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BACKGROUND ELRA is a humanized, BCMA/CD3 bispecific antibody. In the phase 2 registrational MagnetisMM-3 (MM-3) trial, SC ELRA given as 2 step-up priming doses (12 mg on day 1 and 32 mg on day 4 of cycle 1) followed by 76 mg QW in patients (pts) with RRMM naive to BCMA-directed therapy resulted in an ORR of 61.0%. With a median follow-up of 28.4 mos, the median DOR, PFS, and OS were not reached (NR; 2-year rate, 66.9%), 17.2, and 24.6 mos, respectively. Overall, 56.3% of pts had CRS (grade 2, 14.3%; no grade ≥3). Most events occurred after doses 1 (44.5%), 2 (20.2%), and 3 (5.9%); 0.8% (1 pt) had CRS with doses ≥4. Recurrent CRS occurred in 15.1% of pts. The phase 1/2 MagnetisMM-9 (MM-9; NCT05014412) study is examining an alternative priming regimen and longer dosing intervals of ELRA. Here, we present updated MM-9 results. METHODS In MM-9, pts with RRMM and disease refractory to ≥1 IMiD drug, ≥1 PI, and ≥1 anti-CD38 antibody were given SC ELRA as step-up priming doses of 4 and 20 mg on Day 1 and Day 4 of Cycle 1, respectively. After the priming doses, ELRA 76 mg was given QW for the next 6 cycles (Part 1) or for 1 cycle followed by 116 (dose level 1 [DL1]) or 152 (dose level 2 [DL2]) mg Q2W for the next 5 cycles (Part 2A). After 6 cycles, the dosing frequency was decreased from QW to Q2W (Part 1) or from Q2W to Q4W (Part 2) in pts who achieved ≥PR lasting ≥2 months. The recommended phase 2 dose from Part 2A (152 mg, DL2) was evaluated in Part 2B (dose expansion). The primary endpoint for both parts is grade ≥2 CRS rate per ASTCT criteria during Cycle 1. Secondary endpoints include evaluation of efficacy and adverse events. Data cutoff: February 13, 2024. RESULTS Overall, 85 pts (median age, 64.0 y) were treated with ELRA. The majority (72.9%) of pts had R-ISS stage I/II disease (23.5% with R-ISS stage III); 31.8% had high-risk cytogenetics. Median number of prior lines of therapy was 5.0 (range, 1-12). The median treatment duration was 8.6, 2.9, 1.4, and 3.3 mos, in Part 1, Part 2A DL1, Part 2A DL2, and Part 2B, respectively. Median follow-up for pts overall (n=85), in Part 1 (n=33), Part 2A DL1 (n=12), Part 2A DL2 (n=11), and Part 2B (n=29) was 10.4, 23.3, 16.5, 15.2, and 9.3 mos, respectively. Overall, the ORR was 58.8%. For pts in Part 1, Part 2A DL1, Part 2A DL2, or Part 2B, ORR was 66.7%, 58.3%, 36.4%, and 58.6%, respectively; median DOR was NR (9-mo rate: 86.4%), 10.8 mos (9-mo rate: 80.0%), NR (9-mo rate: 100.0%), and NR (9-mo rate: 84.8%); median PFS was 17.1 (9-mo rate: 62.8%), 11.6 (9-mo rate: 50.9%), 1.6 mos (9-mo rate: 40.0%), and NR (9-mo rate: 57.1%); median OS was NR (9-mo rate: 75.8%), 17.4 mos (9-mo rate: 66.7%), NR (9-mo rate: 60.0%), and NR (9-mo rate: 72.4%), respectively. For pts in Part 1, Part 2A DL1, Part 2A DL2, and Part 2B, grade ≥2 CRS rates during Cycle 1 were 15.2%, 16.7%, 27.3%, and 6.9%, respectively. For pts in Part 1, Part 2A DL1, Part 2A DL2, and Part 2B, CRS rates were 30.3%, 33.3%, 36.4%, and 24.1% after dose 1, 27.3%, 25.0%, 36.4%, and 24.1% after dose 2, 15.2%, 33.3%, 27.3%, and 20.7% after dose 3, 6.1%, 8.3%, 0%, and 3.4% after dose 4, and 6.1%, 16.7%, 9.1%, and 0% after doses ≥5. Recurrent CRS occurred in 18.2%, 33.3%, 18.2%, and 17.2% of pts in Part 1, Part 2A DL1, Part 2A DL2, and Part 2B. The most common (>60%) TEAEs (any grade/grade ≥3) were hematologic TEAEs (Part 1, 75.8%/75.8%; Part 2A DL1, 83.3%/83.3%; Part 2A DL2, 81.8%/81.8%; Part 2B, 79.3%/69.0%), infections (Part 1, 78.8%/48.5%; Part 2A DL1, 83.3%/66.7%; Part 2A DL2, 72.7%/63.6%; Part 2B, 62.1%/37.9%), and CRS (Part 1, 60.6%/0%; Part 2A DL1, 83.3%/0%; Part 2A DL2, 90.9%/9.1%; Part 2B, 48.3%/0%). Grade 5 TEAEs not including PD were infection (n=4), fall (n=1), subdural hematoma (n=1), and food poisoning (n=1). CONCLUSIONS The 4/20 mg step-up priming regimen and alternative dosing schedules in MM-9 resulted in similar efficacy vs the step-up regimen used in the registrational MM-3 study (12/32 mg). Responses were durable with encouraging PFS and OS with the alternative step up and longer dosing schedule. While the overall safety signals with the alternative step up regimen are similar to those seen with MM-3, the CRS profile differed with more CRS events after each subsequent dose and a higher prevalence of recurrent CRS. Thus, the optimal ELRA step-up regimen remains 12/32 mg from the original regimen in the MM-3 study. In addition, these early results suggest that reducing the dose frequency of ELRA may not compromise efficacy.

