Guillaume Par

Coutance

Dirigée Par

Jean-Paul Paul Duong

Van Huyen

Alexandre Loupy

Président Du

Leprince Pascal

Analisa Angelini

PH Shaida Varnous

Jignesh Patel

Nicolas Adam

Guillaume Coutance

Pierre-Julien Vially

Fanny Drieux

Philippe Ruminy

Ahmad Abdel Sater

Claire Toquet

Philippe Rouvier

Arnaud François

Marie-Pierre Chenard

Eric Epailly

Romain Guillemain

Sabine Pattier

Arnaud Gay

Jean-Luc Taupin

Marion Rabant

Patrick Bruneval

Allosensitization and allograft rejection in heart transplantation From diagnosis to outcomes

Titre : Allo-immunisation et rejet d'allogreffe en transplantation cardiaque : du diagnostic aux conséquences cliniques. Title : Allosensitization and allograft rejection in heart transplantation: from diagnosis to outcomes.

Ces trois années de thèse auront été une expérience inoubliable au cours de laquelle j'ai pu interagir avec des personnes formidables qui m'ont permis de progresser tant dans le domaine professionnel que personnel. En plus des remerciements formels d'usage pour ce type de document, j'y ajouterai une version personnelle en exprimant à chacun ma reconnaissance de vive voix.

Introduction

L'allo-immunisation dirigée contre le greffon («Donor-Specific Antibodies» -DSA), et son corollaire, le rejet humoral (RH), sont des problématiques cliniques croissantes en transplantation cardiaque du fait de la morbi-mortalité qui leur est associée. Plusieurs points cruciaux de leur prise en charge demeurent non consensuels : #1-le diagnostic anatomopathologique du RH, #2-le rythme de dépistage des complications qui leur sont associées, actuellement non adapté au risque de développement de la maladie (médecine protocolaire en opposition à une médecine personnalisée), et #3-la prise en charge thérapeutique de sous-groupes de patients à haut risque immunologique. Méthodes Dans une première partie, nous avons étudié l'apport de deux techniques de biologie moléculaire comme compagnon au diagnostic anatomopathologique du RH : « Reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) » et « microarray ». Dans une seconde partie, nous avons analysé, par une approche en population (études internationales multicentriques) et appliquant des outils statistiques innovants (modèles mixtes à classes latentes), deux complications majeures de l'allo-immunisation : la maladie coronaire du greffon (n=1301 patients) et les rejets d'allogreffes (n=1053 patients). Notre approche a pour but d'améliorer la modélisation et la stratification du risque de développement de la maladie, première étape vers une individualisation du suivi. Enfin, nous avons étudié l'impact de l'allo-immunisation et du RH dans 2 populations à risque : les patients greffés avec DSA pré-formés et les patients en choc cardiogénique secondaire à un RH. Résultats En appliquant la RT-MLPA à 183 biopsies endomyocardiques, nous avons démontré l'applicabilité de cette technique aux biopsies fixées en paraffine et développé des profils d'expression génique spécifiques des RH (surexpression de CCL4, PLA1A, GNLY, CXCL9, FCGR3 et CXCL11) et des rejets cellulaires (CCL18 et ADAMdec). Nous avons ensuite montré qu'une classification en 4 grades du degré d'inflammation microvasculaire était significativement associée à l'activité moléculaire du rejet humoral et à des paramètres cliniques d'importance (présence de DSA, dysfonction du greffon). Par une approche épidémiologique en population, nous avons montré que l'évolution de la maladie coronaire du greffon pouvait être séparée en 4 trajectoires distinctes et universelles et que les DSA de classe II étaient un facteur de risque indépendant d'apparition et d'aggravation de la maladie. Par une approche similaire, nous avons pu améliorer la modélisation de l'évolution du risque de rejet d'allogreffe au cours du temps et déterminer cinq facteurs de risque indépendant de rejet d'allogreffe au cours de la première année (présence de DSA, âge du receveur, type de greffe, type d'induction, nombre de mismatch HLA). En rapportant les résultats d'un protocole prospectif de prise en charge péri-opératoire des patients greffés avec DSA pré-formés, nous avons montré que ceux-ci présentaient une survie après transplantation similaire aux patients à moindre risque immunologique au prix d'une augmentation de l'incidence des RH. Enfin, nous avons décrit que le pronostic des patients en choc cardiogénique secondaire à un RH était médiocre, avec une mortalité estimée à 82% à 1 an.

Conclusion

La standardisation du diagnostic anatomopathologique du RH basée sur les techniques de biologie moléculaire, l'amélioration de la stratification du risque de complications cliniques de l'allo-immunisation et de la description des évènements cliniques au sein de population à haut risque immunologique représentent autant d'avancées sur la voie d'une amélioration de la prise en charge de l'allo-immunisation et du RH en transplantation cardiaque.

Mots clefs : Transplantation cardiaque, allo-immunisation, rejet d'allogreffe, maladie coronaire du greffon, biologie moléculaire, stratification pronostique Introduction Allosensitization, i.e. the presence anti-HLA Donor-Specific Antibodies (DSA), and its corollary, antibody-mediated rejection (AMR), are major causes of cardiac allograft vasculopathy (CAV), allograft rejection and graft dysfunction, which represent the leading cause of death late after heart transplantation. Several key points of their management remain debated and concern particularly #1-the lack of standardization of the pathologic diagnosis of AMR, #2-the screening protocol of allosensitization-related complications not adapted to the risk of development of the disease (protocol medicine in opposition to personalized medicine), and #3-the clinical management of subgroups of patients at high immunological risk.

Methods

In a first part, we analyzed the contribution of two molecular biology techniques in the standardization of the pathologic diagnosis of AMR: a targeted-gene expression technique, the reverse transcriptase multiplex Ligation-dependent probe amplification (RT-MLPA) and a genome-wide approach using microarrays. In a second part, we analyzed at the population level (multicentric international studies) and using innovative statistical tools (latent classes mixed models), two major complications of allosensitization: CAV (n = 1,301 patients) and allograft rejections (n = 1,053 patients). Our aim was to describe their natural history, epidemiology and risk factors to enable an accurate risk stratification. Finally, in a clinical part, we studied the impact of DSA and AMR in two populations with high immunological risk: patients transplanted with pre-formed DSA and patients with AMR-related cardiogenic shock.

Results

By applying RT-MLPA to 183 endomyocardial biopsies (EMB), we demonstrated the applicability of this technique to paraffin-embedded EMB and developed specific gene expression profiles of AMR (overexpression of CCL4, PLA1A, GNLY, CXCL9, FCGR3 and CXCL11) and cellular rejection (CCL18 and ADAMdec). We then demonstrated that the severity of microvascular inflammation, based on a semi-quantitative evaluation of its extension (none, 1 to 9, 11 to 49 and ≥50% of the capillaries and venules) had biological (association with molecular activity of AMR) and clinical meanings (pAMR grade, DSA, graft dysfunction). In a large multicentric and highly phenotyped prospective cohort of heart transplant recipients, we identified for the first time 4 universal distinct and mutually exclusive CAV trajectories and their respective immune (class II DSA and acute cellular rejection) and non-immune determinants. Our results provide the basis for a trajectory-based assessment of heart transplant patients for early risk stratification and patient monitoring. By a similar approach, we were able to describe the epidemiology of allograft rejection and to identify five independent risk factors for rejection during the first year (DSA, recipient's age, type of transplantation, type of induction therapy, number of HLA mismatches). Finally, by reporting the results of a prospective protocol for perioperative management of preformed DSA based on plasmapheresis and polyvalent immunoglobulins infusions, we showed that those high-risk patients had a similar post-transplant survival compared to other patients at the cost of an increased incidence of AMR. Finally, we reported the poor prognosis of patients with AMR-related cardiogenic shock characterized by a one-year mortality as high as 82%.

Conclusion

The standardization of pathologic diagnosis of AMR using molecular biology techniques as a companion to pathologic analysis, the improvement in the risk stratification of allosensitizationrelated complications and the better description of clinical events in high immunologic risk population represent so many advances in the way of an improvement in the clinical management of DSA and AMR in heart transplantation. Keywords: Heart transplantation, allosensitization, allograft rejection, cardiac allograft vasculopathy, molecular biology, risk stratification.

A Paul, Agathe et Emmanuelle

Merci à mes deux directeurs de thèse, Jean-Paul et Alexandre, pour m'avoir guidé au cours de trois années et offert des opportunités professionnelles incroyables.

Merci à mon chef de service, Pascal Leprince, de me faire l'honneur de diriger ce jury de thèse et de m'offrir une place dans son service auquel je suis tant attaché.

Thanks to Prof. Analisa Angelini and Prof. Maarten Naessens for agreeing to evaluate this work and for their long trip to Paris. I am deeply honored that two experts like you are part of my Ph.D. committee. Thanks to Prof. Jignesh Patel for agreeing to be part of this committee and for his very long trip from LA. I am also very grateful to you for having welcomed me so kindly in your department. I am looking forward to coming back in November. breathlessness, ankle swelling and fatigue) that may be accompanied by signs caused by a structural and/or functional cardiac abnormality, resulting in a reduced cardiac output and/or elevated intracardiac pressures at rest or during stress [START_REF] Ponikowski | ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC). Developed with the special contribution of the Heart Failure Association (HFA) of the ESC[END_REF]. The prevalence of heart failure in industrialized countries is estimated to range between 1% and 2%, making this disease a real public health problem. The therapeutic management of heart failure has been revolutionized over the last twenty years.

Table des illustrations

However, some patients are affected by this disease to such an extent that these treatments remain insufficient.

Intro-A-2. Advanced heart failure

Advanced heart failure refers to these unstable and severe patients. Its definition is based on the association of the persistence of symptoms and / or signs of heart failure despite optimal medical and electrical management and the presence of clinical, biological, electrocardiographic or ultrasound severity criteria [START_REF] Crespo-Leiro | Advanced heart failure: a position statement of the Heart Failure Association of the European Society of Cardiology[END_REF][START_REF] Fang | Advanced (stage D) heart failure: a statement from the Heart Failure Society of America Guidelines Committee[END_REF]. The prevalence of advanced heart failure is estimated at 1% of the heart failure patient population, representing several tens of thousands of patients in Europe or the United States.

The prognosis of these patients is poor. The mortality of patients dependent on inotropic support is estimated to be as high as 75% at 1 year (4), that of INTERMACS 4 patients at 50% (5).

Intro-B. Heart transplantation

Intro-B-1. Epidemiology, post-transplant survival Despite the development of long-term circulatory support techniques, heart transplantation (HTx) remains the reference therapeutic option for patients with advanced heart failure [START_REF] Ponikowski | ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC). Developed with the special contribution of the Heart Failure Association (HFA) of the ESC[END_REF][START_REF] Filippatos | ACC/AHA/HFSA Focused Update of the 2013 ACCF/AHA Guideline for the Management of Heart Failure[END_REF]. Currently, more than 5,000 heart transplants are performed each year worldwide, including nearly 500 procedures in France (Figure 1) [START_REF] Khush | The International Thoracic Organ Transplant Registry of the International Society for Heart and Lung Transplantation: Thirty-fifth Adult Heart Transplantation Report-2018; Focus Theme: Multiorgan Transplantation[END_REF]8). One-year survival is about 88 % and median survival after heart transplantation now exceeds 12 years according to ISHLT data [START_REF] Khush | The International Thoracic Organ Transplant Registry of the International Society for Heart and Lung Transplantation: Thirty-fifth Adult Heart Transplantation Report-2018; Focus Theme: Multiorgan Transplantation[END_REF]. In France, one-year survival is 79% for the 2013-2016 period and median survival after heart transplantation exceeds 10 years (9) (Figure 2). Beyond the first year of transplantation, two major causes account for the majority of deaths: complications of chronic immunosuppression (cancers and infections) and graft-specific diseases: cardiac allograft vasculopathy, allograft rejection and graft dysfunction (Figure 3) [START_REF] Khush | The International Thoracic Organ Transplant Registry of the International Society for Heart and Lung Transplantation: Thirty-fifth Adult Heart Transplantation Report-2018; Focus Theme: Multiorgan Transplantation[END_REF][START_REF] Lund | The Registry of the International Society for Heart and Lung Transplantation: Thirty-fourth Adult Heart Transplantation Report-2017; Focus Theme: Allograft ischemic time[END_REF]. The term "graft dysfunction" is a generic term used in large registers. Aside from the primary graft dysfunction occurring very early after transplantation, this entity reflects both acute dysfunctions associated with allograft rejection or undiagnosed coronary artery disease, as well as chronic dysfunction related to a restrictive evolution of heart allografts or undiagnosed coronary artery diseases. While immunosuppressive induction or maintenance therapies are particularly effective for the prevention and treatment of acute cellular rejection, the therapeutic management of antibody-mediated rejection (AMR) remains much more complex. The incidence of acute cellular rejection has declined in the course of time [START_REF] Shah | Surveillance Endomyocardial Biopsy in the Modern Era Produces Low Diagnostic Yield for Cardiac Allograft Rejection[END_REF]. It now rarely brings severe graft dysfunction and is no longer associated with an increase in cardiovascular mortality, unlike AMR [START_REF] Vaughn | Outcome of antibody-mediated rejection compared to acute cellular rejection after pediatric heart transplantation[END_REF]. In our experience, all the most severe cases of acute graft dysfunction responsible for a cardiogenic shock were related to an AMR highlighting the important role of this type of rejection in the genesis of acute graft dysfunction [START_REF] Coutance | Antibody-mediated rejection induced cardiogenic shock: too late for conventional therapy[END_REF].

Intro-B-3.b. Chronic injury: cardiac allograft vasculopathy Cardiac allograft vasculopathy (CAV) is a common and severe condition following HTx.

It is characterized by an intimal thickening responsible for a diffuse narrowing of the coronary arteries. Despite the progress made in terms of prevention, the incidence of cardiac allograft vasculopathy remains as high as 50% 10 years after transplantation (14). The presence of a CAV is a significant risk factor for morbidity and mortality, even at a subclinical stage diagnosed by an increase in intimal thickness on intravascular ultrasonography [START_REF] Kobashigawa | Multicenter intravascular ultrasound validation study among heart transplant recipients: outcomes after five years[END_REF][START_REF] Potena | Interplay of coronary angiography and intravascular ultrasound in predicting long-term outcomes after heart transplantation[END_REF]. In addition to the usual donors' and recipients' cardiovascular risk factors, numerous experimental and clinical studies, have established a strong link between donor-specific anti-HLA antibodies (DSA) and AMR on the one hand and the occurrence or progression of a CAV on the other hand [START_REF] Hirohashi | Complement Independent Antibody-Mediated Endarteritis and Transplant Arteriopathy in Mice[END_REF][START_REF] Hirohashi | A novel pathway of chronic allograft rejection mediated by NK cells and alloantibody[END_REF][START_REF] Topilsky | Donor-specific antibodies to class II antigens are associated with accelerated cardiac allograft vasculopathy: a threedimensional volumetric intravascular ultrasound study[END_REF][START_REF] Clerkin | The effect of timing and graft dysfunction on survival and cardiac allograft vasculopathy in antibody-mediated rejection[END_REF]. Antibody Dependent Cellular Cytotoxicity (ADCC) appears to have a major role in the pathophysiology of CAV [START_REF] Hirohashi | A novel pathway of chronic allograft rejection mediated by NK cells and alloantibody[END_REF][START_REF] Paul | Genetic and Functional Profiling of CD16-Dependent Natural Killer Activation Identifies Patients at Higher Risk of Cardiac Allograft Vasculopathy[END_REF].

Intro-B-3.c. Chronic injury: graft restrictive physiology Progressive evolution to ventricular filling disorders, characterized by restrictive cardiac transplant physiology, is an important cause of late transplant dysfunction and retransplantation. These abnormalities would account for one-third of cases of heart failure beyond one year of transplantation [START_REF] Ambrosi | Chronic heart failure in heart transplant recipients: Presenting features and outcome[END_REF] and 15% of retransplantations [START_REF] Loupy | Late Failing Heart Allografts: Pathology of Cardiac Allograft Vasculopathy and Association With Antibody-Mediated Rejection[END_REF].

Impairment of the coronary microcirculation is probably an important mechanism of this type of dysfunction. Several studies have established a link between DSA / AMR and restrictive physiology. The presence of DSA was associated with abnormalities of coronary microcirculation and elevation of filling pressures in a Danish study [START_REF] Clemmensen | Donor-specific antibodies are associated with micro-and macrovascular coronary disease, restrictive myocardial damage, and poor outcome in heart-transplanted patients[END_REF]. Most retransplanted patients, whatever the reason, showed signs of AMR, most often subclinical, both on explanted transplants and on endomyocardial biopsies performed during usual follow-up [START_REF] Loupy | Late Failing Heart Allografts: Pathology of Cardiac Allograft Vasculopathy and Association With Antibody-Mediated Rejection[END_REF].

Decreasing late mortality after heart transplantation is an unmet medical need.

The graft-specific diseases (CAV, rejection, late dysfunctions, restrictive physiology) represent the first cause of death beyond the first year of transplant.

Allosensitization and its corollary, the AMR, are at the center of all these graftspecific diseases due to the acute and chronic lesions, both micro and macrovascular, that they might induce [START_REF] Loupy | Antibody-Mediated Rejection of Solid Organ Allografts[END_REF]. Improving the management of allosensitization and AMR is therefore a priority in heart transplantation.

Intro-C. Current issues in the management of DSA and AMR after heart transplantation

The current medical management, which is extremely heterogeneous from one center to another, suffers from three major limitations: i) the difficulty of pathologic diagnosis, ii) the lack of prognostic stratification tool able to help the clinical-decision making, and iii) and the lack of consensus regarding the therapeutic management, direct consequent of the previous issues [START_REF] Chih | A survey of current practice for antibodymediated rejection in heart transplantation[END_REF][START_REF] Colvin | Antibody-mediated rejection in cardiac transplantation: emerging knowledge in diagnosis and management: a scientific statement from the American Heart Association[END_REF][START_REF] Kobashigawa | T he management of antibodies in heart transplantation: An ishlt consensus document[END_REF].

Our objective is to increase our knowledge of these three fundamental points of the management of DSA and AMR in heart transplantation by applying innovative methodologies for cardiac pathology, epidemiology and therapeutic management.

Intro-C-1. Pathologic diagnosis of AMR

The diagnosis of AMR in heart transplantation only relies on the pathologic analysis of an endomyocardial biopsy (EMB), regardless of the presence of DSA or transplant dysfunction [START_REF] Berry | The 2013 International Society for Heart and Lung Transplantation Working Formulation for the standardization of nomenclature in the pathologic diagnosis of antibody-mediated rejection in heart transplantation[END_REF][START_REF] Bruneval | The XIIIth Banff Conference on Allograft Pathology: The Banff 2015 Heart Meeting Report: Improving Antibody-Mediated Rejection Diagnostics: Strengths, Unmet Needs, and Future Directions[END_REF]. Antibody-mediated rejection (AMR) is graded from pAMR0 to pAMR3 according to the presence of #1-microvascular inflammation defined by the intravascular accumulation of activated mononuclear cells (IAMC) within the myocardial capillaries (= histopathologic AMR, pAMR1-H+) and #2-signs of local activation of the complement pathway (C4d, C3d: multifocal or diffuse strong capillaries staining) and/or presence of intravascular macrophages (CD68+, ≥10% of capillaries) defining immunopathologic AMR (pAMR1-I+). The presence of both histopathologic and immunopathologic findings defines pAMR2. The third grade, pAMR3, is reserved to the very rare forms of severe pathologic AMR (interstitial hemorrhage, capillary fragmentation) [START_REF] Berry | The 2013 International Society for Heart and Lung Transplantation Working Formulation for the standardization of nomenclature in the pathologic diagnosis of antibody-mediated rejection in heart transplantation[END_REF][START_REF] Bruneval | The XIIIth Banff Conference on Allograft Pathology: The Banff 2015 Heart Meeting Report: Improving Antibody-Mediated Rejection Diagnostics: Strengths, Unmet Needs, and Future Directions[END_REF].

Although widely used, this classification suffers from a number of inaccuracies [START_REF] Bruneval | The XIIIth Banff Conference on Allograft Pathology: The Banff 2015 Heart Meeting Report: Improving Antibody-Mediated Rejection Diagnostics: Strengths, Unmet Needs, and Future Directions[END_REF].

One of the major limitations is the dichotomization of the diagnosis of microvascular inflammation (present / absent) without clearly defined diagnostic threshold. This directly impacts the problem of interobserver agreement, which is low for acute cellular rejection [START_REF] Crespo-Leiro | Concordance among pathologists in the second Cardiac Allograft Rejection Gene Expression Observational Study (CARGO II)[END_REF], and which has never been evaluated on a large scale for AMR. The large disparities in care between centers are probably due in part to these diagnostic difficulties. Molecular biology techniques have demonstrated their potential relevance in rationalizing the diagnosis of AMR, but several constraints, in particular financial constraints, are a limit to their application in clinical practice [START_REF] Halloran | Building a tissue-based molecular diagnostic system in heart transplant rejection: The heart Molecular Microscope Diagnostic (MMDx) System[END_REF][START_REF] Afzali | Molecular Assessment of Microcirculation Injury in Formalin-Fixed Human Cardiac Allograft Biopsies With Antibody-Mediated Rejection[END_REF].

We hypothesize that i) a technique of molecular biology, the RT-MLPA (Multiplex Ligation-dependent Probe Amplification), which is inexpensive and transposable into the routine of a pathology laboratory, is suitable for the analysis of gene expression on formalin-fixed paraffin-embedded (FFPE) EMB and may be a support for the pathologic diagnosis, and ii) that a semi-quantitative evaluation of microvascular inflammation in four stages is correlated with the molecular activity of AMR and could have a role in the diagnostic standardization and prognostic stratification of AMR (Figure 4).

Intro-C-2. Screening for complications of allosensitization and AMR

Screening for CAV and allograft rejection is based on invasive, iterative and protocolized tests, coronary angiography and endomyocardial biopsy, respectively [START_REF] Costanzo | The International Society of Heart and Lung Transplantation Guidelines for the care of heart transplant recipients. The Journal of heart and lung transplantation: the official publication of the[END_REF][START_REF] Mehra | International Society for Heart and Lung Transplantation working formulation of a standardized nomenclature for cardiac allograft vasculopathy-2010[END_REF]. As the natural history and the risk factors of these complications are not deeply described, the frequency of screening tests is applied in the same way to all patients, regardless of their risk of developing the disease. It results in a poor diagnostic costeffectiveness compared to the risk of complications inherent to these invasive tests [START_REF] Shah | Surveillance Endomyocardial Biopsy in the Modern Era Produces Low Diagnostic Yield for Cardiac Allograft Rejection[END_REF].

We hypothesize that a better knowledge of CAV's and AMR's epidemiology, natural history and risk factors, based on the use of innovative statistical tools and on a population-based approach, would allow a better prognostic stratification of these diseases; a first step toward the rationalization of invasive screening tests (Figure 4). Intro-C-3. Therapeutic management of populations at high immunological risk.

Therapeutic managements of allosensitization and AMR are highly heterogeneous between centers [START_REF] Chih | A survey of current practice for antibodymediated rejection in heart transplantation[END_REF]. Latest guidelines on these topics highlight the lack of consensus and the inability to recommend an accurate medical management in most situations due to the lack of clinical data [START_REF] Colvin | Antibody-mediated rejection in cardiac transplantation: emerging knowledge in diagnosis and management: a scientific statement from the American Heart Association[END_REF][START_REF] Kobashigawa | T he management of antibodies in heart transplantation: An ishlt consensus document[END_REF]. The prognosis and therapeutic management of certain populations at immunological risk have been subject to poor evaluation, especially concerning heart transplantation with pre-formed DSA. Most centers avoid this type of transplantation given the increased risk of post-transplant morbidity and mortality [START_REF] Nwakanma | Influence of pretransplant panel-reactive antibody on outcomes in 8,160 heart transplant recipients in recent era[END_REF][START_REF] Raess | Donor-specific anti-HLA antibodies detected by Luminex: predictive for short-term but not long-term survival after heart transplantation[END_REF][START_REF] Stastny | Antibodies against donor human leukocyte antigens and the outcome of cardiac allografts in adults and children[END_REF][START_REF] Svobodova | Novel insights into pretransplant allosensitization in heart transplant recipients in the contemporary era of immunosuppression and rejection surveillance[END_REF].

We hypothesize that a desensitization protocol, based on plasmapheresis and intravenous polyvalent immunoglobulins (IVIg) infusions, implemented through a prospective protocol and applied at the time of transplantation can enable to minimize the impact of pre-formed DSA on post-transplant morbidity and mortality.

Advances in diagnosis, prognostic stratification and therapeutics concerning

DSA and AMR may have a real clinical impact on the future of cardiac transplant patients and thus constitute a research field of primary importance. We will apply innovative methodologies, in terms of cardiac pathology, epidemiology, patient's care (Figure 4). Whereas the degree of microvascular inflammation is graded according to a semiquantitative histologic scale in kidney transplantation [START_REF] Haas | The Banff 2017 Kidney Meeting Report: Revised diagnostic criteria for chronic active T cell-mediated rejection, antibodymediated rejection, and prospects for integrative endpoints for next-generation clinical trials[END_REF][START_REF] Loupy | The Banff 2015 Kidney Meeting Report: Current Challenges in Rejection Classification and Prospects for Adopting Molecular Pathology[END_REF], the threshold for which IAMC are considered significant for the possibility of AMR in hematoxylin & eosin stained sections has not been formally established in heart transplantation [START_REF] Bruneval | The XIIIth Banff Conference on Allograft Pathology: The Banff 2015 Heart Meeting Report: Improving Antibody-Mediated Rejection Diagnostics: Strengths, Unmet Needs, and Future Directions[END_REF]. This absence of a clear threshold for the diagnosis of AMR might explain in part the important variability of AMR diagnosis and treatment across centers [START_REF] Chih | A survey of current practice for antibodymediated rejection in heart transplantation[END_REF]. Gene expression analysis of the allograft tissue has been increasingly recognized as a powerful tool for the understanding of the pathophysiological pathways of rejection after heart transplantation [START_REF] Lund | The Registry of the International Society for Heart and Lung Transplantation: Thirty-fourth Adult Heart Transplantation Report-2017; Focus Theme: Allograft ischemic time[END_REF][START_REF] Shah | Surveillance Endomyocardial Biopsy in the Modern Era Produces Low Diagnostic Yield for Cardiac Allograft Rejection[END_REF] but their applicability in clinical practice is limited by several flaws.

