FUNCTIONAL CELL COMPARTMENTS IN A RAT MODEL FOR HUMAN ACUTE MYELOCYTIC LEUKAEMIA

Functional cell compartments were studied in a rat model for human acute myelocytic leukaemia (AML). This was done by tracing the distribution of injected ⁵¹Chromium-labelled leukaemic cells in the body. It was concluded that two functional compartments can be distinguished in acute leukaemia, i.e., a rapidly exchangeable pool of cells (including the circulating blood pool, the marginal noncirculating blood pool and the rapidly exchangeable tissue pool; RETP) and a slowly exchangeable tissue pool (SETP). The sizes of these various compartments were roughly quantified at various stages of the disease by calculations based on the principle of isotope dilution and organ weight measurements. As the leukaemia progresses, the size of the SETP increases significantly relative to the size of the RETP. Simultaneously, the exchange rates of leukaemic cells between the organs and the blood decrease. The blood transit time of leukaemic cells was also significantly prolonged, as is the case in human AML.     

Preferential induction of apoptosis of leukaemic cells by farnesol

Farnesol preferentially inhibits proliferation and induces apoptosis of tumour-derived but not non-transformed cell lines. We investigated whether farnesol induces apoptosis of blasts from patients with acute myeloid leukaemia (AML) and leukaemic cell lines, as compared with normal, human primary haemopoietic cells. We show that 30 microM farnesol causes apoptosis of leukaemic cell lines of T- and B-lymphocyte, myeloid or erythroid lineages and primary blasts obtained from patients with AML. However, the same concentration did not kill primary monocytes, or quiescent or proliferating T-lymphocytes. We conclude that farnesol selectively kills AML blasts and leukaemic cell lines in preference to primary haemopoietic cells.     

Immunoreactive calcitonin in leukaemia.

A radioimmunoassay was used to measure concentrations of immunoreactive human calcitonin (HCT) in plasma and leucocytes from patients with various leukaemic and myeloproliferative disorders. Plasma immunoreactive HCT concentrations were increased in 32 out of 33 patients with chronic granulocytic leukaemia (CGL) and in all eight patients with acute myeloid leukamia (AML) at presentation or in relapse. Out of 11 patients with other myeloproliferative disorders, eight had increased plasma immunoreactive HCT concentrations. Buffy-coat-cell extracts and culture media from peripheral leucocytes of patients with CGL also contained increased immunoreactive HCT concentrations. In contrast, plasma from patients with chronic lymphocytic leukaemia, acute lymphoblastic leukaemia, and AML in remission had low or undetectable immunoreactive HCT concentrations. Increased plasma and cellular concentrations of immunoreactive HCT may be a consequence of abnormal proliferation of myeloid cells and might prove to be valuable in predicting relapse in patients with myeloid leukaemias.     

Generation of cytotoxic T lymphocytes from peripheral blood of leukaemic patients

Peripheral blood mononuclear cells from 13 patients with acute leukaemia were used to establish long-term interleukin-2-dependent cytotoxic T lymphocytes. Cells were grown in RPMI medium containing interleukin-2 (IL-2, 100 U/ml) and 2.5% conditioned medium prepared by activating normal lymphocytes with phytohaemagglutinin. Proliferation of IL-2-dependent CD3-positive lymphocytes was seen in 1 of 2 acute lymphocytic leukaemia cases (ALL), 1 of 4 acute myelogeneous leukaemia cases (AML) (M1) and 8 of 8 more differentiated AML. In 2 cases with detectable leukaemic cell markers (1 ALL and 1 AML) passageable cells were developed, that expressed normal T cell phenotypes (namely CD3, CD4, and CD8) at the expense of leukaemic cells. In 1 of 2 cases, long-term IL-2-cultured cells showed specific cytotoxic activity against autologous leukemic cells. The percentage killing against autologous and two allogeneic target cell lines at a 50/1 effector/target (E/T) ratio was 42%, 9% and 19% respectively. Similarly the cytotoxic activity of IL-2 activated from 4 different individuals against conventional tumour targets K562 and Daudi at a ratio of 50/1 was 29%–68% (median=55%) and 34%–78% (median=61%) respectively. It was also found that this killing potential of the activated cells was maintained for as long as culture was continued (median 23 days, range 17–75 days). The mechanism(s) of T cell proliferation at the expense of leukaemic blast cells in the case of a minority of leukaemic patients and the possible clinical therapeutic potential of these cells following in vitro IL-2 activation deserve further investigation.     

