Publications by authors named "Philip F Halloran"

92 Publications

Three-months course of intragraft transcriptional changes in kidney allografts with early histological minimal injury.

Transpl Int 2021 Mar 1. Epub 2021 Mar 1.

Transplant laboratory, Institute for Clinical and Experimental Medicine, Prague, Czech Republic.

The tubulitis with/without interstitial inflammation not meeting criteria for T cell-mediated rejection (minimal allograft injury) is the most frequent histological findings in early transplant biopsies. The course of transcriptional changes in sequential kidney graft biopsies has not been studied yet. Molecular phenotypes were analyzed using the Molecular Microscope Diagnostic System (MMDx) in 46 indication biopsies (median 13 post-operative days) diagnosed as minimal allograft injury and in corresponding follow-up biopsies at 3 months. All 46 patients with minimal injury in early biopsy received steroid pulses. MMDx interpreted indication biopsies as no-rejection in 34/46 (74%), T cell-mediated rejection (TCMR) in 4/46 (9%), antibody-mediated rejection in 6/46 (13%) and mixed rejection in 2/46 (4%) cases. Follow-up biopsies were interpreted by MMDx in 37/46 (80%) cases as no rejection, in 4/46 (9%) as TCMR, and in 5/46 (11%) as mixed rejection. Follow-up biopsies showed a decrease in MMDx-assessed acute kidney injury (p=0.001) and an increase of atrophy-fibrosis (p=0.002). The most significant predictor of MMDx rejection scores in follow-up biopsies was the tubulitis classifier score in initial biopsies (AUC=0.84, p=0.002), confirmed in multivariate binary regression (OR=16, p=0.016). Molecular tubulitis score at initial biopsy has the potential to discriminate patients at risk for molecular rejection score at follow-up biopsy.
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http://dx.doi.org/10.1111/tri.13856DOI Listing
March 2021

Integrating molecular and histologic interpretation of transplant biopsies.

Clin Transplant 2021 Feb 17:e14244. Epub 2021 Feb 17.

Alberta Transplant Applied Genomics Centre, Edmonton, AB, Canada.

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http://dx.doi.org/10.1111/ctr.14244DOI Listing
February 2021

A Randomized Clinical Trial of Anti-IL-6 Antibody Clazakizumab in Late Antibody-Mediated Kidney Transplant Rejection.

J Am Soc Nephrol 2021 Mar 18;32(3):708-722. Epub 2020 Dec 18.

Division of Nephrology and Dialysis, Department of Medicine III, Medical University of Vienna, Vienna, Austria

Background: Late antibody-mediated rejection (ABMR) is a leading cause of transplant failure. Blocking IL-6 has been proposed as a promising therapeutic strategy.

Methods: We performed a phase 2 randomized pilot trial to evaluate the safety (primary endpoint) and efficacy (secondary endpoint analysis) of the anti-IL-6 antibody clazakizumab in late ABMR. The trial included 20 kidney transplant recipients with donor-specific, antibody-positive ABMR ≥365 days post-transplantation. Patients were randomized 1:1 to receive 25 mg clazakizumab or placebo (4-weekly subcutaneous injections) for 12 weeks (part A), followed by a 40-week open-label extension (part B), during which time all participants received clazakizumab.

Results: Five (25%) patients under active treatment developed serious infectious events, and two (10%) developed diverticular disease complications, leading to trial withdrawal. Those receiving clazakizumab displayed significantly decreased donor-specific antibodies and, on prolonged treatment, modulated rejection-related gene-expression patterns. In 18 patients, allograft biopsies after 51 weeks revealed a negative molecular ABMR score in seven (38.9%), disappearance of capillary C4d deposits in five (27.8%), and resolution of morphologic ABMR activity in four (22.2%). Although proteinuria remained stable, the mean eGFR decline during part A was slower with clazakizumab compared with placebo (-0.96; 95% confidence interval [95% CI], -1.96 to 0.03 versus -2.43; 95% CI, -3.40 to -1.46 ml/min per 1.73 m per month, respectively, =0.04). During part B, the slope of eGFR decline for patients who were switched from placebo to clazakizumab improved and no longer differed significantly from patients initially allocated to clazakizumab.

Conclusions: Although safety data indicate the need for careful patient selection and monitoring, our preliminary efficacy results suggest a potentially beneficial effect of clazakizumab on ABMR activity and progression.
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http://dx.doi.org/10.1681/ASN.2020071106DOI Listing
March 2021

Molecular patterns of isolated tubulitis differ from tubulitis with interstitial inflammation in early indication biopsies of kidney allografts.

Sci Rep 2020 12 17;10(1):22220. Epub 2020 Dec 17.

Transplant Laboratory, Institute for Clinical and Experimental Medicine, 140 21, Prague, Czech Republic.

The Banff 2019 kidney allograft pathology update excluded isolated tubulitis without interstitial inflammation (ISO-T) from the category of borderline (suspicious) for acute T cell-mediated rejection due to its proposed benign clinical outcome. In this study, we explored the molecular assessment of ISO-T. ISO-T or interstitial inflammation with tubulitis (I + T) was diagnosed in indication biopsies within the first 14 postoperative days. The molecular phenotype of ISO-T was compared to I + T either by using RNA sequencing (n = 16) or by Molecular Microscope Diagnostic System (MMDx, n = 51). RNA sequencing showed lower expression of genes related to interferon-y (p = 1.5 *10), cytokine signaling (p = 2.1 *10) and inflammatory response (p = 1.0*10) in the ISO-T group than in I + T group. Transcripts with increased expression in the I + T group overlapped significantly with previously described pathogenesis-based transcript sets associated with cytotoxic and effector T cell transcripts, and with T cell-mediated rejection (TCMR). MMDx classified 25/32 (78%) ISO-T biopsies and 12/19 (63%) I + T biopsies as no-rejection. ISO-T had significantly lower MMDx scores for interstitial inflammation (p = 0.014), tubulitis (p = 0.035) and TCMR (p = 0.016) compared to I + T. Fewer molecular signals of inflammation in isolated tubulitis suggest that this is also a benign phenotype on a molecular level.
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http://dx.doi.org/10.1038/s41598-020-79332-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7746707PMC
December 2020

New concepts in chronic antibody-mediated kidney allograft rejection: prevention and treatment.

