dd-cfDNA Levels Research Articles

Donor-Derived Cell-Free DNA for Kidney Allograft Surveillance after Conversion to Belatacept: Prospective Pilot Study

Donor-derived cell-free DNA (dd-cfDNA) is used as a biomarker for detection of antibody-mediated rejection (ABMR) and other forms of graft injury. Another potential indication is guidance of immunosuppressive therapy when no therapeutic drug monitoring is available. In such situations, detection of patients with overt or subclinical graft injury is important to personalize immunosuppression. We prospectively measured dd-cfDNA in 22 kidney transplant recipients (KTR) over a period of 6 months after conversion to belatacept for clinical indication and assessed routine clinical parameters. Patient and graft survival was 100% after 6 months, and eGFR remained stable (28.7 vs. 31.1 mL/min/1.73 m2, p = 0.60). Out of 22 patients, 2 (9%) developed biopsy-proven rejection-one episode of low-grade TCMR IA and one episode of caABMR. While both episodes were detected by increase in creatinine, the caABMR episode led to increase in absolute dd-cfDNA (168 copies/mL) above the cut-off of 50 copies/mL, while the TCMR episode did show slightly increased relative dd-cfDNA (0.85%) despite normal absolute dd-cfDNA (22 copies/mL). Dd-cfDNA did not differ before and after conversion in a subgroup of 12 KTR with previous calcineurin inhibitor therapy and no rejection (12.5 vs. 25.3 copies/mL, p = 0.34). In this subgroup, 3/12 (25%) patients showed increase of absolute dd-cfDNA above the prespecified cut-off (50 copies/mL) despite improving eGFR. Increase in dd-cfDNA after conversion to belatacept is common and could point towards subclinical allograft injury. To detect subclinical TCMR changes without vascular lesions, additional biomarkers or urinary dd-cfDNA should complement plasma dd-cfDNA. Resolving CNI toxicity is unlikely to be detected by decreased dd-cfDNA levels. In summary, the sole determination of dd-cfDNA has limited utility in the guidance of patients after late conversion to belatacept. Further studies should focus on patients undergoing early conversion and include protocol biopsies at least for patients with increased dd-cfDNA.

An Initial Analysis of the Baseline Levels of Dd-cfDNA After Pancreas Transplantation: A Prospective Study From High-volume Centers in the United States.

One hundred eighty-one dd-cfDNA samples (n) were collected from 77 patients (N) up to 132 mo posttransplant. The median dd-cfDNA level among all subjects was 0.28% (0.13%, 0.71%). In simultaneous pancreas-kidney (SPK) transplant recipients, the median dd-cfDNA level was 0.29% (0.13%, 0.71%), and it was 0.23% (0.08%, 0.71%) in pancreas transplant alone (PTA) recipients. When isolating for when without infection or rejection, the median dd-cfDNA level was 0.28% (0.13%, 0.64%) for SPK and 0.20% (0.00%, 0.32%) for PTA. Both transplant types approached 1.0% ≤1 mo posttransplant followed by a decrease in median dd-cfDNA. During episodes of rejection or infection, median dd-cfDNA levels were greater among all transplant types. The mean dd-cfDNA level for all pancreas transplant recipients is <1.0%, consistent with the published kidney transplant rejection threshold (>1.0%), regardless of SPK or PTA. Early posttransplant dd-cfDNA levels are transiently higher than later measurements. Dd-cfDNA elevation also correlates with rejection and infection and thus is a promising biomarker for surveilling pancreas transplant dysfunction.

Droplet digital PCR-based testing for donor-derived cell-free DNA in transplanted patients as noninvasive marker of allograft health: Methodological aspects.

In solid organ transplantation, donor-derived cell-free DNA (dd-cfDNA) is a promising universal noninvasive biomarker for allograft health, where high levels of dd-cfDNA indicate organ damage. Using Droplet Digital PCR (ddPCR), we aimed to develop an assay setup for monitoring organ health. We aimed to identify the least distinguishable percentage-point increase in the fraction of minute amounts of cfDNA in a large cfDNA background by using assays targeting single nucleotide polymorphisms (SNPs). We mimicked a clinical sample from a recipient in a number of spike-in experiments, where cfDNA from healthy volunteers were mixed. A total of 40 assays were tested and approved by qPCR and ddPCR. Limit of detection (LOD) was demonstrated to be approximately 3 copies per reaction, observed at a fraction of 0.002%, and which would equal 6 copies per mL plasma. Limit of quantification (LOQ) was 35 copies per reaction, estimated to 0.038%. The lowest detectable increase in percentage point of dd-cfDNA was approximately 0.04%. Our results demonstrated that ddPCR has great sensitivity, high precision, and exceptional ability to quantify low levels of cfDNA. The ability to distinguish small differences in mimicking dd-cfDNA was far beyond the desired capability. While these methodological data are promising, further prospective studies are needed to determine the clinical utility of the proposed method.

Clinical features and allograft failure rates of pulmonary antibody-mediated rejection categories.

