Bone Marrow Failure Disorders Research Articles

Dioscin alleviates aplastic anemia through regulatory T cells promotion

ABSTRACT Objectives: Aplastic anemia (AA) is one of the immune-mediated bone marrow failure disorders caused by multiple factors, including the inability of CD4 + CD25 + regulatory T cells (Tregs) to negatively regulate cytotoxic T lymphocytes (CTLs). Dioscin is a natural steroid saponin that has a similar structure to steroid hormones. The purpose of this study is to look into the effect of Dioscin on the functions of CD4 + CD25+ Tregs in the AA mouse model and explore its underlying mechanism. Methods: To begin with, bone marrow failure was induced through total body irradiation and allogeneic lymphocyte infusion using male Balb/c mice. After 14 consecutive days of Dioscin orally administrated, the AA mouse model was tested for complete blood counts, HE Staining of the femur, Foxp3, IL-10 and TGF-β. Then CD4 + CD25+ Tregs were isolated from splenic lymphocytes of the AA mouse model, Tregs and the biomarkers and cytokines of Tregs were measured after 24 h of Dioscin intervention treatment in vitro. Results: Dioscin promotes the expression of Foxp3, IL-10, IL-35 and TGF-β, indicating its Tregs-promoting properties. Mechanistically, the administration of Dioscin resulted in the alteration of CD152, CD357, Perforin and CD73 on the surface of Tregs, and restored the expression of Foxp3. Conclusion: Dioscin markedly attenuated bone marrow failure, and promoted Tregs differentiation, suggesting the maintenance of theimmune balance effect of Dioscin. Dioscin attenuates pancytopenia and bone marrow failure via its Tregs promotion properties.

Diagnostic evaluation in bone marrow failure disorders: what have we learnt to help inform the transplant decision in 2024 and beyond?

Aplastic anemia (AA) is the prototypical bone marrow failure syndrome. In the current era of readily available 'molecular annotation', application of comprehensive next-generation sequencing panels has generated novel insights into underlying pathogenetic mechanisms, potentially leading to improvements in personalized therapeutic approaches. New evidence has emerged as to the role of somatic loss of HLA class I allele expression in 'immune-mediated' AA, associated molecular aberrations, and risk of clonal evolution. A deeper understanding has emerged regarding the role of 'myeloid' gene mutations in this context, translating patho-mechanistic insights derived from wider clinical and translational research within the myeloid disorder arena. Here, we review contemporary 'tools' which aid in confirmation of a diagnosis of AA, with an additional focus on their potential in guiding therapeutic options. A specific emphasis is placed upon interpretation and integration of this detailed diagnostic information and how this may inform optimal transplantation strategies.

Five Novel Hemizygous Variants in the PGK1 Gene Associated with Neurological Dysfunction and Mild Hemolytic Anemia in Five Indian Patients

Introduction Phosphoglycerate kinase (ATP: 3-phosphoglycerate 1-phosphotransferase; EC 2.7.2.3; PGK) is the crucial enzyme of the glycolytic pathway, catalyzing1, 3 bis-phosphoglycerate to 3-Phosphoglycerate, generating one molecule of ATP [1]. PGK enzyme is encoded by the PGK1 gene located at Xq13.3, measures 23kb in size, and spans into 11 exons. PGK deficiency is an X-linked disorder associated with a very rare cause of hereditary non-spherocytic hemolytic anemia (HNSHA). PGK1 gene is ubiquitously expressed but the patients exhibit three major symptoms -1) haemolytic anemia 2) myopathy (rhabdomyolysis), 3) mental retardation and various neurological manifestations[1]. The severity of symptoms varies among patients with PGK deficiency. PGK deficiency is reported worldwide, although to our knowledge no case with Indian ancestry has been reported. Method Genomic DNA was extracted from the EDTA blood sample and processed for targeted Next Generation Sequencing (t-NGS) as per the manufacturer's protocol. The t-NGS panel broadly consists of 80 genes of the spectrum of the disorder including congenital hemolytic anemia, bone marrow failure disorders, and iron deficiency anemia. The libraries were sequenced to >80-100X mean coverage on the Illumina sequencing platform. The variants were classified based on ACMG guidelines. The impact of non-synonymous variations was estimated using PolyPhen-2, SIFT, MutationTaster2, CADD Phred, and Mutpred2. In silico effect of the variant was studied by PyMol. Latter the enzyme deficiency was confirmed by standard protocol and parental segregation analysis was performed. Relative quantification (qPCR) was evaluated based on the comparative CT method ( 2 − ΔΔCT). Result We report five male children of <4 years of age with PGK deficiency from Indian origin. Clinically, all of them manifest global development delay and no speech. Autism spectrum disorder is observed in two of the cases, incidence of seizure and convulsion are present in two cases. None except one, presented with haemolytic anaemia and was given one transfusion post an episode of seizure at the age of 2 years. Additionally, a family history of haemolytic anemia with developmental delay and seizure was present in only one case although the patient himself required no transfusion requirement to date. All the patients are routinely followed up for any evidence of a haemolytic crisis, transfusion requirement, or any other clinical symptoms. Detailed phenotypes of all the patients are listed in Table 1. t-NGS revealed five novel hemizygous variants in the PGK1 gene - p. Cys50Trp, p. Gly137Trp, p. Val140Ile, p. Asp159Asn, and p. Arg330Trp. These variants have not been reported in the literature or available databases. DNA Sanger sequencing confirmed the heterozygous state in the mothers. In silico tools confirmed the pathogenicity of the variants. The PGK enzymatic activity was markedly reduced. The PGK1 transcript levels were reduced to 60% in patients as compared to the healthy controls. Discussion To date, only 34 mutations are known to be associated with PGK enzyme deficiency. Amongst them, 50% of variants exhibit symptoms of anaemia whereas the remaining show neurological and /or myopathy. Figure 1 illustrates all 39 mutations reported with PGK deficiency. Only four mutations p.E120K, p.G158R, p.A354P, and p.I371K are known to exhibit all three categories of symptom (PGK-anaemia-neurological-myopathy)[2]. Conclusion NGS allows for the identification of novel genetic variants that may contribute to the development of rare disorders, expanding our knowledge of their underlying mechanisms. This advanced technology also enables researchers to analyze multiple genes simultaneously, increasing the efficiency and speed of diagnosis in unexplained cases of HNSHA.

