Background: Proliferation is a major process in carcinogenesis. RACGAP1 is a protein involved in cell growth regulation and metastasis, Ki67 is a known proliferation marker and TOP2A has a key role in DNA replication and remodeling. The aim of the present study was to explore the prognostic significance of a signature of proliferation markers, such as RACGAP1, Ki67 and TOP2A on disease-free survival (DFS) and overall survival (OS) in high-risk early breast cancer patients. Methods: A total of 1681high-risk breast cancer patients, enrolled in two consecutive phase III trials, were treated with anthracycline-based adjuvant chemotherapy. Formalin-fixed paraffin-embedded tumor tissue samples from 963 of these patients were extracted using a standardized fully automated isolation method for total RNA based on silica-coated magnetic beads, followed by multiplex RT-qPCR for assessing RACGAP1, Ki67 and TOP2A mRNA expression. CALM2 was included in the same reaction, as a reference gene. Results: After a median follow-up of 107 months, 289 patients (30.0%) demonstrated disease progression and 261 (27.1%) patients died. Univariate analysis revealed that poor OS was associated with high RACGAP1 mRNA expression (p=0.0185, log-rank), high Ki67 (p=0.0219), as well as high TOP2A (p=0.0019) mRNA expression, while in multivariate analysis only TOP2A retained significance (Wald’s p=0.008). In an effort to improve prognostic significance, combinations of the expression of two or all three genes were tested, with low mRNA expression of the three genes being associated with improved DFS (HR=0.74, CI=0.56-0.98, p=0.035) and OS (HR=0.60, CI=0.42-0.85, p=0.004). However, in multivariate analysis, none of the combinations retained prognostic significance, except the combination of high RACGAP1 and TOP2A mRNA expression, which was found to be associated with decreased DFS (HR=1.26, CI=0.96-1.63, p=0.092) and OS (HR=1.49, CI=1.10-2.03, p=0.009). Conclusions: High RACGAP1 and TOP2A mRNA expression was found, in multivariate analysis, to be of adverse prognostic significance in high-risk early breast cancer patients treated with anthracycline-containing adjuvant chemotherapy.