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Abstract Introduction: Chronic lymphocytic leukemia (CLL) is marked by impaired T cell responses that limit the efficacy of immunotherapies. CD19-directed chimeric antigen receptor T cell therapy (CART19) received FDA approval to treat CLL in 2024. However, itsefficacy in CLL patients remains low, with durable remission rate of ~20%. Ibrutinib, a Bruton tyrosine kinase inhibitor, has shown synergy with CART19 in preclinical and early clinical studies. We aimed to study the impact of ibrutinib on CART19 utilizing clinical samples from the pivotal clinical trial E1912 (NCT02048813) of ibrutinib/rituximab (IR) vs fludarabine/cyclophosphamide/rituximab (FCR). This trial led to the FDA approval of first line ibrutinib in CLL. Methods: We generated CART19 incorporating either CD28 (28ζ) or 41BB (BBζ) costimulatory domains from T cells isolated from patients with CLL treated with IR or FCR on the E1912 trial. IR samples were collected at baseline (pretreatment), 6- or 12-months during treatment, or at progression. FCR samples were collected at baseline, 15-18 months during treatment, or at progression. For animal experiments, NOD-SCID-γ-/- (NSG) mice were intravenously (i.v.) engrafted with 1×10⁶ luciferase⁺ CD19⁺ JeKo-1 cells. Upon tumor establishment (~10⁸ photons/sec by bioluminescence imaging), mice were randomized and treated i.v. with 1×10⁶ 28ζ or BBζ derived from longitudinal IR- or FCR-treated patient samples. To further assess the impact of prolonged ibrutinib exposure on CART19 function, we chronically stimulated healthy donor-derived 28ζ or BBζ in vitro for 22 days with JeKo-1 cells and 100nM ibrutinib or vehicle control and then tested CART19 functions. Finally, we performed single cell RNA sequencing (scRNAseq) of longitudinal patient T cell samples treated with IR. Results: Ex vivo T cell expansion was significantly enhanced following IR compared to FCR. BBζ generated from IR-treated patients after 6 months of ibrutinib exhibited enhanced in vitro expansion (p=0.016) and antigen-specific proliferation (p=0.038) in the presence of JeKo-1 cells. This corresponded with significantly improved antitumor efficacy and prolonged survival in vivo compared to cells generated at disease progression (p=0.017). In contrast, 28ζ showed no significant differences in efficacy across IR studied treatment timepoints. BBζ from FCR-treated patients did not show differences in tumor control based on treatment timepoints but demonstrated shorter survival compared to BBζ from IR-treated patients. 28ζ from FCR-treated patients generated at disease progression demonstrated significantly reduced tumor control and shorter survival compared to 28ζ produced at baseline (p=0.0002), indicating detrimental effects of FCR treatment on the therapeutic potential of CART products. To further investigate the impact of prolonged ibrutinib exposure, we utilized in vitro models of CART exhaustion. We found that, after repeated antigen stimulation with JeKo-1 cells, ibrutinib-treated BBζ had significantly increased antigen-specific degranulation (p=0.005), and cytokine production (IL-2, p=0.045; TNF-α, p=0.003; IFN-γ, p=0.001), compared to BBζ treated with vehicle control. In contrast, 28ζ did not show improved degranulation or cytokine production in cocultures with ibrutinib. Prolonged ibrutinib exposure led to downregulation of FOXO3, a transcription factor associated with T cell exhaustion which has been shown to cause dysfunction specifically in BBζ. These results further indicate that ibrutinib may be more beneficial to BBζ. Indeed, scRNAseq revealed that after ≥ 6 months of ibrutinib, T cells showed downregulation of inflammatory, oxidative stress, and apoptosis pathways, paralleled by modest activation of MYC and E2F targets. However, at disease progression, T cells under ibrutinib treatment displayed marked upregulation of interferon signaling, inflammation responses, and stress-related programs, indicating a shift toward immune reactivation or dysfunction.Conclusion: Our study shows that BBζ, but not 28ζ, generated 6 months during ibrutinib treatment has superior antitumor activity and persistence compared to similar later timepoints or FCR-treated counterparts. Prolonged ibrutinib exposure enhances BBζ function by modulating stress and apoptotic pathways. These findings highlight the importance of pre-CART treatment timing and support ibrutinib as a potential enhancer of CART cell fitness in CLL.

Abstract Background: VEXAS (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) syndrome is a hemato-inflammatory syndrome due to somatic mutations in UBA1 gene. Recently, DNA methyltransferase inhibitors (specifically, 5-azacitidine) and allogeneic hematopoietic stem cell transplantation (alloHSCT) have shown success in eradication of UBA1 clones and clinical remission in some patients. This study aims to provide a comparative efficacy analysis between DNA methyltransferase inhibitors (hypomethylating agents/HMA) versus alloHSCT in VEXAS syndrome patients. Methods: This is a multi-center, retrospective cohort study. UBA1 mutations were detected either through next generation sequencing [NGS, limit of detection (LOD) 2% variant allele fraction (VAF)] or quantitative droplet digital polymerase chain reaction testing (ddPCR, LOD 0.5% VAF). Outcomes of interest included overall survival (OS), glucocorticoid (GC) discontinuation/reduction in dose (prednisone < 10 mg/day), and complete molecular remission (CMR, defined as an undetectable UBA1 mutation). OS was calculated from the time of diagnosis to death or last follow up. Cumulative incidence of reduction in GC dose was calculated using competing risk analysis after accounting for death. Results: Between years 2020-2024, a total of thirty-nine male patients (median age 67, range 45-82 years) with VEXAS syndrome, necessity for therapy (GC-refractory inflammation and/or progressive marrow failure/myeloid neoplasm) and confirmed UBA1 pathogenic mutation were included. Of these, 18 (46%) received HMA therapy (5-azacitidine 72% and Decitabine 28%) with a median of 4 (range 1-36) cycles. and 21 (54%) underwent alloHSCT (MSD=4, MUD=12, MMUD=2, and haploidentical=3), all received reduced intensity conditioning (Flu/Mel =11, Flu/Bu=5, Flu/Cy/TBI=2, and TBF=3) and 91% received post-transplant cyclophosphamide-based graft-versus-host disease (GVHD) prophylaxis (only 2 patients in alloHSCT group received prior HMA without benefit). Baseline characteristics were well-balanced between HMA and alloHSCT groups; age at treatment (median 70 vs 65 years, p=0.2), and UBA1 M41V subtype (25% vs 40%, p=0.3), cytogenetic and non-UBA1 mutation abnormalities (p=0.4), prior lines of therapy (median 4 in both, p=0.5), time from diagnosis to therapy (median 7 vs 13 months, p=0.15), indication for therapy (p=0.12), Charlson CI scores (median 4 in both, p=0.4) and HCI-CI scores (median 4 in both, p=0.7). At a median follow-up of 45 (range 7-57) months, 11 (28%) deaths (alloHSCT=3 and HMA=8) were documented. Causes of death included infections (n=6), cardiac disease (n=2), poor graft function (n=2) and pulmonary GVHD (n=1). Compared to HMA therapy, alloHSCT group demonstrated higher rates of GC discontinuation (52% vs 6%, p<0.01) and CMR (evaluable= 22, 100% vs 50%, p<0.01). In the HMA group, 50% (n=9) discontinued therapy after a median of 4 months due to infectious complications or lack of benefit. In the alloHSCT group, 3 (14%) patients developed grade 2 acute GVHD (1-skin, 2-gut, no grade 3-4 acute GVHD) and 4 (19%) experienced moderate to severe chronic GvHD (2-lung, 2-mucocutaneous). In multivariable analysis (MVA) adjusted for age and HCT-CI, alloHSCT was identified as the strongest favorable predictor for OS (HR 0.18 95% CI 0.04-0.8) with similar results when analysis was restricted to transplant-eligible patients < 75 years of age (HR 0.1, 95% CI 0.03-0.6). In a 6-month post-treatment landmark analysis (n=31 evaluable), patients who underwent alloHSCT (n=17) exhibited numerically superior GC discontiuation rate (29% vs 14%, p=0.049) and subsequent OS compared to HMA (2-years OS: 84% vs. 61%, median not reached vs 25 months, 95% CI: 0.06–1.7). In a post-treatment competing risk [GC dose reduction (death)] analysis alloHSCT was associated with superior 2-, and 3-year rates of 57% (10%), and 76% (10%), compared to HMA 22% (24%), and 31% (42%) [p<0.01 (0.04)]. AlloHSCT was also associated with higher rate of CMR (evaluable= 21, alloHSCT N=16 and HMA N=5, 100% vs 60%, p=0.01). At the last follow up, none of the patients who achieved CMR had re-emergence of the UBA1 clone (despite early mixed chimerism). Conclusions: This study represents the largest comparative analysis of alloHSCT versus HMA therapy in VEXAS syndrome, and confirms superiority of alloHSCT in achieving molecular remission, GC discontinuation/dose reduction, and overall survival. Prospective clinical trial confirmation is warranted.

Abstract Recent studies have highlighted the important but pleiotropic role of IL-4 in response to chimeric antigen receptor T (CART) cell therapy. IL-4 was identified as a regulator of dysfunction in CD19-targeting CART cells with a CD28 costimulatory domain (CART19-28ζ) through a genome-wide CRISPR knockout screen as well as RNA and ATAC sequencing of baseline and exhausted healthy donor CART19-28ζ cells and pre-infusion CART19-28ζ cell products from responders and non-responders in the ZUMA-1 trial (Stewart et al, Nat Commun., 2024). However, interrogation of a single-cell atlas fromactivated pre-infusion CART cell products from patients with acute lymphoblastic leukemia revealed a beneficial role for IL-4 and other type 2 cytokines in the promotion of long-term response (Bai et al, Nature, 2024). In this report, we aim to further understand how IL-4 can be detrimental to CART cell therapy. To study the regulation of CART19-28ζ cells by IL-4, we performed RNA sequencing on CART19-28ζ cells that were stimulated with CD19+target cells in the presence of either 20 ng/mL hrIL-4 or diluent for 24 hours, one week, or two weeks in an in vitro model for exhaustion as previously described (Stewart et al, Nat Commun., 2024). We observed that hrIL-4 treatment drove the development of a distinct transcriptomic profile after chronic stimulation for one week (up genes = 3253 and down genes = 3645) or two weeks (up genes = 6435 and down genes = 6224) but not following 24 hours of activation (up genes = 0 and down genes = 0). Gene set enrichment analysis on the differentially expressed genes following two weeks of chronic stimulation showed that hrIL-4 treatment pushes CART cells to a more exhausted and less naïve phenotype (FDR = 0.015) and leads to a broad downregulation in cytokine signaling (FDR = 0.012). To investigate transcription factors associated with IL-4-induced CART cell dysfunction, we used BETA analysis with publicly available transcription factor binding sites for STAT6, GATA3, and EOMES and our differential expression data. EOMES (p = 0.039), but not STAT6 (p = 0.999) nor GATA3 (p = 0.999) significantly regulated the differentially expressed genes. In support of these findings, the addition of hrIL-4 