Bone Marrow Failure Disorders Research Articles

A Thrilling Case of Anemia

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare bone marrow failure disorder that presents with triad of peripheral blood cytopenias, hemolytic anemia and thrombosis. The absence of two glycosylphosphatidylinositol (GPI)-anchored proteins, CD55 and CD59, leads to uncontrolled complement activation that accounts for hemolysis and other PNH manifestations. GPI anchor protein deficiency is due to acquired somatic mutations in phosphatidylinositol glycan class A (PIGA) gene.Terminal complement inhibition with eculizumab and allogeneic bone marrow transplantation (BMT) are the only widely effective therapies for patients with classical PNH. Compared to eculizumab and ravulizumab that block the fifth component of complement (C5), pegcetacoplan blocks the third component of complement (C3).

Non-syndromic congenital sideroblastic anaemia; phenotype, and genotype of 15 Indian patients.

Sideroblastic anaemias are a diverse group of congenital and acquired bone marrow failure disorders marked by the presence of ring sideroblasts, ineffective erythropoiesis, and systemic iron overload. Congenital Sideroblastic anaemia (CSA) is mainly caused by gene mutations associated with heme synthesis, iron-sulfur [Fe-S] cluster, and mitochondrial protein synthesis pathways. The most prevalent form of CSA is caused by mutations in the erythroid-specific -amino levulinate synthase (ALAS2) gene, which encodes the first enzyme in the heme synthesis pathway in red blood cells. The second most prevalent form of CSA is caused by a mutation in the Solute carrier family 25 member 38 (SLC25A38) gene, which codes for an erythroid-specific protein of the inner mitochondrial membrane. Additionally, 15-20 genes are altogether associated with CSA. In this study, we aim to identify the CSA patients, understand their genetics and establish genotype-phenotype correlation. We have identified fifteen cases of CSA using our targeted NGS (t-NGS) panel. The major clinical findings in our cohort were microcytic anaemia, ring sideroblasts, and dyserythropoiesis in the bone marrow. Currently, two patients are responsive to pyridoxine, while the rest are on blood transfusion support. We have identified ten variants in three different genes of CSA (ALAS2, SLC25A38 & HSPA9). Five patients harbour four hemizygous variants- p.Ala282Ser, p.Arg170Cys, p.Arg204Gln and exon 2 duplication in the ALAS2 gene. In seven patients, we have identified three homozygous mutations - p.Pro190Arg, p.Arg187Gln and p.Arg134Cys in the SLC25A38 gene. These mutations have been predominantly identified in the European population. Three patients revealed three heterozygous variants p. Thr463Ile, D326Tyr, and Arg284Trp in the HSPA9 gene. PyMoL was used to evaluate the functional effects of these variations and understand their effect on the structure of the protein. We believe that by combining a bone marrow examination with genetic sequencing, CSA patients can acquire a definitive diagnosis.

Telomere length and clonal chromosomal alterations in peripheral blood of patients with severe aplastic anaemia.

Severe aplastic anaemia (SAA) is a rare and life-threatening bone marrow failure disorder. We used data from the transplant outcomes in aplastic anaemia study to characterize mosaic chromosomal alterations (mCAs) in the peripheral blood of 738 patients with acquired SAA and evaluate their associations with telomere length (TL) and survival post-haematopoietic cell transplant (HCT). The median age at HCT was 20.4 years (range = 0.2-77.4). Patients with SAA had shorter TL than expected for their age (median TL percentile for age: 35.7th; range <1-99.99). mCAs were detected in 211 patients (28.6%), with chr6p copy-neutral loss of heterozygosity (6p-CNLOH) in 15.9% and chr7 loss in 3.0% of the patients; chrX loss was detected in 4.1% of female patients. Negative correlations between mCA cell fraction and measured TL (r = -0.14, p = 0.0002), and possibly genetically predicted TL (r = -0.07, p = 0.06) were noted. The post-HCT 3-year survival probability was low in patients with chr7 loss (39% vs. 72% in patients with chr6-CNLOH, 60% in patients with other mCAs and 70% in patients with no mCAs; p-log rank = 0.001). In multivariable analysis, short TL (p = 0.01), but not chr7 loss (p = 0.29), was associated with worse post-HCT survival. TL may guide clinical decisions in patients with SAA.

Analyzing the role of ferroptosis in ribosome-related bone marrow failure disorders: From pathophysiology to potential pharmacological exploitation.

Within the last decade, the scientific community has witnessed the importance of ferroptosis as a novel cascade of molecular events leading to cellular decisions of death distinct from apoptosis and other known forms of cell death. Notably, such non- apoptotic and iron-dependent regulated cell death has been found to be intricately linked to several physiological processes as well as to the pathogenesis of various diseases. To this end, recent data support the notion that a potential molecular connection between ferroptosis and inherited bone marrow failure (IBMF) in individuals with ribosomopathies may exist. In this review, we suggest that in ribosome-related IBMFs the identified mutations in ribosomal proteins lead to changes in the ribosome composition of the hematopoietic progenitors, changes that seem to affect ribosomal function, thus enhancing the expression of some mRNAs subgroups while reducing the expression of others. These events lead to an imbalance inside the cell as some molecular pathways are promoted while others are inhibited. This disturbance is accompanied by ROS production and lipid peroxidation, while an additional finding in most of them is iron accumulation. Once lipid peroxidation and iron accumulation are the two main characteristics of ferroptosis, it is possible that this mechanism plays a key role in the manifestation of IBMF in this type of disease. If this molecular mechanism is further confirmed, new pharmacological targets such as ferroptosis inhibitors that are already exploited for the treatment of other diseases, could be utilized to improve the treatment of ribosomopathies.

Dyskeratosis congenita with squamous cell carcinoma of the tongue: A rare case report.

