CD19 Chimeric Antigen Receptor T-cell Research Articles

Impact of race and social determinants of health on outcomes in patients with aggressive B-cell NHL treated with CAR-T therapy

AbstractHealth care disparities driven by multiple social, economic, and/or environmental factors lead to inequalities in health outcomes. Chimeric antigen receptor (CAR) T-cell (CAR-T) immunotherapy is an effective therapy for relapsed/refractory B-cell non-Hodgkin lymphoma (r/r B-NHL). However, data are limited on the impact of the convergence of race and social determinants of health on outcomes for patients treated with CAR-T therapy. We examined the impact of interactions between race and insurance type on health care use and outcomes in patients treated with CAR-T therapy for aggressive B-NHL. Adult patients with r/r B-NHL treated with CD19 CAR-Ts were identified between 2015 and 2021 across 13 US academic centers. Insurance type, demographic, and clinical data were collected and analyzed via χ2 and Kaplan-Meier analysis. Cox multivariable regression was used to determine the impact of race/ethnicity and other variables on survival. In total, 466 adult patients were included in our analysis. Median follow-up after CAR-T therapy was 12.7 months. Median progression-free survival (mPFS) was longer for Caucasians (11.5 months) than for African Americans (3.5 months; hazard ratio [HR], 1.56 [1.03-2.4]; P = .04) or Asians (2.7 months; HR, 1.7 [1.02-2.67]; P = .04). Differences in median overall survival (mOS) were not significant. For Medicare (n = 206) vs Medicaid (n = 33) vs private insurance (n = 219) vs self-pay (n = 7): mPFS was 15.9 vs 4.2 vs 6.0 vs 0.9 months (P < .001), respectively; and mOS was 31.2 vs 12.8 vs 21.5 vs 3.2 months (P < .001), respectively. Collectively, our multicenter retrospective analysis showed that race and insurance status can affect outcomes for patients treated with CAR-T therapy.

Abstract 4002: Investigating CAR-T cell efficacy and activation in the disseminated NALM6-luc human B-cell acute lymphoblastic leukemia model

Abstract Introduction: Chimeric Antigen Receptor T (CAR-T) cell therapy has emerged as a promising approach for treating hematological malignancies, particularly B-cell acute lymphoblastic leukemia (B-ALL). This study aimed to delve into the activation dynamics and therapeutic efficacy of CD19 CAR-T cells in a disseminated NALM6-luc human B-ALL murine model. Experimental design: NALM6-Luc-mCh-Puro cells were implanted intravenously in female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice. Animals were treated with CD19 CAR-T cells alone and in combination with ibrutinib. The anti-tumor activity was assessed, and the persistence and activation of CAR-T cells in the blood were evaluated using flow cytometry. Additionally, ddPCR analysis was employed to quantify CAR copy numbers, providing insights into the T-cell persistence over time. Results: Treatment with CD19 CAR-T cells exhibited notable anti-cancer activity and that activity was enhanced in combination with ibrutinib, in line with other reports. The increased time to progression was noted as 69.2% for the CAR-T group and 138.4% for the combination of CAR-T and ibrutinib compared to control. Flow cytometry analysis showed that CAR-T cells persisted in the blood throughout the study. The mean CAR-T absolute cell count for both CAR-T and CAR-T plus ibrutinib treated groups increased from 2 cell/µL blood on Day 0 to 11 and 51 cells/µL of blood, respectively, by Day 39. The progressive increase of PD-1+ and LAG-3+ percentage cells correlates positively with tumor burden, indicating a dynamic link between biomarker upregulation and the presence of tumor cells in mice. ddPCR analysis revealed an initial CAR copy decrease in all groups, followed by gradual increase in tumor-bearing mice treated with CD19 CAR-T cells, with a dramatic increase observed by Day 39. Results from CAR-T treated and CAR-T plus ibrutinib treated groups were comparable, showing mean copy numbers per ng of DNA increasing from less than 1 on day 4 to 17.9 and 23.3, respectively, on day 39. Importantly, no changes were noted in non-tumor implanted mice, emphasizing the tumor dependency for CAR-T cell expansion. Conclusion: This study emphasizes the marked anti-cancer activity of CD19 CAR-T cells in the NALM6-Luc-mCh-Puro B-ALL murine model. Comprehensive analysis of CAR-T cell persistence and activation, including insights from ddPCR, provides a robust understanding of therapeutic response dynamics. The observed in vivo anti-cancer efficacy aligns with the persistence of CAR-T cells revealed by flow cytometry and the dynamic expansion captured through ddPCR analysis. Our results indicate ddPCR and flow cytometry as complementary and independent methods for assessing the systemic CAR-T cell persistence, contributing to optimizing therapy for B-cell acute lymphoblastic leukemia. Citation Format: Prashant Pandey, David Draper, Sheri Barnes, Yewei Xing, Derrik Germain, Lauren Kucharczyk, Roys Stacey. Investigating CAR-T cell efficacy and activation in the disseminated NALM6-luc human B-cell acute lymphoblastic leukemia model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4002.

Targeting autophagy overcomes cancer-intrinsic resistance to CAR-T immunotherapy in B-cell malignancies.

Chimeric antigen receptor T (CAR-T) therapy has substantially revolutionized the clinical outcomes of patients with hematologic malignancies, but the cancer-intrinsic mechanisms underlying resistance to CAR-T cells remain yet to be fully understood. This study aims to explore the molecular determinants of cancer cell sensitivity to CAR-T cell-mediated killing and to provide a better understanding of the underlying mechanisms and potential modulation to improve clinical efficacy. The human whole-genome CRISPR/Cas9-based knockout screening was conducted to identify key genes that enable cancer cells to evade CD19 CAR-T-cell-mediated killing. The in vitro cytotoxicity assays and evaluation of tumor tissue and bone marrow specimens were further conducted to confirm the role of the key genes in cancer cell susceptibility to CAR-T cells. In addition, the specific mechanisms influencing CAR-T cell-mediated cancer clearance were elucidated in mouse and cellular models. The CRISPR/Cas9-based knockout screening showed that the enrichment of autophagy-related genes (ATG3, BECN1, and RB1CC1) provided protection of cancer cells from CD19 CAR-T cell-mediated cytotoxicity. These findings were further validated by in vitro cytotoxicity assays in cells with genetic and pharmacological inhibition of autophagy. Notably, higher expression of the three autophagy-related proteins in tumor samples was correlated with poorer responsiveness and worse survival in patients with relapsed/refractory B-cell lymphoma after CD19 CAR-T therapy. Bulk RNA sequencing analysis of bone marrow samples from B-cell leukemia patients also suggested the clinical relevance of autophagy to the therapeutic response and relapse after CD19 CAR-T cell therapy. Pharmacological inhibition of autophagy and knockout of RB1CC1 could dramatically sensitize tumor cells to CD19 CAR-T cell-mediated killing in mouse models of both B-cell leukemia and lymphoma. Moreover, our study revealed that cancer-intrinsic autophagy mediates evasion of CAR-T cells via the TNF-α-TNFR1 axis-mediated apoptosis and STAT1/IRF1-induced chemokine signaling activation. These findings confirm that autophagy signaling in B-cell malignancies is essential for the effective cytotoxic function of CAR-T cells and thereby pave the way for the development of autophagy-targeting strategies to improve the clinical efficacy of CAR-T cell immunotherapy.

