Study of mRNA of WT1, BAALC, EVI1, PRAME and HMGA2 genes in whole blood samples.

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.

[Mutations in the UL97 gene of cytomegalovirus (Herpesvirales: Herpesviridae: Cytomegalovirus: Human betaherpesvirus 5) associated with ganciclovir resistance in recipients of allogeneic hematopoietic stem cells].

INTRODUCTION: Infection caused by cytomegalovirus (CMV) is a serious problem for patients with weakened immunity, including patients with hematopoietic depression. The cases of complications associated with cytomegalovirus require antiviral therapy. However, during the natural mutation process, especially with prolonged use of drugs in suboptimal doses, CMV strains resistant to the action of antiviral drugs (such as ganciclovir, valganciclovir) may occur. Hypothetically, the emergence of resistance in the virus may cause a more aggressive course of infection, the ineffectiveness of antiviral therapy and, as a result, an increase in the number of deaths. In this regard, timely detection of mutations that can potentially lead to the resistance of the virus to antiviral drugs during hematopoietic stem cell transplantation (HSCT), as well as during organ and tissue transplantation, may be important when making a therapeutic decision. We describe three clinical cases for which the dynamics of the appearance of a mutant strain of CMV by the UL97 gene, which correlates with the viral load and clinical picture, is analyzed.The aim of the study was to determine the timing of the occurrence of mutations in CMV phosphotransferase UL97 gene associated with resistance to antiviral drugs in patients with hemoblastoses after allogeneic hematopoietic stem cell (allo-HSCs) transplantation. MATERIAL AND METHODS: The study included 48 samples of CMV DNA isolated from the peripheral blood of three allo-HSCs recipients with CMV infection who were treated in the clinics of the FSBI «National Medical Research Center for Hematology¼ of the Ministry of Health of Russia with oncohematological diseases during 2015-2017. Patients received conditional codes (PR, PD, and FS). Mutations associated with antiviral therapy (AVT) resistance were identified in all patients. Sanger sequencing was used for mutation detection. The obtained DNA sequences were analyzed using Nucleotide BLAST and Genome compiler software. Mutations were searched in MRA mutation resistance analyzer software. The nucleotide sequences were compared with the UL97 reference sequence of the Merlin CMV strain using this software environment. RESULTS AND DISCUSSION: For all patients in whom the virus strains containing C592G (PR), C607F (PD) and C603W (FS) mutations were detected, the timing of the mutation occurrence was determined at days 187, 124 and 1184, respectively. The emergence of mutations with a high resistance factor was shown to be accompanied by an increase in viral load (VL), the appearance of a clinical picture characteristic of CMV infection and a lack of an adequate response to therapy with ganciclovir and its derivatives. CONCLUSION: Using these results, it is proposed to develop the test system based on random polymerase chain reaction (rPCR) to detect mutations in the most frequently encountered codons: M460I/V, C592G, A591V, A594T/V, L595F/S, C603W. Given that the data on the prevalence of these mutations were obtained from foreign sources, it is advisable to conduct similar studies on the frequency of mutations in the UL97 gene among the population of the Russian Federation in order to improve the quality and accuracy of test systems.

Pitfalls in mononucleotide microsatellite repeats instability assessing (MSI) in the patients with B-cell lymphomas.

Analysis of microsatellite instability (MSI) is a routine study in the diagnostics of solid malignancies. The standard for determining MSI is a pentaplex PCR panel of mononucleotide repeats: NR-21, NR-24, NR-27, BAT-25, BAT-26. The presence of MSI is established based on differences in the length of markers in the tumor tissue and in the control, but due to the quasimonomorphic nature of standard mononucleotide loci the use of a control sample is not necessary in the diagnosis of MSI-positive solid tumors. The significance of the MSI phenomenon in oncohematology has not been established. This paper presents the results of a study of MSI in B-cell lymphomas: follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), high-grade B-cell lymphoma (HGBL). We have shown that aberrations of mononucleotide markers occur in these diseases, but the nature of the changes does not correspond to the classical MSI in solid neoplasms. This fact requires further study of the pathogenesis of such genetic disorders. Due to the possibility of ambiguous interpretation of the results of the MSI study for previously uncharacterized diseases, strict compliance with the methodology of parallel analysis of the tumor tissue and the control sample is mandatory.

