Kreuzer K A, Lass U, Nagel S, Ellerbrok H, Pauli G, Pawlaczyk-Peter B, Siegert W, Huhn D, Schmidt C A
Department of Internal Medicine, Division of Hematology and Oncology, Charité-Virchow-Clinic, Humboldt University, Berlin, Germany.
Int J Cancer. 2000 Jun 1;86(5):741-6. doi: 10.1002/(sici)1097-0215(20000601)86:5<741::aid-ijc22>3.0.co;2-1.
Chimeric bcr/abl fusion proteins are thought to be the molecular 'pathogen' of chronic myelogenous leukaemia (CML). Expression levels of the respective fusion RNAs reflect disease progression as well as remission upon therapeutic intervention in CML patients. However, there is no quick and reliable method that would allow the quantitative routine monitoring of bcr/abl hybrid transcripts. A fluorescent probe-based PCR assay (TaqMan) has been described to quantitfy the exact amount of target sequences. We have established TaqMan real-time RT-PCRs for M-bcr/abl (b2a2, b2a3, b3a2, b3a3) and m-bcr/abl (e1a2) fusion transcripts as well as for beta-actin. All PCRs quantified as little as 10 copies/100 ng total cDNA. In order to investigate whether this procedure is appropriate for routine diagnostic monitoring, we performed retrospective measurements on 9 documented CML disease courses. Our data show that ongoing or relapsing CML is paralleled by increasing peripheral levels of bcr/abl fusion RNAs. Furthermore, sucessful anti-leukemic treatment is reflected by decreasing absolute bcr/abl transcript numbers. In contrast with conventional bcr/abl PCR techniques we could distinguish single positive values and gradually increasing copy numbers.
嵌合型bcr/abl融合蛋白被认为是慢性粒细胞白血病（CML）的分子“病原体”。在CML患者中，相应融合RNA的表达水平反映了疾病进展以及治疗干预后的缓解情况。然而，目前尚无快速可靠的方法可用于bcr/abl杂交转录本的定量常规监测。一种基于荧光探针的PCR检测方法（TaqMan）已被描述用于定量目标序列的确切数量。我们已经建立了针对M-bcr/abl（b2a2、b2a3、b3a2、b3a3）和m-bcr/abl（e1a2）融合转录本以及β-肌动蛋白的TaqMan实时逆转录PCR方法。所有PCR检测均可定量低至10拷贝/100 ng总cDNA。为了研究该方法是否适用于常规诊断监测，我们对9个有记录的CML病程进行了回顾性测量。我们的数据表明，持续或复发的CML与外周血中bcr/abl融合RNA水平的升高平行。此外，成功的抗白血病治疗表现为bcr/abl转录本绝对数量的减少。与传统的bcr/abl PCR技术不同，我们能够区分单个阳性值和逐渐增加的拷贝数。
