Vol 22, No 3 (2023)

The main outcomes of allogeneic hematopoietic stem cell transplantation (HSCT) in chemotherapy-refractory acute leukemia remain suboptimal due to a high relapse rate. The incorporation of targeted anti-leukemia agents into the conditioning regimens is a potential approach to improve the efficacy of HSCT. We assessed the safety and potential efficacy of the addition of venetoclax, daratumumab, and plerixafor to the conditioning regimens in children with chemotherapy-refractory acute leukemias who received allogeneic TCRab/CD19-depleted HSCT. We used data from a pilot study, as well as the data of patients from a retrospective cohort who received similar therapy according to the individual indications. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. All 43 patients (33 acute myeloid leukemias (AML), 8 T-cell acute lymphoblastic leukemias (T-ALL) and 2 acute leukemias of ambiguous lineage) had active disease status at the time of transplantation. The preparative regimen included myeloablative conditioning based on either total body irradiation or treosulfan or melphalan. A haploidentical related donor was used as a graft source in 38 cases, while a fully matched related or unrelated donor was used in 5 cases. The engraftment was observed in 93% of cases, no excessive toxicity was noted. MRD-negative complete remission was achieved in 37 patients (86%). The cumulative incidence of grade II–IV acute graft-versus-host disease (GvHD) was 10%, and the cumulative incidence of chronic GvHD was 5%. At 2 years, transplant-related mortality was 7%, relapse incidence was 52%, event-free survival was 41%, and overall survival was 51%. The overall survival rate for the AML group was 58% and 25% for the T-ALL group. Our data show that the addition of targeted agents to the conditioning regimens is safe, however, does not significantly improve the results of HSCT in the study cohort of patients.

The development of inhibitory antibodies against FVIII is the most serious complication associated with the use of FVIII concentrates in hemophilia A patients. There is a need for more research on measures that could reduce the risk of inhibitor formation in previously untreated patients (PUPs) with severe hemophilia A. The purpose of this study was to determine the effectiveness of the prevention of clotting inhibitor development in PUPs (or minimally treated patients) with severe hemophilia A by administering plasma-derived factor VIII concentrate (pdFVIII) at a dose of 25 IU/kg once a week for a year. The study was approved by the Independent Ethics Committee and the Scientific Council of the Belarusian Research Center for Pediatric Oncology, Hematology and Immunology (the Republic of Belarus). Between 2010 and 2022, 56 boys were newly diagnosed with severe hemophilia A. Twenty-one of them received pdFVIII as on-demand treatment to stop bleeding (Group 1). Thirty-five boys received pdFVIII at a dose of 25 IU/kg body weight once a week during the first 50 weeks of treatment for the prevention of inhibitor development (Group 2). The administration of pdFVIII at a dose of 25 IU/kg once a week in the PUPs (or minimally treated patients) contributed to a decrease in the cumulative incidence of inhibitors to 15.9 ± 7.7% (4 out of the 35 patients who had been treated prophylactically) compared with 43.7 ± 11.8% (8 out of the 21 patients who had received hemostatic therapy to stop bleeding) (log-rank test, p = 0.041). Thus, the administration of pdFVIII concentrate at a dose of 25 IU/kg once a week for the first 50 weeks of treatment lead to a decrease (p = 0.009) in the cumulative incidence of inhibitors against the administered coagulation factor VIII to 15.9 ± 7.7%.

