Myeloid Recovery Research Articles

Deciphering bone marrow engraftment after allogeneic stem cell transplantation in humans using single-cell analyses.

BACKGROUNDDonor cell engraftment is a prerequisite of successful allogeneic hematopoietic stem cell transplantation. Based on peripheral blood analyses, it is characterized by early myeloid recovery and T and B cell lymphopenia. However, cellular networks associated with bone marrow engraftment of allogeneic human cells have been poorly described.METHODSMass cytometry and CITE-Seq analyses were performed on bone marrow cells 3 months after transplantation in patients with acute myelogenous leukemia.RESULTSMass cytometric analyses in 26 patients and 20 healthy controls disclosed profound alterations in myeloid and B cell progenitors, with a shift toward terminal myeloid differentiation and decreased B cell progenitors. Unsupervised analysis separated recipients into 2 groups, one of them being driven by previous graft-versus-host disease (R2 patients). We then used single-cell CITE-Seq to decipher engraftment, which resolved 36 clusters, encompassing all bone marrow cellular components. Hematopoiesis in transplant recipients was sustained by committed myeloid and erythroid progenitors in a setting of monocyte-, NK cell-, and T cell-mediated inflammation. Gene expression revealed major pathways in transplant recipients, namely, TNF-α signaling via NF-κB and the IFN-γ response. The hallmark of allograft rejection was consistently found in clusters from transplant recipients, especially in R2 recipients.CONCLUSIONBone marrow cell engraftment of allogeneic donor cells is characterized by a state of emergency hematopoiesis in the setting of an allogeneic response driving inflammation.FUNDINGThis study was supported by the French National Cancer Institute (Institut National du Cancer; PLBIO19-239) and by an unrestricted research grant by Alexion Pharmaceuticals.

Prognostic value of postinduction medullary myeloid recovery by flow cytometry in acute myeloid leukemia.

Risk stratification and treatment response evaluation are key features in acute myeloid leukemia (AML) management. Immunophenotypic and molecular approaches all rely on the detection of persisting leukemic cells by measurable residual disease techniques. A new approach is proposed here by assessing medullary myeloid maturation by flow cytometry through a myeloid progenitor ratio (MPR). The normal MPR range was defined using reference normal bone marrows (n=48). MPR was considered balanced if between 1 and 4 and unbalanced if<1 or>4. MPR was retrospectively assessed at baseline and post-induction for 206 newly diagnosed AML patients eligible for intensive treatment from two different French centers. All AML baseline MPR were unbalanced and thus significantly different from normal MPR (p<0.0001). Patients with an unbalanced MPR after induction had worse 3-year overall survival (OS) (44.4%vs. 80.2%, HR, 2.96; 95% CI, 1.81-4.84, p<0.0001) and 3-year relapse free survival (RFS) (38.7%vs. 64.4%, HR, 2.11; 95% CI, 1.39-3.18, p<0.001). In multivariate analysis, postinduction unbalanced MPR was significantly associated with shorter OS and RFS regardless of the European LeukemiaNet 2010 risk stratification or NPM1/FLT3-ITD status. A balanced postinduction MPR conversely conferred favorable outcomes and reflects medullary myeloid recovery.

Comparison of outcomes for patients with acute myeloid leukemia undergoing haploidentical stem cell transplantation in first and second complete remission.

There was no consensus on whether prognostic advantages existed when transplant conducted at first complete remission (CR1) stage than at second complete remission (CR2) stage for patients with AML who received haploidentical hematological stem cell transplantation (haplo-HSCT). In 768 consecutive AML patients who received haplo-HSCT from January 2014 to December 2017, a 1:2 ratio matched-pair analysis was performed, 69 patients who in CR2 group and 138 CR1 patients were enrolled. Hematopoietic recovery, graft versus host disease (GVHD), relapse, transplant related mortality (TRM), disease-free survival (DFS) and overall survival (OS) were compared in two groups, and further evaluated in low-, intermediate-, and high-risk subgroups. The cumulative incidences of 30-day myeloid recovery and 90-day platelet recovery were comparable in CR1 and CR2 groups. The cumulative incidences of grade II-IV and grade III-IV aGVHD were not significantly different. The cumulative incidences of relapse at 3-year and 5-year in these two groups were 12.4% versus 11.6% (P = 0.880) and 12.4% versus 17.5% (P = 0.322). The cumulative incidences of TRM at 3-year and 5-year were both 10.9% versus 23.2% (P = 0.019). The probability of DFS at 3-year and 5-year were 76.7% versus 65.2% (P = 0.029) and 76.7% versus 59.3% (P = 0.009). The probability of OS at 3-year and 5-year were 81.8% versus 68.1% (P = 0.026) and 76.7% versus 59.3% (P = 0.026). In the intermediate-risk group, TRM was lower in CR1 group, DFS and OS of CR1 group were superior to CR2 group. In conclusion, haplo-HSCT at CR1 stage was of better prognosis for intermediate-risk AML patients than at CR2 stage.

Mitochondrial pyruvate metabolism and glutaminolysis toggle steady-state and emergency myelopoiesis.

