Rapid liquid biopsy assessment through gene profiling from the kidney biopsy transport medium: a technical validation and a proof-of-concept pilot study

Ziyang Li, Marij J.P Welters, Aiko P.J de Vries, Jan Anthonie Bruijn, Hans J Baelde, Jesper Kers
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Abstract

Background: Rapid diagnosis is pivotal in kidney disease for timely and targeted treatment. Conventional microscopic and molecular assessments from biopsy tissues rely on extra sample processing (e.g., formalin-fixation, paraffin-embedding (FFPE)) or an extra biopsy core (e.g., Molecular Microscope Diagnostic System [MMDx]), making same-day diagnosis impractical. Therefore, we introduce a novel and freely accessible material, the biopsy transport medium (BTM), which can serve as a source of biomarkers with high potential and is promising for accelerating the assessment workflow. Methods: Biopsies were cut from tumor-free tissues obtained from nephrectomies to create BTM mimics for homogenization. We optimized the RNA extraction procedure from BTM by investigating crucial steps in the process. We measured the quantity and integrity of the RNA and different biomarkers derived from BTM through qPCR. Additionally, we performed the Banff Human Organ Transplant (B-HOT) panel on BTM from clinical biopsies using the NanoString nCounter platform as a proof-of-concept study. Results: Our results showed that the storage times ranging from 0.5 hours to 24 hours did not significantly affect RNA quality and yield. Differential gene expression analysis on allograft rejection BTM described specific profiles related to rejection. A significant correlation was observed between rejection-related transcripts and the corresponding Banff lesion scores. Conclusion: This study validated that the BTM can provide transcriptomic information relevant to the state of the kidney. The proof-of-concept study demonstrated that BTM has great potential for reflecting the status of the transplanted kidney. Tailored qPCR panels could allow for fast (same-day) molecular diagnosis.
通过肾脏活检运送介质的基因图谱分析进行快速液体活检评估:技术验证和概念验证试点研究
背景:快速诊断对肾脏疾病的及时和有针对性的治疗至关重要。传统的活检组织显微和分子评估依赖于额外的样本处理(如福尔马林固定、石蜡包埋(FFPE))或额外的活检核心(如分子显微诊断系统 [MMDx]),这使得当天诊断不切实际。因此,我们引入了一种可免费获取的新型材料--活组织切片运输培养基(BTM),它可作为一种极具潜力的生物标记物来源,并有望加速评估工作流程。方法从肾切除术中获得的无肿瘤组织中切取活检组织,制成 BTM 模拟物进行均质化。我们通过研究过程中的关键步骤,优化了从 BTM 中提取 RNA 的程序。我们通过 qPCR 测定了从 BTM 提取的 RNA 和不同生物标记物的数量和完整性。此外,作为概念验证研究,我们还使用 NanoString nCounter 平台对来自临床活检的 BTM 进行了班夫人体器官移植 (B-HOT) 分析。结果显示结果表明,储存时间从 0.5 小时到 24 小时不等,对 RNA 质量和产量没有明显影响。异体移植排斥反应的差异基因表达分析 BTM 描述了与排斥反应相关的特定特征。在排斥反应相关转录本和相应的 Banff 病变评分之间观察到了明显的相关性:这项研究验证了 BTM 可以提供与肾脏状态相关的转录组信息。概念验证研究表明,BTM 在反映移植肾状态方面具有巨大潜力。定制的 qPCR 面板可实现快速(当天)分子诊断。
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