Donor-derived Cell-free dd-cfDNA Research Articles

Nivolumab (NIVO) + tacrolimus (TACRO) + prednisone (PRED) +/- ipilimumab (IPI) for kidney transplant recipients (KTR) with advanced cutaneous cancers.

9507 Background: Cancer is a leading cause of death among KTR, but these patients (pts) have been excluded from trials of immune checkpoint inhibitors due to immunosuppression and risk of allograft loss. We report findings from the first prospective clinical trial of NIVO + TACRO + PRED +/- IPI in KTR with selected advanced cutaneous cancers. Methods: The primary composite endpoint was lack of tumor progression per RECIST v1.1 without allograft loss at 16 weeks (W) on NIVO. Adult KTR with advanced melanoma, basal, cutaneous squamous, or Merkel cell carcinoma (MEL, BCC, CSCC, MCC), for whom non-immune therapies were insufficient were eligible. Immunosuppression was standardized to low-dose TACRO (goal trough 2-5 ng/mL) + PRED 5mg daily; pts then received NIVO 480mg IV q4W. Pts with progressive disease (PD) could receive NIVO 3mg/kg + IPI 1mg/kg IV q3W x 4 followed by NIVO 480mg IV q4W. Donor-derived cell-free DNA (dd-cfDNA) levels were measured q2W as a potential predictor of allograft rejection. Results: From 11/2019 - 4/2021, of 12 pts enrolled, 8 pts with CSCC, MCC or MEL were evaluable for response (Table). All pts experienced PD on NIVO; treatment-related allograft loss (TRAL) occurred in 1 pt. 6 pts then received IPI + NIVO. Responses: 2 (33%) with marked tumor regression at 6W and eventual complete response (CR; 1 with TRAL), and 4 (67%) with PD (1 with TRAL). 7/8 pre-NIVO tumor biopsies contained a paucity of infiltrating immune cells. Only 2/5 on-NIVO biopsies demonstrated moderate immune infiltrates; both of these pts later developed a CR to IPI + NIVO. Rejecting allografts contained dense immune responses (plasma cells, CD4+ & CD8+ lymphocytes, PD-1+ lymphocytes, macrophages, PD-L1+ glomerular endothelium, and focal PD-1 & PD-L1 positivity in renal tubules). In 2/3 pts with TRAL, elevations in dd-cfDNA levels occurred 10 and 15 days earlier than increases in weekly serum creatinine levels. TRAL #3 occurred after discontinuation of study therapy (including TACRO) and dd-cfDNA monitoring. Conclusions: In KTR receiving low-dose TACRO + PRED, NIVO augments tumor immune cell infiltration in some pts but is insufficient to mediate tumor regression. Adding IPI can enhance anti-tumor immunity and mediate tumor regression. TACRO + PRED was insufficient to prevent allograft rejection after PD-1 +/- CTLA-4 blockade in 2/8 pts. In pts with TRAL, increased dd-cfDNA levels preceded increased serum creatinine. Based on these findings, we are modifying the trial therapy regimen to augment anti-tumor immunity and preserve allograft function. Clinical trial information: NCT03816332. [Table: see text]

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

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. 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. 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.

Combining donor derived cell free DNA and gene expression profiling for non-invasive surveillance after heart transplantation.

Donor-derived cell free DNA (dd-cfDNA) and gene expression profiling (GEP) offer noninvasive alternatives to rejection surveillance after heart transplantation; however, there is little evidence on the paired use of GEP and dd-cfDNA for rejection surveillance. A single center, retrospective analysis of adult heart transplant recipients. A GEP cohort, transplanted from January 1, 2015 through December 31, 2017 and eligible for rejection surveillance with GEP was compared to a paired testing cohort, transplanted July 1, 2018 through June 30, 2020, with surveillance from both dd-cfDNA and GEP. The primary outcomes were survival and rejection-free survival at 1year post-transplant. In total 159 patients were included, 95 in the GEP and 64 in the paired testing group. There were no differences in baseline characteristics, except for less use of induction in the paired testing group (65.6%) compared to the GEP group (98.9%), P<.01. At 1-year, there were no differences between the paired testing and GEP groups in survival (98.4% vs. 94.7%, P=.23) or rejection-free survival (81.3% vs. 73.7% P=.28). Compared to post-transplant rejection surveillance with GEP alone, pairing dd-cfDNA and GEP testing was associated with similar survival and rejection-free survival at 1year while requiring significantly fewer biopsies.

C53 DONOR–DERIVED CELL–FREE DNA (DD–CFDNA)–BASED ASSESSMENT OF CORONARY ALLOGRAFT VASCULOPATHY IN HEART TRANSPLANTED PATIENTS

