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Introduction. Currently, there is no unequivocal opinion on the optimal list of studies for the genetic diagnosis of oncohematological disorders in children and adults. These discrepancies are due to the limited technological capabilities of laboratories, the rapid development of science, and a significant expansion of the range of new molecular markers, that are attractive, but only for a limited group of patients. Moreover, in modern conditions of limited access to resources, it seems important to bring desires, interests and opportunities to a common denominator. Aim — to develop unified approaches to the cytogenetic and molecular genetic diagnosis of oncohematological diseases in children and adults based on the consensus opinion of the panel of experts. Main findings. The review proposes the arrangement of cytogenetic and molecular genetic diagnostic tests in oncohematological disorders in children and adults into 3 categories depending on the frequency of genetic aberrations, the study complexity and the prognostic impact. Based on this and taking into account the diagnosis and age of patients, the minimal and optimal lists of clinically significant parameters and research markers were identified. The basic preanalytical principles for conducting cytogenetic and molecular genetic studies in oncohematology are pointed out. A brief description of a conventional cytogenetic study and a polymerase chain reaction for the diagnosis of oncohematological diseases is given. The paper also focused on the need for reference diagnostics of cytogenetic and molecular genetic studies in oncohematology. The article is addressed to the specialists in the field of laboratory genetics, clinical laboratory diagnostics, but may also be of interest to hematologists, pediatric oncologists and doctors of related branches.

The effectiveness of treatment for children with B-cell non-Hodgkin lymphomas (B-NHL) has reached 85–90% after the introduction of modern risk-adapted treatment regimens that involve risk group stratification based on tumor stage. Bone marrow involvement is traditionally evaluated using quantitative morphological analysis of tumor cells which has, however, a lower sensitivity compared to molecular genetic methods. In our study, we used next generation sequencing (NGS) to identify tumor-specific V-(D)/J-rearrangements of immunoglobulin genes which can be used as a marker for the evaluation of minimal disseminated disease (MDD) in children with B-NHL. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. Here we demonstrated that NGS allows detection of bone marrow involvement at a sensitivity of 10–6 in patients with Burkitt lymphoma, in whom standard morphological analysis failed to reveal the presence of tumor cells. The detection of molecular MDD can improve tumor staging and risk stratification in children with B-cell non-Hodgkin lymphomas.

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. Despite remarkable improvements in the treatment of pediatric acute lymphoblastic leukemia over last years, relapse still carries a poor prognosis with considerable morbidity and mortality. New immunotherapeutic approaches will change the way we treated our patients and the results we had. Blinatumomab is a bispecific T-cell-engaging antibody indicated for the treatment of relapsed/refractory B-cell lymphoblastic leukemia. The use of Blinatumomab in relapsed B-cell ALL has shown promising effects, especially as a bridging tool to hematopoietic stem cell transplantation. The therapy results for patients in the high risk group remain far from optimal due to refractoriness to chemotherapy, death from infectious complications, as well as acute chemotherapy toxicity. This article demonstrates the results of our experience of using Blinatumomab in children with the high-risk group relapsed B-cell ALL treated according to the ALL-REZ 2016 protocol. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. The efficacy and toxicity of innovational blocks with the use of fludarabine and clofarabine with subsequent Blinatumomab infusion are shown. And we present the efficacy of autologous CD3+ lymphocytes infusion once a week during the continuous blinatumomab therapy. Also we demonstrate the results of using Blinatumomab for the treatment of patients with refractory to the first line therapy relapsed B-lymphoblastic leukemia and patients with a second relapse of B-cell ALL. The first line therapy in these patients was carried out according to the ALL-REZ 2014 protocol. Our results show an improved reduction in minimal residual disease in patients with refractory relapsed B-cell ALL as well as an increased event free survival in children with the high-risk group relapsed B-cell ALL.

