Chimeric antigen receptor (CAR) T-cell (CAR-T) treatment for B-cell acute lymphoblastic leukemia (ALL) induces high initial response rates, but most patients relapse. Low disease burden (often defined as <5% blasts in the bone marrow) is associated with better outcomes. CAR HEMATOTOX (HT) is a score using prelymphodepletion hematologic and inflammatory parameters to predict outcomes in lymphoma. Here, we assess its prognostic utility in a large multicenter adult B-cell ALL cohort. Patients who received brexucabtagene autoleucel across 33 centers in North America were included as part of the Real-World Outcomes Collaborative of CAR-T in Adult ALL (ROCCA) consortium. An independent cohort of 61 patients with ALL treated with an investigational CD19 CAR-T therapy at 1 center was also described. Among 199 ROCCA consortium patients, 43 (22%) patients with HTlow scores had lower rates of delayed neutrophil recovery than those with HThigh scores (26% vs 52%, P =.002) and fewer severe infections (2.5% vs 18.8%, P =.011). They also had higher response rates, overall survival (OS), and event-free survival (EFS), as well as lower nonrelapse mortality and cumulative incidence of relapse. The survival differences remained significant after multivariable adjustment for disease burden and other covariates. In the investigational cohort of 61 patients, patients with HTlow scores had improved OS and EFS, as well as higher peak CAR-T expansion. In summary, CAR HT score is a prognostic factor independent of disease burden in adult ALL. HTlow score is associated with superior outcomes after CD19 CAR-Ts and higher CAR-T expansion in a single-center cohort. These trials were registered at www.clinicaltrials.gov as #NCT01044069 and #NCT01860937.

Brexucabtagene autoleucel (brexu-cel) is an autologous anti-CD19 chimeric antigen receptor T (CAR T) cell therapy approved for adults with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL). Despite encouraging outcomes, the majority of adults relapse following brexu-cel, highlighting the need for strategies to improve the durability of response. One such strategy is consolidative allogeneic stem cell transplant (alloHCT). Here, we report outcomes of adults with B-ALL who underwent consolidative alloHCT following commercial brexu-cel as part of the Real-World Outcomes Collaborative of CAR-T in Adult ALL (ROCCA). We performed a retrospective multicenter analysis of adults with R/R B-ALL who underwent consolidative alloHCT in complete remission after commercial brexu-cel and were registered in ROCCA. ROCCA includes 41 US institutions contributing retrospective data for adults with B-ALL treated with brexu-cel between 2021 and 2025. Patients who received alloHCT after CAR T failure were excluded. Primary endpoints were 12-mo overall survival (OS) and event-free survival (EFS); secondary endpoints included relapse, non-relapse mortality (NRM), and graft-versus-host disease (GVHD). Among 399 brexu-cel-treated patients, 65 underwent consolidative alloHCT, including 56 patients who underwent a first alloHCT and nine who underwent second alloHCT. Among recipients of first alloHCT, the median age was 34 yr, and patients were heavily pretreated with a median of three prior lines of therapy. With a median follow-up of 11 mo post-HCT, the estimated 1-yr post-HCT OS and EFS were 79% (95% CI, 64 to 88) and 66% (95% CI, 51 to 77), respectively. The 1-yr cumulative incidence (CI) of relapse was 19%, and NRM was 13%. Acute grade II to IV GVHD occurred in 18%, grade III to IV in 4%, and moderate-to-severe chronic GVHD in 12%. In univariable analysis, age ≥40 was associated with inferior OS (HR 4.31, 95% CI 1.40 to 13.3) and EFS (HR 3.36, 95% CI 1.43 to 7.91), whereas myeloablative conditioning was associated with improved EFS (HR 0.37, 95% CI 0.15 to 0.93). Among second alloHCT recipients, 1-yr EFS and OS were 59% (95% CI, 19 to 85). The 1-yr CI NRM was 41% and there were no relapses documented among these patients. In this large real-world cohort, consolidative alloHCT after brexu-cel was feasible and associated with encouraging survival, low relapse rates, and acceptable toxicity, particularly among HCT-naive recipients. These findings support alloHCT as a viable consolidation strategy following CAR T-induced remission and highlight the need for prospective studies to refine patient selection, conditioning regimens and transplant approaches in the post-CAR T setting.

In the single-arm Phase Ib/II FELIX study (NCT04404660), obecabtagene autoleucel (obe-cel; CD19-directed autologous CAR T-cell therapy) demonstrated high overall remission rates (ORR) and a favorable safety profile in adults with relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). To contextualize results from FELIX, we compared the efficacy and safety of obe-cel with matched external control arms (ECA) derived from historical trials using propensity score matching. ECAs represented standard of care (SoC) non-CAR T-cell therapies: blinatumomab, inotuzumab ozogamicin, and conventional chemotherapy. The primary endpoint was ORR; secondary endpoints included overall survival (OS). Event-free survival (EFS) and safety were exploratory endpoints. Among the intent-to-treat population in FELIX (n = 107), obe-cel demonstrated significantly higher ORR than non-CAR T-cell therapies (67.3% vs 51.4%; odds ratio 1.9; p = 0.0257). Median OS was longer with obe-cel when censoring for hematopoietic stem cell transplant (15.1 vs 7.0 months; p = 0.0015) and without censoring (13.9 vs 7.8 months; p = 0.0430). EFS was significantly improved with obe-cel (median 9.8 vs 2.5 months; p < 0.0001). Safety profiles were comparable between groups, with similar rates of Grade ≥3 adverse events. Obe-cel offers superior remission rates and survival benefits over current SoC non-CAR T-cell therapies, with an acceptable safety profile; its use could address unmet needs in adult R/R B-ALL.

