Flow Cytometric Measurement of Blood Cells with BCR-ABL1 Fusion Protein in Chronic Myeloid Leukemia.
Flow Cytometric Measurement of Blood Cells with BCR-ABL1 Fusion Protein in Chronic Myeloid Leukemia.
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DOI：
10.1038/s41598-017-00755-y
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发表时间：
2017-04-04
影响因子：
4.6
通讯作者：
Kamali-Moghaddam M
中科院分区：
文献类型：
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作者：
Löf L;Arngården L;Olsson-Strömberg U;Siart B;Jansson M;Dahlin JS;Thörn I;Christiansson L;Hermansson M;Larsson A;Ahlstrand E;Wålinder G;Söderberg O;Rosenquist R;Landegren U;Kamali-Moghaddam M
慢性粒细胞白血病(CML)的特点是t(9；22)(q34；q11)易位，也称为Philadelphia染色体，产生bcr-abl1融合蛋白。目前酪氨酸激酶抑制剂的治疗针对融合蛋白的ABL1结构域，治疗后的微小残留病(MRD)通过融合转录本的实时定量聚合酶链式反应(RQ-PCR)进行监测。在这里，我们描述了一种新的方法来检测和计数bcr-abl1融合蛋白阳性的细胞，方法是将原位邻近连接试验与流式细胞术相结合(PLAFLOW)。通过靶向融合蛋白的BCR和ABL1部分各有一种抗体，并通过滚圈放大产生强烈的荧光信号，PLAFLOW使BCR-ABL1融合阳性细胞的灵敏检测频率低至万分之一。重要的是，流式细胞术的结果与RQ-PCR的结果有很强的相关性，无论是在诊断测试中还是在MRD测量中都是如此。总之，我们相信这种基于流式细胞术的方法可以作为一种有吸引力的方法来对含有BCR-ABL1融合的细胞进行常规测量，也可以同时评估其他细胞表面标志物以及灵敏的纵向随访。
Chronic myeloid leukemia (CML) is characterized in the majority of cases by a t(9;22)(q34;q11) translocation, also called the Philadelphia chromosome, giving rise to the BCR-ABL1 fusion protein. Current treatment with tyrosine kinase inhibitors is directed against the constitutively active ABL1 domain of the fusion protein, and minimal residual disease (MRD) after therapy is monitored by real-time quantitative PCR (RQ-PCR) of the fusion transcript. Here, we describe a novel approach to detect and enumerate cells positive for the BCR-ABL1 fusion protein by combining the in situ proximity ligation assay with flow cytometry as readout (PLA-flow). By targeting of the BCR and ABL1 parts of the fusion protein with one antibody each, and creating strong fluorescent signals through rolling circle amplification, PLA-flow allowed sensitive detection of cells positive for the BCR-ABL1 fusion at frequencies as low as one in 10,000. Importantly, the flow cytometric results correlated strongly to those of RQ-PCR, both in diagnostic testing and for MRD measurements over time. In summary, we believe this flow cytometry-based method can serve as an attractive approach for routine measurement of cells harboring BCR-ABL1 fusions, also allowing simultaneously assessment of other cell surface markers as well as sensitive longitudinal follow-up.