Introduction: There are discrepant definitions of both iron deficiency and anemia in pregnancy. It is recognized that iron requirements increase during pregnancy, particularly in the third trimester, and that deficiencies should be corrected to avoid adverse maternal-fetal outcomes. While low dose iron supplementation is recommended for all pregnant persons (ACOG Bulletin 233), dosing through prenatal vitamins varies widely. Additionally, despite the epidemic of iron deficiency in reproductive-age females globally and availability of high-value biomarkers (e.g., ferritin), current guidelines only recommend screening for iron deficiency during pregnancy in the presence of anemia. As anemia is the terminal manifestation of iron deficiency and reference ranges vary by organization, these guidelines and practices warrant further investigation and discussion. Methods: Retrospective review and analysis of the electronic health record at Mayo Clinic (Rochester, MN) was performed for obstetric deliveries between June 2022 and May 2023. Iron deficiency was defined as a ferritin <50 mcg/L and anemia was defined as a hemoglobin <11 g/dL. Results: A total of 2,141 unique deliveries were identified. The median age at delivery was 31 years old (range: 15 - 45) with 82% identifying as white. Anemia was documented in 42% (n=904) of pregnancies at least once throughout gestation, with an increasing frequency of 2% (n=43) in the first trimester, 11% (n=230) in the second trimester, 17% (n=361) in the third trimester, and 38% (n=803) from admission for delivery through the peri-partum period. There were five documented pRBC transfusions prior to admission for delivery; however, from admission for delivery through the peri-partum period, there were a total of 102 documented pRBC transfusions. In total, 15% (n=318) of pregnancies were screened for iron deficiency through ferritin testing. Ferritin levels were measured 415 times with 23% (n=96) occurring between the last menstrual period and 12 weeks gestational age (WGA), 24% (n=100) occurring between 13-27 WGA, 40% (n=164) occurring between 28-40 WGA, and 13% (n=55) occurring post-partum. Iron deficiency was found in 70% (n=67), 79% (n=79), 80% (n=164), and 78% (n=43) of measured ferritin levels, respectively. Despite the paucity of screening, prescriptions for oral iron increased throughout gestation with supplementation in pregnancy for 8% (n=170) prior to 12 WGA, 14% (n=290) for those 13-27 WGA, and 23% (n=489) for those 28-40 WGA. Similarly, intravenous iron infusions increased throughout gestation with supplementation in pregnancy for 0.3% (n=7) prior to 12 WGA, 0.7% (n=16) for those 13-27 WGA, and 4% (n=95) for those 28-40 WGA. Prenatal vitamins were documented in over 90% of pregnancies throughout gestation, although the majority were non-prescription and contained variable amounts of elemental iron. Conclusions: Iron deficiency in pregnancy is underrecognized and undertreated. It follows that the accuracy of current reference ranges for anemia in pregnancy are suspect without rigorous verification of replete iron stores in the reference population. Given potential implications for blood product utilization and prenatal care, additional studies are warranted to better characterize iron deficiency in pregnancy as well as clinical sequelae for both mothers and infants. Simultaneously, discussions are needed around current guidelines and recommendations for both screening and treating iron deficiency in pregnancy. Providers, institutions, and professional societies should pursue pragmatic quality improvement initiatives and equitable healthcare delivery efforts to optimize care for this vulnerable patient population at high risk of iron deficiency.

