Structure and significance of cytogenetic abnormalities in adult patients with Ph-negative acute lymphoblastic leukemia

Objective. To evaluate occurrence, variety, structural peculiarities and prognostic meaning of cytogenetic abnormalities in adult patients with Ph-negative acute lymphoblastic leukemia (ALL) receiving therapy according to ALL-2009 protocol. Materials and methods. The study included 115 adult patients with firstly diagnosed Ph-negative ALL: 58 male and 57 female aged from 15 to 61 years (mean age 26.5 years), who underwent treatment from September 2009 to September 2015 in National Medical Research Center for Hematology MH RF (n=101) and in hematology departments of regional hospitals (n=14). All patients received therapy of ALL-2009 protocol (ClinicalTrials.gov, NCT01193933). The median follow-up was 24.5 months (0.2-94.4 months). As a part of the study results of a standard cytogenetic assay (SCA) were analyzed and fluorescence hybridization in situ (FISH) with the use of DNA-probes was performed on archived biological material for structural changes in gene locuses MLL/t(11q23), с-MYC/t(8q24), TP53/ deletion 17p13, CDKN2A/ deletion 9p21, translocation t(1;19)/E2A-PBX1 и t(12;21)/ETV6-RUNX1; iAMP21 identification. Results. Karyotype was defined using SCA in 86% of patients. Normal karyotype was found in 48.5% of them, chromosome aberrations in 51.5% (structural changes were found in 19.2%, hyperploidy in 27.2%, and hypoploidy in 5.1%). In 17.2% of patients complex karyotype abnormalities were found. With the use of FISH technique aberrations were found in 67% of patients: 9p21/CDKN2A deletion in 24.3%, MLL/t(11q23) gene abnormalities in 7.8%, 17p13/TP53 deletion in 5.2%, abnormalities of c-MYC/t(8q24) in 1.7%, t(1;19)/E2A-PBX1 in 0.8%, and iAMP21 in 0.8%, other abnormalities (additional signals/absence of signals from gene locuses) in 26.4%, t(12;21)/ETV6-RUNX1 was not found. FISH technique use in addition to SCA allows to increase aberrant karyotype location from 51.5 to 67%. A statistically significant correlation of 9p21/CDKN2A deletion with high serum lactate dehydrogenase activity (p=0.02); MLL/t(11q23) gene abnormalities - with leucocytosis and high blast cells level in blood (p=0.0016), hyperploidy - with normal leukocyte count (p=0.02) was shown. In groups with different cytogenetic abnormalities no statistically significant differences of treatment with ALL-2009 protocol were found (in terms of complete remission, early mortality and treatment resistance). When connection of cytogenetic abnormalities and their combinations with long-term results were analyzed according to ALL-2009 protocol, only two characteristics - MLL/t(11q23) and c MYC/t(8q24) gene abnormalities had a statistically significant influence on disease-free survival (HR - 176.9;

Organisational and methodological challenges of CAR-T manufacturing in the Russian Federation

Despite their widespread clinical implementation, chimeric antigen receptor T-cell (CAR-T) therapy products, including those manufactured by industrial processes, are still not legally available or used in the Russian Federation.The aim of the study was to describe the current challenges associated with specific aspects of CAR-T manufacturing in the Russian Federation and the potential ways to overcome them.This article discusses the regulatory, legal, organisational, and methodological challenges of CAR-T manufacturing. It analyses differences in the interpretation of CAR-T therapy products under national and supranational law. According to Russian Federal Law No. 180-FZ “On Biomedical Cell Products” of 23 June 2016, CAR-T therapy products are considered biomedical cell products. However, according to Decision No. 78 of the Council of the Eurasian Economic Commission “On the Rules of Marketing Authorisation and Assessment of Medicinal Products for Human Use” of 3 November 2016, CAR-T therapy products are considered advanced therapy medicinal products (ATMPs). This article provides a detailed overview of the difficulties in obtaining starting biological materials (i.e. the inability to consider the patient as a donor) and transferring the materials for CAR-T manufacturing (i.e. the inapplicability of national law). In addition, this article describes export aspects specific to biological materials. The authors reckon that CAR-T therapy products should be categorised as ATMPs and that the corresponding active pharmaceutical ingredients, genetically modified autologous lymphocytes, should be defined as starting materials. Therefore, genetically modified autologous lymphocytes should be regulated under the requirements for starting materials for the manufacturing of active pharmaceutical ingredients that are set forth in Decision No. 77 of the Council of the Eurasian Economic Commission “On the Adoption of the Rules of Good Manufacturing Practice of the Eurasian Economic Union” of 3 November 2016. In conclusion, the authors recognise the need for national and supranational law harmonisation. For this task, it is necessary to establish expert groups that will include clinicians, legal experts, and representatives from the relevant authorities and the pharmaceutical industry

