Volume 61, Issue 6, Pages 1245-1256 (June 2012) Genome-wide Analysis of CpG Island Methylation in Bladder Cancer Identified TBX2, TBX3, GATA2, and ZIC4 as pTa-Specific Prognostic Markers  Raju Kandimalla, Angela A.G. van Tilborg, Lucie C. Kompier, Dominique J.P.M. Stumpel, Ronald W. Stam, Chris H. Bangma, Ellen C. Zwarthoff  European Urology  Volume 61, Issue 6, Pages 1245-1256 (June 2012) DOI: 10.1016/j.eururo.2012.01.011 Copyright © 2012 European Association of Urology Terms and Conditions

Fig. 1 Study design. CGI=CpG island; GGMA=Illumina Golden Gate Methylation assay; MI=muscle-invasive. European Urology 2012 61, 1245-1256DOI: (10.1016/j.eururo.2012.01.011) Copyright © 2012 European Association of Urology Terms and Conditions

Fig. 2 Comparison of methylation patterns between bladder cancer subgroups. (A) Unsupervised principal component analysis with all probes present on the 244K Agilent array: The non–muscle-invasive FGFR3 wild type (NMI-WT) bladder tumour group clusters separately from the non–muscle-invasive FGFR3 mutant (NMI-MT) and muscle-invasive (MI) tumours (red, MI; blue, NMI-MT; green, NMI-WT). (B) Venn diagram showing the number of genes hypermethylated in a specific group of bladder tumours. (C) Semisupervised analyses for bladder tumour subgroups by hierarchical clustering of NMI-MT versus NMI-WT tumours, MI versus NMI-WT tumours, and NMI-MT versus MI tumours. (D) Principal component analyses for bladder tumour subgroups as in Figure 2c. European Urology 2012 61, 1245-1256DOI: (10.1016/j.eururo.2012.01.011) Copyright © 2012 European Association of Urology Terms and Conditions

Fig. 3 Identification and validation of probes useful for urine diagnosis. (A) Hierarchical clustering of Illumina Golden Gate Methylation assay data of all the investigated samples. The bladder cancer samples cluster separately from normal blood and urine samples. (B) Heat map showing the probes that are methylated in all tumour subgroups versus blood and urine. (C) Methylation status of CpGs in the GATA2, TBX2, and TBX3 CpG islands as detected with BS-SNaPshot. Depending on whether the probe used is hybridising to the sense or the antisense strand, black/blue peaks represent methylated CpGs (C or G), and red/green peaks represent unmethylated CpGs (T or A) after bisulfite conversion. MI=muscle-invasive; NMI-WT=non–muscle-invasive wild type; NMI-MT=non–muscle-invasive mutation. European Urology 2012 61, 1245-1256DOI: (10.1016/j.eururo.2012.01.011) Copyright © 2012 European Association of Urology Terms and Conditions

Fig. 4 Methylation of specific CpG islands can predict progression to muscle-invasive disease in non–muscle-invasive NMI pTa tumours. Patients without methylation of TBX2, GATA2, ZIC4, and TBX3 have significantly better progression-free survival. (A) Test set: Illumina Golden Gate Methylation assay. (B) Validation set: The differences between the groups were significant, as indicated (log-rank test). The green line indicates methylation, the blue line indicates no methylation, and (+) indicates censored data. European Urology 2012 61, 1245-1256DOI: (10.1016/j.eururo.2012.01.011) Copyright © 2012 European Association of Urology Terms and Conditions

Fig. 5 Comparison of European Organisation for Research and Treatment of Cancer (EORTC) risk scores and methylation scores for predicting progression in the validation set (n=41). (A) Kaplan-Meier plot for progression-free survival according to the EORTC risk score. Based on these scores, patients are classified as low, intermediate, or high risk for progression (p=0.012). (B) Methylation grade improved prediction of progression in the intermediate-risk EORTC group (p=0.007). Methylation grade was determined as 0 (no gene methylated), 1 (one gene methylated), or 2 (two or more genes methylated). European Urology 2012 61, 1245-1256DOI: (10.1016/j.eururo.2012.01.011) Copyright © 2012 European Association of Urology Terms and Conditions