Donor Bone Marrow Transplantation Research Articles

Reduced toxicity matched sibling bone marrow transplant results in excellent outcomes for severe congenital neutropenia.

Severe congenital neutropenia (SCN) is caused by germline mutations, most commonly in ELANE, impacting neutrophil maturation and leading to high risk of life-threatening infections. Most patients with ELANE-mutant SCN can achieve safe neutrophil counts with chronic Granulocyte-Colony Stimulating Factor (G-CSF). However, up to 10% of patients have neutropenia refractory to G-CSF and require allogeneic stem cell transplant. Traditional conditioning for these patients includes busulfan and cyclophosphamide which is associated with significant toxicities. We present five patients with SCN without myeloid malignancy transplanted using a reduced toxicity regimen of busulfan, fludarabine and thymoglobulin. 5 pediatric patients with SCN underwent matched sibling donor bone marrow transplant (MSD-BMT) between 2014-2022 on or per CHP14BT057 (NCT02928991), a prospective, single center trial testing elimination of cyclophosphamide from conditioning in pediatric patients with single lineage inherited BMF syndromes. All patients had MSDs and no evidence of MDS. Conditioning consisted of PK-adjusted busulfan, fludarabine, and thymoglobulin, with calcineurin inhibitor and mycophenolate mofetil GVHD prophylaxis. With median follow-up of 48.4 months, overall and event-free survival were 100%. There was no acute GVHD and one instance of chronic limited GVHD. Patients exhibited >95% donor myeloid chimerism at 5 years post-BMT. Two patients experienced CMV reactivation without end-organ disease, and no other viral reactivation or significant infections occurred. MSD-BMT with reduced toxicity myeloablation for SCN provides excellent outcomes while minimizing toxicity. These data suggest that busulfan, fludarabine, and ATG can be considered an efficacious, low-toxicity standard of care regimen for patients with SCN undergoing MSD-BMT.

Excellent Outcomes Using a Non-Myeloablative Conditioning with Thiotepa and Post -Transplant Cyclophosphamide in Adults Undergoing a Matched Related Donor Bone Marrow Transplant for Severe Sickle Cell Disease

Excellent Outcomes Using a Non-Myeloablative Conditioning with Thiotepa and Post -Transplant Cyclophosphamide in Adults Undergoing a Matched Related Donor Bone Marrow Transplant for Severe Sickle Cell Disease

Prevalence and Outcome of Vexas Syndrome in Unrelated Hematopoietic Stem Cell Transplantation

Introduction Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic (VEXAS) syndrome is a newly identified monogenic disorder defined by somatic UBA1 mutations in hematopoietic progenitor cells, resulting in bone marrow failure (BMF) and systemic inflammation. This syndrome is frequently associated with myelodysplastic syndrome (MDS) or plasma cell dyscrasia. Although high-dose corticosteroids alleviate symptoms and cytopenia, allogeneic hematopoietic stem cell transplantation (HSCT) has been suggested as a potentially curative treatment. This study evaluated the safety and efficacy of unrelated HSCT for treating VEXAS syndrome and its prevalence in patients with hematologic diseases. Methods We conducted retrospective screening for UBA1 mutations in patients with BMF or plasma cell neoplasms who underwent their first HSCT from unrelated donors in Japan between 1995 and 2020. Pre-HSCT blood DNA samples and clinical data were collected from the Japanese Data Center for Hematopoietic Cell Transplantation. Multiplex real-time PCR was used to detect the most common pathogenic UBA1 mutations, including p.M41L (c.121A>C), p.M41V (c.121A>G), and p.M41T (c.122T>C), with detection limits of 0.13%, 0.5%, and 1%, respectively. The presence of these mutations was confirmed using Sanger sequencing and droplet digital PCR. In addition, we examined UBA1 mutations in 2 patients previously diagnosed with MDS but suspected of having VEXAS syndrome based on specific clinical features. Results Among 4340 patients with either BMF or plasma cell neoplasms who underwent unrelated HSCT, 1771 had available pre-HSCT blood DNA samples. The diagnoses of the 1771 patients included MDS (n = 1264), acquired aplastic anemia (n = 375), inherited BMF syndromes (n = 89), plasma cell neoplasms (n = 23), and other acquired BMF syndromes (n = 20). The median patient age at HSCT was 46 (range, 0-75; interquartile range, 20-59) years old, and the male-to-female ratio was 1.6:1. Pathogenic UBA1 mutations were detected in 2 of the 1771 patients (0.11%), both of whom were male MDS patients older than the median age (cases 1 and 2); thus, the prevalence of VEXAS syndrome within this patient subgroup was 0.36%. Both patients received chemotherapy before unrelated donor bone marrow transplantation (BMT), including induction chemotherapy and azacitidine, and achieved complete and partial responses, respectively. In addition, UBA1 mutations were detected in the 2 MDS patients at Kanazawa University Hospital (cases 3 and 4). Case 3 also underwent unrelated BMT after experiencing cytopenia relapse and inflammatory symptoms during corticosteroid reduction. The clinical characteristics and outcomes of the 4 patients with VEXAS syndrome are summarized in Table 1. Variant allele frequencies (VAFs) of UBA1 mutations ranged from 2% to 71%. The 3 patients who underwent unrelated BMT received fludarabine and busulfan-based conditioning regimens and graft-versus-graft disease (GVHD) prophylaxis with tacrolimus + methotrexate or cyclosporine + methotrexate. All 3 patients achieved neutrophil and platelet engraftment. One patient required systemic corticosteroids for acute skin GVHD. One patient died of idiopathic interstitial pneumonia 5 months after BMT. The other 2 patients were alive without relapse, chronic GVHD, or other complications at 18 and 28 months after BMT. Discussion Our results support the curative potential of allogeneic HSCT for VEXAS syndrome without excessive toxicity. The occurrence of transplant-related mortality due to idiopathic interstitial pneumonia may be related to the reported poor outcome of the p.M41V mutation following non-transplant therapy. The low prevalence of VEXAS syndrome in the current cohort may be due to the late age of onset and the rare progression to advanced MDS. The low VAFs of UBA1 mutations in the 2 patients who responded to chemotherapy before HSCT may indicate the efficacy of chemotherapy in selectively eradicating UBA1 mutant clones. Our rapid, sensitive, and cost-effective screening method for VEXAS syndrome can help optimize treatment strategies for this rare disease in future studies.

