Abstract PR06: Comprehensive characterization of the non-tumor microenvironment of relapsed/refractory large B-cell lymphoma identifies patients with greatest benefit from CD19 CAR T-cell therapy
Authors: Li Xubin , Singhal Kartik , Deng Qing , Chihara Dai , Russler-Germain David , Hussein Usama , Foltz Jennifer A. , Henderson Jared , Wilson Ashley , Schmidt Evelyn , Nizamuddin Imran , Dinh Tommy , Sun Ryan , Vega Francisco , Flowers Christopher , Griffith Obi , Fehniger Todd , Griffith Malachi , Green Michael R. 
Published: June 2024

Abstract
Multiple therapies with immune mechanisms of action such as CAR T-cells and bispecific antibodies are now approved for relapsed/refractory large B-cell lymphoma (rrLBCL). The efficacy of these therapies is likely influenced by lymphoma microenvironment (LME) characteristics, but these have not been directly characterized in a comprehensive fashion. We therefore performed single-nucleus multiome (RNA + ATAC), bulk RNA, and WES of 119 biopsies from 114 patients to capture both hematopoietic and non-hematopoietic cell types. After stringent quality control, 970,239 cells remained. Non-B-cell lineages were classified into 76 transcriptionally-distinct cell types using unsupervised clustering. LME archetypes were defined by non-negative matrix factorization (NMF) of non-B cell types. Associations with CAR T-cell outcomes were validated using published Nanostring data from the ZUMA7 clinical trial. NMF clustering identified 5 cellular modules: lymph-node 1 [LN1] and lymph-node 2 [LN2] that are characterized by lymph-node structural cell types, antigen presenting cells and naïve and memory T-cells; T-effector/exhausted [TEX] characterized by high frequencies of effector and exhausted CD8 T-cells; and fibroblast/macrophage 1 [FMAC1] and 2 [FMAC2] that were characterized by high frequencies of macrophage subsets and fibroblast subsets including cancer associated fibroblasts (CAFs). The pairs of LN1 and LN2 modules, and FMAC1 and FMAC2 modules, were correlated and therefore considered collectively in tumor archetype construction, resulting in three major archetypes: LN (33% of tumors), TEX (25% of tumors) and FMAC (42% of tumors). Among these biopsies, 17 were obtained immediately prior to, and 13 were subsequent to CD19 CAR T-cell therapy. Although numbers were limited, these cases suggested that the LN archetype was associated with greater benefit from CAR T cell therapy. We therefore leveraged our bulk RNA-sequencing data plus published Nanostring data from the ZUMA7 study of axicabtagene ciloleucel (axi-cel) in second line rrLBCL to develop a Naïve Bayes classifier for these archetypes. Evaluation of response data from ZUMA7 showed that the greatest benefit for axi-cel compared to chemotherapy was observed within the LN subtype (HR=0.18; P&lt;0.0001), compared to the FMAC (HR=0.4; P&lt;0.0001) and TEX (HR=0.71; P=0.23), with median PFS in the axi-cel arm of 30.16, 4.94, and 4.53 months respectively. Furthermore, LN subtype patients had significantly better outcomes compared to FMAC and TEX subtype patients in the axi-cel arm (HR=0.5, P=0.006) but not in the chemotherapy arm (HR=1.3; P=0.23). Therefore, accurate construction of the rrLBCL LME using direct cell measurements of both hematopoietic and non-hematopoietic cells with single-cell genomics permits the identification of cell types, cell modules, and LME archetypes with important implications for LBCL biology, and may present an opportunity for LME-guided selection of patients most likely to benefit from cellular therapy.
Citation Format: Xubin Li, Kartik Singhal, Qing Deng, Dai Chihara, David Russler-Germain, Usama Hussein, Jennifer A. Foltz, Jared Henderson, Ashley Wilson, Evelyn Schmidt, Imran Nizamuddin, Tommy Dinh, Ryan Sun, Francisco Vega, Christopher Flowers, Obi Griffith, Todd Fehniger, Malachi Griffith, Michael R. Green. Comprehensive characterization of the non-tumor microenvironment of relapsed/refractory large B-cell lymphoma identifies patients with greatest benefit from CD19 CAR T-cell therapy [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PR06.