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Aim: to determine molecular diagnostics routine for different tissue samples in angioimmunoblastic T-cell lymphoma. Materials and methods. Molecular studies were performed for 84 primary AITL patients. The median age was 61 year (29-81); the male to female ratio was 48/36. T-cell and B-cell clonality was assessed by GeneScan analysis of rearranged T-cell receptor (TCRG, TCRB) and immunoglobulin heavy chain genes. For the quantitative determination of cells with RHOA G17V mutation real - time polymerase chain reaction (PCR) with allele - specific LNA modified primers was used. Results. In lymph nodes rearrangements of T-cell receptor genes were determined in 76 (90.5%) of 84 patients and were absent in 8 (9.5%) cases. Identification of the same clonal products of the TCRG and TCRB genes in the lymph node and in peripheral blood and/or bone marrow indicated the prevalence of the tumor process and was observed in 64.7% of patients. Clonal products in peripheral blood and/or bone marrow different from those in the lymph node indicated reactive cytotoxic lymphocyte population and were noted in 58.8% of AITL cases. Simultaneous detection of T- and B-cell clonality in the lymph node was observed in 20 (24.7%) of 81 patients. Cells with RHOA G17V mutation were detected in lymph node in 45 (54.9%) of 82 patients. The use of allele - specific PCR with LNA modified primers revealed presence of the tumor cells in peripheral blood in 100% and in bone marrow in 93.9% of patients with G17V RHOA mutation in the lymph nodes. Conclusion. The validity of different molecular assays performed on certain tissue samples for the diagnosis of angioimmunoblastic T-cell lymphoma has been evaluated. Quantitative allele - specific PCR assay for RHOA G17V mutation based on LNA modified primers possesses sufficient sensitivity for tumor process prevalence evaluation and minimal residual disease monitoring.

Objective. To analyze treatment results of 172 patients with acute myeloid leukemia (AML) aged 18-60 years in National Medical Research Center for Hematology of MHRF. Materials and methods. Inductive and consolidation program for 139 (80%) patients was based on a standardized protocol: 4 courses “7+3” with different anthracycline use (2 courses of daunorubicin, idarubicin, mitoxantrone) and continuous use of cytarabine on the second inductive course. In 20% of patients cytarabine courses at the dose of 1 g/m2 2 times a day for 1-3 days combined with idarubicin and mitoxantrone were used as two consolidation courses. Allogenic bone marrow transplantation was performed in the first complete remission (CR) period in 40% of patients. Results. The frequency of CR achievement in all patients was 78.6%, refractory forms were observed in 13.9% of patients, early mortality - in 7.5% of patients. Seven-year overall survival (OS) rate was 40.7%, relapse free survival (RFS) - 43.2%. When estimating effectiveness depending on cytogenetic risk group it was demonstrated that 5-year OS and RFS in patients with translocation (8; 21) cannot be considered as satisfying, it accounted for 50 and 34%, respectively. At the same time in patients with 16th chromosome inversion (inv16) these characteristics accounted for 68.6 and 63.5%. Acquired results forced reconsidering of the consolidation program in AML patients of this subgroup. The median time to allogenic blood stem cells transplantation (allo-BSCT) in patients with first CR was 6.5 months that was taken as a reference point in landmark analysis of patients in whom allo-BSCT was not performed. Landmark analysis showed that in AML patients of favorable prognosis group allo-BSCT does not significantly reduce the probability of relapse (0 and 36%) and does not influence RFS (33 and 64%). In patients of border-line and poor prognosis allo-BSCT significantly reduces relapse probability (26 and 66%; 20 and 100%) and significantly increases a 7-year RFS (68.7 and 30%; 45.6 and 0%). Allo-BSCT also results in significant RFS increase and reduces the probability of relapse (25 and 78%) in patients in whom CR was achieved only after the second induction course. At the same time allo-BSCT does not influence patients who achieved CR after the first treatment course: 55 and 50%. Conclusion. Multivariate analysis showed that cytogenetic risk group (HR=2.3), time of CR achievement (HR=2.9), and allo-BSCT transplantation (HR=0.16) are independent factors for disease relapse prognosis after achieving CR.

Purpose of the study. A comparative evaluation of the effectiveness of different therapeutic strategies in patients with polycythemia vera (PV) and essential thrombocythemia (ET). Materials and methods. Patients with PV or ET, diagnosed according to the criteria WHO 2016 were included in the study. The primary endpoint - 6 months of therapy (clinical-hematological and molecular responses). The secondary endpoint - 12 months of therapy (clinico-hematologic, molecular, histological responses). Sixty three patients were included in the analysis: the first group consisted of 33 patients who received the therapy with ce-pegiterferone alpha-2b (ce-pegalpha-INF-α-2b), 10 of them received previous treatment; the second group - 23 patients btained hydroxycarbamide; the third group - 7 patients were treated with recombinant interferon alpha therapy (rINFα). In comparison groups, differences in age were revealed: patients receiving hydroxycarbamide therapy were older. Phlebotomy occurred in 36% of patients in the first group, 9% in the second group, and 14% in the third group. Results. By the 6th month of therapy, 43% of the patients receiving the ce-pegalpha-INF-α-2b had complete clinical-hematologic response, 36% had partial clinical-hematologic remission and stabilization of the disease was established in 21% cases. No disease progression occured. By the 12th month of therapy, statistically significant differences in terms of efficacy between the different therapeutic groups (p = 0.2462, Fisher's exact test). In all three groups, the allelic load of JAK2V617F decreased: from 50 to 19%, from 22.3 to 15.8%, from 50 to 7.19%, respectively. The lower the allele load positively correlated with better response to therapy, which was observed in all analyzed groups. Hematologic adverse events (AEs) were more frequently observed in patients receiving ce-pegalpha-INF-α-2b therapy. Local reactions developed on 3-7 days of therapy as a hyperemic macula at the injection site. Both these reactions and hair loss did not influence on patient’s condition. In the second group (patients with hydroxycarbamide therapy) there were changes in the skin and mucous membranes: dry skin, stomatitis, and in older patients new keratomas appeared. The flu-like syndrome was the most common adverse event associated with the therapy of ce-pegalpha-INF-α-2b, which fully relived during the first month of therapy. There was only one case with the flu-like syndrome we observed at the 11th month of therapy. As a rule, the biochemical blood test changes did not influence on patient’s condition, were mostly associated with dietary violations, had a tendency to self-resolution and did not require medical interventions. Serious AEs were reported in one case - pulmonary embolism in a patient treated with rINFα. The reasons for the therapy discontinue in group 1: toxic hepatitis, intolerance, by the request of the patient, inadequate efficacy of therapy; in group 2: skin toxicity, in group 3: thromboses. The conclusion. Treatment of ce-pegalpha-INF-α-2b in patients with PV and ET is highly effective - the most patients pbtained clinical and hematological responses. There were no statistically significant differences in these parameters in comparison with hydroxycarbamide and rINFα. The use of the ce-pegalpha-INF-α-2b had an acceptable safety profile. The estimated therapeutic dose should be calculated according to body weight. To reduce the frequency of hematologic AE, titration of the drug dose is required.

B-cell acute lymphoblastic leukemia (B-ALL) is a diverse group of malignant blood disorders both with regard to the biological properties of the tumor and to therapeutic approaches. Immunophenotyping, molecular genetic techniques, whole-genome sequencing characterize B-ALL as a very diverse group for sensitivity to chemotherapy and prognosis. We present three clinical cases of patients with B-ALL and expected good response to standard therapy, in whom standard protocol treatment failured: refractoriness, persistence of minimal residual disease (MRD), and progression (MRD increase). The remission in these patients was achieved after chemotherapy change to immunological targeted therapy. Nowadays a unified therapeutic approach to all primary patients of the B-ALL is considered generally outdated. Great efforts are carrying out to develop molecular genetic classifications. The molecular dissection of subtypes of B-ALL goes on, and new protocols for selective treatment with targeting are clearly outlined for each subtype of B-ALL.

Thalassemia and qualitative hemoglobinopathy are hereditary disorders of Hb synthesis that lead to change in the Hb conformation or a decrease in the synthesis of structurally normal Hb, and consequently, to erythron pathology. Many variants of Hb are unstable or have altered affinity for oxygen, and, in heterozygous form can be associated with clinical and hematological manifestations (hemolytic anemia, hypochromic microcytic anemia, erythrocytosis). HbD-Punjab [β121 (GH4) Glu → Gln; HBB: C.364G> C] is variant of Hb carrying the amino acid substitution in the 121 position of β-globin chain. In all cases reported so far, patients with HbD-Punjab/β+-thalassemia (IVSI+5 G-C) combination experienced typical thalassemia with hypochromic microcytosis. HbD-Punjab was detected by electrophoresis from 37 to 94% of total Hb. The article describes rare clinical case of the cohabitation of HbD-Punjab/β+-thalassemia (IVSI+5 G-C) in a patient with homozygous variant of Gilbert's syndrome observed in AS Loginov Moscow Clinical Scientific Center.

