alternately processed formalin-fixed, paraffin-embedded specimens of 31 cervical cancers and 31 uterine myomas. Sample no.
RealTime results
INNO-LiPA results
1–17 18–23 24 25–26 27 28 29 30–31 Uterine myomas B1-B31
HPV-16 HPV-18 Other HPVa Other HPVa Other HPVa HPV negativeb HPV-16, other HPVa Other HPVa HPV negative
HPV-16 HPV-18 (possible HPV-39)c HPV-45 HPV-58 (possible HPV-52)c HPV-33 (possible HPV-52, HPV-54)c HPV-73 HPV-16, HPV-51 HPV-31, HPV-33 (possible HPV-52, HPV-54)c HPV negative
a b c
Includes HPV genotypes: HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-66 and HPV-68. HPV-73 is a genotype not targeted by RealTime. The presence of HPV genotype(s) cannot be excluded due to the INNO-LiPA cross-reactive hybridization probes.
In the present study, we evaluated the performance of RealTime in comparison to the INNO-LiPA HPV Genotyping Extra CE test (INNO-LiPA) (Innogenetics, Ghent, Belgium), for detection of HPV in archival FFPE cervical cancer tissue specimens. The INNO-LiPA is the standard HPV genotyping assay, which allows identification of 28 different alpha-HPV genotypes, including all 14 HPVs covered by RealTime. INNO-LiPA is based on the co-amplification of the 65-bp region of the HPV L1 gene and the 270-bp region of the human HLADP1 gene using biotinylated primers, followed by genotyping of the resulting amplicons with a single-typing strip coated with HPV- and internal control-specific oligonucleotide probes. To the best of our knowledge, this study is the first to evaluate the performance of RealTime assay in archival specimens. A total of 62 FFPE tissue specimens collected from the 31 women with histologically confirmed invasive cervical cancer and 31 women with histologically confirmed uterine myomas, all diagnosed between 2004 and 2006, were included in the study (Janˇcar et al., 2009). For each tissue block, three 10 m thick sections were cut and processed for DNA isolation; before sectioning, 2–3 outer sections were discharged. The FFPE blocks of cervical cancer (presumed HPV-positive) and uterine myomas (presumed HPV-negative) were sectioned alternately to control possible sample-to-sample contamination. Additionally, the microtome blade and surface were cleaned extensively using DNAZap (Applied Biosystems, Foster City, CA) after each use. Each specimen was first deparaffinized using xylene and ethanol, as described previously (Silvestre et al., 2009). After removal of paraffin, DNA was extracted using DNA Mini Kit (Qiagen), following the manufacturer’s instructions. Briefly, air-dried sections were incubated with 180 l of Buffer ATL and 20 l of proteinase K overnight on a 56 ◦ C rocking platform. After the addition of 200 l of Bufer AL, samples were incubated for 10 min at 70 ◦ C. Subsequently, 200 l of the lysate were transferred into an m2000sp ASPS Reaction Vessel containing 300 l of Abbott mBulk Lysis Buffer and processed for DNA isolation using the mSample Preparation SystemDNA kit on the Abbott m2000sp instrument, following the manufacturer’s instructions. To extract DNA for INNO-LiPA, 100 l of ethanol was added to the remaining 200 l lysate and DNA extraction proceeded following the DNA Mini Kit tissue protocol. The purified DNA was resuspended in 100 l of Buffer AE and stored at −70 ◦ C until use. RealTime and INNO-LiPA were performed on all 62 samples, following the manufacturers’ instructions. To control for amplicon carry-over contamination, water blanks were placed after every fifth reaction tube in all PCR runs. The 136-bp fragment of human beta-globin, which served as an internal control in RealTime, was amplified successfully from all 62 tissue samples included in the study; the cycle threshold (Ct) values ranged from 20.0 to 28.2 (mean 24.0; median 24.1). The 270-bp fragment of human HLA-DP1 gene, which served as an internal control in the INNO-LiPA, was amplified successfully from 20/31 (64.5%) of the cervical cancer samples and from all 31 ute-