ABSTRACT:Belumosudil was approved by the Food and Drug Administration in the United States for the treatment of relapsed/refractory chronic graft-versus-host disease (cGVHD) based on a randomized phase 2 trial comparing 2 belumosudil doses. The efficacy and safety of belumosudil vs the best available therapy (BAT) has not been studied. Applying rigorous statistical methodology to real-world data, this study estimated the efficacy of belumosudil vs BAT in cGVHD patients whose disease failed to respond to 2 to 5 prior lines of therapy (LOTs). Retrospective data between March 2015 and 2024 were collected across 8 US sites for 196 patients, contributing 113 belumosudil and 245 BAT LOTs. The primary outcome was 6-month overall response rate (ORR), defined as the proportion of complete or partial responses based on 2014 National Institutes of Health consensus criteria, physician assessment, or corticosteroid dose taper of ≥50% without cGVHD progression. Death, relapse, and beginning a new LOT were considered a lack of response. Targeted maximum likelihood estimation (TMLE) was used to estimate the 6-month ORR following belumosudil vs BAT (38.7% vs 26.8%, respectively) or 44.2% improvement with belumosudil (1-sided 95% confidence interval [CI], [4.4 to ∞]; P = .031). TMLE was also used to estimate 1-year failure-free survival when treated with belumosudil (61.2%) or BAT (47.8%), a 13.5% difference (95% CI, 1.5-100; P = .032). Descriptive assessment of safety showed adverse events recorded in 27% of belumosudil and 36% of BAT LOTs. Findings demonstrated that belumosudil improved clinical outcomes compared to BAT in cGVHD patients with 2 to 5 prior LOTs, and safety was consistent with belumosudil's established profile.

Brexucabtagene autoleucel (brexu-cel) is a chimeric antigen receptor T (CAR T) cell therapy approved for adults with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). We studied the impact of social determinants of health (SDoH) on outcomes of adults with B-ALL receiving brexu-cel. This retrospective analysis included adults (≥18 years) with R/R B-ALL treated with brexu-cel between 2021 and 2023. Cox proportional hazards models evaluated the association of race, ethnicity, and SDoH with progression-free survival (PFS) and overall survival (OS). 189 patients received brexu-cel and 57% were male. 55% were non-Hispanic White, 30% Hispanic, 7% non-Hispanic Black, 6% Asian/Pacific Islander, and 2% other/unknown. 43% were referred from private/community-based practices and 35% lived 50 miles or greater from the CAR T center. Health insurance included public (47%) and private (41%). 31% had a high social deprivation index (SDI, 76–99th percentile). Black race was associated with worse OS (HR 3.48; 95% CI 1.01–12.03). There was no difference in PFS (HR 1.03, 95% CI 0.50–2.10) or OS (HR 1.43; 95% CI 0.56–3.65) in Hispanic patients. Outcomes appear independent of SDoH and SDoH did not impact OS. We observed comparable outcomes to non-Hispanic patients.

Background: Obe-cel is an autologous fast off-rate 4-1BB-ζ CD19-directed CAR T-cell therapy with demonstrated clinical efficacy in adult patients with R/R B-ALL from the Phase Ib/II FELIX study (NCT04404660; Roddie et al. NEJM 2024). Following treatment with obe-cel, 78.0% of patients achieved complete remission (CR) or CR with incomplete hematologic recovery (CRi); 24-month event-free survival (EFS) and overall survival (OS) probabilities were 43.0% and 46.0%, respectively. The identification of factors that could predict response and long-term survival outcomes can help clinical decision making. CAR T-cell persistence, as a time-dependent variable, was identified as an important predictive factor for EFS and OS following obe-cel infusion in multivariate analyses (Park et al. EHA 2025). Here, we report the impact of persistence status at Month 3 (M3) on survival outcomes in adult patients with R/R B-ALL who responded to obe-cel, and in patients with deep measurable residual disease (MRD)-negative remission who did not receive post obe-cel stem cell transplant (SCT). Methods: In FELIX, adult (≥18 years) patients with R/R B-ALL underwent lymphodepletion, followed by tumor burden-guided infusions of obe-cel to a total target dose of 410×106 CAR T-cells. Droplet digital polymerase chain reaction (ddPCR) was used to assess CAR transgene levels in peripheral blood samples collected from patients infused with at least one dose of obe-cel (N=127). Vector copy number per diploid genome was determined through duplex detection of the lentiviral vector Ψ region and the endogenous reference gene, RPP30. Samples with measurements above the lower limit of detection (LLoD; 11 copies/reaction) were identified as CAR T-cell positive; measurements below the LLoD indicated loss of CAR T-cell persistence. M3 landmark Kaplan-Meier analyses were conducted to evaluate EFS and OS by persistence status (ongoing vs loss of persistence) in patients who responded to obe-cel and were in ongoing remission beyond M3, and in patients who also had deep MRD-negative remission (defined as <10–6 leukemic cells) by next-generation sequencing (NGS) and had not undergone SCT post obe-cel. Results: As of 18 January 2025, the median study follow up was 32.8 months (range: 19.9–52.8). Of the 99 patients who achieved CR/CRi, 79 (79.8%) had ongoing remission beyond M3 post obe-cel infusion and comprised the subgroup of interest for the analyses reported herein. At M3 post infusion, 60/79 patients (75.9%) had ongoing CAR T-cell persistence, while 19/79 (24.1%) had lost persistence. Median EFS (95% confidence interval) was 44.6 months (18.9–not estimable [NE]) in patients with ongoing persistence at M3 post obe-cel vs 10.1 months (7.2–NE) in patients who had lost persistence. The 24-month EFS probability was 59.9% (46.0–71.3) vs 31.6% (12.9–52.2), and the 24-month OS probability was 69.2% (55.6–79.4) vs 52.6% (28.7–71.9), in patients with ongoing vs loss of persistence at M3, respectively. Among the 79 patients with ongoing remission at M3, 70 did not receive post obe-cel SCT. The median EFS and OS, and 24-month probabilities in these 70 patients, by persistence status at M3, aligned with those reported in the 79 patients with ongoing remission. Among the 70 patients without post obe-cel SCT, 54 had NGS calibration and 49/54 patients (90.7%) achieved deep MRD-negative remission by M3. In these 49 patients, median EFS was not reached vs 9.1 months (4.4–NE), 24-month EFS probability was 56.4% (38.7–70.7) vs 33.3% (7.8–62.3), and 24-month OS probability was 68.4% (50.9–80.7) vs 55.6% (20.4–80.5), in patients with (n=40) and those without persistence (n=9) at M3, respectively. Median OS was not reached for patients with or those without persistence at M3. Conclusions:In patients who remained in remission beyond M3, including those with deep MRD-negative remission and no post obe-cel SCT, ongoing CAR T-cell persistence at M3, measured by ddPCR, was associated with longer EFS and OS compared with loss of persistence. Persistence status at M3 may be a marker for predicting long-term outcomes following obe-cel treatment in patients with R/R B-ALL.

