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Background. The national observational program MPN-QoL-2020 was focused on quality of life (QoL) and symptoms in patients with classical Ph-negative myeloproliferative neoplasms (MPNs) in the Russian Federation, as well as on the perception of the disease and treatment from the patient's and physician's perspective. Aim. To evaluate QoL in patients with different MPNs using new standardized questionnaires, to assess the most common symptoms and their impact on QoL in patients with myelofibrosis (MF), polycythemia vera (PV) and essential throm-bocythemia (ET), and to characterize the perception of the disease and treatment concerns from patients' perspective and their treating physicians' perspective. Materials & Methods. In total 1100 patients with MPNs (MF: n = 355, PV: n = 408, and ET: n = 337; mean age 58 ± 14 years; 61 % women) and 100 hematologists (mean age 42 ± 12 years; 85 % women) from 37 medical centers in 8 Federal districts of the Russian Federation participated in the study. All the patients filled out symptom assessment tool (MPN10), QoL questionnaire for patients with hematological nancies (HM-PRO) and patient's survey checklist; physicians filled out physician's survey checklist and patient record for each patient included in the study. Results. For the first time in Russia in a representative population of MPN patients in the real-world setting, QoL and symptom profiles in patients with different MPNs were characterized and symptom impact on the daily living of MPN patients was identified. MPN patients exhibited QoL impairment: noticeable detriments in physical and emotional functioning, as well as in eating and drinking regimen were found, social functioning was less impaired. More than one third of MPN patients had significant QoL impairment. The vast majority of patients experienced fatigue: 92.6 % MF patients, 83.7 % PI patients, and 82 % ET patients. Symptom prevalence severity differed across different MPNs. Top disease-related symptoms to be resolved were identified from patient's and physician's perspective. Discrepancies in the attitudes of MPN patients and their treating physicians to various aspects regarding the disease and its treatment were found as well as issues needed to be improved in the patient-physician communication were identified. Conclusion. The results of national research program MPN-QoL-2020 allowed to identify the areas of QoL impairment and symptom burden in MPN patients in Russia, to verify areas of concern related to the disease and its treatment in patients with different MPNs, as well as to highlight the unmet needs in this patients' population in our country. The outcomes of the study may contribute to establishing recommendations for improving/maintaining QoL in patients with MPNs and to developing measures aimed to raise awareness of this patients' population about the disease and its treatment.

Background. The introduction of tyrosine kinase inhibitors (TKIs) into real-world clinical practice considerably improved the prognosis for patients with chronic myeloid leukemia (CML). However, during long-term follow-up, almost 1/2 and 2/3 of patients in the chronic phase (CP) discontinue TKI therapy of the first or second line, respectively. According to the Russian and International clinical guidelines, the third-line therapy should include allogeneic hematopoietic stem cell transplantation (allo-HSCT). And yet, some patients on the third-line therapy achieve and sustain optimal response on long-term TKI administration. Up to now, no clear-cut prognostic factors of TKI efficacy in the third-line therapy have been identified. This creates a challenge for treatment decision making after the failures of two lines of TKI therapy. Aim. To assess the efficacy of the third-line TKI therapy in real-world clinical practice and to identify the factors affecting the long-term therapy outcomes in CML-CP. Materials & Methods. The retrospective study enrolled 73 CML-CP patients aged > 18 years, treated with TKIs in the third-line at 5 specialized institutions in Saint Petersburg and Leningrad Region. Among the patients there were 26 men (35 %). The median age of the patients was 51 years (range 25-88 years). Results. With the median (range) third-line TKI therapy duration of 14 (1-120) months, the rate of complete cytogenetic response (CCR) was 30 % (п = 22) in the total cohort. The median time before achieving CCR was 9 (4-25) months. With the median follow-up time from the beginning of third-line TKI therapy till the last visit of 25 (3-136) months, progression to accelerated phase or blast crisis was observed only in 13 (17 %) out of 73 patients. Death was reported in 26 % (n = 19) of cases, among them 5 patients whose death was not CML-associated. At the last visit, 13/73 (18 %) patients were still on third-line TKI therapy. Direct and longterm therapy outcomes, including achievement of CCR and assessment of overall and progression-free survivals, were significantly better in patients with any cytogenetic response (CR) than in those without it or without complete hematologic response. Conclusion. The implementation of TKIs in the third-line CML-CP therapy seems to be suitable for patients with at least some CR, especially if an optimal donor of hematopoietic stem cells is unavailable or if the risk of severe allo-HSCT complications is too high.

Relevance. The relevance of the study is determined by the fact that hopes are placed in the cell therapy for patients with critical limb-threatening (CLI) ischemia as a method of the restoration of blood circulation in the affected limb in patients who cannot undergo surgical or endovascular intervention. Aim. To evaluate the efficiency of allogeneic MSCs for the treatment of critical lower limb ischemia (randomized placebo-controlled study).Materials and methods. The study included 34 patients with critical lower limb ischemia (grade 4 according to Pokrovsky). There were 18 patients in the MSC group, and 16 patients in the placebo group). The groups were comparable concerning age, disease duration, and comorbidities. Allogeneic MSCs (phenotype CD73+, CD90+, CD105+, CD45–, CD34–, CD14–) were injected into the posterior calf muscles. Clinical outcome, ankle pressure, transcutaneous oxygen tension (tcpO2), and pain-free walking distance (PFWD) were evaluated. The patients were followed-up for 12–36 months. According to the clinical outcome in each group, the patients were divided into subgroups with «effect (+)» or «effect (–)». In 2 patients, there was an «uncertain clinical outcome». When analyzing the results, these patients were assigned to one or another subgroup.Results. In the MSC and placebo groups, the clinical outcome assessed as «effect (+)» or «effect (–)» did not differ (OR 1.5; 95 % CI 0.34–6.7). With different variants of group formation and with the assignment of patients with an «uncertain clinical outcome» to a one or another subgroup, the final results neither differed. According to instrumental research methods (PFWD, tcpO2, ankle pressure, angiography), there were no differences in the MSC and placebo groups. Conclusion. With different variants of analysis and group formation, no convincing evidence that allogeneic MSCs can be effective for the treatment of critical lower limb ischemia have been obtained.

