OBJECTIVE: To investigate whether cell preservation solution can prolong the survival time of leukemia cells and increase the survival rate, so as to improve the detection rate of central nervous system leukemia. METHODS: Kasumi cells were added into cerebrospinal fluid (CSF) supernatant with or without cell preservation solution to compare cell viability and biological characteristics at different time point. Wright Giemsa staining was used to compare cell morphology; cell counting, CCK-8 method, and trypan blue staining were used to compare the cell number, and flow cytometry was used to compare the cell viability. The expression of AML-ETO tumor fusion gene was detected by fluorescence quantitative RT-PCR. RESULTS: At different time points (8 h and 24 h), the survival, molecular biological characteristics and RT-PCR result of the cells in CSF with cell preservation solution were significantly better than those in normal cerebrospinal fluid. CONCLUSION Cell preservation solution can effectively improve the survival time and survival rate of leukemic cells, thereby increase the detection rate of CNS leukemia.
Central Nervous System Neoplasms ; Core Binding Factor Alpha 2 Subunit ; Humans ; Leukemia ; RUNX1 Translocation Partner 1 Protein

OBJECTIVETo investigate the clinical significance of RUNX1-RUNX1T1 expression level in bone marrow of patients with acute non-M3 myeloid leukemia (AML non-M3), and to understand the biological characteristics of RUNX1-RUNX1T1 positive AML expressing lymphoid antigens CD19, CD56 and its effect on the initially induced remission rate and prognosis.

METHODSThe expression level of RUNX1-RUNX1T1 in bone marrow of 200 patients with newly diagnosed AML (non-M3) was detected by real-time fluorescent Q-PCR, the expression level of lymphoid antigens was detected by flow cytometry, and the relationship of the initially induced remission rate (CR1) with the overall survival (OS) rate was analyzed, the CR1 and OS differences also were analyzed between CD56 and CD56 patients as well as CD19 and CD17 patients in RUNX1-RUNX1T1 positive patients with AML.

RESULTSThe CD56 patients at the initial diagnosis had lower CR1(P<0.05) in RUNX1-RUNX1T1 positive AML patients, the CR1 of CD19 patients was higher than that in CD19 patients at the initial diagnosis (P<0.05). The OS of CD56 patients was significantly high in comparison with CD56 patients (P<0.05), while the OS between CD19 patients and CD19 patients was not significantly different.

CONCLUSIONThe bone marrow CD56 in RUNX1-RUNX1T1 positive AML patients suggests poor prognosis. The CD19 only correlates with CR1, but does not with OS.

Antigens, CD19 ; CD56 Antigen ; Core Binding Factor Alpha 2 Subunit ; Humans ; Leukemia, Myeloid, Acute ; Mutation ; Prognosis ; RUNX1 Translocation Partner 1 Protein

OBJECTIVE: To analyze the related factors affecting the long-term prognosis of acute myeloid leukemia (AML) children with positive RUNX1-RUNX1T1. METHODS: The clinical data of 63 chlidren with positive RUNX1-RUNX1T1 AML treated by BCH-AML 05 regimen in our hospital from January 2010 to December 2015 were collected and analyzed retrospectively. The level of RUNX1-RUNX1T1 was detected at the time of initial diagnosis (T), after the first induction treatment (T), after the second induction treatment (T), after the first consolidation treatment (T), after the second consolidation treatment (T) and after the third consolidation treatment (T). According to the fusion transcript levels of RUNX1-RUNX1T1 the AML children were divided into low-expression group and high-expression group; the threshold values for grouping were 10 copies/10 β-glucuronidase (GUS), 10 copies/10 GUS, 10 copies/10 GUS, 10 copies/10 GUS, 1 copies/10 GUS and 0 copies respectively. The gained data were enrolled in the statistical analysis. RESULTS: 23 cases of 63 children died during the follow-up period, and the median follow-up time of the remaining 40 children were 30.04 (11-60) months. There were statistically significant differences in CD15 positive rate between low-expression group and high-expression group (P<0.05), however, there were no statistically significant differences in sex, age, FAB typing, platelet (Plt) count, hemoglobin (Hb) and white blood cell (WBC) count and the ratio of bone marrow immature cells at T2 between the two groups (P>0.05). Univariate analysis showed that sex, Plt counts at T and fusion transcript levels at T, T and T correlated with the 5-year overall survival rate (P<0.05). Multivariate analysis showed that fusion transcript level >10 copies/10 GUS at T was an independent risk factor for 5-year overall survival rate (HR=2.13, 95%CI: 1.04-7.78)(P<0.05). CONCLUSION The fusion transcript level after the first induction therapy in RUNX1-RUNX1T1-positive AML children is an independent factor influencing the long-term prognosis.
Child ; Core Binding Factor Alpha 2 Subunit ; Humans ; Leukemia, Myeloid, Acute ; Oncogene Proteins, Fusion ; Prognosis ; RUNX1 Translocation Partner 1 Protein ; Retrospective Studies

