Acute monocytic leukemia: A surface marker study

Thirteen cases of acute monocytic leukemia (AMoL) were studied morphologically, cytochemically, and immunologically in an attempt to determine similarities and/or differences between acute monocytic and lymphocytic leukemias. Single class surface immunoglobulins (IgG kappa) were seen in six cases. These markers are thought to represent serum immunoglobulins attached to the leukemic monocytes via the receptors for the Fc portion of immunoglobulin and are not synthesized by the cells as is the case with B lymphocytes. Receptors for complement were also noted. It appears that surface immunoglobulins and receptors for the Fc portion of the immunoglobulin and complement, which are found on several classes of normal lymphocytes and monocytes, may also be found on leukemic monocytes. Antisera to B lymphocytes, detecting HLA-DRw antigens, also reacted with some of the leukemic monocytes.     

A scanning electron microscopic study of 34 cases of acute granulocytic,myelomonocytic, monoblastic and histiocytic leukemia

This report describes the surface architecture of leukemic cells, as seen by scanning electron microscopy in 34 patients with acute nonlymphoblastic leukemia. Six patients with myeloblastic, 4 with promyelocytic, 10 with myelomonocytic, 8 with monocytic, 4 with histlocytic and 2 with undifferentiated leukemia were studied. Under the scanning electron microscope most leukemic histiocytes and monocytes appeared similar and were characterized by the presence of large, well developed broad-based ruffled membranes or prominent raised ridge-like profiles, resembling in this respect normal monocytes. Most cells from patients with acute promyelocytic or myeloblastic leukemia exhibited narrower ridge-like profiles whereas some showed ruffles or microvilli. Patients with myelomonocytic leukemia showed mixed populations of cells with ridge-like profiles and ruffled membranes whereas cells from two patients with undifferentiated leukemia had smooth surfaces, similar to those encountered in cells from patients with acute lymphoblastic leukemia.It appears that nonlymphoblastic and lymphoblastic leukemia cells (particularly histiocytes and monocytes) can frequently be distinguished on the basis of their surface architecture. The surface features of leukemic histiocytes and monocytes are similar, suggesting that they may belong to the same cell series. The monocytes seem to have characteristic surface features recognizable with the scanning electron microscope and differ from most cells from patients with acute granulocytic leukemia. Although overlap of surface features and misidentification can occur, scanning electron microscopy is a useful adjunct to other modes of microscopy in the study and diagnosis of acute leukemia.     

Monoclonal antibodies to the human CSF-1 receptor (c-fms proto-oncogene product) detect epitopes on normal mononuclear phagocytes and on human myeloid leukemic blast cells

The first monoclonal antibodies (MoAbs) to epitopes in the extracellular domain of the human c-fms proto-oncogene product (receptor for the macrophage colony stimulating factor, CSF-1) were used with flow cytometric techniques to study receptor expression on normal human peripheral blood monocytes, bone marrow cells, and leukemic blasts. On normal cells CSF-1 receptors were restricted in their expression to cells of the mononuclear phagocyte lineage. CSF-1 receptors were detected on leukemic blasts from 15 (30%) of 50 children with acute myeloid leukemia, compared with four (15%) of 26 adults. By contrast, detectable CSF-1 receptors were uniformly absent on blasts from 19 children with acute lymphoblastic leukemia. CSF-1 receptors on normal monocytes and myeloid leukemia cells could be induced to downmodulate by incubation with either human recombinant CSF-1 or phorbol esters, confirming that the receptors had functional ligand- binding sites and responded to transmodulation by inducers of protein kinase C. The numbers of receptors per cell and the percentage of positive cases were highest for leukemic blasts with cytochemical and morphological features of monocytes. However, CSF-1 receptors were also detected on a subset of leukemic blast cells with features of granulocytic differentiation (FAB subtypes M1 through M3). Southern blotting analyses of DNA from 47 cases of acute myeloid leukemia demonstrated no rearrangements within the 32 kb of genomic sequences that contain CSF-1 receptor coding exons or in the 50 kb upstream of the first coding exon. Analysis of the upstream region of the c-fms locus revealed that sequences representing the terminal 112 untranslated nucleotides of c-fms mRNA map 26 kb 5' to the first coding exon, suggesting that at least one c-fms promoter is separated from the receptor coding sequences by a very long intron. Whereas expression of the CSF-1 receptor in myeloid leukemic blasts is not restricted to cells with monocytic characteristics, the apparently aberrant pattern of receptor synthesis in a subset of cases with granulocytic features appears not to be due to chromosomal rearrangements within 50 kb upstream of sequences encoding the receptor.     

