Flow Cytometric Identification of Biclonal Disease Among 5,523 Patients with Chronic Lymphocytic Leukemia and Its Genetic Characterization

Abstract 3608

Chronic lymphocytic leukemia (CLL) is an indolent disease with largely heterogeneous clinical course. While the diagnosis is made based on the characteristic immunophenotype as determined by multiparameter flow cytometry (MFC) another aspect of major clinical importance is the estimation of the prognosis which includes the analysis of chromosomal aberrations, the IGHV mutational status as well as the expression of CD38 and ZAP-70. In general, the methods used to determine these parameters are applied in the assumption of analyzing one homogeneous leukemic population and are evaluated accordingly. The potential presence of subpopulations and even subclones may not always be considered adequately in this regard. We identified 76 out of 5,523 patients (1.4%) in whom MFC identified biclonal disease based on the presence of both kappa- and lambda-light chain restricted leukemic subpopulations. In 45 of these cases fluorescence in situ hybridization (FISH) analysis was performed applying a standard set of probes for the detection of del(6q), del(11q22.3) (ATM), trisomy 12, del(13q14) (D13S25, D13S319), and del(17p13) (TP53). In 17 of the 76 patients a chromosome banding analysis (CBA) was performed and the IGHV mutational status was determined in 38 of 76 patients. The patients` ages ranged from 47.7 to 88.0 years (median, 71.3 years), 50 patients were male. The median WBC count amounted to 19 ×10e9/l (range, 0.6–237 ×10e9/l). In most cases the kappa-light chain restricted subpopulation was larger than the lambda-light chain restricted one. The median values and ranges for the respective percentages of subpopulations amounted to: kappa, 24%, 1%-88%; lambda, 12%, 1%-85%. The respective peripheral blood concentrations amounted to: kappa, 3.68 ×10e9/l, 0.12–142 ×10e9/l; lambda, 2.37 ×10e9/l, 0.01–128 ×10e9/l. The median ratio kappa population/lambda population amounted to 1.9 (range, 0.05–76). FISH analysis identified del(6q) in 2/44 (4.5%) cases, del(11q) in 2/44 (4.5%), trisomy 12 in 7/44 (15.9%), del(13q) in 28/45 (62.2%), del(13q) as sole aberration detected by FISH in 23/43 (53.5%), and del(17p) in 1/45 (2.2%). In three cases more than one aberration was detected by FISH: two cases with del(11q) and del(13q) and one case with trisomy 12 and del(13q). While in two of these three cases the size-ratios of the respective subpopulations were similar in MFC and FISH analysis (1.7:1 vs. 4.3:1 and 2.0:1 vs. 1.5:1) this was not true for the third case (15.5:1 vs. 1.1:1). The further two cases could be considered in line with both methods detecting independent clones. In the latter case the chromosomal aberrations were present in 54% and 59% of the cells and the subpopulations detected by MFC amounted to 62% and 4%. Thus, both chromosomal aberrations must be considered to coexist in one population and not related to the two subpopulations detected by MFC. Overall, however, no clear-cut conclusions can be drawn from FISH results regarding the presence of independent subpopulations and therefore we next focused on the results of CBA. Within the 17 patients analyzed by CBA 12 cases showed an aberrant karyotype (70.6%). In four of these cases more than one clone was identified by differences in the chromosomal aberrations, respectively. In three cases chromosomal evolution was suggested by shared aberrations in both clones and additional aberrations in one of both clones only, respectively. Conversely, in the fourth case two completely different aberration patterns were observed. In 14 out of the 38 patients (36.8%) in whom an IGHV mutational analysis was performed two independent clones were identified by the presence of two different B-cell receptor rearrangements. The presence of biclonal disease had no impact on the clinical outcome of the patients as assessed by time to therapy and overall survival. This data indicates that subpopulations can be identified in a significant number of patients with CLL based on the immunophenotype as well as on the cytogenetic and molecular genetic level. These subpopulations at least in part must be considered as subclones with differing genetic background. These subclones may be associated with differing clinical courses. This data therefore suggests to vigorously screen patients with CLL for subpopulations by MFC and to comprehensively characterize positive cases cytogenetically by CBA and FISH analysis as well as on the molecular genetic level.