Antithymocyte globulin (ATG) prevents graft-versus-host disease (GVHD) in allogeneic hematopoietic cell transplantation (HCT). However, variable ATG exposure impacts transplant outcomes. We aimed to develop a population pharmacokinetic (popPK) model for ATG in myeloablative HCT and evaluate the relationship between area under the time-concentration curve (AUC) and mortality to identify the optimal ATG AUC. We studied 200 adult HCT recipients who received myeloablative conditioning (MAC) and a peripheral blood stem cell graft from 7/8 or 8/8 HLA-matched related or unrelated donors. ATG was given on days -2, -1, and 0. All patients received additional GVHD prophylaxis with methotrexate and cyclosporine. Serum concentration of lymphocyte-binding ATG was determined by flow cytometry in 2,140 samples. For the popPK modeling, the cohort was split into a model development cohort (n = 134) and a validation cohort (n = 66). The modeling was performed using Monolix Suite 2024R1. The relationship between model-estimated AUCs and mortality was evaluated in all 200 patients (combined development and validation subcohorts) using a Cox proportional hazards model. The relationship between model-estimated AUCs and cause-specific outcomes (eg, relapse or acute GVHD [aGVHD]) was evaluated using a competing risk analysis. A two-compartment model with parallel linear and target-mediated elimination best described ATG disposition. Population means, and residual standard errors (RSE%) were 11.78 L (2.05%) for the central volume of distribution (V1), 0.20 L/h (4.36%) for clearance (CL), and 2.20 U/L (7.08%) for the initial ATG-binding capacity of lymphocytes in the central compartment. Lean body weight (LBW) positively correlated with V1, CL, and intercompartmental clearance, while pre-ATG absolute lymphocyte count (ALC) positively correlated with R0_initial. Internal and external validation (using the development and validation subcohorts, respectively) confirmed model stability and robustness. The optimal ATG AUC range was 30 to 45 U·day/L. In multivariate analysis, patients whose AUC was within this range had lower mortality than those whose AUC was outside this range (hazard ratio = 0.46, P = .03). The low mortality of patients with AUC within the range of 30 to 45 U·day/L appeared to be due to both low incidence of grade III to IV aGVHD, which was high in patients with AUC <30 U·day/L, and low incidence of relapse, which was high in patients with AUC >45 U·day/L. This novel model described the pharmacokinetics of ATG in adult HCT recipients following MAC. The model identified LBW and ALC as significant covariates for ATG disposition. Furthermore, we identified the optimal ATG AUC (associated with the lowest mortality). These findings provide the foundation for developing an individualized dosing strategy aimed at improving post-HCT survival.

Objective Autologous hematopoietic cell transplantation (HCT) is an effective treatment for a subset of patients with systemic sclerosis (SSc). Unfortunately, relapse is a significant problem, with no available tests to predict relapse. We studied whether relapse is associated with pre- or post-HCT serum levels of SSc-related autoantibodies. Methods The cohort comprised 38 consecutive evaluable patients with SSc who underwent HCT at a single center and were followed for a median of 33 months. Sixteen patients (42%) relapsed at a median of 14 months post-HCT. Autoantibody levels were determined by immunoassays. Results Regarding pre-HCT autoantibodies, in univariate analyses, the cumulative incidence of relapse (CIR) was lower in anti-RNA polymerase III (ARA)-positive than ARA-negative patients (hazard ratio [HR] 0.21, P = 0.04). Conversely, the CIR was higher among patients with positive anti-Ro52, although this difference was not statistically significant (HR 2.90, P = 0.053). The CIR was similar in patients positive and negative for antitopoisomerase antibody (ATA; ie, Scl-70) or antinuclear antibody (ANA). In bivariate analyses that included older age as a risk factor for relapse, pre-HCT ARA was still associated with relapse (HR 0.21, P = 0.04). This was not the case for Ro52 (HR 2.21, P = 0.16). Regarding post-HCT autoantibody level trajectory, there was no significant difference between patients with vs without relapse. Conclusion Positive ARA pre-HCT is associated with reduced relapse risk, and post-HCT autoantibodies do not appear to be associated with relapse risk.

