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.     

Diisopropylfluorophosphate binding proteins (serine hydrolases) from normal and leukemic hematopoietic cells

Normal and leukemic hematopoietic cell lysates were labeled with [3H]-diisopropylfluorosorophosphate ([3H]-DFP), an active site inhibitor of serine hydrolases. The labeled proteins in the lysates were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by counting of gel segments for radioactivity. The results indicate the presence of distinct [3H]-DFP binding patterns for different normal and leukemic hematopoietic cells; significantly lower labeling in normal or leukemic lymphoid cells compared to myeloid or monocytoid cells; lower labeling in acute myeloblastic leukemia (FAB-M1) as compared to acute myelomonocytic leukemia (FAB-M4), chronic myelomonocytic leukemia or monocytes and an increase in [3H]-DFP binding with cell maturation along granulocytic series. Thus, these patterns could be useful in discriminating acute lymphoblastic leukemia from myeloid/monocytoid types of leukemia and for following maturation of myeloid cells, and perhaps for studying functional or maturation defects in hematopoietic cells in other pathological conditions.     

Analysis of acute myeloid leukemia cells by flow cytometry,introducing a new light-scattering classification

A combined flow-cytometric evaluation of light scattering and the immunophenotype of acute myeloid leukemia (AML) cells from 71 newly diagnosed consecutive patients was conducted. Light-scattering characteristic of AML cells examined by flow cytometry and multiple surface markers were also analyzed using the same samples, to enable a comparison with the French-American-British (FAB) classification. Our AML cases could be classified into three light-scattering classification (LSC) types according to their physical properties on flow cytometry. These were type A, where forward light scattering (FSC) of the leukemic cell population was larger than that of lymphocytes, while side light scattering (SSC) was the same or larger than that of lymphocytes but smaller than that of monocytes; type B, where FSC of the leukemic cell population was larger than that of lymphocytes and SSC spread toward that of monocytes; and type C, where both FSC and SSC of the leukemic cell population spread beyond those of monocytes. Although a clear relationship between the FAB classification and LSC classification by the light-scattering profile of AML was not established, we observed the following findings. The majority of cases were classified as type A (58%), while type B comprised 25% and type C comprised 17%. While CD7 expression on AML cells is considered to be an immature characteristic, CD7 was expressed more frequently among LSC type A cases. Furthermore, all but one of the FAB M1 cases were classified as type A. On the other hand, CD7 was not expressed on type C leukemic cells. The percentage of cases in which more than 60% of leukemic cells possessed another immature surface antigen, CD 34ö, was 13/18 (72%) among FAB M1 cases, much higher than among FAB M2 (35%) or FAB M4 (27%) cases. A negative correlation was observed between mature antigen CD33 and CD34 among the FAB M2 cases. The frequency of CD7 expression was 25% among the total cases, and CD7-positive cases were frequent among FAB M1 and M2, but not among FAB M3 cases. These findings concerning LSC and immunophenotyping indicate that the scattergram pattern analysis may contribute towards more precise immunophenotyping, in that it reflects the maturation stage of each AML case.Abbreviations AML acute myeloid leukemia - LSC light-scattering classification - FAB French-American-British classification - CD cluster of differentiation Partly supported by grants in aid from the Ministry of Education, Science and Culture of Japan (03670325, 04247102, 04454572 and 05670916) and from the Fukuoka Anti-cancer Society     

Effects of human stem cell factor (c-kit ligand) on proliferation of myeloid leukemia cells: heterogeneity in response and synergy with other hematopoietic growth factors.

A novel hematopoietic growth factor, the stem cell factor (SCF), for primitive hematopoietic progenitor cells has recently been purified and its gene has been cloned. In this study we tested the mitogenic activity of recombinant human SCF on myeloid leukemia cells as well as the expression of its receptor. We have investigated the proliferation of 31 myeloid leukemia cell lines as well as fresh myeloid leukemic blasts from 17 patients in a 72-hour 3H-thymidine uptake assay in the presence of various concentrations of recombinant human (rh) SCF alone or in combination with saturating concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, M-CSF, interleukin-3 (IL-3), or erythropoietin (EPO). Only five of 31 lines, but fresh leukemic blasts from 12 of 17 patients with acute myeloid leukemia (AML), significantly responded to SCF. The responding cell lines were of the acute promyelocytic, chronic myeloid, megakaryoblastic, and erythroleukemia origin, the responding blast preparations of all French-American-British subtypes. Synergistic activities of SCF were found with G-CSF, GM-CSF, EPO, and IL-3. To determine the SCF binding sites on leukemic cells, we used 125I-radiolabeled SCF in Scatchard analysis and cross-linking studies. The leukemic cell lines responding to SCF expressed from 2,300 up to 29,000 binding sites per cell. The SCF receptor expression was downregulated in vitro by the presence of its ligand. Cross-linking studies demonstrated a 150-Kd SCF receptor on the surface of all responding myeloid leukemias. This study suggests that SCF may be an important factor for the growth of myeloid leukemia cells, either as a direct stimulus or as a synergistic factor for other cytokines. Furthermore, using polymerase chain reaction analysis of total RNA from the myeloid leukemia lines, we found expression of SCF-mRNA in 17 of 30 lines, suggesting autocrine mechanisms in the growth of a subgroup of leukemic cells by coexpression of SCF and its receptor.     

