Quantification of PML/RAR alpha fusion gene transcripts in patients with acute promyelocytic leukemia by using real-time quantitative PCR / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish and evaluate a real time quantitative PCR (RQ-PCR) method for detection and quantification the PML/RAR alpha fusion gene transcripts in patients with acute promyelocytic leukemia (APL).</p><p><b>METHODS</b>Three pairs of primers and TaqMan probe were designed for detecting the most frequent PML/RAR alpha transcripts (L-form, S-form and V-form) and normal ABL was used as an internal control. A real time PCR condition was established to detect PML/RAR alpha and ABL positive templates with a series of dilutions. To evaluate this assay, bone marrow samples from 6 APL patients were detected.</p><p><b>RESULTS</b>In repeated tests, maximal sensitivities of 10 copies/microL were obtained, while reproducible maximal sensitivity achieved 100 copies/microL. In 10 normal controls, no amplified fluorescent signals were detected. The median absolute and normalized amount of PML/RAR alpha fusion gene transcripts were 4.27 x 10(3)-3.36 x 10(5) copies/50 ng (median 4.33 x 10(4)copies/50 ng) and 29.38%-600.53% (median 48.12%) respectively. One case showed significant decrease of PML/RAR alpha fusion gene transcripts after induction therapy compared to that at the time of diagnosis, while the fusion transcripts significantly increased after relapsed.</p><p><b>CONCLUSION</b>RQ-PCR is a sensitive, reliable quantitative assay and can be used in the diagnosis of APL and measurement of MRD.</p>

Expression of PML-RAR alpha fusion transcripts detection by using RQ-PCR / 中华血液学杂志

<p><b>OBJECTIVE</b>To establish the real time quantitative PCR (RQ-PCR) assay according to 'Europe Against Cancer' (EAC) program and analyze the results of detection and quantification of different PML-RAR alpha transcript isoforms in patients with acute promyelocytic leukemia (APL).</p><p><b>METHODS</b>Three RQ-PCR systems were performed to detect the most frequent PML-RAR alpha transcripts (L-form, S-form and V-form) in 30 APL patients and the RQ-PCR end-point products were identified by electrophoresis.</p><p><b>RESULTS</b>S-form RQ-PCR system could amplify positive signals of three isoforms in all of 30 cases, and V-form RQ-PCR system could do so in both L-form and V-form positive cases, however, L-form RQ-PCR system could only do so in L-form-positive cases. Electrophoresis and sequencing of end-point products amplified by S-form RQ-PCR system revealed three bands in each of L-form (621 bp, 477 bp and 218 bp) and V-form (567 bp, 423 bp and 218 bp) positive patients samples.</p><p><b>CONCLUSIONS</b>RQ-PCR, sensitive and reliable, can be used for monitoring the minimal residual disease in APL patients, however, its results should be interpreted carefully if it is used for detection of PML-RAR alpha fusion transcripts prospectively.</p>