Objectives Diffuse cutaneous systemic sclerosis (dcSSc) is a life-limiting inflammatory disease characterized by progressive fibrosis, vasculopathy and immune dysfunction. Fibroblasts (FB) are the key drivers of fibrosis and adapt epigenetic changes that promote their activation, resistance to apoptosis, and release of pro-fibrotic mediators. Autologous stem cell transplantation (ASCT) is a disease-modifying therapy that improves fibrosis in some dcSSc patients - although its effects on restoring FB function(s) are unknown. We recently identified the cancer-associated glycan, polysialic acid (polySia), in skin sections from patients with dcSSc and polySia levels correlated with fibrosis which normalized post-ASCT.[1] We also showed that dermal sections from patients with dcSSc had increased genomic instability, double-stranded DNA breaks (DSB) and epigenetic activation of the transcription factor FOXO1. We hypothesized that FOXO1 may promote polySia expression to enhance fibrosis, and that targeting polySia may provide anti-fibrotic effects. Methods We measured polySia levels in sera from 22 patients with SSc using a specific polySia ELISA and correlated polySia levels with the severity of skin fibrosis using the modified Rodnan skin score (mRSS). We also used primary dermal FB from skin biopsies of healthy controls (HC, age/sex matched), less severe limited cutaneous SSc (lcSSc), dcSSc and post-ASCT patients (N=4-6/per group) and measured the frequency of DSBs, active (nuclear) FOXO1, and polySia levels using immunofluorescence/confocal microscopy and/or immunoblot (IB). We also quantified polySia, ST8SIA2 levels or pro-fibrotic markers (fibronectin and CTGF) using qRT-PCR and/or IB following FOXO1 pharmacological inhibition, ST8SIA2 knockdown via siRNA, or treatment with a polySia elongation inhibitor, 8-keto-Neu5Ac. Results Total polySia levels correlated with mRSS (p=0.007, rho=0.560) in SSc patients – highlighting its direct link with fibrosis in SSc. We also observed that FB from dcSSc had a 2-3-fold induction in polySia levels and ST8SIA2 expression compared to FB from lcSSc and age/sex matched HC (p=0.02). Importantly, post-ASCT FB had a substantial reduction in ST8SIA2 (p=0.04) and polySia levels. This was associated with increased DSBs and FOXO1 activation exclusively in dcSSc FB. Finally, FOXO1 inhibition, ST8SIA2 siRNA knockdown, or treatment with 8-keto-Neu5Ac resulted in a 1.9-fold reduction in pro-fibrotic markers and total polySia levels (Figure 1). Figure 1: A. Graphical abstract of proposed mechanism. In dcSSc fibroblasts, double-stranded DNA breaks activate the transcription factor FOXO1. Upon activation, FOXO1 upregulates the gene expression of the polySia synthetic enzyme ST8SIA2 , polySia, and fibrotic mediators such as CTGF and fibronectin, thereby promoting fibrotic remodelling and fibrosis in dcSSc. B. Confocal images showing polySia (green), cis-golgi marker GM130 (red), nucleus (DAPI, blue) in fibroblasts from HC, dcSSc and post-ASCT. C. Spearman correlation analysis comparing serum polySia to mRSS in SSc patients (n = 22). Conclusion Our study identifies a novel DSB/FOXO1/polySia pathway as a key driver of fibrosis in SSc. This axis may serve as a biomarker for disease progression and may be indicative of restoration of FB functions post-ASCT. We postulate that targeting the FOXO1/polySia pathway may provide a novel therapeutic approach in dcSSc. References [1.] Khan L. J Autoimmun 2023;140:103110.

Objectives Diffuse cutaneous Systemic Sclerosis (dcSSc) is a life-limiting autoimmune disease with minimal treatment options. Autologous hematopoietic stem cell transplantation (ASCT) is a potent disease-modifying therapy in dcSSc; however, its effects on fibroblasts are unknown. We recently showed that dermal fibroblasts (DFs in patients with dcSSc develop a cancer-like phenotype characterized by genomic instability and increased double-stranded DNA breaks (DSBs).[1] However, little is known about the mechanisms promoting DF survival following the accumulation of genomic mutations. We hypothesized that in dcSSc DF genomic instability results in the accumulation of genomic mutations; and that this results in the activation of Protein Kinase R-like ER Kinase (PERK) and transcription of forkhead box1 (FOXO1) promoting resistance-to-apoptosis and fibrosis (Figure 1). Methods We used whole exome sequencing (WES) to characterize the mutational frequencies and their associated signatures in dcSSc patients who did not undergo ASCT (dcSSc, N=35) and those who did (post-ASCT, N=9). We also generated DFs from dcSSc, post-AHSCT (or age/sex matched healthy controls (HC), (N=8-10 patients/group), and quantified the frequency of DSBs via γ-H2AX levels (immunoblot (IB)), and DSB nuclear foci (confocal microscopy). We measured the relative ROS levels, mitochondrial membrane potential, and phospho-PERK (active) in DFs to mechanistically link DSB with PERK activation using flow cytometry and/or IB, respectively. We also determined the downstream effects of PERK activation on mitochondria (eg, mitochondrial dynamics and biogenesis). Then, we measured FOXO1 activation via nuclear translocation, IB, and expression of its downstream mRNA target SOD2. Finally, mitochondrial-dependent resistance-to-apoptosis was determined at baseline, and following treatment with cyclophosphamide, a PERK or a FOXO1-inhibitor using TUNEL and cleaved caspase 9/3 levels (IB). Results dcSSc patients’ DFs had increased genomic instability and DSBs compared to patients treated with ASCT. dcSSc DFs had increased indicators associated with PERK activation (phospho-PERK, ROS, and mitochondrial membrane potential). This was associated with increased mitochondrial remodeling, mitochondrial biogenesis, and mitochondrial fusion. Importantly, FOXO1 was exclusively activated in dcSSc, but not HC or post-ASCT DFs. Inhibition of PERK or FOXO1 resulted in increased mitochondrial-dependent apoptosis. Conclusion Our study highlights a novel mechanism whereby genotoxic signals in dcSSc promote cell survival via a PERK/FOXO1-dependent axis and associated metabolic remodeling (Figure 1). It also provides mechanistic insights related to how changes to the mutational landscape reduce pro-fibrotic signals in DF after ASCT. Future studies targeting this dysregulated pathway may provide an additional rationale for exploring it therapeutically in patients with dcSSc. References [1.] Gniadecki R. J Autoimmun 2022;131:102847.

