Acute myeloid leukemia with T-lymphoid features: a distinct biologic and clinical entity

We studied the clinical and biologic features of 10 cases of acute leukemia that met standard French-American-British (FAB) criteria for acute myeloid leukemia (AML) but in which the blast cells also expressed the T-cell-associated CD2 surface antigen. All cases had greater than 3% myeloperoxidase and Sudan black B-positive leukemic blasts, and blasts from seven cases contained Auer rods. Reactivity of the cells with a panel of monoclonal antibodies (MAbs) indicated that leukemic cells in all cases expressed myeloid-associated (CD11b, CD13) surface antigens, further supporting the diagnosis of AML. However, blasts from every patient coexpressed the T-cell-associated surface CD2 and CD7 as well as cytoplasmic CD3 antigens. Blasts from five patients expressed surface CD25, whereas blasts from only one expressed surface CD3. Five patients had rearranged T-cell receptor beta-chain genes, whereas only three had rearranged T-cell receptor gamma-chain genes. This pattern of lineage-related gene expression appears to define a distinct subtype of AML with T-lymphoid features (CD2+ AML) and could reflect either aberrant gene expression in leukemic blasts or transformation of a pluripotent stem cell having a flexible pattern of gene expression. Clinically, these 10 patients presented at an older age with a higher leukocyte count and a higher frequency of lymphadenopathy than did children whose blast cells were characteristic of myeloid leukemia. Patients with CD2+ AML also had poorer responses to remission induction therapy (50% v 80% entered complete remission, P = .05). However, each of the five children who failed induction chemotherapy on AML protocols had a striking response to drug combinations usually reserved for lymphoid leukemia. We conclude that this leukemia with mixed lymphoid and myeloid characteristics is a distinct biologic and clinical entity.     

Characterization of a hierarchy in human acute myeloid leukemia progenitor cells

Despite the usual uniform and primitive appearance of cells derived from the leukemic clone in most patients with acute myeloid leukemia (AML), there is considerable heterogeneity among leukemic blasts, particularly with respect to their capacity to proliferate and/or self renew. We have assessed whether these differences in proliferative potential are correlated with the phenotypic changes that characterize normal hematopoiesis, which might suggest an analogous hierarchy of AML progenitors. We have used the ability of primitive AML cells to persist or produce blast colony forming cells (CFU-blast) detected after 2 to 8 weeks in the presence of growth factors in suspension cultures (SC) termed SC-initiating cells (IC), or with stroma in long-term cultures (LTC-IC) as a quantitative assay for a cell that may have primitive characteristics. This SC assay is linear, cell concentration independent, and the frequency of SC-IC by limiting dilution analysis is lower than primary CFU-blast. The average output of CFU-blast after 2 to 8 weeks by individual SC-IC varied between 2 and more than 100 in individual patients. Leukemic blasts were sorted based on their expression of antigens previously found useful to characterize normal progenitor differentiation, and analyzed for the percentage of CFU- blast SC-IC, and leukemic LTC-IC within each fraction. All of these progenitor types were heterogeneous in their expression of CD45RA and CD33, but expressed uniformly low levels of CD15 and differed from normal primitive progenitors in their high expression of HLA-DR. CFU- blast had a significantly higher expression of CD71 and CD38 as compared with SC-IC or leukemic LTC-IC. In patients with CD34+ blasts, the majority of their SC-IC at 4 weeks were CD34+/CD38-; however, patients with CD34- blasts had at least some CD34- progenitors. These results show that while heterogeneity exists between patients, it is possible to physically separate subpopulations of AML cells with different proliferative potentials. It also provides some support for the concept that quantitation of leukemic cells capable of producing CFU-blast for 4 weeks or more in vitro measures a less frequent leukemic progenitor with higher proliferative potential that may be the only relevant cell for maintaining the leukemic clone in vivo.     

Class II-associated invariant chain peptide down-modulation enhances the immunogenicity of myeloid leukemic blasts resulting in increased CD4+ T-cell responses

Background

Disease recurrence in patients with acute myeloid leukemia may be partially explained by the escape of leukemic blasts from CD4⁺ T-cell recognition. The current study investigates the role of aberrant HLA class II antigen presentation on leukemic blasts by determining both the clinical and functional impact of the class II-associated invariant chain peptide (CLIP).

