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 × 10 9 / 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 × 10 9 / 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 TET 2 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.

Introduction . Non-chemotherapy for acute promyelocytic leukaemia (APL) with a combination of all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) provides for a high patient survival rate at lesser toxicity as effectively or superior to standard chemotherapy programmes. Aim  — assessment of the ATO–ATRA risk-adapted exposure protocol in management of de novo acute promyelocytic leucaemia. Materials and methods . A prospective study included 51 primary APL patients aged 18–76 years. The program included remission induction (ATO 0.15 mg/kg intravenously, ATRA 45 mg/m2 orally) for 30–60 days in a low-risk (until remission) and 60 days — in a high-risk cohort that had idarubicin therapy added on days 2 and 4. Remission consolidation was attained with four (low-risk) or five (high-risk) courses. Minimal residual disease was monitored with real-time PCR at all phases. Results . The high-risk cohort was assigned 15 (29.4 %), the low-risk cohort — 36 (70.6 %) patients. Therapy induction till APL morphological remission was performed in 48/51 (94 %) patients. Molecular APL remission was achieved in 47 (92 %) patients, 100 % in the low-risk and 80 % in high-risk cohort. Early mortality was 6 % (n = 3), death in remission — 2 % (n = 1). Differentiation syndrome (DS) occurred in 16 (31.7 %) patients, more frequently in the high-risk vs. low-risk cohort (53.3 % and 22.2 %, respectively, p = 0.05; odds ratio 4.0 [1.1–14.4]). DS developed on days 1–20 (3 days median) of therapy. DS risk factors: a high-risk status, haemorrhagic syndrome and infection at the disease onset. A median follow-up time in survivors was 12.9 months (2.5–34.3), a six-month overall survival — 92 % (95 % CI: 85–100 %). A six-month overall survival was 100 and 73 % in the low- and high-risk cohorts, respectively (95 % CI: 54–100 %, p = 0.001). APL relapse not registered, 47 (92 %) patients survived and achieved the first molecular remission. Conclusion . A differentiated risk-adapted approach to APL therapy with cytostatic treatment added in high-risk patients only provided for a 100 % molecular remission and relapse-free survival. Therapy failures (early mortality and death in remission) affected high-risk patients due to a severe individual condition at the time of APL diagnosis.

Introduction . Non-chemotherapy for acute promyelocytic leukaemia (APL) with a combination of all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) provides for a high patient survival rate at lesser toxicity as effectively or superior to standard chemotherapy programmes. Aim — assessment of the ATO–ATRA risk-adapted exposure protocol in management of de novo acute promyelocytic leucaemia. Materials and methods . A prospective study included 51 primary APL patients aged 18–76 years. The program included remission induction (ATO 0.15 mg/kg intravenously, ATRA 45 mg/m2 orally) for 30–60 days in a low-risk (until remission) and 60 days — in a high-risk cohort that had idarubicin therapy added on days 2 and 4. Remission consolidation was attained with four (low-risk) or five (high-risk) courses. Minimal residual disease was monitored with real-time PCR at all phases. Results . The high-risk cohort was assigned 15 (29.4 %), the low-risk cohort — 36 (70.6 %) patients. Therapy induction till APL morphological remission was performed in 48/51 (94 %) patients. Molecular APL remission was achieved in 47 (92 %) patients, 100 % in the low-risk and 80 % in high-risk cohort. Early mortality was 6 % (n = 3), death in remission — 2 % (n = 1). Differentiation syndrome (DS) occurred in 16 (31.7 %) patients, more frequently in the high-risk vs. low-risk cohort (53.3 % and 22.2 %, respectively, p = 0.05; odds ratio 4.0 [1.1–14.4]). DS developed on days 1–20 (3 days median) of therapy. DS risk factors: a high-risk status, haemorrhagic syndrome and infection at the disease onset. A median follow-up time in survivors was 12.9 months (2.5–34.3), a six-month overall survival — 92 % (95 % CI: 85–100 %). A six-month overall survival was 100 and 73 % in the low- and high-risk cohorts, respectively (95 % CI: 54–100 %, p = 0.001). APL relapse not registered, 47 (92 %) patients survived and achieved the first molecular remission. Conclusion . A differentiated risk-adapted approach to APL therapy with cytostatic treatment added in high-risk patients only provided for a 100 % molecular remission and relapse-free survival. Therapy failures (early mortality and death in remission) affected high-risk patients due to a severe individual condition at the time of APL diagnosis.

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.

