Abstract 4944: Chromogenic triplex characterization of breast and gastrointestinal cancers for biomarker discovery and spatial image analysis

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/orPD-L1-positive breast and GI cancers were stained using CD3/HER2/PD-L1 andCD8/HER2/PD-L1 chromogenic multiplex IHC assays on the BOND RX RUO automated stainer from Leica Biosystems. The assays employed the Chromoplex III Triple Detection RUO system from LeicaBiosystems. 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, expression of PD-L1and TIL markers (CD3, CD8) varied, with a subset of cases showing high CD8+ T-cell infiltration, potentially indicative of a more immunogenic phenotype.​​HALO AI analysis provides insights into proximity ofCD3, CD8 and PDL1 positive infiltrating cells to HER2positive carcinoma cells within breast and stomach carcinoma cases providing valuable spatial analysis using chromogenic multiplexing.​​Visualization of 3 markers simultaneously using ChromoPlex III Triple Detection RUO allows automated spatial analysis of tumor samples.​​In HER2 positive breast cancer and stomach cancer cases higher density of CD3+ cells and CD8+ cells shown to be in closer proximity to HER2 positive cells with the highest number of CD3 or CD8+ cells seen within 10uM of a HER2+ cell. Conclusions: ChromoPlex III Triple Detection RUO can be used as a tool to identify 3 simultaneous markers in FFPE tissue using automated staining (BOND RX). As an example -multiplex chromogenic IHC staining of HER2, PD-L1,and CD3/CD8 provides valuable insights into the interplay between the tumor and the immune cell microenvironment in breast and GI cancers. The use of whole-slide digital pathology (Aperio GT 450 DX) and HALO AI Image Analysis, enables high-throughput assessment of multiplex chromogenic stained slides. HALO AI Image Analysis is a powerful tool for automated quantification of marker expression and proximity analysis. Citation Format: Andrew Fuller, Stacey Margerrison, Scott J. Anderson, Anne Hellebust. Chromogenic triplex characterization of breast and gastrointestinal cancers for biomarker discovery and spatial image analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4944.