Copy number variations of circulating, cell-free DNA in urothelial carcinoma of the bladder patients treated with radical cystectomy: a prospective study

// Armin Soave 1 , Felix K.-H. Chun 1 , Timo Hillebrand 2 , Michael Rink 1 , Lars Weisbach 1 , Bettina Steinbach 3 , Margit Fisch 1 , Klaus Pantel 3 and Heidi Schwarzenbach 3 1 Department of Urology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2 AJ Innuscreen GmbH, Berlin, Germany 3 Department of Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Correspondence to: Heidi Schwarzenbach, email: hschwarzenbach@uke.de Keywords: urothelial carcinoma of the bladder, copy number variation, multiplex ligation-dependent probe amplification (MLPA), cell-free DNA (cfDNA), chromosomal region Received: March 21, 2017      Accepted: April 26, 2017      Published: May 07, 2017 ABSTRACT The aim of the present study was to establish a rapid profiling method using multiplex ligation-dependent probe amplification (MLPA) and characterize copy number variations (CNV) in circulating, cell-free DNA (cfDNA) in 85 urothelial carcinoma of the bladder (UCB) patients treated with radical cystectomy (RC). MLPA was tested for the use of cfDNA extracted from serum and plasma by various commercial extraction kits. Eighteen probes served as reference to control denaturation, ligation and amplification efficiency. MLPA was exclusively suitable for cfDNA extracted from serum. Serum from 72 patients (84.7%) could be analyzed. Thirty-five patients (48.6%) had presence of CNV in cfDNA. The median CNV count in patients with presence of CNV was 2. Predominantly, CNV were located in the genes CDH1, ZFHX3, RIPK2 and PTEN in 15 patients (20.8%), 12 patients (16.7%), 9 patients (12.5%) and 7 patients (9.7%), respectively. CNV in TSG1, RAD21, KIAA0196, ANXA7 and TMPRSS2 were associated with presence of variant UCB histology ( p = 0.029, 0.029, 0.029, 0.029, 0.043, respectively). Furthermore, CNV in miR-15a, CDH1 and ZFHX3 were associated with presence of incidental prostate cancer ( p = 0.023, 0.003, 0.025, respectively). Patients with CNV in KLF5, ZFHX3 and CDH1 had reduced cancer-specific survival, compared to patients without CNV in these genes (pairwise p = 0.028, 0.026, 0.044, respectively). MLPA represents an efficient method for the detection of CNV among numerous genes on various chromosomal regions. CNV in specific genes seem to be associated with aggressive UCB biologic features and presence of incidental prostate cancer, and may have a negative impact on cancer-specific survival.