Angioimmunoblastic T-cell lymphoma (AITL) is a T-cell lymphoma, characterized by abundant polymorphocellular infiltrate of lymph nodes with the small number of tumor CD4+ Tfh-cells. AITL could often be misdiagnosed as reactive processes and other lymphomas, including Hodgkin's lymphoma and diffuse large B-cell lymphoma (DLBCL). We used quantitative allele-specific PCR with LNA (locked nucleotide acid) modified primers (qAS-PCR-LNA) for RHOA G17V mutation assay. Sensitivity of determination (0.02%) was sufficient for minimal residual disease (MRD) monitoring and evaluation of tumor cell number in different tissues. Method proposed demonstrated sensitivity superior to histology and PCR-based clonality determination. RHOA G17V mutation in lymph nodes was detected in 53% (32 of 62) patients with AITL. In control group (n-110) we have revealed RHOA G17V mutation in 3 patients with Hodgkin’s lymphoma (HL) and 1 patient with diffuse large B-cell lymphoma (DLBCL). Three patients with HL had clonal CD4+ T-lymphocytes population with aberrant immunophenotype in blood and clonal rearrangements of TCRG and/or TCRB genes in lymph nodes. We have shown that RHOA G17V can be used as a screening marker for patients with lymphadenopathy to exclude AITL or PTCL NOS. The persistence of tumor cells with RHOA G17V mutation was shown in most patients (12 of 16 -75%) with AITL after the induction chemotherapy and during the maintenance therapy (5 of 7 - 71.4%). Therefore qAS-PCR-LNA can be enrolled into standard protocols for management of patients with AITL to assess the effectiveness and the duration of antitumor therapy.

Having a tropism for erythroid progenitor cells, parvovirus B19 may cause partial red cell aplasia and thrombocytopenia. Early diagnosis of parvovirus B19 infection in immunocompromised patients is needed for timely antiviral therapy. A high-risk group for parvovirus B19 infection includes patients with blood diseases who receive multiple transfusions of blood components; those who have undergone donor organ transplantation and are long taking immunosuppressive drugs; and pregnant women. These patients require careful virological monitoring for major blood-borne viral infections. This paper describes a clinical case of parvovirus B19 infection in a pregnant woman who has undergone kidney transplantation and is continuously taking immunosuppressive medications. Identification of the cause of severe anemia and timely adequate therapy could lead to the recovery of effective erythropoiesis in the patient.

The mutation V617F of gene JAK2 is detected in 95% of patients with genuine polycythemia, in 50% of patients with essential thrombocytemia and idiopathic myelofibrosis. The mutation V617F can be applied as a molecular marker of response to treatment in patients with chronic myeloproliferative diseases associated with this mutation. The technique of quantitative evaluation of V617F (sensitivity up to 0.01%) using polymerase chain reaction is described. This method can be applied to assess the minimal residual disease in patients with chronic myeloproliferative diseases.

The mutation V617F of gene JAK2 is detected in 95% of patients with genuine polycythemia, in 50% of patients with essential thrombocytemia and idiopathic myelofibrosis. The mutation V617F can be applied as a molecular marker of response to treatment in patients with chronic myeloproliferative diseases associated with this mutation. The technique of quantitative evaluation of V617F (sensitivity up to 0.01%) using polymerase chain reaction is described. This method can be applied to assess the minimal residual disease in patients with chronic myeloproliferative diseases.

The incidence of B19 parvovirus (PV B19) was analyzed in patients with hematological diseases, treated on an inpatient or outpatient basis and examined at Hematology Research Center in 2005-2010. Detection of PV B19 DNA in the serum by the real time PCR indicated infection of patients by PV B19. No difference in the incidence of PV B19 DNA in patients of different genders was detected. The incidence of PV B19 marker was 2-fold higher in patients aged less than 40 years vs. those aged over 40. The PV B19 infection was the most incident in patients with acute leukemias. The incidence of PV B19 DNA was higher in the patients with longer hospitalization. Hematological patients constitute a group at a high risk of hemotransmissive viral infections, and hence, their PV B19 infection status should be monitored. Blood components intended for transfusions to patients with immunity disorders should be tested for PV B19 DNA.

Parvovirus (PV) B19 DNA was tested by real time polymerase chain reaction in blood serum of the donors. The PV B19 DNA was detected in 29 (1.9%) of 1521 tested sera. In 3 donors the concentrations of the DNA were higher than 10(5) copy/ml (0.2%), in 5 donors DNA concentrations were 10(4)-10(5) copy/ml (0.3%), and in 21 donors PV B19 DNA concentrations were below 10(4) copy/ml (1.4%). The IgM antibodies were found in 1 donor with the virus concentration higher than 10(4) copy/ml, IgM and IgG together were found in 6 donors with the virus concentrations higher than 10(4) copy/ml, while 1 donor with high concentration of the virus had neither IgM nor IgG antibodies. Only IgG antibodies to PV 619 were detected in 21 donors with PV 619 DNA concentrations below 10(4) copy/ml. The results and analysis of published data indicate that donor blood should be tested for PV B19 DNA and blood of the donors with the virus concentrations higher than 10(4) copy/ml should not be used.

