Setting Of Bone Marrow Transplantation Research Articles

Post-Transplant Cyclophosphamide for the Prevention of Graft-vs.-Host Disease in Allogeneic Hematopoietic Cell Transplantation: A Guide to Management for the Advanced Practitioner.

Cyclophosphamide remains a critical component to haploidentical transplant conditioning regimens. Post-transplant cyclophosphamide (PTCy) emerged as an effective component of graft-vs.-host disease (GVHD) prophylaxis in the nonmyeloablative haploidentical bone marrow transplant setting. The relative ease of administration compared with ex vivo manipulations and efficacy in reducing GVHD has led to increasing PTCy use in transplant centers around the world. The role of PTCy has expanded to haploidentical transplantation with myeloablative conditioning regimens and peripheral blood progenitor cells as the donor source. Moreover, encouraging results in GVHD management have been shown with the use of PTCy alone or in combination with other immunosuppressives in the human leukocyte antigen-matched donor setting. The toxicity profile of cyclophosphamide varies extensively depending on dose, duration, overall drug exposure, and, potentially, pharmacogenetics. This review highlights the pharmacology, pharmacokinetics, and toxic effects of cyclophosphamide and offers practical guidance for clinical application in the post-transplant setting. We summarize data on the management of high-dose cyclophosphamide toxicities and provide insights into the pharmacogenetic implications on drug efficacy and safety data.

Transfusion-Transmitted Disorders 2023 with Special Attention to Bone Marrow Transplant Patients.

Transfusion medicine is traditionally a strong/fundamental part of clinical practice, saving hundreds of millions of lives. However, blood-borne or transmitted infections are a well-known and feared possibility, a risk we relentlessly mitigate. Pathogens are continuously and rather quickly changing, so during the last decade, many, sometimes exotic, new pathogens and diseases were recorded and analyzed, and some of them were proved to be transmitted with transfusions. Blood or blood component transfusions are carried out after cautious preparative screening and inactivation maneuvers, but in some instances, newly recognized agents might escape from standard screening and inactivation procedures. Here, we try to focus on some of these proven or potentially pathogenic transfusion-transmitted agents, especially in immunocompromised patients or bone marrow transplantation settings. These pathogens are sometimes new challenges for preparative procedures, and there is a need for more recent, occasionally advanced, screening and inactivation methods to recognize and eliminate the threat a new or well-known pathogen can pose. Pathogen transmission is probably even more critical in hemophiliacs or bone marrow transplant recipients, who receive plasma-derived factor preparations or blood component transfusions regularly and in large quantities, sometimes in severely immunosuppressed conditions. Moreover, it may not be emphasized enough that transfusions and plasma-derived product administrations are essential to medical care. Therefore, blood-borne transmission needs continued alertness and efforts to attain optimal benefits with minimized hazards.

HLA-haploidentical stem cell transplantation

HLA-haploidentical stem cell transplantations using posttransplant cyclophosphamide (PTCy-haplo) rapidly increased worldwide. In Japan, the number of HLA-haploidentical stem cell transplantation cases exceeded related HLA-matched transplants in 2020. Recent retrospective studies using Japanese registry data have reported comparable transplantation outcomes between PTCy-haplo and HLA-matched unrelated and cord blood transplantations. PTCy-haplo was initially developed in the bone marrow transplantation setting after non-myeloablative conditioning but has recently become widely used in peripheral blood stem cell transplantation and myeloablative conditioning. Peripheral blood stem cell transplantation increases the occurrence of graft-versus-host disease but may have more improved transplant outcomes compared with bone marrow transplantation. Other factors, such as the number of infused cluster of differentiation 34-positive cells, donor age, HLA class II mismatch, HLA-B leader, and reduced PTCy dosage, may also contribute to the outcome of PTCy-haplo transplantations. Furthermore, PTCy has been reportedly effective in related/unrelated HLA-matched transplantation and HLA-mismatched unrelated transplantations. A prospective phase II trial using PTCy in patients who underwent HLA-matched and 1-2 allele-mismatched transplantation is ongoing in Japan. Patient enrollment has already been completed, and the results will be revealed soon. Using PTCy to sufficiently reduce the occurrence of graft-versus-host disease will make performing allogeneic transplants with a higher safety level possible.

