Endomyocardial biopsy (EMB)-based traditional microscopy remains the gold standard for the detection of cardiac allograft rejection, despite its limitation of inherent subjectivity leading to inter-reader variability. Alternative techniques now exist to surveil for allograft injury and classify rejection. Donor-derived cell-free DNA (dd-cfDNA) testing is now a validated blood-based assay used to surveil for allograft injury. The molecular microscope diagnostic system (MMDx) utilizes intragraft rejection-associated transcripts (RATs) to classify allograft rejection and identify injury. The use of dd-cfDNA and MMDx together provides objective molecular insight into allograft injury and rejection. The aim of this study was to measure the diagnostic agreement between dd-cfDNA and MMDx and assess the relationship between dd-cfDNA and MMDx-derived RATs which may provide further insight into the pathophysiology of allograft rejection and injury. This is a retrospective observational study of 156 endomyocardial biopsy (EMB) evaluated with traditional microscopy and MMDx. All samples were paired with dd-cfDNA from peripheral blood prior to EMB (up to 9 days). Diagnostic agreement between traditional histopathology, MMDx, and dd-cfDNA (threshold of 0.20%) were compared for assessment of allograft injury. In addition, the relationship between dd-cfDNA and individual RAT expression levels from MMDx was evaluated. MMDx characterized allograft tissue as no rejection (NR) (62.8%), antibody-mediated rejection (ABMR) (26.9%), T-cell-mediated rejection (TCMR) (5.8%), and mixed ABMR/ TCMR (4.5%). For the diagnosis of any type of rejection (TCMR, ABMR, and mixed rejection), there was substantial agreement between MMDx and dd-cfDNA (76.3% agreement). All transcript clusters (group of gene sets designated by MMDx) and individual transcripts considered abnormal from MMDx had significantly elevated dd-cfDNA. In addition, a positive correlation between dd-cfDNA levels and certain MMDx-derived RATs was observed. Tissue transcript clusters correlated with dd-cfDNA scores, including . For individual transcripts, tissue was significantly correlated with dd-cfDNA in both non-rejection and rejection as assessed by MMDx. Collectively, we have shown substantial diagnostic agreement between dd-cfDNA and MMDx. Furthermore, based on the findings presented, we postulate a common pathway between the release of dd-cfDNA and expression of (a vascular endothelial-specific gene that stabilizes the vasculature) in the setting of antibody-mediated rejection (AMR), which may provide a mechanistic rationale for observed elevations in dd-cfDNA in AMR, compared to acute cellular rejection (ACR).

中文翻译：

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心脏移植中供体来源的游离 DNA 与组织排斥相关转录本之间的关系

基于心内膜心肌活检 (EMB) 的传统显微镜检查仍然是检测心脏同种异体移植排斥反应的金标准，尽管其固有的主观性限制导致读者之间存在差异。现在存在替代技术来监测同种异体移植物损伤并对排斥反应进行分类。供体来源的游离 DNA (dd-cfDNA) 检测现已成为一种经过验证的血液检测方法，用于监测同种异体移植物损伤。分子显微镜诊断系统 (MMDx) 利用移植物内排斥相关转录本 (RAT) 对同种异体移植排斥进行分类并识别损伤。dd-cfDNA 和 MMDx 的结合使用为同种异体移植物损伤和排斥提供了客观的分子洞察。本研究的目的是测量 dd-cfDNA 和 MMDx 之间的诊断一致性，并评估 dd-cfDNA 和 MMDx 衍生 RAT 之间的关系，这可能有助于进一步了解同种异体移植排斥和损伤的病理生理学。这是一项使用传统显微镜和 MMDx 评估的 156 例心内膜心肌活检 (EMB) 的回顾性观察研究。所有样本均在 EMB 之前（最多 9 天）与外周血中的 dd-cfDNA 配对。比较传统组织病理学、MMDx 和 dd-cfDNA（阈值 0.20%）之间的诊断一致性，以评估同种异体移植物损伤。此外，还评估了 dd-cfDNA 与 MMDx 个体 RAT 表达水平之间的关系。MMDx 将同种异体移植组织的特征描述为无排斥 (NR) (62.8%)、抗体介导的排斥 (ABMR) (26.9%)、T 细胞介导的排斥 (TCMR) (5.8%) 和混合 ABMR/TCMR (4.5%) 。对于任何类型的排斥反应（TCMR、ABMR 和混合排斥反应）的诊断，MMDx 和 dd-cfDNA 之间存在显着的一致性（76.3% 一致性）。所有转录物簇（MMDx 指定的基因组组）和 MMDx 中被认为异常的个体转录物的 dd-cfDNA 显着升高。此外，还观察到 dd-cfDNA 水平与某些 MMDx 衍生 RAT 之间呈正相关。组织转录物簇与 dd-cfDNA 评分相关，包括 . 对于个体转录本，通过 MMDx 评估，组织在非排斥和排斥方面与 dd-cfDNA 显着相关。总的来说，我们已经证明了 dd-cfDNA 和 MMDx 之间的诊断一致性。此外，根据所提出的发现，我们假设在抗体介导的排斥反应（AMR）的情况下，dd-cfDNA 的释放和（稳定脉管系统的血管内皮特异性基因）的表达之间存在共同途径，这可能提供与急性细胞排斥 (ACR) 相比，AMR 中观察到的 dd-cfDNA 升高的机制原理。