Neutrophil Engraftment Research Articles

Graft Versus Host Disease Prophylaxis with Tacrolimus, Sirolimus and MMF in Patients Undergoing Reduced Intensity Conditioning Allogeneic Transplantation

I ntroduction GvHD remains the leading cause of toxic morbidity and mortality (NRM) after allo-HSCT. Different prophylaxis schemes have been developed in the context of reduced intensity conditioning (RIC). In 2019, the multicenter phase 3 trial NCT01231412 compared GvHD prophylaxis based on the calcineurin inhibitor (CNI) CSA plus MMF with (experimental arm) or without (control arm) sirolimus. The triple combination was associated with a lower risk of aGvHD and longer survival and, in the authors' opinion, could constitute a new standard (Sandmaier, Lancet Oncol 2019). We present the largest real-world experience to date with the triple prophylaxis scheme in the context of allo-HSCT with RIC from matched-related (MRD), matched-unrelated (MUD) and mismatched URD (mmURD). Material and Methods Prospective study of 159 consecutive patients receiving an allo-HSCT after RIC between 09/2019 and 12/2022 at two centers using the tacro/siro/MMF as GvHD prophylaxis. The primary endpoint was the cumulative incidence of aGvHD grades 2-4. As secondary variables: cGvHD at 1 and 3 years, NRM at +100, +1 year and overall NRM, OS at 1 and 3 years, PFS at 1 and 3 years and GRFS at 1 and 3 years. Results Baseline characteristics are shown in Table 1. Note the high percentage of patients with high/very high rDRI or HCT-CI of 3+. In 38.4% an URD was used as source of progenitor cells and in 20.8% it was a MMURD. The median follow-up was 20 months (3-40). There were 4 (2.5%) graft failures (2 of them secondary). Median neutrophil and platelet engraftment was 16 days (3-175) and 12 days (7-210). The risk of relapse was 31% and 35% at 1 and 3 years. NRM at day +100, + 1 year and overall NRM was 4.4%, 8.17% and 9.4%. The causes of NRM were GvHD +/- infection in 73%, with other causes being TMA (n=1) and SOS (n=1). The cumulative incidence (CI) of grades 2-4 aGvHD 2-4 at day +100 and +180 was 29.7% and 32.3%, including 40% of upper gastrointestinal tract involvement which resolved with topical treatment. The CI of grades 3-4 aGvHD at day +100 and +180 was 12.8% and 16% (3.14% grade 4). The CI of cGvHD at 1 and 3 years was 21.5% and 51.2% and for moderate/severe it was 13.9% and 36.6%. OS at 1 and 3 years was 70.3% and 61%. Median PFS was 35 months, with 1- and 3-year PFS of 60% and 49%. The GRFS at 1 and 3 years was 44% and 32%. The risk of grades 2-4 aGvHD was significantly higher among patients receiving allo-HSCT from MMURD (at d+100 and +180, 35.4% and 39.9%) vs matched donors (at d+100 and +180, 55.5% and 61%, p=0.04). The same variable also influenced on OS (at 1 and 3 years 73.3% vs 64.6% and 57.6% vs 37.8% for matched vs mismatched donors, respectively; p=0.013) (Figure 1). On the other hand, low and intermediate DRI patients had a significantly better OS as compared to high/very high rDRI patients (at 1 and 3 years 80.8% and 80.8% for low vs 86.7% and 78.3% for intermediate vs 56.9% and 55.1% for high and very high; p=0.013) (Figure 1). Conclusions Triple prophylaxis with Tacro-Siro-MMF shows excelent results in terms of NRM, GvHD and survival in a high-risk and elderly population in the context of allo-HSCT from matched-related (MRD) and matched-unrelated (MUD) donors. The subgroup of patients receiving allo-HSCT from MMURD showed a high incidence of overall and grade 3-4 aGvHD with impact on OS, being a group that will probably benefit from other prophylaxis strategies.

Safety and Efficacy of Orca-Q with Haploidentical Donors for the Treatment of Advanced Hematologic Malignancies without the Use of Post-Transplant Cyclophosphamide

Srour, Salhotra, Lowsky and Abedi contributed equally as authors BACKGROUND Allogeneic stem cell transplantation (SCT) can be curative for several high-risk hematologic malignancies, but access was previously limited to patients with a fully matched donor. The introduction of post-transplant cyclophosphamide (PTCy) GvHD prophylaxis has increased the use of haploidentical donors (haplo) for patients who lack an HLA-matched donor. However, clinical outcomes associated with PTCy-based regimens remain challenging due to increased relapse rates (particularly in the setting of reduced intensity conditioning) and the adverse event profile including higher incidence of mucositis, cytokine release syndrome (CRS), delayed engraftment & T cell reconstitution, infections, early cardiotoxicity events and death from organ failure (Dulery 2021, Abboud 2021, Nagler 2022, Hoover 2022, Bolaños-Meade 2023). Additionally, the incidence of chronic GvHD after haplo SCT with PTCy is 24 - 33% at 1 year in various historical cohorts. Orca-Q -an investigational precision engineered cell therapy biologic- represents an alternative strategy which may be administered with single agent prophylaxis and does not require PTCy. The cellular composition of Orca-Q includes enriched CD34+ stem cells, combined with specific T-cell subsets, and is intended to reconstitute the blood and immune systems. METHODS Adult patients with high-risk hematologic malignancies eligible for MAC SCT were enrolled on the haplo donor dose expansion arm of a multicenter phase1 study of Orca-Q (NCT03802695). Haplo was defined as ≥ 4/8 but < 7/8 matched related donor at HLA-A, -B, -C, and -DRB1 typed using DNA-based high-resolution. Patients received MAC conditioning and single agent GVHD prophylaxis with tacrolimus (starting on day -1 and taper day +60). Orca-Q was manufactured centrally at Orca Bio Manufacturing Site in Sacramento, CA from G-CSF mobilized peripheral blood apheresis. RESULTS Orca-Q was successfully manufactured and delivered to all subjects with a vein-to-vein time (time between end of donor apheresis to start of recipient's Orca-T infusion) of < 72 hours. A total of 33 patients (21 AML, 10 ALL, 2 CML) were included. Median age was 43 (range, 21 - 63) years, 73% were male. Median follow-up was 375 (range, 73 - 1384) days. Sixteen patients received TBI-based MAC; 17 received busulfan-based MAC (Table). All patients engrafted with median time of 12.0 (range, 8 - 25) and 15.5 (range, 8 - 79) days for neutrophil and platelet engraftment, respectively. Two patients had secondary graft failure. Two patients had Grade 1 CRS; 1 patient had Grade 2 CRS. Grade 2+ aGvHD through Day 180+ occurred in 15% of patients. Grade 3-4 acute GvHD (aGvHD) was rare with only 1 event of grade 3 aGvHD and no grade 4 aGvHD. No patients had moderate-to-severe cGvHD. Estimated incidence of CTCAE grade 2 and > grade 3 infections were 9% and 15% at 1 year, respectively. 5 patients died (2 from relapsed disease) during the study period. NRM was 9% at 1 year. The 1-year relapse-free survival, GvHD-and-relapse free survival (Figure), and overall survival at 1 year was 82%. CONCLUSIONS Our findings reveal promising safety and efficacy outcomes using Orca-Q cell therapy for haplo-SCT despite the use of MAC with only single-agent tacrolimus, and without PTCy. With median follow-up of approximately 1 year, no patients experienced moderate or severe cGvHD, the low AE profile in the haplo setting remains favorable, and 1 year GvHD-free, relapse-free survival of 83% is very encouraging. This phase 1 study continues to enroll patients across the US. REFERENCES Abboud R, Wan F, Mariotti J, et al. Bone Marrow Transplant. 2021;56(11):2763-2770. Bolaños-Meade GM, Hamadani M, Wu J. N Engl J Med. 2023; 388(25):2338-2348. Duléry R, Mohty R, Labopin M, et al. JACC CardioOncol. 2021; 3(2):250-259. Hoover A, O'Leary D, Cao Q, et al. Blood. 2022; 140 (Supplement 1):282-283 Nagler A, Tsirigotis P. Bone Marrow Transplant. 2022; 57(11):1640-1641.

CR1 Favors PTCy-haplo over UCB in Allogeneic Stem Cell Transplantation for Adult Patients with AL

[Introduction] HLA-haploidentical stem cell transplantation using post-transplant cyclophosphamide (PTCy-haplo) and umbilical cord blood transplantation (UCB) are alternative to HLA-matched hematopoietic stem cell transplantation (HSCT). However, advantage of PTCy-haplo versus UCB has not been determined in patients with acute lymphoblastic leukemia (ALL). Thus, we compared post-HSCT outcomes of PTCy-haplo and UCB in adult patients with ALL to determine patient subgroups who benefit from PTCy-haplo. [Methods] We analyzed data on adult patients (≥16 years) with ALL who underwent HSCT with PTCy-haplo or single-unit UCB between January 2013 and December 2021, using the Japanese nationwide registry database. Endpoints of the analysis included overall survival (OS), disease-free survival (DFS), cumulative incidence of relapse, non-relapse mortality (NRM), engraftments, acute and chronic graft-versus-host disease (GVHD), and infections. In addition to multivariate analysis, propensity score matching was performed as a sensitivity analysis. [Results] We analyzed 1999 patients (PTCy-haplo, 330; UCB, 1669). Median age at HSCT was 44.5 years (range, 16-73) in PTCy-haplo and 45 years (range, 16-74) in UCB group. The proportion of patients who underwent HSCT in the first complete remission (CR1) (PTCy-haplo, 50.3%; UCB, 48.8%) and median time from diagnosis to HSCT (PTCy-haplo, 7.8 months; UCB, 7.7 months) were comparable between the groups. Regarding conditioning, the proportion of those who received myeloablative conditioning (PTCy-haplo, 50.6%; UCB, 54.8%), or conditioning with total body irradiation (PTCy-haplo, 82.4%; UCB, 79.3%) were comparable between the groups. Multivariate analyses showed that PTCy-haplo recipients had comparable 3-year OS (54.1% vs. 52.3%; adjusted hazard ratio [aHR], 0.93; p = 0.51) and DFS (49.7% vs. 47.1%; aHR, 1.06; p = 0.60) with UCB recipients. PTCy-haplo recipients had a significantly higher 3-year relapse rate (32.2% vs. 26.4%; aHR, 1.47; p = 0.01), but had a significantly lower 3 -year NRM (18.1% vs. 26.5%; aHR, 0.70; p = 0.03) as compared to UCB along with significantly faster neutrophil (median, 16 days vs. 20 days) and platelet (median, 30 days vs. 42 days) engraftments, and lower incidence of viral, fungal and bacterial infections. There was no significant difference in cumulative incidence of acute GVHD (30.9% vs. 36.4%, grade II-IV at 100 days; p = 0.15), or chronic GVHD (22.5% vs. 20.9% at 3 years; p = 0.85) between the two groups. These comparable OS and DFS between the two groups were confirmed after minimizing differences in patient characteristics with propensity score matching analysis (Figure 1A). Then, to identify patient subgroups who benefit from PTCy-haplo rather than UCB transplant, we performed subgroup analyses of OS. Significantly favorable effects of PTCy-haplo over UCB were observed in patients with CR1 at HSCT (HR, 0.66; p = 0.03). Indeed, propensity score matching analysis showed that PTCy-haplo recipients had a significantly higher 3-year OS (76.4% vs. 67.6%; HR, 0.57; p = 0.01) and DFS (70.5% vs. 60.6%; HR, 0.66; p = 0.03) than UCB recipients, along with a significantly lower NRM (12.9% vs. 26.3%; HR, 0.39; p < 0.01), and a comparable relapse rate (16.6% vs. 13.1%; HR, 1.41; p = 0.25) with UCB. These results suggest that the low NRM in PTCy-haplo transplant outweighs potential concerns of relapse among patients transplanted in CR1 (Figure 1B). By contrast, in patients with ≥ 2 nd CR or non-CR at HSCT, survival benefits of PTCy-haplo were not observed. [Conclusions] Our results suggest that while both PTCy-haplo and UCB are suitable alternative donor sources for adult ALL patients without HLA-matched donors, in patients with CR1, selection of PTCy-haplo rather than UCB improves post-transplant prognosis.