Accurate and comprehensive genetic characterization of acute myeloid leukemia (AML) is essential for diagnosis, prognostication, and treatment selection. We report here, in 255 adults with AML enrolled in a prospective clinical protocol at 18 major cancer centers across the USA, the results of whole genome DNA-sequencing (WGS) at diagnosis and post-treatment remission. WGS effectively recapitulated, and frequently identified genetic alterations missed by, conventional standard of care clinical testing. These new findings included important prognostic and predictive biomarkers, copy number alterations, regulatory element, splicing, and structural variants including partial tandem duplications within KMT2A. All patients had a pathogenic variant detected at diagnosis, and approximately ten percent also had evidence of a potential inherited myeloid malignancy predisposition. This comprehensive atlas of adult AML genomics provides novel insights into disease biology, creates an evidentiary basis to support clinical testing improvements, and is a resource for both diagnostics and drug development. . Statement of Significance:Acute myeloid leukemia is a diagnostic category encompassing multiple rare hematological malignances. We show, in this nationwide multicenter study, that standardized unbiased whole genome DNA-sequencing and disease-optimized bioinformatics can replicate conventional "standard of care" AML clinical testing results, while also revealing currently underdiagnosed AML disease biology and potential genetic predisposition.

Aberrant activation of the RAS/MAPK signaling limits the clinical efficacy of several targeted therapies in acute myeloid leukemia (AML). In FLT3-mutant AML, the selection of clones harboring heterogeneous RAS mutations drives resistance to FLT3 inhibitors (FLT3i). RAS activation is also associated with resistance to other AML targeted therapies, including the BCL2 inhibitor venetoclax. Despite the critical need to inhibit RAS/MAPK signaling in AML, no targeted therapies have demonstrated clinical benefit in RAS-driven AML. To address this unmet need, we investigated the preclinical activity of RMC-7977, a multi-selective inhibitor of GTP-bound active [RAS(ON)] isoforms of mutant and wild-type RAS in AML models. RMC-7977 exhibited potent antiproliferative and pro-apoptotic activity across AML cell lines with MAPK-activating signaling mutations. In cell line models with acquired FLT3i resistance due to secondary RAS mutations, treatment with RMC-7977 restored sensitivity to FLT3i. Similarly, RMC-7977 effectively reversed resistance to venetoclax in RAS-addicted cell line models with both RAS wild-type and mutant genetic backgrounds. In murine patient-derived xenograft models of RAS-mutant AML, RMC-7977 was well tolerated and significantly suppressed leukemic burden in combination with gilteritinib or venetoclax. Our findings strongly support clinical investigation of broad-spectrum RAS(ON) inhibition in AML to treat and potentially prevent drug resistance due to activated RAS signaling.