Nodal anaplastic ALK-negative large cell lymphoma (nALCL, ALK-) is a Т-cell lymphoma that is characterized by aggressive clinical course and low sensitivity to СНОР (cyclophosphamide, doxorubicin, vincristine, prednisolone) and other chemotherapy regimen. In the article we present a literature review and describe our clinical case of nALCL, ALK-. For the first time a combination of Brentuximab vedotin with modified program NHL-BFM-90 was used as a first-line therapy. As a result of immunochemotherapy a complete antineoplastic effect was obtained. For consolidation of this effect high-dose chemotherapy with following autologous blood stem cell transplantation was performed. The chosen treatment tactics allowed to achieve a complete remission in a medium risk group patient.

Aim. To assess the results of following up patients with chronic myeloid leukemia (CML) and a deep molecular response (MR) without tyrosine kinase inhibitor (TKI) therapy. Subjects and methods. The reasons for TKI discontinuation in 70 patients with CML and a deep MR of more than 1 year’s duration were adverse events, pregnancy, and patients’ decision. Information was collected retrospectively and prospectively in 2008-2016. Results. The median follow-up after TKI therapy discontinuation was 23 months (2 to 100 months). At 6, 12 and 24 months after TKI therapy discontinuation, the cumulative incidence of major MR (MMR) loss was 28, 41 and 48%, respectively; the survival rates without TKI therapy were 69, 50, and 39%, respectively. MMR loss was noted in 28 (88%) patients at 12 months; it was not seen without TKI therapy at 2-year follow-up. Deaths due to CML progression were absent. The Sokal risk group was a reliable factor influencing MMR loss (p ≤ 0.05). The cumulative recovery rate for deep MR after resumption of TKI use was 73 and 100% at 12 and 24 months, respectively, with a median follow-up of 24 months (1 to 116 months). Deep MR recovered at a later time when the therapy was resumed more than 30 days after MMR loss. Conclusion. Safe follow-up is possible in about 50% of the patients with CML and stable deep MRs without TKI therapy. The introduction of this approach into clinical practice requires regular molecular genetic monitoring and organizational activities. Biological factors in maintaining remission after TKI discontinuation need to be separately studied.

Aim. To investigate the nature of mutations in exons 4 and 5 of the uromodulin (UM) gene, including in the area encoding the domain of 8 cysteines (D8C), in patients with multiple myeloma (MM) with the secretion of monoclonal light chains (LC) in cast nephropathy (CN) and without kidney injury. Subjects and methods. The investigation enrolled 24 patients in MM remission, who were observed to have monoclonal LC secretion at onset. Group 1 included 14 patients with CN; Group 2 consisted of 10 patients with normal renal function (a comparison group). The compared groups did not differ in the number of serum and urinary monoclonal LCs. Genomic DNA was extracted from the peripheral blood samples of patients. The nucleotide sequence of exons 4 and 5 of the UM gene was determined by the Sanger method. Results. No differences were found in the frequency of polymorphisms depending on the severity of kidney injury. The missense mutation p.142R>R/Q in the UM gene, which had not been previously described, was discovered. Conclusion. The patients with MM were not found to have statistically significant differences in the frequency and nature of polymorphisms of exons 4 and 5 in the UM gene, including in the area encoding D8C, in CN without kidney injury.

Breast implant-associated anaplastic large-cell lymphoma will be identified as a separate nosological entity in the 2017 adapted WHO classification due to differences in its clinical presentations, pathogenesis, and prognosis with those of nodal and cutaneous anaplastic large-cell lymphomas. The paper gives a review of the literature and describes the authors’ own clinical case of common breast implant-associated anaplastic large-cell lymphoma involving breast tissue, axillary lymph nodes, anterior chest muscles, and bone marrow. The treatment policy chosen by the authors could achieve complete remission.

The aim of the present paper was to evaluate the clinical features and risk of thrombotic events (TE) in patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF), depending on the molecular characteristics of disease. Clinical data and laboratory parameters were analyzed in 50 ET patients and 50 PMF ones who had been followed up at the Department for Standardization of Treatments, National Research Center for Hematology, Ministry of Health of the Russian Federation, from February 2015 to September 2016. The patients with ET and those with PMF were found to have a high risk of TE. The risk for TE in the patients with ET is higher (24% in the entire group) than in those with PMF (14% in the study group). In ET, there is a high thrombosis risk in the detection of JAK2 and CALR gene mutations as compared with triple-negative cases. The PMF patients with JAK2 V617F mutations are at high risk for TE compared to those who are CALR mutation carriers and in triple-negative cases. There was no significant association of TE with high thrombocytosis. A factor, such as age, was found to be of no negative prognostic value in the patients with PMF.

Double-hit lymphoma (DHL) is a rare aggressive B-cell lymphoma with concomitant c-MYC, BCL2 or BCL6 gene rearrangements, which is characterized by the high frequency of extranodal lesions and by resistance to chemotherapy. The median survival does not exceed 18 months in patients with this disease. The majority of DHL is represented by с-MYC/BCL2 cases. The combination of c-MYC/BCL6 occurs rarely (5—8%). The paper describes a case of DHL with concomitant c-MYC and BCL6 gene rearrangements, which mimics diffuse large B-cell lymphoma, leg-type.

Aim. To evaluate the efficiency of interferon (IFN) therapy in patients with essential thrombocythemia (ET) and polycythemia vera (PV). Subjects and methods. A total of 61 patients (41 with ET and 20 with PV) were examined. Prior to study enrolment, 44 (72%) patients with ET or PV received one or other therapy (aspirin was not taken into account). The mean Jak2V617F mutant allele at baseline was 23% (6—54%) in the patients with ET and 40% (11—88%) in those with PV. The median time from diagnosis to enrollment was 49 months. Results. The paper presents the clinical and molecular findings of long-term INF-α therapy in patients with ET or PV. The median follow-up was 52 months. Recombinant IFN-α2 showed its ability to induce complete hematologic remission (ET (76%), PV (70%)) and a complete molecular response. 22 (69%) out of 32 patients were noted to have a smaller number of cells with the Jak2V617F mutation. In the patients with PV and in those with ET, the relative reduction in the proportion of cells with the Jak2V617F mutant gene averaged 85% and 56% of the baseline values, respectively. There was a reduction in the proportion of cells expressing the Jak2V617F mutation in both the ET (from 12 to 2.2%; p=0.001) and PV (from 32.7% to 3.2%) groups (р=0.001). Ten (31%) patients achieved a deep molecular remission (≤2% Jak2V617F allele); among them, 5 patients were not found to have Jak2V617F mutation. The obtained molecular response remained in 7 of the 10 patients untreated for 11 to 86 months. The long-term treatment with IFN-α led to normalization of the morphological pattern of bone marrow in 5 of the 7 PV or ET patients. Conclusion. Significant molecular remissions achieved by therapy with recombinant interferon-α2 confirm the appropriateness of this treatment option in in the majority of patients with ET or PV.

Aim. To identify the clinical features of latent polycythemia vera (PV) as an independent nosological entity. Subjects and methods. The investigation enrolled 81 patients (50 with extensive (manifest) PV and 31 with latent PV) who had visited the Outpatient Department, Hematology Research Center, Ministry of Health of Russia, in 2014 to October 2015. Results. The gender distribution of the patients was statistically comparable in the analyzed groups. The patients with manifest PV were slightly older than those with latent PV: the median age in the compared groups was 56 and 44 years, respectively. Red blood cell counts, hemoglobin concentrations, and packed cell volume were higher in the patients with manifest PV. Blood platelet counts were higher in the latent PV group. There were no differences in the number of white blood cells in the compared groups. All the patients were JAK2 V617F mutation carriers. The JAK2 allele load was significantly higher in the manifest PV group than in the latent PV group. The compared patient groups differed in the rate of thromboses in the history or at diagnosis. In the patients with latent PV, thromboses were detected in 38% of cases versus 16% in those with manifest PV. In latent PV, there were mainly venous thromboses; abdominal vascular thromboses were diagnosed with a high frequency. Arterial thromboses were revealed in only 2 cases. Conclusion. Chronic myeloproliferative disease that is characterized by the JAK2 V617F mutation, borderline hemoglobin counts, and morphological features of a bone marrow trephine biopsy specimen, which are specific for PV, is an independent PV variant, namely: latent PV.

In the past decade, a notable advance has been made in the understanding of the pathogenesis of NK/T-cell lymphomas; however, their diagnosis remains difficult because of their rarity and clinical and morphological variabilities. The paper generalizes the ten-year experience of the Hematology Research Center, Ministry of Health of Russia, in diagnosing and treating hepatosplenic T-cell lymphoma (HSTL), considers the problems of differential diagnosis with other hematological diseases occurring with similar clinical and laboratory symptoms, and lays down current approaches to the diagnosis and treatment of this condition. A clinician’s view of the problem of diagnosis and treatment of this disease is given. HSTL is shown to be a heterogeneous group of diseases differing in a T-cell receptor chain gene rearrangement, the clinical course of the disease, and overall survival (OS). According to our data, 3-year OS was 12%; the median survival was 26 months. Two-year OS for γδ and αβ HSTL was equal to 25 and 70%, respectively. The difference in OS for the variants of HSTL failed to reach statistical significance (because the sample might be insufficient).

