Effects of thrombopoietin on megakaryocyte colony formation from leukemic cells at diagnosis and from marrow cells after induction chemotherapy for acute leukemias

 We have studied the effects of recombinant human thrombopoietin (TPO, mpl ligand) on the megakaryocyte colony formation from control human bone marrow cells, human leukemia cells at diagnosis, and human bone marrow cells after induction chemotherapy for acute leukemias. In the control human bone marrow cells from four adults and nine children who had localized malignancy and histologically normal-looking marrow, TPO alone effectively stimulated megakaryocyte colony formation, and interleukin-3 (IL-3) synergized this. In 17 patients (13 adults and four children) with acute myeloid leukemia (AML) at diagnosis, TPO stimulated leukemic colony formation in only one patient with FAB M7 subtype. In 11 children with acute lymphoblastic leukemia (ALL) at diagnosis, TPO did not enhance leukemic colony formation. After 17 courses of induction chemotherapy, nine for AML and eight for ALL, TPO stimulated megakaryocyte colony formation to a level of 51% of that in the control human bone marrow cells. This may suggest that the administration of TPO to patients with M7 subtype warrants caution, whereas it is probably safe to give TPO at any time to patients with ALL. The administration of TPO to patients with acute leukemias after induction chemotherapy could stimulate megakaryocytopoiesis. Received: July 29, 1997 / Accepted: May 6, 1998     

Growth of normal versus leukemic bone marrow cells in long term culture from acute lymphoblastic and myeloblastic leukemias

Summary The ability of the in vitro long-term bone marrow culture (LTBMC) system to impair the survival of leukemic cells and to enhance the growth of normal progenitors has been studied. Bone marrow cells from 19 acute lymphoblastic leukemia (ALL) and 30 acute myeloid leukemia (AML) patients at diagnosis were grown in LTBMC for 4–10 weeks. In half of the cases the leukemic population declined down to undetectable levels and was replaced by putative normal hemopoietic precursors, both in ALL and in AML. In the remaining cases, leukemic cells persisted throughout the culture time and few if any normal hemopoietic cells were detected. These data led us to extend to the lymphoid compartment the previous observation of decreasing leukemic myeloid blasts in LTBMC. The potential of such cultures as an in vitro purging system for autologous bone marrow transplantation in selected poor-prognosis lymphoid malignancies should be explored, as has been done for acute and chronic myeloid leukemias.     

bcl-2 proto-oncogene expression in normal and neoplastic human myeloid cells.

The present study provides immunobiochemical and molecular data on the differentiation-linked expression of the bcl-2 proto-oncogene in normal and neoplastic myeloid cells. Using a recently developed monoclonal antibody (MoAb) to the bcl-2 molecule, staining of normal bone marrow myeloblasts, promyelocytes, and myelocytes, but neither monocytes nor most polymorphonuclear cells, was demonstrated. By two-color flow cytometric analysis, bcl-2 was evidenced in CD33+ and CD33+/CD34+ myeloid cells as well as in the more primitive CD33-/CD34+ population. The leukemic cell lines HL-60, KG1, GM-1, and K562 were bcl-2 positive together with 11 of 14 acute myeloid leukemias (AML) and three of three chronic myeloid leukemias (CML) in blast crises; six of seven CML were negative. Among myelodysplastic cases, augmentation of the bcl-2 positive myeloblastic compartment was found in refractory anemia with excess of blasts (RAEB) and in transformation (RAEB-t). Western blots of myeloid leukemias and control lymphocytes extracts evidenced an anti-bcl-2 immunoreactive band of the expected size (26 Kd). Moreover, the HL-60 and KG1 cell lines, both positive for the bcl-2 protein, exhibited the appropriate size bcl-2 mRNA (7.5 Kb). These findings clearly indicate that the bcl-2 gene is operative in myeloid cells and that the anti-bcl-2 MoAb identifies its product and not a cross-reactive epitope. Induction of HL-60 differentiation toward the monocytic and granulocytic pathways was accompanied by a marked decrease in bcl-2 mRNA and protein levels; bivariate flow cytometric analysis showed that the fraction becoming bcl-2 negative was in the G1 phase of the cell cycle. These data establish that the bcl-2 proto-oncogene is expressed on myeloid cells and their progenitors and is regulated in a differentiation-linked manner.     

