Clinical features and prognosis of core binding factor acute myeloid leukemia children in South China: a multicenter study / 中华儿科杂志

Objective: To analyze the clinical features, efficacy and prognosis factors of core binding factor (CBF) acute myeloid leukemia (AML) children in South China. Methods: This was a retrospective cohort study. Clinical data of 584 AML patients from 9 hospitals between January 2015 to December 2020 was collected. According to fusion gene results, all patients were divided into two groups: CBF-AML group (189 cases) and non-CBF-AML group (395 cases). CBF-AML group were divided into AML1-ETO subgroup (154 cases) and CBFβ-MYH11 subgroup (35 cases). Patients in CBF-AML group chosen different induction scheme were divided into group A (fludarabine, cytarabine, granulocyte colony stimulating factor and idarubicin (FLAG-IDA) scheme, 134 cases) and group B (daunorubicin, cytarabine and etoposide (DAE) scheme, 55 cases). Age, gender, response rate, recurrence rate, mortality, molecular genetic characteristics and other clinical data were compared between groups. Kaplan-Meier method was used for survival analysis and survival curve was drawn. Cox regression model was used to analyze prognostic factors. Results: A total of 584 AML children were diagnosed, including 346 males and 238 females. And a total of 189 children with CBF-AML were included, including 117 males and 72 females. The age of diagnosis was 7.3 (4.5,10.0)years, and the white blood cell count at initial diagnosis was 21.4 (9.7, 47.7)×109/L.The complete remission rate of the first course (CR1) of induction therapy, relapse rate, and mortality of children with CBF-AML were significantly different from those in the non-CBF-AML group (91.0% (172/189) vs. 78.0% (308/395); 10.1% (19/189) vs. 18.7% (74/395); 13.2% (25/189) vs. 25.6% (101/395), all P<0.05). In children with CBF-AML, the CBFβ-MYH11 subgroup had higher initial white blood cells and lower proportion of extramedullary invasion than the AML1-ETO subgroup, with statistical significance (65.7% (23/35) vs. 14.9% (23/154), 2.9% (1/35) vs. 16.9% (26/154), both P<0.05). AML1-ETO subgroup had more additional chromosome abnormalities (75/154), especially sex chromosome loss (53/154). Compared with group B, group A had more additional chromosome abnormalities and a higher proportion of tumor reduction regimen, with statistical significance (50.0% (67/134) vs. 29.1% (16/55), 34.3% (46/134) vs. 18.2% (10/55), both P<0.05). Significant differences were found in 5-years event free survival (EFS) rate and 5-year overall survival (OS) rate between CBF-AML group and non-CBF-AML group ((77.0±6.4)%vs. (61.9±6.7)%,(83.7±9.0)%vs. (67.3±7.2)%, both P<0.05).EFS and OS rates of AML1-ETO subgroup and CBFβ-MYH11 subgroup in children with CBF-AML were not significantly different (both P>0.05). Multivariate analysis showed in the AML1-ETO subgroup, CR1 rate and high white blood cell count (≥50×109/L) were independent risk factors for EFS (HR=0.24, 95%CI 0.07-0.85,HR=1.01, 95%CI 1.00-1.02, both P<0.05) and OS (HR=0.24, 95%CI 0.06-0.87; HR=1.01, 95%CI 1.00-1.02; both P<0.05). Conclusions: In CBF-AML, AML1-ETO is more common which has a higher extramedullary involvement and additional chromosome abnormalities, especially sex chromosome loss. The prognosis of AML1-ETO was similar to that of CBFβ-MYH11. The selection of induction regimen group FLAG-IDA for high white blood cell count and additional chromosome abnormality can improve the prognosis.

Establishment of leukemia cell model with inducible AML1-ETO expression and its effect on fatty acid metabolism in leukemia cells / 中华血液学杂志

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.