In the past decades, there has been an increase in the incidence of malignancies, including polyneoplasms. Composite synchronous lymphoma characterized by the simultaneous development of several lymphoproliferative diseases occurs very rarely. This paper describes a case of development of 3 different lymphomas in the same patient. Aggressive T-cell lymphoma and indolent B-cell lymphoma are diagnosed during lifetime; primary central nervous system lymphoma is verified only by examination of autopsy material. Three different lymphomas could be identified by an integrated assessment of their clinical picture and by current diagnostic techniques, such as histology, immunohistochemistry, and molecular genetics.

Aim. To determine the immunoglobulin variable heavy chain (IgVH) genes and their somatic mutations and to compare these data with the clinical and laboratory parameters of patients and the outcomes of the disease. Subjects and methods. The investigation enrolled 24 patients (9 men and 15 women whose age was 32 to 77 years (median age, 60 years) with splenic marginal zone B-cell lymphoma (SMZBCL). The latter was diagnosed on the basis of histological and immunohistochemical examinations of a bone marrow trephine biopsy specimen, immunophenotyping of peripheral lymphoid cells or bone marrow aspirates. Results. The mutational status of the IgVH genes was analyzed in all the 24 patients. It was found that the tumor cells contained mutated IgVH genes (the VH1-family genes participated in most cases) in 15 (62.5%) patients with SMZBCL and unmutated ones in 9 (37.5%). There was a tendency towards more common tumor progression in patients with unmutated IgVH genes than in those with mutated ones. Conclusion. The presence of SMZBCL cases with both mutated and unmutated IgVH genes and the higher frequency of the VH1-2 gene are likely to indicate the molecular heterogeneity of the origin of this lymphoma.

The term mastocytosis (MC) encompasses a group of rare diseases characterized by the tumorous proliferation of clonal mast cells and the infiltration of one or several organs. The clinical picture of MC is extremely diverse from skin lesions that can spontaneously regress to the aggressive disease forms associated with organ dysfunction and short survival. Nowadays, the 2008 WHO classification identifies 7 MC subtypes. The disease is diagnosed on the basis of its clinical manifestations and detection of tumorous mast cell infiltrations via morphological, immunohistochemical, immunophenotypic, genetic, and molecular examinations. Abnormal mast cells are characterized by the atypical morphology and pathological expression of CD25 and CD2 antigens. Enhanced serum tryptase activity is a common sign in all MC subtypes. More than 90% of the patients have D816V KIT mutations in the mast cells. This paper reviews the literature. Three cases are described as a clinical example in patients with different MC subtypes.

AIM. To determine the clinical features of multiple primary tumors (MPT) in patients with hemoblastoses, to develop treatment policy for synchronous and metachronous tumors, and to determine the impact of chemotherapy for one disease on the course and prognosis of another one. MATERIALS AND METHODS. The investigation included 20 patients with multiple primary synchronous and metachronous myeloid and lymphoid tumors, who had been followed up at the Outpatient Department of the Hematology Research Center, Ministry of Health of the Russian Federation. The distribution of patients by nosological entities was as follows: 17 (85%) patients with myeloproliferative diseases (MPDs) concurrent with lymphoproliferative diseases (LPDs) and 3 (15%) with two types of MPD. A special group comprised 3 patients who successively developed 3 malignant diseases: cancer/B-cell chronic lymphocytic leukemia (B-CLL)/Ph-positive chronic myeloid leukemia (Ph+CML); cancer/polycythemia vera (PCV)/B-CLL; cancer/essential thrombocythemia (ETC)/multiple myeloma (MM). RESULTS. The Outpatient Department of the Hematology Research Center, Ministry of Health of the Russian Federation, followed up 20 patients with synchronous and metachronous tumors in 1996 to 2013. The patients' age was 42 to 82 years (64 years). The female/male ratio was 1:1.2. Metachronous tumors were 1.5-fold higher than synchronous ones. The time to detection of secondary hemoblastosis averaged 3.3 years; the longest interval was 14 years; the mean coexistence of 2 tumors was 4.8 years (1-11 years). The total length of the follow-up was 8 years (1-19 years). Among them, there were 17 (85%) patients with 2 chronic hematologic tumors with a myeloid or lymphoid phenotype; 3 (15%) of the 20 patients had 3 malignant diseases (cancer/ B-CLL)/Ph+CML, cancer/PCV/B-CLL, cancer/ETC/MM. In the group of 17 patients, 13 (76%) were diagnosed as having Ph-negative MPDs (PCV in 4 patients, primary myelofibrosis in 4, ETC in 4, undifferentiated MPD in1) and 4 (24%) patients had Ph+CML. This patient group was found to have the following LPDs: CLL in 5 (30%), hairy cell leukemia in 1 (5%), paraproteinemic hemoblastoses in 11 (65%). MPD preceded LPD in 8 (47%) patients; the development interval between two tumors averaged 6 years (1 to 14 years). LPD preceded MPD in 3 (18%) patients; the interval averaged 5 years (2 to 17 years). MPD and LPD appeared synchronously in 6 (35%) patients. CONCLUSION. The fact that 2 malignancies or more may occur in one patient determines the need for a careful follow-up of patients with blood system diseases. The activity of one hematologic disease or another is a leading criterion for choosing a therapeutic tactic.

Introduction. Human cytomegalovirus (Orthoherpesviridae: Herpesvirales: Cytomegalovirus: Cytomegalovirus humanbeta 5) (HCMV) is one of the most commonly detected viruses in recipients of allogeneic hematopoietic stem cell (allo-HSCT) transplants. However, the emergence of resistance to antiviral drugs such as ganciclovir (GCV) poses a challenge in managing these patients. This study aims to investigate the prevalence and impact of mutations in the HCMV UL97 gene associated with resistance to GCV on the course of infection among allo-HSCT patients. Materials and methods. The study examined the association between UL97 mutations and the clinical course of HCMV infection in allo-HSCT patients. Genetic sequencing was performed to identify mutations, and their impact on viral replication and resistance to GCV was assessed. Results and discussion. Six mutations were identified (D490A, T502A, C592G, C592F, E596G, C603W). C592G, C592F, E596G, and C603W are associated with resistance to antiviral drugs, while D490A and T502A described for the first time. When comparing patients with wild-type and those carrying the mutant variant, several parameters of peripheral blood were significantly lower in the former group. The median time to peak viral load following allo-HSCT, duration of viremia, and rate of virological response to high-dose therapy also differed significantly between the two groups. Conclusion. It was shown that approximately one third (4 out of 14) of allogeneic stem cell transplant recipients had mutations associated with resistance to GCV. Patients carrying the mutant variant of HCMV had longer viremia and took longer to achieve a negative virological test result after starting high-dose therapy. Performing genotyping may help make more evidence-based therapeutic decisions.

AIM: To characterize the genotype and genotype-phenotype correlations in patients with Gaucher disease (GD) in the Russian Federation. Materials and methods. One hundred adult patients with GD type 1 were examined. Their clinical study encompassed the evaluation of the severity of osteoarticular lesions from instrumental findings. An allele-specific real-time polymerase chain reaction assay was used to screen four most common acid Β-glucoside gene (GBA) mutations (N370S, 84GG, L444P, IVS2+1)/RESULTS: The N370S mutation and the N370S/? genotype where the second allele was presented with the mutation outside the 4 most common GBA gene mutations were found in the Russian patients with GD. Analysis of the clinical manifestations of the disease revealed no association between the genotype under examination and the severity of osteoarticular lesions and supported the unfavorable role of splenectomy (SE) in the development of severe bony disease/CONCLUSION: SE should be carried out in patients with unclear cytopenia and splenomegaly after the diagnosis of GD is excluded. The GD patients undergoing SE should receive emergency enzyme replacement therapy to prevent severe osteoarticular lesions and an irreversible orthopedic defect.

