AIM. To study the fractions of BAALC-expressing (BAALC-e) leukemic hematopoietic stem cells (LHSCs) in acute myeloid leukemia (AML) patients with isolated mutations in the FLT3 gene as well as their combinations with the mutations in the NPM1 gene. MATERIALS & METHODS. The study enrolled adult AML patients with the common element of having isolated FLT3 mutations in the genome (n = 25). The control group (n = 21) consisted of AML patients with mutations in both FLT3 and NPM1. The patients (n = 46) were aged 18–84 years (median 52 years), there were 26 women and 20 men. Non-random chromosomal aberrations, including those of a complex nature (≥ 3 lesions per metaphase), were identified in 13 patients with isolated FLT3 mutation and in 1 patient with both FLT3 and NPM1 mutations. Quantitative real-time PCR was used to measure the level of BAALC, WT1, and EVI1 expressions by the cells in bone marrow aspirate. Thresholds for distinguishing between high and low levels of BAALC and EVI1 expression were considered to be 31 % and 10 %, respectively, and the thresholds for WT1 and FLT3 allele ratio were 250 copies/104 ABL1 copies and 0.5, respectively. РЕЗУЛЬТАТЫ. An increased BAALC expression level roughly reflecting the fraction size of BAALC-e LHSCs was detected in 20/25 (80 %) patients with isolated FLT3 mutations. This was observed together with an increased level of WT1 (n = 22) and EVI1 (n = 7) expression. In all patients with both FLT3 and NPM1 mutations (control group, n = 21), the BAALC and EVI1 expression levels were below the threshold, which did not affect WT1 expression. This observation suggests to question the random nature of the identified decrease of BAALC and EVI1 expressions, which can be hypothetically accounted for by a low count of CD34-positive LHSCs in the bone marrow of AML patients with NPM1 mutations. Serial measurements of these molecular parameters under therapy for AML with FLT3 +/– NPM1 mutations show the feasibility of their use in assessing the therapy efficacy or the need for its correction, if required. CONCLUSION. The data presented in this paper clearly indicate that clinical trials need to intensively apply serial analysis of the fractions of BAALC-expressing leukemic HSCs in AML patients with FLT3 mutations. This approach allows for better molecular monitoring of the therapy efficacy for this challenging category of AML patients.

The discovery of gene BAALC, which is selectively expressed by hematopoietic stem cells, allows to study of pathogenesis and efficacy treatment of human AML and MDS in clinical settings at the level of Stem Cells. To simplify the situation, we collected these cells under a common category "BAALC—expressing Leukemia Stem Cells" and evaluated their burdens by means of RT-qPCR. The chapter contains new findings in this area concerning CBF-positive AML and MDS with isolated 5q-anomaly. The levels of BAALC-e LSCs burdens in patients with CBF+ AML were greatly increased. This index was lower in pediatric patients compared to adults (p = 0.049). In the most patients with MDS it was higher cut off however lower in those with isolated del(5q). Conclusion. These data show correctness of the concept about BAALC-e LSCs, and its perspectives for future studies.

Background. Myelodysplastic syndrome is a group of malignant blood diseases with a high risk of transformation into acute myeloid leukemia. One treatment approach is to target immune checkpoints (ICs) that are overexpressed on tumor cells. To develop these drugs, relevant models are needed for highthroughput screening and study of these biologically active substances, since traditionally used models (mouse and patient biomaterials) are difficult to access, financially and laborintensive, and are characterized by poorly reproducible results.Aim. To develop a model based on a human myeloid cell line with increased expression of L1 and TIM3 to study the activity of ICs inhibitors, the presence of which in the tumor microenvironment in patients with myelodysplastic syndrome and acute myeloid leukemia was associated with a high risk and worse prognosis.Materials and methods. Initial testing of the L1 and TIM3 basal expression level was carried out on cell lines: TH1, HL60, OCIAML2, OCIAML5, KG1, MonoMac1. Induction of IC expression was carried out using interferon γ. Analysis of marker expression was carried out 24 hours after induction of ICs expression and addition of MK2206 using flow cytometry.Results. Basal expression of the studied ICs receptors was absent in all of them, except for KG1; TIM3 was present in 88.4 ± 7.1 % of cells, and L1 – in 88 ± 8.5 %. The addition of interferon γ at a concentration of 50 ng/mL to the MonoMac1 culture led to a significant increase in the proportion of TIM3 and L1 expressing cells (53.3 ± 12.2 and 97.3 ± 1.1 % respectively, compared to 0.1 ± 0.1 and 0.1 ± 0.1 % without interferon γ), and for TH1 only L1 expression (87.5 ± 20 %, control 0.1 ± 0.1 %) was observed at the concentration of interferon γ in a medium of 50 ng/mL, while the proportion of cells expressing TIM3 was 6.9 ± 10 % (control 0.1 ± 0.1 %).Conclusion. The KG1 line, which constantly expresses significant levels of target ICs, as well as TH1 and MonoMac1, which are induced by 50 ng/mL interferon γ, were selected as a model with increased L1 and TIM3 expression based on a human myeloid cell line. The model efficiency was confirmed by the rational response to the IC pathway inhibitor.

