title: [Biological properties and sensitivity to induction therapy of differentiated cells expressing atypical immunophenotype in acute leukemia of children].
abstract: The atypical immunophenotype (expression of determinant from the another cell lines than line of origin) of acute leukaemia blast cells are noted in a part of cases. The characteristics and classification of atypical immunophenotypes are not unified and the clinical significance is not yet fully described. The purpose of the study was: precise description of atypical immunophenotypes and analysis of their frequency in different types of acute leukaemia, analysis of association between expression of atypical immunophenotypes and the level of initial leukocytosis, percentage of blast cells in peripheral blood, expression of CD34, analysis of frequency of multidrug resistance molecule (MDR) expression and association between MDR and immunophenotypes of leukaemia cells, analysis of association between atypical immunophenotypes and proliferation, secretion of cytokines (IL-6, TNF) and spontaneous apoptosis of leukaemia cells, analysis of association between atypical immunophenotypes and sensitivity to induction therapy. The bone marrow samples used for routine diagnosis were the basic source of leukaemia cells for the study. The morphological examination and the immunophenotypes of leukaemia cells were done for classification of leukaemia. The immunophenotype and the expression of MDR determination was performed with flow cytometry after staining the cells with monoclonal antibodies (directly labelled) for CD determinants and MDR. The spontaneous proliferation of leukaemia cells was studied with 3H-Thymidine uptake after 3-days culture in vitro. The type of proliferation (autocrine, paracrine) was defined based on comparison of shorter (3-days) and longer (6-days) culture of leukaemia cells. The percentage of apoptotic leukaemia cells was analysed with flow cytometry after staining of leukaemia cells with propidium iodide in subdiploidal region of DNA profile. The secretion of cytokines (IL-6 and TNF) was determined by ELISA technique in supernatants of leukaemia cells cultured for 24 hr in vitro. The biological activity of TNF was determined in the bioassay using L929 mouse cells line. The effect of induction therapy was estimated base on time of cytoreduction in peripheral blood and time of reaching the haematological remission in bone marrow. The study included 230 children with acute leukaemia: lymphoblastic (ALL)--189 children (ALL-proB--19, common ALL--139, ALL-B--5 and ALL-T--26) and myeloid (AML)--34 children. Moreover, into the study 2 cases of acute undifferentiated leukaemia (AUL) and 3--acute mixed lineage leukaemia (AMLL) and 2--biphenotypic leukaemia were included. The all studies of leukaemia cells had been done before the therapy was installed. Basing on the assay of immunophenotypes the following forms of atypical immunophenotypes were distinguished: immunophenotype incomplete, hyperexpression of determinants, asynchronic immunophenotype, coexpression of determinants from the other line than origin of leukaemia cells, balanced expression of determinants from two cells lines (biphenotypic leukaemia) and three cells lines (mixed lineage leukaemia). The atypical immunophenotypes were observed in: 21.1% ALL-proB cases, 34.5% common ALL cases, 42.3% ALL-T and 58.8% AML. The most common form of atypical immunophenotypes was coexpression of determinants from the other cell line. There were no associations between atypical immunophenotypes and the level of initial leukocytosis and percentage of blast cells in peripheral blood. The expression of CD34, recognised as the one of markers of poorer prognosis, was analysed regarding the leukaemia type and immunophenotype of leukaemia cells. The lowest frequency of CD34 expression was noted in ALL-T (28.5%), the highest one in common ALL (62.3%). The significant association between frequency of CD34 and atypical immunophenotypes was observed in AML and ALL-T. Moreover, in common ALL the expression of CD34 was significantly higher when myeloid determinants were present on common ALL cells (common ALL + My) in comparison to coexpression of lymphoid determinants (common ALL + Ly). The frequency of MDR expression (cases with more than 10% of MDR positive cells) was in range between 16.6% in ALL-proB and 78.9% in AML. The mean percentage of cells expressing MDR was low in ALL-proB (10.7%) and high in ALL-T (39.6%). In ALL the atypical immunophenotype was associated with expression of MDR whereas in AML this association did not appear. The common ALL + My leukaemia cells showed higher ability to proliferation in vitro compare