also significantly upregulated EOMES expression after one (p < 0.0001) and two (p < 0.0001) weeks of chronic stimulation and downregulated the expression of TBX21 (p < 0.0001), a binding competitor of EOMES, following two weeks of chronic stimulation. To evaluate if EOMES is critical for IL-4-induced CART cell dysfunction, we treated either control or EOMES CRISPR knockdown CART cells with 20ng/mL hrIL-4 during chronic stimulation. EOMES knockdown reduced IL-4-induced CART19-28ζ cell dysfunction as seen by an increase in antigen-specific expansion (p = 0.046) and an increase in cytokine production including IL-2 (p < 0.0001), TNF-α (p = 0.006), and IFN-γ (p = 0.020). Having identified that EOMES is at least partially responsible for IL-4-induced CART cell dysfunction, we evaluated EOMES binding sites in human CART19-28ζ cells with CUT&RUN sequencing. Our analysis revealed EOMES binding at hypersensitivity site 2 (HS2) in the IL-4 locus which, in a previous study, was more accessible in both exhausted healthy donor CART19-28ζ cells and pre-infusion CART cell products from non-responders in the ZUMA-1 clinical trial (Stewart et al, Nat Commun., 2024). To determine how HS2 can impact CART cell functions, we generated CART19-28ζ cells from either wildtype (HS2+/+) or HS2-deleted (HS2-/-) murine T-cells and tested them in a syngeneic model of lymphoma generated by engrafting C57BL6 mice with luciferase+ TBL12 cells (1x106, tail vein injection on Day -3) following preconditioning (250mg/kg cyclophosphamide, intraperitoneal injection on Day -4). Following randomization of mice via tumor burden (n = 5 per group), mice were treated with either HS2+/+ or HS2-/- CART19-28ζ cells (1x106 cells, tail vein injection). In this model, HS2-/- CART cells showed enhanced expansion (p = 0.022), increased anti-tumor activity (p = 0.018), and improved overall survival (p = 0.003). Together, our study shows that IL-4 induces a distinct cellular fate in CART19-28ζ cells in the presence of chronic antigen-specific stimulation at least partially through the upregulation of EOMES. Further, the epigenetic regulation of CART cells by the IL-4 enhancer HS2 can significantly influence CART cell activity, and deletion of HS2 can improve CART cell therapy.

Abstract INTRODUCTION Immune checkpoint inhibitors (ICIs) have emerged as a cornerstone of cancer therapy. However, despite their efficacy, ICIs can cause a unique spectrum of autoimmune toxicities known as immune-related adverse events (irAEs). In contrast to irAEs involving other organ systems, hematologic irAEs such as ICI-associated immune thrombocytopenia (ICI-ITP) are less common. As a result, existing data on ICI-ITP are largely confined to isolated case reports, single-center case series, and pharmacovigilance databases lacking granular clinical data. To address this key knowledge gap, we sought to comprehensively characterize the incidence, risk factors, clinical features, treatments, and outcomes associated with ICI-ITP. METHODS We performed a multicenter cohort study of adult patients who initiated ICI therapy at 29 hospitals across 7 major cancer centers in 11 geographically diverse states in the US between 2016-2023. ICI-ITP was defined as a decline in platelet count to <100 x109/L in the absence of an alternative more plausible etiology of thrombocytopenia. Patients with hematologic malignancies were excluded, as were those who received cytotoxic chemotherapy within 30 days prior to thrombocytopenia onset. For each patient identified as having ICI-ITP, we collected data on demographics, comorbidities, medications, laboratory values, treatments, and outcomes. All data were extracted by manual chart review. To identify risk factors for ICI-ITP and to examine its association with death, for each case of ICI-ITP we also collected data on two control patients who initiated ICI therapy at the same site and in the same year but without development of ICI-ITP. We used multivariable logistic regression to identify independent risk factors for ICI-ITP and its recovery (the latter defined according to criteria established by the ITP International Working Group). We used a multivariable Cox model to examine the association between ICI-ITP and its severity with all-cause mortality. RESULTS We identified a total of 214 patients who developed ICI-ITP among 86,648 examined (0.25%). The incidence was highly consistent across sites (range, 0.15–0.30%). Risk factors for ICI-ITP included simultaneous receipt of two different classes of ICIs, additional irAEs, and a lower baseline platelet count. ICI-ITP developed at a median of 8 weeks (IQR, 4–18) following ICI initiation. The median nadir platelet count was 41 x109/L (IQR, 17–64). Six out of 214 patients (2.8%) developed a major bleed, and 16 patients (7.5%) developed a clinically relevant non-major bleed. Cell-bound anti-platelet antibody testing was positive in 6 of 8 patients (75.0%) for whom it was assessed, and median circulating thrombopoietin levels (201 pg/mL [IQR, 178–295]) were similar to those seen in primary ITP. Treatments used included glucocorticoids (49.5%), immune globulin (18.2%), and thrombopoietin receptor agonists (13.6%). Overall, 161 patients (75.2%) recovered from ICI-ITP at a median of 2 weeks (IQR, 1–5) following ICI-ITP onset. Predictors of recovery included additional irAEs and a higher nadir platelet count. Treatment response was observed in 57.0%, 56.4%, and 58.3% of patients within 60 days following initiation of glucocorticoids, immune globulin, and thrombopoietin receptor agonists, respectively. Of 76 patients rechallenged with an ICI, 23 (30.3%) developed recurrent ICI-ITP at a median of 9 weeks (IQR, 2–15); 17 of these 23 patients (73.9%) recovered. Finally, ICI-ITP and its severity were monotonically associated with a higher risk of death (adjusted hazard ratio 2.96 [95% CI, 2.14–4.08] for those with severe ICI-ITP compared to those without ICI-ITP). CONCLUSION In this multicenter study involving patients treated with ICIs at 29 hospitals across the US, we identified 214 patients with ICI-ITP. For the first time, we characterize in detail the incidence, risk factors, clinical features, treatments, and outcomes of ICI-ITP, a relatively novel disease entity. We found that more than half of patients responded to conventional ITP-directed therapies, and that most patients rechallenged did not develop recurrent ICI-ITP. We also found that ICI-ITP was independently associated with an increased risk of death, including a nearly 3-fold increased risk in those with severe ICI-ITP. These findings highlight ICI-ITP as a rare but clinically significant complication of ICI therapy that hematologists must be equipped to recognize and manage.

