Donor-derived Cell-free dd-cfDNA Research Articles

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.

Variability in Donor-Derived Cell-Free DNA Levels Predicts Mortality Risk in Heart Transplant Recipients

<h3>Purpose</h3> Longitudinal surveillance of heart transplant (HTx) recipients using donor-derived cell-free DNA (dd-cfDNA) allows non-invasive detection of molecular injury patterns. We hypothesized that variability in dd-cfDNA levels over time identifies patients at risk of adverse outcomes. <h3>Methods</h3> 72 adult HTx patients monitored with gene expression profiling (GEP) and dd-cfDNA (AlloMap, AlloSure; CareDx Inc., Brisbane, CA) at our center were included. Variability (ASV) was defined as standard deviation of the 3 most recent (3-value) or all (all-value) sequential dd-cfDNA results and scaled x 10 for analysis (ASV*10). We used a Cox proportional hazards model to assess the association between ASV and mortality and a survival classification and regression tree (CART) algorithm to define a threshold for increased mortality risk. <h3>Results</h3> Patients had a median of 6 (IQR 4-9) paired GEP/dd-cfDNA results spaced 40.8 (IQR 31.5 - 66.1) days apart. 48.6% of patients had dd-cfDNA below the assay limit of detection (<0.12%); the remainder had a median dd-cfDNA of 0.17% (IQR: 0.12 - 0.45%) and a mean all-value ASV of 0.24 (SD: 0.40). ASV*10 of patients who died (2.62, SD 2.66) trended higher than of those who survived (0.54, SD 1.10) over a median follow-up of 480 days (IQR 244 - 859). In a univariate Cox model, the 3-value ASV*10 was associated with an increased risk of mortality (HR 1.66, 95% CI 1.14 - 2.41, p<0.009). Only a trend was evident after multivariate adjustment <b>[Figure 1a]</b>. Using a CART algorithm, a 3-value ASV*10 of 2.97 discriminated between patients at high and low risk of mortality after HTx <b>[Figure 1b]</b>. <h3>Conclusion</h3> In this proof-of-concept study evaluating the clinical utility of longitudinal dd-cfDNA results, we found that higher variability in dd-cfDNA over time was associated with an increased risk of mortality after HTx. Additional studies utilizing larger datasets are needed to validate and expand on our findings.

Poke Not Prod: Improving Quality of Life Through Non-Invasive Rejection Surveillance for Heart Transplant Recipients

<h3>Purpose</h3> Gene expression profiling (GEP) and donor-derived cell-free DNA (dd-cfDNA) provide effective non-invasive rejection surveillance for heart transplant (HT) recipients with a trend toward improved quality of life. During the COVID-19 pandemic, rejection monitoring and titration of mediations in HT patients was difficult due to limited health-care resources for endomyocardial biopsy (EMBx). This is the first Canadian study to assess non-invasive rejection surveillance in improving patient satisfaction and reducing anxiety during HT rejection screening. <h3>Methods</h3> Adult HT recipients, at least 6 months post-transplant, were enrolled to have surveillance EMBx replaced by non-invasive rejection testing with GEP and dd-cfDNA. Patients with multiorgan transplant, dialysis, or high rejection risk (recent acute cellular rejection ≥ grade 2R, new graft dysfunction, or heart failure) were excluded. All patients completed the Medical Outcomes Study 12-item Short Form Health Survey (SF-12) and a patient satisfaction survey. Thematic analysis was performed for open-ended responses. <h3>Results</h3> Out of 90 patients screened, 31 had their routine EMBx replaced by non-invasive rejection testing. Based on test results, 89% of EMBx were safely eliminated. On the SF-12, participants had a median physical health score of 43 (40-53) and mental health score of 53 (46-58) out of 100 . Patients' self-reported satisfaction was 90%. Median self-reported anxiety score prior to EMBx was 50 (10-71) versus 2.5 (0-7.5) out of 100 prior to GEP/dd-cfDNA. Four codes ("emotions" (pain, anxiety, fear), "time", "biopsy", "accuracy") were used to uncover two themes of "Superiority to Biopsy" and "Mental or Physical Stress". Patients described feeling much more satisfied and less emotionally distressed with the non-invasive screening compared to EMBx. HT patients reported less fear and anxiety, reduced pain, and enjoyed the simplicity of non-invasive testing. <h3>Conclusion</h3> Non-invasive rejection surveillance screening can positively impact patients' mental health. In this study, non-invasive rejection surveillance eliminated the recovery time and risk of an invasive procedure for HT recipients while reducing anxiety, improving patient satisfaction, and providing an alternative way to screen patients during a period of limited resources due to a global pandemic.

