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Unlabelled: It is well known that patients with leucopenia are susceptible to life-threatening infections due to chemotherapy. The number of neutrophils and monocytes is increasing after administration filgrastim (G-CSF) in patients with the acute lymphoblastic leukemia. CD16 expression was analyzed in order to determine whether monocytes function is also impaired by chemotherapy CD16. The aim of study was to compare the percentage of CD14+CD16+ monocytes in the group of children with the acute lymphoblastic leukemia after administration filgrastim with the group of children with inflammatory state and the group of the healthy children., Materials and Methods: The cell surface expression of CD16 was analyzed by flow cytometry. The percentage of CD16 expression and the mean intensity fluorescence rate of CD16 receptor was studied., Results: Monocytes showed increased CD16 expression in the group of the children with the acute lymphoblastic leukemia in comparison to the other two groups. There were no differences in the mean fluorescence intensity of the CD16 monocytes between groups., Conclusions: The present study demonstrated that G-CSF increased the percentage of monocytes with the CD16 expression, which could be beneficial to the immunological response of the children with the acute lymphoblastic leukemia.

Leukemias are the most common malignancy in children, and acute lymphoblastic leukemia (ALL) accounts for 85% of all childhood leukemias. Sequential monitoring of MRD (minimal residual disease) in a set time points during the induction therapy in ALL proves to be a powerful and independent predictor of treatment outcome. Crucial limitation of MRD monitoring by flow cytometry is immunophenotypic changes seen at relapse. Up to now there are single publications about immunophenotypic changes during treatment of ALL in children. Objective was to assess changes in expression of cell antigens during induction treatment of ALL. From May 2005 to January 2008, from among 78 patients with ALL treated in Oncology and Hematology Department, Children's University Hospital in Krakow according to international treatment protocol ALL IC-BFM-2002, 42 were enrolled in assessment of antigens' cells modulation expression during induction. Finally, 24 boys and 18 girls were eligible for evaluation. For MRD detection 4-colour flow cytometry with FACS Diva Software v.5.1 (BD Immunocytometry Systems) was used. The panel of monoclonal antibodies used for MRD detection was based on ALL IC-BFM-2002 standard, modified by additional antibodies combinations from ALL-BFM-2000 protocol. Identification of leukemia associated phenotype (LAP), used for cell analysis in sequential time points, with a set monoclonal antibodies panel, was possible in all analyzed patients. Superficial and cytoplasmic antigens modulation was observed in most of the patients during MRD monitoring. Changes of antigens were seen mostly in PB on day 8 and on day 15 both in PB and BM. The most common antigen modulation found in children with cALL was: downmodulation of CD10, CD34, CD19, and upmodulation of CD45, CD11a, CD20. Unequivocal character of modulation of CD66c, CD58, CD38 was difficult to define (up and downmodulation). Introduction of treatment monitoring based on flow cytometry MRD measurement could lead to further individualization of therapy and improovement of cure rates in ALL, as well as to reducing side effects of ALL treatment and decrease total cost of patient therapy.

Unlabelled: There is a rising interest in the field of immunotherapy in cancer in the last few years. One of the methods is to prevent the immunosuppression accompanying neoplastic diseases including acute lymphoblastic leukaemia (ALL) in children. In healthy people regulatory T cells (Tregs) prevent the autoimmune, destructive activation of T lymphocytes. However, the hyperfunction of Tregs will lead to impairement of the immune system., The Aim: of our study was to determine the Tregs (including molecules important for their function) in the peripheral blood of children with ALL., Material and Methods: Thirty children were enrolled into our study, the assessment of Tregs was performed with flow cytometry including the following antigens: CD4, CD10, CD19, CD25, CD28, CD45, CD45RA, CD45RO, CD54 (ICAM-1), CD62L, CD69, CD103, CD127, CD134 (OX40), CD152 (CTLA-4), GITR., Results: 1. The percentages of Tregs were higher in the blood of the examined group, however the difference was not statistically significant; 2. In the examined group higher percentage of Tregs with the coexpression of CD45RA, CD134 and CD152 antigens were noted; 3. The percentages of Tregs with coexpression of CD62L (with or without CD103) and CD54 were lower then in the control group; 4. We did not observe differences in Tregs with coexpression of CD11a, CD27, CD28, CD45RO, CD69, CD103, GITR antigens between the examined and the control group., Conclusions: The finding of smaller number and lower percentage of regulatory T cells with coexpression of CD62L or lack expression of CD103 in children with ALL as compared to the control group, may be interpreted as activation of Treg cells and one of the mechanisms of immunosuppression in cancer of children. Further studies in this direction are needed.

