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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.

Introduction. The mutational status of immunoglobulin heavy chain variable region genes (IGHV) is the most important prognostic factor in chronic lymphocytic leukemia (CLL). Furthermore, a significant narrowing of the IGHV gene repertoire is found in CLL and other lymphoproliferative diseases. Aim — to review the publication data on the IGHV genes repertoire and mutational status in CLL and other lymphoproliferative diseases regarding their clinical significance. General information. Nucleotide sequence of rearranged IGHV genes is a unique marker of a tumor clone. CLL patients with unmutated IGHV genes have an extremely unfavorable disease outcome in contrast to the patients with mutated IGHV genes. Patients with mutated IGHV genes benefit from conventional immunochemotherapy, while non-mutated IGHV patients require therapy escalation with new targeted drugs. The study of IGHV genes and stereotyped antigen receptors repertoire makes possible to identify additional groups of CLL patients with specific genetic and clinical features. Stereotype receptors are also detected in other lymphoproliferative diseases, but their clinical significance has not yet been defined. However, stereotyped receptors are found to be disease-specific.

Introduction. Optimal therapy for elderly patients with chronic lymphocytic leukemia (CLL) is the subject of intensive research.Aim – to study the safety of obinutuzumab, as well as the selection of the optimal scheme of its use in patients with CLL, complicated by diabetes mellitus, renal insuffi ciency, cardiac comorbidity.Materials and methods. The study included primary patients with CLL having indications requiring therapy. The inclusion criteria were: Cumulative Illness Rating Scale, CIRS) (CIRS) > 6 and or glomerular fi ltration rate (GFR) < 70 mL/min, the lower limit of GFR was not restricted. The study focused on patients with diabetes mellitus, renal failure and signifi cant cardiac pathology. Patients with Richter’s syndrome, CNS involvement, HBs-antigen, and 17p deletion were not included. In the fi rst cycle obinutuzumab was administered at a dose of 100 or 25 mg on the fi rst day and 900 or 975 mg on the second day, then at a dose of 1000 mg on days 8 and 15. For all subsequent cycles, obinutuzumab was given at a dose of 1000 mg on day 1. The dosage of chlorambucil was 10 mg/m2 from days 1 to 7. Treatment cycles in totals of 6 were repeated every 28 days.Results. The study included 90 patients. Median age was 73.5 years, range – 60–89 years, there were 49 men (54 %) and 41 women (46 %). Twenty-four patients (27 %) had stage C, IGHV unmutated status was detected in 76 % of patients. The median creatinine clearance was 48.6 mL/min (25–110). The median CIRS score was 3 (range – 1–14). Thirty-one patients (34 %) had signifi cant cardiovascular comorbidity (previous myocardial infarction, coronary artery stenting or bypass, HF ≥ II NYHA, peripheral artery disease) as well as hemodynamically signifi cant valvular disease. Fifteen patients (17 %) had diabetes mellitus and 71 patients (79 %) had creatinine clearance < 70 ml/min. Infusion reactions to obinutuzumab grade ≥ II were reported in 29 patients (32 %). Hospitalization on the day of administration or the next day after the fi rst administration was required in 5 cases (5.5 %). Twenty-seven (30 %) patients could not complete 6 cycles. The largest number of patients (14 people, 15.5 %) stopped treatment after 1 course. The causes were the development of persistent cytopenia (n = 4), grade IV reaction to obinutuzumab (n = 3), patient’s refusal (n = 2), infectious complications (n = 2), severe tumor lysis syndrome (n = 1), acute pancreatitis (n = 1) and toxicodermia (n = 1). The leading cause of premature discontinuation on subsequent cycles was persistent neutropenia. Progression during treatment occurred in 3 patients only. Overall survival was signifi cantly predicted by CIRS (maximum discriminatory value of 3, p = 0.013) as well as GFR < 50 mL/min (p = 0.03). No other associations were identifi ed. At least 1 episode of grade III–IV neutropenia occurred in 41 % of patients. Grade IV neutropenia was associated with creatinine clearance < 60 mL/min (p = 0.05), baseline neutrophil level < 2 × 109/L (p = 0.0001), baseline monocyte level < 0.3 × 109/L (p = 0.007) and age > 70 years (p = 0.01). The effectiveness of treatment was evaluated in patients who completed at least 3 cycles of therapy. Complete remission was achieved in 26 patients (35 %), partial remission – in 41 (54 %), stabilization – in 4 (5 %), progression was noted in 3 (4 %). Sixteen patients (18 %) were not available to respond to the assessment. Minimal residual disease < 0.01 % in the bone marrow after completion of treatment was found in 17 patients (19 %), within 0.01–0.9 % – in 25 (28 %) patients. The median follow-up from the date of therapy initiation was 39.7 months (range – 0.6–72 months). The median relapse-free survival was not reached, and 2- and 3-year survival rates were 81 and 62 %, respectively. Poor relapse-free survival signifi cantly correlated with unmutated IGHV genes (HR = 2.4, 95 % CI: 1.12–5.0, p = 0.02) and partial response as opposed to complete response (HR = 3.35; 95 % CI: 1.45–7.7, p = 0.03).Conclusion. The results of our study have practical implications as obinutuzumab is actively integrated into modern treatment regimens. Infusion reactions pose a high risk of complications in elderly patients. The dose of obinutuzumab on day 1 of administration in elderly patients, should not exceed 25 mg. The G-Clb regimen may not be optimal in patients over 75 years of age due to the unpredictable risk of complications. In patients at high risk of neutropenia, it may be appropriate to consider primary prophylaxis. ClbG is an effective regimen that resulted in high rate of MRD-negative responses and prolonged relapse-free survival.

