Pongers-Willemse M J, Verhagen O J, Tibbe G J, Wijkhuijs A J, de Haas V, Roovers E, van der Schoot C E, van Dongen J J
Dept of Immunology, University Hospital Rotterdam/Erasmus University Rotterdam, The Netherlands.
Leukemia. 1998 Dec;12(12):2006-14. doi: 10.1038/sj.leu.2401246.
Analysis of minimal residual disease (MRD) can predict outcome in acute lymphoblastic leukemia (ALL). A large prospective study in childhood ALL has shown that MRD analysis using immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements as PCR targets can identify good and poor prognosis groups of substantial size that might profit from treatment adaptation. This MRD-based risk group assignment was based on the kinetics of tumor reduction. Consequently, the level of MRD has to be defined precisely in follow-up samples. However, current PCR methods do not allow easy and accurate quantification. We have tested 'real-time' quantitative PCR (RQ-PCR) using the TaqMan technology and compared its sensitivity with two conventional MRD-PCR methods, ie dot-blot and liquid hybridization of PCR amplified Ig/TCR gene rearrangements using clone-specific radioactive probes. In RQ-PCR the generated specific PCR product is measured at each cycle ('real-time') by cleavage of a fluorogenic intrinsic TaqMan probe. The junctional regions of rearranged Ig/TCR genes define the specificity and sensitivity of PCR-based MRD detection in ALL and are generally used to design a patient-specific probe. In the TaqMan technology we have chosen for the same approach with the design of patient-specific TaqMan probes at the position of the junctional regions. We developed primers/probe combinations for RQ-PCR analysis of a total of three IGH, two TCRD, two TCRG and three IGK gene rearrangements in four randomly chosen precursor-B-ALL. In one patient, 12 bone marrow follow-up samples were analyzed for the presence of MRD using an IGK PCR target. The sensitivity of the RQ-PCR technique appeared to be comparable to the dot-blot method, but less sensitive than liquid hybridization. Although it still is a relatively expensive method, RQ-PCR allows sensitive, reproducible and quantitative MRD detection with a high throughput of samples providing possibilities for semi-automation. We consider this novel technique as an important step forward towards routinely performed diagnostic MRD studies.
微小残留病（MRD）分析可预测急性淋巴细胞白血病（ALL）的预后。一项针对儿童ALL的大型前瞻性研究表明，以免疫球蛋白（Ig）和T细胞受体（TCR）基因重排作为PCR靶点进行MRD分析，能够识别出规模可观的预后良好和预后不良组，这些组可能会从治疗调整中获益。这种基于MRD的风险组划分是基于肿瘤减少的动力学。因此，在随访样本中必须精确界定MRD水平。然而，目前的PCR方法无法实现简便且准确的定量。我们使用TaqMan技术测试了“实时”定量PCR（RQ-PCR），并将其灵敏度与两种传统的MRD-PCR方法进行比较，即斑点印迹法以及使用克隆特异性放射性探针的PCR扩增Ig/TCR基因重排的液相杂交法。在RQ-PCR中，通过切割荧光内源TaqMan探针，在每个循环（“实时”）测量生成的特异性PCR产物。重排的Ig/TCR基因的连接区决定了ALL中基于PCR的MRD检测的特异性和灵敏度，通常用于设计患者特异性探针。在TaqMan技术中，我们采用相同的方法，在连接区位置设计患者特异性TaqMan探针。我们开发了引物/探针组合，用于对四个随机选择的前体B-ALL中总共三种IGH、两种TCRD、两种TCRG和三种IGK基因重排进行RQ-PCR分析。在一名患者中，使用IGK PCR靶点对12份骨髓随访样本进行了MRD检测。RQ-PCR技术的灵敏度似乎与斑点印迹法相当，但低于液相杂交法。尽管它仍然是一种相对昂贵的方法，但RQ-PCR能够实现灵敏、可重复且定量的MRD检测，且样本通量高，为半自动化提供了可能。我们认为这项新技术是朝着常规进行诊断性MRD研究迈出的重要一步。
