Primary Engraftment Research Articles

Outcomes of older adults undergoing allogeneic hematopoietic cell transplantation with post-transplant cyclophosphamide based prophylaxis.

This study evaluates the feasibility of using post-transplant cyclophosphamide (PTCY) prophylaxis in allo-hematopoietic cell transplantation (HCT) for adults aged 65 and older. PTCY is increasingly used to prevent graft-versus-host disease (GVHD) across all donor types, but concerns remain about potential risks, especially in older patients. Fifty-seven adults aged 65 or older with hematological malignancies, undergoing their first allo-HCT with PTCY prophylaxis between January 2011 and January 2023 were included. Overall, 94.8% of patients achieved primary engraftment. The median durations for neutrophil and platelet engraftments were 19 and 21 days. The day +30 cumulative incidence of bacterial bloodstream infection was 43.9%. No CMV reactivations occurred within the first 100 days after letermovir implementation. The day +180 cumulative incidences of grade II-IV and III-IV acute GVHD, and the 2-year cumulative incidence of moderate/severe chronic GVHD were 26.3%, 10.5%, and 4.8%. Eighteen patients (31.6%) relapsed, and 30 (52.6%) died, with relapse (16.4%) and infection (11.5%) being the main causes of death. The estimated 2-year overall survival, non-relapse mortality, cumulative incidence of relapse, and GVHD-free relapse-free survival rates were 45.5%, 27.1%, 33.9%, and 37.0%. Adults aged 70 or older had similar outcomes to those aged 65-69. This study confirms the safety and feasibility of PTCY-based allo-HCT in older adults.

Daratumumab for De-sensitization of Donor Specific Antibodies: Is it a Quicker and Easier way?

Antibodies directed against donor-specific HLA loci (DSA) has been proved as a main culprit for graft rejection, more specifically in HLA mismatched and haplo-identical transplant settings. There is no standardized regimen to manage the presence of DSAs in allogeneic stem cell transplantations (allo-SCTs). Most widely regimen includes combination of rituximab (anti CD20 antibody), Immunoglobulin (IVIG), plasma exchange, and buffy coat infusion, which is costly and time-consuming. Daratumumab (anti CD38 monoclonal antibody) is an effective therapeutic agent to deplete plasma cells and hence, it has a potential to reduce DSA. It has been utilized widely in solid organ transplantation for this purpose. We used this agent in two haplo-identical transplant patients to eliminate or reduce DSA. We observed definite either reduction or elimination in DSA levels in these cases and we could perform haplo-identical transplantation without much delay and with successful primary engraftment in both scenarios. We emphasize that literature on real-world utilization of daratumumab in allo-SCTs is limited. However, it has been utilized widely in solid organ transplantation for this purpose. Our experience with daratumumab regarding effective reduction of DSA followed by successful engraftment might encourage its use in de-sensitization protocols without much delay in transplantation.

Recent infection with SARS-CoV-2 in donors was associated with a higher incidence of acute graft-versus-host disease in recipients undergoing allogeneic haematopoietic stem cell transplantation.

The global pandemic has resulted in the common occurrence of SARS-CoV-2 infection in the population. In the post-pandemic era, it is imperative to understand the influence of donor SARS-CoV-2 infection on outcomes after allogeneic haematopoietic stem cell transplantation (allo-HSCT). We retrospectively analysed allo-HSCTs from donors with mild SARS-CoV-2 infection or early recovery stage (ERS) (group 1, n = 65) and late recovery stage (group 2, n = 120). Additionally, we included allo-HSCT from donors without prior SARS-CoV-2 infection as group 0 (n = 194). Transplants from donors with different SARS-CoV-2 infection status had comparable primary engraftment and survival rates. However, group 1 had higher incidences of acute graft-versus-host disease (aGvHD), grade II-IV (41.5% vs. 28.1% in group 0 [p = 0.014] and 30.6% in group 2 [p = 0.067]) and grade III-IV (22.2% vs. 9.6% [p = 0.004] in group 0 and 12.2% in group 2 [p = 0.049]). Conversely, the risk of aGvHD in group 2 was similar to that in group 0 (p > 0.5). Multivariable analysis identified group 1 associated with grade II-IV (hazard ratio [HR] 2.307, p = 0.010) and grade III-IV (HR 2.962, p = 0.001) aGvHD, which yielded no significant risk factors for survival. In conclusion, we preliminarily demonstrated donors in the active infection state or ERS of mild SARS-CoV-2 infection were associated with higher incidences of aGvHD in transplants from related donors.

Magnetotactic hepatocytes promote liver repopulation after transplantation

Clinical application of hepatocyte transplantation has been halted due to the low engraftment efficiency and limited proliferation capacity of donor hepatocytes in the host liver. Here, we developed a magneto-plasmonic nanohybrid to spatiotemporally drive the primary hepatocytes into the host liver under the external magnetic field that significantly improves the engraftment efficiency of donor hepatocytes. A magnetic lipid-coated-gold-nanorods/iron-oxide nanocluster (GNR-IO) (LGI) was designed for efficient magneto-actuation and photoacoustic imaging-guided cell tracking during the hepatocyte transplantation. By optimizing the concentration of LGI (100 μg /mL) and the configuration of a magnetic field (260 mT), we were able to increase the engraftment efficiency of primary hepatocytes into the host livers of Fah-/- mice, a model with the genetic deficit of fumarylacetoacetate hydrolase (FAH) resulting in liver injuries, for 3.89-fold of the control by one week of the transplantation. Further assessment of repopulation of hepatocytes in the host liver indicated the LGI (100 μg /mL) + magnetic field (260 mT) group had up to 15 % repopulation of the host liver by three-weeks-post-transplantation and reached full repopulation by 6-weeks-post-transplantation. This was 3 ∼ 5 times more rapid than the Fah-/- mice transplanted with hepatocytes without the LGI and the magnetic field. RNA-sequencing of the hepatocytes incubated with LGI indicated that the loading of LGIs enhanced the migration, survival, and engraftment of hepatocytes by regulating the tumor necrosis factor (TNF)-signaling pathway and reducing the macrophage in host liver. Furthermore, LGI-labeled hepatocytes could be tracked in host livers with photoacoustic imaging for at least one week post transplantation. Most of the LGI were cleared from the host systems via the kidneys by six weeks. The novel strategy established in this study, which utilized nanoparticles combined with the magnetic field to improve the efficiency of primary hepatocyte engraftment and repopulation, can provide a feasible scheme for the clinical application of hepatocyte transplantation in the treatment of liver failure.