Abstract Background Acute graft-versus-host disease (GVHD) is a major cause of non-relapse mortality (NRM) after allogeneic hematopoietic cell transplantation (HCT). High-dose steroids are standard first-line therapy for grade II–IV acute GVHD, whereas systemic treatment (tx) is generally not recommended for grade I. However, in practice many patients (pts) with grade I GVHD receive systemic steroids. A prior GITMO randomized trial (n=173) of grade I GVHD to either systemic steroid tx or “watch and wait” found that preemptive steroid tx reduced progression to grade II but not grade III/IV GVHD, increased serious infections, and resulted in similar chronic GVHD, NRM and survival (Bacigalupo, Haematologica 2017). In this study we analyzed real world evidence for early systemic steroid use and the potential impact of serum biomarkers in grade I GVHD. Methods We analyzed clinical data and serum samples from pts with grade I acute GVHD at 24 Mount Sinai Acute GVHD International Consortium (MAGIC) centers between 2014 and 2022 who were treated according to physician discretion. Pts were divided into a preemptive steroid tx group or a watch-and-wait group (systemic steroids administered only if progression to grade II–IV GVHD). Preemptive steroid use was treated as a time-dependent covariate and a frailty proportional hazards model with random effects was used to adjust for center effects to account for possible heterogeneity in clinical practices across transplant centers. Serum samples collected at the diagnosis of grade I GVHD were analyzed for ST2 and REG3α to calculate validated Ann Arbor (AA) scores (1, 2, 3). Results Grade I GVHD was diagnosed in 1143 pts; 591 (52%) received preemptive tx and 552 (48%) were treated by a watch-and-wait strategy. In the preemptive tx group, steroids were initiated at a median of 0 days and within 7 days for >90%. The median initial dose of steroids was 1 mg/kg of prednisone or its equivalent. In the watch and wait group, only 184 pts (33%) required systemic steroids for progression to grade II-IV GVHD at a median of 14 days from grade I diagnosis. Preemptive steroid tx reduced the risk of grade II–IV acute GVHD (HR, 0.71; 95% CI, 0.56–0.89; P=0.003), but not the risk of grade III/IV acute GVHD (HR, 1.03; 95% CI, 0.69–1.52; P=0.899) or chronic GVHD (HR, 1.03; 95% CI, 0.69–1.52; P=0.899), consistent with the GITMO trial. The 12-month incidence of NRM was higher for preemptive tx vs. watch and wait (13% vs 7%, P=0.002). In multivariate analysis preemptive steroid use independently predicted an increased risk of NRM (HR, 1.63; 95% CI, 1.06–2.50; P=0.026) as did several other known risk factors (older recipient age, umbilical cord blood donor, HCT-CI ≥3, and myeloablative conditioning). Greater NRM in the preemptive tx group was driven primarily by 3-fold more infectious deaths; deaths due to uncontrolled GVHD were similar for both tx groups. The risk for relapse was unexpectedly lower in the preemptive tx group (HR 0.64; 95% CI 0.47-0.88; P=0.006) and overall survival was therefore not different between groups, but risk factors for relapse such as minimal residual disease status were not available precluding further analysis of this finding. Serum was collected in 871 (76%) pts at onset of grade I GVHD for retrospective biomarker analysis. AA scores were AA1 (80%), AA2 (15%), and AA3 (5%). AA scores were highly predictive of progression to grade III/IV GVHD (AA1 vs AA2 vs AA3: 9% vs 14% vs 44%, all P<0.001) and the risk of 12-month NRM (8% vs 16% vs 39%, all P<0.001); this pattern was observed in both tx groups. The negative effect of preemptive steroid tx was most pronounced in pts with grade I/AA1 GVHD who experienced 5-fold more infectious related deaths compared to pts managed with a watch and wait approach. Conclusion Treatment of grade I GVHD with systemic steroids did not reduce the incidence of grade III/IV GVHD or deaths due to uncontrolled GVHD and instead increased the risk of NRM from infections compared to pts who were treated only after GVHD progressed. Importantly, 2/3 of the watch and wait pts never needed systemic steroid tx. These findings remained robust after adjusting for multiple confounders, including center effects, and were consistent with the GITMO trial. MAGIC biomarkers at the onset of grade I GVHD predicted outcomes and a watch and wait strategy may be best for the 80% of pts with AA1 GVHD. Better treatment options are needed for the 20% of pts with grade I and AA2/3 GVHD.

