EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Introduction
Heart transplant remains the definitive treatment for end-stage heart failure, yet acute rejection-induced graft dysfunction substantially affects posttransplant outcomes despite improved survival rates. Although endomyocardial biopsy with histological analysis remains the gold standard for rejection diagnosis per International Society for Heart Lung Transplantation guidelines, its invasive nature and limitations in sensitivity and interobserver variability constrain its utility. Major posttransplant complications include cardiac allograft vasculopathy, acute rejection, malignancy, infections, and renal dysfunction. Notably, 40% of recipients experience acute rejection within the first year, contributing to 12% of early mortality.^1,2 Circulating cell-free DNA (cfDNA), particularly donor-derived cfDNA (dd-cfDNA), has emerged as a promising noninvasive biomarker for graft monitoring. Originating from apoptotic and necrotic cells, cfDNA enters circulation and reflects various pathological states. In transplantation, dd-cfDNA creates a unique genomic signature, with technological advances enabling precise quantification through methods like droplet digital PCR and next-generation sequencing. Clinical studies have reported elevated dd-cfDNA levels (3%-4%) during acute rejection, with detectable increases up to 5 months before clinical diagnosis, establishing a compelling scientific basis for dd-cfDNA as a noninvasive biomarker for early rejection detection. This review comprehensively examined the dynamic alterations in cfDNA profiles after cardiac transplant and evaluated the clinical importance of cfDNA as a biomarker for acute rejection and graft vasculopathy detection.

Biological Basis of Cell-Free DNA and Its Applications
Circulating cell-free DNA comprises fragmented DNA molecules circulating in biological fluids, primarily released through cellular apoptosis and necrosis. In healthy individuals, cfDNA mainly originates from hematopoietic cells, circulating as complex molecular structures influenced by various host factors. Sources of cfDNA include nuclear and mitochondrial DNA from apoptotic leukocytes, neutrophil extracellular traps, and epigenetic modifications, with additional contributions from pathological conditions. Cell-free DNA predominantly exists as 150- to 180-base pair mono-nucleosomal structures, although larger fragments up to 21 kilobase pairs may result from necrosis or active release. With a short half-life (from 16 min to 2.5 h), cfDNA is primarily eliminated through renal excretion or macrophage-mediated degradation. During inflammation, tissue-specific cfDNA levels increase, reflecting organ-specific pathologies. In oncology, circulating tumor DNA enables early cancer detection, treatment guidance, and resistance monitoring. Patients with hepatocellular carcinoma have been reported to show elevated cfDNA levels correlating with tumor burden. Analysis of cfDNA methylation through liquid biopsies has revolutionized cancer diagnostics, enabling early detection and treatment monitoring. In prenatal care, cfDNA analysis facilitates noninvasive testing, detecting chromosomal abnormalities from 8 to 10 weeks of gestation. For transplant monitoring, dd-cfDNA exhibits characteristic patterns: initial elevation posttransplant, decline to baseline, and subsequent increases during rejection or complications. Clinical evidence has supported dd-cfDNA as a reliable noninvasive biomarker, with elevated levels preceding biopsy-confirmed rejection by several weeks, demonstrating its clinical utility. Levels of dd-cfDNA typically exhibit a characteristic pattern: initial elevation posttransplant, followed by gradual decline to baseline levels, with subsequent rapid increases during rejection episodes, ischemia-reperfusion injury, or infectious complications. Clinical evidence has established dd-cfDNA as a reliable noninvasive biomarker for diagnosis of early rejection. Longitudinal monitoring has revealed substantial dd-cfDNA elevation preceding biopsy-confirmed acute rejection by several weeks, underscoring its clinical utility in preemptive rejection management.^3-5

