Inherited thrombocytopenia and platelet disorders with germline predisposition to myeloid neoplasia

Advances in molecular genetic sequencing techniques have contributed to the elucidation of previously unknown germline mutations responsible for inherited thrombocytopenia (IT). Regardless of age of presentation and severity of symptoms related to thrombocytopenia and/or platelet dysfunction, a subset of patients with IT are at increased risk of developing myeloid neoplasms during their life time, particularly those with germline autosomal dominant mutations in RUNX1, ANKRD26, and ETV6. Patients may present with isolated thrombocytopenia and megakaryocytic dysmorphia or atypia on baseline bone marrow evaluation, without constituting myelodysplasia (MDS). Bone marrow features may overlap with idiopathic thrombocytopenic purpura (ITP) or sporadic MDS leading to misdiagnosis. Progression to myelodysplastic syndrome/ acute myeloid leukemia (MDS/AML) may be accompanied by progressive bi‐ or pancytopenia, multilineage dysplasia, increased blasts, cytogenetic abnormalities, acquisition of bi‐allelic mutations in the underlying gene with germline mutation, or additional somatic mutations in genes associated with myeloid malignancy. A subset of patients may present with MDS/AML at a young age, underscoring the growing concern for evaluating young patients with MDS/AML for germline mutations predisposing to myeloid neoplasm. Early recognition of germline mutation and predisposition to myeloid malignancy permits appropriate treatment, adequate monitoring for disease progression, proper donor selection for hematopoietic stem cell transplantation, as well as genetic counseling of the affected patients and their family members. Herein, we describe the clinical and diagnostic features of IT with germline mutations predisposing to myeloid neoplasms focusing on mutations involving RUNX1, ANKRD26, and ETV6.     

Myelokathexis, a congenital disorder of severe neutropenia characterized by accelerated apoptosis and defective expression of bcl-x in neutrophil precursors

Myelokathexis is a congenital disorder that causes severe chronic leukopenia and neutropenia. Characteristic findings include degenerative changes and hypersegmentation of mature neutrophils and hyperplasia of bone marrow myeloid cells. The associated neutropenia can be partially corrected by treatment with granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF). These features led us to propose that accelerated apoptosis of neutrophil precursors might account for the neutropenic phenotype. Blood and bone marrow aspirates were obtained from 4 patients (2 unrelated families) with myelokathexis before G-CSF therapy and from 2 of the affected persons after G-CSF therapy (1 microg/kg per day subcutaneously for 3 weeks). Bone marrow was fractionated using immunomagnetic bead cell sorting into CD34(+), CD33(+)/CD34(-), and CD15(+)/CD34(-)/CD33(- )cell populations. Examination of these cells by flow cytometry and electron microscopy revealed abundant apoptosis in the CD15(+) neutrophil precursor population, characterized by enhanced annexin-V binding, extensive membrane blebbing, condensation of heterochromatin, and cell fragmentation. Colony-forming assays demonstrated significant reduction in a proportion of bone marrow myeloid-committed progenitor cells. Immunohistochemical analysis revealed a selective decrease in bcl-x, but not bcl-2, expression in the CD15(+)/CD34(-)/CD33(-)cell population compared with similar subpopulations of control bone marrow-derived myeloid precursors. After G-CSF therapy, apoptotic features of patients' bone marrow cells were substantially reduced, and the absolute neutrophil counts (ANC) and expression of bcl-x in CD15(+)/CD34(-)/CD33(-)cells increased. The authors concluded that myelokathexis is a disease characterized by the accelerated apoptosis of granulocytes and the depressed expression of bcl-x in bone marrow-derived granulocyte precursor cells. These abnormalities are partially corrected by the in vivo administration of G-CSF. (Blood. 2000;95:320-327)     

Impaired neutrophil maturation in truncated murine G-CSF receptor-transgenic mice