Classic and targeted anti‐leukaemic agents interfere with the cholesterol biogenesis metagene in acute myeloid leukaemia: Therapeutic implications

Despite significant advances in deciphering the molecular landscape of acute myeloid leukaemia (AML), therapeutic outcomes of this haematological malignancy have only modestly improved over the past decades. Drug resistance and disease recurrence almost invariably occur, highlighting the need for a deeper understanding of these processes. While low O₂ compartments, such as bone marrow (BM) niches, are well‐recognized hosts of drug‐resistant leukaemic cells, standard in vitro studies are routinely performed under supra‐physiologic (21% O₂, ambient air) conditions, which limits clinical translatability. We hereby identify molecular pathways enriched in AML cells that survive acute challenges with classic or targeted therapeutic agents. Experiments took into account variations in O₂ tension encountered by leukaemic cells in clinical settings. Integrated RNA and protein profiles revealed that lipid biosynthesis, and particularly the cholesterol biogenesis branch, is a particularly therapy‐induced vulnerability in AML cells under low O₂ states. We also demonstrate that the impact of the cytotoxic agent cytarabine is selectively enhanced by a high‐potency statin. The cholesterol biosynthesis programme is amenable to additional translational opportunities within the expanding AML therapeutic landscape. Our findings support the further investigation of higher‐potency statin (eg rosuvastatin)–based combination therapies to enhance targeting residual AML cells that reside in low O₂ environments.     

Binding of lectins to human leukaemic cells

The interactions of five different lectins: peanut (PNA), lentil (LEN), wheat germ (WGA), soybean (SBA), Asparagus pea (FBP) with leukaemic cells obtained from 31 children: 25 with acute lymphoblastic leukaemia (ALL) and 6 with acute myeloblastic leukaemia (AML) were examined in this study. The relationship of lectin-binding ability to cells cytomorphological, cytochemical and immunological features and its potential clinical application were investigated. It has been shown that PNA and LEN receptors were found in the majority of blast cells. The SBA reacting cells were found only in few patients and FBP binding was not found in studied ALL and AML cells. There was a clear difference in the WGA binding capacity in ALL cells with L1 and L2 characteristics respectively. No differences were found in PNA. WGA and LEN reactivity between PAS negative and PAS positive leukaemic cells. Only PNA of all studied lectins seemed to differentiate T- from B-ALL blast cells. Only WGA binding of ALL cells showed the positive correlation to the risk index value.     

Serological detection of B-cell ("Ia-like") antigens in serum of normal donor and in some sera of leukaemic patients

Some sera from normal donors (1/18) and from leukaemic patients (2/7 with acute myeloid leukaemia [AML], 1/4 with chronic lymphatic leukaemia [CLL], 0/3 with acute lymphoblastic leukaemia [ALL]; with high numbers of leukaemic cells expressing Ia-like p28,33 antigen on the leukaemic cell surface) inhibited the complement mediated cytotoxic activity of highly specific xenogenous anti Ia-like sera (which were prepared by immunization of rabbits with insoluble membrane fractions of B-type lymphoid lines) at a titre 1:4 or less. This effect was not observed with antisera directed against other membrane marker determinants (e.g. T lymphocyte specific antigens). These results suggest that at least a small proportion of membrane bound Ia-like antigens can be released from cell surfaces and in some patients these Ia-like moieties are detectable (by sensitive inhibition assays) in the serum.     

Acute myeloid leukaemia: a paradigm for the clonal evolution of cancer?