Purpose: The aim of the present study was to compare the blast immunophenotype of acute lymphoblastic leukaemia (ALL) at diagnosis and at relapse and to define the most frequent shifts in marker expression., Patients and Methods: Bone marrow samples from 14 patients were analyzed by flow cytometry both at diagnosis and at relapse - in 12 patients with B-cell precursor (BCP)-ALL and in 2 patients with T-ALL., Results: The conversion in blast immunophenotype was observed in 12 out of 14 patients (86%). Antigen CD34 turned out to be the most unstable antigen - the shift in the signal expression was present in 57% of BCP-ALL and in both T-ALL cases. Regarding B-lineage markers, the shifts most frequently concerned CD20 (shifts present in 41.5% of cases) and CD22 (27%). Among the T-lineage markers: CD3, CD4 and CD8 demonstrated the highest incidence of altered signal expression. On the other hand, the most stable antigen included CD19 and CD10 for the BCP-ALL group and CD1a, CD2, CD5, CD7 for T-ALL patients. Expression of HLA-DR, TdT and CD45 antigens remained unchanged in both BCP-ALL and T-ALL groups., Conclusions: The results of the present study support the requirement to monitor at least two different leukaemia specific antigen combinations for detection of MRD to prevent a false-negative result and to increase the effectiveness of monitoring minimal residual disease.

This paper is a review of current diagnostic applications of molecular methods in pediatric hematooncology, including analyses performed at disease presentation, evaluation of prognosis as well as those for assessment of treatment effectiveness. Here we present the examples of important fields of application of molecular methods in pediatric hematooncology, i.e. identification of clinically significant fusion genes in acute lymphoblastic leukemia and monitoring of minimal residual disease in this most frequent childhood malignancy. Moreover, we present the methodology and clinical significance of quantitative analysis of hematopoietic chimerism after allogeneic stem cell transplantation. The methods presented include fluorescent in situ hybridization, reverse transcription, conventional and quantitative polymerase chain reaction, as well as methods of genotyping based on analysis of microsatellite marker polymorphism, single nucleotide polymorphism, and Indel markers.

Monitoring of residual leukemic cells, so called minimal residual disease (MRD) in acute leukemias is of great importance in clinical practice. Especially evaluation of early response to treatment has a important prognostic value, and modifies the therapy schedule in a nowadays used treatment protocols. Methods used for monitoring of MRD consist of molecular techniques, which detect chromosomal aberrations or immunoglobulins and lymphocyte T receptor genes rearrangements, as well as method of flow cytometry, which detect immunophenotype typical for the leucemic clone. The paper shows advantages and disadvantages each of those methods. The best approach to monitoring MRD in ALL during and after the treatment seems to be combination of molecular and immunological techniques.

Objective: Initiation and popularization of routine molecular diagnostics of minimal residual disease (MRD) are currently one of the most urgent challenges in Polish hemato-oncology. The paper is aimed to present preliminary results of identification of immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements and quantitative assessment of MRD levels in Polish children with acute lymphoblastic leukemia (ALL). The results are presented in the context of clinical significance of MRD study, current methodology of MRD assessment and standardization process in Western Europe., Material: DNA isolated from bone marrow / bone marrow mononuclear cells obtained at diagnosis from 26 children (25 B-precursor ALL, 1 T-ALL) aged 1.3-16.5 years., Methods: PCR-heteroduplex analysis, based on standard BIOMED-1 and BIOMED-2 primer combinations and protocols for detection of rearrangements and clonality assessment; sequencing of clonal PCR products and comparison with germline sequences of Ig/TCR genes for identification of the rearranged genes andjunctional regions; real-time quantitative PCR (RQ-PCR) with the use of TaqMan probes for assessment of follow-up MRD levels (in 11 patients)., Results: Clonal TCRG, incomplete TCRD, Vdelta2-Jalpha, TCRB, IGK-Kde and IGH gene rearrangements were detected in 61, 61, 35, 13, 39 and 83% of patients, respectively, which was generally concordant with published data for patients of other European nations., Conclusions: There is an urgent need to broaden the scope of minimal residual disease study in Poland and to develop Polish standards of MRD diagnostics, based on current European experience and standards.