Abstract Background: Parenteral DNA methyltransferase inhibitors (DNMTi) such as azacitidine and decitabine (DEC), including an attenuated 3-day regimen, are recommended in the NCCN Guidelines for patients (pts) with lower-risk myelodysplastic syndromes (LR-MDS) with clinically significant cytopenias. However, parenteral administration requires frequent clinic visits, which imposes a substantial burden on pts, particularly those requiring long-term therapy. Oral DMTIs offer more flexible dosing and maintenance of pts' autonomy. Oral DEC-cedazuridine (DEC-C) is a fixed-dose combination tablet containing DEC (35 mg) and cedazuridine (100 mg). ASTX727-03 (NCT03502668) is a randomized, open-label, multicenter, phase 1/2 clinical trial evaluating safety, efficacy, and pharmacokinetics (PK) of various DEC-C doses and regimens in pts with LR-MDS. Phase 1 identified a 5-day regimen of DEC 10 mg/cedazuridine 100 mg as the optimized low-dose (LD) regimen. Phase 2 compares this LD regimen with the attenuated standard-dose (SD) regimen. Here, we report phase 2 results on efficacy, safety, PK, and pharmacodynamic (PD) data. Methods: Adults (aged ≥18 years) with International Prognostic Scoring System (IPSS) low-risk or Intermediate-1 MDS, ECOG PS 0−2, and ≥1 cytopenia or red blood cell (RBC) transfusion dependence were randomized 1:1 to receive oral DEC-C LD (10 mg x 5 days) or SD (35 mg x 3 days). The primary endpoint was hematologic response. Secondary endpoints included PK, PD (by LINE-1 demethylation), time to bone marrow blasts >5%, leukemia-free survival (LFS), overall survival (OS), and safety. Results: As of October 31, 2024, 81 pts were treated (LD arm: n=40; SD arm: n=41) and completed a median of 8.8 months on treatment. At cutoff, 17 (42.5%) and 12 (28.6%) LD and SD pts, respectively, were still continuing the study; 9 (22.5%) and 6 (14.6%) pts respectively, had discontinued treatment due to hematopoietic stem cell transplant. Median age was 70 (LD) and 75 (SD) years; 75.0% (LD) and 68.3% (SD) had IPSS Int-1 risk MDS and 60.0% vs 41.5% had IPSS-Molecular Lower-Risk MDS (very low, low, or moderate-low). Low-risk prognostic scoring system risk distribution was similar: Category 1 (2.5% vs 4.9%), Category 2 (47.5% vs 46.3%), and Category 3 (50.0% vs 48.8%). After a median follow-up of 29.3 months, median OS was 23.9 months (95% confidence interval [CI]: 13.0, not estimable [NE]) in the LD arm vs 26.0 months (95% CI: 19.4, 28.5) in the SD arm. Median LFS was 23.8 months (95% CI: 13.0, NE) and 25.7 months (95% CI: 17.8, 26.5), respectively. Hematologic improvement per International Working Group 2006 criteria was achieved in 27.5% (LD) and 26.8% (SD) of pts. Among RBC transfusion–dependent pts, 52.4% (LD) and 37.5% (SD) achieved ≥56-day independence and 47.6% (LD) and 20.8% (SD) achieved ≥112-day independence. Pts received a median of 10 (LD) and 9 (SD) cycles. Delayed cycles occurred in 72.5% (LD) vs 82.9% (SD) of pts; dose reductions occurred in 40.0% (LD) and 46.3% (SD) of pts. Both treatment regimens caused a decrease in blood counts, reaching a nadir before recovering in each cycle. Neutropenia was more pronounced during early cycles and was more severe in the SD arm. Blood counts across all lineages remained stable or improved through ≥12 cycles with the LD arm, suggesting a more favorable safety and tolerability profile. Grade ≥3 adverse events (AEs) occurred in 85.0% and 90.2% of pts in the LD and SD arms, respectively; treatment discontinuation due to AEs occurred in 2.5% and 17.1% of pts, respectively. Most commonly reported treatment-emergent AEs were anemia (42.5% LD, 39.0% SD), fatigue (32.5% LD, 43.9% SD), and thrombocytopenia (37.5% LD, 39.0% SD). Three deaths occurred in the trial: 2 in the LD arm, which were both unrelated to study treatment, and 1 in the SD arm (Pseudomonal bacteremia in Cycle 1), which was treatment related. Total AUC0–24 was 199 ng*h/mL in the LD arm and 465 ng*h/mL in the SD arm; AUC for all cycles of the LD arm was approximately half of the SD arm. Maximal % LINE-1 demethylation rate was lower with LD than SD, but narrowed by Cycle 3. Conclusion: LD oral DEC-C demonstrated comparable clinical benefit to SD oral DEC-C, with improved safety and tolerability, including fewer dose modifications and less severe myelosuppression. These findings support LD DEC-C as a potential optimal regimen for pts with LR-MDS. Clinical trial registration: NCT03502668