Donor-Derived Amphiregulin Drives CD4+ T Cell Expansion and Promotes Tissue Pathology after Experimental Allogeneic BMT

Amphiregulin (Areg), a ligand for the epidermal growth factor receptor (EGFR), contributes to cell proliferation and survival. Work in recent years has demonstrated that immune cells such as Tregs and ILC2s can produce Areg, leading to tissue repair after injury. In the bone marrow transplant (BMT) setting, clinical studies have reported that high concentrations of plasma Areg may predict for poor prognosis acute GVHD. However, impacts of Areg/EGFR signaling within the immune system are poorly understood. We thus investigated the role of T cell-derived Areg following allogeneic (allo)-BMT. To evaluate Areg expression in donor T cells, we performed C57BL/6 into B6D2F1 (B6 BDF1) MHC-mismatched allo-BMT and found that donor CD4+ T cells produced Areg post-transplant. We initially hypothesized that T cell-derived Areg may contribute to tissue repair after BMT. To investigate the effects of T cell-derived Areg in the transplant setting, we performed B6 BDF1 allo-BMT using T cells from Aregfl/fl x CD4-Cre (Areg-/-) or Aregfl/fl x Foxp3-Cre (AregΔFoxp3) mice, lacking Areg in all T cells (Areg-/-) or specifically in regulatory T cells (AregΔFoxp3). Unexpectedly, recipients of Areg-/- T cells demonstrated reduced mortality compared to recipients of wild-type (WT) T cells (Figure, left). This observation was recapitulated in a distinct B6 BALB/c allo-BMT model (not shown). Examining the effects of Areg deficiency at the tissue level, recipients of Areg-/- T cells demonstrated reduced GVHD pathology in liver, ileum, and colon ten days post-BMT, while recipients of AregΔFoxp3 T cells showed no difference compared to controls. Consistent with the GVHD histopathology, intestinal stem cell frequencies were substantially preserved in the small intestines from recipients transplanted with Areg-/- T cells compared to recipients receiving WT T cells or AregΔFoxp3 T cells. These data suggested that conventional T cell-derived Areg may contribute to tissue damage in GVHD. To assess the mechanisms by which T cell-derived Areg contributes to GVHD severity, we analyzed donor T cells in spleen, mesenteric lymph nodes, and lamina propria post-BMT. Areg-/- donor CD4+ T cells demonstrated impaired expansion while donor CD8+ T cell expansion remained intact. Areg-/- donor CD4+ T cells also demonstrated a reduced Th1 response as evidenced by decreased IFNg expression. In addition, co-culture in vitro and co-transfer in vivo of WT and Areg-/- CD4+ T cells indicated that the reduced expansion of Areg-/- donor CD4+ T cells was cell-intrinsic and likely occurring in an autocrine Areg-dependent fashion. Notably, these observations were recapitulated following allo-BMT using EGFRfl/fl x CD4-Cre (EGFR-/-) donor T cells: recipients transplanted with EGFR-/- donor T cells demonstrated reduced donor CD4+ T cell expansion and improved survival post-transplant (Figure, right). To further investigate the mechanisms driving Areg-dependent CD4+ T cell expansion, we compared WT and Areg-/-B6 T cells following ex vivo activation with anti-CD3 and anti-CD28 antibodies or activation with allo BALB/c-derived dendritic cells. Compared to WT CD4+ T cells, Areg-deficient CD4+ T cells exhibited reduced proliferation, which was restored by supplementation with recombinant Areg. Additionally, we found that naive CD4+ T cells increased Egfr and Areg mRNA expression upon T cell receptor stimulation. Moreover, Areg inhibition using anti-Areg neutralizing antibodies impaired proliferation of WT naive CD4+ T cells after activation, reducing their expansion upon activation ex vivo. To better understand how Areg was impacting activation-induced CD4+ T cell expansion, we examined downstream targets of EGFR signaling and observed that Areg inhibition resulted in impairment of Akt phosphorylation and reduced glucose uptake in naive CD4+ T cells following activation. Finally, despite the reduction in proliferation and GVHD pathogenicity, Areg-/- donor T cells demonstrated intact GVL activity against A20 lymphoma cells, resulting in an improved overall survival following BMT with tumor challenge. In summary, we found that CD4+ T cell-derived Areg contributes to CD4+ T cell proliferation after activation, leading to increased T cell expansion, tissue damage, and GVHD-related mortality after allo-BMT. Careful inhibition of the T cell Areg/EGFR axis may represent a novel therapeutic target for prevention or amelioration of GVHD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Neutrophils as regulators of macrophage-induced inflammation in a setting of allogeneic bone marrow transplantation.

SummaryClinical data reveal that patients with allogeneic hematopoietic stem cell transplantation (HSCT) are vulnerable to infection and prone to developing severe sepsis, which greatly compromises the success of transplantation, indicating a dysregulation of inflammatory immune response in this clinical setting. Here, by using a mouse model of haploidentical bone marrow transplantation (haplo-BMT), we found that uncontrolled macrophage inflammation underlies the pathogenesis of both LPS- and E.coli-induced sepsis in recipient animals with graft-versus-host disease (GVHD). Deficient neutrophil maturation in GVHD mice post-haplo-BMT diminished modulation of macrophage-induced inflammation, which was mechanistically dependent on MMP9-mediated activation of TGF-β1. Accordingly, adoptive transfer of mature neutrophils purified from wild-type donor mice inhibited both sterile and infectious sepsis in GVHD mice post-haplo-BMT. Together, our findings identify a novel mature neutrophil-dependent regulation of macrophage inflammatory response in a haplo-BMT setting and provide useful clues for developing clinical strategies for patients suffering from post-HSCT sepsis.

