伊马替尼治疗慢性粒细胞白血病患者ABL激酶区点突变的检测

Detection of ABL kinase domain point mutations in chronic myeloid leukemia patients receiving imatinib treatment

目的探讨伊马替尼治疗慢性粒细胞白血病(CML)患者ABL酪氨酸激酶区点突变的发生情况及临床意义。方法采用巢式PCR扩增23例CML患者不同时期的40份骨髓标本的ABL激酶区,对扩增产物纯化、双向测序并进行序列同源性比对。结果7人(30.43%)检测出点突变,共导致5种类型的氨基酸替换:T315I3例,Y253H、E255K、F317L及G321W各1例。慢性期、加速期和急变期患者点突变的例数分别为2例、2例和3例。其中6例患者测序前经400mg/d伊马替尼治疗无效,加大伊马替尼剂量至(600～800)mg/d,随访3～6个月,仅F317L患者获得部分细胞遗传学缓解,另外5例患者均无遗传学反应,且Y253H和1例T315I疾病进展至急变期。G321W为初治慢性期患者,经400mg/d伊马替尼治疗达到完全血液学缓解,BCR-ABL+细胞比例显著下降。结论ABL激酶区点突变是CML患者对伊马替尼耐药的重要原因。不同类型的突变导致的耐药程度不完全相同,且并非所有点突变都会导致耐药的发生。监测ABL激酶区点突变有助于预测疗效并及早调整治疗。

Objective To analyze the frequency and clinical significance of ABL tyrosine kinase point mutations in chronic myeloid leukemia （CML） patients receiving imatinib treatment. Methods Nested reverse transcriptase-polymerase chain reaction （RT-PCR） was performed on 40 bone marrow samples from 23 patients to amplify the ABL kinase domain, followed by direct sequencing and sequence homologous analysis. Results In the 23 patients analyzed, the ABL domain point mutations was detected in 7 patients who presented with 5 types of nucleotide changes, namely T315I（n=3）, Y253H, E255K, F317L and G321W. The incidence of mutations in chronic phase （CP）, accelerated phase （AP） and blast phase （BP） was 25.00%, 40.00% and 30.00%, respectively. For 6 of the 7 patients with mutations who were resistant to imatinib before sequencing, the daily drug dose had been increased to 600-800 mg daily for poor response to 400 mg/day imatinib. During the follow-up for 3-6 months, only the patient with F317L achieved major cytogenetic response （MCR）, and the patient with Y253H and 1 of the 3 with T3151 progressed to BP. The newly diagnosed patient with G321W IN cp achieved a complete hematologic remission and had a significant decrease of the proportion of BCR-ABL-positive cells. Conclusions ABL kinase point mutation is an important mechanism of imatinib resistance. The type of mutations is associated with the level of resistance to imatinib, and detection of ABL kinase point mutations by direct sequencing may help estimate the prognosis and plan for therapeutic strategy adjustment.