Breit T M, Beishuizen A, Ludwig W D, Mol E J, Adriaansen H J, van Wering E R, van Dongen J J
Department of Immunology, Erasmus University/University Hospital Dijkzigt, Rotterdam, The Netherlands.
Leukemia. 1993 Dec;7(12):2004-11.
Polymerase chain reaction (PCR) techniques based on amplification and identification of leukemia-specific DNA sequences provide a sensitive diagnostic method for detection of minimal residual disease (MRD) with a detection limit of 10(-5) to 10(-6) (1-10 malignant cells in 10(6) normal cells). To date, the main leukemia-specific DNA sequences used as PCR targets in detection of MRD are breakpoint fusion regions of chromosome translocations and junctional regions of rearranged immunoglobulin (Ig) or T-cell receptor (TcR) genes. The recently identified tal-1 deletions involving the sil and tal-1 genes, provide a potential MRD-PCR target. tal-1 deletions are site-specific because they are mediated via recombination signal sequences homologous to Ig/TcR genes. In line with this homology, tal-1 deletions also show random insertion and deletion of nucleotides at their breakpoints, resulting in highly variable breakpoint fusion regions. The fusion region diversity can be applied to design patient-specific oligonucleotide probes. Our Southern blot analyses of a large series of 313 acute leukemias with a specific tal-1 deletion probe (SILDB) demonstrated that tal-1 deletions exclusively occur in T-cell acute lymphoblastic leukemia (T-ALL) and not in precursor B-ALL or acute non-lymphocytic leukemias. In addition, we did not detect tal-1 deletions in normal blood cells and normal thymocytes by PCR analysis. The diversity observed in tal-1 deletion fusion regions with an average insertion and deletion of approximately 7 and approximately 6 nucleotides, respectively, allowed us to design fusion-region-specific probes. The specificity of the fusion-region probes was proven and the detection limit of the MRD-PCR technique was tested in a series of dilution experiments. The observed detection limit of 10(-5) indicates that tal-1 deletions in T-ALL represent ideal leukemia-specific PCR targets for detection of MRD.
基于白血病特异性DNA序列扩增和鉴定的聚合酶链反应（PCR）技术为检测微小残留病（MRD）提供了一种灵敏的诊断方法，其检测限为10^(-5)至10^(-6)（10^6个正常细胞中有1 - 10个恶性细胞）。迄今为止，在MRD检测中用作PCR靶点的主要白血病特异性DNA序列是染色体易位的断点融合区域以及重排的免疫球蛋白（Ig）或T细胞受体（TcR）基因的连接区域。最近鉴定出的涉及sil和tal - 1基因的tal - 1缺失，提供了一个潜在的MRD - PCR靶点。tal - 1缺失具有位点特异性，因为它们是通过与Ig/TcR基因同源的重组信号序列介导的。与这种同源性一致，tal - 1缺失在其断点处也显示出核苷酸的随机插入和缺失，导致高度可变的断点融合区域。融合区域的多样性可用于设计患者特异性的寡核苷酸探针。我们用一种特异性tal - 1缺失探针（SILDB）对313例急性白血病进行的Southern印迹分析表明，tal - 1缺失仅发生在T细胞急性淋巴细胞白血病（T - ALL）中，而在前体B - ALL或急性非淋巴细胞白血病中未出现。此外，通过PCR分析我们在正常血细胞和正常胸腺细胞中未检测到tal - 1缺失。在tal - 1缺失融合区域观察到的多样性，平均分别有大约7个核苷酸的插入和大约6个核苷酸的缺失，这使我们能够设计融合区域特异性探针。融合区域探针的特异性得到了证实，并且在一系列稀释实验中测试了MRD - PCR技术的检测限。观察到的10^(-5)的检测限表明，T - ALL中的tal - 1缺失是检测MRD的理想白血病特异性PCR靶点。