Clinical Implications of Germline Predisposition Gene Variants in Patients with Refractory or Relapsed B Acute Lymphoblastic Leukemia

Objectives:Gene variants are important factors in prognosis of the patients with hematological malignancies. In current study, our team investigate the relationship between blood and immune system germline predisposition variants and refractory/relapsed acute B lymphoblastic leukemia in patients. Methods:The study included 75 patients with refractory/recurrent acute B-lymphoblastic leukemia between January 2020 and December 2022. The first group of 41 patients, median age 13.5 years (range: 2-66 years), had not had a bone marrow transplant. The second group of 34 patients with a median age of 28.5 (range: 4-51 years old, once had a bone marrow transplant. More than 700 genetic susceptibility genes related to blood and immune system diseases were detected in all patient samples using high-throughput DNA sequencing technology to detect the type and number of Class I and Class II gene variants. The average sequencing depth was 150×. Class I refers to the gene variants that are clearly significant in guidelines or authoritative research literature. Class II are genetic variants that software analysis suggests may be harmful. Results:In the first group, 41 patients had an average number of Class I genetic susceptibility gene variants of 3.8 and Class II genetic susceptibility gene variants of 14.5 per patient. A total of 94 Class I genetic susceptibility gene variants were found, Thirty-two of them were recurrent more than 3 times that including HIF1A、SERPINE1、CFTR、CHEK2、ELANE、IFIH1、IRF7、JAK3、NCF2、PRF1、TGFBR2、TNFRSF13B、ATM、CYP3A4、ITGA2、NUDT15、THBD、BTLA、DNASE1、MPEG1、ADAMTS13、EP300、FANCA、KIT、BRCA2、F7、MLH1、STK11、TP53、YARS2、CYP2C19、TNFAIP3. The functions of these genes involve in combined immunodeficiency, autoinflammatory disease, complement deficiency, immune deficiency, T-cell dysfunction and antibody deficiency. After remission by CART cell therapy, twenty-three of the 41 patients underwent haploidentical hematopoietic stem cell transplantation, among which 5 patients relapsed after transplantation, three of the five donors of relapsed patients were also found HIF1A and SERPINE1 gene variants. In the second group of 34 patients, the average number of Class I genetic susceptibility gene variants in each patient was 3, and the average number of class 2 genetic susceptibility gene variants was 14. A total of 53 class 1 genetic susceptibility gene variants were found, Eleven of them were recurrent more than 3 times that Including BTLA、MPEG1 、KIT、SERPINE1、STK11、TP53、UGT1A1、EP300、FANCA、YARS2、F7,and four donors also carried the above gene variants, which were EP300. MPEG1, SERPINE1, YARS2 . After 34 patients achieved remission by CART cell therapy, 9 patients underwent the second hematopoietic stem cell transplantation (related half-matched), and 3 of them relapsed after the second transplantation (average 2.3 months). Two of the 3 donors of relapsed patients after second hematopoietic stem cell transplantation were also found BTLA and MPEG1 gene variants. Conclusions:Our results have shown that genetic susceptibility gene variants related to blood and immune system diseases are associated with refractory or relapsed acute B lymphoblastic leukemia. The number of genetic susceptibility gene variants may be related to the relapse of leukemia. 32 Class I genetic susceptibility gene variants may be related to the relapse of leukemia during chemotherapy, and 11 class I genetic susceptibility gene variants may be associated with relapse after bone marrow transplantation. Donor carrying genetic susceptibility gene variants (HIF1A and SERPINE1, EP300, MPEG1, YARS2, BTLA) may be associated with relapse after transplantation. Genetic susceptibility gene variants may also be helpful in the selection of bone marrow transplant donors. Keywords Acute lymphoblastic leukemia, germline predisposition gene, hematopoietic stem cell transplant, , relapse, hematological malignancy

ERG Is a New Predisposition Gene for Bone Marrow Failure and Hematological Malignancy