The prognostic value of cloned genetic mutations detected by second-generation sequencing in RUNX1-RUNX1T1 positive acute myeloid leukemia patients receiving intensive consolidation therapy / 中华血液学杂志

Objective: To investigate the prognostic value of clonal gene mutations detected by second-generation sequencing in patients with positive RUNX1-RUNX1T1 acute myeloid leukemia (AML) who received high-dose chemotherapy or autologous transplantation (intensive consolidation therapy) in the first complete remission (CR(1)) state. Methods: 79 AML patients with positive RUNX1-RUNX1T1 who received intensive consolidation therapy in CR(1) state from July 2011 to August 2017 were analyzed retrospectively. Kaplan-Meier curve and Cox regression model were used to figure out the effect of leukocyte counts at onset and gene mutations for prognosis. Results: C-KIT, FLT3, CEBPA and DNMT3A gene mutations were found in 25 (31.6%) , 6 (7.6%) , 7 (8.9%) and 1 (1.3%) patient among the population. Mutations in C-KIT exon17 and C-KIT exon8 were detected in 19 (24.1%) and 5 (6.3%) cases, respectively, and mutations of FLT3-ITD were confirmed in 5 (6.3%) cases. The higher leukocyte counts presented at onset of leukemia, the shorter overall survival (OS) was seen in these patients (P=0.03) . Patients with C-KIT exon17 mutation had significantly shorter OS (P=0.01) and disease free survival (DFS) (P=0.006) compared with those without gene mutations, and patients with FLT3-ITD gene mutation got the inferior OS (P=0.048) and DFS (P=0.071) . Conclusion: In AML patients with positive RUNX1-RUNX1T1 receiving intensive consolidation therapy, the white blood cell counts at onset of leukemia, C-KIT mutations in exon 17, and FLT3-ITD gene mutations suggest poor prognosis, which would contribute to elaborate risk stratification, personalized treatment and predict prognosis for these patients.

Clinical features and prognosis of core binding factor acute myeloid leukemia in children / 中国当代儿科杂志

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.

An interlaboratory comparison study on the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels / 中华血液学杂志

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.

Related Factors Affecting Long-term Prognosis of AML Children with Positive RUNX1-RUNX1T1 / 中国实验血液学杂志

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 (P0.05). 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 (P10 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.

Detection Rate of Central Nervous System Leukemia Can Be Improved by Cell Preservation Solution / 中国实验血液学杂志

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.

Efficacy Analysis of Unrelated Cord Blood Transplantation for High-Risk Refractory AML1-ETO Positive Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To analyze the clinical outcomes of engraftment, graft-versus-host disease (GVHD) and survival in the patients with AML1-ETO positive acute myeloid leukemia (AML) treated with unrelated umbilical cord blood transplantation (UCBT).@*METHODS@#Forty-Five patients with high-risk refractory AML1-ETO positive AML were treated with a single UCBT in a single center from July 2010 to April 2018. All the patients underwent a myeloablative preconditioning regimen，and cyclosporine A (CSA) combined with mycophenolate mofetil (MMF) was used to prevent GVHD.@*RESULTS@#The median value of total nucleated cells (TNC) in cord blood was 5.21 (1.96-12.68)×10/kg recipient body weight, and that of CD34+ cells was 5.61 (0.56-15.4)×10/kg recipient weight. The implantation rate of neutrophil at 42 d and that of platelet at 120 d were 95.6% and 86.7%, respectively. The median time of absolute neutrophil count (ANC)＞0.5×10/L and platelet 20×10/L were 16 (12-18) d and 37 (17-140) d after transplantation, respectively. The cumulative incidence of Ⅰ -Ⅳ grade acute GVHD (aGVHD) at 100 d after transplantation was 48.9% (95% CI 33.5%-62.6%), Ⅱ-Ⅳ grade aGVHD occurred in 12 cases (33.3%) (95% CI 20%-47.2%) , and Ⅲ-Ⅳ grade a GVHD in 8 cases (20%) (95% CI 9.8% -32.8%). In 5 cases of 40 patients survived over 100 days, the chronic GVHD (cGVHD) occurred after transplantation, among which 4 were localized, and 1 was extensive. 3 patients relapsed, and the 2-year cumulative relapse rate was 9.5% (95% CI 2.4%-22.8%). The median follow-up time was 23.5 (0.9-89.67) months, 10 patients died, 2-year disease-free survival rate (DFS) was 72.7%, and overall survival rate (OS) was 75.5%. Multivariate analysis showed that Ⅲ-Ⅳ. acute GVHD (aGVHD) affected overall survival.@*CONCLUSION@#UCBT is an effective rescue treatment for patients with high-risk refractory AML1-ETO positive AML.

