Sensitive detection and quantification of minimal residual disease in chronic myeloid leukaemia using nested quantitative PCR for BCR-ABL DNA
Sensitive detection and quantification of minimal residual disease in chronic myeloid leukaemia using nested quantitative PCR for BCR-ABL DNA
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DOI：
10.1111/j.1751-553x.2010.01236.x
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发表时间：
2010-12-01
影响因子：
3
通讯作者：
Morley, A. A.
中科院分区：
文献类型：
--
作者：
Bartley, P. A.;Ross, D. M.;Morley, A. A.
越来越多的慢性髓系白血病(CML)患者接受酪氨酸激酶抑制剂治疗后，bcr-abl基因表达水平在灵敏的实时定量逆转录聚合酶链式反应(RT-qPCR)方法中检测不到，而一种测量低水平患者微小残留病(MRD)的方法可能是有价值的。在分离和测序患者特异性bcr-abl断裂点后，建立了一种基于DNA的套式定量聚合酶链式反应方法，并用该方法和一轮RT-qPCR方法对24例CML患者的38份标本进行了MRD检测。用患者DNA与正常DNA的混合实验表明，DNA qPCR可以在大约10(-6)的限度内检测到bcr-abl序列。在两种方法均可检出MRD的22份标本中，DNA定量聚合酶链式反应的结果与同一样本的RT-qPCR结果具有良好的相关性。另16例标本经RT-qPCR检测未检测到bcr-abl基因。在16个样本中，有8个样本在1.1×10(-5)至2.8×10(-4)的范围内检测到bcr-abl DNA，其余8个样本均未检测到bcr-abl。在一名停用伊马替尼的患者中，在顺序样本中观察到MRD几乎增加了1000倍，达到5.2x10(-4)。套式DNA定量聚合酶链式反应比单轮逆转录定量聚合酶链式反应更敏感，可用于监测MRD水平极低的CML患者。
Increasing numbers of patients with chronic myeloid leukaemia (CML) treated with tyrosine kinase inhibitors achieve undetectable levels of BCR-ABL mRNA using sensitive quantitative real-time reverse transcriptase PCR (RT-qPCR) methods and a method to measure minimal residual disease (MRD) in patients with low levels could be of value. Following isolation and sequencing of the patient-specific BCR-ABL breakpoint, a DNA-based nested qPCR assay was established, and MRD was measured by this method and one-round RT-qPCR in 38 samples from 24 patients with CML. Mixing experiments using patient DNA in normal DNA indicated that DNA qPCR could detect BCR-ABL sequences at a limit of approximately 10(-6). In 22 samples in which MRD was detectable by both methods, comparison of the results of DNA qPCR with the results obtained on the same sample by RT-qPCR showed good correlation. In another 16 samples, BCR-ABL mRNA was not detectable by RT-qPCR. In 8 of the 16 samples, BCR-ABL DNA was detected at levels ranging from 1.1 x 10(-5) up to 2.8 x 10(-4) and in the remaining eight samples BCR-ABL was not detected by either method. In one patient, who had stopped imatinib, an almost 1000-fold rise in MRD, to 5.2 x 10(-4) was observed in sequential samples. Nested DNA qPCR was more sensitive than one-round RT-qPCR and could be used for the monitoring of patients with CML with very low levels of MRD.