[Overcoming therapy resistance in prolactinomas: from perspectives to real clinical practice].

The main treatment option of prolactin-secreting pituitary adenomas is dopamine agonist therapy, which demonstrates prolactin level normalizing and reducing the size of an adenoma in the majority of cases. However, significant amount of patients - about 20% - poorly responds even to high doses of dopamine agonists that is explained by the resistance to therapy. The occurrence of pharmacodynamic characteristics is one of the causes responsible for the development of resistance to typical therapy. Clinical manifestations of persistent hyperprolactinemia are due to following pathological factors: hormonal hypersecretion and the mass-effect of pituitary adenoma. Prevention of irreversible changes is possible only with timely detection of resistance and determination of the optimal personalized treatment algorithm.We report a clinical case of dopamine-agonist resistant microprolactinoma. Patient's health stabilisation, normal level of prolactin and reduction in size of adenoma were achieved due to administration of combined treatment with tamoxifen and dopamine agonists. Hyperprolactinaemia occurring because of prolactin-secreting pituitary adenoma and associated adverse effects are significant problem, decreasing quality of life and demographics in general. This underlines the importance of figuring out causes and identifying predictors of the therapy resistance.The results of the study, illustrated by a clinical example, are presented in the present paper.

[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.

[Predictors of severe lesions of the central nervous system in the treatment of blood system diseases]. / Prediktory tyazhelykh porazhenii tsentral'noi nervnoi sistemy pri lechenii zabolevanii sistemy krovi.

OBJECTIVE: To determine the types, frequency and key symptoms of severe lesions of the central nervous system (SLCNS) that occurred in patients with hematological malignancies (HM). MATERIALS AND METHODS: The authors conducted a retrospective exploratory study by analyzing the data of 3.620 patients with HM during the period from 01.01.18 to 31.12.19. Thirty-four patients (14 men and 20 women, median age 39 years), who developed SLCNS during treatment, were selected. For comparison with the main group of patients and exclusion of predictors associated with the development of SLCNS, a comparison group was added (by Kernel matching method). A comparison group consisted of 137 patients (59 men and 78 women, median age - 36 years) and was similar to the main group by clinical and laboratory characteristics. A neurological complication was marked as SLCNS if it was an indication for transfer to the intensive care unit (ICU). Statistical analysis included multivariate analysis - multiple binary logistic regression with stepwise inclusion of variables (that were found in the preliminary contingency table analysis) in the model, with control false results (by the false discovery rate method) and estimating the odds ratio, OR (95% CI). RESULTS: SLCNS in patients with HM developed in 0.94% of cases. The main SLCNS in patients with HM were: epileptic seizure (50.0%, n=17), ischemic stroke (20.6%, n=7), hemorrhagic stroke (17.6%, n=6) and meningoencephalitis (11.8%, n=4). The following independent significant (Wald test p≤0.05) predictors associated with the development of SLCNS in patients with HM during inpatient treatment were identified: antibiotic therapy (when more than 5 drugs are prescribed), OR=2.9 (1.2-7, four); polychemotherapy (if more than 4 drugs are prescribed), OR=2.9 (1.1-7.8); thrombocytopenia (with a platelet count less than 50·109 g/l), OR=2.3 (1.0-5.2) and delirium, OR=3.7 (1.3-10.8), and also the presence of neurological disorders in the patient's history, OR=2.6 (1.1-6.3). CONCLUSION: The main types of SLCNS in patients with HM were: epileptic seizure, ischemic and hemorrhagic strokes, and meningoencephalitis. Four predictors associated with the development of SLCNS in the course of HM treatment were identified: massive antibacterial (with more than 5 drugs) and chemotherapeutic (with more than 4 drugs) effects, thrombocytopenia and manifestation of delirium, as well as one risk factor: a history of neurological disorder. These factors need to be considered and monitored during treatment, because each of them increases the risk of developing SLCNS.

[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.

[Repeated haploidentical allogeneic hematopoietic stem cell transplantation with TCR αß/CD19 depletion in patient with primary myelofibrosis. Case report].

Indications of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with primary myelofibrosis are intermediate-2 and high-risk group of DIPSS (Dynamic International Prognostic Scoring System), beginning of the disease in childhood. The other adverse factors affect engraftment and survival after allo-HSCT, example partialy matched donor. But the result of allo-HSCT from matched related donors and result of allo-HSCT from haploidentical donors are comparable. The method for haploidentical hematopoietic stem cell transplantation is T-cell-depletion. This is clinical case of T-cell-depleted haploidentical hematopoietic stem cell transplantation in patient with primary myelofibrosis, the diagnosis was established in childhood.

