Ex vivo expansion and characterisation of CD34+ cells derived from chronic myeloid leukaemia bone marrow and peripheral blood, and from normal bone marrow and mobilised peripheral blood

Ex vivo culture of CD34+ has the potential to provide large numbers of cells for clinical use in autologous and allogeneic transplantation and for experimental research involving genetic manipulation. We evaluated the ex vivo expansion of CD34+ cells obtained from bone marrow (BM) and peripheral blood (PB) of untreated patients with chronic myeloid leukaemia (CML) in the chronic phase and compared these results with those obtained from BM from normal volunteers (NBM) and peripheral blood after mobilising chemotherapy from patients with non-haematological disorders (MPB). Selected CD34+ cells were stimulated with interleukin 1(beta), interleukin IL-3, interleukin IL-6 and stem cell factor. The proliferation observed in patients with CML was similar to that seen in normal donors. CD34+ cells derived from patients with CML are more differentiated than their normal counterparts, as shown by the coexpression of CD34 and CD33 antigens on day 0 (85.6% for CML-BM and 76.8% for CML-PB). The culture conditions allowed a significant expansion of granulocyte-macrophage colony-forming units (CFU-GM) from NBM (33-fold increase) and MPB (22-fold increase), in contrast with CML-derived BM and PB CD34+ cells (2.3-fold increase). These results indicate that the optimal time to harvest ex vivo expanded cells is dependent on a critical compromise between cell numbers and successful retention of their repopulating potential.     

Antibody therapy for acute myeloid leukaemia

Novel therapies with increased efficacy and decreased toxicity are desperately needed for the treatment of acute myeloid leukaemia (AML). The anti CD33 immunoconjugate, gemtuzumab ozogamicin (GO), was withdrawn with concerns over induction mortality and lack of efficacy. However a number of recent trials suggest that, particularly in AML with favourable cytogenetics, GO may improve overall survival. This data and the development of alternative novel monoclonal antibodies (mAb) have renewed interest in the area. Leukaemic stem cells (LSC) are identified as the subset of AML blasts that reproduces the leukaemic phenotype upon transplantation into immunosuppressed mice. AML relapse may be caused by chemoresistant LSC and this has refocused interest on identifying and targeting antigens specific for LSC. Several mAb have been developed that target LSC effectively in xenogeneic models but only a few have begun clinical evaluation. Antibody engineering may improve the activity of potential new therapeutics for AML. The encouraging results seen with bispecific T cell‐engaging mAb‐based molecules against CD19 in the treatment of B‐cell acute lymphobalstic leukaemia, highlight the potential efficacy of engineered antibodies in the treatment of acute leukaemia. Potent engineered mAb, possibly targeting novel LSC antigens, offer hope for improving the current poor prognosis for AML.     

Expression of multidrug resistance P-glycoprotein in myeloid progenitor cells of different phenotype: comparison between normal bone marrow cells and leukaemia cells

We examined the multidrug resistant P-glycoprotein (P-gp) on normal bone marrow (BM) cells and acute myeloid leukaemia (AML) cells, using newly devised flow cytometric multi-parameter analysis with CD33, CD34 and MRK16 monoclonal antibodies. In both normal BM cells and AML cells, CD34⁺CD33⁻ cells expressed P-gp strongly, CD34⁺CD33⁺ cells moderately, and CD34⁻CD33⁺ cells weakly. Acute promyelocytic leukaemia, mainly expressing CD34⁻CD33⁺ but not CD34⁺CD33⁻ at diagnosis, expressed less P-gp. P-gp expression of AML cells at diagnosis was increased as compared with normal cells of the same phenotype. P-gp expression was more increased in relapsed cases, especially in immature subpopulations.     

Secondary T-acute lymphoblastic leukaemia mimicking blast crisis in chronic myeloid leukaemia

A 34-year-old man with chronic myeloid leukaemia (CML) firstly developed a lymphoid blast crisis of B-cell type. After a second chronic phase which lasted for > 4 years with maintenance chemotherapy of hydroxyurea, 6-mercaptopurine and methotrexate, he developed a T-cell acute lymphoblastic leukaemia of TcR-gammadelta+ type. Cytogenetic analysis revealed disappearance of the t(9;22) translocation and appearance of new abnormalities consistent with the diagnosis secondary acute leukaemia. To our knowledge, secondary leukaemia in CML has not previously been reported.     

