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1.  Isoform-Specific Potentiation of Stem and Progenitor Cell Engraftment by AML1/RUNX1  
PLoS Medicine  2007;4(5):e172.
Background
AML1/RUNX1 is the most frequently mutated gene in leukaemia and is central to the normal biology of hematopoietic stem and progenitor cells. However, the role of different AML1 isoforms within these primitive compartments is unclear. Here we investigate whether altering relative expression of AML1 isoforms impacts the balance between cell self-renewal and differentiation in vitro and in vivo.
Methods and Findings
The human AML1a isoform encodes a truncated molecule with DNA-binding but no transactivation capacity. We used a retrovirus-based approach to transduce AML1a into primitive haematopoietic cells isolated from the mouse. We observed that enforced AML1a expression increased the competitive engraftment potential of murine long-term reconstituting stem cells with the proportion of AML1a-expressing cells increasing over time in both primary and secondary recipients. Furthermore, AML1a expression dramatically increased primitive and committed progenitor activity in engrafted animals as assessed by long-term culture, cobblestone formation, and colony assays. In contrast, expression of the full-length isoform AML1b abrogated engraftment potential. In vitro, AML1b promoted differentiation while AML1a promoted proliferation of progenitors capable of short-term lymphomyeloid engraftment. Consistent with these findings, the relative abundance of AML1a was highest in the primitive stem/progenitor compartment of human cord blood, and forced expression of AML1a in these cells enhanced maintenance of primitive potential both in vitro and in vivo.
Conclusions
These data demonstrate that the “a” isoform of AML1 has the capacity to potentiate stem and progenitor cell engraftment, both of which are required for successful clinical transplantation. This activity is consistent with its expression pattern in both normal and leukaemic cells. Manipulating the balance of AML1 isoform expression may offer novel therapeutic strategies, exploitable in the contexts of leukaemia and also in cord blood transplantation in adults, in whom stem and progenitor cell numbers are often limiting.
The truncated "a" isoform of AML1 is shown to have the capacity to potentiate stem and progenitor cell engraftment, both of which are required for successful clinical transplantation.
Editors' Summary
Background.
Blood contains red blood cells (which carry oxygen round the body), platelets (which help the blood to clot), and white blood cells (which fight off infections). All these cells, which are regularly replaced, are derived from hematopoietic stem cells, blood-forming cells present in the bone marrow. Like all stem cells, hematopoietic stem cells self-renew (reproduce themselves) and produce committed progenitor cells, which develop into mature blood cells in a process called hematopoiesis. Many proteins control hematopoiesis, some of which are called transcription factors; these factors bind to DNA through their DNA-binding domain and then control the expression of genes (that is, how DNA is turned into proteins) through particular parts of the protein (their transcription regulatory domains). An important hematopoietic transcription factor is AML1—a protein first identified because of its involvement in acute myelogenous leukemia (AML, a form of blood cancer). Mutations (changes) in the AML1 gene are now known to be present in other types of leukemia, which are often characterized by overproliferation of immature blood cells.
Why Was This Study Done?
Because of AML1′s crucial role in hematopoiesis, knowing more about which genes it regulates and how its activity is regulated could provide clues to treating leukemia and to improving hematopoietic cell transplantation. Many cancer treatments destroy hematopoietic stem cells, leaving patients vulnerable to infection. Transplants of bone marrow or cord blood (the cord that links mother and baby during pregnancy contains peripheral blood stem cells) can replace the missing cells, but cord blood in particular often contains insufficient stem cells for successful transplantation. It would be useful, therefore, to expand the stem cell content of these tissues before transplantation. In this study, the researchers investigated the effect of AML1 on self-renewal and differentiation of hematopoietic stem and progenitor cells in the laboratory (in vitro) and in animals (in vivo). In particular, they have asked how two isoforms (closely related versions) of AML1 affect the ability of these cells to grow and differentiate (engraft) in mice after transplantation.
What Did the Researchers Do and Find?
The researchers artificially expressed AML1a and AML1b (both isoforms contain a DNA binding domain, but only AML1b has transcription regulatory domains) in mouse hematopoietic stem and progenitor cells and then tested the cells' ability to engraft in mice. AML1a-expressing cells engrafted better than unaltered cells and outgrew unaltered cells when transplanted as a mixture. AML1b-expressing cells, however, did not engraft. In vitro, AML1a-expressing cells grew more than AML1b-expressing cells, whereas differentiation was promoted in AML1b-expressing cells. To investigate whether the isoforms have the same effects in human cells, the researchers measured the amount of AML1a and AML1b mRNA (the template for protein production) made by progenitor cells in human cord blood. Although AML1b (together with AML1c, an isoform with similar characteristics) mRNA predominated in all the progenitor cell types, the relative abundance of AML1a was greatest in the stem and progenitor cells. Furthermore, forced expression of AML1a in these cells improved their ability to divide in vitro and to engraft in mice.
What Do These Findings Mean?
These findings indicate that AML1a expression increases the self-renewal capacity of hematopoietic stem and progenitor cells and consequently improves their ability to engraft in mice, whereas AML1b expression encourages the differentiation of these cell types. These activities are consistent with the expression patterns of the two isoforms in normal hematopoietic cells and in leukemic cells—the mutated AML made by many leukemic cells resembles AML1a. Because the AML1 isoforms were expressed at higher than normal levels in these experiments, the physiological relevance of these findings needs to be confirmed by showing that normal levels of AML1a and AML1b produce similar results. Nevertheless, these results suggest that manipulating the balance of AML1 isoforms made by hematopoietic cells might be useful clinically. In leukemia, a shift toward AML1b expression might slow the proliferation of leukemic cells and encourage their differentiation. Conversely, in cord blood transplantation, a shift toward AML1a expression might improve patient outcomes by expanding the stem and progenitor cell populations.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040172.
Wikipedia has pages on hematopoiesis and hematopoietic stem cells (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The US National Cancer Institute has a fact sheet on bone marrow and peripheral blood stem cell transplantation (in English and Spanish) and information for patients and professionals on leukemia (in English)
The American Society of Hematology provides patient information about blood diseases, including information on bone marrow and stem cell transplantation
doi:10.1371/journal.pmed.0040172
PMCID: PMC1868041  PMID: 17503961
2.  The leukemogenic t(8;21) fusion protein AML1-ETO controls ribosomal RNA genes and associates with nucleolar organizing regions at mitotic chromosomes 
Journal of cell science  2008;121(Pt 23):3981-3990.