Time course of hepatitis B and C virus infection in patients with hematological disorders. T. Ts. Garmaeva, S. M. Kulikov, E. A. Mikhailova, R. E. Igla, T. F. Shmatova, E. G. Gemdzhyan, L. O; Grumbkova, N. G. Yaroslavtseva, A. V. Somova, T. A. Tupoleva, T. V. Makarik, O. A. Glinshchikova, I. S. Fevraleva, A. B. Sudarikov, F. P. Filatov, V. G. Savchenko. Hematology Research Center, Moscow. The patients with hematological disorders refer to a population of a high risk of hepatitis B and C virus infection. The risk factors in these patients are repeated blood transfusions and medical manipulations involving violation of the skin intactness. We carried out a dynamic analysis of hepatitis B and C virus infection in hematological patients during hospitalization and studied the risk of nosocomial infection factors. We found out high infection rates: the incidence of hepatitis B increased 3.5 times (from 14.7 to 51%), incidence of hepatitis C increased 2.7 times (from 7 to 19%), and of coinfection 7-fold (from 2 to 14%). Blood component transfusions were the leading risk factor for nosocomial hepatitis B and C virus infection. Medical manipulations were the second risk factor for hepatitis B virus infection.

Clinical epidemiological characteristics of the incidence of infection by hepatites B and C viruses in hematological patients on admission to hospital. T. Ts. Garmaeva, S. M. Kulikov, E. A. Mikhailova, T. F. Shmatova, E. G. Gemjyan, T. A. Tupoleva, L. O. Grumbkova, N. G. Yaroslavtseva, A. V. Somova, T. V. Makarik, O. A. Glinshchikova, I. S. Fevraleva, A. B. Sudarikov, F. P. Filatov, V. G. Savchenko. Hematology Research Center, Moscow. The incidence of infection by viruses of hepatitis B (HBV) and hepatitis C (HCV) in different population groups is determined by the main factors of viral infection transmission risk in these groups. The aim of this study was clinical and epidemiological characterization of hematological patients, specifically, evaluation of the probability of detection of HBV and HCV markers (in connection with the risk factors) on admission to hospital. The incidence of HBV and HCV infections in hematological patients on admission to hospital was at least 2-fold higher than in the population of Russia. The main significant factors of viral infection risk in hematological patients were history of blood transfusions and medical manipulations, as well as contacts with patients with viral hepatitis B and hepatitis C, and a history of invasive studies. The impact of such well-known risk factors as narcotic abuse and active sexual behavior is extremely negligible in hematological patients. The importance of detecting nonspecific indicators of HBV and HCV infection, hepatic enzymes, and total bilirubin values, improving the detection of potentially infected individuals and donors of blood components, is confirmed.

Hepatites B (HBV), C (HCV) viruses, and parvovirus B19 (PV B19) are particularly hazardous for patients receiving multiple transfusions of blood components during treatment, as donor blood testing for serological markers of viral hepatites alone does not completely rule out the probability of infection, and no testing for PV B19 is carried out. We suggest a diagnostic system based on multiplex chain reaction in the real time mode for simultaneous testing of blood serum and plasma specimens and tissue biopsy specimens for HBV, HCV, and PV B19. A total of 737 patients blood specimens were tested by this multiplex method; HBV was detected in 45 patients, HCV in 113, PV B19 in 25 patients; HBV + HCV were detected in 4 patients, PV B19 + HCV in 1 patient. Our system for complex monitoring of HBV, HCV, and PV B19 will timely detect the infection or will clear out the cause of clinically diagnosed hepatitis.

In this work a new type of the internal standard for the competitive RT-PCR is proposed: the RNA standard on the basis of the retrovirus vector. The internal standard has been developed for the qualitative and quantitative determination of hepatitis C virus RNA in patients' blood serum. The proposed internal standard consists of recombinant retrovirus particles in the culture medium of packing cells, containing the modified sequence of hepatitis virus C used for amplification in PCR. The presence of the selective marker--the gene of resistance to puromycin--in the vector makes it possible to determine the titer of virus particles of the standard in experiments of infecting cell cultures. The proposed retrovirus standard is obtained from the culture of packing cells by collecting the cultivation medium used for growing these cells. The virus particles thus obtained are not pathogenic for humans and animals.