Monitoring Minimal Residual Disease in Leukemia Using Real-time Quantitative Polymerase Chain Reaction for Wilms Tumor Gene (WT1)
Monitoring Minimal Residual Disease in Leukemia Using Real-time Quantitative Polymerase Chain Reaction for Wilms Tumor Gene (WT1)
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DOI：
10.1007/bf02983561
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发表时间：
2003-11
影响因子：
2.1
通讯作者：
H. Tamaki;M. Mishima;M. Kawakami;A. Tsuboi;E. Kim;N. Hosen;K. Ikegame;M. Murakami;T. Fujioka;T. Masuda;Y. Taniguchi;S. Nishida;K. Osumi;T. Soma;Y. Oji;Y. Oka;I. Kawase;H. Sugiyama;H. Ogawa
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文献类型：
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作者：
H. Tamaki;M. Mishima;M. Kawakami;A. Tsuboi;E. Kim;N. Hosen;K. Ikegame;M. Murakami;T. Fujioka;T. Masuda;Y. Taniguchi;S. Nishida;K. Osumi;T. Soma;Y. Oji;Y. Oka;I. Kawase;H. Sugiyama;H. Ogawa
我们以前表明，Wilms肿瘤基因（WT 1）的表达水平，通过定量逆转录聚合酶链反应（RT-PCR）测定，是有用的指标微小残留病（MRD）在白血病和骨髓增生异常综合征。然而，在常规的定量RT-PCR（CQ-PCR）中，RT-PCR必须根据WT 1的表达水平进行不同的循环数。本研究建立了一种新的实时定量RT-PCR（real-time quantitative RT-PCR，RQ-PCR）方法。批内和批间变异性试验结果表明，实时荧光定量WT 1检测方法具有较高的重复性，RQ-和CQ-PCR方法检测的WT 1表达水平具有较强的相关性（r= 0.998）。此外，观察到用3种不同的对照基因（β-肌动蛋白、ABL和甘油醛-3-磷酸脱氢酶）标准化的WT 1转录值之间以及以K562细胞中WT 1表达作为参考的相对WT 1转录值与每微克RNA的绝对WT 1转录拷贝数之间存在强相关性。当在2例bcr-abl阳性急性淋巴细胞白血病患者中同时监测WT 1表达和少量bcr-abl表达时，两个MRD的变化大多是平行的，这表明我们的RQ-PCR方法的可靠性和有效性。结论：建立的RQ-PCR方法简便、可靠，可用于MRD的监测，为aWT 1assay的临床常规应用提供了依据。
We previously showed that Wilms tumor gene (WT1) expression level, measured by quantitative reverse transcriptase polymerase chain reaction (RT-PCR), was useful as an indicator of minimal residual disease (MRD) in leukemia and myelodysplastic syndrome. However, in conventional quantitative RT-PCR (CQ-PCR), RT-PCR must be performed for various numbers of cycles depending onWT1expression level. In the present study, we developed a new real-time quantitative RT-PCR (RQ-PCR) method for quantitatingWT1transcripts. Results of intraassay and interassay variability tests demonstrated that the real-timeWT1assay had high reproducibility.WT1expression levels measured by the RQ- and the CQ-PCR methods were strongly correlated (r= 0.998). Furthermore, a strong correlation was observed amongWT1transcript values normalized with 3 different control genes (β-actin,ABL, andglyceraldehyde-3-phosphate dehydrogenase) and between relativeWT1transcript values withWT1expression in K562 cells as the reference and absoluteWT1transcript copy numbers per microgram RNA. WhenWT1expression andminor bcr-ablexpression were concurrently monitored in 2 patients withbcr-abl-positive acute lymphoblastic leukemia, both MRDs changed mostly in parallel, indicating the reliability and validity of our RQ-PCR method. In conclusion, this RQ-PCR method is convenient and reliable for monitoring MRD and enables routine clinical use of aWT1assay.