Background: Mosunetuzumab (Mosun), a CD20xCD3 bispecific antibody, received accelerated approval for the intravenous treatment of patients (pts) with relapsed/refractory (R/R) follicular lymphoma (FL) who have received ≥2 prior lines of systemic therapy. Mosun can be administered in the outpatient setting with a fixed treatment duration. In an ongoing Phase I/II study, Mosun administered subcutaneously (SC) induced durable responses with a manageable safety profile in pts with previously untreated low-tumor burden FL (LTB; Flinn et al. ASH 2023). Efficacy, safety and pharmacodynamic profiles of Mosun SC are being evaluated in an open-label, multicenter, Phase II study (MorningSun; NCT05207670) in pts with B-cell non-Hodgkin lymphomas, including previously untreated FL. Here we present >8-month follow-up efficacy, safety and biomarker data for Mosun SC in first-line, LTB FL. Methods: Pts had untreated Grade (Gr) 1 or 2 FL, LTB by Groupe d'Etude des Lymphomes Folliculaires criteria, Ann Arbor stage III/IV disease, and Eastern Cooperative Oncology Group performance status (ECOG PS) of 0-2. Mosun SC injection was administered with step-up dosing in Cycle (C) 1 (Day [D] 1, 5mg; D8, 45mg; D15, 45mg), then 45mg on D1 of each 21-day cycle. Hospitalization was not mandatory. Pts with a complete metabolic response (CMR) after C8 could complete therapy. Pts with a partial metabolic response (PMR) or stable disease continued treatment for up to 17 cycles. Dexamethasone premedication was mandatory in C1 and C2 and optional thereafter. The primary endpoint was progression-free survival at 24 months. Exploratory analyses assessed cytokine levels in pts with versus without cytokine release syndrome (CRS) and pharmacodynamic changes in T and B cells in a subgroup of pts. Results: As of February 7, 2024, 65 pts were enrolled in US community (n=41) and academic (n=24) centers. At baseline, median age was 63 years (range: 34-82). Most pts had an ECOG PS of 0 (83.1%) or 1 (15.4%); 29 pts (44.6%) had extranodal involvement and 3 pts (4.6%) had B symptoms. Median duration of follow-up was 8.6 months (95% confidence interval [CI]: 5.7-10.9). Median number of cycles was 8 (range: 1-17). Among 60 pts with ≥1 post-baseline tumor assessment, the objective response rate was 95.0% (95% CI: 86.1-99.0; n=57) and CMR was 83.3% (95% CI: 71.5-91.7; n=50). Median time to response was 2.8 months (range: 2.3-8.3). At data cut-off, responses were ongoing in 48 pts with a CMR and in 5 pts with a PMR. In 64 safety-evaluable pts, the most common adverse event (AE) was injection-site reaction (70.3%; Gr 1, 65.6%; Gr 2, 4.7%). CRS by ASTCT occurred in 30 pts (46.9%; Gr 1, 39.1%; Gr 2, 7.8%); CRS events presented early in C1 (53.1%; Gr1, 45.3%; Gr2, 7.8%) and C2 (Gr1, 1.6%). Nineteen pts received CRS treatment (tocilizumab, 7.8%; steroids, 7.8%; tocilizumab + steroids, 4.7%; fluids, 3.1%; low-flow oxygen, 1.6%); 8 pts had a serious AE and were hospitalized. All CRS events resolved. Other common AEs included headache (45.3%; Gr 1, 29.7%; Gr 2, 15.6%), fatigue (45.3%; Gr 1, 35.9%; Gr 2, 9.4%), maculo-papular rash (31.3%; Gr 1, 14.1%; Gr 2, 15.6%; Gr 3, 1.6%), and increased alanine aminotransferase (28.1%; Gr 1, 21.9%; Gr 2, 4.7%; Gr 3, 1.6%). Gr 3/4 AEs in ≥5% of pts were neutropenia without fever (12.5%; Gr 3, 4.7%; Gr 4, 7.8%) and lymphopenia (7.8%; Gr 3, 1.6%; Gr 4, 6.3%). Suspected immune effector cell-associated neurotoxicity syndrome occurred in 2 pts (confusional state, Gr 1, n=1; Gr 2, n=1); all events resolved. Infections occurred in 30 pts (46.9%; Gr 1, 7.8%; Gr 2, 31.3%; Gr 3, 7.8%) with the most common (≥10%) being COVID-19 (17.2%; Gr 1, 7.8%; Gr 2, 7.8%; Gr 3, 1.6%) and upper respiratory tract infections (12.5%; Gr 1, 3.1%; Gr 2, 9.4%). One pt had a Gr 5 (fatal) AE 13.7 months after treatment was completed (reported as unrelated to Mosun; cause unknown). After the first Mosun dose, cytokine levels significantly increased on C1D2 (interleukin-6, n=44, p≤0.001; interferon-γ and tumor necrosis factor-α, n=41, p≤0.001). This spike in cytokine levels did not differ in pts with (n=19) versus without (n=25) CRS events in C1. Conclusions: Mosun SC monotherapy shows encouraging high efficacy as a fixed-duration treatment in first-line LTB FL. A manageable safety profile was reported, supporting ease of access for patients with drug administration in outpatient settings. Longer follow-up is needed to confirm long-term durability of response and safety.

Introduction: Nodal lymphomas of T-follicular helper cells (n-TFHLs) are considered neoplasms of mature TFH cells and are classified by the World Health Organization (WHO) under one umbrella with three subtypes: angioimmunoblastic-type (n-TFHL-AI), nodal TFHL, follicular type (n-TFHL-F), and nodal TFHL, not otherwise specified (n-TFHL-NOS). TET2 mutations in n-TFHL-AI subtype frequently co-occur with IDH2R172 mutations. Methods: To elucidate the cooperative role of TET2 and IDH2R172K mutation in n-TFHL-AI pathogenesis, we generated a murine model with Tet2 loss and IDH2R172K knock-in mutation in CD4+ T cells (Tet2-/-/IDH2R172K) by crossing Tet2Flox/Flox/IDH2R172K mice with Cd4-Cre transgenic mice. This resulted in Tet2 exon 3 deletion and IDH2R172K knock-in mutation specifically in CD4+ T cells. Murine tumors were comprehensively evaluated through immunohistochemistry and flow cytometry. Transcriptomic (RNA-seq), 5-hydroxymethylcytosine (5hmC), and 5-methylcytosine (5mC) profiling, and genomic (whole exome sequencing [WES]) analyses were conducted on both earlier time points (preneoplastic) and neoplastic cells. Functional validation involved proliferation, T-cell polarization, and Western blotting assays. Results: The transcriptomic analysis (p < 0.05) in n-TFHL-AI (double-mutant) cases showed enrichment of TFH-like signature, IL-12-signaling, NF-ΚB, and downregulation of TH1 cell differentiation (including IFNγ signaling). Evaluation of double-mutant mice revealed an earlier tumor onset than Tet2 mutant mice, with a median survival of 1.3 years compared to 1.7 years (p=0.006), respectively. With chronic stimulation by T cell-dependent antigens (sheep red blood cells [SRBCs]), these mice showed B-cell expansion with a 100% incidence of lymphoid neoplasms, in contrast to PBS-treated mice, where myeloid and lymphoid diseases occurred at an equal ratio. Genomic analysis of murine tumors revealed a unique