Abstract 5185: Flow cytometry-based monitoring of CAR-T cell persistence and immune phenotypes pre- and post-infusion: Challenges in Phase 1/2 hematologic malignancy trials

Abstract Chimeric antigen receptor T-cell (CAR-T) therapies that support hematological malignancies have recently had their treatment efficacy determined by both clinical outcomes and specific assessments of the CAR-T cells themselves. Assay validation typically uses control CAR-T cells spiked into healthy PBMCs, which do not fully replicate the phenotypic and scatter characteristics of patient-derived CAR-T cells after infusion. This discrepancy introduces challenges in gating accuracy and data interpretation. Precision for Medicine (PfM) developed and validated a multiparameter flow cytometry assay to quantify CAR-T persistence and characterize immune phenotypes, including CD4, CD8 subsets, memory, effector differentiation, and activation/exhaustion markers. In the development and validation control CAR-T cells were used to establish the gating strategy. Post-infusion CAR-positive cells exhibited altered scatter profiles and marker expression compared to validation controls. Inter-assay controls ensured baseline reproducibility, but adaptive gating strategies were required for accurate identification of CAR-T populations in patient samples. These adjustments enabled robust persistence tracking and immune profiling. Clinical sample analysis was performed on peripheral blood mononuclear cells (PBMCs) pre- and post-infusion. Differences between validation controls and post-infusion CAR-T cells underscore the need for dynamic and flexible gating strategies. Addressing these challenges is critical for reliable immune monitoring in oncology trials and for correlating CAR-T persistence with clinical outcomes. Citation Format: Jackie Benko, Bhagyaraj Ella, Samantha Splitt, Angelina Bisconte, Rachel Owen, Deborah J. Phippard. Flow cytometry-based monitoring of CAR-T cell persistence and immune phenotypes pre- and post-infusion: Challenges in Phase 1/2 hematologic malignancy trials abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5185.