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Low prevalence of HPV detection and genotyping in non-muscle invasive bladder cancer using single-step PCR followed by reverse line blot

World Journal of Urology volume 33, pages 2145–2151 (2015)Cite this article

Abstract

Purpose

To clarify the role of human papillomavirus (HPV) in non-muscle invasive bladder cancer, HPV-DNA was scrutinized in formalin-fixed, paraffin-embedded (FFPE) bladder cancer tissue using single-step PCR (HPV L1) for HPV detection, followed by reverse line blot (RLB) for genotyping.

Methods

A total of 186 patients who underwent transurethral resection of the bladder due to primary, non-muscle invasive bladder cancer from 2006 to 2009 were reviewed. A positive control group of 22 cervical tissues with cervical carcinoma was included.

Results

Histology confirmed urothelial carcinoma in all patients: primary CIS, pTa, pT1 and pTa + pT1 in 14 (7.5 %), 134 (72 %), 36 (19.4 %) and two (1.1 %) patients, respectively. A total of 119 (63.9 %) of them were classified as low-risk, while 67 (36.1 %) were high-risk cancers. Tumor recurrence and progression (≥pT2) were seen in 79 and 11 patients (mean follow-up 45 months). The presence of HPV-DNA by single-step PCR was detected in four (2.2 %) patients. HPV 16 and HPV 6 were positive in two (1.1 %) and one (0.6 %) patient, respectively In one case, no HPV genotype listed on the RLB assay could be identified. In the control group, the HPV infection rate was 100 %: HPV 16 in 12 (54.6 %) patients, HPV 16/18 in four (18.3 %) patients, HPV 18 in two (9.1 %) patients, HPV 16/45 in one patient (4.5 %), HPV 18/33 in one (4.5 %) patient, HPV 16/33 in one (4.5 %) patient and HPV 33 in one (4.5 %) patient.

Conclusions

Our study demonstrates low prevalence of HPV infection in FFPE bladder cancer tissue, arguing against the etiological role of HPV in non-muscle urothelial carcinogenesis.

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References

  1. Cobos C, Figueroa JA, Mirandola L, Colombo M, Summers G, Figueroa A, Aulakh A, Konala V, Verma R, Riaz J, Wade R, Saadeh C, Rahman RL, Pandey A, Radhi S, Nguyen DD, Jenkins M, Chiriva-Internati M, Cobos E (2014) The role of human papilloma virus (HPV) infection in non-anogenital cancer and the promise of immunotherapy: a review. Int Rev Immunol 33(5):383–401

    CAS  Article  PubMed  Google Scholar 

  2. Tolstov Y, Hadaschik B, Pahernik S, Hohenfellner M, Duensing S (2014) Human papillomaviruses in urological malignancies: a critical assessment. Urol Oncol 32(1):46.e19–46.e27

    CAS  Article  Google Scholar 

  3. Youshya S, Purdie K, Breuer J, Proby C, Sheaf MT, Oliver RT, Baithun S (2005) Does human papillomavirus play a role in the development of bladder transitional cell carcinoma? A comparison of PCR and immunohistochemical analysis. J Clin Pathol 58(2):207–210

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  4. Alexander RE, Hu Y, Kum JB, Montironi R, Lopez-Beltran A, Maclennan GT, Idrees MT, Emerson RE, Ulbright TM, Grignon DG, Eble JN, Cheng L (2012) p16 expression is not associated with human papillomavirus in urinary bladder squamous cell carcinoma. Mod Pathol 25(11):1526–1533

    CAS  Article  PubMed  Google Scholar 

  5. Li N, Yang L, Zhang Y, Zhao P, Zheng T, Dai M (2011) Human papillomavirus infection and bladder cancer risk: a meta-analysis. J Infect Dis 204(2):217–223

    PubMed Central  Article  PubMed  Google Scholar 

  6. Kim SH, Joung JY, Chung J, Park WS, Lee KH, Seo HK (2014) Detection of human papillomavirus infection and p16 immunohistochemistry expression in bladder cancer with squamous differentiation. PLoS One 9(3):e93525

    PubMed Central  Article  PubMed  Google Scholar 

  7. Khaled HM, Bahnassi AA, Zekri AR, Kassem HA, Mokhtar N (2003) Correlation between p53 mutations and HPV in bilharzial bladder cancer. Urol Oncol 21(5):334–341

    CAS  Article  PubMed  Google Scholar 

  8. Ward C, Pedraza J, Kavanagh K, Johannessen I, Cuschieri K (2014) An evaluation of the Qiagen HPV sign for the detection and genotyping of cervical lesions and oropharyngeal squamous cell carcinomas. J Virol Methods 207C:128–132

    Article  Google Scholar 

  9. Hubbard RA (2003) Human papillomavirus testing methods. Arch Pathol Lab Med 127(8):940–945

    CAS  PubMed  Google Scholar 

  10. Lungu O, Wright TC, Silverstein S (1992) Typing of human papillomaviruses by polymerase chain reaction amplification with L1 consensus primers and RFLP analysis. Mol Cell Probes 6:145–152