Diffuse cutaneous systemic sclerosis (dcSSc) is a life-limiting fibrotic disease. We and others have shown that dcSSc fibroblasts accumulate numerous somatic mutations associated with senescence-like features; however, the mechanism(s) enabling their survival remain unclear. Skin biopsies were obtained from lesional tissues from dcSSc (n=10), dcSSc treated with autologous hematopoietic stem cell transplantation (ASCT, n=8) or 7 age/sex-matched healthy controls. Primary dermal fibroblasts were generated from biopsies. Spatial RNA sequencing, immunoblotting, confocal microscopy, and functional assays were used to mechanistically delineate signaling pathways linking DNA-damage with fibroblast survival. dcSSc fibroblasts demonstrated increased pH2AX DNA double-strand-break foci yet remained apoptosis resistant. These cells displayed features of metabolic-stress remodeling, including mitochondrial hyperpolarization, increased reactive oxygen species production, and enhanced mitochondrial biogenesis. Spatial transcriptomics and subsequent biochemical analyses identified activation of a PERK/ATF4/FOXO1 axis, characterized by PERK phosphorylation, selective ATF4 translation, FOXO1 nuclear translocation, and induction of downstream antioxidant and metabolic programs. In contrast, fibroblasts from post-ASCT patients exhibited normalization of DNA-damage markers and mitochondrial parameters without ATF4/FOXO1 activation. Pharmacologic inhibition of either PERK or FOXO1 selectively restored mitochondrial-dependent apoptosis in dcSSc fibroblasts, demonstrating that this axis is required for their survival following extensive genomic injury. dcSSc fibroblasts persist despite substantial genomic injury by engaging a PERK/ATF4/FOXO1 metabolic-adaptation program that suppresses mitochondrial-dependent apoptosis. This survival axis is not present after ASCT. Targeting PERK or FOXO1 restores apoptosis selectively in dcSSc fibroblasts, highlighting its potential use as a therapeutic target for eliminating pathogenic senescence-like fibroblasts in dcSSc. Both ex-vivo skin and in-vitro primary dermal fibroblasts derived from dcSSc patients have a higher frequency of intrinsic DNA damage signals and senescence-associated features; yet they evade mitochondrial-dependent apoptosis. Pathogenic dcSSc fibroblasts rewire their metabolism, characterized by mitochondrial hyperpolarization and elevated ROS. Spatial transcriptomics and functional analyses reveal a PERK/ATF4/FOXO1 stress-adaptation axis that drives fibroblast survival in dcSSc. This maladaptive survival program characterized by increased genotoxic stress, and mitochondrial remodelling is absent in post-ASCT fibroblasts. Targeting PERK or FOXO1 selectively sensitizes dcSSc fibroblasts to apoptosis revealing a potential promising therapeutic strategy in dcSSc.

Background: Cytomegalovirus (CMV) reactivation is a major complication after hematopoietic stem cell transplantation (HSCT). Natural killer (NK) cells help control CMV through killer-cell immunoglobulin-like receptors (KIRs) and their HLA ligands, but donor-derived CMV-specific T-cells may confound the interpretation of NK-mediated effects. Methods: We analyzed 276 HLA-matched (10/10) adults receiving ATG-based T-cell-depleted myeloablative HSCT with a known donor and recipient CMV serostatus. The donor and recipient KIR genotypes were scored by the Cooley B-content score (0-4; ≥2 = high). Clinically significant CMV reactivation (plasma viral load > 25,000 IU/mL, the institutional threshold for pre-emptive therapy) was analyzed with Fine-Gray competing-risks regression, stratified by the donor-recipient serostatus. Results: In seronegative-donor/seropositive-recipient (D-R+) pairs (n = 68), a high donor KIR B-content score was associated with a significantly lower reactivation risk (sub-hazard ratio, 0.46; 95% CI, 0.24-0.91; p = 0.024). No effect was seen in D+R+ pairs (n = 82; SHR, 0.65; p = 0.241); D+R- (n = 28) had too few events to model. A donor Tel-AA/recipient Tel-B+ mismatch was independently associated with a higher reactivation risk (adjusted HR, 2.41; 95% CI, 1.33-4.37; p = 0.004). The overall survival was unaffected in either stratum. Conclusions: A high donor KIR B-content score protects against CMV reactivation in D-R+, but not D+R+, HSCT recipients, consistent with NK dominance when CMV-specific donor T-cells are sparse. A specific donor-recipient telomeric mismatch independently modifies the risk. Donor KIR profiling warrants prospective evaluation in donor-selection algorithms.

