Determination of the optimal cell count for HER2 status assessment in fluorescence in situ hybridization

Human epidermal growth factor receptor 2 (HER2) is a membrane tyrosine kinase encoded by ERBB2, which is amplified in 15%-20% of breast cancers and serves as a critical prognostic and predictive biomarker for targeted therapy. Fluorescence in situ hybridization (FISH) is the reference standard for HER2 gene amplification assessment. The 2023 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines recommend assessing at least 20 cells in FISH but do not specify an optimal count. It remains unclear whether counting additional cells improves interobserver agreement. In this study, we aimed to determine the optimal number of cells to evaluate to accurately assess ERBB2 amplification status. We analyzed 179 invasive breast cancer cases with archival formalin-fixed, paraffin-embedded tissue subjected to HER2 immunohistochemistry and FISH, as well as clinical and pathologic data. Three independent observers selected the specimen areas to assess and determined HER2 status by examining 20, 40, or 60 cells. Interobserver agreement on HER2 status (HER2 positive vs. HER2 negative) was consistently strong across all counting tiers (Fleiss' κ = 0.81-0.87), with no clinically meaningful improvement at higher cell counts. Stratified analysis revealed that unequivocal cases (Groups 1 + 5, approximately 84% of the cohort) maintained excellent concordance (coefficient of variation <5%) at all tiers, while equivocal cases (Groups 2-4) exhibited persistently higher variability regardless of cell count. These findings validate the 20-cell minimum for routine FISH-based HER2 assessment and demonstrate that enhanced concordance in borderline cases requires adjudicatory workflows beyond additional cell counting.