After a brief review of the physiopathology of AMR and the developments of molecular biology in the field or cardiac allograft rejection, we will detail our results concerning the application of two molecular biology techniques to the diagnosis of AMR.

Chapter I-A:

Physiopathology of antibodymediated rejection and developments of molecular biology in heart transplantation.

I-A-1. Pathophysiology of antibody-mediated rejection

The donor vasculature present at the interface between donor tissue and the recipient immune system is the primary target of the alloimmune response. The complex pathophysiology of AMR is more and more understood and implies i) the production of donor-specific antibodies (DSA), mostly anti-HLA antibodies, as an alloimmune response to organ transplantation or to pre-transplant sensitizing events, ii) the binding of these DSA on the donor's endothelial cells, iii) responsible for the activation of both complement-dependent and complement-independent pathways leading to cells damage and allograft rejection.

I-A-2-a. Complement-dependent pathway (Figure 5)

Ever since the discovery by Terasaki et al. of the importance of complementdependent cytotoxicity (CDC) crossmatch to predict hyperacute rejection of allograft, evidence of the central role of the complement activation in the pathophysiology of AMR has been growing [START_REF] Patel | Significance of the positive crossmatch test in kidney transplantation[END_REF].

Classical pathway activation is initiated when plasma C1q binds to the Fc segments of IgM and IgG. The extent of complement activation depends on the antibody isotype, the abundance of the target antigen and density of immunoglobulins, the local concentration of complement regulatory proteins, and the influence of antibodytargeting therapies [START_REF] Loupy | Antibody-Mediated Rejection of Solid Organ Allografts[END_REF]. Activation of the complement cascade results in the formation of a number of biologically active complement fragments. C3b is covalently fixed to cell surfaces and acts as an opsonin. The C3a and C5a fragments act as powerful inducers of the local inflammatory response. Cellular signaling pathways activated after binding of these fragments to endothelial cells (through G protein-coupled receptors) results in the release of pro-inflammatory cytokines, von Willebrand factor and heparan sulfate [START_REF] Stites | The Complement System and Antibody-Mediated Transplant Rejection[END_REF]. The complement activation cascade leads to the assembly of the C5b-9 molecular complex, also called membrane attack complex (MAC). This results from the interaction between C5b, C6 and C7 which anchors this first complex to the membrane and causes the recruitment of C8. The molecular tetrad then leads to the recruitment and polymerization C9 of which forms a pore in the membrane causing osmotic cell lysis. This lytic cellular effect requires a high density of MAC at the membrane level and is considered nowadays as a rare event during AMR. MAC's contribution to complement-mediated endothelial dysfunction is thought to be mediated by its so-called sub lytic activity resulting in endothelial activation via the non-canonical NF-Κb signaling pathway leading to the expression of pro-inflammatory proteins, in particular IL-6 [START_REF] Stites | The Complement System and Antibody-Mediated Transplant Rejection[END_REF][START_REF] Cole | Beyond lysis: how complement influences cell fate[END_REF]. Finally, complement activation and MAC binding to endothelial cells also stimulate the adaptive immune system, which increase interferon production.

γ (IF-γ).

I-A-2-b. Complement-independent pathway (Figure 5)

The cellular component also plays a major role in AMR. Thus, similarly to acute cellular rejection, of which "immunological synapse" between the T cell and the antigen presenting cell in the interstitial sector is the cornerstone of the pathologic process, an "immunological synapse" is also one of the fundamental mechanisms of the AMR, though in the vascular sector, between the Natural Killer (NK) cell and the endothelial cells via FCGRIIIA (Fc gamma receptor, CD16A) and the DSA [START_REF] Loupy | Gene Expression Profiling for the Identification and Classification of Antibody-Mediated Heart Rejection[END_REF]. Activation of the NK cell will result in:

-the production and release of interferon-gamma (IFNG), tumor necrosis factor (TNF), and granulocyte-macrophage colony-stimulating factor (CSF2), increasing i) the « élément sous droit, diffusion non autorisée » I-A-2-a. Genome-wide expression

I-A-2-a-1. Description
Microarray technology has made possible to improve our understanding of the pathophysiology of allograft rejection, by allowing large and a priori-free analyzes of the expression of several tens of thousands of transcripts simultaneously. Our understanding of AMR has been particularly impacted by these techniques [START_REF] Loupy | Gene Expression Profiling for the Identification and Classification of Antibody-Mediated Heart Rejection[END_REF]. Each DNA microarray contains several tens of thousands of probes, which are synthetic DNA sequences specific for the gene whose expression is to be analyzed. After extraction of mRNAs and their retro-transcription into labeled complementary DNAs (cDNAs), these cDNAs are to be put in contact with the chip. During the hybridization step, the cDNAs are to hybridize to the specific sequences of the probe attached to the array. After washing off the unbound cDNAs, the array is then analyzed by a highresolution scanner at the excitation wavelength of the fluorochrome.

I-A-2-a-3. Pathogenesis-based transcripts (PBT)

The comparison of molecular expression profiles between acute cellular rejections, AMR and non-rejection biopsies, allowed the detection of several tens of differentially expressed transcripts [START_REF] Loupy | Gene Expression Profiling for the Identification and Classification of Antibody-Mediated Heart Rejection[END_REF][START_REF] Holweg | Identification and classification of acute cardiac rejection by intragraft transcriptional profiling[END_REF][START_REF] Bodez | Myocardial Gene Expression Profiling to Predict and Identify Cardiac Allograft Acute Cellular Rejection: The GET-Study[END_REF][START_REF] Halloran | Review: The transcripts associated with organ allograft rejection[END_REF]. For example, CD8A (a coreceptor expressed on the surface of the cytotoxic T lymphocyte and allowing interaction with HLA class I molecules), ADAMDEC, CTLA4 (costimulatory signal) are particularly expressed in acute cellular rejection. Conversely, molecules such as CXCL9 and 10 (chemokines promoting the recruitment of NK cells), CCL4, PLA1, are more expressed in AMR (Figure 6). Interestingly, these molecular profiles specific of rejections are comparable between renal and heart transplantations, suggesting the presence of common underlying pathophysiological mechanisms. [START_REF] Halloran | Building a tissue-based molecular diagnostic system in heart transplant rejection: The heart Molecular Microscope Diagnostic (MMDx) System[END_REF][START_REF] Loupy | Gene Expression Profiling for the Identification and Classification of Antibody-Mediated Heart Rejection[END_REF].

« élément sous droit, diffusion non autorisée »

I-A-3. Objectives

The aim of the two following studies was to assess whether a molecular biology approach might help standardizing the diagnosis of AMR in heart transplantation (Figure 7).

We hypothesize i) that RT-MLPA, is applicable to paraffin-fixed EMB and might be a support for the pathologic diagnosis of allograft rejection by identifying a rejectionspecific molecular signature (no rejection, acute cellular rejection, AMR), and ii) that a semi-quantitative evaluation of microvascular cardiac inflammation in four stages is correlated with the molecular activity of AMR and could have a role in the diagnostic standardization and prognostic stratification of AMR (Figure 7).

I-B-1. Molecular biology applied to the diagnosis of heart allograft rejection

Protocol endomyocardial biopsies are a key element of the follow-up of heart transplant recipients [START_REF] Costanzo | The International Society of Heart and Lung Transplantation Guidelines for the care of heart transplant recipients. The Journal of heart and lung transplantation: the official publication of the[END_REF]. The histological analysis of myocardial samples is the gold standard for the diagnosis of heart allograft rejections and guides therapies [START_REF] Berry | The 2013 International Society for Heart and Lung Transplantation Working Formulation for the standardization of nomenclature in the pathologic diagnosis of antibody-mediated rejection in heart transplantation[END_REF][START_REF] Stewart | Revision of the 1990 working formulation for the standardization of nomenclature in the diagnosis of heart rejection[END_REF]. However, this histology-based strategy is limited by several flaws. The reliability of histological analysis is nowadays questioned as inter-observer reproducibility has been reported to be as low as 28% for the diagnosis of acute cellular rejection [START_REF] Crespo-Leiro | Concordance among pathologists in the second Cardiac Allograft Rejection Gene Expression Observational Study (CARGO II)[END_REF]. Besides, the histological grading may be particularly challenging for microvascular inflammation, a key criterion for the diagnosis of antibody-mediated rejection (AMR) [START_REF] Colvin | Antibody-mediated rejection in cardiac transplantation: emerging knowledge in diagnosis and management: a scientific statement from the American Heart Association[END_REF]. Reliable, objective and disease-specific (i.e., rejection versus non-rejection and acute cellular rejection -ACR -versus AMR) markers of rejection are an important unmet clinical need [START_REF] Bruneval | The XIIIth Banff Conference on Allograft Pathology: The Banff 2015 Heart Meeting Report: Improving Antibody-Mediated Rejection Diagnostics: Strengths, Unmet Needs, and Future Directions[END_REF].

Gene expression analysis of the allograft tissue based on pan-genomic approaches has been increasingly recognized as a powerful tool for the understanding of the pathophysiological pathways of rejection after heart transplantation [START_REF] Halloran | Building a tissue-based molecular diagnostic system in heart transplant rejection: The heart Molecular Microscope Diagnostic (MMDx) System[END_REF][START_REF] Loupy | Gene Expression Profiling for the Identification and Classification of Antibody-Mediated Heart Rejection[END_REF]. However, several flaws may limit the impact of these techniques in everyday practice and their widespread use to improve the diagnosis of rejection. Targeted gene expression analysis has emerged as a potentially rapid, easy-to-use, and cost-effective alternative to pan-genomic approaches [START_REF] Adam | Multiplexed color-coded probe-based gene expression assessment for clinical molecular diagnostics in formalin-fixed paraffinembedded human renal allograft tissue[END_REF][START_REF] Smith | RNA expression profiling of renal allografts in a nonhuman primate identifies variation in NK and endothelial gene expression[END_REF]. Reverse Transcriptase-Multiplex Ligasedependent Probe Amplification (RT-MLPA) is an efficient technique of targeted gene relative expression analysis suitable for formalin-fixed paraffin-embedded (FFPE) tissue [START_REF] Bobée | Determination of Molecular Subtypes of Diffuse Large B-Cell Lymphoma Using a Reverse Transcriptase Multiplex Ligation-Dependent Probe Amplification Classifier[END_REF][START_REF] Eldering | Expression profiling via novel multiplex assay allows rapid assessment of gene regulation in defined signalling pathways[END_REF].

The underlying principle of MLPA is to make the amplification step of the hybridized probes dependent on a ligation between the two probes specific for the transcript of interest (Figure 8). In practice, for each transcript of interest, a pair of specific probes must be designed. These two probes must be complementary to two adjacent sequences of the cDNA to be analyzed. Each probe must comprise at one end a PCR primer sequence common to the other pairs of probes, allowing a common amplification step at the end of the reaction using a single primer pair. Spacers are to be added to the 3' end of the probe to obtain lengths of final amplicon specific to each transcript of interest. Finally, the amplification products are analyzed by fragment analysis using an ABI 3130 XL capillary electrophoresis analyzer makes it possible to obtain gene expression profiles. Our study complies with the Declaration of Helsinki and the institutional review board approved the protocol. Written informed consent for data collection and gene expression analysis of a stored EMB was obtained at the time of enrolment in the study.

Baseline donor and recipient characteristics were collected from the prospective national registry CRISTAL database (French National Agency for Organ Procurement).

Immunological and immunosuppressive regimen data were retrospectively collected from the patients' charts. Codes were used to ensure strict donor and recipient anonymity.

I-B-2-c. Histopathology

A review of all the EMB was performed by two pathologists, independently of the initial pathologic diagnosis, clinical, and molecular data. EMB specimens were formalinfixed, paraffin-embedded, and routinely stained with hematoxylin and eosin.

Immunohistochemistry was performed in tissue sections with rabbit monoclonal rabbit anti-C4d and mouse monoclonal anti-CD68 antibodies using the immunoperoxidase method, as previously described [START_REF] Fedrigo | Inflammatory cell burden and phenotype in endomyocardial biopsies with antibody-mediated rejection (AMR): a multicenter pilot study from the AECVP[END_REF]. EMB were evaluated for rejection according to the last ISHLT classification for acute cellular rejection (ACR), and to the last recommendations for the diagnosis of AMR [START_REF] Berry | The 2013 International Society for Heart and Lung Transplantation Working Formulation for the standardization of nomenclature in the pathologic diagnosis of antibody-mediated rejection in heart transplantation[END_REF][START_REF] Bruneval | The XIIIth Banff Conference on Allograft Pathology: The Banff 2015 Heart Meeting Report: Improving Antibody-Mediated Rejection Diagnostics: Strengths, Unmet Needs, and Future Directions[END_REF][START_REF] Stewart | Revision of the 1990 working formulation for the standardization of nomenclature in the diagnosis of heart rejection[END_REF]. Mixed rejection was defined by the presence of ACR ≥2R and pAMR ≥ pAMR2. Histological low-grade rejections were defined as ACR 1R and/or pAMR1(I+) rejections. Histopathology is illustrated in Figure 10-A/C. I-B-2-d. Detection of antibodies against donor-specific HLA molecules HLA typing of transplant recipients was performed by molecular biology using the Innolipa HLA typing kit (Innogenetics, Belgium). Heart transplant donors, HLA-A, -B, -DR, and -DQ tissue-typing was performed using the microlymphocytotoxicity technique with tissue-typing trays (One-Lambda Inc, Canoga Park, CA). Briefly, identification of anti-HLA donor-specific antibodies (DSA) was performed by Luminex SA (LuminexLABScreen Single Antigen,One-Lambda Inc) as previously described [START_REF] Aubert | Antibody-Mediated Rejection Due to Preexisting versus De Novo Donor-Specific Antibodies in Kidney Allograft Recipients[END_REF].

The value of 500 Mean Fluorescence Intensity (MFI) in our laboratory was based on the criteria of validation of negative control sera.

I-B-2-e. RT-MLPA probes design

Fourteen transcripts were included in the RT-MLPA assay. Genes were selected from the Affymetrix and NanoString literature for their performance to diagnose rejection and to distinguish between AMR and ACR [START_REF] Halloran | Building a tissue-based molecular diagnostic system in heart transplant rejection: The heart Molecular Microscope Diagnostic (MMDx) System[END_REF][START_REF] Loupy | Gene Expression Profiling for the Identification and Classification of Antibody-Mediated Heart Rejection[END_REF][START_REF] Afzali | Molecular Assessment of Microcirculation Injury in Formalin-Fixed Human Cardiac Allograft Biopsies With Antibody-Mediated Rejection[END_REF][START_REF] Bodez | Myocardial Gene Expression Profiling to Predict and Identify Cardiac Allograft Acute Cellular Rejection: The GET-Study[END_REF]. They included interferoninduced genes (CXCL11, CXCL9), endothelial activation related genes (PLA1A, ROBO4, ECSCR, TM4SF18, ICAM-1), NK related genes (FCGR3, CCL4, GNLY), cytotoxic T lymphocytes (CD3, CCL18), and antigen presentation (CD74 and ADAMDEC1). A housekeeper gene was added to evaluate the global gene expression level (LDHA). The probe's design has been described by Bobée et al [START_REF] Bobée | Determination of Molecular Subtypes of Diffuse Large B-Cell Lymphoma Using a Reverse Transcriptase Multiplex Ligation-Dependent Probe Amplification Classifier[END_REF]. Briefly, all 5' probes presented a common 5' end tail corresponding to the fluorescent labelled PCR probe illu2-FAM. In addition to the specific region, 3' probes presented a common sequence at their 3' end, a phosphorylated end for the ligation reaction at their 5' extremity and spacer sequences between the specific and common regions to allow the separation of the amplicons during the fragment size analysis. The common sequences were used to amplify all ligation products within a single PCR reaction.

Probes used in the mix are detailed in Table 1.

I-B-2-f. RT-MLPA procedure
The protocol and data processing of the RT-MLPA procedure were used as previously described, using MLPA reagents from MRC-Holland (Amsterdam, The Netherlands) [START_REF] Bobée | Determination of Molecular Subtypes of Diffuse Large B-Cell Lymphoma Using a Reverse Transcriptase Multiplex Ligation-Dependent Probe Amplification Classifier[END_REF]. RNA extraction: All RNAs were extracted from FFPE tissues previously used for routine histopathologic diagnosis of rejection. RNA isolation from paraffin blocks was performed using the 16 LEV FFPE Maxwell kit and the Maxwell 16 automation system (Promega, Madison WI) using the manufacturer's instructions. Reverse transcription:

2 mL of total RNA from the FFPE EMB were added to 3.75 mL of reverse transcription mix containing random hexamer primers. Next, 0.5 mL of Moloney murine leukemia virus reverse transcriptase was added, and the samples were incubated for 15 minutes at 37°C for cDNA synthesis, heated for 2 minutes at 98 °C, and cooled at 4 °C. Ligation:

3 mL of RT-MLPA probe mix was added (1.5 mL of SALSA-MLPA buffer and 1.5 mL of final dilution probe mix) before denaturation at 95°C for 2 minutes and hybridization at 60 °C for one hour. Ligation of the annealed oligonucleotides was performed at 54 °C for 15 minutes, adding 32 mL of ligation mix, and heated for 5 minutes at 98 °C. PCR: 2.5 mL of the ligation mixture was added to 7.5 mL of Salsa PCR master mix containing the labeled forward primer and the unlabeled reverse primer. Fragment analysis: The resulting MLPA amplicons were analyzed by fragment analysis using an ABI 3130 XL capillary electrophoresis analyzer (Applied Biosystems, Foster City, CA).

5' probes CCL4E1L

5'-GTGCCAGCAAGATCCAATCTAGACTCCAGCGCTCTCAGCACCAA-3'

ECSCRE1L 5'-GTGCCAGCAAGATCCAATCTAGACTGGGCTTCCTCCTGTTCCGAG-3' PLA1AE4L 5'-GTGCCAGCAAGATCCAATCTAGAGCCATCCACACAGACACCGACA-3' CD8AE4L 5'-GTGCCAGCAAGATCCAATCTAGATACGACGTGTTTGCAAATGTCCCCG-3' GNLYE4L 5'-GTGCCAGCAAGATCCAATCTAGATACTAAAGAAGATGGTGGATAAGCCCACCCAG-3' CXCL9E1L 5'-GTGCCAGCAAGATCCAATCTAGATATCTTGCTGGTTCTGATTGGAGTGCAAG-3' CD74E2L 5'-GTGCCAGCAAGATCCAATCTAGATACTACTACTGCGCATGAAGCTTCCCAAGC-3' LDHAE2L 5'-GTGCCAGCAAGATCCAATCTAGATTGGCCTGTGCCATCAGTATCTTAATGAAG-3' FCGR3AE5L 5'GTGCCAGCAAGATCCAATCTAGATACTTCAGAGACTGTGAACATCACCATCACTCAAG-3' ICAM1E2L 5'GTGCCAGCAAGATCCAATCTAGATACTTCAGAGACTGTGAACATCACCATCACTCAAG-3' CXCL11E2L 5'GTGCCAGCAAGATCCAATCTAGATACAATGTACCCAAGTAACAACTGTGACAAAATAGAAGTGAT-3' CCL18E2L 5'-GTGCCAGCAAGATCCAATCTAGATACTACTACTACTACTCAGTGCCCCAAGCCAGGTGTCAT ROBO4E15L 5'-GTGCCAGCAAGATCCAATCTAGATACTACTACTACTACAACTCAGTGAGGGTGAGGAGACTCCCAG-3' ADAMDEC1E5L 5'-GTGCCAGCAAGATCCAATCTAGATACTAGAGAGGAAATTACCACGAAACCTGAGAACATG-3' TM4SF18E2L 5'-GTGCCAGCAAGATCCAATCTAGATACTAGTATTTTGAAGGAATCTGTTTCTCAGGCATCATG-3' 3' probes CCL4E2R 5'-Pho-TGGGCTCAGACCCTCCCACCTCCAACCCTTAGGGAACCC-3' ECSCRE2R 5'-Pho-GCCACAACTCCCAGCCCACAATCCAACCCTTAGGGAACCC-3' PLA1AE5R 5'-Pho-ATTTGGGTATTCGGATTCCCGTTGTCCAACCCTTAGGGAACCC-3' CD8AE5R 5'-Pho-GCCTGTGGTCAAATCGGGAGACATACTCCAACCCTTAGGGAACCC-3' GNLYE5R 5'-Pho-AGAAGTGTTTCCAATGCTGCGACCCTACTACTCCAACCCTTAGGGAACCC-3' CXCL9E2R 5'-Pho-GAACCCCAGTAGTGAGAAAGGGTCGCTTCCAACCCTTAGGGAACCC-3' CD74E3R 5'-Pho-CTCCCAAGCCTGTGAGCAAGATGTACTACTCCAACCCTTAGGGAACCC-3' LDHAE3R 5'-Pho-GACTTGGCAGATGAACTTGCTCTTGTTGATTTCCAACCCTTAGGGAACCC-3' FCGR3AE6R 5'-Pho-GTTTGGCAGTGTCAACCATCTCATCATTCTACTATCCAACCCTTAGGGAACCC-3' ICAM1E3R 5'-Pho-GGACTCCAGAACGGGTGGAACTGGTACTACTACTCCAACCCTTAGGGAACCC-3' CXCL11E3R 5'-Pho-TATTACCCTGAAAGAAAATAAAGGACAACGATGCTCCAACCCTTAGGGAACCC-3' CCL18E3R 5'-Pho-CCTCCTAACCAAGAGAGGCCGGCTACTACTACTACTACTATCCAACCCTTAGGGAACCC-3' ROBO4E16R 5'-Pho-GAACAGCGTCTCTCCCATGCCAAGTACTACTACTACTACTCCAACCCTTAGGGAACCC-3' ADAMDEC1E6R 5'-Pho-GAACACTGTTACTATAAAGGAAACATCCTAAATGAAAAGAATTCTTACTTCCAACCCTTAGGGAACCC-3' TM4SF18E3R 5'-Pho-GAACACTGTTACTATAAAGGAAACATCCTAAATGAAAAGAATTCTTACTTCCAACCCTTAGGGAACCC-3' Competitors CD74compL 5'-GCGCATGAAGCTTCCCAAGC-3' LDHAcompL 5'-TGGCCTGTGCCATCAGTATCTTAATGAAG-3' ECSCRcompL 5'-CTGGGCTTCCTCCTGTTCCGAG-3' ROBO4compL 5'-AACTCAGTGAGGGTGAGGAGACTCCCAG-3'

Table 1: Sequences of RTMLPA probes and competitors

All sequences of the RT-MLPA probes and the oligonucleotides used as competitors are provided. The sequences of the primers used for PCR amplification are underlined.

I-B-2-g. Statistical methods

We report means and standard deviations to describe continuous variables, and numbers and percentages for categorical variables. We compared the means and counts with the Student t-test or ANOVA and χ2 test, respectively. Unsupervised

I-B-3-b. RT-MLPA profiles on FFPE endomyocardial biopsies

Examples of RT-MLPA profiles in EMB with a pathologic diagnosis of AMR, ACR and no rejection are given in Figure 10-D/F. AMR was characterized by high expression of CCL4, PLA1A, GNLY, CXCL9, FCGR3, and CXCL11. ACR was characterized by high relative expression of CCL18 and ADAMdec (Figure 11).

Unsupervised hierarchical analysis of these profiles is given in Figure 12-A. Three molecular clusters were delineated, that corresponded to the pathologic diagnosis of the non-rejection, ACR, and AMR, respectively.

I-B-3-e. Disagreement between histology and RT-MLPA

A summary of the disagreements between histopathology and RT-MLPA for the diagnosis of rejection (n=12) and for the type of rejection (n=20) is given in Table 4.

Twelve cases (6.5%) were discordant between histology and molecular diagnosis by LPS1 including eight cases of mNR diagnosis with histological rejection (mostly lowgrade rejection, n = 5 (62.5%): pAMR1(I+): n=4; ACR 1R: n=1) and four cases of hNR with molecular rejection. Interestingly, graft function was severely impaired at the time of EMB in two out of four of the hNR cases with molecular rejection. The histological grade of rejection was significantly associated with the risk of discordance by LPS1, which was higher in case of the histological low-grade rejection (p < 0.01).

Twenty cases (17.2%) were discordant between the histology and molecular diagnosis by LPS2 including 13 cases (65%) of hAMR classified as mACR. The time posttransplant was significantly associated with the risk of discordance by LPS2, which was higher in patients with EMB < six weeks post-transplant (p = 0.02).