Acute myelomonocytic leukemia in children. Possible use of the soft agar culture technique in the differentiation of cellular subtypes.

In 8 children with acute myelomonocytic leukemia (AMML), colony formation in soft agar cultures derived from bone marrow cells was studied in an attempt to differentiate the monocytic (Schilling) from the myelomonocytic (Naegeli) types. The children did not differ markedly in their clinical and morphological parameters. Three in vitro growth patterns were observed: markedly decreased or no growth in 4 cases, extensive growth of granulocytic colonies in 2 cases, and extensive growth of macrophage colonies in the remaining 2. It is suggested that the marrows presenting diminished or no growth patterns are presumably of acute myelogenous leukemia patients with a monocytic component. The excessive granulocytic or macrophage colony growth may be an in vitro indication for an in vivo proliferation of either granulocytic or monocytic leukemic cell lines, and therefore may represent the Naegeli or Schilling variants of AMML respectively. If these observations can be approved in a larger series of AMML patients, this approach can be valuable as another tool in the differential diagnosis of the subtypes of AMML in children.     

Acute myelomonocytic leukemia: an immunoelectron microscopic study

Acute myelomonocytic leukemia (M4; French-American-British classification) is light microscopically defined as the leukemia constituting leukemic cells in both granulocytic and monocytic lineages. Therefore, the characteristics of M4 have not been fully elucidated. The author previously indicated that normal neutrophilic granulocytes could be ultrastructurally differentiated from normal monocytes by the double staining of lactoferrin and lysozyme. In this investigation, the ultrastructural localization of both proteins were observed in order to make the outline of M4 clear. The leukemic cells in acute myeloid leukemia (M2) were also examined in comparison with those in M4. The leukemic cells in M4 showed the double stainability of lactoferrin and lysozyme, and the positive reactions were localized in the cytoplasmic matrix and in the granules. The staining pattern was similar to that in M2. The coexistence of lactoferrin and lysozyme in the leukemic cells in M4, which has ultrastructurally the monocytic characteristics, implied that the leukemic cells also possess the characteristics of the cells in the granulocytic lineage. This suggests that the presence of the various leukemic cells in the granulocytic lineage. This suggests that the presence of the various leukemic cells signifies the diversely abnormal maturations in vivo of the monocytes/granulocytes precursor cell and that M4 consists of not two kinds of distinguishable cells of granulocytic and monocytic lineages but various consecutive cells based on a malignant transformation of the precursor cell.     

Interleukin-6 inhibits the proliferation of B-chronic lymphocytic leukemia cells that is induced by tumor necrosis factor-alpha or -beta

Tumor necrosis factor (TNF-alpha) acts as a growth stimulatory factor on leukemic B lymphocytes from many patients with chronic lymphocytic leukemia (CLL). Because TNF induces production of interleukin-6 (IL-6), which has been shown to be a growth factor for myeloma and other transformed B cells, we examined the possibility that IL-6 mediates the growth-stimulatory effect of TNF on B-CLL cells. In fact, we found that IL-6 is an inhibitor of B-CLL growth. The addition of recombinant human IL-6 markedly decreased the TNF-induced B-CLL growth, and this decrease was even greater when soluble IL-6 receptor, known to act as IL-6 agonist, was added with recombinant IL-6. Conversely, neutralizing monoclonal antibodies to IL-6 and to the IL-6 receptor potentiated the growth stimulation of TNF on B-CLL cells, in line with the possibility that IL-6 functions as a negative feedback regulator of an autocrine TNF action on these B-leukemic cells. Evidence is presented that production of IL-6 by monocytes and B cells of CLL patients is low, suggesting that administration of IL-6 may be beneficial in CLL to reduce the eventual growth stimulation by TNF and, possibly, also the deficiency in platelets and Ig production in this disease.     

