[Detection of PML/RARa transcripts in acute promyelocytic leukemia by real-time quantitative reverse transcription...

This study was purposed to establish a real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) for detection of PML/RARa fusion gene in patients with acute promyelocytic leukemia (APL) 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. The conventional RT-PCR was used to amplify PML/RARa gene from cultured NB4 cells. Standard curves were constructed by modified real-time PCR on standard template after 10-fold serial dilutions of cDNA of 1 microg NB4 cells. The sensitivity, stability and repeatability of this method was determined. The PML/RARa gene transcripts of bone marrows in 4 APL patients before and after treatment and in 1 APL patient relapsed after complete remission were dynamically detected by modified real-time quantitative RT-PCR. The results indicated that the sensitivity of real-time quantitative RT-PCR for detecting PML/RARa fusion gene was about 10(-5) microg cDNA from NB4 cells, the repeatability and reproducibility of this method were satisfactory, intra-and inter-assay coefficients of variation were 2.1% and 3.8%. The copy numbers of PML/RARa transcripte reflecting PML/RARa fusion gene expression level in 4 newly diagnosed patients with APL were 1884, 5533, 1803, 4677 and the median was 3 475. After ATRA + chemotherapy copy numbers of PML/RARa transcript decreased to 40, 135, 79, 29, and mean was 121. Another patient's PML/RARa gene copy number was 8600 at diagnosis, the gene copy number was 730 after therapy for 4 months, although he was in complete remission, but copy number increased to 11 000 when APL relapsed 3 months later. The copy number efficiently reduced to 1200 after ATRA + chemotherapy. It is concluded that the established real-time quantitative RT-PCR method is sensitive, reliable, accurate and repeatable. The efficiency of method was finally tested for patient samples, showing a PML/RARa transcript copy number in APL patients significantly decrease after therapy, and increase at the time of relapse which indicate that changes of fusion gene expression levels coincide with clinical progress of disease. This method can be used to detect the minimal residual disease, assess response to treatment and evaluate prognosis of disease.