The aim of study was to compare the clinical picture and results of laboratory tests according to the acute lymphoblastic leukaemia (ALL) immunophenotype. The observation was carried out on a group of 67 patients treated in the IIIrd Department of Paediatrics and Department of Children Oncology in the Medical Academy of Białystok from January 1994 to April 2001. This group consists of 4 children with pro-B acute lymphoblastic leukaemia, 52 children with pre-B cell ALL, 1 child with B-cell acute lymphoblastic leukaemia and 9 children with T-cell acute lymphoblastic leukaemia. Haemorrhagic diathesis. splenomegaly, enlargement of peripheral lymph nodes as well as higher values of white blood cells count, blasts count, haemoglobin concentration, haematocrit and LDH activity were observed more frequently in patients with T-cell leukaemia than in others.

Unlabelled: In the last years one rises importance of costimulatory molecules in immune response in leukemias. Aim of the study was to assess lymphocytes T function in children with acute lymphoblastic leukemia during remission induction on the grounds of chosen costimulatory and activatory molecules expression. To assess percentages of lymphocytes subpopulations we used tricolor flow cytometry., Results: 1. In the moment of diagnosis and remission induction we noted higher percentages of lymphocytes T with adhesion molecule ICAM-1; 2. During remission induction we observed lower percentage values of lymphocytes T with CD38 coexpression; 3. In the end of remission induction rised the percentage values of lymphocytes T helper with IL-2 receptor expression; 4. In the group of patients with fever/infection we observed higher percentage of activated lymphocytes T (CD3-HLA-DR) comparing to non-infected patients. Summarizing, we suggest lymphocytes T activation during appearance and remission induction of acute lymphoblastic leukemia in children. This confirms participation of cellular immunity in leukemic process.

Analysis of T lymphocytes cytokine profiles allows to differ subpopulations: Th1, Th2, Th3, Tr1. Aim of the study was to assess Th1/Th2 balance in acute lymphoblastic leukemia in children at diagnosis and during/after remission induction, especially during infections. Percentages of lymphocytes T producing IFN-gamma and IL-4 were assessed by flow cytometry. We noted the rise of lymphocytes T helper producing IFN-gamma (Th1) and percentage of lymphocytes T producing IL-4 at the beginning and during remission induction was higher than in control group. During fever/infection we observed the rise of lymphocytes Th1, and no change in Th2 percentage. Summarizing we suggest Th1/Th2 imbalance and Th2 predominance in acute lymphoblastic leukemia in children.

Unlabelled: We analyzed the dynamics of changes in the immune system during maintenance treatment of acute lymphoblastic leukemia (ALL). The study was carried out on the group of 40 children aged 2-15 years (mean +/- 3.46), including 31 lower risk patients (Berlin-Frankfurt-Munich protocol--BFM) and 9 high risk patients (New York protocol--NY). Levels of IgA, IgG, IgM and IgE, and subsets of mononuclear cells using flow cytometry were evaluated in the study group every 1-3 months. During maintenance therapy we found markedly reduced leukocytosis, lymphocytosis, and mean values of IgA, IgM and IgG in comparison to healthy children. A gradual increase was observed in the level of IgG, in the percentage and absolute number of B lymphocytes (CD19+) and in helper/suppressor T-cell ratios. In the group of patients treated according to the NY protocol, a decrease was noted in: lymphocytosis, in the percentage of lymphocytes B, in the levels of IgG and IgM, in the percentage and absolute numbers of T lymphocytes, T-helper cells and CD4/CD8 and CD3 RA/RO ratios, compared to standard risk patients. In the course of infection, a transitory increase was observed in activated T-cells, and a rise was found in the number of T-memory cells and monocytes showing the expression of adhesive molecules., Conclusions: In the course of maintenance treatment in children with ALL, constant suppression of the immune system, of both humoral and cellular responses, can be observed especially in the group of children treated according to the NY protocol; activation of T lymphocytes and monocytes takes place in the course of infection. Lack of this activation may be a marker of poor prognosis during the infection.