Higher Levels of Donor-Derived Cell-Free DNA Are Associated with Acute Cellular Rejection but Not with Severe PGD After Lung Transplantation

<h3>Purpose</h3> Circulating donor-derived cell-free DNA (dd-cfDNA) levels have been proposed as a potential tool for the diagnosis of graft injury (rejection, infection, ischemia/reperfusion injury). In this study, we prospectively investigated dd-cfDNA plasma levels and their association with severe primary graft dysfunction at 72 hours and acute cellular rejection in the first month after lung transplant. <h3>Methods</h3> Thirty-five subjects undergoing double lung transplants at our institution between Jun.2020-Oct.2021 were included in this study. Primary Graft Dysfunction (PGD) was graded according to ISHLT criteria. Our protocol includes a surveillance bronchoscopy with transbronchial biopsy at 3 weeks post-transplant. Acute cellular rejection was diagnosed and graded by a pulmonary pathologist after evaluation of histopathology slides. Blood samples were collected at various time points before and after lung transplant. Dd-cfDNA in samples were determined using AlloSure dd-cfDNA test kits (CareDx, Inc.). <h3>Results</h3> We observed a rapid increase of dd-cfDNA in blood of recipients after lung transplantation compared to baseline. The levels of dd-cfDNA decreased during the first two weeks (Figure A). The peak was observed within 72 hours after transplantation. The peak values of dd-cfDNA varied among subjects and had no correlation with PGD grade 3 occurrences at 72 hours. (Figure B, p=0.85). We observed an association between levels of dd-cfDNA from blood collected at the time of transbronchial biopsy and the histological diagnosis of ACR at 3 weeks. (Figure C, mean 2.7% versus 1.5%, p=0.015) <h3>Conclusion</h3> This preliminary data shows that circulating dd-cfDNA levels are associated with ACR early after transplantation but not with severe PGD at 72hours. Plasma levels of dd-cfDNA may be a less invasive tool to estimate graft rejection after lung transplantation however larger studies are still necessary to better identify thresholds.

Cell-Free DNA to Distinguish High Risk Donor Specific Antibodies in Heart Transplantation

<h3>Purpose</h3> Donor specific antibodies (DSA) develop in a significant fraction of heart transplant recipients (HTR). Enhanced characterization of high-risk DSA may permit early treatment and prevent downstream antibody-mediated rejection (AMR) and allograft failure. We hypothesize that high risk DSA is associated with allograft injury. Here, we compared allograft injury between high and low risk DSA using donor-derived cell-free DNA (ddcfDNA), a sensitive marker shown to detect AMR ∼3 months earlier than biopsy. <h3>Methods</h3> Using the prospective, multicenter study, Genomic Research Alliance for Transplantation (GRAfT), 81 HTR who underwent surveillance and clinically indicated endomyocardial biopsy, and DSA testing were analyzed. <b>Measurement:</b> Serially collected plasma samples (n=860) were assessed for ddcfDNA by shotgun sequencing. <b>Analysis:</b> DSA were categorized as "high risk" if they were associated with AMR or were otherwise considered "low risk". DSA was further categorized by specificity as class II vs. class I. We compared ddcfDNA between groups with generalized mixed linear models; excluding ddcfDNA before Day 14 post-transplant to reduce transplant surgical contribution. <h3>Results</h3> 47% HTR (n=38) developed DSA at a median 24 days (IQR = 6 - 141 days) post-transplant; and include 28 <i>de novo</i> and 11 preformed DSA. 15.8% (n=6) were categorized as high risk DSA and 84.2% (n=32) were low risk DSA. High risk DSA showed 4X higher ddcfDNA levels compared to low risk DSA (mean [95% CI] 0.30[0.22% - 0.39%] vs. 0.07[0.06%-0.10%], <i>p</i> <0.01, Figure A). As compared to patients without DSA, low risk DSA showed similar ddcfDNA (p=0.52, Figure A). We also found higher ddcfDNA in class II than in class I DSA (Figure B). <h3>Conclusion</h3> High risk DSA show higher allograft injury as measured by ddcfDNA. Similarly, <i>de novo</i> and class II DSA are associated with higher allograft injury than class I or preformed DSA. We provide a novel concept that allows for use of ddcfDNA to stratify high-risk DSA in heart transplantation.