Caracterización del gen de fusión RUNX1-RUNX1T1 en pacientes cubanos con leucemia mieloide aguda, 2000-2016 / Characterization of RUNX1-RUNX1T1 fusion gene in Cuban patients with acute myeloid leukemia, 2000-2016

Introducción: el gen de fusión RUNX1-RUNX1T codifica para una proteína quimérica con múltiples efectos en la proliferación, diferenciación y viabilidad de las células leucémicas. Objetivo: describir el comportamiento del RUNX1-RUNX1T1 en pacientes cubanos con dicha enfermedad. Método: Para ello se estudió el gen de fusión RUNX1-RUNX1T1 en 251 pacientes con leucemia mieloide aguda, mediante la reacción en cadena de la polimerasa, en el Instituto de Hematología e Inmunología de La Habana, entre los años 2000 y 2016. Resultados: El 20,3 por ciento (51 pacientes) fue positivo para el gen de fusión RUNX1-RUNX1T1, con una edad comprendida entre los 11 meses y los 80 años, media de 26 años. En los pacientes pediátricos la frecuencia del transcrito fue casi el doble de la de los adultos (29,2 por ciento y 15,3 por ciento, respectivamente) (p= 0,009). Mayor cantidad de pacientes masculinos presentaron el gen quimérico. En menores de 25 años hubo una mayor frecuencia del transcrito (p=0,019) con predominio significativo de la mutación en los adolescentes (p=0,027). Cinco pacientes fueron positivos al RUNX1-RUNX1T1 y a la duplicación interna en tándem del gen FLT3 (12,2 por ciento). Ningún paciente positivo al RUNX1-RUNX1T1 presentó el gen de fusión CBFB-MYH11. La mayor asociación estuvo con la mutación A del gen NPM1 para un 25 por ciento. El debut de la enfermedad se caracterizó por anemia moderada (p= 0,024), trombocitopenia severa (p= 0,004) y gran infiltración medular. La mayor discrepancia entre diagnósticos se concentró entre las variantes morfológicas M2 y M3 (p= 0,000). Conclusiones: En pacientes cubanos la leucemia mieloide aguda con gen de fusión RUNX1-RUNX1T1 positivo, tiene un comportamiento similar a lo descrito internacionalmente con algunas particularidadesen las características hematológicas de presentación de la enfermedad. El estudio molecular es imprescindible para definir el diagnóstico, y la estrategia terapéutica en estos pacientes(AU)

Introduction: The RUNX1-RUNX1T fusion gene codes for a chimeric protein with multiple effects on the proliferation, differentiation and viability of leukemic cells. Objective: To describe the behavior of RUNX1-RUNX1T1 in Cuban patients with this disease. Method: The RUNX1-RUNX1T1 fusion gene was studied in 251 patients with acute myeloid leukemia, through the polymerase chain reaction, at the Institute of Hematology and Immunology of Havana, between 2000 and 2016. Results: The 20.3 percent (51 patients) were positive for the RUNX1-RUNX1T1 fusion gene, with an age between 11 months and 80 years, average of 26 years.In pediatric patients, the transcript frequency was almost twice that of adults (29.2 percent and 15.3 percent , respectively) (p= 0.009). More male patients presented the chimeric gene. There was a higher frequency of the transcript in children under 25 years of age (p= 0.019) with a significant predominance of the mutation in adolescents (p= 0.027).Five patients were positive for RUNX1-RUNX1T1 and for internal tandem duplication of the FLT3 gene (12.2 percent ).No patient positive for RUNX1-RUNX1T1 presented the CBFB-MYH11 fusion gene. The greatest association was with the A mutation of the NPM1 gene for 25 percent . The onset of the disease was characterized by moderate anemia (p= 0.024), severe thrombocytopenia (p= 0.004) and extensive bone marrow infiltration. The greatest discrepancy between diagnoses was concentrated between the morphological variants M2 and M3 (p= 0.000). Conclusions: In Cuban patients, acute myeloid leukemia with a positive RUNX1-RUNX1T1 fusion gene has a behavior similar to that described internationally with some peculiarities in the hematological characteristics of the disease presentation.The molecular study is essential to define the diagnosis, and the therapeutic strategy in these patients(AU)