[Construction and identification of a stable CT26 cell line expressing CD19-FLUC-GFP].

Solid tumors lack well-defined targets for chimeric antigen receptor T-cell (CAR-T) therapy. Therefore, introducing a known target molecule, CD19, into solid tumor cell lines via lentiviral transduction to investigate the cytotoxicity of CD19 CAR-T cells can potentially support CAR-T cell therapy against solid tumors. In this study, a stable colon cancer CT26 cell line, CT26-CD19-FLUC-GFP, expressing CD19, firefly luciferase (FLUC), and green fluorescent protein (GFP), was constructed using a triple-plasmid lentiviral system. The growth characteristics of this cell line were consistent with those of the CT26 cell line. Subsequent flow cytometry analysis confirmed stable expression of CD19 and GFP in CT26-CD19-FLUC-GFP cells after serial passaging up to the 5th, 10th, and 22nd generations. Further validation revealed significantly higher levels of CD19 mRNA and FLUC expression in CT26-CD19-FLUC-GFP cells continuously passaged up to the 22nd generation compared to the control CT26 cells. In comparison to T cells, CD19 CAR-T cells demonstrated substantial cytotoxicity against CT26-CD19-FLUC-GFP cells and MC38-CD19 cells. One week after intraperitoneal implantation of CT26-CD19-FLUC-GFP cells into mice, FLUC expression in the peritoneal region could be detected. These results indicate the successful establishment of a stable CT26 cell line expressing CD19-FLUC-GFP, which can be specifically targeted by CD19 CAR-T cells.

CD19 CAR T-Cell Therapy in Autoimmune Disease — A Case Series with Follow-up

BackgroundTreatment for autoimmune diseases such as systemic lupus erythematosus (SLE), idiopathic inflammatory myositis, and systemic sclerosis often involves long-term immune suppression. Resetting aberrant autoimmunity in these diseases through deep depletion of B cells is a potential strategy for achieving sustained drug-free remission.MethodsWe evaluated 15 patients with severe SLE (8 patients), idiopathic inflammatory myositis (3 patients), or systemic sclerosis (4 patients) who received a single infusion of CD19 chimeric antigen receptor (CAR) T cells after preconditioning with fludarabine and cyclophosphamide. Efficacy up to 2 years after CAR T-cell infusion was assessed by means of Definition of Remission in SLE (DORIS) remission criteria, American College of Rheumatology–European League against Rheumatism (ACR–EULAR) major clinical response, and the score on the European Scleroderma Trials and Research Group (EUSTAR) activity index (with higher scores indicating greater disease activity), among others. Safety variables, including cytokine release syndrome and infections, were recorded.ResultsThe median follow-up was 15 months (range, 4 to 29). The mean (±SD) duration of B-cell aplasia was 112±47 days. All the patients with SLE had DORIS remission, all the patients with idiopathic inflammatory myositis had an ACR–EULAR major clinical response, and all the patients with systemic sclerosis had a decrease in the score on the EUSTAR activity index. Immunosuppressive therapy was completely stopped in all the patients. Grade 1 cytokine release syndrome occurred in 10 patients. One patient each had grade 2 cytokine release syndrome, grade 1 immune effector cell–associated neurotoxicity syndrome, and pneumonia that resulted in hospitalization.ConclusionsIn this case series, CD19 CAR T-cell transfer appeared to be feasible, safe, and efficacious in three different autoimmune diseases, providing rationale for further controlled clinical trials. (Funded by Deutsche Forschungsgemeinschaft and others.)

Clinical features of neurotoxicity after CD19 CAR T-cell therapy in mantle cell lymphoma

AbstractCD19 chimeric antigen receptor (CAR) T-cell therapy has proven highly effective for treating relapsed/refractory mantle cell lymphoma (MCL). However, immune effector cell–associated neurotoxicity syndrome (ICANS) remains a significant concern. This study aimed to evaluate the clinical, radiological, and laboratory correlatives associated with ICANS development after CD19 CAR T-cell therapy in patients with MCL. All patients (N = 26) who received standard-of-care brexucabtagene autoleucel until July 2022 at our institution were evaluated. Laboratory and radiographic correlatives including brain magnetic resonance imaging (MRI) and electroencephalogram (EEG) were evaluated to determine the clinical impact of ICANS. Seventeen (65%) patients experienced ICANS after treatment, with a median onset on day 6. Ten (38%) patients experienced severe (grade ≥3) ICANS. All patients with ICANS had antecedent cytokine release syndrome (CRS), but no correlation was observed between ICANS severity and CRS grade. Overall, 92% of EEGs revealed interictal changes; no patients experienced frank seizures because of ICANS. In total, 86% of patients with severe ICANS with postinfusion brain MRIs demonstrated acute neuroimaging findings not seen on pretreatment MRI. Severe ICANS was also associated with higher rates of cytopenia, coagulopathy, increased cumulative steroid exposure, and prolonged hospitalization. However, severe ICANS did not affect treatment outcomes of patients with MCL. Severe ICANS is frequently associated with a range of postinfusion brain MRI changes and abnormal EEG findings. Longer hospitalization was observed in patients with severe ICANS, especially those with abnormal acute MRI or EEG findings, but there was no discernible impact on overall treatment response and survival.

Adoptive cellular therapy after hematopoietic stem cell transplantation.

Effective cellular therapy using CD19 chimeric antigen receptor T-cells for the treatment of advanced B-cell malignancies raises the question of whether the administration of adoptive cellular therapy (ACT) posttransplant could reduce relapse and improve survival. Moreover, several early phase clinical studies have shown the potential beneficial effects of administration of tumor-associated antigen-specific T-cells and natural killer cells posttransplant for high-risk patients, aiming to decrease relapse and possibly improve survival. In this article, we present an in-depth review of ACT after transplantation, which has the potential to significantly improve the efficacy of this procedure and revolutionize this field.

[Efficacy and safety of chimeric antigen receptor T-cell therapy followed by allogeneic hematopoietic stem cell transplantation in 21 patients with Ph-like acute lymphoblastic leukemia].

