Multiparametric deoxyribonucleic acid and cell cycle analysis of breast carcinomas by flow cytometry. Clinicopathologic correlations

Using intact ethanol-fixed cytokeratin monoclonal (CAM 5.2) and propidium iodide dual-stained cells, we have performed two-color multiparametric flow cytometric (FCM) DNA analysis and S-phase fraction (SPF) determination on 165 mechanically dissociated breast carcinomas. Sixty-seven patients were axillary node positive, 33 patients node negative; 59 had biopsy only and in 8, FCM was performed on tissue from metastatic lesions. Overall, 62% of the tumors contained aneuploid cell populations. Abnormal cellular DNA content (aneuploidy) was significantly correlated with high nuclear grade (p < 0.001), lack of estrogen receptors (p < 0.001), presence of vascular invasion (p < 0.04), high histologic grade (p < 0.04), and tumor size (p < 0.03) but not with patient age (p > 0.07) or axillary node status (p > 0.05). SPF values derived from ungated histograms had a positively skewed frequency distribution (range 2 to 30%, N = 152) with an overall median of 11% (diploid, 8.9%; aneuploid, 15.7%). Higher SPF values were significantlyt correlated with aneuploidy (p < 0.001), presence of necrosis (p < 0.001), lack of estrogen receptor (p < 0.0001), high nuclear grade (p < 0.001), vascular invasion (p < 0.003), tumor size (p < 0.006), and high histologic grade (p < .004) but not the presence of lymph node metastases (p > 0.56). Mean SPF values were significantly higher when calculated from cytokeratin gated DNA histograms (14.1% versus 11.5%, p < 0.001), probably due to exclusion of contaminating stromal/inflammatory cells; and significantly lower when calculated from debris subtracted histograms (7.8% versus 11.4%). Cytokeratin gated and debris subtracted SPF values both had a greater degree of correlation than ungated values with clinicopathologic factors of known prognostic significance. We conclude (a) FCM determined DNA content and cell cycle analysis provides information that correlates with accepted histopathologic and hormonal factors in the identification of high risk patient populations, (b) use of multiparametric analysis with cytokeratin gating significantly refines the biologic relevance of FCM data and (c) SPF may be of greater use than ploidy as a predictor of adverse outcome in carcinoma of the breast.