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).

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.

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

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.

We previously reported a Molecular Microscope system for diagnosis of rejection in heart transplant (MMDx-Heart) in 331 endomyocardial biopsies (EMB) (J Heart Lung Transplant 36 (11):1192-1200, 2017). In the present study we validated these locked algorithms in new EMBs.

The ISHLT system for diagnosing rejection in heart transplant endomyocardial biopsies (EMB) has never been compared to an external standard. A new molecular diagnostic system for kidney (Nat Rev Nephrol 2016;12(9):534) and heart (J Heart Lung Transplant 36:1192, 2017) now permits such comparisons, in both T cell-mediated (TCMR) and antibody-mediated (ABMR) rejection.

In both heart and kidney transplants, rejection is a major cause of graft loss. In kidneys, the principal risk is antibody-mediated rejection (JCI Insight 2 (12), 201710.1172/jci.insight.94197), and molecular rejection predicts better than histologic diagnosis (JASN 26 (7):1711-1720, 2015). Similar comparisons in a heart transplant endomyocardial biopsy population (EMB) have not been performed.

Implantation of a ventricular assist device (VAD) is a known risk factor for development of Human Leukocyte Antigen (HLA) antibodies (Ab). These Ab increase wait list time in heart transplant (HTx) and have been associated with poor outcome post-HTx. Few data exist regarding impact of age on this Ab development. This study aims to determine the class I and II Ab generation from VADs in adult and pediatric patients. This was a single centre review of adult (n=93) and pediatric (n=46) patients who received a long-term VAD between 2005-13. From this cohort, patients were included if: they had both pre- and post-VAD HLA results, were within 1 year post-VAD, pre-HTx, and this was their 1st long-term device. 40 adults and 27 pediatric patients were eligible for analysis. Class I and II PRA were measured by either FlowPRA® or cPRA. cPRA was determined by single antigen beads and was only available in patients with a positive HLA screen. cPRA values were calculated using the Canadian cPRA Calculator. Class I and II PRA values pre-VAD were compared to the 1st sample post-VAD. Sensitization was defined as PRA ≥ 10%. Table 1A describes patient demographics. The median time to 1st HLA sample was 13 days (IQR 7-38) post-VAD. 13 patients were sensitized to class I pre-VAD (7 adults, 6 peds), increasing to 24 patients (15 adults, 9 peds) post-VAD. 3 patients sensitized pre-VAD had PRA <10% post-VAD. Table 1B outlines the class I and II FlowPRA & cPRA pre- and post-VAD. HLA Ab detected early post-VAD implantation appear to be predominantly against class I antigens. Increased class I HLA Ab were detected by FlowPRA in both adults and children, while class II remained unchanged. Only adults had significantly increased cPRA following implantation. As some Ab were detected early post-VAD, it is unclear if they will persist over time and whether the difference between the 2 cohorts represents an amnestic response to previous sensitization. Future studies will examine the long-term Ab response in these patients.

BACKGROUND: Transplantation of sensitized recipients has been associated with increased risk of post transplant complications. In 2010, the Canadian Cardiac Transplant Network (CCTN) created a unique status listing for highly sensitized heart transplant candidates. Status 4S listing requires calculated panel reactive antibody (cPRA) level > 80% as the sole listing criteria and enables geographic expansion of the donor pool by providing national access. In this study, we describe patient characteristics and outcomes of those transplanted as Status 4S in Canada.METHODS: Patients' characteristics and clinical outcomes were retrospectively collected from all I 1 adult heart transplant centers in Canada.RESULTS: Ninety-six patients were listed Status 4S from January 2010 to September 2015. Fifty-two were transplanted as Status 4S. Of these 52 transplants, mean cPRA level was 93.4%, mean age was 47 years, 46% were male, 44% had dilated cardiomyopathy and 17% were re-transplanted for cardiac allograft vasculopathy (CAV). Blood group O comprised 42% and 53% had a left ventricular assist device as a bridge to transplant. Desensitization therapy occurred in 9 patients (17%). Over a mean follow-up period of 28 months (1 week to 5.3 years), 9 patients died (17%). Kaplan Meier 1-year year survival is 86%. Two patients were treated for antibody-mediated rejection (AMR) in the first year post transplant and 33% of patients had at least 1 ISHLT Grade >= 2R cellular rejection in the first year. Twenty-nine percent of patients developed de novo door-specific antibodies and demonstrated no correlation with AMR. Freedom from CAV at 1 year is 88.5% and at 5 years is 81.0%. Fifty-two percent of donor hearts originated from outside the recipients' geographic and organ donation organization.CONCLUSIONS: A national strategy of prioritizing highly sensitized heart transplant recipients has demonstrated effective expansion of the donor pool, acceptable short-term survival, freedom from CAV and low rates of clinically relevant AMR. However, we observed significantly higher rates of cellular rejection and de novo donor-specific antibody development in this population. It is currently unknown whether this will translate into poorer long-term outcome. (C) 2017 International Society for Heart and Lung Transplantation. All rights reserved.