Introduction. Over the past 5 years, signifi cant progress has been achieved in the treatment of patients with Ph-negative acute lymphoblastic leukemia (ALL). Treatment results were compared between two protocols of the Russian multicenter studies «ALL-2009» and «ALL-2016», in which multicomponent high-dose consolidation was not used. The principle of continuity of treatment was observed with modifi cation of doses of cytostatic drugs depending on the depth of cytopenia.Aim – to compare the 5-year results of two studies and to determine the factors of unfavorable prognosis in the treatment of patients with ALL.Materials and methods. The studies were performed from April 2009 to April 2016 (ALL-2009) and from April 2016 to September 2021 (ALL-2016), and 596 patients were included: 330 in ALL-2009 and 266 in ALL-2016. The analysis was performed in March 2022. The median age of patients in ALL-2009 was 28 years (15–55), in ALL-2016 – 32.5 years (18–55). Cytogenetic studies were performed in 242 patients in ALL-2009 (73.3 %) and 236 patients in ALL-2016 (88.7 %). Patients in the ALL-2016 protocol underwent a centralized assessment of minimal residual disease (MRD) by fl ow cytometry on protocol +70 day (after completion of two induction phases), +133 and +190 days. Transplantation of allogeneic stem hematopoietic cells was performed in 7 % of patients in ALL-2009 and in 9 % in ALL-2016.Results. Overall, relapse-free survival (OS, RFS) and the probability of relapse for a period of 3 years from the moment of inclusion of patients in a particular study were 59 %, 63 % and 23 % for ALL-2009, and for ALL-2016 – 64 %, 59 % and 22 %, respectively. For patients with B-cell precursor ALL, two cytogenetic risk groups were formed, in which long-term survival rates differed signifi cantly: the standard group (hyperploid set of chromosomes and normal karyotype) – OS 63 %, RFS 70 %, and high cytogenetic risk (any abnormal karyotype, except for hyperploidy) – OS 49 %, RFS 52 % (р = 0.001, р = 0.0014). In T-ALL, cytogenetic markers had no prognostic value, but the immunophenotype of early T-cell precursor turned out to be an important predictor of poor prognosis (the probability of relapse was 52 % compared with 15 % for all other immunophenotypic variants). According to the results of centralized monitoring of MRD, it was determined that for B-cell precursor ALL, the signifi cant negative factors are the high cytogenetic risk group and positive MRD status at +70 day, and for T-cells, the early immunophenotype and positive MRD status at +133 day.

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.

Introduction. The tactics of therapy for elderly comorbid patients with mantle cell lymphoma with unfavorable prognosis factors (complex karyotype, 17p13 deletion, mutations in the TP53 gene) have not been developed. The use of intensive chemotherapy regimens and transplantation of allogeneic hematopoietic stem cells (allo-HSCT) is impossible due to severe comorbidity in elderly patients. A rational approach is the use of a combination of ibrutinib and venetoclax. As an alternative to allo-HSCT, a new option for elderly patients with poor prognostic factors is Chimeric Antigen Receptor T-cell therapy (CAR-T) cell therapy.Aim — to present the experience of using ibrutinib and venetoclax with CAR-T-cell therapy in the first line of treatment in an elderly patient with MCL with a mutation in the TP53 gene and hyperleukocytosis.Main findings. Patient M., 68 years old. The examination revealed hyperleukocytosis 978 × 109/L, anemia (55 g/L), thrombocytopenia (30 × 109/L), and splenomegaly 250 × 180 mm. According to the results of laboratory studies, the diagnosis of lymphoma from mantle cells with a complex karyotype, deletion 17p13, 13q14 and mutation p.R248W in exon 7 of the TP53 gene (VAF = 26 %) was verified. For cytoreductive purposes, two sessions of leukocytapheresis and prephase with cyclophosphamide (200 mg/m2) and dexamethasone (10 mg/m2) were performed. From day 3, therapy with ibrutinib 420 mg/day and venetoclax 100 mg/day was started. After 2 days, the leukocytes were 0.7 × 109/L, and the size of the spleen decreased, as a result of which the development of tumor lysis syndrome was noted. As a result of intensive therapy, the patient’s condition stabilized, which allowed him to resume treatment. After 7 days, the number of leukocytes was 2.5 × 109/L, neutrophils — 70 %, platelets — 90 × 109/L, hemoglobin — 95 g/L. According to immunophenotyping, the population of B-lymphocytes was 4 %. According to NGS data, the allelic load of the mutation in the TP53 gene is 0.8 %. The patient underwent anti-CD19 CAR-T-cell therapy and achieved complete remission. Three months after therapy, MRD remains-negative remission and the persistence of CAR-T cells is determined.

Introduction. Primary mediastinal lymphoma (PML) is an aggressive lymphoid tumor treatment success of which is determined by induction therapy. To date, none of the standard chemotherapy regimens (CT) have demonstrated an advantage in efficacy. Intensive therapy programs are associated with high toxicity.Aim — to evaluate the efficacy and toxicity of two pilot prospective treatment protocols PML-16 and PML-19 as well as the possibility of using the analysis of freely circulating tumor DNA (ctDNA) to assess MRD in patients with PML.Materials and methods. From January 2016 to January 2022, 34 previously untreated PML patients were included in the study; average age — 32; stage > I — in 60 %; extramediastinal lesions — in 14.7 %; bulky disease — in 73.5 % of patients. Positron emission tomography combined with computed tomography (PET-CT) was performed; ctDNA was determined to assess the completeness of remission.Results. Eighteen patients received treatment according to the PML-16 protocol (6 courses of chemotherapy; 2 blocks of RmNHL-BFM-90 + 4 courses of R-EPOCH). After the end of therapy, all 18 patients achieved PET-negative remission. The next 16 patients received treatment according to the PML-19 protocol (4 courses of chemotherapy; 2 blocks of R-mNHL-BFM-90 + 2 courses of R-EPOCH) in combination with lenalidomide. After the end of therapy, 9 (56 %) patients achieved PET-negative remission; 7 (44 %) retained pathological activity (D4–5 points). After 3 and 6 months 15 (94 %) patients achieved normalization of metabolic activity. Considering the high frequency of false-positive results in patients with PML, a ctDNA study was performed to determine the depth of remission in 15 patients. After the end of therapy, all 15 patients had complete elimination of ctDNA. Of these, 5 (33 %) remained PET-positive at the end of treatment. During further observation, after 3–6 months, in 4 patients the level of metabolic activity decreased to physiological without the use of consolidating therapy. After the end of therapy, one patient suffered the new coronavirus infection, COVID-19. A month later, residual formation of SUVmax 14.2 remained in the mediastinum. The patient is currently under observation. With a median follow-up of 36 months (9 to 76 months) all 34 patients are in remission.Conclusion. The effectiveness of PML-16 made it possible to abandon the consolidation therapy and refuted the idea of the need for 6 courses of CT. The combination of programs based on the application of the principle of high-dose shortpulse induction of remission (R-mNHL-BFM-90) in combination with the prolonged administration of medium doses (R-EPOCH) was crucial in achieving a successful result. The inclusion of lenalidomide in the “PML-19” program made it possible to achieve complete remission in 100 % of cases after 4 courses. The possibility of using DNA analysis to assess MRD in patients with PML was shown.