OBJECTIVE: To study the clinical features and prognosis of core binding factor acute myeloid leukemia (CBF-AML) in children. METHODS: A retrospective analysis was performed from the chart review data of children who were newly diagnosed with CBF-AML in the Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences, from August 2009 to November 2015. According to the type of fusion gene, the children were divided into CBFB-MYH11 and AML1-ETO groups. Clinical features and prognosis were analyzed and compared between the two groups. RESULTS: A total of 91 children with CBF-AML were enrolled in this study, among whom there were 74 (81%) in the AML1-ETO group and 17 (19%) in the CBFB-MYH11 group. Additional chromosomal abnormalities were observed in 38 children (42%), and deletion of sex chromosome was the most common abnormality and was observed in 28 children (31%). After the first course of induction treatment, the complete remission rate was 97% (88/91), the recurrence rate was 29% (26/91), the 5-year event-free survival (EFS) rate was 65%±6%, and the 5-year overall survival (OS) rate was 75%±5%. There were no significant differences between the AML1-ETO and CBFB-MYH11 groups in 5-year EFS rate (62%±7% vs 77%±11%, P>0.05) or 5-year OS rate (72%±6% vs 88%±9%, P>0.05). CONCLUSIONS AML1-ETO is the main type of fusion gene in children with CBF-AML, and deletion of sex chromosome is the most common type of additional chromosomal abnormalities. Children with CBF-AML often have a good prognosis, and the children with AML1-ETO have a similar prognosis to those with CBFB-MYH11.
Child ; Core Binding Factor Alpha 2 Subunit ; Core Binding Factors ; Humans ; Leukemia, Myeloid, Acute ; Oncogene Proteins, Fusion ; Prognosis ; RUNX1 Translocation Partner 1 Protein ; Retrospective Studies

Objective: To investigate the effect of the AML1-ETO (AE) fusion gene on the biological function of U937 leukemia cells by establishing a leukemia cell model that induces AE fusion gene expression. Methods: The doxycycline (Dox) -dependent expression of the AE fusion gene in the U937 cell line (U937-AE) were established using a lentivirus vector system. The Cell Counting Kit 8 methods, including the PI and sidanilide induction, were used to detect cell proliferation, cell cycle-induced differentiation assays, respectively. The effect of the AE fusion gene on the biological function of U937-AE cells was preliminarily explored using transcriptome sequencing and metabonomic sequencing. Results: ①The Dox-dependent Tet-on regulatory system was successfully constructed to regulate the stable AE fusion gene expression in U937-AE cells. ②Cell proliferation slowed down and the cell proliferation rate with AE expression (3.47±0.07) was lower than AE non-expression (3.86 ± 0.05) after inducing the AE fusion gene expression for 24 h (P<0.05). The proportion of cells in the G(0)/G(1) phase in the cell cycle increased, with AE expression [ (63.45±3.10) %) ] was higher than AE non-expression [ (41.36± 9.56) %] (P<0.05). The proportion of cells expressing CD13 and CD14 decreased with the expression of AE. The AE negative group is significantly higher than the AE positive group (P<0.05). ③The enrichment analysis of the transcriptome sequencing gene set revealed significantly enriched quiescence, nuclear factor kappa-light-chain-enhancer of activated B cells, interferon-α/γ, and other inflammatory response and immune regulation signals after AE expression. ④Disorder of fatty acid metabolism of U937-AE cells occurred under the influence of AE. The concentration of the medium and short-chain fatty acid acylcarnitine metabolites decreased in cells with AE expressing, propionyl L-carnitine, wherein those with AE expression (0.46±0.13) were lower than those with AE non-expression (1.00±0.27) (P<0.05). The metabolite concentration of some long-chain fatty acid acylcarnitine increased in cells with AE expressing tetradecanoyl carnitine, wherein those with AE expression (1.26±0.01) were higher than those with AE non-expression (1.00±0.05) (P<0.05) . Conclusion: This study successfully established a leukemia cell model that can induce AE expression. The AE expression blocked the cell cycle and inhibited cell differentiation. The gene sets related to the inflammatory reactions was significantly enriched in U937-AE cells that express AE, and fatty acid metabolism was disordered.
Humans ; U937 Cells ; RUNX1 Translocation Partner 1 Protein ; Leukemia/genetics* ; Core Binding Factor Alpha 2 Subunit/genetics* ; Oncogene Proteins, Fusion/genetics* ; Leukemia, Myeloid, Acute/genetics*

