Validation and Prospective Testing of a High-Sensitivity, Quantitative Analytic Assay for HER2 on Histopathology Slides

The recent approval of antibody-drug conjugates targeting human epidermal growth factor receptor 2 (HER2) (such as trastuzumab deruxtecan [T-DXd]) has led to challenges for the immunohistochemical (IHC) companion diagnostic test because the test was optimized for gene-amplified levels of HER2. Here, we develop and validate an objective test for low-level HER2 expression toward more accurate selection of patients for T-DXd. We validated the high-sensitivity HER2 assay using a mix of the requirements for an IHC assay and that of a ligand-binding assay. Then, we prospectively tested it on 316 core biopsy specimens received by the Yale Pathology Laboratories from August 2022 to August 2023. Using a 40-case breast cancer tissue validation set, we find very high accuracy and precision with a coefficient of variation <10% and define a reportable range for the assay in attomoles per square millimeter. These prospective cases not only show the dynamic range of HER2 expression but also the discordance of Yale Pathology Labs staff pathologist scores with quantitative measurements, especially in the low range of HER2. We find that 6% of the cohort was below the limit of detection of this more sensitive assay, whereas 71% of the IHC 0 cases were above the limit of quantification. Efforts are underway to determine a possible threshold expression level required for T-DXd response. In summary, this assay validation study provides a method for accurate, objective measurement of HER2 and has the potential to improve selection of patients for T-DXd or similarly targeted antibody-drug conjugate therapies in future.