Cell viability of acute myeloid leukaemia blasts in culture correlates with treatment outcome

Despite the advances in understanding the pathophysiology of acute myeloid leukaemia (AML), the cure rate for acute myeloid leukaemia patients remains low. Cytogenetic abnormalities and age are the prognostic factors that guide treatment decisions. However, many AML patients still die. The biological factors that influence treatment outcome are largely unknown. Thus, the objective of our study was to use the in vitro viability test to correlate with treatment outcome. Acute myeloid leukaemia blasts demonstrated differing ability to survive in culture. Our examination of blast phenotype at various days in culture showed two possible growth directions. First, cells underwent maturation by increased expression of CD16 and down-regulated CD34 (a haemopoietic stem cell marker). These cells also appeared to have undergone apoptosis. Alternatively, cells continued to survive in culture and maintained high expression of CD34. An MTT assay was carried out to determine viability after three days of culture. Lower optical density values were obtained for samples that underwent apoptosis and higher values were obtained for samples that survived in culture. Apoptosis was measured by Annexin V/propidium iodide staining. A comparison between results of MTT assay and duration of disease free survival revealed that a higher viability in vitro correlated significantly with shorter survival duration in the patient (R -0.761, p=0.002, n=13). Thus, this study further supports the hypothesis that AML patients with poor survival may be related to having blasts with a biologically more immature or stem cell-like nature.     

The proteasome inhibitors bortezomib and PR-171 have antiproliferative and proapoptotic effects on primary human acute myeloid leukaemia cells

Proteasome inhibitors represent a new class of antineoplastic drugs that are considered in the treatment of haematological malignancies. We compared the effects of the reversible proteasome inhibitor bortezomib (Velcade) and the epoxomicin derivative PR-171, an irreversible inhibitor, on primary human acute myeloid leukaemia (AML) cells. Both drugs inhibited autocrine- and cytokine-dependent proliferation of primary AML blasts when tested at nanomolar levels (0.1-100 nmol/l). The antiproliferative effect was independent of basal chymotrypsin-like proteasome activity (showing a 20-fold variation between patients), genetic abnormalities, morphological differentiation and CD34 expression when testing a large group of consecutive patients (n = 54). The effect was retained in cocultures with bone marrow stromal cells. In addition, both drugs enhanced apoptosis. The effect of PR-171 could be detected at lower concentrations than for bortezomib, especially when testing the influence on clonogenic AML cell proliferation. Both drugs had divergent effects on AML cells' constitutive cytokine release. Furthermore, both drugs caused a decrease in proliferation and viability when tested in combination with idarubicin or cytarabine. An antiproliferative effect on primary human acute lymphoblastic leukaemia cells was also detected. We conclude that nanomolar levels of the proteasome inhibitors tested had dose-dependent antiproliferative and proapoptotic effects on primary AML cells in vitro.     

Therapy-related myelodysplastic syndrome with monosomy 5 after successful treatment of acute myeloid leukemia (M2)

We describe a patient who developed myelodysplastic syndrome over 2 years after achieving complete remission of acute myeloid leukemia (AML). The patient was treated in July 1998 with anthracycline, etoposide, and behenoyl cytarabine chemotherapy for AML (French-American-British classification, M2; World Health Organization classification, AML with maturation) and achieved complete remission. At presentation, no chromosomal abnormalities were detected. In December 2000, the patient's peripheral blood revealed pancytopenia, and his bone marrow was hypocellular with trilineage myelodysplasia and no blasts. Chromosomal analysis revealed complex karyotypic abnormalities, including monosomy 5. The patient was diagnosed with high-grade myelodysplastic syndrome (MDS)/refractory anemia with excess blasts (RAEB) subtype. The pancytopenia progressed rapidly, and he died 2 months after the diagnosis of MDS. Therapy-related MDS and AML (t-MDS/t-AML) developing after treatment for acute leukemia is unusual; the primary leukemia associated with most cases of t-MDS/t-AML is acute promyelocytic leukemia (APL). This unusual case suggests that AML excluding APL should be considered a primary hematologic malignancy for t-MDS/t-AML.     

