Hypocellular Bone Marrow Research Articles

Whole Exome Sequencing of Adult Indians with Apparently Acquired Aplastic Anaemia: Initial Experience at Tertiary Care Hospital

Aplastic anaemia (AA) is a rare hypocellular bone marrow disease with a large number of mutations in the telomerase reverse transcriptase gene (TERT), leading to bone marrow failure. We used our benchmarked whole exome sequencing (WES) pipeline to identify variants in adult Indian subjects with apparently acquired AA. For 36 affected individuals, we sequenced coding regions to a mean coverage of 100× and a sufficient depth was achieved. Downstream validation and filtering to call mutations in patients treated with Cyclosporin A (CsA) identified variants associated with AA. We report four mutations across the genes associated with the AA, TERT and CYP3A5, in addition to other genes, viz., IFNG, PIGA, NBS/NBN, and MPL. We demonstrate the application of WES to discover the variants associated with CsA responders and non-responders in an Indian cohort.

Long-term survival after unrelated donor marrow transplantation for aplastic anaemia after optimized conditioning regimen: a retrospective multicentre cohort study

Almost all acquired severe aplastic anaemia is immune mediated and characterised by hypocellular bone marrow and ≥2 affected haematopoietic lineages. The optimal preparartive regimen for unrelated donor transplantation remains to be established. We aimed to study long-term outcomes after unrelated donor transplantation for severe aplastic anaemia with de-escalation of cyclophosphamide (Cy) dose in steps of 50mg/kg (150, 100, 50, 0mg/kg) in combination with total body irradiation (TBI) 2Gy, anti-thymocyte globulin (ATG) and fludarabine. Ninety-six patients with severe aplastic anaemia aged ≤65 years with adequate organ function enrolled on a trial of human leukocyte antigen (HLA)-matched or 1 HLA-locus mismatched unrelated donor marrow transplantation conducted between 02/2006 and 12/2013 in the United States (NCT00326417). Exclusion criteria were Karnofsky performance status of less than 60, clonal cytogenetic abnormalities and inherited marrow failure syndormes. The primary outcome was day-100 engraftment (achievement of absolute neutrophil recovery to at least 0.5×109/L without subsequent decline) and day-100 survival. The trial determined the lowest effective Cydose as 50mg/kg (n=38) for day-100 engraftment and survival. Cy dose 100mg/kg (n=41) was also acceptable. Accrual to Cy doses 150mg/kg (n=15) and 0mg/kg (n=3) was terminated early for toxicities. The current study is an extended follow up of patients enrolled on the trial (NCT00326477) and includes 76 of 96 patients alive ≥1 year after transplantation. There were 20 deaths in the first year after transplantation (Cy 0mg/kg [n=2], Cy 50mg/kg [n=1], Cy 100mg/kg [n=10], Cy 150mg/kg [n=7]). Patients were followed prospectively from transplantation and data reported using standardized data collection forms until death, loss to follow up or last contact through November 2023. The incidence of graft failure was calculated using the cumulative incidence estimator and the probability of survival using the Kaplan-Meier estimator. The median follow up of the cohort is 8.02 (IQR) 5.16-10.12) years. With Cy 50mg/kg, there was one graft failure and five deaths ≥1 year after transplantation. With Cy 100mg/kg there was only one late death and no graft failure. The 8-year probabilities of survival were 85.0% (95% CI 67.3-93.5) and 75.6% (95% CI 59.4-86.1) after Cy50mg/kg and 100mg/kg, respectively, P=0.31. With Cy 0mg/kg and 150mg/kg, there were no graft failures or death ≥1 year after transplantation. Regardless of Cy dose 12 of 15 patients aged ≥50 years died. Cy 50mg/kg or 100mg/kg with TBI 2Gy, ATG and fludarabine are effective conditioning regimens for unrelated donor marrow transplants for aplastic anaemia. Identification of an optimized transplantation approach for patients aged ≥50 years is needed. US National Institutes of Health.

Clinical Profile of Adults with Inherited Bone Marrow Failure Syndromes: Results of an Ambispective Clinical Single-Center Study