In a randomized pilot trial we compared ATG (standard Arm A, 4.5 mg/kg) with ATG plus PTCy (experimental Arm BE, ATG 4.5 mg/kg, PTCy 100 mg/kg). The primary safety endpoint was overall survival at 100 days. We analyzed 79 patients with AML (n=55) or MDS (n=24). Median age (range) was 59 (19 to 74), 47 (59.5 %) were male. Conditioning was either myeloablative (n=49) or reduced intensity (n=30). Donors were 8/8 HLA-matched. Overall survival at 100 days was 95.0% (37 of 39 patients) vs 94.9% (38 of 40 patients) in arm A vs arm BE (p>0.9). In arms A versus BE, serious adverse events (SAEs) occurred in 26 (65.0%) and 25 (64.1%) patients, graft failure occurred in 3 and 1 patients (p>0.9), median (range) days to neutrophil engraftment were 19 (10-45) versus 22 (15-56) days p=0.007), cytomegalovirus and Epstein-Barr virus reactivations occurred in 11 and 7 patients (p=0.45) and in 6 and 5 patients (p=0.29). Overall survival at 12-months was 72.5% versus 75.8% (p-0.79), cumulative incidence at 6 months of relapse was 15.0% versus 15.7% (p=0.88) and of non-relapse mortality (NRM) 7.5% versus 8.0 (p=0.45). The combination of ATG and PTCy can safely be used in phase III trials.

In a randomized pilot trial we compared ATG (standard Arm A, 4.5 mg/kg) with ATG plus PTCy (experimental Arm BE, ATG 4.5 mg/kg, PTCy 100 mg/kg). The primary safety endpoint was overall survival at 100 days. We analyzed 79 patients with AML (n = 55) or MDS (n = 24). Median age (range) was 59 (19 to 74), 47 (59.5%) were male. Conditioning was either myeloablative (n = 49) or reduced intensity (n = 30). Donors were 8/8 HLA-matched. Overall survival at 100 days was 95.0% (37 of 39 patients) vs 94.9% (38 of 40 patients) in arm A vs arm BE (p > 0.9). In arms A versus BE, serious adverse events (SAEs) occurred in 26 (65.0%) and 25 (64.1%) patients, graft failure occurred in 3 and 1 patients (p > 0.9), median (range) days to neutrophil engraftment were 19 (10-45) versus 22 (15-56) days p = 0.007), cytomegalovirus and Epstein-Barr virus reactivations occurred in 11 and 7 patients (p = 0.45) and in 6 and 5 patients (p = 0.29). Overall survival at 12 months was 72.5% versus 75.8% (p = 0.79), cumulative incidence at 6 months of relapse was 15.0% versus 15.7% (p = 0.88) and of non-relapse mortality (NRM) 7.5% versus 8.0 (p = 0.45). The combination of ATG and PTCy can safely be used in phase III trials.Registration: clinicaltrials.gov (NCT04202835), November 29, 2019.

