Complete Hematologic and Cytologic Remission in M6A Erythroleukemia Using Decitabine Hypomethylation Monotherapy

Abstract Hypomethylating drugs (e.g. Azacitadine, Decitabine) are becoming increasingly attractive choices for the treatment of acute myelogenous leukemia (AML), particularly in populations deemed inappropriate for standard anthracycline-based induction chemotherapy (i.e., 7+3), such as the elderly. Acute erythroleukemia (French-American- British M6) is a rare, heterogeneous disorder involving increased red cell precursors and myeloblasts, that accounts for 3–5% of de novo AML and 20–30% of secondary AML. Three subsets have been described: M6A (myeloblast-rich erythroleukemia); M6B (proerythroblast-rich erythroleukemia); and M6C (myeloblast- and proerythroblast-rich mixed variant). Little data exists on the efficacy of hypomethylation therapy in this uncommon subtype of AML. We report the rapid induction of a complete hematologic and cytologic remission in a patient with the M6A variant erythroleukemia using decitabine monotherapy. A 75 year-old white male with a history of coronary artery disease, was admitted for elective cardiac catheterization because of recurrent exertional chest pain. A pre-catheterization complete blood count (CBC) revealed pancytopenia with a white blood cell (WBC) count of 1400/mm3, hemoglobin of 6.2 g/dL (mean cell corpuscular volume of 113 fL), and a platelet count of 51,000/mm3. The differential showed neutropenia, but was otherwise normal with no circulating blasts. Blood transfusion completely relieved his chest pain, and his catheterization was uneventful. A bone marrow aspirate and biopsy demonstrated a hypocellular marrow (15–20%) with erythroid hyperplasia (70% of total cellularity) and increased blasts (10% of total cellularity, 30% of non-erythroid cells), most compatible with acute M6A erythroid/myeloid leukemia. Cytogenetic studies revealed a normal male karyotype, but a single cell with a 5q- deletion. Subsequent fluorescent in situ hybridization (FISH) analysis using an LSI DNA probe (Vysis Inc.) for detection of the EGR1 gene on chromosome 5q, failed to confirm the 5q deletion, consistent with normal cytogenetics. Because of the patient’s advanced age and co-morbidities, induction with hypomethylation monotherapy was begun with intravenous decitabine (Dacogen®) 20 mg/m2 daily for five consecutive days, repeated every four weeks. Growth factor support (Neupogen® 375 mcg daily) and antibiotic prophylaxis (ciprofloxaxin, acyclovir, fluconazole) were only required after the first cycle of decitabine because neutropenia did not occur with subsequent cycles. WBC and platelet counts normalized shortly after cycle one, while the hemoglobin normalized by cycle five. An interim bone marrow aspirate/biopsy was repeated after cycle three, showing normocellularity (30%), and no evidence of residual leukemia. Other than a localized herpetic reactivation following cycle one, the patient has tolerated hypomethylation therapy extremely well. He has now completed eight cycles of therapy, and remains in complete remission. Epigenetic alterations (such as DNA methylation) may play a role in AML leukemogenesis by inducing the inhibition of tumor suppressor genes. Recent studies have also shown that high estrogen receptor-α and p15INK4B methylation levels in AML patients in clinical remission, were associated with a high risk for leukemia relapse and poor relapse-free survival. However, little data exist for epigenetic dysregulation in the etiology of human erythroleukemia. Cell culture studies using human erythroleukemia cell lines indicate that DNA methylation may have functional consequences for gene activity, including globin gene expression and cellular differentiation. The current case indicates a potential role for DNA methylation in the pathogenesis of human erythroleukemia, and argues for further investigation into this potential mechanism of leukemogenesis. Future studies will include the effect of hypomethylating compounds on the growth and differentiation of human erythroleukemia cells, and the evaluation of DNA methylation levels in bone marrow biopsy samples from M6 patients.

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Selective Inhibitors of Arginine Methyl Transferase 5 (PRMT5) As a Novel Treatment for β-Thalassemia and Sickle Cell Disease.

Blood ◽

10.1182/blood.v120.21.2129.2129 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2129-2129 ◽

Cited By ~ 1

Author(s):

Ian Street ◽

Brendon Monahan ◽

Hendrik Falk ◽

Elizabeth Allan ◽

Ylva Bergman ◽

...

