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Background: Proteomic phenotyping can provide insights into rejection pathophysiology, novel biomarkers, and therapeutic targets., Methods: Within the prospective, multicenter Genomic Research Alliance for Transplantation study, 181 proteins were evaluated from blood drawn at the time of endomyocardial biopsy; protein fold change, logistic regression, and pathway analyses were conducted, with protein discovery adjusted for a 5% false discovery rate., Results: Among 104 adult heart transplant patients (31% female sex, 53% Black race, median age 52 y), 74 had no rejection, 18 developed acute cellular rejection (ACR), and 12 developed antibody-mediated rejection (AMR). Differential expression was found in 2 proteins during ACR (inflammatory proteins CXCL10 and CD5) and 73 proteins during AMR. The most abundant AMR proteins were the heart failure biomarkers N-terminal pro-B-type natriuretic peptide and suppression of tumorigenicity 2. In univariate logistic regression, odds of identifying ACR on endomyocardial biopsy increased with doubling of CXCL10 (odds ratio [OR] 2.2 [95% confidence interval (CI), 1.3-3.6]) and CD5 (OR 4.7 [95% CI, 1.7-12.9]) concentrations, and odds of AMR increased with doubling of N-terminal pro-B-type natriuretic peptide (OR 13.0 [95% CI, 2.7-62.7) and suppression of tumorigenicity 2 (OR 4.8 [95% CI, 2.1-10.7]) concentrations. After multivariable analysis with clinical covariates, these proteins showed similar odds of ACR or AMR on biopsy. Pathway analysis identified T cell-receptor signaling and cell differentiation as key pathways in ACR and cardiovascular disease and cell turnover in AMR., Conclusions: Proteomic analysis reveals unique biomarkers and biological pathway expression in ACR and AMR. Cardiac injury-associated biomarkers were more pronounced in AMR, whereas inflammatory biomarkers were more pronounced in ACR. Proteomic analysis may provide insights into rejection pathophysiology, detection, and therapy., Competing Interests: C.R.d.F. received employment funds (consulting) from Abbott Diagnostics, FujiRebio, Ortho/Quidel, Roche Diagnostics, and Siemens Healthineers. P.S. received unrelated grant support (research funds) paid to Inova from Merck, Bayer, Roche, and Abbott and received consulting fee from Natera, Merck, and Procyrion. All other authors declare no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Heart transplant recipients with donor-specific antibodies (DSAs) have an increased risk for antibody-mediated rejection. However, many patients with graft dysfunction and DSA do not have evidence of antibody-mediated rejection by endomyocardial biopsy (EMB)., Methods: Participants from this prospective, multicenter study underwent serial EMB, echocardiogram, DSA, and donor-derived cell-free DNA evaluations. Outcomes were defined as pAMR+ (pAMR≥1) or DSA+/left ventricle (LV) dysfunction (DSA presence+LVEF drop ≥10% to an LVEF≤50%). Cox regression evaluated the association between antibody-mediated rejection categories and death or sustained (for 3 months) reduction of LVEF to <50%., Results: Two hundred sixteen patients (29% women, 39% Black race, median age 55 [interquartile range, 47-62] years) had 1488 EMB, 2792 DSA, 1821 echocardiograms, and 1190 donor-derived cell-free DNA evaluations. DSAs were present in 86 patients (40%). Fourteen patients had isolated pAMR+ episodes and 8 patients had isolated DSA+/LV dysfunction episodes; 2 patients had pAMR+ and then subsequently DSA+/LV dysfunction with pAMR+. Median %dd-cfDNA was significantly higher at diagnosis of pAMR+ (0.63% [interquartile range, 0.23-2.0]; P =0.0002), or DSA+/LV dysfunction (0.40% [interquartile range, 0.36-1.24]; P <0.0001), compared with patients without these outcomes (0.01% [interquartile range, 0.0001-0.10]). Both pAMR+ and DSA+/LV dysfunction were associated with long-term clinical outcome of death (n=18) or prolonged LV dysfunction (n=10): pAMR+ (hazard ratio, 2.8 [95% CI, 1.03-7.4]; P =0.043); DSA+/LV dysfunction (hazard ratio, 26.2 [95% CI, 9.6-71.3]; P <0.001); composite of both definitions (hazard ratio, 6.5 [95% CI, 2.9-14.3]; P <0.001). Patients who developed pAMR+ or DSA+/LV dysfunction within the first 6 months of transplant were more likely to die within 3 years posttransplant (hazard ratio, 3.9 [95% CI, 1.03-14.6]; P =0.031)., Conclusions: Expanding the characterization of antibody-mediated rejection to include patients with DSA and concurrent allograft dysfunction identified DSA+ patients at risk for death and prolonged LV dysfunction., Competing Interests: Dr Shah performed unrelated grant support paid to the institution from Merck, Bayer, Roche, and Abbott. He performed consulting for Natera, Merck, Ortho Clinical Diagnostics, and Procyrion. The other authors report no conflicts.