PurposeAcute rejection occurs in 20-30% of heart transplant patients during the first year. While acute cellular rejection (ACR) occurs infrequently beyond the first year, surveillance endomyocardial biopsies (EBx) are still routinely performed in many centres. The aim of this analysis was to determine the incidence of late ACR as well as the utility of scheduled surveillance EBx in the detection of late ACR.MethodsUsing medical records at the University of Alberta Hospital, a cohort of adult heart patients (≥ 18 years) who underwent heart transplantation between January 2002 and December 2010 was retrospectively reviewed. All biopsy proven rejection episodes (2R or 3R) were identified. Comparisons between patients with late rejection (>1 year)(LR) and those without late rejection (NLR) were performed.ResultsFrom a total of 193 adult heart transplant recipients during the study period, 106 were followed at University of Alberta hospital for ≥ 2 years (mean F/U of 6.4 years). Patient mean age was 51.4±12.0 y and 75.0% of the patients were males. Mean BMI was 26.3±4.5 kg/m2. 19 patients had episodes of late rejection (LR) while 87 did not (NLR). 100% (19/19) of LR patients were male vs 69.0% (60/87) of NLR patients (p=0.004). The two groups were statistically similar with regards to DM, hypertension, immunosuppressive regimens, CMV infection, incidence of malignancy, renal impairment and vasculopathy. However, LVEF was lower in the LR group (56.5%) than in the NLR group (60.2%) (p=0.04). There were no differences between the peak panel reactive antibody Levels (PRA) of the 2 groups (LR 8.8±15.9% % vs NLR 18.4±28.9% % for Class I, p = 0.1) and (LR 21.1±35.1% vs NLR 12.5± 26.7% for Class II, p= 0.2). ACR in the first year was identified in 31.6% (6/19) of patients in LR group and 6.9% (6/87) in NLR group (p=0.04). Rejection episodes detected by scheduled biopsies represented only 0.51% (5/968) of the total number of EBx performed beyond the first year. Average number of post first year EBx per patient was 11.5±2.3 for LR and 8.6±2.1 for the NLR.ConclusionIn our study, only early ACR was predictive of late rejection. However, more interestingly, our study showed that the overall strategy of routinely scheduled EBx beyond the first year post heart transplantation has very poor diagnostic utility and should be re-evaluated. PurposeAcute rejection occurs in 20-30% of heart transplant patients during the first year. While acute cellular rejection (ACR) occurs infrequently beyond the first year, surveillance endomyocardial biopsies (EBx) are still routinely performed in many centres. The aim of this analysis was to determine the incidence of late ACR as well as the utility of scheduled surveillance EBx in the detection of late ACR. Acute rejection occurs in 20-30% of heart transplant patients during the first year. While acute cellular rejection (ACR) occurs infrequently beyond the first year, surveillance endomyocardial biopsies (EBx) are still routinely performed in many centres. The aim of this analysis was to determine the incidence of late ACR as well as the utility of scheduled surveillance EBx in the detection of late ACR. MethodsUsing medical records at the University of Alberta Hospital, a cohort of adult heart patients (≥ 18 years) who underwent heart transplantation between January 2002 and December 2010 was retrospectively reviewed. All biopsy proven rejection episodes (2R or 3R) were identified. Comparisons between patients with late rejection (>1 year)(LR) and