Systemic sclerosis (SSc) is a life-threatening autoimmune disease with limited treatment options. Autologous stem cell transplantation (ASCT) is the only disease-modifying therapy in SSc; however, its effects on fibroblasts are unknown. We have recently shown that dermal fibroblasts (DFs) from patients with diffuse cutaneous SSc (dSSc) develop a cancer-like phenotype characterized by genomic instability and increased double-stranded DNA breaks (DSBs) associated with resistance-to-apoptosis (Figure 1). In cancer, this is promoted by mitochondrial-dependent metabolic remodelling which is activated by the transcription factor forkhead Box 1 (FOXO1). We hypothesized that metabolic remodeling in dSSc DFs may promote resistance-to-apoptosis via FOXO1, which normalizes post-ASCT. Figure 1. Graphical abstract : In fibroblasts from patients with severe diffuse systemic sclerosis (dSSc), mitochondrial dysfunction results in excessive production of reactive oxygen species (ROS), leading to metabolic stress (1). The elevated ROS levels promotes DNA breaks (2). which activate the transcription factor FOXO1. Upon activation. FOXO1 translocates to the nucleus (3) and binds to the promoter of superoxide dismutase 2 (SOD2) and pyruvate dehydrogenase kinase 4 (PDK4). SOD2 then translocates to the mitochondria, where it reduces mitochondrial ROS into diffusible hydrogen peroxide (H2O2). FOXO1 also promotes metabolic remodelling through PDK4 to increase resistance to apoptosis, and further exacerbate mitochondrial dysfunction. Additionally, nuclear FOXO1 binds to the promoters of fibrotic mediators, driving fibrosis (4). This cycle of mitochondrial dysfunction and FOXOI activation ultimately promotes genomic Instability, senescence and increased ROS (all of which are key pathogenic mechanisms in progressive forms of SSc). This maladaptive mechanism is normalized following ASCT. DFs were generated from healthy control volunteers (HC), limited cutaneous SSc (lSSc), dSSc and post-ASCT (~12 months) patients using 4 mm skin biopsies (N=6-10/group). We quantified the frequency of DSBs and resistance-to-apoptosis via γ-H2AX and/or TUNEL/cleaved caspase 3 (+/− treatment with cyclophosphamide or a FOXO1-inhibitor), respectively. FOXO1 activation was determined by measuring nuclear (active) FOXO1, and expression of its downstream mRNA targets SOD2 and PDK4. Mitochondrial morphology was assessed using confocal microscopy. We also assessed changes in mitochondrial functions, namely mitochondrial dynamics (fusion/fission, immunoblot), biogenesis (qRT-PCR), electron transport chain (ETC) genes (qRT-PCR), and oxygen consumption (XFlux Seahorse analysis). dSSc DF had the highest frequency of DSBs compared to HC, lSSc and post-ASCT. This was associated with increased resistance-to-apoptosis in response to cyclophosphamide, with associated activation of FOXO1. Notably, pharmacological inhibition of FOXO1 in dSSc DF resulted in decreased indicators associated with fibrosis and increased apoptosis. In addition, dSSc DF had an increased frequency of elongated mitochondria and indicators associated with mitochondrial fusion (e.g. OPA1, phospho-DRP1), mitochondrial biogenesis, ETC expression with reduced oxygen consumption rate. Overexpression of constitutively active FOXO1 in HC DF resulted in similar metabolic changes as seen in dSSc. Finally, post-ASCT DF did not have increased DSB, FOXO1 activation, or metabolic reprogramming - suggesting that ASCT may provide some of its beneficial effects by modulating this novel mitochondrial/FOXO1 axis. Our study highlights the critical role of metabolic stress remodeling as a driver of the cancer-like phenotype in severe forms of SSc.[1] It also provides mechanistic insights related to how ASCT may impart some of its beneficial effects. Future studies targeting this pathway may implicate this novel mitochondrial/FOXO1 pathway as a novel therapeutic strategy in patients with SSc. [1.] Gniadecki R. J Autoimmun 2022;131:102847.

Background: Autologous stem cell transplantation (ASCT) remains an important option for patients with late relapses of diffuse large B-cell lymphoma (DLBCL) and in regions where CAR T-cell therapy is inaccessible. Although carmustine, etoposide, cytarabine, and melphalan (BEAM) is widely used for ASCT conditioning, the rising costs, limited availability, and pneumonitis risks of carmustine have reduced its feasibility in some settings. Busulfan-melphalan (BuMel) represents a promising alternative conditioning regimen, though published data are limited. Here, we report our institutional experience with BuMel conditioning, incorporating pharmacokinetic (PK)-guided busulfan dosing to optimize dosing and mitigate toxicity. Methods: This retrospective single-centre study included consecutive patients ≥18 years old who received rituximab (R)-BuMel conditioned ASCT for relapsed/refractory DLBCL between 2012 and 2024. Conditioning consisted of rituximab 375 mg/m2 IV on day -5, busulfan 3.2 mg/kg/day IV on days -4 to -2, and melphalan 140 mg/m2 IV on day -1. PK sampling was performed after the first busulfan dose (at 5 mins, 1, 3, 5, and 7 hrs) and used