Antioxidants As a Novel Treatment to Revert Impaired Angiogenic Potential Driven By Metabolic Alterations Observed in Shwachman-Diamond Syndrome Derived Mesenchymal Stromal Cells

Introduction Shwachman-Diamond Syndrome (SDS) is a rare bone marrow (BM) failure disorder. SDS BM biopsies show tortuous and collapsed vessels, highlighting angiogenic abnormalities. Our group previously demonstrated that SDS BM mesenchymal stromal cells (MSCs) have in vitro and in vivo impaired angiogenic potential compared to healthy donors (HD)-MSCs. Interestingly, recent evidence underlines that pathological angiogenesis could be accompanied by altered metabolism and that antioxidants could represent a novel strategy to target angiogenic defects. Aims We aimed to characterize the metabolic status of SDS-MSCs and to investigate if the treatment with antioxidants may restore their impaired angiogenic potential. Methods BM derived HD- and SDS-MSCs were stimulated with DMEM ± N-Acetylcysteine (NAC, 1mM) or dimethylsulfoxide (DMSO, 0.05% v/v) for 48h. Oxidative phosphorylation (OxPhos) was assayed by oximetry and bioluminescent ATP synthesis assay. Spectrophotometric analyses were performed to evaluate ATP/AMP ratio, malondialdehyde (MDA) level, and Complex IV and lactate dehydrogenase (LDH) activity. The amount of reactive oxygen species (ROS) was quantified by flow cytometry. The angiogenic capability was evaluated by performing in vitro Matrigel angiogenesis assay. Results Firstly, we evaluated OxPhos oxygen consumption and ATP production (n=6). Concerning the I-III-IV mitochondrial complexes pathway, SDS-MSCs consumed 57% less oxygen (p=0.004) and produced 64% less ATP compared to HD-MSCs (p=0.002). Accordingly, the analysis of the II-III-IV complexes pathway demonstrated that the oxygen consumption was reduced by 62% (p=0.002) and the ATP synthesis was 67% lower than HD-MSCs (p=0.002). Furthermore, the P/O ratio, index of OxPhos efficiency, was significantly reduced in SDS-MSCs in both the electron transport chain pathways (p=0.002 for both). Therefore, we demonstrated that Complex IV activity was 61% lower in SDS- vs HD-MSCs (p=0.002), highlighting its role in the SDS OxPhos defect. As for the energetic status, SDS-MSCs showed a low intracellular ATP/AMP ratio (mean=1.1, range=0.8-1.6 vs mean=3.6, range=3.1-4.0, in HDs; p=0.002) that was accompanied by 30% increase of LDH activity in SDS-MSCs (p=0.002), an expression of increased anaerobic glycolysis to compensate the mitochondrial defect. The amount of ROS in SDS-MSCs was increased by 27% compared to HD-MSCs (n=5) and also the SDS-MSCs lipid peroxidation level was significantly higher over HDs (mean=12.7 µM of MDA/mg, range=10.5-14.1µM/mg vs mean=6.6µM/mg, range=5.9-7.4µM/mg; p=0.002). Then we treated SDS-MSCs for 48h with NAC, a broad-range antioxidant (n=5), or with DMSO, which acts at very low concentrations as scavenger, specifically on lipid peroxidation products (n=6). Interestingly, NAC and DMSO stimulated SDS-MSCs increased by 75% the oxygen consumption and by 70% the ATP synthesis in both the electron transport chain pathways, thus resulting in levels comparable to HD-MSCs. The SDS OxPhos restoration was respectively associated to a 53% and 60% increase of Complex IV activity after NAC and DMSO stimulation (p<0.0001 for both). Moreover, antioxidants corrected the SDS energetic defect by restoring ATP/AMP ratio and LDH activity to the levels of HDs. Importantly, SDS-MSCs lipid peroxidation level was drastically reduced after NAC and DMSO treatments (mean=12.7µM/mg, range=10.5-14.1µM/mg vs NAC mean=5.1µM/mg, range=4.3-5.4µM/mg and DMSO mean=6.2µM/mg, range=5.8-6.5µM/mg; p<0.0001 for both). Finally, we approached SDS-MSCs angiogenic defect and showed that SDS-MSCs defective capability to recreate a defined capillary-like network under angiogenic stimuli was completely restored after antioxidant stimulations (n≥5). As shown by ImageJ Angiogenesis Analyzer several angiogenic elements, including branches and segments, were significantly increased in NAC and DMSO stimulated SDS-MSCs that become comparable to HD-MSCs. Conclusions We demonstrated that the altered OxPhos metabolism of SDS-MSCs significantly contributes to their angiogenic defect and, importantly, that antioxidants restored the metabolic alterations and the angiogenic potential in SDS-MSCs, paving the way for new therapeutic strategies.

