An evaluation of the prognostic significance of antigen CD95(Fas/APO-1) expression on the cells of patients with a myelodysplastic syndrome, acute myeloid leukemia and chronic myeloleukemia

AIM: The expression of CD95(Fas/APO-1) antigen was studied on bone marrow cells of 19 MDS patients, peripheral blood blast cells of 15 acute myeloid leukemia (AML) patients, blast cells and granulocytes of 68 patients with chronic myeloid leukemia (CML)--24 in chronic, 9 in accelerated phase and 35 in blastic crisis (BC)--by indirect surface immunofluorescence assay using flow cytometry (FACScan, Becton Dickinson, USA). RESULTS: CD95(Fas/APO-1) antigen was revealed on bone marrow cells of 8 out of 19 (36.8%) MDS patients; the percentage of antigen-positive cells was 38.1 +/- 19.2%; on 45.5 +/- 22.8% of cells in 6(45%) of 15 AML patients. Fas/APO-1 antigen was totally absent in CML chronic stage; its expression was found in 34% (12 of 35) of our patients with CML BC on peripheral blood blasts and in 56% (5 of 9) on peripheral blast cells of CML patients in acceleration phase. CONCLUSION: The data on overall survival of CD95-positive MDS patients suggest that the presence of Fas antigen is a favorable prognostic sign for patients with MDS. The patients from CD95-negative group represent a risk group both for survival and AML transformation. In CML BC group the survival does not depend upon Fas-antigen expression.     

Involvement of APO2 ligand/TRAIL in activation-induced death of Jurkat and human peripheral blood T cells

The interaction of Fas with Fas ligand (FasL) mediates activation-induced cell death (AICD) of T hybridomas and of mature T lymphocytes. The TNF/TNF receptor system also plays a significant role in AICD of mature T cells and in the maintenance of peripheral tolerance. We previously demonstrated that in human Jurkat leukemia cells, AICD is triggered mainly by the rapid release of preformed FasL upon TCR stimulation. In the present work, we show that the cytotoxic cytokine APO2 ligand (APO2L; also known as TRAIL) is constitutively expressed as an intracytoplasmic protein in Jurkat T cells and derived sublines. APO2L is also detected in fresh human peripheral blood mononuclear cells (PBMC) from a significant number of donors, and the amount of both FasL and APO2L substantially increases upon blast generation. A neutralizing anti-APO2L monoclonal antibody (mAb) partially suppresses the cytotoxicity induced by supernatants of phytohemagglutinin (PHA)-prestimulated Jurkat or human PBMC on non-activated Jurkat cells, indicating that APO2L is released by these cells and contributes to AICD. A combination of neutralizing anti-APO2L and anti-Fas mAb blocks around 60 % of the toxicity associated with supernatants from PHA-activated human PBMC. These results show that FasL and APO2L account for the majority of cytotoxic activity released during AICD, and suggest that additional uncharacterized factors may also contribute to this process.     

Vitamin K2 and its derivatives induce apoptosis in leukemia cells and enhance the effect of all-trans retinoic acid

Geranylgeraniol, a polyprenylalcohol composing the side chain of vitamin K2 (VK2), was previously reported to be a potent inducer of apoptosis in tumor cell lines (Ohzumi H et al, J Biochem 1995; 117: 11-13). We examined the apoptosis-inducing ability of VK2 (menaquinone 3 (MK3), MK4 and MK5) and its derivatives such as phytonadione (VK1), as well as polyprenylalcohols with side chains of various lengths including farnesol (C15-OH; FO), geranylgeraniol (C20-OH; GGO), and geranylfarnesol (C25-OH; GFO) toward leukemia cells in vitro. MK3, MK4, MK5 and GFO (at 10 microM) showed a potent apoptosis-inducing activity for all freshly isolated leukemia cells tested and for leukemia cell lines such as NB4, an acute promyelocytic leukemia (APL)-derived cell line and MDS92, a cell line derived from a patient with myelodysplastic syndrome, although there were some differences depending on the cells tested. In contrast, VK1 showed no effect on any of the leukemia cells. The combination of MK5 plus all-trans retinoic acid (ATRA) resulted in enhanced induction of apoptosis in both freshly isolated APL cells and NB4 cells as compared to each reagent alone. These data suggest the possibility of using VK2 and its derivatives for the treatment of myelogenous leukemias, including APL.     