Introduction. Down syndrome (DS) is one of the most common chromosomal abnormalities. Children with DS have an increased risk of developing acute lymphoblastic leukemia (ALL). Standard therapy is usually used to treat ALL in children with Down syndrome, but the outcome is worse than in the general population. The high toxicity of therapy is a particular problem.The purpose of the study – in this study we presents a comparative analysis of the results of therapy for children with DS and ALL (DS-ALL) who received therapy according to the ALL-MB 2008 and ALL-MB 2015 protocols.Materials and methods. The analysis included primary ALL patients, aged 1 to 18 years, who received therapy in Russian and Belarusian clinics participating in the Moscow–Berlin study from January 2008 to December 2020. To analyze the treatment results of DS-ALL patients, a “comparison group” was formed from all patients with ALL registered in the database, using the matched-pair method. Survival was calculated using the Kaplan–Meier method, toxicity analysis and clinical-genetic parameters were investigated using nonparametric statistical methods.Results. The results of therapy both among patients with DS-ALL who received therapy according to ALL-MB 2008 and ALL-MB 2015 in comparison with “sporadic” ALL (non-DS-ALL) are unsatisfactory. The event-free survival rate of patients with DS-ALL in the ALL-MB 2008 group was 61 ± 7 % versus 85 ± 4 % among non-DS-ALL (p = 0.001), in the ALL-MB 2015 group – 67 ± 7 % versus 84 ± 4 % respectively. Overall survival in the ALL-MB 2008 group was 70 ± 7 % in children with DS versus 88 ± 4 % in non-DS (p < 0.001), in the ALL-MB 2015 group – 78 ± 6 % versus 92 ± 3 % respectively (p < 0.001). The risk of therapy-related death was higher in patients with DS: 20.6 ± 6.1 % versus 4.6 ± 2.2 %; p < 0.001 in the ALL-MB 2008 group and 18 ± 4.1 % versus 3.3 ± 1.3 %; p < 0.001 in the ALL-MB 2015 group, without a significant increase in the risk of relapse. The effectiveness of induction therapy among patients with DS treated according to ALL-MB 2008 versus children with DS-ALL treated according to ALL-MB 2015 was 80 % versus 92 % respectively (p = 0.018). The probability of achieving continuous complete remission was also lower in the ALL-MB 2008 group compared to ALL-MB 2015 – 57 % versus 75 %; p < 0.001 respectively. Thus, the results of treatment of DS-ALL according to the ALL-MB 2015 protocol were better than those according to the ALL-MB 2008.Conclusion. The results of therapy for patients with DS-ALL are still unsatisfactory today, this circumstance dictates the need for new approaches to optimize therapy. The main problem for these patients remains the high toxicity of therapy and the associated lethality. Further progress in the treatment of DS-ALL may be associated with the development of new approaches to concomitant therapy, the use of molecular-targeted drugs and immunotherapy, as well as with the study of the molecular genetic characteristics of this subgroup of patients.

Acute lymphoblastic leukemia (ALL) with translocation t(17;19)(q21-q22;p13) TCF3::HLF (E2A::HLF) accounts for less than 1% of childhood B-lineage ALL. Since the first description, patients with this type of ALL are stratified into high-risk group. The disease often has a unique clinical presentation with disseminated intravascular coagulation and hypercalcemia, that are uncommon in other types of B-lineage ALL. This type of ALL is characterized by an extremely poor prognosis despite intensive treatment and hematopoietic stem cell transplantation (HSCT) in the first remission. In the last decade, some new data on the mechanisms of leukemogenesis in this type of ALL made it possible to come closer to understanding the reasons for the high refractoriness to chemotherapeutic agents. Along with the reports on the possible effectiveness of the BCL-2 (venetoclax) and Aurora kinase A (alisertib) inhibitors in this type of ALL, cellular immunotherapy (various chimeric antigen receptor (CAR)-T cell constructs), anti-CD19 (blinatumomab) and anti-CD22 (inotuzumab ozogamicin) monoclonal antibodies appear promising in the treatment of this disease. To date, there are neither published data on direct comparisons of the effectiveness of these methods nor specific recommended therapy protocols for these patients. It is also unclear if the new therapeutic approaches can completely replace HSCT or they only increase relapse-free survival after it. Here, we review the data on this translocation published in the medical literature and present a case report of a 3-year-old boy with this type of leukemia, who did not respond to four-component induction therapy according to the ALL-MB 2015 Protocol and received anti-CD19 CAR-T therapy with the achievement of the first MRD (minimal residual disease)-negative remission, which lasted 11 months. After MRD-relapse and unsuccessful attempt at therapy with autologous CD19/CD22 CAR-T cells, the patient developed an extended isolated bone marrow relapse. He achieved the second MRD-negative remission after reinduction therapy with inotuzumab ozogomycin and received allogeneic HSCT from a related donor. At the time of writing, the patient is in complete molecular remission for 16 months after transplantation. The patient's parents have consented to the use of de-identified clinical information and photos of the patient in scientific research and publications.