Abstract Introduction Cardiac Allograft Vasculopathy (CAV) is a leading cause of long–term graft dysfunction and failure after Heart Transplant (HTx). Early diagnosis is crucial to tailor therapeutic strategies. Despite advancements in cardiac imaging, the standard for CAV diagnosis remains coronary angiography. Donor–derived cell free DNA (dd–cfDNA) is a novel marker of graft injury in solid organ transplants. Elevations in dd–cfDNA levels may reflect CAV development during subclinical inflammation, thus it could represent a non–invasive method of surveillance for CAV rejection in apparently stable post–transplant patients. Given these premises, we decided to evaluate the potential of dd–cfDNA as alternative diagnostic biomarker for CAV. Materials and Methods 31 blood samples from patients who underwent heart transplant at our institution in the last 10 years, were analyzed by Next Generation Sequencing (NGS) according to CareDx Alloseq protocol. The cfDNA was extracted starting from 2mL of plasma (Qiagen). Sequencing was performed on the Illumina Miseq platform. Troponin T (hs–TnT) and NT–proBNP (Brain Natriuretic Peptide) were measured by automated immunoassay analysers. All statistics were performed by STATA software and significance was set at p &amp;lt; 0.05. Results Heart transplant recipients were divided into 2 groups: 16 patients with known CAV status and 15 patients who had not (no–CAV). No significant differences between the two populations were observed, aside from time from HTx (177.28± 84.99 months in CAV vs 93.20 ± 84.39 months in no–CAV; p = 0.033). A significant difference in dd–cfDNA fraction was found between CAV and no–CAV patients (p = 0.03); with mean values of 0,41% e 0,13%, respectively. Neither NT-proBNP (p = 0.08) nor hs–TnT (p = 0.31) concentrations were significantly different between the two groups, but a positive correlation was found between the two biomarkers (r 0.6966; p = 0.0013). A trend of linear relationship emerged between cfDNA and NT-proBNP levels (r 0.4155; p = 0.08). Conclusions With this study, we demonstrated the feasibility of evaluating dd–cfDNA in long–term heart transplanted patients and its possible association with CAV development. Further investigations are warranted to explore a possible association between dd–cfDNA levels other circulating biomarkers and CAV progression. The definition of “homogeneous” subgroups of patients according to a “risk score” will allow to better manage their follow up and improve their prognosis.

Elevated fractional donor-derived cell-free DNA during subclinical graft injury after liver transplantation.

Personalized immunosuppression (IS) promises to improve the balance of necessary control of alloreactivity and dose-dependent adverse effects of long-term IS such as kidney insufficiency, infections, and malignancies. The majority of liver transplantation (LT) recipients exhibit graft injuries (graft inflammation and/or fibrosis) that are not eligible for an IS reduction according to current Banff criteria, even when liver enzymes are normal or only marginally elevated. This cross-sectional study evaluated the noninvasive prediction of such subclinical graft injuries in surveillance liver biopsies via donor-derived cell-free DNA (dd-cfDNA). Absolute and fractional dd-cfDNA increased stepwise from patients without histological signs of rejection (n=26) over subclinical graft injury (n=61), including subclinical T cell-mediated rejection to clinical overt T cell-mediated rejection (n=21). Thus, fractional plasma dd-cfDNA was significantly elevated paired to surveillance biopsies with relevant subclinical graft injury according to 2016 Banff criteria compared with those with minimal or absent histological graft injury. In contrast, the presence of donor-specific anti-human leukocyte antigen antibodies was not associated with the amount of dd-cfDNA. The sensitivity and specificity of fractional dd-cfDNA to noninvasively predict relevant subclinical graft injury was rather limited with 73% and 52% at the cutoff value of 2.1% fractional dd-cfDNA. The positive predictive value of fractional dd-cfDNA above 2.1% was 76% to noninvasively predict subclinical graft injury, calculated on the prevalence of graft injury in our prospective surveillance biopsy program, whereas the negative predictive values was not predictive (47%). In conclusion, dd-cfDNA has a rather limited diagnostic fidelity in addition to other noninvasive markers for the assessment of subclinical graft injury in personalized IS approaches after LT in a cross-sectional setting.

Early Experience Using Donor-derived Cell-free DNA for Surveillance of Rejection Following Simultaneous Pancreas and Kidney Transplantation.

Background.Allograft biopsy is the gold standard for diagnosing graft rejection following simultaneous pancreas and kidney (SPK) transplant. Intraperitoneal biopsies are technically challenging and can be burdensome to patients and the healthcare system. Donor-derived cell-free DNA (dd-cfDNA) is well-studied in kidney transplant recipients; however, it has not yet been studied in the SPK population.Methods.We hypothesized that dd-cfDNA could be utilized for rejection surveillance following SPK transplant. We prospectively collected dd-cfDNA in 46 SPK patients at a single institution.Results.There were 10 rejection events, 5 of which were confirmed with biopsy. The other 5 were treated based on dd-cfDNA and clinical data alone with favorable outcomes. Among all patients who did not have rejection, 97% had dd-cfDNA <0.5%. Dd-cfDNA may also help differentiate rejection from graft injury (ie, pancreatitis) with median values in rejection 2.25%, injury 0.36%, and quiescence 0.18% (P = 0.0006).Conclusions.Similar to kidneys, dd-cfDNA shows promise for rejection surveillance in SPK transplant recipients.

Lack of Usefulness of Donor-Derived Cell-Free DNA as a Biomarker for Cardiac Allograft Vasculopathy: A Prospective Study.

BackgroundCardiac allograft vasculopathy (CAV) remains a major cause of morbidity and mortality among long-term heart transplant recipients. There is an unmet need for a non-invasive biomarker of CAV that could obviate the need to perform surveillance coronary angiograms in these patients. Our aim was to evaluate the performance of Donor-derived Cell Free DNA (dd-cfDNA) as a biomarker of CAV.MethodsWe prospectively measured dd-cfDNA levels in all patients undergoing routine coronary angiography >1 year after heart transplant at a single center. Endpoints included the association between dd-cfDNA levels and the presence CAV, according to several prespecified criteria.ResultsWe included 94 heart transplant recipients, a median of 10.9 years after transplant. Coronary angiogram revealed CAV0, CAV1, CAV2, and CAV3 in 61, 19, 14, and 6% of patients, respectively. Comparison of dd-cfDNA levels in patients with CAV0 and CAV1–2–3 (primary end-point) did not show significant differences (0.92%, IQR 0.46–2.0 vs. 0.46%, IQR 0.075–1.5, p = 0.059), nor did the comparison between patients with stable CAV (no new coronary lesions since previous angiogram, n = 77) and progressive CAV (n = 17); dd-cfDNA values 0.735% (IQR 0.195–2.0) vs. 0.9% (IQR 0.12–1.8), p = 0.76. However, we found an association between NTproBNP levels and CAV degree (p = 0.017). Dd-cfDNA levels did not correlate with NTproBNP (ρ = −0.095).ConclusionIn this study, dd-cfDNA did not perform as a useful biomarker to avoid surveillance coronary angiograms for CAV diagnosis.Clinical Trial NotationPotential Role of Donor-derived Cell Free DNA as a Biomarker in Cardiac Allograft Vasculopathy, NCT 04791852.

