Immunophenotypic features of leukemic stem cells and bulk of blasts in acute myeloid leukemia.

According to the modern concept, leukemic stem cells (LSC) in acute myeloid leukemia (AML) are distinct from the bulk of leukemic cells in bone marrow and peripheral blood of AML patients. Nevertheless, LSC are responsible for managing all the hierarchy of the bulk of leukemic blast populations. This mini-review provides brief information on the distinctive features of LSC and blast cells in cytologically recognized types of AML. The study of different phenotypes of LSC and blast cells in AML with the aid of up-to-date flow cytometric techniques is important both for the deep insight into the mechanisms of leukemogenesis and development of novel strategies of target therapy. The urgent need for extending the diagnostic panel of monoclonal antibodies used for diagnosing AML is beyond doubt.

Overview on association of different types of leukemias with radiation exposure.

Exposure to ionizing radiation is associated with increasing risk of various types of hematological malignancies. The results of major studies on association of leukemias and radiation exposure of large populations in Japan and in Ukraine are analyzed. The patterns of different types of leukemia in 295 Chernobyl clean-up workers diagnosed according to the criteria of up-to-date World Health Organization classification within 10-25 years following Chernobyl catastrophe are summarized. In fact, a broad spectrum of radiation-related hematological malignancies has been revealed both in Life Span Study in Japan and in study of Chernobyl clean-up workers in Ukraine. The importance of the precise diagnosis of tumors of hematopoietic and lymphoid tissues according to up-to-date classifications for elucidating the role of radiation as a causative factor of leukemias is emphasized. Such studies are of high importance since according to the recent findings, radiation-associated excess risks of several types of leukemias seem to persist throughout the follow-up period up to 55 years after the radiation exposure.

Leukemic blast cells and controversies in models of hematopoiesis.

Classical and up-to-date models of hematopoietic lineage determination are briefly reviewed with the focus on myeloid-based models challenging the existence of the common progenitor for T cells, B cells and NK cells. The analysis of immunophenotype of leukemic blast cells seems to be a promising approach for interpreting some controversies in the schemes of normal hematopoiesis. The literature data as well as our own findings in the patients with various types of acute leukemias are in favor of the concept postulating that common myeloid-lymphoid progenitors giving rise to T and B cell branches retain the myeloid potential. The similarity of some immunophenotypic features of blast cells in pro-B acute lymphoblastic leukemia and acute monoblastic leukemia is consistent with monocyte origin postulated in the studies of normal hematopoiesis. Study of acute leukemias may be the challenging area of research allowing for new insight into the origin of hematopoietic cell lineages.

Gene expression profiling of B-CLL in Ukrainian patients in post-Chernobyl period.

BACKGROUND: Genetic mechanisms that result in the development and progression of B-cell chronic lymphocytic leukemia (B-CLL) are mainly unknown. We have analyzed gene expression patterns in Ukrainian B-CLL patients with the aim of identifying B-CLL involved / associated genes in order to shed light on the biology of this pathological entity. MATERIAL AND METHODS: The samples of the peripheral blood and bone marrow of 44 Ukrainian B-CLL patients with no characteristics indicative of unfavorable course of the disease such as CD38 were analyzed morphologically and immunocytochemically according to the new WHO classification. Total RNA was isolated, and gene expression levels were determined by microarray method comparing with the sample from 17 healthy donors. RESULTS: We investigated interactions using the Ingenuity Pathway Analysis (IPA) software and found 1191 network eligible up-regulated genes and 3398 Functions/Pathways eligible up-regulated genes, 1225 network eligible down-regulated genes and 2657 Functions/Pathways eligible down-regulated genes. CONCLUSION: In B-CLL patients, gene networks around MYC, HNF1A and HNF4A, YWHAG, NF-κB1 and SP1 are identified as up-regulated; CEBPA, YWHAG, SATB1 and RB1 -- as down-regulated. G protein coupled receptor signaling, arachidonic acid and linoleic acid metabolisms, calcium signaling, metabolism of xenobiotics by cytochrome P450 are found out as significant up-regulated pathways. EIF2 and Cdc42 signaling, regulation of eIF4 and p70S6k signaling, protein ubiquitination pathway and oxidative phosphorylation are the most significant down-regulated pathways obtained in our study. The involvement of NF-κB gene network and upregulated levels of G protein coupled receptor signaling pathway, which has an important role in transcription of NF-κB, are important and need further examination.

Quantitative real time PCR analysis of apoptosis-related gene expression in leukemias in Ukrainian patients.

