{
  "abstract": "Background The emergence of HER2-targeted antibody-drug conjugates, such as trastuzumab deruxtecan (T-DXd), have transformed the therapeutic landscape for patients with HER2-low breast cancer. However, the biological basis distinguishing HER2-low (IHC 1+ or 2+/FISH-negative) from HER2-0 (IHC 0) tumors, particularly within the hormone receptor-positive (HR+) subset, remains unclear. Understanding the tumor microenvironment (TME) of these subtypes could inform tailored therapeutic strategies.Methods We analyzed treatment-naïve primary HR+ (IHC ER-positive ~90%) breast tumors classified as HER2-low (n=17) or HER2-0 (n=8) using bulk RNA sequencing, single-nucleus RNA sequencing (sNuc-seq), and multiplexed cyclic immunofluorescence (CyCIF). Cellular compositions, gene expression profiles, pathway enrichments, and spatial cell-cell relationships were assessed across tumor, immune, and stromal compartments.Results Bulk and single-nucleus transcriptomic analyses revealed that HER2-0 tumors were enriched in immune-related pathways, including interferon-gamma and inflammatory responses, while HER2-low tumors exhibited upregulation of cell cycle, oxidative phosphorylation, and estrogen signaling. sNuc-seq confirmed increased immune cell infiltration in HER2-0 tumors, with higher frequencies of CD4 + T cells, CD8+ T cells, dendritic cells, and macrophages. Notably, HER2-0 tumors harbored a unique APOE+ tumor-associated macrophage (TAM) subset co-expressing lipid metabolism and antigen presentation genes. Spatial analysis demonstrated enrichment of CD68+APOE+ macrophages in proximity to CD4+ T cells in HER2-0 tumors, suggesting coordinated immune activity. Furthermore, a cytotoxic CD4+ T cell population (CD4-DOCK4+), expressing GZMA, GZMK, and IFN-related genes, was significantly enriched in HER2-0 tumors. Stromal analysis identified a subset of collagen-expressing cancer-associated fibroblasts (C4-CAFs) enriched in HER2-0 tumors, associated with tissue remodeling and angiogenesis pathways. Cell-cell communication analysis revealed increased CAF-endothelial interactions in HER2-0 tumors compared to HER2-low.Conclusions HER2-0 and HER2-low HR+ breast tumors exhibit distinct immune and stromal landscapes. HER2-0 tumors are characterized by immune activation, lipid-associated APOE + macrophages, and cytotoxic CD4+ T cells, along with tissue remodeling CAFs. Conversely, HER2-low tumors display proliferative and immunosuppressive profiles dominated by estrogen signaling. These findings offer mechanistic insights into HER2 spectrum biology and provide a rationale for tailored therapeutic interventions, including the integration of immune-modulatory strategies with HER2-directed ADCs.Acknowledgements This work was supported by the Susan G. Komen Foundation (ASPIRE1039903, CCR18547597; FAPESP 2021/12898-8), The Harvard Ludwig Center, NIH DF/HCC SPORE in Breast Cancer (P50 CA168504), NIH NCI (R37 CA269499), and The Concern Foundation. A.N. is supported by a Canadian Institute of Health Postdoctoral Fellowship (FRN: 194068).Ethics Approval Treatment naive breast tumors used in this study were collected with written informed consent from all patients under DFCI protocol 93-085 with approval from all relevant human research ethics committees (Dana-Farber Cancer Institute Ethics Committee)",
  "authors": [
    {
      "affiliations": [
        "Brigham and Women’s Hospital, Boston, MA, USA",
        "Harvard Medical School, Boston, MA, USA"
      ],
      "name": "Carlos Wanderley"
    },
    {
      "affiliations": [
        "Brigham and Women’s Hospital, Boston, MA, USA",
        "Harvard Medical School, Boston, MA, USA",
        "Laboratory of Systems Pharmacology, Boston, MA, USA"
      ],
      "name": "Daniel E Michaud"
    },
    {
      "affiliations": [
        "Harvard Medical School, Boston, MA, USA"
      ],
      "name": "Kenichi Shimada"
    },
    {
      "affiliations": [
        "Brigham and Women’s Hospital, Boston, MA, USA"
      ],
      "name": "Adam Nelson"
    },
    {
      "affiliations": [
        "Dana-Farber Cancer Institute, Boston, MA, USA"
      ],
      "name": "Jingxin Fu"
    },
    {
      "affiliations": [
        "Brigham and Women’s Hospital, Boston, MA, USA"
      ],
      "name": "Stuart J Schnitt"
    },
    {
      "affiliations": [
        "Dana-Farber Cancer Institute, Boston, MA, USA"
      ],
      "name": "Sara M Tolaney"
    },
    {
      "affiliations": [
        "Dana-Farber Brigham Cancer Center, Boston, MA, USA"
      ],
      "name": "Elizabeth A Mittendorf"
    },
    {
      "affiliations": [
        "Dana-Farber Brigham Cancer Center, Boston, MA, USA"
      ],
      "name": "Romualdo Barroso-Sousa"
    },
    {
      "affiliations": [
        "Dana-Farber Cancer Institute, Boston, MA, USA"
      ],
      "name": "Adrienne Waks"
    },
    {
      "affiliations": [
        "Harvard Medical School, Boston, MA, USA",
        "Dana-Farber Cancer Institute, Boston, MA, USA"
      ],
      "name": "Paolo Tarantino"
    },
    {
      "affiliations": [
        "Brigham and Women’s Hospital, Boston, MA, USA"
      ],
      "name": "Jennifer L Guerriero"
    }
  ],
  "title": "1270 APOE tumor-associated macrophages and CD4-DOCK4 T cells reveal distinct microenvironmental features in HER2-Low and HER2–0 hormone receptor-positive breast cancer",
  "uid": "4bc6fc48-2b1d-5c9c-80bb-bf9ddde11ae8"
}