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Background The recurrence or mortality rate of axillary lymph node-negative invasive breast cancer has been associated with the tumor S-phase fraction, which is measured by DNA flow cytometry. Because many of the studies that established this association were performed using frozen, pulverized tumor specimens, this association could not be tested for independence from the established prognostic factors of histologic and nuclear grading. Methods. Histologic, nuclear, and mitotic grades, DNA ploidy, and S-phase fraction (SPF) were determined from paraffin-embedded tumors obtained from 280 women with node-negative invasive ductal carcinomas using standard grading schemes and flow cytometric techniques. These variables were compared with disease-free and cancer-specific survival (CSS) in univariate and multivariate analyses of these patients. Results. Tumor diameter, SPF, histologic grade, and nuclear grade were significant predictors of disease-free survival (DFS); diameter and SPF had significant associations with CSS. Cox analysis showed histologic grade to be the only independent predictor of relapse, whereas diameter and SPF were independent predictors of mortality. The patients with low nuclear or histologic grade tumors had only a 5% risk of recurrence at 5 years. In contrast, 36% of patients in this series with medium-grade or high-grade high SPF tumors had a 30% risk of recurrence over the same interval. Conclusions. Histopathologic grading and flow cytometric determination of SPF appear to provide additive prognostic information for patients with early invasive ductal carcinomas of the breast. Cancer 1993; 72: 1926-32.

A large group of patients with node-positive breast cancer was divided into a training set (n = 851) and a validation set (n = 432) to demonstrate techniques for integrating steroid hormone receptor status, DNA flow cytometric findings, and other prognostic factors to predict patient survival. Multivariate analyses showed that estrogen receptor status, the number of involved axillary lymph nodes, patient age, S-phase fraction, progesterone receptor status, and tumor size were significant predictors of survival in patients with node-positive breast cancer. Techniques for optimizing and validating a cut point for a new prognostic factor and for examining alternative representations of prognostic factors were demonstrated. Prognostic indexes were created that could be used to identify patients with very good or very poor prognoses. Cancer 1993; 71:2157-62.

Silvano Bosari, M.D.,*,[unkeyable] Arthur K. C. Lee, M.B.B.S.,* Steven R. Tahan, M.D.,[unkeyable] Mary Ann T. Figoni, B.S., M.T. (ASCP),[unkeyable] Brian D. Wiley, B.S.,* Gerald J. Heatley, M.S., Section and Mark L. Silverman, M.D.* Methods. The prognostic significance of flow cytometric analysis in patients with node-negative invasive breast carcinoma was evaluated in a retrospective series of 158 patients with a minimum follow-up study of 9 years. Results. The ploidy status could be assessed in 147 specimens (93%), and the proliferative phase or S-phase fraction (SPF) could be assessed in 136 tumors (86%); 70 tumors (48%) were diploid, 49 tumors (33%) were aneuploid, and 28 tumors (19%) were tetraploid. Ploidy status and SPF were correlated significantly with tumor size, histologic grade, nuclear grade, and mitotic rate. By itself, ploidy was not a statistically significant prognostic factor, although all of the patients with multiploid and hypertetraploid tumors had recurrence of disease. The SPF was related significantly to recurrence of disease (P = 0.04). However, when multivariate analysis of various histopathologic variables was performed, SPF ceased to be a significant prognostic determinant, whereas peritumoral lymphovascular invasion was the most important variable. The combination of tumor size and flow cytometric parameters permitted stratification into three groups with different prognoses at the 9-year follow-up review (P < 0.001). In the low-risk group (diploid tumors [is less than or equal to] 2 cm in diameter with a low SPF or small tetraploid tumors), the recurrence rate was 12%. In the intermediate-risk group (diploid tumors > 2 cm in diameter with a low SPF or aneuploid tumors with a low SPF), the recurrence rate was 21%. In the high- risk group (diploid or aneuploid tumors with a high SPF or large tetraploid tumors), the recurrence rate was 49%. The high-risk group status remained a significant variable in the Cox proportional hazards multivariate analysis model. Conclusions. These results indicate that flow cytometry in breast carcinoma contributes useful but limited prognostic information and stress the importance of using multiple prognostic factors to improve prognostication and optimize patient management. Cancer 1992; 70:1943-1950.