rine myoma FFPE tissues. HPV-DNA was detected in 30/31 (96.8%) of the cervical cancer samples with RealTime, and in none of the uterine myoma specimens (Table 1) – RealTime Ct values ranged from 12.8 to 36.0 (mean 23.0; median 22.8). The single RealTime HPV negative cervical cancer sample contained genotype HPV-73, an HPV genotype which is not targeted by RealTime. Using INNOLiPA, HPV-DNA was identified in all 31 cervical cancer samples and in none of the uterine myoma specimens (Table 1). All water blanks used to control for PCR-contamination were negative for internal control and HPV-DNA with both tests. There was 100% genotyping agreement between the two methods for 14 HPV genotypes that can be identified by both assays (assay-common HPV genotypes) (Table 1). To the best of our knowledge, 10 studies have been published to date evaluating the analytical and clinical performance of RealTime on different patient populations using cervical swab specimens collected in various transport media (Carozzi et al., 2011; Cuzick et al., 2010; Halfon et al., 2010; Huang et al., 2009a,b; Kaliterna et al., 2009; Kitchener et al., 2011; Poljak et al., 2009, 2011; Tang et al., 2009). The results of our study showed that preprocessed FFPE cervical cancer tissue specimens can also be used in conjunction with Abbott DNA extraction and real-time PCR amplification system to detect reliably 14 HPV genotypes with concurrent, separate detection of HPV-16 and HPV-18. In order to minimize the possibility of sample-to-sample contamination, use of a fresh microtome blade after each sectioning is generally recommended when preparing FFPE tissue sections for subsequent molecular analysis. However, this procedure is expensive and time consuming. The absence of amplifiable HPV-DNA in any of the control uterine myoma specimens indicates that the FFPE tissue preparation protocol used in this study – physical removal of tissue/paraffin parts (if any) from microtome surface/blade and subsequent decontamination with DNAZap solution – is sampleto-sample contamination safe, and can hence be used in further studies. Several molecular methods are currently available on the market for the detection and genotyping of hr-HPVs (Poljak and Kocjan, 2010). Although the majority of these methods can reliably detect HPVs in cervical swab specimens, only a few, including RealTime, are potentially suitable for archival clinical specimens, since they target a relatively small portion of HPV genome (less than 160 bp). In addition, RealTime has significantly shorter hands on time in comparison with PCR-based HPV genotyping methods, consisting of DNA amplification and post-PCR analysis step, e.g. reverse blot hybridization. Thus, the observed differences in internal control amplification efficacy between RealTime and INNO-LiPA can be attributed most reasonably to the differences in target amplicon length: 136-bp vs. 270-bp, respectively. The size of the PCR targeted DNA fragment and the time of FFPE block storage were identified in several previous studies as the two most important factors having
B.J. Kocjan et al. / Journal of Virological Methods 175 (2011) 117–119