Design and Methods

The levels of expression of CLIP and HLA-DR on blood and bone marrow samples from 207 patients with acute myeloid leukemia were correlated with clinical outcome. Irradiated CLIP⁻ and CLIP⁺ leukemic blasts were compared for their ability to induce CD4⁺ T cells during mixed leukocyte reactions. To discriminate between these blasts, we down-modulated CLIP expression on myeloid leukemic cell lines by RNA interference of the invariant chain, a chaperone protein critically involved in HLA-DR processing, and performed flow cytometric sorting for their isolation from primary acute myeloid leukemia samples.

Results

We found that patients with leukemic blasts characterized by a high amount of HLA-DR occupied by CLIP (relative amount of CLIP) had a significantly shortened disease-free survival. The clear reductions in amount of HLA-DR occupied by CLIP on blasts of the THP-1 and Kasumi-1 myeloid leukemic cell lines after treatment with invariant chain short interfering RNA resulted in enhanced rates of allogeneic CD4⁺ T-cell proliferation. Similar findings were obtained in an autologous setting, in which there were strong increases in proliferation of remission CD4⁺ T cells stimulated with CLIP⁻-sorted leukemic blasts from HLA-DR⁺ acute myeloid leukemia patients, in contrast to CLIP⁺-sorted leukemic blasts from the same patients.

Conclusions

These data highlight the relevance of CLIP expression on leukemic blasts and the potential of CLIP as a target for immunomodulatory strategies to enhance HLA class II antigen presentation and CD4⁺ T-cell reactivity in acute myeloid leukemia.     

Expression of integrins and examination of their adhesive function in normal and leukemic hematopoietic cells

Adhesion of hematopoietic progenitor cells to marrow-derived adherent cells has been noted for erythroid, myeloid, and lymphoid precursors. In this report, we have characterized very late antigen (VLA) integrin expression on normal CD34+ marrow progenitors, on leukemic cell lines, and on blasts from patients with acute myelogenous or monocytic leukemias. CD34+ progenitor cells expressed the integrin beta 1 chain (CD29), VLA-4 alpha (CD49d), and VLA-5 alpha (CD49e). The myeloid lines KG1 and KG1a also expressed CD49d and CD49e as did the Mo7e megakaryoblastic line. CD29, CD18, and CD11a were also present on each of these cell lines. Only the Mo7e line expressed the cytoadhesins GPIIbIIIa or GPIb. Binding of KG1a to marrow stroma was partially inhibited by antibodies to CD49d and its ligand, vascular cell adhesion molecule (VCAM-1). The majority of leukemic blasts studied expressed CD49d and CD49e as well. Blasts from patients with acute myelomonocytic leukemia consistently bound to stroma at levels greater than 20%, and adhesion to stroma could in some cases be partly inhibited by anti- CD49d. No role for glycosylphosphatidyl-inositol (GPI)-linked structures was demonstrated in these binding assays because the adhesion of leukemic blasts to stroma was not diminished after treatment with phosphatidylinositol-specific phospholipase C (PI-PLC). These studies indicate that CD34+ myeloid progenitors, myeloid leukemic cell lines, and leukemic blasts possess a similar array of VLA integrins. Their functional importance individually or in combination with other mediators of attachment in adhesion, transendothelial migration, and differentiation has yet to be fully elucidated.     

Malignant progenitors from patients with CD87+ acute myelogenous leukemia are sensitive to a diphtheria toxin-urokinase fusion protein