Expression of CD19 and CD56 in AML Patients with RUNX1-RUNX1T1 Mutation and Its Clinical Significance / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>The bone marrow CD56 in RUNX1-RUNX1T1 positive AML patients suggests poor prognosis. The CD19 only correlates with CR1, but does not with OS.</p>

Epigenetic Silencing of Eyes Absent 4 Gene by Acute Myeloid Leukemia 1-Eight-twenty-one Oncoprotein Contributes to Leukemogenesis in t(8;21) Acute Myeloid Leukemia / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The acute myeloid leukemia 1 (AML1)-eight-twenty-one (ETO) fusion protein generated by the t(8;21)(q22;q22) translocation is considered to display a crucial role in leukemogenesis in AML. By focusing on the anti-leukemia effects of eyes absent 4 (EYA4) gene on AML cells, we investigated the biologic and molecular mechanism associated with AML1-ETO expressed in t(8;21) AML.</p><p><b>METHODS</b>Qualitative polymerase chain reaction (PCR), quantitative reverse transcription PCR (RT-PCR), and Western blotting analysis were used to observe the mRNA and protein expression levels of EYA4 in cell lines. Different plasmids (including mutant plasmids) of dual luciferase reporter vector were built to study the binding status of AML1-ETO to the promoter region of EYA4. Chromatin immunoprecipitation assay was used to study the epigenetic silencing mechanism of EYA4. Bisulfite sequencing was applied to detect the methylation status in EYA4 promoter region. The influence of EYA4 gene in the cell proliferation, apoptosis, and cell clone-forming ability was detected by the technique of Cell Counting Kit-8, flow cytometry, and clonogenic assay.</p><p><b>RESULTS</b>EYA4 gene was hypermethylated in AML1-ETO+ patients and its expression was down-regulated by 6-fold in Kasumi-1 and SKNO-1 cells, compared to HL-60 and SKNO-1-siA/E cells, respectively. We demonstrated that AML1-ETO triggered the epigenetic silencing of EYA4 gene by binding at AML1-binding sites and recruiting histone deacetylase 1 and DNA methyltransferases. Enhanced EYA4 expression levels inhibited cellular proliferation and suppressed cell colony formation in AML1-ETO+ cell lines. We also found EYA4 transfection increased apoptosis of Kasumi-1 and SKNO-1 cells by 1.6-fold and 1.4-fold compared to negative control, respectively.</p><p><b>CONCLUSIONS</b>Our study identified EYA4 gene as targets for AML1-ETO and indicated it as a novel tumor suppressor gene. In addition, we provided evidence that EYA4 gene might be a novel therapeutic target and a potential candidate for treating AML1-ETO+ t (8;21) AML.</p>

Construction of SIRT1 promoter expression vector and its activity analysis as well as influence of AML1-ETO on transcriptional regulation of SIRT1 gene / 中国实验血液学杂志

<p><b>OBJECTIVE</b>This study was aimed to analyze the expression and regulation mechanism of SIRT1 in AML1-ETO positive leukemia to find the core promoter.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>The 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.</p>

Effect of autologous peripheral blood stem cell transplantation in 13 patients with AML1/ETO (+) acute myeloid leukemia / 中国实验血液学杂志