NK-cells as innate immunity elements manifest key reactions of antitumor immune response. NKG2D is an activating transmembrane receptor of NK-cells which is responsible for cytotoxicity initiation in response to the binding of specific ligands of genetically modified cells. Selective expression of NKG2D ligands provides a unique perspective on the therapy of wide variety of tumors. Acute myeloid leukemias (AML) are malignant hematological tumors with a high relapse risk. Due to the complexity of AML treatment strategy it is necessary to develop new approaches to tumor elimination using novel genetic constructs. Currently available CAR T-cell drugs with NKG2D receptor are successfully subjected to clinical studies in AML patients and prove their high therapeutic potential.

Aim. To identify mutations in IDH1/IDH2, DNMT3A, and ASXL1 genes responsible for genome epigenetic regulation and their co-occurrence with FLT3, NPM1, and RUNX1 mutations in newly diagnosed adult acute myeloid leukemias (AML). Materials & Methods. The study included 56 patients with newly diagnosed AML treated at the VA Almazov National Medical Research Center. Among them there were 34 men and и 22 women aged 18-76 years (median 46 years). Mutation status of IDH1, IDH2, DNMT3A, and ASXL1 genes of epigenetic regulation was assessed by Sanger sequencing method. Molecular genetic analysis of FLT3, NPM1, and RUNX1-RUNX1T1 genes was performed using commercial kits. Results. Mutations in epigenetic regulation genes were detected in 14 (25 %) out of 56 patients. Mutation prevalence was not associated with risk groups (p = 0.072). IDH1/2 mutations were identified in 15.6 % of patients and were significantly oftener observed concurrent with NPM1 mutations (62.5 %; p = 0.01) compared to patients with wild-type IDH1/2. In most patients IDH1/2 mutations were associated with normal karyotype (p = 0.002). The DNMT3A (R882) mutation was identified in 4 (7.1 %) out of 56 patients within the analyzed group. In 6 patients (11.1 %) ASXL1 mutations were detected co-occurring with RUNX1-RUNX1T1 and FLT3-ITD mutations. Conclusion. Mutations in epigenetic regulation genes are often identified in AML patients and can be concurrent with abnormalities in NPM1, FLT3 и RUNX1 genes.

Aim. To assess the efficacy, safety, and tolerance of gemtuzumab ozogamicin (GO) combined with FLAG/FLAG-Ida chemotherapy or azacitidine in patients with relapsed/refractory acute myeloblastic leukemia (AML) in clinical practice. Materials & Methods. The study included 32 patients (16 men and 16 women). The median age was 44 years (range 23-83 years). Among them there were 15 (46.8 %) patients with refractory and 17 (53.2 %) patients with relapsed AML. GO combined with FLAG/FLAG-Ida was administered to 15 (46.8 %) patients, whereas 17 (53.2 %) patients were treated with GO and azacitidine combination. Therapy safety was assessed according to CTCAE v. 5.0. Results. Overall response rate including complete remission (CR), CR MRD-, CR with incomplete hematologic recovery, and morphologic leukemia-free status was 59.4 % (19/32). Refractoriness was observed in 31.25 % (10/32) of patients. Early mortality was 9.4 % (3/32). Overall response was 64.7 % (11/17) in the azacitidine and 53.3 % (8/15) in the FLAG/FLAG-Ida groups. In 4 (80 %) out of 5 patients with prior to FLAG treatment refractoriness, the response was achieved after GO + azacitidine therapy. In 58.9 % (10/17) of patients who received GO + azacitidine therapy, allogeneic hematopoietic stem cell transplantation (allo-HSCT) could be performed. The incidence of GO infusion complications in the tested groups did not significantly differ (p = 0.72) and was 46.7 % (7/15) (40 % with grade 1/2 and 6.7 % with grade 3) in the GO + FLAG/FLAG-Ida group and 35.3 % (6/17) (29.4 % with grade 1/2 and 5.9 % with grade 4) in the GO + azacitidine group. In the GO + FLAG/FLAG-Ida group 5 (33.3 %) patients experienced serious adverse events (SAE) of sepsis. In the GO + azacitidine group SAEs were reported in 6 (35.3 %) patients: 4 (66.6 %) with sepsis, 1 (16.7 %) with acute cardiovascular failure, and 1 (16.7 %) with acute respiratory failure. The median (range) duration was 23 (10-39) days for neutropenia grade 4, 24 (11-38) days for neutropenia grade 3, 21 (11-41) days for thrombocytopenia grade 4, 26 (16-45) days for thrombocytopenia grade 3, and 25 (22-45) days for thrombocytopenia grade 1/2. Thrombocytopenia duration was longer in patients with GO + FLAG/FLAG-Ida therapy, however, no significant differences were identified. No cases of veno-occlusive liver disease were reported. Median overall survival (OS) for both groups (n = 32) was 31.4 months, median disease-free survival (n = 21) was 13.3 months. In the group of patients with effective treatment, the median OS was not reached. In non-responders, it was 18 months (р = 0.0442). Conclusion. GO combined with FLAG/FLAG-Ida chemotherapy or azacitidine proved effective in relapsed/refractory AML patients. Remission did not appear to be associated with ELN risk, gender, age, CD33 expression, number of prior therapy lines, or number of relapses. GO + azacitidine combination showed efficacy, safety, and good tolerance in patients with prior high-dose chemotherapy refractoriness as well as low ECOG performance status. That allowed for the subsequent allo-HSCT administration to these patients. There was no significant difference between the groups of patients in the incidence of hematologic, non-hematologic toxicity, and time to hematologic recovery. Thrombocytopenia duration was longer in patients with GO + FLAG/FLAG-Ida therapy which is consistent with literature data. GO-based effective treatment in relapsed/refractory AML considerably improves OS: during 36 months of follow-up the median was not reached.