Over the past years, the outcomes of patients with acute myeloid leukemia (AML) have significantly improved due to the use of intensive chemotherapy, more effective supportive therapy, and the availability of allogeneic hematopoietic stem cell transplantation. This article presents the outcomes of children with AML treated in accordance with the AML-MM-2006 protocol. Our study was approved by the Independent Ethics Committee of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. The study included 233 patients with a median age of 6.5 years (7 days – 18 years) who were stratified into the following risk groups: standard risk, intermediate risk, and high risk. The 5-year event-free survival (EFS) was 0.64 ± 0,14, 0.49 ± 0.05, and 0.43 ± 0.05 for standard-risk (n = 12), intermediate-risk (n = 106), and high-risk (n = 115) patients, respectively (p = 0.14), while the 5-year overall survival (OS) was 1.0 year, 0.7 ± 0.05 and 0.55 ± 0.05, respectively (p = 0.001). The OS in the entire cohort was 0.68 ± 0.032. Factors associated with poor prognosis included hyperleukocytosis, the presence of extramedullary lesions, and age < 1 year. The overall survival rates in these patient groups were 0.55 ± 0.08, 0.39 ± 0.09 and 0.49 ± 0.08, respectively. The worst prognosis was for patients with monosomy 7 and t(7;12) whose OS rates were 0.25 ± 0.2 and 0.4 ± 0.2, respectively. For non-responders and patients with relapsed AML, the OS was 0.33 ± 0.08 and 0.54 ± 0.06, respectively. Early death (before remission could be achieved) occurred in 4% of patients, and 3.8% of patients died in first remission. Sixtytwo percent of deceased patients died of disease progression. In the entire cohort of patients, the five-year EFS was 0.53 ± 0.047, the cumulative risk of relapse after 3 years of remission was 40%, the confidence interval was 23–89%.

Contemporary therapy of acute promyelocytic leukemia (APL) is based on the use of all-trans retinoic acid, which is effective against tumor cells harboring RARa gene rearrangements (most common – t(15;17)(q24;q21)/PML::RARa). In several studies, it was suggested to use typical immunophenotypic features of APL (HLA-DR-negativity, etc) for prediction of RARa rearrangements presence. In this study, we aimed to evaluate the range of genetic aberrations that could be found in the HLA-DR-negative acute myeloid leukemia (AML). Our study was approved by the Independent Ethics Committee of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. Among studied 806 pediatric AML patients, HLA-DR-negativity was found in 253 cases. Only in 45.4% of them t(15;17)(q24;q21)/PML::RARa was found, while in remaining 54.6% normal karyotype or other genetic aberrations without RARa involvement. Frequency of the most common immunophenotypic features of APL, such as total CD117, CD33 and MPO expression with the lack of CD34, was higher in patients with t(15;17)(q24;q21)/PML::RARa, although only two thirds of APL cases were found to have all these signs. Moreover, the percentage of cells positive or negative for mentioned antigens varied significantly in APL group. Thus we can conclude, that all “typical” immunophenotypic characteristics of APL including HLA-DR-negativity, are very unspecific and cannot be used for reliable prediction of presence of t(15;17)(q24;q21)/PML::RARa.