Aim. To specify trends in clinical and laboratory manifestations of virus hepatitis B and C (HBV and HCV) in patients with blood diseases from the moment of the first positive specific tests for HBV and HCV markers; to assess effects of HBV and HCV infection on efficacy of treatment of blood disease treatment, i.e. lifespan of patients with hematological diseases. Material and methods. The study enrolled 257 patients: 205 with acute leukemia - AL, 40 with lymphoproliferative diseases, 4 - with CML and 8 - others; 8 healthy bone marrow donors. The patients were admitted to Russian Hematological Research Center in 2004-2006. Follow-up median was 253 days. A total of 7800 biological samples were studied, among them about 4000 tests for HBV DNA and HCV RNA. Results. Positive tests for specific markers of HBV and HCV were absent only in 78 (29.4%) patients. Positive markers of coinfection were detected in 57 (32.8%) of 174 patients with HBV infection and in 81.4% of 70 patients with HCV infection. Probability of detection of HCV markers after positive tests for HBV markers and vice versa is about 3 times higher than probability of their isolated detection. Among patients infected with HBV symptoms of hepatitis B are likely to appear in 56% patients to day 500 of follow-up from the date of the first positive specific test. Median of the interval between the first positive test for HBV markers and probable clinical signs of hepatitis was 30 days. Among patients with HCV infection, 85% develop hepatitis to follow-up day 300 since the date of the first specific positive test. Almost 100% patients infected with two viruses develop hepatitis to follow-up day 600. Median of the interval between the first positive test for HBV and HCV markers and probable hepatitis picture was 47 days. Overall 3-year survival of AL patients was 40%, of patients with lymphoproliferative diseases - 58%. Overall 7-month survival was 75% in AA patients. HBV infection in patients with blood disease is associated with high risk of death, especially in AA and AL. Association between HCV infection and survival is not proved. Conclusion. A high rate of clinical realization of viral hepatitis B and C, especially in coinfection, calls for virological and clinical monitoring of patients with any positive test for HBV and HCV markers.

Aim. to reveal the determinants of the development of iron overload in patients with acute leukemias (AL) and aplastic anemia (AA). Subjects and methods. The investigation included 104 patients, including 64 with various types of AL, 31 with AA, and 9 with myelodysplastic syndromes (MDS). A group affiliation and an erythrocyte phenotype were determined from rhesus system antigens in all the patients and the HFE gene was studied to identify mutations. For control of siderosis, the authors determined serum iron (SI) by a colorimetric technique, by applying the kits of the AGAT firm (Russia), serum ferritin (SF) by an immunoradiometric method, by using the kits of Immunotech (Czechia). The volume of transfusion was estimated in the period of June 2007 to November 2009. Results. There is evidence for a relationship between the higher level of SF and the number of transfusions. SF was 1046.1 μg/l in patients, H63D heterozygous carriers who had received less than 10 packed red blood cell transfusions and 2856 μg/l in those who had 20 transfusions (p < 0.005). HFE gene mutation carriage accelerates iron accumulation and is an additional risk factor for siderosis. In patients with transfusion chimeras and a rare phenotype in terms of rhesus antigens, packed red blood cell transfusion results in a much more increase in iron stores. Conclusion. The most important factor of iron overload acceleration is no specific choice of packed red blood cells for patients with rare combinations of red blood cell antigens and for those with artificially induced chimeras.

Aim. To determine surface and intracellular expression of ACE antigen and Bip shaperon on leukemic dendritic cells (LDC); to study expression of ACE genes and Bip, Calnexin, calreticulin shaperons in LDC at diagnosis of acute myeloid leukemia (AML) under standard and stress cultivation. Material and methods. Expression of ACE antigens and Bip was studied with immunophenotyping and flow cytometry using monoclonal antibodies to shaperon Bip and to CD143), expression of genes of ACE and shaperons Bip, Calnexin, Calreticulin - with polymerase chain reaction (RT-PCR). Dendritic cells (DC) were obtained by culturing of a monoclonal fraction of donor peripheral blood and AML patients in the presence of 180 ng/ml calcium ionophor A23187 (Sigma) for 4 days in parallel at 37°C and 33°C in the atmosphere of 5% CO2. The trial included 9 patients (5 males and 4 females) aged 39-53 years (median 43 years). The control group consisted of 8 healthy donors. Results. Lowering of cultivation temperature did not increase ACE expression. Intracellular shaperon Bip rose insignificantly (1.3-fold) in DC of the controls. ACE and Bip shaperon expression on LDC membrane increased 15- and 11-fold, respectively, while the level of intracellular ACE and Bip decreased 11- and 2-fold, respectively. Expression of the genes was investigated in cultivation temperature lowering from 37 to 33°C and was presented as a logarithmic scale. Changes in expression of the genes Bip, Calnexin, Calreticulin in LDc and DC of the controls were insignificant. ACE expression in LDC significantly differed from ACE gene expression in DC (p = 0.05). Conclusion. LCD and DC of healthy donors are cells which differ by genetic and functional characteristics. Therefore, LDC may response inadequately in development of antitumor immune response. The phenomenon of ACE antigen expression normalization on cell membrane in stress open new opportunities for regulating functional activity of LDC.

Aim. To compare diagnostic efficacy of B-cell clonality determination in application of different electrophoretic methods. Material and methods. B-cell clonality was determined with different techniques in 89 patients having B-cell lymphoma (n = 48), B-cell lymphoma in remission (n = 11), reactive processes (n = 24), lymphogranulomatosis and T-cell lymphoma (n = 6). Healthy donors served control. Clonality was defined by rearrangements of Ig heavy chain genes with usage of primers to FR2 region of Ig gene. Electrophoretic methods were the following: agarose electrophoresis and SSCP. Results. Clonality was detected in the presence of more than 5.9% clonal cells from total count of mononuclear cells or more than 20% of clonal cells from total number of B-lymphocytes in the sample. We achieved detectability of B-clonality in B-cell tumors 87.5% (42 of 48 cases). False negative tests were also investigated with kits of primers to FR1 and FR3 regions, but clonality was not determined. Among reactive processes, non-B-cell tumors and healthy donors clonality was found in 1 of 41 cases. This patient had acute respiratory viral infection. Agarose electrophoresis and SSCP test results coincided. Conlusion. Determination of B-cell clonality with agarose electrophoresis proved to be a simple, reliable, cost-effective and reproducible method of differential diagnosis of tumor and reactive lymphoproliferation. This method is not suitable for verification of tumor remission or assessment of minimal residual disease because of its low sensitivity.

The paper describes a case of a patient with refractory hairy cell leukemia. In spite of the absence of CD25 expression, the disease was classified as a classical form according to the WHO classification (2008), as also confirmed by the detection of BRAFV600E mutation. The disease was characterized by resistance to all lines of therapy (interferon-α, splenectomy, cladribin). Clinical and hematological remission was achieved within 2 months of administration of the BRAF kinase inhibitor vemurafenib.

Background. Current chemotherapy protocols for T-cell acute lymphoblastic leukemia (T-ALL) allow achieving a 5-year overall survival of 60–90 %, but relapsed and refractory forms remain incurable situations.Aim. To determine the significance of immunophenotypic, cytogenetic and molecular markers in adult T-ALL patients receiving therapy according to the ALL-2016 protocol.Materials and methods. From December 2016 to June 2022, 113 patients with primary T-ALL were included in the study. Cytogenetic study was performed in 104 (92 %) patients; anomalies in the IKZF1 and NOTCH1 genes were investigated in 43 (38 %) patients.Results. The worst prognosis was in patients with ETP and near-ETP variants of T-ALL (3-year disease-free survival was 54 % in ETP group, 33 % in near-ETP group vs TI/II – 79 %, TIII – 89 %, TIV – 75 %). In early T-ALL variants, abnormal karyotype was most common (ETP – 80.7 %, near-ETP – 60 %). Aberrations in NOTCH1 gene were found in 53 % of cases (in 23 out of 43 patients), and no mutations were found in IKZF1 gene in our study. In the group with no NOTCH1 abnormalities, the overall survival was significantly worse than in the group with abnormalities (NOTCH1– – 52 % vs NOTCH1+ –81 %; p = 0.05).

Background. Tumor cell proliferation in acute myeloid leukemia (AML) may manifest with high leukocyte counts. In our work, we evaluate the association of high leukocyte counts with individual mutations, as well as their total contribution to the development of leukocytosis in AML. The results obtained should improve our understanding of pathogenic mechanisms leading to the leukocytosis in AML.Aim. To study the genetic landscape of AML with leukocytosis.Materials and methods. The laboratory data of 214 AML patients admitted to the National Medical Research Center for Hematology (Moscow) from 2010 to 2022 were retrospectively examined. Real-time PCR, capillary electrophoresis and NGS (next generation sequencing) methods were used to detect mutations of FLT3, NPM1, CEBPA, IDH1/2, DNMT3A, TET2 genes, and CBFB::MYH11, RUNX1::RUNX1T1 chimeric gene transcripts.Results. Mutations of the FLT3 gene (odds ratio 5.45; p < 0.0001), inv(16)/CBFB::MYH11 (odds ratio 10.03; p = 0.0009) are most associated with leukocyte counts higher than 30 × 109 / L in the debut of AML. Translocation t(8;21)/RUNX1::RUNX1T1 and adverse cytogenetic aberrations, such as -5/del(5q); -7 / del(7q); -17 / abn(17p), complex and monosomic karyotype were significantly associated with leukocyte counts lower than 30 × 109 / L at the time of disease manifestation (p < 0.0001). In the group of patients with intermediate cytogenetic risk bearing only IDH1/2, DNMT3A, and TET2 gene mutations, leukocyte counts at AML debut were significantly lower, whereas the most pronounced leukocytosis was observed in patients with a combination of driver mutations with IDH1/2, DNMT3A, and TET2 gene mutations or FLT3, NPM1, and CEBPA gene mutations.Conclusion. In addition to the individual effect of certain genetic lesions and cytogenetic aberrations on the proliferative potential of tumor cells, there is a total contribution of various types of genetic events to the development of leukocytosis in AML. High leukocyte counts at the time of AML manifestation in patients with intermediate cytogenetic risk can serve as an indirect marker of the presence of a large number of genetic aberrations with a combination of IDH1/2, DNMT3A, and TET2 gene mutations or FLT3, NPM1, and CEBPA gene mutations.