Selective targeting of human lymphokine-activated killer cells by CD3 monoclonal antibody against the interferon-inducible high-affinity Fc gamma RI receptor (CD64) on autologous acute myeloid leukemic blast cells

The potential of the CD3 monoclonal antibody (MoAb) OKT3 to selectively target lymphokine-activated killer (LAK) cells and T-cell clones in vitro against autologous tumor cells was studied using material from patients with acute leukemias (19 acute myeloid leukemias [AML], and 3 acute lymphoblastic leukemias [ALL]). Cytotoxicity mediated by patient LAK cells against AML blasts, but not against ALL cells and autologous Epstein-Barr virus-transformed B cells, was enhanced 1.5-fold to 9.3- fold by OKT3 in all AML patients studied. The following findings suggest that the major target molecule on AML cells for OKT3-coated LAK cells is the high-affinity Fc receptor for IgG (Fc gamma RI; CD64): (1) susceptibility to killing by OKT3-coated effector LAK cells segregated with target cell expression of CD64; (2) preincubation of AML blasts with monomeric OKT3 (murine IgG2a), the Fc portion of which is known to have preferential binding affinity to CD64, resulted in lysis by autologous T cells that were not spontaneously cytotoxic; (3) OKT3- dependent increase in lysis of primary and relapsed AML cells by autologous T-cell clones correlated with the amount of target cell expression of CD64; (4) anti-leukemic cytotoxicity of OKT3-coated T cells could partially be inhibited by monomeric human Ig, the natural ligand of CD64; and (5) expression of CD64 (Fc gamma RI) on fresh AML cells could be increased by interferon-gamma (IFN-gamma) and IFN-alpha translating into further enhancement of lysis by autologous OKT3-coated LAK cells. Nonmalignant CD34+ cells sorted from peripheral blood were found to lack expression of CD64 and hence were not affected by OKT3- triggered T-cell targeting, as detected by colony formation assays. In conclusion, the in vitro data presented provide a rationale for the combined clinical use of recombinant interleukin-2, IFN-gamma, and low doses of CD3 MoAb to eliminate AML cells while sparing nonmalignant hematopoietic progenitor cells, for example, in the setting of purging procedures for autologous bone marrow transplantation.     

Different expression of adhesion molecules on CD34+ cells in AML and B-lineage ALL and their normal bone marrow counterparts.

The expression of adhesion molecules on CD34+ cells in acute myeloid leukemia (AML) and B-lineage acute lymphoblastic leukemia (B-lineage ALL) was compared with that on the myeloid and B-lymphoid CD34+ cells in normal bone marrow. Bone marrow aspirates of 10 patients with AML, 8 patients with B-lineage ALL and of 6 healthy volunteers were examined. The phenotype of the CD34+ cells was determined with a double immunofluorescence method and flow cytometry. CD34+ cells in AML and B-lineage ALL showed a lower expression of VLA-2 and VLA-3 and a higher expression of ICAM-1 and LFA-3 than their normal bone marrow counterparts. AML CD34+ cells had less L-selectin but more VLA-5 on their surface membrane than normal myeloid CD34+ cells. B-lineage ALL CD34+ cells showed an overexpression of LFA-3. In individual patients deficiencies or over-expression of the beta1 integrin chain, VLA-4, PECAM-1 or HCAM also occurred. An abnormal adhesive capacity of the leukemic cells may influence their proliferation, their localisation and apoptosis. An aberrant expression of adhesion molecules may be used for the detection of minimal residual leukemia in these patients.     

