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.

Characteristics of N6-methyladenosine modification patterns in t(8;21) acute myeloid leukemia / 南方医科大学学报

OBJECTIVE@#To investigate the relationship between AML1-ETO (AE) fusion gene and intracellular N6-methyladenosine (m6A) modification pattern in t(8;21) acute myeloid leukemia (AML).@*METHODS@#RNA m6A sequencing was performed in SKNO-1 and AE knockdown SKNO-1 (SKNO-1 siAE) cells using RNA-protein co-immunoprecipitation and high-throughput sequencing (methylated RNA immunoprecipitation sequencing, MeRIP-Seq) to analyze the changes in m6A modification of the entire transcriptome. Transcriptome sequencing (RNA-seq) was performed using high-throughput sequencing. The differentially modified mRNAs were further functionally annotated by Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The changes in m6A-related enzyme expressions were detected using real-time PCR.@*RESULTS@#A total of 26 441 genes were identified in AE knockdown AML cells and AE-expressing cells, containing 72 036 m6A peaks. AE knockdown caused a reduction of the number of intracellular m6A peaks from 37 042 to 34 994, among which 1278 m6A peaks were significantly elevated and 1225 were significantly decreased; 1316 genes with newly emerged m6A modification were detected and 1830 genes lost m6A modification after AE knockdown. The differential peaks were mainly enriched in pathways involving cancer and human T-lymphocytic leukemia virus I. RNA-seq results showed that 2483 genes were up-regulated and 3913 genes were down-regulated after AE knockdown. The combined analysis of MeRIP-Seq and RNA-Seq results revealed relatively high expression levels of m6A-modified genes as compared with the genes without m6A modification (SKNO-1: 0.6116±1.263 vs 2.010±1.655, P < 0.0001; SKNO-1 siAE: 0.5528±1.257 vs 2.067±1.686, P < 0.0001). The m6A modified genes located in the 3'UTR or 5 'UTR had significantly higher expression levels than those located in exonic regions (SKNO-1: 2.177± 1.633 vs 1.333 ± 1.470 vs 2.449 ± 1.651, P < 0.0001; SKNO-1 siAE: 2.304 ± 1.671 vs 1.336 ± 1.522 vs 2.394 ± 1.649, P < 0.05). Analysis of RNA-seq data identified 3 m6A-related enzymes that showed significantly elevated mRNA expression after AE knockdown, namely WTAP, METTL14, and ALKBH5 (P < 0.05), but the results of real-time PCR showed that the expressions of WTAP and ALKBH5 were significantly increased while the expression of METTL14 was lowered after AE knockdown (P < 0.05).@*CONCLUSION@#AE knockdown results in differential expressions of m6A-associated enzymes, suggesting that the AE fusion gene regulates the expression of one or more m6A-associated enzymes to control cellular methylation levels.

Homoharringtonine synergy with oridonin in treatment of t(8; 21) acute myeloid leukemia / 医学前沿

Collaboration of c-KIT mutations with AML1-ETO (AE) has been demonstrated to induce t(8; 21) acute myeloid leukemia (AML). Targeted therapies designed to eliminate AE and c-KIT oncoproteins may facilitate effective treatment of t(8; 21) AML. Homoharringtonine (HHT) features activity against tumor cells harboring c-KIT mutations, whereas oridonin can induce t(8; 21) AML cell apoptosis and AE cleavage. Therefore, studies should explore the efficacy of combination therapy with oridonin and HHT in t(8; 21) AML. In this study, we investigated the synergistic effects and mechanism of oridonin combined with HHT in t(8; 21) AML cell line and mouse model. The two drugs synergistically inhibited cell viability and induced significant mitochondrial membrane potential loss and apoptosis. Oridonin and HHT induced significant downregulation of c-KIT and its downstream signaling pathways and promoted AE cleavage. HHT increased intracellular oridonin concentration by modulating the expressions of MRP1 and MDR1, thus enhancing the effects of oridonin. The combination of oridonin and HHT prolonged t(8; 21) leukemia mouse survival. In conclusion, oridonin and HHTexert synergistic effects against t(8; 21) leukemia in vivo and in vitro, thereby indicating that their combination may be an effective therapy for t(8; 21) leukemia.

