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

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 (R2 = 0.98) and AlloSeq® cfDNA (R2 = 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.

Multi‐centre analytical performance verification of an IVD assay to quantify donor‐derived cell‐free DNA in solid organ transplant recipients

“…High coefficient of correlation with respect to the predicate centralised dd‐cfDNA assay has also been observed at end‐user level based on the results from a large kidney transplant cohort multi‐centre study (data not shown, submitted for publication). This kit assay has also been compared with alternative methods, such as short tandem repeat genotyping by quantitative fluorescence PCR (QF‐PCR), 17 droplet digital PCR, 39 and high‐throughput sequencing 40 using clinical samples. dd‐cfDNA results from these different methods showed mutual agreement with high coefficients of correlation, except agreement between QF‐PCR and NGS <5%, owing to limited sensitivity of QF‐PCR and greater sensitivity of NGS in this range 17 …”

Non‐invasive molecular biomarkers for monitoring solid organ transplantation: A comprehensive overview

“…Strategies to differentiate between the two include using sex‐specific markers in cases where there is a donor–recipient sex mismatch, genetic differences between the donor and the recipient, including single‐nucleotide polymorphisms, amplification of HLA‐specific genes and copy number deletion polymorphisms and tissue‐specific methylation patterns in cfDNA (Gielis et al., 2015) (Figure 2b). Several methods can be used to quantify dd‐cfDNA, including real‐time quantitative PCR (RT‐qPCR), digital droplet PCR, organ‐specific methylation patterns, NGS, mass spectrometry and microarray analysis (Dengu, 2020; Goh et al., 2017; Kueng et al., 2023) (Figure 2c). Studies have shown that dd‐cfDNA can be a useful biomarker for rejection in solid organ transplantation, particularly in cases of acute cellular rejection (ACR) and AMR (Figure 2d).…”

Investigation of Different Library Preparation and Tissue of Origin Deconvolution Methods for Urine and Plasma cfDNA Methylome Analysis

“…After thawing the plasma at 4 • C, cfDNA was isolated with the QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany), according to the protocol provided by the manufacturer, as previously described [17].…”

“…Urinary cfDNA was extracted from 15-50 mL of urine after thawing at room temperature (RT) either with the Quick-DNA Urine Kit (Zymo Research, Irvine, CA, USA) as previously described [17] or using a quaternary ammonium anion exchange resin-based Q Sepharose method based on the method published by Dudley et al [18].…”