In Vivo Potency of Hematopoietic Stem Cells after Long-Term Storage at -80C in DMSO+HES Cryprotectant Mixture: Comparing Engraftment Kinetics and Efficacy Data in First and Second Autologous Stem Cell Transplantation in Myeloma

Background: The use of a cryoprotectant combining both intracellular and extracellular protectants (DMSO+HES) permits large volume hematopoietic stem cell (HSC) cryopreservation in mechanical freezers at -80C without the development of clumping after thawing (Stiff et al, Blood 1983. PMID: 6187518) and was recently utilized for allograft cryopreservation during the COVID-19 pandemic. However, there are competing methods for cryopreservation. Many centers are utilizing vapor phase liquid nitrogen (VPLN) storage protocols with DMSO alone instead. While HSC stored with DMSO alone in VPLN have shown to remain viable after long-term storage (Makhani et al, TCT 2022. PMID:3524877), this has not yet been proven for DMSO+HES stored HSCs at -80C. Here we report on second autologous hematopoietic stem cell transplants (AHSCT2) for relapsed multiple myeloma (RRMM) performed > 2 years after cell storage began at - 80C to determine if there were differences between engraftment and outcomes between initial and long-term storage in DMSO+HES at -80C. Methods: We searched our own institution's records from 1990-2023 to find patients who received two separate AHSCT for MM after only one cell collection prior to first AHSCT (AHSCT1). Prior allogeneic transplants patients were excluded. The primary outcome was neutrophil and platelet engraftment times. Secondary outcomes were progression free survival (PFS) and overall survival (OS). We reviewed patient characteristics, disease state prior to both transplants, engraftment kinetics for both, and survival outcomes. Outcomes were analyzed by Kaplan Meier (KM) and longrank test where applicable, and median PFS (mPFS) and median OS (mOS) were determined by reverse KM. Differences in engraftment were compared using the Kruskal-Wallis test. Results: 35 patients met inclusion criteria, of whom 57% of patients were male. Median age at AHSCT1 was 56.3 years and at AHSCT2 was 60.1 years. Median follow up was 8 years from second transplant and 12.3 years from stem cell collection. Median time from stem cell collection to AHSCT1 was 11 days (range 3-39 days) and to AHSCT2 was 4.6 years (range 2.2-15.8 years). Prior to AHSCT1, 65% of patients were in PR, 20% in VGPR, and 14% in CR. Prior to AHSCT2, 44% were in PR, 38% in VGPR, and 18% in CR. 80% and 83% of patients were conditioned with melphalan alone in AHSCT1 and 2 respectively. Median CD34+ doses were 5.1x10^6/kg and 4.2x10^6/kg (p=0.028) for AHSCT1 and 2 respectively. There was no statistical difference between the engraftment kinetics of the first or second transplant with a median ANC engraftment time of 11 days for both groups (p=0.88), and median platelet engraftment of 17.0 and 17.5 days (p=0.55) for AHSCT1 and AHSCT2 respectively. Between categories of cells stored between 2-5 years and >5 years, there was no statistical difference in ANC (p=0.85) or platelet engraftment (p=0.29). mPFS from AHSCT1 for the overall study was 3.7 years and for AHSCT2 was 3.6 years. The mOS was 4.8 years from AHSCT2. There was a statistical difference in 2-year PFS between ASHCT1 and 2 at 86% and 62% (p=0.0001) respectively. The mPFS differed by disease state prior to AHSCT2 dramatically; those in CR had mPFS of 7.8 years, those in VGPR 4.3 years, and those in PR 1.3 years. Discussion: Our results suggest that HSC stored long-term at -80°C using the DMSO + HES combination does not adversely affect product quality even for cells stored up to 15.8 years. Furthermore, our method is simpler, more cost-effective and poses no risk to the working environment when compared to current freezing method where VPLN storage is mainly used. This is particularly important to expand access to cryopreserved HSC for centers and countries where resources are more limited. These data also add evidence to the efficacy of AHSCT2 as we saw a 2-year PFS of 63% for these patients, which compares favorably to 50% 1-year PFS in AHSCT2 noted in the large CIBMTR retrospective by Dhakal (Leukemia 2021, PMID 32747684). In addition, some patients benefit greatly, best seen in those achieving a CR prior to AHSCT2 who here had a mPFS of 7.8 years. This is an important selection criteria we now evaluate when considering AHSCT2 in RRMM. AHSCT2 is cost effective and compared to recently approved CAR-T and bispecifics is likely a more economical approach in select patients relapsing after long remission following AHSCT1 who achieve deep responses to salvage therapy.

Cryopreservation Changes the Immune Effector Cell Composition of Peripheral Blood Stem Cell Grafts: An Analysis from the DKMS and NMDP Graft Composition Study

The impact of the cellular composition of PBSC grafts on the immune reconstitution as well as graft-versus-host and graft-versus-leukemia effects after allogeneic hematopoietic cell transplantation (alloHCT) is only poorly understood. DKMS and NMDP joined forces to comprehensively characterize the cellular composition of 2,000 PBSC products using a 34-color flow cytometry panel to define immune effector cell subsets of interest and hematopoietic stem cells. In parallel, we have created a biobank of unstimulated donor PBMC and PBSC graft samples for future analyses. As part of this larger effort, we investigated the effect of cryopreservation on the cellular composition of PBSC products. Cryopreservation of PBSC grafts was used frequently during the COVID19-pandemic and facilitated more flexible timelines for transplantation. Still, one third of the US patients and 15% of the German patients continue to receive cryopreserved PBSC grafts. Ongoing studies are investigating the impact of cryopreservation on immune-effector cell function and the outcome of hematopoietic stem cell transplantation. Several observational studies have reported delayed neutrophil and platelet engraftment with cryopreserved products. More recently, less chronic GVHD was reported for patients who had received cryopreserved PBSC products. Cryopreservation affects the cellular composition and hence has the potential to change immune effector cell functions of the graft, but available data on the alterations of cell population frequencies are scarce. We analyzed the cellular composition of 20 fresh grafts and compared them to their frozen and thawed counterparts. Cryopreservation of product samples followed routine clinical procedures using a 10% DMSO containing cryo-medium and a controlled rate freezing. After thawing at 37°C, cells were washed, stained with a 34-parameter phenotyping panel, fixed and analyzed immediately on a full spectrum flow cytometer. Following spectral unmixing and manual spillover correction, proportions of immune cells were assessed using manual gating. First, we investigated the relative frequencies of 10 non-overlapping individual cell populations relative to viable CD45+ cells, including several lymphocyte populations, monocytes, granulocytes, hematopoietic stem cells (HSC) and dendritic cells. Granulocytes showed the largest decrease after cryopreservation and thawing (18.2 % vs 3.5 %; p<0.001). The loss of granulocytes results in an increase of relative frequencies of many other immune cell populations including monocytes (35.4 % vs 44.7 %; p=0.018), B cells (5.8 % vs 9.6 %; p=0.006), and HSC (1.0 % vs 1.5 %; p<0.001). We therefore also calculated changes relative to the viable non-granulocyte cells. By doing so, a significant relative increase of cells was observed for HSC (1.2 % vs 1.6 %; p<0.001), whereas monocyte and B cell frequencies changed only marginally. This suggests that HSC are more resilient to freezing than other cell types. When comparing the proportions of 40 other immune cell (sub-) populations relative to their parent population in the gating hierarchy, we found a profound and significant decrease of the relative frequencies for CD16+ myeloid dendritic cells (mDCs, 68 % vs 30 % of all mDCs; p<0.001) and granulocytic myeloid derived suppressor cells (gMDSCs, 2 % vs 1 % of all MDSCs; p<0.001). In an independent set of analyses performed at Roswell Park Comprehensive Cancer Center, Buffalo, comparable results were obtained with a significant increase of HSC, a significant decrease of CD16+ mDCs, and a trend of a decrease of gMDSCs. As CD16+ mDCs may have a role in the development of chronic GVHD after alloHCT, this decrease may be meaningful. In summary, we found that cryopreservation of PBSC allografts resulted in a decreased relative frequency of granulocytes, and CD16+ mDCs, as well as an increased relative frequency of HSCs. The results provide some insight into the impact of cryopreservation on PBSC graft composition and warrant further efforts to explore the potential mechanisms for the differential outcomes observed using fresh vs cryopreserved PBSCs.

