Genetic characterization of chronic lymphocytic leukemia (CLL) cells by fluorescence in situ hybridization (FISH) has identified genetic aberrations in 80% of CLL patients. In contrast, conventional metaphase cytogenetics detected chromosomal aberrations in only 40–50% of all cases. Immunostimulatory CpG-oligonucleotides (CpG-ODN) in combination with interleukin 2 (IL-2) have recently been reported to induce proliferation in CLL B cells. Therefore, we evaluated this stimulus for its efficacy in metaphase generation for the detection of chromosomal aberrations by cytogenetic banding techniques. In addition these results were compared with data obtained by interphase FISH. For metaphase induction 107 cells were cultured in growth medium with the CpG-ODN DSP30 and IL-2. After 2 days colchicine was added for another 24h before preparation. Of the 133 samples that were included in this study, all cases could be successfully analyzed by interphase FISH with a rate of 79% aberrations like deletions of chromosomes 13q (57%, in 38% as sole abnormality), 11q (17%), trisomy 12 (13%), 6q (7%), 17p (6%) and translocations involving 14q32 (4%). By FISH 55% of all samples showed a single aberration, 22% two aberrations and 2% 3 aberrations. In comparison, 126 cases (95%) could be successfully analyzed by cytogenetic banding techniques. 102 (81%) of the 126 samples showed chromosomal aberrations, which involved all different chromosomes. 9 cases with a normal karyotype in conventional cytogenetics revealed genetic aberrations by FISH. In all but 1 of these cases the aberrant clone represented < 30% of the cell population. In addition to the aberrations that were detected by the FISH probes, a substantial amount of other aberrations was revealed by chromosome banding analysis. Interestingly, 10 cases of a total of 27 cases without abnormalities detected by FISH displayed aberrations in chromosome analysis indicating that the true amount of CLL patients with aberrations exceeds 79%. Overall, 47 samples (37%) showed chromosomal aberrations in chromosome banding analysis in addition to FISH analysis. Compared to FISH analysis, which detected 2 cases with 3 aberrations, metaphase cytogenetics detected 22 cases with 3 or more unbalanced aberrations, which can be considered as a complex aberrant karyotype. Loss of p53 referred to as del(17p13) in FISH has attracted considerable attention, because of the poor clinical outcome of affected patients. In our study, all del(17p13) cases (n=7) displayed either a loss of the short arm of chromosome 17 (n=6) or a complete loss of chromosome 17 (n=1) indicating that chromosomal regions in addition to the p53 locus might be relevant for this phenotype. Furthermore, numerous recurrent aberrations have been identified in this study beyond the aberrations that are detected by FISH. Of note are gains of 2p and 3q, losses in 11q13 and gains in 11q23–25, gains in 13q31–34, gains of 17q21–25 and cases with trisomy 18 and 19, which occurred in parallel to trisomy 12. The results of the present study show that immunostimulatory CpG-oligonucleotides plus interleukin 2 are a simple and cheap stimulus for efficient metaphase induction in CLL. The rate of aberrations detected by conventional banding techniques was even slighty increased compared to FISH analysis, however, the complexity of cytogenetic aberrations is underestimated by FISH analysis in a large portion of CLL cases (37%).

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