The aim of the study was to evaluate the efficacy of hepatitis B prophylaxis in children with acute lymphoblastic leukemia (ALL) and to try to determine the optimal procedure of protection against the infection. The retrospective analysis included 229 patients with ALL divided into three groups depending on the type of anti-HBV prophylaxis. The group 1 (1990-91, 38 patients) received only sporadically passive prophylaxis, in the group 2 (1992-94, 55 patients) passive prophylaxis was regular, and the patients of the group 3 (1995-2001, 138 children) received complete active and passive prophylaxis. Among vaccinated children three subgroups were additionally distinguished: subgroup a--vaccination was completed before the disease, subgroup b--the cycle of vaccination began before and continued during the therapy, subgroup c--the whole cycle of vaccination was performed during the ALL treatment. The efficacy of the prophylaxis was evaluated taking into account the incidence of hepatitis B and the level of anti-HBs antibodies in vaccinated children. Additionally the incidence of hepatitis C was assessed to evaluate the role of unspecific prophylaxis. The incidence of hepatitis B in the group 1, 2, and 3 was: 57.9%, 23.6%, and 0.76%, respectively, and the incidence of hepatitis C: 44.7%, 36.4%, and 5.9%, respectively. The percent of the failure of active prophylaxis in the subgroup a, b, and c was: 29%, 53%, and 93%, respectively. In spite of the reduction of exposure to the infection (unspecific prophylaxis), the role of specific prophylaxis is essential. The program of passive and active prophylaxis used by us is efficient in preventing hepatitis B in children with ALL. However, during the intensive chemotherapy only passive prophylaxis should be used with postponement of the vaccination because of very low response to the vaccination applied in this phase of treatment. Regular control of anti-HBs antibodies' level is essential in all patients with leukemia even in those with initially high level of antibodies.

Unlabelled: We examined immune system at the time of diagnosis and after remission induction in the group of 30 children (aged 6.5 +/- 3.6) with acute pre-B lymphoblastic leukaemia (ALL). The group was divided into standard risk group (treated with BFM protocol, n = 20) and high risk group (New York protocol, n = 10). We measured: episodes of infection, leukocytosis, immunoglobulin concentrations (G, M, A and E), lymphocytes and their subpopulations (CD19+, CD3+, CD3 + HLA-DR+, CD4+, CD8+, CD4 + CD45RA+, CD4 + CD45RO+, CD8 + CD45RA+, CD8 + CD45RO+, CD16 + CD56+)., Results: Immunoglobulin concentrations at the time of diagnosis were normal, and decreased after remission induction only reduction of IgG concentration was statistically significant (p = 0.008). At the time of diagnosis we noted the following differences in examined group compared to control group: higher leukocytosis (p = 0.03), lower lymphocyte count (p = 0.0008), significantly lower lymphocyte subpopulation count (for subpopulations CD19+; CD3+; CD4+; CD8+ and CD16 + 56+). After remission induction comparing to the time of diagnosis we observed: total leukocytosis reduction (p = 0.01), percentage and count CD19+ lymphocytes reduction (adequately p = 0.000007, p = 0.03), increase of lymphocyte CD3+ percentage (p = 0.002) and CD8+ lymphocyte percentage (p = 0.00003)., Conclusions: 1. At the time of diagnosis of acute lymphoblastic leukaemia in children lower counts of all lymphocyte populations are observed. 2. Immune suppression after remission induction in this group of patients concerns mainly humoral response, particularly immunoglobulin G production. 3. Severe infections in patients treated for acute lymphoblastic leukaemia are indication to immunological system assessment and early immunoglobulin supplementations of deficits e.g. immunoglobulin infusions. 4. Humoral immunity impairment in children with ALL is an effect of treatment, not disease.