BACKGROUND. Inherited bone marrow failure syndromes (IBMFS) is a heterogenous group of rare genetically determined diseases with variable hematologic and nonhematologic manifestations. The implementation of highly specific methods of genetic diagnosis advanced the understanding of IBMFS and allowed its application also beyond pediatrics. That presupposes an awareness of clinical features and reference points for recognizing IBMFS in adults. AIM. To describe the clinical profile of adult IBMFS patients. MATERIALS & METHODS. This ambispective single-center study enrolled 35 patients (10 women and 25 men) with IBMFS. Patients were aged 18–51 years (median 26 years). The following IBMFS were identified: congenital dyskeratosis (n = 10; 28 %), Diamond-Blackfan anemia (n = 9; 26 %), Fanconi anemia (n = 7; 20 %), GATA2 deficiency (n = 3; 8 %), Shwachman-Diamond syndrome (n = 1; 3 %), GATA2 deficiency (n = 1; 3 %), amegakaryocytic thrombocytopenia (n = 1; 3 %), bone marrow failure syndrome type 3 (n = 1; 3 %), severe congenital neutropenia (n = 1; 3 %), bone marrow failure with SAMD9 mutation (n = 1; 3 %). These diseases were analyzed in terms of hematologic and nonhematologic manifestations as well as main diagnosis stages and factors that contribute to recognizing IBMFS. RESULTS. Monolinear cytopenia, bilinear cytopenia, and pancytopenia were identified at hematologic onset in 18 (52 %), 6 (17 %), and 11 (31 %) patients, respectively. The median age of patients by hematologic onset was 15 years (range 0–43 years), in 14 (40 %) patients cytopenia was newly diagnosed at the age of > 18 years. In 23 (63 %) patients hypocellular bone marrow was reported, 7 (20 %) and 5 (14 %) patients had pure red cell aplasia and multilineage myelodysplasia, respectively. Chromosomal aberrations were identified in 2 patients. Paroxysmal nocturnal hemoglobinuria clone was detected in none of 27 examined patients. In 12 (34 %) patients, the criteria for non-severe aplastic anemia were met. Temporary partial or complete spontaneous hematologic recovery was observed in 6 (17 %) patients. Abnormalities with partial or complete organ dysfunctions were identified in 14 patients, whereas all patients showed minor congenital defects. All 7 Fanconi anemia patients and 9 out of 10 congenital dyskeratosis patients demonstrated organ damage specific to these diseases. Family history predominantly showing malignant neoplasms in relatives was reported in 15 (43 %) patients. Initial hematological examination yielded suspect of IBMFS in 12 (34 %) patients with the median time to diagnosis of 6 months. In 23 (66 %) patients, hematologic defects with cytopenia were erroneously accounted for by various acquired diseases, which led to a delayed correct diagnosis (median 7 years). The key factors in suspecting IBMFS were organ abnormalities and positive family history. The IBMFS diagnosis was verified by the next-generation sequencing (NGS) in 29 (83 %) patients and by other specific methods in 4 (11 %) patients. In 2 patients, the diagnosis was established on the basis of complete clinical criteria alone. CONCLUSION. IBMFS is a matter of current concern and a difficult-to-recognize clinical challenge in adult hematology patients. Differential diagnosis of acquired and congenital bone marrow failure needs to be performed irrespective of patient’s age. A detailed physical examination of patients, family history, and critical analysis of clinical profile and disease course allow for early suspicion of IBMFS. Suspected IBMFS is an indication for referral of patients to specialized centers and performing genetic diagnostics including NGS.

Growth Charts for Shwachman-Diamond Syndrome at Ages 0 to 18 Years.

Shwachman-Diamond syndrome (SDS) is one of the most common inherited bone marrow failure syndromes. SDS is characterized by hypocellular bone marrow, with a severe impairment of the myeloid lineage, resulting in neutropenia, thrombocytopenia, and, more rarely, anemia. Almost 15% of patients with SDS develop myelodysplastic syndrome or acute myeloid leukemia as early as childhood or young adulthood. Exocrine pancreatic insufficiency is another common feature of SDS. Almost all patients with SDS show failure to thrive, which is associated with skeletal abnormalities due to defective ossification. Considering these observations, it remains unfeasible to use the common growth charts already available for the general population. To address this issue, we report how we drew up growth charts of patients with SDS aged 0 to 18 years. We analyzed height, weight, and body max index (BMI) in 121 Italian patients with SDS. Results indicated that the 50th and 3rd percentiles of weight and height of the pediatric general population correspond to the 97th and 50th percentiles of patients with SDS aged 0-18 years, respectively. In addition, the percentage increment in weight of subjects aged 14-18 years was higher in patients with SDS than in the general population. SDS-specific growth charts, such as those described here, afford a new tool, which is potentially useful for both clinical and research purposes in SDS.

Erratum to: Utility of clinical comprehensive genomic characterization for diagnostic categorization in patients presenting with hypocellular bone marrow failure syndromes.

Erratum to: Utility of clinical comprehensive genomic characterization for diagnostic categorization in patients presenting with hypocellular bone marrow failure syndromes.

Genetic ablation of Immt induces a lethal disruption of the MICOS complex.

The mitochondrial contact site and cristae organizing system (MICOS) is important for crista junction formation and for maintaining inner mitochondrial membrane architecture. A key component of the MICOS complex is MIC60, which has been well studied in yeast and cell culture models. However, only one recent study has demonstrated the embryonic lethality of losing Immt (the gene encoding MIC60) expression. Tamoxifen-inducible ROSA-CreERT2-mediated deletion of Immt in adult mice disrupted the MICOS complex, increased mitochondria size, altered cristae morphology, and was lethal within 12 d. Pathologically, these mice displayed defective intestinal muscle function (paralytic ileus) culminating in dehydration. We also identified bone marrow (BM) hypocellularity in Immt-deleted mice, although BM transplants from wild-type mice did not improve survival. Altogether, this inducible mouse model demonstrates the importance of MIC60 in vivo, in both hematopoietic and non-hematopoietic tissues, and provides a valuable resource for future mechanistic investigations into the MICOS complex.

Clinical Outcomes, Survival, and Predictors in Lower-Risk Myelodysplastic Syndrome Patients Treated with Cyclosporine A

Plain Language SummaryTherapeutic options to improve myelodysplastic syndrome (MDS)-related cytopenias in patients with lower-risk MDS are limited, and cyclosporin A (CSA) is an available option. We retrospectively assessed the effects of CSA on patients with lower-risk MDS at Shuguang Hospital affiliated with Shanghai University of Traditional Chinese Medicine. After statistical analysis, we found that more patients treated with CSA improved cytopenias than those treated without CSA. Patients with hypocellular marrow were more likely to get responses to CSA. Hypocellular marrow was defined as marrow cellularity of <30%. However, CSA did not delay disease progression and prolong survival span. The results of this study suggest that CSA is a safe treatment and can significantly improve cytopenias of a substantial proportion of patients with lower-risk MDS, especially in individuals with hypocellular bone marrow.

