Juvenile myelomonocytic leukemia and chronic myelomonocytic leukemia.

In 1982, chronic myelomonocytic leukemia (CMML) was first classified in the category of myelodysplastic syndromes (MDSs), but it always seemed somewhat out of place compared with the rest of the MDS categories. In the 1990s, many argued that there were two different forms of CMML, a proliferative type and a myelodysplastic type. Then in 2001 the World Health Organization created a new category called the mixed myelodysplastic/myeloproliferative diseases, under which CMML was included. Although we still do not understand much about CMML pathogenesis nor do we have specific therapies for this disease, at least now most agree that it is in an appropriate category such that other areas of investigation can now proceed. On the other hand, we now understand a great deal of the pathogenesis underlying the disease now called juvenile myelomonocytic leukemia (JMML). JMML also fits in the new category of mixed myelodysplastic/myeloproliferative diseases. JMML is an excellent model malignancy for investigating and understanding dysregulated and aberrant signal transduction in the Ras pathway. It has also served as a teaching tool for exploring inherited predispositions to cancer.

Expression of CD1d by myelomonocytic leukemias provides a target for cytotoxic NKT cells.

Natural killer T (NKT) cells with an invariant T-cell receptor for alpha-galactosylceramide (alphaGalCer) that is presented by CD1d have been reported to be cytotoxic for myelomonocytic leukemia cells. However, the necessity for leukemia cell CD1d expression, the role of alphaGalCer, and the cytotoxic mechanisms have not been fully elucidated. We evaluated these issues with myeloid leukemia cells from 14 patients and purified NKT cells that were alphaGalCer/CD1d reactive. CD1d was expressed by 80-100% of cells in three of seven acute myeloid leukemias (AMLs) and by 28-77% of cells in five of six juvenile myelomonocytic leukemias (JMML). CD1d+ AML cells were myelomonocytic or monoblastic types, and CD1d+ JMML cells were differentiated and CD34-. Cytotoxicity required leukemia cell CD1d expression and was increased by alphaGalCer (P<0.0001) and inhibited by anti-CD1d mAb (P<0.001). The perforin/granzyme-B pathway of NKT cells caused up to 85% of cytotoxicity, and TNF-alpha, FASL, and TRAIL mediated additional killing. CD56+ NKT cells expressed greater perforin and were more cytotoxic than CD56 NKT cells without alphaGalCer (P<0.0001), but both subpopulations were highly and equally cytotoxic in the presence of alphaGalCer. We conclude that CD1d expression is stage-specific for myelomonocytic leukemias and could provide a target for NKT-cell-mediated immunotherapy.

Inhibition of juvenile myelomonocytic leukemia cell growth in vitro by farnesyltransferase inhibitors.

Juvenile myelomonocytic leukemia (JMML) is an early childhood disease for which there is no effective therapy. Therapy with 13-cis retinoic acid or low-dose chemotherapy can induce some responses, but neither mode is curative. Stem cell transplantation can produce lasting remissions but is hampered by high rates of relapse. The pathogenesis of JMML involves deregulated cytokine signal transduction through the Ras signaling pathway, with resultant selective hypersensitivity of JMML cells to granulocyte-macrophage colony-stimulating factor (GM-CSF). A JMML mouse model, achieved through homozygous deletion of the neurofibromatosis gene, confirmed the involvement of deregulated Ras in JMML pathogenesis. With this pathogenetic knowledge, mechanism-based treatments are now being developed and tested. Ras is critically dependent on a prenylation reaction for its signal transduction abilities. Farnesyltransferase inhibitors are compounds that were developed specifically to block the prenylation of Ras. Two of these compounds, L-739,749 and L-744, 832, were tested for their ability to inhibit spontaneous JMML granulocyte-macrophage colony growth. Within a dose range of 1 to 10 micromol/L, each compound demonstrated dose-dependent inhibition of JMML colony growth. An age-matched patient with a different disease and GM-CSF-stimulated normal adult marrow cells also demonstrated dose-dependent inhibitory effects on colony growth, but they were far less sensitive to these compounds than JMML hematopoietic progenitors. Even if the addition of L-739,749 were delayed for 5 days, significant inhibitory effects would still show in JMML cultures. These results demonstrate that a putative Ras-blocking compound can have significant growth inhibitory effects in vitro, perhaps indicating a potential treatment for JMML. (Blood. 2000;95:639-645)

Diphtheria toxin fused to granulocyte-macrophage colony-stimulating factor is toxic to blasts from patients with juvenile myelomonocytic leukemia and chronic myelomonocytic leukemia.