La biología molecular en la precisión diagnósticade las leucemias / Molecular biology in the diagnostic accuracy of leukemia

Los estudios de citogenética y biología molecular permiten correlacionar la presencia de determinadas anomalías cromosómicas y moleculares con tipos específicos de leucemias y linfomas. Este conocimiento ha hecho posible el perfeccionamiento progresivo del sistema de clasificación de las enfermedades oncohematológicas. Actualmente la presencia de ciertas anomalías citogenéticas o moleculares son suficientes para identificar algunas de estas entidades y en ocasiones, el diagnóstico cambia después de un análisis integrado de la citomorfología con la citogenética y la biología molecular. Este reporte pretende resaltar la importancia del estudio molecular cuando la citomorfología es compleja y propicia diagnósticos erróneos. Mediante la reacción en cadena de la polimerasa, previa reverso-transcripción del ARN aislado de sangre medular, se estudiaron cuatro biomarcadores: los genes de fusión AML1-ETO, BCR-ABL, CBFβ-MYH11 y PML-RARα. Fueron estudiados 14 pacientes con diagnósticos inicial citomorfológico de: leucemia promielocítica (n= 6), leucemia aguda de linaje indefinido (n= 3) y dudoso entre leucemia mieloide crónica en crisis blástica mieloide y leucemia mieloide aguda (LMA) (n= 5). Al culminar la caracterización molecular todos fueron diagnosticados como LMA. Los resultados ilustran la importancia del estudio molecular en la clasificación de las leucemias, lo cual redunda en que el paciente reciba el tratamiento más adecuado y alcance una mejor respuesta.

Cytogenetic and molecular studies have correlated the presence of certain chromosomal and molecular anomalies with leukemia and lymphomas specific types. These evidences have allowed the progressive improvement of the system of classification of the oncohematological entities. Currently, the presence of certain cytogenetic or molecular anomalies is sufficient to identify specific entities and, in some occasions, the diagnostic changes after an integral analysis of cytomorfologic, cytogenetic and molecular studies. T This report aims to highlight the importance of molecular study when cytomorphology is complex and leads to erroneous diagnoses. Through polymerase chain reaction, prior reverse transcription of the RNA isolated from medullary blood, four biomarkers were studied: the fusion genes AML1-ETO, BCR-ABL, CBFβ-MYH11 and PML-RARα. Fourteen patients with initial diagnosis of: promyelocytic leukemia (n= 6), acute leukemia without lineage definition (n= 3) and chronic myeloid leukemia in myeloid blastic crisis or acute myeloid leukemia (AML) (n= 5) were studied. At the end of the molecular characterization all were diagnosed as AML. These results enlightened the role of the molecular studies in the classification of leukemia, which permit the patient receives the more appropriate treatment and achieve a better response.

Mechanism of decitabine in inhibiting cell proliferation and inducing apoptosis of AML1-ETO+ leukemia cells / 中国现代医生

Objective To study the decitabine inhibits the proliferation and induces apoptosis of AML1-ETO+ leukemia cells and to explore its possible mechanism. Methods Kasumi-1 cells were commonly cultured in RPMI-1640 medium containing 10％ fetal bovine serum. The proliferation of Kasumi-1 cells was determined by CCK8 assay. Flow cytometry was used to detect Kasumi-1 apoptosis. Related protein expression was measured by Western Blot. The expressions of AML1-ETOand miR-193a were measured by RT-PCR. Results Decitabine could inhibit the proliferation of Kasumi-1 cells with concentration effect and time effect, and could induce apoptosis. The apoptosis rates of control group, 24 hours and 48 hours group were (5. 29±0. 88)％ and (9. 83±1. 71)％ and (19. 47±1. 84)％, respectively. Decitabine could decrease the expression of AML1-ETOprotein, and the AML1-ETOprotein ratios of 0. 1, 0. 5 and 1 μmol/Ldecitabine group to the control group were (0. 85±0. 21), (0. 28土0. 06) and (0. 10±0. 07). The ratios of AML1-ETOprotein of 24 hours group, 48 hours group to control group were (0. 31±0. 21) and (0. 24±0. 11). But decitabin did not affect the expression of AML1-ETOmRNA, and the ratios of AML1-ETOmRNAof 24 hours group and 48 hours group to control group were (0. 96±0. 19) and (0. 84±0. 11). The difference was not significant(F=1. 22, P＞0. 05). Decitabine could up-regulate the expression of miR-193a by (3. 61士0. 06) and (6. 99±0. 74) fold respectively at 24 and 48 hours, and decreased the expression of MDM2 and Cyclin Dl. The protein expression ratios of MDM2 and Cyclin D1 of dosing group and control group were (0. 51土0. 19) and (0. 50±0. 10), respectively. Conclusion Decitabine inhibits the proliferation and induces apoptosis of AML1-ETO+ leukemia cells by up-regulating miR-193a to repress the translation of AML1-ETOand inhibiting the expression of MDM2 and Cyclin Dl.

Expression and prognostic significance of CD19 in patients with acute myelogenous leukemia with AML1-ETO positive / 中国免疫学杂志

Objective:To investigate the expression and prognostic significance of CD19 in patients with Acute myelogenous leukemia with AML1-ETO positive. Methods: Clinical data of 66 patients AML with AML1-ETO positive who were newly diagnosed from Jan 2010 to Dec 2015 were collected. To retrospectively analyze the relationship between clinical characteristics and expression of CD19,so dose the prognosis. Results:The positive rate of CD19 expressing in AML with AML1-ETO positive was 50. 0%. There were no statistically significant differences in terms of age,gender,hemoglobin,platelet,percentage of bone marrow blasts,accompanied with chromosome ,gene mutations between patients with and without CD19 expression(P>0. 05). The white blood cell count(WBC) of the CD19 negative group was higher than CD19 positive group,while showed significant difference(P=0. 027). Although the relapse-free survival (RFS) of patients with CD19 expression was higher than those without,no significant difference was calculated (P=0. 105). Patients with CD19 expression had superior overall survival ( OS ) compared to those without CD19 expression ( P = 0. 030 ) . Multivariable analysis for OS identified CD19 positivity as an independent predictor associated with better prognosis. Conclusion: The expression of CD19 in AML with AML1-ETO positive may be an indicator associated with better prognosis.

Gen de fusión AML-1/ETO y mutación NPM-1A en leucemia mieloide crónica en crisis blástica mieloide / AML-1/ETO fusion gene and mutation NPM-1A in myeloid chronic leukemia in myeloid blast crisis

La leucemia mieloide crónica es una neoplasia mieloproliferativa de naturaleza clonal que generalmente y de manera progresiva transita por tres fases: crónica, acelerada y crisis blástica. Alrededor del 80 por ciento de los enfermos con leucemia mieloide crónica son diagnosticados durante la fase crónica, 10 por ciento en fase acelerada y otro 10 por ciento durante la crisis blástica. A la presencia del cromosoma Filadelfia y la formación del gen de fusión BCR/ABL, que se traduce en la proteína quimérica pBCR/ABL, le sigue una gran inestabilidad genómica y la adquisición de alteraciones cromosómicas y moleculares adicionales. Algunas alteraciones moleculares, que suelen estar presentes en otras hemopatías malignas, pueden ser adquiridas durante la progresión de la leucemia mieloide crónica a crisis blástica. Se presenta el caso de un paciente que debutó con una leucemia mieloide crónica en crisis blástica, con positividad del gen de fusión BCR/ABL, el gen de fusión AML-1/ETO y la mutación NPM-1A(AU)

Chronic myeloid leukemia is a clonal myeloproliferative neoplasm which generally and progressively goes through three phases: chronic, accelerated and blast crisis. About 80 percent of patients with chronic myeloid leukemia are diagnosed in chronic phase, 10 % in accelerated phase and 10 percent in blast crisis. The presence of Philadelphia chromosome and the formation of BCR/ABL fusion gene, resulting in the chimeric protein pBCR/ABL, generates a large genomic instability and the acquisition of additional chromosomal and molecular alterations. Some molecular alterations, which are usually present in other hematological malignancies, could be acquired during the progression of chronic myeloid leukemia to blast crisis. This report presents the case of a patient with de novo chronic myeloid leukemia in blast crisis, with positivity of BCR/ABL fusion gene, AML-1/ETO fusion gene and mutation NPM-1A(AU)

Analysis of molecular characteristics and prognosis in acute myeloid leukemia patients with AML1/ETO / 白血病·淋巴瘤

Objective To analyze the molecular characteristics and prognosis in acute myeloid leukemia patients with AML1/ETO.Methods The clinical data of 63 cases of acute myeloid leukemia (AML) patients with AML1/ETO positive were analyzed retrospectively.56 cases of AML patients with AML1/ETO negative in the same period were analyzed as control.Characteristics in morphology,immunology,cytogenetics,molecular biology and the clinical effects of treatment were studied and analyzed.Results M2a was 57.12 ％ (36/63),M2b was 33.33 ％ (21/63) in AML with AML1/ETO.The percent of initial marrow blasts was 0.46±0.16.The positive rate of CD34,CD13,CD33,CD19,CD7 and CD56 was 67.21％,52.46 ％,40.98 ％,63.93 ％,4.92 ％ and 50.82 ％,respectively.The rate of t(8;21) translocation was 82.54 ％.There was 4.76 ％ with additional chromosome abnormality,three cases with EV1 1and one case with MLL/AT9.The overall CR rate,the relapse rate,the 3-year and the 5-year overall survival rate was 71.43 ％,51.11％,(43.01±5.31) ％ and (32.79±3.81) ％,respectively.There was no significant difference compared with the control group (P ＞ 0.05).But extramedullary infiltration,the expression of CD56 and additional chromosome abnormality had statistical effects on overall survival (P ＜ 0.05).Conclusions There has unique characteristics in AML with AML1/ETO.The effects of treatment and the prognosis are affected by many factors,so the efficacy and prognosis of AML with AML1/ETO couldn' t just depend on AML1/ETO.

Gen de fusión AML1-ETO: particularidades en la leucemia mieloide aguda / AML1-ETO fusion gen: characteristics in acute myeloid leukemia

La leucemia mieloide aguda (LMA) es una enfermedad neoplásica de la médula ósea en la que los pacientes con la translocación (8;21) representan un subgrupo con características clínicas y biológicas específicas. Esta alteración citogenética resulta de la fusión de dos genes, dando lugar a una proteína quimérica formada por un dominio N-terminal del gen AML1 y cuatro dominios C-terminales del gen ETO. Esta proteína tiene múltiples efectos en la regulación de la proliferación, la diferenciación y la viabilidad de las células leucémicas. La translocación puede ser detectada como una sola anomalía genética o como parte de anomalías complejas. A diferencia de otros pacientes, el diagnóstico de LMA con t(8;21) puede ser realizado con menos del 20 por ciento de blastos en la médula ósea. La enfermedad se caracteriza por anomalías genéticas adicionales, las células leucémicas muestran un perfil de expresión global de genes y un perfil de microARNs. Usualmente hay un bajo riesgo de recaída de la leucemia después de altas dosis de citosina arabinósido

Acute myeloid leukemia (AML) is a heterogeneous bone marrow malignancy where patients with the cytogenetic t(8;21) abnormality represent a subset with specific clinical and biological characteristics. The translocation results in an in-frame fusion of two genes, resulting in a fusion protein of one N-terminal domain from the AML1 gene and four C-terminal domains from the ETO gene. This protein has multiple effects on the regulation of the proliferation, the differentiation and the viability of leukemic cells. The translocation can be detected as the only genetic abnormality or as part of more complex abnormalities. In contrast to other AML patients, the diagnosis of t(8;21) AML can be made even when less than 20 percent leukemic blasts are present in the bone marrow. The leukemic cells show specific global gene expression and microRNA profiles; and usually there is a low risk of leukemia relapse after high-dose cytarabine therapy

Advances study of t(8;21) acute myeloid leukemia / 白血病·淋巴瘤

One of the sub-types of acute myeloid leukemia(AML) characterized by the translocation of chromosome 8 to chromosome 21 and the expression of AML1-ET0 leukemia genesis fusion gene was proven to have better prognosis. Although remission rate has been improved by the combined chemotherapy primarily containing high dose cytarabine,it seems that target treatment with AML1-ET0 fusion gene/protein should finally cure this disease. In this article,we reviewed the relative studies and clinic target-treatment on AML1-ETO.

