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Abstract Chimeric antigen receptor (CAR) T-cell therapy has demonstrated remarkable response rates in relapsed/refractory aggressive B-cell non-Hodgkin's lymphoma. However, durable remissions occur in only 40% of treated patients, highlighting mechanisms of therapeutic resistance and immune escape. While cytotoxicity is principally executed by CAR T-cells, monocytes shape CAR-T cell efficacy and orchestrate cytokine-driven immunity. In this study, we defined distinct metabolic profiles of T-cell and monocyte subsets based on clinical response. Single-cell RNA sequencing (scRNA-seq) and T-cell receptor sequencing (scTCR-seq) were performed on peripheral blood mononuclear cells at three timepoints: before lymphodepletion (BL), at peak CAR-T expansion (PK), and one-month post-infusion (M1). Data were processed with CellRanger v7.0.1 and analyzed with the immunopipe pipeline (Wang et al., 2025). Seurat v4.3.0 was utilized for unsupervised hierarchical clustering analysis; subsets identified included classical monocytes (CM), intermediate monocytes (IM), non-classical monocytes (NM), and monocytic myeloid derived suppressor cells (mMDSC). Metabolic profiles were compared across patients achieving ≥6-month complete remission (CR, n=16), primary refractory disease (PD1, n=4), or relapsed after a prior response (PD2, n=12). A total of 107035 T cells and 129981 monocytes from 77 serial samples in 32 patients were analyzed. CR patients had a higher T-cell:monocyte ratio than PD1/PD2 at all time points, especially at BL and PK (BL 1.2 vs 0.4, p=0.011; PK 1.3 vs 0.3, p=0.027). Clinical responses were compared with metabolic pathway analysis and identified oxidative phosphorylation (OXPHOS) as the dominant pathway. OXPHOS was consistently enriched in PD cohorts across T-cell subsets, particularly in PD1 vs CR at BL (mucosal-associated invariant T-cell (MAIT) and T peripheral memory (Tpm)), and at PK (CD4/CD8 memory precursor effector cells (MPEC) and activated MPECs (MPECa), CD4 T-helper 1 proliferating (Th1p), CD4 Th2 activated (Th2a), regulatory T-cell (Treg), CD8 naive senescent (CD8NS), CD8 T effector memory (Tem) tumor recirculating (TR), CD8 Tpm, CD8 T resident memory (Trm) TR and MAIT Tpm). By M1, OXPHOS enrichment in CD4/CD8MPECa, CD4Th2a, CD8NS, CD8TemTR reversed, now predominant in CR although CD8Tpm and CD8TrmTR remained enriched in PD1. In the monocyte subsets at BL, PD2 showed OXPHOS enrichment in CD163+ monocytes (mono), IM IL1, mMDSC SIRPA, mMDSC TGFβ, and CM IL6 subsets compared to PD1 and CR, while PD1 showed OXPHOS enrichment in IM CD38, CM TGFβ, and mMDSC HIF1A vs CR and PD2. This persisted at PK. By M1, the pattern reversed and mirrored that of T-cells, with CR patients showing OXPHOS enrichment in CD163+mono, CM IL6, CM TGFβ, IM CD38, mMDSC HIF1A, mMDSC SIRPA and mMDSC TGFβ, and additionally in CM EMT, CM p53, NM, NM CD36 vs PD1/PD2. Glycolysis/gluconeogenesis (GLY) was also enriched across subsets. In T-cells, PD1 vs CR showed GLY enrichment in CD4Th2a, CD8Tpm, CD8Tpm proliferating and MAIT Tpm at PK. In monocytes, GLY was enriched in PD1/2 vs CR at BL (CD163+mono, CM IL6) and PK (CD163+mono, IM CD18, mMDSC SIRPA). By M1, GLY metabolism was enriched in CR vs PD1/PD2 in CD163+mono, CM IL6, mMDSC SIRPA. Additional GLY enriched subsets at CR vs PD1 included CM, CM p53, EMT, IM CD38, IM IL1, mMDSC HIF1A, NM, NM CD36. Inositol phosphate metabolism (IP) was predominantly enriched in PD2 vs CR at BL (CD8NS, CD8Tcm, CD8Tpm activated), PK (CD4Th2, CD8NS, CD8Tem TR, CD8Tpm proliferating, γδT Tem) and M1 (CD4/CD8 MPECa, CD4 Th1/Th2, CD8NS, CD8Tpm, CD8 Tpm proliferating, Treg and MAIT Tpm). Notably, Treg was the only T-cell subset enriched for IP at PK in CR vs PD1/PD2. Additionally, the TCA cycle was enriched in PD1 Tregs and Th2 cells at BL and PK vs CR/PD2. A higher T-cell:monocyte ratio in CR vs PD1/PD2 supports prior evidence that elevated pre-lymphodepletion lymphocyte:monocyte ratios predict better outcomes. We identified distinct metabolic signatures in T-cells and monocytes between CR and PD1/PD2. OXPHOS enrichment in T-cells and monocytes at M1 in CR patients may reflect oxidative programming as a hallmark of sustained anti-tumor immunity. Conversely, GLY enrichment in PD1 T-cells at PK may reflect short-lived effector states lacking persistence. Overall, these findings highlight metabolic profiling as a promising predictive biomarker and therapeutic programming target to enhance CAR T-cell outcomes.

