1. Volume 73, Issue 4, Pages 535-540 (April 2018)
Monitoring Treatment Response and Metastatic Relapse in Advanced Bladder Cancer by Liquid Biopsy Analysis 
Karin Birkenkamp-Demtröder, Emil Christensen, Iver Nordentoft, Michael Knudsen, Ann Taber, Søren Høyer, Philippe Lamy, Mads Agerbæk, Jørgen Bjerggaard Jensen, Lars Dyrskjøt 
European Urology 
Volume 73, Issue 4, Pages (April 2018)
DOI: /j.eururo
Copyright © 2017 European Association of Urology Terms and Conditions

2. Fig. 1
Patient overview and results of liquid biopsy analyses. (A) Patient enrolment and analyses performed. Predesigned assays were used to screen 60 tumour biopsies for hotspot mutations in PIK3CA and FGFR3. In total, 19 patients had at least one hotspot mutation in PIK3CA and FGFR3. Tumour and germline DNA from 24 selected patients was exome sequenced (nine with a hotspot mutation and 15 without), and 81 personalised assays against cancer driver mutations were designed. The cfDNA in longitudinally collected plasma and urine samples from 26 patients (24 exome sequenced and two included due to positive screening without sequencing) was analysed by ddPCR. (B) Levels of ctDNA in disease-free patients and in patients with metastatic relapse. Comparison of ctDNA levels in plasma between patients being disease free and patients experiencing clinical relapse after cystectomy. Each point represents an average of all ctDNA measurements for a specific sample given as copies/ml plasma. The yellow diamond represents the median value. (C) Kaplan–Meier survival estimate. Recurrence-free survival as a function of ctDNA levels during the initial 4 mo after cystectomy. Patients were dichotomised based on the median of the maximum ctDNA level observed after cystectomy. Time was limited to 12 mo as only few events occurred during the remainder of the clinical surveillance after cystectomy. (D) Liquid biopsy analysis summary. Graphical overview of liquid biopsy analyses of 26 patients using personalised assays. Every line represents a patient with circles indicating analysed plasma samples according to the timeline presented at the top. Grouping of patients into “Disease free” and “Clinical relapse” was based on radiographic imaging results at scheduled controls during the disease courses. Patient visits were marked positive if at least one personalised assay was positive. cfDNA=cell-free DNA; ctDNA=circulating tumour DNA; CX=cystectomy; ddPCR=digital droplet polymerase chain reaction; pt.=patient; TUR-B=transurothelial resection-bladder. a Patients were selected based on the number of samples available as well as having a long follow-up time to represent a 1:1 ratio of disease-free patients versus patients with metastatic relapse. b Patients were selected based on availability of tumour biopsies with a high carcinoma cell percentage and a high number of longitudinally collected plasma samples. c Last point matching timeline. d Death. e Relapse after 45 mo.
European Urology  , DOI: ( /j.eururo )
Copyright © 2017 European Association of Urology Terms and Conditions

3. Fig. 2
Monitoring disease courses and treatment efficacy by ctDNA analysis. (A) Monitoring individual disease courses in cfDNA from plasma. Selected disease courses showing the analysis of longitudinally collected plasma are presented. Clinical events displayed on the timeline are explained in the legend. Coloured lines: two to six personalised assays per patient targeting cancer driver mutations are shown as copies ctDNA/ml plasma (left y-axis). Black horizontal bars: measurements of total cfDNA given as copies/ml plasma (right y-axis). The red vertical lines mark time points of metastatic relapse and progression. Specifically, patients 31 and 21 experienced initial disease regression and response to treatment of metastatic disease accompanied by decreasing or steady ctDNA levels. Pt. 2 showed neither progression nor regression, while pt. 54 showed disease progression under immunotherapy. Levels of ctDNA increased successively in pts. 31, 21, and 54 before progression was diagnosed. Pt. 23 showed disease regression and treatment response upon neoadjuvant chemotherapy, accompanied by decreasing ctDNA levels. (B) Comparison of ctDNA levels before, during, and after treatment. Coloured lines represent individual patients. Dots represent average ctDNA values for all utilised markers, while diamonds represent ctDNA values for all individual markers. Time points were defined as follows: pre=at diagnosis and/or before first cycles of chemotherapy, during=before each cycles of chemotherapy except first cycles, and after=after treatment. cfDNA=cell-free DNA; ctDNA=circulating tumour DNA; CX=cystectomy; LN=lymph node; pt.=patient; TUR-B=transurothelial resection-bladder.
European Urology  , DOI: ( /j.eururo )
Copyright © 2017 European Association of Urology Terms and Conditions