Keyword(s):

Dna Methylation ◽

Bone Marrow ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Globin Gene ◽

Cell Disease ◽

Globin Mrna ◽

Cpg Dinucleotides ◽

Radiometric Assay ◽

Adult Bone

Abstract Abstract 2129 The developmental switch in human β-like globin gene subtype from fetal (γ) to adult (β) that begins at birth foreshadows the onset of the hemoglobinopathies, β-thalassemia and sickle cell disease (SCD). In the clinical setting it is established that β-thalassemia and SCD patients with hereditary persistence of fetal hemoglobin mutations enjoy a significant amelioration of disease severity due to the continued expression of γ-globin. This has prompted the search for therapeutic strategies to reverse γ-globin gene silencing. Central to the mechanism of γ-gene silencing is DNA methylation, which marks critical CpG dinucleotides flanking the γ-gene transcriptional initiation site in adult bone marrow erythroid cells. These marks are established by recruitment of DNMT3A, a DNA methyltransferase, to the γ-globin promoter by protein arginine methyltransferase 5 (PRMT5)[Zhao Q et al. Nat Struct Mol Biol. 2009;16(3):304–311]. PRMT5 catalyses the symmetric dimethylation of arginine 3 of Histone 4 (H4R3me2), which serves as a template for direct binding of DNMT3A and the subsequent DNA methylation of the γ-gene promoter. Loss of PRMT5 or its enzymatic activity is sufficient to induce demethylation of the CpG dinucleotides and reactivation of γ-globin gene expression [Rank, G., et al. Blood, 116(9), 1585–92]. Based on these observations we hypothesize that small molecule inhibitors of PRMT5 activity could provide a beneficial treatment for β-thalassemia and SCD. To identify small molecule inhibitors of PRMT5 a high throughput screen (HTS) was performed. Both radiometric and non-radiometric assay formats were developed to support the screening campaign. The radiometric assay format measures the ability of PRMT5 purified from K562 cells to catalyse the labelling of a short peptide based on the N-terminal sequence of Histone H4 by 3H-Methyl-S-Adenosyl-L-methionine (SAM). In contrast, the non-radiometric assay format employs recombinant PRMT5/MEP50 and measures the production of S-adenosyl-L-homocysteine (SAH), which is generated by PRMT5-catalysed methylation of H4 peptide. SAH is measured with Transcreener EPIGEN” and the assay is formatted in 1536-well microtitre plates in a total assay volume of 4 μL. Using these assays, a chemical library of 350,000 lead-like molecules and known pharmacologically active agents was screened to identify inhibitors of PRMT5 methyltransferase activity. A number of compounds with low micromolar or submicromolar inhibitory activity were identified by the HTS campaign, and six were selected for re-synthesis. The inhibitory activity of five of the six compounds was confirmed. To provide an initial appraisal of inhibitor selectivity the five active compounds were subsequently tested against a panel of enzymes consisting of 23 protein and DNA methyltransferases and 12 kinases. These compounds were found to be remarkably selective PRMT5 inhibitors, inhibition of MLL4 being the only significant off-target activity noted for one of the scaffolds. We have established a critical path for selection and progression of new chemical analogues which entails testing the compounds for: i) inhibition of PRMT5, other protein methyl transferases and kinases; ii) the ability to induce expression of γ-globin mRNA in the K562 erythroleukemic cell line; iii) the ability to induce expression of γ-globin mRNA in adult bone marrow erythroid cells; and iv) the induction of γ-globin in vivo in β-YAC mice, a transgenic model which carries the 250-kb human globin locus. In parallel, the physicochemical, metabolism, and pharmacokinetic properties of the most promising compounds are also determined. Medicinal chemistry efforts have now produced molecules with > 100-fold increased inhibitory potency against PRMT5 compared to the original hits, and preliminary results indicate that the more potent compounds have the ability to induce γ-globin mRNA in our cell based models. These early results illustrate the potential of PRMT5 inhibitors as a novel approach for the treatment of β-thalassemia and sickle cell disease. Disclosures: No relevant conflicts of interest to declare.

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A Rare Case of Pediatric Chronic Myelogenous Leukemia Presenting With Severe Thrombocytosis Without Leukocytosis

Pediatric and Developmental Pathology ◽

10.1177/1093526617698601 ◽

2017 ◽

Vol 21 (1) ◽

pp. 100-104 ◽

Cited By ~ 3

Author(s):

Albert N Huho ◽

Niveen Issaq ◽

Ionela Iacobas ◽

Tarek M Elghetany ◽

Dolores López-Terrada ◽

...

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Chronic Myelogenous Leukemia ◽

Bone Marrow Aspirate ◽

Kinase Inhibitor ◽

Fusion Transcript ◽

Myelogenous Leukemia ◽

Log Reduction ◽

Normal White Blood Cell ◽

Limit Of Quantitation

Pediatric chronic myelogenous leukemia is uncommon. We report a pediatric patient with chronic myelogenous leukemia presenting with a normal white blood cell count and no circulating immature myeloid cells. The patient presented with extreme thrombocytosis (platelet count range: 2175–3064 × 109/L) noted incidentally. No splenomegaly was found. Examination of the bone marrow aspirate revealed normal cellularity and normal myeloid: erythroid ratio with marked megakaryocytic hyperplasia. Molecular studies on the bone marrow aspirate detected both the major BCR/ABL1 p210 fusion transcript (9280 copies; p210/ ABL1 ratio: 38.2%) and the minor p190 transcript (below limit of quantitation). The platelet count normalized within 2 weeks after treatment with the second-generation tyrosine kinase inhibitor dasatinib. Follow-up after 3 months revealed a 1.87 log reduction in p210 transcripts compared to diagnosis and no detectable p190 transcripts. This case highlights the need to include BCR/ABL1 fusion testing to accurately diagnose pediatric patients presenting with isolated thrombocytosis.

