Donor-derived Cell-free dd-cfDNA Research Articles

Diagnostic value of donor-derived cell-free DNA to predict antibody-mediated rejection in donor-specific antibody-positive renal allograft recipients.

SummaryCirculating donor‐specific antibodies (DSA) do not necessarily indicate antibody‐mediated rejection (ABMR). Here, we evaluated the diagnostic value of donor‐derived cell‐free DNA (dd‐cfDNA) as an add‐on to DSA detection. The study included two independent cohorts of DSA+ kidney allograft recipients, 45 subclinical cases identified by cross‐sectional antibody screening (cohort 1), and 30 recipients subjected to indication biopsies (cohort 2). About 50% of the DSA+ recipients had ABMR and displayed higher dd‐cfDNA levels than DSA+ABMR− recipients (cohort 1: 1.90% [median; IQR: 0.78–3.90%] vs. 0.52% [0.35–0.72%]; P < 0.001); (cohort 2: 1.20% [0.82–2.50%] vs. 0.59% [0.28–2.05%]; P = 0.086). Receiver operating characteristic (ROC) analysis revealed an area under the curve (AUC) of 0.89 and 0.69 for dd‐cfDNA, and 0.88 and 0.77 for DSA mean fluorescence intensity (MFI), respectively. In combined models, adding dd‐cfDNA to DSA‐MFI or vice versa significantly improved the diagnostic accuracy. Limited diagnostic performance of dd‐cfDNA in cohort 2 was related to the frequent finding of other types of graft injury among ABMR− recipients, like T cell‐mediated rejection or glomerulonephritis. For dd‐cfDNA in relation to injury of any cause an AUC of 0.97 was calculated. Monitoring of dd‐cfDNA in DSA+ patients may be a useful tool to detect ABMR and other types of injury.

Early findings after integration of donor-derived cell-free DNA into clinical care following pediatric heart transplantation.

Endomyocardial biopsy (EMB) is costly and discomforting yet remains a key component of surveillance after pediatric heart transplantation (HT). Donor-derived cell-free DNA (dd-cfDNA) has been histologically validated with high negative predictive value, offering an alternative to surveillance EMB (sEMB). We implemented an alternative surveillance protocol using commercially available dd-cfDNA assays in place of sEMB after pediatric HT. Recipients ≧7months post-HT with reassuring clinical assessment were referred for dd-cfDNA. When not elevated above the manufacturers' threshold, sEMB was deferred. Subsequent clinical status and results of follow-up EMB were analyzed. Over 17months, 58 recipients [34% female, median age at HT 3.1years (IQR 0.6-10.6)] had dd-cfDNA assessed per protocol. Median age was 14.8years (8.4-18.3) and time from HT 6.0years (2.2-11.2). Forty-seven (81%) had non-elevated dd-cfDNA and 11 (19%) were elevated. During a median of 8.7months (4.2-15), all are alive without allograft loss/new dysfunction. Among those with non-elevated dd-cfDNA, 24 (51%) had subsequent sEMB at 12.1months (6.9-12.9) with 23showing no acute rejection (AR): grade 0R/pAMR0 (n=16); 1R(1A)/pAMR0 (n=7). One had AR (grade 2R(3A)/pAMR0) on follow-up sEMB after decreased immunosuppression following a diagnosis of PTLD. All 11 with elevated dd-cfDNA had reflex EMB at 19days (12-32) with AR in 4: grade 1R(1B-2)/pAMR0 (n=3); 1R(1B)/pAMR2 (n=1). dd-cfDNA assessment in place of selected, per-protocol EMB decreased surveillance EMB by 81% in our pediatric HT recipient cohort with no short-term adverse outcomes. Individual center approach to surveillance EMB will influence the utility of these findings.

Association between dd-cfDNA levels, de novo donor specific antibodies, and eGFR decline: An analysis of the DART cohort.