to adjust the third dose to target a total busulfan AUC <13,500 μM·min. Primary outcomes were progression-free survival (PFS) and overall survival (OS) from ASCT. Results: This study included 30 patients with a median age of 59 years (range 19-70) who received R-BuMel conditioned ASCT for relapsed/refractory DLBCL NOS (n=19), high-grade B-cell lymphoma NOS (n=1) or with MYC and BCL2 rearrangements (n=4), transformed indolent B-cell lymphoma (n=3), primary mediastinal B-cell lymphoma (n=2), and T-cell/histiocyte-rich large B-cell lymphoma (n=1). International prognostic index (IPI) score was 3-5 in 19 (63%) patients at diagnosis and 14 (47%) patients at relapse. Primary refractory disease occurred in 12 (40%), early relapse within 12 months of first-line therapy in 5 (17%), and late relapse >12 months in 13 (43%) patients. With a median follow up time of 2.8 years (range 0.2-8.6), the median PFS was 2.6 years and median OS was 3.8 years. Estimated PFS rate was 46% (95% CI 24-65%) and OS rate was 57% (95% CI 34-75%) at 3 years. Inferior PFS was associated with primary refractory disease (HR 4.04, 95% CI 1.34-12.2, p=0.013) and IPI 3-5 at relapse (HR 3.36, 95% CI 1.11-10.12, p=0.031). The median CD34+ cell dose was 7.2×106/kg (range 2.8-40.0). Median time to neutrophil and platelet engraftment was 11 days (range 10-14) and 16 days (range 10-79), respectively. Median ASCT hospital stay was 19 days (range 16-81). Busulfan PK monitoring led to dose reductions in 13 (43%) patients and no increases. Infection-related toxicities during ASCT hospitalization included febrile neutropenia in 24 (80%), bacteremia in 3 (10%), C. difficile colitis in 3 (10%), pneumonia in 2 (7%), and UTI in 1 (3%) patient. Other grade 3-4 organ toxicities included mucositis in 14 (47%), colitis in 7 (23%), atrial fibrillation in 2 (7%), pulmonary edema in 3 (10%), and seizure in 1 (3%) patient. One (3%) patient required ICU admission; none required dialysis. No pneumonitis or veno-occlusive disease was observed. Non-relapse mortality was 3% (95% CI 0-15%) at 3 months and 7% (95% CI 1-20%) at 6 months, due to cardiac arrest (n=1) and aspiration pneumonia (n=1). One (3%) patient developed therapy-related myeloid neoplasm following CAR T-cell therapy for post-ASCT relapse. Estimated 2025 drug acquisition costs per patient were approximately $1,000 CAD for BuMel versus $20,000 CAD for BEAM. Busulfan PK monitoring added approximately $1,500 CAD per patient. Conclusion: These results demonstrate the efficacy and tolerability of PK-guided BuMel conditioning for patients undergoing ASCT for relapsed/refractory DLBCL, with PFS and OS comparable to those historically reported with BEAM. Drug acquisition costs are considerably lower with BuMel compared to BEAM. BuMel may represent a reasonable alternative to BEAM, particularly in settings where carmustine is unavailable or cost-prohibitive or where avoidance of pulmonary toxicity is a priority.

Multiple factors have been described to influence the risk of acute or chronic graft-versus-host disease (aGVHD or cGVHD) after allogeneic hematopoietic cell transplantation (HCT), including underlying chronic myeloid leukemia (CML) and high-dose total body irradiation (TBI). However, the impact of the underlying disease or low-dose TBI on the risk of GVHD in the modern era has not been determined. The objective of this study was to determine risk factors for GVHD in the modern era in the setting of antithymocyte globulin (ATG)-based GVHD prophylaxis. This retrospective study included 1219 patients with hematologic malignancy who underwent first peripheral blood allogeneic HCT using myeloablative fludarabine and busulfan conditioning ± low-dose total body irradiation, along with ATG, cyclosporine, and methotrexate as GVHD prophylaxis. The adjusted cumulative incidence of GVHD was compared between patient subgroups using multivariable competing risks regression. When disregarding the underlying disease, risk factors for grade 2-4 aGVHD were donor type other than matched sibling donor (non-MSD) and lack of low-dose TBI (non-TBI). Risk factors for grade 3-4 aGVHD were non-MSD, non-TBI, and CMV donor negative/recipient positive serostatus (D-R+). Risk factors for moderate-severe cGVHD were ≤9/10 HLA match, non-male/male donor/recipient sex, and non-TBI. In models including the underlying disease, additional significant risk factors were chronic lymphocytic leukemia (CLL) for grade 2 to 4 aGVHD (sub-hazard ratio over acute myeloid leukemia [SHR] 3.16, 95% CI 1.97-5.08, P < .001); CLL and acute lymphoblastic leukemia (ALL) for grade 3-4 aGVHD (SHR for CLL 3.54, 95% CI 1.54-8.17, P = .003 and SHR for ALL 2.26, 95% CI 1.26-4.04, P = .006); and myelofibrosis (MF) for moderate-severe cGVHD (SHR 2.14, 95 CI 1.34-3.41, P = .001). In the modern era when using ATG for GVHD prophylaxis, newly identified risk factors include CLL and non-TBI for grade 2-4 aGVHD; CLL, ALL, and non-TBI for grade 3-4 aGVHD; and MF and non-TBI for moderate-severe cGVHD. These findings, if confirmed in a separate cohort, should be taken into consideration when tailoring the prophylaxis and monitoring of GVHD.

