Virchows Archiv

, Volume 469, Issue 5, pp 547–552 | Cite as

ESR1, ERBB2, and Ki67 mRNA expression predicts stage and grade of non-muscle-invasive bladder carcinoma (NMIBC)

  • Johannes Breyer
  • Ralph M. Wirtz
  • Mark Laible
  • Kornelia Schlombs
  • Philipp Erben
  • Maximilian Christian Kriegmair
  • Robert Stoehr
  • Sebastian Eidt
  • Stefan Denzinger
  • Maximilian Burger
  • Arndt Hartmann
  • Wolfgang Otto
Original Article


Pathological staging and grading are crucial for risk assessment in non-muscle-invasive bladder cancer (NMIBC). Molecular grading might support pathological evaluation and minimize interobserver variability. In this study, the well-established breast cancer markers ESR1, PGR, ERBB2, and MKI67 were evaluated as potential molecular markers to support grading and staging in NMIBC. We retrospectively analyzed clinical data and formalin-fixed paraffin-embedded tissues (FFPE) of patients with NMIBC. Messenger RNA (mRNA) expression of the aforementioned markers was measured by single-step reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) using RNA-specific TaqMan assays. Relative gene expression was determined by normalization to two reference genes (CALM2 and B2M) using the 40−ΔΔCT method and correlated to histopathological stage and grade. Pathological assessment was performed by an experienced uropathologist. Statistical analysis was performed using the SAS software JMP 9.0.0 version and GraphPad Prism 5.04. Of 381 cases of NMIBC, samples of 100 pTa and 255 pT1 cases were included in the final study. Spearman rank correlation revealed significant correlations between grade and expression of MKI67 (r = 0.52, p < 0.0001), ESR1 (r = 0.25, p < 0.0001), and ERBB2 (r = 0.18, p = 0.0008). In Mann-Whitney tests, MKI67 was significantly different between all grades (p < 0.0001), while ESR1 (p = 0.0006) and ERBB2 (p = 0.027) were significantly different between G2 and G3. Higher expression of MKI67 (r = 0.49; p < 0.0001), ERBB2 (r = 0.22; p < 0.0001), and ESR1 (r = 0.18; p = 0.0009) mRNA was positively correlated with higher stage. MKI67 (p < 0.0001), ERBB2 (p = 0.0058), and PGR (p = 0.0007) were significantly different between pTa and pT1. In NMIBC expression of ESR1, ERBB2 and MKI67 are significantly different between stage and grade. This potentially provides objective parameters for pathological evaluation.


NMIBC ERBB2 ESR1 MKI67 Stage Grade 



Estrogen receptor alpha


erb-b2 receptor tyrosine kinase 2/human epidermal growth factor receptor 2


Quantification cycle


Formalin-fixed paraffin-embedded


Gene of interest


Hazard ratio


Messenger ribonucleic acid


Marker of proliferation Ki-67


Progesterone receptor


Reference gene


Reverse transcription quantitative real-time polymerase chain reaction



The authors thank Sefanie Herlein for excellent technical support.

Compliance with ethical standards

Informed consent

Informed consent was obtained from all the individual participants included in the study.


The work on this topic was funded by the German Cancer Aid (Deutsche Krebshilfe), grant number 110,541.

Conflict of interest

RMW and SE are founders of STRATIFYER Molecular Pathology GmbH. RMW is an employee of STRATIFYER Molecular Pathology GmbH. ML and KS are employees of BioNTech Diagnostics GmbH.

Supplementary material

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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Johannes Breyer
    • 1
  • Ralph M. Wirtz
    • 2
    • 3
  • Mark Laible
    • 4
  • Kornelia Schlombs
    • 4
  • Philipp Erben
    • 5
  • Maximilian Christian Kriegmair
    • 5
  • Robert Stoehr
    • 6
  • Sebastian Eidt
    • 3
  • Stefan Denzinger
    • 1
  • Maximilian Burger
    • 1
  • Arndt Hartmann
    • 6
  • Wolfgang Otto
    • 1
  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.BioNTech Diagnostics GmbHMainzGermany
  5. 5.Department of UrologyUniversity of MannheimMannheimGermany
  6. 6.Institute of PathologyUniversity of Erlangen-NurembergErlangenGermany

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