Virus-reactive T cells expanded in aplastic anemia eliminate hematopoietic progenitor cells by molecular mimicry

AbstractAcquired aplastic anemia is a bone marrow failure syndrome characterized by hypocellular bone marrow and peripheral blood pancytopenia. Frequent clinical responses to calcineurin inhibition and antithymocyte globulin strongly suggest critical roles for hematopoietic stem/progenitor cell–reactive T-cell clones in disease pathophysiology; however, their exact contribution and antigen specificities remain unclear. We determined differentiation states and targets of dominant T-cell clones along with their potential to eliminate hematopoietic progenitor cells in the bone marrow of 15 patients with acquired aplastic anemia. Single-cell sequencing and immunophenotyping revealed oligoclonal expansion and effector differentiation of CD8+ T-cell compartments. We reexpressed 28 dominant T-cell receptors (TCRs) of 9 patients in reporter cell lines to determine reactivity with (1) in vitro–expanded CD34+ bone marrow, (2) CD34− bone marrow, or (3) peptide pools covering immunodominant epitopes of highly prevalent viruses. Besides 5 cytomegalovirus-reactive TCRs, we identified 3 TCRs that recognized antigen presented on hematopoietic progenitor cells. T cells transduced with these TCRs eliminated hematopoietic progenitor cells of the respective patients in vitro. One progenitor cell–reactive TCR (11A5) also recognized an epitope of the Epstein-Barr virus–derived latent membrane protein 1 (LMP1) presented on HLA-A∗02:01. We identified 2 LMP1-related mimotopes within the human proteome as activating targets of TCR 11A5, providing proof of concept that molecular mimicry of viral and self-epitopes can drive T cell–mediated elimination of hematopoietic progenitor cells in aplastic anemia.

Guidelines for the diagnosis and management of adult aplastic anaemia: A British Society for Haematology Guideline.

Pancytopenia with hypocellular bone marrow is the hallmark of aplastic anaemia (AA) and the diagnosis is confirmed after careful evaluation, following exclusion of alternate diagnosis including hypoplastic myelodysplastic syndromes. Emerging use of molecular cyto-genomics is helpful in delineating immune mediated AA from inherited bone marrow failures (IBMF). Camitta criteria is used to assess disease severity, which along with age and availability of human leucocyte antigen compatible donor are determinants for therapeutic decisions. Supportive care with blood and platelet transfusion support, along with anti-microbial prophylaxis and prompt management of opportunistic infections remain key throughout the disease course. The standard first-line treatment for newly diagnosed acquired severe/very severe AA patients is horse anti-thymocyte globulin and ciclosporin-based immunosuppressive therapy (IST) with eltrombopag or allogeneic haemopoietic stem cell transplant (HSCT) from a matched sibling donor. Unrelated donor HSCT in adults should be considered after lack of response to IST, and up front for young adults with severe infections and a readily available matched unrelated donor. Management of IBMF, AA in pregnancy and in elderly require special attention. In view of the rarity of AA and complexity of management, appropriate discussion in multidisciplinary meetings and involvement of expert centres is strongly recommended to improve patient outcomes.

Effects of Cyclosporine and Azacitidine on Some Hematologic and ‎Biochemical Parameters of Benzene-Induced Aplastic Anemia in Rats

Aplastic anemia, marked by deficiencies in hematopoietic stem cells, leads to peripheral blood pancytopenia and hypocellular bone ‎marrow. This study aimed to evaluate the therapeutic efficacy of cyclosporine and azacitidine, administered either alone or in combination, in rats with benzene-induced aplastic ‎anemia, focusing on restoring normal blood cell levels and preventing disease complications. Thirty adult female Wistar rats ‎(Rattus ‎norvegicus)‎ were randomly divided into five groups: negative control (C-, untreated), positive control (C+, induced aplastic anemia with ‎distilled water), cyclosporine-treated (CsA, 5.86 mg/kg), azacitidine-treated (Aza, 5.75 mg/kg), and combination-treated (CsA+Aza, 3.68 ‎mg/kg each). Benzene (1940 mg/kg) was administered orally for fifteen days to induce aplastic anemia. Post a 30-day treatment period, ‎evaluations included differential WBC and reticulocyte counts, serum IL-2 levels, and alkaline phosphatase (ALP) activity. Results ‎showed significant improvements in WBC% and reticulocyte% in all treated groups compared to the C+ group, with the combination-‎treated group showing the highest enhancement. IL-2 levels in the combination group were significantly reduced compared to other ‎treatment groups, aligning closely with the negative control. The ALP activity was significantly higher in both the cyclosporine and ‎azacitidine-treated groups compared to the positive control, with the combination group showing a marked increase over the azacitidine ‎group but no significant difference from the cyclosporine group and negative control.‎ In conclusion, the study demonstrates the potential therapeutic benefits of cyclosporine and azacitidine in treating benzene-induced ‎aplastic anemia in rats. The combination therapy, in particular, showed improved efficacy in all tested parameters, suggesting a potential ‎strategy for dose reduction and toxicity mitigation.

