Prospective assessment of a quantitative immunofluorescent assay to measure HER2 and TROP2 concentrations on a single tissue biopsy slide

Abstract With over 700 antibody-drug conjugates (ADCs) in clinical trials and multiple approvals for the same indications, new challenges arise in protein-based diagnostics. In general, higher levels of ADCs target specific proteins are associated with increased response to the drug. We believe a new biomarker category (selective biomarkers) is needed to help the physician decide which ADC best matches the patient’s tumor biology. Toward that goal, we have produced the Troplex™ assay to quantitatively measure concentrations of both HER2 and TROP2 in attomoles/mm 2 on a single tissue slide. In this study, we conducted a prospective assessment of the Troplex assay on 160 breast cancer core biopsies collected from Yale Pathology Labs between 2023 and 2024 to compare relative levels of target expression. Troplex assay demonstrated a linear range for TROP2 between 556 and 21966 amol/mm² and for HER2 between 105 and 7184 amol/mm². The assay measured HER2 levels below the limit of detection (LOD) in 2% (3/160) and above LOD in 98% (157/160) of cases. No significant correlation was found between HER2 and TROP2 expression in this cohort (P=0.8, R²=0.0003, Pearson r=0.017). Efforts are underway to determine the relationship of each ADC target with the ADC drug response. This study represents a proof of concept of a selective biomarker for two ADC targets, validation of the assay and demonstration of the relative levels of each target in real world breast cancer tissue. As more ADCs are approved, we envision similar assays may be built to allow selection of the ADC drug that best matches the patient’s tumor biology.