Higher Molecular Injury at Diagnosis of Acute Cellular Rejection Increases the Risk of Lung Allograft Failure: A Clinical Trial.

Rationale: The association of acute cellular rejection (ACR) with chronic lung allograft dysfunction (CLAD) in lung transplant recipients has primarily been described before consensus recommendations incorporating restrictive phenotypes. Furthermore, the association of the degree of molecular allograft injury during ACR with CLAD or death remains undefined. Objectives: To investigate the association of ACR with the risk of CLAD or death and to further investigate if this risk depends on the degree of molecular allograft injury. Methods: This multicenter, prospective cohort study included 188 lung transplant recipients. Subjects underwent serial plasma collections for donor-derived cell-free DNA (dd-cfDNA) at prespecified time points and bronchoscopy. Multivariable Cox proportional-hazards analysis was conducted to analyze the association of ACR with subsequent CLAD or death as well as the association of dd-cfDNA during ACR with risk of CLAD or death. Additional outcomes analyses were performed with episodes of ACR categorized as "high risk" (dd-cfDNA ⩾ 1%) and "low risk" (dd-cfDNA < 1%). Measurements and Main Results: In multivariable analysis, ACR was associated with the composite outcome of CLAD or death (hazard ratio [HR], 2.07 [95% confidence interval (CI), 1.05-4.10]; P = 0.036). Elevated dd-cfDNA ⩾ 1% at ACR diagnosis was independently associated with increased risk of CLAD or death (HR, 3.32; 95% CI, 1.31-8.40; P = 0.012). Patients with high-risk ACR were at increased risk of CLAD or death (HR, 3.13; 95% CI, 1.41-6.93; P = 0.005), whereas patients with low-risk status ACR were not. Conclusions: Patients with ACR are at higher risk of CLAD or death, but this may depend on the degree of underlying allograft injury at the molecular level. Clinical trial registered with www.clinicaltrials.gov (NCT02423070).

Cell-Free DNA Maps Tissue Injury and Correlates with Disease Severity in Lung Transplant Candidates.

Rationale: Plasma cell-free DNA levels correlate with disease severity in many conditions. Pretransplant cell-free DNA may risk stratify lung transplant candidates for post-transplant complications. Objectives: To evaluate if pretransplant cell-free DNA levels and tissue sources identify patients at high risk of primary graft dysfunction and other pre- and post-transplant outcomes. Methods: This multicenter, prospective cohort study recruited 186 lung transplant candidates. Pretransplant plasma samples were collected to measure cell-free DNA. Bisulfite sequencing was performed to identify the tissue sources of cell-free DNA. Multivariable regression models determined the association between cell-free DNA levels and the primary outcome of primary graft dysfunction and other transplant outcomes, including Lung Allocation Score, chronic lung allograft dysfunction, and death. Measurements and Main Results: Transplant candidates had twofold greater cell-free DNA levels than healthy control patients (median [interquartile range], 23.7 ng/ml [15.1-35.6] vs. 12.9 ng/ml [9.9-18.4]; P < 0.0001), primarily originating from inflammatory innate immune cells. Cell-free DNA levels and tissue sources differed by native lung disease category and correlated with the Lung Allocation Score (P < 0.001). High pretransplant cell-free DNA increased the risk of primary graft dysfunction (odds ratio, 1.60; 95% confidence interval [CI], 1.09-2.46; P = 0.0220), and death (hazard ratio, 1.43; 95% CI, 1.07-1.92; P = 0.0171) but not chronic lung allograft dysfunction (hazard ratio, 1.37; 95% CI, 0.97-1.94; P = 0.0767). Conclusions: Lung transplant candidates demonstrate a heightened degree of tissue injury with elevated cell-free DNA, primarily originating from innate immune cells. Pretransplant plasma cell-free DNA levels predict post-transplant complications.

Emergency myelopoiesis distinguishes multisystem inflammatory syndrome in children from pediatric severe COVID-19.

BACKGROUND: Multisystem inflammatory syndrome in children (MIS-C) is a hyperinflammatory condition caused by recent SARS-CoV-2 infection, but the underlying immunological mechanisms driving this distinct syndrome are unknown. METHODS: We utilized high dimensional flow cytometry, cell-free (cf) DNA, and cytokine and chemokine profiling to identify mechanisms of critical illness distinguishing MIS-C from severe acute COVID-19 (SAC). RESULTS: Compared to SAC, MIS-C patients demonstrated profound innate immune cell death and features of emergency myelopoiesis (EM), an understudied phenomenon observed in severe inflammation. EM signatures were characterized by fewer mature myeloid cells in the periphery and decreased expression of HLA-DR and CD86 on antigen presenting cells. IL-27, a cytokine known to drive hematopoietic stem cells towards EM, was increased in MIS-C, and correlated with immature cell signatures in MIS-C. Upon recovery, EM signatures decreased, and IL-27 plasma levels returned to normal levels. Despite profound lymphopenia, we report a lack of cfDNA released by adaptive immune cells and increased CCR7 expression on T cells indicative of egress out of peripheral blood. CONCLUSIONS: Immune cell signatures of EM combined with elevated innate immune cell-derived cfDNA levels distinguish MIS-C from SAC in children and provide mechanistic insight into dysregulated immunity contributing towards MIS-C, offering potential diagnostic and therapeutic targets.