Classical Ph-negative myeloproliferative neoplasms (MPN) constitute a group of diseases including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. Over the past decade, the approaches to understanding of MPN pathogenesis and therapy have considerably changed. At the same time, etiological factors and pathophysiological mechanisms of disease progress are being thoroughly studied. The improvement of diagnosis methods and new approaches to therapy can reduce complications and mortality risks. The review outlines the current diagnosis methods, such as the molecular genetic one, and provides prognostic scores. Different methods of conservative therapy are assessed. Special attention is paid to quality of life measurement and targeted treatment of patients.

Aim. The observational program was aimed at obtaining data on classical Hodgkin's lymphoma (cHL) incidence in the Russian Federation, therapy options, and clinical outcomes of treatment. The aim of the prospective part of the program was to standardize the approaches to therapy and to compare its outcomes with off-protocol treatment. Materials & Methods. The prospective-retrospective observational program for Hodgkin's lymphoma treatment engaged 32 regional and federal centers. It included 218 patients, 21 out of them were included into the prospective part of the RNWOHG-HD1 (Russian North-West Oncology and Hematology Group - Hodgkin Disease Study 1) program. The median age was 36 years (range 22-87 years). cHL stages I/II were identified in 48 % of patients, III/IV stages were reported in 52 % of patients. The prospective part of the program used escalating protocol in patients with stages I/IIA and without risk factors and de-escalating protocol in patients with advanced stages. Overall (OS) and progression-free (PFS) survivals were analyzed in 160 and 152 patients, respectively. PET-CT was used to assess the response in 33 % of patients. Results. The study used the following first-line chemotherapy regimens: ABVD in 42 %, BEACOPPst in 11 %, BEACOPP-14 in 17 %, BEACOPPesc in 25 %, and EACOPP in 1 % of cases. After the completion of first-line therapy objective response rate was 91 % including 61 % of complete responses. Response structure did not significantly differ in the groups of non-intensive therapy (ABVD and BEACOPPst), intensified regimens (BEACOPP-14, BEACOPPesc, and EACOPP), and treatment according to the RNWOHG-HD1 protocol (91 %, 92 %, and 96 %, respectively; p = 0.7226). In the total cohort the 3-year OS was 97 % (95% confidence interval [95% CI] 94-99 %), PFS was 87 % (95% CI 80-92 %). The 3-year PFS did not differ in ABVD, BEACOPPst, BEACOPP-14, BEACOP-Pesc, and RNWOHG-HD1 recipients (р = 0.37). International Prognostic Score (IPS) yielded significant results in PFS prediction for patients with IPS score of 5-6, but not for those with IPS score of 1-4 (p = 0.0028). Conclusion. The observational program showed that the majority of participating centers use the risk-adapted ABVD/ BEACOPPesc approach which explains no difference in PFS being found with the use of these chemotherapy options. The study demonstrated the need for PET-CT to assess the response since the CT alone cannot distinguish between complete and partial responses in a considerable number of patients. The prospective unified program for cHL treatment may well be implemented in the Russian Federation.

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.

Myeloid sarcoma, also known as chloroma or granulocytic sarcoma, is a rare disease characterized by the proliferation of immature myeloid cells in extramedullary lesions. Chloroma is more commonly observed in patients with acute myeloid leukemias, other myeloproliferative neoplasms, or myelodysplastic syndrome. However, it can also manifest itself as solitary tumor. Sarcoma can develop in different organs and tissues, but most frequently it appears in lymph nodes, soft tissues, and bones. Myeloid sarcoma with primary gynecological lesion is very rarely mentioned. In literature cases of cervical lesions are described. The present article summarizes the literature data concerning different aspects of myeloid sarcoma diagnosis and treatment. The issue under discussion is the role of chemotherapy, radiotherapy, surgery, and bone marrow transplantation in the treatment of this malignant tumor. It appears that whatever the primary tumor localization, the best treatment options are chemotherapy and allogeneic bone marrow transplantation (allo-BMT). A promising trend is the use of novel targeted drugs improving outcomes of treatment. The article provides a case report of a female patient with cervical myeloid sarcoma and concomitant bone marrow involvement, as well as the description of clinical course, diagnosis, and treatment. The patient received chemotherapy with subsequent allo-BMT. The pre-transplant therapy enabled allo-BMT with the deepest response possible. The patient achieved PET-and MRD-negative complete remission of cervical myeloid sarcoma and bone marrow.

