Immunophenotyping Features in Acute Myeloid Leukemia (AML) with NPM1+ and/or FLT3+

Abstract Abstract 4908 Immunophenotyping Features in Acute Myeloid Leukemia (AML) with NPM1 and/or FLT-3 Positive Pervin Topçuoglu, Klara Dalva, Sinem Civriz Bozdag, Önder Arslan, Muhit Özcan, Osman Ýlhan, Hamdi Akan, Meral Beksaç, Günhan Gürman Aim: We aimed to evaluate immunophenotypical (IP) features in AML pts with NPM1+ and/or FLT3+ except on acute promyelocytic leukemia. Patients&Method: Between Nov 2009 and Feb 2011, we retrospectively analyzed IP features by flow cytometry (FCM) in 51 pts (46M;17F) with new diagnosed AML. Median age was 46 years (range: 14–71 ys). The mutations of NMP1 and FLT-3 TKD&ITD were determined by the methods of RQ-PCR or RFLP, respectively in the samples of bone marrow (n=31) or periheral blood (n=20) at the diagnosis. Antigenic expression of leukemic cells was analyzed by four-color FCM (FITC, PE, PerCP&APC) based by Nomdedeu et al researh (Leuk res 2011; 35:163) (Table-1). Results: We detected NMP1+ mutation in 16 patients. Of these, three were associated with mutations of FLT3-ITD (n=2) or -TKD (n=1). Twelve patients had FLT3+ (9 ITD or 3 TKD). More than half of the patients without any mutation were CD15+/CD34+/HLA-DR+ and 11.5% for CD34 negative. Similarly, the patients with FLT-3 positive were mostly CD34+ as the pts w/o any mutations. Contrary, most of the pts with NMP1+ were CD34 negative (56.3%) (Table 1). When evaluated the complete IP in leukemic cells, the expression of CD123 was significantly marked in the patients with NPM1+ and/or FLT3+ than those w/o mutations (p=0.008). While the co-expression of CD7 and CD117 was found in 67% of the pts w/o any mutations, 30% of the pts with NMP1 and/or FLT-3 ITD (p=0.01). CD56 expression was detected in more pts with NMP1+ than those with FLT-3+ (40% vs 8%, p=0.04). Besides, CD36 expression was positive in the all pts with FLT3-ITD than TKD+ (p=0.005). More intensive CD33 expression was seen in NMP1+ pts. The expression of CD64 was similar in all three mutations. Conclusion: Though NMP1 mutation was associated more CD34+ cells, more FLT3+ pts had CD34 positivity. The expression of CD123 was especially associated with the mutations. Aberrant expression of CD56 was in more pts with NPM1+, but CD36 for FLT3-ITD. These data might be a step for a study aiming to show a correlation between the type of mutations combined with IP features of leukemic cells and clinical characteristics or disease course of AML pts. Disclosures: No relevant conflicts of interest to declare.

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Coagulopathy in HLA-DR Antigen-Negative Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v104.11.4457.4457 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4457-4457

Author(s):

Hideki Uchiumi ◽

Takafumi Matsushima ◽

Arito Yamane ◽

Hiroshi Handa ◽

Hiroyuki Irisawa ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

De Novo ◽

Myeloid Cell ◽

Leukemic Cell ◽

Leukemic Cells ◽

Hla Dr ◽

Risk Of Mortality ◽

Acute Myeloid

Abstract Background: HLA-DR antigen is present on hematopoietic progenitors and granulocyte/monocyte, erythrocyte and megakaryocytic precursors but absent at the promyelocytic stage during myeloid cell maturation. In accordance with this, majority of promyelocytic leukemia (APL) cells were negative for HLA-DR. Meanwhile, some of non-APL acute myeloid leukemia (AML) cells is found to express HLA-DR. However, the clinical significance of HLA-DR antigen on AML cells is currently unclear. Purpose: We sought to determine the prevalence and clinical characteristics of negativity in HLA-DR expression by retrospectively analyzing 181 consecutive patients with de novo adult AML. Patients and Methods: AML patients examined in the current study (aged 15–86 years) had been diagnosed between August 1995 and July 2004, and categorized to M0 (8 patients), M1 (35), M2 (74), M3 (20), M4 (25), M5 (15), and M6 (4), based on the FAB classification. Median follow-up time was 19.3 months. Phenotypic analyses of leukemic cells were performed using CD45 gating methods. HLA-DR-negative AML was defined as HLA-DR expression less than 20% of cells in the CD45 leukemic cell gate. Results: Among 181 patients, HLA-DR antigens were not detected on AML cells from 46 patients; 20 with APL and 26 with non-APL (non-APL/DR(−)), the latter of which included M0 (2 patients), M1 (15), M2 (7), M4 (2). Leukemic cells from other non-APL patients were HLA-DR-positive (non-APL/DR(+)). None of non-APL/DR(−) patients had t(15;17) nor PML/RARa rearrangement on cytogenetic analysis. Twenty out of 26 patients with non-APL/DR(−) had normal chromosome, and 6 had abnormal karyotypes. In the non-APL/DR(−) group, various degrees of nuclear folding, convolution, or lobulation were observed in 9 patients. Although treatment response and overall survival rate were similar in the three groups (APL, non-APL/DR(−), and non-APL/DR(+)), both FDP levels at diagnosis (57.3 vs 13.2, p<0.05) and maximal FDP levels (232.6 vs 43.8, p<0.01) were significantly higher in non-APL/DR(−) compared with non-APL/DR(+). The maximal FDP levels in the non-APL/DR(−) patients were comparable to those in the APL patients. FDP levels greater than 40 mg/ml were significantly more prevalent in the non-APL/DR(−) than in the the non-APL/DR(−) group. Logistic regression analysis demonstrated that low HLA-DR expression was an independent risk factor for FDP > 40 mg/ml. Conclusion: Our study suggests that AML with negative HLA-DR antigen tend to be associated with abnormality in coagulation and fibrinolysis even if they are genetically non-APL. We propose that more attention should be paied for HLA-DR expression to avoid a devastating coagulopathy which carries a high risk of mortality unless specifically addressed.

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Acute Myeloid Leukemia with Mutated NPM1 Presenting with Life-Threatening, Either Arterial or Venous, Thromboembolism: a Report of 4 Cases.

Blood ◽

10.1182/blood.v114.22.4135.4135 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4135-4135

Author(s):

Maria Paola Martelli ◽

Lorenzo Brunetti ◽

Luca De Carolis ◽

Elisabetta Agliani ◽

Laura Berchicci ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Venous Thromboembolism ◽

Myeloid Leukemia ◽

Thromboembolic Event ◽

Conflicts Of Interest ◽

The Other ◽

Leukemic Cells ◽

Acute Ischemia ◽

Life Threatening ◽

Acute Myeloid

Abstract Abstract 4135 Acute myeloid leukemia (AML) expressing mutated NPM1 gene and cytoplasmic nucleophosmin (NPMc+ AML) [Falini B et al, NEJM 2005;352:254-266] is a new entity of WHO classification that shows distinctive biological and clinical features. AML with mutated NPM1 usually presents with a high white blood cell count; the bone marrow biopsy is usually markedly hypercellular and leukemic cells frequently show myelomonocytic or monocytic features, with dysplasia and involvement of two or more cell lineages in about 25% of cases. Lack, or low expression, of CD34 in over 90% of cases is the most distinctive immunophenotypic feature of NPM1-mutated AML and is independent of leukemic cell maturation. NPM1 gene mutation without concomitant FLT3-ITD identify a subgroup of AML patients with a favorable prognosis and has been associated with an approximately 50-60% probability of survival at 5 years in younger patients. Here we report 4 out of 41 (10%) patients, admitted at our Hospital in the last year, with new-diagnosed AML with mutated NPM1 presenting with life-threatening thromboembolic (either arterial or venous) events. The main characteristics of these patients are summarized in Table 1. The patients had neither personal nor family history concerning thromboembolism. Hyperleukocytosis was a common feature of the vast majority of NPM1-mutated AML patients at diagnosis. Immunophenotypic analysis did not show a peculiar phenotype in these patients. Table 1 Characteristics of patients with NPM1-mutated AML and thrombosis. Case report no Age Sex (M/F) FAB subtype WBC/mmc Type of thrombosis Site of thrombosis 1 41 F M1 14970 arterial Anterior interventricular branch of left coronary artery 2 56 M M4 93990 arterial external iliac and femoral (right limb) 3 63 M M2 113000 deep venous great saphenous veins (bilateral) 4 73 F M4 190000 deep venous iliac and femoral In two patients (cases 1 and 2), the arterial thromboembolic event (acute myocardial infarction and acute ischemia of right lower limb, respectively) presented about one month before diagnosis of leukemia. In the other 2 patients (cases 3 and 4), deep venous thromboembolism was concomitant with the diagnosis of leukemia. One patient (case 4), who could not initiate chemotherapy for severe concomitant renal failure, died few days after diagnosis. The other patients recovered from the acute event and upon diagnosis of leukemia were promptly treated with standard polychemotherapy which allowed to obtain complete hematological remission associated with complete resolution of the thromboembolic event. The clinical course after chemotherapeutic treatment of the patients outlines the importance and life saving role of early chemotherapy even under adverse circumstances. The pathogenesis of thromboembolic disease in hematological malignancies is complex and multifactorial: tumor cell-derived procoagulant, fibrinolytic or proteolytic factors and inflammatory cytokines affect clotting activation. Other important factors include infectious complications and hyperleukocytosis. However, large vessel thrombosis is a very rare clinical presentation. Our report of severe thromboembolic events at presentation in AML with mutated NPM1 suggests some still unidentified biological features of this leukemia which we are currently investigating. Disclosures: No relevant conflicts of interest to declare.

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CD19 Expression in t(8;21)-Negative Acute Myeloid Leukemia Is Not Restricted to Cytogenetically Aberrant Populations.

Blood ◽

10.1182/blood.v114.22.4128.4128 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4128-4128

Author(s):

Jawad Francis ◽

Avinash Dharmadhikari ◽

Sheila N.J. Sait ◽

George Deeb ◽

Paul K. Wallace ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Normal Karyotype ◽

Leukemic Cells ◽

Aberrant Expression ◽

The Third ◽

Signal Pattern ◽

First Case ◽

Cd19 Expression ◽

Acute Myeloid

Abstract Abstract 4128 Aberrant expression of the B lymphoid marker, CD19, in acute myeloid leukemia (AML) has frequently been associated with t(8;21)(q22;q22). However, AML cases lacking this translocation may occasionally express CD19. We identified eight such cases at Roswell Park Cancer Institute. Three of these cases had noticeable CD19-positive leukemic subsets with the following karyotypic abnormalities. The first case had monosomy X in 6 cells and normal karyotype in 14, the second case had trisomy 22 in 19 cells and normal karyotype in one, and the third case had monosomy 7 in 18 cells and trisomy 18 in two. We therefore asked if CD19 expression is restricted to the karyotypically abnormal leukemic cells. Bone marrow cells were sorted according to the specific immunophenotype. First, a generous mononuclear sorting region was drawn in the bivariate display of forward versus side scatter to exclude debris and aggregates. Next, the CD45-negative to CD45-dim, CD19-positive and CD34-positive leukemic cells were sorted. Fluorescent in situ hybridization (FISH) studies were carried out on the sorted samples using the following commercially available probes: the Centromere enumeration probe CEP X (SpectrumGreen) in the first case; the locus specific BCR (22q11) (SpectrumGreen)/ABL1 (9q34) (SpectrumOrange) dual color, dual fusion, translocation probe in the second, and the locus specific D7S486 (7q31) SpectrumOrange/CEP 7 SpectrumGreen probe in the third. A total of 200 nuclei were assessed in each case. The results are displayed in the Table below. There were no significant differences in the signal pattern between the CD19-positive and CD19-negative leukemic populations. These results indicate that aberrant CD19 expression in t(8;21)-negative AML is not restricted to leukemic cells that harbor specific karyotypic abnormalities. Table FISH results based on CD19 expression Case # Probe Signal Pattern CD19-positive CD19-negative 1 CEP X 1 Green (-X) 20/200 30/200 2 BCR/ABL 3 Green/2 Orange (+22) 163/200 170/200 3 CEP 7 1 Green/1 Orange (-7) 200/200 200/200 Disclosures: No relevant conflicts of interest to declare.

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MYB Overexpression Is Directly Involved in Acute Myeloid Leukemia Pathogenesis and Could Constitute a New Therapeutic Target for Patients with Aberrant Expression of This Gene.

Blood ◽

10.1182/blood.v114.22.2402.2402 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2402-2402 ◽

Cited By ~ 1

Author(s):

Carmen Vicente ◽

Ana Conchillo ◽

Daphnie Pauwels ◽

Iria Vazquez ◽

Laura Garcia-Orti ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Complete Remission ◽

Synergistic Effect ◽

Cell Lines ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Recurrent Event ◽

Aberrant Expression ◽

Acute Myeloid

Abstract Abstract 2402 Poster Board II-379 The MYB proto-oncogene encodes a nuclear transcription factor with an essential role in proliferation, lineage commitment, and differentiation of hematopoietic progenitor cells. Proper levels of MYB are known to be important during hematopoietic cell development, and the Myb gene is a frequent target of retroviral insertions in myeloid, B- and T-cell leukemias in the mouse. Overexpression of MYB in T-acute lymphoblastic leukemia (T-ALL) causes a differentiation block of the T cells, and it has been shown that NOTCH1 mutation and MYB duplication cooperate in the pathogenesis of T-ALL. Our aim was to study the role of MYB in the pathogenesis of acute myeloid leukemia (AML), and to investigate its potential as a target for therapy. We functionally characterized MYB in 15 AML cell lines. Twelve of the 15 cell lines tested had MYB overexpression. Knockdown of MYB by siRNA in these cell lines caused decreased cell viability and proliferation, and reduced the clonogenic capacity, that could be explained in some cell lines by changes on the stage of cell differentiation. These results show that MYB overexpression is involved in the pathogenesis of AML. Moreover, knockdown of MYB in combination with common AML treatments (Idarubicin, Cytarabine and Sorafenib) had a strong synergistic effect on proliferation and viability of cells, suggesting that MYB could be a new target for therapy in AML. These observations prompted us to quantify MYB expression in a cohort of 159 patients with AML at diagnosis. We detected MYB overexpression in 14.5% (23/159) patients, with a higher prevalence within the intermediate prognosis group (17/83, 20.5%), particularly in patients with normal karyotype (NK) (14/62, 22.6%). Interestingly, 33% of patients without FLT-3 ITD and NPM1 mutations had MYB overexpression. To study the prognosis impact of MYB overexpression in AML, we performed a survival analysis in a preliminary series of 100 AML patients at diagnosis. As expected, significant differences in OS according to age, complete remission and cytogenetic prognostic group were found (p<0.01). MYB overexpression had no significant impact in the OS; however, this genetic marker allowed distinguishing a group of patients with a worse outcome within the group that did not get complete remission after treatment. Recently it has been described that MYB duplication causes elevated MYB expression in T-ALL; we detected duplication of MYB in 2 of 13 AML cell lines and in 2 patients with MYB overexpression (2/23, 8.6%). In conclusion, these results show that aberrant expression of MYB is involved in the activation of pathways responsible for the increased proliferative and clonogenic capacity that is characteristic of AML, independently of other genetic aberrations. Moreover, we show that MYB overexpression is a recurrent event in AML, especially in the subgroup of patients with NK, and that MYB could cooperate with other mutations in the leukemic transformation, as described previously in T-ALL. The synergistic effect of combined treatments with MYB knockdown, suggest that MYB silencing could be a new target for therapy in patients with AML and MYB overexpression. Disclosures: No relevant conflicts of interest to declare.

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Unraveling The Leukemic Nature Of hMICL and CD123 Expressing Cells In Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v122.21.2626.2626 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2626-2626 ◽

Cited By ~ 2

Author(s):

Line Nederby ◽

Peter Hokland ◽

Gordon Brown ◽

Maria Hansen ◽

Charlotte Guldborg Nyvold ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Capillary Electrophoresis ◽

Myeloid Leukemia ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Leukemic Cells ◽

Fragment Analysis ◽

Marker Combination ◽

Allelic Burden ◽

Acute Myeloid

Abstract Flow cytometry constitutes a cornerstone in the diagnosis and follow up of acute myeloid leukemia (AML) and it is based on the identification of leukemia-associated immunophenotypes (LAIPs). We have recently demonstrated that the C-type lectin hMICL in combination with CD123 constitute a highly stable and reliable LAIP marker combination at diagnosis and relapse. In addition, we have shown that an hMICL/CD123-based assay is an effective tool for the monitoration of minimal residual disease (MRD) in AML. To what extent hMICL/CD123 marking identifies early leukemic cells is, however, not established. We hypothesized that this could be addressed by studying molecular aberrations in leukemic cell subsets according to their expression of hMICL and CD123. Employing cell sorting and mutational analyses, we here establish the leukemic origin of hMICL and CD123 expressing cell populations. Analyzing diagnostic AML samples with homozygous FLT3-ITD aberrations allowed for verification of pure malignant clones. Five patients with FLT3-ITD allelic burden of >50% (range 77-93%, median 85%) as measured by DNA fragment analysis by capillary electrophoresis on mononuclear cells (MNC) were identified in our local database of 600 cases. We found that 5/5 patients displayed a normal karyotype and carried NPM1 mutations (NPM1 allelic burden 42-48%, median 46%). In contrast, mutations in FLT3-D835, IDH1-R132, c-KIT-D816V or indel mutations in CEBPA and WT-1 exon 7 were absent. From samples of cryopreserved mononuclear cells (bone marrow (n=4) and peripheral blood (n=1)), CD45low/SSClow blast cell subsets with the following immunophenotypes were sorted by FACS: CD34+/hMICL+/CD123+, CD34+/hMICL+/CD123-, CD34+/hMICL-/CD123+, and CD34+/hMICL-/CD123-. In one case of CD34 negative AML the sorted subsets were CD34-/hMICL+/CD123+, CD34-/hMICL+/CD123-, CD34-/hMICL-/CD123+, and CD34-/hMICL-/CD123-. Sorted cell subsets were analyzed for FLT3-ITD and NPM1 mutations using fragment analysis by capillary electrophoresis. The results of the fragment analyses are tabulated in the table below. In all cases the hMICL and CD123 expressing subsets of interest closely approximated 100% FLT3-ITD allelic burden. In contrast, hMICL-/CD123- cells approximated only a 50% FLT3-ITD allelic burden. Of note, an extended search in our AML database, revealed only 9 of 600 patients to have an FLT3-ITD allelic burden >50% (range 52-94%, median 81%) hence indicating a state of either homo- or hemizygosity. Interestingly, with the exception of one case carrying a chromosome 13 duplication, each of these 9 patients also harbored a mutation in the NPM1 gene as the only other known aberration. In conclusion using AML patients with high FLT3-ITD allelic burdens we have been able to show that blasts expressing hMICL and/or CD123 at diagnosis are indeed malignant thus further substantiating the use of these antigens in AML MRD detection. Additionally, a direct relationship between NPM1 and FLT3-ITD homo-/hemizygosity may be suggested in the evolution of the malignant clone.Phenotype of sorted cell subsetNumber of patientsFLT3-ITD allelic burden (%) Min-max (median)NPM1 allelic burden (%) Min-max (median)MNC577-93 (85)42-48 (46)CD45low/SSClow/CD34+/hMICL+/CD123+495-100 (98)48-50 (49)CD45low/SSClow/CD34+/hMICL+/CD123-1*9248CD45low/SSClow/CD34+/hMICL-/CD123+497-100 (99)47-51 (48.5)CD45low/SSClow/CD34+/hMICL-/CD123-436-68 (47)16-38 (25)CD45low/SSClow/CD34-/hMICL+/CD123+110046CD45low/SSClow/CD34-/hMICL+/CD123-19448CD45low/SSClow/CD34-/hMICL-/CD123+110047CD45low/SSClow/CD34-/hMICL-/CD123-17735*Subset only present in one of four patients Disclosures: No relevant conflicts of interest to declare.

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High Expression of Mir-15a Predicts Shorter Survival and Worse Response to Chemotherapy in Patients with Acute Myeloid Leukemia (AML)

Blood ◽

10.1182/blood.v124.21.5330.5330 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5330-5330 ◽

Cited By ~ 1

Author(s):

Aleksandra Butrym ◽

Dagmara Baczynska ◽

Andrzej Tukiendorf ◽

Justyna Rybka ◽

Tadeusz Dobosz ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Reverse Transcriptase ◽

Induction Chemotherapy ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Control Group ◽

Aberrant Expression ◽

Risk Of Death ◽

Response To Chemotherapy ◽

Acute Myeloid

Abstract Background: MicroRNAs (miRNAs) are small non-coding RNA molecules, that control gene expression by targeting messenger RNA (mRNA), via degradation or suppression of translation. Aberrant expression of microRNAs (miRs) has been proved to have a role in acute myeloid leukemias (AML) The aim of the study was to determine expression of miR-15a in acute myeloid leukemia patients before and after chemotherapy and its influence on patient clinical outcome. Methods: miRNAs from isolated leukemic cells were extracted using mirVanaTM miRNA Isolation kit (Ambion Inc., Carlsbad, CA, USA) following the manufacturer's protocol. Reverse transcriptase (RT) reactions were performed for mature miRNA cDNA synthesis in separate tubes using specific stem-loop RT primers and TaqMan® MicroRNATM Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). After microRNA isolation, reverse transcriptase reactions were performed, followed by cDNA amplification. The relative amount of microRNA-15a was normalized according to the reference RNU48 level. Results were considered statistically significant with p-value < 0.05. Results: 95 patients (aged 60.2 ± 15.0, 22–90, Male = 61%) with newly diagnosed AML were included into the study. Samples of the bone marrow for miR-15a expression analysis were collected before start of chemotherapy and repeated after completed induction chemotherapy (40 patients). A control group of 20 matched patients was also taken into account. The analyzed group consisted of: 7 patients with AML M0, 34 had M1, 29 had M2, 14 had M4 and 11 had M5. AML patients has higher miR-15a expression than control group (p=0.005633). The risk of death in AML patients was higher in patients with higher miR-15a expression on diagnosis (p=0.0430), Fig.1. Patients with lower miR-15a expression were more likely to achieve complete remission after induction chemotherapy (p=0.0130). After successful chemotherapy we observed significant drop in miR-15a expression. Conclusions: We proved that miR-15a was upregulated in AML patients and its expression influenced remission achieving and death risk. Figure 1. Survival of AML patients depending on miR-15a expression. Figure 1. Survival of AML patients depending on miR-15a expression. Disclosures No relevant conflicts of interest to declare.

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ETO2-GLIS2 Controls Differentiation Arrest and Self-Renewal through Aberrant Enhancers Regulation in Pediatric Leukemia

Blood ◽

10.1182/blood.v128.22.572.572 ◽

2016 ◽

Vol 128 (22) ◽

pp. 572-572

Author(s):

Cecile Thirant ◽

Cecile K Lopez ◽

Cathy Ignacimouttou ◽

M'Boyba Diop ◽

Lou Le Mouël ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Genetic Alterations ◽

Leukemic Cells ◽

Hematopoietic Progenitors ◽

Pediatric Leukemia ◽

Self Renewal ◽

Megakaryocytic Differentiation ◽

Acute Myeloid

Abstract Deregulated gene expression due to genetic alterations, such as gene fusions affecting transcription and/or epigenetic factors is the hallmark of acute myeloid leukemia and the basis for the differentiation block of hematopoietic progenitors. Acute megakaryoblastic leukemia (AMKL) is a subtype of poor prognosis acute myeloid leukemia (AML) affecting primarily young children. Recently, the ETO2-GLIS2 fusion has been identified in 20-30% of de novo AMKL and associated with the worst prognosis in this subtype of AML. To characterize the transformation induced by ETO2-GLIS2, we first defined the consequences of ETO2-GLIS2 expression on hematopoietic progenitors and the contribution of ETO2 and GLIS2 on differentiation and self-renewal. Using methylcellulose replating assays and phenotype characterization, we show that the GLIS2 moiety drives the megakaryocytic phenotype whereas both the ETO2 and GLIS2 moieties are required for maintaining self-renewal. Global expression profiling and comparison to patients' signature consistently identify ETO2-GLIS2-mediated deregulation of major transcriptional regulators of hematopoiesis and leukemogenesis, including overexpression of the ERG oncogene. ChIP-seq analysis reveals that ETO2-GLIS2 is recruited at normal ETO2 complexes sites and also at GLIS2-specific targets through binding via GLIS2 DNA-binding domain. We demonstrate that ETO2-GLIS2 fusion localize at half of H3K27Ac-dense enhancers, so called super-enhancers, to control transcription of associated genes. We show that interaction of ETO2-GLIS2 with ETO2 complexes is an essential node for the transcriptional control by the fusion at enhancer elements. Indeed, ETO2-GLIS2 dimerizes and interacts with endogenous ETO2 via its NHR2 domains. An NHR2 peptide-interference strategy inhibits oligomerization, reverses the transcriptional activation at enhancers, promotes megakaryocytic differentiation and abrogates human AMKL cells maintenance in vivo. Finally, upregulation of ERG by ETO2-GLIS2 further strengthen enhancers formation as ERG is co-recruited generating a feed forward loop at these elements and its knockdown or genetic inactivation downregulates expression of ETO2-GLIS2 targets required for leukemic cells survival. We propose that the megakaryocytic differentiation arrest and self-renewal controlled by ETO2-GLIS2 results from an imbalance in the expression of master transcription factors imposed by aberrant chromatin structures at enhancers that may be disrupted by targeting the NHR2 interface. Disclosures No relevant conflicts of interest to declare.

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Metabolic Reprogramming of Human Mitochondrial NAD(P)+-Dependent-Malic Enzyme 2 in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2019-123339 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5168-5168

Author(s):

Ching-Tien Peng

Keyword(s):

Acute Myeloid Leukemia ◽

Malic Enzyme ◽

Myeloid Leukemia ◽

Kinase Inhibitors ◽

Conflicts Of Interest ◽

Metabolic Reprogramming ◽

Leukemic Cells ◽

Internal Tandem Duplication ◽

Blast Cells ◽

Acute Myeloid

Yu-Nan Huang1, Kang-Hsi Wu4, Te-fu Weng4, Su-Ching Liu4, Hui-Chih Hung1*, Ching-Tien Peng4,5* FLT3 internal tandem duplication (FLT3-ITD) mutations in patients with acute myeloid leukemia (AML) are usually associated with other mutations resulting in unfavorable outcome. Tyrosine kinase inhibitors (TKI) have shown promising responses, however, these responses are almost transient in therapy-resistant AML. Here, we show that human mitochondrial NAD(P)+-dependent-malic enzyme 2 (ME2) have significantly increased in CD34+ cell of patients with AML. To determine how ME2 establish metabolic reprogramming of leukemogenesis, we performed a comprehensive analysis of metabolism in CRISPR-mediated ME2 knockout leukemic cells (THP-1 and MV4-11) and purified leukemic blast cells (CD34+) derived from patients with AML. We demonstrate that disrupting ME2 signaling exerts potent activities against proliferation, reduced oxidative metabolism and lactate metabolism. We also show that genetic inhibition of RUNX1/FLT3/ME2 markedly repressed AML cell leukemogenesis. In conclusion, our findings provide a rationale for clinical development of this strategy for treating RUNX1 and FLT3-mutated leukemic patients. Disclosures No relevant conflicts of interest to declare.

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A Strong Expression of CD44-6v Correlates With Shorter Survival of Patients With Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v91.9.3401.3401_3401_3413 ◽

1998 ◽

Vol 91 (9) ◽

pp. 3401-3413 ◽

Cited By ~ 4

Author(s):

S. Legras ◽

U. Günthert ◽

R. Stauder ◽

F. Curt ◽

S. Oliferenko ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Protein Expression ◽

Myeloid Leukemia ◽

Surface Glycoprotein ◽

Leukemic Cells ◽

Cell Surface Glycoprotein ◽

Variant Isoforms ◽

Cd34 Cells ◽

First Time ◽

Acute Myeloid

CD44 is a ubiquitous cell-surface glycoprotein that displays many variant isoforms (CD44v) generated by alternative splicing of exons 2v to 10v. The expression of variant isoforms is highly restricted and correlated with specific processes, such as leukocyte activation and malignant transformation. We have herein studied CD44v expression in acute myeloid leukemia (AML) and, for comparison, in normal myelopoiesis. Protein expression of total CD44 and of CD44-3v, -6v, and -9v isoforms has been measured using specific monoclonal antibodies and flow cytometry. The composition of variant exon transcripts has been analyzed by semi-quantitative reverse transcriptase-polymerase chain reaction followed by Southern hybridization with exon-specific probes. Our data show that (1) CD44-6v isoforms are expressed on 12.0% ± 2.5% of normal CD34+ cells; this expression is sharply upregulated through monopoiesis and, inversely, downregulated during granulopoiesis. Also, CD44-3v and CD44-9v isoforms are detected on 10% and 14% of normal monocytes, respectively. (2) Sixty-nine from a total of 95 AML patients display a variable proportion (range, 5% to 80%) of CD44-6v+ leukemic cells. (3) A shorter overall survival characterizes the group of AML patients displaying more than 20% of CD44-6v+ leukemic cells (8 months v 18 months, P < .02). These data suggest, for the first time, that the protein expression of CD44-6v containing isoforms may serve as a new prognostic factor in AML.

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Immunophenotyping of acute myeloid leukemia using monoclonal antibodies and the alkaline phosphatase-antialkaline phosphatase technique

Blood ◽

10.1182/blood.v70.1.83.83 ◽

1987 ◽

Vol 70 (1) ◽

pp. 83-89 ◽

Cited By ~ 34

Author(s):

CA Hanson ◽

KJ Gajl-Peczalska ◽

JL Parkin ◽

RD Brunning

Keyword(s):

Alkaline Phosphatase ◽

Acute Myeloid Leukemia ◽

Monoclonal Antibodies ◽

Myeloid Leukemia ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Platelet Glycoprotein ◽

Specific Marker ◽

Hla Dr ◽

Acute Myeloid

Abstract The leukemic cells from 41 cases of acute myeloid leukemia (AML) and 17 cases of acute lymphocytic leukemia (ALL) were immunophenotyped by the alkaline phosphatase-antialkaline phosphatase (APAAP) immunocytochemical technique utilizing eight monoclonal antibodies (MoAb) reactive with cells of myeloid origin and seven MoAb reactive with lymphoid antigens. Ninety percent of the cases of AML reacted with one or more of the pan-myeloid MoAb, My7, My9, or 20.3. Reactivity of the myeloid panel of MoAb showed some correlation with the French- American-British (FAB) classification of AML. Five of six cases of acute promyelocytic leukemia (APL) were HLA-DR negative; the one HLA-DR- positive APL had a minor population of HLA-DR-negative promyelocytes. OKM5 and/or My4 reacted with 16 of 16 monocytic leukemias. No specific marker of early erythroid development was identified. AP3, a MoAb reactive with platelet glycoprotein (GPIIIa), was specific for acute megakaryoblastic leukemia. Immunocytochemistry was also helpful in classifying seven cases of AML with equivocal or negative routine cytochemistry. Two cases of AML had minor populations of blasts detected by the APAAP technique that were immunologically distinct from the major blast population; these minor populations emerged as the predominant cell type at relapse. Two cases of ALL expressed multiple myeloid and lymphoid antigens. Two other cases that morphologically were ALL reacted with only myeloid MoAb; one consisted entirely of immature basophils on ultrastructural examination. Immunophenotyping results using the APAAP technique were comparable with those obtained with flow cytometry. The APAAP technique is a reliable method for immunophenotyping leukemia that complements other methods of immunologic evaluation. The primary advantages of this method include its use with routinely prepared blood and bone marrow smears and the ability to correlate immunocytochemical reactions with morphology.

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