Acute myeloid leukaemia (AML) is an uncontrolled clonal proliferation of abnormal myeloid progenitor cells in the bone marrow and blood. Advances in cancer genomics have revealed the spectrum of somatic mutations that give rise to human AML and drawn our attention to its molecular evolution and clonal architecture. It is now evident that most AML genomes harbour small numbers of mutations, which are acquired in a stepwise manner. This characteristic, combined with our ability to identify mutations in individual leukaemic cells and our detailed understanding of normal human and murine haematopoiesis, makes AML an excellent model for understanding the principles of cancer evolution. Furthermore, a better understanding of how AML evolves can help us devise strategies to improve the therapy and prognosis of AML patients. Here, we draw from recent advances in genomics, clinical studies and experimental models to describe the current knowledge of the clonal evolution of AML and its implications for the biology and treatment of leukaemias and other cancers.KEY WORDS: Acute myeloid leukaemia, Cancer, Clonal evolution, In vivo models of leukaemia, Mutation     

Studies of the geographic patterns of c-myc expression in bone marrow

C-myc expression was studied semi-quantitatively in bone marrow biopsies obtained from normal individuals, patients with non-malignant haematological disorders and patients with various haematological malignancies. In normal bone marrow and in the bone marrow of patients with non-malignant haematological disorders, cells containing c-myc protein are present in small clones (average 7 +/- 2.5 cells/clone) located in the centre of the histotopographic region of the biopsy. In contrast, c-myc-containing cells are diffusely distributed in the bone marrow of patients with acute myelogenous leukaemia (AML). In the marrow of patients with myelodysplastic syndromes evolving to AML and in patients with AML in early relapse, the clones of cells containing c-myc are larger than those present in normal marrows (average clone size = 17.5 +/- 3.5 cells). Additionally, the proportion of the cells in normal bone marrow which express c-myc protein is less than that present in AML marrows (23.3 +/- 10.17 v. 60.2 +/- 6.17) and the intensity of staining is also less. Non-Hodgkin's lymphoma patients with bone marrow involvement had distribution of c-myc positive cells similar to those with leukaemic infiltration.     

Mathematical model of acute myeloblastic leukaemia: an investigation of the relevant kinetic parameters

A simple model of acute myeloblastic leukaemia (AML) development is introduced, explicitly including cell growth, cell differentiation and cell-cell interaction. Each of these processes is described by a single model parameter. It is hypothesized that the leukaemic cell is characterized by an alteration of only one of these processes. The kinetic behaviour of the AML system is examined separately for possible alterations of each of the three parameters describing the three processes involved. It is shown that, on the basis of the existing data on AML kinetics, the alteration of the growth and cell-cell interaction parameters can be eliminated as a possible source of AML. Thus kinetic data support the modification of the differentiation process as the origin of the AML state. Further, the growth characteristics of normal and leukaemic cells in the presence of each other are analysed. It is shown how the initial growth of leukaemic cells depends on the difference in the differentiation of normal and leukaemic cells and how the same difference determines the decay of normal cells in the presence of the predominantly leukaemic population. Correlations between the kinetic parameters of the normal and leukaemic populations are suggested to characterize the leukaemic state.     

Human leukaemia-associated antigens expressed by acute myelocytic leukaemia cells and their detection by heterologous antisera.

Antisera against human acute myelocytic leukaemias were tested in complement-dependent in-vitro cytotocity tests against leukaemia cells and normal cells as targets. After absorption with erythrocytes and spleen cells from allogeneous donors the antisera reacted with leukaemia cells, but not with leukocytes from bone marrow and the peripheral blood of children in remission, lymphocytes from healthy donors, enriched B-lymphocytes, enriched T-lymphocytes, PHA-induced blasts and cord blood lymphocytes. Extensive cross reactions were obtained in the tests against leukaemia cells. The antisera reacted not only with AML cells, but also with ALL, CLL, and CML cells. It was possible to remove the cross-reactivity with ALL cells through absorption with ALL cells or with fetal tissue, and to remove the cross reactivity with CLL cells through absorption with CLL. A complete absorption of the anti-AML sera was possible with AML and CML cells. After absorption with fetal tissue and CLL cells the antisera showed exclusively specificity for myelocytic leukaemias. Thus, AML cells contain three leukaemia-associated membrane antigen components: an antigen of fetal origin, a "CLL-specific" antigen, and an antigen that occurs on myelocytic leukaemias.     

