Rearrangement of the bcr gene in Philadelphia chromosome-negative chronic myeloid leukemia.

We studied the clinical, hematologic, cytogenetic, and molecular biologic features of seven patients with Philadelphia (Ph1) chromosome-negative chronic myeloid leukemia (CML). In five cases the hematologic findings were indistinguishable from those of patients with classical Ph1-positive disease. Myeloid cells were studied by chromosome-banding techniques. One patient had a masked Ph1 chromosome (with translocation t(4;9;22)), one had a deletion involving chromosome 16, and one had a small minority population of 22q- cells without 9q+ but otherwise normal metaphases; metaphases from the other four patients were entirely normal. DNA prepared from the myeloid cells was digested with the restriction enzymes EcoRI, HindIII, BamHI and BglII. Southern analysis using a 0.6-kb fragment of the breakpoint cluster region (bcr) gene showed the presence in each patient's DNA of a germline fragment together with a rearranged fragment or fragments with at least one of the restriction enzymes. We conclude that genomic changes in the bcr gene characteristic of CML can be present in the absence of a Ph1 chromosome.

Antigenic determinants on myeloid leukaemia colony-forming cells resemble those of normal myeloid progenitor cells and differ from those of circulating blast cells.

We studied the antigenic characteristics of leukaemic colony-forming cells (CFU-L) from the blood of patients with chronic granulocytic leukaemia (CGL) in blastic transformation (BT) and acute myeloid leukaemia (AML) by in vitro culture techniques after complement-mediated lysis with one anti-DR and 10 selected myeloid monoclonal antibodies (McAbs), all of which were cytotoxic in the presence of complement. At the same time we studied the antigenic characteristics of the circulating blast cells from the same patients using in addition one non-complement fixing antibody (BI.3C5) with standard immunofluorescence and immunoalkaline phosphatase techniques. We also used myeloid progenitor cell assays in conjunction with cytotoxic McAbs to investigate the antigenic determinants on Day 7 CFU-GM, Day 14 CFU-GM and BFU-E from the blood of patients with CGL in chronic phase (CP) and from normal bone marrow. We found that two of the McAbs, S4-7 and WGHS29.1, recognized a higher proportion of CFU-L from the blood of AML patients than from patients with CGL-BT. However, the patterns of reactivity for CFU-L from CGL-BT and AML patients with the other McAbs quite closely resembled those observed in CFU-GM and BFU-E from normal individuals and patients with CGL in CP. A McAb with DR specificity and one of the myeloid McAbs, 54/39, recognized both CFU-L from CGL-BT and AML and reacted also with circulating blast cells from the same patients. In contrast, six of the other myeloid McAbs that recognized CFU-L failed to label the corresponding blast cells. We conclude that the antigenic properties of CFU-L in CGL-BT and AML are very similar to, but perhaps not identical with, those of normal CFU-GM and BFU-E. There was a major discrepancy in the antigenic profiles of CFU-L and of the blast cells predominating in the blood.

Purification of haemopoietic progenitor cells from patients with chronic granulocytic leukaemia using percoll density gradients and elutriation.

Purification of haemopoietic progenitor cells from chronic granulocytic leukaemia buffy coat preparations requires a multistep approach using complementary cell separation techniques. In this study Percoll density gradient centrifugation and centrifugal elutriation were used to isolate large numbers of immature progenitor cells. Percoll density gradients were valuable as a first separation step: CFU-GM and CFU-GEMM could be enriched 75-fold in a light density fraction of d less than 1.056 g/ml and the technique could be adapted to cope with more than 10(10) buffy coat leucocytes. Progenitors cells were concentrated 3-fold by elutriation used as single method to separate buffy coat cells or when used to purify further light density Percoll fractions. When Percoll gradients and elutriation were used sequentially, undifferentiated mononuclear cells were enriched to more than 90% purity and between 5% and 40% of these cells formed CFU-GM or BFU-E colonies consisting of more than 40 cells. The enriched fractions were further characterized with monoclonal antibodies. The density and elutriation profiles of these colony forming cells resembled corresponding profiles of cells that reacted with the monoclonal antibody BI-3C5, which recognizes an antigen on primitive haemopoietic progenitor cells. Physical separation methods are a valuable first stage in the attempt to procure relatively pure myeloid progenitor cell populations, whose characteristics can then be further studied at a cellular or molecular level.

Chromosome and cell culture studies in eosinophilic leukaemia.