Abstract Chimeric Antigen Receptor (CAR) T cell therapy is a transformative treatment for hematologic malignancies. However, ex vivo autologous CAR T cell manufacturing presents substantial logistical and clinical challenges. Direct in vivo CAR-T cell engineering offers a new paradigm for CAR T delivery. We recently developed the CD3-targeted lentiviral vector LV-169 which can deliver a CAR transgene to resting niche-resident T cells in lymphoid tissues. LV169 is pseudotyped with a mix of chimeric and nonchimeric VSV-G proteins detargeted (blinded) from the natural LDL receptor and re-targeted to T cells through the display of a CD3-specific scFv. Here we demonstrate that LV169 can specifically deliver a functional aBCMA-CAR transgene to resting human T cells in PBMC cultures from the healthy volunteers and from multiple myeloma patients. For clinical translation, we selected an αBCMA-CAR construct comprising a fully human single heavy chain nanobody linked to 41BB and CD3z cytoplasmic signaling domains. Human T cells expressing this CAR construct have low tonic signaling and efficiently kill BCMA-positive myeloma target cells with similar or lower associated cytokine release compared to a clinically relevant benchmark BCMA-CAR. To further improve LV169 and to minimize CAR protein incorporation in the LV envelope which may lead undesirably to myeloma cell transduction, we placed the CAR transgene under a T-cell specific Lck promoter in reverse orientation relative to the 5' LTR. The transcriptional activity of the Lck promoter was comparable to that of a constitutive EF1a promoter in primary T cells and, in contrast to EF1a had minimal activity in a variety of other tested cell types including the HEK293T producer cell line and the THP-1 monocytic cell line. Western blotting confirmed the absence of CAR protein in LV particles (LVP) generated using the Lck promoter-driven reverse orientation construct. In contrast, high levels of CAR protein were detected in LVP generated using the parental EF1a promoter-driven construct. The LV-incorporated CAR protein mediated off-target gene delivery to BCMA-positive myeloma cells. The therapeutic efficacy of intravenously administered LV169 LV particles carrying BCMA-CAR constructs was tested in the OPM-2 human multiple myeloma xenograft model. NSG mice with knockout of MHC-I and MHC-II, and with advanced disseminated OPM-2-Luc tumors were engrafted with human PBMC one week prior to intravenous LV169-BCMA-CAR administration. Mice were treated with single intravenous injection of LV169 at different doses 3 weeks after tumor engraftment. OPM-2-Luc tumor burden was monitored by regular luciferase imaging. All treated mice cleared their tumors within 28-days following LV administration and the rate of clearance was correlated to the LV particle dose. Even at the 4e8 dose level (by p24) tumors were completely cleared. Eighty four days post-treatment the treated mice remained tumor-free and resisted re-challenge with OPM-2 tumor cells. Circulating CAR-T cells, monitored by flow cytometry, became detectable by 14 dpi and peaked between 21-35 dpi. Following tumor clearance, the number of circulating CAR-T cells declined dramatically. The treatment was well tolerated and analysis of cytokines associated with CAR-T cell cytotoxicity confirmed the absence of a severe cytokine storm. IFNγ was the only cytokine which was elevated during the expansion period of CAR-T cells, while IL-2, IL-6, TNFα and GM-CSF were barely detectable. In summary the characteristics of our new developed LV169-based platform make it suitable for further clinical translation which is underway.