Mathematical Model of Acute Myeloblastic Leukaemia: an Investigation of the Relevant Kinetic Parameters

A simple model of acute myeloblastic leukaemia (AML) development is introduced, explicitly including cell growth, cell differentiation and cell-cell interaction. Each of these processes is described by a single model parameter. It is hypothesized that the leukaemic cell is characterized by an alteration of only one of these processes. the kinetic behaviour of the AML system is examined separately for possible alterations of each of the three parameters describing the three processes involved. It is shown that, on the basis of the existing data on AML kinetics, the alteration of the growth and cell-cell interaction parameters can be eliminated as a possible source of AML. Thus kinetic data support the modification of the differentiation process as the origin of the AML state. Further, the growth characteristics of normal and leukaemic cells in the presence of each other are analysed. It is shown how the initial growth of leukaemic cells depends on the difference in the differentiation of normal and leukaemic cells and how the same difference determines the decay of normal cells in the presence of the predominantly leukaemic population. Correlations between the kinetic parameters of the normal and leukaemic populations are suggested to characterize the leukaemic state.     

Unfavourable clinical implications for HLA-G expression in acute myeloid leukaemia

Human leukocyte antigen-G (HLA-G) molecule exerts multiple immunoregulatory functions that have been suggested to contribute to the immune evasion of tumour cells. Studies on HLA-G expression in malignant haematopoietic diseases are controversial, and the functions of HLA-G on this context are limited. In the current study, HLA-G expression was analysed in different types of patients: de novo acute myeloid leukaemia (AML, n = 54), B cell acute lymphoblastic leukaemia (B-ALL, n= 13), chronic myeloid leukaemia (CML, n= 9) and myelodysplastic syndrome (MDS, n= 11). HLA-G expression was observed in 18.5% cases of AML, 22.2% in CML and 18.2% in MDS, but not in B-ALL patients. In AML, HLA-G-positive patients had a significant higher bone marrow leukaemic blast cell percentage when compared with that of HLA-G-negative patients (P < 0.01). Total T-cell percentage was dramatically decreased in HLA-G-positive patients (P < 0.05). Cytogenetic karyotyping results showed that all HLA-G-positive AML patients (n= 5) were cytogenetically abnormal, which was markedly different from that of HLA-G-negative patients (P < 0.01). Ex vivo cytotoxicity analysis revealed that HLA-G expression in AML leukaemic cells could directly inhibit NK cell cytolysis (P < 0.01). These findings indicated that HLA-G expression in AML is of unfavourable clinical implications, and that HLA-G could be a potential target for therapy.     

Reactivating the ARF-p53 axis in AML cells by targeting ULF

The tumor suppressor ARF plays an essential role in the cellular response to oncogenic stress mainly through activation of p53. Nucleophosmin (NPM), a multifunctional protein, forms a stable protein complex with ARF in the nucleolus and protects ARF from the proteasome-mediated degradation. Notably, NPM is mutated in about one third of acute myeloid leukaemia (AML) patients and these mutations lead to aberrant cytoplasmic dislocation of nucleophosmin (NPM-c). Cytoplasmic NPM mutants lose their abilities to retain ARF in the nucleolus and fail to stabilize ARF. Thus, activation of the ARF-p53 axis is significantly compromised in these AML cells. We have recently identified the ubiquitin ligase of ARF (ULF) as a key factor that controls ARF turnover in human cells. Here, we found that the steady levels of both ARF and p53 are very low in human acute myeloid leukaemia OCI-AML3 cells expressing cytoplamsic dislocated nucleophosmin (NPM-c). As expected, ARF is very unstable and rapidly degraded by proteasome. Nevertheless, ULF knockdown stabilizes ARF and reactivates p53 responses in these AML cells. These results further demonstrate that ULF is a bona fide E3 ligase for ARF and also suggest that ULF is an important target for activating the ARF-p53 axis in human AML cells.     

Decreasedin vitro chemosensitivity of tumour cells in patients suffering from malignant diseases with a poor prognosis

The aim of the study was to assess the predictive value of MTTin vitro assay for evaluation of tumour cell resistance/sensitivity to cytotoxic drugs. We analyzed 105 samples of malignant cells of different origin. The study included patients with a diagnosis of acute and chronic lymphatic leukaemia, acute and chronic myeloid leukaemia, non-Hodgkin lymphoma, carcinoma of the lung, stomach and liver, rhabdomyosarcoma and breast carcinoma. The results demonstrate outstanding chemosensitivity in the majority of childhood acute lymphoblastic leukaemias, medium chemosensitivity of adult haematopoietic malignant diseases and chemoresistance of solid tumour cells. Our preliminary data suggest a good correlation betweenin vitro MTT assay and clinical curability of individual malignant diseases.Abbreviations ALL acute lymphoid leukaemia - AML acute myeloid leukaemia - CML chronic myeloid leukaemia - LCS₅₀ 50% leukaemic cell survival     