In previous studies, we demonstrated that the diphtheria toxin-urokinase fusion protein DTAT was selectively toxic to acute myeloid leukemia (AML) cell lines overexpressing the CD87 urokinase receptor. In the present study, we analyzed the sensitivity of patient leukemic progenitors to DTAT and correlated the sensitivity with CD87 expression.We isolated leukemic blasts by density gradient centrifugation and performed immunophenotyping by flow cytometry and blast sensitivity measurements by inhibition of cell proliferation and colony formation in semisolid media.We found CD87 overexpression in 18 (25%) of 71 patient leukemic blast samples, including 18 (28%) of 64 myeloid malignancies and 0 (0%) of 7 lymphoid malignancies. DTAT was toxic to patient leukemic blasts by both proliferation inhibition (IC50 85% inhibition by 10 nM DTAT in 11/41 evaluable samples). Only AML and chronic myeloid leukemia (CML) blast crisis blasts (18/61 [30%]) were sensitive to DTAT by the proliferation inhibition assay. Lymphoid leukemia and chronic phase CML/chronic myelomonocytic leukemia (CMML) progenitors were insensitive to DTAT by the proliferation inhibition assay (n = 7 and n = 3, respectively). Similarly, normal marrow progenitors were insensitive to DTAT by both proliferation inhibition (n = 2) and colony inhibition (n = 5) assays. The DTAT toxicity measured by both proliferation inhibition assay and colony inhibition assay correlated with CD87 density (p < 0.0001 and p = 0.001, respectively). DTAT toxicity results were similar for leukemic blasts measured by either of the two assays (p = 0.0002).This study provides the first evidence that a urokinase receptor targeted diphtheria fusion protein is toxic to patient AML blasts. The work also suggests that blast proliferation assays yield similar responses to leukemia colony-forming cell colony assays.     

Acute leukemia associated with the t(4;11) chromosome rearrangement: ultrastructural and immunologic characteristics

The acute leukemia associated with the t(4;11) chromosome rearrangement is characterized by relatively consistent clinical features: occurrence primarily in young individuals, hyperleukocytosis, and poor response to therapy. This study describes the morphological, ultrastructural, and immunologic characteristics of the leukemic cells from ten patients with this type of leukemia. The morphological features of the leukemic blasts vary from lymphoid-appearing to monocytic. Ultrastructurally and cytochemically, some of the lymphoid-appearing blasts possess features of myeloid origin. The immunologic phenotype is characteristically E- SIg- CALLA- BA-1- BA-2+ HLA-DR+ and TdT+. These findings suggest that the t(4;11)-associated acute leukemia represents a proliferation of an early myeloid progenitor cell.     

Characterization of Thy-1 (CDw90) expression in CD34+ acute leukemia

Thy-1 (CDw90) is a phosphatidylinositol-anchored cell surface molecule which, when coexpressed with CD34 in normal human bone marrow, identifies a population of immature cells that includes putative hematopoietic stem cells. To date, the characterization of Thy-1 expression has been confined largely to normal tissues and cell lines. In this study, we evaluated the frequency and intensity of Thy-1 expression as defined by reactivity with the anti-Thy-1 antibody 5E10 in 38 cases of CD34+ acute leukemia (21 acute myelogenous leukemia [AML], 8 chronic myelogenous leukemia [CML] in blast crisis, and 9 acute lymphoblastic leukemia [ALL]). In 34 of 38 cases (89%) the CD34+ cells lacked expression of the Thy-1 antigen. High-density Thy-1 expression was found in 1 case of CML in lymphoid blast crisis, and low- density Thy-1 expression was identified on a portion of the leukemic cells in 2 cases of AML with myelodysplastic features, and 1 case of CML in myeloid blast crisis, suggesting a possible correlation between Thy-1 expression and certain instances of stem cell disorders such as CML and AML with dysplastic features. In contrast, the dissociation of Thy-1 and CD34 expression in the majority of acute leukemias studied suggests that the development of these leukemias occurs at a later stage than the hematopoietic stem cell. Characterization of Thy-1 expression in acute leukemia may eventually provide insights into the origin of the disease. In addition, separation of leukemic blasts from normal stem cells based on Thy-1 expression may prove useful in assessing residual disease, as well as in excluding leukemic blasts from stem cell preparations destined for autologous bone marrow or peripheral stem cell transplantation.     

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.     

