Busulfan Conditioning Research Articles

Comparison of busulfan and total body irradiation conditioning on hematopoietic clonal dynamics following lentiviral gene transfer in rhesus macaques

The clonal dynamics following hematopoietic stem progenitor cell (HSPC) transplantation with busulfan conditioning are of great interest to the development of HSPC gene therapies. Compared with total body irradiation (TBI), busulfan is less toxic and more clinically relevant. We used a genetic barcoded HSPC autologous transplantation model to investigate the impact of busulfan conditioning on hematopoietic reconstitution in rhesus macaques. Two animals received lower busulfan dose and demonstrated lower vector marking levels compared with the third animal given a higher busulfan dose, despite similar busulfan pharmacokinetic analysis. We observed uni-lineage clonal engraftment at 1month post-transplant, replaced by multilineage clones by 2 to 3months in all animals. The initial multilineage clones in the first two animals were replaced by a second multilineage wave at 9months; this clonal pattern disappeared at 13months in the first animal, though was maintained in the second animal. The third animal maintained stable multilineage clones from 3months to the most recent time point. In addition, busulfan animals exhibit more rapid HSPC clonal mixing across bone marrow sites and less CD16+ NK-biased clonal expansion compared with TBI animals. Therefore, busulfan conditioning regimens can variably impact the marrow niche, resulting in differences in clonal patterns with implications for HSPC gene therapies.

Lovo-cel (bb1111) Gene Therapy for Sickle Cell Disease: Updated Clinical Results and Investigations into Two Cases of Anemia from Group C of the Phase 1/2 HGB-206 Study

Background: Lovo-cel (bb1111; LentiGlobin for sickle cell disease [SCD]) gene therapy (GT) uses autologous transplantation of hematopoietic stem and progenitor cells (HSPCs) transduced with the BB305 lentiviral vector encoding a modified β-globin gene, which produces an anti-sickling hemoglobin (Hb), HbAT87Q. The ongoing phase 1/2 HGB-206 study (NCT02140554) is the largest clinical trial of GT in SCD to date. We report updated efficacy and safety results for Group C and describe the results of additional investigations in two patients (pts) in Group C with persistent anemia. Methods: Pts (≥12-≤50 yrs of age) with SCD and recurrent severe vaso-occlusive episodes (VOEs) underwent plerixafor mobilization and apheresis followed by myeloablative busulfan conditioning and lovo-cel infusion. Lab evaluations, SCD-related outcomes, and safety were assessed. Data are median (min-max) unless otherwise stated; all timepoints are from lovo-cel infusion. Results: As of July 2021, 35 Group C pts had received a lovo-cel infusion, with a median follow-up of 20.9 months (min-max: 8.5-28.5). Neutrophil and platelet engraftment were achieved on Days 20 (12-35) and 36 (18-136), respectively. The median vector copy number in peripheral blood remained stable in all pts at ≥1 c/dg from 6 months after infusion until the last study visit, and resulted in median HbAT87Q levels of ≥5 g/dL during this period. The median total Hb level increased without transfusions from 8.5 g/dL at baseline to ≥11 g/dL from 6 months through last visit; HbAT87Q contributed at least 40% of total Hb (Fig 1A). Of the 29 pts who could be evaluated, 28 had a complete resolution of severe VOEs after lovo-cel infusion in the 2-yr parent study as of last follow-up, as compared with 3.5 events per yr (1.0-13.5) in the 2 yrs before enrollment (Fig. 1B); no severe VOEs have been reported during the long-term follow up study. Key hemolysis markers approached normal levels. Overall, 15 pts (43%) had ≥1 serious adverse event after lovo-cel infusion; the most frequently reported were abdominal pain, drug withdrawal syndrome (opiate), nausea, vomiting, and dehydration (6% each). No cases of veno-occlusive liver disease, graft failure, replication-competent lentivirus, vector-mediated insertional oncogenesis, or hematologic malignancies were observed. The lovo-cel treatment regimen generally reflected the known side effects of HSPC collection and busulfan conditioning regimen, and/or adverse events commonly seen in the population being evaluated. Further investigations were conducted as of March 2022 in two pts who presented with similar clinical and pathological findings that initially raised suspicion of myelodysplastic syndrome (MDS). Presentation features included neutropenia in one patient; both patients had persistent anemia despite adequate HbAT87Q production, bone marrow morphology showing erythroid specific dysplasia (binucleated or trinucleated cells) consistent with dyserythropoiesis as seen in other hemoglobinopathies, and low level of trisomy 8 (≤7.7%) only observed by fluorescence in situ hybridization analysis. There are no clinical symptoms suggestive of hematologic malignancies, no driver mutations, no clonal process (vector-related or otherwise) as shown by Integration Site Analysis and Next-Generation Sequencing (both subjects had >29,000 Unique Integration Sites with no integration having a relative frequency above 0.425%), and no chromosomal abnormalities on karyotyping among up to 205 metaphases. Both pts have had anemia and one maintained total Hb of 9.8 g/dL at Month 18 without transfusions, and the other required chronic transfusions to maintain 10.3 g/dL at Month 18 with otherwise normal complete blood counts. Both pts experienced a reduction or resolution of severe VOEs after lovo-cel infusion. Of note, these are the only pts in the study to have two α-globin deletions (−α3.7/−α3.7). We propose that α-globin trait may contribute to anemia and dyserythropoiesis after lovo-cel infusion. Summary: In Group C of the HGB-206 study, one-time treatment with lovo-cel resulted in sustained production of HbAT87Qand near-complete resolution of severe VOEs. Investigations to date in both patients with anemia have not confirmed MDS; thus, currently no hematologic malignancies have occurred in Group C. Ongoing long-term follow-up should be continued for all pts receiving GT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Long Term Outcomes of 63 Patients with Transfusion-Dependent β-Thalassemia (TDT) Followed up to 7 Years Post-Treatment with betibeglogene autotemcel (beti-cel) Gene Therapy and Exploratory Analysis of Predictors of Successful Treatment Outcomes in Phase 3 Trials

Background: One-time treatment with beti-cel ex vivo gene therapy (GT) adds functional copies of a modified HBB gene, βA-T87Q, to autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) through transduction with BB305 lentiviral vector (LVV), thereby addressing the underlying genetic cause of Transfusion-Dependent β-Thalassemia (TDT). Here, we evaluate the long-term efficacy and safety profile of beti-cel across phase 1/2, phase 3, and long-term follow-up (LTF) studies. We also present an exploratory analysis of predictors of successful treatment outcomes in patients from phase 3 studies. Methods: Patients with TDT underwent HSPC collection followed by pharmacokinetic-adjusted myeloablative busulfan conditioning and beti-cel infusion. Patients were followed for 2 yrs in the parent phase 1/2 (HGB-204, NCT01745120; HGB-205, NCT02151526) and phase 3 (HGB-207, NCT02906202; HGB-212, NCT03207009) studies; enrollment and all beti-cel infusions have been completed. Patients then entered a 13-yr LTF study, LTF-303 (NCT02633943). Transfusion independence (TI) was defined as weighted average hemoglobin (Hb) ≥ 9 g/dL without packed red blood cell transfusions for ≥ 12 months. An exploratory multivariate analysis was also carried out in patients enrolled in the phase 3 trials to identify predictors of clinical outcomes. Results: As of August 2021, 63 patients had received beti-cel and were followed for a median of 41.4 (9.0-87.5) months across the 4 parent studies and LTF. The median time until neutrophil and platelet engraftment was 23 (13-39) and 45 (19-191) days, respectively, and lymphocyte subsets were generally within normal range after beti-cel infusion. Peripheral blood vector copy number (VCN), levels of GT-derived adult Hb (HbAT87Q), and total Hb were stable and durable across studies and higher in phase 3 vs phase 1/2 studies following optimized beti-cel drug product (DP) manufacturing. HbAT87Q levels were stabilized by Month 6 and contributed to stable levels of total Hb. Total unsupported Hb levels ≥ 9g/dL at Month 6 were significantly associated with achieving TI (p<0.0001) (March 2021 data cut). One-time beti-cel GT was followed by durable TI across ages and genotypes with up to 7 yrs follow-up. A trend towards more patients achieving TI was observed in phase 3 (34/38; 89.5%) (Fig 1A) vs phase 1/2 (15/22; 68.2%). All patients who achieved TI (N=49) continue to remain TI as of last follow-up. Patients who achieved TI had reductions in markers of ineffective erythropoiesis and iron overload. Overall, 21/37 patients who restarted chelation stopped chelation at 24 months; 25% (12/49) of patients who achieved TI utilized phlebotomy for iron removal, including 6 patients who also received iron chelation. Overall, 18% (11/63) of patients experienced ≥1 adverse event (AE) considered related or possibly related to beti-cel; the most common DP-related AEs were abdominal pain (5/63 [8%]) and thrombocytopenia (3/63 [5%]), but none were observed beyond 2 yrs post-infusion, supporting a favorable long-term safety profile. Veno-occlusive liver disease was reported in 11% (7/63) of patients and resolved after appropriate treatment. No malignancies, insertional oncogenesis, vector-derived replication competent lentivirus, or clonal predominance was observed. The safety of the beti-cel treatment regimen largely reflected the known side effects of hematopoietic stem cell collection and the busulfan conditioning regimen. An exploratory multivariate analysis in patients from phase 3 studies (n=37; March 2021 data cut) identified that the percentage of DP cells transduced with the BB305 LVV (%LVV+ cells) was the best predictor of clinical outcomes (Fig 1B). DP VCN and %LVV+ cells are current release assays for beti-cel. Summary: beti-cel is a potentially curative GT for patients with TDT across ages and genotypes through the achievement of TI and normal or near normal Hb levels. An exploratory multivariate analysis demonstrated that DP transduction efficiency was the most predictive attribute of TI, a key endpoint for clinical outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Preliminary Safety and Efficacy Results of EDI001: An Investigator Initiated Trial on CRISPR/Cas9-Modified Autologous CD34+ Hematopoietic Stem and Progenitor Cells for Patients with Transfusion Dependent β-Thalassemia

