Src kinase signaling in leukaemia

Role of Src kinases in acute lymphoblastic leukaemia has been recently demonstrated in leukaemia mouse model. Retained activation of Src kinases by the BCR-ABL oncoprotein in leukaemic cells following inhibition of BCR-ABL kinase activity by imatinib indicates that Src activation by BCR-ABL is independent of BCR-ABL kinase activity and provides an explanation for reduced effectiveness of the BCR-ABL kinase activity inhibitors in Philadelphia chromosome-positive acute lymphoblastic leukaemia. Simultaneous inhibition of kinase activity of both BCR-ABL and Src kinases results in long-term survival of mice with acute lymphoblastic leukaemia. Leukaemic stem cells exist in acute lymphoblastic leukaemia, and complete eradication of this group of cells would provide a curative therapy for this disease.     

Auer bodies in acute lymphocytic leukaemia and B-cell lymphoma: evidence for a common progenitor of myeloid and lymphoid cells?

In a patient with acute lymphocytic leukaemia (pre-T ALL) and another patient with leukaemic generalization of B-cell lymphoma Auer bodies were found in a few immature cells. The diagnosis in both cases was based on clinical grounds, morphology, cytochemistry, and immunological marker analysis of the blasts. Auer bodies are known to be a marker of high significance for acute non-lymphocytic leukaemias. Therefore the findings described suggest mixed leukaemias with either T-cell or B-cell predominance. It provides further evidence for the existence of a common progenitor of myeloid and lymphoid cells.     

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.     

The expression of CD 15 antigen on myeloid leukaemia cells--a new prognostic index for complete remission and for survival

377 untreated acute leukaemia patients were categorized according to FAB and cytochemical criterials and simultaneously phenotyped with the use of 6-21 monoclonal antibodies (MoAb) of VI series (W. Knapp, Vienna). The leukaemia phenotype was compared with the patients outcome after treatment. In adult ANLL patients a positive relationships was proved statistically between the expression of the CD 15 cell differentiation antigen on leukaemic blasts and the CR rate (p less than 0.01, chi 2 test). Also a comparison of the Kaplan-Meier survival curves revealed that the CD 15 positive group of ANLL patients has a better outcome than the CD 15 negative one (p less than 0.01, by Wilcoxon and Log-rank tests). Thus, examination of cell differentiation antigens could be a useful addition to existing risk assignment in acute leukaemia.     

Scanning electron microscope cytochemistry of normal and leukaemic leukocytes

Backscattered Electron Imaging (BEI) is a particular technique which permits to study cytochemical reactions with the Scanning Electron Microscope (SEM). The BEI data pertaining to specific enzymatic activities can be directly correlated to the surface morphology of each individual cell. Leukocytes from 5 normal individuals, 14 patients with acute nonlymphoblastic leukaemia (ANLL), 7 patients with chronic myeloid leukaemia (CML) and 3 patients with acute lymphoblastic leukaemia (ALL) were studied for myeloperoxidase activity, acid phosphatase localization, silver staining of the nuclei and phagocytosis of iron carbonyl in the BEI mode of SEM. Some normal peripheral blood leukocytes which cannot be distinguished by their surface morphology alone were satisfactorily identified with the BEI technique. Leukaemic myeloid cells can be recognized in many cases because of their positive myeloperoxidase reaction, while monocytic elements can be characterized by the presence of surface ruffles, acid phosphatase activity and active phagocytosis. The usefulness of the BEI technique in identifying different blood cell types with the SEM and its possible application to the diagnosis of certain cases of leukaemia are discussed.     

