Staging of leukemic progenitor cells in acute myeloid leukemia by phenotyping with myeloid monoclonal antibodies

We studied the immune phenotype of leukemic progenitor cells (AML-CFU-L) in 16 cases of acute myeloid leukemia (AML) using 12 myeloid monoclonal antibodies (McAbs) in a complement-mediated cytotoxicity assay. On the basis of their reactivities with normal day-14 (D14) and day-7 (D7) myeloid progenitor cells (CFU-GM), these McAbs could be classified into three groups: six McAbs reacted strongly with "early" antigens on D14-CFU-GM, two McAbs reacted only with "late" antigens on D7-CFU-GM, while four McAbs formed an "intermediate" group that reacted both with the "late" antigens on D7-CFU-GM and to some extent with the "early" antigens of D14-CFU-GM. The McAbs all reacted with antigens on AML-CFU-L:McAbs that reacted with "early" antigens reacted consistently strongly with AML-CFU-L, in contrast to McAbs in the two other groups, which displayed greater heterogeneity. On the basis of antigenic phenotype, the predominating CFU-L in AML could thus be placed in one of three "stages." This phenotypic "staging" of AML correlated with the French-American-British (FAB) morphological classification of AML. The AML-CFU-L of patients with FAB morphological types M2, M4, and M5 expressed more differentiated antigenic phenotypes than those of the three patients with M7. The latter reacted poorly with the "intermediate" and "late" McAbs. Our data and those of others suggest that classification based on AML-CFU-L antigenic profile may complement the FAB classification of AML.     

急性髓细胞白血病细胞诱导分化成为树突状细胞的实验研究

目的 :探讨急性髓细胞白血病 (AML)患者的白血病细胞体外是否可诱导分化成为树突状细胞(DC)。方法 :从 11例AML患者的骨髓或外周血中获取非贴壁细胞 ,利用细胞因子 (rhGM CSF、rhIL 4、rhTNF α)联合培养 ,每 12d收获细胞。培养前后分别用倒置显微镜、电镜观察细胞形态 ,用流式细胞术测定细胞表面标志 ,用MTT法检测收获细胞激发混合淋巴细胞反应的能力。结果 :8例AML标本分化成为具有典型树突状形态的细胞 ,培养后的白血病细胞的HLA DR、CD86、CD1a表达较培养前明显增高 ,差异有统计学意义 (P <0 .0 1)。在混合淋巴细胞反应中 ,DC具有强烈激发同种T淋巴细胞增殖的能力 ,且随数量增加而作用增强。M4/M5型AML DC的表面标志的表达率明显高于非M4/M5型AML DC(P <0 .0 1)。结论 :急性髓细胞白血病细胞可以诱导分化成为具有DC形态、表型、功能的细胞。     

In vitro sensitivity of normal and leukemic myeloid clonogenic cells to hyperthermia: absence of selective effect

The in vitro heat sensitivity of myeloid clonogenic cells was tested in 22 normal marrows (granulo-monocytic progenitors) and in 40 marrows of patients with acute myelogenous leukemia (leukemic progenitors). Cells were treated for 1 h at 42 degrees, 43 degrees, 44 degrees, or 45 degrees C prior to plating. A temperature-dependent inhibition of growth was seen without a selective effect on the two kinds of progenitors. Because these two kinds of progenitors have the same heat sensitivity, hyperthermia should not be used alone as a technique for in vitro depletion of residual myeloid leukemic cells.     

Purging of acute myeloid leukaemia cells from stem cell grafts by hyperthermia: enhancement of the therapeutic index by the tetrapeptide AcSDKP and the alkyl-lysophospholipid ET-18-OCH(3)

Hyperthermia has been shown to be a potential purging modality in autologous stem cell transplantation settings owing to its selective toxicity towards leukaemic cells. We describe two approaches to further increase the therapeutic index of the hyperthermic purging modality by using normal murine bone marrow cells and a murine model for acute myeloid leukaemia. First, the tetrapeptide AcSDKP was used to protect the normal haematopoietic progenitor cells against hyperthermic damage. Pretreatment for 8 h at 37 degrees C with 1 x 10(-9) mol/l AcSDKP resulted in a decrease in hyperthermic sensitivity of only normal haematopoietic progenitor cells. This combined treatment protocol revealed a therapeutic index (ratio of surviving fractions of normal vs. leukaemic cells) of > 500, which was considered to be sufficient for purging. This was confirmed in vivo by the survival of lethally irradiated recipients transplanted with purged simulated remission bone marrow (1 x 10(6) normal bone marrow cells and 5 x 10(4) leukaemic cells). A further increase of the therapeutic index cells was achieved by the alkyl-lysophospholipid ET-18-OCH(3). An incubation for 4 h at 37 degrees C with 25 microg/ml in the presence of 5% fetal calf serum preferentially enhanced the cytotoxic effect towards the leukaemic stem cell. The combination of AcSDKP and ET-18-OCH(3) with hyperthermia resulted in a therapeutic index of > 5000. This enabled a reduction of the hyperthermic treatment and will further minimize the toxicity to normal haematopoietic stem cell subsets, while a therapeutic index far above the required value is achieved. This tripartite purging treatment therefore offers a safe and fast purging protocol for the elimination of residual leukaemic cells in autografts.     