Ligation of cell surface CD38 protein with agonistic monoclonal antibody induces a cell growth signal in myeloid leukemia cells

CD38 is expressed during early stages of differentiation in normal and leukemic myeloid cells. Recently, CD38 has been shown to participate in intracellular signal transduction pathways following its ligation with CD38-specific mAbs. In this study we report that ligation of CD38 by one such agonistic mAb (IB4) induced proliferation of cultured leukemic cells in vitro. In HL-60, KG-1A, NB4, and OCI-AML-3 myeloid leukemia cell lines, IB4 mAb induced an increase in the proliferating cell fraction as determined by cell number, clonogenic assay, and flow cytometric analysis. The presence of Ab caused a dose-dependent increase in the number of CFU and an increase in cell divisions. HL-60-Dox cells (a HL-60-doxorubicin-resistant cell line), which have no detectable CD38 expression, failed to respond to IB4 mAb. The effect of CD38 ligation on cell growth was also evaluated in freshly isolated leukemic cells from patients with acute myelogenous leukemia (AML). A significant increase in the proliferating cell fraction (S+G2M) was observed in 50% of the patients incubated with IB4 mAb. In five of the six AML patients, anti-CD38 mAb stimulated the proliferation of AML colony-forming cells. These results suggest that ligation of CD38 can induce the proliferation of leukemic cells and may play a role in the propagation of leukemic cell clones in certain cohorts of AML patients.     

TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in Fas ligand-resistant melanoma cells and mediates CD4 T cell killing of target cells

We have previously shown that melanoma cells were resistant to apoptosis induced by TNF family members Fas ligand (FasL), TNF-alpha, and CD40L. FasL also was not involved in CD4 T cell-mediated killing of melanoma cells. In the present study, we have tested melanoma cells for their susceptibility to apoptosis induced by human TNF-related apoptosis-inducing ligand (TRAIL) and the ability of a mAb against TRAIL to inhibit apoptosis and CD4 CTL-mediated killing of melanoma and Jurkat target cells. The results show that TRAIL-induced apoptosis in cells from 7 of 10 melanoma cell lines tested as well as in Jurkat T cells. Susceptibility to apoptosis was increased in some of the cell lines by treatment with cyclohexamide or actinomycin D. The melanoma cells were resistant to apoptosis induced by FasL, TNF-alpha, and CD40L. mAb M180 against TRAIL inhibited apoptosis induced by TRAIL. It was also found to inhibit CD4 CTL-mediated killing of Jurkat T cells as well as autologous and allogeneic melanoma cells. The degree of inhibition produced by the mAb varied between different clones of CTL and according to the susceptibility of the target cells to TRAIL-induced apoptosis. These results suggest that TRAIL is an important mediator of cell death induced by CTL and may have an important therapeutic role against human melanoma.     

Dose-escalation trial of M195 labeled with iodine 131 for cytoreduction and marrow ablation in relapsed or refractory myeloid leukemias

