van der Velden V H J, Wijkhuijs J M, Jacobs D C H, van Wering E R, van Dongen J J M
Department of Immunology, Erasmus MC, Rotterdam, The Netherlands.
Leukemia. 2002 Jul;16(7):1372-80. doi: 10.1038/sj.leu.2402515.
Several studies have shown that quantitative detection of minimal residual disease (MRD) predicts clinical outcome in childhood acute lymphoblastic leukemia (ALL). In this report we investigated the applicablility of T cell receptor gamma (TCRG) gene rearrangements as targets for MRD detection by real-time quantitative PCR analysis. Seventeen children with precursor-B-ALL and 15 children with T-ALL were included in this study. Using an allele-specific (ASO) forward primer in combination with germline Jgamma reverse primers and Jgamma TaqMan probes, a reproducible sensitivity of < or =10(-4) (defined by strict criteria) was obtained in only four out of 19 (21%) TCRG gene rearrangements in precursor-B-ALL patients and in 10 out of 15 (67%) TCRG gene rearrangements in T-ALL patients. The main reason for not obtaining a reproducible sensitivity of < or =10(-4) in approximately 60% of cases was the non-specific amplification of TCRG gene rearrangements in normal T-lymphocytes. A maximal sensitivity of < or =10(-4) (defined by less strict criteria) was obtained in 42% of TCRG gene rearrangements in precursor-B-ALL patients. The number of inserted nucleotides was significantly higher in T-ALL (mean: 8.5) as compared to precursor-B-ALL (mean: 6.8) and appeared to be the most important predictor for reaching a reproducible sensitivity < or =10(-4). The usage of a touchdown PCR or the usage of an ASO reverse primer in combination with Vgamma member forward primers and TaqMan probes did not clearly improve the overall results. Nevertheless, RQ-PCR analysis of TCRG gene rearrangements in follow-up samples obtained from 12 ALL patients showed the applicability of this method for MRD detection. We conclude that RQ-PCR analysis of TCRG gene rearrangements can be used for the detection of MRD, but that sensitivities might be limited due to non-specific amplification. This method is applicable in the majority of T-ALL patients and in almost half of precursor-B-ALL patients, particularly when used as second-choice target for confirmation of the MRD results obtained via the first-choice target.
多项研究表明，儿童急性淋巴细胞白血病（ALL）中微小残留病（MRD）的定量检测可预测临床结局。在本报告中，我们研究了T细胞受体γ（TCRG）基因重排作为实时定量PCR分析检测MRD靶点的适用性。本研究纳入了17例前体B-ALL患儿和15例T-ALL患儿。在前体B-ALL患者的19个（21%）TCRG基因重排中，只有4个通过使用等位基因特异性（ASO）正向引物与种系Jγ反向引物及Jγ TaqMan探针结合，获得了≤10⁻⁴（由严格标准定义）的可重复灵敏度；在T-ALL患者的15个（67%）TCRG基因重排中，有10个获得了该灵敏度。约60%的病例未获得≤10⁻⁴可重复灵敏度的主要原因是正常T淋巴细胞中TCRG基因重排的非特异性扩增。在前体B-ALL患者42%的TCRG基因重排中获得了≤10⁻⁴（由不太严格的标准定义）的最大灵敏度。与前体B-ALL（平均：6.8）相比，T-ALL中插入核苷酸的数量显著更高（平均：8.5），并且似乎是达到≤10⁻⁴可重复灵敏度的最重要预测指标。使用降落PCR或使用ASO反向引物与Vγ成员正向引物及TaqMan探针结合并未明显改善总体结果。然而，对12例ALL患者随访样本中TCRG基因重排的实时定量PCR分析表明该方法可用于MRD检测。我们得出结论，TCRG基因重排的实时定量PCR分析可用于检测MRD，但由于非特异性扩增，灵敏度可能受限。该方法适用于大多数T-ALL患者和几乎一半的前体B-ALL患者，特别是当用作确认通过首选靶点获得的MRD结果的第二选择靶点时。
