HLA-haploidentical stem cell transplantation in children with inherited bone marrow failure syndromes: A retrospective analysis on behalf of EBMT severe aplastic Anemia and pediatric diseases working parties.

Haploidentical stem cell transplantation (haplo-SCT) represents the main alternative for children with inherited bone marrow failure syndrome (I-BMF) lacking a matched donor. This retrospective study, conducted on behalf of the EBMT SAAWP and PDWP, aims to report the current outcomes of haplo-SCT in I-BMFs, comparing the different in vivo and ex vivo T-cell depletion approaches. One hundred and sixty-two I-BMF patients who underwent haplo-SCT (median age 7.4 years) have been registered. Fanconi Anemia was the most represented diagnosis (70.1%). Based on different T-cell depletion (TCD) approaches, four categories were identified: (1) TCRαß+/CD19+-depletion (43.8%); (2) T-repleted with post-transplant Cyclophosphamide (PTCy, 34.0%); (3) In-vivo T-depletion with ATG/alemtuzumab (14.8%); (4) CD34+ positive selection (7.4%). The cumulative incidences (CI) of neutrophil and platelet engraftment were 84% and 76% respectively, while that of primary and secondary graft failure was 10% and 8% respectively. The 100-day CI of acute GvHD grade III-IV(95% CI) was 13%, while the 24-month CI of extensive chronic GvHD was 4%. After a median follow-up of 43.4 months, the 2-year overall survival(OS) and GvHD/Rejection-free Survival (GRFS) probabilities are 67% and 53%, respectively. The TCR CD3+αß+/CD19+ depletion group showed a significantly lower incidence of both acute and chronic GvHD and higher OS (79%; p0.013) and GRFS (71%; p < .001), while no significant differences in outcomes have been observed by different diagnosis and conditioning regimens. This large retrospective study supports the safety and feasibility of haplo-SCT in I-BMF patients. TCRαß+/CD19+ depletion offers higher chances of patients' survival, with a significantly lower risk of severe a- and c-GvHD in I-BMFs compared to other platforms.

Post-transplant сyclophosphamide after matched donor hematopoietic stem cell transplantation in children with acute leukemia.

INTRODUCTION: The data on post-transplant cyclophosphamide (PTCy) in pediatric acute leukemia after matched allo-HSCT are limited to case series. The present study aimed to assess the results of PTCy-based GVHD prophylaxis in a large cohort of children with acute leukemia after matched allo-HSCT. METHODS: A retrospective analysis of 190 pediatric patients with acute leukemia who had a first allograft between 2008 and 2020 from a matched sibling donor (MSD) or matched unrelated donor (MUD) was carried out. In the MSD setting, GVHD prophylaxis consisted of PTCy alone (n = 28) for the study group, and calcineurin inhibitor (CNI) ± antimetabolite (n = 30) for the control group. In MUD setting, most patients in the study group received GVHD prophylaxis with PTCy+CNI+mycophenolate mofetil (n = 42, 66.7%) or PTCy+CNI+sirolimus (n = 12, 19%). All patients (n = 69) in the control group received ATG+CNI+antimetabolite. RESULTS: After MUD allo-HSCT, the incidences of acute GVHD grade III-IV and moderate/severe chronic GVHD were significantly lower in the PTCy group compared to control (6.6% vs. 35.0% and 12.7% vs. 47.1%, respectively, p < .0001). Five-year GVHD-free, relapse-free survival (GRFS) after MUD allo-HSCT was higher in the PTCy group compared to control (35.1% vs. 7.3%, p < .0001). At the same time, there was no significant difference between both groups after MSD allo-HSCT. CONCLUSIONS: In pediatric acute leukemia, PTCy-based GVHD prophylaxis for MUD allo-HSCT is a feasible and effective option that results in a low incidence of GVHD. Compared to the ATG-based approach, PTCy provides better control of GVHD in children. In pediatric allo-HSCT from MSD, PTCy demonstrates comparable effectiveness to conventional GVHD prophylaxis.

Allogeneic Hematopoietic Stem Cell Transplantation Activity in Inborn Errors of Immunity in Russian Federation.