Introduction. Translocation t(12;21)(p13;q22) is one of the most common structural genetic abnormalities in childhood acute lymphoblastic leukemia (ALL). It cannot be detected by conventional G-banding, so a reverse-transcriptase polymerase chain reaction (RT-PCR) or fluorescent in situ hybridization are used for this purpose. The aim of the study was to evaluate the prognostic significance of qualitative and quantitative detection of ETV6-RUNX1 fusion gene transcript at various time points in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients. Materials and methods. ETV6-RUNX1 fusion gene transcript was revealed by both reverse-transcriptase PCR and quantitative real-time PCR (RQ-PCR) in 34 out of 166 (20.5 %) children with BCP-ALL. Qualitative ETV6-RUNX1-positivity at days 36 and 85 led to unfavorable outcome (lower event-free survival –EFS and higher cumulative incidence of relapse – CIR). While ETV6-RUNX1 status at day 15 did not allow to divide patients with different outcomes. By ROC curve analysis we determined threshold levels (TL) for ETV6-RUNX1/ABL1 ratio at days 0, 15, 36 and 85. Afterwards we adjusted obtained results to 10-fold scale. Results. So practically applicable TL were as follows 500.0 %, 1 %, 0.1 % и 0.01 % for days 0, 15, 36 and 85, respectively. EFS and CIR were both worse in patients with ETV6-RUNX1/ABL1 ratio equal or above defined TL. Moreover, initial ratio ≥500,0 % corresponded to delayed blast clearance at days 15 and 36. We showed good qualitative (84.8 %) and quantitative (R 2 = 0.953) concordance between ETV6-RUNX1/ABL1 ratio and MRD data obtained by flow cytometry at days 15, 36, 85. Of note, defined TL for ETV6-RUNX1/ABL1 at days 15, 36, 85 were equal to prognostically important levels for flow cytometry MRD. Conclusion. Thus, qualitative detection and quantitative value of ETV6-RUNX1 fusion gene transcript showed prognostic significance in the course of treatment in children with BCP-ALL. Based on these results we propose standardization approaches for Moscow – Berlin ALL study group.

We assessed the prognostic significance of IKZF1 gene deletions in 141 pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) on Russian multicenter trial in pediatric clinics of Ekaterinburg and Orenburg. IKZF1 deletions were estimated by multiplex ligation-dependent probe amplification. IKZF1 deletions were revealed in 15 (10.6 %) patients. IKZF1 deletions were associated with age older than 10 years (p = 0.007), initial white blood cell count higher than 30 × 109/l (p = 0.003), t(9;22)(q34.q11) (p = 0.003) and delayed blast clearance: М3 status of bone marrow at day 15 of remission induction (p = 0.003), lack of hematological remission at day 36 (p < 0.001) and high levels of minimal residual disease at days 15, 36 and 85 (p = 0.014; p < 0.001; p = 0.001 correspondingly). Patients with IKZF1 deletions had significantly lower event-free survival (EFS) (0.30 ± 0.15 vs 0.89 ± 0.03; p < 0.001) and overall survival (OS) (0.44 ± 0.19 vs 0.93 ± 0.02; p < 0.001), while cumulative incidence of relapse was higher (0.67 ± 0.18 vs 0.07 ± 0.02; p < 0.001). In the multivariate analysis IKZF1 deletions were associated with decreased EFS (hazard ratio (HR) 4.755; 95 % confidence interval (CI) 1.856–12.185; p = 0.001), and OS (HR 4.208; 95 % CI 1.322–13.393; p = 0.015), but increased relapse risk (HR 9,083; 95 % CI 3.119–26.451; p < 0.001). IKZF1 deletions retained their prognostic significance in the intermediate risk group patients (p < 0.001), but not in standard or high-risk groups. Majority of IKZF1 deletions – 12 (80 %) of 15 – were revealed in the “B-other” group (n = 83). In this cohort of patients IKZF1 deletions led to inferior EFS (HR 6.172; 95 % CI 1.834–20.767; p = 0.003) and higher relapse rate (HR 16.303; 95 % CI 3.324–79.965; p = 0.015). Thus, our results showed that IKZF1 deletions are independent risk factor in BCP-ALL patients.

