Evaluation of urinary cfDNA workflows for the molecular profiling of malignant disease.
Author(s): Eberhard A, Moser T, Ziegler L, Vlachos G, Loibner M, Bauernhofer T, Balic M, Gerger A, Dandachi N, Beichler C, Glawitsch L, Moser M, Graf R, Abuja PM, Schmitz M, Krenz T, Voss T, Mancarella D, Heitzer E
Publication: iScience, 2025, Vol. 28, Page 113632
PubMed ID: 41142998 PubMed Review Paper? No
Purpose of Paper
This paper compared the stability of cell-free DNA (cfDNA) in unpreserved urine and urine preserved with PAXgene or Streck urine preservative during 4 days of room temperature storage. The authors then compared the detection of mutations in breast cancer, prostate cancer and colorectal cancer in patient plasma with that of case-matched urine that was stored for 0 or 3 days unpreserved or preserved with PAXgene or Streck preservatives. Mutations were detected using different platforms for each cancer type investigated.
Conclusion of Paper
Urine cfDNA levels were higher in specimens from women than men (P<0.001), but intra-individual variability was also higher in specimens from women than men. Urine cfDNA levels were more variable when quantified by Qubit than by PCR-based methods. When unpreserved urine was stored for 3 h, DNA yield and DNA fragmentation increased, indicating the release of cellular DNA and degradation of cfDNA. In contrast, cfDNA levels were not affected by when urine in PAXgene or Streck tubes were stored for up to 96 h at room temperature. Consequently, cfDNA levels were lower in unpreserved urine than PAXgene-preserved urine at all timepoints (P<0.001 for all), but were comparable in Streck- and PAXgene-preserved urine. Shallow whole genome sequencing showed that urine cfDNA ranged from 50-500 bp with a 10 bp periodicity, but there was an increase in the number of fragments <150 bp in unpreserved urine specimens. Mutations and copy number variants were detected in preserved urine from cancer patients, but detection was generally at a lower variant allele frequency than in plasma. When urine was not preserved, there was a higher rate of sequencing library failure and a decrease in the detection of mutations using digital PCR.
Studies
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Study Purpose
This study compared the stability of cell-free DNA (cfDNA) in unpreserved urine and urine preserved with PAXgene or Streck urine preservative during 4 days of room temperature storage. Second-morning urine was collected from healthy volunteers (19 women and 20 men) into BD Vacutainer Urine Collection Cups. Urine from 10 women and 10 men was split into PAXgene Urine Liquid Biopsy Tubes and plain tubes, and urine from the remaining 9 women and 10 men was divided between PAXgene Urine Liquid Biopsy Tubes and tubes containing a 1:5 ratio of Streck urine preservative. Urine was then stored for 0, 3, 6, 24, 48 and 96 h at room temperature, centrifuged at 1900 g for 15 min followed by 1900 g for 10 min (PAXgene and unpreserved urine) or 2,700g for 10 min (Streck urine), and the supernatants were frozen at -80°C. DNA was extracted with the QIAsymphony DSP Circulating DNA Kit on a QIAsymphony SP instrument. DNA was quantified using the Qubit dsDNA High Sensitivity Kit, real-time PCR amplification of a 66 bp fragment of the 18S rRNA gene, the digital PCR-based TaqMan RNase P assay and the Quantiplex Pro multiplex assay.
Summary of Findings:
Urine cfDNA levels were higher in urine specimens from women than men (P<0.001), but intra-individual variability was also higher in specimens from women than men. Urine cfDNA levels were more variable when quantified by Qubit than PCR-based methods. When unpreserved urine was stored for 3 h, DNA yield and DNA fragmentation increased, indicating the release of cellular DNA and degradation of cfDNA. In contrast, cfDNA levels were not affected when PAXgene or Streck tubes were stored for up to 96 h at room temperature. Consequently, cfDNA levels in unpreserved urine were lower than PAXgene-preserved urine at all timepoints (P<0.001 for all), but were comparable in Streck- and PAXgene-preserved urine. Shallow whole genome sequencing showed that urine cfDNA ranged from 50-500 bp with a 10 bp periodicity, but there was an increase in the number of fragments <150 bp in unpreserved urine.