Absolute versus Percent of Donor-Derived Cell-Free DNA to Detect Lung Transplantation Rejection

<h3>Purpose</h3> Plasma donor-derived cell-free DNA (ddcfDNA) is a sensitive biomarker for acute rejection (AR). Percent ddcfDNA, expressed as percent of donor DNA to donor plus recipient cfDNA, is reliant on stable recipient cfDNA. However, lung transplant patients are predisposed to kidney and other end-organ injury, which changes recipient cfDNA. In this study, we assessed the test performance of absolute amount of ddcfDNA measured in copies per mL of plasma and %ddcfDNA for detecting lung transplant rejection. <h3>Methods</h3> Prospectively enrolled patients (n=56) were adjudicated for antibody-mediated rejection (AMR) and acute cellular rejection (ACR) using ISHLT consensus guidelines. <b>Measurement</b><i>:</i> Serially collected plasma samples (n=729) were assayed for %ddcfDNA by shotgun sequencing and for absolute ddcfDNA by digital droplet PCR using primers that target donor recipient single nucleotide polymorphisms. <b>Analysis:</b> We assessed the area under the receiver operator characteristics curve (AUROC) of %ddcfDNA and absolute ddcfDNA to detect AR, a composite endpoint of AMR and ACR. <h3>Results</h3> Over the median 19.6 months follow-up, there were 60 episodes of AR (AMR = 31, ACR = 29). ddcfDNA levels, measured as absolute or as %, were highest after transplant surgery and decayed to baseline levels of 74.7 (IQR = 45.7-116.9) Copies/mL or 0.34% (IQR = 0.21%-0.82%), respectively. Levels increased with AR compared to controls for %ddcfDNA (1.17% vs. 0.34%, <i>p = <0.</i>0001) and absolute ddcfDNA (232 versus 74 copies/mL, <i>p = 0.</i>0014). %ddcfDNA showed an AUROC of 0.73 to detect AR, higher than the AUROC of 0.66 for absolute ddcfDNA (<b>Fig.1A</b>). In select patients with increased recipient cfDNA (<b>Fig.1B</b>), absolute ddcfDNA was better to detect AR than %ddcfDNA. <h3>Conclusion</h3> %ddcfDNA performed better than absolute ddcfDNA quantitation to detect AR. However, absolute ddcfDNA detects AR better when concurrent recipient tissue injury occurs. A combination of these strategies may provide the optimal method to diagnose acute rejection.

Donor Specific Allo-Antibody is Significantly Associated with Variability in Donor-Derived Cell-Free DNA Scores in Adult Heart Transplant Recipients