Curr Opin Organ Transplant 2021 Feb;26(1):97-105

Division of Nephrology and Dialysis, Department of Medicine III, Medical University of Vienna, Vienna, Austria.

Purpose Of Review: Chronic antibody-mediated rejection (AMR) is a cardinal cause of transplant failure, with currently no proven effective prevention or treatment. The present review will focus on new therapeutic concepts currently under clinical evaluation.

Recent Findings: One interesting treatment approach may be interference with interleukin-6 (IL-6) signaling to modulate B-cell immunity and donor-specific antibody (DSA) production. Currently, a large phase III randomized controlled trial is underway to clarify the safety and efficacy of clazakizumab, a high-affinity anti-IL-6 antibody, in chronic AMR. A prevention/treatment strategy may be costimulation blockade using belatacept to interfere with germinal center responses and DSA formation. In a recent uncontrolled study, belatacept conversion was shown to stabilize renal function and dampen AMR activity. Moreover, preliminary clinical results suggest efficacy of CD38 antibodies to deplete plasma and natural killer cells to treat AMR, with anecdotal reports demonstrating at least transient resolution of active rejection.

Summary: There are promising concepts on the horizon for the prevention and treatment of chronic AMR. The design of adequately powered placebo-controlled trials to clarify the safety and efficacy of such new therapies, however, remains a big challenge, and will rely on the definition of precise surrogate endpoints predicting long-term allograft survival. Mapping the natural history of AMR would greatly help the understanding of who would derive benefits from treatment.
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http://dx.doi.org/10.1097/MOT.0000000000000832DOI Listing
February 2021

Discovering novel injury features in kidney transplant biopsies associated with TCMR and donor aging.

Am J Transplant 2020 Oct 26. Epub 2020 Oct 26.

Alberta Transplant Applied Genomics Centre, Edmonton, Alberta, Canada.

We previously characterized the molecular changes in acute kidney injury (AKI) and chronic kidney disease (CKD) in kidney transplant biopsies, but parenchymal changes selective for specific types of injury could be missed by such analyses. The present study searched for injury changes beyond AKI and CKD related to specific scenarios, including correlations with donor age. We defined injury using previously defined gene sets and classifiers and used principal component analysis to discover new injury dimensions. As expected, Dimension 1 distinguished normal vs. injury, and Dimension 2 separated early AKI from late CKD, correlating with time posttransplant. However, Dimension 3 was novel, distinguishing a set of genes related to epithelial polarity (e.g., PARD3) that were increased in early AKI and decreased in T cell-mediated rejection (TCMR) but not in antibody-mediated rejection. Dimension 3 was increased in kidneys from older donors and was particularly important in survival of early kidneys. Thus high Dimension 3 scores emerge as a previously unknown element in the kidney response-to-injury that affects epithelial polarity genes and is increased in AKI but depressed in TCMR, indicating that in addition to general injury elements, certain injury elements are selective for specific pathologic mechanisms. (ClinicalTrials.gov NCT01299168).
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http://dx.doi.org/10.1111/ajt.16374DOI Listing
October 2020

Molecular T-cell‒mediated rejection in transbronchial and mucosal lung transplant biopsies is associated with future risk of graft loss.

J Heart Lung Transplant 2020 Dec 26;39(12):1327-1337. Epub 2020 Aug 26.

Department of Medicine, University of Alberta, Edmonton, Alberta, Canada. Electronic address:

Background: We previously developed molecular assessment systems for lung transplant transbronchial biopsies (TBBs) with high surfactant and bronchial mucosal biopsies, identifying T-cell‒mediated rejection (TCMR) on the basis of the expression of rejection-associated transcripts, but the relationship of rejection to graft loss is unknown. This study aimed to develop molecular assessments for TBBs and mucosal biopsies and to establish the impact of molecular TCMR on graft survival.

Methods: We used microarrays and machine learning to assign TCMR scores to an expanded cohort of 457 TBBs (367 high surfactant plus 90 low surfactant) and 314 mucosal biopsies. We tested the score agreement between TBB-TBB, mucosal-mucosal, and TBB-mucosal biopsy pairs in the same patient. We also assessed the association of molecular TCMR scores with graft loss (death or retransplantation) and compared it with the prognostic associations for histology and donor-specific antibodies.

Results: The molecular TCMR scores assigned in all the TBBs performed similarly to those in high-surfactant TBBs, indicating that variation in alveolation in TBBs does not prevent the detection of TCMR. Mucosal biopsy pieces showed less piece-to-piece variation than TBBs. TCMR scores in TBBs agreed with those in mucosal biopsies. In both TBBs and mucosal biopsies, molecular TCMR was associated with graft loss, whereas histologic rejection and donor-specific antibodies were not.

Conclusions: Molecular TCMR can be detected in TBBs regardless of surfactant and in mucosal biopsies, which show less variability in the sampled tissue than TBBs. On the basis of these findings, molecular TCMR appears to be an important predictor of the risk of future graft failure.

Trial Registration: ClinicalTrials.gov NCT02812290.
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http://dx.doi.org/10.1016/j.healun.2020.08.013DOI Listing
December 2020

Impact of belatacept conversion on kidney transplant function, histology, and gene expression - a single-center study.

Transpl Int 2020 Nov 7;33(11):1458-1471. Epub 2020 Sep 7.

Alberta Transplant Applied Genomics Center, Edmonton, AB, Canada.