mutation spectrum in metabolic genes (Ata3d3a, Atp1a3, Ppp4r3a, and Nemf), T- and B-cell interaction (Gnai3), Stat pathway (Jak1), and DNA repair (Zfp451). Transcriptomic analysis of tumors showed downregulation of Tbx21, increased TFH-related genes, and increased expression of Bcl6 with downregulation of its known suppressed genes. Transcriptomic analysis of murine CD4+ T cells isolated at 12 weeks revealed downregulation of genes associated with TH1-cell differentiation and Trp53 signaling, concurrent activation of T-cell receptor (TCR) and MAPK signaling, and API-complex. We demonstrated elevated levels of 2-hydroxyglutarate (2-HG) in double-mutant cells, and enasidenib (IDH2R172 inhibitor) treatment significantly decreased 2-HG levels and increased Tbx21 expression. Mutant cells showed a proliferative advantage in IL-2 and IL-21 in vitro conditions, resulting in enhanced TCR, AKT, ERK1/2, p-65, and inactivation of p-FOXO1 protein. While these cells showed a growth advantage in in vitro culturing with TFH conditions, their growth and polarization were impaired in TH1 conditions, indicating a differentiation block. Tet2-/-/IDH2R172K mice (12-week) with SRBCs treatment revealed a significant induction of B and CD4+ T- cell populations compared to wild-type (WT) mice (p=0.01). In vitro co-culturing experiments demonstrated that Tet2-/-/IDH2R172K mutant CD4+ T-cells confer a substantial proliferative advantage to B-cells (p=0.021). Notably, these double-mutant CD4+ T cells exhibited superior proliferation compared to WT CD4+ T-cells when co-cultured with B-cells isolated from WT or double mutant mice (p=0.004). When Tet2-/-/IDH2R172K neoplastic cells with whole tumor were allografted into NOD scid gamma (NSG) mice, lymphoma developed within three months, but also showed numerous B-cells in the TME. This suggests that the propagation of neoplastic T-cells likely require B cell help. Overall, the TFH program remained intact in transplanted cells, and a global decrease in 5hmC levels and an increase in 5mC levels were observed. Conclusion: Tet2-/-/IDH2R172K murine CD4+ T cells exhibited hyperactive TCR signaling, impaired DNA damage response, and NF-ΚB activation as key oncogenic transitions. Both in vitro co-culturing and the allografting of neoplastic Tet2-/-/IDH2R172K cells showed dependence on B-cells, suggesting that interrupting T- and B-cell interaction could be a therapeutic approach.

Background: Risk stratification for suspected pulmonary emboli (PE) remains an area of active research. Current risk stratification algorithms have moderate discriminatory capabilities and are guideline-recommended, but they are poorly utilized in practice, with most clinicians relying predominantly on their clinical training and personal “gestalt.” Our group recently derived an Artificial Intelligence (AI) algorithm using electrocardiograms (ECG) to predict the presence of PE (AUC 0.69) on computed tomography scans of the pulmonary arteries (CTPA) in a cohort of 79K patients. Aim: Validate the AI-ECG algorithm in a contemporary dataset of patients with suspected PE. Methods: Emergency Department (ED) visits between 9/1/2022 and 12/31/2023 across the Mayo Clinic Enterprise were searched using a unified data platform. ED visits in patients over 18 years of age with a discretely documented Wells' PE or PERC Rule, a D-dimer result, or a CTPA were included. Only CTPA imaging and D-dimers performed within 48 hours of ED admission were eligible to be included. Patients were included in the final cohort if an ECG was performed within 6 hours of ED admission, D-dimer testing, or CTPA. Patients were considered negative for PE if CTPA was negative, and when this wasn't performed, a negative D-dimer (high sensitivity) or PERC rule of 0 was considered negative. A previously derived Artificial Neural Network algorithm to analyze the ECG to predict PE was applied to the dataset for validation. Results: A total of 27,028 ED visits in 24,288 unique patients meet inclusion criteria. Among this group, the first ED visit with a CTPA and an ECG within 6 hours was included, leaving 18,936 patients for the final validation cohort. The mean age of the cohort was 54.7 (SD 18.7), and 57% were male. Among the entire cohort, 13,573 underwent D-dimer testing for which 4,895 (36%) were positive at standard thresholds. A total of 10,486 patients underwent CTPA (55% of patients evaluated), of which 455 were positive for PE (4.3%). Using the AI-ECG model from the derivation cohort applied to this validation cohort resulted in an AUC of 0.69, identical to the derivation cohort. Using ECG model estimates alone, low (0.74%, n=2447, NPV 99.3%), elevated (2.27%, n=15404, NPV, 97.72%), and high-risk (8.02%, n=1085, NPV 91.98%) categories for PE can be created. A negative D-dimer in the elevated risk group increased the negative predictive value (NPV) to >99.9% and could be used to reduce imaging in this group further. Using the proposed ECG to D-dimer cascade risk stratification could further reduce the need for PE imaging for an overall NPV of 99.9% and sensitivity of 95.2%. We then used additional clinical parameters to train a new algorithm with additional clinical data and the AI-ECG algorithm estimates. Multiple machine learning algorithms were then evaluated in a dataset divided into training (2/3) and testing (1/3) datasets, after downsampling (to address class imbalance). The best results were obtained with AI-ECG results and D-dimer: Ada Boost (AUC 0.93, F1 score 0.895) compared to standard logistic regression (AUC 0.79, F1 score 0.746). The addition of age, sex, and oxygen saturation did not improve model performance. Conclusion: We have now validated the previously derived AI-ANN ECG algorithm for PE in a contemporary and independent cohort and demonstrate potential for integration into clinical PE assessment algorithms. Unlike prior risk stratification processes, ECGs are invariably performed in patients with cardiopulmonary symptoms and have the potential to prevent missed or delayed PE diagnosis by proactively identifying high-risk patients. Additionally, we demonstrate that the results of this algorithm can be combined with D-dimer values, resulting in a simple, powerful, predictive algorithm (AUC 0.93) that pairs cardiovascular electrophysiology with laboratory biomarkers and could lead to a new paradigm in clinical risk stratification for PE.

