Monitoring allograft health is an important component of posttransplant therapy. Endomyocardial biopsy is the current gold standard for cardiac allograft monitoring but is an expensive and invasive procedure. Proof of principle of a universal, noninvasive diagnostic method based on high-throughput screening of circulating cell-free donor-derived DNA (cfdDNA) was recently demonstrated in a small retrospective cohort. We present the results of a prospective cohort study (65 patients, 565 samples) that tested the utility of cfdDNA in measuring acute rejection after heart transplantation. Circulating cell-free DNA was purified from plasma and sequenced (mean depth, 1.2 giga–base pairs) to quantify the fraction of cfdDNA. Through a comparison with endomyocardial biopsy results, we demonstrate that cfdDNA enables diagnosis of acute rejection after heart transplantation, with an area under the receiver operating characteristic curve of 0.83 and sensitivity and specificity that are comparable to the intrinsic performance of the biopsy itself. This noninvasive genome transplant dynamics approach is a powerful and informative method for routine monitoring of allograft health without incurring the risk, discomfort, and expense of an invasive biopsy.
The survival rate following lung transplantation is among the lowest of all solid-organ transplants, and current diagnostic tests often fail to distinguish between infection and rejection, the two primary posttransplant clinical complications. We describe a diagnostic assay that simultaneously monitors for rejection and infection in lung transplant recipients by sequencing of cell-free DNA (cfDNA) in plasma. We determined that the levels of donor-derived cfDNA directly correlate with the results of invasive tests of rejection (area under the curve 0.9). We also analyzed the nonhuman cfDNA as a hypothesis-free approach to test for infections. Cytomegalovirus is most frequently assayed clinically, and the levels of CMV-derived sequences in cfDNA are consistent with clinical results. We furthermore show that hypothesis-free monitoring for pathogens using cfDNA reveals undiagnosed cases of infection, and that certain infectious pathogens such as human herpesvirus (HHV) 6, HHV-7, and adenovirus, which are not often tested clinically, occur with high frequency in this cohort.organ transplantation | cell-free DNA | infection | rejection | diagnosis
BackgroundIt remains difficult to predict and to measure the efficacy of pharmacological immunosuppression. We hypothesized that measuring the B-cell repertoire would enable assessment of the overall level of immunosuppression after heart transplantation.Methods and FindingsIn this proof-of-concept study, we implemented a molecular-barcode-based immune repertoire sequencing assay that sensitively and accurately measures the isotype and clonal composition of the circulating B cell repertoire. We used this assay to measure the temporal response of the B cell repertoire to immunosuppression after heart transplantation. We selected a subset of 12 participants from a larger prospective cohort study (ClinicalTrials.gov NCT01985412) that is ongoing at Stanford Medical Center and for which enrollment started in March 2010. This subset of 12 participants was selected to represent post-heart-transplant events, with and without acute rejection (six participants with moderate-to-severe rejection and six without). We analyzed 130 samples from these patients, with an average follow-up period of 15 mo. Immune repertoire sequencing enables the measurement of a patient’s net state of immunosuppression (correlation with tacrolimus level, r = −0.867, 95% CI −0.968 to −0.523, p = 0.0014), as well as the diagnosis of acute allograft rejection, which is preceded by increased immune activity with a sensitivity of 71.4% (95% CI 30.3% to 94.9%) and a specificity of 82.0% (95% CI 72.1% to 89.1%) (cell-free donor-derived DNA as noninvasive gold standard). To illustrate the potential of immune repertoire sequencing to monitor atypical post-transplant trajectories, we analyzed two more patients, one with chronic infections and one with amyloidosis. A larger, prospective study will be needed to validate the power of immune repertoire sequencing to predict rejection events, as this proof-of-concept study is limited to a small number of patients who were selected based on several criteria including the availability of a large number of samples and the absence or presence of rejection events.ConclusionsIf confirmed in larger, prospective studies, the method described here has potential applications in the tailored management of post-transplant immunosuppression and, more broadly, as a method for assessing the overall activity of the immune system.
The development of de novo human leukocyte antigen (HLA) donor specific antibodies (DSA), detected by both cytotoxic or solid phase assays, was considered the major risk factor for cardiac graft failure in heart transplantation.However, it was shown that not all patients with persistent production of DSA suffered loss of their allografts. The ability to activate complement may be an important factor differentiating clinically relevant DSA from non-relevant DSA. Recently, a C1q-binding assay (C1qScreen; One Lambda, Inc. Canoga Park, CA) has been developed to identify complement-fixing HLA antibodies with high sensitivity and specificity.The aim of this study was to investigate the association between C1q-binding ability of HLA-DSA and the clinical outcomes post-transplant to identify clinically significant de novo DSA in heart transplantation. Methods: We enrolled 64 consecutive patients who received heart transplant between May 1999 and January 2015 in our institute. Sixty of 64 patients (93.7%) were screened for the presence of circulating DSA using Luminex Single Antigen Flow Bead assays between June 2014 and August 2015, and patients with post-transplant DSA with mean fluorescence intensity (MFI) > 500 were selected to assess C1q fixation by C1q-binding assays. The clinical outcomes were compared with the results. Results: Of 60 patients, twelve patients were considered as DSA positive (MFI> 500, range 698-5952, class 1: 75% class 2: 17%, class1+2:8%). All of these patients were identified as C1q negative. As the results, we divided all patients into two groups; group DSA+, C1q-(n= 12) and group DSA-(n= 48). Rejection episodes, development of cardiac graft vasculopathy, cardiac function (EF, BNP), cardiac event-free survival and overall mortality were not statistically different between the two groups.
Conclusion:Patients producing C1q-negative DSA had good graft survival, which was comparable to that of DSA negative patients. Adding the assessment of the complement-binding capacity of DSA might redefine the traditional risk stratification of de novo DSA positive patients.
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