1/ MOLECULAR CORRELATES OF ENDOTHELIAL MTOR ACTIVATION IN HEART TRANSPLANT RECIPIENTS
Session: “ Basic and translational immunology” (17:45 to 18:45)
Location: Room 118 + 119 – 18:29 

M Racapé, A Loupy, J Reeve, J Venner , R Guillemain, L Hidalgo, C Lefaucher, X Jouven, P Bruneval, J Duaong Van Huyen, P Halloran 
Background
The detection of phosphorylated effectors of the mTOR pathway such as phosphorylated-S6RP in endothelial cells by immunohistochemistry (IHC) has been associated with Antibody-Mediated allograft Rejection (AMR). The aim of this study was to evaluate the molecular phenotype related to the endothelial detection of pS6RP in heart transplant recipients.
Methods
This case-control study included 41 heart transplant patients from four French referral centers with biopsy proven antibody-mediated rejection (pAMR+) and a matched control group of 30 patients without rejection (pAMR0) based on the updated ISHLT classification. From these patients, 94 endomyocardial biopsies (EMB) had adequate material for microarray analysis and endothelial expression analysis of pS6RP by IHC. We also determined the allograft gene expression profile using the ABMR molecular score in addition to pathogenesis-based transcripts reflecting endothelial activation (DSAST and ENDAT), macrophage burden (QCMAT), gamma-interferon response (GRIT) and NK-cell burden (NKB) (http://atagc.med.ualberta.ca).
Results
Among the 94 EMBs included in the main analyses, 50 were pAMR+ (53.2%) and 44 (46.8%) were pAMR0 normal EMBs. Endothelial expression of pS6RP was observed in 27/50 (54%) of pAMR+ biopsies and 12 out of 44 normal biopsies (27.3%, Fischer's exact: p=0.012). As compared with biopsies without pS6RP labeling, biopsies with pS6RP staining showed increased expression of DSAST (Mann-Whitney: p<0.0001), ENDAT (p=0.0009), QCMAT (p=0.0046), NKB (p=0.0001), GRIT (p=0.0008) and increased ABMR molecular score reflecting AMR injury (p=0.0001).
Conclusion
Endothelial activation of mTOR pathway is associated with AMR and increased expression in transcripts reflecting endothelial activation, macrophage burden, microcirculation and NK burden. Our results suggest the importance of the mTOR pathway activation in AMR injury and the potential interest of using mTOR inhibitors in this setting.

Abstract
B. Afzali, E. Chapman,  M. Racapé, B. Adam,  P. Bruneval, F. Gil, D. Kim, L. Hidalgo, P. Campbell, B. Sis, J. P. Duong Van Huyen, M. Mengel.
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Precise diagnosis of antibody-mediated rejection (AMR) in cardiac allograft endomyocardial biopsies (EMBs) remains challenging. This study assessed molecular diagnostics in human EMBs with AMR. A set of 34 endothelial, natural killer cell and inflammatory genes was quantified in 106 formalin-fixed, paraffin-embedded EMBs classified according to 2013 International Society for Heart and Lung Transplantation (ISHLT) criteria. The gene set expression was compared between ISHLT diagnoses and correlated with donor-specific antibody (DSA), endothelial injury by electron microscopy (EM) and prognosis. Findings were validated in an independent set of 57 EMBs. In the training set (n = 106), AMR cases (n = 70) showed higher gene set expression than acute cellular rejection (ACR; n = 21, p < 0.001) and controls (n = 15, p < 0.0001). Anti-HLA DSA positivity was associated with higher gene set expression (p = 0.01). Endothelial injury by electron microscopy strongly correlated with gene set expression, specifically in AMR cases (r = 0.62, p = 0.002). Receiver operating characteristic curve analysis for diagnosing AMR showed greater accuracy with gene set expression (area under the curve [AUC] = 79.88) than with DSA (AUC = 70.47) and C4d (AUC = 70.71). In AMR patients (n = 17) with sequential biopsies, increasing gene set expression was associated with inferior prognosis (p = 0.034). These findings were confirmed in the validation set. In conclusion, biopsy-based molecular assessment of antibody-mediated microcirculation injury has the potential to improve diagnosis of AMR in human cardiac transplants.

Authors
M. Racapé, A. Loupy, J. Reeve, J. Venner, R. Guillemain, L. Hidalgo, C. Lefaucheur, X. Jouven, P. Bruneval, J. Duong Van Huyen, P. Halloran. 

Abstract

• Purpose: The detection of phosphorylated effectors of the mTOR pathway in endothelial cells by immunohistochemistry (IHC) has been associated with allograft rejection. The aim of this study was to evaluate in heart transplant recipients the molecular phenotype related to the endothelial detection of the phosphorylated effectors of the mTOR pathway, pS6RP.
• Methods: This case-control study included 41 heart transplant patients from four French referral centers with biopsy proven antibody-mediated rejection (pAMR+) and a matched control group of 32 patients without rejection (pAMR0) based on the updated ISHLT classification. From these patients, 93 endomyocardial biopsies (EMB) had adequate material for microarray analysis and IHC. We studied in all EMB the endothelial expression of pS6RP by IHC. We also determined the allograft gene expression profile using the ABMR molecular score in addition to pathogenesis-based transcripts reflecting endothelial activation (DSAST and ENDAT), macrophage burden (QCMAT), gamma-interferon response (GRIT) and NK-cell burden (NKB) (http://atagc.med.ualberta.ca).
• Results: Among the 93 EMBs included in main analyses, 49 were pAMR+ (52.7%) and 44 (47.3%) were pAMR0 normal EMBs. Endothelial expression of pS6RP was observed in 27/49 (55.1%) of pAMR+ biopsies and 12 out of 44 normal biopsies (27.3%, Fischer's exact: p=0.011). As compared with biopsies without endothelial pS6RP labeling, biopsies with positive endothelial pS6RP staining showed increased expression in DSAST (Mann-Whitney: p<0.0001), ENDAT (p=0.0008), QCMAT (p=0.0052), NKB (p=0.0001) and increased ABMR molecular score reflecting interferon-effects, microcirculation stress and NK burden (p=0.0001).
• Conclusion: Endothelial activation of mTOR pathway is associated with antibody-mediated allograft rejection and increased expression in transcripts reflecting endothelial activation, macrophage burden, microcirculation and NK burden. Our results suggest the importance of the mTOR pathway activation in antibody-mediated heart allograft injury and the potential interest of using mTOR inhibitors in this setting.