Background. Up to the present the survival rates of acute myeloid leukemia (AML) patients have remained low. A successful AML management presupposes generating personalized models of the disease. The most promising research activity in this field is creation of AML patient-derived xenograft models using the advanced strain of immunodeficient humanized NSG-SGM3 mice. Aim. To generate AML patient-derived xenograft models using immunodeficient NSG-SGM3 mice. Materials & Methods. The creation of PDX models was based on bone marrow aspirates taken from 4 patients with newly diagnosed AML who were treated at the V.A. Almazov National Medical Research Center. Patient-derived tumor cells were transplanted to NSG-SGM3 mice. Test experiment consisted in injecting AML cells OCI-АМL2 and HL60 in NSG-SGM3 mice. The efficacy of tumor engraftment was evaluated in terms of physical condition of animals and laboratory tests (blood count, blood smear, PCR, and flow cytofluorometry). Results. The engraftment of applied tumor cells derived from AML patients was achieved in half (2 out of 4) of the transplanted tumor samples. In mice with successful transplantation leukocytosis was reported. Blast cells were identified in peripheral blood on Day 30 after transplantation. The mice with injected AML cells OCI-АМL2 and HL60 showed a more aggressive course of disease. Among tested approaches to evaluate tumor engraftment in mouse recipients, the PCR method was marked by highest sensitivity. Conclusion. The use of immunodeficient humanized NSG-SGM3 mice enables successful generation of AML patient-derived xenograft models.

The therapeutic landscape of chronic myeloid leukemia (CML) has profoundly changed over the past 7 years. Most patients with chronic phase (CP) now have a normal life expectancy. Another goal is achieving a stable deep molecular response (DMR) and discontinuing medication for treatment-free remission (TFR). The European LeukemiaNet convened an expert panel to critically evaluate and update the evidence to achieve these goals since its previous recommendations. First-line treatment is a tyrosine kinase inhibitor (TKI; imatinib brand or generic, dasatinib, nilotinib, and bosutinib are available first-line). Generic imatinib is the cost-effective initial treatment in CP. Various contraindications and side-effects of all TKIs should be considered. Patient risk status at diagnosis should be assessed with the new EUTOS long-term survival (ELTS)-score. Monitoring of response should be done by quantitative polymerase chain reaction whenever possible. A change of treatment is recommended when intolerance cannot be ameliorated or when molecular milestones are not reached. Greater than 10% BCR-ABL1 at 3 months indicates treatment failure when confirmed. Allogeneic transplantation continues to be a therapeutic option particularly for advanced phase CML. TKI treatment should be withheld during pregnancy. Treatment discontinuation may be considered in patients with durable DMR with the goal of achieving TFR. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/.

Background. FLT3 gene is an important prognostic molecular marker in acute myeloid leukemia (AML). However, the detection of FLT3 mutations presents a challenge. Aim. To compare techniques used for the detection of FLT3 mutations, and to develop a test-system based on polymerase chain reaction (PCR) for quick and reliable determination of FLT3 mutation status. Materials & Methods. Bone marrow samples obtained from AML patients were subjected to examination. To detect FLT3-ITD и FLT3-TKD mutations PCR was performed with subsequent agarose gel electrophoresis visualization. The results were verified by Sanger sequencing. The data obtained using our test-system were compared with widely applied commercial kit ‘FLT3 Mutation Assay for Gel Detection’ by Invivoscribe. Results. To determine the FLT3 mutation status a PCR test was developed. This technique was validated on 22 bone marrow samples obtained from AML patients. FLT3-ITD mutation was detected in 4 patients, 3 patients showed FLT3-TKD mutation. In 1 patient both mutations were identified. These results fully corresponded to the molecular genetic analysis of FLT3, performed by ‘FLT3 Mutation Assay for Gel Detection’. The chosen technique was validated using Sanger sequencing data analysis. Conclusion. The article offers the review of all existing FLT3 mutation screening techniques and describes the experience of developing the PCR test for FLT3-ITD and FLT3-TKD mutation detection. The chosen technique is affordable and easy to use compared with the others. The present study with its applied nature can provide guidance for both doctors and researchers.

Background. Cytokines are considered as important factors that enhance the efficacy of CAR-T cell therapy. Besides, they are key elements of the pathogenesis of cytokine release syndrome and neurotoxicity in applying the CAR-T technology. However, cytokine effects in the context of CAR-T therapy have not yet been properly studied. Aim. To quantitatively assess cytokine secretion using multiplex assay with co-incubation of anti-CD19 CAR-T lymphocytes with epithelial HeLa and A431 cell lines expressing CD19 on their surface. Materials & Methods. T-lymphocytes were transduced with the lentiviral vector containing anti-CD19-CAR gene. CAR expression was tested based on GFP reporter using flow cytometry. To confirm a specific CAR-T cell activation response to tumor antigen, the levels of interleukin-2, interferon-γ, and tumor necrosis factor-α were measured by means of immunoassay. Cytotoxic activity of CAR-T lymphocytes obtained was examined with their direct co-culturing with target cells. The levels of cytokines isolated prior to and after incubation of targets with CAR-T cells were compared using multiplex assay. Results. The level of some proinflammatory cytokines (interleukin-6, interleukin-1β, interferon-γ) (P < 0.01) increased. The difference in the levels of anti-inflammatory cytokines (interleukin-4, interleukin-10) was inconsiderable, and in the HeLa cell line experiment it was insignificant (p > 0.05). The concentration of granulocyte-macrophage colony-stimulating factor (GM-CSF) was many times higher after incubation with CAR-T lymphocytes (p < 0.01). Conclusion. The trial revealed multiple enhancement of GM-CSF, one of the key elements of the pathogenesis of cy tokine release syndrome and CAR-T-associated neurotoxicity. The results of further studies of GM-CSF can contribute to improving the efficacy of CAR-T therapy with considerably lower toxicity.