We have previously demonstrated that human granulocyte-macrophage colony-stimulating factor fused to a truncated diphtheria toxin (DT388-GM-CSF) is toxic to patient acute myeloid leukemia progenitors bearing the GM-CSF receptor, but not normal marrow progenitors. We now report that exposure of mononuclear cells from five of seven (71%) juvenile myelomonocytic leukemia (JMML) patients and from 12 of 20 (60%) adult chronic myelomonocytic leukemia (CMML) patients to 10(-9) mol/L DT388-GM-CSF for 48 hours in culture reduces the number of cells capable of forming colonies in semisolid medium (colony-forming units-leukemia) 10-fold to 300-fold (1 to 2.5 log decrease). In contrast, normal myeloid progenitors (colony-forming unit-granulocyte-macrophage) from six different donors treated and assayed under identical conditions were consistently insensitive to the same fusion toxin even when treated as highly purified CD34(+) cells. The leukemic progenitors from the two other JMML patients showed intermediate sensitivity to DT388-GM-CSF and the leukemic progenitors from eight of the 20 (40%) CMML patients were not different from normal progenitors. Parallel measurements of the number and affinity of GM-CSF receptors on cells from the same samples showed no consistent differences between JMML, CMML, and normal light density or CD34(+) bone marrow cells. The increased sensitivity of leukemic progenitors from all JMML progenitors and some CMML patients to the fusion toxin is therefore not likely to be explained by an increased density of GM-CSF receptors on these cells. We also examined the DT388-GM-CSF sensitivity of two murine cell lines transfected with cDNAs encoding varying portions of the human GM-CSF receptor and/or beta chains. These studies showed that high-affinity ligand binding was sufficient for DT388-GM-CSF-induced toxicity, as this could occur even in the absence of functional signal transduction and that the background of the host cell had a major influence on the degree to which this decreased the toxicity of DT388-GM-CSF. The selective sensitivity to DT388-GM-CSF of leukemic progenitors from a majority of JMML and CMML patients suggests that this agent could have therapeutic potential for some patients with these diseases.

Mutations of the NF1 gene in children with juvenile myelomonocytic leukemia without clinical evidence of neurofibromatosis, type 1.

Juvenile myelomonocytic leukemia (JMML) is a pediatric myelodysplastic syndrome that is associated with neurofibromatosis, type 1 (NF1). The NF1 tumor suppressor gene encodes neurofibromin, which regulates the growth of immature myeloid cells by accelerating guanosine triphosphate hydrolysis on Ras proteins. The purpose of this study was to determine if the NF1 gene was involved in the pathogenesis of JMML in children without a clinical diagnosis of NF1. An in vitro transcription and translation system was used to screen JMML marrows from 20 children for NF1 mutations that resulted in a truncated protein. Single-stranded conformational polymorphism analysis was used to detect RAS point mutations in these samples. We confirmed mutations of NF1 in three leukemias, one of which also showed loss of the normal NF1 allele. An NF1 mutation was detected in normal tissue from the only patient tested and this suggests that JMML may be the presenting feature of NF1 in some children. Activating RAS mutations were found in four patients; as expected, none of these samples harbored NF1 mutations. Because 10% to 14% of children with JMML have a clinical diagnosis of NF1, these data are consistent with the existence of NF1 mutations in approximately 30% of JMML cases.

Conformationally defined retinoic acid analogues. 4. Potential new agents for acute promyelocytic and juvenile myelomonocytic leukemias.

