Flow Cytometric Immunobead Assay for Detection of BCR-ABL1 Fusion Proteins in Chronic Myleoid Leukemia: Comparison with FISH and PCR Techniques

Chronic Myeloid Leukemia (CML) is characterized by a balanced translocation juxtaposing the Abelson (ABL) and breakpoint cluster region (BCR) genes. The resulting BCR-ABL1 oncogene leads to increased proliferation and survival of leukemic cells. Successful treatment of CML has been accompanied by steady improvements in our capacity to accurately and sensitively monitor therapy response. Currently, measurement of BCR-ABL1 mRNA transcript levels by real-time quantitative PCR (RQ-PCR) defines critical response endpoints. An antibody-based technique for BCR-ABL1 protein recognition could be an attractive alternative to RQ-PCR. To date, there have been no studies evaluating whether flow-cytometry based assays could be of clinical utility in evaluating residual disease in CML patients. Here we describe a flow-cytometry assay that detects the presence of BCR-ABL1 fusion proteins in CML lysates to determine the applicability, reliability, and specificity of this method for both diagnosis and monitoring of CML patients for initial response to therapy. We show that: i) CML can be properly diagnosed at onset, (ii) follow-up assessments show detectable fusion protein (i.e. relative mean fluorescent intensity, rMFI%>1) when BCR-ABL1IS transcripts are between 1–10%, and (iii) rMFI% levels predict CCyR as defined by FISH analysis. Overall, the FCBA assay is a rapid technique, fully translatable to the routine management of CML patients.

慢性髓系白血病（Chronic Myeloid Leukemia, CML）以平衡性易位为特征，该易位使阿贝尔森（Abelson, ABL）基因与断裂点簇集区（breakpoint cluster region, BCR）基因发生并置。由此产生的BCR-ABL1癌基因可促使白血病细胞增殖能力与存活能力增强。随着CML治疗手段的不断进步，临床精准且灵敏地监测治疗反应的能力也持续提升。当前，通过实时定量聚合酶链反应（real-time quantitative PCR, RQ-PCR）检测BCR-ABL1 mRNA转录本水平，是判定关键疗效终点的标准方法。基于抗体的BCR-ABL1蛋白检测技术有望成为RQ-PCR极具吸引力的替代方案。迄今为止，尚无研究评估基于流式细胞术（flow-cytometry）的检测方法在CML患者残留病评估中的临床应用价值。本研究描述了一种可在CML裂解物中检测BCR-ABL1融合蛋白的流式细胞术检测法，以明确该方法用于CML患者诊断及治疗初始反应监测的适用性、可靠性与特异性。研究结果表明：其一，可在发病初期准确诊断CML；其二，当BCR-ABL1IS转录本水平介于1%~10%时，随访评估可检测到融合蛋白（即相对平均荧光强度比值rMFI%>1）；其三，rMFI%水平可预测经荧光原位杂交（Fluorescence In Situ Hybridization, FISH）分析定义的完全细胞遗传学缓解（Complete Cytogenetic Response, CCyR）。总体而言，该流式细胞术检测法（Flow Cytometry-Based Assay, FCBA）是一种快速检测技术，可完全应用于CML患者的常规临床管理。