Nonmyeloablative Conditioning Research Articles

Durable Mixed Chimerism May Permit Subsequent Immunosuppression-Free Intestinal Transplantation – a Proof-of-Principle Study

Intestinal transplantation (ITx) is the definitive treatment for intestinal failure but has the highest rejection rate among solid organ transplants, requiring high doses of immunosuppression with high rates of infection, graft-versus-host disease, and malignancy. Transplant tolerance would overcome the need for long-term immunosuppression. Using non-myeloablative conditioning, our laboratory has developed a novel swine model of hematopoietic stem cell transplantation (HSCT) that produces durable mixed chimerism (MC) and immune tolerance without toxicity. We investigated whether durable MC would promote tolerance of subsequently transplanted donor-matched intestinal allografts without immunosuppression. Using miniature swine with defined MHC, we performed HSCT across an MHC-Class-I haplotype mismatch. Immunosuppression was stopped by day 45. MC was evaluated by flow cytometry, and mixed lymphocyte reaction (MLR) assays were used to evaluate cellular responses. Subsequently, orthotopic ITx was performed without immunosuppression using a donor that was MHC-matched to the HSCT donor. Recipients were observed for four weeks and euthanized for tissue collection and mechanistic assays. After HSCT, the recipients developed durable multilineage MC and apparent deletional tolerance. After ITx, recipients showed no clinical or histological signs of rejection, and chimerism was unchanged. These results demonstrate the potential value of generating durable MC to achieve transplant tolerance.

Long-term outcome after allogeneic stem cell transplantation for GATA2 deficiency: An analysis of 67 adults and children from France and Belgium.

Modalities and timing of haematopoietic stem cell transplant (HSCT) in patients with GATA2 deficiency are still subject to debate. On June 2022, 67 patients (median age 20.6 years) underwent a first allogeneic HSCT among 21 centres. Indications for HSCT were myelodysplastic syndrome (MDS) ≤5% blasts ± immunodeficiency (66%), MDS >5% blasts (15%), acute myeloid leukaemia (19%). Conditioning regimen was myeloablative in 85% and anti-thymocyte globulins were used in 67%. The cumulative incidence (CInc) of acute graft versus host disease (GvHD) grade II-IV and III-IV at day 100 were 42% and 13%, and CInc of chronic and extensive chronic GvHD at 2 years were 42% and 23%. CInc of relapses was 3% and 11% at 1 and 5 years. Overall survival (OS) at 1 and 5 years was 83% and 72% (median follow-up 5.6 years). The factors associated with worse OS in multivariable analysis were the year of HSCT, a history of excess blasts before transplant and peripheral blood stem cell (PBSC) grafts. Age at HSCT, non-myeloablative conditioning and PBSC grafts were associated with increased non-relapse mortality. In conclusion, bone marrow monitoring to identify clonal evolution and perform HSCT before the appearance of excess blast is mandatory.

211At-Labeled Anti-CD45 Antibody as a Nonmyeloablative Conditioning for Canine DLA-Haploidentical Stem Cell Transplantation.

The α-emitter 211At deposits a high amount of energy within a few cell diameters, resulting in irreparable DNA double-strand breaks while minimizing off-target toxicity. We investigated the use of the 211At-labeled anti-CD45 monoclonal antibody (mAb) 211At-CD45-B10 as a nonmyeloablative conditioning regimen for dog-leukocyte-antigen-haploidentical hematopoietic cell transplantation. Methods: Seventeen healthy dogs were injected with either a 0.50 (n = 14) or 0.75 (n = 3) mg/kg dose of anti-CD45 mAb labeled with 211At (8.436-23.199 MBq [0.228-0.627 mCi/kg]) on day -3. Peripheral blood stem cells from dog-leukocyte-antigen-haploidentical donors were given on day 0. Peripheral blood chimerism was calculated by polymerase chain reaction assays, and blood clearance of the radioimmunoconjugate was studied using enzyme-linked immunosorbent assay and radioactivity measurements of serial blood samples. Results: All dogs achieved donor chimerism by day 28 (range, 27%-100%). The hematopoietic engraftment rate was 100%, though engraftment durability was variable. No difference in absorbed dose to blood was seen for the 2 mAb dosing levels studied. Neutropenia (0-29 cells/μL), lymphocytopenia (36-130 cells/μL), and thrombocytopenia (1.5-9 × 103/μL) with prompt recovery were observed. The main adverse nonhematologic event related to 211At-CD45-B10 was mild reversible transaminitis. Graft-versus-host disease was not seen. Twelve of the 17 dogs survived over 30 d, with donor chimerism ranging from 3% to 99%. Conclusion: The results suggest that nonmyeloablative conditioning with 211At-CD45-B10 could be used in haploidentical hematopoietic cell transplantation though with variable engraftment.

Pharmacokinetics of Briquilimab as a Conditioning Agent for Hematopoietic Stem Cell Transplantation in Patients With Severe Combined Immunodeficiency, Myelodysplastic Syndrome, or Acute Myeloid Leukemia

For successful engraftment of donor hematopoietic stem cells (HSC), conditioning with chemotherapy and/or radiation prior to hematopoietic cell transplantation (HCT) has been required to open marrow niche space and minimize the risk of immune rejection. Briquilimab, a humanized IgG1 monoclonal antibody that blocks the interaction between the c-Kit receptor and stem cell factor on various C-Kit expressing tissues including HSC, is a potential nonmyeloablative conditioning agent in clinical development for patients with severe combined immunodeficiency (SCID), myelodysplastic syndromes (MDS), and acute myeloid leukemia (AML). This study aimed to characterize pharmacokinetics (PK) and develop a population PK model of briquilimab after single intravenous infusions of 4 different doses in patients with SCID, MDS, or AML receiving HCT. The PK data was collected from 2 different studies: JAS-BMT-CP-001 and JSP-CP-003. JAS-BMT-CP-001 is a phase 1/2 open-label study of briquilimab as a conditioning agent prior to allogenic HCT in SCID patients. The participants received single intravenous infusions of 0.1, 0.3, 0.6, or 1.0 mg/kg. JSP-CP-003 was a phase 1a/b open-label study of briquilimab in combination with a standard conditioning regimen of low dose total body irradiation and fludarabine in MDS or AML subjects undergoing HCT. The participants received a single intravenous dose of 0.6 mg/kg briquilimab. In both studies, briquilimab PK samples were obtained at pre-treatment, 5 minutes post-end of infusion, 4- and 24-hours post-start of infusion, any time between 2 days and 30 days postinfusion, and on the day of HCT prior to donor cell infusion.The population PK model was developed using the PK data from these 2 clinical studies, and the effect of participants’ baseline characteristics on the briquilimab PK was evaluated. PK simulations were performed using the developed PK model to calculate the time to reach target concentrations for HCT. A total of 49 participants (21 SCID adult and pediatric participants with a median age of 12 yr and 28 MDS/AML adult participants with a median age of 70 yr) were included in the PK analysis. A 2-compartment model with combined linear and non-linear elimination best described the PK of briquilimab. Body weight was determined as the sole covariate of the PK parameters among the explored covariates. For a typical subject with a body weight 70 kg, the estimated parameters for clearance, maximum metabolic rate of Michaelis Menten elimination, Michaelis Menten constant, central volume, peripheral volume, and intercompartmental clearance were 17.6 mL/h, 51,434.8 ng/h, 71.5 ng/mL, 3444.0 mL, 1613.3 mL, and 21.2 mL/h, respectively. The median time to reach target concentrations of 500, 1000, and 2000 ng/mL after a single dose of 0.6 mg/kg was calculated as 12.3, 10.4, and 7.7 days, respectively. The PK of intravenous briquilimab was characterized in subjects with SCID, MDS, or AML receiving HCT, and a population PK model was developed to estimate briquilimab clearance to use as a guide to the timing of donor cell infusion post-briquilimab. Body weight was identified as a significant covariate on elimination and volume of distribution of briquilimab.

