Quantitative Multiplex Immunofluorescence Assay for Trophoblast Cell-Surface Antigen 2 and Human Epidermal Growth Factor Receptor 2 Expression in Breast Cancer: Toward Guiding Patient Selection for Antibody-Drug Conjugate Therapies.

Purpose: Accurate quantification of human epidermal growth factor receptor 2 (HER2) and trophoblast cell-surface antigen 2 (TROP2) expression could aid in identifying patients with cancer likely to benefit from emerging HER2 and TROP2 antibody-drug conjugate (ADC) therapies or potentially help oncologists choose which drug to use first, on the basis of the level of the ADC target in the tumor. We developed a standardized multiplex quantitative immunofluorescence (QIF) assay to simultaneously measure HER2 and TROP2 protein levels in cancer tissue.

Materials and methods: A multiplex QIF assay was optimized on tissue microarrays (TMAs) by selecting optimal antibody clones and concentrations to achieve maximal signal-to-noise ratios. We create and release Qymia, a QuPath extension to enable simultaneous molecular compartmentalization and fluorescence quantification in TMAs and whole-slide images. Calibration curves, generated from cell line microarrays with HER2/TROP2 measured by mass spectrometry, were used to convert QIF signal into protein concentrations (attomoles/mm²). The validated assay was applied to a serial collection of 323 breast cancer specimens in TMA format to characterize HER2 and TROP2 expression distributions.

Results: The assay demonstrated linearity across a wide dynamic range of biomarker expression with strong interassay and interoperator reproducibility. Application to 323 breast cancer TMA specimens revealed a weak inverse correlation between HER2 and TROP2 (r = -0.17; P = .001). HER2 was detectable in approximately 85% of TMA cores, including 51% of triple-negative breast cancer TMA cores. TROP2 was detectable in over 96% of specimens across all subtypes.

Conclusion: This multiplex immunofluorescence assay provides an approach to accurately and precisely measure HER2/TROP2 levels within breast cancer tissue and compare relative levels of target expression in a breast cancer tissue population. This assay is now ready for studies to assess clinical validity and utility.