Analysis of Chromosomal Aberrations and DNA Mutations in Bone Marrow Mesenchymal Stroma Cells from Patients with Myelodysplastic Syndrome Amd Acute Myeloid Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4843-4843
Author(s):  
Olga Blau ◽  
Wolf-Karsten Hofmann ◽  
Igor W. Blau ◽  
Claudia D Baldus ◽  
Florian Nolte ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myelodysplastic Syndrome ◽  
Chromosomal Aberrations ◽  
Myeloid Leukemia ◽  
Hematopoietic Microenvironment ◽  
Dna Mutations ◽  
Healthy Donors ◽  
Mesenchymal Stroma Cells ◽  
Acute Myeloid

Abstract Introduction: The biology of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) is not well understood. In MDS, ineffective hematopoiesis may result from disturbed interactions between hematopoietic cells (HC) and the hematopoietic microenvironment. Bone marrow mesenchymal stroma cells (BMSC) are key components of the hematopoietic microenvironment. BMSC from patients with hematological disorders display functional and quantitative alterations. However, the question whether BMSC in MDS/AML have cytogenetic abnormalities is discussed controversially. Methods: In the present study, we have collected BMSC from 51 MDS and 42 AML patients at the time of initial diagnosis. Chromosome preparation was performed after cell culture for 30 days and analyzed by conventional cytogenetic (G-banding) and by different types of FISH-techniques. Furthermore, FLT3 and NPM1 mutation analysis was performed in HC and BMSC. As a control we have studied BMSC from 25 healthy individuals. Results: Cytogenetic analysis of BMSC was successfully performed in 90 of the 93 cases. Clonal structural chromosomal aberrations, including t(1;7), t(1;10), t(1;2), t(7;9), i(1q), inv(X), del(7q), del(13q), del(17p), and others, were detectable in BMSC of 15% of patients. All cytogenetic markers were confirmed by FISH with specific probes and M-FISH. Interestingly, cytogenetic markers in BMSC differed from the aberrations in HC from the same individual. We have found cytogenetic abnormalities in BMSC from patients presenting with cytogenetic alterations in their HC as well as from those with normal karyotype. In BMSC we could not detect specific mutations of NPM1 and FLT3 (ITD and TKD), independent from the mutation status of HC. For control analysis, BMSC cultures from 25 healthy donors were prepared under the same conditions (time of culture, number and frequency of passages). BMSC from healthy donors did show normal diploid karyotypes and absence of DNA-mutations of NPM1 and FLT3. Conclusions: Our data indicate that BMSC from MDS and AML patients are characterized by genetic instability. Lack of aberrations as detected in HC and appearance of novel clonal rearrangements in BMSC may suggest enhanced genetic susceptibility and potential involvement of BMSC in the pathogenesis of MDS and AML.

Cytogenetic and Molecular Genetic Aberrations in Bone Marrow Mesenchymal Stroma Cells from Patients with Myelodysplastic Syndrome and Acute Myeloid Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 985-985
Author(s):  
Olga Blau ◽  
Wolf-Karsten Hofmann ◽  
Igor W. Blau ◽  
Claudia D. Baldus ◽  
Gundula Thiel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myelodysplastic Syndrome ◽  
Chromosomal Aberrations ◽  
Myeloid Leukemia ◽  
Hematopoietic Microenvironment ◽  
Flt3 Mutations ◽  
Cytogenetic Analyses ◽  
Mesenchymal Stroma Cells ◽  
Acute Myeloid

