{
  "abstract": "Background Chimeric antigen receptor (CAR) T-cell therapy, effective in certain hematologic malignancies, still has the potential risk of T cell malignancy post CAR-T treatment. We investigated FFPE tissue sections from a patient with sarcoma that emerged after CD20.19.22 tri-specific CAR-T cell therapy for mantle cell lymphoma (Clinicaltrials.gov: NCT05418088). We explored whether any links between the emergence of this secondary malignancy and CAR-T cell therapy could be established. This was achieved using spatial RNA and proteomic analysis, pre- and post-radiotherapy, combined with a novel approach to identify CAR-T using sequence-specific oligonucleotide probes.Methods The MACSima™ Imaging Cycling Staining (MICS) system was used to detect and visualize CAR T cells using RNASky probes (MICS-rna) on T cells transduced with CAR lentiviral vector (LV), sarcoma tissue, and human tonsil, followed by sequential protein staining. FFPE sections from the patient sarcoma biopsy, (pre- and post-irradiation) were imaged using 40 protein markers. Multiplex image processing and segmentation, unsupervised UMAP clustering, and target quantitation were performed using MACS® iQ view software.Results MICS-rna detected a 54.9% (± 6.6%; n=3 donors) frequency of CAR-positive cells in the product which correlated with CAR detection by flow cytometry (46.6 ± 11.05%). Total T cells were detected with anti-CD3 antibody in sarcoma and tonsil tissue samples. Spatial analysis of the tumor microenvironment was performed in pre- and post- irradiation tissue with circa 11,000 and 14,000 cells analyzed in the tumor region, respectively. Unbiased agglomerative cell clustering using 40 protein markers and overlaid with UMAPs, identified four subgroups of tumor cells (tumor high proliferation, tumor med proliferation, tumor, and tumor metabolic-active), myeloid cell populations (TGF-β1 + and TGF-β1-), two subgroups of T cells and PD1+ exhausted T cells, stroma cells, vessels-endothelium, and muscle. The lesion contained 38.38% tumor cells in the pre- irradiation tissue, and dropped to 21.4% post- irradiation. Strikingly, post irradiation, T lymphocytes were no longer present and multiple groups of fibroblast-like cells emerged (including CAFs), accounting for 62.09% of the total.Conclusions In situ spatial proteomic and RNA analysis of a secondary sarcoma post cell therapy treatment support the finding that the secondary malignancy was not directly associated with the presence of CAR T cells. This novel methodology allows complex spatial profiling of both the tumor, microenvironment and immune cell infiltrates, providing a precise approach for targeted clinical interventions based on patient biopsies.",
  "authors": [
    {
      "affiliations": [
        "Lentigen Technology Inc., a Miltenyi Biotec Company, Gaithersburg, MD, USA"
      ],
      "name": "Jia-Jye Lee"
    },
    {
      "affiliations": [
        "Lentigen Technology Inc., a Miltenyi Biotec Company, Gaithersburg, MD, USA"
      ],
      "name": "Peirong Hu"
    },
    {
      "affiliations": [
        "Lentigen Technology Inc., a Miltenyi Biotec Company, Gaithersburg, MD, USA"
      ],
      "name": "Kun Luo"
    },
    {
      "affiliations": [
        "Miltenyi Biotec, Inc., Waltham, MA, USA"
      ],
      "name": "Jinling Wang"
    },
    {
      "affiliations": [
        "Miltenyi Biotec, Inc., Waltham, MA, USA"
      ],
      "name": "Emily Neil"
    },
    {
      "affiliations": [
        "Miltenyi Biotec, Inc., Waltham, MA, USA"
      ],
      "name": "Dongju Park"
    },
    {
      "affiliations": [
        "Miltenyi Biotec, Inc., Waltham, MA, USA"
      ],
      "name": "Robert Pinard"
    },
    {
      "affiliations": [
        "Department of Internal Medicine, Division of Hematology, The Ohio State University, Columbus, OH, USA"
      ],
      "name": "Yazeed Sawalha"
    },
    {
      "affiliations": [
        "Department of Internal Medicine, Division of Hematology, The Ohio State University, Columbus, OH, USA"
      ],
      "name": "Nathan Denlinger"
    },
    {
      "affiliations": [
        "Department of Internal Medicine, Division of Hematology, The Ohio State University, Columbus, OH, USA"
      ],
      "name": "Evandro Bezerra"
    },
    {
      "affiliations": [
        "Lentigen Technology Inc., a Miltenyi Biotec Company, Gaithersburg, MD, USA"
      ],
      "name": "Pradyot Dash"
    },
    {
      "affiliations": [
        "The Ohio State University Wexner Medical Center, Columbus, OH, USA"
      ],
      "name": "Ashley Angell Krull"
    },
    {
      "affiliations": [
        "The Ohio State University Wexner Medical Center, Columbus, OH, USA"
      ],
      "name": "Lynn O’Donnell"
    },
    {
      "affiliations": [
        "James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA"
      ],
      "name": "Lapo Alinari"
    },
    {
      "affiliations": [
        "Department of Pathology, The Ohio State University, Columbus, OH, USA"
      ],
      "name": "Daniel Jones"
    },
    {
      "affiliations": [
        "Lentigen Technology Inc., a Miltenyi Biotec Company, Gaithersburg, MD, USA"
      ],
      "name": "Rimas J Orentas"
    },
    {
      "affiliations": [
        "Department of Internal Medicine, Division of Hematology, The Ohio State University – Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, Columbus, OH, USA"
      ],
      "name": "Macros de Lima"
    },
    {
      "affiliations": [
        "Department of Internal Medicine, Division of Hematology, The Ohio State University, Columbus, OH, USA"
      ],
      "name": "Sumithira Vasu"
    }
  ],
  "title": "243 Spatial and multiplex immunofluorescence imaging of secondary sarcoma following CAR T cell therapy for mantle cell lymphoma",
  "uid": "6cf4e01a-f203-5dfd-a030-72183a4d1301"
}