Chromosome Banding Analysis Allows a More Precise Classification of CLL Than Interphase FISH: A Study on 446 Cases.

In CLL data from chromosome banding analysis have been scarce due to the low proliferative activity in vitro. We improved the cultivation technique using an immunostimulatory CpG-oligonucleotide DSP30 and IL-2 leading to a high success rate of chromosome banding analysis in routine diagnostics. Since August 2005 446 CLL were analyzed in parallel with chromosome banding analysis (CBA) and interphase-FISH. Diagnosis of CLL was established by standard criteria based on cytomorphology and immunophenotyping. The FISH panel included probes for the detection of trisomy 12, IGH-rearrangements and deletions of 6q21, 11q22.3 (ATM), 13q14 (D13S25 and D13S319) and 17p13 (TP53). 440/446 (98.7%) cases could be successfully stimulated for metaphase generation and are the basis of this study. 370/440 (84.0%) cases showed chromosomal aberrations in CBA while abnormalities were detected by FISH in 353 of 440 (80.2%) successfully evaluated cases. Overall 452 abnormalities were detected by FISH and 788 abnormalities by CBA. Based on FISH results 277 cases showed 1, 67 cases 2, 8 cases 3 and 1 case 4 abnormalities, respectively. In CBA at least 1 aberration was detected in 177, 2 in 98, 3 in 45, 4 in 19, and 5 or more aberrations in 31 patients. In 31 of 87 cases (35.6%) showing no aberrations in FISH abnormalities were detected in CBA. On the other hand 14 of 70 cases (20.0%) with a normal karyotype demonstrated abnormalities using FISH. In 7 of these cases CBA missed the abnormalities due to the small size of the aberrant clone or insufficient proliferation of the aberrant clone in vitro and in another 7 cases due to the small size of the 13q deletion not visible in CBA. Using CBA, in total 97 balanced translocations, 169 unbalanced translocations leading to gain and/or loss of genetic material, 368 deletions, 77 gains of whole chromosomes, 40 losses of whole chromosomes, and 37 other aberrations were observed. Only 17 of 97 balanced translocations involved the IGH gene. In 28 cases balanced translocations involved the breakpoint 13q14. Although these translocations were reciprocal and seemed balanced in CBA FISH demonstrated a 13q14 deletion in the breakpoint region. Therefore, based on CBA cases with 13q deletion could be subdivided into 3 different categories: 1. del(13q) sole, 2. del(13q) with additional abnormalities and 3. del(13q) due to a reciprocal translocation. This genetic heterogeneity might account for differences in clinical outcome. In cases with TP53 deletions the number of chromosome abnormalities was higher compared to cases without TP53 deletion (mean 5.0 vs 1.5, p<0.0001). In conclusion, CBA offers important information in addition to interphase FISH in CLL. 1) CBA detects chromosome abnormalities which can not be detected with a standard interphase FISH panel. These additional abnormalities could explain heterogeneous clinical outcome. 2) CBA provides new biological insights into different CLL subclasses based on a much more heterogeneous pattern of cytogenetic abnormalities as assumed so far for CLL. Therefore, prospective clinical trials should evaluate the prognostic impact of these additional abnormalities that now can be identified by chromosome banding analysis.