Wang, Zixing

Abstract Background Hypomagnesemia has been correlated with inferior outcomes in patients with large B cell lymphoma (LBCL) undergoing stem cell transplants. As T-cell and myeloid cell dysfunction have been associated with low magnesium conditions, we investigated whether serum magnesium (Mg) levels could predict clinical outcomes in LBCL patients who received chimeric antigen receptor T-cell therapy. Methods Patients with LBCL who received axi-cel under the ZUMA-1 trial or as FDA approved therapy at Mayo Clinic were examined. Serum samples were obtained at specified time points and cytokine analysis was performed. Single cell RNA sequencing was performed on peripheral blood mononuclear cells. The Student T-test, Kruskal Wallis, or Fisher's Exact Tests were used to compare differences in demographics across Mg levels. Survival curves were plotted using the Kaplan-Meier methodology and compared using the Wilcoxon test. Results We found that hypomagnesemia before lymphodepletion chemotherapy predicted for inferior progression-free and overall survival in the pivotal study ZUMA-1 (NCT02348216). These results were validated in an independent cohort of LBCL patients receiving axicabtagene ciloleucel (axi-cel) at Mayo Clinic. Hypomagnesemia correlated with increased inflammatory serum markers and cytokine levels including ferritin, IL-6, IL1Ra, IL-8, and MIP1a. scRNAseq analysis unveiled altered immune interactions between monocytes and T cells with a concordant immune suppressive transcriptome. Conclusions Hypomagnesemia at the time of CAR-T infusion associates with an unfavorable inflammatory profile and decreased response and survival in LBCL patients receiving axi-cel. These findings suggest a potentially actionable prognostic factor for patients with large cell lymphoma undergoing CAR-T.

Abstract (a) Expansion of therapeutic strategies against diffuse large B-cell lymphoma (DLBCL) offers new opportunities to fight aggressive non-Hodgkin's lymphoma. Therapy approaches, such as R-CHOP and stem cell transplant, and CAR-T cell therapies are now available for the treatment of DLBCL. While the role of the tumor microenvironment (TME) during the therapeutic response is now recognized, the spatial organization and the communication between the diverse factors that compose the TME remains to be fully understood. In this study, we describe a new immunogram tool to help clinical researchers measure the tumor microenvironment contribution to drug efficacy or toxicity in DLBCL context. (b) In a previous work, using a high-quality testing and innovative multimodal and integrative approach, we constructed the first Veracyte Biopharma Atlas for DLBCL lesions. Based on this knowledge and the understanding of the immune contexture of tumors, we constructed an immunogram using the scoring of clinically relevant biomarkers. Our immunogram scoring approach is composed of key features driving computational derivation of DLBCL Atlas clusters, which include immune gene signatures, immune cell phenotyping combined with spatial information, and genomic and clinical parameters. (c) Patterns that strongly associated with CAR-T treatment response were determined. In addition, promising results have been obtained in the prediction to CAR-T toxicity related to T-regulatory cells or R-CHOP response with transcriptomic signature. (d) Retrospective analysis of CAR-T clinical trial data and tumor biopsies highlighted the Veracyte Immunogram as a powerful tool to score the patient probability of response to CAR-T. This work provides the framework for identification of predictors of response to therapy, potential targets of interest or biomarkers that could be applied in future clinical trials. Citation Format: Michael D. Mattie, Regis Perbost, Sarah Turcan, Corinne Danan, Frederick L. Locke, Sattva S. Neelapu, David B. Miklos, Caron A. Jacobson, Lazaros J. Lekakis, Yi Lin, Armin Ghobadi, Jenny J. Kim, Zixing Wang, Allen Xue, Simone Filosto, Nathalie Scholler, Jerome Galon. Defining the cancer Immunogram using multimodal analysis to guide immunotherapy in diffuse large B cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5879.