Prevalence of Clinical Events Following High Donor Derived Cell Free DNA in the Absence of Rejection in Heart Transplant Recipients

Purpose Donor-derived cell free DNA (dd-cfDNA) is a noninvasive biomarker used to assess graft injury following heart transplant (HTx). While the relationship between increased levels of dd-cfDNA and acute cellular rejection (ACR) and antibody mediated rejection (AMR) has been well studied, less is known about the frequency of other processes that damage the allograft following an elevated dd-cfDNA measurement. The goal of this analysis was to assess the prevalence of clinical events following a high dd-cfDNA measurement in the absence of rejection. Methods This retrospective study included 223 HTx recipients from the Donor-Derived Cell-Free DNA-Outcomes AlloMap Registry (D-OAR) with elevated dd-cfDNA (AlloSure®), defined as a level ≥0.20% that did not occur within 14 days of biopsy proven ACR (grade ≥2R) or AMR (pAMR≥1). Clinical events included the presence of donor specific antibodies (DSA), cardiac allograft vasculopathy (CAV; ISHLT grade ≥1), graft dysfunction [left ventricular ejection fraction (LVEF) ≤45% or a proportional decrease in LVEF ≥25% from the first D-OAR study visit], ACR, AMR and death. Clinical data were collected for 180 days following the first elevated dd-cfDNA. Allosensitization was defined as a pre-transplant panel reactive antibody ≥10%. Results The study consisted of 68% men and 71% Caucasians (mean ± SD age at HTx 52 ± 14 years), with non-ischemic cardiomyopathy (46%) representing the primary reason for HTx. The median time post-HTx to the first non-rejection elevated dd-cfDNA was 241 days (IQR 154-362). Fifty-seven of the 223 patients (26%), of whom 6 were allosensitized, experienced 70 clinical events in the 180 days post-elevated dd-cfDNA. The presence of at least one DSA (n=36) was the most common clinical event, of which 28/36 (78%) were de novo DSA. Graft dysfunction (n=14), CAV (n=9), AMR (n=5), death (n=4), and ACR (n=2) were the next most common. Median time (IQR) from elevated dd-cfDNA to these events was as follows: DSA, 37 days (15-104); graft dysfunction, 22 days (0-77); AMR, 70 days (42-91); and death, 100 days (75-124). Conclusion Elevated dd-cfDNA levels in the absence of allograft rejection appeared to precede the development of clinically relevant events within the next 6 months. Further studies are needed to determine whether elevated dd-cfDNA should trigger a change in clinical management. Donor-derived cell free DNA (dd-cfDNA) is a noninvasive biomarker used to assess graft injury following heart transplant (HTx). While the relationship between increased levels of dd-cfDNA and acute cellular rejection (ACR) and antibody mediated rejection (AMR) has been well studied, less is known about the frequency of other processes that damage the allograft following an elevated dd-cfDNA measurement. The goal of this analysis was to assess the prevalence of clinical events following a high dd-cfDNA measurement in the absence of rejection. This retrospective study included 223 HTx recipients from the Donor-Derived Cell-Free DNA-Outcomes AlloMap Registry (D-OAR) with elevated dd-cfDNA (AlloSure®), defined as a level ≥0.20% that did not occur within 14 days of biopsy proven ACR (grade ≥2R) or AMR (pAMR≥1). Clinical events included the presence of donor specific antibodies (DSA), cardiac allograft vasculopathy (CAV; ISHLT grade ≥1), graft dysfunction [left ventricular ejection fraction (LVEF) ≤45% or a proportional decrease in LVEF ≥25% from the first D-OAR study visit], ACR, AMR and death. Clinical data were collected for 180 days following the first elevated dd-cfDNA. Allosensitization was defined as a pre-transplant panel reactive antibody ≥10%. The study consisted of 68% men and 71% Caucasians (mean ± SD age at HTx 52 ± 14 years), with non-ischemic cardiomyopathy (46%) representing the primary reason for HTx. The median time post-HTx to the first non-rejection elevated dd-cfDNA was 241 days (IQR 154-362). Fifty-seven of the 223 patients (26%), of whom 6 were allosensitized, experienced 70 clinical events in the 180 days post-elevated dd-cfDNA. The presence of at least one DSA (n=36) was the most common clinical event, of which 28/36 (78%) were de novo DSA. Graft dysfunction (n=14), CAV (n=9), AMR (n=5), death (n=4), and ACR (n=2) were the next most common. Median time (IQR) from elevated dd-cfDNA to these events was as follows: DSA, 37 days (15-104); graft dysfunction, 22 days (0-77); AMR, 70 days (42-91); and death, 100 days (75-124). Elevated dd-cfDNA levels in the absence of allograft rejection appeared to precede the development of clinically relevant events within the next 6 months. Further studies are needed to determine whether elevated dd-cfDNA should trigger a change in clinical management.

