Conventional cytogenetics (CC) of B-cell lymphoproliferations remains difficult because of low mitotic in vitro activity of the leukemic cells. Therefore, mitogen stimulation of B-cells is required to analyze an adequate number of metaphases. Chromosome abnormalities using CC with TPA can be detected in up to 50% of chronic lymphocytic leukemia (CLL), but the development of FISH techniques has allowed the detection of selected chromosome abnormalities in more than 80% of CLL. However, FISH is restricted, since information is only available for the genes/loci for which probes are used. So, for a comprehensive genetic analysis, CC is essential because it provides an overview of all microscopically visible chromosome abnormalities important as prognostic factors. The use of the immunostimulatory CpG-oligonucleotide DSP30 to effectively induce cell cycle progression of CLL cells in vitro has been reported. This proliferation is markedly enhanced upon addition of Interleukine-2 to cultures. To our knowledge and to date, there has been no direct comparison of classical TPA versus DSP30+IL-2. DSP30+IL-2 stimulation has been successfully tested in CLL but no data are available for other lymphoproliferations. We cultured 132 B-cell lymphoproliferations (80 CLL and 52 other B-cell lymphoid neoplasms (BCLN)) in parallel, in presence of TPA or DSP30+IL-2. The objective of this study was to evaluate the suitability of DSP30+IL-2 as a routine B-cell mitogen for metaphase cytogenetics. CC successfully analyzed 94.9% of CLL and 98.1% of BCNL with more than 80% abnormal karyotypes. For CLL, failures of karyotypes were more frequent in cultures with DSP30+IL-2 (14%) than in those with TPA (4%). The rate of failures were similar for BCLN (6% versus 4%). For CLL, there were significantly fewer metaphases in DSP30+IL-2 than in TPA spreads (mean of 50 versus 72 metaphases per slide respectively, p=0.0007), as well as for BCLN (mean of 50 versus 71 metaphases per slide respectively, p=0.009). However, the proportion of abnormal metaphases was significantly higher in DSP30+IL-2 (mean of 59%) compared to TPA cultures (mean of 26%, p=0.00265) for CLL and for BCLN (mean of 57% versus 33%, p=0.0065). Stimulation with DSP30+IL-2 allowed to detect more abnormalities, more abnormal subclones and more complex karyotypes in CLL and in the majority of BCLN. Though FISH exploration using a large probe panel has yielded valuable results in lymphoproliferative diseases, it underestimates the heterogeneity of chromosomal aberrations. Complexity of chromosomal changes, recently associated to unfavorable outcomes, can only be assessed with CC. In conclusion, our results in both CLL and BCLN indicate that the immunostimulatory oligonucleotide DSP30 in combination with IL-2 is an easy and efficient stimulus in metaphase generation for routine chromosomal banding.

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