Noninvasive detection of graft injury after heart transplant using donor-derived cell-free DNA: A prospective multicenter study.

Standardized donor-derived cell-free DNA (dd-cfDNA) testing has been introduced into clinical use to monitor kidney transplant recipients for rejection. This report describes the performance of this dd-cfDNA assay to detect allograft rejection in samples from heart transplant (HT) recipients undergoing surveillance monitoring across the United States. Venous blood was longitudinally sampled from 740 HT recipients from 26 centers and in a single-center cohort of 33 patients at high risk for antibody-mediated rejection (AMR). Plasma dd-cfDNA was quantified by using targeted amplification and sequencing of a single nucleotide polymorphism panel. The dd-cfDNA levels were correlated to paired events of biopsy-based diagnosis of rejection. The median dd-cfDNA was 0.07% in reference HT recipients (2164 samples) and 0.17% in samples classified as acute rejection (35 samples; P = .005). At a 0.2% threshold, dd-cfDNA had a 44% sensitivity to detect rejection and a 97% negative predictive value. In the cohort at risk for AMR (11 samples), dd-cfDNA levels were elevated 3-fold in AMR compared with patients without AMR (99 samples, P = .004). The standardized dd-cfDNA test identified acute rejection in samples from a broad population of HT recipients. The reported test performance characteristics will guide the next stage of clinical utility studies of the dd-cfDNA assay.

Cell-Free DNA and Active Rejection in Kidney Allografts.

Histologic analysis of the allograft biopsy specimen is the standard method used to differentiate rejection from other injury in kidney transplants. Donor-derived cell-free DNA (dd-cfDNA) is a noninvasive test of allograft injury that may enable more frequent, quantitative, and safer assessment of allograft rejection and injury status. To investigate this possibility, we prospectively collected blood specimens at scheduled intervals and at the time of clinically indicated biopsies. In 102 kidney recipients, we measured plasma levels of dd-cfDNA and correlated the levels with allograft rejection status ascertained by histology in 107 biopsy specimens. The dd-cfDNA level discriminated between biopsy specimens showing any rejection (T cell-mediated rejection or antibody-mediated rejection [ABMR]) and controls (no rejection histologically), P<0.001 (receiver operating characteristic area under the curve [AUC], 0.74; 95% confidence interval [95% CI], 0.61 to 0.86). Positive and negative predictive values for active rejection at a cutoff of 1.0% dd-cfDNA were 61% and 84%, respectively. The AUC for discriminating ABMR from samples without ABMR was 0.87 (95% CI, 0.75 to 0.97). Positive and negative predictive values for ABMR at a cutoff of 1.0% dd-cfDNA were 44% and 96%, respectively. Median dd-cfDNA was 2.9% (ABMR), 1.2% (T cell-mediated types ≥IB), 0.2% (T cell-mediated type IA), and 0.3% in controls (P=0.05 for T cell-mediated rejection types ≥IB versus controls). Thus, dd-cfDNA may be used to assess allograft rejection and injury; dd-cfDNA levels <1% reflect the absence of active rejection (T cell-mediated type ≥IB or ABMR) and levels >1% indicate a probability of active rejection.

Clinical usefulness of gene-expression profile to rule out acute rejection after heart transplantation: CARGO II.

