Myelodysplastic Syndrome Stem Cells Research Articles

C002 C-type lectin-like molecule-1 (CLL-1) and lineage infidelity marker expression as assessed by flow cytometry distinguishes normal and aberrant stem cells in myelodysplastic syndromes

C002 C-type lectin-like molecule-1 (CLL-1) and lineage infidelity marker expression as assessed by flow cytometry distinguishes normal and aberrant stem cells in myelodysplastic syndromes

Expression of C-Type Lectin-Like Molecule-1 (CLL-1) on Stem Cells Might Discriminate De Novo Acute Myeloid Leukemia from Acute Myeloid Leukemia Originating from Myelodysplastic Syndromes

It is generally accepted that myelodysplastic syndromes (MDS) most often originate in a multipotent, myelorestricted progenitor population, although primary transformation may occur at the hematopoietic stem cell level. MDS can be classified into low risk and high risk with evolution to acute myeloid leukemia (AML) predominantly in the latter cases. In AML, survival of leukemia-initiating cells, often referred to as leukemic stem cells, after chemotherapy is thought to lead to minimal residual disease and relapse. Hence, in de novo AML a larger size of the stem cell compartment is predictive for poor survival. [Van Rhenen et al.,Clin Cancer Res 2005,11] The monoclonal antibody against the cell surface antigen C-type lectin-like molecule-1, CLL-1, together with lineage infidelity markers enables discrimination of normal and malignant stem cells. [Van Rhenen et al.,Blood 2007,110; Van Rhenen et al.,Leukemia 2007,21] It could be hypothesized that CLL-1 and aberrant marker expression on MDS stem cells together with size of the stem cell compartment may predict leukemic evolution. Therefore, stem cells, defined as CD45dimCD34+CD38−, were analyzed for expression of CLL-1 and aberrant lineage markers in bone marrow samples from 88 MDS patients classified by WHO as 16 RA w/o RS, 42 RCMD w/o RS, 3 MDS-U, 5 hypoplastic MDS, 6 MDS/MPD and CMML, 15 RAEB-1 and 2, 20 AML patients with a known MDS history and 26 healthy controls. Analysis of the CD34+CD38− frequency in all MDS patients and normal controls revealed no significant differences (median 0.0061% vs. 0.0074%, respectively), whereas the frequency of CD34+CD38− cells was 17-fold higher in high risk MDS (RAEB-1 and 2, median: 0.076%) as compared to low risk MDS (median: 0.0046%, p<0.001). Similar as in AML, stem cells were significantly more prevalent within the blast cell fraction (CD45dimSSCint/low) of high risk MDS as compared to low risk MDS (median 0.77% and 0.25%, respectively), reflecting the differences in clinical course in these patients (p=0.040). Regarding CLL-1 expression, a reliable number of stem cells (>20) could be tested in 11/15 high risk RAEB-1 and 2 cases and in 16/73 of the remaining low risk MDS cases. In these cases, median CLL-1 expression on the CD34+CD38− cells was 1.6% (range 0–50) in low risk and 2.0% (range 0–27) in high risk MDS. Median CLL-1 expression on stem cells was 0.0% (range 0–4.7) in normal controls. Nevertheless, expression of lineage infidelity markers, such as CD5, CD7 and CD56, on CD34+CD38− stem cells in MDS strongly suggests that a considerable part of these stem cells is malignant (median 35% in 7/16 patients tested). Our data show that CLL-1 is virtually absent on stem cells in MDS. Remarkably, median CLL-1 expression on stem cells in AML cases that evolved from MDS (7%, range 0–53, n=9) was manifold lower than in de novo AML (median 45% when excluding non de novo AML [Van Rhenen et al.,Blood 2007,110], p=0.034). Detailed analysis of CLL-1 expression in AML had already revealed that CLL-1 expression increases with differentiation (CD34− > CD34+CD38− > CD34+CD38+). [Bakker et al.,Cancer Res 2004,64;Van Rhenen et al.,Blood 2007,110] Thus, our data suggest that the CD34+CD38− cells in high risk MDS and AML with antecedent MDS are more immature than in most de novo AML, which might explain poor prognosis of AML cases with MDS history. To conclude, our data indicate that CLL-1 is a specific marker of de novo AML, while CLL-1-negative AML may have been evolved from a MDS pre-phase that is further characterized by an increasing size of the stem cell compartment upon progression towards AML.