Ghosal Hematodiaphyseal Dysplasia: Implications of Late Diagnosis and Unusual Presentation in Adults

Ghosal Hematodiaphyseal Dysplasia (GHDD) is a rare autosomal recessive disorder characterized by bone modeling failure, resulting in thickening of bone cortices with predominant diaphyseal involvement and responsive anemia to steroids. Late diagnosis of GHDD in adulthood can lead to significant morbidity and suboptimal response to steroids. Moreover, GHDD often mimics more common hematological disorders like myelodysplastic syndrome and myelofibrosis, leading to treatments such as transfusions, splenectomy, and hematopoietic stem cell transplantation. Early diagnosis is critical to enable the patient to receive the appropriate treatment. We present a case series of three Indian patients from different families with genetically proven GHDD. Despite experiencing symptoms from early childhood, all patients were diagnosed in adulthood. None of them were born out of a consanguineous marriage . Two of them, aged 43 and 56, are the oldest reported GHDD patients to date. They initiated steroid treatment in their 5th and 6th decades of life, respectively. Unlike previously reported cases, these two patients required additional drugs like Thalidomide along with steroids, and their response to treatment was not as rapid. The first patient, a 43-year-old male, had a history of anemia since childhood and became transfusion dependent after 15 years of age. He developed bicytopenia and later pancytopenia with hypocellular bone marrow , along with splenohepatomegaly and extramedullary hematopoiesis in the paravertebral and presacral areas, a novel finding in GHDD cases. Whole exome sequencing revealed a missense mutation in the TBXA2 gene. The second patient, a 56-year-old male, had a history of anemia since childhood and underwent splenectomy at 46 due to massive splenomegaly and transfusion dependency. However, his transfusion dependency persisted, and bone marrow biopsy suggested myelofibrosis. However he was negativefor myeloid mutations including JAK2. Subsequently, genetic testing revealed a homozygous missense mutation in the TBXAS1 gene. He also developed paraplegia and bladder incontinence, attributed to extramedullary hematopoiesis, which improved with steroid treatment The third patient a 23 year old man with history of anemia, lower limb tenderness and ecchymosis at 14 months of age. Evaluation then had shown hyperostosis of diaphysis of long bones of lower limbs with sparing of metaphysis and bicytopenia. Was on regular transfusions till 9 years. Re-evaluation with bone marrow studies at 9 years revealed a varying cellular marrow with dysplastic megakaryocytes and diagnosed as pediatric myelodysplatic syndrome ,He was on steroids till 13 years and he did not require further transfusions.He stopped steroids for 1 year following which there was a fall in Hb levels and recurrence of pain in both lower limbs with inability to squat. In view of history of steroid responsive anemia and thickening of diaphysis in X-rays GHDD was suspected and confirmed by genetic testing. He was restarted on steroids and there was a good response to steroids within 4 week with normalisation of hemoglobin levels . Late diagnosis of GHDD in adulthood can lead to severe complications and suboptimal treatment response. These cases highlight the importance of early diagnosis to prevent unnecessary interventions and improve patient outcomes. Unusual presentations, like extramedullary hematopoiesis, can further complicate the diagnostic process. Clinicians should maintain a high index of suspicion for GHDD in cases of refractory anemia or myelofibrosis-like features, especially in adults with an earlier history of anemia. Early recognition and appropriate management are crucial to mitigate morbidity and improve patients' quality of life

SMC4 Loss of Function Is a New Cause of Inherited Bone Marrow Failure

Inherited bone marrow failure syndromes (IBMFS) are a rare, heterogenous group of syndromes that are characterized by cytopenias, bone marrow hypocellularity, and in many cases an increased risk of transformation to myeloid malignancy. Presentation with severe pancytopenias at birth is reported but occurs very rarely, and more often neonatal pancytopenia is attributed to congenital and acquired infections or other acquired causes. We present the case of a preterm neonate with pancytopenia from birth and bone marrow failure of no known infectious, other acquired, or genetic etiology. Antenatal findings were notable for non-immune hydrops fetalis at 30 weeks of gestation prompting delivery. The initial laboratory evaluation revealed a normocytic, reticulocytopenic, severe anemia; leukopenia with an absolute neutrophil count of 500; and severe thrombocytopenia. Viral testing, including parvovirus, rubella, herpes simplex virus, cytomegalovirus, adenovirus, and enterovirus, was negative. Additional testing, including a chromosomal microarray, mitochondrial genome sequencing, chromosome stress testing, neonatal alloimmune thrombocytopenia panel, and telomere length analysis, was non-diagnostic. Bone marrow biopsy showed a remarkably hypoplastic marrow without evidence of dysplasia or cytogenetic abnormalities. The proband underwent matched unrelated donor hematopoietic stem cell transplant, and died approximately one month later from post-transplantation complications including hepatic veno-occlusive disease, thrombotic microangiopathy, CNS and pulmonary hemorrhages, pseudomonal sepsis, and multi-organ dysfunction. Autopsy was significant for microscopic pancreatic atrophy with lipomatous hypertrophy and osteopenia. The proband and parents underwent clinical exome testing which was non-diagnostic and then consented to research reanalysis of the exome data. Research reanalysis identified the infant was compound heterozygous for predicted deleterious variants in the Structural Maintenance of Chromosomes 4 gene (SMC4), inherited in trans from each unaffected parent (c.2478_2479ins10 splice site variant predicted to alter splicing and c.T591G; p.Y197X). Germline variants in SMC4 have not been reported as a cause of human disease; however, somatic variants are observed in several cancers including in acute myeloid leukemia (AML). SMC4 is a highly conserved protein that forms a heterodimer with Structural Maintenance of Chromosomes 2 (SMC2) in the condensin II protein complex. This complex is important for chromosome condensation, cell division, and DNA repair. Within hematopoietic tissue, SMC4 expression is higher in hematopoietic stem and progenitor cells when compared with mature lineage cells. Given the predicted loss of function from both SMC4 variants, we performed RNAseq on proband and control fibroblasts. This showed a roughly 10-fold decrease in total SMC4 transcript levels (Figure 1A). A significant proportion of the transcript present was comprised of an aberrantly spliced transcript characterized by exon 15 skipping. We therefore hypothesized that loss of SMC4 expression leads to a cell intrinsic defect in hematopoiesis. To test this hypothesis, we used CRISPR-Cas9 gene editing to generate knockout of SMC4 in human cord blood CD34+ hematopoietic stem/progenitor cells (HSPCs) before culturing cells in an all-colony forming unit (CFU) assay. Using two independent small guide RNAs, we were able to achieve approximately 80% knock-down. After 14 days in culture, we observed a striking decrease in CFUs when compared with controls, though the composition of colony types was relatively unchanged (Figure 1B). Using next-generation sequencing, we observed a significant decrease in remaining SMC4 genome edits at the end of the culture period, suggesting a relative disadvantage of SMC4-edited cells for survival and/or proliferation. In summary, we describe a preterm infant with congenital bone marrow failure without an identifiable cause and link this phenotype to biallelic loss-of-function variants in SMC4. This is the first study identifying SMC4 as a novel gene that causes inherited bone marrow failure, and further experiments are underway to validate this finding.

