Chimeric Antigen Receptor T-cell Treatment Research Articles

CD4+ CAR-T cell exhaustion associated with early relapse of multiple myeloma after BCMA CAR-T cell therapy

Multiple myeloma is characterized by frequent clinical relapses following conventional therapy. Recently, chimeric antigen receptor T (CAR-T) cells targeting B-cell maturation antigen (BCMA) has been established as a treatment option for patients with relapsed or refractory disease. However, while >70% of patients initially respond to this treatment, clinical relapse and disease progression occur in most cases. Recent studies showed persistent expression of BCMA at the time of relapse, indicating that immune intrinsic mechanisms may contribute to this resistance. While there were no pre-existing T cell features associated with clinical outcomes, we found that patients with a durable response to CAR-T cell treatment had greater persistence of their CAR-T cells compared to patients with transient clinical responses. They also possessed a significantly higher proportion of CD8+ T effector memory cells. In contrast, patients with short-lived responses to treatment have increased frequencies of cytotoxic CD4+ CAR-T cells. These cells expand in vivo early after infusion but express exhaustion markers (HAVCR2 and TIGIT) and remain polyclonal. Finally, we demonstrate that non-classical monocytes are enriched in the myeloma niche and may induce CAR-T cell dysfunction through mechanisms that include TGFβ. These findings shed new light on the role of cytotoxic CD4+ T cells in disease progression after CAR-T cell therapy.

Abstract 1418: Priming of the glioblastoma tumour microenvironment via copper chelation to enhance the efficacy of immunotherapies

Abstract Glioblastoma, a highly aggressive brain tumor with a dismal prognosis necessitates novel therapeutic approaches as current standard treatments yield limited success. Despite the transformative impact of immune checkpoint blockade in various malignancies, its efficacy in glioblastoma remains elusive. Similarly, chimeric antigen receptor T (CAR-T) cell therapy, while successful in hematological malignancies, faces challenges in solid tumors. This study addresses the immunosuppressive barriers hindering the effectiveness of immunotherapies in glioblastoma. Highlighting copper's pivotal role in glioblastoma, our research group demonstrated its elevation and influence on PD-L1 expression. Given the success of copper chelators in treating Wilson's Disease, in which neurological manifestations are alleviated following treatment, we hypothesized that copper chelation therapy could be a promising strategy for glioblastoma, especially when combined with immunotherapies. Flow cytometry analysis revealed a significant increase in the tumor-associated antigen GD2 following treatment with the copper chelator trientine (TETA), suggesting its potential synergy with GD2-targeted immunotherapies. In an immunocompetent murine glioblastoma model, combination therapy with GD2-targeted CAR-T cells and TETA effectively slowed tumor growth, demonstrating enhanced efficacy. Additionally, TETA as a monotherapy significantly improved survival compared to the control. The combination of TETA with anti-PD1 antibody therapy also exhibited promising results in inhibiting tumor growth and subsequently prolonging survival, with some mice demonstrating complete tumor regression. Importantly, TETA decreased copper levels in the brains of the mice, indicating the drug can act on the brain, a crucial therapeutic hurdle that is hampering the efficacy of novel therapies in glioblastoma. Further exploration of immune cell populations using OPAL Multiplex immunohistochemistry following TETA, anti-PD1, and GD2-targeted CAR-T cell treatments will underscore the potential of copper chelation in reshaping the tumor microenvironment. Our findings suggest that copper chelation, with agents like TETA, can be repurposed as a viable anticancer therapy when combined with immunotherapies. This study provides a compelling rationale for further pre-clinical validation, offering a potential breakthrough in the quest for effective glioblastoma treatments. Citation Format: Tyler Shai-Hee, Toni Rose Jue, Riccardo Cazzoli, Jourdin R. Rouaen, Filip Michniewicz, Jessica Bell, Tessa Gargett, Michael P. Brown, Orazio Vittorio. Priming of the glioblastoma tumour microenvironment via copper chelation to enhance the efficacy of immunotherapies [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 1418.

Abstract 5241: Generation of allogeneic CAR-T circumvents functional deficits in patient-derived autologous product for glioblastoma

Abstract Glioblastoma (GBM) is the most common malignant primary brain tumor in adults. Despite an aggressive standard of care that includes maximally safe surgical resection, chemo-radiotherapy, median overall survival remains stagnant at 15 months. However, immunotherapeutic strategies have provided an exciting avenue of exploration to meet clinical need. Chimeric antigen receptor T-cell (CAR-T) therapy has shown promising results in liquid malignancies, but clinical trials in GBM targeting various tumor antigens have not shown durable clinical benefit. While this may be attributable to various tumor-intrinsic immune evasion strategies characteristic of GBM, little work has assessed whether the issue is due to the quality of the CAR-T treatment itself. Currently, CAR-Ts for clinical studies are manufactured in an autologous setting wherein T-cells are extracted from patients, engineered ex-vivo, and subsequently re-infused back. However, peripheral T-cells taken from untreated GBM patients have demonstrated qualitative and functional deficits, which may contribute to suboptimal treatment outcomes. Thus, we aimed to establish whether CAR-Ts generated from GBM patients would show reduced efficacy in comparison to healthy donors using our previously validated CD133 CAR-T. In this work, we show that in-spite of no inherent phenotypic differences, patient derived CAR-Ts shows pre-treatment exhaustion and upon preclinical evaluation using an orthotopic xenograft model of human GBM reduced survival advantage in autologous, patient-derived CD133-targeting CAR-T cell products was observed as compared to the controls. Transcriptomic analysis highlighted a decreased panel-wide enrichment in genes related to T cell and lymphocyte activation, lower prevalence of T cells (including Th1 and CD8+) and higher prevalence of exhausted CD8+ cells in T-cells products derived from GBM donors as compared to healthy donors. To overcome the functional and logistical considerations of autologous therapy, we additionally aimed to generate an “off-the-shelf” allogeneic CD133 CAR-T. Using CRISPR gene editing technology, we generated TCR-knockout CAR-T cells with comparable pre-clinical efficacy to our autologous models. In conclusion, this work highlights the need to reassess autologous CAR-T therapy for GBM and considers allogeneic approaches as promising alternatives. By addressing the inherent deficits in patient-derived CAR-Ts, allogeneic CD133 CAR-Ts may offer a more effective and logistically feasible therapeutic option for treating GBM. Citation Format: Muhammad Vaseem Shaikh, Sabra K. Salim, Jeffrey Wei, William T. Maich, Alisha A. Anand, Oliver Young Tang, Minomi K. Subapanditha, Yujin Suk, Manoj Singh, Zahra Alizada, Benjamin Brakel, Vassil Dimitrov, Zoya Tabunshchyk, Kevin Brown, Parvez Vora, Zev Binder, Chitra Venugopal, Jason Moffat, Sheila K. Singh. Generation of allogeneic CAR-T circumvents functional deficits in patient-derived autologous product for glioblastoma [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 5241.

