慢性髓性白血病患者伊马替尼治疗后ABL激酶区点突变分析

Analysis of ABL tyrosine kinase point mutations in imatinib treated chronic myelogenous leukemia patients

目的研究伊马替尼治疗慢性髓性白血病(CML)患者ABL酪氨酸激酶区点突变发生情况。方法采用巢式PCR扩增30例伊马替尼治疗时处于加速/急变或慢性期后期,伊马替尼临床治疗效果不佳,在治疗不同时间的45例CML患者的骨髓标本,检测bcr/abl融合mRNA的abl激酶区,纯化、测序并进行序列同源性分析。结果13例患者检出点突变(43.3%),类型分别为E255K4例,G250E4例,F359C+G250E、E355G、M244V、Y253H及D276G1各1例。其中12例发生疾病进展,转入加速/急变期,8例疾病进展时表现为纯合型点突变,3例为野生与突变型ABL同时存在(1例无进展时标本)。加速/急变期患者(1.5～30个月,中位5个月)明显早于慢性期后期患者(11～45个月,中位25.5个月)检出点突变(P<0.05)。4/7例随访患者在急变前的7～15个月分别检测到不同程度的点突变。结论ABL激酶区点突变是伊马替尼治疗CML失效的主要原因之一。定期监测ABL激酶区点突变有助于及早采取干预治疗,提高疗效。

Objective To evaluate ABL tyrosine kinase point mutations in imatinib treated chronic myelogenous leukemia （CML）patients. Methods A total of 45 bone marrow samples from 30 CML patients were included in this work. The patients were in accelerated/blast phase （AP/BP） or late-chronic phase （CP） at the start of imatinib and usually showed resistance to imatinib. ABL kinase domain of BCR-ABL allele was amplified by nested reverse transcriptase-polymerase chain reaction technique, followed by direct sequencing and sequence homologous analyzing. Results The ABL point mutation was detected in 13 of 30 patients,12 of them had progressed to advanced phase, The other patient who was in late chronic phase showed point mutation when she was at 45th months of imatinib treatment, but she was still in complete eytogenetie remission at 50th months and is doing well. 4 patients had Glu255Lys mutation and 4 had Gly250Glu, the other types of mutation were Phe359Cys, Glu355Gly, Met244Val, Tyr253His and Asp276Gly, each was tested in one patient. 11/12 patients who progressed to advanced disease and showed point mutation were collected samples in advanced stage, 8 patients showed homozygote mutation, and 3 patients had a mixture of wild and mutant type. In advanced stage patients, mutations were detected in a median of 5 months （ranged 0.5 -30 months） , it appeared much ealier than that in late CP patients（25.5 months, ranged 11 -45months, P 〈 0.05 ）. In 4/7 followed up patients, the intensity of point mutation increased gradually within 7 - 15 months before disease progression. 6 patients did not showed ABL point mutation while their disease were in progression. Conclusions Abl kinase point mutation is one of the main mechanisms of CML secondary resistance to imatinib. Long term regular monitoring of ABL kinase point mutation is necessary during imatinib treatment.