Background In China, it is estimated that there are 15,000-30,000 patients with β Thalassemia Major (β-TM). Our recent Cross-sectional survey of β-TM patients’ conditions in China found that only 7% of patients with β-TM expect to live beyond age 18, and only a fraction of patients have received allogeneic hematopoietic stem cell transplantation. Higher level of fetal hemoglobin (HbF) by downregulation of BCL11A during erythroid differentiation has been reported to ameliorate the clinical severity of the patients with β-TM. Disruption of GATAA motif in DHS h+58 region of BCL11A erythroid enhancer (BEE) by gene editing in CD34+ cells has been shown to significantly induce the production of HbF in patients. An adjacent motif, i.e., the CTG motif within TAL1-GATA1 binding site could potentially also be such a gene-editing target due to naturally occurring SNPs associated with elevated HbF levels (eg rs1427407). ET-01 is an investigational cell therapy product comprised of autologous CD34+ cells that have undergone CRISPR/Cas9-mediated ex vivo editing targeting this motif (Fig 1). Methods The EDI-001 trial (NCT04390971) aimed to evaluate the safety and efficacy of ET-01 in patients with transfusion dependent thalassemia (TDT). Patients (aged 6 to 35 years) with TDT whose total hemoglobin level fell within the range of 4.5g/dL and 7g/dL were eligible. One patient was enrolled and CD34+ cells were collected from the patient by apheresis following mobilization with lenograstim and plerixafor. ET-01 was manufactured by editing at BEE with Cas9 mRNA and a specific single-guide RNA via electroporation. The ET-01 product was infused following myeloablative conditioning on date 18 Nov 2020 (patient age at 12). Afterwards, the patient was followed up per schedule defined in the Protocol, monitoring safety and efficacy endpoints. Results cGMP manufacturing process at clinical scale was developed for ET-01. High editing rate was achieved with stemness well maintained (Fig 2A). Indels pattern analysis showed that in ET-01 four indel types were most prevalent (Fig 2B). Moreover, the percentage of HbF+ cells were significantly increased compared with the unedited CD34+ cells in erythroid differentiation assay (Fig 2C). IND-enabling studies showed that no tumorigenicity nor other toxicities associated with ET-01 were observed in over 400 mice at up to 40 weeks of observation. One patient (β0/β+) was enrolled, for which ET-01 was manufactured under cGMP process and infused at a dose of 23.19 X 10^6 CD34+ cells/kg. Successful hematopoietic reconstitution was observed, in which neutrophil engraftment occurred on day 24 and platelet engraftment on day 37 post ET-01 infusion. The patient experienced a significant increase of HbF production and total Hb, in which the levels of HbF increased from 3.35 g/L at baseline to 89.87 g/L, and the total hemoglobin level reached 110 g/L at month 18, respectively (Fig 3). The patient received her last pRBCs transfusion on day 87 after ET-01 infusion, and achieved transfusion-free for over 15 months. Moreover, we observed that the prevalence of indels changed during the initial few weeks and remained stable in both peripheral blood and bone marrow (Fig 4A). NGS analysis of 42 potential off-target sites, discovered by in silico prediction and DiGenome-seq, showed editing efficiencies below 1%, and no significant change was observed in any of the 42 sites throughout visits (Fig 4B). The patient experienced 62 cases of adverse events, most of which were Grade 1 or 2 in severity. Of these, only one Grade 1 AE was attributed to the ET-01 drug product, caused by the ET-01 preparation component DMSO during infusion, and was gradually relieved by adjusting the infusion rate. Two Grade 3 serious adverse events (SAE) unrelated to ET-01 were reported: enterocolitis and febrile neutropenia, caused by inappropriate diet after apheresis and busulfan conditioning, respectively. Conclusions These data demonstrated that a single-dose of ET-01 composed of autologous CRISPR/Cas9 mediated CD34+ cells led to the timely hematopoietic engraftment, significant and durable increases in HbF production and total Hb, and transfusion-independent for 15 months at the time of data cut-off. Safety profile of ET-01 is consistent with that of autologous HSC transplant including busulfan myeloablative conditioning. These preliminary results support further experimental testing of ET-01 to treat patients with β-TM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Lentiviral Gene Therapy with Low Dose Conditioning for X-Linked SCID Results in Complete Immune Reconstitution and No Evidence of Clonal Expansion

Introduction: Gene therapy (GT) for X-linked severe combined immunodeficiency (SCID-X1) using a gamma (γ) retroviral vector resulted in robust T cell immune reconstitution but also insertional oncogenesis. A multi-institutional trial using a self-inactivating (SIN) γ-retroviral vector resulted in equivalent T cell immune reconstitution but no insertional oncogenesis to date (Hacein-Bey-Abina et al, NEJM 2014). In both trials, conditioning was only rarely used and therefore gene marking in B cells and neutrophils was negligible and reconstitution of NK cells was limited. To improve both the immune reconstitution and safety of GT for SCID-X1, we developed a lentiviral vector (LV) in which a codon optimized IL2RG transgene is driven by the elongation factor 1 alpha short promoter (EFS) and introduced low dose busulfan conditioning to enhance hematopoietic and multilineage expression of IL2RG. An international, multicenter, Phase 1/2 clinical trial (NCT03311503, NCT03601286) is currently underway to evaluate the safety and efficacy of LV mediated gene transfer using this vector. Methods: A total of 14 patients have been enrolled. Two patients were withdrawn due to poor stem cell mobilization and collection and manufacturing are in progress for 3 patients. Nine patients have been infused, 8 receiving mobilized peripheral blood stem cells and 1 receiving bone marrow at an average age of 5.3 months (range 3.2-7.1). Hematopoietic stem cells (CD34+ cells) were transduced with the LV vector (using transduction enhancers in 7 products) and drug products were cryopreserved. Median cell dose was 9.06 x 10e6 CD34+ cells/kg (4.55-17.87) and median vector copy number (VCN) was 0.86 copies/diploid genome(dg) (0.67-2.79). Drug products were thawed and infused after 2 days of busulfan conditioning targeted to an AUC of 30 mg*h/L. All infants tolerated busulfan exposure without any significant toxicity. Results: As of July 2022, all treated patients are alive with robust reconstitution of gene marked T cells with median follow up of 2.2 years (0.2-4.3). T cell reconstitution was rapid with median CD3+ T cell count 552 cells/uL (132-1160) at 3 months. Seven patients evaluable at 1 year had CD3+ (2377-4900 cells/uL) and CD4+ T cell counts (1507-3080 cells/uL) normal for age at 1-year post-GT (Figure 1). Gene marking in T cells was robust averaging 1.55-5.66 copies/dg. Evidence of thymic function includes rising naïve CD4+ T cells and maintenance of a normal CD4/CD8 ratio. Multilineage engraftment of gene modified cells is evident in B cells, neutrophils, and NK cells at last follow-up with stable VCN over time (ranges 0.43-2.05, 0.24-2.11, 1.98-3.61 respectively). In contrast to limited NK cell reconstitution seen in the absence of conditioning in prior trials, NK cell counts have been sustained in 6 of 7 patients with greater than 12 months of follow-up achieving values within the normal range for age (Figure 1). Six of these 7 patients have been able to stop immunoglobulin replacement, 4 of 4 who have completed a primary vaccine series show response, with the results of 2 further patients awaited. Insertion site analysis shows uniformly polyclonal repertoire with no evidence of clonal expansion. Conclusion: In summary, gene therapy using a lentiviral vector and low dose busulfan conditioning resulted in robust and complete immune reconstitution correcting T, NK, and B cell defects characteristic of SCID-X1 with 100% survival and no gene therapy related adverse events. Figure 1 Immune reconstitution in 7 patients treated with >1 year follow-up A) CD3+ T cells. B) CD4+ T cells. C) NK cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Treatment of Individuals with Severe Sickle Cell Disease with OTQ923, an Autologous, Ex Vivo, CRISPR/Cas9-Edited, CD34+ Hematopoietic Stem and Progenitor Cell Product, Leads to Durable Engraftment and Fetal Hemoglobin Induction