A Phase I/II, Intra-Patient Dose-Escalation Study of Bitopertin, a Selective Inhibitor of GlyT1 and Heme Synthesis, for Steroid-Refractory Diamond-Blackfan Anemia

Background: Diamond-Blackfan anemia (DBA) is a heritable bone marrow failure disorder characterized by macrocytic anemia with reticulocytopenia and bone marrow erythroid hypoplasia, usually presenting within the first two years of life. Treatment consists of systemic corticosteroids. Treatment failure or steroid intolerance occurs in approximately half of all cases, necessitating chronic transfusions, which carry the risks of iron overload and infections. Hematopoietic cell transplant remains the sole curative therapy for DBA. In cases where a causative mutation can be identified, with rare exceptions, the affected genes encode ribosomal proteins (RPs), defining DBA as prototypic haploinsufficient ribosomopathy. Erythrocyte hemoglobinization requires balanced heme synthesis and globin translation. Various in vitro and animal models suggest that RP insufficiency results in reduced ribosomal biogenesis, disrupting this balance and leading to excess of heme, resulting in toxicity to developing erythroid cells and premature termination of erythroid differentiation. Bitopertin is an investigational, orally administered, inhibitor of the GlyT1 glycine transporter. Outside the central nervous system (CNS), GlyT1 is expressed almost exclusively on developing erythrocytes. By reducing intracellular glycine, a rate-limiting substrate for heme synthesis, bitopertin slows heme synthesis and thus we hypothesize rebalances hemoglobin synthesis. In preclinical DBA models, bitopertin rescues erythropoiesis. The drug was originally developed for CNS indications, and has been shown to exhibit a favorable safety profile in prior studies with cumulative enrollment of over 4,000 patients and healthy volunteers. It is currently under clinical investigation for the treatment of erythropoietic protoporphyria. Study Design and Methods: This is a phase I/II, intra-patient dose-escalation study of bitopertin for steroid-refractory DBA, conducted at the National Institutes of Health (NIH) Clinical Center in Bethesda, Maryland. Subjects visit the NIH for initial evaluation and eligibility assessment and at the primary endpoint, with interim follow-up conducted via telehealth and local laboratory assessments. Subjects must be 18 years of age or and older, diagnosed with DBA and with clinically significant anemia (hemoglobin < 9/dL and/or requiring 2 units PRBC per 8 weeks). They must have relapsed and/or steroid refractory disease or be intolerant of systemic steroids, and be without significant comorbidities. Ongoing transfusions and chelation therapy are allowed, but other DBA-directed therapy, most notably steroids and growth factors, are not. Each subject will take oral bitopertin daily, escalating the dose monthly (5 mg, 10 mg, 20 mg, 40mg, and 60 mg) until reaching either the study maximum dose of 60 mg, the minimum effective dose (MED), or the maximum tolerated dose (MTD). Subjects will be followed for 8 months (32 weeks), including a minimum of 4 months (16 weeks) at the final dose (MED, MTD or study maximum). Response will be assessed on an ongoing basis and will be defined as an increase in hemoglobin ≥by 1.5 g/dL and/or a reduction in the units of PRBC transfusions by ≥50% from baseline. Furthermore, a robust response will be defined as a hemoglobin ≥10 g/dL and transfusion independence for 8 weeks. Patients with response (robust or non) by the 8-month primary endpoint will be allowed to continue extended therapy for 3 additional years. The primary endpoint is erythroid response. Secondary endpoints include safety as defined by the toxicity profile over 8 months of therapy and on extended therapy, relapse, survival, clonal evolution, health-related quality of life, and ancillary laboratory studies including the impact of bitopertin upon erythroid cell dynamics. The study employs a minmax design. A minimum of 15 patients will be enrolled. If the minimum efficacy threshold is met or surpassed (≥13% response rate), an additional 10 patients will be enrolled. Status: This study has been granted an FDA IND (165778) and is open for enrollment. It is monitored under the NIH Institutional Review Board (001528-H) and is registered with the U.S. National Library of Medicine (NCT05828108). Funding: This study is supported by the NHLBI Division of Intramural Research and in part through a Cooperative Research and Development Agreement (CRADA) with Disc Medicine, Inc. (Watertown, MA).

In Vivo Effects of Porcine and Rabbit Antithymocyte Globulin in Patients with Severe Aplastic Anemia

