{
  "abstract": "Background HER2 and PD-L1 expression serve as key biomarkers in breast and gastrointestinal (GI) cancer research. The tumor immune microenvironment, particularly the presence of tumor-infiltrating lymphocytes (TILs), also plays a critical role in these cancers, with mounting evidence of the importance of the signaling between the tumor and its environment. In this study, we evaluated two multiplex chromogenic immunohistochemistry (IHC) assays, HER2/PD-L1/CD3 and HER2/PD-L1/CD8, to assess the relationships among their expressions and related signaling pathways.Methods Formalin-fixed, paraffin-embedded (FFPE) tissue sections from HER2-expressing and/or PD-L1-positive breast and GI cancers were stained using HER2/PD-L1/CD3 and HER2/PD-L1/CD8 chromogenic multiplex IHC assays on the BOND RX RUO automated stainer from Leica Biosystems. The assays employed distinct chromogens in a sequential staining protocol optimized in the Leica Biosystems Center for Enabling Precision Medicine (Newcastle, UK). Whole-slide imaging was performed on the Aperio GT 450 DX scanner, and HALO® digital image analysis from Indica Labs was used to quantify biomarker expression with the Multiplex IHC module leveraging HALO AI nuclear and membrane segmentation. Relationships between identified tumor cells and immune infiltrates were further interrogated with the Spatial Analysis module.Results In HER2-positive breast cancer, co-expression of PD-L1 and TIL markers (CD3, CD8) varied, with a subset of cases showing high CD8+ T-cell infiltration, potentially indicative of a more immunogenic phenotype.In HER2-low breast cancer, PD-L1 expression was more heterogeneous, and CD3+/CD8+ infiltration patterns provided insights into the immune landscape of these tumors, with potential implications for interplay between these important biomarkers and the neighboring microenvironment.In gastric and gastroesophageal junction (GEJ) adenocarcinoma, HER2+/PD-L1+ tumors exhibited distinct immune profiles, with some cases demonstrating high CD8 density, suggesting possible synergy between HER2-targeted therapy and immune checkpoint blockade.In HER2-amplified colorectal cancer, PD-L1 expression and immune infiltration were variable, highlighting the importance of visualizing the markers together in one assay.Conclusions Multiplex chromogenic IHC staining of HER2, PD-L1, and CD3/CD8 provides valuable insights into the interplay between oncogenic signaling and the tumor immune microenvironment in breast and GI cancers. The use of automated staining (BOND RX RUO automated stainer) and whole-slide digital pathology (Aperio GT 450 DX) enables high-throughput assessment of these biomarkers, with potential applications in biomarker analysis and discovery. This a research study only and is not intended to infer any diagnostic use and/or utility of the PD-L1 (CAL10) RUO BOND RTU Primary.",
  "authors": [
    {
      "affiliations": [
        "Leica Biosystems, Newcastle, UK"
      ],
      "name": "Andrew Fuller"
    },
    {
      "affiliations": [
        "Leica Biosystems, Newcastle, UK"
      ],
      "name": "Claire Bowen"
    },
    {
      "affiliations": [
        "Leica Biosystems, Newcastle, UK"
      ],
      "name": "Mark Burton"
    },
    {
      "affiliations": [
        "Leica Biosystems, Atlanta, GA, USA"
      ],
      "name": "Jack Heath"
    },
    {
      "affiliations": [
        "Leica Biosystems, Newcastle, UK"
      ],
      "name": "Tom McNicholas"
    },
    {
      "affiliations": [
        "Leica Biosystems, Newcastle, UK"
      ],
      "name": "Elizabeth Sullivan"
    },
    {
      "affiliations": [
        "Indica Labs, Albuquerque, NM, USA"
      ],
      "name": "Anne E Hellebust"
    },
    {
      "affiliations": [
        "Leica Biosystems, Newcastle, UK"
      ],
      "name": "Scott J Anderson"
    }
  ],
  "title": "65 Chromogenic triplex characterization of breast and gastrointestinal cancers for biomarker discovery and spatial image analysis",
  "uid": "5be31cbe-cda1-52c0-b624-bb99315cbfe3"
}