Purpose: The Molecular Microscope® Diagnostic System (MMDx) system for interpreting heart transplant endomyocardial biopsies (EMBs) currently uses expression of kidney-derived rejection-associated transcripts (RATs) to identify antibody-mediated and T cell-mediated rejection (ABMR, TCMR), but this also detects recent acute injury. We developed a new model to assess rejection and injury separately.

Purpose: Consistent increase in number of sensitized patients at the waitlist requires strategies to prevent antibody-mediated rejection. In Vienna Daratumumab is used in this cohort, an antibody targeting CD38 at plasma cells, natural killer cells and T-regulatory cells. In the past short-term results confirmed its efficacy in heart transplantation (HT), however mid-term results are still missing.

Purpose: Recent evidence suggests that antibody-mediated rejection (AMR) is not exclusively driven by antibodies, but rather involves a variety of cell types. Hence, a monoclonal antibody against CD38, Daratumumab, which targets two components of the complex mechanisms of ABMR, plasma cells and natural killer cells, has been proposed as a promising therapeutic agent. Previously, we demonstrated that Daratumumab is effective in reducing DSA titers and results in a stable clinical course of sensitized patients.

Purpose: In heart transplants, antibody-mediated and T cell-mediated rejection (ABMR, TCMR) are frequent, but the relationship between findings in initial endomyocardial biopsies (EMBs) and future EMBs are not understood.

Purpose The INTERHEART study previously used microarray assessment of 889 heart transplant biopsies to develop the Molecular Microscope Diagnostic System (MMDx) based on expression of rejection-associated transcripts (RATs). The present study reclassified the rejection-related states in an expanded set of 1320 prospectively collected biopsies from 645 patients from 13 centers. Methods Biopsies were classified by ensembles of classifiers and analyzed for left ventricular ejection fraction (LVEF) and survival. Results New algorithms identified 853 No rejection (NR), 179 ABMR, 76 TCMR, 13 Mixed, 161 possible ABMR (pABMR), and 38 possible TCMR (pTCMR). No rejection was subclassified as NR-Normal 462, NR-Minor 359, and NR-Early-injury 32 (Figure 1A). Compared to NR-Normal, NR-Minor biopsies had mild elevation of many inflammation transcripts (e.g. IFNG-inducible genes) and to a lesser extent parenchymal injury transcripts. NR-minor biopsies were often designated as TCMR1R by histology. In all NR biopsies, NR-Minor scores and histologic TCMR1R increased through the first year, peaking about one year, suggesting that Minor inflammation is a late response to injury, unrelated to rejection. LVEF was similar in NR-Normal and NR-Minor but depressed in TCMR and Early injury. In a 3-year post-biopsy survival analysis, NR-Minor and NR-Normal had similar survival. TCMR and Early-injury were associated with increased graft loss, but many losses were not related to rejection. Surprisingly, 76 hearts with ABMR (149 biopsies) and follow-up data had only 3 losses within 3 years post-biopsy (Figure 1B). Conclusion Many biopsies with molecular no rejection develop minor increases in rejection-related transcripts in the first year, often called TCMR1R by histology, with no apparent effects on function or survival. An unexpected finding was that molecular ABMR was associated with very few graft losses over three years. (ClinicalTrials.gov #NCT02670408).

