Quantitative Imaging Analysis Fluorescence In Situ Hybridization Validation for Clinical HER2 Testing in Breast Cancer

center dot Context.-Quantitative imaging is a promising tool that is gaining wide use across several areas of pathology. Although there has been increasing adoption of morphologic and immunohistochemical analysis, the adoption of evaluation of fluorescence in situ hybridization (FISH) on formalin-fixed, paraffin-embedded tissue has been limited because of complexity and lack of practice guidelines. Objective.-To perform human epidermal growth factor receptor 2 (HER2) FISH validation in breast carcinoma in accordance with the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) 2018 guideline. Design.-Clinical validation of HER2 FISH was performed using the US Food and Drug Administration- California) with digital scanning performed on a PathFusystem. Validation parameters evaluated included z-stacking, classifier, accuracy, precision, software, and hardware settings. Finally, we evaluated the performance of digital enumeration on clinical samples in a real-world setting. Results.-The accuracy samples showed a final concor-dance of 95.3% to 100% across HER2 groups 1 to 5. During clinical implementation for HER2 groups 2, 3, and 4, we achieved a final concordance of 76% (95 of 125). Of these cases, only 8% (10 of 125) had discordances with clinical impact that could be identified algorithmically and triaged for manual review. Conclusions.-Digital FISH enumeration is a useful tool to improve the efficacy of HER2 FISH enumeration and capture genetic heterogeneity across HER2 signals. Ex-cluding cases with high background or poor image quality and manual review of cases with ASCO/CAP group discordances can further improve the efficiency of digital HER2 FISH enumeration.