Restrictive Allograft Syndrome Patients Have Higher Cell-Free DNA Assessed Allograft Injury Prior to Diagnosis

Purpose Restrictive Allograft Syndrome (RAS) and Bronchiolitis Obliterans Syndrome (BOS) are two forms of chronic lung allograft dysfunction (CLAD), an irreversible loss of allograft function and a major cause of death in lung transplant recipients (LTR). Clinical features and trends in allograft injury associated with RAS and BOS remain incompletely understood. This study examines the clinical characteristics and trends of allograft injury, as assessed by donor-derived cell free DNA (ddcfDNA), in RAS and BOS. Methods 369 LTR enrolled in two cohorts were examined for the development of BOS or RAS, as judged by adjudication committees using standardized definitions for CLAD. Demographic and clinical variables were collected prospectively for each patient. Serial plasma samples were collected for ddcfDNA quantification via shotgun sequencing. Results During the median 38 months follow-up (IQR 24.3-49.7), 116 patients (31.4%) developed CLAD, including 83 (71.5%) with BOS and 33 (28.4%) with RAS. There were no significant differences in demographic and baseline clinical factors between the two groups, including sex, race, BMI, pre-transplant diagnosis, or lung allocation score. RAS patients had a higher percentage of positive donor-specific antibodies (80.6%) compared to BOS (60.5%) (p=0.03). RAS patients had higher levels of ddcfDNA than BOS before CLAD diagnosis (p=0.02), with a rise in ddcfDNA levels preceding RAS diagnosis by 3 months (Fig 1a). RAS patients had significantly lower baseline forced expiratory volume in 1 second (FEV1) (p=0.007) and forced vital capacity (FVC) (p=0.001) six months prior to CLAD diagnosis (Fig 1b). Conclusion RAS patients showed more prevalent DSA and significantly lower baseline lung function compared to BOS. RAS patients also showed higher allograft injury measured via ddcfDNA than BOS patients, with a ddcfDNA rise preceding RAS diagnosis by 3 months. This study demonstrates differences in clinical variables and molecular injury between RAS and BOS. Restrictive Allograft Syndrome (RAS) and Bronchiolitis Obliterans Syndrome (BOS) are two forms of chronic lung allograft dysfunction (CLAD), an irreversible loss of allograft function and a major cause of death in lung transplant recipients (LTR). Clinical features and trends in allograft injury associated with RAS and BOS remain incompletely understood. This study examines the clinical characteristics and trends of allograft injury, as assessed by donor-derived cell free DNA (ddcfDNA), in RAS and BOS. 369 LTR enrolled in two cohorts were examined for the development of BOS or RAS, as judged by adjudication committees using standardized definitions for CLAD. Demographic and clinical variables were collected prospectively for each patient. Serial plasma samples were collected for ddcfDNA quantification via shotgun sequencing. During the median 38 months follow-up (IQR 24.3-49.7), 116 patients (31.4%) developed CLAD, including 83 (71.5%) with BOS and 33 (28.4%) with RAS. There were no significant differences in demographic and baseline clinical factors between the two groups, including sex, race, BMI, pre-transplant diagnosis, or lung allocation score. RAS patients had a higher percentage of positive donor-specific antibodies (80.6%) compared to BOS (60.5%) (p=0.03). RAS patients had higher levels of ddcfDNA than BOS before CLAD diagnosis (p=0.02), with a rise in ddcfDNA levels preceding RAS diagnosis by 3 months (Fig 1a). RAS patients had significantly lower baseline forced expiratory volume in 1 second (FEV1) (p=0.007) and forced vital capacity (FVC) (p=0.001) six months prior to CLAD diagnosis (Fig 1b). RAS patients showed more prevalent DSA and significantly lower baseline lung function compared to BOS. RAS patients also showed higher allograft injury measured via ddcfDNA than BOS patients, with a ddcfDNA rise preceding RAS diagnosis by 3 months. This study demonstrates differences in clinical variables and molecular injury between RAS and BOS.

Donor-Derived Cell-Free DNA Plus Tissue Gene Expression Profiling of Lung Allograft Injury

<h3>Purpose</h3> Lung transplantation outcomes are limited by chronic lung allograft dysfunction, of which acute rejection is a significant risk factor. Acute rejection includes acute cellular rejection (ACR) and antibody-mediated rejection (AMR), and historically has been diagnosed through a combination of transbronchial biopsy histopathology, pulmonary function testing, and the presence of serum donor-specific antibodies. Donor-derived cell-free DNA (dd-cfDNA) has recently shown promise as a less invasive biomarker of allograft injury, but, in isolation, dd-cfDNA does not sufficiently delineate ACR from AMR from infection. Here we propose to combine dd-cfDNA analysis of allograft injury with in situ lung tissue gene expression to better characterize different forms of allograft injury following lung transplantation. <h3>Methods</h3> This is a single center retrospective cohort study of at least 20 lung transplant recipients with longitudinally matched plasma and transbronchial biopsies (n=100). All samples have previously been collected and stored. Plasma will be obtained from the institution's Total Transplant Care Protocol (TTCP) Biorepository, while formalin fixed paraffin embedded (FFPE) biopsy specimens will be obtained from the Department of Pathology through the institution's Tissue Archive Service (TAS). Samples will be assigned to specific clinical cohorts based on histopathology, radiology, microbiological studies and pulmonary function tests at the time of sample collection. Plasma dd-cfDNA fractions will be quantified using Allosure Lung (CareDx), and tissue gene expression profiles (GEP) will be captured with the nCounter Gene Expression Assay (Nanostring Technologies). Data will be analyzed with non-parametric tests and multivariate analyses to determine associations between dd-cfDNA levels and tissue gene expression patterns. <h3>Endpoints</h3> 1. Correlate plasma dd-cfDNA levels with acute allograft injury from ACR, AMR, or infection and determine the time course of dd-cfDNA expression during the first year after lung transplantation. 2. Validate the nCounter Gene Expression Assay platform and identify gene(s) associated with different types of acute rejection and/or infection.

Donor-Derived Cell-Free DNA in Cardiac Allograft Rejection Detection

<h3>Purpose</h3> Cardiac allograft rejection is monitored by histological evaluation of surveillance endomyocardial biopsies (EMB). EMB is invasive and may result in complications, such as bleeding, pneumothorax, damage to tricuspid valve, or cardiac tamponade. Furthermore, the biopsies evaluated by pathologists represent only a very small local histological area and their analysis is imprecise due to inter-observer variance. Donor-derived cell-free DNA (dd-cfDNA) detection from peripheral blood has been previously proposed as a feasible alternative and less-invasive method. The purpose of this study is to validate the method of detecting dd-cfDNA in a prospective patient cohort. <h3>Methods</h3> We have prospectively collected clinical data, EMB, and blood samples from all heart transplant recipients operated in our unit. Patients were treated with tacrolimus-based triple-drug immunosuppression including anti-thymocyteglobulin induction therapy. EMB samples were taken at 2, 4, 6, 8, 10, and 12 weeks, and 4, 5, 6, 8, 10, and 12 months after transplantation and subjected to histological evaluation. Simultaneously, selected single-nucleotide polymorphisms were determined from pre-biopsy peripheral blood samples, and the ratio of dd-cfDNA-to-recipient-cfDNA was calculated. With every EMB sampling, an extra study sample is taken for RNA-sequencing analysis. <h3>Results</h3> We have collected data and samples from 52 consecutive heart transplant recipients with the mean follow-up of 19.2±9.2 months and performed 399 dd-cfDNA quantitations. Of these, 10 results were excluded from the analyses due to multiorgan transplantation (heart+kidney), and 5 results were discarded due to insufficient corresponding EMB sample. Our cohort consists of 297 samples with G0R, 97 with G1R, 8 with G2R, and one with G3R histopathological rejection. The corresponding dd-cfDNA values were 0.16%±0.216% for G0R, 0.16%±0.217% for G1R, and 0.28%±0.162% for G2R/G3R. The predictive value of dd-cfDNA for acute rejection failed to reach statistical significance. <h3>Conclusion</h3> The novel detection method for dd-cfDNA shows promising preliminary results with higher values in more severe histopathological rejection classes. The limitations of our current results stem from the low numbers of G2R/G3R rejections.

Should We Be Comforted by a “Negative” Endomyocardial Biopsy? Risk of Future Events with Donor Derived Cell Free DNA in the Setting of Histologic Quiescence

<h3>Purpose</h3> Endomyocardial biopsy (EMB) is considered the gold standard for screening for rejection in heart transplant (HT) recipients. Lack of histological evidence of rejection on EMB has typically been reassuring to clinicians, but the interpretation of elevated levels of donor-derived cell free DNA (dd-cfDNA) in the setting of a "negative" biopsy is unclear. <h3>Methods</h3> Patients from the multi-center Surveillance Using HeartCare Outcomes Registry (SHORE) database who had dd-cfDNA measured within 30 days of a negative EMB (Grades 2R/AMR1 or higher rejection) within the first 100 days of heart transplant (HT) were reviewed. These patients were followed for the subsequent development of significant rejection and <i>de novo</i> donor-specific antibodies (dnDSA) over the next 365 days. <h3>Results</h3> A total of 648 HT recipients with a mean age 57, 74% male, white 64%, 60% of which had PRA < 1% and had a total of 982 paired biopsies with a median dd-cfDNA of 0.05% for those with a Grade 0R and 0.06% for Grade 1R biopsy (Figure 1a). The dd-cfDNA was measured a median of 112 days post-transplant for Grade 0R and 109 days post-transplant for Grade 1R. Despite negative histology on EMB, those with a cfDNA >= 0.20% were at significantly higher risk for the development of significant rejection (14.3% v 5.2%, p<0.01) and dnDSA (11.3% v. 6.8%, p<0.01) over the subsequent year. <h3>Conclusion</h3> Patients with elevated dd-cfDNA, but without prior rejection and negative histology in the first 100 days post-transplant are at increased risk for significant rejection and dnDSA in the subsequent year. The use of dd-cfDNA may be a better method to determine true quiescence and call into question the utility of the EMB as the gold standard for cardiac allograft monitoring.

A Cost Effective Liquid Biopsy Technology Using Donor Derived Cell Free Dnato Diagnose Rejection in Thoracic Organ Transplant Recipients

<h3>Purpose</h3> Solid organ transplant recipients are periodically reviewed to assess the health of transplanted organ. Surgical biopsy is considered as gold standard to monitor rejections as histological changes associated with rejection can be identified by direct microscopy or after immunohistochemical staining with rejection specific biomarker, C4d. However, a number of inconsistencies exist while interpreting the sections as criteria for quantitative scoring of endothelial enlargement and C4d-positivity in patients without clinical evidence of rejection and C4d-negativity in patients with clinical evidence of rejection is not established. Besides the above repeated biopsies can expose the organs to risk of scarring, which in turn can affect its optimal function. <h3>Methods</h3> To mitigate these inconsistencies and risks we have developed a cost effective liquid biopsy technology to diagnose allograft rejection. We first screen an array of single-nucleotide polymorphisms (SNPs) in donor and recipient genomic DNA to discriminate and identify donor specific SNPs. As these SNPs are representative of donor derived cell free DNA (dd-cfDNA), quantification of them by real time PCR in total cell free DNA (total cfDNA) fraction of recipients can be considered as indicator for allograft health. By using this technology we performed a pilot study on 12 heart or lung allograft recipients. 9ml of whole blood collected in PAXGENE tubes from the patients before biopsy were processed to extract total cfDNA and quantify the abundance of dd-cfDNA in the total cfDNA fraction by real time qPCR. <h3>Results</h3> Data analysis indicated presence of dd-cfDNA in 2/12 samples, which correlated with histopathological investigations that identified minimal acute rejection in one patient and cell mediated rejection in the other. One more patient was identified to be undergoing minimal cellular rejection one month post transplant. However, we could not detect dd-cfDNA signal in the plasma. Based on these data, our liquid biopsy technology is able to diagnose rejection in 66.6% (2/3) of patients with a negative prediction at 100% (9/9). <h3>Conclusion</h3> A cost effective Liquid Biopsy Technology using dd-cfDNA quantification by real time qPCR which has excellent correlation with conventional biopsies. Large randomised control studies need to be conducted to further validate this.

Longitudinal Monitoring of Donor Derived Cell Free DNA in Lung Transplant Recipients

<h3>Purpose</h3> The purpose of this study is to evaluate whether longitudinal monitoring of donor-derived cell free DNA (dd-cfDNA) in lung transplant recipients can be used as a marker of lung injury and stability. <h3>Methods</h3> 24 subjects were enrolled in this study measuring dd-cfDNA levels on a monthly basis in the first year after bilateral lung transplant. Blinded clinical adjudications were performed at the same timepoints to categorize allograft function as stable (FEV1 within 10% of prior value) or unstable. When deemed unstable, etiology of unstable graft function was elicited based on available clinical data. We then evaluated correlation between dd-cfDNA results and the clinical impression of allograft function. <h3>Results</h3> Median dd-cfDNA levels in those with clinically stable graft function was 0.28 (IQR 0.16, 0.65), and unstable graft function was 0.35 (IQR 0.22, 0.90) (<i>Figure 1.)</i>. In those with stable graft function, the change in dd-cfDNA decreased over time with a median change of -0.08 (IQR -0.27, +0.08) whereas those with unstable graft function had increases in dd-cfDNA over time with a median change of +0.09 (IQR -0.01, +0.17) <i>(Figure 2.)</i>. <h3>Conclusion</h3> dd-cfDNA does not have sufficient precision to be used as an independent monitor of allograft function using a threshold value in the absence of clinical correlation. Serial trends in dd-cfDNA may identify an unstable graft that is vulnerable. Further analysis of the dataset regarding development of CLAD will refine the utility of this clinical tool.

Impact on Donor Derived Cell Free DNA (dd-cfDNA) of Procurement Using Paragonix SherpaPak™ (SP) Cardiac Transport System versus ICE Transportation

<h3>Purpose</h3> Little is known about the impact of various procurement methods on dd-cfDNA. We aimed to determine if differences exist in dd-cfDNA values when using SherpaPak (SP) compared to historical ice transportation methods. <h3>Methods</h3> A retrospective review of 50 single organ heart transplant (HT) recipients with heart care measurements within 90 days post-transplant were included. Transportation type and long total ischemic time above the upper tertile of 223 minutes with log-transformed dd-cfDNA levels using repeated measures analysis was performed. <h3>Results</h3> 29 recipient hearts were transported on ice and 21 with SP. Recipient and donor characteristics were similar with expected shorter mean and maximum total ischemic times on ICE (197 ± 48 minutes, max. 285 minutes) compared to SP (214 ± 50 minutes, max. 325 minutes). The median dd-cfDNA level was the highest within the first 27 days in both groups (median 0.18 v. 0.20) and were comparable up to 90 days (p=0.10). <b>Fig1A.</b> There were 17 patients with long ischemic times with a mean of 256 minutes. In SP patients, long ischemic times had higher dd-cfDNA compared to short ischemic times (p=0.01). This relationship was not observed in ice patients (p=0.41). In long ischemic time patients, higher dd-cfDNA levels approached near significance when comparing SP v. ice patients (p=0.053). In patients with short ischemic times, dd-cfDNA levels were similar between SP v. ice (p=0.80). <b>Fig1B.</b> The overall cohort with long ischemic times, regardless of transport method, had higher levels of dd-cfDNA within the first 90 days (p=0.012). <h3>Conclusion</h3> Patients with long ischemic times are more likely to have higher dd-cfDNA vs short ischemic times. These results were predominantly driven by the patients in the SP group. Further studies including cost analysis of SP vs ICE modalities are needed to validate these preliminary findings.

Absolute Quantification of Donor Derived Cell Free DNA in Heart Transplant Patients

PurposeCirculating donor-derived cell-free DNA (dd-cfDNA) is a validated biomarker of allograft rejection. Current assays express dd-cfDNA as a percentage of total cell-free DNA (donor fraction). However, a number of factors can influence recipient cfDNA levels (e.g. BMI, infection) unrelated to the allograft. Thus, we assessed the performance of absolute quantification to detect acute rejection (AR) in heart transplant recipients.MethodsWe developed a method to quantify absolute levels of dd-cfDNA expressed as copies/mL. We measured both dd-cfDNA fraction (%) and absolute quantity (copies/mL) using a clinically available SNP-based massively multiplexed PCR dd-cfDNA assay (ProsperaTM, Natera, San Carlos, CA). These values were compared with diagnosis of AR by histology (defined as ACR >1R and pAMR >0) and test performance was assessed for sensitivity, specificity, negative predictive value and positive predictive value.ResultsOut of 447 samples collected from 150 heart-transplant only patients, 29 had AR and 418 did not. Both dd-cfDNA fraction and absolute quantity were significantly higher in samples with AR compared to samples with no AR. Table 1 characterizes the performance of donor fraction and quantification to distinguish AR. Median dd-cfDNA fraction for AR was 0.62% (IQR: 0.09-1.43) and significantly higher than no AR at 0.04% (0.01-0.11, p < 0.01). Median absolute dd-cfDNA for AR was 78.8 copies/mL (5.3-125.6) and significantly higher than no AR at 2.7 copies/mL (1.37-6.90, p=1.67e-09).ConclusionAbsolute quantification of dd-cfDNA may increase the accuracy to discriminate rejection when compared with dd-cfDNA expressed as a percentage of total cfDNA. Circulating donor-derived cell-free DNA (dd-cfDNA) is a validated biomarker of allograft rejection. Current assays express dd-cfDNA as a percentage of total cell-free DNA (donor fraction). However, a number of factors can influence recipient cfDNA levels (e.g. BMI, infection) unrelated to the allograft. Thus, we assessed the performance of absolute quantification to detect acute rejection (AR) in heart transplant recipients. We developed a method to quantify absolute levels of dd-cfDNA expressed as copies/mL. We measured both dd-cfDNA fraction (%) and absolute quantity (copies/mL) using a clinically available SNP-based massively multiplexed PCR dd-cfDNA assay (ProsperaTM, Natera, San Carlos, CA). These values were compared with diagnosis of AR by histology (defined as ACR >1R and pAMR >0) and test performance was assessed for sensitivity, specificity, negative predictive value and positive predictive value. Out of 447 samples collected from 150 heart-transplant only patients, 29 had AR and 418 did not. Both dd-cfDNA fraction and absolute quantity were significantly higher in samples with AR compared to samples with no AR. Table 1 characterizes the performance of donor fraction and quantification to distinguish AR. Median dd-cfDNA fraction for AR was 0.62% (IQR: 0.09-1.43) and significantly higher than no AR at 0.04% (0.01-0.11, p < 0.01). Median absolute dd-cfDNA for AR was 78.8 copies/mL (5.3-125.6) and significantly higher than no AR at 2.7 copies/mL (1.37-6.90, p=1.67e-09). Absolute quantification of dd-cfDNA may increase the accuracy to discriminate rejection when compared with dd-cfDNA expressed as a percentage of total cfDNA.

Dd-cfDNA Levels Are Independent of Allograft Longevity in Stable Lung Transplant Recipients

<h3>Purpose</h3> Donor-derived cell free DNA (dd-cfDNA) is a well-studied biomarker of early allograft injury, though kinetics of dd-cfDNA outside the early period is understudied. We measured levels of plasma dd-cfDNA in stable lung transplant recipients (LTRs) >2-years post-transplant to determine if dd-cfDNA varies with allograft longevity. <h3>Methods</h3> We performed a prospective, observational study of LTRs that were > 2-years post-transplant and had stable lung function. Plasma dd-cfDNA (CareDx) was measured at outpatient visits. Patients were considered at baseline if they were hospital-free in the prior 30-days, FEV1 was >90% baseline, and they were sans fever, cough, or dyspnea. Patients treated with augmented immunosuppression for ALAD / CLAD were excluded. LTRs were categorized into three allograft-age cohorts (2-3, 3-5, or > 5 years post-transplant). Statistics were performed using SSPS (IBM); chi-squared testing and ANOVA was used for binary and continuous data, respectively. <h3>Results</h3> Baseline dd-cfDNA was measured in 59 LTRs meeting inclusion criteria. Overall median dd-cfDNA was 0.45% (IQR 0.30-0.65). (Fig. 1). Median plasma dd-cfDNA was 0.51% (IQR 0.34-0.63), 0.35% (IQR 0.12-0.59), and 0.43% (IQR= 0.29-0.67) for LTRs 2-3years, 3-5 years and >5-years post-transplant, respectively (p=0.91). Baseline demographics were similar between cohorts for all variables except transplant type (Table 1). <h3>Conclusion</h3> Plasma dd-cfDNA is <1% in clinically stable LTRs > 2-years post-transplant and does not vary with allograft survival. Future studies will determine whether changes in dd-cfDNA from baseline can predict CLAD onset.

Metagenomic Next Generation Sequencing (mNGS) Can Complement Fractional Donor-Derived Cell-Free DNA in Lung Allograft Assessment: Pilot Data

<h3>Purpose</h3> Transbronchial biopsy (TBBx) histology and lavage (BAL) for microbiology are standard of care in lung transplant (LT) evaluation, but are inherently flawed. Plasma donor-derived cell-free DNA (dd-cfDNA) and viral DNA/RNA (vDNA/RNA) are promising biomarkers for more accurate assessment of allograft injury. Here we compare TBBx histology, BAL microbiology, dd-cfDNA and vDNA/RNA in matched samples. <h3>Methods</h3> 40 post-LT samples (median 212 days) from 33 patients (median age: 61.0 yrs) were analyzed for dd-cfDNA fraction (Prospera™, Natera) and vDNA/RNA by metagenomic next-generation sequencing (mNGS) (Galileo™-ONE ArcBio) and correlated with clinical-pathologic (TBBx) diagnosis and BAL microbiology including Respiratory Viral Panel (RVP) multiplex PCR testing (BioFire RP2.1™; Biomerieux) <h3>Results</h3> Native lung diseases included: IPF (N=18), COPD (N=17), CF (N=2) and PAH (N=3). mNGS identified vDNA/RNA considered pathologic to LT in 27.5% (11/40) cases, including 2 previously undiagnosed cases of HIV-1, with a median dd-cfDNA of 1.14% [<b>TABLE</b>]. For 9 cases with "negative" BAL/RVP, putative respiratory viral infection was indicated by mNGS. vDNA/RNA was also detected in 3 cases of histologic acute cellular rejection (ACR), one of which had elevated dd-cfDNA. In 2/4 patients with chronic lung allograft dysfunction (CLAD), the dd-cfDNA fraction was elevated and associated with vDNA/RNA. Two stable patients (A0B0/X) with vDNA/RNA had elevated dd-cfDNA. <h3>Conclusion</h3> Detection of clinically significant vDNA/RNA signatures by plasma mNGS testing contradicted clinical-pathologic diagnoses in >25% of patients. Future studies will help better understand and adjudicate discrepant results between TBBx histology, BAL microbiology, dd-cfDNA and mNGS. mNGS testing offers promise for properly assigning clinical-pathologic diagnosis in the assessment of LT health and may support the appropriate adjudication of elevated dd-cfDNA fractions.

Genome-Wide Cell-Free DNA Methylation Analysis Reveals Significant Recipient Tissue Injury in Allograft Rejection

<h3>Purpose</h3> Acute rejection (AR) causes significant allograft injury. However, its systemic effect on recipient tissues remains poorly defined. Donor-derived cell-free DNA (ddcfDNA) is a sensitive marker of allograft rejection, but does not capture any concurrent recipient tissue injury. Herein, we leverage cfDNA epigenetics as marker of tissue injury to profile recipient tissue injury associated with AR. <h3>Methods</h3> cfDNA was extracted in 92 plasma samples from 33 heart transplant recipients (HTR) (19 with AR and 14 stable controls) from the GRAfT study (NCT 02423070), and 19 healthy controls (HC). <b>Measurements:</b> Total cell-free nuclear (ncfDNA) and mitochondrial DNA (mtcfDNA) were assayed via digital-droplet PCR. To quantify recipient cfDNA, we first measured ddcfDNA fraction by shotgun sequencing; recipient cfDNA is the difference of total ncfDNA and ddcfDNA. Next, we performed whole-genome bisulfite sequencing on cfDNA and used tissue-specific DNA methylation signatures to deconvolute recipient tissue sources of cfDNA. Median [interquartile range (IQR)] of cfDNA percent or copies (cp)/mL are reported. <h3>Results</h3> ddcfDNA levels increased 6 fold with AR compared to stable HTR (0.54 [0.32-1.21] % vs. 0.09 [0.03 - 0.12] %, <i>p</i><0.01). ncfDNA, which is >99% recipient-derived, was 5X higher in stable HTR (7997 [3974-9740] cp/mL) than HC (1224 [921-1985] cp/mL, <i>p</i><0.01). With AR, ncfDNA doubled (14949 [8460-24018] cp/mL) compared with stable HTR (<i>p</i><0.01). mtcfDNA tripled with AR than stable HTR, and showed excellent discriminatory performance (AUC = 0.92, 95%CI [0.84-0.99]). Genome-wide methylation analysis showed significantly higher cfDNA from hematopoietic cells, vascular endothelium, hepatocyte and kidney with AR than Stable HTR, Fig.1. <h3>Conclusion</h3> Genome-wide cfDNA methylome analysis shows increased recipient tissue injury with AR. Ongoing studies intend to validate this finding and test if this approach can distinguish antibody-mediated from acute cellular rejection phenotypes.

Comparative Performance Analysis of Donor-Derived Cell-Free DNA to Detect Acute Rejection in Single and Double Lung Transplant Recipients

<h3>Purpose</h3> Plasma donor-derived cell-free DNA (ddcfDNA) is a sensitive biomarker for acute rejection (AR). However, its performance remains unknown for single (SL) vs double lung (DL) transplant recipients, where differences in lung mass may affect interpretation. In this study, we aim to distinguish the performance characteristics of ddcfDNA to detect AR in SL vs DL transplant patients. <h3>Methods</h3> Lung transplant recipients (n=105) enrolled in two prospective cohort studies were adjudicated for acute cellular rejection (ACR) and antibody mediated rejection (AMR). <b>Measurement:</b> 337 serially-collected plasma samples were assayed for ddcfDNA by shotgun sequencing. <b>Analysis:</b> Area-under-the-receiver-operating-characteristic-curves (AUROC) of ddcfDNA and performance characteristics for the detection of AR were calculated separately for both DL and SL transplant recipients; cfDNA values were not doubled for SL transplant as in prior studies. <h3>Results</h3> The AUROC for ddcfDNA to detect AR was 0.86 and 0.89 in DL and SL transplants, respectively. The optimal ddcfDNA threshold of 0.54% for SL transplant showed a 92% sensitivity and 80% specificity for detecting AR. For DL transplant, the optimal threshold of 1.1% showed 78% sensitivity and 83% specificity for detecting AR. When ddcfDNA was doubled for single lung transplant, the 1.1% threshold showed 92% sensitivity and 80% specificity for detecting rejection. The AUROC for detection of AMR was 0.91 for both DL and SL transplants. However, the AUROC for detection of ACR was 0.73 for DL and 0.87 for SL transplants. <h3>Conclusion</h3> Plasma ddcfDNA showed similar accuracy to detect AR in both SL and DL transplant recipients, however, the accuracy of ddcfDNA for the detection of ACR appears higher in SL vs DL transplant recipients. Correcting ddcfDNA levels in SL transplant patients by doubling their value appears to be a reasonable method of preserving the performance characteristics of the test for the detection of acute rejection.