Background: ASCO-CAP recently recommended further changes to evaluation of HER2 gene amplification by FISH. We retrospectively assessed the impact of the guidelines using annotated BCIRG-005, BCIRG-006, and BCIRG-007 clinical trial data for which we have detailed outcomes. Methods:HER2 FISH status of BCIRG-005/006/007 breast cancers was re-evaluated according to 2013/2014 ASCO-CAP guidelines, which designate five different groups according to HER2 FISH ratio and average HER2 gene copy number per tumor cell; group 1 (“ISH-positive”): HER2-to-CEP17 ratio ≥2.0, average HER2 copies ≥4.0; group2 (“ISH-positive”): ratio ≥2.0, copies<4.0; group3 (“ISH-positive”): ratio<2.0, copies ≥6.0; group 4 (“ISH-equivocal”): ratio<2.0, copies ≥4.0 and <6.0; and group 5 (“ISH-negative”): ratio<2.0, copies<4.0. We assessed correlations with HER2 protein, clinical outcomes by disease-free (DFS) and overall survival (OS) and benefit from trastuzumab therapy (hazard ratio). Results: Among 10,468 breast cancers successfully evaluated by FISH for patient trial entry, 40.8% were in ASCO-CAP ISH group 1; 0.7% in group2; 0.5% in group3; 4.1% in group 4 and 53.9% in group 5. Distributions were similar in screened compared to accrued subpopulations. Among accrued patients, FISH group 1 breast cancers were strongly correlated with IHC3+ status (p-value <0.0001); while groups 2, 3, 4 and 5 were not. However, groups 2, 4 and 5 were strongly correlated with IHC0/1+ status (all p-values <0.0001), while group 3 was not. Among patients accrued to BCIRG-005, group 4 was not associated with significantly worse DFS or OS compared to group 5. Among patients accrued to BCIRG-006, only group 1 showed significant benefit from trastuzumab therapy (DFS hazard ratio=0.71, 95% CI: 0.60-0.83; p<0.0001; OS hazard ratio=0.69, 95% CI: 0.55-0.85; p=0.0006), while group 2 did not. Conclusions: Our findings support the original categorizations of HER2-positivity by FISH status (HER2-to-CEP17 FISH ratio ≥2.0 and average HER2 gene copy number ≥4.0) in BCIRG trials and do not support the adoption of the current ASCO-CAP guidelines.