Hemophilia A is the most common severe bleeding disorder caused by various genetic changes in the F8 gene, leading to coagulation factor VIII deficiency. Hemophilia A is characterized by high heterogeneity of genetic defects. The severity of hemophilia A varies depending on the type of genetic defects in the F8 gene. More than 3000 unique variants of the F8 gene are associated with the hemophilia A. Approximately 30% of genetic defects occur de novo. The aim of this study is to determine the spectrum of genetic defects in the F8 gene in children with hemophilia A in Belarus. The study was approved by the Independent Ethics Committee and the Scientific Council of the Belarusian Research Center for Pediatric Oncology, Hematology and Immunology (the Republic of Belarus). The study included 98 patients with hemophilia A, who had been treated or followed up at the Belarusian Research Center for Pediatric Oncology, Hematology and Immunology (the Republic of Belarus). Patients were categorized into 3 groups based on the severity of their disease: severe (n = 82), moderate (n = 3), and mild (n = 13). Twenty (20.4%) patients had a history of inhibitors to factor VIII. For our study, we used venous blood samples. Genomic DNA was isolated from leukocyte suspension (obtained from the whole blood samples) using phenol-chloroform extraction. All severe hemophilia A patients were prescreened for intron 22 and 1 inversions in the F8 gene using inverse and multiplex polymerase chain reaction assays, respectively. Sequencing of F8 coding regions was carried out by next generation sequencing. All clinically relevant variants were confirmed by Sanger sequencing. Genetic testing revealed that 99% of the patients with hemophilia A (n = 97) had pathogenic variants in the F8 gene. Intron 22 and intron 1 inversion mutations within the F8 gene were detected in 45.1% (n = 37) and 1.2% (n = 1) patients with severe hemophilia A, respectively. Two patients had an abnormal pattern of intron 1 inversion, not previously described in the literature. A total of 48 different variants in the F8 gene were detected in 57 patients using next generation sequencing. Eleven of the 48 genetic variants identified have not been previously reported.

Sickle cell disease (SCD) is one of the most prevalent autosomal recessive diseases, characterized by the generation of abnormal hemoglobin S. Our study aimed to assess how the serum level of interferon-gamma affects the health status of patients with SCD in Basrah. A total of 90 participants were enrolled in this study and divided into two main groups: a SCD group and a control group. The SCD group included 30 patients with SCD in steady state and 30 patients with SCD in vasoocclusive crisis; the control group included 30 ageand sexmatched apparently healthy individuals. Approval was obtained from the Research Ethics Committee of the College of Medicine, University of Basrah before conducting the study. Two milliliters of venous blood were drawn from all the participants, and ELISA tests were utilized to determine the levels of serum interferon-gamma. There was a statistically significant increase in the serum level of interferon-gamma among SCD patients (both in steady state and in crisis) compared to the control group (p = 0.05). There were no significant differences in the levels of interferon-gamma between the patients in steady state and during vaso-occlusive crisis (p > 0.05). Interferon-gamma may influence the general health of sickle cell patients and contribute to the cause of inflammation, no matter whether the patient is in stable condition or is experiencing a crisis.

Hemophilia is defined as X-linked recessive bleeding disorder. Recurrent bleeding episodes lead to hemarthrosis.

Objectives: to investigate the levels of serum 25(OH) D and trace elements in children with hemophilia A and B and to identify the possible association of these factors with Hemophilia Joint Health Score (HJHS). This case-control study was conducted among children with hemophilia A and B. A total of 48 cases were recruited from the hematology units at the Menoufia University Hospital (n = 36) and Sohag University Hospital (n = 12) from December 2020 to February 2022. Forty healthy controls were matched to cases on age, sex and socioeconomic status. Serum zinc and magnesium levels in the hemophilia patients were significantly lower than in the controls, while serum alkaline phosphatase levels in the cases were significantly higher than in the controls. Informed consent was obtained from all the children's parents and ethical approval was acquired from the ethical committee (ID: 5/2020PEDI38), Faculty of Medicine, Menoufia University. The levels of phosphorus and calcium were the same in two groups. Serum 25(OH) D levels were deficient in 85.4% of the cases and insufficient in 14.6%. None of the hemophilia patients had sufficient levels of serum 25(OH) D. There was no significant correlation between HJHS and the levels of serum trace elements but there was a significant positive correlation between HJHS and annualized bleeding rate and a significant negative correlation between HJHS and serum vitamin D. There was no significant difference regarding the demographic data except for weight and body mass index. The patients had significantly higher weight and body mass index compared to the control group. The levels of serum vitamin D and trace elements were decreased in hemophilia patients, and these low values were associated with the worst joint health.