Donor-derived cell-free DNA (dd-cfDNA) is a marker of allograft injury in transplant recipients; however, the relationship between dd-cfDNA and other clinical parameters associated with adverse allograft outcomes is not well-characterized. We performed a retrospective analysis of kidney transplant recipients from the DART cohort (ClinicalTrials.gov Identifier: NCT02424227) to evaluate the associations between eGFR decline, de novo donor-specific antibodies (dnDSA), and dd-cfDNA. Both elevated dd-cfDNA (≥1%) and dd-cfDNA variability (≥.34%) in the first post-transplant year were associated with decline in eGFR ≥25% in the second year (21.4%vs. 4.1%, P=.005; 25%vs. 3.6%, P=.002, respectively). Compared to samples from DSA negative patients, samples from patients with concurrent de novo HLA DSAs had higher dd-cfDNA levels (P<.0001). Abnormalities in dd-cfDNA levels are associated with clinical parameters commonly used as surrogate endpoints for adverse allograft outcomes, raising the possibility that molecular injury as characterized by dd-cfDNA could help identify patients at risk of these outcomes.

Longitudinal variance of Donor-Derived Cell-Free DNA (dd-cfDNA) in Stable Kidney Transplant (KTx) patients are influenced by donor/recipient variables.

The longitudinal time-course of dd-cfDNA after kidney transplant (KTx) is not well-described. The cut off values of dd-cfDNA in KTx derive from biopsy-coupled single measurements. Meaningful interpretation necessitates understanding of: (1) time variance of dd-cfDNA levels post-KTx, (2) factors determining biologic variability, and (3) relationship to donor and recipient characteristics. We hypothesized that an understanding of the aforementioned factors would better inform clinical decision-making using dd-cfDNA. One hundred and twenty five KTx patients with dd-cfDNA obtained longitudinally were included. Univariate analyses were directed at inter-patient variability and intra-patient inter-occasion variability of dd-cfDNA. Multivariate linear regression was used in analyses accounting for repeat measures. At 1-month post KTx median dd-cfDNA: (1) were higher in repeat KTx (.57%, P<.001), and dual KTx (1.10%, P=ns) versus a first KTx (.31%); (2) showed a significant difference in donor after cardiac death (DCD [.45%]) versus living related (LRD [.27%]) donors (P=.036). Longitudinal (1-3months) dd-cfDNA measurements showed a significant downtrend for all donor types. Panel-reactive antibodies (PRA) were positively correlated with dd-cfDNA. Repeat Tx, dual Tx, DCD, and PRA are associated with a higher dd-cfDNA. Incorporation of donor/recipient variables and time down post transplant is material for rational interpretation of dd-cfDNA.

Clinical Rationale for a Routine Testing Schedule Using Donor-Derived Cell-Free DNA After Kidney Transplantation.

Kidney transplant recipients require meticulous clinical and laboratory surveillance to monitor allograft health. Conventional biomarkers, including serum creatinine and proteinuria, are lagging indicators of allograft injury, often rising only after significant and potentially irreversible damage has occurred. Immunosuppressive medication levels can be followed, but their utility is largely limited to guiding dosing changes or assessing adherence. Kidney biopsy, the criterion standard for the diagnosis and characterization of injury, is invasive and thus poorly suited for frequent surveillance.Donor-derived cell-free DNA (dd-cfDNA) is a sensitive, noninvasive, leading indicator of allograft injury, which offers the opportunity for expedited intervention and can improve long-term allograft outcomes. This article describes the clinical rationale for a routine testing schedule utilizing dd-cfDNA surveillance at months 1, 2, 3, 4, 6, 9, and 12 during the first year following kidney transplantation and quarterly thereafter. These time points coincide with major immunologic transition points after transplantation and provide clinicians with molecular information to help inform decision making.

Donor derived cell free DNA% is elevated with pathogens that are risk factors for acute and chronic lung allograft injury

Acute and chronic forms of lung allograft injury are associated with specific respiratory pathogens. Donor-derived cell free DNA (ddcfDNA) has been shown to be elevated with acute lung allograft injury and predictive of long-term outcomes. We examined the %ddcfDNA values at times of microbial isolation from bronchoalveolar lavage (BAL). Two hundred and six BAL samples from 51 Lung Transplant Recipients (LTRs) with concurrently available plasma %ddcfDNA were analyzed along with microbiology and histopathology. Microbial species were grouped into bacterial, fungal, and viral and "higher risk" and "lower risk" cohorts based on historical association with downstream allograft dysfunction. Analyses were performed to determine pathogen category association with %ddcfDNA, independent of inter-subject variability. Presence of microbial isolates in BAL was not associated with elevated %ddcfDNA compared to samples without isolates. However, "higher risk" bacterial and viral microbes showed greater %ddcfDNA values than lower risk species (1.19% vs. 0.65%, p < 0.01), independent of inter-subject variability. Histopathologic abnormalities concurrent with pathogen isolation were associated with higher %ddcfDNA compared to isolation episodes with normal histopathology (medians 1.23% and 0.66%, p=0.05). Assessments showed no evidence of correlation between histopathology or bronchoscopy indication and presence of higher risk vs. lower risk pathogens. %ddcfDNA is higher among cases of microbial isolation with concurrent abnormal histopathology and with isolation of higher risk pathogens known to increase risk of allograft dysfunction. Future studies should assess if %ddcfDNA can be used to stratify pathogens for risk of CLAD and identify pathogen associated injury prior to histopathology.