Objective: To investigate the current status and real performance of the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels in China through interlaboratory comparison. Methods: Peking University People's Hospital (PKUPH) prepared the samples for comparison. That is, the fresh RUNX1-RUNX1T1 positive (+) bone morrow nucleated cells were serially diluted with RUNX1-RUNX1T1 negative (-) nucleated cells from different patients. Totally 23 sets with 14 different samples per set were prepared. TRIzol reagent was added in each tube and thoroughly mixed with cells for homogenization. Each laboratory simultaneously tested RUNX1-RUNX1T1 and WT1 transcript levels of one set of samples by real-time quantitative PCR method. All transcript levels were reported as the percentage of RUNX1-RUNX1T1 or WT1 transcript copies/ABL copies. Spearman correlation coefficient between the reported transcript levels of each participated laboratory and those of PKUPH was calculated. Results: ①RUNX1-RUNX1T1 comparison: 9 samples were (+) and 5 were (-) , the false negative and positive rates of the 20 participated laboratories were 0 (0/180) and 5% (5/100) , respectively. The reported transcript levels of all 9 positive samples were different among laboratories. The median reported transcript levels of 9 positive samples were from 0.060% to 176.7%, which covered 3.5-log. The ratios of each sample's highest to the lowest reported transcript levels were from 5.5 to 12.3 (one result which obviously deviated from other laboratories' results was not included) , 85% (17/20) of the laboratories had correlation coefficient ≥0.98. ②WT1 comparison: The median reported transcript levels of all 14 samples were from 0.17% to 67.6%, which covered 2.6-log. The ratios of each sample's highest to the lowest reported transcript levels were from 5.3-13.7, 62% (13/21) of the laboratories had correlation coefficient ≥0.98. ③ The relative relationship of the reported RUNX1-RUNX1T1 transcript levels between the participants and PKUPH was not always consistent with that of WT1 transcript levels. Both RUNX1-RUNX1T1 and WT1 transcript levels from 2 and 7 laboratories were individually lower than and higher than those of PKUPH, whereas for the rest 11 laboratories, one transcript level was higher than and the other was lower than that of PKUPH. Conclusion: The reported RUNX1-RUNX1T1 and WT1 transcript levels were different among laboratories for the same sample. Most of the participated laboratories reported highly consistent result with that of PKUPH. The relationship between laboratories of the different transcript levels may not be the same.
China ; Core Binding Factor Alpha 2 Subunit ; Humans ; Leukemia, Myeloid, Acute ; RUNX1 Translocation Partner 1 Protein ; Real-Time Polymerase Chain Reaction ; Transcription, Genetic ; WT1 Proteins

Humans ; Core Binding Factor Alpha 2 Subunit/genetics* ; Translocation, Genetic ; Leukemia, Myeloid, Acute/genetics* ; Prognosis ; High-Throughput Nucleotide Sequencing ; RUNX1 Translocation Partner 1 Protein/genetics* ; Oncogene Proteins, Fusion/genetics*

OBJECTIVEThis study was aimed to analyze the expression and regulation mechanism of SIRT1 in AML1-ETO positive leukemia to find the core promoter.

METHODSThe real-time RT-PCR was used to detect the expression of SIRT1 in AML1-ETO positive leukemia cell line and clinical samples of leukemia patients, a SIRT1 promoter-luciferase reporter vector was constructed and the promoter activity was evaluated in the 293T cell line. A series of possible core promoter fragments of the SIRT1 5'-untranslated region were amplified by PCR, the PCR products were cloned into XhoI/HindIII-digested pGL3-Basic reporter vector, the poly-cationic compound SuperFect reporter vector complexes were transfected into 293T cells.The dual-luciferase Reporter Assay System was used to quantitate the reporter vector luciferase activity.

RESULTSThe six kinds of promoter fragment of SIRT1 gene were successfully constructed and cloned into the pGL3-Basic reporter vector, which was authenticated by XhoI/HindIII co-digestion and DNA sequencing. The luciferase activity of the promoter construct was significantly higher than that of the pGL3-Basic promoter in 293T cells. The luciferase report gene assay was also used to detect the regulation of AML1-ETO on the transcription activity of SIRT1 promoter. The results showed that the expression level of SIRT1 increased with the increase mens of AML1-ETO, the promoter of SIRT1 could be bound by AML1-ETO.

CONCLUSIONThe SIRT1 promoter-luciferase reporter vector is successfully constructed, the transfection system used in this study can effectively transfer gene in 293T cells. The SIRT1 core promoter possesses higher activity in 293T cells and can promote significantly expression of luciferase reporter gene in 293T cells. The transcription regulation of AML1-ETO on SIRT1 is carried out via promoting its promoter activity.

Cell Line ; Core Binding Factor Alpha 2 Subunit ; Gene Expression Regulation ; Genes, Reporter ; Genetic Vectors ; Humans ; Leukemia ; Luciferases ; Oncogene Proteins, Fusion ; Promoter Regions, Genetic ; RUNX1 Translocation Partner 1 Protein ; Sirtuin 1 ; Transcription, Genetic ; Transfection

OBJECTIVETo investigate the expression of AML1/ETO9a isoform in the acute myeloid leukemia (AML)-M2 patients.

METHODSExpressions of AML1/ETO fusion gene and AML1/ETO9a isoform were detected by using reverse transcriptase-polymerase chain reaction (RT-PCR) in leukemia patients, MDS patients, leukemia cell lines and healthy subjects. Karyotype was studied by R-banding technique.

RESULTIn 30 newly diagnosed AML-M2 patients 15 were found to express AML1/ETO9a isoform, while the rest including 20 AML-M2CR, 18 other subtypes of AML, 5 chronic myelogenous leukemia (CML), 3 myelodysplastic syndromes (MDS), 3 leukemia cell lines (NB4, KG-1, K562) and 5 healthy subjects were AML1/ETO9a negative. Among the 15 AML/ETO9a isoform expressing cases, 13 were demonstrated t(8;21) translocation and AML1/ETO expression.

CONCLUSIONIsoform AML1/ETO9a was correlated to AML/M2, and it may promote the development of leukemia in combination with the AML1/ETO fusion gene.

Adolescent ; Adult ; Aged ; Core Binding Factor Alpha 2 Subunit ; genetics ; metabolism ; Female ; Gene Expression ; Humans ; Karyotyping ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Male ; Middle Aged ; Oncogene Proteins, Fusion ; genetics ; metabolism ; Protein Isoforms ; genetics ; metabolism ; RUNX1 Translocation Partner 1 Protein

This study was aimed to investigate various factors influencing long-term survival in adult AML patients with fusion gene aml1/eto positive. A single institutional retrospective study with long-term follow-up was performed to better define the prognostic factors for AML patients with aml1/eto positive. Newly diagnosed 89 adult AML patients with aml1/eto positive were followed up for 1 to 42 months (median 24 months) from January 2004 to July 2008. Univariate and multivariate analysis of potential factors influencing survival and prognosis were carried out by using Log-Rank and Cox regression method, including sex, age, initial WBC counts, extramedullary leukemic disease, central nervous system leukemia (CNSL), chromosome aberrations, immunophenotype, first induction regimen, chemotherapy course to complete remission (CR), time from induction therapy to CR, negative or positive rate of aml1/eto and allogeneic hematopoietic stem cell transplantation and so on. The results showed that the estimated 5-year overall survival (OS) and relapse-free survival (RFS) were (50.0 +/- 2.3)% and (47.0 +/- 1.9)% respectively in follow-up of 89 patients for 1 - 42 months (mean 24 months). Univariate analysis revealed that initial WBC counts, CNSL, chemotherapy course to CR, time from induction therapy to CR, persistent negative in remission and allogeneic hematopoietic stem cell transplantation were important prognostic factors for long-term surviva1. Multivariate study demonstrated that initial WBC counts, CNSL, CD56 positive, negative or positive rate of aml1/eto, time from induction therapy to CR, persistent negative result of RT-PCR assay in remission and allogeneic hematopoietic stem cell transplantation were all critical factors in relation to OS and RFS. It is concluded that Chinese adult AML patients with fusion gene aml1/eto positive have some different characteristics as compared with patients from other countries, a relatively poor outcome is observed in patients, HSCT should be recommended to adult AML patients.
Adolescent ; Adult ; Core Binding Factor Alpha 2 Subunit ; genetics ; Female ; Humans ; Leukemia, Myeloid, Acute ; genetics ; mortality ; Male ; Middle Aged ; Oncogene Proteins, Fusion ; genetics ; Prognosis ; RUNX1 Translocation Partner 1 Protein ; Retrospective Studies ; Survival Analysis ; Young Adult