Donor types and outcomes of transplantation in myelofibrosis: a CIBMTR study

AbstractWe evaluate the impact of donor types on outcomes of hematopoietic cell transplantation (HCT) in myelofibrosis, using the Center for International Blood and Marrow Transplant Research registry data for HCTs done between 2013 and 2019. In all 1597 patients, the use of haploidentical donors increased from 3% in 2013 to 19% in 2019. In study-eligible 1032 patients who received peripheral blood grafts for chronic-phase myelofibrosis, 38% of recipients of haploidentical HCT were non-White/Caucasian. Matched sibling donor (MSD)–HCTs were associated with superior overall survival (OS) in the first 3 months (haploidentical hazard ratio [HR], 5.80 [95% confidence interval (CI), 2.52-13.35]; matched unrelated (MUD) HR, 4.50 [95% CI, 2.24-9.03]; mismatched unrelated HR, 5.13 [95% CI, 1.44-18.31]; P < .001). This difference in OS aligns with lower graft failure with MSD (haploidentical HR, 6.11 [95% CI, 2.98-12.54]; matched unrelated HR, 2.33 [95% CI, 1.20-4.51]; mismatched unrelated HR, 1.82 [95% CI, 0.58-5.72]). There was no significant difference in OS among haploidentical, MUD, and mismatched unrelated donor HCTs in the first 3 months. Donor type was not associated with differences in OS beyond 3 months after HCT, relapse, disease-free survival, or OS among patients who underwent HCT within 24 months of diagnosis. Patients who experienced graft failure had more advanced disease and commonly used nonmyeloablative conditioning. Although MSD-HCTs were superior, there is no significant difference in HCT outcomes from haploidentical and MUDs. These results establish haploidentical HCT with posttransplantation cyclophosphamide as a viable option in myelofibrosis, especially for ethnic minorities underrepresented in the donor registries.

Prognostic impact of the conditioning intensity on outcomes after allogeneic transplantation for MDS with low blasts: a nationwide retrospective study by the adult MDS working group of the Japan Society for Transplantation and Cellular Therapy.

Poor prognostic factors, such as transfusion dependency and chromosomal risk, need to be considered in the indication of allogeneic hematopoietic cell transplantation (allo-HCT) for patients harboring myelodysplastic syndromes with less than 5% marrow blasts (MDS-Lo). We analyzed the post-transplant outcomes of 1229 MDS-Lo patients who received myeloablative (MAC)(n = 651), reduced-intensity (RIC)(n = 397), and non-myeloablative conditioning (NMAC) regimens (n = 181). The multivariate analysis revealed that the RIC group had better chronic graft-versus-host disease (GVHD)- and relapse-free survival (CRFS) (P = 0.021), and GVHD- and relapse-free survival (GRFS) than the MAC group (P = 0.001), while no significant differences were observed between the NMAC and MAC groups. In the subgroup analysis, the MAC group has better overall survival (P = 0.008) than the RIC group among patients with an HCT-comorbidity index (HCT-CI) score of 0, while the RIC group had better overall survival (P = 0.029) than the MAC group among those with an HCT-CI score ≥3. According to the type of conditioning regimen, total body irradiation 12 Gy-based MAC regimen showed better OS and CRFS than the other MAC regimen, and comparable outcomes to the RIC regimen. In conclusion, the RIC and NMAC regimens are promising options for MDS-Lo patients in addition to the MAC regimen.

Significant impact of antibiotic exposure on GI-GVHD, NRM, and GRFS following allogeneic HCT with non-myeloablative Flu-TBI conditioning

Background Acute gastro-intestinal graft-versus-host disease (GI-GVHD) and non-relapse mortality (NRM) after allogeneic HCT are closely related to loss of microbial diversity and intestinal dominance by single taxa resulting from the use of antibiotics, dietary changes, and mucosal barrier injury. There is a paucity of data on the impact of use of antibiotics in HCT after Flu-TBI-based non-myeloablative (NMA) conditioning where there is absence of mucositis and limited malnutrition. Methods We did a retrospective single-center analysis of patients receiving Flu-TBI-based NMA HCT for a high-grade myeloid malignancy, mostly AML, and MDS, or acute lymphoblastic leukemia (ALL). We analyzed the impact of pre-engraftment antibiotic exposure, prophylactic ciprofloxacin, and or treatment with broad-spectrum cephalosporin/carbapenem, on HCT outcomes, with a focus on the incidence of acute GI-GVHD by day 180 and NRM at 1 year. Results A total of 150 patients were evaluable with a median age of 62 years. Antibiotics were used in 90 patients; 60 prophylactic use only and 30 therapeutic use with or without previous prophylaxis. Antibiotic use resulted in a significant higher incidence of GI-GVHD Stage 1–4; 29% (26/90) versus 5% (3/60) in those not receiving antibiotics (OR 8.1 (95% CI 2.3–28.3), p = 0.001). Use of antibiotics resulted in higher 1-year NRM (19% (17/90) versus 10% (6/60), HR 2.3, p = 0.06), and decreased 2-year GRFS (42% (38/90) versus 55% (33/60), HR 1.7, p = 0.04), but did not impact RFS or OS. Conclusions Use of antibiotics was related to the occurrence of GI-GVHD, NRM, and GRFS in patients receiving truly NMA HCT. Therefore, in the absence of mucositis and low incidence of bacteremia, antibiotics can and should be used restrictively in this setting.

Graft Failure Incidence, Risk Factors, and Outcomes in Patients Undergoing Non-Myeloablative Allogeneic Hematopoietic Cell Transplantation Using Post-Transplant Cyclophosphamide

Graft failure (GF) is a major complication of allogeneic hematopoietic cell transplantation (alloHCT) that results in significant morbidity and mortality. Post-transplant cyclophosphamide (PTCy)-based graft-versus-host disease (GVHD) prophylaxis has emerged as an effective regimen across the spectrum of donor-match settings, but few studies have investigated the characteristics of GF in the setting of PTCy-based GVHD prophylaxis. The objective was to detail the incidence, clinical features, risk factors, and outcomes for patients with primary graft failure (PGF) and secondary graft failure (SGF). In this retrospective study at a single institution, 958 consecutive patients undergoing first nonmyeloablative (NMA) alloHCT with PTCy-based GVHD prophylaxis were analyzed. PGF was defined as a failure to achieve an ANC ≥ 500 cells/m3 by day 30 of transplant in the absence of residual disease. SGF was defined as complete loss of donor chimerism after initial engraftment. The incidences of PGF and SGF were 3.8% (n = 37) and 1.8% (n = 17), respectively. Neither PGF nor SGF were associated with HLA disparity. In a multivariate analysis, risk factors for PGF in this cohort included age ≥ 65 (OR 2.4, 95% CI 1.2 to 4.8, P = .0120), an underlying diagnosis of MDS, MPN, or MDS/MPN overlap (OR 2.8, 95% CI 1.4 to 5.7, P = .0050), post-transplant viremia with HHV-6 (OR 2.9, 95% CI 1.5 to 5.7, P = .0030), and low CD34+ dose (OR 0.7, 95% CI 0.5 to 0.9, P = .0080). Patients with PGF had poor overall survival, driven primarily by a high rate of nonrelapse mortality (59% at 36 months). SGF was associated with use of a bone marrow graft source and a diagnosis of Hodgkin lymphoma. Patients with SGF had excellent clinical outcomes with only one of seventeen patients experiencing relapse and relapse-related mortality. The incidence of PGF and SGF in patients receiving NMA conditioning and PTCy is low and is not impacted by HLA disparities between donors and recipients. PGF is more common in recipients with age ≥ 65, a diagnosis of MDS, MPN, or MDS/MPN-overlap, post-transplant HHV-6 viremia, and low CD34+ cell dose. Low total nucleated cell dose is also a risk factor for PGF in patients receiving a bone marrow graft source. Patients who experience PGF have poor outcomes due to high rates of nonrelapse mortality, whereas patients who experience SGF have excellent long-term outcomes.

Excellent Outcomes Using a Non-Myeloablative Conditioning with Thiotepa and Post -Transplant Cyclophosphamide in Adults Undergoing a Matched Related Donor Bone Marrow Transplant for Severe Sickle Cell Disease

Excellent Outcomes Using a Non-Myeloablative Conditioning with Thiotepa and Post -Transplant Cyclophosphamide in Adults Undergoing a Matched Related Donor Bone Marrow Transplant for Severe Sickle Cell Disease

Allogeneic bone marrow transplantation for patients with treatment-refractory Crohn's Disease

Background & aimsDurable remissions of Crohn's Disease (CD) have followed myeloablative conditioning therapy and allogeneic marrow transplantation. For patients with treatment-refractory disease, we used reduced-intensity conditioning to minimize toxicity, marrow from donors with low Polygenic Risk Scores for CD as cell sources, and protracted immune suppression to lower the risk of graft-versus-host disease (GVHD). Our aim was to achieve durable CD remissions while minimizing transplant-related complications. MethodsDNA from patients and their HLA-matched unrelated donors was genotyped and Polygenic Risk Scores calculated. Donor marrow was infused following non-myeloablative conditioning. Patient symptoms and endoscopic findings were documented at intervals after transplant. ResultsWe screened 807 patients, 143 of whom met eligibility criteria; 2 patients received allografts. Patient 1 had multiple complications and died at day 332 from respiratory failure. Patient 2 had resolution of CD symptoms until day 178 when CD recurred, associated with persistent host chimerism in both peripheral blood and intestinal mucosa. Withdrawal of immune suppression was followed by dominant donor immune chimerism in peripheral blood and resolution of CD findings. Over time, mucosal T-cells became donor-dominant. At 5 years after allografting, Patient 2 remained off all medications but had mild symptoms related to a jejunal stricture that required stricturoplasty at 6 years. At 8 years, she remains stable off medications. ConclusionsThe kinetics of immunologic chimerism after allogeneic marrow transplantation for CD patients depends on the intensity of the conditioning regimen and the magnitude of immune suppression. One patient achieved durable improvement of her previously refractory CD only after establishing donor immunologic chimerism in intestinal mucosa. Her course provides proof-of-principal for allografting as a potential treatment for refractory CD, but an immunoablative conditioning regimen should be considered for future studies.(ClinicalTrials.gov, NCT01570348)

Chapter 12 - Hematopoietic stem cell transplantation for neuromyelitis optica spectrum disorder. Can immune tolerance be reestablished?

Neuromyelitis optica (NMO), which is also referred to as Devic's disease, was originally considered an aggressive subtype of multiple sclerosis (MS) presenting as optic neuritis and/or extensive transverse myelitis in which 50% of patients become blind or in a wheelchair within 5 years of onset. Subsequently, NMO was categorized as one of a spectrum of inflammatory and demyelinating autoimmune disorders that are distinct from multiple sclerosis and termed neuromyelitis optica spectrum disorder (NMOSD). NMOSD differs from multiple sclerosis by its clinical course, presentation, magnetic resonance imaging findings, clinical presentation, serum biomarker prognosis, and response to treatment. More recently, NMOSD has been subdivided according to auto-antibody status as aquaporin 4 (AQP4) seropositive NMO, myelin oligodendrocyte glycoprotein (MOG) antibody-associated disease (MOGAD), and seronegative NMOSD. The only treatment to date that has resulted in treatment-free remissions, now lasting for more than 5-10 years with posttreatment disappearance of anti-AQP4 antibodies, is hematopoietic stem cell transplantation (HSCT) using either an allogeneic (matched sibling or unrelated) donor with a reduced toxicity conditioning regimen or an autologous stem cell source using a nonmyeloablative conditioning regimen of plasmapheresis (PLEX), cyclophosphamide (Cytoxan®), rabbit antithymocyte (ATG), and rituximab (Rituxan®). Post-HSCT long-term resolution of disease activity and disappearance of AQP4 antibodies is consistent with HSCT-induced immune tolerance.

The range of haploidentical transplant protocols in sickle cell disease: all haplos are not created equally.

The ideal curative therapy for sickle cell disease (SCD) must be applicable across all ages and include individuals with strokes and preexisting heart, lung, and kidney disease. Myeloablative, matched sibling donor hematopoietic stem cell transplant (HCT) for children with SCD has shown excellent outcomes over the past 3 decades but has been restricted due to the limited availability of a human leukocyte antigen-matched sibling donor (10%-15%) and increased treatment-related death in adults with myeloablative conditioning. To overcome these 2 significant barriers to curative therapy in SCD, related haploidentical HCT has become an active area of research. The use of related haploidentical donors (first- and second-degree relatives) increases the donor pool to at least 90% of those eligible across the life span. Importantly, most adults, even with strokes or significant comorbidities, can tolerate the nonmyeloablative conditioning regimen without treatment-related death. Since 2013, at least 3 related haploidentical HCT strategies have emerged as potential curative therapies for SCD: (1) a nonmyeloablative, T-cell replete, bone marrow transplant with thiotepa and posttransplant cyclophosphamide with a goal of complete donor chimerism; (2) a nonmyeloablative, in vivo T-cell depletion, using peripheral blood stem cells (PBSCs) with a goal of stable mixed donor-recipient chimerism; and (3) a myeloablative, ex vivo T-cell depletion using PBSCs and advanced-technology graft manipulation, with a goal of complete donor chimerism. We review the similarities, differences, outcomes, and gaps in knowledge with these 3 haploidentical HCT approaches for SCD.

