Detection of Cytogenetic and Molecular Biology in Chronic Myeloid Leukemia Patients
|School||Huazhong University of Science and Technology|
|Course||Department of Hematology|
|Keywords||Leukemia myeloid chronic Philadelphia chromosome Imatinib mesylate Karyotypingchronic myeloid leukemia Imatinib resistance Abl kinase domain pointmutation BCR-ABL fusion gene|
PART ONE Cytogenetic analysis of76cases with chronicmyeloid leukemiaObjective: To investigate clonal evolution of in chronic myeloid leukemia(CML) treatedwith IM．Methods: A total of76CML patients were retrospectively studied． Chromosomepreparation of bone marrow cells was made using direct and short-term culture. Karyotypewas analyzed by G-banding． Results: karyotypes in patients with CML before IMtreatment include typical Ph translocation, variation Ph translocation, Ph+additionalanomaly (Ph+clones with additional chromosomal aberrations, Ph+ACA), Ph-additionalanomaly (Ph-clones with additional chromosomal aberrations, Ph-ACA). A few patientsnot detected ACA before IM treatment could be detected the Ph+ACA or Ph-ACA aftertreatment. The CML patients with ACA after IM treatment may gain a completecytogenetic response.Conclusion: CML patients before and after IM treatment are both likely to be accompaniedby ACA in karyotype, but after treatment ACA clone scales can fall even disappearcompletely, get CCyR. PART TWO Detection and clinical significance of Abl kinasedomain point mutations in chronic myeloid leukemia patientsObjective: To detect the proportion and the type of point mutations within Abl kinasedomain in chronic myeloid leukemia(CML) patients, and analyze their clinicalsignificance；Methods：(1)Collected blood or bone marrow samples of58CML patients，48of chronicphase(CP)、6of accelerated phase(AP) and4of blast crisis(BC) included;(2)Mononuclearcells were get using Lymphocyte Separation Medium named as Ficoll, and then total RNAwas extracted usingTrizol reagent;(3) The synthesis of cDNA with special kit;（4）TwoDNA fragments in Abl kinase domain were amplified using nested PCR: The first roundPCR product (BCR-A) with1475bp amplication length was amplified from thesynthesized cDNA, followed by PCR product (ABL-B) with393bp amplification lengthand PCR product (ABL-C) with482bp amplification length, both of which were thesecond round PCR products amplified from BCR-A amplification. Then both of the ABL-BandABL-C products were purified and bidirection-sequenced, and the sequencing resultswere compared with NCBI database to analyze sequence homologous and to detect thelocation of mutant base, finally find its corresponding amino acid.Result: We detected11patients with Abl kinase domain point mutation in the58CMLpatients in all, namely T212A、K219E、I242V、G250E、E255K、M278V、E282V、K291E、 H295R、T315I、L323P、A344V、N358S、I347V and D363T; according to thedifferent clinical stage of differrent patients, the ratio of CP、AP and BC patients who bearAbl kinase domain point mutation was16.7%、33.3%and25%respectively.Conclusion:(1) one of the important mechanisms of imatinib resistance is Abl kinasedomain point mutation; (2) Different types of point mutations lead to different levels of resistance.(3) Abl kinase domain point mutation can be detected in newly diagnosed CML patients.(4) A few CML patients can simultaneously detect two or more point mutation.