Dual-color dual-hapten in situ hybridization (D-DISH) - Comparison with fluorescence in situ hybridization (FISH) for HER2/neu testing in breast cancer

Context: HER2/neu testing in breast cancer is a mandate due to availability of trastuzumab, a monoclonal antibody targeted against this biomarker. Dual-color dual-hapten in situ hybridization (D-DISH) is a new test for assessment of HER2/neu gene overexpression on light microscopy. Aims: This was a validation study for D-DISH in our laboratory and was conducted to study the concordance between fluorescence in situ hybridization (FISH) and D-DISH for HER2/neu testing in breast cancer. Materials and Methods: In all, 150 cases of invasive breast carcinoma requested for FISH analysis were selected. Immunohistochemistry by Ventana PATHWAY anti-HER2/neu (4B5) antibody, FISH by ZytoLight SPEC ERBB2/CEN17 Dual Color Probe, and D-DISH using the Ventana INFORM HER2 Dual ISH DNA Probe Cocktail Assay was carried out. Statistical Analysis: Cohen's kappa coefficient was used to calculate concordance between FISH and D-DISH assays. The ratios and average number of signals were compared with Lin's concordance correlation coefficient. Results: About 93.1% of the cases showed concordance between FISH and D-DISH results. Cohen's kappa correlation coefficient was 0.836, indicating almost perfect level of agreement. Lin's concordance correlation coefficient (.c) showed moderate strength of agreement for HER2/chromosome 17 ratios between FISH and D-DISH assays (rho c 0.9452). As per the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) 2018 updated guidelines, four of the cases that were nonamplified on FISH showed low-level amplification on D-DISH due to counting errors caused by faint signals or background dust. Genomic heterogeneity and larger red chromosome 17 signals on D-DISH led to discordance of the six cases amplified by FISH. D-DISH failure rate was 3.33%. Conclusion: Overall, D-DISH showed good concordance with FISH but needs expertise for reporting.