AIMS: A non-invasive gene-expression profiling (GEP) test for rejection surveillance of heart transplant recipients originated in the USA. A European-based study, Cardiac Allograft Rejection Gene Expression Observational II Study (CARGO II), was conducted to further clinically validate the GEP test performance. METHODS AND RESULTS: Blood samples for GEP testing (AlloMap(®), CareDx, Brisbane, CA, USA) were collected during post-transplant surveillance. The reference standard for rejection status was based on histopathology grading of tissue from endomyocardial biopsy. The area under the receiver operating characteristic curve (AUC-ROC), negative (NPVs), and positive predictive values (PPVs) for the GEP scores (range 0-39) were computed. Considering the GEP score of 34 as a cut-off (>6 months post-transplantation), 95.5% (381/399) of GEP tests were true negatives, 4.5% (18/399) were false negatives, 10.2% (6/59) were true positives, and 89.8% (53/59) were false positives. Based on 938 paired biopsies, the GEP test score AUC-ROC for distinguishing ≥3A rejection was 0.70 and 0.69 for ≥2-6 and >6 months post-transplantation, respectively. Depending on the chosen threshold score, the NPV and PPV range from 98.1 to 100% and 2.0 to 4.7%, respectively. CONCLUSION: For ≥2-6 and >6 months post-transplantation, CARGO II GEP score performance (AUC-ROC = 0.70 and 0.69) is similar to the CARGO study results (AUC-ROC = 0.71 and 0.67). The low prevalence of ACR contributes to the high NPV and limited PPV of GEP testing. The choice of threshold score for practical use of GEP testing should consider overall clinical assessment of the patient's baseline risk for rejection.

Performance of gene-expression profiling test score variability to predict future clinical events in heart transplant recipients.

BACKGROUND: A single non-invasive gene expression profiling (GEP) test (AlloMap®) is often used to discriminate if a heart transplant recipient is at a low risk of acute cellular rejection at time of testing. In a randomized trial, use of the test (a GEP score from 0-40) has been shown to be non-inferior to a routine endomyocardial biopsy for surveillance after heart transplantation in selected low-risk patients with respect to clinical outcomes. Recently, it was suggested that the within-patient variability of consecutive GEP scores may be used to independently predict future clinical events; however, future studies were recommended. Here we performed an analysis of an independent patient population to determine the prognostic utility of within-patient variability of GEP scores in predicting future clinical events. METHODS: We defined the GEP score variability as the standard deviation of four GEP scores collected ≥315 days post-transplantation. Of the 737 patients from the Cardiac Allograft Rejection Gene Expression Observational (CARGO) II trial, 36 were assigned to the composite event group (death, re-transplantation or graft failure ≥315 days post-transplantation and within 3 years of the final GEP test) and 55 were assigned to the control group (non-event patients). In this case-controlled study, the performance of GEP score variability to predict future events was evaluated by the area under the receiver operator characteristics curve (AUC ROC). The negative predictive values (NPV) and positive predictive values (PPV) including 95 % confidence intervals (CI) of GEP score variability were calculated. RESULTS: The estimated prevalence of events was 17 %. Events occurred at a median of 391 (inter-quartile range 376) days after the final GEP test. The GEP variability AUC ROC for the prediction of a composite event was 0.72 (95 % CI 0.6-0.8). The NPV for GEP score variability of 0.6 was 97 % (95 % CI 91.4-100.0); the PPV for GEP score variability of 1.5 was 35.4 % (95 % CI 13.5-75.8). CONCLUSION: In heart transplant recipients, a GEP score variability may be used to predict the probability that a composite event will occur within 3 years after the last GEP score. TRIAL REGISTRATION: Clinicaltrials.gov identifier NCT00761787.

Donor-derived Cell-free DNA Identifies Antibody-mediated Rejection in Donor Specific Antibody Positive Kidney Transplant Recipients.

BACKGROUND: Elevated levels of donor-derived cell-free DNA (dd-cfDNA) in the plasma of renal allograft recipients indicates organ injury and an increased probability of active rejection. Donor-specific antibodies (DSA) to HLA antigens are associated with risk of antibody-mediated rejection (ABMR). This study assessed the combined use of dd-cfDNA and DSA testing to diagnose active ABMR. METHODS: Donor-derived cell-free DNA was assayed in 90 blood samples with paired DSA and clinically indicated biopsies from 87 kidney transplant patients. Sixteen cases met criteria for active ABMR. Performance characteristics of dd-cfDNA for diagnosis of active ABMR were determined for samples with prior or current positive DSA (DSA+, n = 33). RESULTS: The median level of dd-cfDNA (2.9%) in DSA+ patients with active ABMR was significantly higher than the median level (0.34%) in DSA+ patients without ABMR (P < 0.001). The median level of dd-cfDNA in DSA- patients was 0.29%. The positive predictive value of dd-cfDNA (at 1%) to detect active ABMR in DSA+ patients was 81%, whereas the negative predictive value was 83%. The positive predictive value for DSA+ alone was 48%. CONCLUSIONS: The combined use of dd-cfDNA and DSA testing may improve the noninvasive diagnosis of active ABMR in kidney transplant patients. Patients with dd-cfDNA+/ DSA+ results have a high probability of active ABMR.

