Study On Using The Real Time Polymerase Chain Reaction To Quantitate PML-RARα MRNA In APL

Acute promyelocytic leukemia (APL) accounts for approximately 10%to15% of all cases of acute myeloid leukemia (AML). Using all-trans retinoic acid(ATRA), AS2O3 and chemotherapy can get complete remission(CR) , and the survival rate of 5 years is the first place of all AML. But there is 5% to 15% of APL patients relapse in the end. Relapse of disease still remains as the major problem in clinical management and the minimal residual disease (MRD) has been confirmed to be associated with leukemia relapse in the period of CR in the past decades. MRD was defined small leukemic cells which can not be detected in conventional morphological methods in the period of CR. MRD spreads widely in the body from 100 to 1010. 105 to 1010 is considered it perhaps will relaps. Below 105 the leukemic cells can be controlled by the immunologic mechanism. Thus it is important to investigate and monitor MRD in that consulting the treatment after CR, finding relaps early and extending life span even curability of disease. Conventional cytogenetics is not sensitive for MRD, though it is helpful to diagnose APL. Molecular biology and immunology are the major methods for MRD at present. Sensibility of flow cytometry (FCM) is low ,furthermore immunophenotype often changs in the course of disease. RT-PCR has false positive and it can not quantitate. Thus it has becomed the focus to build a new method reflecting leukemic burden after CR. An available index is urgent to assess response to treatment and the rate of relaps. Real-time PCR can be used to detect leukemic burden and MRD. We applied TaqMan probe PCR system to quantify the dose of PML-RAR alpha fusion transcripts in a series of APL patients at distinct disease stages.To study the value of real-time PCR in diagnose APL and detect MRD, we have to establish a real-time PCR method to detect PML-RAR alpha fusion transcripts at first. We choose TaqMan probe technique. NB4 having t(15;17) was used to establish the standard curves of L-type and GAPDH. S-type was artificial synthesized. The coefficient of correlation(R) of the standard curve is about 0.98. The expression of PML-RARαtranscripts is mormalized using the housekeeping GAPDH gene. This technique allows detection of 10 copies of PML-RAR alpha or GAPDH plasmids. Repeatability and reproducibility of the method were satisfying as intra- and inter- assay. Variation coefficients(CV) were not higher than 5%. Stability is 2.72% and 4.05% respectively. PML-RAR alpha fusion transcripts is about 1×104copies per 1μg RNA, and GAPDH transcripts is about 1×107copies per 1μg RNA.The lowest sensitivities of PML-RARαDoseN obtained in our experiments was thus 0.0025. Though our test, we have established real-time PCR method successfully and verify good reproducibility and repeatability, good stability, high sensitivity and specificity.A total of 36 APL patients, including 24 new-diagnosed APL patients, 2 relapsed patients and 10 patients after CR, were analysed. Real-time Quantitative RT-PCR was used to measure the mormalized dose(DoseN) of PML-RAR alpha fusion transcripts. The results shew: high levels of PML-RAR alpha transcripts above 1×103 were detected in 24 newly diagnosed patients. A large variation of PML-RAR alpha DoseN between individual APL cases from 1528.3 to 528430(median was 4506.95)at presentation was observed in this study. This variation showed no correlation with either the initial WBC count or the percentage of promyelocytes in BM, and it can most likely be ascribed to the expression status of PML-RAR alpha fusion gene at the single cell level in a given patient. PML-RAR alpha DoseN was significantly decreased after the first complete remission, the median was 25.17, while the conventional RT-PCR gave positive results. This suggested real-time PCR is more sensitive. After consolidation chemotherapy, a further decrease of PML-RAR alpha transcript was achieved in most cases investigated. All patients with long disease free survival had a constantly low PML-RARαDoseN below 2×102 and a higher level predicted impending relapse. Hence, we tentatively propose that a PML-RARαDoseN of 2×102 may serve as a realistic'threshold'for molecular remission. This was performed in 6 patients followed-up for 12 months. In 2 patients, a temporary decrease of PML-RAR alpha transcript was realized after CR or consolidation chemotherapy(DoseN from 4306.7 to 167,and 3291.5 to 504.8). An increase of PML-RAR alpha DoseN to2864 and to 3576.3 at 9 and 7 months respectively, after CR, accompanied with clinical relapse. This suggested if PML-RAR alpha DoseN becomes high, it often accompied relapse in short term.Real-time PCR is useful in reflecting leukemic burden, assessing response to treatment and indicating the ultimate clinical outcome or curability of disease. The sample in our study is not enough ,which made it necessary to obtain more sample to test our conclusion.