Inappropriate notch activity and limited mesenchymal stem cell plasticity in the bone marrow of patients with myelodysplastic syndromes

Myelodysplastic syndromes (MDSs) are a heterogeneous group of hematological disorders characterized by ineffective hematopoiesis, enhanced bone marrow apoptosis and frequent progression to acute myeloid leukemia. Several recent studies suggested that, besides the abnormal development of stem cells, microenvironmental alterations are also present in the MDS bone marrow. In this study, we have examined the relative frequencies of stem and progenitor cell subsets of MDS and normal hematopoietic cells growing on stromal cell layers established from MDS patients and from normal donors. When hematopoietic cells from MDS patients were co-cultured with normal stromal cells, the frequency of either early or late cobblestone area-forming cells (CAFC) was significantly lower compared to the corresponding normal control values in 4 out of 8 patients. In the opposite situation, when normal hematopoietic cells were incubated on MDS stromal cells, the CAFC frequencies were decreased in 5 out of 6 patients, compared to normal stromal layer-containing control cultures. Moreover, a soluble Notch ligand (Jagged-1 protein) was an inhibitor of day-35-42 CAFC when normal hematopoietic cells were cultured with normal or MDS stromal cells, but was unable to inhibit MDS stem and early progenitor cell growth (day-35-42 CAFC) on pre-established stromal layers. These findings suggest that in early hematopoietic cells isolated from MDS patients the Notch signal transduction pathway is disrupted. Furthermore, there was a marked reduction in the plasticity of mesenchymal stem cells of MDS patients compared with those of normal marrow donors, in neurogenic and adipogenic differentiation ability and hematopoiesis supporting capacity in vitro. These results are consistent with the hypothesis that when alterations are present in the myelodysplastic stroma environment along with intrinsic changes in a hematopoietic stem/progenitor cell clone, both factors might equally contribute to the abnormal hematopoiesis in MDS.

NR2F6, the Mammalian Homologue of Drosophila Seven Up, Can Initiate Myelodysplasia and Acute Leukemia.