Inb-100: A Pilot Study of Donor Derived, Ex-Vivo Expanded/Activated Gamma-Delta T Cell (EAGD) Infusion Following Haploidentical Hematopoietic Stem Cell Transplantation and Post-Transplant Cyclophosphamide (PTCy)

Background: Gamma-delta (gd) T cells are MHC unrestricted lymphocytes that recognize and lyse malignant cells in allogeneic settings. Although haploidentical transplant with PTCy has reduced the risk of graft-versus-host disease (GvHD); the incidence of relapse remains up to 50% at year 1. Early post-transplant infusion of haploidentical EAGD cells may decrease relapse risk through a graft-versus-leukemia (GvL) effect without severe GvHD. We present updated clinical and correlative data from our Phase I trial using our recommended phase 2 dose (RP2D). Methods: Adults with newly diagnosed or relapsed ALL, CML, AML undergoing first haploidentical transplant with reduced-intensity flu/cy/TBI conditioning received EAGD intravenously within 7 days of neutrophil engraftment. Peripheral blood was collected at EAGD infusion and monthly through day 90, with additional collections every 6 months through 1 year. Primary endpoints include dose-limiting toxicities (DLT), grade (G) 3-4 adverse events including GvHD with secondary endpoints of relapse and overall survival. Biologic parameters included multiparameter flow cytometric immunophenotyping and additional serum cytokine analysis using the Olink ® 48 target panel. Results: 14 subjects were enrolled with 4 treated at Dose Level (DL) 1 of 1 x 10 6 EAGD/kg and 6 subjects treated at the RP2D of DL2 (3 x 10 6 EAGD/kg). Untreated subjects included: One screen failure, a manufacturing failure, one subject who died prior to dosing, and one subject who received an out of study specification product. Treated subjects were 60% male, median age of 68, and primarily AML subjects in CR1. RP2D was defined by an acceptable toxicity profile, no DLT's, prolonged RFS and elevated gd levels. Peripheral lymphodepletion persisted through the first 100 days post-BMT followed by T cell recovery from a CD45+CD27-effector phenotype to central and effector memory phenotype. Notably, DL2 showed significant increases in gd T cell count recovery at days 60, 100 and 180 post-BMT vs. DL1 and historically untreated Haplo/PTCy patients, p = <0.05. NK cells remained within the low normal range and <3% of circulating T cells were Tregs. Preliminary serum cytokine analysis revealed elevated IL-6 and IL-15 in serum post- transplant prior to EAGD infusion, but dropped to normal median levels at day 60, 20-30 days after EAGD infusion. One subject received intermittent hypomethylating therapy for emergence of recipient chimerism. Treatment emergent AE's included transient WBC and ANC decreases (100% each), platelet count decrease and anemia (88.9% each) and maculopapular rash, hypomagnesemia and blood creatine increased (55.6% each). EAGD related toxicities were primarily G1-2, included G1-2 skin acute GvHD and gastrointestinal GvHD, and one case of G3 platelet count decrease. Treatment related serious AE's of G3 nausea and G2 rash were reported. No DLTs, neurotoxicity, cytokine release syndrome or treatment related deaths were reported. Conclusions: Post-BMT infusion of donor-derived, EAGD cells has demonstrated manageable safety with no infusional toxicity or ≥G3 acute GvHD or extensive chronic GvHD. Durable relapse free survival at a maximum of >3 years in subjects with poor risk cytogenetics and adverse clinical risk factors combined with ongoing homeostatic reconstitution of increasing gd T cell effectors post-BMT suggests this therapy may be an effective measure in mitigating relapse after HaploBMT. NCT03533816

Long Term Outcomes and Renal Response Following Autologous Stem Cell Transplantation for Light Chain Deposition Disease:a Retrospective Study on Behalf of the Cmwp of the EBMT

Background: There is a paucity of data concerning long-term outcomes following autologous stem cell transplantation (ASCT) for light chain deposition disease (LCDD). The goal of this retrospective study was to analyze the outcomes following ASCT in LCDD patients in terms of toxicity and efficacy with both hematological and renal responses. Methods: Adult patients who underwent a first ASCT for renal biopsy-proven LCDD (AL amyloidosis excluded) between the years 1995 and 2021 were selected from the EBMT registry. The probability of overall survival (OS) and progression-free survival (PFS) was estimated based on the Kaplan-Meier method and differences analyzed using the log-rank test. Engraftment with death as competing event and relapse and non-relapse mortality (NRM) were modeled using the crude cumulative incidence estimator and compared between groups with Gray's test. eGFR, measured at auto-HCT and 3 6, and 12 months was longitudinally modeled using linear mixed effects models. Results: There were 51 patients in 24 centres. 63% were male. The median year of diagnosis was 2011 (IQR: 2008-2015). The underlying plasma cell disorder was either myeloma (47%) or Monoclonal Gammopathy of Clinical Significance (MGCS, 50%), with 3% missing. The median bone marrow aspirate plasmacytosis was 10% (IQR: 7.8-20) and the immunoglobulin isotypes were as follows: Light Chain only 69% (Kappa 82%, Lambda 18%), IgG 24%, IgA 2%, IgD 2% and non-secretory 2%. Of 17 patients with cytogenetic data, 3 were t(11;14) and one del 17p. All 40 patients with data on disease involvement had renal involvement, and, of these, 3 had cardiac and 2 hepatic involvement. At diagnosis, the median serum creatinine was 233 µmol/L (IQR:159-467), median proteinuria was 1,813 mg/24h (IQR:445-5974) and 16% had bone lesions. 42 of 44 patients with available data had received an induction regimen (93% bortezomib-based) and the hematological responses prior to transplant were as follows: CR 12%, VGPR 29%, PR 31%, MR/SD 16%, and relapse/progression 6% (missing: 6%). A total of 59% were transplanted in or after 2012. The median age at transplant was 55 years (IQR:49-61). The median time from diagnosis to transplant was 7.4 months (IQR:5.5-13.0) and 42% were on dialysis at the time of transplant. The Karnofsky Performance Score (KPS) was >80 in 79%. The Melphalan conditioning dose (mg/m 2) was 100 (23%), 140 (55%) or 200 (21%). The median number of infused CD34+ cells x 10 6/kg was 3.4 (IQR:2.5-4.6) and 33% received G-CSF post-ASCT. A total of 13% received consolidation and 8% maintenance treatment post-ASCT. The median follow-up post-ASCT was 84 months (IQR:46-122). The median time to neutrophil engraftment was 12 days (IQR:11-13) and to platelet engraftment 13 days (11-16). The best hematological response at Day +100 post-ASCT (33% missing) was as follows: CR 44%, VGPR 26% and PR 29%. Mean eGFR at auto-HCT was 48.8 mL/min/1.73m 2 (95% CI 36.8-60.8) in those not on dialysis and 15.2 mL/min/1.73m 2 (95% CI 1.9-28.8) in those on dialysis (difference 33.6 mL/min/1.73m 2, p<0.0001). In both groups there were no significant changes in eGFR after auto-HCT (p=0.99 and p=0.80, respectively). The box-plot figure shows eGFR at auto-HCT, and at 3, 6 and 12 months. At 6 years post-ASCT, OS was 88% (95% CI: 78-98%) and PFS was 44% (95% CI 28-60%). The median OS was not reached. The median PFS was 65 months (95% CI: 45-103). The 2-year cumulative relapse incidence was 17% (95% CI: 6-27%) and the 2-year cumulative incidence of NRM was 2% (95% CI: 0-6%). In univariate analysis, transplant from 2012 onwards was associated with a significantly superior OS (6-year OS 100 vs. 75%; log-rank p=0.05). There was a trend to superior OS in females (6-year OS 100 vs 82%, p=0.05). There was no significant association between KPS, Age, Disease Status at ASCT (VGPR or better vs. Other) or Dialysis Status at ASCT with any outcomes in this small cohort evaluation. Conclusions: ASCT is a feasible option for patients with LCDD even though 42% were on dialysis at the time of transplantation. Overall outcomes are favourable with a low NRM and good long-term OS. There was no improvement in renal function.

Allogeneic Hematopoietic Cell Transplantation with Sequential-Based Conditioning in Refractory / Relapsed Acute Myeloid Leukemia: Experience from a Single Institution

INTRODUCTION: Despite improvements in the treatment and outcomes of patients with acute myeloid leukemia (AML), in nearly 10-20% of the cases, a morphological remission (CR) after administrating induction regimens is not achieved. In other cases, and despite initial CR, early disease relapse may occur, with only a minority of patients durably benefiting from salvage regimens. Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative strategy for patients with relapsed / refractory (R/R) AML. However, the prognostic of these patients after allo-HCT remains dismal, with reported survival rates ranging from 20% to 35% in different publications. Allo-HCT using peripheral blood and sequential-based conditioning regimens has been the transplant platform used at our institution for R/R AML during the last decade. During the study period the use of post-transplant cyclophosphamide (PTCY) combined with tacrolimus (TK) has been implemented on this allo-HCT setting. This study evaluates the results obtained from a single institution obtained from using sequential allo-HCT for patients with R/R AML, and the effectiveness of having implemented the use of PTCY in this type of transplant. Very few studies have evaluated the safety of including PTCY-based in sequential-based conditioning allo-HCTs. METHODS: This study includes the 21 patients with R/R AML who underwent allo-HCT with sequential-based conditioning regimens at our institution between 2011 and 2022. Retrospective data were collected during June 2023. Sequential regimens combined fludarabine (150 mg/m2), cytarabine (2 g/m2 x 5 days), and idarubicin (12 mg/m2 x 3 days) chemotherapy with low doses of Bu (n=3), melphalan (n=17), or treosulfan (n=1). Six adults transplanted after November 2016 received PTCY (50mg/kg/24h) on day +3 and +4 followed by TK from day +5 to day +90 for GvHD prevention. RESULTS: The main baseline characteristics of the study cohort and according to the GVHD prophylaxis are shown in Table 1 . The median age was 53 years (range 31-68), 11 (52%) patients were males, and all patients had R/R AML after having received at least one line of induction chemotherapy with curative intent. Fourteen (66.7%) patients received grafts from HLA-matched donors, and 7 (33.3%) from 9/10 HLA mismatched unrelated donors (MMUD). Baseline characteristics between patients who received PTCY and those who did not were balanced, except for the proportion of allo-HCT performed from MMUD which was higher in the PTCY group (66.7% vs. 20.0% p=0.03). Post-transplant results are shown in Table 2. All patients engrafted (100%). Patients who received PTCY experienced similar median of days to neutrophil engraftment (18 vs. 15 days, P=0.103) and a longer median of time to platelet engraftment (25 vs. 13 days, P=0.046). The day +100 cumulative incidences (CI) of grade II-IV and III-IV aGVHD, and 2-year CI of moderate/severe cGVHD were 33.3%, 0% and 0% for patients who received PTCY-TK, and 40% (p=0.823), 33% (p=0.118), and 40.4% (p=0.099) for those who did not. During the first 2-years after allo-HCT, 8 (38%) out of 21 patients died and 7 (21%) relapsed. The main causes of death were relapse (n=4, 19%) and infection (n=2, 9.5%), and the 2-year OS, PFS, NRM and CIR were 61.5%, 51.9%, 14.3%, and 33.3%. As shown in Table 2 patients who received PTCY had comparable rates of OS (2-y: 50.0% vs. 66.7%, p=0.903) and RFS (2-y: 50.0% vs. 53.3%, p=0.873) and those who did not. In addition, and although no patient who received PTCY died secondary to transplant-related toxicity (2-y 0 vs. 20%, p=0.254), a non-significant trend to higher cumulative incidence of relapse (2-y: 50.0% vs. 26.7%, p=0.282) was observed in this group of patients. Lastly, a non-significant trend to higher GRFS was documented in patients who received PTCY (2-y: 50.0% and 25.0%, p=0.266). CONCLUSIONS: At our institution, 50% of the patients with R/R AML going thought allo-HCT performed using sequential-based conditioning regimens survived after two years. The implementation of PTCY as part of the GVHD prophylaxis results decreased rates clinically relevant GVHD and NRM, resulting on a trend to better GRFS. Although the results provided by this analysis suggest that using PTCY in this allo-HCT is safe and effective, caution is still recommended considering the trend to higher relapse rates observed among these patients and the limited sample size.