Pathology

I-B-4. Discussion

We described a first evaluation of the diagnostic performance of RT-MLPA, a technique of targeted gene expression analysis suitable for FFPE EMB to detect biopsy-proven rejection in heart transplantation. By studying the relative expression of Reliable, objective and disease-specific markers of rejection are an important unmet clinical need in the field of heart transplantation. Gene expression analysis of the allograft tissue based on pan-genomic approaches has been increasingly recognized as a powerful tool for the discovery and the understanding of the pathophysiological pathways of rejection in solid organ transplant [START_REF] Halloran | Review: The transcripts associated with organ allograft rejection[END_REF][START_REF] Halloran | Microarray diagnosis of antibodymediated rejection in kidney transplant biopsies: an international prospective study (INTERCOM)[END_REF][START_REF] Sellarés | Molecular diagnosis of antibody-mediated rejection in human kidney transplants[END_REF]. Recently, the analysis of the expression of a 34-gene set using the NanoString nCounter demonstrated the efficiency of this technology in the field of AMR in heart transplantation [START_REF] Afzali | Molecular Assessment of Microcirculation Injury in Formalin-Fixed Human Cardiac Allograft Biopsies With Antibody-Mediated Rejection[END_REF]. As underlined by the Banff 2017 meeting report, the molecular assessment using new methods of targeted-gene expression of a selected gene-set from FFPE routine biopsies is the next step forward for a multicenter validation of the contribution of molecular pathology in the daily clinical practice [START_REF] Haas | The Banff 2017 Kidney Meeting Report: Revised diagnostic criteria for chronic active T cell-mediated rejection, antibodymediated rejection, and prospects for integrative endpoints for next-generation clinical trials[END_REF]. Within this context, we evaluated the diagnostic performance of RT-MLPA, a new cheap, robust and easy-to-use technique of multiplex PCR able to analyze the profiles of expression of a selectedgene set from FFPE-biopsies [START_REF] Bobée | Determination of Molecular Subtypes of Diffuse Large B-Cell Lymphoma Using a Reverse Transcriptase Multiplex Ligation-Dependent Probe Amplification Classifier[END_REF]. Our aim was to develop a tool able to bring additional value to the gold-standard pathologic assessment of EMB and to help both pathologists and clinicians in case of uncertainty. We wanted to introduce a two-step approach allowing us to discriminate non-rejection from rejection and ACR from AMR.

We believe this approach fits clinical practice. The selection of our 14-transcript set was based on the Affimetrix and NanoString literature. A major interest of our technique is its applicability to FFPE EMB. First, this allows histopathologic assessment of the myocardial samples analyzed, excluding the non-specific lesions such as fibrosis, fibrin clot, previous biopsy site or pericardial tissue. Second, this technique can be included in a standard of care procedure since an extra-bite for a fresh-frozen sample is not required, thus limiting the risk of potential procedural complications. Third, we can analyze simultaneously all myocardial samples of a paraffin block. The focal distribution of the rejection accounts for the high variability of myocardial samples and the need for at least three samples to accurately assess rejection [START_REF] Berry | The 2013 International Society for Heart and Lung Transplantation Working Formulation for the standardization of nomenclature in the pathologic diagnosis of antibody-mediated rejection in heart transplantation[END_REF][START_REF] Stewart | Revision of the 1990 working formulation for the standardization of nomenclature in the diagnosis of heart rejection[END_REF]. Finally, RT-MLPA is a cost-effective, strong and easy-to-use technique based on common instruments and reagents and could thus easily be implemented into routine diagnosis workflows. Using the RT-MLPA procedure and class prediction analysis, the agreement between molecular and pathology diagnoses was high (92.2%, 79.2% and 76.8% for the diagnosis of rejection, the type of rejection and the final diagnosis, respectively). However, we observed several discordant cases. At the level of diagnosis of rejection (LPS1), discordant cases included mainly cases with low-grade of rejection (pAMR1(I+), ACR1R) classified as non-rejection by RT-MLPA. Such lowgrade rejections have previously been identified as molecularly quiescent according to Affimetrix and NanoString nCounter studies [START_REF] Afzali | Molecular Assessment of Microcirculation Injury in Formalin-Fixed Human Cardiac Allograft Biopsies With Antibody-Mediated Rejection[END_REF][START_REF] Loupy | Gene Expression Profiling for the Identification and Classification of Antibody-Mediated Heart Rejection[END_REF][START_REF] Holweg | Identification and classification of acute cardiac rejection by intragraft transcriptional profiling[END_REF]. Moreover, the clinical relevance of isolated subclinical pAMR1(I+) and ACR 1R is debated and international guidelines currently do not recommend their treatment [START_REF] Colvin | Antibody-mediated rejection in cardiac transplantation: emerging knowledge in diagnosis and management: a scientific statement from the American Heart Association[END_REF][START_REF] Costanzo | The International Society of Heart and Lung Transplantation Guidelines for the care of heart transplant recipients. The Journal of heart and lung transplantation: the official publication of the[END_REF]. Other discordant cases included cases with normal histology (hNR) that were classified as molecular rejection.

Interestingly, two of these cases had severe graft dysfunction at the time of biopsy and could correspond to "biopsy-negative" rejections [START_REF] Fishbein | Biopsy-negative cardiac transplant rejection: etiology, diagnosis, and therapy[END_REF]. Disagreements between histology and RT-MLPA for the diagnosis of the type of rejection corresponded mostly to hAMR being classified as mACR. Early post-transplant EMB were at higher risk of disagreement at the LPS2 level. Induction therapy, ischemia-reperfusion lesions, inflammatory post-transplant burden might modify gene expression and make it more difficult to distinguish AMR from ACR.

The present study should be interpreted within the context of its limitations. Our study was not designed to assess the performance of RT-MLPA as compared to the molecular gold standard (microarray analysis). However, the molecular expression comparison between these two techniques has already been performed [START_REF] Bobée | Determination of Molecular Subtypes of Diffuse Large B-Cell Lymphoma Using a Reverse Transcriptase Multiplex Ligation-Dependent Probe Amplification Classifier[END_REF]. The findings of this retrospective cross-sectional case-control study must be further validated. The next step will be to analyze longitudinally the targeted-gene expression profile of rejections and to evaluate the dynamic changes before rejection and after its treatment.

I-B-5. Conclusion

We demonstrated that RT-MLPA is a suitable method to analyze FFPE EMB in the

I-C-2-c. Histology and immunohistochemistry

Endomyocardial biopsies were formalin-fixed, paraffin-embedded, and routinely stained with hematoxylin and eosin. Immunohistochemistry was performed in tissue sections with monoclonal rabbit anti-C4d (Cliniscience, Nanterre, France) and monoclonal anti-CD68 (DakoCytomation, Glostrup, Denmark) using the immunoperoxidase method, as previously described [START_REF] Loupy | Gene Expression Profiling for the Identification and Classification of Antibody-Mediated Heart Rejection[END_REF][START_REF] Fedrigo | Inflammatory cell burden and phenotype in endomyocardial biopsies with antibody-mediated rejection (AMR): a multicenter pilot study from the AECVP[END_REF]. All EMB were graded according to the ISHLT guidelines for acute cellular rejection (ACR) and AMR [START_REF] Berry | The 2013 International Society for Heart and Lung Transplantation Working Formulation for the standardization of nomenclature in the pathologic diagnosis of antibody-mediated rejection in heart transplantation[END_REF][START_REF] Stewart | Revision of the 1990 working formulation for the standardization of nomenclature in the diagnosis of heart rejection[END_REF]. This study was based on the complementary approach of two types of molecular analysis. A pangenomic microarray technology was performed on 213 cases using an additional EMB (that is distinct from the biopsies used for histopathology, either snap frozen or stored in a nucleic acid preservation solution -RNAlater) and Affymetrix GeneChips (Affymetrix, Santa Clara, CA), as previously described [START_REF] Halloran | Building a tissue-based molecular diagnostic system in heart transplant rejection: The heart Molecular Microscope Diagnostic (MMDx) System[END_REF][START_REF] Loupy | Gene Expression Profiling for the Identification and Classification of Antibody-Mediated Heart Rejection[END_REF]. Microarray gene expression results for each EMB were summarized as Pathogenesis Based Transcript (PBT) scores as previously described and validated. AMR and TCMR classifiers were designed as previously described [START_REF] Sellarés | Molecular diagnosis of antibody-mediated rejection in human kidney transplants[END_REF].

The second molecular approach was based on FFPE EMBs, allowing the evaluation of the extension of IAMC and the molecular assessment on the same paraffin block and biopsies. As described in the previous chapter, we applied the RT-MLPA technique to FFPE-EMB. The 213 The microarray EMB were included from one retrospective case-control enriched with rejecting cases and one prospective cohort that reflects the routine biopsy recruitment. The 185 RTMLPA biopsies were included from one retrospective case-control cohort enriched with rejecting cases. This explains the differences observed between the 2 groups, Affymetrix and RTMLPA, regarding the DSA status at the time of the biopsy, type of BEM (protocol/cause) and distribution of histological diagnoses on BEMs.

ATG means anti-thymocyte globulins, DSA: donor-specific antibodies, EMB: endomyocardial biopsy, LVEF: left ventricular ejection fraction, MVI= microvascular inflammation. We then analyzed the molecular signals of selected rejection-associated transcripts in the same EMB that was used for the initial pathologic diagnosis and MVI scoring.

Figure 15 C-D gives the results of RT-MLPA analysis with the geometric mean of transcripts associated with AMR (ROBO4, TM4SF1, PLA1, GNLY, CXCL11, CXCL9, CCL4 and FCGR3; Figure 15C) and ACR (CCL18, ADAMDEC1 and CD8A ; Figure 15D) according to MVI score.

Altogether, these results from independent series and techniques, show that the extension of MVI correlated with the intensity of the AMR-related molecular signals,

but not with the ACR signals.

I-C-3-c. MVI score and ISHLT ACR and pAMR classifications

The detailed distribution of MVI score in the 398 EMB is given in Table 6 and illustrated in Figure 16-A

Risk stratification of Donor-Specified Antibodies and

Antibody-mediated rejection-related complications

In the quest for personalized medicine, heart transplantation must improve its standards for monitoring patients. Current approaches to screening for complications of allosensitization involve iterative invasive protocol tests, whether coronary angiography to detect CAV or endomyocardial biopsies to detect allograft rejection.

Recent epidemiological data have shown that the incidence of these two complications has decreased over time [START_REF] Shah | Surveillance Endomyocardial Biopsy in the Modern Era Produces Low Diagnostic Yield for Cardiac Allograft Rejection[END_REF][START_REF] Tremblay-Gravel | Changes in Outcomes of Cardiac Allograft Vasculopathy Over 30 Years Following Heart Transplantation[END_REF]. The current cost-effectiveness of these screening tests is low and should be compared with their potential complications.

Numerous risk factors/markers for the development of CAV or allograft rejection have been described in the literature, however, the absence of an integrated tool that enables an estimate of the risk of events for an individual makes the passage from a protocol medicine to a personalized medicine impossible.

In a first step towards the improvement of the stratification of the risk of events, we aim to accurately define the epidemiology and natural history of CAV and allograft rejection and to determine the risk factors independently associated with these complications. In order to do this, we will apply innovative methodologies to large cohorts of highly phenotyped heart transplant recipients.

We formulate the following hypotheses: i) that independent risk factors for allograft rejection are identifiable at the time of transplantation in a large population of heart transplants, which would make it possible to assess the risk of rejection early after transplant; and ii) that there are common evolutive profiles of CAV identifiable by latent class mixed models, constituting a first step towards individual risk stratification of CAV development (Figure 18). Chapter II-A: Risk stratification One of the primary objectives of biomedical research is the identification of risk factors for a disease or a clinical event that can stratify the risk of its occurrence, at least at the population level. Many statistical models exist and depend on the type of event or variables that we are trying to explain. Often in the biomedical field, the analyzed criterion is binary (disease / no disease), and the time of occurrence of the event is added to the analysis. In this typical case, the Cox model is the most relevant and frequently used model. This model makes it possible to determine independent risk factors for the occurrence of the disease that are assigned a ß coefficient corresponding to the importance of their effect on the time of occurrence of the event analyzed. We will describe this model in detail in the introduction and will apply it in a first epidemiological study aiming to identifying independent risk factors for biopsyproven rejection during the first year of transplantation.

However, this type of model suffers from several limitations. In particular, the Cox models are limited to the analysis of the occurrence of a first clinical event and cannot apprehend either the recurrence or the evolution of the severity of the disease after its diagnosis. Moreover, the Cox model is not able to fully understand the longitudinal character of iterative test results from the same patient. Statistical models such as generalized linear models can be a means of apprehending these issues by taking into account the correlation between results of repetitive tests performed on the same patient. However, they do not make it possible to take into account the variability of responses between individuals. We will then discuss the interest in mixed models applied to heart transplantation, which allow not only the analysis of longitudinal data, but also the introduction of random effects such as inter-cluster variability, which is the first step towards individual risk stratification. It is this type of model that we will apply to CAV in a second epidemiological study. This model underlies two hypotheses. Firstly, the relationship between the hazard function and covariates is loglinear. There are several methods to evaluate the log linearity deviation of continuous variables (statistics and / or graphs). When the variable does not satisfy the log linearity hypothesis, the common practice is to transform the continuous variable into a categorical variable. Secondly, the ratio of the instant hazards for two subjects 𝑖𝑖 and 𝑗𝑗 of characteristics 𝑍𝑍 𝑖𝑖 and 𝑍𝑍 𝑗𝑗 depends only on 𝑍𝑍 𝑖𝑖 and 𝑍𝑍 𝑗𝑗 and does not depend on time (proportional hazard ratios hypothesis). In other words, if an individual has a risk of death at some initial time point that is twice as high as that of another individual, then at all later times the risk of death remains twice as high. The proportional hazards ratios hypothesis can be verified by a graphical method or a statistical method. The statistical method is the most robust and is the preferred solution. It is based on the Schoenfeld residue analysis. The residues are calculated for each event date and correspond to the difference between the profile of the individual with the event and the profile of the individuals at risk on that date. If residues are distributed in the same way over time, the assumption of proportional hazards is verified. If this is not the case, then: i) the Cox model can be stratified on this variable (loss of the estimation of the impact of this variable on the event), ii) it is possible to introduce a time-dependent covariate (the covariate is then the same variable but with an interaction with time), and iii) it is possible to create a model divided into two parts by considering the analysis separately over two periods.

II-

II-A-1-b. Interpretation of the ß coefficients

The interpretation of the exponentiated coefficients is the risk ratio for the increase in a unit of the specific covariant. The validity of this risk report is based on the assumption of proportional hazard ratios. If z1 is a variable with two modalities, 𝑧𝑧1 = {1/0} among the p explanatory variables, then the risk ratio (RR) to this variable is written as: Hypothesis 𝐻𝐻 0 required testing of the equality of the hazard functions of each modality of the explanatory variable 𝑧𝑧1: { 𝛽𝛽 1 =0 ⇐⇒ 𝑅𝑅𝑅𝑅 𝑧𝑧1=1/𝑧𝑧1=0}. The alternative hypothesis 𝐻𝐻 1 is most often bilateral: {𝛽𝛽 1 ≠0 ⇐⇒ 𝑅𝑅𝑅𝑅 𝑧𝑧1=1/𝑧𝑧1=0}. If 𝐻𝐻 0 is rejected, there is a difference in survival between the two populations defined by the variable 𝑧𝑧1.

𝑅𝑅𝑅𝑅 𝑧𝑧1=1/𝑧𝑧1=0 = ℎ 0 (𝑡𝑡 𝑖𝑖 )exp (𝛽𝛽

II-A-1-c. Discrimination of the statistical model

When testing a new prognostic factor, it is essential to test its additional value. Indeed, if this factor is independently associated with the event, its contribution compared to existing models needs to be determined. The question is whether this new factor provides additional and complementary information and whether its application in clinical practice is relevant. If so, this factor is considered discriminant. The discriminative capacity of a model is defined by its ability to separate individuals who made the event from those who did not, and thus consists of separating individuals with different prognoses. One of the classical methods of evaluating discrimination is the analysis of an ROC curve. This is a graphical representation of the sensitivity of a factor for the event on the y-axis versus 1-specificity on the x-axis. This amounts to evaluating the rate of true positives according to the rate of false positives. The associated index is the area under the ROC curve named AUC. This provides information on the probability that the result of the test for two people with and without the event can make the correct diagnosis. Thus, the test is perfectly discriminating if the AUC is 1. In this case, when evaluating two people the test will always distinguish the person having the disease from the one not having it. A test that is no better than chance will have an AUC of 0.5. To quantify the overall performance of a model, it is essential to take into account its calibration corresponding to the distance between the predicted events and the observed events. These distances between the events observed and predicted are related to the notion of the goodness of fit of a model, with the best models having smaller distances between the predicted results and the observed results. Calibration is essential since it refers to the ability of the model to produce accurate, unbiased predictions in similar patient groups. It is an indispensable preliminary step before the study of model discrimination since a discriminating, but poorly calibrated model has limited clinical value. The calibration can be measured by a graphical method using calibration curves or a statistical method, such as Hosmer-Lemeshow for survival models. The Hosmer-Lemeshow statistical model or its extension for survival data is derived from n balanced groups of patients defined by the quantiles of the probability predicted by the model. The measured bias is defined as the sum of the differences between the mean of the predicted probabilities and the mean of the probabilities observed in each group of similar patients. The calibration of the model is then appreciated by the p-value of the test which is based on this sum. When this sum is less than 0.1, the calibration is not adequate.

II-

Discrimination. As previously defined, discrimination is the ability of the model to discriminate individuals who made the event from those who did not, and thus is the model's ability to separate individuals with different prognoses (see above).

II-A-2. Introduction to the interest in mixed models in heart transplantation

The Cox model is limited to the analysis of a first clinical event and is not the most suitable model for the exploration and analysis of longitudinal data. Models specific to the analysis of longitudinal data, such as generalized linear models, can be a means of understanding the problems of the repetition of tests in the same individual.

However, they do not take into account the variability in responses between individuals and, therefore, do not allow individual risk stratification. Hence, the goal of our research is to be able to generalize our results to all individuals in the population, not just those observed. We will, therefore, want to take into account the inter-cluster variance (intersubject, inter-individual variability). The goal is to allow certain regression parameters to vary from cluster to cluster, via the introduction of random effects. Thus, various sources of the heterogeneity of the population can be taken into account. A mixed model is a statistical model in which we consider both fixed effects, which will intervene at the level of the model mean and random effects, which will intervene at the level of the variance of the model. A model is called mixed when there is at least one factor of each nature. In a second epidemiological study we will apply these models. We will use the particular case of latent classes mixed models, adapted to the analysis of categorical longitudinal data (grade of severity of the CAV according to the ISHLT).

These models postulate the existence of latent variables, which are unobservable directly, but whose effects can be measured or observed. This approach allows the unsupervised categorization, that is, without a priori, of groups of patients. Each class includes a homogeneous group of individuals sharing a common evolutive profile.

II-A-3. Risk factors for allograft rejection after heart transplantation

Several clinical, laboratory, and immunological parameters might help to stratify the risk of allograft rejection after heart transplantation.

The time between transplantation and EMB is an important element of the risk of rejection. The risk of acute cellular rejection is the highest during the first weeks and decreases over time [START_REF] Rizeq | Acute rejection: significance of elapsed time after transplantation[END_REF]. The dynamic risk of AMR is less described but depends on the presence of pre-formed DSA [START_REF] Coutance | Favorable outcome of an exclusively posttransplant prophylactic strategy after heart transplantation in recipients with high immunological risk[END_REF]. The effectiveness of protocol endomyocardial biopsies, has been well demonstrated during the first year, is less certain between 1 and 5 years and is lost for EMB after 5 years of transplantation, outside populations at risk [START_REF] Stehlik | Utility of long-term surveillance endomyocardial biopsy: a multi-institutional analysis[END_REF].

While many studies have investigated the impact of both induction and basal immunosuppression on the risk of allograft rejection following cardiac transplantation [START_REF] Neylan | Immunosuppressive therapy in high-risk transplant patients: dosedependent efficacy of mycophenolate mofetil in African-American renal allograft recipients. U.S. Renal Transplant Mycophenolate Mofetil Study Group[END_REF][START_REF] Beniaminovitz | Prevention of rejection in cardiac transplantation by blockade of the interleukin-2 receptor with a monoclonal antibody[END_REF][START_REF] Cantarovich | Antithymocyte globulin induction allows a prolonged delay in the initiation of cyclosporine in heart transplant patients with postoperative renal dysfunction[END_REF][START_REF] Kobashigawa | Tacrolimus with mycophenolate mofetil (MMF) or sirolimus vs. cyclosporine with MMF in cardiac transplant patients: 1year report[END_REF], there are few data available on the intrinsic risk to the patient. Two important factors have, however, been described. The first is the ethnicity of the recipient.

African-American patients have a higher risk of rejection than Caucasian patients, which justifies appropriate monitoring and immunosuppression protocols [START_REF] Stehlik | Utility of long-term surveillance endomyocardial biopsy: a multi-institutional analysis[END_REF]. A prior history of rejection is an important risk factor for the development of a new episode of rejection. This parameter should be taken into account in order to stratify the risk of subsequent rejection [START_REF] Costanzo | The International Society of Heart and Lung Transplantation Guidelines for the care of heart transplant recipients. The Journal of heart and lung transplantation: the official publication of the[END_REF][START_REF] Hamour | Limited utility of endomyocardial biopsy in the first year after heart transplantation[END_REF]. In fact, late rejections occur most often in patients who have had one or more episodes of rejection early after transplantation [START_REF] Imamura | Late rejection occurred in recipients who experienced acute cellular rejection within the first year after heart transplantation[END_REF]. The risk of AMR depends on the presence or absence of pre-formed DSA [START_REF] Coutance | Favorable outcome of an exclusively posttransplant prophylactic strategy after heart transplantation in recipients with high immunological risk[END_REF], and on the development of DSA de novo after transplantation [START_REF] Chen | Development and Impact of De Novo Anti-HLA Antibodies in Pediatric Heart Transplant Recipients[END_REF][START_REF] Smith | De novo donor HLA-specific antibodies after heart transplantation are an independent predictor of poor patient survival[END_REF].

The stratification of the basal risk of rejection, i.e., at the time of transplantation, has not been studied in large cohorts of patients by analyzing a large number of potential risk factors for rejection. We will analyze the epidemiology and identify the risk factors for rejection in the first epidemiological study.

II-A-4. Risk factors for CAV

The situation of the coronary endothelium, at the border between the donor and the recipient, places it at the center of complex interactions between many immunological and non-immunological factors responsible for chronic aggressions leading to endothelial activation, chronic inflammatory responses, proliferation signals and finally chronic remodeling and diffuse narrowing of the coronary arteries [START_REF] Pober | Interacting mechanisms in the pathogenesis of cardiac allograft vasculopathy[END_REF][START_REF] Mehra | Contemporary concepts in prevention and treatment of cardiac allograft vasculopathy[END_REF]. Factors"[Mesh]). To capture parameters relative to longitudinal changes in CAV, we also used the terms "repeated measure", "repeated measures", "repeated measurements", "trajectory", "trajectories", and "slope." AND ((("Natural

Numerous
History"[Mesh]) OR "Disease Progression"[Mesh]) OR "Latent Class

Analysis"[Mesh]))). To focus the search on Heart transplantation, we used the terms "heart transplantation", "heart transplant". Based on this approach, a comprehensive search strategy was conducted using several databases (Medline, Embase, Cochrane, and Scopus).

The

II-B-1. Introduction

Cardiac allograft rejection remains an important cause of graft loss and death [START_REF] Khush | The International Thoracic Organ Transplant Registry of the International Society for Heart and Lung Transplantation: Thirty-fifth Adult Heart Transplantation Report-2018; Focus Theme: Multiorgan Transplantation[END_REF]. The diagnosis of rejection is based on the histological analysis of a myocardial fragment obtained by endomyocardial biopsy (EMB). The vast majority of the rejections are subclinical, and their diagnosis is based on a large number of iterative protocol EMB, as recommended by international societies [START_REF] Costanzo | The International Society of Heart and Lung Transplantation Guidelines for the care of heart transplant recipients. The Journal of heart and lung transplantation: the official publication of the[END_REF]. EMB is an invasive and non-risk-free procedure [START_REF] Deckers | Complications of transvenous right ventricular endomyocardial biopsy in adult patients with cardiomyopathy: a seven-year survey of 546 consecutive diagnostic procedures in a tertiary referral center[END_REF]. Complications, which although rare at the level of an EMB (approximately 1% of cases, all complications combined), concern 20% of patients due to the repetition of EMB [START_REF] Hamour | Limited utility of endomyocardial biopsy in the first year after heart transplantation[END_REF]. The cost-effectiveness of this tests is low, since it is estimated that less than 10% of biopsies show signs of rejection, most of which are considered to be minimal signs, and that more severe forms of rejection are only present in 2% of the EMB [START_REF] Hamour | Limited utility of endomyocardial biopsy in the first year after heart transplantation[END_REF]. Strategies aimed at limiting the number of EMB during the first year seem feasible, however, the reported experiences have never adapted the screening protocol to the baseline risk of rejection [START_REF] Weckbach | Lower frequency routine surveillance endomyocardial biopsies after heart transplantation[END_REF]. Tools, whether biological or epidemiological, to rationalize the number of protocol BEM are therefore an unmet medical need. Research on non-invasive biomarkers of rejection has been particularly active for more than 15 years. However, only very few of them have been applied in clinical practice. Only the ALLOMAP test is both recommended by the ISHLT guidelines and recognized by the US and European drug regulatory authorities [START_REF] Costanzo | The International Society of Heart and Lung Transplantation Guidelines for the care of heart transplant recipients. The Journal of heart and lung transplantation: the official publication of the[END_REF][START_REF] Pham | Gene-expression profiling for rejection surveillance after cardiac transplantation[END_REF].

ALLOMAP is a score derived from the analysis of the expression of an 11-gene panel in peripheral blood mononuclear cells that enables the detection of acute cellular rejection ≥ 2R with good sensitivity and specificity [START_REF] Pham | Gene-expression profiling for rejection surveillance after cardiac transplantation[END_REF][START_REF] Deng | Noninvasive discrimination of rejection in cardiac allograft recipients using gene expression profiling[END_REF]. However, the limits of this test are numerous (a lack of sensitivity, screening only grade 2R or more cellular rejections, cost). The search for donor-derived cell-free DNA in the blood of the recipient is a particularly promising method that is the subject of an abundant literature, but has not yet been validated on a large scale (94-96).