Our study aimed to assess the prognostic significance of immature platelet fraction (IPF) and its role in deciding whether to transfuse platelets. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. We monitored this hematologic parameter in 6 patients before and after hematopoietic stem cell transplantation (HSCT). For the study, we used whole blood collected in K EDTA tubes. IPF levels were measured by flow cytometry on the automated hematology analyzer SYSMEX XE-2100 (Sysmex, Kobe, Japan). The presence of platelet antibodies was detected using Capture-P Ready-Screen solid phase system for the detection of antibodies to platelets and the NEO Blood Bank Analyzer by Immucor, Inc. It was shown that the use of IPF enabled an early establishment of platelet engraftment and helped to make more a reasoned decision regarding the transfusion of platelets. It was established that if a rise in IPF > 6% was not accompanied by an increase in platelet count, serum platelet antibody testing was needed. IPF can help diagnose platelet engraftment failure following HSCT and thus eliminate the need for bone marrow aspiration. IPF is of great clinical importance; if adopted as a routine hematologic parameter, it can serve as an additional factor in deciding on the need for platelet transfusions and platelet antibody testing and subsequent personalized selection of platelets.

Somatic translocations involving the NTRK genes occur in 0.34–2.2% of all malignant neoplasms in children. TRK inhibitors whose efficacy has been demonstrated in prospective clinical studies expand treatment options for patients with solid tumors harboring NTRK gene rearrangements. The aim of our study was to summarize the first Russian experience with the use of the TRK inhibitor entrectinib in patients with extracranial NTRK fusion-positive solid tumors included in the compassionate use program. This study was approved by the Independent Ethics Committee and the Academic Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. The study included 8 patients who had been treated at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology and the N.N. Blokhin National Medical Research Center of Oncology. The main criteria for inclusion in the compassionate use program were a confirmed rearrangement of either NTRK1/2/3 genes in a solid tumor in patients with unresectable disease for whom no effective standard systemic therapy was available, progressive or recurrent disease during therapy prescribed according to the established diagnosis, risk group and risk stratification criteria, and the infeasibility of non-mutilating radical surgery. The median age at diagnosis was 4.3 months (range 1.2–83.6). The male to female ratio was 1:1. The primary site distribution was as follows: head and neck (n = 6; 75%), chest wall (n = 1; 12.5%), pelvis (n = 1; 12.5%). None of the patients had regional lymph node involvement or distant metastases at diagnosis. The distribution by histology (according to histopathology reports) was as follows: infantile fibrosarcoma (n = 4; 50%), undifferentiated round cell sarcoma, low-grade (n = 1; 12.5%), undifferentiated spindle cell sarcoma, high-grade (n = 1; 12.5%), NTRK-rearranged spindle cell sarcoma, low-grade (n = 1; 12.5%), spindle cell tumor associated with an NTRK rearrangement (n = 1; 12.5%). Immunohistochemistry (IHC) with a pan-Trk monoclonal antibody was performed in 7/8 (87.5%) patients. Pan-Trk IHC was positive in 4/7 (57%) patients. Rearrangements in the NTRK1 and NTRK3 genes were confirmed in all the patients. The final methods used for the detection of fusion transcripts were as follows: reverse transcription polymerase chain reaction (n = 4; 50%) and RNA-based next-generation sequencing (n = 4; 50%). NTRK1 and NTRK3 gene translocations were detected in 3/8 (37.5%) and 5/8 (62.5%) patients, respectively. The following fusion transcripts were identified: ETV6::NTRK3 (n = 4), DIP2C::NTRK3 (n = 1), TPR::NTRK1 (n = 1), TPM3::NTRK1 (n = 1), MYH10::NTRK1 (n = 1). One (12.5%) patient received entrectinib as first-line therapy, other patients (7/8, 87.5%) received entrectinib as secondor subsequent-line therapy. Three (37.5%) patients had undergone surgery before treatment with entrectinib: 2 had R2 resection, 1 had R0/R1 resection (resection margins were not evaluated). None of the patients received radiation therapy. The median duration of entrectinib therapy at the time of analysis was 11.8 months (range 2.3–20.1). Delayed surgery was performed in 2/8 patients; according to the histopathology reports, they achieved grade IV pathomorphosis. Three patients experienced adverse events during treatment with entrectinib. The median time to adverse events was 0.23 months (range 0.2–7.96). Three patients required temporary interruption in treatment to relieve symptoms, a subsequent dose reduction by one dose level was necessary when resuming therapy in two patients. The median follow-up since diagnosis was 19.5 months (range 14.9–75.0). All the patients included in our analysis were alive, three of them had no radiologic evidence of disease. Fifty percent of the patients completed targeted therapy, another 50% of the patients continued treatment with entrectinib. Complete and very good partial response was achieved in 3/8 and 2/8 patients, respectively. Partial response, minor partial response and stable disease were observed in one patient each. These results indicate high efficacy and safety of entrectinib in pediatric patients with extracranial NTRK fusion-positive solid tumors. Further studies are needed to determine the therapeutic potential of TRK inhibitors in the treatment of different solid malignant neoplasms in children and to assess long-term treatment results.