Severe congenital neutropenia (SCN) is a hematopoietic disorder characterized by neutropenia in peripheral blood and maturation arrest of neutrophil precursors in bone marrow. Patients with SCN may evolve to have myelodysplastic syndrome or acute myelocytic leukemia. In approximately 20% of SCN cases, a truncation mutation is found in the cytoplasmic region of the granulocyte colony-stimulating factor receptor (G-CSFR). We then generated mice carrying murine wild-type G-CSFR and its mutants equivalent to truncations at amino acids 718 and 731 in human G-CSFR, those were reported to be related to leukemic transformation of SCN. Although numbers of peripheral white blood cells, red blood cells, and platelets did not differ among mutant and wild-type G-CSFR transgenic (Tg) mice, both of the mutant receptor Tg mice had one third of peripheral neutrophil cell counts compared with wild-type receptor Tg mice. The mutant receptor Tg mice also showed impaired resistance to the infection with Staphylococcus aureus. Moreover, bone marrow of these Tg mice had an increased percentage of immature myeloid cells, a feature of SCN. This maturation arrest was also observed in in vitro cultures of bone marrow cells of truncated G-CSFR Tg mice under G-CSF stimulation. In addition, clonal culture of bone marrow cells of the truncated G-CSFR Tg mice showed the hypersensitivity to G-CSF in myeloid progenitors. Our Tg mice may be useful in the analysis of the role of truncated G-CSFR in SCN pathobiology.     

Effect of hypertransfusion on bone marrow regeneration in sublethally irradiated mice. I. enhanced granulopoietic recovery

Hypertransfusion can enhance recovery from neutropenia in certain clinical and experimental situations. We have studied the pattern of myeloid recovery in mice hypertransfused after receiving 350 rads whole body irradiation. Both hypertransfused and control groups showed the degenerative phase, abortive rise, and regenerative phase that has been described following sublethal irradiation. The blood granulocyte counts in the hypertransfused group returned to normal more rapidly and were maintained at a significantly higher level during the regenerative phase. This difference is not the result of a shift in granulocytes from the marrow granulocyte reserve or marginal granulocyte pool to the circulating pool, but is associated with significantly enhanced bone marrow granulopoiesis. While the total bone marrow cellularity of the hypertransfused mice is less than that of the control mice, the hypertransfused group contains more CFU-GM and myeloid cells during the regenerative phase. The enhanced granulopoiesis is not due to increased colony-stimulating activity (CSA) levels in the hypertransfused mice, as the CSA levels were significantly lower in this group compared to the controls prior to and during the initial phase of granulopoietic recovery. This study suggests that hypertransfusion increases the rate of recovery of myelopoiesis by increasing the number of precursors available for myeloid differentiation from an earlier stem cell compartment.     

The severity of immune neutropenia correlates with the maturational specificity of antineutrophil antibodies

Sera from 15 patients with antineutrophil antibodies against peripheral blood neutrophils were incubated with normal bone marrow, and the reaction with marrow precursors was determined. Serum from controls did not deposit IgG on either peripheral blood or marrow myeloid cells. Serum from patients with moderate immune neutropenia with myeloid hyperplasia deposited IgG on neutrophils, bands and metamyelocytes, but not on earlier precursors. Serum from one patient with severe neutropenia and myeloid hypoplasia deposited IgG on myelocytes and promyelocytes as well as more mature cells. Serum from a patient with antineutrophil antibodies which impaired chemotaxis but who did not have neutropenia deposited IgG only on neutrophils and occasional bands. Immune neutropenia may become increasingly severe as antibodies become directed against earlier precursors.     

Hereditary myeloid malignancies

Myelodysplastic syndromes and acute myeloid leukemia are sporadic for the majority of cases affecting the elderly population. Inherited cases, however, do occur. Genetic predispositions to myeloid malignancies can be classified into three categories: familial cancer syndromes associated with increased risk of various malignancies including myelodysplasia and acute myeloid leukemia such as Li-Fraumeni syndrome and constitutional mismatch repair deficiency (CMMRD); germline mutations conferring a specific increased risk of myelodysplastic syndrome and acute myeloid leukemia such as mutations in ANKRD26, CEBPA, DDX41, ETV6, GATA2, RUNX1, SRP72 genes; and finally primarily pediatric inherited bone marrow failure syndromes such as Fanconi anemia, dyskeratosis congenita, severe congenital neutropenia, Shwachman-Diamond syndrome and Diamond Blackfan anemia. The recognition of these germline syndromes is essential in the management and follow-up of patients. Herein, we review the conditions associated with hereditary myeloid leukemia with a special clinical focus on management and monitoring.     

Ambient air particulates stimulate alveolar macrophages of smokers to promote differentiation of myeloid precursor cells.

