Volume 68, Issue 5, Pages 824-832 (November 2015) A Molecular Pathologic Framework for Risk Stratification of Stage T1 Urothelial Carcinoma  Oliver Patschan, Gottfrid Sjödahl, Gunilla Chebil, Kristina Lövgren, Martin Lauss, Sigurdur Gudjonsson, Petter Kollberg, Pontus Eriksson, Mattias Aine, Wiking Månsson, Mårten Fernö, Fredrik Liedberg, Mattias Höglund  European Urology  Volume 68, Issue 5, Pages 824-832 (November 2015) DOI: 10.1016/j.eururo.2015.02.021 Copyright © 2015 European Association of Urology Terms and Conditions

Fig. 1 Flow diagram for case selection and characterisation. The population-based cohort included 254 patients identified in the Swedish Bladder Cancer Registry as having been diagnosed with primary T1 bladder cancer in the Southern Health Care Region between January 1997 and April 2003. For classification, urobasal (Uro) cases were defined as having at least two of the following three characteristics: urothelial-like histology, pathologic grade 1 or 2 (WHO 1999), or a high CCNB1 labelling index. Staining for smooth muscle α-actin (α-SMA) was performed to establish the integrity of the tumour-stroma interface, and squamous-cell carcinoma–like (SCCL) tumours were distinguished from genomically unstable (GU) tumours by high KRT5 expression. Urothelial-like growth pattern, CCNB1, and KRT5 intensity thresholds were applied as defined by Sjödahl et al [11]. European Urology 2015 68, 824-832DOI: (10.1016/j.eururo.2015.02.021) Copyright © 2015 European Association of Urology Terms and Conditions

Fig. 2 Immunohistochemistry validation of molecular classification of stage T1 tumours. (A) Heat map showing average subtype expression levels determined as tumour cell scores (TCS) for FGFR3, CCND1, TP63, RB1, ERBB2 (Her2), MKI67 (Ki67), CDKN2A (p16), and nuclear E2F3. High (red), intermediate (black), and low (green) average expression levels are indicated. (B) Bar plot illustrating the proportions of tumours with basal stratification of P-cadherin (P-Cad) and KRT5 expression: in the basal cell layer when present (dark blue); throughout the tumour parenchyma (light blue); not present (white). (C) Representative immunohistochemical staining of tumours classified as urobasal (Uro), genomically unstable (GU), and squamous-cell carcinoma–like (SCCL). Areas shown are 0.6mm×0.6mm. nuc.=nuclear. European Urology 2015 68, 824-832DOI: (10.1016/j.eururo.2015.02.021) Copyright © 2015 European Association of Urology Terms and Conditions

Fig. 3 Kaplan-Meier analyses with progression as endpoint. The analyses were performed using the following as grouping variables: (A) urobasal (Uro), genomically unstable (GU), and squamous-cell carcinoma–like (SCCL) tumours; (B) GU/SCCL low-EORTC (WHO 1999) and GU/SCCL high-EORTC (WHO 1999) tumours; (C) GU/SCCL low-CD3 and GU/SCCL high-CD3 tumours; (D) and Uro, GU/SCCL low-EORTC (WHO 1999)/CD3 and GU/SCCL high-EORTC (WHO 1999)/CD3 tumours. The group designated Uro high-EORTC (WHO 1999)/CD3 was omitted from Kaplan-Meier analysis because of its small size (n=4). European Urology 2015 68, 824-832DOI: (10.1016/j.eururo.2015.02.021) Copyright © 2015 European Association of Urology Terms and Conditions

Fig. 4 Progression biomarkers are associated with molecular subtype. The heat map shows mRNA expression of 27 genes (Supplementary Table 1) significantly associated with molecular subtype. The subtype urobasal B (UroB) is included because it was readily identified when using mRNA expression data. Note that some markers for good prognosis (eg, SERPINB5) are also expressed in the poor-prognosis UroB and SCCL tumours, and that the poor-prognosis markers S100A8 and SERPINA1 are found specifically in UroB and SCCL tumours, not in the poor-prognosis GU tumours. European Urology 2015 68, 824-832DOI: (10.1016/j.eururo.2015.02.021) Copyright © 2015 European Association of Urology Terms and Conditions