Objective: To evaluate the efficacy and safety of chimeric antigen receptor T-cell (CAR-T) therapy followed by allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with Ph-like acute lymphoblastic leukemia (Ph-ALL) . Methods: Patients with Ph-ALL who underwent CAR-T therapy followed by allo-HSCT from March 2018 to August 2023 at the First Affiliated Hospital of Soochow University were included, and their clinical data were retrospectively analyzed. Results: Of the 21 patients, 14 were male and 7 were female. The median age at the time of CAR-T therapy was 22 (6-50) years. Seven patients had ABL1-like rearrangements, and 14 had JAK-STAT rearrangements. Prior to CAR-T therapy, 12 patients experienced hematologic relapse; 7 were multiparameter flow cytometry minimal residual disease (MFC-MRD) -positive and 2 were MFC-MRD-negative. CAR-T cells were derived from patients' autologous lymphocytes. Nine patients were treated with CD19 CAR-T cells, and 12 were treated with CD19/CD22 CAR-T cells. After assessment on day 28 after CAR-T therapy, 95.2% of the patients achieved complete remission, with an MRD-negative remission rate of 75%. Nineteen patients developed grade 0-2 cytokine release syndrome (CRS) and 2 patients suffered grade 3 CRS, all cases of which resolved after treatment. All patients underwent allo-HSCT after CAR-T therapy. The median time from CAR-T therapy to allo-HSCT was 63 (38-114) days. Five patients experienced relapse after CAR-T therapy, including four with hematologic relapse and one with molecular relapse. The 3-year overall survival (OS) rates in the ABL1 and JAK-STAT groups were (83.3±15.2) % and (66.6±17.2) %, respectively (P=0.68) . The 3-year relapse-free survival (RFS) rates were (50.0±20.4) % and (55.6±15.4) % in the ABL1 and JAK-STAT groups, respectively. There was no significant difference in 3-year OS or RFS between the two groups. Conclusions: CAR-T therapy followed by allo-HSCT leads to rapid remission in most patients with Ph-ALL and prolongs leukemia-free survival.

The Mechanisms of Altered Blood-Brain Barrier Permeability in CD19 CAR T-Cell Recipients.

Cluster of differentiation 19 (CD19) chimeric antigen receptor (CAR) T cells are a highly effective immunotherapy for relapsed and refractory B-cell malignancies, but their utility can be limited by the development of immune effector cell-associated neurotoxicity syndrome (ICANS). The recent discovery of CD19 expression on the pericytes in the blood-brain barrier (BBB) suggests an important off-target mechanism for ICANS development. In addition, the release of systemic cytokines stimulated by the engagement of CD19 with the CAR T cells can cause endothelial activation and decreased expression of tight junction molecules, further damaging the integrity of the BBB. Once within the brain microenvironment, cytokines trigger a cytokine-specific cascade of neuroinflammatory responses, which manifest clinically as a spectrum of neurological changes. Brain imaging is frequently negative or nonspecific, and treatment involves close neurologic monitoring, supportive care, interleukin antagonists, and steroids. The goal of this review is to inform readers about the normal development and microstructure of the BBB, its unique susceptibility to CD19 CAR T cells, the role of individual cytokines on specific elements of the brain's microstructural environment, and the clinical and imaging manifestations of ICANS. Our review will link cellular pathophysiology with the clinical and radiological manifestations of a complex clinical entity.

Factors associated with long-term outcomes of CD19 chimeric antigen receptor T-cell therapy for relapsedrefractory chronic lymphocytic leukemia 6-year follow-up

Factors associated with long-term outcomes of CD19 chimeric antigen receptor T-cell therapy for relapsedrefractory chronic lymphocytic leukemia 6-year follow-up

Combination of Ibrutinib with CD19 Chimeric Antigen Receptor (CAR) T Cells Suggests Greater Contribution to CAR T-Cell Efficacy Than Other Approved BTK Inhibitors in the Preclinical Setting

Background: CD19-directed CAR T-cell therapy has demonstrated remarkable clinical benefits for patients with B-cell malignancies, but impaired CAR T-cell fitness can result in lack of response or relapse. Rational combinations that enhance T-cell fitness may overcome primary and secondary resistance. Bruton tyrosine kinase inhibitors (BTKi), such as Ibrutinib (Ibr) have been shown to enhance CAR T-cell efficacy (Fraietta et al, Blood 2016;Liu et al, Cancer Sci 2020), but the precise mechanism remains unclear. In addition to BTK, Ibr potently inhibits IL-2-inducible tyrosine kinase (ITK), an important mediator of T-cell signaling. Ibr has been shown to enhance Th1 polarization through suppression of the Th2 differentiation pathway, thereby potentiating a cytotoxic effector response (Qin et al, J Immunother 2020). Consistent with this finding, the Th1 phenotype was more frequent in peripheral blood mononuclear cells of Ibr-exposed patients than in acalabrutinib (Acala)-exposed patients (Scarfò et al, Blood 2021). Therefore, we aimed to assess the potential impact of ITK inhibition (ITKi) on CD19 CAR T-cell function by comparing Ibr to other approved BTKi that display low or no ITKi activity (Estupiñán et al, Front Cell Dev Biol 2021). Methods: CD19-41bb CAR T cells (CAR19) were generated by lentiviral transduction of healthy donor T cells. BTKi additivity to CAR19 function was evaluated in a Luciferase (Luc)+ Ramos, BTKi resistant systemic tumor model in NSG mice co-treated with covalent BTKis Ibr, Acala, zanubrutinib (Zanu), and CAR T cells. Next, to evaluate the impact of ITKi on CAR19 fitness and cytotoxicity, repeated stimulation in an in vitro cytotoxicity assay was performed at low CAR19 effector-to-tumor ratio (1:20) to mimic higher tumor burden stress conditions in the presence of each BTKi, including non-covalent BTKi pirtobrutinib (Pirto) (500 nM). Tumor cells were replenished every 3 days and cytotoxicity was measured on day 7 by luciferase assay. For phenotypic characterization, CAR T cells were repeatedly stimulated by plate-bound anti-Whitlow linker every 3 days. Viability and proliferation were measured by cell counter. Flow cytometry (FACS) was performed on surface and intracellular T-cell phenotypic, activation, and exhaustion markers. Metabolic fitness (glycolysis and ATP production) was characterized using Seahorse (Van der Windt, 2012). Impact of Ibr on signal strength was evaluated in CD19-expressing Jurkat reporter cells (CAR-J19) with knock-in of mKate in frame with Nur77. Ibr was added to coculture of CAR-J19 and tumor cells and upregulation of Nur77 was measured by FACS. Downstream ITK signaling pathway was measured by Western blot. Results: Preliminary in vivo experiments demonstrated that co-administration of each BTKi with CD19 CAR T cells improved tumor control at day 26 (N=5 per group; P≤0.0004 vs CAR T cells alone). In serial killing experiments, addition of Ibr to the coculture increased proliferation (n=2; P&amp;lt;0.05), viability (n=2; P=0.01), and the cytotoxicity of CAR T cells against Ramos cells ( P=0.02 vs CAR19 alone) to a greater extent than other BTKis tested (Fig. 1A). Prolonged stimulation of CAR19 with Ibr led to downregulation of activation and inhibitory markers on CAR19 and maintenance of a naive T-cell phenotype (Fig. 1B) that was confirmed by increased metabolic fitness (n=5; P&amp;lt;0.05). Addition of Ibr to CAR-J19 resulted in a 72% reduction in CAR activation and reduced phosphorylation of ITK, PLCG, and Zap70. Additional data including the impact of ITK knockout on CAR-T cells will be presented. Conclusions: Our study expands on the hypothesized mechanism of action underlying the clinical benefit observed with Ibr in CD19 CAR T-cell-treated patients with chronic lymphocytic leukemia or mantle cell lymphoma (Liu et al, Front Immunol 2022; Wang et al, NEJM 2020). Our data suggest that Ibr enhances CAR T-cell fitness to a greater extent than both covalent and noncovalent BTKi that display reduced or no ITKi activity. Through ITKi, Ibr tempers CAR T-cell activation induced cell death, maintains a naive state and prevents premature exhaustion, thereby enabling more efficacious tumor cell killing over time. Together, these data support the further exploration of Ibr to enhance CD19 CAR T-cell efficacy.