Adhesion molecules on CD 34+ hematopoietic cells in normal human bone marrow and leukemia

Summary Expression of selected adhesion molecules of the integrin and immunoglobulin family was investigated on CD 34+ leukemic cells in 19 AML and 11 ALL cases to evaluate phenotypic differences in adhesive properties of malignant hematopoietic precursor cells in comparison to normal bone marrow CD 34+ cells. Of the 2-integrin family, CD 11a was expressed on > 50% of CD 34+ cells in normal bone marrow and almost all leukemias, whereas CD 11 b and CD 11 c were not expressed on CD 34+ cells in normal bone marrow, but were found on CD 34+ blasts in some leukemias of a heterogeneous immunophenotype. Of the 1-family, CDw 49d (VLA-4) was strongly expressed on normal CD 34+ bone marrow cells and on the blasts of all 30 CD 34+ leukemic samples, whereas CDw 49 b (VLA-2) was absent on CD 34+ cells in normal bone marrow, but detected on CD 34+ cells in a few leukemias which did not constitute a clinical or phenotypic entity according to the FAB classification or immunocytological analysis. The lymphocyte-homing-associated adhesion molecule CD 44 (HCAM) and CD 58 (LFA-3) were expressed on CD 34+ cells in all investigated cases of normal and leukemic bone marrow. ICAM-1 (CD 54), the inducible receptor ligand for CD 11 a/CD 18, although present on CD 34+ cells in normal bone marrow, was lacking on blast cells of some ALL and AML cases. So far, the variable expression of 2-integrins as well as of VLA-2 and of ICAM-1 could indicate distinct differences in cell-cell or cell-matrix adhesion of leukemic cells in ALL and AML patients.     

Stromal-mediated down-regulation of CD13 in bone marrow cells originating from acute myeloid leukemia patients

The metallopeptidase CD13 is expressed on normal myeloid cells of monocytic and granulocytic origin and on the surface of leukemic blasts in most acute myeloid leukemias (AML). To study the mechanisms regulating lineage restricted CD13 expression in AML we determined normalised CD13 mRNA levels in bone marrow cells and peripheral blood cells of 27 AML patients. Cells of bone marrow origin had lower levels of normalised CD13 mRNA than cells of peripheral blood origin, even though fluorescence intensity and fraction of cells expressing CD13 on the surface was unchanged. In particular, AML patients with very low levels of normalised CD13 mRNA in bone marrow cells showed an increase in CD13 mRNA expression in peripheral blood. To evaluate the effects of bone marrow microenvironment on CD13 mRNA expression, we cultured leukemic myeloid cells with and without murine stromal cells. Bone marrow cells with high and low CD13 surface expression that entered the stromal layers all down-regulated CD13 mRNA expression as compared to cells in suspension above. For peripheral blood cells within stromal layers, CD13 mRNA expression was diminished in only 3 out of 6 cases. The ambiguous effect of stromal cells on peripheral blood cells may illustrate a differentiation-dependent response towards stroma. We determined the polyadenylation status of CD13 mRNA for 9 bone marrow aspirates and 7 peripheral blood samples. Polyadenylation was diminished in bone marrow cells from AML patients with low levels of normalised CD13 mRNA, raising the possibility of involvement of mRNA instability in regulation of CD13 mRNA expression in this subgroup of patients.     

Differential expression of the carcinoembryonic antigen-related cell adhesion molecules panCD66, CD66a,CD66c and of sialyl-Lewis x (CD15s) on blast cells of acute leukemias

Expression of CD66 has been reported to occur on blast cells from children with acute lymphoblastic leukemia (ALL), but little is known about the differential expression pattern of panCD66 and other members of the CD66 family on blast cells from patients with acute myeloid leukemia (AML). We have performed flow cytometry immunophenotyping on blast cells from 28 patients with acute myeloid leukemia (AML), 13 patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and 7 patients with T-ALL using monoclonal antibodies (mAbs) against panCD66 (clone D14HD11), CD66a (clone 4.3.17), CD66c (clone 9A6) and CD15s. Expression of the panCD66 mAb was found to be positive in 13 of 28 patients with AML (46%) and in 6 of 13 patients with BCP-ALL (46%) but negative for all the seven patients with T-ALL. In AML, panCD66, CD66a and CD66c were more frequently coexpressed with CD65, CD15 and CD64 than with CD13, CD33 or the two progenitor markers CD34 and CD117. In contrast to CD15, the expression of the sialylated Lewis X (CD15s) was associated with CD117 positivity in the majority of AML cases (64 vs. 85%, P = 0.043). Radioimmunotherapeutic strategies targeting CD66 antigens should consider the heterogeneous expression pattern of CD66 molecules in acute leukemias especially in AML where expression is correlated with mature granulomonocytic cells but not with CD34 and CD117 positive progenitor cells.     