Our hypothesis is that an epidemiological approach allowing adaptation of the biopsy screening protocol to the risk of rejection could make it possible to rationalize the number of protocol biopsies. Although some risk factors for allograft rejection have been identified in the literature, such as the recipient's age, a prior history of rejection, and the presence of DSA, a risk stratification tool incorporating all of these factors has not been developed [START_REF] Coutance | Favorable outcome of an exclusively posttransplant prophylactic strategy after heart transplantation in recipients with high immunological risk[END_REF][START_REF] Stehlik | Utility of long-term surveillance endomyocardial biopsy: a multi-institutional analysis[END_REF][START_REF] Hamour | Limited utility of endomyocardial biopsy in the first year after heart transplantation[END_REF]. As a first step towards personalized medicine in heart transplantation, our goal is to analyze the epidemiology of allograft rejection and to identify independent risk factors for rejection measurable at the time of the transplant.

II-B-2. Materials and methods

III-B-2-a. Patients and design

We performed a retrospective observational multi-center center study. We included all consecutive HTx recipients from two French heart transplantation reference centers (La Pitié-Salpêtrière Hospital, Paris, and Georges Pompidou European Hospital, Paris) transplanted between 2004 and 2016 and who had at least one EMB during the firstyear post-transplant. During the study period, 1,250 patients were transplanted and 197 of these never had any EMB. Thus, 1,053 patients were included in our study.

Patients were followed until January 2019. Our study complies with the Declaration of Helsinki, and the institutional review board approved the protocol. Informed consent was obtained at the time of inscription on the waiting list. Baseline donor and recipient characteristics were obtained from the prospective national registry CRISTAL database (French National Agency for Organ Procurement). We collected prospectively the results of EMB and clinical events during the first year (see the data collection section in the Supplementary Appendix). The design of our study is described in Figure 19. Due to the different practices between the two centers, CD68 and CD163 positivity were not considered as pAMR1(I+).

II-B-2.c. Evolution of practices over time

During the study period, the scheduled number of protocol biopsies during the first year was reduced from 15 to 13 in 2012. In 2009, as described in the third part of this manuscript, a specific prophylactic treatment for transplantation with pre-formed DSA was applied at La Pitié-Salpêtrière Hospital. Patients transplanted with MFI ≥ 1,000 pre-formed DSA were prospectively treated with peri-operative plasmapheresis and IVIg. These elements led us to define three monitoring periods: period #

II-B-3. Results

II-B-3-a. Characteristics of patients and endomyocardial biopsies

A total of 1,053 patients who were transplanted during the study period had at least one EMB during their first-year follow-up and were included in the study. One hundred and twenty-eight patients (12.2%) died during the first year. The median duration of follow-up was 354 days (quartile 1 = 315 days, quartile 3 = 363 days), representing a total follow-up of 868 patient-years. The main characteristics of donors, recipients, transplantation and immunosuppression are described in Table 8. The results of the pathologic analysis of the EMBs are summarized in Table 9. A total of 13,676 BEMs was performed, representing an average of 12.99 ± 5.5 BEM per patient. The distribution of EMBs over time during the first year is described in Figure 20 (overall and rejection EMBs). The number of BEMs per patient decreased from 14.6±6.1 to 10.8±3.7 after the introduction of a new screening protocol in 2012 (Figure 21).

II-B-3-b. Epidemiology of rejection in the first year

The incidence of the first significant rejection during the first year is presented in Figure 22-A 10.

In multivariable analyzes, five variables were independently associated with the risk of rejection during the first year: the recipient's age (per 10-year increment: HR = 0.90, 95% CI = 0.85-0.95, p = 0.003), the type of transplantation (combined vs isolated: HR = 0.25, 95% CI = 0.13-0.47, p <0.001), the type of induction (induction by IL-2R inhibitors compared to induction with thymoglobulin: HR = 1.58, 95% CI = 1.17-2.16, p = 0.003), the number of HLA AB-DR mismatches (for each mismatch increase: HR = 1.17, 95% CI = 1.06-1.29, p = 0.001) and the presence of pre-formed DSA (MFI ≥ 500, HR = 1.46, 95% CI = 1.21-1.75, p <0.001). The results are detailed in Table 11.

The calibration of the model was good (p = 0.91). On the other hand, the discrimination of the model evaluated by a ROC curve proved to be bad (Figure 23-A). The area under the curve of the ROC curve was calculated at 0.64. After multivariable analyzes, we identified three independent risk factors for AMR during the first year: the recipient's age (per 10-year increment: HR = 0.78, 95% CI = 0.66-0.91, p = 0.002), the presence of pre-formed DSA (MFI ≥ 500, HR = 2.01, 95% CI = 1.30-3.12, p = 0.002) and the ischemic time (per 1-hour increment: HR = 1.33, 95% CI = 1.04-1.68, p = 0.021). The discrimination of the model was poor (AUC = 0.66).

II-B-4. Discussion

In a large cohort of heart transplant recipients, we were able to accurately determine the epidemiology of allograft rejection during the first year and to identify five independent risk factors for rejection during this period, which were identifiable at the time of transplantation: the young age of the recipient, an induction therapy based on IL-2R inhibitors compared to anti-thymocyte globulins infusions, an isolated heart transplant compared to a combined transplantation (heart-kidney or heart-liver), the number of A-B-DR HLA mismatches and the presence of pre-formed DSA (MFI ≥ 500).

Cox models, although well calibrated, have been disappointing in terms of discrimination. The main strengths of our study concern the sample size, the fact there was a retrospective analysis of EBM by two expert pathologists, the high level of patients' phenotyping (anti-HLA antibodies screening based on the reference method even for transplantations performed before 2009), and the analysis of many potential risk factors for rejection.

The risk of rejection evolves during the first year. These rejection dynamics have already been described for acute cellular rejection but never for AMR [START_REF] Hamour | Limited utility of endomyocardial biopsy in the first year after heart transplantation[END_REF]. While significant patterns of rejection are found in approximately 10% of EMB achieved in the first 3 months, this probability falls to less than 5% between 6-and 11-months posttransplant. The increased probability of rejection we observed at the end of the first year can have several explanations, such as the development of de-novo DSA and an increase in the time interval between two biopsies thus increasing the risk of more severe rejection due to it being identified later.

The discrimination of a statistical model is its ability to identify patients who will present the disease from others who will not, in our case, the patients who will experience rejection in the first year from those who will not. The discrimination of our models, evaluated by the area under ROC curve, proved to be poor and was lower than 0.65.

Such discrimination does not allow the practical application of this statistical model to a patient's scale. Its diagnostic performance is too limited and the confidence in this model too low to adapt the biopsy screening. Therefore, we did not derive a scoring system from these models. Several explanations for these poor statistical performances can be evoked. First, the diagnosis of rejection might be open to discussion. It is difficult to establish a dichotomization of an endomyocardial biopsy result. The threshold used to define acute cellular rejection ≥ 1R1B is not a consensus.

ISHLT guidelines do not recommend the treatment of subclinical 1R1B rejections [START_REF] Costanzo | The International Society of Heart and Lung Transplantation Guidelines for the care of heart transplant recipients. The Journal of heart and lung transplantation: the official publication of the[END_REF][START_REF] Stewart | Revision of the 1990 working formulation for the standardization of nomenclature in the diagnosis of heart rejection[END_REF]. This definition has the effect of overrepresenting cellular rejections among all rejections in our cohort. A definition threshold ≥ 2R could have been chosen. However, our local experience led us to treat 1R1B rejections, even when sub-clinical. Thus, the epidemiology of grade 2R rejection is different in our cohort compared to North American practices. Moreover, more and more molecular data suggest that 1R1B rejections are molecularly closer to ≥ 2R rejections than to non-rejection biopsies [START_REF] Bodez | Myocardial Gene Expression Profiling to Predict and Identify Cardiac Allograft Acute Cellular Rejection: The GET-Study[END_REF].

Similarly, the threshold for the definition of AMR is questionable. In addition to the diagnostic difficulties of the pAMR1(H +) rejection already described in the first part of the thesis, molecular data suggest that pAMR1(I +) rejections would behave more like biopsies without rejection than pAMR1(H +) or pAMR2-3 rejection biopsies [START_REF] Afzali | Molecular Assessment of Microcirculation Injury in Formalin-Fixed Human Cardiac Allograft Biopsies With Antibody-Mediated Rejection[END_REF][START_REF] Loupy | Gene Expression Profiling for the Identification and Classification of Antibody-Mediated Heart Rejection[END_REF].

Secondly, although we have carried out extensive data collection, our base could benefit from being supplemented by the addition of evolutive data over the first year, such as the modifications in immunosuppressive therapy, the analysis of anticalcineurin dosages, de novo DSA development or the evolution of the MFI of preformed DSA after transplantation. Finally, the statistical models used were not optimal for the analysis of longitudinal data. The Cox model is a model for analyzing the occurrence of a first clinical event and is therefore not the best model for longitudinal data analysis. Mixed models represent the next stage of statistical development to be applied to our cohort. Not only can these models can take into account the recurrence of the disease and the correlation between the results of multiple EMB carried out on the same patient, but they also can incorporate random effects related to the intercluster variation.

Although large and well phenotyped, our cohort has management specificities that could limit the application of our results to foreign cohorts, particularly those from North America. The almost systematic induction by anti-thymocyte globulins and the transplantation with pre-formed DSA are peculiarities of our centers. Internationally, more than half of patients do not receive any induction therapy. The most commonly used induction is IL-2R inhibitors followed by polyclonal induction, accounting for only 22% of patients (compared to 91.6% in our cohort) (14). Similarly, the most prescribed calcineurin inhibitor at the international level is tacrolimus, prescribed for more than 85% of patients worldwide, compared to for only 7.8% in our cohort (14). The small number of tacrolimus patients probably explains why this factor does not appear to be protective in our cohort (HR = 0.86 with a wide confidence interval), despite tacrolimus patients having been shown to have fewer rejections than patients given cyclosporin [START_REF] Kobashigawa | Tacrolimus with mycophenolate mofetil (MMF) or sirolimus vs. cyclosporine with MMF in cardiac transplant patients: 1year report[END_REF].

II-B-4. Conclusion

The risk of allograft rejection evolves during the first year. We identified five independent risk factors for rejection in the first year following heart transplantation: recipient's age, type of induction, type of transplantation, number of HLA mismatches and the presence of pre-formed DSA.

II-C-1. Introduction

Whereas the short term survival after HTx has improved considerably in the last decades, the survival beyond 1 year post-transplant has remained unchanged over years despite important progresses in immunosuppression and patients' care [START_REF] Khush | The International Thoracic Organ Transplant Registry of the International Society for Heart and Lung Transplantation: Thirty-fifth Adult Heart Transplantation Report-2018; Focus Theme: Multiorgan Transplantation[END_REF]8,[START_REF]pdf&usg=AOvVaw1U-4EpkFaOSvGZ2mKtgo_w[END_REF].

In this setting, cardiac allograft vasculopathy (aka CAV), a diffuse intimal thickening leading to progressive narrowing of the coronary arteries, has a prevalence of 50% at ten years after transplantation, making this injury the third cause of late mortality and the leading cause of late allograft dysfunction [START_REF] Khush | The International Thoracic Organ Transplant Registry of the International Society for Heart and Lung Transplantation: Thirty-fifth Adult Heart Transplantation Report-2018; Focus Theme: Multiorgan Transplantation[END_REF][START_REF] Loupy | Antibody-Mediated Rejection of Solid Organ Allografts[END_REF][START_REF] López-Sainz | Late graft failure in heart transplant recipients: incidence, risk factors and clinical outcomes: Heart failure after heart transplantation[END_REF].

The current gold standard for CAV monitoring after HTx relies on protocol angiograms, which guides clinical management of patients including prevention strategies, therapeutic changes, and also potential endpoint for clinical trials (99,100). protocols (derivation cohort) for follow-up data included CAV measurement at one year after transplantation and every two years thereafter as well as at the time of any clinically indication. The American center protocol included CAV measurement at 6month one-year after transplantation and every year thereafter as well as at the time of any clinically indication.

Current approaches for investigation

In the European derivation cohort, a total of 2,742 reports of coronary angiograms were retrospectively and independently reviewed by three senior cardiologists. In case of discrepancies between observers or inconclusive report, the coronary angiograms were reviewed to reach an agreement (n = 454, 16.6%, mostly ISHLT CAV1). In the American validation cohort, a total of 1,968 coronary angiograms were analyzed. Two senior cardiologists graded all coronary angiograms according to the summarized report collected prospectively during patient follow-up.

Circulating donor-specific antibodies against HLA-A, HLA-B, HLA-Cw, HLA-DR, HLA-DQ and HLA-DP were assessed using single-antigen flow bead assays (see the Methods section in the Supplementary Appendix). Heart allograft pathology data were recorded according to the ISHLT classifications in the derivation and validation cohorts (see the Methods section in the Supplementary Appendix) [START_REF] Berry | The 2013 International Society for Heart and Lung Transplantation Working Formulation for the standardization of nomenclature in the pathologic diagnosis of antibody-mediated rejection in heart transplantation[END_REF][START_REF] Stewart | Revision of the 1990 working formulation for the standardization of nomenclature in the diagnosis of heart rejection[END_REF]. During the firstyear post-transplant, a total of 12,803 EMB were performed in the derivation cohort.

II-C-2.c. Outcome measures

The primary outcome was CAV trajectories after transplantation. 13). One hundred and sixty-six out of 1,301 (12.76%) were diagnosed with CAV grade > 0 in the first year post-Tx. The detailed results of coronary angiograms and their evolution across years post-transplant are described in Table 13.

Two hundred and eighteen patients (27.3%) and 89 patients (20.1%) had detectable anti-HLA DSA, either pre-formed or de-novo with 69.81% of DSA being class II, in the derivation and validation cohorts, respectively. Overall, 108 patients (13.3%) and 76 patients (12.5%) from the derivation cohort presented with acute cellular rejection ≥ 2R and AMR ≥ pAMR1, respectively. In the validation cohort, where only the information "treated rejections" was available, 32 (6.6%) and 28 (5.8%) patients presented with treated acute cellular rejection ≥ 2R and treated AMR ≥ pAMR1, respectively. The detailed results of EMB are provided in Table 14. After multivariable analysis, the following independent determinants of GFR trajectories were identified: i) donor age (p<0.001), ii) donor gender (p<0.001), iii)

donor tobacco consumption (p=0.001), iv) recipient LDL-c ≥ 1g/dL one year after transplantation (p<0.001), v) recipient immunological profile as defined by the presence of preexisting or de novo circulating class 2 anti-HLA donor-specific antibodies (p=0.019) and vi) allograftv injury defined by acute cellular rejection ≥ 2R

occurring in the first year post transplant (p=0.028, Table 16).

Table 15: Determinants of CAV trajectories in the derivation cohort

This table shows the association of clinical, immunological, functional and structural parameters associated with CAV trajectories in univariate multinomial regression analysis. The trajectory of reference was trajectory#1, including patients with no CAV at baseline and stable over time. ATG means anti-thymocyte globulins, AMR: antibody-mediated rejection, BMI: body mass index, CMV: cytomegalovirus, DSA: donor-specific antibodies, MCS: mechanical circulatory support. This table shows the association of clinical, immunological, functional and structural parameters associated with CAV trajectories in multivariate multinomial regression analysis. The trajectory of reference was trajectory#1, including patients with no CAV at baseline and stable CAV grade over time.

II-C-3-e. Association between CAV trajectories and overall cause mortality

We found that the trajectories were also associated with patient death. During the follow-up, a total of 198 patients died overall. We found that CAV trajectories 3 and 4

were associated with higher mortality (10- p<0.001, Figure 27).

II-C-3-f. Sensitivity analyzes

Various sensitivity analyses were performed to test the robustness and generalizability of the CAV trajectories and determinants in different subpopulations and clinical scenarios.

i) CAV trajectories and center effect: to take into account a possible bias in the assessment of the determinants of the CAV trajectories, the center was also entered in the final multivariable model and did not modify the set of independent parameters associated with trajectories. When the multivariable model was performed adding to the derivation cohort the external validation cohort, the 6 independent determinants of CAV trajectories identified in primary analyzes remained unchanged. ii) Immune determinants of CAV trajectory: Replacing the variable "DSA at transplant or in the first-year" by "DSA at transplant" did not have any impact on the final model. When histological antibody mediated rejection was forced in the final multivariable model, it showed a trend for association (p=0.07) with CAV trajectory, but was outperformed by the circulating anti HLA DSA status. iii) Consistency of eGFR trajectories according to timing of baseline CAV assessment: We tested and confirmed the robustness of the profiles of CAV trajectories when the CAV evaluation started before 1-year posttransplant (the starting point of our study), with a discrimination of 0.89. We also confirmed the consistency of the trajectories when the baseline CAV was assessed at 2 years after transplantation (discrimination of 0.90).

Last, the similar four-trajectory pattern was found when performing LCCM analyses stratified by countries of the derivation cohort. Overall survival was significantly lower in trajectoryes #3 and #4 compared to trajectories #1 and #2 (p<0.001).

II-C-4. Discussion

In this international study of 1,301 carefully-phenotyped heart transplant recipients with protocol-based repeated assessement of CAV over 10 years, (4,710 coronary angiographies), combined with histology (12,803 biopsies) and immune profiling, we identified for the first time using unsupervised approach 4 distinct trajectories of longterm CAV progression. We demonstrated that the 4 trajectories were consistent in geographically distinct cohorts recruited in Europe and the US, which is remarkable given their distinct allocation systems, patient characteristics and management practices. We found that these trajectories were quite varied for several different conditions related to donor and recipient characteristics, ongoing diseases processes and immunological profile that can be determined at an early stage after transplantation.

II-C-4-a. Study strenghs and novelties

Despite the importance of angiogram evaluation as standard of care after heart transplantation, no study has characterized long-term trajectories using large unselected transplant cohorts with a prespecified longitudinal CAV monitoring together with a patient deep level phenotyping including donor and recipient characteristics, histology, immunology and treatment. Beyond the novel study design, the original advantage of our trajectory approach over traditional analysis is its ability to map the CAV course and classify individuals into distinct, mutually exclusive groups. Hence this approach not only helps to conceptualize the long term trajectories of CAV but also allows us to probe the population heterogeneity and the susceptibility of change in CAV over the life course. Furthermore, we believe that identifying the profiles of evolution in time of a chronic disease with an unsupervised approach and investigating whether these profiles manifest consistently across centers and countries may provide important guidance on the potential universality of the disease, their determinants, which has great implications for future research in particular for counseling patients, and also for choosing therapies.

II-C-4-b. Clinical significance of the CAV trajectories

The four 4 distinct trajectories identified represent different profiles of CAV evolution over time. CAV trajectory #1 is characterized by the absence of CAV overtime, trajectory #2 by a mild and late onset of CAV, trajectory #3 by an early onset with progressive evolution of CAV and trajectory #4 by an early onset with rapid evolution of CAV. From a clinical perspective, those findings might help to refine CAV management. On the one hand, the coronary angiogram monitoring protocol could be adapted to the evolving risk of CAV and therefore to the likelihood of belonging to each trajectory. Based on the determinants identified and the results of baseline coronary angiograms, we build an online interfacing to provide clinicians with a ready-to-use tool that projects the CAV trajectories. Patients with a high likelihood of belonging to trajectory #1 might be monitored less intensively. On the opposite, patients with a high likelihood of belonging to trajectories #3 and #4 , at risk of CAV progression, might benefit from a closer follow-up. On the other hand, these trajectories might improve prognosis stratification after heart transplantation since trajectories #3 and #4 are associated with a significant higher risk of overall mortality.

II-C-4-c. Significance of immune determinants of CAV

This study also emphasizes the interplay between cardiac transplant donor and recipient characteristics, the allograft immune profile, and existing allograft injury and the long-term CAV trajectories. The associations between these characteristics and the trajectories CAV suggest mechanistic explanations. Class II DSA appears to be an important trigger of CAV in our cohort. Both experimental and clinical studies support these immune determinants of CAV. The passive transfer of DSA to immunodeficient mice promote the development of an early and severe CAV, even in complement KO mice [START_REF] Hirohashi | Complement Independent Antibody-Mediated Endarteritis and Transplant Arteriopathy in Mice[END_REF][START_REF] Hirohashi | A novel pathway of chronic allograft rejection mediated by NK cells and alloantibody[END_REF]. Heart transplant recipients with preformed class II DSA were at increased risk of accelerated CAV as detected by consecutive volumetric three-dimensional intravascular ultrasound [START_REF] Topilsky | Donor-specific antibodies to class II antigens are associated with accelerated cardiac allograft vasculopathy: a threedimensional volumetric intravascular ultrasound study[END_REF]. The expression of class II HLA antigens by the endothelial cells requires their activation. Traditional cardiovascular risk factors associated with Trajectory#3, both donor-derived and from the recipient, might contribute to the endothelial inflammation and activation, thus increasing the number of potential class-II DSA targets. More and more data suggest that the Natural-Killer cell (NK) and ADCC plays a critical role in the development of immunological atherosclerosis. Mice treated with a depletive NK cells agent or genetically deficient in mature NK cells had a significantly reduced DSA-induced CAV [START_REF] Hirohashi | A novel pathway of chronic allograft rejection mediated by NK cells and alloantibody[END_REF]. FCGR3A-VV polymorphic variant, which encodes the highly responsive NK cells CD16 Fc receptor, was identified as an independent baseline predictor of cardiac allograft vasculopathy [START_REF] Paul | Genetic and Functional Profiling of CD16-Dependent Natural Killer Activation Identifies Patients at Higher Risk of Cardiac Allograft Vasculopathy[END_REF].

We found that acute cellular rejection ≥ 2R component is an independent risk factor for CAV progression. On the one hand, severe cellular rejection might acutely accelerate CAV by promoting local inflammation and the recruitment of other immunological cells.

On the other hand, cellular rejection has been recognized as a risk factor for the development of de-novo DSA (106). In the French derivation cohort, patients with ≥ 1 episode of acute cellular rejection ≥ 2R were twice as likely to develop de-novo DSA as patients without acute cellular rejection (17/60 (25.3%) compared to 55/315 (14.9%), p = 0.01). Finally, we found marginal associations between antibody mediated rejection and CAV trajectories in our cohort, contrasting with robust association with the presence of preexisting or de novo anti-HLA DSA. DSA is probably a much more sensitive markers of allo-sensitization than AMR. Although acute and severe antibody mediated rejection has been associated with fulminant forms of CAV, this entity is rare and chronic allograft inflammation induced by allo-sensitization is probably a much more trigger of CAV at a population level. Chronic antibody-mediated rejection has not been properly identified in heart allografts in contrary to kidney transplantation. This sub-clinical disease process might not be captured by 1-year histology.

II-C-4-d. Universality of the 4 trajectories profiles and prospects for patient risk stratification and monitoring Remarkably, despite the identification without any preconceptions of 4 CAV trajectories in the derivation cohort, very similar trajectories were detected in North-American independently. This validation of the same set of trajectories for patients in different healthcare systems promotes the idea that the same universal factors drive the evolution of CAV over the world which we further supported by identifying the determinants of CAV trajectories in the derivation cohort, and in the overall pooled derivation and validation cohorts. Furthermore, although the proportions of patients in each trajectory differed somewhat between the cohorts, reflecting intrinsic variability in the demographics and heart transplant practices across nations, our results nonetheless suggest promise for using these profiles to first categorize heart transplant recipients and then tailor the plan for future allograft monitoring and therapy based on the category. Consequently, to make trajectory-based monitoring of patients feasible in contemporary practice, we developed an easy-to-use online interface that allows clinicians to predict the personalized likely future of CAV trajectory of any given patient.

This approach to determining patient trajectories brings wider dimension to the traditional approaches that are either derived from one single angiogram result and selected but limited parameters, or are instead based on few measurements in non protocol based cohorts. Taking into account the evolution of CAV in the long-term could therefore takes advantage of more information about subtle changes and therefore could be an important complement to current medical practices. In addition, given the advancement of technology, especially the growing use of electronic medical records, trajectory assessment will conceivably have improved accuracy and may not be an trajectory approach also holds promise as an outcome measure. Indeed, for most of trials, the duration of follow-up is too brief to expect a large change in CAV.

II-C-4-f. Study limitations

Several limitations of the study should be noted. First, CAV grading is subject to measurement error. However, given our prospective design, any error would have probably attenuated identification of 4 distinct trajectories, whereas we were able to consistently identify the trajectories across different cohorts. Besides, in the derivation cohort, all CAV were reassessed by 2 independent observers. vSecondly, a limited number of trajectories were derived that may not accurately reflect every possible CAV profiles. However, the good discrimination of our trajectory building model indicate that these trajectories can parsimoniously summarize the predominant features of patient allografts in our population without a significant loss of information. Third, some classes -especially trajectory #4 -have relatively high inter-patient variability. Yet, since our goal was to give the overall picture of the CAV in large and diverse cohorts, we considered such variability acceptable. Last, there are unmeasured characteristics of the recipients and allografts such as donor genetic profiles, non HLA antibodies or inflammatory circulatory biomarkers that may well affect CAV trajectories and could be explored in future studies.

II-C-5. Conclusion

In conclusion, this analysis takes an original approach to analyzing and characterizing for the first-time trajectories of long-term CAV after heart transplantation associated with overall mortality. We also found that the identified CAV trajectories and their determinants could be generalizable and transportable across centers in different continents. Our results provide an important new tool for improving the approach to monitoring and risk stratifying heart transplant recipients, potentially paving the way towards therapeutic interventions.

Chapter II-D: Synthesis of epidemiological studies.

By using a population-based approach and applying two different epidemiological approaches, we were able to describe the epidemiology and natural history of allograft rejection and CAV, two important complications of allosensitization after heart transplantation, and to determine their risk factors.

We were able to show that allograft rejection had an evolutionary epidemiology during the first year, with a maximum risk of rejection during the first 3 months followed by a gradual decrease in risk and to identify 5 independent risk factors for rejection: recipient's age, type of induction, type of transplantation (isolated vs combined), number of HLA mismatch and presence of pre-formed DSA.

Using latent class mixed models, we identified four mutually exclusive CAV trajectories derived in a European cohort and validated on a US cohort. We identified five independent risk factors associated with these trajectories: donor age, donor gender, donor smoking, post-transplant dyslipidemia and the presence of class II DSA. (Figure 28).