Background: Black heart transplant patients are at higher risk of acute rejection (AR) and death than White patients. We hypothesized that this risk may be associated with higher levels of donor-derived cell-free DNA (dd-cfDNA) and cell-free mitochondrial DNA., Methods: The Genomic Research Alliance for Transplantation is a multicenter, prospective, longitudinal cohort study. Sequencing was used to quantitate dd-cfDNA and polymerase chain reaction to quantitate cell-free mitochondrial DNA in plasma. AR was defined as ≥2R cellular rejection or ≥1 antibody-mediated rejection. The primary composite outcome was AR, graft dysfunction (left ventricular ejection fraction <50% and decrease by ≥10%), or death., Results: We included 148 patients (65 Black patients and 83 White patients), median age was 56 years and 30% female sex. The incidence of AR was higher in Black patients compared with White patients (43% versus 19%; P =0.002). Antibody-mediated rejection occurred predominantly in Black patients with a prevalence of 20% versus 2% ( P <0.001). After transplant, Black patients had higher levels of dd-cfDNA, 0.09% (interquartile range, 0.001-0.30) compared with White patients, 0.05% (interquartile range, 0.001-0.23; P =0.003). Beyond 6 months, Black patients showed a persistent rise in dd-cfDNA with higher levels compared with White patients. Cell-free mitochondrial DNA was higher in Black patients (185 788 copies/mL; interquartile range, 101 252-422 133) compared with White patients (133 841 copies/mL; interquartile range, 75 346-337 990; P <0.001). The primary composite outcome occurred in 43% and 55% of Black patients at 1 and 2 years, compared with 23% and 27% in White patients, P <0.001. In a multivariable model, Black patient race (hazard ratio, 2.61 [95% CI, 1.35-5.04]; P =0.004) and %dd-cfDNA (hazard ratio, 1.15 [95% CI, 1.03-1.28]; P =0.010) were associated with the primary composite outcome., Conclusions: Elevated dd-cfDNA and cell-free mitochondrial DNA after heart transplant may mechanistically be implicated in the higher incidence of AR and worse clinical outcomes in Black transplant recipients., Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02423070., Competing Interests: Disclosures Dr P. Shah has unrelated grant support paid to the institution from Merck, Bayer, Roche, and Abbott and participated in consulting for Natera, Merck, and Procyrion. Dr K. Shah is a consultant for Eidos and Grant Reviewer for Pfizer. Dr Valantine participated in Board of Directors for CareDx. The other authors report no conflicts.