Aim. To compare different methods of lentivirus concentration in order to select the best way of providing high-level transduction for generating laboratory CAR-T cells. Materials & Methods. Concentration of lentiviral supernatant was carried out by 4 methods: ultrafiltration, ultracentrifugation, polyethylene glycol (PEG), water-soluble non-ionic polymer, precipitation method, and ion-exchange chromatography. Functional viral titer was determined by mCherry reporter protein expression in the transduced HeLa cell line as well as by rapid immunochromatographic (IC) tests. Physical titer was determined by ELISA. Transduction efficiency of healthy donor’s T-lymphocytes was assessed by flow cytometry with respect to signal intensity of reporter protein FusionRed. Functional activity of generated anti-CD19 CAR-T was evaluated by microscopy after co-cultivation with CD19-HeLa cell line as well as subsequent cytokine testing. Results. Lentivirus purification and concentration by ultrafiltration provided the greatest number of transduced cells, i.e. 84.7 %. Methods of ultracentrifugation, PEG precipitation, and ion-exchange chromatography yielded 56.08 %, 74.22 %, and 21.05 % of T-cell transduction, respectively. Results of rapid IC tests were comparable (г = 0.91) with cell line titer data. The mean T-cell transduction efficiency was 59.55 % ± 2.94 %, and its maximum reached 76.26 %. Conclusion. The focus was laid on optimization of CAR-T cell production during the generation of lentiviral vectors and their purification. Ultrafiltration was selected as the best method of lentiviral supernatant concentration to efficiently transduce T-lymphocytes and to generate functional CAR-T cell population.

Aim. To assess the efficacy of targeted therapy with ruxolitinib in patients with myelofibrosis in real clinical practice in Russia. To determine the prognostic value of spleen reduction in the early stages of ruxolitinib treatment and its effect on overall survival. Materials & Methods. The present retrospective study was based on the data of 10 centers of Russia. It included 56 myelofibrosis (primary or post-polycythemic and post-throm-bocythemic) patients who received ruxolitinib. The median age of patients was 56 years (range 26-76 years). Most of them (59 %) were considered intermediate-1 risk according Results. By the start of data collection most of patients (79 %) had been treated with ruxolitinib. In no case therapy was withdrawn for the reason of drug toxicity. On ruxolitinib constitutional symptoms were reversed in 70 %, 87 %, and 98 % of patients by months 1, 3 and 6, respectively. In 36 % and 46 % of patients by months 3 and 6, respectively, > 50 % decrease in spleen size was observed. Overall, in 31 % and 27 % of cases the size of the spleen decreased by less than 25 % by months 3 and 6, respectively. The factors affecting the changes in spleen size have not been identified. The probability of overall survival by years 2 and 5 of follow-up was 97 % and almost 70 %, respectively. This parameter was significantly affected by the extent of spleen size reduction by month 3 of follow-up as well as by its initial size. Conclusion. Ruxolitinib shows high efficacy for both decrease of general myelofibrosis symptoms and reduction in spleen size. The extent of spleen reduction is an important prognostic factor. It seems, that in patients with insufficient spleen reduction an increase in drug dose is advisable. If it is not possible, alternative methods of treatment should be sought.

The treatment of refractory acute myeloid leukemia (AML) patients presents considerable challenges. They are often critically ill. The critical conditions of these patients are mainly associated with severe infectious complications resulting in sepsis as well as with the extramedullary lesions with organ dysfunctions. So far, the obtained data demonstrated the successful use of gemtuzumab ozogamicin, the mechanism of which is likely to be based not only on depletion of CD33-positive tumor cells but also on its immunomodulatory effect. The present article focuses on the fast-effect mechanisms of gemtuzumab ozogamicin and deals with clinical experience of successful use of this drug combined with hypomethylating agents in patients with refractory AML whose condition is critical by the time therapy begins. The use of this drug combination results in fast stabilization of health status, recovery of internal organs, and apyrexia with the decreasing systemic inflammatory response within the first days of therapy. All this together with significantly lower blast count in blood and in bone marrow can bring critically ill patients to recovery.

Prognostic scores support clinicians in selecting risk-adjusted treatments and in comparatively assessing different results. For patients with chronic-phase chronic myeloid leukemia (CML), four baseline prognostic scores are commonly used. Our aim was to compare the prognostic performance of the scores and to arrive at an evidence-based score recommendation. In 2949 patients not involved in any score development, higher hazard ratios and concordance indices in any comparison demonstrated the best discrimination of long-term survival with the ELTS score. In a second step, of 5154 patients analyzed to investigate risk group classification differences, 23% (n = 1197) were allocated to high-risk by the Sokal score. Of the 1197 Sokal high-risk patients, 56% were non-high-risk according to the ELTS score and had a significantly more favorable long-term survival prognosis than the 526 high-risk patients according to both scores. The Sokal score identified too many patients as high-risk and relatively few (40%) as low-risk (versus 60% with the ELTS score). Inappropriate risk classification jeopardizes optimal treatment selection. The ELTS score outperformed the Sokal score, the Euro, and the EUTOS score regarding risk group discrimination. The recent recommendation of the European LeukemiaNet for preferred use of the ELTS score was supported with significant statistical evidence.