Pulmonary antibody-mediated rejection (AMR) consensus criteria categorize AMR by diagnostic certainty. This study aims to define the clinical features and associated outcomes of these recently defined AMR categories. Adjudication committees reviewed clinical data of 335 lung transplant recipients to define clinical or subclinical AMR based on the presence of allograft dysfunction, and the primary endpoints, time from transplant to allograft failure, a composite endpoint of chronic lung allograft dysfunction and/or death. Clinical AMR was subcategorized based on diagnostic certainty as definite, probable or possible AMR if 4, 3, or 2 characteristic features were present, respectively. Allograft injury was assessed via plasma donor-derived cell-free DNA (ddcfDNA). Risk of allograft failure and allograft injury was compared for AMR categories using regression models. Over the 38.5 months follow-up, 28.7% of subjects developed clinical AMR (n=96), 18.5% developed subclinical AMR (n=62) or 58.3% were no AMR (n=177). Clinical AMR showed higher risk of allograft failure and ddcfDNA levels compared to subclinical or no AMR. Clinical AMR included definite/probable (n=21) or possible AMR (n=75). These subcategories showed similar clinical characteristics, ddcfDNA levels, and risk of allograft failure. However, definite/probable AMR showed greater measures of AMR severity, including degree of allograft dysfunction and risk of death compared to possible AMR. Clinical AMR showed greater risk of allograft failure than subclinical AMR or no AMR. Subcategorization of clinical AMR based on diagnostic certainty correlated with AMR severity and risk of death, but not with the risk of allograft failure.

Comparison of methods for donor-derived cell-free DNA quantification in plasma and urine from solid organ transplant recipients.

In allograft monitoring of solid organ transplant recipients, liquid biopsy has emerged as a novel approach using quantification of donor-derived cell-free DNA (dd-cfDNA) in plasma. Despite early clinical implementation and analytical validation of techniques, direct comparisons of dd-cfDNA quantification methods are lacking. Furthermore, data on dd-cfDNA in urine is scarce and high-throughput sequencing-based methods so far have not leveraged unique molecular identifiers (UMIs) for absolute dd-cfDNA quantification. Different dd-cfDNA quantification approaches were compared in urine and plasma of kidney and liver recipients: A) Droplet digital PCR (ddPCR) using allele-specific detection of seven common HLA-DRB1 alleles and the Y chromosome; B) high-throughput sequencing (HTS) using a custom QIAseq DNA panel targeting 121 common polymorphisms; and C) a commercial dd-cfDNA quantification method (AlloSeq® cfDNA, CareDx). Dd-cfDNA was quantified as %dd-cfDNA, and for ddPCR and HTS using UMIs additionally as donor copies. In addition, relative and absolute dd-cfDNA levels in urine and plasma were compared in clinically stable recipients. The HTS method presented here showed a strong correlation of the %dd-cfDNA with ddPCR (R 2 = 0.98) and AlloSeq® cfDNA (R 2 = 0.99) displaying only minimal to no proportional bias. Absolute dd-cfDNA copies also correlated strongly (τ = 0.78) between HTS with UMI and ddPCR albeit with substantial proportional bias (slope: 0.25; 95%-CI: 0.19-0.26). Among 30 stable kidney transplant recipients, the median %dd-cfDNA in urine was 39.5% (interquartile range, IQR: 21.8-58.5%) with 36.6 copies/μmol urinary creatinine (IQR: 18.4-109) and 0.19% (IQR: 0.01-0.43%) with 5.0 copies/ml (IQR: 1.8-12.9) in plasma without any correlation between body fluids. The median %dd-cfDNA in plasma from eight stable liver recipients was 2.2% (IQR: 0.72-4.1%) with 120 copies/ml (IQR: 85.0-138) while the median dd-cfDNA copies/ml was below 0.1 in urine. This first head-to-head comparison of methods for absolute and relative quantification of dd-cfDNA in urine and plasma supports a method-independent %dd-cfDNA cutoff and indicates the suitability of the presented HTS method for absolute dd-cfDNA quantification using UMIs. To evaluate the utility of dd-cfDNA in urine for allograft surveillance, absolute levels instead of relative amounts will most likely be required given the extensive variability of %dd-cfDNA in stable kidney recipients.

Application of plasma donor-derived cell free DNA for lung allograft rejection diagnosis in lung transplant recipients

BackgroundDonor-derived cell-free DNA (dd-cfDNA) has been applied to monitor acute rejection (AR) in kidney and heart transplantation. This study was aimed to investigate the application of dd-cfDNA levels in the diagnosis of AR and chronic lung allograft dysfunction (CLAD) among the lung transplantation recipients (LTRs).MethodsOne hundred and seventy LTRs were enrolled at the First Affiliated Hospital of Guangzhou Medical University between 1 June 2015 and 30 March 2021. Patients were divided into 4 groups: stable group, AR group, infection group and CLAD group. The level of dd-cfDNA was analyzed using target region sequencing and the performance characteristics of dd-cfDNA for diagnosis of AR and CLAD were determined, respectively.ResultsKruskal–Wallis test showed that there were some significant differences in the level of dd-cfDNA (%) among the 4 groups, with p < 0.001. Among them, the level of dd-cfDNA (%) was highest (median 2.17, IQR [1.40–3.82]) in AR group, and higher in CLAD group (median 1.07, IQR [0.98–1.31]), but lower in infection group (median 0.71, IQR [0.57–1.07]) and lowest in stable group (median 0.71, IQR [0.61–0.84]). AUC-ROC curve analysis showed that the threshold of dd-cfDNA for AR was 1.17%, with sensitivity being 89.19% and specificity being 86.47%, and the optimal threshold of 0.89% was determined of CLAD, with sensitivity being 95.00% and specificity of 76.99%.ConclusionsPlasma dd-cfDNA could be a useful tool for the assessment of lung allograft rejection, including AR and CLAD, and holds promise as a noninvasive biomarker for “allograft injury” in both acute and chronic rejection following lung transplantation.

