Pre-infusion plasma proteomics identifies an endothelial-immune priming signature predictive of severe cytokine release syndrome and neurotoxicity following CAR T-cell therapy in relapsed/refractory lymphoma.

BACKGROUND: Severe cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) remain frequent, life-threatening complications of CD19 chimeric antigen receptor (CAR) T-cell therapy and constrain its safety, scalability, and outpatient adoption. Existing predictive models lack sufficient external validation for routine clinical use, and pre-infusion biomarkers that capture host susceptibility before infusion are urgently needed.

METHODS: We applied unbiased mass-spectrometry-based proteomics to pre-infusion biofluids from 98 prospectively-followed adults with relapsed/refractory (r/r) lymphoma at two academic centers (MD Anderson Cancer Center, n = 39, plasma; Moffitt Cancer Center, n = 59, serum). Logistic regression with backward feature selection on the MD Anderson cohort yielded panels for severe (Grade ≥ 2) CRS and ICANS that were locked and tested without refitting on the Moffitt cohort. Patients were stratified into low-, intermediate-, and high-risk tertiles. Ingenuity Pathway Analysis defined upstream regulators and canonical pathways. The 17 CRS-associated and 21 ICANS-associated consensus proteins were classified into mechanistic themes for biological interpretation.

RESULTS: A 5-marker CRS panel (SCRIB, MYL6, MTHFD1L, HSP90B1, MMP2) achieved AUCs of 0.85 (95% CI 0.72-0.98) and 0.76 (0.63-0.89) in the discovery and validation cohorts, respectively. An expanded 8-marker ICANS panel (the CRS panel plus SPOCK2, SLC3A2, CD84) achieved AUCs of 0.91 (0.81-1.00) and 0.67 (0.51-0.84). In the combined dataset, high-risk-tertile patients were 13.84-fold (95% CI 4.21-56.26) and 8.59-fold (2.87-29.09) more likely to develop Grade ≥ 2 CRS and ICANS, respectively. Pathway analysis converged on AKT-driven inflammation and endothelial activation. Functional clustering of the consensus proteins partitioned into mechanistically coherent themes consistent with a dual-anatomy model: severe CRS reflected peripheral macrophage priming and endothelial activation with surplus complement amplification (HSP90B1▴, CSF1▴, MMP2▴, HEG1▴, C3▴) and endotheliopathic coagulation (PROC▾, F7▾), whereas severe ICANS reflected cerebrovascular junction and basement-membrane stripping (CDH5▾, ITGB1▾, FN1▾, brain-enriched SPOCK2▾), hepatic synthetic suppression (TTR▾, APOA2▾, IGFBP3▾), compromised plasma antioxidant capacity (GPX3▾, PON1▾), and inflammasome dis-restraint via DPP9▾. PGLYRP2 and SCRIB depletion were shared by both signatures and identified a common upstream priming substrate.

CONCLUSIONS: Externally validated, pre-infusion proteomic panels predict severe CRS and ICANS following CAR T-cell therapy and define a coherent pre-infusion endothelial-immune priming axis (HSP90B1, MMP2, AKT) with mechanistically interpretable, druggable nodes. The dual-anatomy framework distinguishes peripheral CRS-biased from cerebrovascular ICANS-biased phenotypes downstream of a shared microbiome-host barrier priming substrate, providing a foundation for biomarker-guided risk stratification and cluster-matched prophylactic intervention to enhance the safety and outpatient feasibility of CAR T-cell therapy.