Dyskeratosis congenita is a rare inherited bone marrow failure disorder characterized by a classic triad: nail dystrophy, reticulate skin pigmentation, and oral leukoplakia. Patients with dyskeratosis congenita have also been described as having gastrointestinal, genitourinary, neurological, ophthalmic, pulmonary, and skeletal abnormalities. Also, 80% of patients are affected with oral leukoplakia and these areas are more prone to develop into oral squamous cell carcinoma. We hereby report a rare case of oral squamous cell carcinoma of the tongue in a young male patient with dyskeratosis congenita.

Aberrant early hematopoietic progenitor formation marks the onset of hematopoietic defects in Shwachman-Diamond syndrome.

Shwachman-Diamond syndrome (SDS) is an inherited bone marrow failure disorder that often presents at infancy. Progress has been made in revealing causal mutated genes (SBDS and others), ribosome defects, and hematopoietic aberrations in SDS. However, the mechanism underlying the hematopoietic failure remained unknown, and treatment options are limited. Herein, we investigated the onset of SDS embryonic hematopoietic impairments. We generated SDS and control human-derived induced pluripotent stem cells (iPSCs). SDS iPSCs recapitulated the SDS hematological phenotype. Detailed stepwise evaluation of definitive hematopoiesis revealed defects that started at the early emerging hematopoietic progenitor (EHP) stage after mesoderm and hemogenicendothelium were normally induced. Hematopoietic potential of EHPs was markedly reduced, and the introduction of SBDS in SDS iPSCs improved colony formation. Transcriptome analysis revealed reduced expression of ribosome and oxidative phosphorylation-related genes in undifferentiated and differentiated iPSCs. However, certain pathways (e.g., DNA replication) and genes (e.g., CHCHD2) were exclusively or more severely dysregulated in EHPs compared with earlier and later stages. To our knowledge, this study offers for the first time an insight into the embryonic onset of human hematopoietic defects in an inherited bone marrow failure syndrome and reveals cellular and molecular aberrations at critical stages of hematopoietic development toward EHPs.

Myelodysplastic Syndrome and Hemoglobin Improved by Intravenous Nutrient Therapy: A Case Report

Myelodysplastic syndromes (MDSs) are variations of bone marrow failure disorders that lead to insufficient production of healthy blood cells culminating in some combination of low red blood cells, low white cells, or both. This case report presents a case of increased hemoglobin levels following intravenous (IV) nutrient therapy. J.B. is a 74-year-old male who presented with a chief concern of MDS. He was experiencing extreme fatigue and exhaustion, episodes of shortness of breath and a decline in hemoglobin levels. He was being followed by a hematologist who monitored his complete blood count (CBC) at regular intervals, with primary focus on blood hemoglobin. His hemoglobin levels were significantly lower than the normal range, but never met the threshold for a blood transfusion (&lt; 75 g/dL), his only treatment option. Beginning in November 2021, weekly to biweekly IV nutrient therapy treatments were initiated. In all, 39 IV treatments were given until August 2023. Intravenous nutrient formulas included Myer’s Cocktail, high-dose vitamin C (25 g), Hydrating Myer’s Cocktail and Hydrating Myer’s Cocktail + glutathione. As IV treatments were administered, a trend was observed showing a consistent improvement of hemoglobin levels over time, with the greatest degree of improvement observed after the series of Hydrating Myer’s Cocktails. The patient also reported decreased overall fatigue and reduced recovery time from activity. Delivering a series of IV infusions may have resulted in an improvement of hemoglobin levels in this patient. Further investigation is warranted to understand the potential effects IV nutrient therapy could have on MDS patients with a similar prognosis.

Absence of Neutropenia in Patients With Early Exon Nonsense Mutations in ELANE : Clinical Evidence to Support Gene Therapy Approaches for Severe Congenital Neutropenia.

Severe congenital neutropenia is an inherited bone marrow failure disorder characterized by profoundly low neutrophil counts and promyelocytic maturation arrest in bone marrow. Severe congenital neutropenia is most often caused by heterozygous ELANE mutations. In vitro and mouse xenograft studies using CRISPR/Cas9 have shown that introduction of frameshift/nonsense mutations in mutant ELANE may restore neutrophil counts, providing a model for gene therapy. Here, we present 2 children with inherited nonsense mutations in ELANE analogous to those proposed for gene therapy. Their normal peripheral blood neutrophil counts provide support for this approach through human "experiments of nature."

Generation of iPSC lines and isogenic gene-corrected lines from two individuals with RPS19-mutated Diamond-Blackfan anemia syndrome

Diamond-Blackfan anemia syndrome (DBAS) is an inherited bone marrow failure disorder that typically presents in infancy as hypoplastic anemia and developmental abnormalities in approximately 50% of cases. DBAS is caused by haploinsufficiency in one of 24 ribosomal protein genes, with RPS19 mutations accounting for 25% of cases. We generated iPSC lines from two patients with different heterozygous RPS19 mutations (c.191T > C and c.184C > T) and isogenic lines in which the mutations were corrected by Cas9-mediated homology directed repair.

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 &amp;gt;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 &amp;lt;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.

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 &amp;lt; 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&amp;lt;-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&amp;gt;C and c.183-184TA&amp;gt;CT SBDS mutations. Eleven patients (17.5%) are homozygous for the c.258+2T&amp;gt;C mutation. Four patients (6.3%) were compound heterozygous for c.258+2T&amp;gt;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&amp;gt;C with c. 183-184 TA &amp;gt; 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&amp;lt;1000 cells/ul (79%), platelets &amp;lt;100K cells/ul (43%), and anemia for age (47%). In contrast, bone marrow hypocellularity (&amp;lt;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.

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&amp;lt;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&amp;lt;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 &amp;lt; 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.