Assessment of dd-cfDNA Levels in Clinically Stable Lung Allograft Recipients Beyond the Initial 2 y Posttransplant.

We performed a prospective, single-center, observational study to identify plasma dd-cfDNA levels in clinically stable lung allograft recipients >2 y posttransplant. Fifty-one subjects were enrolled and ≥3 baseline dd-cfDNA measurements were acquired during a median of 252 d. The median baseline percent dd-cfDNA level in our cohort was 0.45% (interquartile range [IQR], 0.26-0.69). There were statistically significant differences in dd-cfDNA based on posttransplant duration (≤5 y posttransplant median 0.41% [IQR, 0.21-0.64] versus >5 y posttransplant median 0.50% [IQR, 0.33-0.76]; P < 0.02). However, the clinical significance of this small change in dd-cfDNA is uncertain because this magnitude of change is within the biologic test variation of 73%. This study is the first to define levels of dd-cfDNA in clinically stable patients who are >2 y post-lung transplant. These findings lay the groundwork for the study of dd-cfDNA as a possible biomarker for CLAD.

Anti-interleukin-6 Antibody Clazakizumab in Antibody-mediated Kidney Transplant Rejection: Effect on Donor-derived Cell-free DNA and C-X-C Motif Chemokine Ligand 10.

We report a secondary endpoint analysis of a phase 2 trial of anti-IL-6 antibody clazakizumab in late AMR (ClinicalTrials.gov, NCT03444103). Twenty kidney transplant recipients were randomized to treatment with clazakizumab or placebo over 12 wk (part A), followed by an extension in which all recipients received clazakizumab through week 52 (part B). Biomarkers were evaluated at day 0 and after 12 and 52 wk, respectively. Fractional dd-cfDNA (dd-cfDNA[%]) did not significantly change under clazakizumab, with no differences between study arms (clazakizumab versus placebo) at week 12 (1.65% [median; interquartile range: 0.91%-2.78%] versus 0.97% [0.56%-2.30%]; P = 0.25) and no significant decrease from weeks 12 to 52 (1.15% [0.70%-2.38%] versus 1.0% [0.61%-1.70%]; P = 0.25). Similarly, urine CXCL10 was not different between groups at week 12 (55.7 [41.0-91.4] versus 60.2 [48.8-208.7.0] pg/mg creatinine; P = 0.44) and did not change over part B (CXCL10 [pg/mg creatinine]: from 58 [46.3-93.1] to 67.4 [41.5-132.0] pg/mL creatinine; P = 0.95). Similar results were obtained for serum CXCL10. There was no association between biomarker levels and resolution of molecular and morphologic AMR activity. Our results suggest that IL-6 blockade does not significantly affect levels of dd-cfDNA[%] and CXCL10. Subtle responses to this therapeutic principle may be overlooked by early biomarker surveillance.

Abstract 10563: Differences in Donor-Derived Cell-Free DNA Levels During Allograft Rejections in Hepatitis C Positive Donor Recipient Heart Transplant Patients

Introduction: Non-invasive quantification of donor-derived cell-free DNA (dd-cfDNA) has recently been shown to correlate with endomyocardial biopsy (EMB) for diagnosis of allograft rejection in heart transplant (HTx) patients. Hepatitis C virus-positive (HCV+) donor hearts are being more utilized with comparable survival outcomes. Hypothesis: The aim of this study is to evaluate the utility of dd-cfDNA monitoring in different types of rejections in recipients of HCV+ donor hearts. Methods: We performed a retrospective review of recipients of HCV+ donor hearts between 2018-2022 with paired dd-cfDNA/GEP and EMB results. The levels of dd-cfDNA/GEP were compared in different rejection types and no rejection for patients using non-parametric comparison. CMR ISHLT ≥ Grade 2R and AMR ≥ Grade 1 from histology were considered as allograft rejection. Results: There were 17 patients who underwent heart transplant from HCV+ donor heart. There was a total of 89 paired samples with dd-cfDNA and EMB. The reason for the biopsy was mostly for routine surveillance (N=68, 76.4%). There was a total of 1 case (1.1%) of grade 2 CMR and 16 cases of pAMR (18%; 10 cases of grade 1, 6 cases of grade 2). Levels of dd-cfDNA were elevated in cases of AMR when compared to no rejection (Median: 0.24% vs. 0.12%, respectively, P&lt;0.001; Figure 1A). There were 57 samples (78%) with dd-cfDNA reference values of &lt;0.12%. In patients with no rejection, dd-cfDNA levels were all below 0.25%. The AUC for elevation of dd-cfDNA and allograft rejection was 0.85 [95% C.I.: 0.72-0.97; P&lt;0.001; Figure 1B]. Conclusions: Non-invasive quantitation of dd-cfDNA levels is elevated in allograft rejection as assessed by traditional microscopy from EMB. There was no false-positive rate of dd-cfDNA in allograft rejection at a threshold of 0.25%. Further studies are indicated to study whether dd-cfDNA levels transiently increase only in the setting of allograft rejection or active hepatitis C viremia at the time of biopsy.

