Reverse transcriptase multiplex ligation-dependent probe amplification in endomyocardial biopsies for the diagnosis of cardiac allograft rejection

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.