Summary There are few substantive methods to measure the health of the immune system, and the connection between immune strength and the viral component of the microbiome is poorly understood. Organ transplant recipients are treated with a post-transplant therapy that combines immunosuppressive and antiviral drugs, offering a window into the effects of immune modulation on the virome. We used sequencing of cell-free DNA in plasma to investigate drug-virome interactions in a cohort of organ transplant recipients (656 samples, 96 patients), and find that antivirals and immunosuppressants strongly affect the structure of the virome in plasma. We observe marked virome compositional dynamics at the onset of the therapy and find that the total viral load increases with immunosuppression, whereas the bacterial component of the microbiome remains largely unaffected. The data provide insight into the relationship between the human virome, the state of the immune system, and the effects of pharmacological treatment, and offer a potential application of the virome state to predict immunocompetence.

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.

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Abstracts S75dd-cfDNA levels between 13 rejection, 14 post-rejection, and 217 non-rejection samples (R vs NR, p= 0.96), and no significant difference in dd-cfDNA levels between biopsy grades (132 OR, 103 1R, and 8 2R samples; p= 0.85 for OR vs ≥ 2R). Of interest was the observation that dd-cfDNA levels rose prior to rejection in 3/5 patients, and decreased post-rejection in 5/8 patients; and patients with only Grade 0 biopsies had consistently low dd-cfDNA levels. Conclusion: With approximately half of this pediatric cohort tested, we found no difference in dd-cfDNA levels between biopsy grades or in pediatric patients with treated acute rejection events. Additional studies are required to determine why the positive results reported in adults were not replicated in children.Purpose: Precise anticoagulation management is critical in minimizing the superimposed risks for bleeding and thromboembolism in CF-LVAD patients. Warfarin dose response is influenced by genetic polymorphisms for warfarin metabolism (Cytochrome P4502C9 [CYP2C9]) and the enzymatic target of vitamin K antagonists (vitamin K epoxide reductase complex 1 [VKORC1]). The purpose of this study is to validate several genomics-based warfarin dosing algorithms in CF-LVAD patients. Methods: This retrospective analysis included all CF-LVAD patients at a single academic institution with genotype data available for CYP2C9 (*1, *2, and *3 [rs1799853 and rs1057910]) and VKORC1 (-1639 G> A [rs9923231]) variants. Several dosing algorithms were tested, including those developed by Sconce et al., Anderson et al., Gage et al., and the International Warfarin Pharmacogenomics Consortium (IWPC), as well as the recommendations from the warfarin US package insert. To validate each algorithm, the predicted warfarin dose was compared against the actual dose at discharge from the index implantation. Actual daily dose was regressed on predicted daily dose, from which R2 values were derived. Results: 121 CF-LVAD patients with warfarin genotype data were identified. The mean age was 57.2 years, 20.7% female, 58.7% ischemic etiology, 56.2% Caucasian, and 32.2% African American. 22 patients (24.4%) carried at least one copy of CYP2C9 variant allele (*2, *3, or both), and 28 patients (31.1%) carried at least one copy of VKORC1 variant allele (A). In our cohort the warfarin dose recommended by the US package insert had the lowest correlation to the actual dose required upon discharge (R2 = 0.084). While the algorithms developed by Sconce and Anderson performed slightly better (R2 = 0.160 and 0.196, respectively), the doses suggested by the Gage and IWPC algorithms produced the highest R2 values (Figure). Conclusion: The Gage and IWPC algorithms were most accurate in predicting warfarin dose requirements in CF-LVAD patients.