Prognostic Impact of Conditioning Intensity on Outcomes after Allogeneic Transplantation for MDS with Low Blasts

Introduction Allogeneic hematopoietic stem cell transplantation (allo-HSCT) provides curative outcomes in patients with myelodysplastic syndromes (MDS). The blast percentage in MDS is one of the important factors to utilize allo-HSCT, and the other disease-related factors, such as transfusion dependency and chromosomal risk, are crucial to consider whether patients harboring MDS with less than 5% of marrow blasts (MDS-Lo) undergo to allo-HSCT. However, the prognostic impacts of conditioning intensity in MDS-Lo patients are still unclear. We here conducted a nationwide retrospective study to clarify the prognostic factor and investigate the optimal conditioning intensity for MDS-Lo. Methods In this study, patients diagnosed as the other than refractory anemia with excess blasts (RAEB)-1 and -2 according to the WHO classification (marrow blasts &amp;lt;5% and peripheral blood blasts &amp;lt;1%) were considered to be MDS-Lo. The clinical data of 1,229 de novo MDS-Lo patients (i) who had stayed MDS-Lo from diagnosis to transplantation and (ii) who underwent their initial allo-HSCT between January 2001 and December 2020 were collected from the Transplant Registry Unified Management Program of the Japan Society for Hematopoietic Cell Transplantation. Cox proportional hazards regression models were used to evaluate variables potentially affecting overall survival (OS), chronic graft-versus-host disease (GVHD)-free survival (CRFS), and GVHD- and relapse-free survival (GRFS). Fine and Gray proportional hazards models were used to evaluate variables potentially affecting cumulative incidences of relapse (CIR) and non-relapse mortality (NRM), using cumulative incidence curves to accommodate competing events. Results In a total of 1,229 patients, 651, 397, and 181 patients received myeloablative conditioning (MAC), reduced-intensity conditioning (RIC), and non-myeloablative conditioning (NMAC) groups, respectively. The estimated 3-year OS rates were 63.3%, 61.0%, and 69.7% in MAC, RIC, and NMAC groups, respectively; the estimated 3-year CRFS rates were 41.1%, 43.9%, and 47.2% in MAC, RIC, and NMAC groups, respectively; the estimated 3-year GRFS rates were 27.5%, 33.4%, and 35.4% in MAC, RIC, and NMAC groups, respectively; the estimated 3-year CIR were 11.9%, 14.2%, and 16.0% in MAC, RIC, and NMAC groups, respectively; and the estimated 3-year NRM were 29.8%, 31.0%, and 24.7% in MAC, RIC, and NMAC groups, respectively. The multivariate analyses revealed four factors affecting lower OS: older recipient's age (Hazard ratio (HR) [95% confidential interval], 1.70 [1.28-2.24]; P&amp;lt;0.001 for 40-59 years)(HR, 2.32 [1.71-3.15]; P&amp;lt;0.001 for 60 years or older), performance status 2-4 at HSCT (HR, 1.84 [1.32-2.57]; P&amp;lt;0.001), the history of infection before HSCT (HR, 2.12 [1.55-2.89]; P&amp;lt;0.001), and high number of red blood cell (RBC) transfusion before HSCT (HR, 1.65 [1.35-2.02]; P&amp;lt;0.001). These factors also negatively affected CRFS, GRFS, and NRM. In addition, poor cytogenetic risk group was associated with worse OS (HR, 1.66 [1.32-2.09]; P&amp;lt;0.001), CRFS (HR, 1.48 [1.22-1.80]; P&amp;lt;0.001), and GRFS (HR, 1.36 [1.14-1.64]; P=0.001) but not CIR. There was no significant difference of OS, CRFS, GRFS, CIR, and NRM in either RIC or NMAC group compared to MAC group. We next performed the subgroups analysis based on the prognostic factors. The subgroup analysis for the comparison between MAC and RIC regimens showed the better OS of MAC regimen among patients with low number of RBC transfusion (HR, 1.33 [1.01-1,75]; P=0.039). The subgroup analysis for the comparison between RIC and NMAC regimens revealed the better OS of NAMC regimen among patients aged &amp;lt;40 years (HR, 0.43 [0.19-0.96]; P=0.038), no history of infection (HR, 0.67 [0.50-0.91]; P=0.009), and good cytogenetic risk (HR, 0.54 [0.38-0.83]; P=0.004). Conclusion This study showed the prognostic factors on the post-transplant outcomes among MDS-Lo patients, and no significant difference of conditioning intensity in the entire population. Furthermore, we found the factors (the number of RBC transfusion, patient's age, the history of infection, and cytogenetic risk) potentially affecting the selection of conditioning intensity. These findings would improve the management for transplant candidates with MDS-Lo.

Improved Gvhd-Relapse Free Survival of Ex Vivo αβtcr/CD19 Depleted Allo-Sct Combined with In Vivo T Cell Depletion with ATG When Compared to T Cell Replete Transplantations with or without the Addition of ATG