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Developmental and Differentiation-Specific Patterns of γ- and β-Globin Promoter DNA Methylation in Primary Human Erythroid Cells.

Blood ◽

10.1182/blood.v108.11.1586.1586 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1586-1586

Author(s):

Christine Richardson ◽

Rodwell Mabaera ◽

Christopher H. Lowrey

Keyword(s):

Dna Methylation ◽

Bone Marrow ◽

Fetal Liver ◽

Globin Gene ◽

Erythroid Differentiation ◽

Erythroid Cells ◽

Globin Promoter ◽

Methylation Patterns ◽

Adult Bone

Abstract Despite years of investigation in a variety of experimental systems, the mechanisms underlying human β-globin locus developmental gene switching remain elusive. Several lines of evidence implicate DNA methylation in this process. As an initial step in studying the role of epigenetic modifications in the human switching process and in determining the mechanisms by which DNA methyltransferase inhibitors reverse the switch, we have characterized the DNA methylation patterns of the individual CpGs in the γ- and β-globin promoters in fetal liver (FL) and adult bone marrow (BM) primary erythroid cells and during in vitro differentiation of adult erythroid cells. Using the bisulfite conversion method we evaluated all CpGs in the ~500 bp regions centered on the γ- and β-globin promoter start sites. Fetal liver (FL) and adult bone marrow (BM) samples were obtained using IRB approved protocols and informed consent procedures. Erythroid cells were purified using anti-glycophorin A (glyA) magnetic beads. Purity was confirmed to be greater than 95%. Samples from five independent BM and FL samples were analyzed and 8–20 bisulfite converted sequences were determined for each promoter in each sample. Our results show that all 8 CpGs between −249 and +210 of the Gγ and Aγ-globin promoters are less than 20% methylated in FL and greater than 80% methylated in BM except for the −158 CpG which is only 40% methylated in BM(p<0.002). The 6 CpGs between −415 and +110 of the β-globin promoter show an inverse pattern with lower levels of DNA methylation in BM. Histone H3 acetylation of the γ-globin promoter, as determined by ChIP analysis, showed a complimentary pattern with higher levels in FL than BM. We next evaluated γ-globin promoter methylation patterns during in vitro erythroid differentiation from CD34+ BM cells. In this experiment, cells were grown with SCF, Flt3 ligand and IL-3 for 7 days and then in EPO for 14 days producing erythroid cells which express 99%HbA and 1% HbF. The initial day 0 CD34+ cells showed 90–100% methylation of all γ promoter sites. By day 3 in culture, before the initiation of erythroid differentiation, methylation at all sites upstream of the promoter had decreased to less than 60% and the CpG at −53 (the site of Stage Selector Protein complex binding) had decreased to less than 20%. The three CpG sites down-stream of the promoter (+6, +17 and +50) remained highly methylated. The pattern was unchanged at day 10, early in erythroid differentiation, when γ-globin mRNA expression was beginning. By day 14, when β-globin expression was peaking, methylation of the upstream promoter had increased back to the 70–100% level at all CpGs. These experiments provide a comprehensive picture of γ- and β-globin promoter methylation during the fetal and adult stages of erythroid development and of the γ-globin promoter during adult erythroid differentiation. The finding of transient γ-promoter hypomethylation during differentiation offers a potential mechanism to explain the transient γ-globin gene expression seen during normal adult erythropoiesis. Our results also raise the possibility that, just as domains of altered histone modification exist in β-globin gene loci, there may also be developmentally-specific domains of DNA methylation.

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The Effects of Environmental Cues on Hematopoietic Stem Cell Colony Fetal Hemoglobin Production.

Blood ◽

10.1182/blood.v116.21.3860.3860 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3860-3860

Author(s):

Jennifer L Nichols ◽

Donald Lavelle ◽

Joseph DeSimone

Keyword(s):