Abstract Direct in vivo T cell engineering offers a new paradigm for CAR T delivery. We have evaluated several approaches to engineer lentiviral (LV) vectors capable of delivering CAR transgenes directly and selectively to T cells in vivo, within the lymphoid niche. Our goal was to develop an LV delivery platform that following intravenous administration can selectively bind and activate T cells, deliver and integrate the (CAR) transgene into genomic DNA, and drive CAR expression on the T cell surface. Further requirements were that the retargeted LV should be associated with minimal off-target transduction of tumor cells and normal host tissues and should be manufacturable at titers sufficient for human application. Our strategy for retargeting has been to modify the trimeric VSV-G protein, which is well established for efficient LV pseudotyping. Of the approaches evaluated, three retargeting platforms have proven to be effective: direct covalent display, indirect covalent display, and G-capping. Direct covalent display. This (LV-169) approach uses chimeric VSV-G protein that contains a CD3-targeting ligand displayed as a N-terminal extension of VSV-G protein that has been mutated to ablate (blind) its interaction with the LDL receptor. Chimeric and nonchimeric blinded G proteins are co- incorporated into LV particles within mixed envelopes which bind to CD3. Upon binding to cell surface CD3, the engineered particles activate T-cells and enter acidifying endosomes, whereupon they trigger membrane fusion and deliver their payloads. LV-169 displaying an anti-human CD3 scFv selectively transduced resting human T cells in PBMC cultures, in fresh human whole blood and following intravenous administration to humanized mice. In immunocompetent Cre reporter mice, surrogate vectors displaying anti-murine CD3 scFv selectively transduced lymph node resident T cells, with off-target transduction being limited to tissue-resident macrophages. To eliminate the incorporation of CAR protein into the LV-169 envelope, which can lead to highly undesirable transduction of tumor cells expressing the CAR target, we placed the CAR transgene in reverse to 5'LTR orientation, downstream of a T-cell specific promoter/enhancer. In vivo efficacy of LV-169-BCMA-CAR was demonstrated in humanized mice bearing disseminated OPM-2 myeloma tumors. Clinical translation of this construct is now underway. Indirect covalent display. In this G-protein engineering approach, a short peptide (SPY-tag) is displayed as an N-terminal extension of the LDLR-blinded VSV-G protein. The SPY-tagged LV vectors are then covalently modified to display a CD3-targeting ligand by simply mixing them at room temperature with a bifunctional fusion protein consisting of the SPY-catcher and the CD3 ligand. Under these conditions, the SPY-catcher and the SPY-tag spontaneously form a covalent isopeptide bond. The CD3 ligand-modified LV particles were shown to specifically activate and transduce T cells ex vivo within human PBMC cultures. G-capping. In the final approach, the VSV-G protein is not genetically modified. Instead, a “capping” protein crosslinker that binds directly to the unmodified G protein on preformed LV particles is used to mask their LDLR binding sites and redirect them to interact with CD3. Mixing of the CD3-targeted capping protein with traditional G-pseudotyped LV particles leads to efficient binding of the cap to the trimeric G proteins. The cap-modified LV are unable to interact with LDLR but interact efficiently with CD3. CD3-capped LV particles were highly specific and efficient in human T cell activation and transduction in PBMC cultures. The association between the G protein and the bound cap is highly stable during freeze-thaw cycles and at room temperature, a critical attribute of this platform to facilitate the manufacture of a clinically and commercially viable LV product. In summary, we have developed three translationally relevant platform technologies for the generation of LV particles capable of delivering CAR transgenes specifically and efficiently to resting niche-resident T cells after intravenous administration. Manufacturing studies are underway and, in the case of the LV-169 platform, clinical translation is already in progress.