Background. Due to changing views on pathogenesis, risk factors and therapy strategies in prognostically favorable CBF-positive acute myeloid leukemias[1] (AML), the expression monitoring of RUNX1/RUNX1T1 or CBFB/MYH11 fusion genes, as an additional evaluation of treatment outcomes, appears to be insufficient. This indicates the need to improve the monitoring of the CBF+ AML course by means of parallel measurements of BAALC expression levels which roughly correlate with the mass of BAALC-expressing leukemia hematopoietic stem cells (BAALC-e LHSC). Aim. To improve the quality of assessing treatment outcomes with due account for expression levels of RUNX1/RUNX1T1 or CBFB/MYH11 fusion genes and the mass of BAALC-e LHSC and on this basis to pave the way for personalized CBF+ AML treatment. Materials & Methods. This study enrolled 39 adult patients aged 20–81 years (median 32 years) and 8 children aged 2–18 years (median 12 years). Among them there were 20 females and 27 males. AML with inv(16)(p13;q22)/t(16;16) was identified in 19 patients, t(8;21)(q22;q22) was detected in 28 patients. BAALC, WT1, RUNX1/RUNX1T1, CBFB/MYH11 expression levels were measured by quantitative real-time PCR and related to the expression of the ABL1 expert gene. Results. In 23 patients, inv(16) and t(8;21) appeared to be isolated. Additional multidirectional chromosomal changes were observed in 24 patients with inv(16) and in 18 patients with t(8;21). All enrolled patients showed increased BAALC expression. In the course of therapy, it was decreasing to the threshold value in 16/18 (89 %) patients. The evaluation of the mean BAALC expression levels in the pooled groups of children and adults with isolated findings of either inv(16) or t(8;21) showed the decrease of the BAALC-e LHSC mass only in children (p = 0.049). The comparison of the mean WT1 expression levels in the pooled groups of children and adults with isolated and additional chromosomal abnormalities revealed their significant decrease in patients with complicated variants (p = 0.023). Conclusion. The case reports provided in this paper show that the molecular monitoring with serial measurements of fusion genes and BAALC gene expression levels in CBF+ AML patients can lay the basis for further improvement of personalized treatment strategies for these patients. In all likelihood, parallel measurements of the above gene expression levels will allow to establish the framework for decision-making concerning treatment extent and timely HSC transplantation.

In preclinical studies, there is a problem of estimating donor chimerism in animal models engrafted with human CD34+ cells. Using digital droplet PCR (ddPCR) more accurate results can be achieved in comparison with routine flow cytometry. The aim of the study was to develop a protocol for determining the level of chimerism by ddPCR in the bone marrow of NBSGW mice.

Оценены результаты трансплантации аллогенных гемопоэтических стволовых клеток (аллоТГСК) у 34 больных с цитогенетически верифицированными вариантами миелодиспластического синдрома (МДС) с трисомией 8 и/или моносомией 7, которые получали лечение в НИИ детской онкологии, гематологии и трансплантологии им. Р.М. Горбачевой с 2013 по 2020 г. В настоящей работе анализу подвергнуты как взрослые, так и детские МДС, причем варианты с двумя дополнительными хромосомными нарушениями или сложным кариотипом не исключались. В исследовании показано: а) необходимо выполнять аллоТГСК при лечении обоих вариантов МДС; б) результаты лечения у больных с трисомией 8 оказались лучше; в) у детей с моносомией 7 отмечается повышенная частота токсических осложнений при аллоТГСК.