Introduction: Extracorporeal photopheresis (ECP) is an established therapy for treatment of heart transplant rejection and is also applied for rejection prevention in the perioperative setting after heart transplantation (HTX). Recently we started a new protocol with avoidance of antibody induction therapy and delay of calcineurin inhibitor (CNI) in high risk patients after HTX incorporating ECP as rejection prophylaxis. Methods: We report our first experience on 20 patients that were treated according to this protocol. Inclusion criteria were: history of cancer (n=5), bridge to transplant via extracorporeal membrane oxygenation (ECMO) (n=5) and ten patients had infections within one month before HTX. Two patients were combined heart-kidney transplants. None of the patients received antithymocyte globuline (ATG) as induction therapy. Immunosuppression consisted of low dose tacrolimus (target range 7-10ng/ml in month 1-3, 5-10ng/ml >3 months) with a delayed start (2 to 7 days post HTX) mycophenolate mofetil (MMF: 2mg/day), and steroids (0.2mg/kg starting on day 7, tapering to 0.03 mg/kg until end of first year). ECP was applied according to the protocol published by M. Barr (1) on days 2 + 3, 5 + 6, 10 + 11, 17 + 18, 27 + 28, 2 times every other week for month 2 and 3, and 2 times every 4 weeks month 4 – 6 after HTX. Routine biopsy protocol was performed in weeks 2,3,4, Months 2,3,6 and 12 and whenever, there were clinical signs of acute rejection. Results: Seventeen of 20 (85%) patients are alive with excellent graft function after a median follow-up of 11,5 (range, 1 – 38) months after HTX. Three patients (15%) showed biopsy proven signs of cellular rejection. Two were successfully treated with iv steroids and one developed steroid resistant rejection and received ATG therapy. One patient showed AMR 1i in a biopsy and received ivIg therapy. All rejections showed no signs of hemodynamic compromise. Three patients developed severe pneumonia (bacterial n=1, fungal n=2), and one a non-sternal wound infection Both patients that developed fungal pneumonia had ECMO support after transplant due to primary graft dysfunction. Both died three weeks and 1,5 months post transplant due to multiorgan failure. One patient with history of cancer showed recurrence of disease and died 13 months after transplantation due to disease progression. Conclusion: Up to now this is the first report on prophylactic ECP with avoidance of induction therapy and CNI delay in HTX patients. Severe infectious complications remain a problem in this high risk group and occur in approximately 20%. However, ECP is a safe and effective strategy for patients at risk for cancer recurrence or sepsis to avoid organ rejection. References: 1. Barr M et al. Photopheresis for the prevention of rejection in cardiac transplantation. Photopheresis Transplantation Study Group. N Engl J Med. 1998 Dec 10;339(24):1744-51.

Parenchymal injury and late changes (atrophy-fibrosis) can be mapped in heart transplant biopsies, and their presentation correlates with low LVEF and lower 3-year survival. Injury is often, but not always, associated with rejection. Severe acute injury and the late fibrosis phenotypes are often associated with TCMR. Thus parenchymal injury is the intermediate phenotype by which rejection mediates disturbed function and survival. ClinicalTrials.gov #NCT02670408.

This is the first report on prophylactic ECP with avoidance of induction therapy and CNI delay in HTX patients. Infectious complications remain a problem in this high risk group and occur in approximately 24%. However, ECP is a safe and effective strategy for patients at risk for cancer recurrence or sepsis to avoid organ rejection.

Background/Introduction:Cardiac transplantation is a lifesaving procedure for patients with end-stage heart failure.As acute rejection is a severe complication patients have to undergo invasive endomyocardial biopsy to rule out rejection episodes especially in the early phase after cardiac transplant.Purpose: This study is focused on comparing extracellular volume (ECV), T1 and T2 mapping via cardiac magnetic resonance imaging (CMR) with results endomyocardial biopsy at early post-transplant follow-ups to provide reference values for rejecting and non-rejecting patients.Methods: Forty-five patients underwent endomyocardial biopsy three months after cardiac transplantation alongside CMR on a 1.5T system for evaluation of graft rejection and function.Left ventricular function (LVEF) was derived from short axis steady-state free precision (SSFP) sequences, myocardial ECV (%) was calculated using T1 pre-and postcontrast maps at three short axis slices using a 16-segment model.T2 maps were acquired from identical planes for edema assessment.CMR findings were compared with histology based on the guideline of the international society for heart and lung transplant (ISHLT).Results: Myocardial biopsy showed no signs of rejection (0A/0R) in thirty-nine patients and mild rejection (1A/1R) in six patients.Mean LVEF was normal in both groups (64.1% vs. 72.7%;p=.082).Mean ECV% (31.8% vs. 31.8%;p=.980), T1 in ms (1063.0 vs. 1079.1;p=.418) and T2 in ms (51.2 vs. 53.0;p=.137) did not differ significantly between patients with no or mild rejection. Conclusion:In the reported patient cohort, there was no significant difference in the observed quantitative CMR markers in patients with mild vs. no transplant rejection, showing comparable pseudonormal values.The reported values may serve as reference values for CMR in the early stage after transplant.Further data from patients with higher rejection scores is needed to establish CMR as a screening tool for transplant rejection.