Targeting metabolism to improve CAR-T cells therapeutic efficacy.

Chimeric antigen receptor T (CAR-T) cell therapy achieved advanced progress in the treatment of hematological tumors. However, the application of CAR-T cell therapy for solid tumors still faces many challenges. Competition with tumor cells for metabolic resources in an already nutrient-poor tumor microenvironment is a major contributing cause to CAR-T cell therapy's low effectiveness. Abnormal metabolic processes are now acknowledged to shape the tumor microenvironment, which is characterized by increased interstitial fluid pressure, low pH level, hypoxia, accumulation of immunosuppressive metabolites, and mitochondrial dysfunction. These factors are important contributors to restriction of T cell proliferation, cytokine release, and suppression of tumor cell-killing ability. This review provides an overview of how different metabolites regulate T cell activity, analyzes the current dilemmas, and proposes key strategies to reestablish the CAR-T cell therapy's effectiveness through targeting metabolism, with the aim of providing new strategies to surmount the obstacle in the way of solid tumor CAR-T cell treatment.

FDA-Approved Chimeric Antigen Receptor (CAR)-T Cell Therapy for Different Cancers-A Recent Perspective.

Cancer is one of the most prevalent diseases in the world, and theirrate of occurencehas beenincreased in recent decades. Current review article, summarizes the novel treatment options Chimeric Antigen Receptor-T (CAR-T) cell therapy for various cancers constitute a major health and development challenge, impacting every aspect of sustainable development quoted by goal 3 good health and well-being of UN sustainable goals. WHO estimates that 70% of cancer deaths occur in low- and middle- income countries (LMICs) by 2030, LMICs are expected to bear the brunt of the expected 24.1million new cancer cases per year. This current review article focuses and discussed about CAR-T cell therapy for various cancers against most prevalent non-communicable disease cancer disease stipulated by WHO and UN sustainable goals. Three literature databases Google scholar, Science Direct, PubMed was utilized to search and collect CAR-T cell treatment options for different cancers published articles sources in between January 2000 and December 2023. There were a total of 18,700 papers found, with 48 of them being found to be eligible focusing various cancer treatment by CAR-T cells utilized for the study. Based on the information gathered, CAR-T cell therapy treating different cancers and their merit and its advantages in heal and improve certain cancers was also discussed in this review article with their detailed molecular mechanisms. This article also gives an insight to utilize CAR-T cell treatment protocols for rejuvenating cancer patient from such ruthless cancer disease condition thereby improving life span of cancer patients and eradication of disease in some cases.

An engineering strategy to target activated EGFR with CAR Tcells.

Chimeric antigen receptor (CAR) Tcells have shown remarkable response rates in hematological malignancies. In contrast, CAR Tcell treatment of solid tumors is associated with several challenges, in particular the expression of most tumor-associated antigens at lower levels in vital organs, resulting in on-target/off-tumor toxicities. Thus, innovative approaches to improve the tumor specificity of CAR Tcells are urgently needed. Based on the observation that many human solid tumors activate epidermal growth factor receptor (EGFR) on their surface through secretion of EGFR ligands, we developed an engineering strategy for CAR-binding domains specifically directed against the ligand-activated conformation of EGFR. We show, in several experimental systems, that the generated binding domains indeed enable CAR Tcells to distinguish between active and inactive EGFR. We anticipate that this engineering concept will be an important step forward to improve the tumor specificity of CAR Tcells directed against EGFR-positive solid cancers.

Chimeric Antigen Receptor-T (CAR-T) Cells as “Living Drugs”: A Clinical Pharmacist Perspective

Background. Chimeric antigen receptor (CAR) T cell therapy, a “living drug” immunotherapy, harnesses the power of T-cells from a patient (autologous) or healthy donor (allogeneic) to target and kill cancer cells and has shown unprecedented outcomes in patients with relapsed and refractory malignancies. Treatment with CAR-T cells requires the application of unique skillsets in recognised specialist centres for successful outcomes and requires management by the multidisciplinary team incorporating the specialist pharmacist. Method. A multimodal research strategy was employed for this literature review whereby PubMed, Google Scholar, Embase, Stella Library Search, EMA website, and EBMT website were sources of information. The search was limited from 2020 onwards with key terms referring to CAR-T cell therapy. Results and Discussion. There are six CAR-T cell products currently approved by the European Medicines Agency (EMA) and Food and Drug Administration (FDA) which target haematological malignancies with abundant clinical trials underway exploring new and improved CAR designs and antigen targets. As CAR-T cell therapy is an advanced therapy medicinal product (ATMP), there is need for an extensive regulatory framework underpinning its safety and efficacy. The clinical pharmacist plays an integral role in the provision of safe and effective CAR-T cell therapy including governance, operational and clinical aspects of treatment. Pharmacists may also be involved through provision of “Qualified Person” (QP) expertise in clinical trials and for release within hospitals under certain circumstances. There is a need for harmonised and accessible guidance on the clinical delivery of ATMPs such as CAR-T cells, with fully delineated responsibilities of pharmacists involving the oversight and supervision of CAR-T cell treatment. Conclusion. There is an unmet need to provide suitable and applicable literature for clinical pharmacists who are involved in the delivery of CAR-T cells. We have provided an overview of T-cell biology and an explanation of CAR-T cell design and the biomanufacturing process. We reviewed the complex and multifaceted treatment cycle requiring considerable logistics, and described the involvement of the clinical pharmacist in each part of this cycle from patient selection to postinfusion care. Finally, we look to the challenges and future opportunities that will require the involvement of the clinical pharmacist.

Mucin-1-Targeted Chimeric Antigen Receptor T Cells Are Effective and Safe in Controlling Solid Tumors in Immunocompetent Host.

The chimeric antigen receptor (CAR) T-cell therapy in solid epithelial tumors has been explored, however, with limited success. As much of the preclinical work has relied on xenograft models in immunocompromised animals, the immune-related efficacies and toxicities may have been missed. In this study, we engineered syngeneic murine CAR T cells targeting the tumor form of human mucin-1 (tMUC1) and tested the MUC1 CAR T cells' efficacy and toxicity in the immunocompetent human MUC1-expressing mouse models. The MUC1 CAR T cells significantly eliminated murine pancreatic and breast cancer cell lines in vitro. In vivo, MUC1 CAR T cells significantly slowed the mammary gland tumor progression in the spontaneous PyVMT×MUC1.Tg (MMT) mice, prevented lung metastasis, and prolonged survival. Most importantly, there was minimal short or long-term toxicity with acceptable levels of transient liver toxicity but no kidney toxicity. In addition, the mice did not show any signs of weight loss or other behavioral changes with the treatment. We also report that a single dose of MUC1 CAR T-cell treatment modestly reduced the pancreatic tumor burden in a syngeneic orthotopic model of pancreatic ductal adenocarcinoma given at late stage of an established tumor. Taken together, these findings suggested the further development of tMUC1-targeted CAR T cells as an effective and relatively safe treatment modality for various tMUC1-expressing solid tumors.

Experiences and perspectives on chimeric antigen receptor (CAR) T-cell therapy among recipients, carers and referrers (RE-TELL): a qualitative study to inform CAR T-cell service design

ObjectivesRE-TELL is a qualitative study, which aims to understand patient, support person, clinician and coordinator experiences and perspectives of chimeric antigen receptor (CAR) T-cell therapy, to inform design of a...

Hematopoietic cell transplantation and cellular therapies in Switzerland. Evolution over 25years. A report from the stem cell transplantation and cellular therapies working groups of the SBST 1997-2021.

The Swiss Blood Stem Cell Transplantation and Cellular Therapy Group (SBST) leads a mandatory national registry for all hematopoietic stem cell transplants (HCT) and cellular therapies. After 25years, information was available for 11,226 patients receiving an HCT (4031 allogeneic and 7195 autologous), including 925 pediatric patients. We compared patient characteristics and outcome by quinquennia 1997-2001, 2002-2006, 2007-2011, 2012-2016, and 2017-2021. There were numerous changes over time. Allogeneic transplant recipients became older (median age 33.7 vs. 54.3) and had more frequently unrelated donors and reduced intensity conditioning in later quinquennia. Similarly, age increased for recipients of autologous HCT (median 48.3 vs. 59.9). We did not see a significant drop in transplant activity during the SARS-CoV-2 pandemic. Analysis of outcome showed overall survival (relative risk (RR) of death 0.664 (0.529-0.832) and progression free survival (RR 0.708 (0.577-0.870) being improved over time comparing the latest to the first quinquennium adjusting for risk factors. Non-relapse mortality decreased in recipients of allogeneic HCT (RR: 0.371 (0.270-0.509)) over time but relapse risks did not. Outcome of autologous HCT improved as well across quinquennia, this improvement was mainly due to decreased relapse risks (RR 0.681 (0.597-0.777)), possibly related to maintenance treatment or rescue treatment for relapse mainly in myeloma patients. Cellular therapies other than allogeneic or autologous HCT, particularly chimeric antigen receptor T-cells (CAR-T) treatment have started to increase after 2019, year of approval of the first commercial CAR-T product in Switzerland. Data on chimeric antigen receptor T-cell treatment are too early for comparative analyses. Detailed analyses of changes over time are presented. This study includes all HCTs, and cellular therapies, data useful for quality assurance programs, health care cost estimation and benchmarking. Between 50% and 60% of patients are long-term survivors after both types of HCT, indicating growing populations of surviving patients requiring long-term care.

CD19 Intron Retention Is Mechanism of CAR-T Treatment Resistance in Non-Hodgkin Lymphoma

Background: Chimeric antigen receptor T-cell (CAR-T) therapy has become a viable treatment option for patients (pts) with relapsed or refractory diffuse large B-cell lymphoma (R/R DLBCL). Transcriptomic markers for CD19 directed CAR-T failure have been characterized in pre-treatment B-cell acute lymphoblastic leukemia (B-ALL) but not in DLBCL. Here, we report the identification of RNA alternative splicing (AS) events associated with CD19 directed CAR-T response in pre-treatment DLBCL pts and of RNA binding proteins (RNABP) regulating these AS events. Methods: To identify putative AS markers for CAR-T treatment outcome, we performed differential AS on bulk RNA-seq samples of pre-treatment tumor biopsies in pts that had a durable response (DR), defined via remission up to 9 months post-treatment, and pts that had a non-durable response (NDR) with rMATS-turbo (Shen et al. PNAS. 2014). Intron retention (IR) events within CD19 were quantified with a secondary, more robust algorithm to ensure reproducibility and accuracy. Survival analyses were done using the survival and survminer libraries in R. Validation assays for the downstream effects CD19 IR were conducted in EJ1, Jeko-1, OCI-Ly3, SU-DHL-6, and Toledo cell lines. The surface protein expression of CD19 was assayed via flow cytometry (FC) followed by Quantibrite PE (BD Biosciences, San Jose, CA) quantification. Total CD19 protein expression was measured via western blot (WB) in the aforementioned cell lines. Analyses of CD19 IR was done via RT-PCR followed by electrophoresis, and bands were quantified with iBright Analysis Software (Thermo Fisher Scientific, Carlsbad, CA). The effect of RNABP was assessed via the creation of CRISPR/Cas9 knockout (KO) cell lines followed by WB of the target gene and quantification of CD19 surface protein expression by FC. Results: Introns 2 and 6 of CD19 are more highly expressed in NDR pts. A combined metric estimating the proportion of normal CD19 isoforms that do not express introns 2 or 6 (normal CD19) was created and DR pts expressed normal CD19 isoforms significantly higher than NDR pts (Figure 1). Normal CD19 isoforms classification performance was assessed via leave one out cross validation and achieved an overall accuracy of 67.6%. Sensitivity (classifying NDR) was 61.1% and specificity (classifying DR) was 81.3%. The expression of normal CD19 relative to the median level within the cohort was significantly associated with progress free survival (Figure 2). The expression of CD19 intron 6 was negatively correlated with CD19 surface protein expression in DLBCL cell lines. Cell lines were sorted by cells that exhibited high CD19 surface expression (bright) and low expression (dim). Cell lines sorted into the dim category expressed higher levels of intron 6 than bright cells. The correlation of RNABP expression with CD19 intron 6 expression was done in this patient cohort and the NCICCR-DLBCL cohort. Six RNABP were significantly correlated in both cohorts (r 2 > 0.2 and p-value <0.01). NOP2, one of the RNABP that was positively correlated with intron 6 expression, was knocked out in Jeko-1 cell lines to validate the putative correlation with CD19 protein expression. The resulting knock out cells exhibited lower levels of intron 6 retention and higher CD19 protein expression by FC and WB than non-transformed cells. Conclusion: Pre-treatment tumors of R/R DLBCL patients who relapse before 9 months following CD19 directed CAR-T therapy have higher expression of CD19 isoforms that retain introns 2 and 6. The upregulated expression of the CD19 isoforms retaining these introns is correlated with CD19 surface protein expression and is a marker for sustained remission following CAR-T treatment. NOP2 is correlated with CD19 intron retention and surface protein expression and could be a target for modulating CD19 expression in conjunction with CAR-T treatment. Figure 1: The expression of normal CD19 isoforms in pre-treatment tumor samples of DR (red) and NDR patients (blue). ΔPSI denotes the difference in medians between the two groups. P-value yielded by a Wilcoxon test between the two groups is reported. Figure 2: Progress free survival (PFS) stratified by expression of normal CD19 isoforms. Red - above cohort median expression of normal CD19 isoforms. Blue - below cohort median expression of normal CD19 isoforms. P-value yielded via log-rank test.

Risk of Bleeding and Thrombosis with CAR T-Cell Therapy: A Scoping Review

Introduction: Chimeric antigen receptor (CAR) T-cell therapy is emerging as a novel and revolutionary therapy in the treatment of relapsed or refractory lymphoma and other hematological malignancies. Patients with hematological malignancies are at risk of bleeding and thrombosis due to multiple risks factors. As there is an ongoing exploration of the incidence and characteristics of bleeding and thrombosis after CAR T-cell therapy, there is no existing published data that incorporates both peer-reviewed and non-peer-reviewed literature. Aim: Assess the risk of bleeding and thrombosis in CAR T-cell recipients for hematological malignancies. Method: We conducted a systematic literature search in MEDLINE, EMBASE, and CENTRAL electronic databases from inception to October 2022 using a combination of subject headings and text words. We included studies that assessed thrombotic and bleeding complications in patients eighteen or older who underwent CAR T-cell therapy for a hematologic malignancy. Our search strategy yielded 14 studies in the form of abstracts and full-text articles. The review process consisted of two levels of screening: (1) a title and abstract review and (2) a full-text review. Result: Of 3354 records, we included 14 studies based on our inclusion criteria [figure 1]. Majorities of the studies were retrospective. Most of the patients in these studies received CAR T-cell therapy for refractory diffuse large B-cell lymphoma. Patients received at least 2 lines of therapy or more before CAR T-cell treatment. Different types of CAR T-cell therapy were used. Based on the included studies, the risk of bleeding was higher than thrombosis [figure 2]. Bleeding sites were variable, but gastrointestinal (GI) bleeding was predominant. In Ma et al, GI bleeding occurred in 11 (42.3%) patients. In Qi et al and Johnsrud et al, bleeding occurred in 44 (11%) and 12 (9.4%) patients respectively. Variable sites of thrombosis were noticed. In Hashmi et al incidence of thrombosis was 10.8%. Although there are some risk factors associated with bleeding and thrombosis in this population, the exact mechanism is still unknown. We think future studies to determine the exact mechanism of this toxicity and to assess the underlying risk factors would be crucial to decrease morbidity and mortality in this population and to develop risk assessment tools.

Early Prediction of Treatment Response By Circulating Tumor DNA Profiling in Patients with Diffuse Large B-Cell Lymphoma Receiving CAR T-Cell Therapy

Introduction Patients with relapsed or refractory diffuse large B-cell lymphoma (r/r DLBCL) are characterized by a particularly poor prognosis. Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy has improved outcomes of patients with r/r DLBCL, yet a substantial proportion of patients still experiences relapse or progression after CAR T-cell treatment. Identifying patients at high risk of CAR T-cell therapy resistance or future lymphoma progression remains challenging. Here, we explored the value of circulating tumor DNA (ctDNA) as a prognostic biomarker at baseline and early into treatment in DLBCL patients undergoing standard-of-care CAR T-cell therapy, without the need for matched tumor genotypes. We further investigated associations between ctDNA and other known clinical risk factors. Methods We applied targeted next-generation sequencing (NGS) to a total of 53 samples from r/r DLBCL patients receiving CAR T-cell therapy (axicabtagene ciloleucel and tisagenlecleucel, n=16) at the University Medical Center Freiburg (Germany), using a modified version of the AVENIO ctDNA analysis workflow (Roche; Research Use Only) that covered ~314kb and targeted 466 distinct genes recurrently mutated in DLBCL (based on the CAPP-Seq workflow, Kurtz DM et al. J Clin Oncol 2018). Tumor genotypes were assessed noninvasively from plasma samples obtained at baseline before lymphodepletion with matched germline controls. Concentrations of ctDNA were quantified at baseline and early after CAR T-cell therapy at day 7-10. Initial treatment response was assessed by conventional PET-CT obtained 4-6 weeks after treatment based on the Lugano criteria. Results At baseline prior to lymphodepletion, we detected somatic mutations in 100% of plasma samples by noninvasive genotyping, with a median of 19.5 mutations per patient (range: 3-97). The most frequently mutated genes were IGHV, IGLL5, BCL2, MYC, BTG1, CREBBP, and TP53. Patients with high baseline ctDNA concentrations (mean allele frequency [AF] >= 1%) showed shorter progression-free survivial (PFS) and overall survival (OS) than patients with ctDNA levels below this threshold (median PFS: 86.5 days vs. not reached, p=0.04, HR: 6.3, 95% CI: 1.7-23.6; median OS: 219 days vs. not reached, p=0.048, HR: 4.1, 95% CI 1.0-16.6, Figure 1). Pretreatment ctDNA levels were significantly correlated with LDH concentrations ( p=0.049, r=0.5). We further found significantly higher ctDNA concentrations in patients with transformed DLBCL (vs. non-transformed, p=0.008) and in those with 3 or more prior treatment lines (vs. 1-2, p=0.04). At day 7-10 after CAR T-cell therapy, ctDNA levels dropped a median of 10-fold compared to the pretreatment time point ( p=0.008). Patients achieving a complete (CMR) or partial metabolic response (PMR) by PET-CT after 4-6 weeks showed a significant decrease of ctDNA ( p=0.001), while those with a stable (SD) or progressive disease (PD) had no significant ctDNA reduction at day 7-10 after CAR T-cell infusion. Vice versa, while 100% of patients with a >1.5-log-fold drop of ctDNA level between the pretreatment and interim time point revealed a CMR or PMR in early PET-CT scans, only 62% of patients with a smaller ctDNA decline responded to treatment according to PET-CT. Conclusions Our data suggests, together with previously published studies (Sworder BJ et al., Cancer Cell, 2023; Frank MJ et al. J Clin Oncol, 2021), that ctDNA profiling may allow robust noninvasive genotyping, accurate risk-stratification, and early prediction of clinical outcomes in r/r DLBCL patients undergoing CAR T-cell therapy. Although limited by small sample size, this pilot study highlights a potential future role of ctDNA measurements for personalized treatment selection and guidance in clinical trials.

Potent and Target Specific Cytotoxicity of PRAME TCR Mimic CAR T Cells in KMT2A-Rearranged AML Patient-Derived Xenograft Model

Chimeric antigen receptor (CAR) T cell therapy was a resounding success in CD19+ ALL despite the fact that it is essential for B-cell maturation because it is not expressed during normal myeloid hematopoiesis. Normal and leukemic B-cells are targeted by CD19+ CAR T-cells without causing myeloid hematopoietic toxicity. Advancing such therapy to acute myeloid leukemia (AML) has been challenging in part due to a paucity of “dispensable” targets or targets whose expression is limited to the leukemic cells. Using a large AML and normal hematopoiesis transcriptome database, we pursued discovery of “AML-Restricted Targets”; genes that are silent in normal hematopoiesis but expressed in AML. This broad discovery effort yielded a library of AML-restricted targets that included PRAME (Preferentially Expressed Antigen in Melanoma), an intracellular protein expressed via MHC on the cell surface, to be a highly expressed in AML including the aggressive KMT2A-rearranged (KMT2A-r) AML. We used a TCR mimic (mTCR) antibody, which recognizes the PRAME peptide/HLA-A2 complex on the tumor cell surface, to develop a PRAME directed TCR mimic CAR T (PRAME mTCRCAR T) for pre-clinical studies. Studies in cell lines and CDX models have shown significant efficacy of this CAR T (Kirkey, Bld Adv. 2022). To conduct final IND-enabling studies, we generated a KMT2A-r HLA-A2+/PRAME+ patient-derived xenograft (PDX) model to treat with PRAME mTCRCAR T-cells. Here we demonstrate the in vivo activity of PRAME mTCRCAR T cells against this unique PDX AML model. VL and VH sequences from the PRAME specific TCR mimic antibody (Pr20) were used to construct the single-chain fragment variable domain into the 41-BB/CD3ζ CAR vector. The PDX model was derived from a PRAME +/HLA-A2 + pediatric patient with KMTA2-r AML. PDX cells were transduced with lentiviral ffluciferase for noninvasive bioluminescent IVIS imaging to monitor leukemic progression. Mice were transplanted with 1x10 6 PDX leukemia cells then 1 week later, PDX leukemia-bearing mice were treated with unmodified T cells or PRAME mTCRCAR T cells at 5x10 6 cells (1:1 CD4:CD8) per mouse. Leukemia burden was measured by IVIS imaging and regular peripheral blood analysis. Mice who received unmodified T cells had rapid disease progression by day 50 and all died by day 105. In contrast, PRAME mTCRCAR T-treated mice rapidly cleared disease and all remained alive and leukemia-free >180 days post-treatment (p=0.001). Average leukemia burden in the control cohort was 3.5% at week 6 and 41.75% at week 11, while no leukemia was detected following CAR T-cell treatment. In addition, the control arm had a marked expansion of leukemia with a 7.75- and 429.6-fold increase in radiance by IVIS at 6 and 11 weeks post-treatment, respectively. No significant increase in radiance was seen in the PRAME mTCRCAR T cell treated group. We further evaluated hematopoietic toxicity of PRAME mTCRCAR T cells in humanized mice. Sub-lethally irradiated NSG-SGM3 mice were reconstituted with HLA-A2+ CD34 selected human cord blood stem cells with simultaneous and allowed to engraft. Human hematopoietic cell engraftment was evaluated following treatment with unmodified T cells vs. PRAME mTCRCAR T cells. Evaluation of human CD45+ cells in the peripheral blood via flow cytometry showed no difference in the levels of hematopoietic engraftment in mice treated with PRAME mTCRCAR T cell (8%) vs. unmodified T cells (6%) vs. an untreated control cohort (5%), demonstrating lack of toxicity of PRAME mTCRCAR T cells against HLA-A2 hematopoiesis. We demonstrate that PRAME mTCRCAR T cells can irradicate AML in a target specific manner in aggressive KMT2A-r AML without toxicity. We show potent efficacy with eradication of leukemia in PDX-bearing mice following treatment with PRAME mTCRCAR T cells resulting in prolonged survival, without hematopoietic toxicity. These results provide strong rationale for advancing this therapeutic approach to clinical development.

Hematopoietic Stem Cell Transplantation for DLBCL: 55,000 Cases from EBMT As a Comparator for CAR T-Cells

Introduction: Autologous (auto-HSCT) and allogeneic (allo-HSCT) stem cell transplantation represent established treatments for patients (pts) with diffuse large B-cell lymphomas (DLBCL). Transplant modalities and results substantially changed over time. Due to the favorable data reported for chimeric antigen receptor T-cell (CART) treatment and other more targeted drugs, the landscape of transplantation for DLBCL is rapidly changing. We provide long-term data for DLBCL transplants reported to the European Society for Blood and Marrow Transplantation (EBMT) over 30 years as a comparator to CAR T-cell therapies. Methods: We conducted a registry-based analysis of auto- and allo-HSCT activities in Europe and other countries reporting to EBMT in patients with DLBCL treated between 1990 and 2022. Patients meeting the following criteria were included: age≥18 years, diagnosis of DLBCL, auto-HSCT as first HSCT or allo-HSCT either as first HSCT or after auto-HSCT. For transplants reported between 1990-2019, information on modalities and outcomes were retrieved from the EBMT database. CART numbers from the first CART treatment reported to EBMT until 2022 were also collected. Results: In total, 50,776 patients received an auto-HSCT and 5,282 an allo-HSCT between 1990 and 2022. Numbers for auto-HSCT increased from 1,021 in 1990-1994 to a maximum of 2,385 in 2019; the number of allo-HSCT rose from 45 to 305 for these time periods. After 2019, transplant numbers experienced a sharp decline with only 1,548 auto- and 124 allo-HSCT performed in 2022, Conversely, the number of CART consistently increased since the first EBMT-recorded infusion in 2014, rising from 537 in 2019 to 809 in 2022. For detailed analyses, 38,979 auto-HSCT and 4,100 allo-HSCT pts (1,753 allo-HSCT as 1 st HSCT; 2,347 after auto-HSCT) between 1990 and 2019 were identified. Important trends for auto-HSCT were an increase of median age at HSCT [42.0yr (1990-1994) to 57.4yr (2015-2019), p<.0001] and better performance status ≥80% [91.3% (1990-1994) vs 93.0%(2015-2019), p=.006]. Peripheral blood (PB) became the dominant stem cell source for auto-HSCT in 1994; numbers of patients prepared with total body irradiation decreased [16.5% (1990-1994) to 0.4% (2015-2019), p<.0001]. For allo-HSCT pts, the following trends emerged: significant increase in median age [35.8yr (1990-1994) to 53.0yr (2015-2019), p<.0001], adoption of PB as the main stem cell source in 1998 and a substantial shift towards reduced intensity conditioning [0% (1990-1994) to 60.6% (2015-2019), p<.0001]. The proportion of unrelated donors and haploidentical donors significantly increased over time [0% (1990-1994) vs 51.2% (2015-2019), p<.0001 and 0% vs 16.2%, p<.0001]. With a median follow-up of 3.7yr (range 0-30.8yr) for autografted pts, 3-yr overall survival (OS) significantly improved over time [57% (1990-1994) to 71% (2015-2019), p<.001], the relapse rate (RI) declined [50% (1990-1994) to 36%(2015-2019), p<.001], but non-relapse mortality (NRM) remained at 4%. Auto-HSCT patients transplanted in first CR/PR between 2016 and 2019 showed superior outcomes compared to pts treated in second or later CR/PR [3-yr PFS: 69% vs 56%, p<.001; 3-yr OS: 79% vs 69%, p<.001]. For allo-HSCT recipients, with a median follow-up of 4.7yr (range 0-28.6yr), significant improvements were noted for 3-yr OS [35% (1990-1999) to 46% (2015-2019), p<.001)], 3-yr PFS ( p<.001) as well as 3-yr RI ( p<.005). However, 1-yr NRM only improved numerically [30% (1990-1994) to 20% (2015-2019), p=.21]. Conclusions: With more than 50,000 patients analyzed, we provide a comprehensive overview of changes in transplantation modalities and results over more than 30 years. We observed major changes in clinical characteristics and substantial improvements in all major outcome parameters. Patients became increasingly older and medically fit and were transplanted earlier in the course of disease. OS and PFS significantly increased, relapse incidence decreased, NRM did not show major evolution, all supposedly attributable to better patient selection and supportive care. With CART entering the clinical arena, the numbers of transplants for DLBCL sharply decreased. Auto- and allo-HSCT remain important modalities for pts failing CART or in countries where CART are not readily available. The outcomes reported here may serve as real-world benchmark data when new modalities like CART are considered.

Efficacy and Safety of Chimeric Antigen Receptor T Cells Therapy Strategy That Dual Targeting CD19 and CD22 to Treat Relapsed/Refractory Acute Lymphoblastic Leukemia in Adults

Background: Chimeric antigen receptor T-cells (CAR-T) targeting CD19 therapy is reported to induce 83-93% response in relapsed/refractory acute lymphoblastic leukemia (r/r-ALL) in adults. However, 30-60% of patients relapse after CAR-T treatment, of which 14-25% CD19-negative relapse. In spite of CAR-T cells persistence, CD19 absent causing tumors that evade CAR-T cells mediated recognition and clearance. For CD19-negative relapse, it is necessary to explore more effective targets. There are some research proved that compared with single-targeted CARs, CD19/CD22 dual-targeted CARs induce more IFN-γ and IL-2 in vitro and eradicate patient-derived xenografts (PDX) produced with CD19-negative relapse of CD19-directing CAR-T treatment. Therefore, our study explored the efficacy and safety of clinical studies on mixture of CD19 CAR-T and CD22 CAR-T cells in the treatment of r/r ALL in adults. Patients and Methods: CD3 + cells were selected from the apheresis PBMC and activated before lentiviral CAR T cells infection. The cells were transduced with a caspase9-inducible, safety-engineered lentivector CAR containing anti-CD19 or -CD22 scFv fused with multiple intracellular signaling domains: CD28/CD27/CD3z-iCasp9. The quality of apheresis cells, gene transfer and T cell proliferation efficiencies, and effective CAR-T infusion dose were quantitatively scored and documented. All enrolled patients expressed CD19 and CD22 positively. All patients received fludarabine (FLU) and cyclophosphamide (CTX) conditioning chemotherapy (FLU 30mg/m 2, d1-3; CTX 500mg/m 2, d1-3) before CAR-T infusions. Results: The study included 16 r/r ALL patients, median age is 26 years, and median of previous theraples is 6, all of patient details can be found in the table (Fig.1). In the first month after CD19/CD22 CAR-T cells infusion, total of 93.8% (15/16) of patients achieved complete response (CR), 1 case (6.25%) was evaluated as non-remission (NR). In the evaluation of safety, 68.75% (11/16) of patients did not experience cytokine release syndrome (CRS), 25% of patients (4/16) occurred grade 1 CRS and 6.25% (1/16) of patients occurred grade 2 CRS, no case of severe CRS defined as≥grade 3 and no any immune effector cell-associated neurotoxicity syndrome (ICANS) occurred. After a median follow-up of 9.9 months, 4 patients maintained CR, while other 11 patients were relapsed, with a duration of response (DOR) of 25% (4/16), median LFS was 6.1 months and median OS were not reached (Fig.2). Conclusions: In summary, CD19/CD22 exhibited a manageable safety but limited antileukemia activity. Our study showed that mixture of CD19 CAR-T and CD22 CAR-T cells infusion is fail to expected to compensate for the treatment of relapsed in r/r ALL and prolonging the leukemia-free survival of patients. The low-efficacy of dual-targeted CAR-T cells may be caused by the CAR19 and CAR22 cells interfere with each other and weaken the CAR-T amplification in vivo. From our data, it seems that the timing of treatment and tumor burden status may play a more important role rather than new targets CAR-T cells infusion for r/r ALL in adults.

Real-World Incidence, Characteristics and Management of Cytokine Release Syndrome Induced By Chimeric Antigen Receptor T-Cell Therapy across Hematologic Malignancies

Background: Cytokine release syndrome (CRS) is a potentially life-threatening, supraphysiologic response following immunotherapy resulting in the activation or engagement of endogenous or infused T cells and/or other immune effector cells. CRS occurs with varying frequency and severity, depending on the underlying disease and immunotherapy used, such as chimeric antigen receptor T-cell (CAR-T) therapy or off-the-shelf T-cell engaging bispecific antibodies. Limited research exists on the diagnosis and management of CRS in the real world. This study describes the real-world incidence, severity and clinical management of CRS induced by commercially available CAR-T therapies for diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL) and multiple myeloma (MM). Methods: Real-world data (RWD) were obtained from the Flatiron Health, US, nationwide electronic health record (EHR)-derived, de-identified database of cancer patients within a network comprised primarily of community cancer practices. The Flatiron Health network also includes practices designated as academic sites by the National Cancer Institute; however, relative proportions of community/academic practices may vary depending on the study cohort. Data as of August 31, 2022, on CAR-T therapy, clinical characteristics and disease outcomes specific to CAR-T therapy, as well as CRS and its clinical management, were abstracted from unstructured documents in EHRs (in addition to routinely available structured data), in a cohort of patients receiving commercial CAR-T therapy. Specifically, data for CRS including treatments and resolution were abstracted based on explicit mention of and attribution to CRS in charts. Results: Table 1 summarizes data by indication. Incidence of CRS ranged from 56% (104/185) in DLBCL to 72% (66/92) in MCL. Excluding categories with small numbers of cases (<15), CRS incidence also varied by individual CAR-T therapy and underlying indication and ranged from 35% for idecabtagene vicleucel in MM to 73% for brexucabtagene autoleucel in MCL. Highest incidence of Grade ≥2 CRS was observed among MCL patients, whereas Grade ≥3 CRS was most commonly observed among FL patients. Time to onset of CRS ranged from 0 to 16 days, with median onset between 3-4 days among lymphoma patients and earlier median onset of 1 day for MM patients. Median time to CRS resolution ranged from 2 to 6 days across indications. While 81-95% of patients received CAR-T therapy at an academic center, 5-14% received CAR-T therapy at a community center. Tocilizumab and corticosteroids were the most commonly used treatments for CRS, including 37-48% use of tocilizumab for Grade 1 CRS (Table 2). Discussion: This study uniquely summarizes RWD on CRS following the use of commercially available CAR-T therapies across multiple hematologic malignancies, based on information from clinical notes abstracted retrospectively from EHRs. Consistent with the timing of approval of commercially available CAR-T therapies, the sample size for MM was limited compared with other malignancies. Safety events are typically under-reported in RWD sources, specifically for CRS when relying on information documented in patients' EHRs, compared with other published literature. In accordance with other RWD on CRS, grading criteria for CRS as well as vasopressor and oxygen use were not consistently reported in patients' EHRs. CAR-T treatments were predominantly administered in academic centers. Limited use in community practices was also observed; however, further information to accurately characterize the size and capabilities of community centers was not available in this database. Data also showed considerable use of tocilizumab to treat low-grade CRS, suggesting the need for additional evidence on its role to treat CRS beyond its current regulatory approval. Increased evidence generation, specifically from usual clinical practice, can contribute to increased awareness and knowledge about CRS diagnosis and management, and in turn realize the maximum potential of T-cell therapeutics.

Resistance Mechanisms Impacting Bispecific Antibody (BsAbs) and Chimeric Antigen Receptor (CAR) T-Cell Therapy Outcomes in Large B Cell Lymphoma (LBCL) Patients

Introduction: BsAbs and CAR T-cell therapy have revolutionized the treatment landscape of patients with relapsed/refractory (R/R) LBCL. Regrettably, less than 40% of patients will attain durable responses, and determinants of response remain unclear. Our aim was to identify tumoral characteristics that can predict resistance to CAR T-cell therapy and BsAbs in LBCL patients. Methods: We retrospectively collected clinical data and obtained pre-treatment formalin-fixed and paraffin-embedded specimens prior CAR T-cell and BsAb treatment from 56 LBCL patients from 4 centers. Targeted NGS was performed to identify variants and copy number variations (CNVs) across a custom gene panel of 200 genes. These include lymphoma related genes, and genes involved in tumor antigen presentation, B and T lymphocyte interaction, and cell death pathway. Variables assessing response where Overall Response Rate (ORR), Complete Response Rate (CRR), Progression-Free Survival (PFS), and Overall Survival (OS). To ascertain each gene's influence on these endpoints, a logistic model was used for ORR and CRR comparisons, while Kaplan-Meier and Cox methods were employed to analyze PFS and OS. Results: A total of 56 patients were included in the study, 36 patients treated with CAR T-cell and 36 with BsAbs (16 patients received both). Samples were obtained for 31 patients prior to CAR T-cell therapy (CAR T-cell cohort) and 34 cases prior to BsAbs (BsAbs cohort). Baseline characteristics of the patient population are shown in Table 1. In summary, median age was 62 years, 57% were male, 42% had a previous indolent lymphoma and median previous lines were 2 (range 1-4). Best response included CRR in 59%, with a median PFS and OS of 13.6 (CI95% 5.98 - Not reached [NR]) and 29.5 (CI95% 15.7 - NR) months, respectively. Median follow-up for the full cohort was 18.7 months (CI95% 15.1 - 24). As per the CAR T-cell cohort, ORR and CRR were 81% and 68%, respectively. Median PFS and OS for CAR-T-cell cohort was 29.4 months (CI95% 5.98 - NR) and NR, respectively. Regarding the BsAb cohort, ORR and CRR were 66% and 50%, respectively. Median PFS and OS was 10 (CI95% 5.16 - NR) and 22.5 (CI95% 14.4 - NR) months, respectively. For the full cohort, the most frequently altered genes, considering both mutations and CNVs, were KMT2D (66%), TP53 (60%), CREBBP (58%), IGLL5 (51%), REL (42%), EZH2 (38%), BCL2 (38%), TNFRSF14 (37%), SGK1 (32%) and KLHL6 (32%) (Figure 1). The most frequent CNVs were gains of 2p16.1 (34%), 3q28 (15%), 6p(14%), 11q (12%), 21q (12%), 3q (10%); and losses of 17p (32%), 6q (26%), 1p36.32 (25%), 13q14.2 (18%), 19p13.3 (18%), 4q35.1(16%), (10q23.31 (12%), 9p21.3 (10%). Focusing on the CAR T-cell cohort (n=31), mutations and/or deletions in EP300 (7%, n=2)( p=0.01) and KLHL6 (n=2, 7%) ( p=0.04) were associated with an inferior PFS. In addition, mutations and/or deletions in KLHL6 ( p=0.02) and EP300 (n=3, 10%) ( p=0.05), and amplifications in MYC (n=3,10%) ( p=0.031) were associated with shorter OS. No differences in PFS and OS were oberved in patients with mutations and/or deletions in TP53 (n=19, 63%). Regarding RHOA patients with mutations or deletions (n=2, 7%) did not present an inferior PFS ( p=0.88) or OS ( p=0.52). The analyzed alterations did not show an impact on response rate. In terms of patients who received BsAbs (n=34), we observed that mutations and/or deletions in TP53 (n=19, 58%) ( p=0.04) , RHOA (n=5, 15%) ( p=0.05) and GNAI2 (n=5,15%) ( p=0.02)were associated with aninferior PFS. Mutations and/or deletions in TP53 (p=0.04) , RHOA (p=0.009) , GNAI2 ( p=0.01) and CD274(PDL1)-PDCD1LG2(PDL2) (12%, n=3) ( p=0.01) were associated with an inferior OS. Regarding MYC, 8 cases were mutated (n=2, 6%), amplified (n=5, 15%) or both (n=1, 3%), but these alterations were not associated with an inferior PFS (p=0.46) or OS ( p=0.27). Again, no impact on response rate was observed for patients carrying alterations. Conclusions: In our study, a pattern of genetic alterations predicting worse OS was observed in patients treated with CAR T-cells ( KLHL6 EP300 and MYC) and BsAbs (TP53, RHOA, PDL1, PDL2, GNAI2). This important information will aid in the development of targeted strategies to overcome resistance and improve treatment outcomes.

Patterns of Use and Impact of Post-Referral and Bridging Therapy on Outcomes in Patients Undergoing CAR T-Cell Therapy for B-Cell Lymphoma: A Single-Center Analysis

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy has changed the treatment landscape for B-cell non-Hodgkin lymphoma (B-NHL) . Treatment is often given after patients have been referred for CAR T-cell therapy while evaluation is underway but prior to leukapheresis, subsequently referred to as post-referral therapy (PRT). Bridging therapy (BT) may be used while CAR T cells are being manufactured. Little has been previously reported on patterns of use of various regimens, optimal choice of treatment, and how use of these treatments impacts patient outcomes when undergoing CAR T-cell treatment. METHODS: Patients aged ≥18 years with B-NHL who underwent apheresis between November 2017 and March 2023 for commercial CAR T-cell therapy at Vanderbilt University Medical Center were identified from an internal database. Patients received axicabtagene ciloleucel (axi-cel), tisagenlecleucel (tisa-cel), or brexucabtagene autoleucel (brexu-cel). The primary endpoint was complete remission (CR) at 30 days post CAR-T. Patients who died prior to that time point were not censored and considered to not have achieved CR. Tumor response after CAR T-cell infusion was assessed per Lugano criteria by the treating clinician. PRT and BT regimens were defined as follows: platinum-based included cisplatin, carboplatin, or oxaliplatin; polatuzumab-based included rituximab with or without bendamustine; non-platinum based included: R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone), R-hyper CVAD (rituximab, hyperfractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone and/or high-dose methotrexate and cytarabine), or R-mini CVD (rituximab, cyclophosphamide, vincristine, dexamethasone); oral targeted agents included: ibrutinib, venetoclax, or lenalidomide based treatments. Differences in baseline characteristics were assessed with chi squared analysis and two sample t-test. Predictors of 30-day CR were identified with logistic regression analysis. RESULTS: This study included 60 patients who were treated with commercial CAR-T with median follow up of 280 days. A total of 9 did not receive any PRT while 51 received one or more lines of PRT. There were no statistically significant differences between those who did or did not receive PRT in median age, sex, diagnosis, disease stage, or product type (Table 1). Elevated LDH (44% vs. 80%, p=0.022) and private insurance (33% vs. 69%, p=0.043) were more common in those who received PRT compared to those who did not. There were no significant differences in the receipt of BT. A detailed summary of types of regimens used in PRT and BT is included in Table 1. For those who received PRT, the median total number of cycles of treatment received including bridging therapy was 3 (range 1-7). The median time from referral to leukapheresis was longer in those who received PRT compared to those who did not (68.5 days vs. 36 days). Fewer patients who received PRT underwent leukapheresis within 45 days (22% vs. 56%, p=0.037). There was no difference in 30-day CR after CAR-T infusion. Figure 1 shows 30-day CR stratified by type of PRT given in the first line. Amongst patients who received platinum-based therapy, 12 (60.0%) achieved a CR at 30 days, while 8 (40.0%) did not. Amongst patients who received polatuzumab-based therapy, 4 (26.7%) achieved a CR at 30 days, while 11 (77.3%) did not. In univariate logistic regression analysis, predictors of achieving CR at 30 days included age (OR 1.04, CI: 1.00-1.09, p=0.033), elevated LDH (OR 0.19, CI: 0.051-0.69, p=0.012), and receipt of platinum-based therapy as first line PRT (OR 4.13, CI: 1.23-13.78, p=0.021). Multivariate analysis showed receipt of platinum-based therapy as first line PRT (OR 5.11, CI: 1.23-21.22, p=0.025) and elevated LDH (OR 0.08, CI: 0.013-0.53, p=0.09) were significant predictors. CONCLUSION: In this retrospective analysis, we provide a detailed overview of the number of lines and types of PRT and BT used in patients undergoing CAR-T cell therapy at a single US academic medical center. While 30-day CR was not impacted by the receipt of PRT, patients who received platinum-based chemotherapy as their first PRT had significantly higher odds of achieving of 30-day CR than those who received other therapies in multivariate analysis. This trend will be important to explore in larger analyses and may provide insight into optimal post-referral treatment choices.

Positron Emission Tomography Evaluation in Relapsed/Refractory B-Cell Lymphoma Patients Treated with Anti-19 Chimeric Antigen Receptor (CAR) T-Cells in the CART-SIE Observational Study

Background: CD19 direct chimeric antigen receptor (CAR) T-cell therapy is an efficacious therapy for patients (pts) affected by relapsed/refractory (R/R) large B-cell lymphomas (LBCL). The role of early evaluation by Positron emission tomography/computed tomography (PET/CT) is still undefined. The study's objective was to analyze the role of early PET/CT according to histology and CAR T-cell product administered. Methods:CART-SIE is an ongoing prospective and retrospective observational study evaluating the outcome of lymphoma pts treated with CAR T-cells. From March 2019 to June 2023, 659 patients were enrolled. In this study, we included only pts with adequate follow-up (30 days) and an FDG-PET/CT before infusion (PET-0) and at least one month (PET-1) after CAR-T cells infusion. A landmark analysis based on PET-1 and PET-3 (3 months evaluation) results was performed for Progression-free survival (PFS) and Overall Survival (OS) estimation. Results: Three hundred twenty-seven pts with R/R LBCL [n= 189 (58%) Diffuse large B-cell lymphomas, n= 59 (18%) High-grade B-cell lymphomas (HGBCL), n= 41 (13%) Primary Mediastinal B-cell lymphomas (PMBCL), n= 38 (11%) Mantle Cell Lymphoma (MCL)] received CAR T-cell treatment with axicabtagene-ciloleucel [n=161(49%), axicel], tisagenlecleucel [n=128 (39%), tisacel] or brexucabtagene autoleucel [n=38(12%), brexucel]. The median age of the pts was 58 years (90 pts were older than 65 years). Most pts [n=280, (85,6%)] were treated with a bridging therapy. Bulky and extranodal disease were observed in 108 (33%) and 177 (54,1%) pts, respectively. Pts who received tisacel were significantly older (p<0.0001), had a longer time from lymphocyte apheresis to the infusion (<0.0001), and did not include PMBCL. The median follow-up time was 12 months [IQR, 6,09-22,37]. At the time of PET-1, 180 pts (55%) showed a PET with DS1-3, 82(25%) had a PET with DS4 (n=61 PR, n=21 SD), and 65(20%) with DS5 (n=13 SD, n=47 PD, n=5 PR). The results of early PET were significantly different according to the histology (p<0.0005): the percentage of DS 4 and DS 5 was 22% and 23% for DLBCL, 30% and 31% for HGBCL, 39% and 7% for PMBCL, and 16% and 0% for MCL, respectively. Overall, 1-year PFS based on PET-1 was 67%, 42%, and 8% for DS1-3, DS4, and DS5 (p<0,0001), respectively. The prognostic role of DS 4 varied with histology: 1-year PFS was 36%, 58%, and 44% for DLBCL, PMBCL, and HGBCL (<0.0001), respectively. The outcome of early PET was significantly influenced by CAR T-cell product (also excluding PMBCL): 1-year PFS for pts with DS1-3 and DS4 at PET-1 was 66% and 51% for axicel and 58,7% and 32% for those treated with tisacel (p<0.0001). The 1-year OS based on PET-1 was 88%, 82%, and 33% for DS1-3, DS4, and DS5 (p<0,0001), respectively. At 90 days, only 219 pts were evaluable; 21 of 82 (26%) DS4 and 5 of 65 (8%) DS5 at PET-1 converted in CR. Overall, 1-year PFS and OS based on PET-3 was 80% and 93% for DS1-3, 64% and 93% for DS4, and 8% and 56% for DS5, respectively. Multivariable analyses identified DS4 and DS5 values on PET-1 and CAR T-cell product Tisacel associated with increasing risk of failure [HR: 1,95 (95%CI;1,01-3,76) for DS4; HR: 9,63 for DS5 (95%CI:6,13-15,13); p< 0,0001; HR: 1,53 for Tisacel vs Axicel; (95%CI:1,06-2,22); p<0.0231]. Conclusion: Early evaluation by PET significantly influenced the prognosis. Estimating the risk of failure must be integrated with CAR T- cell product.