Recognition of leukemic blasts by HLA-DPB1-specific cytotoxic T cell clones: a perspective for adjuvant immunotherapy post-bone marrow transplantation.

There is increasing evidence that the immune response plays a role in the prevention of leukemic relapses after allogeneic bone marrow transplantation (BMT). Producing this effect (referred to as the graft-versus-leukemia reaction or GVL) is a current goal of clinical transplantation. At present, all protocols rely on the injection of donor T cells with unknown specificities. In keeping with this approach, we recently proposed the use of a single allogeneic T cell clone transfected with the HSv-tk gene to target an HLA-DPB1 mismatch in the GVH direction. For this strategy to be successful, HLA-DP antigens must be expressed on leukemic cells, which should be recognised by the HLA-DP-specific T cell clone and subsequently destroyed. In the present study, differential expression of HLA-DR, -DQ and -DP was tested by fluorescence using monoclonal antibodies on a panel of 46 acute myeloid leukemias (AML), 28 acute lymphoblastic leukemias (ALL) and 31 chronic lymphocytic leukemias of B cell origin (B-CLL). The vast majority of leukemic cells expressed HLA-DP antigens although with considerable variability. HLA-DPB1 genotyped leukemic cells were used as target cells for an HLA-DPB1*0401-specific T cell clone. Specific recognition of leukemic blasts was demonstrated for 11 out of 11 B-CLL, 11 out of 19 AML and nine out of 16 ALL. These data show that most leukemic blasts are accessible to direct lysis by allogeneic HLA-DP-specific T cells.     

The generation of immunocompetent dendritic cells from CD34+ acute myeloid or lymphoid leukemia cells

The ability of CD34+ leukemic cells to differentiate to dendritic cells (DCs) was investigated in 18 acute myeloid leukemia (AML) and 4 lymphoid leukemia (ALL) patients. The generation of DCs was determined by the expression of DC-associated CD1a or CD83 (more than 30%) with costimulatory molecules, by CD80 antigens (>20%), and by the exhibition of allostimulatory activity. In the AML patients, allostimulatory mature DCs were generated from 3 of 9 M0 or M1, 2 of 5 M2,2 of 4 M4 or M5, and 3 of 4 ALL (L2) cases. In total, DCs were more efficiently induced from cases expressing over 75% of CD34+ among whole bone marrow mononuclear cells (8 of 12), compared with those under 75% (2 of 10; P < .05). B-cell (CD19), natural killer (NK)-cell (CD56), or T-cell (CD7) lineage markers, which were aberrantly expressed on the blasts, were rarely found on leukemic DCs at the end of the culture period, and myeloid (CD13, CD33), not lymphoid (CD10), markers were shown on ALL-derived DCs. In Philadelphia chromosome-positive ALL or AML patients with t (8;21), DCs were confirmed to be of leukemic origin by fluorescence in situ hybridization analysis.     

Immunophenotyping of acute myeloid leukemia using monoclonal antibodies and the alkaline phosphatase-antialkaline phosphatase technique

The leukemic cells from 41 cases of acute myeloid leukemia (AML) and 17 cases of acute lymphocytic leukemia (ALL) were immunophenotyped by the alkaline phosphatase-antialkaline phosphatase (APAAP) immunocytochemical technique utilizing eight monoclonal antibodies (MoAb) reactive with cells of myeloid origin and seven MoAb reactive with lymphoid antigens. Ninety percent of the cases of AML reacted with one or more of the pan-myeloid MoAb, My7, My9, or 20.3. Reactivity of the myeloid panel of MoAb showed some correlation with the French-American-British (FAB) classification of AML. Five of six cases of acute promyelocytic leukemia (APL) were HLA-DR negative; the one HLA-DR-positive APL had a minor population of HLA-DR-negative promyelocytes. OKM5 and/or My4 reacted with 16 of 16 monocytic leukemias. No specific marker of early erythroid development was identified. AP3, a MoAb reactive with platelet glycoprotein (GPIIIa), was specific for acute megakaryoblastic leukemia. Immunocytochemistry was also helpful in classifying seven cases of AML with equivocal or negative routine cytochemistry. Two cases of AML had minor populations of blasts detected by the APAAP technique that were immunologically distinct from the major blast population; these minor populations emerged as the predominant cell type at relapse. Two cases of ALL expressed multiple myeloid and lymphoid antigens. Two other cases that morphologically were ALL reacted with only myeloid MoAb; one consisted entirely of immature basophils on ultrastructural examination. Immunophenotyping results using the APAAP technique were comparable with those obtained with flow cytometry. The APAAP technique is a reliable method for immunophenotyping leukemia that complements other methods of immunologic evaluation. The primary advantages of this method include its use with routinely prepared blood and bone marrow smears and the ability to correlate immunocytochemical reactions with morphology.     

In vitro sensitivity of leukemic cells to nucleoside derivatives in childhood acute leukemias: good activity in leukemic relapses

Nucleoside analogues such as fludarabine and cladribine are used in therapy of indolent lymphomas and leukemias in adults, while cytarabine is used mainly in protocols for acute leukemias. Mechanisms of their activity is based on inhibition of enzymes involved in DNA, RNA and protein synthesis. The objective of the study was the analysis of in vitro cellular drug sensitivity in childhood acute lymphoblastic (ALL) and myeloid (AML) leukemia. Isolated leukemic cells obtained from 264 patients, including 152 initial ALL, 45 relapsed ALL, 54 initial AML and 13 relapsed AML were tested for cytotoxicity for fludarabine, cladribine, and cytarabine by the MTT assay. Drug concentration lethal to 50% of tested cells was regarded as a value of drug resistance. Three tested nucleoside analogues showed highest cytotoxicity against initial ALL samples. Samples of relapsed ALL and initial AML were more resistant than ALL de novo ones. Unexpectedly, no differences were observed between initial and relapsed AML samples for all tested drugs, what suggests that nucleoside analogues are active drugs in relapsed AML, which is commonly regarded as a resistant disease. All tested drugs presented significant cross-resistance in each of analyzed subgroups. In summary, tested nucleoside analogues presented relatively good activity against childhood leukemias at relapse stage.     

Identification of the CD33-related Siglec receptor, Siglec-5 (CD170), as a useful marker in both normal myelopoiesis and acute myeloid leukaemias

Sialic acid-binding immunoglobulin-like lectin (Siglec)-5 or CD170 is a CD33-related receptor, containing cytoplasmic immune receptor-based tyrosine signalling motifs, that has previously been reported to be myeloid-specific like CD33 and thus may be useful in the characterization of both normal and malignant haemopoiesis. This study showed that Siglec-5 had a distinct expression pattern to CD33 both on normal myeloid cells and in acute myeloid leukaemia (AML). In normal bone marrow and cord blood, myeloid cells predominantly expressed Siglec-5 at the later stages of granulocytic differentiation. Siglec-5 was not expressed at significant levels by CD34+ progenitors either from bone marrow or mobilized peripheral blood. During in vitro myeloid differentiation of cord blood purified CD34+ cells, Siglec-5 was upregulated later than CD33. Siglec-5 expression remained absent or very low on cultured CD34+ cells, unlike CD33, which was present on almost all CD34+ cells by day 4. However, analysis of blasts from 23 patients with AML revealed aberrant expression of Siglec-5 with CD34 in 50% (seven of 14) of patients with CD34+ AML; 61% (14 of 23) of AML cases were positive for Siglec-5 with an increased frequency in the French-American-British subtypes M3-5 (80%) compared with M0-2 (25%). All 13 acute lymphoblastic leukaemic (ALL) samples tested, including a CD33+ ALL, were Siglec-5 negative. These results support the further evaluation of Siglec-5 antibodies in the diagnosis and monitoring of AML.     

Expression of the Fas antigen on primary human leukemia cells

Summary The antigen defined by the monoclonal antibody anti-Fas can mediate apoptosis, is associated with the receptor for tumor necrosis factor, and is expressed on a limited number of human tissues. In this study we analyzed the expression of Fas on primary human leukemic cells and on mononuclear cells from other hematologic disorders. A total of 95 samples of blood or bone marrow were studied by indirect immunofluorescence. These samples included the normal controls, 47 cases of acute myelogenous leukemia (AML), 11 cases of acute lymphoblastic leukemia (ALL), 21 cases of leukemic lymphoma, seven cases of chronic myelogenous leukemia (CML), five cases of plasma cell leukemia or multiple myeloma, and five cases of myelodysplastic or myeloproliferative syndromes. Normal controls were negative without exception. Among AML, 13/47 cases (28%) were positive; among ALL, 1/11 cases (9%) was positive; among leukemic lymphomas, 3/21 cases (14%) were positive. In a case of plasma cell leukemia which strongly expressed the Fas antigen, we demonstrated that the antibody mediates cell lysis, which was synergistically enhanced by the addition of rabbit complement. In patients with AML, Fas positivity had no obvious clinical relevance. Taken together, our results show that approximately 30% of cases of AML and occasionally other leukemias express the Fas antigens, whereas normal controls are negative in our test system. These findings may be useful in the treatment of refractory leukemias or may permit the purging of autologous transplants.Presented in part at the 34th annual meeting of the American Society of Hematology, Anaheim, CA, December 1992     

Clinical significance of standard CD assessment in acute leukemia

The data of detailed studies of immunophenotype of blast cells in 426 patients with acute leukemias are presented. Diagnostic and prognostic significance of different marker expression has been evaluated in groups of patients with ALL and AML. Frequency distribution of T1, T2, T3, pre-B, B, common, Ia and null subvariants identified according to immunoclassification of Baryshinkov et al. was studied in 250 children with ALL. These subvariants differed both in duration of disease (p = 0.0015) and in duration of first complete remission (p = 0.0031). The use of monoclonal antibodies of VI-series in 90 patients with ALL allowed to describe an immunophenotype of the subvariants in detail. The mosaic expression of myeloid antigens CD11, CD14, CD15, CDw65 identified by MoAbs VIM-12, VIM-13, VIM-D5 and VIM-2, respectively, on blast cells of patients with AML was shown. The expression of CD11 (ICO-GM1) or CD15 (ICO-G2) was prognostically unfavorable in children with AML (p = 0.0028). The expression of T-cell markers (E-receptor, CD7, reactivity with anti-T-cell serum) on blasts was prognostically favorable in children with AML (p = 0.003). So the data of immunophenotyping are of great value for accurate diagnosis and prognosis of acute leukemias.     

Immunological typing of acute leukemias: immunoenzymatic staining of fixed cells compared with immunofluorescence staining of unfixed cells in suspension

A panel of 14 monoclonal antibodies (McAb) against hematopoietic cell surface antigens was applied on mononuclear blood or bone marrow cells from 40 cases of acute leukemia in order to compare immunoenzymatic staining (IE) (alkaline phosphatase) of fixed cells with immunofluorescence staining (IF) of unfixed suspended cells. According to the immunological results, 25 cases were phenotyped as ALL and 15 cases as AML. Cases with blast crisis secondary to chronic myelogenous leukemia (CML-BC) were not represented in this series. In all ALL cases the two methods gave an identical antigenic distribution. In 20 our of 21 cases of non-T-cell ALL, a B-cell progenitor origin was demonstrated by a positive staining reaction with the anti-CD19 McAb AB1 or HD37, and in 10 cases additionally with the anti-CD20 McAb B1 or 1F5. In contrast to the results obtained with IF, IE revealed a poor preservation of the AB1 epitope on CD19, whereas the HD37 epitope was equally well demonstrated by both methods. In 15 cases of AML the distribution of positive versus negative cells with IE or IF was identical for all McAb except J5 (anti-CALLA) (CD10) and B1 (CD20). Thus, 10/15 AML cases expressed CALLA with IE compared to 2/15 with IF. The corresponding figures for B1 were 5/15 and 0/15, respectively. Accordingly, normal myeloid precursor cells were CALLA-positive with IE but negative with IF. The discrepancy probably reflects the fact that, whereas both intracytoplasmatic and membrane-bound antigens are exposed in IE, only the latter are in IF. If the alteration of antigenic accessibility after fixation is considered, IE can safely be used for immunophenotyping of acute leukemia.     