Introduction: Blinatumomab (Blin), a CD19xCD3 bispecific T-cell engager, is recommended in the consolidation phase of front-line therapy for Philadelphia (Ph) negative B-cell acute lymphoblastic leukemia (B-ALL) on the basis of the ECOG-ACRIN 1910 (E1910) study. On the E1910 regimen, patients were randomized to receive consolidation chemotherapy with or without Blin if they achieved and maintained an MRD negative complete remission with three courses of BFM-like induction and intensification, with a significant survival advantage for those who received Blin. Due to toxicities of the initial chemotherapy or progression prior to the randomization, only 59% of patients were randomized. This suggests that alternative approaches to initial chemotherapy debulking of leukemia burden, and possibly earlier implementation of Blin, may be preferable. The MD Anderson Cancer Center (MDACC) also has published a regimen incorporating Blin into hyperCVAD (cyclophosphamide, vincristine, doxorubicin, dexamethasone) therapy as induction and consolidation for adults with Ph neg B-ALL. We have used a modification of the hyperCVAD/Blin approach at the University of California San Francisco for adult patients and report here our initial results. Methods: A modification to the MDACC hyperCVAD/Blin regimen was used in which all patients received hyperCVAD cycles (C) and Blin courses in the following order: C1A-C1B-Blin1-Blin2-C2A-C2B-Blin3-Blin4-maintenance (POMP with quarterly blinatumomab). This approach was adopted to ensure all patients follow the same sequence and to provide exposure to Blin after no more than two chemotherapy cycles. Two doses of rituximab were included in each of the first 4 cycles of therapy when CD20 expression was identified on ≥20% of blasts at diagnosis. Intrathecal prophylaxis consisted of alternating doses of methotrexate and cytarabine, with a goal of administering 2 doses per cycle for a minimum of 8 doses. Diagnostic bone marrow biopsies were evaluated by conventional cytogenetics, ALL FISH, and NGS using Foundation One Heme. Remission was assessed by bone marrow biopsy with flow and/or NGS MRD quantification. Results: Twenty-two adult patients (median age 36, range 23-60) initiated treatment with the modified hyperCVAD/Blin regimen from July 2023 to May 2025. The population was 72% male and 64% Hispanic. High risk (HR) B-ALL (68%) was defined as genotypes including Ph-like lesions (36% of total population had CRLF2/JAK2 abnormalities), KMT2A rearrangement (9%), complex karyotype (14%), and TP53 mutations (4.5%), or therapy-related ALL (after prior treatment for multiple myeloma, 4.5%). Median follow-up for the group is 18 months (mos). The complete remission rate after C1A was 82% (53% and 7.7% MRD negative by flow and NGS, respectively), after C1B was 95% (71%/22% MRD negative by flow/NGS), and after completion of one or two cycles of Blin (cycle number dependent on timing of subsequent transplant in those selected to proceed) was 95% (100%/81% MRD negative by flow/NGS). Median RFS/OS have not been reached, with 1 year RFS 66% and OS 94%. In patients with standard risk Ph neg B-ALL, RFS remains 100% with median follow-up 21 months in that subset, with 1 of 7 having undergone CR1 alloHCT due to persistent MRD after Blin. In patients with HR B-ALL, the median RFS is 10.9 mos with median follow-up of 15 months. Eight HR patients (36%) proceeded to alloHCT in CR1 resulting in median RFS 14.8 months and OS 16.5 months. Amongst patients who could not proceed to alloHCT due to lack of adequate social support (n=6), the RFS was reduced at a median 4 months, but OS is 16.6 months with current followup. Conclusions: The addition of Blin to front-line hyperCVAD chemotherapy is associated with excellent RFS in adults with standard-risk ALL, but patients with HR genotypes remain at risk of relapse within one year of treatment initiation. This demonstrates a need for further iteration on this approach to better mitigate the risk of relapse. Because alloHCT remains the best approach to sustain long-term definitive disease control, it remains especially important to navigate patients to potentially curative transplants after achieving deep remission with the first 3-4 cycles of chemo/Blin. In adults unable to proceed to transplant in a timely manner due to limited social support, outcomes remain suboptimal and additional approaches to prevent relapse need further exploration.

The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Hematopoietic Cell Transplantation (HCT) provide expert, multidisciplinary recommendations for autologous and allogeneic HCT based on current evidence. The NCCN Guidelines focus on pretransplant recipient evaluation, hematopoietic cell mobilization, conditioning, and the diagnosis and management of graft-versus-host disease (GVHD), a common complication of allogeneic HCT that is associated with significant morbidity. This selection from the NCCN Guidelines for HCT focuses on the diagnosis, workup, and management of acute and chronic GVHD, as well as important supportive care considerations. J Natl Compr Canc Netw 2025;23(10):e250047 doi:10.6004/jnccn.2025.0047

There remains an unmet clinical need for improved treatment strategies in Acute Myeloid Leukemia (AML). Although radiopharmaceutical therapies targeting non-cancer-selective antigens have shown promise in AML, their clinical utility is often limited by prolonged bone marrow suppression. Using a unique proteomics-based strategy, we recently identified the active conformation of integrin-β2 (aITGB2) as a novel, tumor-selective target for AML. Importantly, this conformational epitope is expressed widely on AML cells but minimally on normal marrow progenitors/healthy tissues. Here we first confirmed widespread aITGB2 expression on AML tumors that was largely independent of tumor genotype or prior therapeutic regimen. We developed diagnostic and therapeutic radiopharmaceuticals targeting aITGB2 utilizing a conformation-specific antibody (clone 7065). PET/CT imaging with 89Zr and 134Ce-labeled 7065 in AML models revealed high target-mediated uptake, greater than that compared to standard of care [18F]-FDG. PET/CT imaging with [89Zr]DFO*-7065 showed reduced binding to normal bone marrow and immune cells in humanized immune system mice compared to [89Zr]DFO*-anti-CD33. For therapy, we developed [225Ac]Macropa-PEG4-7065 using an optimized chelator-linker combination. Treatment with [225Ac]Macropa-PEG4-7065 in Nomo-1 and PDX AML disseminated models delayed tumor growth and improved overall survival compared to controls, including [225Ac]DOTA-anti-CD33, a clinical stage-radioimmunotherapy under evaluation in AML. Relapsed tumors demonstrated persistent aITGB2 expression, supporting continued development of fractionated dosing schemes, and proteomics analysis indicated activation of TCA cycle and carbon metabolism pathways, consistent with therapy-induced stress responses. These findings highlight [89Zr]DFO*-7065 and [225Ac]Macropa-7065 as a promising aITGB2-targeted theranostic pair with potential for imaging and treatment in future clinical translation.

