[Comparisons of conventional and normalized calculation of quantitative real-time RT-PCR detecting PML-RAR alpha fusion gene in patients with acute promyelocytic leukemia].

ABSTRACT

OBJECTIVE: To optimize the calculation of quantitative real time RT-PCR (Q-RT-PCR) of PML-RARalpha in patients with acute promyelocytic leukemia (APL) for molecular monitoring of minimal residual disease (MRD). METHODS: By using both regular reverse transcription polymerase chain reaction (RT-PCR) and Q-RT-PCR, the expression levels of PML-RARalpha transcripts were measured before and after treatment. The conventional Q-RT-PCR calculation was directly compared the post-treatment transcript level with the respective pre-treatment one (DoseN) in the individual patient while the standardized calculation was based on the calculation of standardized pre-treatment DoseN of all patients. RESULTS: In 181 samples from 31 patients, the results of log-reduction of PML-RARa after induction, at the end of consolidation and during maintenance by conventional method were (1.9 +/- 1.9), (4.8 +/- 1.3) and (5.7 +/- 0.4), respectively, while by standardized method were (2.0 +/- 1.9), (4.9 +/- 1.4) and (5.7 +/- 0.1), respectively. Of notice, the result was with significant less variation of the latter methods during maintenance therapy. Moreover, with defined criteria of molecular response (3.0-4.9 log-reduction as minor and > or = 5.0 log-reduction as major molecular response), the standardized method was validated in clinical settings. CONCLUSION: The standardized method is superior to the conventional method for calculation of Q-RT-PCR results. The new method can reduce the individual variation in monitoring the MRD and is feasible even for patients with unavailable pre-treatment samples.