Concurrent acute myeloid leukemia with inv(16)(p13.1q22) and chronic lymphocytic leukemia: molecular evidence of two separate diseases

Acute myeloid leukemia (AML) occurring concurrently with or after untreated chronic lymphocytic leukemia (CLL) is rare. We report a case of a 59-year-old man who was evaluated for anemia, thrombocytopenia, and leukocytosis with circulating blasts. On the basis of the morphology and immunophenotyping results, a preliminary diagnosis of chronic myelomonocytic leukemia with concurrent CLL was considered. Subsequently, cytogenetic analysis of the leukemic blood specimen revealed inv(16)(p13.1q22) with secondary trisomy 22 in a sideline clone. Fluorescence in situ hybridization confirmed the CBFbeta rearrangement associated with inv(16) in myeloblasts and myelomonocytic cells, but not in CLL cells. Therefore, a final diagnosis of AML with inv(16) with concurrent CLL was made. After standard chemotherapy for AML, the patient achieved complete remission for both his AML and CLL. The unique aspects of this case include concomitant AML and CLL, which do not share clonality, complex cytogenetic abnormalities with trisomy 22 as a secondary abnormality associated with inv(16), and achievement of remission for both AML and CLL by AML chemotherapy regimen. This case also represents one of the rare instances where a diagnosis of AML can be established even when the blast percentage in the marrow and blood is less than 20%.     

Tyrosine kinase inhibitor therapy can cure chronic myeloid leukemia without hitting leukemic stem cells

Background

Tyrosine kinase inhibitors, such as imatinib, are not considered curative for chronic myeloid leukemia – regardless of the significant reduction of disease burden during treatment – since they do not affect the leukemic stem cells. However, the stochastic nature of hematopoiesis and recent clinical observations suggest that this view must be revisited.

Design and Methods

We studied the natural history of a large cohort of virtual patients with chronic myeloid leukemia under tyrosine kinase inhibitor therapy using a computational model of hematopoiesis and chronic myeloid leukemia that takes into account stochastic dynamics within the hematopoietic stem and early progenitor cell pool.

Results

We found that in the overwhelming majority of patients the leukemic stem cell population undergoes extinction before disease diagnosis. Hence leukemic progenitors, susceptible to tyrosine kinase inhibitor attack, are the natural target for chronic myeloid leukemia treatment. Response dynamics predicted by the model closely match data from clinical trials. We further predicted that early diagnosis together with administration of tyrosine kinase inhibitor opens the path to eradication of chronic myeloid leukemia, leading to the wash out of the aberrant progenitor cells, ameliorating the patient’s condition while lowering the risk of blast transformation and drug resistance.

Conclusions

Tyrosine kinase inhibitor therapy can cure chronic myeloid leukemia, although it may have to be prolonged. The depth of response increases with time in the vast majority of patients. These results illustrate the importance of stochastic effects on the dynamics of acquired hematopoietic stem cell disorders and have direct relevance for other hematopoietic stem cell-derived diseases.     

c-fms expression is a molecular marker of human acute myeloid leukemias

The c-fms protooncogene product was identified as the CSF-1 or M-CSF receptor, a polypeptide growth factor that plays a major role in myelomonocytic differentiation. This led us to look for expression of c-fms in fresh acute myeloid leukemia (AML) cells, using Northern blot analysis. c-fms expression was found in the leukemic cells of 28 AML patients, regardless of their stage of differentiation, which was assessed in the French-American-British (FAB) classification. However, the level of c-fms expression was especially high in AML of the M5 stage. High levels of expression were not accompanied by either amplification or rearrangements of the c-fms gene in AML cell DNAs. In contrast, c-fms expression was not found in acute lymphoid leukemias, whether of T or B origin. Thus, c-fms expression appears as a specific molecular marker of leukemogenesis in the myeloid lineage.     