Prior studies on belatacept conversion from calcineurin inhibitor (CNI) have been limited by an absence of postconversion surveillance biopsies that could underestimate subclinical rejection, or a case-controlled design. A total of 53 adult patients with allograft dysfunction underwent belatacept conversion (median: 6 months) post-transplant. At a median follow-up = 2.5 years, patient survival was 94% with a death-censored graft survival of 85%. Seven (13%) patients had acute rejection (including 3 subclinical) at median 6 months postconversion. Overall, eGFR improved (P = <0.001) from baseline = 31±15 to 40.2 ± 17.6 ml/min/1.73m by 6 months postconversion, but then stayed stable. This improvement was also observed (P < 0.001) in comparison with a propensity matched control cohort on CNI, where eGFR stayed stable (mean ~ 32ml/min/1.72m ) over 2-year follow-up. Patients converted < 6 months post-transplant were more likely to have a long-term improvement in kidney function. Paired gene expression analysis of 30 (of 53) consecutive pre- and postconversion surveillance biopsies did not reveal changes in inflammation/acute injury; although atrophy-fibrosis score worsened (mean = 0.28 to 0.44; P = 0.005). Thus, improvement in renal function with belatacept conversion occurred early and then sustained in comparison with controls where renal function remained unchanged overtime. We were unable to show molecular signals that could be related to CNI administration and regressed after withdrawal.
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http://dx.doi.org/10.1111/tri.13718DOI Listing
November 2020

Factors associated with kidney graft survival in pure antibody-mediated rejection at the time of indication biopsy: Importance of parenchymal injury but not disease activity.

Am J Transplant 2020 Jun 28. Epub 2020 Jun 28.

Alberta Transplant Applied Genomics Centre, Edmonton, Alberta, Canada.

We studied the relative association of clinical, histologic, and molecular variables with risk of kidney transplant failure after an indication biopsy, both in all kidneys and in kidneys with pure antibody-mediated rejection (ABMR). From a prospective study of 1679 biopsies with histologic and molecular testing, we selected one random biopsy per patient (N = 1120), including 321 with pure molecular ABMR. Diagnoses were associated with actuarial survival differences but not good predictions. Therefore we concentrated on clinical (estimated GFR [eGFR], proteinuria, time posttransplant, donor-specific antibody [DSA]) and molecular and histologic features reflecting injury (acute kidney injury [AKI] and atrophy-fibrosis [chronic kidney disease (CKD)] and rejection. For all biopsies, univariate analysis found that failure was strongly associated with low eGFR, AKI, CKD, and glomerular deterioration, but not with rejection activity. In molecular ABMR, the findings were similar: Molecular and histologic activity and DSA were not important compared with injury. Survival in DSA-negative and DSA-positive molecular ABMR was similar. Multivariate survival analysis confirmed the dominance of molecular AKI, CKD, and eGFR. Thus, at indication biopsy, the dominant predictors of failure, both in all kidneys and in ABMR, were related to molecular AKI and CKD and to eGFR, not rejection activity, presumably because rejection confers risk via injury.
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http://dx.doi.org/10.1111/ajt.16161DOI Listing
June 2020

The Molecular Microscope Diagnostic System meets eminence-based medicine: A clinician's perspective.

Am J Transplant 2020 10 13;20(10):2964-2965. Epub 2020 May 13.

Alberta Transplant Applied Genomics Centre, Edmonton, Alberta, Canada.

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http://dx.doi.org/10.1111/ajt.15940DOI Listing
October 2020

CD38 Antibody Daratumumab for the Treatment of Chronic Active Antibody-mediated Kidney Allograft Rejection.

Transplantation 2021 02;105(2):451-457

Division of Nephrology and Dialysis, Department of Medicine III, Medical University of Vienna, Vienna, Austria.

Background: Late antibody-mediated rejection (AMR) is a major cause of transplant failure. Potential therapeutic targets are plasma cells and natural killer (NK) cells, both expressing high levels of CD38.

Methods: Here, we report the use of CD38 monoclonal antibody daratumumab (9-mo course) in a kidney allograft recipient diagnosed with smoldering myeloma and anti-HLA class II donor-specific antibody-positive chronic active AMR 13 years after transplantation. Patient monitoring included serial HLA single-antigen testing, peripheral blood immune cell phenotyping, as well as follow-up allograft and bone marrow biopsies at 3 and 9 months, including analyses of rejection-related gene expression patterns.

Results: Daratumumab led to persistent CD138+ cell depletion in the bone marrow and blood and substantially decreased NK cells counts in blood and graft tissue. At the same time, donor-specific antibody in serum disappeared, and in vitro alloantibody production by CD138+ cells enriched from bone marrow aspirates was abrogated. A 3-month follow-up biopsy revealed a complete resolution of microcirculation inflammation (g+ptc: 3 to 0) and molecular AMR activity (AMR score: 0.79 to <0.2). The same biopsy showed (subclinical) tubulointerstitial inflammation, which prompted steroid treatment. Over an observation period of 12 months, graft function stabilized.

Conclusions: Targeting CD38 for plasma cell and NK cell depletion may be an effective strategy to counteract AMR. Our results may encourage the design of future trials to clarify the role of this innovative treatment concept in organ transplantation.
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http://dx.doi.org/10.1097/TP.0000000000003247DOI Listing
February 2021

High-activity Classical and Alternative Complement Pathway Genotypes-Association With Donor-specific Antibody-triggered Injury and Renal Allograft Survival.

Transplant Direct 2020 Mar 10;6(3):e534. Epub 2020 Feb 10.

Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Vienna, Austria.

Background: Complement may contribute to donor-specific antibody (DSA)-triggered transplant injury. Here, we investigated whether the intrinsic strength of classical pathway and alternative pathway (AP) relates to the pathogenicity of DSA.

Methods: Classical pathway and AP high-activity genotypes were defined according to C4 gene copy number and the presence of functional polymorphisms in C3 (C3), factor B (fB), and factor H (fH) genes. Associations of these genotypes with blood complement profiles and morphologic/molecular rejection features were evaluated in a cohort of 83 DSA-positive patients (antibody-mediated rejection [AMR], n = 47) identified upon cross-sectional screening of 741 kidney allograft recipients ≥180 days posttransplantation. Associations with long-term graft survival were evaluated in a larger kidney transplant cohort (n = 660) not enriched for a specific type of rejection.