Abstract Introduction: HMA monotherapy (HMAm) continues to be the standard of care for HR-MDS. While the addition of VEN to azacitidine (AZA) improved overall survival (OS) and complete remission (CR) rates among older pts with acute myeloid leukemia (AML) over AZA monotherapy, the recently completed phase III VERONA trial of AZA+/-VEN in HR-MDS failed to show an OS benefit for AZA/VEN over AZA monotherapy. Using the icMDS VALIDATE database, we compared outcomes of pts with HR-MDS treated with HMA or HMA/VEN as frontline therapy. Methods: The icMDS VALIDATE database includes over 4000 adult MDS pts who received HMA-based therapy from 32 international centers in 9 countries. For this analysis, we included pts with HR-MDS defined by International Prognostic Scoring System [IPSS] ≥1.5 or IPSS-revised [IPSS-R] ≥4 (at diagnosis or HMA initiation) and treated with HMAm or HMA/VEN as frontline therapy for HR-MDS. We assessed responses based on IWG 2023 criteria (Zeidan A et al, Blood 2023). Composite complete remission (cCR) was defined as CR+CRbi+CRuni+CRh+CRequivalent. We calculated OS from time of HMA initiation to date of death from any cause. Pts lost to follow-up were censored at the date they were last known to be alive. We used Kaplan-Meier methods to calculate OS and compared OS between different groups using the log-rank test. We used Cox and logistic regression models to adjust for baseline prognostic variables as appropriate. The study was funded by an independent grant from Abbvie. Results: A total of 1907 pts met eligibility and were included (1552 AZA, 221 decitabine, 134 HMA+VEN). 479 pts subsequently underwent allogeneic stem cell transplant (allo-SCT; 430 after HMAm [32.6%]; 49 after HMA/VEN [37.1%]). Our cohort was enriched for pts with adverse disease characteristics including complex karyotype (33.6%) and TP53 mutations (28.7%). Compared to pts treated with HMA/VEN, pts treated with HMAm were more likely to be male (p=0.012), have lower baseline platelet counts (p=0.037), peripheral blood (PB) blasts (p=0.004), or bone marrow blasts (p<0.001), and were less likely to have very high risk IPSS-M (p<0.001), TP53 mutations (p<0.001), or complex karyotype (p=0.020). In unadjusted analyses, the rate of cCR was significantly lower in HMAm treated pts compared to HMA/VEN treated pts (27.7% vs. 48.8%, p<0.001). However, CR rate was not significantly different (11.7% vs. 17.1% for HMAm and HMA/VEN, respectively, p=0.22). After adjusting for age, sex, and IPSS-M, pts treated with HMA/VEN were more likely to achieve cCR compared to HMAm with an odds ratio (OR) of 2.49 (95% CI: 1.56-3.96, p<0.001). When separated by TP53 mutation status, the OR for cCR for HMA/VEN vs HMAm was 4.17 (95%CI: 2.14-8.31; p<0.001) and 1.27 (95%CI: 0.54-2.86; p=0.57) for TP53 wild-type and TP53-mutant pts, respectively. In unadjusted analyses, median OS was 17.4 months for HMAm and 19.6 months for HMA+VEN, and OS did not differ significantly by treatment type (p=0.33). For HMAm treated pts, the OS rates at year 1, 2, 3 were 0.65 (95%CI: 0.63-0.67), 0.38 (95%CI: 0.36-0.40), and 0.27 (95%CI: 0.25-0.29), respectively. For HMA/VEN treated pts, the OS rates at year 1, 2, 3 were 0.64 (95%CI: 0.56-0.73), 0.45 (95%CI: 0.36-0.56), and 0.37 (95%CI: 0.27-0.51), respectively. In a multivariate analysis adjusting for age, sex, PB blast, IPSS-M and allo-SCT as a time-varying covariate, treatment with HMA/VEN vs HMAm did not significantly influence OS (hazard ratio (HR) of OS for HMA/VEN vs HMAm: 0.89, 95%CI: 0.69-1.13, p=0.33). When stratified by TP53 mutation status, HR of HMA/VEN vs. HMAm was favorable for TP53 wild-type (HR: 0.47; 95%CI: 0.29-0.74) but not TP53-mutant pts (HR=0.96; 95%CI: 0.66-1.41). Adjusting for other variables in the model, HR for pts who underwent vs. those who did not undergo allo-SCT were 0.65 (95% CI: 0.49-0.85) and 0.36 (95%CI: 0.24-0.53) in TP53 wild-type and mutant patients, respectively. Conclusion: In this large, international retrospective analysis from the icMDS VALIDATE database, HMA/VEN was associated with higher cCR rates—but not CR rates—compared to HMAm among pts with HR-MDS, though we observed no significant differences in OS. In our analysis, clinical benefit from the addition of VEN was most clear in TP53 wild-type pts, and allo-SCT was associated with improved OS regardless of baseline TP53 status. Our analysis was limited by the relatively small number of HMA/VEN-treated pts and heterogeneous real-world treatment patterns.

