Comparison of methods for donor-derived cell-free DNA quantification in plasma and urine from solid organ transplant recipients

Nicholas Kueng; Séverine Arcioni; Fanny Sandberg; Christian Kuhn; Vanessa Banz; Carlo R Largiadèr; Daniel Sidler; Ursula Amstutz

doi:10.3389/fgene.2023.1089830

Comparison of methods for donor-derived cell-free DNA quantification in plasma and urine from solid organ transplant recipients

Front Genet. 2023 Jan 27:14:1089830. doi: 10.3389/fgene.2023.1089830. eCollection 2023.

Authors

Nicholas Kueng^{1

2}, Séverine Arcioni^{1

3}, Fanny Sandberg^{1

2}, Christian Kuhn⁴, Vanessa Banz⁵, Carlo R Largiadèr¹, Daniel Sidler⁴, Ursula Amstutz¹

Affiliations

¹ Department of Clinical Chemistry, Inselspital, Bern University Hospital and University of Bern, Bern, Switzerland.
² Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland.
³ Division of Medical Genetics, Central Institute of Hospitals, Valais Hospital, Sion, Switzerland.
⁴ Department of Nephrology and Hypertension, Inselspital, Bern University Hospital and University of Bern, Bern, Switzerland.
⁵ Department of Visceral Surgery and Medicine, Inselspital, Bern University Hospital and University of Bern, Bern, Switzerland.

Abstract

In allograft monitoring of solid organ transplant recipients, liquid biopsy has emerged as a novel approach using quantification of donor-derived cell-free DNA (dd-cfDNA) in plasma. Despite early clinical implementation and analytical validation of techniques, direct comparisons of dd-cfDNA quantification methods are lacking. Furthermore, data on dd-cfDNA in urine is scarce and high-throughput sequencing-based methods so far have not leveraged unique molecular identifiers (UMIs) for absolute dd-cfDNA quantification. Different dd-cfDNA quantification approaches were compared in urine and plasma of kidney and liver recipients: A) Droplet digital PCR (ddPCR) using allele-specific detection of seven common HLA-DRB1 alleles and the Y chromosome; B) high-throughput sequencing (HTS) using a custom QIAseq DNA panel targeting 121 common polymorphisms; and C) a commercial dd-cfDNA quantification method (AlloSeq^® cfDNA, CareDx). Dd-cfDNA was quantified as %dd-cfDNA, and for ddPCR and HTS using UMIs additionally as donor copies. In addition, relative and absolute dd-cfDNA levels in urine and plasma were compared in clinically stable recipients. The HTS method presented here showed a strong correlation of the %dd-cfDNA with ddPCR (R ² = 0.98) and AlloSeq^® cfDNA (R ² = 0.99) displaying only minimal to no proportional bias. Absolute dd-cfDNA copies also correlated strongly (τ = 0.78) between HTS with UMI and ddPCR albeit with substantial proportional bias (slope: 0.25; 95%-CI: 0.19-0.26). Among 30 stable kidney transplant recipients, the median %dd-cfDNA in urine was 39.5% (interquartile range, IQR: 21.8-58.5%) with 36.6 copies/μmol urinary creatinine (IQR: 18.4-109) and 0.19% (IQR: 0.01-0.43%) with 5.0 copies/ml (IQR: 1.8-12.9) in plasma without any correlation between body fluids. The median %dd-cfDNA in plasma from eight stable liver recipients was 2.2% (IQR: 0.72-4.1%) with 120 copies/ml (IQR: 85.0-138) while the median dd-cfDNA copies/ml was below 0.1 in urine. This first head-to-head comparison of methods for absolute and relative quantification of dd-cfDNA in urine and plasma supports a method-independent %dd-cfDNA cutoff and indicates the suitability of the presented HTS method for absolute dd-cfDNA quantification using UMIs. To evaluate the utility of dd-cfDNA in urine for allograft surveillance, absolute levels instead of relative amounts will most likely be required given the extensive variability of %dd-cfDNA in stable kidney recipients.

Keywords: biomarker; cell-free DNA; dd-cfDNA; ddPCR; high-throughput sequencing; kidney transplant; liver transplant; urine.

Grants and funding

This study has been funded by the Swiss National Science Foundation grant number 310030_188762, the Hemmi foundation and the Fondation Johanna Dürmüller-Bol. The open access publication fee was paid by the Swiss National Science Foundation.