Abstract Introduction : The biology of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) is heterogeneous. Ineffective hematopoiesis results from complex interactions between hematopoietic cells (HC) and the hematopoietic microenvironment. Bone marrow mesenchymal stroma cells (BMSC) are key components of the hematopoietic microenvironment. The question of whether BMSC from patients with hematological disorders have cytogenetic abnormalities is discussed controversially. Methods: We performed standard cytogenetic analyses (G-banding), FISH, M-FISH, and FLT3 mutation examinations of both HC and BMSC from 56 patients (30 MDS and 26 AML) and 9 healthy individuals. For BMSC selection, mononuclear cells were isolated from fresh bone marrow aspirates at the time of initial diagnosis and were further expanded in cell culture. Results: Clonal cytogenetic aberrations were observed in HC from 14 (46%) MDS and 14 (53%) AML patients. Cytogenetic analyses of BMSC were successfully performed in 50 of the 56 cases. Structural chromosomal aberrations, including t(1;7); t(1;3); t(1;10); t(4;7); t(7;9); t(7;19); i(1q); inv(X); del(1q); del(2q); del(3p); del(4p); del(11q); del(13q); del(17p), and others were detectable in BMSC from 42% of patients. The breakpoints of chromosomes in BMSC were typical for leukemia aberrations. Six patients showed clonal chromosomal markers: t(1;7), t(1;10), t(7;9), inv(X), del(17p), and monosomy 4. Interestingly, cytogenetic markers in BMSC differed to the aberrations in HC from the same individual. No deletions or monosomy of chromosomes 5 or 7 in BMSC (FISH, 500 cells) were found in BMSC, even in those patients, who showed these aberrations in HC. M-FISH confirmed chromosomal aberrations in BMSC. We have found cytogenetic abnormalities in BMSC from patients presenting with cytogenetic alterations in their HC as well as from those with normal karyotype. We did not find structural chromosomal alterations in BMSC cultures of healthy bone marrow donors. Fourteen percent of AML patients showed FLT3 mutations in HC, but no FLT3 mutations were found in BMSC. Conclusion: We showed that BMSC from AML and especially from MDS patients are characterized by genetic instability. The breakpoints of chromosomes in BMSC were typical for leukemia aberrations. The fact that BMSC showed typical chromosomal changes may suggest enhanced genetic susceptibility and potential involvement in the pathophysiology of MDS. Characterization of BMSC may help us to better understand the biology of this disease.

NUP98-HOXD13 Transgenic Mice Develop Myelodysplastic Syndrome, Acute Myeloid Leukemia, and Pre-T Lymphoblastic Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 345-345
Author(s):  
Yingwei Lin ◽  
Christopher Slape ◽  
Zhenhua Zhang ◽  
Peter D. Aplan
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Transgenic Mice ◽  
Myelodysplastic Syndrome ◽  
Myeloid Leukemia ◽  
Lymphoblastic Leukemia ◽  
Gene Rearrangements ◽  
Hematologic Disease ◽  
Amino Terminal ◽  
Acute Myeloid

Abstract The NUP98 gene is located at chromosome 11p15 and encodes the 98 kd component of the nuclear pore complex; this protein normally functions as a docking protein involved in nucleocytoplasmic transport. NUP98 is fused to at least 15 different partner genes by chromosomal translocation in a wide spectrum of hematological malignancies including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myelogenous leukemia (CML), and pre-T lymphoblastic leukemia (pre-T LBL). Over half of the known NUP98 gene fusions involve fusions to a HOX family member; these fusions invariably retain the amino terminal FG repeats of NUP98 and the homeodomain DNA-binding region of the HOX partner. The NUP98-HOXD13 fusion was initially identified in a patient with MDS that subsequently transformed to erythroleukemia, and has subsequently been identified in AML M1 and M2 patients as well. To model this disease in vivo, we generated transgenic mice which expressed the NUP98-HOXD13 (NHD13) fusion from vav regulatory elements. The NHD13 transgene is ubiquitously expressed in hematopoietic tissues such as thymus, spleen, and bone marrow, and is not expressed in other tissues. Serial CBCs from clinically healthy mice aged 4–7 months demonstrated a progressive neutropenia, lymphopenia, anemia, and macrocytosis. Peripheral blood smears showed signs of dysplasia including giant platelets and hypersegmented neutrophils; bone marrow exam showed an increase number of dysplastic binucleate erythroblasts and increased apoptosis, consistent with a diagnosis of MDS. 10/10 (100%) of the NHD13 mice died of hematologic disease by 14 months of age; in contrast, none of the non-transgenic control littermates developed evidence of hematologic disease. We classified the hematologic diseases according to the Bethesda proposals. Three mice died with MDS, two mice had pre-T LBL, two had acute undifferentiated leukemia, one had megakaryocytic leukemia, one had myeloid leukemia with maturation, and one had both pre-T LBL and erythroid leukemia. The malignant blasts from mice with pre-T LBL showed monoclonal T-cell receptor B gene rearrangements and were positive for CD3, 4, and 8. The mouse with megakaryocytic leukemia had serial CBCs documenting a platelet count of 3.2 million/uL, rising to >15million/uL at the time of death. This mouse had CD41+ megakaryocytes and megakaryoblasts invading the liver and spleen, and an osteosclerotic bone marrow reminiscent of chronic idiopathic myelofibrosis (CIMF). The mouse with concurrent pre-T LBL and erythroid leukemia had replacement of the thymus and infiltration of the lung with T-lymphoblasts which had a clonal TCRB gene rearrangement; interestingly, the spleen, liver, and bone marrow of this mouse were invaded with erythroblasts that were negative for CD3 and TCRB gene rearrangements. We conclude that the NHD13 transgene consistently induces an MDS, of variable severity, in these mice. Some mice die of severe anemia due to MDS, and MDS transforms into an acute non-lymphoid leukemia in other mice. Still other mice die of pre-T LBL which we believe evolves in the thymus separately from the MDS. These data demonstrate that the NHD13 fusion gene is transforming in both lymphoid and myeloid cells.

