Citations

Abstract Introduction: The GPRC5D-directed bispecific antibody (BsAb) Talquetamab (Talq) has shown promising results for patients (pts) with relapsed/refractory multiple myeloma (RRMM) in the MonumenTAL-1 clinical trial (Chari et al. NEJM 2022). However, there are limited data on the use of Talq in the real-world setting. Methods: All pts with RRMM who started Talq between 8/2023-3/2025 across 15 US academic centers were included. High-risk cytogenetic abnormalities (HRCA) were defined as del(17p), t(4;14), t(14;16) and/or gain/amplification 1q21. Time-to-event analyses were estimated using the Kaplan-Meier method. Toxicities including CRS and ICANS were graded using CTCAE v5.0 or ASTCT criteria. Results: A total of 484 pts were included, with a median age of 65 years (range 35-89), and 83 pts (17%) were aged ≥75 years. 20% of pts were black. 21% had baseline CrCl<40, 65% had HRCA and 29% had >1 HRCA (i.e Ultra high-risk MM). The median number of prior lines of therapy was 6 (IQR 4, 8). 50% were penta-refractory, 69% received at least one prior BCMA-directed therapy (46% CART; 32% BsAb). Prior to initiation of Talq, 11 pts (2%) had CNS MM, 29 (6%) had PCL and 126 (26%) had true extramedullary disease (EMD). 70% would have been ineligible for the MonumenTAL-1 trial. For most pts (n=405; 84%) the dosing strategy from Cycle 2 was 0.8 mg/kg every 2 weeks. and the 0.4 mg/Kg weekly dosing was used in 28 patients (6%). In 6% of pts, an additional drug was added to Talq, mostly pomalidomide (n=20) or daratumumab (n=10). 101 pts (21%) received Talq as bridging for CART. Median duration of Talq treatment was 102 days. 62% developed CRS (Grade ≥3, 2%; including one Grade 5 event). 15% developed ICANS (Grades 3-4, 4%). Steroids were used in 213 patients (44%), tocilizumab in 145 (30%) and anakinra in 9 (2%). 22 patients (5%) required ICU stay during the first cycle, for a median of 6 days (range 1-31). A total of 200 pts (41%) had infections (median 1 infection each, range 1-6), including 99 (20%) with bacterial infections, 113 (23%) viral infections and 21 (4%) fungal infections. IVIG was used in 261 pts (54%). 79% of patients (n=384) developed Talq-related oral toxicity, 55% (n=269) skin toxicity and 199 (41%) nail toxicity. 157 patients died after a median follow up of 11.5 months (mos), and the leading causes of death were MM progression (n=120) and infection (n=10). 8 pts (1.7%) developed invasive second primary malignancies. The ORR was 73%, including 46% ≥VGPR. The median PFS (mPFS) for the entire cohort was 7.8 mos (95% CI 6.9-9.4) and the 12 months PFS rate was 39%. The median OS had not been reached and the 12 months OS rate was 63%. For pts who would have been eligible for MonumenTAL-1, the mPFS was 12.2 mos (95% CI 9.7 – not reached (NR); for trial-ineligible pts, the mPFS was 6.9 mos (5.8 – 8.0; p=0.003). For BCMA-naive pts, the mPFS was 15.7 mos (11.8-NR); for BCMA-exposed pts, the mPFS was 6.8 mos (6.0-7.8; p=0.0002). For patients who received Talq as bridging prior to CART, the median PFS was NR (14.1 – NR); for those who did not receive Talq as bridging the mPFS was 6.5 mos (5.3-7.6; p<0.0001). In multivariable analyses, worse OS and PFS were associated with trial ineligibility [(HR 1.38, 95% CI 1.03-1.86; p=0.03) and (2.33, 1.31-4.14; p=0.004)], BMI<30 [(1.42,1.06-1.89; p=0.02) and (1.50, 1.01-2.22; p=0.045)] and true EMD at baseline [(1.95, 1.49-2.56; p<0.001) and (1.65, 1.16-2.36; p=0.005). On the other hand, use of Talq as bridging to CART was associated with better PFS (0.27, 0.17-0.43; p<0.001) and OS (0.13, 0.05-2.56; p<0.001). Ultra high-risk MM (1.40, 1.06-1.84; p=0.02) and baseline albumin <3 (1.68, 1.18-2.37; p=0.004) were associated with worse PFS; ECOG ≥2 (1.68, 1.18-2.40; p=0.004), and baseline Plt<50K (2.14, 1.48-3.10; p<0.0001) was associated with worse OS. Conclusions: In this large real-world analysis of 484 Talq recipients, the ORR was comparable to MonumenTAL-1 but the median PFS was shorter. This likely reflects differences in patient selection, with 70% of patients in our cohort being trial-ineligible. Patients who received Talq as pre-CAR-T bridging had superior survival outcomes. Rates of toxicities were similar to MonumenTAL-1, including >70% of patients developing dysgeusia.