Scanning electron microscope cytochemistry of normal and leukaemic leukocytes

Backscattered Electron Imaging (BEI) is a particular technique which permits to study cytochemical reactions with the Scanning Electron Microscope (SEM). The BEI data pertaining to specific enzymatic activities can be directly correlated to the surface morphology of each individual cell. Leukocytes from 5 normal individuals, 14 patients with acute nonlymphoblastic leukaemia (ANLL), 7 patients with chronic myeloid leukaemia (CML) and 3 patients with acute lymphoblastic leukaemia (ALL) were studied for myeloperoxidase activity, acid phosphatase localization, silver staining of the nuclei and phagocytosis of iron carbonyl in the BEI mode of SEM. Some normal peripheral blood leukocytes which cannot be distinguished by their surface morphology alone were satisfactorily identified with the BEI technique. Leukaemic myeloid cells can be recognized in many cases because of their positive myeloperoxidase reaction, while monocytic elements can be characterized by the presence of surface ruffles, acid phosphatase activity and active phagocytosis. The usefulness of the BEI technique in identifying different blood cell types with the SEM and its possible application to the diagnosis of certain cases of leukaemia are discussed.     

The effect of exogenous alkalization on cytokinetics and on the survival rate of mice with lymphoblastic leukemia]

In mice vaccinated with two forms of lymphoblastic leukaemia and alkalized with intravenous administration of sodium bicarbonate, the survival rate, the extent of leukaemic infiltration and the proliferative capacity of cells in the bone-marrow, thymus, spleen, lymphnodes, liver and lungs were investigated. The survival rate in the TAL leukaemia of the AKR stem producing an endogenous acidosis could be significantly prolonged in a statistical way by alkalization. Yet an accelerated expiring rate could be observed after exogenous alkalization in L-1210 leukaemia of the DBA/2J stem producing an endogenous alkalosis. By means of cytological and impulse-cytophotometrical investigations the exogenous alkalization of both forms of leukaemia could be proved to have a direct bearing on the proliferative kinetics. In TAL leukaemia the leukaemic proliferation was inhibited by the exogenously involved correction of the acid-base balance; in the L-1210 leukaemia, however, the pH disturbances were enhanced, thus accelerating the leukaemic proliferation. Consequently, the disturbances of the acid base balance seem to be an essential cofactor in the leukaemia genesis. The exogenous direction of the acid-base balance may be important as a means of treating leukaemia.     

Cytochemical studies in the blastic transformation of chronic granulocytic leukaemia

The cytochemical features of blast cells were studied in 45 patients with blastic phase of chronic granulocytic leukaemia. Various degrees of Sudan black B positivity was characteristic of myeloblastic transformation (23 patients), while in the medullary blast cells of nine patients with myelomonocytic transformation the alpha-naphthyl-acetate esterase showed intensive activity. In two cases the demonstrability of beta-thromboglobulin and factor VIII-related antigen in blast cells showing otherwise PAS, acid phosphatase and alpha-naphthyl-acetate esterase activity referred to megakaryocytic transformation. In six patients with lymphoid blast crisis proliferation of the Sudan negative blast cells with different granular PAS, acid phosphatase and/or beta-glucuronidase positivity was demonstrated. In five cases the cytochemical findings of leukaemic cells indicated biphenotypic and mixed transformation, respectively.     

Leukocyte mobilization in mice by polyanions. Decline of leukocyte mobilizing capacity after transplantation of lymphoma

The leukocyte mobilizing polyanions dextran sulphate (DS) and polymethacrylic acid (PMAA) were administered to AKR and (C57BL x CBA) F1 mice at various times after transplantation of syngeneic lymphoma cells. In nonleukaemic mice DS and PMAA increased the number of circulating leukocytes 3--4-fold. The extent of leukocyte mobilization in leukaemic mice depended on the interval between transplantation of the lymphoma cells and injection of the polyanion. During the development of leukaemia in AKR as well as in (C57BL x CBA) F1 mice the capacity to react upon injection of polyanions with leukocyte mobilization gradually decreased. For DS, this decrease started before the number of leukocytes increased in the peripheral blood. On the other hand, the capacity for PMAA-induced leukocyte mobilization was fully preserved for several more days. In heavily leukaemic mice neither DS nor PMAA could further increase the number of peripheral blood leukocytes. In such mice the distribution pattern of leukaemic blast cells, small lymphocytes, granulocytes and monocytes was also hardly or not affected by injection of the polyanion.     

Human T lymphocyte activation in the presence of acute myelogenous leukaemia blasts; studies of normal polyclonal T cells and T lymphocyte clones derived early after allogeneic bone marrow transplantation

 T cell clones (CD4⁺CD8^–TCRαβ⁺γδ^–) derived from bone marrow transplant recipients were stimulated with phytohaemagglutinin (PHA) +interleukin-2 (IL-2) in the presence of irradiated (50 Gy) peripheral blood mononuclear cells (PBMC) derived from acute leukaemia patients(leukaemic PBMC containing more than 95% blast cells). Leukaemic PBMC could function as accessory cells during mitogenic T cell activation resulting in both T cell proliferation and a broad T cell cytokine response [IL-3, IL-4, IL-10, granulocyte/macrophage-colony-stimulating factor (GM-CSF) tumour necrosis factor α (TNFα) and interferon γ (IFNγ) secretion]. Blockade of IL-1 effects by adding IL-1 receptor antagonist together with PHA+IL-2+leukaemia blasts increased T cell proliferation, whereas IL-6-neutralizing antibodies did not alter T cell proliferation. A qualitatively similar T cell cytokine response and a similar cytokine profile (highest levels detected for GM-CSF and IFNγ) were detected when normal polyclonal T cell lines were stimulated with PHA in the presence of non-irradiated leukaemic PBMC. When leukaemic PBMC derived from 18 acute myelogenous leukaemia patients were cultured with PHA and cells from a polyclonal T cell line, increased concentrations of the T cell cytokines IFNγ and IL-4 were detected for all patients. We conclude that T cell activation resulting in proliferation and a broad cytokine response can take place in the presence of excess acute myelogenous leukaemia blasts. Received: 30 November 1995 / Accepted: 9 January 1996     

Chronic myelogenous leukaemia exosomes modulate bone marrow microenvironment through activation of epidermal growth factor receptor

Chronic myelogenous leukaemia (CML) is a clonal myeloproliferative disorder. Recent evidence indicates that altered crosstalk between CML and mesenchymal stromal cells may affect leukaemia survival; moreover, vesicles released by both tumour and non‐tumour cells into the microenvironment provide a suitable niche for cancer cell growth and survival. We previously demonstrated that leukaemic and stromal cells establish an exosome‐mediated bidirectional crosstalk leading to the production of IL8 in stromal cells, thus sustaining the survival of CML cells. Human cell lines used are LAMA84 (CML cells), HS5 (stromal cells) and bone marrow primary stromal cells; gene expression and protein analysis were performed by real‐time PCR and Western blot. IL8 and MMP9 secretions were evaluated by ELISA. Exosomes were isolated from CML cells and blood samples of CML patients. Here, we show that LAMA84 and CML patients’ exosomes contain amphiregulin (AREG), thus activating epidermal growth factor receptor (EGFR) signalling in stromal cells. EGFR signalling increases the expression of SNAIL and its targets, MMP9 and IL8. We also demonstrated that pre‐treatment of HS5 with LAMA84 exosomes increases the expression of annexin A2 that promotes the adhesion of leukaemic cells to the stromal monolayer, finally supporting the growth and invasiveness of leukaemic cells. Leukaemic and stromal cells establish a bidirectional crosstalk: exosomes promote proliferation and survival of leukaemic cells, both in vitro and in vivo, by inducing IL8 secretion from stromal cells. We propose that this mechanism is activated by a ligand–receptor interaction between AREG, found in CML exosomes, and EGFR in bone marrow stromal cells.     

Ultrastructural cytochemistry of chronic T-cell leukaemias. A study with four acid hydrolases

The ultrastructural localization of four acid hydrolases (acid phosphatase, beta-glucuronidase, beta-glucosaminidase and alpha-naphthylacetate esterase) has been studied in lymphocytes from 16 patients with three types of chronic T-cell leukaemia, namely, T-prolymphocytic leukaemia (T-PLL), T-chronic lymphocytic leukaemia (T-CLL) and adult T-cell lymphoma leukaemia (ATLL). Different patterns of enzyme distribution were observed in the leukaemic T-cells from these disorders. In T-PLL, reactivity for the four acid hydrolases was confined to single or a few large granules. Gall bodies were reactive for beta-glucuronidase, b-glucosaminidase and alpha-naphthylacetate esterase but apparently unreactive for acid phosphatase. In T-CLL, scattered small- to medium-size cytoplasmic granules and many parallel tubular arrays were strongly reactive for acid phosphatase, beta-glucuronidase and beta-glucosidase but showed no reactivity for alpha-naphthylacetate esterase. Intermediate features were observed in ATLL. The observed differences in enzyme reactivity reflect a different content of lysosomal granules in the various types of leukaemic T-cells. They also suggest that similar differences may be found in normal T-lymphocyte subsets.     

Antibody-dependent cellular cytotoxicity (AAZZ) against leukemia cells--K-cell activity of human leukocytes

The activity of human leukocytes from the peripheral blood (PB) and bone-marrow (BM) to take the function as K-cells in the antibody dependent cellular cytotoxicity (ADCC) was tested in a 51Cr-test against mouse leukaemic cells and ALL cells covered with specific heterologous antibodies. Mononuclear PB-leukocytes and granulocytes of healthy donors and patients with leukemia and lymphoma in remission lysed murine and human leukaemic cells in the presence of specific antibodies. There was no lowering of K-cell activity of mononuclear PB-leukocytes of patients with leukaemia and lymphoma in remission under chemotherapy as compared with healthy donors and patients in remission without chemotherapy. There was a good correlation between the percentage of K-cell active mononuclear leukocytes in PB and BM. Attempts of fractionation with peripheral blood leukocytes of healthy donors resulted in the non-adherent mononuclear PB-leukocytes (lymphocytes) and granulocytes being effector cells in ADCC. To a high degree K-cell active lymphocytes could be identified in the non-B-fraction and only slightly in the fraction forming E rosettes.     

Immunologic characterization of T-lymphoproliferative disorders by monoclonal antibodies

A series of 11 mouse monoclonal antibodies reactive with human T lymphoid cells at different stages of differentiation was used for immunological classification of leukaemic cells of 16 patients with T cell lymphoproliferative disorders by using a fluorescence assay. The majority of T-ALL cells had an immature or early thymic phenotype and T lymphoblastic lymphoma had phenotypes corresponding to different levels of more mature stages of T cell differentiation, Two cases of T-CLL and one adult patient with mycosis fungoides had mature T cell phenotypes being T-3+, T-4-, T-8+, cytotoxic/suppressor cell types and one case of T-CLL had T-3+, T-4+, T-8-, "helper/inducer" cell type, too. These results suggested that surface marker analysis in T cell lymphoproliferative disorders may be used as a highly reproducible immunological classification system that will provide additional information about phenotypes of leukaemic cells in connection with morphological analysis and clinical diagnosis.     

Quantitative cytochemistry of blood neutrophils in myelodysplastic syndromes and chronic granulocytic leukaemia

Quantitative cytochemistry of components of blood neutrophil azurophilic granules (myeloperoxidase, chloroacetate esterase, beta-glucuronidase, and acid phosphatase) and specific granules (lactoferrin) has been performed by scanning and integrating microdensitometry in 13 patients with a myelodysplastic syndrome and 11 patients with chronic granulocytic leukaemia. Both patient groups showed a reduction of enzyme activity in azurophilic granules, and also of lactoferrin, consistent with abnormal development of neutrophil granules. These cytochemical changes in blood neutrophils are similar to those found in acute myeloid leukaemia, are consistent with a leukaemic maturation defect, and may be of diagnostic value.     

Leukocyte Mobilization In Mice By Polyanions Decline of Leukocyte Mobilizing Capacity After Transplantation of Lymphoma

The leukocyte mobilizing polyanions dextran sulphate (DS) and polymethacrylic acid (PMAA) were administered to AKR and (C57BL × CBA) F₁ mice at various times after transplantation of syngeneic lymphoma cells. In nonleukaemic mice DS and PMAA increased the number of circulating leukocytes 3–4-fold. the extent of leukocyte mobilization in leukaemic mice depended on the interval between transplantation of the lymphoma cells and injection of the polyanion. During the development of leukaemia in AKR as well as in (C57BL × CBA) F₁ mice the capacity to react upon injection of polyanions with leukocyte mobilization gradually decreased. For DS, this decrease started before the number of leukocytes increased in the peripheral blood. On the other hand, the capacity for PMAA-induced leukocyte mobilization was fully preserved for several more days. In heavily leukaemic mice neither DS nor PMAA could further increase the number of peripheral blood leukocytes. In such mice the distribution pattern of leukaemic blast cells, small lymphocytes, granulocytes and monocytes was also hardly or not affected by injection of the polyanion.     

The nature of blast cells in myelodysplastic syndromes evolving to acute leukaemia

The blast cells from nine patients with an overt acute leukaemia following a previous myelodysplastic syndrome (MDS) are analyzed with a panel of monoclonal antibodies as well as by morphological and cytochemical criteria. By integrating the results obtained with these three approaches the leukaemia in 6 patients was assessed as myeloid-granulocytic and/or monocytic-, in two as mixed- megakaryoblastic/myeloid- and in one as lymphoid. A good correlation between morphology, cytochemistry and immunological markers was observed in 7 out of the 9 cases. In three cases a noteworthy percentage of J5+ cells was detected. The exceptional finding of lymphoid as well as megakaryocytic and myeloid transformations suggests that the target cell for these leukaemias could be a pluripotent stem cell.     

Marrow grown in long-term culture can be used for autologous transplantation in the treatment of acute myeloid leukaemia

Leukaemic cells can be selectively depleted in the conditions of long-term bone marrow culture (LTBMC). Use can be made of LTBMC to purge bone marrow of leukaemic cells in autologous bone marrow transplantation (ABMT).     

Immunological typing of blast cells. Cases with pre-B cell characteristics

Leukemic cells from 32 patients were examined by using conventional immunological markers (E and EAC rosettes, surface immunoglobulins). Additionally, the test for intracytoplasmic IgM, Fc IgG receptor and the presence of light chains were performed. Leukemic blasts of all patients were investigated according to morphological and cytochemical criteria. Lymphoblasts from 3 patients had pre-B cell phenotype: cIgM +, sIg-. Each of 3 patients with pre-B cell characteristics had different diagnosis and different morphological and cytochemical features of the leukemic cells (ALL, NHL and CML). In 24 ALL cases the diagnosis of non-T, non-B ALL, in 4 cases T-ALL and in one B-ALL was established. The correlation of cytochemical results with special reference to acid phosphatase and immunological subclasses of ALL was also analyzed. An important question is raised with regard to diagnostic classification and treatment by finding ALL phenotypes in lymphoproliferative disorders that are not diagnosed as ALL.     

Flow cytometric characterization of chronic lymphocyte leukaemias using orthogonal light scattering and quantitative immunofluorescence

Light scattering properties and antigen distribution of lymphocytes labeled with the monoclonal antibodies CD 5 and CD 20 were determined for 19 patients with a chronic B-cell derived leukaemia. The density of the antigen detected by the monoclonal antibody CD 5 appeared to be considerably lower on malignant B-lymphocytes of the patients as compared with T lymphocytes. A large variation was observed in the amount of receptors for the monoclonal antibodies CD 5 and CD 20 on the malignant cells of the different patients. B-cell chronic lymphocytic leukaemia (B-CLL) patients were clearly distinguishable from leukaemic follicular non Hodgkin lymphoma patients (LF-NHL, formerly lymphosarcoma cell leukaemia) and from a patient with a prolymphocytoid transformation (PLT) of the B-CLL according to the amount of the antigens for CD 5 and CD 20. Within the B-CLL patient population, no relation of progression of the disease with distribution of these antigens could be observed. In one patient the extraordinary phenotype CD 20+, CD 11+, leu 8+, CD 5- of the malignant lymphocytes was observed. An experimentally simple method to differentiate between the various chronic lymphocytic leukaemias (CLL) appeared to be the determination of orthogonal light scattering properties of lymphocytes. In healthy donors one can always distinguish two populations of lymphocytes in the orthogonal light scatter histograms. Lymphocytes of B-CLL patients show one uniform population with a relatively small orthogonal light scattering signal, lymphocytes of our patients with PLT of B-CLL or with LF-NHL show one uniform population with a relatively large orthogonal light scattering signal.