Представлены данные, указывающие на высокую частоту обнаружения гиперэкспрессии гена BAALC, в т. ч. в комбинации с гиперэкспрессией гена WT1, у больных с миелодиспластическими синдромами (МДС) с верифицированными методом FISH нарушениями хромосом. При анализе уровней экспрессии генов BAALC и WT1 у 16 больных МДС (у 6 из них выполнена трансплантация аллогенных гемопоэтических стволовых клеток) повышение экспрессии гена BAALC выявлено у 14. У 2 пациентов уровень экспрессии был близок к пороговому. Низкие уровни экспрессии зафиксированы у больной, имевшей в кариотипе изолированную делецию 5q, а также при ее комбинации со сложным кариотипом. Напротив, самые высокие уровни экспрессии отмечались у больных с нормальным кариотипом и с вовлечением в перестройки локуса 3q26, что было коррелировало с избыточной экспрессией гена EVI1. Поскольку уровень экспрессии гена BAALC, по крайней мере у больных с основными (кроме М3 и М7) FAB-вариантами острых миелоидных лейкозов (ОМЛ), был тесно связан с продуцирующими его клетками-предшественницами лейкозного клона, углубленное изучение этого феномена при МДС представляется важным для понимания тончайших механизмов патогенеза ОМЛ и его рецидивов на уровне клеток-предшественниц.

The present paper provides evidence for a high detection rate of BAALC gene overexpression, also combined with WT1 gene overexpression, in patients with myelodysplastic syndromes (MDS) and FISH-verified chromosome defects. The BAALC and WT1 gene expression profiling of 16 MDS patients (6 out of them received allogeneic hematopoietic stem cell transplantation) showed an increased BAALC expression in 14 patients. The expression level in 2 patients was near the cut-off. Low expression levels were identified in a female patient with isolated 5q deletion in karyotype and also with its combination with complex karyotype. On the other hand, the highest expression levels were reported in patients with normal karyotype and 3q26 locus rearrangement, which was associated with EVI1 gene overexpression. Since the BAALC expression level, at least in patients with the major (except for М3 and М7) FAB-variants of acute myeloid leukemias (AML), was closely associated with BAALC-producing precursor cells of leukemia clone, a profound study of this phenomenon in MDS patients seems to be important for understanding the finest mechanisms underlying the pathogenesis of AML and AML relapses on the level of precursor cells.

The study assessed the outcomes of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in 34 patients with cytogenetically verified variants of myelodysplastic syndrome (MDS) with trisomy 8 and/or monosomy 7, who were treated at the RM Gorbacheva Scientific Research Institute of Pediatric Oncology, Hematology and Transplantation from 2013 to 2020. Both adult and pediatric MDS were analyzed without excluding the variants with two additional chromosomal abnormalities or complex karyotype. The study revealed that а) allo-HSCT should be performed in the treatment of both MDS variants; b) the outcomes of trisomy 8 treatment appeared to be better; c) children with monosomy 7 showed a higher rate of toxic complications in allo-HSCT.

The present review provides the analysis of theoretical background and therapy of prognostically poorest EV/1-positive myeloid leukemias and myelodysplastic syndromes which is performed at the RM Gorbacheva Scientific Research Institute of Pediatric Oncology, Hematology and Transplantation. The focus is on the evidence of the dominating role of EV/1 gene in impaired epigenetic regulation of hematopoiesis and, thus, on the feasibility of allogeneic hematopoietic stem cell transplantation with hypomethylating agents and/ or trans-retinoic acid used for these diseases treatment.

Настоящий обзор посвящен анализу теоретической базы и проводимой в клинике НИИ детской онкологии, гематологии и трансплантологии им. Р.М. Горбачевой терапии наиболее неблагоприятных в прогностическом отношении EVI1-позитивных вариантов миелоидных лейкозов и миелодиспластических синдромов. Основной акцент в работе сделан на доказательстве ведущей роли гена EVI1 в нарушении эпигенетической регуляции гемопоэза и, следовательно, целесообразности использования трансплантации аллогенных гемопоэтических стволовых клеток с гипометилирующими агентами и/или транс-ретиноевой кислотой для лечения этих заболеваний.

Based on the annual UNAIDS reports the number of HIVinfected patients is continually growing since 1983. Antiretroviral Therapy (ART) allows to prolong life expectancy, but the problem of life quality and overall survival is still remaining. Nowadays, in the era of ART, one of the main cause of mortality in HIV-infected patients is malignancies. Lymphomas play one of the key roles in this group of diseases. The treatment of lymphomas includes combined regiments of chemotherapy with a curative potential. High dose chemotherapy with autologous hematopoietic stem cell transplant (auto-HSCT) is the main path of the treatment for relapsed / refractory lymphomas. In the last few years with a development of the genome editing technology auto-HSCT is becoming one of the most promising methods of HIV treatment. The case of “Berlin patient” when allogeneic HSCT from donor with mutation CCR5-delta32 lead to cure from HIV and proof of concept the efficacy of the gene therapy for HIV based on HSCT. Hematopoietic stem cell transplantation with edited autologous HSC (CCR5 knockout by site-specific genome editing tools with engineering nucleases) is a comprehensive treatment for this cohort of patients. On one hand, high dose chemotherapy with auto-HSCT cures the malignancy; on the other hand auto-HSCT works as a delivery method for the edited cells and creates an environment for the HIV eradication. This review is dedicated to HIV and oncology, methods of treatment of hematological malignancies and HIV-infection using genome editing technology based on HSCT.

Modeling of allogeneic bone marrow transplantation and graft-versus-host disease (GVHD) plays a crucial role in the improvement of transplantation procedures and testing novel immunosuppressive drugs. In this study we evaluated the feasibility of busulfan and cyclophosphamide conditioning with posttransplantation cyclophosphamide (150 mg/kg) in donorrecipient pairs C57Bl/6-Balb/с and Balb/с-C57Bl/6. We observed that Balb/с mice as opposed to C57Bl/6 mice can’t be the recipients because of the unacceptably high acute mortality from acute toxicity of chemotherapeutic drug. Histological studies in the C57Bl/6 recipients revealed acute GVHD in 93 % of animals. All animals had only grade I–II severity of GVHD. This model has the potential for future studies in the field of transplantology, nonetheless a decrease in the intensity of prophylaxis is required to augment the manifestations of GVHD.

Allogeneic hematopoietic stem cell transplantation (HSCT) remains the sole universal curative approach for a number of hereditary diseases, such as severe combined immunodeficiency (SCID), severe non-SCID primary immunodeficiencies (non-SCID PID), hematologic diseases and part of lysosomal storage disorders. Unfortunately, to date, HSCT remains a high-risk procedure, especially in cases of poor performance status of the patient and lack of HLA-matched related donors. In those cases the correction of the patients autologous HSCs with gene therapy could be a promising alternative. Current paradigm of HSCT-based gene therapy approaches is based on the utilization of viral vectors, which may lead to the severe complications due to insertion mutagenesis. Throughout the last several years, new technologies of site-specific genome editing with endonucleases such as ZFNs, TALENs, and CRISPR/Cas9 were introduced. These enzymes may induce a DNA doublestranded break, homology-directed repair and insertion of functional copy of gene in precisely targeted locus. This review focuses on the advantages and disadvantages of the genome editing tools utilization that carries the great potential of changing the paradigm of gene therapy in the setting of HSCT.

Reduction of minimal residual disease to undetectable levels is the key criterion for efficiency of allogeneic hematopoietic stem cell transplantation (alloHSCT), along with engraftment of transplanted cells with complete replacement of recipient hematopoiesis, i. e., full posttransplant chimerism. Among different approaches, molecular genetic techniques are preferable, being based on the analysis of highly polymorphic DNA sequences (short tandem repeats, STRs). However, this approach, despite its high specificity, has a limited sensitivity. In this regard, it seems appropriate to introduce more sensitive diagnostic solutions, in particular, analysis of insertion/deletion (InDel) polymorphisms, followed by real-time detection of PCR products. The data obtained upon analysis of several genetic markers have shown higher sensitivity of this method. However, the deviations in the range of 10 to 90 % in evaluation of the cell ratios indicates the feasibility of using this approach just to evaluate the residual populations of recipient cells.

Pathogenesis of acute leukemia is associated with the presence of genetic and functional aberrations in hematopoietic stem cells. However, as one of the possible factors contributing to the progression of disease could be regarded as the pull of the stromal cells of bone marrow. In this paper we evaluated the major functional characteristics of stromal cells: proliferative activity, hematopoiesis supportive and differentiation ability, as well as analysis of the expression of adhesion molecules. Stromal cells derived from acute leukemia patients in comparison with healthy donors are characterized by a high proliferative activity and a pronounced ability to osteogenic and adipogenic differentiation with prominent expression of hematopoietic stem cells homing mediating molecules.