There remain gaps in our knowledge of hereditary and sporadic causes of hematological malignancy (HM) and bone marrow failure (BMF) that prevent optimal diagnosis, disease surveillance and treatment. Here we report the discovery of ERG as a novel predisposition gene for BMF and HM. ERG is a known oncogene, typically via gene-fusions, leading to dysregulated ERG overexpression in blood and solid cancers. We identified a germline ERG ETS domain variant p.Y373C segregating with thrombocytopenia in a mother, who progressed to AML (27 yr) and then therapy-related MDS (35 yr), and in her 2 sons. All three showed copy neutral loss of heterozygosity of all or part of chromosome 21q, including the ERG locus, with the oldest son showing at least 2 somatic genetic rescue (SGR) events. The possibility of causal RUNX1 variants were ruled out, with the smallest somatic cnLOH event beginning within the RUNX1 gene, but not encompassing the RUNT domain where the majority of pathogenic missense variants are located. ERG, a highly constrained gene (LOEUF <0.33), is critical for definitive hematopoiesis, adult hematopoietic stem cell (HSC) function and platelet maintenance. An identical corresponding heterozygous germline variant (p.Y343C) in ERG’s closest gene by homology, FLI1, causes platelet-type bleeding disorder-21 (BDPLT21, OMIM #617443). Through global collaborations, we have identified 15 heterozygous variants in the ERG gene, 13 of which are missense and 2 truncating variants, in 17 individuals with cytopenia and/or HM (mainly myeloid) or lymphedema (Table). Onset of hematological symptoms ranged from birth to 38 years for truncating and constrained ETS domain variants. Of these 15 variants, 12 have been confirmed germline including 2 de novo. Only 4 meiotic transmissions are observed. None of the missense variants in the highly conserved ETS domain of ERG which mediates DNA binding, protein-protein interactions and nuclear localization, are present in gnomAD. We have functionally characterized 19 ERG variants, 12 potentially pathogenic, 1 known mouse pathogenic variant and 3 population controls demonstrating that most ETS domain missense variants display loss-of-function (LOF) characteristics disrupting transcriptional transactivation (Figure), DNA-binding and/or nuclear localization in vitro. Robust preliminary data from ex vivo models of ERG overexpression in mouse fetal liver cells in tissue culture (cytokine-independence), a mouse transplant assay and previous germline mutant Erg mouse models are concordant with ETS domain missense variants being LOF compared to wildtype ERG and benign controls. Together, these data provide clinical, in vitro and ex vivo functional studies implicating LOF variants in hematological disease predisposition. LOF ERG mutations also occur in sporadic cases of HM. Recently, as part of a Genomics England Research Consortium population study, 4 truncating ERG variants were described in 7 individuals across 4 families with 3 meiotic transmissions and a de novo case with primary lymphedema (1) and we add 2 novel missense variants here. One patient showed SGR across the ERG locus in blood. Blood phenotypes were not described. Our results demonstrate that germline ERG variants predispose to diverse cytopenia, BMF and HM in both children and adults. In our family mentioned above, the mother received an unrelated alloHSCT due to t-MDS while her 2 sons with cytopenias continue to be monitored. The natural history of this new syndrome will require careful identification of germline lesions with additional longitudinal studies in more patients and families needed. This ERG syndrome parallels GATA2 deficiency syndrome (HM and lymphedema) and RUNX1 Familial Platelet disorder-myeloid malignancy (thrombocytopenia and HM). Like the well-known disease genes GATA2 and RUNX1, ERG is also a member of the transcription factor heptad involved in HSC maintenance and differentiation. ERG adds to a growing list of genes whose unregulated expression contributes to HM and other cancers. Identification of causal germline ERG variants like those outlined in this study, has direct clinical implications for patient and family management including diagnosis, counselling, surveillance and treatment strategies such as selection of bone marrow transplant donors and potential for targeted therapies including gene and cell therapy. Reference 1. Greene D et al. Nat.Med. 29:679-688 2023

TMI-Based Dose-Escalated Bone Marrow Transplantation Can Help Preserve the Bone Marrow Microenvironment and Reduce Cellular Senescence in Old Mice

Introduction:Senescence is one of the hallmarks of aging and age-related conditions, including but not limited to, inflammaging, frailty, and various age-related diseases. It has been well-established that radiation and chemotherapy can accelerate senescence. While myeloablative conditioning-based allogeneic hematopoietic cell transplant (HCT) is a curative treatment for patients with leukemia. The dose of total body irradiation (TBI) conditioning has to be reduced in older patients to reduce potential high treatment-related mortality, which may still cause accelerated senescence in aged individuals, resulting in poor survival outcomes. We initiated the testing of a novel total marrow irradiation (TMI) based myeloablative conditioning in older patients to enhance the anti-leukemia effect and overall survival. We hypothesize that this new radiation preconditioning strategy may also reduce radiation-induced senescence in aged individuals compared to TBI to reduce treatment-associated side effects and improve the outcomes of allogeneic HCT. We tested this hypothesis in the present study and further investigated the mechanisms of the effects of TMI-based myeloablative and dose-escalated bone marrow transplantation (BMT) on old mice by examining the changes in expression of various senescence markers and senescence-associated secretory phenotype (SASP) in the bone marrow (BM) and selected organs. Method: The radiation treatment regimens were: TMI (12:4), TMI (16:4) and TBI (12:12), and TBI (16:16), were developed as described (Zuro et al., 2021); i.e. (12:4) means the radiation dose to the BM was 12 Gy and the dose to the organs was 4 Gy. The young (8 weeks old) and aged mice (90 weeks old) of host C57BL/6j (JAX000664, CD45.2) were irradiated in 2 fractions 24h apart, and 25 million whole BM cells from donor (JAX002014, CD45.1) were transplanted by i.v. at 24 h after irradiation. For the hematopoietic stem and progenitor cell (HSPC) population study, more mature hematopoietic progenitors (Lin − Sca-1 −c-Kit + or LK cells) and HSPCs (Lin − Sca-1 + c-Kit + or LSK cells) were examined in BM by flow analysis. Cultured bone marrow mesenchymal stem cells (MSCs) were analyzed for the gene expression of the senescence marker CDNK1a (or p21) and SASP factors MMP13 and CXCL12 by qPCR. For senescence study in organs such as the liver, gut, and spleen, senescence-associated beta-galactosidase (SA-β-gal) staining was performed and expression of the CDNK2a (or p16) was analyzed by qPCR. Results: TMI or TBI treatment with young donor BMT repopulated the BM HSPC population without significant bone damage in aged mice (data not shown). While the BM LK and LSK populations significantly reduced in aged mice after TMI or TBI with young donor BMT compared to aged control mice ( Fig 1A), these populations were not different from young mice groups ( Fig 1B). In addition, TMI or TBI treatment with young donor BMT reduced MSC senescence. Cultured MSCs analysis showed the reduction of p21, MMP13, and CXCL12 expression ( Fig 1C-E) after TMI or TBI with young donor BMT in aged compared to aged control without radiation and BMT. However, while TMI treatment and BMT reduced senescence in the organs, mice receiving TBI treatment and BMT showed a higher senescence burden. In the liver, the aged control mice showed a significant increase in SA-β-gal staining, which was decreased in TMI (12:4) and TMI (16:4) mice ( Fig 1F). In contrast, this effect was not observed in aged mice after TBI (12:12) or (16:16) ( Fig 1G). Moreover, the senescence marker p16 expression in the liver and gut was reduced only in TMI (12:4) and TMI (16:4), not in TBI (12:12) or (16:16) ( Fig 1H, I), whereas the p16 expression in the hematopoietic tissue, spleen was decreased in all treatment groups TMI vs. TBI ( Fig 1J). Conclusion: TMI-based dose escalates with young donor BMT in aged mice preserved BM microenvironment and reduced senescence of organs while TBI treatment causes senescence of the organs. Young donor BMT, which combined with TMI or TBI treatment, preserved bone structure and reduced senescence of HSPC and MSC. However, TMI treatment decreased the SA-β-gal staining and p16 expression significantly in organs such as the liver and gut, but not by TBI treatment. This study showed the senescence reduction in both bone marrow and organs after young donor BMT and TMI treatment. For further study, we have a plan to study the mechanism and signaling pathway of senescence reduction.