Interaction of monocytes and T cells in the regulation of normal human megakaryocytopoiesis in vitro: role of IL-1 and IL-2

Autologous or allogeneic peripheral blood T cells can stimulate the human megakaryocyte progenitor cell (CFU-Meg)-derived colony formation in a dose-dependent fashion in agar cultures of nonadherent (NA), T cell-depleted (NT) bone marrow (BM) cells. Low concentrations of monocytes and T cells can collaborate in the stimulation of CFU-Meg colony formation or in the production of megakaryocyte colony stimulating factor (Meg-CSF) by T cells in the presence of mitogens or IL-2. Monocytes alone can produce only negligible Meg-CSF under any conditions. When monocyte conditioned medium (CM) was added to T cell-stimulated NA, NT BM cell cultures, CFU-Meg colony growth was appreciably increased compared with that stimulated by T cells alone. Dose-dependent increase in CFU-Meg colony growth was noted when varying concentrations of IL-1 were added to T cell-stimulated NA, NT cell cultures, although IL-1 itself could support no CFU-Meg colony growth in the absence of T cells. These data suggest that a synergistic interaction between T cells and monocytes during the production of Meg-CSF by T cells could be partly mediated by IL-1. IL-2 was found to stimulate Meg-CSF production by T cells in the presence or absence of mitogens. IL-2-stimulated Meg-CSF production by T cells was augmented by the addition of monocytes. Although IL-2 itself had no stimulatory effect on CFU-Meg colony growth, dramatic augmentation in the CFU-Meg colony number was noted when IL-2 was added to T cell-stimulated NA, NT cell cultures. High concentrations of monocytes and prostaglandin E (PGE) inhibited the CFU-Meg colony formation. These results suggest that IL-1 and IL-2 may play a stimulatory role on the normal human in vitro megakaryocytopoiesis, and may be involved in the development of reactive thrombocytosis and bone marrow megakaryocytic hyperplasia in various inflammatory diseases.     

Alkaline phosphatase-positive leukemic monocytes in a child with acute monocytic leukemia

A subpopulation with alkaline phosphatase activity and neutrophilic granules was found in leukemic monocytes from a child with acute monocytic leukemia (M5B). Almost all leukemic cells were strongly positive for nonspecific esterase and phagocytized opsonized zymosans. These findings suggest that the subpopulation are chimeric cells with characteristics of both monocytic and neutrophilic cell lines. The leukemogenesis in this patient might involve the simultaneous expression of differentiation genes otherwise restricted to either cell lineage.     

Leukemia inhibitory factor/human interleukin for DA cells: a growth factor that stimulates the in vitro development of multipotential human hematopoietic progenitors.

We investigated the in vitro hematopoietic stimulatory activity of leukemia inhibitory factor/human interleukin for DA cells (LIF/HILDA) on bone marrow progenitor populations in 17 normal individuals. In serum-free cultures LIF/HILDA did not induce colony growth. In serum containing media, LIF/HILDA stimulated the growth of colony forming unit (CFU)-MIX and CFU-EO in a dose-dependent fashion and resulted in an increased CFU-MIX and burst forming unit-erythrocytes (BFU-E) colony size. Similar stimulatory effects were seen on a highly purified hematopoietic progenitor population obtained after immunomagnetic depletion of mature myeloid precursors and lymphoid cells. Addition of LIF/HILDA to cultures containing maximally stimulatory concentrations of recombinant human interleukin-3 (rhuIL3), rhuIL3 + rhuIL6, or rhu granulocyte-macrophage colony-stimulating factor (rhu GM-CSF) in serum containing media significantly increased the number of CFU-MIX and eosinophil colonies and increased size and cluster number of CFU-MIX and BFU-E. Depletion of accessory T lymphocytes or monocytes from bone marrow progenitors did not alter the response of hematopoietic precursors to LIF/HILDA. A similar increased colony growth was seen when LIF/HILDA was added to cultures of positively selected CD34/HLA-DR+ or CD34+/HLA-DR- bone marrow hematopoietic progenitor cells stimulated with maximally stimulatory concentrations of rhuIL3 + rhuIL6. LIF/HILDA is a novel cytokine capable of stimulating growth and proliferation of multi-lineage, erythroid, and eosinophil colonies in the presence of serum. LIF/HILDA exerts its activity by direct interaction with highly purified immature bone marrow progenitor cells, has an additive effect when used with other cytokines known to stimulate primitive hematopoietic precursors, and does not require accessory cells.     