Despite improvements in the early diagnosis and treatment of breast cancer, one major question resists resolution: which women with early-stage breast cancer should endure chemotherapeutic treatment when only a few in every hundred actually benefit from this treatment. It would be valuable to successfully predict which women have the best prognosis and therefore do not need adjuvant chemotherapy and which women should receive this therapy to ensure their best chances for survival. One promising method for improving the accuracy of prognosis for breast cancer patients is DNA measurement. In this technique, the DNA content of tumors is measured on a cell-by-cell basis. It may be normal or abnormal, and may indicate that many cells are dividing rapidly or only a few. These measurements may correlate with the patient's prognosis. In most cases, DNA measurement is made by flow cytometry, in which cells are dribbled past lasers one-by-one to obtain optical measurements, which indicate their DNA content. The methods of data analysis most appropriate for breast cancer prognosis are still under investigation; the variability of the technique appears to be great, and different laboratories may obtain different results. The automated flow cytometer does not know if it is looking at a cell nucleus, a clump of cell nuclei, or irrelevant fragments of broken cells; consequently, the resulting data is contaminated by erroneous measurements. In a recent study, researchers attempted to use the scattering of the laser light to reduce the sources of variability in the technique. The way the laser light scatters may be used to distinguish the best measurements from the flawed ones. In examining cells from women with node-positive breast cancer, researchers found that the prognosis was worse when cells had an unusual number of chromosomes (aneuploid), better with a normal number (diploid), and best with exactly twice the normal number of chromosomes (tetraploid). Similarly, patients with indications of greater cell proliferation also had a worse prognosis. However, when the traditional prognostic indicators were included in the statistical analysis, it was found that the prognosis based on both traditional factors and the measurement of DNA was no better than the prognosis based on the traditional factors alone. (Consumer Summary produced by Reliance Medical Information, Inc.)

The widening array of treatment alternatives available for cancer has placed new significance on prognostic indicators. Presumably, better results may be achieved if more aggressive treatments are chosen for patients with a poorer prognosis, while patients with a good prognosis can be spared unnecessarily harsh side effects. In breast cancer, the single most important factor in determining prognosis is the spread of cancer to the lymph nodes. Although patients with negative lymph nodes enjoy a better prognosis, recurrence is still a possibility, and 30 percent of node-negative breast cancer patients will die of their disease within 10 years. If additional prognostic factors could be identified, some of these patients might be saved with more aggressive systemic adjuvant chemotherapy. One prognostic factor that might prove to be of value is the DNA content of tumor cells. Previous studies have shown that an abnormal quantity of DNA, indicative of an aneuploid chromosome content, is an indicator of poorer prognosis for breast cancer. An advantage to the method is that DNA analysis may be quantified by automated devices including the flow cytometer. Using archival tumor specimens, a retrospective DNA analysis was performed for 155 node-negative patients with breast cancer; the median available follow-up at the time of the study was 10 years. It was found that 64 tumors, or 41 percent, measured as aneuploid. Of these 64 patients, 23 had relapsed and 21 had died. Of the remaining 91 patients with an apparently normal DNA complement, only eight have relapsed and six have died. Although the apparent chromosome complement, or ploidy, was related to tumor size and grade, the results could not be accounted for solely on this basis. A Cox multivariate statistical model revealed that tumor grade, tumor size, patient age, and ploidy were all independent predictors of overall survival. The results suggest that DNA analysis is a predictor of risk of recurrence, and may prove to be clinically useful. (Consumer Summary produced by Reliance Medical Information, Inc.)

Flow cytometric analysis of DNA has become a popular method of exploring prognostic variables in cancer. Numerous studies have shown that an abnormal amount of DNA, which generally represents aneuploidy, has prognostic value, as do indices of cellular proliferation. Most studies use ethidium bromide as a quantitative fluorescent stain to measure the amount of DNA; there are other possibilities, however. In particular, the antibody Ki-67 may be used to identify replicating cells. When cells are actively traversing the cell cycle, they express an antigen which is recognized by Ki-67. Cells which are busy doing their job and are not replicating do not express this antigen. To evaluate the usefulness of Ki-67 in the prognosis of breast cancer, specimens from 102 breast cancers were examined for Ki-67 staining, microscopic counts of cell division or mitosis, and standard fluorescent DNA measurements. The staining with Ki-67 correlated with high mitotic counts and high tumor grade. Curiously, Ki-67 correlated with the number of cells in the S-phase of the cell cycle in cells which were aneuploid, but not in cells which were diploid. The authors offer no explanation for this, except to say it may be a problem with ''methodology.'' Nevertheless, the staining with Ki-67 seems to correlate well with measures of cell proliferation such as S-phase fraction and mitotic index, as well as measures of tumor aggressiveness such as DNA aneuploidy and tumor grade. Since the antibody is economical and easy to use, it seems likely to be a useful reagent in the evaluation of breast cancer specimens. (Consumer Summary produced by Reliance Medical Information, Inc.)