Results: In the cohort of DSA-positive subjects, the number of C4 gene copies was related to C4 protein levels in serum and capillary C4d staining, but not AMR activity. Patients with a high-activity AP complotype, which was associated with complement consumption in serum, showed enhanced microcirculation inflammation (median glomerulitis plus peritubular capillaritis score, 2 [interquartile range, 0-4 versus 1 0-2]; = 0.037). In the larger transplant cohort, this complotype was associated with a slightly increased risk of graft loss (hazard ratio, 1.52; 95% confidence interval, 1.02-2.25; = 0.038 and multivariable Cox model, 1.55; 1.04-2.32; = 0.031).

Conclusions: Our study suggests a contribution of complement genetics to the phenotypic presentation of AMR. Future studies will have to clarify whether a possible association of AP strength with graft survival relates to enhanced antibody-triggered injury.
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http://dx.doi.org/10.1097/TXD.0000000000000978DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7056277PMC
March 2020

The molecular diagnosis of rejection in liver transplant biopsies: First results of the INTERLIVER study.

Am J Transplant 2020 08 9;20(8):2156-2172. Epub 2020 Apr 9.

Alberta Transplant Applied Genomics Centre, Edmonton, Alberta, Canada.

Molecular diagnosis of rejection is emerging in kidney, heart, and lung transplant biopsies and could offer insights for liver transplant biopsies. We measured gene expression by microarrays in 235 liver transplant biopsies from 10 centers. Unsupervised archetypal analysis based on expression of previously annotated rejection-related transcripts identified 4 groups: normal "R1 " (N = 129), T cell-mediated rejection (TCMR) "R2 " (N = 37), early injury "R3 " (N = 61), and fibrosis "R4 " (N = 8). Groups differed in median time posttransplant, for example, R3 99 days vs R4 3117 days. R2 biopsies expressed typical TCMR-related transcripts, for example, intense IFNG-induced effects. R3 displayed increased expression of parenchymal injury transcripts (eg, hypoxia-inducible factor EGLN1). R4 biopsies showed immunoglobulin transcripts and injury-related transcripts. R2 correlated with histologic rejection although with many discrepancies, and R4 with fibrosis. R2 , R3 , and R4 correlated with liver function abnormalities. Supervised classifiers trained on histologic rejection showed less agreement with histology than unsupervised R2 scores. No confirmed cases of clinical antibody-mediated rejection (ABMR) were present in the population, and strategies that previously revealed ABMR in kidney and heart transplants failed to reveal a liver ABMR phenotype. In conclusion, molecular analysis of liver transplant biopsies detects rejection, has the potential to resolve ambiguities, and could assist with immunosuppressive management.
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http://dx.doi.org/10.1111/ajt.15828DOI Listing
August 2020

Discrepancy analysis comparing molecular and histology diagnoses in kidney transplant biopsies.

Am J Transplant 2020 05 23;20(5):1341-1350. Epub 2020 Jan 23.

Department of Medicine, Division of Nephrology and Transplant Immunology, University of Alberta, Edmonton, Alberta, Canada.

Discrepancy analysis comparing two diagnostic platforms offers potential insights into both without assuming either is always correct. Having optimized the Molecular Microscope Diagnostic System (MMDx) in renal transplant biopsies, we studied discrepancies within MMDx (reports and sign-out comments) and between MMDx and histology. Interpathologist discrepancies have been documented previously and were not assessed. Discrepancy cases were classified as "clear" (eg, antibody-mediated rejection [ABMR] vs T cell-mediated rejection [TCMR]), "boundary" (eg, ABMR vs possible ABMR), or "mixed" (eg, Mixed vs ABMR). MMDx report scores showed 99% correlations; sign-out interpretations showed 7% variation between observers, all located around boundaries. Histology disagreed with MMDx in 37% of biopsies, including 315 clear discrepancies, all with implications for therapy. Discrepancies were distributed widely in all histology diagnoses but increased in some scenarios; for example, histology TCMR contained 14% MMDx ABMR and 20% MMDx no rejection. MMDx usually gave unambiguous diagnoses in cases with ambiguous histology, for example, borderline and transplant glomerulopathy. Histology lesions or features associated with more frequent discrepancies (eg, tubulitis, arteritis, and polyomavirus nephropathy) were not associated with increased MMDx uncertainty, indicating that MMDx can clarify biopsies with histologic ambiguity. The patterns of histology-MMDx discrepancies highlight specific histology diagnoses in which MMDx assessment should be considered for guiding therapy.
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http://dx.doi.org/10.1111/ajt.15752DOI Listing
May 2020

Molecular phenotyping of rejection-related changes in mucosal biopsies from lung transplants.

Am J Transplant 2020 04 1;20(4):954-966. Epub 2019 Dec 1.

Department of Medicine, University of Alberta, Edmonton, Alberta, Canada.

Diagnosing lung transplant rejection currently depends on histologic assessment of transbronchial biopsies (TBB) with limited reproducibility and considerable risk of complications. Mucosal biopsies are safer but not histologically interpretable. Microarray-based diagnostic systems for TBBs and other transplants suggest such systems could assess mucosal biopsies as well. We studied 243 mucosal biopsies from the third bronchial bifurcation (3BMBs) collected from seven centers and classified them using unsupervised machine learning algorithms. Using the expression of a set of rejection-associated transcripts annotated in kidneys and validated in hearts and lung transplant TBBs, the algorithms identified and scored major rejection and injury-related phenotypes in 3BMBs without need for labeled training data. No rejection or injury, rejection, late inflammation, and recent injury phenotypes were thus scored in new 3BMBs. The rejection phenotype correlated with IFNG-inducible transcripts, the hallmarks of rejection. Progressive atrophy-related changes reflected by the late inflammation phenotype in 3BMBs suggest widespread time-dependent airway deterioration, which was especially pronounced after two years posttransplant. Thus molecular assessment of 3BMBs can detect rejection in a previously unusable biopsy format with potential utility in patients with severe lung dysfunction where TBB is not possible and provide unique insights into airway deterioration. ClinicalTrials.gov NCT02812290.
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http://dx.doi.org/10.1111/ajt.15685DOI Listing
April 2020

Complement Markers in Blood and Urine: No Diagnostic Value in Late Silent Antibody-Mediated Rejection.