Dna Methylation ◽

Bone Marrow ◽

Globin Gene ◽

Gene Promoter ◽

Feeder Layer ◽

Liquid Media ◽

Feeder Layers ◽

Globin Promoter ◽

Cells Cultured ◽

In Cells

Abstract Abstract 3860 Objective. Increased fetal hemoglobin (HbF) levels are associated with increased life span and reduced pain crises in patients with Sickle Cell disease. An increased understanding of the mechanisms controlling HbF expression would be important to develop new therapies to increase HbF. The objective of these experiments was to test the hypothesis that interactions between bone marrow (BM) erythroid progenitor cells and the stromal microenvironment influence HbF expression. Materials and Methods. Baboon CD34+ BM cells were harvested and purified by immunomagnetic column chromatography using the 12.8 anti-CD34 mouse monoclonal antibody and immunomagnetic microbeads conjugated to rat anti-mouse IgM (Miltenyi). CD34+ BM Cells were grown in liquid cultures in Iscove's media containing 30% fetal bovine serum, 200ng/ml stem cell factor, 2u/ml erythropoietin, and 1uM dexamethasone in the presence of an AFT024 mouse fetal liver cell line and an OP9 mouse bone marrow cell line as feeder layers, in methylcellulose media, and in liquid media. Globin chain synthesis in cultures was measured on d11 and d14 by biosynthetic radiolabeling. Cells were incubated overnight in leucine-free α-MEM media containing 50uCi/ml [3H leucine]. Globin chains were separated by High Performance Liquid Chromatography (HPLC), and the radioactivity in each fraction was determined by liquid scintillation counting. To correlate changes in γ-globin expression with DNA methylation of the γ-globin gene promoter, the methylation state of 5 CpG sites within the γ-globin promoter region was determined by bisulfite sequencing. Erythroid cells were purified from cultures on d14 by immunomagnetic column chromatography using mouse anti-baboon RBC monoclonal antibody. DNA was isolated from purified erythroid cells using Qiagen kits. Bisulfite modification was performed using Epitect bisulfite kits. Two rounds of PCR amplification were performed using nested primers flanking 5 CpG residues within the baboon γ-globin gene promoter. Minilysate DNA was prepared from at least 10 independent clones for each sample. Sequence analysis of purified DNA samples was performed at the University of Illinois DNA Sequence facility. Results. Elevated levels of γ-globin chain synthesis were observed in cells cultured in methylcellulose and liquid media in the absence of a feeder layer when compared to cells grown in the presence of feeder layers. On d11, the γ/γ+β chain synthetic ratio was 0.623 in cells cultured in methylcellulose and 0.324 in cells cultured in liquid media, compared to 0.092 in cells cultured in the presence of AFT024 feeder layers and 0.178 in cells cultured in the presence of OP9 feeder layers. On d14, the γ/γ+β chain synthetic ratio was 0.663 in cells cultured in methylcellulose and 0.349 in cells cultured in liquid media, compared to 0.135 in cells cultured in the presence of AFT024 feeder layers and 0.252 in cells cultured in the presence of OP9 feeder layers. The level of DNA methylation (%dmC) of 5 sites in the γ-globin gene promoter negatively correlated with levels of HbF production. On d14, the level of DNA methylation was 79% in cells cultured in methylcellulose, 72% in cells cultured in liquid media, and 97% in cells grown in the presence of AFT024 feeder layers. Conclusions. Cells grown in the presence of AFT024 feeder layers expressed physiologic levels of γ-globin and cells grown in the presence of OP9 feeder layers expressed midrange levels of γ-globin. Significantly increased γ-globin expression was observed in cells cultured in the absence of a feeder layer. The γ-globin promoter in cells grown in the presence of the AFT024 feeder layer exhibited a significantly higher degree of methylation than in cells grown in methylcellulose and liquid media. These results show that interactions between erythroid progenitor cells and the stromal microenvironment can influence both the level of γ-globin expression and the DNA methylation of the γ-globin gene promoter. These results have clinical relevance; if the bone marrow microenvironment could be effectively altered in vivo, methylation of the γ-globin promoter could be decreased and HbF production increased. Further experiments must be performed to determine what is mediating the methylation of the γ-globin promoter when cells are grown on these feeder layers. Disclosures: No relevant conflicts of interest to declare.

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Dynamic Regulation of 5-Hydroxymethylcytosine At the γ-Globin Promoter During Erythroid Differentiation

Blood ◽

10.1182/blood.v120.21.824.824 ◽

2012 ◽

Vol 120 (21) ◽

pp. 824-824

Author(s):

Maria Armila Ruiz ◽

Kestis Vaitkus ◽

Aparna Vasanthakumar ◽

Angela Rivers ◽

Tatiana Kouznetsova ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Bone Marrow ◽

