Volume 71, Issue 6, Pages 961-969 (June 2017) Liquid Biopsy Analysis of FGFR3 and PIK3CA Hotspot Mutations for Disease Surveillance in Bladder Cancer  Emil Christensen, Karin Birkenkamp-Demtröder, Iver Nordentoft, Søren Høyer, Kirstin van der Keur, Kim van Kessel, Ellen Zwarthoff, Mads Agerbæk, Torben Falck Ørntoft, Jørgen Bjerggaard Jensen, Lars Dyrskjøt  European Urology  Volume 71, Issue 6, Pages 961-969 (June 2017) DOI: 10.1016/j.eururo.2016.12.016 Copyright © 2016 European Association of Urology Terms and Conditions

Fig. 1 Overview of patient selection and number of tumours with FGFR3 and PIK3CA mutations. Mutation types are indicated together with the number of urine and plasma samples used for screening analysis. Two patients in the radical cystectomy (Cx) cohort analysed for tumour DNA in plasma and urine harboured both PIK3CA E545K and FGFR3 S249C mutations. * Patient selection was based on availability of liquid biopsies and disease course characteristics (Ta, T1, and progressive disease). NIMBC=non–muscle-invasive bladder cancer. European Urology 2017 71, 961-969DOI: (10.1016/j.eururo.2016.12.016) Copyright © 2016 European Association of Urology Terms and Conditions

Fig. 2 Tumour DNA in urine supernatants and correlation to tumour characteristics. Urinary tDNA level of all samples grouped by (A) stage, (B) grade, (C) tumour size dichotomised at 3cm, and (D) dichotomised European Organisation for Research and Treatment of Cancer (EORTC) risk score. The Wilcoxon rank-sum test was used to calculate p values. Upper p values (T) are based on urinary tDNA levels and lower p values (F) are based on urinary tDNA relative to total urinary DNA. ×=mean level of total urinary DNA; —=mean level of urinary tDNA; T0=no evidence of primary tumour. European Urology 2017 71, 961-969DOI: (10.1016/j.eururo.2016.12.016) Copyright © 2016 European Association of Urology Terms and Conditions

Fig. 3 Urinary tumour DNA levels in patients with non–muscle-invasive bladder cancer (NMIBC). Patients were grouped by the highest tumour stage observed during their disease course. Every box indicates a patient visit and tDNA copies per ml of urine supernatant are represented using the colour code as defined. Stage, therapy, presence of carcinoma in situ, and time since inclusion are displayed for all visits. BCG=bacillus Calmette-Guérin; NA=not available; T0=no evidence of primary tumour. European Urology 2017 71, 961-969DOI: (10.1016/j.eururo.2016.12.016) Copyright © 2016 European Association of Urology Terms and Conditions

Fig. 4 Tumour DNA in urine supernatants and plasma in relation to outcome. (A–C) Non–muscle-invasive bladder cancer (NMIBC) cohort. (A) Urinary tDNA level for all samples throughout the disease courses grouped by outcome. Samples obtained at or after progression were excluded. (B) Urinary tDNA level for all samples grouped by outcome and stage. Samples obtained at or after progression were excluded. (C) Kaplan-Meier survival estimates based on urinary tDNA level for the first sample analysed for every patient. (D–G) Radical cystectomy (Cx) cohort. (D) Urinary tDNA level for all patients grouped by outcome. (E) Kaplan-Meier survival estimates based on urinary tDNA level. (F) Plasma tDNA level for all patients grouped by outcome. (G) Kaplan-Meier survival estimates based on plasma tDNA level. The Wilcoxon rank-sum test was used to calculate p values for DNA levels. Upper p values (T) are based on urinary/plasma tDNA levels and lower p values (F) are based on urinary/plasma tDNA relative to total urinary/plasma DNA. The p values for Kaplan-Meier analysis were calculated using a log-rank test and patients were grouped by the median tDNA level. PFS=progression-free survival; RFS=recurrence-free survival; T0=no evidence of primary tumour; ×=mean level of total urinary/plasma DNA; —=mean level of urinary/plasma tDNA. European Urology 2017 71, 961-969DOI: (10.1016/j.eururo.2016.12.016) Copyright © 2016 European Association of Urology Terms and Conditions