The role of an anti-myeloperoxidase antibody in the diagnosis and classification of acute leukaemia: a comparison with light and electron microscopy cytochemistry

The enzyme myeloperoxidase (MPO) is the hallmark of the myeloid lineage. We have analysed the presence of MPO in blasts from 180 cases of acute leukaemia (103 acute myeloid leukaemia (AML) and 77 acute lymphoid leukaemia (ALL) by means of monoclonal antibodies anti-MPO and immunocytochemistry (alkaline phosphatase anti-alkaline phosphatase method). The aim of the study was to investigate the specificity and sensitivity of this marker compared with MPO cytochemistry by light (LM) and electron microscopy (EM), and with the expression of myeloid antigens. Anti-MPO was positive (greater than 3% blasts) in all but one of the 90 AML positive by LM cytochemistry. Of 13 AML cases negative by MPO cytochemistry, six showed 3-10% blasts reactive with anti-MPO and were also positive with antibodies to CD13 and/or CD33. The presence of MPO was confirmed in four of these by EM. The overall positivity of anti-MPO in AML was 92%. Anti-MPO was negative in all but two ALL (6% and 8% positive blasts). The blasts in these two cases were also CD13, CD33 and MPO positive by EM; both were thus reclassified as biphenotypic. Another two ALL reinterpreted as biphenotypic were negative by MPO cytochemistry and anti-MPO but were MPO positive by EM and with CD13 and/or CD33. We conclude that anti-MPO is a sensitive and specific early marker of myeloid blasts and should be incorporated in the routine immunophenotyping of acute leukaemia.     

Core-binding factor-beta positive acute myeloid leukaemia cells induce T-cell responses

The addition of specific cytokines is a mandatory prerequisite for the generation and subsequent function of leukaemia-derived dendritic cells (DC) believed to induce specific T-cell responses. In this study, we report the ability of blasts derived from cytogenetically classified acute myeloid leukaemia (AML) cells with the inversion of chromosome 16 to stimulate allogeneic and autologous T cells without additional cytokines. They displayed a measurable immunogenic effect. Sixteen of 17 established, stable AML cell lines, growing primary tumour cells from patients with a variety of chromosomal abnormalities, altered their surface marker expression pattern in proliferating culture. They lost the progenitor markers CD33, CD13 and CD34 while significantly increasing expression of the co-stimulatory molecules CD80 and CD86. Four cell lines derived from inv(16) positive blasts mounted allogeneic as well as autologous T cell activation with concomitant expression of CD25 and CD69. Moreover, oligoclonal expanded T cells were able to lyse inv(16) AML blasts in a specific major histocompatibility complex class I-restricted and CD80-dependent manner. AML blasts with karyotypes other than inv(16) activated T cells, but without inducing a significant proliferation. We conclude from this study that AML blasts derived from inv(16) positive patients may be preferential targets for AML immunotherapy strategies.     

Lack of effect of the human GM-CSF analog E21R on the survival of primary human acute myeloid leukemia cells

The granulocyte-macrophage colony-stimulating factor (GM-CSF) analog E21R binds to the GM-CSF receptor complex with low affinity and acts as a competitive antagonist. In addition, it has been reported to be a potent direct activator of apoptosis in primary human acute myeloid leukemia (AML) cells. We have confirmed the ability of E21R to neutralize the biologic effects of GM-CSF and investigated its activity on primary AML blasts. We find that it failed to induce cell death in blast cells from 23 separate AML cases treated in vitro at concentrations of E21R up to 30 microg/mL. Significant cell death resulted in all cases after incubation with cytosine arabinoside. The lack of effect of E21R on AML blasts was unlikely to be due to an absence of functional GM-CSF receptors because 13 cases demonstrated an increase in cell number with the addition of exogenous GM-CSF. These results do not support the use of E21R for the treatment of AML.     