Prophylactic or Preemptive Donor-Derived CD19 CAR-T Cell Infusion for Preventing Relapse in High-Risk B-ALL after Allogeneic Hematopoietic Stem Cell Transplantation

Introduction: Relapse remains a significant challenge in the management of high-risk B-cell acute lymphoblastic leukemia (B-ALL) patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Existing post-transplant relapse prevention strategies for high-risk B-ALL patients after transplantation, particularly in cases of Philadelphia chromosome-negative B-ALL, have limitations and suboptimal efficacy. This study aims to evaluate the efficacy and safety profile of prophylactic or preemptive infusion of donor-derived CD19 chimeric antigen receptor T (CAR-T) cells in high-risk B-ALL patients after allo-HSCT. Methods: Between May 2017 and July 2022, high-risk B-ALL patients with minimal residual disease (MRD) positive or MRD negative status after allo-HSCT were enrolled in clinical trials of donor-derived CD19 CAR-T cell therapy (ChiCTR 2000041025 and ChiCTR-ONN-16009862). The primary outcome was the cumulative incidence of relapse (CIR). Secondary outcomes were progression-free survival (PFS), overall survival (OS) and CAR-T cell treatment-related toxicities. Results: A total of 25 high-risk B-ALL patients who received infusion of donor-derived CD19 CAR-T cells after allo-HSCT were enrolled in the CAR-T cell group, including 8 patients with MRD and 17 patients without MRD. A contemporaneous cohort of 43 high-risk B-ALL patients who did not receive CAR-T cell therapy in MRD-positive or MRD-negative status after transplantation was also included as a control group. The median age of patients enrolled in the CAR-T group was 29 years (range: 9-60 years). The median infused CAR-T cell dose was 2.5×10^6/kg (range: 1.0-5.0×10^6/kg), with a median time from transplantation to CAR-T cell treatment of 122 days (range: 45-315 days). The median follow-up duration was 23.6 months (range: 8.3-60.6 months). Of the 25 patients in CAR-T group, eight MRD-positive patients achieved MRD-negative remission after CAR-T cells treatment (Figure1A). The two-year cumulative incidence of relapse in the CAR-T cell group was significantly lower at 4.8% (95% CI: 0.0%-12.6%) compared to the control group's incidence of 26.6% (95% CI: 11.4%-41.8%, P = 0.03，Figure1B). Additionally, the CAR-T cell group exhibited a higher two-year PFS rate [86.3% (95% CI: 71.8%-100%)] compared to the control group [63.5% (95% CI: 48.0%-79.0%), P = 0.04]. However, the two-year OS rates were comparable between the CAR-T cell group [91.0% (95% CI: 79.0%-100%)] and the control group [79.1% (95% CI: 66.0%-92.2%), P = 0.20]. Adverse events were well tolerated, and no patients experienced grade 3-4 cytokine release syndrome or any grade of immune effector cell-associated neurotoxicity syndrome. Hematological cytopenia was the most severe CAR-T cell related toxicity, but the toxicity was manageable, and only three patients experienced acute graft-versus-host disease following CAR-T cell infusion. Conclusions: Prophylactic or preemptive infusion of CAR-T cells after allo-HSCT demonstrated a reduced incidence of relapse and acceptable adverse events in high-risk B-ALL patients. These findings highlight the potential of CAR-T cell therapy as a promising strategy for relapse prevention in high-risk B-ALL patients after allo-HSCT.

Machine Learning-Based Decision Tree for Identifying and Grading Severity of Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS) Based on Neurological Adverse Events in Patients Treated with CD19 CAR-T

Introduction One of the major challenges associated with Chimeric Antigen Receptor T-cell (CAR-T) therapy is the development of immune effector cell-associated neurotoxicity syndrome (ICANS), which can vary in severity and requires prompt intervention. This study proposes using machine learning in combination with clinical insight to build a decision tree that accurately identifies and grades ICANS severity in patients treated with CD19 CAR-T based on the neurological adverse events (NAEs). Methods We conducted a retrospective analysis of 384 patients that exhibited NAEs after being treated with CD19-targeted CART-cell therapy in the pooled clinical trial data from the Medidata Enterprise Data Store. Of note, this dataset excludes patients without NAEs; these patients were assumed to not experience clinical ICANS. Patient NAEs were reviewed and the highest ICANS grade was assigned by a neuroimmunologist, resulting in the following patient counts: grade 0 (n=61), 1 (n=69), 2 (n=166), 3 (n=75), 4 (n=13). The clinical expert graded each patient based on the NAEs recorded in the first 28 days post infusion during the clinical trial, their CTCAE toxicity and symptom onset day. To process NAEs, we first mapped each of the 219 verbatim terms to the MedDRA hierarchy using a proprietary Medidata coding algorithm. We then aggregated the NAEs to the High Level Group Term (HLGT) level except for the MedDRA term “Neurological Disorders NEC” which was broken down to the High Level Term (HLT) level due to the large size of this group (73 out of 219 terms).The mapping of NAEs to MedDRA groups was reviewed together with the neuroimmunologist. The final dataset included the CTCAE severity of the NAEs mapped to MedDRA groups as features and the expert-labeled ICANS grades as the target variable. The data was then split into train (70%) and test (30%) sets stratifying by study and ICANS grade. We fit a decision tree while performing 5-fold hyperparameter grid search on the train set. The tree with the highest area under the Receiver Operating Characteristic curve one-vs-one (ROCAUC OVO) was selected and evaluated on the test set using an array of classification performance metrics. The model output was evaluated at the ICANS grade and severity (no/moderate/severe) level. The confusion matrices for the test set and the full decision tree were plotted. Results When binning the ICANS grade prediction by severity (grade 0 = no ICANS, grade 1-2 = moderate, grade 3-4 = severe), the model achieves a strong out-of-sample accuracy of 83.6% and ROCAUC OVO of 0.935. Regarding the individual ICANS grade prediction, the model yielded lower out-of-sample accuracy of 54.3%, with the majority of errors between grades 1 and 2. In spite of this, there was still reasonable discrimination based on the out-of-sample ROCAUC OVO values of 0.863. The training algorithm selected six MedDRA groups of NAEs to make predictions with: Headaches, Seizures (incl subtypes), Encephalopathies, Cortical dysfunction NEC, Disturbances in consciousness NEC, and Movement disorders (incl parkinsonism). We observe that patients with only headaches (a separate feature in our dataset) are all labeled as having no ICANS while those with seizures of grade 2 and higher are classified as grade 4. Refer to Figure 1 for the full decision tree. Comparing the neurologist-assigned versus predicted ICANS grades in the out-of-sample patient group (Figure 2) revealed that the majority of errors were bound to +/- one grade. Only three out of 116 patients were mislabeled by two grades. Crucially, none of the grade 1+ patients were predicted by the model to have grade 0. Conclusion Our machine learning-based decision tree accurately predicts the severity and grade of ICANS in CD19 CAR-T treated patients, aiding in clinical decision-making as well as retrospective analyses of ICANS patients. This type of clinical decision support tool has the potential to be a powerful aid to physicians by providing them with earlier physiological insights and data-driven assessments to help them triage patients accordingly. The model's performance highlights its potential clinical utility in improving patient care and outcomes. Further validation across multiple neurologists and prospective studies are warranted to assess its generalizability and refine its predictive capabilities. Acknowledgments We thank the developers of Scikit-learn and Dtreeviz Python packages for open-sourcing their code.

