Early changes in cell‐free DNA levels in newly transplanted heart transplant patients

Abstract Heart transplantation is a well‐established therapy for end‐stage heart failure in children and young adults. The highest risk of graft loss occurs in the first 60 days post‐transplant. Donor fraction of cell‐free DNA is a highly sensitive marker of graft injury. Changes in cell‐free DNA levels have not previously been studied in depth in patients early after heart transplant. A prospective study was conducted among heart transplant recipients at a single pediatric heart center. Blood samples were collected from children and young adult transplant patients at three time points within 10 days of transplantation. DF and total cell‐free DNA levels were measured using a targeted method (myTAIHEART). In 17 patients with serial post‐transplant samples, DF peaks in the first 2 days after transplant (3.5%, [1.9‐10]%) and then declines toward baseline (0.27%, [0.19‐0.52]%) by 6‐9 days. There were 4 deaths in the first year among the 10 patients with complete sample sets, and 3 out of 4 who died had a late rise or blunted decline in donor fraction. Patients who died trended toward an elevated total cell‐free DNA at 1 week (41.5, [34‐65] vs 13.6, [6.2‐22] P = .07). Donor fraction peaks early after heart transplant and then declines toward baseline. Patients without sustained decline in donor fraction and/or elevated total cell‐free DNA at 1 week may have worse outcomes.


K E Y W O R D S
biomarkers, cell-free DNA, early postoperative period, pediatric heart transplant cfDNA also may have diagnostic and prognostic value. TcfDNA is an emerging marker of inflammation and cell turnover with diverse applications in clinical practice. [7][8][9] Herein, we report high-resolution measurements of DF and TcfDNA at three time points during the first week post-transplant using a rapid and economical targeted assay suitable for clinical surveillance. 10

| PATIENTS AND ME THODS
A prospective study to assess the utility of DF cfDNA as a marker for rejection was conducted among heart transplant recipients at a single pediatric heart center. A substudy to characterize early changes in DF and TcfDNA after heart transplant was devised. All patients listed for cardiac transplant at the Herma Heart Center at the CHW were invited to participate in this substudy. Samples were excluded if they (a) were from multi-organ transplant recipients, (b) failed genotyping QC, (c) had incompletely documented collection times, (d) were obtained from patients on ECMO, or (e) the transplant recipient had fewer than two DF results in the first 9 days post-transplant.
Informed consent was obtained for all subjects through a protocol approved by the CHW Institutional Review Board (CHW 10/83, GC 1111).

| Blood sample collection
Three to ten milliliter of anti-coagulated blood was collected to assess circulating levels of cfDNA. Each sample was collected in 10-mL cfDNA Blood Collection Tubes (BCT) tubes (Streck). Samples were immediately coded, de-identified, and delivered to the laboratory for processing. Blood samples were obtained from subjects at three targeted time points following transplantation, characterized as day 1: within 0-2 days; day 4: within 3-6 days; and day 8: within 6-9 days of transplant.

| Plasma processing and DNA extraction
Separation of plasma from whole blood by centrifugation was carried out as previously described. 11

| Total cfDNA analysis
Total cfDNA (TcfDNA) content from plasma was evaluated by quantitative real-time PCR as previously described. 11 PCR analysis was carried out on an Applied Biosystems QuantStudio 7 Flex Real-Time PCR System (Thermo Fisher Scientific). A dilution series of human genomic DNA was used to create a standard curve for quantification.

| Quantitative genotyping
The myTAI-HEART™ assay was used to calculate DF as a percentage of TcfDNA (TAI Diagnostics). 10 The assay quantitatively genotypes a panel of high-frequency single-nucleotide polymorphisms selected for their ability to reliably discriminate between alleles and was performed without a donor sample. 10

| Clinical data collection
Clinical, laboratory, cardiac catheterization, and echocardiographic data were recorded at the time of collection, and data were managed using Research Electronic Data Capture (REDCap) tools hosted at CHW. 12

| Statistical analysis
Data processing was performed in RStudio, statistical analyses include linear modeling, Wilcoxon's rank-sum test, and the Student's t test for difference in means. Data are reported as median and interquartile range.

| RE SULTS
Seventeen patients were identified with at least two samples within the targeted time frame 0-9 days post-transplant. One sample was excluded for failure to pass genotyping QC. Patient characteristics are shown in Table 1

| D ISCUSS I ON
DF of circulating cfDNA is a highly sensitive marker of graft injury following HTx. Monitoring of DF in HTx recipients has been shown in a growing number of studies to be a promising screening tool for rejection. [3][4][5]10 Given the fact that DF can be performed more frequently than biopsy, its availability could empower early intervention and improve graft viability. Our data align with previous reports that DF approaches baseline around 1 week following the heart transplant procedure. 5 This early and predictable decline in DF renders it useable as a screening tool for rejection in the early post-transplant period; this is important as the first few months represent a period of increased risk.

| LI M ITATI O N S
This study should be considered in the context of a few important limitations. This analysis was part of a pilot study focused on the relationship between DF cfDNA and rejection in cardiac transplant recipients; this substudy evaluating early changes in cfDNA following heart transplant included <20 patients. Further, all of the subjects were pediatric or young adult recipients with corresponding young donors. The study cohort as a whole had an uncharacteristically high rate of death. While we could not identify a source of selection bias, it will be important to validate these trends in a larger more representative series of heart transplant recipients. Finally, the draw times for serial sampling was not consistent but rather fell within ranges as described in the manuscript.

ACK N OWLED G M ENTS
The

CO N FLI C T O F I NTE R E S T
A.T-M. and MM own stock and are co-founders of TAI Diagnostics.
KS owns stock and is an employee of TAI Diagnostics. SZ and MH own stock and are consultants to TAI Diagnostics.