Clinical studies to better characterize the outcomes of at-risk populations

As previously discussed in the introduction section, the therapeutic management of allosensitization and AMR is heterogeneous between centers and there is no consensus due to the lack of clinical data and therapeutic trials. Two populations at high immunological risk are particularly concerned by this lack of data: the patients transplanted with preformed DSA and the patients presenting the most severe forms of AMR, who are refractory to usual therapies and characterized by a severely depressed graft function responsible for a cardiogenic shock.

Our objective in this clinical part is to report the experience of our center with these two types of patients in order to better describe and understand their clinical events (Figure 29). The decision to accept a graft was made regardless of the presence of pfDSA. A prophylactic protocol including perioperative management of pfDSA and systematic treatment of subsequent ABMR was applied to patients transplanted with pfDSA.

Perioperative management was adapted to the results of the virtual cross-match and to the level of pfDSA, as evaluated by the mean fluorescence intensity (MFI) of the immunodominant DSA (i.e., DSA with the highest MFI): (1) patients with MFI 500-1000

were treated with IVIg (0.5 g/kg over 4 consecutive days, total dose of 2 g/kg; Privigen;

CSL Behring AG, Bern, Switzerland), ( 2) patients with MFI >1000 were treated with perioperative plasmapheresis sessions (one immediately before HTx, then four sessions over 4 consecutive days; 2/3 fresh frozen plasma and 1/3 albumin, fibrinogen substitution if serum fibrinogen < 2g/L) and IVIg. Initiation of the protocol was based on detection of anti-HLA antibodies in historical sera. The treatment was readjusted 2 to 3 days after HTx according to the MFI results at the time of HTx (Figure 30).

Subsequent biopsy-proven ABMR, even subclinical ABMR, were treated with corticosteroid pulses, plasmapheresis (five sessions) and IVIg (2 g/kg). Of the 523 patients, 241 had no DSA, 88 patients had pfDSA with MFI between 500 and 1000, and 194 patients had pfDSA with MFI >1000 in historical or day 0 sera. The proportion of sensitized patients increased over the study period. The evolution in group repartition between historical sera and day 0 sera is presented in Figure 30.

None of our patients received pre-transplant desensitisation therapies. The median follow-up was 4.06 years (IQR 1-7.98). Survival rates at 1 and 3 years after transplantation were 81.1% (95% CI [START_REF] Chen | Development and Impact of De Novo Anti-HLA Antibodies in Pediatric Heart Transplant Recipients[END_REF][START_REF] Sato | Risk Stratification for Cardiac Allograft Vasculopathy in Heart Transplant Recipients -Annual Intravascular Ultrasound Evaluation[END_REF]) and 73.6% (95% CI [START_REF] Coutance | Favorable outcome of an exclusively posttransplant prophylactic strategy after heart transplantation in recipients with high immunological risk[END_REF][START_REF] Chen | Development and Impact of De Novo Anti-HLA Antibodies in Pediatric Heart Transplant Recipients[END_REF]), respectively, and were not significantly different between groups (Figure 33 and Table 18). In the univariate analyses, donor age, weight and gender, recipient age and creatinine clearance, weight mismatch and ischemic time were associated with the risk of death, regardless of DSA group or DSA MFI (Table 19). In the multivariate analyses, donor age, recipient age, weight mismatch and cold ischemic time remained associated with mortality (Table 20). 18). This difference was driven by a four-fold increase in the risk of biopsy-proven ABMR at 1 year (Figure 34). A total number of 116 biopsy-proven ABMR, mostly subclinical (93%), were diagnosed in 73 patients during the entire follow-up. In the multivariate analyses, only DSA status at transplantation and cold ischemic time remained independent risk factors for death or rejection. Rejection-free survival was no longer significantly different between groups when defining rejection as ACR ≥2R and/or pAMR≥2 (Table 18). Patients with preformed donor-specific anti-HLA antibody (pfDSA) mean fluorescence intensity (MFI) levels >1000 had a four-fold increase in the risk of biopsy-proven antibody-mediated rejection 1 year following heart transplantation (p<0.001).

Table 18. Primary, secondary and safety endpoints.

The risk factors for PGD were analyzed in the subgroup of 412 patients without pre-HTx ECMO for the reasons discussed above. The rate of PGD was similar between groups, although we observed a non-significant increase in the prevalence among patients with pfDSA MFI >1000 (Table 18). However, after the multivariate analyses, only the cold ischemic time, donor gender and donor norepinephrine use >3 mg/h were associated with PGD (Hosmer-Lemeshow test: p=0.81). Results remained unchanged when including also in the analysis pre-HTx ECMO-patients. There was no difference in CAV incidence between the groups ( ). However, this did not translate into a significant increase in surgical revisions for bleeding. The risk of infectious complications, as assessed by the incidence of blood stream infections within the first 15 days after HTx, was not significantly different between groups (overall 8.6%; Table 18).

III-A-3-f. Exploratory analyses in patients with preformed donor-specific anti-HLA antibodies with a mean fluorescence intensity greater than 1000

In this group, we evaluated the impact of the MFI level on the day of transplant, representative of immunodominant DSA, on outcomes. While survival did not significantly differ between subgroups (p = 0.37; figure 35-A), patients with MFI >5000 experienced worse rejection-free survival than other patients (p = 0.01; Figure 35-B) due to a higher rate of early ABMR. This association remained significant once adjusted for cold ischemic time. Although not statistically significant, we observed a trend toward an increase in the risk of PGD in patients with MFI>5000 (rate of PGD = 39.2%, 37.1%, 35.2% and 47.6% for MFI<1000, 1000-3000, 3000-5000 and > 5000, respectively). The incidence of CAV did not significantly differ between groups.

In patients with pfDSA MFI >1000, survival and rejection-free survival did not significantly differ between patients with or without pre-transplant sensitizing event (p = 0.99 and 0.25, respectively).

III-A-4. Discussion

We report a retrospective analysis of a prospective, single-center experience in the management of sensitised heart transplant recipients. We applied a post-transplant protocol to patients with pfDSA consisting of IVIg +/-perioperative plasmapheresis sessions combined with induction therapy, with systematic treatment of subsequent ABMR. None of the patients had received pre-transplant desensitisation therapies. We observed a significant decrease in MFI (both immunodominant and cumulative) after the peri-operative management. We found that overall survival, CAV-free survival and the incidence of PGD were not significantly different between groups. However, rejection-free survival was significantly worse in patients with pfDSA MFI greater than 1000, evidenced by a four-fold increased risk of early ABMR. Perioperative plasmapheresis increased the risk of requiring a transfusion during the first week after HTx but had no impact on the incidence of bloodstream infections.

Management of the increasing number of sensitised heart transplant candidates is challenging. Pre-transplant desensitisation therapy is the most widely used strategy, and data suggest that lowering PRA before HTx improves prognosis after transplantation (116). While different protocols have been evaluated, robust clinical trials are still lacking. Several issues and concerns might limit the widespread application of such pre-transplant therapies [START_REF] Colvin | Antibody-mediated rejection in cardiac transplantation: emerging knowledge in diagnosis and management: a scientific statement from the American Heart Association[END_REF]110). Firstly, as they require a HLAcompatible donor, sensitised patients have longer waiting times, and subsequently, worse outcomes (111). Secondly, potential over-immunosuppression induced by these therapies might increase the risk of infectious complications. Thirdly, this strategy is costly because desensitisation protocols are based on expensive medications, and sensitised patients might require a bridge-to-transplant assist-device strategy due to the expected long waiting time. Finally, the initiation of these anti-HLA antibody therapies is based on the PRA, which evaluates the potential risk of being transplanted with DSA, but is not based on the real DSA, potentially leading to overtreatment. Rather than using pre-transplant desensitisation therapies, we established a post-transplant prophylactic strategy consisting of perioperative management of pfDSA and systematic treatment of ABMR. Our decision to perform HTx was made regardless of the presence of pfDSA. We believe that this protocol might improve access to transplantation of sensitised patients.

A recent ISHLT consensus stated that desensitization likely confers a benefit to the patients [START_REF] Kobashigawa | T he management of antibodies in heart transplantation: An ishlt consensus document[END_REF]. The conventional goal of desensitization therapy is to achieve a negative crossmatch before proceeding with a transplant. As emphasized by ISHLT experts, there is, however, an alternative strategy whereby transplantation proceeds despite a positive virtual crossmatch and therapies are initiated to mitigate the impact of DSAs [START_REF] Kobashigawa | T he management of antibodies in heart transplantation: An ishlt consensus document[END_REF]. We found that an exclusively post-transplant management of pfDSA resulted in a neutralization of the risk of DSA on post-HTx survival and might be a therapeutic option for sensitized patients.

The largest experience of solid organ transplantation with pfDSA comes from kidney transplantation. Lefaucheur et al. found that 8-year graft survival was significantly worse among patients with pfDSA compared with non-sensitised patients (117). While de novo DSA has been consistently associated with worse outcomes after HTx, the impact of pfDSA is less clear (118). Retrospective, single-center studies without a posttransplant prophylactic protocol suggest that they may be associated with worse outcomes, particularly CAV and ABMR; however, their impact on survival or graft survival is highly debated in the field of heart transplantation [START_REF] Topilsky | Donor-specific antibodies to class II antigens are associated with accelerated cardiac allograft vasculopathy: a threedimensional volumetric intravascular ultrasound study[END_REF][START_REF] Raess | Donor-specific anti-HLA antibodies detected by Luminex: predictive for short-term but not long-term survival after heart transplantation[END_REF]119). In our cohort, we observed similar survival but a four-fold higher risk of biopsy-proven ABMR at 1 year in transplant patients with pfDSA MFI greater than 1000. Chronic rejection is widely recognized as a progressive process that poses significant risks of allograft loss and mortality after organ transplant [START_REF] Loupy | Antibody-Mediated Rejection of Solid Organ Allografts[END_REF]. The main feature of chronic AMR across solid organ transplants is chronic vascular lesions. In cardiac allografts, these vascular injuries manifest as CAV. Furthermore, AMR has been associated with CAV and cardiovascular mortality (120). However, while late and symptomatic ABMR are associated with a higher risk of CAV progression, the ABMR in our study were early and mostly subclinical [START_REF] Clerkin | The effect of timing and graft dysfunction on survival and cardiac allograft vasculopathy in antibody-mediated rejection[END_REF]. We did not observe a higher incidence of CAV in sensitised patients, although a longer follow-up period is necessary to detect such potential chronic complications of ABMR and DSA (median follow-up in this study of 4.06 years).

As pointed out in a recent review, the choice of a clear cut-off value of MFI to predict post-transplant outcomes is highly controversial (121). When our protocol was set up in 2009, there was very little data on MFI cut-off values in the field of HTx. The presence of weak antibodies is likely to reflect the presence of memory antibody-producing cells, which are dormant pre-transplant but may be re-activated after transplant (121). Based on this, our local cut-off point was intentionally low in order to minimize immunological risks. Moreover, Svobodova et al. demonstrated that pfDSA MFI greater than 1000 is an independent risk factor for early ABMR after HTx [START_REF] Svobodova | Novel insights into pretransplant allosensitization in heart transplant recipients in the contemporary era of immunosuppression and rejection surveillance[END_REF]. International experts reported that the most frequently cited MFI cut-off for identifying the antigens to avoid is 5000. We found a similar cut-off value in our exploratory analysis of the subgroup of sensitised patients: worse rejection-free survival, trend toward an increase in the risk of PGD. These findings raise caution concerning the use of this protocol in patients with MFI >5000.

More in depth characterization of pfDSA may have allowed for better prognosis stratification of post-transplant outcomes. There is growing evidence that suggests that simply using MFI values as the sole gauge for antibody strength is not sufficient. The ability of DSA to bind to C1q and the IgG isotype provides additional prognosis value (122,123). Kobashigawa et al. reported that using four antibody detection assays might optimize the selection of unacceptable antigens (124).

Although limited by the small sample size, our data suggest that patients with high MFI at transplant that remain high despite therapies are at increased risk of early events. This result emphasizes the need for DSA monitoring after immunosuppressive therapies. Reinforcement of therapies should be considered in poor or nonresponders.

The results of the current study led to several changes to our practice. Firstly, due to the four-fold increase in the risk of ABMR, we reserve this protocol for patients requiring emergency access to HTx. Secondly, we treat patients transplanted with pfDSA MFI of >1000 with tacrolimus as a calcineurin inhibitor therapy since 2017 as it significantly decreases rejection compared to cyclosporine [START_REF] Kobashigawa | Tacrolimus with mycophenolate mofetil (MMF) or sirolimus vs. cyclosporine with MMF in cardiac transplant patients: 1year report[END_REF]. Finally, despite the uncertainties regarding the ability of rituximab to prevent and/or treat ABMR, we are currently considering using this medication in patients with pfDSA MFI >5000 on top of the current therapy.

Compared to international cohorts, our patients have several particularities. Firstly, only 13.4% of our patients were under long-term mechanical circulatory support at the time of transplantation compared to the 42.9% of patients reported in the most recent ISHLT registry [START_REF] Lund | The Registry of the International Society for Heart and Lung Transplantation: Thirty-fourth Adult Heart Transplantation Report-2017; Focus Theme: Allograft ischemic time[END_REF]. Our data are consistent with French national data. Previous and current allocation policies in France give high-priority HTx access to patients under ECMO and/or inotropes, which might explain this important difference. Twenty percent of our patients were under ECMO support at the time of transplantation compared to 1% in the 2017 ISHLT registry [START_REF] Lund | The Registry of the International Society for Heart and Lung Transplantation: Thirty-fourth Adult Heart Transplantation Report-2017; Focus Theme: Allograft ischemic time[END_REF]. Second, alloimmune sensitization was almost twice more prevalent in our cohort in comparison to international registries, probably due to differences in definitions (pfDSA MFI >1000 in our cohort compared to PRA of >10% in registries.

It is important to highlight that re-transplantations were excluded from our study. Most of re-transplantations were combined heart-kidney transplantations and were treated with pre-transplant desensitization therapies according to our kidney-transplant team protocol. Since more than half of re-transplantation patients received pre-transplant desensitization therapies before transplantation, including re-transplanted patients in the analysis might have induced selection bias.

The present study should be interpreted within the context of its limitations. Firstly, our study does not include a control group for comparison, thus the independent impact of our prophylactic strategy on outcomes cannot be analyzed. However, a randomized clinical trial could not have been performed due to ethical issues, as data published before the initiation of this protocol suggested that pfDSA is correlated with graft loss after HTx [START_REF] Stastny | Antibodies against donor human leukocyte antigens and the outcome of cardiac allografts in adults and children[END_REF]. Secondly, as this is a single center experience, external validation is required. Moreover, as discussed above, the MFI threshold was made as low as 1000, which could explain, in part, the good prognosis we observed in our sensitised patients.

The number of patients with very high MFI (e.g., >10,000) was too low in our cohort to determine a value above which the transplantation should not be performed. Similarly, our exploratory analyses of the subgroup of patients with MFI >5000 are undeniably underpowered due to the small number of patients. Thirdly, the lack of C1q assay did not allow us to provide an in-depth characterization of pfDSA. Fourthly, we limited treatment of DSA to the peri-transplant period and to the AMR episodes whereas it has been widely demonstrated that IVIg-based treatment protocol shows a higher clearance efficacy if IVIg infusions are repeated over time (125-127). Repeated infusion of IVIG with a lower dosage may reduce subsequent incidence of ABMR.

However, IVIg shortage and costs are two important limits for a wider use of IVIg.

Fifthly, we acknowledge that post-transplant confounding factors that were not tested in the model might have influenced the risk of transfusions during the first week posttransplant. Sixthly, as discussed above, longer follow-up is necessary to monitor CAV progression potentially associated with pfDSA and ABMR. Finally, PRA is not routinely performed at our center, thus our strategy is not based on this result.

III-A-5. Conclusion

III-B-1. Introduction

The clinical spectrum of AMR ranges from asymptomatic subclinical AMR to acute cardiogenic shock. Subclinical AMR has been associated with increased cardiovascular mortality (128). Allograft dysfunction at the time of AMR is an independent risk factor for the development of de novo CAV and death [START_REF] Clerkin | The effect of timing and graft dysfunction on survival and cardiac allograft vasculopathy in antibody-mediated rejection[END_REF]. Late symptomatic AMR is associated with poor short-term prognosis: high mortality, fulminant forms of cardiac allograft vasculopathy (CAV) and persistent left ventricular (LV) dysfunction [START_REF] Coutance | Late antibody-mediated rejection after heart transplantation: Mortality, graft function, and fulminant cardiac allograft vasculopathy[END_REF]129).

As underlined by a scientific statement from the American Heart Association, the widely used term of "hemodynamic compromise" lacks uniformity, and the specific criteria for dysfunction may range from decreased ejection fraction to cardiogenic shock requiring inotropic support [START_REF] Colvin | Antibody-mediated rejection in cardiac transplantation: emerging knowledge in diagnosis and management: a scientific statement from the American Heart Association[END_REF]. Cardiogenic shock is the most severe form of allograft dysfunction. The prognosis of AMR with cardiogenic shock is suspected to be poor but data are scarce in the literature.

In this retrospective cohort we aimed to analyze the characteristics, the treatment and the prognosis of patients treated for cardiogenic shock due to biopsy-proven AMR at our center. Treatment of AMR included intravenous methylprednisolone infusion (500 to 1000 mg per day for 3 days), 5 plasmapheresis (1 plasmapheresis on 5 consecutive days) and high dose IVIg (Privigen CSL Berhing Laboratories, Pennsylvania, USA -total dose = 2 g / kg administered during 4 consecutive days). When required, treatment could be intensified with more plasmapheresis, rabbit anti-thymocyte globulins (rATG, Thymoglobuline, Sanofi-Genzyme, Massasuchets, United States, 1.5 mg/kg per day during 3 days) and/or anti-CD20 therapy (Rituximab, Mabthera, Roche pharmaceuticals, Basel, Switzerland -375 mg/m2, 2 to 4 injections). Eculizumab (Soliris, Alexion Pharmaceuticals, Connecticut) could be used as a salvage therapy.

III-B-2. Methods

III-B-2-f. Endomyocardial biopsies (EMB)

EMBs were performed according to current standard using either right jugular vein or femoral approach. Biopsies were processed and examined according to current standards (2). Our routine EMB protocol includes 13 EMB during the first year, then every 4 months for 2 years, followed by every 6 months for 2 years. III-B-3-d. Survival after AMR Survival after AMR is described in Figure 36. In hospital and one-year mortality were 76 % and 82 % respectively. Causes of death are detailed in Table 21. The leading cause of death was refractory cardiogenic shock but infectious complications during hospitalization in the intensive care unit and multiple organ failure (MOF) were common. Among patients discharged from hospital, one patient presented a sudden cardiac death at home 10 months after AMR whereas the last EMB was considered as normal and coronary angiography performed after AMR revealed smooth coronary arteries (CAV 0).

III-B-4. Discussion

We described a cohort of 17 patients treated for cardiogenic shock due to AMR, including 11 patients requiring ECMO support. AMR was mostly due to high-level de novo class II DSA. Our results suggest that AMR with cardiogenic shock is a rare condition associated with very poor short-term prognosis (in-hospital mortality = 76 %).

Major causes of deaths were refractory allograft dysfunction and infectious complications. Patients discharged from hospital recovered allograft function, but one case of rapidly progressive CAV and one case of sudden cardiac death occurred during follow-up.

Acute allograft dysfunction is a well-known risk factor of death in patients diagnosed with AMR. Our purpose was not to compare sub-clinical AMR to symptomatic AMR.

We aimed to point out the particularly poor prognosis of CS due to AMR to emphasize the need for an early-standardized aggressive immunosuppressive protocol in this high emergency context.

The poor prognosis of cardiogenic shock due to AMR observed in our cohort suggests that an early intensification of immunosuppressive treatment should strongly be considered. Whereas most centers would treat symptomatic AMR similarly, the treatment of AMR resistant to conventional therapies is highly controversial. Rituximab and bortezomib have been used for the treatment of cardiac allograft AMR. However, the literature in cardiac allograft AMR is mostly based on case reports (131). A randomized trial in acute kidney AMR failed to show any additional effect of rituximab in patients receiving plasmapheresis, IVIg, and steroids (132). Similarly, bortezomib was not proven to be superior to placebo for the treatment of late kidney AMR (133). Since all of these drugs only prevent the production of new DSA and are not able to remove circulating antibodies, their use in the high emergency context of AMR with cardiogenic shock is questionable. Complement activation is the predominant effector pathway of acute AMR [START_REF] Colvin | Antibody-mediated rejection in cardiac transplantation: emerging knowledge in diagnosis and management: a scientific statement from the American Heart Association[END_REF]. Therefore, the blockade of antibody-mediated complement activation is an attractive target in the context of severe AMR. Eculizumab is a C5 inhibitor that is approved for use in paroxysmal nocturnal hemoglobinuria and has been used as rescue therapy in the treatment of kidney AMR resistant to conventional therapies (134) with mixed results (135). A human plasma-derived C1 inhibitor that is approved for use in patients with hereditary angioedema has been tested in a phase 2b double blind randomized trial for the treatment of kidney AMR with promising results (136). The prompt use of complement inhibitors could have the advantage of immediate effects by stopping cellular injury, providing a therapeutic window to allow conventional treatment modalities to be effective (134). Only one patient of our cohort was treated with eculizumab (patient 1) and experience in cardiac transplantation is very limited. The significant cost associated with the use of eculizumab might limit its use.

Despite the single center design of our study, significant discrepancies do exist in the treatment of patients. Their prognosis could greatly benefit from a standardized immunosuppressive protocol. We believe that corticosteroids pulses, plasmapheresis and IVIg remain the basic treatment. The high rate of infections complications and the poor effect on allograft function we observed with ATG and rituximab emphasized the need for an immediately effective and well-tolerated drug. In this way, the use of complement inhibitor should be discussed as soon as possible. However, the lack of a standardized treatment protocol and the small number of patients in our study limits how strong any association between immunosuppressive treatment and outcomes can be made.

Several factors may have worsened the prognosis of our patients beyond the severity of AMR. First, more than one-third of our patients had mixed rejection. This condition reflects a complex interplay between cellular and humoral processes and is associated with worse outcomes (137). Secondly, almost half of our patients required renal replacement therapy, a well-known risk factor for poor outcomes in cardiogenic shock (138). Finally, the effect of timing of AMR might be important. Most of our patients suffered from late AMR (> 12 months post-transplant), a condition that has been associated with poor outcomes [START_REF] Clerkin | The effect of timing and graft dysfunction on survival and cardiac allograft vasculopathy in antibody-mediated rejection[END_REF][START_REF] Coutance | Late antibody-mediated rejection after heart transplantation: Mortality, graft function, and fulminant cardiac allograft vasculopathy[END_REF].

Graft dysfunction during AMR was an independent predictor of progression of CAV after AMR in a large cohort [START_REF] Clerkin | The effect of timing and graft dysfunction on survival and cardiac allograft vasculopathy in antibody-mediated rejection[END_REF]. Fulminant form of cardiac allograft vasculopathy has been described following late AMR [START_REF] Coutance | Late antibody-mediated rejection after heart transplantation: Mortality, graft function, and fulminant cardiac allograft vasculopathy[END_REF]. Rapidly progressive forms of CAV might have been underdiagnosed in our cohort since only 3 patients (18 %) had a coronary angiogram after AMR, including one case of rapidly progressive CAV (patient 13). We suggest that in case of persistent allograft dysfunction despite aggressive immunosuppressive regimen, a coronary angiogram should be discussed to rule out an ischemic participation to the persistent allograft dysfunction.

Chapter III-C: Conclusion of the clinical studies.

These clinical data allow us to better understand the clinical events of two populations at high immunological risk (Figure 37).

On the one hand, an exclusively post-transplant strategy based on the management of pre-formed DSA using plasmapheresis and IVIg was associated with favorable post-transplant outcomes, with no impact on post-transplant survival, but at the cost of an increased incidence of subclinical AMR and bleeding complications.

On the other hand, since graft dysfunction is so advanced in AMR-related cardiogenic shocks, usual and widely accepted therapies (plasma exchange, IVIg) are poorly effective and an early intensification of immunosuppression is mandatory in these patients (Figure 37). To answer these questions, we applied three complementary approaches: a molecular, an epidemiological and a clinical approach. Our results provide new information in the field of alloimmunization in heart transplantation that could contribute to the evolution of clinical practices.

Discussion of results and future directions

Molecular biology as a companion to the pathologic diagnosis of rejection Our results raise the question of the place of molecular biology in the field of heart allograft rejection. Molecular biology has often been described as the new gold standard for the diagnosis of rejection, neglecting the central role of pathology in diagnosticating rejections and guiding therapies. We believe that pathologic and molecular results should be analyzed as a whole since each approach brings valuable and additional information allowing an in-depth histo-molecular characterization of rejection. We believe that the results from our molecular analyses make it possible to apprehend molecular biology as a companion to the pathologic diagnosis of allograft rejection. The good agreement between the unsupervised molecular diagnosis based on RT-MLPA gene expression profiles and the pathologic diagnosis is an important point. The discrepancies between these two diagnoses are just as much important, both for the cases of histological non-rejection classified as molecular rejection (biopsy-negative rejection with a high prevalence of allograft dysfunction), and for the cases of histological rejections classified as molecular non-rejection (lowgrade rejections). Molecular biology has also allowed us to validate in-situ a histological classification of microvascular inflammation. Its extension, categorized into four classes, correlated with the degree of molecular activity of AMR. These two studies illustrate how pathologic analysis and molecular biology can jointly help to standardize the diagnosis of AMR, a necessary first step towards improving the management of this pathology.