Clonal Hematopoiesis Is Associated with Severe Cytokine Release Syndrome in Patients Treated with Chimeric Antigen Receptor T-Cell (CAR-T) Therapy

Background: Among patients receiving CD19 or B-cell maturation antigen (BCMA) CAR-T therapy, inflammation pre- and post-CAR-T infusion is attributed in the development of toxicities such as cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and prolonged cytopenias. Clonal hematopoiesis (CH), the clonal expansion of hematopoietic stem cells with somatic mutations, has been associated with inflammatory disorders such as rheumatoid arthritis and graft-versus-host disease, as well as clonal cytopenias of undetermined significance. In the setting of CAR-T, others have reported an association between CH and both ICANS and treatment-associated myeloid neoplasms. Herein, we examined the prevalence of pre-CAR-T CH in a predominantly transplant-naïve cohort of recipients with non-Hodgkin lymphoma (NHL) or multiple myeloma (MM), and assessed the relationship between CH and CAR-T related outcomes including CRS, ICANS, prolonged cytopenia, progression-free survival (PFS), and overall survival (OS). Methods: This study included 62 patients with NHL or MM who underwent CD19 or BCMA CAR-T therapy from 2017 to 2022 at City of Hope and had available pre-CAR-T cryopreserved peripheral blood mononuclear cells (PBMCs). DNA was isolated with QIAamp DNA Mini Kit (Qiagen) from PBMC samples (94% collected &amp;lt;30d of CAR-T infusion), on which we performed targeted exome sequencing (108 pre-defined gene panel with 1000x sequencing depth) to determine the presence of CH (variant allele frequency [VAF] ≥2%). Multivariable logistic regression was used to examine the association between CH (Yes/No) and absolute neutrophil count (ANC) recovery at day +60 (Y/N), maximum grade CRS and ICANS, grade &amp;lt;2 vs 2+, and OS and PFS at 1y. Covariables considered were age at CAR-T, baseline ANC, sex, race, CAR-HEMATOTOX (Rejeski et al. 2021), bridging therapy (Y/N), and number of prior lines of therapy. ANC recovery was defined as grade 2 or better for 3 measurements without growth factor support &amp;gt;2 weeks. Those without ANC recovery and who started new treatment before day +60 were excluded from that analysis. Results: Overall, the median age was 64y (range 21y-85y); 37 (60%) had de novo diffuse large B-cell lymphoma, 17 (27%) had transformed follicular lymphoma, 2 (3%) had MM, 2 (3%) had mantle cell lymphoma, and the remaining patients had other transformed indolent lymphomas. Median prior lines of therapy was 3 (1-12), and only 6% had previously undergone autologous hematopoietic cell transplantation. Systemic bridging therapy prior to CAR-T was required in 27 (44%), and 8 (13%) received radiation as bridging. All received fludarabine and cyclophosphamide for lymphodepleting chemotherapy. Axicabtagene ciloleucel was the most common (76%) CAR-T product, followed by tisagenlecleucel in 16% of patients. Overall, 51 (82%) and 29 (47%) patients experienced CRS and ICANS, respectively. had delayed ANC recovery. One-year PFS was 50% and 1y OS was 73%. Clonal hematopoiesis: 15 (24%) patients had at least one pathogenic CH mutation; 4 (6.5%) had ≥2 CH mutations. Of the 28 CH mutations identified, the most common were DNMT3A (18%), CHEK2 (11%), PMS2 (7%) and ASXL1 (7%); 29% of mutations had VAFs &amp;gt;10%. Outcomes after CAR-T: Patients with CH were significantly more likely to develop grade ≥2 CRS (60% vs. 28%, p=0.03) compared to those without CH (odds ratio [OR] 3.9, 95% CI 1.2-13.2; p=0.027). Accounting for baseline ANC (which was unexpectedly higher among the CH cohort and associated with delayed ANC recovery, p=0.02) patients with CH did not have a significantly different rate of delayed ANC recovery compared to those without CH (adjusted OR 0.37, 95% CI 0.09-1.5; p=0.17). There was no association between CH and ICANS, nor with 1y PFS and OS. Conclusion: CH was frequent (24%) in this cohort of CAR-T recipients and was associated with a higher risk of ≥2 CRS after CAR-T. Additional validation studies are currently underway, which may set the stage for consideration of pre-CAR-T CH as a biomarker for risk stratification towards more proactive CRS prophylaxis Acknowledgements: Funding through the CoH Hematological Malignancies Program Pilot Project Award. The authors acknowledge the work provided by CoH Center for Informatics, notably Research Informatics, and the utilization of the POSEIDON platform and the Honest Broker process.

Safety and Efficacy of CD22/ CD19 CAR-T and Auto-HSCT “Sandwich” Strategy As Consolidation Therapy for Ph Negative B Cell Acute Lymphoblastic Leukemia