A central conundrum in the biology of myelodysplastic syndrome (MDS) is the observation that haematopoietic cells derived from the MDS clone have an increased disposition to apoptosis, yet gain clonal dominance in the bone marrow (BM). One explanation for this apparent paradox could be that MDS stem cells attain clonal dominance by acquiring an augmented probability of self-renewal. We therefore sought to identify novel genes associated with self-renewal that are overexpressed in both MDS and AML. By analyzing the growth of individual clonal siblings derived from low passage cultures of OCI-AML4 we found the expression of the orphan nuclear receptor NR2F6 to be four-fold higher in leukaemia cells with high proliferative capacity, compared to those which spontaneously lose proliferative ability. NR2F6 mRNA transcripts were more abundant in patients (n=37) with MDS, CMML, and AML compared to normal BM (n=16; p<0.0003 MDS, <0.03 CMML, <0.0009 AML), validating the clinical relevance of this gene and suggesting that deregulation of NR2F6 might be an early event in disease progression. We studied the effects of NR2F6 on differentiation and proliferation in vitro in the monoblastic cell line U937. Induction of differentiation of U937 cells resulted in a decrease in NR2F6 expression, while forced expression of NR2F6 reduced the doubling time of U937 cells, and inhibited induction of differentiation by retinoic acid. We next studied the effects of NR2F6 on the behaviour of primary haematopoietic cells. Overexpression of NR2F6 in murine bone marrow cells resulted in a significant reduction in the number of BFU-E and CFU-GM and the size of erythroid and myeloid colonies, consistent with the idea that NR2F6 inhibits maturation of normal BM. These results were corroborated by immunophenotyping of BM cultured in suspension, which showed that NR2F6 overexpression resulted in a significant reduction in mature monocytes and granulocytes. In addition, serial replating of haematopoietic colonies revealed greatly extended replating potential in NR2F6-overexpressing BM, consistent with augmented self-renewal capacity. Finally, we assessed the effects of NR2F6 on haematopoiesis in vivo by bone marrow transplantation. Competitive repopulation of lethally irradiated murine hosts with NR2F6-transduced bone marrow cells resulted in successful engraftment and augmented self-renewal as evidenced by increased engraftment in some recipients as well as increased colony formation in serial replating experiments. Recipients of NR2F6-transduced grafts had hypercellular BM, with abnormal localization of immature precursors (ALIP) and an increase in the percentage of blasts. Strikingly, ∼30% of recipients of secondary transplants of NR2F6-transduced bone marrow cells developed acute leukaemia, characterized by infiltration by blasts of bone marrow, peripheral blood, spleen and liver, and by haematopoietic failure. These data establish the importance of NR2F6 in the regulation of haematopoietic cell self-renewal and differentiation. This suggests that deregulated expression of NR2F6 is an important step in the pathogenesis of human MDS and AML and supports the hypothesis that acquisition of augmented self-renewal capacity helps the MDS stem cell gain clonal dominance. Furthermore, the NR2F6 transplant chimera system provides a mouse model of MDS, and the transformation of MDS to AML.

The molecular signature of MDS stem cells supports a stem-cell origin of 5q− myelodysplastic syndromes

AbstractGlobal gene expression profiling of highly purified 5q-deleted CD34+CD38−Thy1+ cells in 5q− myelodysplastic syndromes (MDSs) supported that they might originate from and outcompete normal CD34+CD38−Thy1+ hematopoietic stem cells. Few but distinct differences in gene expression distinguished MDS and normal stem cells. Expression of BMI1, encoding a critical regulator of self-renewal, was up-regulated in 5q− stem cells. Whereas multiple previous MDS genetic screens failed to identify altered expression of the gene encoding the myeloid transcription factor CEBPA, stage-specific and extensive down-regulation of CEBPA was specifically observed in MDS progenitors. These studies establish the importance of molecular characterization of distinct stages of cancer stem and progenitor cells to enhance the resolution of stage-specific dysregulated gene expression.

Reduced intensity conditioning in unrelated donor transplantation for refractory cytopenia in childhood

Myelodysplastic syndromes (MDS) are a heterogenous group of acquired hematopoietic stem cell disorders. Refractory cytopenia (RC) is the most common subtype of childhood MDS and hematopoietic stem cell transplantation (HSCT) is the only curative treatment. HSCT following a myeloablative preparative regimen is associated with a low probability of relapse and considerable transplant-related mortality. In the present European Working Groups of MDS pilot study, we investigated whether a reduced intensity conditioning regimen (RIC) is able to offer reduced toxicity without increased rates of graft failure or relapse. Nineteen children with RC were transplanted from an unrelated donor following RIC consisting of fludarabine, thiotepa and anti-thymocyte globulin. Three patients experienced graft failure. Neutrophil and platelet engraftment occurred at a median time of 23 and 30 days, respectively. Cumulative incidence of grade II-IV and grade III and IV acute graft-versus-host disease (GVHD) was 0.48 and 0.13, respectively; three patients developed extensive chronic GVHD. Although infections were the predominant complications, only one patient with extensive chronic GVHD died from infectious complications. Overall and event-free survival at 3 years were 0.84 and 0.74, respectively. In conclusion, our results were comparable to those of patients treated with myeloablative HSCT. Long-term follow-up is needed to demonstrate the expected reduction in long-term sequelae.

