Introduction

To characterize genomic aberrations of circulating, cell-free DNA (cfDNA) in bladder cancer patients treated with radical cystectomy (RC), we established a new and rapid profiling method using multiplex ligation-dependent probe amplification (MLPA). In a single reaction, MLPA allows analyzing genomic variations in 43 chromosomal regions containing 37 genes.

Methods

We prospectively enrolled 85 bladder cancer patients treated with RC without neoadjuvant chemotherapy at our institution between 2011 and 2014. Blood samples were obtained from all patients preoperatively. Serum and plasma were prepared from 6ml whole blood in all patients. We extracted cfDNA from serum and plasma using various DNA extraction kits (QiAmp DNA Blood Mini kit, Qiagen, Hilden, Germany; QiAmp Circulating Nucleic Acid kit, Qiagen; NucleoSpin Plasma XS kit, Macherey Nagel, Dueren, Germany; PME free-circulating DNA Extraction kit, Analytik Jena, Germany). Following extraction of DNA with the applied commercial kits, we tested MLPA in serum and plasma, respectively. Eighteen probes served as references to control denaturation, ligation and amplification efficiency.

Results

To identify genomic aberrations, MLPA was suitable in cfDNA extracted from serum, but not in cfDNA extracted from plasma. Serum from 72 patients (84.7%) could be analyzed. In 35 patients (48.6%), one to 6 deleted and/or amplified chromosomal regions were detected. Most changes were located in the genes E-cadherin, ZFHX3 (both amplifications in chromosome 16), RIPK2 (deletion in chromosome 8) and PTEN (deletion in chromosome 10) in 15 patients (20.8%), 12 patients (16.7%), 9 patients (12.5%) and 7 patients (9.7%), respectively. Copy number variations of various genes were associated with presence of variant histology (TSG1, RAD21, KIAA0196, ANXA7; all p-values<0.029), pathologic tumor grade (TSG1, KIAA0196, RAD21, ANXA7, TMPRSS2; all p-values<0.029), presence of incidental prostate cancer (EZH2, miR-15a, CDH1, ZFHX3; all p-values<0.024), as well as increased risk of disease recurrence (RIPK2; p=0.038), overall (KLF5; p=0.033) and cancer-specific mortality (KLF5, ZFHX3; all p-values<0.022).

Conclusions

In serum cfDNA, MLPA represents an efficient method for the detection of genomic aberrations among numerous chromosomal regions and genes. Copy number variations of various genes are associated with aggressive pathologic bladder cancer features and seem to have a negative impact on disease recurrence and survival.

Funding

None