Abstract LB465: Establishing quantitative image analysis driven multiplex immunofluorescence panels for retrospective clinical research

Abstract Multiplex immunofluorescence (mIF) is increasingly used in spatial biology to profile protein expression within the tumor microenvironment and to characterize immune cell populations in situ. Unlike conventional single-marker immunohistochemistry (IHC), mIF enables concurrent visualization of multiple biomarkers on a single formalin-fixed, paraffin-embedded (FFPE) tissue section, allowing for comprehensive assessment of cellular phenotypes, spatial interactions, and architectural organization. This capability supports deeper investigation of tumor-immune dynamics and offers value in predicting response to immunotherapy. To leverage the powerful capabilities of mIF, Discovery developed and validated “off the shelf” panels. Individual biomarkers were initially optimized as single-plex IHC assays prior to integration into mIF panels. Panel development involved evaluation of fluorophore-marker pairing to maintain signal integrity and minimize spectral overlap. Epitope stability under repeated retrieval and stripping cycles was assessed to inform optimal staining sequence. Antibody titration studies were performed to establish appropriate dynamic range and linearity. Following panel finalization, analytical sensitivity was assessed across selected tumor types to quantify signal intensity and spatial complexity. Analytical precision was evaluated to confirm consistent performance. The resulting panels were validated for research use only (RUO) and applied to retrospective clinical patient samples. In this study, Discovery employed a tyramide signal amplification-based mIF workflow to evaluate several panels containing up to eight biomarkers, including T-cell markers (e. g. CD3, CD8, and FoxP3), immune checkpoint markers (e. g. PD-L1), and macrophage markers (e. g. CD68 and CD163), in both tumor and normal tissue samples. Multispectral imaging and machine learning-based image analysis enabled accurate cell segmentation, phenotyping, and spatial context analysis at subcellular resolution. This work highlights Discovery’s mIF service capabilities as a powerful tool for in-depth spatial characterization of the tumor immune microenvironment. The validated RUO panels offer a reproducible and customizable approach for biomarker discovery and translational research, with potential applications in companion diagnostics. Future studies will focus on evaluating assay performance in clinical trial settings and further investigating the potential of mIF to inform immunotherapy response. Citation Format: Salome Tchotorlishvili, Brigham Rhoads, Sarah Gordon, Luke Szyszkiewicz, Jacob Lewis, Karen Kirchner, Noreen McBrearty. Establishing quantitative image analysis driven multiplex immunofluorescence panels for retrospective clinical research abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts) ; 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86 (8Suppl): Abstract nr LB465.