Background : Rituximab is a monoclonal antibody that targets CD20 and can significantly improve the prognosis of patients with CD20-positive B-cell non-Hodgkin lymphoma (B-NHL). Nonetheless, a significant number of patients experience disease progression or relapse post-rituximab therapy. CD19-chimeric antigen receptor (CAR) T cells have demonstrated remarkable efficacy in B-NHL treatment. However, few studies have focused on the efficacy and safety of CD20 CAR-T cells in rituximab-refractory/relapsed (R/R) B-NHL patients, particularly those treated with rituximab for a short time. Here, we conducted a prospective study, aimed to assess the long-term remission and survival in patients with rituximab-R/R B-NHL after treatment with U16 (CD20 CAR-T cells). Methods : This first-in-human study is an open-label, single center investigator-initiated trial (IIT) to determine the safety and efficacy of CD20 CAR-T in R/R B-NHL after previous rituximab treatment failure (no. ChiCTR2000036350). CD20 CAR-T was administered at a target dose of 1.0×107 CAR-T cells/kg after a 3-day conditioning chemotherapy with fludarabine and cyclophosphamide. The primary objective was to assess the safety and tolerability of CD20 CAR-T. Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) were graded according to ASTCT 2019 criteria. The secondary objectives were to evaluate efficacy and pharmacokinetics (PK). Response was assessed per Lugano 2014 criteria. Results:A total of 16 patients with R/R B-NHL were enrolled.The median age was 49 years (range, 28-66). Median number of prior lines of therapy was 3 (range, 2-5). All patients had received rituximab therapy; of these, 9 had done so within the 3 months before CAR-T infusion. CRS occurred in all patients, with 11 patients (68.75%) experiencing grade 1 or 2 CRS and 5 patients (31.25%) experiencing grade 3 CRS. No patient developed grade 4 CRS , and only one patient had ICANS.The most common grade 3 or higher adverse events observed after infusion included leukopenia (93.3%), anemia (40%) and thrombocytopenia (60%). An overall response rate of 100% was observed among enrolled patients, with 11 patients (68.75%) achieving complete remission(CR) and 5 patients (32.25%) achieving partial remission(PR) within 3 months. The median follow-up time was 11.7 months(range 0.69-47.47). The median overall survival was not reached, and the median progression-free survival was 14.5 months. 9 patients remained CR or PR for 12 months, with 7 of them continuing in CR for up to 24 months. The pharmacokinetics of CD20 CAR-T cells were assessed using quantitative PCR to determine the expansion and persistence ability. Peak CAR amplification were detected 7-14 days after infusin. Patients 1, 2 and 4 achieved long-term remission, and persistent positive CAR-T cell copy numbers were detected for 15 months, 30 months and 26 months, respectively. However, patients 7, 8 and 12 relapsed within 3 months, and the CAR-T cell copies of all three patients were reduced to fewer than 1×103 copies/μg of DNA within 3 months. The qPCR results indicated that the maintenance of efficacy was closely related to the persistence of CAR-T cells. Notably, patients who received rituximab less than 3 months before CAR-T infusion had a poorer prognosis, with higher relapse rates (77.8% vs. 28.6%) and shorter progression-free survival, regardless of the degree of response. Conclusion : Long-term follow-up demonstrated that CD20 CAR-T therapy can produce a deep and durable response with a favorable safety profile in patients with rituximab-refractory/relapsed B-NHL. Notably, patients who had received rituximab within 3 months had a poorer prognosis.

