P1382: circulating car-t cells monitoring of kinetics and exhaustion markers as predictive factors in b-cell malignancies

Topic: 24. Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research Background: CAR-T cell therapy has proven to be a disruptive treatment in the hematology field. Tisagenlecleucel and Axicabtagene ciloleucel showed impressive efficacy in relapsed/refractory B-cell lymphoma; however, less than 50% of patients maintain a long-term response and predictors of outcome are inconsistently defined. Tisa-cel and Axi-cel kinetics have been poorly investigated in real life settings so it becomes necessary to evaluate biomarkers that predict response or toxicity. Real life evaluations assessing the immunophenotypic composition of CAR-T cell infusion products and analyzing how these specific features affect CAR-T cell expansion and persistence in vivo and thus outcome, are scarce. Aims: Here we aimed to optimize the detection of CD19 CAR-T cells in blood and to identify early biomarkers that could predict patient outcomes. Methods: CD19 CAR-T cells were monitored by flow cytometry and digital PCR from peripheral blood in 48 patients with diffuse large B-cell lymphoma, transformed follicular lymphoma, primary mediastinal B cell lymphoma or B-cell acute lymphoblastic leukemia. Flow cytometry and digital PCR approaches were performed for determining cell kinetics (n=48) at days 5, 7, 11, 14, and 20 (±1) after CAR-T cells infusion. In parallel, immunophenotype characterization of CAR- T lymphocytes was conducted at the day of CAR-T expansion in blood. Results: After testing different commercially available antibodies for the detection of CD19 CAR-T cells, CD19 protein conjugated with biotin and identified by streptavidin showed the highest resolution in contrast to Antigen CD19 labeled FITC (stain index of 22.19 vs 4.64, p=0.0286). The median day of peak expansion of CAR-T cells was 7 days (range of 5-14 days) after infusion, reaching a median of 119 CAR+ cells/uL (range of 2.4.-1019 CAR+ cells/uL) in blood. Kinetics of CD19 CAR-T cell expansion in blood was also performed by digital PCR with similar results (peak expansion at day 7 post-infusion). Besides, correlation was observed between peak CAR-T cell expansion in blood by flow cytometry and digital PCR and more severe CRS (p=0.0261 and p=0.0224, respectively). Analysis of immune-phenotypic profile of overall circulating T lymphocytes displayed no significant differences in the distribution of naive/memory/effector from CD4+ compartment. However, a decrease in effector CD8+ CAR-T subpopulations was observed compared to non-modified T-cells. Moreover, CAR-T cells showed a significantly higher expression levels of activation (CD69+), proliferation (Ki67+), cytotoxicity (CD107a+) and exhaustion (PD1+TIM3+, PD1+LAG3+) markers in comparison to T lymphocytes (p<0.05). Overall, 31.6% (6/19) patients reached complete remission while 47.4% (9/19) achieved partial response at the first month after CAR-T therapy with event free survival (EFS) of 73% at 1 year (median follow-up of 1 year). Remarkably, cases with lower expression levels of PD-1 and LAG-3 (i.e <5.2%) in CD4+CAR+ T cells at the day of peak expansion had a worse outcome (EFS at 1 year 43% vs 77% for patients with low vs high expression of the above exhaustion markers, p=0.04) (Figure 1A). Similarly, patients with a highly cytotoxicity pattern (i.e CD107a expression >6%) within CD8+CAR+ T cells had a shorter EFS (40%) as compared to those with <6% of CD8+CAR+CD107a+ (89%) (p=0.06) (Figure 1B). Summary/Conclusion: In summary, in our cohort, the median day of CD19 CAR-T cell peak expansion as assessed by flow cytometry and digital PCR was 7 days after infusion. Markers of exhaustion within the CAR+ population allows an early identification of prognostic groups in order to guide therapeutic actions.Keywords: Cytometry, CAR-T, Lymphoma, Acute lymphoblastic leukemia