Donor-derived Cell-free DNA Kinetics Post-kidney Transplant Biopsy.

Background.Donor-derived cell-free DNA (dd-cfDNA) has generated interest as a biomarker for kidney injury including transplant (KT) rejection. It is possible that the KT biopsy procedure can cause the release of dd-cfDNA, therefore affecting the reliability of this assay in the postbiopsy period. We evaluated the effect of KT biopsy on the kinetics of dd-cfDNA.Methods.We conducted a single-arm prospective study. Samples were collected from 16 adult KT recipients undergoing KT biopsy. All participants had samples drawn within 8 h before the biopsy (prebiopsy), within 20 min (hour 0), 2 h (hour 2), and 24–48 h (hours 24–48) after the biopsy. We evaluated the change in dd-cfDNA from the prebiopsy time point to the following 3 time points after the biopsy.Results.At hour 0 and hour 2, there was a significantly larger log dd-cfDNA mean score compared with the prebiopsy score (least square mean estimate 0.4 [0.17-0.63] and 0.39 [0.09-0.68], respectively). By 24–28 h postbiopsy, there was no significant difference in log dd-cfDNA mean score compared with the prebiopsy score (least square mean estimate −0.21 [−0.6 to 0.19]).Conclusions.Mechanical injury from a KT biopsy can transiently increase circulating dd-cfDNA. The increase resolves by 24–48 h after the biopsy. Providers should wait 48 h postbiopsy to obtain dd-cfDNA levels to establish the correct baseline to be used for monitoring.

Liquid biopsies: donor-derived cell-free DNA for the detection of kidney allograft injury.

In kidney transplantation, the use of minimally invasive damage biomarkers that are more sensitive and specific than plasma creatinine will be crucial to enable early, actionable detection or exclusion of structural kidney damage due to acute or chronic rejection. Donor-derived cell-free DNA (dd-cfDNA), which can be quantified, for example, through next-generation sequencing, droplet digital PCR and quantitative PCR, is a candidate biomarker with great potential for enabling comprehensive monitoring of allograft injury. dd-cfDNA has a favourable overall diagnostic performance for the detection of rejection and its high negative predictive value might be especially useful for avoiding unnecessary biopsies. Elevated dd-cfDNA levels have been shown to be detectable before graft injury can be clinically identified using current diagnostic methods. Moreover, dd-cfDNA falls rapidly to baseline levels after successful treatment for rejection owing to its short half-life. dd-cfDNA can detect graft injury caused by immune activation owing to insufficient immunosuppression and might therefore also help guide immunosuppression dosing. The fractional abundance of dd-cfDNA can be affected by changes in the recipient cfDNA (for example, due to infection or physical exercise) but the use of absolute quantification of dd-cfDNA overcomes this limitation. Serial dd-cfDNA determinations might therefore facilitate cost-effective personalized clinical management of kidney transplant recipients to reduce premature graft loss.

HLA-DRB1 mismatch-based identification of donor-derived cell free DNA (dd-cfDNA) as a marker of rejection in heart transplant recipients: A single-institution pilot study