Trem-Cel, a CRISPR/Cas9 Gene-Edited Allograft Lacking CD33, Shows Rapid Primary Engraftment with CD33-Negative Hematopoiesis in Patients with High-Risk Acute Myeloid Leukemia (AML) and Avoids Hematopoietic Toxicity during Gemtuzumab Ozogamicin (GO) Maintenance Post-Hematopoietic Cell Transplant (HCT).

Trem-Cel, a CRISPR/Cas9 Gene-Edited Allograft Lacking CD33, Shows Rapid Primary Engraftment with CD33-Negative Hematopoiesis in Patients with High-Risk Acute Myeloid Leukemia (AML) and Avoids Hematopoietic Toxicity during Gemtuzumab Ozogamicin (GO) Maintenance Post-Hematopoietic Cell Transplant (HCT).

Inhibition of Enhancer of Zeste Homolog 2 Induces Blast Differentiation, Impairs Engraftment and Prolongs Survival in Murine Models of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is the malignant proliferation of immature myeloid cells characterized by a block in differentiation. As such, novel therapeutic strategies to promote the differentiation of immature myeloid cells have been successful in AML, although these agents are targeted to a specific mutation that is only present in a subset of AML patients. In the current study, we show that targeting the epigenetic modifier enhancer of zeste homolog 2 (EZH2) can induce the differentiation of immature blast cells into a more mature myeloid phenotype and promote survival in AML murine models. The EZH2 inhibitor EPZ011989 (EPZ) was studied in AML cell lines, primary in AML cells and normal CD34+ stem cells. A pharmacodynamic assessment of H3K27me3; studies of differentiation, cell growth, and colony formation; and in vivo therapeutic studies including the influence on primary AML cell engraftment were also conducted. EPZ inhibited H3K27me3 in AML cell lines and primary AML samples in vitro. EZH2 inhibition reduced colony formation in multiple AML cell lines and primary AML samples, while exhibiting no effect on colony formation in normal CD34+ stem cells. In AML cells, EPZ promoted phenotypic evidence of differentiation. Finally, the pretreatment of primary AML cells with EPZ significantly delayed engraftment and prolonged the overall survival when engrafted into immunodeficient mice. Despite evidence that EZH2 silencing in MDS/MPN can promote AML pathogenesis, our data demonstrate that the therapeutic inhibition of EZH2 in established AML has the potential to improve survival.

Hematopoietic stem cell transplantation for B-thalassemia major with alemtuzumab

While matched related donor (MRD) allogeneic hematopoietic stem cell transplantation (HSCT) is a curative option for transfusion-dependent beta-thalassemia (TDT), the use of alternative sources has increased, resulting in the exploration of novel transplant-conditioning regimens to reduce the contribution of graft-versus-host disease (GVHD) and graft failure (GF) to transplant-related morbidity and mortality. Alemtuzumab is a CD52 monoclonal antibody that has been successfully incorporated into myeloablative conditioning regimens for other hematologic conditions, yet there have been limited studies regarding the use of alemtuzumab in HSCT for TDT. The purpose of this study was to evaluate engraftment, incidence of GVHD, and transplant related morbidity and mortality in patients with TDT who received alemtuzumab in addition to standard busulfan-based conditioning. The primary endpoint was severe GVHD-free, event-free survival (GEFS). Our cohort included 24 patients with a median age of 6.8 years (range 1.5-14.9). Eleven patients received a 10/10 MRD HSCT, eleven 10/10 unrelated donor (UD), and two mismatched UD. All patients achieved primary engraftment. For all patients, 5-year GEFS was 77.4% and 5-year overall survival (OS) was 91%. The 5-year cumulative incidence of GF (attributed to poor graft function) without loss of donor chimerism was 13.8% (95% CI: 4.5, 35.3). We report low rates of significant acute GVHD grade II–IV (12.5%) and chronic GVHD (4.4%). Younger age and MRD were associated with significantly improved GEFS, OS and EFS. Our results show that the use of alemtuzumab promotes stable engraftment, may reduce rates of severe GVHD, and results in acceptable GEFS, OS, and EFS.

Trem-Cel, a CRISPR/Cas9 Gene-Edited Allograft Lacking CD33, Shows Rapid Primary Engraftment with CD33-Negative Hematopoiesis in Patients with High-Risk Acute Myeloid Leukemia (AML) and Avoids Hematopoietic Toxicity during Gemtuzumab Ozogamicin (GO) Maintenance Post-Hematopoietic Cell Transplant (HCT)