Aryl hydrocarbon receptor–targeted therapy for CD4+ T cell–mediated idiopathic pneumonia syndrome in mice

AbstractWe previously demonstrated that interferon γ (IFN-γ) derived from donor T cells co-opts the indoleamine 2,3-dioxygenase 1 (IDO1) → aryl hydrocarbon receptor (AHR) axis to suppress idiopathic pneumonia syndrome (IPS). Here we report that the dysregulated expression of AP-1 family genes in Ahr−/− lung epithelial cells exacerbated IPS in allogeneic bone marrow transplantation settings. AHR repressed transcription of Jund by preventing STAT1 from binding to its promoter. As a consequence, decreased interleukin-6 impaired the differentiation of CD4+ T cells toward Th17 cells. IFN-γ– and IDO1-independent induction of Ahr expression indicated that the AHR agonist might be a better therapeutic target for IPS than the IDO1 activator. We developed a novel synthetic AHR agonist (referred to here as PB502) that potently inhibits Jund expression. PB502 was highly effective at inducing AHR activation and ameliorating IPS. Notably, PB502 was by far superior to the endogenous AHR ligand, L-kynurenine, in promoting the differentiation of both mouse and human FoxP3+ regulatory CD4+ T cells. Our results suggest that the IDO1-AHR axis in lung epithelial cells is associated with IPS repression. A specific AHR agonist may exhibit therapeutic activity against inflammatory and autoimmune diseases by promoting regulatory T-cell differentiation.

Why the HIV Reservoir Never Runs Dry: Clonal Expansion and the Characteristics of HIV-Infected Cells Challenge Strategies to Cure and Control HIV Infection.

Antiretroviral therapy (ART) effectively reduces cycles of viral replication but does not target proviral populations in cells that persist for prolonged periods and that can undergo clonal expansion. Consequently, chronic human immunodeficiency virus (HIV) infection is sustained during ART by a reservoir of long-lived latently infected cells and their progeny. This proviral landscape undergoes change over time on ART. One of the forces driving change in the landscape is the clonal expansion of infected CD4 T cells, which presents a key obstacle to HIV eradication. Potential mechanisms of clonal expansion include general immune activation, antigenic stimulation, homeostatic proliferation, and provirus-driven clonal expansion, each of which likely contributes in varying, and largely unmeasured, amounts to maintaining the reservoir. The role of clinical events, such as infections or neoplasms, in driving these mechanisms remains uncertain, but characterizing these forces may shed light on approaches to effectively eradicate HIV. A limited number of individuals have been cured of HIV infection in the setting of bone marrow transplant; information from these and other studies may identify the means to eradicate or control the virus without ART. In this review, we describe the mechanisms of HIV-1 persistence and clonal expansion, along with the attempts to modify these factors as part of reservoir reduction and cure strategies.

Cigarette Smoke and E-Cigarette Aerosols Lead to Clonal Expansion of Tet2 -/- and Dnmt3a R878H Cells In Vivo

