Flow cytometric analysis of DNA of tumor cells rapidly provides information on cell kinetics and tumor ploidy. Human B-cell lymphomas, however, often contain high numbers of nonneoplastic cells, mainly T-lymphocytes, which may hamper the accurate measurement of cell cycle phases and ploidy level of these tumors. The neoplastic cells in each B-cell lymphoma express a single immunoglobulin light chain. Therefore, we labeled surface immunoglobulin light chains to discriminate between predominantly neoplastic B-cells and nonneoplastic cells in the same tissues. Using this label as well as antibodies against nonneoplastic T-cells, we performed multiparameter correlated flow cytometric analysis of 52 human B-cell lymphomas measuring cellular DNA in neoplastic and nonneoplastic populations from the same tissues without physical separation of cells. Comparison of cellular DNA of immunoglobulin light chain-bearing neoplastic cells with that of nonneoplastic cells from the same tumor enabled us to detect DNA changes (aneuploidy) in almost 80% of the lymphomas, an incidence higher than observed previously by conventional DNA analysis of unseparated cells. These ploidy changes were confirmed by comparing in the same tumor the DNA of normal T-cells with that of predominantly neoplastic cells. The proportion of neoplastic cells in the synthetic phase of the cell cycle (S-fraction) varied widely from tumor to tumor. Lymphomas with high neoplastic S-fractions (higher than 10%) were mostly hyperdiploid tumors and historically corresponded to intermediate- and high-grade unfavorable lymphomas. Tumors with low neoplastic S-fractions (less than 5%) were predominantly diploid and near diploid, historically low-grade lymphomas. Six lymphomas showed two discrete cell populations bearing the same immunoglobulin light chain but containing different amounts of DNA suggesting the presence of two neoplastic clones in the same tumor (biploidy). In two patients in whom the lymphoma relapsed at 17 and 34 months, respectively, after the initial biopsies, repeat tumor samples were obtained. Despite an increase in the neoplastic S-fraction, no change in ploidy level was observed in either case. Light scatter analysis suggested a relationship between cell size and genomic size; large cells in these tumors were mostly presynthetic aneuploid cells. The ability to measure DNA, antigens, and cell size in individual cells in a rapid, correlated manner is a unique attribute of flow cytometry. The approach utilized in this study enabled us to analyze cell cycle fractions and ploidy changes in lymphomas with a precision and resolution that is extremely difficult to attain by any other method.

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Supported by Grants CA-23393 from the National Cancer Institute and ACS-81-67 from the American Cancer Society. A preliminary report on this work was presented at the UCLA Symposium on “B and T Cell Tumors: Biological and Clinical Aspects,” March 1982, Squaw Valley, CA (50).

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