Studies from our laboratory have shown that exposure to air pollution particles smaller than 10 microm (PM10) induced a systemic inflammatory response that includes the release of granulocytes from the bone marrow. In the present study we tested the hypothesis that mediators released from human alveolar macrophages (AM) exposed to PM10 accelerate the maturation of granulocyte precursors. Human myeloid precursor cells (HL60 cells) were incubated with the supernatant from AM exposed to PM10. Phagocytosis of PM10 by AM resulted in the production of cytokines, particularly interleukin-6 (IL-6) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (P < .05). The supernatant from AM exposed to PM10 did not influence myeloid cell proliferation but promoted cell differentiation as measured by surface GD11b and CD14 expressions compared to control supernatant (P < .05). This effect of exposed-AM supernatants on myeloid cell differentiation was blocked by anti-IL-6 monoclonal antibodies (CD11b and CD14; P < .05) and anti-GM-CSF monoclonal antibodies (CD14, P < .01). We conclude that human AM exposed to PM10 produce mediators, particularly IL-6 and GM-CSF that promote the differentiation of bone marrow myeloid cells and we speculate that these cytokines are involved in the release of granulocytes from the bone marrow associated with exposure to air pollution particulates.     

Chronic neutropenia of childhood: frequent association with parvovirus infection and correlations with bone marrow culture studies

Summary. Children with neutropenia of more than 3 months duration often have evidence of immune-mediated destruction of mature neutrophils and variable abnormalities of myeloid precursors in their bone marrow. These patients often have anti-neutrophil antibodies which persist for several months. To further investigate the aetiology of neutropenia in such patients, bone marrow cells were evaluated for the presence of common viruses. Fifteen of 19 patients tested had evidence for parvovirus infection by PCR amplification of bone marrow DNA with parvovirus specific primers. Of these 15, six also had serologic evidence of parvovirus infection. Anti-neutrophil antibodies were identified in nine of 12 patients with parvovirus infection. Bone marrow culture studies done on six patients revealed varying degrees of myeloid and erythroid inhibition by patient plasma. These studies indicate that parvovirus may be a common cause of immune-mediated neutropenia in children.     

Cell Kinetics in Human Cyclic Neutropenia

Cell kinetics have been studied at several periods in a patient with typical cyclic neutropenia. Peripheral blood granulocyte labelling showed an increased destruction in the preleucopenic phase. Bone marrow colony-forming cells and blood leucocyte colony forming activity were normal or above the normal range. The major abnormalities were found in sequential studies of bone marrow proliferation measured by ‘in vitro’ ³H-thymidine flash labelling. A fall in the labelling index of promyelocytes was observed in the second part of the cycle. Incubation of normal human bone marrow cells with patient's granulocytes showed a marked decrease in ³H-thymidine incorporation, compared with incubation with the same number of normal granulocytes. Human cyclic neutropenia seems to be due to a factor secreted by abnormal polymorphonuclears inhibiting myeloid proliferation.     

G-CSF stimulated donor granulocyte collections for prophylaxis and therapy of neutropenic sepsis

Background: The administration of granulocyte colony-stimulating factor (G-CSF) increases the granulocyte count in normal donors and enables the collection of large numbers of mature myeloid cells by leukapheresis. This has potential value in the treatment of sepsis unresponsive to antibiotics in patients with severe neutropenia. Aim: To evaluate the tolerability of granulocyte collections in normal donors receiving G-CSF, the optimal method of collection and the clinical factors influencing the efficacy of granulocyte infusions. Methods: Analysis of the outcome of 55 granulocyte collections from 26 donors for progressive bacterial or fungal sepsis in neutropenic patients (n-8) or as prophylaxis in patients with recent fungal infections undergoing allogeneic bone marrow transplantation (BMT) (n=3). Results: G-CSF was well tolerated in most donors. Fatigue occurred commonly after the second collection. The median WCC per 200–220 mL bag was 351X10⁹7L. Collections were optimised with the use of a sedimenting agent (dextran) and a deepened interface setting on the cell separator. There was only a weak correlation between the number of granulocytes infused and the increment in the patient, but levels were usually maintained 0.5X 10⁹7L for the next 24 hours. The infusions were successful in three septic patients without multi-organ dysfunction and prophylactically, in two patients with localised fungal infections undergoing MBT The infusions were not beneficial in patients with septicaemia and established organ dysfunction or with extensive pulmonary aspergillosis. Conclusions: G-CSF mobilised granulocyte collections are feasible and the preliminary evidence suggests that the infusion of these cells may be useful early in the prophylaxis or treatment of severe neutropenic sepsis.     