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

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

Combining donor-derived cell-free DNA and donor specific antibody testing as non-invasive biomarkers for rejection in kidney transplantation

Donor specific anti-HLA antibodies (DSA) and donor-derived cell-free DNA (dd-cfDNA) have lead to substantial progress in the non-invasive monitoring of the renal allograft by being able to detect or rule out subclinical rejection and guide immunosuppressive changes. In this study we sought to analyze the clinical, de novo DSA (dnDSA) and histological determinants of dd-cfDNA levels. The study included a cohort of stable renal function kidney transplant (KT) recipients who underwent anti-HLA dnDSA and dd-cfDNA testing between September 2017-December 2019. Statistical models were constructed to detect association with predictors of dd-cfDNA levels and other clinical characteristics. 171 renal allograft recipients were tested for dd-cfDNA and dnDSA at a median 1.06 years posttransplant (IQR: 0.37–4.63). Median dd-cfDNA was 0.25% (IQR: 0.19–0.51), 18.7% of patients having a dd-cfDNA ≥ 1%. In a multivariate linear regression model the presence of dnDSA MFI ≥ 2500 was the best independent determinant of dd-cfDNA level (p < 0.001). Among patients tested, 54 had concurrent dd-cfDNA determination at the time of an allograft biopsy. dd-cfDNA had an AUC of 0.82 (95% CI 0.69–0.91; p < 0.001) and of 0.96 (95% CI 0.87–0.99) to discriminate any rejection and ABMR, respectively. After multivariate adjustment, the models that included ABMR (R = 0.82, R2 = 0.67, p < 0.001), or ptc (R = 0.79, R2 = 0.63, p < 0.001) showed the best correlation with dd-cfDNA level. We are confirming a strong association of dd-cfDNA with dnDSA and underlying alloimmune-mediated injury in renal allograft recipients in a cohort of patients with unsuspecting clinical characteristics for rejection and excellent allograft function. Our findings support the need for noninvasive biomarker surveillance in KT recipients and we propose that dd-cfDNA may complement dnDSA screening.

343.9: Longitudinal Monitoring of Pancreas and Kidney Transplant Recipients Using Donor-Derived Cell-Free DNA

Introduction: Donor-derived cell-free DNA (dd-cfDNA) is validated for allograft surveillance in kidney transplant recipients; however, the utility in patients undergoing pancreas and kidney (PTx) transplantation has not been well studied. Pancreatic rejection remains a major clinical concern, yet diagnosis relies on pancreatic biopsy which is associated with significant risk of procedural complications. Early identification of allograft injury is imperative to optimize treatment and intervention. Here, we describe dd-cfDNA levels in PTx transplant recipients in the setting of clinical stability and immunological events. Methods: PTx recipients were monitored longitudinally with dd-cfDNA (AlloSure, CareDx) at the discretion of the treating physician. dd-cfDNA was collected in tandem with standard of care laboratory testing including serum creatinine, amylase, lipase, DSA and BK PCR. Pancreatic graft dysfunction was defined by elevations in serum amylase/lipase or dysregulated glucose control. The reference population was defined by stable allograft function in the absence of immunological or clinical events. Immunological events were defined as clinically suspected allograft rejection or documented subtherapeutic immunosuppression. Results: A total of 16 PTx patients were monitored longitudinally with dd-cfDNA, including 15 SPK recipients and 1 pancreas after kidney transplant (Table 1). 12 patients met criteria for the stable reference population with a median dd-cfDNA level of 0.22% (IQR 0.12 – 0.36%, n=248). There was a trend towards increased variability in dd-cfDNA levels in the first 2 months post-transplant, after which there was no significant difference in dd-cfDNA stratified by time (Figure 1A). 4 patients developed 6 independent immunological events, which were associated with a significantly increased median dd-cfDNA level of 1.15% (IQR 0.15 – 1.85%) compared to 0.22% in the reference population (p = 0.03, Figure 1B). Conclusions: In stable PTx recipients, dd-cfDNA levels remain low post-transplant and reflect the reference ranges validated in kidney transplant alone. Early variability in dd-cfDNA levels may be explained by recovery from ischemia reperfusion injury. Significant increases in dd-cfDNA were associated with immunological events including clinically suspected allograft rejection, highlighting its utility in longitudinal graft surveillance.