Apoptosis mediated by the interaction of cytotoxic T lymphocyte with blast cell via Fas receptor/Fas ligand (FasL) pathway is a one of the mechanisms of immunological leukemia surveillance. There is few data on possible blocking of Fas receptor by soluble form of Fas (sFasL) present in serum and the role of blast cells as the source of this ligand. Forty-eight patients with de novo diagnosis of acute leukemia, 32 with myeloblastic (AML) and 16 with acute lymphoblastic leukemia (ALL) were studied. Fas expression on bone marrow leukemic blasts was determined by flow cytomertic immunofluorescent analysis and serum concentration of sFasL assessed at presentation, in remission and in relapse. Blasts of all patients expressed Fas at variable degree (0.8 to 100%). Fas expression was significantly higher on myelo--than on lymphoblasts. There was no relation between degree of Fas expression at diagnosis and remission rate. Concentration of sFasL in acute leukemia patients at diagnosis was significantly higher than in healthy control group, decreased to normal values in remission and rose again in relapse. There was a negative correlation between Fas expression on blasts and sFasL concentration at the time of diagnosis. Results obtained suggest that blast cells could be the source of soluble FasL in acute leukemia patients and that sFasL serum concentration could be used for monitoring of disease activity.

The improvement in acute lymphoblastic leukemia (ALL) children treatment results in the need of examining their immune system after therapy. The purpose of the research was the immune system evaluation in children with low risk ALL diagnosis, during the treatment and twelve months after therapy. The examined material included 41 children. A group of 16 patients was examined during the above the treatment, immediately after intensive chemotherapy. The other 25 patients were examined twelve months after the completion of therapy. The lymphocyte immune phenotype was examined with FACScan flow cytometer using monoclonal antibodies. The study confirmed the intensive chemotherapy significantly decreases the number of leukocytes, lymphocytes and their subpopulations. The period of 12 months after cessation of the chemotherapy is sufficient to recover the immune system function in all examined low risk ALL children.

Unlabelled: One of the side effects of antineoplastic treatment is immunosuppression. A consequence of this are frequent infections, sometimes with fatal outcome. The immunological system "answers" to infections with changes in number and quality of cells participating in the inflammatory process. The aim of the study was to investigate the subpopulations of mononuclear cells in patients with neoplastic (haematologic) diseases during infections. We studied 16 children with acute lymphoblastic leukaemia and with lymphomas (Hodgkin and non-Hodgkin). Among our patients we note 25 episodes of infections requiring hospitalization. Mean percent values of mononuclear antibodies: CD14-PE/CD45-FITC, CD3-FITC/CD19-PE, CD4-FITC/CD8-PE, CD3-FITC/CD16+CD56-PE, CD14-PerCP/CD54-PE, CD3-PerCP/CD54RA-FITC/CD45RO-PE, CD3-PerCP/HLA-DR, CD14-PerCP/HLA-DR. In the examination carried out during acute infection, we found higher mean percent values of CD3+ HLDAR+, CD14+ CD54+ and CD3+ CD45RO+ cells than in analogous values in the remaining examinations. After the infection we found higher percent of CD3 CD45RA+/CD3+CD45RO+ cells. In a patient with cytomegalovirus pneumonitis our examination showed a depression of HLA-DR antigens on monocytes during acute infections (10th day of treatment) and return to normal values after 20 days of treatment., Conclusions: (1) The most sensitive expression of infection in children with neoplastic disease was the variable percent of HDLA-DR molecules on T lymphocytes. (2) Presence of activation indicators on T lymphocytes and monocytes can testify that there is persistent function of the immunological system in spite of immunosuppression in patients with neoplastic disease. (3) Depression in percent value of monocytes with HLA-DR antigens in the course of cytomegalovirus pneumonitis confirms its immunosuppressive influence on the human organism.

