Accessible Requires Authentication Published by De Gruyter December 2, 2015

Comparative analysis of prostate cancer specific biomarkers PCA3 and ERG in whole urine, urinary sediments and exosomes

Rianne J. Hendriks, Siebren Dijkstra, Sander A. Jannink, Martijn G. Steffens, Inge M. van Oort, Peter F.A. Mulders and Jack A. Schalken

Abstract

Background:PCA3 and ERG are mRNA-based prostate cancer (PCa) specific biomarkers that can be detected in urine. However, urine is a complex substrate that can be separated in several fractions. In this study we compared the levels of PCa-specific biomarkers (PCA3 and ERG) and KLK3 as prostate-specific reference gene in three urine substrates–whole urine, urinary sediment (cell pellet) and exosomes–and evaluated the influence of performing a digital rectal examination (DRE) prior to urine sampling.

Methods: First-voided urine samples were prospectively obtained before and after DRE from 29 men undergoing prostate biopsies. The urine was separated in whole urine, cell pellet and exosomes and the biomarker levels were measured with RT-qPCR.

Results: PCa was identified in 52% (15/29) of men. In several samples the mRNA levels were below the analytical limit of detection (BDL). The biomarker levels were highest in whole urine and significantly higher after DRE in all substrates. In PCa patients higher levels of PCA3 and ERG were found in all urine substrates after DRE compared to non-PCa patients.

Conclusions: This is the first study in which urinary PCa-specific biomarker levels were compared directly in three separate urine fractions. These results suggest that whole urine could be the urine substrate of choice for PCa-diagnostics based on analytical sensitivity, which is reflected directly in the high informative rate. Moreover, the significant positive effect of performing a DRE prior to urine sampling is confirmed. These findings could be of influence in the development of PCa-diagnostic urine tests.


Corresponding author: Prof. Dr. Jack A. Schalken, Department of Urology, Radboud University Medical Center, Geert-Grooteplein Zuid 10, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands, Phone: +31 24 36 14 146, E-mail: ; and Department of Research and Development, NovioGendix, Nijmegen, The Netherlands

References

1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA-A Cancer J Clin 2015;65:87–108. Search in Google Scholar

2. Salagierski M, Schalken JA. Molecular diagnosis of prostate cancer: PCA3 and TMPRSS2:ERG gene fusion. J Urol 2012;187:795–801. Search in Google Scholar

3. Draisma G, Etzioni R, Tsodikov A, Mariotto A, Wever E, Gulati R, et al. Lead time and overdiagnosis in prostate-specific antigen screening: importance of methods and context. J Natl Cancer I 2009;101:374–83. Search in Google Scholar

4. Truong M, Yang B, Jarrard DF. Toward the detection of prostate cancer in urine: a critical analysis. J Urol 2013;189:422–9. Search in Google Scholar

5. Hessels D, Schalken JA. Urinary biomarkers for prostate cancer: a review. Asian J Androl 2013;15:333–9. Search in Google Scholar

6. Bussemakers MJ, van Bokhoven A, Verhaegh GW, Smit FP, Karthaus HF, Schalken JA, et al. DD3: a new prostate-specific gene, highly overexpressed in prostate cancer. Cancer Res 1999;59:5975–9. Search in Google Scholar

7. de Kok JB, Verhaegh GW, Roelofs RW, Hessels D, Kiemeney LA, Aalders TW, et al. DD3(PCA3), a very sensitive and specific marker to detect prostate tumors. Cancer Res 2002;62:2695–8. Search in Google Scholar

8. Hessels D, Klein Gunnewiek JM, van Oort I, Karthaus HF, van Leenders GJ, van Balken B, et al. DD3(PCA3)-based molecular urine analysis for the diagnosis of prostate cancer. Eur Urol 2003;44:8–15; discussion -6. Search in Google Scholar

9. Groskopf J, Aubin SM, Deras IL, Blase A, Bodrug S, Clark C, et al. APTIMA PCA3 molecular urine test: development of a method to aid in the diagnosis of prostate cancer. Clin Chem 2006;52:1089–95. Search in Google Scholar

10. Gittelman MC, Hertzman B, Bailen J, Williams T, Koziol I, Henderson RJ, et al. PCA3 molecular urine test as a predictor of repeat prostate biopsy outcome in men with previous negative biopsies: a prospective multicenter clinical study. J Urol 2013;190:64–9. Search in Google Scholar

11. Tomlins SA, Rhodes DR, Perner S, Dhanasekaran SM, Mehra R, Sun XW, et al. Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. Science 2005;310:644–8. Search in Google Scholar

12. Perner S, Mosquera JM, Demichelis F, Hofer MD, Paris PL, Simko J, et al. TMPRSS2-ERG fusion prostate cancer: an early molecular event associated with invasion. Am J Surg Pathol 2007;31:882–8. Search in Google Scholar