232.7: Elevated Donor-Derived Cell-Free DNA Associates With Subsequent Decline of Allograft Function

Introduction: Donor-derived cell-free DNA (dd-cfDNA) emerged as a candidate biomarker for detecting graft injury, particularly from antibody-mediated rejection, and is currently proposed to complement donor-specific antibodies (DSAs) as an alloimmune-mediated injury surveillance method. We sought to investigate the impact of an initial increase of dd-cfDNA level on subsequent decline in graft function. Materials: The study included all kidney transplant (KT) recipients that underwent dd-cfDNA testing as part of their clinical care between September 2017 and December 2019 at our center. Only patients with a follow-up of at least 12 months were included in this analysis. An elevated dd-cfDNA was defined as a level over 0.5%. Results: Of the 171 KT recipients tested for dd-cfDNA, 49 were followed for at least 12 months since initial testing. The study cohort had an initial serum creatinine and eGFR of 1.64 ± 0.58 mg/dl and 48 ± 21 ml/min/1.73m2, respectively. Overall, the absolute and percentual decline of eGFR were -0.12 ml/min/month (IQR, -0.43 to 0.56) and -4.26% (IQR, -20.5% to 17%), with 26.5% and 10.2% of patients having an eGFR decline of more than 15% and 30%, respectively. Fifteen patients (30.6%) had an elevated dd-cfDNA (over 0.5%) and had a similar baseline allograft function compared to those without an elevated dd-cfDNA level. After a median follow-up period of 15.3 months (IQR:13.6-19), patients with elevated dd-cfDNA had a faster decline of eGFR [absolute eGFR decline, -0.21 ml/min/month (IQR, -0.36 to 0.22); percentual GFR decline, -8.7% (IQR, -25% to 4.4%)], compared to those without elevated dd-cfDNA [absolute eGFR decline, +0.03 ml/min/month (IQR, -0.5 to 0.6); percentual GFR decline, +1.14% (IQR, -14% to 28%)](p=0.09)(Figure 1). Similarly, there was a tendency for a higher percentage of patients with elevated dd-cfDNA for a decline of eGFR greater than 15% (33.3% vs. 23.5%, p=0.5) or 30% (13.3% vs. 8.8%, p=0.6), respectively. In addition, patients with DSAs and an elevated dd-cfDNA showed a greater percentual eGFR decline (-9.1±16.5%), compared to those with DSAs and normal dd-cfDNA (+3.3±21.1%), or to those without DSAs and normal dd-cfDNA (+2.3±18.05%). In multivariable logistic regression analysis, an elevated dd-cfDNA level was independently associated with the subsequent percentual decline of eGFR (OR, 0.96; 95%CI, 0.93 to 1.00; p=0.05). Conclusion: We have shown that despite initial stable allograft function, a higher dd-cfDNA level associates with subsequent decline of eGFR. Thus, the initial identification of subclinical graft injury could associate with long-term graft outcomes, expanding the clinical utility of dd-cfDNA.

232.11: Longitudinal Surveillance of a Kidney Transplant Recipient During Pregnancy Using Quantification of Fetal and Donorderived Cell-Free DNA

Introduction: Following kidney transplantation, women of childbearing age often experience rapid restoration of fertility as the feedback mechanisms of the hypothalamic-pituitary-gonadal axis normalize. Although rates of rejection during pregnancy are comparable to the non-pregnant transplant population, subtle indications of allograft injury such as increases in serum creatinine or proteinuria can be masked by pregnancy related changes. Here, we describe a novel approach of allograft surveillance using quantification of fetal and donor-derived cell-free DNA (dd-cfDNA) throughout gestation and postpartum. Methods: The patient was monitored longitudinally with dd-cfDNA (AlloSure, CareDx) pre-pregnancy, during gestation and postpartum. For this analysis, specific SNPs were selected that are heterozygous in kidney donor and homozygous (same allele) in recipient and fetal cells. An average minor allele frequency (MAF) was calculated from these selected SNPs. This average represents half of the expected donor cfDNA% due to Hardy-Weinberg principle based on Mendelian genetics. Hence, calculated average MAF was multiplied by factor of 2 to calculate the kidney donor percentage. Results: A 39 year old female with a living related kidney transplant (transplanted September 2018) conceived in February 2021. The patient had a baseline creatinine of 1.4 mg/dl and median dd-cfDNA level of 0.44%. dd-cfDNA levels remained stable throughout pregnancy (median 0.42%, IQR 0.31-0.44%) and were consistent with pre-pregnancy baseline, in keeping with absence of allograft injury. The percentage of fetal cfDNA saw an exponential rise in the first trimester, plateaued in the second trimester and showed a rapid decay post-delivery. Allograft function remained stable throughout pregnancy until development of pre-eclampsia with an acute rise in serum creatine to 2.1 mg/dl, prompting delivery at 31 weeks (Figure 1). Conclusions: The estimated percent of fetal fraction of cfDNA increased with gestational age and rapidly declined post-delivery. Throughout pregnancy, dd-cfDNA levels remained consistent with pre-pregnancy baseline in the absence of graft dysfunction, rejection, or clinical events. This case study suggests that longitudinal monitoring of fetal and dd-cfDNA contributions during pregnancy in kidney transplant recipients is a feasible, novel approach for allograft surveillance in this high-risk patient population.

