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. 2024 Jun 25;8(12):3314-3326.
doi: 10.1182/bloodadvances.2024012637.

CAR19 monitoring by peripheral blood immunophenotyping reveals histology-specific expansion and toxicity

Mark P Hamilton  1   2   3 , Erin Craig  4 , Cesar Gentille Sanchez  1 , Alain Mina  1 , John Tamaresis  4 , Nadia Kirmani  1 , Zachary Ehlinger  1 , Shriya Syal  1 , Zinaida Good  1   4 , Brian Sworder  3 , Joseph Schroers-Martin  3 , Ying Lu  4 , Lori Muffly  1   2 , Robert S Negrin  1   2 , Sally Arai  1   2 , Robert Lowsky  1   2 , Everett Meyer  1   2 , Andrew R Rezvani  1   2 , Judith Shizuru  1   2 , Wen-Kai Weng  1   2 , Parveen Shiraz  1   2 , Surbhi Sidana  1   2 , Sushma Bharadwaj  1   2 , Melody Smith  1   2 , Saurabh Dahiya  1   2 , Bita Sahaf  1 , David M Kurtz  3 , Crystal L Mackall  1   2 , Robert Tibshirani  4 , Ash A Alizadeh  3   5   6 , Matthew J Frank  1   2 , David B Miklos  1   2
Affiliations

CAR19 monitoring by peripheral blood immunophenotyping reveals histology-specific expansion and toxicity

Mark P Hamilton et al. Blood Adv. .

Abstract

Chimeric antigen receptor (CAR) T cells directed against CD19 (CAR19) are a revolutionary treatment for B-cell lymphomas (BCLs). CAR19 cell expansion is necessary for CAR19 function but is also associated with toxicity. To define the impact of CAR19 expansion on patient outcomes, we prospectively followed a cohort of 236 patients treated with CAR19 (brexucabtagene autoleucel or axicabtagene ciloleucel) for mantle cell lymphoma (MCL), follicular lymphoma, and large BCL (LBCL) over the course of 5 years and obtained CAR19 expansion data using peripheral blood immunophenotyping for 188 of these patients. CAR19 expansion was higher in patients with MCL than other lymphoma histologic subtypes. Notably, patients with MCL had increased toxicity and required fourfold higher cumulative steroid doses than patients with LBCL. CAR19 expansion was associated with the development of cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome, and the requirement for granulocyte colony-stimulating factor 14 days after infusion. Younger patients and those with elevated lactate dehydrogenase (LDH) had significantly higher CAR19 expansion. In general, no association between CAR19 expansion and LBCL treatment response was observed. However, when controlling for tumor burden, we found that lower CAR19 expansion in conjunction with low LDH was associated with improved outcomes in LBCL. In sum, this study finds CAR19 expansion principally associates with CAR-related toxicity. Additionally, CAR19 expansion as measured by peripheral blood immunophenotyping may be dispensable to favorable outcomes in LBCL.

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Conflict of interest statement

Conflict-of-interest disclosure: M.P.H. served on an advisory board for Kite Pharmaceuticals. Z.G. is an inventor on 2 patent applications; holds equity in Boom Capital Ventures; and is a consultant for Mubadala Ventures. S.B. reports consulting for Allogene. C.L.M. is a founder, equity holder, consultant, and director of Cargo Therapeutics and Link Cell Therapies; reports equity in Lyell Immunopharma and royalties from the National Institutes of Health for CAR22; and consults for Immatics, Ensoma, Mammoth, Adaptimmune, and Bristol Myers Squibb. L.M. reports research support from Adaptive Biotechnologies and Servier Laboratories, and consults for Amgen and Pfizer. P.S. reports research support from Kite Pharma-Gilead. S. Sidana reports consulting for Janssen. D.M.K. reports consultancy for Roche, Adaptive Biotechnologies, and Genentech, and equity ownership interest in Foresight Diagnostics. A.A.A. reports consulting for and having stock options in CiberMed; consulting fees and research funding from Celgene; holding stock options in CAPP Medical; consulting fees, honoraria, and travel funding from Roche; holding stop options in Forty Seven; holding stock options in Syncopation Life Sciences; consulting fees and travel fund from Gilead; consulting fees from and holding stock options in Foresight Diagnostics; honoraria from Jannsen; and consulting for Lymphoma research foundation. M.J.F. reports consulting for Kite Pharma-Gilead, Adaptative Biotechnologies, and Cargo Therapeutics, and research support from Kite Pharma-Gilead, Allogene Therapeutics, Cargo Therapeutics, and Adaptative Biotechnologies. D.B.M. reports consulting for Kite Pharma-Gilead, Juno Therapeutics-Celgene, Novartis, Janssen, and Pharmacyclics, and research support from Kite Pharma-Gilead, Allogene Therapeutics, Cargo Therapeutics, Pharmacyclics, Miltenyi Biotec, and Adaptive Biotechnologies. The remaining authors declare no competing financial interests.

