Background: CD19-directed CAR-T cell therapies have significantly improved outcomes in relapsed/refractory (R/R) B-cell malignancies, including acute lymphoblastic leukemia (B-ALL) and non-Hodgkin lymphoma (B-NHL). Despite their efficacy, these therapies are associated with serious early toxicities, particularly cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), driven by aberrant cytokine surges. Predictive biomarkers for these complications are urgently needed to enable early risk stratification and guide supportive care. While cytokine kinetics have been well-characterized in commercial CAR-T products such as tisagenlecleucel and axicabtagene ciloleucel, no published studies to date have described cytokine profiles in recipients of NexCAR19 (Actalycabtagene autoleucel), a novel CD19-directed CAR-T cell product developed in India. This is the first pilot study to conduct such an analysis.

Aim: To evaluate whether early post-infusion cytokine profiling predicts toxicity and clinical outcomes in patients receiving NexCAR19 for R/R B-cell malignancies.

Methods: This prospective, single-center study included 11 patients (8 B-ALL, 3 B-NHL) with complete cytokine data, selected from a larger cohort of 23 patients infused with NexCAR19 between January 2024 and July 2025. Disease burden was classified by marrow blast percentage in B-ALL and metabolic tumor volume (MTV) on baseline PET-CT in B-NHL (≥65 cc considered high). Cytokine analysis was performed only in patients who developed fever within the first 7 days post-infusion, targeting the window of peak inflammatory response. Plasma samples were analyzed using the Randox Evidence MultiSTAT platform, a fully automated biochip-based immunoassay system providing simultaneous quantification of IL-1β, IL-2, IL-6, IFN-γ, TNF-α, MCP-1, and ferritin. Results were available within 30 minutes. Clinical and laboratory toxicities were documented per ASTCT and CTCAE v5.0 guidelines. Associations between cytokine levels and outcomes were assessed using Spearman's rank correlation and exact permutation tests.

Results: Median age was 26 years (range: 18–50); 7 patients were male. ECOG performance status >2 was noted in 3 patients. CRS occurred in all patients, with Grade ≥2 in 6 (55%). ICANS occurred in 1 patient (9.1%), HLH-like syndrome in 4 (36.3%), and documented infections in 4 (36.3%). Grade ≥3 cytopenias were universal (100% neutropenia), with 91% experiencing thrombocytopenia and 55% anemia. Median ANC <500 duration was 21 days (range: 12–35). Four patients (36.4%) died during follow-up (median: 5.6 months, range: 0.4–18 months).

Key cytokine correlations:

  • IL-1β Median value is 2pg/ml (IQR:1-2) : Strong correlation with mortality and overall toxicity (r = 0.78, p = 0.001)

  • IL-2 2.7pg/ml (IQR:2.0-5.8): Strongly associated with HLH syndrome (r = 0.72, p = 0.001)

  • IL-6 53pg/ml (IQR:29-456): Correlated with both adverse outcomes (r = 0.86, p = 0.001) and Grade ≥3 anemia (r = 0.62, p = 0.04)

  • IFN-γ 12pg/ml (IQR:7-22) : Associated with infection and sepsis (r = 0.64, p = 0.04)

  • TNF-α 16pg/ml (IQR:7-21) : Correlated with EASIX score (r = 0.74, p = 0.01); showed a trend with transaminitis (r = 0.58, p = 0.06)

  • MCP-1 193pg/ml (IQR:136-489): Linked to HLH diagnosis and requirement of immunosuppressive therapy (r = 0.63, p = 0.04)

  • Ferritin 794ng/ml (IQR:197-2045): Correlated with HLH status (r = 0.60, p = 0.05)

Conclusion: Early post-infusion cytokine profiling in NexCAR19 recipients demonstrated predictive utility for key toxicities and adverse clinical outcomes. Elevated IL-1β, IL-2, IL-6, and MCP-1 levels were significantly associated with HLH, cytopenias, infections, and mortality. The rapid turnaround of the point-of-care cytokine panel supports its feasibility in real-time clinical decision-making. These findings suggest a potential role for cytokine-guided risk stratification and preemptive interventions in CAR-T programs. Validation with longitudinal anayses in larger, multicenter cohorts is warranted to confirm clinical applicability and optimize patient outcomes.

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2025
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