The pursuit of research aimed at standardizing pathologic diagnosis is a priority objective. The contribution of molecular biology as a companion to pathology is an important area of research. International kidney allograft rejection classifications now incorporate these molecular tools in the diagnosis of allograft rejection, particularly for AMR [START_REF] Haas | The Banff 2017 Kidney Meeting Report: Revised diagnostic criteria for chronic active T cell-mediated rejection, antibodymediated rejection, and prospects for integrative endpoints for next-generation clinical trials[END_REF]. Despite its satisfactory diagnostic performance, RT-MLPA suffers from the limitation of the number of transcripts of interest that can be analyzed in the same sample. Despite its cost, we believe that the molecular biology technique developed by NanoString technologies is a promising technique that might be applicable in clinical practice. This technique allows not only the use of FFPE EMB, but also offers the possibility of simultaneously analyzing several hundred transcripts of interest, to provide an absolute gene expression and do without any amplification step during the procedure. We have already begun to apply this technique to FFPE BEMs in a retrospective case-control study with encouraging initial results. We are currently performing a multicenter study with a centralized review of pathology to validate a restricted molecular signature of each type of allograft rejection based on NanoString analyses.

Integrative epidemiology

The unit in which I work has developed over time a multidisciplinary and transversal scientific approach by mixing clinical and statistical investigations. The mathematical tool is used in an integrative thought process coupled with multiple questions, intuitions, and clinical investigations. We have called this approach integrative epidemiology. Our approach uses the statistical and epidemiological tool as a means of first-line investigation, generating new hypotheses and new directions of research. This approach, applied to large multicenter cohorts of very well phenotyped patients, allowed us to better describe the epidemiology, natural history, and risk factors of two important complications of alloimmunization, namely allograft rejections and CAV.

Although our results provide new insights into the epidemiology, natural history, and determinants of allograft rejection and CAV, they do not yet allow us to achieve an individual risk stratification of events that would allow the development of personalized medicine in heart transplantation. Consideration of the notion of futile tests is of primary importance in our field. What is the probability that a protocol biopsy performed at 8 months after transplant is abnormal when the 8 previous biopsies were normal? What is the probability that a coronary angiography performed 5 years after transplantation will show evidence of CAV when the 2 previous coronary angiograms were normal, and the patient has no risk factors for developing the disease? The application of mixed models to assess these issues will allow us to access the individual prediction level.

As previously discussed, these models allow us to understand the longitudinal character of the data and to introduce random effects. We are currently applying mixed logistic models to the risk of rejection modeling in the first year. The first results are promising. We will now continue to characterize an optimal model that allows access to an individual stratification of the risk of rejection. We are also working on the development of joint models, which associates a mixed model with a Cox model. We wish to apply this approach to graft coronary artery disease in order to individually define the likelihood of finding abnormalities in a coronary angiogram based on the results of previous coronary angiography and the risk factors for disease development.

Clinical studies

The analysis of the results of the application of a prospective protocol for the treatment of pre-formed DSA at the time of the transplant that we report is a first step towards a better definition of the optimal management of these patients. We were able to show that the risk of AMR was particularly high in patients with high MFI values on the day of the transplant, especially if MFI did not significantly decrease after specific treatment. Intensification of immunosuppression might be indicated in these very highrisk patients. Comparing the experience of other centers could help define optimal management of such patients. We are currently working towards this goal with the team of Dr. J. Kobashigawa and J. Patel of Cedars-Sinai Medical Center, the world's leading heart transplant center. This team has developed an innovative approach to cardiac transplantation with a very high immunological risk by combining prolonged complement inhibitor therapy (Eculizumab) with the standard of care including polyclonal induction, high dose infusion of IVIg, and a triple basal immunosuppression.

The first results seem very encouraging. I will have the opportunity to continue the comparison between the cohorts of our two centers during a postdoctoral year that I will perform within this prestigious team.

S3. Pathology

Endomyocardial biopsies were performed, processed and examined according to current standards. The routine EMB protocol from the European derivation cohort included 13 protocolar EMBs during the first year (every 10 days from day 15 to M2, every month from M2 to M6 and every month and a half from M6 to M12). Additional biopsies were performed in case of clinical indication. Serial sections were cut from formalin-fixed paraffin-embedded EMB specimens and stained with hematoxylin, eosin and saffron for diagnosis. Immunofluorescence for C4d was performed (frozen section; C4d monoclonal Quidel Corporation 1/100; Polyclonal rabbit anti mouse FITC Dako).

Only capillary staining for C4d was assessed. The EMBs were classified as cellular (0 to 3R) or ABMR (pAMR 0 to 3) according to the ISHLT guidelines.

For the purpose of this study, a systematic retrospective analysis of all abnormal EMBs (i.e. ACR ≥ 1R1B and/or pAMR ≥ 1), for-cause EMBs and pre-specified protocolar EMBs (first, M1, M3, M6 and M12) was performed by expert pathologists, independently of the initial pathological diagnosis, clinical, and CAV data in the French derivation cohort.

  Merci à Patrick Bruneval et Marion Rabant, de me faire l'honneur de leur présence dans ce jury. Cela a été un plaisir d'échanger avec vous pendant ces années. Merci à Xavier Jouven pour ses conseils et son encadrement au cours de cette thèse ainsi que de son soutien. Merci à Shaida Varnous, dont je ne serai jamais assez reconnaissant de m'avoir tant appris dans le domaine de la transplantation et dans tant d'autres d'ailleurs. Table des matières ACKNOWLEDGEMENTS.......................................................................................................... PUBLICATIONS ........................................................................................................................ AVERTISSEMENT ................................................................................................................... INTRODUCTION ...................................................................................................................... INTRO-A. ADVANCED HEART FAILURE: DEFINITION AND EPIDEMIOLOGY ............................................................... INTRO-B. HEART TRANSPLANTATION ............................................................................................................. INTRO-C. CURRENT ISSUES IN THE MANAGEMENT OF DSA AND AMR AFTER HEART TRANSPLANTATION ................... PART I. ROLE OF MOLECULAR BIOLOGY TO IMPROVE THE DIAGNOSIS OF ANTIBODY-MEDIATED REJECTION. ................................................................................................................................ CHAPTER I-A: PHYSIOPATHOLOGY OF ANTIBODY-MEDIATED REJECTION AND DEVELOPMENTS OF MOLECULAR BIOLOGY IN HEART TRANSPLANTATION. ............................................................................................................ I-A-1. PATHOPHYSIOLOGY OF ANTIBODY-MEDIATED REJECTION ......................................................................... I-A-2. MOLECULAR BIOLOGY IN HEART ALLOGRAFT REJECTION........................................................................... I-A-3. OBJECTIVES ..................................................................................................................................... CHAPTER I-B: APPLICATION OF MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION TO ENDOMYOCARDIAL BIOPSIES. ...................................................................................................................................... I-B-1. MOLECULAR BIOLOGY APPLIED TO THE DIAGNOSIS OF HEART ALLOGRAFT REJECTION .................................... I-B-2. MATERIAL AND METHODS .................................................................................................................. I-B-3. RESULTS .......................................................................................................................................... I-B-4. DISCUSSION ..................................................................................................................................... I-B-5. CONCLUSION ................................................................................................................................... CHAPTER I-C: EXTENSION OF INTRAVASCULAR ACTIVATED MONONUCLEAR CELLS CORRELATES WITH ANTIBODY-MEDIATED REJECTION MOLECULAR ACTIVITY, DONOR-SPECIFIC ANTIBODIES AND GRAFT FUNCTION. ..................... I-C-1. INTRODUCTION ................................................................................................................................ I-C-2. PATIENTS AND METHODS ................................................................................................................... I-C-3. RESULTS .......................................................................................................................................... I-C-4. DISCUSSION ..................................................................................................................................... II-B-1. INTRODUCTION ............................................................................................................................... II-B-2. MATERIALS AND METHODS ............................................................................................................... II-B-3. RESULTS ......................................................................................................................................... II-B-4. DISCUSSION .................................................................................................................................... CHAPTER II-C: IDENTIFICATION AND CHARACTERIZATION OF TRAJECTORIES OF CARDIAC ALLOGRAFT VASCULOPATHY AFTER HEART TRANSPLANTATION: A POPULATION BASED STUDY. ............................................................. II-C-1. INTRODUCTION ............................................................................................................................... II-C-2. PATIENTS AND METHODS .................................................................................................................. II-C-3. RESULTS ......................................................................................................................................... II-C-4. DISCUSSION .................................................................................................................................. II-C-5. CONCLUSION ................................................................................................................................ CHAPTER II-D: SYNTHESIS OF EPIDEMIOLOGICAL STUDIES. ....................................................................... PART 3. CLINICAL STUDIES TO BETTER CHARACTERIZE THE OUTCOMES OF AT-RISK POPULATIONS ................................................................................................................................................. CHAPTER III-A: FAVORABLE OUTCOME OF AN EXCLUSIVELY POST-TRANSPLANT PROPHYLACTIC STRATEGY AFTER HEART TRANSPLANTATION IN RECIPIENTS WITH HIGH IMMUNOLOGICAL RISK. ........................................................ III-A-1. HEART TRANSPLANTATION WITH PRE-FORMED ANTI-HLA DONOR-SPECIFIC ANTIBODIES ........................... III-A-2. PATIENTS AND METHODS ............................................................................................................... III-A-3. RESULTS ..................................................................................................................................... III-A-4. DISCUSSION ................................................................................................................................. III-A-5. CONCLUSION ............................................................................................................................... CHAPTER III-B: ANTIBODY-MEDIATED REJECTION INDUCED CARDIOGENIC SHOCK: TOO LATE FOR CONVENTIONAL THERAPY. .................................................................................................................................... III-B-1. INTRODUCTION ............................................................................................................................ III-B-2. METHODS ................................................................................................................................... III-B-3. RESULTS ...................................................................................................................................... III-B-4. DISCUSSION ................................................................................................................................. III-B-5. CONCLUSION ............................................................................................................................... CHAPTER III-C: CONCLUSION OF THE CLINICAL STUDIES. .......................................................................... CONCLUSION ........................................................................................................................ DISCUSSION OF RESULTS AND FUTURE DIRECTIONS ......................................................................................... BIBLIOGRAPHY ..................................................................................................................... SUPPLEMENTARY APPENDIX ............................................................................................

FIGURE 1 : 15 FIGURE 2 : 15 FIGURE 3 : 16 FIGURE 4 . 20 FIGURE 5 : 27 FIGURE 7 : 33 FIGURE 10 : 35 FIGURE 11 : 40 FIGURE 12 : 41 FIGURE 13 : 50 FIGURE 15 : 53 FIGURE 16 : 55 FIGURE 17 : 60 FIGURE 18 : 62 FIGURE 19 :

 1152153164205277331035114012411350155316551760186219 FIGURE 1: EVOLUTION OF THE NUMBER OF HEART TRANSPLANTATIONS. (A) ISHLT DATA, (B) "AGENCE DE LA BIOMÉDECINE"............................................................................................... 15 FIGURE 2: POST-TRANSPLANT SURVIVAL. (A) ISHLT DATA, (B) "AGENCE DE LA BIOMÉDECINE". ...................................................................................................................................................... 15 FIGURE 3: DYNAMIC OF CAUSES OF DEATH AFTER HEART TRANSPLANTATION .................... 16 FIGURE 4. ALLOSENSITIZATION AND ALLOGRAFT REJECTION IN HEART TRANSPLANTATION: FROM DIAGNOSIS TO OUTCOMES. ......................................................................................... 20 FIGURE 5: PATHOPHYSIOLOGY OF ANTIBODY-MEDIATED REJECTION (25) ............................. 25 FIGURE 6: T-CELL MEDIATED REJECTION AND ANTIBODY-MEDIATED REJECTION-SELECTIVE TRANSCRIPTS (51). .................................................................................................................... 27 FIGURE 7: LACK OF STANDARDIZATION IN THE DIAGNOSIS OF ANTIBODY-MEDIATED REJECTION: STRATEGIES BASED ON MOLECULAR BIOLOGY TECHNIQUES TO REFINE AMR DIAGNOSIS. ........................................................................................................................ 30 FIGURE 8: PRINCIPLE OF MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION. ......... 32 FIGURE 9: FLOWCHART OF THE RT-MLPA STUDY ......................................................................... 33 FIGURE 10: HISTOPATHOLOGY AND RT-MLPA PROFILES ............................................................ 35 FIGURE 11: EXPRESSION OF TRANSCRIPTS ACCORDING TO HISTOLOGICAL DIAGNOSIS .... 40 FIGURE 12: UNSUPERVISED HIERARCHICAL AND CLASS PREDICTION ANALYSIS .................. 41 FIGURE 13: CLASS PREDICTION ANALYSIS .................................................................................... 42 FIGURE 14: SEMI-QUANTITATIVE ASSESSMENT OF MICROVASCULAR INFLAMMATION USING THE MVI SCORE.......................................................................................................................... 50 FIGURE 15: MOLECULAR CORRELATES OF EXTENSION OF MICROVASCULAR INFLAMMATION MVI SCORE. ................................................................................................................................. 53 FIGURE 16: RELATIONSHIP BETWEEN EXTENSION OF MICROVASCULAR INFLAMMATION (MVI SCORE) AND ISHLT CLASSIFICATIONS OF REJECTION, MFI OF IMMUNODOMINANT DSA AND IMPAIRED GRAFT FUNCTION AT THE TIME OF ENDOMYOCARDIAL BIOPSY. .......... 55 FIGURE 17: SUMMARY OF RESULTS OF MOLECULAR BIOLOGY STUDIES. ............................... 60 FIGURE 18: OBJECTIVES OF THE PART 2: RISK STRATIFICATION. ............................................. 62FIGURE 19: DESIGN OF THE STUDY. ................................................................................................ 73

FIGURE 20 : 75 FIGURE 21 : 75 FIGURE 22 :

 2075217522 FIGURE 20: REPARTITION OF (A) OVERALL EMB AND (B) REJECTING-EMB ACCORDING TO THE TIME POST-TRANSPLANT. ........................................................................................................ 75 FIGURE 21: EVOLUTION OF THE MEAN NUMBER OF EMB PER PATIENT PERFORMED DURING THE FIRST-YEAR POST TRANSPLANT..................................................................................... 75 FIGURE 22: EPIDEMIOLOGY OF ALLOGRAFT REJECTION DURING THE FIRST YEAR AFTER HEART TRANSPLANTATION ...................................................................................................... 78

FIGURE 23 : 80 FIGURE 24 . 87 FIGURE 25 . 94 FIGURE 26 : 95 FIGURE 27 :FIGURE 28 :FIGURE 29 :FIGURE 30 :FIGURE 31 :FIGURE 36 :

 2380248725942695272829303136 FIGURE 23: DISCRIMINATION OF REGRESSION MODELS. A : COX MODEL. B: LOGISTIC REGRESSION MODEL. ............................................................................................................... 80 FIGURE 24. DESIGN OF THE STUDY. ................................................................................................ 87 FIGURE 25. DISTINCT PROFILES OF CAV TRAJECTORIES IDENTIFIED POST-TRANSPLANTATION IN THE EUROPEAN DERIVATION COHORT (N=815). .......................... 94 FIGURE 26: CARDIAC ALLOGRAFT VASCULOPATHY TRAJECTORIES: EXTERNAL VALIDATION IN THE AMERICAN COHORT. .................................................................................................... 95 FIGURE 27: DESCRIPTION OF OVERALL MORTALITY ACCORDING TO THE CAV TRAJECTORY IN THE OVERALL COHORT (N=1,301). .................................................................................... 101 FIGURE 28: SYNTHESIS OF THE RESULTS OF THE EPIDEMIOLOGICAL STUDIES. ................. 107 FIGURE 29: BETTER UNDERSTANDING OF OUTCOMES OF AT-RISK POPULATIONS. ............ 108 FIGURE 30: STUDY PROTOCOL. ..................................................................................................... 111 FIGURE 31: MEAN FLUORESCENCE INTENSITY OF DONOR-SPECIFIC ANTIBODIES.............. 115 FIGURE 32. EVOLUTION OF MFI AFTER PERI-OPERATIVE MANAGEMENT IN PATIENTS WITH PREFORMED DONOR-SPECIFIC ANTI-HLA ANTIBODY (PFDSA) MEAN FLUORESCENCE INTENSITY (MFI) LEVELS >1000. ............................................................................................. 116 FIGURE 33. PRIMARY AND SECONDARY OUTCOMES. ................................................................ 117 FIGURE 34. INCIDENCE OF BIOPSY-PROVEN ANTIBODY-MEDIATED REJECTION (ABMR) (DEATHS NOT SHOWN). .......................................................................................................... 117 FIGURE 35. EXPLORATORY ANALYSIS IN THE GROUP OF PATIENTS WITH PREFORMED DONOR-SPECIFIC ANTI-HLA ANTIBODY (PFDSA) MEAN FLUORESCENCE INTENSITY (MFI) LEVELS >1000. .......................................................................................................................... 121 FIGURE 36: SURVIVAL AFTER ANTIBODY-MEDIATED REJECTION WITH CARDIOGENIC SHOCK. .................................................................................................................................................... 133 FIGURE 37. SUMMARY OF THE RESULTS OF CLINICAL STUDIES. ............................................ 137

Figure 1 :

 1 Figure 1: Evolution of the number of heart transplantations. (A) ISHLT data, (B) "Agence de la Biomédecine".

Figure 2 :

 2 Figure 2: Post-transplant survival. (A) ISHLT data, (B) "Agence de la Biomédecine".

Figure 3 :

 3 Figure 3: Dynamic of causes of death after heart transplantation

↓ Figure 4 .

 4 Allosensitization and allograft rejection in heart transplantation: from diagnosis to outcomes. Part I: role of molecular biology to improve the diagnosis of antibody-mediated rejection. Part II: Epidemiologic approaches to enhance prognostic stratification of CAV and allograft rejections. Part III: Clinical studies to improve the understanding of at-risk subpopulation -transplantation with pre-formed DSA & AMR-induced cardiogenic shock. AMR means Antibody-mediated rejection, CAV: cardiac allograft vasculopathy, DSA: donor-specific antibodies, IAMC: intravascular activated mononuclear cells, RT-MLPA: Retro-Transcription Multiplex Ligation-dependent Probe Amplification. Part I. Role of molecular biology to improve the diagnosis of antibody-mediated rejection.

  cytotoxicity of surrounding monocytes (TNF-α, IL-1, IL-6) and ii) the expressions of CXCL9 and CXCL10, which facilitates the recruitment of new NK cells, -the production and release of pro-inflammatory chemokines supporting monocytes recruitment (CCL3 and CCL4), -the release of the content of NK cells granules (perforin, granulysin granzymes A and B) responsible for endothelial lesions via the antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism.

Figure 5

 5 Figure 5 summarizes current knowledge of the pathophysiology of AMR. In the era in which molecular tools play an important role in alloimmune pathology, knowledge of the different cellular and soluble effectors involved in AMR, shown below, is essential to understand the interpretation of transcriptomics data.

Figure 5 :

 5 Figure 5: Pathophysiology of antibody-mediated rejection[START_REF] Loupy | Antibody-Mediated Rejection of Solid Organ Allografts[END_REF] This figure summarizes the pathophysiology of antibody-mediated rejection in solid organ transplantation. Left side: complement-independent pathways; right side: complement-dependent pathways.

Figure 6 :

 6 Figure6: T-cell mediated rejection and antibody-mediated rejection-selective transcripts[START_REF] Halloran | Review: The transcripts associated with organ allograft rejection[END_REF].The dots represent individual microarray probe sets. The x axis illustrates the false discovery rate-adjusted P value for the association of each transcript with the diagnosis of rejection, with the fold change on the y axis. Red: top 30 TCMR-selective, blue: top 30 ABMR-selective, black: top 30 rejection-associated.

Figure 7 :

 7 Figure 7: Lack of standardization in the diagnosis of antibody-mediated rejection: strategies based on molecular biology techniques to refine AMR diagnosis. This figure summarizes the development of two techniques of molecular biology applied in our studies aimed at standardizing the diagnosis of AMR in heart transplantation. AMR means antibody-mediated rejection, EMB: endomyocardial biopsy, RT-MLPA: Reverse Transcriptase-Multiplex Ligase-dependent Probe Amplification.

Figure 8 :

 8 Figure 8: Principle of Multiplex Ligation-dependent Probe Amplification. Synthesis of the 5 steps of RT-MLPA : reverse transcription, hybridation, ligation, amplification and fragment size analysis. Example of application of RT-MLPA to the analysis of the expression of a 3-transcript set. Each couple of probes contains a specific sequence of the gene to analyze (green), a common primer sequence (red and orange) and a spacer of variable length (yellow).

Figure 9 :

 9 Figure 9: Flowchart of the RT-MLPA study 183 endomyocardial biopsies corresponding to 183 heart allograft recipients were selected for the study and randomized into a training (n=113) and a validation series (n=70). hACR: histological diagnosis of acute cellular rejection, hAMR: histological diagnosis of antibody-mediated rejection, hMixed: histological diagnosis of mixed rejection and hNR: histological diagnosis of non-rejection.

Figure 10 :

 10 Figure 10: Histopathology and RT-MLPA profiles A-C. Histopathology. A-Antibody-mediated rejection: Microcirculation inflammation defined by intravascular activated mononuclear cells accumulation in capillaries. B-Acute cellular rejection: perivenular lymphocytic infiltrates. C-Non-rejection EMB with no myocardial inflammation. D-F. RT-MLPA profiles. Molecular profile of antibody-mediated rejection (D) with high expression of CCL4, PLA1A, GNLY, CXCL9, FCGR3 and CXCL11 and of acute cellular rejection (E) with high expression of ADAMdec and CCL18 as compared to nonrejecting control EMB (F).

  clustering analysis and dendrograms of the molecular profiles obtained after RT-MLPA procedures were performed with the hcluster module of the amap package in the R software. A class prediction analysis was developed to assign each molecular profile obtained by RT-MLPA with a molecular diagnosis: mNR (molecular non-rejecting), mACR (molecular ACR) and mAMR (molecular AMR). A two-step linear predictor score (LPS) method was applied to train two consecutive Bayesian predictors. The first (LPS1) was built to identify rejection cases (rejection vs mNR) and the second (LPS2) to discriminate mAMR from mACR cases. Each LPS was first developed in the training series that was randomly divided into two subgroups. The first subgroup (⅔ of the profiles = training set) and the second subgroup (⅓ of the profiles = validation set) were used to create the model and to assess its internal validation, respectively. External validation was then performed using the validation series (Figure9). Statistical significance was set at p ≤ 0.05. All tests were two-sided. Statistical analyses were performed using STATA 15.0 software (StataCorp LP, College Station, TX) and R software.I-B-3. ResultsI-B-3-a. Patients characteristics and histopathologyThe clinical and histopathologic characteristics of the 183 included patients are summarized in Table2. In brief, patients were mostly males (66%) and mean age at transplant was 46.7 ± 14 years old. Almost three-quarters of the patients received an induction therapy based on anti-thymocyte globulin (ATG) infusions. The median time between heart transplantation and EMB was 60.7 ± 15 months, and 16% of EMB were performed in the six weeks following heart transplantation. Most of the patients were on triple immunosuppressive therapy at the time of EMB (calcineurin inhibitors, mycophenolate mofetil, and corticosteroids) and an anti-HLA DSA was present in 41.5% of the cases. Baseline characteristics were similar between the training and the validation series.

Figure 11 :

 11 Figure 11: Expression of transcripts according to histological diagnosis CCL4, PLA1A, CXCL9, FCGR3, CXCL11, and TN4SF18 were overexpressed in hAMR biopsies compared to histological non-rejection. CD8A, CXCL9, CCL18, and ADMAdec were overexpressed in hACR biopsies compared to histological non-rejection. ECSCR, ROBO4, and TM4SF18 were under expressed in histological rejection, either AMR or ACR, compared to histological non-rejection. hAMR: histological antibody-mediated rejection (n=82), hACR: histological cellular rejection (n=48), hNR: histological non-rejection (n=48). Mixed rejection (n=5) is not shown.

Figure 12 :

 12 Figure 12: Unsupervised hierarchical and class prediction analysis A. Unsupervised hierarchical analysis of RT-MLPA analysis from the 183 EMB. Histological diagnosis is provided on the upper line (Pathology). Three clusters of molecular patterns are delimited by the relative expression of the 14 selected transcripts. The lower line (RTMLPA) provides the molecular diagnosis according to the two steps in the class prediction analysis. Unclassified: Cases unclassified by LPS1. Unclassified rejection: cases classified as rejection by LPS1 but unclassified by LPS2 (see below). B. The principle of the RT-MLPA prediction model for rejection. The RT-MLPA assay consists of two consecutive linear prediction scores (LPS). The first LPS is designed to identify rejection cases among all EMB, whereas the second is designed to discriminate mAMR from mACR. LPS means Linear prediction score, mACR: molecular acute cellular rejection, mAMR: molecular antibody-mediated rejection, mNR: molecular non-rejection.

Figure 13 :

 13 Figure 13: Class Prediction Analysis A and B: Development and internal validation of LPS1. The most discriminant transcripts between mNR and rejection were ROBO4, ECSCR, TM4SF18, CCL4, CXCL11, and CXCL9. C and D: Development and internal validation of LPS2. The most discriminant transcripts between mAMR and mACR were CCL18, CD74, CD8A, ADAMDEC1, PLA1A, TM4SF18, and CXCL11. The confidence threshold to classify a sample was set at 90 percent.