Introduction The aim of allogeneic stem cell transplantation (allo-SCT) is to reach graft-versus-host disease, relapse free survival (GRFS). To reduce the incidence of graft-versus-host disease (GVHD), we have developed an allo-SCT platform combining ex vivo αβTCR/CD19 depletion with in vivo T-cell depletion through administration of antithymoglobulin (ATG). Here we compare GRFS to historical cohorts of T-cell replete allo-SCT. Methods Adults with hematological malignancies and transplanted between 2011 and 2022 were included in this retrospective analysis. Written informed consent was obtained in accordance with the JACIE guidelines. Clinical data was extracted from the EBMT registry. 3 cohorts were identified. Cohort A: Allo-SCT of unmanipulated peripheral blood derived mononuclear cells (PBMCs) of matched related donors (MRD) after non-myeloablative (NMA) or myeloablative (MA) conditioning without ATG. Cohort B: Allo-SCT of unmanipulated PBMCs of 10/10 or 9/10 matched unrelated donors (MUD) after NMA or MA conditioning with ATG. Patients in cohorts A &amp; B received dual immunosuppression with cyclosporin (CsA) and mycophenolic acid (MMF). Cohort C: Allo-SCT of αβTCR/CD19 depleted PBMCs of MRD and MUD (10/10 and 9/10) after ATG and a myeloablative conditioning as previously described[1]. Patients in cohort C received 28 days of MMF. Cumulative incidence (CI) of GVHD was defined as time to onset of GVHD, with relapse and death as competing events. Overall survival (OS) was defined as time to death from any cause. CI of relapse was defined as time to relapse, with death as a competing event. Non-relapse mortality (NRM) was defined as time to death, without relapse or progression. Event free survival (EFS) was defined as the time to relapse, graft failure or death. GRFS was defined as the time to relapse, aGVHD 3-4 or extensive cGVHD, graft failure or death. CI of CMV and EBV reactivations were calculated with relapse and death as competing events. Results 341 patients were included (cohort A: N=63; cohort B: N=150; cohort C: N=128) (table 1). In T cell replete allo-SCT (A&amp;B) ATG was administered to recipients of MUD and MA conditioning was administered to patients &amp;lt; 40 years with acute leukemia. In cohort C, all patients received ATG and an MA conditioning, regardless of age or underlying malignancy. This explains differences donor types and intensity of conditioning between the cohorts (table 1). Other clinical characteristics were comparable. Two year OS (A=66.7%; B=58.7%; C=63.8%) and EFS (A=57.1%; B=52%; C=55.7%) was comparable between the cohorts. Two year CI of relapse was also comparable (A=30%; B=23%; C=28%). Two year NRM appeared higher in B, but this difference did not reach significance (A= 9.8%; B=24%; C=15%). The CI of aGVHD grade 2-4 and 3-4 at day 100 was significantly higher in T cell replete allo-SCT with MUD (2-4; A=23%; B=37%; C=20% (p=0.002); 3-4: A=5%; B=16%; C=4.7% (p=0.043)). The incidence of extensive cGVHD was significantly higher in T cell replete allo-SCT of MRD. (A= 25%; B=13%; C= 2.3% (p=&amp;lt; 0.001)). The low incidence of grade 3-4 aGVHD and extensive cGVHD without an increase in relapse or NRM, translated into a superior 2 year GRFS in patients receiving ATG combined with an αβTCR/CD19 allograft (C=55.6%) as compared to T cell replete allo-HSCT of MRD (A=38.1%) and MUD (B=40.8%) (Figure 1, p=0.04). This difference remained significant in a multivariate analysis. Conclusion We demonstrate that αβTCR/CD19 depletion combined with ATG, with a myeloablative conditioning and a very short course of immunosuppression results in very low incidences of life-threatening GVHD and an improved GRFS as compared to T cell replete allo-SCT for adult patients up to the age of 70. This is likely to positively impact the long term quality of life in survivors of allo-SCT[2]. In addition, this platform provides a window for additional early post allo-interventions to further improve the control of underlying hematological malignancies. 1. de Witte, M.A., et al., alphabeta T-cell graft depletion for allogeneic HSCT in adults with hematological malignancies. Blood Adv, 2021. 5(1): p. 240-249. 2. Oerlemans, S., et al. Relapse and severe Graft-versus-Host Disease have a negative impact on long-term symptoms and quality of life of patients three years after allogeneic haematopoietic stem cell transplantation. Annual meeting EBMT 2022.

Excellent Outcomes of HLA Matched Related Donor Transplant for Adults with Severe Sickle Cell Disease Using a Non-Myeloablative Conditioning with Thiotepa and Post-Transplant Cyclophosphamide: Multi-Center International Experience

Introduction: The validated curative treatment for sickle cell disease (SCD) is allogeneic hematopoietic stem-cell transplantation (HSCT). Despite paucity of available matched sibling donors (10-14%), they are preferred due to less immunogenic complications like graft versus host disease (GVHD) and graft failure (GF) using alternative donors. In-vivo T-cell depletion by post-transplant cyclophosphamide (PTCy) has been shown to be most effective in GVHD control and a non-myeloablative regimen minimizes the toxicity associated with HSCT in this vulnerable population. In 2013, the multi-institutional Vanderbilt Haploidentical Learning Collaborative showed the improved outcomes of their non-myeloablative regimen for related haploidentical BMT for SCD (ClinicalTrials.gov identifier NCT01850108). We used a similar non-myeloablative approach to improve donor engraftment rates, while minimizing GVHD. Material and methods: This study was conducted at three international centers using similar conditioning from our experience in multi-institutional Vanderbilt Global Haploidentical Learning Collaborative to optimize haploBMT for SCD. The conditioning regimen included ( Figure3): rabbit anti-thymoglobulin (ATG) 4.5 mg/kg, thiotepa 10 mg/kg, fludarabine 150 mg/m2, cyclophosphamide 29 mg/kg and total body irradiation (TBI) 200 cGy. GVHD prophylaxis included PTCy 100 mg/kg, mycophenolate mofetil (MMF), and sirolimus. Stem cell source included bone marrow (BM) mobilized with filgrastim (5-10 μg/kg/d x 3-5 days) and peripheral blood (PB). Primary graft failure (GF) was defined as &amp;lt;5% donor myeloid/lymphoid chimerism or &amp;lt;5% donor cells by whole blood (WB) chimerism by day+28 posttransplant. Secondary graft rejection was defined &amp;lt;5% donor myeloid/lymphoid chimerism or &amp;lt;5% donor cells by whole blood (WB) chimerism with prior documentation of &amp;gt;5% donor cells by day +28. The Event free survival (EFS) was defined by survival with no GF, no death and/or no chronic GVHD. This cohort includes 4/22 patients (18.2%) had prior graft failure from a previous bone marrow transplants. Results: Here we report the outcome of 22 SCD patients (100% HbSS genotype), with a median age of 26.5 (15-41), transplanted from 10/10 MRD 86.4 % (n= 19) or single antigen mismatch related donor (MMRD) (9/10) 13.6% (n=3). The stem cell source was PB in 54.5% (12/22), and BM in 45.5% (10/22). Median CD34 stem cells dose was 6 X 10 6/Kg (1.2 - 10.25). Neutrophils and platelets engrafted at a median of 21.0 (15 - 26) and 25.0 (12 - 45) days, respectively. Demographic and clinical characteristics of participants summarized in Table 1. No primary or secondary graft failure was observed in the participants based on the last follow-up and time of analysis of this report. For participants with durable engraftment, the median WB chimerism values for D+28 and D+365 engraftment was 99.25% (n=14; IQR: 52 - 100) and 96.5% (n=16; IQR: 52 - 100), respectively. The median follow-up time was 14.6 months (n=22; IQR: 0.5-45.44). The overall incidence of acute GVHD grade III-IV was 4.5% (n= 1/22), and no incidence of chronic GVHD. There was no transplant related mortality. A Kaplan-Meier based probability of overall survival (OS) was 100%, Figure 1. All patients were off immunosuppression at 1-year post transplant. EFS as defined in the method was 100%, Figure 2. The EFS of free acute GVHD grade III-IV was 95.5%. Conclusion: This result shows the superiority of Thiotepa based non-myeloablative conditioning and PTCy for MRD transplants in adults with severe SCD. It not only minimizes the toxicity and GVHD but provides optimal donor engraftment mitigating against risk of secondary myeloid neoplasms from mixed chimerism. This needs further validation in a larger cohort.