Combining Treg Therapy With Donor Bone Marrow Transplantation: Experimental Progress and Clinical Perspective.

Donor-specific tolerance remains a goal in transplantation because it could improve graft survival and reduce morbidity. Cotransplantation of donor hematopoietic cells to achieve chimerism is a promising approach for tolerance induction, which was successfully tested in clinical trials. However, current protocols are associated with side effects related to the myelosuppressive recipient conditioning, which makes it difficult to introduce them as standard therapy. More recently, adoptive cell therapy with polyclonal or donor-specific regulatory T cells (Treg) proved safe and feasible in several transplant trials, but it is unclear whether it can induce tolerance on its own. The combination of both approaches-Treg therapy and hematopoietic cell transplantation-leads to chimerism and tolerance without myelosuppressive treatment in murine models. Treg therapy promotes engraftment of allogeneic hematopoietic cells, reducing conditioning requirements and enhancing regulatory mechanisms maintaining tolerance. This review discusses possible modes of action of transferred Treg in experimental chimerism models and describes translational efforts investigating the potent synergy of Treg and chimerism.

Abstract 28 A Novel Chemically Defined Culturing System to Support Hematopoietic Stem Cell Expansion

Abstract Introduction Hematopoietic stem cells (HSCs) are known to be multipotent and can reconstitute the entire blood and immune system following HSC transplantation (HSCT). However, the current HSCTs have many unresolved issues including difficulties in finding donors for bone marrow transplantation, preparing a sufficient HSC dose in cord blood transplantation, and the risk of graft-versus-host disease (GvHD) for all allogeneic HSCTs. In recent years, there have been substantial efforts to research and understand the biology of HSCs to develop cell therapy products that meet these challenges and provide an alternative to traditional HSCTs. Several culturing methods have been developed to stimulate HSC expansion ex vivo, but the use of biological materials, such as albumin, has not been ideal for pharmaceutical manufacturing, and it has been difficult inhibiting differentiation of HSCs into lineage-committed cells while culturing in a cytokine-filled medium. We have developed a novel albumin-free culturing system that supports proliferation of long-term HSCs ex vivo by replacing cytokines and albumin with chemical agonists and a caprolactam-based polymer. Objectives The objective of this study is to characterize our expanded cell population as part of an analysis to determine whether the population reconstitutes bone marrow and is therefore suitable for development of a cell therapy product. Methods Here we report the in vitro and in vivo characterization of proliferated HSCs using our novel culturing system compared with a nicotinamide system which is known to support HSC proliferation ex vivo. A colony forming assay, cell surface marker analysis using a flow cytometer, and in vivo repopulating analysis using the NOD/Shi-scid IL-2Rγnull (NOG) mouse were conducted. Results Surface marker analysis of the proliferated population shows more than 70% remaining as undifferentiated CD34+. Of which, a higher number of CD201+CD90+CD45RA- exists compared with the nicotinamide-cultured population. Colony forming unit assays indicate that our proliferated cell population contains a higher number of multipotent progenitors. The repopulating cell assay also shows that the proliferated cells have bone marrow reconstitution capability in bone marrow-ablated NOG mice. Discussion We believe that our chemically defined culturing system can selectively proliferate HSCs while restraining their differentiation and will contribute to the development of novel HSC therapy products.

