Jang Moon Kyoo, Oluwayiose Oladele, Redekar Neelam, Andargie Temesgen E, Park Woojin, Alnababteh Muhtadi, Hill Tom, Phipps Kellie, Kong Hyesik, Tian Xin, Luikart Helen I, Solomon Michael A, Shah Palak, Valantine Hannah A, Khush Kiran, Agbor-Enoh Sean
medRxiv. 2025 Mar 5:2025.03.04.25323391. doi: 10.1101/2025.03.04.25323391.
Antibody-mediated rejection (AMR) remains the major risk factor for allograft loss across all solid organ transplantation. Unfortunately, its diagnosis relies on biopsy, an invasive gold standard that often sample unaffected allograft tissue leading to missed diagnosis. Plasma donor-derived cell-free DNA (dd-cfDNA) is noninvasive biomarker that has high sensitivity but low specificity for AMR diagnosis. This proof-of-concept study assessed the utility of cell-free chromatin immunoprecipitation (cfChIP) as a surrogate for gene expression to detect cardiac AMR and the associated pathobiology.
The discovery GRAfT multicenter cohort of heart transplant patients ( NCT02423070 ) identified AMR, acute cellular rejection (ACR), and stable controls based on biopsy and dd-cfDNA results. Plasma cfChIP-sequencing was performed to identify peaks, associated genes and pathobiological pathways. Plasma from an external cohort (GTD, NCT01985412 ) was also analyzed to verify pathways identified. Digital droplet PCR (ddPCR) assays targeting differential regions were constructed to test the diagnostic performance of cfDNA to detect AMR/ACR from stable controls (rejection-specific assays) or AMR from ACR (AMR-specific assays).
The cohort included 21 AMR, 28 ACR, and 45 stable controls from GRAfT and GTD, and 23 healthy controls. cfChIP detected expected active genes, including housekeeping genes and gene targets of transplant immunosuppressive drugs but not inactive genes. Unsupervised clustering of the discovery GRAfT cohort assigned 95% of samples correctly as AMR, ACR or stable control. Differential analysis identified pathobiological pathways of AMR such as neutrophil degranulation and complement activation. The pathways were consistent in GTD samples. Rejection-specific assays detected AMR/ACR from controls with AUC of 0.78 - 0.95. AMR-specific assays detected AMR from ACR with AUC of 0.71 - 0.85, sensitivities of 0.73 - 0.94 and specificities of 0.73 - 0.80.
This study provides valuable preliminary data supporting the use of cfChIP to detect AMR and the associated pathobiological pathways.
抗体介导的排斥反应（AMR）仍然是所有实体器官移植中移植物丢失的主要风险因素。不幸的是，其诊断依赖于活检，这是一种侵入性的金标准，常常对未受影响的移植组织进行采样，导致漏诊。血浆供体来源的游离DNA（dd-cfDNA）是一种非侵入性生物标志物，对AMR诊断具有高敏感性但低特异性。这项概念验证研究评估了游离染色质免疫沉淀（cfChIP）作为检测心脏AMR及相关病理生物学的基因表达替代方法的效用。
心脏移植患者的发现队列GRAfT多中心研究（NCT02423070）根据活检和dd-cfDNA结果确定了AMR、急性细胞排斥反应（ACR）和稳定对照组。进行血浆cfChIP测序以识别峰值、相关基因和病理生物学途径。还分析了来自外部队列（GTD，NCT01985412）的血浆以验证所识别的途径。构建靶向差异区域的数字液滴PCR（ddPCR）检测方法，以测试cfDNA从稳定对照组中检测AMR/ACR（排斥特异性检测）或从ACR中检测AMR（AMR特异性检测）的诊断性能。
该队列包括来自GRAfT和GTD的21例AMR、28例ACR和45例稳定对照组，以及23例健康对照组。cfChIP检测到了预期的活性基因，包括管家基因和移植免疫抑制药物的基因靶点，但未检测到非活性基因。发现队列GRAfT的无监督聚类将95%的样本正确分类为AMR、ACR或稳定对照组。差异分析确定了AMR的病理生物学途径，如中性粒细胞脱颗粒和补体激活。这些途径在GTD样本中是一致的。排斥特异性检测从对照组中检测AMR/ACR的曲线下面积（AUC）为0.78 - 0.95。AMR特异性检测从ACR中检测AMR的AUC为0.71 - 0.85，敏感性为0.73 - 0.94，特异性为0.73 - 0.80。
本研究提供了有价值的初步数据，支持使用cfChIP检测AMR及相关病理生物学途径。