[Multipotent mesenchymal stromal cells application for acute graft versus host disease treatment].

AIM: Analysis of the effectiveness of the MSCs aministration as the second- or third-line therapy of acute GVHD (aGVHD) resistant to glucocorticosteroid treatment. MATERIALS AND METHODS: The study included 35 patients who received MSCs obtained from the bone marrow of healthy donors as a treatment of steroid-resistant aGVHD. The clinical parameters of patients, MSCs cultural characteristics, the MSC expression profile for various genes including those involved in immunomodulation, expression of cells surface markers, the source of MSCs, as well as the frequency and number of MSC administrations were analyzed. RESULTS: Response to therapy was achieved in 74% of cases, a complete response was reached in 13 (37%) patients, partial response/clinical improvement was demonstrated in 13 (37%). This treatment was ineffective in 9 patients. The prediction of a group of patients with good response to MSC therapy turned to be impossible. The differences between the effective and ineffective for the GVHD treatment MSCs samples were found. The effective ones were characterized with a decreased total MSCs production and an increase in the main histocompatibility complex and PDL-1 antigens expression. CONCLUSION: These data allow to select optimal samples for aGVHD treatment that can improve clinical results. aGVHD treatment with MSCs has shown efficacy comparable to other treatment approaches. Given the low percentage of complications and the absence of significant adverse effects, MSC therapy seems to be one of the optimal approaches to the treatment of resistant forms of GVHD.

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.

Recombinant MHC Tetramers for Isolation of Virus-Specific CD8+ Cells from Healthy Donors: Potential Approach for Cell Therapy of Posttransplant Cytomegalovirus Infection.

Patients undergoing allogeneic hematopoietic stem cell transplantation have a high risk of cytomegalovirus reactivation, which in the absence of T-cell immunity can result in the development of an acute inflammatory reaction and damage of internal organs. Transfusion of the virus-specific donor T-lymphocytes represents an alternative to a highly toxic and often ineffective antiviral therapy. Potentially promising cell therapy approach comprises transfusion of cytotoxic T-lymphocytes, specific to the viral antigens, immediately after their isolation from the donor's blood circulation without any in vitro expansion. Specific T-cells could be separated from potentially alloreactive lymphocytes using recombinant major histocompatibility complex (MHC) multimers, carrying synthetic viral peptides. Rapid transfusion of virus-specific T-cells to patients has several crucial advantages in comparison with methods based on the in vitro expansion of the cells. About 30% of hematopoietic stem cell donors and 46% of transplant recipients at the National Research Center for Hematology were carriers of the HLA-A*02 allele. Moreover, 94% of Russian donors have an immune response against the cytomegalovirus (CMV). Using recombinant HLA-A*02 multimers carrying an immunodominant cytomegalovirus peptide (NLV), we have shown that the majority of healthy donors have pronounced T-cell immunity against this antigen, whereas shortly after the transplantation the patients do not have specific T-lymphocytes. The donor cells have the immune phenotype of memory cells and can be activated and proliferate after stimulation with the specific antigen. Donor lymphocytes can be substantially enriched to significant purity by magnetic separation with recombinant MHC multimers and are not activated upon cocultivation with the antigen-presenting cells from HLA-incompatible donors without addition of the specific antigen. This study demonstrated that strong immune response to CMV of healthy donors and prevalence of HLA-A*02 allele in the Russian population make it possible to isolate a significant number of virus-specific cells using HLA-A*02-NLV multimers. After the transfusion, these cells should protect patients from CMV without development of allogeneic immune response.

Long-term survival of donor bone marrow multipotent mesenchymal stromal cells implanted into the periosteum of patients with allogeneic graft failure.

The present study involved three patients with graft failure following allogeneic hematopoietic stem cell transplantation (allo-HSCT). We obtained multipotent mesenchymal stromal cells (MSCs) from the original hematopoietic cell donors and implanted these cells in the periosteum to treat long-term bone marrow aplasia. The results showed that in all patients endogenous blood formation was recovered 2 weeks after MSC administration. Donor MSCs were found in recipient bone marrow three and 5 months following MSC implantation. Thus, our findings indicate that functional donor MSCs can persist in patient bone marrow.