Transplant Direct 2019 Jul 27;5(7):e470. Epub 2019 Jun 27.

IIIrd Department of Internal Medicine and MTA-SE Research Group of Immunology and Hematology, Research Laboratory, Hungarian Academy of Sciences and Semmelweis University, Budapest, Hungary.

Background: Antibody-mediated rejection (AMR) is a major cause of kidney allograft failure. Its molecular mechanisms are multifaceted and may include a role of complement activation via the classical pathway. Here, we investigated whether noninvasive complement monitoring adds predictive power to the diagnosis of AMR in the setting of donor-specific antibody (DSA) positivity.

Methods: In this cross-sectional study, 741 kidney transplant recipients with stable graft function ≥180 days posttransplantation were screened for the presence of human leukocyte antigen (HLA) alloantibodies. Eighty-three of 111 DSA-positive recipients underwent protocol biopsies and were tested for blood and urinary levels of complement proteins (C1q, C4, C3) and activation products (C4d, C3a, C5a, C5b-9).

Results: Forty-seven recipients were diagnosed with AMR, and 21 were C4d-positive. While biopsy-confirmed AMR (and C4d) associated with DSA-binding strength (IgG mean fluorescence intensity of the immunodominant DSA versus AMR; area under the receiver operating characteristic curve: 0.76), tested complement markers did not have any predictive value for rejection (area under the receiver operating characteristic curve: 0.49-0.56). There were, however, tight correlations between complement activation products in urine and protein/creatinine ratio ( = 0.44-0.64; < 0.001). Analysis of death-censored graft survival over a median of 60 months revealed no independent associations with levels of complement markers in blood or urine.

Conclusions: Complement patterns in blood and urine failed to identify AMR in late biopsies and may have no relevant diagnostic value in this particular context.
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http://dx.doi.org/10.1097/TXD.0000000000000915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6616143PMC
July 2019

Non-HLA agonistic anti-angiotensin II type 1 receptor antibodies induce a distinctive phenotype of antibody-mediated rejection in kidney transplant recipients.

Kidney Int 2019 07 15;96(1):189-201. Epub 2019 Mar 15.

Clinic for Nephrology and Critical Care Medicine, Campus Virchow-Klinikum, Charité Universitätsmedizin Berlin, Germany; Center for Cardiovascular Research, Medical Faculty of the Charité Berlin, Berlin, Germany.

Anti-angiotensin II type 1 receptor (AT1R) antibodies have been associated with allograft rejection. We hypothesized that circulating AT1R antibodies might identify kidney transplant recipients at increased risk of allograft rejection and loss who are not identified by the HLA system. We prospectively enrolled 1845 kidney transplant recipients from two centers. Donor-specific HLA antibodies (DSAs) and AT1R antibodies were measured at the time of the first acute rejection episode or at 1 year post-transplant. Allograft biopsy was performed to evaluate the rejection phenotype and to assess for endothelial activation. Overall, 371 (20.1%) participants had AT1R antibodies, 334 (18.1%) had DSAs, and 133 (7.2%) had both. AT1R antibodies were associated with an increased risk of allograft loss (adjusted HR 1.49, 95% CI 1.07-2.06 for AT1R antibodies alone and 2.26, 95% CI 1.52-3.36 for AT1R antibodies and DSAs). Participants with AT1R antibodies had a higher incidence of antibody-mediated rejection (AMR) compared with participants without AT1R antibodies (25.0% vs. 12.9%). Among 77 participants with histological features of AMR but without DSAs, 51 (66.2%) had AT1R antibodies. Compared to participants with prototypical DSA-mediated rejection, those with AT1R antibody-associated rejection had a higher prevalence of hypertension, more vascular rejection with arterial inflammation, higher levels of endothelial-associated transcripts, and lack of complement deposition in allograft capillaries. Thus, AT1R antibodies may identify kidney transplant recipients at high risk of allograft rejection and loss, independent of the HLA system. Recognition of complement-independent AT1R antibody-mediated vascular rejection could lead to the development of new treatment strategies to improve allograft survival.
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http://dx.doi.org/10.1016/j.kint.2019.01.030DOI Listing
July 2019

The therapeutic challenge of late antibody-mediated kidney allograft rejection.

Transpl Int 2019 Aug 7;32(8):775-788. Epub 2019 May 7.

Alberta Transplant Applied Genomics Centre (ATAGC), University of Alberta, Edmonton, AB, Canada.

Late antibody-mediated rejection (ABMR) is a cardinal cause of kidney allograft failure, manifesting as a continuous and, in contrast with early rejection, often clinically silent alloimmune process. While significant progress has been made towards an improved understanding of its molecular mechanisms and the definition of diagnostic criteria, there is still no approved effective treatment. In recent small randomized controlled trials, therapeutic strategies with promising results in observational studies, such as proteasome inhibitor bortezomib, anti-C5 antibody eculizumab, or high dose intravenous immunoglobulin plus rituximab, had no significant impact in late and/or chronic ABMR. Such disappointing results reinforce a need of new innovative treatment strategies. Potential candidates may be the interference with interleukin-6 to modulate B cell alloimmunity, or innovative compounds that specifically target antibody-producing plasma cells, such as antibodies against CD38. Given the phenotypic heterogeneity of ABMR, the design of adequate systematic trials to assess the safety and efficiency of such therapies, however, is challenging. Several trials are currently being conducted, and new developments will hopefully provide us with effective ways to counteract the deleterious impact of antibody-mediated graft injury. Meanwhile, the weight of evidence would suggest that, when approaching using existing treatments for established antibody-mediated rejection, "less may be more".
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http://dx.doi.org/10.1111/tri.13436DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6850109PMC
August 2019

Generating automated kidney transplant biopsy reports combining molecular measurements with ensembles of machine learning classifiers.