<h3>Purpose</h3> In this single-center retrospective pilot study heart transplant (HTx) patients were non-invasively monitored by donor-derived cell-free DNA (dd-cfDNA). We hypothesized that longitudinal dd-cfDNA variability scores correlate with preformed and de novo (dn) donor specific HLA allo-antibody (DSA). <h3>Methods</h3> 72 adult HTx patients with at least 3 dd-cfDNA results were included (AlloSure; CareDx Inc., Brisbane, CA). We determined dd-cfDNA variability (ASV) by standard deviation (SD) of longitudinal dd-cfDNA results, scaled x10 (ASV*10), and DSA by single antigen class I/II and MFI >1000 for HLA-A, B, DR, DQB/A and >2000 for HLA-C and DPB/A. <h3>Results</h3> Age at HTx was 49.1 years (SD 14.3). Study enrollment occurred at 270 days +/-SD 420.2 post-HTx. Biopsy-proven rejection (ACR ≥ 2R and/or pAMR ≥ 1) was detected in 16 (22 %), preformed DSA in 9 (12.5%), dnDSA in 13 (18%) and preformed + dn in 2 (2.7%) patients while 48 (66.7%) were DSA negative. Median time from HTx to dnDSA was 329d (range: 39-827). Including all values, mean ASV*10 was highest in patients with dnDSA (3.03 ± 4.74) compared to those with no DSA (0.65 ± 2.04) or preformed DSA (1.36 ± 3.40, p=0.001; <b>Table 1</b>). Patients who developed dnDSA showed a significant increase of ASV*10 after dnDSA detection (1.50 ± 2.06, p = 0.014), but not before dnDSA detection (1.24 ± 1.49, p=0.380) in comparison to patients with no DSA (0.65 ± 2.04, <b>Table 1</b>). <h3>Conclusion</h3> These findings highlight the value of longitudinal assessment of dd-cfDNA and DSA in HTx recipients. Our results show that increased dd-cfDNA variability is associated with the presence of preformed and dnDSA. Further, ASV*10 is significantly increased after identification of dnDSA, further supporting the mechanistic role of dnDSA in mediating poor outcomes after HTx. Confirmation in larger data sets may further advance the utility of longitudinal non-invasive surveillance using dd-cfDNA and dnDSA in transplant recipients.

Rationale and Design of a Randomized Controlled Trial of Donor-Derived Cell-Free DNA to Detect Rejection in Cardiac Transplantation (DETECT)

<h3>Purpose</h3> Endomyocardial biopsy (EMB) for rejection surveillance in cardiac transplantation is invasive, costly, associated with complications, variable in interpretation and thus an imperfect gold standard. Donor-derived cell-free DNA (dd-cfDNA) is an emerging biomarker for detection and surveillance of cardiac allograft rejection. Elevated circulating levels of dd-cfDNA are associated with acute cellular and antibody-mediated rejection in observational studies, but a clinical strategy trial to establish its usefulness in decreasing or eliminating EMB has not been conducted. We describe the rationale and design of the randomized clinical trial, Donor-Derived Cell-free <b>D</b>NA to D<b>ET</b>ect R<b>E</b>jection in <b>C</b>ardiac <b>T</b>ransplantation (DETECT) to test the hypothesis that dd-cfDNA monitoring is non-inferior to standard of care (SOC) surveillance EMB during year 1 post-transplant. <h3>Methods</h3> DETECT is a prospective, multicenter, open-label, randomized controlled trial in 600 adult heart transplant recipients at 40 transplant centers, randomized 1:1 30 days post-transplant to surveillance with dd-cfDNA (Prospera, Natera, San Carlos, CA) (Intervention) versus EMB surveillance (SOC) and followed for 1 year. Rejection surveillance in both arms will be performed per the institution's SOC EMB surveillance schedule. <h3>Results</h3> The primary endpoint is the composite of treated rejection +/- graft dysfunction, any graft dysfunction, listing for or undergoing re-transplantation or death. Secondary endpoints include number of heart biopsies performed, development of de novo donor specific antibodies and cost of care. The study has 80% power to demonstrate non-inferiority if the primary endpoint rate in the experimental arm is no greater than 0.27 (non-inferiority margin of 0.15, assuming a control rate of 0.22). Sample size re-estimation will occur once 80% of subjects are enrolled or 50% reach 1 year follow-up, whichever occurs first in order to maintain 80% power. If the non-inferiority hypothesis is met, analysis for superiority of surveillance with dd-cfDNA will be performed. <h3>Conclusion</h3> The DETECT trial will establish the efficacy and safety of dd-cfDNA as a primary clinical monitoring strategy for cardiac allograft rejection in an effort to decrease or eliminate invasive surveillance biopsies in adult heart transplant recipients.