To define the metabolic requirements of hematopoiesis, we examined blood lineages in mice conditionally deficient in genes required for long-chain fatty acid oxidation (Cpt2), glutaminolysis (Gls), or mitochondrial pyruvate import (Mpc2). Genetic ablation of Cpt2 or Gls minimally impacted most blood lineages. In contrast, deletion of Mpc2 led to a sharp decline in mature myeloid cells and a slower reduction in T cells, whereas other hematopoietic lineages were unaffected. Yet MPC2-deficient monocytes and neutrophils rapidly recovered due to a transient and specific increase in myeloid progenitor proliferation. Competitive bone marrow chimera and stable isotope tracing experiments demonstrated that this proliferative burst was progenitor intrinsic and accompanied by a metabolic switch to glutaminolysis. Myeloid recovery after loss of MPC2 or cyclophosphamide treatment was delayed in the absence of GLS. Reciprocally, MPC2 was not required for myeloid recovery after cyclophosphamide treatment. Thus, mitochondrial pyruvate metabolism maintains myelopoiesis under steady-state conditions, while glutaminolysis in progenitors promotes emergency myelopoiesis.

ANALYSIS OF B-LYMPHOCYTE MATURATION KINETICS WITH A 10-COLOR BCP-ALL MINIMAL RESIDUAL DISEASE DETECTION TUBE BY FLOW CYTOMETRY: A COMPARISON OF REGENERATING BONE MARROW BEFORE AND AFTER HEMATOPOIETIC STEM CELL TRANSPLANTATION

Minimal residual disease (MRD) measurement in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has been consolidated in the literature as the strongest adverse prognostic factor for overall and disease-free survival in both child and adult studies. However, only recently has multiparametric flow cytometry (MFC) been fully standardized with the addition of new markers and acquisition of a higher number of cellular events, which can improve the objectivity and sensitivity of the test. This prospective study aims to test a single 10-color flow cytometric assay compared to standardized 8-color two tubes in patients with BCP-ALL before bone marrow transplantation, follow by analysis of the kinetic of medullary B-cell recovery after transplant. Prospective analysis of 75 consecutive patients (pcts) transplanted between 2019/January and 2022/May, with MRD evaluation in the month before HSCT, day30, day100 and day360. Flow cytometry was performed on bone marrow samples after bulk-lysis with ammonium chloride when necessary, and subsequent staining with antibody panels. A 10-color tube included the backbone markers CD19, CD45, CD34, CD10, and CD20, in addition to CD38 and CD81 to improve separation between normal/reactive and malignant BCP, and four discriminatory markers placed in the same tube (CD66c and CD123 in PE, CD73 in BV605, and CD304 in APC-R700). This tube was compared to the standardized EuroFlow®method, which was composed of two 8-color tubes previously described. FACSCantoII™and FACSLyric™flow cytometry's were used. Data were analyzed with Infinicyt. The achieved sensitivity and percentages of lymphoblasts, normal mature and precursor B-cells, plasmatic cells and stromal cells were compared before and after transplantation. Statistical analyses were performed in SPSS Statistics v.20.0 software. 34 ALL (42.6%), being 26 BCP-ALL and 8 high-risk T-ALL, and 41 AML (57.4%), median age 31.6 (4.1-62.5 years) for ALL and 42.9 (0.6-74.2 years) for AML; 45(60%) in first complete remission (CR1), 21 CR2 (28%), one CR3 and 7 active diseases. Among 64 patients in morphologic remission, we found 38(59.4%) MRD negative and 26(40.6%) MRD positive before transplant. Pos-transplantation MRD and maturation analysis was possible in 26 pcts at d30, 24 at d60, 57 at d100 and 18 at d360. All but two samples had more than 9.000.000 cell events acquired, median lower limit of detection (LoD) was 0,0002% and lower limit of quantification (LoQ) was 0,0005% in both assays. All patients had the same percentages of lymphoblasts, normal mature and precursor B-cells, plasmatic cells, in the two different assays. The posttransplant B-cell recovery kinetic was similar between BCP-ALL, T-cell ALL and myeloid leukemia recovery after transplantation, and different between adults and children at day100 and day360, but not in day30. This study showed that this 10-color single tube strategy achieves the same sensitivity as the 8-color assay and allows correct identification of MRD, achieving maximum sensitivities and specificities in a single tube, and still maintaining the best discrimination between pathological B cells and normal B cell precursors. B-cell maturation kinetics were similar between leukemia types, and slightly different between adults and children. Additional studies and a larger sample size are needed to confirm these findings.

Assessment of safety and immunogenicity of MHC homozygous iPSC-derived CD34+ hematopoietic progenitors in an NHP model

AbstractAdministration of ex vivo expanded somatic myeloid progenitors has been explored as a way to facilitate a more rapid myeloid recovery and improve overall survival after myeloablation. Recent advances in induced pluripotent stem cell (iPSC) technologies have created alternative platforms for supplying off-the-shelf immunologically compatible myeloid progenitors, including cellular products derived from major histocompatibility complex (MHC) homozygous superdonors, potentially increasing the availability of MHC-matching cells and maximizing the utility of stem cell banking. However, the teratogenic and tumorigenic potential of iPSC-derived progenitor cells and whether they will induce alloreactive antibodies upon transfer remain unclear. We evaluated the safety and efficacy of using CD34+CD45+ hematopoietic progenitors derived from MHC homozygous iPSCs (iHPs) to treat cytopenia after myeloablative hematopoietic stem cell (HSC) transplantation in a Mauritian cynomolgus macaque (MCM) nonhuman primate (NHP) model. We demonstrated that infusion of iHPs was well tolerated and safe, observing no teratomas or tumors in the MCMs up to 1 year after HSC transplantation and iHP infusion. Importantly, the iHPs also did not induce significant levels of alloantibodies in MHC-matched or -mismatched immunocompetent MCMs, even after increasing MHC expression on iHPs with interferon-γ. These results support the feasibility of iHP use in the setting of myeloablation and suggest that iHP products pose a low risk of inducing alloreactive antibodies.