Donor-derived cell-free DNA (dd-cfDNA) is considered a reliable marker of organ damage with potential applications in the follow-up of transplant recipients. In this work we present an assay based on the donor-recipient HLA-mismatch (human leukocyte antigen) at the HLA-DRB1 locus to monitor rejection by quantifying the percentage of dd-cfDNA using a droplet digital PCR (polymerase chain reaction) technique. A panel of probes targeting the HLA-DRB1 locus and covering >85% genetic variability was validated and used to assess dd-cfDNA levels in a prospective cohort of 19 adult heart transplant recipients (mean age 50.9±14.8 years). The assay was carried out on a total of 232 liquid biopsies collected at the same time as endomyocardial biopsy (EMB) during routine post-transplant follow-up. Results show a significant increase of dd-cfDNA related to ischemia-reperfusion injury (2.22±2.09%) and to acute cellular rejection (1.71±3.10%) compared to stable conditions (0.43±1.04%, p < 0.0001). On the contrary, no increase was observed during infections or vascular complications, underlining the potential role of this biomarker for rejection monitoring. With a cut-off of 0.11%, the test showed 70.8% specificity (95% CI, 58.17% - 81.40%) and 64.2% sensitivity (95% CI, 49.80% - 76.86%) in discriminating acute rejection from no rejection. These data demonstrate that this HLA mismatch-based droplet digital PCR method is effective for monitoring rejection in heart transplant recipients. Compared to next generation sequencing approaches, it is far more flexible, less expensive and provides faster results.

Donor-Derived Cell-Free DNA in Kidney Transplantation: Origins, Present and a Look to the Future.

Since its first detection in 1948, donor-derived cell-free DNA (dd-cfDNA) has been employed for a myriad of indications in various medical specialties. It has had a far-reaching impact in solid organ transplantation, with the most widespread utilization in kidney transplantation for the surveillance and detection of allograft rejection. The purpose of this review is to track the arc of this revolutionary test—from origins to current use—along with examining challenges and future prospects though the lens of transplant nephrology.

Donor-derived cell-free DNA: An independent biomarker in kidney transplant patients with antibody-mediated rejection

IntroductionAntibody-mediated rejection (ABMR) is a major cause of kidney transplant failure which requires donor-specific antibodies (DSA) for a definitive diagnosis. Donor-derived cell-free DNA (ddcfDNA) is an emerging biomarker used to assess kidney allograft injury. However, current data is limited to predict the accuracy of ddcfDNA in ABMR diagnosis. This study was conducted to compare the performance of DSA with plasma ddcfDNA for the diagnosis of ABMR. MethodsIn this retrospective single-center observational study, we enrolled 50 kidney transplant recipients who were diagnosed with the suspicion of rejection between June 2018 and May 2019 at the Jinling Hospital. Plasma ddcfDNA was measured by using a novel target region capture sequencing methodology. A total of 37 patients who were tested with DSA and biopsy were divided into four subgroups (ABMR+/DSA+, ABMR+/DSA-, ABMR-/DSA+, ABMR-/DSA-) for the distribution of ddcfDNA (%) by ABMR and DSA. ResultsThe median level of ddcfDNA in biopsy showed that the ABMR group (1.66%, IQR 1.34–3.76%) was significantly higher than the median level (0.63%, IQR 0.43–0.74%) in non-ABMR (p < 0.001). With a ddcfDNA cutoff of 0.96%, the AUC was 0.90 (95%CI, 0.86–0.95), which was associated with a sensitivity of 90.5% (95%CI, 69.6–98.8%) and specificity of 96.6% (95%CI, 82.2–100%), a PPV of 95% (95%CI, 73.4–99.2%) and NPV of 93.3% (95%CI, 78.9–98.1%) were also observed. Among the four subgroups, ddcfDNA had no significant difference in both DSA+ group and DSA-group (p > 0.05). In the diagnosis of ABMR, the specificity, sensitivity, PPV and NPV of DSA were 50%, 74.1%, 41.7%, 80%, respectively. ConclusionsddcfDNA levels in the blood could highly distinguish (biopsy-supported) ABMR occurrence, irrespective of whether this method is accompanied by DSA or not.

Donor-Derived Cell-Free DNA (ddcf-DNA) and Acute Antibody-Mediated Rejection in Kidney Transplantation.

Monitoring kidney transplant recipients for evidence of allograft rejection is essential to lower the risk of graft loss. The traditional method relies on serial checks in serum creatinine with a biopsy of the allograft if dysfunction is suspected. This is invasive, labor-intensive and costly. As such, there is widespread interest in the use of biomarkers to provide a noninvasive approach to detecting allograft rejection. One such biomarker is donor-derived cell-free DNA (ddcf-DNA). Here, we review the methodology for the determination of the amount/fraction of ddcf-DNA, evaluate the available data of its use in kidney transplantation and render an opinion in the clinical decision-making of these patients.