Am J Transplant 2019 10 10;19(10):2719-2731. Epub 2019 Apr 10.

Alberta Transplant Applied Genomics Centre, Alberta, Canada.

We previously reported a system for assessing rejection in kidney transplant biopsies using microarray-based gene expression data, the Molecular Microscope Diagnostic System (MMDx). The present study was designed to optimize the accuracy and stability of MMDx diagnoses by replacing single machine learning classifiers with ensembles of diverse classifier methods. We also examined the use of automated report sign-outs and the agreement between multiple human interpreters of the molecular results. Ensembles generated diagnoses that were both more accurate than the best individual classifiers, and nearly as stable as the best, consistent with expectations from the machine learning literature. Human experts had ≈93% agreement (balanced accuracy) signing out the reports, and random forest-based automated sign-outs showed similar levels of agreement with the human experts (92% and 94% for predicting the expert MMDx sign-outs for T cell-mediated (TCMR) and antibody-mediated rejection (ABMR), respectively). In most cases disagreements, whether between experts or between experts and automated sign-outs, were in biopsies near diagnostic thresholds. Considerable disagreement with histology persisted. The balanced accuracies of MMDx sign-outs for histology diagnoses of TCMR and ABMR were 73% and 78%, respectively. Disagreement with histology is largely due to the known noise in histology assessments (ClinicalTrials.gov NCT01299168).
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http://dx.doi.org/10.1111/ajt.15351DOI Listing
October 2019

An integrated molecular diagnostic report for heart transplant biopsies using an ensemble of diagnostic algorithms.

J Heart Lung Transplant 2019 06 6;38(6):636-646. Epub 2019 Feb 6.

Alberta Transplant Applied Genomics Centre, University of Alberta, Edmonton, Alberta, Canada; Department of Medicine, University of Alberta, Edmonton, Alberta, Canada. Electronic address:

Background: We previously reported a microarray-based diagnostic system for heart transplant endomyocardial biopsies (EMBs), using either 3-archetype (3AA) or 4-archetype (4AA) unsupervised algorithms to estimate rejection. In the present study we examined the stability of machine-learning algorithms in new biopsies, compared 3AA vs 4AA algorithms, assessed supervised binary classifiers trained on histologic or molecular diagnoses, created a report combining many scores into an ensemble of estimates, and examined possible automated sign-outs.

Methods: We studied 889 EMBs from 454 transplant recipients at 8 centers: the initial cohort (N = 331) and a new cohort (N = 558). Published 3AA algorithms derived in Cohort 331 were tested in Cohort 558, the 3AA and 4AA models were compared, and supervised binary classifiers were created.

Results: A`lgorithms derived in Cohort 331 performed similarly in new biopsies despite differences in case mix. In the combined cohort, the 4AA model, including a parenchymal injury score, retained correlations with histologic rejection and DSA similar to the 3AA model. Supervised molecular classifiers predicted molecular rejection (areas under the curve [AUCs] >0.87) better than histologic rejection (AUCs <0.78), even when trained on histology diagnoses. A report incorporating many AA and binary classifier scores interpreted by 1 expert showed highly significant agreement with histology (p < 0.001), but with many discrepancies, as expected from the known noise in histology. An automated random forest score closely predicted expert diagnoses, confirming potential for automated signouts.

Conclusions: Molecular algorithms are stable in new populations and can be assembled into an ensemble that combines many supervised and unsupervised estimates of the molecular disease states.
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http://dx.doi.org/10.1016/j.healun.2019.01.1318DOI Listing
June 2019

Molecular assessment of rejection and injury in lung transplant biopsies.

J Heart Lung Transplant 2019 05 6;38(5):504-513. Epub 2019 Feb 6.

Department of Medicine, University of Alberta, Edmonton, Alberta, Canada; Alberta Transplant Applied Genomics Center, Edmonton, Alberta, Canada. Electronic address:

Background: Improved understanding of lung transplant disease states is essential because failure rates are high, often due to chronic lung allograft dysfunction. However, histologic assessment of lung transplant transbronchial biopsies (TBBs) is difficult and often uninterpretable even with 10 pieces.

Methods: We prospectively studied whether microarray assessment of single TBB pieces could identify disease states and reduce the amount of tissue required for diagnosis. By following strategies successful for heart transplants, we used expression of rejection-associated transcripts (annotated in kidney transplant biopsies) in unsupervised machine learning to identify disease states.

Results: All 242 single-piece TBBs produced reliable transcript measurements. Paired TBB pieces available from 12 patients showed significant similarity but also showed some sampling variance. Alveolar content, as estimated by surfactant transcript expression, was a source of sampling variance. To offset sampling variation, for analysis, we selected 152 single-piece TBBs with high surfactant transcripts. Unsupervised archetypal analysis identified 4 idealized phenotypes (archetypes) and scored biopsies for their similarity to each: normal; T-cell‒mediated rejection (TCMR; T-cell transcripts); antibody-mediated rejection (ABMR)-like (endothelial transcripts); and injury (macrophage transcripts). Molecular TCMR correlated with histologic TCMR. The relationship of molecular scores to histologic ABMR could not be assessed because of the paucity of ABMR in this population.

Conclusions: Molecular assessment of single-piece TBBs can be used to classify lung transplant biopsies and correlated with rejection histology. Two or 3 pieces for each TBB will probably be needed to offset sampling variance.
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http://dx.doi.org/10.1016/j.healun.2019.01.1317DOI Listing
May 2019

Molecular phenotype of kidney transplant indication biopsies with inflammation in scarred areas.

Am J Transplant 2019 05 13;19(5):1356-1370. Epub 2018 Dec 13.

Alberta Transplant Applied Genomics Centre, Edmonton, Alberta, Canada.