Globin Gene ◽

Erythroid Differentiation ◽

Dna Demethylation ◽

Pcr Analysis ◽

Erythroid Precursor ◽

Erythroid Precursors ◽

Globin Promoter

Abstract Abstract 824 DNA methylation is a key element responsible for γ-globin gene repression in adult erythroid cells. Our laboratory previously observed that the γ-globin gene promoter region was demethylated in a progressive manner as γ-globin expression was activated during erythroid differentiation of primary baboon pre-switch fetal liver cells and, to a lesser extent, of adult baboon bone marrow (BM) cells (Singh et al Exp Hematol 35:48-55, 2007). The mechanism responsible for DNA demethylation of the γ-globin promoter during erythroid differentiation remains unknown. Recent studies have shown that DNA demethylation in the early embryo is mediated by the “sixth base” 5-hydroxymethylcytosine (5-hmC) whose formation from 5-methylcytosine is catalyzed by the enzymatic activity of the three TET proteins. To investigate the hypothesis that 5-hmC mediates DNA demethylation of the γ-globin promoter during erythroid differentiation, levels of 5-hmC at Msp I (CCGG) sites within the γ- and ϵ-globin promoters and γ-globin IVS II region in DNA isolated from peripheral WBC, purified terminal erythroid precursors, and FACS-purified adult bone marrow subpopulations enriched for different stages of differentiation (CD117+ CD36-, CD117+ CD36+, and CD117-CD36+), were analyzed using a T4 glucosylase-MspI quantitative real time PCR assay. Levels of 5-hmC associated with the γ-globin promoter MspI site were 11.6 fold higher (p<.0001) in terminal erythroblasts (n=9) compared to peripheral blood WBC (n=4) while 5-hmC levels associated with the ϵ-globin promoter were 11.8 fold higher (p<.0001) in terminal erythroid precursors (n=13) compared to peripheral WBC (n=4). Levels of 5-hmC associated with the γ-globin promoter (n=9) were 9.4 fold higher (p<.0001) than with the IVS II region of the γ-globin gene (n=8) in terminal erythroid precursors suggesting that elevated levels of 5-hmC within the β-globin gene complex in terminal erythroid precursors may be localized to promoter regions. Genomic 5-hmC levels, analyzed by HPLC-MS, were similar in WBC and terminal erythroid precursors. Within purified BM subpopulations enriched for different stages of erythroid differentiation, levels of 5-hmC associated with the γ-globin promoter were 3.2 to 4.1 fold higher in the CD117+CD36+ subpopulation enriched in erythroid colony forming cells (n=4) than in CD117+CD36- (n=3; p<.01), more differentiated CD117-CD36+ (n=3; p<.02), and terminal erythroid precursor (p<.001) subpopulations. Similar differences in levels of 5-hmC associated with the ϵ-globin promoter were also observed between these subpopulations. Enrichment of 5-hmC within the β-globin locus in the CD117+CD36+ and terminal erythroid precursors compared to peripheral WBC was confirmed by 5-hmC affinity selection and PCR analysis. In addition, similar differences in genomic 5-hmC levels between these subpopulations were also observed by HPLC-MS analysis. Bisulfite sequence analysis showed that the changes in 5-hmC levels at the γ-globin promoter were temporally associated with loss of DNA methylation within the γ-globin promoter as erythroid differentiation progressed. TET gene expression analysis showed that TET2 expression was 3 fold less (p<.01) while TET3 expression was >7 fold higher (p<.05) in terminal erythroid precursors compared to the CD117+CD36- subpopulation. These results strongly suggest that differences in 5-hmC associated with the ϵ- and γ-globin promoters are the result of wider dynamic alterations of genomic 5-hmC levels during erythroid differentiation that may be mediated by differences in TET gene expression and support the hypothesis that 5-hmC is involved in the mechanism responsible for DNA demethylation of the γ-globin promoter during erythroid differentiation. Disclosures: Godley: Celgene: Research Funding.

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Study of myelodysplastic features in patients with myelodysplastic syndromes by multicolor flow cytometry

Oncohematology ◽

10.17650/1818-8346-2019-13-4-75-88 ◽

2019 ◽

Vol 13 (4) ◽

pp. 75-88

Author(s):

O. Yu. Davydova ◽

I. V. Galtseva ◽

E. N. Parovichnikova ◽

A. V. Kokhno ◽

N. M. Kapranov ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Myelodysplastic Syndromes ◽

Bone Marrow Aspirate ◽

Screening Method ◽

Control Group ◽

Allogeneic Bone ◽

Multicolor Flow Cytometry ◽

Color Flow ◽

5Q Deletion

Background . Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal diseases of the hematopoiesis system characterized by dismyelopoiesis and cytopenia, the presence of cytogenetic aberrations and a high risk of transformation into acute myeloid leukemias. Diagnosis of MDS requires a comprehensive approach and mandatory performance of cytological, cytochemical and cytogenetic studies of bone marrow aspirate, as well as histological examination of trephine biopsy. However, in some cases it is necessary to undergo a diagnostic test that would allow verification of the MDS. The study of bone marrow aspirate by multicolor flow cytometry (MFC) can be considered as an additional diagnostic criterion in the diagnosis of MDS.The objective of the study was to estimate the incidence of myelodysplastic features in patients with various forms of MDS by the MFC method. Materials and methods . The study included 79 patients with MDS: 8 with MDS with 5q deletion, 33 with MDS without excess blast cells and 38 with excess of blasts. A bone marrow aspirate test was performed by 6-color flow cytometry. The control group included 35 donors of allogeneic bone marrow. The analysis resulted in a conclusion on the Ogata score scale, the Wells prognostic scale and the combined Ogata–Wells scale. When using the screening method, the presence of two or more cytometric signs of MDS was detected in 60 (75.9 %) of 79 MDS patients. Wells score was higher in MDS group with an excess of blast than in others. Using the combined Ogata–Wells scale, cytometric aberrations were found in 70 (88.6 %) of 79 MDS patients. In patients with MDS with an excess of blasts, the incidence of increased CD34+ and/or CD117+ myeloid cells was higher than in MDS patients without an excess of blasts and an MDS with a 5q deletion. The frequency of abnormal cytometric parameters (anomalous expression of CD34, CD117, CD56+ myeloblasts) in these groups did not differ. In patients with isolated 5q deletion and MDS without excess of blasts, an increased proportion of CD7+CD34+ cells was more often detected than in MDS with an excess of blasts.Conclusion . Thus, cytometric abnormalities in MDS are common, even in patients without excess of blasts. The MFC method can be used as an additional diagnostic method in the initial diagnosis of MDS.