Expression of the leucocyte common antigen (LCA,CD45) isoforms RA and RO in acute haematological malignancies: possible relevance in the definition of new overlap points between normal and leukaemic haemopoiesis

Summary. The membrane expression of CD45RA and CD45RO on fresh leukaemic cells taken from 529 cases of acute haemopoietic malignancies, including 117 B-origin acute lymphoblastic leukaemia (B-origin ALL), 3 7 T-origin acute lymphoblastic leukaemia (T-origin ALL), 297 de novo acute myeloid leukaemia (AML), 42 refractory anaemia with excess of blasts in transformation (RAEB-T) and 36 myeloid blastic phase of chronic myelogenous leukaemia (CML-BP-my), was analysed. B-origin ALLs were characterized by the lack of the RO isoform along with the consistent presence of RA. Conversely, a differential expression of the two isoforms was detected in different subsets of T-origin ALL, in that T-stem cell leukaemias (T-SCL: CD7⁺, CD4^?, CD8^?, CD1^?) preferentially expressed CD45RA whereas conventional T-acute lymphoblastic leukaemias (T-ALL: CD7⁺, CD4⁺ and/or CD8⁺ and/or CD1+) were consistently marked by CD45RO. Within myeloid malignancies, most of AMLs displayed CD45RA, while a substantial group of CML-BP-my preferentially exhibited CD45RO. As a general rule, a reciprocal exclusion of the two isoforms was observed in AML as well as in ALL. Nevertheless, a frequent coexpression of CD45RA and CD45RO was observed in CD14⁺ AML. In vitro treatment with all-trans retinoic acid (ATRA) was able to promote a switch from CD45RA to CD45RO expression in 2 7 de novo AML, independently from morphological subtyping. To our knowledge, this is the first report on CD45 isoform expression in a large series of patients with acute leukaemia. The knowledge of the differential expression of CD45RA and CD45RO can ameliorate our classificative approach to haematological malignancies, as well as disclose new multiple overlap points between normal and leukaemic cell differentiation.     

The murine monoclonal antibody, 14A2.H1, identifies a novel platelet surface antigen

A murine monoclonal antibody 14A2.H1, raised against acute myeloid leukaemia cells, identifies a previously undescribed 27 kDa platelet surface glycoprotein which is expressed at low copy number (10(3)/platelet). MAb 14A2.H1 caused aggregation of platelets which was dependent on Fc gamma RII. Binding of the antibody to platelets was not altered by activation by thrombin or phorbol ester. In haemopoietic cell populations the antibody bound to megakaryocytes, monocytes (weakly), several myeloid leukaemic cell lines and fresh myeloid leukaemic blasts from some patients. Lymphocytes, lymphoid cell lines, neutrophils and haemopoietic progenitor cells were negative. Expression of the antigen was not restricted to haemopoietic cells as epithelial cells in tonsillar crypts and endothelial cells were positive.     

Telomerase activity in acute myelogenous leukaemia: clinical and biological implications

We examined telomerase activity in myeloid leukaemic cell lines, normal haemopoietic cells, and leukaemic blasts from acute myelogenous leukaemia (AML) patients. Normal bone marrow mononuclear (BMNC) cells expressed low telomerase activity. Higher telomerase activity was detected in 10 myeloid leukaemic cell lines compared to normal BMNC cells. Treatment with 1,25(OH)₂D₃, and vitamin D₃ analogues, EB1089 and KH1060, reduced telomerase activity in vitamin D₃-sensitive HL-60 cells, whereas vitamin D₃ insensitive K562 cells did not change its activity. This down-regulation of telomerase activity by EB1089 was associated with induction of p21 protein. The rank order of telomerase activity was leukaemic CD34⁻ cells > leukaemic CD34⁺ cells > normal CD34⁻ cells > normal CD34⁺ cells. Telomerase activity was positive in all of the AML patients tested; however, heterogeneity of telomerase activity was found amongst this group. Therefore we compared telomerase activity with clinical response. Unexpectedly, we found that a higher rate of complete remission was noted in AML patients with higher telomerase activity. No association between telomerase activity and biological parameters including percentage of S-phase, cytotoxicity to cytosine arabinoside and percentage of CD34⁺ cells in AML blasts was found. These results suggest that telomerase activity in AML patients is detected with high frequency, but is heterogenous. Expression level of telomerase activity may have a clinical implication in AML patients regarding clinical response.     