those without late rejection (NLR) were performed. Using medical records at the University of Alberta Hospital, a cohort of adult heart patients (≥ 18 years) who underwent heart transplantation between January 2002 and December 2010 was retrospectively reviewed. All biopsy proven rejection episodes (2R or 3R) were identified. Comparisons between patients with late rejection (>1 year)(LR) and those without late rejection (NLR) were performed. ResultsFrom a total of 193 adult heart transplant recipients during the study period, 106 were followed at University of Alberta hospital for ≥ 2 years (mean F/U of 6.4 years). Patient mean age was 51.4±12.0 y and 75.0% of the patients were males. Mean BMI was 26.3±4.5 kg/m2. 19 patients had episodes of late rejection (LR) while 87 did not (NLR). 100% (19/19) of LR patients were male vs 69.0% (60/87) of NLR patients (p=0.004). The two groups were statistically similar with regards to DM, hypertension, immunosuppressive regimens, CMV infection, incidence of malignancy, renal impairment and vasculopathy. However, LVEF was lower in the LR group (56.5%) than in the NLR group (60.2%) (p=0.04). There were no differences between the peak panel reactive antibody Levels (PRA) of the 2 groups (LR 8.8±15.9% % vs NLR 18.4±28.9% % for Class I, p = 0.1) and (LR 21.1±35.1% vs NLR 12.5± 26.7% for Class II, p= 0.2). ACR in the first year was identified in 31.6% (6/19) of patients in LR group and 6.9% (6/87) in NLR group (p=0.04). Rejection episodes detected by scheduled biopsies represented only 0.51% (5/968) of the total number of EBx performed beyond the first year. Average number of post first year EBx per patient was 11.5±2.3 for LR and 8.6±2.1 for the NLR. From a total of 193 adult heart transplant recipients during the study period, 106 were followed at University of Alberta hospital for ≥ 2 years (mean F/U of 6.4 years). Patient mean age was 51.4±12.0 y and 75.0% of the patients were males. Mean BMI was 26.3±4.5 kg/m2. 19 patients had episodes of late rejection (LR) while 87 did not (NLR). 100% (19/19) of LR patients were male vs 69.0% (60/87) of NLR patients (p=0.004). The two groups were statistically similar with regards to DM, hypertension, immunosuppressive regimens, CMV infection, incidence of malignancy, renal impairment and vasculopathy. However, LVEF was lower in the LR group (56.5%) than in the NLR group (60.2%) (p=0.04). There were no differences between the peak panel reactive antibody Levels (PRA) of the 2 groups (LR 8.8±15.9% % vs NLR 18.4±28.9% % for Class I, p = 0.1) and (LR 21.1±35.1% vs NLR 12.5± 26.7% for Class II, p= 0.2). ACR in the first year was identified in 31.6% (6/19) of patients in LR group and 6.9% (6/87) in NLR group (p=0.04). Rejection episodes detected by scheduled biopsies represented only 0.51% (5/968) of the total number of EBx performed beyond the first year. Average number of post first year EBx per patient was 11.5±2.3 for LR and 8.6±2.1 for the NLR. ConclusionIn our study, only early ACR was predictive of late rejection. However, more interestingly, our study showed that the overall strategy of routinely scheduled EBx beyond the first year post heart transplantation has very poor diagnostic utility and should be re-evaluated. In our study, only early ACR was predictive of late rejection. However, more interestingly, our study showed that the overall strategy of routinely scheduled EBx beyond the first year post heart transplantation has very poor diagnostic utility and should be re-evaluated.