We recently synthesized several conformationally constrained retinoic acid (RA) analogues [8-(2'-cyclohexen-1'-ylidene)-3, 7-dimethyl-2,4,6-octatrienoic acids with different alkyl substituents at 2' (R1) and 3' (R2) positions on the cyclohexene ring] (Muccio et al. J. Med. Chem. 1996, 39, 3625) as cancer chemopreventive agents. UAB8 (R1 = Et; R2 = iPr), which contains sufficient steric bulk at the terminal end of the polyene chain to mimic the trimethylcyclohexenyl ring of RA, displayed biological properties similar to those of RA. To explore the efficacy of this retinoid in acute promyelocytic leukemia (APL) and juvenile myelomonocytic leukemia (JMML), we evaluated UAB8 isomers in in vitro assays which measure the capacity of retinoids to inhibit aberrant myeloid colony growth from blood or bone marrow cells obtained from human JMML patients and in assays measuring the potential of retinoids to differentiate NB4 cells (an APL cell line). Both (all-E)- and (13Z)-UAB8 were 2-fold more active than RA in the NB4 cell differentiation assay; however, only (all-E)-UAB8 had comparable activity to the natural retinoids in the JMML cell assays. These results were compared to the biological effectiveness of a new retinoid, UAB30 [8-(3', 4'-dihydro-1'(2'H)-naphthalen-1'-ylidene)-3,7-dimethyl-2,4, 6-octatrienoic acid], which had different nuclear receptor binding and transactivational properties than UAB8. Relative to (all-E)-RA and (all-E)-UAB8, (all-E)-UAB30 bound well to RARalpha but did not activate transcription-mediated RARalpha homodimers, even though it was effective in RARbeta- and RARgamma-mediated transactivational assays. In APL assays, this retinoid had much reduced activity and was only moderately effective in JMML assays and in cancer chemoprevention assays.

Juvenile myelomonocytic leukemia: molecular understanding and prospects for therapy.

Juvenile myelomonocytic leukemia is a rare but deadly myeloproliferative disorder of early childhood that infrequently progresses to acute leukemia. The pathogenesis of this leukemia has been linked to deregulated signal transduction, resulting in growth factor hypersensitivity. Several other myeloproliferative disorders appear to share growth factor hypersensitivity as a common pathophysiological mechanism and thus this leukemia serves as an important model. New treatment modalities, such as retinoid therapy, are emerging for juvenile myelomonocytic leukemia. Further understanding of deregulated signal transduction should pave the way for even more rationally designed therapy for this leukemia and related disorders.

Sickle cell acute chest syndrome associated with parvovirus B19 infection: case series and review.

Acute Chest Syndrome (ACS) continues to be a major source of morbidity and mortality among patients with sickle cell disease. It is characterized by the presence of pleuritic chest pain, fever, raises on lung auscultation, and pulmonary infiltrates on chest x-ray [Castro et all: Blood 84:643-649]. The pathophysiology of this disorder remains poorly understood leading to the descriptive term "Acute Chest Syndrome" designated by Charache et al. [Arch Intern Med 139:67-69, 1979]. Typical bacterial pathogens are seldom isolated in adults, although they play a significant role in the pathogenesis of this entity in children. Until recently, the technology to accurately study viral infection as a precipitating cause of ACS has been unavailable. Parvovirus B19 is being increasingly recognized as an important human pathogen, and has been established as the cause of transient "aplastic crisis" in patients with sickle cell diseases [Saarien et al: Blood 67:-11411-11417, 1986; Young: Sem Hematol 25:159-172, 1988]. We present three patients with hemoglobin SC variant of sickle cell disease who developed ACS in association with acute parvovirus B19 infection, one of which died of respiratory failure. Parvovirus B19 infection was established by polymerase chain reaction for parvovirus B19 DNA, and the presence of parvovirus B19 specific IgM antibodies. These cases suggest that parvovirus B19 may be associated with more than self-limited illness in patients with sickle cell disease, and that this ubiquitous virus may merit further study as a precipitating cause of ACS.