Flt3 Receptor Inhibition Abolishes Constitutive NFκB Activation in High-Risk Myelodysplastic Syndrome and Acute Myeloid Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2434-2434
Author(s):  
Jennifer Grosjean ◽  
Lionel Ades ◽  
Simone Bohrer ◽  
Pierre Fenaux ◽  
Guido Kroemer
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Plasma Membrane ◽  
High Risk ◽  
Myelodysplastic Syndrome ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Constitutive Activation ◽  
Nf Kappab ◽  
Acute Myeloid

Abstract High-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are characterized by the constitutive activation of the anti-apoptotic transcription factor NF-kappaB, via the activation of the IKK complex. We show that constitutive activation of the receptor tyrosine kinase Flt3 is responsible for IKK activation and this activation of the NF-kappaB pathway was found to involve a not yet described phosphorylation of the IKK and IkBa complex involving tyrosine residues compared to serine residues in the classical NF-kappaB pathway. Chemical inhibition or knockdown of Flt3 with small interfering RNAs abolished NF-kappaB activation in MDS and AML cell lines, as well as in primary CD34+ bone marrow cells from patients, causing mitochondrial apoptosis. Epistatic analysis involving the simultaneous inhibition of Flt3 and IKK indicated that both kinases act via the same anti-apoptotic pathway. An IKK2 mutant with a constitutive kinase activity and a plasma membrane-tethered mutant of NEMO that activates IKK1/2 prevented the cytocidal action of Flt3 inhibition. IKK2 and Flt3 physically associated in MDS and AML cells and Flt3 inhibition caused the release of IKK2 from a preferential association with the plasma membrane. Flt3 inhibition only killed CD34+ bone marrow cells from high-risk MDS and AML patients, in correlation with the blast numbers and the NF-kappaB activity, yet had no lethal effect on healthy CD34+ cells or cells from low-risk MDS. These results suggest that Flt3 inhibitors might exert an anti-neoplastic effect in high-risk MDS and AML through inhibition of constitutive NF kappaB activation.

Mesenchimal Stroma Cells From Patients with Myelodysplastic Syndrome and Acute Myeloid Leukemia Show Distinct Cytogenetic and DNA-Mutation Data as Compared with Bone Marrow Leukemic Blasts.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4697-4697
Author(s):  
Olga Blau ◽  
Wolf-Karsten Hofmann ◽  
Claudia D Baldus ◽  
Gundula Thiel ◽  
Florian Nolte ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Control Analysis ◽  
Npm1 Mutation ◽  
Dna Mutation ◽  
Healthy Donors ◽  
Acute Myeloid

Abstract Abstract 4697 Bone marrow mesenchymal stroma cells (BMSC) are key components of the hematopoietic microenvironment. BMSC from patients with acute myeloid leukemia (AML) and myelodisplasic syndrome (MDS) display functional and quantitative alterations. To gain insight into these questions, we carried out cytogenetic analyses, FISH, FLT3 and NPM1 mutation examinations of both hematopoietic (HC) and BMSC derived from 53 AML and 54 MDS patients and 35 healthy donors after in vitro culture expansion. Clonal chromosomal aberrations were detectable in BMSC of 12% of patients. Using FISH we have assume that cytogenetic markers in BMSC were always distinct as the aberrations in HC from the same individual. 17% and 12% of AML patients showed FLT3 and NPM1 mutations in HC, respectively. In BMSC, we could not detect mutations of NPM1 and FLT3, independent from the mutation status of HC. For control analysis, BMSC cultures from 35 healthy donors were prepared under the same conditions. BMSC from healthy donors did show normal diploid karyotypes and absence of specific DNA-mutations of NPM1 and FLT3. Our data indicate that BMSC from MDS and AML patients are not a part of malignant clone and characterized by genetic aberrations. Lack of aberrations as detected in HC and appearance of novel clonal rearrangements in BMSC may suggest enhanced genetic susceptibility and potential involvement of BMSC in the pathogenesis of MDS and AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Acute Myeloid Leukemia with Basophilic Differentiation Transformed from Myelodysplastic Syndrome