Levels of Cell-Free DNA After Heart Transplant
As a result of continuous cellular turnover, cfDNA is continuously released into circulation after transplant organ implantation (Table 1). This biological process enables longitudinal monitoring of dd-cfDNA levels, establishing its utility as a diagnostic biomarker for disease states. Under physiological homeostasis, cfDNA concentrations are maintained within a tightly regulated low range. In healthy individuals, baseline circulating cfDNA concentrations typically range from 0 to 100 ng/mL. Single-center study data demonstrated that elevated dd-cfDNA levels serve as an early detection marker for acute rejection after heart transplant, with substantial elevation preceding histological confirmation by endomyocardial biopsy. The pivotal multicenter D-OAR study, involving 740 patients, revealed that the mean dd-cfDNA level was 0.12% in patients without pathological antibody-mediated rejection grade 1. A cohort study of 52 transplant recipients demonstrated a median donor fraction of 0.08% at 14 days posttransplant. Notably, this value increased significantly to 0.19% during rejection episodes, with corresponding elevation in absolute dd-cfDNA levels from a baseline of 8.8 copies/mL to 23 copies/mL during rejection. The concentration of blood dd-cfDNA typically peaks immediately after solid-organ transplant and subsequently follows a characteristic decline pattern. Postoperative monitoring revealed a mean dd-cfDNA level of 3.8% at 24 hours after heart transplant, decreasing to <1% by postoperative day 7. A multicenter prospective observational study reported median cfDNA levels of 0.43% in the acute cellular rejection (ACR) group versus 0.10% in healthy controls, demonstrating a progressive decline after treatment. Importantly, patients experiencing cardiovascular events (including cardiac arrest, mechanical support requirement, or mortality) exhibited significantly elevated dd-cfDNA levels compared with event-free patients, with values of 2.11% versus 0.31% at baseline and 0.51% versus 0.22% at day 14, respectively. In a separate analysis of 171 heart transplant recipients with over 1800 dd-cfDNA measurements, researchers observed an initial elevation in dd-cfDNA levels posttransplant, followed by a characteristic phase 1 logarithmic decay pattern, reaching 0.13% by postoperative day 28. Hidestrand and colleagues performed quantitative polymerase chain reaction (qPCR) with 94 single-nucleotide polymorphisms (SNPs) to quantify (dd-cfDNA) donor-derived cell-free DNA in 32 cardiac transplant recipients. At a rejection threshold of 1%, the assay detected all rejection episodes (sensitivity: 100%; specificity: 84%).^6-9

Relationship Between Donor-Derived Cell-Free DNA and Rejection
Relationship with acute rejection after heart transplant: Cardiac allograft rejection is classified into ACR and antibody-mediated rejection (AMR), potentially leading to graft dysfunction, failure, and increased mortality. Although conventional noninvasive diagnostics often lack sufficient sensitivity, dd-cfDNA has emerged as a promising biomarker for graft injury in transplantation. Elevated dd-cfDNA levels have demonstrated a predictive value for acute rejection, often preceding histological confirmation by endomyocardial biopsy. For optimal assessment of dd-cfDNA as a biomarker of acute rejection, plasma sample collection should precede endomyocardial biopsy procedures. In a multicenter study of 740 patients, use of dd-cfDNA achieved 44% sensitivity and 97% negative predictive value at a 0.2% threshold. Multiple studies have consistently correlated elevated dd-cfDNA with acute rejection episodes. In a seminal 2011 study from Snyder and colleagues, a 1.7% diagnostic threshold with 0.84 area under the curve (AUC) was identified for detecting grade ?2R/3A rejection, and a subsequent study of 65 recipients established a 0.25% threshold with 0.83 AUC, 58% sensitivity, and 93% specificity. Recent advancements in fluid-based assays have enhanced noninvasive monitoring of rejection. Agbor-Enoh and colleagues demonstrated 81% sensitivity and 85% specificity for ACR 2R/AMR detection at 0.92 AUC, with AMR showing 5-fold higher dd-cfDNA levels and distinct molecular characteristics compared with ACR. In a longitudinal study of 87 recipients, Knüttgen and colleagues reported 76% sensitivity and 83% specificity at a 0.35% threshold. Other studies established diagnostic thresholds of 0.11% (64.2% sensitivity, 70.8% specificity), 0.13% (86% sensitivity, 67% specificity), and 0.1% (92% sensitivity, 56% specificity), 0.15% (65% sensitivity, 93% specificity). Variability in studies likely stemmed from inconsistent protocols and sample size heterogeneity. Although dd-cfDNA has shown a high sensitivity for graft injury, its specificity is limited by concurrent conditions like viral infections. Clinical interpretation requires correlation with additional diagnostic parameters.^10-15