PURPOSE: Allogeneic hematopoietic stem cell transplantation (HSCT) is an established therapy for many inborn errors of immunity (IEI). The indications for HSCT have expanded over the last decade. The study aimed to collect and analyze the data on HSCT activity in IEI in Russia. METHODS: The data were collected from the Russian Primary Immunodeficiency Registry and complemented with information from five Russian pediatric transplant centers. Patients diagnosed with IEI by the age of 18 years and who received allogeneic HSCT by the end of 2020 were included. RESULTS: From 1997 to 2020, 454 patients with IEI received 514 allogeneic HSCT. The median number of HSCTs per year has risen from 3 in 1997-2009 to 60 in 2015-2020. The most common groups of IEI were immunodeficiency affecting cellular and humoral immunity (26%), combined immunodeficiency with associated/syndromic features (28%), phagocyte defects (21%), and diseases of immune dysregulation (17%). The distribution of IEI diagnosis has changed: before 2012, the majority (65%) had severe combined immunodeficiency (SCID) and hemophagocytic lymphohistiocytosis (HLH), and after 2012, only 24% had SCID and HLH. Of 513 HSCTs, 48.5% were performed from matched-unrelated, 36.5% from mismatched-related (MMRD), and 15% from matched-related donors. In 349 transplants T-cell depletion was used: 325 TCRαß/CD19+ depletion, 39 post-transplant cyclophosphamide, and 27 other. The proportion of MMRD has risen over the recent years. CONCLUSION: The practice of HSCT in IEI has been changing in Russia. Expanding indications to HSCT and SCID newborn screening implementation may necessitate additional transplant beds for IEI in Russia.

Studying the Plastic Deformation of Cu-Ti-C-B Composites in a Favorable Stress State.

Composites with a copper matrix attract the attention of researchers due to their ability to combine high ductility, heat conductivity, and electrical conductivity of the matrix with the high hardness and strength of the reinforcing phases. In this paper, we present the results of studying the effect of thermal deformation processing of a Сu-Ti-C-B composite produced by self-propagating high-temperature synthesis (SHS) on its ability to deform plastically without failure. The composite consists of a copper matrix and reinforced particles of titanium carbide TiC (sized up to 1.0 µm) and titanium diboride TiB2 (sized up to 3.0 µm). The composite hardness is 60 HRC. Under uniaxial compression, the composite starts to deform plastically at a temperature of 700 °C and a pressure of 100 MPa. Temperatures ranging between 765 and 800 °C and an initial pressure of 150 MPa prove to be the most effective condition for composite deformation. These conditions enabled a true strain of 0.36 to be obtained without composite failure. Under higher strain, surface cracks appeared on the specimen surface. The EBSD analysis shows that dynamic recrystallization prevails at a deformation temperature of at least 765 °C; therefore, the composite can plastically deform. To increase the deformability of the composite, it is proposed to perform deformation under conditions of a favorable stress state. Based on the results of numerical modeling by the finite element method, the critical diameter of the steel shell is determined, which is sufficient for deformation of the composite with the most uniform distribution of the stress coefficient k. Composite deformation in a steel shell under a pressure of 150 MPa, at 800 °C, is experimentally implemented until a true strain of 0.53 is reached.

Risk factors for a severe disease course in children with SARS-COV-2 infection following hematopoietic cell transplantation in the pre-Omicron period: a prospective multinational Infectious Disease Working Party from the European Society for Blood and Marrow Transplantation group (EBMT) and the Spanish Group of Hematopoietic Stem Cell Transplantation (GETH) study.

Risk factors for severe SARS-Cov-2 infection course are poorly described in children following hematopoietic cell transplantation (HCT). In this international study, we analyzed factors associated with a severe course (intensive care unit (ICU) admission and/or mortality) in post-HCT children. Eighty-nine children (58% male; median age 9 years (min-max 1-18)) who received an allogeneic (85; 96%) or an autologous (4; 4%) HCT were reported from 28 centers (18 countries). Median time from HCT to SARS-Cov-2 infection was 7 months (min-max 0-181). The most common clinical manifestations included fever (37; 42%) and cough (26; 29%); 37 (42%) were asymptomatic. Nine (10%) children following allo-HCT required ICU care. Seven children (8%) following allo-HCT, died at a median of 22 days after SARS-Cov-2 diagnosis. In a univariate analysis, the probability of a severe disease course was higher in allo-HCT children with chronic GVHD, non-malignant disease, immune suppressive treatment (specifically, mycophenolate), moderate immunodeficiency score, low Lansky score, fever, cough, coinfection, pulmonary radiological findings, and high C-reactive protein. In conclusion, SARS-Cov-2 infection in children following HCT was frequently asymptomatic. Despite this, 10% needed ICU admission and 8% died in our cohort. Certain HCT, underlying disease, and SARS-Cov-2 related factors were associated with a severe disease course.

Extracorporeal Photopheresis in Children with Chronic Graft-Versus-Host Disease.