Background. The effectiveness of therapy for acute T-lymphoblastic leukemia (T-ALL) and T-lymphoblastic lymphoma (T-LBL) has significantly improved over the past decades, including through the implementation of hematopoietic stem cell transplantation (HSCT). The Russian multicenter ALL-2009 study (ClinicalTrials.gov NCT01193933) showed that performing autologous HSCT (auto-HSCT) in patients with T-ALL/LBL improves long-term results. However, the study was non-randomized and the need for auto-HSCT in clinical practice requires careful study.Aim. To assess the significance of auto-HSCT in patients with T-ALL/LBL in the framework of ALL-2016 multicenter, prospective, randomized study (ClinicalTrials.gov NCT03462095).Materials and methods. The study included 109 adult patients with T-ALL/LBL (m:f 82:27). Median age was 31 (18– 52) years. T-ALL was diagnosed in 88 (81 %) patients, T-LBL in 21 (19 %) patients. All patients are treated according to the ALL-2016 protocol. Using the web platform, upon completion of induction therapy (+70 days), all T-ALL/LBL patients who achieved clinical and hematological remission were randomized to the auto-HSCT arm or chemotherapy alone (CT). Centralized monitoring of minimal residual disease (MRD) in bone marrow samples was performed by multicolor flow cytometry at the control points according to the ALL-2016 protocol. Statistical analysis was performed using SAS 9.4.Results. 87 patients with T-ALL/LBL were randomized: 44 to the auto-HSCT arm and 43 to the CT arm. Further analysis included 25 patients who underwent auto-HSCT and 36 patients receiving only CT. Three-year relapse-free survival in T-ALL is estimated at 62 % (auto-HSCT) vs. 81 % (CT) (p = 0.3422), and in T-LBL – 67 % (auto-HSCT) vs. 79 % (CT) (p = 0.59). MRD persistence on the +70th day of therapy according to the ALL-2016 protocol was determined in 40 % of patients (autoHSCT) and in 67 % (CT) (p = 0.057). In a multivariate analysis, it was determined that T-ALL from early T-cell precursor (ETP-variant) and MRD persistence after the end of II phase induction are the main risk factors for relapse in the treatment according to the ALL-2016 protocol.Conclusion. Performing auto-HSCT both in patients with T-ALL and T-LBL, and with MRD persistence on day +70 according to the ALL-2016 protocol did not improve long-term results. The development of new programs for the treatment of patients with MRD persistence, as well as the ETP variant of T-ALL, is of current interest.

Allogeneic hematopoietic stem cells transplantation is one of the effective methods of treating patients with diseases of the blood system.Establishment of complete (100%) donor chimerism is among of the main indicators of successful transplantation in such cases. Monitoring chimerism makes it possible both to assess the graft acceptance, and also potentially predict the risk of developing primary/secondary graft failure, relapse, and graft-versus-host disease.The purpose of this review is to summarize the main concepts associated with chimerism after allogeneic hematopoietic stem cell transplantation; consideration of the need to study chimerism in various cell populations, as well as the relationship between chimerism and the development of various immunological complications.

Preclinical studies showed synergic anti-tumor activity of Rituximab and Fludarabin. Comparison of efficacy of FC with FCR regimens administered in first line therapy and their influence on disease-free and overall survival in patients with B-cell lymphocytic leukemia were the main goals of this retrospective study. It has been demonstrated that inclusion of Rituximab into the FC regimen improves outcome of patients with B-cell lymphocytic leukemia without significant increase of toxicity.

Background. Multiple myeloma complicated by extramedullary plasmacytoma is an unfavorable variant of the disease. It remains unknown what triggers tumor transformation. The review presents literature data on the pathogenesis of extramedullary disease, as well as a clinical example of a comprehensive study of the tumor substrate.Aim. To study the molecular and biological characteristics of the tumor substrate of the bone marrow and extramedullary plasmacytoma using various research methods.Materials and methods. A 55-year-old patient was admitted to National Medical Research Center for Hematology with a diagnosis of multiple myeloma occurring with extramedullary plasmacytoma of the retroperitoneal space. dNA was isolated from samples of different localization (blood plasma, Cd138+ bone marrow cells, plasmacytoma and buccal epithelial cells). The profile of short tandem dNA repeats (STR) from the obtained samples was studied by multiplex polymerase chain reaction followed by fragment analysis. fluorescent in situ hybridization (fISH) of bone marrow Cd138+ cells was performed using various dNA probes. Comparative genomic hybridization on a microarray (arrayCGH) plasmacytoma dNA was also performed. The mutation profile of the KRAS, NRAS, BRAF genes was studied by Sanger sequencing in tumor samples of various localizations.Results. The induction therapy (vCd (bortezomib + cyclophosphamide + dexamethasone), vRd (bortezomib + lenalidomide + dexamethasone), daratumumab therapy) was ineffective, death occurred 4 months after the first clinical manifestations appeared. Comparison of STR markers of circulating cell-free tumor dNA (cfdNA), Cd138+ bone marrow cells, and plasmacytoma revealed the largest number of involved loci exactly in plasmacytoma’ dNA. A mutation in the NRAS gene was found only in plasmacytoma’ dNA. This indicates the presence of another clone of tumor cells in the extra-medullary plasmacytoma. Molecular karyotyping of plasmacytoma using the arrayCGH method revealed rearrangements of many chromosomes. 1p32.3 bi-allelic deletion, amplification of 1q21, 8q24/MyC rearrangements and del17p13 were confirmed by arrayCGH molecular karyotyping and fISH studies in bone marrow and plasmacytoma.Conclusion. A comprehensive molecular genetic study of the extramedullary plasmacytoma’ substrate is necessary to understand the pathogenesis mechanisms and, on this basis, to develop differentiated therapeutic approaches.

T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) is an aggressive hematological disease. Modern polychemotherapy protocols allow achieving a 5-year overall survival of 60–90 % in different age groups, however, relapses and refractory forms of T-ALL remain incurable. Over the past decades, the pathogenesis of this variant of leukemia has been studied in many trials, and it has been found that various signaling pathways are involved in the multi-step process of leukemogenesis. This opens the way for targeted therapy.In this review, we provide an update on the pathogenesis of T-ALL, opportunities for introducing targeted therapies, and issues that remain to be addressed.

Introduction of the immunophenotyping in 70-ties and 80-ties of the past century has led to significant revision of conception of hematological malignancies. A new era in developement of medicine and oncology in particular is related to functional genomics. This field of molecular biology attempts to make use of the vast wealth of data produced by genomic projects for studying gene functions and interactions. Functional genomics focus on the dynamic aspects such as gene transcription, mRNA translation, and protein-protein interactions, as opposed to the static aspects of the genomic information such as DNA sequence or structure. Several genomic technologies have been developed to study gene expression. Gene expression profiling on DNA microarrays has been particularly useful in analyzing expression of thousands genes in parallel. Modern microarrays contain comprehensive probe sets which are able to measure the majority of genes encoded in human genome. Here we focus on microarray technology, discuss its clinical applications and briefly review some results obtained by this method in researches of hematological malignancies.

Background. 6-mercaptopurine (6-MP) is a drug that is included in the treatment protocols for children and adults with acute lymphoblastic leukemias/lymphomas (ALL/LBL). It is known that individual differences in 6-MP tolerance can be explained by the TPMT and NUDT15 polymorphisms.Aim. To determine 6-MP toxicity profile in adult patients with Ph-negative ALL/LBL treated by ALL-2016 protocol, depending on the TPMT and NUDT15 polymorphisms.Materials and methods. The study included 54 adult patients with Ph-negative ALL/LBL (40 male and 14 female). The median age was 31 (18-51) years. T-ALL/LBL was diagnosed in 29 patients, B-ALL/LBL - in 22, acute leukemia with a mixed immunophenotype - in 3. All patients received treatment according to the multicenter study ALL-2016 (ClinicalTrials.gov, NCT03462095). polymorphisms in NUDT15 (*2, *3) and TPMT (*2, *3A, *3B, *3C) genes were detected using the allele-specific real-time polymerase chain reaction. Genomic DNA was extracted from patients peripheral blood samples. On the induction and consolidation therapy by the protocol, the received and proper 6-MP doses were calculated for all the patients. Drug toxicity was evaluated based on clinical and laboratory data.Results. TPMT and NUDT15 polymorphisms were detected in 11 (20 %) patients, more often in B-ALL - 7 (32 %) of 22 (p 0.05).Conclusion. There were no differences in the received dose of 6-MP and the incidence of toxicity in adult patients between Ph-negative ALL/LBL with or without TPMT and NUDT15 polymorphisms treated according to ALL-2016 protocol (p >0.05). further studies including evaluation of 6-MP metabolites concentrations are required for a more complete understanding of the metabolism of this drug.

