急性早幼粒细胞白血病PML-RARα融合基因实时定量RT-PCR标化法和常规法计算的比较

Comparisons of conventional and normalized calculation of quantitative real-time RT-PCR detecting PML-RARα fusion gene in patients with acute promyelocytic leukemia

目的改进实时定量RT—PCR的检测计算方法，提高定量RT—PCR在急性早幼粒细胞白血病（APL）微量残留病变监测的临床应用价值。方法采用实时定量RT—PCR和常规定性RT—PCR检测31例APL患者在治疗前后融合基因PML—RARα的表达水平，对两种计算方法进行比较：常规按照患者治疗前后自身比较计算（常规法）和治疗前标准化基线水平计算（标化法）。结果31例患者采用两种定量计算方法结果近似，标化法计算结果示患者在治疗缓解、巩固治疗和维持治疗期间融合基因PML—RARα转录本对数下降值分别为（2．0±1．9）、（4．9±1．4）和（5．7±0．1），常规法计算结果为（1．9±1．9）、（4．8±1．3）和（5．7±0．4）。在维持治疗阶段标化法定量RT—PCR的结果显示患者个体差异更小。按照标化法计算结果，次要分子生物反应（对数值≥3）和主要分子生物反应（对数值≥5）标准仍然适用。结论采用标化法计算实时定量RT—PCR结果能较好的对APL患者的微量残留病变进行监测，一定程度上降低了患者个体差异对检测结果的影响，同时适用于治疗前标本缺如的患者。

Objective To optimize the calculation of quantitative real time RT-PCR （Q-RT-PCR） of PML-RARα 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-RARα transcripts were measured before and after treatment. The conventional Q-RT-PCR calculation was directly compared the post-treatment transcript leveI 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-RARα 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 〉15.0 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 pretreatment samples.