In kidney transplant biopsies, inflammation in areas of atrophy-fibrosis (i-IFTA) is associated with increased risk of failure, presumably because inflammation is evoked by recent parenchymal injury from rejection or other insults, but some cases also have rejection. The present study explored the frequency of rejection in i-IFTA, by using histology Banff 2015 and a microarray-based molecular diagnostic system (MMDx). In unselected indication biopsies (108 i-IFTA, 73 uninflamed IFTA [i0-IFTA], and 53 no IFTA), i-IFTA biopsies occurred later, showed more scarring, and had more antibody-mediated rejection (ABMR) based on histology (28%) and MMDx (45%). T cell-mediated rejection (TCMR) was infrequent in i-IFTA based on histology (8%) and MMDx (16%). Twelve i-IFTA biopsies (11%) had molecular TCMR not diagnosed by histology, although 6 were called borderline and almost all had histologic TCMR lesions. The prominent feature of i-IFTA biopsies was molecular injury (eg, acute kidney injury [AKI] transcripts). In multivariate analysis of biopsies >1 year posttransplant, the strongest associations with graft loss were AKI transcripts and histologic atrophy-scarring; i-IFTA was not significant when molecular AKI was included. We conclude that i-IFTA in indication biopsies reflects recent/ongoing parenchymal injury, often with concomitant ABMR but few with TCMR. Thus, the application of Banff i-IFTA in the population of late biopsies needs to be reconsidered.
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http://dx.doi.org/10.1111/ajt.15178DOI Listing
May 2019

Exploring the cardiac response to injury in heart transplant biopsies.

JCI Insight 2018 10 18;3(20). Epub 2018 Oct 18.

Alberta Transplant Applied Genomics Centre, Edmonton, Alberta, Canada.

Background: Because injury is universal in organ transplantation, heart transplant endomyocardial biopsies present an opportunity to explore response to injury in heart parenchyma. Histology has limited ability to assess injury, potentially confusing it with rejection, whereas molecular changes have potential to distinguish injury from rejection. Building on previous studies of transcripts associated with T cell-mediated rejection (TCMR) and antibody-mediated rejection (ABMR), we explored transcripts reflecting injury.

Methods: Microarray data from 889 prospectively collected endomyocardial biopsies from 454 transplant recipients at 14 centers were subjected to unsupervised principal component analysis and archetypal analysis to detect variation not explained by rejection. The resulting principal component and archetype scores were then examined for their transcript, transcript set, and pathway associations and compared to the histology diagnoses and left ventricular function.

Results: Rejection was reflected by principal components PC1 and PC2, and by archetype scores S2TCMR, and S3ABMR, with S1normal indicating normalness. PC3 and a new archetype score, S4injury, identified unexplained variation correlating with expression of transcripts inducible in injury models, many expressed in macrophages and associated with inflammation in pathway analysis. S4injury scores were high in recent transplants, reflecting donation-implantation injury, and both S4injury and S2TCMR were associated with reduced left ventricular ejection fraction.

Conclusion: Assessment of injury is necessary for accurate estimates of rejection and for understanding heart transplant phenotypes. Biopsies with molecular injury but no molecular rejection were often misdiagnosed rejection by histology.TRAIL REGISTRATION. ClinicalTrials.gov NCT02670408FUNDING. Roche Organ Transplant Research Foundation, the University of Alberta Hospital Foundation, and Alberta Health Services.
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http://dx.doi.org/10.1172/jci.insight.123674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237487PMC
October 2018

Belatacept rescue for delayed kidney allograft function in a patient with previous combined heart-liver transplant.

Am J Transplant 2018 10 3;18(10):2613-2614. Epub 2018 Aug 3.

Division of Nephrology, Virginia Commonwealth University, Richmond, VA, USA.

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http://dx.doi.org/10.1111/ajt.15003DOI Listing
October 2018

Letter to AJT editor re: Nankivell et al.

Am J Transplant 2018 03 3;18(3):765-766. Epub 2018 Feb 3.

University of Alberta, Edmonton, Alberta, Canada.

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http://dx.doi.org/10.1111/ajt.14653DOI Listing
March 2018

A Randomized Trial of Bortezomib in Late Antibody-Mediated Kidney Transplant Rejection.

J Am Soc Nephrol 2018 02 14;29(2):591-605. Epub 2017 Dec 14.

Division of Nephrology and Dialysis, Department of Medicine III,

Late antibody-mediated rejection (ABMR) is a leading cause of kidney allograft failure. Uncontrolled studies have suggested efficacy of the proteasome inhibitor bortezomib, but no systematic trial has been undertaken to support its use in ABMR. In this randomized, placebo-controlled trial (the Bortezomib in Late Antibody-Mediated Kidney Transplant Rejection [BORTEJECT] Trial), we investigated whether two cycles of bortezomib (each cycle: 1.3 mg/m intravenously on days 1, 4, 8, and 11) prevent GFR decline by halting the progression of late donor-specific antibody (DSA)-positive ABMR. Forty-four DSA-positive kidney transplant recipients with characteristic ABMR morphology (median time after transplant, 5.0 years; pretransplant DSA documented in 19 recipients), who were identified on cross-sectional screening of 741 patients, were randomly assigned to receive bortezomib (=21) or placebo (=23). The 0.5-ml/min per 1.73 m per year (95% confidence interval, -4.8 to 5.8) difference detected between bortezomib and placebo in eGFR slope (primary end point) was not significant (=0.86). We detected no significant differences between bortezomib- and placebo-treated groups in median measured GFR at 24 months (33 versus 42 ml/min per 1.73 m; =0.31), 2-year graft survival (81% versus 96%; =0.12), urinary protein concentration, DSA levels, or morphologic or molecular rejection phenotypes in 24-month follow-up biopsy specimens. Bortezomib, however, associated with gastrointestinal and hematologic toxicity. In conclusion, our trial failed to show that bortezomib prevents GFR loss, improves histologic or molecular disease features, or reduces DSA, despite significant toxicity. Our results reinforce the need for systematic trials to dissect the efficiency and safety of new treatments for late ABMR.
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http://dx.doi.org/10.1681/ASN.2017070818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5791086PMC
February 2018

Review: The transcripts associated with organ allograft rejection.

Am J Transplant 2018 04 23;18(4):785-795. Epub 2017 Dec 23.