Background: Allogeneic HCT is the standard of care for eligible patients with AML harboring high-risk features such as adverse-risk cytogenetics or measurable residual disease (MRD), however, the majority of these patients still relapse post-HCT and have very poor outcomes. The use of maintenance therapies intended to control post-HCT relapse is often limited by hematopoietic toxicity towards normal engrafted cells. GO (tradename: Mylotarg TM) is an anti-CD33 antibody-drug conjugate targeting CD33+ AML, but its use is limited by on-target, off-tumor hematopoietic toxicity toward CD33+ myeloid and progenitor cells. Tremtelectogene empogeditemcel (trem-cel; formerly VOR33) is a hematopoietic stem and progenitor cell product, manufactured from CD34+ cells isolated from a patient-matched donor, that has been modified by CRISPR/Cas9 gene-editing to lack CD33. Trem-cel was developed to protect normal hematopoietic cells from post-HCT CD33-directed therapies and allow exclusive targeting of residual leukemia. Methods: VBP101(NCT04849910) is a first-in-human Phase 1/2 open-label multicenter trial to establish the safety of using trem-cel as a donor allograft for CD33+ AML patients who are at high risk of relapse and undergoing myeloablative HCT followed by treatment with GO. Eligible patients (18-70 y) must have CD33+ AML with high-risk features for relapse, such as adverse-risk cytogenetics or MRD, and a 10/10 human leukocyte antigen (HLA)-matched related or unrelated donor. Donors undergo mobilization with G-CSF and plerixafor prior to apheresis. Trem-cel is manufactured from isolated CD34+ cells and cryopreserved. Patients undergo either a busulfan- or TBI-based myeloablative conditioning regimen prior to transplantation with trem-cel. After engraftment and recovery of a sufficient CD33-negative neutrophil count, patients begin maintenance therapy with GO in a 3+3 dose escalation strategy starting at 0.5 mg/m 2 every 28d for 4-8 cycles. Results: To date, 6 patients between 32-68 y (median 63.5 y) have been treated with trem-cel at a median dose of 5.2 x 10 6 CD34+ cells/kg (2.6 - 7.6) and CD33 editing efficiency of 88% (80 - 91). Primary neutrophil engraftment occurred in all patients after a median of 10 days (9 - 11) and platelet recovery occurred after a median of 16 days (15-22) excluding one patient with documented anti-platelet antibody (Fig 1). At the day 28 assessment, full peripheral blood (PB) myeloid chimerism was achieved in all patients and CD33 expression by flow was absent in a median of 94% of neutrophils (86-99) and 92% of monocytes (82 - 94), respectively. Comparable CD33 edits were detected in donor cells across myeloid and lymphoid lineages. GO dosing is ongoing at 0.5 mg/m 2 dose and neutrophil and platelet counts remain stable, suggesting protection from GO-mediated hematotoxicity. Following the 0.5 mg/ m 2 dose, the exposures of GO were predictively higher in the context of CD33-negative hematopoiesis relative to GO doses of 1-2 mg/m 2 (C max) and 4-5 mg/m 2 (AUC), respectively, in CD33+ relapsed/refractory AML patients. During GO dosing, the percent of CD33-negative cells increased in the PB and bone marrow (BM) across cell lineages consistent with enrichment of edited donor cells. Patients are followed for safety, hematologic protection from GO, disease status, and persistence of CD33 editing.

Human Pro-T-Cell Manufacturing in Vitro Is a Safe Procedure for Hematopoietic Stem Cell Transplantation with Delayed T-Cell Reconstitution: Interim Results from 2 Different Clinical Trials

Introduction: Alternative donor Hematopoietic stem cell transplantation (HSCT) strategies are followed by a prolonged period of immune deficiency due to delayed T-cell reconstitution. Either Umbilical Cord (CB) blood transplant or CD34+ selected haplo-transplant provided similar T-cell lymphocytes (CD3+ and CD4+) recoveries at any time point, and recipients had significantly fewer CD4+ T-cells during the first six months compared to HLA-matched donors. The GMP generation of Human T lymphoid Progenitors (HTLP) that are competent to rapidly become in vivo differentiated functional and thymic-educated T-cells may overcome the post-transplant delayed immune reconstitution. Method: HTLP safety and efficacy to accelerate immune reconstitution is currently evaluated in 2 multicentric and single-arm clinical trials: after umbilical cord blood in adult hematologic malignancy (Eudract 2019-004883-23) and after partially HLA compatible CD34+ immune-selected allogeneic hematopoietic stem cell transplantation in Severe Combined Immune Deficiency (SCID) patients (Eudract 2018-001029-14). Results: We enrolled and treated six patients, 4 SCID and 2 adults with malignancy, with an average follow-up of 8.00 (range, 1.60 to 37.7) months. HTLP cell product was infused between D7 to D14 post-HSCT for SCID patients after ensuring the decrease of the ATG level while meaning for the adult patient; the expanded pro-T cells were injected at D0. The Pro-T manufacturing process allows to robustly expand CD34+ cells to HTLP with a median of 79.7% (range 54.6; 98.9) of differentiation (CD7+ CD3-) and a median fold of proliferation of 14.7 (range 8.80; 18.5). No chromosome instability was induced during the expansion. Patients received increasing doses ranging from 0.1 to 0.5 10 6 HTLP/kg. The amount of CD3+/kg was less than 11.2 (range 0; 588)/ kg. No early or mid-term toxicity due to the administration of HTLP has been reported for evaluable patients (6/6). One SCID patient died on D53 because of severe VOD. Primary engraftment was confirmed in 4 out of 5 patients. Interestingly, despite the gradual increase of doses, there was no aGVHD over grade >2. Preliminary efficacy results suggest that circulating CD3+ CD4+ TCRαβ+ T cells > 50/μl before M4 depended on the increased infused dose of HTLP and an intrathymic paracrine effect. Notably, there was no viral infection disease or relapse during the outcome. Discussion conclusion: First infusions of HTLP in both clinical trials confirmed the safety and reproducible GMP manufacturing. HLTP offers an exciting perspective for improving immune reconstitution in alternative hematopoietic transplants.

TCRαβ +/CD19 + - Depleted HLA-Haploidentical Hematopoietic Stem Cell Traansplantation in Pediatric Patient with Fanconi Anemia

Background Allogeneic hematopoietic stem cell transplantation (HSCT) currently represents the only proven treatment able to rescue bone marrow failure and prevent the occurrence of clonal evolution in patients with Fanconi Anemia (FA). If HSCT from HLA-identical related donor (MRD) is reported to be associated with better outcomes, this is not available for most patients. On the other hand, the identification of a fully matched unrelated donor (MUD) may be unsuccessful, or require times incompatible with a prompt treatment of the haematological condition. For patients who lack a MRD or MUD, the preferred donor choice is debated. HLA partially matched haploidentical donor (haplo) is virtually immediately available for all patients and haplo-HSCT has been reported over the last few years as a promising alternative for those patients. Following the favorable results already reported from our group of a cohort of pediatric and young adult FA patients underwent haplo-HSCT, based on the selective depletion of TCRαβ + and CD19 + cells graft manipulation, here we present the outcome of an enlarged cohort of patients who received HSCT in 3 different centers. Methods Patients diagnosed with FA and eligible for an allogeneic transplantation, but lacking a MRD or MUD, and with a suitable HLA-haploidentical donor were eligible to this study. The conditioning regimen included intravenous fludarabine, cyclophosphamide and single-dose total body irradiation for most patients. Pretransplant anti-T-lymphocyte globulin was administered in all patient to control in vivo bidirectional donor-recipient alloreactivity. Apheresis and graft manipulation were performed as previously reported (Li Pira, Biol Blood Marrow Transplant 2016). Results Thirty-five patients (19 males and 16 females) underwent haplo-HSCT between September 2011 and April 2023 in 3 different centers (Bambino Gesù Children's Hospital, Rome, Italy; Policlinico San Matteo, Pavia, Italy; Tokyo; National Center for Child Health and Development, Children's Cancer Center, Tokyo, Japan). Twenty-four patients have been previously reported (Strocchio, Blood Adv 2021). Details on patients and donor characteristics, graft composition and conditioning regimen are listed in table 1. One patient with clonal evolution and 1 with AML received fludarabine and AraC 3 weeks before the start of the conditioning. Primary engraftment was achieved in 33 patients (94.3%), with median time for neutrophil and platelet recovery of 12 days (range, 9-20 days) and 10 days (range, 7-14 days), respectively. Primary graft failure occurred in 2 patients and both were successfully transplanted from the other haploidentical parent. The monitoring of chimerism analysis confirmed a stable engraftment of donor hemopoiesis in all engrafted patients. Within 33 evaluable patients, 9 (27.3%) experienced grade 1-2 aGvHD (7 with stage I-II skin-only involvement, 1 with stage I lower gastrointestinal involvement) at a median time of 35.5 days (range, 16-67 days) after HSCT. One patient developed late-onset skin only grade 2 aGvHD at day +211 after HSCT. One patient (3%) experienced grade IV aGvHD (liver and gastrointestinal involvement) at day + 100 from HSCT, 4 weeks after the infusion of adenoviral-specific donor T cells; he is currently receiving ruxolitinib and the complete resolution of GvHD was obtained. Only one patient (3%), developed cGvHD with mild skin involvement. Thirteen patients (39.4%) experienced one or more viral infections or reactivations (6 cytomegalovirus, 4 herpesvirus-6, 2 adenovirus, 1 parainfluenzae); the specific antiviral treatment was administered with a complete resolution of infection in all cases. With a median follow up of 5.5 years (range, 0.6-11.4 years) all the patients are alive and with resolution of hematological complications. Except for the already reported vulvar intraepithelial neoplasia, no additional posttransplant malignancy was observed Conclusions Our data obtained from enlarged cohort of patients previously reported, confirm excellent results in terms of survival and a good rate of engraftment also in a multicenter setting. The low incidence of acute and chronic GvHD, a particularly detrimental complication in patients with DNA repair disorder, makes this approach attractive in this patient population. In FA patients lacking a standard donor, TCRab +/CD19 + haplo-HSCT is confirmed to be a promising strategy.

Inhibiting Mitochondrial RNA Degradosome Complex SUV3 and PNPT1 Increases dsRNA in the Cytoplasm, Triggers Viral Mimicry Response and Sensitizes AML Cells to Immune Mediated Killing

Eukaryotic cells have two separate genomes; nuclear DNA organized in chromosomes, and circular mitochondrial DNA located within mitochondria. Mitochondrial DNA is comprised of a double-stranded circular genome that is 16.6 kB in length, lacks introns, and encodes two rRNAs, 22 t-RNAs and 13 of the 90 proteins in the mitochondrial respiratory chain. To maintain homeostasis, mitochondria possess RNA degradation machinery to regulate mitochondrial RNA turnover. The ATP-dependent helicase, SUV3 (encoded by the gene SUPV3L1) and the exonuclease PNPase (encoded by the gene PNPT1) function in a complex to degrade mitochondrial dsRNA. We identified PNPT1 and SUPV3L1 through an unbiased in-silico screen using bioinformatics platform to identify novel targets in AML. Our analysis revealed a strong correlation between these genes and ontologies of exogenous dsRNA, response to viruses, and RNA catabolic processes. PNPase and SUV3 protein were increased in 7/7 AML patient samples and 10/10 of AML cell lines compared to the normal hematopoietic cells by immunoblotting. Analysis of the TARGET AML dataset revealed that AML patients with increased expression of SUPV3L1 (p = 0.051, p= 0.045) and PNPT1 (p = 0.0013, p = 0.018) had decreased overall survival and event-free survival respectively. We knocked down or knocked out PNPT1 and SUPV3L1 with shRNA or sgRNA in AML cells, to study the importance of these genes in AML. Genetic knockdown or knockout of PNPT1 or SUPV3L1 decreased the growth and viability of OCI-AML2, TEX, K562, U937, NB4 and 8227 leukemia cells. Moreover, SUPV3L1 & PNPT1 ranked top 5.2% and 7.4% of essential genes in 26 leukemia cell lines in CRISPR screens and 2.7% and 4.9% in RNAi screens (https://depmap.org/portal). Knockdown of PNPT1 & SUPV3L1 also reduced the clonogenic growth of AML cells. Demonstrating the functional importance of PNPT1 & SUPV3L1 on leukemia-initiating cells in vivo, genetic knockdown of PNPT1 and SUPV3L1 significantly reduced engraftment of TEX cells into the marrow of immune-deficient mice. Finally, primary AML cells with SUPV3L1 knockdown had reduced engraftment into the marrow of immune-deficient mice. Whereas, the knockdown of SUPV3L did not alter the primary engraftment of CD34+ enriched cord blood cells. Mechanistically, knockdown of PNPT1 and SUPV3L1 in OCI-AML2 cells increased levels of cytoplasmic dsRNA 3-4 fold compared to control. Knockdown of PNPT1 and SUPV3L1 also increased cytoplasmic dsRNA in 143B cells, but not Rho(0) 143B cells that lack mitochondrial DNA, demonstrating a mitochondrial source for the increased dsRNA. Increased cytoplasmic dsRNA can mimic viral infection and trigger a type 1 Interferon response. Knockdown of PNPT1 or SUPV3L1 increased expression of genes (INFgR1, ICAM, IRF7 & JAK/STAT) associated with a type 1 interferon response compared to control. We also observed that PNPT1 and SUPV3L1 knockdown in AML cells sensitized the leukemic cells to T-cell mediated killing upon co-culturing with double negative T cells. In addition, using an immunocompetent Balbc mouse model, we demonstrated endogenous T cells were required to observe the greatest reduction in tumor burden after SUPV3L1 knockdown. In summary, the mitochondrial RNA degradosome complex SUPV3L1 and PNPT1 are overexpressed in AML and are essential for AML cells and stem/progenitors. These enzymes regulate the levels of mitochondrial dsRNA and their inhibition leads to a viral mimicry response and heightened sensitivity to immune-mediated killing.

