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DNA in situ sensitivity to denaturation as a marker of human breast tumors.

DNA content and in situ sensitivity to denaturation were analyzed by flow cytometry of individual cell nuclei isolated from 40 breast carcinomas, nine fibroadenomas, and 14 samples of normal breast tissue. The extent of DNA denaturation induced by acid was expressed as alpha t, which represents the fraction of DNA staining metachromatically red with the fluorochrome acridine orange. In all cases of normal breast tissue DNA was very sensitive to denaturation and the frequency distribution of alpha t values was unimodal with over 90% of cells having alpha t above 0.6. All fibroadenomas were diploid; four had unimodal alpha t as in normal tissue and five had a bimodal distribution with an additional peak below 0.6. Twenty-seven adenocarcinomas (67%) had a DNA index above 1.0; of these 24 had bimodal alpha t distributions. Among 13 diploid carcinomas 10 had bimodal alpha t distributions. Statistically significant differences were observed in alpha t distributions of normal versus tumor breast tissue (P less than 0.005). In normal tissue and in all tumors a predominant proportion of cells with S and G2 + M DNA content were characterized by DNA resistant to denaturation (alpha t below 0.6). Of interest, the diploid cells from aneuploid tumors which may represent reactive host cells often displayed bimodal distributions of alpha t. These results may be interpreted in light of earlier studies demonstrating increased resistance of DNA to denaturation in diffuse chromatin of proliferating and/or transcriptionally active cells, and greater sensitivity to denaturation of DNA in condensed chromatin of quiescent cells. Thus, the presence of the second peaks representing cells with low alpha t values in breast tumors may indicate a high proportion of proliferating cells, whereas high alpha t populations may represent quiescent and differentiating (condensed chromatin) or dying (pycnotic nuclei) cells. It is likely that the low alpha t diploid cells detected in aneuploid tumors may represent the reactive (transcriptionally active and/or proliferating) infiltrating host cells (i.e., lymphocytes, monocytes) whose presence may also be of prognostic value. The data suggest that a DNA denaturability assay may be useful to characterize tumor and infiltrating host cell populations.