In long-term bone marrow cultures derived from tumor necrosis factor-deficient mice the total cell production and the total duration of hemopoiesis are increased (the latter is comparable with mouse life span). Telomerase activity in cells of nonadherent fraction of long-term bone marrow cultures from tumor necrosis factor-deficient mice increases with time and peaks after 1-year culturing. Karyotyping of nonadherent and adherent cells of long-term bone marrow cultures revealed instability of nonadherent cells and hyperploidy of the stromal sublayer cells, which attested to the presence of a neoplastic transformation. However, cell differentiation is not blocked in long-term bone marrow cultures. The nonadherent fraction of long-term bone marrow cultures from tumor necrosis factor-deficient mice cannot be cultured without exogenous growth factors; in the presence of growth factors the cells proliferate, but cannot be passaged; stromal sublayer cells cannot be passaged as well. Intraperitoneal and intravenous injections of nonadherent cells to recipients with normal and radiation-attenuated immunity induced no tumor growth. Hence, peculiar dynamics of long-term bone marrow cultures from tumor necrosis factor-deficient mice cannot be explained by neoplastic transformation.

The clinical condition and treatment of a driver who was overexposed to an unshielded 137 Cs source that was in his truck for about 5 months is described. Bone marrow and blood changes are presented as he progressed from pancytopaenia to myelodysplasia with features of refractory anaemia and then anaemia with an excess of blasts and transformation to frank myelomonocytic leukemia. He died about 22 months after the discovery of the source from an overwhelming hepatitis B infection concurrent with immunosuppression following cytostatic treatment for the leukaemia.

The discovery in 1995 of a 48 GBq (1.3 Ci) 137 Cs source in the door pocket of a truck is described. The truck driver had been seriously irradiated to an average whole-body dose of approximately 8 Gy. His exposure was, however, inhomogeneous and protracted over about 5 months. The reconstruction of his dose is described in this paper. A measurement by electron spin resonance on a tooth provided a fixed point cumulative dose estimate of 7.7 Gy and from this the dose distribution to other parts of his body was calculated by considering the source-site distances for a driver in a seated posture. Biological dosimetry by extensive conventional and fluorescence in situ hyridisation cytogenetic studies was also carried out on peripheral blood lymphocytes. Good agreement in dose estimates from both cytogenetic endpoints and the physical calculations was obtained.