Haematological & molecular profile of acute myelogenous leukaemia in India.

Background & objectives: Recurrent balanced translocations are generally recognized to be a major parameter for prognostication in acute myeloid leukaemia (AML). The chromosomal translocation t(15;17) results in PML/RARα fusion gene, t(8;21) results in AML1/ETO fusion gene and Inv 16 generates CBFβ/MYH11 fusion gene. Patients with these mutations have a good prognosis unlike abnormalities in chromosome 5 or 7 or FLT3 genes. Therefore, we screened the AmL patients for known specific genetic abnormalities that could lead to more definitive prognoses. Methods: A total of 113 AML patients were evaluated at diagnosis based on routine morphology and cytochemistry and classified according to the WHO criteria. The distribution of AML subtypes was M1(1), M2(32), M3(57), M4(14), M5(1), M6(1) and seven cases where morphological subtype could not be classified. RT-PCR was performed to identify PML/RARα, AML1/ETO, CBFβ/MYH11 and FLT3 internal tandem duplication (ITD). Results: Of the 57 patients with M3 subtype, 55 had the PML-RARα fusion transcript. The prevalence of bcr3 (short isoform) was higher (62%) than that of bcr1 (long isoform) (38%) and no correlation was found with age, sex or white blood cell count. FLT3 internal tandem duplication (ITD) mutations were more frequent in patients with APL than in other AML subtypes (17.5 vs. 8.9%), the frequency greater in patients with bcr3 isoform (70%) than in those with in bcr1 isoform (30%). Patients with FLT3/ITD mutations had a significantly higher median white cell count than those without these mutations (55 x 109/l vs. 6.3 x 109/l; P<0.001). More patients with FLT3/ITD mutations died early (53%) than those without these mutations (16%) (P<0.01). AML1-ETO fusion transcript was detected in 16 of 56 patients with no correlation with clinical or haematological parameters. Interpretation & conclusion: The results of the present study showed presence of bcr3 (short isoform) higher than bcr1 (long isoform). FLT3 internal tandem duplication (ITD) mutation was predominant in acute promyelocytic leukaemia patients with bcr3 isoform. Thus, patients with APL who have FLT3 mutation appear to have a poorer prognosis. Therefore, rapid identification of specific translocations at diagnosis is important for prognostic purposes and their detection should be incorporated into routine assessment.

A case of biphenotypic acute leukemia with expression of the AML1-ETO gene rearrangement / 대한내과학회지

Biphenotypic acute leukemia (BAL) is a subtype of acute leukemia that expresses two different immunophenotypic lineages, most commonly myeloid and either B- or T-lymphoid lineages. This entity has been defined by a scoring system proposed by the European Group for the Immunological Characterization of Leukemias (EGIL). The prognosis of BAL is regarded as being worse than either acute lymphoid or myeloid leukemia that does not show lineage ambiguity. However, a treatment strategy for BAL has not yet been established. We experienced a case of BAL with the t(8;21) translocation, a favorable cytogenetic rearrangement in acute myeloid leukemia (AML). The patient was successfully treated with cytarabine and anthracycline for induction and consolidation. The quantitative value of the AML1-ETO gene decreased after achieving complete hematologic remission. Thus, the AML1-ETO gene rearrangement in BAL may be associated with an acceptable response to the treatment strategy for AML.

Real-time RT-PCR for detection and quantification of AML1/ETO leukemia fusion gene / 白血病·淋巴瘤

