Vol 23, No 2 (2024)

Hematopoietic stem cell transplantation (HSCT) is known to be most effective in cancer patients in remission. In this paper, we analyzed a cohort of children with refractory acute myeloid leukemia (AML) in order to study the effectiveness of HSCT in such patients. 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 and was conducted in accordance with the principles of the Declaration of Helsinki. All the patients and/or their legal representatives signed an informed consent form for participation in the study. Our retrospective analysis included 69 patients with refractory AML (induction failure (n = 31), refractory relapse (n = 38)) whose median age was 9.4 (1.1–22) years and who had undergone HSCT between February 2012 and January 2020, with the median follow-up of 5.47 (1.9–8.9) years. Fifty patients were transplanted from haploidentical donors, 10 – from matched related donors, 9 – from matched unrelated donors. All the patients received treosulfan-based conditioning and either melphalan or thiotepa. Fifty-five cases received TCR_ab⁺/CD19⁺-depleted HSCs (CliniMACS), 11 patients received native bone marrow and 2 – unrelated umbilical cord blood. For post-transplant relapse prevention, 21 patients were treated with hypomethylating agents in combination with bortezomib and 48 patients received modified donor lymphocyte infusions. Primary engraftment was achieved in 66 out of 69 patients (3 patients had died before engraftment). By Day +30, 86% of patients showed complete chimerism. The cumulative incidence of grade II–IV acute graft-versus-host disease was 42%, chronic graft-versushost disease was diagnosed in 17 pts. In the entire cohort, transplant-related mortality was 7.8% and cumulative incidence of relapse was 53%. NK cell recovery by Day +30 was significantly associated with decreased incidence of relapse: patients whose absolute NK cell counts were below the median had a cumulative incidence of relapse of 76% versus 43% in patients with NK cell counts above the median (p = 0.013). At a median follow-up of 5.5 years, the event-free survival was 37 ± 11%, and the overall survival was 42 ± 10%. Remission was achieved in 86% of the patients, while long-term overall survival reached about 40%. Our findings suggest that allogeneic HSCT provides a reasonable chance of cure in children with refractory AML and creates a solid basis for further improvement. Tumor burden in the bone marrow before conditioning and early post-transplant NK cell counts in the blood were found to be the most significant prognostic factors in HSCT.

A decrease in T cell alloreactivity following hematopoietic stem cell transplantation (HSCT) can be achieved via the depletion of TCR_ab⁺ and CD19⁺ lymphocytes or post-transplant therapy with high-dose cyclophosphamide (PTCy). The purpose of this study was to analyze HSCT outcomes according to the type of lymphocyte depletion. In our non-randomized retrospective study, we analyzed the results of 118 HSCTs performed in 107 patients with malignant blood disorders at the Center for Pediatric Oncology and Hematology of the Regional Children’s Clinical Hospital (Yekaterinburg) from 2010 to 2023. The patients were divided into 2 groups: those who had received ex vivo TCR_ab⁺/CD19⁺ depletion using the CliniMACS technology (n = 75; Group 1) and those who had received in vivo depletion using PTCy at a dose of 50 mg/kg (n = 43; Group 2). The study was approved by the Independent Ethics Committee and the Scientific Council of the Institute of Medical Cell Technologies (Yekaterinburg). Informed consent for the research and treatment was obtained from each patient and their legal representatives. Even though the patients from the TCR_ab⁺/CD19⁺ depletion group showed a better engraftment rate (median time: 12 days) than the subjects from the PTCy group (median time: 18 days) (p < 0.0001), there was no statistically significant difference in the cumulative incidence of cytomegalovirus and herpesvirus type 6 infection reactivation post-transplant between the two study groups. The cytomegalovirus reactivation rate was 0.467 and 0.466 in Group 1 and 2 respectively (p = 0.89), while for herpesvirus type 6 the reactivation rate was 0.360 and 0.240 respectively (p = 0.13). There were no significant differences in the cumulative incidence of acute graft-versus-host disease (GVHD) in the two groups by Day +100 (0.294 and 0.419 in Group 1 and 2 respectively; p = 0.77); neither was there any difference in the incidence of severe acute GVHD (0.067 and 0.093 respectively; p = 0.11). The incidence of chronic GVHD was 0.12 in the TCR_ab⁺/CD19⁺ depletion group and 0.26 in the PTCy group (p = 0.11). The event-free survival, overall survival and cumulative incidence of relapse did not differ between the studied groups either. The two methods of the depletion of unwanted T-cell populations showed a comparable effectiveness. The choice between them should be based on factors related to the biological characteristics of the disease and the clinical status of a patient. The availability of different depletion methods at a single transplant center helps to choose the optimal treatment option for patients who are in need of HSCT.

