The goal of this observational study is to develop and validate a molecular heart rejection diagnostic system based on targeted transcriptome as a novel monitoring companion tool for heart allograft precision diagnostics applicable to formalin-fixed paraffin-embedded endomyocardial biopsies.

The primary outcome will be the biopsy-proven rejection, that will be predicted with molecular classifiers (cellular and antibody-mediated rejection scores).

Heart transplantation (HTx) remains the most valuable therapeutic option for patients with end-stage heart failure refractory to optimal medical therapy. Despite major improvements in immunosuppression and transplant care, acute and chronic rejection-induced allograft injuries remain one of the leading causes of mortality and morbidity after heart transplantation, thus limiting recipients' life expectancy. An improvement in the overall management of rejection remains an unmet medical need. As a first step, a precise diagnosis of rejection is crucial to guide patient care and optimize management.

Nowadays, the diagnosis of cardiac rejection relies exclusively on the pathological assessment of endomyocardial biopsies (EMBs) by identifying and grading cellular infiltrates and myocardial damage. While important advances have been made in the standardization of the rejection diagnosis, pathology remains an imperfect gold-standard, particularly due to the inter-observer variability, sample bias and the use of qualitative or semi-quantitative scales that oversimplify complex phenotypes. Additionally, disease severity, degree of myocardial injury and progression stage are crucial pieces of information poorly captured by the current working formulations. All these limits represent major barriers to achieve a precise and reliable diagnosis of rejection.

In this context, gene expression profiling analysis of fresh myocardial samples retrieved during an extra-core biopsy and using a whole transcriptome approach arose as a potential objective companion tool of pathology to refine the diagnosis of rejection. However, important drawbacks have limited the routine clinical applicability of whole-transcriptome based molecular diagnosis including extra-core sampling bias, low reproducibility, technical and analytical heaviness, with costs and sample turn-around that is not compatible with a clinical setting.

Recent technologies may overcome this limitation by allowing analysis of the same tissue used for histology assessment. Targeted molecular profiling applicable to formalin-fixed paraffin-embedded (FFPE) endomyocardial biopsies may allow the implementation of molecular diagnosis into the clinical routine. Recently, we have shown that the Banff Human Organ Transplant Panel (B-HOT) panel, a consortium-approved consensual targeted panel including 770 genes, developed by the Banff Molecular Diagnostics Working Group, accurately captured key molecular patterns of antibody-mediated rejection (AMR) in heart allograft biopsies and may serve as a proxy to whole transcriptome-based analysis.

The aim of the present study is therefore to identify gene expression signatures for AMR and acute cellular rejection in heart transplantation and to develop and validate a molecular heart rejection diagnostic system based on targeted transcriptome as a novel monitoring companion tool for heart allograft precision diagnostics applicable to FFPE-EMB.

Inclusion Criteria:

• heart transplant recipients ≥ 18 years old
• at least one endomyocardial biopsy performed as part of routine clinical care
• biopsy performed at least ≥ 1 month post transplant and less that 10-year post-transplant
• written informed consent

Exclusion Criteria:

• age below 18 years old

We designed an international multicenter study, building a deep phenotyped cohort of heart transplant recipients recruited at 4 referral centers (Pitié-Salpêtrière and Georges Pompidou hospitals in Paris, Center Gallucci of the University General Hospital of Padova and the Cedars Sinai Medical Center in Los Angeles, USA).