Detection Of The BCR-ABL Fusion Gene Levels In Chronic Myeloid Leukemia Patients By Real-time Quantitative RT-PCR And Mutation Analysis In The Imatinib Resistant Patients

Objective Detecting the BCR-ABL mRNA levels in chronic myeloid leukemia patients of different clinical status by real-time fluorescent quantitation PCR(RQ-PCR) approach;and to research point mutations in the Abl kinase domain of BCR-ABL gene in Imatinib resistant patients.Method Collecting the bone marrow of 26 cases patients in chonic phase (10 new diagnosed and 16 treated with Imatanib already)and 19 cases patients in accelerated or blast phase,then detecting the expression of BCR-ABL mRNA transcript level in these patients by RQ-PCR;ABL kinase domain of the BCR-ABL fusion gene of 10 CML-CP patients and 13 Imanitib resistanted patients was amplified by nested reverse transcriptase- polymerase chain reaction technique, followed by direct sequencing the amplification product and sequence homologous analyzing.Results (1)The median bcr/abl mRNA expression level in 26 chonic phase CML patients was 0.72×10~4 [(0.13-2.4)×10~4]copies per 10~5 karyocytes ,and the differences were obviously; The median bcr/abl mRNA expression level was 3.6×104 [(0.26-4.7)×10~4]copies per 10~5 karyocytes and 4.3×10~4 [(0.18-8.3)×10~4]copies per 10~5 karyocytes in 8 patients in accelerated phase and 11 patients in blast crisis,respectively. The bcr/abl mRNA expression level in accelerated and blast crisis were significantly higher than that in chronic phase. Analyzed by statistics methods,the difference was significant(P﹤0.05) ,but the difference was no significant between the accelerated phase and blast crisis patients(P﹥0.05). The bcr/abl mRNA expression level of the 16 patients treated with Imatinib were detected before treatment and after the 6th,12th months respectively.In the 6th month,11 patients achieved MCyR,8 patients achieved CCyR;In the 12th month,all patients achieved MCyR and 12 patients achieved CCyR. The bcr/abl mRNA transcript level were descended more than 2 and 3 logarithm respectively compared to after treatment of the patients achieved MCyR and CCyR. The bcr/abl mRNA transcript level detected in the 12th month were continuing descending of the 8 patients achieved CCyR in the 6month.(2)The ABL point mutations were detected in 6 of 13 patients who develop imatinib resistance(6/13 46.2 % ) , we find four kinds of point mutationspresented in all patients tested E255V, Q252H,E279K and M351T. 3 patients had Q252H,the other mutation was E255K, E279K,M351T,each was tested in one person.3 patients(1 patient was treated in AP and 2 patients were treated in BP)showed homozygote mutation and 3 patients had a mixture of wild and mutant type.No point mutation was found in the 10 CML-CP patients during Imatinib treatment.Conclusion (1)RQ-PCR is a exact and reliable method with good sensitivity. To the Imatinib treated patients, continuous quantitative observation of BCR-ABL mRNA is much more valuable than a single consequence; and the BCR-ABL mRNA levels descended greatly in the CCyR patients.(2)Abl kinase point mutation is one of the main mechanisms of CML secondary resistance to imatinib.There is high frequency of point mutations clustered within the adenosine triphosphate—binding region of BCR/ABL in patients with chronic myeloid Leukemia. Long term regular monitoring of ABL kinase point mutation is necessary during imatinib treatment.Itˊs good for patients to switch to another therapeutic strategy when the mutations are detected.