Blood Hematopoietic Stem Cell Transplantation Research Articles

Analysis of Factors Affecting Hematopoietic Stem Cell Mobilization Efficiency and Early Hematopoietic Reconstruction Indicators during Autologous Peripheral Blood Hematopoietic Stem Cell Transplantation.

Purpose To analyze the factors affecting the mobilization efficiency of hematopoietic stem cells and hematopoietic reconstruction indicators during autologous peripheral hematopoietic stem cell transplantation. Methods The clinical data of 54 patients who underwent autologous peripheral blood hematopoietic stem cell mobilization and transplantation at Xuzhou Central Hospital from May 2016 to April 2023 were retrospectively analyzed. The gender, age, disease type, mobilization regimen, number of chemotherapy sessions, G-CSF (granulocyte colony-stimulating factor) dosage, and platelet number at the time of collection were also collected. Moreover, the relationship between these indicators with mobilization results and hematopoietic reconstruction was analyzed. Results Results showed that age, disease type, and number of collections were significantly related to the mobilization results (number of CD34+ hematopoietic stem cells). Furthermore, multivariate analysis showed that the number of collections was an independent factor affecting mobilization efficiency. Similarly, age, platelet value at the time of collection, CD34+ stem cell value during collection, white blood cell count, and number of chemotherapy times were significantly related to the time of megakaryocytic hematopoietic reconstruction. Multifactor analysis found that age and platelet count were independent factors affecting the reconstruction time of the megakaryocytic system. However, no factor was related to the time of granulocyte hematopoietic reconstruction. Conclusion Platelet count and age when collecting hematopoietic stem cells are closely related to megakaryocytic hematopoietic reconstruction and are key indicators of early hematopoietic reconstruction after autologous hematopoietic stem cell transplantation.

Advance of research on the role of BCL11A in the occurrence and treatment of β-Thalassemia

β-Thalassemia is a single-gene disease caused by mutations in β-globin and has a distinct geographical characteristics. Current treatment for patients with moderate to severe thalassemia has mainly relied on long-term blood transfusion and/or hematopoietic stem cell transplantation. B cell lymphoma/leukemia 11A (BCL11A) as a transcriptional repressor plays a vital role in monitoring γ/β hemoglobin switching, maintaining the normal function of hematopoietic stem cells, and regulating erythrocyte differentiation and lymphocyte development. With the rapid progress in gene editing technology, the BCL11A as a therapeutic target for β-thalassemia has shown promising results. This article has systematically summarized the regulatory mechanism and therapeutic potential of the BCL11A, with an aim to provide new ideas for the treatment of β-thalassemia.

Comparable outcomes of allogeneic peripheral blood versus bone marrow hematopoietic stem cell transplantation from a sibling donor for pediatric patients.

Traditionally, bone marrow (BM) has been preferred as a source of stem cells (SCs) in pediatric hematopoietic SC transplantation (HSCT); however, the use of peripheral blood SCs (PBSC) has recently increased. With advancing graft-versus-host disease (GVHD) prophylaxis, whether the BM is still a better SC source than PB in sibling donor HSCT remains controversial. Here, we compared the results of BM transplantation (BMT) and PBSC transplantation (PBSCT) in pediatric patients with malignant or non-malignant diseases receiving sibling HSCT using a total of 7.5mg/kg of anti-thymocyte globulin (ATG). We retrospectively reviewed children who received HSCT from a sibling donor between 2005 and 2020 at Seoul National University Children's Hospital. Of the 86 patients, 40 underwent BMT, and 46 underwent PBSCT. Fifty- six patients had malignant diseases, whereas thirty patients had non-malignant diseases. All conditioning regimens comprised ATG. Busulfan-based myeloablative conditioning regimens were administered to patients with malignant diseases and approximately half of those with non-malignant diseases. The remaining half of the patients with non-malignant diseases were administered cyclophosphamide-based reduced- intensity conditioning regimens. According to studies conducted at our center, all BM donors received G-CSF before harvest to achieve early engraftment. In all 86 patients (47 males and 39 females), the median age at the time of HSCT was 11.4 (range, 0.7 - 24.6) years. The median follow-up period was 57.9 (range, 0.9-228.6) months, and the corresponding values for those with BM and PBSC were 77 (range, 2.4-228.6) months and 48.7 (range, 0.9-213.2) months, respectively. Engraftment failure occurred in one patient with BM and no patient with PBSC. The cumulative incidence of acute GVHD with grades II-IV was higher in PBSC (BM 2.5%, PBSC 26.1%, p = 0.002), but there was no significant difference in those with grades III-IV acute GVHD (BM 0%, PBSC 6.5%, p = 0.3703) and extensive chronic GVHD (BM 2.5%, PBSC 11.6%, p = 0.1004). There were no significant differences in treatment-related mortality (TRM) (BM 14.2%, PBSC 6.8%, p = 0.453), 5-year event-free survival (EFS) (BM 71.5%, PBSC 76.2%, p = 0.874), and overall survival (OS) rates (BM 80.8%, PBSC 80.3%, p = 0.867) between BM and PBSC in the univariate analysis. In the multivariate analysis, which included all factors with p < 0.50 in the univariate analysis, there was no significant prognostic factor for EFS or OS. There was no significant difference in the relapse incidence between BM and PBSC among patients with malignant diseases (BM 14.2%, PBSC 6.8%, p = 0.453). Additionally, there were no significant differences in the TRM, 5-year EFS, and OS rates between malignant and non-malignant diseases nor between the busulfan-based myeloablative regimen and reduced-intensity chemotherapy using cyclophosphamide. In this study, we showed no significant differences in EFS, OS, TRM, and GVHD, except for acute GVHD grades II-IV, between BMT and PBSCT from sibling donors, using ATG (a total of 7.5mg/kg). Therefore, PB collection, which is less invasive for donors and less labor-intensive for doctors, could also be considered an acceptable SC source for sibling donor HSCT in children.

