Measures of the proliferative activity of tumor cells have prognostic value in patients with node-negative breast cancer. We studied 367 women in southern Sweden who had undergone surgical resection for such cancer. Tumor specimens were analyzed with DNA flow cytometry in order to estimate both the DNA content (ploidy) and the fraction of cells in the synthetic phase of the cell cycle (S phase). The median duration of follow-up was four years; 28 percent of the patients received adjuvant therapy, usually with tamoxifen (n = 83). A multivariate analysis based on complete data on 250 patients included the following covariates: age (greater than or equal to 75, 50 to 74, and less than or equal to 49 years), tumor size (less than or equal to 20 vs. greater than 20 mm), concentration of estrogen and progesterone receptors (less than 10 vs. greater than or equal to 10 fmol per milligram of protein), ploidy (diploid vs. nondiploid), and S-phase category (fraction of cells in S phase: less than 7.0 percent, 7.0 to 11.9 percent, and greater than or equal to 12 percent). The S-phase fraction yielded the most prognostic information, followed by progesterone-receptor status and tumor size. A prognostic model based on these three variables identified 37 percent of the patients as constituting a high-risk group with a fourfold increased risk of distant recurrence. In the remaining 63 percent of the patients, the five-year overall survival rate (92 +/- 4 [+/- SE] percent) did not differ from the expected age-adjusted rate for Swedish women. We conclude that a prognostic index that includes indicators of the proliferative activity of tumor cells may be able to identify women with node-negative breast cancer in whom the risk of recurrence is sufficiently low that adjuvant chemotherapy can be avoided.

Flow cytometric DNA measurements yield the amount of DNA for each of a large number of cells. A DNA histogram normally consists of a mixture of one or more constellations of GdG,-, S-, G2/Mphase cells, together with internal standards, debris, background noise, and one or more populations of clumped cells. We have modelled typical DNA histograms as a mixed distribution with Gaussian densities for the Go/G1 and G2/M phases, an S-phase density, assumed to be uniform between the Go/G1 and G2/M peaks, observed with a Gaussian error, and with Gaussian densities for standards of chicken and trout red blood cells. The debris is modelled as a truncated exponential distribution, and we also have included a uniform background noise distribution over the whole observation interval. We have explored a new approach for maximum-likelihood analyses of complex DNA histograms by the application of the EM algorithm. This algorithm was used for four observed DNA histograms of varying complexity. Our results show that the algorithm works very well, and it converges to reasonable values for all parameters. In simulations from the estimated models, we have investigated bias, variance, and correlations of the estimates.Key terms: DNA-histogram analysis, maximum-likelihood estimation, cell-cycle compartments The DNA distribution, consisting of the Go/G1, S, and G2/M compartments of a cell population, provides useful information about the ploidy and the proliferation activity. These two indices, which express the modal DNA value and the total cell proliferation activity, respectively, of the cell population, have been measured in several investigations and shown to be of prognostic value in some malignancies (reviewed in 3,4). Flow cytometry (FCM) enables rapid quantification of DNA content of individual cells, giving the DNA distribution based on several thousand single cells in a couple of minutes (11,18,24,26,29,34). Therefore, FCM instruments have become important tools in producing raw data for the calculation of the ploidy and proliferation indices as well as other valuable cell characteristics.Many models and computer programs have been developed for the analysis of 9,[14][15][16][17][20][21][22]27,35), but most of these programs are limited to DNA histograms based on a cell population with just one DNA stemline and with DNA quantified with a resolution of 100-250 channels. In these cases, special efforts have been made to calculate the fraction of S-phase cells, particularly in synchronously growing cell cultures (41), but no attempt has been made to calculate the DNA index (DI) in these applications. The DI is here defined as the ratio between the modal Go/G1 DNA value of the cell population and a standard diploid peak (25). A comparison of various mathematical methods for DNA histogram analyses made by Baisch et al. (2) showed discrepancies in the estimates of the histogram compartments when the methods were applied to either a simulated histogram or a n experimentally derived DNA histogram based on a single cell stem line.Wit...

Summary The use of continuous prognostic variables is clinically impractical, and arbitrarily chosen cut-off points can result in a loss of prognostic information. Here we report findings from a study of primary breast cancer, showing how the prognostic value of the fraction of cells in the S-phase of the cell cycle (SPF), as measured by flow cytometry, can be affected by the SPF cut-off level(s) adopted. It was possible to evaluate the SPF in 566 (94%) of 603 consecutive cases where fresh frozen specimens were available in a tumour bank at our department. Clinically, all patients were without distant spread at the time of diagnosis, and the median duration of follow-up was 4 years. Using different survival end-points and x2 values for each cut-off level, two optimal cut-off points, at the 7% and 12% levels, were consistently obtained for the SPF. The aim of the current study was to elicit optimal cut-off levels for the SPF in primary breast cancer, as determined with FCM.Correspondence: H. Sigurdsson. Received 3 January 1990; and in revised form 9 July 1990. Patients and methodsIn the health care region of southern Sweden hormone receptor analyses are routinely performed on specimens from patients with primary breast cancer, any residual tumour specimens being stored in a tumour bank at the Oncology Department at University Hospital in Lund.The study included 603 consecutive cases where specimens were available in the tumour bank, and which fulfilled the following inclusion criteria: (1) tumour sample from primary breast cancer (cancer in situ excluded); (2) diagnosed during the period between September 1982 and January 1986; (3) clinically without signs of distant metastasis at the time of diagnosis; (4) sufficient tumour specimen to yield a DNA histogram; and (5) tumour cells microscopically identified in a cytopathologic investigation of all imprints used for making cell suspensions for DNA analysis.The median age of the patient population was 63 years (range 26-97). Tumour size was taken from the pathological report and usually determined on a unfixed specimen. A median of ten axillary lymph nodes was investigated. The distribution of cases by tumour size was as follows: 0-20mm, n=250 (41%); 21-50mm, n=317 (53%); and > 51 mm, n = 36 (6%). Distribution by axillary lymph node status was: node-negative, n = 303 (50%); node-positive, n = 277 (46%); and no axillary staging performed, n = 23 (4%).Laboratory methods Tumour samples were taken from biopsies originally obtained for steroid receptor analysis. Fresh specimens from the macroscopic mammary tumours were taken by the examining pathologist, except in a few cases where it was done during surgery. The specimens were stored frozen (-70°C), and later analysed (in a single laboratory) at the Oncology Department in Lund. Flow