Background: HER2-directed therapies are approved for the treatment of patients with HER2-positive invasive breast cancer as defined by HER2 protein overexpression, or HER2 gene amplification based on HER2/chromosome enumeration probe (CEP17) ratio ≥ 2.2. Above this accepted HER2 status determination, however, it is still unknown whether the efficacy of HER2-directed therapy in early breast cancer increases with increasing HER2/CEP17 ratios. Therefore, the purpose of the presented work is to evaluate whether quantitative assessment of the HER2/CEP17 ratio predicts pathological complete response (pCR) and event-free survival (EFS) of patients treated with neo-adjuvant HER2-based regimen in the prospective phase III NeoALTTO trial. Methods: 455 women with HER2-positive early breast cancer, who had received neo-adjuvant trastuzumab and/or lapatinib for 6 weeks and then together with 12 cycles of weekly paclitaxel were included in this analysis. The HER2/CEP17 ratio in the primary tumor samples was correlated with pCR and survival outcome. Results: The Median HER2/CEP17 ratio in NeoALTTO was 5.1 (range: 1.1 – 100.0), and ratios were not associated with age, hormone receptor status, or any of the other clinicopathological variables analyzed. The log HER2/CEP17 ratio significantly predicted for pCR in both uni-variate (OR: 1.83; 95% CI: 1.11 - 3.01, p = 0.0176) and multivariate analysis (OR: 1.79; 95% CI: 1.07 - 2.99, p = 0.0257). Higher HER2/CEP17 ratios were, however, not associated with improved EFS (adjusted HR = 0.79; p = 0.3537). A pCR prediction model which included HER2/CEP17 ratio, treatment arm, and hormone receptor status improved the predictive strength of treatment arm alone from a ROC AUC value of 0.60 to 0.69. Conclusions: In patients treated with HER2-based neoadjuvant therapy, quantitative analysis of the readily available pre-treatment HER2/CEP17 ratio by FISH is predictive of pCR but not EFS.