Use of surface markers to identify a subset of acute myelomonocytic leukemia cells with progenitor cell properties

The expression of differentiation-associated surface antigens was used to identify subsets of human acute myelomonocytic leukemia cells. The leukemic colony-forming cells expressed antigens characteristic of very immature myeloid cells (Ia, MY7), while the majority of leukemic cells also expressed an antigen (MY4) characteristic of later myeloid cells. The highly proliferative MY4 negative colony-forming cells may serve as progenitor cells for the less proliferative MY4 + leukemic cells.     

The origin of neoplastic mast cells in systemic mastocytosis with AML1/ETO-positive acute myeloid leukemia

OBJECTIVE: Systemic mastocytosis with associated clonal hematological non-mast cell lineage disease (SM-AHNMD) is a distinct entity that was defined by World Health Organization. Systemic mastocytosis with acute myeloid leukemia (AML) is frequently seen among SM-AHNMD. However, the pathogenesis or origin of neoplastic mast cells has not been fully elucidated in this category of diseases. METHODS: We examined KIT mutation, chimeric status, and AML1/ETO mRNA concerning mast cells and immature hematopoietic cells of the bone marrow in a patient with systemic mastocytosis with AML1/ETO-positive AML following allogeneic hematopoietic stem cell transplantation (HSCT). RESULTS: Mast cells of the patient displayed KIT D816Y mutation, and were derived from the recipient. In contrast, immature hematopoietic cells as defined by CD34+ CD117+ were derived from the donor, which did not possess detectable KIT D816Y mutation. The ratio of AML1/ETO to 18S rRNA of the mast cells was 7.53, whereas that of immature hematopoietic cells was 1.67. CONCLUSIONS: In a patient with SM-AHNMD who underwent allogeneic HSCT, the major source of the detectable AML1/ETO mRNA of the bone marrow after transplantation was neoplastic mast cells with KIT mutation, which were thought to be derived from CD34+ CD117+ immature leukemic cells of the recipient.     

Terminal differentiation surface antigens of myelomonocytic cells are expressed in human promyelocytic leukemia cells (HL60) treated with chemical inducers

The expression of two surface antigens present on the cell membrane of both human granulocytes and monocytes was studied during the process of myelomonocytic differentiation using two monoclonal antibodies (B9.8.1 and B13.4.1). These surface antigens are not present on immature myeloid cells nor on nonmyeloid hematopoietic cells, but can be detected when the cells are terminally differentiated. Among the bone marrow cells, B13.4.1 binds to metamyelocytes and B9.8.1 to metamyelocytes and a fraction (30%) of myelocytes. HL60 human promyelocytic leukemia cells did not react with such monoclonal antibodies. However, when such cells were induced to differentiate in vitro into mature myeloid elements by treatment with retinoic acid or dimethyl sulfoxide, 70%--90% of the differentiated cells expressed both surface antigens. Cell sorting studies on these treated HL60 cells indicated that myelocytes and metamyelocytes were the most immature cells expressing such markers. Expression of the two surface antigens was also observed when HL60 cells were induced to differentiate into monocyte/macrophage cells by treatment with the tumor promoter 12-O- tetradecanoyl-phorbol-13-acetate. Thus, human promyelocytic leukemia cells induced to differentiate in vitro by treatment with specific chemical agents express membrane antigens in the same pattern as normal bone marrow myeloid cells at the corresponding stage of differentiation.     

A retroviral mutagenesis screen reveals strong cooperation between Bcl11a overexpression and loss of the Nf1 tumor suppressor gene

NF1 inactivation occurs in specific human cancers, including juvenile myelomonocytic leukemia, an aggressive myeloproliferative disorder of childhood. However, evidence suggests that Nf1 loss alone does not cause leukemia. We therefore hypothesized that inactivation of the Nf1 tumor suppressor gene requires cooperating mutations to cause acute leukemia. To search for candidate genes that cooperate with Nf1 deficiency in leukemogenesis, we performed a forward genetic screen using retroviral insertion mutagenesis in Nf1 mutant mice. We identified 43 common proviral insertion sites that contain candidate genes involved in leukemogenesis. One of these genes, Bcl11a, confers a growth advantage in cultured Nf1 mutant hematopoietic cells and causes early onset of leukemia of either myeloid or lymphoid lineage in mice when expressed in Nf1-deficient bone marrow. Bcl11a-expressing cells display compromised p21(Cip1) induction, suggesting that Bcl11a's oncogenic effects are mediated, in part, through suppression of p21(Cip1). Importantly, Bcl11a is expressed in human chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia samples. A subset of AML patients, who had poor outcomes, of 16 clusters, displayed high levels of BCL11A in leukemic cells. These findings suggest that deregulated Bcl11a cooperates with Nf1 in leukemogenesis, and a therapeutic strategy targeting the BCL11A pathway may prove beneficial in the treatment of leukemia.     