Background: The Phase Ib/II FELIX study (NCT04404660) evaluated obe-cel, a CD19-directed autologous CAR T-cell therapy with a fast off-rate binding domain designed to improve persistence and minimize severe immunotoxicity, in adults with relapsed/refractory B-cell acute lymphoblastic leukemia. Patients achieved a high overall remission rate (ORR) with low incidence of Grade ≥3 cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome. While CAR T-cell therapy has been used for over a decade, the association between specific drug product (DP)/leukapheresis product (LP) phenotypes and clinical outcomes remains poorly characterized. This analysis investigated how DP phenotypes correlate with treatment outcomes following infusion with obe-cel. Methods: Phenotypic markers of memory, activation, and exhaustion were assessed via an 18-parameter flow cytometry panel in CD3+, CD4+, or CD8+ CAR+ cells from LP (47 phenotype features) and DP (48 phenotype features) samples in FELIX. Memory subsets were defined by CCR7/CD45RA expression; activation by CD25 and HLADR; and exhaustion by LAG3, TIM3, PD-1, or TIGIT positivity. Univariate Cox regression analysis (UVA) in the Phase Ib discovery cohort (n=16) was used to identify OS-associated features (p<0.1), which were validated in the Phase II validation cohort (n=107) and tested for association with event-free survival (EFS) and ORR, in the combined cohort (n=123). Multivariate analysis (MVA) models of the combined cohort were used to evaluate features associated with outcomes after adjustment for key clinical covariates that were previously described in Park et al. EHA 2025. The OS MVA model included: Philadelphia chromosome status, prior inotuzumab ozogamicin (InO), bone marrow blasts at lymphodepletion, and CAR T-cell persistence (time-varying). The EFS MVA model substituted prior InO with the number of prior lines of therapy and prior stem cell transplant. All continuous features were scaled and centered, therefore odds ratios (OR) and hazard ratios (HR) reflect one standard deviation from the mean. Results: In the DP samples, 15 phenotypic features were associated with OS in the discovery cohort (p<0.1) and nine were confirmed in the validation cohort (p<0.1).The validated features associated with longer OS included: a higher percentage of central memory cells (Tcm; in CD3+, CD4+, and CD8+ cells), one T-cell activation phenotype (CD25+ HLADR+ in CD8+ cells), and four exhaustion phenotypes (measured by combinations of TIM3, PD-1 and LAG3). Having a higher percentage of terminal effector memory (TEMRA) T-cells in the DP was associated with shorter OS. None of the nine phenotypic features validated in the DP samples were also validated in the LP samples. In the LP samples (n=122), only the expression of CD25+ HLADR+ in CD4+ cells was associated with shorterOSin both the Phase Ib and II cohorts (UVA). In the UVA of the combined cohort (n=123), having a higher Tcm percentage in CD4+ cells in the DP was significantly associated with improving EFS (HR: 0.735, p=0.035) and ORR (OR: 2.061, p=0.026). A higher Tcm percentage in CD4+ cells in the DP was also an independent predictor of longer OS (HR: 0.673, p=0.008) when adjusted for previously identified key clinical covariates. In the LP, cell phenotypes only showed a weak correlation with those in the DP (median Spearman's rho: 0.173, range: –0.033–0.594). A higher percentage of CD25+ HLADR+ CD4+ cells in the LP independently predicted worse clinical outcomes (EFS HR: 1.510, p=1.54x10-6; OS HR: 1.248, p=0.008) after adjusting for key clinical covariates. Conclusions: A higher Tcm percentage in the DP samples was an independent predictor of positive clinical outcomes, including OS, following obe-cel infusion. While the T-cell phenotype composition in the LP was weakly correlated with that in the DP, CD25+ HLADR+ CD4+ cells in the LP independently predicted less favorable clinical outcomes. However, other factors (e.g. tumor characteristics) are also likely to affect outcomes; therefore, further investigations are needed to better understand and predict favorable clinical outcomes, and to potentially guide studies of additional cell manipulations during CAR T-cell manufacturing.

Background Despite recent therapeutic advances, 5-year survival for AML in adults remains ~35% (SEER estimate). Immunotherapies have shown limited efficacy in AML largely due to the lack of surface antigens that clearly distinguish malignant cells from normal hematopoietic counterparts. Conventional transcriptomics and proteomics have only found targets lacking clear distinction between AML, myeloid cells, and hematopoietic stem and progenitor cells (HSPCs). Here, we employed high-resolution surface proteomics of primary leukemia bone marrow aspirates to identify both tumor-enriched canonical surface proteins as well as a new class of “non-canonical” surface protein targets, i.e. typically intracellular proteins aberrantly presented on the AML cell surface. Methods We used periodate-based cell surface-capture (CSC) and lysine-targeting (sulfo-NHS) labeling strategies, analyzed with both DDA and DIA proteomic methods, to profile surface proteins in newly diagnosed (n=35), relapsed (n=25), and remission (n=16) primary AML samples. An independent cohort (n=38) was used for target validation, with assessment in peripheral blood mononuclear cells and HSPCs for off-tumor expression. Plasma membrane localization of non-canonical targets was validated using flow cytometry and Western blotting. Interactors of canonical and non-canonical membrane proteins were evaluated through a recently-described multiscale photocatalytic proximity labeling strategy (Lin et al., Science (2024)). For promising AML-specific target DC-SIGN/CD209 we synthesized an MMAE-based antibody-drug conjugate (ADC). Results Canonical cell surfaceome analysis revealed a distinctive expression of potential targets including CD209, CD300LB, LILRA1 amongst >150 significantly upregulated surface proteins in AML as compared to remission samples. Single-cell RNA-seq analysis of publicly available datasets supported AML specificity of these targets. Flow cytometry validated expression of CD209 across 5 cell line models and primary AML (n=15), with protein expression independent of genomic alterations. In non-canonical surfaceome analysis, we identified a preponderance of RNA binding proteins, consistent with our recent findings in other cell lines (Perr et al, Cell (2025)). However, we also identified many Golgi-, ER- and Mitochondrial-annotated proteins as possible AML-specific cell surface antigens given absence in remission sample surface proteomic data. Nine of these non-canonical surface antigens were confirmed to be surface-localized on AML cells by flow cytometry (n=13). Flow cytometry confirmed particularly strong expression of GORASP2 on the AML cell surface, whereas HSPC flow cytometry showed no expression of either CD209 or GORASP2. Further in silico analysis, Western blotting, and super-resolution imaging confirmed localized clusters of GORASP2, a canonical Golgi body protein, on the AML plasma membrane. Multiscale photocatalytic-based proximity labeling proteomics validated that GORASP2 interacted with a similar set of canonical membrane proteins as known surface antigens ITGB2 and CD209. An antibody-drug conjugate developed against CD209 demonstrated strong preclinical efficacy both in vitro and in vivo versus AML cell line models. The in vitro ADCcytotoxic assay targeting CD209 showed prominent efficacy with IC50 ranging 1.5nM to 3nM in various AML cell lines. Ex vivo primary AML cytotoxicity assays showed killing in an antigen dependent manner. Treatment with CD209 ADC in Nomo-1 AML disseminated murine model suppressed tumor growth and improved overall survival compared to controls. Conclusion Our study provides a comprehensive surfaceome landscape of AML, identifying both canonical and non-canonical membrane proteins as promising immunotherapy targets. The successful validation of these targets across multiple AML lines and primary samples, with absent expression on healthy cells or HSPCs, validates their potential as AML-specific immunotherapy targets. Preclinical efficacy of a CD209-targeting ADC underscores the ability to develop novel AML-selective therapies based on this data. This work will also drive fundamental investigation into new areas of AML tumor biology and surface protein trafficking.