Abstract Background Paroxysmal nocturnal hemoglobinuria (PNH) is a rare blood disorder characterized by uncontrolled terminal complement activation leading to intravascular hemolysis (IVH), thrombosis, and premature mortality. The complement C5 inhibitor (C5i) ravulizumab provides immediate, complete, and sustained inhibition of terminal complement activity through an 8-weekly weight-based dosing interval, resulting in control of IVH, reduced morbidity (thrombosis rate) and mortality (improved survival). Pivotal trials of ravulizumab have shown durable efficacy and safety (Kulasekararaj et al. Ann Hematol 2025). While real-world evidence supporting its use is accumulating, further data are needed to more comprehensively characterize its effectiveness and safety in routine clinical practice.Aims To evaluate the real-world effectiveness and safety of ravulizumab among US patients with PNH over 1 year, based on physician chart review data. Methods Medical chart data from 127 US patients with PNH who started ravulizumab between June 1, 2021 and May 31, 2023 were randomly selected and abstracted by 50 hematologists/oncologists from academic (n=25) or community (n=25) settings. Included patients were aged ≥18 years on the date of ravulizumab initiation (index), had ≥12 months of medical data pre- and post-index, and continued ravulizumab for ≥12 months, unless the patient died. Complement inhibitor (Ci)-experienced patients had previous eculizumab or pegcetacoplan use pre-index; Ci-naive patients had no previous use of any Ci pre-index. Outcomes included changes in lactate dehydrogenase (LDH), hemoglobin (Hb), and absolute reticulocyte count (ARC) up to 12 months post-index. Breakthrough IVH (BT-IVH) events (defined as LDH ≥2 × upper limit of normal [ULN; 246 U/L] and ≥1 of the following: anemia, dyspnea, hemoglobinuria, or fatigue) were assessed from index to earliest of death, loss to follow-up, ravulizumab discontinuation, or chart abstraction. Event rate was calculated per 10 person-years (PY).Results The sample included 69 Ci-naive (mean [standard deviation; SD] age: 51.4 [14.1] years; 59.4% male) and 58 Ci-experienced patients (mean [SD] age: 45.0 [14.7] years; 51.7% male; previous eculizumab: n=52; previous pegcetacoplan: n=6). In both respective cohorts, ravulizumab was associated with significant reductions in mean (SD) LDH level at month 12 (−348 [424] U/L and −20 [75] U/L; p<0.001 and p<0.05), with improvements observed from 1-month post-index. Similarly, mean (SD) Hb levels significantly increased over 12 months (2.2 [1.8] g/dL and 1.3 [1.5] g/dL, respectively; p<0.001 for both), with improvements seen as early as 1-month post-index. Significant reductions in mean (SD) ARC were observed 1-month post-index and were maintained for up to 12 months in Ci-naive patients (−45 [52] × 103/μL; p<0.001). This effect was not observed in Ci-experienced patients (−7 [85] × 103/μL; p=0.536). Over a median follow-up of 1.8 years (Ci-naive) and 1.6 years (Ci-experienced), 4 BT-IVH events occurred in 4 Ci-naive patients (5.8%; event rate: 0.30 per 10 PY), and 7 events occurred in 5 Ci-experienced patients (8.6%; event rate: 0.69 per 10 PY). All events were resolved without transfusion, dose modification, or discontinuation of ravulizumab, and no events resulted in thrombosis. Summary/Conclusion These real-world data from US adults with PNH showed that ravulizumab was associated with early and sustained significant and clinically meaningful improvements in hematological markers for PNH, in both Ci-naive patients and those with prior eculizumab or pegcetacoplan use. Ravulizumab treatment was associated with a low incidence of BT-IVH events, and all events were resolved without the need for transfusion, dose adjustment, or treatment discontinuation. Importantly, no thrombotic events occurred during these events. Despite the known differences in data collection between clinical trial and real-world populations, these findings align with an analysis of long-term clinical trial data from Kulasekararaj et al. (2025), which reported BT-IVH in 14.8% of C5i-naive (event rate: 1.0 per 10 PY) and 7.8% of eculizumab-experienced patients (event rate: 0.33 per 10 PY) with PNH over a follow-up period of up to 6 years (median: 3.9 and 2.7 years, respectively). Overall, this analysis supports the use of ravulizumab, with only 6–7 infusions per year, as an effective treatment for patients with PNH in the real-world setting.

Abstract Background: Mosunetuzumab, a CD20xCD3 bispecific antibody, can be administered in the outpatient setting for a fixed duration. Mosunetuzumab by intravenous (IV) administration is FDA- and EMA-approved in relapsed/refractory (R/R) FL after ≥2 prior lines of therapy. Interim results from the Phase II MorningSun study (NCT05207670) demonstrated promising efficacy and manageable safety of mosunetuzumab SC in pts with previously untreated HTB FL (Flinn et al. ASCO 2025). We report 12-month follow-up efficacy and safety data of mosunetuzumab SC in pts with previously untreated HTB FL in the MorningSun study, including pts who received optional maintenance therapy. Methods: Pts with previously untreated HTB FL, per Groupe D'Etude des Lymphomes Folliculaires criteria, and Eastern Cooperative Oncology Group performance status of 0–2 were enrolled, primarily from United States (US) community sites. Mosunetuzumab SC was administered as step-up dosing in Cycle (C)1 (Day [D]1, 5mg; D8, 45mg; D15, 45mg) followed by 45mg on D1 of each 21-day cycle, for up to 17 cycles (1 year). Pts with a partial or complete metabolic response (CMR) after C17 could receive optional mosunetuzumab maintenance treatment (45mg every 8 weeks for up to 1 year). The primary endpoint was progression-free survival (PFS) rate at 1 year. Key secondary endpoints included objective response rate (ORR), duration of response (DOR), duration of complete response (DOCR) and safety. Exploratory analysis of ctDNA levels was performed using the AOA-NHL assay. Results: As of February 10, 2025, 103 pts were enrolled (n=82 pts from community sites, n=21 pts from academic sites). Overall, the median age was 65 years (range: 24–86), 51.5% of pts were female and most pts had Ann Arbor stage III/IV