2017 ◽  
Vol 2017 ◽  
pp. 1-6 ◽  
Author(s):  
Yasuhiro Tanaka ◽  
Atsushi Tanaka ◽  
Akiko Hashimoto ◽  
Kumiko Hayashi ◽  
Isaku Shinzato
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myelodysplastic Syndrome ◽  
Myeloid Leukemia ◽  
Bone Marrow Failure ◽  
Immunosuppressive Treatment ◽  
Flow Cytometric Analysis ◽  
Chromosomal Analysis ◽  
Monosomy 7 ◽  
Acute Myeloid

Myelodysplastic syndrome (MDS) terminally transforms to acute myeloid leukemia (AML) or bone marrow failure syndrome, but acute myeloid leukemia with basophilic differentiation has been rarely reported. An 81-year-old man was referred to our department for further examination of intermittent fever and normocytic anemia during immunosuppressive treatment. Chromosomal analysis showed additional abnormalities involving chromosome 7. He was diagnosed as having MDS. At the time of diagnosis, basophils had not proliferated in the bone marrow. However, his anemia and thrombocytopenia rapidly worsened with the appearance of peripheral basophilia three months later. He was diagnosed as having AML with basophilic differentiation transformed from MDS. At that time, monosomy 7 was detected by chromosomal analysis. We found that basophils can be confirmed on the basis of the positivity for CD203c and CD294 by flow cytometric analysis. We also found by cytogenetic analysis that basophils were derived from myeloblasts. He refused any chemotherapy and became transfusion-dependent. He died nine months after the transformation. We should keep in mind that MDS could transform to AML with basophilic differentiation when peripheral basophilia in addition to myeloblasts develops in patients with MDS.

scholarly journals Development of Somatic NRAS Mutation Associated with Rapid Transition from Germline GATA2 Mutation Associated Myelodysplastic Syndrome to Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3616-3616 ◽  
Author(s):  
Yanqin Yang ◽  
Yubo Zhang ◽  
Jun Zhu ◽  
Catherine E. Lai ◽  
Jingrong Tang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Myelodysplastic Syndrome ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Bone Marrow Aspirate ◽  
Nras Mutation ◽  
Trisomy 8 ◽  
Acute Myeloid

Abstract There is increasing recognition of the role of inherited germline predisposition for myeloid disorders such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The additional somatic genetic events required for development of a malignant phenotype are however poorly understood. A 25 year old woman was referred to the NHLBI hematology branch in March 2014 for a seven year history of pancytopenia. Her medical history included recurrent pneumonias, oral ulcers, severe varicella infection and arthralgias. Prior bone marrow examinations at ages 21 and 23 at outside institutions reported normocellular marrow, tri-lineage hematopoiesis and mild dyspoiesis. Cytogenetics were remarkable for trisomy 8 in 80% (aged 21) or 90% (aged 23) of metaphases. Previously unrecognized lymphedema was noted on examination. Peripheral blood counts showed WBC 2.28 K/ul [normal range: 3.98-10.04], HGB 9.9 g/dL [11.2-15.7], PLT: 67 K/ul [173-369], ALC: 0.36 K/ul [1.18-3.74] and AMC: 0.06 [0.24-0.86]. Peripheral blood flow cytometry demonstrated decreased CD3+ CD4+ (T) cells, CD19+ (B) cells and NK cells. HLA-DR15 negative. Bone marrow examination showed trilineage hematopoiesis, 50-60% cellularity, mild erythroid predominance and mildly increased, mildly atypical megakaryocytes. Blasts less than 5%. Bone marrow flow cytometry revealed severely decreased B-cells and monocytes, absent B-cell precursors, absent dendritic cells, inverted CD4:CD8 ratio, and atypical myeloid maturation pattern. Cytogenetics demonstrated stable trisomy 8 in 90% of metaphases. On the basis of this assessment the diagnosis of MDS was confirmed. Sanger sequencing revealed a GATA2 L375S mutation in the second zinc finger of known pathogenic significance. Four months later she developed increased fatigue and easy bruising with worsening thrombocytopenia (PLT: 10K/ul). Bone marrow was dramatically changed; now markedly hypercellular (90-100%) with diffuse sheets of immature cells consistent with blasts having fine chromatin, distinct or prominent nucleoli, and visible cytoplasm. Blasts were positive for CD33, CD56, CD64, CD123, and CD163; and were negative for CD34, CD14, and myeloperoxidase. Cytogenetics showed a new trisomy 20 in 65% of metaphases, in addition to previously seen trisomy 8 in 100%. A diagnosis of acute monoblastic leukemia (M5a subtype) was made. At both clinic visits bone marrow aspirate was collected on an IRB approved research sample acquisition protocol. Whole exome sequencing of 1ug DNA was performed using Agilent SureSelect v5 Exome enrichment Kits on an Illumina HiSeq 2000 with 100-bp paired-end reads (Macrogen, Rockville, MD). Data was mapped to hg19 (BWA) and processed using an in-house pipeline (Samtools/Picard/GATK/VarScan/Annovar). Mean read depth of target regions was 157 and 149. There was high correlation between both samples with the exception of a NRAS:NM_002524:exon3:c.C181A:p.Q61K mutation (57 of 180 reads) seen only in the later sample. Confirmatory ultra-deep sequencing for NRAS was performed using Illumina TruSight Myeloid Sequencing Panel on an Illumina MiSeq. No evidence of the NRAS Q61K mutation was found in the earlier March MDS bone marrow sample even when sequenced to a depth greater than 1750 reads (see figure). The mutation was confirmed in the August AML sample at a variant allele frequency of 35%. If heterozygous this would reflect a clone size of 70%, consistent with data from both cytogenetics (new trisomy 20 in 65% of metaphases) and the 76% blasts documented by bone marrow aspirate smear differential. We report here the rapid progression to AML in a patient with germline GATA2 MDS associated with development of a new trisomy 20 karyotype and a NRAS Q61K mutation. The NRAS mutation was not detectable after the patient achieved a complete remission following induction chemotherapy further supporting this association. This NRAS mutation has been implicated in the pathogenesis of multiple cancers by constitutive activation of proliferative signaling. GATA2 associated MDS is a high-risk pre-leukemic condition with the potential for rapid evolution to AML. This is the first report of acquired somatic mutations in the RAS/RTK signaling pathway in the context of germline GATA2 insufficiency associated with acute leukemic transformation. Figure 1. Figure 1. Disclosures Townsley: Novartis: Research Funding; GSK: Research Funding.

Measurement of HSP90 and HSP70 in CD34-Positive Cells: Lower Levels in Myelodysplastic Syndrome Than in Acute Myeloid Leukemia and Correlation with Clinical Behavior.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3426-3426
Author(s):  
M. Gorre ◽  
I. Jilani ◽  
R. Chang ◽  
H. Chan ◽  
R. Urcia ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myelodysplastic Syndrome ◽  
Myeloid Leukemia ◽  
Binding Capacity ◽  
Rational Approach ◽  
Specific Cell ◽  
Cd34 Cells ◽  
Shock Proteins ◽  
Acute Myeloid

Abstract Heat shock proteins (HSPs) are molecular chaperones involved in binding and regulating the levels of several client cellular proteins. HSPs are induced by stress and play a role in the modulation of apoptosis and proliferation. Expression of HSPs by acute myeloid leukemia (AML) cells has been reported to correlate with more aggressive disease. Here we used a flow cytometry approach to quantify the expression of HSP90 and HSP70 in specific cell populations in bone marrow. Quantification using QuantiBRITE and PE (phycoerythrin)-labeled antibodies with a 1:1 ratio allowed us to specifically measure the antibody binding capacity in 100 CD34+ cells (molecules/100 CD34+ or CD3+ cells). Using this approach, we compared bone marrow samples from patients with myelodysplastic syndrome (MDS) and patients with AML. This approach can also be used to monitor patients treated with therapeutic agents that target HSPs, such as 17-allylamino-17-demethoxygeldamycin (17AAG). The percentage of CD34+ cells expressing HSP90 (P=0.008) and HSP70 (P<0.001) was significantly lower in MDS (n=20) than in AML (n=33) patients. Similarly, the percentage of CD3+ cells expressing HSP90 and HSP70 was significantly higher in MDS than in AML patients (P<0.01). This suggests that the environment in the bone marrow (cytokines, chemokines, other factors) may affect the levels of HSPs in neoplastic and normal cells in a similar fashion. In patients with MDS, higher levels of HSP90 were associated with shorter survival (P=0.03). However, after achieving remission (CR), MDS patients who expressed high levels of HSP90 had significantly longer remission duration (CRD) (P=0.03). These findings not only confirm that the environment and blasts in patients with MDS are different from those in patients with AML, but also suggest that therapy targeting HSPs may be a rational approach in patients with MDS. Figure Figure

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