Preexistence and evolution of imatinib mesylate-resistant clones in chronic myelogenous leukemia detected by a PNA-based PCR clamping technique
Preexistence and evolution of imatinib mesylate-resistant clones in chronic myelogenous leukemia detected by a PNA-based PCR clamping technique
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DOI：
10.1007/s00277-003-0644-y
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发表时间：
2003-05-01
影响因子：
3.5
通讯作者：
Dörken, B
中科院分区：
文献类型：
--
作者：
Kreuzer, KA;le Coutre, P;Dörken, B
最近，Abl序列内的各种突变被描述为对伊马替尼与Bcr/Abl结合产生负面影响，导致慢性髓性白血病（CML）细胞的细胞耐药。到目前为止，对于这些突变是预先存在的还是在伊马替尼治疗下发生的知之甚少，因为目前的突变分析受到约1:2（50%）至1:5（20%）的低灵敏度的限制。通过将基于肽核酸（PNA）的DNA夹紧与荧光杂交探针检测相结合，我们开发了一种新的高灵敏度技术，用于检测Bcr/Abl激酶结构域内已知的突变。用这种方法，我们调查了19例在伊马替尼治疗前和治疗期间难治性CML。通过PNA夹夹野生型Abl，我们可以有效提高对Bcr/Abl突变Thr315Ile、Glu255Lys和Tyr253His的检测灵敏度，在500个阴性（0.2%）中检测到I突变cDNA分子。我们在一个病例中观察到治疗前可检测到Gly255Lys突变。通过对口腔互换的DNA分析，可以排除遗传多态性。在两个病例中，已知突变的克隆进化在治疗过程中逐渐发展。在另一个病例中，最初可检测到的Tyr253His突变在治疗开始后消失，但在伊马替尼治疗6周后再次观察到。预先存在的和发展中的与不良预后相关的Bcr/Abl突变可以通过本技术安全地检测到。这可能有助于CML的风险分层，并可作为其他恶性疾病个体化分子监测和治疗策略的模型。
Recently, various mutations within the Abl sequence have been described that negatively affect imatinib binding to Bcr/Abl resulting in cellular resistance of chronic myeloid leukemia (CML) cells. So far, little is known as to whether these mutations are preexisting or develop under imatinib therapy as current mutation analyses are limited by a low sensitivity of approximately 1:2 (50%) to 1:5 (20%). By combining peptide nucleic acid (PNA)-based DNA clamping with a fluorescence hybridization probe assay, we developed a new and highly sensitive technique for the detection of known mutations within the Bcr/Abl kinase domain. With this approach we investigated 19 cases of CML refractory to imatinib treatment before and during therapy. By clamping of wild-type Abl through PNA we could effectively enhance the detection sensitivity for the Bcr/Abl mutations Thr315Ile, Glu255Lys, and Tyr253His such that I mutant cDNA molecule could be detected in 500 negatives (0.2%). We observed in one case that a Gly255Lys mutation was detectable before treatment. By DNA analysis of buccal swaps, a genetic polymorphism could be excluded. In two cases clonal evolution of known mutations developed gradually under treatment. In another case an initially detectable Tyr253His mutation disappeared after therapy onset but was again observed after 6 weeks of imatinib treatment. Preexisting and evolving Bcr/Abl mutations associated with an unfavorable prognosis could be safely detected by the presented technique. This may facilitate risk stratification in CML and may serve as a model for individualized molecular monitoring and therapeutic strategies in other malignant diseases.