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.

中文翻译：

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逆转录酶多重连接依赖性探针扩增在心内膜活检中诊断心脏同种异体移植排斥反应。

背景技术分子生物学已经成为组织学用于心脏移植后排斥反应诊断的潜在伴侣。逆转录酶多重连接依赖探针扩增（RT-MLPA）是一种靶向基因表达分析技术，适用于福尔马林固定石蜡包埋（FFPE）活检。我们的目的是评估RT-MLPA对心脏移植中同种异体移植排斥反应的诊断。方法我们进行了横断面，病例对照，多中心研究。在选择14个转录本的组（内皮负担，自然杀伤细胞，干扰素-γ途径，效应T细胞和抗原呈递）后，将RT-MLPA应用于183 FFPE心肌内膜活检（EMB），随机分为训练（n = 113）和验证（n = 70）系列。进行了两步分类预测分析（线性预测分数-LPS1：排斥与非排斥； LPS2：抗体介导的排斥[AMR]与急性细胞排斥[ACR]）。进行了病理学与RT-MLPA之间一致性的研究。结果总体上，分析了48个ACR，82个AMR，5个混合排斥和48个非排斥EMB。通过无监督的层次分析（分子不排斥，ACR和AMR）描绘了三个分子簇。AMR的特征是CCL4，GNLY，FCGR3，CXCL11和ACR的高表达是CCL18和ADAMdec的高表达。在测试，验证和总体队列研究中，RT-MLPA和组织病理学在最终诊断中分别占EMB的82.2％，67.7％和76.8％。在组织学上低度排斥反应和移植后早期EMB病例中，意见分歧的情况更为普遍。结论RT-MLPA是一种适用于FFPE EMB的靶向基因表达分析的技术，与心脏异体移植排斥的组织学诊断具有良好的总体一致性。