Background. The study of genetic predictors of non-Hodgkin’s lymphomas prognosis is one of the most relevant areas of oncohematology. It is extremely interesting to search for integral markers that reflect the most important stages of tumor pathogenesis. DNA repair system plays one of the key roles in genomic instability. Aberrations of microsatellite repeats such as microsatellite instability (MSI), in particular elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is characteristic for mismatch repair system and loss of heterozygosity (LOH) is an integral feature of genomic instability.Objective. Analysis of MSI, EMAST, LOH significance in follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), and high-grade B-cell lymphoma (HGBL) patients.Materials and methods. The study was performed by multiplex PCR and fragment analysis with diagnostic panels COrDIS Plus and COrDIS MSI in 85 FL patients, 32 DLBCL patients, and 37 HGBL patients.Results. The frequency of LOH in the general FL group was 40/81 (49.4 %), MSI – 10/82 (12.2 %), EMAST – 15/81 (18.5 %). The frequency of LOH in the HGBL group was 21/31 (67.8 %), MSI – 11/37 (29.7 %), EMAST – 13/31 (41.9 %). The frequency of LOH in the DLBCL group was 18/29 (62.0 %), MSI – 5/32 (15.6 %), EMAST – 14/32 (43.8 %). When considering the morphological types of FL, it was noted that a higher frequency of genetic aberrations was characteristic of lymphomas with a more aggressive morphology (p

Background. Genetic instability, an important phenomenon involved in oncogenic transformation and tumor progression, is associated with the insufficiency of the multicomponent DNA repair complex, in particular, the nucleotide mismatch repair (MMR) system. The MMR defect manifests itself as abnormalities in DNA microsatellite repeats, or microsatellite instability (MSI). In the studies of colorectal cancer, the role of MSI in prognostication of the disease, and defining the choice of specific therapy with immune checkpoint inhibitors has been proven.However, in lymphatic system tumors, the significance of this phenomenon is poorly understood. Determination of genetic instability in the onset of follicular lymphoma, a disease characterized by a heterogeneous course, may have prognostic value.Objective: to determine the genetic instability at the onset of follicular lymphoma.Materials and methods. Here we report an analysis of 24 microsatellite repeats and amelogenin loci in tumor cells of 46 follicular lymphoma patients.Results. In the studied cohort, lesions in microsatellite repeats were presented by MSI in 9 cases (19.6 %) and the loss of heterozygosity (LOH) in 19 cases (41.3 %). Most frequent lesions were found for the SE33 marker located at the q14 locus of chromosome 6. A significant association was shown between MSI and the double-hit follicular lymphoma group with rearrangements of the MYC and BCL2/BCL6 genes.Conclusion. Thus, our data indicate that the MSI phenomenon might be involved in the pathogenesis of the lymphatic tumors and particularly follicular lymphoma. However further studies on the expanded cohorts of patients are required to define the possible prognostic value of MSI in lymphatic tumors.

Introduction. Аngioimmunoblastic T-cell lymphoma (AITL) is a T-cell lymphoma, characterized by abundant polymorphocellular infiltrate of lymph nodes with the small number of tumor CD4+ T-cells. AITL could often be misdiagnosed as reactive processes and other lymphomas, including Hodgkin’s lymphoma. Molecular methods of determining clonality are used to confirm the diagnosis. TCR gene rearrangements detected in the lymph nodes may not coincide with those detected in the bone marrow (BM), blood or skin, that makes their interpretation difficult. Recently discovered point somatic RHOA (Ras homolog gene family, member A) Gly17Val mutation is present in 53–71 % of angioimmunoblastic T-cell lymphomas.The aim of the study was to evaluate the number of RHOA Gly17Val mutated cells in different tissues of AITL patients and to correlate it with corresponding T-cell clonality results.Materials and methods. TCRG and TCRB gene rearrangements were PCR-amplified according to BIOMED-2 standardized protocol and analyzed by capillary electrophoresis on ABI Prism 3130. Gly17Val mutation was analyzed by quantitative allele-specific (qAS) TaqMan Real-Time PCR assay. Lymph nodes (LN) and skin biopsies, blood and BM samples were studied for 40 patients with AITL.Results. The clonal TCR gene rearrangements were found in 37 (92 %) of 40 patients in LN and in 26 (96 %) of 28 patients in BM, but they were not matched in length in LN and BM in 12 (46 %) of 26 patients. RHOA (Gly17Val) mutation in LN was revealed in 60 % (24 of 40) patients. Number of cells with RHOA mutation was highest in the LN (in average 26.7 % of the total cells), while in the BM RHOA mutation were undetectable (in 7 patients), or detected in 10 patients in a low quantity (in average 2 % of the total cells). Skin, blood and BM samples with the T-cell clonality peaks that differ from those found in the LN were also negative for the presence of cells having RHOA Gly17Val mutation. Also, in four patients we showed that clonal products in BM and blood that do not coincide with LN refer to CD8+ lymphocytes.Conclusions. The results of the study demonstrate that the quantitative determination of cells with the mutation of RHOA Gly17Val must be taken into account in staging the disease and interpreting the results of T-cell clonality assay. Clonal cells with rearrangements not matching those identified for the LN should be considered reactive.

DNA samples from patients with hairy cell leukemia (HCL) and splenic marginal zone B-cell lymphoma (MZBCL) for BRAF and MAP2K1 activating mutations were analyzed. V600E mutation of BRAF was detected in 39 (98 %) of 40 patients with hairy cell leukemia, and no patient with MZBCL. In none of the patients in this study any other activating mutations than V600E in exons 11 and 15 of BRAF gene were revealed. MAP2K1 Q56P mutation characterized by IGHV4-34 expression was detected in 1 (2 %) of 40 patients with HCL. In none of the patients with MZBCL activating mutations in the 2, 3 or 11 exons of MAR2K1 gene have been identified, including those with IGHV4-34 expression.

Mutation status of the heavy chain variable region genes has long been known as an important factor in long‑term prognosis in B‑cell chronic lymphocytic leukemia (B‑CLL). A more detailed study of the gene sequences of immunoglobulin heavy chain (IgVH) led to the discovery of stereotyped antigen receptors (SAR) — receptors that have the same set of VH‑, D‑ and JH‑genes used. Cells with SARs have been found almost in a quarter of all B‑CLL cases. This phenomenon is not observed in other lymphatic tumors. In our study, we confirmed and extended the basic observations concerning the repertoire of IgVH in B‑CLL. Differences in the B‑CLL IgVH gene repertoirs between Russia, Вelarus and other countries are also analysed and discussed.

Introduction. Infection caused by cytomegalovirus (CMV) is a serious problem for patients with weakened immunity, including patients with hematopoietic depression. The cases of complications associated with cytomegalovirus require antiviral therapy. However, during the natural mutation process, especially with prolonged use of drugs in suboptimal doses, CMV strains resistant to the action of antiviral drugs (such as ganciclovir, valganciclovir) may occur. Hypothetically, the emergence of resistance in the virus may cause a more aggressive course of infection, the ineffectiveness of antiviral therapy and, as a result, an increase in the number of deaths. In this regard, timely detection of mutations that can potentially lead to the resistance of the virus to antiviral drugs during hematopoietic stem cell transplantation (HSCT), as well as during organ and tissue transplantation, may be important when making a therapeutic decision. We describe three clinical cases for which the dynamics of the appearance of a mutant strain of CMV by the UL97 gene, which correlates with the viral load and clinical picture, is analyzed. The aim of the study was to determine the timing of the occurrence of mutations in CMV phosphotransferase UL97 gene associated with resistance to antiviral drugs in patients with hemoblastoses after allogeneic hematopoietic stem cell (allo-HSCs) transplantation. Material and methods. The study included 48 samples of CMV DNA isolated from the peripheral blood of three allo-HSCs recipients with CMV infection who were treated in the clinics of the FSBI «National Medical Research Center for Hematology» of the Ministry of Health of Russia with oncohematological diseases during 2015–2017. Patients received conditional codes (PR, PD, and FS). Mutations associated with antiviral therapy (AVT) resistance were identified in all patients. Sanger sequencing was used for mutation detection. The obtained DNA sequences were analyzed using Nucleotide BLAST and Genome compiler software. Mutations were searched in MRA mutation resistance analyzer software. The nucleotide sequences were compared with the UL97 reference sequence of the Merlin CMV strain using this software environment. Results and discussion. For all patients in whom the virus strains containing C592G (PR), C607F (PD) and C603W (FS) mutations were detected, the timing of the mutation occurrence was determined at days 187, 124 and 1184, respectively. The emergence of mutations with a high resistance factor was shown to be accompanied by an increase in viral load (VL), the appearance of a clinical picture characteristic of CMV infection and a lack of an adequate response to therapy with ganciclovir and its derivatives. Conclusion. Using these results, it is proposed to develop the test system based on random polymerase chain reaction (rPCR) to detect mutations in the most frequently encountered codons: M460I/V, C592G, A591V, A594T/V, L595F/S, C603W. Given that the data on the prevalence of these mutations were obtained from foreign sources, it is advisable to conduct similar studies on the frequency of mutations in the UL97 gene among the population of the Russian Federation in order to improve the quality and accuracy of test systems.

The objective: to perform quantitative analysis of SARS-CoV-2 viral load (VL) levels in lung tissues in deceased patients with COVID-19 and to evaluate its association with the nature of histological changes in the lungs and the duration of stay in ICU till the lethal outcome.Subjects and Methods. Sections of formalin-fixed and paraffin-embedded lung tissues of 36 deceased patients with COVID-19 were used. The SARS-CoV-2 viral load was quantitatively assessed using the original qPCR. VL was calculated using the following formula: copies SARS-CoV-2 / copies ABL1 × 100, expressed as the ratio of the true number of SARS-CoV-2 cDNA copies per 100 copies of ABL1 gene cDNA.Results. In cases with no histological changes typical of diffuse alveolar lung injury (DAI), the detection rate of SARS-CoV-2 RNA and the average level of the SARS-CoV-2 viral load were 62.5% (5 out of 8 observations) and 104.75 (range 0-313) copies of SARS-CoV-2 cDNA per 100 copies of human ABL1 gene cDNA. The average level of the SARS-CoV-2 viral load in the lungs with prevailing histological changes characteristic of the proliferative and exudative phases of DAI differed by 60 times and amounted to 909 (18-2,657) and 54,924 (834-250,281) copies of SARS-CoV-2 cDNA per 100 copies of human ABL1 cDNA, respectively. The average duration of stay in the intensive care unit in the group of patients with exudative and proliferative phases of DAI was 10.64 (1-22) and 8.14 (1-21) bed-days, respectively. The detection rate of the SARS-CoV-2 RNA in patients with diffuse alveolar lung injury was 100%.

Background. LiFraumeni syndrome (LFS) is a rare, autosomal dominant, hereditary disorder that is characterized by an increased risk for certain types of cancer, acute lymphoblastic leukemia (ALL), particularly. Germline TP53 mutations are associated with LFS. Genetic counseling and follow-up is essential for patients with LFS and their relatives. Special therapeutic approaches are needed for treatment of oncological disease in these patients. The article presents a series of clinical cases of patients with ALL and SLF, considers general issues of diagnosis and treatment of adult patients with this hereditary genetic syndrome. Aim. Describe clinical observations of patients with acute lymphoblastic leukemia (ALL) and LFS and consider general issues of diagnosis and treatment of adult patients with LFS and ALL. Materials and methods. TP53 gene mutations were screened using Sanger sequencing in 180 de novo patients with Ph-negative (B- and T-cell) and Ph-positive ALL treated by Russian multicenter protocols (ALL-2009, ALL-2012, ALL-2016) at the National Research Center for Hematology, Moscow, Russia, and at the hematology departments of regional clinics of Russia (multicenter study participants). Results. TP53 gene mutations were found in 7.8% (n=14) of de novo ALL patients. In patients, whose biological material was available TP53 gene mutational status was determined in non-tumor cells (bone marrow and peripheral blood during remission, bone marrow samples after allogeneic hematopoietic stem cells transplantation and in tissue of non-hematopoietic origin) for discriminating germline mutations. The analysis included 5 patients (out of 14 with TP53 mutations), whose non-tumor biological material was available for research. Germline status was confirmed in 4 out of 5 B-cell ALL (n=3), T-cell ALL (n=1) investigated patients. Conclusion. Practical value of the research is the observation that the greater part of TP53 gene mutations in patients with Ph-negative B-cell ALL are germinal and associated with LFS.

Thrombotic complications are the most significant factors determining the prognosis in myeloproliferative neoplasms. Markers for assessing the risk of thrombosis are the number of leukocytes, platelets, hemoglobin level, hematocrit, age, molecular status, history of thrombosis, obesity, arterial hypertension, hyperlipidemia, hereditary or acquired thrombophilia. The pathogenesis of thrombosis in patients with myeloproliferative neoplasms is complex and multifactorial. In most cases, the etiological factor remains unknown. Currently, antiplatelet and anticoagulant therapy is carried out on an individual basis. The algorithm for primary and secondary (after thrombosis) prevention requires development and testing. We present a clinical case of repeated arterial and venous thrombotic complications in a patient with primary myelofibrosis.

Aim. To analyze the results of treatment in patients with acute myeloid leukemia (AML) within protocols AML-17 and modified AML-17 (mOML-17) as part of two consecutive pilot studies in order to develop the best treatment strategy for AML patients aged below 60 years. Materials and methods. The study included 89 AML patients who were aged below 60 years and received treatment within the AML-17 and mOML-17 protocols. Cytogenetic and molecular genetic studies were performed in all patients. The presence of mutations in the FLT3, NPM1, CEBPa genes was assessed by fragment analysis. 35 patients underwent a study for mutTP53, mutRUNX1 using next generation sequencing (NGS). The minimum residual population of tumor cells was evaluated by multicolor flow cytometry. Statistical analysis was performed using the procedures of the SAS 9.3 package. Results. Complete remission (CR) was achieved in 89.7% of patients treated with intensive chemotherapy (CT) courses and in 52.4% of patients treated with low-dose CT courses. 8.8% of intensively treated patients were refractory to therapy, and 38% did not respond to low-dose exposure. The early mortality rate was 3%. The overall survival and disease-free 3-year survival for patients included in 2 consecutive studies was were 60% and 67%, respectively. The level of minimal residual disease (MRD) after the first course of induction CT was an important prognostic indicator. The three-year relapse-free survival for patients in whom CR was achieved after the first course of induction CT and in whom MRD was not detected (MRD-negative status was obtained) was 90% compared to 43% for patients who were MRD positive after the first course of induction CT (p=0.00001). Conclusion. The key factor that significantly affects the long-term results of therapy is the rate of MRD after the first course of induction CT.

The prevalence of multiple primary tumors has significantly increased last time. The question of choosing the optimal tactics of therapy today not fully resolved. Particular interest is the simultaneous detection of two neoplasms of similar origin in one study biopsy material. This publication presents a case of simultaneous diagnosis of myeloid sarcoma and mantle cell lymphoma in a 65-year-old patient, which required use of two different chemotherapy protocols. This example shows the need to use an extended diagnostic approach at all stages of the therapy, which allows choosing right tactics of therapy and achieving complete remission of two neoplasms.

Aim.This study conducted the possibilities of diffusion-weighted magnetic resonance imaging of the whole body diffusion WB-MRI (in comparison with positron emission tomography with computed tomography PET/CT) in assessing the volume and prevalence of the tumor, as well as determining bone marrow (BM) damage (for various cytological types) in the diagnosis and staging of the disease in patients with FL. Materials and methods.A prospective comparative search study included 15 patients (4 men and 11 women, with a median age of 53 years) with newly diagnosed FL. Patients have not received antitumor chemotherapy previously. After the diagnosis was established, all patients (with the blindness of both the cases themselves and some specialists regarding the results of other specialists) were examined by PET/CT and diffusion WB-MRI, after which a BM examination was performed (histological examination and determination of B-cell clonality in BM puncture by PCR). Using the diffusion WB-MRI method, the prevalence of tumor lesion (nodal and extranodal foci) in each patient was estimated, and the total tumor volume was calculated, BM lesion was detected, and BM lesion volume was calculated. For lesions of different localization, the measured diffusion coefficient (DC) of the diffusion WB-MRI and the standardized rate of accumulation of the radiopharmaceutical in tissues (SUV) of the PET/CT method were determined and compared with each other (for the same areas). Statistical analysis was performed using the estimate of agreement (by Cohens kappa coefficient and asymptotic test) of the results of the compared methods. Results.Estimates of the prevalence of tumor damage (lymph nodes and extranodal foci) using the diffusion WB-MRI and PET/CT methods were the same. High DC and SUV were observed in the peripheral lymph nodes, extranodal foci and bulky, low DC and SUV in the foci of BM. All 4 methods successfully determined BM damage, however, the diffusion WB-MRI had comparatively less negative results. The highest values of SUV and CD were noted in cases of the 3 grade of FL. Using the diffusion WB-MRI method, the prevalence of tumor lesion was assessed in each patient (nodal and extranodal foci were detected) and the total tumor volume was calculated, BM lesion detection was performed, and the volume of BM lesion was calculated. It is important to note that with the help of diffusion WB-MRI, it was possible to measure separately the total tumor volume (462025 cm3) and separately the volume of bulky (251358 cm3). The diffusion WB-MRI allowed us to differentiate the volume of tumor tissue (reduced as a result of treatment) and residual (fibrous-adipose) tissue in residual formations (which averaged 21% of the initial volume). The predictors of a poor antitumor response were the maximum SUV values (more than 14.0) and the minimum DC values (0.510-3mm2/s) in the BM foci. Conclusion.The diffusion WB-MRI allows for detailed visualization of BM lesions and surrounding soft tissues both in the debut of the FL and in the process of tracking the effectiveness of chemotherapy, which makes it possible to use it along with PET/CT. Diffusion WB-MRI allows to separately evaluate the volume of true tumor tissue and residual tissue. Cases of the 3 grade of FL (including the transformation of FL into diffuse B-large cell lymphoma) are isolated due to low DC values (and high SUV values) in the tumor tissue. BM foci of FL lesion also have (in comparison with nodal and extranodal foci) lower DC values. The predictors of a poor antitumor response were high (from 14.0 or more) SUV valuesin the tumor (and especially in bulky), and low (about 0.5103mm2/s) DC values of BM foci. The PET/CT and diffusion WB-MRI have proven to be reliable diagnostic tools for establishing the stage of FL and detecting BM damage. Diffusion WB-MRI for FL is an informative first-line diagnostic method that allows regular monitoring of the disease and early detection of foci of relapse and disease progression.