Background: Severeaplastic anemia (SAA) is a bone marrow failure disorder mediated by T lymphocytes, resulting in severe deficiency of hematopoietic stem cells. Horse antithymocyte globulin (H-ATG) plus cyclosporin was the first-line therapy for SAA patients in the absence of an HLA-matched sibling 1. The inhibitory effect of Rabbit-ATG (R-ATG) on lymphocytes is more deeply than that of H-ATG, and the recovery of CD4 T cells is slower, resulting in poor efficacy in SAA patients 2. Porcine ATG (P-ATG) and R-ATG are two available immunosuppressive choices in China mainland, the first-line formulation of ATG remains unknown. Methods: From November 2020 through September 2022, we performed a prospective, multi-center, non-randomized trial comparing two ATG formulations. 15 patients received R-ATG therapy and 45 patients treated with P-ATG were collected by prospective registration (ChiCTR2200055696) in the Chinese Eastern Collaboration Group of Anemia (CECGA). The pharmacokinetics, dynamic changes of peripheral blood cells were evaluated. Hematologic responses were also evaluated at 3, 6 months after treatment. Results: In both P-ATG-treated and R-ATG-treated patients, ATG reached highest concentration 5 days after treatment (1268.4 ± 381.1 ug/ml and 641.2 ± 448.5 ug/ml for P-ATG and R-ATG, respectively), and gradually decreased. At week 2, week 3 and 1 month from the peak values when be assessed, the P-ATG concentration decreased by 70.3%, 81.7%, and 85.4%, while the R-ATG concentration decreased by 68.9%, 88.5%, and 92.2%, respectively. R-ATG showed more powerful effects in depleting peripheral blood lymphocytes than P-ATG, while P-ATG displayed rapid recovery of T cells (CD3 + T cells, CD4 + T cells and CD8 + T cells) (P-ATG vs R-ATG, CD3 + T cells: 30.18 ± 6.04% vs 0.49 ± 0.37%, p=0.0036; 60.58 ± 8.02% vs 7.16 ± 7.16%, p=0.0016; 77.83 ± 3.17% vs 59.67 ± 7.93%, p= 0.015 for day 3, week 1 and week 3, respectively; CD4 + T cells: 9.13 ± 2.20% vs 0.03 ± 0.02%, p=0.013, 24.49 ± 4.21% vs 1.75 ± 1.75%, p=0.0076; 38.49 ± 3.02% vs 17.62 ± 3.79%, p=0.0007 for day 3, week 1 and week 3, respectively; CD8 + T cells: 16.77 ± 4.15 vs 0.10 ± 0.067, p=0.015; 27.55 ± 3.56% vs 4.94 ± 4.94%, p=0.0036; 33.80 ± 2.19% vs 35.67 ± 6.20, p=0.72 for day 3, week 1 and week 3, respectively). There were lower number of neutrophils in patients treated with R-ATG than those treated with P-ATG during the first week after immunosuppressive therapy (P-ATG vs R-ATG, Day 3: 3.46 ± 0.49 vs 1.29 ± 0.56, p=0.02; Day 5: 3.73 ± 0.56 vs 1.12 ± 0.55, p=0.02). The overall response rates were comparable between P-ATG and R-ATG at third month and sixth month (60% vs 40%, p=0.24; 60% vs 60%, p=1). Early infection rates (within 1 month) showed no significant differences in two formulations (P-ATG vs R-ATG, 33.33% vs 46.67%, p=0.76). There were five deaths in patients treated with P-ATG and three deaths in R-ATG-treated patients (P-ATG vs R-ATG, 11.1% vs 20.0%, p=0.40). Conclusion: P-ATG and R-ATG showed different pharmacokinetics and effects on neutrophils, lymphocyte subsets, which may lead to diverse immunosuppression. These two kinds of globulins showed comparable overall response rate after treatment. Reference 1. Scheinberg P, Nunez O, Weinstein B, et al. Horse versus rabbit antithymocyte globulin in acquired aplastic anemia. N Engl J Med. 2011;365(5):430-438. 2. Feng X, Scheinberg P, Biancotto A, et al. In vivo effects of horse and rabbit antithymocyte globulin in patients with severe aplastic anemia. Haematologica. 2014;99(9):1433-1440.

Clinical Characteristics of Shwachman-Diamond Syndrome in China - Result from Childhood Bone Marrow Failure Diseases Register of China Alliance for Blood Diseases(cBMFR-CABD)

OBJECTIVE This study aimed to identify the clinical features of 73 children diagnosed with SDS from 22 centres in China. METHODS Childhood Bone Marrow Failure Diseases Register of China Alliance for Blood Diseases (cBMFR-CABD) was a bidirectional multicenter cohort study that collected data from 22 paediatric blood disease treatment centres from all over China. Patients were enrolled in the sub-centres from 1.1.2011 that were aged < 18 years at diagnosis and met the diagnostic criteria for an inherited bone marrow failure disorder. The clinical data for the 73 SDS patients analyzed in this study were collected between 1.1.2011 and 31.12.2022. RESULTS Among the 73 SDS children, 38 were male and 35 were female. Short stature (height<-2SD) was found in 53.4 % (39/73) patients. Chronic diarrhea was observed in 24.7 % (18/73) patients. In children evaluated for intellectual and physical malformations, 7 patients (17.3 %,6/52) had mental retardation and 14 (20.5 %, 15/73) patients had physical deformities. Genetic testing was available in 63 patients. seventeen (27.0 %) patients carry bi-allelic c.258+2T>C and c.183-184TA>CT SBDS mutations. Eleven patients (17.5%) are homozygous for the c.258+2T>C mutation. Four patients (6.3%) were compound heterozygous for c.258+2T>C and exon 2 deletions. One patient carries bi-allelic mutation of DNAJC21. This patient had a history of repeated infections and pancytopenia.Bone marrow smear shows multilineage dysplasia. Bone marrow results were available for evaluation in 54 patients including 67 % (36/54) BMF, 30 %(16/54) MDS, 4 % (2/54) AML.TP53 mutation testing was available in 15 patinets with eight (53.3%) patients carry TP53 mutation.Karyotype analysis was available in 36 patinets with 5 patients were complex karyotype. Among the 56 patients available for follow-up, 17 patients underwent haematopoietic stem cell transplantation, 5 patients died (one infection,three transplant-related death and one relapse). Among the 39 non-HSCT patients, 3 patients died of AML transformation. Three-year overall survival of all patients were 91.7%(95%CI 99.5 %~ 83.9% ), BMF patients were 100.0%(95%CI 100.0%~100.0% ), MDS/AML patients were 67.7%(95%CI 94.0%~ 41.4% ). CONCLUSION Our results provide the clinical features of the largest SDS cohort in China. A high incidence of short stature was observed. Bi-allelic c.258+2T>C with c. 183-184 TA > CT SBDS mutations were the most common mutation type. HSCT is an effective treatment modality in SDS. However, AML transformation is still a major risk factor for children with SDS.