Tissue-restricted T cell alloresponses across HLA barriers: selection and identification of leukemia-restricted CTL in HLA-mismatched stimulator-responder pairs

Exploiting the graft-versus-leukemia (GVL) effect in mismatched transplants requires its separation from graft-versus-host disease (GVHD). We generated leukemia-specific cytotoxic T lymphocytes (CTL) in three haplotype-mismatched, two class I-mismatched and two single HLA-A locus-matched stimulator-responder pairs. Six patients with chronic myelogenous leukemia and one patient with acute myeloid leukemia transformed from MDS were studied. CTL generated after 10 days stimulation with unselected leukemic peripheral blood mononuclear cells inhibited leukemic CFU-GM colony growth (>85% at 10:1 effector:target ratio) with no third-party colony inhibition. In five pairs, responders were cultured separately with leukemia cells, PHA-B or LCL from the stimulator. After 2-4 restimulations, the T cell repertoire was examined by flow analysis using Vbeta-specific antibodies. Test cultures (but not controls) showed preferential expansion of 1-4 Vbeta families either common to two or more stimulators or unique to a particular stimulator. Notably, we elicited leukemia-specific TCR Vbeta expansions on four out of five occasions. In two pairs, responder cells selected for the appropriate leukemia-specific Vbeta family were shown to have leukemia-specific cytotoxicity. These leukemia-restricted T-cells were CD8+ or CD4+ and CD25+ or CD57+. The results support the development of strategies to selectively deplete GVHD and conserve GVL reactivity in mismatched transplants.     

The expression of myeloid-specific antigens on myeloid leukemia cells: correlations with leukemia subclasses and implications for normal myeloid differentiation

The expression of three distinct myeloid-specific cell surface antigens detected by monoclonal antibodies (PMN 6, PMN 29, and AML-2-23) on acute and chronic myeloid leukemia cells is correlated with blast cell morphology and normal myeloid cell antigen display. In studies on normal peripheral blood cells, monoclonal antibodies PMN 6 and PMN 29 have previously been shown to react exclusively with neutrophils while AML-2-23 reacts with both neutrophils and monocytes. The present report demonstrates that these antigens are absent from blast cells of patients with acute myelocytic leukemia (AML) classified as M1 and M2 in the French-American-British system and chronic myelocytic leukemia in myeloid blast crisis. However, leukemia cells with myelomonocytic morphology (M4) expressed all three antigens, while cells with pure monocytic features (M5) were generally only positive for AML-2-23. Based on the absence of these antigens on both leukemic and normal myeloblasts and granulocyte-monocyte progenitors and their characteristic patterns of display on more differentiated leukemic and normal cells, we propose a modified concept of normal myelopoiesis. In this hypothesis, the myeloblast is an uncommitted cell that gives rise to a series of intermediate precursors that acquire committment to either the granulocytic or monocytic lineage marked by the acquisition of specific cell surface markers.     

Antibody-dependent cellular cytotoxicity of leukemic cells from the patients with acute myeloid leukemia to human erythrocytes coated with anti-D antiserum

We examined the antibody-dependent cellular cytotoxicity (ADCC) of normal leukocytes from healthy volunteers, immature neutrophils from 4 patients with chronic myelocytic leukemia (CML) and leukemic cells from 22 patients with acute myeloid leukemia, employing human erythrocyte targets. Ability to mediate ADCC was demonstrated only in monocytes among normal leukocytes and in leukemic cells from all the 6 patients with acute myelomonocytic or monocytic leukemia (AMMoL, AMoL). CML immature neutrophils and leukemic cells from 14 patients with acute myeloblastic or promyeloblastic leukemia displayed very low levels of ADCC. In the remaining 2 patients with acute myeloblastic leukemia, the ADCC levels were high. We described the usefulness of this ADCC assay for distinguishing AMMoL or AMoL from other types of acute myeloid leukemia.     