PURPOSE: Mouse monoclonal antibody (mAb) M195 (anti-CD33) is reactive with most myeloid leukemia cells, monocytes, and hematopoietic progenitors, but not with other hematopoietic cells or stem cells nor with nonhematopoietic human tissues. A therapeutic dose-escalation study of M195 labeled with iodine 131 was conducted in patients with relapsed or refractory myeloid leukemias. METHODS: Twenty-four patients (16 relapsed or refractory acute myeloid leukemias, five blastic myelodysplastic syndromes [MDS], two chemotherapy-related secondary leukemias, and one blastic chronic myelogenous leukemia [CML]), including seven who had failed to respond to prior bone marrow transplantation (BMT), received from 50 mCi/m2 to 210 mCi/m2 of 131I-M195 in divided doses. RESULTS: In 22 patients, whole-body gamma-imaging demonstrated marked uptake of antibody into all areas of bone marrow. Twenty-three patients (96%) demonstrated decreases in peripheral-blood cell counts, with decreased percentage of bone marrow blasts seen in 83% of cases. Eighty-nine percent of bone marrow biopsies examined quantitatively demonstrated substantial decreases in the number of blasts, with greater than 99% of blasts killed in some patients. The two cases that failed to demonstrate leukemic cytoreduction occurred in the first two dose levels. For 131I doses of 135 mCi/m2 or greater, pancytopenia was profound and lasted for at least 12 days. Eight patients had sufficient marrow cytoreduction to proceed to BMT. Three of these achieved marrow remission, one of 6+, and one of 9 months' duration. Two patients in blastic phase temporarily reverted to their original myelodysplastic states. Thirty-seven percent of assessable patients developed human anti-mouse antibody (HAMA). In two patients with HAMA who were re-treated, plasma 131I-M195 levels could not be maintained and no therapeutic effect resulted. Significant nonhematologic toxicity (hepatic) was seen in one patient and the maximum-tolerated dose (MTD) was not reached. CONCLUSION: These data suggest that safe leukemic cytoreduction can be achieved with 131I-M195 even in multiply relapsed or chemotherapy-refractory leukemias. This agent may be useful as part of a preparative regimen for BMT.     

Proteins and cells on PEG immobilized silicon surfaces

Functional adhesion of blood and marrow leukemic cells from 14 acute myeloid leukemia patients presenting with hyperleukocytosis was evaluated by performing cytoadhesion assays on purified (extracellular matrix proteins) and non-purified supports (MRC5 fibroblastic cell line). Results, in 30-min chromium release assay, show a mean +/- S.D. adhesion to fibronectin, collagen, and laminin respectively of 30 +/- 17%, 20 +/- 13%, 25 +/- 17% for blood leukemic cells and 18 +/- 11%, 11 +/- 10%, 11 +/- 8% for marrow leukemic cells. These differences between blood and marrow cells were statistically significant (respectively P = 0.005, P = 0.01 and P = 0.002), while no difference was noted regarding adhesion to non-purified supports. The higher adhesion of blood blast cells to purified supports was observed regardless of CD34 expression. No significant difference was observed in the expression of cell surface VLA-molecules (CD29, CD49b, CD49d, CD49e, CD49f) between blood and marrow blast cells. The addition of GM-CSF or G-CSF induced increased adhesion of marrow blasts and decreased adhesion of blood blasts leading to a loss of the difference between blood and marrow cells. In a 60-min chromium release assay, marrow blasts adhered even more than blood leukemic cells to fibronectin. In contrast, marrow blasts from 'aleukemic' acute myeloid leukemia patients did not show any modification regarding their adhesion to extracellular matrix proteins when co-cultured with growth factors.     

Replication errors in hematological neoplasias: genomic instability in progression of disease is different among different types of leukemia

Genetic alteration, including genomic instability, is an ultimate step toward the malignant process. One approach to delineating replication errors in cancer cells is to determine the alterations of microsatellites, which are short, repeated nucleotide sequences existing throughout the genomes. We used a fluorescent system to assess microsatellite changes in seven loci (D2S123, D3S643, D5S107, LPL, D17S261, TP53, and D18S34) of 73 consecutive patients with various hematological neoplasias. De novo acute leukemia patients had a low frequency (<1%) of microsatellite alterations at each locus, and none of them demonstrated multiple microsatellite changes. In chronic myeloid leukemia patients, no microsatellite instability was detected in the chronic phase, whereas a relatively high frequency (25%) of multiple microsatellite changes was evident in the blastic phase, and half of these patients had multiple microsatellite changes. About 50% of the patients with myelodysplastic syndrome (MDS) and post-MDS acute myeloid leukemia (post-MDS AML) had microsatellite alterations. We next compared microsatellite alterations in two different hematological phases (MDS and post-MDS AML phases); 5 of 11 patients with post-MDS AML had de novo appearance of microsatellite instability during disease progression. This indicates that genomic instability at multiple microsatellite loci could occur either before or after leukemic transformation in MDS patients. We concluded that genomic instability in chronic myeloid leukemia might be linked to blastic transformation in combination with cytogenetic changes. In contrast, MDS patients had replication errors as a relatively early genetic event as well as a late genetic event. These results suggest that the involvement of genomic instability in the progression of disease is different among various types of leukemia.     