This study was purposed to analyse the clinical efficacy of autologous peripheral blood stem cell transplantation (APBSCT) in 13 Patients with AML1/ETO (+) acute myeloid leukemia, and to evaluate the role of quantitative detecting the AML1/ETO gene in treatment of AML patients. A total of 13 patients with AML1/ETO (+) acute myeloid leukemia treated with APBSCT from August 2007 to November 2012 were retrospectively analyzed. The median follow-up time was 26 (7.8-75.8)months. Kaplan-Meier analysis was used to calculate the overall survival (OS), leukemia-free survival (LFS) and cumulative relapse rate (RR). Log rank method was used to perform univariate analysis. The results showed that the 3 year-OS, LFS, and RR were (70.5 ± 15.3)%, (51.3 ± 16.7)%, 48.7%, respectively. The AML1/ETO expression level in 4 cases out of 5 relapsed patients was quantified during and after therapy, and the result showed that AML1/ETO expression level significantly increased before morphological relapse. In univariate analysis, there was no statistic significance in terms of age, sex, count of white blood cells at diagnosis, interval from diagnosis to transplantation, count of MNC for infusion. It is concluded that APBSCT has good therapeutic effect on AML1/ETO (+) AML, and regular quantitative monitoring of AML1/ETO expression level can predict early recurrence. Allogeneic hematopoietic stem cell transplantation after relapse may contribute to obtain opportunity to achieve the long-term survival for intermediate and high risk patients.

Clinical efficacy of decitabine combined with modified CAG regimen for relapsed-refractory acute myeloid leukemia with AML1-ETO⁺ / 中国实验血液学杂志

This study was aimed to investigate the clinical characteristics of relapsed-refractory acute myeloid leukemia (AML) with AML1-ETO⁺, and its therapeutic efficacy and side effects when decitabine combined with modified CAG regimen was used. Clinical data of 5 cases of AML with AML1-ETO⁺ from January 2013 to Agust 2013 were analyzed retrospectively. The analyzed data included age, sex, initial symptoms, peripheral blood and bone marrow characteristics. Meanwhile, the therapeutic effecacy and side effects of decitabine combined with modified CAG regimen were evaluated. The 5 patients were with median age of 35 (17-43) years. Among these 5 patients, 2 patients were relapsed and other 3 patients were relapsed-refractory patients, their median white blood cell count was 12.55 (7.8-66.55) × 10⁹/L, median platelets count was 44 (20-72) × 10⁹/L, median hemoglobin level was 110 (77-128) g/L, median lactate dehydrogenase level was 312.9 U/L (123.6-877.8) at the initial diagnosis. The results showed that after decitabine combined with modified CAG regimen was administered, 4 patients achieved complete remission, 1 patient did not achieve remission, the overall remission rate was 80% (4/5). The main side effects of this regimen was myelosuppression, these were no new lung infection and other serious complications, one case without complete remission treated with FLAG once again died of heart failure when being mobilized for transplantation. It is concluded that according to preliminary results of decitabine combined with modified CAG regimen for relapsed and refractory AML patients with AML1-ETO⁺ displays higher remission rate and lower side effects, which worthy to further explore for clinal application.

FLT3 gene overexpression and its clinical significance in acute myeloid leukemia with AML1/ETO fusion gene positive / 中国实验血液学杂志

This study was aimed to investigate the expression levels of FLT3 gene in AML-M2 patients carrying AML1/ETO fusion gene, and analyze its relation with clinical and laboratorial features and prognosis. RQ-PCR method was used to detect the expression level of FLT3 in bone marrow of 21 AML-M2 patients with AML1/ETO(+). The correlation of the expression level of FLT3 with clinical features, other laboratorial examinations and disease prognosis were analyzed. The results showed that gene expression level of FLT3 (FLT3 gene/ reference gene) in patients at initial diagnosis were 1.65%-261.5%. The expression level of FLT3 over 35% was defined as high expression group (12 cases) , while the expression level below 35% was defined as low expression group (9 cases) . The proportion of patients with extramedullary infiltration in high expression group was higher than that in low expression group (25% vs 0%, P = 0.2286). The proportion of patients at initial diagnosis with white blood cell count > 10×10(9)/L in high expression group was higher than that in low expression group (66.67% vs 22.22%), but there was no statistical significance (P = 0.0805). No significant difference was observed at the age (P = 0.1369) and the rate of bone marrow blasts (P = 0.6923) between the above mentioned two groups. The differences in complete remission rate (66.67% vs 88.89%, P = 0.3383), the relapse rate (66.67% vs 22.22%,P = 0.0805) and the mortality rate (50% vs 22.22%, P = 0.3666) between the two group were not significant, but there was a clear trend that the low expression group has a higher CR rate and a lower relapse rate and mortality rate. It is concluded that FLT3 gene high expression in AML-M2 patients with AML1/ETO(+) have a higher rate of relapse and hence poor prognosis. Therefore, detection of FLT3 expression level in routine clinical practice is important for patient's risk stratification, prognostic evaluation and effective treatment selection.

