Virchows Archiv

, Volume 470, Issue 3, pp 267–274 | Cite as

In stage pT1 non-muscle-invasive bladder cancer (NMIBC), high KRT20 and low KRT5 mRNA expression identify the luminal subtype and predict recurrence and survival

  • Johannes Breyer
  • Ralph M. Wirtz
  • Wolfgang Otto
  • Philipp Erben
  • Maximilian C. Kriegmair
  • Robert Stoehr
  • Markus Eckstein
  • Sebastian Eidt
  • Stefan Denzinger
  • Maximilian Burger
  • Arndt Hartmann
  • on behalf of the BRIDGE Consortium
Original Article


Differential expression of cytokeratins (CK) is a characteristic feature of chemoresistant luminal (KRT20) and chemosensitive intrinsic aggressive basal (KRT5) subtypes in muscle-invasive bladder cancer (MIBC). We investigated mRNA expression of KRT5 and KRT20 and its predictive value in stage pT1 bladder cancer. In retrospective analysis of clinical data and formalin-fixed paraffin-embedded tissues (FFPE) of patients with stage pT1 NMIBC who underwent transurethral resection of the bladder, a single-step RT-qPCR was used to measure mRNA expression. Furthermore, immunohistochemical (IHC) staining of CK20, panCK, and MIB1 was performed. Valid measurements were obtained from 231 samples out of a series of 284 patients. Spearman correlation revealed significant associations between mRNA and protein expression of KRT20/CK20 (ρ 0.6096, p < 0.0001) and MKI67/MIB1 (ρ 0.5467, p < 0.0001). A positive correlation was found between MKI67 and KRT20 expression (ρ 0.3492, p < 0.0001), while MKI67 and KRT5 were negatively correlated (ρ −0.1693, p = 0.01). High KRT20 expression (≥40.26) was significantly associated with worse recurrence free survival (RFS) (p = 0.001), progression-free survival (PFS) (p = 0.0003), and cancer specific survival (CSS) (p = 0.0414). The combination of high KRT20 expression and low KRT5 expression (<36.83) was associated with unfavorable RFS (p = 0.0038) and PFS (p = 0.0003) and proved to be the only independent predictor for RFS (p = 0.0055) and PFS (p = 0.0023) in multivariate analysis. KRT20 mRNA determination was superior to CK20 protein estimation with regard to RFS and PFS prediction. KRT20 and KRT5 mRNA quantification can predict recurrence and progression of stage pT1 NMIBC reflecting basal and luminal subtypes of MIBC and is superior to CK20 protein expression determined by IHC.


KRT5 KRT20 Cytokeratin Molecular subtypes mRNA NMIBC 



Cancer-specific survival


Formaline fixed paraffin embedded


Gene of interest


Hazard ratio


Messenger ribonucleic acid


Overall survival


Progression-free survival


Reference gene


Recurrence-free survival


Reverse transcription quantitative real time polymerase chain reaction



We would like to thank Stefanie Herlein, Elke Veltrup, and Silke Claas for excellent technical support.

Compliance with ethical standards

Informed consent was obtained from all the individual participants included in the study. The study was conducted after approval of the local ethics committee.


This study was funded by the German Cancer Aid (Deutsche Krebshilfe (DKH)), grant number 110541.

Competing interests

RMW and SE are founders of STRATIFYER Molecular Pathology GmbH. RMW is an employee of STRATIFYER Molecular Pathology GmbH.

Supplementary material

428_2017_2064_Fig4_ESM.gif (94 kb)
Suppl. Figure 1

(GIF 93 kb)

428_2017_2064_MOESM1_ESM.tif (33 kb)
High resolution image (TIFF 32 kb)
428_2017_2064_Fig5_ESM.gif (223 kb)
Suppl. Figure 2

(GIF 222 kb)

428_2017_2064_MOESM2_ESM.tif (45 kb)
High resolution image (TIFF 45 kb)
428_2017_2064_Fig6_ESM.gif (219 kb)
Suppl. Figure 3

(GIF 219 kb)

428_2017_2064_MOESM3_ESM.tif (113 kb)
High resolution image (TIFF 113 kb)


  1. 1.
    Antoni S, Ferlay J, Soerjomataram I, Znaor A, Jemal A, Bray F (2016) Bladder cancer incidence and mortality: a global overview and recent trends. Eur Urol. doi: 10.1016/j.eururo.2016.06.010 PubMedGoogle Scholar
  2. 2.
    Babjuk M, Böhle A, Burger M et al (2016) EAU guidelines on non-muscle-invasive urothelial carcinoma of the bladder: update 2016. Eur Urol. doi: 10.1016/j.eururo.2016.05.041 Google Scholar
  3. 3.
    Prout GR Jr, Barton BA, Griffin PP, Friedell GH (1992) Treated history of noninvasive grade 1 transitional cell carcinoma. The National Bladder Cancer Group. J Urol 148:1413–1419PubMedGoogle Scholar
  4. 4.
    Hong YM, Loughlin KR (2008) Economic impact of tumor markers in bladder cancer surveillance. Urology 71:131–135CrossRefPubMedGoogle Scholar
  5. 5.
    Sylvester RJ, van der Meijden A, Oosterlinck W et al (2006) Predicting recurrence and progression in individual patients with stage Ta, T1 bladder cancer using EORTC risk tables: a combined analysis of 2596 patients from seven EORTC trials. Eur Urol 49:466–475CrossRefPubMedGoogle Scholar
  6. 6.
    Cancer Genome Atlas Research Network (2014) Comprehensive molecular characterization of urothelial bladder carcinoma. Nature 507:315–222CrossRefGoogle Scholar
  7. 7.
    Choi W, Porter S, Kim S et al (2014) Identification of distinct basal and luminal subtypes of muscle-invasive bladder cancer with different sensitivities to frontline chemotherapy. Cancer Cell 25:152–165CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Damrauer JS, Hoadley KA, Chism DD et al (2014) Intrinsic subtypes of high-grade bladder cancer reflect the hallmarks of breast cancer biology. Proc Natl Acad Sci U S A 111:3110–3115CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Sjodahl G, Lauss M, Lovgren K et al (2012) A molecular taxonomy for urothelial carcinoma. Clin Cancer Res 18:3377–3386CrossRefPubMedGoogle Scholar
  10. 10.
    Hedegaard J, Lamy P, Nordentoft I et al (2016) Comprehensive transcriptional analysis of early-stage urothelial carcinoma. Cancer Cell 30(1):27–42CrossRefPubMedGoogle Scholar
  11. 11.
    Choi W, Czerniak B, Ochoa A et al (2014) Intrinsic basal and luminal subtypes of muscle-invasive bladder cancer. Nat Rev Urol. 11:400–410CrossRefPubMedGoogle Scholar
  12. 12.
    Schaafsma HE, Ramaekers FCS, van Muijen GNP et al (1989) Distribution of cytokeratin polypeptides in epithelia of the adult human urinary tract. Histochemistry 91:151–159CrossRefPubMedGoogle Scholar
  13. 13.
    Moll R, Löwe A, Laufer J et al (1992) Cytokeratin 20 in human carcinomas: a new histodiagnostic marker detected by monoclonal antibodies. Am J Pathol 140:427–447PubMedPubMedCentralGoogle Scholar
  14. 14.
    Reis-Filho JS, Simpson PT, Martins A, Preto A, Gärtner F, Schmitt FC (2003) Distribution of p63, cytokeratins 5/6 and cytokeratin 14 in 51 normal and 400 neoplastic human tissue samples using TARP-4 multi-tumor tissue microarray. Virchows Arch 443(2):122–132CrossRefPubMedGoogle Scholar
  15. 15.
    Ho PL, Kurtova A, Chan KS (2012) Normal and neoplastic urothelial stem cells: getting to the root of the problem. Nat Rev Urol 9:583–594CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Dadhania V, Zhang M, Zhang L et al (2016) Meta-analysis of the luminal and basal subtypes of bladder cancer and the identification of signature immunohistochemical markers for clinical use. EBioMedicine. doi: 10.1016/j.ebiom.2016.08.036 PubMedPubMedCentralGoogle Scholar
  17. 17.
    Desai S, Lim SD, Jimenez RE et al (2000) Relationship of cytokeratin 20 and CD44 protein expression with WHO/ISUP grade in pTa and pT1 papillary urothelial neoplasia. Mod Pathol 13(12):1315–1323CrossRefPubMedGoogle Scholar
  18. 18.