Background:Environmental factors must play a significant role in the emergence of clonal hematopoiesis since only a fraction of individuals harboring clonal hematopoiesis of indeterminate potential (CHIP) mutations develop hematologic malignancy. Mouse models of chronic inflammation have demonstrated clonal expansion of Tet2 and Dnmt3a knockout hematopoietic cells while Ppm1d mutated clones exhibit clonal dominance in response to cytotoxic DNA damaging chemotherapy stress. Epidemiologic studies have associated smoking behavior with clonal hematopoiesis but the leukemogenic effects of cigarette smoke on hematopoietic stem cells (HSCs) are poorly defined. In addition, the exploding use of electronic (e)-cigarettes has led to significant concern on their detrimental health effects plus studies of E-cigarettes on the hematopoietic system are non-existent. Here, we investigated the role of cigarette smoke and E-cigarette aerosols in promoting clonal expansion of common CHIP mutations. We hypothesize that one or more specific somatic CHIP mutation displays a fitness advantage in the presence of cigarette smoke and/or e-cigarette aerosols.Methods:Competitive bone marrow transplant assays were used to determine the development of clonal expansion in response to cigarette smoke and E-cigarette aerosols using Tet2 knockout (Tet2 -/-), Dnmt3a R878H and Jak2 V617F genetically modified mice. Lethally irradiated recipient mice in the CD45.1/2 background were transplanted with whole bone marrow cells from wild type (WT) (CD45.1) and mutant (CD45.2) mice. Ratio of cells transplanted were 1:10 for Tet2 -/- and WT; 1:5 for Dnmt3a R878H and WT and 1:1 for Jak2 V617F and WT. Transplanted mice were exposed to cigarette smoke or E-cigarette aerosols using a nose-only inhalation exposures system for 2 hours/day, 4 days/week for 2 or 3 months. Control mice were exposed to room air using the nose-only inhalation approach.Results:After 2 months of exposure, we observed that Tet2 -/- cells had significantly expanded in the smoke group (paired t-test, p<0.05) while there was no significant difference in the air group (Fig. 1A). Furthermore, this increase in Tet2 -/- cells was more pronounced in the myeloid cell subset (Fig. 1B). While the knock-in mouse model of Jak2 V617F does not display a competitive advantage in a lethally irradiated bone marrow transplant setting, we observed persisting levels of Jak2 V617F mutant cells following smoke exposure but significantly reduced levels in the air group, illustrating that the mutant cells prevail in a smoke environment (Fig. 1C). During sacrifice of the Jak2 V617F transplanted and exposed mice, long-term HSCs in the bone marrow exhibited a trend towards increased bromodeoxyuridine (BrdU) incorporation and increased DNA damage as determined by H2AX staining (Fig 1D). Meanwhile, E-cigarette aerosol exposure of mice transplanted with Dnmt3a R878H cells, displayed increased levels of circulating mutant cells compared to the air group (Fig. 1E) (repeated measures, 2-way ANOVA).Conclusion:In vivo exposure of mouse models of CHIP to cigarette smoke and E-cigarette aerosols promotes mutant cell expansion over time. Our data indicate that more than one mutation is selected by environmental factors in the form of tobacco products. This data is important to guide us in preventive medicine and early detection of clonal hematopoiesis. Future research is aimed at deciphering the molecular responses of WT cells to cigarette smoke and E-cigarette aerosols and strategies to preserve WT stem cell fitness in the context of smoking and E-cigarette usage. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Efficacy of Oral Cryotherapy in the Prevention of Oral Mucositis Associated with Cancer Chemotherapy: Systematic Review with Meta-Analysis and Trial Sequential Analysis.

Background: This review aimed to evaluate the efficacy of oral cryotherapy in the prevention of chemotherapy-induced oral mucositis using meta-analysis and trial sequential analysis, as well as to assess the quality of the results by the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach. Methods: A comprehensive search of three databases including Medline, Embase and Central was performed to identify randomized controlled trials that used oral cryotherapy for the prevention of chemotherapy-induced oral mucositis. The primary outcome was the incidence of oral mucositis for trials employing oral cryotherapy as the intervention for the prevention of oral mucositis. The meta-analysis was performed using the random-effects model and random errors of the meta-analyses were detected by trial sequential analysis. Results: A total of 14 RCTs with 1577 participants were included in the present meta-analysis. Patients treated with oral cryotherapy were associated with a significantly lower risk of developing oral mucositis of any grade (risk ratio (RR), 0.67 (95% CI: 0.56–0.81, p < 0.05)). Findings from the subgroup analyses showed that oral cryotherapy significantly reduced the risk of oral mucositis in patients undergoing bone marrow transplantation (RR 0.69, CI: 0.54–0.89, p < 0.05) as well as chemotherapy (RR 0.66, CI: 0.58–0.75, p < 0.05). Findings from the trial sequential analysis suggested that the evidence on oral cryotherapy as a preventive intervention for oral mucositis in patients with solid malignancies receiving conventional chemotherapy was conclusive. Conclusion: Oral cryotherapy is effective in preventing oral mucositis in patients undergoing chemotherapy for the management of solid malignancies. The use of oral cryotherapy in preventing oral mucositis in bone marrow transplantation settings showed promising efficacy, but the evidence is not conclusive and requires more high-quality randomized controlled trials.

The E3 Ligase Itch Is a Regulator of Bone Marrow Mesenchymal Stem Cells and the Hematopoietic Stem Cell Niche