Purpose In heart and kidney transplants, rejection is a major cause of graft loss. In kidneys, antibody-mediated rejection (ABMR) is more important than T cell-mediated rejection (TCMR), and molecules predict graft loss better than histology (JASN 26 (7):1711-1720, 2015). We examined the relative importance of ABMR vs TCMR in heart transplant endomyocardial biopsies (EMBs), and the molecules predicting graft survival. Methods The INTERHEART population includes 1219 transplant biopsies from 8 centers in Canada, USA, Australia and Europe. Gene expression was studied using microarrays, selecting the most recent biopsy per patient. Random forest classifiers were used to assess predictive accuracy and determine the importance of molecular predictors, including gene sets and scores from analyses in a reference set of 889 EMBs. Results We studied 3-year survival in 484 patients with follow-up times. Graft failure occurred in 60 patients. Median follow-up was 435 days; biopsies were mainly for indications. Surprisingly, TCMR was a greater short-term hazard than ABMR. The molecular archetype clusters for TCMR and injury (Fig. 1) had the highest risk of graft failure. Fig. 2 combines these clusters since they have similar characteristics. Conclusion Unlike kidneys, graft loss (particularly within one year) after EMB is highly associated with TCMR but not ABMR. TCMR may reflect failure of immunosuppression or non-adherence. This difference between the heart and renal transplant populations raises the possibility that TCMR is relatively more destructive, and ABMR less destructive, in heart than in kidney transplants. ClinicalTrials.gov # NCT02670408

INTERHEART (ClinicalTrails.gov NCT02670408). Introduction We previously developed a molecular diagnostic system for assessment of rejection phenotypes in endomyocardial biopsies (EMB) based on expression of rejection-associated transcripts (RAT) derived in kidney (JHLT 36:1192, 2017). This system used archetypal analysis to identify 3 rejection-related molecular phenotypes among the biopsies (A1NoRejection, A2TCMR, A3ABMR), with each biopsy assigned scores relating them to the molecular phenotypes. We now explored whether there was another dimension beyond rejection that reflected acute parenchymal injury. Materials & Methods 889 single-piece EMBs from 462 heart transplant recipients at 8 centres in North America, Europe, and Australia were analyzed on Affymetrix microarrays. We used 2 methods to assess injury: archetypal analysis to assign groups, and expression of previously defined injury-repair transcripts (IRRAT). EMBs with A2TCMR scores ≥ 0.3 and A3ABMR scores ≥ 0.5 in the previously published 3 archetype model were designated “molecular rejection.” EMBs with A4 scores ≥ 0.4 in a new 4 archetype model were designated “molecular injury.” The rejection and injury designations were not mutually exclusive. Results EMBs characterized by archetypal analysis using 4 rather than 3 archetypes were distributed by principal component analysis based on RAT expression (Figure 1). PC1 reflected rejection, and PC2 reflected ABMR vs. TCMR (Figure 1A). The new group (A4) had an “acute injury” phenotype that was most apparent in PC3 (Figure 1B). Figure. No caption available. A4 had high expression of macrophage transcripts and IRRAT. The median IRRAT score in EMBs with high injury and low rejection scores was high compared to relatively normal biopsies (0.79 vs. -0.19) (Table 1). Median IRRAT scores in EMBs with high rejection and low injury were also elevated, albeit to a lesser extent (0.21 vs. -0.19). The expression of rejection transcripts was somewhat elevated in acute injury without rejection, reflecting overlap between inflammatory processes activated in rejection and non- rejection-related injury. Table. No title available. Many A4 EMBs were taken very early post-transplant (median time 30 days), probably reflecting injury induced in donation/implantation. Histology sometimes misdiagnosed rejection in A4 EMBs because of the macrophage infiltration: of 17 EMBs with high molecular injury and no molecular rejection, 9 were called rejection by histology. Conclusion Unsupervised analysis with 4 archetypes discovered a new group, acute injury, with high expression of macrophage and injury transcripts and early time post-transplant. Thus hearts often have an early acute injury phenotype that is inflamed, more so than in kidney transplants. Some of these biopsies have molecular rejection but some do not. Some with no molecular rejection are called rejection by histology, apparently reflecting injury-induced inflammation. We conclude that a molecular approach that independently assesses rejection and injury is needed for EMBs. Transcriptome Sciences, Inc.

We previously developed a molecular diagnostic system for heart transplant biopsies, based on expression of rejection-associated transcripts derived in kidney transplants (J Heart Lung Transplant 36:1192, 2017). We now expanded this to 889 biopsies and examined the relationship to histology in 768 with histology assessments.