Biospecimens
Preservative Types
- Streck/BCT
- None (Fresh)
- PAXgene
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Automated electrophoresis/Bioanalyzer DNA Real-time qPCR DNA Fluorometry DNA Digital PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Patient gender Female
Male
Biospecimen Preservation Type of fixation/preservation Streck cfDNA urine presevative
PAXgene
None (fresh)
Storage Storage duration 0 h
3 h
6 h
24 h
48 h
96 h
Fluorometry Specific Technology platform Real-time PCR of 18S rRNA gene
Qubit
Digital PCR based TaqMan RNase P assay
Quantiplex Pro multiplex assay
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Study Purpose
This study compared the detection of mutations in breast cancer, prostate cancer and colorectal cancer in patient plasma with those in case-matched urine that was stored for 0 or 3 days unpreserved or preserved with PAXgene or Streck preservatives. Mutations were detected using different platforms for each cancer type investigated. Blood and urine were collected from 13 women with ER-positive breast cancer and known PIK3CA mutations, 19 patients with colorectal cancer (10 men and 9 women, 49-73 years old), and 30 (20 retrospective, and 10 prospective) men with metastatic prostate cancer (54-88 years). Blood was collected into PAXgene Blood ccfDNA Tubes and plasma was isolated by two centrifugations at 1900 g for 20 min at room temperature. Urine was collected into PAXgene Urine Liquid Biopsy Cups and then, when possible, aliquots were left unpreserved or preserved with PAXgene or Streck urine preservatives. Urine aliquots were centrifuged immediately (0.5-2h) and after 3 days at room temperature. Urine was centrifuged at 1900 g for 15 min, followed by 1900 g for 10 min (PAXgene and unpreserved urine) or 2,700g for 10 min (Streck urine); supernatants were frozen at -80°C until cfDNA isolation. cfDNA was isolated from plasma and urine supernatants using either the QIAamp Cirulating Nucleic Acid Kit or the QIAsymphony PAXgene Blood ccfDNA Kit and stored at -20°C. DNA was extracted with the QIAsymphony DSP Circulating DNA Kit on a QIAsymphony SP instrument. cfDNA isolated from the urine of colorectal cancer patients was concentrated using the using a MinElute 96 UF PCR Purification Kit. cfDNA was quantified using the Qubit dsDNA High Sensitivity Kit, real-time PCR amplification of a 66 bp fragment of the 18S rRNA gene, the digital PCR-based TaqMan RNase P assay and the Quantiplex Pro multiplex assay. PIK3CA mutations were identified in specimens from patients with breast cancer using the next-generation sequencing-based SIMSen-Seq assay and the digital PCR-based QIAcuity Multitarget LNA Mutation Detection assays. Homologous recombination repair mutations were detected in plasma and urine from prostate cancer patients using custom QIAseq next-generation sequencing panels that included ATM, BARD1, BRCA1, BRCA2, BRIP1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, RAD54L, RB1, PTEN and TP53. Clinically relevant mutations were identified in specimens from colorectal cancer patients using the next-generation sequencing AVENIO ctDNA Targeted Kit. Additionally, shallow whole genome sequencing (sWGS) libraries were prepared from specimens of 12 patients with prostate cancer using the TruSeq DNA Nano Sample Preparation Kit and were on sequenced Illumina NextSeq or NovaSeq 6000 instruments.
Summary of Findings:
The authors report that the SiMSen-Seq PIK3CA assay failed to generate results for most urine specimens from patients with breast cancer, which they attribute to lower cfDNA levels and fragment size relative to those in plasma. Using the dPCR PIK3CA assay, mutations were detected in 5 of 9 plasma specimens, but mutations weren’t detected or were at the limit of detection in urine specimens. The effect of stabilization on the detection of mutant PIK3CA was unclear, with some cases showing decreased detection in preserved urine on day 0 compared to unpreserved urine, but other cases showing an increase in detection on day 0 and 3 when specimens were preserved. Wildtype PIK3CA was detected in urine at similar copy numbers to plasma. When processed immediately, urine preserved with Streck or PAXgene had 6-7-fold more copies of wildtype PIK3CA than unpreserved urine. Wildtype PIK3CA remained relatively stable in preserved urine but declined to just 16% of initial levels in unpreserved urine stored for 3 days. As a result, urine stored for 3 days with either Streck or PAXgene preservative had 81-87-fold more copies of wildtype PIK3CA than urine stored for 3 days without preservatives (P=0.0159 and P=0.0097, respectively).
In retrospectively analyzed specimens from patients with prostate cancer, at least one mutation in homologous recombination repair (HRR) genes was detected in 18 of the 20 plasma specimens, and the same mutation was detected in the Streck-preserved urine specimen in 9 of those patients. Sequencing libraries were successfully prepared from all preserved urine specimens but only 6 of 8 unpreserved urine specimens. Nevertheless, for all specimens with successful library generation, the concentration, total reads, mean read per unique molecular identifier (UMI), mean UMI depth, and number of variants identified were comparable regardless of stabilization method (unpreserved, Streck or PAXgene) and storage duration (0 versus 3 days). The authors conclude that preserved urine was capable of producing high-quality sequencing data and allowed for mutation detection. Shallow whole genome sequencing detected somatic copy number alterations (SCNA) in 3 of 12 plasma specimens, with two having the same SCNA found in the matched PAXgene- and Streck-preserved urine specimens (one at a similar VAF and the other at a lower VAF).
While all plasma and preserved urine specimens from colorectal cancer patients produced high-quality sequencing libraries, library preparation was only successful in 2 of the 5 unpreserved urine specimens. Importantly, both of these unpreserved urine specimens had a lower sequencing depth and the error rate was much higher than for preserved urine or plasma specimens in unpreserved aliquots stored for 3 days. The error rate and the number of low-level variants (VAF <0.5%) were higher in preserved urine specimens than in plasma specimens, and this was particularly true when <50 ng cfDNA was used as input for the library. When the same variants were detected in plasma and urine from a patient, the VAF was lower in the urine specimen. Several variants were detected at low levels in only one urine specimen, which the authors state likely represents technical errors. Interestingly, some mutations detected at VAFs below the limit of detection were found in both Streck- and PAXgene-preserved specimens, indicating they may be true variants despite their absence in plasma and the low VAF.
Biospecimens
Preservative Types
- PAXgene
- None (Fresh)
- Streck/BCT
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Real-time qPCR DNA Digital PCR DNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Biospecimen location Plasma
Urine
Biospecimen Preservation Type of fixation/preservation Streck cfDNA urine presevative
PAXgene
None (fresh)
Storage Storage duration 0 days
3 days