Chronic idiopathic neutropenia: Granulopoietic assessment by both marrow culture and granulocyte kinetics

Granulopoietic assessment was made in 14 patients with chronic idiopathic neutropenia (CIN) whose neutrophils were consistently less than 1.5 × 10⁹/ liter and in whom there was absence of splenomegaly and recent drug ingestion. Granulopoiesis was studied using a combination of bone marrow culture in nutrient agar and granulocyte kinetics. Agar colony growth assessed bone marrow concentration of granulocyte progenitor cells (GPC) and the proportion of GPC in DNA synthesis by in vitro ³HTdR suicide. Granulocyte kinetics with in vitro DF³²P labelling of patient granulocytes measured granulocyte half-life (T^1/2), turnover rate (GTR), and the circulating, marginated, and total blood granulocyte pools. The results indicated that either GPC concentration or the proportion of GPC in DNA synthesis was outside the normal range in all but one patient and decreased in ten out of 14 patients. CIN was also characterized by reduced total, circulating, and marginated blood granulocyte pools, reduced GTR, and normal granulocyte half-life. The neutropenia appeared to be due to a variety of intra-marrow causes, including either a reduction in the GPC compartment, a reduction in GPC proliferation, a maturation arrest, or a reduced amplication during granulopoiesis. Increased granulocyte utilization, intra-vascular destruction or excessive margination could be excluded as possible causes of CIN in this series. Although GPC parameters tended to be reduced, suggesting a production defect, there were signs in nine patients that the bone marrow was attempting to compensate for the peripheral neutropenia. It is suggested that for a complete assessment of granulopoiesis in man, granulocyte kinetic studies need to be combined with quantitative studies of the bone marrow granulocyte progenitor compartment using the agar colony system.     

G-CSF enhances the proliferation and mobilization, but not the maturation rate, of murine myeloid cells

Objectives: Whether G‐CSF enhances the maturation of neutrophilic granulocytes or just accelerates the mobilization of mature and maturing granulocytes from bone marrow to blood, or both, is not clear. Using an in vivo culture system where such mobilization cannot take place, we previously showed that G‐CSF did not accelerate maturation. To further clarify the role of G‐CSF, we now have examined its effect on murine granulopoiesis in situ. Methods: Murine bone marrow precursors in S‐phase were labeled with BrdU, and hematopoiesis stimulated by the long‐acting G‐CSF compound pegfilgrastim (peg‐G‐CSF). Performing flow cytometric analysis of incorporated BrdU and the granulocyte maturation antigen Gr1, we investigated the cell flux from the proliferative to the non‐proliferative granulocyte compartments in bone marrow and further from bone marrow to blood. Results: Peg‐G‐CSF mobilized neutrophils from bone marrow to blood and markedly increased their concentration in blood for several days. It also increased the proliferation of precursor cells. Newly produced, less mature granulocytes (Gr1⁺BrdU⁺) travelled faster to blood in treated mice than in controls. The flow cytometric and cell density analyses of the bone marrow cells showed that peg‐G‐CSF skewed the population toward less mature cells, mainly because of the mobilization of granulocytes to blood. Conclusions: Collectively, our data do not support the notion that G‐CSF accelerates murine granulocyte maturation per se.     

Studies on the Proliferative Capacity of the In Vitro Colony Forming Cell in Normal Human Bone Marrowt†

Summary: The agar colony system which assays a committed myeloid stem cell compartment in murine marrow has been applied to the study of human bone marrow. The linear relationship between the number of nucleated marrow cells plated and the number of colonies obtained, together with the number and type of cells in the colonies is explored. The colony forming cell (CFC) in normal human bone marrow, when its cell cycle characteristics are explored in vitro using radioactive thymidine, has been shown to arise from a precursor compartment which is actively proliferating. On this basis there is a similarity with the murine system and it is postulated that the CFC in man monitors the committed myeloid stem cell compartment. Normal values have been established for the concentration of CFC and for thymidine kill in normal human bone marrow cell suspensions under our conditions of growth. The value of the system for the quantitation of granulopoiesis at the myeloid stem cell level in man is emphasised and its possible role in the study of neutropenia is discussed.     