Introduction Knowledge of epidemiologic risk factors, clonal changes associated with MDS development from precursor states, and evolution from one MDS state to another (lower- (LR) or higher-risk (HR) disease or leukemia (AML)) are largely limited to retrospective studies, which may not reflect the true MDS spectrum. The MDS NHS is an established prospective cohort of patients (pts) with suspected MDS enrolled at diverse US sites. Extensive clinical data and biospecimens are collected at baseline and throughout follow-up (F/U), in real time, as pts are followed for life. Here, we assess rates and potential predictors of progression providing a foundation for precise stratification across the MDS spectrum, which may ultimately guide intervention. Methods At baseline, all MDS NHS pts have central pathology review and those assigned to MDS, MDS/MPN overlap, AML<30% blasts, or at-risk of MDS (AR-MDS: CCUS and other at-risk) are followed prospectively. We compared AR-MDS with LR-MDS (MDS and MDS/MPN overlap with IPSS-R ≤ 3.5) and HR-MDS (MDS and MDS/MPN overlap with IPSS-R > 3.5). We defined disease progression as any F/U diagnosis of worsening disease or documented disease-related death. Rates of disease progression were compared between groups using survival analysis methods for cumulative incidence incorporating non-disease death as a competing risk. Associations between progression and predictor variables were explored using univariate tests of association and multivariable survival (Cox regression) models for time-to-progression and logistic models for probability of progression (yes/no). Results The MDS NHS enrolled 2051 pts during June 2016-February 2024, with 54% (1111, above categories) followed prospectively. Median F/U is 1.7 yrs (IQR: 0.8-3.1 yrs, max: 7.9 yrs). For the current analysis, 582 MDS and 505 AR-MDS pts were included. Compared to AR-MDS, MDS patients were more often male (70% v 61%, p<0.01), older (median age: 75 v 72 years, p<0.01), and had lower baseline hemoglobin (median: 10.1 v 11.9 g/dL, p<0.001), platelets (median: 124 x109 v 146 x109 cells/L, p<0.001), and absolute neutrophil counts (median: 1.9 x109 vs 2.8 x109 cells/L, p<0.001). F/U time was similar among AR-MDS and MDS (median: 1.9 vs 1.6 yrs), with both groups having a 75th percentile of 3 yrs and maximum of 7 yrs. A total of 331 deaths were observed (30%), with 145 (44%) of these due to causes related to MDS (myeloid disease and/or cardiovascular disease). Overall, 18.5% had disease progression, with annual rates of 8% (MDS) and 2% (AR-MDS) (p<0.01). Among HR-MDS, progression rates were greatest in the 1st year following diagnosis (33%) and declined thereafter, whereas progression rates remained between 5-8% for LR-MDS and 1-3% for AR-MDS over 5 yrs of F/U. AR-MDS had similar rates of progression within the first 3 yrs from diagnosis as MDS with the lowest IPSS-R category. Among AR-MDS, low hemoglobin (hazard ratio, HR [95% CI]=3.2 [1.4-7.3], p<0.01) was significantly associated with progression. Among MDS, low hemoglobin (HR=4.0 [2.2-7.0], p<0.001), low platelet count (HR=3.1 [1.7-5.6], p<0.001) and higher numbers of mutations (HR=1.13 [1.0-1.2], p<0.01) were significantly associated with progression. Due to the low rate of progression in AR-MDS, meaningful comparisons of mutations could not be completed. In MDS, univariate analyses showed that somatic mutation prevalence in gene pathways for transcription, cell cycle and RAS signaling, and in genes TP53, BCOR, SF3B1, DDX41, PTPN11, SETBP1, and JAK2 were significantly associated with the probability of progression (p<0.05 in all). In multivariable models, adjusting for patient age, sex, and MDS risk level, the probability of progression (PP) was significantly higher among those with vs without somatic mutations in BCOR (PP=63% vs 29%, p=0.004), PTPN11 (PP=67% vs 29%, p=0.009), SETBP1 (PP=61% vs 30%, p=0.02), JAK2 (PP=59% vs 30%, p=0.02), DDX41 (PP=56% vs 30%, p=0.03) and TP53 (PP=43% vs 29%, p=0.04). Conclusions In this large prospective cohort of MDS and pts at risk for MDS, the annual rate of any disease progression was 8% among MDS and 2% among AR-MDS, lower than some retrospective series. Specific cytopenias and genetic factors associated with progression were identified. The MDS NHS provides an incomparable opportunity to address the critical clinical challenges of management and prevention of MDS progression across its spectrum.

Introduction: Myelodysplastic syndromes (MDS) occur due to the acquisition of mutational events over time. Tobacco smoking is a risk factor for MDS and has been associated with distinct genetic mutations in solid cancers. We investigated the association between smoking intensity and duration and the number and types of genetic mutations in MDS using a large prospective cohort. Methods: The prospective NHLBI/NCI National MDS Natural History Study includes individuals with suspected or recently diagnosed MDS enrolled between June 2016-August 2023. Patient (Pt) bone marrow samples underwent central pathology review, resulting in diagnoses including MDS, MDS/myeloproliferative neoplasm overlap (MDS/MPN), or precursor conditions, such as clonal cytopenia of undetermined significance (CCUS). Self-reported smoking data were collected at enrollment. Smoking intensity (i.e., light, medium, and heavy) was defined per CDC definitions. Included mutations were those manually annotated to be pathogenic for MDS. Mutation frequency and prevalence, as well as associations with smoking history and other key covariates, were assessed using univariate tests of association and multivariable regression models for counts and proportions. Rates of disease progression were compared between groups using survival analysis methods for cumulative incidence incorporating non-disease death as a competing risk. Results: Of 1898 evaluable pts, 52% (N=979) had a history of cigarette smoking [MDS: 58% (252/436), MDS/MPN: 57% (52/91), CCUS: 57% (230/405)], and 18% still smoked at diagnosis. The mean ± SD cigarettes per day was 15.8 ± 12.5 and the mean ± SD smoking years was 29.8 ± 16.9 years. Compared to non-smokers, smokers were more often males (68% v 54%, p<0.001) in the 70-79 year age group (45% v 34%, p<0.001). Similar proportions of those with and without a smoking history had MDS (41% v 37%, respectively). Within MDS, there was no association between smoking and MDS prognostic risk per revised international prognostic scoring system. Compared to light smokers [1-10 pack-years (PY)], heavy smokers (30+ PY) were more often male (81% v 62%, p<0.001) and had higher rates of cardiovascular disease (38% v 20%, p<0.001). In