Bone Marrow Quality Index: A Predictor of Acute Graft-versus-Host Disease in Hematopoietic Stem Cell Transplantation for Thalassemia

Bone marrow continues to be the preferred source of stem cells in allogenic transplantations for non-malignant disorders. Granulocyte-colony stimulating factor (G-CSF)-primed bone marrow (BM) is associated with low rates of acute GVHD and allows reduced collection volumes while ensuring speedy engraftment. However, variability in bone marrow harvest quality is a concern. This report analyses the utility of a novel indicator - Bone Marrow Quality Index (BMQI), which is a ratio of marrow cell count to peripheral blood cell counts, to predict acute GVHD. We analysed 184 consecutive first matched related donor bone marrow transplants for thalassemia using G-CSF-primed bone marrow over 6 years from March-2017 to April-2023 across 2 centres in India. Bone Marrow Quality Index (BMQI) was defined as the ratio of WBC count of G-CSF primed bone marrow to Peripheral WBC count within 24 hours of harvest. EBMT criteria were used for grading GVHD. Log-rank test was used to assess the impact of BMQI on GVHD. Chi-squared test was used to compare categorical data and Wilcoxon test was used to compare the numeric data. Cox proportional-hazards model was used to investigate the association of BMQI vis-à-vis other factors on acute GVHD. Of the 184 patients, 19 had a BMQI <0.9 and 18 between 0.9 and 1. The remaining 147 had BMQI >1. The proportion of patients with acute GVHD grade II-IV was 37%, 22% and 12% respectively for patients who received BMQI <0.9, 0.9-1 and >1 (p= 0.018). Patients who received BMQI <0.9 had a 3.1 times higher chance (95% CI being 0.9 to 10.6) of developing aGVHD II-IV and in those with BMQI 0.9-1 this was 2 times (95% CI being 0.5 to 6.6) higher. BMQI was the significant predictor associated with GVHD hazard (p=0.014). BMQI seems to be the most relevant and controllable predictor of acute GVHD. BMQI is a novel, informative, and very simple indicator which could influence GVHD prophylaxis decision-making. Our indicator is accurately measurable, inexpensive, precise, and timely; further it does not involve any sophisticated equipment, thus may be widely applicable. Prior knowledge of poor marrow quality may help intensify prophylaxis and monitoring for acute GVHD besides triggering review of collection procedures.

Treatment of Omenn syndrome using multiple immunosuppressive strategies: a case series

Introduction: Omenn syndrome represents a therapeutic challenge as a state of florid immune dysregulation born out of autoreactive oligoclonal T cells due to hypomorphic variants in genes associated with severe combined immunodeficiency (SCID).Aims: To describe the therapeutic strategies used in the care of three patients with Omenn syndrome from 2013 to 2022. All had negative maternal engraftment studies and received immunosuppression to control autoreactive T cells. All patients ultimately received myeloablative conditioning with anti-thymocyte globulin (ATG), busulfan, and fludarabine for transplant. All patients are currently alive and well.Case 1: 2-month-old female with adenosine deaminase (ADA)-deficient SCID on pegylated ADA replacement developed respiratory distress, rash, and diarrhea in the setting of rising CD3+/CD4+/CD45RO+ cell count. Treated with high-dose systemic steroids. Due to continued CD3+ increase, systemic tacrolimus was added. She clinically improved and underwent matched unrelated donor cord blood transplant at 5 months of age.Case 2: A male with RAG1-SCID developed generalized erythroderma and rapidly rising CD3+/CD4+/CD45RO+ cell and absolute eosinophil counts soon after birth. Systemic steroids were given with clinical improvement. With taper of steroid, ATG 1 mg/kg was given. Initially, the absolute lymphocyte count (ALC) improved; however, within 5 days, his rash worsened and ALC rose. This necessitated re-initiation of high-dose steroids and addition of tacrolimus until matched related donor bone marrow transplant (MRD BMT) at 2 months old.Case 3: 9-day-old male with RAG1-SCID developed diffuse maculopapular rash, lymphadenopathy, and elevated memory T cells and eosinophils. Initiated high-dose systemic steroids and cyclosporine A (CSA) with quick clinical improvement. CSA discontinued due to toxicity and rising ALC and eosinophils. Given ATG 3 mg/kg/dose for 4 doses over 2 weeks with pattern of quick drop then rapid rise in ALC. Treated with steroids and tacrolimus until MRD BMT at 2 months old. ConclusionsWe describe consistent disease control with immunosuppression by systemic steroids and tacrolimus prior to transplant and suggest ATG as a potential adjunctive consideration in poorly-controlled disease. A growing body of knowledge and experience in optimal Omenn syndrome treatment strategies is imperative to improving outcomes.

Efficacy and safety of allogeneic hematopoietic cell transplantation in acute myeloid leukemia patients aged &gt; 65 years with unfavorable cytogenetics

Unrelated donor bone marrow transplantation (UR-BMT), unrelated donor cord blood stem cell transplantation (UR-CBT), and haploidentical peripheral blood stem cell transplantation (Haplo-PBSCT) are the main alternative stem cell sources for allogeneic hematopoietic cell transplantation (HCT) in Japan. The present study aimed to identify factors associated with the outcomes of UR-BMT, UR-CBT, and Haplo-PBSCT in older patients with acute myeloid leukemia (AML) and intermediate- or poor-risk cytogenetics to improve the clinical efficacy and safety of allogeneic HCT. We retrospectively analyzed data for 448 AML patients aged > 65 years who received UR-BMT (n = 102), UR-CBT (n = 250), or Haplo-PBSCT (n = 96) between 2014 and 2020. Overall survival (OS) in the UR-BMT group was superior (P = 0.033) to that in the other groups. However, all patients without complete remission (non-CR) who had Karnofsky performance status (KPS) < 80 at HCT and poor-risk cytogenetics died within 1 year after HCT. Multivariate Cox regression analysis identified KPS <80 at HCT and poor-risk cytogenetics as independent predictors of worse OS in non-CR patients. KPS < 80 may be an alternative indicator for non-CR AML patients with poor-risk cytogenetics during the selection of HCT, alternative treatments, or best supportive therapy, and the optimal KPS is important for the success of HCT.

