In children with acute lymphoblastic leukemia (ALL), relapse is still the leading cause of treatment failure occurring in 10–15% of cases. Overall survival after relapse plateaus at 50–60%, whereas event-free survival after second and third relapse is approximately 25% and 15%, respectively. The introduction of new immunotherapeutic agents such as blinatumomab (a bispecific T-cell engager), inotuzumab ozogamicin (InO; a CD22 + monoclonal antibody) and a chimeric antigen T-cell receptor targeted to CD19 + can significantly increase the effectiveness of treatment for relapsed ALL and help patients achieve remission faster and thus shorten the time to allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the toxicity of these novel agents and their impact on the results of allo-HSCT are still to be investigated. Our study included 55 patients with refractory B-cell ALL aged from 3 to 17 years (the median age was 10 years). The study was approved by the Independent Ethics Committee and the Scientific Council of the I.P. Pavlov First Saint Petersburg State Medical University. The patients were divided into two groups based on whether they received inotuzumab ozogamicin or not: InO+ group ( n = 24; 43.6%) and InO– group ( n = 31; 56.4%). The majority of the patients underwent haploidentical HSCT ( n = 53; 96.4%); 1 (1.8%) patient received HSCT from a matched related donor, and 1 (1.8%) from a matched unrelated donor. Conditioning regimens before allo-HSCT included: myeloablative conditioning ( n = 20; 36.4%), reduced toxicity myeloablative conditioning ( n = 5; 9.1%), and reduced intensity conditioning ( n = 30; 54.5%). Acute graft-versus-host disease prophylaxis with post-transplant cyclophosphamide was given to 49 (87.7%) recipients; 6 (12.3%) patients received seroprophylaxis. Basic combined immunosuppressive therapy consisting of a calcineurin inhibitor and an mTOR inhibitor was used in 35 (63.6%) cases, and single m-TOR inhibitor treatment was administered to 20 (36.4%) patients. In the InO+ group, 21 (87.5%) patients achieved complete remission with incomplete hematologic recovery before allo-HSCT: 5 (23.8%) patients had minimal residual disease (MRD), and 16 (76.2%) patients were MRD negative. In the InO– group, remission with incomplete hematologic recovery before allo-HSCT was achieved in 15 (48.4%) patients: 3 (9.7%) cases were MRD positive and 12 (38.7%) were MRD negative ( p = 0.003). All the patients underwent allo-HSCT, regardless of response to prior therapy. Engraftment was achieved in the InO+ group in 20 (83.3%) children in a median of 22 days (D+22) and in the InO– group in 25 (80.6%) children in a median of 19 days (D+19). Relapse was observed in 11 (55%) patients in the InO+ group and in 15 (60%) patients in the InO– group at a median of 164 days and 203 days post-transplant, respectively ( p = n. s.). In the InO+ group, 5 (31.25%) out of 16 patients in complete remission with incomplete hematologic recovery and negative MRD status relapsed after allo-HSCT within a median of 105 days (D+58 – D+169). In the InO–, 6 (50%) out of 12 patients in complete remission with incomplete hematologic recovery and negative MRD status relapsed within a median of 296 days (D+108 – D+929). Due to the small number of patients in the groups, a correlation and regression analysis showed a weak correlation between the use of InO before allo-HSCT and the occurrence of post-transplant relapse (Pearson's contingency coefficient was 0.178). Loss of the HLA haplotype at relapse was found in 1 (4.2%) patient from the InO+ group and in 2 (6.5%) patients from the InO– group ( p = n. s.). Transplant-associated thrombotic microangiopathy was diagnosed in 6 (25%) recipients in the InO+ group and in 3 (9.7%) recipients in the InO– group. Eight (32%) patients in the InO+ group and 3 (9.7%) patients in the InO– group had clinical manifestations of sinusoidal obstruction syndrome. Our study suggests the effectiveness of inotuzumab ozogamicin for the treatment of relapsed B-ALL in children before allo-HSCT. Patients with large tumor burden and high expression of CD22 + would benefit the most from therapy with InO. The application of reduced intensity conditioning regimen after CD22 + directed monoclonal antibody therapy significantly improves the overall survival rates by reducing early transplant-related mortality and makes it possible to use adoptive immunotherapy as a next line of treatment. Current allo-HSCT protocols and approaches to acute graft-versus-host disease prevention help control the development of severe complications in the early post-transplant period. Our study showed that adoptive immunotherapy via donor lymphocyte infusions can be applied in patients treated with InO who experience loss of the HLA haplotype at relapse after allo-HSCT.

