Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
J Hematol Oncol. 2011 Feb 8;4:7. doi: 10.1186/1756-8722-4-7.
BCR-ABL kinase domain (KD) mutation is the major mechanism contributing to suboptimal response to tyrosine kinase inhibitors (TKI) in BCR-ABL-positive chronic myeloid leukemia (CML) patients. T315I mutation, as one of the most frequent KD mutations, has been shown to be strongly associated with TKI resistance and subsequent therapeutic failure. A simple and sensitive method is thus required to detect T315I mutation at the earliest stage.
A single-tube allele specific-polymerase chain reaction (AS-PCR) method was developed to detect T315I mutation in a mixture of normal and mutant alleles of varying dilutions. Denaturing high performance liquid chromatography (DHPLC) and direct sequencing were performed as a comparison to AS-PCR.
T315I mutant bands were observed in the mixtures containing as low as 0.5-1% of mutant alleles by AS-PCR. The detection sensitivity of DHPLC was around 1.5-3% dilution whereas sequencing analysis was unable to detect below 6.25% dilution.
A single-tube AS-PCR is a rapid and sensitive screening method for T315I mutation. Detection of the most resistant leukemic clone in CML patients undergoing TKI therapy should be feasible with this simple and inexpensive method.
BCR-ABL 激酶结构域（KD）突变是导致 BCR-ABL 阳性慢性髓性白血病（CML）患者对酪氨酸激酶抑制剂（TKI）治疗反应不佳的主要机制。T315I 突变是最常见的 KD 突变之一，已被证明与 TKI 耐药和随后的治疗失败密切相关。因此，需要一种简单而敏感的方法在最早阶段检测 T315I 突变。
开发了一种单管等位基因特异性聚合酶链反应（AS-PCR）方法，用于检测不同稀释度的正常和突变等位基因混合物中的 T315I 突变。变性高效液相色谱（DHPLC）和直接测序作为 AS-PCR 的比较方法。
通过 AS-PCR，在含有低至 0.5-1%突变等位基因的混合物中观察到 T315I 突变带。DHPLC 的检测灵敏度约为 1.5-3%的稀释度，而测序分析无法检测到低于 6.25%的稀释度。
单管 AS-PCR 是一种快速灵敏的 T315I 突变筛选方法。用这种简单廉价的方法，应该可以在接受 TKI 治疗的 CML 患者中检测到最耐药的白血病克隆。
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