Infusion of Non-HLA-Matched Off-the-Shelf Ex Vivo Expanded Cord Blood Progenitors in Patients Undergoing Cord Blood Transplantation: Result of a Phase II Clinical Trial.

Recipients of myeloablative cord blood transplants (CBT) are known to experience delayed hematopoietic recovery and an increased risk of transplant related mortality (TRM). We developed methods for ex vivo expansion and cryopreservation of CB stem and progenitor cells. 15 patients with hematologic malignancies were enrolled in this single center phase II trial between September 2010 and August 2012 to assess the safety of infusing a non-HLA-matched expanded CB product to bolster a conventional CBT. On the day of transplant, an infusion of the expanded CB product followed the primary graft (1 or 2 unmanipulated CB units). All patients engrafted. Median time to neutrophil and platelet recovery was 19 and 35 days, respectively. Early myelomonocytic recovery was almost entirely due to cells arising from the non-HLA-matched expansion product and were no longer detected at day 14 in all but 2 patients. The probability of 3-years disease free survival was 86%. No TRM was observed throughout the study period, and only 2 patients relapsed. All patients presented with grade II acute graft-versus-host disease (aGVHD) at a median time of 32 days, with no grade III-IV aGVHD observed. At 2 years only 2 patients remain on immunosuppressive therapy for mild chronic GVHD. This phase II safety study demonstrate that infusion of an off-the-shelf non-HLA-matched expanded CB product in addition to a conventional CB graft was safe and led to sustained myeloid recovery. Based on these encouraging results, a prospective multicenter randomized trial utilizing this product has been conducted and results will be soon released. ClinicalTrials.gov Identifier: NCT01175785.

Clinical Translation of SC-DARIC33: A Pharmacologically Controlled CD33-Targeted Anti-AML CAR T Cell Product Regulated By Low Nanomolar Concentrations of Rapamycin

Background: Acute myeloid leukemia (AML) chimeric antigen receptor (CAR) T cell therapies are at early stages of testing in human clinical trials. We previously described the design of a CD33-specific dimerizing agent regulated immunoreceptor complex (DARIC33) that, in the presence of rapamycin (RAPA), switches from an “OFF” to “ON” state that activates T cells in response to tumor antigen. Here, we describe RAPA controlled activation and anti-AML activity of DARIC33 using human AML xenograft NSG mouse models and GMP-compliant T cell manufacturing methodologies. We find that low nanomolar whole blood concentrations of RAPA, below levels used for immunosuppression, are needed for DARIC33 to become active in vivo and exhibit potent CD33-specific anti-AML activity.Methods: Clinical scale, DARIC33 and control cell products were manufactured by Seattle Children's Therapeutics (SC-DARIC33) by stimulating equal numbers of CD4+ and CD8+ T cells with anti-CD3/CD28 microbeads in closed gas-permeable culture vessels followed by lentiviral vector transduction and expansion in serum-free media supplemented with IL7, IL15 and IL21. The activation and anti-AML activity of SC-DARIC33 assayed in vitro by measuring cytokine production or lysis of chromium labeled target cells in the presence or absence of RAPA. NSG mice inoculated with either 1x10 6 luciferase expressing MV4-11 (CD33+) or 0.5x10 6 Raji cells expressing a huCD33 transgene (Raji.CD33, CD19+/CD33+) were treated with SC-DARIC33 and RAPA. Concentrations of RAPA in mouse blood were quantified by LC-MS/MS (Charles River).Results: Donor matched SC-DARIC33 and control CD19 CAR T cell products exhibited similar surface markers of engraftment fitness (CD62L+CD45RA+) and capacity for anti-tumor (CD27+CCR7+) effector function. Following coculture of SC-DARIC33 and Raji.CD33 cells without RAPA, concentrations of IL-2, TNF-α or IFN-γ were not increased in comparison to Raji.CD33 cells cocultured with mock T cell products. However, when exposed to 1 nM RAPA and Raji.CD33 targets, SC-DARIC33 produced cytokines in quantities similar to (Fig A). To determine the concentration of RAPA required to activate SC-DARIC33 in patients, DARIC33 T cells, CD33+ AML targets, and graded concentrations of RAPA were added to allogeneic human whole blood samples and plasma was recovered after 24 hours of incubation. RAPA addition increased IFN-g release with an apparent EC50 = 2.6nM (Fig B).Anti-AML SC-DARIC33 activity and whole blood [RAPA WB] in tumor bearing mice were determined and compared to pediatric RAPA pharmacokinetic models to select appropriate clinical RAPA dose schedules. NSG mice inoculated with Raji.CD33 tumors treated with either 1x10 7 CD19 CAR T cells or 3x10 7 SC-DARIC33 T cells, followed by RAPA 0.1 mg/kg IP QOD, exhibited stringent control of tumor growth, demonstrating a 3:1 cell dose equivalency (Fig C). AML progression was also inhibited when NSG mice were inoculated with MV411 AML cells and subsequently treated with 10 7 SC-DARIC33 followed by 0.01 mg/kg RAPA QD (Fig D). Blood samples from tumor bearing mice obtained on day 15, 2 hours post RAPA administration, showed [RAPA WB] = 2.3 ± 1.3 ng/mL, overlapping with the in vitroand indicating very low concentrations of RAPA effectively modulate the “OFF-to-ON” state transition. Population PK models simulating various RAPA doses and schedules in pediatric patients found oral daily dosing of 0.5 mg/m 2 RAPA will achieve [RAPA WB] = 1-3 ng/mL in most patients (Fig E).Conclusion: Evaluation of GMP cell products and RAPA PK demonstrate that very low doses of RAPA are sufficient to regulate SC-DARIC33. To establish safety of SC-DARIC33 in humans, an upcoming phase 1 trial clinical trial evaluating SC-DARIC33 in pediatric AML patients will test escalating cell doses followed by low-dose RAPA administration from post T cell infusion days 2-21 using a Bayesian optimal interval (BOIN) design. Peripheral blood samples will be monitored for CD33+ myeloid cell recovery after cessation of RAPA dosing. These data will establish safety and support the feasibility of SC-DARIC33 CAR T cells to be reversibly modulated in an “OFF-ON-OFF” fashion by intermittent low-dose RAPA administration. [Display omitted] DisclosuresPrice: bluebird, bio: Current Employment. Zhang: bluebird, bio: Current Employment. Sundaram: bluebird, bio: Current Employment. Lewis: bluebird, bio: Current Employment. Bilic: C4 Therapeutics: Current Employment. Xia: bluebird, bio: Current Employment. Krostag: bluebird, bio: Ended employment in the past 24 months. So: bluebird, bio: Current Employment. Martin: bluebird, bio: Current Employment. Leung: bluebird, bio: Ended employment in the past 24 months. Astrakhan: bluebird, bio: Current Employment. Pogson: bluebird, bio: Current Employment. Jarjour: bluebird, bio: Current Employment. Jensen: bluebird, bio: Research Funding.