Poke Not Prod: First Canadian Experience Using Donor-Derived Cell Free DNA to Replace Endomyocardial Biopsy During COVID-19

PurposeAfter a heart transplant (HT), non-invasive methods for rejection surveillance minimize the need for endomyocardial biopsies (EMBx). We describe the first experience with combined use of genetic expression profiling (GEP) and donor-derived cell-free DNA (dd-cfDNA) testing in Canada as part of a quality improvement project to minimize patient risk during the COVID pandemic.MethodsAdult outpatients at least 6 months after HT were screened from May 2021 to July 2021 to have their routine EMBx replaced by a combination of GEP and dd-cfDNA. Demographics, modification of immunosuppression (IS) and outcomes (hospital admission, rejection, and need for EMBx) were collected.ResultsAmong 90 patients, 31 (33%) were enrolled, and 37 non-invasive tests were performed. The median time after HT was 2 years and patients were predominantly Caucasian (52%) and male (68%). 53% had a history of acute cellular rejection during the first year and 32% had cardiac allograft vasculopathy. Of the tests performed, 23 (60%) were - GEP / - dd-cfDNA, 10 (27%) were + GEP / - dd-cfDNA, 4 (11%) were - GEP / + dd-cfDNA and none were + GEP / + dd-cfDNA. Being bridged with a VAD (OR = 5.5, p=0.034) and a history of a previously treated CMV (OR = 16.0, p=0.003) were associated with a positive GEP and a negative dd-cfDNA result. Having received a COVID vaccine in the last 3 months did not affect GEP results (GEP was positive in 23.8% after vaccination vs 33.3% in non-vaccinated patients, p=0.690; average GEP score 29.8 vs 30.7, p=0.673). The 4 patients with a + dd-cfDNA (range 0.19 - 0.81%) underwent an EMBx with no significant cellular or antibody mediated rejection, thus avoiding 89% of the EMBx. No unscheduled clinic visits, emergency department or hospital admissions were recorded. After non-invasive testing, the IS was reduced in 16 cases (43.2%). IS was reduced in in 59% of patients with negative concordant tests (- GEP / - dd-cfDNA), 30% in patients with + GEP / - dd-cfDNA and no reduction in IS occurred in those with + dd-cfDNA.ConclusionThe combination of GEP and dd-cfDNA for rejection surveillance allowed for a marked reduction in EMBx (89%) and for a personalized downtitration of IS without adverse events in the short term. The use of non-invasive rejection surveillance testing was an effective strategy to avoid hospital contact for HT recipients during the COVID-19 pandemic.

Prognostic Implications and Characteristics of Low dd-cfDNA Results in Heart Transplant Patients with Biopsy Proven Rejection

<h3>Purpose</h3> Donor-derived cell-free DNA (dd-cfDNA) has emerged as an important noninvasive marker of heart transplant allograft injury. The characteristics and prognostic implications of low level dd-cfDNA in patients with histological evidence of acute rejection is not known. Although considered the gold standard, endomyocardial biopsy is limited to focal tissue sampling and is subject to interobserver variability in its interpretation. <h3>Methods</h3> Patients enrolled in the Surveillance HeartCare Outcomes Registry (SHORE) with biopsy proven acute cellular rejection (ACR) and antibody mediated rejection (AMR) who had low levels of dd-cfDNA (<0.15%) were evaluated. A targeted amplification, next generation-sequencing assay (AlloSure®; CareDx, Inc.) was used to detect dd-cfDNA. <h3>Results</h3> The SHORE registry had 2029 patients in total. Patients with ACR 1R were excluded. Only those patients who had a dd-cfDNA level within 30 days of biopsy (mean 2.7 days) were included. 61 patients (male 78%, median age 48 years) had low dd-cfDNA and endomyocardial biopsy evidence of ACR ≥2R and/or AMR ≥1R. Mean duration of the biopsy from the time of transplant was 196.31 days (IQR 88-257 days). ACR was seen in 36 patients (35=2R, 1=3R) and AMR was seen in 27 patients (21=pAMR 1(H+), 6=pAMR2). 2 patients had both ACR and AMR. The mean ejection fraction (EF) at the time of the rejection was mostly preserved (ACR 2R=EF 62.8±5.6%,ACR 3R=EF 47.5%, and AMR 1=EF 60.9±6%, AMR 2=EF 61±4.7%). 2 patients (3.3%) died 330±31days after the initial diagnosis of rejection. None of the patients developed graft dysfunction at one year follow up. One patient developed coronary artery vasculopathy and 7 patients developed <i>de novo</i> DSA. <h3>Conclusion</h3> Patients with low dd-cfDNA and biopsy-confirmed rejection typically had lower grades of rejection and maintained preserved left ventricular function on echocardiography. There was no immediate death after rejection or graft dysfunction. In this preliminary analysis, low dd-cfDNA despite biopsy evidence of rejection appears to have favorable prognosis. Discordance between light-based histology and molecular histology has shown better correlation with dd-cfDNA levels, and so consideration of dd-cfDNA in treatment and immunosuppression management requires ongoing evaluation.

