Abstract 5245: Identification and quantification of somatic structural variations in tumor, urine and plasma from bladder cancer patients
Authors: Birkenkamp-Demtröder Karin , Nordentoft Iver , Christensen Emil , Hoyer Søren , Reinert Thomas , Vang Søren , Bjerggaard Jensen Jørgen , Ørntoft Torben F. , Dyrskjøt Lars 
Published: August 2015

Abstract
Bladder tumors can be divided into two clinically divergent groups, non-muscle invasive (NMIBC) and muscle invasive bladder tumors (MIBC). NMIBCs have a recurrence rate of 50-70%, while MIBCs often develop metastases. No molecular biomarkers are yet available to directly guide treatment and follow-up regimes for patients with BC.
Aim of this study was to identify tumor specific, large somatic structural variations (SSV) in DNA from tumors and quantify SSVs in DNA from urine and in circulating free DNA (cfDNA) in plasma from successive visits during treatment and follow-up.
Methods: Whole genome paired-end (WGS) or mate-pair (MPS) sequencing on HiSeq2000 and Nextseq sequencers. SSVs called by algorithms “CREST” or “Breakdancer”. cfDNA was extracted from 1-3 ml plasma. Quantification of SSVs was done by droplet digital PCR (ddPCR) using probes covering the breakpoint.
We selected six patients with progressive/metastatic disease, followed for 4 to 19 years with consecutive sampling of tumors, urine and plasma samples (4 to 27 visits). WGS was carried out on DNA from germ-line and tumors from four patients (57x and 45x read depth). CREST identified 20-226 SSVs per tumor. PCR assays (n = 170, 21-64 per patient) showed validation rates of 60-75% (CTX), 75-90% (ITX), 88-100% (INS) and 50% (INV) among the eight tumors. MPS was carried out on DNA from two patients (22-30x physical read depth). Breakdancer identified 82 and 120 SSVs, 24 and 13 were potential somatic with a validation rate of 69% and 79%. For each patient, 3-5 fluorescent probes covering the breakpoint were used to quantify the level of tumor specific by ddPCR. SSVs were quantified in tumor DNA from several visits. Their frequency varied considerably (0.08-0.54) between the individual tumors, indicating a large inter-tumor heterogeneity. SSVs detected in DNA from urine pellets were correlated to the presence of tumor and treatment for some cases. Inter-individual cfDNA level ranged from 600-3500 genomic equivalents (GEs) per ml plasma. SSVs were identified in plasma. The level of ctDNA ranged between 5-300 GEs/ml plasma and an increase in ctDNA was already detectable up to 21 months before progression to muscle invasive disease. For metastasized disease, a 42-fold increase of the ctDNA level was observed compared to localized disease. No SSVs were detected in cfDNA from patients cured from BC (3 and 7 years post-recurrence and 5 years post-cystectomy).
Conclusions: Tumor derived SSVs were identified and quantified in cfDNA from plasma of patients with progressive and metastatic BC. Detection of SSVs in cfDNA was possible at early time points before progression and metastasis of the disease were discovered by cystoscopy or image diagnostic. The tumor specific and highly sensitive detection and quantification of SSVs in cfDNA from plasma may offer a unique tool to monitor disease course, residual tumor burden, disease progression and treatment response in BC.
Citation Format: Karin Birkenkamp-Demtröder, Iver Nordentoft, Emil Christensen, Søren Hoyer, Thomas Reinert, Søren Vang, Jørgen Bjerggaard Jensen, Torben F. Ørntoft, Lars Dyrskjøt. Identification and quantification of somatic structural variations in tumor, urine and plasma from bladder cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5245. doi:10.1158/1538-7445.AM2015-5245