Infusion of iPSC-Derived CD34 + Hematopoietic Progenitors Following Myeloablative HSC Transplantation and Assessment of Their Immunogenicity in MHC-Defined Nonhuman Primate Model

Bone marrow suppression and associated neutropenia that renders the patient more susceptible to infection is a major complication and limiting factor for current chemotherapy. Strategies aimed at neutrophil reconstitution using transfusions or accelerating neutrophil recovery with G-CSF show a limited effect in reducing the rate of infection. To overcome these limitations, administration of ex vivo expanded somatic hematopoietic progenitors (HPs) has been explored as a way to facilitate a more rapid myeloid recovery and improve overall survival following myeloablation. Recent advances in reprogramming and induced pluripotent stem cell (iPSC) technologies have created alternative platforms for off-the-shelf supply of immunologically compatible HPs, including cellular products derived from MHC homozygous superdonors which can increase the degree of MHC matching and may provide a maximum utility for stem cell banking. In these studies, we developed Mauritian cynomolgus macaque (MCM) model to evaluate the utility and safety of CD34 +CD45 + hematopoietic progenitors derived from iPSCs (iHPs) generated from MHC homozygous animals in the treatment of cytopenia following myeloablative stem cell transplantation. MCM iPSCs were generated from MHC homozygous animals and used to generate iHPs in coculture with OP9. Three groups of MCMs underwent myeloablative total body irradiation (TBI) followed by transplantation of cryopreserved autologous CD34 + HSPCs. Four days after autologous HSPC transplantation, animals received transfusion of 30x10 6/kg cryopreserved iCD34 + cells tagged with eGFP or tdTomato from homozygous MHC-matched iPSCs (group 2) or homozygous MHC mismatched iPSCs (group 3). We have demonstrated that infusion of iHPs is safe and well tolerated. No teratoma or tumor formation was observed in animals one year after infusion of iHPs. Although we detected few iPSC derived hematopoietic cells in the blood within 1-week post-infusion, we did not see any significant differences in blood counts or other peripheral blood parameters between all animal groups. Intriguingly we found footprints of iPSC-derived cells in the colon, lymph node, skin and brain specimens collected 25 days after iHP infusion from one animal. This suggests a possibility of migration and retention within these tissues of iHP and iHP-derived myeloid cells. To assess immunogenicity, MHC homozygous iHPs were infused into immunocompetent MCMs across different MHC barriers. 10x10 6/kg of cryopreserved iCD34+ from homozygous iPSCs were transfused every week for 3 weeks, into animals that were divided into 4 groups namely autologous (group I), MHC matched homozygous (group II), MHC matched heterozygous (group III) and MHC mismatch (group IV). Overall, these studies revealed low immunogenicity of MHC homozygous iHPs in MHC matched homozygous and heterozygous animals, while weak immune response was detected following iHP infusion in some MHC mismatched animals. DisclosuresSlukvin: Cynata Therapeutics: Consultancy, Current equity holder in publicly-traded company.

First-Line Unrelated Double-Unit Umbilical Cord Blood Transplantation for Acquired Severe Aplastic Anemia

We analyzed the outcomes of 14 patients with severe aplastic anemia (SAA) who received first-line double-unit cord blood transplantation (dUCBT). Patients' median age was 24.5 years (range, 10-44 years). The median numbers of infused nucleated and CD34+ cells were 5.48×107/kg (range, 3.33-7.96×107/kg) and 2.30×105/kg (range, 0.86-3.97×105/kg), respectively. One patient died 5 days after transplantation. Three of the 13 patients acquired autologous myeloid recovery. Neutrophil engraftment was observed in 10 patients (76.29%), and the median time of neutrophil recovery was 19 days (range, 15-40 days). Platelet engraftment was observed in 7 cases (53.8%), and 3 patients experienced platelet graft failure. The median time of platelet recovery was 32 days (range, 22-80 days). The cumulative incidence of grade II-IV acute graft-vs-host disease (GVHD) was 38.5%. One patient demonstrated mild chronic GVHD. After a median follow-up of 61 months (range, 18-102 months), 6 patients were alive. The predicted 5-year overall survival and GVHD-free, failure-free survival rates were 42.9% ± 13.2% and 14.3% ± 9.4%, respectively. The first-line dUCBT for SAA is still primarily evaluated through multicenter prospective clinical trials by an optimal conditioning regimen, cell dose, and other graft and transplantation-related factors.