Abstract Introduction: Elranatamab, a BCMA-CD3 bispecific antibody, has shown efficacy in relapsed multiple myeloma (MM), but real-world data on its safety and effectiveness are limited. Methods: We conducted a retrospective, multi-center study of patients (pts) treated with commercial elranatamab from 8/2023–3/2025 at 9 U.S. academic centers. Logistic and Cox regression were used for binary and time-to-event outcomes, respectively. Multivariable analysis (MVA) adjusted for prior lines of therapy (LOTs), baseline Hgb (per g/dL), and LDH (per 100 U/L) as continuous variables; and for BCMA exposure, extramedullary disease (EMD; extraosseous soft tissue lesions), and ECOG ≥2 as binary variables. Results: Of 130 pts, median age was 71 yrs (range 39–95); 57% were female, 82% white, 13% black, and 35% had ECOG ≥2 (11% ≥3). Median LOTs was 6; 91% were triple-class refractory, 49% penta-refractory, 49% BCMA-exposed (median 15.5 mo [IQR 11.8–20.5] since last exposure), and 49% had prior anti-BCMA CAR-T. Most recent BCMA exposures included: CAR-T (84%), TCE (9.4%), and ADC (6.3%). Only 22% met MagnetisMM-3 cohort A eligibility (which excluded BCMA-exposed pts). High-risk cytogenetics (del(17p), t(4;14), t(14;16), or amp(1q)) were present in 44% (25%, 13%, 6.3%, and 7.9%, respectively). EMD was seen in 22%. Median treatment duration was 4.0 mo (95% CI 2.93–6.3) overall and 7.9 mo (95% CI 5.83–NA) with ≥VGPR. Discontinuation due to toxicity or non-relapse mortality occurred in 12% but was significantly lower among those receiving IVIg (5% vs. 17%, p=0.031); IVIg was given in 47%. The overall response rate (ORR) was 65%, including ≥VGPR in 46% and ≥CR in 36%, consistent with MagnetisMM-3 (ORR 61%, ≥CR 35%; Lesokhin et al. Nat Med. 2023). After median follow-up of 7.5 mo, median PFS was 4.3 mo (95% CI 3.4–11.3), OS was 14.6 mo (95% CI 8.6–NA), and DOR was 12.2 mo (95% CI 6.1–NA). In contrast, MagnetisMM-3 (Prince et al., ASH 2024) reported median PFS and OS of 17.2 and 24.6 mo, respectively, with DOR not reached despite 28.4 mo of follow-up. In univariate analysis, ECOG ≥2 was associated with lower ORR (45% vs. 71%; OR 0.34, 95% CI 0.16–0.73, p=0.006), shorter OS (4.3 mo vs. NA; HR 2.41, 95% CI 1.33–4.35, p=0.004), and shorter PFS (1.7 vs. 6.5 mo; HR 1.77, 95% CI 1.09–2.87, p=0.020). Prior BCMA-directed therapy was linked to reduced odds of ≥VGPR (OR 0.38, 95% CI 0.19–0.77, p=0.008) and ≥CR (OR 0.43, 95% CI 0.20–0.89, p=0.025). Higher baseline LDH was linked to shorter OS (HR 1.37, 95% CI 1.23–1.53, p<0.001) and PFS (HR 1.31, 95% CI 1.18–1.44, p<0.001). Higher baseline Hgb was associated with higher ORR (OR 1.46, 95% CI 1.20–1.81, p<0.001) and ≥CR (OR 1.54, 95% CI 1.26–1.92, p<0.001). Among BCMA-exposed pts, recent exposure (<1 [n=15] vs. ≥1 yr [n=32]) was associated with shorter OS (4.1 mo vs. NA; HR 3.66, 95% CI 1.26–10.6, p=0.017). In MVA, higher baseline LDH remained independently associated with shorter OS (aHR 1.36, 95% CI 1.19–1.54, p<0.001) and PFS (aHR 1.27, 95% CI 1.13–1.43, p<0.001); higher Hgb with greater odds of ≥CR (aOR 1.44, 95% CI 1.14–1.85, p=0.003) and longer PFS (aHR 0.84, 95% CI 0.72–0.99, p=0.034); and BCMA exposure with lower odds of ≥CR (aOR 0.32, 95% CI 0.10–0.91, p=0.037). CRS occurred in 40% (grade ≥2 in 12.3%, grade 3 in 2.3%), and ICANS in 17% (grade ≥2 in 7.7%). These rates contrast with MagnetisMM-3 (no grade ≥3 CRS, 3.4% ICANS). ICANS risk was lower in BCMA-exposed pts (9.4% vs. 24%; OR 0.32, 95% CI 0.11–0.85, p=0.029), and decreased with more LOTs (OR 0.73, 95% CI 0.55–0.92, p=0.016) and higher Hgb (OR 0.71, 95% CI 0.52–0.94, p=0.023). Tocilizumab and steroids were used in 36% (treatment 28%, prophylaxis 9.2%) and 23%, respectively. Steroid use was less common in BCMA-exposed pts (11% vs. 34%; OR 0.23, 95% CI 0.08–0.60, p=0.004) and declined with more LOTs (OR 0.68, 95% CI 0.52–0.85, p=0.002). Infections occurred in 40% of the cohort, more common in pts with CrCl <40 mL/min (59% vs. 36%; HR 2.58, 95% CI 1.02–6.80, p=0.049). IVIg was linked to improved infection-free survival (HR 0.59, 95% CI 0.37–0.93, p=0.023). Conclusions: Elranatamab demonstrated meaningful activity in real-world relapsed MM, though PFS and OS were shorter than in MagnetisMM-3, likely reflecting an older, frailer population with higher disease burden. ICANS was more frequent than in MagnetisMM-3. Baseline anemia and elevated LDH, markers of poor marrow reserve and higher disease burden, were strongly linked to worse outcomes.