Graft-versus-host disease (GVHD) remains the main life-threatening immunologic complication of hematopoietic stem cell transplantation. Despite modern pharmacological approaches for preventing and treating GVHD, there remains a need for new approaches to cure GVHD. Currently, more and more clinical experience is emerging globally in the field of using regulatory T-cell (T_reg) therapies for the treatment of refractory GVHD. Manufacturing cell products for T_reg therapies has a wide range of protocol variations. We have developed an approach of T_reg manufacturing for cell therapy and present data from our experience in manufacturing a haploidentical T_reg cell product by combining CD25⁺ immunomagnetic selection with closed system flowbased cell sorting methods. 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. The study describes the processes of producing 9 T_reg cell products. According to the manufacturing protocol described here, it is possible to obtain a cell product that meets the quality control requirements necessary for approval for clinical use. Quality control includes the assessment of the cell composition, viability, and microbiological safety of the product, and is performed at all major stages of production. The final cell product is characterized by consistently high levels of FoxP3-expressing T_reg (median: 98%), with a median cell viability of 99.1%, and has a high potential for functional efficacy. Thus, the protocol for producing T_reg cell products by combining CD25⁺ immunomagnetic selection with flow cytometry-based cell sorting methods can be used for the clinical treatment of GVHD.

The use of reduced toxicity conditioning regimens in patients with primary immunodeficiencies (PID) leads to the reduction of toxic effects of hematopoietic stem cell transplantation (HSCT). Currently, HSCT should result not only in disease control, but also in the improvement of the quality of life. We report the experience of HSCT in PID with TCRab+/CD19+ graft depletion after conditioning regimen containing treosulfan in combination with thiotepa as a second alkylating agent. 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. A group of 79 PID patients aged 0.5–17.6 years (the median age was 3.1 years) received HSCT from HLA matched related (n = 5), unrelated (n = 34) and haploidentical (n = 40) donors. The incidence of acute graft-versus-host disease (GVHD) ≥ grade II was 21%, grade III GVHD was observed in 3.8%, and none of the patients had GVHD grade IV. There were no cases of severe toxicity, including venoocclusive disease and thrombotic microangiopathy. The incidence of primary and secondary graft failure was 15.4%. The overall survival was 82.3%. There was no statistical difference between overall survival rates of patients who underwent transplantation from different types of donors (p = 0.164). All deaths were transplant-related and were due to infections. The use of thiotepa in reduced toxicity conditioning regimen is effective and safe, and can be considered as an option for HSCT in PID.

Today, results of allogeneic hematopoietic stem cell transplantation (allo-HSCT) are very encouraging but nevertheless, 25–75% of patients still develop complications, and mortality rates reach as high as 10–19%. Two major complications of allo-HSCT are graft-versus-host disease (GVHD) and viral infections. The diagnosis of intestinal GVHD remains a challenge. Incorrect interpretation of gastrointestinal lesions may lead to grave consequences. The development of endoscopic criteria for GVHD is a major focal point in foreign literature. In our study, we included 33 patients aged 1–17 years with signs of enterocolitis and suspected isolated intestinal GVHD who had received allo-HSCT at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of Ministry of Healthcare of Russia between 2020 and 2023. 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 of Ministry of Healthcare of Russia. All the patients underwent white-light colonoscopy with i-scan and, if necessary, chromoendoscopy; as well as colonic mucosal biopsy for further histopathological evaluation and intestinal infection testing. Statistical analysis was performed using the IBM SPSS Statistics 23 software. Viruses were identified in the colonic mucosal samples of 84.8% of the patients. The most common pathogens were: HHV-6 (in 50% of the cases), norovirus (46.4%) and adenovirus (35.7%). The high incidence of colonic viral infections in the children with GVHD is likely the result of virus reactivation during intensive immunosuppressive therapy following allo-HSCT. In 78.8% of the study patients, the colonic mucosa had orange-peel appearance, which led us to consider such mucosal changes to be the main criterion for the endoscopic diagnosis of GVHD in children after allo-HSCT, regardless of the presence or absence of viral infections. Our study demonstrated a fairly high effectiveness of the chromoendoscopy advanced imaging technology. Further research on ways to improve methods for GVHD differential diagnosis is required.

