Flow cytometry (CFM) allows the rapid measurement of antigens and nucleic acids in individual cells. DNA analysis by FCM is particularly useful because it provides rapid information on cell cycle kinetics and tumor ploidy. B-cell lymphomas are kinetically heterogeneous tumors composed of immunoglobulin-bearing lymphocytes. Curiously, these tumor often contain large numbers of normal T-cells which hamper the precise analysis of DNA in tumor B-cells. By labelling cell surface immunoglobulins, we measured DNA in the neoplastic cells of B-cell lymphomas without interference from T-cells. The DNA synthetic fraction for the neoplastic cells correlated remarkabley well with histologic type and it should be an useful indicator of tumor proliferative activity. By comparing the DNA content of tumor B-cells with that of non-neoplastic T-cells from the same tissue, we detected minimal (2%) aneuploidy in neoplastic cells. We also observed two neoplastic populations with different DNA content in approximately 10% of the tumors suggesting ciclonality. In the present study we propose to answer the following: What is the nature of aneuploidy detected by FCM in the B-cell lymphomas? Does it only reflect genomic size or is it in part due to binding properties of the fluorescent dyes to neoplastic or cycling cells? Is the conversion of low grade lymphomas into high grade lymphomas accompanied by ploid or kinetic changes? What is the nature of the """"""""biclonality"""""""" observed in 10% of B-cell lymphomas? Since most of the second """"""""clones"""""""" in these cases are near-tetraploid, do they represent arrested or cycling cells? If they are cycling, is the DNA synthetic rate of the aneuploid cells higher or lower than that of the diploid cells of the same tumor? Are multiple """"""""clones"""""""" related to previous therapy? Is there a relationship between T-cell subset distribution within the lymph node and the kinetic properties of the neoplastic B-cells? We plan to answer these questions as follows: Modal chromosome number determined on metaphases obtained in direct preparations of tumor cells or in normal and tumor cells induced by stimulation with B-cell mitogens will be compared with the DNA index obtained by FCM. Various DNA fluorescent probes will be tested to determine if ploidy changes reflect DNA binding properties of the fluorescent dyes. DNA indices and kinetics properties of recurrent tumors will be compared with those obtained in previous biopsies. Simultaneous analysis of cellular DNA content and bromodeoxyuridine incorporation will be performed to establish if aneuploid cells are cycling and their rate of DNA synthesis. Correlations will be made between therapy prior to biopsy and tumor ploidy. T-lymphocyte subsets within the tumors will be analyzed to assess if a relationship exists between T-cell distribution and kinetic properties of the neoplastic B-cells.
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