This study introduces a rapid, cost-effective, and efficient method for in ovo xenografting of patient-derived acute lymphoblastic leukemia (ALL) cells, encompassing both B-cell and T-cell lineages. Using fertilized chicken embryos, we injected patient-derived B-ALL and T-ALL cells into the vasculature of embryos 11 days post-fertilization (11dpf). Remarkably, four days following the injection, the engrafted human leukemia cells exhibited significant survival, proliferation, and vascular colonization within the developing chicken embryo. By 15dpf, we detected a notable increase in CD10/CD19+ B-ALL and CD4/CD8+ T-ALL cells in blood samples from the embryo's vasculature, confirming successful engraftment. This system facilitates efficient and reproducible assessment of leukemia cell behavior in a living organism without the considerable costs and ethical constraints associated with other animal models. This rapid approach provides a high-throughput and biologically relevant in vivo platform for evaluating potential therapeutic compounds. The in ovo patient-derived xenograft (PDX)-ALL system presented here offers a promising tool for preclinical drug screening, mechanistic studies, and potentially for personalized medicine approaches in leukemia research.

Survivors of allogeneic hematopoietic cell transplantation (allo-HCT) are known to be at risk of late toxicities. The spectrum of late toxicities may be impacted by changing allo-HCT practices: In contemporary allo-HCT practice, conditioning with high-dose total body irradiation is infrequent, the incidence of chronic GVHD is declining, and older adults receive allo-HCT more frequently. Few studies have examined the burden of comorbidities and the quality of life (QoL) of recent long-term survivors of allo-HCT, and even fewer have included a biological sibling control group for comparison. We set out to quantify and compare the burden of comorbidities and the QoL of a largely contemporary group of survivors of allo-HCT versus their biological siblings. We further aimed to understand the association of transplant-related variables, demographic variables, and comorbidities, with QoL amongst recipients. We conducted a cross-sectional study comparing QoL and comorbidity burden between allo-HCT recipients and their biological siblings. In addition, we built multivariable models to understand predictors of physical health (PH) and mental health (MH)-related QoL amongst recipients. Recipients without active chronic GVHD or relapse were enrolled at 1 of 2 survivorship clinics alongside their siblings. We used PROMIS Global Health to assess QoL and the post-transplant multimorbidity index to evaluate comorbidities. In total, 391 recipients were enrolled. Of these, 106 recipients had a total of 154 siblings enrolled for comparison. The 106 recipients experienced significantly more comorbidities versus their siblings: 3 or more comorbidities were observed in 51.9% of recipients versus 33.1% of siblings (P = .002), while at least 1 severe comorbidity was observed in 24.5% versus 13.0%, respectively (P = .02). In spite of this, PH and MH-related QoL of recipients was similar to that of siblings: PH QoL median T-score 49.9 (IQR 45.8 to 57.4) versus 50.7 (IQR 46.3 to 54.4), respectively (P = .77), and MH QoL median T-score 50.9 (IQR 44.8 to 54.6) versus 51.6 (IQR 45.3 to 55.1), respectively (P = .58). Social functioning was rated as very good or excellent by 65.1% of recipients versus 68.6% of siblings (P = .33). Amongst the entire cohort of 391 recipients, the number of comorbidities was strongly associated with MH and PH-related QoL as well as social functioning, whereas transplant-related variables such as prior cGVHD, receipt of low dose TBI, graft and donor type, were not. Survivors of allo-HCT continue to experience excess comorbidities versus their biological siblings. Despite this, survivors enjoy QoL and social functioning that are comparable to their siblings and general population norms. Amongst survivors, the number of comorbidities is strongly associated with QoL and social functioning while transplant-related variables are not.

Antithymocyte globulin (ATG; Thymoglobulin) infusion may result in infusional side effects (ISEs) resembling cytokine release syndrome. This study aimed to identify cytokines associated with ISEs, factors predicting ISEs, and the impact of ISEs on hematopoietic cell transplantation (HCT) outcomes. We studied 211 allogeneic HCT recipients who received 3 infusions of ATG, on days -2, -1, and 0. The focus was on the first infusion. ISE was defined as a maximum temperature ≥38°C, maximum heart rate >125/minute, minimum systolic blood pressure <90 mmHg, or supplemental oxygen use between the start of the first infusion and the start of the second infusion. In 158 of the 211 patients, we determined the post-first infusion serum levels of 34 cytokines using Luminex and compared the levels in patients with ISEs and patients without ISEs using a signed-rank test with Bonferroni correction for multiple comparisons. In all 211 patients, we compared overall survival (OS), relapse-free survival (RFS), and moderate to severe chronic graft-versus-host disease (cGVHD)- and relapse-free survival (GRFS) between patients with ISEs and those without ISEs using Cox regression, as well as in the incidences of acute GVHD (aGVHD), chronic GVHD (cGVHD), relapse, and nonrelapse