Abstract Patients with myeloproliferative neoplasms (MPNs) are inherently at increased risk of both thrombotic and bleeding events due to the nature of the condition. Disease management often includes anticoagulants, antiplatelet agents, and/or cytoreductive therapy to mitigate these risks. On the contrary, these therapies can also increase the risk of bleeding, presenting a clinical challenge in balancing the prevention of thrombosis with the management of bleeding complications. The relationship between treatment type, thrombotic events, and bleeding risk remains underexplored in large, real-world populations. This Swedish population-based study aimed to describe how the risk of bleeding in patients living with MPN differs by treatment strategy, and how timing of thrombotic events influences this risk. A total of 15,495 patients diagnosed with MPN between 2006 and 2021, aged 18–90, were identified through the Swedish Cancer Register and the Swedish Patient Register. Bleeding rates were estimated using Cox proportional hazards models with time-split survival data. Treatment categories were defined as follows: no therapy, anticoagulant monotherapy (e.g., warfarin, direct oral anticoagulants [DOACs]); antiplatelet monotherapy (e.g., aspirin, clopidogrel); cytoreductive monotherapy (e.g., hydroxyurea, interferon); and combination therapy, defined as any combination of at least two of the three aforementioned therapies. All treatment categories were compared to anticoagulant monotherapy. Both treatment exposure and thrombotic status were modelled as time-varying covariates, allowing individuals to contribute across multiple thrombosis and treatment combinations over time. Models were adjusted for age at diagnosis, sex, year of diagnosis, and MPN subtype. The cohort was 52% female, with a median age at diagnosis of 71 years (IQR 60–79). The most common MPN subtypes were polycythemia vera (39%) and essential thrombocythemia (38%), followed by MPN-unclassifiable (16%) and primary myelofibrosis (7%). A history of thrombosis at MPN diagnosis was present in 22% of patients, and 19% had a history of bleeding. Among the 10,487 patients receiving combination therapy, the majority included antiplatelet agents: 57% received antiplatelet + cytoreductive therapy, 12% received antiplatelet + anticoagulant therapy, and 22% received all three therapies. Only 8% received anticoagulant + cytoreductive therapy without antiplatelet agents. Patients receiving no therapy had the lowest bleeding rate overall (HR 0.63, 95% CI 0.53–0.75), likely reflecting individuals with less severe disease or contraindications to treatment. Among patients with no thrombotic event, those receiving antiplatelet monotherapy had a 35% lower rate of bleeding compared to those on anticoagulant monotherapy (HR 0.65, 95% CI 0.56–0.76). Combination therapy was associated with a reduced rate of similar magnitude (HR 0.68, 95% CI 0.59–0.78), while cytoreductive monotherapy showed no significant difference (HR 0.96, 95% CI 0.76–1.21). In patients with a prior thrombotic event, antiplatelet monotherapy remained protective (HR 0.59, 95% CI 0.43–0.79), and combination therapy was associated with a slightly lower bleeding rate compared to anticoagulant monotherapy (HR 0.78, 95% CI 0.61–0.99). The estimate for cytoreductive monotherapy in this group (HR 2.04, 95% CI 0.89–4.68) was based on a small number of patients and had wide confidence intervals, limiting interpretability. These findings suggest that bleeding risk in MPN patients is influenced by both treatment type and thrombotic history. Antiplatelet monotherapy and combination therapy were associated with lower bleeding rates than anticoagulant monotherapy, both in patients with and without prior thrombosis, though the effect was more pronounced in those without prior thrombosis. By accounting for the complete patient pathway and modelling treatment and thrombotic status as time-varying exposures, a more accurate representation of real-world treatment dynamics is presented here. Given that the majority of combination therapy regimens included antiplatelet agents, these results underscore the potential protective role of antiplatelet therapy in managing bleeding risk. These insights may support clinical decision-making, and guide personalized treatment strategies and risk assessment in patients diagnosed with MPN.

Abstract Background: The functional importance of bone marrow adipocytes (BMA) is frequently overlooked even though they are critical components of the BM (bone marrow) microenvironment. BMA have been proposed to play a supportive role within the hematopoietic niche and to directly sustain hematopoietic stem cell (HSC) survival as well as to preferentially support leukemia cell survival and proliferation (Sabbah Cell Commun Signal 2023). Although significant ex-vivo data implicates direct and indirect crosstalk between BMA and neoplastic HSCs (Venti Front Endocrinol 2022), the possible role of BMA in myeloproliferative neoplasms and their dynamics following therapy have yet to be characterized. Adiponectin, an anti- inflammatory adipokine that maintains metabolic homeostasis through regulation of insulin sensitivity, lipid metabolism and pro-inflammatory signaling is low in MPN patients and increases with IFN-α therapy (Avcu Int J Hematol. 