Primary myelofibrosis is a myeloproliferative neoplasm that occurs de novo, characterized by clonal proliferation of stem cells, abnormal expression of cytokines, bone marrow fibrosis, hepatosplenomegaly as a result of extramedullary hematopoiesis, symptoms of tumor intoxication, cachexemia, peripheral blood leukoerythroblastosis, leukemic progression and low survival. Primary myelofibrosis is a chronic incurable disease. The aims of therapy: preventing progression, increasing overall survival, improving quality of life. The choice of therapeutic tactics is limited. Allogenic hematopoietic stem cell transplantation is the only method that gives a chance for a cure. The role of mutations in a number of genes in the early identification of candidates for allogeneic hematopoietic stem cell transplantation is being actively studied. The article describes the clinical case of the detection ofASXL1gene mutations in a patient with prefibrous primary myelofibrosis. The diagnosis was established on the basis of WHO criteria 2016. The examination revealed a mutation ofASXL1. Interferon alfa therapy is carried out, against the background of which clinico-hematological remission has been achieved. Despite the identified mutation, the patient is not a candidate for allogeneic hematopoietic stem cell transplantation. Given the unfavorable prognostic value of theASXL1mutation, the patient is subject to active dynamic observation and aggressive therapeutic tactics when signs of disease progression appear.

Issue.The study of activating mutations (NRAS,KRAS,FLT3,JAK2,CRLF2genes) of RAS/RAF/MEK/ERK and JAK/STAT signaling pathways in B-cell acute lymphoblastic leukemia (B-ALL) in adult patients which are included in Russian multicenter clinical trials. Materials and methods.Within the multicenter study there were 119 adult patients included withde novoB-ALL. The study was considered as prospective and retrospective. The group withBCR-ABL1-negative B-ALL consisted of up to 93 patients (45 male and 48 female, at the age of 17 to 59, the median age 31), they were treated according to the protocols ALL-2009, ALL-2016. The median follow-up lasted for 19 months (1119). The group withBCR-ABL1-positive B-ALL with up to 26 patients (10 male and 16 female, at the age of 23 to 78, the median age 34 years) was included in the study as well. The treatment was carried out according to the protocols ALL-2009 and ALL-2012 in combination with tyrosine kinase inhibitors. The median follow-up lasted for 23 months (4120). The molecular analysis of activating mutations inNRAS,KRASgenes (RAS/RAF/MEK/ERK signaling pathway) andJAK2,CRLF2genes (JAK/STAT signaling cascade) was performed via Sanger sequencing. The internal tandem duplications (ITDs) inFLT3gene were studied by fragment analysis. The evaluation of CRLF2 expression was fulfilled via flow cytometry. Results.Activating mutations inNRAS,KRAS,FLT3genes were found in 22 (23.6%) patients withBCR-ABL1-negative B-ALL. In total, 23 mutations were revealed in theNRAS(n=9),KRAS(n=12), andFLT3(n=2) genes, according to statistics that was significantly more frequent than withBCR-ABL1-positive B-ALL, these genes mutations were not identified in patients (p=0.007). The frequency of mutations detection inKRASandNRASgenes in patients withBCR-ABL1-negative B-ALL was comparable as 12.9% (12 of 93) to 9.7% (9 of 93), respectively (p=0.488). One patient was simultaneously revealed 2 mutations in theKRASgene (in codons 13 and 61).FLT3-ITD mutations were detected in 3.5% (2 of 57) cases ofBCR-ABL1-negative B-ALL. In patients withBCR-ABL1-positive B-ALLFLT3-ITD mutations were not assessed. Violations in the JAK/STAT signaling cascade were detected in 4 (4.3%) patients withBCR-ABL1-negative B-ALL. They were represented by the missense mutations ofJAK2gene (n=3) and the overexpression of CRLF2 (n=2); in one patient were detected the overexpression of CRLF2 and a mutation inJAK2gene simultaneously. No mutations were found inCRLF2gene. In patients withBCR-ABL1-positive B-ALL noJAK2mutations were detected. As long as analyzing demographic and clinical laboratory parameters between groups of patients with and without mutations, there were no statistically significant differences obtained. In the analyzed groups of patients, long-term therapy results did not differentiate according to the mutations presence inNRAS,KRAS,FLT3,JAK2genes. Also, substantive differences were not shown in the rate of the negative status achievement of the minimum residual disease between patients with and without activating mutations in the control points of the protocol (on the 70th, 133rd and 190th days). Conclusion.NRAS,KRAS,FLT3,JAK2activating mutations do not affect the long-term results of the therapy and the rate of the negative status achievement of the minimum residual disease in patients withBCR-ABL1-negative B-ALL treated by the Russian multicenter clinical trials.

Abstract Background Epstein-Barr virus is associated with many human hematopoietic neoplasms; however, Epstein-Barr virus-positive mucosa-associated lymphoid tissue lymphoma is extremely rare. In routine clinical practice, detection of mucosa-associated lymphoid tissue lymphoma and diffuse large B-cell lymphoma in a tissue sample presumes a clonal relation between these neoplasms and that diffuse large B-cell lymphoma developed by transformation of the mucosa-associated lymphoid tissue lymphoma. However, evidence to support this presumption is sparse and controversial. Assessment of the clonal relationship of the lymphoid components of a composite lymphoma is important for understanding its pathogenesis and correct diagnosis. Case presentation We present an unusual case of composite lymphoma (Epstein-Barr virus-positive mucosa-associated lymphoid tissue lymphoma/Epstein-Barr virus-negative diffuse large B-cell lymphoma) in the parotid salivary gland of a 62-year-old Caucasian woman with Sjögren’s syndrome and rheumatoid arthritis. Simultaneous occurrence of mucosa-associated lymphoid tissue lymphoma and diffuse large B-cell lymphoma in the parotid salivary gland led us to initially assume a clonal relationship between diffuse large B-cell lymphoma and mucosa-associated lymphoid tissue lymphoma. Epstein-Barr virus was detected by in situ hybridization and polymerase chain reaction in the mucosa-associated lymphoid tissue lymphoma, but not in diffuse large B-cell lymphoma, suggesting that these lymphomas were not clonally related. Fragment analysis of frame region 3 polymerase chain reaction products from microdissected mucosa-associated lymphoid tissue lymphoma and diffuse large B-cell lymphoma components revealed different clonal pattern rearrangements of the immunoglobulin heavy chain gene. Conclusions Our patient’s case highlights the importance of assessing the clonal relationships of the lymphoid components of a composite lymphoma and Epstein-Barr virus screening in mucosa-associated lymphoid tissue lymphoma in patients with autoimmune disease.

Objective was to study genetic markers of thrombophilia in patients with hemophilia, which can affect the course of the disease and contribute to thrombotic complications. Material and methods. The study included 96 patients with severe hemophilia: 75 (78.1 %) – hemophilia A, 16 (16.7 %) – hemophilia B, 5 (5.2 %) – hemophilia with inhibitor form. All patients were with severe hemophilic arthropathyand and underwent knee or hip replacement. The average age of patients was 39.6 years. All patients were examined for markers of thrombophilia. Results. Ninety three patients had either a heterozygous or homozygous form of thrombophilia marker polymorphism. One of thrombophilia markers was present in 15 (15.6 %) patients and in 78 (81.3 %) there was a combination of several markers. In patients with hemophilia B homozygous mutations in the MTHFR gene (A1298C and C677T) were more than 2 times more frequent than in patients with hemophilia A. Conclusion. The frequency of occurrence of polymorphism of FV (G1691A), MTHFR (C677T) and PAI-1 in the studied group of patients with hemophilia is higher than in the general Russian population.