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 TPMT, NUDT15 polymorphisms only at consolidation IV (p = 0.01). we didn't find a correlation between the 6-MP toxicity and the polymorphisms in our patients (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.

Introduction. Von Willebrand disease (vWD) is caused by von Willebrand factor (vWF) dysfunction resulting from pathogenic variants in the vWF gene coding the vWF protein. vWD type 2N is of particular interest, as it is characterized by almost normal vWF antigen level (Ag:vWF) and vWF loss of ability to bind FVIII and protect it from premature clearance, which leads to a low FVIII coagulation activity (FVIII:C). Therefore, the same phenotype occurs in patients with 2N type of vWD and hemophilia A.Aim — to identify patients with 2N type vWD using molecular genetic methods.Methods. Data from the medical histories of vWD patients were used. The major parameter in consideration was FVIII:C to vWF:Ag ratio, which is expected to be below 0.7 in type 2N of vWD. Pathogenic variants in exons and exon-intron junctions of the vWF gene were identified by Sanger sequencing. Due to recessive inheritance of type 2N, verification of the 2N vWD diagnosis required the identification of two pathogenic variants.Results. Three patients were considered as suffering from type 2N of vWD according to hemostasis parameters (FVIII:C/vWF:Ag < 0.7). One patient with a preliminary hemophilia A diagnosis was included after sequencing of the F8 gene, which showed no alterations, so 2N type of vWD was suspected. In all cases, sequencing of the relevant functional regions of the vWF gene led to verification of vWD type 2N. One woman (patient # 4) had a homozygous pathogenic variant p.Arg854Gln (c.2561 G>A) associated with type 2N vWD. One woman (patient # 3) was a compound heterozygote for the pathogenic variant p.Arg816Trp (c.2446 C>T) associated with type 2N and a newly described insertion c.2098_2099insG, that leads to a frameshift. The woman with FVIII:C/vWF:Ag < 0.7 (patient # 1) and the patient # 2 with preliminary hemophilia А diagnosis were both compound heterozygotes for the same combination of pathogenic variants — c.2435delC and p.Thr791Met (c.2372 C>T). Pathogenic variant p.Thr791Met is associated with type 2N, while the deletion c.2435delC should lead to allele disabling.Conclusion. Molecular methods allow more precise differentiation of type 2N from other types of vWD and hemophilia A.

Introduction. 13q14 deletion is the most common chromosomal abnormality in chronic lymphocytic leukemia (CLL), and as the sole abnormality determines the most favorable prognosis of the disease. Using molecular genetic methods two subtypes of 13q14 deletion were identifi ed based on the size of the lost chromosomal material: small (type I) with the involvement of the D13S319 segment containing MIR15A/MIR16-1 and DLEU1 genes and large (type II) containing centromeric region of 13q14 involving RB1 gene. Data on the impact of type I and II deletions on the course of CLL are controversial.Aim — to evaluate the prognostic signifi cance of different variants of 13q14 deletion in CLL.Patients and methods. The study enrolled two cohorts of CLL patients. Cohort 1: 256 patients who were studied by FISH with DNA probes for detection of 13q14/D13S319, 11q23/ATM, 17p13/TP53 deletions, and trisomy 12 before immunochemotherapy. 101 patients with identifi ed 13q14/D13S319 deletion were analyzed with a DNA probe for RB1 locus for determination of deletion size (type I or type II). Cohort 2: 28 patients at different stages of the disease with deletion 13q14 detected by FISH were studied by using combination of standard and molecular cytogenetic methods (mFISH, mBAND, arrayCGH) to clarify the structure of 13q abnormalities.Results. In Cohort 1 chromosomal aberrations were detected in 75 % of patients: 13q deletion — 52 % (isolated — 36 % of all cases and 48 % of cases with deletion), 11q deletion — 19 %, +12 — 13 %, 17p deletion — 6 %. 13q14 deletion type I was detected in 56 %, type II — in 44 % of patients. Type II deletion correlated with the presence of 11q deletion (p = 0.05). Isolated deletions of type I and II were found in 61 and 39 %, respectively. Biallelic deletion was identifi ed in 12.7 % of patients with 13q deletion. Statistically signifi cant differences in OS were obtained in type I and II groups of patients with isolated 13q14 deletions: median OS was not reached and made 67.5 months, respectively, p = 0.05. In Cohort 2 structural abnormalities of chromosome 13 by conventional cytogenetic analysis (CCA) were identifi ed in 50 % of cases: 13q deletion — 11 cases; translocations involving 13q14 — 6 cases. In 5 cases with biallelic deletion identifi ed by FISH, 13q14 deletion by CCA was detected in two patients, and only in one allele.Conclusion. In general, 13q14 deletion is a cytogenetic factor of favorable prognosis for CLL but its structure is heterogeneous. Loss of tumor suppressor RB1 (type II deletion) negatively affects OS in patients treated with immunochemotherapy

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%.

Introduction. Among main curative substances in acute lymphoblastic leukaemia/lymphoma (ALL/LBL) is 6-mercaptopurine (6-MP). However, the severity of adverse reactions (ADRs) to this drug varies considerably among patients, which is sometimes conditioned by individual single nucleotide polymorphisms in key 6-MP metabolism enzyme genes.Aim — a literature review on the role of TPMT and NUDT15 gene variants in 6-MP metabolism in ALL/LBL.Main findings. The TPMT and NUDT15 genes encode enzymes mediating key steps of the 6-MP metabolism. The metabolites determine the 6-MP therapeutic and toxic properties, with ADRs emerging when their concentrations alter. A number of TPMT and NUDT15 single nucleotide polymorphisms are associated with varied activities of the encoded enzymes, and their allelic combinations condition functional and non-functional phenotypes. Non-functional variant carriers more likely develop toxicity on 6-MP treatment compared to functional phenotypes. Non-functional TPMT/NUDT15 carriers should have the 6-MP dosage reduced to minimise emerging ADRs.