Impact of graft-versus-host disease prophylaxis on immune reconstitution in patients after allogeneic hematopoietic stem cell transplantation

The graft-versus-host disease (GVHD) is among the most common complications after hematopoietic stem cell transplantation (allo-HSCT). The main tools for GVHD prevention remain calcineurin inhibitors (cyclosporin A, tacrolimus), methotrexate, mycophenolate mofetil. Upon implementation of reduced-intensity conditioning regimens, antithymocyte globulin was widely introduced. However, negative effects upon reconstitution of T-cell immunity have been noted, thus increasing risk of severe infectious complications and disease relapse. With extended practice of HSCT from alternative (partially matched or haploidentical) donors, cyclophosphamide was increasingly used. Our aim was to study reconstitution of immune cell subpopulations in the patients undergoing bone marrow transplantation (BMT), when using different GVHD prophylaxis regimens, including the schedules with post-transplant CP usage. The study concerned 44 cases classified into 2 groups. The first one included patients with standard immunosuppressive therapy, antithymocyte therapy, cyclosporine A, methotrexate, mycophenolate mofetil. The second group included the patients who received CP as immunosuppressive drug combined with other treatments (cyclosporine A, methotrexate, mycophenolate mofetil). At specified control terms, (D+14, +30, +60, +90) the blood leukocyte subpopulations were assayed by means of multicolor flow cytometry. Absolute counts of CD4+ cells in HSCT recipients treated with CP post-BMT proved to be sufficiently lower at D+14 and +30, than in those treated with classical immunosuppressive therapy. However, at later terms, (D+60, +90), these differences were not observed. Moreover, in CP-treated bone marrow recipients, absolute numbers of CD8+ cells was significantly higher, compared to the patients who received conventional GVHD prophylaxis. Reconstitution of the studied lymphocyte populations in hematopoietic cell recipients did not depend on the GVHD prophylaxis regimen. Usage of CP combined with bone marrow as a source of stem cells, brings about sufficient decrease of some cell populations (CD4+; CD8+; NK cells) at early terms post-transplant. Administration of CP combined with hematopoietic stem cells as the source of hematopoietic graft seems to be more reasonable

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

Синдром VEXAS: на рубеже смены представлений об известных заболеваниях

This article presents the first case of VEXAS syndrome identified in the Russian Federation as well as characteristics of currently known clinical manifestations and treatment approaches. The clinical observation described is an impressive example of how the identification of a new pathogenic mutation can change the understanding of the classification, diagnosis and treatment of previously known immunoinflammatory diseases. Thus, in refractory forms of relapsing polychondritis, neutrophilic dermatosis, atypical forms of vasculitis, inflammatory joint diseases or undifferentiated systemic inflammatory syndrome, especially when associated with macrocytic anemia and myelodysplastic syndrome, VEXAS syndrome should be suspected and genetic testing should be performed to exclude the autoinflammatory nature of the existing condition.В статье приведен первый случай синдрома VEXAS, выявленный в Российской Федерации, а также характеристика известных на настоящий момент клинических проявлений и подходов к его терапии. Описанное клиническое наблюдение является ярким примером того, как выявление новой патогенной мутации может изменить представление о классификации, диагностике и терапии ранее известных иммуновоспалительных заболеваний. Так, при рефрактерных формах рецидивирующего полихондрита, нейтрофильного дерматоза, нетипичных формах васкулита, воспалительных заболеваний суставов или недифференцированном системном воспалительном синдроме, особенно при ассоциации с макроцитарной анемией и миелодиспластическим синдромом, необходима настороженность в отношении синдрома VEXAS и проведение генетического исследования для исключения аутовоспалительной природы имеющегося состояния