Mitochondrial thioredoxin reductase regulates major cytotoxicity pathways of proteasome inhibitors in multiple myeloma cells.

It is generally accepted that intracellular oxidative stress induced by proteasome inhibitors is a byproduct of endoplasmic reticulum (ER) stress. Here we report a mechanism underlying the ability of proteasome inhibitors bortezomib (BTZ) and carfilzomib (CFZ) to directly induce oxidative and ER stresses in multiple myeloma (MM) cells via transcriptional repression of a gene encoding mitochondrial thioredoxin reductase (TXNRD2). TXNRD2 is critical for maintenance of intracellular red-ox status and detoxification of reactive oxygen species. Depletion of TXNRD2 to the levels detected in BTZ- or CFZ-treated cells causes oxidative stress, ER stress and death similar to those induced by proteasome inhibitors. Reciprocally, restoration of near-wildtype TXNRD2 amounts in MM cells treated with proteasome inhibitors reduces oxidative stress, ER stress and cell death by ~46%, ~35% and ~50%, respectively, compared with cells with unrestored TXNRD2 levels. Moreover, cells from three MM cell lines selected for resistance to BTZ demonstrate elevated levels of TXNRD2, indirectly confirming its functional role in BTZ resistance. Accordingly, ectopic expression of TXNRD2 in MM cell xenografts in immunocompromised mice blunts therapeutic effects of BTZ. Our data identify TXNRD2 as a potentially clinically relevant target, inhibition of which is critical for proteasome inhibitor-dependent cytotoxicity, oxidative stress and ER stress.

[Treating patients with acute myeloid leukemias (AML) according to the protocol of the AML-01.10 Russian multicenter randomized trial: the coordinating center's results].

AIM: To make a randomized comparison of 2 consolidation treatment options (two patient groups): 2 cycles of cytarabine in average (Ig/m2 in Group 2) and standard (100 mg/mi2 in Group 1) doses in combination with idarubicin (8-12 mg/m2) and mitoxantrone (10 mg/m2), after two 7+3 induction cycles of daunorubicin (60 mg/mi2) and subsequent 6 cycles of maintenance therapy. SUBJECTS AND METHODS: In January 2010 to October 2013, a Russian multicenter trial was conducted to treat patients with acute myeloid leukemias (AML) in accordance with the AML-01.10 protocol (ClinicalTrials.gov Identifier: NCT01587430). The trial enrolled 243 AML patients from 21 centers, including 71 patients (median age 38 years) from the State Hematology Center, Ministry of Health of the Russian Federation; 35 and 36 patients were randomized to Groups 1 and 2, respectively. The randomized groups were balanced by basic clinical and laboratory parameters. Favorable, intermediate, and high cytogenetic prognoses were in 14 (21.9%), 40 (62.5%), and 10 (15.6%) patients, respectively. RESULTS: Prior to treatment, 2 patients died; one patient refused treatment. Fifty-eight (85.3%) of the 68 patients achieved complete remission (CR); early deaths was in 2 (2.9%) and resistance in 8 (11.8%). Four (6.9%) patients died during CR. Protocol deviations (doses, intervals, and the number of cycles) were recorded in 12 (20.7%) of the 58 patients. Other 8 (11.8%) patients were switched to low-dose cytarabine because of complications, withdrawn from the protocol and not included into the analysis of randomized comparison. Twenty allogeneic bone marrow transplantations (allo-BMT) (7 related, 12 unrelated, and 1 haploidentical) were performed; of them 15 allo-BMTs were done during first CR. In the 68 patients, 3-year overall survival (OS) was 45.6%; relapse-free survival (RFS) was 41.5%. OS was 64.6% in Group 1 and 58.3% in Group 2; RFS was 62 and 38.8% in Groups 1 and 2, respectively (p>0.5). In the favorable, intermediate, and high prognosis groups, OS was 79.5, 60, and 31.1% and RFS was 81.8, 41.3, and 33.3%, respectively (p=0.1). The consolidation treatment option unchanged survival rates in the above risk groups. Unachieved CR after the first cycle considerably decreased RFS (33.9% versus 60%) and served as an indication for allo-BMT during first CP (RFS without BMT was 0; that with BMT was 78%). CONCLUSION: No differences were found between both consolidation options according to long-term results. Protocol deviations were recorded in one-third of the patients. While implementing the protocol, the efficiency of treatment was high. Allo-BMT during first CR substantially increased RFS if CP was not achieved after the first cycle.