Flow cytometric quantification and immunophenotyping of leukemic stem cells in acute myeloid leukemia

Leukemic stem cells (LSCs) are root of clonal growth in acute myeloid leukemia (AML) and responsible for the propagation of leukemic blasts (LBs). LSCs are considered as CD34?+?CD38- population among LBs and often express as CD123, CD44, or CD184, which are rarely expressed on normal hematopoietic stem cells and could be the potential therapeutic targets. Using multi-color flow cytometry, we analyzed the proportions of CD34?+?CD38- LSCs and expression of CD123, CD44, and CD184 on LSCs in 63 patients with AML. The median proportion of LSCs was 1.3?% (0.0-33.1?%) at the time of diagnosis. Of all patients, 74.6?% of them had CD123-positive LSCs, all patients had CD44-positive LSCs, and 85.7?% had CD184-positive LSCs, respectively. The proportions of LSCs were significantly lower in the complete remission (CR) group compared with non-CR group (P?=?0.006). The lower proportions of LSCs in CR group indicated that measurement of the proportion of LSCs might be helpful to predict the prognosis of AML.     

Expression of a myeloid marker on TdT-positive acute lymphocytic leukemic cells: evidence by double-fluorescence staining

The expression of a myeloid-specific antigen was detected on TdT- positive blast cell populations in two cases of childhood acute lymphocytic leukemia. Double-fluorescence staining by using the monoclonal antibody, VIM-D5, which is specific for cells of myeloid origin, in combination with TdT antiserum revealed that a distinct portion of the blast cells carried both markers. The finding represents the first direct demonstration of this specific biphenotype in leukemic cells and was interpreted as the abnormal expression of a myeloid antigen on lymphoid blast cells.     

Purging leukemic cells from simulated human remission marrow with alkyl- lysophospholipid

Alkyl-lysophospholipids (ALP) are analogues of 2- lysophosphatidylcholine that have been reported to have selective antitumor activity. These compounds could potentially be useful in purging bone marrow of leukemic cells in autologous marrow transplantation in acute leukemia. To determine the efficacy of pharmacological purging by ALP, we have designed a human assay system to mimic the conditions expected in the clinical setting of autotransplantation using remission marrow. A simulated remission marrow (SRM) was prepared by mixing normal marrow cells and HL60 cells in a ratio of 1,000:1. The effect of cryopreservation on ALP-treated normal, HL60, and SRM cells was examined. In separate experiments, ALP significantly reduced the number of clonogenic HL60 cells with no effect on normal marrow progenitors. The effect of ALP was more apparent after cryopreservation. Incubation of HL60 cells with 50 micrograms/mL ALP for four hours followed by cryopreservation resulted approximately in a 3 log reduction of clonogenic HL60 cells. ALP also selectively purged the small number of leukemic cells from SRM. In SRM, the data suggested that ALP had indirect cytotoxic activity on leukemic cells by enhancing the cytotoxic activity of monocytes in addition to its direct effect. We found no evidence that clonogenic HL60 cells decreased because of induction of differentiation by ALP. These data indicated that treatment of marrow cells with ALP offers an efficient means to eliminate leukemic cells from the graft.     

Application of hyperthermia to the treatment of human acute leukemia: purging human leukemic progenitor cells by heat

The application of hyperthermia to the treatment of neoplastic disease has focused on solid tumors. Since the hyperthermic sensitivity of human acute leukemia cells is not known, we have studied the in vitro response of human leukemic progenitor cells (L-CFU) to hyperthermia using a quantitative assay system for L-CFU. Human L-CFU were found to be more sensitive than committed normal myeloid progenitor cells to hyperthermic killing (41 to 42 degrees C). In addition, in the five acute myelogenous leukemic patients studied, it was shown that their leukemic progenitor cells--all types were studied according to the French-American-British diagnosis--were unable to form colonies when exposed to a temperature of 42 degrees C for 60 minutes, whereas the residual normal clones suppressed by the leukemic cell population were found to recover and to form more colonies in vitro as compared with untreated leukemic marrows. This strongly suggests that in vitro hyperthermia may selectively purge residual leukemic cells, especially L-CFU in stored remission bone marrow before autologous bone marrow transplantation.     