Aim. To assess the efficacy of FLAG/FLAG-Ida regimen and to identify factors that influence remission, duration of disease-free survival (DFS) and overall survival (OS) of patients with relapsed/refractory acute myeloid leukemia (AML). Materials & Methods. The trial included 54 patients (28 men and 26 women), median age was 37 years (range 18–70 years). 27 (50 %) out of 54 patients had refractory AML and 27 (50 %) patients had relapsed AML. FLAG and FLAG-Ida regimens were administered as induction therapy. 37 (68.5 %) patients received bone marrow transplantation. Molecular genetic and cytogenetic examinations were performed prior to therapy and on the 28th day after the start of treatment. WT1 gene expression was evaluated on the 14th–16th day of treatment. Results. Complete remission (CR) was achieved in 42 (77.8 %) out of 54 patients. Refractoriness to therapy was observed in 9 (16.7 %) out of 54 patients, mortality was 5.5 % (3/54). Remission rate was higher in patients with relapsed AML compared with refractory AML: 85.2 % (23/27) and 70.4 % (19/27), respectively. On the 14th–16th day of treatment patients with blast cell count ≥ 10 % in bone marrow (BM) showed significantly lower CR rate (60 %) compared with the group of patients with < 10 % blast cells in BM (89.6 %; p = 0.024) and shorter DFS (median 7.6 vs. 17.6 months, respectively; p = 0.03). Median DFS in patients with WT1 expression reduction to < 1 log on the 14th–16th day was 5 vs. 18 months in patients without WT1 expression reduction (p = 0.01). DFS varied in groups of patients with blast cell count < 10 % in BM on the 14th–16th day of treatment based on the level of WT1 expression reduction (p = 0.04). MRD-negative patients (57.1 %) showed significantly longer DFS and OS compared with MRD-positive patients (median DFS was 17.6 vs. 5.2 months, respectively, p = 0.02; median OS was 19 vs. 6.9 months, p = 0.0002). Median DFS and OS were different only in ELN low- and high-risk groups (median not reached vs. 5.2 months, respectively, p = 0.039; median not reached vs. 10.2 months, p = 0.039). Conclusion. FLAG and FLAG-Ida are effective and safe regimens in the treatment of relapsed/refractory AML. Achieving remission depends on neither the risk group nor the time of relapse occurrence. The blast cell count in BM on the 14th–16th day of FLAG/FLAG-Ida treatment is a prognostic factor determining achievement and duration of remission. WT1 expression level in the early post-induction period is a sensitive DFS marker. MRD status and molecular genetic risk (ELN) group affiliation are essential prognostic factors determining DFS and OS.

Aim. To assess the rate of WT1 gene overexpression and its clinical value in Ph-negative myeloproliferative disorders (MPD). Materials & Methods. The trial included 72 patents with Ph-negative MPD. Among them there were patients with primary myelofibrosis (MF; n = 32), post-polycythemia vera MF (n = 7), polycythemia vera (PV; n = 17), and essential thrombocythemia (ET; n = 16) with median age of 57 years (range 19-78 years). Median (range) time from diagnosis to the date of evaluating WT1 expression in PV, ET, and MF was 9.4 (0-309), 14.4 (0-55), and 21.4 months (0-271 months), respectively. WT1 expression in terms of WT1 copies/104 ABL copies was measured by quantitative PCR. Results. WT1 gene overexpression is revealed solely in patients with MF (in 34/39; 87 %). In PV/ET no WT1 gene overexpression was observed. Median WT1 expression in MF was 230/104 ABL copies (range 42.2-9,316.45/104 ABL copies). Sensitivity and specificity of WT1 gene overexpression in MF with respect to PV/ET were 87 % and 100 %, respectively. A distinct correlation was identified between WT1 gene expression level and spleen size, duration of the disease, blast cell count, and DIPSS risk group. WT1 gene expression level could be correlated neither with age and sex, nor with MF mutation status and leucocyte, thrombocyte, and haemoglobin levels. Conclusion It appears that due to a high specificity and sensitivity of WT1 gene expression in MF it can be used as a marker for differential diagnosis of Ph-negative MPD. A correlation between WT1 gene expression and tumor mass in MF cannot be excluded. It is advisable to analyze the dynamics of WT1 expression level to predict the efficacy of current targeted therapy.

Aim. To assess the efficacy of FLAG/FLAG-Ida regimen and to identify factors that influence remission, duration of disease-free survival (DFS) and overall survival (OS) of patients with relapsed/refractory acute myeloid leukemia (AML). Materials & Methods. The trial included 54 patients (28 men and 26 women), median age was 37 years (range 18-70 years). 27 (50 %) out of 54 patients had refractory AML and 27 (50 %) patients had relapsed AML. FLAG and FLAG-Ida regimens were administered as induction therapy. 37 (68.5 %) patients received bone marrow transplantation. Molecular genetic and cytogenetic examinations were performed prior to therapy and on the 28th day after the start of treatment. WT1 gene expression was evaluated on the 14th-16th day of treatment. Results. Complete remission (CR) was achieved in 42 (77.8 %) out of 54 patients. Refractoriness to therapy was observed in 9 (16.7 %) out of 54 patients, mortality was 5.5 % (3/54). Remission rate was higher in patients with relapsed AML compared with refractory AML: 85.2 % (23/27) and 70.4 % (19/27), respectively. On the 14th-16th day of treatment patients with blast cell count ≥ 10 % in bone marrow (BM) showed significantly lower CR rate (60 %) compared with the group of patients with < 10 % blast cells in BM (89.6 %; p = 0.024) and shorter DFS (median 7.6 vs. 17.6 months, respectively; p = 0.03). Median DFS in patients with WT1 expression reduction to < 1 log on the 14th-16th day was 5 vs. 18 months in patients without WT1 expression reduction (p = 0.01). DFS varied in groups of patients with blast cell count < 10 % in BM on the 14th-16th day of treatment based on the level of WT1 expression reduction (p = 0.04). MRD-negative patients (57.1 %) showed significantly longer DFS and OS compared with MRD-positive patients (median DFS was 17.6 vs. 5.2 months, respectively, p = 0.02; median OS was 19 vs. 6.9 months, p = 0.0002). Median DFS and OS were different only in ELN low- and high-risk groups (median not reached vs. 5.2 months, respectively, p = 0.039; median not reached vs. 10.2 months, p = 0.039). Conclusion. FLAG and FLAG-Ida are effective and safe regimens in the treatment of relapsed/refractory AML. Achieving remission depends on neither the risk group nor the time of relapse occurrence. The blast cell count in BM on the 14th-16th day of FLAG/FLAG-Ida treatment is a prognostic factor determining achievement and duration of remission. WT1 expression level in the early post-induction period is a sensitive DFS marker. MRD status and molecular genetic risk (ELN) group affiliation are essential prognostic factors determining DFS and OS.

