Application of multiplex rt-PCR assay for screening rare or cryptic chromosome translocations in de novo patients with acute myeloid leukemia / 中国实验血液学杂志

This study was aimed to investigate the clinical feasibility of using multiplex PT-PCR assay for screening rare/cryptic chromosome translocations in patients with de novo acute myeloid leukemia. For 126 patients with de novo AML-M4/M5 without common chromosome translocations including t(15;17), t(8;21) and t(16;16), 3 parallel multiplex RT-PCR assays were set up to detect 6 mll-related gene rearrangements (mll/af10, mll/af17, mll/ell, mll/af9, mll/af6 and mll/enl) with low detection rate and 4 rare fusion genes (dek/can, tls/erg, aml1/mds (evi1) and npm/mlf1). The results showed that 11 patients with positive result from 126 patients were detected which involved in 5 molecular abnormalities. Among them, 10 cases were AML-M5 (16.67%), 1 cases AML-M4 (1.51%). The marker chromosomes were observed in 2 cases out of 11 cases through conventional karyotyping analysis, the karyotyping analysis in 1 case was not performed because this case had 1 mitotic figure only, no any cytogenetic aberrations were found in other 8 cases through R-band karyotyping analysis. It is concluded that multiplex RT-PCR designed in this study can quickly, effectively and accurately identify the rare/cryptic chromosome translocations and can be used in clinical detection.

Comparisons of conventional and normalized calculation of quantitative real-time RT-PCR detecting PML-RAR alpha fusion gene in patients with acute promyelocytic leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>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).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

FLT3 internal tandem duplication in patients with acute promyelocytic leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze the mutation of FLT3 internal tandem duplication (FLT3-ITD) in bone marrow cells from patients with newly-diagnosed acute promyelocytic leukemia (APL).</p><p><b>METHODS</b>The mutation of FLT3-ITD in bone marrow mononuclear cells (MNCs) from 103 APL patients were screened by polymerase chain reaction (PCR) and the clinical features of ITD positive patients were analyzed.</p><p><b>RESULTS</b>FLT3-ITD mutations were identified in 19.4% (20/103) patients. It was associated with short/variant form of PML-RAR alpha isoforms (P < 0.0001). Among the 20 patients with FLT3-ITD mutation, 16 presented with short, 2 with variant and 2 with long form of PML-RAR alpha isoforms. Patients with FLT3-ITD mutation also presented significantly higher initial peripheral white blood cell count (WBC) (P < 0.01), especially in those with short/variant PML-RAR alpha isoforms (P = 0.015). For patients with long form PML-RAR alpha, there was no significant difference in initial WBC. Out of FLT3-ITD positive patients, 18/20 (90%) obtained complete remission and 16 evaluable patients (2 lost follow-up) remained in first remission in a median follow-up of 26 (11-47) months.</p><p><b>CONCLUSION</b>FLT3-ITDs are frequently identified in patients with newly diagnosed APL. FLT3-ITD mutation is associated with short/variant form of PML-RAR alpha fusion gene and higher initial WBC. No significant impact on treatment outcome was observed with a limited follow-up.</p>

Study of mutation and single nucleotide polymorphism of PDGFRbeta and SHIP gene in acute myeloid leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the significance of mutation and single nucleotide polymorphism (SNP) of class III receptor tyrosine kinases such as PDGFRbeta and SHIP in acute myeloid leukemia (AML) patients.</p><p><b>METHODS</b>Screening of the mutation and SNP of PDGFRbeta and SHIP by genomic PCR, RT-PCR, directly sequencing and Mass-ARRAY system was carried out in 273 AML patients.</p><p><b>RESULTS</b>The mutations of PDGFRbeta R685C and SHIP Q1153L were detected for the first time in AML patients. The positivity ratio was 0.73% and 0.36% respectively.</p><p><b>CONCLUSION</b>The mutations of PDGFRbeta R685C and SHIP Q1153L may contribute to leukemogenesis of AML.</p>

Detection of MRD and its clinical application--review / 中国实验血液学杂志

With recent development and progress achieved in the diagnosis and treatment of leukemia, relapse of disease still remains as the major problem in clinical management and the minimal residual disease (MRD) has been confirmed to be associated with leukemia relapse in the past decades. Due to the low sensitivity, morphology-based assays have limitation in the MRD monitoring. With the development of molecular assays, especially the real-time RT-PCR method have been a sensitive, precise and reliable tool to MRD detection. Numerous clinical studies demonstrated that the existence of MRD reflects the clinical and molecular response, and remain as a major prognostic factor for leukemia. Another important application of MRD detection is to evaluate the efficacy of different therapy at molecular level. In this paper, the different methods and their clinical application for MRD detection were systemically reviewed and it is confident that the establishment of standardized MRD detection system will be important in the clinical prevention for relapse of leukemia.

Clinical observation of the short-term efficacy of the treatment with combination of all-trans retinoic acid (ATRA) and arsenic trioxide (As2O3) in newly diagnosed acute promyelocytic leukemia (APL) / 中华血液学杂志

<p><b>OBJECTIVE</b>To study whether all-trans retinoic acid (ATRA) combined with arsenic trioxide (As(2)O(3)) in acute promyelocytic leukemia (APL) treatment could further improve the clinical and molecular remission rate.</p><p><b>METHOD</b>Thirty one newly-diagnosed APL patients of whom 15 were males, 16 females and median age 35.4 years entered into the study. They were treated with ATRA 25 mg x m(-2) x d(-1) combined with As(2)O(3) 0.16 mg x kg(-1) x d(-1) until complete remission (CR). The doses were adjusted according to white blood cell (WBC) counts, occurrence of RA syndrome and the status of liver function. CR rate, time of reaching clinical and molecular remission and side effects were observed.</p><p><b>RESULT</b>Two patients died 2 approximately 3 days after the treatment due to intracranial hemorrhage, and 29 (93.5%) achieved CR. The average time for achieving CR was 25.1 +/- 3.9 days. Hyperleukocytosis emerged in 66.5% and hepatic damages in 65.5% of the patients, they were ameliorated within one week after reduction of the As(2)O(3) dose or its suspension. The PML/RAR alpha fusion gene that was positive in all 29 patients before treatment turned negative only in 3 cases (10.3%) after obtaining CR (CR1) and in 10/13 cases (77%) after consolidation treatment. Up to now (1-8 months follow-up), all 29 patients remain in CR1.</p><p><b>CONCLUSION</b>ATRA combined with As(2)O(3) in de novo APL treatment can yield a high CR rate without intolerable side effects. Long term effect needs further observation.</p>