Donor-Derived Cell-Free DNA for the Detection of Heart Allograft Injury: Impact of Timing of the Liquid Biopsy

<h3>Purpose</h3> In heart transplant recipients, donor-derived cell-free DNA (ddcfDNA) is a potential biomarker for acute rejection (AR), in that increased values may indicate rejection. Blood plasma sampling around the endomyocardial biopsy (EMB) seems a practical approach for the assessment of ddcfDNA. Nevertheless, the EMB procedure itself might also induce injury and therefore a rise in ddcfDNA values. Here, the effect of the EMB procedure was evaluated by pairwise comparing ddcfDNA values before EMB to ddcfDNA values after EMB. <h3>Methods</h3> Plasma samples from in total 15 heart transplant recipients were obtained pre-EMB and post-EMB. A droplet digital PCR (ddPCR) method was used for measuring ddcfDNA, making use of single-nucleotide polymorphisms (SNPs) that allowed both absolute quantification in copies/mL as well as relative quantification of ddcfDNA (ddcfDNA% of total cfDNA). <h3>Results</h3> The median (IQR) pre-EMB ddcfDNA concentration was 7.5 (3.0-14.5) copies/mL and increased significantly to 9.6 (5.4-20.8) copies/mL post-EMB, corresponding to a 1.28-fold increase (n=113; p=0.007). DdcfDNA% increased significantly from 0.08% (0.00-0.14) pre-EMB to 0.10% (0.02-0.20) post-EMB, corresponding to a 1.31-fold increase in ddcfDNA% (n=113; p=0.03). DdcfDNA concentration and ddcfDNA% demonstrated a comparable fold increase after the EMB procedure. This suggests that variations in total recipient cfDNA due to the EMB procedure is small or even absent. <h3>Conclusion</h3> The EMB procedure causes iatrogenic injury to the heart allograft that results in an increase in ddcfDNA% and ddcfDNA concentration. For the assessment of ddcfDNA as marker for AR in heart transplant recipients, collection of plasma samples before the EMB procedure is therefore essential.

Elevated Cell Free DNA in Respiratory Viral Infection and Associated Lung Allograft Dysfunction

<h3>Purpose</h3> Respiratory viral infection (RVI) is common in lung transplant recipients (LTRs), and a risk for subsequent chronic lung allograft dysfunction (CLAD). Approaches to identify such high risk for post RVI - CLAD are lacking. Our prior research showed viruses thought to be high risk are associated with elevated donor-derived cell-free DNA (%ddcfDNA), a sensitive marker of allograft injury. We hypothesize that high risk RVI are associated with greater allograft injury. We assessed if %ddcfDNA identifies events in which RVI leads to decline in lung function. <h3>Methods</h3> This study included 39 LTRs enrolled in the prospective Genomic Research Alliance for Transplantation with RVI from January 2016 - March 2019 and 1 year of follow up. Serially collected plasma samples were assayed for %ddcfDNA by shotgun sequencing. We defined significant allograft injury as %ddcfDNA values within 7 days of RVI ≥ 1 % (cases) or < 1% (controls). We examined type of RVI, concurrent histopathology, and lung function decline defined as percent change from personal best. <h3>Results</h3> We identified 59 RVI events at a median of 274 days post-transplant (IQR: 142, 425) including 38 controls (%ddcfDNA <1%) and 21 cases (%ddcfDNA ≥ 1%) (medians 0.47% and 3.30%, <i>p < 0.001*</i>). Demographics, personal best FEV₁, and histopathology were not different between cases and controls. At RVI diagnosis, cases showed significantly greater %FEV₁ decline than controls (medians -13.38% and -1.83%, <i>p = 0.006*).</i> Cases also had significantly greater %FEV₁ decline at 90 (medians -7.97% and 0.91%, <i>p = 0.04*</i>) and 365 (medians -20.05% and 1.09%, <i>p = 0.04*</i>) days post RVI. In contrast, no difference in %FEV₁ decline was seen at any timepoint when RVIs were grouped by abnormal (n=17) vs normal (n=27) histopathology. <h3>Conclusion</h3> We found that in subjects experiencing RVI, %ddcfDNA may discriminate between those who will recover their lung function and those who will experience sustained decline, a predictive utility not seen with histopathology or pulmonary function testing.

Performance of Donor-Derived Cell-Free DNA (dd-cfDNA) for Discriminating Acute Lung Allograft Injury in a Multicenter Cohort

Purpose Histological patterns of acute allograft injury including perivascular acute rejection (AR), lymphocytic bronchiolitis (LB), organizing pneumonia (OP) and acute lung injury (ALI) are frequent after lung transplant and have been associated with worse outcomes. Circulating dd-cfDNA has been proposed as a biomarker to discriminate acute allograft injury in lung recipients. We sought to determine the performance characteristics of three plasma dd-cfDNA thresholds for distinguishing acute lung allograft injury in a prospective, multicenter cohort. Methods The cohort was drawn from the Clinical Trials in Organ Transplantation-20 study and comprised 126 first adult lung recipients with 320 plasma samples that paired with a lung biopsy performed early within the first posttransplant year. Total cfDNA was isolated from ∼1mL of plasma using automated nucleic acid purification and the dd-cfDNA fraction determined at CareDx using next generation sequencing and validated bioinformatics. Biopsies were binned into typical sampling timeframes of 1, 3, and 6 months posttransplant. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for discriminating between a biopsy with vs. without acute allograft injury (defined as any of AR, LB, OP or ALI) was estimated at each timeframe for each of the following dd-cfDNA thresholds; 0.5%, 0.85%, and 1.0%. Results The Table reports the performance characteristics of each dd-cfDNA threshold at each early posttransplant timeframe. At 1month posttransplant the NPV, indicating the probability of a biopsy without acute allograft injury having a concurrent plasma dd-cfDNA value below the threshold, was poor for all evaluated thresholds. However, by 6months posttransplant the NPV improved, in particular for the 0.5% threshold. Conclusion In this prospective real-world cohort, a dd-cfDNA threshold of 0.5% achieved a high NPV for acute histological allograft injury at 6 months posttransplant. Histological patterns of acute allograft injury including perivascular acute rejection (AR), lymphocytic bronchiolitis (LB), organizing pneumonia (OP) and acute lung injury (ALI) are frequent after lung transplant and have been associated with worse outcomes. Circulating dd-cfDNA has been proposed as a biomarker to discriminate acute allograft injury in lung recipients. We sought to determine the performance characteristics of three plasma dd-cfDNA thresholds for distinguishing acute lung allograft injury in a prospective, multicenter cohort. The cohort was drawn from the Clinical Trials in Organ Transplantation-20 study and comprised 126 first adult lung recipients with 320 plasma samples that paired with a lung biopsy performed early within the first posttransplant year. Total cfDNA was isolated from ∼1mL of plasma using automated nucleic acid purification and the dd-cfDNA fraction determined at CareDx using next generation sequencing and validated bioinformatics. Biopsies were binned into typical sampling timeframes of 1, 3, and 6 months posttransplant. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for discriminating between a biopsy with vs. without acute allograft injury (defined as any of AR, LB, OP or ALI) was estimated at each timeframe for each of the following dd-cfDNA thresholds; 0.5%, 0.85%, and 1.0%. The Table reports the performance characteristics of each dd-cfDNA threshold at each early posttransplant timeframe. At 1month posttransplant the NPV, indicating the probability of a biopsy without acute allograft injury having a concurrent plasma dd-cfDNA value below the threshold, was poor for all evaluated thresholds. However, by 6months posttransplant the NPV improved, in particular for the 0.5% threshold. In this prospective real-world cohort, a dd-cfDNA threshold of 0.5% achieved a high NPV for acute histological allograft injury at 6 months posttransplant.