Hematopoietic stem cells (HSCs) reside in a complex cellular microenvironment within the bone marrow. Bone marrow mesenchymal stromal/stem cells (BM-MSCs) have emerged as an important cellular component of this hematopoietic stem cell niche. BM-MSCs have been implicated in physiological hematopoiesis as well as in hematological malignancies. The intracellular regulatory network that governs BM-MSC homeostasis and their function within the HSC niche remains poorly understood. Posttranslational modification of proteins through ubiquitylation has been proven an integrative component in the regulation of quiescence, proliferation, self-renewal and differentiation of stem cells. We have previously demonstrated that the E3 ubiquitin ligase Itch is a negative regulator of HSC homeostasis and function (Rathinam et al., Nat.Immunol., 2011). Notably, patients suffering from a rare ITCH deficiency syndrome display morphologic abnormalities with dysmorphic features, suggesting a role for this E3 ligase within the skeletal tissue and mesenchymal lineage cells (Lohr et al., Am.J.Hum.Genet., 2010).In the present study, we identify Itch as a critical regulator of BM-MSCs. Itch-/- mice exhibit an at least two-fold reduction of MSCs (immunophenotypically identified as CD45-TER119-CD31-PDGFRα+Sca-1+CD90+) in the BM. Consistently, Colony Forming Unit-Fibroblasts (CFU-F) assays revealed a remarkable reduction of MSCs in the BM (p=0.0392). Moreover, in-vitro culture studies identified a hyperproliferative phenotype of Itch mutant MSCs. However, further studies revealed that Itch deficiency leads to reduced differentiation of MSCs into osteoblasts, both in-vivo and in-vitro, and therefore resulting in osteopenic bone phenotype. Reciprocal and Serial Bone marrow transplantation studies suggested an alteration of the niche for HSCs in Itch deficient BM. Interestingly, Itch-/- recipients exhibited a five-fold increase in their donor (wild-type) hematopoietic stem and progenitor cell compartment. Molecular and biochemical studies documented an increase of signals mediated by Notch in Itch deficient MSCs and co-localization of Itch and Notch-1 intracellular domain. Of note these findings gain importance, as Notch signaling has been previously implicated in the regulation of MSC differentiation into osteoblasts, as well as in the regulation of the HSC niche.In summary, our studies identified Itch as a positive regulator of BM-MSC homeostasis. Our results suggest that the Itch-Notch axis within the HSC niche may serve as a potential pharmaceutical target, especially in the setting of bone marrow transplantation in humans. Treatment of the transplant recipient with an Itch inhibitor may avoid graft failure or rescue poor hematopoietic recovery. DisclosuresSchünemann:German Academic Exchange Service: Other: scholarship; Boehringer Ingelheim Fonds: Other: travel grant.

CD94 Ex Vivo Cultures in a Bone Marrow Transplantation Setting.

Complementary, marrow donor-derived peripheral blood T-lymphocyte infusions enable consistent hematopoietic engraftment in lethally irradiated dog leukocyte antigen (DLA)-haploidentical littermate recipients, but at the cost of severe graft versus host disease (GVHD). Here, we explored whether CD94-selected and in vitro-expanded natural killer (NK) cells could be substituted for T-lymphocytes for enhancing marrow engraftment without causing severe GVHD. Five dogs were conditioned with 700 cGy total body irradiation followed by infusion of DLA-haploidentical donor marrow and CD94-selected, in vitro-expanded NK cells. NK cells were infused at a median of 140 000 (range 78 000-317 000) cells/kg. Four dogs rejected their marrow grafts, whereas 1 dog fully engrafted and developed GVHD. We observed an increase in peripheral blood NK cells after infusion of CD94-selected, ex vivo-expanded NK in 2 dogs. Peripheral blood lymphocyte counts peaked at day 7 or 8 posttransplant in the 4 rejecting dogs, whereas in the fully engrafted dog, lymphocyte counts remained stable at suboptimal levels. Our study indicates NK cells can be expanded in vitro and safely infused into DLA-haploidentical recipients. Within the range of CD94-selected and expanded cells infused we concluded that they failed to both uniformly promote engraftment and avert GVHD.

Long-term mid-onset dietary restriction rejuvenates hematopoietic stem cells and improves regeneration capacity of total bone marrow from aged mice.

Currently, the world's aging population is expanding rapidly, leading to a rise in aged hematopoietic cell transplantation (HCT) recipients and aged donors. However, the age of donors is negatively related to the prognosis after transplantation due to functional decline in hematopoietic stem cells (HSCs) during aging. Previously, we showed that an early‐onset dietary restriction (DR) significantly retards early aging of HSCs. However, the effects of a mid‐onset DR on HSCs remain unknown. In the current study, we performed 30% DR in 15‐ to 18‐month‐old mice (equivalent to 50–60 human years) for short‐term (4 months) and long‐term (9 months). We show that DR reduces and rectifies the imbalance of the HSC pool in aged mice. Short‐term DR improves hematopoietic reconstitution in purified HSC transplantations, but not in bone marrow transplantations. Intriguingly, long‐term mid‐onset DR improves the hematopoietic regeneration of aging HSCs with a particular enhancement of lymphoid outputs even in total bone marrow transplantation settings. Mechanistically, long‐term DR rejuvenates the aberrantly regulated mitochondrial pathways in aging HSCs and is accompanied by increased quiescence and reduced DNA damage signaling in HSCs. Short‐term DR showed a similar trend of rescuing these aging hallmarks but to a much lesser extent. Together, the current study suggests that mid‐onset DR ameliorates the function of aging HSCs and long‐term DR even improved hematopoietic reconstitution in bone marrow transplantation, which could potentially have considerable implications in HCT of humans when only old donors are available.

Rapid Antibody Testing for SARS-CoV-2 in Asymptomatic and Paucisymptomatic Healthcare Professionals in Hematology and Oncology Units Identifies Undiagnosed Infections.

Rapid Antibody Testing for SARS-CoV-2 in Asymptomatic and Paucisymptomatic Healthcare Professionals in Hematology and Oncology Units Identifies Undiagnosed Infections.

The Role of Antioxidants in Ameliorating Cyclophosphamide-Induced Cardiotoxicity.

The chemotherapeutic and immunosuppressive agent cyclophosphamide has previously been shown to induce complications within the setting of bone marrow transplantation. More recently, cardiotoxicity has been shown to be a dose-limiting factor during cyclophosphamide therapy, and cardiooncology is getting wider attention. Though mechanism of cyclophosphamide-induced cardiotoxicity is not completely understood, it is thought to encompass oxidative and nitrative stress. As such, this review focuses on antioxidants and their role in preventing or ameliorating cyclophosphamide-induced cardiotoxicity. It will give special emphasis to the cardioprotective effects of natural, plant-derived antioxidants that have garnered significant interest in recent times.

Reduction in Routine Labs in the Inpatient BMT Setting: A Quality Improvement Initiative

<h3>Introduction</h3> Common labs, such as a daily complete blood count (CBC) or a daily basic metabolic panel (BMP), represent possible healthcare waste. The Global Aim of this quality improvement initiative was to reduce lab draws and overall healthcare costs through implementation of routine standardized lab practices for patients undergoing bone marrow transplant (BMT). Our Smart Aim was to decrease the median number of labs per hospitalized BMT patient per week by 10%. <h3>Methods</h3> Using the Model for Improvement, we identified several opportunities to decrease the frequency of routine labs. This included: 1) daily CBC and BMP were obtained on all patients from the start of the preparative regimen through discharge; 2) daily CBC and BMP were obtained on all patients upon readmission; 3) there was no standardized process to clinically evaluate lab frequency. We addressed these opportunities through Plan-Do-Study-Act (PDSA) testing. Interventions included: 1) implementing a mindful mechanism to determine lab frequency prior to the start of chemotherapy; 2) weekly scheduled assessment and discussion of lab frequency during rounds; 3) implementation of a readmission order set with decreased frequency of lab monitoring. Finally, we used standardized run charts to measure the impact of the interventions. <h3>Results</h3> From January 2019 to June 2019, the median number of weekly labs for patients admitted to the BMT unit decreased from 54 to 49. These gains have been sustained for an additional four months. <h3>Discussion</h3> Using standardized processes, we demonstrated a substantial decrease in the number of weekly labs obtained on patients admitted to the BMT unit. We believe these interventions and measurements can be used in all transplant centers and can influence outcomes.

Thymus Regeneration Is Dependent on Distinct Mesenchymal Stromal Cell Populations

Background The regenerative ability of the thymus is an important factor in determining the outcome of bone marrow transplantation. However, the currently employed cytoreductive regimens invariably damage the thymic stroma, thus impeding recovery of T lymphopoiesis. Additionally, the thymic niche is poorly defined. Thymic epithelial cells have been extensively characterized, but our understanding of how other stromal cell types contribute to T lymphopoiesis is limited. We therefore set out to further define the thymic niche under homeostasis and regeneration. Results Using single-cell RNA-sequencing, we demonstrated that the thymic stromal cell compartment is composed of 10 stromal cell subsets. A specific subset of periostin expressing mesenchymal stromal cells (Postn+ MSCs) were found to be enriched in T cell promoting factors such as BMP2, BMP4, Ccl19 and Flt3 ligand (Fig. 1A). To elucidate the functional role of Postn+ MSCs in thymus regeneration, thymic stromal cells were isolated 3 days post-irradiation and transplantation and sequenced. Although the subsets classified as MSC generally persist following irradiation, the Postn+ MSCs were significantly reduced at a time when thymus seeding progenitors typically enter the tissue (Fig 1B). The secretion of chemokines and cytokines was also found to be faulty in the Postn+ MSC subset following transplantation, including significant reductions in Bmp2 and Cxcl14 (Fig 1C). In addition, there was a significant increase in a separate class of pro-adipogenic MSCs (Fig 1B), suggesting that the slow regeneration of the thymus after a transplantation could in part be due to this imbalance in MSC subtypes. Testing this hypothesis, thymic MSC subsets were adoptively transferred into irradiated and transplanted hosts. Specific subsets increased influx of thymocyte progenitors and aided in endothelial cell recovery (Fig 1D) consistent with regeneration of the thymic microenvironment. Furthermore, the transferred MSCs persisted and improved T cell numbers in the circulation up to 16 weeks post-transplantation (Fig 1E). To further investigate the clinical relevance of the MSC compartment, single-cell RNA-sequencing was performed on thymus stromal cells from human samples. Similarly, to what was observed in the murine tissue, human Postn+ MSC were found to express high levels of CCL19 and BMP4. Conclusion These data indicate that specific mesenchymal cell subsets in the thymus are important mediators of thymus regeneration. Moreover, adoptive transfer of MSC subsets may enable improved T cell recovery in the setting of bone marrow transplantation and perhaps other settings of T cell deficiency. Disclosures Scadden: Novartis: Other: Sponsored research; Bone Therapeutics: Consultancy; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Editas Medicine: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Fog Pharma: Consultancy; Red Oak Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy, Equity Ownership; Clear Creek Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; LifeVaultBio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

In-Vivo Adsorption of Iso-Haemagglutinin (IHA) Antibodies By Donor Type Red Cell Transfusion during Conditioning Is a Safe and Effective Method to Overcome Major ABO Incompatibility-Related Acute Hemolytic Reactions in Stem Cell Transplant Using Bone Marrow As Stem Cell Graft Source

Introduction: ABO blood group incompatibility is not a barrier to performing allogeneic stem cell transplant, but may result in life-threatening acute hemolytic reactions as well as pure red cell aplasia. As stem cell product manipulation is cumbersome and may entail cell loss, attempts have been made in the past to reduce IHA titers in-vivo either by donor type red cell transfusion or using frozen plasma in peripheral blood stem cell (PBSC) transplants (Scholl et al. Transfusion 2005; Damodar et al. BMT 2005). The efficacy of such strategies have not been described in Bone marrow transplant (BMT) setting. We are reporting the effectiveness and safety of donor type red cell infusion during conditioning as a method of reducing acute hemolytic reaction during transplant while using unmanipulated marrow as stem cell source (BMT). Materials and Methods: We retrospectively analyzed 241 consecutive allogeneic BMTs for beta thalassemia major, between August 2015 and July 2019 out of which 82 were ABO mismatched transplants, either major (n=30) or minor (n=40), or bidirectional (n =12) mismatched. Infusion of donor type red blood cell during conditioning after the infusion of Anti-thymocyte globulin (ATG) and post-transplant complication of acute hemolysis were determined by retrospective review of individual medical records. When there is a major ABO incompatibility and IHA titers against the donor were &gt; 1:64, a single unit of donor type Packed Red Blood cell (PRBC) was divided into 4 aliquots, irradiated and administered over 4 days at increasing incremental volumes once daily over 4 days if tolerated (Day 1 - 5 ml, Day 2 - 10ml, Day 3- 20-30 ml, Day 4 - 40-60 ml) (Fig.1). Patients were watched carefully for febrile reactions and hemoglobinuria and mild reactions were tolerated. If no clinical evidence of severe hemolytic reaction, bone marrow was infused without manipulation on the day of transplant. Results: Out of 30 patients with major ABO incompatibility, 13 patients had titers more than 1:64 (highest was 1:2048) and hence received donor type PRBC infusion in small incremental doses. Eight patients showed evidence of some hemolysis (4 during infusion of donor type PRBC aliquot and 4 showed increase in indirect bilirubin with marrow infusion) which was managed conservatively with hydration. None of the patients developed severe hemolytic or anaphylactic reaction at the time of marrow infusion. Post infusion of donor type blood, titers were checked in 7 patients. 6 patients had significant reduction in titers (all were less than 1:32) except for 1(titers increased to 1:4096), which was not considered clinically relevant as he tolerated 100mls of donor type PRBC. He also tolerated marrow infusion without any evidence of severe hemolytic reaction. Four more patients with bidirectional mismatch had IHA titers against the donor more than 1:64, hence the same procedure was followed. One of them had mild hemoglobinuria during donor type PRBC infusion and 3 patients had mild hemoglobinuria with rise in indirect bilirubin at the time of marrow infusion. All patients were managed conservatively with hydration. Conclusion: Our experience demonstrated that donor-type PRBC infusion as a method of in-vivo adsorption of IHA antibodies against donor is safe and effective in preventing acute hemolysis in major ABO-mismatched stem cell transplants even in the context where bone marrow is used as graft source. This simple method in addition to avoiding the problems related to product manipulation can also be safely and easily performed in resource limited settings. Disclosures No relevant conflicts of interest to declare.

