Abstract PS4-01-26: Development and Validation of TROP2/HER2-low/Ki67/HR multiplex-immunofluorescence panel with membrane identification: enabling improved Antibody Drug Conjugate therapy selection

Abstract Background Antibody-drug conjugates (ADCs) have emerged as a promising therapeutic class for solid tumors, especially in breast cancer (BC), with a focus on agents targeting HER2 and Trop2. Currently, two ADC drugs targeting HER2: Trastuzumab emtansine (T-DM1) and fam-trastuzumab deruxtecan-nxki (T-DXd), and two targeting Trop2: Sacituzumab govitecan (SG) and datopotomab deruxtecan-dlnk (Dato-DXd) have gained approval by the FDA in treating metastatic BC, with additional ADCs in development. Despite the advances, challenges remain in patient selection including identifying the HER2 “ultra-low” patient subgroup, characterizing HER2/Trop2 co-expression, and ensuring reliable evaluation of biomarkers on the cell membrane surface. Here, we present the fully validated Opal™ multiplex-immunofluorescence (mIF) BC-ADC panel, leveraging the IVD-grade Akoya PhenoImager HT imaging system. The BC-ADC panel provides simultaneous assessment of BC standard-of-care biomarkers with Ki67 and hormone receptor (HR), ADC-specific targets HER2 and TROP2, together with a proprietary membrane marker cocktail facilitating specific biomarker membrane evaluation. Methods A proprietary membrane cocktail, comprised of multiple membrane-specific biomarkers, was developed to specifically label and visualize cell membranes. The membrane cocktail performance supporting accurate cell definition and subcellular localization was assessed against ground truth boundaries created by board-certified pathologists. Biomarkers TROP2, HER2, Ki67, ER, PR and the membrane cocktail, plus DAPI were tested for staining order effects and spectral unmixing, then developed into an single slide Opal™ 6-color multiplex immunofluorescence panel. HER2-positive/HER2-negative breast cancer tissue was utilized for assay development and verification. Board-certified pathologists evaluated panel validation according to the pre-set acceptance criteria including: absence of spectral crosstalk or umbrella effect by antibody drop-test; assay robustness by inter/intra-day triplicate precision studies, and biomarker sensitivity and specificity confirmed by equivalency between “gold-standard” IHC and mIF. HER2-negative breast cancer samples utilized for testing contained samples spanning HercepTest score 0+ (null or ultra-low), score 1+ (low), and score 2+ (low). Assay HER2 signal sensitivity was evaluated by pathologists to differentiate HER2-negative scores qualitatively. Results The membrane cocktail consistently delineates cell membrane in both tumor and stromal compartments. The finalized mIF assay met the predefined validation criteria as assessed by board-certified pathologists. Antibody drop-testing revealed no umbrella effect or spectral crosstalk was observed between channels. The precision study demonstrated high reproducibility in inter-day comparison and intra-day comparison. Strong equivalency of each biomarker was observed between IHC and the mIF panel. Pathologist review confirmed the high sensitivity in differentiating HER2 expression levels across HER2-negative cases: null/ultra-low/low. Conclusion The validated 5-plex/6-color BC-ADC mIF panel demonstrated robust analytical performance: high sensitivity and specificity across all biomarkers, especially in HER2-negative samples, stratifying HER2 low and ultra-low subgroups. The mIF panel showed reliable isolation of markers and robust assay performance. Results also suggested that the custom membrane cocktail can enhance the subcellular evaluation of biomarkers. The study will be supplemented by an independent patient verification cohort to support forthcoming clinical-utility studies. Citation Format: Y. Liu, M. Parrella, S. Schwaegerle, S. Berry, C. Betts, M. Landers. Development and Validation of TROP2/HER2-low/Ki67/HR multiplex-immunofluorescence panel with membrane identification: enabling improved Antibody Drug Conjugate therapy selection [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS4-01-26.