HER2 signals were visualized as discrete black metallic silver dots while CEN 17 signals were detected as slightly larger red dots. Sequential hybridization and signal detection for HER2 and CEN 17 ISH demonstrated both DNA targets in the same cells. Interpretation of HER2 and CEN 17 BDISH slides was conducted by 4 observers using a conventional brightfield microscope without oil immersion objectives. Then, the BDISH performance was evaluated with 94 routinely processed breast cancer tissues. The BDISH specificity was optimized with formalin-fixed, paraffin-embedded xenograft tumors, MCF7 (non-amplified HER2 gene) and BT-474 (amplified HER2 gene). Detection of HER2 and CEN 17 signals was accomplished with the silver acetate, hydroquinone, and H 2O 2 reaction with horseradish peroxidase (HRP) and the fast red and naphthol phosphate reaction with alkaline phosphatise (AP), respectively. The BDISH assay was developed with the nick translated dinitrophenyl (DNP)-labeled HER2 DNA probe and DNP-labeled CEN 17 oligoprobe on the Ventana BenchMark ® XT slide processing system. Our objective was to develop an automated brightfield double in situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) and test the assay performance with dual color HER2 FISH evaluated breast carcinomas.
Furthermore, FISH slides cannot be archived for long term storage and review.
However, current HER2 FISH procedures are labor intensive, manual methods that require skilled technologists and specialized fluorescence microscopy.
Human epidermal growth factor receptor 2 ( HER2) fluorescence in situ hybridization (FISH) is a quantitative assay for selecting breast cancer patients for trastuzumab therapy.