Организационно-методические проблемы производства CAR-T в Российской Федерации

Despite their widespread clinical implementation, chimeric antigen receptor T-cell (CAR-T) therapy products, including those manufactured by industrial processes, are still not legally available or used in the Russian Federation.The aim of the study was to describe the current challenges associated with specific aspects of CAR-T manufacturing in the Russian Federation and the potential ways to overcome them.This article discusses the regulatory, legal, organisational, and methodological challenges of CAR-T manufacturing. It analyses differences in the interpretation of CAR-T therapy products under national and supranational law. According to Russian Federal Law No. 180-FZ “On Biomedical Cell Products” of 23 June 2016, CAR-T therapy products are considered biomedical cell products. However, according to Decision No. 78 of the Council of the Eurasian Economic Commission “On the Rules of Marketing Authorisation and Assessment of Medicinal Products for Human Use” of 3 November 2016, CAR-T therapy products are considered advanced therapy medicinal products (ATMPs). This article provides a detailed overview of the difficulties in obtaining starting biological materials (i.e. the inability to consider the patient as a donor) and transferring the materials for CAR-T manufacturing (i.e. the inapplicability of national law). In addition, this article describes export aspects specific to biological materials. The authors reckon that CAR-T therapy products should be categorised as ATMPs and that the corresponding active pharmaceutical ingredients, genetically modified autologous lymphocytes, should be defined as starting materials. Therefore, genetically modified autologous lymphocytes should be regulated under the requirements for starting materials for the manufacturing of active pharmaceutical ingredients that are set forth in Decision No. 77 of the Council of the Eurasian Economic Commission “On the Adoption of the Rules of Good Manufacturing Practice of the Eurasian Economic Union” of 3 November 2016. In conclusion, the authors recognise the need for national and supranational law harmonisation. For this task, it is necessary to establish expert groups that will include clinicians, legal experts, and representatives from the relevant authorities and the pharmaceutical industry.Несмотря на широкое внедрение в клиническую практику терапии Т-клетками с химерным антигенным рецептором (chimeric antigen receptor T-cell, CAR-T), на территории Российской Федерации данные препараты до сих пор официально не представлены и не используются, в том числе и произведенные промышленным (индустриальным) способом.Цель работы — описание текущих проблем, связанных с особенностями производства CAR-T в Российской Федерации, и потенциальных путей их решения.Рассмотрены трудности, связанные c регуляторными, юридическими и организационно-методическими аспектами производства CAR-T. Проанализированы расхождения в трактовке понятия CAR-T в национальном и наднациональном праве: как биомедицинского клеточного продукта согласно Федеральному закону от 23.06.2016 № 180-ФЗ «О биомедицинских клеточных продуктах» и как высокотехнологического лекарственного препарата (ВТЛП) согласно Решению Совета Евразийской экономической комиссии от 03.11.2016 № 78 «О Правилах регистрации и экспертизы лекарственных средств для медицинского применения». Подробно рассмотрены трудности на этапе получения исходного биологического материала (невозможность рассматривать пациента как донора биологического материала); на этапе передачи биологического материала для производства CAR-T (невозможность применения национального права); особенности экспорта биологического материала. По мнению авторов статьи, CAR-T следует относить к ВТЛП, активной фармацевтической субстанцией (АФС) которых являются генетически модифицированные аутологичные лимфоциты, а последние должны быть определены как «исходный материал», и к ним должны применяться требования как к исходному материалу для производства АФС, указанные в Решении Совета Евразийской экономической комиссии от 03.11.2016 № 77 «Об утверждении Правил надлежащей производственной практики Евразийского экономического союза». Сделано заключение о необходимости гармонизации национального и наднационального права, что требует формирования экспертных групп, объединяющих врачей-клиницистов, специалистов в области права, представителей профильных ведомств и фармацевтической индустрии

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

Objective. 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 and 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

