CAR T expansion and systemic inflammation: diverging impacts on large B-cell lymphoma therapy in the multicenter CART SIE study

Abstract

Chimeric antigen receptor (CAR) T expansion has been linked to anti-tumor response in relapsed/refractory large B-cell lymphoma both in clinical trials and smaller real-world studies. Here, we present the largest multicenter real-world analysis to date, evaluating 262 patients treated with tisagenlecleucel or axicabtagene ciloleucel in second or subsequent relapse. Our findings underscore the complementary roles of multiparameter flow cytometry and droplet digital polymerase chain reaction in monitoring CAR T cells. While droplet digital polymerase chain reaction accurately quantifies transgene copies, multiparameter flow cytometry provides critical phenotypic details, revealing CAR T-cell subpopulations that are associated with its efficacy. Consistent with prior studies, we confirm the association of CAR T expansion with response rates, progression-free survival, and toxicities. However, we reveal that expansion alone does not ensure efficacy. Elevated markers of systemic inflammation, such as ferritin and C-reactive protein, are linked to poorer outcomes despite robust expansion. These markers correlate with reduced cytotoxic CD8+ T cells with central memory features among in vivo expanded CAR T-cell populations, with similar associations observed in manufactured and leukapheresis products. Importantly, patients with high baseline inflammation who achieved significant expansion demonstrated progression-free survival outcomes comparable to those with limited expansion, highlighting the negative impact of inflammation on CAR T-cell efficacy. Interestingly, ferritin and C-reactive protein levels were similar among responding patients, regardless of differences in CAR T expansion. Collectively, our findings indicate that systemic inflammation is associated with the phenotypic quality of T and CAR T cells. While functional validation is warranted, these results underscore the need to address inflammatory pathways to improve treatment outcomes.

Figure 1.

CAR T expansion monitoring by multiparameter flow cytometry and droplet digital polymerase chain reaction. Longitudinal chimeric antigen receptor (CAR) T expansion kinetics assessed by both (A) multiparameter flow cytometry (MFC) and (B) droplet digital polymerase chain reaction (ddPCR) at the indicated time points, on peripheral blood (PB) samples from 20 lymphoma patients. (C) Scatter dot plots showing the Spearman correlation between CAR T enumerations performed by MFC and ddPCR (N=114 paired samples).

Figure 2.

Study of tisa-cel and axi-cel infusion products using multiparameter flow cytometry and droplet digital polymerase chain reaction. Dot plots showing the differences in the percentage of chimeric antigen receptor-positive (CAR⁺) cells among (A) CD3⁺ and (B) CD45⁺ cells between tisagenlecleucel (tisa-cel) or axicabtagene ciloleucel (axi-cel) infusion products (N=30). (C) Dot plot showing the difference in CAR copy number/mg DNA between tisa-cel or axi-cel infusion products (N=30). (D) Dot plot showing the difference in CAR copy number/CAR T cell between tisa-cel and axi-cel infusion products (N=30). The calculation was performed as described in the Online Supplementary Methods. P values were calculated applying the Mann-Whitney test; ****P<0.0001. MFC: multiparameter flow cytometry; ddPCR: droplet digital polymerase chain reaction.

Figure 3.

CAR T expansion significantly impacts day 90 response and progression-free survival. (A) Magnitude of chimeric antigen receptor (CAR) T expansion within the first month after infusion (AUC_0-30) in responders (RE) and non-responders (NR) by day 90 (N=235). Exact median values are reported. P value was calculated applying the Mann-Whitney test; **P<0.01. (B) Kaplan-Meier curve showing progression-free survival (PFS) according to CAR T expansion (N=262). Patients were dichotomized into strong and poor expanders based on the AUC_0-30. Comparisons were made applying the log-rank test. (C) Forest plot reporting the results of a multivariable logistic model assessing potential clinical and biological risk factors for response at day 90 (N=160). (D) Forest plot showing results of multivariate Cox regression model assessing potential clinical and biological risk factors for progression-free survival (PFS) (N=147). CRP: C reactive protein; LDH: lactate dehydrogenase; ASCT: autologous stem cell transplantation; HGBCL: high grade B-cell lymphoma; DLBCL: diffuse large B-cell lymphoma; PMBCL: primary mediastinal B-cell lymphoma; OR: odds ratio; HR: hazard ratio; NA: not applicable.

Figure 4.

Patients expanding CAR T cells but not responding to therapy are characterized by high levels of inflammatory markers. Graphs showing pre infusion values of (A) ferritin (N=107), (B) C-reactive protein (CRP) (N=110) and (C) normalized lactate dehydrogenase (LDH) (N=94) in expanders who responded (exp-RE) and expanders who did not respond by day 90 (exp-NR). LDH levels were divided by the respective upper limit of normal (ULN), to generate normalized ratios. Ratios >1 correspond to LDH levels higher than ULN. Graphs showing cumulative levels of (D) ferritin and (E) CRP within the first months after infusion (AUC_0-30, N=52). In (A-E), exact median values are reported. P values were calculated applying the Mann-Whitney test; **P<0.01; ***P<0.001; ****P<0.0001. (F) Forest plot reporting the results of a multivariable Cox model assessing potential risk factors for progression-free survival (PFS) in expander patients (N=72). (G) Kaplan-Meier curve showing PFS according to chimeric antigen receptor (CAR) T expansion and pre infusion levels of ferritin and CRP (N=205). Patients were dichotomized into strong and poor expanders based on the median AUC_0-30. High ferritin/CRP: patients with both ferritin and CRP levels higher than the median (406 ng/mL and 9 mg/L, respectively); low ferritin/CRP: patients with both ferritin and CRP levels lower than the median. Comparisons were made applying the log-rank test. HR: hazard ratio.

Figure 5.

High levels of inflammatory markers impact T-cell composition in infusion products, at peak expansion and in leukapheresis. Levels of chimeric antigen receptor-positive (CAR⁺)CD8⁺ T central memory (T_CM) cells in infusion products (IP) (N=49) based on inflammatory markers at infusion (A), CAR⁺CD8⁺ T_CM cells at peak expansion (C_max, N=25) based on inflammatory markers at infusion (B), CD8⁺ T-stem cell memory (T_SCM) cells in leukapheresis (LK) products (N=40) based on inflammatory markers at infusion (C), CD8⁺ T_SCM cells in LK products (N=40) based on ferritin levels at leukapheresis (D). Patients were dichotomized based on ferritin and C-reactive protein (CRP) values before infusion. High ferritin/CRP: patients with both ferritin and CRP levels higher than the median (406 ng/mL and 9 mg/L, respectively); low ferritin/ CRP: patients with both ferritin and CRP levels lower than the median for (A-C). In (D) patients were dichotomized based on ferritin values (437 ng/L) measured around the time of leukapheresis (+/-7 days). In (A-D) exact median values are reported. P values were calculated applying the Mann–Whitney test; *P<0.05. (E) Kaplan-Meier curve showing progression-free survival (PFS) according to the levels of CAR⁺CD8⁺ in infusion products (IP) and pre-infusion levels of ferritin and CRP (N=205). High ferritin/CRP: patients with both ferritin and CRP levels higher than the median; low ferritin/CRP: patients with both ferritin and CRP levels lower than the median. High CAR⁺CD8⁺ in IP: CAR⁺CD8⁺ levels in IP higher than the median; low CAR⁺CD8⁺ in IP: CAR⁺CD8⁺ levels in IP lower than the median. Comparisons were made applying the log-rank test.