Abstract PAX5 is genetically altered in over 30% of B-ALL cases, with point mutations representing the second most common type of alteration. These point mutations are highly enriched in the DNA binding (paired) domain and have been recognized as founder events in PAX5 P80R and PAX5alt B-ALL subtypes, which account for over 12% of B-ALL. However, the molecular features and underlying mechanisms of these mutations in B-ALL remain largely undefined, limiting the development of targeted therapies and resulting in poor outcomes, especially in adult patients. We systematically investigated the functional roles and oncogenic mechanisms of PAX5 paired domain mutations. Integrated CRISPR screening and genomic profiling revealed that the paired domain is critical for PAX5 function. Disruptions in this region significantly reduced cell viability in B-ALL cell lines, and analysis of 2,955 B-ALL samples showed that recurrent PAX5 missense mutations are highly enriched in this domain, further underscoring its functional importance. Functional analysis of knock-in mice with PAX5 mutations revealed that R38H and R140L severely impair DNA binding and block B-cell differentiation, while P34L and P80R retain binding ability but alter binding specificity and gene regulation. Mice carrying homozygous (P34L, R38H, P80R, R140L) or heterozygous (R38H, P80R) mutations developed spontaneous B-ALL within one year. To model co-occurring mutations observed in patients, Pax5 R38H/P34L and R38H/R140L mice were generated. These compound mutations exhibited greater leukemogenic potential than single mutants. Transcriptomic analysis of leukemia samples showed consistent up-regulation of metabolic pathways and Myc target genes. Notably, biallelic PAX5 alterations and additional genetic lesions in signaling pathways were detected in all PAX5R38H/+ and PAX5P80R/+ leukemias, faithfully recapitulating the genetic alteration features of patient B-ALL driven by PAX5 mutations. Among Pax5-mutant mouse strains, Pax5P80R/+ mice develop B-ALL with short latency and complete penetrance, making them an ideal model to study the mutational evolution and leukemogenesis of B-ALL. To characterize the stepwise leukemogenesis process, we performed serial bone marrow aspirations in the same Pax5P80R/+ mice. In all 17 mice analyzed, we identified early preleukemic clones with only Pax5 WT deletions, followed by the emergence of leukemic clones harboring Jak1/3 mutations. Single-cell RNA/BCR-seq revealed that Pax5 WT allele deletions relieve metabolic constraints, thereby promoting clonal expansion of preleukemic B cells blocked at the pre-B stage. These changes coincide with downregulation of metabolic checkpoint genes and upregulation of glycolytic enzymes, promoting genetic instability and facilitating malignant transformation. Clinically, PAX5 P80R patients have better outcomes than PAX5alt cases. Using our mouse models, we observed that leukemic cells driven by the PAX5 P80R mutation exhibit greater sensitivity to dexamethasone compared to the ones carrying other PAX5 mutations in PAX5alt subtype, potentially due to their heightened dependence on glucose uptake and glycolysis, suggesting that metabolic rewiring driven by PAX5 mutants may influence therapeutic response. Interestingly, we found that an aberrant isoform of MEGF10 (aMEGF10) is highly and specifically expressed in PAX5 P80R B-ALL. This N-terminal truncated isoform encodes a protein that retains the intracellular immunoreceptor tyrosine-based activation motifs (ITAMs). Using CUT&Tag, we showed that PAX5 P80R directly binds to the aMEGF10 promoter. By engineering human CD34⁺ cells, we established a humanized PAX5 P80R leukemia model that recapitulates the gene expression profile of patient samples, including robust activation of aMEGF10. Functionally, aMEGF10 binds to and activates SYK, thereby promoting leukemic cell proliferation. Importantly, this oncogenic effect depends on intact ITAM motifs and SYK signaling, revealing a novel targetable vulnerability in PAX5 P80R leukemias. Overall, the study establishes that PAX5 DNA binding domain mutations contribute to leukemogenesis through stepwise loss of PAX5 WT alleles and acquisition of signaling pathway mutations, involving impaired differentiation, metabolic deregulation, and novel oncogene activation. These findings not only deepen the understanding of PAX5-driven leukemias but also highlight aMEGF10–SYK signaling as a promising therapeutic target.

Abstract Introduction: Patients (pts) with soft tissue plasmacytomas noncontiguous with bone (true extramedullary disease [EMD]) have poor outcomes with standard therapies shown by low overall response rates (ORRs), which are not durable. Talquetamab (Tal; anti-GPRC5D×CD3) and teclistamab (Tec; anti-BCMA×CD3) are first-in-class bispecific antibodies (BsAbs) approved as monotherapies for triple-class exposed (TCE) relapsed/refractory multiple myeloma (RRMM). Primary analysis from the RedirecTT-1 (NCT04586426) dedicated phase 2 EMD cohort (March 2025 data cut; median follow-up [mFU] 12.6 mo) showed Tal + Tec elicited an ORR of 78.9% (assessed by independent review committee) and a 12-mo progression-free survival (PFS) of 61.0%. Here, we report updated efficacy, using investigator assessment, and safety results from the phase 2 RedirecTT-1 EMD cohort with Tal + Tec. Importantly, we report EMD location and a novel analysis of tumor burden as a prognostic indicator of ORR. Methods: Pts had TCE RRMM and true EMD defined as ≥1 nonradiated soft tissue plasmacytoma noncontiguous with bone ≥2 cm in 1 dimension (with or without paramedullary plasmacytomas). Nonsecretory/oligosecretory disease was permitted. Prior CAR-T and non-BCMA/-GPRC5D BsAb therapies were permitted. Pts received Tal 0.8 mg/kg + Tec 3.0 mg/kg Q2W, preceded by step-up doses, with a permitted switch to monthly dosing at investigator's discretion after cycle 6 or after cycle 4 with confirmed ≥VGPR. Response was assessed per IMWG; EMD response was assessed by PET-CT or MRI whole-body scans. Tumor burden was assessed by total EMD tumor volume. Results: As of July 2025, 90 pts received Tal + Tec, with a median follow-up of 16.3 mo (range 0.5–23.7). Baseline characteristics were as previously reported; 39% had nonsecretory/oligosecretory disease, 20% had prior anti-BCMA CAR-T therapy, and 9% had prior BsAb therapy (all anti-FcRH5). The median number of plasmacytomas was 2 (range 1–7); 17 (18.9%), 29 (32.2%), and 74 (82.2%) pts, respectively, had ≥1 nodal, organ, or soft tissue EMD, while 11 (12.2%) also had ≥1 additional paramedullary plasmacytoma. Of 268 EMDs across all pts, 95 (35.4%) were nodal EMD, 77 (28.7%) were organ EMD (most commonly liver), and 64 (23.9%) were soft tissue EMD; 32 (11.9%) were paramedullary. Baseline EMD tumor volume was <25 cm2in 43 (47.8%) pts, 25–50 cm2 in 21 (23.3%) pts, and >50 cm2 in 26 (28.9%) pts. Per investigator assessment, ORR (95% CI) was 77.8% (67.8–85.9); 50.0% had a ≥CR. Median duration of response (DOR) was not estimable (NE; 12-mo DOR rate, 60.1%), 12-mo PFS was 55.6%, and median overall survival (OS) was NE (12-mo OS rate 73.8%). ORR was 90.7% (77.9–97.4; ≥CR 60.5%) in pts with EMD tumor volume <25 cm2, 66.7% (43.0–85.4; ≥CR 52.4%) for 25–50 cm2, and 65.4% (44.3–82.8; ≥CR 30.8%) for >50 cm2. Common adverse events (AEs) included CRS (77.8%; no grade [gr] 3/4) and neutropenia (72.2%; gr 3/4 62.2%). Taste changes (78.9%), non-rash skin AEs (68.9%), and nail AEs (55.6%) were all gr 1/2, and rash AEs (30.0%) were mostly gr 1/2. ICANS occurred in 11 (12.2%) pts (gr 3, 1.1%; gr 4, 1.1%). Infections occurred in 72 (80.0%) pts (gr 3/4 40.0%), most commonly upper respiratory tract infection (26.7%; gr 3/4 5.6%); IVIG was highly recommended to prevent and manage hypogammaglobulinemia and infection. Ten (11.1%) pts discontinued Tal + Tec due to treatment-emergent AEs (4 pts due to infections and 6 due to gr 5 AEs). Two pts discontinued Tal only due to AEs. In total, 11 (12.2%) pts had gr 5 AEs (6 due to infections), 6 of which were deemed to be drug related by investigators. Data will be updated with an additional 4 mo of follow-up for the presentation. Conclusions: With longer follow-up in pts with TCE RRMM with true EMD regardless of baseline tumor characteristics, Tal + Tec efficacy exceeded all approved therapies, including T-cell redirecting and cellular therapies, noting limitations of cross-study comparisons. Lower total EMD tumor volume was associated with a higher ORR but low pt numbers in each group and lack of statistical testing limits robust interpretation. The safety profile of Tal + Tec was generally consistent with each monotherapy; AEs were not exacerbated with the combination. These data continue to highlight the clinical benefit of the novel combination of Tal + Tec in pts with true EMD, a population with high disease burden and significant unmet need.

Abstract Introduction: Lisocabtagene maraleucel (liso-cel) was approved in the United States for the treatment of CLL in 3/2024 for patients who have received two prior lines of therapy including a covalent BTK inhibitor and a BCL-2 inhibitor. Data regarding outcomes of liso-cel is limited to reports from the registrational TRANSCEND-CLL 004 clinical trial which had strict inclusion and exclusion criteria. This work investigates the real-world efficacy and safety of liso-cel for patients with CLL and its efficacy in specific sub-populations. Methods: This is a multi-institutional retrospective study (Collaborative Assessment of Real-World Evidence of Lisocabtagene Maraleucel in Patients with Relapsed/Refractory CLL/SLL [CARE CAR-T CLL]) of patients with CLL who received commercial liso-cel in the United States. Patients with Richter transformation were excluded. Response was defined by iwCLL criteria. Patients who did not undergo a marrow biopsy but met other criteria for a complete response (CR) were categorized as unconfirmed CR (CRu). CRS and ICANS were graded per ASTCT criteria. Results: A total of 30 patients were identified with a median age of 67 years (range 44 – 80). Twenty of thirty patients (67%) had either del17p and/or mutated TP53, and 8/30 (27%) had complex karyotype (≥ 3 abnormalities) at time of liso-cel infusion. Patients received a median of 6 prior lines of therapy (range 1 - 12), and 24 (80%) were previously treated with chemotherapy. All patients had previous exposure to a covalent BTK inhibitor and a BCL-2 inhibitor. Twenty-seven (90%) of patients had prior exposure to pirtobrutinib. Twenty-nine patients (96.7%) received bridging treatment that was stopped after leukapheresis with 1 patient in CR and 25 patients in PR at time of liso-cel. Eighteen patients (60%) were treated with pirtobrutinib as the last line of treatment prior to liso-cel. Median length of time on pirtobrutinib prior to liso-cel infusion was 4 months (interquartile range [IQR], 2 – 8 months). Twelve (66.7%) patients stopped pirtobrutinib between leukapheresis and lymphodepletion, and 6 continued after liso-cel. Best CR/CRu rate was 60.0% (CR, n = 17/30; CRu, n = 1/30), and best overall response rate (ORR) was 83.3% (25/30) with median time to best response of 30 days (IQR, 28 – 30 days). Of the 18 patients who received pirtobrutinib as their last line of therapy, 13 (72%) achieved a CR/CRu. Meanwhile, of the 12 patients who did not receive pirtobrutinib as their last line of therapy, only 5 (28%) achieved a CR. Measurable residual disease testing (MRD) by flow cytometry was done in 14/30 patients, and 9 patients (64%) had achieved undetectable MRD (uMRD). MRD by next generation sequencing (NGS) was done in 17 patients, and 7 (41%) patients had uMRD. Twenty-seven of 30 patients (90%) were alive at time of data collection. Median follow-up was 3.3 months (IQR, 1.2 – 8.9 months). Twenty-six patients (87%) experienced any grade cytokine release syndrome (CRS), and 3 patients experienced grade 3 CRS (10%). Twelve patients (40%) experienced any grade immune effector cell-associated neurotoxicity syndrome (ICANS), and 4 patients (13%) experienced grade 3 ICANS. There were no grade 4 or 5 CRS or ICANS. One patient experienced grade 3 immune effector cell-associated hemophagocytic lymphohistiocytosis-like syndrome (IEC-HS). At day 30, patients had a median absolute neutrophil count of 1.7K/μL (IQR, 0.8 – 3.6K/μL), hemoglobin of 11.7 g/dL (IQR, 8.6 – 13.1 g/dL), and platelet count of 72K/μL (IQR, 29 – 119K/μL). Conclusion: Real-world analysis of liso-cel for patients with relapsed/refractory CLL showed CR and ORR of 60.0% and 83.3%, respectively. Despite having a heavily pre-treated and chemotherapy-exposed population and with over half the patients having high-risk features, these response rates are higher than what was described in the TRANSCEND-CLL 004 clinical trial. These results are limited by the short follow-up period and the variability in response assessment typical of a retrospective study. As we increase our sample size, we will also be interested if the use of the non-covalent BTK inhibitor pirtobrutinib prior to liso-cel is associated with a higher CR rate, mimicking the findings of the sub-cohort analysis of TRANSCEND-CLL 004 where the concurrent use of ibrutinib led to higher CR rates. Further updates and patients from additional collaborators will be presented at the meeting.

