[About possible additions to actual scheme of normal blood formation on the basis of study of leukemic blast cells].

The issue of introduction of number of additions into actual scheme of blood formation is discussed. The long standing experience of laboratory diagnostic of oncologic hematological diseases in adults and children and the analysis of published data about normal blood formation are involved into consideration. The existence is surmised of common oligo-linear precursors for B-lymphocytes and monocytes, natural killer cells and monocytes and common cell-precursor of T-lymphocytes and dendrite cells as well. At the same time, the issue concerning the existence of human common cell-precursor of lymphization capable of differentiating into Band T-lymphocytes and natural killer cells is disputable.

RAPID LOW-COST DETECTION OF TYPE 2CALR MUTATION BY ALLELE-SPECIFIC RT-PCR FOR DIAGNOSIS OF MYELOPROLIFERATIVE NEOPLASMS.

BACKGROUND: Approximately 15% to 24% of essential thrombocythemia (ET) and 25-35% of primary myelofibrosis cases carry a mutation in the calreticulin (CALR) gene. Sanger sequencing, qPCR, high resolution melt or targeted next generation sequencing usually used to detect these mutations are expensive and require costly equipment. Nevertheless, type 1 CALR mutations are detectable by using polymerase chain reaction (PCR) and agarose gel electrophoresis. AIM: To offer the use of the allele-specific reverse transcription (RT) PCR for rapid low-cost detection of the type 2 mutation in the CALR gene. MATERIALS AND METHODS: Allele-specific primers designed for detecting type 2 mutation (5-bp insertion; c.1154_1155 ins TTGTC) of the CALR gene were used for allele-specific RT-PCR analysis of cDNA of the patient with JAK2-, MPL-negative ET, whose mutation in CALR gene has been identified by Sanger sequencing. RT-PCR samples were analyzed by agarose gel electrophoresis. RESULTS: The type 2 mutation (K385fs*47 ins5) in CALR gene was detected by Sanger sequencing in JAK2- and MPL-negative ET patient. The cDNA obtained was then re-analyzed by using allele-specific RT-PCR with newly designed primers. Normal and type 2 mutation alleles of the CALR gene were detected by gel electrophoresis. The results of allele-specific RT-PCR were consistent with the data of Sanger sequencing. CONCLUSION: Allele-specific RT-PCR analysis may be used for the fast low-cost detection of the major type 2 mutation (ins 5) of the CALR gene in patients with MPNs.

Immunodeficiency-associated lymphoproliferative disorders and lymphoid neoplasms in post-COVID-19 pandemic era.

The 2017 revision of WHO Classification of tumors of hematopoietic and lymphoid tissues contains separate chapters on the immunodeficiency-associated lymphoproliferative disorders. In this mini-review, the brief description of pathological, immunophenotypical and clinical features of lymphoid neoplasms associated with primary immune disorders, HIV infection, those arising in post-transplant setting and other lymphoproliferative disorders (excluding those induced by radiation) is given. The heterogeneous spectrum of these lymphoid malignancies is specified by the nature of those factors that are capable to induce immune suppression or chronic antigenic stimulation of immune system. Taking into account the full swing of SARS-CoV-2 pandemic and our ignorance of the ability of this virus to induce the sustained stimulation of immune system, we could not exclude the high risk of autoimmune diseases and lymphoid neoplasms in the long-term post-pandemic period. In this context, the role of angiotensin-converting enzyme 2  as well as some recently reported cell receptors for SARS-CoV-2 cell entry should be considered as far as some of them (CD147, CD26) could be tumor-associated antigens.

Long-term exposure to low doses of ionizing radiation and COVID-19 pandemic: oncohematological aspects.

For more than 35 years after Chornobyl catastrophe, about 5 million people in Ukraine, Republic of Belarus and Russian Federation inhabit the territories that are residually contaminated with long-lived radionuclides such as 137Cs, 90Sr. The previous studies of the Reference Laboratory operating at RE Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology allowed specifying the effects of the protracted low dose irradiation on the state of the hematopoietic and lymphoid tissues resulting in the increased proportion of the B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma and acute myeloid leukemia among the patients referred from the contaminated areas of Ukraine. Since the beginning of 2020, these effects of radiation were superimposed by the factors associated with COVID-19 pandemic. SARS-CoV-2 infection is associated with the significant impact on hematopoiesis and immune system. Particular attention should be given to the role of such combined burden in the development of the immunodeficiency-associated lymphoid neoplasms. The extensive studies of the combined effects of low dose irradiation and COVID-19 within the large affected populations could be made a priority in future endeavors of epidemiologists and oncohematologists.

Aberrant expression of placental-like alkaline phosphatase in chronic myeloid leukemia cells in vitro and its modulation by vitamin E.

Placental-like alkaline phosphatase (PLAP) is expressed by many tumors and can be detected in sera of patients with various cancers. Its aberrant expression has been considered to be potentially useful as tumor marker. However, the biological background of the role of this aberrant alkaline phosphatase (AP) in cancer is still unclear. The expression of various forms of AP in cells of chronic myeloid leukemia (CML) has not yet been studied. AIM: To analyze the expression patterns of various AP forms in cells originated from CML patients in blast crisis and to modify their expression by vitamin E. MATERIALS AND METHODS: RNA extracted from leukemic cells was converted to cDNA and real-time reverse transcription polymerase chain reaction was performed using SYBR Green protocol with primers to tissue non-specific alkaline phosphatase (TNAP), intestinal alkaline phosphatase and CCAAT-enhancer-binding proteins alpha (C/EBPα). To analyze the modulation of expression of APs and C/EBPα, CML cells were incubated with 100 µM vitamin E. RESULTS: We have observed the aberrant expression of mRNA intestinal alkaline phosphatase in CML cells that upon sequencing demonstrated the significant alignment with PLAP sequence while no gene homology with tissue placental alkaline phosphatase (PAP) was revealed. Vitamin E decreases mRNA PLAP expression and increases mRNA TNAP expression. Moreover, along with down-regulation of aberrant PLAP and up-regulation of TNAP, vitamin E increases C/EBPα mRNA expression. CONCLUSION: The loss of TNAP in CML may contribute to pathogenesis of this disease. PLAP may be considered as a putative target in differentiation therapies in myeloid neoplasms. Our findings suggest the potential role of vitamin E as the inducer of differentiation potential of leukemic cells in CML.

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.

Vitamin Е activates expression of С/EBP alpha transcription factor and G-CSF receptor in leukemic K562 cells.

BACKGROUND: Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder associated with the activity of BCR-ABL fusion oncogene. Tyrosine kinase inhibitors are the current treatment of CML, but secondary mutations finally contribute to therapy resistance and blast crisis of the disease. The search for the novel compounds for the effective control of CML is now in the spotlight. The progression of CML to blast crisis is correlated with down-modulation of C/EBP alpha. Therefore, C/EBP alpha may be considered as a putative target in differentiation therapies in myeloid leukemias. The aim of the study was to assess the potential of vitamin E as the possible inducer of C/EBP alpha expression in BCR-ABL-positive CML K562 cells. MATERIALS AND METHODS: RNA extracted from K562 cells cultured with valproic acid or vitamin E was converted to cDNA, RT-PCR reactions were carried out using HotStarTaq DNA polymerase with primers for C/EBP alpha and granulocyte colony-stimulating factor receptor (G-CSFR). RESULTS: We have not found detectable expression of C/EBP alpha in K562 cells. Upon 48-h culture with vitamin E at a dose of 100 µM, K562 cells expressed both C/EBP alpha and G-CSFR. CONCLUSION: Vitamin E restored the expression of C/EBP alpha mRNA in chronic myelogenous leukemia K562 cells. In this setting, G-CSFR expression in vitamin E treated K562 cells seems to suggest the activation to granulocytic differentiation. It should be further elucidated whether such effects of vitamin E on C/EBP alpha transcription factor are direct or mediated indirectly due to antioxidant properties of vitamin E.

Several aspects of descriptive epidemiology of hematological malignancies in adult population of Ukraine, Belarus and Russian Federation after Chornobyl accident.

Chornobyl impact on the health of adult population in Ukraine, Belarus and Russian Federation was a subject of several studies. However, the studies of the effects of Chornobyl on leukemia in adult populations in post-Soviet countries are scarce and the results are contradictory up to present. The results of the epidemiological studies of the oncohematological consequences of Chornobyl accident are briefly reviewed with particular focus on pre-Chornobyl and post-Chornobyl trends in leukemia incidence in Ukraine, Belarus and Russian Federation as well as in small territories of these countries with various levels of radionuclide contamination. This article is a part of a Special Issue entitled "The Chornobyl Nuclear Accident: Thirty Years After".

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.

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.

Oligosaccharide-binding molecules on the surface of human hemopoietic and lymphoid cells.

High oligosaccharide specificity for binding carbohydrate probes (biotinylated polyacrylamide with carbohydrates attached) with human hemopoietic and lymphoid cells is shown. Of 15 probes studied those bearing blood group trisaccharides, A and B, bound most intensely. In addition, transformed (leukemic and lymphoid) cells interacted more strongly than normal ones.

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.

Monoclonal antibodies IPO-3 and IPO-10 against human B cell differentiation antigens.

Monoclonal antibodies (mAbs) IPO-3 and IPO-10 were generated following immunization of a BALB/c mice with human cell lines RPMI-1788 and Daudi respectively. The reactivity of these mAbs was studied by indirect immunofluorescence technique with 10 human cell lines, blood cells of healthy persons and patients with the malignant lymphoproliferative diseases. Studies of normal and neoplastic B cells suggest that mAbs IPO-3 and IPO-10 which recognize antigens are B lineage restricted. The presence of an antigen defined by the mAb IPO-10 appears to include most but not all the stages of B cell differentiation, whereas IPO-3 detected antigen is not represented on resting B cells and has a very limited expression on activated B lymphocytes. The results obtained with mAbs IPO-3 and IPO-10 are discussed in relation to other known B cell surface markers.

Leukemia of BALB/c mice induced by cellfree filtrate from transplantable Thurzo--Svec rat leukemia.

Leukemia was produced in adult BALB/c mice inoculated with cellfree filtrate derived from cells of transplantable Thurzo-Svec rat leukemia. The leukemogenic activity of 20% filtrates was 10(8) ID 50 per ml. Leukemia induced in mice with rat materials was serially transplantable by cellfree filtrates of leukemic tissues and plasma to the same strain of mice.

Monoclonal antibodies of IPO series against B cell differentiation antigens in leukemia and lymphoma immunophenotyping.

Monoclonal antibodies (mAbs) of IPO series were developed following immunization with human B cell lines RPMI-1788, Daudi, and spleen cells from a patient with hairy cell leukemia. Reactivity of these mAbs was studied on 19 human cell lines, mononuclear cells of 50 healthy persons and 142 patients with leukemias and lymphomas. It was shown that mAbs IPO-3, IPO-10 and IPO-24 define B cell-specific antigens expressed at different stages of maturation. MAb IPO-3 reacted with activated B lymphocytes. MAb IPO-10 defined the antigen which appears on B cell progenitors following HLA-DR and proceeding CD19, CD10, CD22, CD37; cy mu and CD20 and have been lost during terminal differentiation. The antigen detected by mAb IPO-24 was expressed throughout B cell ontogeny from pre-B cell until the B-blasts. MAb IPO-4 detected an antigen of activated T and B lymphocytes. These mAbs are useful tools in the leukemia and lymphoma phenotypic characterization and classification.

[Surface membrane antigens and receptors of the Reed-Sternberg and Hodgkin's cells in lymphogranulomatosis]. / Antigeny i retseptory poverkhnostnykh membran kletok Berezovskogo-Sternberga i Khodzhkina pri limfogranulematoze.

The results obtained provide evidence concerning the nature of the Hodgkin and Reed-Sternberg cells. The data based on application of new methods developed during last years such as different enzymo- and immunocytochemical techniques, monoclonal antibody and lectin application and molecular studies of gene rearrangement are presented.

[Lectin receptors--histochemical markers in the study of leukemias and lymphomas]. / Retseptory lektinov--gistokhimicheskie markery v izuchenii leikozov i limfom.

Data are presented on histochemical determination of lectin receptors on surface membranes of different haematopoietic and lymphoid cells of healthy persons and of patients with leukemias and lymphomas.

[Determination of subpopulations of normal and malignant transformed lymphocytes in blood smears by an immunoenzyme histochemical method using monoclonal antibodies]. / Opredelenie subpopuliatsii normal'nykh i zlokachestvenno transformirovannykh limfotsitov v mazkakh krovi immunofermentnym gistokhimicheskim metodom s ispol'zovaniem monoklonal'nykh antitel.

The data on lymphocyte subpopulations in blood smears are presented. They can be revealed by the double PAP-method and monoclonal antibodies ICO--1.02, 10, 11, 13, LT--1.8, 1B4, IPO--3, 4, 5, 10 and BL--TH4, DR 1, DR 4.

[The expression of an antigen interacting with monoclonal antibody IB4 on the surface membranes of normal human leukocytes and in lymphoproliferative diseases]. / Izuchenie ékspressii antigena, vzaimodeistvuiushchego s monoklonal'nym antitelom IB4, na poverkhnostnykh membranakh leikotsitov cheloveka v norme i pri limfoproliferativnykh zabolevaniiakh.

Monoclonal antibody (MAb) 1B4 of IgG3 isotype reacting with 55 +/- 10% peripheral blood lymphocytes was obtained. MAb 1B4 is bound with 100% of B and NK cells and 50-70% of T cells in blood. The most part of CD8+ cells (60-100%) and approximately 50% of CD4+ cells belong to 1B4+ population. T-ALL cells do not express 1B4 antigen. Analysis of reactivity of MAb 1B4 with transformed malignant cells has shown that 1B4 antigen appeared in the process of differentiation of B lymphocytes at the state of pre-B cells and disappeared with activation of B lymphocytes and transition to plasma cells.

[Use of monoclonal antibodies and lectins in the study of subpopulations of lymphoid cells in normal state and in leukemia]. / Ispol'zovanie monoklonal'nykh antitel i lektinov dlia izucheniia subpopuliatsii limfoidnykh kletok v norme i pri leikozakh.

The heterogeneity of cell populations with peanut agglutinin receptors in reactive tonsilar tissue has been stated. It contains B lymphocytes (HLA-DR+, CD 10+, IPO-10+ cells), part of T-cells (CD 4+), but no CD 8+ lymphocytes. PNA+ CD 10+ cells are the normal analogues of centrocytic non-Hodgkin's lymphomas.