Study of differentiation of fresh human leukemic cells by low dose cytosine arabinoside (LD Ara-C) and 12-O-tetradecanoylphorbol-13-acetate (TPA)

The effect of LD Ara-C (10(-8) mol/l) (Ara-C), TPA (1.6 x 10(-7) mol/l) and 13-cis-retinoic acid (RA) (10(-6) mol/l) on the differentiation in liquid culture of bone marrow cells from 5 patients with acute lymphoblastic, 17 patients with acute myelogenous leukemia, 1 patient in myeloid and 1 in lymphoid crisis of chronic granulocytic leukemia was studied. Ara-C induced morphological and cytochemical differentiation into monocytic cells in 2 cases (M1, M5 type). TPA induced convincing morphological and cytochemical features of maturation into monocytic cells in 4 cases (two M1, one M2, and one M5 type) and into differentiated myeloid cells in 2 cases (M1, M4 type). RA in one case (M2 type) out of three AML studied induced cytochemical and immunocytochemical features of maturation. The results of the study indicate that although TPA is a better inducer of blast cell differentiation than Ara-C, however, neither is a potent differentiation agent of leukemic blasts in liquid culture. The heterogeneity of leukemic blasts within the same type of leukemia was confirmed by their different response to differentiating agents.     

Fas ligand is highly expressed in acute leukemia and during the transformation of chronic myeloid leukemia to blast crisis.

Fas ligand (FasL) induces apoptosis in susceptible Fas-bearing cells and is critically involved in regulating T-cell immune responses. It is highly expressed in several human malignancies, and a role in the suppression of antitumor immune responses has been suggested. We evaluated FasL expression in leukemia and normal hematopoietic cells. By Western blotting, all acute leukemic cell lines (n = 9) and primary samples of acute leukemic marrow (n = 4) revealed high levels of FasL. In contrast, much weaker signals were observed in samples of normal marrow (n = 5), and either weak or intermediate expression was seen in chronic myeloid leukemia (CML) in chronic phase (n = 7). Additional leukemic samples were examined by immunohistochemistry. Staining for FasL was negative in 7 of 9 cases of chronic-phase CML, whereas all cases of CML in blast crisis (n = 6), acute lymphoblastic leukemia (n = 6), and acute myeloid leukemia (n = 11) stained strongly in 60 to 100% of nucleated cells. FasL+ leukemic cell lines did not trigger Fas-mediated apoptosis in either Jurkat cells or activated human T lymphocytes, possibly related to the intracellular location of the ligand. Western analysis of normal marrow subpopulations revealed that most FasL in marrow mononuclear cells was expressed by CD7+ lymphocytes. FasL also was strongly expressed in CD34+ hematopoietic progenitor cells from both normal and chronic-phase CML marrow, suggesting a correlation with primitive maturation stage. In summary, high levels of FasL expression were associated with aggressive biologic behavior in leukemia, including transformation of CML to blast crisis. This could potentially represent a response to loss of proapoptotic Fas signaling, which is known to occur in acute leukemic blasts.     

5'-(3')-nucleotidase mRNA levels in blast cells are a prognostic factor in acute myeloid leukemia patients treated with cytarabine

We analyzed cytosolic 5'-(3')-nucleotidase (dNT-1) mRNA expression by quantitative polymerase chain reaction at diagnosis in leukemic blasts from 114 patients with acute myeloid leukemia (AML) treated with ara-C. Our results show that low dNT-1 mRNA expression in leukemic blasts at diagnosis is correlated with a worse clinical outcome and suggest that this enzyme may have a role in sensitivity to ara-C in AML patients.     

High CD33 expression levels in acute myeloid leukemia cells carrying the nucleophosmin (NPM1) mutation

The CD33 antigen is expressed on the blast cells of most cases of acute myeloid leukemia and represents a suitable tumor-associated target antigen for antibody-based therapies. The aim of this study was to investigate the relationship between the CD33 levels quantified by mean fluorescence intensity and antibody binding capacity, and the presence/absence of NPM1 and FLT3 gene mutations in 99 newly diagnosed acute myeloid leukemia cases. The CD33 intensity evaluated as mean fluorescence intensity and antibody binding capacity was significantly higher in the NPM1-mutated acute myeloid leukemia cases compared to the NPM1-unmutated cases (P=0.0001 and P=0.0088, respectively). On the contrary, FLT3 gene mutations did not influence the levels of CD33 expression on the leukemic cells. These results establish a rational basis for the therapeutic use of anti-CD33 antibodies in NPM1-mutated acute myeloid leukemia patients.     