Background Chronic graft-versus-host disease (cGVHD) is a pleomorphic clinical entity that causes significant morbidity and mortality after allogeneic hematopoietic cell transplant (alloHCT). The clinical heterogeneity of cGVHD poses challenges for standardized diagnosis and treatment. While assessment tools such as the NIH cGVHD scoring system are invaluable for enrolling patients in clinical trials and assessing treatment responses, these clinical tools capture only a subset of cGVHD manifestations and rely on clinician interpretation. Unbiased, comprehensive approaches for capturing cGVHD are needed to better reflect the full spectrum of clinical symptoms and to facilitate improved diagnosis, treatment and assessment of response. Methods To better understand the depth and diversity of cGVHD manifestations, we developed a large language model (LLM) pipeline to extract symptoms and clinical events from unstructured progress notes. The pipeline combines extraction and self-reflection steps with retrieval-augmented generation using the CTCAE term database, enabling accurate extraction of diverse clinical descriptions to a standardized vocabulary. Using data from a de-identified electronic health record (EHR) database, we applied this pipeline to 14,276 outpatient hematology progress notes written for a single-center cohort of 523 alloHCT recipients (median 24 notes per patient; range 1–127) between day +100 and three years post-transplant. Results Our pipeline identified 52,060 events with a median of 3 events per note and 79 cumulative events per patient. Internal validation of the pipeline against manual annotations by hematologists and hematology fellows demonstrated that the LLM accurately identified events with a recall of 93% and a precision of 55% for events in the same organ system. Lower precision relative to recall reflected extraction of symptoms documented but not clearly attributed to cGHVD in the progress notes. External validation, based on concordance with medication prescriptions, showed that extracted symptoms were associated with a 5.04-fold increase in new prescriptions for appropriate supportive medications compared to the cohort baseline. Our pipeline identified high rates of known cGVHD manifestations in the cohort with 61% of patients experiencing at least one of eye symptoms, oral involvement, or skin hyperpigmentation. To identify additional cGVHD manifestations, we analyzed which symptoms significantly co-occurred with symptoms specific to cGVHD (i.e. ocular, oral, genitourinary, and sclerotic manifestations). At a false discovery rate of 5% we found symptoms frequently occurring outside of the NIH scoring system included muscle cramps (19%) and nasal symptoms (5%). Our analysis also identified known rare manifestations of cGVHD including pericarditis (1.3%) and capillary leak syndrome (0.9%). We next studied how these symptoms that are not scorable using the NIH scoring system correlated with response to immunosuppression. From our cohort, we identified 281 progress notes for 143 patients that were written in the two weeks prior to the patient being started on systemic corticosteroids. Of these patients, 58 were steroid refractory/dependent and received a second line immunosuppressant such as ruxolitinib (76% of patients), belumosudil (14%), or ibrutinib (16%). Presence of any non-scorable symptom (i.e. muscle symptom, nasal/sinus symptoms, serositis, or capillary leak syndrome) in the two weeks leading up to the start of corticosteroid therapy occurred more frequently in patients who required additional immunosuppression (11/58, 19.0%) compared those who received prednisone alone (6/85, 7.1%) with an odds ratio of 0.32 (pvalue 0.04). Conclusions Our initial findings demonstrate that LLMs can be a powerful tool for capturing the clinical heterogeneity of and quantifying the symptom burden of cGVHD in an unbiased manner. This approach enabled the construction of a comprehensive database of events post-transplant. We anticipate that this database will facilitate future investigation into distinct patterns of cGVHD activity, creating well-calibrated metrics of disease severity, and predicting response to immunosuppressive therapy.