Galectin-9 alleviates acute graft-versus-host disease after haplo-hematopoietic stem cell transplantation by regulating regulatory T cell/effector T cell imbalance.

Acute graft-versus-host disease (aGVHD) arises from the imbalance of host T cells. Galectin-9 negatively regulates CD4 effector T cell (Th1 and Th17) function by binding to Tim-3. However, the relationship between Galectin-9/Tim-3 and CD4+ T subsets in patients with aGVHD after Haplo-HSCT (haploidentical peripheral blood hematopoietic stem cell transplantation) has not been fully elucidated. Here, we investigated the role of Galectin-9 and CD4+ T subsets in aGVHD after haplo-HSCT. Forty-two patients underwent Haplo-HSCT (26 without aGVHD and 16 with aGVHD), and 20 healthy controls were included. The concentrations of Galectin-9, interferon-gamma (IFN-γ), interleukin (IL)-4, transforming growth factor (TGF)-β, and IL-17 in the serum and culture supernatant were measured using enzyme-linked immunosorbent assay or cytometric bead array. The expression levels of Galectin-9, PI3K, p-PI3K, and p-mTOR protein were detected by western blot analysis. Flow cytometry was used to analyze the proportions of CD4+ T cell subsets. Bioinformatics analysis was performed. In patients with aGVHD, regulatory T (Treg) cells and Galectin-9 decreased, and the Th1, Th17, and Treg cells were significantly imbalanced. Moreover, Treg and Galectin-9 were rapidly reconstituted in the early stage of patients without aGVHD after Haplo-HSCT, but Th17 cells were reconstituted slowly. Furthermore, Tim-3 upregulation on Th17 and Th1 cells was associated with excessive activation of the PI3K/AKT pathway in patients with aGVHD. Specifically, in vitro treatment with Galectin-9 reduced IFN-γ and IL-17 production while augmenting TGF-β secretion. Bioinformatics analysis suggested the potential involvement of the PI3K/AKT/mTOR pathway in aGVHD. Mechanistically, exogenous Galectin-9 was found to mitigate aGVHD by restoring the Treg/Teffs (effector T cells) balance and suppressing PI3K. Galectin-9 may ameliorate aGVHD after haplo-HSCT by modulating Treg/Teffs balance and regulating the PI3K/AKT/mTOR pathway. Targeting Galectin-9 may hold potential value for the treatment of aGVHD.

Hematopoietic progenitor cell count as a potential quantitative marker in apheresis products during allogeneic stem cell transplantation.

The quality and quantity of hematopoietic stem cells in apheresis products are essential to the success of peripheral blood hematopoietic stem cell transplantation (PB-HSCT). While the flow cytometry measurement of CD34+ cells as a golden standard for stem cell count is labor and cost-intensive, hematopoietic progenitor cell number evaluated by XN Sysmex series automated hematology analyzers (XN-HPC) is suggested as a surrogate marker. We evaluated the correlation and consistency of XN-HPC and CD34+ cell count in apheresis samples from both allogeneic donors and autologous patients during PB-HSCT. Good correlation and consistency were observed between XN-HPC and CD34+ cell counts in harvests collected from healthy donors (R = .852) rather than autologous patients (R = .375). Subgroup analysis showed that the correlation was especially poor when autologous patients used plerixafor as an additional mobilizer or were diagnosed with multiple myeloma (MM). In the setting of allogeneic transplantation, the correlation coefficients were even better in samples from non-first-round apheresis (R = .951), with high white blood cell (WBC) counts (R = .941), or having successful engraftment within 2 weeks (R = .895). ROC analysis suggested that an optimal XN-HPC count of 1127 × 106 /L best predicted a sufficient yield of CD34+ stem cells, with diagnostic sensitivity and specificity being 92% and 72%, respectively (AUC = 0.852). XN-HPC is a sufficient quantitative marker for stem cell assessment of harvest yield in allogeneic but not autologous HSCT.

Efficacy of Triple Prophylaxis for Prevention of Graft-Versus-Host Disease in Matched Sibling Allogeneic Peripheral Blood Hematopoietic Stem Cell Transplantation in Pediatric Patients

BACKGROUND: Graft-versus-host disease (GVHD) continues to compromise the overall success of allogenic hematopoietic stem cell transplantation. It is the most important cause of morbidity and non-relapse mortality after allogeneic hematopoietic stem cell transplantation for malignant disease. In adults, post-transplant cyclophosphamide (Cy-PT) has been shown to be a feasible, economically accessible, and effective strategy to reduce the incidence of GVHD in matched sibling hematopoietic stem cell transplantation in combination with a calcineurin inhibitor plus mycophenolate mofetil (MMF). AIM: Demonstrate the clinical benefit of Cy-PT plus a calcineurin inhibitor combined with MMF for GVHD prophylaxis in HLA-matched, related peripheral blood stem cell transplants in pediatric patients with malignant hematologic neoplasms compared to standard therapy in historical controls. METHODS: A retrospective study was conducted on 22 pediatric patients with malignant hematologic neoplasms who underwent HLA-matched related peripheral blood allogenic stem cell transplantation (alloSCT) between July 2012 and December 2022. A comparison was made between two groups, one with triple prophylaxis using Cy-PT, cyclosporine (CsA), and MMF, and a historical cohort that received standard GVHD prophylaxis based on CsA and methotrexate (MTX). Patients were identified from a hospital registry. Descriptive and inferential statistics will be reported, using SPSS version 25 for analysis. RESULTS: Twenty-two patients received HLA-matched alloSCT from first-degree relatives genotypically identical in HLA-A, HLA-B, and HLA-DRB1 alleles. The demographic characteristics of both groups are summarized in the table. No patient experienced primary graft failure, sinusoidal obstructive syndrome, or hemorrhagic cystitis. Acute GVHD grade II-IV did not develop in patients who received Cy-PT, whereas in the CsA/MTX prophylaxis group, 1 (9%) grade IV patient was observed who died due to this cause before day 100. Two patients (18%) developed Moderate/Severe GVHD in the CsA/MTX group, and 1 (9%) had moderate GVHD in the Cy-PT group. Two-year overall survival was similar between the two groups (Cy-PT 53% and CsA/MTX 67%), as was event-free survival (PTCy 48% vs. CsA/MTX 61%). CONCLUSION: Peripheral blood represents a feasible option in our setting due to rapid graft engraftment, short hospital stays, and low incidence of primary graft failure, but with a higher incidence of GVHD as reported in the literature. We explored the efficacy and safety of triple prophylaxis for HLA-matched alloSCTs from peripheral blood using Cy-PT + CsA + MMF in a small group of patients, with outcomes slightly better than standard prophylaxis. These preliminary results motivate us to continue with this new regimen.