Chronic graft versus host disease (cGVHD) remains a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). It significantly decreases survival and quality of life. The present study demonstrates retrospective data on extracorporeal photopheresis (ECP) in children with cGVHD. A total of 42 children with steroid-refractory cGVHD were enrolled in the study. The majority of patients had acute leukemia (n = 32, 76%). All patients received ECP as second (n = 18, 43%) or third (n = 24, 57%) line of therapy. Initial ECP schedule consisted of bimonthly regimen for two consecutive days with possibility of further tapering according to response. Any concurrent treatment administered before ECP could be continued if considered necessary. Complete response to ECP was registered in seven (17%) patients and partial response in 24 (57%). Overall response according to organ involvement was as follows: skin (n = 24, 75%), mucous membranes (n = 16, 73%), liver (n = 8, 80%), gut (n = 4, 80%), lungs (n = 2, 22%) and joints (n = 2, 67%). Five-year overall, progression-free and failure-free survival was 57%, 56% and 30%, respectively. Non-relapse mortality at 5 years was 14%. We didn't observe any clinically significant complications in children that could be attributed to the procedure. ECP remains important and safe treatment option in children with cGVHD.

Stem Cell Transplantation for Diamond-Blackfan Anemia. A Retrospective Study on Behalf of the Severe Aplastic Anemia Working Party of the European Blood and Marrow Transplantation Group (EBMT).

Data on stem cell transplantation (SCT) for Diamond-Blackfan Anemia (DBA) is limited. We studied patients transplanted for DBA and registered in the EBMT database. Between 1985 and 2016, 106 DBA patients (median age, 6.8 years) underwent hematopoietic stem cell transplantation from matched-sibling donors (57%), unrelated donors (36%), or other related donors (7%), using marrow (68%), peripheral blood stem cells (20%), both marrow and peripheral blood stem cells (1%), or cord blood (11%). The cumulative incidence of engraftment was 86% (80% to 93%), and neutrophil recovery and platelet recovery were achieved on day +18 (range, 16 to 20) and +36 (range, 32 to 43), respectively. Three-year overall survival and event-free survival were 84% (77% to 91%) and 81% (74% to 89%), respectively. Older patients were significantly more likely to die (hazard ratio, 1.4; 95% confidence interval, 1.06 to 1.23; P < .001). Outcomes were similar between sibling compared to unrelated-donor transplants. The incidence of acute grades II to IV of graft-versus-host disease (GVHD) was 30% (21% to 39%), and the incidence of extensive chronic GVHD was 15% (7% to 22%). This study shows that SCT may represent an alternative therapeutic option for transfusion-dependent younger patients.

mTOR kinase inhibitor pp242 causes mitophagy terminated by apoptotic cell death in E1A-Ras transformed cells.

mTOR is a critical target for controlling cell cycle progression, senescence and cell death in mammalian cancer cells. Here we studied the role of mTOR-dependent autophagy in implementating the antiprolifrative effect of mTORC1-specific inhibitor rapamycin and ATP-competitive mTOR kinase inhibitor pp242. We carried out a comprehensive analysis of pp242- and rapamycin-induced autophagy in ERas tumor cells. Rapamycin exerts cytostatic effect on ERas tumor cells, thus causing a temporary and reversible cell cycle arrest, activation of non-selective autophagy not accompanied by cell death. The rapamycin-treated cells are able to continue proliferation after drug removal. The ATP-competitive mTORC1/mTORC2 kinase inhibitor pp242 is highly cytotoxic by suppressing the function of mTORC1-4EBP1 axis and mTORC1-dependent phosphorylation of mTORC1 target--ULK1-Ser757 (Atg1). In contrast to rapamycin, pp242 activates the selective autophagy targeting mitochondria (mitophagy). The pp242-induced mitophagy is accompanied by accumulation of LC3 and conversion of LC3-I form to LC3-II. However reduced degradation of p62/SQSTM indicates abnormal flux of autophagic process. According to transmission electron microscopy data, short-term pp242-treated ERas cells exhibit numerous heavily damaged mitochondria, which are included in single membrane-bound autophagic/autolysophagic vacuoles (mitophagy). Despite the lack of typical for apoptosis features, ERas-treated cells with induced mitophagy revealed the activation of caspase 3, 9 and nucleosomal DNA fragmentation. Thus, pp242 activates autophagy with suppressed later stages, leading to impaired recycling and accumulation of dysfunctional mitochondria and cell death. Better understanding of how autophagy determines the fate of a cell--survival or cell death, can help to development of new strategy for cancer therapy.

Sustained activation of DNA damage response in irradiated apoptosis-resistant cells induces reversible senescence associated with mTOR downregulation and expression of stem cell markers.