Anemia is a common comorbidity in children with chronic kidney disease (CKD) and is associated with adverse outcomes. Erythroferrone (ERFE) is a hepcidin inhibitor whose synthesis is stimulated by erythropoietin, which increases iron absorption and mobilization. Aim of the study: to assess the levels of ERFE hormone in children with CKD on regular hemodialysis. This case–control study was carried out at Menoufia University Hospital and included 70 subjects: 38 healthy individuals (controls) and 32 children with CKD on regular dialysis (cases). The study was approved by the Faculty of Medicine Ethics Committee at Menoufia University. All children were subjected to full history taking, complete clinical examination, blood tests such as complete blood count, reticulocyte count, serum iron, ferritin, and total iron binding capacity, liver and renal function tests, and an immunoassay to measure human ERFE. There was a statistically significant difference in the levels of ERFE between the cases and controls (p < 0.001). There was a significant, strong correlation between the levels of hemoglobin and serum iron and the level of ERFE (r = –0.655, p < 0.001). There was no significant correlation between the administered dose of exogenous erythropoietin and the level of ERFE (p = 0.460). Serum ERFE levels in the children with CKD on regular hemodialysis were significantly higher than in the controls and were negatively correlated with hemoglobin and iron levels. There was no significant correlation between ERFE levels and both serum ferritin and total iron binding capacity levels.

Reticulocyte hemoglobin content (RET-He) is a promising marker of iron deficiency (ID) in newborns. Objective: to determine the diagnostic value of RET-He as a marker of ID in premature newborns with very low birth weight (VLBW). We conducted a single-center retrospective cohort study, which included 66 premature infants admitted to the National Medical Research Center for Obstetrics, Gynecology and Perinatology named the Academician V.I. Kulakov of the Ministry of Healthcare of the Russian Federation. Data were obtained from January 2016 to December 2018. The gestational age ranged from 29 to 32 weeks. Laboratory examination included blood tests on the 1^st and 3^rd day of life, then every 10–14 days until the day of life, then every 10–14 days the Institute of Neonatology and Pediatrics;  discharge from hospital, and the measurements of serum iron, ferritin, transferrin on the 7^th until the discharge from hospital. This clinical study was approved by the Biomedical Research Ethics Committee (Minutes No.12 of 17 November 2016) and the Scientific Council (Minutes No.19 of 29 November 2016) of the National Medical Research Center for Obstetrics, Gynecology and Perinatology named after the Academician V.I. Kulakov of Ministry of Healthcare of the Russian Federation. RET-He was the highest at birth and declined gradually thereafter in premature newborns reaching the lowest values after 3 weeks of life (median (interquartile range) 28.4 (25.8–34.8) pg (on the 1st day of life – 40.0 (35.7–41.9) pg and 33.5 (29.2–36.6) pg at the time of discharge). A low RET-He level was associated with low reticulocytes, with no changes in hemoglobin. There was a positive correlation between RET-He and MCH. D-He decreased from 1 to 42 days of life as a marker of increasing anemia. There was a negative correlation between RET-He and Hypo-He (p < 0.005). Starting from 42 days of life, or by the time of discharge, 32% of premature infants (n = 21) had a low ferritin level and 77% (n = 51) of premature infants had a low RET-He level, of which 21 infants developed ID (a positive correlation between RET-He and ferritin after 42 days of life (r = 0.34, p = 0.046)). There was no correlation between RET-He and ferritin in newborns without ID. Also, there were no correlations between RET-He and iron and RET-He and transferrin. After 42 days of life, RET-He less than 28.4 pg was a marker of ID (sensitivity 83.3% and specificity 93.7%). Low RET-He, D-He, RBC-Hе and high microR, Hypo-He were the earliest markers of ID in premature infants which predicted a decrease in serum iron and ferritin levels. RET-He, D-Не and Hypo-He are biomarkers with accurate diagnostic value of ID in premature infants with VLBW.