Current Treatment Options: Impact of Cytogenetics on the Course of Myelodysplasia

The heterogeneity of myelodysplastic syndromes (MDS) has driven the search for unifying biologic and clinical features that would stratify patients into distinct prognostic and therapeutic subgroups. Cytogenetics has been shown to impact the course of myelodysplasia. Despite the presence of non-random cytogenetic abnormalities in approximately 50% of MDS patients, it is significant that only a proportion of metaphases may contain the abnormality. Clonality studies however show that the karyotypically normal metaphases are still part of the MDS clone. This would suggest that the chromosomal abnormality may not be the initiating lesion in MDS, and that the gross karyotypic changes represent clonal evolution in a genetically unstable population. Yet, as will be described below, specific cytogenetic abnormalities are associated with clinically and biologically distinct forms of the disease, most notable in the response of del(5q) patients to lenalidomide. One possible explanation for the appearance of non-random mutational events could relate to the interaction of MDS cells with their microenvironment. Whatever the initiating lesion in the MDS stem cell, the end result is a clonal expansion where the marrow becomes populated by the monoclonal progeny of this cell. Interaction of these cells with a microenvironment which has been shown to be rich in pro-apoptotic cytokines such as tumor necrosis factor alpha (TNFa), leads to increased genetic instability. Hypoxia mediated decrease in DNA repair enzymes could further accelerate mutational events culminating in accumulation of multiple chromosomal abnormalities. Some of these chromosomal changes are associated with increased sensitivity to specific drugs. Lenalidomide has shown a high degree of efficacy in MDS patients with del(5q), although the target for the drug is unknown since a small but significant subset of MDS patients without del(5q) abnormality also respond to the drug. In contrast, the molecular target for imatinib mesylate is known; mutations in tyrosine kinase receptor family of genes found in patients with t(5;12) and del(4q12) make these individuals sensitive to the drug. Patients with isolated trisomy 8 have an immune component to the disease phenotype which can be targeted by cyclosporine and or anti-thymocyte globulin (ATG), especially in the presence of a PNH (paroxysmal nocturnal hemoglobinurea) clone. In the absence of these specific cytogenetic abnormalities described above, the two FDA approved hypomethylating agents 5 azacytidine and decitabine should be considered as therapeutic alternatives.

Avancées dans la prise en charge des syndromes myélodysplasiques

Objectives Myelodysplastic syndromes (MDS) are a group of clonal hematologic disorder, which combine ineffective hematopoiesis and evolution to acute myeloid leukemia. Significant progress has been made in the understanding of the disease pathogenesis, diagnostics and classification. Promising new agents and innovative therapeutic strategies are currently used. In this article we will review these achievements and their impact on the treatment of MDS. Current knowledge and key points The pathogenesis of MDS involves abnormalities of the MDS clone itself such as abnormal apoptosis, signalling or epigenetic regulation and abnormalities of the microenvironment such as immune deregulation or increased angiogenesis, which represent potential therapeutic targets. There is currently no standard treatment for MDS and allogeneic stem cell transplantation remains the only curative strategy. However, besides conventional chemotherapy and growth factors, new agents including hypomethylating agents, antiangiogenic drugs, immune modulatory agents have proved effective. Future prospects and projects The interesting results achieved with these new agents show that it is necessary to continue investigation in order to improve therapeutic strategies in MDS.

