Evaluation of analytical accuracy of HER2 status in patients with breast cancer: Comparison of HER2 GPA with HER2 IHC and HER2 FISH

Human epidermal growth factor receptor 2 (HER2) gene status and overexpression, occurring in ~ 13.6% of primary breast cancers, is essential for identifying patients likely to benefit from biological treatment. In this method of evaluation study, we tested and compared the HER2 gene-protein assay (GPA) with routine HER2 immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). The GPA was evaluated using 67 formalin-fixed paraffin-embedded (FFPE) HER2 equivoval IHC (2+) breast cancer tissue samples. Overall, agreement between GPA silver in situ hybridization (SISH) and FISH was 91.9% (57/62). Regression analysis revealed slightly higher, but non-significant difference in HER2/chromosome enumeration probe 17 (CEP17) ratio for GPA as compared to FISH (p = 0.074). Intraclass correlation coefficients (ICCs) of 0.94 and Spearman´s rank correlation coefficients of 0.93 (p < 0.0001) for FISH and GPA SISH suggested strong inter-observer association for methods with one observer counting on average 0.23 significant higher for GPA SISH (p = 0.014). Intra-observer IHC method reproducibility was 52.6% (κ = 0.3122, p = 0.004) and 79.7% (κ = 0.6428, p = 0.9197), suggesting fair significant and substantial non-significant difference between tests for reviewers. Inter-observer reproducibility for IHC methods was 53%. While inter-observer reproducibility for experienced IHC interpretation suggested significant differences (κ = 0.3636, p = 0.0332), unexperienced interpretation of IHC GPA suggested fair non-significant difference between reviewers (κ = 0.3101, p = 0.0747). Using FISH as reference, the diagnostic indices for GPA SISH were as follows: sensitivity 100%, specificity 95% and accuracy 92%. Inaccuracy between tests was in 80% of cases due to ISH categorization as equivocal by one of the methods. IHC results highlight that it may be beneficial with a method for simultaneously visualization of HER2 gene and protein status.