Cryostorage of peripheral blood hematopoietic stem cells in transplantology: current status and prospects

Peripheral blood hematopoietic stem cell (HSC) transplantation is a well-established procedure for the treatment of hematological, cancer and autoimmune diseases. In cancer patients, HSC transplantation allows the use of high-dose cytotoxic drugs in combination with radiation therapy during treatment, which provides a pronounced antitumor effect. The hematological toxicity of such treatment is eliminated by the sequential introduction of stem cells, which contribute to hematopoiesis restoration. Before transplantation, peripheral blood HSCs are subjected to collection and cryopreservation for further storage. An important requirement for cryopreservation is viable HSCs responsible for hematopoietic restoration. The aim of the review was to analyze the literature devoted to the influence of various methods of cryopreservation of human peripheral blood HSCs on the preservation of cell viability after thawing, as well as the development of adverse events in patients. Issues related to the use of various cryoprotectants, as well as methods for storing HSC grafts, are considered. The presented data indicate the need for further study of the effect of cryoprotectants on the human body and the cellular composition of the graft and improvement of protocols for HSC cryopreservation.

Day 100 Natural Killer Cell/CD14+HLA-DRDIM ratio and survival in lymphoma post-autologous peripheral blood hematopoietic stem cell transplantation.

The infusion of autograft Natural Killer Cells (NKC)/CD14+ HLA-DRDIM ratio is a predictor of survival in lymphoma patients undergoing autologous peripheral blood hematopoietic stem cell transplantation (APBHSCT). This study evaluated if the Day 100 NKC/CD14+ HLA-DRDIM ratio still functions as a prognostic immune-biomarker. This was a retrospective, single-institution, cohort analysis including 107 patients in this study that had clinical assessment at Day 100 post-APBHSCT from our prior phase III trial. We evaluated the prognostic ability of the Day 100 NKC/CD14+ HLA-DRDIM ratio to predict overall survival (OS) and progression-free survival (PFS) using Cox regression model for outcome analysis and survival by Kaplan-Meier method. The median follow-up from day 100 was 94.7 months (range 4.83-158.1 months) for the entire cohort. Patients with a Day 100 NKC/CD14+ HLA-DRDIM ratio ≥1.67 experienced better OS and PFS versus those with a Day 100 NKC/CD14+ HLA-DRDIM ratio <1.67: median OS was not reached versus 49.7 months, the 5-year OS rates were 91% (95% CI, 81%-96%) versus 40% (95% CI, 27%-55%), p<.0001, respectively; and median PFS was not reached versus 23.5 months, the 5-year PFS rates were 66% (95% CI, 55%-81%) versus 21% (95% CI, 15%-40%), p<.0001, respectively. Day 100 NKC/CD14+ HLA-DRDIM ratio was an independent predictor for OS and PFS in the multivariate analysis. Day 100 NKC/CD14+ HLA-DRDIM ratio is a prognostic immune-biomarker in lymphoma patients post- APBHSCT.

ABO Incompatibility Haploidentical Peripheral Blood Stem Cell Transplantation Combined with a Single Cord Blood Unit for Severe Aplastic Anemia Patients

Background: For young severe aplastic anemia (SAA) patients without HLA matched donors, haploidentical hematopoietic stem cell transplantation can be used as an alternative selection. The grafts of classic Beijing protocol in China is a combination of bone marrow and peripheral blood stem cell. For patients with major or minor blood type incompatibility, bone marrow stem cells need to undergo red blood cell or plasma removal treatment. There was rare reports about haploidentical peripheral blood stem cell combined with a single unrelated cord blood unit for ABO incompatibility haploidentical stem cell transplantation in SAA patients. Objective: To observe the efficacy of ABO incompatibility haploidentcial peripheral blood hematopoietic stem cell transplantation combined with a single unrelated cord blood unit for SAA patients. Method s : Fifty-five SAA patients underwent stem cell transplantation from August 2018 to February 2022 in the First Affiliated Hospital of Xi'an Jiaotong Univeristy were divided into two groups based on donor-recipient ABO compatibility, the donor-recipient ABO matched group（BM+PB group）using haploidentical bone marrow and peripheral blood stem cell as grafts, and donor-recipient ABO mismatched group（CB+PB group）, using unrelated cord blood and haploidentical peripheral blood stem cell as grafts. The differences of hematopoietic reconstitution, acute and chronic GVHD, CMV and EB virus infection, PFS, and OS between the two groups were compared. Result s : There were 30 cases in CB+PB group and 27 cases in the BM+PB group. Except for differences in donor-recipient ABO compatibility, the other characteristics of the two groups of patients were comparable((P&amp;gt;0.05). One patient in the BM+PB group had primary graft failure, while the rest of the patients were successfully implanted. There were no significant statistical differences in neutrophil,platelet and red blood cells recovery rates, the incidence of acute and chronic GVHD, CMV, EB virus infection, and PTLD between the two groups of patients(P&amp;gt;0.05). There was no significant statistical difference in OS between the two groups of patients(P&amp;gt;0.05)(Figure 1). Conclusion: For SAA patients with donor-recipient ABO incompatibility could use haploidentical peripheral blood cell combined with unrelated cord blood for transplantation which had compared outcome with haploidentcal bone marrow and peripheral blood cell transplantation.

