{
  "abstract": "Background Metastatic BRAFV600E mutant colorectal cancer (CRC) has a poor prognosis, even with targeted molecular therapies. We previously found that the combined BRAF/MEK/PD1 inhibition showed an overall response rate of 24.3%, and induced upregulation of an interferon-stimulated gene (ISG) program in malignant cells. 1 However, the mechanism underlying this response has not been well studied.Methods We analyzed over 300,000 immune and stromal cells from 23 paired pretreatment and day 15 on-treatment tumor biopsies profiled by scRNAseq. Progression-free survival greater than 6 months was defined as a clinical response. We performed hierarchical clustering to annotate fine-grained cell subtypes for T and myeloid cells, and tested the association of subtype abundance with treatment and response to identify associated subtypes. With the cell subtypes identified in scRNAseq analysis, we tracked their TCR repertoire in tumor and blood respectively to analyze the clonal expansion pattern, especially for the associated subtypes.We next analyzed over 200,000 cells from 16 biopsies profiled by spatial transcriptomics (Xenium) with a custom 480-gene panel. We performed cell segmentation, cell type annotation integrative of scRNAseq data, tissue segmentation, cell type abundance analysis within spatial context and spatial colocalization analysis.Results Single-cell RNA sequencing analysis of 23 paired pretreatment and day 15 on-treatment tumor biopsies revealed increased abundance of CXCL10+ macrophages (FC = 3.4, p = 0.0039) and activated CD8 T cells, particularly HOBIT+ CD8 T cells (FC = 1.6, p=0.0098) for responders with treatment.T-cell receptor analysis showed activated CD8 T cells exhibited robust expansion on-treatment in several responders, though this expansion is not sustained and these patients ultimately relapsed.Spatial transcriptomic analysis revealed these cell types, especially CXCL10+ macrophages and activated CD8 T cells were spatially colocalized with each other, and showed broadly increasing abundance at multiple distances from the stromal/neoplastic epithelium interface with treatment, indicating the potential de novo formation and expansion of immunity hubs in response to the combined therapy.Conclusions Together, these results show that even in late-stage BRAFV600E CRC, combined BRAF/MEK/PD-1 inhibition can induce an immune response and remodel the tumor immune microenvironment systematically. Ongoing work will explore the biological mechanism driving immune remodelling in response to the combined therapy by performing spatially differentially expressed gene (DEG) analysis and ligand receptor analysis to identify relevant genes, pathways and cell-cell communication networks.Trial Registration This study was approved by the DF/HCC IRB as protocol 18-144 (see also ClinicalTrials.gov NCT03668431).Reference Tian J, Chen J H, Chao S X, et al. Combined PD-1, BRAF and MEK inhibition in BRAFV600E colorectal cancer: a phase 2 trial[J]. Nature Medicine 2023;29(2):458–466.Ethics Approval This study was approved by the DF/HCC IRB as protocol 18-144 (see also ClinicalTrials.gov NCT03668431).",
  "authors": [
    {
      "affiliations": [
        "Brigham and Women’s Hospital, Boston, MA, USA"
      ],
      "name": "Hongcheng Yao"
    },
    {
      "affiliations": [
        "Massachusetts General Hospital Cancer Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA"
      ],
      "name": "Vjola Jorgji"
    },
    {
      "affiliations": [
        "Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, MA, USA"
      ],
      "name": "Alexander Tang"
    },
    {
      "affiliations": [
        "Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, MA, USA"
      ],
      "name": "David Lieb"
    },
    {
      "affiliations": [
        "Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, MA, USA"
      ],
      "name": "Sherry Chao"
    },
    {
      "affiliations": [
        "Department of Biomedical Informatics, Harvard Medical School, Boston, MA, USA"
      ],
      "name": "Daniel Stein"
    },
    {
      "affiliations": [
        "School of Medicine, Yale University, New Haven, CT, USA"
      ],
      "name": "Maxwell Spurrell"
    },
    {
      "affiliations": [
        "Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, MA, USA"
      ],
      "name": "Liad Elmelech"
    },
    {
      "affiliations": [
        "School of Medicine, University of Pennsylvania, Philadelphia, PA, USA"
      ],
      "name": "Joshua Pirl"
    },
    {
      "affiliations": [
        "Massachusetts General Hospital Cancer Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA"
      ],
      "name": "Jun Tian"
    },
    {
      "affiliations": [
        "Massachusetts General Hospital Cancer Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA"
      ],
      "name": "Moshe Sade-Feldman"
    },
    {
      "affiliations": [
        "Gladstone-UCSF Institute of Genomic Immunology, Gladstone Institutes, San Francisco, CA, USA"
      ],
      "name": "Karin Pelka"
    },
    {
      "affiliations": [
        "Massachusetts General Hospital Cancer Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA"
      ],
      "name": "Ryan Corcoran"
    },
    {
      "affiliations": [
        "Massachusetts General Hospital Cancer Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA"
      ],
      "name": "Nir Hacohen"
    },
    {
      "affiliations": [
        "Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA"
      ],
      "name": "Ilya Korsunsky"
    },
    {
      "affiliations": [
        "Feinberg School of Medicine, Northwestern University, Cambridge, MA, USA"
      ],
      "name": "Jonathan Chen"
    }
  ],
  "title": "498 Integrated scRNAseq, TCR repertoire and spatial transcriptomic analysis reveals immune remodeling induced by combined BRAF/MEK/PD-1 inhibition in metastatic BRAFV600E colorectal cancer",
  "uid": "5dba6433-675f-5df2-b464-bda795e517bb"
}