Functional response of leukaemic blasts to stromal cell-derived factor-1 correlates with preferential expression of the chemokine receptor CXCR4 in acute myelomonocytic and lymphoblastic leukaemia

The chemokine stromal cell-derived factor-1 (SDF-1) that is released by bone marrow (BM) stromal cells and contributes to stem cell homing may also play a role in the trafficking of leukaemic cells. We analysed SDF-1-induced intracellular calcium fluxes in leukaemic blasts from the peripheral blood of patients with newly diagnosed acute myeloid leukaemia (AML) and lymphoblastic leukaemia (B-lineage ALL), determined the effect of BM stromal cell-conditioned medium on in vitro transendothelial migration (TM) and measured expression of the SDF-1 receptor, CXCR4, by flow cytometry. AML FAB M1/2 blasts did not show calcium fluxes and TM was not stimulated. In myelomonocytic AML (M4/5), however, SDF-1 induced significant calcium fluxes and TM was increased twofold by the conditioned medium. M3 and M4 blasts with eosinophilia (M4eo) showed intermediate activity and M6 blasts showed no functional activity. In ALL, strong calcium fluxes and increased TM (2.5-fold) were observed. Accordingly, expression of CXCR4 was low in undifferentiated (M0) AML, myeloid (M1/2) AML and erythroid (M6) AML, but high [mean fluorescence (MF) > 50] in promyelocytic (M3) AML, myelomonocytic (M4/5) AML and B-lineage ALL. We conclude that, in AML, SDF-1 is preferentially active in myelomonocytic blasts as a result of differentiation-related expression of CXCR4. Functional activity of SDF-1 and high expression of CXCR4 in B-lineage ALL is in accordance with the previously described activity of SDF-1 in early B cells. SDF-1 may contribute to leukaemic marrow infiltration, as suggested by increased CXCR4 expression and migratory response in BM-derived blasts compared with circulating cells.     

Growth stimulatory effect of thrombopoietin on the blast cells of acute myelogenous leukaemia

Thrombopoietin stimulated blast colony formation in 11/20 acute myelogenous leukaemia (AML) patients studied. The FAB subtypes of the blasts responding to thrombopoietin were not restricted to those of the megakaryocyte lineage, but also included M1–M5 AML blasts. The morphology of colony cells produced by megakaryocytic blasts showed megakaryocytoid features, whereas colony cells produced by M1–M5 AML blasts remained myeloblasts. An increase in CD41 was observed in the cells of colonies produced by blasts from the megakaryocyte lineage involving leukaemia and chronic myeloid leukaemia in blastic crisis. Thrombopoietin receptor was observed on leukaemic blasts which formed colonies following incubation with thrombopoietin.     