CD45 gating of acute leukemia

Flow cytometry is a powerful tool for immunophenotyping of acute leukemia. Gating of leukemic cells is currently performed on the two dimensional display of forward scatter (FSC) and side scatter (SSC). However, this gating method can not discriminate leukemic cells from contaminated normal cells. Therefore, we used a CD45 monoclonal antibody to detect leukemic cells (CD45dlm cells) in combination with the SSC parameter. This CD45-SSC gating method was very useful for immunotyping of AML and ALL, especially leukemia with a low percentage of blasts. We recommend this new gating method for more accurate immunophenotyping of acute leukemia.     

CD40 expressed on thymic epithelial cells provides costimulation for proliferation but not for apoptosis of human thymocytes

Human thymic epithelial cells express CD40, so we examined the possible role of CD40 in activation of thymocytes. We observed that both CD4+CD8- and CD4-CD8+ thymocytes proliferate after stimulation by anti-CD3 mAb in the presence of cultured thymic epithelial cells. Costimulation of CD4+ thymocytes by thymic epithelial cells is partly inhibited by an anti-CD40 mAb, but this mAb has no effect on costimulation of CD8+ thymocytes. The selective costimulatory ability of CD40 for CD4+ thymocytes was confirmed in experiments in which thymocytes were stimulated with anti-CD3 in the presence of murine P815 cells transfected with CD40 cDNA. The level of costimulation induced by P815-CD40 was comparable with that induced by P815 cells expressing CD80 (B7.1). Treatment of thymocytes with the Ca2+ ionophore ionomycin and the phorbol ester PMA or with anti-CD3 mAb resulted in up-regulation of the CD40 ligand, suggesting that this molecule is involved in CD40-mediated costimulation of human thymocytes. Costimulation of thymocytes by CD80 strongly increased anti-CD3-induced death of fetal thymocytes. In contrast, costimulation by CD40 did not increase anti-CD3-mediated apoptosis of these thymocytes. To confirm that CD40 does not affect anti-CD3-induced cell death, we established a variant of the Jurkat T leukemic cell line that constitutively expresses CD40L and analyzed the sensitivity of this cell line for activation-induced apoptosis. In contrast to CD80, CD40 failed to increase anti-CD3-mediated apoptosis in CD40L+ Jurkat cells, whereas both CD40 and CD80 strongly increased IL-2 production induced by anti-CD3. These findings suggest that costimulation by CD40 is involved in clonal expansion of CD4+ thymocytes but not in activation-induced cell death.     

Monocyte-mediated lysis of acute myeloid leukemia cells in the presence of the bispecific antibody 251 x 22 (anti-CD33 x anti-CD64)

