| Objective To establish the real time quantitative PCR (RQ-PCR) assay and analyze its results in detection and quantification the isoforms (Long-form, Short-form and Variant-form) and the specific isomers (bcr1/2, P4R3, P46R3, bcr3 and P2R3) of PML/RARαfusion transcripts in patients with acute promyelocytic leukemia (APL).Methods Three pairs of primer and TaqMan probe were designed according to 'Europe Against Cancer' (EAC) program for detecting the most frequent isoforms (Long-form. Short-form and Variant-form) of PML/RARαtranscripts. To evaluate the utility of this assay, bone marrow samples from 51 newly diagnosed APL patients were detected and the RQ-PCR end-point products were identified by electrophoresis and sequencing. Specific primers of different isoforms (bcr1/2, P4R3, P46R3, bcr3 and P2R3) were designed and the isomer-specific RQ-PCR method for detecting each isoform was established. To evaluate the utility of this assay, bone marrow samples from 45 newly diagnosed APL patients were detected.Results In 51 newly diagnosed APL patients, there are 33 cases with Long-form PML/RARαtranscripts, 3 with Variant-form PML/RARαtranscripts and 15 with Short-form PML/RARαtranscripts. There was significant negative correlation between initial WBC count and the age of APL patients (R =-0.376, P=0.014), mainly seen in the patients with Long-form and the Variant-form (R=-0.51, .P=0.005). The WBC count is higher in the Short-form group than in the Long-form group (P=0.041). However, there was no statistical difference in the sex, age, Hb, Plt, proportion of the blast and promyelocyte in the bone marrow, the time of achieving hematologic complete remission and molecular complete remission between the Long-form patients and the Short-form patients.In repeated tests, maximal sensitivity of 10 copies/μl for each 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.In 36 cases with Long-form or Variant-form transcripts, the median absolute and normalized amount of PML/RARαtranscripts were 3.75×10~2-6.20×10~5 copies/50 ng (median 7.24×10~3 copies/50 ng) and 0.51%-676.87% (median 9.31%) respectively. In 15 cases with Short-form transcripts, the median absolute and normalized amount of PML/RARαtranscripts were 2.26×10~3-2.33×10~6 copies/50 ng (median 1.17×10~5 copies/50 ng) and 12.04%-802.68% (median 95.26%) respectively. The normalized amount of Short-form PML/RARαtranscripts is significantly higher than that of Long-form (P＜0.01). Among 33 cases with follow-up data, neither of the time of achieving hematologic complete remission nor molecular complete remission was correlated with initial WBC, the proportion of blast and promyelocyte in the bone marrow and the expression level of the PML/RARαtranscripts.Short-form RQ-PCR system recommended by the 'EAC' could amplify positive signal in all of 51 cases, Variant-form RQ-PCR system could amplify positive signal in both Long-form and Variant-form positive cases, however, Long-form RQ-PCR system could only amplify positive signal in Long-form-positive cases. Electrophoresis and sequencing of end-point products amplified by short form RQ-PCR system revealed three bands in Long-form (621 bp, 477 bp and 218 bp) and Variant-form (567 bp, 423 bp and 218 bp) positive patients. There were P4R3 and P46R3 isoforms in Long-form and Variant-form positive cases. There was P2R3 isoform in Short-form positive cases.According to the results mentioned above, we further established the RQ-PCR to detect six specific isoforms of PML/RARαtranscripts. In repeated tests, maximal sensitivity of 10 copies/μl for each RQ-PCR was also 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 can not amplify any other isoform.In 31 cases with Long-form and Variant-form transcript, the median absolute and normalized amount of bcr1/2 isoform were 1.73×10~2-7.65×10~4 copies/50 ng (median 2.14×10~4 copies/50 ng) and 0.86%-208.01% (median 11.61%) respectively. The median absolute and normalized amount of P4R3 isoform were 7.3×10~2-1.38×10~5 copies/50 ng (median 1.46×10~4 copies/50 ng) and 0.71%-266.19% (median 18.13%) respectively. The median absolute and normalized amount of P46R3 isoform were 1.65×10~2-1.05×10~5copies/50 ng (median 1.41×10~4 copies/50 ng) and 0.53%-222.90% (median 8.65%) respectively. In 14 cases with Short-form transcript, the median absolute and normalized amount of bcr3 isoform were 7.53×10~2-3.22×10~5 copies/50 ng (median 6.07×10~4 copies/50 ng) and 11.91%-326.55% (median 58.03%) respectively. The median absolute and normalized amount of P2R3 isoform were 48.87-1.39×10~4 copies /50 ng (median 8.72×10~2copies/50 ng) and 0.02%-57.71% (median 0.93%) respectively. The expression level of bcr3 isoform was significantly higher than that of bcr1/2 (P=0.001). In the Long-form positive patients, there is no statistical difference in the expression level of bcr1/2, P4R3 and P46R3 (P=0.344). The expression level of bcrl/2 was correlated with P46R3 (R=0.73, P<0.001), and also with the P4R3 (R=0.5, P=0.04), but there is no correlation between bcr1/2 and P4R3. 14 cases with Short-form transcript contained low level of P2R3 isoform, which was significantly lower than that of bcr3 (P <0.001). However, there was no correlation between them (P=0.714). Neither of the time of achieving hematologic complete remission nor molecular complete remission was correlated with the expression level of each isoform.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 relapsed.Conclusion RQ-PCR for detecting the isoforms of PML/RARαfusion transcripts is a sensitive, reliable quantitative assay and can be used in the diagnosis of APL and measurement of MRD.
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