Objective To set up real-time quantitative RT-PCR technique and measure leukemia fusion gene transcripts in patients with AML of FAB-M_2 subtype,and also to investigate the positive rate in patients and the relationship between the AMLI/ETO mRNA levels and the response rate after chemical therapy.Methods The plasmid containing the AMLI/ETO fusion gene sequences were constructed from myeloid cell lines Kasumi-1 (expressing AML1/ETO)to establish the standard curves,A TaqMan based realtime quantitative RT-PCR was performed to measure aberrant fusion gene transcripts in 45 samples of peripheral blood(PB) or bone marrow (BM) from 25 newly diagnosed patients with AML-M_2.All these 25 patients were diagnosed by the FCM(flow cytometry)and bone marrow molecular cytogenetics,and received the induce remission therapy with MA (Mitoxantrone+Ara-C).Results As a result,the AML1/ETO fusion gene transcripts were detected in 7(28%)out of 25 AML-M_2 patients (the ratios of AML1/ETO/ABL vary from 0.01 to 19.2),in which 5 patients were found t(8;21)(q22;q22).The transcript level of AML1/ETO fusion gene varied from the clinical situation of patients.These 7 patients with AML1/ETO fusion gene got complete remission(CR) after the first MA therapy,and the fusion gene reduced by 3 log in AML1/ETO/ABL.Only 11 patients got CR in 18 patients without AML1/ETO fusion gene.By following up these 7 patients with AML1/ ETO fusion gene kept persistent CR for 6 months.Conclusion It was concluded that real-time quantitative PCR is a reliable,innovative and promising technology with high sensitivity and speciality.It has potential clinical value for diagnosis,tumor typing,treatment selection,measuring the tumor load,monitoring fusion gene expression level and evaluating therapeutic strategy.It is worthy to apply in the clinical practice.

Research on AML1/ETO fusion gene on 76 patients diagnosed with acute myelogenous leukemia

Background: Chromosome mutation type t(8;21) has quite a high frequency in acute myelogenous leukemia, which accounted for about 15% among adult patients. From 2001, the WHO has a new classification for acute myelogenous leukemia based on genetic mutations. Form had AML1/ETO were arranged into genetic mutation group with better prognosis and ability to fully recover after chemotherapy with a high dose of cytarabin. Objective: Study AML1/ETO fusion gene on the patients diagnosed with Acute Myelogenous Leukemia (AML), as well as the clinical features and some haematologic parameters of the AML1/ETO positive group. Subject and methods: 76 patients with AML were treating in the National Institute of Hematology & Blood Transfusion and the Department of Hematology & Blood Transfusion of Bach Mai Hospital from April 2007 to July 2008. These patients were studied for clinical examination, morphology and RNA were extracted from leukemic cells and PCR for AML1/ETO fusion transcript was performed. Results and conclusions: The incidence of AML1/ETO positive in the AML patients was 24%. The incidence of AML1/ETO positive in AML-M2 was 28%. In the AML1/ETO positive group: median age was 26.94+/-9.22; rate of severe anemia, hemorrhage, fever, infection, hepatomegaly, splenomegaly, lymphadenopathy and gum hypertrophy was 44%, 33%, 28%, 11%, 44%, 28%, 17% and 6%, respectively. Median hemoglobin, WBC, platelet, bone marrow cell count, % blast in peripheral blood and in bone marrow was 84.41+/-28.97 g/l, 29.42+/-31.36 g/l, 42.12+/-33.83 g/l, 215.93+/-134.42 g/l, 56.21+/-26.58% and 65.14+/-16.12%, respectively.

Value of interphase fluorescence in situ hybridization in diagnosing acute myeloid leukemia M2 and M3 / 肿瘤研究与临床

Objective To investigate the value of interphase fluorescence in situ hybridization(FISH)technique and the detection of fusion gene in the diagnosis of acute myeloid leukemia(AML)M2 and M3 Methods FISH was used to detect the AML1/ETO fusion gene and/or PML/RARα fusion gene in incipient cases including 9 AML-M2, 12 AML-M3 and 10 AML undetermined as AML-M2 or AML-M3 primarily diagnosed by routine morphology though bone marrow,cytochemical staining and immunophenotyping,which can help diagnose and guide clinical therapy.Results 4 of 9 AML-M2 cases were AML1/ETO positive.Among 12 AML-M3 cases,10 were PML/RARα positive.1 case was detected AML1/ETO fusion gene.In 10 untonfirmed M3 or M2,3 case8 showed AML1/ETO,5 showed PMIJRARot fusion gene and the rest showed neither of the genes.Conclusion As a new technique of the molecular genetics,FISH is accurate, rapid and efficient.It would be of significance not only at diagnosis of AML,but also for subsequent clinical decision-making.

Morphological characteristics of leukemia cells in acute myeloblastic leukemia with t(8;21)(q22;q22): possible predictability of t(8;21).