Relationship with cardiac allograft vasculopathy after heart transplant: Cardiac allograft vasculopathy (CAV) persists as the primary etiology of long-term graft failure and late mortality in heart transplant recipients. Despite previous associations between peripheral biomarkers of inflammation/angiogenesis and CAV, reliable noninvasive diagnostic markers remain elusive. Although dd-cfDNA has shown excellent utility for excluding cellular rejection, its efficacy as a CAV biomarker requires further validation. In a cohort study of 65 patients stratified by dd-cfDNA levels (?0.12% vs <0.12%), prevalence of CAV was shown in 63% and 35% of high and low dd-cfDNA groups, respectively. The study reported that elevated dd-cfDNA levels correlated with moderate to severe CAV progression, suggesting CAV as a potential etiology for dd-cfDNA elevation in rejection-free patients, although confirmatory studies are warranted. In a single-center prospective study of 94 heart transplant recipients (median 10.9 y posttransplant), dd-cfDNA levels were measured during routine 1-year coronary angiography. Angiographic findings demonstrated CAV distribution as follows: 61% with CAV0, 19% with CAV1, 14% with CAV2, and 6% with CAV3. No significant differences emerged in dd-cfDNA levels between CAV0 and CAV1-3 groups (0.92% [interquartile range, 0.46-2.0] vs 0.46% [interquartile range, 0.075-1.5]) or between stable and progressive CAV cases (0.735% vs 0.9%). In a pediatric study of 66 patients that used a donor-genotyping-independent approach, comparable dd-cfDNA levels were shown between CAV and non-CAV groups (0.27% vs 0.55%). Despite these findings, dd-cfDNA has retained important potential as a CAV development biomarker.^16-18

Limitations
Although dd-cfDNA has shown important potential for monitoring of rejection in cardiac transplant recipients, several limitations persist, including technological limitations in detection methodologies, ongoing debate on absolute threshold values and clinical superiority, and insufficient evidence for establishing optimized quantitative diagnostic criteria. Plasma cfDNA concentrations have shown substantial biological variability, with limited consensus regarding physiological baseline levels in healthy populations. Standardization and optimization of clinical implementation remain crucial, particularly given the inability of dd-cfDNA analysis to differentiate between various genetic mechanisms underlying immune-mediated injury. The development of multianalyte diagnostic panels incorporating dd-cfDNA with complementary biomarkers may enhance diagnostic accuracy and clinical utility. In multiorgan transplant scenarios, dd-cfDNA testing can detect graft injury but lacks organ-specific localization capability.^19,20

Conclusions
Circulating cell-free DNA serves as a noninvasive biomarker for allograft injury and demonstrates important potential as a safe and accurate monitoring tool for acute rejection in heart transplant recipients. Nevertheless, additional large-scale, prospective, multicenter clinical trials are needed to establish robust evidence-based medical guidelines. The cfDNA-based liquid biopsy technology is poised to play a pivotal role in advancing noninvasive diagnostic approaches for posttransplant rejection, potentially transforming the clinical paradigm from reactive management to proactive prevention. Current clinical evidence has shown that blood-based diagnostic models exhibit high negative predictive values and particular sensitivity in detection of antibody-mediated rejection. However, additional validation studies are required to establish clinically applicable thresholds for routine implementation. Although cfDNA analysis can effectively identify patients who would benefit most from preoperative biopsy, further research is necessary to elucidate the underlying mechanisms responsible for the observed discrepancy in positive results between adult and pediatric populations.^21-23

Volume: 23 Issue: 4 April 2025

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