Automated imaging workflow for FISH-stained tissue microarrays enables assessment of FGFR2 translocations in a large intrahepatic cholangiocarcinoma patient cohort

BACKGROUND: Fibroblast growth factor receptor-2 (FGFR2) translocations are frequently observed in intrahepatic cholangiocarcinoma (iCCA) and represent actionable therapeutic targets. While fluorescence in situ hybridization (FISH) is commonly used for their detection, its applicability to large cohorts, particularly on tissue microarrays (TMAs), is limited by time-consuming and error-prone manual scoring due to core disorientation. We therefore sought to develop an automated imaging workflow to enable efficient and scalable assessment of FGFR2 translocations in FISH-stained TMAs. METHODS: Tumor samples from 86 iCCA patients were obtained to construct TMAs, which underwent FISH staining for the detection of FGFR2 translocations. Slides were imaged with an automated inverted wide-field fluorescence microscope. First, a whole-slide overview scan at low resolution was acquired to identify TMA cores. Subsequently, a high-resolution automated scan was performed across the identified regions of interest (ROI) using a custom-built general analysis script with optimized parameters for single-cell analysis. Images were stored with positional metadata for downstream evaluation. RESULTS: Of 86 cases, 85 yielded images with sufficient quality for FGFR2 translocation analysis. Interphase FISH evaluation of the generated images revealed that 8.2% (7/85) of patients tested positive, consistent with reported frequencies. CONCLUSIONS: We developed an automated imaging workflow for high-throughput acquisition and archiving of FISH-stained TMAs for FGFR2 translocation assessment in iCCA. By enabling standardized digital imaging, precise core identification, and permanent image preservation, the workflow facilitates efficient and reproducible review of FISH images from large TMA cohorts in both diagnostic and translational research settings. It is readily adaptable to other FISH-based assays and staining protocols, extending its utility beyond FGFR2 testing.