A Napaac Survey to Understand Practices in Diagnosis and Treatment of Children with Moderate Aplastic Anemia

Among patients with acquired aplastic anemia, approximately 20% do not meet current classification as severe based on established criteria that reflect blood counts and marrow cellularity. For patients with moderate aplastic anemia (MAA), there is no set of defined diagnostic criteria, or clear standard-of-care for management. Little is known about the natural history of spontaneous recovery versus progression to severe bone marrow failure (BMF), response to conventional immunosuppressive treatment (IST), or evolution to myelodysplasia (MDS). Thus, institutional and individual practice can range from observation to immunosuppression or hematopoietic stem cell transplantation (HSCT). Recent studies of this patient population have begun to identify individuals harboring pathogenic mutations in genes known to cause BMF. To better understand population characteristics and management practices for patients with MAA, a survey was conducted through the North American Pediatric Aplastic Anemia Consortium (NAPAAC), consisting of 52 centers with expertise in BMF across North America. A total of 65 surveys were completed by pediatric hematology-oncology providers (55 attending physicians, 5 nurse practitioners and 5 fellows). Across all groups, there was marked variability of the MAA definition. Cytopenias were the defining criteria for diagnosis, including ANC<1000 cells/ul (79%), platelets <100K cells/ul (43%), and anemia for age (47%). In contrast, bone marrow hypocellularity (<50% cellularity) was considered by less than half (48%) of the respondents to be necessary for diagnosis. The combination of hypocellular bone marrow plus two cytopenias was considered the most common diagnostic definition of MAA (60% of participants). Although genetic testing has begun to transform diagnosis for many hematological diseases and can alter treatment decisions for pediatric BMF disorders, only 51% currently consider next generation sequencing (NGS) and 4% consider whole exome/whole genome sequencing as part of the initial work-up for MAA. Conventional cytogenetics and FISH are used more frequently in the initial work-up (86% and 73%, respectively). The two main triggers for treatment were dependence on red blood cell- (92%) or platelet (87%) transfusions, followed by persistent cytopenias (54%) and cytogenetic abnormalities (53%). Where available, acquired somatic variants triggered treatment in 30% of responses. Interestingly, infections were a less frequent trigger for treatment (35%). The most common management approaches were observation with transfusions (79%), single agent thrombopoietin receptor agonists (71%) or ATG+Cyclosporine based immune suppressive therapy (IST, 61%). Progression to severe aplastic anemia (SAA, 97%), myelodysplastic syndrome (MDS, 97%), or refractoriness to IST (61%) were the most common indications for HSCT.Availability of a matched sibling donor (MSD, 12%) was an infrequent indication for HSCT. A growing body of work, albeit largely in adult patients, suggests that patients with MAA represent a heterogenous group of individuals, some with genetic predisposition to BMF and inherent risk of myelodysplastic evolution. We identified that half of the responding NAPAAC practitioners use NGS panels routinely, suggesting a need for increased awareness to consider this work-up at initial diagnosis. Future studies are important to determine the yield of standard NGS testing, as the diagnosis of inherited BMF syndromes alter the strategy for long term surveillance of patients and genetic counseling for the family, as well as the use of related donor HSCT. We observed variability regarding the diagnostic criteria and management of children with MAA by providers who are members of NAPAAC. The heterogeneous responses to our survey suggest the need to establish management guidelines for uniform work-up and treatment, and to facilitate research of this understudied population. PC, LO: contributed equally as first authors. PK, JD: contributed equally as senior authors

Dyskeratosis Congenita: A Case Report

Dyskeratosis Congenita (DC) is a rare inherited bone marrow failure disorder characterized by lacy reticulated skin pigmentation, nail dystrophy and mucosal leukoplakia. In DC bone marrow failure is the main cause of premature death with an additional predisposition to malignancy. DC results from progressive shortening of telomeres resulting in DNA replication. X-linked recessive is the main mode of inheritance. Most of the patients respond to Androgen therapy. This is a case report of a 5-year-old boy presented with features of bone marrow failure and had abnormality of skin, oral mucosa and nail. His complete blood count with peripheral blood film showed pancytopenia and no blast cell. Hypocellular marrow was present in his Bone marrow examination. Chromosomal analysis- fluorescence in situ hybridization (FISH) revealed very short telomere of chromosome. Microarray genotyping showed DKC1 gene abnormality. BANGLADESH J CHILD HEALTH 2022; VOL 46 (1) : 39-41

Bone marrow plasma metabonomics of idiopathic acquired aplastic anemia patients using 1H nuclear magnetic resonance spectroscopy.

Idiopathic acquired aplastic anemia (AA) is a bone marrow failure disorder where aberrant T-cell functions lead to depletion of hematopoietic stem and progenitor cells in the bone marrow (BM) microenvironment. T-cells undergo metabolic rewiring, which regulates their proliferation and differentiation. Therefore, studying metabolic variation in AA patients may aid us with a better understanding of the T-cell regulatory pathways governed by metabolites and their pathological engagement in the disease. To identify the differential metabolites in BM plasma of AA patients, AA follow-up (AAF) in comparison to normal controls (NC) and to identify potential disease biomarker(s). The study used 1D 1H NMR Carr-Purcell-Meiboom-Gill (CPMG) spectra to identify the metabolites present in the BM plasma samples of AA (n = 40), AAF (n = 16), and NC (n = 20). Metabolic differences between the groups and predictive biomarkers were identified by using multivariate analysis and receiver operating characteristic (ROC) module of Metaboanalyst V5.0 tool, respectively. The AA and AAF samples were well discriminated from NC group as per Principal Component analysis (PCA). Further, we found significant alteration in the levels of 17 metabolites in AA involved in amino-acid (Leucine, serine, threonine, phenylalanine, lysine, histidine, valine, tyrosine, and proline), carbohydrate (Glucose, lactate and mannose), fatty acid (Acetate, glycerol myo-inositol and citrate), and purine metabolism (hypoxanthine) in comparison to NC. Additionally, biomarker analysis predicted Hypoxanthine and Acetate can be used as a potential biomarker. The study highlights the significant metabolic alterations in the BM plasma of AA patients which may have implication in the disease pathobiology.

Real-world outcomes with immunosuppressive therapy for aplastic anemia in patients treated at the University of Michigan.