BACKGROUND: The complete medical consequences of the long-term exposure of population to ionizing radiation in post-Chernobyl period are still a controversial issue. The molecular biological analysis of malignant diseases of hematopoietic and lymphoid tissues in contaminated territories requires the precise diagnosis based on criteria of novel classifications. AIM: To analyze the relative gene expression of six apoptosis-related genes in different types of tumors of hematopoietic and lymphoid tissues in patients living in areas of Ukraine contaminated with radionuclides in post-Chernobyl period. MATERIAL AND METHODS: The samples of the peripheral blood and bone marrow of 189 Ukrainian leukemia patients and 16 patients with reactive lymphocytosis were analyzed morphologically and immunocytochemically for precise delineation of the main forms and cytological variants of hematological malignancies according to new WHO classification. Expression of six apoptosis-related genes was analyzed in the individual samples of 9 different groups of malignant diseases of hematopoietic and lymphoid tissues and one group of patients with reactive lymphocytosis by quantitative RT-PCR. Expression of genes was assessed relative to that in control group of healthy donors. RESULTS: Up-regulation of six analyzed apoptosisrelated genes is observed in all groups of leukemia. In most groups of leukemia being analyzed, BCL-2 up-regulation level is superior to that of BAX. Prominent MYC up-regulation is observed in B-lymphoblastic leukemia/lymphoma, non-Hodgkin's lymphoma, and T-lymphoblastic leukemia/lymphoma groups. In myelodysplastic/myeloproliferative neoplasms, the striking up-regulation of Fas-1 and P38MAPK is evident. Practically all the groups of leukemia are characterized by stable high ratios of P53 up-regulation. CONCLUSION: In Ukrainian patients, up-regulation of six analyzed apoptosis-related genes is observed practically in all types of malignant diseases of hematopoietic and lymphoid tissues under study. Microarray-based analysis of these samples would be of great importance in terms of elucidating genomic interactions in leukemias and their possible association with ionizing radiation.

Mature B-cell neoplasms in Chernobyl clean-up workers of 1986-1987: summary of cytomorphological and immunocytochemical study in 25 years after Chernobyl accident.

The data on the verified cases of mature B-cell neoplasms (chronic lymphocytic leukemia - CLL, B-prolymphocytic leukemia, non-Hodgkin's lymphoma in leukemization phase and multiple myeloma - MM; 146 cases in total) in the consecutive group of Ukrainian clean-up workers within 10-25 years after Chernobyl accident are summarized. B-cell neoplasms represent the most prevalent group among all diagnosed neoplasms of hematopoietic and lymphoid tissues in clean-up worker patients under study (49.4%). MM percentage in the patients of Chernobyl clean-up worker group turned out to be significantly higher than in the patients of the general populations studied at the same period. While the percentage of B-CLL is similar in clean-up worker patients and patients of general population, the trend towards younger age of patients with mature B-cell neoplasms in clean-up worker group is evident. The current concepts on the possible association between mature B-cell neoplasms (mainly B-CLL) and radiation exposure are briefly outlined. Only the precise diagnosis of hematopoietic malignancies combining with large-scale analytical epidemiological studies with careful dose assessment and long-term follow-up may represent the basis for resolving the question whether mature B-cell neoplasms may be radiogenic.

Immunocytochemical markers in acute leukaemias diagnosis.

The study included 1742 patients with acute myeloblastic leukaemias (AML) and acute lymphoblastic leukaemias (ALL), Kyiv city residents and patients from 20 regions of Ukraine. Bone marrow and blood smears were sent at diagnosis to Reference Center. The analysis was based on May-Grünvald-Giemza (MGG) stain and cytochemical reactions (MPO, acNSE, CAE, AP, PAS). Immunocytochemical techniques (APAAP, LSAB) and broad panel of monoclonal antibodies (MoAbs) against lineage specific and differentiation antigens of leukocytes were employed for immunophenotyping of leukemic blast cells directly in blood and bone marrow smears. Different types of AML were defined by the expression of the cell surface and cytoplasmic antigens. Immunocytochemical study was required especially in diagnosing of AML with minimal differentiation, acute megakaryoblastic leukaemia, acute erythroid leukaemia and acute leukaemias of ambiguous lineage. Acute lymphoblastic leukaemias was broadly classified into B-lineage and T-lineage ALL. According to the degree of B-lymphoid differentiation of the blast cells four subtypes of B-lineage ALL were established. T-lineage ALL observed in patients were also divided into four subtypes. Immunocytochemical examination was required to diagnose AL of ambiguous lineage with no clear evidence of lineage differentiation (acute undifferentiated leukaemia) or those with blasts that express markers of more than one lineage (mixed phenotype acute leukaemias).

Study of morphocytochemical and immunophenotypic features of acute leukemia stem cells.

The immunophenotypic profile of hematopoietic stem cells (HSC) and hematopoietic precursor cells as well as leukemic stem cells (LSC) has been extensively studied in several laboratories worldwide. The results of our studies suggest that the standard panel for classification of acute leukemias should be supplemented with several new markers allowing us to identify more precisely the different forms of the leukemias being of the closely related origin, for example AML M6b and AML M7. The common bipotent LSC in AML M7 of low grade and AML M6b may exist analogous to precursor cell common for megakaryocytopoiesis and erythropoiesis. We have also found the similarity between blast cells in pro-B-ALL [t (4;11), 11q23] and AML M5a [t (9;11), 11q23]. Such similarity of immunophenotype and cytogenetic abnormalities in blast cells in pro-B-ALL and AML M5a may be considered as hint explaining the cases of AML M5a as a recurrence of leukemia in children with originally diagnosed pro-B-ALL.

Sensitivity of normal and malignant human lymphocytes to 5-aminolevulinic acid-mediated photodynamic damage.

AIM: To compare the sensitivity of normal and malignant human lymphocytes to 5-aminolevulinic acid (ALA) - mediated photodynamic damage. METHODS: Blood lymphocytes isolated by Ficoll-sodium metrizoate density gradient from healthy donors (6) and hematologic patients (20) with different forms of lympholeukemia, and also transformed lymphocytes of human B-cell (Raji, Namalwa) and T-cell (MT-4, HUT-78) lines were investigated. Diagnoses of chronic lymphoproliferative disorders were made on the grounds of morphological, cytochemical and immunocytochemical studies of peripheral blood and bone marrow cells, with immunophenotype determination by monoclonal antibodies to differentiation antigens of T, B lymphocytes and NK cells and immunocytochemical ABC-AP method. Cells of leukemic B- and T-cell lines were cultured in standard RPMI-1640 medium. For photodynamic treatment, the cells were incubated with ALA and then irradiated by a helium-neon laser (wavelength of 633 nm). The number of dead cells was determined in 20 h with trypan blue dye exclusion test. RESULTS: The striking difference in responsiveness to ALA-mediated photodynamic treatment (ALA-PDT) between normal lymphocytes and cells isolated from lymphatic leukemia patients was established. A bulk of leukemic cells (mean for 10 patients with B-CLL - 62.06 -/+ 4.03%) were destroyed under the lowest ALA-PDT doses tested: 1 mM ALA, irradiation dose of 25 J/cm(2). However, it was virtually impossible to attain any appreciable damage of lymphocytes from healthy donors even with the highest treatment doses (5 mM ALA, 150 J/cm(2)). High sensitivity to ALA-PDT of malignant lymphocytes was confirmed in experiments with human T- and B-cell leukemic cell lines, and in these experiments, an anomalous reaction to the treatment of Raji cells was also detected. The mechanisms of the difference between normal and malignant lymphocytes are discussed in terms of altered heme-synthesis processes in malignant cells. CONCLUSIONS: 1) It is shown for the first time that blood lymphocytes from lymphatic leukemia patients are highly sensitive to the damage with ALA-PDT while lymphocytes of normal donors are practically not damaged. 2) Transformed lymphocytes of human T-cell lines are more sensitive than lymphocytes of B-cell lines. 3) Lymphocytes of the Raji line display anomalous dose-effect dependence with ALA-PDT. 4) It is proposed to evaluate the drastic difference in ALA-PDT responsiveness of normal and malignant lymphocytes as a possible simple and low-traumatic test for B-CLL screening among the elderly people.

Structure of leukemias and lymphomas in children of Ukraine in post-Chernobyl period.

The structure of hematopoietic malignancies in post-Chernobyl period among pediatric patients in Kyiv city and 24 regions of Ukraine especially those born in 1986 and 1987 and the infants at the age below 1 year is reviewed taking into account the data of the Reference Laboratory obtained in 1993-2004 and based on the modern diagnostic technologies in accordance with FAB, WHO, EGIL, ICD-10 and ICD-O-2 classifications.

Acute leukemias in children from the city of Kiev and Kiev region after the Chernobyl NPP catastrophe.

During 1993-1997, 247 cases of childhood acute leukemia (AL) were analyzed among inhabitants of the city of Kiev and Kiev region, excluding the most contaminated areas belonging to the strict control zone. The criteria of an FAB classification supplemented by immunophenotyping data were applied. The AL pattern was shown to be quite typical except for several peculiar features characteristic of this regional group of patients, especially the absence of age peaks in children with acute myelogenous leukemias (AML), increased frequency of the T1 variant in T-cell acute lymphoblastic leukemia (ALL), and higher levels of M4 and M5 variants in AML. A typical variant of M5a-AML with minimal signs of differentiation was found.

Immunocytochemical staining of cells in 153 pleural effusions with a panel of monoclonal antibodies and lectins.

A panel of markers, including MAbs to different epitopes of CEA, B 6.2, detection of AP activity and lectin receptors (PNA, HPL, SBA, LAL, LcL) for cell identification in pleural effusions, is proposed. Using immunocytochemical methods cancer cells were determined in all 80 cytological positive and in 13 from 21 cytologically negative cancer effusions. The reaction was not observed in 45 benign effusions. The panel was unsuccessful to determine the tumor cell origin. The immunophenotypic features of reactive transformed lymphocytes in effusions were described and the criteria for diagnosis of B-cell and T-cell NHL were present. By means of these criteria lymphomatous cells in serous effusions of seven patients were revealed.