The clinical course of the novel coronavirus disease (COVID-19) in patients with oncological and hematological diseases after hematopoietic stem cell transplantation (HSCT), are of special interest. To further investigate the problem, a two-center study was undertaken at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology and the R.M. Gorbacheva National Research Institute for Pediatric Oncology, Hematology and Transplantation between January 2020 and January 2023. This was a retrospective-prospective, non-randomized, non-interventional study that included children aged 0–19 years with oncological and hematological diseases and primary immunodeficiencies who had undergone allogeneic HSCT and subsequently contracted COVID-19. 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. COVID-19 cases were confirmed by polymerase chain reaction testing and classified as asymptomatic, mild, moderate, severe, and critical. The study included 105 patients with a median age of 9 years; male patients were predominant (the male-to-female ratio was 1.8:1). The primary diseases were hematological malignancies (73%), benign hematological diseases (14%) and primary immunodeficiencies (13%). The most common clinical symptoms of COVID-19 were fever, gastrointestinal symptoms, and respiratory symptoms; 40% of COVID-19 cases were asymptomatic. Lymphopenia was found to be a risk factor for severe COVID-19. The patients without immune reconstitution had a longer persistence of the COVID-19 virus than those with immune reconstitution (17 days versus 13 days), however, no significant differences were obtained (p = 0.7). There were also no significant differences in the severity and outcomes of COVID-19 between the patients with immune reconstitution and those without reconstitution. There was no effect of therapy on the duration of COVID-19, and there was no association between the type of treatment and the duration of the disease. The overall survival rate in the allo-HSCT recipients who had been diagnosed with COVID-19 was 88%, which was lower than in the non-recipients (88% vs 94%; p = 0,077).

Today, the use of anti-CD19 biomedical cell products (BMCPs) for the treatment of B-cell malignancies yields impressive results and is becoming ever more popular. Several bioreactors have been developed that allow the manufacturing of high-quality cell products for clinical use. Choosing an appropriate bioreactor is an important step in this process. The aim of this study was to characterize and compare immunophenotypic and functional properties of anti-CD19 BMCPs manufactured using the automated CliniMACS Prodigy system (Miltenyi Biotec, Germany) and the manual G-Rex 10M-CS platform (Wilson Wolf, USA). The manufacturing of BMCPs and subsequent CAR T-cell therapy were carried out at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of Ministry of Healthcare of Russia. We used T cells from patients who had undergone HSCT as well as from autologous and allogeneic donors. In this study, we employed 26 anti-CD19 BMCPs manufactured using the automated CliniMACS Prodigy system in accordance with the GMP requirements as well as 25 cell products produced with the G-Rex platform. 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. Quality control was ensured throughout the entire manufacturing cycle and included assessment of cell composition and survival, transduction efficiency, cell expansion, expression of exhaustion markers, and CD19-specific antitumor activity. Our research showed that both manufacturing platforms generate stable high-quality products with sufficient cell expansion, viability and T cell transduction for subsequent CAR-T therapy. However, the median transduction efficiency of the BMCPs produced using the CliniMACS Prodigy platform was statistically significantly higher than that of the BMCPs manufactured using the G-Rex bioreactor (41% vs 26%). The study showed that in the anti-CD19 BMCPs, there was a predominance of Tcm subpopulation over Tem subpopulation, a low expression of exhaustion markers and a pronounced CD19-specific activity. Nevertheless, the percentage of Tcm cells in the BMCPs manufactured using the CliniMACS Prodigy platform was statistically significantly higher than in the BMCPs produced using the G-Rex bioreactor (86.7% CD8⁺ Tcm cells and 82.3% CD4⁺ Tcm cells for CliniMACS Prodigy vs 69.0% CD8⁺ Tcm cells and 72.0% CD4⁺ Tcm cells for G-Rex). Despite the lower number of anti-CD19 CAR-T cells in the final cell products obtained with the G-Rex bioreactor, in all processes this amount was sufficient for subsequent CAR-T therapy. Thus, the CliniMACS Prodigy and G-Rex platforms can be used to produce high-quality anti-CD19 BMCPs.