Dendritic cells fused with core binding factor-beta positive acute myeloid leukaemia blast cells induce activation of cytotoxic lymphocytes

Several reports have described various strategies of dendritic cell (DC) vaccination to induce specific T-cell responses in patients with acute myeloid leukaemia (AML). About 50-60% of AML cases blasts have chromosomal abnormalities, such as inv(16) or t(8,21), which could encode for leukaemia-specific antigenic peptide sequences, possibly presented in the context of self-major histocompatibility complex (MHC) molecules. As the co-culture of AML blasts with T lymphocytes seldom resulted in T-cell stimulation, we fused AML blasts with autologous DC to enhance this effect. The fusion cells expressed MHC class I and II, CD40, B7-1, B7-2, CD209 and several adhesion molecules. In a mixed lymphocyte hybrid reaction, the fusion cells induced the proliferation of autologous T cells. Moreover, in the special case of fusion cells established from AML blasts with the chromosomal abnormality inv(16), the autologous T lymphocytes could be primed to induce cytotoxicity against up to 70% autologous AML blasts in a effector:target ratio of 20:1. Blocking assays demonstrated that the lysis was chiefly mediated by CD8(+), CCR7(-) T lymphocytes, which could be further expanded in the form of effector memory CD8(+) T cells by repeated co-cultures with the autologous fusion cells.     

Poor response to intensive chemotherapy in de novo acute myeloid leukaemia with trilineage myelodysplasia

Summary. The findings of morphologically dysplastic features in haemopoietic cells in de novo acute myeloid leukaemia (AML) has been named AML with trilineage myelodysplasia (AML/TMDS). We analysed the clinical data, karyotypes, and treatment outcomes of 230 de novo AML patients treated with the Japan Adult Leukaemia Study Group AML-87 protocol. 40 (17%) patients had AML/TMDS. Platelet count was significantly higher ( F =0·006) and bone marrow blasts were fewer ( P =0·01) in the AML/TMDS group than in the AML without TMDS. Abnormal karyotype was shown in 12/30 patients (40%) analysed.
The complete remission (CR) rate for AML/TMDS was significantly lower than AML without TMDS (63% v 81%) ( P =0·01). The overall survival curves showed that the 40 patients with TMDS had a significantly worse survival than the 190 without TMDS ( P =0·0005). AML/TMDS also showed significantly worse disease-free survival (DFS) ( P =0·0001).
Multivariate analysis revealed that the absence of TMDS in AML was the most significant factor in obtaining CR ( P =0·01) and a significant factor in predicting longer DFS ( P =0·04). Our data suggest that AML/TMDS responds poorly to intensive chemotherapy. Further study is required to determine the best treatment strategy for AML/TMDS and the biological differences between AML/TMDS and other types of AML.     

The relationship between bcl-2 expression and response to chemotherapy in acute leukaemia

Summary. Immunocytochemistry was used to assess bcl-2 expression in blasts obtained from the bone marrow of 28 patients with acute lymphoblastic leukaemia (ALL) (16 children and six adults at presentation and three children and three adults on relapse) and 20 with acute myeloid leukaemia (AML) (19 adults and one child, 13 with de novo AML, 11 at presentation and two on relapse, and seven secondary to myelodysplasia or chronic myeloid leukaemia). Slides were examined both for the percentage of positive cells and for the intensity of staining using a five-point scale. There was a statistically significant increase in both the percentage of positive cells seen (P < 0.002) and the intensity of staining (P < 0.01) between samples obtained at relapse and those at presentation in ALL. There was a significantly greater intensity of staining in cells from patients with ALL (P < 0.05) and AML (P < 0.05) who failed to achieve remission after chemotherapy than in those who responded. The intensity of staining in cases of secondary AML was lower than that in de novo disease (P < 0.01). These results suggest that expression of bcl-2 may be an important prognostic feature in both de novo AML and in ALL, but not in secondary AML.     

