Bcr/abl Gene Expression In Leukemia By Using Fluorogenic Probe Quantitative RT-PCR Method

Objective To establish an objective and quantitative criterion for the assessment of bcr/abl mRNA fusion gene expression level and prognosis of leukemia and minimal residual disease (MRD) in leukemia. Provide a useful tool for leukemia diagnosis and minimal residual disease inspectation.Methods: Expression of bcr/abl' gene was measured with fluorogenic probe quantitative RT-PCR (FQ-RT-PCR) in 34 cases of leukemia at different disease-developing stage. Nineteen patients with chronic myeloid leukemia (CML), 15 with acute leukemia (ALL) were tested. Mononudear cells were obtained from bone marrow and/or peripheral blood. Total RNA of them was prepared and calculated by optical densitometry. cDNA was synthesized by reverse transcription (RT). PCR amplification was run in Micro-Amp reaction tubes placed in rack of PE 5700 Sequence Detection System. The reaction condition was: 93℃ 2min and 93 C30s, 55C60s, 40cycles. The results and subsequent calculations were performed by PE5700 sequence detection system.Results Expression of bcr/abl was revealed in 17 of 19 CML and none of 12 normal controls. Eleven patients were obtained at diagnosis and/or blastic transformation and its median level of expression was 2. 60× 104copies/ul. Eight patients were obtained after a -interferon therapy was 2.43× 102copies/ul. There is significance difference between two groups. ( p<0. 05 . 2 of 15 ALL were detected bcr/abl mRNA. Four patients with CML and 2 ALL were follow-up. Follow-up data were obtained in 6 bcr/abl positive cases, bcr/abl gene expression level was markedly decreased after CR whereas highly increased when replased or blastic transformation, bcr/abl gene expression level and the kinetic of bcr/abltranscripts was correlated well with the clinical outcome and prognosis of leukemia.Conclusion The FQ-RT-PCR method is sensitive, specific, reliable and accurate. The results expressed in copies were easy for evaluation and comparative. This method can minimize the risk of contamination. FQ-RT-PCR assay may detect kinetic evolution of bcr/abl transcripts during the course of the disease and has the potential to predict prognosis on the basis of assistance with early treatment decisions. It is more suitable for the diagnosis and follow-up with the t (9;22) translocation.