death using Fine-Gray regression. At least 1 ISE occurred in 93 patients (44%). Median levels of the following cytokines were significantly higher in the patients with ISEs: interleukin-1 receptor antagonist (IL1-RA) (30,166 pg/mL versus 6394 pg/mL; P < .001), interleukin-6 (IL-6) (188 pg/mL versus 49 pg/mL; P < .001), and interferon gamma-induced protein-10 (IP-10) (106 pg/mL versus 70 pg/mL; P = .004). Patients with ISEs and those without ISEs did not differ in terms of weight; body mass index; day -2 leukocyte, neutrophil, monocyte, or lymphocyte count; post-first infusion ATG level; or ATG area under the time-concentration curve. There were no significant between-group differences in OS (hazard ratio [HR], 0.83; P = .43), RFS (HR, 0.85; P = .38), or GRFS (HR, 1.01; P = .98). There also were no significant differences in the rates of grade II-IV aGVHD, grade III-IV aGVHD, moderate to severe cGVHD, relapse, or nonrelapse death. IL-6, IP-10, and IL1-RA appear to be involved in the pathogenesis of ISEs; however, ISEs appear to have no significant impact on HCT outcomes. © 2025 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

Background: Anti-thymocyte globulin (ATG, or ATLG) and post-transplant cyclophosphamide (PTCy) are used for the prevention of graft-versus-host disease (GVHD). Two ongoing randomized trials (ISRCTN50290131; NCT05153226) are comparing these agents for efficacy and safety, but the safety and efficacy of the combination is unknown and has not been well studied. We conducted a randomized pilot trial of a combination of these agents to assess safety, with the aim of an expansion phase III trial. Methods: Following CONSORT guidelines, we conducted a randomized pilot trial to compare ATG (standard Arm A, 4.5 mg/kg) with ATG plus PTCy (experimental Arm BE, ATG 4.5 mg/kg, PTCy 50 mg/kg x 2) with short term feasibility endpoints at 100 days but otherwise following a phase III design. Patients consented to two years follow-up in anticipation of an expansion to phase III, as described in the CONSORT extension for pilot trials (Abbade LPF et al (2018) DOI 10.1186/s40409-018-0142-2). Overall survival was calculated using the Kaplan-Meier estimate; the log-rank test was used to compare treatment differences. We randomized a pre-planned convenience sample of 79 eligible and transplanted patients. Median (range) age was 59 (19 to 74), 47 (59.5 %) were male. Patients had either AML (n=55) or MDS (n=24) and were transplanted using either myeloablative (n=49) or reduced intensity (n=30) conditioning. Donors, either sibling (n=28) or unrelated (n=51), were 8/8 HLA-matched. The primary feasibility endpoints included: (1) recruitment of the intended sample size, (2) 100-day survival of the experimental arm B to be at least 90% compared with the standard arm A, (3) complete data on 95% of recipients at 12 months and (4) completion of the trial within budget. A variety of secondary endpoints, both efficacy and safety, were included. Objectives 1, 3, and 4 were fulfilled and this abstract describes the safety and efficacy outcomes related to objective (2). Results at day 100: Forty-one patients were randomized to Arm A and thirty-eight patients to Arm BE. At the time of data lock (May 14, 2024), all patients had been followed for at least 100 days. Thirty-nine (95.1%) patients in Arm A and 36 (94.7%) in Arm B survived to 100 days (primary endpoint). Acute graft versus host disease (AGVHD) was reported in 15 and 6 patients in Arms A and BE respectively, of which 6 and 3 had grades II-IV and 3 and 0 had grades III-IV. At 100 days, 3 patients in Arm A and 3 patients in Arm BE were receiving systemic corticosteroids. Serious Adverse Effects (SAEs) were experienced by 31 patients in Arm A and 25 in Arm BE. Graft failure at 28 days occurred in 2 patients in each arm, with all patients achieving engraftment eventually. Median (range) days to engraftment was 19 (13-45) for Arm A and 22.5 (15-56) for Arm BE. Cytomegalovirus reactivations occurred in 10 and 7 patients in Arm A and Arm BE respectively and Epstein-Barr virus reactivations occurred in 7 and 4 patients in Arm A and Arm BE respectively. Results Follow Up: Median follow up of living patients was 23.6 months (range 3.19- 25.92) for Arm A patients and 22.9 months (range 1.64-25.07). Overall survival between the arms was similar at 12-months; 68.9% (standard error (SE) 7.5%) in the standard arm A and 75.4% (SE 7.6%) in the experimental arm BE (p = 0.52). Relapse or disease progression were seen in 7 patients in Arm A and 7 patients in Arm BE. AGVHD was reported in 18 patients in Arm A and 9 in Arm BE (grades II-IV 11 and 5, grades III-IV 5 and 1), with late (>100 days) AGVHD occurring in 3 and 2 cases in Arms A and BE respectively. CGVHD has been reported to date in 7 patients in Arm A and 4 patients in Arm BE, moderate or severe grade in 2 patients and 2 patients respectively. Conclusion: The addition of PTCy to ATG did not result in excessive adverse events in this pilot trial (to day 100), and long-term results remained consistent. The combination of ATG and PTCy can be used safely in future trials to further prevent graft-versus-host disease. Based on the demonstrated safety and feasibility of this combination, and encouraging signals of efficacy, planning for an expansion to a full phase III trial with a primary endpoint of GVHD, relapse free survival is underway.