Mesenchymal stem cells in myelodysplastic syndromes: phenotypic and cytogenetic characterization

Bone marrow-derived mesenchymal stem cells (MSC) have been defined as primitive, undifferentiated cells, capable of self-renewal and with the ability to give rise to different cell lineages, including adipocytes, osteocytes, fibroblasts, chondrocytes, and myoblasts. MSC are key components of the hematopoietic microenvironment. Several studies, including some from our own group, suggest that important quantitative and functional alterations are present in the stroma of patients with myelodysplasia (MDS). However, in most of such studies the stroma has been analyzed as a complex network of different cell types and molecules, thus it has been difficult to identify and characterize the cell(s) type(s) that is (are) altered in MDS. In the present study, we have focused on the biological characterization of MSC from MDS. As a first approach, we have quantified their numbers in bone marrow, and have worked on their phenotypic (morphology and immunophenotype) and cytogenetic properties. MSC were obtained by a negative selection procedure and cultured in a MSC liquid culture medium. In terms of morphology, as well as the expression of certain cell markers, no differences were observed between MSC from MDS patients and those derived from normal marrow. In both cases, MSC expressed CD29, CD90, CD105 and Prolyl-4-hydroxylase; in contrast, they did not express CD14, CD34, CD68, or alkaline phosphatase. Interestingly, in five out of nine MDS patients, MSC developed in culture showed cytogenetic abnormalities, usually involving the loss of chromosomal material. All those five cases also showed cytogenetic abnormalities in their hematopoietic cells. Interestingly, in some cases there was a complete lack of overlap between the karyotypes of hematopoietic cells and MSC. To the best of our knowledge, the present study is the first in which a pure population of MSC from MDS patients is analyzed in terms of their whole karyotype and demonstrates that in a significant proportion of patients, MSC are cytogenetically abnormal. Although the reason of this is still unclear, such alterations may have an impact on the physiology of these cells. Further studies are needed to assess the functional integrity of MDS-derived MSC.

Bmi-1 Expression Predicts the Progression of Malignant Hematopoietic Stem Cells in Myelodysplastic Syndrome.

Bmi-1 is a member of the Polycomb group (PcG) of transcriptional repressor genes, which could have an essential role in embryogenesis and be expressed restrictedly in the stem cells and proliferating cells. Some studies have recently reported that Bmi-1 is required to regulate the adult self-renewing hematopoietic stem cells. Others have shown that overexpressed Bmi-1 gene could cause the neoplastic proliferation of cells. A series of evidence on Bmi-1 suggests that it might be closely associated with the progression of the hematopoietic malignancies. Myelodysplastic syndrome (MDS) is a clonal bone marrow disorder characterized by aberrant hematopoiesis susceptible to acute leukemia. This category is quite heterogeneous with a variable clinical course and prognosis. Therefore, it is difficult to decide when to initiate therapies because some patients survive for years without treatment whereas others come to die in rapid progress in overt leukemia. Thus, we investigated whether the expression of Bmi-1 protein in CD34+ cells could be correlated with disease progression of MDS. Primary cells from patients with RA, RAEB, and MDS-AML, were stained with anti-CD34 antibody-PE and then fixed in PFA followed by staining with anti-Bmi-1-antibody-FITC. These cells were subjected to flow cytometric analysis. Bmi-1 expression in CD34+ cells was preferentially seen in RAEB and MDS-AML compared with RA (13.86%±6.40% in RA(n=4), 63.05%±8.19% in RAEB(n=6), 73.99%±33.40% in MDS-AML(n=6)). Patients were dead with >70% at Bmi-1 expression level in CD34+. Moreover, two patients with RA at >10% of Bmi-1 positivity made disease progression to RAEB. One patient with RAEB at <10% at the Bmi-1 expression in CD34+ is still alive. Furthermore, we are dissecting gene profiling of cells from different types of MDS. These results show that the expression of Bmi-1 can distinguish the proliferating malignant stem cells from resting cells by using flow cytometry. Alternatively, IPSS in MDS patients is closely correlated with Bmi-1 expression of CD34+ cells. It may lead to the prediction of prognosis in patients with MDS in quality. Taken together, Bmi-1 expression could predict the disease progression and prognosis of MDS. Finally, Bmi-1 gene might be a target to control leukemogenesis by immunotherapy or gene therapy.