SUMMARY
RUNX1/AML1 is required for definitive hematopoiesis and is frequently targeted by chromosomal translocation in acute myeloid leukemias (AML). The t(8;21) related AML1-ETO fusion protein blocks differentiation of myeloid progenitors. Here, we show by immunofluorescence microscopy that during interphase, endogenous AML1-ETO localizes to nuclear microenvironments distinct from those containing native RUNX1/AML1 protein. At mitosis, we clearly detect binding of AML1-ETO to nucleolar organizing regions (NORs) in AML derived Kasumi-1 cells and binding of RUNX1/AML1 to NORs in Jurkat cells. Both RUNX1/AML1 and AML1-ETO occupy ribosomal DNA repeats during interphase, as well as interact with the endogenous RNA Pol I transcription factor UBF-1. Promoter cytosine methylation analysis indicates that RUNX1/AML1 binds to rDNA repeats that are more highly CpG methylated than those bound by AML1-ETO. Down-regulation by RNA interference reveals that RUNX1/AML1 negatively regulates rDNA transcription, while AML1-ETO is a positive regulator in Kasumi-1 cells. Taken together, our findings identify a novel role for the leukemia-related AML1-ETO protein in epigenetic control of cell growth through upregulation of RNA Pol I-mediated ribosomal gene transcription, consistent with the hyper-proliferative phenotype of myeloid cells in AML patients.
doi:10.1242/jcs.033431
PMCID: PMC2904240  PMID: 19001502
acute myelogenous leukemia; Runx1; ribosomal DNA transcription; RNA polymerase I; UBF1; nucleolar organizing region
3.  Differences in Platelet Function In Patients with Acute Myeloid Leukaemia and Myelodysplasia Compared to Equally Thrombocytopenic Patients with Immune Thrombocytopenia 
Background
Severe thrombocytopenia is a major risk factor for haemorrhage, and yet platelet function and bleeding risk at low platelet counts are poorly understood because of limitations of platelet function testing at very low platelet counts.
Objectives
To examine and compare platelet function in severely thrombocytopenic patients with acute myeloid leukaemia (AML) or myelodysplasia (MDS) to patients with immune thrombocytopenia (ITP).
Methods
Whole blood flow cytometric measurement of platelet activation and platelet reactivity to agonists was correlated with the immature platelet fraction (IPF) and bleeding symptoms.
Results
Compared with patients with ITP, patients with AML/MDS had smaller platelets, lower IPF, and substantially lower platelet surface expression of activated GPIIb/IIIa and GPIb both with and without addition of ex vivo ADP or TRAP. In both ITP and AML/MDS, increased platelet surface GPIb on circulating platelets and expression of activated GPIIb/IIIa and GPIb on ex vivo activated platelets correlated with a higher IPF. Whereas platelet reactivity was higher for AML/MDS patients with bleeding than those with no bleeding, platelet reactivity was lower for ITP patients with bleeding than those with no bleeding.
Conclusions
AML/MDS patients have lower in vivo platelet activation and ex vivo platelet reactivity than patients with ITP. The proportion of newly-produced platelets correlates with the expression of platelet surface markers of activation. These differences might contribute to differences in bleeding tendency between AML/MDS and ITP. This study is the first to define differences in platelet function between AML/MDS patients and ITP patients with equivalent degrees of thrombocytopenia.
doi:10.1111/j.1538-7836.2011.04506.x
PMCID: PMC3210015  PMID: 21920014
Autoimmunity; bleeding; flow cytometry; haemorrhage; thrombocytopenia; thrombopoiesis
4.  Significance of Neuropilin-1 Expression in Acute Myeloid Leukemia 
Turkish Journal of Hematology  2013;30(3):300-306.
Objective: Neuropilin-1 is a vascular endothelial growth factor receptor that acts as a mediator of angiogenesis. Its importance in hematological malignancies such as acute myeloid leukemia (AML) remains to be elucidated. The aim of this study was to evaluate the significance of neuropilin-1 expression in AML patients by both flow cytometry and real-time polymerase chain reaction (PCR) in regard to its diagnostic and prognostic values.
Materials and Methods: Bone marrow aspirates of 44 patients with de novo AML and 12 relapsed AML patients were examined in this study. Ten subjects with nonhematological malignancy serving as the control group were also included.
Results: Neuropilin-1 expression by flow cytometry showed a highly significant increase in de novo and relapsed AML patients with a mean of 27.1±17.5% and 21.5±16.6%, respectively, compared to control group’s mean of 3.4±1.9%. A cut-off value of 6% was established as differentiating patients from the control group. By real-time PCR, no statistical significance was found in de novo and relapsed AML patients with a mean of 1.9±3.6 IU/L and 0.3±0.2 IU/L, respectively, compared to the control group’s mean of 0.3±0.1 IU/L. Neuropilin-1 surface expression by flow cytometry showed a significant correlation with total leukocyte count and a negative correlation with hemoglobin level in de novo AML patients. In relapsed AML patients, positive significant correlations were found with age, bone marrow blast percentage, and CD14. Neuropilin-1 mRNA level by real-time PCR showed a positive significant correlation with peripheral blood blast percentage and CD117 and a negative correlation with hemoglobin level in de novo AML patients. In relapsed patients, a positive correlation was found with lactate dehydrogenase.
Conclusion: Neuropilin-1 can be used as a tool for diagnosis and prognosis in AML patients.
Conflict of interest:None declared.
doi:10.4274/Tjh.2011.0005
PMCID: PMC3878529  PMID: 24385810
Neuropilin-1; Acute myeloid leukemia
5.  Both SEPT2 and MLL are down-regulated in MLL-SEPT2 therapy-related myeloid neoplasia 
BMC Cancer  2009;9:147.
Background
A relevant role of septins in leukemogenesis has been uncovered by their involvement as fusion partners in MLL-related leukemia. Recently, we have established the MLL-SEPT2 gene fusion as the molecular abnormality subjacent to the translocation t(2;11)(q37;q23) in therapy-related acute myeloid leukemia. In this work we quantified MLL and SEPT2 gene expression in 58 acute myeloid leukemia patients selected to represent the major AML genetic subgroups, as well as in all three cases of MLL-SEPT2-associated myeloid neoplasms so far described in the literature.
Methods
Cytogenetics, fluorescence in situ hybridization (FISH) and molecular studies (RT-PCR, qRT-PCR and qMSP) were used to characterize 58 acute myeloid leukemia patients (AML) at diagnosis selected to represent the major AML genetic subgroups: CBFB-MYH11 (n = 13), PML-RARA (n = 12); RUNX1-RUNX1T1 (n = 12), normal karyotype (n = 11), and MLL gene fusions other than MLL-SEPT2 (n = 10). We also studied all three MLL-SEPT2 myeloid neoplasia cases reported in the literature, namely two AML patients and a t-MDS patient.
Results
When compared with normal controls, we found a 12.8-fold reduction of wild-type SEPT2 and MLL-SEPT2 combined expression in cases with the MLL-SEPT2 gene fusion (p = 0.007), which is accompanied by a 12.4-fold down-regulation of wild-type MLL and MLL-SEPT2 combined expression (p = 0.028). The down-regulation of SEPT2 in MLL-SEPT2 myeloid neoplasias was statistically significant when compared with all other leukemia genetic subgroups (including those with other MLL gene fusions). In addition, MLL expression was also down-regulated in the group of MLL fusions other than MLL-SEPT2, when compared with the normal control group (p = 0.023)
Conclusion
We found a significant down-regulation of both SEPT2 and MLL in MLL-SEPT2 myeloid neoplasias. In addition, we also found that MLL is under-expressed in AML patients with MLL fusions other than MLL-SEPT2.
doi:10.1186/1471-2407-9-147
PMCID: PMC2689242  PMID: 19445675
6.  NPM1 Gene Type A Mutation in Bulgarian Adults with Acute Myeloid Leukemia: A Single-Institution Study 
Turkish Journal of Hematology  2014;31(1):40-48.