Aplastic anemia (AA) is a rare bone marrow failure disorder that is treated with either allogeneic stem cell transplant or immunosuppressive therapy (IST) consisting of antithymocyte globulin (ATG), cyclosporine (CSA), and eltrombopag. While outcomes are favorable in younger patients, older patients (>60) have significantly worse long-term survival. The dose of ATG is often reduced in older patients and those with multiple comorbidities given concerns for tolerability. The efficacy and safety of dose-attenuated IST in this population is largely undescribed. We performed a retrospective review of patients with AA treated with IST. Our analysis was confounded by changes in practice patterns and the introduction of eltrombopag. We identified 53 patients >60 years old, of which, 20 received dose-attenuated IST, with no statistically significant difference in overall survival between full and attenuated dose cohorts. Overall response rates in both cohorts were similar at 6 months at 71% and 68%. There were more documented infectious complications in the full dose cohort (13 vs. 3). This supports the consideration of dose-attenuated IST in older patients with concerns about tolerance of IST. Lastly, our data confirmed favorable outcomes of younger patients receiving IST, especially in combination with eltrombopag.

Retrospective Evaluation of Patients Who Underwent Allogeneic Stem Cell Transplantation for Bone Marrow Failure

Background: Bone marrow failure is a disease that develops due to different etiologies. Aplastic anaemia (AA) and hypocellular myelodysplastic syndrome (HMDS) are the most common bone marrow failure disorders. Treatment options include supportive therapy, immunosuppressive therapy, and allogeneic hematopoietic stem cell transplantation (allo-HCT). Allo-HCT is the only curative treatment option. This study aimed to retrospectively evaluate the demographic characteristics, treatment, and transplantation results of patients who underwent Allo-HCT for bone marrow failure.
 Material and Methods: This single-centre retrospective study enrolled 11 patients (9 with severe AA and 2 with HMDS) who underwent allo-HCT for bone marrow failure. The patients' records until 17.08.2023 were analysed. Age, gender, diagnosis, donor age and gender, type of transplantation, pre-transplant ferritin levels, time to transplantation, volume of infused product, number of CD34+ cells in the infused product, post-transplant engraftment times, discharge time, transplant-related complications, post-transplant follow-up and overall survival times were obtained.
 Results: Eleven patients underwent 12 allo-HCTs for bone marrow failure. Seven patients were male, and four were female. The median age was 40, and seven patients were ≥40 years old at the time of transplantation. Eleven transplants were performed from HLA fully matched siblings and one from a 9/10 matched sibling donor. Bone marrow was used as a stem cell source in 8 transplants and peripheral blood in 4 transplants. The conditioning regimen was fludarabine/cyclophosphamide/anti-thymocyte globulin in all patients. The median time from diagnosis to transplantation was five months. The median time for neutrophil engraftment was 23 days. The median platelet >20.000/mm3 engraftment time was 16 days. A statistically significant positive correlation was found between ferritin levels and platelet >20.000/mm3 engraftment (days) (r=0.653, p=0.040) and platelet >50.000/mm3 engraftment (days) (r=0.720, p=0.029). There was a statistically significant negative correlation between the number of infused CD34 positive cells (10⁶/kg) and platelet >50.000/mm3 engraftment (days) (r=-0.670, p=0.024). Patients were discharged in a median of 23 days. Acute graft versus host disease (GvHD) was observed in one patient, while chronic GvHD was not observed in any patient. The median overall survival time was 48 months, and the median post-transplant follow-up was 37 months. Secondary malignancy and MDS were not detected in any patient during the follow-up period. All 11 patients who underwent Allo-HCT from a matched sibling donor are alive and continue to have a complete hematological response. There was no increase in mortality and morbidity in patients aged 40 years and older.
 Conclusions: In patients with severe AA and high-risk HMDS without comorbidities between the ages of 40 and 50, allo-HCT should be considered as first-line treatment in the presence of an HLA-matched sibling donor.

Severe Aplastic Anaemia in a 14-year-old boy

Aplastic anemia is a disorder of bone marrow failure characterized by pancytopenia and hypocellular bone marrow. We report a case of severe aplastic anemia in a 14-year-old boy.

Deciphering treatment patterns in non-severe/moderate aplastic anemia: an international observational study

Non-severe aplastic anemia is a rare bone marrow failure disorder characterized by variable degrees and combination of cytopenias, with limited data on management and outcome. We describe a large multicentric series of 259 patients, focusing on clinical and molecular features, treatment, evolution, and survival. The majority required treatment with cyclosporine (CyA) alone (N = 84) or in combination with anti-thymocyte globulin (ATG,44) or eltrombopag (20), eltrombopag alone (10), or others (25) including androgens. Similar outcomes were observed across different strategies, with a 6-month overall response rate of 73% for CyA, 74% for ATG plus CyA, 68% for CyA plus eltrombopag, 87% for eltrombopag, and 79% for others. Notably, 56 patients (39%), mainly receiving CyA plus eltrombopag, achieved a trilineage response (p = 0.02). Progression to myeloid neoplasms was limited (8%) and not related to mutational status. Hemolytic PNH developed in 10% of cases, being predicted by detection of small clones at diagnosis. Survival was negatively impacted by age, male gender, LDH, platelets/erythrocyte transfusion need, and somatic mutations by NGS, and positively by higher neutrophils at diagnosis, PNH clones, and trilineage response at 6 and 12 months. Multivariable analysis confirmed the detrimental role of age and the favorable association with PNH clone and trilineage response at 6 months.

