Establishing The Technology Terrace Of Detecting PML-RARα Transcript Applied To Patients Of APL Using Rela-Time Reverse-Transcription Polymerase Chain Reaction

Background and Purpose: Acute promyelocytic leukemia (acute promyelocytic leukemia,APL) is a special Leukemia,accounting for adult acute myeloid leukemia 10%,which is the first successful application of induced differentiation and the molecular markers for tumor-specific treatment with significant effect of blood malignancies. Combined treatment with all-transretinoic acid (ATRA) and arsenic trioxide (arsenic trioxide,ATO As₂O₃) is highly successful in APL,providing long-term complete remissions and apparent cure rates to approximately 70% .However,disease relapse has been a major contributor to treatment failure and overall survival in patients,and the root cause of relapse is residual leukemia cells in patient,namely,acute leukemia minimal residual disease (MRD). Detection of PML/RARαtranscripts by molecular techniques constitutes an availably tool for MRD and predicting evolution in malignant diseases of the blood system. This study was purposed to establish a real-time quantitative reverse transcript polymerase chain reaction (RQ-PCR) for detection of PML/RARαfusion gene transcrips in patients with acute promyelocytic leukemia and to explore the relationship between the expression level of PML /RARa fusion gene transcript and the clinical status or efficacy of the therapy in APL. Methods: The conventional RT-PCR was used to amplify PML/RARαgene from cultured NB4 cells. Standard curves were constructed by modified real-time PCR on standard template after 10-fold serial dilutions of cDNA and ABL control gene with Taq Man probe. The reliability,repeatability and sensitivity were determined. The PML/RARαmRNA transcript levels of three periods of initial treatment,induction treatment approaching CR and after consolidation treatment in Initial 14 cases of L-APL patients were dynamically monitored by modified real-time quantitative RT-PCR. The normalized quotient (NQ ) of PML /RARαmRNA was calculated as followings: NQ = PML/RARαmRNA the copy number/ABL mRNA copy number of×10⁵.Results: The results of this experiment set up a real-time quantitative PCR method to detect the L-type PML/RARαfusion gene,the gene mapping of quantitative PCR standard curve,with regression coefficient of 0.99876. The experiment can detect the 10-5ugNB₄ cell cDNA in PML/RARαin fusion gene,and its determination of repeatability and stability of the Ct value of coefficients of variation were 1.77% and 2.11%. The average level copy numbers of PML/RARαtranscription reflecting PML /RARa fusion gene expression level in 14 newly diagnosed patients with APL was 3731 copies,when approaching to CR by using AS₂O₃ dual-induced therapy,PML / RARαfusion gene in the average level was 231 copies , After two cycles of consolidation treatment with ATRA and chemotherapy sequentially,the median was116 copies. One patient′s PML/RARαgene copy number was 5872 at diagnosis,the gene copy number was 267 after dual-induced therapy . After two cycles of sequential treatment while he is in complete remission at this period,but the transcription level of copy number slightly increased to 284,but NQ increased to 6247 when APL relapsed 80 days later. After ATRA and chemotherapy treatment,transcript levels gradually decreased to 1030 copies. Conclusion: It is concluded that the established real-time quantitative RT-PCR method is sensitive,reliable,accurate and repeatable,can quantitatively PML/RARαfusion gene copy number; can be used for acute promyelocytic leukemia diagnosis and the detection of minimal residual disease. After treatment of patients with PML/RARαfusion gene transcript levels decreased significantly over time and heighten when disease relapse .The expression levels of clinical disease progression with the same therapeutic relationship,can be used to monitored PML/RARαfusion gene copy number dynamically,is useful for monitoring minimal residual disease of leukemia,the evaluation to determine the efficacy and prognosis.