ONE OR MULTIPLE SAMPLINGS FOR FLOW CYTOMETRIC DNA ANALYSES IN BREAST-CANCER PROGNOSTIC IMPLICATIONS

Flow cytometric assessments of DNA ploidy status and the S-phase fraction (SPF) have been shown to yield prognostic information in breast cancer. The aim of the present investigation was to elucidate the reproducibility of results with regard to tumor DNA heterogeneity, and to ascertain whether the prognostic value of DNA measurements might be enhanced by analyzing two pieces of a tumor instead of one. Agreement with regard to ploidy status (diploid versus non-diploid) was obtained in 90% of cases (71/79) when two adjacent sections of the tumor were investigated, and in 77% of cases (10/13) when four biopsies from different quadrants of the tumor specimen were investigated. The corresponding figures for agreement in SPF (divided into three categories, < 7.0%, 7.0-11.9%, and greater-than-or-equal-to 12%) were 75% (59/79; 2-sample series) and 55% (7/13; 4-biopsy series). The main reason for variance in ploidy results was the difficulties in distinguishing near diploid cell populations. Discrepancies in SPF categories could be explained by minor fluctuations in SPF values near the cut-off levels, or by variance in ploidy status, the fraction of non-diploid nuclei, and background noise due to cell debris. There was a stepwise increase in recurrence rate (RR) among patients with increasing SPF category (RR: 20%, 41%, and 53%). Patients whose SPF categories varied, from low or intermediate in one part of the tumor to high in another, seemed to have a poor prognosis (RR = 57%). We conclude that the variation in ploidy status and SPF between different parts of the same tumor specimen is largely to be explained by uncertainty in the interpretation of the DNA histograms and by difficulties inherent in the estimation of SPF, rather than by the existence of DNA tumor heterogeneity. Evaluation of a different part of the same tumor specimen may be of value, especially in the case of SPF, as this may yield additional prognostic information. These findings also emphasize the need for good quality control of FCM DNA analysis.