Background: Recent evidence indicates that a subset of axillary node-negative (ANN) breast cancer patients can benefit from adjuvant therapy. Reliable prognostic markers are needed, however, to help clinicians identify these patients and arrive at more rational treatment decisions. Purpose : Mutations of the p 53 tumor suppressor gene often result in overexpression of the p 53 protein. In this study, we evaluated the prognostic significance of p 53 protein overexpression in patients with ANN breast cancer. We also studied the association between the tumor cell proliferation rate and overexpression of the p 53 and c-erbB-2 proteins, both of which have been implicated in cell. cycle control. The c-erbB-2 protein is the product of the ERBB2 gene. Methods: Two hundred eighty-nine ANN cases were randomly selected from a population-based cohort of patients who had not received any kind of adjuvant chemotherapy or endocrine therapy. Overexpression of the p 53 and c-erbB-2 proteins was studied immunohistochemically in archival paraffin-embedded tumor samples, using the CM-1 polyclonal and the TAb 250 monoclonal antibodies, respectively. The tumor cell proliferation rate was measured as the S-phase fraction by DNA flow Statistical analyses were per-Jorma Isola, * Tapir Visakorpi, Kaija Holli, Olli-P. Kallioniemiformed using BMDP software. Results: High-level p 53 protein overexpression, found in 41 of the 289 tumors, was most common in tumors with high his-tologic grade, negative estrogen receptor status, c-erbB-2 protein over-expression, DNA index greater than 1.3, or high S-phase fraction. The lowest S-phase levels were found in tumors with neither p 53 nor c-erbB-2 protein overexpression; the highest levels were seen in tumors showing overexpression of both proteins ( P <.0001). Both p 53 and c-erbB-2 overexpression, as well as tumor size, had independent prognostic value in multivariate analysis. Eight-year survival of patients with p 53 protein over-expression was 56% compared with 81% in patients with no overexpression (relative risk, 3.7; P <.0001). If the S-phase fraction was included in a Cox regression analysis, however, only the tumor size and the S-phase fraction emerged as independent predictors of survival. Conclusions: Over-expression of the p 53 and c-erbB-2 proteins indicates a high malignant potential in ANN breast cancer, but it is not a significant prognostic factor independent of the cell proliferation rate. The correlation between overexpression of these proteins and an increased S-phase fraction suggests that they may confer a proliferative advantage to cancer cells in vivo. [J Natl Cancer Inst 84:1109–1114,1992]