Ruxolitinib for Glucocorticoid-Refractory Acute Graft-versus-Host Disease

BACKGROUND: Acute graft-versus-host disease (GVHD) remains a major limitation of allogeneic stem-cell transplantation; not all patients have a response to standard glucocorticoid treatment. In a phase 2 trial, ruxolitinib, a selective Janus kinase (JAK1 and JAK2) inhibitor, showed potential efficacy in patients with glucocorticoid-refractory acute GVHD. METHODS: We conducted a multicenter, randomized, open-label, phase 3 trial comparing the efficacy and safety of oral ruxolitinib (10 mg twice daily) with the investigator's choice of therapy from a list of nine commonly used options (control) in patients 12 years of age or older who had glucocorticoid-refractory acute GVHD after allogeneic stem-cell transplantation. The primary end point was overall response (complete response or partial response) at day 28. The key secondary end point was durable overall response at day 56. RESULTS: A total of 309 patients underwent randomization; 154 patients were assigned to the ruxolitinib group and 155 to the control group. Overall response at day 28 was higher in the ruxolitinib group than in the control group (62% [96 patients] vs. 39% [61]; odds ratio, 2.64; 95% confidence interval [CI], 1.65 to 4.22; P<0.001). Durable overall response at day 56 was higher in the ruxolitinib group than in the control group (40% [61 patients] vs. 22% [34]; odds ratio, 2.38; 95% CI, 1.43 to 3.94; P<0.001). The estimated cumulative incidence of loss of response at 6 months was 10% in the ruxolitinib group and 39% in the control group. The median failure-free survival was considerably longer with ruxolitinib than with control (5.0 months vs. 1.0 month; hazard ratio for relapse or progression of hematologic disease, non-relapse-related death, or addition of new systemic therapy for acute GVHD, 0.46; 95% CI, 0.35 to 0.60). The median overall survival was 11.1 months in the ruxolitinib group and 6.5 months in the control group (hazard ratio for death, 0.83; 95% CI, 0.60 to 1.15). The most common adverse events up to day 28 were thrombocytopenia (in 50 of 152 patients [33%] in the ruxolitinib group and 27 of 150 [18%] in the control group), anemia (in 46 [30%] and 42 [28%], respectively), and cytomegalovirus infection (in 39 [26%] and 31 [21%]). CONCLUSIONS: Ruxolitinib therapy led to significant improvements in efficacy outcomes, with a higher incidence of thrombocytopenia, the most frequent toxic effect, than that observed with control therapy

MINIMAL RESIDUAL DISEASE ASSESMENT IN PATIENTS WITH ACUTE MYELOID LEUKEMIA BY MULTICOLOUR FLOW CYTOMETRY (LITERATURE REVIEW)

Patients with acute myeloid leukemia (AML) have a high risk of relapse. Determination of the presence of residual tumor cells during the remission of AML allows predicting the onset of relapse. Slow decrease of blast cells after induction courses is associated with a high probability of relapse. There are two most sensitive methods for determining minimal residual disease (MRD): molecular (polymerase chain reaction – PCR, droplet digital PCR, next generation sequencing – NGS) and immunophenotypic (multicolor flow cytometry – MFC). Both methods have advantages and disadvantages. The molecular diagnostic method is more sensitive, but it takes more time to get the result (from several days). Measurement of MRD by PCR is applicable in 40–50 % of AML patients, and by MFC – in 90 % of patients. The MFC method is based on the identification of antigens combination that is characteristic of tumor cells and is not found on normal hematopoietic cells. There are several drawbacks of the MFC method: the change of tumor clone immunophenotype during treatment, the inadequate difference in the antigen profiles of tumor cells and normal cells, difficulties in evaluating the MRD status in low cellularity bone marrow or blood sample. In AML patients the effect of MRD, measured by the MFC method, on long-term treatment outcomes was well-demonstrated. Evaluation of early MRD status shows tumor chemosensitivity and therapy efficacy. Despite the different approaches in MRD detection, thresholds and the combination of monoclonal antibodies, the lack of standardization, “positive” MRD values in early or later stages of therapy worsen the disease-free (DFS) and overall survival (OS) in AML patients. So far, the principles of MRD-directed therapy have not been developed, but protocols exist with the use of targeted drugs in combination with standard chemotherapy that help to reduce the MRD level. It is proved that the intensification of treatment does not affect the quantitative value of MRD and the long-term results of therapy. New prospective studies are needed to search for universal MRD markers, to create a standardized panel of monoclonal antibodies and to develop a specific therapy strategy in accordance with the MRD values