Generation of cytokine-induced killer cells from leukaemic samples with in vitro cytotoxicity against autologous and allogeneic leukaemic blasts

Cytokine-induced killer (CIK) cells are CD3(+)CD56(+) non-major histocompatibility complex (MHC)-restricted immune effector cells. The present report demonstrates that it was possible to expand CIK cells obtained at diagnosis from patients with acute leukaemia. The percentage of CD3(+)CD56(+) CIK cells generated following culture ranged between 7.6% and 65% (median of 35.3%) and these cells were able to kill the human natural killer target K562 cells. Although the same effector cells were able to lyse autologous acute myeloid leukaemia (AML) target cells, they were not able to lyse autologous acute lymphoblastic leukaemia target cells. Pre-absorption of the CIK effector cells by K562 cells did not completely abrogate the cytotoxicity of CIK cells against autologous blasts in 9 out of 12 samples tested. Moreover, it was observed that the cytotoxicity generated by the CIK effector cells against allogeneic leukaemic blasts was similar to that against autologous blasts. The present study suggests the potential application of CIK cells in the immunotherapy of AML, either in minimal disease state, as donor lymphocyte infusion in relapse post allogeneic transplant, or in cases of chemotherapy refractory leukaemia.     

Biological and clinical relevance of matrix metalloproteinases 2 and 9 in acute myeloid leukaemias and myelodysplastic syndromes

We analysed by immunocytochemistry metalloproteinase (MMP)-2 and MMP-9 expression in bone marrow cells from 54 acute myeloid leukaemia (AML) patients, 153 myelodysplastic syndrome (MDS) patients, and 52 non-haemopathic subjects, in order to evaluate whether MMP expression abnormalities were associated with relevant laboratory or clinical findings. In normal samples MMP-2 was detected in rare myeloid cells, MMP-9 in most maturing myeloid cells. In MDS MMP-2 myeloid levels were higher than in controls (P < 0.0001); MMP-2 and MMP-9 were often co-expressed. Also many erythroblasts expressed MMP-2. There was a positive correlation between MMP-2 erythroblast expression and erythroid dysplasia (P = 0.002) and an inverse correlation between MMP-2 or MMP-9 myeloid expression and blast cell percentage (P = 0.05 and P = 0.04 respectively). High MMP levels in myeloid cells were associated with longer overall survival (P = 0.03) and evolution-free survival (P = 0.04). In AML MMP-2 levels were lower than in MDS (P < 0.0001) and MMP-9 levels lower than in MDS and controls (P < 0.0001). MMP levels did not predict response to therapy. The release of active MMPs was detected by colorimetric analysis in cell cultures from representative MDS and AML cases. In conclusion, we have demonstrated an abnormal MMP expression in AML as well as in MDS. The production and release of these enzymes may influence haematopoietic cell behaviour. In MDS, the detection of MMP deregulated expression may be important also from the clinical point of view: it may provide a useful tool for diagnosis, prognosis and a possible target for experimental treatments.     

Relapse after allogeneic bone marrow transplantation for acute leukaemia: a survey by the E.B.M.T. of 117 cases

A multi-centre retrospective analysis on 117 patients relapsing after bone marrow transplantation (BMT) for acute leukaemia was carried out by the Leukaemia Working Party of the European Group for Bone Marrow Transplantation (E.B.M.T.). Forty-one patients had acute myeloid leukaemia (AML) and 76 had acute lymphoblastic leukaemia (ALL). Relapse occurred between 3 and 30 months after BMT and where investigated the leukaemia was found to have relapsed in recipient cells. In 10 cases the relapse was associated with new cytogenetic abnormalities. 74 patients received further treatment for leukaemia. Of these 21 out of 50 with ALL and 11 out of 24 with AML achieved a complete remission and had a median survival of 12 months compared with a median survival of 4 months for untreated patients or patients not achieving complete remission (P less than 0.001). Factors predictive for successful remission induction were a long interval between bone marrow transplant and relapse in ALL patients; and isolated extramedullary relapse. Presenting blast count, karyotype and remission status and number at the time of BMT were not predictive. Donor bone marrow was shown to be responsible for haemopoietic recovery occurring in the 21 out of 31 patients tested who achieved remission using donor karyotype or red blood cell antigens as markers. Nine patients received a second bone marrow transplant but only one became a long-term survivor. The results show that chemotherapy can usually prolong survival in selected patients with acute leukaemia relapsing after BMT but further BMT has a poor outlook.