建立RQ-PCR方法检测急性早幼粒细胞白血病患者６种不同PML/RARα异构体

［摘要］目的: 建立检测急性早幼粒细胞白血病（acute promyelocytic leukemia, APL）患者PML/RARα融合基因６种特异性异构体（bcr1/2、P4R3、P46R3、bcr3及P2R3）的实时定量PCR（RQ-PCR）技术，并评价其特异性。方法: 设计不同异构体的特异性引物和探针，建立异构体特异性RQ-PCR方法对６种特异性异构体PML/RARα阳性模板进行扩增，评价异构体定量检测的特异性，并检测10例APL患者中不同异构体的含量。结果： 所建立的各异构体特异性RQ-PCR法最大敏感性均达10拷贝/μL，但其重复性较差，108~102拷贝/μL阳性模板的重复性良好，正常对照及空白对照无扩增信号，每个异构体特异性RQ-PCR均不能扩增其他异构体。5例长型（bcr1）和1例变异型（bcr2）阳性APL患者中bcr1/2异构体表达量为7.71%~89.71%（中位数13.70%），P4R3异构体表达量为0.71%~16.26%（中位数4.48%），P46R3异构体表达量为3.57%~14.09%（中位数6.33%）。4例短型（bcr3）患者中bcr3异构体表达量为21.43%~197.04%（中位数100.69%），P2R3异构体表达量为0.34%~9.07%（中位数2.45%）。1例短型患者经诱导治疗缓解后bcr3和P2R3异构体转录本明显下降，复发时又明显升高。结论： 检测PML/RARα异构体特异性RQ PCR方法敏感、可靠，可用于APL患者不同异构体的定量检测和微小残留病灶的随访监测。

Quantification of six different PML/RARα isoforms in patients with acute promyelocytic leukemia using quantitative real time PCR

Objective: To establish a quantitative real time PCR(RQ-PCR) method for detecting the six different PML/RARα isoforms(bcr1/2,P4R3,P46R3,bcr3,P2R3) in acute promyelocytic leukemia (APL) patients, and evaluate the specificity of isoform-specific RQ-PCR. Methods: Specific primers and probe of different isoforms were designed and isoform-specific RQ-PCR method for detecting each isoform was established. To evaluate the utility of this assay, bone marrow samples from 10 APL patients were detected. Results: In repeated tests, maximal sensitivity of 10 copies/μL for each isoform-specific RQ-PCR was obtained, however, the reproducible maximal sensitivities achieved 100 copies/μL. In the normal controls and the no-template control, any amplified fluorescent signals were not detected. Each isoform-specific RQ-PCR could not amplify other isoforms. In five bcr1-positive cases and one bcr2 positive case, the expression level of bcr1/2, P4R3 and P46R3 isoforms was 7.71%-89.71% (median 13.70%), 0.71%-16.26% (median 4.48%),3.57%-14.09% (median 6.33%), respectively. In four bcr3-positive cases, the expression level of bcr3 and P2R3 isoforms was 21.43%-197.04% (median 100.69%) and 0.34%-9.07% (median 2.45%). The expression level of bcr3 and P2R3 isoforms significantly decreased in one case achieving complete remission after induction therapy compared to that in initial diagnosis, and significantly increased after relapse. Conclusion: Isoform-specific RQ-PCR method for six PML/RARα isoforms was a sensitive, reliable quantitative assay and could be used in the detection of the six different PM/RARα isoforms of APL and the measurement of minimal residual disease.