Abstract Background: CD19-directed CART cell (CART19) therapy has transformed the treatment of B-cell malignancies but is limited by life-threatening toxicities, including cytokine release syndrome (CRS) and immune effector cell–associated neurotoxicity syndrome (ICANS), as well as low durable remission rates. Our understanding of the pathogenesis of CART-associated toxicities is limited, and although current treatments are effective, refractory cases remain challenging. Methods and Results: To investigate CRS/ICANS pathogenesis and identify new therapeutic targets, we analyzed plasma cytokine kinetics within the first month of CART19 cell therapy in 25 relapsed/refractory (r/r) large B-cell lymphoma (LBCL) and 14 r/r follicular lymphoma (FL) patients (NCT02030834). In this cohort, 22 (88%) patients developed any-grade CRS (73% LBCL, 27% FL) and 4 (16%) developed any-grade ICANS (50% LBCL, 50% FL). Across 30 analyzed cytokines, IL-5 emerged as the second most enriched cytokine based on the median peak-to-baseline (Day 0) ratio in patients who experienced CRS vs. those who did not. The IL-5 ratio was 6.6-fold higher (p<0.05) and 11.9-fold higher (p=0.093) in patients who experienced CRS or ICANS, respectively. Moreover, IL-5 was elevated in patients experiencing CRS on Days 1 and 2 (p<0.05). Prompted by our results, we evaluated the effect of IL-5 neutralization with a monoclonal antibody (mAb) on CART-associated toxicity in a patient-derived B-acute lymphoblastic leukemia (B-ALL) xenograft model. After engraftment (average of 110 human CD19+ cells/µL peripheral blood), NSG mice were randomized based on tumor burden and treated with 3.5×10⁶ CART19 plus either IL-5 mAb or IgG control (10 mg/kg, weekly), or no treatment. Mice receiving IL-5 mAb showed reduced weight loss (p<0.005, Days 13–16) and neuroscore (p<0.05, Day 15), as assessed by a daily, blinded 18-point assessment, vs. the IgG group. Additionally, while CRS-associated cytokines (GM-CSF, IFN-γ, MIP-1β) were significantly elevated in the IgG group (p<0.05) vs. the untreated group, they were not in the IL-5 mAb group. In this model, all mice treated with CART19 cells were able to clear the tumor burden. However, in a follow-up challenge model generated by engrafting NSG mice with 1x106 luciferase+ JeKo-1 cells, mice treated with a combination of 1x106CART19 cells + IL-5 mAb showed reduced weight loss (p<0.0001) and enhanced overall survival (p<0.01) vs. mice treated with CART19 + IgG. Next, we evaluated IL-5 neutralization in an immunocompetent toxicity model. BALB/c mice were treated with 10×10⁶ murine untransduced T cells, CART19 + IL-5 mAb, or CART19 + IgG (10 mg/kg, weekly). Mice receiving IL-5 mAb exhibited significantly reduced weight loss (p<0.05, Day 1) and lower neuroscore (p<0.05, Days 1, 6, and 8) vs. those receiving IgG control, with a consistent trend observed on other days, supporting a protective role for IL-5 blockade in mitigating CART-associated toxicities. To explore the direct effects of IL-5 on CART19 cells, we disrupted either IL-5 (IL-5KO-CART19) or its receptor (IL-5RαKO-CART19) using CRISPR-Cas9. In NSG mice engrafted with 1×10⁶ luciferase+NALM6 cells, mice receiving 1×10⁶ IL-5KO-CART19 vs. IL-5WT-CART19 showed a trend toward reduced weight loss (p=0.08), significantly increased antitumor activity by Day 20 (p<0.05), and improved overall survival (p<0.05), indicating that IL-5 production can significantly impair CART19 cell efficacy. We next evaluated changes in IL-5Rα gRNA representation in a published genome-wide CRISPR screen (PMID: 39266501). CART19 cells with IL-5Rα-targeting gRNAs were enriched following chronic stimulation, indicating a protective effect for IL-5 pathway interruption. In subsequent studies, IL-5RαKO-CART19 cells showed enhanced killing of JeKo-1 cells following 48 hours of co-culture (p<0.05) and reduced co-expression of multiple inhibitory markers (p<0.01) following chronic stimulation with JeKo-1 cells for one week vs. IL-5RαWT-CART19 cells, suggesting an improvement in CART19 cell activity and phenotype following disruption of IL-5Rα. Conclusion: Our data identify IL-5 as a previously unrecognized mediator of CART–associated toxicities and show significant improvement of CART19 activity with disruption of IL-5 signaling either indirectly with an IL-5 neutralizing antibody or directly with CRISPR gene editing. Thus, IL-5 could serve as a dual-purpose target to improve both safety and efficacy of CART cell therapy.