Platelet-type bleeding disorder-20 is a rare inherited thrombocytopenia caused by mutations in the SLFN14 gene. We report a case of a female patient with SLFN14 mutation, macrothrombocytopenia, severe hemorrhagic syndrome and a positive family history who was followed up from the age of 17 to 19. The 3-year follow-up showed a tendency towards partial normalization of platelet counts (from 47 to 82 × 109/L) and morphology. Platelet size and granularity as well as the density of glycoprotein (GP) membrane receptors such as GP Ib/V/IX and GP IIb/IIIa decreased. GP IIb/IIIa activation was impaired and there were no positive changes over time. The dense granules indicators were stably elevated. The parameters of a-granules (assessed by P-selectin expression) did not differ from the control group. The proportion of procoagulant phosphatidylserine-positive platelets at rest was increased and the potential to form procoagulant platelets upon activation was reduced. As the patient grew older, her bleeding disorder symptoms abated and she showed a tendency towards normalization of platelet laboratory parameters. All investigations were performed after obtaining informed consent from the patient and her parents in accordance with the Declaration of Helsinki.

Primary immunodeficiencies are congenital genetically determined immune disorders. Recent advances in molecular genetic technologies have enabled a simultaneous analysis of a large number of genes in a patient. The purpose of this study was to analyze the mutational spectrum in DNA samples collected from patients with various types of primary immunodeficiencies. In this study, we applied next-generation sequencing technology using a panel developed at the Belarusian Research Center for Pediatric Oncology, Hematology and Immunology and consisting of 290 genes that are associated with primary immunodeficiencies according to the existing literature. The testing was carried out in 96 patients with a clinical history suggesting a primary immunological defect. As a result, 37.5% of cases (36/96 patients) were found to harbor genetic defects that lead to disorders of the immune system.

Shwachman–Diamond syndrome is a rare genetic disorder with an autosomal recessive inheritance pattern. Most often (in more than 90% of cases) this disease is caused by biallelic pathogenic variants in the highly conserved SBDS gene located on the long arm of chromosome 7. However, approximately 10% of patients with the clinical phenotype of Shwachman–Diamond syndrome lack mutations in SBDS but have pathogenic variants in other genes, such as DNAJC21 or EFL1. Shwachman–Diamond syndrome is a multisystemic disorder characterized by exocrine pancreatic insufficiency, protein-energy undernutrition, delayed physical development, cognitive disorders, anomalies of the skeletal system, and immunological disorders. In addition to the described symptoms, Shwachman–Diamond syndrome is characterized by the presence of bone marrow failure (most often neutropenia and anemia), as well as an increased risk of cytogenetic abnormalities and a predisposition to myelodysplastic syndromes and acute myeloid leukemia. In this review, the authors summarize the spectrum of hematological disorders observed in Shwachman–Diamond syndrome, as well as describe the molecular mechanisms underlying them.

Multicolor flow cytometry is now routinely used in laboratory practice for minimal residual disease (MRD) monitoring in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). This article describes the methodology of MRD detection in BCP-ALL using flow cytometry as recommended by the Russian-Belarusian multicenter group for pediatric acute leukemia studies. This wellharmonized approach includes recommendations for the choice of monoclonal antibodies, sample preparation, cytometer setup, flow cytometry data analysis and interpretation as well as for reporting. These guidelines allow application of multicolor flow cytometry for MRD monitoring in BCP-ALL in children and adults both in local laboratories and in multicenter settings in prospective clinical trials.