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

Outcomes of Hematopoietic Cell Transplantation for Patients with Refractory Cytopenia with Multilineage Dysplasia: A Comparison Children with Adolescents and Young Adults

Background: The 2008 World Health Organization (WHO) classification proposed a new entity in childhood myelodysplastic syndrome (MDS), refractory cytopenia of childhood (RCC). The spectrum of patients with RCC is wide, ranging from patients with severe hypocellular bone marrow (BM) and mild dysplasia (hypocellular RCC) to those with normocellular BM and distinct dysplasia meeting the criteria for refractory cytopenia with multilineage dysplasia (RCMD) defined for adults with MDS. The term of RCMD was revised to MDS with multilineage dysplasia in the 2017 WHO classification. In Japan, children who meet the criteria for RCMD have been diagnosed as RCMD distinguished from hypocellular RCC. Although hematopoietic cell transplantation (HCT) is a curative treatment for RCMD in children, there have been few reports of the outcomes of HCT for children with RCMD. Patients and Methods: To clarify the outcomes of HCT for children with RCMD, we retrospectively analyzed the clinical data in children (&amp;lt;16 years) with RCMD who underwent the first HCT from 2010 to 2021 and registered in the Japan Society for Hematopoietic Cell Transplantation Registry, and compared with those in adolescents and young adults (AYA; ≥16, &amp;lt;40 years) with RCMD who underwent the first HCT during the same study period. During the study period, 195 patients with RCMD (children, 69; AYA, 126) underwent HCT. The stem cell source was BM in 138 (children, 58; AYA, 80), peripheral blood (PB) in 27 (children, 2; AYA, 25), and cord blood (CB) in 30 patients (children, 9; AYA, 21). Myeloablative conditioning (MAC) regimen was defined as the use of total body irradiation (TBI) ≥8 Gy (MAC-TBI) and the administration of busulfan (BU) &amp;gt;8 mg/kg (MAC-BU). All other regimens were included in reduced intensity conditioning (RIC) regimen. Results: We retrospectively analyzed 195 patients with RCMD (male, 105; female, 90). The median age at HCT was 8 years (range, 0-15) in children and 29 years (range, 16-39) in AYA. Of the 195 patients, 20 (29%) and 52 (41%) had abnormal karyotypes in children and AYA, respectively. Of the patients who underwent HCT, 68 were from related (children, 22; AYA, 46), and 127 from unrelated (children, 47; AYA, 80) donors. MAC-BU was used for 46 (children, 9; AYA, 37), MAC-TBI for 51 (children, 1; AYA, 50), and RIC for 98 (children, 59; AYA, 39) patients. Of the 195 patients, 8 (children, 3; AYA, 5) developed primary graft failure, 16 (children, 5; AYA, 11) secondary graft failure, 10 (children, 3; AYA, 7) disease relapse, and 11 (children, 2; AYA, 9) secondary tumor. Twenty-four (children, 5; AYA, 19) patients died of HCT-related complications and four AYA of disease relapse. The 5-year overall survival (OS) rate was significantly higher in children (93%) than in AYA (82%) (p = 0.04). The 5-year event-free survival (EFS) rate was 76% in children and 64% in AYA. The 1-month cumulative incidence (CI) of neutrophil engraftment was 93% in children and 94% in AYA. The 5-year CI of relapse was 6% in AYA and 2% in children. The 5-year CI of treatment-related mortality (TRM) was 16% in AYA and 7% in children. The 5-year CI of graft failure was 12% in children and 12% in AYA. The 3-year CI of chronic graft-versus-host disease (GVHD) was significantly higher in AYA (37%) than in children (21%) (p = 0.04), while there was no difference in the 3-month CI of acute GVHD (gradeⅡ-Ⅳ) between children (35%) and AYA (39%). In children, the 5-year OS and EFS rates were significantly lower in CB (67% and 42%) than in BM/PB (97% and 81%) (p &amp;lt; 0.01 and p &amp;lt; 0.01), respectively, while the 5-year OS and EFS rates were not different between CB (91% and 76%) and BM/PB (80% and 62%) in AYA, respectively. Of the 59 children who underwent HCT using RIC, 43 without abnormal karyotypes showed 95% and 16 with abnormal karyotypes showed 88% in the 5-year OS rate. Even in AYA, there was no difference in the 5-year OS rate between RIC and MAC in both groups with (RIC, n = 14, 76%; MAC, n =38, 72%) and without (RIC, n = 25, 81%; MAC, n = 49, 90%) abnormal karyotypes. Conclusions: HCT for children with RCMD showed better OS rate than that for AYA with RCMD. HCT using RIC regimen provided good outcomes for RCMD in children with or without abnormal karyotypes. Even in AYA, HCT using RIC regimen showed OS rate comparable to MAC regimen in both groups with and without abnormal karyotypes.