Abstract Background: A randomized multicenter trial (BMT CTN 1703) demonstrated that after reduced intensity conditioning HCT, PT-Cy-based GVHD prophylaxis led to significant reductions in acute and chronic GVHD, but was associated with significantly more Grade 2 (moderate) infections and similar overall survival vs Tac/MTX. A linked mechanistic study (BMT CTN 1801) found that PT-Cy led to pan-T cell depletion and reduced T cell receptor (TCR) repertoire diversity vs Tac/MTX. To discover the molecular mechanisms driving the association of PT-Cy with increased infections, we examined pathogen-specific T cell reconstitution in BMT CTN 1801 patients (n=165 PT-Cy, n=159 Tac/MTX). Methods: We performed deep TCR sequencing on 2,369 blood samples collected pre-HCT (recipient baseline) and on Days +7, 14, 28, 63, 100, 180, 270, 365, 730, and from the graft infusion product. We then applied a novel set of TCR classifiers (Adaptive Biotechnologies) capable of identifying pathogen-specific clones associated with CMV, EBV, SARS-CoV-2, HSV-1, HSV-2, parvovirus, RSV, influenza A, rhinovirus, adenovirus, HHV-6B, norovirus and Toxoplasma. Single-cell (sc) TCR/RNA-sequencing on a sample subset (n=26) was performed to determine the phenotype of pathogen-specific T cells. Results. By linking scRNA-seq with pathogen-specific TCR barcoding, we identified CD4 and CD8 memory T cells as their predominant T cell reservoir. We found that the total number of pathogen-specific singleton TCRs was significantly lower with PT-Cy, starting early post-HCT and persisting through 2 years. For example, at Day +14, PT-Cy patients had 7.8-fold fewer pathogen-specific singleton TCRs vs Tac/MTX (mean of 6.9 vs 54.1, p<0.001). Consistent differences between PT-Cy and Tac/MTX were observed for TCRs specific for each pathogen individually. Patients who developed Grade 3 (severe) viral infections had significantly fewer pathogen-specific singleton TCRs at Day +14 compared to those who had no viral infections (mean of 4.0 vs 32.5, p=0.03). Leveraging intrinsic TCR barcoding of the HCT infusion (“graft”) and of the patient's baseline samples (“recipient”), we could assign the graft/recipient origin of the pathogen-specific cells with high fidelity. This analysis revealed pronounced differences in the origin of pathogen-specific T cells with PT-Cy vs Tac/MTX, especially prior to Day +100. The absolute number of pathogen-specific TCRs originating from the graft was significantly higher with Tac/MTX vs PT-Cy. This difference was observed from day +7 through 6 months post-HCT, with up to 7.1-fold more graft-derived pathogen-specific TCRs in Tac/MTX vs PT-Cy (p<0.001 at each timepoint before day +100). Within patients who received Tac/MTX, the balance of graft vs recipient pathogen-specific clones significantly favored graft clones (for example, a mean of 14.9 graft vs 8.6 recipient pathogen-specific TCRs at Day+14, p=0.02). By contrast, with PT-Cy, in addition to having substantially fewer pathogen-specific TCRs overall, the proportion of these TCRs was not significantly different between graft and recipient clones (mean of 2.1 graft vs 3.3 recipient pathogen-specific TCRs at Day+14, p=0.42). These data uncover that, in addition to the reduction in pathogen-specific T cells with PT-Cy, there was a relative skewing of the anti-microbial T cell reservoir towards recipient cells that persisted after conditioning. Conclusions: PT-Cy led to in vivo T cell depletion across all T cell subsets, which prominently included clones tracked from the allograft itself into the HCT recipient, with a relative sparing of recipient T cell clones that survived transplant conditioning. While the mechanism for relative sparing of recipient cells has not yet been determined, these conditioning-exposed cells may be less proliferative than newly infused graft T cells. The depletion of graft T cells included CD4 and CD8 memory cells specific for infections, which may have contributed to relative deficits in protective immunity against infection in patients receiving PT-Cy. Importantly, these data suggest that with PT-Cy, persisting recipient TCRs may play a key role in preserving anti-infectious immunity. Taken together, these results provide a mechanistic basis for key post-transplant clinical outcomes of the landmark BMT CTN 1703 study. They also underscore the importance of recipient immune status with PT-Cy, creating the opportunity for targeted infection risk assessment in these patients.

Abstract Background: The recently published BLAST clinical and BLAST molecular (BLAST-Mol) risk models for chronic myelomonocytic leukemia (CMML) incorporate clinical and molecular variables, respectively, for prediction of overall survival (OS), while BLAST-BT (blast transformation) risk model was predictive of BT free survival (BTFS) [Blood 2025;doi:]. In the current study, the Mayo Clinic (USA) CMML cohort used in the aformentioned study was expanded from 457 to 600 patients and merged with an external cohort of 501 patients from the Humanitas Research Hospital (HRH; Italy) in order to validate the BLAST, BLAST-Mol and BLAST-BT risk models. Methods: CMML diagnosis and sub-categorization were according to the International Consensus Classification, which included CMML-1 (BM/PB blasts <10%/<5%) vs. CMML-2 (BM/PB blasts ≥10%/≥5%) and CMML-MP (leukocytes ≥13 x 10⁹/L) vs. CMML-MD (leukocytes <13 x 10⁹/L) [Blood 2022;140:1200]. The BLAST score is based on clinical risk factors including PB blasts ≥2%, leukocytes ≥13 x 109/L, and severe or moderate anemia resulting in BLAST low, intermediate, and high risk groups. BLAST-Mol also accounts for mutations and karyotype and identifies PHF6 and TET2 as “favorable” and DNMT3A, U2AF1, BCOR, SETBP1, ASXL1, NRAS, PTPN11, RUNX1, TP53, as “unfavorable” mutations with adverse karyotype also considered unfavorable. BLAST-BT variables include absence of PHF6MUT, BM blasts ≥10%, presence of DNMT3AMUT, PB blasts ≥2%, leukocyte count ≥13 x 109 /L, and ASXL1MUT. OS analyses were censored for allogeneic stem cell transplant (ASCT). Conventional statistical methods were employed, using JMP Pro 18.0.0 software SAS Institute, Cary, NC, USA. Results: A total of 1,101 patients (median age 73 years; 67% males) with newly-diagnosed CMML were considered: Mayo cohort 600 and HRH cohort 501. Compared to the HRH cohort, the Mayo cohort was more likely to include CMML-MP (44% vs. 28%; p<0.01), CMML-1 (87% vs. 