[Optimization of mobilization regimes of blood hemopoietic stem cells in patients with multiple myeloma].

AIM: To determine an optimal cyclophosphamide dose in the mobilization scheme providing adequate collection of CD34+ cells in patients with multiple myeloma (MM), to optimize the time of initiation of granulocytic colony-stimulating factor (G-CSF) administration, to study effects of induction therapy schemes on results of mobilization and collection of CD34+ cells. MATERIAL AND METHODS: Department of hemoblastoses chemotherapy and bone marrow transplantation of the Russian Hematological Center performed mobilization of autologous blood hemopoietic stem cells (BHSC) in 93 MM patients treated in 2001-2010. This was done with cyclophosphamide and G-CSF. The former was used in 59 cases in a dose 6 g/m2, in 34 cases - 4 g/m2. RESULTS: Myelotoxic agranulocytosis after cyclophosphamide administration developed in all the patients and was observed for 3-10 days (median 5 days). Agranulocytosis ran without documented infections in 51 (54.8%) patients, with febril fever - in 42 (45.2%) patients. Cepticemia, pneumonia, necrotic enteropathy, stomatitis, herpetic lesion of the skin were registered in 9, 4, 11, 14 and 6 cases, respectively. Severe thrombocytopenia (< 30 x 10(9)/l) occurred more frequently in administration of 6 g/m2 cyclophosphamide. It was corrected with 2-5 transfusions of thromboconcentrates, only 1 transfusion was needed after the dose 4 g/m2. Collection of CD34+ cells started in leukocyte level over 3.5 x 10(9)/l on mobilization day 12-20 (median day 15). The day of the first leukocytapheresis did not depend on the day of the first introduction of G-CSF. Duration of G-CSF administration was significantly shorter in the start of its use after leukocyte count decrease under 1.0 x 10(9)/l. Conduction of 1 to S (median 2) leukocytapheresis was needed for collection of BHSC. Sufficient for 2 autotransplantations number of BHSC were stored in 90 of 93 patients. Cyclophosphamide administration in a dose 6 g/m2 allowed collection of cells sufficient for one autotransplantation for the first leukapheresis in 52 (88.1) patients. A total number of CD34+ cells over 4 x 10(6) cells/kg were collected in 56 (94.9%) patients. In administration of cyclophosphamide in a dose 4 g/m2 mobilization was effective in all 34 patients. The first leukapheresis provided sufficient for one autotransplantation number of cells in 29 (85.3%) patients. CONCLUSION: Administration of high cyclophosphamide doses in combination with G-CSF is an effective and safe method of BHSC mobilization providing collection of adequate number of CD34+ cells for double autotransplantation in 96.8% patients. Cost effective is the start of G-CSF administration in the fall of leukocytes under 1.0 x 10(9)/l. Cyclophosphamide dose 4 g/m2 provides collection of CD34+ cells number sufficient for two autotransplantations in moderate thrombocytopenia and in less number of substitute transfusions in the absence of serious toxic complications.

[A case of legionellesis pneumonia verified by isolation of Legionella pneumophila serogroup 1 from bronchoalveolar lavage fluid treated with levofloxacine and tigecycline].

A male patient received non-chemotherapeutic drugs which induced deep neutropenia complicated with sepsis, bilateral pneumonia, acute respiratory insufficiency. Artificial pulmonary ventilation was applied. The examination of bronchoalveolar lavage showed the presence of the culture L. pneumophila (serogroup 1) in a concentration 2 x 10(3) CFU/ml. Antibacterial therapy with levofloxacin in a dose 1000 mg/day was conducted. In a week not only L.pneumophila but also Acinetobacter baumanii was isolated from bronchoalveolar lavage. Tigecyclin was added to levofloxacin treatment. Two air cavities were found in the left lung. The treatment reduced the size of these cavities, infiltrative changes in the lungs and respiratory insufficiency regressed. The patient was discharged from hospital This case is the first case in Russia of L.pneumophila isolation from bronchoalveolar lavage. The case is also characterized by use of tigecycline for treatment of combined legionella and akinetobacterial infection and cavities in the lungs in legionella pneumonia.