Clinical importance of bone marrow monocytic nodules in patients with myelodysplasia: retrospective analysis of 21 cases

Bone marrow monocytic nodules (MNs) can occur in various myeloid disorders. This retrospective review identified 21 patients with myelodysplasia who had unusual and distinct MNs. Eight patients had chronic myelomonocytic leukemia (CMML); 4 had acute myeloid leukemia (AML); and 9 had myelodysplastic/myeloproliferative diseases. In each case, the cells forming MNs expressed strong CD68. MNs appeared to persist even after aggressive chemotherapy, including conventional chemotherapy for 2 AML patients and high-dose chemotherapy preceding allogeneic bone marrow transplantation for 1 CMML patient. Thirteen of 21 patients (62%) died, and acute leukemic transformation was the main cause of death in 3 of 8 patients with CMML. The median survival of the 20 patients with appropriate follow-up was 9.8 months. Our findings demonstrate that MNs are associated with CMML, AML, myelodysplastic syndromes, and myeloproliferative diseases and suggest that MNs are resistant to intensive chemotherapy and patients with bone marrow MNs have a poor prognosis.     

Uncommon phenotypes of acute myelogenous leukemia: basophilic, mast cell, eosinophilic, and myeloid dendritic cell subtypes: a review

The potential of the transformed (leukemic) multipotential hematopoietic cell to differentiate and mature along any myeloid lineage forms the basis for the phenotypic classification of acute and chronic myelogenous leukemia. Although most cases of leukemia can be classified phenotypically by the dominant lineage expressed, the genotype within each phenotype is heterogeneous. Thus, covert genetic factors, cryptic mutations, and/or polymorphisms may interact with the seminal transforming genetic mutations to determine phenotype. The phenotype usually is expressed sufficiently to determine the lineage that is dominant in the leukemic clone by light microscopic examination, by cytochemistry of blood and marrow cells, and by immunophenotyping.The basis for the frequency of the AML phenotypes is unclear, although there is a rough concordance with the frequency of marrow precursor cells of different lineages. The least common AML phenotypes are a reflection of the least common blood or marrow cell lineages: acute basophilic, acute mast cell, acute eosinophilic, and acute myeloid dendritic cell leukemia. We discuss the features of these uncommon phenotypes and review the criteria used for their diagnosis.     

Basophils (Bsp-1+) derive from the leukemic clone in human myeloid leukemias involving the chromosome breakpoint 9q34

The monoclonal antibody (MoAb) Bsp-1 was used to purify basophilic cells from leukemic blood of five patients with Philadelphia chromosome (Ph') positive chronic myeloid leukemia (CML) and two patients with acute myeloid leukemia (AML) characterized by the chromosomal translocation t(6;9)(p23;q34). When cultured, Bsp-1 positive cells from all CML and AML patients showed the same clonal karyotype changes observed in diagnostic buffy coat preparations, indicating that the basophilic cells were of leukemic origin. In contrast, T lymphocytes from four of five CML patients cultured in the presence of interleukin- 2 (IL-2) showed a normal karyotype and were therefore not derived from the leukemic clone. Bsp-1 staining correlated with toluidine blue- positive basophils in chronic phase CML and with toluidine blue- negative blast cells expressing an immature myeloid phenotype in blast crisis CML and AML. Chromosome in situ hybridization showed that the ABL oncogene was translocated from chromosome 9 to chromosome 22 in the CML patients but remained on chromosome 9 in the AML patients. These results indicate that the breakpoint at 9q34 in CML is 5' of ABL, whereas the breakpoint at 9q34 in AML is 3' of ABL. Field inversion gel electrophoresis showed that the 9q34 breakpoint was not within 200 kb 3' of ABL in one of the AML patients, nor was there any rearrangement of the PIM oncogene locus at 6p21.     