81%; p<0.01), ASXL1MUT (48% vs. 35%; p<0.01), SRSF2MUT (43% vs. 33%; p<0.01), NRASMUT (15% vs 9%; p<0.01), PHF6MUT (7% vs. 4%; p<0.01), and less likely to harbor TET2MUT (50% vs. 58%; p<0.01), and SF3B1MUT (4% vs. 10%; p<0.01) [Table 1]. At a median follow-up of 55 months (range 0-202), 686 (62%) deaths, 218 (20%) BTs, and 142 (13%) ASCTs were documented. CPSS-mol risk categories at diagnosis included low (N=190; median OS 67 months), intermediate-1(N=271; median 38 months), intermediate-2 (N=407; median 26 months), and high (N=231; median 16 months; p<0.01) [AUC at 5 years 0.75]. BLAST clinical risk distribution was low (N=356; median OS 65 months), intermediate (N=393; median 30 months), and high (N= 352; median 16 months; p<0.01) [Figure 1; AUC at 5 years 0.79]. The corresponding AUCs calculated for the Mayo and HRH cohorts, separately, were 0.81 and 0.76, respectively. BLAST-Mol risk assignments included low (N=357; median OS 66 months), intermediate (N=512; median 27 months) and high (N=232; median 15 months; p<0.01) [Figure 2; AUC at 5 years 0.79]. The corresponding AUCs for the Mayo and HRH cohorts were 0.81 and 0.77, respectively. The 3-year cumulative incidence of BT (accounting for death from another cause) stratified by BLAST-BT risk model included 8% for very low risk (N=54), 11% for low risk (N=332), 20% for intermediate-risk (N=281), 27% for high risk (N=250), and 34% for very high risk (N=176) [p<0.01; Figure 3]. Multivariable analysis of the combined Mayo-Humanitas cohort (N=1,101) identified age >65 years (HR 1.7, 95% CI 1.3-2.1), CMML-2 subtype (HR 2.0; 95% CI 1.6-2.5), and a number of other mutations or co-mutations with BLAST-Mol-independent and CPSS-mol-independent prognostic contribution (to be elaborated further at the time of meeting presentation). Conclusions: The current study validates the predictive performance of the BLAST, BLAST-Mol and BLAST-BT risk models in a large two-center international cohort of patients with CMML. The possibility of further enhancement of the BLAST-Mol model is suggested by the demonstration of independent prognostic relevance from additional mutations and co-mutations.

Abstract BACKGROUND: DNA methyltransferase inhibitors (DNMTi) are US FDA approved for the management of CMML. These agents are associated with an overall response rate (ORR) of 40-50%, with a true complete remission (CR) rate of <20%. TET2 mutations are seen in 60% of CMML, with TET2 being a ferrous and oxoglutarate dependent DNA-dioxygenase that oxidizes 5mC to generate 5hmC, 5fC and 5caC. Ascorbic acid (AA) is critical for TET activity and can enhance TET 1/2/3 dioxygenase function by binding to the catalytic domain and when given in high doses, can result in glutathione depletion and free radical induced cell death. In a pilot study (Xie et al. Blood 2024), we demonstrated that high dose IV AA can be clinically effective and induce changes in differentially methylated regions (DMR) involving hematopoietic enhancers and improve cytopenias in TET2-mutant (mt) patients [CCUS]. We expanded this study to combine high dose IV AA with DNMTi to enhance the demethylation effect and produce durable responses in new diagnosed, treatment requiring, TET2mt CMML patients. METHODS: At Mayo Clinic, the LS1781 (NCT03418038) clinical trial was amended to include Arm E, combing high dose IV AA with standard of care decitabine in newly diagnosed TET2mt CMML patients that needed therapy. IV AA was dosed at 1 g/kg on days 1, 3 and 5 (per 28-day cycle, max dose 100 gm) through a central line, based on demonstrated safety from phase 1 studies. Standard dosing was adopted for decitabine, and patients were then asked to take oral AA, 1 gm daily from days 6-28. Key inclusion criteria included a WHO-defined diagnosis of CMML with at least one detectable TET2 mutation requiring therapy with adequate performance status (ECOG PS <2) and organ function (creatinine clearance > 55 mL/min). Response was evaluated by the MDS/MPN International Working Group Criteria (Savona et al. Blood 2015). We measured pre- and post-therapy (4 cycles) bone marrow variant allele fractions (VAF) through next generation sequencing assays, transcriptomic changes through RNA seq, and DNA methylation changes using the Illumina Methylation EPIC array. RESULTS: Between February 2024 and July 2025, a total of six patients were enrolled. The median age at enrollment was 73 years (range: 65-80); all Caucasian with 3 (50%) males. All patients had a diagnosis of CMML-1 with a median bone marrow (BM) blast percentage of 3 (0-10), median hemoglobin 13 (9.5-14.5) g/dL, median WBC count of 5.7 (2.1-12.6) x 109/L, absolute monocyte count 1.4 (0.7-2.7) x 109/L, and platelet count of 92.5 (32-123) x 109/L. TET2 (100%, 4 patients > 1 TET2 mutation at mean VAF of 36%), SRSF2 (67%), and KRAS (33%) were the most frequent molecular abnormalities. Neutropenia (without infection/sepsis) was the only treatment-associated adverse effect seen in 3 (50%, all grade 3) patients. Response was assessed in patients who completed at least 4 cycles (n=5) of therapy, with all (100%) patients meeting criteria for either a complete response (n=2, 40%), optimal marrow response (n=1, 20%) or clinical benefit (platelet response, n=2, 40%). In five patients who had NGS assessment pre-and post-therapy, there was a >50% reduction in TET2mt VAF, with one patient loosing the KRAS G12D clone in its entirety. Furthermore, we identified significant changes in DNA methylation associated with treatment with >85% of the CpGs being hypomethylated post-treatment. RNA-sequencing on patients pre- and post-treatment, identified key transcriptomic changes, with a decrease in expression of leukemogenic genes. Integration of the differential methylation data with transcriptomic changes identified several genes in which changes in DNA methylation correlated with a change in gene expression. These genes include regulators of blood cell development and inflammatory response including GATA2, BCL2, and HLA-DPA. Several leukemia-associated genes were downregulated including MEIS1, TCL1A, and SETBP1. CONCLUSION: In TET2mt CMML, high dose IV AA acid enhances TET activity and synergizes with DNMTi with encouraging clinical responses in all patients and with no new safety signals. On completion of this study, we plan a randomized phase 2 trial comparing high dose IV AA with decitabine vs decitabine alone in newly diagnosed TET2/IDHmt CMML.