Evaluation of Circulating Endothelial Cells (CECs) As Marker of Endothelial Damage in Allo-Transplanted Patients at High Risk of Hepatic Veno-Occlusive Disease/Sinusoidal Obstruction Syndrome (VOD/SOS): The Cecinvod Study

INTRODUCTION Sinusoidal obstruction syndrome (SOS), also known as veno-occlusive disease (VOD), is a potentially fatal complication after allogeneic stem cell transplantation (alloSCT). Identifying a predictive biomarker for VOD has been challenging. Since endothelial injury is considered one of the main pathogenic factors for VOD onset, with the CECinVOD prospective study we aimed to evaluate Circulating Endothelial Cells (CEC) in allo-transplanted patients (pts) at higher risk to develop VOD. METHODS From October 2020 to November 2022, 150 pts have been enrolled in the CECinVOD study from 11 Italian Bone Marrow Transplantation Units. All pts must be older than 18 years and undergoing myeloablative alloSCT. CECs were detected using the CellSearch system, the FDA-approved immunomagnetic selection approach incorporating ferrofluid nanoparticles (anti CD146) and fluorophore-labelled antibodies (anti CD105, CD45 and DAPI). CEC were defined as CD146+, CD105+, DAPI+ and CD45-. CEC were collected at the following timepoints: before conditioning regimen (T0), at the end of conditioning regimen and before alloSCT (T1), at the time of neutrophils engraftment (T2), and 7-10 days after engraftment (T3). In pts who developed VOD, additional timepoints were collected as follows: at any time of suspected or proven VOD onset (T4), and then weekly during Defibrotide treatment (T5-T8). SOS/VOD was defined according to the 2016 European Group for Blood and Marrow Transplantation criteria. RESULTS Pts' main characteristics are summarized in Table 1. Six out of 150 pts (4%) developed VOD during the follow up (4 “severe”, and 2 “very severe”). All pts were treated with Defibrotide, obtaining a complete remission in 5 of them, while 1 pt died due to VOD complications. Pts receiving TBI-based regimen were more likely to develop VOD compared to those receiving Treosulfan (10 to 14 g/m2) or Busulfan ev (9.6 to 12.8 mg/kg) (p 0.08). Similarly, higher baseline levels of bilirubin were associated with a higher incidence of VOD (p 0.08). Considering the CECs analysis, 615 samples were evaluated. At the enrollment, CECs levels were not related with any of clinical characteristics analyzed, except for the number of previous treatments. Indeed, those pts with 2 or more previous lines of treatments had higher levels of CECs (OR=0.53; p<0.001). Considering the different timepoints, conditioning regimen and alloSCT result in higher levels of CECs. Thus, CEC were higher at T1 than at T0 (p 0.02), as well as they were even higher at T2 than at T1 (p<0.0001) (Fig.1). Conversely, no significant differences have been observed between T3 and T2. No other clinical characteristic was correlated with CEC counts at the different timepoints. Pts who developed VOD had higher median levels of CEC at all the timepoints analyzed, but this difference has not achieved statistical significance, probably due to the low number of pts in the VOD group. At VOD onset, the pts always had an increase in CEC levels compared with the previous timepoint. After defibrotide treatment, the CEC levels increased in the first week, while they progressively decreased during the VOD treatment (T6 and T7, -50,7% and -71,5%, respectively). Recently, another endothelial activation marker has been considered: the Easix score. We didn't find any relationship between the Easix score at transplant and the VOD onset, even if a trend may be observed. We also investigated whether the CEC levels may be related with Easix score, but we didn't find any relationship at each timepoint. Interestingly, in a subgroup of pts CECs have been observed as a cluster of multiple cells. This data is firstly described in allo-SCT. Its functional significate remains unclear, but we found a relationship between CECs cluster and the number of CECs (p < 0.001). CONCLUSIONS The incidence of VOD in our prospective study was low with respect to the one reported in the literature. This can be explained by the changes in alloSCT from the past (better selection of the pts, lower use of TBI, higher use of reduced intensity regimen) and the retrospective nature of most of the previous reports. We show that CECs can be considered reliable marker of endothelial damage in alloSCT pts, highlighting the impact of previous treatments, the conditioning regimen, and allo-SCT itself. Increased CEC level may be helpful to confirm VOD diagnosis, as well as their monitoring may be useful to evaluate the response to the treatment for VOD.

Post-Transplant Cyclophosphamide, Tacrolimus or Cyclosporine a and Mycophenolate Mofetil Compared to Anti-Thymocyte Globulin tacrolimus or Cyclosporine a and Methotrexate Combinations As Graft- versus -Host Disease Prophylaxis Post Allogeneic Stem Cell Transplantation from Sibling and Unrelated Donors in Patients with Acute Myeloid Leukemia: a Study on Behalf of the Acute Leukemia Working Party of the European Society for Blood and Marrow

Background: Recently, the BMT CTN 1703 phase III study compared post-transplant cyclophosphamide with tacrolimus with mycophenolate mofetil (PTCy/TAC/MMF) to TAC /methotrexate (MTX) as graft- versus-host disease (GVHD) prophylaxis post allogeneic transplantation (HSCT), demonstrating a lower incidence of severe acute (a) GVHD and chronic (c) GVHD and better GVHD-free, relapse-free survival (GRFS). The control arm did not include anti-thymocyte globulin (ATG), used in many centers for GVHD prophylaxis. Methods: The study aim was to compare PTCy with TAC or cyclosporine A (CSA) and MMF (PTCy/TAC or CSA & MMF) to ATG combined with TAC or CSA and MTX (ATG/TAC or CSA & MTX) in acute myeloid leukemia (AML) patients (pts) undergoing HSCT from matched siblings (MSD) or 9-10/10 unrelated donor (UD) in first complete remission (CR1). Statistical tests included a multivariate analysis (MVA) adjusting for potential confounding factors using a Cox proportional-hazards regression model for main outcomes. . Results: 6050 pts met the inclusion criteria, 402 received PTCy/TAC or CSA & MMF and 5648 received ATG/TAC or CSA & MTX as GVHD prophylaxis. Median follow-up was 23.4 (IQR, 20.3-24.9) and 41.8 (IQR, 39.6-43.3) months (p<0.0001). The median year of the transplant was 2018 (2010-2020) and 2016 (2007-2020) (p<0.0001). Pts in the PTCy/TAC or CSA & MMF group were younger, with a median age of 48.7 (range 18-5.6) versus 51.5 (8-77.8) years (p=0.024). The diagnosis was de novo AML in 84.1% vs 85.3% and secondary (s) AML in 15.9% vs. 14.7% (p=0.49). The cytogenetic risk was categorized as intermediate (70.9% vs. 67.1%), adverse (22.2% vs. 25.7 %), and favorable (6.9% vs. 7.2%) for pts in the PTCy/TAC or CSA & MMF and ATG/TAC or CSA & MTX groups, respectively (p=0.35) (data missing for 2214 pts). Karnofsky performance status (KPS) did not differ between the groups. There was a higher frequency of pt cytomegalovirus (CMV) seropositivity and female (F) donor to male (M) pt combination in the PTCy/TAC or CSA & MMF group, 77.8% vs. 71.8% (p=0.009) and 18.4% vs. 14.4% (p=0.029). More pts in the PTCy/TAC or CSA & MMF group received reduced intensity conditioning (RIC) 51.5% versus 41.1% in the ATG/TAC or CSA & MTX group, respectively (p<0001). Day 60 neutrophil engraftment (ANC >0.5 x 10 9/L) was 98.7% vs. 98.6% (p=0.84). Day 180 incidence of a GVHD grade II-IV and III-IV was 21.2% vs. 20.4% (p=0.92) and 8.1% vs. 6% (p=0.1), in pts receiving PTCy/TAC or CSA & MMF versus the ATG/TAC or CSA & MTX GVHD prophylaxis, respectively. The 2-year (y) total and extensive chronic (c) GVHD were 33.7% vs. 30% (p=0.09) and 10.7 % vs. 11.2% (p=0.81), respectively. GVHD was the cause of death in 11.6% vs. 13.9% of pts who died. In the MVA, both aGVHD (grade II-IV or III-IV) and cGVHD (total or extensive) did not differ between the groups with hazard ratios (HRs) =1.15 (95% CI 0.86-1.53, p=0.35), HR=0.87 (95% CI 0.56-1.34, p=0.52), HR=0.91 (95% CI 0.7-1.18, p=0. 47 and HR=1.51 (95% CI 0.96-2.36, p=0.074). Two-y NRM was significantly lower in pts that received PTCy/TAC or CSA & MMF versus ATG/TAC or CSA & MTX for GVHD prophylaxis, HR=1.57 (95% CI 1.07-2.3, p=0.022). Other HSCT outcome parameters did not differ between the groups. The HR for 2-y RI was 0.99 (95% CI 0.77-127, p=0. 93). The HRs for 2-y leukemia-free survival (LFS), overall survival (OS), and GRFS were HR=1.15 (95% CI 0.94-1.42, p<0.18), HR=1.18 (95% CI 0.94-1.49, p=0.16) and HR=1.12 (95% CI 0.93-1.36, p=0.22), respectively. Donor type and conditioning regimen were poor prognostic factors for grade II-IV, III-IV aGVHD, and total and extensive cGVHD. For cGVHD, additional poor prognostic factors were F donor to M pt combination and pt CMV seropositivity. Poor prognostic factors for LFS, OS, and GRFS were 9/10 UD, age (by 10 y), sAML, adverse-risk cytogenetics, lower KPS, and pt CMV seropositivity. For NRM, factors were the same apart from cytogenetics risk which was not a prognostic factor. In addition, time from diagnosis to HSCT was a prognostic factor for NRM and RI. Other poor prognostic factors for RI were lower KPS and pt CMV seropositivity. Conclusions: In this registry-based retrospective analysis, comparing PTCy in combination with TAC or CSA and MMF to ATG in combination with TAC or CSA and MTX as GVHD prophylaxis, we observed a similar incidence and severity of both aGVHD and cGVHD. NRM was significantly lower with the PTCy-based GVHD prophylaxis, while all other transplant outcome parameters were similar.