an inverse effect on the PCR amplification efficacy (Gillio-Tos et al., 2007; Greer et al., 1991; Poljak et al., 2000; Shibata, 1994). In conclusion, this pilot study showed that the RealTime assay is a reliable, sensitive, and specific diagnostic tool for the detection and partial genotyping of targeted HPV genotypes in FFPE cervical cancer tissue specimens. Acknowledgments ˇ The authors thank Jasna Sinkovec for providing and histological reviewing of the FFPE tissue samples used in the study as well as Nina Gale and her staff for tissue blocks sectioning. The authors also thank Robert Kroˇselj for performing the RealTime assay. References Bouvard, V., Baan, R., Straif, K., Grosse, Y., Secretan, B., El Ghissassi, F., BenbrahimTallaa, L., Guha, N., Freeman, C., Galichet, L., Cogliano, V., WHO International Agency for Research on Cancer Monograph Working Group, 2009. A review of human carcinogens—Part B: biological agents. Lancet Oncol. 10, 321–322. Carozzi, F.M., Burroni, E., Bisanzi, S., Puliti, D., Confortini, M., Giorgi Rossi, P., Sani, C., Scalisi, A., Chini, F., 2011. Comparison of Clinical Performance of Abbott RealTime High Risk HPV Test with That of Hybrid Capture 2 Assay in a Screening Setting. J. Clin. Microbiol. 49, 1446–1451. Chen, W., Zhang, X., Molijn, A., Jenkins, D., Shi, J.F., Quint, W., Schmidt, J.E., Wang, P., Liu, Y.L., Li, L.K., Shi, H., Liu, J.H., Xie, X., Niyazi, M., Yang, P., Wei, L.H., Li, L.Y., Li, J., Liu, J.F., Zhou, Q., Hong, Y., Li, L., Li, Q., Zhou, H.L., Bian, M.L., Chen, J., Qiao, Y.L., Smith, J.S., 2009. Human papillomavirus type-distribution in cervical cancer in China: the importance of HPV 16 and 18. Cancer Causes Control 20, 1705–1713. Cuschieri, K.S., Cubie, H.A., 2005. The role of human papillomavirus testing in cervical screening. J. Clin. Virol. 32 (Suppl. 1), S34–S42. Cuzick, J., Ambroisine, L., Cadman, L., Austin, J., Ho, L., Terry, G., Liddle, S., Dina, R., McCarthy, J., Buckley, H., Bergeron, C., Soutter, W.P., Lyons, D., Szarewski, A., 2010. Performance of the Abbott RealTime high-risk HPV test in women with abnormal cervical cytology smears. J. Med. Virol. 82, 1186–1191. Gilbert, M.T., Haselkorn, T., Bunce, M., Sanchez, J.J., Lucas, S.B., Jewell, L.D., Van Marck, E., Worobey, M., 2007. The isolation of nucleic acids from fixed, paraffinembedded tissues—which methods are useful when? PLoS One 2, e537. Gillio-Tos, A., De Marco, L., Fiano, V., Garcia-Bragado, F., Dikshit, R., Boffetta, P., Merletti, F., 2007. Efficient DNA extraction from 25-year-old paraffin-embedded tissues: study of 365 samples. Pathology 39, 345–348. Greer, C.E., Peterson, S.L., Kiviat, N.B., Manos, M.M., 1991. PCR amplification from paraffin-embedded tissues: effects of fixative and fixation time. Am. J. Clin. Pathol. 95, 117–124. Halfon, P., Benmoura, D., Agostini, A., Khiri, H., Penaranda, G., Martineau, A., Blanc, B., 2010. Evaluation of the clinical performance of the Abbott RealTime High-Risk HPV for carcinogenic HPV detection. J. Clin. Virol. 48, 246–250. Hannisdal, K., Schjølberg, A., De Angelis, P.M., Boysen, M., Clausen, O.P., 2010. Human papillomavirus (HPV)-positive tonsillar carcinomas are frequent and have a favourable prognosis in males in Norway. Acta Otolaryngol. 130, 293–299. Huang, S., Tang, N., Mak, W.B., Erickson, B., Salituro, J., Li, Y., Krumpe, E., Schneider, G., Yu, H., Robinson, J., Abravaya, K., 2009a. Principles and analytical performance of Abbott RealTime High Risk HPV test. J. Clin. Virol. 45 (Suppl. 1), S13–17. Huang, S., Erickson, B., Tang, N., Mak, W.B., Salituro, J., Robinson, J., Abravaya, K., 2009b. Clinical performance of Abbott RealTime High Risk HPV test for detection of high-grade cervical intraepithelial neoplasia in women with abnormal cytology. J. Clin. Virol. 45 (Suppl. 1), S19–S23. Janˇcar, N., Kocjan, B.J., Poljak, M., Lunar, M.M., Bokal, E.V., 2009. Distribution of human papillomavirus genotypes in women with cervical cancer in Slovenia. Eur. J. Obstet. Gynecol. Reprod. Biol. 145, 184–188.