    CAS  Article  PubMed  Google Scholar 

  11. Laconi S, Greco M, Pellegrini-Bettoli P, Rais M, Laconi E, Pani P (2001) One-step detection and genotyping of human papillomavirus in cervical samples by reverse hybridization. Diagn Mol Pathol 10:200–206

    CAS  Article  PubMed  Google Scholar 

  12. Mills A, Balasubramaniam R, Longacre TA, Kong CS, Pinsky BA (2013) Laboratory-developed L1 sequencing and type-specific, real-time polymerase chain reaction for the detection and typing of human papillomaviruses in formalin-fixed, paraffin-embedded tissues. Arch Pathol Lab Med 137(1):50–54

    CAS  Article  PubMed  Google Scholar 

  13. Steenbergen RD, Snijders PJ, Heideman DA, Meijer CJ (2014) Clinical implications of (epi)genetic changes in HPV-induced cervical precancerous lesions. Nat Rev Cancer 14(6):395–405

    CAS  Article  PubMed  Google Scholar 

  14. Conesa-Zamora P, Doménech-Peris A, Orantes-Casado FJ, Ortiz-Reina S, Sahuquillo-Frías L, Acosta-Ortega J, García-Solano J, Pérez-Guillermo M (2009) Effect of human papillomavirus on cell cycle-related proteins p16, Ki-67, Cyclin D1, p53, and ProEx C in precursor lesions of cervical carcinoma: a tissue microarray study. Am J Clin Pathol 132(3):378–390

    Article  PubMed  Google Scholar 

  15. Gould VE, Schmitt M, Vinokurova S, Reddy VB, Bitterman P, Alonso A, Gattuso P (2010) Human papillomavirus and p16 expression in inverted papillomas of the urinary bladder. Cancer Lett 292(2):171–175

    CAS  Article  PubMed  Google Scholar 

  16. Piaton E, Casalegno JS, Advenier AS, Decaussin-Petrucci M, Mege-Lechevallier F, Ruffion A, Mekki Y (2014) p16INK4a overexpression is not linked to oncogenic human papillomaviruses in patients with high-grade urothelial cancer cells. Cancer Cytopathol 122(3):211–220. doi:10.1002/cncy.21376

    CAS  Article  PubMed  Google Scholar 

  17. Steinestel J, Cronauer MV, Müller J, Al Ghazal A, Skowronek P, Arndt A, Kraft K, Schrader M, Schrader AJ, Steinestel K (2013) Overexpression of p16(INK4a) in urothelial carcinoma in situ is a marker for MAPK-mediated epithelial-mesenchymal transition but is not related to human papillomavirus infection. PLoS One 8(5):e65189

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  18. Alexander RE, Williamson SR, Richey J, Lopez-Beltran A, Montironi R, Davidson DD, Idrees MT, Jones CL, Zhang S, Wang L, Rao Q, Pedrosa JA, Kaimakliotis HZ, Monn MF, Koch MO, Cheng L (2014) The expression patterns of p53 and p16 and an analysis of a possible role of HPV in primary adenocarcinoma of the urinary bladder. PLoS One 9(4):e95724

    PubMed Central  Article  PubMed  Google Scholar 

  19. Pierik A, Zwanenburg C, Moerland E, Broer D, Stapert H, van den Brule AJ (2011) Rapid genotyping of human papillomavirus by post-PCR array-based hybridization techniques. J Clin Microbiol 49(4):1395–1402

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  20. Shigehara K, Sasagawa T, Kawaguchi S, Nakashima T, Shimamura M, Maeda Y, Konaka H, Mizokami A, Koh E, Namiki M (2011) Etiologic role of human papillomavirus infection in bladder carcinoma. Cancer 117(10):2067–2076

    CAS  Article  PubMed  Google Scholar 

  21. Brunner A, Verdorfer I, Prelog M, Mayerl C, Mikuz G, Tzankov A (2008) Large-scale analysis of cell cycle regulators in urothelial bladder cancer identifies p16 and p27 as potentially useful prognostic markers. Pathobiology 75(1):25–33

    CAS  Article  PubMed  Google Scholar 

  22. Spruck CH 3rd, Ohneseit PF, Gonzalez-Zulueta M, Esrig D, Miyao N, Tsai YC, Lerner SP, Schmütte C, Yang AS, Cote R et al (1994) Two molecular pathways to transitional cell carcinoma of the bladder. Cancer Res 54(3):784–788

    CAS  PubMed  Google Scholar 

  23. Lopez-Beltran A, Escudero AL, Vicioso L, Muñoz E, Carrasco JC (1996) Human papillomavirus DNA as a factor determining the survival of bladder cancer patients. Br J Cancer 73(1):124–127

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  24. López-Beltrán A, Escudero AL, Carrasco-Aznar JC, Vicioso-Recio L (1996) Human papillomavirus infection and transitional cell carcinoma of the bladder. Immunohistochemistry and in situ hybridization. Pathol Res Pract 192(2):154–159

    Article  PubMed  Google Scholar 

  25. Heidegger I, Pichler R, Müller B, Klocker H, Oswald D, Haid B, Zelger B, Horninger W, Oswald J (2013) Is real-time PCR the correct method to evaluate the incidence of human papillomavirus in prepuces of asymptomatic boys and men? World J Urol 32(5):1199–1204. doi:10.1007/s00345-013-1190-4

