Department of Hematology and Central Hematological Laboratory, Inselspital, Bern University Hospital, University of Bern, 3010 Bern, Switzerland.
Department for BioMedical Research, University of Bern, 3008 Bern, Switzerland.
Int J Mol Sci. 2024 Aug 6;25(16):8556. doi: 10.3390/ijms25168556.
In this study, we present the design, implementation, and successful use of digital droplet PCR (ddPCR) for the monitoring of chimeric antigen receptor T-cell (CAR-T) expansion in patients with B-cell malignancies treated with different CAR-T products at our clinical center. Initially, we designed a specific and highly sensitive ddPCR assay targeting the junction between the 4-1BB and CD3ζ domains of tisa-cel, normalized with , and validated it using blood samples from the first tisa-cel-treated patient in Switzerland. We further compared this assay with a published qPCR (quantitative real-time PCR) design. Both assays showed reliable quantification of CAR-T copies down to 20 copies/µg DNA. The reproducibility and precision were confirmed through extensive testing and inter-laboratory comparisons. With the introduction of other CAR-T products, we also developed a corresponding ddPCR assay targeting axi-cel and brexu-cel, demonstrating high specificity and sensitivity with a limit of detection of 20 copies/µg DNA. These assays are suitable for CAR-T copy number quantification across multiple sample types, including peripheral blood, bone marrow, and lymph node biopsy material, showing robust performance and indicating the presence of CAR-T cells not only in the blood but also in target tissues. Longitudinal monitoring of CAR-T cell kinetics in 141 patients treated with tisa-cel, axi-cel, or brexu-cel revealed significant expansion and long-term persistence. Peak expansion correlated with clinical outcomes and adverse effects, as is now well known. Additionally, we quantified the CAR-T mRNA expression, showing a high correlation with DNA copy numbers and confirming active transgene expression. Our results highlight the quality of ddPCR for CAR-T monitoring, providing a sensitive, precise, and reproducible method suitable for clinical applications. This approach can be adapted for future CAR-T products and will support the monitoring and the management of CAR-T cell therapies.
在本研究中，我们介绍了数字液滴 PCR（ddPCR）的设计、实施和成功应用，用于监测我们临床中心接受不同 CAR-T 产品治疗的 B 细胞恶性肿瘤患者嵌合抗原受体 T 细胞（CAR-T）的扩增。最初，我们设计了一种针对 tisacel 的 4-1BB 和 CD3ζ 结构域连接的特异性和高灵敏度的 ddPCR 检测方法，并用瑞士首例 tisacel 治疗患者的血液样本进行了验证。我们还将该检测方法与已发表的 qPCR（定量实时 PCR）设计进行了比较。两种检测方法均能可靠地定量 CAR-T 拷贝数，最低可检测到 20 拷贝/µg DNA。通过广泛的测试和实验室间比较，证实了重复性和精密度。随着其他 CAR-T 产品的引入，我们还开发了针对 axi-cel 和 brexu-cel 的相应 ddPCR 检测方法，具有高特异性和灵敏度，检测限为 20 拷贝/µg DNA。这些检测方法适用于多种样本类型（包括外周血、骨髓和淋巴结活检材料）的 CAR-T 拷贝数定量，表现出稳健的性能，并表明 CAR-T 细胞不仅存在于血液中，而且存在于靶组织中。对 141 例接受 tisacel、axi-cel 或 brexu-cel 治疗的患者的 CAR-T 细胞动力学进行纵向监测显示出显著的扩增和长期持续存在。峰值扩增与临床结果和不良事件相关，这是众所周知的。此外，我们还定量了 CAR-T mRNA 表达，显示与 DNA 拷贝数高度相关，并证实了活跃的转基因表达。我们的结果强调了 ddPCR 在 CAR-T 监测中的质量，提供了一种敏感、精确和可重复的方法，适用于临床应用。这种方法可以适应未来的 CAR-T 产品，并将支持 CAR-T 细胞治疗的监测和管理。