Long-Term Outcome of a Unique Haploidentical High-Dose Peripheral Stem Cell Transplantation Protocol for Hematologic Malignancies: A Prospective Single-Center Study

Background Although many reports have established the efficacy of post-transplantation cyclophosphamide (PTCy) or ATG for GVHD prophylaxis to in vitro non-T cell-depleted (non-TCD) haplo-HSCT and achieved a good clinical effect, the overall results of NRM, RI, DFS, and OS still need to be improved, especially for patients with high-risk or R/R hematologic malignancies. We speculated that if GVHD could be controlled well, higher doses of in vitro non-TCD peripheral blood stem cells (PBSCs) as a graft source for haplo-HSCT may be able to better balance engraftment, immune reconstitution, infection, and graft versus leukemia (GVL) effect to improve clinical outcomes. Apparently, due to the increased risk of GVHD as a major concern, the research of haplo-HSCT using ultra-high doses of in vitro non-TCD PBSCs as a graft source is now scarce. To this end, we constructed a unique haploidentical high-dose peripheral stem cell transplantation (haplo-HDPSCT) protocol. A prospective study was performed in our center using this protocol to treat patients with hematologic malignancies. Methods From January 2006 to December 2018, a total of 346 patients with hematologic malignancies less than 50 years old were enrolled. 205 patients who had no matched sibling donor (MSD) enrolled in the haplo-HDPSCT group, and 141 patients were enrolled in the MSD-PSCT group. Patients in two groups were comparable except for several characteristics such as more young and high-risk patients in the haplo-HDPSCT group. All patients were followed up until December 2022 with a median follow-up time of 66.1 months. The MSD-PSCT protocol was performed with the classical standard process. Our haplo-HDPSCT protocol was designed adopting high-dose in vitro non-TCD PBSCs as graft, Ara-C+Bu/Cy+rATG as the conditioning regimen, and Basiliximab plus short-term low-dose GCs added to standard GVHD prophylaxis. The long-term outcomes of the two groups were analyzed and compared. The primary endpoint was aGVHD, and the second endpoints were engraftment, infection, RI, DFS, and OS. Results In the haplo-HDPSCT group(n=205), the infused median mononuclear cells(MNCs), CD34+ cells, and CD3+ cells were 15.63×10 8/kg, 8.59×10 6/kg and 5.39×10 8/kg, respectively, which significantly higher than those in the MSD-PSCT group(n=141). Only 3 of 205 patients in the haplo-HDPSCT group developed primary graft failure (PGF) and the primary engraftment rate was 98.5%. The incidence of grades II-IV and III-IV aGVHD within 100 days was similar between the two groups (37.1% vs. 27.3%, P=0.109; and 11.7% vs. 8.7%, P=0.507). The 3-year cumulative incidence of localized cGVHD was similar between the two groups (50.3% vs 49.3%, P=0.357), but the extensive cGVHD in the haplo-HDPSCT group was significantly lower than that in the MSD-PSCT group (10.9% vs 26.9%, P=0.035). The 3-year cumulative RI and NRM were also similar between the two groups (RI: 31.1%vs 27.3%, P=0.625; NRM: 16.1% vs 11.5%, P=0.394). No significant differences in 3- and 5-year OS (69.6% and 66.9% vs. 74.9% and 73.2%, P=0.282) or 3- and 5-year DFS (67.2% and 63.7% vs. 72.0% and 69.4%, P=0.215) were observed in the haplo-HDPSCT group and the MSD-PSCT group. The CMV disease occurrence was 3.1% in the haplo-HDPSCT group. The EBV-DNAemia rate was 10.4% in the haplo-HDPSCT group and 5.7% in the MSD-PSCT group(P=0.836). Only two patients in the haplo-HDPSCT group developed PTLD. Conclusions This study demonstrates that aGVHD is not high in our haplo-HDPSCT protocol and the long-term follow-up shows that this protocol may bring benefits including high engraftment rate, less relapse and viral infection, which implies that higher doses of in vitro non-TCD PBSCs as a graft source for haplo-HSCT protocol is feasible if appropriate GVHD prophylaxis is adopted.

Outcomes of Tcrαβ and CD19-Depleted Hematopoietic Stem Cell Transplantation from Matched, Mismatched Unrelated and Haploidentical Donors for Pediatric Patients with Hematological Malignancy: A Meta-Analysis

Introduction: TCR αβ+/CD19+ depletion in hematopoietic stem cell transplantation (HSCT) is an emerging graft manipulation technique to help overcome the HLA barrier in the mismatched or haploidentical transplant setting and reduce or prevent graft-versus-host disease. Limited research has explored the impact of various donor sources, and type of malignancy on the outcomes of TCR αβ+/CD19+ depleted HSCT for acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and myelodysplastic syndrome (MDS). Therefore, we performed a comprehensive meta-analysis of published clinical studies using individual patient data (IPD) to address this scientific gap. Methods: This meta-analysis combined patient-level data from studies involving AML/MDS and ALL patients treated with TCR αβ+/CD19+ depleted HSCT. By utilizing the IPDfromKM technique, we reconstructed time-to-event data from aggregated Kaplan-Meier curves, specifically focusing on overall survival (OS), disease-free survival (DFS), and GVHD-free, relapse-free survival (GRFS). Additionally, we conducted parallel meta-analyses to calculate the pooled incidences of GVHD, graft failure, mortality, as well as CMV and EBV reactivation, and sinusoidal obstruction syndrome (SOS) using aggregate data. All analyses were performed using STATA software. Our comprehensive literature search included publications up to May 2023, excluding studies with significant sample overlap. Results: The meta-analysis incorporated 817 children with AML/MDS or ALL from 12 studies. Successful primary engraftment was observed in 95% of patients (95% CI: 92 - 98), with an average time to neutrophil and platelet engraftment of 12 days. The incidence of acute GVHD grades I-II was 24% (95% CI: 14 - 34), while grades III and IV were 11% (95% CI: 3.0 - 0.18). Chronic GVHD was observed in 19% of patients (95% CI: 11 - 28). The relapse and graft failure rates were 25% (95% CI: 19 - 32) and 6% (95% CI: 2.0 - 9.0), respectively. The all-cause mortality was 34% (95% CI: 29 - 38), with HSCT-related mortality at 11% (95% CI: 8.0 - 13), while relapse-related mortality accounted for 22% (95% CI: 17 - 28). At a median follow-up of 2.55 years, the 1-year and 5-year OS rates were 74.7% (95% CI: 60.6 - 78.3) and 64.4% (95% CI: 59.9 - 68.5), respectively. No significant differences in OS were found between ALL and AML/MDS patients (HR= 0.99, 95% CI: 0.48 - 2.02, P=0.98) or between different donor types (Figure 1A). The DFS rates at 1- and 5-year were 72.8% (95%CI: 67.6 - 77.3) and 61.8% (95%CI: 56.1 - 67.0), respectively. No significant differences in DFS were observed between ALL and AML/MDS patients (HR = 0.74 95%CI: 0.48 - 1.14, P=0.17) or between different donor types (Figure 1B). The overall 1- and 5-year GRFS rates were 71.1% (95% CI: 64.1 - 77.4) and 63.0% (95% CI: 55.3 - 69.8), respectively. The overall incidence of CMV and EBV reactivation was 33% (95% CI: 19 - 46) and 16% (95% CI: 0 - 38), respectively. Conclusions: Our meta-analysis confirms the effectiveness and safety of TCRαβ and CD19-depleted HSCT for hematological malignancies, demonstrating high engraftment success, low graft failure, and a manageable incidence of acute and chronic GVHD. The consistently favorable outcome data across different donor types and malignancies support the broad applicability of this form of graft manipulation.