Introduction: Belumosudil (BEL) is a ROCK2 inhibitor approved by the US FDA for the treatment of steroid-refractory chronic graft-versus-host disease (cGVHD) after ≥2 prior lines of therapy (LOT) in patients 12 years or older. While clinical trials such as ROCKstar demonstrated high response rates and steroid-sparing effects, real-world treatment patterns, sequencing, and optimal timing of BEL initiation in the treatment sequence remain unclear. This study investigates real-world BEL use patterns by LOT across different practice settings. Methods: The study was conducted using two complementary databases: ROCKreal (academic transplant centers) and MarketScan (mixed academic/community settings) in patients receiving BEL LOTs 3-6. Patient eligibility criteria included age ≥12 years, documented cGVHD diagnosis, and BEL initiation in LOTs 3-6 between March 1, 2015 - March 27, 2024. The MarketScan analysis required continuous enrollment for ≥6 months prior to first BEL initiation, with exclusion of patients with prior cancer relapse. Primary objectives included characterizing treatment patterns and sequencing prior to BEL initiation across both databases. Secondary objectives included describing patient characteristics and comparing overall response rates (ORR) at 6-months for BEL use in ROCKreal database across subsequent LOTs and assessing reasons for switching to BEL. Results: The current analysis included 113 patients from ROCKreal. The most common treatment prior to BEL was ruxolitinib (RUX) alone or in combination with calcineurin inhibitors (CNIs) or glucocorticoids (GCs), which was consistent across all LOTs. In Marketscan, there were 87 patients initiating BEL and a similar proportion had prior exposure to RUX. In the ROCKreal database, concurrent RUX and BEL was used in 21% and 33% in LOT 3 and LOTs 4-6, respectively. In MarketScan, concurrent use of RUX and BEL was observed in 39% and 43% of patients in BEL LOT 3 and LOTs 4-6, respectively, though the duration of concurrent therapy remains unknown. Primary reasons for switching to BEL in ROCKreal were lack of efficacy (39.6%), physician decision (27.3%), or adverse events (18.0%). In ROCKreal, patients in BEL LOTs 4-6 had similar organ involvement at BEL initiation (median [inter quartile range, IQR]): 3.0 [2.0, 4.0] compared to BEL LOT 3: 3.0 [2.0, 3.5], though the upper quartile was slightly higher in later lines. The most commonly involved organs were skin (69% in BEL LOT 3, 80% in LOTs 4-6), eyes (65% in LOT 3, 64% in LOTs 4-6), and muscles/joints/fascia (35% in LOT 3, 64% in LOTs 4-6). Pulmonary involvement was present in 33% of LOT 3 and 30% of LOTs 4-6 patients. Comorbidities were common, with pulmonary (56% in LOT 3, 43% in LOTs 4-6) and cardiovascular (41% in LOT 3, 3% in LOTs 4-6) conditions being prevalent. In the ROCKreal data, patients initiating BEL in LOT 3 or LOTs 4-6 both had responses exceeding the clinically meaningful threshold of 30%; (ORR at 6 months 42.9% vs. 35.7%). Additional analyses of the MarketScan data are ongoing. Conclusions: These real-world data demonstrate consistent use of BEL across practice settings, including frequent use of BEL subsequent to treatment with RUX and GCs. The extent of concurrent use of BEL and RUX remains to be further elucidated before the frequency of this approach in real-world management of advanced cGVHD can be fully characterized. In spite of common use of RUX in pre-BEL LOT, ROCKreal data confirm clinically significant 6 month response rates in patients receiving BEL in LOT 3 and LOTs 4-6. There may be a trend toward better responses earlier in the treatment sequence, but this analysis did not adjust for potentially confounding factors. The data from this study to date do not permit discrimination of differences in response rates in patients treated with BEL alone or in combination with other agents, but this merits further exploration.

Introduction: Tyrosine Kinase Inhibitors (TKI) are standard additions to first-line therapy for patients (pts) with Philadelphia chromosome-positive (Ph+) B-cell acute lymphoblastic leukemia (B-ALL). Ponatinib offers superior efficacy compared to dasatinib or imatinib when combined with hyperCVAD (cyclophosphamide, vincristine, doxorubicin, and dexamethasone) based on outcomes from experience at the MD Anderson Cancer Center. Real-world evidence from other centers evaluating outcomes with this regimen, including measurable residual disease (MRD) responses, may help inform optimal use of this approach. Previous studies primarily utilized flow cytometry to assess MRD. Next-generation sequencing (NGS), a more sensitive and specific method to quantify MRD, has become more commonly utilized and is recommended to more reliability evaluate treatment efficacy. Methods: This retrospective cohort study included pts aged ≥ 18 years with newly diagnosed Ph+ B-ALL who received hyperCVAD with ponatinib at the University of California San Francisco between January 1, 2016, and August 1, 2024. Pts were excluded if they received a non-hyperCVAD chemoimmunotherapy for more than 1 cycle or single agent TKI with or without steroids for more than 4 weeks before starting hyperCVAD (n=7). Use of non-ponatinib TKI was allowed for up to 2 cycles before starting ponatinib. Eight pts started dasatinib before switching to ponatinib. Ponatinib was started at 45mg (n=13) or 30mg (n=10) and decreased to 30mg after achievement of complete remission (CR) for those who started at 45mg. The dose was further decreased to 15mg after the achievement of complete molecular response (CMR). Ponatinib was started at 15mg in 2 pts based on pre-existing hepatic dysfunction. The primary outcomes included overall response rates after induction therapy, including rates of CR, CR with incomplete count recovery, CMR, and MRD negativity assessed by flow cytometry, polymerase chain reaction, and NGS. The secondary outcomes included overall survival (OS), relapse-free survival (RFS), the percentage of pts who received blinatumomab for MRD positivity or chemotherapy intolerance and/or allogenic hematopoietic stem cell transplant (alloHCT) and related outcomes, central nervous system relapse, toxicities, and changes in TKI treatment. Results: Twenty-three pts were included in the study. The median age was 48 (39-55) and 61% were male. Initial white blood cell count was 30.7 x 103/µL (9.9-135) and 10 pts (43.5%) received cytoreductive therapy (typically hydroxyurea or corticosteroids) prior to induction. A median number of 7 cycles of hyperCVAD with a median number of 12 intrathecal chemotherapies for CNS prophylaxis were administered. CD20 positivity ≥20% was present in 18 (78.3%) of whom 14 (77.8%) received Rituximab. All pts achieved complete remission or complete remission with incomplete recovery by the completion of cycle 2. Complete molecular response by BCR-ABL1 PCR was achieved in 36.8% after cycle 1 and 71.4% after cycle 2. Forty percent of pts achieved MRD negativity by NGS after cycle 1, 66.7% by month 3, and 100% by month 6. With the median follow-up of 38.3 months (12.3-82), 2 pts died, resulting in a 3-year OS of 95.5 % (95% confidence interval (CI) 71.9-99.4). Five pts relapsed, including 3 isolated CNS relapses, leading to a 3-year RFS of 81.5% (95 % CI 57.7-92.7). Five pts (22%) received blinatumomab for MRD positivity (median 2.5 cycles), 4 (17.4%) switched to blinatumomab based treatment for chemotherapy intolerance (median 2.5 cycles prior to switch), and 1 (4.3%) switched electively. Four pts (17.4%) received alloHCT — 1 (4.3%) for CR1 consolidation and 3 (13%) in CR2. Six pts (26%) switched ponatinib to a different TKI for adverse events, including thromboembolic events (8.6%), vascular toxicity (8.6%), hepatotoxicity (4.3%), and cardiotoxicity (4.3%). Conclusions: HyperCVAD with ponatinib demonstrated excellent efficacy and safety outcomes that validate findings at the institution that originally explored this approach. A minority of patients (26%) experienced toxicities requiring ponatinib to be changed to an alternative TKI. Furthermore, we demonstrate that the majority of pts can avoid need for alloHCT, with use of blinatumomab reserved for pts with MRD persistence or chemotherapy intolerance. This finding is important for centers that practice in resource-restricted contexts where front-line blinatumomab may not be generally possible.