    Alsheikh A, Mohamedali Z, Jones E, Masterson J, Gilks CB (2001) Comparison of the WHO/ISUP classification and cytokeratin 20 expression in predicting the behavior of low-grade papillary urothelial tumors. World/Health Organization/Internattional Society of Urologic Pathology. Mod Pathol 14(4):267–272CrossRefPubMedGoogle Scholar
  19. 19.
    Bertz S, Otto W, Denzinger S et al (2014) Combination of CK20 and Ki-67 immunostaining analysis predicts recurrence, progression, and cancer-specific survival in pT1 urothelial bladder cancer. Eur Urol 65:218–226CrossRefPubMedGoogle Scholar
  20. 20.
    Harnden P, Mahmood N, Southgate J (1999) Expression of cytokeratin 20 redefines urothelial papillomas of the bladder. Lancet 353(9157):974–977CrossRefPubMedGoogle Scholar
  21. 21.
    Christoph F, Müller M, Schostak M, Soong R, Tabiti K, Miller K (2004) Quantitative detection of cytokeratin 20 mRNA expression in bladder carcinoma by real-time reverse transcriptase-polymerase chain reaction. Urology 64(1):157–161CrossRefPubMedGoogle Scholar
  22. 22.
    Güdemann CJ, Weitz J, Kienle P et al (2000) Detection of hematogenous micrometastasis in patients with transitional cell carcinoma. J Urol 164(2):532–536CrossRefPubMedGoogle Scholar
  23. 23.
    Okegawa T, Kinjo M, Nutahara K, Higashihara E (2004) Value of reverse transcription polymerase chain assay in peripheral blood of patients with urothelial cancer. J Urol 171(4):1461–1466CrossRefPubMedGoogle Scholar
  24. 24.
    Ribal MJ, Mengual L, Marín M et al (2006) Molecular staging of bladder cancer with RT-PCR assay for CK20 in peripheral blood, bone marrow and lymph nodes: comparison with standard histological staging. Anticancer Res 26:411–419PubMedGoogle Scholar
  25. 25.
    Gazquez C, Ribal MJ, Marín-Aguilera M et al (2012) Biomarkers vs conventional histological analysis to detect lymph node micrometastases in bladder cancer: a real improvement? BJU Int 110(9):1310–1316CrossRefPubMedGoogle Scholar
  26. 26.
    Retz M, Rotering J, Nawroth R et al (2011) Long-term follow-up of bladder cancer patients with disseminated tumour cells in bone marrow. Eur Urol 60(2):231–238CrossRefPubMedGoogle Scholar
  27. 27.
    Lerner SP, McConkey DJ, Hoadley KA et al (2016) Bladder cancer molecular taxonomy: summary from a consensus meeting. Bladder Cancer 2(1):37–47CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Volkmer JP, Sahoo D, Chin RK et al (2012) Three differentiation states risk-stratify bladder cancer into distinct subtypes. Proc Natl Acad Sci U S A 109(6):2078–2083CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Denzinger S, Fritsche HM, Otto W, Blana A, Wieland WF, Burger M (2008) Early versus deferred cystectomy for initial high-risk pT1G3 urothelial carcinoma of the bladder: do risk factors define feasibility of bladder-sparing approach? Eur Urol 53:146–152CrossRefPubMedGoogle Scholar
  30. 30.
    Park J, Song C, Shin E, Hong JH, Kim CS, Ahn H (2013) Do molecular biomarkers have prognostic value in primary T1G3 bladder cancer treated with bacillus Calmette-Guerin intravesical therapy? Urol Oncol 31:849–856CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Johannes Breyer
    • 1
  • Ralph M. Wirtz
    • 2
    • 3
  • Wolfgang Otto
    • 1
  • Philipp Erben
    • 4
  • Maximilian C. Kriegmair
    • 4
  • Robert Stoehr
    • 5
  • Markus Eckstein
    • 5
  • Sebastian Eidt
    • 3
  • Stefan Denzinger
    • 1
  • Maximilian Burger
    • 1
  • Arndt Hartmann
    • 5
  • on behalf of the BRIDGE Consortium
  1. 1.Department of UrologyUniversity of RegensburgRegensburgGermany
  2. 2.STRATIFYER Molecular Pathology GmbHCologneGermany
  3. 3.Institute of Pathology at the St Elisabeth Hospital Köln-HohenlindCologneGermany
  4. 4.Department of UrologyUniversity Hospital of MannheimMannheimGermany
  5. 5.Institute of PathologyUniversity of Erlangen-NurembergErlangenGermany

Personalised recommendations