Germline Variant in ZCCHC8 Is Associated with Pulmonary Fibrosis, Bone Marrow Failure, Liver Inflammation, Early Grey Hair and Short Telomer Length

Introduction Telomere biology disorders (TBD) encompass rare genetic conditions that affect telomere function. TBD is associated with a wide range of symptoms, including hematologic, dermatologic, pulmonary, hepatic manifestations. Research is continuously identifying novel variants and genes causative of TBD. In a recent study, Gable et al. (2019) detected a germline missense variant in ZCCHC8 (NM_017612.5:c.557C&amp;gt;T; p.(Pro186Leu)) in a single family with pulmonary fibrosis. This ZCCHC8 variant was associated with short telomere length (TL) and pulmonary fibrosis. To the best of our knowledge, no other families or individuals have been reported to have pathogenic variants in ZCCHC8 and TBD . However, in this abstract, we present data from a second family discovered to harbor a pathogenic variant in ZCCHC8 and to have symptoms of TBD. Methods Family members provided informed consent for genetic testing and underwent trio-based whole-genome sequencing (WGS). DNA isolated from fkin-derived fibroblasts was analyzed in the proband, while DNA from whole blood was used for the parents. To measure TL, we employed Computel (Nersisyan L. et al., 2015) and compared the mean TL to a cohort of patients who also underwent WGS to estimate a relative mean TL. Results: The proband was a 14-year-old male who had pancytopenia and elevated ALAT levels at presentation. A bone marrow biopsy showed a hypocellular bone marrow. A liver biopsy revealed mild centrilobular, pericellular, and portal fibrosis, along with mild inflammation in the portal space, these findings were interpreted as liver regeneration following acute hepatitis. Despite thorough investigations, no explanation for the findings was found. The proband had a history of psychogenic non-epileptic seizures. When the patient was around 20 years of age his hair started to gray. At present, the proband is 23 years old and has not experienced any progression of symptoms. Family history revealed that the proband's father and two second-degree relatives on the paternal side of the family had developed pulmonary fibrosis in the fifth and sixth decade of life as well as early grey hair. Thus, the inheritance pattern of disease related to TBD was autosomal dominant. WGS revealed a heterozygous pathogenic variant in ZCCHC8 (NM_017612.5c.557C&amp;gt;T (p.P186L)) present in the proband and the father which was absent in the mother. The variant was the same as reported by Gable et al. (2019). The variant is not present in gnomAD or COSMIC and it is the only reported pathogenic or likely pathogenic ZCCHC8 missense variant in ClinVar to date. Both the proband and the father exhibited short relative mean TL by Computel estimation. Subsequently, Flow-FISH analysis confirmed that the proband's telomere length was below the 1st percentile. Conclusion Here we present a family with a heterozygous germline variant in ZCCHC8 and TBD including pulmonary fibrosis, pancytopenia, early graying of the hair, and liver disease. Thus, we provide strong evidence of an association between TBD and ZCCHC8.

Bone Marrow Cellularity at Diagnosis Predicts Outcomes of Patients with Myelodysplastic Syndrome with Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only known curative therapy for myelodysplastic syndromes (MDS). The decision of allo-HSCT depends on prognostic scoring models such as revised international prognostic scoring system (IPSS-R) which include cytogenetic abnormalities, cytopenia, and % of bone marrow (BM) blasts. Recently, the prognostic role of genomic profile in MDS patients is arising. However, it is hard to conduct the next generation sequencing to all MDS patients in the real-world. Therefore, we aimed to evaluate the prognostic impact of BM cellularity at diagnosis as a new clinical prognostic factor of MDS with allo-HSCT. A total of 122 patients with MDS patients who underwent allo-HSCT between July 2009 to August 2022 in single center were analyzed retrospectively. Overall survival (OS) was analyzed using the Kaplan-Meier survival curves according to the BM cellularity at diagnosis. The potential prognostic parameters were evaluated for OS by uni-and multivariate cox-regression analysis. All patients were eligible for allo-HSCT and median age of 61.5 years (range: 19-71). Median follow-up duration was 36.9 (IQR 17.6-72.4) months. The 3-year and 5-year OS were 60.1% (95% CI 51.6-69.0) and 52.5% (95% CI 42.8-61.3), respectively. IPSS-R risk group was classified as very low (6, 4.9%), low (15, 12.2%), intermediate (40, 32.8%), high (37, 30.3%) and very high (24, 19.7%). Seventeen patients (13.9%) had complex karyotype at diagnosis. Seventy-seven patients (63.1%) were treated with hypomethylating agents before allo-HSCT. Most patients (70, 57.4%) had less than 5% BM blasts at allo-HSCT. The majority (74, 60.7%) received reduced intensity conditioning. OS significantly differed between patients with hypercellular BM and normo- to hypocellular BM at diagnosis by the log-rank test ( P &amp;lt;0.001) (Figure 1). In multivariate analysis for OS, complex karyotype at diagnosis ( P&amp;lt;0.001; HR 6.00; 95% CI 3.08-11.66), age ( P=0.04; HR 1.03; 95% CI 1.00-1.06) and hypercellular BM at diagnosis ( P=0.005; HR 2.16; 95% CI 1.26-3.72) were associated with worse outcome (Table 1). In this study, we demonstrated the BM hypercellularity at diagnosis independently predicts the inferior outcome of MDS patients with allo-HSCT. Although further validation with a larger cohort would be needed, it could be helpful to decide the treatment strategy of transplantation eligible MDS patients.