Immunotherapy using bispecific antibodies (BsAb) to direct immune effector cells toward target tumor cells has been shown to be effective in a number of studies. Several immune trigger molecules have been characterized. Among them, FcgammaRI appears to play an important role in antibody-dependent cellular cytotoxicity. It is expressed mainly on monocytes, macrophages, and neutrophils under certain clinical situations. The expression of FcgammaRI can be regulated by a variety of cytokines, primarily by IFN-gamma. Recent studies have shown that granulocyte-colony-stimulating factor (G-CSF) and granulocyte-macrophage-colony stimulating factor (GM-CSF) can increase the number of the FcgammaRI-positive monocytes, increase the expression of FcgammaRI on circulating neutrophils after in vivo infusion, and greatly enhance the cytotoxic activity of circulating neutrophils. CD33 is a glycoprotein expressed on the cell surface of mature monocytes, myeloid progenitor cells, and myeloid leukemic blasts, but not on the earliest hematopoietic progenitor cells and other normal tissues. Herein, we report the construction of a BsAb, 251 x 22, by conjugating an anti-CD33 mAb (mAb 251) to an anti-FcgammaRI mAb (mAb 22). The BsAb 251 x 22 is capable of enhancing the cytotoxicity of several leukemia cell lines by cytokine-activated monocytes. Our data also show that G-CSF- and GM-CSF-stimulated monocytes can mediate cytotoxicity of target leukemia cells comparable to that of IFN-gamma-stimulated monocytes. The expression of FcgammaRI on monocytes after 24-h in vitro incubation with G-CSF and GM-CSF was increased, although not significantly. Prolonged incubation of monocytes with G-CSF for 48 h significantly increased the FcgammaRI expression. Because humanized anti-CD33 and anti-FcgammaRI mAb are available, and because GM-CSF and G-CSF have been used widely for patients after chemotherapy to stimulate the recovery of myeloid hematopoiesis, additional clinical development of this project is feasible. A BsAb comprised of humanized anti-CD33 and anti-FcgammaRI could have clinical application in the treatment of myeloid leukemia, especially in the management of minimal residual disease.     

Fas/APO-1 (CD95) expression in myelodysplastic syndromes

Increased apoptosis of myeloid precursors appears to contribute to the pathophysiology of cytopenias in myelodysplastic syndromes (MDS). Fas /APO-1(CD95) is a cell surface protein inducing an apoptotic signal after its binding to Fas ligand or to a functional anti-Fas antibody. Here we studied Fas expression by immunocytochemistry on marrow slides from 30 cases of MDS. Increased Fas expression in erythroblasts and/or immature granulocytes, compared to controls, was seen in 12 (40%) of the cases. In addition, in 16 of the 18 cases with > or = 5% marrow blasts, a variable proportion of blasts expressed Fas. Increased apoptosis was found by morphological analysis and/or TUNEL technique in marrow cells from 8 of the 26 cases analyzed (31%) The ability of Fas antigen to trigger apoptosis was studied after addition of a functional anti Fas antibody in 5 of the patients with Fas overexpression. Addition of this antibody, however, only lead to mild increase of apoptosis in immature granulocytes (but not other myeloid cells) in 2 of the 5 cases. Thus, increased Fas expression is seen in myeloid and/or blast cells in the majority of MDS cases. However, the relationship between this finding and increased apoptosis in MDS still remains to be established.     

Effect of a vitamin D3 analog, EB1089, on hematopoietic stem cells from normal and myeloid leukemic blasts

The seco-steroid 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) induces differentiation and inhibits clonal proliferation of HL-60 cells. We analyzed the effect of a novel vitamin D3 analog, EB1089, on normal myeloid and leukemic cells as well as CD34+ cells. EB1089 showed an extraordinary inhibition of clonal growth of HL-60 cells (ED50 = 5 x 10(-11) M) and AML blast cells (ED50 = 9 x 10(-10) M) compared to 1,25(OH)2D3 without suppression of growth of normal human bone marrow CFU-GM. The CD34+ cells from acute myeloid leukemia (AML) blasts were inhibited in a dose-dependent fashion by 1,25(OH)2D3 with an ED50 of 1.2 x 10(-9) M; and even more strikingly, 10(-10) M of EB1089 inhibited all clonal growth of human CD34+ leukemic colony-forming cells. In contrast, both EB1089 and 1,25(OH)2D3 (10(-8) M) showed little or only mild inhibition of CD34+ clongenic hematopoietic cells from normal human peripheral blood (PB); and in liquid culture, EB1089 stimulated the proliferation of normal human CD34+ cells about 2.5 times as compared to control cultures. In order to evaluate the potential use of EB1089 for purging leukemic cells from normal CD34+ progenitor cells for PB stem cell transplantation (PBSCT), normal human PB mononuclear cells (PBMNC) were contaminated with HL-60 cells, and then CD34+ cells purified and treated with EB1089. We found that CD34+ purification and EB1089 purging was able to eliminate approximately 100% of HL-60 leukemic cells with no toxicity to normal CD34+ hematopoietic progenitor cells. These data suggested that purification of CD34+ cells and ex vivo treatment with EB1089 might provide an effective therapeutic approach for PBSCT.     