Day 100 Inhibitory KIR2DL2 and Activating NKp30 Natural Killer Cell Receptors Predicts Survival Post-Autologous Stem Cell Transplantation in Lymphomas

Introduction: Our group previously published that the infusion autograft inhibitor KIR2DL2 (CD158b+CD337-) and activating NKp30 (CD158b- CD337+) Natural killer (NK) cells receptors were predictors of clinical outcomes in lymphoma patients undergoing autologous peripheral blood hematopoietic stem cell transplantation (APBHSCT) (Porrata et al. Leukemia Research 2019; 81: 1-9). To assess if these subsets of NK cells still hold clinical relevance, we set up to investigate their prognostic ability to predict clinical outcomes at Day 100 post-APBHSCT. Methods: 107 lymphoma patients who participated in our double-blind phase III trail (Porrata et al, Biology of Blood and Marrow Transplantation 2016; 22(6); 1017-1023) and had clinical assessment at Day 100 post-APBHSCT were included in this study. Nkp30 and KIR2DL2 at Day 100 were assessed by flow cytometry analysis. Results: With a median follow-up for the entire cohort from Day 100 was 94.7 months (range: 4.83-158.1 months). Figure 1 shows the overall survival (OS) and progression-free survival (PFS) based on the absolute numbers of KIR2DL2 and Nkp30 NK cells. At Day 100, patients with a KIR2DL2 &amp;lt; 0.08 and Nkp30 ≥0.19 experienced superior OS and PFS compared with patients with a Day 100 KIR2DLD2 ≥0.08 and NKp30 &amp;lt; 0.19. In the multivariate analysis both the KIR2DL2 [OS: HR = 1.767, 95%CI, 1.302-30.31, p &amp;lt; 0.04; and PFS: HR = 2.663, 95%CI, 1.108-6.403, p &amp;lt; 0.03] and Nkp30 [OS: HR = 2.594, 95% CI, 1.239-5.432, p &amp;lt; 0.01; and PFS: HR = 3.676, 95% CI, 1.875-7.206, p &amp;lt; 0.01] were independent predictors for OS and PFS. Conclusion: Day 100 inhibitory KIR2DL2 and activating NKp30 NK cells are prognostic immune-biomarkers in lymphoma patients undergoing APBHSCT.

Phase I/II Study of Reduced Dosing of Post-Transplantation Cyclophosphamide (PTCy) after HLA-Identical Peripheral Blood Hematopoietic Stem Cell Transplantation

Introduction Post-transplant cyclophosphamide (PTCy)-based prophylaxis for acute graft versus host disease (aGvHD) has become a standard protocol in HLA haploidentical bone marrow transplantation. Preliminary evidence indicates that compared to the standard dosage (50 mg/kg on days +3 and +4 post-transplantation), a regimen of half the typical cyclophosphamide dosage is both feasible and effective. However, the optimal dosage and timing of PTCy for aGvHD prophylaxis in a HLA identical setting, using peripheral stem cells as the graft, remain unknown. This study aims to investigate the efficacy and safety of a reduced PTCy dosage regimen in patients undergoing peripheral blood stem cell transplants (PBSCT) with HLA indentical donors. Methods This prospective, single-center phase I/II study enrolled patients scheduled for PBSCT with HLA identical donors. The aGVHD prophylaxis regimen comprised PTCy on days +3 and +4, followed by cyclosporine initiation from day 5 post-transplant. In the phase I stage, an initial cohort of five patients received PTCy at a dosage of 50 mg/kg/day on days +3 and +4 (designated as dose level 1, DL1). This was followed by a 3+3 dose de-escalation design with subsequent dose levels as follows: DL2, 50 mg/kg on day +3 and 25 mg/kg on day +4; and DL3, 25 mg/kg on both day +3 and +4. The dose-limiting toxicity for this de-escalation was defined as the occurrence of grade III-IV aGVHD within the first 100 days post-transplantation. The phase II stage comprised an expansion cohort, which was based on the optimal dose identified during phase I. aGvHD classification was established using the Mount Sinai Acute GVHD International Consortium (MAGIC) criteria, while chronic GvHD (cGvHD) was assessed per the latest National Institutes of Health Consensus on cGvHD. Patients were closely followed-up at +1, +2, +3, +4, +6, +9, +12, +18, and +24 months post-transplant. Results In the first stage, a total of 11 patients (5 in DL1, 3 in DL2, and 3 in DL3) were included, and no patient developed grade III-IV aGVHD. Based on this, DL3 was selected as the PTCy dose for phase II, which comprised 18 patients. Overall, 29 patients (17 females, 12 males) with a median age of 39 years (range 17-65) were included, and the median follow-up duration was 12.7 months (range 2.2-22.1). Patient and disease characteristics are summarized in Table 1. By day +30 post-ASCT, all patients achieved complete remission (CR) and minimal residual disease (MRD) clearance. No patient developed grade III-IV aGVHD within 100 days post-transplantation. There was no significant difference in the neutrophil and platelet engraftment interval among the three groups (Figure 1A, B). Among the 21 patients who received the DL3 dose of PTCy, the estimated cumulative incidence of grade II to IV aGVHD at day +100 was recorded at 28.6%, and the incidence of aGVHD at day +100 presented no difference among the three DL groups (Figure 1C). In addition, 1 patient in DL3 developed grade IV intestinal aGVHD on day +141 and died one month later. The estimated 1-year cumulative incidence of hemorrhagic cystitis (HC) and cytomegalovirus (CMV) reactivation was 19.3% and 19%, respectively (Figure 1D). Five patients (all in DL3) experienced relapse, with a median interval of 6.7 months and an estimated 1-year cumulative incidence of 10.6%. A total of 5 fatalities occurred during our study (1 in DL1, and 4 in DL3), 1 due to relapse, 2 from COVID-19-associated pulmonary aspergillosis, 1 from severe septicemia and 1 due to grade IV aGVHD as mentioned above. The estimated 6-month cumulative incidence of NRM was 9.5%, presenting no difference among the three DL groups (Figure 1E). Among the 20 evaluable patients, the estimated 1-year cumulative incidence of chronic GVHD (cGVHD) and moderate cGVHD was 37.3% and 16% (Figure 1F), respectively, with no severe cGVHD observed. No cases of patient relapse were reported. The estimated 1-year progression-free survival (PFS) and overall survival (OS) were 61.2% and 80.4%, respectively (Figure 1G). Conclusion The findings of this study substantiate that a two-day regimen of PTCy at 25 mg/kg/day serves as an effective and safe prophylactic measure against aGVHD, comparable to the standard dosing protocol. However, to definitively establish whether the de-escalated PTCy dosage provides superior outcomes to the standard regimen, longer-term follow-up and comparative studies are warranted.