13. Hessels D, Smit FP, Verhaegh GW, Witjes JA, Cornel EB, Schalken JA. Detection of TMPRSS2-ERG fusion transcripts and prostate cancer antigen 3 in urinary sediments may improve diagnosis of prostate cancer. Clin Cancer Res 2007;13:5103–8. Search in Google Scholar

14. Smit FP, Salagierski M, Jannink S, Schalken JA. High-resolution ERG-expression profiling on GeneChip exon 1.0 ST arrays in primary and castration-resistant prostate cancer. BJU Int 2013;111:836–42. Search in Google Scholar

15. Braun M, Goltz D, Shaikhibrahim Z, Vogel W, Bohm D, Scheble V, et al. ERG protein expression and genomic rearrangement status in primary and metastatic prostate cancer–a comparative study of two monoclonal antibodies. Prostate Cancer P D 2012;15:165–9. Search in Google Scholar

16. Park K, Tomlins SA, Mudaliar KM, Chiu YL, Esgueva R, Mehra R, et al. Antibody-based detection of ERG rearrangement-positive prostate cancer. Neoplasia 2010;12:590–8. Search in Google Scholar

17. Donovan MJ, Noerholm M, Bentink S, Belzer S, Skog J, O;Neill V, et al. A molecular signature of PCA3 and ERG exosomal RNA from non-DRE urine is predictive of initial prostate biopsy result. Prostate Cancer P D 2015. Search in Google Scholar

18. Leyten GH, Hessels D, Jannink SA, Smit FP, de Jong H, Cornel EB, et al. Prospective multicentre evaluation of PCA3 and TMPRSS2-ERG gene fusions as diagnostic and prognostic urinary biomarkers for prostate cancer. Eur Urol 2014;65:534–42. Search in Google Scholar

19. Nilsson J, Skog J, Nordstrand A, Baranov V, Mincheva-Nilsson L, Breakefield XO, et al. Prostate cancer-derived urine exosomes: a novel approach to biomarkers for prostate cancer. Brit J Cancer 2009;100:1603–7. Search in Google Scholar

20. van Gils MP, Hessels D, van Hooij O, Jannink SA, Peelen WP, Hanssen SL, et al. The time-resolved fluorescence-based PCA3 test on urinary sediments after digital rectal examination; a Dutch multicenter validation of the diagnostic performance. Clin Cancer Res 2007;13:939–43. Search in Google Scholar

21. Marks LS, Fradet Y, Deras IL, Blase A, Mathis J, Aubin SM, et al. PCA3 molecular urine assay for prostate cancer in men undergoing repeat biopsy. Urology 2007;69:532–5. Search in Google Scholar

22. Sokoll LJ, Ellis W, Lange P, Noteboom J, Elliott DJ, Deras IL, et al. A multicenter evaluation of the PCA3 molecular urine test: pre-analytical effects, analytical performance, and diagnostic accuracy. Clin Chim Acta 2008;389:1–6. Search in Google Scholar

23. Laxman B, Morris DS, Yu J, Siddiqui J, Cao J, Mehra R, et al. A first-generation multiplex biomarker analysis of urine for the early detection of prostate cancer. Cancer Res 2008;68:645–9. Search in Google Scholar

24. Mitchell PJ, Welton J, Staffurth J, Court J, Mason MD, Tabi Z, et al. Can urinary exosomes act as treatment response markers in prostate cancer? J Transl Med 2009;7:4. Search in Google Scholar

25. Jansen FH, Krijgsveld J, van Rijswijk A, van den Bemd GJ, van den Berg MS, van Weerden WM, et al. Exosomal secretion of cytoplasmic prostate cancer xenograft-derived proteins. Mol Cell Proteomics 2009;8:1192–205. Search in Google Scholar

26. Dijkstra S, Birker IL, Smit FP, Leyten GH, de Reijke TM, van Oort IM, et al. Prostate cancer biomarker profiles in urinary sediments and exosomes. J Urol 2014;191:1132–8. Search in Google Scholar

27. Miranda KC, Bond DT, McKee M, Skog J, Paunescu TG, Da Silva N, et al. Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease. Kidney Int 2010;78:191–9. Search in Google Scholar

28. Zhou H, Yuen PS, Pisitkun T, Gonzales PA, Yasuda H, Dear JW, et al. Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery. Kidney Int 2006;69:1471–6. Search in Google Scholar

29. Li M, Rai AJ, Joel DeCastro G, Zeringer E, Barta T, Magdaleno S, et al. An optimized procedure for exosome isolation and analysis using serum samples: Application to cancer biomarker discovery. Methods 2015;87:26–30. Search in Google Scholar

30. Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 2007;9:654–9. Search in Google Scholar

Supplemental Material:

The online version of this article (DOI: 10.1515/cclm-2015-0599) offers supplementary material, available to authorized users.

Received: 2015-6-25
Accepted: 2015-10-22
Published Online: 2015-12-2
Published in Print: 2016-3-1

©2016 by De Gruyter