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Background  Dual in-situ hybridization (DISH) assay is a relatively new assay for evaluating Human Epidermal Growth Factor Receptor 2 (HER2) genomic amplification. Optimization protocol for the assay is not yet well established, especially for archival tissues. Although there is a recommended nominal protocol, it is not suited for formalin-fixed and paraffin-embedded (FFPE) samples that were archived for long periods.    Findings  In a study on local population of mucinous epithelial ovarian cancer, we developed a series of optimization protocols based on the age of samples to improve success of the DISH assay. A decision workflow was generated to facilitate individualization of further optimization protocols. The optimizations were evaluated on 92 whole tissue sections of FFPE mucinous ovarian tumors dating from 1990 to 2011. Overall, 79 samples were successfully assayed for DISH using the series of optimization protocols. We found samples older than 1 year required further optimization beyond the nominal protocol recommended. Thirteen samples were not further assayed after first DISH assay due to inadequately preserved nuclear morphology with no ISH signals throughout the tissue section.    Conclusion  The study revealed age of samples and storage conditions were major factors in successful DISH assays. Samples that were ten years or less in age, and archived in-house were successfully optimized, whereas older samples, which were also archived off-site, have a higher frequency of unsuccessful optimizations. The study provides practical and important guidelines for the new DISH assay which can facilitate successful HER2 evaluation in ovarian cancers and possibly other cancers as well.

Optimizing Ventana chromogenic dual in-situ hybridization for mucinous epithelial ovarian cancer