{
  "abstract": "Background Antibody-drug conjugates (ADCs) are a rapidly expanding class of therapeutics targeting tumor-associated antigens. Accurate, spatially resolved detection of multiple ADC targets in tumor tissues is essential to support research into target expression and potential mechanisms of response or resistance. Multiplex immunofluorescence (mIF) offers a powerful approach to visualize co-expression of ADC targets alongside immune markers in situ. Here we describe the development and early applications of a mIF assay and workflow designed for flexible profiling of ADC-relevant biomarkers in FFPE tissues.Methods A ready-to-use 4-plex immunofluorescence detection kit (Research Use Only) was deployed on the BOND RX from Leica Biosystems for automated staining of formalin-fixed, paraffin-embedded (FFPE) tissue sections. Slides were imaged using the Aperio VERSA digital scanner. Marker panels included combinations of HER2, Trop2, folate receptor alpha (FRα), and cMET—key targets in current and emerging ADC pipelines—along with CD8 to characterize tumor-infiltrating lymphocytes (TILs). Tumor types included NSCLC (HER2, Trop2, FRα, cMET), HER2+ breast carcinoma (HER2, Trop2, cMET, CD8), and high-grade serous ovarian carcinoma (FRα, Trop2, cMET, CD8). Multispectral analysis was performed to evaluate marker co-localization and spatial relationships between tumor and immune compartments, and further image analysis is ongoing to quantify these analyses.Results Initial multiplex staining demonstrated high-quality, specific signal for all included markers, with clear cellular localization and minimal background. Stained slides were independently reviewed by three board-certified pathologists, who confirmed that staining intensity, localization, and interpretability were similar to conventional single-marker chromogenic immunohistochemistry. Observed expression patterns were consistent with known tumor biology. Spatial profiling of tumor and immune markers further highlighted the assay’s potential for evaluating co-expression and spatial relationships within the tumor microenvironment. Image analysis revealed correlations between TIL number and ADC target expression across tissue types. Further quantification is being performed to confirm these observations and translate them for future studies.Conclusions The workflow highlighted here enables staining of ADC-relevant biomarkers and immune markers in a single FFPE section. The assay shows potential to support translational research efforts into ADC development strategies. Future work will expand the number of panels and tissues evaluated and explore integration with high-content image analysis for quantitative spatial biomarker assessment.",
  "authors": [
    {
      "affiliations": [
        "Leica Biosystems, Newcastle, UK"
      ],
      "name": "Stacey Lindsay"
    },
    {
      "affiliations": [
        "Leica Biosystems, Newcastle, UK"
      ],
      "name": "Jessica Baumann"
    },
    {
      "affiliations": [
        "Leica Biosystems, Newcastle, UK"
      ],
      "name": "Mark Burton"
    },
    {
      "affiliations": [
        "Leica Biosystems, Atlanta, GA, USA"
      ],
      "name": "Jack Heath"
    },
    {
      "affiliations": [
        "Leica Biosystems, Newcastle, UK"
      ],
      "name": "Elizabeth Sullivan"
    },
    {
      "affiliations": [
        "Leica Biosystems, Newcastle, UK"
      ],
      "name": "Rachel C Jones"
    }
  ],
  "title": "1233 Multiplex immunofluorescence workflow for high-throughput in situ profiling of immune targets in FFPE tissues",
  "uid": "fa5e4668-7664-57c9-b54c-71f46f46d496"
}