Introduction: Increased levels of fetal hemoglobin (HbF; encoded by γ-globin [HBG1/HBG2]) are protective against complications of sickle cell disease (SCD). BCL11A is a transcription factor that binds to HBG1/HBG2 promoters and produces a γ-to-β globin (fetal-to-adult hemoglobin) switch. Inhibiting BCL11A binding to its target can reverse the switch and induce HbF expression in adult red blood cells (RBCs). OTQ923 is an autologous, ex vivo, CRISPR/Cas9-edited, CD34+ cellular product with a targeted disruption of the HBG1/HBG2 promoters on chromosome 11. Targeting this region in preclinical models led to HbF induction, phenotypically recapitulating hereditary persistence of fetal hemoglobin (a naturally occurring condition whose co-inheritance ameliorates SCD severity). This approach is more targeted than complete elimination of BCL11A expression in erythrocyte precursors for treating hemoglobinopathies. Additionally, unlike lentiviral vector gene addition, induction of endogenous γ-globin transcription could concur with reduction of βS-globin expression to avoid α- and β-like globin chain imbalance. Methods: Subjects with severe SCD, defined by a history of stroke, recurrent vaso-occlusive events (VOE), acute chest syndrome (ACS), chronic transfusions, recurrent priapism or red cell alloimmunization, were eligible to participate in the adult cohort (≥18-≤40 years old) of this clinical trial (NCT04443907). OTQ923 was manufactured from cryopreserved, peripheral blood derived CD34+ cells, collected after plerixafor mobilization. Prior to infusion, participants received myeloablative busulfan. Primary endpoints are to evaluate engraftment, long-term safety, and efficacy of HbF expression. Results: As of the data cut-off (July 8, 2022), 2 participants had received OTQ923, with follow-ups of 9 months (mos) and 3 mos, respectively. Here, we present initial clinical data from these 2 participants; additional data will be presented at the meeting. Both participants were homozygous for the sickle mutation. Participant 1 is a 22-year-old male with a history of silent cerebral infarcts, ACS, and priapism. At study entry, he was receiving chronic transfusions. He received 2.8x106 CD34+ cells/kg of OTQ923 (2 combined manufacturing batches). Participant 2 is a 21-year-old male with a history of ACS, and a silent cerebral infarct and was receiving hydroxyurea. He received 5.99x106 CD34+ cells/kg of OTQ923 (3 combined manufacturing batches). Time to neutrophil engraftment (the first of 3 consecutive days of absolute neutrophil count above 500 cells/mL after cell infusion) was 26 and 20 days for Participants 1 and 2, respectively. No OTQ923-related adverse events (AEs) were reported in either participant. All observed AEs were consistent with busulfan conditioning. Participants received RBC transfusions around conditioning and have received none after engraftment. For Participant 1, HbF level increased in the 6 mos after infusion and was stable at data cut-off (22.1% HbF at 9 mos after infusion). Induction of HbF was pancellular, with an F-cell percentage around 91% at 6 mos after infusion (Fig. 1A). Participant 2 had achieved an HbF level of 15.9% at 3 mos after infusion, the latest evaluable timepoint at data cut-off. Neither participant had any SCD-related VOEs after infusion of the cellular product. The proportion of edited alleles in the peripheral blood of Participant 1 was maintained at over 60% until Mo 6, which was the last evaluable timepoint (Fig. 1B). The indels were diverse, except for a predominant, large 5 kb deletion arising from simultaneous double-stranded DNA cleavage at HBG1 and HBG2 on-target sites, similar to previous reports. Conclusion: Preliminary data demonstrate clinically meaningful increases in HbF and evidence of benefit for both Participants 1 and 2 after OTQ923 infusion. Edited alleles were stable and maintained in the peripheral blood for >6 mos. The safety profile of OTQ923 was as expected after myeloablative busulfan and autologous hematopoietic stem cell transplantation. Taken together, these data suggest that this approach is safe and offers a potentially curative option for patients with severe SCD. Using cryopreserved, CD34+ cells as a starting material offers the advantage of centralizing manufacturing, scaling up the process to allow for improved patient access around the globe. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The NSG-hIL6 Mouse As a Novel Murine Patient Derived Xenograft Model of Plasma Cell Dyscrasias for Study of Disease Biology and Therapeutic Development

Background Plasma cell dyscrasias include pre-malignant and malignant conditions spanning the spectrum from monoclonal gammopathy of undetermined significance (MGUS) to smoldering multiple myeloma (SMM) to active multiple myeloma (aMM) and include the disorders AL amyloidosis and plasma cell leukemia (PCL). Though the last 2 decades has yielded effective therapies against these disorders, there remain no reliably curative treatments, and complications due to multiple myeloma are most often the cause of death. A major reason for the lack of curative therapies is the dearth of relevant research models in which to study these diseases and test new drug candidates. Cell lines that do not reflect the biology of mainstream myeloma are currently the mainstay of disease modeling. Plasma cells do not survive well outside the bone marrow and to this point, little success has been made using primary tumors from patients to model disease. Thus, there is a significant need for more relevant models to build the next, hopefully curative, generation of therapeutics for these diseases. Research Significance We present a novel immunodeficient murine model able to engraft plasma cell dyscrasias from primary patient tumors and the ability to study basic biology and test efficacy of cellular therapies, representing a significant advance in this field. Methods The NSG-h(IL6) mouse, developed by Dr. Shultz, builds on the background NSG mouse that lacks adaptive immunity and has limited innate immunity. It expresses a BAC with a piece of human chromosome 7, containing the human IL-6 gene and all associated local promoter and enhancer elements. With the expertise of the stem cell and xenograft core facility (SCXC), mice were preconditioned with busulfan, and 1e6 mononuclear cells (MNCs) isolated from human myeloma patient marrow aspirate were injected into the murine femur. Blood and urine were sampled every 4-5 weeks and analyzed for the presence of human antibodies by ELISA. Deceased mice were sent for histology and their spleens and marrow analyzed for the presence of clonal plasma cells by flow cytometry. Serum protein electrophoresis (SPEP) and complete blood counts (CBC) were noted as hallmarks of disease. Samples from patients with MGUS, SMM, aMM (both newly diagnosed and relapsed/refractory), PCL and AL amyloidosis were engrafted into mice as above. Mice were treated with untraduced T-cells (UTD) or BCMA-directed CARTs 10 weeks after engraftment of active myeloma and blood was assayed for level of immunoglobulins to follow response. Results Analysis of murine serum and urine by ELISA revealed the presence of rising titers of human antibodies in all engrafted mice and the presence of an M-spike on SPEP. NSG-hIL6 mice conditioned with busulfan showed greater engraftment than non-IL6 containing mice and non-conditioned mice (7 vs 2 vs 0, p<0.005). Light chain specific ELISA showed a clear predominance of one light chain, congruent with the patient sample that was engrafted. Flow cytometric analysis of engrafted murine bone marrow showed the presence of light chain restricted plasma cells. Histology showed the presence of plasma cells in the marrow, spleen and liver of engrafted animals with IHC positivity for specific light chain and CD138. CBC showed pancytopenia (p<0.005) with anemia being most significant in engrafted vs. unengrafted animals. All fresh samples and nearly all frozen samples of MGUS, SMM, aMM, PCL and AL amyloidosis were able to be engrafted at high rates (>50% of mice). Mice injected with column separated CD138+ cells alone showed no engraftment vs. mice injected with total MNCs from the marrow of the same myeloma (3 vs 0, p<0.005 ). BCMA directed CARTs significantly decreased circulating antibody compared to UTD controls (p<0.005). Conclusion Using the NSG-hIL6 mouse, busulfan conditioning and intrafemur injections, we have been able to engraft all the major plasma cell dyscrasias as denoted by the presence of monoclonal human antibody in these mice and presence of light chain restricted plasma cells congruent with the patient's paraprotein. We have preliminarily shown that the ability to engraft disease is dependent on human supporting cells. Additionally, treatment with BCMA directed CARTs is showing a response. This simple model will allow for further preclinical testing of new agents against multiple myeloma and associated plasma cell neoplasms and allow for study of basic biology of these diseases. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Relevance of Monitoring Thymic Function and Recovery in Interpreting Immune Reconstitution Post Hematopoietic Cell Transplantation in Inborn Errors of Immunity

Introduction Allogeneic hematopoietic cell transplantation (HCT) is the treatment of choice for many inborn errors of immunity (IEI). Impaired recovery of thymic function predicts poor clinical outcomes after HCT, especially in pediatric patients with IEI. CD31+CD4+CD45RA+ T cells are recognized as recent thymic emigrants (RTE) or true naïve T cells, and indicators of thymic output in HCT recipients. Reduced intensity-conditioning (RIC) and reduced toxicity-conditioning (RTC) regimens are increasingly used to limit organ toxicity and late effects of HCT. However, it is not known if they facilitate thymic recovery. Furthermore, graft-versus-host disease (GVHD) and its treatment can lead to prolonged thymic dysfunction. Methods Pediatric normal values and percentiles for a flow cytometry based RTE assay were developed. In this prospective study, we used RIC with fludarabine, melphalan, alemtuzumab, RTC with busulfan, fludarabine, alemtuzumab or myeloablative conditioning regimen (MAC) with busulfan, cyclophosphamide, alemtuzumab in patients with IEI. For GVHD prophylaxis cyclosporine A or tacrolimus together with mycophenolate mofetil were used. We assessed RTE immune reconstitution at 100 days, 6 months, 1 year and 2 years after HCT. Institutional review board of the University of Minnesota approved the study protocol. We hypothesized that thymic-derived immune reconstitution is negatively affected by occurrence of acute GvHD, age at transplantation (negatively with higher age) and type of conditioning (negatively with more intensive conditioning). Linear mixed model was performed for the repeated measures of RTE (after square root transformation) with covariates time (discrete), age at transplant (continuous), conditioning regimen, acute GHVD and all the second-order interactions with time. Results Between January 1st 2012 and December 31st 2021 45 patients with IEI were transplanted. Two patients did not receive the conditioning regimen per protocol, one patient received alemtuzumab only and two patients received no conditioning. These five patients were excluded from the final analysis. Thirteen (32%) of 40 patients were female. Median age at transplantation was 2.64 years (IQR 0.89 to 10.09). Median time to neutrophil engraftment was 12 days (IQR 10 to 14). Median follow-up time was 46.5 months (IQR 22.8 to 62.7). The overall survival was 82.5% (95%CI 66.8% to 91.2%). 13 (32%) received RIC, 23 (57%) RTC and 4 (10%) MAC. 10 (25%) patients had a matched related donor, 24 (60%) had a matched unrelated donor and 6 (15%) received an umbilical cord blood transplant. 7 (17.5%) patients had Chronic Granulomatous Disease, 5 (12.5%) Hemophagocytic lymphohistiocytosis, 5 (12.5%) Severe congenital neutropenia, 4 (10.0%) CD40 Ligand deficiency, 4 (10.0%) Severe combined immunodeficiency, 4 (10.0%) immunodeficiency not otherwise specified, 3 (7.5%) Wiskott-Aldrich syndrome, and 8 (20%) other primary immunodeficiency as underlying diagnosis. Patients with more intensive conditioning had lower RTE absolute values at one- and two-years post-transplant than patients with less intensive conditioning regimen (Figure 1A). Patients with acute GHVD occurring in the first 100 days after transplantation had lower RTE absolute values at one- and two-years post-transplant compared to patients without GVHD (Figure 1B). In the linear mixed model for the repeated measures of RTE (after square root transformation), we found at one-year post-transplantation a significant difference with increasing age (p<0.0001), between any grade acute GVHD and no acute GVHD (p=0.03) and between MAC and RIC (p=0.01) and RTC and RIC (p<0.01) (Table 1). These results remained robust at two-year follow-up as well. Conclusions Serial measurement of RTE is an useful immune reconstitution marker for assessment of thymic function after HCT. In patients with IEI, age at transplantation, occurrence of acute GHVD and intensity of conditioning regimen were strongly associated with slower thymic-derived immune reconstitution. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Easix Score Predicts Overall Survival and Late Relapse in Patients Undergoing Reduced Intensity Conditioned Allogeneic Stem Cell Transplantation for Myeloid Disease

Background Allogeneic hematopoietic stem cell transplantation (alloSCT) is an effective treatment for myeloid malignancies, however outcomes post-transplant are variable and predictive scores limited. The Endothelial Activation and Stress Index (EASIX) measured at key timepoints pre- and post-transplant has been shown to predict outcomes in alloSCT, including overall survival, non-relapse mortality and incidence of sinusoidal obstructive syndrome (SOS). It can be readily calculated using standard biomarkers (LDH (U/L) x creatinine (mg/dL)/platelets (x109/L). We sought to examine its predictive role in a homogenous population of patients. Methods We conducted a retrospective national review of 117 patients undergoing reduced-intensity alloSCT for myeloid disease including AML (n=56), MDS (n=35) and MPN (n=26). All included received fludarabine, busulfan and rabbit anti-thymocyte globulin (Flu/Bu/ATG) conditioning from 2015-2020. We calculated the EASIX score on the day prior to conditioning, on day 0, at onset of acute graft versus host disease and 1 year post transplant. Univariate analysis was conducted in the STATA statistical package. Results At the pre-conditioning time-point the mean EASIX was 3.11 (0.3-17.54). Increased scores correlated with and were reliably predictive of reduced overall survival (p=0.034) (fig. 1). Higher scores at this time-point did not predict chronic GVHD or relapse, however an increased rate of acute GVHD was seen in those with lower EASIX (p=0.012). At day 0 the mean EASIX was 15.38 (1.07-179.02), however we could not demonstrate a predictive effect on SOS as no events occurred in this cohort. Nor did we identify a predictive value at this point for GVHD risk or relapse. At onset of acute GVHD (n=67, mean 3.52, 0.44-51.43), the EASIX score was predictive of overall survival (p=0.027) but did not predict risk of chronic GVHD or relapse. At 1 year post-transplant (n=88, mean 2.09, 0.48-13.59) a higher score showed a trend towards decreased overall survival (p=0.1) but was associated with an increased risk of subsequent relapse (p=0.01)(fig. 2). Conclusions The EASIX score is an easily applicable clinical tool using real world biomarkers readily available at serial time-points in transplant recipients. When applied to our homogenously treated cohort it retains its value in predicting overall mortality at the key time points of pre-conditioning and at onset of GVHD. Adoption of this simple score may be of benefit in risk stratifying the transplant process for reduced intensity conditioned allograft recipients, particularly when combined with established prediction tools for non-relapse mortality such as the HCT-CI. In addition to this, we have shown that the EASIX score may be a useful predictive tool for late relapse in those patients who are alive at 1 year post transplant. We suspect that this effect is primarily reflective of those patients with persistent thrombocytopenia and raised LDH, both markers of potential disease activity. Importantly, the use of this score in the reduced intensity population may be particularly relevant due to the increasing age of transplant eligibility and its use as a surrogate of endothelial dysregulation and potential for end organ complications through the transplant process. To assess the underlying T cell mediated and cytokine secretome effects on endothelial dysfunction, an observational study is ongoing at our institution, examining the interplay of immunomodulatory therapy, graft T cell content, early T and NK cell recovery and the cytokine secretion patterns at various time points before, during and after transplant in reduced intensity conditioned patients with myeloid disease. We plan to correlate these data with serial EASIX assessments to identify the determinants of the underlying biological process and identify potential time-points and biological targets for future study.

