Pfitzner T, Engert A, Wittor H, Schinköthe T, Oberhäuser F, Schulz H, Diehl V, Barth S
Department I of Internal Medicine at the University of Cologne, Germany.
Leukemia. 2000 Apr;14(4):754-66. doi: 10.1038/sj.leu.2401706.
Several new therapeutic approaches for the treatment of monoclonal B cell lymphomas are currently being investigated. In parallel with new therapeutic modalities, more sensitive diagnostic methods are needed. These methods should be highly sensitive in detecting very low amounts of malignant cells and should be specific for the malignant clone. In addition, these methods should allow the quantification of residual tumor cells. In this study a new real-time polymerase chain reaction (LightCycler) was evaluated to quantify residual tumor cells in monoclonal B cell malignancies. This technology combines the advantages of rapid cycling PCR with the online detection of PCR products using fluorescent dyes. Our assay is based on immunoglobulin heavy chain (IgVH)-specific PCR with allele-specific primers complementary to hypervariable CDRII and CDRIII regions. A set of framework region III (FRIII)-specific hybridization probes was used for detection of the specific amplification product, and IgVH copy number was quantified with the cloned IgVH sequence as an external standard. The approach was evaluated with the Hodgkin lymphoma cell line L428 in order to quantify L428 dilutions. L428 cells mixed with peripheral blood mononuclear cells (PBMNCs) were detected and quantified with a sensitivity of one cell within 1 x 10(5) PBMNCs. Sample DNA from the peripheral blood and from the bone marrow of two patients with B-CLL was analyzed in the new set up at different time points before and after therapy. Statistically significant changes in IgVH copy numbers were documented in both patients. We conclude that this technology offers an additional opportunity to detect and quantify residual tumor cells in B-CLL over several log steps with a high sensitivity. The kinetics of residual tumor cell counts in B-CLL can be analyzed by this method.
目前正在研究几种治疗单克隆B细胞淋巴瘤的新方法。与新的治疗方式并行，需要更灵敏的诊断方法。这些方法应在检测极少量恶性细胞时具有高度敏感性，并且应对恶性克隆具有特异性。此外，这些方法应能够对残留肿瘤细胞进行定量。在本研究中，对一种新的实时聚合酶链反应（LightCycler）进行了评估，以定量单克隆B细胞恶性肿瘤中的残留肿瘤细胞。该技术将快速循环PCR的优势与使用荧光染料在线检测PCR产物相结合。我们的检测基于免疫球蛋白重链（IgVH）特异性PCR，使用与高变CDRII和CDRIII区域互补的等位基因特异性引物。一组框架区域III（FRIII）特异性杂交探针用于检测特异性扩增产物，并以克隆的IgVH序列作为外标定量IgVH拷贝数。为了定量L428稀释液，用霍奇金淋巴瘤细胞系L428对该方法进行了评估。检测并定量了与外周血单核细胞（PBMNCs）混合的L428细胞，在1×10（5）个PBMNCs中检测单个细胞的灵敏度为1。在治疗前后的不同时间点，对两名B-CLL患者外周血和骨髓的样本DNA进行了新方法分析。两名患者的IgVH拷贝数均有统计学上的显著变化。我们得出结论，该技术提供了一个额外的机会，以高灵敏度在几个对数级上检测和定量B-CLL中的残留肿瘤细胞。该方法可分析B-CLL中残留肿瘤细胞计数的动力学。