The serological detection of leukemia-associated antigens in chronic leukemia: Correlation with disease status

Changes in the expression of leukemia-associated antigens (LAA) on the peripheral blood cells of patients with chronic lymphocytic leukemia (CLL) and chronic myelogenous leukemia (CML) were monitored over a period of several months with a battery of heterologous antisera in a complement-dependent microcytotoxicity test system. The sera were prepared by immunization of rabbits and monkeys with human CLL, CML, or acute lymphoblastic, myeloblastic, or myelomonocytic leukemic cells, or cell extracts. Following extensive adsorption with normal human erythrocytes and leukocytes, these sera did not exhibit reactivity with peripheral blood monuclear cells, nor, in most cases, with enriched B-lymphocyte preparations from healthy donors. Cells from CLL patients reacted mainly with adsorbed antisera raised against lymphocytic leukemic cells, but there was significant cross-reactivity with antisera raised against myelomonocytic leukemia cells; the number and intensity of positive reactions correlated with leukemic cell counts. Cells from patients in the chronic stage of CML reacted almost exclusively with adsorbed antisera to myeloid leukemic cells. However, blastic transformation of CML was regularly accompanied and on one occasion preceded by the appearance of reactivity with antisera to lymphocytic leukemic cells. During remission, peripheral blood cells from most CML patients were LAA-negative; positive serologic reactions were often followed by major hematologic and clinical deterioration within a few months. These observations suggest that serological monitoring of LAA may represent a useful adjunct to present methods of evaluating the course and prognosis of chronic leukemias. Serological reactions may also have some utility in the differential diagnosis of leukemia.     

Remission maintenance in acute myeloid leukemia: impact of functional histamine H2 receptors expressed by leukemic cells

Post-consolidation immunotherapy with histamine dihydrochloride and interleukin-2 has been shown to improve leukemia-free survival in acute myeloid leukemia in a phase III trial. For this study, treatment efficacy was determined among 145 trial patients with morphological forms of acute myeloid leukemia as defined by the French-American-British classification. Leukemia-free survival was strongly improved in M4/M5 (myelomonocytic/monocytic) leukemia but not in M2 (myeloblastic) leukemia. We also analyzed histamine H₂ receptor expression by leukemic cells recovered from 26 newly diagnosed patients. H₂ receptors were typically absent from M2 cells but frequently expressed by M4/M5 cells. M4/M5 cells, but not M2 cells, produced reactive oxygen species that triggered apoptosis in adjacent natural killer cells. These events were significantly inhibited by histamine dihydrochloride. Our data demonstrate the presence of functional histamine H₂ receptors on human AML cells and suggest that expression of these receptors by leukemic cells may impact on the effectiveness of histamine-based immunotherapy.Key words: acute myeloid leukemia, remission maintenance, histamine H₂ receptors     

Bone marrow mesenchymal stromal cells non-selectively protect chronic myeloid leukemia cells from imatinib-induced apoptosis via the CXCR4/CXCL12 axis

Background

Residual chronic myeloid leukemia disease following imatinib treatment has been attributed to the presence of quiescent leukemic stem cells intrinsically resistant to imatinib. Mesenchymal stromal cells in the bone marrow may favor the persistence and progression of leukemia by preserving the proliferation and self-renewal capacities of the malignant progenitor cells.

Design and Methods

BV173 or primary chronic myeloid leukemia cells were co-cultured with human mesenchymal stromal cells and imatinib-induced cell death was then measured. The roles of pro-and anti-apoptotic proteins and chemokine CXCL12 in this context were evaluated. We also studied the ability of BV173 cells to repopulate NOD/SCID mice following in vitro exposure to imatinib and mesenchymal stromal cells.

Results

Whilst imatinib induced dose-dependent apoptosis of BV173 cells and primary chronic myeloid leukemia cells, co-culture with mesenchymal stromal cells protected both types of chronic myeloid leukemia cells. Molecular analysis indicated that mesenchymal stromal cells reduced caspase-3 activation and modulated the expression of the anti-apoptotic protein Bcl-XL. Furthermore, chronic myeloid leukemia cells exposed to imatinib in the presence of mesenchymal stromal cells retained the ability to engraft into NOD/SCID mice. We observed that chronic myeloid leukemia cells and mesenchymal stromal cells express functional levels of CXCR4 and CXCL12, respectively. Finally, the CXCR4 antagonist, AMD3100 restored apoptosis by imatinib and the susceptibility of the SCID leukemia repopulating cells to the tyrosine kinase inhibitor.

Conclusions

Human mesenchymal stromal cells mediate protection of chronic myeloid leukemia cells from imatinib-induced apoptosis. Disruption of the CXCL12/CXCR4 axis restores, at least in part, the leukemic cells’ sensitivity to imatinib. The combination of anti-CXCR4 antagonists with tyrosine kinase inhibitors may represent a powerful approach to the treatment of chronic myeloid leukemia.     

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.