Imaging recommendations for the follow-up of heart transplant recipients (HTRs) lack evidence justifying their prognostic value. Cardiovascular magnetic resonance imaging (CMRI) can characterize heart structure and function and has prognostic value in many myocardial diseases. We hypothesized that CMRI evaluation of cardiac allografts would predict adverse events. We performed CMRI on 60 HTRs evaluating biventricular size, function and myocardial scar. We performed survival analysis to identify independent predictors of cardiovascular (CV) death or hospitalization. Participants had a mean age of 51 ± 14 years, mean graft age of 3.5 years (±4) and 75% are male. Median follow-up time was 4.9 years with 22 CV hospitalizations and 7 CV deaths. A multivariable survival analysis of imaging and clinical variables identified myocardial scar (hazard ratio [HR] of 10.7, p = 0.005), right ventricular end- diastolic volume index (RVEDVI; 1.1/mL/m(2) , p = 0.001), graft age (HR = 1.2/year, p = 0.004) and previous allograft rejection (HR = 4.4, p = 0.006) as predictive of time to CV death or hospitalization. CMRI-derived myocardial scar and RVEDVI are independently associated with CV outcomes in HTRs.

Cytomegalovirus (CMV) infection is common among heart transplant recipients and has adverse direct and indirect host effects. Little is known about the effects of CMV infection on cardiac allograft morphology or function. Cardiovascular MRI (CMR) is the gold standard for assessment of cardiac morphology and function. We hypothesized that history of CMV infection would be associated with myocardial scar, adverse ventricular remodeling, and reduced function. We retrospectively ascertained the pre-transplant recipient and donor CMV serology and post-transplant CMV infection (documented by CMV DNAemia) of heart transplant recipients who had undergone CMR as part of a separate research protocol. At our institution CMV surveillance is performed as part of a standardized post-transplant protocol and data were retrieved up to the time of their CMR scan. CMR variables of cardiac morphology and function were compared between recipients with and without previous CMV infection and across donor-recipient pre-transplant serology groups. Forty six participants (mean age 51 +/- 15 yrs, 74% male, mean time since transplantation 3.7 +/- 4 years) were analyzed, of whom eighteen (39%) had >= 1 documented CMV infection. Previous CMV infection was associated with increased left ventricular (LV) mass (176 vs 143 g, p = 0.006), LV wall thickness (11 vs 9 mm, p = 0.002), LV mass to volume ratio (1.2 vs 1.0, p = 0.01), and reduced diastolic function (E/E` ratio 10 vs 7, p = 0.04). A greater number of CMV infections was associated with reduced right ventricular ejection fraction (RVEF) (r = -0.52, p = 0.03) and larger right ventricular end diastolic volume (r = 0.48, p = 0.05). Higher peak CMV DNAemia was also associated with reduced RVEF (r = -0.49, p = 0.04). There was no association between CMV infection and myocardial scarring. There were no significant differences in cardiac morphology across pre-transplant donor and recipient CMV sero-status groups. Heart transplant recipients with post-transplant CMV infection demonstrate morphologic and functional allograft changes on CMR. CMR may be helpful in characterizing the indirect effects of CMV infection on cardiac allografts.

Technology to assess the panel reactive antibody (PRA) has evolved rapidly. The purpose of this study was to determine the effect of introducing solid phase PRA screening on adult orthotopic heart transplant (OHT) outcomes at our institution.

Histopathology of endomyocardial biopsies (EMB) is the standard rejection surveillance for heart transplants. However, ISHLT consensus criteria for interpreting biopsies are arbitrarily defined. Gene expression offers an independent re-evaluation of existing diagnostic systems. We performed histologic and microarray analysis on 105 EMB from 45 heart allograft recipients. Histologic lesions, diagnosis and transcripts were compared to one another, time posttransplantation, indication for biopsy and left ventricular ejection fraction (LVEF). Histologic lesions presented in two groups: myocyte-interstitial and microcirculation lesions. Expression of transcript sets reflecting T cell and macrophage infiltration, and γ-interferon effects correlated strongly with each other and with transcripts indicating tissue/myocardium injury. This molecular phenotype correlated with Quilty (p < 0.005), microcirculation lesions (p < 0.05) and decreased LVEF (p < 0.007), but not with the histologic diagnosis of rejection. In multivariate analysis, LVEF was associated (p < 0.03) with γ-interferon inducible transcripts, time posttransplantation, ischemic injury and clinically indicated biopsies, but not the diagnosis of rejection. The results indicate that (a) the current ISHLT system for diagnosing rejection does not reflect the molecular phenotype in EMB and lacks clinical relevance; (b) the interpretation of Quilty lesions has to be revisited; (c) the assessment of molecules in heart biopsy can guide improvements of current diagnostics.