The laboratory systems for chromosomal analysis or the detection of fusion genes are generally not available in Indonesia. Therefore, bone marrow (BM) morphological analysis should be developed and applied to get an accurate diagnosis. In this study the BM smears of eight (8) cases of acute myeloblastic leukemia (AML) which had already been known to have t(8;21)(q22;q22), were morphologically evaluated in order to find out the characteristics, which might be used to predict t(8;21)(q22;q22) or the presence of AML1-ETO(MTG8) fusion gene. All of the cases belonged to AML-M2. The morphological characteristics, indicative of t(8;21) were pink colored cytoplasm in mature neutrophil (75%), neutrophilic myelocytes or metamyelocytes without granules or with scarce granules (2.3%), eosinophilia (eosinophilic myelocytes and metamyelocytes) (above 5%), myelocytes with abundant granules 8.5%, and low percentage of type I blasts (below 10%). These characteristics were not observed in AML-M2 cases without t(8;21) or AML1-ETO(MTG8). The myelocytes with abundant granules have not been described so far, while other characteristics were in line with the findings of Nakamura et al (Leukemia 1997;11:651-55).

Cases of extramedullary relapse after alloBMT in AML1/ETO positive AML / 대한내과학회지

The occurrence of granulocytic sarcoma as a pattern of relapse after allogenic bone marrow transplantation (allo-BMT) is rare. We report two AML1/ETO positive patients with granulocytic sarcoma as a pattern of relapse after allo-BMT. The first case is 39-year-old woman who had lower back pain without any abnormal peripheral blood exam. MRI showed a paraspinal mass, and the pathologic report was granulocytic sarcoma. The second case is 34-year-old woman who had breast humps without any abnormal peripheral blood exam. CT showed multiple breast masses, and the pathologic report was granulocytic sarcoma. We believe that this is the first cases report of extramedullary relapse after allo-BMT in AML1/ETO positive AML in Korea. These cases suggest that we should be aware of the possibility of an extramedullary relapse in bone marrow transplant recipients with AML1/ETO positive AML.

Prognostic Value of AML1/ETO Fusion Transcripts in Patients with Acute Myelogenous Leukemia

BACKGROUND: The t (8; 21) (q22; q22), which produces the fusion gene AML1/ETO, is associated with relatively good prognosis and, in particular, with a good response to cytosine arabinoside. Analysis of t (8; 21) positive leukemic blasts has shown characteristic morphological and immunological features. We performed this study to investigate the incidence of AML1/ETO rearrangement in adult acute myelogenous leukemia (AML), especially in M2 subtype, to make a comparison of clinical, morphological and immunophenotypic characteristics between AML1/ETO rearrangement positive and negative group in patients with AML and to analyze the correlation with other biological parameters. METHODS: From May 1995 to Sept. 2000, fifty-nine patients with AML, including twenty-nine AML-M2, were studied. RNAs were extracted from leukemic cells and reverse transcriptase mediated polymerase chain reaction (RT-PCR) for AML1/ETO fusion transcript was done. Chromosome study, immunophenotypic and clinical characteristics were analyzed and statistical analysis was done. RESULTS: The incidence of AML1/ETO fusion transcripts was 22.0% in AML and 44.8% in AML-M2. The morphologic finding of bone marrow in AML-M2 showed higher incidence of Auer rods, large blast with prominent golgi and abnormal granules in AML1/ETO positive patients. There was no significant difference of immunophenotype. AML patients with AML1/ETO had a tendency of higher complete remission rate (81.8% vs 56.6%, p=0.13). The overall survival (median; 82.2 weeks vs 34.4 weeks, p=0.02) and progression free survival (median; 50.9 weeks vs 20.4 weeks, p=0.02) of AML1/ETO positive group were longer than those of the negative group in AML. AML-M2 patients with AML1/ETO rearrangement had also a tendency of longer overall survival and progression free survival, although there was no significant difference between both groups. CONCLUSION: Our data suggest that AML1/ETO rearrangement is detected frequently in AML, especially M2, and is a favorable prognostic factor. Thus, molecular diagnostic approaches should be used routinely to identify patients with this genetic subtype of AML.

