Takenokuchi M, Yasuda C, Takeuchi K, Nakamachi Y, Mukai M, Kondo S, Kumagai S, Saigo K, Murayama T, Koizumi T, Tatsumi E
Department of Clinical Laboratory, Kobe University Hospital, Kobe, Japan.
Clin Lab Haematol. 2004 Apr;26(2):107-14. doi: 10.1111/j.1365-2257.2004.00587.x.
A quantitative nested reverse transcriptase polymerase chain reaction (QN-RT-PCR) method was developed using a plasmid cDNA containing the AML1/ETO (MTG8) fusion transcript from Kasumi-1 cells, an acute-myelogenous leukemia cell line with the t(8;21) translocation. In this method, the plasmid was detectable at a concentration of 10(-17) m. The fusion transcript in a mixture of 10(7) Rice94 (Burkitt lymphoma cell line) cells containing two Kasumi-1 cells was detectable at 10(-17) m. In a previously published real-time PCR method, the plasmid containing the fusion transcript was detectable at 10(-16) m or higher, and 20 or more Kasumi-1 cells were detectable in 10(7) Rice94 cells. Thus, this QN-RT-PCR method is more sensitive than the real-time PCR. When the same samples were examined by real-time PCR and our QN-RT-PCR method, in one patient in clinical remission after chemotherapy and allogeneic-bone marrow transplantation (BMT), the transcript was detected by QN-RT-PCR 60 days prior to hematological relapse, in contrast to 10 days before hematological relapse by real-time PCR. The transcript level was below 10(-17) m (undetectable) with this QN-RT-PCR in patients in clinical remission after chemotherapy and BMT, while it was 10(-15)-10(-16) m in patients in clinical remission after chemotherapy alone. The quantitative difference of the transcript level in minimal residual disease (MRD) between these two different types of clinical remission was estimated to be at least 10(2)-fold. This QN-RT-PCR method is useful for predicting hematological relapse and for quantitatively estimating MRD in different types of clinical remission.
利用一个含有来自Kasumi-1细胞（一种具有t(8;21)易位的急性髓性白血病细胞系）的AML1/ETO（MTG8）融合转录本的质粒cDNA，开发了一种定量巢式逆转录聚合酶链反应（QN-RT-PCR）方法。在该方法中，质粒在浓度为10^(-17) m时可被检测到。在含有两个Kasumi-1细胞的10^7个Rice94（伯基特淋巴瘤细胞系）细胞的混合物中，融合转录本在10^(-17) m时可被检测到。在先前发表的实时PCR方法中，含有融合转录本的质粒在10^(-16) m或更高浓度时可被检测到，并且在10^7个Rice94细胞中可检测到20个或更多的Kasumi-1细胞。因此，这种QN-RT-PCR方法比实时PCR更灵敏。当用实时PCR和我们的QN-RT-PCR方法检测相同样本时，在一名化疗和异基因骨髓移植（BMT）后处于临床缓解期的患者中，QN-RT-PCR在血液学复发前60天检测到转录本，而实时PCR在血液学复发前10天检测到。在化疗和BMT后处于临床缓解期的患者中，用这种QN-RT-PCR检测转录本水平低于10^(-17) m（不可检测），而在仅化疗后处于临床缓解期的患者中，转录本水平为10^(-15)-10^(-16) m。这两种不同类型临床缓解期微小残留病（MRD）中转录本水平的定量差异估计至少为10^2倍。这种QN-RT-PCR方法可用于预测血液学复发以及定量估计不同类型临床缓解期的MRD。