Differential nucleolus organizer activity in normal and leukemic bone marrow

A cytochemical technique that uses silver nitrate staining has been used to study the nucleolus organizer activity in bone marrow cells from 13 patients with acute lymphocytic leukemia (ALL), 11 patients with chronic myelogenous leukemia (CML), 7 patients with acute myelogenous leukemia (AML), and 4 healthy persons. Our results indicate that the nucleolus organizer region (NOR) activity was significantly lower in the control group than in the ALL patients. The NOR activity level was significantly lower in both the CML patients in chronic phase and the AML patients than in the ALL group and similar to the control group. These disease-related differences in NOR activity as detected by silver staining can be used as diagnostic procedure in evaluating human leukemias.     

Detection of p53 oncogene in acute-leukemia cells by immunoperoxidase technique

Expression of p53 oncogene in blast cells may have prognostic importance in acute leukemia. Simple and reliable methods which could detect enhanced p53 expression in leukemia cells would be important for follow-up studies of leukemia patients in remission. We used immunoperoxidase (IP) technique with an anti-p53 monoclonal antibody PAb421 to study the expression of p53 in leukemia cells. The expression of p53 was studied in 9 cell lines and 17 de novo acute leukemia (9 acute myeloid leukemia [AML], 8 acute lymphoblastic leukemia [ALL]) patients. The expression of p53 was demonstrated in non-T non-B cells and Burkitt's lymphoma cell lines, but neither in two myeloid leukemia cell lines nor in normal lymphoid cells after mitogenic stimulation. p53 expression was demonstrated in 7 cases (2 AML, 5 ALL) but only in ALL cases the percentage of positive of cells was over 20%. Bone marrow cells from patients were studied also after short-term culture (AML patients); in 1 case the number of PAb421-positive cells rose significantly after culture. These data suggest that IP staining with PAb421 can be used to demonstrate high p53 expression in B cell leukemias.     

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.     

CD7+, CD4-, CD8- acute leukemia: a syndrome of malignant pluripotent lymphohematopoietic cells

Following our initial observation of in vivo conversion of CD7+, CD4-, CD8- acute lymphoblastic leukemia (ALL) cells from lymphoid to myeloid lineages (Proc Natl Acad Sci (USA) 81:253, 1984) we have studied eight additional cases of ALL with this leukemic cell phenotype. The CD7+, CD4-, CD8- phenotype was associated with a distinct clinical entity with those affected predominantly male (either less than 35 years or greater than 65 years of age), with frequent mediastinal and/or thymic masses, skin and CNS disease, high peripheral WBC counts, and bone marrow blasts that were morphologically L1 or not ascribable to a specific lineage. These patients did not respond to conventional chemotherapeutic regimens for either acute lymphoid or myeloid leukemias. No common karyotype or T-cell gene rearrangement pattern could be defined. Importantly, seven of eight patient's leukemic cells studied were capable of multilineage (myeloid, erythroid, monocytoid, megakaryocytoid, and lymphoid) differentiation in vitro. Data is presented suggesting that CD7+, CD4-, CD8- leukemias, in many instances, are leukemias of immature hematopoietic cells. The development of novel therapeutic approaches to this form of leukemia will be necessary to alter its poor prognosis.