Abstract Introduction Blast transformation (BT) in CMML occurs in 15-30% of patients (pts) with a median overall survival (OS) <6 months (mo). While clonal evolution can be observed, biological mechanisms of BT remain unknown for many pts. Using paired samples from chronic phase (CP) and at BT, this study defines the cytogenetic and molecular landscape of BT-CMML. Methods BT-CMML pts from Mayo Clinic (n=153, 31%), MD Anderson (n=195, 40%), and 7 centers of the Groupe Francophone des Myélodysplasies (n=145, 29%) were included after IRB approval. ICC 2022 criteria defined CMML and AML. Pt data were collected at initial diagnosis or referral at CP and again at BT. Clonal dominance was inferred from a natural log-fold difference of mutation variant allele fractions (VAF). OS was censored at allogeneic stem cell transplant (alloSCT). Single-cell multiomic analyses used the Tapestri platform (Mission Bio). Statistics used R. Results Among 1,883 cases of CMML with a median follow up of 96 mo, 493 (26%) underwent BT. The median age at CP and BT was 69 and 71 years, respectively. BT-CMML pts were predominantly male (69%) and had proliferative CMML (53%) or CMML-1 (69%) in CP; 39% and 33% were considered high-risk by CPSS-Mol and BLAST-Mol criteria, respectively. The median time to BT was 16 mo (interquartile range 7-35 mo) and was not different in males vs females (p=0.60). Compared to their CP parameters (p<0.001 each), pts at BT had lower median hemoglobin (10.8 vs 9.2 g/dL) and platelets (100 vs 44 x109/L) but higher leukocytes (13.9 vs 16.3 x109/L), peripheral blasts (0% vs 14%), and bone marrow blasts (6% vs 32%). Blasts exhibited mostly monocytic (57%) or immature myeloid (40%) differentiation. Abnormal karyotypes (AK) were more prevalent at BT (25% vs 62%, p<0.001). Among 318 pts with paired karyotype data, 221 (69%) had a normal karyotype (NK) in CP, of which 123 (56%) remained normal at BT and 98 (44%) acquired new abnormalities. Conversely, 97 (31%) pts had an AK in CP, of which 67 (69%) acquired abnormalities or increased clonal metaphases (6%), 23 (24%) remained stable, and 7 (7%) reverted to a NK at BT. Overall, 46% acquired and 8% lost a clonal cytogenetic abnormality, while 46% remained unchanged. Numeric (37%) and structural (30%) changes were most common. Complex (12% vs 3%) and monosomal (5% vs 2%) karyotypes were more frequent at BT than in CP. Only 6 (2%) pts acquired an AML-defining rearrangement at BT. Mutational data were available for 248 paired cases. The number of somatic mutations was higher at BT (median 3 vs 4, p<0.001). Mutations in RUNX1 (27% vs 39%), NRAS (23% vs 33%), KRAS (11% vs 17%), FLT3 (6% vs 15%), NPM1 (0% vs 11%), IDH2 (5% vs 11%), and CEBPA (3% vs 10%) were more frequent at BT (all p<0.05). At BT, the average VAF of the dominant clone increased in 32%, decreased in 35%, and remained unchanged (delta ≤5%) in 33%. The most frequent dominant clones both in CP and at BT had mutations in TET2 (8% vs 4%) or co-dominant mutations involving SRSF2-TET2 (7% vs 3%). Mutational clonal evolution was observed in 72%, with mutations gained in 45% and lost from the dominant clone in 19%. The most gained mutations at BT were in NRAS (15%) and RUNX1 (13%). Single cell proteogenomic analyses in 4 selected CP and BT pts shed light on the impact of NRAS and RUNX1 mutations on clonal hierarchies and differentiation. Among 196 fully evaluable cases, a new cytogenetic and/or molecular aberration was uniquely gained in 106 (54%) cases and lost in 9 (5%), with 40 (20%) both gaining and losing a genetic alteration. Forty-one (21%) patients had no identifiable genetic change between CP and BT, suggesting driver alterations outside of that detected by routine analyses. Median OS was 31 mo from CMML diagnosis and 5.6 mo from BT. Compared to supportive care, treatment in CP did not prolong median time to BT (14 vs 17 mo, p=0.91) or OS (35 vs 29 mo, p=0.05), regardless of intensity. After BT, median OS was 1.2 mo with supportive care (n=83), 8.7 mo with a hypomethylating agent (HMA, n=39), 10 mo with HMA plus venetoclax (n=37), 7.8 mo with chemotherapy (n=91), and 31 mo in pts who underwent alloSCT (n=57). Conclusions In this large BT-CMML cohort, cytogenetic (46% gained, 8% lost) or mutational (45% gained, 19% lost) driver events were identified in 79% of pts, with 21% having no identifiable changes by conventional testing. Whole genome/transcriptome analyses are underway in this subgroup to identify novel drivers of BT.