High-Dose of Epoietin Alfa in Patients with Low-Risk Myelodysplastic Syndromes (MDS): A Single Institution Experience.

Several studies showed that the response rate to standard dose of recombinant Human Erythropoietin (rHuEPO) in MDS patients is generally low, with only few cases presenting a significant increase of haemoglobin (Hb) levels. So, currently, the interest has focused on the use of high dose rHuEPO. The rationale for using high dose rHuEPO was elucidated: residual normal stem cells and/or abnormal clone of MDS stem cells, unresponsive to low levels of endogenous EPO, might respond to high doses of rHuEPO. The aim of this study was to assess the efficacy and safety of high dose rHuEPO treatment. EPO alfa 40.000 IU was given subcutaneously twice weekly for 4 weeks. Twenty-five patients with low-risk MDS (17 RA and 8 RARS) and Hb levels ≤ 10 g/dL were included in this study; sixteen patients were female and 9 male; mean age of enrolled patients was 74 years (range 66 – 85). Twenty-two of 25 patients completed the scheduled treatment and were evaluated for response. At 4 weeks eighteen of 22 patients (81%) showed a Hb mayor response (Hb increase ≥ 2 g/dL); Hb mean value at baseline was 8,15 g/dL (range 7 – 10), at 4 weeks was 13,15 g/dL (range 10 – 14,6). In 4 of 22 patients (19%) the high dose rHuEPO did not induce an increase of Hb levels after 4 weeks of treatment; in addition, these patients needed of RBC transfusions to maintain Hb levels ≥ 8 g/dL. The failure of treatment with rHuEPO occurred in patients with diagnosis of RARS. In our study there were no statistically significant differences between the group of patients with erythroid hyperplasia and the group of patients with normal percent of bone marrow erythroid cells (P = 0,4); no significant difference was noted in response rates between patients with RBC pre-treatment transfusion need and those with stable anaemia without prior transfusion (P = 0,09). In our study, Hb mayor response occurred also in one patient with marked marrow fibrosis. In this study all patients presented defective endogenous EPO production related to their degree of anaemia, with serum EPO levels ≤ 100 mU/ml (mean value 43,5; range 6 – 98). The responder patients need continuous maintenance treatment to maintain their response; EPO alfa 40.000 IU was given subcutaneously once a week; at 12 weeks overall response rate was 77%: 13/18 patients maintained their mayor response, 4/18 patients showed decreased Hb levels in comparison to initial response (Hb decrease > 1 < 2 g/dL), 1 patient progressed on RAEB. Hb mean value at 12 weeks was 11,8 g/dL (range 9,2 – 13,5). The median duration of maintenance of the erythroid response was 7,5 months (range 2 – 24 months). Treatment with high dose of rHuEPO is well tolerated; only one adverse event of arterial hypertension of moderate severity was reported as possible episode related to treatment. In conclusion, our study shows that, in low-risk MDS patients with defective endogenous EPO production, EPO alfa 40.000 IU, given subcutaneous twice weekly for 4 weeks, induces rapid, significant and persistent increase of Hb, without important adverse events; continuous maintenance treatment with 40.000 IU/w is necessary for the majority of the responding patients to maintain their response.

Involvement and functional impairment of the CD34+CD38−Thy-1+ hematopoietic stem cell pool in myelodysplastic syndromes with trisomy 8