Functional expression of Fas (CD95) in acute myeloid leukemia cells in the context of CD34 and CD38 expression: possible correlation with sensitivity to chemotherapy

Clinical studies of bone marrow transplantation (BMT) suggest that the immune system contributes to the eradication of acute myeloid leukemia (AML). A recent study also showed that the Fas (CD95/APO1) mediates apoptotic signal from cytotoxic T lymphocytes. Sixty-four patients with AML were studied for the expression of Fas in the context of CD34 and CD38 coexpression. The clinical relevance of Fas expression and function on AML was also investigated. Fas was expressed on 2% to 98% of AML cells (2% to 20% in 11 patients, 20% to 50% in 20 patients, 50% to 80% in 24 patients, and 80% to 98% in nine patients). Only 44.4% of patients with AML M1 (French-American-British [FAB] classification) were Fas+ (>/=20% of leukemia cells expressed Fas), whereas 89.1% of patients with AML M2, M3, M4, M5 were Fas+ (P < .01). Among 43 CD34+ patients (>/=20% leukemia cells were CD34+), 34 were Fas+, and 19 of 21 CD34- patients were Fas+ (P = NS). Thirteen cases were studied for their expression of Fas in the context of CD34 and CD38 using three-color analysis. Fas is expressed at a high level in the gated CD34+CD38+/- and CD34+CD38+ population. In 10 AML samples, Fas was expressed at a higher level in CD34+/CD38+ population than in CD34+/CD38+/- or CD34- cell populations. Fas-induced apoptosis by anti-Fas monoclonal antibody (MoAb) was determined by morphologic features and colorimetric DNA fragmentation assay. Induction of apoptosis was found in 14 of 24 cases. However, no statistically significant correlation was observed between Fas expression and induction of apoptosis. Leukemia colony-forming unit assays suggested that in some cases, Fas-induced apoptosis occurred in the clonogenic cell populations. Parameters such as laboratory and clinical data at initial diagnosis were correlated with Fas expression and only response to initial induction chemotherapy showed significant correlation with Fas expression (P < .05). We conclude that the majority of AML cells exhibit variable expression of Fas, and apoptosis could be induced by anti-Fas MoAb in some cases. Our results suggest the Fas-mediated apoptosis may be clinically relevant, whereas the issue of clonogenic leukemia cells and Fas expression needs further studies.     

The effect of ascorbic acid and ferric ammonium citrate on iron uptake and storage in lens epithelial cells

Fas antigen (CD95) is a membrane receptor that plays a major role in induction of apoptosis. In surface conjunctival epithelial cells the expressions of Fas, Fas ligand, the apoptotic marker APO2.7 and of HLA DR class II antigen, a membrane marker known to be expressed in inflammatory conditions were investigated. Impression cytology specimens were collected in 65 patients: 20 normal ones, 15 contact lens wearers, 20 receiving chronic topical antiglaucoma treatment and 10 with nonspecific chronic conjunctivitis. Cells were processed for flow cytometry, using monoclonal antibodies to Fas, Fas ligand, APO2.7, HLA DR antigens and a negative isotypic control. Percentages of positive cells were recorded and levels of fluorescence quantified using fluorescent beads at standardized fluorescence intensities. In addition, a human conjunctival cell line was incubated with anti-Fas stimulating antibodies in order to test Fas-induced apoptosis in vitro. Fas was found in all specimens in most of the conjunctival cells, but quantitation of levels of fluorescence showed a significantly higher expression in pathologic eyes than in normal ones. Fas ligand and APO2.7 were variably expressed by conjunctival cells, but in a significantly higher percentage of cells in pathological eyes than in normal ones. In these eyes a strong expression of HLA DR was also observed, whereas normal eyes showed lowest levels. Highly significant correlations were found between Fas, Fas ligand, APO2.7 and HLA DR levels. Anti-Fas antibodies in vitro induced strong apoptosis in epithelial cells as confirmed by APO2.7 expression and DAPI staining. This study confirms that conjunctival epithelial cells normally express Fas antigen, and more inconstantly its ligand, as do corneal ones or keratinocytes. Fluorescence quantitation by flow cytometry showed much higher expression in inflammatory eyes than in normal ones, and demonstrated a strong correlation between apoptotic and inflammatory pathways in the ocular surface.     

