Volume 72, Issue 3, Pages 461-469 (September 2017) Prognostic Impact of a 12-gene Progression Score in Non–muscle-invasive Bladder Cancer: A Prospective Multicentre Validation Study  Lars Dyrskjøt, Thomas Reinert, Ferran Algaba, Emil Christensen, Daan Nieboer, Gregers G. Hermann, Karin Mogensen, Willemien Beukers, Mirari Marquez, Ulrika Segersten, Søren Høyer, Benedicte P. Ulhøi, Arndt Hartmann, Robert Stöhr, Sven Wach, Roman Nawroth, Kristina Schwamborn, Cane Tulic, Tatjana Simic, Kerstin Junker, Niels Harving, Astrid C. Petersen, Jørgen B. Jensen, Bastian Keck, Marc-Oliver Grimm, Marcus Horstmann, Tobias Maurer, Ewout W. Steyerberg, Ellen C. Zwarthoff, Francisco X. Real, Núria Malats, Per-Uno Malmström, Torben F. Ørntoft  European Urology  Volume 72, Issue 3, Pages 461-469 (September 2017) DOI: 10.1016/j.eururo.2017.05.040 Copyright © 2017 European Association of Urology Terms and Conditions

Fig. 1 Patient enrolment and 12-gene expression assay performance. (A) Flow chart of patient enrolment and exclusion. (B) Heat map of the expression of the 12 genes included in the assay for the first tumours included from each patient (n=750) together with clinical and histopathological characteristics. The distribution of the continuous progression score and associated cut-off values are shown in the bottom panel. Samples are sorted according to the progression score. Colour coding: Stage: Ta, white; T1+CIS, black. Grade: low grade+PUNLMP, white; high grade, black. BCG treatment: no, white; yes, black. CIS any time in disease course: no, white; yes, black. Cystectomy: no, white; yes, black. Progression to MIBC: no (white), yes (black); EORTC risk score: low (white), intermediate (grey), high (black). MIBC=muscle-invasive bladder cancer; CIS=carcinoma in situ; PUNLMP=papillary urothelial neoplasm of low malignant potential; MIBC=muscle-invasive bladder cancer; EORTC=European Organisation for Research and Treatment of Cancer; RT-qPCR=real-time qualitative polymerase chain reaction; pts=patients; Ct=cycle threshold. European Urology 2017 72, 461-469DOI: (10.1016/j.eururo.2017.05.040) Copyright © 2017 European Association of Urology Terms and Conditions

Fig. 2 Correlation of the progression score and outcome in non–muscle-invasive bladder cancer. Kaplan-Meier estimates of cumulative incidence of progression as a function of molecular classification of the first tumour included tumour for (A) all patients, (B) EORTC high-risk patients (risk >6), and (C) EORTC intermediate-risk patients (risk >0 and ≤6). No progression events occurred in the EORTC low-risk group (risk=0). Cumulative incidence curves to the left are based on cut-offoptimal and cumulative incidence curves to the right on cut-off90% sensitivity. Blue and red lines indicate patients classified as low risk and high risk, respectively. The number of progression events (Prog) is indicated for each patient group. EORTC=European Organisation for Research and Treatment of Cancer. European Urology 2017 72, 461-469DOI: (10.1016/j.eururo.2017.05.040) Copyright © 2017 European Association of Urology Terms and Conditions

Fig. 3 Combined analysis of continuous EORCT risk score and 12-gene progression score. (A) Nomogram for 2-yr and 5-yr PFS probability according to combined EORTC risk and progression scores. (B) Receiver operating characteristic curves depicting sensitivity and specificity for two-year PFS estimates for continuous EORTC risk score and progression score separately and in combination. (C) Nomogram calibration plot with 95% confidence intervals comparing observed and predicted two-year PFS for the combined risk calculation. EORTC=European Organisation for Research and Treatment of Cancer; PFS=progression-free survival. European Urology 2017 72, 461-469DOI: (10.1016/j.eururo.2017.05.040) Copyright © 2017 European Association of Urology Terms and Conditions

Fig. 4 Correlation of progression scores obtained from analysis of paired fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tumours. The cut-offoptimal values (broken lines) are shown for FFPE and FF samples. Samples that showed different dichotomised progression scores between FF and FFPE are marked in red. European Urology 2017 72, 461-469DOI: (10.1016/j.eururo.2017.05.040) Copyright © 2017 European Association of Urology Terms and Conditions