Cost-Effectiveness of Unrelated Umbilical Cord Blood Transplantation versus HLA-Haploidentical Related Bone Marrow Transplantation: Evidence from BMT CTN 1101

BMT CTN 1101 was a Phase III randomized controlled trial comparing reduced-intensity conditioning followed by double unrelated umbilical cord blood transplantation (UCBT) versus HLA-haploidentical related donor bone marrow transplantation (haplo-BMT) for patients with high-risk hematologic malignancies. Here we report the results of a parallel cost-effectiveness analysis of these 2 hematopoietic stem cell transplantation (HCT) techniques. In this study, 368 patients were randomized to unrelated UCBT (n=186) or haplo-BMT (n=182). We estimated healthcare utilization and costs using propensity score-matched haplo-BMT recipients from the OptumLabs Data Warehouse for trial participants age <65 years and Medicare claims for participants age ≥65 years. Weibull models were used to estimate 20-year survival. EQ-5D surveys by trial participants were used to estimate quality-adjusted life-years (QALYs). At a 5-year follow-up, survival was 42% for haplo-BMT recipients versus 36% for UCBT recipients (P=.06). Over a 20-year time horizon, haplo-BMT is expected to be more effective (+.63 QALY) and more costly (+$118,953) for persons age <65 years. For those age ≥65 years, haplo-BMT is expected to be more effective and less costly. In one-way uncertainty analyses, for persons age <65, the cost per QALY result was most sensitive to life-years and health state utilities, whereas for those age ≥65, life- years were more influential than costs and health state utilities. Compared to UCBT, haplo-BMT was moderately more cost-effective for patients age <65 years and less costly and more effective for persons age ≥65 years. Haplo-BMT is a fair value choice for commercially insured patients with high-risk leukemia and lymphoma who require HCT. For Medicare enrollees, haplo-BMT is a preferred choice when considering costs and outcomes.

Alternative donor BMT with posttransplant cyclophosphamide as initial therapy for acquired severe aplastic anemia

AbstractSevere aplastic anemia (SAA) is a marrow failure disorder with high morbidity and mortality. It is treated with bone marrow transplantation (BMT) for those with fully matched donors, or immunosuppressive therapy (IST) for those who lack such a donor, which is often the case for underrepresented minorities. We conducted a prospective phase 2 trial of reduced-intensity conditioning HLA-haploidentical BMT and posttransplantation cyclophosphamide (PTCy)-based graft-versus-host (GVHD) prophylaxis as initial therapy for patients with SAA. The median patient age was 25 years (range, 3-63 years), and the median follow-up time was 40.9 months (95% confidence interval [CI], 29.4-55.7). More than 35% of enrollment was from underrepresented racial/ethnic groups. The cumulative incidence of grade 2 or 4 acute GVHD on day 100 was 7% (95% CI, not applicable [NA]-17), and chronic GVHD at 2 years was 4% (95% CI, NA-11). The overall survival of 27 patients was 92% (95% CI, 83-100) at 1, 2, and 3 years. The first 7 patients received lower dose total body irradiation (200 vs 400 cGy), but these patients were more likely to have graft failure (3 of 7) compared with 0 of 20 patients in the higher dose group (P = .01; Fisher exact test). HLA-haploidentical BMT with PTCy using 400 cGy total body irradiation resulted in 100% overall survival with minimal GVHD in 20 consecutive patients. Not only does this approach avoid any adverse ramifications of IST and its low failure-free survival, but the use of haploidentical donors also expands access to BMT across all populations. This trial was registered at www.clinicaltrials.gov as NCT02833805.

222 - Matched Sibling Donor Bone Marrow Transplant with Busulfan + Fludarabine Conditioning Results in Excellent Outcomes for Severe Congenital Neutropenia

222 - Matched Sibling Donor Bone Marrow Transplant with Busulfan + Fludarabine Conditioning Results in Excellent Outcomes for Severe Congenital Neutropenia

Artificial intelligence-assisted diagnosis of hematologic diseases based on bone marrow smears using deep neural networks

Background and objectivesThe morphological examination of bone marrow (BM) cells is essential in both diagnosing and treating various hematologic diseases. However, it is still done manually with a heavy workload. An artificial intelligence-assisted diagnosis support system of BM cells is highly required to reduce the workloads of examiners and improve the reproducibility of the results. MethodsIn this paper, we proposed an artificial intelligence-assisted diagnosis support system of morphological examination based on bone marrow smears including cells detection, classification and prediction of leukemia types. For cell detection, we trained the novel YOLOX-s model to locate cells precisely and obtain single cell images. For cell classification, we regarded it as a fine- grained classification task and proposed a novel architecture called MLFL-Net utilizing multi-level features. Furthermore, we predicted the leukemia types on a dataset including 40 normal people (BM transplantation donors) and 40 patients of different kinds of acute leukemia according to the World Health Organization (WHO) standard. ResultsWe constructed a large-scale data set of 11,788 fully-annotated micrographs from 728 smears and 131,300 expert-annotated single cell images. With the data set, the detection model achieved 0.9797 AUC and 4.33% box placement error. For cell classification, the total accuracy of our proposed MLFL-Net reached 89.53% which outperformed all the other related models in identifying cell categories. In the meantime, we took acute leukemia as an example to explore the leukemia types prediction procedure of hematological disease. It generated the same diagnostic prediction as the experts gave for 92.5 percent of the cohort. ConclusionThis Artificial Intelligence-assisted system can be implemented to aid in clinical decision making and accelerate diagnosis. The method will contribute to promote the intelligence and modernization of BM cytomorphology, which has vital significance of the development of the medical career.