The Area Under Tacrolimus Concentrations on Day 14-28 Is the Independent Predictor of the Development of Chronic Gvhd in Patients without Severe Acute Gvhd after Unrelated Allogeneic HSCT

Introduction: In unrelated allogeneic hematopoietic stem cell transplantation (UR-SCT), chronic graft-versus-host disease (GVHD) is a critical complication that affects not only patients' quality of life but also post-transplant outcome. Acute GVHD is a major risk factor for the subsequent development of chronic GVHD. The combination of tacrolimus and short-term methotrexate (TAC+sMTX) has been widely used as a GVHD prophylaxis for UR-SCT in Japan. Previous reports have shown that tacrolimus blood concentrations early after transplantation are associated with the development of acute GVHD, but not with that of chronic GVHD. On the other hand, among UR-SCT recipients who did not develop severe acute GVHD, the relationship between tacrolimus blood concentrations and the development of chronic GVHD remains unclear. Methods: To examine the association between tacrolimus blood concentrations and post-transplant outcomes, the area under the tacrolimus concentration on day 0-14 (AUTC 0-14) and day 14-28 (AUTC 14-28) was calculated simply as the sum of the area of the trapezoid for the corresponding period ( Figure). Tacrolimus was initiated by continuous intravenous administration from day -1 in all patients, and no patients were switched to oral administration prior to day 28. Short-term methotrexate was administered at 10 mg/m 2 on day 1 and 7 mg/m 2 on day 3, day 6, and day 11. Among all 151 adult patients with hematopoietic malignancies who underwent first UR-SCT with GVHD prophylaxis of TAC+sMTX between 2010 and 2022 at the Jikei University Hospital, the following were excluded from the analysis: developing grade III-to-IV acute GVHD, using prednisolone or methyl-prednisolone ≥0.5 mg/kg within day 100, engraftment failure, death before day 100, and relapse before day 100. Results: A total of 68 patients were included in the analysis. Median age was 50.5 (range, 16-68) years. Twenty-seven (40%) patients had acute myeloid leukemia, and 18 (27%) had acute lymphoblastic leukemia. Fifty-five (81%) were in complete remission/chronic phase/early stage. The donor source consisted of 42 (62%) bone marrow transplantation (BMT), 20 (29%) cord blood transplantation (CBT), and 6 (9%) peripheral blood stem cell transplantation (PBSCT). BMT recipients transplanted from 22 (52%) HLA 8/8 matched donor, 19 (45%) HLA 7/8 matched donor, and one (3%) HLA 6/8 matched donor. CBT recipients transplanted from 15 (75%) HLA 4/6 matched donor and 5 (25%) HLA 5/6 matched donor. PBSCT recipients transplanted from 5 (83%) HLA 8/8 matched donor and one (17%) HLA 7/8 matched donor. Fifteen (22%) patients were administered additional low-dose anti-thymocyte globulin (ATG) as GVHD prophylaxis. Fifteen male patients transplanted from female donors (22%). Thirty-nine (57%) patients received myeloablative conditioning regimen. The median time from UR-SCT to neutrophil engraftment was 19 days (range, 11-34). Regarding acute GVHD, 46 patients (68%) developed none, 13 patients (19%) developed grade I, and 9 patients (13%) developed grade II. The median observation period for all 68 patients was 48.2 months (4.0 years). The 4-year cumulative incidence (CI) of chronic GVHD was 51%, of which 24% were moderate-to-severe. The CI of moderate-to-severe chronic GVHD was not associated with age, disease status, donor source, HLA incompatibility, ATG use, female to male, intensity of conditioning regimen, and grade I-to-II acute GVHD development. AUTC 14-28 <200 was the only risk factor (AUTC 14-28<200: 32% vs AUTC 14-28≥200: 11%; p=0.047). Multivariate analysis also showed that AUTC 14-28 <200 was the independent factor for development of moderate-to-severe chronic GVHD ( p=0.04). In all 68 patients, 4-year CI of relapse was 20%, 4-year CI of non-relapse mortality was 10%, 4-year GVHD-free, relapse-free, survival (GRFS) was 50%, and 4-year Overall survival was 80%, all of which did not differ between AUTC 14-28 <200 and AUTC 14-28 ≥200 groups. Conclusion: In UR-SCT, our data showed that AUTC 14-28 <200 may be an independent risk factor for developing moderate-to-severe chronic GVHD, even in patients who did not develop severe acute GVHD.

Dynamics of Fecal Antibiotic-Resistance Genes after Fecal Microbiota Transplantation in Allogeneic Hematopoietic Cell Transplant Recipients and AML Patients: Results from a Randomized Placebo-Controlled Trial

Intestinal colonization with antibiotic-resistant bacteria has been associated with increased risk of systemic infections after allogeneic hematopoietic cell transplantation (HCT) and anti-leukemia induction chemotherapy. Fecal microbiota transplantation (FMT) has been used with success in gut decolonization from select antibiotic-resistant pathogens in small series. Large-scale data using expanded panels of antibiotic-resistance genes (ARGs) and long-term effects of FMT are unavailable. We performed a correlative analysis of samples from a prospective trial to address this knowledge gap. We conducted a randomized double-blind trial of oral, encapsulated, third-party fecal microbiota transplantation (FMT) versus placebo at the time of neutrophil recovery in two independent cohorts of patients with AML undergoing induction chemotherapy or allogeneic transplant recipients, with the primary objective of reducing infections (ClinicalTrials.gov identifier: NCT03678493; J. Clin. Oncol 2023 May 26). 100 patients (HCT: 74, AML: 26) were randomized between FMT vs. placebo, each given as 5 oral capsules taken at once a the time of neutrophil engraftment. FMT was safe and ameliorated intestinal dysbiosis, but the trial did not meet its primary endpoint. Longitudinal pre- and post-treatment stool samples were collected at the following timepoints: baseline (before starting conditioning; T0), pre-dose 1 (T1), day 10 post-dose 1 (T2), day 28 post-dose 1 (T3), and at the end of study follow up (9 months; T4). All stool samples underwent 16S rRNA gene sequencing for the pre-planned microbiota endpoints of the trial. 226 samples with remaining DNA were used for microfluidic qPCR to simultaneously detect and quantify 47 ARGs including those for beta-lactams, quinolones, vancomycin, and carbapenem resistance. All samples were run in duplicate and their average gene content for each ARG (gene copies/microL DNA) was calculated using standard curves of the respective genes. Both qualitative (presence/absence; Fisher's exact test) and quantitative (Wilcoxon's test) analyses were performed, with a 0.05 threshold for P values defining statistical significance. The small number of T3 samples (n = 27) did not allow a meaningful comparison between the two arms; these samples were not included in analysis. As expected from the randomized design, there was no difference between the FMT and placebo arms in the number of ARGs at T0 or T1 (median [range]; T0: 29 [1-45] vs. 31 [13-39], P = 0.33; T1: 25 [5-40] vs. 26 [9-43], P = 0.52). There was a trend for more ARGs at T2 in the FMT arm (28 [1-42] vs. 22 [12-36], P = 0.07). At T4, there were fewer ARGs in the FMT arm compared to placebo (23 [9-38] vs. 32 [20-40], P < 0.01; Fig. 1 left panel). In quantitative analysis comparing the two arms at each post-treatment timepoint, sul1 (sulfonamide), floR (chloramphenicol), ctxm32 (beta-lactam), qnrB (quinolone), tetS (tetracycline), and aadA5 (aminoglycoside) were more abundant in the FMT arm at T2. At T4, blaKPC (carbapenem) and aadD (aminoglycoside) were more abundant in the placebo arm ( Fig. 1 right panel). Given our findings at T2, we evaluated whether ARGs in post-treatment samples were also present in pre-treatment samples from the same patient. In the case of FMT, de novo appearance of an ARG post-treatment could indicate ARG transmission via FMT. Although donor stool was screened for a panel of clinically relevant ARGs and pathogens, some ARGs may be found in healthy individuals and could have been present in the FMT product. Using a per-ARG Fisher's exact test, no ARG appeared de novo post-FMT more frequently than after placebo, arguing against ARG transmission via FMT. Findings from this largest randomized trial of FMT in AML patients and HCT recipients to date indicate long-term efficacy of FMT in antibiotic-resistant gut decolonization. A temporary early phase of reduced colonization resistance after FMT, possibly due to drastic, rapidly changing rewiring of microbiota networks, seems to occur, followed by formation of more stable communities resistant to ARG colonization. Clinical translation of these findings requires further research.

Haploidentical Hematopoietic Cell Transplant with Post-Transplant Cyclophosphamide Expedites Donor-Derived Immune Reconstitution in a Pediatric Severe Aplastic Anemia Cohort

Severe aplastic anemia (SAA) is a life-threatening, idiopathic process involving the immune-mediated destruction of hematopoietic stem cells. Treatment involves immunosuppressive therapy (IST) and/or allogeneic hematopoietic cell transplantation (HCT). The latter has recently been optimized with use of a post-transplant cyclophosphamide (PTCy) based graft-versus-host disease (GvHD) platform, permitting successful HCT with only half-HLA-matched haploidentical donors. In pediatrics, this platform results in >90% overall survival with low rates of GvHD and graft failure. However, reports on donor chimerism and immune reconstitution are limited. We hypothesized that a PTCy platform would expedite CD3 donor engraftment and CD4 T cell recovery. In a phase III clinical trial, we treated three cohorts of pediatric (age </=25 years) patients with SAA requiring alloHCT: MUD, MSD, and HAPLO. The MUD cohort included n=15 recipients of 7-8/8 HLA-matched unrelated donor grafts (n=5 7/8, n=10 8/8) with median age 14 years (range 2-17 years), following cyclophosphamide (Cy) 50 mg/kg, fludarabine (Flu) 200 mg/m 2, rabbit anti-thymocyte globulin (rATG) 9 mg/kg total dose rabbit and low dose total body irradiation (TBI) at 200 cGy. The MSD cohort included n=9 recipients of 8/8 HLA-matched sibling donor grafts with median age 15 years (range 12-24 years), following Cy 200 mg/kg and 9 mg/kg rATG. The HAPLO cohort included n=4 recipients of 4/8 HLA-matched related donor grafts with median age 8.5 years (range 2-21 years) following Cy 29 mg/kg, Flu 150 mg/m 2, 9 mg/kg rATG, and low dose TBI at 200 cGy. GvHD prophylaxis consisted of 30 days of mycophenolate mofetil, and 100 days of a calcineurin inhibitor (followed by an 8-10 week taper), with the HAPLO cohort additionally receiving post-transplant Cy (PTCy) 50 mg/kg/day on days +3 and +4. Peripheral blood lymphocyte counts and peripheral blood and bone marrow donor chimerism were evaluated on days +100, +180 and +365. Recipient characteristics were largely equivalent between cohorts, with 43% receiving IST prior to HCT. Clonality was overrepresented in the HAPLO cohort (2 of 4, 1 with monosomy 7 myelodysplastic syndrome and 1 with a large PNH clone), with only 2 of 15 in the MUD and none in the MSD cohorts. Active controlled infections at the time of HCT were additionally overrepresented in the HAPLO cohort, with disseminated pseudomonas in 1. The MUD cohort had 1 with Rhizopus invasive sinusitis (none in the MSD cohort). Achievement of neutrophil engraftment was statistically significantly delayed in the HAPLO cohort at a median of 17 days compared to 12 for MSD and 14 for MUD, p=0.004), however there were no episodes of primary or secondary graft failure. With only a single late death from intracranial hemorrhage in the MUD cohort, OS was equivalent at 2 years (100% in MSD and HAPLO cohorts, 93.3% in MUD). GvHD rates were low with only 1 (MUD) experiencing grade II acute GvHD, and 1 (MUD) experiencing mild chronic GvHD. Immune reconstitution varied by cohort with statistically significantly greater CD3 peripheral blood donor chimerism (p<0.001) as well as CD4 (p=0.04) and CD8 (p=0.03) T cell counts over time for the HAPLO recipients (CD3 chimerism in Fig 1, CD4 count in Fig 2; 2-way ANOVA fixed effects model). Considering a CD4 >200 cell/uL as clinically meaningful, we assessed the proportion of each cohort achieving this milestone at each timepoint. While each cohort had an increase in proportion achieving CD4>200 over time, the HAPLO cohort reached 50% with CD4 >200 at day +100, compared to 11% MUD and 0% MSD (p=0.09). At day +180, the proportion with CD4 >200 were 36% MUD, 25% MSD and 75% HAPLO (1 yr: 90% MUD, 100% MSD and HAPLO). Donor availability remains a challenge to successful transplant for patients with SAA, and haploidentical matches overcome donor limited accessibility. PTCy as GvHD prophylaxis is associated with low incidences of graft failure and severe GvHD. While PTCy might be anticipated to inhibit donor T cells and immune reconstitution, our data, albeit limited by small cohorts, demonstrates superior and more efficient CD3 donor engraftment and CD4 T cell count recovery following HAPLO HCT with PTCy compared to non-PTCy based MUD and MSD cohorts.

Optimized Double-Unit Cord Blood Transplantation Mitigates Transplant-Related Mortality Resulting in High Progression-Free Survival in Adults with Hematologic Malignancies

Background : Double-unit cord blood transplantation (dCBT) with intermediate intensity conditioning (Cy50/ Flu150/ Thio10/ TBI400cGy) and cyclosporine-A (CSA)/ mycophenolate mofetil (MMF) graft-versus-host disease (GVHD) prophylaxis has been associated with high progression-free survival (PFS) in adult patients (pts) with hematologic malignancies ( Barker JN et al, Blood Advances 2020). Now, our Center has investigated multiple refinements to further mitigate transplant-related mortality (TRM). Herein, we have analyzed transplant outcomes in these adult dCBT pts to establish an optimized dCBT platform. Methods : Consecutive adult first allograft pts with high-risk hematologic malignancies who underwent first allografts with intermediate intensity dCBT between 3/2018-8/2022 were analyzed. Optimizations involved further refined unit selection placing highest priority on unit quality (i.e. optimized banking practices) & CD34+ cell dose over HLA-match, ensuring therapeutic (CSA) levels (> 250 ng/mL) by day 0, prompt treatment of pre-engraftment syndrome (PES) & letermovir prophylaxis in CMV seropositive pts from day +7. Also, we have investigated 2 strategies to mitigate acute graft-versus-host disease (aGVHD): pts early in the study period received CSA, MMF & tocilizumab (8 mg/kg on day -1) prophylaxis ( Toci Group), whereas recent pts ( Recent Group) received CSA/ MMF with prompt therapy of clinically diagnosed aGVHD. Results : Of 74 patients (Table), 49 were in the Toci Group, & 25 in the Recent Group. Most pts were transplanted for acute leukemia (n = 55, 74%) & over half (n = 39, 53%) had non-European ancestry. Distribution of diagnoses, CMV serostatus, HCT-CI & graft characteristics were similar between the groups but Recent Group pts tended to be younger (38 vs. 47 years, p = 0.071). Toci Group pts had delayed neutrophil recovery [engraftment incidence 94% (95%CI: 87-99) at a median of 25 days (range 16-40), one graft failure], but low rates of PES (n = 19, 39%). The day-100 incidence of grade II-IV & III-IV aGVHD were 69% (95%CI: 56-82) & 10% (95%CI: 2-19), respectively. One pt (false negative CMV-serostatus) who did not receive letermovir had CMV infection. One-year TRM was 16% (95%CI: 6-27) with a 1-year relapse rate of 6% (95%CI: 3-13). TRM was due to infection (n = 3), aGVHD (n = 3, 1 with concurrent COVID-19 infection), multiorgan failure (n = 2) & graft failure (n = 1). With a median follow-up of 47 months (range 28-62), the 1-year OS was 84% (95%CI: 73-94) & 1-year PFS was 78% (95%CI: 66-89). By contrast, the engraftment incidence in Recent Group pts was 100% with a faster median neutrophil recovery of 19 days (range 11-36, p = 0.002) & all engrafted platelets at a median of 36 days (range 24-92). However, most Recent group pts developed PES [n = 20 (80%), p < 0.001] although this responded promptly to short course corticosteroids. Day 100 grade II-IV aGVHD was also higher [92% (95%CI: 81-99), p = 0.009], although grade III-IV aGVHD was low [8% (95%CI: <1-19)] & similar to tocilizumab pts. Day 100 incidence of CMV was 0%. With a median follow-up of 17 months (range 6-50), the 1-year TRM in Recent Group pts is 0% with a 1-year relapse incidence of 13% (95%CI: 7-27). Consequently, the 1-year OS & PFS are high at 96% (95%: 89-99) & 87% (95%CI: 74-99), respectively (Figure). Conclusions: Optimized dCBT with intermediate intensity conditioning, graft selection that prioritizes unit quality & CD34+ dose, CSA/ MMF prophylaxis with prompt therapy of PES & aGVHD, & letermovir CMV prophylaxis mitigates TRM in young & middle-aged adults. Thus, combined with relatively low relapse rates, the PFS & OS are high. Tocilizumab has been abandoned due to its notable association with delayed neutrophil engraftment & lack of survival advantage. Letermovir is highly effective completely preventing CMV infections & may contribute to the reduced aGVHD-related mortality recently (as compared to the pre-tocilizumab era), since avoidance of CMV antiviral therapy toxicities greatly facilitates aGVHD prevention & therapy. Given the rapid availability of cryopreserved unmanipulated CB grafts & frequent limitations of URD availability for minority populations, optimized dCBT is a highly attractive curative therapy for acute leukemias & other high-risk myeloid malignancies warranting prospective multi-center investigation.

Comparison of Non-Relapse Mortality after Haploidentical Hematopoietic Stem Cell Transplantation with Post-Transplant Cyclophosphamide Versus Single Umbilical Cord Blood Transplantation for Hematologic Disease

Introduction: Unrelated cord blood transplantation (UCBT) and haploidentical stem cell transplantation with post-transplant cyclophosphamide as graft-versus-host disease (GVHD) prophylaxis (PTCy-haplo) are alternative options for patients with hematologic disease lacking a human leukocyte antigen (HLA)-matched donor. Non-relapse mortality (NRM) after UCBT may vary depending on the cord blood CD34+ cell count, but no study has accounted for CD34+ cell count when comparing the outcomes after UCBT and PTCy-haplo. Therefore, we analyzed transplant outcomes after UCBT and PTCy-haplo in patients with hematologic disease from data of a nationwide registry that included cord blood CD34+ cell count. Methods: Patients with hematologic disease receiving first UCBT or PTCy-haplo between 2014 and 2020 were identified from the nationwide registration data of the Japanese Data for Hematopoietic Cell Transplantation. Acute GVHD-related mortality was defined as death caused primarily by GVHD or occurred secondary to GVHD, and infection-related mortality as death due to graft failure or infection but not acute GVHD. Patients treated by UCBT were stratified by the median CD34+ cell count for comparison (0.84 × 10 5/kg). Results: A total of 7,499 patients were enrolled and classified into 3 groups: PTCy-haplo (n = 1,142), UCBT with high CD34+ cell count (UCB-H; ≥ 0.84 × 10 5/kg, n = 3,185), and UCBT with low CD34+ cell count (UCB-L; < 0.84 × 10 5/kg, n = 3,172). Patients in the PTCy-haplo group were slightly younger than UCB-H and UCB-L patients (median age: 54 vs. 56 and 55 years). The PTCy-haplo group also included a greater proportion of males than the UCB-H group (62.4% vs. 50.0%) but not the UCB-L group (64.2%). A greater proportion of PTCy-haplo patients had a PS score of 0 (53.2% vs. 46.2% and 45.6%), a hematopoietic cell transplantation-specific comorbidity index of 0 (57.6% vs. 52.7% and 54.4%) and donor-specific anti-HLA antibody (2.7% vs. 1.0% and 1.6%), received reduced-intensity conditioning (58.0% vs. 40.5% and 41.6%), calcineurin inhibitor with mycophenolate mofetil (88.5% vs. 38.6% and 38.4%), and underwent transplantation in the later period (82.0% vs. 54.6% and 50.7%). There were no significant differences in disease-risk index between groups. At 2 years post-transplantation, NRM and overall survival (OS) were significantly better in the PTCy-haplo group than the UCB-H and UCB-L groups (NRM: 18.8% vs. 21.6% and 25.3%, P < 0.001; OS: 56.2% vs. 53.3% vs. 49.6%, P < 0.001). Neutrophil engraftment rate at 42 days was also significantly higher in the PTCy-haplo group than the UCB-H and UCB-L groups (93.7% vs. 87.5% and 82.9%, P < 0.001). The PTCy-haplo group less frequently developed grade II-IV (26.4% vs. 37.3% and 34.4%, P < 0.001) and III-IV acute GVHD (7.0% vs. 12.2% and 10.5%, P < 0.001). While neither the differences in NRM nor that in OS remained significant between PTCy-haplo and UCB-H groups in multivariable analysis (NRM: hazard ratio [HR], 1.18, 95% confidence interval [CI], 0.99-1.41, P = 0.073; OS: HR, 1.05, 95% CI, 0.94-1.17, P = 0.42), significant differences were maintained between PTCy-haplo and UCB-L groups (NRM: HR, 1.35, 95% CI, 1.13-1.61, P = 0.001; OS: HR, 1.12, 95% CI, 1.00-1.26, P = 0.042). Regarding the cause-specific mortality, infection-related mortality was also significantly lower in the PTCy-haplo group compared to UCB-H and UCB-L groups (8.1% vs. 11.1% and 13.6%, P < 0.001) while GVHD-related mortality did not differ among groups (5.0% vs. 5.2% and 4.9%, P = 0.69). Conclusion: UCBT-H resulted in similar NRM and OS as patients receiving PTCy-haplo, whereas outcomes were worse in UCBT-L. These findings indicate that high CD34+ cell count in cord blood is a critical criterion for alternative donor selection.