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Kaliterna, V., Lepej, S.Z., Vince, A., 2009. Comparison between the Abbott RealTime High Risk HPV assay and the Hybrid Capture 2 assay for detecting highrisk human papillomavirus DNA in cervical specimens. J. Med. Microbiol. 58, 1662–1663. Kitchener, H.C., Blanks, R., Cubie, H., Desai, M., Dunn, G., Legood, R., Gray, A., Sadique, Z., Moss, S., MAVARIC Trial Study Group, 2011. MAVARIC – a comparison of automation-assisted and manual cervical screening: a randomised controlled trial. Health Technol. Assess. 15, iii–iv, ix–xi, 1–170. Kleter, B., van Doorn, L.J., Schrauwen, L., Molijn, A., Sastrowijoto, S., ter Schegget, J., Lindeman, J., ter Harmsel, B., Burge, M., Quint, W., 1999. Development and clinical evaluation of a highly sensitive PCR-reverse hybridization line probe assay for detection and identification of anogenital human papillomavirus. J. Clin. Microbiol. 37, 2508–2517. ˜ Munoz-Cadavid, C., Rudd, S., Zaki, S.R., Patel, M., Moser, S.A., Brandt, M.E., Gómez, B.L., 2010. Improving molecular detection of fungal DNA in formalin-fixed paraffinembedded tissues: comparison of five tissue DNA extraction methods using panfungal PCR. J. Clin. Microbiol. 48, 2147–2153. Odida, M., de Sanjose, S., Sandin, S., Quiros, B., Alemany, L., Lloveras, B., Quint, W., Kleter, B., Alejo, M., van Doorn, L.J., Weiderpass, E., 2010. Comparison of human papillomavirus detection between freshly frozen tissue and paraffin embedded tissue of invasive cervical cancer. Infect. Agent Cancer 5, 15. Poljak, M., Seme, K., Gale, N., 1998. Detection of human papillomaviruses in tissue specimens. Adv. Anatomic. Pathol. 5, 216–234. Poljak, M., Seme, K., Gale, N., 2000. Rapid extraction of DNA from archival clinical specimens: our experiences. Pflug. Arch. Eur. J. Physiol. 439 (Suppl.), R42–R44. Poljak, M., Kovanda, A., Kocjan, B.J., Seme, K., Janˇcar, N., Vrtaˇcnik-Bokal, E., 2009. The Abbott RealTime High Risk HPV test: comparative evaluation of analytical specificity and clinical sensitivity for cervical carcinoma and CIN 3 lesions with the Hybrid Capture 2 HPV DNA test. Acta Dermatovenerol. Alp. Panonica Adriat. 18, 94–103. Poljak, M., Kocjan, B.J., 2010. Commercially available assays for multiplex detection of alpha human papillomaviruses. Expert Rev. Anti Infect. Ther. 8, 1139–1162. Poljak, M., Oˇstrbenk, A., Seme, K., Uˇcakar, V., Hillemanns, P., Vrtaˇcnik-Bokal, E., Janˇcar, N., Klavs, I., 2011. Comparison of clinical and analytical performance of the Abbott RealTime High Risk HPV test and Hybrid Capture 2 in population-based cervical cancer screening. J. Clin. Microbiol., doi:10.1128/JCM.00012-11. Rubin, M.A., Kleter, B., Zhou, M., Ayala, G., Cubilla, A.L., Quint, W.G., Pirog, E.C., 2001. Detection and typing of human papillomavirus DNA in penile carcinoma: evidence for multiple independent pathways of penile carcinogenesis. Am. J. Pathol. 159, 1211–1218. Schiffman, M., Clifford, G., Buonaguro, F.M., 2009. Classification of weakly carcinogenic human papillomavirus types: addressing the limits of epidemiology at the borderline. Infect. Agent Cancer 4, 8. Shibata, D., 1994. Extraction of DNA from paraffin-embedded tissue for analysis by polymerase chain reaction: new tricks from an old friend. Hum. Pathol. 25, 561–563. Silvestre, O., Borzacchiello, G., Nava, D., Iovane, G., Russo, V., Vecchio, D., D’Ausilio, F., Gault, E.A., Campo, M.S., Paciello, O., 2009. Bovine papillomavirus type 1 DNA and E5 oncoprotein expression in water buffalo fibropapillomas. Vet. Pathol. 46, 636–641. Snijders, P.J., Heideman, D.A., Meijer, C.J., 2010. Methods for HPV detection in exfoliated cell and tissue specimens. APMIS 118, 520–528. Tan, S.E., Garland, S.M., Rumbold, A.R., Tabrizi, S.N., 2010. Human papillomavirus genotyping using archival vulval dysplastic or neoplastic biopsy tissues: comparison between the INNO-LiPA and linear array assays. J. Clin. Microbiol. 48, 1458–1460. Tang, N., Huang, S., Erickson, B., Mak, W.B., Salituro, J., Robinson, J., Abravaya, K., 2009. High-risk HPV detection and concurrent HPV 16 and 18 typing with Abbott RealTime High Risk HPV test. J. Clin. Virol. 45 (Suppl. 1), S25–S28. zur Hausen, H., 2009. Papillomaviruses in the causation of human cancers – a brief historical account. Virology 384, 260–265.
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
Comparison of the Abbott RealTime High Risk HPV test and INNO-LiPA HPV Genotyping Extra test for the detection of human papillomaviruses in formalin-fixed, paraffin-embedded cervical cancer specimens Boˇstjan J. Kocjan, Katja Seme, Mario Poljak ∗ Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Zaloˇska 4, 1105 Ljubljana, Slovenia
a b s t r a c t Article history: Received 2 February 2011 Received in revised form 29 March 2011 Accepted 5 April 2011 Available online 12 April 2011 Keywords: HPV Genotyping INNO-LiPA Abbott RealTime Archival specimens
Comparative evaluation of Abbott RealTime and Innogenetics INNO-LiPA on alternately processed formalin-fixed, paraffin-embedded specimens of 31 cervical cancers and 31 uterine myomas showed complete agreement in the detection of 14 assay-common HPV genotypes and partial genotyping of HPV-16 and HPV-18. The tissue preparation protocol was shown to be sample-to-sample contamination safe. © 2011 Elsevier B.V. All rights reserved.
Cervical cancer is the second most frequent cancer in women worldwide. Persistent infection with high-risk alpha-human papillomaviruses (hr-HPV) is a necessary although not sufficient etiological factor for the development of cervical cancer and its immediate precursors (Bouvard et al., 2009; Schiffman et al., 2009). In addition to cervical cancer, hr-HPVs, the most frequent being HPV-16, play the leading etiological role in the development of anal cancer and a substantial proportion of vaginal, penile, vulvar and oropharyngeal cancers (zur Hausen, 2009). Formalin-fixation and subsequent paraffin/paraplast embedding (FFPE) is a standard method for long-term preservation of tissue specimens in pathological departments worldwide. These specimens, used routinely for histopathological diagnoses, also represent an invaluable resource for etiological and epidemiological studies of HPV and other viral infections in cases in which fresh or frozen tissue is not available (Janˇcar et al., 2009; Poljak et al., 1998; Tan et al., 2010). However, the detection of viruses in FFPE specimens is often challenging, due to substantial degradation of viral DNA/RNA as a result of excessive fixation or tissue ageing or the presence of substances that inhibit PCR amplification ˜ or proteinase K (Gilbert et al., 2007; Gillio-Tos et al., 2007; MunozCadavid et al., 2010; Tan et al., 2010). Since the fragmentation of DNA influences significantly the efficiency of PCR amplification,
∗ Corresponding author. Tel.: +386 1 543 7453; fax: +386 1 543 7418. E-mail address: [email protected] (M. Poljak). 0166-0934/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2011.04.006
HPV methods that amplify a relatively small portion of the viral genome are the most suitable when working with archival specimens (Cuschieri and Cubie, 2005; Greer et al., 1991; Poljak et al., 1998, 2000; Poljak and Kocjan, 2010; Snijders et al., 2010). SPF10 and GP5+/GP6+ primers amplifying the 65-bp and 150-bp portions of the HPV L1 gene, respectively, have been used most frequently for detection of HPV in FFPE specimens (Chen et al., 2009; Hannisdal et al., 2010; Janˇcar et al., 2009; Kleter et al., 1999; Odida et al., 2010; Rubin et al., 2001). The Abbott RealTime High Risk HPV test (RealTime) (Abbott, Wiesbaden, Germany) is a novel real-time PCR-based assay based on concurrent individual genotyping for HPV-16 and HPV-18 and pooled detection of 12 HPV genotypes: HPV-31, HPV-33, HPV35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-66 and HPV-68. The assay was launched on the European market in January 2009. RealTime is performed on a fully automated nucleic acid preparation instrument m2000sp and the real-time PCR instrument m2000rt using a modified GP5+/GP6+ primer mix; co-amplification of a 136-bp region of human beta-globin is used as an internal process control for sample adequacy, DNA extraction and amplification (Huang et al., 2009a). The assay is validated for use with cervical specimens collected with ThinPrep PreservCyt solution (Hologic, Madison, WI), SurePath Preservative Fluid (BD, Frankli Lakes, NJ) and Cervi-Collect Specimen Collection Kit (Abbott Molecular) (Huang et al., 2009a) but, in our experience, cervical specimens collected in Digene Specimen Transport Medium (Qiagen, Hilden, Germany) are also appropriate (Poljak et al., 2009).