In Vitro Induction of Granulocyte Differentiation in Hematopoietic Cells from Leukemic and Non-Leukemic Patients

Human spleen-conditioned medium can induce the formation in vitro of large granulocyte colonies from normal human bone marrow cells. The granulocyte colonies contained cells in various stages of differentiation, from myeloblasts to mature neutrophile granulocytes. Human spleen-conditioned medium also induced colony formation with rodent bone-marrow cells, whereas rodent spleen-conditioned medium induced colony formation with rodent bone marrow but not with human cells.This in vitro system has been used to determine the potentialities for cell differentiation in bone-marrow and peripheral blood cells from patients with a block in granulocyte differentiation in vivo. The cloning efficiency, colony size, and number of mature granulocytes in bone-marrow colonies from patients with congential neutropenia, whose bone marrow contained only 1% mature granulocytes, were not less than in people whose bone marrow had the normal level of about 40% mature granulocytes. The cloning efficiency of peripheral blood cells from patients with acute myeloid leukemia was 350 times higher, with 10 times larger colonies, than the cloning efficiency of peripheral blood cells from normal people. The cytochemical properties and number of mature granulocytes in colonies from the leukemic patients were the same as in colonies from non-leukemic people.The results indicate that a block in cell differentiation in vivo, in these cases with neutropenia and acute myeloid leukemia, was overcome in vitro, in the presence of an inducer in the conditioned medium. In patients with chronic myeloid leukemia, colony formation was induced only in some of the cases. This indicates that there are blast cells with different potentialities for the development of colonies in different patients.     

Investigating severe neutropenia with a simple bone marrow immunofluorescence test

Severely neutropenic patients who are suspected of having circulating autoantibodies to neutrophils and/or myeloid precursors present a diagnostic problem. This report describes the use of a simple bone marrow immunofluorescence test (BMIFT) to demonstrate autoantibodies reactive with the patient's own bands, neutrophils and myeloid precursors represented on fresh-frozen bone marrow aspirate smears. Positive results were seen in two of 13 evaluable marrows from children with primary neutropenia and in eight of 44 evaluable marrows from adults with primary or secondary neutropenia. The BMIFT is simple enough to perform in most laboratory environments and provides serological information to support a diagnosis of immune neutropenia when other means of investigation are precluded.     

A novel G6PC3 gene mutation in severe congenital neutropenia: pancytopenia and variable bone marrow phenotype can also be part of this syndrome

Glucose‐6‐phosphatase catalytic subunit 3 (G6PC3) deficiency is a newly described syndrome characterized by severe congenital neutropenia associated with multiple organ abnormalities including cardiac and urogenital malformations. The underlying pathophysiology of increased apoptosis of myeloid cells and of neutrophil dysfunction in G6PC3 deficiency involves disturbed glucose metabolism, increased endoplasmic reticulum stress and deficient protein folding. Here, we report a new case of G6PC3 deficiency caused by a novel homozygous G6PC3 gene mutation p.Trp59Arg. The patient showed pancytopenia and a variable bone marrow phenotype with maturation arrest and vacuolization in myeloid lineage cells and a normocellular marrow, respectively. She also showed persistent lymphopenia with low CD4 T‐ and CD19 B‐cell counts. Lymphopenia and even pancytopenia as well as a variable bone marrow phenotype can be part of this syndrome. These clinical findings in a patient with chronic neutropenia should alert the clinician to consider a diagnosis of G6PC3 deficiency.     

Growth factor receptor profile of CD34 cells in normal bone marrow, cord blood and mobilized peripheral blood