Alberta Transplant Applied Genomics Centre, Edmonton, AB, Canada.

The molecular mechanisms operating in human organ transplant rejection are best inferred from the mRNAs expressed in biopsies because the corresponding proteins often have low expression and short half-lives, while small non-coding RNAs lack specificity. Associations should be characterized in a population that rigorously identifies T cell-mediated (TCMR) and antibody-mediated rejection (ABMR). This is best achieved in kidney transplant biopsies, but the results are generalizable to heart, lung, or liver transplants. Associations can be universal (all rejection), TCMR-selective, or ABMR-selective, with universal being strongest and ABMR-selective weakest. Top universal transcripts are IFNG-inducible (eg, CXCL11 IDO1, WARS) or shared by effector T cells (ETCs) and NK cells (eg, KLRD1, CCL4). TCMR-selective transcripts are expressed in activated ETCs (eg, CTLA4, IFNG), activated (eg, ADAMDEC1), or IFNG-induced macrophages (eg, ANKRD22). ABMR-selective transcripts are expressed in NK cells (eg, FGFBP2, GNLY) and endothelial cells (eg, ROBO4, DARC). Transcript associations are highly reproducible between biopsy sets when the same rejection definitions, case mix, algorithm, and technology are applied, but exact ranks will vary. Previously published rejection-associated transcripts resemble universal and TCMR-selective transcripts due to incomplete representation of ABMR. Rejection-associated transcripts are never completely rejection-specific because they are shared with the stereotyped response-to-injury and innate immunity.
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http://dx.doi.org/10.1111/ajt.14600DOI Listing
April 2018

Complement-Activating Anti-HLA Antibodies in Kidney Transplantation: Allograft Gene Expression Profiling and Response to Treatment.

J Am Soc Nephrol 2018 02 17;29(2):620-635. Epub 2017 Oct 17.

Paris Translational Research Center for Organ Transplantation, Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche-S970, Paris, France.

Complement-activating anti-HLA donor-specific antibodies (DSAs) are associated with impaired kidney transplant outcome; however, whether these antibodies induce a specific rejection phenotype and influence response to therapy remains undetermined. We prospectively screened 931 kidney recipients for complement-activating DSAs and used histopathology, immunostaining, and allograft gene expression to assess rejection phenotypes. Effector cells were evaluated using human cell cultures. Additionally, we assessed the effect of complement inhibition on kidney allograft rejection phenotype and the clinical response to complement inhibition in 116 independent kidney recipients with DSAs at transplant receiving rejection prophylaxis with eculizumab or standard of care (plasma exchange and intravenous Ig) at ten international centers. The histomolecular rejection phenotype associated with complement-activating DSA was characterized by complement deposition and accumulation of natural killer cells and monocytes/macrophages in capillaries and increased expression of five biologically relevant genes (, , , , and ) indicative of endothelial activation, IFN response, CD16-mediated natural killer cell activation, and monocyte/macrophage activation. Compared with standard of care, eculizumab specifically abrogated this histomolecular rejection phenotype and associated with a decreased 3-month rejection incidence rate in patients with complement-activating DSAs (56%; 95% confidence interval [95% CI], 38% to 74% versus 19%; 95% CI, 8% to 35%; =0.001) but not in those with noncomplement-activating DSAs (9%; 95% CI, 2% to 25% versus 13%; 95% CI, 2% to 40%; =0.65). In conclusion, circulating complement-activating anti-HLA DSAs are associated with a specific histomolecular kidney allograft rejection phenotype that can be abrogated by complement inhibition.
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http://dx.doi.org/10.1681/ASN.2017050589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5791056PMC
February 2018

A molecular biopsy test based on arteriolar under-hyalinosis reflects increased probability of rejection related to under-immunosuppression.

Am J Transplant 2018 04 10;18(4):821-831. Epub 2017 Nov 10.

Alberta Transplant Applied Genomics Centre Edmonton, Edmonton, AB, Canada.

Calcineurin inhibitor immunosuppressive drugs induce changes such as arteriolar hyalinosis (ah) in kidney transplants, raising the possibility that molecular changes in biopsies related to histologic ah can provide information about drug exposure. We hypothesized that molecular changes associated with less-than-expected hyalinosis might highlight a subpopulation of patients with under-immunosuppression/nonadherence at intermediate times of biopsy posttransplant (TxBx). Using gene expression data from 562 indication biopsies, we developed a molecular classifier for predicting the expected ah lesions (M ) at a particular TxBx. M -scores increased linearly with log(TxBx), but some biopsies had lower scores than expected for TxBx. The deviation of individual M -scores below the predicted regression line of M -scores vs TxBx is defined as "low hyalinosis index." Low hyalinosis indices were frequent in biopsies between 3 months and 3 years posttransplant, particularly among biopsies lacking histologic hyalinosis (ah0), and were associated with T cell-mediated rejection and a subset of recent-onset antibody-mediated rejection without glomerular double contours. In patients with medical records available for review, low hyalinosis indices were frequently associated with physician-recorded concerns about nonadherence (suspected or proven). We conclude that the M classifier and hyalinosis index identify indication biopsies with rejection for which the possibility of patient nonadherence should be considered.
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http://dx.doi.org/10.1111/ajt.14532DOI Listing
April 2018

Response by Loupy et al to Letters Regarding Article, "Gene Expression Profiling for the Identification and Classification of Antibody-Mediated Heart Rejection".

Circulation 2017 08;136(7):698-699

From Paris Translational Research Centre for Organ Transplantation, INSERM, UMR-S970, France (A.L., L.H., J.P.D.); Paris Descartes University and Hôpital Necker, Assistance Publique - Hôpitaux de Paris, France (A.L., L.H., J.P.D., P.B.); Alberta Transplant Applied Genomics Centre; University of Alberta, Edmonton, Canada (P.F.H.); and Cardiology Department and Intensive Care, Georges Pompidou Hospital, Paris, France (X.J.).

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http://dx.doi.org/10.1161/CIRCULATIONAHA.117.029146DOI Listing
August 2017