2006). IFN-α has also been implicated in inhibiting adipogenesis via JAK-STAT1 signaling (Lee Biochim Biophys Act. 2016). A single institutional study of IFN-α in patients with essential thrombocythemia (ET) and polycythemia vera (PV) confirmed the potential for BM histopathologic responses and a correlation with molecular responses (Masarova Exp Hematol Oncol 2017). The aim of this analysis was to characterize the change in BM cellularity and fat composition with pegylated IFN α-2a (Pegasys; PEG) and the relationship with both hematologic and molecular response from tissue collected in prospective, global clinical trials of PEG in patients with ET/PV. Methods: A total of 24 subjects from the phase 2 MPN-RC 111 (Yacoub Blood 2019) salvage PEG study and 16 subjects from the MPN-RC 112 (Mascarenhas Blood 2022) phase 3 study comparing PEG to HU with available clinical, BM H&E slides, laboratory and molecular data were included in this retrospective analysis of prospective clinical trial data. We assigned a semi-quantitative score for changes in cellularity, fat distribution, and megakaryocyte (MK) histo-topography for changes from baseline, at 12, 24 and 36 months when available. Changes in BMA content and para-trabecular distribution were assessed on whole slide digital images with machine learning models. BMA content was quantified with a QuPath pixel classifier that distinguished adipocytes from other marrow components (Bankhead Scientific Reports 2017). Para-trabecular adipocyte distribution was assessed by leveraging UNI2-h, a self-supervised Vision Transformer (ViT-H/14) model and successor to UNI (Mahmood Nature 2024). UNI2-h was pre-trained on 200 million image tiles from over 300,000 H&E and IHC slides. In this study, UNI2-h was adapted to distinguish between high and low para-trabecular adiposity using image tiles centered on trabeculae. Trabeculae with a surrounding layer of adipocytes were labeled as ‘high’ adiposity, while ‘low’ para-trabecular fat distribution was defined by the direct contact of cellular marrow with trabeculae, lacking surrounding adipocytes. Results: A total of 40 PEG-treated ET (n=18) and PV (n=22) patients were included in this analysis with a median age of 64 years (26-85), 77% JAK2V617F+, 23% CALRmut+, and 40% treatment-naïve. The median JAK2V617F variant allele fraction (VAF) was 26% (range 3-96%). A complete hematologic response (CHR) was obtained with PEG in 58%, 61% attained >20% reduction in driver VAF and 48% attained a >50% reduction in VAF (MR) within 24 months of baseline. We observed an increase in BMA content and notable increase in para-trabecular distribution (confirmed by morphology and ML, independently) in 93% and 53% of PEG treated patients attaining a MR versus only 31% and 6% who did not attain MR, respectively (P Value=0.0004). There was more notable improvement towards normalization of MK histo-topography and orderly erythropoiesis in MR (86.6%) than in non-MR (31.2%) (P value=0.001). Increase in BMA content, BMA para-trabecular distribution, or improvement in MK histo-topography were not associated with achievement of CHR or changes in CALR VAF in this small cohort. Cytokine correlation with BMA dynamics is currently ongoing and will be reported at the meeting. Conclusion: Treatment of MPN patients with IFN-α resulted in reproducible changes in BM cellularity including MK histo-topography, erythropoiesis, BMA number and distribution. Changes in BMA were closely associated with reduction in JAK2V617F VAF following PEG treatment.

Abstract Background Several BsAbs with different treatment (Tx) approaches have been approved by the FDA for Tx of lymphoma, including: fixed-duration and less frequently dosed mosunetuzumab (mosun) and glofitamab (glofit) (Option 1 Tx approach); and treat to progression and more frequently dosed epcoritamab (epcor) (Option 2 Tx approach). Differences in Tx approaches have implications for time spent receiving care, known as “time toxicity”. We previously reported that mosun and glofit were associated with about 2 weeks lower time toxicity vs epcor over the first year in R/R FL and DLBCL, based on registrational trial protocols (Torka et al. Blood 2024; Ma et al. Value in Health 2025). In this study, we assessed time toxicity from the perspective of patients (pts) receiving BsAbs for R/R FL or DLBCL. Methods An online survey was conducted aiming for input from 120 pts with R/R FL or DLBCL, who have received BsAb for ≥1 month in the US. Time toxicity was measured by pts recalling time spent on healthcare contacts related to BsAb Tx, cancer management, and any other reasons in the past 30 days. Pts' preferences were assessed through direct elicitation of choice between 2 hypothetical Tx profiles with different Tx approaches and time toxicity, assuming the same efficacy and safety. The survey was pre-tested and refined based on input from 3 BsAb pts with R/R DLBCL to ensure relevance and comprehension. Descriptive analyses were performed on pts characteristics, mean and standard deviation (SD) of time spent per pt per month, and the proportion of pts preferring the convenience of different Tx profiles. Results At this interim analysis, 101 pts receiving BsAbs responded to the survey: 48 pts with R/R FL (mosun n=28; epcor n=20) and 53 pts with R/R DLBCL (glofit n=32; epcor n=21). Most pts with R/R FL lived in urban/suburban areas (87.5% vs 12.5% in rural areas) and had a shorter one-way travel time to receive care vs pts in rural areas (mean±SD: 28.2±13.5 vs 60.3±30.2 minutes). The median age was 58 years for both mosun and epcor. Mean±SD Tx duration was 6.2±1.8 months for mosun vs 4.5±1.3 months for epcor. In the past 30 days, pts on mosun had fewer BsAb Tx visits vs epcor (mean±SD: 1.0±0.2 vs 2.0±1.2), and fewer all-cause healthcare contacts vs epcor (mean±SD: 1.7±0.8 vs 3.5±2.0). The mean±SD time spent per BsAb Tx visit was 5.0±1.4 hours for mosun vs 3.8±1.8 hours for epcor. Less time was spent per pt per month on recovery at home after Tx visit for mosun vs epcor (22.7±16.5 vs 43.2±47.6 hours). Less time