A review of HLA matching to improve clinical outcome in bone marrow transplants and the role of haplotyping in unrelated donor transplants

When the first allogeneic bone marrow transplants in humans was attempted, little was known of the major histocompatibility complex (HLA) and its role in ensuring success of the graft. Therefore, many of the early attempts resulted in failure. During the 1970s just a small number of alleles were detected serologically and the mixed lymphocyte reaction (MLR) served as a surrogate for class II matching, the DR series of alleles not being formally serologically defined until 1978. Nevertheless, selection of nuclear family members who were two HLA haplotype matched became possible using serology and the MLR and transplant successes were reported. The breakthrough in technology was undoubtedly the introduction of DNA techniques, particularly DNA sequencing. This enabled fully matched unrelated individuals to be found as potential bone marrow donors. Genetic phasing or haplotyping of unrelated donors who are found on bone marrow registries has remained elusive until recently. The development of individual chromosome sequencing will permit haplotype matching in the absence of family studies and will expedite studies aimed at identifying other genes which are important matching factors. AIM: The aim of this review is to outline the technological advances that have been made in HLA genotyping over the last 50 years. The progress from serology to DNA sequence as part of the matching algorithm is one of the reasons clinical results have improved so much during this period. This review outlines the DNA techniques that have been utilised for this purpose, concluding with a discussion of the value of haplotype matching in unrelated donor transplants, where the donor has been sourced from a bone marrow registry. Haplotype matching represents one of the last remaining hurdles to be overcome in unrelated donor bone marrow transplants.

The DNA-Sensor AIM2 Promotes BCR-Activated B Cells in Chronic Graft-Versus-Host-Disease

Chronic graft-versus-host-disease (cGVHD) remains a common and poorly understood cause of late non-relapse morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HCT). T cells and IgG-producing B cells mediate cGVHD development and perpetuation, but molecular mechanisms driving immune pathology require further study. We have shown that B cells from cGVHD patients are B Cell Receptor (BCR)-activated and primed for survival. In a mouse model of bone-marrow transplantation (BMT) with cGVHD manifestations, BCR signaling is promoted by alloantigens and BAFF (Jia et al., Blood 2021). BCR signaling is a viable therapeutic target in cGVHD, but other targetable pathways that promote pathological BCR-activated B cells remain largely unknown. Our single cell RNA-sequencing study of B cells from the peripheral blood (PB) of HCT patients identified 'Absent in Melanoma 2' (AIM2) as a prime candidate for further study (ASH abstract 2019, Poe et al., manuscript submitted). AIM2 was a signature gene in a BCR-activated subset after HCT, and AIM2 transcripts were significantly increased in a pre-germinal center (GC) B cell subset in cGVHD vs. no cGVHD patients (adjusted p=0.049). AIM2 incites inflammasome-mediated pyroptotic cell death in myeloid cells, but the function of AIM2 in B cells is largely unknown. AIM2 is expressed by CD27+ antigen (Ag)-experienced B cells in healthy individuals (HI) (Svensson et al., PlosOne 2017) and emerging evidence suggests that AIM2 promotes antibody production (Yang et al., Signal Transduct Target Ther 2021). We hypothesize that AIM2 promotes pathological BCR-activated B cells and anti-host IgG production in cGVHD. Using intracellular flow cytometry (IFC) and PB samples from HI (n=27), we first confirmed that the AIM2 protein is mainly expressed by CD27+ Ag-experienced B cells. Specifically, 88% and 99% of the IgD+ pre- and IgD- post-GC CD27+ B cells expressed AIM2, respectively. In HCT, we found a significantly higher proportion of AIM2+ cells in the circulating CD27+ IgD- post-GC B cells from cGVHD patients (n=7) vs. no cGVHD patients (n=5) (p=0.02). We also found significant overexpression of AIM2 transcripts in B cells from mice with cGVHD manifestations vs. no cGVHD mice (n=5/group, p=0.03, Figure 1A). To determine if Ag-activation together with other extrinsic factors typically encountered by B cells after HCT induced AIM2 expression by purified CD27- B cells from HI, we used IFC. The proportion of AIM2+ CD27- B cells increased after BCR +/- Toll-like receptor 7 or 9 stimulation only when IFNγ was present. Next, we studied the function of AIM2 in B cells. Unlike myeloid cells, CD27+ B cells from HI did not undergo pyroptotic cell death after stimulation with double stranded DNA. This lack of pyroptosis was not due to a deficiency in the inflammasome components and B cells had the capacity to undergo pyroptosis after exposure to the NLRP1/3 activator, nigericin. To assess effects of AIM2 on specific IgG production, we used AIM2 KO mice in a hapten-carrier immunization model (NP-KLH + alum i.p.). AIM2-KO mice had larger splenic GC vs. WT controls at day 8 post-immunization (p<0.0001). GC enlargement paradoxically associated with lower anti-NP IgG levels (p=0.03). Finally, having previously shown that BM+Spl recipients have a significantly higher proportion of GL7+ BCR-activated blood B cells (Jia et al., Blood 2021), we asked whether AIM2 promoted GL7+ B cells in cGVHD. To address this, we used AIM2-floxed mice, kindly supplied by Genentech, to breed AIM2fl/fl Mb1Cre/+ conditional KO mice (AIM2-CKO), deficient in AIM2 only in B cells. We then used these AIM2-CKO vs. AIM2fl/fl Mb1+/+ control littermates mice which are AIM2-sufficient as BMT donors. At day 24 post-BMT, recipients of BM+Spl from AIM2-CKO mice had a significantly lower percentage of mature (CD19+ CD93-) GL7+ B cells in PB vs. recipients of BM+Spl from the AIM2-sufficient littermates (n=10/group, p=0.004, Figure 1B). This was not due to baseline B cell differences in donor CKO mice. These results are being confirmed at later time points in ongoing, additional experiments. Our data show that AIM2 promotes IgG-producing B cells and circulating GL7+ B cells, suggesting that AIM2 plays a novel inflammasome-independent role in cGVHD B cell pathology. We are actively investigating how AIM2 effects alloantibody production and cGVHD development, so that therapeutic targets can be identified. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Comparable survival outcomes with haploidentical stem cell transplantation and unrelated bone marrow transplantation.