Haploidentical Stem Cell Transplantation Using Total Body Irradiation (600 cGy) and Fludarabine with ATG in Upfront and Salvage Setting in Adult Patients with Severe Aplastic Anemia

Background: Haploidentical stem cell transplantation from a related mismatched donor (Haplo-SCT) has extended into adult patients with severe aplastic anemia (SAA) with advances in controlling graft failure and GVHD. However, because of concerns for morbidity and mortality, Haplo-SCT has been used as salvage treatment after the failure of immunosuppressive therapy (IST). In Haplo-SCT for SAA patients, the achievement of successful engraftment without GVHD is fundamental for better long-term survival with good quality of life. We optimized the dose of rabbit ATG and fractionated TBI (fTBI) with fixed dose of fludarabine (Flu, 150mg/m 2/day) using step-by-step dose de-escalation (Lee et al, Am J Hematol, 2018) and reached fTBI 600 cGy/Flu/intermediate-dose ATG (5 mg/kg). This protocol showed an overall survival rate of 91.0% after a median follow-up of 32.3 months in a study of 47 patients (Lee et al, BBMT, 2020). Here, we evaluated the feasibility of this protocol in both upfront and salvage setting for Haplo-SCT in adult patients with SAA. Methods: We analyzed 68 consecutive patients who underwent Haplo-SCT between Oct. 2014 and Jan. 2023. Fifty-six (82%) patients had salvage Haplo-SCT after the failure of standard IST (ATG + CsA) (n=36) and CsA monotherapy (n=20). Twelve (18%) patients underwent upfront Haplo-SCT. All patients received a conditioning regimen of 600 cGy fTBI (200 cGy, 3 times) and Flu (30 mg/m 2/day) for 5 days. GVHD prophylaxis consisted of ATG/tacrolimus/methotrexate (MTX). ATG (Thymoglobulin ®,2.5 mg/kg/day) was administered on days -2 and -3. All patients received PBSC. Results: The median age was 34.0 years (17-61) and 30 (44%) patients had very SAA (VSAA) at transplantation. Haploidentical donors are composed of mother (n=10), father (n =8), sibling (n=30), offspring (n=17), and others (n=3). All patients achieved primary engraftment. The cumulative incidence of acute GVHD (grade ≥2) and chronic GVHD (≥ moderate) was 26.5% at 100 days and 9.3% at 4 years, respectively. With a median follow-up of 54 months, the 4-year overall survival (OS) and failure-free survival (FFS) was 93.9% and 92.4%, respectively. 4-year OS of upfront and salvage Haplo-SCT was 100 and 94.7%, respectively (P = 0.352). The 4-year GFFS was 78.9%, and there was no significant difference between upfront and salvage Haplo-SCT (83.3% vs 78.1%, P = 0.709). In the subgroup analysis, 4-year GFFS of patients who failed the standard IST (ATG+CsA) excluding CsA monotherapy also showed no statistical difference compared to the others (85.8% vs 71.3%, P = 0.143). Furthermore, after adjusting for the potential factors affecting GFFS (patient's age, donor type, and comorbidity index), no different GFFS was shown between upfront and salvage Haplo-SCT. Conclusions: Our results suggest that Haplo-SCT with fTBI 600 cGy/Flu/ATG-5 can be an effective alternative option in both upfront and salvage settings, when fully matched donors are not available.

Comparison of Stem Cell Transplantation Using Unrelated, Haploidentical, and Sibling Donors for Patients with Acquired Severe Aplastic Anemia: A Single-Center Retrospective Cohort Study

The preferred donor (haploidentical vs. matched unrelated donor) choice in patients with acquired severe aplastic anemia (SAA) who lack a human leukocyte antigen (HLA)-matched sibling donors (MSDs) and fail upfront immunosuppressive treatment (IST) therapy is unknown. We retrospectively investigated SAA patients (n=58) who underwent allogeneic stem cell transplantation (allo-SCT) between January 2012 and October 2022. The 5-year overall survival (OS) (87.5% ± 11.7% vs. 98.0% ± 6.5% vs. 83.3% ± 7.6%, respectively, p= 0.926) and 5-year failure-free survival (FFS) (60.0% ± 18.2% vs. 87.0% ± 7.0% vs. 78.3% ± 8.6%, respectively, p= 0.222) were comparable among the unrelated donors (URD; n=8), haploidentical donors (HID; n=25), and MSD cohorts (n=25). Multivariate analysis revealed that primary engraftment failure independently predicted OS, and secondary graft failure predicted FFS among SAA patients who underwent allo-SCT, although donor type and age did not predict the outcomes. An urgent second SCT for patients with engraftment failure may be an effective salvage treatment. Summarily, our findings showed that an alternative donor SCT is indicated for eligible SAA patients without MSDs even if these patients are aged ⩾ 40 years.