Updated Findings of a Phase II Study of Ruxolitinib Pre-, during- and Post-Hematopoietic Stem Cell Transplantation for Patients with Primary or Secondary Myelofibrosis

Background: Despite tremendous progress in the treatment of myelofibrosis in the last decade, the only curative therapy remains allogeneic hematopoietic cell transplantation (HCT). There are now three FDA-approved JAK inhibitors (JAKi) to treat symptomatic splenomegaly and constitutional symptoms in myelofibrosis, although these agents do not prevent disease progression. Discontinuation of JAKi before (pre) HCT is challenging as patients can experience return of symptoms. Thus, ruxolitinib is often continued during HCT in an off-label fashion; however, little is known about the safety and efficacy of this approach. Furthermore, there is increasing use of ruxolitinib after (post) HCT, following its approval in treating refractory graft-versus-host disease (GVHD). Methods: We previously reported the interim results of a phase II, multicenter, investigator-initiated trial investigating ruxolitinib given pre-, during- and for 1-year post-HCT for patients with primary or secondary MF (NCT03427866). Here we present the results of the study with full accrual of 43 patients. The primary endpoint was 1-year GVHD-free, relapse-free survival (GRFS). Secondary endpoints included overall (OS) and progression free survival (PFS), engraftment, and incidence of acute and chronic GVHD. Patients were treated with reduced intensity conditioning with fludarabine (30mg/m 2/d x 5 days) and melphalan (100 or 140mg/m 2 x 1). Patients received peripheral blood stem cell grafts from either 7/8 or 8/8 HLA-matched donors. GVHD prophylaxis consisted of standard tacrolimus and methotrexate. Results: 44 patients with myelofibrosis were enrolled and 43 underwent HCT between 9/2018 and 5/2023. Median age was 66 (range 46-75), 37% were female (n=16), and most patients had DIPSS intermediate-2/high risk (n=36), 60% (n=27) were receiving ruxolitinib prior to enrolling on the study. Most (n=35) received 8/8 matched unrelated transplants and had measurable splenomegaly by either ultrasound or CT (n=40) prior to HCT. At baseline, 25 patients had JAK2 mutation, 8 had CALR and 5 had MPL mutations, 13 patients had ASXL1 mutations (Figure 1). No differences in outcomes were observed by pre-HCT mutational status. At day 100, all but one patient had undetectable mutations by next generation sequencing. The most common grade 3/4 hematologic adverse events (AE) were anemia (n=12), thrombocytopenia (14), neutropenia (n=11). Non-hematologic grade 3/4 events included hypertriglyceridemia (n=3). Median time to neutrophil engraftment was 15 days (range 4-38), one patient did not engraft. Median time to platelet (>20x10 9) engraftment was 25 days (range 11-145). At day 30, 2 patients had a donor-cell chimerism below 90% (one ultimately relapsed) and at day 100, 2 patients had chimerism below 90 (one ultimately relapsed). With a median follow up among survivors of 13 months (range 0.7 - 26), 1-year GRFS was 74%. OS, PFS and cumulative incidence of NRM and disease relapse were, 86%, 79%, 10%, and 10%, respectively (Figure 2). The cumulative incidence of grade III-IV acute GVHD at 6 months was 2.4%. Only one patient had grade IV acute GVHD. One year moderate/severe chronic GVHD was 11%In univariable analysis, thrombocytopenia of <40 x 10 9/L pre-HCT was associated with worse OS (1-yr OS: 64% vs 92%, p=0.008). Splenomegaly (defined as >17 cm) was associated with worse PFS (1-yr PFS: 100% vs 64%, p=0.046). There was an improvement in OS in patients that received ruxolitinib and achieved response pre-HCT compared to those that did not but this did not achieve statistical significance (1-yr OS 100% vs 74%, p=0.054). Discussion: This study is the first, multicenter trial to test ruxolitinib use pre-, during- and post-HCT in patients with myelofibrosis. Our results demonstrate safety and feasibility of this approach. Favorable rates of engraftment were observed with ongoing ruxolitinib administration even through hematopoietic nadir. We continue to demonstrate favorable outcomes of PFS, OS and GRFS with a low incidence of severe acute and chronic GVHD. Our results strongly support the use of ruxolitinib in the peri- and post-transplant setting for patients with myelofibrosis undergoing HCT.

Outcomes of Attenuated Dose Fludarabine and Melphalan Based Conditioning Regimen in Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplant for Acute Leukemia and Myelodysplastic Syndrome

Introduction: Fludarabine combined with melphalan 140 mg/m2 (FluMel140) is a commonly used reduced-intensity conditioning (RIC) regimen in patients undergoing matched related (MRD) and unrelated donor (MUD) allogeneic stem cell transplant (alloSCT). Ciurea et al Blood 2020, described outcomes of Flu 160 mg/m2 and Mel 100 mg/m2. In this study, we describe our experience of attenuated melphalan dosing 100-120 mg/m2 versus 140 mg/m2 with fludarabine 125 mg/m2 (attenuated FluMel versus regular dose FluMel). Methods: We retrospectively reviewed patients from November 2016 to April 2023 with AML, ALL or MDS undergoing alloSCT who received fludarabine and melphalan. Patients who received the lower dose of melphalan were considered to be the attenuated FluMel cohort, whereas patients receiving the melphalan 140 mg/m2 were considered to be the control cohort. Kaplan-Meier and log-rank tests were used to estimate OS. NRM and relapse incidence (RI) was calculated using competing risk analysis. R 4.2.0 (R Foundation for Statistical Computing) was used for statistical analyses. Results: A total of 134 patients [92 (68.7%) males] were evaluated. Thirty-seven (27.6%) patients received attenuated FluMel and 97 (72.4%) patients received regular dose FluMel (controls). Among the patients in the attenuated FluMel cohort, 5 (13.5%) patients also received fludarabine at doses less than 125 mg/m 2 [median 20 mg/m 2 (range 87.5-112.5 mg/m 2]. Median age at transplant was 65 years (range 30 - 76 years). The most common disease was AML [75 (56.0%) patients] ( Table 1). Patients in the attenuated FluMel group had comparable iothalamate clearance compared to control cohort (clearance <65 ml/min/1.73 m2: 18.9% vs. 12.4%, P = 0.52). Patients in the attenuated FluMel group had comparable rates of high/very high DRI to those in control cohort (51.4% vs. 43.3%, P = 0.91). Compared to the control cohort, patients in the attenuated FluMel group were less likely to have a matched related donor (21.6% vs. 45.4%, P = 0.02) and were more likely to receive PTCy for GVHD prophylaxis (37.8% vs. 16.5%, P = 0.02). Among the patients receiving attenuated FluMel, the median melphalan dose administered was 100 mg/m2. The median time to neutrophil engraftment was 19 days (IQR 16-21 days), and median time to platelet engraftment was 18 days (range 12-25 days). Eleven (29.7%) patients developed post-alloSCT mucositis, and 3 (8.1%) patients developed grade 3-4 mucositis. All the patients in the attenuated FluMel group achieved 100% CD33 donor chimerism at day +30 (n=35) and day +100 (n=28) after alloSCT. Similarly, 26 (74.3%, n = 35) patients achieved ≥ 95% CD3 donor chimerism at day +30 after alloSCT and 23 (82.1%, n = 28) patients achieved ≥ 95% CD3 donor chimerism at day +100 after alloSCT. Median follow-up of the entire cohort after alloSCT was 2.3 years (95% CI 2.1-2.5 years). Median follow-up was 1.5 years in the attenuated FluMel cohort and 2.5 years for the control cohort ( P<0.001) . Survival was similar among patients receiving attenuated FluMel vs. control cohort (2-year OS 74.7% vs. 65.0%, P = 0.28). At 2 years post-alloSCT, the attenuated FluMel and control cohort had similar non-relapse mortality (19.9% vs. 24.4%, P=0.58) and relapse (22% vs. 15.2%, P=0.51). The attenuated FluMel was not associated with an increased incidence of relapse (HR 1.36, 95% CI 0.50-3.73, P=0.55) or NRM (HR 0.74, 95% CI 0.28-1.95, P = 0.55). GVHD-free relapse free (GRFS) survival was also similar among the attenuated Flu/Mel and control cohort (1-year GRFS rate 41.1% vs. 45.6%, P = 0.8). Among the patients receiving PTCy for GVHD prophylaxis, the 2-year OS in patients receiving attenuated FluMel was higher than the control group, but it was not statistically significant (2-year OS rate: 90% vs. 55%, P=0.37, Figure 1). Compared to the control group, the attenuated FluMel group had similar rates of NRM at 100 days (10% vs. 12.5%), 1 year (NA vs. 25%) and at 2 years after alloSCT (NA vs. 25%, P=0.54). GRFS at 1 year was also similar between the attenuated FluMel and control group (68.6% vs. 43.8%, P = 0.69). Conclusion: The attenuated dosed FluMel conditioning is a safe and effective regimen for elderly patients with acute leukemia and MDS, and is non-inferior to regular dose FluMel. Among patients receiving PTCy for GVHD prophylaxis, the lower dose of melphalan was non-inferior to regular dose FluMel for survival outcomes. Larger studies are needed to confirm our findings.