216.9: Urinary Extracellular Vesicle DNA Cargo Reflects Kidney Allograft Injury

Introduction: Following allograft injury, donor-derived cell-free DNA (dd-cfDNA) levels increase. Numerous research studies have demonstrated increased dd-cfDNA levels in blood and urine as biomarkers of kidney allograft injury in patients with transplanted kidney. Extracellular vesicles (EVs) are nanosized particles released by almost all cells. Because of the anatomical proximity of kidney EV-producing cells and urine, (dd)DNA containing urinary EVs (uEVs) are potentially relevant biomarkers of allograft injury. The aim of our study was to explore uEVs and their DNA cargo, to reveal their association with allograft injury and finally to compare their biomarker potential with (dd)cfDNA found directly in the urine of patients with transplanted kidney. Method: We collected a second morning urine sample from 40 patients before surveillance or for-cause biopsy. We also collected blood and biopsy samples for donor-recipient genotyping. Considering histological analysis, we divided patients into normal histology, rejection injury (combination of antibody- (ABMR) and T-cell mediated rejection (TCMR)), and non-rejection injury group. First, we isolated uEVs (using protocol based on size-exclusion chromatography), their DNA cargo (uEV-DNA), and cfDNA from urine. We used nanoparticle tracking analysis to determine the concentration and size of uEVs. For DNA parameter analysis, including yield, copy number, integrity index, and donor-derived fraction, we used fluorometry and digital droplet PCR. We applied DNase assay and electron microscopy (TEM) immunogold labelling technique to investigate whether DNA was present inside or outside the uEVs. Results: The median uEV concentration was 8.47x1010/ mmol U-creatinine. The concentration was similar in all patient groups, whereas the size parameter differed significantly, as patients with rejection and non-rejection injury had significantly larger uEVs compared with patients with normal histology (177.5 nm and 174.1 nm vs. 160.7 nm, respectively; P=0.045). DNA co-isolated with uEVs and in several parameters correlated with urinary cfDNA. uEV-DNA and cfDNA yields (Figure 1A) and DNA copy number (Figure 1B) were significantly higher in both allograft injury groups. For uEV-DNA, the copy number of donor-derived DNA was significantly higher in patients with ABMR than in patients with TCMR (3044.3 vs. 680.9 copies, respectively; P=0.036). Regarding the long/short gene amplicon ratio, uEV-DNA (green) was less fragmented compared with cfDNA (yellow; Figure 1C). The TEM DNA labelling showed that the DNA was bound to the EV surface. uEV-DNA characteristics correlated with interstitial inflammation, inflammation in areas of fibrosis, and combination of glomerulitis and peritubular capillaritis Banff scores. Conclusion: In kidney transplant recipients, DNA bound to the surface of uEVs reflects allograft injury, particularly allograft rejection and is therefore a suitable potential biomarker for kidney allograft injury.Slovenian Research Agency Grants P3-0323, P1-0170, P4-0165, P4-0407.

232.1: Donor-derived Cell-Free DNA Testing in Kidney Transplantation: Do One-month Values Have Any Prognostic Significance?

Introduction: Percutaneous renal allograft biopsy is the current standard for detecting acute rejection after kidney transplantation (KT). Noninvasive tests such as plasma-based donor-derived cell-free DNA (dd-cfDNA) assays have been proposed as an alternative to biopsy. We sought to examine the value of dd-cfDNA testing at one month following KT in different donor and recipient categories. Methods: We performed a single center retrospective review of all adult KT patients (pts) who underwent dd-cfDNA testing between January 2018 and August 2021. The diagnosis of acute rejection was based on histopathology obtained from percutaneous, ultrasound-guided needle biopsies. Our center’s standard practice included dd-cfDNA testing and performing surveillance kidney biopsies at 1 month following KT. The dd-cfDNA test was considered elevated if >1.0%. Results: During the study period, 3157 dd-cfDNA tests were performed in 695 de novo pts. Higher mean 1-month dd-cfDNA levels were noted in the following categories: Retransplants (n=30, 1.8±2.1); highly sensitized pts (PRA 98-100%, n=27, 1.6±2.0; for highly sensitized retransplants, n=18, 2.0±2.3; for highly sensitized primary KTs, n=9, 0.7±0.5); and pts who underwent early reoperations (n=16, 0.85±0.9). In all other KT categories, mean 1-month dd-cfDNA levels were lower and similar: DCD donor KTs (n=53, 0.56±0.4), living donor KTs (n=51, 0.47±0.4); acute kidney injury (AKI) donor KTs (n=23, 0.47±0.6); standard criteria donor (SCD) KTs (n=91, 0.47±0.4); expanded criteria donor (ECD) KTs (n=44, 0.46±0.5); dual KTs (n=16, 0.45±0.4); and pts with delayed graft function (DGF, n=50, 0.46±0.4). In 58 primary KT pts with negative 1-month surveillance biopsies, corresponding mean 1-month dd-cfDNA levels were 0.49±0.4. In 10 pts with positive 1-month surveillance biopsies, corresponding mean 1-month dd-cfDNA levels were 1.5±1.8. In 26 pts with a 1-month dd-cfDNA level >1.0, the subsequent incidence of acute rejection was 27%; in 22 pts with a 1-month dd-cfDNA level ≥2.0, the subsequent incidence of acute rejection was 50% (mean follow-up 14 months). Conclusions: We noted a bimodal distribution of 1-month dd-cfDNA levels. Higher mean 1-month dd-cfDNA levels (range 0.72-2.2) were associated with retransplants, high PRA pts, and pts with early reoperations; 22-44% of these pts had 1-month dd-cfDNA levels >1.0. Conversely, primary KT alone pts (DCD, living donor, AKI donor, SCD, ECD, dual KT, those with DGF) had lower 1-month dd-cfDNA levels (range 0.45-0.56) and only 7-14% had 1-month dd-cfDNA levels >1.0. One-month surveillance biopsy results correlated with corresponding dd-cfDNA levels. Elevated 1-month dd-cfDNA levels occurred in pts at a higher risk for acute rejection, suggesting that these pts need to be monitored more closely.