[Development of program therapy for patients with acute myeloid leukemia under the age of 60 years, based on the principles of differentiated effects].

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

[Li-Fraumeni syndrome in adult patients with acute lymphoblastic leukemia].

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

[Multiple primary tumor of hematopoietic tissue: myeloid sarcoma in combination with mantle cell lymphoma. Case report].

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.

[Thrombosis in patients with myeloproliferative neoplasms. Case report].

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.

[Detection of activating mutations in RAS/RAF/MEK/ERK and JAK/STAT signaling pathways].

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

[Informativeness of whole-body diffusion-weighted magnetic resonance imaging and positron emission tomography with computed tomography in follicular lymphoma].

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

[The prognostic value of ASXL1 mutation in primary myelofibrosis. Literature review and clinical case description].

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.

[Molecular diagnosis angioimmunoblastic T-cell lymphoma].

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

Results of program acute myeloid leukemia therapy use in National Medical Research Center for Hematology of the Ministry of Health of Russian Federation.

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

Cepeginterferon alfa-2b in the treatment of chronic myeloproliferative diseases.

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

First experience of using Brentuximab vedotin and modified program NHL-BFM-90 in the front-line treatment of patient with anaplastic large-cell lymphoma: a case report and a review of literature.

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

Coinheritance of HbD-Punjab/ß+-thalassemia (IVSI+5 G-C) in patient with Gilbert's syndrome.

Thalassemia and qualitative hemoglobinopathy are hereditary disorders of Hb synthesis that lead to change in the Hb conformation or a decrease in the synthesis of structurally normal Hb, and consequently, to erythron pathology. Many variants of Hb are unstable or have altered affinity for oxygen, and, in heterozygous form can be associated with clinical and hematological manifestations (hemolytic anemia, hypochromic microcytic anemia, erythrocytosis). HbD-Punjab [ß121 (GH4) Glu → Gln; HBB: C.364G> C] is variant of Hb carrying the amino acid substitution in the 121 position of ß-globin chain. In all cases reported so far, patients with HbD-Punjab/ß+-thalassemia (IVSI+5 G-C) combination experienced typical thalassemia with hypochromic microcytosis. HbD-Punjab was detected by electrophoresis from 37 to 94% of total Hb. The article describes rare clinical case of the cohabitation of HbD-Punjab/ß+-thalassemia (IVSI+5 G-C) in a patient with homozygous variant of Gilbert's syndrome observed in AS Loginov Moscow Clinical Scientific Center.

Diagnostics and treatment challenges of Ph-like acute lymphoblastic leukemia: a description of 3 clinical cases.

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

[Allele-specific polymerase chain reaction and electrophoretic detection in the detection algorithm clinically significant somatic mutations in the gene of calreticulin (calr).]

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".

[Uromodulin gene polymorphisms in patients with cast nephropathy in multiple myeloma]. / Polimorfizmy gena uromodulina u bol'nykh kast-nefropatiei pri mnozhestvennoi mielome.

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

[Clinical features of essential thrombocythemia and primary myelofibrosis, depending on the molecular characteristics of disease]. / Klinicheskie osobennosti éssentsial'noi trombotsitemii i pervichnogo mielofibroza v zavisimosti ot molekuliarnykh kharakteristik zabolevaniia.

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

[Diffuse large B-cell lymphoma with concomitant c-MYC and BCL6 gene rearrangements with primary skin involvement: A case report and a review of literature]. / Diffuznaia V-krupnokletochnaia limfoma s sochetannoi rearanzhirovkoi genov c-MYC i BCL6 s pervichnym porazheniem kozhi: sobstvennoe nabliudenie i obzor literatury.

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