Cell-Free DNA in Pediatric Solid Organ Transplantation Using a New Detection Method of Separating Donor-Derived from Recipient Cell-Free DNA

Abstract

Background The use of cell-free DNA (cfDNA) as a noninvasive biomarker to detect allograft damage is expanding rapidly. However, quantifying the low fraction of donor-derived cfDNA (ddcfDNA) is challenging and requires a highly sensitive technique. ddcfDNA detection through unique donor single nucleotide polymorphisms (SNPs) is a recent new approach, however there are limited data in pediatric solid organ transplant (SOT) recipients. Methods We developed an assay using a combination of 61 SNPs to quantify the ddcfDNA accurately using a custom R script to model for both the patient and donor genotypes requiring only a single sample from the allograft recipient. Performance of the assay was validated using genomic DNA (gDNA), cfDNA and donor samples where available. Results The R “genotype-free” method gave results comparable to when using the known donor genotype. applicable to both related and unrelated pairs and can reliably measure ddcfDNA (limit of blank, below 0.12%; limit of detection, above 0.25%; limit of quantification 0.5% resulting in 84% accuracy). 159 pediatric SOT recipients (kidney, heart, and lung) were tested without the need for donor genotyping. Serial sampling was obtained from 82 patients. Conclusion We have developed and validated a new assay to measure the fraction of ddcfDNA in the plasma of pediatric SOT recipients. Our method can be applicable in any donor-recipient pair without the need for donor genotyping and can provide results in 48 h at a low cost. Additional prospective studies are required to demonstrate its clinical validity in a large cohort of pediatric SOT recipients.

Use of a donor‐derived cell‐free DNA assay to monitor treatment response in pediatric renal transplant recipients with allograft rejection

“…Over the past five years, pediatric recipients have accounted for only 39 Lee and colleagues showed that the majority of dd-cfDNA in plasma was derived from leukocytes undergoing natural apoptosis and did not correlate with biopsy findings of rejection. 40 While these studies focus on the utilization of dd-cfDNA in…”

“…Furthermore, multiple studies have questioned the utility of dd‐cfDNA in the diagnosis of rejection. Perka et al suggest that elevations in systemic cfDNA in response to other stressors may lead to low fractions of dd‐cfDNA, resulting in missed diagnoses of rejection 39 . Lee and colleagues showed that the majority of dd‐cfDNA in plasma was derived from leukocytes undergoing natural apoptosis and did not correlate with biopsy findings of rejection 40 …”

Non‐invasive fetal genotyping for maternal alleles with droplet digital PCR: A comparative study of analytical approaches

“…Each cfDNA sample was then tested with the ddPCR assay for the relevant pathogenic variant, and results were analyzed using Quantasoft (v1.7.4). The fetal fraction was determined via ddPCR for the ZFY locus 29 or a paternally inherited SNP 21,30 . Identification of informative SNPs was performed using NGS and ddPCR and is described in more detail in the Supplementary Methods.…”

Assessment of donor cell engraftment after hematopoietic stem cell transplantation for sickle cell disease: A review of current and future methods

“…This requires prior knowledge of donor and recipient genotypes, which can be costly, time‐consuming, and labor‐intensive. Recently, methods of computational analysis have emerged to estimate donor and recipient DNA fractions without sequencing both genomes 62,63 …”

“…Recently, methods of computational analysis have emerged to estimate donor and recipient DNA fractions without sequencing both genomes. 62,63 Liu et al investigated the potential of ABO sequence differences to differentiate donor-and recipient-derived DNA, noting that 40%-50% of HSCT are ABO-incompatible. 64 In addition, a given ABO phenotype can be derived from multiple different genotypes, which, in theory, leads to an exceedingly low possibility of donor and recipient having completely identical ABO alleles.…”