Correction for chromosome-17 is critical for the determination of true Her-2/neu gene amplification status in breast cancer

Purpose: Trastuzumab is the cornerstone for treatment of women with HER2-overexpressing breast cancer, both in the adjuvant and in the metastatic settings. The accurate assessment of HER2 is, therefore, critical to identifying patients who may benefit from trastuzumab-based therapy. This project aimed to determine the optimal scoring method for fluorescence in situ hybridization (FISH) assay. Methods: FISH assay was done on 893 samples of breast cancer. Three scoring methods were evaluated: Her2/CEP17 >= 2, Her2 > 4, or Her2 > 6. Protein and gene expression were evaluated by immunohistochemistry (n = 584) and mRNA/assay/nucleic acid sequence-based amplification (NASBA; n = 90). Results: Samples were divided into five groups based on FISH results: disomic amplified and nonamplified, polysomic amplified, nonamplified, and discordant (10.8% of cases, mostly positive with Her2 > 4 scoring, but negative with the others). Her2/CEP17 >= 2 and Her2 > 6 scoring methods showed the best association (a) with regard to FISH scoring (n = 0.906, P < 10(-6)) and (b) between FISH and immunohistochemistry (3+ as positive; kappa > 0.650, P < 10(-6)) or NASBA (kappa > 0.536, P < 10(-6)). Polysomy had an effect on Her:2 copy number (P < 10-6), but had no effect on protein and mRNA content. Therefore, within the discordant subgroup, for which additive Her-2 gene copies are due to high polysomy, protein and mRNA levels were similar to those of the nonamplified samples. For this subgroup, the best concordance between FISH/immunohistochemistry/NASBA was observed with the Her2/CEP17 ratio and Her-2 > 6 scoring (68% and 58% perfect matches, respectively). No perfect matches were observed using the Her2 > 4 scoring method. Conclusion: Correction for chromosome-17 is the method of choice for clinical practice; Her-2 > 6, but not Her-2 > 4, could be used as an alternative.