Introduction: Chimeric antigen receptor T-cell (CAR-T) therapy has a high relapse rate in B cell acute lymphoblastic leukemia (B-ALL). CAR T-cells target multi-antigen or CAR-T combined with other strategy may reduce the relapse post CAR-T. The main purpose of this study is to observe the safety and efficacy of CD22/CD19 CAR-T and Auto-HSCT “Sandwich” strategy in Ph negative (Ph -) B-ALL patients. Methods: A total of 19 newly diagnosed Ph - B-ALL patients were enrolled in this study (NCT05470777). These patients received induction and consolidation chemotherapy according to standard protocols. Autologous lymphocytes were collected at week 4 after induction chemotherapy. Afterwards, autologous CAR T-cells targeting CD22 and CD19 (CAR-T 1, co-stimulatory molecule was 4-1BB and infusion dose was 5×10 6/kg, respectively) were sequential infused after lymphodepletion chemotherapy with fludarabine and cytophosphamide. Autologous stem cells mobilization and collection were performed at 6-8 weeks after CAR T-cells infusion. After conditioned with modified BuCy regimen, autologous stem cells transplantation (auto-HSCT) was conducted. Sequential infusion of CD22 and CD19 CAR-T cells was performed on the second day after transplantation (CAR-T 2, co-stimulatory molecules and dose as CAR-T 1). No maintenance therapy was performed after auto-HSCT. Measurable detectable disease (MRD) was monitored by flow cytometry and IGHV leader-based next generation sequencing. Persistence of CAR T-cells were detected using real time quantitative RT-PCR (qPCR). Results: Three of the enrolled patients were Ph-like B-ALL. 10 (52.6%) patients had poor risk factors according to NCCN guidelines. Grade 1-2 cytokine release syndrome (CRS) occurred in four (21.1%) and seven (36.8%) patients after CAR-T 1 and CAR-T 2, respectively. No grade 3-4 CRS and immune effector cell associated neurotoxicity syndrome (ICANS) occurred. After CAR-T therapy, all patients achieved MRDnegative complete remission (MRD -CR). Until the last follow-up on July 1 st, 2023, the median follow-up was 16 months (range, 7 - 32 months). 84.2% of enrolled patients remain MRD -CR. Duration of continuous MRD -CR was detected in 2 patients for more than 2 years and in 9 patients for more than 1 year. The median overall survival (OS) and leukemia-free survival (LFS) in all 19 patients are not reached. Three (3/19, 15.8%) patients experienced antigen-positive relapse. One Ph-like B-ALL patient relapsed at 10 months after auto-HSCT and underwent allogeneic HSCT. Two patients with Ph - B-ALL relapsed at 5 and 6 months after auto-HSCT and were treated with blinatumomab. All of them achieved MRD -CR again after treatment. All patients are alive and CAR-T cells are detectable in all patients at the last follow-up (range, 5-28 months). Conclusions: Our preliminary study demonstrated that CD22/CD19 CAR-T and Auto-HSCT “Sandwich” strategy as a consolidation strategy showed favorable safety and efficacy in Ph - B-ALL. CD22/CD19 CAR-T resulted in deeper remission before transplantation. The new strategy may benefit patients from LFS and OS.

Zanubrutinib Combined Regimen As Bridging Treatment on Promising Clinical Outcome of CD19 Chimeric Antigen Receptor T-Cell Therapies in Relapsed or Refractory Large B-Cell Lymphoma

Background Relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL) shows poor clinical outcomes. Anti-CD19 chimeric antigen receptor-T cells (CAR-T) therapy is emerging as the second-line therapy of choice, but in real-world studies its efficacy and long term follow is not promising. Effective bridging therapy can reduce tumor burden and improve CAR-T efficacy. Zanubrutinib, a selective Bruton tyrosine kinase (BTK) inhibitors, combined regimens have established therapeutic activity by targeting B-cell receptor signaling and have individually shown efficacy in patients with R/R DLBCL. Thus, the present study evaluated the efficacy and safety of zanubrutinib-combined regimen as bridging treatment in CD19 CAR-T therapy. Methods This retrospective study includes all patients participant in the clinical trail (NCT 02537977) between January 2020 and December 2022. All 21 heavily treated patients with r/r DLBCL who received zanubrutinib-combined regimens as bridging therapy are included. Patient clinical characteristics were collected upon patient enrollment. Efficacy outcomes included overall response rate (ORR), progression-free survival (PFS), and overall survival (OS), whereas safety outcomes included incidence of adverse events. The zanubrutinib combined regimens are zanubrutinib with IMIDs, chemotherapy and/or rituximab. Results: Of the 21 evaluable patients, median age of patients is 57 year old and median treatment line is 3. 19 patients (90%) are non-GCB subtype and 18 patients(86%) are IPI score &amp;gt;3. 14 patients(67%) are double expression and 7 patients (33%) have TP53 mutation. Objective response rate(ORR) is 81%; of whom, 10 patients(48%) got complete remission(CR) and 7 patients(33%) got partial remission(PR). With a median follow-up of 25 months, the median PFS was 12.8 months and median OS was not yet reached. 8 CR patients and 3 PR patients maintain long-term remission and are still under followup. 18 patients (86%) has grade 1-2 cytokines release syndrome(CRS) and 3 patients(14%) has grade 1 immune effector cell-associated neurotoxicity syndrome (ICANS). No severe CRS and &amp;gt;1 grade ICANS were observed. The most common hematologic toxicities included grade ≥3 neutropenia (90%) and grade 3/4 thrombocytopenia (71%). No patients had grade ≥3 non hematologic toxicities. Conclusion: Zanubrutinib-combined regimens are effective and safe as bridging therapy in CD19 CAR-T therapy. More prospective studies are further command.

Methylated Whole Genome Cell-Free DNA Sequencing before Chimeric Antigen Receptor T-Cell Therapy for Large B-Cell Lymphoma Predicts Treatment Outcomes

Introduction More than half of patients with relapsed or refractory large B-cell lymphoma (R/R LBCL) will experience progressive disease after CD19 chimeric antigen receptor T-cell therapy (CART). Circulating tumor DNA profiling has been used to prognosticate patients before CART, but mainly has focused on identification of somatic genomic aberrations. Epigenetic changes, specifically aberrant DNA methylation, can alter transcriptional regulation of genes important for treatment resistance. Altered methylation features can be detected from cell-free DNA (cfDNA) in patients with LBCL and are associated with outcomes after frontline immunochemotherapy. In the current study, we used whole genome bisulfite sequencing (WGBS) to analyze the methylation landscape in cfDNA from patient plasma before CART and identified novel methylation signatures associated with poor treatment outcomes. Methods Patients with R/R LBCL who received standard-of-care CART with plasma samples drawn before lymphodepletion (LD) or at cell infusion were identified. The primary endpoint was 3-month overall response after CART. cfDNA was extracted from 3-5 ml of plasma and cfDNA libraries were prepared using a Zymo-Seq cell free WGBS kit. Each library was sequenced to 300 - 600 million read pairs at a read length of 150 bp, enabling an average CpG coverage of 7-13X per detected CpG. Unique alignment rate among all libraries were about or above 82%. Reads were adapter trimmed with Trim Galore and aligned to reference genome hg19 using Bismark. Methylation calling was performed by MethylDackel. Differentially methylated cytosines (DMC, FDR ≤0.05) and regions (DMR) from WGBS data with an absolute methylation difference of ≥ 0.1 between 3-month responders and non-responders were identified using DSS. Functional enrichment analysis of genes overlapped by DMRs was performed by gprofiler2. Results A total of 22 patients with plasma collected before LD (n=21) or at cell infusion (n=1) who received CART were identified. Median follow-up was 21.5 months (95% CI 12.5-NR). Three-month response was evaluable in 21 patients (1 pending evaluation). Overall response rate (ORR) was 77% (8 CR, 9 PR) and 55% (9 CR, 2 PR) at one- and 3-months post-CART, respectively. Median PFS was 6.5 months (95% CI 3.0-NR), and median OS was 57.7 months (95% CI 7.7-NR). Patient and disease characteristics are described in Table 1. Differential methylation analysis identified 22,815 DMCs, of which 1,043 were hypomethylated and 21,772 were hypermethylated in non-responders. The global average methylation values across 25 million cytosines were 0.807 and 0.786 for non-responders and responders, respectively (p=0.265). A total of 582 DMRs were identified, of which 34 were hypomethylated and 548 were hypermethylated in non-responders (Figure 1). Of these, 281 (48%) overlapped intergenic regions, 265 (46%) gene bodies, and 33 (6%) promoter regions. DMRs overlapping DNA repair gene PARP1 (areaStat 88.2), B-cell receptor signaling genes SYK (27.2), FYN (21.4), and PTPN6 (20.9), polycomb repressor complex genes JARID2 (25.2) and BCOR (21.4), tumor suppressor WT1 (47.1), immune mediators NRP1 (80.0), TRAF3 (56.7), PAK3 (28.4), IRAK2 (28.7), and TNFS14 (23.6), and non-coding RNAs MIR4520 (95.8) and VTRNA-2 (23.1) were hypermethylated in non-responders and IL17RA (-33.2) hypomethylated. Functional enrichment analysis on overlapped genes also identified common molecular functions involving guanyl nucleotide exchange factors (p=0.0007), GTPase regulation (p=0.001), and cGMP binding (p=0.007). Discussion This proof-of-concept study is the first to perform WGBS on pretreatment plasma from patients with R/R LBCL to associate methylation signatures with post-CART outcomes. We identified that non-responders were enriched in hypermethylated DMRs compared to responders, overlapping with genes important to lymphomagenesis, immune function, and GTP energy metabolism. Further work will expand this cohort, compare with matched-tissue to find tumor-specific markers, and identify candidate DMR targets for targeted bisulfite sequencing from pre- and post-treatment plasma samples to assess response. By identifying high-risk methylation signatures in the plasma of CART recipients, we may be able to identify patients to be prioritized for novel treatment strategies such as combination with hypomethylating agents to improve outcomes.