Detection of Residual Leukemia with Reverse Transcription-polymerase Chain Reaction from Patients with AML1/ETO Positive Acute Myeloid Leukemia in Remission / 대한혈액학회지

BACKGROUND: One of the most frequent cytogenetic abnormality in acute myeloid leukemia (AML) is t(8;21) (q22;q22), with rearrangement of the AML1 gene on chromosome 21q22 and the ETO gene on chromosome 8q22. In adult AML1/ETO-associated leukemia patients, chemotherapy alone results in cure rates that are comparable to or better than those achieved with allogenic bone marrow transplantation. Despite the relatively good prognosis of AML1/ETO fusion transcript, relapse of leukemia remains the most common cause of treatment failure. Monitoring minimal residual disease (MRD) in leukemia has two main aims : to assess the effectiveness of treatment and to detect early signs of relapse. Reverse transcription-polymerase chain reaction (RT-PCR)- based methods is the rapid and sensitive method in the identification of this molecular abnormality. The purpose of this study is to ensure the usefulness of the RT-PCR technique for detecting MRD in AML1/ETO-associated leukemia patients in remission and to establish the correlation of the serial detection of AML1/ETO fusion transcripts after complete remission and long-term outcome. METHODS: From the bone marrow aspirates of 25 AML1/ETO positive AML patients, serial detection of AML1/ETO fusion trascripts was performed using RT-PCR. RESULTS: AML1/ETO fusion transcripts were positive in 14 cases who did not show t(8;21). In serial assay, AML1/ETO fusion transcripts was positive in 9 cases and negative in 13 cases at 10 weeks after complete remission. AML1/ETO fusion transcripts (+) group has 107.4+/-18.2 months suvival and AML1/ETO fusion transcripts (-) group has 47.3+/-18.0 months survival. However, there is no significance (P=0.11). CONCLUSION: This study suggests that the early negative conversion of AML1/ETO fusion transcript may be the good prognostic predictor. The RT-PCR technique is useful for detecting minimal residual disease in leukemia patients in remission and it may improve the therapeutic strategy for leukemia.

Prognostic value of AML1/ETO fusion transcripts in patients with acute myelogenous leukemia / 대한내과학회지

BACKGROUND: The t (8;21) (q22;q22), which produces the fusion gene AML1/ETO, is associated with relatively good prognosis and, in particular, with a good response to cytosine arabinoside. Analysis of t (8;21) positive leukemic blasts has shown characteristic morphological and immunological features. We performed this study to investigate the incidence of AML1/ETO rearrangement in adult AML, especially in M2 subtype, to make a comparison of morphologic, immunophenotypic and clinical characteristics between AML1/ETO rearrangement positive and negative group in patient with AML and to analyze the correlation with other biological parameters. METHODS: From May 1995 to Sep. 2000, fifty-nine patients with AML including twenty-nine AML-M2 were studied. RNAs were extracted from leukemic cells and reverse transcriptase mediated polymerase chain reaction (RT-PCR) for AML1/ETO fusion transcript was done. Chromosome study, immunophenotypic, and clinical characteristics were analysed and statistical analysis was done. RESULTS: The male to female ratio was 32:27 in AML and 17:12 in AML-M2. The median age was 43 years (range 14-86) in AML and 43 years (range 14-77) in AML-M2. The incidence of AML1/ETO fusion transcripts was 22.0% in AML and 44.8% in AML-M2. The morphologic finding of bone marrow in AML-M2 showed higher incidence of Auer rods, large blast with prominent golgi and abnormal granules in AML1/ETO positive patients. There was no significant difference of immunophenotype. AML patients with AML1/ETO rearrangement had a tendency of higher complete remission rate (81.8% vs 56.6%, p=0.13). The overall survival (median 82.2 weeks vs 34.4 weeks, p=0.02) and progression free survival (median 50.9 weeks vs 20.4 weeks, p=0.02) of AML1/ETO positive group were longer than those of negative group in AML. AML-M2 patients with AML1/ETO rearrangement had also a tendency of longer overall survival and progression free survival, although there was no significant difference between both group (median OS 82.4 weeks vs 15.6 weeks, p=0.07, median PFS 50.9 weeks vs 16.0 weeks, p=0.09). CONCLUSION: Our data suggest that AML1/ETO rearrangement is detected frequently in AML, especially M2, and is a favorable prognostic factor. Thus, molecular diagnostic approaches should be used routinely to identify patients with this genetic subtype of AML.