Simultaneous quantitative measurement of mRNA of the WT1, BAALC, EVI1, PRAME and HMGA2 genes in whole blood samples reflects the specific pathological proliferative activity in acute leukemia and their ratio is promising as a diagnostic marker. The transcriptome profile of acute leukemia cells is usually assessed using NGS or microarray techniques after a preliminary procedure for isolation of mononuclear cells. However, the results of using the multiplex PCR reaction for the simultaneous determination of all above mRNAs in whole blood samples have not been published so far. Determination of mRNA of WT1, BAALC, EVI1, PRAME and HMGA2 genes in venous blood level samples by multiplex RT-PCR. The study included 127 blood samples from patients who diagnosis of acute leukemia was subsequently confirmed. In the comparison group, 87 samples of patients without oncohematological diagnosis were selected, including 31 samples (K1) with a normal blood formula and 56 samples (K2) with a violation of the cellular composition - anemia, leukocytosis and thrombocytopenia. RNA isolation and reverse transcription were performed using the Ribozol-D and Reverta-L kits (TsNIIE, Russia). Determination of the mRNA expression level of the WT1, BAALC, EVI1, PRAME and HMGA2 genes by multiplex real-time PCR using a homemade multiplex PCR kit. The mRNA level was characterized by high interindividual variation and did not correlate with the rate of circulating leukocytes or blood blasts. Expression of WT1 mRNA was observed in whole blood only in one patient from the control group and in 112 (88%) patients with leukemia and was combined with a decrease in the level of HMGA2 mRNA expression and BAALC mRNA values. In contrast to the control groups, patients with leukemia had higher levels of BAALC mRNA in AML and ALL, increased PRAME mRNA in AML and APL, but lower levels of HMGA2 in APL.

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.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.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).The key factor that significantly affects the long-term results of therapy is the rate of MRD after the first course of induction CT.Цель. Проанализировать результаты терапии больных острыми миелоидными лейкозами (ОМЛ) по протоколам ОМЛ-17 и модифицированному ОМЛ-17 (мОМЛ-17) в рамках двух последовательных пилотных исследований с целью разработки оптимальной химиотерапевтической стратегии в лечении больных ОМЛ в возрасте до 60 лет. Материалы и методы. В исследование включены 89 больных ОМЛ в возрасте моложе 60 лет, получавших терапию согласно протоколам ОМЛ-17 и мОМЛ-17. Цитогенетическое и молекулярно-генетическое исследования осуществляли всем больным. Оценивали наличие мутаций в генах FLT3, NPM1, CEBPa методом фрагментного анализа. У 35 больных выполнили исследование на mutTP53, mutRUNX1 методом секвенирования нового поколения (next generation sequencing NGS). Минимальную остаточную популяцию опухолевых клеток оценивали методом многоцветной проточной цитометрии. Статистический анализ проводили с помощью процедур пакета SAS 9.3. Результаты. Полная ремиссия (ПР) достигнута у 89,7% больных, пролеченных по интенсивным программам, и у 52,4% больных, получивших низкодозное воздействие. Рефрактерными к терапии оказались 8,8% больных, пролеченных интенсивно, и 38% не ответили на низкодозное воздействие. Показатель ранней летальности составил 3%. Общая и безрецидивная 3-летняя выживаемость всех больных, включенных в 2 последовательных исследования, составили 60 и 67% соответственно. Значимым стал показатель минимальной остаточной болезни (МОБ) после 1-го курса индукционной терапии. Трехлетняя безрецидивная выживаемость тех больных, у кого ПР достигнута после 1-го индукционного курса и МОБ не выявлена (получен МОБ-негативный статус), составила 90% в сравнении с 43% у тех, у кого при достижении ПР после 1-го курса определялся МОБ-позитивный статус (р=0,00001). Заключение. Ключевой фактор, существенно влияющий на долгосрочные результаты терапии, это показатель МОБ после 1-го курса индукции.

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.Тромботические осложнения значимые факторы, определяющие прогноз при миелопролиферативных новообразованиях. Маркерами, позволяющими оценить риск развития тромбозов, являются количество лейкоцитов, тромбоцитов, уровень гемоглобина, гематокрит, возраст, молекулярный статус, тромбозы в анамнезе, ожирение, артериальная гипертензия, гиперлипидемия, наследственная или приобретенная тромбофилия. Патогенез тромбозов у больных с миелопролиферативными новообразованиями сложный и многофакторный. В большинстве случаев этиологический фактор остается неизвестным. В настоящее время терапия антиагрегантами и антикоагулянтами проводится в индивидуальном порядке. Алгоритм первичной и вторичной (после состоявшегося тромбоза) профилактики требует разработки и апробации. Мы представляем клиническое наблюдение повторные артериальные и венозные тромботические осложнения у больного с первичным миелофиброзом.

The development of National clinical guidelines on diagnosis and treatment of Ph-negative myeloproliferative neoplasms comes in response to the need to standardize the approach to diagnosis and treatment. The availability of clinical guidelines can facilitate the choice of adequate treatment strategy, provides practicing physicians with exhaustive and up-to-date information on advantages and shortcomings of different treatment methods as well as lets health professionals better assess expected extents of treatment required by patients. In 2013 a working group was formed to develop and formulate clinical guidelines on the treatment of myeloproliferative neoplasms. These guidelines were first published in 2014, afterwards they were revised and republished. The dynamic development of current hematology presupposes constant updating of knowledge and implementation of new diagnosis and treatment methods in clinical practice. In this context clinical guidelines present a dynamic document to be continuously amended, expanded, and updated in accordance with scientific findings and new requirements of specialists who deal directly with this category of patients. The present edition is an upgraded version of clinical guidelines with updated information on the unification of constitutional symptoms assessment using MPN-SAF TSS questionnaire (MPN10), on applying prognostic scales in primary myelofibrosis, assessing therapy efficacy in myeloproliferative neoplasms, revising indications for prescription, on dose correction, and discontinuation of targeted drugs (ruxolitinib). The guidelines are intended for oncologists, hematologists, healthcare executives, and medical students.

Introduction. Mutations in the TP53 gene in patients with mantle cell lymphoma (MCL TP53+) are associated with a low response to intensive chemotherapy (CT) and adverse outcomes. Allogeneic haematopoietic stem cells transplantation (allo-HSCT) is a curative approach in MCL-TP53+ patients.Aim. Efficacy and safety assessment of allo-HSCT in MCL-TP53+ patients.Main findings. During 2016–2020, allo-HSCT in MCL TP53+ was performed in three patients. Two of them were grafted from HLA-identical unrelated donors, and one — from a haploidentical donor. Pre-transplant conditioning was “fludarabine + treosulfan + melphalan” in one case, and “fludarabine + busulfan” — in the other two. In three patients, leukocyte and platelet counts were recovered at days +18 and +20, +17 and +21, +19 and +16 after allo-HSCT, respectively. Acute graft-versushost disease (aGVHD) was observed in all patients (grade I — in 2 patients, grade IV — in 1 patient). One patient developed chronic GVHD (cGVHD) of moderate grade. All three patients exhibited complete remission and 100% donor chimerism in allo-HSCT follow-up of 6, 15 and 40 months, respectively.