Expression of Fas antigen in acute myeloid leukaemia is associated with therapeutic response to chemotherapy

Flow cytometric immunofluorescent analysis was used to assess Fas antigen (CD95) expression in blasts obtained from the bone marrow of 30 patients with acute myeloid leukaemia. The percentage of positive cells in each sample was highly variable. Fas antigen expression did not correlate with age, FAB subtype, white blood cell counts, or CD34 expression. Low expression of Fas was associated with a low complete remission rate after induction chemotherapy (62.5% in cases with <20% positive cells v 92.9% in cases with ≥ 20% positive cells, P <0.01). The main cause for not achieving remission was resistant disease. Our results suggest that the quantitation of Fas expression can be predictive of treatment outcome in acute myeloid leukaemia.     

BCR-ABL1 tyrosine kinase sustained MECOM expression in chronic myeloid leukaemia

MECOM oncogene expression correlates with chronic myeloid leukaemia (CML) progression. Here we show that the knockdown of MECOM (E) and MECOM (ME) isoforms reduces cell division at low cell density, inhibits colony-forming cells by 34% and moderately reduces BCR-ABL1 mRNA and protein expression but not tyrosine kinase catalytic activity in K562 cells. We also show that both E and ME are expressed in CD34(+) selected cells of both CML chronic phase (CML-CP), and non-CML (normal) origin. Furthermore, MECOM mRNA and protein expression were repressed by imatinib mesylate treatment of CML-CP CD34(+) cells, K562 and KY01 cell lines whereas imatinib had no effect in non-CML BCR-ABL1 -ve CD34(+) cells. Together these results suggest that BCR-ABL1 tyrosine kinase catalytic activity regulates MECOM gene expression in CML-CP progenitor cells and that the BCR-ABL1 oncoprotein partially mediates its biological activity through MECOM. MECOM gene expression in CML-CP progenitor cells would provide an in vivo selective advantage, contributing to CML pathogenesis.     

New insights into the biology of Philadelphia-chromosome-positive acute lymphoblastic leukaemia using a combination of May-Grünwald-Giemsa staining and fluorescence in situ hybridization techniques at the single cell level

It is sometimes difficult to discriminate chronic myeloid leukaemia (CML) in lymphatic blast crisis from Ph-chromosome positive acute lymphoblastic leukaemia (ALL). Previous studies have suggested that ALL is restricted to the lymphatic lineage only, whereas CML involves all cell lineages.
In four cases of Ph-positive ALL we combined the standard May-Grünwald-Giemsa staining with FISH at the single cell level and were able to demonstrate that  98% of lymphatic blasts carried the Philadelphia chromosome. Erythropoiesis was not involved when this technique was applied. Using immunological identification of single cells (FICTION), we detected the t(9;22) in 100% of CD19-positive B lymphoblasts in all four cases, in some CD3-positive T cells in two patients, and in  98% of CD34-positive precursor cells. However, in two out of four patients the myeloid cell compartment was involved in the malignant transformation, unequivocally demonstrated not only by the combination of MGG and FISH but also by FICTION using the antibodies CD13 and CD33. The observation that both patients with myeloid cell lineage involvement had a myeloid co-expression on their blasts and a better survival supports the concept of a separate, biologically determined subgroup in Ph-positive ALL, leading to further investigations, and individually tailored treatment strategies.     