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B.J. Kocjan et al. / Journal of Virological Methods 175 (2011) 117–119
Table 1 Results of comparative evaluation of RealTime and INNO-LiPA assays on alternately processed formalin-fixed, paraffin-embedded specimens of 31 cervical cancers and 31 uterine myomas. Sample no.
RealTime results
INNO-LiPA results
1–17 18–23 24 25–26 27 28 29 30–31 Uterine myomas B1-B31
HPV-16 HPV-18 Other HPVa Other HPVa Other HPVa HPV negativeb HPV-16, other HPVa Other HPVa HPV negative
HPV-16 HPV-18 (possible HPV-39)c HPV-45 HPV-58 (possible HPV-52)c HPV-33 (possible HPV-52, HPV-54)c HPV-73 HPV-16, HPV-51 HPV-31, HPV-33 (possible HPV-52, HPV-54)c HPV negative
a b c
Includes HPV genotypes: HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-66 and HPV-68. HPV-73 is a genotype not targeted by RealTime. The presence of HPV genotype(s) cannot be excluded due to the INNO-LiPA cross-reactive hybridization probes.
In the present study, we evaluated the performance of RealTime in comparison to the INNO-LiPA HPV Genotyping Extra CE test (INNO-LiPA) (Innogenetics, Ghent, Belgium), for detection of HPV in archival FFPE cervical cancer tissue specimens. The INNO-LiPA is the standard HPV genotyping assay, which allows identification of 28 different alpha-HPV genotypes, including all 14 HPVs covered by RealTime. INNO-LiPA is based on the co-amplification of the 65-bp region of the HPV L1 gene and the 270-bp region of the human HLADP1 gene using biotinylated primers, followed by genotyping of the resulting amplicons with a single-typing strip coated with HPV- and internal control-specific oligonucleotide probes. To the best of our knowledge, this study is the first to evaluate the performance of RealTime assay in archival specimens. A total of 62 FFPE tissue specimens collected from the 31 women with histologically confirmed invasive cervical cancer and 31 women with histologically confirmed uterine myomas, all diagnosed between 2004 and 2006, were included in the study (Janˇcar et al., 2009). For each tissue block, three 10 m thick sections were cut and processed for DNA isolation; before sectioning, 2–3 outer sections were discharged. The FFPE blocks of cervical cancer (presumed HPV-positive) and uterine myomas (presumed HPV-negative) were sectioned alternately to control possible sample-to-sample contamination. Additionally, the microtome blade and surface were cleaned extensively using DNAZap (Applied Biosystems, Foster City, CA) after each use. Each specimen was first deparaffinized using xylene and ethanol, as described previously (Silvestre et al., 2009). After removal of paraffin, DNA was extracted using DNA Mini Kit (Qiagen), following the manufacturer’s instructions. Briefly, air-dried sections were incubated with 180 l of Buffer ATL and 20 l of proteinase K overnight on a 56 ◦ C rocking platform. After the addition of 200 l of Bufer AL, samples were incubated for 10 min at 70 ◦ C. Subsequently, 200 l of the lysate were transferred into an m2000sp ASPS Reaction Vessel containing 300 l of Abbott mBulk Lysis Buffer and processed for DNA isolation using the mSample Preparation SystemDNA kit on the Abbott m2000sp instrument, following the manufacturer’s instructions. To extract DNA for INNO-LiPA, 100 l of ethanol was added to the remaining 200 l lysate and DNA extraction proceeded following the DNA Mini Kit tissue protocol. The purified DNA was resuspended in 100 l of Buffer AE and stored at −70 ◦ C until use. RealTime and INNO-LiPA were performed on all 62 samples, following the manufacturers’ instructions. To control for amplicon carry-over contamination, water blanks were placed after every fifth reaction tube in all PCR runs. The 136-bp fragment of human beta-globin, which served as an internal control in RealTime, was amplified successfully from all 62 tissue samples included in the study; the cycle threshold (Ct) values ranged from 20.0 to 28.2 (mean 24.0; median 24.1). The 270-bp fragment of human HLA-DP1 gene, which served as an internal control in the INNO-LiPA, was amplified successfully from 20/31 (64.5%) of the cervical cancer samples and from all 31 ute-