Endothelial cell activation by myeloblasts: molecular mechanisms of leukostasis and leukemic cell dissemination

Leukostasis and tissue infiltration by leukemic cells are poorly understood life-threatening complications of acute leukemia. This study has tested the hypothesis that adhesion receptors and cytokines secreted by blast cells play central roles in these reactions. Immunophenotypic studies showed that acute myeloid leukemia (AML) cells (n = 78) of the M0 to M5 subtypes of the French-American-British Cooperative Group expressed various amounts of adhesion receptors, including CD11a, b, c/CD18, CD49d, e, f/CD29, CD54, sCD15, and L-selectin. The presence of functional adhesion receptors was evaluated using a nonstatic adhesion assay. The number of blast cells attached to unactivated endothelium increased by 7 to 31 times after a 6-hour exposure of endothelium to tumor necrosis factor (TNF)-alpha. Inhibition studies showed that multiple adhesion receptors--including L-selectin, E-selectin, VCAM-1, and CD11/CD18--were involved in blast cell adhesion to TNF-alpha-activated endothelium. Leukemic cells were then cocultured at 37 degrees C on unactivated endothelial cell monolayers for time periods up to 24 hours. A time-dependent increase in the number of blasts attached to the endothelium and a concomitant induction of ICAM-1, VCAM-1, and E-selectin were observed. Additional experiments revealed that endothelial cell activation by leukemic myeloblasts was caused by cytokine secretion by blast cells, in particular TNF-alpha and IL-1 beta, and direct contacts between adhesion receptors expressed by blast cells and endothelial cells. Thus, leukemic cells have the ability to generate conditions that promote their own adhesion to vascular endothelium, a property that may have important implications for the pathophysiology of leukostasis and tissue infiltration by leukemic blast cells. (Blood. 2001;97:2121-2129)     

Resistance of leukemic blasts to lymphokine activated killer (LAK)-mediated cytotoxicity is not related to their adhesion properties.

The expression of adhesion molecules on blasts from 14 patients with acute myeloid leukemia (AML) was investigated by immunofluorescence and flow cytofluorometry. All tested blast populations expressed CD18/CD11a complex [leukocyte function antigen-1 (LFA-1)] and CD29 (very-late antigen (VLA)) and the majority were positive for CD54 [intercellular adhesion molecule-1 (ICAM-1), 78.6%] and CD56 [neural cell adhesion molecule (NCAM), 64.3%]. The expression of two other alpha chains of CD18/CD11b and CD11c varied considerably (64.3% and 42.8% of positive cases, respectively). Only one case (AML-M4) showed a weak expression of the activated platelet antigen CD41b. None of the tested blasts expressed the vitronectin receptor (CD61/CD51). No significant correlation between the expression of adhesion molecules and the FAB type of leukemia could be found. All tested blast populations were completely resistant to NK-mediated cytotoxicity and relatively resistant to LAK-mediated cytotoxicity in the standard 51Cr release assay. While no statistically significant correlation of the results in cytotoxicity assays with the expression of adhesion molecules or the expression of HLA-DR antigen could be observed, 2 out of 3 completely resistant cases lacked ICAM-1. These results show that even leukemic blasts which express all of the tested adhesion molecules can still be resistant to LAK-mediated cytotoxicity.     

RHAMM/HMMR (CD168) is not an ideal target antigen for immunotherapy of acute myeloid leukemia

Background

Criteria for good candidate antigens for immunotherapy of acute myeloid leukemia are high expression on leukemic stem cells in the majority of patients with acute myeloid leukemia and low or no expression in vital tissues. It was shown in vaccination trials that Receptor for Hyaluronic Acid Mediated Motility (RHAMM/HMMR) generates cellular immune responses in patients with acute myeloid leukemia and that these responses correlate with clinical benefit. It is not clear however whether this response actually targets the leukemic stem cell, especially since it was reported that RHAMM is expressed maximally during the G2/M phase of the cell cycle. In addition, tumor specificity of RHAMM expression remains relatively unexplored.

Design and Methods

Blood, leukapheresis and bone marrow samples were collected from both acute myeloid leukemia patients and healthy controls. RHAMM expression was assessed at protein and mRNA levels on various sorted populations, either fresh or after manipulation.

Results

High levels of RHAMM were expressed by CD34⁺CD38⁺ and CD34^- acute myeloid leukemia blasts. However, only baseline expression of RHAMM was measured in CD34⁺CD38^- leukemic stem cells, and was not different from that in CD34⁺CD38^- hematopoietic stem cells from healthy controls. RHAMM was significantly up-regulated in CD34⁺ cells from healthy donors during in vitro expansion and during in vivo engraftment. Finally, we demonstrated an explicit increase in the expression level of RHAMM after in vitro activation of T cells.

Conclusions

RHAMM does not fulfill the criteria of an ideal target antigen for immunotherapy of acute myeloid leukemia. RHAMM expression in leukemic stem cells does not differ significantly from the expression in hematopoietic stem cells from healthy controls. RHAMM expression in proliferating CD34+ cells of healthy donors and activated T cells further compromises RHAMM-specific T-cell-mediated immunotherapy.Key words: leukemic stem cell, acute myeloid leukemia, cell therapy and immunotherapy, HMMR, RHAMM     

Analysis of peroxidase-negative acute unclassifiable leukemias by monoclonal antibodies. 1. Acute myelogenous leukemia and acute myelomonocytic leukemia

In this study, pretreatment peripheral and/or bone marrow blasts from 12 patients with acute unclassifiable leukemia (AUL) expressing the myeloid-related cell-surface antigen (CD 11) were isolated for further analysis. Despite a lack of myeloperoxidase (MPO) activity, 1 patient's blasts contained cytoplasmic Auer rods. The circulating blasts from another patient expressed MPO while maintaining the same surface phenotype during 20 months of clinical follow-up. In addition, the blasts from 3 cases demonstrated both myelomonocytic and monocyte-specific surface antigens, whereas the remaining 9 cases completely lacked any monocyte-specific antigen detectable by monoclonal antibodies, Mo2, My4 and Leu M3 (CD 14). The first case eventually was diagnosed as acute myelomonocytic leukemia and the second as acute myelogenous leukemia by means of immunophenotypic analysis using flow cytometry (FACS IV). In addition, the presence of MPO protein was identified in the cytoplasm of blast cells from 5 patients with AUL by means of a cytoplasmic immunofluorescence test using a monoclonal antibody (MA1). Our study indicates that non-T, non-B AUL expressing OKM1 (CD 11) antigens include acute leukemias which are unequivocally identifiable as being of either myeloid or myelomonocytic origin. However, further investigations, including immunophenotypic and cytoplasmic analysis, ultrastructural cytochemistry and gene analysis with molecular probes (tests applicable to normal myeloid cells), are necessary in order to determine the actual origin of blasts and to recognize the differentiation stages of the various types of leukemic cells from patients with undifferentiated forms of leukemia.     

Expression of the hematopoietic growth factor receptor FLT3 (STK- 1/Flk2) in human leukemias

Normal expression of the hematopoietic growth factor receptor FLT3 (STK- 1@Flk2) is limited to CD34+ stem/progenitor cells. We have evaluated the expression of FLT3 by RNase protection assay and Western blotting in 161 primary bone marrow (BM) samples from patients with leukemia. FLT3 RNA was found to be expressed at a higher level than in normal BM controls in 33 of 33 B-lineage acute leukemias, 11 of 12 acute myeloid leukemias (AMLs), and 3 of 11 T-cell acute leukemias (T-ALLs). Expression of FLT3 RNA was also observed in some cases of blast crisis CML. The FLT3 signal resulted from expression on the leukemic blasts, and was not caused by increased FLT3 expression on normal CD34+ stem/progenitor cells in the leukemic samples. To determine if FLT3 protein was also overexpressed, proteins were extracted from leukemic BM samples and screened by Western blotting with anti-FLT3 antisera. FLT3 protein was not detected in normal BM controls, but was found in 14 of 14 B-lineage ALLs, 36 of 41 AMLs, and 1 of 4 T-ALLs. Stimulation of patient samples with FLT3 ligand resulted in autophosphorylation of the FLT3 receptor, suggesting the receptor is functional in these cells. These data show that FLT3 RNA and protein are aberrantly expressed by AML and ALL cells in that CD34 expression and FLT3 expression are no longer synchronous, and suggest the possibility that overexpression of FLT3 could play a role in the survival and/or proliferation of malignant clones in acute myeloid and lymphoid leukemias.