Carcinoid of the appendix. Observations on 4 cases

Myelodysplastic syndrome (MDS) is a group of hematopoietic disorders characterized by peripheral cytopenias in the presence of normo- or hypercellular dysplastic marrow. It has been suggested that premature intramedullary apoptosis may contribute to this phenomenon. We used terminal dUTP nick-end labeling (TUNEL) of bone marrow biopsy specimens and cytocentrifuge preparations from patients with MDS and a variety of other hematopoietic disorders to determine whether there is increased intramedullary apoptosis in MDS and whether any such effect is specific to MDS. TUNEL labeling of bone marrow from 24 patients with MDS revealed significant positivity in 10 of 11 patients with refractory anemia (RA), five of seven with RA and excess of blasts (RAEB), all three patients with RAEB in transformation (RAEB-t), and all three patients with RA with ring sideroblasts (RARS). The percent of positive cells ranged from 5 to 50% but showed no apparent correlation with morphological subtype. In a series of 29 patients with acute leukemia, 17 showed significant positivity (13 of 13 with myeloid disease: three M1, seven M2, one M3, two M4; four of 16 patients with lymphoid disease: one Burkitt-type lymphoma, two null acute leukemia, and one common acute lymphoid leukemia). Intramedullary apoptosis was associated with myeloid or early committed progenitor cells and was highest in secondary acute myeloid leukemia (AML). Normal bone marrow samples from 12 individuals showed no evidence of apoptosis. Our results suggest that an increased level of intramedullary apoptosis is apparent in both patients with MDS and those with AML; those with secondary AML have the highest levels. The relative absence of such findings in lymphoid malignancy suggests that the apoptotic pathways are different in this lineage.     

Analysis of myeloid and lymphoid markers on the surface and in the cytoplasm of mononuclear bone marrow cells in patients with myelodysplastic syndrome

Mononuclear cells of the bone marrow (BM) of patients in various subgroups of the myelodysplastic syndrome (MDS) were studied by flow cytometry for the expression of myeloid and lymphoid markers both on the surface and in the cytoplasm. A significantly higher percentage of the BM cells of MDS patients reacted with monoclonal antibodies (mAbs) to myeloid antigens (CD13, CD15 and CD33) by cytoplasmic staining as compared with cell surface staining. The percentage of BM cells expressing CD34 was markedly elevated in patients with RAEB-T. A distinct finding in MDS patients was the expression of myeloid antigens on mononuclear BM cells. The proportion of individuals whose mononuclear BM cells were positive for surface reactivity with anti-CD13 and anti-CD33 mAbs was highest among RAEB-T patients while none of the patients with RA expressed these surface antigens. Cytoplasmic staining significantly increased the percentage of CD13+ and CD33+ BM cells among RAEB and RAEB-T patients. The proportion of individuals whose BM cells possessed myeloid antigens was increased by cytoplasmic staining in all subgroups of MDS. The BM of a considerable proportion of RAEB-T and RAEB patients showed cells which coexpressed the CD7 and CD3 lymphoid markers along with the CD13 and CD33 myeloid antigens. The present study indicates the importance of comparative surface and cytoplasmic immunophenotyping with CD13 and CD33 mAbs for the diagnosis of subgroups of MDS. The coexpression of CD3 and CD7 with markers of the myeloid lineage may reflect derangement of the differentiation of pluripotent stem cells characteristic for MDS.     