Juvenile chronic myelogenous leukemia.

Juvenile chronic myelogenous leukemia (JCML) is an aggressive myeloproliferative disorder of childhood that differs both clinically and pathologically from adult type, Philadelphia chromosome positive chronic myelogenous leukemia, and from the other myeloproliferative disorders that are more common in adulthood. The disease can have widely varying clinical presentations and shares many features with the monosomy 7 syndrome and chronic myelomonocytic leukemia. With no specific marker chromosome, establishing the diagnosis can be difficult, and relies on a constellation of clinical, pathologic, and laboratory findings. This article discusses the differential diagnosis of JCML with an emphasis on the pathologic findings and laboratory data that are particularly important for confirming the diagnosis. The sensitivity, specificity, and clinical utility of cell culture colony assays are reviewed. Finally, current knowledge of the biology of JCML and some of the controversies regarding this disease are discussed.

A pilot study of isotretinoin in the treatment of juvenile chronic myelogenous leukemia.

BACKGROUND: Juvenile chronic myelogenous leukemia (CML) is a rare myeloproliferative disease of infants and young children for which there is no effective therapy other than allogeneic bone marrow transplantation. In vitro, isotretinoin (13-cis-retinoic acid) attenuates both the spontaneous proliferation of leukemic peripheral-blood progenitor cells (granulocyte-macrophage colony-forming units) and their selective hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). We conducted a pilot study to evaluate the clinical efficacy of isotretinoin in juvenile CML. METHODS: To be eligible the patients had to have newly diagnosed untreated disease, leukocytosis with monocytosis, marrow with less than 25 percent blasts, hepatosplenomegaly, no chromosomal abnormalities, and negative viral cultures and antibody titers. Isotretinoin was administered orally in single daily doses of 100 mg per square meter of body-surface area. When possible, patients subsequently underwent bone marrow transplantation. RESULTS: Ten children (median age, 10 months) were enrolled in the study. In all 10 there was spontaneous colony formation of leukemic progenitor cells in vitro. In the eight patients tested there was hypersensitivity to GM-CSF. The only toxic effect of isotretinoin therapy was cheilitis in two patients. Four children had disease progression. Two children had complete responses to isotretinoin (normalization of the white-cell count and disappearance of organomegaly), three had partial responses (more than a 50 percent reduction in the white-cell count and degree of organomegaly), and one had a minimal response (more than a 50 percent reduction in the white-cell count, but a 26 to 50 percent reduction in the degree of organomegaly). The median duration of response was 37 months (range, 6 to 83). Three of the four children who had a complete or partial response and who did not undergo bone marrow transplantation were alive 36 to 83 months after the diagnosis of juvenile CML. The spontaneous colony formation in vitro was reduced in samples from the five patients in whom this factor was reassessed during treatment. There was also a reduction in the hypersensitivity of leukemic progenitor cells to GM-CSF in the two patients retested. CONCLUSIONS: Isotretinoin can induce durable clinical and laboratory responses in patients with juvenile CML.

Cloning and sequencing of the cDNAs encoding two alternative splicing-derived variants of the alpha subunit of the granulocyte-macrophage colony-stimulating factor receptor.

Two distinct cDNA clones, corresponding to alternative mRNA splicing variants of the alpha subunit of the granulocyte-macrophage colony-stimulating factor receptor (GM-CSF-R alpha) were isolated from human blood mononuclear cells by RT-PCR. The first one lacks 179 bp, and the second one lacks 136 bp of the major variant, GM-CSF-R alpha 1. Both of variants result in shifts in the reading frame and are expected to encode 377- and 285-amino-acid membrane anchoring and soluble receptor isoforms, respectively.

Familial and congenital polycythemia in three unrelated families.