Effect of Two Conditionings on Relapse in Myelodysplastic Syndrome and Secondary Acute Myeloid Leukemia Undergoing Allogeneic Hematopoietic Cell Transplantation

Background The relapse rate remains high in patients with myelodysplastic syndrome (MDS)-refractory anemia with excess blasts (RAEB) and secondary acute myeloid leukemia evolving from MDS (sAML) undergoing allogeneic hematopoietic cell transplantation (allo-HCT). We investigated whether the addition of granulocyte colony-stimulating factor (G-CSF) and decitabine to busulfan plus cyclophosphamide (BuCy) conditioning would reduce relapse in this population. Methods We did an open-label, randomised phase 3 trial at six hospitals in China. Eligible patients (aged 14-65 years) had a diagnosis of MDS-RAEB or sAML, were willing to undergo allo-HCT, and had an Eastern Cooperative Oncology Group performance status of 0-2 and HCT-comorbidity index of 0-2. Patients were randomly assigned 1:1 to receive G-CSF and decitabine plus BuCy (G-DAC-BuCy) or BuCy conditioning. Randomisation was done with permuted blocks (block size four) and implemented through an interactive web-based randomisation system. The primary endpoint was the 2-year cumulative incidence of relapse (CIR) in the intention-to-treat population. This trial is registered with ClinicalTrials.gov, NCT02744742. Findings Between April 18, 2016 and September 30, 2019, 202 patients were enrolled and randomly assigned to receive G-DAC-BuCy (n=101) or BuCy conditioning (n=101). Median follow-up was 32.4 (range, 1.0-64.5) months after randomization. The 2-year CIR was 10.9% (95% CI: 5.8-17.9%) and 24.8% (16.8-33.5%) in the G-DAC-BuCy and BuCy groups (HR=0.387, 95% CI: 0.190-0.786; P=0.011). Within 100 days post-transplantation, the most common grade 3-4 adverse effects (AEs) in the G-DAC-BuCy and BuCy groups were infections (34 [33.7%] and 32 [31.7%]), acute graft-versus-host disease (30 [29.7%] and 30 [29.7%]), and gastrointestinal toxicity (28 [27.7%] and 29 [28.7%]). Eleven patients in the G-DAC-BuCy and 13 in the BuCy group died of AEs. Interpretation G-DAC-BuCy conditioning can prevent relapse compared with BuCy conditioning and is well tolerated, suggesting its potential to become a standard conditioning in patients with MDS-RAEB and sAML undergoing allo-HCT.

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.

UBTF tandem Duplications Account for a Third of Advanced Pediatric MDS without Genetic Predisposition to Myeloid Neoplasia

Introduction: Recurrent tandem duplications (TD) in exon 13 of the upstream binding transcription factor gene (UBTF) have recently been identified in 4% of pediatric AML (Umeda et al. 2022). UBTF-TD AMLs were associated with adolescent age, normal karyotype, trisomy 8, as well as FLT3-ITD and WT1 mutations. Response to chemotherapy was poor and probability of overall survival (OS) at 5 years only 44%. Here, we evaluate the role of UBTF-TD in pediatric MDS. Methods: We studied 104 consecutive patients (pts) diagnosed with primary MDS with excess blasts below the age of 18 years enrolled in the prospective studies 98 or 2006 of the European Working Group of MDS in Childhood (EWOG-MDS) in Germany between July 1, 1998 and June 30, 2021. Pts with MDS secondary to chemo- or radiation therapy, acquired aplastic anemia and pts with classical inherited bone marrow failure disorder had been excluded from the analysis. High molecular weight DNA was extracted from stored bone marrow (BM) samples or archived smears. Screening for UBTF-TD was performed by PCR using 6-FAM-labelled primers covering exon 13 of the UBTF gene followed by high resolution fragment analysis. For samples showing aberrant signals indicating UBTF-TDs, Sanger sequencing confirmed the presence of a mutation. All UBTF mutant samples were also analyzed using targeted next generation sequencing (Archer Myeloid panel) for the presence of co-occurring alterations. Results:UBTF-TDs were detected in 25 of the 104 pts (24%; 16 males/9 females), while 79 pts (76%; 45 males, 34 females) showed no UBTF mutation (UBTF-wt). Variant allelic frequencies of UBTF-TD ranged from 11%-79% (median 41%). Median age at diagnosis in UBTF-TD and UBTF-wt pts was not different (11.0 vs. 9.8 years). UBTF-TD pts had higher absolute neutrophil counts (2.7 vs. 0.9 x109/L, p=0.01) and more often normoblasts on peripheral blood smears (92% vs 43%, p<0.01). Blast percentage in BM was similar with <10% blasts in 40% and 38% of UBTF-TD and wt pts, respectively. BM cellularity was normal or increased in 92% of UBTF mutated pts compared to 72% of UBTF-wt pts (p<0.01). Cytogenetic analyses revealed striking differences according to UBTF mutational status with the following karyotypes (TD vs wt): normal (72% vs 33%), -7 (0% vs 30%), del(7q) (0% vs 2%), +8 (24% vs 9%), +8 plus other (0% vs 4%), random (4% vs 7%) and structural complex (0% vs 7%). Probability of OS at 5 years for pts with UBTF-TD was inferior (0.50, 0.28-0.72) to that of pts with UBTF-wt (0.73, 0.62-0.84; p=0.03). Of the 104 pts, 30 pts had a genetic predisposition to myeloid neoplasia, i.e. GATA2 deficiency (n=15), RUNX1 deficiency (n=7), SAMD9/SAMD9L disease (n=6) or neurofibromatosis type 1 (n=2). Of note, none of the pts with predisposition harbored a UBTF-TD. To study the impact of UBTF-TD on outcome of hematopoietic stem cell transplantation (HSCT), pts with germline disease were excluded; of the 72 pts available for HSCT analysis (25 UBTF-TD and 47 UBTF-wt). Therapy prior to HSCT in UBTF-TD and UBTF-wt pts was comparable and consisted of AML-type therapy in 32% vs 21%, less intensive chemotherapy 8% vs 15% and no therapy in 60% vs 64% pts, respectively. Type of donor, stem cell source and conditioning regimen did not differ between the 2 groups, 74% of pts received a busulfan, cyclophosphamide and melphalan conditioning. Event-free survival (EFS) and relapse incidence (CIR) at 5 years of the total cohort were 0.55 (0.39-0.71) and 0.23 (0.15-0.36), respectively. There was no difference in EFS between the 25 UBTF-TD (0.43, 0.22-0.64) and 47 UBTF-wt pts (0.60, 0.45-0.75). However, in pts with normal karyotype, presence of an UBTF-TD was associated with an inferior EFS (0.35 vs. 0.77, p=0.03) and higher CIR compared to wt pts (0.36 vs. 0.06, p=0.03). Co-occurrence of WT1 mutations among UBTF mutated pts was associated with higher CIR (p=0.03); other mutations were less frequent (Figure). Discussion: We identified UBTF-TD in a third of all pediatric pts with advanced primary MDS and absence of germline predisposition. In addition, we noted UBTF-TD at a high frequency in pediatric pts with MDS-related AML (3/15 pts UBTF-TD). In regards to the recent discovery in AML, we conclude that UBTF-TD mutations define a novel subgroup of myeloid neoplasia presenting frequently as pediatric MDS. The high CIR following HSCT particularly in patients with concurrent WT1 mutations outlines an urgent need for new therapeutic strategies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Frequency and Clinical Significance of Mixed T-Cell Chimerism Following Myeloablative Busulfan and Cyclophosphamide Conditioning in HLA -Matched Donor Transplants for Myeloid Malignancies: An Analysis of 179 Patients Transplanted at a Single Center

Myeloablative busulfan and cyclophosphamide (BuCy2) is one of the most used preparative regimens for allogeneic hematopoietic cell transplantation for myeloid malignancies from HLA-identical (MRD) and 8 of 8 HLA-A, B, C and DRB1 allele matched unrelated (MUD) donors. However, mixed T-cell chimerism may be relatively common during the early post-engraftment phase using this regimen. The significance of early incomplete donor T-cell chimerism following BuCy2 allotransplants with respect to post-transplant outcomes is unclear. Methods: We analyzed 179 consecutive patients who underwent allotransplants for myeloid malignancies using myeloablative BuCY2 conditioning at our center. Cyclophosphamide was administered at 60mg/m2/d x 2 and busulfan was administered over four days with pharmacokinetically targeted exposure (usually 4.9 mgxhr/L) based on test dosing. Tacrolimus/methotrexate was used for GVHD prophylaxis. Uniform institutional supportive care protocols were employed for all patients. Lineage specific chimerism on T-cell (CD3) and myeloid (CD33) peripheral blood (PB) cells separated using immunomagnetic beads was performed monthly through d 100 following transplant. Patients with < 50% donor CD3 chimerism on d30 were scheduled to receive low dose DLI if available (starting at 5-10 x 10e5 CD3 cells /kg) while maintaining therapeutic tacrolimus levels. No intervention was performed if d30 donor CD3 chimerism was > 50%. Outcomes were analyzed using data extracted from our institutional database where information had been prospectively entered. GVHD diagnosis and grading were determined by a single dedicated practitioner. Results: Patient characteristics were: median age 50 (18-66), male 51%; diagnosis AML (65%), CML (23%) MDS/MPS (12%); MRD (52%) MUD (48%); graft source- PBSC 81%, BM18%, both 1%; DRI - low 12%, intermediate 57%, high 25%, very high 6%; donor CMV pos 51%, neg 49%; year of transplant- 2004-2012 (35%), 2013-2016 (32%) 2017-2021(33%). Median follow-up for surviving patients was 84 months (6-213). Kaplan-Meier (K-M) estimates for survival and relapse-free survival at 5 years for all patients were 66% (95%CI 58-73%) and 59% (51-66%). Median (range) donor chimerism on d 30 for CD3 cells was 80% (6-100%) and for CD33 cells was 100% (84-100%). Median CD3 chimerism on d 60, d 100 and d 180 were 86%(7-100%), 91% (8-100%) and 95% (12-100%) respectively. Twenty-four patients (13%) had CD3 <50% on d30 of which 9 received DLI. On multiple regression modeling (Table1) the following factors were associated with higher d 30 CD3 chimerism - diagnosis (AML vs other) (difference 0.104, p=0.001), DRI (high/v.high vs low/intermediate)(diff 0.070, p=0.03) and graft CD3 dose (>15.5x 10e7/kg vs < 15.5) (diff 0.105, p<0.001) For patients who had > 80% vs <80% donor CD3 chimerism on d 30, 5-year K-M estimates of survival and RFS were 56% vs 76% (p=0.003) and 54% vs 64% (p=0.16). Five-year cumulative incidences of NRM and relapse were 20% vs 8% (p=0.010) and 26% vs 28% (p=0.85). Cumulative incidences of grade 2-4 and grade 3-4 acute GVHD at 6 months were 49% vs 27% (p=0.002) and 20% vs 4% (p=0.001) respectively. Cumulative incidences of moderate to severe and severe chronic GVHD at 3 years were 81% vs 64% (p=0.017) and 53% vs 34% (p=0.008). On a Cox proportional-hazards model adjusting for significant patient, disease and transplant covariates, d 30 CD3 chimerism > 80% was associated with a significantly higher risk of NRM (HR 3.42, p=0.007), aGVHD gd 2-4(HR1.79, p=0.013), aGVHD gd 3-4 (HR 2.66, p=0.008) and severe cGVHD (HR 1.84, p=0.011). However, d 30 CD3 chimerism was not significantly associated with relapse, survival and relapse-free survival (Fig 1). Conclusions: These data suggest that while pharmacokinetically-targeted BuCy2 as administered is almost always myeloablative (CD33 chimerism 100%), early mixed T-cell chimerism is relatively common (CD3 chimerism < 80% in 50% of patients). d30 CD3 chimerism ≥80% is not associated with lower relapse rates or better survival outcomes, but is associated with higher rates of acute and chronic GVHD. No intervention to improve CD3 chimerism appears necessary for patients with d 30 CD3 chimerism between 50 and 80%. Patients with ≥ 80% CD3 chimerism at d 30 may benefit from more aggressive measures to prevent acute and chronic GVHD Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High-Dose Idarubicin Plus Busulfan As Conditioning Regimen to Autologous Stem Cell Transplantation Improves Prognosis in Patients with Favorable and Intermediate-Risk AML

Introduction: The optimal post-remission therapy (PRT) for patients with favorable and intermediate-risk acute myeloid leukemia (AML) remains uncertain. We retrospectively analyzed and compared the efficacy of high-dose idarubicin plus busulfan conditioning regimen combined with autologous stem cell transplantation (ASCT) and intermediate- to high-dose cytarabine consolidation chemotherapy for post-remission treatment (PRT) in patients with favorable- and intermediate-risk AML. Methods: A total of 116 patients with favorable- and intermediate-risk AML according to 2022 ELN risk stratification who were treated with ASCT (n=58) or intermediate- to high-dose cytarabine regimen (n=58) were enrolled in this study. All the patients received IA regimen (idarubicin 10-12 mg/m2 on days 1 to 3 and cytarabine 100 mg/m2/d on days 1 to 7) as induction and complete remission (CR) was obtained in all the patients after one course of induction. Fifty-eight patients were subjected to 2-4 courses of intermediate- to high-dose cytarabine (cytarabine 2-3 g/m2 twice daily on days 1-3). High-dose idarubicin plus busulfan conditioning regimen (I-Bu; idarubicin 20 mg/m2 continuous i.v. from days -13 to -11, busulfan in intravenous formulation: 3.2 mg/m2 or oral formulation: 4.0 mg/m2 from day -5 to -2) combined with ASCT was given to the other 58 patients 2-3 courses of intermediate- to high-dose cytarabine and mobilization and collection of peripheral blood stem cells. Clinical outcomes were retrospectively analyzed for patients belonging to the two treatment arms. Results: The median age was 38 (14-60 years) and 32 years (14-59 years) in ASCT and chemotherapy cohort, respectively. After a median follow-up of 68 months (1-200 months), and the median overall survival (OS) has not been reached in both groups. The 5-year and 10-year OS in the ASCT group was 77.5% and 71.5%, and 5-year and 10-year disease-free survival (DFS) was 73.9% and 71.1%, respectively. The 5-year OS and DFS in the chemotherapy group were 53.5% and 52.4%, respectively. The median OS and DFS of the patients in ASCT group were significantly better than those in chemotherapy group (OS: p=0.009, HR=0.444, CI 0.237 to 0.831; DFS：p=0.018, HR=0.494,CI 0.268 to 0.911). No treatment-related early death (death within 8 weeks after treatment) was observed in the ASCT group. All the patients experienced severe bone marrow suppression and severe (grade 3 or 4) hematological toxicity, and we did not observe severe (grade 3 or 4) cardiotoxic complications during or after ASCT in all the patients. Conclusions: ASCT with I-Bu regimen is possibly a promising PRT for patients with favorable- and intermediate-risk AML in the first CR, improving prognosis of these patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Induction of Fetal Hemoglobin and Reduction of Clinical Manifestations in Patients with Severe Sickle Cell Disease Treated with Shmir-Based Lentiviral Gene Therapy for Post-Transcriptional Gene Editing of BCL11A: Updated Results from Pilot and Feasibility Trial

Background Induction of fetal hemoglobin (HbF) by repressing BCL11A could reduce or eliminate sickle cell disease (SCD) clinical manifestations. The BCH-BB694 lentiviral vector (LVV) encodes a shRNA targeting BCL11A embedded in a microRNA scaffold (shmiR) allowing erythroid-specific knockdown to induce γ-globin expression and concomitantly and coordinately repress β-sickle globin expression (Brendel et al. JCI 2016). The safety and efficacy of gene therapy (GT) with BCH-BB694-transduced autologous CD34+ cells in patients with SCD is being investigated in a pilot and feasibility trial (NCT03282656), which has now completed enrollment of all 10 planned patients. Preliminary data in the first 6 patients described robust HbF induction and a favorable safety profile (Esrick et al. NEJM 2021). Additional follow-up data including all ten pilot trial patients is discussed here with longest follow-up now > 4 years. Methods Patients with severe SCD were screened for eligibility. Autologous CD34+ cells were collected via apheresis after plerixafor mobilization (Esrick and Manis et al. Blood Adv 2018) and then transduced ex vivo with the BCH-BB694 shmiR LVV. Gene modified cells were infused into subjects who had received myeloablative busulfan conditioning. Patients were monitored for adverse events (AEs), hemoglobin (Hb), HbF fraction, percent F cells, hemolysis markers, and SCD clinical manifestations. Results Ten patients (7-25 years of age at enrollment) received GT, with median follow-up of 30.5 (2-50) months. There were no Grade 3 or 4 AEs associated with mobilization, collection or infusion. Median product VCN, % transduced cells, and CD34+ cell dose in the 10 patients were 3.5 (1.8-6.9) copies/diploid genome (c/dg), 96% (62-100%), and 5.7 (3.6-8.8) x 106 CD34+ cells/kg, respectively. Engraftment of neutrophils occurred on day 22 (18-30) and of platelets on day 32 (25-62). One patient (patient 10) has low post-GT peripheral VCN, HbF, and percent F cells; most recently 0.23 c/dg, 11.2%, and 37.3% respectively. Although improved, this was considered a GT failure and the patient has been started on new SCD-directed therapies due to ongoing SCD clinical manifestations. Subsequent data described here exclude patient 10. At the most recent study visit, the 7 untransfused patients with follow up > 6 months had mean total Hb of 10.5 (8.8-12.2) g/dL, HbF/(F+S) of 30.3% (21.4 - 42.4%), percent F cells of 70.6% (58.8-79.8%), and HbF per F cell of 12 (10.1-14.4) pg (Table 1). The lowest Hb response was seen in patient 11 who has 2-gene deletion alpha thalassemia in combination with SCD. Five patients with follow-up > 6 months (patients 4, 6, 8, 9, and 11) had frequent severe vaso-occlusive events (ACS, priapism, or pain requiring inpatient or ED opioids) as indication for eligibility, with a median of 6 (3-13) events (Table 2). From 6 months after GT through present, these 5 patients have had a median of 1 (0-1) event. Three patients with follow-up > 6 months (patients 2, 3, and 7) were on chronic transfusion regimens pre-GT. Patient 3 has continued chronic transfusions due to pre-existing moyamoya with goal of maintaining HbS at pre-GT levels. Patients 2 and 7 have received no post-GT transfusions. Patients 2, 3, and 7 have had no SCD clinical events. Mean lactate dehydrogenase and absolute reticulocyte count at last study visit were 346 (215-389) U/L and 241 (200-360) 109/L, respectively, indicating decreased but persistent mild hemolysis in all patients. There have been no instances of graft failure, replication competent lentivirus detection, or clonal dominance in any patient. Six patients with ≥2 years post GT have had bone marrow biopsies at the 2 year timepoint without evidence of myelodysplasia. Targeted sequencing of ~95 genes frequently mutated in hematologic malignancies has been performed on peripheral blood samples every 6 months since early 2021 in patients with ≥6 months from GT. One patient had a pathogenic mutation detected in the DNMT3A gene, which was present in a stored pre-GT sample and has been present at a stable VAF <4% at 6, 9, and 12 months post-GT. Summary Post-transcriptional gene editing of BCL11A appears safe with stable induction of broadly distributed HbF and significant mitigation of VOE events in severe SCD in all but one treated patient. Based on the results of this pilot study, a phase 2, multi-institutional trial is now open for enrollment using the same vector and approach (NCT05353647). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Interim Results from an Ongoing Phase 1/2 Study of Lentiviral-mediated Ex-vivo Gene Therapy for Pediatric Patients with Severe Leukocyte Adhesion Deficiency-I (LAD-I)

Background: Leukocyte adhesion deficiency-I (LAD-I) results from mutations in ITGB2 encoding the β2-integrin CD18. Severe LAD-I (<2% CD18+ polymorphonucleocytes [PMNs]) causes severe and often refractory infections, impaired wound healing, and frequent childhood mortality. Allogeneic hematopoietic stem cell transplant is potentially curative but limited by donor availability, graft-versus-host disease, and graft failure. RP-L201-0318 (NCT03812263) employs autologous CD34+ cells transduced with a lentiviral vector carrying ITGB2. Methods: Patients ≥3 months old with severe LAD-I were enrolled. Hematopoietic stem and progenitor cells (HSPCs) were collected via apheresis after mobilization with granulocyte-colony stimulating factor and plerixafor and transduced ex-vivo with Chim-CD18-WPRE-LV. Myeloablative therapeutic drug monitoring (TDM) busulfan conditioning preceded RP-L201 infusion. Patients are followed for safety and efficacy including survival to age 2 and ≥1-year post-infusion, peripheral blood [PB] PMN CD18 expression, PB vector copy number [VCN], neutrophilia improvement, decrease in infections/hospitalizations, and resolution of skin/periodontal abnormalities. Results: Nine patients (ages 5 months to 9 years) received RP-L201, all with follow-up of 3 to 24 months. RP-L201 cell doses ranged from 2.8x106 to 10x106 CD34+ cells/kg with a drug product VCN of 1.8 to 3.8. All 9 patients demonstrated PMN CD18 restoration with median expression of 56.3% and sustained, stable genetic markings with median peripheral blood mononuclear cell VCN of 1.53. At 1 year, the overall survival (OS) rate is 100% per Kaplan-Meier estimate. Neutrophilia resolved uniformly. Hospitalizations and severe infections were significantly reduced following therapy. The safety profile has been highly favorable with no RP-L201-related serious adverse events (SAEs); insertion site analyses indicate polyclonal integration patterns. Conclusion: RP-L201 confers durable reversal of the severe LAD-I phenotype with improved clinical course in 9 of 9 pediatric patients.

Long-Term Patient-Reported Outcomes Following Treatment with betibeglogene autotemcel in Patients with Transfusion-Dependent β-Thalassemia

Introduction: Patients (pts) with transfusion-dependent β-thalassemia (TDT) have increased morbidity and mortality and reduced quality of life (QoL) compared with the general population owing to the mental, physical, and time demands of chronic transfusions and iron chelation. Autologous gene therapy with betibeglogene autotemcel (beti-cel) therapy uses a modified β-globin gene that establishes stable production of functional adult hemoglobin (Hb), thus allowing for transfusion independence (TI). Pts with TDT were treated with beti-cel in 2 completed phase 1/2 studies (HGB-204, HGB-205) and 2 ongoing phase 3 studies as of Aug 2021 (HGB-207, HGB-212). After 2 years of follow-up, pts could enroll in a long-term follow-up study (LTF-303 [NCT02633943]) for up to an additional 13 years. QoL improvement in the 1-2 years post beti-cel treatment was previously described. Here, we evaluate long term (3 years post-treatment) patient reported outcomes after beti-cel treatment in pts with TDT enrolled in LTF-303. Methods: Pts with TDT underwent hematopoietic stem progenitor cell collection followed by myeloablative busulfan conditioning and beti-cel infusion. TI is defined as weighted average Hb ≥9 g/dL without packed red blood cell transfusions for ≥12 months. Generic health related QoL (HRQoL) over time was assessed using the Pediatric Quality of Life Inventory (PedsQL; range, 0-100) for pediatric and adolescent pts (<18 years old [YO]). Short Form-36 Health Survey Physical Component Summary and Mental Component Summary (SF-36 PCS and MCS, respectively; range 0-100), and the EuroQol (EQ-5D-3L) visual analog scale (VAS; range 0-100) composite index score (range 0-1) for adult (≥18 YO) pts. HRQoL was assessed at baseline (BL), Months (M) 6, 12, 18, 24 (in the parent HGB studies), and M 36, 48, and 60 (in LTF-303) after beti-cel infusion. Pts were excluded from HRQoL analyses if BL or M36 HRQoL assessment was missing as of data cut (Aug 2021). Higher scores indicated improvement in HRQoL. A bespoke questionnaire assessing β-thalassemia and pt-centric aspects of daily living (i.e., ability to work or attend school, physical activity, and perceived benefit from treatment) was completed at M 36. Results: As of August 18, 2021, 57 pts were enrolled in LTF-303 (phase 1/2: 22 pts; phase 3: 35 pts), of which 26 (45.6%) were pediatric / adolescent pts and 31 (54.4%) were adult pts. Among pts <18 YO with PedsQL data who achieved TI, the mean (standard error (SE)) PedsQL total score (population norm = 80.9) increased from 77.4 (3.4) at BL (n=19) to 92.1 (4.2) at M 36 (n=4). Among adult pts with SF-36 data who achieved TI, the mean (SE) SF-36 PCS and MCS scores (US general population norm = 50) increased from 53.8 (1.4) and 50.9 (1.7), respectively, at BL (n=12) to 55.7 (1.7) and 56.3 (1.4) at M 36 (n=9), respectively. The mean (SE) EQ-5D-3L Composite Index and VAS scores increased from 0.92 (0.04) and 85.2 (3.0), respectively, at BL (n=12), to 0.95 (0.03) and 91.4 (2.2) at M 36 (n=9) (Table 1). Data from the M 36 testimonial questionnaire (Figure 1) showed the ability to seek employment or be employed, increased from 68.8% (11/16) of pts at BL to 93.8% (15/16) at M 36. There was a reduction in school absences at M 36 (44.4% [4/9] missing school) compared with BL (83.3% [15/18] missing school). 50% (10/20) of pts still required management of disease symptoms at M 36. 80% (16/20) of pts reported improvement in physical activity at M 36. 100% (19/19) of pts reported an overall benefit from undergoing treatment with beti-cel. Conclusions: Published data shows beti-cel is potentially curative in pts with TDT, defined as having TI with normal or near-normal Hb. It was previously shown that with high-range QoL at BL, QoL improved at 1-2 years post-beti-cel. The updated data indicates durability of QoL improvement through year 3. A trend towards a positive impact on employment, school attendance, physical activity, and other aspects of life is also observed. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Lovo-cel gene therapy for sickle cell disease: Treatment process evolution and outcomes in the initial groups of the HGB-206 study.

lovo-cel (bb1111; LentiGlobin for sickle cell disease [SCD]) gene therapy (GT) comprises autologous transplantation of hematopoietic stem and progenitor cells transduced with the BB305 lentiviral vector encoding a modified β-globin gene (βA-T87Q ) to produce anti-sickling hemoglobin (HbAT87Q ). The efficacy and safety of lovo-cel for SCD are being evaluated in the ongoing phase 1/2 HGB-206 study (ClinicalTrials.gov: NCT02140554). The treatment process evolved over time, using learnings from outcomes in the initial patients to optimize lovo-cel's benefit-risk profile. Following modest expression of HbAT87Q in the initial patients (Group A, n=7), alterations were made to the treatment process for patients subsequently enrolled in Group B (n=2, patients B1 and B2), including improvements to cell collection and lovo-cel manufacturing. After 6months, median Group A peripheral blood vector copy number (≥0.08 c/dg) and HbAT87Q levels (≥0.46 g/dL) were inadequate for substantial clinical effect but stable and sustained over 5.5 years; both markedly improved in Group B (patient B1: ≥0.53 c/dg and ≥2.69 g/dL; patient B2: ≥2.14 c/dg and ≥6.40 g/dL, respectively) and generated improved biologic and clinical efficacy in Group B, including higher total hemoglobin and decreased hemolysis. The safety of the lovo-cel for SCD treatment regimen largely reflected the known side effects of HSPC collection, busulfan conditioning regimen, and underlying SCD; acute myeloid leukemia was observed in two patients in Group A and deemed unlikely related to insertional oncogenesis. Changes made during development of the lovo-cel treatment process were associated with improved outcomes and provide lessons for future SCD GT studies.

CT-275 Incidence and Characteristics of Chronic GVHD in Haploidentical Stem-Cell Transplantation With Post-Transplantation Cyclophosfamide: Experience of the Spanish Group of Hematopoietic Transplantation and Cell Therapy (GETH-TC)

Chronic graft-versus-host-disease (cGVHD) is a relevant cause of late morbidity and mortality related to allogenic-stem-cell transplantation in long-term survivors. GVHD prophylaxis using post-transplantation cyclophosphamide (PTCy) has improved haploidentical stem-cell transplantation (haplo-HSCT) outcomes; however, data related to characteristics, risk factors, or cGVHD outcomes are limited. In this study, we analyze the incidence, risk factors and disability related to cGVHD in patients undergoing haplo-HSCT PTCy. Retrospective multicenter analysis of 389 consecutive patients that underwent haplo-HSCT-PTCy at 7 Spanish transplant centers from 2007 to 2020. cGVHD diagnosis was made following the NIH criteria and Disability related to cGVHD was defined as Fatobene et al criteria. Median age was 48 years (5-74), and 62% were males. AML and HL were the most common indications for transplantation (37%, 22%). 36% received a previous transplant. The median follow-up was 35 months (3-102). Cumulative incidence of aGVHD and aGVHD III-IV at day +180 was 66% and 20%. The 2-year OS was 59%, 2-year PFS 54%, 2 year-GRFS was 45%. 95 patients developed cGVHD, cumulative incidence of cGVHD and cGVHD moderate-severe was 34% and 10% at 3 years. 42 patients required systemic treatment. In the univariate analysis of risk factors for cGVHD in patients alive on day +100, female donor (p=0.034), female donor and male recipient (p=0.041), DRI low (p=0.026), myeloablative conditioning (p=0-047), previous aGVHD (p=0.014), and the use of radiotherapy in conditioning (p=0.004) were identified. Busulfan conditioning was identified as a protective factor (p=0.036). In multivariate analysis, the presence of previous GVHD (p=0.031) and the protective power of conditioning with busulfan (p=0.036) remains statistically significant. cGVHD according to NIH at 3 years, moderate cGVHD showed a higher OS (p=0.054), higher PFS (p=0.004) and lower relapse than the rest of the groups (p=0.035). Receiving one line of treatment (p=0.043) and use of bone marrow were the only risk factors identified for these patients. Haplo-HPT with Cy-post shows a low, but not negligible incidence of cGVHD, and its use represents an alternative for some patients. The development of moderate cGVHD is a protective factor in this transplant modality. The characterization of predictive factors of this subgroup is relevant given the results.

Phenytoin as seizure prophylaxis in hematopoietic stem cell transplantation with busulfan conditioning.

BackgroundPhenytoin is widely used as primary seizure prophylaxis in hematopoietic stem cell transplantation in patients undergoing myeloablative conditioning with busulfan. Because of the negative side effects of phenytoin, we abandoned phenytoin use in these patients. To assess the effect of this change, we performed a retrospective cohort study on all patients receiving busulfan.MethodsWe included 139 patients who underwent conditioning with busulfan for hematopoietic stem cell therapy. We registered the use of phenytoin, as well as the occurrence of seizures, until 7 days after busulfan administration. We compared seizure incidence between patients who received phenytoin and those who did not.ResultsOf the 43 patients who received phenytoin prophylaxis, four patients (9.3%) had a seizure during the conditioning regimen, of which two patients had cerebral non-Hodgkin lymphoma. Furthermore, all these 4 patients had very high levels of phenytoin (intoxication). Of the 96 patients that did not receive phenytoin prophylaxis, three patients (3.1%) had a seizure, and one of these patients had an undefined cerebral lesion. Phenytoin did not relate to seizure prevention in a logistic regression analysis.ConclusionWe conclude that phenytoin prophylaxis in patients treated with busulfan is obsolete and possibly harmful, as phenytoin intoxication can occur. We recommend discontinuing the use of phenytoin as primary seizure prophylaxis in these patients.