Volume 191, Issue 1, Pages (January 2014)

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Volume 191, Issue 1, Pages 261-269 (January 2014) Validation Study of a Noninvasive Urine Test for Diagnosis and Prognosis Assessment of Bladder Cancer: Evidence for Improved Models Lourdes Mengual, María José Ribal, Juan José Lozano, Mercedes Ingelmo-Torres, Moisés Burset, Pedro Luís Fernández, Antonio Alcaraz The Journal of Urology Volume 191, Issue 1, Pages 261-269 (January 2014) DOI: 10.1016/j.juro.2013.06.083 Copyright © 2014 American Urological Association Education and Research, Inc. Terms and Conditions

Figure 1 Project outline. DNA microarray and TaqMan arrays were used to screen gene expression profiles in bladder cancer9 and identify gene set expression signature to detect and stratify UCC.12 BW, bladder washings. RT-qPCR, real-time quantitative PCR. Gray boxes represent current study. The Journal of Urology 2014 191, 261-269DOI: (10.1016/j.juro.2013.06.083) Copyright © 2014 American Urological Association Education and Research, Inc. Terms and Conditions

Figure 2 Diagnostic performance of 12-gene model in validation set of 207 samples. A, heat map shows 12-gene expression signature for UCC diagnosis. Red pixels represent increased mRNA in urine samples. Green pixels represent decreased mRNA in urine samples. Rows indicate individual genes. Columns indicate experimental samples. Blue bars indicate control. Red bars indicate tumor. B, multidimensional scaling plot. Red dots represent tumor. Blue dots represent control. C, ROC curves in training set of 211 samples and validation set of 207 with 0.525 cutoff. n T/n C, number of tumors/number of controls. PPV, positive predictive value. NPV, negative predictive value. D, SN of 12-gene signature in UCC risk groups in validation set. Ta Gx and Tx LG samples were excluded from analysis. The Journal of Urology 2014 191, 261-269DOI: (10.1016/j.juro.2013.06.083) Copyright © 2014 American Urological Association Education and Research, Inc. Terms and Conditions

Figure 3 Prognostic performance of 12 + 2 gene set model in validation set of 95 samples. A, heat map shows 12 + 2 gene signature to predict UCC tumor aggressiveness in validation set. Ta Gx sample was excluded from analysis. Red pixels represent increased mRNA in urine samples. Green pixels represent decreased mRNA in urine samples. Rows indicate individual genes. Columns indicate experimental samples. Red bars indicate HG. Blue bars indicate LG. B, multidimensional scaling plot of HG (red dots) and LG (blue dots) tumor samples. C, ROC curves of 12 + 2 gene expression signature in training set of 97 samples and validation of 95 with 0.525 cutoff. The Journal of Urology 2014 191, 261-269DOI: (10.1016/j.juro.2013.06.083) Copyright © 2014 American Urological Association Education and Research, Inc. Terms and Conditions

Figure 4 Diagnostic performance of new gene set signatures in enlarged training set of 404 samples, including 190 tumors and 214 controls. A, overall diagnostic performance of 4 new gene set signatures. B, SN of 4-gene expression signatures in UCC risk groups. Ta Gx and Tx LG samples were excluded from analysis. PPV, positive predictive value. NPV, negative predictive value. ER, error rate. C, influence on UCC diagnosis of each study gene. Bar height above reference line indicates statistically significant association (p <0.05). Select genes for diagnostic gene signatures mostly matched those with higher bars in influence plot. D, ROC curves of 4 new diagnostic gene expression signatures in enlarged set using LOOCV. The Journal of Urology 2014 191, 261-269DOI: (10.1016/j.juro.2013.06.083) Copyright © 2014 American Urological Association Education and Research, Inc. Terms and Conditions

Figure 5 Prognostic performance of new gene set signatures in enlarged training set of 192 samples, including 76 LG and 116 HG tumors. A, overall prognostic performance. PPV, positive predictive value. NPV, negative predictive value. ER, error rate. B, influence on UCC aggressiveness of each study gene. Bar height above reference line indicates statistically significant association (p <0.05). Select genes for GS_P6 mostly matched those with higher bars in influence plot. C, ROC curves of 2 new prognostic gene expression signatures using LOOCV. The Journal of Urology 2014 191, 261-269DOI: (10.1016/j.juro.2013.06.083) Copyright © 2014 American Urological Association Education and Research, Inc. Terms and Conditions