Image cytometric bcl-2:bax and bcl-2:bcl-x ratios in invasive breast carcinoma: Correlation with prognosis

Abstract

Background: The bcl-2 family of proteins are important regulators of apoptosis. Some of the members, such as bcl-2 and bcl-xL, inhibit cell death, whereas others, such as bax and bcl-xS, promote cell death. We evaluated the ratios of bcl-2:bax and bcl-2:bcl-x expression by image cytometry in invasive breast carcinoma to determine prognostic significance. Design: Five-micron sections of formalin-fixed, paraffin-embedded tissue from 88 invasive breast carcinomas were immunostained using steam antigen retrieval, an avidin biotin-complex technique with automated stainer and primary antibodies against bcl-2 (1/160; Dako, Carpenteria, CA), bax (1/1,500; PharMingen, San Diego, CA), and bcl-x (1/1,500; PharMingen). Positive controls were tonsil (bcl-2) and normal breast (bax and bcl-x) tissue samples. Immunostain was measured in 15 high power fields as percentage positive area (PPA) in nuclei and cytoplasm using the CAS 200 image analyzer (Becton Dickinson, San Jose, CA). Results: Median follow-up was 105 months (range 11-130). Significantly improved disease-free survival was found in patients with a bcl-2:bcl-x ratio ≥ 1 by univariate and multivariate analyses. The bcl-2:bax ratio was not predictive of overall or disease-free survival. A significant difference in overall and disease-free survival was found between carcinomas with positive and negative bcl-2 expression by univariate analysis; by multivariate analysis, bcl-2 expression was an independent prognostic factor for disease-free survival. The 5-year survival rates were 77% and 50% in patients with bcl-2-positive and bcl-2-negative carcinomas, respectively. Conclusion: A bcl-2:bcl-x ratio ≥ 1, assessed by image cytometry, is significantly associated with improved disease-free survival in patients with invasive breast carcinoma. Significantly increased overall and disease-free survival is associated with positive bcl-2 expression. © 2002 Wiley-Liss, Inc.