Optimized mitogen stimulation induces proliferation of neoplastic B cells in chronic lymphocytic leukemia: significance for cytogenetic analysis

We tested the effects of interleukin-2 (IL-2), human recombinant tumor necr osis factor alpha (TNF-alpha), Staphylococcus cus aureus Cowan I (SAC), TPA , and their combinations, using a standard thymidine incorporation assay, i n order to identify an optimal mitogen combination (OMC) for 24 consecutive patients with B-cell chronic lymphocytic leukemia (B-CLL). The combination that induced the highest thymidine incorporation was chosen as the OMC for each patient. Among 14 mitogen combinations tested, there were six differe nt OMCs, of which the most frequent was TNF-alpha + IL2. It was the OMC in 9 of 24 cases. The other OMCs were TNF-alpha + TPA1 (5/24), SAC + IL-2 (5/2 4), TPA1 + IL-2 (3/24), TPA10 + IL-2 (1124), and TNF-alpha + TPA10 + IL-1 ( 1/24). The mitogenic power of the selected OMC in each case was then evalua ted both by the combination of immunophenotyping and molecular cytogenetic techniques known as MAC (Morphology, Antibody, Chromosomes) and standard ch romosome analysis, After OMC stimulation, the levels of DNA synthesis and B -cell proliferation (mitotic index) were, on average, 10-fold higher than t hose observed after standard TPA stimulation (P< 0.0001). The proportion of mitotic B cells exceeded the proportion of mitotic T cells in 70.1 % of th e cases after OMC stimulation, After TPA stimulation, 7.7 % +/- 2.5 % of al l mitoses were B-cell mitoses, whereas after OMC stimulation this proportio n rose to 57.9 % +/- 5.3 %. The frequency of clonal chromosomal aberrations increased from 46 % after TPA stimulation to 79 % after OMC stimulation. T he clonal aberrations del(6q), del(11q), and/or del(13q) were observed in 2 6%, 32%, and 42 % of the patients with the respective clonal chromosomal ab errations, whereas the corresponding frequencies after TPA stimulation were only 4%, 21 %, and 17 %. When the lineage involvement of cells with clonal chromosomal aberrations from three patients was analyzed, the aberrations were found to be restricted to B cells only, and in one patient to a minor subset of B cells. The results demonstrate that an individually chosen OMC induces a high rate of proliferation in neoplastic B cells. We found deleti ons in 6q, 11q, and 13q at higher frequencies than reported previously, mos t probably as a result of an improved mitogenic response. The identificatio n of an optimal mitogen stimulation for each patient, prior to chromosome a nalysis, can well be expected to reduce the rate of false-normal results in the future, This is essential for accurate evaluation of the prognostic si gnificance of chromosomal aberrations in B-CLL.