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.

It was established that high level of BCL-2 antigen inhibited suicidal cell death and prevented apoptosis induced by antitumor drugs. Due to the fact that evaluation of BCL-2 expression in bone marrow and peripheral blood cells could be an important prognostic factor in patients with malignant hematopoietic diseases, we decided to study the number of BCL-2+ nuclear cells and assess this antigen expression in the cells. Using monoclonal FITC-conjugated antibody, antigen BCL-2 was found in peripheral blood cells in children with both lymphoblastic (ALL) and non-lymphoblastic leukemia (NLL). At the same time the percentage of PCNA+ blood nuclear cells was investigated. The cells were analysed with a flow cytometer. The study carried out in a group of 12 ill and 7 healthy children showed significantly increased percentage of lymphocytes and neutrophiles PCNA+ and BCL-2+ in ALL, in comparison with a healthy control group. Exclusively an increased percentage of BCL-2+ lymphocytes and neutrophiles was observed in NLL patients. Due to a relatively small group of investigated patients we can not make a certain conclusion but preliminary analysis suggests a correlation between a high number of BCL-2+ cells and a long time without remission.

Immunophenotype of peripheral blood lymphocytes was studied in 10 children after the cessation of treatment acute lymphoblastic leukemia and in 3 after non Hodgkin's lymphoma non-B. They all were in complete remission. Peripheral blood lymphocytes cells were analysed by flow cytometric analysis. Estimate surface antigens were estimated: CD3, CD19, CD4, CD8, CD3-16+ 56+. The results were compared with data obtained in children not treated because of neoplastic diseases. Our results showed that the absolute number of lymphocytes T(CD3+), TS (CD8+) and NK cells were significantly increased in the study group.

Total number of 160 children with ALL, 98 boys and 62 girls, aged from 0.5 to 18 years was included in the study. Apoptotic cells death acc. to annexin V and ICAM-1 levels in serum and cell culture supernatants according to conventional antibody sandwich ELISA Genzyme assay were studied. Blood samples were drawn from children at different stages of their disease: at the time of diagnosis, during intensive therapy (induction, consolidation), during maintenance therapy and after completing the treatment. Thirty seven healthy children served as the control group. ICAM-1 before therapy was higher than that obtained in control group of healthy children (5.619 v 2.53 g/l). ICAM-1 decrease (3.228) after starting of chemotherapy of ALL was noticed. It was found that in children with ALL during the whole period of therapy the ICAM-1 serum levels were significantly lower than that observed in the control group of healthy children (p < 0.005). After cessation of the therapy ICAM-1 grew up (6.27 g/l). Therapeutics that modulate the regulation of apoptosis and/or expression of sICAM and cytokines production provide a new opportunity for the treatment of childhood ALL.

The clinical efficacy of empiric antibiotic treatment regimen for infection in 38 neutropenic patients with acute leukemia during induction and intensification therapy was evaluated. The therapy, which was applied in 74 episodes of fever in patients with neutrophil count less then 0.5 G/l, consisted of three sets of antibiotics (gentamycin + carbenicillin or azlocillin, amikacin + cephradine++ or cefuroxime, netilmicin+cefotaxime or ceftazidime) used one after another in case of persistence of fever in spite of 72 hours of treatment: In addition, flucytosine was applied in case of stable fever after 72 hours of antibiotic therapy. Flucytosine was replaced by amphotericin after 72 hours of ineffective treatment. The response rate of 68% and 96% was observed for patients during induction and intensification chemotherapy respectively. Low clinical efficiency of gentamicin and carbenicillin/azlocillin during induction treatment indicates that the therapy with antibiotics of broader range of activity is needed for this group of patients.

