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This report describes a companion flow cytometry study (Cancer and Leukemia Group B (CALGB)--8869) using tumors derived from patients enrolled in a large randomized clinical trial (CALGB-8541) performed on 1,572 patients with early stage, node-positive breast cancer. The CALGB initiated an adjuvant breast cancer trial in 1985 to determine if dose intensification (dose of drug per unit time) of chemotherapy was related to relapse-free and overall survival. Patients were randomized by pretreatment clinical variables to one of three different dosage regimens of chemotherapy. Using a tumor enrichment procedure, 442 paraffin-embedded blocks were analyzed by flow cytometry, and S-phase fraction (SPF) was analyzed by three different methods. Ploidy analysis was performed using standard procedures. Tissue from 90% of the patients was suitable for ploidy analysis, whereas only 68% could be assessed for SPF. With a median follow-up time of 80 months, our results show that ploidy status had no clinical utility, whereas high SPF predicted poorer overall survival. The rectangular fit model for SPF was more predictive of outcome than both the area fit model and a computer fit model (modfit) for SPF. In univariate analysis, patients with a low SPF (10%) had a better prognosis than those patients with a high SPF (10%), but they responded equally well to the different treatment regimens. Patients with high SPF (10%) had longer relapse-free and overall survival to high dose chemotherapy compared to low or standard dose chemotherapy. Multivariate analysis indicated that treatment intensity as well as the number of positive nodes, tumor size, steroid receptor status, and c-erb B-2 expression were significant in predicting overall and disease-free survival. The multivariate analysis, however, revealed that SPF was significant in predicting overall but not disease-free survival, but there was no longer any relationship among SPF, dose intensity, and outcome.

This is the consensus statement regarding the clinical utility of DNA cytometry in breast cancer from the DNA Cytometry Consensus Conference held in Prout's Neck, Maine, USA, on October 1–4, 1992. Guidelines for clinical DNA cytometry generated at that meeting also appear in this issue of Breast Cancer Research and Treatment.

Leukemia blasts isolated from bone marrow aspirates of 44 adults with acute leukemia were incubated for 1 h with 0.008-32 microM bromodeoxyuridine (Brd-Urd). After dual labeling with monoclonal anti-BrdUrd antibodies and propidium iodide, the cells were analyzed by flow cytometry. Percent labeled cells and intensity of labeling were similar over concentrations of BrdUrd ranging from 0.8-32 microM--a 40-fold range. Therefore, despite potential interpatient variability in nucleoside pharmacokinetics, commonly used doses of BrdUrd which are intended to achieve steady-state plasma concentrations in the 8.0 microM range can be expected to provide a reliable estimate of the S-phase fraction.

Estrogen receptor (ER) analysis of breast cancer tissue has been shown to be very useful in predicting which patients will respond to hormone therapy and have a better prognosis. The ER assay is, however, tedious and time consuming. Measurement of ER by flow cytometry would be rapid and based on either an average fluorescence-E2 probe intensity per cell or the percentage of the ER+ cells per cell suspension. Analysis of E2 modified structures for relative binding affinity to the ER determined by competition studies and for fluorescence uptake into cell suspensions determined by flow cytometry was performed. Lack of high affinity to the ER and purity of the compound were major problems for the fluorescein-labeled estrogen in fluorescein-E2 compounds demonstrated by HPLC very little estradiol derivative in the parent compounds compared to total components present. A second type of fluoresceinated estrogen which has a peptide bond between the steroid and the chromophore was also tested. It was less contaminated but was unable to get into the cell and showed no binding activity to the ER. A pure plant fluorescent estrogen, coumestrol, has Ka of 6 × 108 M−1 for the ER and is a single component as determined by HPLC. Specific fluroescent uptake of coumestrol was performed on ER+ and ER− viable cell suspensions. When these coumestrol-cell suspensions were excited at 350–360 nm and the blue emission was measured using flow cytometry, the result was a fluroescence uptake that was not highly displaceable by exess nonfluorescence E2 probes. Further studies on the modification of several parameters of the incubation step to enhance specific fluorescent uptake were also unsuccessful. Coumestrol was, however, a good competitor for both cellular and nuclear uptake of 3H-E2.

The cell cycle kinetics of normal mammary tissue and cancer mammary tissues were studied by flow cytometry to determine the effect of estrogen and progesterone and antiestrogens on proliferative activity. The proliferative activity was higher in rat mammary tissue during the estrus and pregnant stages compared to the nonestrus or lactational stages. Upon 5 days administration of estrogen and progesterone to the lactating animal, the low proliferative activity was increased to the values found in the pregnant stage. Steroidal effect on proliferative activity was also shown using organ cultures of normal rat tissues and the increase in proliferative activity can be reversed by the addition of 10(-6) M retinoic acid but not by retinyl acetate. In a human mammary tumor cell line (MCF-7), the cell kinetic activity was significantly depressed by antiestrogens (i.e., tamoxifen) as determined by both flow cytometry and tritiated thymidine uptake studies. Finally, a human tumor model system was developed with the MCF-7 cell line in estrogenized nude mice. Flow cytometry of the resulting tumor indicates a tetraploid tumor similar to the cell culture. Cell kinetic analysis of these nude mice tumor cells demonstrates the high proliferative activity of the estrogenized animals compared to the tamoxifen-treated animals. In conclusion, the use of flow cytometry to rapidly measure cell kinetic activity of mammary tissue has been demonstrated. The effects of estrogen or estrogen and progesterone hormones seem to increase the proliferative activity, while antiestrogens decrease this activity. Findings are observed in both in vivo and in vitro conditions or normal tissues, mammary cancer tissue, and cultured cells.

The purpose of this technical report is to determine the reproducibility of flow cytometry data for ploidy and cell cycle kinetics using paraffin-embedded blocks of breast cancer tissue. One block from each of 39 tumors was studied in this report with each block having multiple sections analyzed independently. All of these sections gave ploidy analyses, while only 34 gave cell kinetic values. The standard deviation for the DNA index value in the multiple analysis study was less than 0.1 in all but three cases. The cell kinetic values gave larger variability, and the actual values were dependent on the method of analysis. Comparison of the variability for each method of analysis could not predict which procedure was superior. These results would indicate that ploidy is a reproducible value, while cell kinetic parameters should only be used as an indicator of proliferative activity that has been normalized to the mean or median of a large set of observations processed and analyzed by the same procedure.