Background. Haploidentical bone marrow transplantation (BMT) can be a reliable alternative if a fully matched donor is not available. The main challenges after BMT are a relapse of major disease, graft-versus-host disease (GVHD), and infections. Azacitidine possesses antileukemic effect together with immunomodulating properties and being administered soon after BMT can significantly improve the outcome. Aim. To study azacitidine effect on the outcome of haploidentical BMT in patients with acute myeloid leukemia (AML) in the early post-transplantation period. Materials & Methods. The trial included 18 AML patients who received haploidentical BMT at VA Almazov National Medical Research Center. In all patients MRD-negative remission was achieved on the 30th day after BMT. Azacitidine therapy was initiated not earlier than 2 months after BMT with a complete engraftment of transplant and no GVHD. Azacitidine 100 mg/day was administered on D1-D5 every 28 days within a year after BMT. When a molecular relapse was detected, donor lymphocytes were additionally infused during every other cycle of therapy. Results. Eleven patients received preventive azacitidine treatment, 7 patients were included in control group. Median onset of azacitidine treatment after haploidentical BMT was 4 months (range 2-10 months), median number of azacitidine courses was 3.5 (range 1-9). During azacitidine treatment acute GVHD was identified in 5 (45.4 %) patients. In 4 of them an exacerbation of earlier GVHD was detected (3 with cutaneous form and 1 with intestinal form), and only in 1 patient de novo acute intestinal GVHD was discovered. Conclusion. Azacitidine treatment of AML patients after haploidentical allo-BMT is safe and well tolerated. Preventive azacitidine treatment after haploidentical BMT improves overall survival of AML patients.

Ruxolitinib, a potent Janus kinase 1/2 inhibitor, has demonstrated durable improvements in patients with myelofibrosis. In this analysis of the Phase 3b JUMP study, which included patients aged ≥18 years with a diagnosis of primary or secondary myelofibrosis, we assessed the safety and efficacy of ruxolitinib in patients stratified by Dynamic International Prognostic Scoring System (DIPSS) risk categories. Baseline characteristic data were available to assess DIPSS status for 1844 of the 2233 enrolled patients; 60, 835, 755, and 194 in the low‐, intermediate (Int)‐1‐, Int‐2‐, and high‐risk groups, respectively. Ruxolitinib was generally well tolerated across all risk groups, with an adverse‐event (AE) profile consistent with previous reports. The most common hematologic AEs were thrombocytopenia and anemia, with highest rates of Grade ≥3 events in high‐risk patients. Approximately, 73% of patients experienced ≥50% reductions in palpable spleen length at any point in the ≤24‐month treatment period, with highest rates in lower‐risk categories (low, 82.1%; Int‐1, 79.3%; Int‐2, 67.1%; high risk, 61.6%). Median time to spleen length reduction was 5.1 weeks and was shortest in lower‐risk patients. Across measures, 40%–57% of patients showed clinically meaningful symptom improvements, which were observed from 4 weeks after treatment initiation and maintained throughout the study. Overall survival (OS) was 92% at Week 72 and 75% at Week 240 (4.6 years). Median OS was longer for Int‐2‐risk than high‐risk patients (253.6 vs. 147.3 weeks), but not evaluable in low‐/Int‐1‐risk patients. By Week 240, progression‐free survival (PFS) and leukemia‐free survival (LFS) rates were higher in lower‐risk patients (PFS: low, 90%; Int‐1, 82%; Int‐2, 46%; high risk, 15%; LFS: low, 92%; Int‐1, 86%; Int‐2, 58%; high risk, 19%). Clinical benefit was seen across risk groups, with more rapid improvements in lower risk patients. Overall, this analysis indicates that ruxolitinib benefits lower‐risk DIPSS patients in addition to higher risk.

Objectives: To the best of our knowledge, data from Gemtuzumab ozogamicin in Acute Myeloid Leukemia (AML) patients with failure of organ functions and poor performance status are extremely lacking. Moreover, the fast recovery from organ failure, after Gemtuzumab ozogamicin administration, has never been reported. This study aimed to demonstrate the efficacy and rapid response of Gemtuzumab ozogamicin in refractory acute myeloid leukemia (AML) patients with pulmonary and kidney failure and poor performance status. Three refractory AML patients, with organ dysfunction, are described. One patient was pre-treated with intensive chemotherapy, and two other patients progressed during Azacitidine treatment. Two patients had respiratory failure grade 2 and one patient suffered from acute kidney insufficiency. Two patients were highly febrile with an elevated С-Reactive Protein (CRP) level. The WHO performance status of three was measured in all patients. Gemtuzumab ozogamicin administration was performed in three patients, followed by a further switch to Gemtuzumab ozogamicin + Azacitidine or “7+3” treatment. Results: Gemtuzumab ozogamicin administration resulted in abrupt fever cessation in two febrile patients simultaneously with a rapid decrease in CRP level and fast resolution of respiratory failure. Recovery of kidney function was noticed rapidly in patients with renal insufficiency. The WHO performance status was elevated in all three patients. No adverse grade II−III effects were noticed. Further treatment made two patients eligible for intensive chemotherapy, one patient underwent allogeneic stem cell transplantation, and the patient with kidney failure obtained complete remission. Conclusions: Gemtuzumab ozogamicin therapy appeared to be safe and highly efficacious in relapsed/refractory AML patients with organ dysfunction, like pulmonary or renal failure and poor performance status, and may contribute to rapid recovery from organ failures.

