Flow Cytometry Tracks CAR T-Cell Therapy Persistence in Aggressive LBCL

A new single-center analysis offers one of the most detailed real-world evaluations to date of how flow cytometry can be used to monitor chimeric antigen receptor (CAR) T cell expansion, persistence, and toxicity risk in patients with aggressive large B-cell lymphoma (LBCL). The study was recently published in Hematological Oncology.¹

The findings, based on 45 patients treated with commercial CAR T products, provide real-world evidence for integrating flow cytometry into routine CAR T follow-up. Although its prognostic value for survival requires further validation, its utility for toxicity risk assessment and longitudinal immune tracking positions it as a valuable component of CAR T patient management.

Though less sensitive than molecular assays, flow cytometry offers practical advantages for real-time monitoring, explained the researchers, noting the tool’s ability to flag early, high-risk expansion profiles that could help clinicians anticipate well known side effects of CAR T-cell treatment, allocate monitoring resources, and guide preemptive management strategies.

Despite transforming outcomes for many patients with relapsed or refractory LBCL, CAR T-cell treatment yields responses and toxicities that vary considerably from one patient to another. Early expansion of CAR T cells is essential to achieve an antitumor effect, yet this same immune activation can trigger serious inflammatory complications commonly associated with CAR T-cell treatment, such as cytokine release syndrome (CRS) and immune effector cell–associated neurotoxicity syndrome (ICANS).

Most data linking expansion kinetics to clinical outcomes come from a controlled setting via clinical trials, leaving limited insight into real-world variability. The current analysis aimed to fill that gap by using flow cytometry, one of the most widely available and accessible laboratory tools, to characterize CAR T behavior from the time of infusion through 12 months of follow-up.

Among the 45 evaluable patients, the majority had advanced disease and a high prognostic risk profile. Most (89%) received axicabtagene ciloleucel (axi-cel) (Yescarta; Kite Pharma), and the remainder were treated with tisagenlecleucel (tisa-cel) (Kymriah; Novartis). The study found that both products produced rapid expansion in peripheral blood, but with distinct kinetic profiles. Axi-cel reached peak expansion earlier—typically by day 7—with substantially higher median peak levels than tisa-cel. Tisa-cel expanded more modestly and peaked later, around day 10.

These early expansion dynamics were meaningful clinically. Patients with higher CAR T cell expansion were more likely to develop immune-related toxicity. CRS occurred in nearly 87% of the cohort, and patients with grade 2 CRS displayed substantially higher expansion levels than those without CRS. ICANS followed a similar pattern; patients who developed neurotoxicity had markedly higher peak percentages of CAR T cells within the lymphocyte population than those who did not. These associations reinforce the concept that early, robust expansion drives both therapeutic activity and inflammatory toxicity.

The analysis also examined whether expansion corresponded with treatment response or survival outcomes. Responders tended to have numerically higher expansion peaks, as well as greater overall exposure captured by area-under-the-curve calculations. Although these differences did not reach statistical significance, likely due to sample size, progression-free survival at 6 months was higher among patients whose CAR T expansion exceeded 39% of circulating lymphocytes. This suggests that, at least for axi-cel, rapid expansion in the first week may be more prognostic of benefit than the absolute peak magnitude.

“Although we observed only a trend between CAR T cell expansion and clinical response or survival, our findings suggest that FC monitoring can provide clinically useful insights into CAR T cell–treated patients,” wrote the researchers.

The study is not the first to characterize the relationship between CAR T-cell expansion and response to treatment. For example, a 2023 report detailed how CAR T-cell expansion is linked to both efficacy and side effects related to treatment.²

Beyond the initial expansion window, the study characterized CAR T persistence over the first year.¹ While overall levels declined steadily, a substantial proportion of patients still had detectable CAR T cells at month 6, and a small subset maintained detectable levels at month 12. B-cell aplasia, a pharmacodynamic marker of ongoing CAR T activity, was present in most patients at the 6-month mark. These findings confirm long-term persistence but highlight broad heterogeneity in immune recovery patterns.

Cytopenias were another notable observation. Nearly two-thirds of the patients experienced prolonged cytopenia beyond the first month, most commonly pancytopenia. These patients displayed higher median expansion early after infusion, hinting that an intense inflammatory milieu may disrupt hematopoiesis. Although not statistically significant, the pattern aligns with other reports linking expansion intensity to delayed marrow recovery.

References

1. Zduniak A, Martinet J, Lévêque E, et al. Routine monitoring of CAR-T-cells expansion and persistence in patients with aggressive large B-cell lymphoma by flow cytometry: a single-center experience. Hematol Oncol. Published online October 7, 2025. doi:10.1002/hon.70139

2. Baur K, Buser A, Jeker LT, et al. CD4+ CAR T-cell expansion is associated with response and therapy related toxicities in patients with B-cell lymphomas. Bone Marrow Transplant.