344.2: Delta Changes in Donor-Derived Cell-Free DNA (dd-cfDNA) Complement the Donor Fraction in Kidney Transplant Surveillance

Introduction: Delta changes in dd-cfDNA levels over time may identify patients with evolving alloimmune injury, providing complementary information to thedonor fraction. We explored the dd-cfDNA trajectories preceding biopsy-proven rejection (BPAR) events among kidney transplant recipients enrolled in theKidney allograft Outcomes AlloSure Registry (KOAR, NCT03326076) and undergoing longitudinal surveillance with dd-cfDNA. Methods: In the primary analysis, we compared the change in dd-cfDNA from baseline to the time of BPAR (for-cause or surveillance biopsies). We identifiedpatients with first-time rejection events and at least 3 prior dd-cfDNA results; the index result was obtained ≤30 days before BPAR; we then selected the lowestof two preceding results as the patient-specific baseline and compared this with the index result. We then analyzed patients with rejection (first or subsequent, atleast 90 days apart) and ≥2 dd-cfDNA results, including one within 30 days of the index biopsy. The reference change value (RCV) was calculated using areference population of patients with stable allograft function, at least 3 dd-cfDNA measurements, and no significant clinical events. Results: A total of 28 patients with BPAR met criteria for the primary analysis; among these, median baseline dd-cfDNA was 0.22% (IQR: 0.19 - 0.35) and mediandd-cfDNA at the time of rejection was 1.35% (IQR: 0.75 - 2.75), representing a 491% (IQR: 177 - 1133) increase between these results, obtained 71.5 (IQR: 46 -113) days apart (Table 1A).51 events met criteria for the second analysis where a median increase of 253% (IQR: 72 - 821%) between sequential dd-cfDNAvalues obtained 69 days (IQR: 45 - 108) apart preceded biopsy-proven rejection events (Table 1B). The calculated RCV for the stable reference population withinKOAR was 56.3%. 39 of these 51 events (76%) demonstrated increases greater than this RCV. Conclusions: Longitudinal surveillance with dd-cfDNA allows the integration of delta changes and trajectories over time, allowing earlier identification ofevolving allograft injury. Significant changes from an established baseline and between sequential values ahead of biopsy-proven rejection highlight how longitudinal surveillance may improve diagnostic performance.

216.3: Enhanced Histological Yield and Actionable Findings When Biopsy Is Guided by Donor-derived Cell-free DNA (dd-cfDNA)

Introduction: Implementation of molecular biomarkers in kidney transplantation has expanded the amount of information available to clinicians before deciding topursue biopsy. We evaluated how use of donor-derived cell-free DNA testing (dd-cfDNA) impacts histologic biopsy yield. Methods: 1663 kidney transplant (KTx) recipients enrolled in the Kidney allograft Outcomes AlloSure Registry (KOAR, NCT03326076) were followed with dd-cfDNA post-transplant; testing was obtained either as part of a surveillance strategy or for-cause; indications for biopsy and histologic diagnoses were captured. This analysis included only for-cause biopsies. Results: 65 biopsies (from 59 pts) driven by elevated dd-cfDNA levels were compared to 540 biopsies (392 pts) obtained for causes other than elevated dd-cfDNA. Patient age (55 vs 56 years), percent deceased donor (84.75% vs 78.83%), and biopsy timing post-transplant (123 vs 105.5 days) did not differ betweengroups (Table 1). Among dd-cfDNA-guided biopsies, yield was enriched for rejection (48% vs 29%) and included fewer cases of ATI/ATN (12% vs 22%)compared to biopsies obtained for other causes (p<0.05) (Figure 1). Among dd-cfDNA-guided biopsies with rejection (ABMR, TCMR, or Mixed), median dd-cfDNA was 1.46% (IQR: 1.15 - 2.87). Among biopsies not performed due to elevated dd-cfDNA but with paired results available (n = 267), median dd-cfDNA was0.24%. Within this group, patients with rejection had median dd-cfDNA of 0.82% and demonstrated an increase of 133% from the preceding result. Conclusions: Dd-cfDNA surveillance enhances the histologic yield for actionable results in for-cause allograft biopsies. A low dd-cfDNA result can obviate the need for biopsy even in the presence of other clinical factors that are routinely used to guide the biopsy decision.