Ruxolitinib is a potent Janus kinase (JAK) 1/JAK2 inhibitor approved for the treatment of myelofibrosis (MF). Ruxolitinib was assessed in JUMP, a large (N = 2233), phase 3b, expanded-access study in MF in countries without access to ruxolitinib outside a clinical trial, which included patients with low platelet counts (

Bosch, Francesc/0000-0001-9241-2886 WOS: 000523481800012 PubMed: 31455851 GREEN (NCT01905943) is a nonrandomized, open-label, single-arm, phase 3b study investigating the safety and efficacy of obinutuzumab alone or in combination with chemotherapy in chronic lymphocytic leukemia (CLL). We report the preplanned subgroup analysis of 140 previously untreated, fit CLL patients who received obinutuzumab plus fludarabine and cyclophosphamide (G-FC). The primary endpoint was safety and tolerability. Efficacy was the secondary endpoint. Obinutuzumab 1000 mg was administered intravenously on Day (D)1 (dose split D1.2), D8 and D15 of Cycle (C)1, and D1 of C2-6 (28-day cycles). Standard intravenous/oral doses of fludarabine and cyclophosphamide were administered on D1-3 of C1-6. Overall, 87.1% of patients experienced grade >= 3 adverse events (AEs), including neutropenia (67.1%) and thrombocytopenia (17.1%). Serious AEs were experienced by 42.1% of patients. Rates of grade >= 3 infusion-related reactions and infections were 19.3% and 15.7%, respectively. Overall response rate was observed in 90.0%, with 46.4% of patients achieving complete response (CR; including CR with incomplete marrow recovery). Minimal residual disease negativity rates were 64.3% in peripheral blood and 35.7% in bone marrow (intent-to-treat analysis). After a median observation time of 25.6 months, 2 year progression-free survival was 91%. Frontline G-FC represents a promising treatment option for fit patients with CLL. F. Hoffmann-La Roche Ltd.Hoffmann-La Roche This study was sponsored by F. Hoffmann-La Roche Ltd.

Background. The most promising variant of adoptive immunotherapy of the B-line oncohematological diseases includes the use of cells with the chimeric antigen receptor (CAR T-cells), that showed extraordinary results in clinical studies. Aim. To manufacture CAR T-cells for the clinical use and to study their cytotoxicity in vitro. Methods. Human T-lymphocytes were transduced by the lentiviral vector containing anti-CD19-CAR, RIAD, and GFP genes. The T-cell transduction efficacy was assessed on the basis of GFP protein signal by flow cytometry. Propidium iodide was used to analyse the cell viability. Cytotoxic activity of the manufactured CAR T-cells was studied in the presence of the target cells being directly co-cultivated. Analysis of the number and viability of CAR T-cells and cytokine expression was performed by flow cytometry. Results. The viability of the transduced T-cells and GFP expression reached 91.87 % and 50.87 % respectively. When cultured in the presence of IL-2 and recombinant CD19 (the target antigen), the amount of CAR-T after 120 h of the process was 1.4 times larger compared with the period of 48 h. In the cytotoxic test of co-cultivation CART with the K562-CD19+ cells the percentage of CAR-T increased to 57 % and 84.5 % after 48 h and 120 h of exposure respectively. When cultured with the K562 cells (test line not expressing CD19) the number of CAR T-cells decreased to 36.2 % within 48 h while the number of K562 cells increased to 58.3 %. The viability of target cells in the experimental and control groups was 3.5 % and 36.74 % respectively. Comparison of IL-6 level in the control and experimental groups revealed that the differences are insignificant, as opposed to the level of other cytokines (IFN-y, IL-2, TNF) which proved to be different in both groups. Conclusion. The present work resulted in the production of anti-CD19 CAR T-cells with adequate viability. The in vitro model demonstrated their cytotoxicity. Manufacturing of CAR T-cells for clinical use is the first step of the development of adoptive immunotherapy in the Russian Federation.

Background. In patients with chronic lymphocytic leukemia (CLL) the eradication of minimal residual disease (MRD) is a prognostic factor of overall survival (OS) and progressionfree survival (PFS). IGHV mutational status has also independent prognostic value. Aim. To analyse the impact of mutational status and MRD eradication in CLL patients after first-line standard BR (ben-damustine + rituximab) immunochemotherapy. Materials & Methods. The prospective study included patients with immunophenotypically confirmed CLL who had not previously received anticancer therapy. All patients were treated by BR combination from 2012 to 2015. MRD level was determined in 109 patients after completing the 3rd and the 6th treatment courses. IGHV mutational status data were available for 98 patients. IGHV mutational status was evaluated in accordance with ERIC recommendations. MRD was assessed by standardized method of 4-color flow cytometry. Results. MRD negativity was achieved in 37 (34 %) out of 109 patients. MRD eradication correlated with the best PFS (p = 0.04). IGHV mutational status had a statistically significant impact on PFS (p = 0.02). In patients with MRD-nega-tive response and IGHV mutation no unfavorable events occurred during the period of monitoring. Conversely, PFS rates in MRD-negative patients having no IGHV mutation and in MRD-positive patients with mutation were significantly worse. MRD eradication resulted in statistically significant improvement of PFS rates after completing 3 treatment courses, compared with the cases with MRD persistence regardless of residual malignant clone level (p = 0.01). Conclusion. BR therapy as first-line treatment statistically improved PFS in patients who achieved MRD-negative remission after completing the 3rd treatment course. PFS was significantly higher in MRD-negative patients with IGHV mutation after 6 treatment courses. MRD negativity resulting from 6 BR therapies in patients having no / GHV mutation was not accompanied by PFS improvement. It follows that by itself MRD negativity cannot be considered to be a universal prognostic factor.