CD19-directed CART cells (CD19-CAR) have demonstrated remarkable clinical results in patients suffering from refractory or relapsed lymphoma and acute lymphoblastic leukemia. In order to further optimize follow-up, to explain treatment failure, and to control adverse events biomarkers for monitoring of response are urgently needed. Peak expansion and persistence are correlated with response rates and severity of side effects. However, no standardized method or commercially assay for CD19-CAR measurement is established yet. In this study, two primer-probe assays for digitaldroplet PCR (ddPCR) were designed and subsequently explored on 54 samples collected from seven patients after CD19-CAR treatment with axi-cel over time. Detection and quantification of CART cells were feasible and reliable for all patients included. Peak expansion measured with our assay significantly correlated with the grade of neurologic adverse events but not with cytokine release syndrome. All patients with loss of CARsignal eventually had disease progression. In summary, our novel assay allows monitoring of CART cells in vivo and may add to safety and efficacy of CART treatment.

Hematotoxicity after chimeric antigen receptor (CAR)-T cell therapy is associated with infection and death but management remains unclear. We report results of 31 patients receiving hematopoietic stem cell boost (HSCB; 30 autologous, 1 allogeneic) for either sustained severe neutropenia of grade 4 (<0.5 x109/l), sustained moderate neutropenia (less than or equal to 1.5 x109/l) and high risk of infection, or neutrophil count less than or equal to 2.0 x109/l and active infection. Median time from CAR-T cell therapy to HSCB was 43 days and median absolute neutrophil count at time of HSCB was 0.2. Median duration of neutropenia prior to HSCB was 38 days (range, 7-151). Overall neutrophil response rate (recovery or improvement) was observed in 26 patients (84%) within a median of 9 days (95% confidence interval, 7-14). Time to response was significantly associated with the duration of prior neutropenia (p=0.007). All non-responders died within the first year after HSCB. One-year overall survival for all patients was 59% and significantly different for neutropenia ≤38 days (85%) versus neutropenia >38 days prior to HSCB (44%; p=0.029). In conclusion, early or prophylactic HSCB showed quick response and improved outcomes for sustained moderate to severe neutropenia after CAR-T.

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We present the case of a 64-year-old man diagnosed with large B-cell lymphoma who relapsed twice after standard-of-care therapy. Due to persisting cytopenia, Next generation sequencing analysis was performed, revealing a small TP53-mutated clone. As a third-line therapy, the patient was treated with CAR-T cells, which resulted in complete remission. However, this treatment also led to the expansion of the TP53-mutated clone and therapy-related myelodysplasia with a complex aberrant karyotype. This case may serve as a paradigmatic example of clonal hematopoietic progression in a patient undergoing CAR-T cell therapy, especially in the context of a TP53-mutated clone.