rine myoma FFPE tissues. HPV-DNA was detected in 30/31 (96.8%) of the cervical cancer samples with RealTime, and in none of the uterine myoma specimens (Table 1) – RealTime Ct values ranged from 12.8 to 36.0 (mean 23.0; median 22.8). The single RealTime HPV negative cervical cancer sample contained genotype HPV-73, an HPV genotype which is not targeted by RealTime. Using INNOLiPA, HPV-DNA was identified in all 31 cervical cancer samples and in none of the uterine myoma specimens (Table 1). All water blanks used to control for PCR-contamination were negative for internal control and HPV-DNA with both tests. There was 100% genotyping agreement between the two methods for 14 HPV genotypes that can be identified by both assays (assay-common HPV genotypes) (Table 1). To the best of our knowledge, 10 studies have been published to date evaluating the analytical and clinical performance of RealTime on different patient populations using cervical swab specimens collected in various transport media (Carozzi et al., 2011; Cuzick et al., 2010; Halfon et al., 2010; Huang et al., 2009a,b; Kaliterna et al., 2009; Kitchener et al., 2011; Poljak et al., 2009, 2011; Tang et al., 2009). The results of our study showed that preprocessed FFPE cervical cancer tissue specimens can also be used in conjunction with Abbott DNA extraction and real-time PCR amplification system to detect reliably 14 HPV genotypes with concurrent, separate detection of HPV-16 and HPV-18. In order to minimize the possibility of sample-to-sample contamination, use of a fresh microtome blade after each sectioning is generally recommended when preparing FFPE tissue sections for subsequent molecular analysis. However, this procedure is expensive and time consuming. The absence of amplifiable HPV-DNA in any of the control uterine myoma specimens indicates that the FFPE tissue preparation protocol used in this study – physical removal of tissue/paraffin parts (if any) from microtome surface/blade and subsequent decontamination with DNAZap solution – is sampleto-sample contamination safe, and can hence be used in further studies. Several molecular methods are currently available on the market for the detection and genotyping of hr-HPVs (Poljak and Kocjan, 2010). Although the majority of these methods can reliably detect HPVs in cervical swab specimens, only a few, including RealTime, are potentially suitable for archival clinical specimens, since they target a relatively small portion of HPV genome (less than 160 bp). In addition, RealTime has significantly shorter hands on time in comparison with PCR-based HPV genotyping methods, consisting of DNA amplification and post-PCR analysis step, e.g. reverse blot hybridization. Thus, the observed differences in internal control amplification efficacy between RealTime and INNO-LiPA can be attributed most reasonably to the differences in target amplicon length: 136-bp vs. 270-bp, respectively. The size of the PCR targeted DNA fragment and the time of FFPE block storage were identified in several previous studies as the two most important factors having
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