117 cases of infant acute lymphoblastic leukemia without Down syndrome (aged from 1 to 365 days) were included in the current study. Rearrangements of 11q23 (MLL) were revealed in 74 (63.2 %) patients. Among this group the most common rearrangement was t(4;11) Введение Ген MLL (myeloid/lymphoid or mixed lineage leukemia), располагающийся на длинном плече 11-й хромосомы в регионе 11q23, состоит из 37 экзонов [1] и кодирует гистоновую метилтрансферазу. Перестройки с вовлечением района 11q23 и участием гена MLL встречаются примерно в 10 % всех случаев острого лимфобластного лейкоза (ОЛЛ) и в 3 % случаев острого миелоидного лейкоза (ОМЛ) [2, 3]. Наиболее частыми генами-партнерами MLL являются AF4, MLLT1 (ENL), MLLT3 (AF9), MLLT4 (AF6), MLLT10 (AF10). Суммарно они встречаются более чем в 80 % всех случаев MLL-позитивных острых лейкозов у детей и взрослых [4]. Среди всех возрастных категорий наиболее часто перестройки гена MLL выявляются у детей первого года жизни с ОЛЛ [5, 6], где частота их выявления может достигать 79 % [7, 8]. Несмотря на относительную редкость ОЛЛ у детей данной возрастной группы, он представляет большой интерес для исследователей как из-за неблагоприятного прогноза [9-13], так и из-за особенностей биологии опухоли и наличия широкого спектра перестроек гена MLL [6, 14], которые могут возникать in utero [10, 15]. По механизму образования все перестройки 11q23 (MLL) можно разделить на инверсии, делеции, внутренние тандемные повторы, вставки, транслокации с вовлечением 3 и более хромосом, а также комбинации различных вариантов, например транслокация с одновременной делецией части гена MLL [4]. Наиболее часто встречаются реципрокные транслокации, на долю которых приходится более 80 % всех случаев перестроек 11q23 (MLL) как при ОЛЛ, так и при ОМЛ [16]. На сегодняшний день на молекулярном уровне охарактеризовано 64 транслокации [4]. Столь большое число перестроек затрудняет проведение молеку лярно-генетической диагностики и верификацию генапартнера[12]. Необходимо отметить, что особую ценность приобретает выявление перестроек гена MLL в свете того, что они представляют собой удобную мишень для мониторирования минимальной остаточной болезни (МОБ) методом полимеразной цепной реакции (ПЦР). Ранее, в рамках протокола MLL-Baby [17], нам удалось показать прогностическую значимость выявления МОБ данным способом [18], а также раннего достижения молекулярной ремиссии у пациентов с наличием различных перестроек гена MLL [19]. Целью данной работы являлась характеристика перестроек гена MLL (11q23) у детей первого года жизни с ОЛЛ. Материалы и методы Проанализированы данные 117 пациентов с ОЛЛ без синдрома Да уна в возрасте от 1 до 365 дней (медиана 211 дней). Их подробная характеристика представлена в табл. 1. Диагноз ОЛЛ у станавливался на основании стандартных цитоморфологических показателей [20], дополненных данными иммунофенотипирования согласно критериям группы EGIL (European Group for the Immunological Characterization of Leukemias) [21]. Все пациенты получали терапию по одному из следующих химиотерапевтических протоколов: MLL-Baby, ALL-MB-2002, ALL-MB-2008, ALL-BFM 90, ALL-MB 91, CO ALL в детских онкогематологических клиниках Российской Федерации (q21;q23)/, MLL-AF4 detected in 63.5 % cases, less frequently was found t(11;19)(q23;p13)/MLL-MLLT1 (in 18.9 % cases), t(10;11) (p12;q23)/MLL-MLLT10 and t(1;11)(p32;q23)/ML L-EPS15 (each one in 6.8 %), t(9;11)(p22;q23)/MLL-MLLT3 in 2.7 %. Children under 6 months of age had significantly higher incidence of 11q23 (ML L) rearrangements in comparison with infants olde r than 6 months (84.0 % vs. 47.8 %, p

Culturing of suspension fraction of a long-term bone marrow culture derived from tumor necrosis factor (TNF)-deficient mice for 75 days produced cells forming adherent cell strains. The cells of all strains expressed RNA of various stromal differentiating markers in various combinations. The cells of many strains simultaneously expressed genes encoding products characteristic of different differentiation lineages. The derived strains maintained hemopoiesis for 10 weeks. RNA-analysis of gene expression by cells of these strains showed that they express a set of various growth factors and cytokines. It was hypothesized that suspension fraction of long-term bone marrow culture derived from TNF-deficient mice includes immature stromal precursor cells, which were never detected in long-term bone marrow culture derived from wild-type mice.

Detection of the t(15;17) or PML/RARa rearrangment is important for analysis of clinical trials involving patients with suspected acute promyelocytic leukemia (APL). According to the majority of reports the translocation t(15;17)(q22;ql2–21) is detected in 70% to 100% of cases [1]. PML/RARa fusion transcript from add(15q) is beleived to play crucial role in leukemogenesis of this type of leukemia [2]. It’s presence remains the predictor of favourable prognosis and good response to modern modalities of therapy, especially ATRA [3].

A comparison of chromosomal abnormalities in bone marrow leukaemic cells and of stable and unstable aberrations in lymphocytes of patients with hematological malignancies who live in areas with or without contamination by the Chernobyl nuclear accident has been made using FISH and G-banding. Healthy residents of these areas comprised the control group. No systematic cytogenetic differences of leukaemic cells between patients from contaminated and uncontaminated areas were observed. Lymphocyte aberrations, however, were generally higher in all subjects from contaminated areas. Comparison has been made with specific cytogenetic features of leukaemic cells and a high level of stable aberrations in lymphocytes of patients with secondary leukaemias that had developed after chemo- and/or radio-therapy.