A significant proportion of breast cancers with HER2 amplification have simultaneous amplification of topoisomerase II-alpha (topoII). Amplification of HER2 and topoII was assayed using a novel chromogenic in situ hybridisation (CISH) method. HER2 and topoII amplification status and the response to preoperative doxorubicin chemotherapy were analysed in 67 locally advanced breast cancer patients. Response to chemotherapy was increased in the cases with coamplification of HER2 and topoII (18/19), whereas the response rate was significantly decreased in the cases without HER2 and topoII amplification (17/36). The 12 cases with HER2 amplification alone showed an intermediate response rate (9/12). The findings of the current study indicate that topoII amplification may play a role in determining chemosensitivity of breast cancers to doxorubicin chemotherapy.