Distinct Temporal Dynamic Serum Metabolite Profiles Characterize Treatment Response and Early Toxicity after B-Cell Lymphoma Directed CAR-T-Cell Therapy

Introduction: CD19 chimeric antigen receptor T-cell therapy (CD19.CAR-T) has profoundly improved treatment options for patients with relapsed/refractory B-cell lymphomas (r/r BCL), but is also associated with distinct toxicities. To elucidate host-intrinsic immunometabolic factors contributing to clinical benefit or immunotoxicity, we recently demonstrated that an increase of metabolically high-risk adipose tissue mass is associated with an elevated cytokine release syndrome (CRS) risk but also with improved survival (Haematologica 2022, CIR 2023). To gain further insights into the underlying immunometabolic mechanisms, we analyzed temporal dynamic changes of serum metabolites in r/r BCL patients during the first 14 days of CD19.CAR-T treatment. Methods: In this single-center analysis, we studied patients receiving CD19.CAR-T for r/r large BCL or mantle cell lymphoma (MCL) between 01/2019 and 12/2021 in a real-world setting. All patients were consecutively treated in our department (LMU University Hospital) and serum was prospectively collected in the morning hours from fasted patients beginning at the day of CAR-T infusion (D0), and 3-5 and 14 days later. Using liquid chromatography mass-spectrometry, we quantitatively analyzed a total of 305 water-soluble metabolites and 734 lipids. Metabolomic data was integrated with clinical patient records including CRS, 1-year progression-free survial (PFS) and amount of visceral adipose tissue (VAT). Results: In total, we measured 170 serum samples from 55 r/r LBCL and 4 r/r MCL patients, of which 52 had serial samples at all three time points (D0: 55, D3-5: 59, D14: 56). The following CAR-T products were administered: axi-cel (n = 26), tisa-cel (n = 29) and brexu-cel (n = 4). 25 (42.4%) patients developed a CRS grade ≥ 2, while 34 patients had a CRS grade 0-1. The best 90-day overall response rate was 52.5%, with a complete remission rate of 42.4%. At 1 year, 67.8% of patients progressed. From the 1039 metabolites measured, 525 metabolites passed quality control. Metabolite values were normalized and auto-scaled for further analysis. A partial least square differential analysis based on the collected time points led to a clear separation of samples with uric acid, desoxy-ribose-5-phosphate (dRibose-P), essential amino acids and (phospho)lipids having the strongest impact. Whereas 56 metabolites showed a significant increase from D0 to D14 led by uric and essential amino acids, 34 metabolites decreased over the course of treatment with the strongest reduction in dRibose-P and sphingosine phosphate. Performing a metabolic pathway enrichment analysis based on the most altered metabolite levels, we detected 22 significantly affected metabolic pathways and programs including aerobic glycolysis, urea cycle, amino acid and lipid metabolism (FDR &amp;lt; 0.1, Fig. 1A). To further delineate metabolite changes in regards to CRS, 1-year PFS and amount of VAT, we conducted a multivariate empirical bayes analysis of variance to specifically detect metabolites with differential temporal dynamics and considered all metabolites with an FDR &amp;lt; 0.1. Patients who developed a CRS ≥ 2° showed increased levels of kynurenine, phosphatidylethanolamides and lysophosphatidylserines, and decreased levels of ornithine and phosphatidylinositols. 1-year progressors showed a significant increase of several phosphatidylinositols beginning at D3-5 after CAR-T infusion, and decreased levels of phosphatidylcholines. For VAT, we used a threshold of 144 cm² which was derived from previously conducted computer tomography segmentation analyses. As expected, VAT cohorts mostly differed in fatty acid metabolism-associated metabolites including (acyl)carnitines, cholesterol esters, triglycerides and xanthine. In order to identify metabolites being associated with multiple outcomes, we looked for mutual metabolites among distinct cohort profiles. Here, one example is phosphatidylinositol-(22:5/16:0), which was increased in CRS 0-1° and 1y-progressors ( Fig. 1B). Conclusions: Our study highlights that CD19.CAR-T treatment is accompanied by broad changes in the serum metabolome of r/r BCL patients, which can be used to profile patients being affected by CAR-T-related immunotoxicity and their response to treatment. In ongoing analyses, we evaluate the use of these profiles for predictive models, and assess their impact on CAR-T cell function in-vitro.

Development and Validation of a Clinical Prediction Model for the Efficacy of CD19 CAR T-Cell Therapy in DLBCL

