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DNA in Situ Sensitivity to Denaturation as a Marker of Human Breast Tumors¹

Memorial Sloan-Kettering Cancer Center, New York, New York 10021

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 _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 _t values was unimodal with over 90% of cells having _t above 0.6. All fibroadenomas were diploid; four had unimodal _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 _t distributions. Among 13 diploid carcinomas 10 had bimodal _t distributions. Statistically significant differences were observed in _t distributions of normal versus tumor breast tissue (P < 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 (_t below 0.6). Of interest, the diploid cells from aneuploid tumors which may represent reactive host cells often displayed bimodal distributions of _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 _t values in breast tumors may indicate a high proportion of proliferating cells, whereas high _t populations may represent quiescent and differentiating (condensed chromatin) or dying (pycnotic nuclei) cells. It is likely that the low _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.

¹ Supported by NCI Grants P30-CA-08748, R01-CA-28704, and R37-CA-23296.

² Present address: Immunobiology Research Institute, Route 22 East, P. O. Box 999, Annandale, NJ 08801.

³ Present address: Department of Pathology, School of Medicine, Szczecin, Poland.

⁴ To whom requests for reprints should be addressed, at Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021.

Received 2/15/89. Revised 8/ 1/89. Accepted 8/18/89.