with common ALL without atypical immunophenotype. The opposite results were observed in AML. AML leukaemia cells with coexpression of lymphoid determinants (AML + Ly) showed lower proliferation in vitro than AML without atypical immunophenotype. The autocrine type of proliferation was observed frequently in AML (35.3% of cases) than in ALL (14.2%). This type of spontaneous proliferation was observed only when the leukaemia cells without changes in immunophenotype had been cultured. The low level in common ALL and high in AML of spontaneous release of IL-6 and TNF were noted. AML leukaemia cells without changes in immunophenotype released significantly higher amount of these cytokines than AML cells with atypical immunophenotypes (AML + Ly). The above observations suggested that coexpression of myeloid determinants in ALL and lymphoid determinants in AML were leading to changes of some biological properties of these cells. The ALL + My leukaemia cells behaved similarly to myeloid leukaemia cells, while AML + Ly cells showed features of lymphoid leukaemia cells. The common ALL and AML leukaemia cells with atypical immunophenotype showed higher percentage of apoptotic cells (16.1% and 16.9% respectively) comparing to common ALL and AML without changes in immunophenotype (9.0% and 9.2% respectively). The weak negative association of MDR expression and apoptosis suggested the indirect inhibiting influence of MDR on ability of cells to undergo into the apoptosis process. In common ALL and AML with typical immunophenotype of leukaemia cells and ALL-T the level of apoptosis was associated positively with the spontaneous proliferation, whereas this relation was negative in AML with atypical immunophenotype. There were no differences of the time of cytoreduction of leukaemia cells in peripheral blood in B cell origin ALL and AML with or without changes in immunophenotype of blastic cells. In ALL-T + My the time of cytoreduction was significantly longer. However, the expression of CD10 in ALL-T had no effect on cytoreduction time. The expression of MDR in ALL-T with typical immunophenotype was independent marker associated with elongation of cytoreduction time. The time of reaching the complete haematological remission was analysed in 186 children with ALL (ALL-proB--18 children, common ALL--137 children, ALL-T--26) and only 19 children with AML. The longest period of time for reaching the remission was observed in AML, shortest--in ALL-T. In common ALL and ALL-T the expression of myeloid determinants was associated with significant elongation of time of reaching the remission. In the majority of AML cases with coexpression lymphoid determinants, the complete remission was reached. The time needed for the reaching of remission was similar in AML with or without coexpression of lymphoid determinants. The results of this study suggest that coexpression of determinants from the other cell line modify the biological properties of leukaemia cells into the cells from the line of origin of these additional determinants. In ALL the combined expression of MDR and atypical immunophenotype of leukaemia cells were associated with poorer response to induction therapy. In AML the combined expression of CD34 and atypical immunophenotype were associated with response to induction therapy by reaching the complete remission, but without any influence on the time of reaching this remission. The results of analysis of cytoreduction time and time of reaching the remission improved the usefulness of these parameters for the estimation of response to the induction therapy. The clinical importance of these observations consist in characterisation of leukaemia cells potentially resistant to the induction therapy what may suggest the modification and individualization of the induction therapy.
mesh_terms: D000293:Adolescent; D000818:Animals; D018952:Antigens, CD34 / Q000276:immunology; D017209:Apoptosis; D002648:Child; D002675:Child, Preschool; D016207:Cytokines / Q000378:metabolism; D018432:Drug Resistance, Multiple / Q000276:immunology; D006801:Humans; D016130:Immunophenotyping; D007223:Infant; D015470:Leukemia, Myeloid, Acute / Q000276:immunology* / Q000473:pathology / Q000628:therapy*; D051379:Mice; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000276:immunology; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000276:immunology* / Q000473:pathology / Q000628:therapy*; D012074:Remission Induction
chemical_list: D018952:Antigens, CD34; D016207:Cytokines; D027425:ATP-Binding Cassette, Sub-Family C Proteins
vernacular_title: Właściwości biologiczne i wrazliwość na leczenie indukcyjne komórek rozrostowych o nietypowym immunofenotypie w ostrych białaczkach u dzieci.