Donor-derived cell-free DNA accurately detects acute rejection in lung transplant patients, a multicenter cohort study

Acute rejection, which includes antibody-mediated rejection and acute cellular rejection, is a risk factor for lung allograft loss. Lung transplant patients often undergo surveillance transbronchial biopsies to detect and treat acute rejection before irreversible chronic rejection develops. Limitations of this approach include its invasiveness and high interobserver variability. We tested the performance of percent donor-derived cell-free DNA (%ddcfDNA), a non-invasive blood test, to detect acute rejection. This multicenter cohort study monitored 148 lung transplant subjects over a median of 19.6 months. We collected serial plasma samples contemporaneously with TBBx to measure %ddcfDNA. Clinical data was collected to adjudicate for acute rejection. The primary analysis consisted of computing the area-under-the-receiver-operating-characteristic-curve of %ddcfDNA to detect acute rejection. Secondary analysis determined %ddcfDNA rule-out thresholds for acute rejection. ddcfDNA levels were high after transplant surgery and decayed logarithmically. With acute rejection, ddcfDNA levels rose six-fold higher than controls. ddcfDNA levels also correlated with severity of lung function decline and histological grading of rejection. %ddcfDNA area-under-the-receiver-operating-characteristic-curve for acute rejection, AMR, and ACR were 0.89, 0.93, and 0.83, respectively. ddcfDNA levels of <0.5% and <1.0% showed a negative predictive value of 96% and 90% for acute rejection, respectively. Histopathology detected one-third of episodes with ddcfDNA levels ≥1.0%, even though >90% of these events were coincident to clinical complications missed by histopathology. This study demonstrates that %ddcfDNA reliably detects acute rejection and other clinical complications potentially missed by histopathology, lending support to its use as a non-invasive marker of allograft injury.

Extremely High Cell-free DNA Levels Observed in Renal Allograft Patient With SARS-CoV-2 Infection.

Beyond its widely recognized morbidity and mortality, coronavirus disease 2019 poses an additional health risk to renal allograft recipients. Detection and measurement of donor-derived cell-free DNA (dd-cfDNA), expressed as a fraction of the total cell-free DNA (cfDNA), has emerged as a noninvasive biomarker for allograft rejection. Here, we present a case report of a patient who was infected with severe acute respiratory syndrome coronavirus 2, 11 mo post–kidney transplant. The patient was serially monitored using an analytically and clinically validated massively multiplex PCR-based dd-cfDNA assay to assess allograft injury and risk for rejection. Over the course of infection, low dd-cfDNA fractions were observed (below the 1% cutoff) and were accompanied by unusually highly elevated levels of total cfDNA, which gradually declined as the infection resolved. The case study highlights the variability in total cfDNA levels during and after viral infection, and the need to consider both total and dd-cfDNA levels when clinically interpreting the results for allograft rejection. Furthermore, the study highlights the importance of serial testing, wherein an interplay between total cfDNA and dd-cfDNA can inform the optimization of a patient’s immunosuppressive treatment regimen in response to infection.

Identification of cell-free DNA methylation patterns unique to the human left ventricle as a potential indicator of acute cellular rejection.

Increased levels of donor-derived cell-free DNA (dd-cfDNA) in recipient plasma have been associated with rejection after transplantation. DNA sequence differences have been used to distinguish between donor and recipient, but epigenetic differences could also potentially identify dd-cfDNA. This pilot study aimed to identify ventricle-specific differentially methylated regions of DNA (DMRs) that could be detected in cfDNA. We identified 24 ventricle-specific DMRs and chose two for further study, one on chromosome 9 and one on chromosome 12. The specificity of both DMRs for the left ventricle was confirmed using genomic DNA from multiple human tissues. Serial matched samples of myocardium (n=33) and plasma (n=24) were collected from stable adult heart transplant recipients undergoing routine endomyocardial biopsy for rejection surveillance. Plasma DMR levels increased with biopsy-proven rejection grade for individual patients. Mean cellular apoptosis in biopsy samples increased significantly with rejection severity (2.4%, 4.4% and 10.0% for ACR 0R, 1R, and 2R, respectively) but did not show a consistent relationship with DMR levels. We identified multiple DNA methylation patterns unique to the human ventricle and conclude that epigenetic differences in cfDNA populations represent a promising alternative strategy for the non-invasive detection of rejection.