Introduction: Acute myeloid leukemia (AML) with FLT3-internal tandem duplication (ITD) mutations represent ~25% of all AML cases and are associated with high relapse rates, even after allogeneic hematopoietic cell transplantation (HCT). A common strategy to mitigate relapse is the use of FLT3-ITD tyrosine kinase inhibitor maintenance (TKIm) therapy post-HCT. Recently, the MORPHO trial found an improvement in relapse-free survival (RFS) in patients (pts) with pre- or post-HCT detectable measurable residual disease (MRD) treated with post-HCT gilteritinib maintenance. Real-world data evaluating this approach remain limited. Herein, we report our experience with FLT3i maintenance at the University of California, San Francisco. Methods: We conducted a retrospective analysis including pts with FLT3-ITD mutated AML who underwent their first HCT between January 2017 and June 2024 while in a complete remission (CR) or a morphologic leukemia free state with a known MRD status at the time of HCT. MRD-negativity (MRD-) was assessed either by multiparameter flow cytometry (<0.01%) or FLT3 next generation sequencing assessment (allelic sensitivity 5 x 10-5). The primary endpoint was 3-year overall survival (OS), while secondary endpoints included 3-year relapse-free survival (RFS), TKIm completion rate as per treating physician, and incidence of TKIm discontinuation. Baseline characteristics and toxicity data were summarized with descriptive statistics and survival analyses were performed via the Kaplan Meier method. Results: In total, 58 pts were included, of whom 32 (55%) received TKIm post-HCT. The median age at HCT was 54.5 years (range, 19-79), 32 (55%) were female, and 4 (7%) had secondary AML. Forty-seven (81%) patients were in CR1, 4 (7%) were in CR2, and 41 (71%) were MRD- pre-HCT. Thirty-nine (67%) received myeloablative conditioning (MAC), 56 (97%) received peripheral blood stem cells, and 39 (67%) had an 8/8 matched unrelated or sibling donor. Fourteen (44%) received gilteritinib, 13 (41%) received sorafenib, and 5 (16%) received midostaurin as their first TKI post-HCT. TKIm was started at a median of 83.5 days (range, 38-412) post-HCT. The median follow-up time for the entire cohort was 3.76 years (IQR, 2.18-6.62). Among pts who received TKIm, 24 (75%) received MAC while 15 (58%) received MAC in the no TKIm group (p= 0.26). Among pts who were MRD-, 22 pts (54%) received TKIm, 18 (82%) of whom received MAC, while 19 (46%) did not receive TKIm, 14 (74%) of whom received MAC (p-value for MAC between TKI and no TKI groups: 0.71). Of the 17 pts who were MRD+, 10 (59%) pts received TKIm, 6 (60%) of whom received MAC, while 7 (41%) did not receive TKIm, of whom only 1 (14%) received MAC (p-value for MAC between TKI and no TKI groups: 0.13). The 3-year OS and RFS for the entire cohort was 69.7% (95% CI, 58.5-82.9) and 60.1% (48.2-74.9), respectively. Among pts who were MRD-, the 3-year OS in pts who received TKIm vs no TKIm was 95.5% (87.1-100) vs 66.2% (47.3-92.8) (p=0.046), while the 3-year RFS was 80.7% (65.4-99.8) vs 73.7% (56.3-96.4) (p=0.2), respectively. Among pts who were MRD+ pre-HCT, the 3-year OS in those who received TKIm vs no TKIm was 46.7% (19-100) vs 14.3% (2.33-87.7) (p=0.027), while the 1-year RFS was 50% (26.9-92.9) vs 0% (p <0.001), respectively. Of the 13 pts who received sorafenib, 3 (23%) switched to a second TKI at a median of 105 days while 1 pt each (20% and 7.7%) initially treated with midostaurin or gilteritinib switched to a 2nd TKI at 370 and 54 days, respectively. The median cumulative exposure of TKIm was 312 days (range, 28-1393). For gilteritinib, sorafenib, and midostuarin as the first TKI, the median cumulative exposures for each TKI were 250 days (range, 28-1393), 241 days (range, 28-886), and 363 days (range, 128-676), respectively. Twelve (38%) completed predetermined TKIm and 5 (16%) are receiving ongoing TKIm at the time of study cutoff. Reasons for discontinuation include: cytopenias (n=4, 13%), gastrointestinal intolerance (n=4, 13%), lack of insurance (n=2, 6%), relapse (n=2, 6%), graft-vs-host disease (n=1, 3%), peripheral neuropathy (n=1, 3%), and transaminitis (n=1, 3%). Conclusions: Despite limitations, including limited sample size, we observed an improvement in OS in patients who received post-HCT TKIm, irrespective of pre-HCT MRD status and conditioning regimen intensity. Consistent TKIm post-HCT is challenging due to the high discontinuation rate from toxicity.