was spent per pt per month on Tx and cancer management for mosun vs epcor (mean±SD: 30.5±16.9 vs 56.9±59.3 hours). Most pts with R/R DLBCL lived in urban/suburban areas (88.7% vs 11.5% in rural areas) and had a shorter one-way travel time to receive care vs pts in rural areas (mean±SD: 31.4±14.1 vs 103.3±19.4 minutes). The median age was 67 years for glofit and 66 years for epcor. The mean±SD Tx duration was 6.1±2.0 months for glofit vs 5.9±1.8 months for epcor. In the last 30 days, pts on glofit had fewer BsAb Tx visits vs epcor (mean±SD: 1.1±0.2 vs 1.7±0.5), and fewer all-cause healthcare contacts vs epcor (mean±SD: 2.2±1.3 vs 3.0±1.1). Irrespective of the route of administration, the time spent per BsAb Tx visit was similar (mean±SD: 5.1±1.6 hours for mosun; 5.6±1.2 hours for epcor). Less time was spent per pt per month on recovery at home after Tx visit for glofit vs epcor (mean±SD: 34.3±23.8 vs 69.3±39.4 hours). Less time was spent per pt per month on Tx visits and cancer management for glofit vs epcor (mean±SD: 43.0±25.9 vs 85.0±44.2 hours). Regardless of which BsAb pts received, most pts (93%) preferred the convenience of Option 1 Tx approach with less time required over Option 2 Tx approach with more time required. Conclusions This is the first study to directly measure pt-reported time toxicity from pts with R/R FL and DLBCL receiving BsAbs in a real-world setting. Fixed-duration less frequently dosed mosun and glofit (Option 1 Tx approach) had less pt-reported time toxicity, with less healthcare contact and less time spent per pt per month vs treat to progression more frequently dosed epcor (Option 2 Tx approach). Over 90% of pts preferred the convenience of a fixed-duration, less frequently dosed, and less time required Tx option, suggesting that time toxicity is a significant factor in shared Tx decision-making for pts with R/R FL and DLBCL. Analysis is ongoing to comprehensively measure pt-reported time toxicity.

Abstract Background: Routine laboratory testing prior to chemotherapy administration is a cornerstone of hematology and oncology practice, aimed at ensuring the safety and efficacy of treatment. However, over time, standardized protocols may accumulate excessive or low-yield laboratory tests that offer limited value to clinical decision-making. In 2018, Mayo Clinic implemented standardized treatment protocols using Epic Beacon, an electronic health record module that integrates chemotherapy regimens with associated laboratory testing and clinical workflows. While well-intentioned, these protocols may include redundant tests that delay treatment initiation, increase patient discomfort and anxiety, and burden providers with excessive in-basket communications. We hypothesize that many laboratory tests currently embedded within prechemotherapy workflows are unnecessary and can be safely omitted without compromising patient care. Methods: We conducted a retrospective cohort study evaluating the pre-treatment laboratory testing associated with the R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) chemotherapy protocol within the Mayo Clinic Rochester Division of Hematology. The study included 100 patients diagnosed with diffuse large B-cell lymphoma who received treatment between January 1 and December 31, 2024. Patients were excluded if they had incomplete medical records or received treatment outside the Mayo Clinic system. Data was abstracted via chart review using the Mayo Data Explorer. We analyzed complete blood counts (CBC), which include 12 individual tests, and comprehensive metabolic panels (CMP), which include 16 individual tests, obtained prior to each treatment cycle. For each cycle, we assessed the proportion of abnormal laboratory results and the proportion of abnormal results deemed clinically relevant which were defined as those that prompted a treatment delay, dose reduction or early termination of a cycle. Results: The cohort comprised 100 patients with a mean age of 68.2 years (SD 13.4) and a median age of 71 years (IQR 58.75–77); 57 were male and 43 were female. Based on available documentation, disease stage was classified as limited (stage I/II; n=41) or extensive (stage III/IV; n=59). A total of 519 R-CHOP cycles were administered, including 419 subsequent cycles (cycles 2–6). Fifteen patients discontinued treatment early, although none of the discontinuations were attributable to abnormalities in pre-infusion laboratory testing. Among the 419 subsequent cycles, 34 (8.1%) experienced treatment delays, with only one delay resulting from a clinically significant laboratory abnormality (hyperglycemia). Additionally, 23 patients underwent regimen modification (dose reduction or drug holding), but none of these were due to pre-infusion lab abnormalities.Of the 411 comprehensive metabolic panels (CMPs) obtained prior to these cycles, 315 (76.6%) contained at least one abnormal value, yielding a total of 786 abnormal results. Similarly, 405 of 415 (97.6%) complete blood counts (CBCs) demonstrated at least one abnormality, generating 2,696 abnormal values in total. Despite the high frequency of abnormal findings, the overall clinical yield was minimal, with only 1 of 826 pre-infusion laboratory panels (0.12%) leading to a modification in clinical management. Conclusion: Routine pre-infusion laboratory testing prior to R-CHOP chemotherapy frequently identifies abnormal results. However, these abnormalities are rarely clinically significant and do not affect treatment decisions. These findings support the reconsideration of current testing protocols to reduce unnecessary testing, enhance workflow efficiency, and minimize patient and provider burden without compromising safety.