The postischemic inflammatory response plays a significant role in the pathogenesis of acute ischemic stroke (IS). It has been established that acute IS is accompanied by aseptic inflammation, which induces the activation of costimulatory molecules in the process of innate immunity response to brain tissue damage. The constantly progressive destruction of neuronal antigens contributes to an increase in the volume of the ischemic lesion. Evidence continues to accumulate indicating an important role of NLRP3-mediated inflammation in the pathogenesis of IS. It has been shown that autophagy is involved in the inflammatory cascade in acute IS. Many of the anti-inflammatory mechanisms mediated by autophagy in acute IS involve the key autophagic proteins Beclin-1, LC3, and p62. Experimental studies have shown that autophagy suppresses the activation of NLRP3 inflammation. Data on cross interactions between apoptosis and autophagy in the pathogenesis of IS are still controversial. The aim of the study was to evaluate the relationship between biomarkers of autophagy, inflammation, and apoptosis in the dynamics of the acute period of atherothrombotic IS. The article presents the results of a dynamic study of the serum concentration of the key autophagy biomarkers Beclin-1, LC3 and p62, apoptosis indicators Bcl-2 and p53, pro-inflammatory cytokines IL-1β, TNFα, IL-8, IL-18 which are involved in postischemic neuroinflammation. A statistically significant increase in the studied parameters was established in comparison with the control group. The maximum increase in the studied biomarkers is noted on the 1st day after the development of ischemia in patients with a severe course of the disease. The relationship between autophagy activity, apoptosis biomarkers, and some indicators of the systemic inflammatory response in patients with moderate and severe atherothrombotic stroke was revealed. The results obtained confirm the literature data on the involvement of autophagy in the regulation of the postischemic inflammatory response.

Acute myeloid leukemia (AML) is the second most common type of leukemia in children and accounts for up to 20 % of all leukemias. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective, and sometimes the only therapeutic option in high-risk patients with AML. Graft-versus-host disease (GVHD) is a major complication of allo-HSCT and the main cause of transplant-related mortality. GVHD prophylaxis in children includes calcineurin inhibitors, either alone or in combination with other immunosuppressants, which can lead to grade II–IV acute GVHD in 40–85 % of cases. Alternatively, GVHD can be prevented with high-dose cyclophosphamide (50 mg/kg/day) administered on days +3, +4 after allo-HSCT, either alone or in combination with other immunosuppressive drugs depending on HLA compatibility of the donor. The aim of this study was to evaluate outcomes after allo-HSCT from an unrelated donor with GVHD prophylaxis with post-transplant cyclophosphamide (PTC) in children in their first and second remission of AML in comparison with a historical control group. We retrospectively analyzed patient outcomes after 53 first-time allo-HSCTs from HLA-matched (n = 40) and partially-matched (8–9/10) (n = 13) unrelated donors performed in pediatric patients (aged 0 to 18 years) in their 1 st or 2 nd remission of AML at the R. M. Gorbacheva Research Institute for Pediatric Oncology, Hematology and Transplantation from 2008 to 2018. The study was approved by the Independent Ethics Committee and the Scientific Council of the I. P. Pavlov First Saint Petersburg State Medical University of Ministry of Healthcare of the Russian Federation. Our group of interest included 26 patients preventively treated