Clinical and in vitro evidence supports the concept that human epidermal growth factor receptor-2 ( HER2 ) gene amplification prediction of response to anthracycline-based chemotherapy in breast cancer is not a direct effect of HER2 overexpression, but the result of coamplification of topoisomerase II-alpha ( TOP2A ). We investigated the relationship of TOP2A to HER2 genomic alterations by fluorescence in situ hybridization (FISH) and the correlations with polysomic states for chromosome 17 (CEP17). One hundred thirty-eight cases of breast cancer HER2 gene amplified by 2-color FISH ( HER2 /CEP17) were reevaluated with a 3-color probe set ( HER2 /CEP17/ TOP2A ) to investigate the frequency of coamplification and deletion of TOP2A . TOP2A was never amplified in the absence of HER2 amplification and was coamplified with HER2 in 68 (50%) of 137 cases; HER2 gene copy number was higher than the TOP2A copy number ( P < .01). Of the 137 cases with HER2 amplification, 23 (16%) showed a monoallelic deletion of TOP2A . Of the 43 cases not amplified for HER2 , 27 (63%) were CEP17 eusomic, 13 (30%) polysomic, and 3 (7%) monosomic. Of the HER2 nonamplified cases, 2 (5%) showed monoallelic deletion of both the HER2 and TOP2A . The current study demonstrates the complex interrelationship between the HER2 and TOP2A genes in breast cancer. The clinical implications of TOP2A amplification and deletion in breast cancer need to be further defined. If TOP2A gene dosage can be confirmed to correlate with tumor responsiveness to anthracycline-based therapy in the clinical setting, FISH testing for TOP2A status may be warranted to aid in the selection of the most appropriate therapy.