Currently, Autologous CD19 chimeric antigen receptor T-cell therapy (CART) has become the most important treatment modality for relapsed or refractory diffuse large B-cell lymphoma (r/r DLBCL) beyond conventional multi-line treatments. However, it is worth noting that over 60% of CD19 CAR-T recipients will eventually experience relapse. A substantial amount of clinical research has been conducted to explore the reasons for the ineffectiveness or relapse of CAR-T therapy. Based on a comprehensive analysis of clinical data, we selected various combinations of predictive factors and used the TRIPOD guidelines to construct a logistic regression model for predicting the efficacy of CD19 CAR-T therapy. This study was conducted as a retrospective cohort study, including patients with confirmed diagnosis of DLBCL who underwent CD19 CAR-T therapy and met the following criteria: (1) aged 18 years or older but not exceeding 80 years; (2) voluntarily participated in the study and signed an informed consent form. The exclusion criteria for this study were: (1) addition of other chemotherapy drugs or targeted drugs within 90 days before assessing the best response after infusion, except for those who achieved complete response (CR) or progressive disease (PD); (2) death caused by non-tumor progression factors within 90 days after infusion. After the inclusion and exclusion criteria, a total of 54 patients with DLBCL undergoing CD19 CART at Shanghai Tongji Hospital of Tongji University ultimately participated in this study from January 1, 2019, to April 31, 2023. The derivation cohort consisted of 45 patients enrolled in clinical trials (NCT02537977, NCT03154775, CTR20201986, CTR20200561). The median age of the patients in the derivation cohort was 60 years (IQR: 50-67 years). Out of the 45 participants, 25 (55.6%) were male, and 29 (64.4%) had the non-germinal center B-cell (non-GCB) subtype, 1 had grade 3-4 Cytokine Release Syndrome (CRS). The validation cohort consisted of 9 patients involved in real-world studies. The median age of the patients in the validation cohort was 55 years (IQR: 52.5-69 years). Out of the 9 participants, 5 (55.6%) were male, and 6 (66.7%) had the non-GCB subtype, 2 had grade 3-4 CRS. The demographic and clinical characteristics of the derivation cohort were similar to those of the validation cohort. Based on the best CD19 CART response at 90 days, we divided the derivation cohort into two groups, CR and non-CR. Then, we selected a series of candidate predictive factors from baseline data such as clinical features, genetic features, and laboratory features before CART (Table 1). The results of univariate and multivariate logistic regression analysis were used to select specific factors (Gender, ECOG, TP53) as model prediction indicators based on p-values (p&amp;lt;0.05). There was no significant correlation between these factors (p&amp;gt;0.1). A score nomogram (Model1) was plotted using the regression coefficients of each predictive factor as weights (Fig.1 A), C-index (Model1) =0.872 (Fig.1 C). To facilitate better clinical use, we also constructed a risk nomogram (Model2) to assess the degree of risk (Fig.1 D). Model2 considers three factors: Male, ECOG≥2, TP53 altered. If a person has not any of these risk factors, they are categorized as low risk. If there is one risk factor present, they are deemed to be at moderate risk, whereas having two or more risk factors classifies them as high risk. C-index (Model2) =0.845 (Fig.1 F). Finally, the model's clinical efficacy was validated using real-world CART-treated patients, and the C-index was calculated as Model1=0.875 (Fig.1 G), Model2=0.7 (Fig.1 H), confirming the clinical impact of the model. In conclusion, this study has successfully developed an easily implementable clinical prediction model. For the first time, we have used CART clinical studies as the derivation cohort and real-world results as the validation cohort. Our scoring system provides clinicians with accurate and objective efficacy information, helping them identify the appropriate population for CAR-T therapy in DLBCL.

Day +3/Day 0 Ferritin Ratio: A Simple Point-of-Care Index Predictive of Severe Immune Effector Cell-Associated Neurotoxicity Syndrome after CD19 CAR T-Cell Therapy

Introduction: Chimeric antigen receptor (CAR) T-cell therapy is transforming the management of patients with relapsed/refractory (R/R) B- and plasma-cell malignancies. However, a major limitation of CAR T-cell therapy is the development of severe, sometimes life-threatening, toxicities such as immune effector cell-associated neurotoxicity (ICANS) and cytokine release syndrome (CRS). While the management of CRS has dramatically improved with the early use of tocilizumab and corticosteroids, how to best predict and treat ICANS remains poorly understood, with up to 30% of patients developing severe ICANS after CD19 CAR T-cell therapy. Since we had previously observed features overlapping with hemophagocytosis lymphohistiocytosis in patients with severe CRS and ICANS, we hypothesized that the early kinetics of ferritin and soluble IL-2 receptor alpha [sIL-2Rα] post infusion could predict the development of severe ICANS. Methods: We retrospectively analyzed data from 195 patients with R/R B-cell malignancies previously treated on a phase I/II clinical trial (NCT01865617) of an investigational defined 1:1 CD8+:CD4+ composition CD19 CAR-T product (JCAR014; training set). CRS and ICANS severity were graded using Lee 2014 and CTCAE 4.03 criteria, respectively. Logistic regression was used to predict the occurrence of severe ICANS. Ferritin was measured using an enzyme immunoassay. sIL-2Rα was measured using Luminex®. Goodness of fit was assessed using the Akaike information criterion (AIC). Independent test set included 203 patients treated at our center with the standard-of-care CD19 CAR T-cell products axicabtagene or lisocabatagene maraleucel. Discrimination and calibration were assessed in both the training and test set. Results: In the training set (n=195), median age was 55 (range: 20-76). Disease type was as follows: acute lymphoblastic leukemia, n=64 (32.8%); B-cell non-Hodgkin lymphoma, n=82 (42.1%); chronic lymphocytic leukemia, n= 47 (24.1%). We observed any grade and grade ≥3 CRS in 134 (68.7%) and 27 (13.8%), respectively, and any grade and grade ≥3 ICANS in 72 (38.8%) and 37 (19.0%), respectively. Longitudinal analyses of serum ferritin (Figure 1) and sIL-2Rα values using locally estimated scatterplot smoothing suggested that the rate of increase of both analytes was markedly higher in patients with grade ≥3 ICANS compared to grade 0-2 ICANS. We applied univariate logistic regression to estimate the association between serum ferritin levels at early timepoints, select ratios of ferritin and sIL-2Rα values, and the occurrence of severe ICANS. The strongest association was observed between the ferritin day +3/day 0 ratio and severe ICANS (OR per log10 increase, 175; 95% CI, 19.1-2668; p &amp;lt; 0.001). We could further improve our model by modeling the ferritin day +3/day 0 ratio with a restricted cubic spline to allow for non-linear effects (unsplined model, AIC = 129, C-index, 0.74; splined model, AIC = 127, C-index, 0.78). Ferritin day +3/day 0 ratios of 3, 5, and 10 were associated with an 81%, 95%, and 99% probability of grade ≥3 ICANS, respectively (Figure 2). The splined model was associated with near-perfect calibration corrected for optimism using bootstrapping. We next evaluated the predictive ability of the ferritin day +3/day 0 ratio to predict severe ICANS in our independent test set. Predictions in the test set based on our splined model retained good discriminative ability with a C-index of 0.64, although with suboptimal calibration. In a refitted logistic regression model in the test set, the ferritin day +3/day 0 ratio remained strongly associated with the development of severe ICANS (OR = 18.8 per log10 increase, 95%CI, 2.48-142.01, p = 0.004). Conclusion: We validated the ability of a simple point-of-care index (day +3/day 0 ferritin ratio) to predict the development of severe ICANS after CD19 CAR T-cell therapy in our training and test set. Since serum ferritin levels can be quickly measured by most clinical laboratories, the day +3/day 0 ferritin ratio could become a practical and useful tool for practitioners taking care of CAR T-cell patients. The early identification of patients at high risk of developing severe ICANS days prior to symptoms onset could enable the evaluation of targeted prophylactic interventions in future clinical trials. We plan to refine our modeling approach to further improve the calibration in test set.