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The Effect of Decitabine on Covalent Histone Modifications of Chromatin Associated with the ε-,γ -, and β-Globin Gene Promoters in the Baboon (P. anubis).

Blood ◽

10.1182/blood.v104.11.378.378 ◽

2004 ◽

Vol 104 (11) ◽

pp. 378-378

Author(s):

Donald Lavelle ◽

Kestas Vaitkus ◽

Maria Hankewych ◽

Mahipal Singh ◽

Joseph DeSimone

Keyword(s):

Dna Methylation ◽

Bone Marrow ◽

Globin Gene ◽

Post Treatment ◽

Gene Promoters ◽

Rna Pol Ii ◽

Pol Ii ◽

Pre Treatment ◽

Globin Promoter ◽

Covalent Histone Modifications

Abstract Treatment with the DNA demethylating drug decitabine increased HbF and F cells to therapeutically significant levels in patients with sickle cell disease in clinical trials. To gain more insight into the mechanism of action of this drug and to increase our understanding of the relationship between DNA methylation and chromatin structure we have determined the effect of decitabine on DNA methylation and covalent histone modifications of chromatin associated with the ε-, γ-, and β-globin gene promoters in purified erythroid bone marrow-derived cells obtained from 2 baboons (P. anubis) pre- and post-treatment. Baboons were phlebotomized for 10 days to maintain a hematocrit of 20 followed by subcutaneous decitabine administration (0.52mg/kg/day; 10–13 days). Erythroid cells (30–50 X 106) were purified from pre and post-treatment bone marrow apirates by immunomagnetic columns using an anti-baboon RBC antibody. The level of DNA methylation of 3 sites in the ε-globin promoter and 5 sites in the γ-globin promoter was determined by sequence analysis of ten cloned PCR products of each sample following bisulfite modification. Levels of ac-H3, ac-H4, dimethyl H3 ly4 (H3-dimeK4), dimethyl H3 ly79 (H3-meK79, dimethyl H3 ly36 (H3-meK36), and RNA pol II associated with the ε-, γ-, and β-globin promoters were determined by chromatin immunoprecipitation of sheared, formaldehyde fixed chromatin followed by real time PCR. In pre-treatment samples the level of DNA methylation of the γ-globin promoter (75%, 59.5%) was lower than the ε-globin promoter (84.%, 83.3%) and was associated with a small increase in HbF (5.4%, 9.0%). Following decitabine treatment HbF increased 8–10 fold (51.1%, 76%) and the level of DNA methylation of the ε-globin promoter (30.5%, 38.8%) and γ-globin promoter (24.3%, 25.0%) decreased 2–3 fold. Binding of RNA pol II to βthe -promoter was 2–3 fold higher than to the γ-promoter in pre-treatment samples. Following decitabine treatment the level of pol II associated with the γ-globin promoter was at least tenfold higher than with the β-globin promoter. In pre-treatment samples the level of ac-H3 and ac-H4 associated with the β-globin promoter was 2–4 fold higher than with the γ-globin promoter and 50 fold higher than with the ε-globin promoter. Following decitabine treatment the level of ac-H3 associated with the γ-promoter was 3–4 fold higher than with the β-promoter, while the level of ac-H4 associated with the γ-promoter increased to a level equivalent to that bound to the β-promoter. While no difference in the pattern of H3-dimeK4 associated with the ε-, γ-, and β-globin promoters was observed between pre- and post-treatment samples, minor changes in H3-meK79 and H3-meK36 were observed. These results demonstrate that decitabine treatment decreases DNA methylation of both the ε- and γ-globin promoters, greatly increases binding of RNA pol II to the γ-globin promoter, and increases the levels of ac-H3 and ac-H4 in chromatin associated with the γ-globin promoter. These experiments illustrate the usefulness of the baboon model to investigate the mechanism of pharmacologic reactivation of HbF synthesis at the molecular level.

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Establishment and Characterization of a New Human Ph+ Erythroleukemic Cell Line, VES.

Blood ◽

10.1182/blood.v106.11.4558.4558 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4558-4558

Author(s):

Mirna Golemovic ◽

Lana Rnjak ◽

Klara Dubravcic ◽

Sanja Davidovic-Mrsic ◽

Ivana Franic ◽

...

Keyword(s):

Bone Marrow ◽

Cell Line ◽

Multiparameter Flow Cytometry ◽

Fish Analysis ◽

Suitable Model ◽

Short Period ◽

Erythroleukemia Cell ◽

Erythroleukemic Cell ◽

Erythroleukemic Cell Line

Abstract Continuous human malignant hematopoietic cell lines are invaluable tool for hematological research. Here we report on a new erythroleukemic cell line termed VES that was established from the bone marrow mononuclear cells (BMNC) of a 22-year-old woman with Ph+ CML during her second post-transplant period. Actually, after unsuccessful allo-transplantation, the patient received auto-transplant that also failed to engraft. At the time of initiation of BMNC into culture for the analysis of stromal cell capacity, her bone marrow was tested Ph-negative by both FISH and PCR. Instead of establishment of an adherent stromal layer in vitro, blast-like cells appeared in the supernatant on day 27. These blast cells expressed highly amplified bcr/abl locus as shown by FISH, whereas the patient’s bone marrow was still Ph-negative at the time. Following a very short period, the cultured cells started to express stable features of erythroleukemia cell line. Optimal growth was obtained by suspending the cells at concentration of 0.3x106 cells/ml in IMDM containing 10% FBS, 1% penicillin-streptomycin and 1% L-glutamine solution. After 3 days of culture, cell concentration varied between 1.3 and 1.8x106 cells/ml. VES cells were tested negative for the presence of EBV virus. For analysis of clonogenicity, VES cells at concentration of 103/ml were grown in a serum-free methylcellulose medium without cytokines (H4236; StemCell Tech., Canada). Following 7-day culture, 103 VES cells produced 251 ±42 and after 14 days 431 ±30 GF-independent colonies. Furthermore, on day 14 of the cell culture it was possible to observe reddish color of the colonies indicating the presence of hemoglobin. Cytological examination of cytospin preparations indicated homogenous population of leukemia blasts (>80%) the majority of them (>90%) being glycophorinA positive and MPO negative. Imunophenotyping and multiparameter flow cytometry revealed proerythroblastic lineage-associated profile: GlyA/CD235a+, CD11b+, CD15+, CD29+, CD33+, and CD117+. Conventional cytogenetics revealed complex karyotype with multiple numerical/structural abnormalities (MAKA), while metaphase FISH revealed the following aberrations: t(9;22), 5q31, 7q31, +8, +6. Since FISH analysis detected highly amplified bcr/abl locus, we tested VES cells’ sensitivity to imatinib (Gleevec). Treatment with imatinib for 3 days (MTS assay) inhibited proliferation of VES cells with IC50 value of 0.2mM. Following 14 days of culture in methylcellulose medium with addition of 0.2mM imatinib, the clonogenic potential of VES cells was reduced by 55% in relation to untreated control. We demonstrated that imatinib induced apoptosis in VES cells in time- and dose-dependant manner, assessed with AnnexinV and PI staining, while there were no significant changes observed in the cell cycle except for a mild increase in cells in G0/G1 phase. Although further analyses are required, we believe that VES cells represent a new Ph+ erythroleukemia cell line and a suitable model for studying Ph+ malignant hematopoiesis in vitro.

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Detection of Minimal Residual Disease in Precursor B Lymphoblastic Leukemia (B-ALL) Patients by a Novel Epigenetic DNA Methylation Biomarker.

Blood ◽

10.1182/blood.v106.11.4524.4524 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4524-4524

Author(s):

Michael X. Wang ◽

Deiter Duff ◽

Huidong Shi ◽

Kristen H. Taylor ◽

Barbara A. Gruner ◽

...

Keyword(s):

Dna Methylation ◽

Bone Marrow ◽

Minimal Residual Disease ◽

Bone Marrow Aspirate ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Receptor Gene ◽

Cell Receptor ◽

Specific Pcr ◽

Minimal Residual

Abstract Background: Increasing evidence indicates that the minimal residual disease in childhood precursor B lymphoblastic leukemia (B-ALL) can predict the clinical outcome of the patients. With the combination of multicolor flow cytometry and B-cell receptor gene rearrangement or gene translocation analysis, the majority of minimal residual disease can be detected at the 10−5 level. However, these methods may suffer from high cost and complexity and result in false negatives due to antigen shift or lack of genetic markers. Aberrant DNA hypermethylation in CpG islands of tumor suppressor genes is a hallmark of many human malignancies including B-ALL. Since the DNA methyltransferase 1 (DNMT1) is a component of replisome at the DNA replication fork, the methylation in cytosine is precisely duplicated in each cell cycle. Thus the DNA methylation status is maintained and can be detected in the minimal residual leukemic cells if the original clone has DNA methylation markers. Methods: We developed a new method for detection of minimal residual disease in B-ALL patients using a specific DNA methylation marker in the promoter region of the tumor suppressor gene DLC-1 (deleted in liver cancer -1). The quantitative real-time methylation specific PCR (qMSP) was utilized as a primary detection method and it was validated via combined bisulfite restriction analysis (COBRA). The qMSP uses two specifically designed amplification primers which are complementary only for bisulfite-treated methylated DNA sequences and an additional fluorogenic probe specificlly hybridizing methylated target amplicons. Results: By using highly sensitive qMSP assay, we demonstrated that DNA methylation of the DLC-1 gene promoter occurred in 15 out of 20 (75 %) of B-ALL patient bone marrow aspirate specimens and 2 (NALM-6 and MN60) out of 3 B-ALL cell lines. DLC-1 methylation was detected in multiple sequential specimens (up to 6 time points) from 3 childhood B-ALL patients and is comparable with morphologic, flow cytometry and B-cell receptor gene rearrangement (in 5 specimens) assessment. In addition, DLC-1 methylation in different types of specimens collected at the same time point including frozen bone marrow aspirate, unstained bone marrow aspirate slides, fresh peripheral blood, and unstained peripheral blood smear showed complete consistency. Finally, the analytic sensitivity of qMSP was determined by a series of dilution of tumor cell DNA (MN60) in normal human genomic DNA. Ten ng of tumor DNA can be detected in 1 μg of normal DNA. The co-efficient of variation (CV) of the intra- and inter- measurements was about 0.5% and 1.5% by Ct values, respectively. Conclusion: We have developed a novel real-time methylation specific PCR method to detect minimal residual disease in B-ALL patients. The method is sensitive, quantitative, simple and fast, and has the potential to be used for routine clinical minimal residual disease detection in majority of B-ALL patients. The analytic sensitivity and specificity of this method are compatible with flow cytometric and molecular analysis. A study with larger number of B-ALL patient specimens using this novel method is currently underway.