Human T lymphocyte activation in the presence of acute myelogenous leukaemia blasts: studies of allostimulated interferon-γ secretion

 Normal peripheral blood mononuclear cells (PBMC responders) were cultured together with non-irradiated allogeneic PBMC (more than 95% leukaemia blasts) derived from patients with acute leukaemia (referred to as leukaemic PBMC stimulators). Cytokine secretion was determined as cytokine concentrations in supernatants. Both normal PBMC and enriched CD4⁺ and CD8⁺ T cells responded to allostimulation with interferon (IFNγ) secretion. Interleukin-1 (IL-1) receptor antagonist and IL-2-neutralizing antibodies decreased IFNγ secretion. Exogenous IL-1β, IL-2 and IL-7 increased allostimulated IFNγ secretion, whereas decreased levels were seen in the presence of IL-6, IL-10 and granulocyte-colony-stimulating factor (G-CSF). During allorecognition IFNγ -neutralizing antibodies decreased acute myelogenous leukaemia (AML) blast secretion of G-CSF. We conclude that (i) both CD4⁺ and CD8⁺ T cells show allostimulated cytokine secretion in response to allogeneic stimulator cells containing a dominating population of native, cytokine-secreting leukaemia blasts, and (ii) IFNγ released during this response can modulate the function of allogeneic AML blasts. Received: 4 June 1996 / Accepted: 15 October 1996     

In vitro proliferation of normal and leukaemic human leukocytes controlled by an inhibitory endopeptide

GI-3, an endogenous inhibitory fraction isolated from leukocytes, selectively inhibits the proliferation of granuloid precursor cells in a non-toxic manner. Its active principle was determined as an acidic chlor-tolidine positive decapeptide [ 3 ]. The in vitro effect on normal and acute leukaemic human bone marrow and blood cells was examined. A dose dependent inhibition by GI-3 of 3H-TdR incorporation into myeloid cells of normal bone marrow was found, the sensitivity of human cells being higher than that of rat cells. The proliferation of the target leukaemic bone marrow and blood cells (AML, AMMoL) was also decreased by the endogenous inhibitor in a dose dependent manner in untreated subjects as well as in patients in remission or relapse. The rate of inhibition of leukaemic of well-known cytostatics (adriamycin hydrochloride, dianhydrogalactitol) applied for comparison. Beyond its direct cytostatic effect, GI-3 could be used in the differential diagnosis of blastic leukaemias, complementing the routine cytochemical methods.     

Reactivating the ARF-p53 axis in AML cells by targeting ULF

The tumor suppressor ARF plays an essential role in the cellular response to oncogenic stress mainly through activation of p53. Nucleophosmin (NPM), a multifunctional protein, forms a stable protein complex with ARF in the nucleolus and protects ARF from the proteasome-mediated degradation. Notably, NPM is mutated in about one third of acute myeloid leukaemia (AML) patients and these mutations lead to aberrant cytoplasmic dislocation of nucleophosmin (NPM-c). Cytoplasmic NPM mutants lose their abilities to retain ARF in the nucleolus and fail to stabilize ARF. Thus, activation of the ARF-p53 axis is significantly compromised in these AML cells. We have recently identified the ubiquitin ligase of ARF (ULF) as a key factor that controls ARF turnover in human cells. Here, we found that the steady levels of both ARF and p53 are very low in human acute myeloid leukaemia OCI-AML3 cells expressing cytoplamsic dislocated nucleophosmin (NPM-c). As expected, ARF is very unstable and rapidly degraded by proteasome. Nevertheless, ULF knockdown stabilizes ARF and reactivates p53 responses in these AML cells. These results further demonstrate that ULF is a bona fide E3 ligase for ARF and also suggest that ULF is an important target for activating the ARF-p53 axis in human AML cells.Key words: ARF, ubiquitination, ULF, p53, NPM, B23, NPM-c