Endothelial dysfunction and proinflammatory state determine severe hematotoxicity and adverse outcome of CAR-T therapy

Abstract Hematotoxicity with prolonged cytopenia is the most relevant high-grade toxicity after chimeric antigen receptor (CAR)-T therapy. It is associated with higher risk for infections, impaired quality of life, increased healthcare burden, and contributes substantially to mortality. Despite its clinical relevance, predictive biomarkers, particularly those reflecting underlying mechanisms, remain scarce. In this study, we employed the immune-related adverse outcome pathway framework to delineate hematotoxicity into defined, measurable key events and to identify mechanistically informed biomarkers to enhance risk stratification and guide patient management. This European initiative between four CAR-T cell centers included 78 patients with relapsed/refractory multiple myeloma (MM) or diffuse large B-cell lymphoma treated with ide-cel (n=31), cilta-cel (n=23), or axi-cel (n=24). Peripheral blood was collected before apheresis, at baseline (i.e. before lymphodepletion), early post-infusion and day 14 after CAR-T therapy. Flow cytometry analysis and multiplex cytokine profiling of 42 different analytes were performed. Associations between variables were analyzed using spearman correlation and logistic regression. Multiple testing was controlled using a false discovery rate threshold of <0.1. The impact of our markers on adverse outcomes was studied in univariate and multivariate logistic regression analysis. Receiver operating characteristic (ROC) analysis were used to test the prognostic value of markers on adverse outcomes. Hematotoxicity and infections were the most frequent and clinically relevant toxicities. Grade ≥3 neutropenia occurred in 99% of patients, with 40% experiencing prolonged neutropenia ≥14 days. Prolonged grade ≥4 neutropenia was significantly associated with mortality, life-threatening infections, and poor peak CAR-T expansion. Severe infections (grade ≥3) occurred in 31%, with higher incidence of any grade infections following BCMA-targeted CAR-T products. Hematotoxicity showed no association with age, sex, number of prior therapy lines, prior autologous or allogeneic stem cell transplantation, disease entity, or high-risk profiles. Endothelial dysfunction emerged as a key event of hematotoxicity, evidenced by an imbalance of endothelial cytokines such as ANG1, soluble selectins and VCAM-1 (sVCAM-1) early post-infusion in patients developing prolonged cytopenias. At baseline, markers of endothelial dysfunction (sVCAM-1; r=0.46; p<0.0001) and pro-inflammation (sIL-2R; r=0.39; p=0.0004) demonstrated the strongest association with prolonged neutropenia, outperforming conventional lab markers such as CRP and ferritin. Furthermore, elevated baseline sVCAM-1 or sIL-2R were significantly associated with reduced overall survival (p=0.0009 and p=0.025), independent of disease entity or high-risk profiles in cox regression analysis. While no impact on progression-free survival was observed, elevated baseline sVCAM-1 was associated with reduced CD8 CAR-T expansion and a trend toward inferior response rates, particularly in MM. Patients with elevated baseline values of our markers experienced longer grade 4 neutropenia (6.6 vs. 10.3 days, p = 0.019, sIL-2R; 6.0 vs. 12.1 days, p = 0.0016, sVCAM-1), more frequent aplastic neutrophil recovery (5% vs. 30%, p = 0.007, both), and higher rates of severe infections (22.4% vs. 55%, p = 0.011, both). Finally, ROC analysis confirmed the prognostic value of both markers for the adverse outcomes of prolonged neutropenia, severe infections and death. Incorporating sVCAM-1 and sIL-2R into existing models such as the CAR-HEMATOTOX improved predictive performance for nearly all endpoints and across CAR-T products. Importantly, both markers retained consistent and independent prognostic value across almost all endpoints, even after adjusting for key clinical variables including CAR-T product type, prior stem cell transplantation, and indicators of disease burden such as bulk or elevated LDH. This study identifies endothelial dysfunction and a proinflammatory state as key risk factors of hematotoxicity and adverse outcomes following CAR-T therapy. sVCAM-1 and sIL-2R are suitable molecular markers to detect these unfavorable states and reflect distinct pathophysiological key events within the immune-related adverse outcome pathway of hematotoxicity. These markers improve risk assessment and will be implemented in clinical routine for informed decision-making and to guide interventions.