ALBERT

Description: Juvenile chronic myelogenous leukemia (JCML) is a rare myeloproliferative disorder of early childhood that is clinically and cytogenically distinct from the well-recognized adult type of chronic myeloid leukemia. Unlike the adult disease, growth of hematopoietic progenitors from peripheral blood (PB) occurs in the absence of exogenous stimulus even at low cell densities. This so-called “spontaneous”growth can be abrogated by adherent cell depletion and appears to depend on production of endogenous growth factors. We studied seven children with JCML to determine the nature of endogenous stimulators. With isolated PB mononuclear cells (PBMNCs) and a 3H- thymidine (3H-TdR) incorporation assay, JCML cells were shown to incorporate high levels of 3H-TdR when cultured in the absence of stimulus even at low cell densities. When neutralizing antisera prepared against each of the four known colony-stimulating factors (CSFs), GM-CSF, G-CSF, M-CSF, and interleukin-3 (IL-3), as well as antisera against interleukin-1 (alpha and beta) and tumor necrosis factor (TNF) were added to these cultures, only the antisera against recombinant human GM-CSF (rhGM-CSF) consistently resulted in significant inhibition of cell proliferation, achieving up to 72% inhibition of 3H-TdR incorporation in one case. Monoclonal antibodies (MoAbs) against rhGM-CSF resulted in a similar and highly significant degree of inhibition. A marked inhibitory effect of rhGM-CSF antiserum on “spontaneous” growth of PB CFU-GM derived colonies in semisolid medium was also demonstrated in four of five patients studied (87% to 90% inhibition). Production of growth factors by highly enriched JCML monocytes was variable. When initially studied in five of the seven patients, the monocytes from three of the patients revealed increased release of IL-1-like activities; two patients had levels similar to those of controls. One patient with normal levels when initially studied was later shown to have markedly increased amounts of IL-1-like activities in a second preparation of monocyte-conditioned medium (MCM). High levels of GM-CSF were detected in the initial MCM from one patient, but this may have indirectly reflected elevated IL-1-like activities present in the MCM. IL-3 and M-CSF levels were either low or undetectable in the patients studied as compared with MCM prepared with normal adult monocytes. These results clearly implicate GM-CSF as the primary endogenous regulator of JCML cell proliferation in culture and suggest that this malignant myeloproliferative disease may in part result from paracrine stimulation of marrow progenitor cells by growth factors/cytokines secreted by the malignant monocytes.

Description: 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.

Description: 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.

Description: Nonspecific esterase (NSE) is used to identify normal and leukemic mononuclear phagocytes cytochemically. It can be demonstrated by the hydrolysis of alpha-naphthyl acetate or butyrate. NSE from leukapheresed leukemic monocytes were extracted with several types of detergent and grouped into two isoelectric point (pl) categories based on the ease of solubility: pl 5.7–6.3 (8 bands) greater than or equal to pl 6.6–7.6 (6 bands). Normal monocytes yielded only the pl 5.7–6.3 isozymes. Isozymes from both leukemic and normal monocytes were inhibited similarly by sodium fluoride, pH less than 4.7, and a serine active site inhibitor. All isozymes were bound by Sepharose- concanavalin A (Con-A) and displayed similar substrate preferences and pH v activity slopes. Under the conditions of detergent solubilization, the smallest mol wt species retaining enzymatic activity was 50 +/- 5 kd. Despite these similarities, the isozymes of pl 5.7 through 6.3 and pl greater than 6.3 exhibited different degrees or types of inhibition by phenylmethylsulfonyl fluoride (PMSF), resistance to heat and antigenic character. Thus, the esterase isozymes represent two families of glycoproteins, both of them probable cell surface enzymes, resembling classical liver carboxyl or B-esterases (EC 3.1.1 1).

Description: 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.

Description: 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.

Description: Nonspecific esterase (NSE) is used to identify normal and leukemic mononuclear phagocytes cytochemically. It can be demonstrated by the hydrolysis of alpha-naphthyl acetate or butyrate. NSE from leukapheresed leukemic monocytes were extracted with several types of detergent and grouped into two isoelectric point (pl) categories based on the ease of solubility: pl 5.7–6.3 (8 bands) greater than or equal to pl 6.6–7.6 (6 bands). Normal monocytes yielded only the pl 5.7–6.3 isozymes. Isozymes from both leukemic and normal monocytes were inhibited similarly by sodium fluoride, pH less than 4.7, and a serine active site inhibitor. All isozymes were bound by Sepharose- concanavalin A (Con-A) and displayed similar substrate preferences and pH v activity slopes. Under the conditions of detergent solubilization, the smallest mol wt species retaining enzymatic activity was 50 +/- 5 kd. Despite these similarities, the isozymes of pl 5.7 through 6.3 and pl greater than 6.3 exhibited different degrees or types of inhibition by phenylmethylsulfonyl fluoride (PMSF), resistance to heat and antigenic character. Thus, the esterase isozymes represent two families of glycoproteins, both of them probable cell surface enzymes, resembling classical liver carboxyl or B-esterases (EC 3.1.1 1).

Description: 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.

Description: Juvenile chronic myelogenous leukemia (JCML) is a rare myeloproliferative disorder of early childhood that is clinically and cytogenically distinct from the well-recognized adult type of chronic myeloid leukemia. Unlike the adult disease, growth of hematopoietic progenitors from peripheral blood (PB) occurs in the absence of exogenous stimulus even at low cell densities. This so-called “spontaneous”growth can be abrogated by adherent cell depletion and appears to depend on production of endogenous growth factors. We studied seven children with JCML to determine the nature of endogenous stimulators. With isolated PB mononuclear cells (PBMNCs) and a 3H- thymidine (3H-TdR) incorporation assay, JCML cells were shown to incorporate high levels of 3H-TdR when cultured in the absence of stimulus even at low cell densities. When neutralizing antisera prepared against each of the four known colony-stimulating factors (CSFs), GM-CSF, G-CSF, M-CSF, and interleukin-3 (IL-3), as well as antisera against interleukin-1 (alpha and beta) and tumor necrosis factor (TNF) were added to these cultures, only the antisera against recombinant human GM-CSF (rhGM-CSF) consistently resulted in significant inhibition of cell proliferation, achieving up to 72% inhibition of 3H-TdR incorporation in one case. Monoclonal antibodies (MoAbs) against rhGM-CSF resulted in a similar and highly significant degree of inhibition. A marked inhibitory effect of rhGM-CSF antiserum on “spontaneous” growth of PB CFU-GM derived colonies in semisolid medium was also demonstrated in four of five patients studied (87% to 90% inhibition). Production of growth factors by highly enriched JCML monocytes was variable. When initially studied in five of the seven patients, the monocytes from three of the patients revealed increased release of IL-1-like activities; two patients had levels similar to those of controls. One patient with normal levels when initially studied was later shown to have markedly increased amounts of IL-1-like activities in a second preparation of monocyte-conditioned medium (MCM). High levels of GM-CSF were detected in the initial MCM from one patient, but this may have indirectly reflected elevated IL-1-like activities present in the MCM. IL-3 and M-CSF levels were either low or undetectable in the patients studied as compared with MCM prepared with normal adult monocytes. These results clearly implicate GM-CSF as the primary endogenous regulator of JCML cell proliferation in culture and suggest that this malignant myeloproliferative disease may in part result from paracrine stimulation of marrow progenitor cells by growth factors/cytokines secreted by the malignant monocytes.