Growth factors regulate the proliferation and differentiation of hemopoietic cells. Their effect on hemopoietic precursors differs according to the ontogenic source of the cells. Cord blood and mobilized blood CD34(+) cells have a higher sensitivity for growth factors than bone marrow CD34(+) cells. This could be due to a higher expression of growth factor receptors. Therefore, we examined the expression of receptors for stem cell factor (SCF), interleukin-6 (IL-6), IL-3, granulocyte colony-stimulating factor (G-CSF) and IL-7 on the CD34(+) cells of cord blood, mobilized peripheral blood and bone marrow. The receptors were detected with monoclonal antibodies and flow cytometry. The majority of the CD34(+) cells in bone marrow clearly expressed SCFR; they showed a moderate positivity for IL-3Ralpha and a weak staining for G-CSFR and IL-6 Ralpha. Less than 10% of the cells were IL-7R positive. Cord blood CD34(+) cells showed a higher expression of SCFR and a lower positivity for G-CSFR and IL-6Ralpha. Mobilized blood CD34(+) cells showed a lower expression of SCFR and G-CSFR, and a higher positivity for IL-3Ralpha. This was not solely due to the presence of more myeloid precursors in mobilized blood, as the growth factor receptor profile did not correspond to that of early or late myeloid CD34(+) precursors in normal bone marrow. Changes induced by the mobilization procedure occurred as well. In conclusion, the higher sensitivity for growth factors of hemopoietic precursors in cord blood and mobilized blood cannot be explained by a general increase of the growth factor receptor expression on the CD34(+) cells.     

Impaired survival of bone marrow hematopoietic progenitor cells in cyclic neutropenia

Cyclic neutropenia (CN) is a congenital hematopoietic disordercharacterized by remarkably regular oscillations of blood neutrophils from near normal to extremely low levels at 21-day intervals. Recurringepisodes of severe neutropenia lead to repetitive and sometimeslife-threatening infections. To investigate the cellular mechanism ofCN, the ultrastructure and the proliferative and survivalcharacteristics of bone marrow-derived CD34⁺ earlyprogenitors, CD33⁺/CD34 myeloid progenitors,and CD15⁺ neutrophil precursors from CN patients andhealthy volunteers were studied. The ultrastructural studies showedprofound apoptotic features in bone marrow progenitor cells in CN.Colony-forming assays demonstrated a 75% decrease in the number ofearly myeloid-committed colonies compared with controls. Long-termculture-initiating cell assays demonstrated a 6-fold increase inproduction of primitive progenitor cells in CN. To determine whetheraccelerated apoptosis might account for the underproduction of myeloidprogenitors, the hematopoietic subpopulations were labeled withfluorescein isothiocyanate-annexin V and analyzed by flow cytometry.Short-term culture of CN cells resulted in apoptosis of approximately65% of CD34⁺ cells, 80% ofCD33⁺/CD34 cells, and more than 70% ofCD15⁺ cells, as compared with 20%, 7%, and 15% apoptosisin respective control subpopulations. Evidence of accelerated apoptosisof bone marrow progenitor cells was observed in all 8 patientsparticipating in the study, regardless of the stage in the CN cycle inwhich bone marrow aspirations were obtained. Granulocytecolony-stimulating factor therapy of CN patients significantly improvedsurvival of bone marrow progenitor cells. These data indicate thatineffective production of neutrophils is due to accelerated apoptosisof bone marrow myeloid progenitor cells in CN.     

Mice expressing a neutrophil elastase mutation derived from patients with severe congenital neutropenia have normal granulopoiesis

Severe congenital neutropenia (SCN) is a syndrome characterized by an isolated block in granulocytic differentiation and an increased risk of developing acute myeloid leukemia (AML). Recent studies have demonstrated that the majority of patients with SCN and cyclic neutropenia, a related disorder characterized by periodic oscillations in the number of circulating neutrophils, have heterozygous germline mutations in the ELA2 gene encoding neutrophil elastase (NE). To test the hypothesis that these mutations are causative for SCN, we generated transgenic mice carrying a targeted mutation of their Ela2 gene ("V72M") reproducing a mutation found in 2 unrelated patients with SCN, one of whom developed AML. Expression of mutant NE mRNA and enzymatically active protein was confirmed. Mice heterozygous and homozygous for the V72M allele have normal numbers of circulating neutrophils, and no accumulation of myeloid precursors in the bone marrow was observed. Serial blood analysis found no evidence of cycling in any of the major hematopoietic lineages. Rates of apoptosis following cytokine deprivation were similar in wild-type and mutant neutrophils, as were the frequency and cytokine responsiveness of myeloid progenitors. The stress granulopoiesis response, as measured by neutrophil recovery after cyclophosphamide-induced myelosuppression, was normal. To define the leukemogenic potential of V72M NE, a tumor watch was established. To date, no cases of leukemia have been detected. Collectively, these data suggest that expression of V72M NE is not sufficient to induce an SCN phenotype or leukemia in mice.