Objective: Mutations of the nucleophosmin (NPM1) gene are considered as the most frequent acute myeloid leukemia (AML)-associated genetic lesion, reported with various incidences in different studies, and type A (NPM1-A) is the most frequent type. However, since most series in the literature report on the features of all patients regardless of the type of mutation, NPM1-A(+) cases have not been well characterized yet. Therefore, we evaluated the prevalence of NPM1-A in Bulgarian AML patients and searched for an association with clinical and laboratory features.
Materials and Methods: One hundred and four adults (51 men, 53 women) were included in the study. NPM1-A status was determined using allele-specific reverse-transcription polymerase chain reaction with co-amplification of NPM1-A and β-actin and real-time quantitative TaqMan-based polymerase chain reaction. Patients received conventional induction chemotherapy and were followed for 13.2±16.4 months.
Results: NPM1-A was detected in 26 (24.8%) patients. NPM1-A mutation was detected in all AML categories, including in one patient with RUNX1-RUNX1T1. There were no differences associated with the NPM1-A status with respect to age, sex, hemoglobin, platelet counts, percentage of bone marrow blasts, splenomegaly, complete remission rates, and overall survival. NPM1-A(+) patients, compared to NPM1-A(-) patients, were characterized by higher leukocyte counts [(75.4±81.9)x109/L vs. (42.5±65.9)x109/L; p=0.049], higher frequency of normal karyotype [14/18 (77.8%) vs. 26/62 (41.9%); p=0.014], higher frequency of FLT3-ITD [11/26 (42.3%) vs. 8/77 (10.4%); p=0.001], and lower incidence of CD34(+) [6/21 (28.8%) vs. 28/45 (62.2%); p=0.017]. Within the FLT3-ITD(-) group, the median overall survival of NPM1-A(-) patients was 14 months, while NPM1-A(+) patients did not reach the median (p=0.10).
Conclusion: The prevalence of NPM1-A mutation in adult Bulgarian AML patients was similar to that reported in other studies. NPM1-A(+) patients were characterized by higher leukocyte counts, higher frequency of normal karyotypes and FLT3-ITD, and lower incidence of CD34(+), supporting the idea that the specific features of type A mutations might contribute to the general clinical and laboratory profile of NPM1(+) AML patients.
doi:10.4274/Tjh.2013.0023
PMCID: PMC3996648  PMID: 24764728
Acute myeloid leukemia; NPM1 gene type A mutation; FLT3-ITD; allele-specific polymerase chain reaction
7.  Transcription factor RUNX1 promotes survival of acute myeloid leukemia cells 
The Journal of Clinical Investigation  2013;123(9):3876-3888.
RUNX1 is generally considered a tumor suppressor in myeloid neoplasms. Inactivating RUNX1 mutations have frequently been found in patients with myelodysplastic syndrome (MDS) and cytogenetically normal acute myeloid leukemia (AML). However, no somatic RUNX1 alteration was found in AMLs with leukemogenic fusion proteins, such as core-binding factor (CBF) leukemia and MLL fusion leukemia, raising the possibility that RUNX1 could actually promote the growth of these leukemia cells. Using normal human cord blood cells and those expressing leukemogenic fusion proteins, we discovered a dual role of RUNX1 in myeloid leukemogenesis. RUNX1 overexpression inhibited the growth of normal cord blood cells by inducing myeloid differentiation, whereas a certain level of RUNX1 activity was required for the growth of AML1-ETO and MLL-AF9 cells. Using a mouse genetic model, we also showed that the combined loss of Runx1/Cbfb inhibited leukemia development induced by MLL-AF9. RUNX2 could compensate for the loss of RUNX1. The survival effect of RUNX1 was mediated by BCL2 in MLL fusion leukemia. Our study unveiled an unexpected prosurvival role for RUNX1 in myeloid leukemogenesis. Inhibiting RUNX1 activity rather than enhancing it could be a promising therapeutic strategy for AMLs with leukemogenic fusion proteins.
doi:10.1172/JCI68557
PMCID: PMC3754260  PMID: 23979164
8.  Multivariate and subgroup analyses of a randomized, multinational, phase 3 trial of decitabine vs treatment choice of supportive care or cytarabine in older patients with newly diagnosed acute myeloid leukemia and poor- or intermediate-risk cytogenetics 
BMC Cancer  2014;14:69.
Background
Compared with younger patients, older adults with acute myeloid leukemia (AML) generally have poorer survival outcomes and less benefit from clinical trials. A recent phase 3 trial demonstrated a trend toward improved overall survival (OS) with decitabine, a hypomethylating agent, compared with treatment choice of either cytarabine or supportive care (7.7 months, 95% CI: 6.2–9.2 vs 5.0 months, 95% CI: 4.3–6.3, respectively) in older adults with newly diagnosed AML. The current analyses investigated prognostic factors for outcomes in this trial and examined OS and responses in prespecified subgroups.
Methods
A multivariate Cox proportional hazards model was used to investigate effects of demographic and baseline characteristics, including age, sex, cytogenetic risk, AML type, ECOG Performance Status, geographic region, bone marrow blasts, platelets, and white blood cells on OS, based on mature data. Similar analyses were conducted with a logistic regression model to predict response rates. Prespecified subgroup analyses were performed for OS and response rates, also using mature data.
Results
Patient characteristics that appeared to negatively influence OS included more advanced age (hazard ratio [HR] 1.560 for ≥75 vs <70 years; p = 0.0010), poorer performance status at baseline (HR 0.771 for 0 or 1 vs 2; p = 0.0321), poor cytogenetics (HR 0.699 for intermediate vs poor; p = 0.0010), higher bone marrow blast counts (HR 1.355 for >50% vs ≤50%; p = 0.0045), low baseline platelet counts (HR 0.775 for each additional 100 × 109/L; p = 0.0015), and high white blood cell counts (HR 1.256 for each additional 25 × 109/L; p = 0.0151). Regarding geographic regions, patients from Western Europe had the longest median OS. Response rates favored decitabine for all subgroups investigated, including patients ≥75 years (odds ratio 5.94, p = 0.0006).
Conclusion
Response to decitabine in AML is associated with known prognostic factors related to both patient demographics and disease characteristics.
Trial registration
ClinicalTrials.gov identifier NCT00260832
doi:10.1186/1471-2407-14-69
PMCID: PMC3928608  PMID: 24498872
Decitabine; Acute Myelocytic Leukemia; Elderly; Treatment
9.  Five distinct biological processes and 14 differentially expressed genes characterize TEL/AML1-positive leukemia 
BMC Genomics  2007;8:385.
Background
The t(12;21)(p13;q22) translocation is found in 20 to 25% of cases of childhood B-lineage acute lymphoblastic leukemia (B-ALL). This rearrangement results in the fusion of ETV6 (TEL) and RUNX1 (AML1) genes and defines a relatively uniform category, although only some patients suffer very late relapse. TEL/AML1-positive patients are thus an interesting subgroup to study, and such studies should elucidate the biological processes underlying TEL/AML1 pathogenesis. We report an analysis of gene expression in 60 children with B-lineage ALL using Agilent whole genome oligo-chips (44K-G4112A) and/or real time RT-PCR.
Results
We compared the leukemia cell gene expression profiles of 16 TEL/AML1-positive ALL patients to those of 44 TEL/AML1-negative patients, whose blast cells did not contain any additional recurrent translocation. Microarray analyses of 26 samples allowed the identification of genes differentially expressed between the TEL/AML1-positive and negative ALL groups. Gene enrichment analysis defined five enriched GO categories: cell differentiation, cell proliferation, apoptosis, cell motility and response to wounding, associated with 14 genes -RUNX1, TCFL5, TNFRSF7, CBFA2T3, CD9, SCARB1, TP53INP1, ACVR1C, PIK3C3, EGFL7, SEMA6A, CTGF, LSP1, TFPI – highlighting the biology of the TEL/AML1 sub-group. These results were first confirmed by the analysis of an additional microarray data-set (7 patient samples) and second by real-time RT-PCR quantification and clustering using an independent set (27 patient samples). Over-expression of RUNX1 (AML1) was further investigated and in one third of the patients correlated with cytogenetic findings.
Conclusion
Gene expression analyses of leukemia cells from 60 children with TEL/AML1-positive and -negative B-lineage ALL led to the identification of five biological processes, associated with 14 validated genes characterizing and highlighting the biology of the TEL/AML1-positive ALL sub-group.
doi:10.1186/1471-2164-8-385
PMCID: PMC2211320  PMID: 17956600
10.  Minimal Residual Disease-Directed Therapy for Childhood Acute Myeloid Leukemia: Results of the AML02 Multicenter Trial 
The lancet oncology  2010;11(6):543-552.
Summary
Background
We sought to improve outcome of childhood acute myeloid leukemia (AML) by applying risk-directed therapy based on the genetic abnormalities of the leukemic cells and measurements of minimal residual disease (MRD) as determined by flow cytometry during treatment.
Methods
From October 13, 2002 to June 19, 2008, 232 patients with de novo AML (n=206), therapy- or myelodysplasia-related AML (n=12), or mixed-lineage leukemia (n=14) were enrolled at eight centers. Block, nonblinded randomization, stratified by cytogenetic or morphologic subtype, assigned patients to high-dose (18 g/m2, n=113) or low-dose (2 g/m2, n=117) cytarabine (A), given together with daunorubicin (D) and etoposide (E) (Induction I); achievement of MRD negative status was the primary endpoint. Induction II consisted of ADE with or without gemtuzumab ozogamicin (GO); consolidation therapy included three additional courses of chemotherapy or hematopoietic stem cell transplantation (HSCT). Levels of MRD were used to allocate GO and determine the timing of Induction II; both MRD and genetic abnormalities at diagnosis were used to determine final risk classification. Low-risk patients (n=68) received 5 courses of chemotherapy, whereas high-risk patients (n=79), as well as standard-risk patients (n=69) with matched sibling donors, were eligible for HSCT (performed in 48 high and 8 standard-risk patients). All randomized patients (n=230) were analyzed for the primary endpoint. The other analyses were limited to the 216 patients with AML, excluding mixed-lineage leukemia. This trial, closed to accrual, is registered with ClinicalTrial.gov, number NCT00136084.
Findings
The complete remission rates were 80% (173 of the 216) after Induction I and 94% (203 of 216) after Induction II. Induction failures included two toxic deaths and 10 cases of resistant leukemia. The introduction of high-dose cytarabine did not significantly lower the rate of MRD positivity after Induction I therapy (34% vs. 42%, P=0.17). The cumulative incidences of grade 3 or greater infection were 79.3% ± 4.0% and 75.5% ± 4.2% for patients treated on the high-dose or low-dose arms. The 3-year estimates (± SE) of event-free and overall survival were 63.0% ± 4.1% and 71.1% ± 3.8%, respectively. Achievement of MRD < 0.1% after Induction II identified a large group of patients (80%) with a cumulative incidence of relapse of only 17% ± 3%. Post-Induction I MRD ≥ 1% was the only independent adverse prognostic factor that was statistically significant (P < 0.05) for both event-free (HR, 2.41; CI 1.36–4.26; P=0.003) and overall survival (HR, 2.11; CI 1.09–4.11; P=0.028).
Interpretation
Our findings suggest that the use of targeted chemotherapy and HSCT, in the context of a comprehensive risk-stratification strategy based on genetic features and MRD findings, can improve the outcome of childhood AML.
doi:10.1016/S1470-2045(10)70090-5
PMCID: PMC3171799  PMID: 20451454
11.  Accelerated Telomere Shortening Precedes Development of Therapy-Related Myelodysplasia or Acute Myelogenous Leukemia After Autologous Transplantation for Lymphoma 
Journal of Clinical Oncology  2009;27(5):791-798.
Purpose
Therapy-related myelodysplasia or acute myelogenous leukemia (t-MDS/AML) is a lethal complication of autologous hematopoietic stem-cell transplantation (aHCT) for Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL). Here, we investigated the hypothesis that accelerated telomere shortening after aHCT could contribute to the development of t-MDS/AML.
Patients and Methods
A prospective longitudinal cohort was constructed to investigate the sequence of cellular and molecular abnormalities leading to development of t-MDS/AML after aHCT for HL/NHL. This cohort formed the sampling frame for a nested case-control study to compare changes in telomere length in serial blood samples from patients who developed t-MDS/AML with matched controls who did not develop t-MDS/AML.
Results
An initial increase in telomere length at day 100 after aHCT was followed by an accelerated telomere shortening in t-MDS/AML patients when compared with controls. These telomere alterations preceded the onset of t-MDS and were independent of other known risk factors associated with development of t-MDS/AML on multivariate analysis. Additionally, we observed reduced generation of committed progenitors in patients who developed t-MDS/AML, indicating that these telomere alterations were associated with reduced regenerative capacity of hematopoietic stem cells.
Conclusion
The development of t-MDS/AML after aHCT is associated with and preceded by markedly altered telomere dynamics in hematopoietic cells. Accelerated telomere loss in patients developing t-MDS/AML may reflect increased clonal proliferation and/or altered telomere regulation in premalignant cells. Genetic instability associated with shortened telomeres may contribute to leukemic transformation in t-MDS/AML.
doi:10.1200/JCO.2008.17.1033
PMCID: PMC2645091  PMID: 19124806
12.  Prognostic value of miR-96 in patients with acute myeloid leukemia 
Diagnostic Pathology  2014;9:76.
Objective
Aberrant expression of miRNA (miR)-96 is associated with tumorigenesis and tumor progression in several solid cancers. However, little is known about the expression and prognostic value of miR-96 in acute myeloid leukemia (AML). Therefore, the aim of this study was to investigate the correlation of miR-96 expression with clinicopathological features and prognosis of AML.
Methods
Real-time quantitative RT-PCR assay was performed to evaluate the expression levels of miR-96 in mononuclear cells from bone marrow or peripheral blood specimens in 86 patients with newly diagnosed AML.
Results
Compared with normal controls, miR-96 expression was significantly downregulated in patients with newly diagnosed AML (P < 0.001). In analysis of 14 diagnosis/CR-paired samples, the expression level of miR-96 was found markedly elevated in patients after treatment than before (P < 0.001). Moreover, lower levels of miR-96 were associated with a higher white blood cell count, bone marrow blast count (P < 0.001 and 0.022, respectively), and lower hemoglobin and platelet count (P = 0.036 and 0.033, respectively). Although the low-expression group seemed to have a lower CR rate (53.85% vs 70.0%), there was no significant difference between the two groups (P = 0.213). The low-expression group had a lower relapse-free survival (RFS) (P = 0.038) and overall survival (OS) (P = 0.022) compared with the high-expression group during a median follow-up of 20 months.
Conclusion
Our data demonstrated that the expression of miR-96 was downregulated in newly diagnosed AML patients and associated with leukemic burden, as well as RFS and OS. This suggests that miR-96 detection might become a potential biomarker of prognosis and monitoring in AML.
Virtual slides
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1434808553949498
doi:10.1186/1746-1596-9-76
PMCID: PMC3975266  PMID: 24678958
miR-96; Acute myeloid leukemia; Real-time quantitative RT-PCR assay; Prognosis
13.  Treatment of Acute Myeloid Leukemia with 20–30% Bone Marrow Blasts 
The transition of patients with ≥20% <30% bone marrow (BM) blast from the FAB category of myelodysplasia to the family of acute myeloid leukemia (AML) according to the recent WHO classification has not resolved the argument as to whether the natural history and responsiveness to therapy of these diseases is comparable to that of AML with > 30% BM blast. These controversies are even more manifest when it comes to elderly patients in whom concern for intensive chemotherapy (IC) related toxicity is the critical determinant for the therapeutic choice. In fact, due to concerns of treatment-related morbidity and mortality associated with delivery of IC, approximately only 30% of all patients ≥65 years are considered eligible for this approach. Therefore, a great deal of attention has been dedicated to alternative agents such as hypomethylators (azacitidine and decitabine). Actually, these agents have shown efficacy with reduced toxicity when administered to elderly patients with 20–30% BM blasts and not eligible for IC. In the present review, we will discuss the clinical results achieved in the treatment of elderly patients with 20%–30% BM blasts AML using intensive chemotherapy (IC) or hypomethylating agents. Overall, our survey of the literature suggests that only controlled, randomized, clinical trials will answer the question as to whether hypomethylating agents has the potential to substitute for IC even in elderly patients with an optimal functional status.
doi:10.4084/MJHID.2013.032
PMCID: PMC3684350  PMID: 23795270
14.  Pretreatment long interspersed element (LINE)-1 methylation levels, not early hypomethylation under treatment, predict hematological response to azacitidine in elderly patients with acute myeloid leukemia 
OncoTargets and therapy  2013;6:741-748.
Background
Epigenetic modulations, including changes in DNA cytosine methylation, are implicated in the pathogenesis and progression of acute myeloid leukemia (AML). Azacitidine is a hypomethylating agent that is incorporated into RNA as well as DNA. Thus, there is a rationale to its use in patients with AML. We determined whether baseline and/or early changes in the methylation of long interspersed element (LINE)-1 or CDH13 correlate with bone marrow blast clearance, hematological response, or survival in patients with AML treated with azacitidine.
Methods
An open label, phase I/II trial was performed in 40 AML patients (median bone marrow blast count was 42%) unfit for intensive chemotherapy treated with azacitidine 75 mg/m2/day subcutaneously for 5 days every 4 weeks. Bone marrow mononuclear cell samples were taken on day 0 (pretreatment) and day 15 during the first treatment cycle; LINE-1 and CDH13 methylation levels were quantified by methylation-specific, semiquantitative, real-time polymerase chain reaction.
Results
Treatment with azacitidine significantly reduced LINE-1 but not CDH13 methylation levels over the first cycle (P < 0.0001). Absolute LINE-1 methylation levels tended to be lower on day 0 (P = 0.06) and day 15 of cycle 1 (P = 0.03) in patients who went on to achieve subsequent complete remission, partial remission or hematological improvement versus patients with stable disease. However, the decrease in LINE-1 methylation over the first treatment cycle did not correlate with subsequent response (P = 0.31). Baseline methylation levels of LINE-1 or CDH13 did not correlate with disease-related prognostic factors, including cytogenetic risk, relapsed/refractory AML, or presence of NPM1 or FLT3 mutations. No correlation was observed between LINE-1 or CDH13 methylation levels and overall survival.
Conclusion
Analysis of baseline LINE-1 methylation levels may help identify elderly AML patients who are most likely to respond to azacitidine therapy.
doi:10.2147/OTT.S45459
PMCID: PMC3699298  PMID: 23836986
DNA methylation; acute myeloid leukemia; azacitidine; clinical response; CDH13; LINE-1 methylation
15.  Correlations of hematological parameters with bone marrow findings in chronic lymphoproliferative disorders associated with hepatitis viruses 
Journal of Medicine and Life  2013;6(4):464-471.
Abstract
Background. Hepatitis B and C viruses’ infections are often associated with hematological disorders in evolution, suggesting that these viruses have a tropism for peripheral blood and/or bone marrow cells.
Aim. To analyze the hematological parameters and bone marrow findings in a group of patients diagnosed with chronic lymphoproliferative disorders (CLD) and hepatitis viruses B, C, D infections, which were included in the research grant (acronym LIMFO-VIR) between December 2007 and May 2010 in the Hematology Department of the Emergency University Hospital of Bucharest.
Methods and results. Patients were diagnosed by using immunopathology according to the WHO criteria. The analyzed group included 42 patients (both sexes), with the mean age of 60,35 years. The most frequent hematologic disease was non-Hodgkin’s lymphoma 30/42 (71,42%), followed by chronic lymphocytic leukemia (16,66%) and Hodgkin’s lymphoma (7,14%). Hepatitis viruses were distributed: 17/42 (40,47%) patients with HBV, 22/42 (52,38%) with HCV and 3/42 (7,14%) had a double/triple association of viruses. Most of the patients had an indolent type of disease - 27/42 (64,28%), whereas 15/42 (35,71%) had an aggressive one, pattern found both in the HBV and HCV infected groups. An abnormal bone marrow result was revealed in 32/42 (76,19%) patients, 19 (59,37%) of them being HCV infected. Myelodysplasia was found in 6/42(14,28%) patients, the majority being HCV infected, all having an indolent form of CLD. The antiviral therapy did not influence the hematological parameters (no significant differences were found between the groups with/without an antiviral therapy).
Discussions. Patients with hepatitis virus infections may associate neutropenia and thrombocytopenia; the mechanisms are thought to involve hypersplenism, autoimmune processes and antiviral therapy. We excluded the influence of chemotherapy, as the study was performed before the treatment. In our group, patients whether HBV or HCV infected, presented an isolated cytopenia. The abnormal bone marrow cellularity (increased or decreased) and dysplasia were found especially in the HCV group. There are studies showing no association between myelodysplasia and hepatitis viruses; others found a strong relation of these. One of the mechanisms of myelodysplasia could be a dysregulation of the immune system.
Conclusions. Bone marrow/peripheral blood features correlate with the type of viral infection and HCV is more prone to develop additional hematological changes than HBV. The degree of bone marrow involvement by CLDs influences these features. We considered mandatory to perform a bone marrow analysis at the diagnosis of CLDs to stage and to establish if other bone marrow changes were present, a crucial aspect for therapy and outcome of the disease. The association between the hepatitis viruses – myelodysplasia- autoimmunity seems to have a role in the lymphoproliferative disorders etiology.
Abbreviations: CLD – chronic lymphoproliferative disorders; NHL- non-Hodgkin’s lymphoma, CLL- chronic lymphocytic leukemia, HL- Hodgkin’s lymphoma, MDS – myelodysplastic syndrome, AML – acute myeloid leukemia
PMCID: PMC4034312  PMID: 24868264
hepatitis viruses; chronic lymphoproliferative disorders; myelodysplasia; cytopenia
16.  Characteristics of hematologic malignancies with coexisting t(9;22) and inv(16) chromosomal abnormalities 
Blood research  2014;49(1):22-28.
Background
The coexistence of t(9;22)(q34;q11.2) and inv(16)(p13q22) chromosomal abnormalities is extremely uncommon, and only a small number of such cases have been reported. Here, we characterized 7 cases of hematologic malignancy exhibiting t(9;22) and inv(16) coexistence.
Methods
We reviewed the cytogenetic data for hematologic malignancies treated at the Catholic Blood and Marrow Transplantation Center between January 2004 and June 2013. We identified 7 cases exhibiting t(9;22) and inv(16) coexistence. In addition, we analyzed mutations in the IKZF1, NPM1, FLT3, N-RAS, K-RAS, c-KIT, and TP53 genes.
Results
Four cases of chronic myelogenous leukemia (CML; 1 chronic phase, 2 accelerated phase, and 1 blast phase) and 3 cases of acute myeloid leukemia (AML; 1 de novo and 2 therapy-related) were identified. The percentages of circulating blasts and bone marrow eosinophils were higher in AML cases than in CML cases (53% vs. 5% and 30% vs. 5.5%, respectively). The proportions of each chromosomal abnormality were used along with follow-up karyotyping results to identify secondary changes. In BCR/ABL, a p210 fusion transcript was associated with CML, whereas a p190 fusion transcript was associated with AML. One patient with AML harbored 2 mutations: c-KIT D816V and TP53 E11Q. All patients except 1 with CML blast phase sustained clinical remission after treatment, which included an imatinib mesylate regimen.
Conclusion
This study shows that observations of bone marrow morphology, initial and follow-up cytogenetic studies, and karyotyping of BCR/ABL1 and CBFB/MYH11 provide valuable information for characterizing hematologic malignancies exhibiting t(9;22) and inv(16) coexistence.
doi:10.5045/br.2014.49.1.22
PMCID: PMC3974952  PMID: 24724063
Chronic myelogenous leukemia; Acute myeloid leukemia; t(9;22); BCR/ABL1; inv(16); CBFB/MYH11
17.  Chromosomal Minimal Critical Regions in Therapy-Related Leukemia Appear Different from Those of De Novo Leukemia by High-Resolution aCGH 
PLoS ONE  2011;6(2):e16623.
Therapy-related acute leukemia (t-AML), is a severe complication of cytotoxic therapy used for primary cancer treatment. The outcome of these patients is poor, compared to people who develop de novo acute leukemia (p-AML). Cytogenetic abnormalities in t-AML are similar to those found in p-AML but present more frequent unfavorable karyotypes depending on the inducting agent. Losses of chromosome 5 or 7 are observed after alkylating agents while balanced translocations are found after topoisomerase II inhibitors. This study compared t-AML to p-AML using high resolution array CGH in order to find copy number abnormalities (CNA) at a higher resolution than conventional cytogenetics. More CNAs were observed in 30 t-AML than in 36 p-AML: 104 CNAs were observed with 63 losses and 41 gains (mean number 3.46 per case) in t-AML, while in p-AML, 69 CNAs were observed with 32 losses and 37 gains (mean number of 1.9 per case). In primary leukemia with a previously “normal” karyotype, 18% exhibited a previously undetected CNA, whereas in the (few) t-AML with a normal karyotype, the rate was 50%. Several minimal critical regions (MCRs) were found in t-AML and p-AML. No common MCRs were found in the two groups. In t-AML a 40kb deleted MCR pointed to RUNX1 on 21q22, a gene coding for a transcription factor implicated in frequent rearrangements in leukemia and in familial thrombocytopenia. In de novo AML, a 1Mb MCR harboring ERG and ETS2 was observed from patients with complex aCGH profiles. High resolution cytogenomics obtained by aCGH and similar techniques already published allowed us to characterize numerous non random chromosome abnormalities. This work supports the hypothesis that they can be classified into several categories: abnormalities common to all AML; those more frequently found in t-AML and those specifically found in p-AML.
doi:10.1371/journal.pone.0016623
PMCID: PMC3038855  PMID: 21339820
18.  Successful treatment with low-dose decitabine in acute myelogenous leukemia in elderly patients over 80 years old: Five case reports 
Oncology Letters  2013;5(4):1321-1324.
The incidence of acute myelogenous leukemia (AML) in patients over 80 years old is >20 times greater than that observed in younger patients. Previously, no standard treatment protocol for elderly patients with AML existed, however the development of hypomethylating agents, including decitabine, has brought about promising results in AML. In the present study, we report on the usage of a lower than routine dosage of decitabine in patients over 80 years old with AML. Since January 2010, 5 patients diagnosed with AML over the age of 80 years old received treatment with decitabine in our hospital. Decitabine was administered at a dose of 10–15 mg/m2 and repeated every other day for a total of 5 days. This cycle was repeated for ∼6 weeks. The 5 patients received a total of 19 cycles of treatment with decitabine. No patient achieved complete or partial remission. An antileukemic effect was observed in 25% of courses (3/12). An increase in platelet count of >20×109/l was observed in 26.3% (5/19) of cycles compared with previous treatment. An increase in hemoglobin concentration of >20 g/l was observed in 36.8% (7/19) of cycles in comparison to previous treatment, four of which achieved normal hemoglobin levels. One patient became red blood cell transfusion-independent. The median survival time was 19.8±4.8 months. Survival time from decitabine administration to mortality was 13.2±5.1 months. The main side-effect was bone marrow suppression with grade III–IV thrombocytopenia, grade III–IV leukocytopenia, grade III–IV neutropenia and anemia accounting for 94.7% (18/19), 47.4% (9/19), 89.5% (17/19) and 21.1% (4/19), respectively. Severe infection or bleeding was not observed and no patient stopped treatment due to adverse effects. In conclusion, extremely low-dose decitabine may be used safely in elderly patients and achieved longer survival times than reported previously in AML patients aged 80 and above. It is suggested that complete remission may not be the primary objective, while improvement of quality of life may be a better choice in AML patients over 80 years old. The cases observed in our study were limited, so more cases are required for further study.
doi:10.3892/ol.2013.1139
PMCID: PMC3629021  PMID: 23599787
decitabine; acute myelogenous leukemia; methylation; elderly
19.  RUNX1 Mutations Are Associated With Poor Outcome in Younger and Older Patients With Cytogenetically Normal Acute Myeloid Leukemia and With Distinct Gene and MicroRNA Expression Signatures 
Journal of Clinical Oncology  2012;30(25):3109-3118.
Purpose
To determine the association of RUNX1 mutations with therapeutic outcome in younger and older patients with primary cytogenetically normal acute myeloid leukemia (CN-AML) and with gene/microRNA expression signatures.
Patients and Methods
Younger (< 60 years; n = 175) and older (≥ 60 years; n = 225) patients with CN-AML treated with intensive cytarabine/anthracycline-based first-line therapy on Cancer and Leukemia Group B protocols were centrally analyzed for RUNX1 mutations by polymerase chain reaction and direct sequencing and for established prognostic gene mutations. Gene/microRNA expression profiles were derived using microarrays.
Results
RUNX1 mutations were found in 8% and 16% of younger and older patients, respectively (P = .02). They were associated with ASXL1 mutations (P < .001) and inversely associated with NPM1 (P < .001) and CEBPA (P = .06) mutations. RUNX1-mutated patients had lower complete remission rates (P = .005 in younger; P = .006 in older) and shorter disease-free survival (P = .058 in younger; P < .001 in older), overall survival (P = .003 in younger; P < .001 in older), and event-free survival (P < .001 for younger and older) than RUNX1 wild-type patients. Because RUNX1 mutations were more common in older patients and almost never coexisted with NPM1 mutations, RUNX1 mutation–associated expression signatures were derived in older, NPM1 wild-type patients and featured upregulation of genes normally expressed in primitive hematopoietic cells and B-cell progenitors, including DNTT, BAALC, BLNK, CD109, RBPMS, and FLT3, and downregulation of promoters of myelopoiesis, including CEBPA and miR-223.
Conclusion
RUNX1 mutations are twice as common in older than younger patients with CN-AML and negatively impact outcome in both age groups. RUNX1-mutated blasts have molecular features of primitive hematopoietic and lymphoid progenitors, potentially leading to novel therapeutic approaches.
doi:10.1200/JCO.2011.40.6652
PMCID: PMC3732007  PMID: 22753902
20.  Prognosis and Therapy When Acute Promyelocytic Leukemia and Other “Good Risk” Acute Myeloid Leukemias Occur as a Therapy-Related Myeloid Neoplasm 
Treatment for a pre-existing condition using chemotherapy, radiation therapy, immunosuppressive therapy, or a combination of these modalities may lead to the devastating complication of therapy-related myelodysplastic syndrome or acute myeloid leukemia (t-MDS/t-AML), collectively known as therapy-related myeloid neoplasm (t-MN). This disorder arises as a direct consequence of mutational events induced by the primary treatment. The outcomes for these patients have been historically poor compared to people who develop AML de novo. Currently comprising 10–20% of all cases of AML, t-MN is relatively resistant to conventional leukemia therapies, and is associated with s ort survival times. Median life expectancy from diagnosis is about 8–10 months in most series. Although the spectrum of cytogenetic abnormalities in t-AML is similar to AML de novo, the frequency of unfavorable cytogenetics, such as a complex karyotype or deletion or loss of chromosomes 5 and/or 7, is considerably higher in t-MN. Two distinct groups of patients with t-MN have been described. The more common subtype, seen in about 75% of patients, typically occurs 5–7 years after first exposure to alkylating agents or radiation, is often preceded by a myelodysplastic syndrome (MDS), and is frequently accompanied by clonal cytogenetic abnormalities such as the loss of all or part of chromosomes 5 or 7. Mutations of the P53 tumor suppressor gene are also common. The risk is related to total cumulative exposure over time to alkylating agents. In contrast, among individuals who develop t-AML after treatment with topoisomerase II inhibitors, the latency period to the development of t-AML is often only 1–3 years, antecedent MDS is rare, and gene rearrangements involving MLL at 11q23 or RUNX1/AML1 at 21q22 are common. It is now well recognized that APL and other subtypes of AML with balanced translocations sometimes occur as therapy-related myeloid neoplasms (t-MN) in patients who have previously received cytotoxic therapy or ionizing radiation therapy (RT). The most of this review will focus on these “good risk” leukemias, i.e. those with APL or inv(16)/t(16;16) or t(8;21).
doi:10.4084/MJHID.2011.032
PMCID: PMC3152454  PMID: 21869918
21.  Acute myeloid leukemia with t(7;21)(p22;q22) and 5q deletion: a case report and literature review 
The gene RUNX1 at chromosome 21q22 encodes the alpha subunit of Core binding factor (CBF), a heterodimeric transcription factor involved in the development of normal hematopoiesis. Translocations of RUNX1 are seen in several types of leukemia with at least 21 identified partner genes. The cryptic t(7;21)(p22;q22) rearrangement involving the USP42 gene appears to be a specific and recurrent cytogenetic abnormality. Eight of the 9 cases identified in the literature with this translocation were associated with acute myeloid leukemia (AML), with the remaining case showing refractory anemia with excess blasts, type 2. Herein, we present a patient with two preceding years of leukopenia and one year of anemia prior to the diagnosis of AML, NOS with monocytic differentiation (myelomonocytic leukemia) whose conventional cytogenetics showed an abnormal clone with 5q deletion. Interphase FISH using LSI RUNX1/RUNXT1 showed three signals for RUNX1. FISH studies on previously G-banded metaphases showed the extra RUNX1 signal on the short arm of chromosome 7. Further characterization using the subtelomeric 7p probe showed a cryptic 7;21 translocation. Our case and eight previously reported leukemic cases with the t(7;21)(p22;q22) appear to share similar features including monocytic differentiation, immunophenotypic aberrancies (often with CD56 and/or CD7), and a generally poor response to standard induction chemotherapy. About 80% of these cases had loss of 5q material as an additional abnormality at initial diagnosis or relapse. These findings suggest that t(7;21) may represent a distinct recurrent cytogenetic abnormality associated with AML. The association between the t(7;21) and 5q aberrancies appears to be non-random, however the pathogenetic connection remains unclear. Additional studies to evaluate for RUNX1 partner genes may be considered for AML patients with RUNX1 rearrangement and 5q abnormalities; however knowledge of the prognostic implications of this rearrangement is still limited.
doi:10.1186/2162-3619-3-8
PMCID: PMC4012275  PMID: 24646765
AML; Acute myeloid leukemia; t(7;21); t(7;21)(p22;q22); RUNX1; USP42
22.  Detection of FLT3 Oncogene Mutations in Acute Myeloid Leukemia Using Conformation Sensitive Gel Electrophoresis 
FLT3 (fms-related tyrosine kinase 3) is a receptor tyrosine kinase class III that is expressed on by early hematopoietic progenitor cells and plays an important role in hematopoietic stem cell proliferation, differentiation and survival. FLT3 is also expressed on leukemia blasts in most cases of acute myeloid leukemia (AML). In order to determine the frequency of FLT3 oncogene mutations, we analyzed genomic DNA of adult de novo acute myeloid leukemia (AML). Polymerase chain reaction (PCR) and conformation-sensitive gel electrophoresis (CSGE) were used for FLT3 exons 11, 14, and 15, followed by direct DNA sequencing. Two different types of functionally important FLT 3 mutations have been identified. Those mutations were unique to patients with inv(16), t(15:17) or t(8;21) and comprised fifteen cases with internal tandem duplication (ITD) mutation in the juxtamembrane domain and eleven cases with point mutation (exon 20, Asp835Tyr). The high frequency of the flt3 proto-oncogene mutations in acute myeloid leukemia AML suggests a key role for the receptor function. The association of FLT3 mutations with chromosomal abnormalities invites speculation as to the link between these two changes in the pathogenesis of acute myeloid leukemiaAML. Furthermore, CSGE method has shown to be a rapid and sensitive screening method for detection of nucleotide alteration in FLT3 gene. Finally, this study reports, for the first time in Saudi Arabia, mutations in the human FLT3 gene in acute myeloid leukemia AML patients.
doi:10.3390/ijms9112194
PMCID: PMC2635624  PMID: 19330068
AML; Flt3; ITD; CSGE; Mutational analysis
23.  Over-Expression of CYP2E1 mRNA and Protein: Implications of Xenobiotic Induced Damage in Patients with De Novo Acute Myeloid Leukemia with inv(16)(p13.1q22); CBFβ-MYH11 
Environmental exposure to benzene occurs through cigarette smoke, unleaded gasoline and certain types of plastic. Benzene is converted to hematotoxic metabolites by the hepatic phase-I enzyme CYP2E1, and these metabolites are detoxified by the phase-II enzyme NQO1. The genes encoding these enzymes are highly polymorphic and studies of these polymorphisms have shown different pathogenic and prognostic features in various hematological malignancies. The potential role of different cytochrome p450 metabolizing enzymes in the pathogenesis of acute myeloid leukemia (AML) in an area of active interest. In this study, we demonstrate aberrant CYP2E1 mRNA over-expression by quantitative real-time polymerase chain reaction in 11 cases of de novo AML with inv(16); CBFβ-MYH11. CYP2E1 mRNA levels correlated with CBFβ-MYH11 transcript levels and with bone marrow blast counts in all cases. CYP2E1 over-expression correlated positively with NQO1 mRNA levels (R2 = 0.934, n = 7). By immunohistochemistry, CYP2E1 protein was more frequently expressed in AML with inv(16) compared with other types of AML (p < 0.001). We obtained serial bone marrow samples from two patients with AML with inv(16) before and after treatment. CYP2E1 mRNA expression levels decreased in parallel with CBFβ-MYH11 transcript levels and blast counts following chemotherapy. In contrast, CYP1A2 transcript levels did not change in either patient. This is the first study to demonstrate concurrent over-expression of CYP2E1 and NQO1 mRNA in AML with inv(16). These findings also suggest that a balance between CYP2E1 and NQO1 may be important in the pathogenesis of AML with inv(16).
doi:10.3390/ijerph9082788
PMCID: PMC3447587  PMID: 23066397
CYP2E1; NQO1; AML with inv(16); de novo AML; CBFB-MYH11; benzene; xenobiotic induced damage
24.  Elevated serum endostatin levels is associated with favorable outcome in acute myeloid leukemia 
Introduction
Endostatin is the C-terminal antiangiogenic fragment of the extracellular matrix protein collagen XVIII, and is generated by tumor-derived proteases. The levels and the prognostic relevance of serum endostatin in AML patient is not fully clear.
Aim
To evaluate serum levels of endostatin in acute myeloid leukemia patients before chemotherapy and after achieving complete remission and to correlate endostatin levels with patients outcome.
Materials and methods
Serum samples from 30 adult patients (22 males and 8 females, median age 37, range 19–66 years) with AML had been taken before chemotherapy was administered. In addition 20 out of 30 patients were reinvestigated again at complete remission (CR). Ten samples from healthy normal persons of matched age and sex were evaluated as a reference control group. Serum endostatin levels were determined using enzyme linked immune sorbent assay (ELISA).
Results
Endostatin serum levels were not significantly different in the pretreatment AML patients as compared to that in normal controls (P>0.05). In AML patients the baseline endostatin levels were significantly lower than at CR (P=0.001). No significant correlation were detected between pretreatment serum endostatin levels and age, peripheral blood white cell counts, platelet counts, bone marrow blast cell counts, blast cell distribution ratio. The prognostic value of sE was also evaluated by dividing AML patients into high and low sE groups using the 75 percentile sE levels of the patients group as cutoff. The authors found that patients group in the high sE group survived for significantly longer time than those patients in the low sE group.
Conclusions
Elevated endostatin levels at AML diagnosis is a good prognostic marker for patients’ outcome. Wide scale study is recommended in order to establish the clinical value of this study.
doi:10.1007/s12288-008-0015-x
PMCID: PMC3453162  PMID: 23100932
AML; Endostatin; Prognosis; Angiogenesis
25.  Prevalence of ETV6/RUNX1 Fusion Gene in Pediatric Patients with Acute Lymphoblastic Leukemia in Iran 
Iranian Journal of Pediatrics  2013;23(6):681-686.
Objective
ETV6/RUNX1 (also known as TEL/AML1) is the most frequent gene fusion in childhood acute lymphoblastic leukemia (ALL). Sixty-three patients were enrolled in this study to explore the distribution of this gene in Iranian population.
Methods
This study used 63 peripheral blood and bone marrow (PB/BM) samples from children with ALL. Immunophenotyping of PB and BM samples were performed using flow cytometry to illustrate the lineage. Moreover, reverse transcriptase polymerase chain reaction (RT-PCR) technique was used to amplify transcripts of leukemia-specific chromosome fusion gene ETV6/RUNX1 and to monitor the expression levels of the ETV6/RUNX1 in patients according to Van Dongen et al protocol.
Findings
On the basis of French-American-British (FAB) classification, 47 individuals had ALL-L1. The incidence of ETV6/RUNX1 fusion gene in this study was 34.9%. The laboratory and clinical features of twenty two ETV6/RUNX1 positive ALL cases were similar to those of other studies. The most positive cases of ETV6/RUNX1 fusion gene had the early pre B ALL and pre B ALL immunophenotypes.
Conclusion
The ETV6/RUNX1 fusion gene is a common genetic anomaly in Iranian childhood ALL patients and the prevalence of the ETV6/RUNX1 fusion gene is similar to that of ALL patients in other countries. However early pre B cells were the most common type in studied patients.
PMCID: PMC4025127  PMID: 24910748
Acute Lymphoblastic Leukemia; Reverse Transcriptase; ETV6/RUNX1 Fusion; Polymerase Chain Reaction

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