Dynamic Response of Donor-Derived Cell-Free DNA Following Treatment of Acute Rejection in Kidney Allografts.

The quantification of rejection treatment efficacy has been insufficient using traditional markers due, in part, to the lagging response of serum creatinine and histologic alterations on biopsy specimens. Donor-derived cell-free DNA (dd-cfDNA) is a molecular marker of injury that may assess allograft injury after rejection. Retrospective review of the DART study identified 70 patients who had a clinically indicated biopsy, simultaneous dd-cfDNA measurement, and at least one follow-up dd-cfDNA within 3 months post-treatment. Thirty-five patients had no biopsy-proven rejection and no rejection treatment (NR), 16 patients had no biopsy-proven rejection but did receive rejection treatment (CR), 9 patients had diagnosis of ABMR/mixed rejection on biopsy and received rejection treatment (ABMR), and 10 patients had diagnosis of TCMR and received rejection treatment (TCMR). The CR, ABMR, and TCMR groups combined to form a rejection (R) group. In the R group, median dd-cfDNA values at baseline and 1 month were 0.62% and 0.35% (n=21 pairs, p=0.34), and at baseline and 2-3 months were 0.77% and 0.21% (n=23 pairs, p=0.002). In TCMR, median dd-cfDNA values at baseline and 1 month were 1.13% and 0.37% (n=5 pairs, p=0.63), and at baseline and 2-3 months were 0.25% and 0.12% (n=9 pairs, p=0.004). In ABMR, median dd-cfDNA values at baseline and 1 month were 1.61% and 1.2 % (n=6 pairs, p>0.99), and at baseline and 2-3 months were 3.85% and 1.32% (n=6 pairs, p=0.09). In CR, median dd-cfDNA values at baseline and 1 month were 0.31% and 0.29% (n=10 pairs, p=0.38), and at baseline and 2-3 months were 0.38% and 0.17% (n=8 pairs, p=0.31). Lastly, in NR, median dd-cfDNA values at baseline and 1 month were 0.23% and 0.18% (n=21 pairs, p=0.10), and at baseline and 2-3 months were 0.33% and 0.17% (n=26 pairs, p=0.003). Changes in serum creatinine across 1 month and 2-3 months following rejection were similar. dd-cfDNA may be a useful dynamic biomarker to assess the health of the kidney allograft following rejection treatment.

Donor Derived Cell Free DNA as a Risk Factor for Initiating De-Novo Donor Specific Antibodies in Heart Transplantation