Granulocyte Transfusion Therapy as a Bridge to Hematopoietic Stem Cell Transplant in a Child with Aplastic Anemia and Refractory Perirectal Abscess

Granulocyte Transfusion Therapy as a Bridge to Hematopoietic Stem Cell Transplant in a Child with Aplastic Anemia and Refractory Perirectal Abscess

Irisin protected hemopoietic stem cells and improved outcome of severe bone marrow failure

Acquired aplastic anemia (AA) is a bone marrow failure (BMF) disease, characterized by fatty bone marrow (BM) and BM hypocellularity resulted from auto-immune dysregulated T cells-mediated destruction of BM haemopoietic stem cells (HPSC). The objective of this study was to investigate potential therapeutic effect of irisin, a molecule involved in adipose tissue transition, on AA mouse model. Our results showed that the concentration of irisin in serum was lower in AA patients than in healthy controls, suggesting a role of irisin in the pathogenesis of AA. In the AA mice, irisin administration prolonged the survival rate, prevented or attenuated peripheral pancytopenia, and preserved HPSC in the BM. Moreover, irisin also markedly reduced BM adipogenesis. In vitro results showed that irisin increased both cell proliferation and colony numbers of HPSC. Furthermore, our results demonstrated that irisin upregulated the expression of mitochondrial ATPase Inhibitory Factor 1 (IF1) in HPSC, inhibited the activation of mitochondrial fission protein (DRP1) and enhanced aerobic glycolysis. Taken together, our findings indicate novel roles of irisin in the pathogenesis of AA, and in the protection of HPSC through stimulation of proliferation and regulation of mitochondria function, which provides a proof-of-concept for the application of irisin in AA therapy.

Preliminary Results of Nathali-01: A First-in-Human Phase I/IIa Study of UCART20x22, a Dual Allogeneic CAR-T Cell Product Targeting CD20 and CD22, in Relapsed or Refractory (R/R) Non-Hodgkin Lymphoma (NHL)

Background: UCART20x22 is a dual targeted allogeneic CAR T-cell product candidate generated from healthy donor T-cells. Donor-derived T-cells are transduced with a bicistronic lentiviral vector to express CARs targeting CD20 and CD22. TALEN ® gene editing technology is used to inactivate the TRAC and CD52 genes to minimize graft-versus-host disease (GvHD) and allow for the use of alemtuzumab (CLLS52, an anti-CD52 monoclonal antibody) in the lymphodepletion (LD) regimen, respectively. UCART20x22 previously demonstrated robust and sustained activity in vitro and in vivo (Aranda-Orgilles B, et al. Cancer Immunol Res 2023). Here we present the initial preliminary results from the NatHaLi-01 trial (NCT05607420), the first-in-human Phase 1/2a dose-finding and expansion study evaluating UCART20x22 in R/R B-cell NHL. Methods: The primary endpoints are safety, tolerability, and determining the recommended phase 2 dose of UCART20x22. Additional endpoints include anti-lymphoma activity and cellular kinetics of UCART20x22. Key eligibility criteria are age 18-80y, lymphoma expression of CD20 and/or CD22 by local assessment, and ≥ 2 prior treatment regimens including autologous CD19 CAR T-cell therapy (CAR19), if eligible. After LD with FCA (fludarabine 30 mg/m 2 × 3d, cyclophosphamide 0.5g/m 2 × 3d, CLLS52 12 mg on D1, 24 mg on D2, D3), a single infusion of UCART20x22 is administered at a flat dose level ([DL1] 50 x 10 6 cells; [DL2] 150 x 10 6 cells; and [DL3] 450 x 10 6 cells). Results: As of 01 July 2023, 3 pts were enrolled and treated at DL1. Pt 1 is a 76yo female with double-expressor diffuse large B-cell lymphoma (DLBCL) relapsed after R-CHOP, radiation therapy, and polatuzumab vedotin with bendamustine/rituximab (BR) and was considered ineligible for CAR19 due to recent high dose bendamustine. Pt 2 is a 65yo female with R/R triple-hit lymphoma transformed from follicular lymphoma who was previously treated with radiation therapy, BR, dose-adjusted R-EPOCH, and two separate CAR19 treatments. Pt 3 is an 18yo female with R/R DLBCL transformed from marginal zone lymphoma who previously received chemoimmunotherapy, venetoclax, ibrutinib, BR, CAR19, obinutuzumab, glofitamab, tafasitamab, lenalidomide, and an experimental epigenetic modifier. Cytokine release syndrome (CRS) occurred in all patients. Pt 1 had G1 CRS and received tocilizumab (toci) x3 and dexamethasone (dex) x1. Pt 2 had G2 CRS and received toci and dex x1. Pt 3 had G1 CRS and received toci x1. All pts' CRS resolved with treatment. No immune effector cell associated neurotoxicity (ICANS) or GvHD was observed. There were no UCART20x22 dose limiting toxicities (DLTs), and there was 1 CLLS52 SAE and 1 DLT. The SAE was G3 CMV reactivation and was managed with IV antivirals. The DLT was in Pt 2, who entered the study with G1/2 cytopenias and a hypocellular bone marrow and had prolonged bone marrow aplasia after treatment thought to be due to cumulative chemotherapy exposure. This pt proceeded to allogeneic hematopoietic stem cell transplant (HSCT) from a matched donor while in complete remission from UCART20x22 treatment. Responses were assessed per Lugano criteria beginning on D28. At D28, all 3 pts responded with 2 CRs and 1 PR. Host lymphocytes remained suppressed through D28 for all pts. UCART20x22 expansion was observed by flow cytometry in 3/3 pts and was initially detected on D7 followed by peaks between D9-14. Peak expansion ranged from 16-682 cells/μL and was predominantly CD8 T-cells. Ferritin levels and increases in serum cytokines, including IFNγ, TNFα, IL-2, and IL-6, correlated closely with UCART20x22 expansion. Summary: Treatment with UCART20x22 at DL1 has been safe and well-tolerated, with no UCART20x22 DLTs or ICANS, and CRS was &amp;lt;G3 in all pts. There was 1 DLT of prolonged bone marrow aplasia in a pt with impaired hematopoietic reserve at study entry. UCART20x22 was active at DL1, with all 3 pts responding, including 2 CRs in pts who had previously received CAR19. UCART20x22 expansion was observed in all pts. These initial data support the continued study of UCART20x22 in R/R NHL, and updated results will be presented.