#3585 FAT1 ANTIGEN IN PATIENTS WITH MEMBRANOUS NEPHROPATHY AFTER HEMATOPOIETIC STEM CELL ALLOTRANSPLANTATION: A CASE SERIES

Abstract Background and Aims Nephrotic syndrome (NS) is a rare complication of haematopoietic stem cell transplantation (HSCT). When it occurs, it usually develops after cessation of immunosuppressive therapy, which is why it is commonly denominated as a renal form of chronic graft-versus-host disease. Membranous nephropathy (MN) is the most common pathological finding. The protocadherin FAT1 antigen is a novel antigen identified only in patients with MN who have undergone allogeneic HSCT. PLA2R and NELL1 antigens can also be found, even though they are rare and not specific to HSCT-associated MN. The aim of this study was to present a case series of allogeneic HSCT-associated MN, the treatment, outcomes and presence of a novel antigen specifically detected in this population. Method All patients with full blown NS due to MN after HSCT in UHC Zagreb were enrolled. Kidney biopsy was performed and examined by light, immunofluorescence and electron microscopy. Biopsy samples were sent to Renal Pathology Laboratory, Department of Laboratory Medicine and Pathology, Mayo Clinic for detection of FAT1 antigen in biopsy tissue samples Results In last 30 years there had been 1302 HSCT in UHC Zagreb and three of them developed MN. The first two patients were treated with allogeneic HSCT for acute myeloid leukaemia (AML). Both developed NS after cessation of GVHD prophylaxis, after 1 and 8 months respectively. The first patient's kidney function was preserved, while the second patient's was reduced. Kidney biopsy showed MN. Thorough examination found no secondary cause of the disease. In the first patient, complete remission (CR) was achieved after 5 months of steroids, 2 boluses of cyclophosphamide (CP) and cyclosporine treatment. The second patient reached partial remission (PR) after 6 months of steroid treatment and oral CP. Immunosuppression was terminated due to infectious complications. CR was achieved after an additional 8 months while the patient was not taking immunosuppressants. Both patients remained in CR during the follow-up period. The third patient with acute lymphoblastic leukaemia (ALL) underwent a related peripheral blood HSCT. Sixteen years later, he developed NS with preserved renal function and was diagnosed with MN. Apart from thyroid disease, no secondary cause was detected and he was treated with rituximab. Partial remission (PR) was achieved after 24 months of therapy. Anti-PLA2R antibodies were negative in all three cases. Tissue samples from patients 1 and 2 were positive for FAT1 antigen using laser microdissection and tandem mass spectrometry (MS/MS) of glomeruli. The third patient was negative for FAT1. Clinical and biopsy findings in our cohort of HSCT-associated MN are summarised in Tables 1 and 2. Conclusion MN can occur at different points in time, sometimes even years, after HSCT. It can be successfully treated with steroids and immunosuppressants, achieving long-term remission. Most patients with HSCT-associated MN are FAT1-positive, although it is not the only antigen associated with MN in HSCT patients. In the future, serum testing for anti-FAT1 antibodies in patients with HSCT could be of similar significance in diagnosing FAT1-associated MN as PLA2R antibodies are for PLA2R-associated MN.

Therapeutic efficacy of hematopoietic stem cell transplantation for Wiskott-Aldrich syndrome in 60 children