Final Results from Phase 1 Study of Briquilimab, an Anti-CD117 Monoclonal Antibody, in Combination with Low Dose Irradiation and Fludarabine Conditioning, Shows Durable Remissions in Older Adults with Acute Myeloid Leukemia in Complete Remission and Myelodysplastic Syndrome Undergoing Allogeneic Hematopoietic Cell Transplantation

Background: Allogeneic hematopoietic cell transplantation (HCT) with non-myeloablative conditioning (NMA) is a potential cure for AML and MDS in older/frail patients. NMA is associated with better tolerability but higher relapse rates compared to more intensive regimens. In a phase 1 study (NCT#04429191), briquilimab (JSP191), a first-in-class monoclonal antibody (mAb) that inhibits stem cell factor binding to CD117 (c-Kit) to deplete hematopoietic stem and progenitor cells (HSPC), in combination with standard NMA conditioning of low dose total body radiation (TBI) and fludarabine (Flu) for HCT, was evaluated in older adults with AML and MDS. Pre-clinical experiments suggest briquilimab synergizes with low dose TBI to deplete normal and malignant HSPC to facilitate donor cell engraftment. Final 1-year efficacy endpoints for all subjects enrolled in this study are presented. Methods: 32 subjects, median age 70 yrs (range 62-79), with AML in morphologic CR (CR1 or CR2+) (n=13), MDS (n=16), or AML not in CR (n =3), and HLA-matched related or unrelated donors were enrolled. Following infusion of briquilimab 0.6 mg/kg, serum levels were assessed to determine start of Flu at 30 mg/m 2/day on Transplant Day (TD)-4, -3, -2, and TBI 2-3 Gy on TD0. Peripheral blood grafts were infused on TD0 (10-14 days after JSP191). GVHD prophylaxis was tacrolimus, sirolimus, and mycophenolate mofetil. Primary endpoints were safety, tolerability, and briquilimab pharmacokinetics. Secondary endpoints included engraftment, chimerism, measurable residual disease (MRD) clearance, acute GVHD (aGVHD), chronic GVHD (cGVHD), NRM, RFS, and OS at 1 year. Results: Summary of main efficacy outcomes is presented in Fig 1. 10 of 13 AML in CR and 14 of 16 MDS subjects had MRD at pre-HCT screening assessed by cytogenetics, flow cytometry, and/or next generation sequencing. There were no infusion toxicities and no briquilimab-related SAEs. Consistent with our previous reports, briquilimab exhibited predictable clearance prior to donor cell infusion, and all subjects engrafted, with neutrophil recovery between TD+19 and TD+26. Compared to baseline marrow, briquilimab alone depleted HSPC with a mean decrease of 62.4±22.7% prior to Flu/TBI (Fig 2). The 28 evaluable subjects at TD+90 achieved median donor myeloid chimerism of 99% (range 96-100%) and median total chimerism of 95% (range 81-100%). There was one secondary graft failure without relapse, who received a 2 nd HCT. Among 13 AML in CR subjects, by 1 year, there were 3 relapses, 1 NRM (GVHD), 9 subjects were alive, and 8 were MRD negative. The 1 subject who was MRD positive at 1 year had only a detectable DNMT3A mutation at low level. 1 of 13 AML in CR subjects had TP53+ disease at screening and this subject was alive and MRD negative at 1 year post-AHCT. Among 16 MDS subjects, by 1 year, there were 5 disease progression/relapses, 1 subject withdrew consent due to persistent disease 3 months post-HCT, 1 NRM (sepsis), 8 subjects were alive, and 6 were MRD negative. 5 of 16 MDS subjects had TP53+ disease, 3 of whom had persistent disease/progressed/relapsed prior to 1 year post-HCT, and the other 2 of whom had MRD positive disease/molecular relapse at 1 year post-HCT. Median time to MRD negativity in all subjects who converted from MRD-positive disease to MRD-negative disease was TD+28. All 3 subjects with AML not in CR had disease progression before 4 months post-AHCT. There was no apparent correlation between level of HSPC depletion at the assessed timepoint (Fig 2) and relapse or survival. Of the 32 enrolled subjects, 4 had MAGIC Grade 2 aGVHD, 1 had MAGIC grade 3-4 aGVHD, 13 had mild/moderate cGVHD, and none had severe cGVHD. Conclusions: These results demonstrate that targeting CD117 with briquilimab together with TBI/Flu as a novel conditioning regimen is safe, well-tolerated, facilitates full donor myeloid chimerism, and sustained clearance of MRD in older adults with AML in CR and MDS without TP53 undergoing NMA AHCT.

Role of Measurable Residual Disease (MRD) Testing for the Prediction of Late Relapses Following Allogeneic Hematopoietic Cell Transplantation in Adult Acute Myeloid Leukemia

BACKGROUND: Measurable residual disease (MRD) before and/or shortly after allogeneic hematopoietic cell transplantation (HCT) is an independent indicator of high post-HCT relapse risk in acute myeloid leukemia (AML). However, many relapses, particularly among patients with MRD, occur within the first 3 months after allografting, and the role of MRD testing in the identification of patients at risk of relapses occurring at later times is unknown. METHODS: To address this uncertainty, we studied all adults ≥18 years with AML or MDS/AML based on 2022 International Consensus Classification criteria who received a first allograft while in first or second morphologic remission at a single institution between 4/2006 and 3/2023 and underwent MRD testing via multiparameter flow cytometry during the pre-HCT evaluation. In line with the performance characteristics of the assay, any detectable MRD was considered positive. RESULTS: One thousand two hundred and sixty-two patients with AML (n=1,112 [88%]) or MDS/AML (n=150 [12%])underwent allograft following myeloablative (n=732 [58%]) or non-myeloablative (n=530 [42%]) conditioning regimens. Of these, 260 (21%) tested positive for MRD during the pre-HCT evaluation. With a median follow-up of 60.6 (range: 1.9-196.3) months after HCT among survivors, there were 589 deaths, 381 relapses, and 259 NRM events contributing to the probability estimates for relapse, overall survival (OS), relapse-free survival (RFS), and non-relapse mortality (NRM) in this cohort. 239/1,262 patients (19%) experienced disease recurrence (n=181 [14%]) and/or death (n=89 [7%]) within the first 100 days after allografting. Relapse ( P&amp;lt;0.001) and death ( P=0.002) but not NRM ( P=0.29) before day +100 were more common among patients with pre-HCT MRD. Of patients alive without relapse at day +100, 93% (954 of 1,023) underwent bone marrow restaging studies between day 70 and 100 post-HCT. Among these 954 patients, 146 (15%) had MRD before HCT, whereas only 25 (3%) had MRD at day +70-100 testing. The latter had a significantly higher 3-year relapse risk (37% vs. 18%; P=0.002), and worse RFS (17% vs. 68%; P&amp;lt;0.001) OS (16% vs. 72%; P&amp;lt;0.001) and NRM (46% vs. 14%; P&amp;lt;0.001) relative to the 929 patients without MRD; for results of univariate regression models, see Table 1. Importantly, however, while 130 of the 146 patients (89%) with MRD before HCT tested negative for MRD at day +70-100, their outcomes were substantially worse compared to patients who tested negative for MRD before HCT and at day +70-100, with 3-year relapse risk of 38% vs. 15% ( P&amp;lt;0.001), 3-year RFS of 47% vs. 71% ( P&amp;lt;0.001), and 3-year OS of 53% vs. 75% ( P&amp;lt;0.001), whereas 3-year NRM estimates were similar (14% vs. 14% ( P=0.21). Qualitatively similar results were obtained when the cohort was restricted to patients with AML, and also when comparing patients undergoing myeloablative conditioning with those undergoing non-myeloablative conditioning, with a statistically significantly higher relapse risk and lower RFS and OS but not NRM for patients with MRD before HCT but no MRD at day +70-100 compared to those who tested negative for MRD before HCT and at day +70-100 (Table 1). CONCLUSION: For AML and MDS/AML patients alive without relapse at day +100 after allografting, MRD testing at day +70-100 post-HCT identifies patients at increased risk of relapse and worse survival, but only a small number of patients will test positive at this post-HCT timepoint. While a high proportion of patients with MRD before HCT will test negative for MRD at day +70-100, their outcomes are substantially inferior to patients without MRD both before and at day +70-100 after HCT. These data suggest that all patients with pre-HCT MRD should be considered for pre-emptive therapeutic strategies, ideally in the setting of well-controlled clinical trials, given their high risk of disease recurrence regardless of the post-HCT MRD information.

Jak2V617F Bone Marrow Exposure Leads to Dysfunction of Normal NK Cells

Introduction: We have previously demonstrated a functional impairment of NK cells in the Jak2V617F murine model and peripheral blood cells from patients with Myeloproliferative Neoplasms, with emphasis on the increased frequency of immature NK cells and reduced cytotoxicity of the more mature CD27-CD11b+ fraction. It is not known whether MPN initiating cells are intrinsically resistant to tumor immunity or whether they can modify NK cells of the leukemic microenvironment, leading to dysfunction. Aims: To clarify these mechanisms, the present work aimed to model the leukemic exposure of normal NK cells and verify their modifications using a Jak2V617F chimeric transplant model. Methods: For transplantation, a non-myeloablative conditioning (single sublethal irradiation of 400cGy) was performed to partially preserve the recipient's hematopoiesis (50-60% of donor engraftment). 5 x 10 6 total bone marrow (BM) cells from Jak2VF or Jak2WT control animals (both C57BL/6, CD45.2) were injected retro-orbitally into wild-type mice (C57BL/6, CD45.1, N=6 per group). Engraftment was evaluated by flow cytometry according to CD45.2/CD45.1 differential ratio. After engraftment was confirmed, frequency, receptors, and functional subtypes of exposed NK cells were evaluated. NK cytotoxicity against YAC-1 target cells was evaluated after culture with serum and supernatant of whole BM from chimeric animals. Results: The frequency of host NK cells (CD45.1) exposed to Jak2VF BM was lower than that of NK cells exposed to Jak2WT BM (Jak2WT 44.05% vs Jak2VF 19.76%, p&amp;lt;0.0001). Evaluation of functional subtypes showed that host NK cells had a lower frequency of immature secretory (Jak2WT 44.32% vs Jak2VF 30.70%, p=0,0407) and higher frequency of tolerant (Jak2WT 12.43% vs Jak2VF 24.34%, p=0.0016) and cytotoxic (Jak2WT 7.15% vs Jak2VF 19.30%, p&amp;lt;0.0001) NK cells when exposed to leukemia than when exposed to normal BM. In comparison, secretory immature NK cells from the leukemic BM (CD45.2) were low numbered as compared to Jak2WT NK cells (Jak2WT 73.30% vs Jak2VF 35.74%, p&amp;lt;0.0001) and cytotoxic NK cells were more frequent (Jak2WT 7.28% vs Jak2VF 19.16%, p=0.0003). In addition, host NK cells presented decreased expression of the activating receptor DNAM-1 when exposed to leukemia than when exposed to normal BM (Jak2WT 51.20% vs Jak2VF 46.10%, p=0.0006), whereas CD244 and NKG2D activating receptors were not differentially expressed. In contrast, the expression of TIGIT (but not NKG2A) inhibitory receptor was increased after leukemia exposure (Jak2WT 14.83% vs 46.10%, p=0.0003). Regarding the effect of the leukemic medium on NK cells, cytotoxicity was not altered when WT NK cells were exposed to serum or BM supernatant from chimeric transplanted animals. However, NK cells exposed to Jak2VF chimeric serum were activated according to CD69 expression (Jak2WT 0.060% vs Jak2VF 0.30%, p=0.0114) but presented lower expression of the CD107a degranulation marker (Jak2WT 91.92% vs Jak2VF 62.34%, p=0.0075) than NK cells exposed to Jak2WT cells. Conclusions: Our findings demonstrated that leukemia exposure led to altered NK cell frequency and maturation distribution as demonstrated by reduced immature and an increased tolerant and cytotoxic fraction. Changes in the expression profile of activating and inhibitory receptors were also observed. Therefore, we concluded that the leukemic environment leads to NK dysfunction and that strategies that aim to control NK maturation and the interaction between stem cell ligands and functional NK receptors can be explored to restore immunosurveillance in JAK2V671F Myeloproliferative Neoplasms.

Favorable Fertility Outcome in a Large Cohort of Sickle Cell Disease Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation

Introduction: Fertility preservation is an important consideration for individuals with sickle cell disease (SCD) who may face reproductive challenges. While SCD itself does not directly affect fertility, certain complications and its treatment such as hematopoietic cell transplantation (HCT) can impact the reproductive health in SCD patients. HCT is a potential curative therapy for SCD, however, one of the negative outcomes is the premature gonadal failure and infertility. Both male and female patients have different risk factors that may impact the fertility post-transplant. Therefore, these patients should undergo fertility preservation counselling pre transplant. In this study, we report the fertility outcome of large transplant cohort undergoing Non-myeloablative (NMA) conditioning regimen. Methods: We retrospectively collected the data for all SCD patients who were ≥ 14 years old and who underwent allogeneic HCT in our center from 2015 until 2022 with a minimum of 1 year follow up post-transplant. All patients received hydroxyurea for 2 to 3 months pre- transplant at maximum tolerated dose. The conditioning regimen was alemtuzumab 1 mg /kg divided over 5 days from day - 7 to day -3 and Total Body Irradation 200 cGy on day -2 (with testicular shielding for male patients). Graft versus host disease prophylaxis with sirolimus started from day -1 and continued for at least 1 year post-transplant. We started the fertility program in October 2019 where all patients get counselled for fertility preservation methods. Male patients undergo sperm cryopreservation after holding hydroxyurea for 3 months and female patients were referred to a specialized oncology fertility clinic for either oocyte cryopreservation or treatment with Gonadotropin-releasing hormone (GnRH) agonists pre transplant. Primary objective of the study was to assess post-transplant fertility (defined by either ability to conceive for married patients, or development of azoospermia for male patient or secondary amenorrhea for female patients). Secondary objectives were to assess pre transplant fertility, assess the success of different fertility preservations methods and finally to assess for risk factors (pre- transplant and transplant) that affect the fertility potential. Results: We identified a total of 200 SCD patients who underwent HCT. Out of these 200 patients, 107 (54%) were included in our fertility program. The median post-transplant follow up was 3.5 years (1 - 7.5). One hundred and twelve patients (56%) were male, and eighty eight (44%) were female patients. All patients were on hydroxyurea pre transplant with average dose 1000 mg and a duration of less than 2 years in 47 (23.5%) patients, 2 to 5 years in 94 (47%) patients and more than 5 years in 59 (39.5%) patients. Other baseline characteristics are shown in table 1. In the pre-transplant fertility assessment; 58 patients were married and 48 (82%) of them had children pre transplant. 36 male patients attempted sperm cryopreservation and were successful in 33 (91%). 39 female patients received Leuprolide (GNRH agonists) pre-transplant with no complications and 6 female patients attempted oocyte cryopreservation and all were successful. One patient developed cerebral venous thrombosis during the oocyte cryopreservation course. For the post-transplant fertility assessment; out of 57 married patients 26 (45%) were able to conceive. From these 26 pregnancies; 20 ended with successful live-births, 1 ended with abortion and 5 are still pregnant. 6 patients were able to conceive twice and one patient had one twin babies. For male patients 74 did semen analysis at minimum 1 year post-transplant; 35 (47%) had azoospermia and 39 (53%) had viable sperms. Majority recovered spermatogenesis after 1 year (24 patients), at 2 years (9 patients) and after 3 years (3 patients). Three male patients had Azoospermia at one year but was able to conceive successfully without repeating the test. For female patients 71 (80.7%) were able to regain menses and 17 (19.3%) developed secondary amenorrhea. Table 2 shows the pre and post-transplant fertility measures outcome. In conclusion In this study, we have shown the fertility outcomes of male and female patients with SCD undergoing NMA conditioning HCT can be preserved in a significant proportion of patients. All adult patients should be counselled for fertility preservation methods before undergoing curative allogeneic HCT.

A Novel and Cost-Efficient Measurable Residual Disease (MRD) Detection Assay Using a Single Molecule Molecular Inversion Probe-Based Next Generation Sequencing (NGS) Assay Predicts Overall Survival in Patients with Acute Myeloid Leukemia Undergoing Allogeneic Stem Cell Transplantation

Introduction Relapse remains the main reason for poor outcomes of patients (pts) with acute myeloid leukemia (AML). Allogeneic hematopoietic stem cell transplantation (HSCT) is the preferred post-remission treatment in pts at high risk of relapse. The presence of measurable residual disease (MRD) after intensive frontline treatment or before or after HSCT has emerged as an important predictor of relapse. Various methods are used for MRD detection, including flow cytometry and molecular assays detecting individual gene mutations (mut) such as NPM1mut. Next generation sequencing (NGS)-based assays covering multiple genes commonly mutated in AML may allow sensitive MRD detection in a broad range of pts with diverse genetic characteristics. Methods We established a targeted, error corrected NGS MRD assay utilizing single-molecule molecular inversion probes (smMIPs). The smMIP protocol uses a hybridization-capture approach to selectively enrich and amplify both DNA strands of 92 regions in 32 genes in a single reaction. Unique molecular identifiers (UMIs) facilitate computational correction of sequencing errors to enable reliable detection of variants ≥0.5% variant allele frequency (VAF), with the additional advantage of relatively low reagent and sequencing costs of &amp;lt; $100 per sample. The smMIP assay was used to evaluate MRD status on pre-HSCT bone marrow samples from 98 AML pts who received an HSCT in first (n=83) or second (n=15) complete remission with or without count recovery. Median age at HSCT was 59 (range 20-75) years, 42% were female; 70% had de novo AML, 19% had an antecedent myeloid neoplasm and 11% had AML post cytotoxic therapy. European LeukemiaNet (ELN)-2022 risk groups at diagnosis were favorable in 11%, intermediate in 33%, adverse in 20% and unknown in 22%. All pts had received intensive induction chemotherapy. Twenty-nine percent received a myeloablative conditioning regimen (MAC), while 23% received reduced intensity conditioning (RIC) and 48% a non-myeloablative (NMA) conditioning regimen. Donors were matched related (18%), haploidentical (6%), or matched (55%) or mismatched (21%) unrelated. The median follow-up for pts alive was 34.8 months. Results Overall, we detected 190 variants in 71 (72%) of 98 pre-HSCT remission samples. The most commonly affected genes were DNMT3A (in 37% of pts), TET2 (21%), PPM1D (12%), IDH2 (10%) and TP53 (5%). Twenty-nine percent of pts had only variants affecting the clonal hematopoiesis-related genes DNMT3A, TET2 or ASXL1 (‘DTA‘), while 44% of patients had ≥1 variant involving a non-DTA gene. MRD-positive pts, defined as those with ≥1 non-DTA variant, had similar baseline and HSCT-related characteristics (includingage, sex, ELN risk group, de novo vs. secondary AML) compared to pts without non-DTA variants. Overall survival (OS) was significantly inferior in NGS MRD-positive pts compared to those with no non-DTA variant (Figure A, 5-year OS, 49% vs. 85%, P=.011). MRD-positive pts also had a non-significant higher cumulative incidence of relapse at 5 years (46% vs. 24%, P=.14), while non-relapse mortality was similar in both groups (P=.72). We next performed multivariable analyses, considering variables associated with OS at an univariable P&amp;lt;.10 (MRD status, patient sex, therapy-related AML, conditioning intensity), and using stepwise backward variable selection to identify factors significantly associated with OS (Figure B). In the final multivariable model, pre-HSCT MRD detection remained associated with inferior OS (hazard ratio 2.51; 95% confidence interval, 1.07 - 5.91; P=.035). Female sex associated with favorable OS, while myeloablative conditioning (MAC) showed a borderline significant association with superior OS. Of note, we did not detect an interaction between conditioning intensity and detection of MRD with regard to OS, i.e. the prognostic association of MRD detection was not different between pts receiving myeloablative or less-intensive conditioning. However, this analysis was limited by the small patient number. Conclusion Detection of MRD by targeted NGS using the smMIPs approach provides prognostic information in pts undergoing allogeneic HSCT, including those without established molecular markers such as NPM1mut. This method is applicable to a large proportion of AML pts and is cost-efficient for broad clinical use. S.M.K. and T.H., and M.J. and K.H.M. contributed equally to this abstract.