Outcomes of Unrelated Cord Blood Transplantation for 18 Patients with Severe Aplastic Anemia

Objective To evaluate the outcomes of unrelated cord blood transplantation (UCBT) for severe aplastic anemia (SAA).Methods The clinical data of 18 SAA patients, including 9 very severe aplastic anemia (vSAA), received UCBT from Sep 2009 to Feb 2017 were analyzed retrospectively. Three patients received a single unrelated cord blood unit, fifteen patients received double unrelated cord blood units. One patient was 4/6 HLA match, nine patients were 5/6 HLA match, eight patients were 6/6 HLA match. The conditioning regimens were as follow: 1 patient received modified BU/CY- based regimen, 1 patient received fludarabine (Flu) + antithymocyte globulin (ATG) + cyclophosphamide (CY) + total body irradiation (TBI) regimen, 16 patients received fludarabine (Flu) + antithymocyte globulin (ATG) + cyclophosphamide (CY) regimens. Prophylaxis for graft-versus host disease (GVHD): cyclosporine (CsA) plus short-term methotrexate (MTX).Results There was1 patient early death on day +2, 6 patients experienced primary graft rejection, but 5 of them acquired autologous myeloid recovery, 11 patients were engrafted successfully. The median days of neutrophils(ANC)above 0.5 × 109/L and platelets (PLT) more than 20 × 109/L were 18.5(9~29)days and 26.5(20~48)days, respectively. Six patients (54.5%) developed acute GVHD (aGVHD), 1 for grade I aGVHD, 3 for grade Ⅱ aGVHD, 2 for grade Ⅳ aGVHD. Of 9 patients, 2 occurred chronic GVHD (cGVHD), 1 for mild cGVHD, 1 for severe cGVHD. After a median follow-up of 56(4 ∼ 72)months, 9 patients (50.0%) died, out of which one died of severe aGVHD, the reasons of death were GVHD, severe infection, posttransplant lymphoproliferative disorders (PTLD) and transplant-associated thrombotic microangiopathy (TMA). The 5-year estimated overall survival was 46.9% ± 12.3%. No patient relapsed.Conclusion UCBT is an effective therapy for SAA patients who lack an appropriate matched related donor. DisclosuresNo relevant conflicts of interest to declare.

Longitudinal Analysis of COVID-19 Patients Shows Age-Associated T Cell Changes Independent of Ongoing Ill-Health.

ObjectivesThe immunological and inflammatory changes following acute COVID-19 are hugely variable. Persistent clinical symptoms following resolution of initial infection, termed long COVID, are also hugely variable, but association with immunological changes has not been described. We investigate changing immunological parameters in convalescent COVID-19 and interrogate their potential relationships with persistent symptoms.MethodsWe performed paired immunophenotyping at initial SARS-CoV-2 infection and convalescence (n=40, median 68 days) and validated findings in 71 further patients at median 101 days convalescence. Results were compared to 40 pre-pandemic controls. Fatigue and exercise tolerance were assessed as cardinal features of long COVID using the Chalder Fatigue Scale and 6-minute-walk test. The relationships between these clinical outcomes and convalescent immunological results were investigated.ResultsWe identify persistent expansion of intermediate monocytes, effector CD8+, activated CD4+ and CD8+ T cells, and reduced naïve CD4+ and CD8+ T cells at 68 days, with activated CD8+ T cells remaining increased at 101 days. Patients >60 years also demonstrate reduced naïve CD4+ and CD8+ T cells and expanded activated CD4+ T cells at 101 days. Ill-health, fatigue, and reduced exercise tolerance were common in this cohort. These symptoms were not associated with immune cell populations or circulating inflammatory cytokines.ConclusionWe demonstrate myeloid recovery but persistent T cell abnormalities in convalescent COVID-19 patients more than three months after initial infection. These changes are more marked with age and are independent of ongoing subjective ill-health, fatigue and reduced exercise tolerance.

Combining Mobilizing Agents with Busulfan to Reduce Chemotherapy-Based Conditioning for Hematopoietic Stem Cell Transplantation.

In the context of hematopoietic stem cell (HSC) transplantation, conditioning with myelo- and immune-ablative agents is used to eradicate the patient’s diseased cells, generate space in the marrow and suppress immune reactions prior to the infusion of donor HSCs. While conditioning is required for effective and long-lasting HSC engraftment, currently used regimens are also associated with short and long-term side effects on extramedullary tissues and even mortality. Particularly in patients with severe combined immunodeficiency (SCID), who are generally less than 1-year old at the time of transplantation and often suffer from existing comorbidities. There is a pressing need for development of alternative, less toxic conditioning regimens. Hence, we here aimed to improve efficacy of currently used myeloablative protocols by combining busulfan with stem-cell niche-directed therapeutic agents (G-CSF or plerixafor) that are approved for clinical use in stem cell mobilization. T, B and myeloid cell recovery was analyzed in humanized NSG mice after different conditioning regimens. Increasing levels of human leukocyte chimerism were observed in a busulfan dose-dependent manner, showing comparable immune recovery as with total body irradiation in CD34-transplanted NSG mice. Notably, a better T cell reconstitution compared to TBI was observed after busulfan conditioning not only in NSG mice but also in SCID mouse models. Direct effects of reducing the stem cell compartment in the bone marrow were observed after G-CSF and plerixafor administration, as well as in combination with low doses of busulfan. Unfortunately, these direct effects on the stem population in the bone marrow were not reflected in increased human chimerism or immune recovery after CD34 transplantation in NSG mice. These results indicate moderate potential of reduced conditioning regimens for clinical use relevant for all allogeneic transplants.

MBCL-23. PRELIMINARY ANALYSIS OF TREATMENT-RELATED TOXICITIES DURING INDUCTION CHEMOTHERAPY FOR PATIENTS ON THE HEAD START 4 TRIAL

The currently active, prospective multi-center Head Start 4 (HS4) trial for CNS embryonal tumors differs from prior HS I-III trials by utilizing absolute phagocyte count (APC) as a measure of myeloid recovery instead of absolute neutrophil count. The aim of this study was to determine if utilization of APC resulted in unanticipated treatment-related toxicities during induction chemotherapy for patients enrolled on HS4. Review of the RedCap database was conducted for treatment-related CTCAE grade 3 and 4 toxicities. Data were summarized descriptively. Nonparametric statistical methods were used for comparisons. At the time of this most recent analysis, a total of 180 induction cycles were completed for the 57 patients enrolled. Of the 57 patients, nine voluntarily discontinued therapy after completing a median of three cycles each. These patients had a higher number of documented infections (59% versus 24%, p=0.0004). Veno-occlusive disease (VOD) occurred in five patients, three of whom voluntarily discontinued therapy. Since the protocol amendment utilizing milligram per kilogram dosing for patients less than six years of age, there have been no documented episodes of VOD. The overall toxicities for this cohort were comparable to those reported for induction chemotherapy in HS I-II trials. The toxic death rate is lower for HS4 compared to HS I-II (0.018% versus 4.7–6%) (Chi et al 2004). Other than the high rate of infection, possibly associated with shorter duration of the immediately prior cycles, the use of APC as part of a dose-compression strategy in HS4 does not appear associated with more significant toxicities.

Outcome of allogeneic hematopoietic stem cell transplantation for hypoplastic myelodysplastic syndrome.

The prognosis of patients with hypoplastic myelodysplastic syndrome (hMDS) after receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains unclear. This study aimed to evaluate the outcomes of patients with hMDS after allo-HSCT. Between September 2013 and October 2019, a total of 20 consecutive patients with hMDS and 1 patient with clonal cytopenia of undermined significance (CCUS) who underwent allo-HSCT, which included procedures with 9 matched sibling donors, 2 matched unrelated donors, 4 mismatched unrelated donors and 6 haploidentical donors, were enrolled in this study. The median time for myeloid engraftment was 11days (range 9-17days), and that for platelet engraftment was 10days (range 7-17days). The cumulative incidence (CI) of myeloid and platelet recovery was 95.2 ± 6.0% and 90.5 ± 7.3%, respectively. The CI rates were 40.0 ± 11.3% for grades II-III acute graft-versus-host disease (GVHD), 36.8 ± 11.5% for chronic GVHD and 23.8 ± 9.6% for nonrelapse mortality. No patients experienced relapse. Sixteen surviving patients were followed up for a median of 1113days (range 110-2305days), and the overall survival and relapse-free survival rates were both 72.7 ± 10.6%. This limited retrospective analysis suggests that patients with hMDS had a favorable survival after allo-HSCT.

Engraftment kinetics after transplantation of double unit cord blood grafts combined with haplo-identical CD34+ cells without antithymocyte globulin.

Double unit cord blood (dCB) transplantation (dCBT) is associated with high engraftment rates but delayed myeloid recovery. We investigated adding haplo-identical CD34+ cells to dCB grafts to facilitate early haplo-identical donor-derived neutrophil recovery (optimal bridging) prior to CB engraftment. Seventy-eight adults underwent myeloablation with cyclosporine-A/mycophenolate mofetil immunoprophylaxis (no anti-thymocyte globulin, ATG). CB units (median CD34+ dose 1.1 × 105/kg/unit) had a median 5/8 unit-recipient HLA-match. Haplo-identical grafts had a median CD34+ dose of 5.2 × 106/kg. Of 77 evaluable patients, 75 had sustained CB engraftment that was mediated by a dominant unit and heralded by dominant unit-derived T-cells. Optimal haplo-identical donor-derived myeloid bridging was observed in 34/77 (44%) patients (median recovery 12 days). Other engrafting patients had transient bridging with second nadir preceding CB engraftment [20/77 (26%), median first recovery 12 and second 26.5 days] or no bridge [21/77 (27%), median recovery 25 days]. The 2 (3%) remaining patients had graft failure. Higher haplo-CD34+ dose and better dominant unit-haplo-CD34+ HLA-match significantly improved the likelihood of optimal bridging. Optimally bridged patients were discharged earlier [median 28 versus 36 days]. ATG-free haplo-dCBT can speed neutrophil recovery but successful bridging is not guaranteed due to rapid haplo-identical graft rejection.

Demonstration of the Safety of Post Induction Infusion of Ex-Vivo Expanded Cord Blood Progenitor Cells in Pediatric Patients with Relapsed/Refractory Acute Myelogenous Leukemia

Introduction AML therapy requires intensive and highly immunosuppressive cytotoxic therapy leading to prolonged neutropenia. Prolonged neutropenia leads to highly invasive bacterial and fungal infections causing high rates of infection related morbidity and mortality. Even with improved antimicrobial and antifungal therapy, infectious complications remain a common cause of treatment related morbidity and mortality. There is a large need for strategies to reduce the toxicity of AML therapy and infectious complications in particular. We proposed that post induction infusion of non HLA-matched, off-the-shelf (OTS) ex-vivo expanded cord blood progenitor cells may provide rapid, short term bridging hematopoiesis and truncated period of neutropenia. Here we describe results of a pilot study assessing the safety of infusing this OTS product following FLAG chemotherapy in pediatric patients. Methods Between March 2013 and July 2016, 7 pediatric patients with relapsed/refractory AML or acute leukemia of ambiguous lineage (ALAL) were enrolled in this pilot study to evaluate the use of the OTS product as supportive care following FLAG chemotherapy (n=4 at Fred Hutchinson Cancer Research Center/Seattle Children's Hospital and n=3 at Children's Healthcare of Atlanta). All patients received 1 round of FLAG chemotherapy: total dose of 35 mcg/kg of G-CSF, 150 mg/m2 of Fludarabine, and 10 g/m2 of Cytarabine (Ara-C). OTS products were infused no sooner than 24 hours after last dose of Ara-C. The primary study endpoint was safety of infusing a single OTS product as measured by a. infusion toxicity (&gt; grade 3), b. allo-immune platelet refractoriness, c. treatment related mortality, d. delayed marrow recovery beyond day 42, and e. transfusion related GVHD. Chimerism studies were done on cycle days 15 and 22 (+/- 1 day). To monitor for alloimmunization, pre- and post-treatment panel reactive antibody (PRA) tests were conducted. Bone marrow evaluations were performed at the end of the chemotherapy cycle. Absolute neutrophil count (ANC) of both 100 and 500 were used to assess myeloid recovery. Infectious complications were collected. Results Table 1 shows demographics, disease characteristics and disease status at the time of study enrollment. There were no infusional toxicities reported. No alloimmunization, treatment related mortality, delayed marrow recovery, or transfusion related GVHD occurred in any patient. No adverse events were attributed to the OTS product. Chimerism results showed lack of persistence of OTS products after cycle day 15 (7 days post-infusion of OTS product). The median TNC dose infused was 7.66x107 (range 4.8-9.84 x107), and the median CD34+ cell dose was 9.58x106 (range 8.89-17.6 x106) (Table I). Six of the 7 patients demonstrated an ANC &gt; 100. One patient relapsed prior to count recovery with 33% blasts in the marrow. The median time to reach an ANC &gt; 100 was cycle day 30 (range 25-35). Five patients recovered an ANC &gt; 500. The patient who did not reach this point relapsed on cycle day 33 with 80% blasts in the marrow. For these 5 patients, the median time to reach an ANC &gt; 500 was cycle day 38 (range 28-42) (Fig 1). Of the 7 patients enrolled on the study, 3 achieved complete remission (CR), 3 had persistent disease after initial response, and 1 had treatment failure. Infectious complications occurred in 4 patients and included 3 bacteremias in 3 patients (Strep viridans, E. coli, and Staph aureus), one RSV URI (in patient with concurrent Strep viridans bacteremia) and a single case of C. difficile colitis. There were no induction deaths with all patients completing induction therapy and all but one receiving further treatment. Overall, all study patients met pre-specified safety criteria. Conclusion These preliminary data demonstrate safety of the use of OTS products in the setting of intensive chemotherapy in pediatric patients with relapsed/refractory high-risk AML. More comprehensive randomized studies will enable evaluation of efficacy of OTS products in decreasing infection related morbidity and mortality. Given demonstrated safety of this product, its use in newly diagnosed AML should be considered to eliminate the substantial infection related toxicities. Disclosures Qayed: Bristol-Myers Squibb: Honoraria. Milano:ExCellThera: Research Funding; Amgen: Research Funding. Delaney:Nohla Therapeutics: Employment, Equity Ownership; Biolife Solutions: Membership on an entity's Board of Directors or advisory committees.

Low-dose post-transplant cyclophosphamide and anti-thymocyte globulin as an effective strategy for GVHD prevention in haploidentical patients

BackgroundLow-dose post-transplant cyclophosphamide (PTCy) in conjunction with anti-thymocyte globulin (ATG) appears as a potentially effective graft-versus-host disease (GVHD) prevention strategy in haploidentical hematopoietic cell transplant (haplo-HCT). Our study aims to assess the efficacy of this regimen.MethodsWe extended our prospective study in patients treated with low-dose PTCy (14.5 mg/kg on days 3 and 4) in ATG/granulocyte colony-stimulating factor (G-CSF)-based regimen and compared the results to the contemporary cohort of patients without low-dose PTCy (ATG cohort). Both study cohort and control are transplanted from maternal donor or collateral relatives.ResultsWe identified 239 consecutive patients (ATG-PTCy cohort = 114; ATG cohort = 125). All patients but one in ATG cohort achieved myeloid engraftment by day 30 post-HCT. We found that both the cumulative incidence of 100-day grade III–IV aGvHD and non-relapse-mortality (NRM) in the ATG-PTCy cohort was significantly reduced than that in the ATG group (5% vs 18%; P = 0.003; and 6% vs 15%; P= 0.045); the 2-year cumulative incidences of relapse and overall survival were comparable between the two cohorts (13% vs 14%; P = 0.62; and 83% vs 77%; P = 0.18, respectively). Furthermore, GVHD-free, relapse-free survival (GRFS) was significantly improved in the ATG-PTCy arm (63% vs 48%; P = 0.039). In multivariate analysis, the joint treatment resulted in lower grade II–IV acute GVHD (HR 0.58; P = 0.036), grade III–IV aGvHD (HR 0.28; P = 0.006), chronic GVHD (HR 0.60; P = 0.047), NRM (HR 0.26; P = 0.014), and higher GRFS (HR 0.59; P = 0.021) but slower myeloid and platelet recovery (HR 0.29 and 0.30; both P < 0.001).ConclusionsThese results suggested that ATG/PTCy (low-dose) can reduce both acute and chronic GVHD as compared with standard ATG-based prophylaxis using maternal donor or collateral relatives at particular high GVHD risk.

S1625 REDUCTION OF GRAFT‐VERSUS‐HOST DISEASE USING DUAL LYMPHOCYTE SUPPRESSION WITH ANTI‐THYMOCYTE GLOBULIN AND LOW‐DOSE POST‐TRANSPLANT CYCLOPHOSPHAMIDE IN HAPLOIDENTICAL PATIENTS AT HIGH GVHD RISK

Background:In the setting of haplo HCT, further reducing GVHD merits examination, especially after HCT from maternal donor or collateral relatives with high risk of GVHD occurrence. With the aim to reduce the risk of GVHD without increasing the incidence of relapse, the combined use of antithymocyteglobulin (ATG) and post‐transplant cyclophosphamide (PTCy) has become feasible. However, the combination regimen has not been validated.Aims:There have been only two studies on the combination of ATG and high‐dose PTCy for hematological malignancy without control cohort or with short follow‐up. Our previous single‐center preliminary report on 40 patients seems to support the feasibility of the combined use of ATG/granulocyte colony‐stimulating factor (G‐CSF) and low‐dose PTCy (14.5 mg/kg on days 3 and 4 after HCT). Thus, these results need to be confirmed on a larger number of patients with hematological malignancies.Methods:In an attempt to assess the efficacy of this strategy in Haplo, we conducted a multicenter analysis in patients transplanted from maternal donor or collateral relatives and treated with ATG‐PTCy (low‐dose) regimen, and compared the results to contemporary cohort of patients with same characteristics who received ATG without low‐dose PTCy (ATG cohort). We identified 162 consecutive patients (ATG‐PTCy cohort = 73; ATG cohort = 89) between 2016 and July 2018.Results:All patients but one in ATG cohort achieved myeloid engraftment by day 30 post‐HCT. We found that both the cumulative incidence of 100‐d severe acute GVHD and 2‐y total chronic GVHD in ATG‐PTCy cohort was significantly lower than that in ATG cohort (5% vs 20%; P = 0.007; and 26% vs 49%; P = 0.001). And severe GVHD was the most common cause of death (33%) in the ATG cohort as compared to none in the ATG‐PTCy cohort. Furthermore, TRM was significantly lower in ATG‐PTCy cohort (4% vs 14%; P = 0.039).In contrast, myeloid recovery was significantly slower (median, 15d vs 12d; p &lt; 0.0001) and 100‐d platelet recovery was significantly lower in the ATG‐PTCy group (87% vs 98%; P = 0.0002). The 100d cumulative incidence of CMV reactivation in ATG‐PTCy cohort was significantly higher than that in ATG cohort (73% vs 33%; P &lt; 0.001). The 100‐d cumulative incidence of EBV reactivation and PTLD was comparable (25% vs 17%; P = 0.21; and 4% vs 2%; P = 0.49).In multivariate analysis, the combined treatment resulted in lower severe acute GVHD (Hazard Ratio 0.18; P = 0.004), chronic GVHD (HR 0.39; P = 0.010), and TRM (HR 0.24; P = 0.023) but slower myeloid and platelet recovery (HR 0.27 and 0.30; both P &lt; 0.001). No significant differences were found in relapse rate and disease‐free survival according to treatment group. A propensity score technique was used to confirm results of main analysis.Summary/Conclusion:Unmanipulated haploidentical transplantation with ATG/G‐CSF and low‐dose PTCy as a GVHD prevention strategy results in lower rates of both acute and chronic GVHD without influencing the GVL effect for patients transplanted from maternal donor or collateral relatives with high risk of GVHD occurrence.image

Successful donor engraftment and repair of the blood-brain barrier in cerebral adrenoleukodystrophy

AbstractAdrenoleukodystrophy (ALD) is caused by mutations within the X-linked ABCD1 gene, resulting in the inability to transport acylated very long chain fatty acids (VLCFAs) into the peroxisome for degradation. VLCFAs subsequently accumulate in tissues, including the central nervous system. Up to 40% of boys develop a severe progressive demyelinating form of ALD, cerebral ALD, resulting in regions of demyelination observed on brain magnetic resonance imaging that are associated with a “garland ring” of gadolinium contrast enhancement. Gadolinium enhancement indicates blood-brain barrier (BBB) disruption and an active inflammatory disease process. Only hematopoietic cell transplant (HCT) has been shown to halt neurologic progression, although the mechanism of disease arrest is unknown. We evaluated imaging- and transplant-related biomarkers in 66 males who underwent HCT. In 77% of patients, gadolinium contrast resolved by 60 days post-HCT. We determined that time to neutrophil recovery and extent of donor chimerism correlated significantly with time to contrast resolution post-HCT. Graft failure was associated with a significantly slower rate of contrast resolution (P < .0001). Time to neutrophil recovery remained significant in multivariate analysis with other biomarkers (P = .03). Our data suggest that robust donor myeloid recovery is necessary for timely repair of the BBB.