    Article  PubMed  Google Scholar 

  26. Klinglmair G, Pichler R, Zelger B, Dogan HS, Becker T, Esterbauer J, Riccabona M, Loidl W, Horninger W, Oswald J (2013) Prevalence of the human papillomavirus (HPV) expression of the inner prepuce in asymptomatic boys and men. World J Urol 31(6):1389–1394

    Article  PubMed  Google Scholar 

  27. Biedermann K, Dandachi N, Trattner M, Vogl G, Doppelmayr H, Moré E, Staudach A, Dietze O, Hauser-Kronberger C (2004) Comparison of real-time PCR signal-amplified in situ hybridization and conventional PCR for detection and quantification of human papillomavirus in archival cervical cancer tissue. J Clin Microbiol 42(8):3758–3765

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  28. Karlsen F, Kalantari M, Chitemerere M, Johansson B, Hagmar B (1994) Modifications of human and viral deoxyribonucleic acid by formaldehyde fixation. Lab Invest 71:604–611

    CAS  PubMed  Google Scholar 

  29. Baay MF, Quint WG, Koudstaal J, Hollema H, Duk JM, Burger MP, Stolz E, Herbrink P (1996) Comprehensive study of several general and type-specific primer pairs for detection of human papillomavirus DNA by PCR in paraffin-embedded cervical carcinomas. J Clin Microbiol 34(3):745–747

    PubMed Central  CAS  PubMed  Google Scholar 

  30. Zappacosta R, Colasante A, Viola P, D’Antuono T, Lattanzio G, Capanna S, Gatta DM, Rosini S (2013) Chromogenic in situ hybridization and p16/Ki67 dual staining on formalin-fixed paraffin-embedded cervical specimens: correlation with HPV-DNA test, E6/E7 mRNA test, and potential clinical applications. Biomed Res Int 2013:453606. doi:10.1155/2013/453606

    PubMed Central  Article  PubMed  Google Scholar 

  31. Yavuzer D, Karadayi N, Salepci T, Baloglu H, Bilici A, Sakirahmet D (2011) Role of human papillomavirus in the development of urothelial carcinoma. Med Oncol 28(3):919–923

    Article  PubMed  Google Scholar 

  32. Panagiotakis GI, Papadogianni D, Chatziioannou MN, Lasithiotaki I, Delakas D, Spandidos DA (2013) Association of human herpes, papilloma and polyoma virus families with bladder cancer. Tumour Biol 34(1):71–79

    CAS  Article  PubMed  Google Scholar 

  33. Knowles MA (1992) Human papillomavirus sequences are not detectable by Southern blotting or general primer-mediated polymerase chain reaction in transitional cell tumours of the bladder. Urol Res 20(4):297–301

    CAS  Article  PubMed  Google Scholar 

  34. De Gaetani C, Ferrari G, Righi E, Bettelli S, Migaldi M, Ferrari P, Trentini GP (1999) Detection of human papillomavirus DNA in urinary bladder carcinoma by in situ hybridization. Clin Pathol 52(2):103–106

    Article  Google Scholar 

  35. Lu QL, Lalani E-N, Abel P (1997) Human papillomavirus 16 and 18 infection is absent in urinary bladder carcinomas. Eur Urol 31(4):428–432

    CAS  PubMed  Google Scholar 

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Acknowledgments

The authors want to thank Julia Heppke for excellent technical assistance. This research was supported by a grant from the Medical Research Foundation Tyrol (MFF Tyrol, Project Number 255). The funders had no role in study design, data collection and analysis, or decision to publish.

Conflict of interest

The authors had no conflicts of interest to declare.

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Affiliations

  1. Division of Experimental Urology, Department of Urology, Medical University of Innsbruck, Innsbruck, Austria

    Renate Pichler, Zoran Culig, Sebastian List, Isabel Heidegger, Helmut Klocker, Wolfgang Horninger & Hannes Steiner

  2. Section of Virology, Department of Hygiene, Microbiology and Social Medicine, Innsbruck, Austria

    Wegene Borena & Dorothee Von Laer

  3. Division of General Pathology, Department of Pathology, Medical University Innsbruck, Innsbruck, Austria

    Georg Schäfer, Claudia Manzl & Andrea Brunner

  4. Department of Medical Statistics, Informatics and Health Economics, Müllerstrasse 44A-6020, Innsbruck, Austria

    Sabrina Neururer

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  1. Renate Pichler
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Correspondence to Andrea Brunner.

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Pichler, R., Borena, W., Schäfer, G. et al. Low prevalence of HPV detection and genotyping in non-muscle invasive bladder cancer using single-step PCR followed by reverse line blot. World J Urol 33, 2145–2151 (2015). https://doi.org/10.1007/s00345-015-1539-y

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  • DOI: https://doi.org/10.1007/s00345-015-1539-y

Keywords

  • Bladder cancer
  • Human papillomavirus
  • Infection
  • Carcinogenesis
  • PCR
  • Hybridization