We retrospectively compared outcomes of unrelated donor bone marrow transplant (UBMT) and HLA-haploidentical peripheral blood stem cell transplantation using post-transplant cyclophosphamide (PTCy-haploPBSCT) using the Japanese registry data. Recipients of first HCT for acute leukemia and myelodysplastic syndromes between 2012 and 2015 were included. The analyzed subjects comprised UBMT recipients with 8/8 matched HLA alleles (n = 1470), 7/8 matched alleles (n = 859), 6/8 matched alleles (n = 186), and recipients of PTCy-haploPBSCT (n = 133). In multivariate analyses with 8/8 matched UBMT as the reference, PTCy-haploPBSCT showed similar overall mortality, decreased risk of non-relapse mortality (NRM), increased risk of relapse, and decreased risk of grade II-IV acute graft-versus-host disease (GVHD) and chronic GVHD. Adjusted probabilities for 8/8 matched UBMT, PTCy-haploPBSCT, and 7/8 and 6/8 matched UBMT groups at 2 years post-transplant were 61%, 60%, 58%, and 52% for overall survival, 23%, 28%, 21%, and 19% for relapse, and 20%, 7%, 24%, and 33% for NRM. PTCy-haploPBSCT was associated with remarkably low NRM, contributing to survival outcomes that were comparable to 8/8 matched UBMT. The higher relapse rate in the PTCy-haploPBSCT group might be associated with the higher proportion of high-risk patients. PTCy-haploPBSCT may be a viable alternative when HLA-matched related donors are not available.

Serial Analysis of the T-Cell Receptor β-Chain Repertoire in People Living With HIV Reveals Incomplete Recovery After Long-Term Antiretroviral Therapy.

Long-term antiretroviral therapy (ART) in people living with HIV (PLHIV) is associated with sustained increases in CD4+ T-cell count, but its effect on the peripheral blood T-cell repertoire has not been comprehensively evaluated. In this study, we performed serial profiling of the composition and diversity of the T-cell receptor β-chain (TRB) repertoire in 30 adults with HIV infection before and after the initiation of ART to define its long-term impact on the TRB repertoire. Serially acquired blood samples from 30 adults with HIV infection collected over a mean of 6 years (range, 1-12) years, with 1-4 samples collected before and 2-8 samples collected after the initiation of ART, were available for analysis. TRB repertoires were characterized via high-throughput sequencing of the TRB variable region performed on genomic DNA extracted from unsorted peripheral blood mononuclear cells. Additional laboratory and clinical metadata including serial measurements of HIV viral load and CD4 + T-cell count were available for all individuals in the cohort. A previously published control group of 189 TRB repertoires from peripheral blood samples of adult bone marrow transplant donors was evaluated for comparison. ART initiation in PLHIV was associated with a sustained reduction in viral load and a significant increase in TRB repertoire diversity. However, repertoire diversity in PLHIV remained significantly lower than in the control group even after long-term ART. The composition of TRB repertoires of PLHIV after ART also remained perturbed compared to the control cohort, as evidenced by large persistent private clonal expansions, reduced efficiency in the generation of TRB CDR3 amino acid sequences, and a narrower range of CDR3 lengths. Network analysis revealed an antigen-experienced structure in the TRB repertoire of PLHIV both before and after ART initiation that was quite distinct from the structure of control repertoires, with a slight shift toward a more naïve structure observed after ART initiation. Though we observe significant improvement in TRB repertoire diversity with durable viral suppression in PLHIV on long-term ART, the composition and structure of these repertoires remain significantly perturbed compared to the control cohort of adult bone marrow transplant donors.

Interrogation of RDEB Epidermal Allografts after BMT Reveals Coexpression of Collagen VII and Keratin 15 with Proinflammatory Immune Cells and Fibroblasts

Recessive dystrophic epidermolysis bullosa (RDEB) is a devastating genodermatosis characterized by dysfunctional collagen VII protein resulting in epithelial blistering of the skin, mucosa, and gastrointestinal tract. There is no cure for RDEB, but improvement of clinical phenotype has been achieved with bone marrow transplantation and subsequent epidermal allografting from the bone marrow transplant donor. Epidermal allografting of these patients has decreased wound surface area for up to 3 years after treatment. This study aimed to determine the phenotype of the epidermal allograft cells responsible for durable persistence of wound healing and skin integrity. We found that epidermal allografts provide basal keratinocytes coexpressing collagen VII and basal stem cell marker keratin 15. Characterization of RDEB full-thickness skin biopsies with single-cell RNA sequencing uncovered proinflammatory immune and fibroblast phenotypes potentially driven by the local environment of RDEB skin. This is further highlighted by the presence of a myofibroblast population, which has not been described in healthy control human skin. Finally, we found inflammatory fibroblasts expressing profibrotic gene POSTN, which may have implications in the development of squamous cell carcinoma, a common, lethal complication of RDEB that lacks curative treatment. In conclusion, this study provides insights into and targets for future RDEB studies and treatments.