Selection and Interpretation of Molecular Diagnostics in Heart Transplantation.

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.

European Society for Organ Transplantation (ESOT) Consensus Statement on the Use of Non-invasive Biomarkers for Cardiothoracic Transplant Rejection Surveillance.

While allograft rejection (AR) continues to threaten the success of cardiothoracic transplantation, lack of accurate and repeatable surveillance tools to diagnose AR is a major unmet need in the clinical management of cardiothoracic transplant recipients. Endomyocardial biopsy (EMB) and transbronchial biopsy (TBBx) have been the cornerstone of rejection monitoring since the field's incipience, but both suffer from significant limitations, including poor concordance of biopsy interpretation among pathologists. In recent years, novel molecular tools for AR monitoring have emerged and their performance characteristics have been evaluated in multiple studies. An international working group convened by ESOT has reviewed the existing literature and provides a series of recommendations to guide the use of these biomarkers in clinical practice. While acknowledging some caveats, the group recognized that Gene-expression profiling and donor-derived cell-free DNA (dd-cfDNA) may be used to rule out rejection in heart transplant recipients, but they are not recommended for cardiac allograft vasculopathy screening. Other traditional biomarkers (NT-proBNP, BNP or troponin) do not have sufficient evidence to support their use to diagnose AR. Regarding lung transplant, dd-cfDNA could be used to rule out clinical rejection and infection, but its use to monitor treatment response is not recommended.

Plasma Cell-Free DNA Predicts Survival and Maps Specific Sources of Injury in Pulmonary Arterial Hypertension.

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.

Preemptive treatment of de novo donor-specific antibodies in lung transplant patients reduces subsequent risk of chronic lung allograft dysfunction or death.

The development of donor-specific antibodies after lung transplantation is associated with downstream acute cellular rejection, antibody-mediated rejection (AMR), chronic lung allograft dysfunction (CLAD), or death. It is unknown whether preemptive (early) treatment of de novo donor-specific antibodies (dnDSAs), in the absence of clinical signs and symptoms of allograft dysfunction, reduces the risk of subsequent CLAD or death. We performed a multicenter, retrospective cohort study to determine if early treatment of dnDSAs in lung transplant patients reduces the risk of the composite endpoint of CLAD or death. In the cohort of 445 patients, 145 patients developed dnDSAs posttransplant. Thirty patients received early targeted treatment for dnDSAs in the absence of clinical signs and symptoms of AMR. Early treatment of dnDSAs was associated with a decreased risk of CLAD or death (hazard ratio, 0.36; 95% confidence interval, 0.17-0.76; P < .01). Deferring treatment until the development of clinical AMR was associated with an increased risk of CLAD or death (hazard ratio, 3.00; 95% confidence interval, 1.46-6.18; P < .01). This study suggests that early, preemptive treatment of donor-specific antibodies in lung transplant patients may reduce the subsequent risk of CLAD or death.

Cell-Free DNA to Detect Heart Allograft Acute Rejection.

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.

Elevated cell-free DNA in respiratory viral infection and associated lung allograft dysfunction.

Respiratory viral infection (RVI) in lung transplant recipients (LTRs) is a risk for chronic lung allograft dysfunction (CLAD). We hypothesize that donor-derived cell-free DNA (%ddcfDNA), at the time of RVI predicts CLAD progression. We followed 39 LTRs with RVI enrolled in the Genomic Research Alliance for Transplantation for 1 year. Plasma %ddcfDNA was measured by shotgun sequencing, with high %ddcfDNA as ≥1% within 7 days of RVI. We examined %ddcfDNA, spirometry, and a composite (progression/failure) of CLAD stage progression, re-transplant, and death from respiratory failure. Fifty-nine RVI episodes, 38 low and 21 high %ddcfDNA were analyzed. High %ddcfDNA subjects had a greater median %FEV1 decline at RVI (-13.83 vs. -1.83, p = .007), day 90 (-7.97 vs. 0.91, p = .04), and 365 (-20.05 vs. 1.09, p = .047), compared to those with low %ddcfDNA and experienced greater progression/failure within 365 days (52.4% vs. 21.6%, p = .01). Elevated %ddcfDNA at RVI was associated with an increased risk of progression/failure adjusting for symptoms and days post-transplant (HR = 1.11, p = .04). No difference in %FEV1 decline was seen at any time point when RVIs were grouped by histopathology result at RVI. %ddcfDNA delineates LTRs with RVI who will recover lung function and who will experience sustained decline, a utility not seen with histopathology.

