Spatial Tissue Analysis of Secondary Sarcoma Following CAR T Cell Therapy for B Cell Lymphoma
Authors: Schneider Dina , Lee Jia-Jye , Wang Jinling , Pinard Robert , Hu Peirong , Luo Kun , Tran Ngoc , Sawalha Yazeed , Denlinger Nathan , Bezerra Evandro , Dash Pradyot , Krull Ashley Angell , O'Donnell Lynn , Alinari Lapo , Jones Dan , de Lima Marcos , Vasu Sumithira 
Published: December 2024

Introduction
The appearance of secondary malignancies in patients receiving CAR T cell therapies has recently prompted regulatory scrutiny. Whereas the apparent frequency of such events is deemed low, and their risk is greatly outweighed by therapy's benefits, it is important to understand whether a causal link between the emergence of the secondary malignancies and CAR T cell therapy may exist. Herein, we interrogated FFPE tissue sections from a patient with sarcoma that emerged after CD20.19.22 tri-specific CAR-T cells therapy for mantle cell lymphoma (Clinicaltrials.gov identifier: NCT05418088), to reveal the state of the tumor, stroma, their interactions with the immune compartment, and to assess the possibility of CAR-T cells involvement in the emergence of this malignancy.
Patient History and Methods
The patient is a 63-year-old male with mantle cell lymphoma and was treated with CD20.19.22 tri-specific CAR-T cells. He achieved a durable remission that has lasted 18 months. On protocol-specified month 12 imaging, an incidental finding of indeterminate nodule was noted on CT and found to be FDG-avid on PET. Biopsy showed an undifferentiated Pleomorphic Sarcoma, FNCLCC Grade III/III [high grade], BRAF and KRAS negative. The patient underwent wide surgical excision and neoadjuvant radiation. Sarcoma was deemed unrelated to investigational treatment.Diagnostic sarcoma tissue was obtained for histopathology and MACSima analysis. Sarcoma FFPE sections were interrogated by MACSima™ Imaging Cyclic Staining (MICS) protein analysis of the tumor, stroma, and immune tissue compartments, in combination with specifically amplified oligonucleotide detection probe (RNAsky®) analysis to investigate the presence of CAR mRNA transcripts in T cells residing in the sarcoma tissue. Spatial data analysis and visualization was performed on MACS iQ View software.
RNASky probe specific for the CD20.19.22 tri-specific CAR-T cells cells sequence was validated on normal donor T cells transduced with CD20.19.22 CAR lentiviral vector after CAR T cell surface expression was confirmed by flow cytometry. CAR T cells and untransduced T cell controls were stained with CAR-specific RNASky probe, and positive control probes for housekeeping genes PSMB4 and VCP in a MACSwell 24 plate, and counterstained with DAPI to visualize cell nuclei.
FFPE tissue sections were subjected to protein analysis using a 41-marker custom antibody panel. Agglomerative hierarchical clustering revealed distinct cellular compartments of sarcoma cells with varying proliferative potential, muscle and endothelial vascular compartments, stromal compartment, monocytes, inhibitory myeloid cells, and several T cell sub-populations.
Results
The specificity of the RNASky probe was first confirmed in vitro by CAR-specific punctate staining in CAR-positive T cells generated from a healthy donor, but not in untransduced matched T cells control. When applied to the FFPE sarcoma tissue section and to an unrelated human tonsil tissue specimen as a control, no appreciable CAR-specific staining was observed in CD3+ T cells in the sarcoma tissue specimen.
Interactions of a subset of CD11c+ PD-L1+ cells with CD8+PD-1+ T cells were identified in the sarcoma tissue, highlighting an opportunity for checkpoint blockade intervention. Likewise, high abundance of TGF beta in the Collagen-1+ ECM stromal compartment underscored an opportunity for therapeutic blockade of TGF beta. By contrast, CD3+CD4+FoxP3+ Tregs were scarce, indicating minimal involvement in this tumor lesion.
Conclusions
The combined MACSima MICS and RNASky analysis of the tumor FFPE specimen identified sarcoma-typical tumor and stromal features, and moderate T cell infiltration. We uncovered the presence of PD-1-dependent and TGF beta-dependent mechanisms of immune resistance, however we detected no significant presence of CAR-positive T cells in the clinical specimen. This novel methodology allows for comprehensive tumor and immune microenvironment profiling to identify potential therapeutic targets. Studies are underway to validate these preliminary findings.