Figures

None
Graphical abstract
Figure 1.
Figure 1.
CAR-FACS analysis measures CAR19 expansion across histologic subtypes over 7 orders of magnitude. (A-B) PFS and OS curves in Stanford’s CAR19 cohort for LBCL, FL, and MCL are representative of published results. Dotted lines represent respective median time to event. Only the first 30 months of follow-up are illustrated. (C) Percent and absolute plots of longitudinal CAR19 expansion in the first month demonstrates CAR19s expand over 7 orders of magnitude with CD8 dominance. Dotted lines represent theoretical limits of detection based on prior assay calibration. (D) Absolute CD4 and CD8 CAR19 counts are highly correlated during expansion. (E) There is a small relative increase in CD8 CAR19 expansion at D14 and D21 with increasing persistence of the CD4 CAR19 at D28 (Wilcoxon test). ∗P < .05, ∗∗P < .01.
Figure 1.
Figure 1.
CAR-FACS analysis measures CAR19 expansion across histologic subtypes over 7 orders of magnitude. (A-B) PFS and OS curves in Stanford’s CAR19 cohort for LBCL, FL, and MCL are representative of published results. Dotted lines represent respective median time to event. Only the first 30 months of follow-up are illustrated. (C) Percent and absolute plots of longitudinal CAR19 expansion in the first month demonstrates CAR19s expand over 7 orders of magnitude with CD8 dominance. Dotted lines represent theoretical limits of detection based on prior assay calibration. (D) Absolute CD4 and CD8 CAR19 counts are highly correlated during expansion. (E) There is a small relative increase in CD8 CAR19 expansion at D14 and D21 with increasing persistence of the CD4 CAR19 at D28 (Wilcoxon test). ∗P < .05, ∗∗P < .01.
Figure 2.
Figure 2.
CAR19 expansion is dependent on lymphoma histology. (A) MCL histology has increased D14, D21, and D28 CAR19 expansion relative to LBCL based on a multivariate linear mixed effects model of longitudinal data. (B) CAR19 AUC in MCL is greater than LBCL with almost fourfold higher CAR19 exposure (Wilcoxon test). (C-D) Breakdown of the LBCL histology indicates there are differences between HGBCL, TFL, and other LBCL pathologies in CAR19 expansion. These differences are not significant in mixed effects modeling (C) but there is a significant AUC difference between HGBCL and other lymphoma histologic subtypes when directly compared (D; Wilcoxon test). (E) Greater LDH is associated with significantly more persistence of CAR19s on D21 and D28 using a multivariate mixed effects model. Plot is representative of statistical output that uses continuous values. (F) Discrete analysis of CAR19 expansion (Wilcoxon test) by AUC based off prelymphodepletion LDH greater than the upper limit of normal does not demonstrate statistically significant total exposure differences in CAR19 expansion. (G) Greater age is associated with reduced CAR19 persistence on D21 and D28 using the same modeling. Plot is representative of statistical results that uses continuous values. (H) Discrete modeling of total CAR19 exposure over the first 28 days does not demonstrate significant differences in AUC using a standard age threshold (Wilcoxon test). However, highly significant effect sizes in mixed effects modeling for both LDH and age were modest. ∗P < .05; ∗∗P < .01, ∗∗∗P < 0.001; ns; error bars are standard error of the mean; plots are representative of modeling results. Significance values in line charts represent the interaction between CAR19 expansion and day; graphs are only representative of the continuous statistics. ns, not significant.
Figure 3.
Figure 3.
CAR19 expansion is associated with CAR19-mediated toxicity. (A) CAR19 expansion defined by AUC is significantly associated with the development of severe ICANS. (B) Lower CAR19 CD4:CD8 ratio is also associated with the development of severe ICANS. (C) Increased CAR19 expansion is associated with more severe CRS. (D) Increased CAR19 expansion is associated with the need for G-CSF after D14. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. All significance values represent Wilcoxon testing.
Figure 4.
Figure 4.
The interaction between pretreatment risk by LDH and CAR19 expansion classifies patients with high and low risks. (A) Pretreatment LDH strongly correlates with pretreatment ctDNA. (B) Results of lasso regression incorporating the interaction between prelymphodepletion LDH (greater or less than data set median) and prelymphodepletion CAR AUC (greater or less than data set median). Expected values selected by the lasso based on prior publications include prelymphodepletion LDH, pre-LD CRP, and bridging therapy. In addition, patients with high LDH and low CAR19 expansion (CARlow/LDHhigh) are selected as doing worse, whereas patients with low LDH and low CAR19 expansion (CARlow/LDHlow) are selected as having improved outcomes. (C) Kaplan-Meier estimate of patients stratified by LDH and CAR expansion demonstrate superior outcomes by PFS in patients with low LDH and low CAR19 expansion.
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Comment in

  • CAR T-cell expansion: harmful or helpful?
    Lionel AC, Neelapu SS. Lionel AC, et al. Blood Adv. 2024 Jun 25;8(12):3311-3313. doi: 10.1182/bloodadvances.2024013146. Blood Adv. 2024. PMID: 38916899 Free PMC article. No abstract available.

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