Treosulfan-Based Conditioning Prior to Allogeneic Hematopoietic Stem Cell Transplantation (alloHSCT) for Patients with Myelodysplastic Syndrome (MDS): Comparative Analysis of a Randomized Controlled Trial and Real-World Data

Introduction MDS is a clonal myeloid disorder manifested by diverse genotypes and phenotypes, characterized by ineffective hematopoiesis. AlloHSCT is the only potential curative therapy for MDS. Myeloablative conditioning regimens are integral part of alloHSCT that allow engraftment and provide a therapeutic effect. Treosulfan has myeloablative and immunosuppressive properties and provides less transplantation related mortality than some of the traditionally used myeloablative conditioning regimens like busulfan-based treatments. In our analysis, we compared the results from the MDS subgroup of the randomized controlled trial (RCT) MC-FludT.14/L (NCT00822393) with real-world data (RWD) of patients treated at the University Hospital Münster and the University Hospital Dresden. Methods MDS patients conditioned with treosulfan (30 g/m²) and fludarabine in the RCT (June 2013 to January 2018) and the data obtained from 2 hospitals (RWD) (August 2017 to February 2023) were compared. Baseline characteristics, overall survival, relapse-free survival, relapse (or progression), non-relapse mortality, time to engraftment, and safety profiles between the RCT and RWD were compared. A sensitivity analysis was conducted using propensity score matching (PSM). Additionally, subgroup analyses evaluated the clinical effectiveness of treosulfan by baseline morphologic blast count and the revised International Prognostic Scoring System (IPSS-R). Results Overall, 195 MDS patients (RCT 84, RWD 111) with at least 12 months follow-up were analyzed. Median age was 62 years (range 39-76 years). Baseline characteristics only differed between the RCT and RWD with respect to age, IPSS-R categories, Hematopoietic Cell Transplantation Co-Morbidity Index total score (HCT-CI), and neutrophil count, indicating some heterogeneity between the RCT and RWD patients. In the PSM analysis, 106 (RCT 53, RWD 53) patients were matched to address the heterogeneity. The Kaplan-Meier estimates for overall survival (OS) at 2 years were 73% (95% CI 62-81%) and 72% (95% CI 62-81%) for the RCT and RWD (Figure 1), and for relapse-free survival (RFS) at 2 years were 68% (95% CI 57-77%) and 67% (95% CI 56-76%), respectively. Cumulative incidence of relapse at 2 years was 11% (95% CI 4-18%) and 15% (95% CI 7-23%), and for non-relapse mortality (NRM) was 21% (95% CI 12-30%) and 18% (95% CI 10-26%) for the RCT and RWD, respectively. In addition, cumulative incidence of neutrophil engraftment at 28 days was 93% (95% CI 87-98%) and 94% (95% CI 89-98%) for the RCT and RWD, while the median engraftment time was later in the RCT (19 days vs 15 days). Cumulative incidence of platelet engraftment at 28 days was 89% (95% CI 83-96%) and 93% (95% CI 88-98%) for the RCT and RWD, with the same median time 14 days. Further, the PSM sensitivity analysis showed comparable efficacy between the RCT and RWD. Interestingly, bone marrow blast count prior to conditioning and IPSS-R subgroups only had minor, statistically not significant impact on survival endpoints (Table 1). The number of deaths were 27 (32%) and 28 (25%) in the RCT and RWD, respectively. The most frequent cause of death was transplantation related (20% vs 14%). One patient experienced primary graft failure in the RCT and none in the RWD. More patients had acute GVHD with at least grade 2 in the RCT (27%) than in RWD (18%). Conclusions The efficacy and safety of treosulfan-based conditioning therapy prior to alloHSCT in post-authorization real world setting was comparable with the results obtained from the RCT. Our data suggest that bone marrow blast count is no strong predictor for survival after alloHSCT. Therefore, we conclude that the approved treosulfan-based conditioning therapy for patients with MDS, including those with increased blasts, has promising outcomes after alloHSCT.

Risk Factors and Prognostic Model for II-IV aGVHD in Patients with Engraftment Syndrome after Haploidentical Hematopoietic Stem Cell Transplantation

Background Engraftment syndrome (ES), characterized by noninfective fever, rash, noncardiogenic pulmonary edema, hepatic dysfunction and weight gain, is a common complication after haplo-HSCT. Although ES has been reported in the previous literature, there is still no consensus on the risk factors, outcomes and treatment of ES, as there is not only a lack of uniformity described by authors but also conflicting evidence between studies. One meta-analysis found that patients with ES have significantly higher odds of developing aGVHD than patients without ES. However, a strategy for identifying high-risk patients with ES for aGVHD remains unclear although important for early aGVHD prevention and the improvement of patient outcomes. Here, we retrospectively analyzed a large cohort of posthaplo-HSCT ES patients and described the risk factors, outcomes and prognostic model for II-IV aGVHD in patients with ES. Methods A total of 384 patients with ES after haplo-HSCT at Peking University People's Hospital between 2013 and 2022 were retrospectively identified. We first performed a nested case‒control study to identify the risk factors for ES, and three controls were randomly selected for each case according to the time of transplant and the length of follow-up. The cohort of ES patients was further divided into a derivation cohort of 258 patients and a validation cohort of 126 patients. We used a logistic regression model to perform univariate and multivariate analyses to determine the risk factors and prognostic factors for II-IV aGVHD in patients with ES after haplo-HSCT. The prognostic model performance was assessed by evaluating the discrimination [area under the curve (AUC)], calibration (calibration plot), and net benefit [decision curve analysis (DCA)]. Results In 384 patients diagnosed with ES after haplo-HSCT, the median diagnostic time of ES was 11 days after haplo-HSCT, close to the time of neutrophil engraftment. Among them, 252 (65.6%) patients developed aGVHD, and 123 (32.0%) suffered II-IV aGVHD. The 3-year survival rate of ES patients who developed II-IV aGVHD was 17.1% (21/123) compared with 4.3% (11/252) in those without II-IV aGVHD (p<.001).Through multivariable logistic binary regression models, six prognosis risk factors were selected，including age at HSCT≤25 years(p=0.008; odds ratio [OR], 4.47; 95% confidence interval [CI], 1.54-14.30), obesity (BMI≥28) at HSCT(p=0.039; OR, 12.60; 95% CI, 1.30-194.14), previous pregnancy history (p=0.09; OR, 3.42; 95% CI, 0.83-18.45), time from diagnosis to transplantation≥6 months (p=0.027; OR, 2.73; 95% CI, 0.97-8.50), a high dose of MNC administration (≥9×10 8)(p=0.003; OR, 4.39; 95% CI, 1.70-12.41) and a high dose platelet transfusion (≥2u) during conditioning regimen (p=0.007; OR, 4.04; 95% CI, 1.51-11.73). According to the regression coefficient, 2 scores were assigned for obesity at HSCT, and 1 score was assigned for the other risk factors. Hence, a prognostic model for II-IV aGVHD termed TOPMAP ( time from diagnosis to transplantation, obesity, previous pregnancy history, high-dose MNC administration, age, and high-dose platelet transfusion) was constructed. Patients were stratified into 3 risk groups: low risk (0-2), intermediate risk (3-4), and high risk (5-7), which showed an incidence of II-IV aGVHD of 15.9% for low risk, 39.2% for intermediate risk, and 83.3% for high risk (P<0.001). The AUCs of the prognostic scoring model were 0.816 (95% CI 0.727-0.903) and 0.813 (0.759-0.868) for the internal validation and validation cohorts, respectively. The calibration plots showed a high agreement between the predicted and observed outcomes. Decision curve analysis indicated that patients with engraftment syndrome could benefit from the clinical application of the prognostic model. Conclusion Here, we reported the largest study on ES posthaplo-HSCT and found risk factors for ES onset. We developed the first straightforward scoring model (designated TOPMAP) that incorporates clinical and laboratory risk factors to predict the high-risk group for II-IV aGVHD in patients with ES after haplo-HSCT. This model can effectively help clinicians identify ES patients early, sectionalize them according to risk for II-IV aGVHD, carry out aGVHD prevention and improve patient outcomes.

A Modified Busulfan/Cyclophosphamide Preconditioning Regimen Incorporating Selinexor for Allogeneic Hematopoietic Stem Cell Transplantation in Relapsed/Refractory Hematologic Malignancies

Objective: To investigate the safety and efficacy of a modified busulfan (Bu)/cyclophosphamide (Cy) conditioning regimen incorporating Selinexor for allogeneic hematopoietic stem cell transplantation in relapsed/refractory hematologic malignancies. Methods: We retrospectively evaluated the clinical outcomes of patients with hematologic malignancies who had received allo-HSCT with conditioning incorporating Selinexor at the Institute of Hematology and Blood Disease Hospital. Results: Between September 2022 and June 2023, 13 adult patients with high-risk hematologic malignancies were enrolled including 6 males and 7 females with a median age of 49 (13-58) years. There were 7 patients with acute myeloid leukemia (AML), 4 with myelodysplastic syndromes (MDS)/AML, 1 with multiple myeloma (MM) and 1 with extramedullary plasmacytoma. Six patients were in complete remission (CR), four in CR1, two in ≥CR2, five in non-remission (NR), and two patients were in a progressive disease status (PD) prior to conditioning. Three patients received matched-sibling donors transplant, while ten received related haploid hematopoietic stem cell transplantation. All patients had received myeloablative conditioning regimen incorporating Selinexor. Eleven patients received the conditioning: Bu (3.2mg/kg/d*2d); Melphalan (Mel, 60mg/m 2/d*2d), Fludarabine(Flu 30mg/m 2/d*3d) or CLA (5mg/m 2/d*3d), Cy (40mg/kg/d*2d), Selinexor 60mg/d*2d. The other 2 patients received the conditioning: Decitabine (DAC, 20mg/m 2/d*5d), Bu (3.2mg/kg/d*3d), Cy (40mg/kg/d *2d), Flu 30mg/m2/d*3d, Selinexor 60mg/d*2d. The median amount of infused mononuclear cell and CD34 + stem cell were 11.72×10 8/kg(range, 9.66 to 14) and 2.92×10 6/kg(range, 2.35 to 9.61), respectively. All the 13 patients achieved neutrophil engraftment with a median time of 12 (range, 9 to 18) days and 9 patients achieved platelet engraftment with a median time of 13 (range, 11 to 30) days. Four patients were still experiencing prolonged isolated thrombocytopenia. All patients achieved complete donor chimerism and minimal residual disease (MRD) negative at 30 days post-transplantation.The cumulative incidence of cytomegalovirus (CMV) reactivation was 15.4%, while Epstein-Barr virus (EBV) reactivation was not observed. Six patients (46.2%) experienced mucosal barrier injury, comprising three cases of oral mucosal ulcer, one case of digestive tract hemorrhage, and two cases of hemorrhagic cystitis. The cumulative incidence of II-IV and III-IV acute GVHD was 23.1% and 15.4%. The median follow-up for all the participants was 93 (40-316) days after transplantation. Only one patient was dead due to Thrombotic Microangiopathy (TMA) 78 days after transplant. One patient experienced molecular relapse 156 days after transplant, and another patient experienced hematology relapse 20 days after transplant. The two relapsed patients had received donor lymphocyte infusions after chemotherapy. The other 10 patients were still in CR with complete donor chimerism and MRD negative. The cumulative transplant-related mortality (TRM) and relapse rates are 7.7% (1 case) and 15.4% (2 cases), respectively. Conclusions: Incorporating Selinexor into the modified BU/CY conditioning regimen for allogeneic HSCT in patients with relapsed/refractory hematologic malignancies demonstrated a favorable safety profile. Ongoing prospective trials will provide valuable insights into the potential benefits of Selinexor in patients undergoing HSCT.