Abstract Background: The human epidermal growth factor receptor 2(HER2) proto-oncogene is overexpressed or amplified in approximately 15%-25% of invasive breast cancers. Approximately 35% of HER2-amplified breast cancers have coamplification of the topoisomerase II-alpha (TOP2A) gene encoding an enzyme that is a major target of anthracyclines. Hence, the determination of genetic alteration (amplification or deletion) of both genes is considered as an important predictive factor that determines the response of breast cancer patients to treatment. The aims of this study are to determinate TOP2A status gene amplification in a set of Iraqi patients with breast cancer that have had an equivocal (2+) and positive HER2/neu by immunohistochemistry (IHC) and to compare the results with estrogen receptor (ER) and progesterone receptor (PR) and HER2/neu status. Patients and methods: A cross-sectional prospective study done on 53 patients with invasive breast carcinoma. Twenty-six out of total 53 cases were positive HER2/neu (3+), the remaining 27 equivocal HER2-IHC (2+) cases reanalyzed using dual-color chromogenic in situ hybridization (ZytoVision) probe kit for further identification of HER2/neu gene amplification. Using chromogenic in situ hybridization (CISH), TOP2A gene status determination was done for all cases. Results: There is a direct significant correlation between TOP2A gene amplification and HER2/neu positivity, P < 0.05 in that 15 (39.4%) out of 38 positive HER2/neu cases were associated with topoisomerase gene amplification. Regarding relation of topoisomerase gene to hormone receptor status (ER and PR), there was a significant negative relationship between the gene and ER receptor status. The higher level of gene amplification was noticed in ER and PR negative cases in about 13 (43.3%) and 14 (48.2%) for ER and PR, respectively. Conclusion: TOP2A gene status has a significantly positive correlation with HER2/neu status while it has a significantly negative correlation with hormone receptor status.