univariate analyses, smokers and non-smokers had similar numbers of mutations (mean ± SD: 2.0 ± 2.7 in both groups). Pts with CCUS had the least number of mutations (1.5 ± 1.2), followed by MDS (2.3 ± 1.8), and MDS/MPN overlap (3.3 ± 1.8). Within disease groups, smokers and non-smokers had similar numbers of mutations. Mutation prevalence was higher among smokers v non-smokers in gene pathways for chromatin modification (15% v 11%, p<0.01) and RNA splicing (26% v 19%, p<0.001) and for individual genes ASXL1 (12% v 8%, p<0.01), SF3B1 (9% v 6%, p<0.05), U2AF1 (6% v 3%, p<0.05) and ZRSR2 (2% v 1%, p<0.05). In multivariable regression models, after adjustment for sex, age and disease group, smokers had significantly more mutations on average than non-smokers (2.0 v. 1.4, p=0.04). Moreover, the number of mutations increased significantly with PY smoked (p=0.006), and those at the 75th and 90th percentiles of PY had 1.8 and 3.5 times the number of mutations, respectively, compared to non-smokers, indicating a dose-response relationship. After adjustment, mutation prevalence increased with PY smoked in the gene pathways for chromatin modification and RNA splicing (p<0.03 for both) and for the genes ASXL1 and ETNK1 (p<0.05 in both cases). The five-year cumulative incidence of disease progression was significantly higher among long-term smokers compared to non-smokers/those with shorter smoking history (mean [95% CI] proportion progressed, 20+ years vs <20 years smoking: 27% [19%-36%] vs 18% [13%-24%], p < 0.05). Overall survival was significantly lower for smokers compared to non-smokers for CCUS pts [HR 1.91, 95% CI (1.03-3.55), p=0.04] but not for MDS [HR 1.21, 95% CI (0.53-1.30), p=0.41]. Conclusions: In this prospective, national cohort, tobacco smoking was associated with distinct genetic mutations related to MDS, suggesting smoking potentially contributes to its multi-step molecular pathogenesis. A dose-response relationship was also demonstrated, as was an association with progression and survival. Our data suggest that pts with a new MDS diagnosis who are also smokers should be counseled to stop smoking, as it appears to contribute to the acquisition of new genetic mutations that can lead to progression.

Introduction The pathogenic role of somatic non-coding (nc) single nucleotide variants (SNVs) is underexplored in multiple myeloma (MM) and additional characterization of these regions has the potential to provide significant new insights into disease progression. To investigate the role of ncSNVs and their relationship with MM pathogenesis, we have characterized a novel series of WGS from 294 cases of MGUS, SMM, and MM using computational tools optimized to identify functional driver SNVs in these regions. We hypothesize the ncSNVs that co-localize to sites of super-enhancers (SEs) active in plasma cells (PC) are sites of high functional impact SNVs and these mutations lead to the dysregulated expression of genes that should be silent at the terminally differentiated stage of PCs, providing a selective advantage that shapes the fitness of the MM genomic landscape. Methods We analyzed 294 high-coverage matched tumor/normal pairs (80x tumor and 40x normal) derived from 21 MGUS, 86 SMM, 167 NDMM and 40 RRMM. In addition to previously sequenced samples, newly sequenced patients were obtained from NYU Langone Health, Memorial Sloan Kettering Cancer Center, University of Alabama at Birmingham and University of Miami following IRB-approved informed consent. DNA was extracted from CD138+ purified bone marrow plasma cells using standard approaches. We used the MGP1000 (https://github.com/pblaney/mgp1000) to process sequencing data which employs a union-consensus mechanism for determining the final set of variants. We collected enhancer elements (E/SEs) active in GC B-cells, ABC-DLBCL and GBC-DLBCL (Bal, et al. Nature, 2022) and PC specific E/SEs (Loven, et al. Cell 2013). FunSeq2 was used to score ncSNVs. GRAM was used to predict how variants alter TF binding and gene expression in a subset of 6 cases with paired WGS in our series and will expand this analysis on a validation set of 263 NDMM and 93 RRMM cases from University-Hospital Heidelberg enrolled in the GMMG-HD6 clinical trial. Results We identified 1,952,232 high-quality SNVs with 18,199 (0.93%) accumulating in 280 focal sites we termed “mutationally enriched regions” or MERs. To discern SNVs in MERs with the highest potential for imparting a selective advantage, we clustered mutations based on the density of FunSeq2 impact scores which uses reference data contexts on sequence conservation, modification of transcription factor (TF) binding, and gene linkage to regulatory elements and network hubs from pan-cancer studies. We show that MERs are enriched for ncSNVs with high impact scores (mean=2.57) compared to random similarly-sized regions of the genome (Fisher's exact test P<9.7e-12, OR=10.10 [3.88-37.40]). The high impact ncSNVs (hi-ncSNVs) in MERs (n=220) account for 8.09% of all hi-ncSNVs and are present in 46.3% of cases (136/294) with some patients having up to 7. The proportion of cases carrying a MER hi-ncSNVs was similar across disease stages (MGUS=42.9%, SMM=46.5%, MM=40.7%, and RR=47.5%). Analysis of the VAF of MER hi-ncSNVs shows a minor shift towards more clonal versus subclonal mutations (53.6% vs 46.4% VAF>=0.25) suggesting some high impact mutations are acquired in the GC but additional hi-ncSNV are acquired later in PC development, providing evidence that hi-ncSNVs accumulate in MERs throughout disease progression. We mapped these hi-ncSNVs to associated genes then intersected the loci with E/SEs active in GCBs and PCs to determine their functional context. This approach identified 12 genes affected by hi-ncSNVs including the previously identified nc-drivers NEAT1, MIR142, ZCCHC7, and IGLL5 as well as the PC differentiation genes IRF8, BACH2, proliferation genes RHOH, SPRED2, known coding MM drivers CCND1, ZFP36L1, and chr1q genes BTG2 and CROCC. We predicted the effect of ncSNVs on expression through modulation of TF binding at this set of putative nc drivers in MM and identified a trend towards increase in expression of SPRED2, CCND1, NEAT1, and IGLL5 compared to non-mutated cases (P=0.089). We will present data on the validation of this set of genes on a larger set of data derived from the Heidelberg group. Conclusions We identified a set of 12 putative nc driver genes in MM that modify expression and have the potential to significantly alter the pathogenesis of MM by providing a selective survival advantage. We will present the impact of these by clinical stage and on outcome.

