Nicolas Adam1, Guillaume Coutance2, Pierre-Julien Viailly3, Fanny Drieux3, Philippe Ruminy4, Ahmad Abdel Sater3, Claire Toquet5, Philippe Rouvier6, Arnaud François7, Marie-Pierre Chenard8, Eric Epailly9, Romain Guillemain10, Sabine Pattier11, Arnaud Gay12, Shaida Varnous6, Jean-Luc Taupin13, Marion Rabant1, Alexandre Loupy14, Patrick Bruneval15, Jean Paul Duong Van Huyen16. 1. Paris Translational Research Centre for Organ Transplantation, INSERM, UMR-S970, Paris, France; Pathology department, Hôpital Necker, Assistance Publique - Hôpitaux de Paris and Paris Descartes University, Paris, France. 2. Paris Translational Research Centre for Organ Transplantation, INSERM, UMR-S970, Paris, France; Department of cardio-thoracic surgery, La Pitié-Salpêtrière, Assistance Publique des hôpitaux de Paris, Paris, France. 3. INSERM U1245, Centre Henri Becquerel, Rouen, France. 4. Pathology department, La Pitié Salpétrière, Assistance Publique - Hôpitaux de Paris, Paris, France. 5. Pathology department, Nantes University, Nantes, France. 6. Department of cardio-thoracic surgery, La Pitié-Salpêtrière, Assistance Publique des hôpitaux de Paris, Paris, France. 7. Pathology department, Rouen, France. 8. Pathology department, Strasbourg, France. 9. Thoracic Surgery departments, Strasbourg, France. 10. Cardiovascular surgery and heart transplant department Georges Pompidou Hospital, Assistance Publique - Hôpitaux de Paris, Paris, France. 11. Cardiovascular surgery and heart transplant department, Nantes, France. 12. Cardiovascular surgery and heart transplant department, Rouen, France. 13. Histocompatibility laboratory, Saint Louis hospital, Paris, France. 14. Paris Translational Research Centre for Organ Transplantation, INSERM, UMR-S970, Paris, France. 15. Paris Translational Research Centre for Organ Transplantation, INSERM, UMR-S970, Paris, France; Pathology, cardiology and heart transplant department Georges Pompidou Hospital, Assistance Publique - Hôpitaux de Paris, Paris, France. 16. Paris Translational Research Centre for Organ Transplantation, INSERM, UMR-S970, Paris, France; Pathology department, Hôpital Necker, Assistance Publique - Hôpitaux de Paris and Paris Descartes University, Paris, France; Pathology department, Nantes University, Nantes, France. Electronic address: jean-paul.duong@aphp.fr.
Abstract
BACKGROUND: Molecular biology has emerged as a potential companion to histology for the diagnosis of rejection after heart transplantation. Reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) is a technique of targeted gene expression analysis suitable for formalin-fixed paraffin-embedded (FFPE) biopsies. Our aim was to assess RT-MLPA for the diagnosis of allograft rejection in heart transplantation. METHODS: We performed a cross-sectional, case-control, multicenter study. After the selection of a 14-transcript panel (endothelial burden, Natural killer cells, interferon-γ pathway, effector T-cells, and antigen presentation), RT-MLPA was applied to 183 FFPE endomyocardial biopsies (EMB), randomized into a training (n = 113) and a validation (n = 70) series. A two-step class prediction analysis was developed (Linear prediction score-LPS1: rejection vs non-rejection; LPS2: antibody-mediated rejection [AMR] vs acute cellular rejection [ACR]). A study of the agreement between pathology and RT-MLPA was performed. RESULTS: Overall, 48 ACR, 82 AMR, 5 mixed rejection, and 48 non-rejection EMBs were analyzed. Three molecular clusters were delineated by unsupervised hierarchical analysis (molecular non-rejection, ACR, and AMR). AMR was characterized by the high expression of CCL4, GNLY, FCGR3, CXCL11 and ACR by the high expression of CCL18 and ADAMdec. RT-MLPA and histopathology agreed in the final diagnosis in 82.2%, 67.7%, and 76.8% of the EMB in the test, validation, and overall cohort, respectively. Disagreement cases were more common in the case of histologic low-grade rejection and early post-transplant EMB. CONCLUSIONS: RT-MLPA is a suitable technique for targeted gene expression analysis on FFPE EMB with a good overall agreement with the histologic diagnosis of heart allograft rejection.
BACKGROUND: Molecular biology has emerged as a potential companion to histology for the diagnosis of rejection after heart transplantation. Reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) is a technique of targeted gene expression analysis suitable for formalin-fixed paraffin-embedded (FFPE) biopsies. Our aim was to assess RT-MLPA for the diagnosis of allograft rejection in heart transplantation. METHODS: We performed a cross-sectional, case-control, multicenter study. After the selection of a 14-transcript panel (endothelial burden, Natural killer cells, interferon-γ pathway, effector T-cells, and antigen presentation), RT-MLPA was applied to 183 FFPE endomyocardial biopsies (EMB), randomized into a training (n = 113) and a validation (n = 70) series. A two-step class prediction analysis was developed (Linear prediction score-LPS1: rejection vs non-rejection; LPS2: antibody-mediated rejection [AMR] vs acute cellular rejection [ACR]). A study of the agreement between pathology and RT-MLPA was performed. RESULTS: Overall, 48 ACR, 82 AMR, 5 mixed rejection, and 48 non-rejection EMBs were analyzed. Three molecular clusters were delineated by unsupervised hierarchical analysis (molecular non-rejection, ACR, and AMR). AMR was characterized by the high expression of CCL4, GNLY, FCGR3, CXCL11 and ACR by the high expression of CCL18 and ADAMdec. RT-MLPA and histopathology agreed in the final diagnosis in 82.2%, 67.7%, and 76.8% of the EMB in the test, validation, and overall cohort, respectively. Disagreement cases were more common in the case of histologic low-grade rejection and early post-transplant EMB. CONCLUSIONS: RT-MLPA is a suitable technique for targeted gene expression analysis on FFPE EMB with a good overall agreement with the histologic diagnosis of heart allograft rejection.
Authors: Michael Mengel; Alexandre Loupy; Mark Haas; Candice Roufosse; Maarten Naesens; Enver Akalin; Marian C Clahsen-van Groningen; Jessy Dagobert; Anthony J Demetris; Jean-Paul Duong van Huyen; Juliette Gueguen; Fadi Issa; Blaise Robin; Ivy Rosales; Jan H Von der Thüsen; Alberto Sanchez-Fueyo; Rex N Smith; Kathryn Wood; Benjamin Adam; Robert B Colvin Journal: Am J Transplant Date: 2020-06-27 Impact factor: 9.369