Outcome of first or second transplantation using unrelated umbilical cord blood without ATG conditioning regimen for pediatric bone marrow failure disorders

BackgroundUnrelated umbilical cord blood transplantation (UCBT) for bone marrow failure (BMF) disorders using conditioning regimens without Anti-Thymocyte Globulin (ATG) has been used as an alternative transplantation for emerging patients without matched-sibling donors. Experience with this transplant modality in children is limited, especially as a secondary treatment for transplant failure patients. ProcedureWe retrospectively reviewed 17 consecutive bone marrow failure patients who underwent unrelated umbilical cord blood transplantation in our center and received conditioning regimens of Total Body Irradiation (TBI) or Busulfan (BU) + Fludarabine (FLU) + Cyclophosphamide (CY). ResultsAmong the 17 BMF patients, 15 patients were treated with first cord blood transplantation and another 2 with secondary cord blood transplantation because of graft failure after first haploidentical stem cell transplantation at days +38 and +82.All patients engrafted with a median donor cell chimerism of 50 % at days +7 (range, 16 %–99.95 %) and finally rose to 100 % at days +30. Median time to neutrophil engraftment was 19 days (range, 12–30) and time to platelet engraftment was 32 days (range, 18–61). Pre-engraftment syndrome (PES) was found in 16 patients (94.11 %, 16/17). Cumulative incidence of grades II to IV acute GVHD was 58.8 % (95 % CI: 32.7–84.9 %), and 17.6 % (95 % CI: 2.6–37.9 %) of patients developed chronic GVHD. The 3-year overall survival (OS) and failure-free survival (FFS) rates were 92.86 ± 6.88 %. ConclusionUCBT is an effective alternative treatment for bone marrow failure pediatric patients. TBI/BU + FLU + CY regimen ensure a high engraftment rate for unrelated umbilical cord blood transplantation, which overcomes the difficulty of graft failure. Secondary salvage use of cord blood transplantation may still be useful for patients who have failed after other transplantation.

Effects of ruxolitinib on murine regulatory T cells are immune-context dependent

Aplastic anemia is a bone marrow failure (BMF) disorder characterized by pancytopenia and hypocellular marrow from an immune-mediated etiology. Regulatory T cells (Tregs) prevent autoimmunity by suppressing autoreactive T cells. We recently demonstrated the efficacy of ruxolitinib (RUX), a JAK 1/2 inhibitor, in attenuating murine BMF. Herein, we investigated the changes of Tregs in the context of RUX treatment for murine BMF. Tregs are conventionally identified by surface expression of CD4 and CD25, in addition to intracellular transcription factor FoxP3. RUX promoted the expansion of Tregs in BMF mice defined by increased expression of FoxP3 in CD4 T cells but suppressed expression of activation marker CD25 in CD4 and CD8 T cells. In this context, CD25 is no longer a reliable surface marker for Tregs. We observed strong co-expression of FoxP3 with surface marker GITR instead of CD25 in RUX-treated BMF mice. Fluorescence-activated cell sorting (FACS)-sorted CD4+GITRhi cells showed high FoxP3 expression and intact suppressive function in vitro, suggesting GITR to be a surrogate marker for Tregs. In contrast to its expansive effect on Tregs in BMF condition, RUX suppressed Tregs in normal and sublethal irradiation conditions, indicating that the effects of RUX on Tregs are immune-context dependent.

Racial disparities in the outcomes of allogeneic bone marrow transplant: A 5-year nationwide inpatient study.

e19060 Background: Allogeneic bone marrow transplantation (ABMT) is a life-saving procedure in hematological malignancies and bone marrow failure disorders. Despite advances in the field, literature proposes racial disparities in the outcomes of ABMT. This study aims to investigate this difference among distinct racial cohorts. Methods: We identified patients who underwent ABMT for malignant and nonmalignant hematologic diseases from 2016-2020 using the nationwide inpatient sample (NIS) database. We compared the racial differences in outcomes during the hospitalization, including immunologic and hematologic complications, associated infections, length of stay, and mortality rate. Baseline characteristics were compared using a T-test and Chi-Square. Multivariate regression analysis was applied to estimate differences in outcomes. We used STATA Version 17.0 Software for data analysis. The p-value was set at p < 0.05 for statistical significance. Results: A retrospective study was conducted on a cohort of 14,599 adults who underwent ABMT. Out of which 30% (4,379) developed complications. The mean age for ABMT was 54, and the average length of stay was 29 days. In this cohort, 79.5% (11,325) were whites, 4.2% (610) were blacks, 8.3% (1,185) were Hispanics, and 7.8% (1,125) were others. When comparing the outcomes among racial groups, invasive fungal infections 15.5% (95), mucositis 36% (220), septic shock 9% (55), and respiratory failure 14.7% (90) were more common in blacks (p < 0.05). Neutropenia 41.3% (490) and cytomegalovirus (CMV) infection 12.2% (145) were more common in Hispanics (p < 0.05). However, no significant difference was noted in immunologic complications (graft versus host disease (GVHD), bone marrow rejection (BMR), bone marrow failure (BMF)), pancytopenia, clostridium difficile infection (CDI), and mortality (p < 0.05). Conclusions: Our study shows that blacks had a higher prevalence of invasive fungal infections, mucositis, septic shock, and respiratory failure, while Hispanics reported a higher prevalence of neutropenia and CMV infection. However, no significant differences were noted in GVHD, BMR, BMF, pancytopenia, CDI, and mortality rates. Therefore, it can be concluded that while there are some differences in post-ABMT complications among racial groups, overall, the rates of major complications and mortality do not significantly differ.

Current use of androgens in bone marrow failure disorders: a report from the Severe Aplastic Anemia Working Party of the European Society for Blood and Marrow Transplantation.