Bronchoalveolar Lavage Fluid (BALF) as a Compartment for Donor-Derived Cell-Free DNA (dd-cfDNA) Assessment in Lung Recipients

<h3>Purpose</h3> Circulating dd-cfDNA has been proposed as a biomarker to discriminate acute lung rejection. It is unknown whether BALF may represent a more sensitive compartment for dd-cfDNA analysis in lung recipients. Additionally, the relative contribution of the recipient or donor to the BALF cfDNA content remains uncertain. We sought to understand the feasibility of distinguishing dd-cfDNA in the BALF and determine the greatest contributor of BALF cfDNA. <h3>Methods</h3> The cohort was drawn from the Clinical Trials in Organ Transplantation-20 study and comprised 126 first adult lung recipients with paired BALF and plasma samples (n=320 each). Total cfDNA was isolated from ∼1mL of sample using automated nucleic acid purification. The % cfDNA contributed by the donor vs. recipient was distinguished in plasma and BALF samples using a next generation sequencing (NGS) assay that measures single nucleotide polymorphisms (SNPs) followed by bioinformatics algorithms. Because these algorithms were developed on plasma, for each BALF sample the SNP typing from the paired plasma sample was used to verify the accuracy of the donor vs. recipient cfDNA contribution in the BALF. <h3>Results</h3> Applying standard QC filters, 168 BALF samples "passed" and generated a dd-cfDNA result while 96 "failed" and did not generate a dd-cfDNA result. 45 samples yielded insufficient total cfDNA to support NGS while 11 generated a potentially invalid dd-cfDNA result. To understand reasons underlying inability to estimate BALF dd-cfDNA, we compared the volume of BALF used for total cfDNA isolation and the total cfDNA yield from the BALF in passed vs. failed samples. The BALF volume was similar between these groups, however the total cfDNA yield was notably less among samples that failed (median total cfDNA 0.38ng/uL in failed vs. 1.42ng/uL in passed samples). Considering only passed BALF samples, the median (25^th, 75^th percentile) fraction of cfDNA contributed by the donor was 6.95% (2.40%, 14.00%), with the remainder contributed by the recipient. <h3>Conclusion</h3> Our data point to challenges in consistently generating dd-cfDNA data from the BALF and suggest the dilute nature of BALF may be a contributing factor. Additionally, these results indicate that the recipient, rather than the donor, is the largest contributor to the BALF cfDNA content potentially reflecting intragraft recipient immune cells.

Use of a donor-derived cell-free DNA assay to monitor treatment response in pediatric renal transplant recipients with allograft rejection.

Detection of donor-derived cell-free DNA (dd-cfDNA) reliably identifies allograft rejection in pediatric and adult kidney transplant (KT) recipients. Here, we evaluate the utility of dd-cfDNA for monitoring response to treatment among pediatric renal transplant recipients suffering graft rejection. 58 pediatric transplant recipients were enrolled between April 2018 and March 2020 and underwent initial dd-cfDNA testing to monitor for rejection. Allograft biopsy was performed for dd-cfDNA scores >1.0%. Patients with histologically proven rejection formed the study cohort and underwent appropriate treatment. Results of dd-cfDNA, serum creatinine (SCr), biopsy findings, and treatment outcomes were evaluated. Standard statistical analyses were applied. Nineteen of 58 (31%) patients had dd-cfDNA score >1.0%, of which 18 (94.7%) had biopsy-proven rejection. Median dd-cfDNA value was 1.90% (interquartile range 1.43%-3.23%), and biopsy results showed 11 patients (61.1%) with antibody-mediated rejection (AMR), 2 patients (11.1%) with T-cell mediated rejection (TCMR), and 5 patients (27.7%) with mixed AMR/TCMR. SCr at time of biopsy was 1.28±1.09mg/dl. Following treatment, dd-cfDNA scores decreased for all types of rejection but still remained >1.0% in both AMR (1.50% [0.90%-3.10%]) and mixed (1.40% [0.95%-4.15%]) groups. Repeat dd-cfDNA values were <1.0% for patients with TCMR (0.20%-0.28%). SCr showed minimal change from pre-treatment levels regardless of rejection subtype. Patients with TCMR may be reliably followed by dd-cfDNA; however, it remains unclear whether persistently elevated dd-cfDNA levels in AMR is a reflection of ongoing subclinical rejection or an inherent limitation of the assay's utility.