The number of heart transplants performed annually in the United States and worldwide continues to increase, but there has been little change in graft longevity and patient survival over the past 2 decades. The reference standard for diagnosis of acute cellular and antibody-mediated rejection includes histologic and immunofluorescence evaluation of endomyocardial biopsy samples, despite invasiveness and high interrater variability for grading histologic rejection. Circulating biomarkers and molecular diagnostics have shown substantial predictive value in rejection monitoring, and emerging data support their use in diagnosing other posttransplant complications. The use of genomic (cell-free DNA), transcriptomic (mRNA and microRNA profiling), and proteomic (protein expression quantitation) methodologies in diagnosis of these posttransplant outcomes has been evaluated with varying levels of evidence. In parallel, growing knowledge about the genetically mediated immune response leading to rejection (immunogenetics) has enhanced understanding of antibody-mediated rejection, associated graft dysfunction, and death. Antibodies to donor human leukocyte antigens and the technology available to evaluate these antibodies continues to evolve. This review aims to provide an overview of biomarker and immunologic tests used to diagnose posttransplant complications. This includes a discussion of pediatric heart transplantation and the disparate rates of rejection and death experienced by Black patients receiving a heart transplant. This review describes diagnostic modalities that are available and used after transplant and the landscape of future investigations needed to enhance patient outcomes after heart transplantation., Competing Interests: Disclosures Dr deFilippi reports consulting for Abbott Diagnostics, FujiRebio, Ortho/Quidel, Roche Diagnostics, and Siemens Healthineers. Dr Khush is a CareDx scientific advisor, speaker, and grant recipient. Dr Shah reports unrelated grant support paid to Inova from Merck, Bayer, Roche, and Abbott and consulting for Natera, Merck, and Procyrion. The other authors report no relevant disclosures.

Background: Cell-free DNA (cfDNA) is a noninvasive marker of cellular injury. Its significance in pulmonary arterial hypertension (PAH) is unknown., Methods: Plasma cfDNA was measured in 2 PAH cohorts (A, n=48; B, n=161) and controls (n=48). Data were collected for REVEAL 2.0 (Registry to Evaluate Early and Long-Term PAH Disease Management) scores and outcome determinations. Patients were divided into the following REVEAL risk groups: low (≤6), medium (7-8), and high (≥9). Total cfDNA concentrations were compared among controls and PAH risk groups by 1-way analysis of variance. Log-rank tests compared survival between cfDNA tertiles and REVEAL risk groups. Areas under the receiver operating characteristic curve were estimated from logistic regression models. A sample subset from cohort B (n=96) and controls (n=16) underwent bisulfite sequencing followed by a deconvolution algorithm to map cell-specific cfDNA methylation patterns, with concentrations compared using t tests., Results: In cohort A, median (interquartile range) age was 62 years (47-71), with 75% female, and median (interquartile range) REVEAL 2.0 was 6 (4-9). In cohort B, median (interquartile range) age was 59 years (49-71), with 69% female, and median (interquartile range) REVEAL 2.0 was 7 (6-9). In both cohorts, cfDNA concentrations differed among patients with PAH of varying REVEAL risk and controls (analysis of variance P ≤0.002) and were greater in the high-risk compared with the low-risk category ( P ≤0.002). In cohort B, death or lung transplant occurred in 14 of 54, 23 of 53, and 35 of 54 patients in the lowest, middle, and highest cfDNA tertiles, respectively. cfDNA levels stratified as tertiles (log-rank: P =0.0001) and REVEAL risk groups (log-rank: P <0.0001) each predicted transplant-free survival. The addition of cfDNA to REVEAL improved discrimination (area under the receiver operating characteristic curve, 0.72-0.78; P =0.02). Compared with controls, methylation analysis in patients with PAH revealed increased cfDNA originating from erythrocyte progenitors, neutrophils, monocytes, adipocytes, natural killer cells, vascular endothelium, and cardiac myocytes (Bonferroni adjusted P <0.05). cfDNA concentrations derived from erythrocyte progenitor cells, cardiac myocytes, and vascular endothelium were greater in patients with PAH with high-risk versus low-risk REVEAL scores ( P ≤0.02)., Conclusions: Circulating cfDNA is elevated in patients with PAH, correlates with disease severity, and predicts worse survival. Results from cfDNA methylation analyses in patients with PAH are consistent with prevailing paradigms of disease pathogenesis.

