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Background The emergence of molecular systems offers opportunities for improving the assessment of rejection in heart transplant biopsy specimens. The present study developed a microarray-based system for assessing heart transplant endomyocardial biopsy (EMB) specimens. Methods We analyzed 331 protocol or for-cause EMB specimens from 221 subjects in 3 centers (Edmonton, Bologna, and Paris). Unsupervised principal component analysis (PCA) and archetype analysis used rejection-associated transcripts (RATs) shown in kidney transplants to be associated with antibody-mediated rejection (ABMR) or T cell–mediated rejection (TCMR), or both. To compare EMB specimens to kidney biopsy specimens, rejection status in both was simplified to TCMR, ABMR, or no rejection. Results The pattern of RAT expression was similar in EMB and kidney specimens, permitting use of RATs to assign scores and group (“cluster”) membership to each EMB, independent of histology. Three clusters emerged in EMB specimens, similar to kidney specimens: TCMR, ABMR, and no rejection. This permitted each EMB specimen to be given 3 scores and assigned to 1 cluster by its highest score. There was significant agreement between molecular phenotype—archetype scores or clusters—and both histologic diagnoses and donor-specific antibody. Area under curve estimates for predicting histologic TCMR, ABMR, and no rejection by molecular assessment were lower in EMB specimens than in kidney specimens, reflecting more uncertainty in EMB specimens, particularly in histologic diagnosis of TCMR. Conclusions Rejection-associated transcripts can be used to estimate the probability of TCMR and ABMR in heart transplant specimens, providing a new dimension to improve the accuracy of diagnoses and an independent system for recalibrating the histology guidelines.

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

Purpose Gene expression profiling (GEP) of endomyocardial biopsies (EMB) by the MMDx system has been shown to estimate the probability of T-cell mediated (TCMR) and antibody-mediated rejection (AMR), improving the histology-based diagnoses in heart transplant (HT) recipients. Second generation analysis allowed to identify an additional phenotype characterised by molecules related to tissue injury (INJ), such as de-differentiation pathways, macrophages transcripts, and heart transcripts. The clinical correlates of this novel profile and its association with ISHLT pathology grading is under development. In this study we analyse the association of MMDx novel profiles with cardiac function and with ISHLT grades for TCMR and AMR. Methods 254 EMBs from 121 patients were analysed with MMDx platform. EMBs were performed according to standard protocol during the first 5 years after transplant and by clinical indication later on. Right heart catheretization was performed at the time of EMB, which were graded by 1990 and 2006 ISHLT classifications for TCMR and 2014 guidelines for AMR. Results Prevalence of molecular phenotypes varied during time course: INJ was frequent in the first months, absent during the first 5 years, and rose again in late for-cause EMBs; AMR peaked early and late after transplant, in line with DSA time-course; TCMR probability was meaningful in the early months only. Cardiac function also varied across the three profiles: INJ was significantly associated with low cardiac index (CI) and high right atrial pressure, AMR with elevated wedge pressure and low CI, and TCMR with low CI only. Overall, GEP was in agreement with pathology readings in most cases, being normal in 90% of the 0 graded EMB, and abnormal in over 70% of the 2R or pAMR2. While current 1R grade showed abnormal GEP only in 37% of cases, the old 1B grade had abnormal GEP in 60% of cases, mostly associated with AMR profile. INJ profile was found in 54% of the 2R or greater EMBs. Conclusion GEP reveals variable molecular patterns during transplant time course; INJ phenotype appears associated mainly with low CI in the context of post-operative injury and severe mixed rejection, while AMR seems to increase markers of graft stiffness. Despite good overall agreement with pathology readings, current ISHLT grading is inaccurate, with the “old” 1B grade being highly associated with AMR molecular profiling.

Purpose Chronic lung allograft dysfunction (CLAD) is a clinical/functional diagnosis that cannot be defined by histology in transbronchial biopsies (TBBs). Moreover, understanding the biology of CLAD is crucial for prevention and potential treatment. We studied gene expression associated with CLAD in TBBs, but also studied mucosal biopsies from the third bronchial bifurcation (3BMBs). Methods 223 highly alveolated TBBs (54 CLAD, 169 no CLAD) and 182 3BMBs (43 CLAD, 139 no CLAD) were collected from 7 centers and processed on microarrays. The top 100 non-overlapping genes associated with a diagnosis of CLAD prior to biopsy were identified, annotated, and used for gene set analysis and gene ontology (GO) biological process analysis. Results The top genes associated with CLAD reflected de-differentiation (expression loss), injury (expression gain), and inflammation in both TBBs and 3BMBs. These associations were stronger in 3BMBs. Pathway analysis using the top genes also suggested parenchymal de-differentiation (e.g. loss of transcriptional regulation and mitochondrial function in TBBs) and inflammatory pathway activation, particularly in 3BMBs (Table 1). Gene sets previously annotated in atrophy-scarring, including immunoglobulin transcripts (plasma cells), were elevated in CLAD in TBBs and 3BMBs, but 3BMBs also showed more inflammation-related changes (e.g. interferon gamma effects). Conclusion Molecular changes associated with CLAD were apparent in TBBs and especially in 3BMBs, and indicate that CLAD is a combination of atrophy/scarring, function loss, and, particularly in 3BMBs, inflammation. Molecular assessment of 3BMBs is a particularly promising opportunity to dissect CLAD biology and potentially gain insight into the BOS variant. ClinicalTrials.gov: NCT02812290

s S235 Methods: An analysis of all LTR at a single center from 10/2011-12/2013 was conducted; re-transplant and multi-organ recipients were excluded. RIFLE criteria was applied and AKI within 72 hours post-operatively was categorized as Risk (SCr 1.5x baseline), Injury (SCr 2x baseline), Failure (SCr 3x baseline), Loss (renal replacement therapy [RRT] > 4 weeks), and End-Stage disease (RRT > 3 months). Outcomes were assessed at 1 year and risk factors were compared between LTR with and without AKI. Results: Sixty-two LTR were included; the cohort was largely white males with IPF (45%) or COPD (35%). A total of 26 patients met the criteria for AKI: 17 (30.6%) Risk, 7 (11.3%) Injury, 2 (3.2%) Failure. Two patients who met AKI criteria for Failure progressed to end-stage disease. Mortality at 1 year (15% vs. 0%, p= 0.015) was significantly higher in LTRs with early AKI (Figure 1); ICU and hospital LOS were also prolonged (Table 1). Patients who had higher LAS and bilateral lungs were more likely to develop AKI. Peri-operative factors increasing ischemic injury including use of CPB, decreased intra-op SpO2, and duration of vasopressors were all significantly associated with AKI. Comorbidities and patient demographics did not appear to influence development of early AKI. Tacrolimus initiation was delayed in the AKI cohort as expected, and while not statistically significant, there was an increased cumulative dose of diuretics in the AKI cohort. Conclusion: Early AKI was shown to have worse 1 year mortality rates and longer hospital/ICU LOS. Ischemic risk factors in the peri-operative period was associated with development of AKI in LTR.