  14 selected transcripts (endothelial burden, NK cells, interferon-γ pathway, effector Tcell and antigen presentation) on 183 EMB, the unsupervised analysis identified three clusters of gene expression. A two-step class prediction analysis was developed to assign each molecular profile obtained by RT-MLPA with a molecular diagnosis: molecular non-rejection (mNR), mACR and mAMR. Overall agreement between RT-MLPA (mNR, mACR and mAMR) and histopathology (hNR, hACR and hAMR) were 92.2%, 79.2% and 76.8% for the diagnosis or rejection, the type of rejection and the final diagnosis, respectively.

  setting of a clinical transplant practice. A signature of 14 transcripts could distinguish between ACR, AMR, and absence of rejection. Integration of molecular assays to conventional histopathology represent the next step for the development of a reliable gold standard of rejection diagnosis.Chapter I-C: Extension of intravascular activated mononuclear cells correlates with antibodymediated rejection molecular activity, donorspecific antibodies and graft function.I-C-1. IntroductionThe 2013 International Society for Heart and Lung Transplantation working formulation for pathologic diagnosis of heart allograft AMR (pAMR classification) includes the presence of microvascular inflammation (MVI) as a major diagnostic criterion. Thus, the term "intravascular activated mononuclear cells" (IAMC) was chosen for cardiac MVI to take into account both swollen activated endothelial cells and intravascular inflammatory cells accumulation in microvascular lumens. In the pAMR classification, significant IAMC was defined as "more than occasional focal aggregates or scattered isolated foci of intravascular activated mononuclear cells" and thus no definitive threshold for IAMC has been proposed. Experiences from kidney transplantation have suggested that the assessment of the severity of MVI on kidney allograft biopsies (glomerulitis and peritubular capillaritis) is of interest not only to define a threshold for the pathologic diagnosis but also to evaluate the disease activity.A similar evaluation of MVI could be a valuable tool in heart transplantation. As a prerequisite, we conducted the present study to evaluate the biological and clinical meanings of the extension of MVI in endomyocardial biopsies (EMB) using a semiquantitative score. Our aim was to determine whether the MVI extension scale might be associated with AMR molecular activity, current rejection classifications, DSA and graft function at the time of EMB.I-C-2. Patients and methodsI-C-2-a. Study designAn assessment of the extension of microvascular inflammation using a semiquantitative scoring system was retrospectively performed in 398 endomyocardial biopsies from 268 heart transplant recipients. All EMB had molecular studies, either by microarray analysis (n=213)[START_REF] Halloran | Building a tissue-based molecular diagnostic system in heart transplant rejection: The heart Molecular Microscope Diagnostic (MMDx) System[END_REF][START_REF] Loupy | Gene Expression Profiling for the Identification and Classification of Antibody-Mediated Heart Rejection[END_REF], or by targeted gene expression analysis on FFPE EMB (see chapter RT-MLPA). The extension of MVI was then compared i) to molecular rejection signatures, ii) to histopathology according to the last ISHLT classifications, iii) to the DSA status and iv) to the left ventricular ejection fraction (LVEF) at the time of the EMB.I-C-2-b. PatientsTwo-hundred and sixty-eight heart recipients from five referral French centers transplanted between January 2004 and June 2017 were included. All recipients underwent routine protocol EMB according to a predetermined schedule or for-cause EMB. Data were collected for donors' and recipients' gender and age, primary heart disease, cold ischemic time, and induction therapy. Donor specific antibody was evaluated by Luminex technology (Luminex LABScreen Single Antigen, One-Lambda Inc) at the time of the endomyocardial biopsy as previously described[START_REF] Loupy | Gene Expression Profiling for the Identification and Classification of Antibody-Mediated Heart Rejection[END_REF]. An MFI value ≥ 500 were considered positive. We selected the highest rank of class I or II DSA MFI (immunodominant DSA). Left ventricular (LV) dysfunction at the time of EMB was defined as an unknown left ventricular ejection fraction (LVEF) < 50 % (1) and/or a drop in LVEF ≥ 15 % compared to baseline LVEF.

  I-C-2-d. Evaluation of the extension of MVI: MVI score All EMB (n=398) were assessed retrospectively for the extension of MVI by 3 pathologists, two senior heart transplant pathologists and a fellow transplant pathologist. The extension of MVI was evaluated semi-quantitatively using the percentage of the area with microvascular inflammation in capillaries and venules as described in Figure 14: score 0 representing 0% of MVI on the area (negative MVI); score 1 representing 0-10% of MVI on the area (minimal MVI); score 2 representing 11-50% of MVI on the area (focal MVI); score 3 representing >50% of MI on the area (diffuse MVI, Figure 14). Area of previous biopsy site, of replacement fibrosis and in close contact with foci of cellular rejection were excluded for the evaluation of MVI. All EMB were assessed independently by the 3 readers who were blinded for clinical data and for the initial histopathology diagnosis. In case of discrepancies between the readers, the slides were reviewed by the 3 readers altogether to achieve a final consensual score. I-C-2-e. Molecular pathology

Figure 14 :

 14 Figure 14: Semi-quantitative assessment of microvascular inflammation using the MVI score. A: MVI score 0, absence of microvascular inflammation (insert show no inflammatory cell in capillaries); B: MVI score 1, minimal microvascular inflammation defined by presence of inflammatory cells in the capillaries and venules in 10 and less percent of the surface of the biopsy; C: MVI score 2, focal microvascular inflammation defined by presence of inflammatory cells in the capillaries and venules in more than 10 percent and less than 50 percent of the surface of the biopsy; D: MVI score 3, diffuse microvascular inflammation defined by presence of inflammatory cells in the capillaries and venules in 50 and more percent of the surface of the biopsy. Inserts in B-D show similar numbers of inflammatory cells in the most affected vessel. (H&E stain x50 and x200 in insert).

  Figure 15 gives the results of the molecular analysis according to the extension of MVI (MVI score) using microarrays (15-A/B) or RT-MLPA (15-C/D) analysis. We first tested whether the selected PBT, AMR and TCMR classifiers were correlated to the extension of MVI (MVI score). Regarding microarrays analysis, as shown in the Principal Component Analysis (Figure 15-A), most of the cases with MVI grade 0 and MVI grade 1 clusterized on the left side of the PCA while MVI grade 2 and MVI grade 3 clusterized on the right side of the PCA and were strongly driven by the PBT associated with AMR (ENDAT, DSAST, GRIT1, NK and QCMAT) and the AMR classifier (probABMR). Values of individual PBT and classifiers according to MVI score are given in Figure 15-B. ENDAT, DSAST, GRIT1, QCMAT, NK and TCB values strongly increased with the MVI score. MVI score was significantly associated with AMR classifier (probABMR) but not with ACR classifier (probTCMR).

Figure 15 :

 15 Figure 15: Molecular correlates of extension of microvascular inflammation MVI score. A-B: Microarray analysis: A: Unsupervised principal component (PC) analysis of MVI based on molecular profiles. Each dot represents one of 231 EMB samples for the microarray analysis. They are colored by their MVI scores and distributed by their molecular features as determined using principal component analysis. The principal component analysis is based on six PBT (ENDAT, DSASR, GRIT1, NK, QCMAT and TCB) and one classifier score (probABMR). PC1, principal component 1 (accounts for x% of total molecular variance); PC2, principal component 2 (accounts for x% of total molecular variance); B: Individual results for intragraft expression of pathogenesis based transcripts (PBT: DSAST, ENDAT, GRIT1, QCMAT, TCB) and AMR and ACR classifiers (probABMR and probTCRM) according to MVI score (MVI 0: n=158; MVI 1: n=26; MVI 2: n=38; MVI 3: n=10; median and SEM) ; C-D: RT-MLPA analysis: C: Median of geometric mean of AMR-associated transcripts according to MVI score; D: Median of geometric mean of ACR-associated transcripts according to MVI score (MVI 0: n=109; MVI 1: n=18; MVI 2: n=23; MVI 3: n=35; median and min-max values). Statistical significances are *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001.

Figure 16 :

 16 Figure 16: Relationship between Extension of microvascular inflammation (MVI score) and ISHLT classifications of rejection, MFI of immunodominant DSA and impaired graft function at the time of endomyocardial biopsy. A: Distribution of MVI grades according to rejection categories based on ISHLT ACR and pAMR classifications in 398 EMB: MVI is strongly associated with pAMR1(h+) and pAMR2/3 categories and was absent in pure cellular rejection ACR 2R/3R. (Details (n in each categories and statistical significance) are presented in Table 5); B: Relationship between MVI score and DSA: Evaluation of DSA concomitant to the EMB was available in 281 biopsies. MFI of the Immunodominant DSA (MFI ID DSA) were grouped according to 4 categories (negative, 500-2,999, 3000-9,999 and >10,000). The percentage of each MFI ID DSA category is given for each MVI score. High MVI scores (MVI 2 and MVI 3) are enriched in cases with high MFI ID DSA (3000-9,999 and >10,000 categories), (p<0.001, the number of cases is given on the top of each column). C: Relationship between MVI score and graft function: Assessment of cardiac function by measurement of Left ventricular ejection fraction (LVEF) at the time of EMB was available in 378 cases. The percentage of cases with cardiac dysfunction (LVEF below 50) is given in each MVI score (p<0.001, the number of cases is given on the top of each column).

  This study must be interpreted in the context of its limitations. Although multicentric for the selection of cases, MVI scoring was only performed by 2 senior readers from the same research team. Thus, the question of reproducibility of the MVI score should be further evaluated in prospective unselected studies, together with the determination of a cut-off of MVI for the diagnosis of AMR and the assessment of the evolution of MVI score after rejection therapies.I-C-4. ConclusionThis study demonstrated that the extension of MVI evaluated according to a semiquantitative score was associated with AMR molecular activity, DSA and graft dysfunction. The MVI score gave additional information to the current pAMR classification, although our study was not designed to answer the question of the prognostic impact on graft survival of the MVI score.Chapter I-D: Synthesis of part 1 -molecular biology in heart transplantation.Through the application of two distinct molecular biology approaches, we have been able to demonstrate the potential contribution of these techniques to the diagnosis of heart allograft rejection, particularly AMR. We have shown (i) that RT-MLPA, a technique for the analysis of expression profiles of preselected transcripts, was applicable to FFPE-EMB, that the concordance between unsupervised molecular and pathologic diagnoses was good and that the discordant cases could provide materials for discussion in the difficult diagnosis of allograft rejection; and (ii) that these molecular biology techniques could be of interest to refine the pathologic diagnosis of AMR by highlighting the link between the extension of microvascular inflammation in EMB and AMR molecular activity, thus validating a pathologic 4-scale classification of microvascular inflammation (Figure17).These studies illustrate our belief that molecular pathology and histopathology could be complementary to progress towards a histo-molecular phenotype of rejection that might be one of the keys to a better understanding and a homogenization of the diagnosis of AMR across centers.

Figure 17 :

 17 Figure 17: Summary of results of molecular biology studies.

Figure 18 :

 18 Figure 18: Objectives of the part 2: risk stratification.

A- 1 .

 1 Identification of prognostic factors using Cox regression model II-A-1-a. Cox semi-parametric model This model was introduced in 1972 by David Cox and remains the most used model in right-censored survival analyzes and medical studies. This model takes into account several explanatory variables to explain the occurrence of an event (parametric part) without making assumptions on the survival distributions and the hazard function (nonparametric part). The model expresses the hazard function as the product of a basic hazard function ℎ 0 (𝑡𝑡) common to all individuals and considered as an unknown function, and a parametrically explicit regression function, exp (𝛽𝛽 ′ 𝑍𝑍) where 𝛽𝛽 ′ is a pvector of unknown regression coefficient. The proportional risk model of Cox is written as: ℎ(𝑡𝑡, 𝑍𝑍) = ℎ 0 (𝑡𝑡) exp (𝛽𝛽 ′ 𝑍𝑍)

  A-1-d. Construction of prognostic tools based on integrative models After having identified the factors independently associated with the event and the additive value of new factors, the final step is the development of adequate prediction tools to enable risk stratification. A score is created according to the ß coefficients attributed by the final multivariate model to each variable. These coefficients represent the relative and individual contribution of the variables to the survival distribution. II-A-2-e. Statistical model performance The analysis of the performance of the model involves the study of its calibration and discrimination, already discussed in the previous paragraphs.

  risk factors for the development of CAV have been described. This literature has been enriched by the use of endocoronary imaging techniques, which are able to detect with a high sensitivity minimal changes in intimal thickness between two tests. Several syntheses of this abundant literature have been published (82). These CAV risk factors are traditionally categorized into (i) traditional cardiovascular risk factors of the donor responsible for transmitted atherosclerosis (83) (age, gender, diabetes, tobacco consumption etc. (84,85)); (ii) traditional cardiovascular risk factors of the recipient after transplantation (especially dyslipidemia (86)); (iii) non-immunological transplant-specific factors (ischemic time, cause of death of the donor, CMV infection (87,88)); and iv) immunological transplant-specific factors (acute cellular rejections, AMR, DSA (19,20,84,89)).Some authors highlight that most of the studies analyzing CAV risk factors ultimately focused on the study of a limited number of potential factors, limiting the impact of their results[START_REF] Langstraat | Coronary artery disease in heart transplantation: new concepts for an old disease[END_REF]. The epidemiological approach usually applied to the determination of risk factors for CAV is dichotomous (presence or absence of the disease, evolution or no change in intimal thickness), sometimes taking into account the delay in onset of the disease but neglecting the evolutionary profiles of the disease.We therefore conducted a systematic review of the published literature for studies of trajectory-based assessment of CAV in heart transplant recipients. We searched PubMed for publications from January 1st, 2008 until June 30th, 2019 using the terms (((cardiac allograft vasculopathy) OR ((cardiac allograft vasculopathy) AND "Risk

Figure 19 :

 19 Figure 19: Design of the study. Pre-transplant, day of transplant, and early post-transplant data were collected to identify the risk factors for biopsyproven rejection during the first-year post-transplant. II-B-2.b. Anti-HLA antibodies screening and endomyocardial biopsies

Figure 20 :

 20 Figure 20: Repartition of (A) overall EMB and (B) rejecting-EMB according to the time post-transplant.

Figure 21 :

 21 Figure 21: Evolution of the mean number of EMB per patient performed during the first-year post transplant.

Figure 22 :

 22 Figure 22: Epidemiology of allograft rejection during the first year after heart transplantation A-Incidence of first significant-rejection (ACR ≥ 1R1B and / or AMR ≥ pAMR1). B-Incidence of the first acute cellular rejection ≥ 1R1B. C-Incidence of first antibody-mediated rejection ≥ pAMR1. D-Dynamic prevalence of rejection during the first year according to the time post-transplant. Interpretation: for example, the probability of finding significant signs of rejection on an EMB performed 3 months after the transplantation is about 10%, at 8 months post-transplant, about 5%.

Figure 23 :

 23 Figure 23: ACR ≥ 1R1B and/or AMR ≥ pAMR1: discrimination of the Cox model.

II-B- 3

 3 -e. Sensitivity analyses Different sensitivity analyzes were conducted. First, a stratification over the period (2004 to 2008, 2009 to 2011 and 2012 to 20016) did not change the final model, including the effect of pre-formed DSA on the risk of rejection: recipient's age (per 10year increment: HR = 0.92, 95% CI = 0.85-0.97, p = 0.005), type of transplant (combined compared to isolated heart transplants: HR = 0.25, 95% CI = 0.14-0.48, p <0.001), type of induction (IL-2R inhibitors compared to ATG: HR = 1.58, 95% CI = 1.16-2.14, p = 0.004), number of HLA A-B-DR mismatches (per 1 mismatch increment: HR = 1.17, 95% CI = 1.06-1.29, p = 0.001) and presence of pre-formed DSA (HR = 1.60, 95% CI = 1.32-1.95, p <0.001). Similarly, the stratification on the center did not modify the final multivariate model, either in terms of statistical significance or the significance of the effect (HR and OR).

  CAV have been limited by registry data including convenience samples with limited number of measures for a single patient. Importantly no study to date has been primarily designed towards a prospective protocol based and standardized assessments of CAV together with thoroughgoing patient characterization including immunological and biological profile, histological phenotypes and information on treatments. To avoid making any prior assumptions on the number and shapes of CAV trajectories, we used a contemporary unsupervised trajectory-based approach known as latent class mixed modeling (101). This model has already demonstrated its clinical relevance in different medical settings such as depression, atherosclerosis, or disability by revealing unrecognized profiles of patients and by contributing to a deeper understanding of the evolution in time of chronic diseases (102-104).

II-C- 3

 3 -b. Identification and characterization of CAV trajectories in the derivation and external validation cohorts Derivation cohort. We identified 4 mutually exclusive CAV trajectories over 10 years after transplantation (Figure 25); the model showed a good discrimination of 0.92 (0.96, 0.83, 0.93 and 0.88 for trajectory#1,#2,#3 and #4, respectively) and entropy of 0.82, meaning that the model adequately separated the trajectories. External validation cohort. Latent class mixed models were applied in the independent external validation cohort from US. The best fitting model identified 4 profiles of CAV trajectories and confirmed the consistency of the 4 profiles previously demonstrated in the derivation cohort (Figure 26). In this geographically distinct cohort, the model showed an excellent discrimination of 0.97 (0.93, 0.97, 0.88 and 0.92 for trajectory#1,#2,#3 and #4, respectively). CAV trajectory#1 was composed of patients with no CAV at baseline that remained stable over time (n=823, 63.26%, CAV slope per year = 0.00±0.01). CAV trajectory#2 was composed of patients with no CAV at baseline, with late onset increase of CAV overtime starting at around four years post-transplant (n=79, 6.07%, overall CAV slope per year = 0.11±0.12). CAV trajectory#3 was composed of patients with intermediate CAV grade at 1 year who experienced moderate increase during follow-up (n=261, 20.06%, CAV slope per year = 0.18±0.16) Patients from CAV trajectory#4 presented with a pattern of intermediate baseline CAV and acceleration over time (n=138, 10.61%, CAV slope per year = 0.40±0.50).

Figure 25 .

 25 Figure 25. Distinct profiles of CAV trajectories identified post-transplantation in the European derivation cohort (n=815). This figure represents the main profiles CAV grades identified with latent class mixed models. Each patient, represented by an individual CAV trajectory, is assigned to the class for which the membership probability is the highest. Discrimination = 0.92. CAV trajectory#1 was composed of patients with no CAV at baseline that remained stable over time; CAV trajectory#2 was composed of patients with no CAV at baseline, with slight increase of CAV overtime starting around four years post-transplant; CAV trajectory#3 was composed of patients with intermediate CAV grade at 1 year who experienced moderate increase during follow-up; Patients from CAV trajectory#4 presented with a pattern of intermediate baseline CAV and acceleration over time. Thick lines represent latent class trajectory; thin lines represent CAV individual patient trajectory.

Figure 26 :

 26 Figure 26: Cardiac allograft vasculopathy trajectories: external validation in the American cohort.Latent class mixed models were applied to the American validation cohorts. CAV profiles identified in these independent analyses were similar to those identified in the derivation cohort with an excellent discrimination of 0.97. Thick lines represent latent class trajectory; thin lines represent CAV individual patient trajectory.

  p=0.02), 4 immunological parameters (presence of preexisiting or de novo anti-HLA DSA: p=0.007; class II immunodominant DSA: p=0.025; cellular rejection grade≥ 2R occurring during the first year post transplant: p=0.042; episode of AMR occurring during the first year: p=0.049).

  II-C-3-d. Projection of patient individual 10 years CAV trajectories based on determinants assessed at the time of transplant and in the first year after transplantBased on the determinants identified in previous analyzes, we built an online interfacing to provide clinicians with a ready-to-use tool that projects the /transplantpredictionsystem.shinyapps.io/personalized_CAV_trajectories/).Clinicians can enter individual parameters of one patient and 4 likelihoods of belongingfor each trajectory are provided, corresponding to the personalized likely future CAV progression of the patient, as well as the corresponding allograft survival and the mean slope of CAV per year of the predominant CAV trajectory.

Figure 27 :

 27 Figure 27: Description of overall mortality according to the CAV trajectory in the overall cohort (n=1,301).

  overwhelming task for clinical practitioners to perform in the future. II-C-4-e. Clinical trials Although not validated by American and European drug regulatory authorities, CAV is a major clinical endpoints for past and ongoing clinical trials in heart transplantation since this pathology represents one the leading cause of death or retransplantation late after transplant (JHLT 2018 annual report). Endocoronary imaging modalities, either intravascular ultrasound or optical coherence tomography, have the ability to detect early coronary lesions while the coronary angiogram remains normal. The change in maximal intimal thickness from baseline to one year or from one to five year post-transplant has been associated with mortality, nonfatal major adverse cardiac events and development of angiographic CAV (15,16). All not-withdrawn CAV-focused studys registered on clinicaltrials.com (n=18) have an IVUS/OCT endpoint. However, endocoronary imaging procedures have specific complications and are not available in all centers.(107) Moreover, coronary angiography coupled with assessment of cardiac aloograft function maintains the highest level of evidence for CAV monitoring according to ISHLT guidelines.(108) We propose the possibility that the latent class

Figure 28 :

 28 Figure 28: Synthesis of the results of the epidemiological studies.

Figure 29 :

 29 Figure 29: Better understanding of outcomes of at-risk populations.

Figure 30 :

 30 Figure 30: Study protocol. Study protocol and evolution of repartition in the group with preformed donor-specific anti-HLA antibodies (pfDSA) between historical sera and day 0 serum. The initiation of treatment was based on the mean fluorescence intensity (MFI) values of immunodominant DSA in historical sera and readjusted according to the results for day 0 serum. SOC means Standard of Care.

Figure 32 .

 32 Figure 32. Evolution of MFI after peri-operative management in patients with preformed donor-specific anti-HLA antibody (pfDSA) mean fluorescence intensity (MFI) levels >1000. Evolution of MFI after peri-operative management in patients with preformed donor-specific anti-HLA antibody (pfDSA) mean fluorescence intensity (MFI) levels >1000. Evolution of MFI of DSA between day 0 and postplasmapheresis sera (A, B, n = 139, missing values: n = 55) and between day 0 and one-month post-transplant sera (C, D, n = 153 patients, missing values: n = 41). (A, C: immunodominant DSA; B, D: cumulative MFI of DSA).Categories are determined according to the MFI at the day of transplant (immunodominant or cumulative). The evolution of MFI after plasmapheresis and at one-month is described in each subgroup.

Figure 33 .

 33 Figure 33. Primary and secondary outcomes. (A) Survival after heart transplantation (n = 523). (B) Biopsy-proven rejection-free survival (cellular rejection ≥2R and antibody-mediated rejections; n = 523). Survival was not significantly different between groups (p=0.92). Rejection-free survival was worse in patients with pfDSA MFI >1000 (p=0.05).

Figure 34 .

 34 Figure 34. Incidence of biopsy-proven antibody-mediated rejection (ABMR) (deaths not shown).

Figure 35 .

 35 Figure 35. Exploratory analysis in the group of patients with preformed donor-specific anti-HLA antibody (pfDSA) mean fluorescence intensity (MFI) levels >1000. (A) Overall survival after heart transplantation. (B) Biopsy-proven rejection-free survival (cellular rejection ≥2R and antibody-mediated rejection) according to day 0 MFI (n = 194). While survival did not significantly differ between subgroups (p = 0.37), patients with MFI >5000 experienced worse rejection-free survival than other patients (p = 0.01).

A

  post-transplant prophylactic strategy including perioperative management of DSA and systematic treatment of subsequent ABMR achieved favorable outcomes after HTx in patients transplanted with pfDSA.Chapter III-B: Antibody-mediated rejection induced cardiogenic shock: too late for conventional therapy.Article published in Clinical Transplantation 2018 May;32[START_REF] Shah | Left ventricular assist devices versus medical management in ambulatory heart failure patients: An analysis of INTERMACS Profiles 4 and 5 to 7 from the ROADMAP study[END_REF]:e13253[START_REF] Coutance | Antibody-mediated rejection induced cardiogenic shock: too late for conventional therapy[END_REF]

  III-B-2-a. PatientsWe performed a retrospective, single center, observational study. We included all consecutive patients with (i) biopsy proven diagnosis of AMR and (ii) severe allograft dysfunction responsible for cardiogenic shock requiring inotropic support and / or temporary mechanical circulatory support. Our institutional review board approved the protocol and informed consent was obtained.III-B-2-b. ObjectivesOur objectives were to analyze the characteristics, treatment and prognosis of patients treated for cardiogenic shock due to biopsy-proven AMR requiring inotropic support and / or temporary mechanical circulatory support at our center.III-B-2-c. Definition of AMR and cardiogenic shockThe diagnosis of acute AMR was based on the presence of histological evidence of AMR on endomyocardial biopsies according to ISHLT guidelines (pAMR 1 to 3) (112). Cardiogenic shock was defined according to ESC guidelines on acute and chronic heart failure as hypotension (SBP ≤ 90 mmHg) despite adequate filling status with signs of hypoperfusion (cold sweated extremities, oliguria, mental confusion, dizziness, narrow pulse pressure; laboratory measures: metabolic acidosis, elevated serum lactate, elevated serum creatinine) (1). All patients received inotropic support based on dobutamine infusion. In case of cardiac arrest or refractory cardiogenic shock with escalating inotropic requirements a temporary mechanical circulatory supportextracorporeal membrane oxygenation (ECMO) -was implanted usually through femoral vein and artery according to current standards (bridge to recovery strategy) (130). III-B-2-d. Immunology The detection of pre and post-transplant anti HLA antibodies was based on Luminex Single Antigen beads technology (One Lambda, Canoga Park, CA). The fluorescence of each bead is detected by a reader (LABscan) and recorded as the normalized Mean Fluorescence Intensity (MFI). Beads showing MFI > 500 were considered as a positive result. HLA typing of heart recipient was performed by low-resolution class I HLA-A, -B, and class II HLA-DRB1, -DQB1 PCR-SSO (LABType, One Lambda). Donors HLA-A, B, DR, DQ typing was performed by CDC (One lambda tissue-typing trays) and controlled by molecular biology. Our routine monitoring protocol includes DSA quantification: #1 at 3, 6, 12 months post-transplant and then once a year in patients transplanted with pre-formed DSA and #2 every year in other patients. III-B-2-e. Immunosuppressive protocol Immunosuppressive therapy at the time of transplantation was based on an induction therapy with anti-thymocyte globulin (rabbit ATG, Thymoglobuline, Genzyme, Lyon, France). Prophylactic immunosuppressive treatment usually included calcineurin inhibitors, mycophenolate mofetil and corticosteroids with posology as recommended by ISHLT guidelines (115).