Binding characteristics and proliferative action of purified granulocyte colony-stimulating factor (G-CSF) on normal and leukemic human promyelocytes

The binding of purified 125I-labeled murine granulocyte colony stimulating factor (125I-G-CSF) to normal and leukemic human cells was examined. Normal neutrophils and their precursors demonstrated specific labeling with 125I-G-CSF, whereas eosinophils, lymphocytes, and erythroid cells did not. Normal human promyelocytes demonstrated the highest binding among hemopoietic cells. Human myeloid leukemic cells also demonstrated consistent specific labeling with 125I-G-CSF. Normal promyelocytes and chronic myeloid leukemia promyelocytes demonstrated only transient clonal proliferation in vitro when stimulated by G-CSF, but this was not always the case with acute promyelocytic leukemic cells. The qualitative responsiveness of normal and leukemic cells to G-CSF was very similar despite heterogeneity in receptor numbers on individual cells. A subset of acute promyelocytic leukemic cells appeared unresponsive to stimulation by GM-CSF.     

Leukemia cell lines can replace monocytes for mitogen-induced T-lymphocyte responses: this accessory function is dependent upon their differentiation stage.

Highly purified peripheral T lymphocytes do not proliferate in response to phytohemagglutinin A or concanavalin A, unless adherent HLA-DR+ monocytes are added as accessory cells. The accessory function (AF) of monocytes is mediated through the release of interleukin-1 (IL-1). We here report that cells from three human leukemic lines--K562, HL-60, and U-937--could exert AF and efficiently replace monocytes in a 72-hr mitogen-stimulated proliferation assay. This AF was clearly related to precise maturational stages of these cells, since the hematopoietic precursor K562 cells spontaneously exerted high AF but lost this property when treated with differentiation inducers such as sodium butyrate or phorbol 12-myristate 13-acetate (PMA). On the other hand, untreated HL-60 and U-937 cells exhibited no spontaneous AF, but they acquired this function when induced to differentiate either along the granulocytic pathway (dimethyl sulfoxide-treated HL-60 cells) or along the monocytic pathway (PMA-treated HL-60 and U-937 cells). Supernatants from PMA-triggered K562 or HL-60 cells allowed the proliferative response of murine thymocytes to phytohemagglutinin A and were therefore shown to contain IL-1. Analysis of phenotypical markers showed that AF and IL-1 production were not restricted to cells of the monocytic lineage. No HLA-DR antigen could be detected on K562 and HL-60 cells. Thus, the expression of HLA-DR antigens is not required for AF and IL-1 production in response to mitogens. Human leukemia cell lines could provide useful sources of human IL-1.     

Cytochemical and immunophenotypic heterogeneity in acute promyelocytic leukemia

The leukemic promyelocytes in 37 cases of acute promyelocytic leukemia (APML; FAB, M3) were examined for their cytochemical property. Thirty-two cases (86%) showed strong myeloperoxidase (MPO), chloroacetate esterase (Es-chl) and Sudan black B (SBB) positivity, suggesting a pure neutrophilic differentiation of the leukemic cells. However, in 5 out of 37 cases, a strong, diffuse alpha-naphthyl acetate esterase (Es-a) positivity, which was sensitive to sodium fluoride treatment was observed in addition to strong MPO, Es-chl and SBB positivity. This suggested monocytic differentiation of a proportion of APML cases. In 31 cases, surface marker studies were carried out with the help of a panel of monoclonal antibodies consisting of two panmyeloid antibodies (GM 58/8 and 1G10), one anti-HLA-DR antibody (7.2) and one 'myeloid' antibody (5F1) with restricted reactivity with monocytes (CD14). The purpose of including the monoclonal antibodies 5F1 and 7.2 was to determine if a correlation could be established between strong Es-a positivity and reactivity of the leukemic promyelocytes with 5F1 and 7.2 in individual cases. All the 5 cases with 'monocytoid' cytochemistry were unreactive with 5F1, and only one case in this group showed 15% 7.2-positive cells. The lack of immunophenotypic support for the monocytic cytochemistry of the 5 cases of APML suggests that the monocytic phenotype of leukemic promyelocytes is both aberrant and incomplete. Since normal promyelocytes are purely neutrophilic, this could be a manifestation of an 'intralineage infidelity' in APML, similar to that observed in other types of acute leukemia. The clinical significance of 'monocytic' phenotype of APML is not clear; cases with monocytic differentiation did not show different clinical and hematological features when compared to the more common, pure neutrophilic variety.     