肿瘤坏死因子相关凋亡诱导配体联合柔红霉素诱导急性髓系白血病细胞凋亡的研究

目的探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)联合柔红霉素(DNR)诱导急性髓系白血病细胞凋亡的作用。方法分离21例急性髓系白血病患者的骨髓或外周血单个核细胞,每例分为4组进行观察：空白对照组、TRAIL组、DNR组、TRAIL DNR组,共同培养6、12、24小时。通过MTr比色法测定细胞杀伤率、DNA电泳及流式细胞仪检测细胞凋亡。结果TRAIL和柔红霉素可协同降低急性髓系自血病细胞活力,抑制细胞增殖,诱导细胞凋亡,其作用随培养时间延长及药物浓度增加而增强。结论TRAIL与柔红霉素具有协同作用,能高效杀灭肿瘤细胞。TRAIL是一种有望应用于临床的新型生物制剂。     

肿瘤坏死因子相关凋亡诱导配体联合阿糖胞苷诱导急性髓细胞性白血病细胞凋亡的研究

目的：探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)联合阿糖胞苷(Ara-C)诱导急性髓细胞性白血病(AML)细胞凋亡的作用。方法：分离39例AML患者的骨髓单个核细胞，每例分为4组进行观察：空白对照组、TRAIL组、Ara-C组、TRAIL加Ara-C组，共同培养12、24和48h。通过MTT比色法测定细胞杀伤率及流式细胞仪检测细胞凋亡。结果：TRAIL和Ara-C可协同降低AML细胞活力，抑制细胞增殖，诱导细胞凋亡，其作用随培养时间延长及药物浓度增加而增强。结论：TRAIL与Ara-C具有协同作用，能高效杀灭肿瘤细胞，可能是一种有临床应用潜能的新型抗白血病的生物制剂。     

BCR-ABL-transformed GMP as myeloid leukemic stem cells

During blast crisis of chronic myelogenous leukemia (CML), abnormal granulocyte macrophage progenitors (GMP) with nuclear β-catenin acquire self-renewal potential and may function as leukemic stem cells (Jamieson et al. N Engl J Med, 2004). To develop a mouse model for CML-initiating GMP, we expressed p210^BCR-ABL in an established line of E2A-knockout mouse BM cells that retain pluripotency in ex vivo culture. Expression of BCR-ABL in these cells reproducibly stimulated myeloid expansion in culture and generated leukemia-initiating cells specifically in the GMP compartment. The leukemogenic GMP displayed higher levels of β-catenin activity than either the nontransformed GMP or the transformed nonGMP, both in culture and in transplanted mouse BM. Although E2A-deficiency may have contributed to the formation of leukemogenic GMP, restoration of E2A-function did not reverse BCR-ABL-induced transformation. These results provide further evidence that BCR-ABL-transformed GMP with abnormal β-catenin activity can function as leukemic stem cells.     

Indirect induction of differentiation in myeloid leukemic cells by lipid A

Normal myeloid and MGI(+)D(+) clones of myeloid leukemic cells can be induced for Fc and complement component 3 rosettes, lysozme, and mature macrophages and granulocytes by a protein with macrophage- and granulocyte-inducing (MGI) activity, whereas MGI(+)D(-) clones can be induced by this protein for rosettes and lysozme but not mature cells. Lipopolysaccharides (LPS) from different bacteria induced the appearance of rosettes, lysozyme, and macrophages in some MGI(+)D(+) clones but did not induce any of these changes in MGI(+)D(-) clones. Lipid A gave the same results as LPS. Incubation of MGI(+)D(+) cells with LPS also induced an MGI activity detectable in the culture medium. This activity behaved like MGI in inducing (i) rosettes, lysozyme, and mature cells in MGI(+)D(+) leukemic cells including a clone resistant to LPS, (ii) rosettes and lysozyme in MGI(+)D(-) leukemic cells, and (iii) differentiation of normal myeloid cells to mature macrophages and granulocytes. This activity was induced in MGI(+)D(+) cells by LPS before the induction of rosettes or lysozyme. The results indicate that the lipid A portion of LPS indirectly induces differentiation of MGI(+)D(+) myeloid leukemic cells by inducing MGI protein. It is suggested that induction of specific regulatory proteins may be a more general mechanism for the induction of differentiation by surface-acting compounds.     

Near-tetraploid minimally differentiated acute myeloid leukemia with extensive erythrophagocytosis by leukemic blasts

 Numerical change of chromosomes is common in acute myeloid leukemia (AML). However, a chromosome number as high as near-tetraploidy is very rare, especially in minimally differentiated AML (AML-M0). Erythrophagocytosis by reactive or malignant histiocytes is common in malignant hematological diseases; however, erythrophagocytosis by leukemic blasts is also very rare, especially in AML-M0. We report here the first case of AML-M0 with both of these unique characteristics: a near-tetraploid karyotype and erythrophagocytosis by leukemic blasts. Received November 12, 1998 / Accepted: June 28, 1999     

A self-assembled leucine polymer sensitizes leukemic stem cells to chemotherapy by inhibiting autophagy in acute myeloid leukemia

Chemotherapy is the primary treatment option for acute myeloid leukemia (AML), but leukemic stem cells (LSC) can survive chemotherapy for disease recurrence and refractory. Here, we found that AML cells obtained from relapsed patients had increased autophagy levels than de novo AML cells. Furthermore, doxorubicin (DOX) treatment stimulated autophagy in LSC by repressing the mTOR pathway, and pharmaceutical inhibition of autophagy rendered chemoresistant LSC sensitive to DOX treatment in MLL-AF9 induced murine AML. Moreover, we developed a self-assembled leucine polymer, which activated mTOR to inhibit autophagy in AML cells by releasing leucine. The leucine polymer loaded DOX (Leu-DOX) induced much less autophagy but more robust apoptosis in AML cells than the DOX treatment. Notably, the leucine polymer and Leu-DOX were specifically taken up by AML cells and LSC but not by normal hematopoietic cells and hematopoietic stem/progenitor cells in the bone marrow. Consequently, Leu-DOX efficiently reduced LSC and prolonged the survival of AML mice, with more limited myeloablation and tissue damage side effects than DOX treatment. Overall, we proposed that the newly developed Leu-DOX is an effective autophagy inhibitor and an ideal drug to efficiently eliminate LSC, thus serving as a revolutionary strategy to enhance the chemotherapy efficacy in AML.     

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     

T-cell acute lymphoblastic leukemia with severe leukopenia: evidence for suppression of myeloid progenitor cells by leukemic blasts

A patient with T-cell acute lymphoblastic leukemia presented with leukopenia due to neutropenia, no circulating blasts and normal hemoglobin level. Marrow leukemic T lymphoblasts inhibited in vitro normal CFU-GM colony growth and released an activity that stimulated normal BFU-E growth. This patient demonstrates that immature T cells may modify hematopoietic stem cell growth both in vitro and in vivo.     

Stimulation of autologous proliferative and cytotoxic T-cell responses by "leukemic dendritic cells" derived from blast cells in acute myeloid leukemia

Effective presentation of tumor antigens is fundamental to strategies aimed at enrolling the immune system in eradication of residual disease after conventional treatments. Myeloid malignancies provide a unique opportunity to derive dendritic cells (DCs), functioning antigen-presenting cells, from the malignant cells themselves. These may then co-express leukemic antigens together with appropriate secondary signals and be used to generate a specific, antileukemic immune response. In this study, blasts from 40 patients with acute myeloid leukemia (AML) were cultured with combinations of granulocyte-macrophage colony-stimulating factor, interleukin 4, and tumor necrosis factor alpha, and development to DCs was assessed. After culture, cells from 24 samples exhibited morphological and immunophenotypic features of DCs, including expression of major histocompatibility complex class II, CD1a, CD83, and CD86, and were potent stimulators in an allogeneic mixed lymphocyte reaction (MLR). Stimulation of autologous T-cell responses was assessed by the proliferative response of autologous T cells to the leukemic DCs and by demonstration of the induction of specific, autologous, antileukemic cytotoxicity. Of 17 samples, 11 were effective stimulators in the autologous MLR, and low, but consistent, autologous, antileukemic cytotoxicity was induced in 8 of 11 cases (mean, 27%; range, 17%-37%). This study indicates that cells with enhanced antigen-presenting ability can be generated from AML blasts, that these cells can effectively prime autologous cytotoxic T cells in vitro, and that they may be used as potential vaccines in the immunotherapy of AML.