for GVHD with 50 mg/kg of cyclophosphamide on days +3 and +4 in combination with calcineurin inhibitors (cyclosporin A – 2 (7.7 %) patients, tacrolimus – 24 (92.3 %) patients), the mTOR inhibitor sirolimus (5 (19.2 %) patients) or mycophenolate mofetil (21 (80.8 %) patients). The historical control group was made up of 27 patients whose GVHD prophylaxis was based on antithymocyte globulin used in combination with calcineurin inhibitors (tacrolimus – 5 (18.5 %) patients, cyclosporin A – 21 (77.8 %) patients) or the mTOR inhibitor sirolimus (1 (3.7 %) patients) or methotrexate (25 (92.6 %) patients), or mycophenolate mofetil (2 (7.4 %) patients). The groups were matched for diagnosis, age, disease status before allo-HSCT, the matched-to-partially-matched donor ratio, the source of hematopoietic stem cells and conditioning regimen intensity (myeloablative conditioning regimen (MAC) or reduced intensity conditioning regimen (RIC)). The median age at the time of allo-HSCT was 8.6 (0.97–18) years in the PTC group and 6.55 (1.42–17.76) years in the historical control group. In the PTC group, 21 (80.8 %) patients were diagnosed with primary AML and 5 (19.2 %) – with secondary AML, while the historical control group included 22 (81.5 %) and 5 (18.5 %) patients with primary and secondary AML respectively. Disease status at the time of allo-HSCT: 21 (80.8 %) patients treated with PTC were in the 1 st complete clinical and hematologic remission (CCHR) and 5 (19.2 %) – in the 2 nd CCHR; among the controls, there were 19 (70.4 %) cases of the 1 st CCHR and 8 (29.6 %) cases of the 2 nd CCHR. In the PTC group, 18 (69.2 %) patients underwent allo-HSCT from 10/10 fully HLA gene-matched donors and 8 (30.8 %) – from 9/10 HLA-matched donors. In the historical control group, 19 (70.4 %) patients had allo-HSCT from 10/10 fully HLA gene-matched donors, 4 (14.8 %) – from 9/10 matched donors, and 1 (3.7 %) – from an 8/10 matched donor. In the PTC group, MAC was used in 14 (53.8 %) patients, RIC – in 12 (46.2 %) patients. In the control group, MAC and RIC were used in 14 (51.9 %) and 13 (48.1 %) patients respectively. In the group treated with PTC, hematopoietic stem cells were derived from the bone marrow in 14 (53.8 %) patients, from the peripheral blood – in 12 (46.2 %) patients. In the historical group, bone marrow was used in 13 (48.1 %) patients and peripheral blood - in 14 patients (51.9 %). The median graft cellularity (CD34+ × 10 6 /kg) in the PTC group was 4.60 (1.7–10.9) × 10 6 /kg, in the historical group – 6.60 (1.0–13.2) × 10 6 /kg. The overall and relapse-free 5-year survival rates were higher in the PTC group than in the historical control group: 83.3 % (95 % confidence interval (CI) 60.9–93.5) vs 59.3 % (95 % CI 38.6–75.0), p = 0.0327 and 76.9 % (95 % CI 55.7–88.9) vs 48.1 % (95 % CI 28.7–65.2), respectively, p = 0.0198. The cumulative incidence of grade II–IV acute GVHD and grade III–IV acute GVHD by day +125 and of moderate and severe chronic GVHD, and the 2-year transplant-related mortality were significantly lower in the PTC group compared to the controls: 15.4 % (95 % CI 4.8–31.5) vs 51.8 % (95 % CI 31,9–68.5), p = 0.004; 7.7 % (95 % CI 1.3–21.7) vs 33.3 (95 % CI 16.8–50.9), p = 0.026; 23.4 % (95 % CI 9.5-41.0) vs 58.6 % (95 % CI 33.8–76.8), p = 0.022; 3.8 % (95 % CI 0.3–16.4) vs 25.9 % (95 % CI 11.5–43.1), p = 0.0232, respectively. GVHD-related mortality was higher in the historical control group than in the PTC group (3.8 % vs 18.5 %, p = 0.192). Thus, PTC-based GVHD prophylaxis was shown to be more effective in managing acute and chronic GVHD compared to antithymocyte globulin, with better overall, relapse-free and GVHD-free relapse-free survival rates and low transplant-related mortality.

Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative therapy for patients with juvenile myelomonocytic leukemia (JMML).The optimal pre-transplantation therapy with maximum efficacy and minimal toxic complications remains a matter of debate.This retrospective study demonstrates single center experience of pre-transplant therapy by hypomethylating agent in children with JMML.Overall, 33 patients were included in analysis.Improved outcome was observed in patients that received pre-transplant AML-like therapy+hypomethylating agents (HMA), AML-like therapy, low-dose chemotherapy ±HMA, only HMA (OS=83%, 44%, 43%, 0%).We have demonstrated that loss of heterozygosity (LoH) of HLA is an important determinant of sensitivity to adoptive immunotherapy and indication for second allo-HSCT.

Relapse of acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains one of the main causes of reduced long-term survival. Modern methods for predicting the risk of AML relapse after allo-HSCT take into account the data on the pre-transplant level of minimal residual disease (MRD) determined by flow cytometry and molecular biological studies of recurrent genetic abnormalities, which are currently widespread in clinical practice. Recent studies of the expression of genes characteristic of leukemic stem cells (LSCs) have shown prognostic significance for children with AML in relation to treatment response and the risk of relapse. The study of LSC persistence in order to predict the risk of recurrence after allo-HSCT in children with AML in addition to standard MRD detection methods may be of great importance. The aim of the work was to evaluate the impact of MRD status, both using classic methods and taking into account the genes characteristic of LSC, on the results of allo-HSCT in children with AML. The study was approved by the Independent Ethics Committee and the Scientific Council of the I.P. Pavlov First Saint Petersburg State Medical University of Ministry of Healthcare of the Russian Federation. To assess MRD using standard diagnostic methods, we analyzed the data of 95 children with AML in their 1 st –2 nd remission (cohort 1). MRD status was negative in 67 (70.6 %) patients; in 28 (29.4 %) children, MRD status was positive according to molecular genetic studies and / or immunophenotyping results. For pre-transplant evaluation of the expression of genes characteristic of LSC, we investigated bone marrow samples of 50 patients (cohort 2) using real-time polymerase chain reaction. The DNMT3B , GPR56 , CD34 , SOCS2 , SPINK2 , FAM30A , and ABL genes were studied by real-time polymerase chain reaction, followed by calculation of the pLSC6 value using the formula: DNMT3b × 0.189 + GPR56 × 0.054 + CD34 × 0.0171 + SOCS2 × 0.141 + SPINK2 × 0.109 + FAM30A × 0.0516. At the time of allo-HSCT, 37 (74 %) children with AML were in their 1 st or 2 nd remission of the disease, 13 (26 %) were out of the 1 st –2 nd remission. With a median follow-up of 5 years in the group of patients with a positive MRD status, determined by standard methods (cohort 1), overall survival (OS) was 67.9 % vs 73.1 % for patients with a negative MRD status (p = 0.83). The cumulative incidence of relapse was 50 % and 22 %, respectively; p = 0.012. When assessing the level of expression of genes characteristic of LSC (cohort 2), a pLSC6 level was above the median in 18/37 (49 %) patients. The results of linear regression showed that the pre-transplant level of expression of genes characteristic of LSC was not associated with the number of blasts/MRD (odds ratio 1.002; 95 % confidence interval 0.979–1.025). One-year OS rates did not differ significantly in children in the 1st–2nd remission of AML, depending on pLSC6 level: 84.2% in patients with low pLSC6 and 72.2 % – with high pLSC6 (p = 0.4), event-free survival in the corresponding groups – 68.4 % and 61.1 %, respectively (p = 0.34). The cumulative incidence of early relapse after allo-HSCT in the group of AML patients with a high pLSC6 score was significantly higher than in children with a low pLSC6 score before allo-HSCT (22 % and 0 %, respectively; p = 0.03). MRD does not have a statistically significant effect on OS. However, MRD positivity before allo-HSCT increases cumulative incidence of relapse. The level of expression of genes characteristic of LSC, determined before allo-HSCT, showed a prognostic significance in relation to the development of early AML relapse after allo-HSCT.