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.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.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).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.Цель.Изучение активирующих мутаций генов (NRAS,KRAS,FLT3,JAK2,CRLF2) сигнальных каскадов RAS/RAF/MEK/ERK и JAK/STAT при В-клеточном остром лимфобластном лейкозе (В-ОЛЛ) у взрослых больных, включенных в российские многоцентровые исследования. Материалы и методы.В многоцентровое исследование включены 119 взрослых пациентов с впервые установленным В-ОЛЛ. Исследование носило проспективный и ретроспективный характер. Группа сBCR-ABL1-негативным В-ОЛЛ составила 93 больных (48 женщин и 45 мужчин от 17 до 59 лет, медиана возраста 31 год), им проводили терапию по протоколам ОЛЛ-2009, ОЛЛ-2016. Медиана наблюдения составила 19 мес (1119). В группу сBCR-ABL1-позитивным В-ОЛЛ включены 26 пациентов (16 женщин и 10 мужчин от 23 до 78 лет, медиана возраста 34 года). Лечение проводили по протоколам ОЛЛ-2009 и ОЛЛ-2012 в сочетании с ингибиторами тирозинкиназ. Медиана наблюдения составила 23 мес (4120). Молекулярный анализ активирующих мутаций для геновNRAS,KRAS(сигнальный путь RAS/RAF/MEK/ERK) и геновJAK2,CRLF2(сигнальный путь JAK/STAT) проводился методом секвенирования по Сэнгеру. Внутренние тандемные повторы (ITD) генаFLT3 рецепторной внутриклеточной тирозинкиназы, приводящие к запуску целого каскада реакций, относящихся к различным сигнальным путям, включая RAS/RAF/MEK/ERK и JAK/STAT, исследованы методом фрагментного анализа. Экспрессию белка CRLF2 оценивали методом проточной цитофлуориметрии. Результаты.Активирующие мутации в генахNRAS,KRAS,FLT3обнаружены у 22 (23,6%) больныхBCR-ABL1-негативным В-ОЛЛ. В общей сложности выявлено 23 мутации в генахNRAS(n=9),KRAS(n=12),FLT3(n=2), что статистически значимо чаще, чем приBCR-ABL1-позитивном В-ОЛЛ, где мутации этих генов не выявлены ни у одного больного (р=0,007). Частота обнаружения мутаций в генахKRASиNRASу больныхBCR-ABL1-негативным В-ОЛЛ сопоставима 12,9% (12 из 93) и 9,7% (9 из 93) соответственно (р=0,488). У одного больного выявлено одновременно 2 мутации в генеKRAS(в кодонах 13 и 61). МутациюFLT3-ITD детектировали в 3,5% (2 из 57) случаевBCR-ABL1-негативных В-ОЛЛ. У больныхBCR-ABL1-позитивным В-ОЛЛ мутациюFLT3-ITD не оценивали. Нарушения в сигнальном каскаде JAK/STAT обнаружены у 4 (4,3%) больныхBCR-ABL1-негативным В-ОЛЛ. Они представлены миссенс-мутациями генаJAK2(n=3) и гиперэкспрессией CRLF2 (n=2), у одного больного обнаружены одновременно гиперэкспрессия CRLF2 и мутация в генеJAK2. В генеCRLF2мутации не выявлены. У больныхBCR-ABL1-позитивным В-ОЛЛ мутаций генаJAK2не выявлено. При анализе демографических и клинико-лабораторных показателей между группами больных с мутациями и без таковых статистически значимых различий не получено. В анализируемых группах больных долгосрочные результаты терапии в зависимости от наличия мутаций в генахNRAS,KRAS,FLT3,JAK2не различались. Также не показано значимых отличий в скорости достижения негативного статуса минимальной остаточной болезни между пациентами с активирующими мутациями и без в контрольные сроки протокола (на 70, 133 и 190-й день). Заключение.Активирующие мутации геновNRAS,KRAS,FLT3,JAK2не влияют на долгосрочные результаты терапии и скорость достижения негативного статуса минимальной остаточной болезни у больныхBCR-ABL1-негативным В-ОЛЛ при применении протоколов российского многоцентрового исследования.

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.Первичный миелофиброз (ПМФ) представляет собой миелопролиферативное новообразование, которое возникает de novo, характеризуется клональной пролиферацией стволовых клеток, аномальной экспрессией цитокинов, фиброзом костного мозга, гепатоспленомегалией как следствие экстрамедуллярного гемопоэза, симптомами опухолевой интоксикации, кахексией, лейкоэритробластозом в периферической крови, лейкемической прогрессией и невысокой выживаемостью. ПМФ является хроническим неизлечимым заболеванием. Цели терапии: предупреждение прогрессии, увеличение общей выживаемости, улучшение качества жизни. Выбор лечебной тактики ограничен. Трансплантация аллогенных гемопоэтических стволовых клеток (алло-ТГСК) единственный метод, дающий шанс на излечение. В настоящее время активно изучается роль мутаций ряда генов в раннем выявлении кандидатов для алло-ТГСК. В статье представлено описание клинического случая выявления мутации гена ASXL1 у больного префиброзным ПМФ. Диагноз установлен на основании критериев Всемирной организации здравоохранения 2016 г. При обследовании выявлена мутация ASXL1. Проводится терапия интерфероном альфа, в результате которой достигнута клинико-гематологическая ремиссия. Несмотря на выявленную мутацию, пациент не является кандидатом для алло-ТГСК. Учитывая неблагоприятное прогностическое значение мутации ASXL1, пациент подлежит активному динамическому наблюдению и агрессивной терапевтической тактике при появлении признаков прогрессирования заболевания.