AbstractClonality studies of mature cells suggest that the primary transformation event in myelodysplastic syndrome (MDS) most frequently occurs in a myeloid-restricted progenitor, a hypothesis supported by recent studies of purified CD34+Thy1+hematopoietic stem cells (HSCs) in cases with trisomy 8 (+8). In contrast, we recently demonstrated that a lymphomyeloid HSC is the target for transformation in MDS cases with del(5q), potentially reflecting heterogeneity within MDS. However, since +8 is known to frequently be a late event in the MDS transformation process, it remained a possibility that CD34+CD38−Thy1+ HSC disomic for chromosome 8 might be part of the MDS clone. In the present studies, although a variable fraction of CD34+CD38−Thy1+ cells were disomic for chromosome 8, they did not possess normal HSC activity in long-term cultures and nonobese diabetic–severe combined immunodeficiency (NOD-SCID) mice. Mixing experiments with normal CD34+CD38− cells suggested that this HSC deficiency was intrinsic and not mediated by indirect mechanisms. Furthermore, investigation of 4 MDS cases with combined del(5q) and +8 demonstrated that the +8 aberration was always secondary to del(5q). Whereas del(5q) invariably occurs in CD34+CD38−Thy-1+ HSCs, the secondary +8 event might frequently arise in progeny of MDS HSCs. Thus, CD34+CD38−Thy1+ HSCs are invariably part of the MDS clone also in +8 patients, and little HSC activity can be recovered from the CD34+ CD38−Thy1+ HSC. Finally, in advanced cases of MDS, the MDS reconstituting activity is exclusively derived from the minor CD34+CD38−HSC population, demonstrating that MDS stem cells have a similar phenotype as normal HSCs, potentially complicating the development of autologous transplantation for MDS.

Cytogenetic analysis of CD34+ subpopulations in AML and MDS characterized by the expression of CD38 and CD117.

It has been supposed in de novo AML that malignant transformation occurs at the level of committed progenitors. Recent data of our group and others provide evidence that in AML malignant transformation may regularly occur at the level of stem cells. These cells can be discriminated by function and specific surface molecules. CD34, a glycophosphoprotein, is a cellular surface antigen characteristically expressed by stem cells. CD34+ stem cells can be further subdivided by the expression of additional surface molecules like CD38 and CD117. In this article we present results from cytogenetic examinations of FACS-isolated stem cell subpopulations in eight patients (four AML and four MDS). Six of them displayed clonal karyotype abnormalities at the time of first diagnoses in the native bone marrow (5q-; 5q- and complex abnormalities; +8; inv(16) and +8; i(17q) and -21; i(21q)). We used CD117, the receptor for the stem cell factor (also KIT oncogene) as a new cellular surface marker. CD34+/CD117+/- stem cell subpopulations were examined in two patients with AML and three patients with MDS. We found leukemic stem cells in every type of stem cell subpopulation examined (CD34+/CD38-, CD34+/CD38+, CD34+/CD117-, CD34+/CD117+). Secondary, progression-associated chromosome abnormalities likewise were demonstrable in CD34+ cells. In three patients a mosaic of normal and abnormal metaphases was found in the highly purified stem cell subpopulations. We conclude that in AML and MDS stem cells are the target of leukemogenic genetic defects. CD117 as a new marker to isolate different CD34+ subpopulations was not sufficient to discriminate between normal and leukemic stem cells. Our findings have implications for autologous stem cell transplantation, high-dose chemotherapy and the pathogenetic concept of leukemogenesis.

A novel factor‐dependent human myelodysplastic cell line, MDS92, contains haemopoietic cells of several lineages

A novel long-term cultured interleukin (IL)-3-dependent human myelodysplastic cell line, MDS92, was shown to contain several myeloid-lineage cells such as neutrophils, macrophages, eosinophils, and a small number of megakaryocyte-lineage cells. Therefore this cell line possesses at least bipotential characteristics of myeloid- and megakaryocyte-lineages. Granulocyte colony-stimulating factor clearly promoted the neutrophil alkaline phosphatase activity of MDS92 cells. To the contrary, the incidence and growth of CD41-positive cells were hardly affected by the addition of IL-6, IL-11, c-mpl ligand (thrombopoietin, TPO) or erythropoietin. TPO slightly supported the growth of CD34-positive cell fraction, but not CD41-positive cell fraction of MDS92 cells in combination with IL-3 or Steel factor. This cell line will be a useful tool for the study of MDS stem cells, but the mechanism of commitment of differentiation in MDS stem cells remains unknown.

