Li Ai-Hong, Forestier Erik, Rosenquist Richard, Roos Göran
Department of Medical Biosciences and Pathology, Umeå University, Umeå, Sweden.
Exp Hematol. 2002 Oct;30(10):1170-7. doi: 10.1016/s0301-472x(02)00892-5.
Clone specific immunoglobulin (Ig) and T-cell receptor (TCR) gene sequences can be used as molecular targets for detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL). Real-time quantitative PCR (RQ-PCR) with no need for post-PCR processing is an attractive approach for detection and quantification of specific DNA or RNA sequences. In the present study we evaluated a real-time PCR-based technology for MRD quantification in children with precursor-B ALL.
DNA samples from 36 children with newly diagnosed precursor-B ALL were available for molecular analysis. All patients were uniformly treated according to the Nordic Society of Pediatric Hematology and Oncology (NOPHO) protocols from 1992. A real-time PCR assay was applied for MRD quantification using LightCycler technology and the SYBR green fluorescent dye for detection of clone-specific Ig and TCR gene rearrangements as target sequences. The specificity of the PCR products was verified by melting curve analysis.
Thirty-four of the 36 children with precursor-B ALL (94%) displayed at least one clonal Ig heavy chain (IgH) or TCR gene sequence useful as a molecular target. These clone-specific targets were successfully applied for real-time PCR quantification in all but one patient. Melting curve analysis was important for identifying all specific PCR products. In 32 pediatric precursor-B-ALL patients an MRD level >/=10(-3) at day 29 during induction treatment was significantly correlated with later bone marrow relapse (p = 0.0025).
Real-time PCR using clone-specific primers and the SYBR green dye for detection is a feasible technique for identifying patients at risk for relapse. This approach provides an easily applicable tool for detection of IgH/TCR gene rearrangements in the routine setting. Melting curve analysis allowed clear distinction between specific rearrangements and unspecific background signals.
克隆特异性免疫球蛋白（Ig）和T细胞受体（TCR）基因序列可作为检测急性淋巴细胞白血病（ALL）微小残留病（MRD）的分子靶点。无需PCR后处理的实时定量PCR（RQ-PCR）是检测和定量特定DNA或RNA序列的一种有吸引力的方法。在本研究中，我们评估了一种基于实时PCR的技术用于前体B-ALL儿童的MRD定量。
36例新诊断的前体B-ALL儿童的DNA样本可用于分子分析。所有患者均按照1992年北欧儿科血液学和肿瘤学会（NOPHO）方案进行统一治疗。应用实时PCR检测法，使用LightCycler技术和SYBR绿色荧光染料，以克隆特异性Ig和TCR基因重排作为靶序列来定量MRD。通过熔解曲线分析验证PCR产物的特异性。
36例前体B-ALL儿童中有34例（94%）显示至少有一个克隆性Ig重链（IgH）或TCR基因序列可作为分子靶点。除一名患者外，这些克隆特异性靶点均成功用于实时PCR定量。熔解曲线分析对于识别所有特异性PCR产物很重要。在32例儿科前体B-ALL患者中，诱导治疗第29天时MRD水平≥10⁻³与随后的骨髓复发显著相关（p = 0.0025）。
使用克隆特异性引物和SYBR绿色染料进行实时PCR检测是识别有复发风险患者的可行技术。这种方法为在常规情况下检测IgH/TCR基因重排提供了一种易于应用的工具。熔解曲线分析能够清晰区分特异性重排和非特异性背景信号。