Neither Conditioning Chemotherapy Nor GvHD-Prophylaxis: CD7 CAR-T Treatment Bridging to Haplo-HSCT

BACKGROUND: Relapsed/refractory (R/R) acute leukemia patients ineligible for allo-HSCT have poor prognosis. CAR-T therapy has shown promising therapeutic outcomes for these patients, but simultaneously confers the risk of pancytopenia and potential tumor relapse. Bridging CAR-T therapy with allo-HSCT has the potential to restore hematopoiesis and consolidate long-term efficacy. However, GvHD remains a major impediment, and the myeloablative conditioning regimen eradicates residual CAR-T cells and compromises CAR-mediated antitumor function. Furthermore, the implementation of conditioning regimen and GvHD-prophylaxis could lead to severe toxicities. Novel strategies are therefore warranted to preserve CAR-T cells, enhance antitumor potency and minimize complications. METHODS: In this study, we presented 10 patients (enrolled between November 2021 and June 2023) with R/R CD7-positive acute leukemia who proceeded directly to haploidentical HSCT (haplo-HSCT) after CAR-T therapy. All patients received allogeneic CD7 CAR-T therapy, manufactured from either haploidentical donors (n=8) in a phase I clinical trial of donor-derived CAR-T cells (NCT04599556) or (n=1) universal CD7 CAR-T cells (RD13-02) therapy (NCT05716113) or as a compassionate use program after the end of a phase I clinical trial of universal CD7 CAR-T cells (RD13-01) (NCT04538599). CAR-T cell doses were at 2´10 6(haploidentical donor, n=8), 4´10 6 (RD13-02, n=1) or 5´10 6 (RD13-01, n=1) cells/kg. After achieving complete remission with incomplete hematological recovery (CRi), all patients underwent haplo-HSCT without conditioning regimen or GvHD prophylaxis. The primary end point was efficacy, safety, hematopoiesis recovery, and donor chimerism. RESULTS: All patients relapsed from multiple lines of chemotherapy or immunotherapy (median, 7; range, 4-13), were ineligible for allo-HSCT and received palliative care. After CAR-T infusion, 9 patients achieved MRD-negative CRi, and 1 achieved MRD-positive CRi, on day 15 after CD7 CAR-T therapy. 9 patients developed grade 1-2 cytokine release syndrome, and no neurological toxicity was reported. All patients exhibited grade 4 pancytopenia and bone marrow hypocellularity and were directly bridging to haplo-HSCT. The median time from CAR-T infusion to haplo-HSCT was 19 (range, 15-89) days. One patient died of HHV6 infection in central nervous system on day 13 after haplo-HSCT and was not evaluable for the following safety and efficacy. 3 patients experienced short-term grade II acute GvHD, which was resolved completely after low dose steroid, anti-TNF-α or ruxolitinib therapy. One patient developed PTLD at 2.1 months post-HSCT. In 9 evaluable patients, 8 showed full donor chimerism at 1-month post-HSCT, and 1 showed autologous hematopoiesis recovery. At a median follow-up of 10.2 (range, 2.2-20.7) months after CAR-T therapy, 6 patients remained in MRD-negative CR with normal hematopoiesis (including the patient with autologous hematopoietic recovery), 1 had CD7-negative relapse at 5.6 months, 1 developed CD7-negative relapse at 4.3 months and died at 4.8 months, and 1 died from septic shocks at 3.7 months. OS, PFS, NRM, and CIR at 1 year were 64.3% (95% CI, 38.5%-100%), 51.4% (95% CI, 26.2%-100%), 22.9% and 25.7%, respectively. Peripheral normal T cells in 8/10 of patients remained CD7-negative at the last follow-up, illustrating a well-maintained CAR-T function post-HSCT. CONCLUSIONS: Our findings demonstrate a novel strategy to simultaneously achieve CAR-T persistence and complete HSC engraftment, while reducing the risks of GvHD, providing valuable insights into combining cellular therapy with allo-HSCT for the treatment of R/R acute leukemia patients ineligible for conventional allo-HSCT.