The Investigation Of Minimal Residual Disease (MRD) In Acute Promyelocytic Leukemia

Objectives: In China, acute myeloid leukemia (AML) is the major part of leukemia, which is one of the tenth frequency malignancies. Minimal residual disease (MRD) is defined that there are residual leukemia cells in the range of 106-108 in the patients who are in complete remission (CR). Now, a large number of studies have shown that detection of MRD, significantly correlates with clinical outcome in many hematologic malignancies. How to improve the detection of MRD has become an important study for leukemia therapy. Recently, the application of retinoic acid and arsenic trioxide has made it possible for patients to achieve CR. However, relapse still occurs in part of patients. Some prospective research indicated that it is useful for monitoring of MRD for predicting relapse. Real-time quantitative PCR can monitor the PCR products in the whole amplification. The method is very specific, accurate and automatic. Acute promyelocytic leukemia is characterized by the presence of the t(15;17),which produces the PML-RARαfusion gene. We have established a real-time quantitative PCR RNA substances for the detection of PML-RARαfusion gene. Using this method, we were able to detect MRD in 25 patients in diagnosis, CR or relapse. Moreover, we successfully follow-up analyzed 6 patients. Flow cytometry could monitor MRD by detecting tumor-associated immunophenotypes. We detect the MRD in patients with the combination of CD15/CD117/CD33/HLA-DR by calculating the MRD%. At the same time, we analyze three apoptosis-realated protein Bax,Bcl-2 and Bcl-xl expression in minimal residual disease in APL and get information of patients by the ratio of Bax/Bcl-2 and antiapoptosis index. Fluorescence in situ hybridization is a method which apply fluorescence signed probes and chromosomes.Moreover, it could display fusion gene directly and reliably. In this study, the PML-RARαfusion gene is the molecular marker in monitoring the MRD in APL. We analyzed real-time quantitative PCR, flow cytometry and fluorescence in situ hybridization in monitoring the MRD and investigate whether the protocol is meaningful for clinical diagnosis.Methods:1. Establishing a real-time quantitative PCR RNA substances for detection of PML-RARαfusion gene and ABL control gene in acute promyelocytic leukemia. Using the total RNA extracted from a human APL cell line NB4 as a template, the PML-RARαfusion gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) with a pair of specific primers. The amplified fragment of PML-RARαfusion gene was subcloned into cloning vector PMD-18T and extracted the plasmid. We have developed a method by transcription in vitro after the plasmid was digested by HindⅢ. On the other hand, the ABL control gene RNA substance was achieved directly by transcription in vitro. The standard curves were made by real-time quantitative PCR reaction using the dilutions of RNA as the template. 2. We have accessed the PML-RARαfusion gene relative copies in the patients of diagnosis, CR or relapse. 3. Using FCM detect the MRD in patients with the combination of CD15/CD117/CD33/HLA-DR by calculating the MRD%.4.Analyzing three apoptosis-realated protein Bax,Bcl-2 and Bcl-xl expression in minimal residual disease in APL and get information of patients by the ratio of Bax/Bcl-2 and antiapoptosis index. 5. Monitoring the MRD by using the molecular marker that is the PML-RARαfusion gene.Results:1.We could get good standard curves from the two RNA substances. 2. Real-time quantitative PCR could monitor the MRD in patients(P<0.05). 3. At diagnosis, the PML-RARαfusion gene expression level was high. When the patients became CR, the level was decreased. If the patient with a high level PML-RARαduring therapy and could subsequent relapse. 4.The patient is still in CR with MRD%>1, which indicated the patient with poor outcome. It is similar to the result of real-time quantitative PCR. 5. The APL patients with low Bax/Bcl-2 ratio or high antiapoptosis index have a poor clinical outcome. 6. The APL patients who are positive in Fluorescence in situ hybridization may be associatedwith relapse.Conclusion: The assessment of PML-RARαexpression level might be helpful in identifying patients with higher relapse risk and clinical diagnosis.