Biologic characteristics of acute leukemia after myelodysplastic syndrome

Ten patients with acute leukemia after primary myelodysplastic syndrome (MDS-AL) were examined to clarify the biologic nature of the leukemic cells in comparison with that of de novo acute myelocytic leukemia (AML). The morphologic and cytochemical features of the leukemic cells from all these patients corresponded well to those of de novo AML, and they were diagnosed with MDS-AML. Phenotypically, the frequent expression of the lymphocyte activation antigens, CD25 and CD30, was characteristic in MDS-AML. The in vitro response of MDS-AML cells to various growth factors was similar to that of de novo AML cells. Transforming growth factor beta 1 (TGF beta 1) suppressed growth factor- dependent colony formation by normal bone marrow cells, MDS bone marrow cells, and de novo AML cells, but did not inhibit colony formation by MDS-AML cells. The number of TGF beta 1 high-affinity binding sites of MDS-AML samples (< 5 to 47 sites/cell) was markedly lower than that in de novo AML samples (120 to 221 sites/cell). Our results indicate that the reduced TGF beta 1 may represent disregulation of the proliferation system in MDS-AML cells. This is thought to occur during the MDS phase, and may be related to the poorer response shown to conventional chemotherapy of AML.     

Autocrine growth mechanisms of the progenitors of blast cells in acute myeloblastic leukemia

Autocrine growth mechanisms of leukemic blast progenitors in acute myeloblastic leukemia (AML) were investigated. Colony formation of leukemic blast progenitors was observed in 14 of 14 patients tested when purified blast cell fraction depleted of both T cells and monocytes was plated in methylcellulose without any colony-stimulating factor (CSF). However, there existed a minimal cell density required to initiate blast progenitor growth with marked patient-to-patient variation. To clarify the role of cell density on the spontaneous growth of blast progenitors, we tested whether leukemic cells produced and secreted some stimulatory humoral factor(s). Production of colony- stimulating activity (CSA) by blast cells was observed in 17 of 18 patients tested. Following further depletion of monocytes, the CSA levels decreased markedly in 14 patients, indicating that blast cells with monocytoid differentiation were responsible for CSA production. We also confirmed granulocyte colony-stimulating factor (G-CSF) and/or granulocyte macrophage-colony-stimulating factor (GM-CSF) production by leukemic blasts using specific immunologic assays. When leukemic cells were divided into nonadherent nonphagocytic cell fraction and adherent cell fraction, only nonadherent nonphagocytic cells showed clonogenecity and adherent blast cells lacked the colony-forming capacity. The results indicate that there are at least two blast cell subpopulations in AML: one is proliferating subpopulation with self- renewal capacity and the other is supporting subpopulation with functions such as CSF production. The quite intimate relationship between these two blast cell subpopulations in AML may play an important role on the growth of leukemic blast progenitors in vitro.     

Human natural killer cells can inhibit clonogenic growth of fresh leukemic cells

The effect of allogenic human natural killer (NK) cells on fresh leukemic cells from three patients was investigated. The low levels of leukemic target cell lysis in the conventional 51Cr-release assay contrasted with a pronounced inhibitory effect on the colony growth of the clonogeneic leukemic target cells (L-CFC). The ability of allogeneic lymphocytes to inhibit L-CFC increased if they were pretreated with interferon (IFN), which also increased their NK activity, monitored in parallel cytotoxicity assay, against K562. Furthermore, cell separation procedures, based on differences in density among nonadherent lymphocytes, revealed that only NK cell containing fractions were inhibitory. We have also compared the susceptibility to NK-mediated L-CFC inhibition of IFN pretreated leukemic target cells with that of nontreated target cells. As in the case of NK lysis in general, this pretreatment of target cells abolished the presumably NK-mediated L-CFC inhibition. In conclusion, these data provide the first indication that NK cells can inhibit the in vitro growth of fresh clonogenic leukemia cells from patients with nonlymphocytic leukemia. The identity of NK cells as effector is strongly suggested by Percoll separation and responsiveness to interferon; the final proof awaits more sophisticated purification of these cells.