Detection Of Abl Kinase Domain Point Mutations In Chronic Myeloid Leukemia Patients Who Develop Imatinib Resistance And Its Clinical Significance

Objective (1)To detect the ratio of point mutations within Abl kinase domain inchronic myeloid leukemia(CML) patients who develop imatinib resistance in themechanisms of imatinib resistance、 the type of point mutations and its clinicalsignificance;(2) Analyze the type of BCR-ABL fusion genes in imatinib-resistant CMLpatients and whether the type of BCR-ABL fusion genes is different between imatinib-resistant CML patients and non-resistant patients.Methods (1)Collected bone marrow or blood samples of32imatinib-resistant CMLpatients including18of chronic phase(CP)、8of accelerated phase(AP) and6of blastcrisis(BC), and collected bone marrow or blood samples of30CML patients who had notdevelop imatinib resistance as control group;(2)Mononuclear cells were get using Ficoll,Trizol reagent was used to extract total RNA;(3) The synthesis of cDNA;(4)We detecteddifferent type of BCR-ABL fusion genes by polymerase chain reaction(PCR) in32imatinib-resistant CML patients and analyzed, in the control group,we analyzed the typeof BCR-ABL fusion genes in30CML patients who had not develop imatinib resistance;(5)An864bp cDNA fragment in Abl kinase domain was amplified using nested polymerasechain reaction, the product was then purified and direct-sequenced, sequence homologousanalysis was performed against NCBI database to detect the mutant base and itscorresponding amino acid.Result (1)12patients who had Abl kinase domain point mutation were detected in the 32imatinib-resistant CML patients, namely T315I(2)、Y253H(2)、M351T(2)、M244V(1)、F311I(1)、M388L(1)、G250E(1)、I293T(1) and M290T(1),among them, M290T mutationwas reported firstly; according to the clinical stage, the proportion of CP、 AP and BCpatients bearing the mutation was38.89%、37.5%and33.3%respectively;(2)In the32imatinib-resistant CML patients, the number of patients who expressed b3a2、b2a3、b2a2/b3a2was17、14and1respectively, and among them, in the12imatinib-resistantpatients who had Abl kinase domain point mutation, the number of patients who expressedb3a2、b2a3、 b2a2/b3a2was7、4and1respectively; in the30CML patients of controlgroup, the number of patients who expressed b3a2、b2a3、 b2a2/b3a2was13、5and3, inaddition, there was one patient who expressed b2a2/e20a2, and we could not detectBCR-ABL fusion gene in8patients.Conclusion (1) Abl kinase domain point mutation is one of the important mechanismsof imatinib resistance;(2) The level of resistance to imatinib varies among different types ofmutations;(3)The construct of the type of BCR-ABL fusion genes is differentbetween imatinib-resistant patients and patients who have not developed imatinibresistance; the proportion of BCR-ABL fusion genes is similar between imatinib-resistantCML patients who have Abl kinase domain point mutation and who do not have Abl kinasedomain point mutation.