Purpose of Review: Recent evidence indicates that plasma donor-derived cell-free DNA (dd-cfDNA) is a sensitive biomarker for the detection of underlying allograft injury, including rejection and infection. In this review, we will cover the latest evidence revolving around dd-cfDNA in lung transplantation and its role in both advancing mechanistic insight into disease states in lung transplant recipients as well as its potential clinical utility., Recent Findings: Plasma dd-cfDNA increases in the setting of allograft injury, including in primary graft dysfunction, acute cellular rejection, antibody-mediated rejection and infection. Dd-cfDNA has demonstrated good performance characteristics for the detection of various allograft injury states, most notably with a high negative-predictive value for detection of acute rejection. Elevated levels of dd-cfDNA in the early posttransplant period, reflecting molecular evidence of lung allograft injury, are associated with increased risk of chronic lung allograft dysfunction and death., Summary: As a quantitative, molecular biomarker of lung allograft injury, dd-cfDNA holds great promise in clinical and research settings for advancing methods of posttransplant surveillance monitoring, diagnosis of allograft injury states, monitoring adequacy of immunosuppression, risk stratification and unlocking pathophysiological mechanisms of various disease., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose of Review: Endomyocardial biopsy (EMB), the current gold standard for cardiac allograft monitoring is invasive, may have a low sensitivity and is associated with significant variability in histopathologic interpretation. Fortunately, on-going research is identifying noninvasive biomarkers that address some of these limitations. This review provides an update on noninvasive blood-based methods for rejection surveillance and diagnosis in heart transplantation., Recent Findings: Recent studies highlight good test performance to detect acute rejection for donor-derived cell-free DNA (dd-cfDNA) and microRNAs (miR). dd-cfDNA is sensitive, nonspecific, and has a high negative predictive value for acute cellular and antibody-mediated rejection. Clinical utility trials are being planned to test its role as a rule-out test for acute rejection as compared to the EMB. miRs may have an added advantage as it may phenotype the subtypes of rejection alleviating the need for an EMB or permitting the initiation of targeted therapy while awaiting the results of the EMB., Summary: In this review, we discuss recent advances in the field of noninvasive biomarkers to detect allograft rejection after heart transplant. We provide a perspective of additional studies needed to prove their clinical utility and bring these biomarkers to widescale clinical use., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: After heart transplantation, endomyocardial biopsy (EMBx) is used to monitor for acute rejection (AR). Unfortunately, EMBx is invasive, and its conventional histological interpretation has limitations. This is a validation study to assess the performance of a sensitive blood biomarker-percent donor-derived cell-free DNA (%ddcfDNA)-for detection of AR in cardiac transplant recipients., Methods: This multicenter, prospective cohort study recruited heart transplant subjects and collected plasma samples contemporaneously with EMBx for %ddcfDNA measurement by shotgun sequencing. Histopathology data were collected to define AR, its 2 phenotypes (acute cellular rejection [ACR] and antibody-mediated rejection [AMR]), and controls without rejection. The primary analysis was to compare %ddcfDNA levels (median and interquartile range [IQR]) for AR, AMR, and ACR with controls and to determine %ddcfDNA test characteristics using receiver-operator characteristics analysis., Results: The study included 171 subjects with median posttransplant follow-up of 17.7 months (IQR, 12.1-23.6), with 1392 EMBx, and 1834 %ddcfDNA measures available for analysis. Median %ddcfDNA levels decayed after surgery to 0.13% (IQR, 0.03%-0.21%) by 28 days. Also, %ddcfDNA increased again with AR compared with control values (0.38% [IQR, 0.31-0.83%], versus 0.03% [IQR, 0.01-0.14%]; P <0.001). The rise was detected 0.5 and 3.2 months before histopathologic diagnosis of ACR and AMR. The area under the receiver operator characteristic curve for AR was 0.92. A 0.25%ddcfDNA threshold had a negative predictive value for AR of 99% and would have safely eliminated 81% of EMBx. In addition, %ddcfDNA showed distinctive characteristics comparing AMR with ACR, including 5-fold higher levels (AMR ≥2, 1.68% [IQR, 0.49-2.79%] versus ACR grade ≥2R, 0.34% [IQR, 0.28-0.72%]), higher area under the receiver operator characteristic curve (0.95 versus 0.85), higher guanosine-cytosine content, and higher percentage of short ddcfDNA fragments., Conclusions: We found that %ddcfDNA detected AR with a high area under the receiver operator characteristic curve and negative predictive value. Monitoring with ddcfDNA demonstrated excellent performance characteristics for both ACR and AMR and led to earlier detection than the EMBx-based monitoring. This study supports the use of %ddcfDNA to monitor for AR in patients with heart transplant and paves the way for a clinical utility study. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02423070.