Platelet Phenotype Prediction from Whole Genome Sequencing in 621 Sickle Cell Disease Patients

Introduction: Patients with sickle cell disease (SCD) are at increased risk of alloimmunization. Platelet refractoriness is a serious known complication and often seen in SCD patients who are heavily transfused and/or in the bone marrow transplantation (BMT) setting. Next generation sequencing (NGS) is an emerging and promising genotyping strategy in the context of blood typing, due its high throughput and its ability to detect both known and novel variants in the patient and donor population. Here we describe an algorithm to predict common and rare human platelet antigens (HPA) from NGS data, and its validation through Sanger sequencing. Design/Methods: Whole genome sequencing (WGS) was performed on stored blood samples from 621 SCD patients enrolled in 2 IRB-approved clinical studies. Our open source software RyLAN (Red Cell and Lymphocyte Antigen prediction from NGS) was utilized to translate WGS data into predicted RBC and platelet phenotypes. The 29 genomic variants interpreted by RyLAN in 6 HPA genes were correlated with Sanger sequencing. Results: Our study cohort consisted of 621 SCD patients (485 HbSS, 21 HbSβ0, 29 HbSβ+, 84 HbSC, 1HbS O Arab, and 1 HbSD). The mean age was 34.3 years, and 46% were male. Previous red cell transfusions were recorded in 62% of patients, and 3% were documented as never transfused. RyLAN software was executed as a singularity container in multithreaded mode, completing analysis of all 621 .bam WGS files in 18 hours. RyLAN predicted 237 unique extended platelet phenotype combinations in this cohort, with an average read depth of 33 in genomic areas of interest. Predictions for 10 platelet antigens in 26 participants, including rare phenotypes like HPA-25bw+ and HPA-13bw+, were confirmed by bidirectional Sanger. Conclusions: We describe an efficient, open-source algorithm used to interpret 6 HPA genes from WGS in a large SCD cohort. WGS, in conjunction with the RyLAN algorithm, demonstrated 100% accuracy in predicting common and rare HPA genomic variants. Future studies are needed to refine WGS algorithms in SCD, and to examine the possible value of this technology in HPA alloantibody identification workups, optimal platelet product allocation, and donor recruitment. Disclosures No relevant conflicts of interest to declare.

Next-Generation Sequencing Analysis in Posttransplant Relapsed Acute Myeloid Leukemia Reveals Clonal Evolution and Donor-Derived Germline Variant Indicating Bone Marrow Chimerism

Abstract The era of next-generation sequencing (NGS) has led to a deeper understanding of the genetic complexity and heterogeneity of this disease, in addition to revealing mechanisms of disease relapse. Clinical NGS is becoming routine in clinical practice in both solid organ and hematologic malignancies to identify molecular markers of disease that might assist with diagnosis, prognosis, and the treatment of cancer. These tumor-specific markers also enable treatment response monitoring, as they serve as clonal markers unique to the disease. Continuous molecular monitoring also allows identification of disease recurrence with potentially new actionable mutations. This practice is complicated in the setting of allogeneic bone marrow transplant, as the admixtures of donor and recipient DNA pose unique challenges to NGS interpretation. This case highlights the importance of systematic methodological interpretation of NGS results to better understand the clinical significance and impact of new mutations discovered posttransplant and reveals another potential application of NGS for bone marrow engraftment analysis.

2021 - CHOLINERGIC INFLUENCE OF BONE MARROW VASCULAR MICROENVIRONMENT INFLUENCES HEMATOPOIETIC STEM CELL MOBILIZATION

The regulation of HSC movement between the bone marrow (BM) microenvironment and the circulation remains an important clinical target in the setting of BM transplantation. The influence of parts of the nervous system, the sympathetic arm in particular, on BM niche function and mobilization have been investigated. However, the basal role of the cholinergic system in regulating HSC mobilisation is yet to be elucidated. We show that the cholinergic system and specifically the nicotinic -7 acetylcholine receptor (Chrna7) mediates alterations in the structure and function of the BM sinusoidal endothelial cells as well as perivascular stromal cells (LepR+). Inhibition of this receptor with MLA leads to a loss of integrity in the BM sinusoidal structures along with a loss in perivascular niche stem cell factor production. Blocking Chrna7 leads to mobilisation of functional HSCs without skewing that is typically present in G-CSF mobilization. This is suggested by single cell RNA sequencing of MLA mobilised peripheral HSCs that indicates a more balanced genetic lineage signature compared to those mobilised with G-CSF. In addition, administering MLA during G-CSF treatment leads to a synergetic effect, while stimulation of the Chrna7 receptor with GTS-21 blunts G-CSF induced HSC mobilisation. These findings suggest a novel role for the peripheral cholinergic system in HSC mobilisation via niche cell alterations that provides a G-CSF independent alternative route for BM transplantation.