Three families with polycythemia inherited through apparently different modes are described. Secondary causes of polycythemia were ruled out. Erythropoietin (EPO) levels were normal or low, even after phlebotomy. In vitro erythroid colony growth in standard assay cultures containing EPO was normal; however, in the absence of added EPO, a few progenitors from most of the affected individuals were able to generate recognizable colonies of mature erythroblasts, although these were smaller and proportionately less numerous than seen in polycythemia vera (PV). To search for EPO-receptor changes as a possible pathophysiologic mechanism, we examined, by Southern blot analysis, genomic DNA samples from affected and nonaffected family members, as well as three patients with PV. Two different probes, derived from the human EPO-receptor, were used. We found no evidence for chromosomal rearrangements or gene amplification in hereditary polycythemia or PV patients. Further, no nucleotide sequences were found that were homologous to the Friend spleen focus-forming virus glycoprotein gp55, which has been shown to bind to and activate the murine EPO-receptor. Functional studies examining number and binding affinity of the EPO-receptor on erythroid progenitors from three hereditary polycythemia patients demonstrated no abnormalities. We conclude that the mechanism(s) for the erythrocytosis in familial and congenital polycythemia and in PV may not involve the EPO-receptor and, therefore, may result from alterations of postreceptor responses.

Transplantation of umbilical cord blood after myeloablative therapy: analysis of engraftment.

The possibility that umbilical cord and placental blood from an HLA-identical sibling might produce stable donor-derived lymphohematopoietic engraftment was tested in a patient with juvenile chronic myelogenous leukemia (JCML). After conditioning with high-dose busulfan and cyclophosphamide, cryopreserved umbilical cord blood, containing 0.5 x 10(8) nucleated cells/kg and 2.7 x 10(4) colony forming units-granulocyte, macrophage (CFU-GM)/kg, was infused. A leukocyte count greater than 1,000/microL, absolute neutrophil count (ANC) greater than 500/microL, and platelet count greater than 20,000/microL (untransfused) were observed on days 39, 39, and 47 after transplantation, respectively. Donor cell engraftment was documented in the peripheral blood and bone marrow by cytogenetic analysis, restriction fragment length polymorphism (RFLP), and polymerase chain reaction (PCR) as early as day 21. Furthermore, the donor origin of each lymphohematopoietic lineage (ie, CD5+ T cells, CD19/20+ B cells, CFU-GM, and burst-forming unit-erythrocyte [BFU-E]) was confirmed. On day 200, assays of the peripheral blood and bone marrow showed an abnormal proliferation of CFU-GM at low seeding densities in the absence of exogenous growth factors, as well as a hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF), both pathophysiologic characteristics of JCML. Recurrent disease was confirmed histologically on day 225. Together, these results demonstrate that umbilical cord blood contains sufficient numbers of hematopoietic stem cells necessary for the engraftment of leukemia patients treated with myeloablative therapy and that the detection of "spontaneous" CFU-GM and hypersensitivity to GM-CSF after treatment is a marker of residual or recurrent disease in patients with JCML.

The role of monocyte-derived hemopoietic growth factors in the regulation of myeloproliferation in juvenile chronic myelogenous leukemia.