CD66 expression in acute leukaemia

Antibodies against CD66 identify antigens from the carcinoembryonic antigen (CEA) family of proteins, which belong to the immunoglobulin gene superfamily. Despite being usually restricted to cells of myeloid or monocytic origin, CD66 expression has also been reported in blasts from children with B-cell lineage acute lymphocytic leukaemia (ALL). An analysis of the CD66 expression was undertaken in a series of acute leukaemia patients. Antigenic expression was analysed using triple combinations of monoclonal antibodies (mAbs) in forty-five patients. The CD66 Kat4 fluorescein isothiocyanate clone was purchased from Dako (Glostrup, Denmark). CD66 was expressed in 2 of 29 patients with AML (acute myeloblastic leukemia) (6.8%) and in 8 of 12 patients with B-cell lineage ALL (66.7%; P <0.001); in blast crisis (BC) of chronic myelocytic leukaemia (CML), CD66 was expressed in two patients with lymphoid BC but not in the two with myeloid BC. The co-expression of CD66 with other myeloid antigens was observed in all CD66+ ALL/Ly-BC cases tested: CD 13 in six patients, CD33 in seven and CD117 in two patients. The CD66 expression is more frequent in ALL than in AML. Furthermore, we analysed minimal residual disease (MRD) in eight patients in complete remission. CD66 expression was associated with an abnormal B-cell differentiation pattern and with increases in CD34/CD19+ cells in all but one case. These findings suggest that an aberrant expression of CD66 could be used to investigate MRD in ALL. The association between CD66 reactivity and bcr-abl in adult ALL remains to be investigated. Received: 31 May 1999 / Accepted: 10 November 1999     

Cancer in relatives of children with myelodysplastic syndrome, acute and chronic myeloid leukaemia

Familial myelodysplastic syndrome (MDS) has been claimed to account for as many as one third of children with MDS, especially among those showing monosomy 7. The present study is the first to provide population-based estimates of the risk of haematological and other malignancies in relatives of children with MDS. The study was extended to include children with acute myeloid leukaemia (AML) and chronic myeloid leukaemia (CML). The index group consisted of 46 children with MDS, 62 with AML, and eight with CML, which is thought to represent all myeloid leukaemias in Danish children, 1980–91. By linkage to the Central Population Register we identified parents (230), siblings (231), grandparents (151), aunts and uncles (132) and cousins (140). Information on the cancer incidence was obtained from the Danish Cancer Registry. 27 cancers were observed versus 26.7 expected (relative risk 1.0). Leukaemia in relatives was observed in only one family. None of 11 children with MDS and monosomy 7 had family members affected by leukaemia. We found no evidence of an increased overall risk of cancer in the relatives. The risk of familial MDS may be considerably lower than previously estimated.     

Absence of leukaemic CD34+ cells in acute myeloid leukaemia is of high prognostic value: a longstanding controversy deciphered

Primary resistance and relapses after initial successful treatment are common in acute myeloid leukaemia and therefore outcome remains poor. More accurate risk group stratification and effective personalized risk adapted treatment are necessary to improve outcome. In the last two decades, controversial results have been published concerning the prognostic relevance of CD34 expression. In this study of 706 acute myeloid leukaemia patients, we established a new flow cytometric‐based CD34‐definition, without use of cut‐off values. We discriminated CD34‐positive (n = 548) and CD34‐negative (n = 158) patients by the presence or absence of neoplastic CD34+ cells, respectively. CD34‐status was defined using aberrant immunophenotypes and validated using molecular phenotypes. This new definition of CD34 enables strong prediction of treatment outcome in the entire patient group and in several risk subgroups. Previously observed discrepancies in prognostic impact of CD34 protein expression using cut‐offs (5–20%) can now entirely be explained by considering the number of CD34‐negative cases. In the total patient group, the absence of neoplastic CD34‐positive cells is paralleled by low levels of minimal residual disease, suggesting relative therapy sensitivity and explaining longer survival. Overall, we present CD34 surface expression as a relatively simple, powerful and independent predictor of clinical outcome, now warranting incorporation in acute myeloid leukaemia risk stratification.     

Lack of expression of the chondroitin sulphate proteoglycan neuron-glial antigen 2 on candidate stem cell populations in paediatric acute myeloid leukaemia/abn(11q23) and acute lymphoblastic leukaemia/t(4;11)

It has increasingly been acknowledged that only a few leukaemic cells possess the capability to renew themselves and that only these self-renewing leukaemic stem cells are able to initiate relapses. Therefore, these leukaemic stem cells should be the target cells for therapy and for minimal residual disease (MRD) detection. Because of its presence on blasts of 11q23-rearranged high-risk leukaemic patients, neuron-glial antigen 2 (NG2) is thought to be a valuable marker for detecting leukaemic stem cells. Six acute myeloid leukaemia (AML)/abn(11q23) and three acute lymphoblastic leukaemia (ALL)/t(4;11) samples were analysed by four-colour flow cytometry for NG2 expression on primitive cell populations. Candidate leukaemic cell populations were defined by the antigen profiles CD34+CD38- in AML and CD34+CD19-CD117+ in ALL. Surprisingly, in all patients these candidate stem cell populations were shown to lack expression of NG2. Instead, a correlation between the expression of the myeloid differentiation marker CD33 and increasing levels of NG2 on maturing cells could be demonstrated. Similarly, in ALL patients CD34+CD19+ cells showed a higher expression of NG2 mRNA compared with CD34+CD19-. Thus, NG2 appears to be upregulated with differentiation and not to be expressed on primitive disease-maintaining cells. This hampers the clinical use of NG2 as a therapeutic target and as a sensitive marker for MRD detection.     