  EMB after 5 years of transplantation are performed when clinically indicated. Standard serial sections were cut from formalin-fixed paraffin-embedded EMBs and stained with hematoxylineosin-safran for diagnosis. Immunofluorescence for C4d was performed on all reference and post-AMR EMBs. Only capillary staining for C4d was assessed. All EMB were retrospectively reviewed by an expert pathologist (blinded review, inclusion of control biopsies) and classified as cellular (0 to 3R) and AMR (pAMR 0 to 3) according to the ISHLT guidelines (112,113).III-B-2-g. Cardiac allograft vasculopathyAt our institution, routine coronary angiograms are performed one year after heart transplantation and then every two years or in case of unexplained LV dysfunction.Staging of CAV was performed according to the recommended ISHLT nomenclature[START_REF] Mehra | International Society for Heart and Lung Transplantation working formulation of a standardized nomenclature for cardiac allograft vasculopathy-2010[END_REF].III-B-2-h. Statistical analysesStatistical analyses were performed with Stata/IC 14.2 (StataCorp). Results are presented as median and inter-quartile range (IQR) for continuous variables and as number and percentage for categorical variables. Cumulative survival curves for the time-to-event analyses were constructed according to the Kaplan-Meier method.Statistical significance was determined if the null hypothesis could be rejected at the p < 0.05 level.III-B-3. ResultsIII-B-3-a. Characteristics of included patientsSeventeen patients were diagnosed with AMR with cardiogenic shock requiring dobutamine infusion and/or ECMO support at our center. Their characteristics are detailed in Table21. Patients were transplanted from 1994 to 2015. Rejections were diagnosed from 2008 to 2016. During this period of time, 740 heart transplantations were performed at our center (13 of 17 patients of our cohort were transplanted during this interval). 883 patients are currently followed in our transplant unit. The incidence of AMR induced cardiogenic shock could then be estimated around 1.7% of heart transplant recipients (=13/740). Patients were mostly males (n = 12/17, 70 %). Median age at diagnosis was 55.7 years (17-69 years). Median time between heart transplantation and AMR was 21.5 months (10-64 months). Four patients were transplanted with MFI > 3,000 preformed DSA. Only 2 patients (12 %) had past history of biopsy proven AMR. All patients were treated with calcineurin inhibitors (CNI) and corticosteroids (CS); most of them received mycophenolate mofetil on top of CNI and CS (82 %) at the time of rejection. Eight patients (47%) required renal replacement therapy during the treatment of AMR induced cardiogenic shock. III-B-3-b. Diagnosis and treatment of AMR According to the inclusion criteria, all patients had severe graft dysfunction at the time of diagnosis of AMR. Median LVEF at diagnosis was 28 % (5 to 34 %). All patients had circulating anti-HLA DSA. Immunodominant DSA was a high-level class II de novo DSA in 76 % of cases (n = 13/17). Pre-formed DSA were present at the time of rejection in patients 1 and 2 but with lower MFI values compared to de-novo DSA. Median MFI of immunodominant DSA and cumulative MFI of DSA were respectively 10,895 (6,375-15,321) and 18,275[START_REF] Shah | Surveillance Endomyocardial Biopsy in the Modern Era Produces Low Diagnostic Yield for Cardiac Allograft Rejection[END_REF]907). All EMBs were performed for acute allograft dysfunction (for-cause EMBs). Histological analyses of EMB revealed features of AMR in all patients (pAMR 2: n = 3; pAMR1 (H+): n = 11; pAMR1 (I+): n = 3). Two patients had concomitant 2R3A acute cellular rejection and 5 patients 1R cellular rejection.Treatments of AMR are detailed in Table21. In brief, all patients received high dose IV corticosteroids pulses, plasmapheresis (5 to 15 plasmaphereses per patient), and high dose IVIg. Two patients died before the initiation of plasmapheresis and five before IVIg. On top of this standard regimen, 12 patients received add-on therapies: 10 patients were treated with rATG and 6 received at least one infusion of rituximab. Four of the six patients with "dobutamine infusion-only" group did not receive any complementary treatment. III-B-3-c. ECMO support Eleven patients presented with refractory cardiogenic shock requiring ECMO support at the diagnosis of AMR or during follow-up (LV decline despite treatment). Eight patients died under ECMO support after a median time of circulatory support of 13 days (3 to 30 days). ECMO was successfully weaned in two patients after respectively 13 and 45 days of support. The remaining patient died shortly after total artificial heart surgery. ↓ Table 21: Characteristics of included patients. ACR means Acute Cellular Rejection, AMR: Antibody-Mediated Rejection, ATG: anti-thymocyte globulin, CAV: Cardiac Allograft Vasculopathy, DSA: donor-specific anti-HLA antibodies, EF: Ejection Fraction, EMB: Endomyocardial Biopsy, ID: Immunodominant, LVEF: Left Ventricular Ejection Fraction, MFI: mean fluorescence intensity, MI: Myocardial Infarction, MOF: multi-organ failure, yr: year, TAH: Total Artificial Heart, AZA: azathioprine, CS: corticosteroids, CYA: Cyclosporine A, EVL: Everolimus, MMF: Mycophenolate mofetil, TAC: Tacrolimus, TAH: total artificial heart.

Figure 36 :

 36 Figure 36: Survival after antibody-mediated rejection with cardiogenic shock. Time from AMR is indicated in days.

Figure 37 .

 37 Figure 37. Summary of the results of clinical studies.

Table 2 :

 2

Characteristics of patients and endomyocardial biopsies

hACR means histological diagnosis of acute cellular rejection, mAMR: histological diagnosis of antibody-mediated rejection, hMixed rejection: histological diagnosis of mixed rejection, IS regimen: immunosuppressive regimen.

Table 3

 3 details the results of the agreement between the histopathology and RT-MLPA in the training series (n=97), validation series (n=57), and overall cohort (n=154) after exclusion of the cases left unclassified by the class prediction analysis. RT-MLPA and histopathology agreed for the diagnosis of rejection in 91.8%, 82.9%, and 92.2% of the EMB in the training, validation, and overall cohort, respectively. In rejecting cases, an agreement for the type of rejection (AMR or ACR) was achieved in 86.7%, 66.7%, and 79.2% of the EMB in the test (n=60), validation (n=36), and overall cohort (n=96), respectively. Finally, RT-MLPA and histopathology agreed for a final diagnosis (No rejection, AMR, or ACR) in 82.2%, 66.7%, and 76.8% of the EMB in test, validation and overall cohort, respectively.

	Agreement for the diagnosis of rejection
	-Test series (n=97)	89 (91.8%)
	-Validation series (n=57)	53 (82.9%)
	-Overall (n=154)	142 (92.2%)
	Agreement for the type of rejection
	-Test series (n=60)	52 (86.7%)
	-Validation series (n=36)	24 (66.7%)
	-Overall (n=96)	76 (79.2%)
	Agreement for the final diagnosis
	-Test series (n=90)	74 (82.2%)
	-Validation series (n=48)	32 (66.7%)
	-Overall (n=138)	106 (76.8%)

Table 3 :

 3 Agreement between histopathology and RT-MLPA

Table 4 :

 4

Details of disagreement between histopathology and RT-MLPA

ACR means acute cellular rejection, AMR: antibody-mediated rejection, mACR: molecular diagnosis of acute cellular rejection, mAMR: molecular diagnosis of antibody-mediated rejection, mNR: molecular diagnosis of nonrejection.

Table 5 :

 5 Characteristics of patients and of endomyocardial biopsies.

Table 6 :

 6 -transplant (p=0.46). After adjustment for the presence of DSA, ACR, the delay between transplantation and the type of EMB, the MVI grade remained an

	time of the biopsy. Forty-eight patients (48/378, 12.7%) were diagnosed with left
	ventricular (LV) dysfunction. The percentage of cases with LV dysfunction increased
	with the extension of MVI (5.8%, 18.9%, 18.5%, and 37% for MVI score 0, 1, 2 and 3,
	respectively, p<0.001, p for trend < 0.001, Figure 16-C). Table 7 gives the main

Clinical and pathologic characteristics according to MVI score. ACR means acute cellular rejection, AMR: antibody-mediated rejection, EMB: endomyocardial biopsy, ID DSA: immunodominant donor-specific antibody, MVI: microvascular inflammation. * Effect of ACR grade in each sub-group of AMR grade. ** Effect of AMR grade. I-C-3-e. MVI score and graft function Finally, we assessed the association between the MVI score and graft function at the characteristics of EMB with concomitant LV dysfunction. Graft dysfunction was more frequent in "for cause" EMB and was significantly associated with MVI grade (p<0.001), pAMR grade (p<0.001), and the presence of DSA (p<0.001), but not with ACR (p=0.28) and time post

Table 7 :

 7 MVI, recognized at the microscopic level reflects pathogenic processes resulting from DSA interaction with the graft endothelium. These processes include endothelial activation, gamma-interferon response, and leukocyte infiltration with high levels of macrophages, T-cells and NK-cells transcripts. Interestingly, the level of expression of these molecular markers are up-regulated as early as MVI 1 and raised with MVI scores 2 and further increased with MVI score 3, suggesting that histological and molecular activities evolve in parallel. This would substantiate the acceptance of the MVI score as a marker of activity of heart allograft AMR. This is reinforced by the association of the MVI score with C4d and CD68 staining and by the fact that EMB with high MVI scores had higher MFI of immunodominant DSA. Finally, MVI score was associated with graft dysfunction as assessed by a LVEF below 50% at the time of the index EMB.In the present study, we did not analyze the cellular composition of the inflammatory infiltrate, which might be important point for a precise histopathologic phenotyping. We have previously demonstrated that the type of inflammatory cells within the microvascular compartment may greatly vary in both heart and kidney AMR with various proportions of macrophages, T and B lymphocytes and NK cells. Such a heterogeneity in the cell infiltrate may reflect different subtypes of AMR and should be investigated in the future. Recent advances in histopathology, such as multiplex immunofluorescence analysis, that allows analysis of several labelled cell types on the same slide, cellular compartmentalization and computer assisted cell counting, may be of great interest to progress on this particular point. Correlation with molecular analysis performed on the same EMB will be warranted.

	The present study demonstrates that the severity of MVI, based on a semi-quantitative
	evaluation of its extension has biological and clinical meanings. MVI score was
	associated with AMR molecular activity, pAMR classification, DSA and graft function

Clinical and pathologic characteristics according to the presence of a left ventricular dysfunction at the time of endomyocardial biopsy.

ACR means acute cellular rejection, AMR: antibody-mediated rejection, EMB: endomyocardial biopsy, DSA: donorspecific antibody, MVI: microvascular inflammation.

I-C-4. Discussion

at the time of EMB. Using two complementary approaches, pangenomic (performed on an additional EMB) and targeted gene transcriptomics (done on the same EMB), we confirm that Currently, the endomyocardial biopsy serves as a primary diagnostic tool for the diagnosis of AMR according to the 2013 ISHLT pAMR classification. This is a 4 grades composite classification: pAMR0 (no AMR), pAMR1(i+) (immunopathogenic AMR), pAMR1(h+) (histologic AMR), pAMR2 (proven AMR) and pAMR3 (severe AMR), considering IAMC and immunohistochemical criteria (C4d, CD68). Although the ISHLT Consensus has taken important steps to improve the pathologic diagnosis of antibodymediated rejection, an important gap remains in terms of histologic diagnosis accuracy and clinical-pathologic correlations to determine the indications for a therapeutic intervention. Except for the nowadays rare pAMR3 grade that has been associated with survival, the pAMR classification has not been associate with graft outcome. In a previous study, we have not been able to differentiate the molecular signatures from pAMR1(H+) and pAMR2/3 biopsies, probably reflecting the heterogeneity in terms of MVI severity. In the present study we systematically evaluated the distribution of MVI in EMB graded for both pAMR and ACR ISHLT classifications. We found that MVI score was correlated to the pAMR classification but not to the ACR classification as it transplantation, the severity of MVI would be of importance to accurately phenotype cardiac allograft AMR.

  1) exp(∑ 𝛽𝛽 𝑗𝑗𝑧𝑧 𝑖𝑖,𝑗𝑗

	𝑝𝑝 𝑗𝑗=2 ℎ 0 (𝑡𝑡 𝑖𝑖 ) exp(∑ 𝛽𝛽 𝑗𝑗𝑧𝑧 𝑖𝑖,𝑗𝑗 𝑝𝑝 𝑗𝑗=2	)	)	= exp (𝛽𝛽 1 ) ∀𝑡𝑡

𝑖𝑖

Patients with the explanatory variable {𝑧𝑧1=1} have an exp (𝛽𝛽 1 )-fold risk of failure compared to those with the explanatory variable {𝑧𝑧1=0}. The relative risk for variable 𝑧𝑧1 is said to be adjusted for all other variables, i.e. with all other variables constant. It is constant over time since it only depends on the regression coefficient 𝛽𝛽 1 .

  search yielded 249 articles. Ninety-eight did not address CAV, including studies on other solid organ transplants (n=46), other fields of heart transplantation (n=46) and

	Chapter II-B: Epidemiology and risk factors of
	biopsy-proven rejection during the first-year post-
	transplant.
	animal models or in vitro studies (n=8). Sixty-six studies did not include longitudinal
	CAV data, including case-reports, reviews, editorials (n=36), and cross-sectional This work has been presented as full oral communication at the 2019 ISHLT annual
	studies (n=30), leaving 85 studies that studied the progression of CAV. Among them, meeting (Orlando, April 2019) and at the 2019 ESOT meeting (Copenhagen,
	60 studies considered CAV as a dichotomous endpoint (presence or absence), September 2019).
	neglecting the severity and subsequent progression of CAV and 24 small retrospective
	studies compared the evolution of the mean of IVUS-derived parameters between
	groups at two points, neglecting the longitudinal characteristics of CAV data and

subsequent CAV progression. Finally, no studies were derived from large patient cohorts with systematic monitoring and a specific design aimed at risk stratification and protocol repeated CAV assessment. Furthermore, none investigated CAV trajectories, none integrated a large spectrum of potential immune and non-immune risk factors and none were validated in large cohorts with different transplant allocation systems and management practice.

Our aim was to apply an innovative statistical model able to analyze the longitudinal data of coronary angiograms in a large cohort of patients allowing us to unmask the evolutive profiles of CAV over time.

Table 9 :

 9 Diagnosis of EMB

	The most commonly diagnosed type of rejection was 1R1A acute cellular rejection (n
	= 4,077, 29.8%). Significant acute cellular rejection signs ≥ 1R1B were found in 896
	BEMs (6.6%) and patterns of AMR ≥ pAMR1 were found in 351 BEMs (2.6%). Overall,
	significant signs of cellular rejection and/or AMR were present in 1,150 BEM (8.4%).
	At the patient level, 457 (43.4%) had at least one episode of cellular rejection ≥ 1R1B,
	195 (15.5%) at least one AMR episode ≥ pAMR1 and 520 (49.4%) at least one episode
	of significant rejection (≥ 1R1B and/or ≥ pAMR1) in the first year. The comparison of
	patients' characteristics between the rejection and non-rejection groups is described
	in Table 8.

Table 8: Baseline characteristics of included patients ATG means anti-thymocyte globulins, BMI = body mass index, DSA = donor-specific antibodies, ECMO = extra corporeal membrane oxygenation, eGFR = estimated glomerular filtration rate, MCS = mechanical circulatory support.

Table 11 :

 11 Risk factors for biopsy-proven rejection during the first year: multivariable Cox model analysis.

Table 12 :

 12 Baseline characteristics of patients (European derivation and American validation cohorts).

	All patients	European	American
	(4 centers, n=1,301)	derivation cohort	validation cohort	p
		(3 centers, n=815)	(1 center, n=486)
	N	N	N

* Only the information "treated rejection" was available in the US validation cohort. ** The retrospective analysis of the histologic criteria of antibody-mediated rejection pAMR1(H+) was only performed in the two French centers. ATG means anti-thymocyte globulins, AMR: antibody-mediated rejection, BMI: body mass index, CMV: cytomegalovirus, DSA: donor-specific antibodies, IQR: interquartile range, MCS: mechanical circulatory support, MMF: mycophenolate mofetil.

Table 13 :

 13 Characteristics of coronary angiogramsThe coronary angiograms were graded according to the ISHLT guidelines[START_REF] Mehra | International Society for Heart and Lung Transplantation working formulation of a standardized nomenclature for cardiac allograft vasculopathy-2010[END_REF]. Significantly higher grades of cardiac allograft vasculopathy were diagnosed in the European derivation cohort (p<0.001). CA means coronary angiograms, CAV: cardiac allograft vasculopathy.

Table 14 .

 14 Detailed classification of endomyocardial biopsies in the European derivation cohort

	Endomyocardial biopsies were graded according to the ISHLT guidelines (29,59).
	* The retrospective analysis of the histologic criteria of antibody-mediated rejection pAMR1(H+) was only performed
	in the two French centers (Pitié-Salpêtrière & Georges Pompidou European Hospital, n=609).
	ACR means acute cellular rejection, AMR: antibody-mediated rejection.

Table 15

 15

	. We identified 17 significant variables

mellitus: p=0.07; statins therapy: 0.09; LDLc≥1g/L: p=0.002; creatinine clearance:

cell mediated rejection ≥ 2R during the 1 st year

				1.91	2.14	<0.001
				(1.62-2.25)	(1.73-2.65)
	Gender	-	1.64	1.82	3.69	<0.001
	(ref=female)		(0.89-3.00)	(1.23-2.71)	(2.11-6.46)
	Tobacco	-	1.40	1.73	2.22	0.001
			(0.82-2.39)	(1.20-2.50)	(1.40-3.51)
	1-year post-transplant
	cardiovascular risk factor
	LDL-c ≥ 1g/L	-	1.87	1.72	1.78	0.009
			(0.80-2.35)	(1.19-2.47)	(1.13-2.80)
	Immunology and histology
	Anti-HLA DSA					0.019
	pre-formed+ 1-year post HTx
	(ref=None)
	Class I	-	0.47	0.87	1.03
			(0.14-1.59)	(0.44-1.70)	(0.48-2.23)
	Class II	-	0.45	1.93	1.49
			(0.17-1.17)	(1.23-3.02)	(0.83-2.68)
	Acute T--	2.39	1.29	1.98	0.028
			(1.22-4.68)	(0.75-2.21)	(1.07-3.65)

Table 16 :

 16 Factors associated CAV trajectories in multivariate analyses.

Table 17 .

 17 Characteristics of included patients.LVAD means left ventricular assist device; MCS, mechanical circulatory support, TAH, total artificial heart.

	All patients	No pfDSA	MFI 500-1000	MFI > 1000	p-value
	N=523	N=241	N=88	N=194

Table 18

 18

	). After multivariate analyses, only

Table 19 .

 19 Univariate analysis (Cox regression): all-cause mortality.

BMI means Body Mass Index, ECMO: extra-corporeal membrane oxygenation, HTx: heart transplantation, ID DSA: Immunodominant donor-specific antibodies, MCS: mechanical circulatory support, MFI: mean fluorescence intensity.

Table 20 .

 20 Multivariate analysis (Cox regression): all-cause mortality.

	perioperative plasmapheresis sessions remained an independent risk factor for
	postoperative PRBC transfusion (OR = 3.97, 95% CI [2.3, 6.85], p < 0.001; other
	significant risk factors: pre-transplant and post-transplant ECMO support, prior history
	of cardiac surgery, long-term mechanical circulatory support at transplant; Hosmer-
	Lemeshow test: p=0.83

III-A-3-e. Safety endpoints

Transplanted patients with pfDSA MFI >1000 (i.e., plasmapheresis group) received significantly more PRBC and platelet concentrates during the first week following HTx compared to other patients (5.2 ± 6.8 compared to 8.5 ± 7.2 units of PRBC in patients with plasmapheresis, p < 0.001; Table

18

). After multivariate analyses, undergoing

pAMR1(H+), No. of EMB (% of EMB) -90 (0.96) -pAMR2/3, No. of EMB (% of EMB) -69 (0.74) -

Coutance Guillaume -Thèse de doctorat -2019

Acknowledgements

Appendix for the list of parameters assessed for the derivation cohort).

CAV angiograms were recorded per center protocol for all patients after transplantation. CAV was graded according to the current ISHLT classification as CAV 0 (not significant), 1 (mild), 2 (moderate) and 3 (severe) [START_REF] Mehra | International Society for Heart and Lung Transplantation working formulation of a standardized nomenclature for cardiac allograft vasculopathy-2010[END_REF]. European centers

Derivation of post-transplantation CAV trajectories

CAV trajectories were identified over 10 years after transplantation using latent class mixed models (101). Latent class mixed models characterize trajectories in repeated measurements, with the assumption that several underlying subpopulations (i.e, the latent classes) can be detected (104). This approach requires neither the same number of measures per patient nor the same timepoints of measurement. CAV grading at baseline (1-year after transplantation) and CAV slopes were specific to each subpopulation identified. We compared the linear trajectory models with non-linear models, including quadratic (t² effect) and splines, and confirmed the suitability of linear models used for trajectory identification. Furthermore, we tested different link functions to identify the best fitting model. At model convergence, each patient was assigned posterior likelihoods of belonging to each CAV trajectory. Patients were assigned the class to which they had the highest likelihood of belonging.

Definition of the optimal number of CAV trajectories

The number of CAV trajectories was defined according to i) the Bayesian Information Criterion (BIC) and the Akaike Information Criterion (AIC), ii) the discrimination (i.e, the ability of the model to specifically classify patients in the CAV trajectories , iii) the entropy (i.e, the ability of the model to identify distinct CAV trajectories) and iv) the interpretability of the model, as previously published (see further details in Supplementary appendix) (102,104,105).

Determinants of CAV trajectories

In the derivation cohort, the associations between CAV trajectories and clinical, histological, functional, and immunological parameters at the time of transplantation, during the first year and at one year post-transplant were assessed using multinomial logistic regression. Parameters associated with trajectories in the univariate analysis with p-value < 0.10 were thereafter included in the multivariable model. Stepwise backward elimination was performed to obtain the final multivariable.

We used R (version 3.2.1, R Foundation for Statistical Computing) and STATA (version 14, Data Analysis and Statistical Software) for the descriptive and survival analyses.

Values of p<0.05 were considered statistically significant, and all tests were 2-tailed.

III-A-3-b. Donor-specific antibodies

Patients with pfDSA MFI >1000 were younger and were more likely to be female, under mechanical circulatory support and to have had a pre-transplant sensitizing event (Table 17). The median MFI value for immunodominant DSA at day 0 was 1536 (776-2816) in this group. One-quarter of patients had pfDSA MFI >3000. The repartition of immunodominant DSA MFI and cumulative MFI is presented in Figure 31. Seventeen patients had a retrospective positive CDC-LCT crossmatch (8.7%). We observed a significant decrease in the amount of DSA, as assessed by the MFI of the immunodominant DSA and cumulative MFI, between day 0 serum and postplasmapheresis serum and between day 0 serum and 1-month serum (p < 0.001 for all comparisons, Figure 32).

In patients transplanted without pfDSA (n = 241), 187 (77.6%) had at least one screening for de novo DSA (dnDSA) during the first year. Among them, a dnDSA was detected in 54 patients (28.9%). Anti-HLA antibodies not directed against the donor HLA were present on historical and/or day 0 sera in 108 patients (57.8%) and was a significant risk factor for dnDSA development (incidence of dnDSA at one-year: 40.8 and 12.7% in patients with and without pre-formed anti-HLA antibodies not directed against the donor, respectively, p<0.001).

Animal models of AMR underline the early endothelial and platelets activation following the passive transfer of donor specific antibodies (139). In our cohort, all patients were under anti-platelets agents (low dose aspirin) but only patients under ECMO support received curative anticoagulation. However, as underlined by an AHA scientific statement, systemic anticoagulation is probably required in severe AMR to reduce the risk of intra-vascular thrombosis [START_REF] Clerkin | The effect of timing and graft dysfunction on survival and cardiac allograft vasculopathy in antibody-mediated rejection[END_REF].

Refractory cardiogenic shock due to AMR is considered a contra-indication to cardiac retransplantation at our institution. Survival after retransplantation for acute rejection has been reported to be as low as 30 % at one year (140). The analysis of the UNOS database suggests that retransplantation under ECMO support is also associated with poor prognosis. These critical patients could be considered for a bridge-to-transplant strategy with long term ventricular assist devices since survival after retransplantation for patients bridged has improved considerably over time (141). Timing is crucial in this strategy. On the one hand, cardiac allograft recovery after AMR can take weeks. On the other hand, long term ECMO support is associated with potentially life-threatening complications and poor outcomes (142).

We acknowledge some notable limitations in our study. First, the small sample size of our cohort is a major limit. Second, this cohort represents a single center experience with very similar treatments used. It would be interesting to analyze data from others centers with different protocols. Finally, despite exhaustive research, retrospective data collection might have induced selection bias.

III-B-5. Conclusion

Cardiogenic shock due to AMR occurred mostly in patients without history of biopsy proven AMR who developed high-level de novo class II DSA. Our results suggest that AMR with cardiogenic shock has a very poor short-term prognosis despite aggressive immunosuppressive therapies and mechanical circulatory support. Despite a significant incidence of infectious complications, an early intensification of immunosuppressive therapies should strongly be considered in case of cardiogenic shock due to AMR. Among patients discharged from hospital one case of rapidly progressive CAV and one sudden cardiac death occurred during follow-up.

Supplementary appendix S1. Data collection procedures

Baseline clinical data on the donors and recipients from Paris and Leuven were obtained from the national registry CRISTAL (Agence de la Biomédecine, French National Agency for Organ Procurement) and Eurotransplant registry, respectively.

Codes were used to ensure strict donor and recipient anonymity and blinded assays.

Informed consent was obtained from the participants at the time of transplantation.

Anonymized data from these registries are prospectively entered by dedicated staff at In patients with multiple anti-HLA DSA, the immunodominant anti-HLA DSA (the one with the highest MFI) was selected for analysis. HLA typing of the recipients was performed via molecular biology (Innolipa HLA typing kit, Innogenetics, Belgium). For all heart transplant donors, tissue typing was initially performed using the microlymphocytotoxicity technique with One Lambda Inc. tissue typing trays at transplantation and was confirmed by molecular biology.