disease (91.3%) and a FLIPI score ≥2 (78.6%). In total, 69 pts completed initial treatment (17 cycles), 33 discontinued (primarily due to progressive disease [PD; n=18] and adverse events [AEs; n=5]), and 1 pt was continuing treatment. After 17 cycles, 68 pts were eligible for maintenance treatment, of whom 46 received maintenance, and 15 were continuing maintenance treatment at the time of data cut-off. Median duration of follow-up was 22.3 months (95% confidence interval [CI]: 20.9–24.1). The 12-month PFS rate was 85.5% (95% CI: 76.2–91.3). The ORR was 87.4% (95% CI: 79.4–93.1); 64.1% of pts had a CMR. The 12-month OS rate was 91.9% (95% CI: 84.5–95.9); the 12-month DOR and DOCR rates were 89.6% (95% CI: 80.2–94.7) and 91.1% (95% CI: 79.4–96.2), respectively. Of the 46 pts who received maintenance, no pts experienced PD, and 1 pt died (reason unknown). Of the 22 pts who were eligible but did not receive maintenance, 1 pt had PD. The most common AEs (≥30%) were injection-site reaction (67.0%), fatigue (44.7%), cytokine release syndrome ([CRS] 34.0%), headache (32.0%), and nausea (32.0%). Grade (Gr) ≥3 AEs and serious AEs were reported in 47.6% and 35.9% of pts, respectively. Five pts experienced Gr 5 AEs: COVID-19 pneumonia (n=2) and cardiogenic shock, unexplained death, pneumonia (n=1 each). Infections occurred in 77.7% of pts and were predominantly Gr 1/2 (Gr 1: 10.7%; Gr 2: 47.6%; Gr 3: 12.6%; Gr 4: 3.9%), with the most common infection being COVID-19 (24.3%). Of 3 (2.9%) Gr 5 infections reported, 1 occurred during initial treatment (COVID-19), 1 during maintenance (pneumonia), and 1 occurred 2.8 months after completion of treatment (COVID-19). AEs were reported in 80.4% of pts in the maintenance phase, mostly Gr 1/2 events. No CRS events occurred during maintenance treatment. Infections were reported in 45.7% of pts during maintenance, with the majority being Gr 1/2 events (37.0%), and were similar types of infection as during initial therapy. ctDNA levels were assessed in a subset of pts who achieved a CMR; 86.4% (19/22) of pts were minimal residual disease (MRD) negative (p-value cut-off 0.005).Conclusions: Longer follow-up of mosunetuzumab SC with optional maintenance treatment continues to demonstrate promising PFS and OS rates, and a manageable safety profile in pts with previously untreated HTB FL. Exploratory analysis of ctDNA in a subset of CMR pts showed MRD negativity in 86.4%, thus supporting fixed treatment duration of mosunetuzumab SC. Overall, these data support the outpatient administration of mosunetuzumab SC in US community practices and further evaluation of mosunetuzumab-based treatment in first-line HTB FL.

Abstract Introduction The treatment of newly diagnosed multiple myeloma (NDMM) has rapidly evolved. Quadruplet induction combining an anti-CD38 monoclonal antibody, lenalidomide, a proteasome inhibitor, and dexamethasone has become a standard of care. In this prospective randomized phase 2 trial, we evaluated the efficacy and safety of induction with a four-drug combination of daratumumab (Dara), lenalidomide (R), ixazomib (I), and dexamethasone (d) (Dara-RId) in older, transplant ineligible or deferred patients, followed by maintenance with Dara RI versus R for up to 2 years. We hypothesized that this quadruplet would offer improved tolerability with less peripheral neuropathy in this older population as well as the convenience of largely oral therapy. Herein, we present the initial results of the induction phase of the study. Methods Transplant-ineligible or deferred patients with NDMM were eligible to participate. This was a multicenter trial sponsored by the Alliance Foundation Trials, LLC, AFT-41 (NCT04009109). Each cycle of treatment was 28 days. All patients received 12 cycles of induction therapy with daratumumab 1800 mg subcutaneously on the conventional schedule, lenalidomide 15 mg orally (po) on days 1-21, ixazomib 4 mg po on days 1, 8, 15, and dexamethasone weekly. After 12 cycles, patients received maintenance based on prior randomization (1:1) to Dara-RI or R for up to 2 years. In maintenance, R was reduced to 10 mg and I was reduced to 3 mg. Randomization assignment occurred prior to starting induction. Stratification was based on presence of high-risk features defined as ISS stage III or high-risk FISH cytogenetics (del 17, t (14;16), t (14;20), t (4;14), del (1p), or gain (1q)). The primary endpoint was progression-free survival (PFS). Secondary endpoints included objective response rate (ORR) and adverse events. Results We enrolled 79 patients across 7 sites from October 2020 until December 2023. Accrual was affected by the COVID19 pandemic, leading to early closure to accrual in December 2023. Median age was 74 years (range 63-86); ≥75 years of age (45.6%); female (59.5%); white (83.5%), African American (7.6%), Asian (1.3%), and Hispanic ethnicity (6.3%). Additional baseline characteristics included ISS staging I (41.8%), II (35.4%), and III (22.8%) with high-risk features in 37% by ISS stage III and/or high-risk FISH. The 12-month PFS was 92% (95% confidence interval [CI] 86.1-98.4%). The ORR (≥ partial response [PR]) during induction was 92.4% (PR 22.8%; very good partial response [VGPR] 46.8%; complete response 15.2%; stringent complete response 7.6%). Notable grade ≥3 adverse events included neutropenia (16.5%), infections (11.4%), anemia (10.1%), thrombocytopenia (8.9%), and syncope (7.6%). There was no grade ≥3 peripheral neuropathy (grade 1, 15%; grade 2, 14%). No treatment-related mortality was reported. Sixty-one patients went on to maintenance therapy and seven patients discontinued treatment for adverse events during induction (8.9%). Conclusions Dara-RId therapy in NDMM appears to be efficacious with an ORR of 92.4%; VGPR or better of 69.6% and 12-month PFS of 92%. The combination was generally tolerated well with manageable toxicity, despite 45.6% of patients being ≥75, offering the convenience of an oral proteasome inhibitor, ixazomib, that minimizes the risk of peripheral neuropathy for older, transplant-ineligible or deferred patients. Follow-up is ongoing to determine the benefit of maintenance with Dara-RI v. R alone in this population. Support: AFT, Celgene, Janssen, Takeda.