Juvenile chronic myelogenous leukemia (JCML) is a rare pediatric malignancy characterized by marked hepatosplenomegaly, leukocytosis with prominent monocytosis, elevated fetal hemoglobin, no Philadelphia chromosome, and generally a poor prognosis. In vitro, JCML peripheral blood granulocyte-macrophage progenitors (granulocyte-macrophage colony-forming units, CFU-GM) demonstrate the unique characteristic of "spontaneous" proliferation at very low cell densities in the absence of exogenous growth factors. The "spontaneous" CFU-GM proliferation can be abolished by prior adherent cell (monocyte) depletion, suggesting a paracrine mode of cellular proliferation. Although previous studies using a [3H]thymidine ([3H]TdR) incorporation assay suggested an important role for granulocyte-macrophage colony-stimulating factor (GM-CSF) in JCML, many non-growth factor-related reasons for [3H]TdR incorporation and the relatively low level of inhibition of [3H]TdR uptake left those conclusions open to question. Therefore, we performed clonal CFU-GM assays, which more specifically reflect cytokine effects on CFU-GM, using JCML peripheral blood mononuclear cells (PBMNC) and neutralizing antibodies against GM-CSF, granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating (M-CSF), interleukin 3 (IL-3), interleukin 1 alpha (IL-1 alpha), interleukin 1 beta (IL-1 beta), interleukin 4 (IL-4), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF alpha), and interferon gamma (IFN gamma). Cultures containing anti-GM-CSF alone inhibited "spontaneous" JCML CFU-GM by 87% +/- 9% (mean +/- standard error of the mean [SEM]). No other anti-cytokine antibody produced a significant inhibition of CFU-GM growth. Various combinations of antibodies, excluding anti-GM-CSF, failed to demonstrate any synergistic inhibitory effects upon CFU-GM. Because this apparent paracrine cellular stimulation could be due to excessive cytokine production, by monocytes or other accessory cells, we examined cytokine levels in conditioned media from various JCML cell populations using enzyme-linked immunosorbent assays (ELISAs). Monocytes from only a minority of JCML patients produced higher than normal quantities of GM-CSF, G-CSF, IL-1 beta, IL-6, and/or TNF alpha, but no obvious pattern could be discerned. Further, only 7 of 15 JCML monocyte-conditioned media (MCM) had elevated GM-CSF, and 6 of 15 JCML patients had normal levels of all nine cytokines tested. The monocyte depletion experiments and the inhibition experiments with anti-cytokine antibodies taken together demonstrate clearly that the "spontaneous" growth of JCML CFU-GM in vitro critically depends on at least one monocyte-derived growth factor, GM-CSF.(ABSTRACT TRUNCATED AT 400 WORDS)

Urticaria pigmentosa and preleukemia: evidence for reactive mast cell proliferation.

A 64-year-old man had urticaria pigmentosa and myelodysplasia (refractory anemia with excess blast cells; partial trisomy 8 syndrome) without increased numbers of marrow mast cells. Clonal marrow assays in agar demonstrated normal to increased colony-forming units of granulocytes/macrophages. In long-term liquid cultures containing mast cell growth factor (interleukin 3), his marrow cells proliferated after 3 weeks to produce abnormal myeloid precursors similar to those in the corresponding marrow aspirate specimen. Cells with basophilic-staining granules were less abundant in comparison with normal marrow specimens cultured similarly. These results suggest that the mast cells in this patient are not of the same clone as the preleukemic marrow cells, although the possible marrow-cell origin of urticaria pigmentosa mast cells cannot be excluded. Previous reports suggest that urticaria pigmentosa without systemic mastocytosis occurs as a nonspecific abnormality in a variety of myeloid, lymphoid, and nonhematologic malignancies. Our data also support this hypothesis that urticaria pigmentosa is a reactive process rather than a manifestation of clonal proliferation of the primary malignancy.

Selective hypersensitivity to granulocyte-macrophage colony-stimulating factor by juvenile chronic myeloid leukemia hematopoietic progenitors.

Juvenile chronic myelogenous leukemia (JCML) is a good model for the study of myeloproliferation because JCML hematopoietic progenitor cells grow in vitro at very low cell densities without the addition of exogenous stimulus. Previous studies have demonstrated that this proliferation is dependent on granulocyte-macrophage colony-stimulating factor (GM-CSF), and that removal of monocytes from the cell population before culture eliminates this "spontaneous" myeloproliferation, suggesting a paracrine role of monocyte stimulation. However, subsequent studies have shown that increased GM-CSF production from the JCML monocytes is not a consistent finding and therefore not a plausible sole mechanism. In examining hematopoietic growth factor dose-response curves, both JCML GM and erythroid nonadherent progenitor cell populations displayed a marked and selective hypersensitivity to GM-CSF. Responses to interleukin-3 and G-CSF were identical to control dose-response curves. This is the first demonstration of a myeloid leukemia in which hypersensitivity to a specific growth factor appears to be involved in the pathogenesis of the disease.