伊马替尼耐药的慢性粒细胞白血病患者ABL激酶区点突变检测的意义

Examination of point mutations within the ABL kinase domine region of BCR/ABL in patients with chronic myeloid leukemia who develop imatinib resistance

目的研究发生伊马替尼(IM)耐药的慢性粒细胞白血病(CML)患者 BCR/ABL 融合基因 ABL 激酶区发生点突变的情况。方法采集11例(血液学耐药7例,遗传学耐药4例)共计17份发生 IM 耐药的 CML 患者 IM 治疗前后的骨髓,采用半筑巢式扩增长片段逆转录-PCR(RT-PCR)的方法,应用分别位于 BCR 基因和 ABL 基因的引物进行2次 PCR,扩增 BCR/ABL 基因 ABL 激酶区周围863 bp 碱基,进行纯化、测序序列同源性分析。结果本研究共发现3种突变,即 G250E、E255K和 T315I。其中,血液学耐药发生突变的频率为4/7,95%可信区间为18%～90%;而遗传学耐药发生突变的频率为1/4,95%可信区间为1%～81%。所有患者发生耐药前均未发生点突变。结论发生IM 耐药的 CML 患者 BCR/ABL 基因 ABL 激酶区周围存在高频率的点突变。通过对影响与 IM 结合的突变情况进行早期检测,有利于在发生耐药前进行治疗干预,为患者提供更有效的治疗选择。

Objective To investigate the point mutations within the adenosine triphosphate-binding region of BCR/ABL in patients with chronic myeloid leukemia who develop imatinib resistance. Methods We collected a total of 17 bone marrow samples obtained from 11 patients who showed hematology resistance （ n = 7 ） or cytogenetic refractoriness （ n = 4）. A long semi-nest PCR method was used to amplify the ABL kinase domain of the BCR/ABL allele. After two rounds of PCR reactions, we got a fragment of 863 bases The PCR products were purified and followed by sequencing. Results In total, we find three point mutations presented in all patients tested G250E, E255K and T315I. The mutation rate of hematology resistance is 4/ 7 ,and 95% confidence interval was 8%-90%, while mutation rate of cytogenetic refractoriness 1/4,95% confidence interval 1%-81%. For those patients whose samples were available, no single mutation were determined before imatinib resistance emerged. Conclusions There is high frequency of point mutations clustered within the adenosine triphosphate-binding region of BCR/ABL in patients with chronic myeloid leukemia. It＇s good for patients to switch to another therapeutic strategy when the mutations are detected.