Mixed Chimerism Research Articles

Post-Transplant Cyclophosphamide Associated with Increased Early Mixed Donor Chimerism in Myeloid Malignancies; A Single-Center Experience

Post-Transplant Cyclophosphamide Associated with Increased Early Mixed Donor Chimerism in Myeloid Malignancies; A Single-Center Experience

G-CSF Improves Stable Engraftment in Adults with Sickle Cell Disease Undergoing Nonmyeloablative Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation (HSCT) is curative in patients with sickle cell disease (SCD). HSCT using nonmyeloablative conditioning leads to a mixed chimerism that improves the SCD phenotype but may be complicated by the continued need for immunosuppression and graft loss. Granulocyte colony stimulating factor (G-CSF) may stimulate progenitor cells and is used post-HSCT to reduce the duration of neutropenia and the risk of neutropenic fever. G-CSF has been generally avoided in patients with SCD due to its risk of promoting vaso-occlusion (PMID 19513902). Starting in 7/2016, our institution administered G-CSF after day +5 post-HSCT on the days when the ANC was < 500 x103/µL. In this study, we evaluated whether G-CSF is safe and can improve the duration of neutropenia, infection risk, and donor chimerisms in adults with SCD undergoing non-myeloablative HSCT. We included 34 adults with SCD that underwent allogeneic HSCT from an HLA-matched sibling donor in our institution and were conditioned with alemtuzumab and total body irradiation (TBI) (PMID 33534948) between 11/2011 and 8/2021. Medical records were individually reviewed for data extraction. Linear and categorical outcomes were compared by G-CSF use with the Kruskal-Wallis and chi-square test, respectively. We also compared the Kaplan-Meier curves for discontinuation of immunosuppression with the log-rank method. Median and interquartile range (IQR) are provided. The median age of our cohort was 33 years (IQR, 26 - 45 years), 18 (53%) patients were female, and 30 (88%) had hemoglobin SS genotype. We did not observe statistically significant differences in baseline characteristics between the SCD patients that received G-CSF versus did not receive G-CSF for median age (37 years vs. 32 years), female gender (69% vs. 43%), hemoglobin SS genotype (85% vs. 90%) or median CD34+ dose (8.6 vs. 8.1 x 106/kg), respectively. In the 13 SCD patients that received G-CSF, the median day when it was initiated was day +10 (IQR, 7 - 17) and for a median of 3 days (IQR, 2 - 7 days). The maximum pain score during hospitalization was not significantly different in the SCD group that received G-CSF (median 8, IQR, 7 - 10) compared to those that did not receive G-CSF (median 10, IQR, 9 - 10). Median time to neutrophil engraftment (G-CSF: day 16 vs. no G-CSF: day 22) and duration of neutropenia (G-CSF: 7 days vs. no G-CSF: 9 days) were relatively similar between the 2 groups, while the incidence of neutropenic fevers trended lower with G-CSF treatment (8% vs. 29%, respectively, P=0.1). Whole blood or myeloid chimerism values were significantly higher in the SCD patients that received G-CSF at day +30, +90, +180, and 1 year post-HSCT (Figure 1A). Secondary graft loss was not observed after we implemented G-CSF while 4 out of 21 (19%) of patients experienced secondary graft failure who did not receive post-HSCT G-CSF. Furthermore, in those patients with stable engraftment, we observed a trend for more patients who received G-CSF being successfully tapered off immunosuppression (11/13, 85%) versus those that did not receive G-CSF (10/17, 59%) (Figure 1B). In conclusion, administration of G-CSF to patients with SCD undergoing HSCT conditioned with alemtuzumab/TBI was associated with significantly higher whole blood or myeloid donor chimerism values. This benefit was achieved without a significant increase in pain scores during the hospitalization for HSCT and was associated with trends for a greater proportion of patients discontinuing immunosuppression and less secondary graft loss. Future studies investigating the effects of G-CSF on donor myeloid progenitor cells and on T-cell clearance may guide strategies to improve outcomes for curative therapies in adults with SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Acrobat: A Multicenter Prospective Study to Monitor Chimerism in Post Allogeneic Hemopoietic Cell Transplant Patients with AML, ALL, or MDS for Prediction of Engraftment and Relapse

BACKGROUND: Allogeneic hematopoietic cell transplantation (HCT) remains the treatment of choice for leukemia patients, yet its success is limited at least in part by disease relapse, which can be as high as 50% or more at two years post-HCT. While engraftment post-HCT is routinely measured by semi-quantitative fluorescent PCR of short tandem repeats (STRs), its low sensitivity (1-5%) limits its usefulness as a routine relapse monitoring tool. We have developed an ultra-sensitive, accurate, and precise microchimerism monitoring solution, AlloHeme that can potentially improve allogeneic HCT patient surveillance for engraftment and relapse. NGS-based AlloHeme can accurately measure microchimerism (mC, defined as <1% recipient DNA) with a limit of detection down to 0.035%. We hypothesized that detection of microchimerism and precise measurement of increasing mixed chimerism (iMC) using a highly sensitive test can predict disease relapse in patients post allo-HCT. OBJECTIVES: To study the role of mC and iMC measured by AlloHeme on relapse detection in patients with acute leukemias and myelodysplastic syndrome, we designed a prospective multicenter clinical trial - ACROBAT (Assessment of Chimerism in Recipients of Allogeneic Bone Marrow / Hematopoietic Cell Transplantation using AlloHeme Test). METHODS: The ACROBAT study will enroll approximately 260 allo-HCT recipients with AML, ALL and MDS across about 10 centers in the US. All patients will undergo serial chimerism monitoring in the first two years post-HCT. All AlloHeme testing will be conducted at CareDx's CLIA-certified/CAP-accredited laboratory. Chimerism will be monitored in the whole blood as well as for its constituent CD3+, CD15+ and CD33+ cellular subtypes. Bone marrow (whole bone marrow and CD34+ sub-type) chimerism will also be evaluated at specific timepoints. The study will determine the predictive value of mC and iMC for relapse detection and the lead time to relapse. Univariable and multivariable sub-distributional hazard regression models and joint models of longitudinal and time-to-event data will be used to determine the impact of mC and iMC measured at different time points on relapse post-HCT. A time-dependent ROC curve will be used to investigate the predictive ability of AlloHeme, MRD, and chimerism by STR for relapse detection. CONCLUSIONS: The ACROBAT study is a multicenter prospective clinical study designed to evaluate the role of increasing mixed chimerism and microchimerism for early relapse detection in patients who have undergone allo-HCT for AML, ALL or MDS.

A Model-Informed Precision Busulfan Dosing in Pediatric Patients Undergoing Hematopoietic Cell Transplantation- Real-World Data

Targeted i.v. busulfan dosing in pediatric non-malignant and malignant disorders undergoing HCT has been challenging due to the unpredictable PK of busulfan in young children. In 59 children with b-thalassemia undergoing HCT, we have previously reported that Bu TDM is feasible, and targeted IV Bu resulted in better EFS than untargeted oral bu in these patients (Balasubramanian et al., TCT 2019). However, the incidence of graft rejection was still high, probably due to age-related clearance of busulfan in very young children (Gaziev J et al. Blood. 2010). Precision dosing models result in less inter-patient variability in systemic exposure and better treatment outcomes (Shukla P et al. Front Pharmacol. 2020). We carried out a pilot study of precision busulfan dosing in pediatric patients in collaboration with InsightRX(California, USA). We aimed to evaluate the performance of the InsightRX precision dosing model for busulfan in young patients undergoing HCT and compare with the incidence of mixed chimerism on days 30, 60, and 90 post HCT [> 5% recipient cells], the time to neutrophil and platelet engraftment and the incidence of regimen related toxicities. Thirteen patients were enrolled in the study from June 2021 to May 2022, and the demographics are listed in Table 1. Initial Bu dosing was based on EMA guidelines (Body weight-based dosing, Palmer J et al. BBMT. 2016). Busulfan levels were monitored after doses 1, 5, 9, and 13 using a validated LC-MS/MS method (Desire et al., 2013). The values were entered in the InsightRX TDM algorithm, and a cumulative AUC of 80-90mg.hr/L was targeted. The mean Bu dose per EMA guideline and the actual dose received after precision dosing was compared. Based on InsightRX prediction, ten patients received higher cumulative Bu dosing, and 3 received lower cumulative Bu dosing compared to fixed weight-based dosing (Fig 1A). All patients had lower Bu AUC (<4.6 mg*hr/L) after the 1st dose, while at the end of the 13th dose, most patients achieved therapeutic Bu AUC (Median: 4.8 (3.6-5.8 mg*hr/L, Fig 1B). The interpatient variability in systemic exposure was only 12% with precision dosing, compared to 33% with the trapezoidal rule-based method (Balasubramanian et al., TCT 2019). Irrespective of the busulfan formulation, GST genotype, and underlying disease, the total cumulative target AUC was achieved in all the patients; two patients rejected the graft (one rejected within day+90 and the other rejected late after day+90, both had unrelated donor source and lowered cumulative Bu AUC compared to the other patients), and one developed Sinusoidal obstruction syndrome (despite lower cumulative Bu AUC, therefore cannot be attributed to Bu exposure). Targeting higher cumulative Bu exposure using a model-informed precision dosing software resulted in better HCT outcomes. Our study suggests the feasibility of improved precision of dosing of busulfan in young children undergoing HCT resulting in improved outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

First Documentation of Cell-to-Cell Peroxisome Transfer in Hematopoietic Stem and Progenitor Cells: A Method to "Relieve Stress"?

Cell to cell communication is key to survival. Recently it has been demonstrated that intercellular transfer of mitochondria occurs and can aid in marrow recovery after irradiation. Peroxisomes are a single membrane bound organelle responsible for various functions including lipid metabolism and protecting the cell from oxidative damage by reactive oxygen species (ROS). We have created the first mouse model in which peroxisomes can be tracked in living tissues using GFP tagged with a peroxisome targeting signal sequence (PTS), under the control of the Rosa promoter. Such GFP-PTS animals showed widespread peroxisomal GFP expression in all organs including hematopoietic stem and progenitor cells (HSPC) such as LSK-SLAM. Marrow stroma cell lines were established from GFP-PTS animals. In vitro peroxisome transfer from stroma to HSPC showed a transfer rate of up to 0.5 %, which could be achieved only when cell-to-cell contact took place; the use of transwells inhibited transfer. When HSPCs were simulated to proliferate, peroxisome transfer dropped by 50.1% (p<0.0001). The ability of HSPC to take up peroxisomes in vivo was tested in a model of hematopoietic cell transplant (HCT). Using GFP-PTS mice as recipients with wild-type (WT) marrow donors, we found that after busulfan or radiation (9 Gy) conditioning, peroxisome transfer could occur into WT adoptively HSPC within 48 hours after HCT at a rate of 1%. At 3 months post HCT, bone marrow mononuclear cells (BMMC) and Lin-SCA1+CKIT+ (LSK) were 100% donor and maintained GFP+peroxisomes at a frequency of 0.9% which were transferred from surrounding niche/stroma cells. To test HSPC to HSPC peroxisome transfer, sublethal conditioning (4.5 Gy) of WT was used to generate mixed chimerism models with marrow from GFP-PTS mice used as donor. Total peripheral or marrow engraftment was 45-55% donor as expected at 3 months post-HCT. WT BMMC could take up peroxisomes at a 0.9% frequency, while LSK had a significantly higher peroxisome uptake of 3.9% (p=0.02) indicating greater efficiency. Results were similar if GFP-PTS+ animals were recipients in a sublethal transplant setting. Remarkably, we could also demonstrate that peroxisome transfer can occur between GFP-PTS donor hematopoietic cells and WT cells in the liver, spleen, thymus, and brain after HCT. The genetic deletion of peroxisomes (via pex1 knockout) in animals resulted in ROS levels 10.3-fold over that of wild-type in PBMC, BMMC, and LSK (p<0.0001) as assessed by CellROX assay. Such animals had a severe defect in growth, had normal hematopoiesis, and increased radiation sensitivity indicating the importance of peroxisomes in reducing oxidative stress. To test the hypothesis that peroxisome transfer is utilized in situations of increased oxidative stress, pex1 KO stroma lines were exposed to increasing doses of radiation followed by incubation with GFP-PTS+ BMMC. Radiation treatment enhanced peroxisome transfer 5.1-fold over no-irradiated control (p<0.01), and cells receiving donor peroxisomes had greater viability (84% vs 68% viable, p<0.01) and reduced levels oxidative stress 24.8% compared to cell not receiving peroxisomes (p=0.03). The entire system was reproduced in a zebrafish model expressing GFP-PTS. Sublethally irradiated WT zebrafish engrafted donor GFP-PTS+ marrow at 50% and showed a significantly high rate of peroxisome uptake of into WT HSPS of 30% at 3 months post-HCT, suggesting some species may perform peroxisome transfer at much greater rates than others. In conclusion, we have demonstrated, for the first time, that peroxisome transfer can occur between a wide variety of cell types in vitro and in vivo, and HSPC-to-HSPC peroxisome transfer seems to be the most robust. The ability of peroxisome transfer to rescue cells from radiation-induced ROS and death was demonstrated in vitro while in vivo experiments are proceeding. There may be other far-reaching effects in terms of altering cellular metabolism occurring as a result of peroxisome transfer remaining to be discovered.

Immunosuppression Boost With Mycophenolate Mofetil for Mixed Chimerism in Thalassemia Transplants

Declining mixed chimerism (MC) portending impending graft failure is an undesirable outcome. However, for hemoglobinopathies in a stable state of MC, residual host cells persist without rejection in 30% to 40% of patients after hematopoietic stem cell transplantation (HSCT). Early detection and level of MC have been attributed to be significant in predicting the outcome of MC. Common clinical approach on MC is removal of immunosuppression. We retrospectively evaluated MC in transfusion dependent thalassemia patients who underwent HSCT in our institution between September 2013 and January 2022 to determine the outcome of MC on the basis of our approach of immunosuppression boost in comparison to conventional approach of immunosuppression tapering. Among 90 patients, 22 (24.4 %) had MC at some time point after transplantation with a median follow-up of 496 (67-1492) days. Immunosuppression withdrawal was done in 12 (54.5%) patients, whereas immunosuppression boost was given in 8 (36.3%) patients. In the immunosuppression withdrawal group, 2 (16.6%) patients evolved to complete chimerism, 5(41.6%) patients had persistent MC (PMC), whereas 5 (41.6%) patients had secondary rejection. All these rejections were at median of 186 (89-251) days after transplantation. In the immunosuppression boost group, all patients (n=8) had PMC with no secondary rejection until median follow-up of 255(97-812) days after transplantation. We acknowledge that we need more experience with our unconventional approach of immunosuppression boost to obtain statistical significance in comparison to the conventional approach of tapering of immunosuppression.

Bone marrow chimerism breaks the barrier to pancreatic islet transplantation.

In their recent Cell Reports paper, Chang and colleagues report on a successful strategy to achieve durable mixed hematopoietic chimerism that promotes the engraftment and long-term function of pancreatic islet allotransplants in fully immunocompetent mice without immunosuppression.

Hematopoietic stem cell Transplantation in Children with very Early Onset Inflammatory Bowel Disease Secondary to Monogenic Disorders of immune-dysregulation.

Background: Very early-onset inflammatory bowel disease (VEOIBD) is defined as IBD in children under six years of age. We present outcome data of hematopoietic stem cell transplantation (HSCT) in the above children. Patients and methods: We performed a retrospective study in children under six years of age who underwent HSCT for VEOIBD with an identified monogenic disorder from December 2012 to December 2020. Results: Of the 25 children included, the underlying diagnosis was IL10R deficiency (n = 4), Wiskott-Aldrich syndrome (n = 4), Leukocyte adhesion defect (n = 4), Hyper IgM syndrome (n = 3), Chronic granulomatous disease (n = 2), and one each with XIAP deficiency, severe congenital neutropenia, Omenn syndrome, Hyper IgE syndrome, Griscelli syndrome, MHC Class II deficiency, LRBA deficiency, and IPEX syndrome. Donors included a matched family donor in 10(40%); a matched unrelated donor in 8 (32%), haploidentical in 7 (28%) (T depleted 16%, T replete with post-transplant cyclophosphamide12%). Conditioning was myeloablative in 84% ofHSCTs. We documented engraftment in 22 (88%) children, primary graft failure in 2 children (8%), mixed chimerism in 6 (24%) children with mortality in 4/6 children. Children with a sustained chimerism of > 95% did not have recurrence of any features of IBD. Overall survival was 64%, with a median follow-up of 55 months. Mixed chimerism was associated with a significantly increased risk of mortality (p-value = 0.001). Conclusions: VEOIBD caused by monogenic disorders can be offered HSCT. Early recognition, optimal supportive care, and complete chimerism are essential components to achieving survival.

Antibody based conditioning for allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapeutic option for many patients with hematological malignancies and nonmalignant hematopoietic disorders. To achieve stable engraftment of donor hematopoietic stem cells (HSCs), recipient HSC deletion is needed to create space for incoming donor HSCs and donor HSCs must escape immune rejection by the recipient. Conventional allo-HSCT requires high dose of irradiation and/or chemotherapy to produce sufficient host stem cell and immune system ablation to permit donor HSC engraftment. However, these procedures also result in nonspecific tissue injury that can cause short- and long-term adverse effects as well as incite and amplify graft-versus-host-disease (GVHD). The delivery of targeted radiotherapy to hematopoietic tissues with the use of a radioimmunoconjugate (ROIC) as a part of transplant preparative regimen has shown clinical benefits. ROIC clinical data provide evidence for decreased relapse without increased transplant-related mortality by delivering higher targeted radiation to sites of malignancy than when given in a nontargeted fashion. An alternative approach to allo-HSCT has been developed and tested in preclinical mouse models in which nonmyeloablative preconditioning with low dose of the alkylating agent (busulfan) or lower systemic dose of irradiation combined with co-stimulatory pathway blockade (CTLA4-Ig, anti-CD40L monoclonal antibody) and/or immunosuppressive drugs have been used. Under these conditions, mixed chimerism and transplantation tolerance to fully MHC mismatched donor marrow was observed. Recently, several novel proof-of-concept antibody-mediated preconditioning methods have been developed that can selectively target hematopoietic stem and immune cells with minimal overall toxicity. Antibody-drug-conjugate (ADC) combined with reduced intensity conditioning or high dose ADC as single dose monotherapy have shown promise for allo-HSCT in preclinical models. The purpose of the current review is to discuss the literature exploring antibody-based conditioning that includes native antibody, radiolabeled antibody conjugates, and ADC for allo-HSCT.

CT-314 Multicenter Long-Term Follow Up of Allogeneic Hematopoietic Stem Cell Transplantation With Omidubicel: A Pooled Analysis of Five Prospective Clinical Trials

Allogeneic hematopoietic stem cell transplantation (allo-HCT) with umbilical cord blood (UCB) is limited by the low number of hematopoietic stem cells. Omidubicel is an ex-vivo expanded stem cell product derived from UCB. Prospective clinical trials have demonstrated faster engraftment and fewer infections with omidubicel compared to UCB, but long-term outcomes are unknown. This is a pre-specified pooled secondary analysis of long-term outcomes with allo-HCT using omidubicel from 5 multicenter prospective trials. Patients transplanted with omidubicel in one randomized phase III and four single-arm trials between 2006 and 2020 were followed for up to 10 years post-transplant. Twenty-six international academic transplant centers. Among 116 patients transplanted with either standalone omidubicel (n=92) or omidubicel with supplementary UCB (n=24), 97 (83.6%) engrafted with omidubicel, 11 (9.5%) with UCB, 2 mixed chimerism, 1 unevaluable, and 5 (4.3%) primary graft failure. This study included all patients aside from those engrafted with UCB (n=105). Median age was 42 (range, 2-62), 52.4% male, 30.5% non-white, and 30.5% high/very-high disease risk indices. Most common diseases were AML (40.1%), ALL (26.7%), MDS (13.3%), and sickle cell disease (7.6%). Median follow-up was 22.0 months (range, 0.3-122.5). The 3-year OS and DFS were 62.5% (95%CI, 53.4-73.2) and 56.2% (95%CI, 47.0-67.1), respectively. Common causes of death included disease relapse (n=16) and infection (n=11). Three-year cumulative incidences of chronic GVHD and relapse were 37.8% (95%CI, 27.9-47.6) and 24.3% (95%CI, 16.1-33.3), respectively. Durable trilineage hematopoiesis was observed for up to 10 years. Similarly, median numbers of lymphoid subsets, including CD3+, CD4+, CD8+ T cells, CD19+ B cells, and CD16+/CD56+ NK cells were maintained within normal range for up to 8 years. Secondary graft failure was noted in 5 patients, 3 of whom underwent a second allo-HCT. Secondary hematologic malignancies included donor-derived myeloid neoplasm (dd-MN) (n=1; 40 months post-transplant) and PTLD (n=2; 17 and 20 months), with one death attributed to PTLD. Notably, there was also one case of dd-MN in the control UCB group of the phase III study. Omidubicel demonstrated durable long-term hematopoiesis and immune competence. One case of dd-MN was observed with both omidubicel and control UCB.

CT-368 Discordance of Chimerism in the Blood and Bone Marrow as a Possible Sign of Adverse Outcome After Allo-HSCT

DNA isolated from bone marrow (BM) or peripheral blood (PB) of patients receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT) is used to assess hematopoietic chimerism. Both sources are presumably equivalent for this purpose. Here, we report several allo-HSTC cases with BM chimerism not matching that of PB and vice versa. To investigate the possible association between the disparity of chimerism in paired PB and BM samples and disease features in post-transplant acute myeloid leukemia patients. This study analyzed the STR profiles in the DNA of paired archival BM and PB samples of 43 patients who received allo-HSCT in the National Medical Research Center for Hematology (Moscow, Russia) from 2016 to 2022. Paired BM and PB samples were collected with a time difference of one day or less. Chimerism was assessed by STR-PCR. STR profiles were evaluated by PCR with a COrDIS Plus multiplex kit (Gordiz Ltd, Russia). Patients with mixed hematopoietic chimerism at certain time points during monitoring were included. In 8 patients out of 43 (18.6%), a disparity of chimerism between PB and BM samples was found. In 3 patients (7%) in the early stages after allo-HSCT, chimerism measured in the PB sample was ≥2 times higher than in BM. In 5 (11.6%) patients, the proportion of recipient DNA in BM was twice as high as in PB. In 2 patients, this phenomenon might be due to donor lymphocytes transfusion before measuring chimerism. For 3 patients, no simple explanation for this phenomenon is evident so far. However, all 5 relapsed. Four patients died from the progression of the disease, and 1 underwent a second transplant and maintains remission. The predominance of recipient DNA in PB samples compared to BM, noted in the early stages after allo-HSCT, may not correspond to the real picture due to delayed hematopoietic reconstitution. The study of chimerism in sorted cell populations might provide an explanation for host predominance in BM over PB. Probably, such patients relapsing in the early stages after allo-HSCT could have chimerism split between certain cell lineages. Further studies of chimerism in paired PB and BM samples on extended cohorts of post-transplant patients are required.

Development and characterization of a preclinical total marrow irradiation conditioning-based bone marrow transplant model for sickle cell disease

Sickle cell disease (SCD) is a serious global health problem, and currently, the only curative option is hematopoietic stem cell transplant (HCT). However, myeloablative total body irradiation (TBI)-based HCT is associated with high mortality/morbidity in SCD patients. Therefore, reduced-intensity (2–4 Gy) total body radiation (TBI) is currently used as a conditioning regimen resulting in mixed chimerism with the rescue of the SCD disease characteristic features. However, donor chimerism gradually reduces in a few years, resulting in a relapse of the SCD features, and organ toxicities remained the primary concern for long-term survivors. Targeted marrow irradiation (TMI) is a novel technique developed to deliver radiation to the desired target while sparing vital organs and is successfully used for HCT in refractory/relapsed patients with leukemia. However, it is unknown if TMI will be an effective treatment for a hematological disorder like SCD without adverse effects seen on TBI. Therefore, we examined preclinical feasibility to determine the tolerated dose escalation, its impact on donor engraftment, and reduction in organ damage using our recently developed TMI in the humanized homozygous Berkley SCD mouse model (SS). We show that dose-escalated TMI (8:2) (8 Gy to the bone marrow and 2 Gy to the rest of the body) is tolerated with reduced organ pathology compared with TBI (4:4)-treated mice. Furthermore, with increased SCD control (AA) mice (25 million) donor BM cells, TMI (8:2)-treated mice show successful long-term engraftment while engraftment failed in TBI (2:2)-treated mice. We further evaluated the benefit of dose-escalated TMI and donor cell engraftment in alleviating SCD features. The donor engraftment in SCD mice completely rescues SCD disease features including recovery in RBCs, hematocrit, platelets, and reduced reticulocytes. Moreover, two-photon microscopy imaging of skull BM of transplanted SCD mice shows reduced vessel density and leakiness compared to untreated control SCD mice, indicating vascular recovery post-BMT.

Increased incidence of hematologic malignancies in SCD after HCT in adults with graft failure and mixed chimerism

Lawal et al report on a 45-fold increase in secondary hematologic malignancy in 120 patients following hematopoietic stem cell transplantation (HSCT) for sickle cell disease (SCD), comparable to what has been reported following gene therapy. Notably, the cohort is enriched for older patients and for haploidentical transplant recipients with mixed chimerism following HSCT. These data further support the idea that pre-existing premalignant myeloid clones undergo clonal selection in the setting of nonmyeloablative HSCT and contribute to secondary malignancy.

215.9: Achievement of Persistent Mixed Chimerism in Recipients of Matched and Mismatched Living Donor Kidney Transplants in a Tolerance Induction Protocol

Purpose: The induction of tolerance to kidney transplants after development of specific unresponsiveness of recipient immune cells to donor alloantigens allows elimination of maintenance immunosuppressive (IS) drugs while preventing graft loss due to rejection. We have previously reported successful induction of mixed chimerism and tolerance in recipients of fully HLA-matched living donor kidney and hematopoietic cell (HCT) transplants. This work continues in HLA-mismatched transplants, wherein achieving persistent mixed chimerism is more challenging. Methods: Living donors underwent hematopoietic stem cell mobilization and leukopheresis 6 weeks before kidney donation. The recipient conditioning regimen for the HCT consisted of 10 doses of total lymphoid irradiation (TLI) and 5 doses of anti-thymocyte globulin (ATG) following kidney transplant, with HCT on day 11. The HCT consisted of CD34+ hematopoietic progenitor cells and CD3+ cells. Twenty-nine patients received fully HLA-matched living donor transplants. Twenty-five patients received HLA-mismatched (1-haplotype matched) living donor transplants with an escalating dose of CD3+ cells to enhance HCT engraftment. Six patients recently received HLA-mismatched living donor transplants with substitution of a dose of total body irradiation (TBI) for the final dose of TLI. Results: Of the 29 fully HLA-matched living donor transplants, complete IS drug withdrawal was achieved in 24, all of whom had at least 6 months of persistent mixed chimerism. Of the 24, 17 are alive, well and have been off IS drugs from 4 to 14 years without evidence of rejection. Two off IS drugs died due to cardiovascular events at 2 and 3 years. Two returned to IS drugs after loss of chimerism at 1 year followed by acute rejection at 4 years off IS drugs, and 3 after relapse of kidney disease at 1, 5 and 6 years off IS drugs. In the 25 1-haplotype matched living donor transplants, mixed whole blood chimerism persisted in 10 (mean 20% chimerism) at the end of the 1st year posttransplant while immunosuppression was reduced to tacrolimus monotherapy. Chimerism was lost during the 2nd year in the 5 who were withdrawn from monotherapy; they returned to IS drug(s). Mixed chimerism without rejection has persisted in 3 maintained on IS monotherapy, for up to 5 years. The recent addition of a dose of TBI markedly increased levels and stability of mixed chimerism during the 1st year posttransplant. Conclusions: The loss of chimerism during withdrawal of IS drug monotherapy in the HLA-mismatched transplants may have been due to the low level of whole blood chimerism, a contrast to the HLA-matched. A very low single dose of TBI added to the TLI/ATG conditioning regimen increased early chimerism. The stability of high levels of mixed chimerism after complete IS drug withdrawal during the 2nd year posttransplant is the subject of continuing investigation.

231.2: Selective Bcl-2 Inhibition Promotes Hematopoietic Chimerism and Allograft Tolerance Without Myelosuppressive Complications

Introduction: Hematopoietic stem cell transplantation (HSCT) has been an only approach that reproducibly achieved renal allograft tolerance in humans. However, severe myelosuppression and toxicities after myeloablative conditioning have hampered wider clinical application. To achieve hematopoietic stem cell (HSC) engraftment, it is essential to make space in the bone marrow (BM) niches by depleting host HSCs. This has been achievable only by nonselective treatments such as irradiation or chemotherapeutic drugs. Therefore, a novel approach more selectively capable of depleting HSCs is needed to widen the clinical application of HSCT. Based on murine studies that showed successful induction of mixed chimerism and allograft tolerance without irradiation by Bcl-2 inhibition, we evaluated an FDA approved Bcl-2 inhibitor (Venetoclax, Vntx) in our mixed chimerism inducing conditioning regimen. Methods: Cynomolgus monkeys received kidney and bone marrow transplantation after conditioned with various nonmyeloablative regimens which included total body irradiation (TBI), thymic irradiation (TI) and anti-thymocyte globulin (ATG), followed by a short post-transplant course of costimulatory blockade (CoB) and Cyclosporine. The study groups were consisted of 3Gy TBI, no Vntx (Group A), 1.5Gy TBI, no Vntx (Group B), TBI 1.5Gy with Vntx(10 mg/kg x 11 on days -4 to 6) (Group C), TBI 1.5Gy with Vntx but Belatacept as CoB (Group D), no TBI with Vntx (Group E), no TI with Vntx (Group F), no CoB with Vntx (Group G). Results: Most recipients treated with 3Gy TBI (Group A) developed mixed chimerism and long-term immunosuppression (I.S.)-free renal allograft survival (Table 1), but suffered from transient but severe pancytopenia. With reduced TBI (1.5Gy), all recipients failed to develop chimerism, and 4/5 developed acute rejection. By adding Vntx, despite the dose of TBI being reduced to half (1.5Gy), all six recipients (Group C) developed significantly superior chimerism (Figure 1) than regimens without Bcl-2 inhibition (Groups A and B), and 5/6 achieved long-term I.S.-free allograft without pancytopenia (Figure 2). With Belatacept in place of anti-CD154 mAb, 2/3 achieved chimerism and long-term I.S. free allograft survival. The current studies also revealed that a minimal non-toxic dose of TBI, TI and CoB remain to be essential for long-term I.S.-free renal allograft survival. Without TBI (Group E) or CoB (Group G), the recipients failed to develop chimerism and rejected allografts despite administration of Vntx. Without TI (Group F), all recipients developed mixed chimerism but failed to achieve tolerance. Conclusion: Bcl-2 inhibition with Vntx significantly promoted mixed chimerism and renal allograft tolerance without myelosuppressive complications. A minimal non-toxic dose of TBI, TI, and CoB were still required for chimerism and allograft tolerance induction even with Bcl-2 inhibition.

Haploidentical stem cell transplantation for patients with sickle cell disease: current status

For patients with high-risk sickle cell disease (SCD) without any available matched sibling or unrelated donor, haploidentical stem cell transplantation (haploHCT) expands the availability of this life-saving intervention to nearly all patients who may benefit from HCT. The greatest challenge in haploHCT has been the significant risk of graft failure. Developing a treatment modality which sustains engraftment without increasing the incidence of debilitating graft-versus-host disease (GvHD) remains the ultimate goal. A number of modifications have been explored to overcome the high incidence of graft rejection and severe GvHD including: (1) ex-vivo T-cell depletion (via CD34+ selection, CD3+/CD19+, or TCRαβ+/CD19+ depletion), and (2) in vivo T-cell depletion using unmanipulated grafts followed by post-transplant cyclophosphamide (PTCy) for GvHD prophylaxis. Furthermore, the presence of donor-specific anti-HLA antibodies (DSA) has been associated with an increased risk of both graft failure and poor graft function. Several approaches for desensitization ameliorate this risk when a suitable donor without DSA is not available. In addition to advances in supportive care, the recent demonstration that stable mixed chimerism post-HCT sufficiently sustains symptom-free status has opened the door for less toxic treatment approaches yielding excellent survival outcomes. Though late effects remain uncertain, the goal of finding the least toxic conditioning regimen while providing the highest rate of donor engraftment draws closer within reach. In this review, the authors aim to present the latest findings, challenges, and treatment modalities of this life-saving modality.

Efficacy of a Second Allogeneic Hematopoietic Cell Transplant in Relapsed Acute Myeloid Leukemia: Results of a Systematic Review and Meta-Analysis

Allogeneic hematopoietic cell transplantation (allo-HCT) remains the only known treatment modality that can offer the possibility of cure for acute myeloid leukemia (AML). Unfortunately, relapse and disease progression still occur in more than one third of cases even when patients are allografted in complete hematologic remission (CR). Treatment of AML relapsing after a first allo-HCT is particularly challenging. A second allo-HCT is offered to patients considered fit for the procedure, but reported outcomes have been conflicting. To perform a systematic review and meta-analysis to assess the totality of evidence on the role of a second allo-HCT in patients with AML, we performed a comprehensive literature search using PUBMED/MEDLINE and EMBASE on August 25, 2021, and extracted clinical outcome data relating to benefits (CR, overall survival [OS], and progression-free/disease-free survival [PFS/DFS]) and harms (acute and chronic graft-versus-host disease, non-relapse mortality [NRM], and relapse). The search identified 821 studies. Only 20 studies (n = 2772 patients) met our inclusion criteria. A second allo-HCT resulted in pooled CR, OS, PFS/DFS, NRM and relapse rates of 67%, 34%, 30%, 27%, and 51%, respectively. OS was 2-fold higher when the second allo-HCT was performed in CR (38% versus 17%) and 3-fold higher in patients who had a later relapse from the first allo-HCT (34% versus 10%). There was no apparent difference in pooled OS (hazard ratio = 1.01; 95% confidence interval, 0.78-1.31; P = .94) whether the same original donor or a different one was used. Notwithstanding several limitations apart from the high heterogeneity among included studies, this analysis shows that a second allo-HCT is a reasonable treatment option for relapsed AML. The procedure appears to be more effective when performed in CR and in patients who had a later relapse from the first allo-HCT. The high pooled relapse rates exceeding 50%, even when receiving the second allo-HCT in CR is worrisome and emphasizes the need to incorporate new therapies whether as post-transplantation maintenance or consolidation to mitigate relapse risk. This analysis was limited to patients receiving a second allo-HCT for the sole purpose of treating AML relapse. Accordingly, we caution against extrapolating these findings to other indications such as treatment of graft failure, poor graft function, or mixed donor chimerism.

CD19+ lineage chimerism, an early biomarker after anti-CD19 CAR-T cell therapy in patients previously receiving a hematopoietic stem cell transplantation.

Treatment targeting CD19 by a chimeric antigen receptor expressed on T cells (anti-CD19 CAR-T) has led to a breakthrough in the management and treatment of relapsed and refractory B- cell acute lymphoblastic leukemia (B-ALL). After infusion, the efficacy of anti-CD19 CAR-T is monitored by bone marrow negative minimal residual disease and the absence of peripheral CD19+ B lymphocytes (B-cell aplasia). In patients who have received an allogenic Hematopoietic Stem Cell Transplantation (HSCT) prior to treatment with anti-CD19 CAR-T, monitoring lineage-specific chimerism could be helpful. We found that on 4 patients who received anti-CD19 CAR-T cells after HSCT and achieved early complete response, CD19+ lineage mixed chimerism but not CD3+ lineage mixed chimerism monitored by molecular techniques anticipated earlier than B-cell aplasia determined by flow cytometry, lack of effectiveness of anti-CD19 CAR-T and leukemia relapse. Donor lymphocyte infusions (DLIs) did not prevent relapse but recovered CD3+ full donor chimerism. We suggest that continuous lineage chimerism analysis should be done routinely in patients who receive anti-CD19 CAR-T cells after HSCT and achieve complete remission because it can support early treatment intervention. However, the role of DLI in this setting is unclear, so further prospective studies should be developed.

Immune tolerance induction through haematopoietic chimerism after kidney donation

A 57-year-old man with end-stage kidney disease from type 2 diabetes was assessed at our specialist unit for a kidney transplant to be donated by his brother. The patient was unsensitised and blood group O; his 59-year-old brother was the same blood type and two-haplotype HLA-matched. Both the patient and his brother were enrolled into our immunological tolerance protocol, which combines a haematopoietic stem-cell transplant with a kidney transplant with the goal of inducing sustained mixed chimerism—a state where the recipient's haematopoietic system has a mixture of both host and donor cells.

Busulfan and cyclophosphamide‐based conditioning regimen still holds the promise of being a safe and efficacious regimen for allogeneic transplantation in patients with transfusion‐dependent thalassemia, even in high risk

Busulfan and cyclophosphamide (BuCy)-based regimen has been used as a standard myeloablative chemotherapy for haematopoietic stem cell transplantation in thalassemia. However, treosulfan-based conditioning regimen has emerged due to concerns of toxicities. We retrospectively analysed the safety and efficacy of fludrabine/Bu/Cy/antithymocyte globulin (ATG) versus treosulfan/thiotepa/fludrabine regimens for Hematopoietic Stem Cell Transplant (HSCT) in transfusion-dependent thalassemia (TDT) conducted at our institute (2013-2021). In 75 patients, 36 (48%) received Flu/Bu/Cy/ATG whereas 39 (52%) received Treo/Thio/Flu. Median age was 6 (1-12) and 9 (1-15) years, respectively. Number of patients with Classes I, II, and III were 14, 10, and 12 in Flu/Bu/Cy/ATG versus 2, 19, and 18 in Treo/Thio/Flu group, respectively. Graft was growth factor mobilized bone marrow in Flu/Bu/Cy/ATG versus peripheral blood stem cell in Treo/Thio/Flu group. Mean stem cell dose was 3.82 (2.2-9.1) versus 5 (1.65-8.01)106 /kg in Flu/Bu/Cy/ATG versus Treo/Thio/Flu group, respectively. Neutrophils and platelets engrafted at a median of 16 (14-21) and 16 (9-47) days in Flu/Bu/Cy/ATG and 15 (10-20) and 13 (9-41) days in Treo/Thio/Flu group. Median duration of follow-up was 28 (23-32.9) months. Five (6.6%) patients had rejection (all secondary). Venoocclusive disease was observed in 2 (5.7%) versus 4 (10.3%) patients (p=.047), respectively. Flu/Bu/Cy/ATG had 4 (11.4%) patients with acute GVHD versus 15 (38.5%) patients which had significant impact on survival (p=.038). We observed chronic GVHD in 4 (11.4%) and 11 (28.2%) patients, respectively, with significant impact on survival (p=.031). Four (5.1%) patients had TRM in Treo/Thio/Flu group, in contrast to none in Flu/Bu/Cy/ATG group. Mixed chimerism was common in Flu/Bu/Cy/ATG {20 (57.1%)} versus Treo/Thio/Flu group {12 (30.1%)}. Five-year Event Free Survival (EFS) and OS of entire cohort were 87% + 4% and 94% + 3%, respectively. Estimated TFS, EFS, OS of Flu/Bu/Cy/ATG versus Treo/Thio/Flu was 97.1% + 2.9% versus 89.2% + 5.1% (p=.251), 97 + 3% versus 80.7 + 6% (p=.041) and 100% versus 90.4 + 5% (p=.067), respectively. In our experience, Flu/Bu/Cy/ATG regimen is safe and effective even in high-risk TDT. However, one needs to be vigilant for mixed chimerism.