336.3: Clinical Utility of Donor-Derived Cell-Free DNA During the Period of Recovery of Renal Function After Kidney Transplantation

Background: The transplant of kidneys with high KDPI, and the incidence and duration of delayed graft function (DGF) are risk factors associated with incomplete recovery of kidney function (IRF). In both situations, kidney biopsies are essential to exclude the diagnosis of acute rejection (AR). Purpose: This study evaluates the clinical utility of monitoring donor-derived cell-free DNA (dd-cfDNA), a biomarker of kidney injury, in recipients of high Kidney Donor Profile Index (KDPI) transplants during the period of kidney function recovery. Methods: This is a single center, prospective, cohort study in recipients of high KDPI kidneys who developed delayed graft function (DGF), defined as the need for dialysis within the first week after transplantation. All patients received a single 3 mg/kg anti-thymocyte globulin dose, tacrolimus, mycophenolate, and prednisone. Blood samples were obtained at 14 (D14) and 30 (D30) days to measure the percentage (%) of dd-cfDNA. The dd-cfDNA > 1% is associated with increased probability for acute rejection (AR). Results: This preliminary analysis includes data from 69 patients. The median KDPI was 73% [IQR 47-87] and the mean cold ischemia time was 27± 7 hours. The incidence of DGF was 89.8% with a median duration of 6 (IQR 2 – 9) days. At D14 the median GFR was 12 ml/min/1.73 m2 (IQR 6-21) and the median dd-cfDNA levels were 0.43% (IQR 0.02-0.78), with 19 patients (28%) showing dd-cfDNA >1%. Surveillance biopsies during DGF were performed in 42 (61%) patients showing one T-cell mediated rejection IA (dd-cfDNA of 0.41%) and 4 borderline changes (dd-cfDNA 0.82%, 0.85%, 0.98%, 1.90%). Using dd-cfDNA >1% to guide the indication of the surveillance biopsy, we would have avoided 22 (52%) biopsies but missed 1 (2%) patient with AR. At D30, the median GFR was 36 ml/min/1.73 m2 (IQR 28-48) and the median dd-cfDNA levels were 0.40% (IQR 0.01-0.66), with 12 patients (19%) showing dd-cfDNA >1%. Surveillance biopsies performed in 21 (30.43%) patients with incomplete recovery of kidney function showed 4 borderline changes (dd-cfDNA 0.17%, 0.66%, 0.67%, and 2.80%). Using dd-cfDNA >1% to guide the indication of the surveillance biopsy, we would have avoided 12 (57%) biopsies without missing any AR episodes. Overall, 5 (7%) patients showed dd-cfDNA >1% at D14 and D30, but none of them showed biopsy confirmed AR. Conclusions: This preliminary analysis suggests that monitoring dd-cfDNA may assist clinical decision during the period of DGF and in kidney transplant recipients with incomplete recovery of graft function.

230.5: Antibody Response and Molecular Graft Surveillance in Kidney Transplant Recipients Following SARS-CoV-2 Vaccination

Introduction: Preliminary studies suggest that kidney transplant recipients (KTRs) show diminished humoral responses to SARS-CoV-2 vaccination. Although reports of allograft rejection around the time of SARS-CoV-2 vaccination have been rare, there is no recommended framework for monitoring for potential vaccine-related allograft injury. Here, we describe an approach for longitudinal assessment of immunogenicity and safety of SARS-COV-2 vaccination in KTRs. Methods: KTRs eligible for SARS-CoV-2 vaccination were identified through medical records, beginning March 12, 2021. Baseline and weekly blood samples were collected for routine assessment, SARS-CoV-2 spike protein antibody titers, dd-cfDNA (AlloSure, CareDx) and gene expression profiling (GEP) (AlloMap, CareDx) for 12 weeks. HLA DSA testing was performed at baseline, 2 weeks after completion of vaccine doses and at week 12. Antibody response was defined as a 10-fold increase in total binding IgG titers. Results: 49 KTRs were identified for analysis. Patient demographics are summarized in Table 1. 10 patients (20.4%) demonstrated a spike antibody response following completion of the vaccine series. Patients with a prior history of COVID-19 were more likely to mount a spike antibody response (n=8, 53.3%) compared to those with no reported history of COVID-19 (n=2, 5.8%). The odds ratio was 18.3 (95% CI 3.2, 105.0, p=0.0005). Median dd-cfDNA levels did not differ between pre- and post-vaccination (0.23% versus 0.21% respectively; Table 2). There was no significant difference between pre- and post-vaccination GEP scores (9.85 versus 10.4 respectively; Table 2). No patients developed clinically significant DSA, eGFR decline or allograft rejection following vaccination. Conclusions: Quantitative antibody responses were strongly associated with a diagnosis of prior SARS-CoV-2 infection. Stability of eGFR, dd-cfDNA, GEP profiles and lack of allosensitization reinforce the safety profile of SARS-CoV-2 vaccination in KTRs. Further studies are needed to better understand immunogenicity in SARS-CoV-2 naïve individuals, including whether cellular responses are protective in the absence of humoral responses.