Introduction: Survival of children with relapsed/refractory (r/r) acute myeloid leukemia (AML), myelodysplastic syndromes (MDS) and juvenile myelomonocytic leukemia (JMML) remains dismal, and successful treatment approaches are limited. Myeloid leukemias frequently have high telomerase expression, and neoplastic progenitor cells have relatively shorter telomeres compared to normal tissues due to the balance between frequent malignant cell division and compensatory increased telomerase activity. In one study, the 5-year overall survival (OS) in children with AML and lower telomerase activity was 88% compared to 43% for those with higher activity, suggesting potential prognostic and therapeutic importance of telomerase in this population (Verstovsek Cancer 2003). Imetelstat is a first-in-class telomerase inhibitor approved by the FDA in 2024 for the treatment of adult patients with low-to-intermediate-1 risk myelodysplastic syndromes (LR-MDS) with transfusion-dependent anemia who are r/r/ineligible for erythropoiesis-stimulating agents. . Imetelstat has also been shown to drive AML cells into ferroptosis (Bruedigam Nature Cancer 2024). The Children's Oncology Group (COG) previously conducted the ADVL1112 Phase 1 study of imetelstat monotherapy in children with r/r solid tumors or lymphomas (Thompson CCR 2013). Imetelstat was administered intravenously on Days 1 and 8 of a 21-day cycle. The recommended phase 2 dose (RP2D) was 268 mg/m2. Imetelstat was well-tolerated with no grade >3 non-hematologic toxicities and one dose-limiting thrombocytopenia observed in a subject treated at the RP2D. In preclinical studies, imetelstat was active against leukemia stem cells in pediatric AML patient derived xenograft models (Barwe J Clin Med, 2022). Methods: PEPN2312 is an open-label, multi-center phase 1 study of imetelstat in combination with fludarabine/high dose cytarabine (FLA) for patients with AML in second or greater relapse, refractory to re-induction after first relapse, or r/r MDS or JMML (NCT06247787). The primary objective of this study is to estimate the maximum tolerated dose (MTD) and/or RP2D of imetelstat in combination with FLA in this patient population. Secondary objectives include toxicity assessment, pharmacokinetics, clinical response/minimal residual disease assessments per standard criteria, and OS. Exploratory analyses will be conducted to analyze telomerase activity in peripheral blood mononuclear cells, conduct pharmacodynamic studies, and evaluate any changes in cytogenetic and mutational abnormalities. Eligible patients must be between the ages of 1 to 18 at the time of study enrollment with Lansky/Karnofsky performance status of ≥50. Patients with leukemia must have platelet count >25,000/µL and hemoglobin >8 g/dL (transfusions allowed). Patients known to have a congenital bone marrow failure syndrome are excluded. This study will use the Rolling 6 design to estimate the MTD/RP2D. The starting dose of imetelstat (DL1) is 212 mg/m2 IV over 2 hours (80% of the single-agent RP2D) given on days 1 and 8 of a 28-day cycle in combination with FLA administered days 2-6. Premedication for infusion reactions with an antihistamine and corticosteroid are required prior to each dose of imetelstat. Patients may receive up to 2 cycles of therapy in the absence of progressive disease or unacceptable toxicity as graded by CTCAE version 5.0. Response assessment will be performed via morphology and central flow cytometry analysis for patients with AML and standard response criteria for patients with JMML or MDS (Niemeyer Haematologica 2015; Zeidan Blood 2023). One dose escalation to 268 mg/m2 (DL2) will be explored if DL1 is deemed safe. Intra-patient dose escalation is not permitted. Pharmacokinetic sampling will be performed during cycle 1. Once the MTD/RP2D is determined, further assessment of safety and preliminary activity of imetelstat and FLA chemotherapy will occur in an expansion cohort of up to 6 additional patients treated at the MTD/RP2D. PEPN2312 was activated in July 2024 and will enroll at 21 participating COG/PEP-CTN sites.