Cells respond to genotoxic stress by activating the DNA damage response (DDR). When injury is severe or irreparable, cells induce apoptosis or cellular senescence to prevent transmission of the lesions to the daughter cells upon cell division. Resistance to apoptosis is a hallmark of cancer that challenges the efficacy of cancer therapy. In this work, the effects of ionizing radiation on apoptosis-resistant E1A + E1B transformed cells were investigated to ascertain whether the activation of cellular senescence could provide an alternative tumor suppressor mechanism. We show that irradiated cells arrest cell cycle at G 2/M phase and resume DNA replication in the absence of cell division followed by formation of giant polyploid cells. Permanent activation of DDR signaling due to impaired DNA repair results in the induction of cellular senescence in E1A + E1B cells. However, irradiated cells bypass senescence and restore the population by dividing cells, which have near normal size and ploidy and do not express senescence markers. Reversion of senescence and appearance of proliferating cells were associated with downregulation of mTOR, activation of autophagy, mitigation of DDR signaling, and expression of stem cell markers.

Rapamycin induces pluripotent genes associated with avoidance of replicative senescence.

Primary rodent cells undergo replicative senescence, independent from telomere shortening. We have recently shown that treatment with rapamycin during passages 3-7 suppressed replicative senescence in rat embryonic fibroblasts (REFs), which otherwise occurred by 10-14 passages. Here, we further investigated rapamycin-primed cells for an extended number of passages. Rapamycin-primed cells continued to proliferate without accumulation of senescent markers. Importantly, these cells retained the ability to undergo serum starvation- and etoposide-induced cell cycle arrest. The p53/p21 pathway was functional. This indicates that rapamycin did not cause either transformation or loss of cell cycle checkpoints. We found that rapamycin activated transcription of pluripotent genes, oct-4, sox-2, nanog, as well as further upregulated telomerase (tert) gene. The rapamycin-derived cells have mostly non-rearranged, near-normal karyotype. Still, when cultivated for a higher number of passages, these cells acquired a chromosomal marker within the chromosome 3. We conclude that suppression mTORC1 activity may prevent replicative senescence without transformation of rodent cells.

Suppression of replicative senescence by rapamycin in rodent embryonic cells.

The TOR (target of rapamycin) pathway is involved in aging in diverse organisms from yeast to mammals. We have previously demonstrated in human and rodent cells that mTOR converts stress-induced cell cycle arrest to irreversible senescence (geroconversion), whereas rapamycin decelerates or suppresses geroconversion during cell cycle arrest. Here, we investigated whether rapamycin can suppress replicative senescence of rodent cells. Mouse embryonic fibroblasts (MEFs) gradually acquired senescent morphology and ceased proliferation. Rapamycin decreased cellular hypertrophy, and SA-ß-Gal staining otherwise developed by 4-6 passages, but it blocked cell proliferation, masking its effects on replicative lifespan. We determined that rapamycin inhibited pS6 at 100-300 pM and inhibited proliferation with IC(50) around 30 pM. At 30 pM, rapamycin partially suppressed senescence. However, the gerosuppressive effect was balanced by the cytostatic effect, making it difficult to suppress senescence without causing quiescence. We also investigated rat embryonic fibroblasts (REFs), which exhibited markers of senescence at passage 7, yet were able to slowly proliferate until 12-14 passages. REFs grew in size, acquired a large, flat cell morphology, SA-ß-Gal staining and components of DNA damage response (DDR), in particular, γH2AX/53BP1 foci. Incubation of REFs with rapamycin (from passage 7 to passage 10) allowed REFs to overcome the replicative senescence crisis. Following rapamycin treatment and removal, a fraction of proliferating REFs gradually increased and senescent phenotype disappeared completely by passage 24.

p21Waf1 is required for complete oncogenic transformation of mouse embryo fibroblasts by E1Aad5 and c-Ha-ras oncogenes.

Cyclin-dependent kinase inhibitor p21(Waf1) is known to have alternative functions associated with positive regulation of proliferation, actin cytoskeleton remodeling and suppression of apoptosis. The goal of the present study was to assess the role of p21(Waf1) in the establishment of the transformed phenotype of mouse embryo fibroblasts with stable expression of E1Aad5 and c-Ha-ras complementary oncogenes. Herein, we demonstrate that E1A/c-Ha-Ras-transformed p21(Waf1)-null fibroblasts possess some characteristic features of transformed cells, such as loss of contact inhibition, high saturation density, shortened cell cycle, inability to undergo cell-cycle arrest after DNA damage and serum deprivation, but, at the same time, they are not completely transformed in that they are unable to proliferate at clonal density, are anchorage-dependent, retain a fibroblast-like morphology with pronounced actin cytoskeleton and show reduced migration and invasion. Our data support the concept of p21(Waf1) "tumor suppressor" having alternative oncogenic functions in the cytoplasm and for the first time indicate that p21(Waf1) can be indispensable for complete oncogenic transformation.