Biological Variation of Donor-Derived Cell-Free DNA in Renal Transplant Recipients: Clinical Implications.

BACKGROUND: Previous studies have demonstrated that donor-derived cell-free DNA (dd-cfDNA) found in circulating blood of transplant recipients may serve as a noninvasive biomarker of allograft rejection. To better interpret the clinical meaning of dd-cfDNA, it is essential to understand the biological variation of this biomarker in stable healthy recipients. This report establishes the biological variation and clinical reference intervals of dd-cfDNA in renal transplant recipients by using an analytically validated assay that has a CV of 6.8%. METHODS: We sampled venous blood at patient surveillance visits (typically at posttransplant months 1-4, 6, 9, and 12) in a 14-center observational study. Patients with stable renal allograft function spanning ≥3 serial visits were selected. We used AlloSure®, a targeted next-generation sequencing-based approach, to measure dd-cfDNA in the plasma and computed the intraindividual CV (CVI) and interindividual CV (CVG), the index of individuality (II), and reference change value (RCV). RESULTS: Of 93 patients, 61% were men, 56% were Caucasian, mean age was 49 years, and 63% were deceased donor kidney recipients. Of 380 blood samples, the dd-cfDNA median value was 0.21% (interquartile range 0.12%-0.39%) and the 97.5th percentile was 1.20%. In 18 patients with an average of 4.1 tests, the CVI was 21%, CVG was 37%, II was 0.57, and RCV was 61%. CONCLUSIONS: In a renal transplant recipient, a dd-cfDNA level above 1.2% is out of range and potentially abnormal. A serial increase of up to 61% in level of dd-cfDNA in a patient may be attributable to biological variation.Clinicaltrials.gov Identifier: NCT02424227.

Validation of a Clinical-Grade Assay to Measure Donor-Derived Cell-Free DNA in Solid Organ Transplant Recipients.

The use of circulating cell-free DNA (cfDNA) as a biomarker in transplant recipients offers advantages over invasive tissue biopsy as a quantitative measure for detection of transplant rejection and immunosuppression optimization. However, the fraction of donor-derived cfDNA (dd-cfDNA) in transplant recipient plasma is low and challenging to quantify. Previously reported methods to measure dd-cfDNA require donor and recipient genotyping, which is impractical in clinical settings and adds cost. We developed a targeted next-generation sequencing assay that uses 266 single-nucleotide polymorphisms to accurately quantify dd-cfDNA in transplant recipients without separate genotyping. Analytical performance of the assay was characterized and validated using 1117 samples comprising the National Institute for Standards and Technology Genome in a Bottle human reference genome, independently validated reference materials, and clinical samples. The assay quantifies the fraction of dd-cfDNA in both unrelated and related donor-recipient pairs. The dd-cfDNA assay can reliably measure dd-cfDNA (limit of blank, 0.10%; limit of detection, 0.16%; limit of quantification, 0.20%) across the linear quantifiable range (0.2% to 16%) with across-run CVs of 6.8%. Precision was also evaluated for independently processed clinical sample replicates and is similar to across-run precision. Application of the assay to clinical samples from heart transplant recipients demonstrated increased levels of dd-cfDNA in patients with biopsy-confirmed rejection and decreased levels of dd-cfDNA after successful rejection treatment. This noninvasive clinical-grade sequencing assay can be completed within 3 days, providing the practical turnaround time preferred for transplanted organ surveillance.