PurposeWe hypothesize that subclinical inflammation, resulting in molecular injury to the cardiac allograft, may contribute to sensitization of the recipients’ immune system. Antigenic presentation of donor-derived nucleic acids, as reflected by elevated donor derived cell free DNA (dd-cfDNA) levels, may lead to leukocyte activation, promoting cytokine release and donor specific antibody (DSA) formation, with elevated dd-cfDNA levels seen prior to detection of de-novo DSAs in kidney and lung transplantation. We aimed to assess the relationship between DSA and dd-cfDNA in cardiac transplantation.MethodsThis was a retrospective cohort analysis examining a subset of the Donor‐Derived Cell‐Free DNA‐Outcomes AlloMap Registry (D‐OAR) data, focusing on dd‐cfDNA and DSA results. 67 patients were identified, providing 284 samples, with corresponding dd-cfDNA (AlloSure) and DSA results. Samples that had associated rejection were excluded.ResultsOf the 67 patients, 28 (42%) had negative DSA, with a median dd-cfDNA = 0.06% (IQR 0.03% - 0.08%), 27 patients (40%) had pre-formed DSA with a median dd-cfDNA = 0.09% (IQR 0.06% - 0.18%) and 12 patients (18%) had de-novo DSA formation, median dd-cfDNA = 0.34%, (IQR 0.11% - 0.45%) [Figure]. DSA negative patients had significantly lower dd-cfDNA levels than patients who were DSA positive (p< 0.001), while patients with de-novo DSA had significantly higher dd-cfDNA than patients who had pre-formed DSA. (p= 0.001). Elevations in dd-cfDNA appeared a median of 20 days (range 2-28) prior to DSA testing. Multivariate logistic regression analysis identified dd-cfDNA an independent predictor of de-novo DSA formation, when controlling for recipient race, age, donor demographics and HLA mismatch (1-6) (p < 0.001).ConclusionHigher dd-cfDNA levels are associated with de-novo DSA formation. Further studies are required to determine whether this is a causal relationship and if dd-cfDNA acts as a trigger for DSA formation. We hypothesize that subclinical inflammation, resulting in molecular injury to the cardiac allograft, may contribute to sensitization of the recipients’ immune system. Antigenic presentation of donor-derived nucleic acids, as reflected by elevated donor derived cell free DNA (dd-cfDNA) levels, may lead to leukocyte activation, promoting cytokine release and donor specific antibody (DSA) formation, with elevated dd-cfDNA levels seen prior to detection of de-novo DSAs in kidney and lung transplantation. We aimed to assess the relationship between DSA and dd-cfDNA in cardiac transplantation. This was a retrospective cohort analysis examining a subset of the Donor‐Derived Cell‐Free DNA‐Outcomes AlloMap Registry (D‐OAR) data, focusing on dd‐cfDNA and DSA results. 67 patients were identified, providing 284 samples, with corresponding dd-cfDNA (AlloSure) and DSA results. Samples that had associated rejection were excluded. Of the 67 patients, 28 (42%) had negative DSA, with a median dd-cfDNA = 0.06% (IQR 0.03% - 0.08%), 27 patients (40%) had pre-formed DSA with a median dd-cfDNA = 0.09% (IQR 0.06% - 0.18%) and 12 patients (18%) had de-novo DSA formation, median dd-cfDNA = 0.34%, (IQR 0.11% - 0.45%) [Figure]. DSA negative patients had significantly lower dd-cfDNA levels than patients who were DSA positive (p< 0.001), while patients with de-novo DSA had significantly higher dd-cfDNA than patients who had pre-formed DSA. (p= 0.001). Elevations in dd-cfDNA appeared a median of 20 days (range 2-28) prior to DSA testing. Multivariate logistic regression analysis identified dd-cfDNA an independent predictor of de-novo DSA formation, when controlling for recipient race, age, donor demographics and HLA mismatch (1-6) (p < 0.001). Higher dd-cfDNA levels are associated with de-novo DSA formation. Further studies are required to determine whether this is a causal relationship and if dd-cfDNA acts as a trigger for DSA formation.

The Impact of Grade 1R Endomyocardial Biopsies on Donor-Derived Cell-Free DNA, Higher-Grade Rejection and Development of Donor Specific Antibodies

<h3>Purpose</h3> Recurrent episodes of mild rejection early post-transplant may lead to subsequent higher severity rejections and adverse outcomes, but there is a paucity of data on the prognosis of these patients. Elevations in donor-derived cell-free DNA (dd-cfDNA) and the development of donor specific antibodies (DSA) in kidney, heart, and lung transplantation have been described. We evaluated the association of AlloSure (dd-cfDNA) during episodes of acute cellular rejection (ACR) ISHLT Grade 1R with subsequent development of higher-grade rejections and DSA in heart transplantation. <h3>Methods</h3> This is a retrospective analysis of 484 patients who developed ACR Grade 1R (< 365 days post-transplant) paired with dd-cfDNA in the <i>Surveillance HeartCare® Outcomes Registry</i> (SHORE). dd-cfDNA levels were divided into 4 quartiles (median dd-cfDNA; Q1 = 0.10%, Q2 = 0.12%, Q3 = 0.15%, Q4 = 0.32%). The number of recurrent rejections (ISHLT Grade 2R/3R) and DSA were compared across quartiles using the Cochran-Armitage test (p<0.05). <h3>Results</h3> There were 933 episodes of Grade 1R biopsies. The median dd-cfDNA in 72 for-cause biopsies was 0.16% and in 860 surveillance biopsies was 0.12%, p=0.8979. Grade 1R rejection was not associated with later development of higher grades of ACR or dd-cfDNA quartiles (Table). Sixty-seven patients developed DSA subsequent to a 1R rejection and a higher incidence of DSA was associated with increased dd-cfDNA (median dd-cfDNA in Q1=8.7%, Q2=10.9%, Q3=18.9%, Q4=18.3%) (p = 0.006). <h3>Conclusion</h3> Grade 1R biopsies did not progress to higher grade rejections. There was no significant elevation in dd-cfDNA with Grade 1R biopsies. With increasing dd-cfDNA there was a significant trend toward DSA development following Grade 1R biopsies. Further analysis with longer follow-up to assess recurrent rejection and larger sample size of DSA is warranted.