Four children with the acute leukemia are presented. Their blasts shown the presence of 2 cellular lines markers. Coexistence of markers in the blasts was detected with the technique of double staining the blasts from the bone marrow with: alkaline phosphatase-anti-alkaline phosphatase, and peroxidase with the use of monoclonal antibodies series. Analysis of blasts phenotype with monoclonal antibodies confirm the occurrence of leukemias different from the normally programmed cellular line. Deviations of leukemic cells phenotype may be explained with the fact that leukemogenesis is not an absolute block of cells differentiation but combines maturation disorders and proliferation enabling expression normally absent antigens. It confirms the concept of line preservation and presentation of "earlier frozen" phenotype, and explains the occurrence of leukemias in which blasts present phenotype of one line which does not comply with cell differentiation pattern. Further genotypic studies are necessary to clarify pathogenesis and origin of such blasts. Consequently examination of the larger group of patients with hybrid leukemias will enable conclusions concerning prognostic value of such findings and necessity of introduction of the special therapies.

Transplantation of allogeneic bone marrow was done in 4 patients during the second remission of acute lymphoblastic leukaemia (3 cases) and B-cell non-Hodgkin lymphoma with dissemination to bone marrow (1 case). The engraftment and complete haematological reconstruction was achieved.

In the time period from February to October 1988 the indicators of hepatitis virus B infection, transaminase activity, and bilirubin level were studied in 89 children with acute leukaemias and malignant lymphomas. The age of the children was from 7 months to 16 years. Fifteen children were examined before starting the treatment with cytostatics, 48 during intensive chemotherapy, and 26 during maintenance treatment. HBV markers were found in 53 children during intensive therapy and maintenance treatment. Out of 36 children with negative HBV infection markers 31 were examined before starting the treatment with cytostatics or during the first 6 months of treatment. Nine out of 53 children in whom HBV infection markers were found had aminotransferase activity raised over 100 u/l and/or bilirubin level over 5 mg/dl and in nine cases transaminase activity and bilirubin level were raised only slightly (below 100 u/l and below 5 wg/dl). In 53 children the results were normal. The high incidence of HBV infection in children with acute leukaemias and lymphomas indicates the necessity of routine prophylactic treatment in this group of patients.

In the paper recent advances in the etiology and pathogenesis of acute lymphoblastic leukaemia are reviewed. Role of enzymes and cytogenetic abnormalities, oncogenes and gene rearrangements in the biology of this leukaemia is presented. Progress in chemotherapy, immunotherapy and bone marrow transplantation is also discussed.

The one-direction radial diffusion method has been used in studies of the blood sera from 75 subjects and the kappa and lambda immunoglobulin types levels have been determined. The kappa:lambda (K:L) index was also calculated. The index values in sera of 20 healthy subjects remained in the range from 1.58:1 to 2.14:1 (mean: 1.86:1). The normal index values have been stated in 5/5 sera of patients with bacterial pneumonia. In 12/12 sera from patients with plasma cell myeloma the significant abnormalities of the index values have been noted. Abnormal index values have also been stated in 9/10 patients with acute leukemia, 4/9 patients with chronic lymphocytic leukemia, 7/8 patients with lupus erythematosus and 4/4 patients with autoimmune hemolytic anemia. The quantitative determination of particular immunoglobulin types may be useful in the diagnostics of the immunoglobulin types may be useful in the diagnostics of the immunoglobulin monoclonal synthesis. The abnormalities of the K:L index observed in patients with leukemias and autoimmune disease suggest that these diseases are frequently characterized by the monoclonal synthesis of immunoglobulins.

An analysis of infection incidence within one year after completion of the treatment for the acute lymphoblastic leukemia was performed in the group of 24 children. Infections incidence was related to the selected immunological parameters, mainly T4 and T8 lymphocyte subpopulations assayed with monoclonal antibodies. T4/T8 cells ratio (Ix) was determined. It was found that moderate decrease in the total lymphocyte count and Ix exist in children one year after completion of the treatment. This index is more significantly lower in children with more frequent infections while the absolute numbers of T4 and T8 lymphocytes are relatively increased. The obtained results suggest that no significant immunosuppression is observed in children affecting the number and the course of infections.

A case of Varicella-zoster virus re-infection in a 3.5-year child with the acute lymphoblastic leukemia is presented. This report aims at emphasizing a possibility of re-activation of the virus in younger and younger children with immunodeficiency. A clinical course may imitate chicken-pox creating diagnostic problems.