Effect of AML1-ETO fusion protein on the expression of BCL-2 / 中国实验血液学杂志

This study was aimed to investigate the effect of AML1-ETO fusion protein on the anti-apoptotic gene BCL-2 in leukemic cells and to explore its role in leukemogenesis. The apoptotic levels of U937-WT, U937-Mock and U937-A/E1-4 cells were examined by flow cytometry. And cleaved caspase-3 protein expression was detected by Western blot. BCL-2 gene expression both in AML1-ETO-expressing cells or U937 nonexpressing cells and in leukemia cells of AML patients with or without t(8;21) was assessed by quantitative PCR. The chromatin immunoprecipitation (ChIP)-based PCR was used to investigate the direct interaction between the AML1-ETO and BCL-2 promoter in AML1-ETO positive leukemia cell line. The results indicated that in U937-A/E cells but not in U937-WT or U937-Mock cells, apoptotic cells statistically significantly increased, and AML1-ETO expression also significantly enhanced activation of caspase-3. AML1-ETO-expressing cell subclones displayed significantly low levels of BCL-2 mRNA in comparison with the non-transfected U937. In primary bone marrow cells of acute myeloid leukemia containing AML1-ETO, levels of BCL-2 mRNA were markedly lower as compared with other acute myeloid leukemias lacking this translocation. The enriched regions in transfected cells were located within BCL-2 promoter. It is concluded that BCL-2 is the direct target gene of AML1-ETO. AML1-ETO can down-regulate the expression of BCL-2.

Dexamethasone and vorinostat cooperatively promote differentiation and apoptosis in Kasumi-1 leukemia cells through ubiquitination and degradation of AML1-ETO / 中华血液学杂志

<p><b>OBJECTIVE</b>To probe the effects of dexamethasone (DEX) combined with histone deacetylase (HDAC) inhibitor vorinostat on inhibiting proliferation and inducing differentiation and apoptosis in Kasumi-1 leukemia cells, and its possible mechanisms in order to provide a theoretical basis for the treatment of AML1-ETO positive AML.</p><p><b>METHODS</b>The cell survival, differentiation and apoptosis rates were tested by MTT or flow cytometry analysis after Kasumi-1 cells were treated by DMSO, DEX (20 nmol/L), vorinostat (1 μmol/L) or DEX (20 nmol/L) in combination with vorinostat (1 μmol/L). WB and IP-WB were performed to detect AML1-ETO and its ubiquitination.</p><p><b>RESULTS</b>Treatment with the combination of DEX and vorinostat for 48 h led to statistically significant differences of inhibited proliferation [(42.06±8.20)%], increased differentiation [(52.83±8.97)%] and apoptosis [(52.92±2.53)%] of Kasumi-1 cells when compared with vorinostat [(33.82±9.41)%, (43.93±9.04)% and (42.98±3.01)%, respectively], DEX [(17.30±3.49)%, (22.53±4.51)% and (19.57±2.17)%, respectively] or control [(6.96±0.39)%, (21.73±2.03)% and (6.96±0.39)%, respectively]. Also significant ubiquitination and decreased AML1-ETO protein in Kasumi-1 cells after the combination treatment over single agent or control were observed.</p><p><b>CONCLUSION</b>The results indicated that DEX and vorinostat could synergistically inhibit the Kasumi-1 cells proliferation, induce Kasumi-1 cells differentiation and apoptosis through ubiquitination and degradation of AML1-ETO.</p>

C-kit mutation in acute myeloid leukemia patients with AML1-ETO fusion gene and its clinical significance / 中国实验血液学杂志