Comparison of donor-derived cell-free DNA between single versus double lung transplant recipients.

Plasma donor-derived cell-free DNA (dd-cfDNA) is a sensitive biomarker for the diagnosis of acute rejection in lung transplant recipients; however, differences in dd-cfDNA levels between single and double lung transplant remains unknown. We performed an observational analysis that included 221 patients from two prospective cohort studies who had serial measurements of plasma dd-cfDNA at the time of bronchoscopy and pulmonary function testing, and compared dd-cfDNA between single and double lung transplant recipients across a range of disease states. Levels of dd-cfDNA were lower for single vs. double lung transplant in stable controls (median [IQR]: 0.15% [0.07, 0.44] vs. 0.46% [0.23, 0.74], p < .01) and acute rejection (1.06% [0.75, 2.32] vs. 1.78% [1.18, 5.73], p = .05). Doubling dd-cfDNA for single lung transplant to account for differences in lung mass eliminated this difference. The area under the receiver operating curve (AUC) for the detection of acute rejection was 0.89 and 0.86 for single and double lung transplant, respectively. The optimal dd-cfDNA threshold for the detection of acute rejection was 0.54% in single lung and 1.1% in double lung transplant. In conclusion, accounting for differences in dd-cfDNA in single versus double lung transplant is key for the interpretation of dd-cfDNA testing in research and clinical settings.

Cell-free DNA in lung transplantation: research tool or clinical workhorse?

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.

Noninvasive biomarkers in heart transplant: 2020-2021 year in review.

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.

A gentler approach to monitor for heart transplant rejection.

Despite developments in circulating biomarker and imaging technology in the assessment of cardiovascular disease, the surveillance and diagnosis of heart transplant rejection has continued to rely on histopathologic interpretation of the endomyocardial biopsy. Increasing evidence shows the utility of molecular evaluations, such as donor-specific antibodies and donor-derived cell-free DNA, as well as advanced imaging techniques, such as cardiac magnetic resonance imaging, in the assessment of rejection, resulting in the elimination of many surveillance endomyocardial biopsies. As non-invasive technologies in heart transplant rejection continue to evolve and are incorporated into practice, they may supplant endomyocardial biopsy even when rejection is suspected, allowing for more precise and expeditious rejection therapy. This review describes the current and near-future states for the evaluation of heart transplant rejection, both in the settings of rejection surveillance and rejection diagnosis. As biomarkers of rejection continue to evolve, rejection risk prediction may allow for a more personalized approach to immunosuppression.

Sex-specific patterns of donor-derived cell-free DNA in heart transplant rejection: An analysis from the Genomic Research Alliance for Transplantation (GRAfT).

BACKGROUND: Noninvasive methods for surveillance of acute rejection are increasingly used in heart transplantation (HT), including donor-derived cell-free DNA (dd-cfDNA). As other cardiac biomarkers differ by sex, we hypothesized that there may be sex-specific differences in the performance of dd-cfDNA for the detection of acute rejection. The purpose of the current study was to examine patterns of dd-cfDNA seen in quiescence and acute rejection in male and female transplant recipients. METHODS: Patients enrolled in the Genomic Research Alliance for Transplantation who were ≥18 years at the time of HT were included. Rejection was defined by endomyocardial biopsy with acute cellular rejection (ACR) grade ≥2R and/or antibody-mediated rejection ≥ pAMR 1. dd-cfDNA was quantitated using shotgun sequencing. Median dd-cfDNA levels were compared between sexes during quiescence and rejection. The performance of dd-cfDNA by sex was assessed using area under the receiver operator characteristic (AUROC) curve. Allograft injury was defined as dd-cfDNA ≥0.25%. RESULTS: One hundred fifty-one unique patients (49 female, 32%) were included in the analysis with 1,119 available dd-cfDNA measurements. Baseline characteristics including demographics and comorbidities were not significantly different between sexes. During quiescence, there were no significant sex differences in median dd-cfDNA level (0.04% [IQR 0.00, 0.16] in females vs 0.03% [IQR 0.00, 0.12] in males, p = 0.22). There were no significant sex differences in median dd-cfDNA for ACR (0.33% [0.21, 0.36] in females vs 0.32% [0.21, 1.10] in males, p = 0.57). Overall, median dd-cfDNA levels were higher in antibody-mediated rejection (AMR) than ACR but did not significantly differ by sex (0.50% [IQR 0.18, 0.82] in females vs 0.63% [IQR 0.32, 1.95] in males, p = 0.51). Elevated dd-cfDNA detected ACR/AMR with an AUROC of 0.83 in females and 0.89 in males, p-value for comparison = 0.16. CONCLUSIONS: There were no significant sex differences in dd-cfDNA levels during quiescence and rejection. Performance characteristics were similar, suggesting similar diagnostic thresholds can be used in men and women for rejection surveillance.