Abstract Introduction: Cilta-cel is a BCMA-directed CAR T-cell therapy that has shown remarkable efficacy in patients with RRMM. As real-world (RW) experience with cilta-cel continues to expand, and with the recent FDA guidance reducing the recommended proximity to the CAR T center from four to two weeks post-infusion, there is growing interest in identifying patients at low risk for severe early complications who may be safely managed in the outpatient setting and potentially ‘discharged’ home earlier. Methods Data were collected from 769 patients with RRMM treated with cilta-cel at 15 institutions in the U.S. via the Multiple Myeloma Immunotherapy Consortium.Logistic regression was used to identify predictors of patients who could be managed in the outpatient setting (low-risk group). Based on expert consensus within the Consortium, low-risk patients were defined as: grade ≤1 CRS, no ICANS, and no IEC-HS. The remaining patients were categorized as high-risk. We evaluated baseline characteristics with missingness < 20% as predictors, including age, sex, race, and ethnicity, ECOG performance status (PS) at time of lymphodepletion (LD), extramedullary disease, bone marrow plasma cell (BMPC) percentage, high-risk cytogenetics, bridging therapy including response, penta-refractory status, number of prior lines of therapy, prior anti-BCMA therapy, baseline C-reactive protein (CRP), and baseline ferritin. The cohort was restricted to patients with complete data on all predictors (n=455) and split into a training and testing cohort (80/20 split, n=364/91). Bidirectional stepwise regression was used to build the predictive model using variables with significance of p<0.15 selected for entry. The best model was selected based on the AIC (Akaike Information Criterion) and a significance level of p<0.05. Results Among the 455 patients with complete data included in the final model, 290 (64%) were categorized as low-risk and 165 (36%) as high-risk. Compared to the low-risk group, high-risk patients were more likely to have an ECOG PS ≥2 at time of LD (20% vs 9%, p<0.001), high-risk cytogenetics (47% vs 35%, p=0.009), and penta-refractory disease status (34% vs 24%, p=0.031). High-risk patients also had a higher median BMPC percentage (15% vs 5%, p<0.001), and higher baseline pre-LD CRP and ferritin. Overall response rates were high, at 95% vs 90% (p=0.047) for the low vs high risk groups, with complete response or better achieved in 73% vs 66% of patients, respectively (p=0.14). With a median follow-up of approximately 11 months, median progression-free survival was not reached (95% CI: 27-NA) in the low-risk group and 18.2 months (95% CI: 13.1-NA) in the high-risk group. In terms of safety, non-ICANS neurotoxicity (NINT) occurred less frequently in the low-risk group (6.6% vs 13%, p=0.015). Specifically, Parkinsonism was observed in 1.7% vs 6.1% (p=0.013), and cranial nerve palsy in 4.5% vs 5.5% (p=0.7), respectively. Non-relapse mortality (NRM) was lower in the low-risk group (4.2%) compared to the high-risk group (14%; p < 0.001). The most common causes of NRM in the low- and high-risk groups were acute toxicities (0% vs 36%, including CRS, IEC-HS, and ICANS), delayed toxicities (0% vs 8%, including colitis, NINT, and Parkinsonism), infections (50% vs 48%), and other unrelated causes (50% vs 8%). The best model to predict low-risk patients that could be managed in the outpatient settingincluded BMPC percentage and ECOG PS at LD. In the training cohort, patients with a higher pre-LD BMPC percentage and an ECOG PS ³2 were less likely to be in the low-risk group(OR per 1-unit change in BMPC percentage = 0.99, 95% CI=0.98-0.99, p<0.001 and OR for ECOG PS 2-4 = 0.38, 95% CI=0.20-0.72, p=0.003). The AUC for this model was 0.663 in the training cohort and 0.659 in the testing cohort. Conclusion In this large, multi-institutional cohort of patients with RRMM treated with cilta-cel, we identified clinical factors associated with lower risk for early severe toxicities, supporting outpatient administration and the potential for earlier discharge in selected patients, particularly those who are fit and have low disease burden. Our findings provide a practical framework to guide risk stratification and outpatient CAR T management strategies, particularly in light of evolving regulatory guidance and growing RW experience with cilta-cel.