Children with Down syndrome (DS, OMIM #190685) have an increased risk for a hematopoietic malignancy.Down syndrome-associated AML (DS-AML) is diagnosed in children under 4 years, being often characterized as acute megakaryoblastic leukemia (AMegL), with somatic mutations in the GATA1 gene, and a relatively favorable course of the disease.This disease is commonly preceded by a stage of bone marrow failure with morphological myelodysplasia of erythroid and megakaryocytic cells.Additional chromosomal abnormalities in the DS-MDS/AML karyotypes are described in 20-35% of patients.To date the published data on the cytogenetic changes that occur during the evolution of DS-AML with an assessment of their prognostic impact are still insufficient. Case presentationsWe present three clinical observations of DS-MDS/AML in children under 4 years old.In all patients, DS-AML was preceded by DS-MDS.One patient was diagnosed with AMegL with a non-mutated GATA1 gene.In two others, the M0-AML and M4-AML FAB variants were diagnosed, and in both, the pathogenic GATA1 mutations were found.In all patients, cytogenetic and molecular cytogenetic analyzes, in addition to constitutive trisomy 21, revealed unbalanced translocations that led to the formation of one or two additional copies of duplicated segments 1q25-q44 / 1q32-q44, 11q13-q25 in the genome and trisomy of chromosome 11 thus resulting in dismal outcomes of the disease. ConclusionOur findings suggest that partial trisomy of the chromosome 1 with a duplication of segments 1q25-1q44 or 1q32-q44 in combination with complete or partial (11q13-q25) trisomy of the chromosome 11 may be indicative for a poor prognosis in DS-MDS/AML.

Juvenile myelomonocytic leukemia (JMML) is a rare and aggressive myeloproliferative/myelodysplastic neoplasm of early childhood characterized by activation of the Ras signaling pathway. Allogeneic hematopoietic stem cell transplantation (alloHSCT) is the only proven curative treatment for JMML. However, the 5-year overall survival is about 52–64%. In this work, we analyzed 4 clinical cases of patients with relapses of JMML with loss of heterozygosity in HLA (LoH) after allo-HSCT. The patients' parents gave their consent to the use of their children's data, including photographs, for research purposes and in publications. Two patients received a second allo-HSCT from an alternative donor, two patients – from the same donor. A positive result in the form of a durable remission was observed in one patient who underwent a second allo-HSCT from an alternative donor and restored HLA genetic heterozygosity. At the same time, immunotherapy with infusions of donor lymphocytes led to the development of graft-versus-host disease without potentiating the antileukemic effect. Thus, a second allo-HSCT from an alternative donor for the treatment of relapsed JMML with HLA LoH is necessary to restore the “graft-versus-JMML” response. The study was approved by the Independent Ethics Committee and the Scientific Council of the I.P. Pavlov First Saint Petersburg State Medical University of Ministry of Healthcare of the Russian Federation.

There is a limited number of publications on the incidence of different mutations in the BCR-ABL kinase domain, the efficacy and safety of therapy in children and adolescents with resistant forms of chronic myeloid leukemia (СML).Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is still the only method able to cure the disease completely in pediatric and adolescent patients with CML, however, being associated with life-threatening complications.This article analyzes the course, treatment and outcomes of chronic myeloid leukemia in 3 pediatric patients pre-treated with tyrosine kinase inhibitors (TKI) who exhibited the T315I mutation in BCR-ABL kinase domain.