Differential CD95 expression and function in T and B lineage acute lymphoblastic leukemia cells

CD95 (Fas/APO-1) is a cell surface receptor able to trigger apoptosis in a variety of cell types. The expression and function of the CD95 antigen on leukemic blasts from 42 patients with B lineage and 53 patients with T lineage acute lymphoblastic leukemia (ALL) were investigated using immunofluorescence staining and apoptosis assays. The CD95 surface antigen was expressed in most ALL cases, with the T lineage ALL usually showing a higher intensity of surface CD95 expression as compared with the B lineage ALL cells (relative fluorescence intensity, RFI: 4.8 +/- 0.47 vs 2.2 +/- 0.23, respectively, P < 0.01). Functional studies disclosed that upon oligomerization by anti-CD95 monoclonal antibodies the CD95 protein was either not able to initiate apoptosis of leukemic cells (75% of cases) or induced low rates of apoptosis (20% of cases). Only in 5% of cases did the apoptosis rate exceed the 20% level of the CD95-specific apoptosis. Most of the CD95-sensitive cases were found among T lineage ALLs (38% of T lineage vs 10% of B lineage ALLs). Overall, the extent of CD95-induced apoptosis did not correlate with the expression level of CD95. Similarly, no significant correlation between expression level and functionality of CD95 in human leukemia cell lines of B and T cell origin could be observed. Bcl-2 protein has been associated with prolonged cell survival and has been shown to block partially CD95-mediated apoptosis, but for ALL cells no correlation between bcl-2 expression and spontaneous or CD95-mediated apoptosis could be found. The results obtained in this study indicate that, despite constitutive expression of CD95, the ALL cells are mainly resistant to CD95-triggering. More detailed investigations of the molecular mechanisms involved in the intracellular apoptotic signal transduction, such as interactions of the bcl-2 and the other members of the bcl-2 family, and functionality of the interleukin-1beta converting enzyme (ICE) like-proteases, may give new insights into key events responsible for the resistance or sensitivity to the induction of apoptosis in acute leukemia.     

Radiological reasoning and its computer-based simulation. Reasons to use computer-based diagnostic systems developed on shells

p53 overexpression was studied immunohistochemically in paraffin-embedded bone marrow biopsies using a recently described technique for antigen retrieval based on microwave oven heating of paraffin sections. Using a monoclonal antibody (PAb1801) that reacts with human cellular p53, nuclear staining was detected in 7/11 (63%) therapy-related myelodysplastic syndromes and in 3/4 (75%) therapy-related acute myeloid leukemias. Conversely, staining for p53 was seen only in 9/40 (22%) cases of "primary" hematologic conditions (P < 0.007); these included myelodysplastic syndromes [#2], acute myeloid leukemia [#4], and chronic granulocytic leukemia in accelerated phase or blast crisis [#3]. Biopsies of normal controls and of chronic granulocytic leukemia in stable phase were consistently p53(-). Nine of the 10 karyotyped p53(+) acute myeloid leukemia/myelodysplastic syndrome cases showed complex cytogenetic findings with frequent involvement of chromosome 5 and/or 7. Only four of the 33 karyotyped p53(-) cases showed similar cytogenetic changes. Chromosome 17 involvement was present in four of 13 (31%) cytogenetically assessed p53+ cases, but in none of the p53(-). In univariate analysis, p53 expression in both MDS and AML was significantly associated with shorter survival. The frequent overexpression of p53 in therapy-related myelodysplastic syndromes, therapy-related acute myeloid leukemias and in accelerated phase/blast crisis, chronic granulocytic leukemia and its strong association with complex karyotypes suggests an important role of this gene in the pathogenesis of these leukemic conditions.