MOZ/TIF2-induced acute myeloid leukaemia in transgenic fish

The inv(8)(p11q13) chromosomal abnormality, described in acute myeloid leukaemias (AML), fuses the histone acetyl-transferase (HAT) MYST3 (MOZ) gene with another HAT gene, NCOA2 (TIF2). We generated a transgenic zebrafish in which the MYST3/NCOA2 fusion gene was expressed under control of the spi1 promoter. An AML developed in 2 of 180 MYST3/NCOA2-EGFP-expressing embryos, 14 and 26 months after injection of the fusion gene in a one-cell embryo, respectively. This leukaemia was characterised by an extensive invasion of kidneys by myeloid blast cells. This model, which is the first zebrafish model of AML, demonstrates the oncogenic potency of MYST3/NCOA2 fusion gene.     

Grading of symptoms in hyperleukocytic leukaemia: a clinical model for the role of different blast types and promyelocytes in the development of leukostasis syndrome

OBJECTIVE: Patients with hyperleukocytic leukaemia were graded according to the severity of symptoms possibly caused by leukostasis to evaluate the effectiveness of therapy and to test the relative contribution of blast type and count of blasts and promyelocytes in the development of leukostasis syndrome. METHODS: Ninety-five patients (59 male, 36 female, median age 52 yr) with hyperleukocytic leukaemia [leukocytes above 50 x 10(9)/L, 48 acute myeloid leukaemia (AML), 31 chronic myeloid leukaemia (CML), 13 acute lymphoblastic leukaemia (ALL), three chronic myelomonocytic leukaemia (CMML)] were grouped according to the presence or absence and severity of neurologic, pulmonary and other symptoms into four categories (no, possible, probable and highly probable leukostasis syndrome). Age, white blood count (WBC), haemoglobin, blast count and total of blasts plus promyelocytes of these groups were compared by Mann-Whitney U-test. RESULTS: Patients with myeloid leukaemia (AML M1/M2, CML) which scored as highly probable leukostasis showed significantly higher WBC (P = 0.011), lower haemoglobin (P = 0.004), higher peripheral blast counts (P = 0.004) and higher total of peripheral blasts plus promyelocytes (P < 0.001) compared with the lower probability groups. In leukaemia involving the monocytic lineage (AML M4/M5, CMML) no significant differences were found in any of these factors between patients with highly probable leukostasis and the other patients. CONCLUSIONS: Our results show that a four-stage clinical grading scale is a valuable tool for analysing hyperleukocytic patient populations and evaluate the effectiveness of therapy more precisely. We further demonstrate that the mechanisms of leukostasis are different in myeloid leukaemia as compared with leukaemia with involvement of the monocytic lineage.     

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.     

Neoplastic circulating endothelial-like cells in patients with acute myeloid leukaemia

Accumulating evidence suggests that angiogenesis may play a key role in the pathogenesis of leukaemic disorders. Several studies have shown that bone marrow-derived endothelial cells (EC) may contribute to tumour angiogenesis and that in the peripheral blood of cancer patients there is an increased amount of circulating ECs (CECs) that may participate to new vessel formation. In this report, we showed that, in seven acute myeloid leukaemia (AML) patients with known cytogenetic abnormalities, CEC levels were significantly increased in comparison with controls and that a significant proportion of these CECs carried the same chromosomal aberration as blast cells (20-78%, mean value 42.1% of CECs). Most of CECs (mean value 74.4%) displayed immunophenotypic features of endothelial progenitor cells as they expressed CD133, a marker gradually lost during EC differentiation and absent on mature EC. These findings suggest a possible direct contribution of AML-related CECs to tumour vasculogenesis and possibly to the spreading and progression of the disease.