Objective: To evaluate the therapeutic efficacy of hematopoietic stem cell transplantation (HSCT) for Wiskott-Aldrich syndrome (WAS), and to analyze the factors related to the outcomes. Methods: The clinical data of 60 children with WAS received HSCT in Shanghai Children's Medical Center from January 2006 to December 2020 were retrospectively analyzed. All cases were treated with a myeloablative conditioning regimen with busulfan and cyclophosphamide, and a graft-versus-host disease (GVHD) prevention regimen based on cyclosporine and methotrexate. Implantation, GVHD, transplant-related complications, immune reconstitution and survival rate were observed. Survival analysis was performed by Kaplan-Meier method, and Log-Rank method was used for univariate comparison. Results: The 60 male patients had main clinical features as infection and bleeding. The age at diagnosis was 0.4 (0.3, 0.8) years, and the age at transplantation was 1.1 (0.6, 2.1) years. There were 20 cases of human leukocyte antigen matched transplantation and 40 mismatched transplantation; 35 patients received peripheral blood HSCT, and 25 cord blood HSCT. All cases were fully implanted. The incidence of acute GVHD (aGVHD) was 48% (29/60) and only 2 (7%) developed aGVHD of grade Ⅲ; the incidence of chronic GVHD (cGVHD) was 23% (13/56), and all cases were limited. The incidence of CMV and EBV infection was 35% (21/60) and 33% (20/60) respectively; and 7 patients developed CMV retinitis. The incidence of sinus obstruction syndrome was 8% (5/60), of whom 2 patients died. There were 7 cases (12%) of autoimmune hemocytopenia after transplantation. Natural killer cells were the earliest to recover after transplantation, and B cells and CD4+T cells returned to normal at about 180 days post HSCT. The 5-year overall survival rate (OS) of this group was 93% (95%CI 86%-99%), and the event free survial rate (EFS) was 87% (95%CI 78%-95%). EFS of non-CMV reactivation group is higher than that of CMV reactivation group (95% (37/39) vs.71% (15/21), χ2=5.22, P=0.022). Conclusions: The therapeutic efficacy of HSCT for WAS is satisfying, and the early application of HSCT in typical cases can achieve better outcome. CMV infection is the main factor affecting disease-free survival rate, which can be improved by strengthening the management of complications.

5612627 MORTALITY, CHRONIC MORBIDITY AND INTENSITY OF TREATMENTS IN A NATURAL HISTORY COHORT OF SICKLE CELL DISEASE

Background: While many advances have been made in the understanding of Sickle Cell Disease (SCD) in the past decades, the natural history of the disease is often elusive due to lack of long-term prospective data collection (1). The SCD Natural History Study is a longitudinal follow-up study. During the 15 years of the study, the Italian Association of Pediatric Hematology Oncology (AIEOP) guidelines were used to standardize treatment with Hydroxyurea (HU), red blood cell transfusion and Hematopoietic Stem Cell Transplantation (HSCT) as well as management of acute and chronic complications (2). Aims: To describe mortality, chronic morbidity and intensity of treatments in a pediatric natural history cohort. Methods: Since 2007 all pediatric patients with SCD followed in the Pediatric SCD Reference Center of Padua were enrolled. Data on physical examination, acute/chronic complications and laboratory parameters were prospectively recorded at every outpatient and inpatient visit since diagnosis. Results: 197 children and adolescents with SCD were enrolled and 182 patients had clinical, hematological and organ damage monitoring information as of 31/12/2020. The mean follow-up is 79.8 months (range 2.1-298.6) (IQR 36.9-126.3). Females were 51%, mean age at diagnosis was 39.0 months (range 0-228.4), with only 31% diagnosed before the first year of life and 18% before the 2nd year, due to the lack of national universal newborn screening. 83% were HbSS/SB°, 14.8% SC, 2.2% SB +; 83% came from Sub-Saharan Africa, 9.3% from Europe. 44.5% had chronic comorbidities, the most frequent being: neurological manifestations, asthma and wheezing, gallbladder stones. As of 12/31/2020, 117 were in follow-up and treatment, 57 had transferred abroad or to other national centers, 4 had been lost to follow-up and 4 deceased (3 due to Pneumococcus septic shocks, 1 for a trauma accident). 67% of children required disease-modifying therapies in various combinations (44% HU, 2.2% transfusion regimen, 10% HU and transfusions, 10.8% HSCT after having performed HU and/or transfusion regimen) showing the need for intensive treatments to control the symptoms of the disease (mainly recurrent Vaso-Occlusive Crisis or Acute Chest Syndrome, Anemia or Cerebral Vasculopathy); 16/182 required enrollment in clinical trials with new drugs due to lack of persistent response to HU or chronic transfusion. The treatment with HU, although prescribed according to the national guidelines only to children with clinical manifestations (for anemia, VOC or ACS), was effective in increasing Hemoglobin (9 vs 8.3 g/ dl, p <0, 0001), reducing hemolysis indices and lowering the incidence of neurological complications overall (abnormal/conditional TCD or presence of silent cerebral infarcts on MRI or stenosis on MRA) with EFS 89.1 (95% CI 81.2-93.8) in those who performed HU within 5 years of diagnosis vs 65.6 (95% CI 51.2-76.6) in those who did not (p<0,0001) (Figure 1).However, 11% of the children still present significant neurological complications 5 years after diagnosis in spite of undergoing HU, thereby in need of more intensive treatment to reduce the cerebrovascular complications which in our cohort appear to develop early. Conclusion: Long-term data collection in our setting indicates that, in spite of being offered highly specialized monitoring and treatment options, a subgroup of children still displays significant organ damage and would require intensified treatment in early age.

Efficacy of Activated Autologous Hematopoietic Stem Cell Transplantation in the Treatment of Acute Myeloid Leukemia

To compare the efficacy of activated autologous bone marrow and peripheral blood hematopoietic stem cell transplantation (Auto-HSCT) and matched sibling donor allogeneic hematopoietic stem cell transplantation (MSD-HSCT) for the first complete remission of adult acute myeloid leukemia (AML-CR1). For 86 adult patients with first complete remission of AML who underwent auto-HSCT (41 cases) and MSD-HSCT (45 cases) in our hospital from June 2012 to June 2020, the patients were treated with modified MAC ［Malflane 160 mg/(m2·d), -3 days, Ara-C 2 g/(m2·2), -3 days 21∶00, -2 days 9∶00, CTX 60 mg/(kg·d),-3 d, -2 d］, the stem cells were activated by IL-2 (1 000 U/ mL), IFN-α (100 U/ mL) and IFN-γ (100 U/ml). The overall survival (OS), leukemia free survival (LFS), cumulative incidence of recurrence (CIR) and non-recurrence mortality (NRM) of patients with different types of transplantation were compared. The 3-year OS rates of Auto-HSCT group and MSD-HSCT group were 75% and 69.5%, and the 3-year LFS rates were 70.6% and 82.4%, respectively. There was no statisticaly significant difference in the 3-year OS rates of low risk, medium risk and high risk patients in the Auto-HSCT and MSD-HSCT group (90.2% vs 87.5%, 68.4% vs 68.8%, 28.6% vs 53.3%), the LFS rates of low risk, medium risk and high risk patients in the auto-HSCT and MSD-HSCT group were 90.2% and 87.5%(P=0.838), 71.8% and 91.7%(P=0.184), 0 and 67.5%(P=0.027), respectively. The NRM of Auto-HSCT and MSD-HSCT group were 4.9% and 20% (P=0.036), and CIR were 24.4% and 13.3% (P=0.188). Univariate analysis showed that the survival time of patients was significantly correlated with the number of CR courses and disease risk stratification (P=0.005, P=0.000). Cox multivariate analysis showed that disease risk stratification was an independent risk factor affecting OS (P=0.001). For adult patients with primary AML-CR1, Auto-HSCT is safe and effective. In the absence of sibling donor, Auto-HSCT can be regarded as an effective post-remission treatment for patients with intermediate risk AML-CR1.

Downregulation of circBIRC6 and circCORO1C during differentiation of human cord blood-derived CD34+ cells

BackgroundUmbilical cord blood hematopoietic stem cells (UCB HSCs) transplantation has attracted the attention of many researchers due to the low probability of GVHD and HLA mismatching; however, the use of this resource for adults has been limited due to the inadequate number of CD34+ HSC from any individual donor. Recent reports indicate that circular RNAs (circRNAs) are involved in stem cell properties. MethodsIn this study, the expression of several candidate circRNAs, including circCORO1C and circBIRC6 in CD34+ HSCs was investigated by using quantitative RT-PCR. ResultsThe results showed that the expression levels of circBIRC6 and circCORO1C and simultaneously, the expression of transcription factors such as NANOG and SOX2 were significantly downregulated during differentiation. ConclusionTaken together, these findings suggested that expression of circBIRC6 and circCORO1C were downregulated during differentiation of CD34+ HSCs.

T Cell-Depleted Peripheral Blood versus Unmanipulated Bone Marrow in Matched Sibling Transplantation for Aplastic Anemia

Bone marrow (BM) is the recommended stem cell source for hematopoietic stem cell transplantation (HSCT) from matched sibling donors (MSDs) in patients with severe aplastic anemia (SAA) for its superior survival and graft-versus-host disease (GVHD) outcomes compared to recipients of unmanipulated peripheral blood (PB) HSCT. Nevertheless, no studies comparing BM with ex vivo T cell-depleted (TCD) PB have been reported to date. The aim of the present study was to compare the transplantation outcomes of MSD HSCT recipients with SAA using PB (with partial ex vivo TCD targeted cell dose grafts) with those of MSD HSCT recipients with SAA using unmanipulated BM. We performed a matched-pair analysis of MSD-HSCT using TCD PB in a single institution with unmanipulated BM MSD-HSCT in the United States between 2013 and 2019 reported to the Center for International Blood and Marrow Transplant Research. We compared 23 recipients of TCD PB HSCT for SAA (cases) and 69 recipients of unmanipulated BM grafts (controls) matched for age, Karnofsky Performance Status, Hematopoietic Cell Transplantation-Specific Comorbidity Index, time from diagnosis to transplantation, and recipient cytomegalovirus serostatus. We found significantly faster neutrophil and platelet recovery in the TCD PB cohort (P < .001 and P=.03, respectively), as well as a lower incidence of grade II-IV acute GVHD (0% versus 17%; P=.05) and similar overall survival (96% versus 97% at 3 years; P=.8). Our study shows that TCD PB can be considered a safe source for MSD-HSCT in patients with SAA, with potential advantages in engraftment and GVHD that could challenge the standard with BM. These findings provide a basis for future research in a prospective controlled clinical trial.

The level of Tim-3+CD8+ T cells can serve as a potential marker for evaluating the severity of acute graft-versus-host disease after haplo-PBSCT.

Early and accurate diagnosis of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation is crucial for the prognosis of patients. This study identified a potential biomarker for the severity of aGVHD after human leukocyte antigen (HLA)-haploidentical peripheral blood hematopoietic stem cell transplantation (haplo-PBSCT). We included 20 healthy subjects and 57 patients who underwent haplo-PBSCT. Of these patients, 22 developed aGVHD after haplo-PBSCT. The results showed that patients with aGVHD had significantly increased levels of Tim-3+/Perforin+/Granzyme B+CD8+ T cells, but significantly decreased Galectin-9. The differences in Galectin-9 and Tim-3+/Granzyme B+CD8+ T cells between grade I-II aGVHD and III-IV aGVHD were also significant. In vitro, the apoptosis of CD8+ T cells from aGVHD patients was significantly increased after Tim-3/Galectin-9 pathway activation, which decreased Granzyme B secretion. As revealed by univariate analysis, the level of Tim-3+CD8+ T cells was a risk factor for severe aGVHD. ROC analysis demonstrated that high levels of Tim-3+CD8+ T cells had a significant diagnostic value for severe aGVHD, with an area under the curve of 0.854 and cut-off value of 14.155%. In conclusion, the binding of Tim-3 with exogenous Galectin-9 can promote apoptosis of CD8+ T cells and affect the secretion of Granzyme B. Tim-3+CD8+ T cells have the potential to serve as immunological markers for assessing the severity of aGVHD after haplo-PBSCT and identifying patients at a higher risk for severe aGVHD.

The impacts of total body irradiation on umbilical cord blood hematopoietic stem cell transplantation.

Umbilical cord blood hematopoietic stem cells are commonly used for hematopoietic system reconstitution in recipients after umbilical cord blood transplantation (UCBT). However, the optimal conditioning regimen for UCBT remains a topic of debate. The exact impact of total body irradiation (TBI) as a part of conditioning regimens remains unknown. The aim of this study was to evaluate the impacts of TBI on UCBT outcomes. This was a multi-institution retrospective study. A retrospective analysis was conducted on the outcomes of 136 patients receiving UCBT. Sixty-nine patients received myeloablative conditioning (MAC), in which 33 underwent TBI and 36 did not, and 67 patients received reduced-intensity conditioning (RIC), in which 43 underwent TBI and 24 did not. Univariate and multivariate analyses were conducted to compare the outcomes and the post-transplant complications between patients who did and did not undergo TBI in the MAC subgroup and RIC subgroup, respectively. In the RIC subgroup, patients who underwent TBI had superior overall survival (adjusted hazard ratio [aHR] = 0.25, 95% confidence interval [CI]: 0.09-0.66, p = 0.005) and progression-free survival (aHR = 0.26, 95% CI: 0.10-0.66, p = 0.005). However, in the MAC subgroup, there were no statistically significant differences between those receiving and not receiving TBI. In the setting of RIC in UCBT, TBI utilization can improve overall survival and progression-free survival. However, TBI does not show superiority in the MAC setting.

Distinct Patterns in the Reconstitutions of Treg and T-Helper Subsets after Autologous Hematopoietic Stem Cell Transplantation in Juvenile-Onset Systemic Sclerosis Patients

Systemic sclerosis (SSc) is a refractory autoimmune disease characterized by diffuse fibrosis and vasculopathy affecting the skin, joints, and internal organs. Most immunosuppressive drugs have failed to show long-term benefit in randomized trials. In recent years, autologous hematopoietic stem cell transplantation (ASCT) has been performed to treat multiple sclerosis and SSc, however, the reconstitutions of immune components post-ASCT in SSc have not been fully described. Subjects and Methods: We tested a total of 13 samples from 4 consenting SSc patients (3 females, 1 male) receiving IRB-approved immunoablation (TBI 600cGy) with lung shielding and Cytoxan (n=3) or Thiotepa (n=1) +/- Alemtuzumab followed by autologous CD34+ selected, cryopreserved peripheral blood hematopoietic stem cells transplantation. Their median age was 18.5-years-old (range 15.2- to 21.4-years-old). Peripheral blood samples were collected at baseline pre-ASCT, and 3, 6, 12, 24-months post-ASCT. By flow cytometry, we examined lymphocyte and monocyte subset recovery with additional focus on the FOXP3+Treg and T-helper subset reconstitution. Results and Discussion: All 4 subjects experienced significant improvement in several clinical and quality of life parameters, especially in their skin disease evident within weeks after ASCT. T-cell lymphopenia was profound at 3-months; however, the recovery was evident by 6-months reaching pre-ASCT levels by 2-years post-ASCT (Fig 1A). Natural killer (NK) lymphocytes and monocytes attained the normal values by 3-months and after (Fig 1A). The FOXP3+Tregs that appeared post-ASCT displayed distinct features. There was an increased percentage of FOXP3+Tregs in total CD4+T cells during the early stage (3- to 6-months) post-ASCT (Fig 1B), leading to an increase in the ratio of Treg/Tcon (Fig 1B). The higher proportions of proliferating Tregs as measured by Ki67+FOXP3+CD4+/CD25+/CD127- T cells (Fig 1C) likely led to their increased numbers. This trend was sustained until the last evaluation at 2-years post-ASCT (Fig 1C). In contrast, CD4+Tcons showed a relatively lower proliferation rate, leading to a higher ratio of Ki67+Treg/Ki67+Tcon compared to pre-ASCT values (Fig 1C). Notably, there was an increased proportion of Tregs expressing the skin homing marker cutaneous lymphocyte-associated antigen (CLA) at 3-months lasting until the last test at 2-years post-ASCT (Fig 1D), whereas CLA+/CD4+Tcons were less plentiful. The reconstitutions of T-helper (Th) subsets were also examined. Due to lymphopenia at the early stage post-ASCT, Th subsets were identified according to their chemokine receptor expression (Mahnke et alCytometry 2013;83(5):439-440). Gradually decreased numbers of Th2, Th9, and Th17 were observed in the peripheral pool (Fig 1E) contributing to a decline of their proportions in total CD4+T (Fig 1F). Nevertheless, absolute number of Th1 and Th22 cells returned to levels seen pre-ASCT without any recurrence or flare of SSc symptoms. The CD45RO+ memory phenotype was dominant during the early stages post-ASCT in all subsets (data not shown). Summary: Successful reconstitution of lymphocytes and monocytes post-ASCT was observed in available SSc patients. Post-ASCT circulating FOXP3+Tregs displayed distinct features where the increased number led to an elevated Treg/Tcon ratio in the peripheral pool. Amongst Tregs, the skin homing CLA+ subset was further enriched correlating with improved skin scores in all patients. Improved peripheral proliferation and increased expression of CLA+ Tregs may provide protection from skin disease recurrence despite parallel Tcon reconstitution. The decrease of the Th2/Th9/Th17 subsets in the peripheral pool post-ASCT possibly contributes to the clinically less autoimmune phenotype. These results need to be validated in future patients. Acknowledgements: We thank all participating patients, nurses, physicians, and scientists in this study for their contributions. The study was supported by the "Idea Development Award" from the Department of Defense - Scleroderma Research Program (Award number W81XWH-21-1-0782 and W81XWH-21-1-0783). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal