CAR-T Cell Infusion Research Articles

Reduction of Metabolic Active Tumor Volume Prior to CAR T-Cell Therapy Improves Survival Outcomes in Patients with Large B-Cell Lymphoma

Introduction CD19-targeted chimeric antigen receptor (CAR) T-cell therapy has become the standard 2 nd or 3 rd line therapy for patients with relapsed or refractory Large B-cell lymphoma (r/r LBCL). As part of this treatment bridging therapy is frequently used between T-cell apheresis and CAR T-cell infusion in order to limit disease progression, diminish lymphoma-related symptoms and/or reduce tumor volume. Bridging therapy has shown to be feasible, safe and effective in patients prior to infusion. In addition, a lower tumor volume is associated with better treatment outcomes and less toxicity. However, the exact reduction and dynamics in metabolic active tumor volume (MATV) are unknown. Here we investigated the relation of changes in pre-apheresis (baseline) MATV, based on 18F-FDG PET scans, and pre-lymphodepleting chemotherapy (pre-LD) MATV with CAR T-cell outcomes and toxicities in patients with r/r LBCL. Methods Baseline and pre-LD MATVs and PET characteristics were calculated semi-automatically of patients who were treated with CAR T-cell therapy. A Standardized Uptake Value (SUV) threshold of 2.5 was used to maintain robustness and avoid MATV underestimation. Bridging strategies consisted of radiotherapy, chemotherapy, or a combination of both. Primary endpoints were duration of response (DOR) and overall survival (OS). Secondary endpoints included cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). The Youden index was used to identify the optimal cut-off for dividing patients into low- and high-MATV groups to predict DOR at 2 years. Two-year DOR and OS rates were estimated using Kaplan-Meier curves and the log-rank test was utilized to compare the differences among low- and high-MATV patient groups. Univariable analysis was performed with Cox regression for DOR and OS, and with logistic regression for CRS and ICANS. Results Seventy-four patients with LBCL and treated with CAR T-cell therapy were included in the study. For all patients baseline 18F-FDG PET scans were available, whereof in 68 patients the pre-LD scans. The median baseline MATV was 180 cc (IQR 64 - 426) with a median SUV max of 18.8 (IQR 11.9 - 28.1) and increased to 241 cc (IQR 75 - 525) at pre-LD, with a median SUV max of 21.0 (IQR 12.8 - 31.3). The optimal cut-offs for low- and high-MATV patient groups at baseline and pre-LD were 190 cc and 480 cc, respectively. Patients with a low MATV baseline had a better DOR (Figure 1A) and OS compared to high MATV baseline patients (both p < 0.001). Patients with a high MATV baseline that was reduced to a low MATV pre-LD showed a significant improvement in DOR (Figure 1B) and OS, compared to patients with a high MATV pre-LD ( p = 0.036 for DOR; p = 0.015 for OS). Univariable analysis showed that a high baseline MATV was associated with a worse DOR (HR 6.43 CI [2.15 - 19.20]; p < 0.001) and OS (HR 6.97 CI [2.33 - 20.80]; p < 0.001). A high pre-LD MATV was also associated with a worse DOR (HR 4.86 CI [1.98 - 12.00]; p < 0.001) and OS (HR 5.87 [2.41 - 14.30]; p < 0.001). Other significant associations with DOR and OS were observed for pre-LD lactate dehydrogenase (LDH), the IPI score for DOR exclusively, and gender and bridging therapy for OS exclusively. Conclusions We showed that patients with a low MATV compared to a high MATV at baseline have better survival outcomes and less toxicity. Additionally, reducing tumor volume by applying bridging therapy in patients with a high baseline MATV, results in a significant better DOR and OS. Reducing the MATV before CAR T-cell infusion is therefore essential in CAR T-cell therapy patient care and provides a rationale for the use of more intensive bridging therapies.

A Multicenter Study of CAR-T Cell Therapy for Relapse of B-ALL Post Allogeneic Stem Cell Transplantation

BACKGROUND: Patients with acute B-cell lymphoblastic leukemia (B-ALL) relapsed after allogeneic stem cell transplantation (allo-HSCT) have very poor prognosis. Chimeric antigen receptor T (CAR-T) cells targeting CD19 have been developed as one of the effective immunotherapies for relapsed/refractory B-ALL. The safety and efficacy of CAR-T cells in patients relapsed after allo-HSCT remain unclear. METHODS: This study analyzed 60 B-ALL patients relapsed after allo-HSCT treated with CAR-T cells between October 2015 and February 2022 from four Chinese centers. Their pre-treatment patient characteristics, treatment regimens, efficacy, adverse effects, and follow-up data were comprehensively analyzed. RESULTS: One patient refused efficacy evaluation. 7 patients died before day 28 after CAR-T infusion. A total of 52 were assessed for efficacy within 28 days after CAR-T infusion.76.92% (40/52) patients achieved complete remission (CR) at Day 28 after CAR-T infusion, including 38 patients who achieved MRD-negative CR. 4 of the 40 patients relapsed at a median follow-up date of 12.1 months. The 1-year OS and EFS of all patients were 52.8% ± 7.0% and 51.5% ± 7.0%, respectively, and the 2-year OS and EFS were 40.1% ± 8.6% and 45.1% ± 8.6%, respectively. Multifactorial analysis showed that patients with severe CRS had worse OS and EFS, and other factors including age, gender, site of CAR-T tumor involvement, type of previous transplantation, previous transplantation type, CAR-T product and CRS grade, etc., did not affect the long-term survival of patients. The incidence of CRS was 80.00%, the incidence of grade ≥3 CRS was 20.83%, and the median time of CRS was 2 days after CAR-T cell infusion (range: 0-34 days). 3 patients (5.00%) died due to multiorgan failure caused by severe CRS, and the rest of the patients' CRS was controlled, and the grade of CRS was related to the proportion of blast cells in the bone marrow prior to CAR-T cell infusion., independent of the type of transplantation and the source of CAR-T cells. The incidence of ICANS was 5.00%. The incidence of aGvHD was 30.00%, and patients who had received prior haploidentical allogeneic HSCT were significantly more likely to develop aGvHD after CAR-T cell infusion than those who had received prior matched allogeneic HSCT. The occurrence of aGvHD after CAR-T treatment was not associated with the occurrence of CRS (p = 1), the presence of aGvHD after prior transplantation (p = 0.3484), the presence of cGvHD after prior transplantation (p = 0.3167), the presence of cGvHD at the time of CAR-T infusion (p = 0.5136), and the presence of prior donor lymphocyte infusion (p = 0.7404), and whether CAR-T cells were from autologous or donor (p = 0.4819). CONCLUSIONS: CAR-T cells are safe and effective in treating B-ALL patients with post-transplant relapse, and donor-derived CAR-T cells do not exacerbate CRS with aGVHD, and there is a high incidence of aGVHD after CAR-T therapy in patients with HLA-haplotype-matched transplantation.

Real World Experience with a Zuma -1 Cohort 4 Adopted Approach to CRS and Icans in CAR-T Recipients

Introduction: Axicabtagene ciloleucel (axi-cel) is a chimeric antigen receptor T-cell (CAR-T) therapy approved for treatment of relapsed/refractory (R/R) large B cell lymphoma (LBCL). The registrational phase I/II ZUMA-1 study Cohorts 1 and 4 addressed toxicity management in terms of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) whereby tocilizumab and corticosteroids were deployed earlier in Cohort 4.(1,2) Although Cohort 4 consisted of only 41 patients, it demonstrated that earlier intervention resulted in significantly fewer cases of grade ≥3 CRS (2%) and ICANS (17%) and a lower cumulative dose of steroids. Furthermore, efficacy as determined by overall response rate (ORR) and complete response (CR) did not appear to be negatively impacted by earlier use of tocilizumab and corticosteroids. However, given the small number of patients in Cohort 4, concern has remained that earlier intervention may have a deleterious impact on CAR-T cell expansion, persistence, and efficacy. During the COVID-19 pandemic, with overwhelmed Intensive Care Units (ICUs) across the country, we adopted a Cohort 4 guided approach to CAR-T toxicity management with the hope of decreasing the need for ICU level of care following CAR-T infusion. Herein, we present our single-institution, real-world experience since implementing an early intervention strategy with Cohort 4 and compare it with our previous Cohort 1 axi-cel recipients in terms of toxicity (CRS, ICANS, ICU utilization) and efficacy. Methods: 119 patients receiving axi-cel CAR-T therapy for R/R LBCL are included in this single-institution, retrospective study with 70 in Cohort 1 and 49 in Cohort 4. The transition from a Cohort 1 to Cohort 4 approach occurred in March 2021. Thus, for Cohort 1 the data cut-off was February 2021 and for Cohort 4 it was after April 2021. The COVID pandemic and the lack of the ICU capacity was the initial chief rationale for transitioning to Cohort 4. This study was conducted following IRB approval and in accordance with the Declaration of Helsinki. R version 1.3.1099 (R-studio) was used for all statistical modeling. All statistical tests were 2-sided and conducted at the 0.05 level of significance. Results: The median age was 62 (22-88) years-old, 35% female gender, 76% advanced stage III-IV disease, 22% with bulky ≥10cm disease, 71% with diagnosis of Diffuse LBCL, and 26% with more than three lines of previous therapy. The median follow-up time at analysis was 1.5 years. Laboratory parameters and comorbidities did not significantly differ between groups except for a higher incidence of renal insufficiency in the Cohort 4 group (28.6% vs 4.3%, p<0.001) (see Table 1). The median maximum grade CRS was significantly lower for Cohort 4 (2 vs 1, p<0.001) although the median grade of ICANS was not different. The utilization of tocilizumab and siltuximab was similar in both groups. The duration of steroid use was comparable in both; however, the total steroid use (dexamethasone equivalent) was notably lower in Cohort 4 (110 mg vs 190 mg, p=0.026). ICU admission following CAR-T infusion was much lower in Cohort 4 (16.3% vs 60%, p<0.001). Most patients (>80%) were discharged home after CAR-T therapy and did not need placement in a rehabilitation center or long-term facility. The ORR and ongoing responses were similar in both groups with nearly 90% achieving a response and 60% with an ongoing response. The cumulative incidence of relapse, PFS, and OS were not significantly different between the groups (see Figure 1). Conclusion: The use of an early toxicity intervention strategy based upon ZUMA-1 Cohort 4 in the real world results in significantly lower high-grade CRS, decreased cumulative steroid dose, and an 73% reduction in ICU utilization without significantly impacting efficacy in terms of incidence of relapse, PFS, or OS. Our real-world experience with the adoption of a Cohort 4 strategy suggests that patient morbidity may be greatly attenuated without compromising CAR-T therapy response. However, the need for longer follow-up to evaluate the impact early use of steroids has on CAR-T persistence and PFS at >5 years remains.

Efficacy and Safety of CD19-Specific CAR-T Cell Therapy Following Immunochemotherapy in Newly Diagnosed B-Cell Lymphoma Associated Hemophagocytic Lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) represents a grave and potentially fatal systemic inflammatory condition. Lymphoma associated hemophagocytic lymphohistiocytosis (LAHS), as a common type in secondary HLH, suffers the worst outcome among sHLH. B cell Lymphoma associated hemophagocytic lymphohistiocytosis (B-LAHS) typically presents as high-risk lymphoma and is associated with a poorer prognosis compared to B cell lymphoma without HLH with standard first-line chemoimmunotherapy. As a result, there is an urgent need for more effective therapeutic strategies. Recent advancements in chimeric antigen receptor (CAR) T-cell therapy have demonstrated remarkable responses in relapsed or refractory B-cell non-Hodgkin lymphoma (B-NHL), as well as in some high-risk newly diagnosed B-cell lymphoma. However, There have been no clinical reports regarding the application of CAR-T cell treatment as part of first-line therapy in B-LAHS. We assessed the effectiveness and safety of autologous anti-CD19 CAR-T cell therapy following an immunochemotherapeutic regimen based on R-DEP (Rituximab-doxorubicin-etoposide-methylprednisolone) as a first-line treatment option for patients with B-LAHS. As of 1 July 2023, a cohort of 13 patients diagnosed with B-LAHS（with 3 cases enrolled before registration for clinical trials）was recruited for evaluation and met the criteria for assessing treatment efficacy at our institution. The overall response rate (ORR) for HLH was 100% (with 7 cases achieving complete response and 6 cases achieving partial response) prior to infuse CAR-T cells and the ORR for lymphoma also was 100% (with 10 cases achieving complete response and 3 cases achieving partial response) on the time of a month after CAR-T cell infusion. As of the follow-up date on 1 July 2023 (with a median follow-up time of 14 months), all 13 patients were still alive, 12 cases maintained sustained remission, while 1 case experienced relapse at 16 months after CAR-T cell infusion. The median overall survival (OS) has not been reached, and the median progression-free survival (PFS) was 31.5 months. To provide comparison, we included a group of 26 patients diagnosed with B-LAHS who did not receive CAR-T cell therapy during the same period. The non-CAR-T group demonstrated a significantly lower median OS of 5.93 months and a median PFS of 4.43 months, highlighting the superior prognosis of the CAR-T group (p < 0.001). Regarding adverse events, the CAR-T group exhibited a cytokine release syndrome (CRS) incidence of 53.8% (7/13) and an immune effector cell-associated neurotoxicity syndrome (ICANS) incidence of 30.7% (4 /13), all of which were classified as grade 1. None of the patients experienced non-hematologic toxicity of grade ≥3. While all 13 patients developed cytopenias, hematologic toxicity reactions ≥ grade 3 were observed in 76.9% (10 /13) of patients, but blood cell counts recovered within 3 weeks. Robust expansion of CAR T-cells occurred in all patients, with a median time to peak expansion of 8 days. Importantly, no exacerbation of hemophagocytic syndrome occurred following CAR-T cell infusion, and no patients succumbed to adverse events. In conclusion, our findings suggest that CD19 CAR-T cell treatment exhibits high efficacy as part of first-line therapy for B-LAHS, with a manageable safety profile.

Low Tmtv Influences Response and Outcomes in R/R DLBCL 3L+ Patients Treated with CAR-T Cells : First Results of FDG-PET/CT Analysis in the French Descar-T Registry

Context : DESCAR-T is a French national registry designed by LYSA/LYSARC including real-life patients receiving commercial CAR T-cells for hematologic malignancies. We initiated a project to conduct a centralized review of Fluorodeoxyglucose Positron Emission Tomography and Computed Tomography (FDG-PET/CT) performed before CAR T-cells infusion, and during follow-up, to evaluate the impact of several metabolic imaging parameters at baseline and response assessment. Materials and methods : Patients with relapsed/refractory (R/R) DLBCL who initiated at least a third line of therapy (3L+) from the DESCAR-T registry and treated with commercial anti-CD19 CAR T-cells, with at least one central reviewed PET before CAR T-cell infusion (PRE-CAR T), at one month (M1) or three months (M3) after infusion, were included. For each visit, Total Metabolic Tumor Volume (TMTV) and SUVmax of the tumor were measured. Deauville score (DS) and response according to 2014 Lugano classification were registered on follow-up FDG-PET/CT. TMTV optimal cut-off for survival prediction (PFS and OS) was determined using X-tile analysis and association of DS with prognosis was studied. Progression-free survival (PFS) was defined as the time between CAR T-cell infusion and progression during follow-up. Overall survival (OS) was defined as the time between CAR T-cell treatment and death during follow-up. Results : Our analysis included FDG-PET/CT data from 212 R/R DLBCL patients (59 treated with tisagenlecleucel and 153 with axicabtagene ciloleucel). Median follow-up was 24.1 months and overall response rate was 47.1% with 112 patients presenting progression or relapse. On PRECAR-T FDG-PET/CT, the median SUVmax was 16.1 (25th-75th percentiles, 8.55-25.30) and median TMTV was 43.91 cm3 (25th-75th percentiles, 12.79-140.43 cm3). Tumor burden significantly affected survival using a TMTV optimal cut-off of 30 cm3. It was associated with a shorter PFS in patients with PRECAR-T TMTV ≥30 cm3 compared with <30 cm3 (median of 3 vs 22.2 months; p=0.0001; respectively) and a shorter OS (median of 9.4 vs 32 months ; p=0.0001; respectively). Regarding metabolic response at M1, reported in 192 patients, DS 1-3 (n=120; 62.5%) was significantly associated with better PFS and OS than DS4-5 (n=72; 37.5%) (for PFS: median of 1.3 vs 21.8 months ; p<0.0001; for OS: median of 4.5 vs 30.6 months; p<0.0001). Of note, considering only DS5 (n=46; 24%) as unequivocally insufficient response onM1 evaluation, identified patients with the poorest outcomes (for PFS: median of 0 month vs 21.2 months ; p<0.0001; for OS: median of 4.5 months vs 31.1 months; p<0.0001). Conclusion : Low PRECAR-T TMTV influences response and retains its predictive and prognostic value in R/R DLBCL patients treated with anti-CD19 CAR T-cells. Moreover, metabolic response assessed with DS is significantly associated with patients' outcome.

BCMA CAR-T-Cell Therapy in Combination with Long-Term Pomalidomide Is a Safe and Effective Treatment for Relapsed/Refractory Multiple Myeloma

Background: B-cell maturation antigen (BCMA)-targeted chimeric antigen receptor T-cell (CAR-T-cell) therapy has exhibited remarkable efficacy in refractory or relapsed multiple myeloma (R/R MM), but recurrence and rapid progression of disease are still observed in some cases within a short time after treatment. Long-term pomalidomide therapy, which potentiates T-cell functionality and has direct antimyeloma effects, might enhance the efficacy of BCMA CAR-T-cell therapy. Here, we present the efficacy of this combination regimen for R/R MM. Methods: We performed a single-center prospective clinical study that was registered with the Chinese Clinical Trial Registration Center (ChiCTR2000036350). Patients with relpased or refractory multiple myeloma receiving BCMA CAR-T cell infusion were randomized to receive long-term pomalidomide treatment (4 mg/day) one month after infusion. The primary endpoint is overall survival (OS) in R/R MM with long-term pomalidomide administration compared to those without pomalidomide therapy after CAR T-cell infusion. The secondary endpoints include time to progression (TTP) and therapeutic response. Also, the copy numbers of CAR were assessed after infusion. Results: A total of 14 R/R MM patients who received BCMA CAT T-cell therapy, were included in this study. The median age of the enrolled R/R MM patients was 59.5 years (44-71). Five patients had high-risk cytogenetic abnormalities (IGH rearrangement, 1q21 amplification, and P53 deletion) at the time of initial diagnosis. In addition, high-risk genetic alterations were newly detected in patient 2 during first-line therapy. All patients had previously received 3 to 6 lines of treatment with proteasome inhibitors (PIs), immunomodulatory drugs (IMiDs), or anti-CD38 monoclonal antibodies. As shown in Figure 1, all patients had demonstrated progressive disease prior to CAR T-cell infusion. The objective response rate (ORR) of BCMA-CART was 100%. Three months following CAR-T-cell infusion, of the 6 patients receiving pomalidomide, all patients (6/6) achieved VGPR (very good partial response) or CR (complete response）, while only 5 patients (5/8) who did not receive pomalidomide treatment achieved VGPR or better. At a median follow-up of 19.7 months, for the 8 patients who did not receive pomalidomide administration, the median TTP (time to progression) was 4.5 (1-14) months, while the TTP was not reached in patients who received long-term pomalidomide treatment. Moreover, the dynamic abundance of circulating BCMA CAR-T cells was monitored as shown in Figure 2. In patients who received pomalidomide treatment after CAR-T-cell therapy, the CAR copy numbers of circulating CAR-T cells peaked 8 to 10 days after CAR-T-cell infusion, with a peak copy number of up to 10 6. In addition, BCMA CAR-T cells persisted (>1*10 3 copies/µg DNA) for 3 months in vivo. Without pomalidomide administration, BCMA CAR-T cells persisted for up to 2.5 months in the peripheral circulation (results not shown). Conclusion: Our results confirm the efficacy of the long-term oral pomalidomide administration (4 mg/day) after BCMA CAR T-cell infusion in patients with R/R MM. Notably, this combination regieme reduced the recurrence rate and prolonged progression-free survival for R/R MM patients, providing a promising option for treating R/R MM. In addition, oral administration of pomalidomide did not significantly prolong the persistence of BCMA CAR T-cells in vivo. However, data from studies with larger sample sizes are required to draw valid conclusions.

Phenotypic, Transcriptomic and Proteomic Characteristics of CAR T-Cell Dysfunction Are Associated with Inferior CAR T-Cell Expansion and Treatment Failure in r/r B-NHL

Introduction: Despite the curative potential of CAR T-cells, a significant number of patients fail to respond or relapse early. Recently, we and others showed that early expansion failure, and particularly CAR T-cell levels at day 7, are strong predictors for treatment resistance (Blumenberg et al, Blood Supplement 2022; Locke et al, Blood Adv 2020). Here we dissected early characteristics of CAR T-cell dysfunction based on immune checkpoint expression, transcriptomic signatures and cytokine profiles and investigated their link to CAR T-cell expansion failure and patient outcome of CD19-targeted CAR T-cell therapy. Methods: Patients with r/r B-NHL who underwent treatment with axi-cel or tisa-cel between January 2019 and November 2021 in the third- or later-line setting at the LMU in Munich were included (n=55). EDTA-anticoagulated peripheral blood and serum was collected. CD19 CAR and immune checkpoint expression (IC; PD-1, TIM-3, LAG-3 and CD224) was assessed by multiparameter flow cytometry on day 14 after CAR T-cell infusion. A next-generation targeted single-cell proteogenomics approach (BD Rhapsody TM) to analyze the transcriptome of distinct CAR T-cell subpopulations on day 7 and day 28 after CAR T-cell infusion. A total of 92 human immuno-oncology related proteins were simultaneously measured using the Olink® Immuno-Oncology panel on day 14 after infusion. Signatures of CAR T-cell dysfunction have been compared between patients with low (loEx) or high (hiEx; < vs ≥ 19 CAR T-cells/µl at day 7 post infusion)CAR T-cell expansion and were put into context of clinical outcome measures. Results: CAR T-cell levels at day 7 were inversely correlated to co-expression of PD-1 (r = -0.4923, p = 0.0011, n = 41), TIM-3 (r = -0.3634, p = 0.0195, n = 41) and LAG-3 (r = -0.5412, p = 0.0003, n = 41) on CAR T-cells at day 14, suggestive of an association of early expansion failure and dysfunction of CAR T-cells. Indeed, loEx (n=26) showed higher frequencies of CAR T-cells coexpressing several IC, including LAG-3 + (p=0.0036), LAG-3 +PD-1 + (p = 0.0048), or LAG-3 +TIM-3 + (p = 0.0065) compared to hiEx (n=15) on day 14 after transfusion. Conversely, we detected higher frequencies of non-dysfunctional CAR T-cell phenotypes negative for several IC, such as CD244 +PD-1 -TIM-3 - (p = 0.0006), CD244 +LAG-3 -PD-1 -TIM-3 - (p = 0.001) and CD244 +LAG-3 +PD-1 -TIM-3 - (p = 0.011) in hiEx. Next, we applied uniform manifold approximation and projection (UMAP) data to examine the surface expression of IC on CAR T-cells at day 14 post-infusion according to treatment response. Unsupervised clustering revealed a higher abundance of CAR T-cell clusters positive for several IC (CD244 +LAG-3 -PD-1 +TIM-3 +) in NR (n = 16) compared to R (n = 23). By contrast, CAR T-cell clusters containing CAR T-cells expressing no IC were more abundant in R compared to NR. In addition, we found significantly increased levels of a highly dysfunctional CAR T-cell phenotype positive for all four IC in NR compared to R at day 14 post infusion (p = 0.0178, n = 46). When specifically comparing the transcriptome of CAR T-cells between NR (n = 6) and R (n = 6), immune inhibitory and exhaustion-related genes were upregulated in NR, such as GZMK, KLRC1 and NR4A2 (figure 1, cluster 1). Conversely, CAR T-cells in R overexpressed genes related to T-cell effector function and cell differentiation, such as TIMP-1, TNFDF10 and MYC (figure 1, cluster 0). Consistent with these findings, a multiplexed proteomics assay for immunomodulatory serum factors in day 14 samples revealed an upregulation of proteins linked to an immune inhibitory and inflamed milieu in patients with highly dysfunctional CAR T-cells (e.g., sPD-L1 and sLAG-3, p = 0.0235 and p = 0.0264, respectively, n = 46) as well as in non-responding patients (e.g., sPD-L1 and IL-6, p = 0.0036 and p = 0.0053, respectively, n = 55). Conclusion: We were able to characterize a phenotypic, transcriptomic and proteomic signature of CAR T-cell dysfunction in post-infusion samples of both patients experiencing CAR T-cell expansion failure as well as lack of radiographic response. Our data show that treatment failure might be predicted not only by CAR T-cell kinetics but also by early assessment of the CAR T-cell phenotype, transcriptome and secretome, and thus enables identification of patients in need of salvage treatment.

Role of Microenvironment in Influencing the Activity of CD19 CAR T Cells in B-Cell Precursor Acute Lymphoblastic Leukemia (B-ALL)

Background: As a therapy based on immune cells, CAR T cells treatment elicits an acute response involving both innate and adaptive immunity which is followed by a process of resolution. A major interest is to identify, within the tumor microenvironment (TME), factors that influence the activity and potency of anti-CD19 CAR T cells. The B-ALL TME provides factors that protect leukemia and, conversely, suppress effector T cells. However, studies on the interaction between the bone marrow (BM) TME and CAR T cells are still limited. We hypothesized that the immunological niche of the BM reacts to CAR T-cell-mediated inflammation by modulating the immune response through the activation of inhibitory pathways and molecules. Methods: We conducted an academic phase I/II study (NCT03389035) with donor-derived anti-CD19 CAR T cells generated with Sleeping Beauty transposon (CARCIK-CD19) in B-ALL patients relapsed after alloHSCT. Hematology laboratory data, CAR T,and CD3+ cell kinetics were analyzed in 43 pediatric and adult B-ALL patients who underwent commercial and investigational anti-CD19 CAR T cell treatment. The scRNA-seq libraries of BM samples were generated using Chromium Single Cell 5′ Reagent Kit of the 10x Genomics (v3.1). Validation was performed in 20 pre-/post- CAR-T matched BM samples by a 30-color flow cytometry panel. Results: Following the peak of expansion, CAR T-cell counts decreased in the third and four-week post-infusion. A similar dynamic of expansion and contraction was observed in CD3+ T cells, total white blood cells (WBC), and monocytes, indicating that a widespread resolution of CAR T-driven inflammation occurs during the first month after infusion. Interestingly, the intensity of the response seems to differ depending on the cell product used. To elucidate the mediators involved in the resolution of CAR T-cell-mediated inflammation, the single-cell transcriptome of patients' BM cells at early time points post-CAR T-cell infusion (1-2 months) was compared to pre-treatment samples at the moment of disease relapse and the infusion products. Eighteen sequencing libraries were analyzed, 6 libraries per patient, for a total of 71407 high-quality cells at an average of 65000 reads per cell. Unbiased clustering of BM cells and infusion products was performed by UMAP embedding. Notably, extensive integration of the dataset coming from the individual patients was observed.The more representative clusters were classified into infusion product, CD4 and CD8 endogenous population, B cells, myeloid cells, pDC, NK, and NK-T cells. We observed profound changes in the composition of BM after CAR T cells infusion compared to pre-treatment samples. Complete disappearance of the B cells associated cluster was observed after treatment. CAR T-cell treatment generated a remodeling of the BM microenvironment, with an increase in the myeloid cells (specifically monocytes), NK, NK-T, and exhausted CD8+ T cell populations. After CAR T-cell infusion, myeloid cells displayed a higher resemblance to myeloid-derived suppressor cells (MDSCs). GeneSet Enrichment Analysis (GSEA) showed significant enrichment in pathways associated with immunosuppression both in the myeloid compartment and in endogenous T cells and in CAR T cells. Of note, the genes involved are also strictly correlated to the generation of terminally exhausted T cells and the emergence of MDSCs. Spectral flow cytometry-based experiments validated our observation of an increase of monocytes, MDSC-like cells, NK, NK-T, and CD8 terminal cells. Modeling intercellular communication using NicheNet suggested the induction of immunosuppressive genes in myeloid cells by both CAR T cells and endogenous T cells. Using the same tool with all niches as the sender and T cells as the receiver, we observed that myeloid cells induced significant signaling in both CAR T cells and endogenous T cells. Conclusions: Through sc-RNAseq and spectral flow cytometry, we have characterized changes in the BM TME after CAR T cell therapy. Specifically, CAR T cells-mediated myeloid activation is associated with pathways of immune dysregulation that may dampen CAR T cell expansion and antagonize the effects of the therapy. Furthermore, these data support the hypothesis that transcriptomic interrogation can infer pathways relevant to the communication between CAR T cells and the TME.

Early Autologous Hematopoietic Stem Cells (AHSC) Boost (HCB) to Overcome Immune Effector Cell-Associated Hematotoxicity (ICAHT) after CAR T-Cell Therapy for Lymphoma: A Single Center Experience

Sustained cytopenias designed as Immune Effector Cell-Associated Hematotoxicity (ICAHT) represent one of the most frequent side effect of CAR-T-cell therapy (30 to 90 % patients in recent real life studies) resulting in significant morbidity, mortality and resources consumption. We report single center experience of early reinjection of previously cryopreserved AHSC (HCB) in that setting. From July 2021 until January 2023, 27 patients received CAR T-cell therapy (25 Axi-Cel, 2 Brexu-Cel) after Fludarabine and Cyclophosphamide lymphodepletion for relapsed/refractory large B-cell lymphomas (24), mantle lymphoma (2), or follicular lymphoma (1). Median age was 62 years (19-75). CAR-HEMATOTOX score was ≥ 2 for 46% of patients. All patients received Filgrastim from D0. Hematopoietic recovery was longitudinally evaluated until D90 or censoring for relapse and/or subsequent cytotoxic treatment: severe (CTAE grade ≥ 3) neutropenia, anemia or thrombopenia occurred respectively for 88, 44 and 76% of patients until D28, and 60, 24 and 60% until D90. Median duration of severe neutropenia was 7 days (0-39) and 28 days for severe thrombocytopenia (0-118). Prolonged neutropenia occurred in 18/25 evaluable patients, mostly with a biphasic course (intermittent recovery and aplastic phenotype for 14 and 4 patients respectively). CAR-HEMATOTOX score was significantly correlated with sustained thrombopenia (p = 0,005) but only marginally with ICAHT (p = 0,054) and not with severe neutropenia (p = 0,44) . We observed significant correlation of severe ICAHT with progressive disease at CAR T-cell infusion (p = 0,015), platelets nadir < 30 giga/l before D28 (p < 0,005) and CAR T-cell expansion (> 200/µl) at D7 (p = 0,023). 7 patients received HCB (no conditioning) at a median D24 (15-41) post CAR T-cell infusion: after D14 for patients with aplastic phenotype, or for cytopenic recurrence for patients with biphasic course. Filgrastim was pursued or reintroduced until neutrophil recovery (> 1 giga/l) which occurred after a median of 10 days (4-16); platelet recovery (> 20 giga/l and > 50 giga/l) occurred after a median of respectively 12 (8-14) and 17 (11-22) days. If we consider D0 as CAR T-cell infusion, neutrophil recovery occurred at median D37 (22-49) and platelet recovery (> 50 giga/l) at median D37 (26-63). Median complete hematopoietic reconstitution (defined as PMNC > 1 giga/l, and Hb > 100 g/l, and PLT > 100 giga/l) was significantly shorter (median 23 days, 14-30) for patients receiving HCB compared with patients with prolonged cytopenia (> 28 days) who do not receive HCB (60 days, p = 0,018). Transfusion and hematopoietic growth factors (HGF) consumption was significantly reduced after HCB but we observe no difference between 2 groups considering hospitalization, EFS or OS. 2 patients receiving HCB experienced late recurrence of cytopenia (after D90) requiring intermittent HGF support but no transfusion or hospitalization. Discussion - Perspectives Recent case series highlighted clinical feasibility and safety and of this approach across a broad population of pediatric and adult patients: we validate this strategy in a more homogeneous and systematic practice. As study population is small, we failed to demonstrate that earlier application of an HCB is associated with superior survival outcomes as shown by others. We agree with recent updated recommendation of experts panel considering application of HCB without prior conditioning chemotherapy for grade ≥ 3 ICAHT beyond D14 (so considered early) if AHSC graft is readily available. Prophylactic AHSC harvest in high-risk candidates has been proposed but collection process may add to already high logistic burden and costs of CAR T-cell therapy in the lymphoma setting. Conversely, cryopreserved ASHC is mainly available for patients with myeloma and this approach would be readily proposed and useful for around 20% patients as recently described. Gagelmann N et al. Hematopoietic stem cell boost for persistent neutropenia after CAR T-cell therapy: a GLA/DRST study. Blood Adv. 2023;7(4):555 Rejeski K et al, Immune Effector Cell-Associated Hematotoxicity (ICAHT): EHA/EBMT Consensus Grading and Best Practice Recommendations. Blood. 2023 doi: 10.1182/blood.2023020578 Davis JA et al. Efficacy and safety of CD34+ Stem Cell Boost for Delayed hematopoietic Recovery After BCMA Directed CAR T-cell Therapy. Transplant Cell Ther. 2023 doi: 10.1016/j.jtct.2023.05.012

Myeloid Derived Suppressor Cell Kinetics in Peripheral Blood Correlate with Efficacy and Toxicity of CAR T Cell Therapy in Relapsed or Refractory B Cell Lymphoma

Introduction. CAR T-cell therapy is potentially curative for patients with relapsed or refractory B-cell lymphoma (BCL). However, about 60% of patients fail CAR T-cell therapy. Immune-related adverse events (irAE) like cytokine release syndrome (CRS) and immune effector cell associated neurotoxicity syndrome (ICANS) occur in an inflammatory state. Myeloid-derived suppressor cells (MDSCs) expand during acute inflammation as steady-state hematopoiesis switches to emergency myelopoiesis. We hypothesized that the acute inflammatory conditions created by CAR T-cell therapy may alter MDSC kinetics and investigated MDSC expansion in the context of CAR T-cell efficacy and toxicity in BCL. Methods. Patients with BCL receiving standard-of-care CAR T-cell therapy were enrolled in a prospective sample collection study. Blood samples were collected before lymphodepletion (baseline), prior to and at early intervals after CAR T-cell infusion. Viable white blood cell (WBC) and MDSC counts were evaluated by multicolor flow cytometry. Phenotypically, MDSCs were defined as immature myeloid cells (CD11b + CD33 + HLA-DR -/low IL-4R +) and divided into monocytic (M-MDSC, CD14 +) and polymorphonuclear (PMN-MDSC, CD15 +) subsets. Clinical response was assessed by Lugano criteria at 1, 3 and 6 months. Incidence and severity of CRS and ICANS were recorded. Cell counts were analyzed by descriptive statistics and reported as mean ± standard error or fold-change from baseline. Kinetics of MDSCs in blood and correlation with clinical outcomes were evaluated by Wilcoxon matched pair signed rank t-test and Mann Whitney U-test. Results. Forty-three BCL patients (41 large BCL, 2 mantle cell lymphoma) were enrolled in the study. Median age was 64 (range 27-83), 32.6% were female. Racial and ethnic minority groups represented 14% of enrolled subjects. One patient developed grade 5 ICANS and was not evaluable for response. Thirty-seven (86%) developed leukopenia and all received granulocyte colony stimulating factor (G-CSF) starting on day 5 post-CAR T infusion. WBC count fell sharply from baseline following lymphodepletion (7.7E +05±8.6E +04 baseline vs 2.9E +05±9.2E +04/mL, p<0.0001) and continued to decline 2-3 days post-CAR T infusion (baseline vs 1.4E +05±2.1E +04/mL, p<0.0001) before returning to baseline within 1 week post-CAR T (i.e. within 3 days of G-CSF). MDSCs followed similar kinetics early on but significantly increased from baseline levels by 1-week post-CAR T (3.4E +03±5.8E +02 baseline vs 8.5E +03±1.7E +02 /mL, p<0.0001). This effect was mainly driven by an exuberant expansion of M-MDSC observed in 33/43 patients (median 6.1-fold increase from baseline at 1 week, range 0.5-4301). Seven patients (16.3%) had progressive disease (PD) at 1 month, 6 (13.9%) additional patients progressed at 3 months and 4 (9.3%) more progressed at 6 months after CAR T-cell infusion. M-MDSC expansion 1-week post-CAR T was only observed in 3/7 refractory patients compared with 28/35 responding patients (Fisher's exact test, p=0.0635). Patients who progressed at 3 months had poorer M-MDSC retention compared to patients with sustained response (1.7E +02±6.1E +01 PD vs 1.4E +03±2.4E +01/mL at 2-week post-infusion, p<0.1). Similarly, patients who progressed at 6 months had lower M-MDSC compared with sustained responders (2.0E +02±8.1E +01 PD vs 4.5E +02±1.4E +02 ml at 1-month post-infusion, p<0.05). Thirteen patients (30.2%) developed > grade 2 CRS and had lower PMN-MDSC retention rate 2-3 days post-CAR-T compared with those with no/low-grade CRS (4.4E +02±2.6E +02 vs 1.4E +03±3.9E +02/mL, p<0.05). Similarly, patients who had ICANS > grade 2 (n=14, 32.5%) had lower PMN-MDSC early retention compared to those with no or low-grade ICANS (5.0E +02±2.5E +02 vs 1.5E +03±4.1E +02/mL, p<0.05). Further analyses are currently underway to bridge these findings with the kinetics of CAR T-cells and T-cell repertoire post-CAR T. Conclusions. The proinflammatory milieu after CAR-T and the use of G-CSF can lead to significant expansion of M-MDSCs. Rather than an impediment to CAR T therapy, M-MDSC expansion and retention are associated with favorable clinical response and PMN-MDSC with a lower risk of developing high grades CRS or ICANS. Whether MDSCs play a mechanistic role remains to be elucidated, but they may have a role as a biomarker in predicting outcomes after CAR T-cell therapy in BCL.

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.

Characteristics of Central Nervous System Relapse after CD19 CAR-T Cell Therapy in R/R NHL

Background CD19-targeted chimeric antigen receptor T (CAR-T) cell therapy has shown remarkable antitumor activity against relapsed/refractory (R/R) non-Hodgkin lymphoma (NHL). However, NHL with central nervous system (CNS) relapse carries a poor prognosis and high mortality. We reported an analysis of 80 patients with NHL who underwent CAR-T cell therapy, and described the characteristics including the international prognostic index (IPI) and other clinical variables to evaluate the outcomes of CNS relapse in NHL patients treated with CAR-T cell therapy. Methods A total of 80 patients with NHL treated with CD19 CAR-T cell therapy in our center between April 2017 and November 2022 was retrospectively analyzed. Patient characteristics, laboratory parameters, and clinical outcomes were collected. CNS relapse was diagnosed by cerebrospinal fluid cytology, MRI or PET-CT. Results Baseline characteristics of all 80 patients are summarized in Table 1. The median age was 58.5 (range, 10 to 79) years, and 57.5% of patients were male. Overall, 67 (83.8%) patients achieved complete remission (CR) or partial remission (PR) after CD19 CAR-T therapy. Seven (8.43%) patients developed isolated CNS relapse after CAR-T cell therapy, all of which were heavily pre-treated with multiple lines of chemotherapy prior to CAR-T cell therapy, and one of them had received autologous stem cell transplant before infusion of CAR-T cells. Only 4 of 7 patients with isolated CNS relapse achieved CR (n=4), and the median time to isolated CNS relapse from CAR-T cell infusion was 6.3(range, 1.53 to 25.8) months. Thirty-seven patients (46.2%) developed systemic recurrence without CNS involvement, with the median time from CAR-T to relapse of 3.2 (range, 0.467-30.8) months. The other 36 patients remained in remission at the last follow-up. During the median follow-up of 13.5 months of the whole cohort, the number of recorded deaths in isolated CNS relapse, systemic only recurrence, and no relapse patients was 5 (71.4%), 22 (59.4%), 1(2.78%), respectively. The 1-year OS and 1-year PFS of patients with isolated CNS recurrence was significantly lower compared to the no relapse group (OS, 35.7% [95%CI 12.1-100] vs 97.22% [95%CI 92-100], P<0.001; PFS, 28.6% [95%CI 8.86-92.2] vs 100% [95%CI 100-100], P<0.001), but showed no difference compared to the systemic only recurrence group(OS, 53.3% [95%CI 39.3-72.4], P=0.79; PFS, 13.0% [95%CI 5.53-30.5], P=0.17). Conclusion In conclusion, patients with CNS relapse showed significant difference in 1-year outcomes with no relapse group but not systemic relapse group in our study, which suggested the importance of effective prophylaxis against CNS relapse. Further investigation is necessary to better define the characteristics of patients who will benefit from prophylaxis. Acknowledgements We gratefully thank all the participation from patients enrolled in clinical trials (ChiCTR1800015575; NCT03118180; NCT04532281; NCT04532268; NCT04213469).

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.

Invasive Fungal Infections after CAR T-Cell Therapy for B-Cell Lymphoma: A Study from the French Descart Registry

Introduction: Chimeric antigen receptor (CAR) T cell therapy has emerged as an important treatment option for patients with B-cell malignancies. Despite its major clinical impact, CAR T cell therapy holds a potential risk of increased susceptibility to infections, including invasive fungal infections (IFI). Since data about the prevalence and impact of fungal infections in this population are limited, we aimed to provide a comprehensive overview of IFI using real-world data from the national DESCAR-T registry. Methods: We performed a multi-center retrospective cohort study, quering the French DESCAR-T registry (NCT04328298), to describe type, frequency and outcomes of IFI in adults with refractory or relapsed (R/R) B cell lymphomas. In addition, healthcare providers were contacted to declare any missing cases. Demographic, biological and clinical characteristics were collected retrospectively. Each IFI case was reviewed by an infectious disease specialist and a microbiologist, and classified according to the European Organization for Research and Treatment of Cancer (EORTC) criteria (Donnelly et al. Clinical Infect Dis 2020). Results: Among the 1144 patients included in the registry from 2018 to 2022, we identified 57 suspected IFI cases. After exclusion of 25 patients not adjudicated for IFI, 32 patients (3%) were identified as having presented a proven (n = 14), probable (n = 12), or possible (n = 6) IFI. DLBCL was the primary diagnosis for 30 patients and axi-cel was the most frequent CAR T product (23/32, 72%). Seven patients (22%) had received a previous hematopoietic stem cell transplantation. All patients experienced cytokine release syndrome (including 8 grade ≥ 3) and 21 (66%) experienced immune effector cell-associated syndrome (including 13 grade ≥ 3). Overall, 27 (84%), 21 (66%), and 5 (16%) received tocilizumab (median 960mg, range 120-2880mg), corticosteroids, and anakinra, respectively. Among known risk factors for IFI, grade 4 neutropenia and late neutropenia (< 1g/L) at day 28 after infusion were identified in 25 (78%) and 14 (44%) patients respectively. Overall, 8 (25%) patients received antifungal prophylaxis, posaconazole was used for 4 of them. Over half of the IFI cases occurred between 0 and 30 days after CAR T-cell infusion (18/32, 56%), with a median onset time of 29.5 days (IQR 16.5 - 79.5). The most frequently observed infections were invasive Aspergillosis (n = 13), with pulmonary involvement in 10/13 (77%) of patients, leading to a fatal issue in 3/13 patients (23%). Other mold infections included Mucormycosis (n = 5), with pulmonary involvement in 4/5 patients and a fatal outcome in 1/5, as well as Fusariosis (n = 1). Yeast infections (n = 12) were mostly identified as Candidemia (92%) with a mortality rate of 3/12 (27%). The median overall survival of patients with IFI was 186.5 days (IQR 79-506), 7 deaths being related to IFI and 3 to lymphoma relapse. Median progression-free survival was 94 days (IQR 31 - 252). Conclusions: This study represents one of the largest investigations about IFI in B-cell lymphoma patients treated with CAR T-cells. Even though IFI represent a rare complication after CAR T-cell therapy, the related-mortality is high. Selective antifungal prophylaxis may benefit high-risk patients, while rapid preemptive diagnostic workup, particularly for lung involvement, is mandatory. Analysis to define potential risk factors for IFI are ongoing.

The Post-CAR Prognostic Index (PC-PI): A New Prognostic Score to Predict Overall Survival in Large B-Cell Lymphoma Patients Progressing after Chimeric Antigen Receptor T-Cell Therapy

Introduction: Chimeric antigen receptor (CAR) T-cell therapy fails to achieve durable responses in 60% of relapsed/refractory (R/R) large B-cell lymphoma (LBCL) patients in the third or later line setting. There is no standard treatment after CAR T-cell therapy progression and a wide range of outcomes are observed in this patient population. Besides the interval between CAR T-cell infusion and progressive disease (PD), data regarding prognostic factors at time of progression are scarce. Our aim was to develop a new prognostic tool to predict overall survival (OS) after CAR T-cell therapy progression with easily-available markers from routine clinical practice. Methods: First, we performed a retrospective data collection at 12 Spanish centers of R/R LBCL patients who progressed after CAR T-cell therapy in the third or later line setting from September 2018 until June 2022 (training cohort, TC). We analyzed a total of 15 variables, including pre-CAR T-cell therapy characteristics (gender, histology, primary refractory disease, previous hematopoietic transplant, number of previous lines) and values collected at time of progression to CAR T-cells (age, stage, extranodal sites, ECOG, hemoglobin, neutrophils, platelets, LDH, best response to CAR T-cells, time from CAR T-cell infusion to PD). The primary endpoint was OS from date of progression to CAR T-cell therapy. A stratified Cox model was used to estimate hazard ratios (HRs) using post-progression treatment as a stratification factor. We used LASSO regression with minimum lambda to identify which variables had the highest prognostic impact on OS. Additionally, the factors with a lower contribution were eliminated to create a parsimonious model. The C-statistic was used to evaluate its discrimination. We examined the performance of the International Prognostic Index (IPI) score and Revised IPI (R-IPI) in this setting. Finally, we tested the score in an external validation cohort (VC) which included a comparable patient population from 3 European countries. Results: Among the 216 LBCL patients included in the TC, most were male (66%), had an ECOG of 1 (48%) and stage IV disease (71%) at time of CAR T-cell therapy progression. Median time from CAR T-cell infusion to PD was 2.5 months (95CI% 1.9-2.9) and median follow-up from progression was 15 months. Salvage treatment was classified into 3 subgroups, including immunotherapy or targeted agents (43%), chemotherapy or radiotherapy (20%) and palliative care (38%). To build the prognostic score, a total of 5 variables were selected. Each marker received 1 point (given the similar HR [1.48-1.77]), if they met defined criteria: ECOG (>0), hemoglobin (<10 g/dL), LDH (>2 x upper normal limit), number of extranodal sites (>1) and time from CAR T-cell infusion to PD (<4 months). Patients with 0-1 points were classified as low risk, 2 points as intermediate-low risk, 3 points as intermediate-high risk and 4 or 5 points as high risk. In the TC, the 4 risk groups showed statistically significant differences in OS (Figure 1). In the low-risk group (n=39 [18%]), the median OS [mOS] was not reached; in the intermediate-low risk (n=56 [26%]) mOS was 7.3 months (HR=2.89, p=0.002); in the intermediate-high risk (n=57 [26%]) mOS was 4.9 months (HR=4.81, p<0.001) and in the high risk (n=64 [30%]) mOS was 1.8 months (HR=6.69, p<0.001). In terms of post-relapse therapies, both the chemo/radiotherapy and the immunotherapy groups showed a balanced patient distribution, from low to high risk (32%, 35%, 14% and 19% vs 22%, 32%, 30% and 16%, respectively). The VC included 204 patients with a similar patient distribution in the 4 prognostic risk groups (35%, 25%, 12%, 27%). The mOS for each of these groups was 15.2, 5.3, 2.9 and 0.9 months, respectively. Each group had distinct OS outcomes when compared with all the other risk groups (p<0.05 for each comparison) (Figure 2). Finally, our model presented a C-index of 0.712 for the TC and 0.811 for the VC, outperforming both the IPI (0.647 [TC] and 0.691 [VC]) and R-IPI (0.632 [TC] and 0.683 [VC]). Conclusions: The Post-CAR Prognostic Index (PC-PI) is a clinically useful tool for OS prediction and risk-adapted treatment planning in LBCL patients progressing after CAR T-cell therapy. In addition, our results will help stratification in clinical trials which include patients with prior CAR T-cell therapy.

Efficacy and Safety of the Second CAR-T Therapy in Patients with Refractory/Relapsed Acute B-Cell Lymphoblastic Leukemia

Background: Chimeric antigen receptor T (CAR-T) cells therapy has shown significant efficacy in relapsed or refractory B-cell acute lymphoblastic leukemia (R/R B-ALL), but a substantial proportion of patients still relapse after a period of remission. The efficacy of a second CAR-T cell infusion is uncertain. The aim of this study was to explore the efficacy and safety of the second CAR-T therapy in R/R B-ALL patients. Methods: Between August 2018 and October 2022, 35 patients with R/R B-ALL successfully received the second CAR-T treatment. Among them, 18 patients received single CD19 or CD22 CAR-T therapy, 14 patients received tandem CD19/CD22 CAR-T therapy, and 3 patients received sequential CD19 and CD22 CAR-T therapy. Results: The complete remission (CR) rates were 55.6% (10/18) in patients who received CD19 or CD22 CAR-T therapy, 85.7% (12/14) in patients who received tandem CD19/CD22 CAR-T therapy, and 33.3% (1/3) in patients who received sequential CD19 and CD22 CAR-T therapy (tandem CD19/CD22 vs. others, P=0.045). Tandem CD19/CD22 CAR-T therapy remained one of the significant favorable factors in multivariate logistic regression analysis of CR rate in all patients (hazard ratio, 0.128; 95% CI, 0.017-0.974). A total of 5 patients with severe cytokine release syndrome (Grade ≥ 3) were observed, including 2 patients in the single CD19 CAR-T group, 2 patients in the tandem CD19/CD22 CAR-T group, and 1 patient in the sequential CD19 and CD22 CAR-T group. Of the 23 patients who achieved CR, 4 patients bridged to allogeneic hematopoietic stem cell transplantation (allo-HSCT) and 3 patients received decitabine (DAC) consolidation.With the median follow-up of 25.5 months (range, 1.1 to 33.5), the 2-year overall survival, leukemia-free survival (LFS) and cumulative incidence of relapse rates were 23.7%, 30.5% and 69.5%, respectively. Multivariate Cox regression analyses showed that a better LFS related to the absence of high-risk cytogenetics and genetic characteristics, DAC combination with fludarabine and cyclophosphamide lymphodepletion regimen, and bridging allo-HSCT or DAC consolidation. Conclusions: Our study showed that the second infusion of tandem CD19/CD22 CAR-T therapy obtains a better response than other infusion strategies. Bridging allo-HSCT or DAC consolidation had a significant survival benefit in patients with CR after the second CAR-T therapy.

Chimeric Antigen Receptor T-Cell Therapy in Elderly Patients with Relapsed or Refractory Large B-Cell Lymphoma: A Multicenter Study

Background: Anti-CD19 chimeric antigen receptor T-cell (CART) therapy is a standard of care in patients (pts) with relapsed or refractory (RR) large B-cell lymphoma (LBCL). However, it remains underutilized in elderly pts due to limited efficacy and safety data. We therefore evaluated real world outcomes of CART in such pts. Methods: Seven academic centers participated in this retrospective study. Pts aged ≥65 years with RR LBCL who received a commercial CART product were included. Baseline characteristics, treatment data, CART toxicity, and clinical outcomes were collected. Progression-free survival (PFS; time from infusion to disease progression or death) and overall survival (OS; time from infusion to death from any cause) were analyzed using Kaplan-Meier method. Cumulative incidences of non-relapse mortality (NRM) and disease relapse/progression were estimated accounting for competing risks. Statistical analyses were performed in JMP v15 and XLSTAT v2022.1.2. Results: 209 pts were identified. Baseline characteristics and treatment pattern are shown in Table. Median age at infusion was 71 years (range 65-89), and 25 (12%) were ≥80 years. 131 (63%) were male, 188 (90%) were white, and 27 (13%) had ECOG PS of ≥2. The median HCT-CI was 1 (range 0-6), and 39 (22%) pts had a score of ≥3. 165 (84%) had stage III-IV disease, 131 (70%) had extranodal involvement, 24 (18%) had bulky disease, and 24 (11%) had high grade B-cell lymphoma (HGBL; double- or triple-hit lymphoma, n=15; HGBL, NOS, n=9). The median number of prior lines of therapy was 2 (range 1-8), and 99 (48%) pts had ≥3 prior lines of therapy, 45 (22%) had prior bendamustine, and 44 (21%) had prior stem cell transplant (SCT; auto-SCT, n=41; allo-SCT, n=2; both auto- and allo-SCT, n=1). Bridging therapy was given to 100 (52%) pts; among those, 57 had chemotherapy, 20 had radiotherapy, 6 had both chemotherapy and radiotherapy, 10 had lenalidomide-based regimen, and 7 had CD20 antibody and/or corticosteroids. Axicabtagene ciloleucel (axi-cel) was most frequently used (63%, n=131), followed by lisocabtagene maraleucel (liso-cel) (24%, n=49) and tisagenlecleucel (tisa-cel) (13%, n=27). Cytokine release syndrome (CRS) occurred in 75% of pts, and a minority (6%) had grade ≥3. Immune effector cell associated neurotoxicity syndrome (ICANS) occurred in 55%, and 29% had grade ≥3. The management of CRS and ICANS included corticosteroids in 101 (49%) pts, tocilizumab in 116 (56%) pts, and other biologic agents (anakinra, siltuximab, basiliximab) in 8 (4%) pts. Day 30 response was evaluable in 195 pts with an objective response rate (ORR) of 84%, and a complete response rate (CR) of 52%. 82% of the responders continued to have a response at 3 month follow up. The ORR/CR rates were 86%/53% for axi-cel, 64%/44% for tisa-cel, and 89%/52% for liso-cel, respectively. At a median follow up of 19.1 months (95% CI 16.1-23.6) after infusion, the median PFS was 6.7 months (95% CI 5.1-11.5), with 6- and 12-month PFS rates of 54% (95% CI 47-61) and 43% (95% CI 35-50), respectively. The median OS was 16.5 months (95% CI 13.2-26.7), and the 6-and 12-month OS rates were 70% (95% CI 64-77) and 61% (95% CI 54-68), respectively. The NRM rate was 17% (95% CI 11-24) at 1 year, primarily due to infections including 5 COVID-19 related deaths. In univariate analysis, ECOG PS ≥2, bulky disease, elevated LDH, and the need for bridging therapy were associated with inferior PFS and OS. No significant difference in PFS and OS was seen based on age at infusion with a median PFS of 6.4 vs 9.6 vs 6.4 vs 6.0 months ( P=0.93) (Figure) and OS of 16.4 vs 19.2 vs 28.5 vs 12.7 months ( P=0.85) in age groups ≥65-69 vs ≥70-74 vs ≥75-79 vs ≥80-89. In addition, there was no statistically significant difference in PFS and OS based on type of CART with a median PFS of 6.4 vs 5.6 vs 12.7 months ( P=0.16) and OS of 15.1 vs vs 16.1 vs NR months ( P=0.24) in pts treated with axi-cel vs tisa-cel vs liso-cel. Conclusions: In this large multicenter study of elderly pts with RR LBCL, CART therapy demonstrated favorable efficacy and safety profile comparable to that in pivotal registration trials. Survival outcomes appeared similar regardless of patient age, including those aged ≥80 years, and type of CART product used. Similar to younger pts, high tumor burden and poor performance status at CART infusion were associated with inferior survival. This analysis confirms that older pts should not be excluded from receiving CART therapy solely due to chronological age.

Phase I Study of a CD19-Directed CAR-T Cell Therapy for Relapsed/Refractory Mantle Cell Lymphomas(MCL)

Background: IM19 is an autologous, CD19-directed, chimeric antigen receptor (CAR) T-cell product, which is manufactured in China using a commercial process. We aimed to evaluate the efficacy and safety of IM19 in patients with relapsed or refractory mantle cell lymphomas in a Phase I clinical study. Methods: A phase 1 clinical trial has been launched to evaluate the safety and efficacy of IM19 for the treatment of relapsed/refractory MCL. We enrolled adult patients (aged ≥18 years) with relapsed or refractory MCL who had received at least 2 prior treatment regimens(including BTK inhibitor). Patients were assigned to one of two target dose levels of IM19 as they were sequentially tested in the trial (100×10⁶ CAR+ T cells [one or two doses], 200×10⁶ CAR+ T cells [one or two doses]). Leukapheresed patients could receive bridging chemotherapy based on investigator assessment of disease burden and tumor progression risk. Lymphodepleting (LD) chemotherapy was administered over 3 days, including fludarabine (30 mg/m2 i.v. daily) and cyclophosphamide (300 mg/m 2 i.v. daily), followed 2 days later by intravenous infusion of IM19 at the assigned dose. The first three subjects in each dose group will receive the second LD chemotherapy and infusion of IM19 CAR-T cells for consolidation treatment (with the same dosage as the first treatment) if the efficacy evaluation after the first infusion shows no progression and no DLT occurs. The investigators can determine the start time of consolidation treatment based on the patient's tolerance, but no later than 60 days after the first infusion. Efficacy was assessed at 1, 3, 9, 12, 18, and 24 months post first infusion. Safety events were monitored from beginning of LD through 2 years follow-up. The primary endpoints were adverse events, dose-limiting toxicities, and the objective response rate. Results: Between Mar 3, 2022, and May 5, 2023, Five patients underwent leukapheresis for manufacture of CAR+ T cells (IM19) and all received bridging chemotherapy during the production of IM19. ALL 5 patients received LD and first infusion of IM19 at a dose of 100*10^6 CAR+ cells and no DLTs were observed. CRS was reported only in 1 patient(grade 1) and no patients developed ICANS. ALL 5 patients achieved CR on 28 days after first infusion. The median maximum CAR-T cells expansion (Cmax) was 156×10⁶ CAR+ cells/L, and median area under the curve from 0-28 days post-first infusion (AUC0-28d) was 860 day×CAR+ cells/L. 2 patients received the second LD chemotherapy and infusion of IM19 CAR-T cells for consolidation treatment (with the same dosage as the first treatment) at 44th days post first infusion. No DLTs，CRS or ICANS were observed after second infusion. Meanwhile, CAR-T cells can not be detected at 1, 4, 7, 10, 14, 21 and 28 days post second infusion by Flow cytometry in 2 patients. One patient maintained complete remission after 9 months of follow-up and another patient experience PD at 7th month after first infusion. For three patients who received single infusion of IM19, one maintained complete remission after 6 months of follow-up，one progressed at 3th month after infusion and one are still waiting for second efficacy evaluation. Conclusions: Initial data from this Phase I study demonstrate that use of IM19 resulted in a high objective response rate, with a low incidence of grade 3 or worse cytokine release syndrome and neurological events in patients with relapsed or refractory MCL. We observed that CAR-T cells cannot expand in vivo after secondary infusion. Therefore, secondary infusion may not be necessary.

Early Progressions of Undetermined Significance (EPUS) in the Month Following CAR-T Cells for B-Cell NHL Is Mainly Related to Pseudoprogression

INTRODUCTION - Relapsed-refractory B-cell non-Hodgkin's lymphomas (R/R-B-NHL) treatment remains challenging. In this context, CAR T-cell therapy has shown sustained efficacy in 40-60% of patients. Early progressions and pseudo-progression have been described after CAR T-cell therapy, but data are scarce. This study aims to assess incidence, risk factors and outcomes of early progression in patients treated with CAR T-cells for R/R B-NHL. PATIENTS & METHODS - All patients were consecutively treated at the University Hospital of Montpellier with a commercial CAR T-cells for R/R-B-NHL between December 2019 and June 2022 and included in the CARTBANK cohort (ID RCB: 2019-A03032-55, NCT04290000). Cases were retrospectively reviewed for early progression. No antitumor treatment was initiated before the day 28 evaluation. Early Progressions of Undetermined Significance (EPUS) were defined as either: self-reported symptom of lymphoma (possible EPUS), clinically assessed progression (probable), or measured through imaging procedure (highly probable) after CAR T-cell infusion and before day 28 (D28) post evaluation. Pseudo-progression was defined as EPUS with partial or complete response on D28 evaluation. On the contrary, hyper-progression was defined as EPUS with confirmed progression on D28 evaluation. RESULTS - A total of 105 patients were included in the study, with a median age of 65 years (interquartile range: 55;70]) and 70% were male. Diffuse large B-cell lymphoma (DLBCL) was the most common histology (89%), followed by follicular lymphoma (6%) and mantle cell lymphoma (5%). Patients had received a median of 2 previous lines of anti-cancer therapy [2;3]. Axicabtagene-ciloleucel was used in 77% of the cases, tisagenlecleucel in 18%, and brexucabtagene-autoleucel in 5%. Overall 18 patients (17%) had EPUS: 2 possible EPUS (11%), 6 (33%) probable EPUS and 10 (56%) highly probable EPUS (panel A). D28 evaluation revealed that most of EPUS were pseudo-progressions (n=13/18, 72%). Hyper-progression was observed in one patient (6%) and 4 patients (22%) died before first metabolic assessment and remained unclassified (panel B). The cause of death was suspected of progression for three of them and sepsis for one. All EPUS occurred before day 21 (median: 7 days [3;8]). Comparison of patients with and without EPUS showed that no clear risk factors for EPUS were identified. EPUS occurrence had no impact on PFS nor OS. The blood expansion of CAR-T cells and their level of exhaustion were monitored by multi-parametric flow cytometry. Regarding these pharmacokinetic parameters, no significant difference was obtained between patients with and without EPUS. Analysis of data regarding total metabolic volume is ongoing and will be presented. DISCUSSION - In this cohort, EPUS is observed in 17% of patients after CAR T-cells infusion for R/R B-NHL. Most of EPUS are related to pseudo-progression. These results should be confirmed in a dedicated study. Thus, early treatment before first evaluation should be avoided.

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.