Plasma Donor-Derived Cell-Free DNA Levels are Not Affected by Prednisone Dose nor Time after Heart Transplant: Pilot Data from DOAR and SHORE

<h3>Purpose</h3> Gene expression profiling (GEP; AlloMap®) scores can be impacted in the presence of >20 mg/day of prednisone due to effects on steroid-responsive genes (IL-1R2, ITGAM, FLT3) and is not validated for use prior to 55 days post-heart transplant. Donor-derived cell-free DNA (dd-cfDNA) can be used as early as 14 days after heart transplant (HT), but the impact of early use and the impact of steroid dosing has not yet been demonstrated. <h3>Methods</h3> The dd-cfDNA (AlloSure®) from the <i>Donor-Derived Cell-Free DNA Outcomes AlloMap Registry</i> (D-OAR) and <i>Surveillance Heart Care Outcome Registry</i> (SHORE) registries had 17 visits < 55 days and 106 visits 55 - 90 days post-HT. dd-cfDNA was also as analyzed by Kruskal-Wallis for prednisone dose ranges: 0-5, >5-10, >10-15, >15-20, and >20 mg/day (n=1113, 542, 203, 82, and 30). <h3>Results</h3> For those < 55 and >= 55 days post-HT the median dd-cfDNA result was 0.15% (IQR: 0.15-0.15%) and 0.15% (0.15-0.19%), respectively (p=NS). When stratified by prednisone doses (0-5], (5-10], (10-15], (15-20], and (>20, there was also no difference in median dd-cfDNA: 0.12%, 0.12%, 0.15%, 0.15%, and 0.15%, respectively, p=0.7. <h3>Conclusion</h3> This preliminary data suggests that dd-cfDNA levels are not significantly affected when assessed earlier than 55 days post-HT or by varying prednisone dosage. Implementation of dd-cfDNA as an early biomarker of allograft injury and rejection may represent a valuable clinical tool during the early post-HT period.

Racial Disparities in Gene Expression Profiling but Not Donor-Derived Cell-Free DNA after Heart Transplant

<h3>Purpose</h3> Recipient race remains a powerful risk factor for mortality after heart transplantation (HT), with African Americans (AA) having worse survival than Caucasians. The aim of this analysis was to evaluate racial differences in gene expression profiling (GEP) and donor derived cell free DNA (dd-cfDNA) after HT using the Surveillance HeartCare Outcomes Registry (SHORE). <h3>Methods</h3> SHORE is an ongoing registry with >1200 patients enrolled at 60 HT centers in the United States. The registry includes HT recipients aged ≥15 years and ≥55 days post-transplant who had routine GEP and dd-cfDNA testing for rejection surveillance. Since patient sampling is from multiple centers, a linear mixed effects model, an extension of the linear model, was used to accommodate both fixed and random effects in the data. <h3>Results</h3> Among 956 recipients included in the analysis, 198 (20.7%) were AA, median age 57.0 years [0, 74.0] and 73.9% male. Overall, median GEP values demonstrated a borderline difference (p=0.07, Kolmogorov Smirnov test) for AA versus non-AA patients (Figure). This is evident by the cumulative distribution function, where a slightly higher rise in slope is observed for the non-AA cohort. Using linear mixed effects models, there was a statistically significant difference in GEP score when grouped by months post-transplant, demonstrating an early difference between AA and non-AA patients between months 2-5, but not subsequently. For dd-cfDNA analysis, median values were similar between AA (0.12%; Range 0.1-3.9%) and non-AA (0.12%; 0.1-3.5%) patients within the first year post-transplant (p=0.87, 2-sided K-S test) (Figure). <h3>Conclusion</h3> AA race is associated with higher GEP scores early post-transplant. This may be related to differential expression of the MARCH8 and FLT3 genes following HT, as previously described. Further analysis of specific GEP clusters and covariates across racial groups may shed light on these observed differences. In contrast, dd-cfDNA values do not appear to be influenced by race.