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Aberrant CpG Island Methylation in AML Is Associated with Disease Progression and Relapse.

Blood ◽

10.1182/blood.v108.11.564.564 ◽

2006 ◽

Vol 108 (11) ◽

pp. 564-564

Author(s):

Heike Kroeger ◽

Jaroslav Jelinek ◽

Carlos E. Bueso-Ramos ◽

Jean-Pierre J. Issa

Keyword(s):

Dna Methylation ◽

Bone Marrow ◽

Disease Progression ◽

Peripheral Blood ◽

Blood Cells ◽

Cpg Islands ◽

Myelogenous Leukemia ◽

Peripheral Blood Cells ◽

Z Score ◽

Methylation Index

The role of DNA methylation in relapse and progression of acute myelogenous leukemia (AML) is incompletely understood. We studied DNA methylation of 9 promoter-associated CpG islands of genes frequently hypermethylated in leukemic cell lines. These were NOR1, NPM2, HIN1, SLC26A4, CDH13, PGRA, PGRB, OLIG2 and the tumor suppressor gene p15INK4b. We examined bone marrow and/or peripheral blood cells collected at the time of diagnosis and at the first relapse from 32 patients (13 females, 19 males) with AML. The median age was 58 years (20–68), the median survival was 18 months (8–80), the median blast count was 64% (20–98), and 10 patients had additional solid tumors and/or lymphatic/hematologic malignancies. Bisulfite treatment of DNA, followed by PCR and pyrosequencing were used to quantitatively measure levels of cytosine methylation in promoter-associated CpG islands. We analyzed methylation data for individual genes and for a methylation index derived after Z-score (z = [value –mean]/standard deviation) transformation to equalize absolute differences between individual genes and we used paired t-tests for statistical analysis. Abnormal hypermethylation (≥10%) in bone marrow and peripheral blood cells at diagnosis was detected in all 9 investigated genes, with a range of 7/29 (24%) for HIN1 to 24/32 (75%) for CDH13. On average, an increase in methylation between diagnosis and relapse was detected in all genes, and was significant for CDH13 (mean 10%, p=0.0006), SLC26A4 (mean 7%, p=0.0012), HIN1 (mean 8%, p=0.0037), NPM2 (mean 7%, p=0.0073), p15INK4b (mean 13%, p=0.0081), NOR1 (mean 4%, p=0.0124), PGRB (mean 6%, p=0.0144), PGRA (mean 9%, p=0.0275), and OLIG2 (mean 3%, p=0.0732). When analyzed by change in methylation status: negative (methylation below 10%) turning positive (methylation ≥ 10%) and vice versa, of 238 analyses, 39 (16%) showed a negative to positive switch, 15 (6%) showed a positive to negative switch, and the remaining 184 (77%) were either positive or negative unchanged. Finally, when analyzed in individual patients, an increase in methylation was noted in 29 of 32 patients (91%). The median increase in methylation index between diagnosis and relapse calculated as a delta-Z-score was 30% (range from −10% to 147%), and was highly significant (p<0.0001). In summary, abnormal hypermethylation in bone marrow and/or peripheral blood cells from AML patients was detected in all investigated genes at diagnosis. Methylation levels further increased at relapse of the disease in 29 of 32 patients in 1 to 8 of 9 investigated genes. Based on quantitative analyses, we propose that methylation of CDH13, PGRB, PGRA and OLIG2 CpG islands are early markers for AML, while hypermethylation of HIN1, NPM2 and p15INK4b CpG islands is associated with disease progression and predominantly appears at relapse. Thus, aberrant hypermethylation is clearly associated with disease progression and relapse in AML, and likely mediates drug resistance in this setting. Increase of Methylation Index in Relapse Increase of Methylation Index in Relapse

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