Abstract Background: Treatment of multiple myeloma (MM) has advanced dramatically over the past decade with median overall survival (OS) for newly diagnosed MM patients (pts) now exceeding 10 years. However, outcomes in pts with high-risk disease remain poor. While most pts with high-risk disease are identified through baseline cytogenetic risk stratification, presence of extramedullary disease, or R-ISS stage, there is also a population of pts who may or may not have these traditional high-risk factors who relapse earlier than expected and have similarly poor outcomes. These pts are often delineated as “functional high-risk,” but a standardized definition of this population in the era of modern myeloma therapy is lacking. Methods: Using the ASH Research Collaborative (ASH RC) Data Hub, we retrospectively searched 7,249 pts across 10 participating institutions for those with newly diagnosed multiple myeloma (NDMM) treated with a triplet or quadruplet induction regimen (with or without autologous stem cell transplant) who had response data available. We evaluated duration of response from first line therapy (PD1) on post-PD1 clinical outcomes such as: time to next treatment (TTNT), time to subsequent progression and/or death (PFS2), and OS. We also analyzed characteristics of those who did vs. did not progress at prespecified time points of interest (12-, 18-, 24-, and 36-months) from the start of induction. We utilized recursive partitioning analyses to iteratively explore all possible cutpoints and identify which one(s) provide the greatest differentiation in the risk of subsequent progression and/or death. Results: We identified 1,305 pts who met the eligibility criteria. Of these, 264 had PD1 data including 201 who had response follow-up (f/u) after PD1. Median f/u after PD1 in this population was 3.2 years, and median PFS2 f/u after PD1 (n=201) was 4.4 years. Months to PD1 was significantly associated with PFS2 (p=0.038). All prespecified time points examined showed inferior PFS2 compared to those with PD1 >36 mos, although each subgroup was not statistically significant, likely due to limited numbers in each group: PD1 <12 mos HR=1.44 (n=43; 95% CI: 0.90 – 2.30; p=0.126), PD1 12-24 mos HR=1.65 (n=30; 95% CI: 0.95 – 2.85; p=0.073), and PD1 24-36 mos HR=1.58 (n=52; 95% CI: 0.98 – 2.53; p=0.059). Similarly, timing of PD1 was also associated with OS post-PD1; pts with PD1 <12 mos had significantly lower OS compared to those with PD1 >36 mos (HR=2.35, 95% CI: 1.27 – 4.32; p=0.006). Our recursive partitioning analysis identified 30-months at the cutoff that produced the greatest differential in outcomes between those above vs. below the landmark. Approximately 23% of the pts analyzed had PD1 within 30 mos, and these pts had significantly lower OS compared to those with PD1 ≥30 mos (HR=1.99, 95% CI: 1.21 – 3.27; p=0.006). Similarly, pts who progressed within 30 mos of starting 1L therapy (n=98) had significantly shorter PFS2 (median=12.5 mos vs 22.8 mos; HR=1.55, 95% CI: 1.09 – 2.20; p=0.014) than those who did not (n=103). Those pts who progressed within 30 mos were more likely to have a higher R-ISS stage at diagnosis (p=0.048), but were not more likely to have high-risk cytogenetics (p=0.66) compared to pts with PD1 ≥30 mos. Interestingly, receiving SCT as part of 1L therapy was not significantly associated with improved PFS2 (HR=0.89, 95% CI: 0.62 – 1.29; 0.54) but was significantly associated with improved OS (HR=0.53, 95% CI: 0.31 – 0.90; 0.018) by univariate analysis. Conclusion: Our study is the first to use a large, multicenter, cohort to evaluate the definition of functional high-risk MM in the era of novel induction therapies. Our data suggest that pts who progress within 30 mos of induction on triplet or quadruplet regimens (irrespective of whether they underwent stem cell transplant) have poorer outcomes than those who progress at 30 mos or later, and that progression prior to 30 mos represents functional high-risk disease. These pts represent a clear unmet medical need, and additional focus should be taken to identify them early with the goal of designing trials to optimize their subsequent therapies.