The differential diagnosis of hereditary spherocytosis is a great challenge because of the similar clinical and laboratory signs it shares with other hereditary hemolytic anemias as well as due to the limited availability of molecular genetic testing. The development of easy-to-perform laboratory tests for the differential diagnosis of hereditary hemolytic anemias remains as relevant as ever. Here, a method of measuring red blood cell filterability for the diagnosis of hereditary spherocytosis is proposed for the first time. The aim of our study was to compare red blood cell filterability measurement with other diagnostic tests for hereditary spherocytosis as well as to assess its specificity and sensitivity. We included 30 patients (18 girls and 12 boys, with the median age of 8.6 years) with hereditary spherocytosis and 15 patients (9 girls and 6 boys, with the median age of 10 years) with other hereditary hemolytic anemias (pyruvate kinase deficiency (n = 14) and stomatocytosis (n = 1)). The diagnostic work-up for hereditary hemolytic anemia included a complete blood count test using an automated hematology analyzer, an osmotic resistance analysis before and after 24 hours of incubation at 37°С, erythrocytometry with sphericity index calculation, EMA test (eosin-5-maleimide binding assay) and red blood cell filterability measurement using artificial filters with cylindrical pores 3-5 μm in diameter. 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. The parents of all of the enrolled children signed a voluntary informed consent form for peripheral blood collection and diagnostic testing. In all the cases of hereditary spherocytosis diagnosed in accordance with the relevant clinical recommendations, red blood cell filterability was very low (0–0.31 units). It was higher only in 3 instances, reaching 0.47, 0.64 and 0.82 units, but in two of these cases there were no genetic data available, and the remaining patient was found to harbor the SPTA1 c.433999C>T mutation which was characterized both as spherocytosis and elliptocytosis. Red blood cell filterability in the group of the patients with other hemolytic anemias equalled 0.55 to 0.86 units (with the median of 0.77 units). The sensitivity of the RBC filterability measurement method in diagnosing hereditary spherocytosis was 93% (with 100% specificity), while the EMA test had a sensitivity of 89% and specificity of 95%. Our comparative study showed that red blood cell filterability measurement and the EMA test have similar sensitivity and specificity in diagnosing hereditary spherocytosis but the former is much cheaper and easier to perform since it does not require expensive equipment and can be carried out at any laboratory.

Treatment of non-Hodgkin lymphomas (NHL) in children using risk-based chemotherapy protocols is currently effective in 80–95% of cases, even in advanced stages of the disease. However, relapsed/refractory forms of NHL (which are less common) have an extremely unfavorable course with low survival rates. The addition of autologous and allogeneic hematopoietic stem cell transplantation (HSCT) to a comprehensive treatment program for relapsed/refractory forms of NHL can improve treatment results due to the antitumor effect of chemotherapy drugs of the conditioning regimen and the graft-versus-tumor effect, which is, however, less significant than in leukemia. Moreover, post-transplant complications after allogeneic HSCT in some cases can offset its positive results in NHL; therefore, to reduce toxicity, especially in severe somatic status of the patient, preference is often given to reduced-intensity conditioning regimens. This article presents two clinical cases. In one case, autologous HSCT was carried out for the first relapse of Burkitt's lymphoma. However, the patient developed a second relapse and underwent allogeneic HSCT from a haploidentical donor. In the second case, HSCT from an unrelated HLA identical donor was carried out in a patient with relapsed anaplastic large cell lymphoma. Both patients received reduced-intensity conditioning regimens. This approach helped to avoid the development of severe post-transplant complications, ensuring successful engraftment and achievement of donor hematopoietic chimerism. Early after transplantation, the patient with relapsed Burkitt's lymphoma developed a second tumor – acute T-lymphoblastic leukemia, from which the patient died. Despite treatment with targeted drug crizotinib, the second patient showed lymphoma progression, which resulted in death. The patients' parents gave consent to the use of their children's data, including photographs, for research purposes and in publications.

Chronic graft-versus-host disease (GVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation (HSCT) caused by immune dysregulation leading to multisystem involvement resulting in tissue sclerosis. This is a long-term complication that can significantly affect the quality of life of HSCT recipients due to secondary immunodeficiency associated with combined immunosuppressive therapy, impaired organ function and even disability. In addition to active prophylaxis for chronic GVHD, regular follow-up of patients is necessary for early detection of signs and symptoms of GVHD to enable timely and effective treatment. Here, we present a brief overview of novel approaches to diagnosis, classification, and staging of chronic GVHD, as well as current prophylaxis and treatment options.

Classic hyper-IgE syndrome (due to a mutation in the STAT3 gene) is a primary immunodeficiency associated with multisystem disorder affecting organs and tissues. To date, there are only few published cases of hematopoietic stem cell transplantation for this disease, which does not allow us to fully assess the main effects of transplantation in this group of patients. This article presents a clinical case of hematopoietic stem cell transplantation from an unrelated donor with TCRab/CD19 depletion in a patient with hyper-IgE syndrome, which was carried out at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. The patient's parents gave consent to the use of their child's data, including photographs, for research purposes and in publications. At 5 years after treatment, the patient has a good graft function and no signs of immunodeficiency, and infectious complications remain controlled. However, despite accompanying bisphosphonate therapy and the absence of osteopenia signs according to densitometry, the patient still has musculoskeletal disorders, associated with spontaneous long bone fractures and severe joint deformities.

The improvement of treatment outcomes for children with high-risk neuroblastoma is one of the most significant challenges in current pediatric oncology/hematology practice. Treatment outcomes in these patients need to be improved, both in terms of achieving remission and in terms of toxicity profile. At the present time, a number of approaches have been adopted to achieve better outcomes, including the intensification of treatment by using tandem high-dose multiagent chemotherapy followed by autologous hematopoietic stem cell transplantation as a part of treatment. Tandem high-dose chemotherapy with autologous hematopoietic stem cell transplantation has been shown to be effective and safe in several studies. The purpose of this literature review is to present research data showing the feasibility and safety of this treatment strategy in practice.

A thrombus is a heterogeneous structure consisting of platelets in different functional states. Flow cytometry is one of the most promising tools for the diagnosis of platelet state. However, its optimization and standardization are the subjects of heated debate. How to properly activate and label platelets in order to assess their functional status? In this work, we would like to briefly highlight this issue and propose the hypothesis that several levels/types of platelet activation correspond to various positions in the thrombus and various physiological meanings. One should use this entire necessary and sufficient set of activation levels in order to draw a conclusion about how the patient’s platelets “feel”.

This article presents a brief overview of publications on pediatric aplastic anemia (AA) and closely related conditions. Here we consider the pathophysiology of AA, which includes three main mechanisms of bone marrow destruction resulting in aplasia: direct injury, immune mediated destruction and bone marrow failure resulting from inherited and clonal disorders. New aspects of inherited bone marrow failure syndromes, inborn errors of immunity and myelodysplastic syndromes are highlighted as the most common conditions included in the spectrum of differential diagnosis of AA in children. A comprehensive algorithm for the diagnosis of AA in children is presented, including standard laboratory tests and additional modern molecular and genetic techniques that contribute to a better understanding of this heterogeneous group of diseases and determine approaches to the choice of therapy. The purpose of the review is to provide pediatricians and pediatric hematologists with an updated information of this rare, heterogeneous condition based on an analysis of the latest literature data.