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.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.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.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 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.Распространенность первично-множественных опухолей в последнее время значимо выросла, но вопрос выбора оптимальной тактики терапии при данной нозологии по-прежнему остается нерешенным. Особый интерес представляет одномоментное, или симультанное, выявление двух новообразований родственного происхождения в одном и том же очаге или очагах поражения. В данной публикации представлен случай одномоментной диагностики миелоидной саркомы и лимфомы из клеток мантийной зоны у больной 65 лет, потребовавший последовательного применения двух различных подходов и схем химиотерапии. Этот пример демонстрирует необходимость внедрения расширенного диагностического подхода с применением гистологических, иммуногистохимических, цитогенетических, молекулярных методов исследования на всех этапах диагностики и лечения, что позволяет своевременно выбрать правильную тактику терапии и достичь полной ремиссии при длительных сроках наблюдения по двум новообразованиям при первично-множественных опухолях.

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.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.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).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.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.Обоснование. Синдром ЛиФраумени (СЛФ) редкое доминантно наследуемое заболевание, характеризующееся генетической предрасположенностью к опухолям различной природы, в том числе к опухолям кроветворной ткани. Генетической основой СЛФ являются мутации гена TP53. Пациентам с СЛФ и их родственникам требуются генетическое консультирование с последующим наблюдением и выбор оптимальной терапевтической тактики в случае развития онкологического заболевания. Цель. Описать серию клинических наблюдений пациентов с острым лимфобластным лейкозом (ОЛЛ) и СЛФ и рассмотреть общие вопросы диагностики и лечения взрослых больных с этим наследственным генетическим синдромом. Материалы и методы. Исследование мутаций гена TP53 выполнено 180 больным de novo Ph-негативным (В- и Т-клеточным) и Ph-позитивным ОЛЛ, лечение которым проводили по протоколам российской исследовательской группы (ОЛЛ-2009, ОЛЛ-2012, ОЛЛ-2016) в ФГБУ НМИЦ гематологии и региональных клиниках участниках многоцентровых исследований. Результаты. Мутации гена TP53 обнаружены у 7,8% (n=14) больных de novo ОЛЛ. Для доказательства герминального характера мутаций и подтверждения СЛФ статус гена TP53 оценивали в ремиссии на образцах костного мозга и периферической крови, на образцах костного мозга после трансплантации аллогенных гемопоэтических стволовых клеток и в тканях некроветворного происхождения. Всего в анализ включены 5 больных (из 14, у которых обнаружены мутации), неопухолевый биологический материал которых доступен для исследования. Герминальный характер мутаций, свидетельствующий о наличии СЛФ, подтвержден у 4 из 5 В-клеточный ОЛЛ (n=3), Т-клеточный ОЛЛ (n=1) обследованных пациентов. Заключение. Таким образом, важным практическим выводом работы является наблюдение, что основная часть мутаций гена TP53 у больных Ph-негативным В-клеточным ОЛЛ носит герминальный характер и ассоциирована с CЛФ.

Standard therapy in hairy cell leukemia (HCL) is often impossible at the time of deep neutropenia/agranulocytosis with or without infectious complications; it is thus a complex therapeutic problem. Vemurafenib has been used to treat resistant HCL since 2012. Because vemurafenib does not have a myelotoxic effect, we thought that it could be used to treat HCL associated with deep neutropenia/agranulocytosis with or without the development of infectious complications as a preliminary stage before treatment with cladribine. We conducted a retrospective analysis of treatment with vemurafenib followed by a standard course of cladribine provided to 22 patients with deep neutropenia/agranulocytosis with or without infectious complications at diagnosis. Vemurafenib was provided to 22 patients with HCL. The response to therapy was evaluated by complete blood cell count (absolute neutrophil count [ANC], hemoglobin concentration, platelet count, absence of hairy cells), spleen size (assessed by ultrasound), and reduce infectious complications. After that, a standard course of cladribine was provided. Among the 22 patients, the male/female sex ratio was 2:1, and median (range) age was 52 (24-78) years. There were 7 patients with severe infectious manifestations admitted to the intensive care unit, including 1 patient during extracorporeal membrane oxygenation. The median (range) ANC at diagnosis was 0.3 (0.04-0.7) × 10

Objective. To identify mutations in UL97 gene associated with antiviral drug resistance in recipients of allogeneic hematopoietic stem cells transplants (allo-HSCT). Materials and Methods. A total of 9 HCMV DNA samples were studied. These samples were received from the blood of 8 allo-HSCT recipients who were infected with HCMV and undergoing treatment at NMRC of Hematology (Russia) over the period of 2016 to 2017. Sanger sequencing was used to find mutations. The sequenced part of the virus DNA was analyzed using nucleotide BLAST and Genome compiler. Mutations were identified using MRA program which compared the obtained nucleotide sequence with the reference sequence of UL97 gene from Merlin strain. Results. Rate of detection of viruses with mutations that may lead to drug resistance is relatively high – 3 of 8 patients. The following mutations were identified: C592G, C607F and C603W. The obtained data shows that the presence and characteristics of mutations affect the viral load and the time when HCMV DNA is identified in blood. Conclusions. Obtained data show that presence of mutations impacts the course infection, leading to higher viral load and longer persistence of viral DNA in blood samples. Identified mutations had different resistance factor, which also impacted on the pattern of infection.

The detection of somatic mutations in the 9 exon of the calreticulin gene (CALR) is regulated by the clinical recommendations as a diagnostic criterion for chronic Ph-negative myeloproliferative neoplasms (MPN). Some methods of nucleic acids testing are used to identify CALR gene mutations with different requirements for special skills of personnel and expensive equipment. The purpose of this work is to compare the results of the detection of CALR gene mutations in venous blood samples by allele-specific RT-PCR with subsequent electrophoresis, fragment analysis and Sanger- or pyro- sequencing. We used 1284 blood samples of patients with suspected MPN and 20 blood donor samples. Mutations in the CALR gene of the I and II type were identified using PCR-RT with the original primers and TaqMan probes. Also, all samples were tested for mutations in the CALR gene by electrophoretic detection of PCR results in an agarose gel. The use of allele-specific RT-PCR followed by electrophoretic detection made it possible to determine clinically significant mutations in the CALR gene in 81 venous blood samples of JAK2- and MPL-negative patients, including 42 cases of type I mutation, 33 cases of type II mutation and 8 rare CALR mutations. Mutations in the 9 exon of the CALR gene were not detected in any of the 20 blood donor samples or in 121 blood samples of patients with polycythemia vera. In randomly selected 20 negative samples, CALR gene mutations were also not detected using Sanger sequencing. All positive samples were confirmed by fragment analysis, as well as with Sanger- sequencing and pyro- sequencing. The described combined approach to detect mutations of the CALR gene in peripheral blood samples can be used in clinical diagnostic laboratories that have a standard set of equipment for electrophoresis of nucleic acids and a PCR-RT. We also propose a confirmatory test based on the pyrosequencing of DNA using the system of genetic analysis "PyroMark Q24".