Abstract Background: Conventional prognostic factors for LBCL were not associated with outcomes in the pivotal ZUMA-1 study of axi-cel in relapsed LBCL (Neelapu et al. NEJM. 2017); however, other attributes like chimeric antigen receptor (CAR) T-cell fitness and composition (CCR7+CD45RA+ T cells), reduced preTx tumor burden, and immune tumor microenvironment (TME) with presence of activated CD8+PD-1+LAG-3+/-TIM-3- T cells were associated with efficacy (Locke et al. Blood Adv. 2020; Galon et al. ASCO 2020. #3022). Here, we evaluated preTx immune cell phenotypes in premanufacturing apheresis (premfg aph) material, comprising peripheral blood mononuclear cells, to determine associations with product attributes, immune TME features, and clinical efficacy in ZUMA-1. Methods: Evaluable samples from patients (pts) in Phase (Ph) 1 and Ph2 Cohorts (C) 1-3 were analyzed (NCT02348216; Ph1 and Ph2 C1+2, ≥2-y follow-up; C3, ≥6-mo follow-up). Memory T, myeloid, NK, NKT, and B cells in premfg aph material (n=101, excluding C3) were characterized by flow cytometry (FC). PreTx immune TME was analyzed by multiplex IHC (n=18) and gene expression analysis (n=30) as previously described (Rossi et al. AACR 2018. #LB-016; Galon et al. ASCO 2020. #3022). CAR T-cell fitness was analyzed by doubling time, viability during manufacturing, and product T-cell phenotypes by FC (n=145). Associations between these covariates, and with routine hematology tests, were performed by Spearman rank correlation or Wilcoxon tests. Effects on survival were assessed by Kaplan-Meier with optimized cutpoint selection. Results: The percentage (%) of naive (CCR7+CD45RA+) T helper (Th; CD4+CD127+CD25low) cells coexpressing CD27 and CD28 (median, 1%; range, 0.01%-15.8%; IQR, 0.3%-3.8%) in aph associated positively with axi-cel efficacy. The % of intermediate monocytes (IMs; CD14+CD16+; median, 1.8%; range, 0.003%-16.7%; IQR, 1%-3%) in aph associated negatively with efficacy. The % of circulating CD27+CD28+ naive Th cells associated positively with an enriched preTx immune TME T-cell signature, % CCR7+CD45RA+ product T cells, objective response rate, PFS, and OS. An increased % of IMs associated directly with negative predictive markers (preTx serum levels of LDH, IL-6, and CRP) and inversely with TME T-cell signature, PFS, and OS. The premfg ratio of CD27+CD28+ naive Th cells/IMs associated directly with CAR T-cell expansion and efficacy. Conclusions: This work points to a link between the pre-existing state of the immune system, reflected in premfg aph, and immune TME, as well as product attributes influencing axi-cel efficacy in LBCL. These data bear practical implications towards the development of predictive biomarkers for axi-cel efficacy. [JB and JC contributed equally.] Citation Format: Justin Budka, Justin Chou, Vicki Plaks, Francesca Milletti, Zixing Wang, Frederick L. Locke, Sattva S. Neelapu, David B. Miklos, Caron A. Jacobson, Lazaros J. Lekakis, Yi Lin, Armin Ghobadi, Zahid Bashir, Nathalie Scholler, Jérôme Galon, John M. Rossi, Adrian Bot. Pretreatment (PreTx) immune cell phenotypes in peripheral blood associated with the tumor immune contexture, product attributes, and durable clinical efficacy in patients with large B-cell lymphoma (LBCL) treated with axicabtagene ciloleucel (axi-cel) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT166.

3022 Background: Axi-cel is a US and EU-approved autologous anti-CD19 chimeric antigen receptor (CAR) T cell therapy for pts with relapsed/refractory large B cell lymphoma after ≥ 2 prior therapies. In ZUMA-1 (NCT02348216), the objective response rate was 83% (58% complete response rate; Locke et al. Lancet Oncol. 2019). T cell-related biology (Immunosign 21; Immunoscore) measured pretreatment in the tumor microenvironment (TME) was associated with response to axi-cel (Rossi et al. AACR 2018. #LB-016; Rossi et al. AACR 2019. #CT153). This expanded analysis characterized the pretreatment TME immune contexture and examined associations between immune cell subsets and response. Methods: In ZUMA-1, pts received axi-cel at a target dose of 2.0 × 106 CAR T cells/kg. Archival pretreatment tumor biopsy samples were analyzed by multiplex immunohistochemistry (Brightplex). Two panels were developed and applied to assess T cell (CD3, CD8, FoxP3, PD-1, LAG-3, TIM-3) and myeloid cell (CD11b, CD14, CD15, LOX1, S100A9, CD68) subsets (n = 14 total). The association between T cell and myeloid cell subset density, prespecified immune scores (Immunosign 21; Immunoscore), and objective response was evaluated. T test values were based on Brightplex analysis. Results: Pretreatment tumor biopsy samples from 18 pts were analyzed (14 objective responders and 4 nonresponders). The pretreatment TME comprised all major myeloid and T cell subsets, with diverse distribution across samples analyzed. The median TME density of monocytes (CD11b+ CD15− CD14+; 1215 cells/mm2) and macrophages (CD68+; 530 cells/mm2) was greater than that of the total CD8+ T cell subset (312 cells/mm2). The pretreatment Immunosign 21 and Immunoscore scores associated positively with the density of all major T cell subsets and some myeloid subsets. The density of activated CD8+ T cells (PD-1+ LAG-3+/− TIM-3−) was most significantly associated with clinical response versus other T cell subsets. The density of nonactivated CD8+ T cells (PD-1− LAG-3− TIM-3−) and exhausted CD8+ T cells (PD-1+ LAG-3+ TIM-3+) were not significantly associated with response. Additional characterization of the immune contexture and correlative analysis of cell subsets will be presented. Conclusions: These results suggest that a TME associated with increased density of activated PD-1+ LAG-3+/− TIM-3− CD8+ T cells, measurable pretreatment, facilitates clinical response in pts post–axi-cel.