Impact of SARS-CoV-2 infection on graft composition and early transplant outcomes following allogeneic hematopoietic stem cell transplantation

Objective: To assess the feasibility of using donors with novel coronavirus disease 2019 (COVID-19) for allogeneic hematopoietic stem cell transplantation (allo-HSCT) when there are no other available donors and allo-HSCT cannot be delayed or discontinued. Methods: Seventy-one patients with malignant hematological diseases undergoing allo-HSCT between December 8, 2022, and January 10, 2023, were included. Of these, 16 received grafts from donors with mild COVID-19 (D-COVID(+) group) and 55 received grafts from donors without COVID-19 (D-COVID(-) group). The graft compositions were compared between the two groups. Engraftment, acute graft-versus-host disease (aGVHD), overall survival (OS), and relapse were also evaluated. Results: There were no serious side effects or adverse events in the D-COVID(+) group. The mononuclear cell dose and CD34(+) cell dose were comparable between the two groups, and no additional apheresis was required. There were no significant differences in the lymphocyte, monocyte, and T-cell subset doses between the two groups. The median natural killer cell dose in the D-COVID(+) group was significantly higher than that in the D-COVID(-) group (0.69×10(8)/kg vs. 0.53×10(8)/kg, P=0.031). The median follow-up time was 72 (33-104) days. All patients achieved primary engraftment. The 60-day platelet engraftment rates in the D-COVID(+) and D-COVID(-) groups were 100% and (96.4±0.2) %, respectively (P=0.568). There were no significant differences in neutrophil (P=0.309) and platelet (P=0.544) engraftment times. The cumulative incidence of grade 2-4 aGVHD was (37.5±1.6) % vs. (16.4±0.3) % (P=0.062), and of grade 3-4 aGVHD was 25.0% ±1.3% vs. 9.1% ±0.2% (P=0.095) in the D-COVID(+) and D-COVID(-) groups, respectively. The probabilities of 60-day OS were 100% and 98.1% ±1.8% (P=0.522) in the D-COVID(+) and D-COVID(-) groups, respectively. There was no relapse of primary disease during the study period. Conclusion: When allo-HSCT cannot be delayed or discontinued and no other donor is available, a donor with mild COVID-19 should be considered if tolerable. Larger sample sizes and longer follow-up periods are required to validate these results.

Treatment With the Combination of Rituximab and Intravenous γ Globulin Is Effective in Promoting Engraftment in Donor-Specific Antibody Positive Patients Receiving Myeloablative Conditioning Haploidentical Stem Cell Transplantation

Donor-specific anti-HLA antibodies (DSAs) are a major cause of engraftment failure in patients receiving haploidentical stem cell transplantation (HaploSCT). Effective procedures are needed for those who demand urgent transplantation and have no other donor options. We here retrospectively analyzed 13 patients with DSAs successfully treated with desensitization of rituximab and intravenous γ globulin (IVIg) before HaploSCT from March 2017 to July 2022. All 13 patients had DSA mean fluorescence intensity >4000 of at least 1 loci before desensitization. Of the 13 patients, 10 patients were with the initial diagnosis of malignant hematological diseases, and 3 were diagnosed with aplastic anemia. Patients were treated with 1 (n = 3) or 2 (n = 10) doses of rituximab (375 mg/m2 for 1 dose). All patients receive the same total dose of 0.4 g/kg of IVIg within 72 hours before haploidentical stem cell administration to neutralize the remaining DSA. All patients achieved neutrophil engraftment, and 12 patients achieved primary platelet engraftment. The patient with primary platelet engraftment failure received purified CD34-positive stem cell infusion nearly 1 year after transplantation and achieved platelet engraftment thereafter. The estimated 3-year overall survival is 73.4%. Although further studies on larger numbers of patients are needed, it is clear that the combination of IVIg and rituximab is an effective way to clear DSA and has a strong effect on promoting engraftment and survival for patients with DSA. It is a practical and adaptable combination of treatment options.

Abstract B040: Leader cells mediate tumor engraftment and promote immunosuppression to drive ovarian cancer progression in vivo

Abstract Epithelial ovarian cancer (EOC) is most frequently diagnosed at an advanced stage, and is characterized by its high recurrence rate and almost-universal acquisition of chemoresistance. Metastasis of EOC is driven by heterogenous spheroids that passively disseminate throughout the peritoneal cavity, promoting tumor spread and recurrence. We previously identified a sub-population of highly motile and invasive “Leader cells” (LCs) that express the basal epithelial protein KRT14 as an absolute determinant of invasive potential. We demonstrated KRT14 knockout in EOC cells does not impact viability or proliferation, however completely ablates their ability to invade through Matrigel in 2D and 3D format in vitro. Although a role for LCs in mesothelial clearance and invasion has been established, these mechanisms remain poorly understood. Here we show a critical role for KRT14+ LCs in immunosuppression and tumor-immune privilege in vivo. In the ID8 syngeneic EOC mouse model, tumor-specific LC (KRT14) loss (LCKO) impaired ovarian intrabursal primary tumor engraftment and subsequent metastatic spread. Immune phenotyping by flow cytometry revealed that mice bearing LCKO tumors displayed decreased proportions of immunosuppressive tumor-adjacent Tregs and M2 macrophages, and improved CD8+ T-cell/Treg ratios. Conversely, forced LC-overexpression (LCOE) in EOC tumors accelerated metastasis, and promoted tumor-immune evasion via upregulated secretion of suppressive chemokines including CCL22 and CCL5. In addition to their critical role in migration and invasion, the current study provides in vivo evidence that leader cells are mediators of immune suppression in ovarian cancer. This work supports the basis that defining novel strategies to target LCs to promote anti-tumor immunity and limit metastatic progression may be a useful strategy for improved epithelial ovarian cancer therapy. Citation Format: Amy L. Wilson, Brittany R. Doran, Bashirah Basri, Laura R. Moffitt, Magdalena Plebanski, Andrew N. Stephens, Maree Bilandzic. Leader cells mediate tumor engraftment and promote immunosuppression to drive ovarian cancer progression in vivo [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B040.

Fludarabine- and Low-Dose Cyclophosphamide-Based Conditioning Regimens for Unmanipulated Hematopoietic Cell Transplantation from Unrelated Donors and Matched Siblings in Patients with Fanconi Anemia: Results from the Cbmtr

Hematopoietic cell transplantation (HCT) is the only option to cure hematopoietic complications in Fanconi anemia (FA). We have analyzed 98 unmanipulated HCT from matched siblings and unrelated donors. Overall, the outcomes were favorable, with 100% primary engraftment, 76.7% (66.3-84.3%) probability of three-year overall survival (OS), and 75.9% (65.5-83.5%) of event-free survival (EFS). The incidence of acute graft-versus-host disease (GvHD) was 38.8% (27.1-50.3%) for grade II-IV and 23.4% (11.2-38.2%) for grade III-IV. The incidence of chronic GvHD was 27.7% (13.2-43.7%) for all grades and 13.0% (1.4-34.1%) for moderate-to-severe disease. Comparing different conditioning regimens, patients conditioned with fludarabine (Flu) and low-dose cyclophosphamide (Cy) achieved 100% probabilities of OS and EFS, which was superior to those with additional busulfan (Bu) or irradiation (P=.024 and .020, respectively). No significant difference was found in the incidences of GvHD across conditioning regimens. We demonstrated that high-dose Bu (> 6.4 mg/kg vs. ≤ 6.4 mg/kg) was associated with an increased risk of death [hazard ratio (HR)=6.705 (2.384-18.855), P<.001] and events [HR=5.434 (2.060-14.334), P=.001]. In conclusion, unmanipulated HCT from matched siblings and unrelated donors achieved favorable engraftment, OS, and EFS in FA patients. Our data suggested that conditioning regimens based on Flu and low-dose Cy provided excellent survival. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Phase II Study of Myeloablative 8/8- or 7/8-Matched Allotransplantation with Post-Transplant Cyclophosphamide, Tacrolimus, and Mycophenolate Mofetil: Marked Reduction in Gvhd Risk without Increased Relapse Risk Compared to Historical Cyclosporine/Methotrexate

Introduction Allogeneic hematopoietic cell transplantation (HCT) is the only potentially curative treatment for many high-risk hematologic malignancies. Myeloablative conditioning (MAC) is currently the standard of care for young and fit patients; however, graft-versus host disease (GVHD) continues to be a major limitation to the success of HCT, increasing post-transplant morbidity and mortality. An ideal HCT is one that combines strategies to reduce the incidence and severity of GVHD, without compromising graft versus-tumor effect. We hypothesized that the GVHD prophylaxis regimen consisting of post-transplant cyclophosphamide (PTCy), tacrolimus (Tac) and mycophenolate mofetil (MMF) would reduce the incidence of chronic GVHD in patients receiving a standard MAC HCT without an increase in risk of malignant relapse. Methods This is an analysis of a phase II study at the University of Minnesota using a MAC regimen of either: 1. total body irradiation (TBI, total dose 1320 cGy administered twice a day from days -4 to -1) or 2. Busulfan 3.2mg/kg daily (cumulative AUC 19,000 - 21,000 μmol/min/L) plus fludarabine 160mg/m2 days -5 to -2 for patients unable to receive further radiation, followed by a GVHD prophylaxis regimen of PTCy (50mg/kg days +3 and +4), Tac and MMF (beginning day +5). The primary endpoint is cumulative incidence of chronic GVHD requiring systemic immunosuppressive treatment at 1 year post-transplant. Eligibility included: age ≤ 60 years, malignant or non-malignant diagnosis, matched related (MRD) or unrelated (MUD) donor with either a bone marrow (BM) or filgrastim-mobilized peripheral blood (PB) graft. We compared those results to our previous MAC protocol which utilized a regimen of either cyclophosphamide (60mg/kg days -6 and -5)/TBI (1320 cGy administered twice a day from days -4 to -1) or busulfan (0.8 mg/kg/dose IV in 4 daily doses days -9 to -6 [total 12.8 mg/kg])/ cyclophosphamide (50 mg/kg days -5 to -2) and GVHD prophylaxis consisted of cyclosporine and methotrexate. Results Through June 2022, we treated 125 patients with a median follow up of 472 days post-transplant. Of those, 48% were female and 22 (17.6%) were ≤ 18 years old with median age at HCT of 38 years (range, 1-59 yrs; Interquartile range [IQR], 22-50 yrs). Donor source was 8/8 MRD in 69 patients (55.2%), 8/8 MUD in 44 (35.2%), and 7/8 MUD in 12 (9.6%). Graft source was bone marrow in 43 patients (34.4%) and PB in 82 (65.6%). Preparative regimen was TBI in 89.6% of patients. 124 of 125 patients achieved primary neutrophil engraftment by 42 days, median 16 days (range, 13-30). 119 of 125 achieved platelet engraftment by 6 months, median 25.5 days (range, 14-98). 91 patients (72.8%) required total parenteral nutrition (TPN) for oral mucositis and regimen-related toxicities during their initial transplant admission. Cumulative incidence of Grade II-IV acute GVHD by day +100 was 16% (95% confidence interval (CI): 9-23%); Grade III-IV acute GVHD was 4% (CI: 0-8%). No patients experienced grade IV GVHD and there were no deaths due to GVHD. At 1 year, only 3 of 105 patients developed chronic GVHD requiring immune suppression, for a cumulative incidence of 4% (CI: 0-8%). Two-year cumulative incidence of relapse was 25% (CI: 15-36%). Two-year overall survival was 80% (CI: 69-87%) and two-year GVHD-free relapse free survival (GRFS) was 57% (CI: 45-67%). Kaplan-Meier curves comparing overall survival and GRFS between this cohort and our previous MAC protocol without PTCy are shown in Figure 1. Figure 2 shows the comparison between these two consecutive cohorts in incidence of acute and chronic GVHD, transplant-related mortality (TRM) and relapse. Discussion Myeloablative HCT with a TBI or Busulfan/Fludarabine-based preparative regimen, followed by a GVHD prophylaxis regimen of PTCy, Tac, and MMF results in extremely low incidence of chronic GVHD, the primary objective of this clinical trial. Importantly, this regimen is feasible and effective for pediatric and adult patients. Further improvement in outcomes could potentially be achieved by incorporating post-transplant relapse mitigating strategies as well as supportive care measures to decrease regimen-related toxicities. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal