Université de Lille, Faculté de Médecine, Centre Hospitalier Universitaire Lille, Laboratoire de Virologie ULR3610, Lille, France.
Université de Lille, Centre Hospitalier Universitaire Lille, Department of Hematology, Lille, France.
J Mol Diagn. 2021 Apr;23(4):447-454. doi: 10.1016/j.jmoldx.2020.12.004. Epub 2020 Dec 30.
Axicabtagene ciloleucel or axi-cel [CD19 chimeric antigen receptor (CAR) T cell] has been recently approved for refractory/relapsed diffuse large B-cell lymphoma and primary mediastinal B-cell lymphoma. Proliferation of CAR T cells after infusion and their persistence have been reported as important factors. Laboratory tools are needed for the monitoring of patients. We developed a vector-based, simple, and accurate real-time quantitative PCR (qPCR) to measure axi-cel vector copy number in peripheral blood samples. Primers and probe targeting the 5'LTR region of the gammaretroviral vector (mouse stem cell virus) were designed for amplification. To generate standard curves, mouse stem cell virus plasmid was subcultured and quantified using droplet digital PCR. The method was applied to quantify vector copy number in blood samples from patients treated with axi-cel. The limit of quantification of the qPCR assay was established at 2.2 copies/μL in DNA eluate. The qPCR method was well correlated with flow cytometry findings; however, the assay appeared to be more sensitive than flow cytometry. The kinetics observed in blood samples from treated patients were in agreement with previously reported findings. In conclusion, we developed a sensitive and accurate qPCR assay for the quantification of transgenic CAR T cells, which can be a useful additional tool for the monitoring of patients treated with axi-cel.
阿基仑赛（axicabtagene ciloleucel，axi-cel[CD19 嵌合抗原受体（CAR）T 细胞]）最近已被批准用于治疗难治/复发性弥漫性大 B 细胞淋巴瘤和原发性纵隔 B 细胞淋巴瘤。CAR T 细胞输注后的增殖及其持续存在被认为是重要因素。需要实验室工具来监测患者。我们开发了一种基于载体的、简单且准确的实时定量 PCR（qPCR）方法，用于测量外周血样本中的 axi-cel 载体拷贝数。针对γ逆转录病毒载体（小鼠干细胞病毒）5'LTR 区域设计了引物和探针，用于扩增。为了生成标准曲线，用微滴数字 PCR 对小鼠干细胞病毒质粒进行传代和定量。该方法应用于定量 axi-cel 治疗患者的血液样本中的载体拷贝数。qPCR 检测的定量下限在 DNA 洗脱物中设定为 2.2 拷贝/μL。qPCR 方法与流式细胞术结果相关性良好；然而，该检测方法似乎比流式细胞术更灵敏。从治疗患者的血液样本中观察到的动力学与先前报道的结果一致。总之，我们开发了一种用于定量转基因 CAR T 细胞的敏感且准确的 qPCR 检测方法，这可能是监测 axi-cel 治疗患者的有用辅助工具。