This study was aimed to investigate the c-kit mutation in acute myeloid leukemia (AML) patients with AML1-ETO and analyze its relation with clinical and laboratorial features and prognosis. PCR and sequencing methods were used to detect the c-kit 17 exon mutations in 31 AML patients with AML1-ETO. The relation of the c-kit mutation with clinical features, results of laboratorial examination and prognosis of disease were analyzed. The results showed that the c-kit mutation was found in 14 out of 31 AML patients and the mutation frequency was 45.16%. Male patients had a higher incidence of c-kit mutation than that of female patients (P = 0.020). The proportion of patients with newly diagnosed white blood cell>10×10(9)/L and with extramedullary infiltration in mutated group were higher than those in unmutated group respectively. No significant difference was observed at the age (P = 0.437) and the rate of bone marrow blasts(P = 0.510) between the above mentioned two groups. The difference in complete remission rate (64.29% vs 80%, P = 0.344)and relapse rate (58.33% vs 21.43%, P = 0.054) between c-kit mutated and c-kit unmutated groups were not significant. While the c-kit mutated group had a significant higher death rate as compared with c-kit unmutated group (57.14% vs 20%, P = 0.039). It is concluded that the c-kit mutation is frequent in AML patients with AML1-ETO and the c-kit mutated patients have a poor prognosis. It is important to detect c-kit mutation in routine clinical practice for patient's risk stratification, evaluation of prognosis and selection of effective treatment.

Expression of survivin in patients with acute myeloid leukemia / 中国实验血液学杂志

Objective of this study was to detect the expression of Survivin in acute myeloid leukemia (AML) and investigate the relationship of its expression levels with clinical variates and its correlations with BCL-2 ,Bcl-xL and MCL-1. The expression of Survivin, BCL-2, Bcl-xL and MCL-1 were measured by immunohistochemistry in bone marrow biopsy of 63 newly diagnosed AML patients, and the relationship between its expression level and clinical parameters (age, sex, WBC count, diagnosis, prognosis), especially fusion protein AML1/ETO was investigated. The results showed that the expression level of Survivin in newly diagnosed AML patients was higher than that of normal controls (P < 0.01), the expression levels of Survivin did not correlate with age, sex, and WBC counts of patients and so on. There was no significant difference of Survivin expression between different NCCN prognosis groups, either between patients with AML1/ETO or FLT3-ITD mutation and the other patients. Survivin positive patients were got to have lower CR rate but higher relapse rate, however that was not significant in statistics. Indeed, the cumulative survivin rate of Survivin positive patients were lower than that of Survivin negative patients (P = 0.04). Finally, positive correlation between Survivin and MCL-1 was also observed (r = 0.639, P = 0.000). It is concluded that overexpression of Survivin are closely related with occurrence and development of acute leukemia, and may be used as an indicator of prognosis evaluation. In addition, Survivin and MCL-1 may have a relationship of cooperation or interaction.

Monitoring the expression ratio of AML1-ETO9a isoform in t(8;21) acute myeloid leukemia and its significance / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the expression ratio of AML1-ETO9a (AE9a) isoform in t(8;21) acute myeloid leukemia (AML) and its clinical significance.</p><p><b>METHODS</b>Bone marrow samples from 44 newly diagnosed t(8;21) AML patients co-expressed AE9a and AE were screened by RT-PCR. The alteration of the AE9a expression ratio was monitored during follow-up by using quantitative real-time RT-PCR (qPCR).</p><p><b>RESULTS</b>The expression level of AE9a was markedly lower than that of AE in these patients. There was a positive correlation between the expression level of AE9a and AE in most of bone marrow samples. The transcript level of both AE9a and AE was decreased in the 44 patients after one course of standard chemotherapy, but the percentage of AE9a expression level was increased in comparison with that before treatment (P < 0.05). After one course of standard chemotherapy treatment, the percentage of AE9a in incomplete remission (ICR) patients was significantly higher than that in CR patients (P < 0.05). Relapsed patients had a higher AE9a ratio than the unrelapsed patients (P < 0.05). During the remission, the percentage of AE9a in 11/17 relapsed patients obviously elevated even while the expression of AE fusion gene at low level.</p><p><b>CONCLUSIONS</b>AE9a and AE co-expressed in most of AML patients with t(8;21) translocation. The expression level of AE9a was lower than that of AE, and there is a positive correlation between the expression level of these two isoforms. The sensitivity of AE9a gene to the standard chemotherapy is less than that of the AE fusion gene. Monitoring the AE9a to AE ratio during the CR can predict the early relapse of the disease compared to monitoring the AE alone.</p>