A cytogenetic analysis was carried out on bone marrow cells from 11 patients who presented with hypereosinophilia and the clinical features of the idiopathic hypereosinophilic syndrome. One of these patients was found to have trisomy 8 affecting the myeloid series, including eosinophils. In this patient, marrow eosinophils also showed asynchrony of nuclear-cytoplasmic maturation, and there were increased numbers of myeloid progenitor cells in the blood. Six months later, blast cell transformation occurred, and he died soon afterwards. These findings show that abnormalities in the karyotype of bone marrow cells and culture of blood progenitor cells may help to identity eosinophilic leukaemia among patients who present with features of the idiopathic hypereosinophilic syndrome.

Megakaryoblastic transformation of myelofibrosis with expression of the c-sis oncogene.

We describe a case of primary myelofibrosis which terminated in an acute megakaryoblastic leukaemia with massive marrow fibrosis and osteosclerosis. The megakaryocyte lineage of the terminal phase was confirmed by ultrastructural and surface marker studies of the blast cells. The leukaemic phase was associated with the presence of large numbers of progressively more immature megakaryocyte progenitors in the peripheral blood. The expression of c-sis mRNA in these blast cells was significantly higher than in normal mononuclear cells. Activation of the c-sis protooncogene leading to increased production of platelet-derived growth factor could be related to the progressive fibrosis observed.

Separation of human blast progenitors from granulocytic, erythroid, megakaryocytic, and mixed colony-forming cells by "panning" on cultured marrow-derived stromal layers.

Primitive myeloid progenitor cells will adhere to stromal feeder layers of human bone-marrow-derived adherent cells grown in the presence of methylprednisolone (MP+ layers). These progenitors form colonies of blast cells on the MP+ stromal layers, but not on stromal layers grown in the absence of MP (MP- layers). The present study was designed to determine whether this failure of colony formation was caused by inability of the progenitors to adhere to the MP- layers or inability to proliferate in their presence. We also compared the capacities of the blast progenitors to adhere to MP+ and MP- stromal cells with those of mixed (GEMM-CFC), erythroid (BFU-E), megakaryocytic (Mk-CFC), and granulocyte-macrophage (GM-CFC) colony-forming cells. Incubation of bone marrow mononuclear cells with MP+ stromal layers removed 90% of the blast progenitors, but did not remove the majority of the GEMM-CFC, BFU-E, Mk-CFC, and GM-CFC; incubation of bone marrow mononuclear cells with MP- stromal layers did not remove the blast progenitors or the GEMM-CFC, BFU-E, Mk-CFC, and GM-CFC. Thus, the blast progenitors adhere to MP+ stromal feeder layers, but not to MP- stromal layers. In this respect they differ from the other more mature colony-forming cells that do not show any marked tendency to adhere to either type of stromal layer.

Colony formation by primitive haemopoietic progenitors in cocultures of bone marrow cells and stromal cells.

Human bone marrow contains a class of human haemopoietic progenitor cells that adhere to cultured marrow stromal cells and form colonies of blast cells. These progenitor cells are found in the non-adherent mononuclear fraction of normal human bone marrow. They are not in active cell cycle and do not express Ia-like (HLA-DR) antigens but appear to be capable of self-renewal in vitro. These properties indicate that they should be classified as members of the primitive haemopoietic progenitor cell compartment.

Proliferation in liquid culture of megakaryocytes from the blood of patients with primary myelofibrosis and other myeloproliferative disorders.

Using a short term liquid system we have shown that blood from some patients with primary myelofibrosis (PMF) and chronic granulocytic leukaemia (CGL) in megakaryoblastic transformation (CGL-Mk) gives rise to large numbers of progenitor cells committed to the megakaryocyte (Mk) lineage. As assessed by indirect immunofluorescence the number of cells reacting with three antiplatelet monoclonal antibodies, C17, J15 and AN51, increases during the culture period. There is no equivalent increase in cultures from the blood of normal individuals or patients with essential thrombocythaemia (ET). Furthermore plasma-free supernatants from cultures of the cells from patients with PMF and CGL-Mk stimulate the rate of proliferation of fibroblasts from normal bone marrow. These data provide further evidence for the involvement of the Mk lineage in PMF and CGL and suggest that the excess fibrosis seen in these conditions may be caused by a factor emanating from Mks.

Myeloid progenitor cells in the circulation of patients with myelofibrosis and other myeloproliferative disorders.

