[Detection of PML/RARalpha gene transcripts in 46 newly diagnosed acute promyelocytic leukemia patients by real-time quantitative reverse-transcription polymerase chain reaction].
In order to explore the application of real-time quantitative reverse-transcription polymerase chain reaction (Q-PCR) for detecting PML/RARalpha gene transcripts in patients with acute promyelocytic leukemia (APL), the bone marrow samples from 46 newly diagnosed APL patients were collected for analysis. Three plasmids containing cDNA fragments of the bcr1-, bcr3-form PML/RARalpha and ABL control gene were constructed respectively. The ABI Prism 7500 Sequence Detection System using Taqman fluorogenic probes was used to quantify target gene. PML/RARalpha mRNA was detected by Q-PCR in 46 APL patients and 40 non-APL patients. The normalized quotient (NQ) of PML/RARalpha mRNA was calculated as followings: NQ = PML/RARalpha mRNA copy numbers/ABL mRNA copy numbers. Immunophenotype of acute promyelocytic leukemia was determined by four-color flow cytometry. The results showed that the coefficients of variation (CV) of inter-day assay and intra-day assay by using Q-PCR were 1.58% and 0.88% respectively. Q-PCR could detect reproducibly 5 copies of target gene per 100 ng RNA and no pseudopositive results were found. The median NQ of PML/RARalpha mRNA was 0.450 (0.084 - 1.082) in 46 APL patients. There was no indication of any correlation of PML/RARalpha mRNA type with age, sex, hemoglobin, platelet count, percentage of promyelocytes in bone marrow detected by morphology or flow cytometry, PML/RARalpha NQ, or signs of clinically diagnosed coagulation/bleeding disorders. Compared with bcr1-form cases, bcr3-form cases had more M(3v) phenotype (42.9% vs 9.4%, P = 0.015) and higher WBC count (9.35 x 10(9)/L vs 2.15 x 10(9)/L, P = 0.038). APL cells could be classified into large side scatter population (L-SSC) and non-large side scatter population (NL-SSC) in CD45/SSC histogram of flow cytometry. 87.50% patients with bcr1-form showed L-SSC phenotype and 64.29% patients with bcr3-form showed NL-SSC phenotype. It is concluded that a sensitive Q-PCR method is established. The median NQ of PML/RARalpha mRNA was 0.450 in newly diagnosed APL patients. There was no significant difference about PML/RARalpha mRNA expression of both bcr3-form and bcr1-form APL patients. Type of PML/RARalpha transcripts is related with the morphology and immunophenotype.
Zhu
Hong-Hu
HH
Institute of Hematology, People Hospital, Peking University, Beijing 100044, China.
Liu
Yan-Rong
YR
Qin
Ya-Zhen
YZ
Li
Jin-Lan
JL
Chang
Yan
Y
Wang
Ya-Zhe
YZ
Shan
Fu-Xiang
FX
Jiang
Bin
B
Lu
Dao-Pei
DP
D000293:Adolescent; D000328:Adult; D000368:Aged; D002648:Child; D005260:Female; D016313:Genes, abl / Q000235:genetics; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D015514:Oncogene Proteins, Fusion / Q000032:analysis* / Q000235:genetics; D010641:Phenotype; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D018168:Receptors, Retinoic Acid / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D015514:Oncogene Proteins, Fusion; D012333:RNA, Messenger; D018168:Receptors, Retinoic Acid; C093476:promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein