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.

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.

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.

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".

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.

Photodynamic responsiveness of human leukemia Jurkat/A4 cells with multidrug resistant phenotype.

UNLABELLED: Photodynamic therapy (PDT) is considered as a possible alternative approach to overcoming multidrug resistance (MDR). Analysis of cross-resistance to PDT in cells with different MDR pathways and resistance levels seems to be advantageous for elucidating the general mechanisms of cancer cell resistance to various treatment modalities. AIM: The aim of the study was to clarify whether the Jurkat/A4 leukemia cells with MDR phenotype are cross-resistant to PDT. METHODS: Human T-cell acute lymphoblastic leukemia line Jurkat and Jurkat/A4 subline with MDR phenotype were used. 5-Aminolevulinic acid (ALA) and Photolon (a complex of chlorine-e6 and polyvinylpyrrolidone; PL) or gold nanocomposite of PL were applied as photosensitizers. The cells were pretreated with photosensitizers and exposed to laser radiation at corresponding wavelengths. The phototoxicity was assessed in trypan blue exclusion test. The hypodiploid cell fraction was analyzed by flow cytometry of propidium iodide-stained cells. Expression of genes related to PDT resistance was analyzed by microarray technique with Affymetrix U133A chips. RESULTS: ALA-mediated PDT resulted in dose-dependent cell death in both lines, the relative photodynamic efficacy in Jurkat/A4 cells being inferior to that in the parental Jurkat cells. There was no correlation between phototoxicity and apoptosis induction both in Jurkat and Jurkat/A4 cells. PL-mediated general phototoxicity in Jurkat cells amounted up to 75% at the maximal photosensitizer dose with about 40% of apoptotic death fraction. PL-phototoxicity in Jurkat/A4 cells was considerably lower. In contrast to Jurkat cells, PL-gold composite did not increase the efficacy of photosensitization as compared to free PL in Jurkat/A4 cells. CONCLUSIONS: Multidrug-resistant Jurkat/A4 cells exhibit reduced sensitivity to phototoxic effect in comparison with parental Jurkat cells independently of nature of the photosensitizer being assayed.

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 study of Bcr and Bcr-Abl localization in K562 cells.

AIM: To obtain polyclonal antibodies against recombinant proteins recognizing Bcr domain and fusion region of Bcr-Abl and analyze the patterns of intracellular distribution of Bcr and Bcr-Abl proteins in K562 cells of chronic myelogenous leukemia. METHODS: The coding sequences of DH and PH domains of Bcr-Abl were cloned, and the recombinant proteins were expressed in E. coli. The rabbit polyclonal antibodies were produced and used for immunocytochemical study of Bcr and Bcr-Abl localization in K562 cells. RESULTS: The gene constructs containing sequences coding for DH and PH domains of Bcr-Abl have been obtained. The antibodies with relative specificity to corresponding recombinant proteins differ by the patterns of their intracellular reactivity with Bcr- and Bcr-Abl related structures. While Bcr protein is located predominantly perinuclearly, antibody against hybrid Bcr-Abl protein is reacted with the structures in cell periphery, namely on cell membranes. CONCLUSION: Antibodies against DH and PH domains of Bcr-Abl react with proteins located differently in chronic myelogenous leukemia cells. The difference in intracellular localization of Bcr and Bcr-Abl may be attributable to the different domains interacting with different multiprotein complexes.

Apoptosis and content of mobile lipid domains in human leukemia K-562 cells induced to differentiate by quercetin or dimethyl sulfoxide.

The apoptotic index, cell cycle progression and caspase-3 activation in K-562 cells induced to differentiate by DMSO or quercetin have been studied. Quercetin treatment of K-562 cells was accompanied by cell cycle arrest in G2/M and apoptosis with caspase-3 activation. In contrast, DMSO-induced differentiation was accompanied by the complete cell cycle arrest in G1/G0 with negligible caspase-3 activation. In spite of the appearance of benzidine-positive cells and the decreased CD71 level in K-562 cells after exposure to quercetin, the analysis of 1H NMR spectra revealed the overall balance in favor of apoptosis, namely the increase in the content of NMR-visible mobile lipid domains and the decreased intensity of choline-containing metabolites.

[Detection of early apoptotic changes in cells in vitro using nuclear magnetic resonance spectroscopy].

The technique of proton magnetic resonance spectroscopy (1H MRS) was used as the sensitive express method for early specific detection of the apoptotic cells. The technique allows recognition of the changes in signal intensities corresponding to methylene (CH2) and methyl (CH3) protons of the mobile lipid domains (MLD) and choline, which are characteristic of apoptotic rather than of necrotic cells. A strong linear correlation between MLD content (calculated as CH2/CH3 signal intensity ratio) and the number of apoptotic cells in Namalwa or MT4 cell lines has been shown for any inducer of apoptosis used in the study. MLD content estimated by 1H MRS technique correlated significantly with apoptotic cells numbers (r = 0.992) recorded by conventional techniques. The increase in MLD content was registered as early as 60 min after the addition of etoposide coinciding with the time course of caspase-3 activation.

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.

[Apoptosis of human malignant lymphoid NAMALWA cells induced by resveratrol and quercetin].

The effects of naturally-occuring polyphenols, resveratrol and quercetin, on cell viability and apoptosis were studied in Namalwa B-cell lymphoma line. Apoptotic cells were identified using DNA flow cytometric analysis and 1H NMR spectroscopy. The effects of the agents on the cell cycle kinetics and activation of caspase-3 were examined. Both resveratrol and quercetin induced apoptosis in Namalwa cells as demonstrated by the increased number of hypodiploid cells, elevated level of mobile lipid domains and caspase-3 activation. Treatment with 40 microM of resveratrol for 48 h resulted in time-dependent cell-cycle arrest at G0/G1. In contrast, upon quercetin treatment Namalwa cells accumulated in G2/M. Obtained results suggest that resveratrol and quercetin induced caspase-dependent apoptosis in human malignant lymphoid cells in vitro. These findings provide a rationale for further studies of in vivo effects of those polyphenols.

Analysis of 1H NMR-detectable mobile lipid domains for assessment of apoptosis induced by inhibitors of DNA synthesis and replication.

Cell membrane rearrangements coincident with apoptosis may contribute to the increase in the ratio of methylene (CH(2) at 1.3 ppm) to methyl (CH(3) at 0.9 ppm) resonance signal intensity as observed by proton nuclear magnetic resonance ((1)H NMR). We studied CH(2) and CH(3) resonances in cultured cell lines treated with etoposide and fludarabine or bioflavonoid quercetin. Etoposide treatment (10 microM, 18 h) resulted in 3.3 fold increase of the CH(2)/CH(3) signal intensity ratio and 6.4 fold decrease in choline signal of MT4 cells. Incubation of Namalwa cells with fludarabine (3 microM, 72 h) increased the CH(2)/CH(3) signal intensity ratio by 2.4 fold and choline resonance intensity was unchanged. Quercetin treatment (30 microM, 1.5 month) increased CH(2)/CH(3) ratio by 2.1 fold. Necrotic cell death upon ethanol (20%) or DMSO (30%) treatment did not change the CH(2)/CH(3) signal intensity ratio. (1)H NMR-based study of mobile lipid domains is sensitive for detection of early engagement into apoptosis.

Effects of etoposide and fludarabine in subtoxic doses on karyotype of human malignant lymphoid Namalwa cells.

We studied changes in the karyotype of transplanted Namalwa cells induced by DNA-damaging antitumor preparations etoposide and fludarabine in subtoxic doses. The relative number of cells containing increased number of chromosomes and the incidence of chromatid aberrations with primary damage to chromosomes 2, 5, 11, 16, and 17 increased. Cytogenetic changes developed even after short-term incubation of cells with antitumor preparations and were observed during further culturing in a medium not containing etoposide or fludarabine.