Evaluation of Urine cfDNA for Non-Invasive Genomic Characterization in Multiple Myeloma: A Comparative Study with Serum and Bone Marrow Samples
Authors: Neuse Carl Jannes , Schliemann Christoph , Khandanpour Cyrus , Lenz Georg , Ghobrial Irene , Bustoros Mark 
Published: December 2024

Background
Cell-free DNA (cfDNA) sequencing has emerged as a promising tool for detecting cancer and monitoring treatment response. Blood-based cfDNA analyses hold the potential to detect diverse classes of somatic mutations in multiple myeloma (MM) with minimal invasiveness. We sought to evaluate the potential of genomic characterization of MM patients from urine samples as a truly non-invasive source of cfDNA. Here we developed a protocol for collection of urine cfDNA and compared it to serum cfDNA, and bone marrow tumor samples by performing next generation sequencing on these different compartments to assess the detection of known genomic aberrations in MM.
Methods
We prospectively collected serum-cfDNA, urine-cfDNA and bone marrow mononuclear cell (BMMC) DNA from 8 patients, created genomic libraries and performed ultra-low pass whole genome sequencing (ULP-WGS) on those samples. The results were then compared to the current gold standard of genomic aberration detection, fluorescence in situ hybridization (FISH). Of those 8 patients, 4 were newly diagnosed and 4 were relapsed. A preservative was added to the urine samples to limit cfDNA degradation. A centrifugal concentrator (Vivacell 100, Sartorius) was used to increase cfDNA yield in the urine. The cfDNA was extracted from the concentrated urine using a urine-cfDNA purification kit with protease pretreatment (Neo Gene Star). Serum-cfDNA was extracted from the serum using a ccfDNA mini kit (Qiagen). Both kits extract cfDNA through magnetic bead selection. BMMCs were isolated using density gradient media (Ficoll-Paque) and BMMC-DNA was obtained using a DNA blood mini kit (Qiagen). Presence of cfDNA in the samples was confirmed with a bioanalyzer (Agilent) and a dsDNA quantification assay (Invitrogen). Genomic libraries were created from serum-cfDNA, urine-cfDNA and BMMC-DNA using a KAPA library quantification kit for Illumina platforms (Roche). The libraries were pooled and sequenced with HiSeq2500 machines (Illumina) at the Broad Institute of MIT and Harvard with a coverage of 0.1X for ultra-low-pass whole genome sequencing (ULP-WGS). Sequencing results were analyzed using the ichorCNA tool to estimate the tumor fraction and predict large-scale copy number variations (CNVs). CNVs identified via ULP-WGS were compared to FISH-analysis results.
Results
Presence of DNA at the target length of 120-200 base pairs in the urine samples was confirmed via bioanalyzer. Indication of low cfDNA concentration in the urine samples was met with a revised protocol that included urine concentration. After implementation of a centrifugal concentrator, higher concentration of DNA in the urine at the target length of 120-200 base pairs, was confirmed via bioanalyzer.
The median tumor fraction in the BMMC-DNA was 6.1% (range: 0.5% to 20%), in the serum-cfDNA 3.9% (range: 3.2% to 45%) and in the urine-cfDNA 1.6% (range: 1% to 4.3%). In total, the FISH-analysis returned 13 CNVs. ULP-WGS led to the identification of 12 CNVs in the BMMC-DNA, 11 CNVs in the serum-cfDNA and 9 CNVs in the urine-cfDNA. In two patients a CNV, confirmed by FISH, could be detected in the urine-cfDNA that had not been found in the BMMC-DNA or the serum-cfDNA. In two different patients, ULP-WGS of BMMCs, serum-cfDNA and urine-cfDNA found precisely those CNVs which were also identified via FISH. Amplification 5q was only be detected in urine-cfDNA but not in BMMC-DNA nor serum-cfDNA. Amplification 1q was detected in BMMC-DNA and serum-cfDNA, but not in urine-cfDNA. Amplifications 3, 9, 11 and 19 was detected in all compartments.
Conclusion
We provide the first proof of concept study illustrating the presence of tumor derived cfDNA in urine samples of MM-patients as well as the feasibility of isolating it. By combining the ULP_WGS results of BMMC-DNA, serum-cfDNA and urine-cfDNA, genomic characterization of MM was possible with a precision similar to FISH-analysis. We observed that the tumor fraction is relatively low and CNV detection correlated with increased tumor-fraction and burden. However, this could be due to the comparatively small sample size of only 8 patients. We plan to add more samples and perform more comprehensive exome sequencing to detect both CNVs and somatic mutations characteristic to MM. All in all, we show that urine-cfDNA represents a promising biomarker in MM opening novel avenues for monitoring progression of disease or response to therapy in MM-patients.