Introduction: Loss of heterozygosity (LOH) was described for many malignancies including leukemia. Using chromosomal microarray analysis (CMA) we have shown preliminary (Risinskaya et al. HemaSphere, 2019, V.3, No S1, P.782) that LOH in STR loci in tumor cells with the normal karyotype represents the loss of extended chromosomal areas and could be associated with uniparental disomy of the entire chromosomal arm or the entire chromosome. Uniparental disomy results not only in the elimination of the wild type allele, but also may lead to the duplication of the mutant allele of certain candidate tumor gene(s). Retrospective analysis of archival samples have shown that in most cases STR loci absent at the relapse of disease were already lost at the onset of disease. We noted LOH at STR loci is especially frequent for relapsed ALL patients and could be used as prognostic factor for possible poor clinical outcome. The aim: To identify LOH in the blast cells of the patients with ALL at the onset of disease and to analyze therapy outcome relative to the patients without LOH. We expect LOH in the de novo ALL to be an unfavorable factor, and those patients possibly should be treated like high-risk group. Patients and Methods: This study includes a comparative analysis of the STR profiles of the DNA of tumor and normal cells from bone marrow samples on a cohort of 32 patients with de novo diagnosed Ph-negative ALL undergoing treatment according to the "RALL-2016" regimen at the National Research Center for Hematology (Moscow, Russia). Inclusion criteria: de novo diagnosed Ph-negative acute lymphoblastic leukemia (ALL) patients, 18-55 years old, intermediate risk group without MLL translocation t(4;11)(q21;q23), treatment by RALL-2016 protocol. Exclusion criteria: other diagnosis, adult patients older than 55 years, MLL translocation t(4;11)(q21;q23), pretreatment or treatment by other protocol. The presence of blast cells in bone marrow samples was confirmed morphologically. DNA was isolated from patient bone marrow samples taken at different disease stages. Control DNA samples were taken from blood of patients in complete remission and/or from buccal epithelium (buccal swab). STR-profiles were assessed by PCR with COrDIS Plus multiplex kit for amplification of 19 polymorphic STR-markers and amelogenin loci (Gordiz Ltd, Russia). The fragment analysis was performed on ABI3130 Genetic Analyzer. The data processing was accomplished using GeneMapper v.4-0 software. A multivariate survival analysis was used to assess the independent impact of the LOH as risk factor against the background of the abnormal tumor karyotype as standard factor for this cohort of patients. We have chosen Failure Free Survival (FFS) as primary endpoint for short-time follow-up and small patient sample size. Death from any reasons, relapse and second leukemia were chosen as failure events, survival time interval starts from begin of treatment. SAS V9/4 was used for statistical analysis of the data. Results: Of the 32 patients six were found with LOH in the certain STR loci (19%). Of these six, two were resistant to therapy and died from disease progression. One patient with LOH is currently in relapse and one patient with multiple LOH has a second leukemia (Fig.1a). Only two from six patients are "Failure Free" now, whereas only three patients from 26 in group without LOH died (two from infectious complications and one from GvHD after BMT). P value < 0,05 (Fig.1b). Conclusions: We have found statistically significant association of clinical failures with the LOH in STR loci measured at the onset of ALL. We assume that LOH is an unfavorable prognostic factor for de novo ALL patients and could be used for risk stratification and choice of adequate therapy implying allogeneic hematopoietic stem cells transplantation or modern innovative therapy approaches. Disclosures No relevant conflicts of interest to declare.

The article presents the data on the studies contributing to improving the differential diagnostics of T-cell and B-cell cutaneous lymphomas including large-plaque parapsoriasis and T-cell and B-cell cutaneous pseudolymphomas as well as frequency of their transformation into malignant cutaneous lymphomas. There was a study of 101 patients using the polymerase chain reaction method to determine the T-cell and B-cell lymphocyte clonality by genes of g and ƒ chains in the T-cell receptor and immunoglobulin heavy chain genes. Monoclonality was determined in 40 of 46 cases in patients with T-cell cutaneous lymphomas and in three of four cases in patients with B-cell cutaneous lymphomas. Monoclonality was revealed in one of 14 cases of large-plaque parapsoriasis and one of two cases of T-cell cutaneous pseudolymphoma. In all of the 24 cases of chronic benign dermatoses, five cases of small-plaque parapsoriasis and ten skin tissue samples obtained from healthy donors, polyclonal lymphocytes were revealed. So, the obtained results make it possible to consider the method to be an important addition in the field of diagnosing lymphoproliferative skin diseases.

A photokinetic method of detection of fluorescence resonance energy transfer (FRET) between special fluorescent labels is applied to study time-averaged spatial distribution of labeled proteins in protein assemblies. Prolonged irradiation of a sample at the absorption maximum of the energy donor initiates FRET-sensitized fluorescence photobleaching of the energy acceptor label, which was monitored by steady-state fluorimetric measurements. Kinetics of the acceptor photobleaching and kinetics of decreasing the efficiency of FRET from donors to unbleached acceptors were determined. The FRET efficiency was found from measuring sensitization of acceptor fluorescence. Analysis of the photokinetic data permits to estimate the time-averaged distribution of acceptors on donor-acceptor distances in the range of characteristic distances of FRET. Dynamic processes influencing donor-acceptor distances can be also investigated by the method. Application of the method is demonstrated by the studies of a complex of biotinylated IgM with streptavidin and aggregates composed of concanavalin A and sodium dodecyl sulphate. A new thiadicarbocyanine dye was used as the acceptor label. R-phycoerythrin and tetramethylrhodamine isothiocyanate were the donor labels. In the IgM-streptavidin complex, 16% of acceptors most contributed to FRET provided 90% of FRET efficiency, whereas acceptors made about the same time-averaged contribution to FRET in the concanavalin A aggregates.

PCR-based clonality assay of rearranged T-cell receptor genes gamma and beta (TCRG and TCRB) in a number of cases could be essential to discriminate between cutaneous T-cell lymphomas and reactive lymphoproliferative lesions in the skin. However, extraction of good-quality DNA from skin specimens (especially formalin-fixed paraffin-embedded) remains a challenge. Common procedures, being labour-intensive and time-consuming and requiring toxic solvents such as phenol and chloroform, still may end up with DNA sample of insufficient quality. We herewith present a simple and efficient method for DNA isolation based on ammonia extraction of tissue, followed by neutralization and simultaneous salting out of proteins with acetic acid. We have analysed 30 samples - 24 fresh (16 skin, two spleen and six lymph node) and six paraffin-embedded. Standard procedure (proteinase K digestion, followed by phenol/chloroform extraction) has been carried out simultaneously. We observed good PCR signal for TCRG rearrangements in 30 samples processed with the new protocol and only in 20 extracted with proteinase K/phenol/chloroform. For TCRB, the success rate was 29 of 30 with the new protocol, compared to 11 of 30 with conventional protocol. The proposed method of DNA extraction should improve the value of T-cell clonality assay, because insufficient DNA quality and quantity may bias analysis towards monoclonality and therefore cause false-positive results.

In the present study we have analyzed superinduction of TNF-alpha mRNA and enhancement of TNF-alpha gene transcription by cycloheximide (Chx) in human blood monocytes isolated by continuous Percoll gradient and activated in vitro. In the same monocyte cultures, we have compared the rate of gene transcription of TNF-alpha, IL-1 beta, IL-8, and the P53-antioncogene under the influence of plastic adherence, Staphylococcus aureus Cowan 1 (SAC), and Chx added at different times of monocyte culture. It was shown that the cytokine genes have low or negligible transcriptional activity in freshly isolated monocytes, whereas P53 gene transcription was constant in freshly isolated and in vitro-stimulated cells. Transcription of the IL-1 beta and IL-8 genes was induced by adherence and was not more enhanced by SAC. Transcription of the TNF-alpha gene was not induced by adherence. Chx added at the beginning of the monocyte culture did not block TNF-alpha or IL-1 beta gene transcription. IL-8 gene transcription, however, was abrogated by Chx. Addition of SAC to monocyte culture containing Chx caused significant enhancement of TNF-alpha gene transcription. Addition of Chx after 2.5 or 4 h of SAC activation caused "superinduction" of TNF-alpha mRNA and enhancement of TNF-alpha gene transcription. The data imply that TNF-alpha gene transcription in activated human monocytes might be regulated by both positive and negative regulatory factors that differ in their stability and protein synthesis dependence. In addition, results demonstrate that TNF-alpha, IL-1 beta, IL-8, and p53 genes in human monocytes are differently regulated.

To compare diagnostic efficacy of B-cell clonality determination in application of different electrophoretic 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.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.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.

Abstract 2090 BACKGROUND. Coinfection with hepatitis B virus (HBV) and hepatitis C virus (HCV) in immunocompromised patients (pts) with hematological malignancies is not well-studied problem. Early recognition of HBV and HCV infectioning incidence in such pts is difficult due to influence of cytostatic and immunosuppressive therapy and possible mutual HBV or HCV inhibitions. Independence of HBV and HCV infection events has to be discussed. AIM. To determine the prevalence of HBV and HCV co-infection in pts with hematological malignancies to check the possible correlation between the incidence of the infections. PATIENTS AND METHODS. All 265 patients treated in hematology department during 2004–2006 years were prospectively continuously monitored and examined for markers HBV and HCV (HBsAg, anti-HCV, DNA HBV, RNA HCV, HBeAg, anti-HBs, anti-HBc, anti-HBe) in serum, plazma, blood cells, bone marrow, cerebrospinal fluid) and clinico-biochemical symptoms of liver dysfunction were registered approximately every 3 weeks until 2008 year. Liver biopsy was performed on 64 pts in 2004–2006 yy, 23 pts esch with HBV and HCV immunohistology. Approximately 80% of pts were with acute leukemias (AL) and aplastic anemias (AA). Male; 47% (n=125), female; 53% (n=140). The median age was 38 years (range, 15 to 79). RESULTS. At the beginning of treatment and monitoring 17 of 265 pts (6.4%) had positive markers of HCV and the number of pts with positive markers of HCV increased up to 70 (26%) to the end of monitoring, and for HBV markers increased from 23 pts (8.7%) to 174 pts (65.3%). We found, that many pts were co-infected with HBV and HCV, and their total number increased from 5 pts (1,9%) äî 37 pts (14%). Positive tests of HBV and HCV co-infection was found in 57 pts of 265, that is 32.8% of 174 HBV-positive pts and 81.4% of 70 HCV-positive pts, odds ratio (OR=2.9) (95%CI =1.5–5.7), xi square significance p=0.0012. More complicate longitude analysis did not show that the incidence rate of second infection is significantly higher than the first. That means that the correlation between first and second infections is still questionable. Coinfection with HBV and HCV resulted in clinical presentation of hepatitis B and/or hepatitis C in most cases and cumulative incidence rate rises up to 100% at 630 day after the first appearance of any positive marker. CONCLUSION. The high prevalence of HBV and HCV coinfection of pts with hematological malignancies is attributed to common risk factors for infection transmission after transfusions of blood components and parenteral medical procedures. For pts with hematological malignancies after first detection of positive test for single marker of HBV and/or HCV it is very reasonable to intensify the monitoring of HBV and HCV markers and to follow clinical signs. Disclosures: No relevant conflicts of interest to declare.

The present study was undertaken to assess the presence of tumor necrosis factor (TNF)-alpha mRNA and protein in circulating human blood monocytes and to study the TNF-alpha gene expression in human monocytes isolated by continuous Percoll gradient fractionation. The technique of RNA isolation directly from the blood samples was used to study TNF-alpha mRNA expression in circulating human blood leukocytes. It was shown that human blood leukocytes of healthy donors contained no presynthesized pool of TNF-alpha mRNA as well as no TNF-alpha protein. It was found that early pretreatment with cycloheximide interferes with TNF-alpha mRNA induction by Staphylococcus aureus.

The mechanisms of interaction of nuclear and mitochondrial genes in biogenesis of mitochondria are discussed in this review. Brief characterization of yeast mitochondrial genes and their products is presented. The mechanism of nuclear and mitochondrial control of expression of the mosaic genes in mitochondria is described. The data on the processing of imported mitochondrial proteins synthesized on cytoplasmic ribosomes are presented. The possibility of existence of common proteins encoded for by common genes and possessing similar functions in the cytoplasm and mitochondria is discussed. A hypothesis is put forward considering the role of common proteins in coordination of nuclear and mitochondrial genes' expression in biogenesis of mitochondria.

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.