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// Markus Eckstein 1, * , Ralph M. Wirtz 2, 3, * , Carolin Pfannstil 1, * , Sven Wach 4, * , Robert Stoehr 1, * , Johannes Breyer 6, * , Franziska Erlmeier 7, * , Cagatay Gunes 8, * , Katja Nitschke 5, * , Wilko Weichert 7, * , Wolfgang Otto 6, * , Bastian Keck 4, * , Sebastian Eidt 3, * , Maximilian Burger 6, * , Helge Taubert 4, * , Bernd Wullich 4, * , Christian Bolenz 8, * , Arndt Hartmann 1, * and Philipp Erben 5, * 1 Institute of Pathology, University of Erlangen-Nuremberg, Erlangen, Germany 2 STRATIFYER Molecular Pathology GmbH, Cologne, Germany 3 Institute of Pathology at The St. Elisabeth Hospital Koln-Hohenlind, Cologne, Germany 4 Department of Urology, University of Erlangen-Nuremberg, Erlangen, Germany 5 Department of Urology Mannheim, University of Heidelberg, Mannheim, Germany 6 Department of Urology, University of Regensburg, Regensburg, Germany 7 Institute of Pathology, Technical University Munich, Munich, Germany 8 Department of Urology, University of Ulm, Ulm, Germany * On behalf of the BRIDGE-Consortium Germany Correspondence to: Markus Eckstein, email: markus.eckstein@uk-erlangen.de Keywords: bladder cancer; PD-L1; checkpoint inhibitors; molecular therapy stratification; immunohistochemistry Received: October 26, 2017 Accepted: February 10, 2018 Epub: February 19, 2018 Published: March 13, 2018 ABSTRACT Background: Immunohistochemical PD-L1 assessment is currently used to identify responders towards checkpoint inhibitors although it is limited by inter-observer effects. Here, we conducted a multi-center round robin test to prove the possibility of assessing the PD-L1 status by gene expression to avoid inter-observer effects. Patients and methods: Gene expression of PD-L1 was analyzed in a total of 294 samples (14 cases non-muscle invasive and muscle-invasive bladder cancer; MIBC) in seven centers by a RT-qPCR kit and compared with immunohistochemical scoring of three pathologists (DAKO, 22c3). Both assays were compared towards prognosis prediction in a cohort of 88 patients with MIBC. Results: PD-L1 gene expression revealed very high inter center correlation (centrally extracted RNA: r = 0.68–0.98, p ≤ 0.0076; locally extracted RNA: r = 0.81–0.98, p ≤ 0.0014). IHC Inter-observer concordance was moderate to substantial for immune cells (IC), fair for combined IC/ tumor cell (TC) (IC: κ = 0.50–0.61; IC + TC: κ = 0.50), and fair for TC scoring (κ = 0.26–0.35). Gene expression assessment resulted in more positive cases (9/14 cases positive vs. 6/14 cases [IHC]) which could be validated in the independent cohort. Positive mRNA status was associated with significantly better overall and disease-specific survival (5-year OS: 50% vs. 26%, p = 0.0042, HR = 0.48; 5 year DSS: 65% vs. 40%, p = 0.012, HR = 0.49). The 1% IHC IC cut-off also revealed significant better OS (5 year OS: 58% vs. 31%, p = 0.036, HR = 0.62). Conclusion: Gene expression showed very high inter-center agreement. Gene expression assessment also resulted in more positive cases and revealed better prognosis prediction. PD-L1 mRNA expression seems to be a reproducible and robust tool for PD-L1 assessment.