Androgens represent the historical therapeutic backbone of bone marrow failure (BMF) syndromes. However, their role has rarely been analyzed in a prospective setting, and systematic and long-term data regarding their usage, effectiveness and toxicity in both acquired and inherited BMF are currently unavailable. Here, taking advantage of a unique disease-specific international dataset, we retrospectively analyzed the largest cohort so far of BMF patients who received androgens before or in the absence of an allogeneic hematopoietic cell transplantation (HCT), re-evaluating their current use in these disorders. We identified 274 patients across 82 European Society for Blood and Marrow Transplantation (EBMT) affiliated centers: 193 with acquired (median age 32 years) and 81 with inherited (median age 8 years) BMF. With a median duration of androgen treatment of 5.6 and 20 months, respectively, complete and partial remission rates at 3 months were 6% and 29% in acquired and 8% and 29% in inherited disorders. Five-year overall survival and failure-free survival (FFS) were respectively 63% and 23% in acquired and 78% and 14% in inherited BMF. Androgen initiation after second-line treatments for acquired BMF, and after >12 months post diagnosis for inherited BMF were identified as factors associated with improved FFS in multivariable analysis. Androgen use was associated with a manageable incidence of organ-specific toxicity, and low rates of solid and hematologic malignancies. Sub-analysis of transplant-related outcomes after exposure to these compounds showed probabilities of survival and complications similar to other transplanted BMF cohorts. This study delivers a unique opportunity to track androgen use in BMF syndromes and represents the basis for general recommendations on this category of therapeutics on behalf of the Severe Aplastic Anemia Working Party of the EBMT.

Engineered human Diamond-Blackfan anemia disease model confirms therapeutic effects of clinically applicable lentiviral vector at single-cell resolution.

Diamond-Blackfan anemia is a rare genetic bone marrow failure disorder which is usually caused by mutations in ribosomal protein genes. In the present study, we generated a traceable RPS19-deficient cell model using CRISPR-Cas9 and homology-directed repair to investigate the therapeutic effects of a clinically applicable lentiviral vector at single-cell resolution. We developed a gentle nanostraw delivery platform to edit the RPS19 gene in primary human cord bloodderived CD34+ hematopoietic stem and progenitor cells. The edited cells showed expected impaired erythroid differentiation phenotype, and a specific erythroid progenitor with abnormal cell cycle status accompanied by enrichment of TNFα/NF-κB and p53 signaling pathways was identified by single-cell RNA sequencing analysis. The therapeutic vector could rescue the abnormal erythropoiesis by activating cell cycle-related signaling pathways and promoted red blood cell production. Overall, these results establish nanostraws as a gentle option for CRISPR-Cas9- based gene editing in sensitive primary hematopoietic stem and progenitor cells, and provide support for future clinical investigations of the lentiviral gene therapy strategy.

Abstract A51: Divergent effects of MDS predisposing SAMD9L point mutations, in humans, mice, and cellular models

Abstract We previously showed that germline SAMD9 and SAMD9L (SAMD9/9L) disorders are the most common predisposition to childhood myelodysplastic syndromes with bone marrow failure (BMF). Heterozygous mutations in both genes are growth inhibitory and undergo negative selection by acquiring compensatory rescue events in blood. Here, we describe the first report of SAMD9L genetic rescue occurring during embryonal development and resulting in germline triple-allelic mosaicism (1 wildtype (WT) and 2 mutant alleles), where only one mutant allele was transmitted to each of the two diseased children. In a family of 2 affected siblings with hypocellular BMF, the Index case carried germline heterozygous SAMD9L V1512M mutation and her brother was heterozygous for V1512L mutation. The clinical presentation for Index with V1512M was more severe compared to her brother with V1512L mutation. Genetic assessment of the parents showed the asymptomatic mother to be a triple-allelic mosaic, carrying WT SAMD9L allele at ~50% frequency in all tissues, while both V1512M and V1512L mutant alleles “competed” in their allelic distribution depending on the tissue origin. Single-cell DNA sequencing on her peripheral blood (PB) revealed 3 independent diploid clones: V1512M in 14%, V1512L in 68%, and WT (due to revertant UPD7q) in 18% of cells. Because her parents were SAMD9L WT, one of the mutations likely arose de novo and underwent failed embryonic rescue attempt leading to a second mutation. Towards studying the effect on hematopoiesis in vitro, we knocked in V1512M and V1512L mutations in inducible pluripotent stem cells (iPSC). For in vivo functional validation, we created constitutive mouse models with ortholog mutations (V1507M and V1507L). Mutant hematopoietic progenitor cells from iPSC had severely compromised proliferative capacity and yielded fewer erythroid and myeloid cells. This effect was more severe in V1512M vs. V1512L mutants. The divergent mutational phenotypes were also replicated in our mouse models: two-thirds (13/19) of the heterozygous V1507M (V1507Mhet) pups died before 4 weeks of age. In contrast, V1507Lhet mice had overall survival equal to WT mice, while one-third of homozygous V1507L (V1507Lhom) mice showed decreased survival post 19 weeks of age. At baseline, V1507Mhet mice showed severe growth retardation with multisystemic issues, which were absent in the V1507L mutant models. PB cytopenia (anemia, B-cell lymphopenia) though observed in both V1507Mhet and V1507Lhom mice, the degree of severity was high in the V1507Mhet than in the V1507Lhom model. Meanwhile, V1507Lhet mice had blood counts similar to WT. In summary, V1512M compared to V1512L mutation resulted in a more severe phenotype in all analyzed model systems. This observation showed how different mutational permutations of the same amino acid can exert divergent phenotypic effects in SAMD9L BMF disorder. Therefore, together with our genetic analysis, we could postulate that the V1512L mutation arose as a failed rescue attempt of V1512M during embryogenesis. Citation Format: Sushree S Sahoo, Charnise Goodings, Shondra M. Pruett-Miller, Maria A Lillo, Lei Han, Baranda Hansen, Ti-Cheng Chang, Tim Lammens, Mattias Hofmans, Marta Derecka, Barbara De Moerloose, Marcin W Wlodarski. Divergent effects of MDS predisposing SAMD9L point mutations, in humans, mice, and cellular models [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A51.