Bone marrow cellularity in myeloid stem cell disorders: Impact of age correction

We have reviewed the initial diagnostic bone marrow aspirate and biopsy specimens performed on the same date on 92 patients with acute myeloid leukaemia (AML), 100 patients with myelodysplastic syndrome (MDS), 24 patients with chronic granulocytic leukaemia (CGL), 19 patients with polycythemia vera (PV) and essential thrombocythemia (ET). An excellent assessment of cellularity by aspirate and biopsy was found. The estimation of BM cellularity for each group was utilized with and without age adjustment based on normal marrow biopsies. Without correcting the BM cellularity for age it was observed that the median BM cellularity was 50% in AML, CGL, PV and ET. In contrast, the median BM cellularity was estimated at 40% for MDS. In the age group 70 years and beyond the median BM cellularity was not changed in CGL, PV and ET, and only slightly decreased (35%) in MDS. However, a trend from hypercellularity to normocellularity was observed in patients with AML in this age group. By utilizing anatomic comparisons with normal age the corrected data disclosed that all patients with CGL, PV and ET, 63% of patients with AML and only 35% of patients with MDS had hypercellular BM according to their age, while only two patients with AML and seven patients with MDS were found to be truly hypocellular by age. The optimal cut-off value for definition of hypocellular AML and hypocellular MDS, and differences between MDS and other myeloid stem cell disorders in terms of BM cellularity have been discussed.

Variable growth in vitro of chromosomally different stem cells in myelodysplastic syndromes

Ten patients with myelodysplastic syndromes were investigated using in vitro colony assay of bone marrow cells and chromosome analysis of single colonies. The result was compared with conventional cytogenetic analysis of bone marrow cells. The chromosome abnormalities included were 5q−, +8, −7, 11q−, −Y and one complex karyotype. Erythroid colony formation was reduced in eight patients, while the number of granulocyte-macrophage colony-forming units was normal or increased. Cytogenetic examination of single colonies showed that both chromosomally normal and abnormal stem cells had colony forming ability. The proportion of cytogenetically abnormal colonies varied between 25 and 100% of analysed colonies. No further clones than those found in direct cytogenetic analysis were revealed after in vitro growth. One patient with a 5q− abnormality and one patient with a −7 abnormality showed a significantly lower proportion of cytogenetically abnormal colonies than the proportion of abnormal cells in the direct bone marrow chromosome preparation. One patient with a +8 abnormality showed a growth advantage of the +8 clone in comparison with the chromosomally normal clones, but this was not statistically significant. In two patients a chromosomally changed stem cell gave rise to both erythroid and myeloid colonies. The FAB-class did not seem to influence the growth of either chromosomally normal or abnormal colonies.

Convulsions during Anaesthesia: Atropine in Childhood

Abstract Comprehensive preclinical studies of Myelodysplastic Syndromes (MDS) have been elusive due to limited ability of MDS stem cells to engraft current immunodeficient murine hosts. We developed a novel MDS patient-derived xenotransplantation model in cytokine-humanized immunodeficient mice that for the first time provides efficient and faithful disease representation across all MDS subtypes. MISTRG MDS patient-derived xenografts (PDX) reproduce patients9 dysplastic morphology with multi-lineage representation, including erythro- and megakaryopoiesis. MISTRG MDS-PDX replicate the original sample9s genetic complexity and can be propagated via serial transplantation. MISTRG MDS-PDX demonstrate the cytotoxic and differentiation potential of targeted therapeutics providing superior readouts of drug mechanism of action and therapeutic efficacy. Physiologic humanization of the hematopoietic stem cell niche proves critical to MDS stem cell propagation and function in vivo. The MISTRG MDS-PDX model opens novel avenues of research and long-awaited opportunities in MDS research.