We have measured the numbers of myeloid progenitor cells in the circulation of patients with myelofibrosis (MF) and other myeloproliferative disorders. In general, progenitor cell numbers were increased in the circulation of patients with MF compared with controls. The mean increases were 9-fold for the multilineage progenitor cells (CFU-GEMM), 13-fold for the erythroid progenitor cells (BFU-E), 37-fold for the granulocyte-macrophage progenitor cells (CFU-GM) and 167-fold for the megakaryocyte progenitors (CFU-MK). Splenectomized patients generally had reduced numbers of circulating progenitor cells. In the CFU-MK assay, mature megakaryocytes cultured from patients with MF regularly showed large vacuoles in the nucleus and cytoplasm, unlike control cells. The increased colony formation in patients with MK, involving especially CFU-MK colonies, is consistent with the hypothesis that MF is a primary myeloproliferative disorder in which a megakaryocyte-derived factor predisposes to the formation of marrow fibrosis.

A role for 1,25-dihydroxyvitamin D3 in control of bone-marrow collagen deposition?

It is suggested that 1,25-dihydroxyvitamin D3 (1,25-[OH]2D3), the active hormonal metabolite of vitamin D3, inhibits the formation of fibrous tissue (mainly collagen) in bone-marrow and also increases its degradation. 1,25-(OH)2D3 inhibits the proliferation of megakaryocytes which normally promote collagen synthesis. The hormone also directly antagonises collagen synthesis. Degradation of fibrous tissue is mediated by monocytes and macrophages, which contain collagenase, and the number and activity of these cells is increased by 1,25-(OH)2D3. Thus the various actions of this hormone contribute collectively to a reduction in collagen content; conversely a deficiency of 1,25-(OH)2D3 may allow abnormal accumulation of collagen in the marrow.

Antigenic expression and proliferative status of multilineage myeloid progenitor cells (CFU-GEMM) in normal individuals and patients with chronic granulocytic leukaemia.

We assayed the number of multilineage myeloid progenitor cells (CFU-GEMM) in the blood and marrow of patients with newly diagnosed chronic granulocytic leukaemia (CGL). The mean number of CFU-GEMM in the blood was increased 600-fold and CFU-GEMM in the marrow was doubled in the CGL patients compared with normal. A complement-fixing monoclonal antibody with HLA-DR specificity inhibited the proliferation of CFU-GEMM from CGL blood to a greater extent than that of comparable cells in normal marrow. Using a hydroxyurea 'suicide' method we found that the proportion of CFU-GEMM in proliferative cycle was higher in CGL blood than in normal marrow. We conclude that (1) CFU-GEMM numbers are greatly increased in the blood of patients with CGL, (2) CFU-GEMM express HLA-DR antigens on their surface, and (3) the apparently increased expression of the antigen on CFU-GEMM from CGL blood in comparison with CFU-GEMM from normal marrow may parallel the relatively higher proportion of CGL CFU-GEMM in cell cycle.

Effects of human marrow stromal cells on proliferation by human granulocytic (GM-CFC), erythroid (BFU-E) and mixed (Mix-CFC) colony-forming cells.

We have investigated the effects of the major components of bone marrow stroma (fibroblasts, fat cells, macrophages and endothelial cells) on colony-forming haemopoietic precursor cells. Selective cultures of the different stromal cell types, grown to confluence, were used as underlayers for agar or methylcellulose cultures containing bone marrow cells. In different experiments, colony-stimulating factor (CSF) was added to stimulated granulocyte-macrophage colony-forming cells (GM-CFC), erythropoietin was used to induce erythroid burst (BFU-E) formation or erythropoietin and medium conditioned by leucocytes in the presence of phytohaemagglutinin (PHA-LCM) was added to induce the formation of colonies of mixed myeloid lineage (Mix-CFC). Fibroblasts grown from human marrow enhanced granulopoiesis when CSF was present in the cultures but suppressed the formation of BFU-E and mixed colonies. Treatment of the cultures with methylprednisolone induced the formation of fat cells in the fibroblast cultures and prevented both the fibroblast-mediated enhancement of granulopoiesis and the fibroblast-mediated suppression of erythropoiesis. Stromal macrophages reduced granulocyte colony formation but had little effect on the proliferation of BFU-E or mixed colony-forming cells. Endothelial cells stimulated granulopoiesis by releasing CSF into the culture supernatant; supernatant from endothelial cell cultures had no marked effects on either BFU-E or Mix-CFC. We conclude that different components of marrow stroma have contrasting effects on erythropoiesis and granulopoiesis; thus, marrow stroma may regulate the expression of stem cell differentiation in vivo.

Iridescent virus type 22 DNA.

Double stranded DNA extracted from iridescent virus type (IV 22) was characterized by its buoyant density in CsCl, thermal denaturation profile and guanine plus cytosine content. The DNA was linear with a molecular weight of 130--142 x 10(6) determined by reassociation kinetics, contour length measurements and restriction endonuclease analysis.